Liposomal Components Research Articles

Liposomes – metabolically active drug transport systems: classification, components, preparation methods, and stabilization. Part 1 (review)

Introduction. This review article is focused on the modern classification of liposomes, preparation methods, stabilization and the role of structural components, visualization, and pharmacokinetics. Part 1 discusses the first three aspects mentioned above.Text. Depending on the size and number of bilayers, liposomes are classified into simple, long-circulating, cationic, immuno-liposomes, and sterically stabilized. The lipid components of liposomes can have a pronounced effect on target organs and tissues. Metabolites of the main components of liposomes have their biological activity, depending on their combination and dosage. All of the above indicates the promise of using liposomes not only as carriers of drugs but also as independent effectors that can have a significant impact on human metabolism in various diseases. The advantages and limitations of methods for preparing liposomes, and the features for manufacturing stealth liposomes are discussed. A special section of the article is dedicated to liposome stabilization.Conclusions. The information discussed in the review article may be useful in the development of pharmaceutical formulations in liposomes. Liposomes are not only promising nanocontainers for targeted drug delivery, but also metabolically active complexes with a wide spectrum of activity.

Liposomes have a direct effect on multiple myeloma: a Mendelian randomization study.

Multiple myeloma (MM), a malignant disease of plasma cells originating in the bone marrow, is influenced significantly by genetic factors. Although plasma liposomes have been linked to MM, the nature of their potential causal relationship remains to be elucidated. This study aims to explore this relationship using Mendelian randomization (MR) analysis. Liposome-associated genetic instrumental variables (IVs) were identified from plasma lipidomics data of 7,174 Finnish individuals within a Genome-Wide Association Study (GWAS) pooled database. A MM pooled dataset was sourced from a GWAS meta-analysis encompassing 150,797 individuals, including 598 MM patients and 218,194 controls. These IVs underwent MR analysis, adhering to strict criteria for correlation, independence, and the exclusion of confounders. The inverse variance weighted (IVW) method, MR-Egger method, weighted median (WM) method, and simple median were utilized for MR analysis assessment, alongside Cochran's Q test, MR-Egger intercept, MR-Pleiotropy Residual Sum and Outlier (MR-RESSO) method, and leave-one-out analysis for evaluating heterogeneity, multiplicity, and instrumental bias. The study identified 88 significant, independent single nucleotide polymorphisms (SNPs) as IVs for MR analysis, each with an F-statistic value above 10, indicating robustness against weak instrument bias. IVW analysis revealed associations between six plasma liposome components and MM risk (p < 0.05). Phosphatidylinositol (16:0_18:1) serum levels (odds ratio [OR] = 1.769, 95% confidence interval [CI]: 1.132-2.763, p = 0.012) and triacylglycerol (56:4) levels (p = 0.026, OR = 1.417, 95% CI: 1.042-1.926) were positively correlated with the risk of multiple myeloma development. Phosphatidylethanolamine (18:0_20:4) (p = 0.004, 95% CI: 0.621-0.916, OR = 0.754), phosphatidylcholine (18:2_20:4) (p = 0.004, OR = 0.680, 95% CI: 0.519-0.889), sterol ester (27:1/18:3) levels (p = 0.013, OR = 0.677, 95% CI: 0.498-0.922), and phosphatidylcholine (O-18:2_20:4) levels (OR = 0.710, 95% CI: 0.517-0.913, p = 0.033) were negatively associated with the risk of developing multiple myeloma. The Cochran's Q test did not detect statistical method heterogeneity, nor did the MR-RESSO test or the MR-Egger intercept detect horizontal pleiotropy; leave-one-out analyses confirmed the absence of bias from individual SNPs. Our findings suggest a complex relationship between plasma liposome components and MM risk. Elevated serum levels of triacylglycerol and phosphatidylinositol are positively associated with MM risk, while certain phospholipids and sterol esters offer a protective effect. This study provides valuable insights into the clinical relevance of liposomes in the pathology of multiple myeloma.

A Reliable Quantification of Cholesterol and 25-Hydroxycholesterol in Liposomal Adjuvant Formulation by Liquid Chromatography High-Resolution Tandem Mass Spectrometry.

Cholesterol, as one of the major components of liposomes, plays a critical role in modulating membrane bilayer permeability, fluidity, and structural stability. Controlling these quality attributes is essential to maintaining the efficacy and fitness of the liposomes in various applications. However, during the manufacture and storage of liposomes, cholesterol has a propensity to undergo oxidative degradation. Hence, an analytical tool that is capable of determining not only the identity and quantity of cholesterol but also its associated degradants is a prerequisite to effective process control and product quality and safety assessments. In this view, a new liquid chromatography electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method with parallel reaction monitoring (PRM) was developed and qualified to accurately quantify cholesterol and monitor the formation of 25-hydroxycholesterol degradant in liposomal drug formulations without the use of an isotopic internal standard (IS). The method was qualified according to the FDA Quality Guidance for Industry: Q2(R1). Study results showed that the method presents good specificity for cholesterol and 25-hydroxycholesterol detection in the liposomal matrix, good sensitivity characterized by LOD/LOQ in the nanomolar range, and accuracy within the range of 80 to 120%. The described method enables accurate evaluation of in-process and product release samples of Army Liposome Formulation with QS21 (ALFQ).

Replacing cholesterol with asiatic acid to prolong circulation and enhance anti-metastatic effects of non-PEGylated liposomes

Cholesterol is an indispensable component of most liposomes, heavily influencing their physical and surface properties. In this study, cholesterol in non-PEGylated liposomes was replaced by its analog, asiatic acid (AA), to generate liposomes with an alternative composition. These AA liposomes are generally smaller and more rigid than conventional liposomes, circulate longer in the body, and accumulate more in primary tumors and lung metastases in vivo. On the other hand, as an active ingredient, AA can decrease TGF-β secretion to inhibit the epithelial-mesenchymal transition (EMT) process, increase the sensitivity of tumor cells to doxorubicin (DOX), and synergize with DOX to enhance the immune response, thus improving their antitumor and anti-metastasis efficiency. Based on this rationale, DOX-loaded AA liposomes were fabricated and tested against triple-negative breast cancer (TNBC). Results showed that compared with conventional liposomes, the DOX-AALip provided approximately 28.4% higher tumor volume reduction with almost no metastatic nodules in the mouse model. Our data demonstrate that AA liposomes are safe, simple, and efficient, and thus in many situations may be used instead of conventional liposomes, having good potential for further clinical translational development.

CASA Based Assessment of Kangayam bull semen processed with different extenders and Conception rate following AI

Semen extenders are the essential components of cryopreservation of semen. Identification of suitable and ready to use semen extender is the need of hour for the rapid propagation of Kangayam cows through artificial insemination. Kangayam bull, aged 5 years was utilized for the study. The semen was collected twice in a week and each time two ejaculates were collected. A total of 24 ejaculates were utilized for this study. After assessing the general macroscopical examination the semen was subjected to computer aided semen analysis (CASA) for assessing the kinematic parameters in fresh diluted semen, pre freeze and post thawed such as sperm motility, progressive individual motility, VAP, VSL, ALH and LIN were analyzed using CASA. Among all the three extenders, the sperm kinematic parameters during fresh diluted, pre freeze and post thawed semen stages were higher in group III (liposome based extender) followed by group I (TEYG) and in group II (soya bean lecithin based extender). Among the three extenders used, the components of liposome based extender was better and it was able to maintain the structural integrity, plasma membrane integrity and motility in the sperms of the frozen thawed semen than the other two extenders (TEYG and soya bean lecithin based extenders). Further the fertility rate was evaluated by the in vivo insemination of 60 Kangayam cows after 12 hours of onset of estrum. The fertility rate observed was 45.00, 40.00 and 65.00 per cent in groups I, II and III, respectively. The overall conception rate recorded in Kangayam cow was 50.00 per cent. Based on the findings, it was concluded that liposome based extender may be used for cryopreservation of Kangayam bull semen in order to maximize the fertility rate.

Non-clinical evaluation of YN001-a promising drug for atherosclerotic therapy by targeted delivery system

Abstract Background Atherosclerosis (AS) is a systemic disease and the common cause of heart attacks, strokes and peripheral vascular disease collectively referred to as cardiovascular diseases (CVD). In AS plaques, CD44 is upregulated and functionally activated on vascular endothelial cells, smooth muscle cells, and macrophages. YN001 is a liposome encapsulated Rosuvastatin calcium which was initially approved for the reduction of cholesterol levels and slowing the progression of atherosclerosis. YN001 is being developed for the treatment of AS by targeted enrichment of Rosuvastatin in the local atherosclerotic plaques by CD44 active targeting ability of HPD, one of the components of liposome. Purpose Investigating pharmacology, PK and toxicity of YN001 to evaluate its clinical use potential for AS patients. Results As expected, YN001 could bind to CD44 with KD as 485.5pm while there’s no binding affinity of non-targeted liposome (encapsulate Rosuvastatin without HPD). In stimulated human macrophages in which CD44 was overexpressed, YN001 showed enhanced uptake and higher cholesterol effluxion ability than Rosuvastatin, meanwhile more MCP-1 reduction was also observed. In ApoE AS mice model, single YN001 administration showed much higher drug concentrations in the plaque of aortas compared to Rosuvastatin (oral or iv) or non-targeted liposome which demonstrated targeted enrichment ability by HPD. With repeat treatment of YN001 at 5mg/kg in AS mice model, atherosclerotic plaque weight decreasing and plaque area reduction were observed, following MCP-1 reduction. YN001 may down-regulate the expression of inflammatory signaling pathways, such as chemokine-related signaling pathway to reduce the inflammatory response of atherosclerotic plaques to decrease plaque levels. In pharmacokinetic studies in rats and dogs, YN001 had much higher exposure in plasma and prolonged existence compared to Rosuvastatin i.v administration. A comprehensive safety evaluation package was performed in rats and dogs including safety pharmacology, general toxicity, genotoxicity, immunotoxicity and local tolerance. Results showed YN001 has promising safety with NOAEL as 40mg/kg in both or rats and dogs. Conclusion YN001 demonstrated excellent efficacy in AS model, a favorable pharmacokinetic and safety profiles in rats and dogs. These support its FDA IND approval and phase I study is ongoing to investigate further character in clinical.Efficacy and pharmacokinetics resultsSafety evaluation

Preparing Size-Controlled Liposomes Modified with Polysaccharide Derivatives for pH-Responsive Drug Delivery Applications.

The liposome particle size is an important parameter because it strongly affects content release from liposomes as a result of different bilayer curvatures and lipid packing. Earlier, we developed pH-responsive polysaccharide-derivative-modified liposomes that induced content release from the liposomes under weakly acidic conditions. However, the liposome used in previous studies size was adjusted to 100-200 nm. The liposome size effects on their pH-responsive properties were unclear. For this study, we controlled the polysaccharide-derivative-modified liposome size by extrusion through polycarbonate membranes having different pore sizes. The obtained liposomes exhibited different average diameters, in which the diameters mostly corresponded to the pore sizes of polycarbonate membranes used for extrusion. The amounts of polysaccharide derivatives per lipid were identical irrespective of the liposome size. Introduction of cholesterol within the liposomal lipid components suppressed the size increase in these liposomes for at least three weeks. These liposomes were stable at neutral pH, whereas the content release from liposomes was induced at weakly acidic pH. Smaller liposomes exhibited highly acidic pH-responsive content release compared with those from large liposomes. However, liposomes with 50 mol% cholesterol were not able to induce content release even under acidic conditions. These results suggest that control of the liposome size and cholesterol content is important for preparing stable liposomes at physiological conditions and for preparing highly pH-responsive liposomes for drug delivery applications.

In vivo confocal Raman spectroscopy investigation of glabridin liposomes dermal penetration process in human skin

Encapsulating glabridin with liposome technology can contribute to the bioavailability and application in cosmetic formulations. At the same time, the small particle size of liposomes makes it feasible to penetrate the stratum corneum and enhance the dermal delivery of glabridin. Confocal Raman spectroscopy (CRS) can be employed to monitor the dermal penetration of cosmetic actives for topical applications. The method of CRS allows qualitatively and semi-quantitatively in vivo analysis of cosmetic actives based on the position and intensity of peaks in the Raman spectrum. In this work, CRS was used to evaluate epidermal penetration of glabridin and glabridin-containing liposome components. First, glabridin liposomes (GL), glabridin liposome essence (GLE), and glabridin essence (GE) were evenly applied on the forearms of six randomly-selected volunteers. Then, in vivo dermal penetration and of delivery mechanism of glabridin was clinically assessed at 0.5 h and 2 h with the non-invasive devices, confocal Raman spectrometer. The findings indicated that the glabridin transported by liposomes could penetrate the stratum corneum and reach deep layers of human skin, while the content of penetrated glabridin increased with time. Compared to the penetration of GLE and GE through the skin, it was found that the dermal penetration of liposome-encapsulated glabridin in the essence dosage form was 3.8 times higher than that of unencapsulated glabridin. From qualitative and semi-quantitative perspectives, it was demonstrated that glabridin liposomes have good skin permeability and a gradual release effect and the liposome encapsulation can efficiently enhance the poor dermal absorption and bioavailability of glabridin. We believe this in vivo confocal Raman spectroscopy research is benefit for revealing the permeability of glabridin and liposome formulations in cosmetics.

Mass spectrometry study of ascorbyl palmitate as an agent for nanosomes formation

Background. Study of properties and intermolecular interactions of biologically active compounds which can be used for the purposes of transmembrane drugs delivery is a topical task of modern molecular biophysics. Ascorbyl Palmitate (AP) as a fat-soluble form of vitamin C has recently attracted attention as a promising agent for formation of nanosomes for the “fat insoluble” drug molecules transfer through membranes. However, AP is not sufficiently characterized by up-to-date soft ionization mass spectrometric techniques. Objectives. The aim of the present work is to characterize AP and its intermolecular interactions by a number of mass spectrometric techniques: Electrospray Ionization (ESI), Laser Desorption/Ionization (LDI) and Matrix-Assisted Laser Desorption/Ionization (MALDI). The comparison of these techniques applicability to the study of AP intermolecular interactions as a drug delivery assisting agent is scheduled. Methods. ESI mass spectra are obtained with triple quadrupole Micromass Quattro mass spectrometer. LDI and MALDI experiments are performed by Autoflex II mass spectrometer. Results. In the ESI experiments in the positive ion mode abundant peaks of protonated and cationized AP molecules as well as the peaks of AP clusters nAP•H+ and nAP•Na+ (n=2÷4) are revealed in the mass spectra. This result testifies to the formation of stable noncovalent complexes of the AP molecules in the polar media and confirms the AP ability of formation nanosomes for drug delivery. Analysis of LDI and MALDI mass spectra of AP in positive and negative ion modes shows that in the presence of molecular ions of AP, the peaks of AP dimers or larger AP clusters are not recorded. The ESI probing of the model system containing AP and dipalmitoylphosphatidylcholine (DPPC) reveals stable AP•DPPC•H+ complex which models the AP intermolecular interactions with the phospholipid components of biomembranes and/or liposomes under AP functioning as a drug delivery assisting agent. Conclusions. The current study demonstrates the applicability of all tested mass spectrometric techniques for AP identification in solutions and solid phase, while for the purpose of examining of the AP noncovalent complexes formation and study of AP interactions with biomolecules the ESI is defined as the most effective technique.

Peculiarities of the filtration process of liposomal emulsion for the production of eye drops based on a peptide complex

Liposomes are increasingly being studied and implemented as drug delivery systems in the form of eye drops. An important stage in the production of a liposomal preparation in the form of eye drops is the stage of sterilization filtration before filling the medicinal product into a container for final dosage. It is assumed that liposome components interact with the membrane and clog it due to their unique physicochemical properties. Therefore, a deeper understanding of the sterilization filtration of liposomes is needed to make appropriate decisions regarding the choice of filters for sterilization.Resistance and permeability values calculated from the Darcy equation are well suited for comparing different filter membranes or process parameters. Aim. To substantiate the choice of optimal brands of filter membranes and parameters of the liposomal emulsion filtration process for the production of eye drops based on a peptide complex. Materials and methods. The study object was a liposomal emulsion for the production of eye drops based on a peptide complex. A prepared liposomal emulsion of eye drops based on a peptide complex and membrane filters of various brands were used for filtration. Filtration tests were performed on a Zero-T scaling unit manufactured by Sartorius, Germany. For all measurements, the filtrate weight was controlled using a balance. Filtration experiments were evaluated using Zero-T 2.0 software from Sartorius Stedim Biotech, Germany. At the first stage, the recorded data of the results was saved to a file. Based on the data obtained using the Excel table processor, the filtration flow J°, the initial resistance of the membrane R°zag and the throughput Ṽ were calculated according to the calculated measurement time: the beginning of T10 % and the end of T80 %. Results and discussion. According to the results obtained, the optimal brands of sterilizing membranes were determined; it was shown that by increasing the pressure drop, the volumetric throughput was significantly improved by more than 18 times (from 0.7 to 4.1 bar), and in another experiment – by more than 10 times (from 0.3 to 2.1 bar). In addition, the benefit of using a higher pressure drop to filter liposomes through various sterilizing membranes was shown. Conclusions. The obtained values of the initial resistance of the membrane showed that the geometrical aspects and properties of the material of the Supor® (Pall) and Sartopore 2® (Sartorius) membrane brands have a lower ability to block the membrane during filtration and provide the filtration resource due to greater throughput. It has been also shown that the use of a higher pressure drop increases the productivity of the process.

High-Performance Liquid Chromatography Coupled with Tandem Mass Spectrometry Method for the Identification and Quantification of Lipids in Liposomes.

Liposomes are spherical, closed vesicles consisting of at least one lipid bilayer with a water chamber and are widely used to encapsulate bioactive molecules. Lipid membranes, composed of different types of lipids or lipophilic components, determine whether liposomes can achieve the desired purpose and determine the overall quality of liposomes. Thus, the quantification of lipid components and encapsulated molecules is essential to characterize and control the quality of liposomes. Moreover, multicomponent simultaneous determination is the preferred method for lipid component analysis in liposomes. Therefore, the present work describes an analytical methodology for the simultaneous determination of commonly used lipids in liposome formulations, using h igh-performance liquid chromatography coupled with a tandem mass spectrometry (MS) detector (HPLC-MS/MS). HPLC-MS/MS consists of a rapid and highly efficient chromatographic separation of the liposomal components with a C18 column and the subsequent detection of the ingredients through an MS detector, along with an accurate mass fragmentation pattern. The analytical process mainly includes lipid extraction, solution preparation, the optimization of chromatographic conditions, and method validation. We hope this analytical methodology is valuable and efficient and can be applied to the analysis of multiple types of lipids in liposomes, such as raw material quality analysis, formulation study, overall quality control, etc.

Photoresponsive liposomes: an alternative of ELISA for the detection of low quantities of target substances.

Photoresponsive liposome development is needed to serve as a facile alternative to ELISA which is ineffective for detecting small levels of biomarkers due to low detection sensitivity. TheUS20210396744 patent application outlines novel photoresponsive liposomes for the detection of target substances with the aid of light. Although versatile, there may be possible stability issues that can be avoided with the appropriate selection of liposome components. Furthermore, the clinical success of this technology depends on many parameters like plasma stability, efficient loading of photosensitive components in the membrane and immobilization of molecular recognition elements to the membrane. Despite several challenges, they possess enormous potential to become a non-invasive tool for the detection of target substances.

Lipocalin 2, synthesized using a cell-free protein synthesis system and encapsulated into liposomes, inhibits the adhesion of Porphyromonas gingivalis to human oral epithelial cells.

Lipocalin 2 (LCN2), a glycoprotein expressed in epithelial cells and leukocytes, has an antibacterial effect and plays a role in innate immunity. The delivery of LCN2 encapsulated in liposomes to oral epithelium may be useful to prevent oral infectious diseases. This study aimed to investigate the inhibitory effect of LCN2, artificially synthesized using a cell-free protein synthesis (CFPS) system, on the adhesion of Porphyromonas gingivalis to oral epithelial cells in order to approach oral healthcare using LCN2. LCN 2 was synthesized using a CFPS system and assayed by Western blotting, mass spectrometry and enzyme-linked immunosorbent assay (ELISA). The bilayer liposomes were prepared by the spontaneous transfer method using 1,2-dioleoyl-sn-glycero-3 phosphocholine (DOPC), 3-sn-phosphatidylcholine from Egg Yolk (Egg-PC), and 1,2-dioleoyl-sn-glycero-3 phosphoethanolamine (DOPE). The cellular and medium fractions derived from the culture of oral epithelial cells with liposome-encapsulated LCN2 were assayed by Western blotting and ELISA. The effect of the synthesized LCN2 on adhesion of the labeled P.gingivalis to oral epithelial cells was investigated as an evaluation of its antibacterial activity. The synthesized LCN2 protein was identified by Western blotting; its amino acid sequence was similar to that of recombinant LCN2 protein. The additions of DOPE and octa-arginine in the outer lipid-layer components of liposome significantly increased the delivery of liposomes to epithelial cells. When oral epithelial cells were cultured with the synthesized and liposome-encapsulated LCN2, LCN2 was identified in the cellular and medium fractions by Western blotting and its concentration in the cellular fraction from the culture with the synthesized LCN2 was significantly higher than that of a template DNA-free protein. The synthesized LCN2 and liposome-encapsulated LCN2 significantly inhibited the adhesion of P.gingivalis to oral epithelial cells compared with template DNA-free protein. LCN2 was artificially synthesized by a CFPS system, encapsulated in liposomes, and delivered to oral epithelial cells, and demonstrated an antibacterial action against P.gingivalis. This approach may become a useful model for oral healthcare.

Effect of Distigmasterol-Modified Acylglycerols on the Fluidity and Phase Transition of Lipid Model Membranes.

Plant sterols are known for their health-promoting effects, lowering blood cholesterol levels and alleviating cardiovascular disease. In this work, we continue our research on the asymmetric acylglycerols in which fatty acid residues are replaced by two stigmasterol residues in sn-1 and sn-2 or sn-2 and sn-3 positions as new thermostable carriers of phytosterols for their potential application in foods or as components of new liposomes in the pharmaceutical industry. The aim of this manuscript was to compare and analyze the effects of four distigmasterol-modified acylglycerols (dStigMAs) on the fluidity and the main phase transition temperature of the model phospholipid membrane. Their properties were determined using differential scanning calorimetry (DSC), steady-state fluorimetry and attenuated total reflectance Fourier-transform infrared spectroscopy (ATR-FTIR). The determination of the effect of the tested compounds on the mentioned physicochemical parameters of the model membranes will allow for the determination of their properties and stability, which is essential for their practical application. The results indicated that all compounds effect on the physicochemical properties of the model membrane. The degree of these changes depends on the structure of the compound, especially the type of linker by which stigmasterol is attached to the glycerol backbone, as well as on the type of hydrocarbon chain.

Elevating Phospholipids Production Yarrowia lipolytica from Crude Glycerol.

Phospholipids (PLs) are a class of lipids with many proven biological functions. They are commonly used in lipid replacement therapy to enrich cell membranes damaged in chronic neurodegenerative diseases, cancer, or aging processes. Due to their amphipathic nature, PLs have been widely used in food, cosmetic, and pharmaceutical products as natural emulsifiers and components of liposomes. In Yarrowia lipolytica, PLs are synthesized through a similar pathway like in higher eukaryotes. However, PL biosynthesis in this yeast is still poorly understood. The key intermediate in this pathway is phosphatidic acid, which in Y. lipolytica is mostly directed to the production of triacylglycerols and, in a lower amount, to PL. This study aimed to deliver a strain with improved PL production, with a particular emphasis on increased biosynthesis of phosphatidylcholine (PC). Several genetic modifications were performed: overexpression of genes from PL biosynthesis pathways as well as the deletion of genes responsible for PL degradation. The best performing strain (overexpressing CDP-diacylglycerol synthase (CDS) and phospholipid methyltransferase (OPI3)) reached 360% of PL improvement compared to the wild-type strain in glucose-based medium. With the substitution of glucose by glycerol, a preferred carbon source by Y. lipolytica, an almost 280% improvement of PL was obtained by transformant overexpressing CDS, OPI3, diacylglycerol kinase (DGK1), and glycerol kinase (GUT1) in comparison to the wild-type strain. To further increase the amount of PL, the optimization of culture conditions, followed by the upscaling to a 2 L bioreactor, were performed. Crude glycerol, being a cheap and renewable substrate, was used to reduce the costs of PL production. In this process 653.7 mg/L of PL, including 352.6 mg/L of PC, was obtained. This study proved that Y. lipolytica is an excellent potential producer of phospholipids, especially from waste substrates.

The role of glutathione conjugation on the transcellular transport process of PEGylated liposomes across the blood brain barrier

Notwithstanding the growing evidence of improved drug delivery efficiency to the brain by ligand modification of PEGylated liposomes, the comprehensive knowledge of their transport processes and payload across the BBB is yet to be revealed. Herein, this study sought to understand the glutathione (GSH) ligand effect on transcellular transport mechanisms of liposomes through the blood–brain barrier (BBB) by comparing PEGylated liposomes (PEG-L) and GSH PEGylated liposomes (GSH-PEG-L). Endocytosis and exocytosis of liposomes including the role of secreted extracellular vesicles (EVs) of brain endothelial cells (BECs) were assessed. Furthermore, pharmacokinetics and brain distribution analysis of gemcitabine loaded liposomes were carried out in healthy rats to ascertain the in vivo applicability. Our findings suggested that the presence of GSH increased the cellular uptake of liposomes by up to 3-fold in human brain microvascular endothelial cells depending on the dose but not in astrocytes. The cell exposure to liposomes particularly GSH-PEG-L dramatically increased the cell secretion of small and microvesicles with liposomal components, though different liposomes preferred different vesicles for exocytosis. This correlated with GSH-PEG-L transport efficiency of 4 % across the in vitro BBB model in 24 h, 1.7-fold higher than that of PEG-L (p < 0.05). In rats, while PEG-L and GSH-PEG-L showed similar pharmacokinetic profiles and prolonged circulation properties, 3.8 % of the total injected dose (ID) of gemcitabine was found in the brain of the GSH-PEG-L group at 8 h post-injection, compared with 2.8 % ID in the PEG-L group. A brain: blood concentration ratio of 1.27 ± 0.12 indicated that an active transport mechanism to cross the BBB for GSH-PEG-L. Overall, this study revealed that GSH augmented the transcellular transport efficiency of liposomes through BBB to improve targeted brain delivery by enhancing cellular uptake and vesicular exocytosis route of BECs.

2-(2-Cholesteroxyethoxyl)ethyl-3′-S-glutathionylpropionate (COXP) for brain-targeting liposomes

The blood-brain barrier (BBB) prevents a drug from reaching its therapeutic concentration in the brain resulting in a failure for the treatment of most brain diseases. Various approaches have been explored to develop brain-targeting agents that can help a drug or a drug delivery system to cross the BBB to reach the brain. A ligand or ligand analog of a BBB transporter or BBB receptor has been commonly employed to develop a brain targeting agent.We reported early 2-(2-cholesteroxyethoxyl)ethyl-3′-S-glutathionylpropionate (COXP) as a novel glutathione (GSH) transporter-mediated brain targeting agent based on the over-expression of GSH transporters at the BBB. COXP was obtained by connecting a molecule of GSH with a molecule of cholesterol via a two-ethylene glycol unit. COXP can self-assemble resulting in the formation of brain targeting micelles.Since cholesterol is one of the major components of liposomes, this work was designed to investigate if COXP could be used as a component of liposomes to prepare brain-targeting liposomes (COXP liposomes). Our results demonstrated that COXP liposomes effectively delivered DiR, a fluorescence tracking agent, to the brain of a mouse. The whole-body imaging of mice showed that the fluorescence intensity of DiR in the brain could reach 11 times higher for DiR delivered by COXP liposomes than that delivered by a cationic liposome which served as a control. The results show COXP's potential for developing novel brain-targeting liposomes.

Development and single-particle analysis of hybrid extracellular vesicles fused with liposomes using viral fusogenic proteins.

Extracellular vesicles (EVs) have potential biomedical applications, particularly as a means of transport for therapeutic agents. There is a need for rapid and efficient EV‐liposome membrane fusion that maintains the integrity of hybrid EVs. We recently described Sf9 insect cell‐derived EVs on which functional membrane proteins were presented using a baculovirus‐expression system. Here, we developed hybrid EVs by membrane fusion of small liposomes and EVs equipped with baculoviral fusogenic proteins. Single‐particle analysis of EV‐liposome complexes revealed controlled introduction of liposome components into EVs. Our findings and methodology will support further applications of EV engineering in biomedicine.

PEGylated Liposomes Accumulate in the Areas Relevant to Skin Toxicities via Passive Extravasation across "Leaky" Endothelium.

PEGylated liposome is the cornerstone platform for modern drug delivery. Unfortunately, as exemplified by PEGylated liposomal doxorubicin (aka Doxil), altered doxorubicin pharmacokinetics causes off-target accumulation in the skin, including the palms and feet, leading to severe dose-limiting toxicity. In addition to Doxil, other nanoparticles and PEGylated liposomes exhibit significant deposition in the skin, but mechanisms of accumulation are poorly understood. Using ex vivo imaging and ex vivo confocal microscopy, we show that PEGylated liposomes in mice accumulate predominantly in the areas subject to mechanical stress/pressure. Blood vessels in foot skin appear to be especially leaky, exhibiting burst-like extravasations. Using high-resolution confocal microscopy and liposomes labeled with different dyes in the membrane and/or interior, two modes of extravasation were observed: (1) as intact liposomes; (2) as separated liposomal components. On the other hand, stable cross-linked iron oxide nanoworms extravasated only as intact nanoparticles. There was no colocalization between liposomes and exosomal marker CD81, excluding the role of exocytosis. Also, in situ perfusion of formalin-fixed foot skin with labeled liposomes revealed that the extravasation is mediated by passive, energy-independent diffusion and not by leukocyte "hitchhiking". These findings improve our understanding of extravasation pathways of nanocarriers in the areas relevant to skin pathologies and could lead to strategies to prevent and treat liposome-induced skin toxicities.

Incorporation of Toll-Like Receptor Ligands and Inflammasome Stimuli in GM3 Liposomes to Induce Dendritic Cell Maturation and T Cell Responses.

Cancer vaccination aims to activate immunity towards cancer cells and can be achieved by delivery of cancer antigens together with immune stimulatory adjuvants to antigen presenting cells (APC). APC maturation and antigen processing is a subsequent prerequisite for T cell priming and anti-tumor immunity. In order to specifically target APC, nanoparticles, such as liposomes, can be used for the delivery of antigen and adjuvant. We have previously shown that liposomal inclusion of the ganglioside GM3, an endogenous ligand for CD169, led to robust uptake by CD169-expressing APC and resulted in strong immune responses when supplemented with a soluble adjuvant. To minimize the adverse effects related to a soluble adjuvant, immune stimulatory molecules can be incorporated in liposomes to achieve targeted delivery of both antigen and adjuvant. In this study, we incorporated TLR4 (MPLA) or TLR7/8 (3M-052) ligands in combination with inflammasome stimuli, 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC) or muramyl dipeptide (MDP), into GM3 liposomes. Incorporation of TLR and inflammasome ligands did not interfere with the uptake of GM3 liposomes by CD169-expressing cells. GM3 liposomes containing a TLR ligand efficiently matured human and mouse dendritic cells in vitro and in vivo, while inclusion of PGPC or MDP had minor effects on maturation. Immunization with MPLA-containing GM3 liposomes containing an immunogenic synthetic long peptide stimulated CD4+ and CD8+ T cell responses, but additional incorporation of either PGPC or MDP did not translate into stronger immune responses. In conclusion, our study indicates that TLRL-containing GM3 liposomes are effective vectors to induce DC maturation and T cell priming and thus provide guidance for further selection of liposomal components to optimally stimulate anti-cancer immune responses.