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• Published: 09 July 2024

Investigating the impact of 2-OHOA-embedded liposomes on biophysical properties of cancer cell membranes via Laurdan two-photon microscopy imaging

• Xuehui Rui 1 ,
• Yukihiro Okamoto 1 ,
• Shuichiro Fukushima 2 ,
• Nozomi Morishita Watanabe 1 &
• Hiroshi Umakoshi 1  

Scientific Reports volume  14 , Article number:  15831 ( 2024 ) Cite this article

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• Drug delivery
• Membrane biophysics

2-Hydroxyoleic acid (2-OHOA) has gained attention as a membrane lipid therapy (MLT) anti-cancer drug. However, in the viewpoint of anti-cancer drug, 2-OHOA shows poor water solubility and its effectiveness still has space for improvement. Thus, this study aimed to overcome the problems by formulating 2-OHOA into liposome dosage form. Furthermore, in the context of MLT reagents, the influence of 2-OHOA on the biophysical properties of the cytoplasmic membrane remains largely unexplored. To bridge this gap, our study specifically focused the alterations in cancer cell membrane fluidity and lipid packing characteristics before and after treatment. By using a two-photon microscope and the Laurdan fluorescence probe, we noted that liposomes incorporating 2-OHOA induced a more significant reduction in cancer cell membrane fluidity, accompanied by a heightened rate of cellular apoptosis when compared to the non-formulated 2-OHOA. Importantly, the enhanced efficacy of 2-OHOA within the liposomal formulation demonstrated a correlation with its endocytic uptake mechanism. In conclusion, our findings underscore the significant influence of 2-OHOA on the biophysical properties of cancer plasma membranes, emphasizing the potential of liposomes as an optimized delivery system for 2-OHOA in anti-cancer therapy.

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Introduction.

Plasma membrane fluidity is an important factor that influences cancer cell adhesion and migration. One of the most important properties of cancer cells is altered lipid metabolism, and consequently, abnormal cell membrane composition 1 . The composition and fluidity of the of cancer cell plasma membranes vary across different cell types. For instance, glioma cells exhibit higher fluidity than normal brain cells 2 . Increased cancer cell membrane fluidity correlates with higher metastases rate, while lower fluidity hinders the motility of cancer cells during the epithelial–mesenchymal transition (EMT) 3 , 4 . Modulation of membrane lipid composition and organization is actively being developed as an effective therapeutic strategy for cancer treatment, and is recognized as membrane lipid therapy (MLT).

2-OHOA (2-hydroxyoleic acid) is the world’s first conditionally approved MLT drug for the treatment of solid tumors 5 . Researchers have focused on 2-OHOA because of its unique ability to activate sphingomyelin synthase (SMS), leading to an abundance of sphingomyelin (SM) content in cell plasma membranes 6 , 7 . This augmentation of SM levels significantly reduces cancer cell plasma membrane fluidity and prompts the translocation of Ras and the capping of Fas receptor, which subsequently inhibits downstream cell signaling pathways, resulting in the apoptosis and autophagy of cancer cells 8 , 9 , 10 . Exploiting this SMS activation capability, 2-OHOA has been developed for the treatment of solid tumors, with a particular emphasis on its effectiveness in glioma treatment.

Although 2-OHOA is non-toxic anticancer drug, its single-chain lipid structure makes it challenging to dissolve in aqueous solutions. The current method of administering 2-OHOA is through oral delivery, which requires high dosage levels and limits its anti-cancer efficacy. To overcome this obstacle, previous researchers have developed 2-OHOA into various nano drug delivery system (nano-DDS) 11 , 12 , 13 .

To assess the potential of the nano-DDS for delivering 2-OHOA, we embedded 2-OHOA within DOPC-based liposomes. The liposome formulation is a widely utilized nano-drug delivery system, well-researched for its properties and performance. The large inner water pool of liposomes provides high drug loading capacity, potentially facilitating co-delivery of 2-OHOA with other hydrophilic drug payloads 13 . The essential characteristics of the liposomes, including hydrodynamic diameter, ζ-potential and water trapping volume, were investigated. Given that, a significant proportion of nanoparticles tends to accumulate in the liver following intravenous injection due to the clearance of reticuloendothelial system (RES) 14 , and considering the clinical trial focus of 2-OHOA on glioma, this study leveraged both hepatoma (HepG-2) and glioma (NP-8) cells as model cells for 2-OHOA-embedded liposomes treatment. Previous studies have primarily concentrated on elucidating the anti-cancer mechanism of 2-OHOA and exploring the associated cellular signaling pathways 6 , 7 , 8 , without a comprehensive examination of the variations in cell membrane biophysical properties induced by 2-OHOA. This study focused on examining variations in the polarity, fluidity, and lipid packing of cancer cell membranes following treatment with 2-OHOA-embedded liposomes. The overarching objective is to assess the therapeutic efficacy of these liposomes, particularly focusing on the modulation of membrane biophysical properties in the context of 2-OHOA’s anti-cancer potential.

In this study, Laurdan (6-Dodecanoyl-2-Dimethylaminonaphthalene) fluorescence probe was employed to examine variations in cellular membrane polarity/fluidity subsequent to treatment with 2-OHOA-embedded liposomes via two-photon microscopy imaging as well as fluorescence photometer. Laurdan is frequently used as a membrane probe because of its large excited-state dipole moment, which allows it to report the extent of water penetration into the bilayer surface as a result of the dipolar relaxation effect 15 . When solubilized in a lipid bilayer structure, Laurdan senses the environment and its spectrum shifts according to the water content of the bilayer. Water penetration has been correlated with membrane fluidity and lipid bilayer packing 16 , 17 , 18 . Compared to other fluorescent probes used for cell membrane analysis (e.g., DPH, Diphenylhexatriene; Nile Red), Laurdan exhibits dual fluorescence spectrum properties. Its emission spectrum is highly sensitive to changes in membrane phase and polarity. By measuring Laurdan’s fluorescence spectrum, we can distinguish between liquid crystalline and gel phases of membranes. This property is not commonly found in many other fluorescent probes. Additionally, Laurdan’s fluorescence properties facilitate precious quantitative analysis. The generalized polarization ( GP ) value of Laurdan-stained cells was calculated to quantify the cell membrane polarity/fluidity variations. In general, a high GP value indicates low membrane polarity or reduced membrane fluidity, whereas a low GP value implies the opposite. Microscopy can provide a unique tool for the study of membrane heterogeneity, and two-photon excitation has the additional advantages of inducing less damage to live cells and the significantly decreased photo-bleaching of Laurdan.

Another environment-sensitive fluorescent probes, LipiORDER, was used to corroborate Laurdan imaging results. LipiORDER is a pyren-based solvatochromic fluorescent dye which can be inserted into the lipid bilayer and changes its fluorescent properties in response to the environment 19 . Generally, the liquid ordered (Lo) phase is a highly packed lipid bilayer with low fluidity, whereas the liquid disordered (Ld) phase is a sparsely packed lipid bilayer with high fluidity. Based on the lipid packing of the lipid bilayer membrane, LipiORDER changes its fluorescent color from green on the Lo membrane to red on the Ld membrane. The lipid packing of the cell membrane can be approximated and compared by quantifying the LipiORDER red/green fluorescence intensity ratios (R/G ratio) 20 .

These methodologies enabled the assessment and visualization of alterations in cell membrane polarity, fluidity and lipid packing status, facilitating the evaluation and refinement of various formulations of 2-OHOA-embedded liposomes.

Result and discussion

Characterization of 2-ohoa-embedded liposomes.

After the liposome formulation, the DOPC amount was quantified using LabAssay Phospholipid kit (supplementary information Table S-1 ). The particle size, polydispersity index (PDI) and ζ-potential results are shown in Fig.  1 a–c. Compared with DOPC-only liposomes, the presence of 2-OHOA in liposomes (9-1, 7-3, 5-5 and 3-7 formulations) slightly reduced mean hydrodynamic diameters. This phenomenon is attributed to the fact that, 2-OHOA incorporation decreases the packing order and increases the fluidity of liposomal membranes through intercalation between phosphorylcholine (PC) molecules; the kinked structure of the 2-OHOA hydrocarbon residue, akin to oleic acid, is believed to intensify this effect 13 , 21 . Increasing membrane fluidity can promote the formation of smaller liposomes 22 . In the absence of DOPC lipid, 2-OHOA formed particles with a hydrodynamic diameter at 1634.0 ± 180.4 nm and a PDI of 1, indicating that 2-OHOA alone could not form stable nano-sized particles in aqueous solution at a neutral pH.

2-OHOA-embedded liposome characterization results. ( a ) Hydrodynamic diameter; ( b ) PDI and ( c ) ζ-potential. ( d ) Water trapping volume of 10 mM different liposomes. For DLS and ζ-potential investigation, all the liposome samples were diluted into 100 μM using D-PBS and the measurement were carried out at pH 7.4 and 25 °C, error bars represent ±  s.d , n  = 3.

Liposomes composed solely of DOPC exhibit ζ-potential value of − 2.40 ± 0.90 at a pH of 7.4. However, even low concentrations of incorporated 2-OHOA significantly reduced the ζ-potential of the liposomes. Moreover, ζ-potential of the 2-OHOA-embedded liposomes displayed a descending trend that aligns with the ratios of the 2-OHOA component. This is because of the carboxylic groups from 2-OHOA dissociate in aqueous solution, resulting in a negative surface charge 23 . Considering the structural stability of liposomes, the DOPC-only, 9-1, 7-3 and 5-5 liposome formulations were selected for the following research.

Water trapping efficiency is an important liposomal property in relation to the use of liposomes as drug carriers. The water trapping assay results (Fig.  1 d) confirmed the existence of the inner aqueous phase of the prepared liposomes. Compared to the DOPC-only liposomes, the 2-OHOA-embedded liposomes showed a reduction of water trapping volume, which was attributed to the decreased particle size.

Laurdan staining and investigation

Investigating the heterogenicity of cell membrane fluidity.

In this study, the fluctuation in cancer cell membrane fluidity was chosen as the key parameter for assessing the MLT performance of the 2-OHOA-embedded liposomes. Different cells exhibit distinct membrane fluidity characteristics 24 , 25 , 26 . Using Laurdan GP quantification offers an efficient method for analyzing these fluidity variations. Cell membrane GP values can be influenced by factors, such as fluorescence acquisition settings and cell culture conditions 27 , 28 . To ensure consistency and establish a reliable baseline for cell status, all operations and measurement settings were consistently maintained.

As shown in Fig.  2 , both HepG-2 and NP-8 cells exhibited heterogeneous GP patterns, consisting of ordered (higher GP values) and fluidic (lower GP values) membrane regions. The calculated average GP value of HepG-2 cell membrane was approximately 0.32, while the NP-8 cell membrane had an average GP value of approximately 0.2. A lower average GP value indicates a higher fluidity of the NP-8 cell membrane. Particularly, the high- GP regions are mainly distributed in the cell–cell contact regions and the cell margins, reflecting the condensed structure of the cell membranes 29 .

Two-photon microscopy Laurdan GP images and GP histograms of NP-8 (upper row) and HepG-2 (lower row) cells. From left to right, the first column displayed the Laurdan fluorescence images of blue channel (436/20 nm); the second column displayed the Laurdan fluorescence images of cyan channel (495/25 nm); the third columns display the pseudo-colored GP images; and, the fourth column displayed the pixel GP histograms obtained from the corresponding GP images. In the GP images, the orange represents maximum GP (1.0) and pure blue represents minimum GP (− 1.0). Scale bars represent 40 μm. In the GP histograms, the distribution of pixel- GP was deconvoluted by fitting two Gaussian distributions (blue and red lines) to the experimental data (black line).

Laurdan fluorescence signals obtained from two-photon microscopy exhibit multiple pixel populations, corresponding to different membrane environments or lipid bilayer phases. To enable quantification and comparison, the pixel GP values derived from the GP images were normalized and presented in the form of histograms, referred to as GP histograms. The pixel count for a specific GP indicates the region of interest (ROI) associated with that particular population. The GP histogram of NP-8 cells (ranging approximately from − 0.5 to 0.8) displayed a broader distribution than that of HepG-2 cells (around − 0.1 to 0.8). This broader range of pixel GP values in NP-8 cells indicates greater diversity in cell membrane fluidity compared to HepG-2 cells.

Deconvolution helps to separate these pixel populations obtained from the GP images, providing a clearer understanding of the distinct lipid environments within the cellular membrane 29 . The GP histograms derived from the GP images were deconvoluted into two Gaussian distributions (Fig.  2 ). These distributions were categorized into a low- GP peak (illustrated by the red line), and a high- GP peak (illustrated by the blue line). The low- GP peaks are associated with the relatively fluidic membrane regions (shown as green in the GP images), and the high- GP peaks are associated with the more ordered membrane regions (shown as orange in the GP images). The deconvolution results are presented in Table S-2 . Both NP-8 and HepG-2 cells exhibited a border distribution at low- GP peaks (compared according to the full width at half maximum, FWHM), indicating the presence of a predominantly fluidic membrane on the cells, as evidenced by the high-coverage area of the green pseudo-color regions on the GP images. The low- GP peaks of the NP-8 cell membrane were centered at 0.116 ± 0.015, whereas those of the HepG-2 cells were centered at 0.279 ± 0.030. However, concerning the high- GP peaks, NP-8 showed a center at 0.481 ± 0.24, and HepG-2 cells showed a center at 0.500 ± 0.058, with no apparent difference. Furthermore, there was no statistically significant difference in the high- GP region coverage (calculated according to the area under curve, AUC) between NP-8 (20.54 ± 6.98%) and HepG-2 (22.32 ± 7.20%). These findings imply that the difference in membrane fluidity between NP-8 and HepG-2 cells is mainly attributed to variations in the fluidic membrane (Ld) regions, rather than in ordered membrane (Lo) regions.

Laurdan two-photon microscopy revealed heterogeneous membrane fluidity among cell membranes, and distinct characteristics between NP-8 and HepG-2 cells. The higher membrane fluidity and heterogenicity observed in the NP-8 cell membrane can be attributed to a significantly lower sphingomyelin (SM) content in glioma cell membranes, as reported in previous studies 6 , 30 . SM interacts favorably with cholesterol and establishes the co-localization of SM and cholesterol in cell plasma membranes. The formed SM/cholesterol-rich domains are more ordered than the surrounding phase in biological membranes 31 , 32 , 33 . SM could also reduce the lateral heterogeneity in cholesterol-containing membranes. Specifically, unsaturated SM is able to accommodate both phosphorylcholine and cholesterol, forming a single phase, and maintaining membrane lipids in a homogeneous phase 34 . The reduced level of SM in glioma cells is considered be associated with its higher membrane fluidity and lateral heterogenicity.

Evaluating the influence of DOPC liposome on cell membrane fluidity

In this study, the liposome formulations utilized DOPC lipid. Thus, to minimize any additional impact on cell membranes, it is crucial to confirm the influence of DOPC lipid on cell membrane fluidity. HepG-2 and NP-8 cells were treated with varying concentrations of DOPC-only liposomes, and cell membrane fluidity was monitored over 48 h using Laurdan and a fluorescence spectrometer; the results are shown in Fig.  S-1 a. For concentrations up to 500 μM and a treatment duration of 48 h, DOPC-only liposomes did not induce significant changes in either HepG-2 or NP-8 cell membrane GP values. This observation is supported by two-photon microscopy images of Laurdan-stained cells (Fig.  S-1 b,c). These findings justified the exclusion of the influence of DOPC lipids on cell membrane fluidity. Thus, 2-OHOA was considered as the primary factor influencing cancer cell membrane fluidity in this study.

Investigating the impact of 2-OHOA-embedded liposome on cell membranes

Following a 24 h treatment with 2-OHOA-embedded liposomes (containing 100 μM 2-OHOA) or free 2-OHOA (100 μM), noticeable abundant high- GP regions (depicted in orange) emerged in both NP-8 and HepG-2 cell membranes, as shown in Fig.  3 a. The calculated average GP values of cell membranes are summarized in Fig.  3 b. Notably, NP-8 cells exhibited a more pronounced elevation in the average GP values than HepG-2 cells, indicating a more substantial impact of 2-OHOA on the NP-8 cell membrane. Specifically, for NP-8 cells, after a 24 h treatment with 2-OHOA, an increased SM concentration was observed (Fig.  S-8 ). Analysis of normalized pixel GP histograms revealed distinct patterns of GP value elevation in the HepG-2 and NP-8 cell membranes. As shown in Fig.  3 c, NP-8 cell GP histograms exhibited a pronounced rightward shift after treatments. Conversely, in the case of HepG-2 cells, the GP histograms showed a subtle rightward shift.

Laurdan two-photon microscopy imaging results: ( a ) GP images of NP-8 cells and HepG-2 cells with and without treatments. Scale bars represent 40 μm. ( b ) Average GP values calculated from the GP images. ( c ) GP histograms obtained from the GP images. ( d ) Summarized low- GP peak centers of deconvoluted results before and after treatment. In treatment groups, cells were incubated for 24 h with either 100 μM DOPC, 2-OHOA-embedded liposomes containing 100 μM 2-OHOA or 100 μM free 2-OHOA. Error bars represent ±  s.d , ( n  = 4‒8). ns : no significant difference; ***: p  < 0.001; ****: p  < 0.0001.

After deconvolving the GP histograms, a summary of the low- GP peak centers is provided in Fig.  3 d. The low- GP peak shifting pattern reveals variations in high-fluidity membrane regions. After treatment, the NP-8 GP histograms exhibited a remarkable rightward shift. Additionally, as shown in Table S-2 , the high- GP peak centers showed a slight rightward shift after treatment, accompanied by a significant increase in high- GP coverage (indicative of ordered membrane abundance). These findings suggest that, after 2-OHOA-embedded liposome treatment, NP-8 cells demonstrated a notable reduction in fluidity within the high-fluidity (Ld) membrane regions and an abundance of membrane areas exhibiting liquid-ordered (Lo) characteristics. For HepG-2, post 2-OHOA embedded liposome treatments, both low- GP peaks and high- GP peaks showed a subtle rightward shift. However, the high- GP coverage did not show a significant increase. The GP value variations of HepG-2 cell membranes were more moderate compared to NP-8 cells. Comparing the impacts of different liposome formulations, the 9-1 liposomes induced the most significant average GP increase (from ~ 0.2 to ~ 0.43) and the most pronounced shift in low- GP peak center (from ~ 0.12 to ~ 0.32) in NP-8 cells. Meanwhile, the 5-5 liposome induced a slightly higher average GP increase in HepG-2 cells compared to other formulations.

To assess the enhancement of liposome formulation, variations in the cell membrane Laurdan GP after formulated or non-formulated 2-OHOA treatments were investigated using a fluorescence spectrometer. The 9-1 liposome was chosen as the optimized formulation for NP-8 cells, and the 5-5 liposome was chosen as the optimized formulation for HepG-2 cells. Cells were incubated with varying concentrations of 2-OHOA-embedded liposomes or free 2-OHOA for 24 h, and the GP variation results are shown in Fig.  S-2 . Generally, the cell membrane GP elevation showed a 2-OHOA dose-dependent manner. Whereas the liposome formulations induced a more pronounced GP increase in both NP-8 and HepG-2 cells, this result is consistent with the two-photon microscopy observation results, reaffirming the enhancement of 2-OHOA performance after liposome formulation.

It is noteworthy that, an abundance of lipid droplets (LDs) in HepG-2 cells was observed after treatment with 2-OHOA-embedded liposomes or free 2-OHOA. The LDs were stained with Lipi-Red, a fluorescence probe designed for lipid droplet visualization, and observed by fluorescence microcopy (Fig.  S-3 a). After 2-OHOA treatment, a marked abundance of LDs was observed in HepG-2 cells. However, this phenomenon was not observed in NP-8 cells. A similar occurrence was reported in specific cell lines exposed to 2-OHOA 8 , 35 . This may be attributed to the structural similarity between 2-OHOA and oleic acid (OA), both of which belong to the monounsaturated omega-9 fatty acid category. An excess influx of OA into HepG-2 cells appears to trigger an interaction between LD and mitochondria, predominantly fostering LD growth 36 . 2-OHOA was found to induce a similar influence on HepG-2 cells. Notably, the lipid droplets attached on HepG-2 plasma membranes showed an extremely high GP value (0.5–0.8), contributing to the elevated GP values observed in HepG-2 cells after treatment (Fig.  S-3 b). This helps to explain the HepG-2 cells GP histograms showing an increase at GP  ~ 0.6 after 2-OHOA treatment (Fig.  3 c), which is attributed to the abundance of LDs.

In general, Laurdan two-photon microscopy serves as a powerful tool for visualizing the cell membrane fluidity variation. Treatment with 2-OHOA significantly increased the Laurdan GP values in both NP-8 and HepG-2 cells, with NP-8 cells exhibiting greater sensitivity. The distinct patterns in GP value elevation reveal the varied responses of NP-8 and HepG-2 cell to 2-OHOA treatments. The reduction in NP-8 cell membrane GP value is attributed to the overall decrease in plasma membrane fluidity, whereas HepG-2 cells exhibited reduction in a plasma membrane fluidity accompanied with an abundance of lipid droplets. Comparatively, the liposome formulation intensified the impact of 2-OHOA on NP-8 and HepG-2 cells, exhibiting promising enhancements in GP value alterations.

LipiORDER staining and investigation

To further validate the alterations in cell membrane lipid packing following treatment, we used another solvatochromic fluorescence probe, LipiORDER, to visualize changes in cell membrane lipid packing statues. When incorporated into the cell membrane, LipiORDER senses lipid packing and exhibit a fluorescent color shift, transitioning from green on liquid-ordered (Lo) membrane to red on liquid-disordered (Ld) membrane. Lipid packing in the cell membrane can be approximated and compared by quantifying the LipiORDER red/green fluorescence intensity (R/G) ratio. Figure  4 a,b depicts the pseudo-colored R/G ratio images of NP-8 and HepG-2 cells before and after treatments. The average R/G ratios were summarized in Fig.  4 c. Although both Laurdan GP value and LipiORDER R/G ratio provide insights into membrane properties, they offer complementary information rather than directly measuring the same aspect. Therefore, understanding both GP value and R/G ratio can provide a more comprehensive understanding of the membrane characteristics, including both fluidity and lipid packing order. Correlation analysis between cell GP value and R/G ratio variations before and after 2-OHOA treatments (summarized in Fig.  4 d) revealed a linear relationship, solidifying the proportional relation between cell membrane fluidity/polarity and cell membrane lipid packing.

LipiORDER R/G ratio images of ( a ) NP-8 cells and ( b ) HepG-2 cells before and after treatments, green color represents Lo phase (low fluidity), and red color represents Ld phase (high fluidity), magnification is 40 times; Scar bar = 40 μm. ( c ) Summarized R/G ratio results of acquired images, error bars represent ±  s.d , n  = 5. (d) The GP value—R/G ratio correlations of NP-8 and HepG-2 cells with or without treatments. ( ● ): Blank control; (■): freee-2-OHOA treated; (▲): 9-1 liposome treated; ( ▼ ): 7-3 liposome treated; (♦): 5-5 liposome treated. Error bars represent ±  s.d , n  = 3. In treatment groups, cells were incubated for 24 h with 100 μM DOPC, 2-OHOA-embedded liposomes containing 100 μM 2-OHOA or 100 μM free 2-OHOA.

Initially, both HepG-2 and NP-8 cells displayed heterogeneous plasma membrane lipid packing (Fig.  4 a,b), comprising Lo phase (green) and Ld phase (red). NP-8 cells generally exhibited a more disordered membrane lipid packing than HepG-2 cells. Treatment with DOPC-only liposomes did not induce noticeable changes in average R/G ratios. While, after 2-OHOA treatments (formulated or non-formulated), significant R/G ratio decreases were observed in both HepG-2 and NP-8 cells, indicating an increase in membrane lipid packing. Also, NP-8 and HepG-2 cells exhibited distinct R/G ratio variations after 2-OHOA treatments. NP-8 cells showed an abundance of Lo phase on the cell plasma membrane after 2-OHOA treatments (Fig.  4 a). Whereas HepG-2 cells showed a reduced R/G ratio in plasma membrane, accompanied with a marked green colored region in cytoplasm (Fig.  4 b), which is associated with the abundant lipid droplets stained by LipiORDER. As mentioned previously, 2-OHOA treatment induced significant abundance of LDs in HepG-2 cells, but not observed in NP-8 cells. The LDs exhibit intense green fluorescence when staining with LipiORDER 19 , 37 . Notably, in comparison to two-photon microscopy, fluorescence microscopy captures a thicker section, leading to the detection of more cytoplasmic LDs and resulting in a pronounced decrease in the average R/G ratio. Furthermore, analysis of the R/G ratio across various groups (Fig.  4 c) reveled that, compared to the non-formulated 2-OHOA treatment, the liposome formulated 2-OHOA induced more significant R/G ratio decrease. Specifically, 9-1 liposomes induced the most pronounced R/G ratio decrease in NP-8 cells and the 5-5 liposomes induced most significant R/G ratio decrease in HepG-2 cells. This trending is closely aligning with the Laurdan GP investigation results, solidified that, the liposome formulation significantly enhanced the impact of 2-OHOA on cancer cells.

In summary, the LipiORDER R/G ratio results are consistent with the Laurdan GP value results. Treatment with 2-OHOA-embedded liposomes, as well as free 2-OHOA, notably improved the packing of cancer cell membrane lipids, effectively reducing cell membrane fluidity. However, distinct patterns of R/G ratio variation were also observed in NP-8 and HepG-2 cells. The abundance of LDs induced by 2-OHOA treatment significantly contributed to a decrease in R/G ratio in HepG-2 cells. Furthermore, different liposome formulations resulted in varying extents of R/G ratio decrease in both NP-8 and HepG-2 cells. These findings underscore the impact of 2-OHOA on cancer cell membrane lipid packing, while also suggesting that different liposome formulations, despite containing the same amount of 2-OHOA, exhibited diverse effects on cancer cell membrane properties. In the subsequent study, we will investigate the factors that influence the performance differences among various 2-OHOA-embedded liposome formulations.

Cellular internalization efficacy and endocytic mechanism

Considering the diverse effects induced by various liposome formulations on cell membrane fluidities, we hypothesize that differences in cell internalization efficacy and cellular uptake mechanisms are likely contributors to variations in 2-OHOA performance (as illustrated in Fig.  S-4 ). The efficiency of cellular internalization and endocytic mechanisms of nanoparticles (NPs) are dependent on various factors, including the physicochemical and surface properties of NPs 38 , 39 . Importantly, different cell types may utilize distinct endocytic pathways for internalization of the same NPs 40 , 41 . To improve the drug delivery efficacy of 2-OHOA, it is crucial to evaluate both cellular internalization efficiency and the endocytic mechanism.

The results of cellular internalization efficiency are presented in Fig.  5 a,b. After a 6-h incubation, liposomes of different formulations did not exhibit significant differences in cellular internalization efficiency in NP-8 cells. However, for HepG-2 cells, the 5-5 liposome showed a slightly enhance internalization efficacy, potentially contributing to a slightly higher impact of the 5-5 liposome on HepG-2 cells. To assess the endocytic mechanism of 2-OHOA-embedded liposomes, methyl-β-cyclodextrin (MβCD) was utilized to inhibit the caveolin-mediated endocytosis pathway, and chlorpromazine was used to inhibit the clathrin-mediated endocytosis pathway 42 . As shown in Fig.  5 c,d, the cellular endocytic pathway of liposomes varied depending on the cell type and liposome formulation. In NP-8 cells, endocytosis of these liposomes was primarily caveolae-dependent. Inhibition of clathrin reduced the internalization of DOPC-only and 9-1 liposomes, but it did not affect the uptake of 7-3 liposomes and 5-5 liposomes, suggesting that 7-3 liposomes and 5-5 liposomes are not internalized by NP-8 cells via the clathrin-dependent endocytosis pathway. In HepG-2 cells, both DOPC-only liposomes and 9-1 liposomes displayed a similar clathrin-dependent endocytosis pathway. Moreover, with an increase in the ratio of the 2-OHOA compound, the liposomes exhibited heightened endocytosis-dependent internalization by HepG-2 cells.

Liposome internalization characterization results. ( a , b ) Histograms of NP-8 and HepG-2 cellular internalization efficacy; ( c , d ) NP-8 and HepG-2 internalization efficacy with and without endocytosis inhibitions. Error bars represent ±  s.d ( n  = 3).

The summarized results, including liposome endocytic ratios and cell membrane GP variations, are presented in Table 1 . Interestingly, considering the previously mentioned variations in cell membrane fluidity after treatment, the 9-1 liposomes induced a more pronounced membrane fluidity reduction in NP-8 cells than the 7-3 and 5-5 liposomes. Meanwhile, the 5-5 liposomes exhibited an enhanced impact on HepG-2 cells. Notably, cell GP variations were heightened with an increase in liposome endocytic ratio. These outcomes indicate that the endocytosis of 2-OHOA-embedded liposomes intensifies the influence of 2-OHOA on cancer cells, leading to a more significant reduction in cell membrane fluidity. Typically, in the process of caveolae-mediated endocytosis, nanoparticles do not fuse with lysosomes after their entry into cells; via this endocytic pathway, the drug payload can be delivered to endoplasmic reticulum (ER) or Golgi apparatus, increasing the accumulation of drugs in ER or Golgi apparatus 43 , 44 . 2-OHOA has been reported as a sphingomyelin synthase (SMS) activator. SMS has two subtypes: SMS1 and SMS2. Partial SMS2 is localized to the plasma membrane, whereas most SMSs are localized in the inner plasma trans-Golgi network (TGN) rather than in the cytoplasm 45 . The endocytosis pathway associated with 2-OHOA-embedded liposomes is believed to promote interactions between 2-OHOA and SMS, leading to enhanced SM abundance, ultimately reduce the cell membrane fluidity ( GP increase).

These results support the hypothesis that the varied effects of different liposome formulations on cancer cells are associated with diverse cellular uptake mechanisms. The endocytic uptake pathway is believed to amplify the influence of 2-OHOA-embedded liposomes on cancer cells.

Anticancer performance of 2-OHOA-embedded liposomes

Based on previous results, the 9-1 liposome induced more dramatic membrane fluidity changes in NP-8 cells than free 2-OHOA. While the 5-5 liposomes induced greater membrane fluidity changes in HepG-2 cells. Moreover, analysis via the MTT assay on normal cells (OUMS-36T cell line) demonstrated that various liposome formulations exhibited comparable and modest inhibition on OUMS-36T at different concentrations (Fig.  S-5 ), indicating low cytotoxicity of 2-OHOA on healthy cells. To evaluate the anticancer performance enhancement of the liposome formulation on cancer cells, an apoptosis assay was conducted and the results are shown in Fig.  S-6 . Compared to non-formulated 2-OHOA, the 9-1 liposome induced a higher cellular apoptotic ratio, particularly the late apoptotic ratio in NP-8 cells, suggesting superior anticancer efficacy of the liposome formulations. Similar results were observed in HepG-2 cells.

Notably, previous studies have reported augmentations in the fluidity of various cell membranes during apoptosis induced by specific agents 46 , 47 , 48 . During apoptosis process, cells undergo a loss of membrane lipid asymmetry. SM, predominantly abundant in the outer leaflet of the cell membrane, is transferred to the inner leaflet, leading to a depletion of the Lo phase 49 . However, in this study, we did not observe an elevation in cancer cell membrane fluidity following 2-OHOA treatment, despite the induction of apoptosis in these cells. The prevalence of the Lo phase on the cancer cell membrane following 2-OHOA treatment is presumed to overshadow the anticipated increase in fluidity during apoptosis.

From our data, it is evident that the liposome formulation not only enhanced the impact of 2-OHOA on cancer cell membrane fluidity but also augmented the anticancer performance of 2-OHOA compared to non-formulated 2-OHOA.

Using Laurdan two-photon microscopy, we demonstrated that both 2-OHOA and 2-OHOA-embedded liposomes effectively reduced the fluidity of NP-8 and HepG-2 cell membranes. The liposome formulation generally intensified the impact of 2-OHOA on both NP-8 and HepG-2 cell membrane fluidity, although distinct patterns were observed in the descending membrane fluidity. LipiORDER fluorescence microscopy investigation further validated the variations in cell membrane lipid packing after 2-OHOA-embedded liposome treatment. Our investigation was extended to exploring cellular internalization efficacy and endocytic mechanisms, revealing an endocytosis-dependent enhancement of 2-OHOA-embedded liposome performance. Additionally, the enhanced anticancer performance of the liposome formulation was validated.

These findings substantiate the hypothesis that the liposome formulation not only addresses the solubility challenges of 2-OHOA, but also enhances its therapeutic efficacy as a membrane lipid therapy drug. Future research endeavors should explore a range of lipid-based nano-drug delivery systems to identify optimal formulations for delivering 2-OHOA. To advance these findings, it would be beneficial to conduct tests in animal models, focusing specifically on understanding the endocytic pathways of nanoparticles.

Materials and methods

1,2-dioleoyl- sn -glycero-3-phosphocholine (DOPC), 2-hydroxyoleic acid (2-OHOA), sphingomyelin (Brain, Porcine) and 1,2-dipalmitoyl- sn -glycero-3-phosphoethanolamine- N -(7-nitro-2-1,3-benzoxadiazol-4-yl) (16:0 NBD PE) were purchased from Avanti Polar Lipids. 6-Dodecanoyl-2-Dimethylaminonaphthalene (Laurdan) was purchased from Thermo Fisher Scientific. LipiORDER was purchased from Funakoshi. BD Pharmingen™ FITC Annexin V Apoptosis Detection Kit I was purchased from BD Biosciences. Calcein was purchased from TCI Chemicals. 3-( N -Morpholino) propanesulfonic Acid (MOPS free acid), Sodium acetate, Ethylenediamine- N , N , N′ , N′ -tetraacetic acid (EDTA-2Na), and Lipi-Red was purchased from Dojindo Laboratories. Chloroform, Dimethyl sulfoxide (DMSO), Fetal bovine serum (FBS), Eagle’s Minimum Essential Medium (E-MEM), Dulbecco’s Modified Eagle Medium (D-MEM), Trypsin (0.25 w/v%, EDTA solution with Phenol Red), LabAssay Phospholipid kit, MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide), Penicillin–Streptomycin solution, Anhydrous Cobalt (II) Chloride (CoCl 2 ) and D-PBS were purchased from Fujifilm Wako Pure Chemical.

Preparation and characterization of liposomes

Liposomes preparation.

Liposomes were prepared using the thin-film hydration-extrusion method. In brief, DOPC and 2-OHOA were first dissolved in chloroform and mixed in varying ratios in 100 mL round-bottom flasks. These lipid solutions underwent vacuum evaporation at 60 °C, followed by maintenance under high vacuum conditions at room temperature for 24 h. Following the vacuum step, D-PBS was added to the flasks to hydrate lipid films. The resulting vesicle suspensions were subjected to 4 cycles of freezing at − 80 °C and thawing at 65 °C. After the freeze–thaw process, the suspensions were extruded 13 times through a polycarbonate membrane with an average pore diameter of 200 nm, using an extruder (LiposoFast LF-1, Avestin, Canada).

The DOPC:2-OHOA ratios were 10:0, 9:1, 7:3, 5:5, 3:7, 1:9, and 0:10 (molar ratios). For brevity, liposomes fabricated from different formulations and 2-OHOA-only particles were named as follows: DOPC-only liposomes, 9-1 liposomes, 7-3 liposomes, 5-5 liposomes, 3-7 liposomes, 1-9 liposomes, and 2-OHOA-only particles. To investigate the cellular internalization efficiency and uptake mechanism, 1 mol% 16:0 NBD-PE was incorporated into the liposome formulations before the vacuum-evaporation step.

Particle size & ζ-potential characterization

Hydrodynamic particle size, polydispersity index (PDI), and ζ-potential were investigated using a Zetasizer (ZEN5600, Malvern, UK). Various liposome suspensions were diluted to a concentration of 100 μM using D-PBS. These measurements were conducted in triplicate at a temperature of 25 °C.

Evaluation of trapped water volume in liposomes

The trapped water volume in liposomes was investigated using the calcein-CoCl 2 quenching method, following a previously reported method with slight modifications 50 . Briefly, after the preparation of lipid thin films in round bottom flasks, the lipid thin films were hydrated using MOPS buffer (20 mM MOPS free acid, 5 mM sodium acetate, and 1 mM EDTA) containing 10 μM calcein. When the calcein concentration was lower than 20 μM, a linear concentration-fluorescence intensity relation was observed. The liposome preparation procedure was consistent with that previously described. The prepared liposomes containing calcein were diluted for 10 times before fluorescence spectrometer measurements. The calcein fluorescence intensity (FI) in the suspension was measured using a fluorescence spectrometer (FP-8500, Jasco, Japan), with excitation at 495 nm and emission at 515 nm. The total calcein FI was measured and named as \({FI}_{total}\) . After addition of CoCl 2 solution at a final concentration of 0.25 mM, the calcein in outer aqueous phase was quenched, then FI was measured and namely \({FI}_{in}\) . Then 10 (v/v) % Triton X-100 solution was added into the sample at a final concentration of 1 (v/v) % to disrupt the liposomes structure and the FI was measured and referred to as \({FI}_{TX}\) , which is the FI of CoCl 2 -calcein equilibrium statues. The dilution factors (DF) were calculated using volume ratios. Water trapped volume (% of total volume) was calculated using Eq. ( 1 ).

In this equation, \({DF}_{1}\) is the dilution factor after addition of CoCl 2 solution, \({DF}_{2}\) is the dilution factor after addition of Triton X-100 solution.

Cell culture and treatment

Cell culture.

HepG-2, NP-8, and OUMS-36T cells obtained from the Japanese Collection of Research Bioresources (JCRB) were used in this study. HepG-2 was cultured in E-MEM media; NP-8 and, OUMS-36T cells were cultured in D-MEM media. All cell culture media were supplemented with 10% v/v FBS and streptomycin-penicillin. Cells were cultured at 37 °C in in a humidified atmosphere containing 5% CO 2 .

Cell viability was evaluated using the MTT assay. Briefly, OUMS-36T cells were seeded in 96-well plates at a concentration of 5000 cells/well. After 48 h incubation, the cells were washed with D-PBS before adding fresh medium containing different liposomes. The liposome concentrations were set at 100, 200, 300, 400, and 500 μM (calculated based on the total lipid amount). Following 24 and 48 h of incubation, the cells were washed again and incubated for 4 h with fresh medium containing 0.5 mg/mL MTT. After incubation, the 96-well plates were centrifuged at 1000 × g for 5 min, and the media was carefully aspirated. Subsequently, DMSO was added to the wells, followed by a 30-min incubation to dissolve the formazan crystals. The optical densities (OD) of the resulting solutions were measured at 570 nm using a spectrophotometer (xMark™ Microplate Absorbance Spectrophotometer, Bio-Rad, USA). The cell viability was calculated at OD 570 nm .

Cellular internalization assay and endocytosis inhibition

To investigate the cellular internalization efficacy of different liposomes, each liposome formulation was doped with 1 mol % of 16:0 NBD-PE during the preparation procedure. The cells were exposed to 100 μM 16:0 NBD-PE labeled liposomes 6 h. After treatment, the cells were thoroughly washed and collected for analysis using flow cytometer (Applied Biosystems Acoustic focusing cytometer, Attune, USA).

To confirm the endocytic mechanism of liposomes, endocytosis was inhibited using 10 μg/mL chlorpromazine, an inhibitor of clathrin-mediated endocytosis; or 2.0 mM methyl-β-cyclodextrin (MβCD), an inhibitor of caveolae-mediated endocytosis. Each endocytosis inhibitor was added to culture medium for 30 min before the addition of 16:0 NBD-PE labeled liposomes.

Apoptosis assay

NP-8 and HepG-2 cells were treated with 2-OHOA-embedded liposome (containing 100 μM 2-OHOA) or non-formulated 2-OHOA (100 μM) for 48 h. The apoptosis rate was assessed using FITC Annexin V Apoptosis Detection Kit (BD Pharmingen™, BD Biosciences, USA) according to the manufacturer’s instructions.

Cell staining and investigation

Laurdan was dissolved in DMSO at a concentration of 1 mM as a stock solution. To measure the steady-state Laurdan fluorescence spectrum in cell membrane, HepG-2 and NP-8 cells were seeded in 6-well plates. After 24 h treatments with different liposomes or free 2-OHOA (firstly dissolved in DMSO at a concentration of 20 mM as stock solution, then diluted in cell culture media for cell treatment), the culture media was carefully removed, and the cells were gently washed with D-PBS. Subsequently, fresh pre-heated media containing 10 μM Laurdan was introduced into the wells and incubated for 30 min in a cell culture incubator shielded from light. Following Laurdan staining, cells were washed and detached using trypsin. The collected cells were suspended in D-PBS and analyzed using a fluorescence spectrometer (FP-8500, Jasco, Japan). Steady-state Laurdan spectra were obtained with an excitation wavelength of 345 nm, and emission was collected in the range of 400–600 nm. In the blank control group, cells from 3 replicate wells were stained with Laurdan and analyzed. For each treatment group (including each liposome formulation and free 2-OHOA treatment), cells from 5 replicate wells were stained with Laurdan and analyzed. Each replicate of cell samples was measured 3 times, and the Laurdan spectra were averaged across the 3 measurements.

The Laurdan steady-state fluorescence spectra data from fluorescence spectrometer were collected and analyzed. The \({GP}_{s}\) value ( GP value calculated according to steady-state Laurdan spectra) was calculated according to the Eq. ( 2 ):

where \({I}_{440}\) and \({I}_{490}\) represent the fluorescence intensity at 440 and 490 nm, respectively.

For two-photon microscopy observations, cells were initially cultured in 35 mm Φ glass-bottom dishes. Laurdan staining was performed as previously described. Following staining, the samples were observed under a two-photon microscopy. To maintain the temperature and CO 2 concentration of the cell samples during microscopy observation, the glass bottom dishes were placed in a living cell imaging chamber equipped with a stage-top incubator (INUB-PPZI, Tokai Hit, Japan) to sustain a 37 °C and 5% CO 2 environment. Two-photon fluorescence images of the Laurdan-labeled cells were obtained with an inverted microscopes (Eclipse TE2000-U, Nikon, Japan) with a × 60 water-immersion objective (Plan Apo VC 60 × , NA = 1.2, Nikon, Japan). A Ti–sapphire laser (Chameleon Vision II, Coherent, USA) with a repetition rate of 80 MHz and pulse width of 140 femtosecond (fs) was used as the excitation laser. The wavelength peak was tuned to 780 nm and the power was adjusted to 100 mW. The group delay dispersion (GDD) was adjusted to 14,000 femtosecond squared (fs 2 ). Laurdan emission from the cell samples were filtered through 436/20 nm (blue) and 495/25 nm (cyan) bandpass filters. The fluorescence intensity of two channels were detected using a laser-scanning fluorescence detector (D-Eclipse C1, Nikon, Japan). The relative sensitivities of the two channels were determined using 100 μM Laurdan in DMSO (spectrum shown in Fig.  S-7 ), and the calibration factor (G-factor) was calculated (refer to supplementary information). Two-photon microscopy images of Laurdan-stained cell membrane were analyzed using the imageJ software (ImageJ 1.53t. https://imagej.net/ij/ ). Laurdan GP images were acquired by calculating the GP value of each pixel. The \({GP}_{m}\) ( GP value calculated according to Laurdan two-photon microscopy images) of each pixel, was calculated according Eq. ( 3 ).

In this equation, \({I}_{blue}\) is the fluorescence intensity of the blue channel and \({I}_{cyan}\) is the fluorescence intensity of the cyan channel; \({G}_{Laurdan}\) is the Laurdan calibration factor (G factor). The GP values of pixels were obtained using image J software and the GP histograms were deconvoluted using Origin software (Origin 2023 v.10.0. https://www.originlab.com/ ). For blank control group and each treatment group (including each liposome formulation and free 2-OHOA), 3 replicate plates of cell samples were stained with Laurdan and imaged. 3 to 5 images were obtained from each plate of cells, with each image generated by averaging 4 scanning frames.

LipiORDER staining and imaging

LipiORDER was dissolved in DMSO at a concentration of 10 μM as a stock solution. Cells cultured in 35 mm Φ glass-bottom dishes were carefully washed with D-PBS, followed by incubation with 300 nM of LipiORDER in D-PBS for 15 min. After the staining period, the cells were rinsed with D-PBS and subsequently examined using a fluorescence microscopy (BX53, Olympus, Japan) equipped with image-splitting optics (W-View Gemini A12801-01, Hamamatsu, Japan). For excitation, a light filter with a wavelength of 388/38 nm was used. The emitted light was directed through a dichroic mirror, separating it into green channel (510/84 nm) and red channel (> 570 nm). Images from both the green and red channels were captured with an exposure time of 200 ms. The ratiometric analysis of LipiORDER fluorescence images was performed using the ImageJ software (ImageJ 1.53t. https://imagej.net/ij/ ). The backgrounds of the green and red channel images were first subtracted, and the R/G ratio images were acquired by calculating the fluorescence intensity ratio between red channel image and green channel image according to Eq. ( 4 ).

In this equation, the \({I}_{Red}\) is the LipiORDER fluorescence intensity from red channel, the \({I}_{Green}\) is the LipiORDER fluorescence intensity from green channel. For blank control group and each treatment group (including each liposome formulation and free 2-OHOA treatment), 3 plates of cell samples were stained with Laurdan for fluorescence microscopy imaging. 3 to 5 images were obtained from each plate of cells (exposure time of 200 ms).

Lipid droplets staining and imaging

Lipi-Red was dissolved in DMSO at a concentration of 1 mM and used as a stock solution. Cells cultured in glass bottom dishes were stained with Lipi-Red probe, according to the manufacturer’s instructions. After staining, the cells were rinsed carefully with D-PBS and observed using fluorescence microscopy (BX53, Olympus, Japan) at an excitation wavelength of 530–550 nm and the emission fluorescence signal was detected at wavelength > 575 nm.

Cell membrane sphingomyelin quantification

Sphingomyelin from cell membranes was quantified using a high-performance thin-layer chromatography (HPTLC)-densitometry method. Following the treatment, the cell membrane lipids were extracted using the Folch method. Phospholipids extracted from the cell membrane were quantified using the LabAssay Phospholipid Kit. Lipids were separated via HPTLC on Whatman silica gel-60 plates, using a mobile phase consisting of chloroform/ethanol/water/tri-ethylamine in a volume ratio of 30:35:7:35. Following HPTLC separation, the plates were air-dried, sprayed with a solution containing 8% (wt/vol) H 3 PO 4 and 10% (wt/vol) CuSO 4 , and then charred at 180 °C for 10 min. Subsequently, the plates were imaged using a ChemiDoc system, and sphingomyelin (SM) amount was compared based on the staining density.

Data availability

Data will be available upon request. The corresponding authors should be contacted for any data required for the conducted study.

Szlasa, W., Zendran, I., Zalesińska, A., Tarek, M. & Kulbacka, J. Lipid composition of the cancer cell membrane. J. Bioenerg. Biomembr. 52 , 321–342 (2020).

Article   CAS   PubMed   PubMed Central   Google Scholar  

Hattori, T. et al. Membrane phospholipid composition and membrane fluidity of human brain tumour: A spin label study. Neurol. Res. 9 , 38–43 (1987).

Article   CAS   PubMed   Google Scholar  

Nakazawa, I. & Iwaizumi, M. A role of the cancer cell membrane fluidity in the cancer metastases: An ESR study. Tohoku J. Exp. Med. 157 , 193–198 (1989).

Edmond, V. et al. Downregulation of ceramide synthase-6 during epithelial-to-mesenchymal transition reduces plasma membrane fluidity and cancer cell motility. Oncogene 34 , 996–1005 (2014).

Article   PubMed   Google Scholar  

Lopez, J. et al. A phase 1/2A trial of idroxioleic acid: First-in-class sphingolipid regulator and glioma cell autophagy inducer with antitumor activity in refractory glioma. Br. J. Cancer 129 , 811–818 (2023).

Barceló-Coblijn, G. et al. Sphingomyelin and sphingomyelin synthase (SMS) in the malignant transformation of glioma cells and in 2-hydroxyoleic acid therapy. Proc. Natl. Acad. Sci. 108 , 19569–19574 (2011).

Article   ADS   PubMed   PubMed Central   Google Scholar  

Martin, M. L. et al. Sustained activation of sphingomyelin synthase by 2-hydroxyoleic acid induces sphingolipidosis in tumor cells. J. Lipid Res. 54 , 1457–1465 (2013).

Article   ADS   CAS   PubMed   PubMed Central   Google Scholar  

Terés, S. et al. 2-hydroxyoleate, a nontoxic membrane binding anticancer drug, induces glioma cell differentiation and autophagy. Proc. Natl. Acad. Sci. 109 , 8489–8494 (2012).

Llado, V. et al. Minerval induces apoptosis in Jurkat and other cancer cells. J. Cell. Mol. Med. 14 , 659–670 (2010).

Miyaji, M. et al. Role of membrane sphingomyelin and ceramide in platform formation for Fas-mediated apoptosis. J. Exp. Med. 202 , 249–259 (2005).

Prajapati, R., Gontsarik, M., Yaghmur, A. & Salentinig, S. PH-responsive nano-self-assemblies of the anticancer drug 2-hydroxyoleic acid. Langmuir 35 , 7954–7961 (2019).

Antoniou, A. I. et al. 2-hydroxyoleic acid as a self-assembly inducer for anti-cancer drug-centered nanoparticles. Pharmaceuticals 16 , 722 (2023).

Jang, E.-J. et al. 2-hydroxyoleic acid-inserted liposomes as a multifunctional carrier of anticancer drugs. Drug Deliv. 24 , 1587–1597 (2017).

Zhang, Y.-N., Poon, W., Tavares, A. J., McGilvray, I. D. & Chan, W. C. W. Nanoparticle-Liver Interactions: Cellular uptake and hepatobiliary elimination. J. Control. Release 240 , 332–348 (2016).

Parasassi, T. & Gratton, E. Membrane lipid domains and dynamics as detected by Laurdan fluorescence. J. Fluoresc. 5 , 59–69 (1995).

Parasassi, T. & Gratton, E. Packing of phospholipid vesicles studied by oxygen quenching of Laurdan fluorescence. J. Fluoresc. 2 , 167–174 (1992).

Harris, F. M., Best, K. B. & Bell, J. D. Use of laurdan fluorescence intensity and polarization to distinguish between changes in membrane fluidity and phospholipid order. Biochim. Biophys. Acta (BBA) Biomembr. 1565 , 123–128 (2002).

Article   CAS   Google Scholar  

Okamoto, Y., Kishi, Y., Suga, K. & Umakoshi, H. Induction of chiral recognition with lipid Nanodomains produced by polymerization. Biomacromolecules 18 , 1180–1188 (2017).

Valanciunaite, J. et al. Polarity mapping of cells and embryos by improved fluorescent solvatochromic pyrene probe. Anal. Chem. 92 , 6512–6520 (2020).

Okamoto, Y., Hamaguchi, K., Watanabe, M., Watanabe, N. & Umakoshi, H. Characterization of phase separated planar lipid bilayer membrane by fluorescence ratio imaging and scanning probe microscope. Membranes 12 , 770 (2022).

Notman, R., Noro, M. G. & Anwar, J. Interaction of oleic acid with dipalmitoylphosphatidylcholine (DPPC) bilayers simulated by molecular dynamics. J. Phys. Chem. B 111 , 12748–12755 (2007).

Zhao, J. & Mao, S. Tuning the membrane fluidity of liposomes for desirable in vivo fate with enhanced drug delivery. Advances in Biomembranes and Lipid Self-Assembly , 67–106 (2021). https://doi.org/10.1016/bs.abl.2021.11.003 .

Olechowska, K., Mach, M., Hąc-Wydro, K. & Wydro, P. The influence of 2-hydroxyoleic acid—An anticancer drug—On model membranes of different fluidity modulated by the cholesterol content. J. Mol. Liq. 283 , 756–762 (2019).

Sanchez, S. A., Tricerri, M. A. & Gratton, E. Laurdan generalized polarization fluctuations measures membrane packing micro-heterogeneity in vivo. Proc. Natl. Acad. Sci. 109 , 7314–7319 (2012).

Noutsi, P., Gratton, E. & Chaieb, S. Assessment of membrane fluidity fluctuations during cellular development reveals time and cell type specificity. PLoS One 11 , e0158313 (2016).

Article   PubMed   PubMed Central   Google Scholar  

Batta, G. et al. Alterations in the properties of the cell membrane due to glycosphingolipid accumulation in a model of Gaucher disease. Sci. Rep. 8 , 157 (2018).

Pokorna, S. et al. Laurdan in live cell imaging: Effect of acquisition settings, cell culture conditions and data analysis on generalized polarization measurements. J. Photochem. Photobiol. B Biol. 228 , 112404 (2022).

Levitan, I. Evaluating membrane structure by Laurdan Imaging: Disruption of lipid packing by oxidized lipids. New Methods and Sensors for Membrane and Cell Volume Research , 235–256 (2021). https://doi.org/10.1016/bs.ctm.2021.10.003 .

Gaus, K. et al. Visualizing lipid structure and raft domains in living cells with two-photon microscopy. Proc. Natl. Acad. Sci. 100 , 15554–15559 (2003).

Terés, S. et al. Normalization of sphingomyelin levels by 2-hydroxyoleic acid induces autophagic cell death of SF767 cancer cells. Autophagy 8 , 1542–1544 (2012).

St. Clair, J. W., Kakuda, S. & London, E. Induction of ordered lipid raft domain formation by loss of lipid asymmetry. Biophys. J. 119 , 483–492 (2020).

Dorrance, A. Increased membrane sphingomyelin and arachidonic acid in stroke-prone spontaneously hypertensive rats. Am. J. Hypertens. 14 , 1149–1153 (2001).

Slotte, J. P. & Ramstedt, B. The functional role of sphingomyelin in cell membranes. Eur. J. Lipid Sci. Technol. 109 , 977–981 (2007).

Maté, S. et al. N-nervonoylsphingomyelin (C24:1) prevents lateral heterogeneity in cholesterol-containing membranes. Biophys. J. 106 , 2606–2616 (2014).

Marcilla-Etxenike, A. et al. 2-hydroxyoleic acid induces ER stress and autophagy in various human glioma cell lines. PLoS One 7 , e48235 (2012).

Eynaudi, A. et al. Differential effects of oleic and palmitic acids on lipid droplet-mitochondria interaction in the hepatic cell line hepg2. Front. Nutr. 8 , 901 (2021).

Article   Google Scholar  

Sot, J. et al. Phase-selective staining of model and cell membranes, lipid droplets and lipoproteins with fluorescent solvatochromic pyrene probes. Biochim. Biophys. Acta (BBA) Biomembr. 1863 , 183470 (2021).

Behzadi, S. et al. Cellular uptake of nanoparticles: Journey inside the cell. Chem. Soc. Rev. 46 , 4218–4244 (2017).

Augustine, R. et al. Cellular uptake and retention of nanoparticles: Insights on particle properties and interaction with cellular components. Mater. Today Commun. 25 , 101692 (2020).

Xia, T., Kovochich, M., Liong, M., Zink, J. I. & Nel, A. E. Cationic polystyrene nanosphere toxicity depends on cell-specific endocytic and mitochondrial injury pathways. ACS Nano 2 , 85–96 (2007).

dos Santos, T., Varela, J., Lynch, I., Salvati, A. & Dawson, K. A. Effects of transport inhibitors on the cellular uptake of carboxylated polystyrene nanoparticles in different cell lines. PLoS One 6 , e24438 (2011).

Un, K., Sakai-Kato, K., Oshima, Y., Kawanishi, T. & Okuda, H. Intracellular trafficking mechanism, from intracellular uptake to extracellular efflux, for phospholipid/cholesterol liposomes. Biomaterials 33 , 8131–8141 (2012).

Kou, L., Sun, J., Zhai, Y. & He, Z. The endocytosis and intracellular fate of nanomedicines: Implication for rational design. Asian J. Pharm. Sci. 8 , 1–10 (2013).

Le, P. U. & Nabi, I. R. Distinct caveolae-mediated endocytic pathways target the Golgi apparatus and the endoplasmic reticulum. J. Cell Sci. 116 , 1059–1071 (2003).

Gault, C. R., Obeid, L. M. & Hannun, Y. A. An overview of sphingolipid metabolism: From synthesis to breakdown. In Advances in Experimental Medicine and Biology (eds Chalfant, C. & Del Poeta, M.) 1–23 (Springer, 2010). https://doi.org/10.1007/978-1-4419-6741-1_1 .

Chapter   Google Scholar  

Bailey, R. W. et al. Sequence of physical changes to the cell membrane during glucocorticoid-induced apoptosis in S49 lymphoma cells. Biophys. J. 96 , 2709–2718 (2009).

Jourd’heuil, D., Aspinall, A., Reynolds, J. D. & Meddings, J. B. Membrane fluidity increases during apoptosis of sheep ileal Peyer’s Patch B cells. Can. J. Physiol. Pharmacol. 74 , 706–711 (1996).

Fujimoto, K., Iwasaki, C., Kawaguchi, H., Yasugi, E. & Oshima, M. Cell membrane dynamics and the induction of apoptosis by lipid compounds. FEBS Lett. 446 , 113–116 (1999).

Oncul, S. et al. Liquid ordered phase in cell membranes evidenced by a hydration-sensitive probe: Effects of cholesterol depletion and apoptosis. Biochim. Biophys. Acta (BBA) Biomembr. 1798 , 1436–1443 (2010).

Oku, N., Kendall, D. A. & MacDonald, R. C. A simple procedure for the determination of the trapped volume of liposomes. Biochim. Biophys. Acta (BBA) Biomembr. 691 , 332–340 (1982).

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Acknowledgements

The author (X. R) thanks the Japanese Government for the MEXT scholarship. This work was supported by JSPS KAKENHI (23K17862, 21H04628) and the Grant-in-Aid for Transformative Research Areas (A) “Material Symbiosis” (23H04075) from MEXT, Japan. One of the authors (Y.O.) thanks the Multidisciplinary Research Laboratory System in Osaka University, JKA and its promotion funds from KEIRIN RACE, Tateisi Science and Technology Foundation, Toyota Physical and Chemical Research Institute for financial support of this research, and the R3 Institute for Newly-Emerging Science Design, Osaka University for two-photon microscope experiments, and also thanks Dr. Yosuke Niko (Kochi University) for valuable comments on LipiORDER.

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X.R.: conceptualization, methodology, investigation, data analysis, data validation, data curation, writing-original draft preparation. Y.O.: supervision, conceptualization, methodology, data analysis, data validation, writing-review and editing. S.F.: assembled the microscope, methodology, data analysis, writing-review; N.M.W.: supervision, conceptualization, data validation, writing-review and editing. H.U.: supervision, conceptualization, writing-review and editing. All authors (X.R., Y.O., S.F., N.M.W. and H.U.) reviewed the manuscript.

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Rui, X., Okamoto, Y., Fukushima, S. et al. Investigating the impact of 2-OHOA-embedded liposomes on biophysical properties of cancer cell membranes via Laurdan two-photon microscopy imaging. Sci Rep 14 , 15831 (2024). https://doi.org/10.1038/s41598-024-65812-9

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Liposome: classification, preparation, and applications

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• Volume 8 , article number  102 , ( 2013 )

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Liposomes, sphere-shaped vesicles consisting of one or more phospholipid bilayers, were first described in the mid-60s. Today, they are a very useful reproduction, reagent, and tool in various scientific disciplines, including mathematics and theoretical physics, biophysics, chemistry, colloid science, biochemistry, and biology. Since then, liposomes have made their way to the market. Among several talented new drug delivery systems, liposomes characterize an advanced technology to deliver active molecules to the site of action, and at present, several formulations are in clinical use. Research on liposome technology has progressed from conventional vesicles to ‘second-generation liposomes’, in which long-circulating liposomes are obtained by modulating the lipid composition, size, and charge of the vesicle. Liposomes with modified surfaces have also been developed using several molecules, such as glycolipids or sialic acid. This paper summarizes exclusively scalable techniques and focuses on strengths, respectively, limitations in respect to industrial applicability and regulatory requirements concerning liposomal drug formulations based on FDA and EMEA documents.

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Introduction

Liposomes are small artificial vesicles of spherical shape that can be created from cholesterol and natural non-toxic phospholipids. Due to their size and hydrophobic and hydrophilic character(besides biocompatibility), liposomes are promising systems for drug delivery. Liposome properties differ considerably with lipid composition, surface charge, size, and the method of preparation (Table  1 ). Furthermore, the choice of bilayer components determines the ‘rigidity’ or ‘fluidity’ and the charge of the bilayer. For instance, unsaturated phosphatidylcholine species from natural sources (egg or soybean phosphatidylcholine) give much more permeable and less stable bilayers, whereas the saturated phospholipids with long acyl chains (for example, dipalmitoylphos phatidylcholine) form a rigid, rather impermeable bilayer structure[ 1 – 3 ].

It has been displayed that phospholipids impulsively form closed structures when they are hydrated in aqueous solutions. Such vesicles which have one or more phospholipid bilayer membranes can transport aqueous or lipid drugs, depending on the nature of those drugs. Because lipids are amphipathic (both hydrophobic and hydrophilic) in aqueous media, their thermodynamic phase properties and self assembling characteristics influence entropically focused confiscation of their hydrophobic sections into spherical bilayers. Those layers are referred to as lamellae[ 4 ]. Generally, liposomes are definite as spherical vesicles with particle sizes ranging from 30 nm to several micrometers. They consist of one or more lipid bilayers surrounding aqueous units, where the polar head groups are oriented in the pathway of the interior and exterior aqueous phases. On the other hand, self-aggregation of polar lipids is not limited to conventional bilayer structures which rely on molecular shape, temperature, and environmental and preparation conditions but may self-assemble into various types of colloidal particles[ 5 ].

Liposomes are extensively used as carriers for numerous molecules in cosmetic and pharmaceutical industries. Additionally, food and farming industries have extensively studied the use of liposome encapsulation to grow delivery systems that can entrap unstable compounds (for example, antimicrobials, antioxidants, flavors and bioactive elements) and shield their functionality. Liposomes can trap both hydrophobic and hydrophilic compounds, avoid decomposition of the entrapped combinations, and release the entrapped at designated targets[ 6 – 8 ].

Because of their biocompatibility, biodegradability, low toxicity, and aptitude to trap both hydrophilic and lipophilic drugs[ 9 ] and simplify site-specific drug delivery to tumor tissues[ 10 ], liposomes have increased rate both as an investigational system and commercially as a drug-delivery system. Many studies have been conducted on liposomes with the goal of decreasing drug toxicity and/or targeting specific cells[ 11 – 13 ].

Liposomal encapsulation technology (LET) is the newest delivery technique used by medical investigators to transmit drugs that act as curative promoters to the assured body organs. This form of delivery system proposal targeted the delivery of vital combinations to the body. LET is a method of generating sub-microscopic foams called liposomes, which encapsulate numerous materials. These ‘liposomes’ form a barrier around their contents, which is resistant to enzymes in the mouth and stomach, alkaline solutions, digestive juices, bile salts, and intestinal flora that are generated in the human body, as well as free radicals. The contents of the liposomes are, therefore, protected from oxidation and degradation. This protective phospholipid shield or barrier remains undamaged until the contents of the liposome are delivered to the exact target gland, organ, or system where the contents will be utilized[ 14 ].

Clinical medication keeps an enormously broad range of drug molecules at this time in use, and new drugs are added to the list every year. One of the main aims of any cure employing drug is to increase the therapeutic index of the drug while minimizing its side effects. The clinical usefulness of most conservative chemotherapeutics is restricted either by the incapability to deliver therapeutic drug concentrations to the target soft tissue or by Spartan and harmful toxic side effects on normal organs and tissues. Different approaches have been made to overcome these difficulties by providing the ‘selective’ delivery to the target area; the ideal solution would be to target the drug alone to those cells, tissues, organs that are affected by the disease. Selected carriers, for instance colloidal particulates and molecular conjugates, can be appropriate for this determination. Colloidal particulates result from the physical incorporation of the drug into a particulate colloidal system, for instance reverse micelles, noisome, micro- and nano-spheres, erythrocytes, and polymers and liposomes. Among these carriers, liposomes have been most studied. Their attractiveness lies in their composition, which makes them biodegradable and biocompatible. Liposome involves an aqueous core entrapped by one or more bilayers composed of natural or synthetic lipids. They are composed of natural phospholipids that are biologically inert and feebly immunogenic, and they have low inherent toxicity. Furthermore, drugs with different lipophilicities can be encapsulated into liposomes: strongly lipophilic drugs are entrapped almost totally in the lipid bilayer, intensely hydrophilic drugs are located entirely in the aqueous compartment, and drugs with intermediary logP effortlessly partition between the lipid and aqueous phases, both in the bilayer and in the aqueous core[ 15 ].

The present review will briefly explain the characteristics of liposomes and explore the related problems and solutions proposed, with a focus on liposome preparation, characterizations, affecting factors, advantages, and disadvantages. In particular, we return to the literature relating to high-stability, long-circulating liposomes (stealth liposomes), and their field of application.

Classification of liposomes

The liposome size can vary from very small (0.025 μm) to large (2.5 μm) vesicles. Moreover, liposomes may have one or bilayer membranes. The vesicle size is an acute parameter in determining the circulation half-life of liposomes, and both size and number of bilayers affect the amount of drug encapsulation in the liposomes. On the basis of their size and number of bilayers, liposomes can also be classified into one of two categories: (1) multilamellar vesicles (MLV) and (2) unilamellar vesicles. Unilamellar vesicles can also be classified into two categories: (1) large unilamellar vesicles (LUV) and (2) small unilamellar vesicles (SUV)[ 16 ]. In unilamellar liposomes, the vesicle has a single phospholipid bilayer sphere enclosing the aqueous solution. In multilamellar liposomes, vesicles have an onion structure. Classically, several unilamellar vesicles will form on the inside of the other with smaller size, making a multilamellar structure of concentric phospholipid spheres separated by layers of water[ 17 ].

Methods of liposome preparation

General methods of preparation.

All the methods of preparing the liposomes involve four basic stages:

Drying down lipids from organic solvent.

Dispersing the lipid in aqueous media.

Purifying the resultant liposome.

Analyzing the final product.

Method of liposome preparation and drug loading

The following methods are used for the preparation of liposome:

Passive loading techniques

Active loading technique.

Passive loading techniques include three different methods:

Mechanical dispersion method.

Solvent dispersion method.

Detergent removal method (removal of non-encapsulated material) [ 18 , 19 ].

Mechanical dispersion method

The following are types of mechanical dispersion methods:

1.1. Sonication.

1.2. French pressure cell: extrusion.

1.3. Freeze-thawed liposomes.

1.4. Lipid film hydration by hand shaking, non-hand. shaking or freeze drying.

1.5. Micro-emulsification.

1.6. Membrane extrusion.

1.7. Dried reconstituted vesicles[ 18 , 19 ].

Sonication is perhaps the most extensively used method for the preparation of SUV. Here, MLVs are sonicated either with a bath type sonicator or a probe sonicator under a passive atmosphere. The main disadvantages of this method are very low internal volume/encapsulation efficacy, possible degradation of phospholipids and compounds to be encapsulated, elimination of large molecules, metal pollution from probe tip, and presence of MLV along with SUV[ 18 ].

There are two sonication techniques:

Probe sonication. The tip of a sonicator is directly engrossed into the liposome dispersion. The energy input into lipid dispersion is very high in this method. The coupling of energy at the tip results in local hotness; therefore, the vessel must be engrossed into a water/ice bath. Throughout the sonication up to 1 h, more than 5% of the lipids can be de-esterified. Also, with the probe sonicator, titanium will slough off and pollute the solution.

Bath sonication. The liposome dispersion in a cylinder is placed into a bath sonicator. Controlling the temperature of the lipid dispersion is usually easier in this method, in contrast to sonication by dispersal directly using the tip. The material being sonicated can be protected in a sterile vessel, dissimilar the probe units, or under an inert atmosphere [ 20 ].

French pressure cell: extrusion

French pressure cell involves the extrusion of MLV through a small orifice[ 18 ]. An important feature of the French press vesicle method is that the proteins do not seem to be significantly pretentious during the procedure as they are in sonication[ 21 ]. An interesting comment is that French press vesicle appears to recall entrapped solutes significantly longer than SUVs do, produced by sonication or detergent removal[ 22 – 24 ].

The method involves gentle handling of unstable materials. The method has several advantages over sonication method[ 25 ]. The resulting liposomes are rather larger than sonicated SUVs. The drawbacks of the method are that the high temperature is difficult to attain, and the working volumes are comparatively small (about 50 mL as the maximum)[ 18 , 19 ].

Freeze-thawed liposomes

SUVs are rapidly frozen and thawed slowly. The short-lived sonication disperses aggregated materials to LUV. The creation of unilamellar vesicles is as a result of the fusion of SUV throughout the processes of freezing and thawing[ 26 – 28 ]. This type of synthesis is strongly inhibited by increasing the phospholipid concentration and by increasing the ionic strength of the medium. The encapsulation efficacies from 20% to 30% were obtained[ 26 ].

Solvent dispersion method

Ether injection (solvent vaporization).

A solution of lipids dissolved in diethyl ether or ether-methanol mixture is gradually injected to an aqueous solution of the material to be encapsulated at 55°C to 65°C or under reduced pressure. The consequent removal of ether under vacuum leads to the creation of liposomes. The main disadvantages of the technique are that the population is heterogeneous (70 to 200 nm) and the exposure of compounds to be encapsulated to organic solvents at high temperature[ 29 , 30 ].

Ethanol injection

A lipid solution of ethanol is rapidly injected to a huge excess of buffer. The MLVs are at once formed. The disadvantages of the method are that the population is heterogeneous (30 to 110 nm), liposomes are very dilute, the removal all ethanol is difficult because it forms into azeotrope with water, and the probability of the various biologically active macromolecules to inactivate in the presence of even low amounts of ethanol is high[ 31 ].

Reverse phase evaporation method

This method provided a progress in liposome technology, since it allowed for the first time the preparation of liposomes with a high aqueous space-to-lipid ratio and a capability to entrap a large percentage of the aqueous material presented. Reverse-phase evaporation is based on the creation of inverted micelles. These inverted micelles are shaped upon sonication of a mixture of a buffered aqueous phase, which contains the water-soluble molecules to be encapsulated into the liposomes and an organic phase in which the amphiphilic molecules are solubilized. The slow elimination of the organic solvent leads to the conversion of these inverted micelles into viscous state and gel form. At a critical point in this process, the gel state collapses, and some of the inverted micelles were disturbed. The excess of phospholipids in the environment donates to the formation of a complete bilayer around the residual micelles, which results in the creation of liposomes. Liposomes made by reverse phase evaporation method can be made from numerous lipid formulations and have aqueous volume-to-lipid ratios that are four times higher than hand-shaken liposomes or multilamellar liposomes[ 19 , 20 ].

Briefly, first, the water-in-oil emulsion is shaped by brief sonication of a two-phase system, containing phospholipids in organic solvent such as isopropyl ether or diethyl ether or a mixture of isopropyl ether and chloroform with aqueous buffer. The organic solvents are detached under reduced pressure, resulting in the creation of a viscous gel. The liposomes are shaped when residual solvent is detached during continued rotary evaporation under reduced pressure. With this method, high encapsulation efficiency up to 65% can be obtained in a medium of low ionic strength for example 0.01 M NaCl. The method has been used to encapsulate small, large, and macromolecules. The main drawback of the technique is the contact of the materials to be encapsulated to organic solvents and to brief periods of sonication. These conditions may possibly result in the breakage of DNA strands or the denaturation of some proteins[ 32 ]. Modified reverse phase evaporation method was presented by Handa et al., and the main benefit of the method is that the liposomes had high encapsulation efficiency (about 80%)[ 33 ].

Detergent removal method (removal of non-encapsulated material)

The detergents at their critical micelle concentrations (CMC) have been used to solubilize lipids. As the detergent is detached, the micelles become increasingly better-off in phospholipid and lastly combine to form LUVs. The detergents were removed by dialysis[ 34 – 36 ]. A commercial device called LipoPrep (Diachema AG, Switzerland), which is a version of dialysis system, is obtainable for the elimination of detergents. The dialysis can be performed in dialysis bags engrossed in large detergent free buffers (equilibrium dialysis)[ 17 ].

Detergent (cholate, alkyl glycoside, Triton X-100) removal of mixed micelles (absorption)

Detergent absorption is attained by shaking mixed micelle solution with beaded organic polystyrene adsorbers such as XAD-2 beads (SERVA Electrophoresis GmbH, Heidelberg, Germany) and Bio-beads SM2 (Bio-RadLaboratories, Inc., Hercules, USA). The great benefit of using detergent adsorbers is that they can eliminate detergents with a very low CMC, which are not entirely depleted.

Gel-permeation chromatography

In this method, the detergent is depleted by size special chromatography. Sephadex G-50, Sephadex G-l 00 (Sigma-Aldrich, MO, USA), Sepharose 2B-6B, and Sephacryl S200-S1000 (General Electric Company, Tehran, Iran) can be used for gel filtration. The liposomes do not penetrate into the pores of the beads packed in a column. They percolate through the inter-bead spaces. At slow flow rates, the separation of liposomes from detergent monomers is very good. The swollen polysaccharide beads adsorb substantial amounts of amphiphilic lipids; therefore, pre-treatment is necessary. The pre-treatment is done by pre-saturation of the gel filtration column by lipids using empty liposome suspensions.

Upon dilution of aqueous mixed micellar solution of detergent and phospholipids with buffer, the micellar size and the polydispersity increase fundamentally, and as the system is diluted beyond the mixed micellar phase boundary, a spontaneous transition from polydispersed micelles to vesicles occurs.

Stealth liposomes and conventional liposomes

Although liposomes are like biomembranes, they are still foreign objects of the body. Therefore, liposomes are known by the mononuclear phagocytic system (MPS) after contact with plasma proteins. Accordingly, liposomes are cleared from the blood stream.

These stability difficulties are solved through the use of synthetic phospholipids, particle coated with amphipathic polyethylene glycol, coating liposomes with chitin derivatives, freeze drying, polymerization, microencapsulation of gangliosides[ 17 ].

Coating liposomes with PEG reduces the percentage of uptake by macrophages and leads to a prolonged presence of liposomes in the circulation and, therefore, make available abundant time for these liposomes to leak from the circulation through leaky endothelium.

A stealth liposome is a sphere-shaped vesicle with a membrane composed of phospholipid bilayer used to deliver drugs or genetic material into a cell. A liposome can be composed of naturally derived phospholipids with mixed lipid chains coated or steadied by polymers of PEG and colloidal in nature. Stealth liposomes are attained and grown in new drug delivery and in controlled release. This stealth principle has been used to develop the successful doxorubicin-loaded liposome product that is presently marketed as Doxil (Janssen Biotech, Inc., Horsham, USA) or Caelyx (Schering-Plough Corporation, Kenilworth, USA) for the treatment of solid tumors. Recently impressive therapeutic improvements were described with the useof corticosteroid-loaded liposome in experimental arthritic models. The concerning on the application of stealth liposomes has been on their potential to escape from the blood circulation. However, long circulating liposome may also act as a reservoir for prolonged release of a therapeutic agent. Pharmacological action of vasopressin is formulated in long circulating liposome[ 37 , 38 ].

Drug loading in liposomes

Drug loading can be attained either passively (i.e., the drug is encapsulated during liposome formation) or actively (i.e., after liposome formation). Hydrophobic drugs, for example amphotericin B taxol or annamycin, can be directly combined into liposomes during vesicle formation, and the amount of uptake and retention is governed by drug-lipid interactions. Trapping effectiveness of 100% is often achievable, but this is dependent on the solubility of the drug in the liposome membrane. Passive encapsulation of water-soluble drugs depends on the ability of liposomes to trap aqueous buffer containing a dissolved drug during vesicle formation. Trapping effectiveness (generally <30%) is limited by the trapped volume delimited in the liposomes and drug solubility. On the other hand, water-soluble drugs that have protonizable amine functions can be actively entrapped by employing pH gradients[ 39 ], which can result in trapping effectiveness approaching 100%[ 40 ].

Freeze-protectant for liposomes (lyophilization)

Natural excerpts are usually degraded because of oxidation and other chemical reactions before they are delivered to the target site. Freeze-drying has been a standard practice employed to the production of many pharmaceutical products. The overwhelming majority of these products are lyophilized from simple aqueous solutions. Classically, water is the only solvent that must be detached from the solution using the freeze-drying process, but there are still many examples where pharmaceutical products are manufactured via a process that requires freeze-drying from organic co-solvent systems[ 14 ].

Freeze-drying (lyophilization) involves the removal of water from products in the frozen state at tremendously low pressures. The process is normally used to dry products that are thermo-labile and would be demolished by heat-drying. The technique has too much potential as a method to solve long-term stability difficulties with admiration to liposomal stability. Studies showed that leakage of entrapped materials may take place during the process of freeze-drying and on reconstitution. Newly, it was shown that liposomes when freeze-dried in the presence of adequate amounts of trehalose (a carbohydrate commonly found at high concentrations in organism) retained as much as 100% of their original substances. It shows that trehalose is an excellent cryoprotectant (freeze-protectant) for liposomes. Freeze-driers range in size from small laboratory models to large industrial units available from pharmaceutical equipment suppliers[ 41 ].

Mechanism of transportation through liposome

The limitations and benefits of liposome drug carriers lie critically on the interaction of liposomes with cells and their destiny in vivo after administration. In vivo and in vitro studies of the contacts with cells have shown that the main interaction of liposomes with cells is either simple adsorption (by specific interactions with cell-surface components, electrostatic forces, or by non-specific weak hydrophobic) or following endocytosis (by phagocytic cells of the reticuloendothelial system, for example macrophages and neutrophils).

Fusion with the plasma cell membrane by insertion of the lipid bilayer of the liposome into the plasma membrane, with simultaneous release of liposomal content into the cytoplasm, is much rare. The fourth possible interaction is the exchange of bilayer components, for instance cholesterol, lipids, and membrane-bound molecules with components of cell membranes. It is often difficult to determine what mechanism is functioning, and more than one may function at the same time[ 42 – 44 ].

Fusogenic liposomes and antibody-mediated liposomes in cancer therapy

It has been infrequently well-known that a powerful anticancer drug, especially one that targets the cytoplasm or cell nucleus, does not work due to the low permeability across a plasma membrane, degradation by lysosomal enzymes through an endocytosis-dependent pathway, and other reasons. Thus, much attention on the use of drug delivery systems is focused on overcoming these problems, ultimately leading to the induction of maximal ability of anti-cancer drug. In this respect, a new model for cancer therapy using a novel drug delivery system, fusogenic liposome[ 45 ], was developed.

Fusogenic liposomes are poised of the ultraviolet-inactivated Sendai virus and conventional liposomes. Fusogenic liposomes effectively and directly deliver their encapsulated contents into the cytoplasm using a fusion mechanism of the Sendai virus, whereas conventional liposomes are taken up by endocytosis by phagocytic cells of the reticuloendothelial system, for example macrophages and neutrophils. Thus, fusogenic liposome is a good candidate as a vehicle to deliver drugs into the cytoplasm in an endocytosis-independent manner[ 45 ].

Liposomal drug delivery systems provide steady formulation, provide better pharmacokinetics, and make a degree of ‘passive’ or ‘physiological’ targeting to tumor tissue available. However, these transporters do not directly target tumor cells. The design modifications that protect liposomes from unwanted interactions with plasma proteins and cell membranes which differed them with reactive carriers, for example cationic liposomes, also prevent interactions with tumor cells. As an alternative, after extravasation into tumor tissue, liposomes remain within tumor stroma as a drug-loaded depot. Liposomes ultimately become subject to enzymatic degradation and/or phagocytic attack, leading to release of drug for subsequent diffusion to tumor cells. The next generation of drug carriers under development features directs molecular targeting of cancer cells via antibody-mediated or other ligand-mediated interactions[ 17 , 45 ].

Applications of liposomes in medicine and pharmacology

Applications of liposomes in medicine and pharmacology can be divided into diagnostic and therapeutic applications of liposomes containing various markers or drugs, and their use as a tool, a model, or reagent in the basic studies of cell interactions, recognition processes, and mode of action of certain substances[ 43 ].

Unfortunately, many drugs have a very narrow therapeutic window, meaning that the therapeutic concentration is not much lower than the toxic one. In several cases, the toxicity can be reduced or the efficacy can be enhanced by the use of a suitable drug carrier which alters the temporal and spatial delivery of the drug, i.e., its biodistribution and pharmacokinetics. It is clear from many pre-clinical and clinical studies that drugs, for instance antitumor drugs, parceled in liposome demonstration reduced toxicities, while retentive enhanced efficacy.

Advances in liposome design are leading to new applications for the delivery of new biotechnology products, for example antisense oligonucleotides, cloned genes, and recombinant proteins. A vast literature define the viability of formulating wide range of conservative drugs in liposomes, frequently resultant in improved therapeutic activity and/or reduced toxicity compared with the free drug. As a whole, changed pharmacokinetics for liposomal drugs can lead to improved drug bioavailability to particular target cells that live in the circulation, or more prominently, to extravascular disease sites, for example, tumors. Recent improvements include liposomal formulations of all- trans -retinoic acid[ 46 , 47 ] and daunorubicin[ 48 – 51 ], which has received Food and Drug Administration consent as a first-line treatment of AIDS-related advanced Kaposi's sarcoma. Distinguished examples are vincristine, doxorubicin, and amphotericin B[ 38 ].

The benefits of drug load in liposomes, which can be applied as (colloidal) solution, aerosol, or in (semi) solid forms, such as creams and gels, can be summarized into seven categories[ 44 ] (Table  2 ):

Liposomes in parasitic diseases and infections

From the time when conventional liposomes are digested by phagocytic cells in the body after intravenous management, they are ideal vehicles for the targeting drug molecules into these macrophages. The best known instances of this ‘Trojan horse-like’ mechanism are several parasitic diseases which normally exist in the cell of MPS. They comprise leishmaniasis and several fungal infections.

Leishmaniasis is a parasitic infection of macrophages which affects over 100 million people in tropical regions and is often deadly. The effectual dose of drugs, mostly different antimonials, is not much lower than the toxic one. Liposomes accumulate in the very same cell population which is infected, and so an ideal drug delivery vehicle was proposed[ 52 ]. Certainly, the therapeutic index was increased in rodents as much as several hundred times upon administration of the drug in various liposomes. Unexpectedly, and unfortunately, there was not much interest to scale up the formulations and clinically approve them after several very encouraging studies dating back to 1978. Only now, there are several continuing studies with various anti-parasitic liposome formulations in humans. These formulations use mostly ionosphere amphotericin B and are transplanted from very successful and prolific area of liposome formulations in antifungal therapy.

The best results reported so far in human therapy are probably liposomes as carriers foramphotericin B in antifungal therapies. This is the drug of choice in dispersed fungal infections which often in parallel work together with chemotherapy, immune system, or AIDS, and is frequently fatal. Unfortunately, the drug itself is very toxic and its dosage is limited due to its ionosphere and neurotoxicity. These toxicities are normally related with the size of the drug molecule or its complex. Obviously, liposome encapsulation inhibits the accumulation of drug in these organs and radically reduces toxicity[ 53 ]. Furthermore, often, the fungus exists in the cells of the mononuclear phagocytic system; therefore, the encapsulation results in reduced toxicity and passive targeting. These benefits, however, can be associated with any colloidal drug carrier. Certainly, similar improvements in therapy were observed with stable mixed micellar formulations and micro-emulsions[ 54 ]. Additionally, it seems that many of the early liposomal preparations were in actual fact liquid crystalline colloidal particles rather than self-closed MLV. Since the lives of the first terminally ill patients (who did not rely to all the conventional therapies) were saved[ 53 ], many patients were very effectively treated with diverse of amphotericin B formulations.

Comparable methods can be achieved in antiviral and antibacterial therapies[ 55 ]. Most of the antibiotics, however, are orally available; liposome encapsulation can be considered only in the case of very potent and toxic ones which are administered parenterally. The preparation of antibiotic-loaded liposomes at sensibly high drug-to-lipid ratios may not be easy because of the interactions of these molecules with bilayers and high densities of their aqueous solutions which often force liposomes to float as a creamy layer on the top of the tube. Several other ways, for instance, topical or pulmonary (by inhalation) administration are being considered also. Liposome-encapsulated antivirals (for example ribavirin, azidothymidine, or acyclovir) have also shown to reduce toxicity; currently, more detailed experiments are being performed in relation to their efficacy.

Liposomes in anticancer therapy

Numerous different liposome formulations of numerous anticancer agents were shown to be less toxic than the free drug[ 56 – 59 ]. Anthracyclines are drugs which stop the growth of dividing cells by intercalating into the DNA and, thus, kill mainly rapidly dividing cells. These cells are not only in tumors but are also in hair, gastrointestinal mucosa, and blood cells; therefore, this class of drug is very toxic. The most used and studied is Adriamycin (commercial name for doxorubicin HCl; Ben Venue Laboratories, Bedford, Ohio). In addition to the above-mentioned acute toxicities, its dosage is limited by its increasing cardio toxicity. Numerous diverse formulations were tried. In most cases, the toxicity was reduced to about 50%. These include both acute and chronic toxicities because liposome encapsulation reduces the delivery of the drug molecules towards those tissues. For the same reason, the efficiency was in many cases compromised due to the reduced bioavailability of the drug, especially if the tumor was not phagocytic or located in the organs of mononuclear phagocytic system. In some cases, such as systemic lymphoma, the effect of liposome encapsulation showed enhanced efficacy due to the continued release effect, i.e., longer presence of therapeutic concentrations in the circulation[ 60 – 62 ], while in several other cases, the sequestration of the drug into tissues of mononuclear phagocytic system actually reduced its efficacy.

Applications in man showed, in general, reduced toxicity and better tolerability of administration with not too encouraging efficacy. Several different formulations are in different phases of clinical studies and show mixed results.

Conclusions

Liposomes have been used in a broad range of pharmaceutical applications. Liposomes are showing particular promise as intracellular delivery systems for anti-sense molecules, ribosomes, proteins/peptides, and DNA. Liposomes with enhanced drug delivery to disease locations, by ability of long circulation residence times, are now achieving clinical acceptance. Also, liposomes promote targeting of particular diseased cells within the disease site. Finally, liposomal drugs exhibit reduced toxicities and retain enhanced efficacy compared with free complements. Only time will tell which of the above applications and speculations will prove to be successful. However, based on the pharmaceutical applications and available products, we can say that liposomes have definitely established their position in modern delivery systems.

Sahoo SK, Labhasetwar V: Nanotech approaches to drug delivery and imaging. DDT 2003, 8: 24.

Article   Google Scholar  

Gabizon A, Goren D, Cohen R, Barenholz Y: Development of liposomal anthracyclines: from basics to clinical applications. J Control Release 1998, 53: 275–279. 10.1016/S0168-3659(97)00261-7

Allen TM: Liposomes. Opportunities in drug delivery. Drugs 1997, 54(Suppl 4):8–14.

Chrai SS, Murari R, Imran A: Liposomes: a review. Bio Pharm 2001, 14(11):10–14.

Google Scholar  

Andreas W, Karola VU: Liposome technology for industrial purposes. J Drug Deliv 2011, 2011: 9.

Atrooz OM: Effects of alkylresorcinolic lipids obtained from acetonic extract of Jordanian wheat grains on liposome properties. Int J Biol Chem 2011, 5(5):314–321.

Benech RO, Kheadr EE, Laridi R, Lacroix C, Fliss I: Inhibition of Listeria innocua in cheddar cheese by addition of nisin Z in liposomes or by in situ production in mixed culture. Applied Environ Microbiol 2002, 68: 3683–3690. 10.1128/AEM.68.8.3683-3690.2002

Shehata T, Ogawara K, Higaki K, Kimura T: Prolongation of residence time of liposome by surface-modification with mixture of hydrophilic polymers. Int J Pharm 2008, 359: 272–279. 10.1016/j.ijpharm.2008.04.004

Johnston MJ, Semple SC, Klimuk SK, Ansell S, Maurer N, Cullis PR: Characterization of the drug retention and pharmacokinetic properties of liposomal nanoparticles containing dihydrosphingomyelin. Biochim Biophys Acta 2007, 1768: 1121–1127. 10.1016/j.bbamem.2007.01.019

Hofheinz RD, Gnad-Vogt SU, Beyer U, Hochhaus A: Liposomal encapsulated anti-cancer drugs. Anticancer Drugs 2005, 16: 691–707. 10.1097/01.cad.0000167902.53039.5a

Omri A, Suntres ZE, Shek PN: Enhanced activity of liposomal polymyxin B against Pseudomonas aeruginosa in a rat model of lung infection. Biochem Pharmacol 2002, 64: 1407–1413. 10.1016/S0006-2952(02)01346-1

Schiffelers RM, Storm G, Bakker-Woudenberg IA: Host factors influencing the preferential localization of sterically stabilized liposomes in Klebsiella pneumoniae - infected rat lung tissue. Pharm Res 2001, 18: 780–787. 10.1023/A:1011080211226

Stano P, Bufali S, Pisano C, Bucci F, Barbarino M, Santaniello M, Carminati P, Luisi PL: Novel camptothecin analogue (gimatecan)-containing liposomes prepared by the ethanol injection method. J Liposome Res 2004, 14: 87–109. 10.1081/LPR-120039794

Hemanthkumar M, Spandana V: Liposomal encapsulation technology a novel drug delivery system designed for ayurvedic drug preparation. IRJP 2011, 2(10):4–7.

Maria Laura I, Franco D, Cattel L: Stealth liposomes: review of the basic science, rationale, and clinical applications, existing and potential. Int J Nanomedicine 2006, 1(3):297–315. 10.2217/17435889.1.3.297

Amarnath S, Sharma US: Liposomes in drug delivery: progress and limitations. Int J Pharm 1997, 154: 123–140. 10.1016/S0378-5173(97)00135-X

Shaheen SM, Shakil Ahmed FR, Hossen MN, Ahmed M, Amran MS, Ul-Islam MA: Liposome as a carrier for advanced drug delivery. Pak J Biol Sci 2006, 9(6):1181–1191.

Riaz M: Liposome preparation method. Pak J Pharm Sci 1996, 9(1):65–77.

Himanshu A, Sitasharan P, Singhai AK: Liposomes as drug carriers. IJPLS 2011, 2(7):945–951.

Kataria S, Sandhu P, Bilandi A, Akanksha M, Kapoor B, Seth GL, Bihani SD: Stealth liposomes: a review. IJRAP 2011, 2(5):1534–1538.

Mayer LD, Bally MB, Hope MJ, Cullis PR: Techniques for encapsulating bioactive agents in to liposomes. Chem Phys Lipids 1986, 40: 333–345. 10.1016/0009-3084(86)90077-0

Song H, Geng HQ, Ruan J, Wang K, Bao CC, Wang J, Peng X, Zhang XQ, Cui DX: Development of polysorbate 80/phospholipid mixed micellar formation for docetaxel and assessment of its in vivo distribution in animal models. Nanoscale Res Lett 2011, 6: 354. 10.1186/1556-276X-6-354

Zhang Y: Relations between size and function of substance particles. Nano Biomed Eng 2011, 3(1):1–16.

Mozafari MR: Liposomes: an overview of manufacturing techniques. Cell Mol Biol Lett 2005, 10(4):711–719.

Hamilton RL, Guo LSS: Liposomes preparation methods. J Clin Biochem Nut 1984, 7: 175.

Pick U: Liposomes with a large trapping capacity prepared by freezing and thawing of sonicated phospholipid mixtures. Arch Biochem Biophys 1981, 212: 186–194. 10.1016/0003-9861(81)90358-1

Ohsawa T, Miura H, Harada K: Improvement of encapsulation efficiency of water-soluble drugs in liposomes formed by the freeze-thawing method. Chem Pharm Bull 1985, 33(9):3945–3952. 10.1248/cpb.33.3945

Liu L, Yonetaini T: Preparation and characterization of liposome-encapsulated haemoglobin by a freeze-thaw method. J Microencapsulation 1994, 11(4):409–421. 10.3109/02652049409034258

Deamer D, Bangham AD: Large volume liposomes by an ether vaporization method. Biochim Biophys Acta 1976, 443(3):629–634.

Schieren H, Rudolph S, Findelstein M, Coleman P, Weissmann G: Comparison of large unilamellar vesicles prepared by a petroleum ether vaporization method with multilamellar vesicles: ESR, diffusion and entrapment analyses. Biochim Biophys Acta 1978, 542(1):137–153. 10.1016/0304-4165(78)90240-4

Batzri S, Korn ED: Single bilayer liposomes prepared without sonication. Biochim Biophy Acta 1973, 298(4):1015–1019. 10.1016/0005-2736(73)90408-2

Szoka F Jr, Papahadjopoulos D: Procedure for preparation of liposomes with large internal aqueous space and high capture by reverse-phase evaporation. Proc Natl Acad Sci USA 1978, 75(9):4194–4198. 10.1073/pnas.75.9.4194

Handa T, Naito S, Hiramatsu M, Tsuboi M: Thermal SiO and H 13 CO + line observations of the dense molecular cloud G0.11–0.11 in the Galactic Center Region. Astrophys J 2006, 636: 261–266. 10.1086/497881

Daemen T, Hofstede G, Ten Kate MT, Bakker-Woudenberg IAJM, Scherphof GL: Liposomal doxorubicin induced toxicity: depletion and impairment of phagocytic activity of liver macrophages. Int Cancer 1995, 61: 761–721. 10.1002/ijc.2910610604

Kirby CJ, Gregoriadis G: A simple procedure for preparing liposomes capable of high encapsulation efficiency under mild conditions. In Liposome Technology . 1st edition. Edited by: Gregoriadis G. Boca Raton: CRC; 1984:19–27.

Alpes H, Allmann K, Plattner H, Reichert J, Rick R, Schulz S: Formation of large unilamellar vesicles using alkyl maltoside detergents. Biochim Biophys Acta 1986, 862: 294. 10.1016/0005-2736(86)90231-2

Gabizon AA: Stealth liposomes and tumor targeting: one step further in the quest for the magic bullet. Clin Cancer Res 2001, 7: 223.

Romberg B, Hennink WE, Storm G: Sheddable coatings for long-circulating nanoparticles. Pharm Res 2008, 25(1):55–71. 10.1007/s11095-007-9348-7

Mayer ID, Madden TM, Bally MU, Cullis PR: pH gradient-mediated drug entrapment in liposomes. In Liposome Technology . 2nd edition. Edited by: Gregoriadis G. Boca Raton: CRC Press; 1993:27–44.

Arcadio C, Cullis PR: Recent advances in liposomal drug-delivery systems. Curr Opin Biotechnol 1995, 6: 698–708. 10.1016/0958-1669(95)80115-4

Awada A, Gil T, Sales F, Dubuisson M, Vereecken P, Klastersky J, Moerman C, de Valeriola D, Piccart MJ: Prolonged schedule of temozolomide (Temodal) plus liposomal doxorubicin (Caelyx) in advanced solid cancers. Anticancer Drugs 2004, 15: 499–502. 10.1097/01.cad.0000127331.29310.8a

Babai I, Samira S, Barenholz Y, Zakay-Rones Z, Kedar E: A novel influenza subunit vaccine composed of liposome-encapsulated haemagglutinin/neuraminidase and IL-2 or GM-CSF. I. Vaccine characterization and efficacy studies in mice. Vaccine 1999, 17: 1223–1238. 10.1016/S0264-410X(98)00346-6

Banerjee R, Tyagi P, Li S, Huang L: Anisamide-targeted stealth liposomes: a potent carrier for targeting doxorubicin to human prostate cancer cells. Int J Cancer 2004, 112: 693–700. 10.1002/ijc.20452

Baselga J, Metselaar JM: Monoclonal antibodies: clinical applications: monoclonal antibodies directed against growth factor receptors. In Principles and Practice of Biological Therapy of Cancer . Edited by: Rosenburg SA. Philadelphia: Lippincott; 2000:475–489.

Kunisawa J, Mayumi T: Fusogenic liposome delivers encapsulated nanoparticles for cytosolic controlled gene release. J Control Release 2005, 105: 344–353. 10.1016/j.jconrel.2005.03.020

Parthasarathy R, Sacks PC, Harris D, Brock H, Mehta K: Interaction of liposorae-associated all-trans-retinoic acid with squamous carcinoma cells. Cancer Chemother Pharmacol 1994, 34: 527–534. 10.1007/BF00685666

Mehta K, Sadeghi T, McQueen T, Lopez-Berestein G: Liposome encapsulation circumvents the hepatic clearance mechanisms of all- trans -retinoic acid. Leuk Res 1994, 18: 587–596. 10.1016/0145-2126(94)90040-X

Gill PS, Espina M, Muggia F, Cabriales S, Tulpule A, Esplin JA, Liebman HA, Forssen E, Ross ME, Levine AM: Phase I/II clinical and pharmacokinetic evaluation of liposomal daunorubicin. J Clin Oncol 1995, 13: 996–1003.

Gauglianone P, Chan K, DelaFfor-Weiss E, Hanixh R, Jeffers S, Sharma D, Muggia F: Phase I and pharmacologic study of liposomal daunorubicin (DaunoXome). Invest New Drugs 1994, 12: 103–110. 10.1007/BF00874439

Eckardt JR, Campbell E, Burris HA, Weiss CR, Rodriguez CI, Fields SM, Thurman AM, Peacock NW, Cobb P, Rothenbeig ML: A phase II trial of DaunoXome, liposome-encapsulated daunorubicin, in patients with metastatic adenocarcinoma of the colon. Am Clin Oncol 1994, 17: 498–501. 10.1097/00000421-199412000-00009

Schurmann D, Dormann A, Grunewald T, Ruf B: Successful treatment of AIDS-related pulmonary Kaposi's sarcoma with liposomal daunorubicin. Eur Respir J 1994, 7: 824–825. 10.1183/09031936.94.07040824

New RRC, Chance SM, Thomas SC, Peters W: Nature antileishmanial activity of antimonials entrapped in liposomes. Nature 1978, 272: 55–58. 10.1038/272055a0

Lopez-Berestein G, Fainstein V, Hopter R, Mehta KR, Sullivan M, Keating M, Luna M, Hersh EM: Liposomal amphotericin B for the treatment of systemic fungal infections in patients with cancer. J Infect Diseases 1985, 151: 704–710. 10.1093/infdis/151.4.704

Lasic DD: Mixed micelles in drug delivery. Nature 1992, 355: 279–280. 10.1038/355279a0

Svenson CE, Popescu MC, Ginsberg RC: Liposome treatments of viral, bact and protozoal infections. Crit Rev Microbiol 1988, 15: S1-S31. 10.3109/10408418809104463

Gabizon A: Liposomes as a drug delivery system in cancer therapy. In Drug Carrier Systems . Edited by: Roerdink FHD, Kron AM. Chichester: Wiley; 1989:185–211.

Storm G, Roerdink FH, Steerenberg PA, de Jong WH, Crommelin DJA: Influence of lipid composition on the antitumor activity exerted by doxorubicin containing liposomes in a rat solid tumor model. Cancer Res 1987, 47: 3366–3372.

Akbarzadeh A, Asgari D, Zarghami N, Mohammad R, Davaran S: Preparation and in vitro evaluation of doxorubicin-loaded Fe 3 O 4 magnetic nanoparticles modified with biocompatible co-polymers. Int J Nanomedicine 2012, 7: 511–526.

Akbarzadeh A, Zarghami N, Mikaeili H, Asgari D, Goganian AM, Khiabani HK, Samiei M, Davaran S: Synthesis, characterization, and in vitro evaluation of novel polymer-coated magnetic nanoparticles for controlled delivery of doxorubicin. Nanotechnol Sci Appl 2012, 5: 13–25.

Akbarzadeh A, Samiei M, Davaran S: Magnetic nanoparticles: preparation, physical properties, and applications in biomedicine. Nanoscale Res Lett 2012, 7: 144. 10.1186/1556-276X-7-144

Valizadeh A, Mikaeili H, Samiei M, Mussa Farkhani S, Zarghami N, Kouhi M, Akbarzadeh A, Davaran S: Quantum dots: synthesis, bioapplications, and toxicity. Nanoscale Res Lett 2012, 7: 480. 10.1186/1556-276X-7-480

Akbarzadeh A, Samiei M, Joo SW, Anzaby M, Hanifehpour Y, Nasrabadi HT, Davaran S: Synthesis, characterization and in vitro studies of doxorubicin-loaded magnetic nanoparticles grafted to smart copolymers on A549 lung cancer cell line. J Nanobiotechnology 2012, 10: 46. 10.1186/1477-3155-10-46

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Acknowledgments

The authors thank the Department of Medical Nanotechnology, Faculty of Advanced Medical Science of Tabriz University for all the support provided. This work is funded by the 2012 Yeungnam University Research Grant.

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Abolfazl Akbarzadeh, Rogaie Rezaei-Sadabady, Soodabeh Davaran, Nosratollah Zarghami & Kazem Nejati-Koshki

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SWJ conceived the study and participated in its design and coordination. NZ participated in the sequence alignment and drafted the manuscript. AA, RRS, SD, YH, MS, MK, and KNK helped in drafting the manuscript. All authors read and approved the final manuscript.

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Akbarzadeh, A., Rezaei-Sadabady, R., Davaran, S. et al. Liposome: classification, preparation, and applications. Nanoscale Res Lett 8 , 102 (2013). https://doi.org/10.1186/1556-276X-8-102

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Chrysin-loaded PEGylated liposomes protect against alloxan-induced diabetic neuropathy in rats: the interplay between endoplasmic reticulum stress and autophagy

• Mahran Mohamed Abd El-Emam 1 ,
• Amany Behairy 2 ,
• Mahmoud Mostafa 3 ,
• Tarek khamis 4 , 5 ,
• Noura M. S. Osman 6 ,
• Amira Ebrahim Alsemeh 7 &
• Mohamed Fouad Mansour 1  

Biological Research volume  57 , Article number:  45 ( 2024 ) Cite this article

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Diabetic neuropathy (DN) is recognized as a significant complication arising from diabetes mellitus (DM). Pathogenesis of DN is accelerated by endoplasmic reticulum (ER) stress, which inhibits autophagy and contributes to disease progression. Autophagy is a highly conserved mechanism crucial in mitigating cell death induced by ER stress. Chrysin, a naturally occurring flavonoid, can be found abundantly in honey, propolis, and various plant extracts. Despite possessing advantageous attributes such as being an antioxidant, anti-allergic, anti-inflammatory, anti-fibrotic, and anticancer agent, chrysin exhibits limited bioavailability. The current study aimed to produce a more bioavailable form of chrysin and discover how administering chrysin could alter the neuropathy induced by Alloxan in male rats.

Chrysin was formulated using PEGylated liposomes to boost its bioavailability and formulation. Chrysin PEGylated liposomes (Chr-PLs) were characterized for particle size diameter, zeta potential, polydispersity index, transmission electron microscopy, and in vitro drug release. Rats were divided into four groups: control, Alloxan, metformin, and Chr-PLs. In order to determine Chr- PLs’ antidiabetic activity and, by extension, its capacity to ameliorate DN, several experiments were carried out. These included measuring acetylcholinesterase, fasting blood glucose, insulin, genes dependent on autophagy or stress in the endoplasmic reticulum, and histopathological analysis.

According to the results, the prepared Chr-PLs exhibited an average particle size of approximately 134 nm. They displayed even distribution of particle sizes. The maximum entrapment efficiency of 90.48 ± 7.75% was achieved. Chr-PLs effectively decreased blood glucose levels by 67.7% and elevated serum acetylcholinesterase levels by 40% compared to diabetic rats. Additionally, Chr-PLs suppressed the expression of ER stress-related genes (ATF-6, CHOP, XBP-1, BiP, JNK, PI3K, Akt, and mTOR by 33%, 39.5%, 32.2%, 44.4%, 40.4%, 39.2%, 39%, and 35.9%, respectively). They also upregulated the miR - 301a - 5p expression levels by 513% and downregulated miR - 301a - 5p expression levels by 65%. They also boosted the expression of autophagic markers (AMPK, ULK1, Beclin 1, and LC3-II by 90.3%, 181%, 109%, and 78%, respectively) in the sciatic nerve. The histopathological analysis also showed that Chr-PLs inhibited sciatic nerve degeneration.

The findings suggest that Chr-PLs may be helpful in the protection against DN via regulation of ER stress and autophagy.

Graphical Abstract

Diabetes mellitus (DM) refers to a group of metabolic disorders characterized by high glucose levels in the blood and insufficient production or effectiveness of insulin by the pancreas [ 1 ]. According to the World Health Organization, the global population affected by diabetes currently exceeds 400 million individuals. However, this number is projected to rise significantly and reach 552 million by 2030 [ 2 ]. Diabetic neuropathy (DN), which includes allodynia, hyperalgesia, and spontaneous pain, is a severe complication of the disease. About half to two-thirds of people with diabetes would develop diabetic neuropathy [ 3 ]. Controlling DN entails mostly maintaining normal blood sugar levels and treating symptoms [ 4 ]. There has been limited progress in alleviating DN-related chronic pain since undesirable side effects accompany many pharmacological medications, and therapeutic expectations remain largely unmet [ 5 ]. So, it is crucial to develop new therapy strategies for DN.

Endoplasmic reticulum (ER) stress may contribute significantly to DN’s development. ER stress is distinguished by the buildup of imperfect proteins within the ER stress, which can hinder its ability to fold proteins properly [ 6 ]. Unfolded protein response (UPR), which regulates translation, enhances protein folding, and affects inflammation in abnormal conditions, is triggered by ER stress in cells [ 7 ]. UPR is initiated by three canonical UPR mediators (sensors), including inositol-requiring enzyme 1α (IRE1α), protein kinase R-like ER kinase (PERK), and activating transcription factor 6 (ATF6) pathways [ 8 ]. These mediators attach to binding immunoglobulin protein (BiP) in an inactive state. Stress causes BiP to separate from the ER and aid in protein folding, activating PERK and ATF6 sensors [ 9 ]. IRE1α, an evolutionarily conserved ER stress sensor, initiates X-box-binding protein 1’s unconventional mRNA splicing, resulting in XBP1s, an active transcription factor that enhances the ER’s capacity to handle and remove unfolded proteins [ 10 ]. IRE1 mediates ER stress by promoting the phosphorylation of C-jun NH2-terminal kinase (JNK). PERK, a key factor in the UPR response, is activated by phosphorylating the α-subunit of eIF2α, which hinders the assembly of the 80 S ribosome and protein synthesis [ 11 ]. Additionally, under ER stress, ATF6 exports to the Golgi apparatus, cleaves at Sites 1 and 2, and translocates to the nucleus for transcription of UPR target genes, including CCAAT-enhancer-binding protein homologous protein (CHOP) [ 12 ]. Conversely, continuous activation of ER stress leads to cellular breakdown, such as in the case of high blood sugar levels.

Autophagy is a natural response to stress that assists in the breakdown of pathogens, denatured proteins, and impaired organelles within the lysosomes [ 13 ]. There was a robust mechanistic association between ER stress and autophagy [ 14 ]. JNK has been connected to IRE1 activation, which could result in autophagy caused by Beclin-1 [ 15 ]. In addition, during ER stress, autophagy activation is dependent on PERK-eIF-2, indicating a connection between autophagy and the UPR signalling pathway. Mammalian target of rapamycin (mTOR) prevents AMP-activated protein kinase (AMPK) from interacting with UNC-51-like kinase 1 (ULK1), which in turn prevents autophagy [ 16 ]. The PI3K/AKT/mTOR pathway is primarily studied for its regulation of autophagy, involving PI3K, AKT, and mTOR as key molecules. PI3K is a cytoplasmic lipid kinase that can phosphorylate phosphatidylinositol at the D3 position [ 17 ]. The regulatory component maintains the catalytic subunit in a low-activity state during physiological circumstances. External stress triggers the phosphorylation of the SH2 domain of the p85, releasing the restriction on the p110 and activating PI3K and its downstream signaling pathways [ 18 ]. AKT is phosphorylated by PKC-1 and mTOR2 upon PI3K activation, transforming into p-AKT and transported to the cell membrane [ 19 ]. By transducing signals to mTOR, p-AKT inhibits autophagy physiologically by activating the ubiquitin–proteasome pathway and regulating genes associated with autophagy or other downstream substrates [ 20 ]. The PI3K/AKT/mTOR signaling pathway is involved in signal transmission, autophagosome movement, and vesicle fusion in the autophagy process [ 21 ]. Thus, regulation of the PI3K/AKT/mTOR signaling pathway is crucial for maintaining autophagy's homeostasis. Autophagy dysfunction and ER stress were both found to be associated with diabetes [ 22 ]. Interventions aiming to normalize ER stress and autophagy may be proposed as an effective means of limiting DN development.

Various researchers are now studying natural therapies for various medical issues. Chrysin (Chr) 5,7-dihydroxyflavone, is a flavonoid abundant in passiflora, chamomile, oroxylum and honey, and propolis. It possesses considerable antioxidant activity and various pharmacological advantages, such as antidiabetic, anti-inflammatory, and protective effects on the heart and liver [ 23 ]. Nevertheless, the inadequate bioavailability and low solubility of chrysin, which varies between 2 and 5 µg/mL, pose significant challenges to its use in therapeutics [ 24 ]. In addition, chrysin undergoes many pre-systemic metabolism, including glucuronidation and sulphation in the intestine and liver [ 25 ]. For this reason, researchers investigated potential carriers that could overcome these obstacles and enhance the bioavailability of drugs exhibiting suboptimal pharmacokinetics. Drug delivery systems based on nanotechnology are the most popular for this purpose [ 26 ].

Liposomes are lipid-based nanocarriers that bring drugs to their target sites [ 27 ]. The bioavailability of poorly soluble medications in water can be boosted by encapsulating them in liposomes [ 28 ]. Using liposomes, which are believed to be biocompatible, biodegradable, non-toxic, and immunogenic, reduces the risk of adverse drug reactions during administration [ 29 ]. Furthermore, liposomes can improve the pharmacokinetic features of a drug, providing a way to achieve the optimal concentrations required for its intended action [ 30 ]. Liposomes’ half-lives in the bloodstream can be increased from a few minutes to several hours thanks to hydrophilic polymers like PEG employed as surface coatings [ 31 ]. In addition to blocking the reticuloendothelial system from ingesting and opsonizing liposomes, PEG boosts their solubilization power, decreases their aggregation, and lowers their immunogenicity [ 32 ]. PEG lengthens the time it takes for blood to circulate and causes more liposomes to accumulate in damaged tissues [ 33 ]. This PEG technology is used in the pharmaceutical product Doxil®, which has a potent antitumor action [ 34 ].

This work hypothesized that administering Chr-PLs to rats with DN may reduce the ER stress response and promote autophagy by controlling the related genes. The influences of Chr-PLs on insulin, fasting blood glucose level (FBG), and acetylcholinesterase in diabetic rats were investigated to test the hypothesis. The effects of Chr-PLs on ER and autophagy-related markers in diabetic rats were further analyzed by measuring their mRNA, microRNAs (miRNAs), and protein expression levels. Furthermore, Chr-PLs’ influence on the histopathology of the sciatic nerve was demonstrated. The findings of this research offer a distinct perspective on managing ER and autophagy effects and the effective management of peripheral neuropathy by novel Chr-PLs.

Materials and methods

Alloxan, polyethylene glycol 4000 (PEG 4000 ), and cholesterol were provided by Sigma-Aldrich (St. Louis, MO, USA). Lipoid GmbH (Ludwigshafen, Germany) supplied saturated phosphatidylcholine derived from soybean. Chrysin (99.2%) was purchased from Axenic (Oak Park, Melbourne, Australia).

We obtained healthy adult male Sprague–Dawley rats from the experimental animal unit at the College of Veterinary Medicine at Zagazig University, Egypt. These rats weighed between 200 and 250 g . Before the experiment, the rats had a standard commercial food and water diet. Additionally, they were given 2 weeks to acclimate to the laboratory environment, which maintained a temperature of approximately 25 °C.

Preparation of a nano-liposomal formulation for chrysin

The solvent injection method was utilized to produce chrysin-loaded PEGylated liposomes (Chr-PLs), as described elsewhere [ 35 , 36 ]. To develop the organic phase, saturated phosphatidylcholine from soybean, cholesterol, chrysin, and PEG 4000 were added to 100% ethyl alcohol in the following proportions: 13:3:1:1 (w/w). The temperature of the solution was increased to 60–70 °C. However, an aqueous phase consisting of a 0.9% sucrose solution was maintained at the same temperature while agitated with a magnetic stirrer (1000 rpm). In order to enable the assembly of liposomes, the organic phase was infused into the aqueous phase via a 25G syringe. Following the injection procedure, the mixture was maintained at 60–70 °C for 20–30 min in order to facilitate the evaporation of ethyl alcohol [ 37 ]. Particle size diameter, polydispersity score, zeta potential, transmission electron microscope (TEM), and an in vitro release study were all used to characterize the newly generated Chr-PLs formulation. The produced liposomal suspension's particle size diameter, polydispersity score, and zeta potential were measured using the Malvern Zetasizer Nano (Malvern Instruments Ltd., Worcestershire, UK) as previously reported [ 35 , 38 ]. After the liposomal systems were lysed, the entrapment efficiency of the resulting systems was assessed, as previously reported [ 39 ]. In brief, 1 ml of Chr-PLs was centrifuged at 10,000 rpm for 60 min (Hermle, Essen, Germany) and the pellet was resuspended for three times to remove the unentrapped drug. Chr-PLs were mixed with acetonitrile (1:4) and sonicated for 20 min. The amount of Chr was determined spectrophotometrically at 267 nm (Shimadzu, Kyoto, Japan) and the entrapment efficiency was calculated as follows:

An in vitro release study was conducted according to Abd El-Emam et al. [ 39 ]. Briefly, an aliquot of Chr-PLs was placed within the sample compartment of an established Franz diffusion cell. The release medium was composed of PBS (7.4)—ethanol mixture (65:35) which was placed into the reservoir chamber. The two chambers were separated using A nitrocellulose membrane (12–14 kDa MWCO). The system was operated at 60 rpm and 37 °C. Two milliliters of reservoir medium were obtained at regular intervals and subjected to UV examination at a wavelength of 267 nm to quantify the amount of Chr released. The volume of the reservoir medium was restored using an equivalent volume of PBS-ethanol mixture maintained at 37 °C.

Experimental design

Induction of diabetes.

Rats were injected with 150 mg/kg of alloxan solution intraperitoneally (i.p.) following an overnight fast to induce diabetes [ 40 ]. For 48 h, the rats were given sucrose to avoid their deaths from the rise in insulin. After 72 h, a Glucometer was used to assess glucose levels in blood samples collected from the tail veins of the rats. Only diabetic rats (defined as having a fasting blood glucose level of 250 mg/dL or more) were employed to proceed with the experiment.

Experimental groups

Forty rats were randomly assigned into four groups (n = 10). Control group, Alloxan group (Alloxan-treated rats), metformin group (Alloxan-treated rats received metformin orally, 100 mg/kg BWt, once a day for 21 successive days), and Chr-PLs group (5 mg/kg BWt, i.p., every other day for 21 consecutive days) [ 41 ].

Following the last treatment, the rats were fasted overnight. Body weight was recorded, and Blood samples were collected through a cardiac puncture while the rats were under diethyl ether anesthesia. Blood was drawn into tubes without an anticoagulant to separate the serum and tubes containing sodium fluoride to separate plasma. A small cross-section from sciatic nerves was promptly removed and fixed in a 10% formaldehyde solution for histopathological and immunohistological analysis.

Biochemical analysis

Insulin levels were determined using the rat insulin ELISA technique (Catalogue Number ERINS, Thermo Fisher Scientific, Waltham, USA), while FBG levels were evaluated using the glucose oxidase method (Agape Diagnostics Ltd., Kochi, India). The blood acetylcholinesterase levels of each group were determined using a colorimetric kinetic assay (Biodiagnostic, Giza, Egypt). The HOMA-IR value was determined by employing the subsequent equation [ 42 ]:

while HOMA-β was determined by employing the subsequent equation[ 42 ]:

Real-time RT-PCR

Total RNA was extracted from the sciatic nerve sample using the TRIzolTM reagent kit following the manufacturer’s instructions (Invitrogen, Thermofisher Scientific, Waltham, MA, USA). As was previously reported, 500 ng of total RNA was used for transcription, producing mRNA [ 43 , 44 ]. In this case, miRNA transcription was performed using TaqManTM Small RNA Assays (Thermofisher Scientific, Waltham, MA, USA) on ten ng of RNA according to the manufacturer’s guidelines. Primers specific to miRNAs, stem-loops, and the universal reverse primer were all designed with the help of http://genomics.dote.hu:8080/mirnadesigntool (viewed on 10 September 2020) assay design software [ 45 ]. Sangon Biotech (Beijing, China) kindly supplied a list of the primers used in this investigation (Table  1 ). For real-time PCR, we used the Maxima SYBR Green/Rox qPCR 2× Master Mix from Thermofisher Scientific in Waltham, MA, USA. Each gene’s relative expression was calculated using the 2 –ΔΔCt technique, with mRNA and miRNA normalized to housekeeping GAPDH and U6, respectively [ 46 ].

Histopathological assessment of the sciatic nerve

Samples of the sciatic nerve were gathered and subsequently conserved in a solution of buffered neutral formalin, with a concentration of 10%, for 48 h. Next, dehydration occurred by raising the alcohol content of the samples. After being washed in xylene, they were embedded in paraffin. The tissue blocks were preserved in paraffin and subsequently sliced into 5 µm thick sections using a microtome (Leica RM 2155, England). The sections were dewaxed and stained with hematoxylin and eosin (H&E) [ 47 ].

Immunohistochemical examination

The tissue sections, with a thickness of 5 μm and deparaffinization, were immersed in a solution containing 3% hydrogen peroxide (H 2 O 2 ) for 30 min. The samples were then re-incubated at 37 °C for an additional hour. For this phase, we followed the manufacturer’s instructions to treat the slices with anti-Beclin 1 (MA5-15,825; Invitrogen, USA, 1:200), anti-LC3 (ab192890; Abcam, Cambridge, UK, 1:2000), and anti-p62 (ab91526; Abcam, Cambridge, UK, 1:1000). Following a thorough wash in PBS, the sections underwent treatment using a secondary antibody and the HRP Envision kit (DAKO) for 20 min. Once the slices were cleaned thoroughly with PBS, they were incubated with diaminobenzidine for 10 min. Afterward, the samples underwent a process of dehydration, followed by washing with xylene. Subsequently, they were counterstained with hematoxylin and placed under a cover slip for further examination using a microscope. The analysis was completed using the technique adopted by Elsayed et al. [ 48 ]. A set of seven non-overlapping separate fields was selected randomly. Selected areas were then investigated to determine the average percentage of tissue stained positive for Beclin 1, LC3, and p62 using immunohistochemistry for each tissue slice within a sample. Tissue slices were imaged with Full HD microscopic imaging equipment (Leica Microsystems Ltd.; Wetzlar, Germany) and analyzed with Leica Application software version 3.7.5.

Statistical analysis

The data was analyzed using the GraphPad Prism software, specifically version 10.0.1, developed in San Diego, CA, USA. A one-way ANOVA was employed to examine the data, followed by the Tukey–Kramer test. The significance level of 0.05 was chosen as the threshold for determining statistical significance.

Characterization of Chr-PLs

Chr-PLs were successfully fabricated using an ethanol injection methodology and evaluated for their particle size diameter, polydispersity score, zeta potential, TEM, and in vitro drug release. Within the nanoscale range, the average particle diameter of Chr-PLs was 134.6 ± 21.45 nm detected by Zetasizer nano (Malvern, UK) (Fig.  1 A). Liposomal particles were also more evenly distributed, as seen by their polydispersity score of 0.275 ± 0.073. Furthermore, liposomal vesicles exhibit a slightly negative surface charge, measuring − 21.1 ± 1.72 mV. Images obtained by transmission electron microscopy (Jeol, Tokyo, Japan) showed that the Chr-PLs were nearly spherical, and the average particle diameter was like that found using Zetasizer nano (Fig.  1 B). The liposomal vesicular systems also showed improved chrysin trapping capability, with an efficiency of 90.48 ± 7.75%. In vitro release assay demonstrates the cumulative release rate of Chr-PLs, as shown in Fig.  1 C. Within the first six hours, 36.7% of preloaded Chr were released; after that, after 24 h, this percentage rose steadily to over 57.6%. By contrast, the Chr-free drug only released 23.98% of the loaded amount after 6 h, slightly increasing to 27.95% after 24 h.

Characterization of Chr-PLs formulation. Chr-PLs were characterized for particle size diameter ( A ), TEM ( B ), and in vitro drug release ( C ). The preparation of Chr-PLs was accomplished using the ethanol injection approach. The spherical shape and nano-size dimensions of Chr-PLs were confirmed by its characterization. Drug release was found to be improved after formulation into nanoliposomes, according to in vitro release measurements. In Figure C , squares indicate Chr-PLs and circles indicate Chr

Chr-PLs enhance body weight index and serum level of acetylcholinesterase in alloxan-induced diabetes in rats

Next, we looked at how alloxan-induced diabetes in rats affected body weight and acetylcholinesterase levels to determine the impact of Chr-PLs. Alloxan markedly decreased body weight by 20.9% and elevated acetylcholinesterase levels by 199% compared to the control group. Diabetic rats’ body weight was significantly increased by 18.9%, and serum acetylcholinesterase was significantly lowered by 40% after injecting Chr-PLs with Alloxan (Fig.  2 A and B ). Results for the metformin group were comparable to those for the Chr-PLs group.

Influence of Chr-PLs administration on the body weight and serum AChE level in alloxan-induced diabetes in rats. The body weight ( A ) and serum AChE level ( B ) were measured for the different groups. ***P < 0.001, *P < 0.05 vs. control group. ## P < 0.01, # P < 0.05 vs. alloxan group, ns indicates nonsignificant. Data are presented as the mean ± SEM (n = 10)

Chr-PLs enhance blood levels of FBG and insulin, and the scores of HOMA-IR and HOMA-β in alloxan-induced diabetes in rats

The antidiabetic effects of Chr-PLs were also evaluated by measuring blood levels of FBG and insulin and the scores of HOMA-IR and HOMA-β. In contrast to the control group, it was revealed that after receiving Alloxan, FBG increased by 279%, and HOMA-IR increased by 99.7%. In comparison, plasma insulin levels decreased by 47.3%, and HOMA-β levels rose by 92.6%. FBG and HOMA-IR were reduced by 67.7% and 57.6%, respectively, when Alloxan and Chr-PLs were administered concurrently. In contrast, plasma insulin and HOMA-β significantly increased by 31.4% and 532%, respectively, compared to the alloxan group. Similar outcomes were seen in the metformin-treated group as in the Chr-PLs group (Fig.  3 A–D).

Influence of Chr-PLs administration on the levels of FBG, insulin, HOMA-IR, and HOMA-β in alloxan-induced diabetes in rats. The levels of FBG ( A ) and insulin ( B ) in blood and the scores for HOMA-IR ( C ) and HOMA-β ( D ) were measured for the different groups. ****P < 0.0001, ***P < 0.001, **P < 0.01 vs. control group. #### P < 0.0001, ### P < 0.001, ## P < 0.01, # P < 0.05 vs. alloxan group, ns indicates nonsignificant. Data are presented as the mean ± SEM (n = 10)

Chr-PLs downregulate mRNA expression levels of ER stress genes in the sciatic nerve in alloxan-induced diabetic rats

Subsequently, the influence of Chr-PLs on the mRNA expression levels of ER stress genes (ATF6 signaling pathway) was examined in an alloxan-induced DN model. After administering Alloxan, mRNA expression levels of all examined ER genes were markedly increased. The expression levels of activating transcription factor 6 (ATF6), C/EBP homologous protein (CHOP), X-box-binding protein 1 (XBP-1), binding immunoglobulin protein (BIP), and Jun N-terminal kinase (JNK) were increased by 650%, 817%, 782%, 337%, and 474%, respectively, in the sciatic nerve. However, compared to the alloxan group, Chr-PLs resulted in a considerable suppression of mRNA expression levels of ATF6, CHOP, XBP-1, BIP, and JNK by 33%, 39.5%, 32.2%, 44.4%, and 40.4%, respectively. The Chr-PLs group showed better results than the metformin-treated group (Fig.  4 A–E).

Influence of Chr-PLs administration on the mRNA expression of ER stress markers in the sciatic nerve in alloxan-induced diabetes in rats. The mRNA levels of ER stress markers, including ATF6 ( A ), CHOP ( B ), XBP-1 ( C ), BIP ( D ), and JNK ( E ) were measured for the different groups. ****P < 0.0001, ***P < 0.001, **P < 0.01 vs. control group. #### P < 0.0001 vs. alloxan group. $$$ P < 0.001, $$ P < 0.01 vs. metformin group, ns indicates nonsignificant. Data are presented as the mean ± SEM (n = 10)

Chr-PLs enhance mRNA expression levels of autophagy-dependent markers in the sciatic nerve in alloxan-injected rats

The impact of Chr-PLs administration on the expression levels of autophagy-dependent genes was also investigated. The influence of Chr-PLs treatment on PI3K/Akt1/mTOR pathway genes in the sciatic nerve in alloxan-injected rats was evaluated. Alloxan administration resulted in a notable rise in the expression levels of phosphoinositide 3-kinase (PI3K), Akt isoform 1 (Akt1), and Mammalian target of rapamycin (mTOR) by 486%, 760%, and 581%, respectively, in the sciatic nerve in contrast to the control group. The administration of Chr-PLs resulted in a noteworthy reduction of 39.2%, 39%, and 35.9% in the mRNA expression levels of PI3K, Akt1, and mTOR, respectively, compared to the group induced with Alloxan (Fig.  5 A–C). Furthermore, as shown in Fig.  5 A–C, the results obtained by the Chr-PLs group were superior to those obtained by the metformin group.

Influence of Chr-PLs administration on the mRNA expression of autophagy-dependent markers in the sciatic nerve in alloxan-induced diabetes in rats. The mRNA levels of ER stress markers, including PI3K, Akt, and mTOR, were measured for the different groups. ****P < 0.0001, ***P < 0.001 vs. control group. #### P < 0.0001 vs. alloxan group. $$$$ P < 0.0001 vs. metformin group. Data are presented as the mean ± SEM (n = 10)

The mRNA levels of AMP-activated protein kinase (AMPK), unc-51 like autophagy activating kinase 1 (ULK-1), bcl-2 interacting protein 1 (beclin 1), and microtubule-associated proteins 1A/1B light chain 3B (LC3) in the sciatic nerve of alloxan-injected rats were considerably lower than those in the control group by 78%, 87.8%, 79.5%, and 71.5%, respectively. However, administering Chr-PLs to alloxan-induced diabetic rats reversed the downregulation of autophagy-dependent gene expression. Sciatic nerve mRNA levels for AMPK, ULK-1, beclin 1, and LC3 were all significantly increased in the Chr-PLs group compared to the alloxan group by 90.3%, 181%, 109%, and 78%, respectively. The outcomes for Chr-PLs were superior to those of the metformin group (Fig.  6 A–D).

Influence of Chr-PLs administration on the mRNA expression of autophagy-dependent markers in the sciatic nerve of alloxan-induced diabetes in rats. The mRNA levels of AMPK ( A ), ULK-1 ( B ), beclin 1 ( C ), and LC3 ( D ) were measured for the different groups. Data are presented as the mean ± SEM (n = 10). ****P < 0.0001, ***P < 0.001, *P < 0.05 vs. control group. #### P < 0.0001, ## P < 0.01 vs. alloxan group. $$$$ P < 0.0001, $$$ P < 0.001 vs. metformin group

Chr-PLs modulate miRNA expression levels of miR-301a-5p and miR-30e-5p in the sciatic nerve in alloxan-induced diabetic rats

The location of the 3′ UTR region for miRNA binding to the targeted mRNA is shown in (Fig. 7 ). Theinfluence of Chr-PLs treatment on miRNA expression levels of miR-301a-5p and miR-30e-5p in thesciatic nerve in alloxan-injected rats was evaluated. The influence of Chr-PLs treatment on miRNA expression levels of miR - 301a - 5p and miR - 30e - 5p in the sciatic nerve in alloxan-injected rats was evaluated. Alloxan administration resulted in a notable drop in the expression levels of miR - 301a - 5p by 84% and a marked rise in the expression levels of miR - 30e - 5p by 732% in the sciatic nerve compared to the control group. The administration of Chr-PLs resulted in a noteworthy upregulation in the miR - 301a - 5p expression levels by 513% and downregulation in the miR - 301a - 5p expression levels by 65% compared to the group induced with Alloxan (Fig.  8 A, B ). Furthermore, as shown in Fig.  8 , the results obtained by the Chr-PLs group were superior to those obtained by the metformin group.

location of the 3′ UTR region for miRNA binding to the targeted mRNA, ( A , B ) miR-301a-5p, ( C , D ) miR-30e-5p

Influence of Chr-PLs administration on the expression of miR-301a-5p ( A ), and miR-30e-5p ( B ) in the sciatic nerve of alloxan-induced diabetes in rats. ****P < 0.0001, **P < 0.01 vs. control group. #### P < 0.0001, ### P < 0.001, ## P < 0.01 vs. alloxan group. $$$ P < 0.001 vs. metformin group

Chr-PLs restore histological changes in the sciatic nerve in alloxan-injected rats

Longitudinal sections of the peripheral nerve bundles of the control group stained with hematoxylin and eosin revealed many well-organized myelinated intact axons. The nodes of Ranvier are visible between adjacent internodes of the myelin sheath. The nuclei of Schwann cells can be seen through the vacuolated myelin sheaths (Fig. 9 A). Contrarily, myelin sheath degeneration and nerve fiber separation were observed in multiple stained nerve slices taken from alloxan-treated rats. Cellular infiltrations could be noticed (Fig. 9 B). Most myelin sheath integrity was restored with normal Schwann cell nuclei in metformin-treated diabetic rat tissue slices. However, there was still some evidence of nerve fiber disconnection and cellular infiltration (Fig. 9 C). Sciatic nerves of Chr- PLs-treated diabetic rats appear similar to those of the control group, which is an intriguing finding (Fig. 9 D).

Influence of Chr-PLs administration on histopathological changes in the sciatic nerve of alloxan-induced diabetes in rats. Histopathological examination of the sciatic nerve of control ( A ), Alloxan ( B ), metformin ( C ), and Chr-PLs ( D ). (o) indicates myelin sheath degeneration; (*) indicates separation between the nerve fibers; and (IF) indicates mononuclear infiltrating cells. Schwann cell nuclei, myelin sheath axons, and node of Ranvier can be observed

The changes in autophagy markers in the nerve fibers were estimated by measuring the immunohistochemical expression of beclin 1, LC3, and p62. Beclin 1 (Fig. 10 A) and LC3 (Fig. 11 A) exhibited positive expressions in the nerve fibers of the control group. However, beclin 1 (Fig. 10 B) and LC3 (Fig. 11 B) showed weak expression in the alloxan-treated group. However, P62 expression was poorly expressed in the control group (Fig. 12 A) and strongly expressed in the alloxan group (Fig. 12 B). The findings imply that hindering the process of autophagy could potentially enhance the expression of p62. Chr-PLs treatment resulted in increased expression of beclin 1 (Fig. 10 D) and LC3 (Fig. 11 D) and decreased expression of p62 (Fig. 12 D). The results obtained via the administration of Chr-PLs were comparable to those of the anti-hyperglycemic drug metformin (see Figs. 10 - 12 ).

Representative microscopic images displaying the levels of Beclin 1 immuno-expression in the sciatic nerve fibers of alloxan-induced diabetes in rats. Expression of Beclin 1 in the sciatic nerve of control ( A ), Alloxan ( B ), metformin ( C ), and Chr-PLs ( D ). Positive staining was indicated by a brown-yellow color (Scale bar; 50 µm and magnification power; 400 ×)

Representative microscopic images displaying the levels of LC3 immuno-expression in the sciatic nerve fibers of alloxan-induced diabetes in rats. Expression of LC3 in the sciatic nerve of control ( A ), Alloxan ( B ), metformin ( C ), and Chr-PLs ( D ). Positive staining was indicated by a brown-yellow color (Scale bar; 50 µm and magnification power; 400 ×)

Representative microscopic images displaying the levels of P62 immuno-expression in the sciatic nerve fibers of alloxan-induced diabetes in rats. Expression of P62 in the sciatic nerve of control ( A ), Alloxan ( B ), metformin ( C ), and Chr-PLs ( D ). Positive staining was indicated by a brown-yellow color (Scale bar; 50 µm and magnification power; 400 ×)

Insufficient insulin synthesis, action, or both characterize the metabolic disorder known as diabetes. This results in persistently high blood sugar levels and difficulty metabolizing carbohydrates, proteins, and fats [ 49 ]. Nephropathy and neurologic consequences are included in this illness [ 50 ]. In this study, our findings demonstrate the significant involvement of the ER stress and autophagy signaling pathway in the capacity of Chr-PLs to safeguard against advancing diabetic neuropathy. Diabetes was produced in rats via the administration of Alloxan. Insufficient insulin production and high blood sugar levels result from the preferential uptake of the oxygenated pyrimidine alloxan by pancreatic β-cells [ 51 ]. The biopharmaceutical classification system (BCS) assigns Chr a class II classification, indicating its low water solubility and restricted bioavailability [ 52 ]. One way to overcome this challenge has been to suggest incorporating chrysin into suitable delivery systems. Chr was thus included in PEGylated liposomal systems to improve its pharmacokinetic profile and offer a longer half-life [ 53 ]. The effective encapsulation of chrysin into liposomal systems improved its biological activity against diabetes and allowed for better formulation into aqueous preparations.

The mitigation of alloxan-induced diabetic neuropathy effects may be influenced by the different routes by which metformin and Chr-PLs are administered. Metformin is typically administered orally. The administration pathway can influence the bioavailability and pharmacokinetics of these drugs. Metformin's widespread use faces issues like low bioavailability, high dose, frequent dosage, gastrointestinal side effects, and poor absorption due to its cationic biguanide structure [ 54 ]. Chrysin liposome, on the other hand, may have higher bioavailability when administered intraperitoneal due to bypassing the gastrointestinal tract. Ahmed, et al. [ 55 ] found that gold nanoparticles administration by intraperitoneal route resulted in a more significant anti-fibrotic effect against hepatic Schistosoma mansoni infection than oral administrated nanoparticles. Therefore, chrysin liposome administered intraperitoneal may have a more potent effect on the target tissues than orally administered metformin and this serves a part of our study design which mainly focuses on comparing the potential effect of intraperitoneal injected chrysin liposome comparing standard orally administrated metformin already used in market.

Metformin primarily acts by reducing hepatic glucose production and improving insulin sensitivity. The antidiabetic effect of metformin is primarily achieved by inhibiting hepatic gluconeogenesis [ 56 ]. Metformin is widely recognized for targeting hepatic mitochondria and inhibiting mitochondrial respiratory chain complex I, which is reversible and weak [ 57 ]. Furthermore, the slight rise in intracellular AMP levels triggered by metformin inhibits AMP-regulated hepatic gluconeogenesis-related enzymes, including fructose-1,6-bisphosphatase and adenylate cyclase. This, in turn, results in a reduction in the production of glucose in the liver and activates the cellular energy sensor AMPK [ 58 , 59 ]. Chrysin liposome exerts antioxidant, anti-inflammatory, and antiapoptotic effects, potentially modulating various cellular pathways. It is a flavonoid with a diphenylpropane skeleton system that exerts significant anti-oxidant and anti-inflammatory properties due to its hydroxyl substituent position. It upregulates the transcription factor Nrf2, a crucial transcription factor responsible for anti-oxidant effects [ 60 ]. Chrysin's anti-inflammatory properties have been demonstrated to be neuroprotective after cerebral ischemia by modulating estrogen receptors [ 61 ]. It also mitigates dopamine depletion and safeguards against the neurodegeneration of dopaminergic neurons in the brain [ 62 ]. It can limit neuroinflammation by modulating JNK and NF-κB expression and attenuating PI3K/AKT/mTOR and NLRP3 inflammasome pathways [ 63 , 64 ]. Chrysin-loaded magnetic PEGylated silica nanospheres in animals reduce Aβ-induced memory impairment by reducing lipid peroxidation levels and increasing antioxidant molecules, thereby promoting neuroprotection [ 65 ].

The effective encapsulation of chrysin into liposomal systems improved its biological activity against diabetes and allowed for better formulation into aqueous preparations. One extremely prevalent feature of DM is abnormal body weight loss. Based on our findings, Alloxan causes a considerable decrease in body weight levels. These results aligned with those obtained by Tasnin et al. [ 66 ], who found a reduction in the body weight of diabetic rats. However, treatment with Chr-PLs significantly improved weight loss. Diabetes-related cognitive deficits, memory loss, and neurophysiological abnormalities were found to include AChE [ 67 ]. Consistent with the findings of Okesola et al. [ 68 ], the results demonstrated an elevation of serum AChE in alloxan-treated rats. Treatment with Chr-PLs, on the other hand, considerably reduced serum AChE levels. Streptozocin-induced diabetic foot ulcers in rats were found to be improved by chrysin treatment, as reported by Liu et al. [ 69 ]. Here, the administration of Chr-PLs to diabetic rats suppressed AChE activity, which could lessen acetylcholine hydrolysis and alleviate neuronal damage. The most notable change in diabetic sciatic nerves, as revealed by histologic examination, is the numerous losses of myelin sheath axons, accompanied by dissociation of nerve fibers and cellular infiltration. The neurodegenerative alterations in the sciatic nerve of diabetic rats were, however, alleviated by treatment with Chr-PLs. The results matched those of Pathak et al. [ 70 ], who showed that alloxan administration displayed abnormal sciatic nerve fibers and substantial axonal edema while decreasing axonal degeneration in the chrysin nanoemulsion group. These results indicate the regenerative characteristics of Chr-PLs.

Diabetes-induced rats exhibited a reduction in blood insulin and HOMA-β, whereas the FBG and HOMA-IR increased. The same findings were found by Gharib et al. [ 67 ]. In diabetic rats, an increase in the HOMA-IR score and a considerable fall in fasting blood insulin levels are indicative of an insulin-resistant state. Serum insulin and HOMA-β levels were significantly increased in Chr-PLs-treated diabetic rats, whereas FBG and HOMA-IR were markedly decreased. These results were coordinated with the results obtained by Salama et al. [ 50 ], and Kim et al. [ 71 ]. Increased peripheral uptake of glucose, suppression of hepatic glucose production [ 71 ], and improved insulin sensitivity may all contribute to chrysin’s powerful hypoglycemic impact.

When several stressors disrupt the ER’s regular activity, the resulting accumulation of unfolded and misfolded proteins in the ER’s lumen is known as ER stress [ 7 ]. Protein kinase RNA-like ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring enzyme 1 (IRE1) are the three regulatory mechanisms that control UPR. They regulate various cellular processes, such as autophagy, apoptosis, the antioxidant response, and inflammation [ 72 ]. Eukaryotic initiation factor-2 (eIF2) is phosphorylated by PERK during an ER stress response, activating CCAAT-enhancer-binding protein homologous protein (CHOP) [ 73 ]. Dissociation of the chaperone protein BiP from the initiation factor IRE1 triggers trans-autophosphorylation and the processing of unspliced, inactive XBP1 mRNA (uXBP1) into the spliced, active sXBP1, which stimulates transcription of specific target genes [ 74 ]. The formation of vacuoles or autophagosomes requires IRE1-mediated activation of JNK. The adaptive responses produced by ER autophagy offset the damaging consequences of ER stress, prolong cell viability, and delay apoptosis. However, prolonged high blood sugar levels interfere with the autophagy mechanism, leading to cell death [ 75 ]. Autophagy indicators have involved Beclin 1, LC3A/B, and p62 expression levels [ 74 ]. Phosphorylation of the autophagy-related protein UNC-51-like kinase 1 (ULK1) by the AMP-activated protein kinase (AMPK) stimulates autophagy [ 76 ]. The PI3K/Akt/mTOR pathway inhibition activates autophagy and suppresses cancer cell proliferation [ 77 ].

The present investigation observed an increase in the levels of markers associated with ER stress and autophagy in rats with diabetes. These markers included ATF6, CHOP, XBP-1, BIP, JNK, PI3K, Akt, mTOR and p62. However, there was marked downregulation in the AMPK, ULK-1, beclin 1, and LC3 levels. Our results were confirmed by a decrease in the expression levels of miR - 301a as a target for XBP-1 which augments ER stress response and a rise in the expression levels of miR - 30e - 5p as a target for Beclin1 which inhibits autophagy [ 69 ]. Both defective UPR and impaired autophagy have been associated with the onset of many forms of neurodegeneration [ 78 ]. Methamphetamine, for instance, can cause glial cells to experience ER stress by activating the UPR and leading to elevated PERK phosphorylation, ATF6 expression, and p-IRE1 enzyme activity [ 79 ].

Additionally, BiP, CHOP, and XBP-1 mRNA expression levels are raised [ 80 ]. Insulin resistance induces overexpression of ER stress indicators such as p-eIF2, ATF4, CHOP, sXBP1, p-IRE1, and p-ASK1 at the mRNA or protein level, causing neuronal cell death in neuroblastoma cells [ 81 ]. Autophagy was also impaired in a mouse model of neuropathic pain, as evidenced by the downregulation of LC3 and Beclin 1 and increased p62 protein expression following spinal nerve ligation [ 82 ]. ER stress was significantly reduced, and autophagy was enhanced after the administration of Chr-PLs. This finding agreed with Kang et al. [ 83 ], who treated glucose-stimulated human RPE cells with chrysin and observed that ER stress-related sensors such as BiP, ATF6, and the IRE1 pathway were less induced. In a study using a D-galactose model of aging in mice, Salama et al. [ 50 ] found that chrysin prevented neurodegeneration, boosted mitochondrial autophagy and biogenesis, and reduced oxidative stress and neuroinflammation. The increased ER stress response and the suppression of autophagy in the alloxan model of DN may be the outcomes of hyperglycemia’s enhancement of oxidative stress.

Ultimately, our investigation shows that Chr-PLs protect rats from diabetic neuropathy produced by Alloxan. Attenuation of ER stress and induction of autophagy regulation are probably one of the mechanisms by which these effects work. The underlying processes and therapeutic potential of Chr-PLs in treating diabetic neuropathy require further research.

Availability of data and materials

The datasets utilized and analyzed in the ongoing investigation can be made available by the author responsible for correspondence.

Abbreviations

Acetylcholinesterase

Akt isoform 1

AMP-activated protein kinase

Activating transcription factor 6

Bcl-2 interacting protein 1

Binding immunoglobulin protein

• Chrysin PEGylated liposomes

C/EBP homologous protein

Diabetic neuropathy

Diabetes mellitus

Endoplasmic reticulum

Fasting blood glucose

Homeostasis model assessment of insulin resistance

The Homeostasis Model Assessment of Beta Cell Function

Jun N-terminal kinase

Microtubule-associated proteins 1A/1B light chain 3B

Mammalian target of rapamycin

Polyethylene glycol4000

Phosphoinositide 3-kinase

Transmission electron microscopy

Unc-51 like autophagy activating kinase 1

Unfolded protein response

X-box-binding protein 1

Al-Haddad R, Karnib N, Assaad RA, Bilen Y, Emmanuel N, Ghanem A, et al. Epigenetic changes in diabetes. Neurosci Lett. 2016;625:64–9.

Article   CAS   PubMed   Google Scholar  

Alomari G, Al-Trad B, Hamdan S, Aljabali AAA, Al Zoubi MS, Al-Batanyeh K, et al. Alleviation of diabetic nephropathy by zinc oxide nanoparticles in streptozotocin-induced type 1 diabetes in rats. IET Nanobiotechnol. 2021;15:473–83.

Article   PubMed   PubMed Central   Google Scholar  

Bhattacharya D, Mukhopadhyay M, Bhattacharyya M, Karmakar P. Is autophagy associated with diabetes mellitus and its complications? A review. EXCLI J. 2018;17:709.

PubMed   PubMed Central   Google Scholar  

Callaghan BC, Cheng HT, Stables CL, Smith AL, Feldman EL. Diabetic neuropathy: clinical manifestations and current treatments. Lancet Neurol. 2012;11:521–34.

Kumar A, Mittal R. Nrf2: a potential therapeutic target for diabetic neuropathy. Inflammopharmacology. 2017;25:393–402.

Andhavarapu S, Mubariz F, Arvas M, Bever C, Makar TK. Interplay between ER stress and autophagy: a possible mechanism in multiple sclerosis pathology. Exp Mol Pathol. 2019;108:183–90.

Zhang K, Kaufman RJ. The unfolded protein response: a stress signaling pathway critical for health and disease. Neurology. 2006;66:S102.

Credle JJ, Finer-Moore JS, Papa FR, Stroud RM, Walter P. INAUGURAL ARTICLE by a recently elected academy member: on the mechanism of sensing unfolded protein in the endoplasmic reticulum. Proc Natl Acad Sci USA. 2005;102:18773.

Article   CAS   PubMed   PubMed Central   Google Scholar  

Lane JD, Korolchuk VI, Murray JT, Smith M, Wilkinson S. ER homeostasis and autophagy. Essays Biochem. 2017;61:625–35.

Article   Google Scholar  

Jiang Y, Tao Z, Chen H, Xia S. Endoplasmic reticulum quality control in immune cells. Front Cell Dev Biol. 2021;9: 740653.

Shi GJ, Li ZM, Zheng J, Chen J, Han XX, Wu J, et al. Diabetes associated with male reproductive system damages: onset of presentation, pathophysiological mechanisms and drug intervention. Biomed Pharmacother. 2017;90:562–74.

Ye J, Rawson RB, Komuro R, Chen X, Davé UP, Prywes R, et al. ER stress induces cleavage of membrane-bound ATF6 by the same proteases that process SREBPs. Mol Cell. 2000;6:1355–64.

Matsuzawa-Ishimoto Y, Hwang S, Cadwell K. Autophagy and Inflammation. Annu Rev Immunol. 2018;36:73–101.

Rashid HO, Yadav RK, Kim HR, Chae HJ. ER stress: autophagy induction, inhibition and selection. Autophagy. 2015;11:1956.

Shi D, Zhou L, Shi H, Zhang J, Zhang J, Zhang L, et al. Autophagy is induced by swine acute diarrhea syndrome coronavirus through the cellular IRE1-JNK-Beclin 1 signaling pathway after an interaction of viral membrane-associated papain-like protease and GRP78. PLoS Pathog. 2023;19: e1011201.

Kim J, Kundu M, Viollet B, Guan KL. AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Nat Cell Biol. 2011;13:132–41.

Xu Z, Han X, Ou D, Liu T, Li Z, Jiang G, et al. Targeting PI3K/AKT/mTOR-mediated autophagy for tumor therapy. Appl Microbiol Biotechnol. 2020;104:575–87.

Lucas CL, Chandra A, Nejentsev S, Condliffe AM, Okkenhaug K. PI3Kδ and primary immunodeficiencies. Nat Rev Immunol. 2016;16:702–14.

Xue JF, Shi ZM, Zou J, Li XL. Inhibition of PI3K/AKT/mTOR signaling pathway promotes autophagy of articular chondrocytes and attenuates inflammatory response in rats with osteoarthritis. Biomed Pharmacother. 2017;89:1252–61.

Wei C, Pan Y, Zhang Y, Dai Y, Jiang L, Shi L, et al. Overactivated sonic hedgehog signaling aggravates intrauterine adhesion via inhibiting autophagy in endometrial stromal cells. Cell Death Dis. 2020;11(9):755.

Zheng XY, Yang SM, Zhang R, Wang SM, Li GB, Zhou SW. Emodin-induced autophagy against cell apoptosis through the PI3K/AKT/mTOR pathway in human hepatocytes. Drug Des Devel Ther. 2019;13:3171–80.

Lee YH, Kim J, Park K, Lee MS. β-cell autophagy: mechanism and role in β-cell dysfunction. Mol Metab. 2019;27:S92-103.

Article   CAS   PubMed Central   Google Scholar  

Ramírez-Espinosa JJ, Saldaña-Ríos J, García-Jiménez S, Villalobos-Molina R, Ávila-Villarreal G, Rodríguez-Ocampo AN. Chrysin induces antidiabetic, antidyslipidemic and anti-inflammatory effects in athymic nude diabetic mice. Molecules. 2018;23:67.

Komath S, Garg A, Wahajuddin M. Development and evaluation of chrysin-phospholipid complex loaded solid lipid nanoparticles—storage stability and in vitro anti-cancer activity. J Microencapsul. 2018;35:600–17.

Galijatovic A, Otake Y, Walle UK, Walle T. Extensive metabolism of the flavonoid chrysin by human Caco-2 and Hep G2 cells. Xenobiotica. 1999;29:1241–56.

Dong D, Quan E, Yuan X, Xie Q, Li Z, Wu B. Sodium oleate-based nanoemulsion enhances oral absorption of chrysin through inhibition of UGT-mediated metabolism. Mol Pharm. 2017;14:2864–74.

Che J, Najer A, Blakney AK, McKay PF, Bellahcene M, Winter CW, et al. Neutrophils enable local and non-invasive liposome delivery to inflamed skeletal muscle and ischemic heart. Adv Mater. 2020;32:2003598.

Article   CAS   Google Scholar  

Najlah M, Suliman AS, Tolaymat I, Kurusamy S, Kannappan V, Elhissi AMA, et al. Development of injectable PEGylated liposome encapsulating disulfiram for colorectal cancer treatment. Pharmaceutics. 2019;11:610.

Laouini A, Jaafar-Maalej C, Limayem-Blouza I, Sfar S, Charcosset C, Fessi H. Preparation, characterization and applications of liposomes: state of the art. J Colloid Sci Biotechnol. 2012;1:147–68.

Bourquin J, Milosevic A, Hauser D, Lehner R, Blank F, Petri-Fink A, et al. Biodistribution, clearance, and long-term fate of clinically relevant nanomaterials. Adv Mater. 2018;30:1704307.

Maranhão RC, Vital CG, Tavoni TM, Graziani SR. Clinical experience with drug delivery systems as tools to decrease the toxicity of anticancer chemotherapeutic agents. Expert Opin Drug Deliv. 2017;14:1217–26.

Article   PubMed   Google Scholar  

Zheng JC, Lei N, He QC, Hu W, Jin JG, Meng Y, et al. PEGylation is effective in reducing immunogenicity, immunotoxicity, and hepatotoxicity of α-momorcharin in vivo. Immunopharmacol Immunotoxicol. 2012;34:866–73.

Bulbake U, Doppalapudi S, Kommineni N, Khan W. Liposomal formulations in clinical use: an updated review. Pharmaceutics. 2017;9:12.

Banda M, Nyirenda J, Muzandu K, Sijumbila G, Mudenda S. Antihyperglycemic and antihyperlipidemic effects of aqueous extracts of lannea edulis in alloxan-induced diabetic rats. Front Pharmacol. 2018;9:1099.

Salem GA, Mohamed AAR, Khater SI, Noreldin AE, Alosaimi M, Alansari WS, et al. Enhancement of biochemical and genomic pathways through lycopene-loaded nano-liposomes: alleviating insulin resistance, hepatic steatosis, and autophagy in obese rats with non-alcoholic fatty liver disease: involvement of SMO, GLI-1, and PTCH-1 genes. Gene. 2023;883:147670.

Tang L, Li K, Zhang Y, Li H, Li A, Xu Y, et al. Quercetin liposomes ameliorate streptozotocin-induced diabetic nephropathy in diabetic rats. Sci Rep. 2020;10:1–8.

Google Scholar  

Alaaeldin E, Mostafa M, Mansour HF, Soliman GM. Spanlastics as an efficient delivery system for the enhancement of thymoquinone anticancer efficacy: fabrication and cytotoxic studies against breast cancer cell lines. J Drug Deliv Sci Technol. 2021;65: 102725.

Mostafa M, Alaaeldin E, Aly UF, Sarhan HA. Optimization and characterization of thymoquinone-loaded liposomes with enhanced topical anti-inflammatory activity. AAPS PharmSciTech. 2018;19:3490–500.

Abd El-Emam MM, Mostafa M, Farag AA, Youssef HS, El-Demerdash AS, Bayoumi H, et al. The potential effects of quercetin-loaded nanoliposomes on amoxicillin/clavulanate-induced hepatic damage: targeting the SIRT1/Nrf2/NF-κB signaling pathway and microbiota modulation. Antioxidants. 2023;12:1487.

Đurašević S, Nikolić G, Zaletel I, Grigorov I, Memon L, Mitić-Ćulafić D, et al. Distinct effects of virgin coconut oil supplementation on the glucose and lipid homeostasis in non-diabetic and alloxan-induced diabetic rats. J Funct Foods. 2020;64: 103601.

Salama A, Asaad G, Shaheen A. Chrysin ameliorates STZ-induced diabetes in rats: possible impact of modulation of TLR4/NF-κβ pathway. Res Pharm Sci. 2022;17:1.

Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, Turner RC. Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia. 1985;28:412–9.

Abd El-Hakim YM, Abdel-Rahman Mohamed A, Khater SI, Hamed Arisha A, Metwally MMM, Nassan MA, et al. Chitosan-stabilized selenium nanoparticles and metformin synergistically rescue testicular oxidative damage and steroidogenesis-related genes dysregulation in high-fat diet/streptozotocin-induced diabetic rats. Antioxidants (Basel). 2020;10:1–17.

El-Shetry ES, Mohamed AAR, Khater SI, Metwally MMM, Nassan MA, Shalaby S, et al. Synergistically enhanced apoptotic and oxidative DNA damaging pathways in the rat brain with lead and/or aluminum metals toxicity: expression pattern of genes OGG1 and P53. J Trace Elem Med Biol. 2021;68:126860.

Czimmerer Z, Hulvely J, Simandi Z, Varallyay E, Havelda Z, Szabo E, et al. A versatile method to design stem-loop primer-based quantitative PCR assays for detecting small regulatory RNA molecules. PLoS ONE. 2013;8: e55168. https://doi.org/10.1371/journal.pone.0055168 .

Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25:402–8.

Bancroft J, Layton C. The Hematoxylin and Eosin. In: Suvarna SK, Layton C, Bancroft JD, editors. Theory and practice of histological techniques, chapter 10 and 11. 7th ed. Philadelphia: Churchill Livingstone of Elsevier; 2013. p. 179–220.

Elsayed HE, Ebrahim HY, Mady MS, Khattab MA, El-Sayed EK, Moharram FA. Ethnopharmacological impact of Melaleuca rugulosa (Link) Craven leaves extract on liver inflammation. J Ethnopharmacol. 2022;292:115215.

Khan N, Sultana S. Abrogation of potassium bromate-induced renal oxidative stress and subsequent cell proliferation response by soy isoflavones in Wistar rats. Toxicology. 2004;201:173–84.

Salama A, Elgohary R. Influence of chrysin on D-galactose induced-aging in mice: up regulation of AMP kinase/liver kinase B1/peroxisome proliferator-activated receptor-γ coactivator 1-α signaling pathway. Fundam Clin Pharmacol. 2023;37:947–59.

Lenzen S. The mechanisms of alloxan- and streptozotocin-induced diabetes. Diabetologia. 2008;51:216–26.

Baidya D, Kushwaha J, Mahadik K, Patil S. Chrysin-loaded folate conjugated PF127-F68 mixed micelles with enhanced oral bioavailability and anticancer activity against human breast cancer cells. Drug Dev Ind Pharm. 2019;45:852–60.

Kim JY, Kim JK, Park JS, Byun Y, Kim CK. The use of PEGylated liposomes to prolong circulation lifetimes of tissue plasminogen activator. Biomaterials. 2009;30:5751–6.

Shurrab NT. Metformin: a review of its therapeutic efficacy. Obes Med. 2020. https://doi.org/10.1016/j.obmed.2020.100186 .

Ahmed SAA, Gad SEM, Eida OM, Makhlouf LM. Anti-fibrotic effect of oral versus intraperitoneal administration of gold nanoparticles in hepatic schistosoma mansoni-infected mice. Acta Parasitol. 2024;69:190–202.

Lamoia TE, Shulman GI. Cellular and molecular mechanisms of metformin action. Endocr Rev. 2021;42:77–96.

Yoval-Sánchez B, Ansari F, Lange D, Galkin A. Effect of metformin on intact mitochondria from liver and brain: concept revisited. Eur J Pharmacol. 2022;931: 175177.

Hunter RW, Hughey CC, Lantier L, Sundelin EI, Peggie M, Zeqiraj E, et al. Metformin reduces liver glucose production by inhibition of fructose-1-6-bisphosphatase. Nat Med. 2018;24:1395–406.

Foretz M, Hébrard S, Leclerc J, Zarrinpashneh E, Soty M, Mithieux G, et al. Metformin inhibits hepatic gluconeogenesis in mice independently of the LKB1/AMPK pathway via a decrease in hepatic energy state. J Clin Invest. 2010;120:2355–69.

Zhang Y, Zhao J, Afzal O, Kazmi I, Al-Abbasi FA, Altamimi ASA, et al. Neuroprotective role of chrysin-loaded poly(lactic-co-glycolic acid) nanoparticle against kindling-induced epilepsy through Nrf2/ARE/HO-1 pathway. J Biochem Mol Toxicol. 2021;35: e22634.

Shooshtari MK, Farbood Y, Mansouri SMT, Badavi M, Khorsandi LS, Dehcheshmeh MG, et al. Neuroprotective effects of chrysin mediated by estrogenic receptors following cerebral ischemia and reperfusion in male rats. Basic Clin Neurosci. 2021;12:149–62.

Del Fabbro L, Rossito Goes A, Jesse CR, de Gomes MG, Cattelan Souza L, Lobo Ladd FV, et al. Chrysin protects against behavioral, cognitive and neurochemical alterations in a 6-hydroxydopamine model of Parkinson’s disease. Neurosci Lett. 2019;706:158–63.

Li TF, Ma J, Han XW, Jia YX, Yuan HF, Shui SF, et al. Chrysin ameliorates cerebral ischemia/reperfusion (I/R) injury in rats by regulating the PI3K/Akt/mTOR pathway. Neurochem Int. 2019;129:104496.

Liao T, Ding L, Wu P, Zhang L, Li X, Xu B, et al. Chrysin attenuates the NLRP3 inflammasome cascade to reduce synovitis and pain in KOA rats. Drug Des Devel Ther. 2020;14:3015.

Nday CM, Eleftheriadou D, Jackson G. Magnetic chrysin silica nanomaterials behavior in an amyloidogenic environment. Hell J Nucl Med. 2019;22:42.

PubMed   Google Scholar  

Tasnin MN, Islam A, Islam M, Hossain MI, Matiar M. A study on the antidiabetic property of a mixed herbal food. World J Pharm Pharm Sci. 2019;8:70.

CAS   Google Scholar  

Gharib E, Montasser Kouhsari S, Izad M. Punica granatum L. fruit aqueous extract suppresses reactive oxygen species-mediated p53/p65/miR-145 expressions followed by elevated levels of irs-1 in alloxan-diabetic rats. Cell J. 2018;19:520–7.

Okesola MA, Ajiboye BO, Oyinloye BE, Osukoya OA, Owero-ozeze OS, Ekakitie LI, et al. Effect of Solanum macrocarpon Linn leaf aqueous extract on the brain of an alloxan-induced rat model of diabetes. J Int Med Res. 2020;48:1–10.

Liu X, Li Q, Sun L, Chen L, Li Y, Huang B, et al. miR-30e-5p regulates autophagy and apoptosis by targeting beclin1 involved in contrast-induced acute kidney injury. Curr Med Chem. 2021;28:7974–84.

Pathak R, Sachan N, Kabra A, Alanazi AS, Alanazi MM, Alsaif NA, et al. Isolation, characterization, development and evaluation of phytoconstituent based formulation for diabetic neuropathy. Saudi Pharm J. 2023;31: 101687.

Kim SM, Imm JY. The effect of chrysin-loaded phytosomes on insulin resistance and blood sugar control in type 2 diabetic db/db mice. Molecules. 2020;25:5503.

Urra H, Dufey E, Avril T, Chevet E, Hetz C. Endoplasmic reticulum stress and the hallmarks of cancer. Trends Cancer. 2016;2:252–62.

Cubillos-Ruiz JR, Bettigole SE, Glimcher LH. Tumorigenic and immunosuppressive effects of endoplasmic reticulum stress in cancer. Cell. 2017;168:692–706.

Gardner BM, Walter P. Unfolded proteins are Ire1-activating ligands that directly induce the unfolded protein response. Science. 2011;333:1891–4.

Yang S, Xia C, Li S, Du L, Zhang L, Hu Y. Mitochondrial dysfunction driven by the LRRK2-mediated pathway is associated with loss of Purkinje cells and motor coordination deficits in diabetic rat model. Cell Death Dis. 2014;5: e1217.

Schmitz KJ, Ademi C, Bertram S, Schmid KW, Baba HA. Prognostic relevance of autophagy-related markers LC3, p62/sequestosome 1, Beclin-1 and ULK1 in colorectal cancer patients with respect to KRAS mutational status. World J Surg Oncol. 2016;14:1.

Zhao J, Zhu J, Lv X, Xing J, Liu S, Chen C, et al. Curcumin potentiates the potent antitumor activity of ACNU against glioblastoma by suppressing the PI3K/AKT and NF-κB/COX-2 signaling pathways. Onco Targets Ther. 2017;10:5471.

Cai Y, Arikkath J, Yang L, Guo ML, Periyasamy P, Buch S. Interplay of endoplasmic reticulum stress and autophagy in neurodegenerative disorders. Autophagy. 2016;12:225.

Shah A, Kumar A. Methamphetamine-mediated endoplasmic reticulum (ER) stress induces type-1 programmed cell death in astrocytes via ATF6, IRE1α and PERK pathways. Oncotarget. 2016;7:46100–19.

Tungkum W, Jumnongprakhon P, Tocharus C, Govitrapong P, Tocharus J. Melatonin suppresses methamphetamine-triggered endoplasmic reticulum stress in C6 cells glioma cell lines. J Toxicol Sci. 2017;42:63–71.

Song J, Kim OY. Melatonin modulates neuronal cell death induced by endoplasmic reticulum stress under insulin resistance condition. Nutrients. 2017;9:593.

Berliocchi L, Russo R, Maiarù M, Levato A, Bagetta G, Corasaniti MT. Autophagy impairment in a mouse model of neuropathic pain. Mol Pain. 2011;7:1744-8069-7–83.

Kang MK, Lee EJ, Kim YH, Kim DY, Oh H, Il Kim S, et al. Chrysin ameliorates malfunction of retinoid visual cycle through blocking activation of AGE-RAGE-ER stress in glucose-stimulated retinal pigment epithelial cells and diabetic eyes. Nutrients. 2018;10:1046.

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Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB). The authors received no financial assistance for conducting research, writing, and publishing this work.

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Mahran Mohamed Abd El-Emam & Mohamed Fouad Mansour

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Amany Behairy

Department of Pharmaceutics, Faculty of Pharmacy, Minia University, Minia, 61519, Egypt

Mahmoud Mostafa

Department of Pharmacology, Faculty of Veterinary Medicine, Zagazig University, Zagazig, 44519, Egypt

Tarek khamis

Laboratory of Biotechnology, Faculty of Veterinary Medicine, Zagazig University, Zagazig, 44519, Egypt

Department of Human Anatomy and Embryology, Faculty of Medicine, Port Said University, Port Said, Egypt

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Human Anatomy and Embryology Department, Faculty of Medicine, Zagazig University Egypt, Zagazig, Egypt

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Each author contributed to the study’s conception; MMA and MFM contributed to the design, performed the experiments, analyzed the data, wrote, reviewed, and edited the manuscript. TK and AB performed the experiments and analyzed the data. MMA wrote, reviewed, and edited the manuscript. MM prepared the liposomal formulation, performed statistical analysis, and reviewed and edited the manuscript. The final paper was read and approved by all authors.

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Correspondence to Mahran Mohamed Abd El-Emam .

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Abd El-Emam, M.M., Behairy, A., Mostafa, M. et al. Chrysin-loaded PEGylated liposomes protect against alloxan-induced diabetic neuropathy in rats: the interplay between endoplasmic reticulum stress and autophagy. Biol Res 57 , 45 (2024). https://doi.org/10.1186/s40659-024-00521-1

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DOI : https://doi.org/10.1186/s40659-024-00521-1

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Methods of liposomes preparation: formation and control factors of versatile nanocarriers for biomedical and nanomedicine application.

1. Introduction

2. structural features and main control factors of liposomes, 3. conventional methods for the preparation of liposomes.

• Dissolution of lipids in an organic solvent;
• Drying-down of the resultant lipidic solution from the organic solvent;
• Hydrating the lipid with an aqueous media (followed by agitation/stirring);
• Downsizing (and/or change in lamellarity);
• Post-formation processing (purification, sterilization);
• Characterization of the final nanoformulation product.

3.1. Thin-Film Hydration (TFH) Method (Bangham Method)

3.2. detergent removal (depletion) method, 3.3. solvent injection method, 3.3.1. ethanol injection method, 3.3.2. ether injection method, 3.4. reverse-phase evaporation method, 4. downsizing and post-formation processing, 4.1. sonication method, 4.2. extrusion method, 4.3. high-pressure homogenization method, 5. novel technologies for liposome preparation, 5.1. freeze-drying (lyophilization) method, 5.2. dense gas technology: supercritical fluid-assisted methods, 5.2.1. supercritical reverse-phase evaporation (sc-rpe) method, 5.2.2. supercritical anti-solvent (sas) method, 5.2.3. rapid expansion of a supercritical solution (ress) method, 5.2.4. supercritical-assisted liposome formation (superlip) method, 5.2.5. depressurization of an expanded liquid organic solution into aqueous suspension (delos) method, 5.3. microfluidic (channel) methods, 5.4. membrane contactor method, 6. drug loading in liposome nanoformulations, 7. post-formation processing of liposomes, 7.1. purification of liposomes nanoformulations, 7.2. sterilization of liposomes, 7.3. microfluidic lab-on-chip nanodevices for the combined formation, drug loading, and purification of liposomes, micro- and nanofabrication techniques, 8. characterization methods of liposome nanocarriers, 8.1. small-angle x-ray/neutron scattering (saxs/sans) and diffraction techniques, 8.2. electron microscopy and atomic force microscopy (afm) techniques, 8.3. light (fluorescence and confocal) microscopy techniques, 8.4. dynamic light scattering technique, 8.5. zeta (ζ) potential technique, 8.6. other complementary characterization techniques, 9. technology transfer and regulatory perspectives, the quality by design (qbd) method, 10. conclusions, author contributions, institutional review board statement, informed consent statement, data availability statement, conflicts of interest.

• Bozzuto, G.; Molinari, A.A. Liposomes as nanomedical devices. Int. J. Nanomed. 2015 , 10 , 975–999. [ Google Scholar ] [ CrossRef ] [ Green Version ]
• Allen, T.M.; Cullis, P.R. Liposomal drug delivery systems: From concept to clinical applications. Adv. Drug Deliver. Rev. 2013 , 65 , 36–48. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Lombardo, D.; Calandra, P.; Barreca, D.; Magazù, S.; Kiselev, M.A. Soft interaction in liposome nanocarriers for therapeutic drug delivery. Nanomaterials 2016 , 6 , 125. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Lee, M.-K. Liposomes for Enhanced Bioavailability of Water-Insoluble Drugs: In Vivo Evidence and Recent Approaches. Pharmaceutics 2020 , 12 , 264. [ Google Scholar ] [ CrossRef ] [ PubMed ] [ Green Version ]
• Ewert, K.K.; Scodeller, P.; Simón-Gracia, L.; Steffes, V.M.; Wonder, E.A.; Teesalu, T.; Safinya, C.R. Cationic Liposomes as Vectors for Nucleic Acid and Hydrophobic Drug Therapeutics. Pharmaceutics 2021 , 13 , 1365. [ Google Scholar ] [ CrossRef ]
• Boons, G.-J. Liposomes Modified by Carbohydrate Ligands Can Target B-Cells for the Treatment of B-Cell Lymphomas. Expert Rev. Vaccines 2010 , 9 , 1251–1256. [ Google Scholar ] [ CrossRef ] [ Green Version ]
• Kumar Giri, T.; Giri, A.; Kumar Barman, T.; Maity, S. Nanoliposome is a Promising Carrier of Protein and Peptide Biomolecule for the Treatment of Cancer. Anti-Cancer Agents Med. Chem. 2016 , 16 , 816–831. [ Google Scholar ] [ CrossRef ]
• Blanken, D.; Foschepoth, D.; Serrão, A.C.; Danelon, C. Genetically controlled membrane synthesis in liposomes. Nat. Commun. 2020 , 11 , 4317. [ Google Scholar ] [ CrossRef ]
• Xia, Y.; Xu, C.; Zhang, X.; Ning, P.; Wang, Z.; Tian, J.; Chen, X. Liposome-based probes for molecular imaging: From basic research to the bedside. Nanoscale 2019 , 11 , 5822–5838. [ Google Scholar ] [ CrossRef ]
• Shao, X.R.; Wei, X.Q.; Zhang, S.; Fu, N.; Lin, Y.-F.; Cai, X.-X. Effects of Micro-environmental pH of Liposome on Chemical Stability of Loaded Drug. Nanoscale Res. Lett. 2017 , 12 , 504. [ Google Scholar ] [ CrossRef ] [ Green Version ]
• Lombardo, D.; Calandra, P.; Caccamo, M.T.; Magazù, S.; Kiselev, M.A. Colloidal stability of liposomes. AIMS Mater. Sci. 2019 , 6 , 200–213. [ Google Scholar ] [ CrossRef ]
• Pattni, B.S.; Chupin, V.V.; Torchilin, V.P. New Developments in Liposomal Drug Delivery. Chem. Rev. 2015 , 115 , 10938–10966. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Gu, Z.; Da Silva, C.G.; Van der Maaden, K.; Ossendorp, F.; Cruz, L.J. Liposome-Based Drug Delivery Systems in Cancer Immunotherapy. Pharmaceutics 2020 , 12 , 1054. [ Google Scholar ] [ CrossRef ]
• Salehi, B.; Calina, D.; Docea, A.O.; Koirala, N.; Aryal, S.; Lombardo, D.; Pasqua, L.; Taheri, Y.; Salgado Castillo, C.M.; Martorell, M.; et al. Curcumin’s Nanomedicine Formulations for Therapeutic Application in Neurological Diseases. J. Clin. Med. 2020 , 9 , 430. [ Google Scholar ] [ CrossRef ] [ PubMed ] [ Green Version ]
• Ba, S.; Qiao, M.; Jia, L.; Zhang, J.; Zhao, X.; Hu, H.; Chen, D. Construction of Hierarchical-Targeting pH-Sensitive Liposomes to Reverse Chemotherapeutic Resistance of Cancer Stem-like Cells. Pharmaceutics 2021 , 13 , 1205. [ Google Scholar ] [ CrossRef ]
• Bobo, D.; Robinson, K.J.; Islam, J.; Thurecht, K.J.; Corrie, S.R. Nanoparticle-based medicines: A review of FDA approved materials and clinical trials to date. Pharm. Res 2016 , 33 , 2373–2387. [ Google Scholar ] [ CrossRef ]
• Bulbake, U.; Doppalapudi, S.; Kommineni, N.; Khan, W. Liposomal Formulations in Clinical Use: An Updated Review. Pharmaceutics 2017 , 9 , 12. [ Google Scholar ] [ CrossRef ]
• Lamichhane, N.; Udayakumar, T.S.; D’Souza, W.D.; Simone, C.B., 2nd; Raghavan, S.R.; Polf, J.; Mahmood, J. Liposomes: Clinical Applications and Potential for Image-Guided Drug Delivery. Molecules 2018 , 23 , 288. [ Google Scholar ] [ CrossRef ] [ Green Version ]
• Noble, G.T.; Stefanick, J.F.; Ashley, J.D.; Kiziltepe, T.; Bilgicer, B. Ligand-targeted liposome design: Challenges and fundamental considerations. Trends Biotechnol. 2014 , 32 , 32–45. [ Google Scholar ] [ CrossRef ]
• Dutta, S.; Moses, J.A.; Anandharamakrishnan, C. Encapsulation of Nutraceutical Ingredients in Liposomes and Their Potential for Cancer Treatment. Nutr. Cancer 2018 , 70 , 1184–1198. [ Google Scholar ] [ CrossRef ]
• Subramani, T.; Ganapathyswamy, H. An overview of liposomal nano-encapsulation techniques and its applications in food and nutraceutical. J. Food Sci. Technol. 2020 , 57 , 3545–3555. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Lee, W.; Im, H. Theranostics Based on Liposome: Looking Back and Forward. Nucl. Med. Mol. Imaging 2019 , 53 , 242–246. [ Google Scholar ] [ CrossRef ]
• Seleci, M.; Ag Seleci, D.; Scheper, T.; Stahl, F. Theranostic Liposome-Nanoparticle Hybrids for Drug Delivery and Bioimaging. Int. J. Mol. Sci. 2017 , 18 , 1415. [ Google Scholar ] [ CrossRef ] [ PubMed ] [ Green Version ]
• Cheng, X.; Gao, J.; Ding, Y.; Lu, Y.; Wei, Q.; Cui, D.; Fan, J.; Li, X.; Zhu, E.; Lu, Y.; et al. Multi-Functional Liposome: A Powerful Theranostic Nano-Platform Enhancing Photodynamic Therapy. Adv. Sci. 2021 , 8 , e2100876. [ Google Scholar ] [ CrossRef ]
• Fahmy, S.A.; Azzazy, H.M.E.-S.; Schaefer, J. Liposome Photosensitizer Formulations for Effective Cancer Photodynamic Therapy. Pharmaceutics 2021 , 13 , 1345. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Fobian, S.-F.; Cheng, Z.; ten Hagen, T.L.M. Smart Lipid-Based Nanosystems for Therapeutic Immune Induction against Cancers: Perspectives and Outlooks. Pharmaceutics 2022 , 14 , 26. [ Google Scholar ] [ CrossRef ]
• Sackmann, E. Physical basis of self-organization and function of membranes: Physics of vesicles. In Handbook of Biological Physics ; Lipowsky, R., Sackmann, E., Eds.; Elsevier: Amsterdam, The Netherlands, 1995; Volume 1, pp. 213–303. [ Google Scholar ]
• Katsaras, J.; Gutberlet, T. Lipid Bilayers. Structure and Interactions ; Springer: Berlin/Heidelberg, Germany, 2000. [ Google Scholar ]
• Helfrich, W. Elastic properties of lipid bilayers: Theory and possible experiments. Z. Nat. C 1973 , 28 , 693–703. [ Google Scholar ] [ CrossRef ]
• Akbarzadeh, A.; Rezaei-Sadabady, R.; Davaran, S.; Joo, S.W.; Zarghami, N.; Hanifehpour, Y.; Samiei, M.; Kouhi, M.; Nejati-Koshki, K. Liposome: Classification, preparation, and applications. Nanoscale Res. Lett. 2013 , 8 , 102. [ Google Scholar ] [ CrossRef ] [ Green Version ]
• Israelachvili, J.N. Physics of Amphiphiles: Micelles, Vesicles and Microemulsions ; Degiorgio, V., Corti, M., Eds.; North-Holland: Amsterdam, The Netherlands, 1985. [ Google Scholar ]
• Israelachvili, J.; Wennerström, H. Role of hydration and water structure in biological and colloidal interactions. Nature 1996 , 379 , 219–225. [ Google Scholar ] [ CrossRef ]
• Lombardo, D.; Munaò, M.; Calandra, P.; Pasqua, L.; Caccamo, M.T. Evidence of pre-micellar aggregates in water solution of amphiphilic PDMS-PEO block copolymer. Phys. Chem. Chem. Phys. 2019 , 21 , 11983–11991. [ Google Scholar ] [ CrossRef ]
• Ishida, T.; Harashima, H.; Kiwada, H. Liposome clearance. Biosci. Rep. 2012 , 22 , 197–224. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Kraft, J.C.; Freeling, J.P.; Wang, Z.; Ho, R.J.Y. Emerging research and clinical development trends of liposome and lipid nanoparticle drug delivery systems. J. Pharm. Sci. 2014 , 103 , 29–52. [ Google Scholar ] [ CrossRef ] [ PubMed ] [ Green Version ]
• Merino, M.; Zalba, S.; Garrido, M.J. Immunoliposomes in clinical oncology: State of the art and future perspectives. J. Control. Release 2018 , 275 , 162–176. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Luzzati, V.; Tardieu, A. Lipid Phases: Structure and Structural Transitions. Annu. Rev. Phys. Chem. 1974 , 25 , 79–94. [ Google Scholar ] [ CrossRef ]
• Nagle, J.F.; Tristram-Nagle, S. Structure of lipid bilayers. Biochim. Biophys. Acta. 2000 , 1469 , 159–195. [ Google Scholar ] [ CrossRef ] [ Green Version ]
• Lesieur, P.; Kiselev, M.A.; Barsukov, L.I.; Lombardo, D. Temperature-induced micelle to vesicle transition: Kinetic effects in the DMPC/NaC system. J. Appl. Cryst. 2000 , 33 , 623–627. [ Google Scholar ] [ CrossRef ] [ Green Version ]
• Kiselev, M.A.; Lombardo, D. Structural characterization in mixed lipid membrane systems by neutron and X-ray scattering. Biochim. Biophys. Acta Gen. Subj. 2017 , 1861 , 3700–3717. [ Google Scholar ] [ CrossRef ]
• Rawicz, W.; Olbrich, K.C.; Mcintosh, T.; Needham, D.; Evans, E. Effect of Chain Length and Unsaturation on Elasticity of Lipid Bilayers. Biophys. J. 2000 , 79 , 1–12. [ Google Scholar ] [ CrossRef ] [ Green Version ]
• Cullis, P.R.; De Kruijff, B. Lipid polymorphism and the functional roles of lipids in biological membranes. Biochim. Biophys. Acta BBA Rev. Biomembr. 1979 , 559 , 399–420. [ Google Scholar ] [ CrossRef ]
• Mouritsen, O.G. Lipids, curvature, and nano-medicine. Eur. J. Lipid Sci. Technol. 2011 , 113 , 1174–1187. [ Google Scholar ] [ CrossRef ] [ Green Version ]
• Lombardo, D.; Kiselev, M.A.; Magazu, S.; Calandra, P. Amphiphiles Self-Assembly: Basic Concepts and Future Perspectives of Supramolecular Approaches. Adv. Cond. Matter Phys. 2015 , 2015 , 151683. [ Google Scholar ] [ CrossRef ] [ Green Version ]
• Kashapov, R.; Gaynanova, G.; Gabdrakhmanov, D.; Kuznetsov, D.; Pavlov, R.; Petrov, K.; Zakharova, L.; Sinyashin, O. Self-Assembly of Amphiphilic Compounds as a Versatile Tool for Construction of Nanoscale Drug Carriers. Int. J. Mol. Sci. 2020 , 21 , 6961. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Lombardo, D.; Longo, A.; Darcy, R.; Mazzaglia, A. Structural properties of nonionic cyclodextrin colloids in water. Langmuir 2004 , 20 , 1057–1064. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Schäfer, K.; Kolli, H.B.; Killingmoe Christensen, M.; Bore, S.L.; Diezemann, G.; Gauss, J.; Milano, G.; Lund, R.; Cascella, M. Supramolecular packing drives morphological transitions of charged surfactant micelles. Angew. Chem. Int. Ed. 2020 , 59 , 18591–18598. [ Google Scholar ] [ CrossRef ]
• Mallamace, F.; Beneduci, R.; Gambadauro, P.; Lombardo, D.; Chen, S.H. Glass and percolation transitions in dense attractive micellar system. Phys. A Stat. Mech. Appl. 2001 , 302 , 202–219. [ Google Scholar ] [ CrossRef ]
• Chen, S.H.; Mallamace, F.; Faraone, A.; Gambadauro, P.; Lombardo, D.; Chen, W.R. Observation of a re-entrant kinetic glass transition in a micellar system with temperature-dependent attractive interaction. Eur. Phys. J. E Soft Matter 2002 , 9 , 283–286. [ Google Scholar ] [ CrossRef ]
• Nakhaei, P.; Margiana, R.; Bokov, D.O.; Abdelbasset, W.K.; Kouhbanani, M.A.J.; Varma, R.S.; Marofi, F.; Jarahian, M.; Beheshtkhoo, N. Liposomes: Structure, Biomedical Applications, and Stability Parameters With Emphasis on Cholesterol. Front. Bioeng. Biotechnol. 2021 , 9 , 705886. [ Google Scholar ] [ CrossRef ]
• Lee, S.-C.; Lee, K.-E.; Kim, J.-J.; Lim, S.-H. The Effect of Cholesterol in the Liposome Bilayer on the Stabilization of Incorporated Retinol. J. Liposome Res. 2005 , 15 , 157–166. [ Google Scholar ] [ CrossRef ]
• Kaddah, S.; Khreich, N.; Kaddah, F.; Charcosset, C.; Greige-Gerges, H. Cholesterol modulates the liposome membrane fluidity and permeability for a hydrophilic molecule. Food Chem. Toxicol. 2018 , 113 , 40–48. [ Google Scholar ] [ CrossRef ]
• Hernández-Caselles, T.; Villalaín, J.; Gómez-Fernández, J.C. Influence of liposome charge and composition on their interaction with human blood serum proteins. Mol. Cell. Biochem. 1993 , 120 , 119–126. [ Google Scholar ] [ CrossRef ]
• Jobin, M.L.; Alves, I.D. On the importance of electrostatic interactions between cell penetrating peptides and membranes: A pathway toward tumor cell selectivity? Biochimie 2014 , 107 , 154–159. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Pöyry, S.; Vattulainen, I. Role of charged lipids in membrane structures—Insight given by simulations. Biochim. Biophys. Acta 2016 , 1858 , 2322–2333. [ Google Scholar ] [ CrossRef ]
• Claessens, M.M.; van Oort, B.F.; Leermakers, F.A.; Hoekstra, F.A.; Cohen Stuart, M.A. Charged lipid vesicles: Effects of salts on bending rigidity, stability, and size. Biophys. J. 2004 , 87 , 3882–3893. [ Google Scholar ] [ CrossRef ] [ PubMed ] [ Green Version ]
• Miller, C.R.; Bondurant, B.; McLean, S.D.; McGovern, K.A.; O’Brien, D.F. Liposome-cell interactions in vitro: Effect of liposome surface charge on the binding and endocytosis of conventional and sterically stabilized liposomes. Biochemistry 1998 , 37 , 12875–12883. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Campbell, R.B.; Ying, B.; Kuesters, G.M.; Hemphill, R. Fighting cancer: From the bench to bedside using second generation cationic liposomal therapeutics. J. Pharm. Sci. 2009 , 98 , 411–429. [ Google Scholar ] [ CrossRef ]
• Inglut, C.T.; Sorrin, A.J.; Kuruppu, T.; Vig, S.; Cicalo, J.; Ahmad, H.; Huang, H.C. Immunological and Toxicological Considerations for the Design of Liposomes. Nanomaterials 2000 , 10 , 190. [ Google Scholar ] [ CrossRef ] [ Green Version ]
• Gref, R.; Lück, M.; Quellec, P.; Marchand, M.; Dellacherie, E.; Harnisch, S.; Blunk, T.; Müller, R.H. ‘Stealth’ corona-core nanoparticles surface modified by polyethylene glycol (PEG): Influences of the corona (PEG chain length and surface density) and of the core composition on phagocytic uptake and plasma protein adsorption. Colloids Surf. B Biointerfaces 2000 , 18 , 301–313. [ Google Scholar ] [ CrossRef ]
• Immordino, M.L.; Dosio, F.; Cattel, L. Stealth liposomes: Review of the basic science, rationale, and clinical applications, existing and potential. Int. J. Nanomed. 2006 , 1 , 297–315. [ Google Scholar ]
• Mohamed, M.; Abu Lila, A.S.; Shimizu, T.; Alaaeldin, E.; Hussein, A.; Sarhan, H.A.; Szebeni, J.; Ishida, T. PEGylated liposomes: Immunological responses. Sci. Technol. Adv. Mater. 2019 , 20 , 710–724. [ Google Scholar ] [ CrossRef ] [ Green Version ]
• Thi, T.T.H.; Pilkington, E.H.; Nguyen, D.H.; Lee, J.S.; Park, K.D.; Truong, N.P. The Importance of Poly(ethylene glycol) Alternatives for Overcoming PEG Immunogenicity in Drug Delivery and Bioconjugation. Polymers 2020 , 12 , 298. [ Google Scholar ] [ CrossRef ] [ Green Version ]
• Dai, Y.; Wang, B.; Sun, Z.; Cheng, J.; Zhao, H.; Wu, K.; Sun, p.; Shen, Q.; Li, M.; Fan, Q. Multifunctional Theranostic Liposomes Loaded with a Hypoxia-Activated Prodrug for Cascade-Activated Tumor Selective Combination Therapy. ACS Appl. Mater. Interfaces 2019 , 11 , 39410–39423. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• New, R.C.C. Preparation of liposomes. In Liposomes: A Practical Approach ; New, R.C.C., Ed.; Oxford University Press: New York, NY, USA, 1990; pp. 33–104. [ Google Scholar ]
• Zhang, H. Thin-Film Hydration Followed by Extrusion Method for Liposome Preparation. In Liposomes. Methods in Molecular Biology ; D’Souza, G., Ed.; Humana Press: New York, NY, USA, 2017; Volume 1522. [ Google Scholar ] [ CrossRef ]
• Xiang, B.; De-ying, C. Liposome-Based Drug Delivery Systems. In Biomaterial Engineering ; Lu, W.-L., Qi, X.-R., Eds.; Springer: Berlin/Heidelberg, Germany, 2018. [ Google Scholar ] [ CrossRef ]
• Lasch, J.; Weissig, V.; Brandl, M. Preparation of liposomes. In Liposomes: A Practical Approach , 2nd ed.; Torchilin, V., Weissig, V., Eds.; Oxford University Press: New York, NY, USA, 2003; pp. 3–29. [ Google Scholar ]
• Jeon, M.; Kim, G.; Lee, W.; Im, H.-J. Development of theranostic PEGylated liposomal Au-liposome for effective tumor passive targeting and photothermal therapy. J. Nucl. Med. 2020 , 61 (Suppl. 1), 1076. [ Google Scholar ]
• Wang, G.; Yang, Y.; Yi, D.; Yuan, L.; Yin, P.-H.; Ke, X.; Jun-Jie, W.; Tao, M.-F. Eudragit S100 prepared pH-responsive liposomes-loaded betulinic acid against colorectal cancer in vitro and in vivo. J. Liposome Res. 2021 , 13 , 1–15. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Jiskoot, W.; Teerlink, T.; Beuvery, E.C.; Crommelin, D.J. Preparation of liposomes via detergent removal from mixed micelles by dilution. The effect of bilayer composition and process parameters on liposome characteristics. Pharm. Weekbl. Sci. 1986 , 8 , 259–265. [ Google Scholar ] [ CrossRef ]
• Ollivon, M.; Lesieur, S.; Grabielle-Madelmont, C.; Paternostre, M. Vesicle reconstitution from lipid-detergent mixed micelles. Biochim. Biophys. Acta 2000 , 1508 , 34–50. [ Google Scholar ] [ CrossRef ] [ Green Version ]
• Leng, J.; Egelhaaf, S.U.; Cates, M.E. Kinetics of the micelle-to-vesicle transition: Aqueous lecithin-bile salt mixtures. Biophys. J. 2003 , 85 , 1624–1646. [ Google Scholar ] [ CrossRef ] [ Green Version ]
• Kiselev, M.A.; Lombardo, D.; Lesieur, P.; Kisselev, A.M.; Borbely, S.; Simonova, T.N.; Barsukov, L.I. Membrane Self Assembly in Mixed DMPC/NaC Systems by SANS. Chem. Phys. 2008 , 345 , 173–180. [ Google Scholar ] [ CrossRef ]
• Kiselev, M.; Janich, M.; Hildebrand, A.; Strunz, P.; Neubert, R.; Lombardo, D. Structural transition in aqueous lipid/bile salt [DPPC/NaDC] supramolecular aggregates: SANS and DLS study. Chem. Phys. 2013 , 424 , 93–99. [ Google Scholar ] [ CrossRef ]
• Niroomand, H.; Venkatesan, G.A.; Sarles, S.A.; Mukherjee, D.; Khomami, B. Lipid-Detergent Phase Transitions during Detergent-Mediated Liposome Solubilization. J. Membr. Biol. 2016 , 249 , 523–538. [ Google Scholar ] [ CrossRef ]
• Juan-Colás, J.; Dresser, L.; Morris, K.; Lagadou, H.; Ward, R.H.; Burns, A.; Tear, S.P.; Johnson, S.D.; Leake, M.C.; Quinn, S.D. The Mechanism of Vesicle Solubilization by the Detergent Sodium Dodecyl Sulfate. Langmuir 2020 , 36 , 11499–11507. [ Google Scholar ] [ CrossRef ]
• Majeed, S.; Ahmad, A.B.; Sehar, U.; Georgieva, E.R. Lipid Membrane Mimetics in Functional and Structural Studies of Integral Membrane Proteins. Membranes 2021 , 11 , 685. [ Google Scholar ] [ CrossRef ]
• Neves, P.; Lopes, S.C.; Sousa, I.; Garcia, S.; Eaton, P.; Gameiro, P. Characterization of membrane protein reconstitution in LUVs of different lipid composition by fluorescence anisotropy. J. Pharm. Biomed. Anal. 2009 , 49 , 276–281. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Lasic, D.D. Mechanisms of liposome formation. J. Liposome Res. 1995 , 5 , 431–441. [ Google Scholar ] [ CrossRef ]
• Gouda, A.; Sakr, O.S.; Nasr, M.; Sammour, O.A. Ethanol injection technique for liposomes formulation: An insight into development, influencing factors, challenges and applications. J. Drug Deliv. Sci. Technol. 2020 , 61 , 102174. [ Google Scholar ] [ CrossRef ]
• Jaafar-Maalej, C.; Diab, R.; Andrieu, V.; Elaissari, A.; Fessi, H. Ethanol injection method for hydrophilic and lipophilic drug-loaded liposome preparation. J. Liposome Res. 2009 , 20 , 228–243. [ Google Scholar ] [ CrossRef ]
• Guimarães, D.; Noro, J.; Loureiro, A.; Lager, F.; Renault, G.; Cavaco-Paulo, A.; Nogueira, E. Increased Encapsulation Efficiency of Methotrexate in Liposomes for Rheumatoid Arthritis Therapy. Biomedicines 2020 , 8 , 630. [ Google Scholar ] [ CrossRef ]
• Deamer, D.; Bangham, A.D. Large volume liposomes by an ether vaporization method. Biochim. Biophys. Acta 1976 , 443 , 629–634. [ Google Scholar ] [ CrossRef ]
• DDeamer, D.W. Preparation and properties of ether-injection liposomes. Ann. N. Y. Acad. Sci. 1978 , 308 , 250–258. [ Google Scholar ] [ CrossRef ]
• Hasan, M.; Hamiduzzaman; Jahan, I.; Hasan, A. Asaduzzaman Formulation Development, Characterization and In-Vitro Evaluation of Tamoxifen Loaded Liposomes. J. Pharm. Res. Int. 2020 , 32 , 64–82. [ Google Scholar ] [ CrossRef ]
• Hauschild, S.; Lipprandt, U.; Rumplecker, A.; Borchert, U.; Rank, A.; Schubert, R.; Förster, S. Direct preparation and loading of lipid and polymer vesicles using inkjets. Small 2005 , 1 , 1177–1180. [ Google Scholar ] [ CrossRef ]
• Szoka, F., Jr.; Papahadjopoulos, D. Procedure for preparation of liposomes with large internal aqueous space and high capture by reverse-phase evaporation. Proc. Natl. Acad. Sci. USA 1978 , 75 , 4194–4198. [ Google Scholar ] [ CrossRef ] [ PubMed ] [ Green Version ]
• Pidgeon, C.; McNeely, S.; Schmidt, T.; Johnson, J.E. Multilayered vesicles prepared by reverse-phase evaporation: Liposome structure and optimum solute entrapment. Biochemistry 1987 , 26 , 17–29. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Shi, N.-Q.; Qi, X.-R. Preparation of Drug Liposomes by Reverse-Phase Evaporation. In Liposome-Based Drug Delivery Systems. Biomaterial Engineering ; Lu, W.L., Qi, X.R., Eds.; Springer: Berlin/Heidelberg, Germany, 2021. [ Google Scholar ] [ CrossRef ]
• Béalle, G.; Di Corato, R.; Kolosnjaj-Tabi, J.; Dupuis, V.; Clément, O.; Gazeau, F.; Wilhelm, C.; Ménager, C. Ultra Magnetic Liposomes for MR Imaging, Targeting, and Hyperthermia. Langmuir 2012 , 28 , 11834–11842. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Do, H.D.; Ménager, C.; Michel, A.; Seguin, J.; Korichi, T.; Dhotel, H.; Marie, C.; Doan, B.-T.; Mignet, N. Development of Theranostic Cationic Liposomes Designed for Image-Guided Delivery of Nucleic Acid. Pharmaceutics 2020 , 12 , 854. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Liu, C.; Liu, Y.-Y.; Chang, Q.; Shu, Q.; Shen, N.; Wang, H.; Xie, Y.; Deng, X. Pressure-Controlled Encapsulation of Graphene Quantum Dots into Liposomes by the Reverse-Phase Evaporation Method. Langmuir 2021 , 37 , 14096–14104. [ Google Scholar ] [ CrossRef ]
• Mendez, R.; Banerjee, S. Sonication-Based Basic Protocol for Liposome Synthesis. Methods Mol. Biol. 2017 , 1609 , 255–260. [ Google Scholar ] [ CrossRef ]
• Olson, F.; Hunt, C.A.; Szoka, F.; Vail, W.; Papahadjopoulos, D. Preparation of liposomes of defined size distribution by extrusion through polycarbonate membranes. Biochim. Biophys. Acta 1979 , 557 , 9–23. [ Google Scholar ] [ CrossRef ]
• Schneider, T.; Sachse, A.; Rößling, G.; Brandl, M. Generation of contrast-carrying liposomes of defined size with a new continuous high pressure extrusion method. Int. J. Pharm. 1995 , 117 , 1–12. [ Google Scholar ] [ CrossRef ]
• Ong, S.S.G.; Chitneni, M.; Lee, K.S.; Ming, L.C.; Yuen, K.H. Evaluation of Extrusion Technique for Nanosizing Liposomes. Pharmaceutics 2016 , 8 , 36. [ Google Scholar ] [ CrossRef ]
• Hamilton, R.L., Jr.; Goerke, J.; Guo, L.S.; Williams, M.C.; Havel, R.J. Unilamellar liposomes made with the French pressure cell: A simple preparative and semiquantitative technique. J. Lipid. Res. 1980 , 21 , 981–992. [ Google Scholar ] [ CrossRef ]
• Brandl, M.; Bachmann, D.; Drechsler, M.; Bauer, K.H. Liposome preparation by a new high pressure homogenizer Gaulin Micron Lab 40. Drug Dev. Ind. Pharm. 1990 , 16 , 2167–2191. [ Google Scholar ] [ CrossRef ]
• Li, C.; Deng, Y. A novel method for the preparation of liposomes: Freeze drying of monophase solutions. J. Pharm. Sci. 2004 , 93 , 1403–1414. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Franzé, S.; Selmin, F.; Samaritani, E.; Minghetti, P.; Cilurzo, F. Lyophilization of Liposomal Formulations: Still Necessary, Still Challenging. Pharmaceutics 2018 , 10 , 139. [ Google Scholar ] [ CrossRef ] [ Green Version ]
• Kiselev, M.A.; Lesieur, P.; Kisselev, A.M.; Lombardo, D.; Killany, M.; Lesieur, S.; Ollivon, M. A sucrose solutions application to the study of model biological membranes. Nucl. Instrum. Methods Phys. Res. A 2001 , 470 , 409–416. [ Google Scholar ] [ CrossRef ] [ Green Version ]
• Kiselev, M.; Lesieur, P.; Kisselev, A.M.; Lombardo, D.; Killany, M.; Lesieur, S. Sucrose solutions as prospective medium to study the vesicle structure: SAXS and SANS study. J. Alloys Compd. 2001 , 328 , 71–76. [ Google Scholar ] [ CrossRef ] [ Green Version ]
• Crowe, J.H.; Spargo, B.J.; Crowe, L.M. Preservation of dry liposomes does not require retention of residual water. Proc. Natl. Acad. Sci. USA 1987 , 84 , 1537–1540. [ Google Scholar ] [ CrossRef ] [ Green Version ]
• Stark, B.; Pabst, G.; Prassl, R. Long-term stability of sterically stabilized liposomes by freezing and freeze-drying: Effects of cryoprotectants on structure. Eur. J. Pharm. Sci. 2010 , 41 , 546–555. [ Google Scholar ] [ CrossRef ]
• Meure, L.A.; Foster, N.R.; Dehghani, F. Conventional and dense gas techniques for the production of liposomes: A review. AAPS PharmSciTech 2008 , 9 , 798–809. [ Google Scholar ] [ CrossRef ] [ Green Version ]
• Maja, L.; Željko, K.; Mateja, P. Sustainable technologies for liposome preparation. J. Supercrit. Fluids 2000 , 165 , 104984. [ Google Scholar ] [ CrossRef ]
• Castor, T.P.; Chu, L. Methods and Apparatus for Making Liposomes Containing Hydrophobic Drugs. U.S. Patent 5,776,486, 7 July 1998. [ Google Scholar ]
• Otake, K.; Imura, T.; Sakai, H.; Abe, M. Development of a new preparation method of liposomes using supercritical carbon dioxide. Langmuir 2001 , 17 , 3898–3901. [ Google Scholar ] [ CrossRef ]
• Imura, T.; Gotoh, T.; Otake, K.; Yoda, S.; Takebayashi, Y.; Yokoyama, S.; Takebayashi, H.; Sakai, H.; Yuasa, M.; Abe, M. Control of physicochemical properties of liposomes using a supercritical reverse phase evaporation method. Langmuir 2003 , 19 , 2021–2025. [ Google Scholar ] [ CrossRef ]
• Otake, K.; Shimomura, T.; Goto, T.; Imura, T.; Furuya, T.; Yoda, S.; Takebayashi, Y.; Sakai, H.; Abe, M. Preparation of liposomes using an improved supercritical reverse phase evaporation method. Langmuir 2006 , 22 , 2543–2550. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Aburai, K.; Yagi, N.; Yokoyama, Y.; Okuno, H.; Sakai, K.; Sakai, H.; Sakamoto, K.; Abe, M. Preparation of liposomes modified with lipopeptides using a supercritical carbon dioxide reverse-phase evaporation method. J. Oleo Sci. 2011 , 60 , 209–215. [ Google Scholar ] [ CrossRef ] [ PubMed ] [ Green Version ]
• Lesoin, L.; Crampon, C.; Boutin, O.; Badens, E. Preparation of liposomes using the supercritical anti-solvent (SAS) process and comparison with a conventional method. J. Supercrit. Fluids 2011 , 57 , 162–174. [ Google Scholar ] [ CrossRef ]
• Lesoin, L.; Crampon, C.; Boutin, O.; Badens, E. Development of a continuous dense gas process for the production of liposomes. J. Supercrit. Fluids 2011 , 60 , 51–62. [ Google Scholar ] [ CrossRef ]
• Soh, S.H.; Lee, L.Y. Microencapsulation and Nanoencapsulation Using Supercritical Fluid (SCF) Techniques. Pharmaceutics 2019 , 11 , 21. [ Google Scholar ] [ CrossRef ] [ Green Version ]
• Türk, M. Particle synthesis by rapid expansion of supercritical solutions (RESS): Current state, further perspectives and needs. J. Aerosol Sci. 2022 , 161 , 105950. [ Google Scholar ] [ CrossRef ]
• Chakravarty, P.; Famili, A.; Nagapudi, K.; Al-Sayah, M.A. Using Supercritical Fluid Technology as a Green Alternative During the Preparation of Drug Delivery Systems. Pharmaceutics 2019 , 11 , 629. [ Google Scholar ] [ CrossRef ] [ Green Version ]
• Wen, Z.; Liu, B.; Zheng, Z.; You, X.; Pua, Y.; Li, Q. Preparation of liposomes entrapping essential oil from Atractylodes macrocephala Koidz by modified RESS technique. Chem. Eng. Res. Des. 2010 , 88 , 1102–1107. [ Google Scholar ] [ CrossRef ]
• Zhang, Q.; Ou, C.; Ye, S.; Song, X.; Luo, S. Construction of nanoscale liposomes loaded with melatonin via supercritical fluid technology. J. Microencapsul. 2017 , 34 , 687–698. [ Google Scholar ] [ CrossRef ]
• Jiao, Z.; Wang, X.; Han, S.; Zha, X.; Xia, J. Preparation of vitamin C liposomes by rapid expansion of supercritical solution process: Experiments and optimization. J. Drug Deliv. Sci. Technol. 2019 , 51 , 1–6. [ Google Scholar ] [ CrossRef ]
• Trucillo, P.; Campardelli, R.; Reverchon, E. A versatile supercritical assisted process for the one-shot production of liposomes. J. Supercrit. Fluids 2019 , 146 , 136–143. [ Google Scholar ] [ CrossRef ]
• Trucillo, P.; Martino, M.; Reverchon, E. Supercritical Assisted Production of Lutein-Loaded Liposomes and Modelling of Drug Release. Processes 2021 , 9 , 1162. [ Google Scholar ] [ CrossRef ]
• Trucillo, P.; Cardea, S.; Baldino, L.; Reverchon, E. Production of liposomes loaded alginate aerogels using two supercritical CO 2 assisted techniques. J. CO 2 Util. 2020 , 39 , 101161. [ Google Scholar ] [ CrossRef ]
• Meure, L.A.; Knott, R.; Foster, N.R.; Dehghani, F. The Depressurization of an Expanded Solution into Aqueous Media for the Bulk Production of Liposomes. Langmuir 2009 , 25 , 326–337. [ Google Scholar ] [ CrossRef ]
• Zhao, L.; Temelli, F. Preparation of liposomes using a modified supercritical process via depressurization of liquid phase. J. Supercrit. Fluids 2015 , 100 , 110–120. [ Google Scholar ] [ CrossRef ]
• Boloix, A.; Feiner-Gracia, N.; Köber, M.; Repetto, J.; Pascarella, R.; Soriano, A.; Masanas, M.; Segovia, N.; Vargas-Nadal, G.; Merlo-Mas, J.; et al. Engineering pH-Sensitive Stable Nanovesicles for Delivery of MicroRNA Therapeutics. Small 2021 , 18 , 2101959. [ Google Scholar ] [ CrossRef ]
• Merlo-Mas, J.; Tomsen-Melero, J.; Corchero, J.-L.; González-Mira, E.; Font, A.; Pedersen, J.N.; García-Aranda, N.; Cristóbal-Lecina, E.; Alcaina-Hernando, M.; Mendoza, R.; et al. Application of Quality by Design to the robust preparation of a liposomal GLA formulation by DELOS-susp method. J. Supercrit. Fluids 2021 , 173 , 105204. [ Google Scholar ] [ CrossRef ]
• Van Swaay, D.; Demello, A. Microfluidic methods for forming liposomes. Lab Chip 2013 , 13 , 752–767. [ Google Scholar ] [ CrossRef ]
• Jahn, A.; Vreeland, W.N.; DeVoe, D.L.; Locascio, L.E.; Gaitan, M. Microfluidic directed formation of liposomes of controlled size. Langmuir 2007 , 23 , 6289–6293. [ Google Scholar ] [ CrossRef ]
• Carugo, D.; Bottaro, E.; Owen, J.; Stride, E.; Nastruzzi, C. Liposome production by microfluidics: Potential and limiting factors. Sci. Rep. 2016 , 6 , 25876. [ Google Scholar ] [ CrossRef ] [ Green Version ]
• Al-Amin, M.D.; Bellato, F.; Mastrotto, F.; Garofalo, M.; Malfanti, A.; Salmaso, S.; Caliceti, P. Dexamethasone Loaded Liposomes by Thin-Film Hydration and Microfluidic Procedures: Formulation Challenges. Int. J. Mol. Sci. 2020 , 21 , 1611. [ Google Scholar ] [ CrossRef ] [ PubMed ] [ Green Version ]
• Shum, H.C.; Lee, D.; Yoon, I.; Kodger, T.; Weitz, D.A. Double Emulsion Templated Monodisperse Phospholipid Vesicles. Langmuir 2008 , 24 , 7651–7653. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Funakoshi, K.; Suzuki, H.; Takeuchi, S. Formation of giant lipid vesiclelike compartments from a planar lipid membrane by a pulsed jet flow. J. Am. Chem. Soc. 2007 , 129 , 12608–12609. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Bruna, G.; Carvalho, B.G.; Ceccato, B.T.; Michelon, M.; Han, S.W.; de la Torre, L.G. Advanced Microfluidic Technologies for Lipid Nano-Microsystems from Synthesis to Biological Application. Pharmaceutics 2022 , 14 , 141. [ Google Scholar ] [ CrossRef ]
• Zhang, G.; Sun, J. Lipid in Chips: A Brief Review of Liposomes Formation by Microfluidics. Int. J. Nanomed. 2021 , 16 , 7391–7416. [ Google Scholar ] [ CrossRef ]
• Jaafar-Maalej, C.; Charcosset, C.; Fessi, H. A new method for liposome preparation using a membrane contactor. J. Liposome Res. 2010 , 21 , 213–220. [ Google Scholar ] [ CrossRef ]
• Laouini, A.; Jaafar-Maalej, C.; Sfar, S.; Charcosset, C.; Fessi, H. Liposome preparation using a hollow fiber membrane contactor—Application to spironolactone encapsulation. Int. J. Pharm. 2011 , 415 , 53–61. [ Google Scholar ] [ CrossRef ]
• Laouini, A.; Charcosset, C.; Fessi, H.; Holdich, R.; Vladisavljević, G. Preparation of liposomes: A novel application of microengineered membranes-investigation of the process parameters and application to the encapsulation of vitamin E. RSC Adv. 2013 , 3 , 4985–4994. [ Google Scholar ] [ CrossRef ] [ Green Version ]
• Delma, K.L.; Lechanteur, A.; Evrard, B.; Semdé, R.; Piel, G. Sterilization methods of liposomes: Drawbacks of conventional methods and perspectives. Int. J. Pharm. 2021 , 597 , 120271. [ Google Scholar ] [ CrossRef ]
• Soares, G.; Learmonth, D.A.; Vallejo, M.; Davila, S.P.; González, P.; Sousa, R.A.; Oliveira, A. Supercritical CO 2 technology: The next standard sterilization technique? Mater. Sci. Eng. C 2019 , 99 , 520–540. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Hood, R.R.; Vreeland, W.N.; DeVoe, D. Microfluidic remote loading for rapid single-step liposomal drug preparation. Lab Chip 2014 , 14 , 3359–3367. [ Google Scholar ] [ CrossRef ] [ PubMed ] [ Green Version ]
• Kastner, E.; Verma, V.; Lowry, D.; Perrie, Y. Microfluidic-controlled manufacture of liposomes for the solubilisation of a poorly water soluble drug. Int. J. Pharm. 2015 , 485 , 122–130. [ Google Scholar ] [ CrossRef ] [ PubMed ] [ Green Version ]
• Dimov, N.; Kastner, E.; Hussain, M.; Perrie, Y.; Szita, N. Formation and purification of tailored liposomes for drug delivery using a module-based micro continuous-flow system. Sci. Rep. 2017 , 7 , 12045. [ Google Scholar ] [ CrossRef ] [ Green Version ]
• Yadav, S.; Sharma, A.K.; Kumar, P. Nanoscale Self-Assembly for Therapeutic Delivery. Front. Bioeng. Biotechnol. 2020 , 8 , 127. [ Google Scholar ] [ CrossRef ]
• Lombardo, D.; Calandra, P.; Pasqua, L.; Magazù, S. Self-Assembly of Organic Nanomaterials and Biomaterials: The Bottom-Up Approach for Functional Nanostructures Formation and Advanced Applications. Materials 2020 , 13 , 1048. [ Google Scholar ] [ CrossRef ] [ Green Version ]
• Calandra, P.; Caschera, D.; Liveri, V.T.; Lombardo, D. How self-assembly of amphiphilic molecules can generate complexity in the nanoscale. Colloids Surf. A Physicochem. Eng. 2015 , 484 , 164–183. [ Google Scholar ] [ CrossRef ]
• Zemb, T.; Lindner, P. Neutron, X-rays and Light Scattering Methods Applied to Soft Condensed Matter ; North-Holland: Amsterdam, The Netherlands, 2002. [ Google Scholar ]
• Di Cola, E.; Grillo, I.; Ristori, S. Small Angle X-ray and Neutron Scattering: Powerful Tools for Studying the Structure of Drug-Loaded Liposomes. Pharmaceutics 2016 , 8 , 10. [ Google Scholar ] [ CrossRef ]
• Fitter, J.; Gutberlet, T.; Katsaras, J. Neutron scattering in Biology. In Techniques and Applications ; Springer: Berlin/Heidelberg, Germany, 2006. [ Google Scholar ]
• Belloni, L. Interacting monodisperse and polydisperse spheres. In Neutron X-ray and Light Scattering ; Lindner, P., Zemb, T., Eds.; Elsevier Science Publishers B. V.: New York, NY, USA, 1991. [ Google Scholar ]
• Schmidt, P.W. Modern Aspects of Small-Angle Scattering ; Brumberger, H., Ed.; Kluwer Academic: Dordrecht, The Netherlands, 1995. [ Google Scholar ]
• Lombardo, D.; Calandra, P.; Kiselev, M.A. Structural Characterization of Biomaterials by Means of Small Angle X-rays and Neutron Scattering (SAXS and SANS), and Light Scattering Experiments. Molecules 2020 , 25 , 5624. [ Google Scholar ] [ CrossRef ]
• Kinnun, J.J.; Scott, H.L.; Ashkar, R.; Katsaras, J. Biomembrane Structure and Material Properties Studied with Neutron Scattering. Front. Chem. 2021 , 9 , 642851. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Kiselev, M.A.; Zemlyanaya, E.V.; Ryabova, N.Y.; Hauss, T.; Dante, S.; Lombardo, D. Water distribution function across the curved lipid bilayer: SANS study. Chem. Phys. 2008 , 345 , 185–190. [ Google Scholar ] [ CrossRef ]
• Dorrell, M.; Heberle, F.A.; Katsaras, J.; Lyman, E.; Sodt, A.J. Nanoscale Structure of Lipid Bilayers Revealed by In-Silico and Experimental Small Angle Neutron Scattering. Biophys. J. 2019 , 116 , 90a. [ Google Scholar ] [ CrossRef ] [ Green Version ]
• Kiselev, M.; Zemlyanaya, E.V.; Ryabova, N.Y.; Hauß, T.; Almásy, L.; Funari, S.S.; Zbytovská, J.; Lombardo, D. Influence of ceramide on the internal structure and hydration of the phospholipid bilayer studied by neutron and X-ray scattering. Appl. Phys. A 2014 , 116 , 319–325. [ Google Scholar ] [ CrossRef ]
• Amenitsch, H.; Bernstorff, S.; Kriechbaum, M.; Lombardo, D.; Mio, H.; Rappolt, M.; Laggner, P. Performance and first results of the ELETTRA high-flux beamline for small-angle X-ray scattering. J. Appl. Crystallogr. 1997 , 30 , 872–876. [ Google Scholar ] [ CrossRef ]
• Narayanan, T.; Sztucki, M.; Van Vaerenbergh, P.; Léonardon, J.; Gorini, J.; Claustre, L.; Sever, F.; Morse, J.; Boesecke, P. A multipurpose instrument for time-resolved ultra-small-angle and coherent X-ray scattering. J. Appl. Crystallogr. 2018 , 51 , 1511–1524. [ Google Scholar ] [ CrossRef ] [ Green Version ]
• Turco Liveri, V.; Lombardo, D.; Pochylski, M.; Calandra, P. Molecular association of small amphiphiles: Origin of ionic liquid properties in dibutyl phosphate/propylamine binary mixtures. J. Mol. Liq. 2018 , 263 , 274–281. [ Google Scholar ] [ CrossRef ]
• Koch, M.H.; Vachette, P.; Svergun, D.I. Small-angle scattering: A view on the properties, structures and structural changes of biological macromolecules in solution. Q. Rev. Biophys. 2003 , 36 , 147–227. [ Google Scholar ] [ CrossRef ] [ Green Version ]
• Weyerich, B.; Brunner-Popela, J.; Glatter, O. Small-angle scattering of interacting particles. II. Generalized indirect Fourier transformation under consideration of the effective structure factor for polydisperse systems. J. Appl. Crystallogr. 1999 , 32 , 197–209. [ Google Scholar ] [ CrossRef ]
• Micali, N.; Scolaro, L.M.; Romeo, A.; Lombardo, D.; Lesieur, P.; Mallamace, F. Structural properties of methanol-polyamidoamine dendrimer solutions. Phys. Rev. E 1998 , 58 , 6229–6235. [ Google Scholar ] [ CrossRef ]
• Sachs, J.; Petrache, H.; Woolf, T. Interpretation of small angle X-ray measurements guided by molecular dynamics simulations of lipid bilayers. Chem. Phys. Lipids 2003 , 26 , 211–223. [ Google Scholar ] [ CrossRef ]
• Kucerka, N.; Nagle, J.F.; Sachs, J.N.; Feller, S.E.; Pencer, J.; Jackson, A.; Katsaras, J. Lipid bilayer structure determined by the simultaneous analysis of neutron and X-ray scattering data. Biophys. J. 2008 , 95 , 2356–2367. [ Google Scholar ] [ CrossRef ] [ PubMed ] [ Green Version ]
• Lombardo, D.; Calandra, P.; Bellocco, E.; Laganà, G.; Barreca, D.; Magazù, S.; Wanderlingh, U.; Kiselev, M.A. Effect of anionic and cationic polyamidoamine (PAMAM) dendrimers on a model lipid membrane. Biochim. Biophys. Acta 2016 , 1858 , 2769–2777. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Lombardo, D.; Calandra, P.; Magazù, S.; Wanderlingh, U.; Barreca, D.; Pasqua, L.; Kiselev, M.A. Soft nanoparticles charge expression within lipid membranes: The case of amino terminated dendrimers in bilayers vesicles. Colloids Surf. B Biointerfaces 2018 , 170 , 609–616. [ Google Scholar ] [ CrossRef ]
• Lombardo, D.; Calandra, P.; Caccamo, M.T.; Magazù, S.; Pasqua, L.; Kiselev, M.A. Interdisciplinary approaches to the study of biological membranes. AIMS Biophys. 2020 , 7 , 267. [ Google Scholar ] [ CrossRef ]
• Klang, V.; Valenta, C.; Matsko, N.B. Electron microscopy of pharmaceutical systems. Micron 2013 , 44 , 45–74. [ Google Scholar ] [ CrossRef ]
• Bibi, S.; Kaur, R.; Henriksen-Lacey, M.; McNeil, S.E.; Wilkhu, J.; Lattmann, E.; Christensen, D.; Mohammed, A.R.; Perrie, Y. Microscopy imaging of liposomes: From coverslips to environmental SEM. Int. J. Pharm. 2011 , 417 , 138–150. [ Google Scholar ] [ CrossRef ]
• Ruozi, B.; Belletti, D.; Tombesi, A.; Tosi, G.; Bondioli, L.; Forni, F.; Vandelli, M.A. AFM, ESEM, TEM, and CLSM in liposomal characterization: A comparative study. Int. J. Nanomed. 2011 , 6 , 557–563. [ Google Scholar ] [ CrossRef ] [ Green Version ]
• Helvig, S.; Azmi, I.D.M.; Moghimi, S.M.; Yaghmur, A. Recent advances in cryo-TEM imaging of soft lipid nanoparticles. AIMS Biophys. 2015 , 2 , 116–130. [ Google Scholar ] [ CrossRef ]
• Almgren, M.; Edwards, K.; Karlsson, G.B. Cryo transmission electron microscopy of liposomes and related structures. Colloids Surf. A Physicochem. Eng. 2000 , 174 , 3–21. [ Google Scholar ] [ CrossRef ]
• Spyratou, E.; Mourelatou, E.A.; Makropoulou, M.; Demetzos, C. Atomic force microscopy: A tool to study the structure, dynamics and stability of liposomal drug delivery systems. Expert Opin. Drug Deliv. 2009 , 6 , 305–317. [ Google Scholar ] [ CrossRef ]
• Robson, A.L.; Dastoor, P.C.; Flynn, J.; Palmer, W.; Martin, A.; Smith, D.W.; Woldu, A.; Hua, S. Advantages and Limitations of Current Imaging Techniques for Characterizing Liposome Morphology. Front. Pharmacol. 2018 , 9 , 80. [ Google Scholar ] [ CrossRef ] [ PubMed ] [ Green Version ]
• Bouvrais, H.; Pott, T.; Bagatolli, L.A.; Ipsen, J.H.; Meleard, P. Impact of membrane-anchored fluorescent probes on the mechanical properties of lipid bilayers. Biochim. Biophys. Acta 2010 , 1798 , 1333–1337. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Mertins, O.; Dimova, R. Insights on the interactions of chitosan with phospholipid vesicles. Part II: Membrane stiffening and pore formation. Langmuir 2013 , 29 , 14552–14559. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Murphy, D.B.; Davidson, M.W. Confocal laser scanning microscopy. In Fundamentals of Light Microscopy and Electronic Imaging , 2nd ed.; Murphy, D.B., Ed.; John Wiley and Sons: New York, NY, USA, 2012; pp. 265–305. [ Google Scholar ] [ CrossRef ]
• Solomon, M.A. Determination of the Subcellular Distribution of Liposomes Using Confocal Microscopy. Methods Mol. Biol. 2017 , 1522 , 119–130. [ Google Scholar ] [ CrossRef ]
• Bruun, K.; Hille, C. Study on intracellular delivery of liposome encapsulated quantum dots using advanced fluorescence microscopy. Sci. Rep. 2019 , 9 , 10504. [ Google Scholar ] [ CrossRef ]
• Sattler, E.C.; Maier, T.; Hoffmann, V.S.; Hegyi, J.; Ruzicka, T.; Berking, C. Noninvasive in vivo detection and quantification of Demodex mites by confocal laser scanning microscopy. Br. J. Dermatol. 2012 , 167 , 1042–1047. [ Google Scholar ] [ CrossRef ]
• Vaghasiya, K.; Sharma, A.; Kumar, K.; Ray, E.; Adlakha, S.; Katare, O.P.; Hota, S.K.; Verma, R.K. Heparin-Encapsulated Metered-Dose Topical “Nano-Spray Gel” Liposomal Formulation Ensures Rapid On-Site Management of Frostbite Injury by Inflammatory Cytokines Scavenging, ACS Biomater. Sci. Eng. 2019 , 5 , 6617–6631. [ Google Scholar ] [ CrossRef ]
• Berne, B.J.; Pecora, R. Dynamic Light Scattering ; John Wiley: New York, NY, USA, 1976. [ Google Scholar ]
• Brown, W. Dynamic Light Scattering: The Method and some Applications ; Clarendon Press: Oxford, UK, 1993. [ Google Scholar ]
• Hunter, R.J. Foundations of Colloid Science ; Oxford University Press: New York, NY, USA, 1986; Volume I–II. [ Google Scholar ]
• McNeil-Watson, F.; Tscharnuter, W.; Miller, J. A new instrument for the measurement of very small electrophoretic mobilities using phase analysis light scattering (PALS). Colloids Surf. A 1998 , 140 , 53–57. [ Google Scholar ] [ CrossRef ]
• Carvalho, P.M.; Felício, M.R.; Santos, N.C.; Gonçalves, S.; Domingues, M.M. Application of Light Scattering Techniques to Nanoparticle Characterization and Development. Front. Chem. 2018 , 6 , 237. [ Google Scholar ] [ CrossRef ]
• Mazzaglia, A.; Angelini, N.; Darcy, R.; Donohue, R.; Lombardo, D.; Micali, N.; Sciortino, M.T.; Villari, V.; Scolaro, L.M. Novel heterotopic colloids of anionic porphyrins entangled in cationic amphiphilic cyclodextrins: Spectroscopic investigation and intracellular delivery. Chemistry 2003 , 9 , 5762–5769. [ Google Scholar ] [ CrossRef ]
• Mazzaglia, A.; Angelini, N.; Lombardo, D.; Micali, N.; Patané, S.; Villari, V.; Scolaro, L.M. Amphiphilic cyclodextrin carriers embedding porphyrins: Charge and size modulation of colloidal stability in heterotopic aggregates. J. Phys. Chem. B 2005 , 109 , 7258–7265. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Lebrón, J.A.; López-López, M.; García-Calderón, C.B.; Rosado, I.V.; Balestra, F.R.; Huertas, P.; Rodik, R.V.; Kalchenko, V.I.; Bernal, E.; Moyá, M.L.; et al. Multivalent Calixarene-Based Liposomes as Platforms for Gene and Drug Delivery. Pharmaceutics 2021 , 13 , 1250. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Grabielle-Madelmont, C.; Lesieur, S.; Ollivon, M. Characterization of loaded liposomes by size exclusion chromatography. J. Biochem. Biophys. Methods 2003 , 56 , 189–217. [ Google Scholar ] [ CrossRef ]
• Nieh, M.P.; Heberle, F.A.; Katsaras, J. Characterization of Biological Membranes Structure and Dynamics ; De Gruyter: Berlin, Germany, 2019. [ Google Scholar ]
• Pignatello, R.; Musumeci, T.; Basile, L.; Carbone, C.; Puglisi, G. Biomembrane models and drug-biomembrane interaction studies: Involvement in drug design and development. J. Pharm. Bioallied Sci. 2011 , 3 , 4–14. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Pignatello, R. Drug-Biomembrane Interaction Studies, the Application of Calorimetric Techniques ; Elsevier: Amsterdam, The Netherlands, 2013. [ Google Scholar ]
• Abraham, T.; Lewis, R.N.; Hodges, R.S.; McElhaney, R.N. Isothermal titration calorimetry studies of the binding of a rationally designed analogue of the antimicrobial peptide gramicidin s to phospholipid bilayer membranes. Biochemistry 2005 , 44 , 2103–2112. [ Google Scholar ] [ CrossRef ] [ Green Version ]
• Zhang, X.M.; Patel, A.B.; de Graaf, R.A.; Behar, K.L. Determination of liposomal encapsulation efficiency using proton NMR spectroscopy. Chem. Phys. Lipids 2004 , 127 , 113–120. [ Google Scholar ] [ CrossRef ]
• Cruciani, O.; Mannina, L.; Sobolev, A.; Cametti, C.; Segre, A. An improved NMR study of liposomes using 1-palmitoyl-2-oleoyl-sn-glycero-3-phospatidylcholine as model. Molecules 2006 , 11 , 334–344. [ Google Scholar ] [ CrossRef ] [ Green Version ]
• Sainz, V.; Conniot, J.; Matos, A.I.; Peres, C.; Zupanǒiǒ, E.; Moura, L.; Silva, L.C.; Florindo, H.F.; Gaspar, R.S. Regulatory aspects on nanomedicines. Biochem. Biophys. Res. Commun. 2015 , 468 , 504–510. [ Google Scholar ] [ CrossRef ]
• Raval, N.; Maheshwari, R.G.; Kalyane, D.; Youngren-Ortiz, S.R.; Chougule, M.B.; Tekade, R.K. Importance of Physicochemical Characterization of Nanoparticles in Pharmaceutical Product Development ; Elsevier BV: Amsterdam, The Netherlands, 2019; pp. 369–400. [ Google Scholar ] [ CrossRef ]
• Danaei, M.; Dehghankhold, M.; Ataei, S.; Davarani, F.H.; Javanmard, R.; Dokhani, A.; Khorasani, S.; Mozafari, M. Impact of Particle Size and Polydispersity Index on the Clinical Applications of Lipidic Nanocarrier Systems. Pharmaceutics 2018 , 10 , 57. [ Google Scholar ] [ CrossRef ] [ PubMed ] [ Green Version ]
• Németh, Z.; Pallagi, E.; Dobó, D.G.; Csóka, I. A Proposed Methodology for a Risk Assessment-Based Liposome Development Process. Pharmaceutics 2020 , 12 , 1164. [ Google Scholar ] [ CrossRef ]
• European Medicine Agency (EMA). Reflection Paper on the Data Requirements for Intravenous Liposomal Products Developed with Reference to an Innovator Liposomal Product. 2013. Available online: https://www.ema.europa.eu/en/documents/scientific-guideline/reflection-paper-data-requirements-intravenous-liposomal-products-developed-reference-innovator_en.pdf (accessed on 18 February 2022).
• Food and Drug Administration (FDA). Liposome Drug Products Chemistry, Manufacturing, and Controls; Human Pharmacokinetics and Bioavailability; and Labeling Documentation Guidance for Industry. 2018. Available online: http://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/default.htm (accessed on 18 February 2022).
• Sabir, F.; Katona, G.; Pallagi, E.; Dobó, D.G.; Akel, H.; Berkesi, D.; Kónya, Z.; Csóka, I. Quality-by-design-based development of n-propyl-gallate-loaded hyaluronic-acid-coated liposomes for intranasal administration. Molecules 2021 , 26 , 1429. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Porfire, A.; Achim, M.; Barbalata, C.; Rus, I.; Tomuta, I.; Cristea, C. Pharmaceutical Development of Liposomes Using the QbD Approach. Liposomes Adv. Perspect 2019 , 2019 , 1–20. [ Google Scholar ] [ CrossRef ] [ Green Version ]
• Lu, B.; Ma, Q.; Zhang, J.; Liu, R.; Yue, Z.; Xu, C.; Li, Z.; Lin, H. Preparation and characterization of bupivacaine multivesicular liposome: A QbD study about the effects of formulation and process on critical quality attributes. Int. J. Pharm. 2021 , 598 , 120335. [ Google Scholar ] [ CrossRef ] [ PubMed ]
• Xu, X.; Khan, M.A.; Burgess, D.J. A quality by design (QbD) case study on liposomes containing hydrophilic API: I. Formulation, processing design and risk assessment. Int. J. Pharm. 2011 , 419 , 52–59. [ Google Scholar ] [ CrossRef ]

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Lombardo, D.; Kiselev, M.A. Methods of Liposomes Preparation: Formation and Control Factors of Versatile Nanocarriers for Biomedical and Nanomedicine Application. Pharmaceutics 2022 , 14 , 543. https://doi.org/10.3390/pharmaceutics14030543

Lombardo D, Kiselev MA. Methods of Liposomes Preparation: Formation and Control Factors of Versatile Nanocarriers for Biomedical and Nanomedicine Application. Pharmaceutics . 2022; 14(3):543. https://doi.org/10.3390/pharmaceutics14030543

Lombardo, Domenico, and Mikhail A. Kiselev. 2022. "Methods of Liposomes Preparation: Formation and Control Factors of Versatile Nanocarriers for Biomedical and Nanomedicine Application" Pharmaceutics 14, no. 3: 543. https://doi.org/10.3390/pharmaceutics14030543

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ORIGINAL RESEARCH article

Hyaluronic acid-modified liposomes potentiated in-vivo anti-hepatocellular carcinoma of icaritin.

• 1 Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand
• 2 Department of Pharmacy, The Affiliated Hospital of Southwest Medical University, Luzhou, China
• 3 Key Laboratory of Medical Electrophysiology, Ministry of Education, School of Pharmacy, Southwest Medical University, Luzhou, China
• 4 Suining First People’s Hospital, Suining, China
• 5 Cancer Research Unit of Associated Medical Sciences (AMS-CRU), Chiang Mai University, Chiang Mai, Thailand
• 6 Center of Excellence in Pharmaceutical Nanotechnology, Faculty of Pharmacy, Chiang Mai University, Chiang Mai, Thailand
• 7 Department of General Surgery (Thyroid Surgery), The Affiliated Hospital of Southwest Medical University, Luzhou, China
• 8 Central Nervous System Drug Key Laboratory of Sichuan Province, Luzhou, China

Introduction: Icaritin (ICT), a promising anti-hepatocellular carcinoma (HCC) prenylated flavonoid, is hindered from being applied due to its low water solubility and high lipophilicity in poorly differentiated HCC which is associated with upregulation of CD44 isoforms. Thus, hyaluronic acid (HA), a natural polysaccharide with high binding ability to CD44 receptors, was used to formulate a modified liposome as a novel targeted ICT-delivery system for HCC treatment.

Methods: The ICT-Liposomes (Lip-ICT) with and without HA were prepared by a combined method of thin-film dispersion and post-insertion. The particle size, polydispersity (PDI), zeta potential, encapsulation efficacy (%EE), drug loading content (%DLC), and in vitro drug release profiles were investigated for physicochemical properties, whereas MTT assay was used to assess cytotoxic effects on HCC cells, HepG2, and Huh7 cells. Tumor bearing nude mice were used to evaluate the inhibitory effect of HA-Lip-ICT and Lip-ICT in vivo .

Results: Lip-ICT and HA-Lip-ICT had an average particle size of 171.2 ± 1.2 nm and 208.0 ± 3.2 nm, with a zeta potential of −13.9 ± 0.83 and −24.8 ± 0.36, respectively. The PDI resulted from Lip-ICT and HA-Lip-ICT was 0.28 ± 0.02 and 0.26 ± 0.02, respectively. HA-Lip-ICT demonstrated higher in vitro drug release when pH was dropped from 7.4 to 5.5, The 12-h release rate of ICT from liposomes increased from 30% at pH7.4 to more than 60% at pH5.5. HA-Lip-ICT displayed higher toxicity than Lip-ICT in both HCC cells, especially Huh7with an IC 50 of 34.15 ± 2.11 μM. The in vivo tissue distribution and anti-tumor experiments carried on tumor bearing nude mice indicated that HA-Lip- ICT exhibited higher tumor accumulation and achieved a tumor growth inhibition rate of 63.4%.

Discussion: The nano-sized Lip-ICT was able to prolong the drug release time and showed long-term killing HCC cells ability. Following conjugation with HA, HA-Lip-ICT exhibited higher cytotoxicity, stronger tumor targeting, and tumor suppression abilities than Lip-ICT attributed to HA-CD44 ligand-receptor interaction, increasing the potential of ICT to treat HCC.

GRAPHICAL ABSTRACT | Schematic overview of the hyaluronic acid-modified liposomes loaded with icaritin (HA-Lip-ICT) and its proposed anti-tumor mechanism.

1 Introduction

Liver cancer stands as the 6th most prevalent malignancy globally, ranking second among cancer related fatalities. Hepatocellular carcinoma (HCC), responsible for 80% of primary liver cancer cases, which has a bad prognosis with less than 12% of patients having a 5-year overall survival rate ( Chen et al., 2016 ). Nowadays, the foremost methods in treating HCC are surgery and transplantation. Nonetheless, the majority of HCC cases reach inoperable advanced stages where surgical intervention becomes unfeasible, particularly in regions like China ( Torre et al., 2015 ; Chen et al., 2016 ). Moreover, following surgical resection, the outlook for HCC in the long term remains severe, and the persistent risk of cancer relapse or metastasis poses a substantial hurdle ( El-Serag and Rudolph, 2007 ). Conventional chemotherapy drugs, like cisplatin and its counterparts, consistently yield poor responses in advanced-stage HCC. Moreover, acquired chemotherapy resistance and side effects hinder the long-term use of these therapeutic compounds ( Singh et al., 2014 ). Hence, there’s urgent need to find new anti-cancer agents comes from natural plants ( Cragg and Newman, 2005 ). Natural compounds are very important sources for exploration of anti-cancer drugs ( Newman, 2008 ).

Icaritin (ICT), a prenylated flavonoid, comes from plants named Epimedium genus within the Berberidaceae family. Herba Epimedii extracts are widely applied in traditional Chinese medicine due to their tonic and aphrodisiac characteristics ( Sze et al., 2010 ). ICT is presently in the phase three clinical trial stage for treating advanced HCC, supported by a robust foundation of preclinical and clinical evidence ( Bailly, 2020 ). The drug’s anti-tumor efficacy originates from its ability to influence multiple signaling factors within cancer cells, primarily the chemokine receptor CXCR4, NF-κB and transcription factors STAT3, as well as the estrogen receptor splice variant ERα36 ( Tiong et al., 2012 ). Recent research has associated additional factors, such as various microRNAs, the production of ROS, and the regulation of sphingosine kinase-1 ( Lu et al., 2017 ). Furthermore, ICT interacts with the RAGE-HMGB1 pathway and manages the crosstalk between apoptosis and autophagy, thereby enhancing its anti-cancer capabilities ( Liu et al., 2019 ). Additionally, ICT triggers substantial changes in the tumor’s surroundings, fostering an immune response ( Hao et al., 2019 ). Taken together, these diverse biochemical and cellular features provide a strong activity profile for ICT, making it a valuable option for treating HCC. However, the clinical application of ICT for cancer treatment is hindered by its low water solubility and limited bioavailability. It is crucial to develop new strategies aimed at increasing aqueous solubility and enhancing the bioavailability of ICT ( Chang et al., 2012 ; Y; Li et al., 2013 ).

Nanotechnology has facilitated the enhancement of the efficacy of drugs in liver cancer treatment. Using nanocarriers as drug delivery systems is a popular strategy for delivering hydrophobic drugs to specific tissues more effectively and enhancing the dissolution of drugs ( Sabit et al., 2022 ). This can be achieved through the utilization of diverse nanocarrier-mediated drug delivery systems, which subsequently enable reduced drug dosages to achieve higher therapeutic effects, minimize systemic toxicity risks, prolong drug release over days following a single administration, and improve specific targeting to cancer cells ( Mitchell et al., 2021 ). Among these, liposomes stand out as the most promising for clinical applications. Generally, liposomes were used to enhance drug dissolution by enclosing poorly soluble drugs within the hydrophobic bilayer ( Lee, 2020 ). Furthermore, liposomes demonstrated favorable biodegradability and biocompatibility, thus avoiding harm to healthy cells ( Cheng et al., 2020 ).

Modified liposomes have been introduced with various specific ligands to enable additional functions. Hyaluronic acid (HA) holds significant potential as a natural material for liposomal functionalization due to its strong affinity to CD44 receptors, which show increased expression in cancerous cells ( Akim et al., 2021 ; Antonio and Trídico, 2021 ). As a result of this affinity, liposomes can be delivered to cancer cells via receptor-ligand interactions ( Kesharwani et al., 2021 ). HA has found extensive application in formulating dendrimers, micelles and liposomes because of its remarkable hydrophilicity and biocompatibility ( Patra et al., 2018 ; Majumder et al., 2019 ; Su and Peter, 2020 ). As a result, utilizing a hydrophilic HA layer on liposomes provides them with physiological stability and imparts active targeting potential to liposomal nanocarriers, facilitating their internalization into cancer cells through HA/CD44 interactions.

It has been reported that the upregulation of CD44 correlates with poorly differentiated HCC and reduced survival rates ( Endo and Terada, 2000 ). CD44 is a cell surface receptor known to have higher expression levels in several solid tumors than in normal tissues. Previous studies indicated the significance of CD44 in sustaining cancer stem cells (CSCs) and its role in governing oxidative stress levels in human HCC cell lines, such as Huh7 ( Asai et al., 2019 ). Utilizing the specific interaction between CD44 and HA, a targeted drug delivery system can be developed for treating HCC.

Hence, our primary objective was to create a cholesterol-tri(ethyleneglycol)-hyaluronic acid conjugate (HA-Chol) to fabricate hyaluronic acid-decorated liposomes (HA-Lip) for the purpose of precisely delivering ICT. Cholesterol was chosen as the molecule for conjugation with HA due to its prominent role for the liposome fluidity and there’s a free secondary hydroxyl group in its structure, it offers versatility for chemical alterations ( Ohvo-Rekilä et al., 2002 ). We hypothesized that hyaluronic acid-coated liposomes containing ICT (HA-Lip-ICT) could effectively avoid the low water solubility and low bioavailability of ICT. Moreover, it would enhance in vivo tumor targeting and demonstrate remarkable anti-tumor efficacy against liver cancer through CD44-mediated endocytosis. In this study, the characteristics of HA-Lip-ICT were explored and its anti-tumor efficacy on multiple HCC cell models was assessed, including cytotoxicity, cell cycle arrest and apoptosis. Consequently, the intrinsic molecular mechanism was examined by which HA-Lip-ICT triggers cell cycle arrest and initiates apoptosis using Western blotting. Finally, in vivo biodistribution analysis, preliminary safety evaluations and tumor growth inhibition assessments were conducted using BALB/C nude mice bearing Huh7 tumor models.

2 Materials and methods

2.1 materials.

Icaritin was purchased from Plant Origin Biological Co., Ltd. (Nanjing, China; purity = 98%). Standard icaritin was offered by Chengdu Purechem-Standard Biotech Co., Ltd. (Chengdu, China; purity ≥98%). Cholesterol was given by Solarbio Co. (Beijing, China). Hyaluronic acid (HA, [0.5–1.5] × 10 6  Da) was obtained from Shanghai Macklin Biochemical Technology Co., Ltd. (Shanghai, China). The Annexin V-FITC/PI apoptosis detection kit was purchased from Solarbio Life Sciences (Beijing, China). Fetal bovine serum (FBS) and trypsin were obtained from Gibco-BRL (New York, United States). Additionally, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide (DiR) and 4′,6-diamidino-2-phenylin-dole dihydrochloride (DAPI) were obtained from Biyuntian Biotechnology (Shanghai, China). Coumarin-6 was acquired from Ruixi Biotechnology (Xi’an, China). Rabbit polyclonal anti-GAPDH, goat anti-mouse immunoglobulin G (IgG), goat anti-rabbit IgG, as well as rabbit polyclonal antibodies against CD44 were obtained from ABclonal Technology (Wuhan, China). The hyaluronic acid-modified cholesterol (HA-Chol) synthesis routes are shown in Supplementary Figure S1 and the details are listed in it too.

2.2 Cell culture

The HCC cell lines of human origin (Huh7 and HepG2), along with the normal human liver cell line (L02), were procured from The Chinese Academy of Sciences’ Cell Bank in Shanghai, China, and cultured in DMEM medium containing 10% FBS, 100 IU/mL penicillin and 100 μg/mL streptomycin at 37°C with the condition of 5% CO 2 .

2.3 Formulation and characterization of liposomes encapsulating ICT

Liposomes were formulated through a combined method involving mechanical extrusion and thin-film dispersion techniques, using key ingredients, such as soybean lecithin, cholesterol and HA-Chol. Lipids and ICT were dissolved in 10 mL chloroform and methanol (1:1 v/v) then the mixture was transferred to a round-bottom flask. Organic solvent was eliminated with vacuum rotary evaporator till a thin film formed. The thin film was additionally subjected to vacuum drying overnight to ensure there’s no organic solvent. Then, phosphate-buffered solution (PBS, pH 7.4) was used to rehydrate the dried thin film. Lastly, the liposomal suspensions were extruded through the Avanti ® Mini-Extruder with a 200-nm polycarbonate membrane to get the desired size-distribution liposomes. The empty liposomes (empty Lip) without ICT were prepared similarly without the addition of ICT. HA-Lip-ICT was formulated using post-insertion method wherein the HA-Chol conjugate was embedded into the lipid bilayer (L. Cheng et al., 2014 ). An exact amount of HA-Chol conjugate (10% of the total lipid content) was incubated with the liposomes mentioned above for 30 min at 50°C.

Liposome morphology was observed using transmission electron microscopy (TEM, Hitachi HT-7700, Japan). Particle size and the zeta potential of liposomes were assessed by DLS (Zetasizer Nano ZS90, Malvern Instruments, Malvern, United Kingdom). Fourier transform infrared (FTIR) spectra were obtained using an IRAffinity-1S system (Shimadzu Technology, Kyoto, Japan) at frequencies of 500–4000 cm –1 .

Encapsulation efficiency (EE) and drug loading content (DLC) were determined using ultrafiltration tubes (MWCO = 3 kDa). Unencapsulated ICT (W free ) was measured using HPLC. The mobile phase were water and acetonitrile (20:80, v/v) and detected at 270 nm. EE and DLC were calculated according to the below formula:

The accumulative release patterns in vitro of free ICT, Lip-ICT and HA-Lip-ICT were assessed via the dialysis technique under controlled conditions at 37°C, involving PBS, pH 7.4, and pH 5.5 PBS buffers containing 0.2% Tween 80 (w/v). In brief, 1.0 mL solutions of free ICT, Lip-ICT and HA-Lip-ICT at the concentration of 5 mg/mL were encapsulated within dialysis bags (MWCO = 3 kDa) and put in the release medium. Subsequently, these dialysis bags were immersed in 50 mL of PBS buffer under gentle agitation at 100 rpm. At predefined intervals (0.5, 1, 2, 4, 6, 8, 10, 12, 24, 36, 48, 60, and 72 h), 1.0 mL release medium was taken out and replaced with same amount of fresh medium. The concentrations of the released ICT were quantified using HPLC. Each sample was replicated in triplicate.

2.4 Cellular uptake and binding abilities in HCC cells

To assess the cellular uptake and binding efficacy of HA-Lip in HCC cells, a fluorescent dye called Coumarin 6 (Cou 6) was incorporated within the liposomes. For confocal microscopy analysis, Huh7 and HepG2 cells were seeded onto cell slides at the density of 1 × 10 5 cells/well. After incubated for 24 h at 37°C, the cells were washed with PBS and 2.0 mL of serum-free DMEM with free Cou 6, Lip-Cou 6 and HA-Lip-Cou 6 (final Cou 6 concentration at 100 ng/mL) were added to each well, free HA was used to block the interaction between HA-Lip-ICT and CD44 Following 1 h of incubation at 37°C, the cells were washed with PBS, fixed using 4% paraformaldehyde, and treated with DAPI for cell nucleus staining. Fluorescence images were taken using an LSM710 laser confocal microscope (Zeiss, Germany).

For the assessment of cellular uptake via flow cytometry, Huh7 and HepG2 cells were seeded at a density of 2 × 10 5 cells/well in 6-well culture plates and incubated overnight at 37°C. The cells were then given serum-free medium containing 100 ng/mL of free Cou6, Lip-Cou6, and HA-Lip-Cou6 at a final concentration, followed by 1 h of incubation at 37°C. Afterwards, the cells were washed 3 times with PBS, detached using trypsinization, and collected in 0.5 mL of PBS. The mean fluorescence intensity of Cou 6 was measured using a FACScan flow cytometer (BD FACSCalibur, United States).

2.5 In vitro cytotoxicity analysis

The potential cytotoxic effects of free ICT, empty Lip, Lip-ICT and HA-Lip-ICT on Huh7 cells, HepG2 cells and L02 cells were assessed using the MTT colorimetric assay. Initially, Huh7, HepG2 and L02 cells (1.0 × 10 4 cells/well) were seeded into 96-well plates and cultured at 37°C with 5% CO 2 overnight. Subsequently, the cells were exposed to various concentrations (ranging from 0 to 50 μM) of free ICT, Lip-ICT and HA-Lip-ICT, followed by an additional 48 h of incubation. Empty Lip or 0.25% DMSO (vehicle control, VC) was used as control. After removing the medium (100 μL), a solution of MTT dye (15 μL) was added and incubated in the dark for 4 h. The supernatant was abandoned and DMSO (100 µL) was added to dissolve the formazan crystals. Optical density measurements were conducted using a microplate reader at 490 nm. Cell viability was calculated and the values for the 50% and 20% inhibitory concentrations (IC 50 and IC 20 ) were determined. The chemotherapeutic drug 5-fluorouracil (5-Fu) was used as a positive control in this experiment.

2.6 Apoptosis test

The assessment of apoptosis for free ICT, Lip-ICT and HA-Lip-ICT in Huh7 and HepG2 cells was carried out using the FITC-Annexin V/propidium iodide (PI) method. In brief, Huh7 and HepG2 cells were seeded into 6-well plates at a density of 5.0 × 10 4 cells/well and then cultured at 37°C with 5% CO 2 . After incubated overnight, the cells were treated with free ICT, Lip-ICT and HA-Lip-ICT at IC 50 values of free ICT for another 48 h of incubation with saline as control. Subsequently, the cells were harvested and stained with Annexin V-FITC and PI for 15 min. The analysis of cell apoptosis was conducted using flow cytometry.

2.7 Cell cycle analysis

Huh7 and HepG2 cells were cultured in 6-well plates at a density of 5.0 × 10 4 cells/well and maintained at 37°C with 5% CO 2 . Following overnight incubation, non-cytotoxic doses of free ICT, Lip-ICT and HA-Lip-ICT corresponding to the IC 20 value of free ICT were added to the Huh7 and HepG2 cells with saline as control. The cells were then further incubated for 48 h. Subsequently, the cells were collected, rinsed with PBS, and fixed using 70% ethanol. The fixed cells were stored at 4°C overnight. Then, Rnase A was introduced and incubated for 45 min at 37°C, followed by PI staining in the dark for 30 min. The distribution of cell cycle phases was analyzed using flow cytometry with total of 50000 events.

2.8 Acute toxicity analysis of HA-Lip-ICT in mice

To assess potential in vivo toxicity and provide guidance for evaluating anti-tumor effectiveness, an acute toxicity study of HA-Lip-ICT was conducted in normal Kunming mice by a single intravenous injection of the tested formulations, with the treated mice being observed for 7 days. After an adaptation period, the animals were randomly divided into six groups (n = 5), which included 5-Fu (125 mg/kg), empty Lip, ICT (5 mg/kg), Lip-ICT (5 mg/kg) and HA-Lip-ICT (5 mg/kg), while saline was used as the negative control and 5-Fu served as the positive control. All mice were injected 0.2 mL of various formulations and their body weights were measured every day. The levels of creatine kinase (CK), lactate dehydrogenase (LDH), alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CREA) and blood urea nitrogen (BUN) were assessed through automated analysis using a biochemistry analyzer (ADVIA 2400, Siemens, Munich, Germany) in the Affiliated Hospital of Southwest Medical University (Luzhou, China). On the 7th day following treatment, the mice were sacrificed. The heart, liver, spleen, lungs, and kidneys were promptly removed, rinsed with saline and then fixed in 10% formalin and stained for hematoxylin and eosin (H&E) pathological analysis.

2.9 Distribution of HA-Lip-ICT in Huh7 tumor-bearing mice

A Huh7 tumor-bearing nude mice model was induced by subcutaneously injecting 0.1 mL of Huh7 cell suspension (2.5 × 10 6 cells) into the right flank of female BALB/c nude mice aged 6–8 weeks. Once the tumors reached a mean size of 100 mm 3 , mice were separated into two groups (n = 5) and were administered DiR-encapsulated liposomes (Lip-DiR) and HA-modified DiR-loaded liposomes (HA-Lip-DiR) via tail vein injection at a dose of 500 μg/kg DiR. Fluorescence imaging was conducted at 3, 8, and 12 h post-injection using an IVIS imaging system (Kodak, PerkinElmer, Waltham, MA, United States). Subsequently, the mice were sacrificed, and the major organs and tumors were removed for ex vivo fluorescence imaging.

2.10 In vivo antitumor efficacy

Huh7 tumor-bearing nude mice were established following the aforementioned procedure ( Zhuo et al., 2015 ). Once the tumor size reached 100 mm 3 , the mice were divided randomly into 5 groups (5 mice per group), each containing five mice. Subsequently, intravenous administrations of saline and different ICT (5 mg/kg) formulations were administered every 2 days for a total of 7 doses. Tumor volume measurements were taken at 2-day intervals by measuring both the longest axis (L) and the shortest axis (W) of the tumor using a vernier micrometer. On day 14, all the animals were euthanized, and the tumors were retrieved, and their weights were recorded. The anti-tumor activity was assessed through tumor growth inhibition (TGI) by measuring the mean tumor weight (MTW) of the treated groups (TG) compared to the saline group (CG). Both tumor volume and TGI were calculated with the below formula:

The tumor tissues were then subjected to a TUNEL assay to evaluate tumor cell apoptosis, Ki-67 staining for tumor cell proliferation assessment, and immunohistochemistry AFP staining to measure HCC treatment efficacy (H. Li et al., 2021 ).

2.11 Statistical analysis

All results were performed in triplicate independent experiments and data were represented as mean ± SD. A student’s t-test or one-way analysis of variance (ANOVA) were applied to test for significance in the experiments. The statistical differences were considered significant at p < 0.05 and very significant at p < 0.001.

3.1 Preparation and characterization of HA-Lip-ICT

The synthesis of hyaluronic acid-modified cholesterol (HA-Chol) was confirmed using NMR ( Supplementary Figures S2–4 ) The particle size, PDI, zeta potential, encapsulation efficiency and drug loading content were evaluated. The obtained results indicated that the particle size of all formulations demonstrated within the range of approximately 160–200 nm, with PDI values less than 0.3 ( Figures 1A,B ). The zeta potential of HA-Lip-ICT was observed to decrease to −24.8 ± 0.36 mV ( Figure 1B ), which is the most negatively charged. It can be seen from the FTIR spectra ( Supplementary Figure S5 ) that HA-Lip, ICT and HA-Lip-ICT exhibited wide bands around 3268.81, 3407.32, and 3420.661 cm −1 , respectively, which have been confirmed from the O-H bonds stretching ( Pawlikowska-Pawlęga et al., 2013 ). In ICT, the C=O and C–O bonds vibrations led to the absorbance of 1638.73 and 1399.34 cm −1 , respectively. When ICT was incorporated within liposomes, those peaks shifted toward smaller wave numbers (1628.36 and 1396.15 cm −1 , respectively). The encapsulation efficiencies for HA-Lip-ICT and Lip-ICT were 80% and 76%, respectively. The drug loading contents were 3.9% and 2.3%, respectively ( Figure 1C ). Morphological examination through TEM revealed that both HA-Lip-ICT and Lip-ICT exhibited spherical shapes with a uniform size distribution, while no aggregation occurred ( Figure 1D ). Cumulative release of ICT from Lip-ICT and HA-Lip-ICT were investigated using a dialysis method at 37°C and the resulting release curves were depicted in Figure 1E . Comparing with free ICT, both Lip-ICT and HA-Lip-ICT displayed a 2-phase release pattern with a relatively fast initial release then a slow sustained release phase. Within the first 12 h, approximately 36.6% of ICT was released from Lip-ICT, while this proportion was approximately 29.8% for HA-Lip-ICT under pH 7.4 conditions. After 24 h, the accumulated release quantities of ICT from HA-Lip-ICT and Lip-ICT were 45.8% and 63.6% under pH 5.5 conditions, respectively.

Figure 1 . Characterization of empty Lip, Lip-ICT and HA-Lip-ICT, including (A) distribution of the hydrodynamic size detected by DLS, (B) zeta potential and PDI distribution, (C) encapsulation efficiency and drug loading content, (D) TEM micrographs, and (E) accumulative release profile of free ICT in vitro and ICT from Lip-ICT and HA-Lip-ICT in pH 7.4 and pH 5.5 PBS at 37°C during 72 h. Data are shown as mean ± SD (n = 3). ** p < 0.01.

The storage stability of HA-Lip-ICT was assessed at 4°C ( Supplementary Table S1 ) and 25°C ( Supplementary Table S2 ), respectively. The particle size and PDI of HA-Lip-ICT did not change significantly after 10 days at 4°C, and almost no drug was released at that temperature. In contrast, the particle size and PDI increased significantly and the EE declined after storage at 25°C for 5 days. These results suggest that HA-Lip-ICT are stable only at low temperatures and for a limited time.

3.2 Cellular uptake and binding abilities in HCC cells

As depicted in Figure 2 , uptake rate in HA-Lip-Cou 6 group exhibited an approximately two-fold increase compared with the Lip-Cou 6 group in Huh7 cells while there were no significant changes in HepG2 cells. The uptake rate in HA + HA-Lip-Cou 6 group was decreased to approximately 63% to HA-Lip-Cou 6 group in Huh7 cells while there were no notable changes in HepG2 cells. The confocal microscopy pictures in Figures 2A,B and their quantification results in Figures 2E,F yielded similar outcomes correlated to the data from flow cytometry in Figures 2C,D . Specifically, the considerably intensified green fluorescence in the HA-Lip-Cou 6 group compared with Lip-Cou 6 group confirmed a significant enhancement in cellular uptake. After CD44 receptor blocking through pre-treatment using free HA, the intracellular Cou 6 intensity in the HA-Lip-Cou 6 group exhibited a remarkable reduction.

Figure 2 . Cellular uptake abilities of free Cou 6, Lip-Cou 6, HA + HA-Lip-Cou 6 and HA-Lip-Cou 6 on Huh7 cells and HepG2 cells post incubation at 37°C for 1 h. Concentration of Cou 6 was 100 ng/mL. Laser scanning confocal microscopy images of (A) Huh7 cells and (B) HepG2 cells treated with different formulations. Blue and green indicate the fluorescence of DAPI and Cou 6. Cou 6 uptake in (C) Huh7 cells and (D) HepG2 cells was determined and the semi-quantitative analysis of Cou 6 uptake based on flow cytometry was plotted. (E, F) Quantitative analysis of Cou 6 uptake based on confocal microscopy. Each bar shows the average fluorescence intensity with standard deviation (n = 3), * p < 0.05, ** p < 0.01, # p < 0.05, ## p < 0.01, ### p < 0.001 vs. control group.

3.3 In vitro cytotoxicity assay

Cell viability was evaluated using an MTT assay to assess the cytotoxicity of two different ICT liposomes and free ICT. The survival rates of Huh7 cells, HepG2 cells and L02 cells are presented in Figure 3 . The growth inhibitory effects of the ICT formulations exhibited a dose-dependent pattern. Among these, free ICT displayed the highest cytotoxicity at 48 h, with IC 50 values of 8.4 ± 0.8 μM and 28.6 ± 8.4 μM in Huh7 cells and HepG2 cells, respectively. Both Lip-ICT and HA-Lip-ICT exhibited growth inhibition on HCC cells. Specifically, in Huh7 cells, the IC 50 values were 34.2 μM and >50 μM for HA-Lip-ICT and Lip-ICT, respectively. In HepG2 cells, IC 50 values were both >50 μM, respectively. Significantly, HA-Lip-ICT demonstrated greater cytotoxicity than Lip-ICT. The drug 5-Fu at 0.19 μM, which was used as a positive control, demonstrated around 70%–80% of cytotoxicity on HCC cell lines. In addition, HA-Chol showed no cytotoxicity on Huh7, HepG2 or L02 cells ( Supplementary Figure S6 ), which correlated with the previous study ( Song et al., 2019 ).

Figure 3 . Cell viability of Huh7, HepG2 and L02 cells after incubation with (A) free ICT and (B) different liposome formulations at various concentrations for 48 h ** p < 0.01, ## p < 0.01 vs. control group.

3.4 Cell apoptosis induction and cell cycle arrest study

Apoptosis induction in Huh7 and HepG2 cells by free ICT, Lip-ICT and HA-Lip-ICT was assessed using Annexin V-FITC and PI staining. The chemotherapeutic drug 5-Fu used was as a positive control. The HA-Lip-ICT group demonstrated a three-fold increase in the apoptosis ratio compared with the Lip-ICT group in Huh7 cells ( Figure 4A ), while there was no significant change between these two groups observed in HepG2 cells ( Figure 4B ).

Figure 4 . Effect of ICT in various Lip-ICT formulations on apoptosis induction of (A) Huh7 cells and (B) HepG2 cells after 48 h of treatment. The chemotherapeutic drug 5-Fu was used as positive control. Data are represented as mean ± SD, n = 3. ** p < 0.01, ### p < 0.001 vs. control group.

The apoptosis mechanism of HA-Lip-ICT involved in studying the cell cycle arrest of treated Huh7 and HepG2 cells was further investigated using various formulations. As depicted in Figure 5A , the percentage of cells in the G 0 /G 1 phase significantly increased to 61% in the HA-Lip-ICT group, compared with 51% in the Lip-ICT group ( p < 0.05) in Huh7 cells, which correlated with the previous study (S. Wang et al., 2019 ). However, there was no remarkable change observed in HepG2 cells between these two groups ( Figure 5B ).

Figure 5 . Effect of ICT in various Lip-ICT formulations on cell cycle progression of (A) Huh7 cells and (B) HepG2 cells after 48 h of treatment. The chemotherapeutic drug 5-Fu was positive control. Data are represented as mean ± SD, n = 3. * p < 0.05; # p < 0.05, ## p < 0.01 vs. control group.

3.5 HA-Lip-ICT showed no acute toxicity in vivo

The in vivo toxicity of HA-Lip-ICT was assessed in normal Kunming mice. Following treatment with various formulations, all treated mice exhibited a steady increase in body weight, except for the 5-Fu group ( Figure 6A ), wherein mice treated with 5-Fu showed a significant weight decrease of 11.9% ( p < 0.01). Furthermore, serum biochemical analyses were conducted to evaluate the marker of liver function (ALT and AST), heart function (CK and LDH) and kidney function (Creatinine and BUN) ( Figure 6B ). Apart from 5-Fu treated group, serum biomarker analysis results for other groups fell inside the healthy range, showing no remarkable differences. After treated with 5-Fu, ALT and AST levels notably increased when compared with saline treated group ( p < 0.05). As for kidney function marker (BUN), all treatment groups exhibited normal data without obvious differences when compared with saline treated group ( p > 0.05). Finally, histological examination of tissue sections through H&E staining revealed no typical necrosis, including nuclear fragmentation, shrinkage, or dissolution in normal mice, indicating there were no obvious pathological alterations in the major organs across all groups ( Figure 6C ).

Figure 6 . Biosafety of the formulations. (A) Body weights of mice after various formulations treatments (n = 5). (B) Biochemical analysis of ALT and AST, LDH, BUN, Creatinine and CK in normal mice treated with different formulations (n = 5). ALT: alanine aminotransferase; AST: aspartate aminotransferase; LDH: lactate dehydrogenase; BUN: blood urea nitrogen; CK: creatine kinase. (C) H&E-staining images of major organs post treatment. * p < 0.05, ** p < 0.01.

3.6 Biodistribution in vivo

The distribution in vivo and tumor-binding efficacy of formulations were measured using near-infrared fluorescence imaging ( Figure 7A ). As early as 3 h after the administration of HA-Lip-DiR, a clear fluorescence signal was found in the tumor tissue. This signal continued to increase, reaching its peak at 8 h, and remained visible throughout the 12-h monitoring period. Conversely, the Lip-DiR treated group exhibited just a faint fluorescence signal at the location of tumor after 3 h. Throughout the imaging duration, HA-Lip-DiR demonstrated substantially superior and more sustained accumulation in the tumor tissue compared with Lip-DiR. The ex vivo fluorescence images of removed tumors ( Figure 7B ) corroborated the findings from the in vivo imaging. Quantitative analysis of fluorescence intensity revealed that the tumor fluorescence intensity of HA-Lip-DiR was 1.9-fold greater than that of Lip-DiR ( Figure 7C ).

Figure 7 . In vivo targeting of DiR-loaded Lip and HA-Lip liposomes to nude mice bearing Huh7 tumors by NIRF imaging. (A) In vivo fluorescence images of mice at different time points after treatment, (B) Images of dissected tumors and organs taken ex vivo 12 h after injection. (C) Semi-quantitative analysis of the ex vitro fluorescence intensity. Data are shown as mean ± SD (n = 6). *** p < 0.001.

3.7 Efficacy against tumor in vivo

The anti-tumor effectiveness is showed in Figure 8 , wherein the different ICT formulations demonstrated a notable degree of tumor inhibition compared with the fast tumor growth observed in the saline-treated group. In comparison to the saline group, the free ICT, Lip-ICT, and HA-Lip-ICT groups exhibited tumor growth inhibition rates of 32.5%, 40.4%, and 63.4%, respectively. Notably, HA-Lip-ICT exhibited superior antitumor efficacy compared with all other ICT treatments. The HA-Lip-ICT group exhibited significantly smaller average tumor sizes and increased tumor inhibition rates ( Figures 8A–C ), further confirming its enhanced antitumor efficacy.

Figure 8 . Anti-tumor effect of HA-Lip-ICT on Huh7 xenograft model. (A) The tumor volume and (B) tumor-growth inhibition rate were measured and (C) the representative images of tumor tissue from each group were presented. * p < 0.05, ** p < 0.01, # p < 0.05, ## p < 0.01, ### p < 0.001 vs. control group (n = 5). (D) AFP, Ki-67 and TUNEL images of tumor tissues and their quantification results (E–G) . The brown colors in the images indicate AFP-positive, TUNEL-positive or Ki-67-positive cells.

To assess the antitumor efficacy at the cellular level, the results were showed in ( Figure 8D ) and their quantification results ( Figures 8E–G ). Local tumor cell apoptosis was evaluated using the TUNEL staining assay. The HA-Lip-ICT group showed a higher dUTP-positive rate when compared with other groups, consistent with the tumor volume inhibition results. Ki-67, a marker of cell proliferation, was significantly downregulated in mice treated with 5-Fu and HA-Lip-ICT, indicating that HA-Lip-ICT successfully suppressed the growth of cancer cells in vivo , similar to 5-Fu. Moreover, the AFP-positive area, which reflects oncofetal protein expression, was notably reduced in animals treated with 5-Fu and HA-Lip-ICT. This result suggests a greater curative effect on HCC, which was observed in Huh7 xenografts following treatment with 5-Fu and HA-Lip-ICT.

4 Discussion

Icaritin exhibits a multifaceted mechanism of action against liver cancer, including inducing apoptosis through a caspase-dependent pathway, inhibiting cell proliferation by blocking the cell cycle and inducing cell cycle arrest, modulating various signaling pathways such as PI3K/AKT, MAPKs, and JAK/STATs, inhibiting tumor angiogenesis by suppressing VEGF and other angiogenic factors, enhancing the immune response by increasing cytokine production and activating immune cells, inhibiting the growth and self-renewal of cancer stem cells, and modulating the tumor microenvironment by suppressing pro-inflammatory cytokines and promoting immune cell infiltration ( Yang et al., 2019 ; Yin et al., 2020 ; Zhang et al., 2020 ; Lu et al., 2022 ; Reyes-Hernández et al., 2024 ). Despite its potential, the clinical application of icaritin for cancer treatment is currently limited by its low water solubility and restricted bioavailability.

Hyaluronic acid-modified liposomes have shown promise in targeted drug delivery for cancer. These liposomes can be designed to target CD44-overexpressing cancer cells, which are prevalent in lung and colorectal cancers. Studies have shown their effectiveness in delivering ursolic acid to lung cancer cells, co-delivering doxorubicin and paclitaxel, and carrying 5-fluorouracil to colorectal cancer cells. This versatility highlights the potential of this technology for various cancer drug delivery applications ( Song et al., 2019 ; Mansoori et al., 2020 ; Ma et al., 2023 ).

To boost the anti-tumor efficacy of icaritin (ICT) on liver cancer, hyaluronic acid (HA)-modified liposomes (Lip) were utilized. The tumor-specific effect of ICT can be achieved by active targeting via the receptor-ligand interaction between HA and CD44 plus passive targeting by accumulation around tumors due to the Enhanced Permeability and Retention (EPR) effect. The prepared HA-Lip-ICT displayed a spherical shape with a narrow size distribution fell in the range of 160–200 nm. The slightly increased particle size compared with non-modified liposomes suggested successful HA coverage on the liposome surface. Earlier investigations have indicated that nanoparticles with smaller sizes (<200 nm), less than the pore size of leaky vasculatures and hindered lymphatic drainage, are conducive to the accumulation and localization of nanoparticles at solid tumor sites through the enhanced permeability and retention (EPR) effect ( Yang et al., 2007 ; Duan et al., 2013 ; Ravar et al., 2016 ; Lei et al., 2019 ). Smaller nanoparticles (around 100 nm), especially those with specific structures like PEGylated liposomes, exhibited enhanced resistance to reticuloendothelial system (RES) phagocytosis due to the dense PEG layer on their surfaces ( Barua and Mitragotri, 2014 ). As the presence of carboxyl groups from HA molecules on the surface of HA-modified liposomes, the zeta potential of HA-Lip-ICT was observed to decrease to −24.8 ± 0.36 mV, the reversal in potential of HA-Lip-ICT induces repulsive forces among liposomes, promoting a stable system and preventing aggregation or deposition on vessel walls. In contrast to non-modified liposomes, HA-modified liposomes demonstrated potential in evading RES phagocytosis, thus extending circulation time in the bloodstream ( Song et al., 2019 ). So, hydrophilic HA holds promise as an alternative to PEG molecules in achieving prolonged circulation in liposomes. The FTIR result indicated that C=O and C–O groups of ICT participated in hydrogen bonds formation with soybean lecithin of liposomes due to their peaks shifted toward smaller wave numbers, which confirmed the successfully incorporated of ICT ( Zhou et al., 2014 ).

The release outcomes in normal physiological pH of the bloodstream at pH 7.4 can be attributed to the presence of an HA layer on the liposome outer layer, which contributed to the delayed release of ICT from the liposomes. Upon wrapping the liposomes with HA, the HA molecules rapidly adsorb water in an aqueous environment, causing them to expand and create a dense hydration membrane on the shell of liposomes. This process reduces the phospholipid bilayer’s fluidity and permeability ( Ossipov, 2010 ; Tiantian et al., 2014 ). The compact, water-attracting HA structure encasing liposomes act like a barrier, limiting dispersion of ICT from the liposomes into the media. The gradual release profile exhibited by HA-Lip-ICT demonstrates its exceptional stability, preventing fast ICT release in the bloodstream and promoting the accumulation of liposomes at tumor sites. While the acidic environment of the tumor at pH 5.5, the observation could be attributed to the instability of the liposome structure. HA is susceptible to hydrolysis in an acidic environment, causing the polymer chains to undergo random scission, ultimately degrading the integrity of the liposome structure ( Kim et al., 2012 ; Nascimento et al., 2016 ). Another possible explanation is that the hydrolysis of HA exposes the lipid bilayer, while the cholesterol segment within the HA-Chol conjugate remains embedded in the bilayers. Excessive cholesterol content can undermine bilayer stability. Consequently, the loss of the protective hydrophilic layer enhances the phospholipid bilayer’s fluidity and permeability, facilitating the release of the drug from the liposomes ( Song et al., 2019 ).

As for the cellular uptake results, given that HA-Lip-Cou 6 and Lip-Cou 6 shared similar constituents, except for the presence of HA, the distinction in uptake could be directly attributed to HA. The primary uptake route of HA-Lip-Cou 6 involves CD44 facilitated endocytosis. Previous study also postulated that augmentation in cellular uptake could be attributed to the presence of CD44 receptors on the cell surface ( Misra et al., 2015 ). So, the superior uptake level of HA-Lip-Cou 6 may come from the higher expression level of CD44 in Huh7 cells than in HepG2 cells ( Supplementary Figure S7 ) ( Cannito et al., 2022 ). Previous research has suggested that the existence of rivaling HA within the growth medium reduces the cellular uptake of HA-conjugated nanoparticles. In order to support this idea, a receptor competition experiment was created ( Tran et al., 2014 ). The heightened cell uptake rate induced by HA coating was notably decreased when there is free HA, suggesting a competitive interaction between free HA and HA-Lip-Cou 6 for the same CD44 receptor. This outcome indicated the pivotal role of the HA-CD44 interaction in facilitating enhanced cellular uptake. These findings suggested heightened uptake by cells observed with HA-Lip-Cou 6 is facilitated by the HA-CD44 binding between the liposomes and Huh7 cells. However, the main findings are based solely on one CD44 high HCC cell line (Huh7), and a second CD44-high HCC cell line or a CD44 knockout line of Huh7 cells and a CD44-overexpressing line of HepG2 maybe needed to be used to provide more robust evidence for the efficacy of HA-Lip-ICT.

In vitro cytotoxicity results, the IC 50 level of ICT in Huh7 cells is much lower than that in HepG2 cells, this different effect might be attributed to its rapid cellular uptake through passive diffusion and an optimum concentration in Huh7 cells according to previous studies ( Wang et al., 2020a ; Liu et al., 2021 ). While the enhanced cytotoxicity of HA-Lip-ICT could be attributed to the increasing cellular uptake of ICT facilitated by HA-CD44-mediated endocytosis in Huh7 cells.

The apoptosis results suggested that HA-Lip-ICT enhanced the apoptosis-inducing capacity of ICT, particularly by targeting CD44 overexpressed cells. Additionally, the apoptosis ratio of the HA-Lip-ICT group was lower than that of the free ICT group. This could be attributed to the rapid cellular uptake of free ICT via passive diffusion, which might not involve sustained release mechanisms ( Wang et al., 2020a ; Liu et al., 2021 ). Furthermore, the elevated G 0 /G 1 phase in the HA-Lip-ICT group could potentially be attributed to the promotion of internalization through HA-CD44 interactions. The potential mechanisms of HA-Lip-ICT may be the generation of reactive oxygen species (ROS), which damage cellular components and disrupt cellular processes, triggering apoptosis and cell cycle arrest ( Ma et al., 2023 ). Additionally, upregulation of p53 and apoptosis-related proteins in the transforming growth factor-β signaling (ARTS) pathway, leading to cytochrome-c release, caspase-3 activation, and mitochondrial apoptosis ( Ma et al., 2023 ). Furthermore, causing cell cycle arrest at the G 0 /G 1 phase, inhibiting cell proliferation and promoting apoptosis ( Mansoori et al., 2020 ).

The in vivo biosafety findings suggest that both ICT and liposomes did not cause significant systemic toxicity in healthy mice. These findings suggested that the treatments did not lead to noticeable hepatic, cardiac or renal disorders. And the H&E result further confirms the favorable biocompatibility and biosafety of ICT or various liposomes in this study.

These in vivo biodistribution outcomes highlight the significant enhancement in liposome delivery to tumors achieved through HA modification. Hydrophilic HA coating on HA-Lip-DiR contributed to enhanced serum stability, preventing fast non-specific clearance by the reticuloendothelial system (RES) organs, such as spleen and liver. So, it could extend the circulation time in blood ( Liang et al., 2015 ; M; Zhang et al., 2021 ). Moreover, HA functionalization facilitated targeted binding and internalization at the tumor site through receptor-ligand interacted endocytosis for HA-Lip-DiR. This targeted binding allowed for extensive penetration into the tumor tissue, surpassing the effects of enhanced permeability and retention (EPR) alone.

The best in vivo anti-tumor effect of HA-Lip-ICT can be attributed to both the passive targeting mediated by the EPR effect and the active targeting facilitated by the interaction between HA and CD44, as well as the accelerated release of ICT caused by the faint acidic tumor microenvironment ( Song et al., 2019 ; Wang et al., 2020b ). Furthermore, this study demonstrated that even at a relatively low dose (5 mg/kg), the therapeutic effectiveness of ICT in vivo for liver cancer was prominent after encapsulation into HA-coated liposomes. The exceptional anti-tumor effect can be credited to the strong tumor-targeting effectiveness of HA-Lip-ICT, achieved through a combination of passive and active targeted delivery methods. The presence of HA on the surface of liposome improved its selective uptake via HA/CD44 receptor-mediated endocytosis. Therefore, in conjunction with previous research, the potential of ICT highlighted in this study, particularly when delivered through its HA-modified liposomal nanocarrier, indicates a promising approach for liver cancer treatment due to its potent antitumor effects.

5 Conclusion

A novel approach was introduced using a nanoplatform of liposomes modified with hyaluronic acid (HA) to enhance the uptake of the antitumor agent ICT for liver cancer. These HA-modified liposomes, featuring a negatively charged surface, displayed high efficiency in encapsulating ICT and had a compact particle size. In vitro , it was observed that HA-Lip-ICT significantly improved intracellular uptake, increased cytotoxicity, induced apoptosis, and caused cell cycle arrest when compared to Lip-ICT. This improvement can be attributed to the involvement of HA in facilitating CD44 receptor-mediated endocytosis. Functionalizing with HA greatly enhanced the liposomes’ ability to target tumors. In vivo , HA-Lip-ICT demonstrated superior antitumor effectiveness with minimal systemic side effects than free ICT and Lip-ICT due to the faint acidic tumor microenvironment. In summary, HA-Lip-ICT holds promising potential as an effective strategy for targeted drug delivery, enhancing the treatment of liver cancer. Additionally, the anti-tumor mechanism of HA-Lip-ICT requires further investigation.

Data availability statement

The original contributions presented in the study are included in the article/ Supplementary Material , further inquiries can be directed to the corresponding authors.

Ethics statement

Ethical approval was not required for the studies on humans in accordance with the local legislation and institutional requirements because only commercially available established cell lines were used. The animal study was approved by the Animal Ethics Committee of Southwest Medical University. The study was conducted in accordance with the local legislation and institutional requirements.

Author contributions

XS: Data curation, Formal Analysis, Investigation, Methodology, Visualization, Writing–original draft. ZH: Data curation, Formal Analysis, Investigation, Methodology, Visualization, Writing–original draft. RL: Data curation, Formal Analysis, Investigation, Methodology, Visualization, Writing–original draft. ZL: Conceptualization, Funding acquisition, Methodology, Project administration, Resources, Supervision, Writing–review and editing. SC: Supervision, Visualization, Writing–review and editing. SA: Supervision, Visualization, Writing–review and editing. JJ: Conceptualization, Funding acquisition, Methodology, Project administration, Resources, Supervision, Writing–review and editing. ST: Conceptualization, Methodology, Supervision, Visualization, Writing–review and editing. ZZ: Conceptualization, Funding acquisition, Methodology, Project administration, Resources, Supervision, Writing–review and editing.

The author(s) declare that financial support was received for the research, authorship, and/or publication of this article. This work was supported by grants from the Sichuan Science and Technology Program (2022YFS0627), the Cooperative Scientific Research Project of Chunhui Plan of the Ministry of Education of China (202200618), the university level research fund of Southwest Medical University (2021ZKQN086), Central Nervous System Drug Key Laboratory of Sichuan Province (210020-01SZ), Luzhou Science and Technology Program (2023JYJ023 and 2020-JYJ-47). The research was partially supported by the Center of Excellent in Pharmaceutical Nanotechnology, Chiang Mai University, Thailand.

Acknowledgments

All authors are thankful for getting help and support from the Key Laboratory of Medical Electrophysiology, Ministry of Education, School of Pharmacy, Southwest Medical University, Luzhou, Sichuan, China.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphar.2024.1437515/full#supplementary-material

Akim, A., Zafar, M. N., Safdar, N., Sung, Y. Y., and Muhammad, T. S. T. (2021). Understanding hyaluronan receptor (CD44) interaction, HA-CD44 activated potential targets in cancer therapeutics. Adv. Pharm. Bull. 11 (3), 426–438. doi:10.34172/apb.2021.050

PubMed Abstract | CrossRef Full Text | Google Scholar

Antonio, C. R., and Trídico, L. A. (2021). The importance of interaction between hyaluronic acid and CD44 receptor. Surg. Cosmet. dermatology 13. doi:10.5935/scd1984-8773.2021130006

CrossRef Full Text | Google Scholar

Asai, R., Tsuchiya, H., Amisaki, M., Makimoto, K., Takenaga, A., Sakabe, T., et al. (2019). CD44 standard isoform is involved in maintenance of cancer stem cells of a hepatocellular carcinoma cell line. Cancer Med. 8 (2), 773–782. doi:10.1002/cam4.1968

Bailly, C. (2020). Molecular and cellular basis of the anticancer activity of the prenylated flavonoid icaritin in hepatocellular carcinoma. Chemico-Biological Interact. 325, 109124. doi:10.1016/j.cbi.2020.109124

Barua, S., and Mitragotri, S. (2014). Challenges associated with penetration of nanoparticles across cell and tissue barriers: a review of current status and future prospects. Nano today 9 (2), 223–243. doi:10.1016/j.nantod.2014.04.008

Cannito, S., Bincoletto, V., Turato, C., Pontisso, P., Scupoli, M. T., Ailuno, G., et al. (2022). Hyaluronated and PEGylated liposomes as a potential drug-delivery strategy to specifically target liver cancer and inflammatory cells. Molecules 27 (3), 1062. doi:10.3390/molecules27031062

Chang, Q., Wang, G. N., Li, Y., Zhang, L., You, C., and Zheng, Y. (2012). Oral absorption and excretion of icaritin, an aglycone and also active metabolite of prenylflavonoids from the Chinese medicine Herba Epimedii in rats. Phytomedicine 19 (11), 1024–1028. doi:10.1016/j.phymed.2012.05.017

Chen, W., Zheng, R., Baade, P. D., Zhang, S., Zeng, H., Bray, F., et al. (2016). Cancer statistics in China, 2015. CA A Cancer J. Clin. 66 (2), 115–132. doi:10.3322/caac.21338

Cheng, L., Huang, F. Z., Cheng, L. F., Zhu, Y. Q., Hu, Q., Li, L., et al. (2014). GE11-modified liposomes for non-small cell lung cancer targeting: preparation, ex vitro and in vivo evaluation. Int. J. Nanomedicine 9, 921–935. doi:10.2147/ijn.S53310

Cheng, R., Liu, L., Xiang, Y., Lu, Y., Deng, L., Zhang, H., et al. (2020). Advanced liposome-loaded scaffolds for therapeutic and tissue engineering applications. Biomaterials 232, 119706. doi:10.1016/j.biomaterials.2019.119706

Cragg, G. M., and Newman, D. J. (2005). Plants as a source of anti-cancer agents. J. Ethnopharmacol. 100 (1-2), 72–79. doi:10.1016/j.jep.2005.05.011

Duan, X., Xiao, J., Yin, Q., Zhang, Z., Yu, H., Mao, S., et al. (2013). Smart pH-sensitive and temporal-controlled polymeric micelles for effective combination therapy of doxorubicin and disulfiram. ACS Nano 7 (7), 5858–5869. doi:10.1021/nn4010796

El-Serag, H. B., and Rudolph, K. L. (2007). Hepatocellular carcinoma: epidemiology and molecular carcinogenesis. Gastroenterology 132 (7), 2557–2576. doi:10.1053/j.gastro.2007.04.061

Endo, K., and Terada, T. (2000). Protein expression of CD44 (standard and variant isoforms) in hepatocellular carcinoma: relationships with tumor grade, clinicopathologic parameters, p53 expression, and patient survival. J. Hepatology 32 (1), 78–84. doi:10.1016/s0168-8278(00)80192-0

Hao, H., Zhang, Q., Zhu, H., Wen, Y., Qiu, D., Xiong, J., et al. (2019). Icaritin promotes tumor T-cell infiltration and induces antitumor immunity in mice. Eur. J. Immunol. 49 (12), 2235–2244. doi:10.1002/eji.201948225

Kesharwani, P., Chadar, R., Sheikh, A., Rizg, W. Y., and Safhi, A. Y. (2021). CD44-Targeted nanocarrier for cancer therapy. Front. Pharmacol. 12, 800481. doi:10.3389/fphar.2021.800481

Kim, E., Yang, J., Park, J., Kim, S., Kim, N. H., Yook, J. I., et al. (2012). Consecutive targetable smart nanoprobe for molecular recognition of cytoplasmic microRNA in metastatic breast cancer. ACS Nano 6 (10), 8525–8535. doi:10.1021/nn300289u

Lee, M. K. (2020). Liposomes for enhanced bioavailability of water-insoluble drugs: in vivo evidence and recent approaches. Pharmaceutics 12 (3), 264. doi:10.3390/pharmaceutics12030264

Lei, M., Ma, G., Sha, S., Wang, X., Feng, H., Zhu, Y., et al. (2019). Dual-functionalized liposome by co-delivery of paclitaxel with sorafenib for synergistic antitumor efficacy and reversion of multidrug resistance. Drug Deliv. 26 (1), 262–272. doi:10.1080/10717544.2019.1580797

Li, H., Liu, Y., Jiang, W., Xue, J., Cheng, Y., Wang, J., et al. (2021). Icaritin promotes apoptosis and inhibits proliferation by down-regulating AFP gene expression in hepatocellular carcinoma. BMC Cancer 21 (1), 318. doi:10.1186/s12885-021-08043-9

Li, Y., Sun, S., Chang, Q., Zhang, L., Wang, G., Chen, W., et al. (2013). A strategy for the improvement of the bioavailability and antiosteoporosis activity of BCS IV flavonoid glycosides through the formulation of their lipophilic aglycone into nanocrystals. Mol. Pharm. 10 (7), 2534–2542. doi:10.1021/mp300688t

Liang, D. S., Su, H. T., Liu, Y. J., Wang, A. T., and Qi, X. R. (2015). Tumor-specific penetrating peptides-functionalized hyaluronic acid-d-α-tocopheryl succinate based nanoparticles for multi-task delivery to invasive cancers. Biomaterials 71, 11–23. doi:10.1016/j.biomaterials.2015.08.035

Liu, D., Zhang, Q., Wang, J., Guan, S., Cai, D., and Liu, J. (2021). Inhibition of growth and metastasis of breast cancer by targeted delivery of 17-hydroxy-jolkinolide B via hyaluronic acid-coated liposomes. Carbohydr. Polym. 257, 117572. doi:10.1016/j.carbpol.2020.117572

Liu, L., Zhao, Z., Lu, L., Liu, J., Sun, J., and Dong, J. (2019). Icariin and icaritin ameliorated hippocampus neuroinflammation via mediating HMGB1 expression in social defeat model in mice. Int. Immunopharmacol. 75, 105799. doi:10.1016/j.intimp.2019.105799

Lu, P. H., Chen, M. B., Liu, Y. Y., Wu, M. H., Li, W. T., Wei, M. X., et al. (2017). Identification of sphingosine kinase 1 (SphK1) as a primary target of icaritin in hepatocellular carcinoma cells. Oncotarget 8 (14), 22800–22810. doi:10.18632/oncotarget.15205

Lu, Y., Gao, Y., Yang, H., Hu, Y., and Li, X. (2022). Nanomedicine-boosting icaritin-based immunotherapy of advanced hepatocellular carcinoma. Mil. Med. Res. 9 (1), 69. doi:10.1186/s40779-022-00433-9

Ma, T., Zhou, J., Li, J., and Chen, Q. (2023). Hyaluronic acid-modified liposomes for ursolic acid-targeted delivery treat lung cancer based on p53/ARTS-mediated mitochondrial apoptosis. Iran. J. Pharm. Res. IJPR 22 (1), e131758. doi:10.5812/ijpr-131758

Majumder, J., Taratula, O., and Minko, T. (2019). Nanocarrier-based systems for targeted and site specific therapeutic delivery. Adv. Drug Deliv. Rev. 144, 57–77. doi:10.1016/j.addr.2019.07.010

Mansoori, B., Mohammadi, A., Abedi-Gaballu, F., Abbaspour, S., Ghasabi, M., Yekta, R., et al. (2020). Hyaluronic acid-decorated liposomal nanoparticles for targeted delivery of 5-fluorouracil into HT-29 colorectal cancer cells. J. Cell. Physiology 235 (10), 6817–6830. doi:10.1002/jcp.29576

Misra, S., Hascall, V. C., Markwald, R. R., and Ghatak, S. (2015). Interactions between hyaluronan and its receptors (CD44, RHAMM) regulate the activities of inflammation and cancer. Front. Immunol. 6, 201. doi:10.3389/fimmu.2015.00201

Mitchell, M. J., Billingsley, M. M., Haley, R. M., Wechsler, M. E., Peppas, N. A., and Langer, R. (2021). Engineering precision nanoparticles for drug delivery. Nat. Rev. Drug Discov. 20 (2), 101–124. doi:10.1038/s41573-020-0090-8

Nascimento, T. L., Hillaireau, H., Vergnaud, J., and Fattal, E. (2016). Lipid-based nanosystems for CD44 targeting in cancer treatment: recent significant advances, ongoing challenges and unmet needs. Nanomedicine (Lond) 11 (14), 1865–1887. doi:10.2217/nnm-2016-5000

Newman, D. J. (2008). Natural products as leads to potential drugs: an old process or the new hope for drug discovery? J. Med. Chem. 51 (9), 2589–2599. doi:10.1021/jm0704090

Ohvo-Rekilä, H., Ramstedt, B., Leppimäki, P., and Slotte, J. P. (2002). Cholesterol interactions with phospholipids in membranes. Prog. Lipid Res. 41 (1), 66–97. doi:10.1016/s0163-7827(01)00020-0

Ossipov, D. A. (2010). Nanostructured hyaluronic acid-based materials for active delivery to cancer. Expert Opin. Drug Deliv. 7 (6), 681–703. doi:10.1517/17425241003730399

Patra, J. K., Das, G., Fraceto, L. F., Campos, E. V. R., Rodriguez-Torres, M. D. P., Acosta-Torres, L. S., et al. (2018). Nano based drug delivery systems: recent developments and future prospects. J. Nanobiotechnology 16 (1), 71. doi:10.1186/s12951-018-0392-8

Pawlikowska-Pawlęga, B., Misiak, L. E., Zarzyka, B., Paduch, R., Gawron, A., and Gruszecki, W. I. (2013). FTIR, (1)H NMR and EPR spectroscopy studies on the interaction of flavone apigenin with dipalmitoylphosphatidylcholine liposomes. Biochimica Biophysica Acta 1828 (2), 518–527. doi:10.1016/j.bbamem.2012.10.013

Ravar, F., Saadat, E., Gholami, M., Dehghankelishadi, P., Mahdavi, M., Azami, S., et al. (2016). Hyaluronic acid-coated liposomes for targeted delivery of paclitaxel, in-vitro characterization and in-vivo evaluation. J. Control. Release 229, 10–22. doi:10.1016/j.jconrel.2016.03.012

Reyes-Hernández, O. D., Figueroa-González, G., Quintas-Granados, L. I., Hernández-Parra, H., Peña-Corona, S. I., Cortés, H., et al. (2024). New insights into the anticancer therapeutic potential of icaritin and its synthetic derivatives. Drug Dev. Res. 85 (2), e22175. doi:10.1002/ddr.22175

Sabit, H., Abdel-Hakeem, M., Shoala, T., Abdel-Ghany, S., Abdel-Latif, M. M., Almulhim, J., et al. (2022). Nanocarriers: a reliable tool for the delivery of anticancer drugs. Pharmaceutics 14 (8), 1566. doi:10.3390/pharmaceutics14081566

Singh, S., Singh, P. P., Roberts, L. R., and Sanchez, W. (2014). Chemopreventive strategies in hepatocellular carcinoma. Nat. Rev. Gastroenterology Hepatology 11 (1), 45–54. doi:10.1038/nrgastro.2013.143

Song, M., Liang, Y., Li, K., Zhang, J., Zhang, N., Tian, B., et al. (2019). Hyaluronic acid modified liposomes for targeted delivery of doxorubicin and paclitaxel to CD44 overexpressing tumor cells with improved dual-drugs synergistic effect. J. Drug Deliv. Sci. Technol. 53, 101179. doi:10.1016/j.jddst.2019.101179

Su, S., and Peter, M. K. (2020). Recent advances in nanocarrier-assisted therapeutics delivery systems. Pharmaceutics 12 (9), 837. doi:10.3390/pharmaceutics12090837

Sze, S. C., Tong, Y., Ng, T. B., Cheng, C. L. Y., and Cheung, H. P. (2010). Herba Epimedii: anti-oxidative properties and its medical implications. Molecules 15 (11), 7861–7870. doi:10.3390/molecules15117861

Tiantian, Y., Wenji, Z., Mingshuang, S., Rui, Y., Shuangshuang, S., Yuling, M., et al. (2014). Study on intralymphatic-targeted hyaluronic acid-modified nanoliposome: influence of formulation factors on the lymphatic targeting. Int. J. Pharm. 471 (1-2), 245–257. doi:10.1016/j.ijpharm.2014.05.027

Tiong, C. T., Chen, C., Zhang, S. J., Li, J., Soshilov, A., Denison, M. S., et al. (2012). A novel prenylflavone restricts breast cancer cell growth through AhR-mediated destabilization of ERα protein. Carcinogenesis 33 (5), 1089–1097. doi:10.1093/carcin/bgs110

Torre, L. A., Bray, F., Siegel, R. L., Ferlay, J., Lortet-Tieulent, J., and Jemal, A. (2015). Global cancer statistics, 2012. CA A Cancer J. Clin. 65 (2), 87–108. doi:10.3322/caac.21262

Tran, T. H., Choi, J. Y., Ramasamy, T., Truong, D. H., Nguyen, C. N., et al. (2014). Hyaluronic acid-coated solid lipid nanoparticles for targeted delivery of vorinostat to CD44 overexpressing cancer cells. Carbohydr. Polym. 114, 407–415. doi:10.1016/j.carbpol.2014.08.026

Wang, J., Liu, D., Guan, S., Zhu, W., Fan, L., Zhang, Q., et al. (2020a). Hyaluronic acid-modified liposomal honokiol nanocarrier: enhance anti-metastasis and antitumor efficacy against breast cancer. Carbohydr. Polym. 235, 115981. doi:10.1016/j.carbpol.2020.115981

Wang, J., Qian, Y., Xu, L., Shao, Y., Zhang, H., Shi, F., et al. (2020b). Hyaluronic acid-shelled, peptide drug conjugate-cored nanomedicine for the treatment of hepatocellular carcinoma. Mater. Sci. Eng. C 117, 111261. doi:10.1016/j.msec.2020.111261

Wang, S., Wang, Q., Wang, H., Qin, C., Cui, X., Li, L., et al. (2019). Induction of ROS and DNA damage-dependent senescence by icaritin contributes to its antitumor activity in hepatocellular carcinoma cells. Pharm. Biol. 57 (1), 424–431. doi:10.1080/13880209.2019.1628073

Yang, T., Choi, M. K., Cui, F. D., Kim, J. S., Chung, S. J., Shim, C. K., et al. (2007). Preparation and evaluation of paclitaxel-loaded PEGylated immunoliposome. J. Control. Release 120 (3), 169–177. doi:10.1016/j.jconrel.2007.05.011

Yang, X.-J., Xi, Y.-M., and Li, Z.-J. (2019). Icaritin: a novel natural candidate for hematological malignancies therapy. BioMed Res. Int. 2019 (1), 4860268. doi:10.1155/2019/4860268

Yin, L., Qi, X. W., Liu, X. Z., Yang, Z. Y., Cai, R. L., Cui, H. J., et al. (2020). Icaritin enhances the efficacy of cetuximab against triple-negative breast cancer cells. Oncol. Lett. 19 (6), 3950–3958. doi:10.3892/ol.2020.11496

Zhang, C., Sui, X., Jiang, Y., Wang, X., and Wang, S. (2020). Antitumor effects of icaritin and the molecular mechanisms. Discov. Med. 29 (156), 5–16.

PubMed Abstract | Google Scholar

Zhang, M., Gao, S., Yang, D., Fang, Y., Lin, X., Jin, X., et al. (2021). Influencing factors and strategies of enhancing nanoparticles into tumors in vivo . Acta Pharm. Sin. B 11 (8), 2265–2285. doi:10.1016/j.apsb.2021.03.033

Zhou, W., Liu, W., Zou, L., Liu, C., Liang, R., et al. (2014). Storage stability and skin permeation of vitamin C liposomes improved by pectin coating. Colloids Surfaces B Biointerfaces 117, 330–337. doi:10.1016/j.colsurfb.2014.02.036

Zhuo, Z., Hu, J., Yang, X., Chen, M., Lei, X., Deng, L., et al. (2015). Ailanthone inhibits Huh7 cancer cell growth via cell cycle arrest and apoptosis in vitro and in vivo . Sci. Rep. 5 (1), 16185. doi:10.1038/srep16185

Keywords: hepatocellular carcinoma, liposomes, icaritin, hyaluronic acid, CD44

Citation: Sun X, He Z, Lu R, Liu Z, Chiampanichayakul S, Anuchapreeda S, Jiang J, Tima S and Zhong Z (2024) Hyaluronic acid-modified liposomes Potentiated in-vivo anti-hepatocellular carcinoma of icaritin. Front. Pharmacol. 15:1437515. doi: 10.3389/fphar.2024.1437515

Received: 23 May 2024; Accepted: 25 June 2024; Published: 11 July 2024.

Reviewed by:

Copyright © 2024 Sun, He, Lu, Liu, Chiampanichayakul, Anuchapreeda, Jiang, Tima and Zhong. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

*Correspondence: Singkome Tima, [email protected] ; Zhirong Zhong, [email protected]

† These authors have contributed equally to this work

Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.

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Liposomes: Biomedical Applications

Affiliation.

• 1 Department of Nuclear Medicine, Molecular Imaging & Therapeutic Medicine Research Center, Research Institute of Clinical Medicine of Jeonbuk National University and Biomedical Research Institute of Jeonbuk National University Hospital, Jeonju, Korea.
• PMID: 33537216
• PMCID: PMC7840352
• DOI: 10.4068/cmj.2021.57.1.27

Liposomes, with their flexible physicochemical and biophysical properties, continue to be studied as an important potential a critical drug delivery system. Liposomes have overcome the challenges of conventional free drug therapy by encapsulating therapeutic agents, thereby improving in vivo biodistribution and reducing systemic toxicity. New imaging modalities and interpretation techniques, as well as new techniques for targetable system formulation technique, and tumor environmental information, have affected the search for a means of overcoming the difficulties of conventional liposome formulation. In this review, we briefly discuss how liposomal formulation has been applied across the biomedical field, particularly as a therapy, and the role it may play in the future, when paired with new developments in diagnosis and theranostics. The biological challenges that still remain and the translational obstacles are discussed.

Keywords: Drug Delivery Systems; Liposomes; Neoplasms; Precision Medicine.

© Chonnam Medical Journal, 2021.

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Conflict of interest statement

CONFLICT OF INTEREST STATEMENT: None declared.

FIG. 1. Liposome (A) and micelle (B)…

FIG. 1. Liposome (A) and micelle (B) structure.

FIG. 2. Structure of MLV (A), LUV…

FIG. 2. Structure of MLV (A), LUV (B), and SUV (C). MLV: multilamellar vesicles, LUV:…

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Liposomes: structure, biomedical applications, and stability parameters with emphasis on cholesterol, pooria nakhaei.

1 School of Medicine, Tehran University of Medical Sciences, Tehran, Iran

Ria Margiana

2 Department of Anatomy, Faculty of Medicine, Universitas Indonesia, Depok, Indonesia

3 Cipto Mangunkusumo Hospital, The National Referral Hospital, Central Jakarta, Indonesia

4 Master’s Programme Biomedical Sciences, Faculty of Medicine, Universitas Indonesia, Depok, Indonesia

Dmitry O. Bokov

5 Institute of Pharmacy, Sechenov First Moscow State Medical University, Moscow, Russia

6 Laboratory of Food Chemistry, Federal Research Center of Nutrition, Biotechnology, and Food Safety, Moscow, Russia

Walid Kamal Abdelbasset

7 Department of Health and Rehabilitation Sciences, College of Applied Medical Sciences, Prince Sattam Bin Abdulaziz University, Al Kharj, Saudi Arabia

8 Department of Physical Therapy, Kasr Al-Aini Hospital, Cairo University, Giza, Egypt

Mohammad Amin Jadidi Kouhbanani

9 Department of Medical Nanotechnology, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Czechia

Rajender S. Varma

10 Regional Centre of Advanced Technologies and Materials, Czech Advanced Technology and Research Institute, Palacký University in Olomouc, Olomouc, Czechia

Faroogh Marofi

11 Department of Hematology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran

Mostafa Jarahian

12 Toxicology and Chemotherapy Unit (G401), German Cancer Research Center, Heidelberg, Germany

Nasrin Beheshtkhoo

Ebrahim Mostafavi , Stanford University, United States

Liposomes are essentially a subtype of nanoparticles comprising a hydrophobic tail and a hydrophilic head constituting a phospholipid membrane. The spherical or multilayered spherical structures of liposomes are highly rich in lipid contents with numerous criteria for their classification, including structural features, structural parameters, and size, synthesis methods, preparation, and drug loading. Despite various liposomal applications, such as drug, vaccine/gene delivery, biosensors fabrication, diagnosis, and food products applications, their use encounters many limitations due to physico-chemical instability as their stability is vigorously affected by the constituting ingredients wherein cholesterol performs a vital role in the stability of the liposomal membrane. It has well established that cholesterol exerts its impact by controlling fluidity, permeability, membrane strength, elasticity and stiffness, transition temperature (Tm), drug retention, phospholipid packing, and plasma stability. Although the undetermined optimum amount of cholesterol for preparing a stable and controlled release vehicle has been the downside, but researchers are still focused on cholesterol as a promising material for the stability of liposomes necessitating explanation for the stability promotion of liposomes. Herein, the prior art pertaining to the liposomal appliances, especially for drug delivery in cancer therapy, and their stability emphasizing the roles of cholesterol.

Introduction

Increasing advances in nanotechnology and nanoscience have raised great hopes in the field of biomedicine. Due to their unique, multifaceted and flexible properties, nanomaterials circumvents many challenges in diverse fields of medicine, including health, diagnosis, and treatment ( Liu et al., 2020 ; Naskar and Kim, 2021 ), nanoliposomes being one of the most widely used nanoparticles in biomedicine. Liposomes are lipid bilayer spherical membranes that provide both hydrophilic and hydrophobic environments. Adjustability, flexibility, variety of ingredients, ease of functionalization, tunability of the number of layers/sizes, biocompatibility, and biodegradability have turned liposomes into incredible structures in medicine, especially drug delivery ( Aguilar-Pérez et al., 2020 ; Trucillo et al., 2020 ; Kashapov et al., 2021 ), most notable use of these structures being in cosmetics and drug delivery. Consequently, various liposome-based products have been commercialized to date, with the approval from United States Food and Drug Administration (FDA) ( Yuba, 2020 ; Barenholz, 2021 ). Liposomes, a type of lipid-based nanoparticle, have a great assortment each of which in turn offers unique properties. However, there are still barriers and challenges related to lipid nanoparticles, the most critical being their stability ( Yu et al., 2021 ). In the present review, an attempt has been made to comprehensively deliberate liposomes in terms of structure, function, and stability starting initially with an introduction to lipid structures.

Lipid Nanoparticles

Lipid molecules are one of the crucial elements of life and lipid-based nanoparticles comprise a broad range according to their application, components, shape, and fabrication methods; they possess numerous advantages over polymeric-based nanoparticles. Besides topical usage, agent delivery is ascribed to as a significant application of lipid-based nanoparticles. Being a physiological analog to the cellular membrane, liposomes have superior biocompatibility compared to polymeric-based nanoparticles, leading to a more acceptable biomedical application choices ( Müller et al., 2000 ).

As highly adaptable nanoparticles, lipidic nanoparticles can be deployed for a wide varieties of delivery with a bit of condescension. Liposomes and niosomes, as the phospholipid and amphipathic lipids constituents, respectively, are the most known lipid-based formulation. According to previous studies, liposomes could be engineered purposefully with favorable parameters. However, kinetic stability is the primary limitation of vesicular lipid-based nanoparticles, including liposomes ( Battaglia and Ugazio, 2019 ). The most applicable lipid-based nanoparticles are presented in Table 1 .

Lipid-based structures.

Note: DMPC, Dipalmitoyl phosphatidylcholine; DOPC, Dioleoyl-sn-glycero-3-phosphocholine; DOPE, Dioleoyl phosphatidylethanolamine; DOPEPC, Dioleoyl phosphatidylethanolamine phosphatidylcholine; DPPC, Dipalmitoyl phosphatidylcholine; DSPC, Distearoyl-sn-glycero-3-phosphocholin; DSPE, Distearoyl-sn-glycero-3-phosphorylethanolamine; EPC, Ethanolamine phosphatidylcholine; HSPC, Hydro Soy phosphatidylcholine; PC, phosphatidylcholine; POPC, Palmitoyl-oleoyl-sn-glycero-phosphocholine; PS, Phosphatidylserine; SDC, Sodium deoxycholate; SDS, Sodium dodecyl sulfate; SPC, Sphingosyl phosphorylcholine.

Liposome Structure

Liposome structure has been initially described by the British hematologist, Alec D Bangham in 1961. From the terminology point of view, liposome consists of “Lipos” and “Soma,” attributed to fat and body, respectively. The cell membrane’s bilayer lipid structure has been determined using electron microscope images, proving their unmistakable resemblance to plasmalemma ( Dua et al., 2012 ; Hashemzadeh et al., 2020a ). Liposomes were recruited as a drug carrier for the first time in the early 1990s. It has since been concluded that the low percentages inclusion of lipid-bonded polymers (called polymer-lipids) in liposomes’ structure could increase blood circulation in vivo . Structurally, liposomes are concentric bleeder vesicles in which a membranous lipid bilayer surrounds an aqueous volume. Typically, the bilayer lipid membrane comprise phospholipids containing a hydrophobic tail and a hydrophilic head ( Rovira-Bru et al., 2002 ).

According to the phospholipid’s properties, the final structure represents an amphiphilic feature ( Dua et al., 2012 ). Due to the unique structure, both natural and synthetic phospholipids-based liposomes are considered vesicles or drug-carrying systems. The spherical or multilayered spherical design of the fabricated liposomes is highly reliant on the amount and the kind of lipid components. According to a concentric form, the bilayer lipid formation arrangement constitutes an equal number of water chambers ( Chetoni et al., 2004 ; Choi and Maibach, 2005 ; Pavelić et al., 2005 ).

In view of the utmost similarity to the cell membrane, the liposomes have been described as an appropriate membrane model to reveal the fundamental nature of cell membranes with assorted appliances ( Wong et al., 2001 ; Laouini et al., 2012 ). The self-assembly of diacyl-chain phospholipids in aqueous solutions can form spherical bilayer structures referred to as liposomes. Because of encapsulating an extensive aqueous environment, liposomal structures can load almost any type of hydrophilic molecules ( Lebègue et al., 2015 ; Vakili-Ghartavol et al., 2020 ; Wu et al., 2021 ). Liposome’s internal hydrophilic part can protect loaded drugs from the host body’s destructive factors that ultimately minimize the unwanted side effects. Besides the internal aqueous environment, hydrophobic substances can also be embedded between the lipid membranes or adsorb on the liposome surface ( Xu et al., 2007 ; Silverman et al., 2013 ; Chen et al., 2014 ; Eloy et al., 2014 ; Jain et al., 2014 ). The essential liposome-like structures are presented in Table 2 .

Liposome-like structures.

The Packing Parameter

As described, liposomes are lipid-based structures that constitute one or more bilayer phospholipids which can encapsulate aqueous media. Liposome formation begins by dispersing phospholipids in water, leading to interactions between phospholipids and water ( Anwekar et al., 2011 ), PP being a critical criterion determining the formation of liposome.

PP is described as the ratio between the cross-section of the amphiphile hydrophobic part (hydrocarbon chains of phospholipids or the hydrocarbon rings of sterols) and the cross-section of the hydrophilic part (the amphiphile head group). Liposome-forming lipids are considered amphiphilic structures with a PP of 0.74–1.0. In this regard, HSPC (PP: 0.8) and DSPE–PEG (PP: 0.487) have been addressed as liposome and non-liposome forming lipids, respectively. For DSPE-PEG, the low PP implied the presence of an extensive polar head group due to the large (45 mer) polyethylene glycol (PEG) moiety that prevents liposome-structure formation. The head group in this molecule is highly flexible ( Nagarajan, 2002 ; Garbuzenko et al., 2005 ; Barenholz, 2016 ; Maritim et al., 2021 ). The most common phospholipids and cholesterol deployed to prepare liposomes with respect to their transition temperature (Tm) and molecular weights are presented in Table 3 .

The common compounds deployed to prepare liposomes.

Types of Liposomes

Liposomes are highly versatile compounds and they can be fabricated with various combinations, their diversity and properties vary in structure, size, shape, and surface properties. One of the classification types of liposomes is founded on their size and amount of layers, for example, unilamellar and multilamellar liposomes. They can be subdivided into four main categories based on structural parameters: multilamellar liposomes/vesicles (MLV), oligolamellar vesicles (OLV), multilamellar liposomes/vesicles (MVV), and unilamellar vesicles (ULV). Furthermore, the ULV can be divided into giant unilamellar liposomes/vesicles (GUV) groups, large unilamellar liposomes/vesicles (LUV), medium unilamellar vesicles (MUL), and small unilamellar liposomes/vesicles (SUV), and based on size ( Walde and Ichikawa, 2001 ; Gabriëls and Plaizier-Vercammen, 2003 ; Wagner et al., 2006 ; Drulis-Kawa and Dorotkiewicz-Jach, 2010 ; Baykal-Caglar et al., 2012 ; Garg and K. Goyal, 2014 ).

Despite the aforementioned classification of various liposomes, many liposome characteristics cannot be deduced, including synthesis techniques or their applications. Since the introduction of liposomes as lipid-carrying structures, many methods have been proposed for their fabrication. Each has some advantages and disadvantages that can be exploited depending on the practical function. The process of liposome making strongly affects the final properties, so special attention should be paid to the construction method. Liposomes are referred to as a very miscellaneous structure in terms of their charge and size, the ultimate size and electrical charge of the fabricated liposome being highly reliant on the manufacturing method and the types of phospholipid deployed. In this regard, liposomes synthesis methods can be categorized as dehydration and rehydration (DRV), reverse phase evaporation (REV), particularly for SUL, OLV, and MLV liposomes, vesicles prepared by extrusion technique (VET), and frozen and thawed (FAT) for MLV preparation ( Zhang and Pawelchak, 2000 ; Scott and Jones, 2001 ; Xia and Xu, 2005 ; Zaru et al., 2007 ; Akbarzadeh et al., 2013 ; Pradhan et al., 2015 ). Assorted liposome types, according to the fabrication methods, are presented in Table 4 .

Liposome classification based on the synthesis method.

Regarding the production of liposome, in addition to the fabrication method, drug loading should also be considered. In general, drug loading is performed via two standard procedures, passive and active loading, which affects the amount and quality of the loaded drug and some extent on the liposome properties. In the active loading method, known as the remote loading method, the drug molecules are loaded into the fabricated liposome. The pH gradient and electrical potential difference across the liposomal membrane are the most known underlying mechanisms dictating the active drug loading ( Figure 1 ). The active loading method has provided various advantages over passive loading methods, including high efficiency and loading capacity, decreasing the loaded drug’s leakage, and reducing the drug’s shrinking during storage. Another outstanding feature of this method is the possibility of drug loading after carrier formation due to the use of the flexibility of constitutive lipid. It is also possible to prevent the degradation of the biologically active compounds during the preparative process ( Barratt, 2003 ; Anwekar et al., 2011 ; Agrawal et al., 2012 ; Burton et al., 2015 ).

The active and passive drug loading on the liposomes.

It has been well known that liposome characteristics are highly dependent on lipid composition. Surface charge, particle size, and the preparation method are among the most outstanding influenced features by lipid combination. Besides, the effective properties of bilayer structure, including rigidity, fluidity, and electrical charge, can be determined through bilayer component selection. In this regard, natural-based liposomes fabricated from unsaturated phosphatidylcholine species, including egg or soybean phosphatidylcholine, have provided bilayer structures with highly permeable and low stable properties. However, saturated-phospholipids-based liposomes such as dipalmitoyl phosphatidylcholine lead to rigid and almost impermeable bilayer structures ( AllenLiposomes, 1997 ; Sahoo and Labhasetwar, 2003 ). The most common liposomal system, in terms of its composition constituent, are presented in Table 5 .

liposome classification according to composition constituents.

Application of Liposomes

As highly versatile nanoparticles, liposomes have been considered for many biomedical applications ( Figure 2 ). Liposome as cholesterol and natural-non-toxic phospholipids-based spherical shape vesicles has provided many biomedical opportunities, especially for drug delivery ascribed to their biocompatibility, appropriate size, and suitable hydrophobic and hydrophilic character. Besides, the cosmetics industry has been vigorously affected by liposome formulations as they proposed many unique properties for drug carriers ( Figueroa-Robles et al., 2020 ; Matole et al., 2020 ). On the other hand, the great potential application of liposomes should not be underestimated in the food and farming industries. Numerous studies have been conducting regarding liposome encapsulation to develop appropriate delivery systems to entrap unstable compounds. The ensnared invertebrate ingredients such as antimicrobials, antioxidants, hydrophobic, and hydrophilic compounds in liposome particles could be used to targeted delivery and prevent composition and functionality disruption ( Benech et al., 2002 ; Shehata et al., 2008 ; Atrooz, 2011 ).

Diverse biomedical applications of liposome-based structure.

The nanoliposome is a favorable particle to develop cancer drug delivery systems due to its unique properties, including biocompatibility, biodegradability, and hydrophilic and lipophilic drug loading capability. Liposome-based structures have owned the most commercial delivery systems worldwide. There is various ongoing research regarding improvement drug toxicity and specific targeting with liposomes ( Akbarzadeh et al., 2013 ).

In view of the admirable properties mentioned for liposomes, it has been extensively studied in drug delivery to cancerous and tumor tissues via two main approaches in terms of design to target tumor tissues: passive targeting and active targeting ( Figure 3 ). Passive targeting is reliant on the physiological characteristics of the tumor and the size of the nanoparticles. Cancer cells overexpress vascular endothelial growth factor (VEGF) due to their very high metabolism, which leads to excessive angiogenesis in tumor tissue. The vascular pores in the tumor tissue are larger than normal tissue, and the appropriate size of the liposomes enable them to circulate for a longer time in the circulatory system, so the anti-cancer drug nanosystem could target the tumor tissue ( Zhu et al., 2017 ; Jeon et al., 2020 ; Liu et al., 2021 ). In addition, after the drug delivery system enters the cancerous tissue due to defects in the lymphatic system, the retention time of nanoparticles increases, which is not possible for the small drug molecules ( Attia et al., 2019 ). In this method, the nanosystem is also coated with a biocompatible PEG polymer, which causes the escape of the reticuloendothelial (RES) system and increases the circulation time in the circulatory system; effect of PEG is through shielding liposomes from opsonization ( Suk et al., 2016 ; Nunes et al., 2019 ).

Passive targeting and active targeting. Liposomes can be surface functionalized to dedicate stealth through PEGylation and to meliorate receptor-mediated endocytosis by utilizing targeting ligands such as antibodies, peptides, proteins, carbohydrates, Aptamer, and various other small molecules. PEGylation prolongs liposomal circulation half-life in vivo . Types of drugs based on whether they are hydrophobic or hydrophilic can be encapsulated into the aqueous lumen, incorporated into the lipid bilayer, or conjugated to the liposome surface.

Despite the relatively good performance of passive targeting in increasing drug delivery to cancerous tissues, the optimum amount may still not reach the target tissue, or the drug may leak into normal tissues. Hence, researchers are using the active targeting method to enhance drug delivery to the target tissue. The basis of this method can be attributed to the functionalization of the liposome surface. Cancer cells need more nutrients because of their distinct metabolism. Therefore, some surface receptors are overexpressed on the surface of these cells. Targeted drug delivery to the cancerous tissue can be performed using this feature through specifically functionalizing liposome surface ( Dana et al., 2020 ; Montaseri et al., 2020 ; Raj et al., 2021 ).

A comprehensive overview of liposomes’ applications in biomedicine is summarized in Table 6 according to the type of liposomes used.

The use of liposome in biomedical applications.

Advantages and Disadvantages of Liposomes

Like any other carrier, liposomes have provided some advantages and disadvantages, too. The benefits of liposomes have been mentioned briefly in this text. As amphiphilic and non-ionic structures particle, liposomes offer an exceptional opportunity to deliver both water and lipid-soluble drugs. This feature has a significant priority in the pharmaceutical industry to develop new formulations ( Abdelkader et al., 2014 ; Joshi et al., 2016 ). On the other hand, their inimitable structure frame enables researchers to fabricate both sustained and targeted drug delivery systems through controlling permeability, rigidity, size, and surface functionalization ( Daraee et al., 2016 ; Jain and Jain, 2016 ). Although liposomes can be administered through different routes, they are composed of biocompatible ingredients ( Mansoori et al., 2012 ). One of the main limitations of drug distribution systems is the importance of biodegradable drug transport, which can be overcome through liposomal delivery that prevents drug oxidation ( Manconi et al., 2016 ). It has been reported that liposomes can promote drug pharmacokinetics by eliminating and circulation life ( Bhatt et al., 2018 ).

Despite all advantages, liposome-based structures have some limitation that prevents their widespread clinical use. The most significant obstacle is attributed to their physical and chemical stabilities ( Bakker-Woudenberg, 2002 ). Among other essential impediments, low solubility in aqueous solutions ( Li et al., 2019 ), short half-life time in body environment ( Kshirsagar et al., 2005 ), high production cost ( Noble et al., 2014 ), challenges in various tissue targeting due to the relatively large size of liposomes may cause ( Santos et al., 2005 ), leakage and fusion of the loaded drugs ( Joly et al., 2011 ), phospholipids oxidation and hydrolysis ( Jain and Jain, 2016 ), rapidly detection by RES system ( Daraee et al., 2016 ), and allergic reactions to some liposomal compounds ( Mansoori et al., 2012 ), can be mentioned.

Liposome Stability

One of the most critical challenges in liposome application is attributed to the relative instability in aqueous dispersions. The physical and chemical instability of liposomes can lead to unwanted side effects and efficacy reduction ( Scrimgeour et al., 2005 ; Toh and Chiu, 2013 ); oxidation and hydrolysis are two primary mechanisms in the degradation of liposomes that cause chemical instability ( Carlson et al., 2006 ; Jung et al., 2006 ; Frenzel and Steffen-Heins, 2015 ). The oxidation process is very probable due to free radicals in fatty acids as an intrinsic compound where unsaturated fatty acids are more susceptible than the saturated fatty acids to this mechanism ( Anderson and Omri, 2004 ; Tan et al., 2016 ). In the presence of acid- or baseـ catalyst, this process can occur either at the 1-acyl or at the 2-acyl position, which subsequently leads to lysophospholipids through free fatty acids generation, eventually undergoing hydrolysis to free fatty acids and creating phosphoglycerols ( Patel and Panda, 2012 ; Frenzel and Steffen-Heins, 2015 ). The combination of membrane bilayers, aggregation, reduced retaining of encapsulated materials, and alteration can be mentioned as the causes for physical instability in liposomes ( Karmali and Chaudhuri, 2007 ; Shim et al., 2013 ; Rahdar et al., 2019 ).

The administrated liposome fate and stability can be strongly affected by liposomes’ physicochemical characteristics, including bilayer membrane composition, size, rigidity, and charge ( Portet and Dimova, 2010 ; Ghanbarzadeh et al., 2013 ; Ib and Corredig, 2013 ). The surface charge of liposomes can be positive, negative, or without electrical charge based on functional groups on liposomes’ surface at the environmental pH. As such, net-charge liposomes have represented the highest tendency to accumulate in the target tissue following systematic administration due to the reticuloendothelial system’s low clearance. On the other hand, cationic liposomes are often used to transport negatively charged nucleic acids ( Xia and Xu, 2005 ; El-Samaligy et al., 2006a ; Demetzos, 2008 ; Fujisawa et al., 2012 ). The methods used to prepare liposomes significantly affect physical properties, such as the coating of active compounds’ size and efficiency. Various phenomena such as aggregation and mixing can affect material properties, such as particle size and particle size distribution, attributed to the accumulation of liposomal particles. It has been reported that particle size reduction can be used for achieving bioactive compounds with optimal bioavailability due to increasing specific surface area ( Campbell et al., 2001 ; Miao et al., 2002 ; Ulrich, 2002 ; Silva et al., 2011 ). The smaller the liposomal size, easier it is to cross the membrane, but it can affect the liposomal properties, including decreasing the amount and efficiency of drug loading and reducing liposome stability by increasing the surface energy. The smaller the particle size distribution, the more uniform is the liposomes’ size and the more similar would be the products’ overall characteristics ( Marsh, 2001 ; Yamauchi et al., 2007 ; Drin et al., 2008 ).

Another critical factor affecting the stability of liposomes is referred to lipid composition. Studies have shown that controlling solute retention by liposomes and circulation half-life can be strongly affected by liposomal membrane fluidity and composition manipulation. One of the most critical barriers to prevent the transfer of liposomal drugs from the bench scale to the pharmaceutical market is their physical and chemical instability during production and storing. The mechanically robust and well-filled bilayers diminish the oxidizing and hydrolyzing agents that eventually improve the structure stability through size distribution ( Kunzelmann-Marche et al., 2002 ; Liu and Krieger, 2002 ; López-Revuelta et al., 2006 ; Giulimondi et al., 2019 ); composition of the bilayer membrane is one of the influential and essential factors in liposome stability. The lipid selection during liposome fabrication should be commensurate with the carrier’s composition ( Jiménez-Escrig and Sánchez-Muniz, 2000 ; Scheffer et al., 2005 ; Zhao et al., 2015 ; Ricci et al., 2016 ).

The Effect of Cholesterol on Liposome Membrane Stability

Cholesterol is an organic sterol molecule with amphiphilic nature. Structurally, this molecule has a hydroxyl group that can form hydrogen bonds with phospholipids and bulky steroid ring with a flexible carbohydrate. Cholesterol is a 27-carbon molecule found in the eukaryotic cell membrane and its concentration in the cell membrane is about 30–50 mol% of the entire lipid compounds. Various vital roles have been attributed to cholesterol, including membrane permeability regulation, elasticity and stiffness, and membrane strength. Cholesterol is the most used sterol in the formulation of liposomes which can prevent liposome aggregation and improve the liposomal membrane’s stability ( Jiménez-Escrig and Sánchez-Muniz, 2000 ; Scheffer et al., 2005 ; Sun et al., 2007 ; Ricci et al., 2016 ; Trucillo et al., 2017 ).

In view of the spherical 3-D structure, liposomes have shown more realistic fluidity and cell membrane mobility than lipid monolayers. Sterols, such as ergosterol, stigmasterol, lanosterol, β-sitosterol and cholesterol, have been added to liposomes to modulate membrane fluidity and improve the stability of the phospholipids bilayer and decrease permeation of the encapsulated active compounds. The sterol molecule is located inside the phospholipid’s bilayer. The sterol carbohydrate tail at C17 intermingles with the hydrophobic fatty acyl chains, while the sterol hydroxyl group attaches to the hydrophilic head group of phospholipids ( Socaciu et al., 2000 ; Sodt et al., 2015 ; Zhao et al., 2015 ; Giulimondi et al., 2019 ). Cholesterol plays a vital role in the liposome’s composition. It is one of the most critical structural components in the mammalian cell plasma membrane. It has been proven that the fluidity and permeability of the artificial vesicle are strongly affected by cholesterol through hydrogen binding to fatty acids that eventually lead to increasing the cohesiveness and mechanical strength. Cholesterol is a critical regulator of lipid bilayer dynamics, and it is essential for the normal functioning of the cells. The reduction of passive permeability to small molecules results from cholesterol interaction with membrane phospholipids, increasing the membrane cohesion. According to experimental studies, cholesterol adding to liposome bilayers can prevent lipid exchange that can be counted as an additional stabilizing effect ( Sun et al., 2007 ; Ricci et al., 2016 ; Trucillo et al., 2017 ).

The Relationship Between Cholesterol and Transition Temperature

Another significant factor in the arrangement of bilayers in the liposome is the degree of saturation in phospholipids and the chain length. The presence of sterols such as cholesterol, β-sitosterol, and related plant sterols can lead to the pre-transition temperature peak’s disappearance. The gel-to-liquid crystalline phase Tm has been reported to decrease by addition of the sterols. It can be stated that the orientation of cholesterol in the lipid bilayer has a determinative effect on the reduction of head group accumulation. In the low Tm, cholesterol leads to the crystallization of the hydrocarbon chains into the inflexible crystalline gel phase. While in the high Tm, the rigid cholesterol molecule can limit the movement of the hydrocarbon chains. It can be concluded that, by increasing the sterol concentration, decreases the enthalpy of the main phase transition ( Silva et al., 2011 ; Wu et al., 2012 ; Ricci et al., 2016 ).

For example, in the case of the PC molecule, it has been stated that cholesterol at concentrations of more than 25% leads to an order-liquid phase, which is essential for the mobility of membrane compounds; inferred that cholesterol at high concentrations causes the decouples of positional and conformational degrees of freedom of phospholipid molecules. On the other hand, at low cholesterol concentrations below 10%, its effect on the bilayer phospholipid is very different; only a slight decrease in the Tm, the main phase transfer occurs. This means that cholesterol at low concentrations cannot disrupt the crystalline order, nor can it ultimately induce the acyl chains in the liquid phase. Cholesterol, therefore, functions as a surfactant molecule, which causes the formation of lipid domains thus increasing the dynamic membrane heterogeneity ( Trandum et al., 2000 ; Pinilla et al., 2020 ).

The Role of Cholesterol in Retention of Drugs and Modulation of Phospholipid Packing

The cholesterol content has a critical role in drug retention as it implies its effect by raising the phospholipid packing, leading to bilayer permeability reduction to non-electrolytic and dielectrolysis solvents ( Dos Santos et al., 2002 ; Johnston et al., 2007 ). It is believed that the underlying mechanism in a biological milieu may be related to the phospholipid and high-density lipoproteins exchange of the low-cholesterol or no-cholesterol content liposomes while in cholesterol-encompassing liposomes, the phospholipid motility has been limited that prevents lipoproteins loss ( Kunzelmann-Marche et al., 2002 ; Liu and Krieger, 2002 ; López-Revuelta et al., 2006 ).

The phospholipid packing, the membrane fluidity, and the surface charge of liposomes can be modulated and adapted by the cholesterol content, affecting the particle size, encapsulation efficiency, and final morphology ( Zhao et al., 2015 ; Ohvo-Rekilà et al., 2002 ). Studies have shown that cholesterol as a nonionic molecule has revealed interesting effects on the zeta potential, especially increasing the highest zeta potential of cationic liposomes. The proposed mechanism is reliant on the membrane structure transition and the molecular filling state for the cholesterol-induced charge boosting of cationic liposomes. Cholesterol can induce the phase transition from the crystalline state to the liquid-ordered (LO) phase ( Lv et al., 2006 ; Aramaki et al., 2016 ). Increasing the amount of cholesterol content in the membrane of liposomes can increase the mean size of liposomes. Due to the hydrophobic nature, cholesterol structure can easily interact with the phospholipid hydrophobic acyl chain through hydrogen bonds and hydrophobic interactions. The ring structure of cholesterol is comparatively inflexible, and its presence stabilizes the protracted straight-chain arrangement of saturated fatty acids via van der Waals interactions ( Lee et al., 2005 ).

The Role of Cholesterol-Containing Liposomes in Plasma Stability

Cholesterol has a very significant effect on the structural stability of liposomes in plasma ( Figure 4 ). It can reduce the interaction of liposomes with several proteins, leading to less susceptibility to phospholipase, reduce the phospholipid’s loss of high-density lipoproteins, change the membrane enzyme activities, inhibit digestion by macrophages, and inhibit fusion with specific cell types. By changing some liposome features through composition, mostly cholesterol modification, blood clearance and tissue distribution of intravenously injected liposomes can be expected. The cholesterol modification of liposomes has an inhibitory effect on the reticuloendothelial uptake. This modification reduces their interaction with several proteins and probably with serum components, which may affect liposomes’ tissue uptake ( D’Avanzo et al., 2011 ; Johnstone et al., 2001 ; Moghimi and Patel, 2002 ). The cholesterol-based liposomal products are presented in Table 7 .

The liposome preparation and the role of cholesterol in its stability.

Cholesterol-based liposomal products.

Note : CF, carboxyfluorescein; CLX, celecoxib; EPC, Ethanolamine phosphatidylcholine; PUFA, Polyunsaturated fatty acids; DPPC, Dipalmitoyl phosphatidylcholine; PEG-DSPE, Distearoyl-sn-glycero-3-phosphoethanolamine-Poly(ethylene glycol glycol, HSPC: Hydrogenated soybean phosphatidylcholine; LC-Biotin-DPPE, N((biotinyl)amino)hexanoyl)dipalmitoyl-l-α-phosphatidylethanolamine; SPC, Sphingosyl phosphorylcholine; PE, phosphatidyl-ethanolamine; DSPC, Distearoyl L-3-phosphatidylcholine; HPC, hydrogenated soybean phosphatidylcholine; SM, sphingomyelin; DMPC, dimyristoylphosphatidylcholine; DMPG, dimyristoylphosphatidylglycerol; PC, phosphatidylcholine; OQCMC, octadecyl quaternized carboxymethyl chitosan; DPTAP, 1,2-dipalmitoyl-3-trimethylammonium-propane; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; EPC, Egg yolk phosphatidylcholine; FCCP, carbonyl cyanid- p -trifluoro-methoxyphenyl hydrazone; α-DPPC, α-dipalmitoylphosphatidylcholine; SA, stearylamine; DP, dicetyl phosphate; DCP, Dicetyl phosphate.

The Optimum Cholesterol Concentration for Liposome’s Stability

The importance of cholesterol in liposome stability has been described earlier and schematically shows in Figure 3 . However, the optimum concentration of cholesterol to attain a suitable formulation has not yet been elucidated. To make stable and regulated drug discharge means, lipids and cholesterol ratio screening arrangements in different studies can help. For the preparation of liposomes, some phospholipids are blended with varying molar ratios of cholesterol. The results of numerous studies have shown that the maximum amount of cholesterol that can be integrated into reconstructed bilayers is assumed to be ∼50 mol%. The most frequently used proportion is the 2: 1 ratio (e.g., two parts of lipids and one part of cholesterol) or 1: 1 ratio; although the underlying reasons for using these ratios is still unknown ( Marsh, 2001 ; Liang et al., 2007 ; Briuglia et al., 2015 ).

The essential mechanisms of the liposomal interaction with cells according to both, in vitro and in vivo studies have been summarized as:

• 1- Particular interfaces with cell-surface components such as electrostatic bonds and imprecise interactions, including feeble hydrophobic bonds
• 2- Endocytosis caused by cells of the reticuloendothelial system (RES), comprising neutrophils and macrophages
• 3- Combination with the plasma cell membrane by inclusion of the lipid bilayer of the liposome into the plasma membrane ( Akbarzadeh et al., 2013 )

Adjusting cholesterol levels can be a very influential factor in controlling the liposomal stability. This factor can be crucial in designing liposomes for practical use in biological systems in vivo and in vitro ( Epstein et al., 2008 ). The most important commercial liposomes are depicted in Table 8 .

Commercial cholesterol-based liposomes.

DSPG, Distearoyl-sn-glycero-3-phosphoglycerol.

Clinical Trials

Unlike most nanoparticles, which encounter serious challenges in entering the clinic for various reasons, including safety issues, liposomes are well accepted in the clinic, first liposome-based drug being approved was Doxil ® [( Anselmo and Mitragotri, 2015 ; Bulbake et al., 2017 ; Singh et al., 2020 )], a liposome-based Doxorubicin formulation that has been FDA approved in 1995 for the United States market to treat ovarian cancer and AIDS-related Kaposi’s sarcoma. Subsequently, various other liposomal-based drugs have been commercialized, such as DaunoXome ® , for the delivery of daunorubicin, approved in 1996 to manage advanced HIV-associated Kaposi’s sarcoma and several different formulations ( Khadke et al., 2020 ).

Currently, many efforts are underway to develop lipid formulations for entry in to the clinics in various fields. To examine the clinical phase studies of liposome-based structures, the term “liposome” was searched from the PubMed database pertaining to various studies related to the clinical phases in the last year. In one study, the effect of liposomal amphotericin B formulation as an antifungal agent was investigated in patients with hematological malignancies with neutropenia and persistent fever ( Yoshida et al., 2020 ). Because fungal infections subsequent to the chemotherapy in patients with neutropenia are considered a severe complication, and as the use of common antifungal drugs is highly toxic, so eliminating this life-threatening complication is very important. The usage of 3 mg/kg/day liposomal amphotericin B concentration for patients was compared with itraconazole; results showed no difference between the liposomal amphotericin B and itraconazole regarding the efficacy and safety in antifungal therapy in hematological malignancy patients ( Yoshida et al., 2020 ).

Liposomal compounds are known to be very effective in treating cancer. Recently, another liposomal system has been proposed for the treatment of acute myeloid leukemia wherein the Vyxeos liposome in phase III clinical was examined. This liposomal system comprise two different topoisomerase II inhibitors known as daunorubicin and cytarabine, which included 1 and 0.44 mg in every 1 unit of liposome formulation. The study was performed on elderly patients with untreated acute myeloid leukemia where a higher survival rate for patients treated with the liposomal formulation was discerned than for standard chemotherapy, although some side effects have been reported for the use of this formulation ( Tzogani et al., 2020 ).

Health and medical issues and problems have always been one of the main areas of interest for scientists. Applying new methods for solving medical problems requires introducing new and efficient materials and tools that address related questions and issues. Liposomes have been extensively studied since their introduction, and their potential biomedical use has been well demonstrated. The unique properties of liposomes, including biocompatibility, biodegradability, amphiphilic nature, low toxicity, non-ionicity, sustained release, and active targeting, have made them one of the most widely used nanoparticles. Currently, the most commercialized nanoparticles in the field of drug delivery and cosmetics are related to liposomes. However, they still have some shortcomings that need to be addressed for clinical and pharmaceutical use. As mentioned earlier, since Doxil ® introduced in 1995, many efforts have been made to bring these versatile nanomaterials to the clinic; however, their structure still needs to be optimized to reduce some complications. One approach to increase liposomes’ efficiency, especially concerning cancers therapy, is the use of RGD which can be anchored to liposome surfaces or via self-assembly means ( Cheng and Ji, 2019 ). This strategy has been proposed to reduce the side effects of liposomal drugs, as the clinical trial sometimes suffers from, including acute infusion reaction and hand-foot syndrome (HFS). Hence future studies must include new procedures to reduce safety problems ( He and Tang, 2018 ; Cheng and Ji, 2019 ). Since varying combinations of liposomes can provide unique and distinctive properties, exciting suggestions have been propositioned in the field of novel drug delivery systems, such as nose-to-brain direct drug transport using liposome formulation based on DTE and DTP ingredients ( Hong et al., 2019 ).

However, one of the most critical challenges that liposomes encounter is their physical and chemical instability. Various factors such as environmental conditions, manufacturing method, components characteristics, lipid type, and the absence or presence of cholesterol affect liposomes’ stability. The vital need for the development of liposomes with high stability significantly impacts their clinical application. Cholesterol has been shown to increase liposome stability through various mechanisms, including increased retention time, modulating phospholipid packing, Tm, and plasma stability. However, the optimal amount of cholesterol has not yet been identified. Future field research should be based on the principle of finding the optimal amount of cholesterol in liposome production. Notably, cholesterol still tend to maintain membrane’s fluidity as its concentration is increased and decreased. Therefore, the amount of cholesterol and the type of liposome constituents need further investigation. In this context, simulation and computational studies may prove to be very helpful. Hashemzadeh et al. (2020a) have investigated DSPC and DPSM effect on liposome stability through a simulation study which revealed that DSPC preserves its structure shape due to the cylindrical geometric structure and small-size head group, while the DPSM was causing liposome turnover into micelle structure because of its conical geometric design with the larger head group. Such studies regarding cholesterol’s effect on liposome stability via simulation investigations would be highly desirable.

Author Contributions

All authors contributed to the conception and the main idea of the work. MJ, NB, FB, HK, AH, and FM drafted the main text, figures, and tables. MJ supervised the work and provided the comments and additional scientific information. MS and AC-A also reviewed and revised the text. All authors read and approved the final version of the work to be published.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s Note

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• Abd El-Fattah A. I., Fathy M. M., Ali Z. Y., El-Garawany A. E.-R. A., Mohamed E. K. (2017). Enhanced Therapeutic Benefit of Quercetin-Loaded Phytosome Nanoparticles in Ovariectomized Rats . Chemico-Biological Interactions 271 , 30–38. 10.1016/j.cbi.2017.04.026 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Abdelkader H., Alani A. W. G., Alany R. G. (2014). Recent Advances in Non-ionic Surfactant Vesicles (Niosomes): Self-Assembly, Fabrication, Characterization, Drug Delivery Applications and Limitations . Drug Deliv. 21 ( 2 ), 87–100. 10.3109/10717544.2013.838077 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Abu-Dahab R., Schäfer U. F., Lehr C.-M. (2001). Lectin-functionalized Liposomes for Pulmonary Drug Delivery: Effect of Nebulization on Stability and Bioadhesion . Eur. J. Pharm. Sci. 14 ( 1 ), 37–46. 10.1016/s0928-0987(01)00147-6 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Agarwal R., Katare O. P., Vyas S. P. (2001). Preparation and In Vitro Evaluation of Liposomal/niosomal Delivery Systems for Antipsoriatic Drug Dithranol . Int. J. Pharm. 228 ( 1-2 ), 43–52. 10.1016/s0378-5173(01)00810-9 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Agrawal M. M., Jawade S., Khan S. (2012). A Review on Liposome . Int. J. Adv. Res. Pharm. Bio Sci. 2 ( 1 ), 453–465. [ Google Scholar ]
• Aguilar-Pérez K., Avilés-Castrillo J., Medina D. I., Parra-Saldivar R., Iqbal H. (2020). Insight into Nanoliposomes as Smart Nanocarriers for Greening the Twenty-First century Biomedical Settings . Front. Bioeng. Biotechnol. 8 , 1441. 10.3389/fbioe.2020.579536 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Ainbinder D., Paolino D., Fresta M., Touitou E. (2010). Drug Delivery Applications with Ethosomes . J Biomed. Nanotechnol 6 ( 5 ), 558–568. 10.1166/jbn.2010.1152 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Akabori K., Nagle J. F. (2015). Structure of the DMPC Lipid Bilayer Ripple Phase . Soft matter 11 ( 5 ), 918–926. 10.1039/c4sm02335h [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Akbarzadeh A., Rezaei-Sadabady R., Davaran S., Joo S. W., Zarghami N., Hanifehpour Y., et al. (2013). Liposome: Classification, Preparation, and Applications . Nanoscale Res. Lett. 8 ( 1 ), 102. 10.1186/1556-276X-8-102 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Al-Remawi M., Elsayed A., Maghrabi I., Hamaidi M., Jaber N. (2017). Chitosan/lecithin Liposomal Nanovesicles as an Oral Insulin Delivery System . Pharm. Dev. Technol. 22 ( 3 ), 390–398. 10.1080/10837450.2016.1213745 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Alizadeh E., Akbarzadeh A., Eslaminejad M. B., Barzegar A., Hashemzadeh S., Nejati-Koshki K., et al. (2015). Up Regulation of Liver-Enriched Transcription Factors HNF4a and HNF6 and Liver-specific MicroRNA (miR-122) by Inhibition of Let-7b in Mesenchymal Stem Cells . Chem. Biol. Drug Des. 85 ( 3 ), 268–279. 10.1111/cbdd.12398 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• AllenLiposomes T. M. (1997). Liposomen . DrugsSuppl 54 , 8–14. 10.2165/00003495-199700544-00004 [ CrossRef ] [ Google Scholar ]
• Alving C. R., Rao M., Steers N. J., Matyas G. R., Mayorov A. V. (2012). Liposomes Containing Lipid A: an Effective, Safe, Generic Adjuvant System for Synthetic Vaccines . Expert Rev. Vaccin. 11 ( 6 ), 733–744. 10.1586/erv.12.35 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Anderson M., Omri A. (2004). The Effect of Different Lipid Components on the In Vitro Stability and Release Kinetics of Liposome Formulations . Drug Deliv. 11 ( 1 ), 33–39. 10.1080/10717540490265243 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Anselmo A. C., Mitragotri S. (2015). A Review of Clinical Translation of Inorganic Nanoparticles . Aaps J. 17 ( 5 ), 1041–1054. 10.1208/s12248-015-9780-2 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Anwekar H., Patel S., Singhai A. (2011). Liposome-as Drug Carriers . Int. J. Pharm. Life Sci. , 2.(7) [ Google Scholar ]
• Aoki H., Mizuno M., Natsume A., Tsugawa T., Tsujimura K., Takahashi T., et al. (2001). Dendritic Cells Pulsed with Tumor Extract-Cationic Liposome Complex Increase the Induction of Cytotoxic T Lymphocytes in Mouse Brain Tumor . Cancer Immunol. Immunother. 50 ( 9 ), 463–468. 10.1007/s002620100220 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Aramaki K., Watanabe Y., Takahashi J., Tsuji Y., Ogata A., Konno Y. (2016). Charge Boosting Effect of Cholesterol on Cationic Liposomes . Colloids Surf. A: Physicochemical Eng. Aspects 506 , 732–738. 10.1016/j.colsurfa.2016.07.040 [ CrossRef ] [ Google Scholar ]
• Arora R. (2016). Advances in Niosome as a Drug Carrier: a Review . Asian J. Pharmaceutics (Ajp) Free full text articles Asian J Pharm , 1.(1) [ Google Scholar ]
• Ashok B., Arleth L., Hjelm R. P., Rubinstein I., Önyüksel H. (2004). In Vitro characterization of PEGylated Phospholipid Micelles for Improved Drug Solubilization: Effects of PEG Chain Length and PC Incorporation . J. Pharm. Sci. 93 ( 10 ), 2476–2487. 10.1002/jps.20150 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Asprea M., Tatini F., Piazzini V., Rossi F., Bergonzi M., Bilia A. (2019). Stable, Monodisperse, and Highly Cell-Permeating Nanocochleates from Natural Soy Lecithin Liposomes . Pharmaceutics 11 ( 1 ), 34. 10.3390/pharmaceutics11010034 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Atrooz O. M. (2011). Efects of Alkylresorcinolic Lipids Obtained from Acetonic Extract of Jordanian Wheat Grains on Liposome Properties . Int. J. Biol. Chem. 5 ( 5 ), 314–321. 10.3923/ijbc.2011.314.321 [ CrossRef ] [ Google Scholar ]
• Attia M. F., Anton N., Wallyn J., Omran Z., Vandamme T. F. (2019). An Overview of Active and Passive Targeting Strategies to Improve the Nanocarriers Efficiency to Tumour Sites . J. Pharm. Pharmacol. 71 ( 8 ), 1185–1198. 10.1111/jphp.13098 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Attwood S., Choi Y., Leonenko Z. (2013). Preparation of DOPC and DPPC Supported Planar Lipid Bilayers for Atomic Force Microscopy and Atomic Force Spectroscopy . Ijms 14 ( 2 ), 3514–3539. 10.3390/ijms14023514 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Awasthi V. D., Garcia D., Goins B. A., Phillips W. T. (2003). Circulation and Biodistribution Profiles of Long-Circulating PEG-Liposomes of Various Sizes in Rabbits . Int. J. Pharm. 253 ( 1-2 ), 121–132. 10.1016/s0378-5173(02)00703-2 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Bae Y., Jung M. K., Mun J. Y., Mallick S., Song S. J., Kim D. M., et al. (2018). DQAsomes Nanoparticles Promote Osteogenic Differentiation of Human Adipose-Derived Mesenchymal Stem Cells . Bull. Korean Chem. Soc. 39 ( 1 ), 97–104. 10.1002/bkcs.11355 [ CrossRef ] [ Google Scholar ]
• Bakker-Woudenberg I. A. J. M. (2002). Long-circulating Sterically Stabilized Liposomes as Carriers of Agents for Treatment of Infection or for Imaging Infectious Foci . Int. J. Antimicrob. Agents 19 ( 4 ), 299–311. 10.1016/s0924-8579(02)00021-3 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Barenholz Y. (2016). Chapter 13. Doxil - the First FDA-Approved Nano-Drug: from Basics via CMC, Cell Culture and Animal Studies to Clinical Use . Nanomedicines: Des. Deliv. Detect. 51 , 315–345. 10.1039/9781782622536-00315 [ CrossRef ] [ Google Scholar ]
• Barenholz Y. (2021). Doxil - the First FDA-Approved Nano-Drug: From an Idea to a Product . Handbook harnessing Biomater. nanomedicine , 463–528. Jenny Stanford Publishing. 10.1201/9781003125259-16 [ CrossRef ] [ Google Scholar ]
• Barnes L. D., Giuliano E. A., Ota J. (2010). Cellular Localization of Visudyne as a Function of Time after Local Injection in an In Vivo Model of Squamous Cell Carcinoma: an Investigation into Tumor Cell Death . Vet. Ophthalmol. 13 ( 3 ), 158–165. 10.1111/j.1463-5224.2010.00775.x [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Barratt G. (2003). Colloidal Drug Carriers: Achievements and Perspectives . Cell Mol. Life Sci. CMLS 60 ( 1 ), 21–37. 10.1007/s000180300002 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Battaglia L., Ugazio E. (2019). Lipid nano- and microparticles overview of patent-related research . J. Nanomater. 2019 , 2834941. 10.1155/2019/2834941 [ CrossRef ] [ Google Scholar ]
• Baykal-Caglar E., Hassan-Zadeh E., Saremi B., Huang J. (2012). Preparation of Giant Unilamellar Vesicles from Damp Lipid Film for Better Lipid Compositional Uniformity . Biochim. Biophys. Acta (Bba) - Biomembranes 1818 ( 11 ), 2598–2604. 10.1016/j.bbamem.2012.05.023 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Benech R.-O., Kheadr E. E., Laridi R., Lacroix C., Fliss I. (2002). Inhibition of Listeria Innocua in Cheddar Cheese by Addition of Nisin Z in Liposomes or by In Situ Production in Mixed Culture . Appl. Environ. Microbiol. 68 ( 8 ), 3683–3690. 10.1128/aem.68.8.3683-3690.2002 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Benesch M., Urban C. (2008). Liposomal Cytarabine for Leukemic and Lymphomatous Meningitis: Recent Developments . Expert Opin. Pharmacother. 9 ( 2 ), 301–309. 10.1517/14656566.9.2.301 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Bennett W. F. D., MacCallum J. L., Hinner M. J., Marrink S. J., Tieleman D. P. (2009). Molecular View of Cholesterol Flip-Flop and Chemical Potential in Different Membrane Environments . J. Am. Chem. Soc. 131 ( 35 ), 12714–12720. 10.1021/ja903529f [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Benvegnu T., Lemiègre L., Cammas-Marion S. (2009). New Generation of Liposomes Called Archaeosomes Based on Natural or Synthetic Archaeal Lipids as Innovative Formulations for Drug Delivery . Ddf 3 ( 3 ), 206–220. 10.2174/187221109789105630 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Benz M., Gutsmann T., Chen N., Tadmor R., Israelachvili J. (2004). Correlation of AFM and SFA Measurements Concerning the Stability of Supported Lipid Bilayers . Biophysical J. 86 ( 2 ), 870–879. 10.1016/s0006-3495(04)74162-4 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Bern C., Adler-Moore J., Berenguer J., Boelaert M., den Boer M., Davidson R. N., et al. (2006). Liposomal Amphotericin B for the Treatment of Visceral Leishmaniasis . Clin. Infect. Dis. 43 ( 7 ), 917–924. 10.1086/507530 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Bhatt P., Lalani R., Vhora I., Patil S., Amrutiya J., Misra A., et al. (2018). Liposomes Encapsulating Native and Cyclodextrin Enclosed Paclitaxel: Enhanced Loading Efficiency and its Pharmacokinetic Evaluation . Int. J. pharmaceutics 536 ( 1 ), 95–107. 10.1016/j.ijpharm.2017.11.048 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Briuglia M.-L., Rotella C., McFarlane A., Lamprou D. A. (2015). Influence of Cholesterol on Liposome Stability and on In Vitro Drug Release . Drug Deliv. Transl. Res. 5 ( 3 ), 231–242. 10.1007/s13346-015-0220-8 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• B.T.S. Thirumamagal B., Zhao X. B., Bandyopadhyaya A. K., Sureshbabu Narayanasamy S., Johnsamuel J., Tiwari R., et al. (2006). Receptor-targeted Liposomal Delivery of boron-containing Cholesterol Mimics for boron Neutron Capture Therapy (BNCT) . Bioconjug. Chem. 17 ( 5 ), 1141–1150. 10.1021/bc060075d [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Bulbake U., Doppalapudi S., Kommineni N., Khan W. (2017). Liposomal Formulations in Clinical Use: An Updated Review . Pharmaceutics 9 ( 2 ), 12. 10.3390/pharmaceutics9020012 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Burton A. J., Giguère S., Berghaus L. J., Hondalus M. K., Arnold R. D. (2015). Efficacy of Liposomal Gentamicin against Rhodococcus Equi in a Mouse Infection Model and Colocalization with R. Equi in Equine Alveolar Macrophages . Vet. Microbiol. 176 ( 3-4 ), 292–300. 10.1016/j.vetmic.2015.01.015 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Campbell R. B., Balasubramanian S. V., Straubinger R. M. (2001). Influence of Cationic Lipids on the Stability and Membrane Properties of Paclitaxel‐containing Liposomes . J. Pharm. Sci. 90 ( 8 ), 1091–1105. 10.1002/jps.1063 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Caracciolo G., Pozzi D., Amenitsch H., Caminiti R. (2005). Multicomponent Cationic Lipid−DNA Complex Formation: Role of Lipid Mixing . Langmuir 21 ( 25 ), 11582–11587. 10.1021/la052077c [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Cardenia V., Waraho T., Rodriguez‐Estrada M. T., Julian McClements D., Decker E. A. (2011). Antioxidant and Prooxidant Activity Behavior of Phospholipids in Stripped Soybean Oil‐in‐Water Emulsions . J. Am. Oil Chem. Soc. 88 ( 9 ), 1409–1416. 10.1007/s11746-011-1807-y [ CrossRef ] [ Google Scholar ]
• Carlson D. L., Than K. D., Roberts A. L. (2006). Acid- and Base-Catalyzed Hydrolysis of Chloroacetamide Herbicides . J. Agric. Food Chem. 54 ( 13 ), 4740–4750. 10.1021/jf0530704 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Chen C., Johnston T. D., Jeon H., Gedaly R., McHugh P. P., Burke T. G., et al. (2009). An In Vitro Study of Liposomal Curcumin: Stability, Toxicity and Biological Activity in Human Lymphocytes and Epstein-Barr Virus-Transformed Human B-Cells . Int. J. Pharm. 366 ( 1-2 ), 133–139. 10.1016/j.ijpharm.2008.09.009 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Chen C., Han D., Cai C., Tang X. (2010). An Overview of Liposome Lyophilization and its Future Potential . J. controlled release 142 ( 3 ), 299–311. 10.1016/j.jconrel.2009.10.024 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Chen J.-H., Ling R., Yao Q., Li Y., Chen T., Wang Z., et al. (2005). Effect of Small-Sized Liposomal Adriamycin Administered by Various Routes on a Metastatic Breast Cancer Model . Endocr. Relat. Cancer 12 ( 1 ), 93–100. 10.1677/erc.1.00871 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Chen J., Lu W.-L., Gu W., Lu S.-S., Chen Z.-P., Cai B.-C., et al. (2014). Drug-in-cyclodextrin-in-liposomes: A Promising Delivery System for Hydrophobic Drugs . Expert Opin. Drug Deliv. 11 ( 4 ), 565–577. 10.1517/17425247.2014.884557 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Chen X., Wang X., Wang Y., Yang L., Hu J., Xiao W., et al. (2010). Improved Tumor-Targeting Drug Delivery and Therapeutic Efficacy by Cationic Liposome Modified with Truncated bFGF Peptide . J. controlled release 145 ( 1 ), 17–25. 10.1016/j.jconrel.2010.03.007 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Chen Y., Sun J., Lu Y., Tao C., Huang J., Zhang H., et al. (2013). Complexes Containing Cationic and Anionic pH-Sensitive Liposomes: Comparative Study of Factors Influencing Plasmid DNA Gene Delivery to Tumors . Int. J. Nanomedicine 8 , 1573–1593. 10.2147/IJN.S42800 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Chen Y., Wu Q., Zhang Z., Yuan L., Liu X., Zhou L. (2012). Preparation of Curcumin-Loaded Liposomes and Evaluation of Their Skin Permeation and Pharmacodynamics . Molecules 17 ( 5 ), 5972–5987. 10.3390/molecules17055972 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Cheng M. H. Y., Harmatys K. M., Charron D. M., Chen J., Zheng G. (2019). Stable J‐Aggregation of an aza‐BODIPY‐Lipid in a Liposome for Optical Cancer Imaging . Angew. Chem. 131 ( 38 ), 13528–13533. 10.1002/ange.201907754 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Cheng Y., Ji Y. (2019). RGD-modified Polymer and Liposome Nanovehicles: Recent Research Progress for Drug Delivery in Cancer Therapeutics . Eur. J. Pharm. Sci. 128 , 8–17. 10.1016/j.ejps.2018.11.023 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Chetoni P., Rossi S., Burgalassi S., Monti D., Mariotti S., Saettone M. F. (2004). Comparison of Liposome-Encapsulated Acyclovir with Acyclovir Ointment: Ocular Pharmacokinetics in Rabbits . J. Ocul. Pharmacol. Ther. 20 ( 2 ), 169–177. 10.1089/108076804773710849 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Chibowski E., Szcześ A. (2016). Zeta Potential and Surface Charge of DPPC and DOPC Liposomes in the Presence of PLC Enzyme . Adsorption 22 ( 4-6 ), 755–765. 10.1007/s10450-016-9767-z [ CrossRef ] [ Google Scholar ]
• Choi M. J., Maibach H. I. (2005). Liposomes and Niosomes as Topical Drug Delivery Systems . Skin Pharmacol. Physiol. 18 ( 5 ), 209–219. 10.1159/000086666 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Christensen D., Foged C., Rosenkrands I., Lundberg C. V., Andersen P., Agger E. M., et al. (2010). CAF01 Liposomes as a Mucosal Vaccine Adjuvant: In Vitro and In Vivo Investigations . Int. J. pharmaceutics 390 ( 1 ), 19–24. 10.1016/j.ijpharm.2009.10.043 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Chupin V., Boots J. W. P., Killian J. A., Demel R. A., de Kruijff B. (2002). Thermotropic Phase Behavior of Monoglyceride-Dicetylphosphate Dispersions and Interactions with Proteins: a 2H and 31P NMR Study . Biophysical J. 82 ( 2 ), 843–851. 10.1016/s0006-3495(02)75446-5 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Collier M. A., Bachelder E. M., Ainslie K. M. (2017). Electrosprayed Myocet-like Liposomes: an Alternative to Traditional Liposome Production . Pharm. Res. 34 ( 2 ), 419–426. 10.1007/s11095-016-2072-4 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Cordeiro C., Wiseman D. J., Lutwyche P., Uh M., Evans J. C., Finlay B. B., et al. (2000). Antibacterial Efficacy of Gentamicin Encapsulated in pH-Sensitive Liposomes against an In Vivo Salmonella enterica Serovar Typhimurium Intracellular Infection Model . Antimicrob. Agents Chemother. 44 ( 3 ), 533–539. 10.1128/aac.44.3.533-539.2000 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Crommelin D. J. A., van Hoogevest P., Storm G. (2020). The Role of Liposomes in Clinical Nanomedicine Development. What Now? Now what? . J. Controlled Release 318 , 256–263. 10.1016/j.jconrel.2019.12.023 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Csiszár A., Hersch N., Dieluweit S., Biehl R., Merkel R., Hoffmann B. (2010). Novel Fusogenic Liposomes for Fluorescent Cell Labeling and Membrane Modification . Bioconjug. Chem. 21 ( 3 ), 537–543. 10.1021/bc900470y [ PubMed ] [ CrossRef ] [ Google Scholar ]
• D'Avanzo N., Hyrc K., Enkvetchakul D., Covey D. F., Nichols C. G. (2011). Enantioselective Protein-Sterol Interactions Mediate Regulation of Both Prokaryotic and Eukaryotic Inward Rectifier K+ Channels by Cholesterol . PLoS One 6 ( 4 ), e19393. 10.1371/journal.pone.0019393 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Daemen T., Demare A., Bungener L., Dejonge J., Huckriede A., Wilschut J. (2005). Virosomes for Antigen and DNA Delivery . Adv. Drug Deliv. Rev. 57 ( 3 ), 451–463. 10.1016/j.addr.2004.09.005 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Dana P., Bunthot S., Suktham K., Surassmo S., Yata T., Namdee K., et al. (2020). Active Targeting Liposome-PLGA Composite for Cisplatin Delivery against Cervical Cancer . Colloids Surf. B: Biointerfaces 196 , 111270. 10.1016/j.colsurfb.2020.111270 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Daraee H., Etemadi A., Kouhi M., Alimirzalu S., Akbarzadeh A. (2016). Application of Liposomes in Medicine and Drug Delivery . Artif. Cell Nanomedicine, Biotechnol. 44 ( 1 ), 381–391. 10.3109/21691401.2014.953633 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Davidsen J., Rosenkrands I., Christensen D., Vangala A., Kirby D., Perrie Y., et al. (2005). Characterization of Cationic Liposomes Based on Dimethyldioctadecylammonium and Synthetic Cord Factor from M. tuberculosis (Trehalose 6,6'-Dibehenate)-A Novel Adjuvant Inducing Both strong CMI and Antibody Responses . Biochim. Biophys. Acta 1718 ( 1-2 ), 22–31. 10.1016/j.bbamem.2005.10.011 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Davidson E. M., Haroutounian S., Kagan L., Naveh M., Aharon A., Ginosar Y. (2016). A Novel Proliposomal Ropivacaine Oil . Anesth. Analgesia 122 ( 5 ), 1663–1672. 10.1213/ane.0000000000001200 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Dayan N., Touitou E. (2000). Carriers for Skin Delivery of Trihexyphenidyl HCl: Ethosomes vs. Liposomes . Biomaterials 21 ( 18 ), 1879–1885. 10.1016/s0142-9612(00)00063-6 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Demetzos C. (2008). Differential Scanning Calorimetry (DSC): a Tool to Study the thermal Behavior of Lipid Bilayers and Liposomal Stability . J. liposome Res. 18 ( 3 ), 159–173. 10.1080/08982100802310261 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Deng H., Song K., Zhao X., Li Y., Wang F., Zhang J., et al. (2017). Tumor Microenvironment Activated Membrane Fusogenic Liposome with Speedy Antibody and Doxorubicin Delivery for Synergistic Treatment of Metastatic Tumors . ACS Appl. Mater. Inter. 9 ( 11 ), 9315–9326. 10.1021/acsami.6b14683 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Deng M., Tu N., Bai F., Wang L. (2012). Surface Functionalization of Hydrophobic Nanocrystals with One Particle Per Micelle for Bioapplications . Chem. Mater. 24 ( 13 ), 2592–2597. 10.1021/cm301285g [ CrossRef ] [ Google Scholar ]
• Deniz A., Sade A., Severcan F., Keskin D., Tezcaner A., Banerjee S. (2010). Celecoxib-loaded Liposomes: Effect of Cholesterol on Encapsulation and In Vitro Release Characteristics . Biosci. Rep. 30 ( 5 ), 365–373. 10.1042/bsr20090104 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Devrim B., Kara A., Vural İ., Bozkır A. (2016). Lysozyme-loaded Lipid-Polymer Hybrid Nanoparticles: Preparation, Characterization and Colloidal Stability Evaluation . Drug Dev. Ind. Pharm. 42 ( 11 ), 1865–1876. 10.1080/03639045.2016.1180392 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Dokka S., Toledo D., Shi X., Castranova V., Rojanasakul Y. (2000). Oxygen Radical-Mediated Pulmonary Toxicity Induced by Some Cationic Liposomes . Pharm. Res. 17 ( 5 ), 521–525. 10.1023/a:1007504613351 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Domínguez A. R., Hidalgo D. O., Garrido R. V., Sánchez E. T. (2005). Liposomal Cytarabine (DepoCyte) for the Treatment of Neoplastic Meningitis . Clin. Transl Oncol. 7 ( 6 ), 232–238. 10.1007/bf02710168 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Dos Santos N., Mayer L. D., Abraham S. A., Gallagher R. C., Cox K. A. K., Tardi P. G., et al. (2002). Improved Retention of Idarubicin after Intravenous Injection Obtained for Cholesterol-free Liposomes . Biochim. Biophys. Acta (Bba) - Biomembranes 1561 ( 2 ), 188–201. 10.1016/s0005-2736(02)00345-0 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Drin G., Morello V., Casella J.-F., Gounon P., Antonny B. (2008). Asymmetric Tethering of Flat and Curved Lipid Membranes by a Golgin . Science 320 ( 5876 ), 670–673. 10.1126/science.1155821 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Drulis-Kawa Z., Dorotkiewicz-Jach A. (2010). Liposomes as Delivery Systems for Antibiotics . Int. J. Pharm. 387 ( 1-2 ), 187–198. 10.1016/j.ijpharm.2009.11.033 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Dua J., Rana A., Bhandari A. (2012). Liposome: Methods of Preparation and Applications . Int. J. Pharm. Stud. Res. 3 ( 2 ), 14–20. [ Google Scholar ]
• Ducat E., Deprez J., Gillet A., Noël A., Evrard B., Peulen O., et al. (2011). Nuclear Delivery of a Therapeutic Peptide by Long Circulating pH-Sensitive Liposomes: Benefits over Classical Vesicles . Int. J. pharmaceutics 420 ( 2 ), 319–332. 10.1016/j.ijpharm.2011.08.034 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Düzgünes N., Simões S., Slepushkin V., Pretzer E., Rossi J. J., De Clercq E., et al. (2001). Enhanced Inhibition of HIV-1 Replication in Macrophages by Antisense Oligonucleotides, Ribozymes and Acyclic Nucleoside Phosphonate Analogs Delivered in pH-Sensitive Liposomes . Nucleosides Nucleotides Nucleic Acids 20 ( 4-7 ), 515–523. 10.1081/NCN-100002327 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Dynarowicz-Łątka P., Seoane R., Minones J., Jr, Velo M., Minones J. (2003). Study of Penetration of Amphotericin B into Cholesterol or Ergosterol Containing Dipalmitoyl Phosphatidylcholine Langmuir Monolayers . Colloids Surf. B: Biointerfaces 27 ( 2-3 ), 249–263. [ Google Scholar ]
• El-Samaligy M. S., Afifi N. N., Mahmoud E. A. (2006). Evaluation of Hybrid Liposomes-Encapsulated Silymarin Regarding Physical Stability and In Vivo Performance . Int. J. Pharm. 319 ( 1-2 ), 121–129. 10.1016/j.ijpharm.2006.04.023 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• El-Samaligy M. S., Afifi N. N., Mahmoud E. A. (2006). Increasing Bioavailability of Silymarin Using a Buccal Liposomal Delivery System: Preparation and Experimental Design Investigation . Int. J. Pharm. 308 ( 1-2 ), 140–148. 10.1016/j.ijpharm.2005.11.006 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Ellis M., Bernsen R., Ali-Zadeh H., Kristensen J., Hedström U., Poughias L., et al. (2009). A Safety and Feasibility Study Comparing an Intermittent High Dose with a Daily Standard Dose of Liposomal Amphotericin B for Persistent Neutropenic Fever . J. Med. Microbiol. 58 ( 11 ), 1474–1485. 10.1099/jmm.0.012401-0 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Eloy J. O., Claro de Souza M., Petrilli R., Barcellos J. P. A., Lee R. J., Marchetti J. M. (2014). Liposomes as Carriers of Hydrophilic Small Molecule Drugs: Strategies to Enhance Encapsulation and Delivery . Colloids Surf. B: Biointerfaces 123 , 345–363. 10.1016/j.colsurfb.2014.09.029 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Epstein H., Gutman D., Cohen-Sela E., Haber E., Elmalak O., Koroukhov N., et al. (2008). Preparation of Alendronate Liposomes for Enhanced Stability and Bioactivity: In Vitro and In Vivo Characterization . Aaps J. 10 ( 4 ), 505–515. 10.1208/s12248-008-9060-5 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Evjen T. J., Nilssen E. A., Barnert S., Schubert R., Brandl M., Fossheim S. L. (2011). Ultrasound-mediated Destabilization and Drug Release from Liposomes Comprising Dioleoylphosphatidylethanolamine . Eur. J. Pharm. Sci. 42 ( 4 ), 380–386. 10.1016/j.ejps.2011.01.002 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Fang J.-Y., Fang C.-L., Liu C.-H., Su Y.-H. (2008). Lipid Nanoparticles as Vehicles for Topical Psoralen Delivery: Solid Lipid Nanoparticles (SLN) versus Nanostructured Lipid Carriers (NLC) . Eur. J. Pharmaceutics Biopharmaceutics 70 ( 2 ), 633–640. 10.1016/j.ejpb.2008.05.008 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Fang J.-Y., Hung C.-F., Hwang T.-L., Huang Y.-L. (2005). Physicochemical Characteristics Andin Vivodeposition of Liposome-Encapsulated tea Catechins by Topical and Intratumor Administrations . J. Drug Target. 13 ( 1 ), 19–27. 10.1080/10611860400015977 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Farzad N., Barati N., Momtazi-Borojeni A. A., Yazdani M., Arab A., Razazan A., et al. (2019). P435 HER2/neu-Derived Peptide Conjugated to Liposomes Containing DOPE as an Effective Prophylactic Vaccine Formulation for Breast Cancer . Artif. Cell Nanomedicine, Biotechnol. 47 ( 1 ), 664–672. 10.1080/21691401.2019.1576702 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Fattal E., Couvreur P., Dubernet C. (2004). "Smart" Delivery of Antisense Oligonucleotides by Anionic pH-Sensitive Liposomes . Adv. Drug Deliv. Rev. 56 ( 7 ), 931–946. 10.1016/j.addr.2003.10.037 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Faustino C. M. C., Calado A. R. T., Garcia-Rio L. (2011). Mixed Micelle Formation between Amino Acid-Based Surfactants and Phospholipids . J. Colloid Interf. Sci. 359 ( 2 ), 493–498. 10.1016/j.jcis.2011.04.016 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Felnerova D., Viret J.-F., Glück R., Moser C. (2004). Liposomes and Virosomes as Delivery Systems for Antigens, Nucleic Acids and Drugs . Curr. Opin. Biotechnol. 15 ( 6 ), 518–529. 10.1016/j.copbio.2004.10.005 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Figueroa-Robles A., Antunes-Ricardo M., Guajardo-Flores D. (2020). Encapsulation of Phenolic Compounds with Liposomal Improvement in the Cosmetic Industry . Int. J. Pharm. 593 , 120125. 10.1016/j.ijpharm.2020.120125 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Franzé S., Selmin F., Samaritani E., Minghetti P., Cilurzo F. (2018). Lyophilization of Liposomal Formulations: Still Necessary, Still Challenging . Pharmaceutics 10 ( 3 ), 139. 10.3390/pharmaceutics10030139 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Freedman M., Chang E. H., Zhou Q., Pirollo K. F. (2009). Nanodelivery of MRI Contrast Agent Enhances Sensitivity of Detection of Lung Cancer Metastases . Acad. Radiol. 16 ( 5 ), 627–637. 10.1016/j.acra.2008.12.002 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Frenzel M., Steffen-Heins A. (2015). Whey Protein Coating Increases Bilayer Rigidity and Stability of Liposomes in Food-like Matrices . Food Chem. 173 , 1090–1099. 10.1016/j.foodchem.2014.10.076 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Fuchsluger T. A., Hintschich C., Steuhl K.-P., Meller D. (2006). Adjuvante topische Interferon-α-2b-Therapie bei epithelialen Tumoren der Augenoberfläche . Ophthalmologe 103 ( 2 ), 124–128. 10.1007/s00347-005-1249-8 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Fujisawa T., Miyai H., Hironaka K., Tsukamoto T., Tahara K., Tozuka Y., et al. (2012). Liposomal Diclofenac Eye Drop Formulations Targeting the Retina: Formulation Stability Improvement Using Surface Modification of Liposomes . Int. J. Pharm. 436 ( 1-2 ), 564–567. 10.1016/j.ijpharm.2012.07.024 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Gabriëls M., Plaizier-Vercammen J. (2003). Physical and Chemical Evaluation of Liposomes, Containing Artesunate . J. Pharm. Biomed. Anal. 31 ( 4 ), 655–667. 10.1016/s0731-7085(02)00678-7 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Garbuzenko O., Barenholz Y., Priev A. (2005). Effect of Grafted PEG on Liposome Size and on Compressibility and Packing of Lipid Bilayer . Chem. Phys. Lipids 135 ( 2 ), 117–129. 10.1016/j.chemphyslip.2005.02.003 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Garg T., K. Goyal A. (2014). Liposomes: Targeted and Controlled Delivery System . Ddl 4 ( 1 ), 62–71. 10.2174/22103031113036660015 [ CrossRef ] [ Google Scholar ]
• Ghanbarzadeh S., Valizadeh H., Zakeri-Milani P. (2013). The Effects of Lyophilization on the Physico-Chemical Stability of Sirolimus Liposomes . Adv. Pharm. Bull. 3 ( 1 ), 25–29. 10.5681/apb.2013.005 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Ghazizadeh E., Neshastehriz A. (2020). Different Liposome Patterns to Detection of Acute Leukemia Based on Electrochemical Cell Sensor . Analytica Chim. Acta . [ PubMed ] [ Google Scholar ]
• Giulimondi F., Digiacomo L., Pozzi D., Palchetti S., Vulpis E., Capriotti A. L., et al. (2019). Interplay of Protein corona and Immune Cells Controls Blood Residency of Liposomes . Nat. Commun. 10 ( 1 ), 3686–3711. 10.1038/s41467-019-11642-7 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Goto T., Morishita M., Nishimura K., Nakanishi M., Kato A., Ehara J., et al. (2006). Novel Mucosal Insulin Delivery Systems Based on Fusogenic Liposomes . Pharm. Res. 23 ( 2 ), 384–391. 10.1007/s11095-005-9175-7 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Gross N., Ranjbar M., Evers C., Hua J., Martin G., Schulze B., et al. (2013). Choroidal Neovascularization Reduced by Targeted Drug Delivery with Cationic Liposome-Encapsulated Paclitaxel or Targeted Photodynamic Therapy with Verteporfin Encapsulated in Cationic Liposomes . Mol. Vis. 19 , 54–61. [ PMC free article ] [ PubMed ] [ Google Scholar ]
• Gullapalli R. R., Demirel M. C., Butler P. J. (2008). Molecular Dynamics Simulations of DiI-C18(3) in a DPPC Lipid Bilayer . Phys. Chem. Chem. Phys. 10 ( 24 ), 3548–3560. 10.1039/b716979e [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Hadavi R., Jafari S. M., Katouzian I. (2020). Nanoliposomal Encapsulation of Saffron Bioactive Compounds; Characterization and Optimization . Int. J. Biol. Macromolecules 164 , 4046–4053. 10.1016/j.ijbiomac.2020.09.028 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Hahn V., Friedt W. (2012). Important Characters by Recombinant DNA Technology . Prog. Bot. Stuctural Bot. Physiol. Genet. Taxonomy Geobotany/Fortschritte der Botanik Struktur Physiologie Genetik Systematik Geobotanik 53 , 181. [ Google Scholar ]
• Hashemzadeh H., Javadi H., Darvishi M. H. (2020). Study of Structural Stability and Formation Mechanisms in DSPC and DPSM Liposomes: A Coarse-Grained Molecular Dynamics Simulation . Sci. Rep. 10 ( 1 ), 1837–1910. 10.1038/s41598-020-58730-z [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Hashemzadeh H., Javadi H., Darvishi M. H. (2020). Study of Structural Stability and Formation Mechanisms in DSPC and DPSM Liposomes: A Coarse-Grained Molecular Dynamics Simulation . Sci. Rep. 10 ( 1 ), 1837. 10.1038/s41598-020-58730-z [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Hathout R. M., Mansour S., Mortada N. D., Guinedi A. S. (2007). Liposomes as an Ocular Delivery System for Acetazolamide: In Vitro and In Vivo Studies . Aaps Pharmscitech 8 ( 1 ), E1–E12. 10.1208/pt0801001 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• He J., Evers D. L., O’Leary T. J., Mason J. T. (2012). Immunoliposome-PCR: a Generic Ultrasensitive Quantitative Antigen Detection System . J. nanobiotechnology 10 ( 1 ), 26. 10.1186/1477-3155-10-26 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• He K., Tang M. (2018). Safety of Novel Liposomal Drugs for Cancer Treatment: Advances and Prospects . Chemico-Biological Interactions 295 , 13–19. 10.1016/j.cbi.2017.09.006 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Hilmer S. N., Cogger V. C., Muller M., Le Couteur D. G. (2004). The Hepatic Pharmacokinetics of Doxorubicin and Liposomal Doxorubicin . Drug Metab. Dispos 32 ( 8 ), 794–799. 10.1124/dmd.32.8.794 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Hirano M., Nakamura S., Mitsunaga F., Okada M., Shimuzu K., Imamura T. (2002). Transfer of Maternally Administered Fusogenic Liposome-DNA Complexes into Monkey Fetuses in a Pregnancy Model . J. Gene Med. 4 ( 5 ), 560–566. 10.1002/jgm.289 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Ho J. A., Zeng S. C., Huang M. R., Kuo H. Y. (2006). Development of Liposomal Immunosensor for the Measurement of Insulin with Femtomole Detection . Anal. Chim. Acta 556 ( 1 ), 127–132. 10.1016/j.aca.2005.08.074 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Homann H.-H., Rosbach O., Moll W., Vogt P. M., Germann G., Hopp M., et al. (2007). A Liposome Hydrogel with Polyvinyl-Pyrrolidone Iodine in the Local Treatment of Partial-Thickness Burn Wounds . Ann. Plast. Surg. 59 ( 4 ), 423–427. 10.1097/sap.0b013e3180326fcf [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Hong S.-S., Oh K. T., Choi H.-G., Lim S.-J. (2019). Liposomal Formulations for Nose-To-Brain Delivery: Recent Advances and Future Perspectives . Pharmaceutics 11 ( 10 ), 540. 10.3390/pharmaceutics11100540 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Hosny K. M. (2010). Ciprofloxacin as Ocular Liposomal Hydrogel . Aaps Pharmscitech 11 ( 1 ), 241–246. 10.1208/s12249-009-9373-4 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Hu Q., Bally M. B., Madden T. D. (2002). Subcellular Trafficking of Antisense Oligonucleotides and Down-Regulation of Bcl-2 Gene Expression in Human Melanoma Cells Using a Fusogenic Liposome Delivery System . Nucleic Acids Res. 30 ( 16 ), 3632–3641. 10.1093/nar/gkf448 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Hua S. (2013). Targeting Sites of Inflammation: Intercellular Adhesion Molecule-1 as a Target for Novel Inflammatory Therapies . Front. Pharmacol. 4 , 127. 10.3389/fphar.2013.00127 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Huang F.-Y. J., Hung C.-C., Chang C.-W., Chao J.-H., Hsieh B.-T. (2018). Evaluation of Injectable Chitosan-Based Co-cross-linking Hydrogel for Local Delivery of 188Re-LIPO-DOX to Breast-Tumor-Bearing Mouse Model . Anticancer Res. 38 ( 8 ), 4651–4659. 10.21873/anticanres.12770 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Huh S. Y., Shin J. W., Na J. I., Huh C. H., Youn S. W., Park K. C. (2010). Efficacy and Safety of Liposome-Encapsulated 4-N-Butylresorcinol 0.1% Cream for the Treatment of Melasma: A Randomized Controlled Split-Face Trial . J. Dermatol. 37 ( 4 ), 311–315. 10.1111/j.1346-8138.2010.00787.x [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Husain S., Capitano B., Corcoran T., Studer S. M., Crespo M., Johnson B., et al. (2010). Intrapulmonary Disposition of Amphotericin B after Aerosolized Delivery of Amphotericin B Lipid Complex (Abelcet; ABLC) in Lung Transplant Recipients . Transplantation 90 ( 11 ), 1215–1219. 10.1097/tp.0b013e3181f995ea [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Ib G., Corredig M. (2013). Storage Stability and Physical Characteristics of tea-polyphenol-bearing Nanoliposomes Prepared with Milk Fat Globule Membrane Phospholipids . J. Agric. Food Chem. 61 ( 13 ), 3242–3251. [ PubMed ] [ Google Scholar ]
• Ichikawa K., Hikita T., Maeda N., Takeuchi Y., Namba Y., Oku N. (2004). PEGylation of Liposome Decreases the Susceptibility of Liposomal Drug in Cancer Photodynamic Therapy . Biol. Pharm. Bull. 27 ( 3 ), 443–444. 10.1248/bpb.27.443 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Ichikawa K., Hikita T., Maeda N., Yonezawa S., Takeuchi Y., Asai T., et al. (2005). Antiangiogenic Photodynamic Therapy (PDT) by Using Long-Circulating Liposomes Modified with Peptide Specific to Angiogenic Vessels . Biochim. Biophys. Acta (Bba) - Biomembranes 1669 ( 1 ), 69–74. 10.1016/j.bbamem.2005.02.003 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Jacoby G., Cohen K., Barkan K., Talmon Y., Peer D., Beck R. (2015). Metastability in Lipid Based Particles Exhibits Temporally Deterministic and Controllable Behavior . Sci. Rep. 5 , 9481. 10.1038/srep09481 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Jain A., Jain S. K. (2016). In Vitro release Kinetics Model Fitting of Liposomes: An Insight . Chem. Phys. Lipids 201 , 28–40. 10.1016/j.chemphyslip.2016.10.005 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Jain A. K., Thanki K., Jain S. (2014). Solidified Self-Nanoemulsifying Formulation for Oral Delivery of Combinatorial Therapeutic Regimen: Part I. Formulation Development, Statistical Optimization, and In Vitro Characterization . Pharm. Res. 31 ( 4 ), 923–945. 10.1007/s11095-013-1213-2 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Jain M., Zellweger M., Frobert A., Valentin J., Bergh H. v. d., Wagnières G., et al. (2016). Intra-Arterial Drug and Light Delivery for Photodynamic Therapy Using Visudyne: Implication for Atherosclerotic Plaque Treatment . Front. Physiol. 7 , 400. 10.3389/fphys.2016.00400 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Jain N., Gupta B. P., Thakur N., Jain R., Banweer J., Jain D. K., et al. (2010). Phytosome: a Novel Drug Delivery System for Herbal Medicine . Int. J. Pharm. Sci. Drug Res. 2 ( 4 ), 224–228. [ Google Scholar ]
• Jeon M., Kim G., Lee W., Im H-J. (2020). Development of Theranostic PEGylated Liposomal Au-Liposome for Effective Tumor Passive Targeting and Photothermal Therapy . Soc. Nucl. Med 61 , 1076. [ Google Scholar ]
• Jimbo T., Sakuma Y., Urakami N., Ziherl P., Imai M. (2016). Role of Inverse-Cone-Shape Lipids in Temperature-Controlled Self-Reproduction of Binary Vesicles . Biophysical J. 110 ( 7 ), 1551–1562. 10.1016/j.bpj.2016.02.028 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Jiménez-Escrig A., Sánchez-Muniz F. J. (2000). Dietary Fibre from Edible Seaweeds: Chemical Structure, Physicochemical Properties and Effects on Cholesterol Metabolism . Nutr. Res. 20 ( 4 ), 585–598. 10.1016/s0271-5317(00)00149-4 [ CrossRef ] [ Google Scholar ]
• Johnston M. J. W., Semple S. C., Klimuk S. K., Ansell S., Maurer N., Cullis P. R. (2007). Characterization of the Drug Retention and Pharmacokinetic Properties of Liposomal Nanoparticles Containing Dihydrosphingomyelin . Biochim. Biophys. Acta (Bba) - Biomembranes 1768 ( 5 ), 1121–1127. 10.1016/j.bbamem.2007.01.019 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Johnstone S. A., Masin D., Mayer L., Bally M. B. (2001). Surface-associated Serum Proteins Inhibit the Uptake of Phosphatidylserine and Poly(ethylene Glycol) Liposomes by Mouse Macrophages . Biochim. Biophys. Acta (Bba) - Biomembranes 1513 ( 1 ), 25–37. 10.1016/s0005-2736(01)00292-9 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Joly F., Ray-Coquard I., Fabbro M., Donoghoe M., Boman K., Sugimoto A., et al. (2011). Decreased Hypersensitivity Reactions with Carboplatin-Pegylated Liposomal Doxorubicin Compared to Carboplatin-Paclitaxel Combination: Analysis from the GCIG CALYPSO Relapsing Ovarian Cancer Trial . Gynecol. Oncol. 122 ( 2 ), 226–232. 10.1016/j.ygyno.2011.04.019 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Joshi S., Hussain M. T., Roces C. B., Anderluzzi G., Kastner E., Salmaso S., et al. (2016). Microfluidics Based Manufacture of Liposomes Simultaneously Entrapping Hydrophilic and Lipophilic Drugs . Int. J. pharmaceutics 514 ( 1 ), 160–168. 10.1016/j.ijpharm.2016.09.027 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Jung H.-S., Ishii H., Shimanouchi T., Umakoshi H., Kuboi R. (2007). Immobilized-liposome Sensor System for Detection of Proteins under Stress Conditions . Membrane 32 ( 5 ), 294–301. 10.5360/membrane.32 [ CrossRef ] [ Google Scholar ]
• Jung J. H., Ree M., Kim H. (2006). Acid-and Base-Catalyzed Hydrolyses of Aliphatic Polycarbonates and Polyesters . Catal. Today 115 ( 1-4 ), 283–287. 10.1016/j.cattod.2006.02.060 [ CrossRef ] [ Google Scholar ]
• Jung S.-Y., Holden M. A., Cremer P. S., Collier C. P. (2005). Two-Component Membrane Lithography via Lipid Backfilling . ChemPhysChem. 6 ( 3 ), 423–426. 10.1002/cphc.200400540 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Jung S. H., Jung S. H., Seong H., Cho S. H., Jeong K. S., Shin B. C. (2009). Polyethylene Glycol-Complexed Cationic Liposome for Enhanced Cellular Uptake and Anticancer Activity . Int. J. Pharm. 382 ( 1-2 ), 254–261. 10.1016/j.ijpharm.2009.08.002 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Park J. W., Benz C. C., Martin F. J. (2004). in Future Directions of Liposome-And Immunoliposome-Based Cancer Therapeutics. Seminars in Oncology (Elsevier; ). [ PubMed ] [ Google Scholar ]
• Kafle A., Akamatsu M., Bhadani A., Sakai K., Kaise C., Kaneko T., et al. (2020). Phase Behavior of the Bilayers Containing Hydrogenated Soy Lecithin and β-sitosteryl Sulfate . Langmuir . 10.1021/acs.langmuir.0c00472 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Kang S. H., Cho H.-J., Shim G., Lee S., Kim S.-H., Choi H.-G., et al. (2011). Cationic Liposomal Co-delivery of Small Interfering RNA and a MEK Inhibitor for Enhanced Anticancer Efficacy . Pharm. Res. 28 ( 12 ), 3069–3078. 10.1007/s11095-011-0569-4 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Karmali P. P., Chaudhuri A. (2007). Cationic Liposomes as Non-viral Carriers of Gene Medicines: Resolved Issues, Open Questions, and Future Promises . Med. Res. Rev. 27 ( 5 ), 696–722. 10.1002/med.20090 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Kashapov R., Ibragimova A., Pavlov R., Gabdrakhmanov D., Kashapova N., Burilova E., et al. (2021). Nanocarriers for Biomedicine: From Lipid Formulations to Inorganic and Hybrid Nanoparticles . Ijms 22 ( 13 ), 7055. 10.3390/ijms22137055 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Kaur G., Garg T., Rath G., Goyal A. K. (2016). Archaeosomes: an Excellent Carrier for Drug and Cell Delivery . Drug Deliv. 23 ( 7 ), 2497–2512. 10.3109/10717544.2015.1019653 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Khadke S., Roces C. B., Donaghey R., Giacobbo V., Su Y., Perrie Y. (2020). Scalable Solvent-free Production of Liposomes . J. Pharm. Pharmacol. 72 ( 10 ), 1328–1340. 10.1111/jphp.13329 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Khosravi-Darani K., Zahed O., Aarabi S. (2018). Antioxidant activity of Zataria multiflora boiss. Essential oil encapsulated in nanoliposome in broth media and minced beef . Pak. J. Biotechnol. 15 ( 2 ), 365–375. [ Google Scholar ]
• Kidd P. M. (2009). Bioavailability and Activity of Phytosome Complexes from Botanical Polyphenols: the Silymarin, Curcumin, green tea, and Grape Seed Extracts . Altern. Med. Rev. 14 ( 3 ), 226–246. [ PubMed ] [ Google Scholar ]
• Kitayama H., Takechi Y., Tamai N., Matsuki H., Yomota C., Saito H. (2014). Thermotropic Phase Behavior of Hydrogenated Soybean Phosphatidylcholine-Cholesterol Binary Liposome Membrane . Chem. Pharm. Bull. 62 ( 1 ), 58–63. 10.1248/cpb.c13-00587 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Kleusch C., Hersch N., Hoffmann B., Merkel R., Csiszár A. (2012). Fluorescent Lipids: Functional Parts of Fusogenic Liposomes and Tools for Cell Membrane Labeling and Visualization . Molecules 17 ( 1 ), 1055–1073. 10.3390/molecules17011055 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Kshirsagar N. A., Pandya S. K., Kirodian G. B., Sanath S. (2005). Liposomal Drug Delivery System from Laboratory to Clinic . J. Postgrad. Med. 51 Suppl 1 ( 5 ), S5–S15. [ PubMed ] [ Google Scholar ]
• Kunisawa J., Masuda T., Katayama K., Yoshikawa T., Tsutsumi Y., Akashi M., et al. (2005). Fusogenic Liposome Delivers Encapsulated Nanoparticles for Cytosolic Controlled Gene Release . J. controlled release 105 ( 3 ), 344–353. 10.1016/j.jconrel.2005.03.020 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Kunzelmann-Marche C., Freyssinet J.-M., Martínez M. C. (2002). Loss of Plasma Membrane Phospholipid Asymmetry Requires Raft Integrity . J. Biol. Chem. 277 ( 22 ), 19876–19881. 10.1074/jbc.m200324200 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Laouini A., Jaafar-Maalej C., Limayem-Blouza I., Sfar S., Charcosset C., Fessi H. (2012). Preparation, Characterization and Applications of Liposomes: State of the Art . J Coll. Sci. Biotechnol. 1 ( 2 ), 147–168. 10.1166/jcsb.2012.1020 [ CrossRef ] [ Google Scholar ]
• Lebègue E., Anderson C. M., Dick J. E., Webb L. J., Bard A. J. (2015). Electrochemical Detection of Single Phospholipid Vesicle Collisions at a Pt Ultramicroelectrode . Langmuir 31 ( 42 ), 11734–11739. 10.1021/acs.langmuir.5b03123 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Lee C. M., Flynn R., Hollywood J. A., Scallan M. F., Harrison P. T. (2012). Correction of the ΔF508 Mutation in the Cystic Fibrosis Transmembrane Conductance Regulator Gene by Zinc-Finger Nuclease Homology-Directed Repair . BioResearch open access 1 ( 3 ), 99–108. 10.1089/biores.2012.0218 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Lee J., Kim H.-J., Kim J. (2008). Polydiacetylene Liposome Arrays for Selective Potassium Detection . J. Am. Chem. Soc. 130 ( 15 ), 5010–5011. 10.1021/ja709996c [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Lee S. C., Lee K. E., Kim J. J., Lim S. H. (2005). The Effect of Cholesterol in the Liposome Bilayer on the Stabilization of Incorporated Retinol . J. Liposome Res. 15 ( 3-4 ), 157–166. 10.1080/08982100500364131 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Li J., Wang X., Zhang T., Wang C., Huang Z., Luo X., et al. (2015). A Review on Phospholipids and Their Main Applications in Drug Delivery Systems . Asian J. Pharm. Sci. 10 ( 2 ), 81–98. 10.1016/j.ajps.2014.09.004 [ CrossRef ] [ Google Scholar ]
• Li M., Du C., Guo N., Teng Y., Meng X., Sun H., et al. (2019). Composition Design and Medical Application of Liposomes . Eur. J. Med. Chem. 164 , 640–653. 10.1016/j.ejmech.2019.01.007 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Liang L. P. T. H. J., Chung T. W., Liu Y. Y. H. D. Z. (2007). Liposomes Incorporated with Cholesterol for Drug Release Triggered by Magnetic Field . J. Med. Biol. Eng. 27 ( 1 ), 29–34. [ Google Scholar ]
• Liang X. F., Wang H. J., Luo H., Tian H., Zhang B. B., Hao L. J., et al. (2008). Characterization of Novel Multifunctional Cationic Polymeric Liposomes Formed from Octadecyl Quaternized Carboxymethyl Chitosan/cholesterol and Drug Encapsulation . Langmuir 24 ( 14 ), 7147–7153. 10.1021/la703775a [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Lira R. B., Seabra M. A. B. L., Matos A. L. L., Vasconcelos J. V., Bezerra D. P., de Paula E., et al. (2013). Studies on Intracellular Delivery of Carboxyl-Coated CdTe Quantum Dots Mediated by Fusogenic Liposomes . J. Mater. Chem. B 1 ( 34 ), 4297–4305. 10.1039/c3tb20245c [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Liu B., Krieger M. (2002). Highly Purified Scavenger Receptor Class B, Type I Reconstituted into Phosphatidylcholine/cholesterol Liposomes Mediates High Affinity High Density Lipoprotein Binding and Selective Lipid Uptake . J. Biol. Chem. 277 ( 37 ), 34125–34135. 10.1074/jbc.m204265200 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Liu W. L., Zou M. Z., Qin S. Y., Cheng Y. J., Ma Y. H., Sun Y. X., et al. (2020). Recent Advances of Cell Membrane‐Coated Nanomaterials for Biomedical Applications . Adv. Funct. Mater. 30 ( 39 ), 2003559. 10.1002/adfm.202003559 [ CrossRef ] [ Google Scholar ]
• Liu X., Madhankumar A. B., Miller P. A., Duck K. A., Hafenstein S., Rizk E., et al. (2016). MRI Contrast Agent for Targeting Glioma: Interleukin-13 Labeled Liposome Encapsulating Gadolinium-DTPA . Neuro Oncol. 18 ( 5 ), 691–699. 10.1093/neuonc/nov263 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Liu Y., Castro Bravo K. M., Liu J. (2021). Targeted Liposomal Drug Delivery: a Nanoscience and Biophysical Perspective . Nanoscale Horiz. 6 ( 2 ), 78–94. 10.1039/d0nh00605j [ PubMed ] [ CrossRef ] [ Google Scholar ]
• López-Pinto J. M., González-Rodríguez M. L., Rabasco A. M. (2005). Effect of Cholesterol and Ethanol on Dermal Delivery from DPPC Liposomes . Int. J. pharmaceutics 298 ( 1 ), 1–12. 10.1016/j.ijpharm.2005.02.021 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• López-Revuelta A., Sánchez-Gallego J. I., Hernández-Hernández A., Sánchez-Yagüe J., Llanillo M. (2006). Membrane Cholesterol Contents Influence the Protective Effects of Quercetin and Rutin in Erythrocytes Damaged by Oxidative Stress . Chemico-biological interactions 161 ( 1 ), 79–91. 10.1016/j.cbi.2006.03.004 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Lowis S., Lewis I., Lewis I., Elsworth A., Weston C., Doz F., et al. (2006). A Phase I Study of Intravenous Liposomal Daunorubicin (DaunoXome) in Paediatric Patients with Relapsed or Resistant Solid Tumours . Br. J. Cancer 95 ( 5 ), 571–580. 10.1038/sj.bjc.6603288 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Lu F. S. H., Nielsen N. S., Baron C. P., Jensen L. H. S., Jacobsen C. (2012). Physico-chemical Properties of marine Phospholipid Emulsions . J. Am. Oil Chem. Soc. 89 ( 11 ), 2011–2024. 10.1007/s11746-012-2105-z [ CrossRef ] [ Google Scholar ]
• Lv H., Zhang S., Wang B., Cui S., Yan J. (2006). Toxicity of Cationic Lipids and Cationic Polymers in Gene Delivery . J. Controlled Release 114 ( 1 ), 100–109. 10.1016/j.jconrel.2006.04.014 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Maeda N., Takeuchi Y., Takada M., Sadzuka Y., Namba Y., Oku N. (2004). Anti-neovascular Therapy by Use of Tumor Neovasculature-Targeted Long-Circulating Liposome . J. controlled release 100 ( 1 ), 41–52. 10.1016/j.jconrel.2004.07.033 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Manconi M., Marongiu F., Castangia I., Manca M. L., Caddeo C., Tuberoso C. I. G., et al. (2016). Polymer-associated Liposomes for the Oral Delivery of Grape Pomace Extract . Colloids Surf. B: biointerfaces 146 , 910–917. 10.1016/j.colsurfb.2016.07.043 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Mannino R., Lu R. (2018). Cochleates Made with Soy Phosphatidylserine . Google Patents . [ Google Scholar ]
• Mansoori M., Agrawal S., Jawade S., Khan M. (2012). A Review on Liposome . Int. J. Adv. Res. Pharm. Bio-sciences 2 ( 4 ), 453–464. [ Google Scholar ]
• Maritim S., Boulas P., Lin Y. (2021). Comprehensive Analysis of Liposome Formulation Parameters and Their Influence on Encapsulation, Stability and Drug Release in Glibenclamide Liposomes . Int. J. Pharmaceutics 592 , 120051. 10.1016/j.ijpharm.2020.120051 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Marsh D. (2001). Elastic Constants of Polymer-Grafted Lipid Membranes . Biophysical J. 81 ( 4 ), 2154–2162. 10.1016/s0006-3495(01)75863-8 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Matole V., Shegaonkar A., Kumbhar S., Thorat Y., Hosmani A. (2020). Need of Liposomes as a Novel Drug Delivery System . Res. J. Pharm. Dosage Forms Technology 12 ( 4 ), 285–294. 10.5958/0975-4377.2020.00047.6 [ CrossRef ] [ Google Scholar ]
• Matsuoka S., Murata M. (2002). Cholesterol Markedly Reduces Ion Permeability Induced by Membrane-Bound Amphotericin B. . Biochim. Biophys. Acta (Bba) - Biomembranes 1564 ( 2 ), 429–434. 10.1016/s0005-2736(02)00491-1 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Mehnert W., Mäder K. (2012). Solid Lipid Nanoparticles . Adv. Drug Deliv. Rev. 64 , 83–101. 10.1016/j.addr.2012.09.021 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Metselaar J. M., Wauben M. H. M., Wagenaar-Hilbers J. P. A., Boerman O. C., Storm G. (2003). Complete Remission of Experimental Arthritis by Joint Targeting of Glucocorticoids with Long-Circulating Liposomes . Arthritis Rheum. 48 ( 7 ), 2059–2066. 10.1002/art.11140 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Meure L. A., Foster N. R., Dehghani F. (2008). Conventional and Dense Gas Techniques for the Production of Liposomes: a Review . Aaps Pharmscitech 9 ( 3 ), 798. 10.1208/s12249-008-9097-x [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Miao L., Nielsen M., Thewalt J., Ipsen J. H., Bloom M., Zuckermann M. J., et al. (2002). From Lanosterol to Cholesterol: Structural Evolution and Differential Effects on Lipid Bilayers . Biophysical J. 82 ( 3 ), 1429–1444. 10.1016/s0006-3495(02)75497-0 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Moghimi S. M., Hunter A. C., Murray J. C. (2001). Long-circulating and Target-specific Nanoparticles: Theory to Practice . Pharmacol. Rev. 53 ( 2 ), 283–318. [ PubMed ] [ Google Scholar ]
• Moghimi S. M., Patel H. M. (2002). Modulation of Murine Liver Macrophage Clearance of Liposomes by Diethylstilbestrol. The Effect of Vesicle Surface Charge and a Role for the Complement Receptor Mac-1 (CD11b/CD18) of Newly Recruited Macrophages in Liposome Recognition . J. Control. Release 78 ( 1-3 ), 55–65. 10.1016/s0168-3659(01)00481-3 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Moghimi S. M., Szebeni J. (2003). Stealth Liposomes and Long Circulating Nanoparticles: Critical Issues in Pharmacokinetics, Opsonization and Protein-Binding Properties . Prog. lipid Res. 42 ( 6 ), 463–478. 10.1016/s0163-7827(03)00033-x [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Montaseri H., Kruger C. A., Abrahamse H. (2020). Review: Organic Nanoparticle Based Active Targeting for Photodynamic Therapy Treatment of Breast Cancer Cells . Oncotarget 11 ( 22 ), 2120–2136. 10.18632/oncotarget.27596 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Mozafari M. R. (2005). Liposomes: an Overview of Manufacturing Techniques . Cell Mol Biol Lett 10 ( 4 ), 711–719. [ PubMed ] [ Google Scholar ]
• Mozafari M. R., Reed C. J., Rostron C. (2007). Cytotoxicity Evaluation of Anionic Nanoliposomes and Nanolipoplexes Prepared by the Heating Method without Employing Volatile Solvents and Detergents . Pharmazie 62 ( 3 ), 205–209. [ PubMed ] [ Google Scholar ]
• Müller R. H., Mäder K., Gohla S. (2000). Solid Lipid Nanoparticles (SLN) for Controlled Drug Delivery - a Review of the State of the Art . Eur. J. Pharm. Biopharm. 50 ( 1 ), 161–177. 10.1016/s0939-6411(00)00087-4 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Müller R. H., Radtke M., Wissing S. A. (2002). Solid Lipid Nanoparticles (SLN) and Nanostructured Lipid Carriers (NLC) in Cosmetic and Dermatological Preparations . Adv. Drug Deliv. Rev. 54 , S131–S155. 10.1016/s0169-409x(02)00118-7 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Müller R., Petersen R., Hommoss A., Pardeike J. (2007). Nanostructured Lipid Carriers (NLC) in Cosmetic Dermal Products☆ . Adv. Drug Deliv. Rev. 59 ( 6 ), 522–530. 10.1016/j.addr.2007.04.012 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Nagarajan R. (2002). Molecular Packing Parameter and Surfactant Self-Assembly: The Neglected Role of the Surfactant Tail† . Langmuir 18 ( 1 ), 31–38. 10.1021/la010831y [ CrossRef ] [ Google Scholar ]
• Nakanishi T., Hayashi A., Kunisawa J., Tsutsumi Y., Tanaka K., Yashiro-Ohtani Y., et al. (2000). Fusogenic Liposomes Efficiently Deliver Exogenous Antigen through the Cytoplasm into the MHC Class I Processing Pathway . Eur. J. Immunol. 30 ( 6 ), 1740–1747. 10.1002/1521-4141(200006)30:6<1740::aid-immu1740>3.0.co;2-u [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Naskar A., Kim K.-s. (2021). Potential novel food-related and biomedical applications of nanomaterials combined with bacteriocins . Pharmaceutics 13 , 86. 10.3390/pharmaceutics13010086 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Nasrabadi H. T., Abbasi E., Davaran S., Kouhi M., Akbarzadeh A. (2016). Bimetallic Nanoparticles: Preparation, Properties, and Biomedical Applications . Artif. Cell Nanomedicine, Biotechnol. 44 ( 1 ), 376–380. 10.3109/21691401.2014.953632 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Nasti T. H., Khan M. A., Owais M. (2006). Enhanced Efficacy of pH-Sensitive Nystatin Liposomes against Cryptococcus Neoformans in Murine Model . J. Antimicrob. Chemother. 57 ( 2 ), 349–352. 10.1093/jac/dki454 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Noble G. T., Stefanick J. F., Ashley J. D., Kiziltepe T., Bilgicer B. (2014). Ligand-targeted Liposome Design: Challenges and Fundamental Considerations . Trends Biotechnology 32 ( 1 ), 32–45. 10.1016/j.tibtech.2013.09.007 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• North S., Butts C. (2005). Vaccination with BLP25 Liposome Vaccine to Treat Non-small Cell Lung and Prostate Cancers . Expert Rev. Vaccin. 4 ( 3 ), 249–257. 10.1586/14760584.4.3.249 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Nunes S. S., Fernandes R. S., Cavalcante C. H., da Costa César I., Leite E. A., Lopes S. C. A., et al. (2019). Influence of PEG Coating on the Biodistribution and Tumor Accumulation of pH-Sensitive Liposomes . Drug Deliv. Transl. Res. 9 ( 1 ), 123–130. 10.1007/s13346-018-0583-8 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Ohvo-Rekilà H., Ramstedt B., Leppimäki P., Slotte J. P. (2002). Cholesterol Interactions with Phospholipids in Membranes . Prog. lipid Res. 41 ( 1 ), 66–97. 10.1016/s0163-7827(01)00020-0 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Okamoto Y., Masum S. M., Miyazawa H., Yamazaki M. (2008). Low-pH-induced Transformation of Bilayer Membrane into Bicontinuous Cubic Phase in Dioleoylphosphatidylserine/monoolein Membranes . Langmuir 24 ( 7 ), 3400–3406. 10.1021/la7036795 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Otake K., Imura T., Sakai H., Abe M. (2001). Development of a New Preparation Method of Liposomes Using Supercritical Carbon Dioxide . langmuir 17 ( 13 ), 3898–3901. 10.1021/la010122k [ CrossRef ] [ Google Scholar ]
• Pan L. G., Tomás M. C., Añón M. C. (2002). Effect of sunflower Lecithins on the Stability of Water-In-Oil and Oil-In-Water Emulsions . J. Surfact Deterg 5 ( 2 ), 135–143. 10.1007/s11743-002-0213-1 [ CrossRef ] [ Google Scholar ]
• Panahi Y., Farshbaf M., Mohammadhosseini M., Mirahadi M., Khalilov R., Saghfi S., et al. (2017). Recent Advances on Liposomal Nanoparticles: Synthesis, Characterization and Biomedical Applications . Artif. Cell Nanomedicine, Biotechnol. 45 ( 4 ), 788–799. 10.1080/21691401.2017.1282496 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Papagiannaros A., Hatziantoniou S., Dimas K., Papaioannou G. T., Demetzos C. (2006). A Liposomal Formulation of Doxorubicin, Composed of Hexadecylphosphocholine (HePC): Physicochemical Characterization and Cytotoxic Activity against Human Cancer Cell Lines . Biomed. Pharmacother. 60 ( 1 ), 36–42. 10.1016/j.biopha.2005.09.001 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Pastorino F., Brignole C., Marimpietri D., Cilli M., Gambini C., Ribatti D., et al. (2003). Vascular Damage and Anti-angiogenic Effects of Tumor Vessel-Targeted Liposomal Chemotherapy . Cancer Res. 63 ( 21 ), 7400–7409. [ PubMed ] [ Google Scholar ]
• Patel N., Panda S. (2012). Liposome Drug Delivery System: a Critic Review . JPSBR 2 ( 4 ), 169–175. [ Google Scholar ]
• Pavelić Z., Skalko-Basnet N., Jalsenjak I. (2005). Characterisation and In Vitro Evaluation of Bioadhesive Liposome Gels for Local Therapy of Vaginitis . Int. J. Pharm. 301 ( 1-2 ), 140–148. 10.1016/j.ijpharm.2005.05.022 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Pawar A., Bothiraja C., Shaikh K., Mali A. (2015). An Insight into Cochleates, a Potential Drug Delivery System . RSC Adv. 5 ( 99 ), 81188–81202. 10.1039/c5ra08550k [ CrossRef ] [ Google Scholar ]
• Pawar H. A., Bhangale B. D. (2015). Phytosome as a Novel Biomedicine: a Microencapsulated Drug Delivery System . J. Bioanal. Biomed. 7 ( 1 ), 6–12. [ Google Scholar ]
• Peravali R., Brock R., Bright E., Mills P., Petty D., Alberts J. (2014). Enhancing the Enhanced Recovery Program in Colorectal Surgery - Use of Extended-Release Epidural Morphine (DepoDur) . Ann. Coloproctol. 30 ( 4 ), 186. 10.3393/ac.2014.30.4.186 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Phuphanich S., Maria B., Braeckman R., Chamberlain M. (2007). A Pharmacokinetic Study of Intra-CSF Administered Encapsulated Cytarabine (DepoCyt) for the Treatment of Neoplastic Meningitis in Patients with Leukemia, Lymphoma, or Solid Tumors as Part of a Phase III Study . J. Neurooncol. 81 ( 2 ), 201–208. 10.1007/s11060-006-9218-x [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Pinilla C. M. B., Reque P. M., Brandelli A. (2020). Effect of Oleic Acid, Cholesterol, and Octadecylamine on Membrane Stability of Freeze-Dried Liposomes Encapsulating Natural Antimicrobials . Food Bioproc. Technol 13 ( 4 ), 599–610. 10.1007/s11947-020-02419-8 [ CrossRef ] [ Google Scholar ]
• Portet T., Dimova R. (2010). A New Method for Measuring Edge Tensions and Stability of Lipid Bilayers: Effect of Membrane Composition . Biophysical J. 99 ( 10 ), 3264–3273. 10.1016/j.bpj.2010.09.032 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Portilla S., Fernández L., Gutiérrez D., Rodríguez A., García P. (2020). Encapsulation of the Antistaphylococcal Endolysin LysRODI in pH-Sensitive Liposomes . Antibiotics 9 ( 5 ), 242. 10.3390/antibiotics9050242 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Pradhan B., Kumar N., Saha S., Roy A. (2015). Liposome: Method of Preparation, Advantages, Evaluation and its Application . J. Appl. Pharm. Res. 3 ( 3 ), 01–08. [ Google Scholar ]
• Puras G., Mashal M., Zárate J., Agirre M., Ojeda E., Grijalvo S., et al. (2014). A Novel Cationic Niosome Formulation for Gene Delivery to the Retina . J. Controlled Release 174 , 27–36. 10.1016/j.jconrel.2013.11.004 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Qiang L., Yi J., Fu-De C. (2004). Melanoma Vaccine Based on the Vector of Membrane Fusogenic Liposomes . Pharmazie 59 ( 4 ), 263–267. [ PubMed ] [ Google Scholar ]
• Qiao C., Liu J., Yang J., Li Y., Weng J., Shao Y., et al. (2016). Enhanced Non-inflammasome Mediated Immune Responses by Mannosylated Zwitterionic-Based Cationic Liposomes for HIV DNA Vaccines . Biomaterials 85 , 1–17. 10.1016/j.biomaterials.2016.01.054 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Radwan Almofti M., Harashima H., Shinohara Y., Almofti A., Baba Y., Kiwada H. (2003). Cationic Liposome-Mediated Gene Delivery: Biophysical Study and Mechanism of Internalization . Arch. Biochem. Biophys. 410 ( 2 ), 246–253. 10.1016/s0003-9861(02)00725-7 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Rahdar A., Sayyadi K., Sayyadi J., Yaghobi Z. (2019). Nano-gels: A Versatile Nano-Carrier Platform for Drug Delivery Systems: A Mini Review . Nanomedicine Res. J. 4 ( 1 ), 1–9. [ Google Scholar ]
• Réthoré G., Montier T., Le Gall T., Delepine P., Cammas-Marion S., Lemiègre L., et al. (2007). Archaeosomes Based on Synthetic Tetraether-like Lipids as Novel Versatile Gene Delivery Systems . Chem. Commun. 20 , 2054–2056. 10.1039/b618568a() [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Ricci M., Oliva R., Del Vecchio P., Paolantoni M., Morresi A., Sassi P. (2016). DMSO-induced Perturbation of Thermotropic Properties of Cholesterol-Containing DPPC Liposomes . Biochim. Biophys. Acta (Bba) - Biomembranes 1858 ( 12 ), 3024–3031. 10.1016/j.bbamem.2016.09.012 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Rizvi I., Nath S., Obaid G., Ruhi M. K., Moore K., Bano S., et al. (2019). A Combination of Visudyne and a Lipid-Anchored Liposomal Formulation of Benzoporphyrin Derivative Enhances Photodynamic Therapy Efficacy in a 3D Model for Ovarian Cancer . Photochem. Photobiol. 95 ( 1 ), 419–429. 10.1111/php.13066 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Rosenkrands I., Agger E. M., Olsen A. W., Korsholm K. S., Andersen C. S., Jensen K. T., et al. (2005). Cationic Liposomes Containing Mycobacterial Lipids: a New Powerful Th1 Adjuvant System . Infect. Immun. 73 ( 9 ), 5817–5826. 10.1128/iai.73.9.5817-5826.2005 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Rovira-Bru M., Thompson D. H., Szleifer I. (2002). Size and Structure of Spontaneously Forming Liposomes in lipid/PEG-Lipid Mixtures . Biophysical J. 83 ( 5 ), 2419–2439. 10.1016/s0006-3495(02)75255-7 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Raj S., Khurana S., Choudhari R., Kesari K. K., Kamal M. A., Garg N., et al. (Editors) (2021). Specific Targeting Cancer Cells with Nanoparticles and Drug Delivery in Cancer Therapy. Seminars in Cancer Biology (Elsevier; ). [ PubMed ] [ Google Scholar ]
• Saadat E., Amini M., Khoshayand M. R., Dinarvand R., Dorkoosh F. A. (2014). Synthesis and Optimization of a Novel Polymeric Micelle Based on Hyaluronic Acid and Phospholipids for Delivery of Paclitaxel, In Vitro and In-Vivo Evaluation . Int. J. Pharm. 475 ( 1-2 ), 163–173. 10.1016/j.ijpharm.2014.08.030 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Sahoo S. K., Labhasetwar V. (2003). Nanotech Approaches to Drug Delivery and Imaging . Drug Discov. Today 8 ( 24 ), 1112–1120. 10.1016/s1359-6446(03)02903-9 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Saliba A.-E., Vonkova I., Deghou S., Ceschia S., Tischer C., Kugler K. G., et al. (2016). A Protocol for the Systematic and Quantitative Measurement of Protein-Lipid Interactions Using the Liposome-Microarray-Based Assay . Nat. Protoc. 11 ( 6 ), 1021–1038. 10.1038/nprot.2016.059 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Samuni A. M., Lipman A., Barenholz Y. (2000). Damage to Liposomal Lipids: protection by Antioxidants and Cholesterol-Mediated Dehydration . Chem. Phys. Lipids 105 ( 2 ), 121–134. 10.1016/s0009-3084(99)00136-x [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Sankhyan A., Pawar P. (2012). Recent Trends in Niosome as Vesicular Drug Delivery System . J. Appl. Pharm. Sci. 2 ( 6 ), 20–32. [ Google Scholar ]
• Santos H. d. L., Lopes M. L., Maggio B., Ciancaglini P. (2005). Na,K-ATPase Reconstituted in Liposomes: Effects of Lipid Composition on Hydrolytic Activity and Enzyme Orientation . Colloids Surf. B: Biointerfaces 41 ( 4 ), 239–248. 10.1016/j.colsurfb.2004.12.013 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Scheffer L., Solomonov I., Jan Weygand M., Kjaer K., Leiserowitz L., Addadi L. (2005). Structure of Cholesterol/ceramide Monolayer Mixtures: Implications to the Molecular Organization of Lipid Rafts . Biophysical J. 88 ( 5 ), 3381–3391. 10.1529/biophysj.104.051870 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Scott M. J., Jones M. N. (2001). The Interaction of Phospholipid Liposomes with Zinc Citrate Particles: a Microcalorimetric Investigation. Colloids and Surfaces A: . Physicochemical Eng. Aspects 182 ( 1-3 ), 247–256. 10.1016/s0927-7757(00)00827-x [ CrossRef ] [ Google Scholar ]
• Scrimgeour C., Gao Y., Oh W. Y., Shahidi F. (2005). Chemistry of Fatty Acids . Bailey's Ind. oil fat Prod. , 1–40. 10.1002/047167849x.bio005 [ CrossRef ] [ Google Scholar ]
• Semple S. C., Leone R., Wang J., Leng E. C., Klimuk S. K., Eisenhardt M. L., et al. (2005). Optimization and Characterization of a Sphingomyelin/cholesterol Liposome Formulation of Vinorelbine with Promising Antitumor Activity . J. Pharm. Sci. 94 ( 5 ), 1024–1038. 10.1002/jps.20332 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Seo J. W., Qin S., Mahakian L. M., Watson K. D., Kheirolomoom A., Ferrara K. W. (2011). Positron Emission Tomography Imaging of the Stability of Cu-64 Labeled Dipalmitoyl and Distearoyl Lipids in Liposomes . J. controlled release 151 ( 1 ), 28–34. 10.1016/j.jconrel.2011.01.008 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Shah K. A., Date A. A., Joshi M. D., Patravale V. B. (2007). Solid Lipid Nanoparticles (SLN) of Tretinoin: Potential in Topical Delivery . Int. J. Pharm. 345 ( 1-2 ), 163–171. 10.1016/j.ijpharm.2007.05.061 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Shehata T., Ogawara K., Higaki K., Kimura T. (2008). Prolongation of Residence Time of Liposome by Surface-Modification with Mixture of Hydrophilic Polymers . Int. J. Pharm. 359 ( 1-2 ), 272–279. 10.1016/j.ijpharm.2008.04.004 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Shim G., Han S.-E., Yu Y.-H., Lee S., Lee H. Y., Kim K., et al. (2011). Trilysinoyl Oleylamide-Based Cationic Liposomes for Systemic Co-delivery of siRNA and an Anticancer Drug . J. controlled release 155 ( 1 ), 60–66. 10.1016/j.jconrel.2010.10.017 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Shim G., Kim M.-G., Park J. Y., Oh Y.-K. (2013). Application of Cationic Liposomes for Delivery of Nucleic Acids . Asian J. Pharm. Sci. 8 ( 2 ), 72–80. 10.1016/j.ajps.2013.07.009 [ CrossRef ] [ Google Scholar ]
• Shoji J. i., Tanihara Y., Uchiyama T., Kawai A. (2004). Preparation of Virosomes Coated with the Vesicular Stomatitis Virus Glycoprotein as Efficient Gene Transfer Vehicles for Animal Cells . Microbiol. Immunol. 48 ( 3 ), 163–174. 10.1111/j.1348-0421.2004.tb03502.x [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Shrivastava S., Lole K. S., Tripathy A. S., Shaligram U. S., Arankalle V. A. (2009). Development of Candidate Combination Vaccine for Hepatitis E and Hepatitis B: a Liposome Encapsulation Approach . Vaccine 27 ( 47 ), 6582–6588. 10.1016/j.vaccine.2009.08.033 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Silindir M., Erdoğan S., Özer A. Y., Doğan A. L., Tuncel M., Uğur Ö., et al. (2013). Nanosized Multifunctional Liposomes for Tumor Diagnosis and Molecular Imaging by SPECT/CT . J. Liposome Res. 23 ( 1 ), 20–27. 10.3109/08982104.2012.722107 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Silva C., Aranda F. J., Ortiz A., Martínez V., Carvajal M., Teruel J. A. (2011). Molecular Aspects of the Interaction between Plants Sterols and DPPC Bilayers . J. Colloid Interf. Sci. 358 ( 1 ), 192–201. 10.1016/j.jcis.2011.02.048 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Silverman J. A., Reynolds L., Deitcher S. R. (2013). Pharmacokinetics and Pharmacodynamics of Vincristine Sulfate Liposome Injection (VSLI) in Adults with Acute Lymphoblastic Leukemia . J. Clin. Pharmacol. 53 ( 11 ), 1139–1145. 10.1002/jcph.155 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Silverman J. A., Deitcher S. R. (2013). Marqibo (Vincristine Sulfate Liposome Injection) Improves the Pharmacokinetics and Pharmacodynamics of Vincristine . Cancer Chemother. Pharmacol. 71 ( 3 ), 555–564. 10.1007/s00280-012-2042-4 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Simões S., Moreira J. N., Fonseca C., Düzgüneş N., de Lima M. C. P. (2004). On the Formulation of pH-Sensitive Liposomes with Long Circulation Times . Adv. Drug Deliv. Rev. 56 ( 7 ), 947–965. 10.1016/j.addr.2003.10.038 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Singh D., Dilnawaz F., Sahoo S. K. (2020). Challenges of Moving Theranostic Nanomedicine into the Clinic . Future Med. 10.2217/nnm-2019-0401 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Sioud M., Sørensen D. R. (2003). Cationic Liposome-Mediated Delivery of siRNAs in Adult Mice . Biochem. biophysical Res. Commun. 312 ( 4 ), 1220–1225. 10.1016/j.bbrc.2003.11.057 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Socaciu C., Jessel R., Diehl H. A. (2000). Competitive Carotenoid and Cholesterol Incorporation into Liposomes: Effects on Membrane Phase Transition, Fluidity, Polarity and Anisotropy . Chem. Phys. Lipids 106 ( 1 ), 79–88. 10.1016/s0009-3084(00)00135-3 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Sodt A. J., Pastor R. W., Lyman E. (2015). Hexagonal Substructure and Hydrogen Bonding in Liquid-Ordered Phases Containing Palmitoyl Sphingomyelin . Biophysical J. 109 ( 5 ), 948–955. 10.1016/j.bpj.2015.07.036 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Sugita T., Yoshikawa T., Gao J.-Q., Shimokawa M., Oda A., Niwa T., et al. (2005). Fusogenic Liposome Can Be Used as an Effective Vaccine Carrier for Peptide Vaccination to Induce Cytotoxic T Lymphocyte (CTL) Response . Biol. Pharm. Bull. 28 ( 1 ), 192–193. 10.1248/bpb.28.192 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Suk J. S., Xu Q., Kim N., Hanes J., Ensign L. M. (2016). PEGylation as a Strategy for Improving Nanoparticle-Based Drug and Gene Delivery . Adv. Drug Deliv. Rev. 99 ( Pt A ), 28–51. 10.1016/j.addr.2015.09.012 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Sun M., Northup N., Marga F., Huber T., Byfield F. J., Levitan I., et al. (2007). The Effect of Cellular Cholesterol on Membrane-Cytoskeleton Adhesion . J. Cel. Sci. 120 ( 13 ), 2223–2231. 10.1242/jcs.001370 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Tabatabaei Mirakabad F. S., Akbarzadeh A., Milani M., Zarghami N., Taheri-Anganeh M., Zeighamian V., et al. (2016). A Comparison between the Cytotoxic Effects of Pure Curcumin and Curcumin-Loaded PLGA-PEG Nanoparticles on the MCF-7 Human Breast Cancer Cell Line . Artif. Cell Nanomedicine, Biotechnol. 44 ( 1 ), 423–430. 10.3109/21691401.2014.955108 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Tan C., Feng B., Zhang X., Xia W., Xia S. (2016). Biopolymer-coated Liposomes by Electrostatic Adsorption of Chitosan (Chitosomes) as Novel Delivery Systems for Carotenoids . Food hydrocolloids 52 , 774–784. 10.1016/j.foodhyd.2015.08.016 [ CrossRef ] [ Google Scholar ]
• Thompson A. K., Haisman D., Singh H. (2006). Physical Stability of Liposomes Prepared from Milk Fat Globule Membrane and Soya Phospholipids . J. Agric. Food Chem. 54 ( 17 ), 6390–6397. 10.1021/jf0605695 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Toh M.-R., Chiu G. N. C. (2013). Liposomes as Sterile Preparations and Limitations of Sterilisation Techniques in Liposomal Manufacturing . Asian J. Pharm. Sci. 8 ( 2 ), 88–95. 10.1016/j.ajps.2013.07.011 [ CrossRef ] [ Google Scholar ]
• Torchilin V. P. (2006). Recent Approaches to Intracellular Delivery of Drugs and DNA and Organelle Targeting . Annu. Rev. Biomed. Eng. 8 , 343–375. 10.1146/annurev.bioeng.8.061505.095735 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Trandum C., Westh P., Jørgensen K., Mouritsen O. G. (2000). A Thermodynamic Study of the Effects of Cholesterol on the Interaction between Liposomes and Ethanol . Biophysical J. 78 ( 5 ), 2486–2492. 10.1016/s0006-3495(00)76793-2 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Trucillo P., Campardelli R., Reverchon E. (2020). Liposomes: From Bangham to Supercritical Fluids . Processes 8 ( 9 ), 1022. 10.3390/pr8091022 [ CrossRef ] [ Google Scholar ]
• Trucillo P., Campardelli R., Reverchon E. (2017). Supercritical CO 2 Assisted Liposomes Formation: Optimization of the Lipidic Layer for an Efficient Hydrophilic Drug Loading . J. Co2 utilization 18 , 181–188. 10.1016/j.jcou.2017.02.001 [ CrossRef ] [ Google Scholar ]
• Tzogani K., Penttilä K., Lapveteläinen T., Hemmings R., Koenig J., Freire J., et al. (2020). EMA Review of Daunorubicin and Cytarabine Encapsulated in Liposomes (Vyxeos, CPX-351) for the Treatment of Adults with Newly Diagnosed, Therapy-Related Acute Myeloid Leukemia or Acute Myeloid Leukemia with Myelodysplasia-Related Changes . Oncologist 25 ( 9 ), e1414–e20. 10.1634/theoncologist.2019-0785 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Ulrich A. S. (2002). Biophysical Aspects of Using Liposomes as Delivery Vehicles . Biosci. Rep. 22 ( 2 ), 129–150. 10.1023/a:1020178304031 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Vakili-Ghartavol R., Rezayat S. M., Faridi-Majidi R., Sadri K., Jaafari M. R. (2020). Optimization of Docetaxel Loading Conditions in Liposomes: Proposing Potential Products for Metastatic Breast Carcinoma Chemotherapy . Sci. Rep. 10 ( 1 ), 5569. 10.1038/s41598-020-62501-1 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Van Slooten M. L., Storm G., Zoephel A., Küpcü Z., Boerman O., Crommelin D. J. A., et al. (2000). Liposomes Containing Interferon-Gamma as Adjuvant in Tumor Cell Vaccines . Pharm. Res. 17 ( 1 ), 42–48. 10.1023/a:1007514424253 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Varypataki E. M., Benne N., Bouwstra J., Jiskoot W., Ossendorp F. (2017). Efficient Eradication of Established Tumors in Mice with Cationic Liposome-Based Synthetic Long-Peptide Vaccines . Cancer Immunol. Res. 5 ( 3 ), 222–233. 10.1158/2326-6066.cir-16-0283 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Veneti E., Tu R. S., Auguste D. T. (2016). RGD-targeted Liposome Binding and Uptake on Breast Cancer Cells Is Dependent on Elastin Linker Secondary Structure . Bioconjug. Chem. 27 ( 8 ), 1813–1821. 10.1021/acs.bioconjchem.6b00205 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Wagner A., Platzgummer M., Kreismayr G., Quendler H., Stiegler G., Ferko B., et al. (2006). GMP Production of Liposomes-A New Industrial Approach . J. liposome Res. 16 ( 3 ), 311–319. 10.1080/08982100600851086 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Walde P., Ichikawa S. (2001). Enzymes inside Lipid Vesicles: Preparation, Reactivity and Applications . Biomol. Eng. 18 ( 4 ), 143–177. 10.1016/s1389-0344(01)00088-0 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Wang Y., Tu S., Li R., Yang X., Liu L., Zhang Q. (2010). Cholesterol Succinyl Chitosan Anchored Liposomes: Preparation, Characterization, Physical Stability, and Drug Release Behavior . Nanomedicine: Nanotechnology, Biol. Med. 6 ( 3 ), 471–477. 10.1016/j.nano.2009.09.005 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Wang Y., Wang S., Firempong C. K., Zhang H., Wang M., Zhang Y., et al. (2017). Enhanced Solubility and Bioavailability of Naringenin via Liposomal Nanoformulation: Preparation and In Vitro and In Vivo Evaluations . Aaps Pharmscitech 18 ( 3 ), 586–594. 10.1208/s12249-016-0537-8 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Weissig V. (2015). DQAsomes as the Prototype of Mitochondria-Targeted Pharmaceutical Nanocarriers: Preparation, Characterization, and Use . Mitochondrial Med. Springer , 1–11. 10.1007/978-1-4939-2288-8_1 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Weng C.-S., Wu C.-C., Chen T.-C., Chen J.-R., Huang C.-Y., Chang C.-L. (2019). Retrospective Analysis of Comparative Outcomes in Recurrent Platinum-Sensitive Ovarian Cancer Treated with Pegylated Liposomal Doxorubicin (Lipo-Dox) and Carboplatin versus Paclitaxel and Carboplatin . Cmar 11 , 9899–9905. 10.2147/cmar.s217329 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Wibroe P. P., Ahmadvand D., Oghabian M. A., Yaghmur A., Moghimi S. M. (2016). An Integrated Assessment of Morphology, Size, and Complement Activation of the PEGylated Liposomal Doxorubicin Products Doxil, Caelyx, DOXOrubicin, and SinaDoxosome . J. Controlled Release 221 , 1–8. 10.1016/j.jconrel.2015.11.021 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Wong J. P., Nagata L. P., Dale R. M. (2001). Liposome-encapsulated Nucleic Acid-Based Drugs and Vaccines as Novel Antiviral Agents . Recent Dev. Antivir. Res. , 175–191. [ Google Scholar ]
• Wu R. G., Dai J. D., Wu F. G., Zhang X. H., Li W. H., Wang Y. R. (2012). Competitive Molecular Interaction Among Paeonol-Loaded Liposomes: Differential Scanning Calorimetry and Synchrotron X-ray Diffraction Studies . Int. J. Pharm. 438 ( 1-2 ), 91–97. 10.1016/j.ijpharm.2012.08.052 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Wu X., Dai X., Liao Y., Sheng M., Shi X. (2021). Investigation on Drug Entrapment Location in Liposomes and Transfersomes Based on Molecular Dynamics Simulation . J. Mol. Model. 27 ( 4 ), 1–10. 10.1007/s00894-021-04722-3 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Xia S., Xu S. (2005). Ferrous Sulfate Liposomes: Preparation, Stability and Application in Fluid Milk . Food Res. Int. 38 ( 3 ), 289–296. 10.1016/j.foodres.2004.04.010 [ CrossRef ] [ Google Scholar ]
• Xu Q., Tanaka Y., Czernuszka J. T. (2007). Encapsulation and Release of a Hydrophobic Drug from Hydroxyapatite Coated Liposomes . Biomaterials 28 ( 16 ), 2687–2694. 10.1016/j.biomaterials.2007.02.007 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Xu X., Khan M. A., Burgess D. J. (2011). A Quality by Design (QbD) Case Study on Liposomes Containing Hydrophilic API: I. Formulation, Processing Design and Risk Assessment . Int. J. Pharm. 419 ( 1-2 ), 52–59. 10.1016/j.ijpharm.2011.07.012 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Yamauchi M., Tsutsumi K., Abe M., Uosaki Y., Nakakura M., Aoki N. (2007). Release of Drugs from Liposomes Varies with Particle Size . Biol. Pharm. Bull. 30 ( 5 ), 963–966. 10.1248/bpb.30.963 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Yang T., Cui F.-D., Choi M.-K., Lin H., Chung S.-J., Shim C.-K., et al. (2007). Liposome Formulation of Paclitaxel with Enhanced Solubility and Stability . Drug Deliv. 14 ( 5 ), 301–308. 10.1080/10717540601098799 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Yang Z.-Z., Li J.-Q., Wang Z.-Z., Dong D.-W., Qi X.-R. (2014). Tumor-targeting Dual Peptides-Modified Cationic Liposomes for Delivery of siRNA and Docetaxel to Gliomas . Biomaterials 35 ( 19 ), 5226–5239. 10.1016/j.biomaterials.2014.03.017 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Yavlovich A., Singh A., Tarasov S., Capala J., Blumenthal R., Puri A. (2009). Design of Liposomes Containing Photopolymerizable Phospholipids for Triggered Release of Contents . J. Therm. Anal. Calorim. 98 ( 1 ), 97–104. 10.1007/s10973-009-0228-8 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Yoshida I., Saito A. M., Tanaka S., Choi I., Hidaka M., Miyata Y., et al. (2020). Intravenous Itraconazole Compared with Liposomal Amphotericin B as Empirical Antifungal Therapy in Patients with Neutropaenia and Persistent Fever . Mycoses 63 ( 8 ), 794–801. 10.1111/myc.13100 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Yoshikawa T., Imazu S., Gao J.-Q., Hayashi K., Tsuda Y., Okada N., et al. (2006). Non-methylated CpG Motif Packaged into Fusogenic Liposomes Enhance Antigen-specific Immunity in Mice . Biol. Pharm. Bull. 29 ( 1 ), 105–109. 10.1248/bpb.29.105 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Yoshikawa T., Okada N., Tsujino M., Gao J.-Q., Hayashi A., Tsutsumi Y., et al. (2006). Vaccine Efficacy of Fusogenic Liposomes Containing Tumor Cell-Lysate against Murine B16BL6 Melanoma . Biol. Pharm. Bull. 29 ( 1 ), 100–104. 10.1248/bpb.29.100 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Yu F., Tang X. (2016). Novel Long-Circulating Liposomes Consisting of PEG Modified β-Sitosterol for Gambogic Acid Delivery . j nanosci nanotechnol 16 ( 3 ), 3115–3121. 10.1166/jnn.2016.12405 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Yu J. Y., Chuesiang P., Shin G. H., Park H. J. (2021). Post-processing Techniques for the Improvement of Liposome Stability . Pharmaceutics 13 ( 7 ), 1023. 10.3390/pharmaceutics13071023 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Yu T., Ji P., Zhao W. M. (2015). Preparation of Baicalein Liposome-Lyophilized Powder and its Pharmacokinetics Study . Zhong Yao Cai 38 ( 11 ), 2404–2407. [ PubMed ] [ Google Scholar ]
• Yuba E. (2020). Development of Functional Liposomes by Modification of Stimuli-Responsive Materials and Their Biomedical Applications . J. Mater. Chem. B 8 ( 6 ), 1093–1107. 10.1039/c9tb02470k [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Yuba E., Kanda Y., Yoshizaki Y., Teranishi R., Harada A., Sugiura K., et al. (2015). pH-sensitive Polymer-Liposome-Based Antigen Delivery Systems Potentiated with Interferon-γ Gene Lipoplex for Efficient Cancer Immunotherapy . Biomaterials 67 , 214–224. 10.1016/j.biomaterials.2015.07.031 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Zaru M., Mourtas S., Klepetsanis P., Fadda A. M., Antimisiaris S. G. (2007). Liposomes for Drug Delivery to the Lungs by Nebulization . Eur. J. Pharmaceutics Biopharmaceutics 67 ( 3 ), 655–666. 10.1016/j.ejpb.2007.04.005 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Zhang J., Chen Y., Li X., Liang X., Luo X. (2016). The Influence of Different Long-Circulating Materials on the Pharmacokinetics of Liposomal Vincristine Sulfate . Int. J. Nanomedicine 11 , 4187–4197. 10.2147/IJN.S109547 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Zhang J., Tian H., Li C., Cheng L., Zhang S., Zhang X., et al. (2013). Antitumor Effects Obtained by Autologous Lewis Lung Cancer Cell Vaccine Engineered to Secrete Mouse Interleukin 27 by Means of Cationic Liposome . Mol. Immunol. 55 ( 3-4 ), 264–274. 10.1016/j.molimm.2013.02.006 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Zhang J. A., Pawelchak J. (2000). Effect of pH, Ionic Strength and Oxygen burden on the Chemical Stability of EPC/cholesterol Liposomes under Accelerated Conditions. Part 1: Lipid Hydrolysis . Eur. J. Pharm. Biopharm. 50 ( 3 ), 357–364. 10.1016/s0939-6411(00)00127-2 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Zhao L., Temelli F., Curtis J. M., Chen L. (2015). Preparation of Liposomes Using Supercritical Carbon Dioxide Technology: Effects of Phospholipids and Sterols . Food Res. Int. 77 , 63–72. 10.1016/j.foodres.2015.07.006 [ CrossRef ] [ Google Scholar ]
• Zhao X. B., Muthusamy N., Byrd J. C., Lee R. J. (2007). Cholesterol as a Bilayer Anchor for PEGylation and Targeting Ligand in Folate‐receptor‐targeted Liposomes . J. Pharm. Sci. 96 ( 9 ), 2424–2435. 10.1002/jps.20885 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Zhao Y.-Z., Liang H.-D., Mei X.-G., Halliwell M. (2005). Preparation, Characterization and In Vivo Observation of Phospholipid-Based Gas-Filled Microbubbles Containing Hirudin . Ultrasound Med. Biol. 31 ( 9 ), 1237–1243. 10.1016/j.ultrasmedbio.2005.05.007 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Zhu R., Wang Z., Liang P., He X., Zhuang X., Huang R., et al. (2017). Efficient VEGF Targeting Delivery of DOX Using Bevacizumab Conjugated SiO2@LDH for Anti-neuroblastoma Therapy . Acta Biomater. 63 , 163–180. 10.1016/j.actbio.2017.09.009 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Zupančič Š., Kocbek P., Zariwala M. G., Renshaw D., Gul M. O., Elsaid Z., et al. (2014). Design and Development of Novel Mitochondrial Targeted Nanocarriers, DQAsomes for Curcumin Inhalation . Mol. Pharm. 11 ( 7 ), 2334–2345. 10.1021/mp500003q [ PubMed ] [ CrossRef ] [ Google Scholar ]