Liposome Preparation Method Research Articles

An Extensive Review on Novel Liposomes : Classification, Methodology, Characterization, Current Formulations.

Liposomes are vesicles consisting of a phospholipid, hydrophobic drug, hydrophobic tail, hydrophilic tail, cholesterol, targeting agents, positive and negatively charged lipids, and a drug encapsulated in the center of the phospholipid group having a spherical shape. The phospholipid consists of an equal number of aqueous membranes, making the liposomes an important nanocarrier for the drug delivery to the targeted site. Various liposome-based products have recently been approved and are in clinical trials. This review will discuss the structure, classification, types, and method of liposome preparation and various marketed liposomal products.

Fabrication and characterization of hydroquinone in liposomal gel for transdermal drug delivery

This study aimed to formulate a gel for hydroquinone dermal therapy using liposomes to maintain the active agents' concentration in the skin's deepest layers. Cholesterol was incorporated to enhance the liposome's bilayer characteristics, increasing microviscosity, membrane stability, and blister rigidity. Various methods for liposome preparation exist, but the film hydration method, being the most common, was utilized here. Results for formulation HL6, which had lower levels of Lecithin Cholesterol and rotation speed, revealed a vesicle size of 180.4 nm, a Zeta potential of -37.5 mV, and an entrapment efficiency of 69.10±1.52%. In-vitro drug release data for formulations F1, F2, and F3 within 30 minutes showed hydroquinone release rates of 97.75±0.28%, 98.92±0.56%, and 94.45±0.36%, respectively. The order of drug release was F2 > F1 > F3, with F2 demonstrating the maximum release rate. The study concludes that liposomal gel is an effective transdermal drug delivery system for therapeutic molecules. Lipid vesicles, such as liposomes, are among the best mechanisms for delivering medications to their intended locations while minimizing their dissemination to non-target tissues. This liposomal gel-based formulation shows significant potential for effectively treating acne by maintaining high concentrations of active agents in the skin's deepest layers and ensuring a controlled and sustained drug release.

Effect of lipid composition on the characteristics of liposomes prepared using the polyol dilution method

Polyol dilution (PD) is a liposome preparation method suitable for mass production in the cosmetics industry. For PD-liposomes, lecithin is the primary material of choice because of its cost advantage. However, despite the importance of understanding the effect of lipid composition on the physicochemical properties of PD-liposomes in terms of formulation design, this has not yet been elucidated. In this study, PD-liposomes were prepared using hydrogenated egg yolk lecithin (EL) and its main components, 1,2-distearoyl-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-glycero-3-phosphocholine (DPPC), and 1,2-distearoyl-glycero-3-phosphoethanolamine (DSPE). The compound 1,3-butylene glycol (BG), which is widely used in the cosmetics industry, was used as a representative polyol. Herein, we aimed to clarify the effect of lipid composition on PD-liposomes. The results showed that the particle size, dispersion stability, shape, and phase state of the lipid bilayer in PD-liposomes could be controlled by changing the composition of the hydrophilic and hydrophobic groups of the lipids. In particular, when PE was added to a system consisting of PC molecules, a significant decrease in particle size, improvement in dispersion stability, and shape changes were observed, indicating a significant effect on the physicochemical properties of the PD-liposomes. In addition, adding BG induced the formation of an interdigitated structure in the lipid bilayer when PC was the only hydrophilic group component; adding even a small amount of PE suppressed the formation of this structure. Furthermore, we confirmed that premixing BG and lipids enhanced the miscibility of different types of lipid molecules. The findings of this study are expected to contribute to the design of highly functional PD-liposomes by enabling the selection and design of appropriate lipid compositions for practical applications.

Influence of liposome preparation methods on average lipid composition and concentration of two-component liposomes

Influence of liposome preparation methods on average lipid composition and concentration of two-component liposomes

Role of Liposome as Novel Carrier Molecule

Liposomes were discovered by Alec D Bangham in the year 1960.The term liposome means lipid body. They are categorized as a drug delivery system in which drug is targeted to a particular tissue. Liposomes can range from 25nm to 500nm in size. Usually, they have a sphere shape vesicles. They may be having one phospholipid bi- layer or more. They are widely used as drug targeting delivery system because they can penetrate tissues more precisely than free drugs and they have similarity between their lipid bilayer and cell membrane. They are also preferred more because of their drug entrapment efficiency. Liposomes are biocompatible i.e. they are capable to entrap both hydrophilic and lipophilic drug hence they solve the problem of choosing a suitable outer covering layer for the formulation as, they are suitable for both hydrophilic and lipophilic drug molecules. Liposomes are the modern world technology to deliver the drugs to a specific targeted organs or tissue. There exist a lot of methods for preparation of liposomes and they are classified according to their structure and methods of preparation which are discussed in brief in this review article, along with liposome advantages and disadvantages, handling of liposomes, evaluation parameters and liposome applications.

Simple Method for the Creation of a Bacteria-Sized Unilamellar Liposome with Different Proteins Localized to the Respective Sides of the Membrane.

Artificial cells are membrane vesicles mimicking cellular functions. To date, giant unilamellar vesicles made from a single lipid membrane with a diameter of 10 μm or more have been used to create artificial cells. However, the creation of artificial cells that mimic the membrane structure and size of bacteria has been limited due to technical restrictions of conventional liposome preparation methods. Here, we created bacteria-sized large unilamellar vesicles (LUVs) with proteins localized asymmetrically to the lipid bilayer. Liposomes containing benzylguanine-modified phospholipids were prepared by combining the conventional water-in-oil emulsion method and the extruder method, and green fluorescent protein fused with SNAP-tag was localized to the inner leaflet of the lipid bilayer. Biotinylated lipid molecules were then inserted externally, and the outer leaflet was modified with streptavidin. The resulting liposomes had a size distribution in the range of 500-2000 nm with a peak at 841 nm (the coefficient of variation was 10.3%), which was similar to that of spherical bacterial cells. Fluorescence microscopy, quantitative evaluation using flow cytometry, and western blotting proved the intended localization of different proteins on the lipid membrane. Cryogenic electron microscopy and quantitative evaluation by α-hemolysin insertion revealed that most of the created liposomes were unilamellar. Our simple method for the preparation of bacteria-sized LUVs with asymmetrically localized proteins will contribute to the creation of artificial bacterial cells for investigating functions and the significance of their surface structure and size.

Amphotericin B liposomal formulation: applicable preparation methods, challenges, and tips

Objective This study was intended to explore and evaluate the appropriate methods for preparation of Amphotericin B (AmB) liposomes with acceptable characteristics. Significance This project provides pre-formulations for industrial manufacturing of liposomal AmB which confers improved properties, besides reduced toxicity compared with the plain drug. Methods At first, Solubility screening tests were performed, and in the following, three liposome preparation methods including ethanol injection, solvent evaporation, and solvent-free method were examined. In the following, the physicochemical characteristics of the prepared liposomes as well as size, size distribution, zeta potential (ZP), morphology, drug loading, loading capacity, physicochemical stability, and drug-lipid interaction studies were investigated. HPLC was applied for analyzing AmB. Results In all three methods, liposomes with acceptable characteristics were obtained. The size range of liposomes was 150.3 to 263.9 nm and polydispersity index ≤0.32. In morphologic evaluations, the liposomes have appeared as spherical and separate vesicles. A physical loading of AmB without specific interaction between components was achieved. The lyophilized powder in the solvent-free method was physicochemically stable for 6 months without changes in appearance; the remaining drug after 6-month storage at 25 °C and 60% RH, accounts for 91.5 ± 0.5% compared with the initial drug loaded in liposomes, and degradation pattern follows a linear order. Conclusion As a result, AmB-loaded liposomes were prepared in three applicable methods. The solvent-free method can be considered the most economical and environmental-friendly.

Ethanol Injection Method for Liposome Preparation.

Ethanol injection method is one of the preferred methods for liposome preparation due to its advantages including rapidity, safety, and reproducibility. This method involves the injection of phospholipid solution of ethanol into a stirred aqueous solution. Due to the diffusion of ethanol in aqueous solution, the dissolved phospholipids precipitate to form bilayer phospholipid fragments, which further fuse to form closed liposomal structures. After evaporation of ethanol, the liposomes can be finally obtained. In this chapter, we will describe the details of ethanol injection method for preparing liposomes and discuss issues that need to be considered during the fabrication process.

Preparation of DRV Liposomes.

Dried reconstituted vesicle (DRV) liposomes are formulated under mild conditions. The method has the capability to entrap substantially higher amounts of hydrophilic solutes, compared to other passive-loading liposome preparation methods. These characteristics make this liposome type ideal for entrapment of labile substances, such as peptides, proteins, or DNA's (or other nucleotides or oligonucleotides), or in general biopharmaceuticals and sensitive drugs. In this chapter, all possible types of DRV liposomes (in respect to the encapsulated molecule characteristics and/or their applications in therapeutics) are introduced, and preparation methodologies (for each type) are described in detail.

Microfluidic paclitaxel-loaded lipid nanoparticle formulations for chemotherapy

Nanoparticle technology has promising effects on multiple therapeutic purposes, particularly in controlling drug delivery as Drug Delivery System. The unique properties of nanoparticles significantly enhance drug delivery, efficiency, and toxicity. For cancer therapy, controlling chemotherapy delivery can increase the drug concentration in the desired locations, improve drug efficacy, and limit drug toxicity. Liposomes are used in this project to encapsulate paclitaxel due to their ability to carry hydrophobic molecules, low toxicity, and prolonged half-life. Among the multiple liposome preparation methods, microfluidic technology was used to produce liposomes. Microfluidics excels in other conventional methods by offering a high-level control of the process's parameters, which help control particle size, size distribution, and physiochemical properties. This project aims to produce paclitaxel-loaded liposomes with a diameter below 200 nm with low polydispersity index, high homogeneity, and good stability. Different lipid types (DMPC, DPPC, DSPC, and DOPC) were used with different ratios to investigate their impact on empty liposome formulation. Alongside changing the different microfluidic parameters including the total flow ratio and flow rate ratio to study their effects on liposomes' physiochemical properties. The obtained formulations were tested to analyse different physiochemical properties (DLS, FTIR) and stability studies. DMPC and DPPC are determined to study their encapsulation efficiency and in vitro drug release of paclitaxel at total flow rate 1 ml min−1 and 1:4 flow rate ratio. The paclitaxel-loaded liposomes are subjected to the same physiochemical characteristics and stability study. Promising encapsulation efficiency was reported from both DPPC and DMPC, and sustained drug release was observed.

Liposomal Drug Delivery and Its Potential Impact on Cancer Research.

Liposomes are one of the most versatile drug carriers due to their functional properties, such as higher biocompatibility, the ability to encapsulate hydrophilic and hydrophobic products, and higher biodegradability. Liposomes are a better and more significant nanocarrier for cancer therapy. The key to developing a better cancer-targeted nanocarrier is the development of targeted liposomes using various approaches. Several traditional and novel liposome preparation methods are briefly discussed in this mini-review. The current state of liposome targeting, active and passive liposome targeting in cancer therapy, ligand directed targeting (antibody, aptamer, and protein/peptide-mediated targeting), and other miscellaneous approaches such as stimuli-responsive liposome-based targeting, autophagy inhibition mediated targeting, and curcumin loaded liposomal targeting are all discussed within. All of this gathered and compiled information will shed new light on liposome targeting strategies in cancer treatment and will pique the interest of aspiring researchers and academicians.

Novel microfluidic swirl mixers for scalable formulation of curcumin loaded liposomes for cancer therapy

Liposomes have been widely used in nanomedicine for the delivery of hydrophobic and hydrophilic anticancer agents. The most common applications of these formulations are vaccines and anticancer formulations (e.g., mRNA, small molecule drugs). However, large-scale production with precise control of size and size distribution of the lipid-based drug delivery systems (DDSs) is one of the major challenges in the pharmaceutical industry. In this study, we used newly designed microfluidic swirl mixers with simple 3D mixing chamber structures to prepare liposomes at a larger scale (up to 320 mL/min or 20 L/h) than the commercially available devices. This design demonstrated high productivity and better control of liposome size and polydispersity index (PDI) than conventional liposome preparation methods. The microfluidic swirl mixer devices were used to produce curcumin-loaded liposomes under different processing conditions which were later characterized and studied in vitro to evaluate their efficiency as DDSs. The obtained results demonstrated that the liposomes can effectively deliver curcumin into cancer cells. Therefore, the microfluidic swirl mixers are promising devices for reproducible and scalable manufacturing of DDSs.

Methods of Liposomes Preparation: Formation and Control Factors of Versatile Nanocarriers for Biomedical and Nanomedicine Application.

Liposomes are nano-sized spherical vesicles composed of an aqueous core surrounded by one (or more) phospholipid bilayer shells. Owing to their high biocompatibility, chemical composition variability, and ease of preparation, as well as their large variety of structural properties, liposomes have been employed in a large variety of nanomedicine and biomedical applications, including nanocarriers for drug delivery, in nutraceutical fields, for immunoassays, clinical diagnostics, tissue engineering, and theranostics formulations. Particularly important is the role of liposomes in drug-delivery applications, as they improve the performance of the encapsulated drugs, reducing side effects and toxicity by enhancing its in vitro- and in vivo-controlled delivery and activity. These applications stimulated a great effort for the scale-up of the formation processes in view of suitable industrial development. Despite the improvements of conventional approaches and the development of novel routes of liposome preparation, their intrinsic sensitivity to mechanical and chemical actions is responsible for some critical issues connected with a limited colloidal stability and reduced entrapment efficiency of cargo molecules. This article analyzes the main features of the formation and fabrication techniques of liposome nanocarriers, with a special focus on the structure, parameters, and the critical factors that influence the development of a suitable and stable formulation. Recent developments and new methods for liposome preparation are also discussed, with the objective of updating the reader and providing future directions for research and development.

Moxifloxacin Liposomes: Effect of Liposome Preparation Method on Physicochemical Properties and Antimicrobial Activity against Staphylococcus epidermidis.

The aim of this study was the development of optimal sustained-release moxifloxacin (MOX)-loaded liposomes as intraocular therapeutics of endophthalmitis. Two methods were compared for the preparation of MOX liposomes; the dehydration–rehydration (DRV) method and the active loading method (AL). Numerous lipid-membrane compositions were studied to determine the potential effect on MOX loading and retention in liposomes. MOX and phospholipid contents were measured by HPLC and a colorimetric assay for phospholipids, respectively. Vesicle size distribution and surface charge were measured by DLS, and morphology was evaluated by cryo-TEM. The AL method conferred liposomes with higher MOX encapsulation compared to the DRV method for all the lipid compositions used. Cryo-TEM showed that both liposome types had round vesicular structure and size around 100–150 nm, while a granular texture was evident in the entrapped aqueous compartments of most AL liposomes, but substantially less in DRV liposomes; X-ray diffraction analysis demonstrated slight crystallinity in AL liposomes, especially the ones with highest MOX encapsulation. AL liposomes retained MOX for significantly longer time periods compared to DRVs. Lipid composition did not affect MOX release from DRV liposomes but significantly altered drug loading/release in AL liposomes. Interestingly, AL liposomes demonstrated substantially higher antimicrobial potential towards S. epidermidis growth and biofilm susceptibility compared to corresponding DRV liposomes, indicating the importance of MOX retention in liposomes on their activity. In conclusion, the liposome preparation method/type determines the rate of MOX release from liposomes and modulates their antimicrobial potential, a finding that deserves further in vitro and in vivo exploitation.

Liposome Preparation by 3D-Printed Microcapillary-Based Apparatus.

Liposomal encapsulation serves as the basis for the engineering of biomimetic and novel synthetic cells. Liposomes are normally formed using such methods as thin film rehydration (TFH), density-mediated reverse emulsion encapsulation (REE), or one of many microfluidics-based approaches-with the latter of these two methods being used mainly for the encapsulation of various lumen constituents such as cell-free protein expression reactions. Here, we describe the simultaneous formation and encapsulation of liposomes and various cell-mimetic lumen chemistries, respectively, using a 3D-printable microcapillary-based microfluidics device based off of the droplet-shooting and size-filtration (DSSF) liposome preparation method.

Biomimetic Recognition-Based Bioorthogonal Host–Guest Pairs for Cell Targeting and Tissue Imaging in Living Animals

Biomimetic Recognition-Based Bioorthogonal Host–Guest Pairs for Cell Targeting and Tissue Imaging in Living Animals

Basic Methods for Preparation of Liposomes and Studying Their Interactions with Different Compounds, with the Emphasis on Polyphenols.

Studying the interactions between lipid membranes and various bioactive molecules (e.g., polyphenols) is important for determining the effects they can have on the functionality of lipid bilayers. This knowledge allows us to use the chosen compounds as potential inhibitors of bacterial and cancer cells, for elimination of viruses, or simply for keeping our healthy cells in good condition. As studying those effect can be exceedingly difficult on living cells, model lipid membranes, such as liposomes, can be used instead. Liposomal bilayer systems represent the most basic platform for studying those interactions, as they are simple, quite easy to prepare and relatively stable. They are especially useful for investigating the effects of bioactive compounds on the structure and kinetics of simple lipid membranes. In this review, we have described the most basic methods available for preparation of liposomes, as well as the essential techniques for studying the effects of bioactive compounds on those liposomes. Additionally, we have provided details for an easy laboratory implementation of some of the described methods, which should prove useful especially to those relatively new on this research field.

Enhancement of in vitro antitumour activity of epirubicin in HER2+ breast cancer cells using immunoliposome formulation.

Epirubicin (EPI) is one of the potent breast cancer (BC) chemotherapeutic agents, but its adverse effects limit its efficacy. Herein, EPI was selected to be loaded in liposomal carrier, which has been targeted by a monoclonal antibody, Herceptin. The preparation process of liposomes was a modified ethanol injection method followed by Herceptin conjugation. The in vitro cell toxicity and cellular uptake of optimum formulation against HER2+ and HER2− cancer cell lines were evaluated. The results showed that the drug loading (DL%) and encapsulation efficiency (EE%) of liposome preparation method yielded 30.62% ± 0.49% and 62.39% ± 8.75%, respectively. The average size of naked liposomes (EPI‐Lipo) and immunoliposomes (EPI‐Lipo‐mAb) was 234 ± 9.86 and 257.26 ± 6.25 nm, with a relatively monodisperse distribution, which was confirmed by SEM micrographs. The release kinetic followed Higuchi model for both naked and immunoliposomes. In vitro cytotoxicity study on three different BC cell lines including BT‐20, MDA‐MB‐453 and MCF‐7 demonstrated higher toxicity of EPI in the Herceptin conjugated form (EPI‐Lipo‐mAb) in comparison with the free EPI and EPI‐Lipo in HER2 overexpressing cell line. In addition, the cellular uptake study showed a higher uptake of immunoliposomes by MCF‐7 cells in comparison with naked liposomes. In conclusion, these data show that the targeted delivery of EPI to breast cancer cells can be achieved by EPI‐Lipo‐mAb in vitro, and this strategy could be used for breast cancer therapy with further studies.

Compositional inhomogeneity of drug delivery liposomes quantified at the single liposome level

Liposomes are the most used drug delivery vehicle and their therapeutic function is closely linked to their lipid composition. Since most liposome characterization is done using bulk techniques, providing only ensemble averages, the lipid composition of all liposomes within the same formulation are typically assumed to be identical. Here we image individual liposomes using confocal microscopy to quantify that liposomal drug delivery formulations, including multiple component mixtures mimicking Doxil, display more than 10-fold variation in their relative lipid composition. Since liposome function is tightly regulated by the physicochemical properties bestowed by the lipid composition, such significant variations could render only a fraction of liposomes therapeutically active. Additionally, we quantified how this degree of compositional inhomogeneity was modulated by liposome preparation method, the saturation state of the membrane lipid, and whether anti-fouling polyethylene glycol (PEG) conjugated lipids were added to the initial lipid mix or inserted after liposome formation. We believe the insights into the factors governing the degree of inhomogeneity offers the possibility for producing more uniform liposomal drug delivery systems, potentially increasing their therapeutic efficacy.

Abstract 2877: Advanced properties of Vitamin D3 nanocarrier in cancer chemotherapy

Abstract [Purpose] Vitamin D3 (VD3) is used to treat osteoporosis and causes rickets, osteomalacia and hypocalcemia as deficiencies. On the other hand, it has been reported that large intake of VD3 reduce carcinogenesis risk in colorectal cancer, and VD3 itself has an anticancer effect. However, overdose of VD3 causes severe hypercalcemia as an adverse reaction. Therefore, it was expected that liposomalization of VD3 could improve this problem. VD3 encapsulated liposome as a drug nanocarrier is possible to accumulate of VD3 in a tumor as a target site without adverse reaction. In this study, the optimal preparation of VD3 encapsulated liposomes, their antitumor effects without adverse reaction were examined. [Methods] Cholecalciferol as VD3 encapsulated liposome by thin film method with different amount of cholesterol (CHO) was prepared. L-α-Distearoylphosphatidylcholine, CHO, L-α-distearoylphosphatidyl-DL-glycerol and 1-monomethoxypolyethyleneglycol-2,3-distearoylglyerol were dissolved using chloroform/methanol (4:1,(v/v)). After empty liposome preparation, VD3 is added to this suspension and incubated to encapsulate VD3 (Basic Method). The other method used ethanol as solvent and VD3 was added with the constituent lipids (Modified method). Cytotoxic effect was examined using tumor cell with overexpression of VD3 receptor. In P388 leukemia cell suspension, cytotoxicity by VD3 liposomes or VD3 solution were determined by the WST-8 assay. P388 leukemia cells with VD3 liposomes or VD3 solution were incubated and the intracellular VD3 concentration was measured. In vivo experiment, P388 leukemia cells bearing BDF1 mice were administered VD3 liposomes at 1st, 4th and 7th day. On the next day after final administration, tumor weight and blood calcium concentration were measured. [Results and Discussion] The particle size and zeta potential of VD3 liposomes were same level in some ratio of CHO in lipid composition. The encapsulated amount of VD3 in liposomes increased with the decrease of CHO amount. This may be due to the similar structure of CHO and VD3. VD3 encapsulated level of liposome by modified method was showed high amount. Because VD3 is lipophilicity, the high encapsulated amount of VD3 may have when the addition of VD3 with the formation of lipid film. VD3 receptor expression of P388 leukemia cells was higher than that of B16F10 melanoma cells by Western blots. In P388 leukemia cells, VD3 solution was not have cytotoxicity whereas VD3 liposome showed significant cytotoxicity. Furthermore, VD3 concentration in P388 leukemia cells was slight in VD3 solution group. In contrast, its concentration in VD3 liposome group quickly elevate and maintain a high concentration. In tumor bearing mice after VD3 liposome treatment, the tumor weight was decreased and the blood calcium concentration had lower level than that of control level. Namely, it was suggested that VD3 liposomes has a superior antitumor effect without the increase of adverse reaction. In conclusion, the optimum method of VD3 liposome preparation and its usefulness in novel cancer treatment were clarified. Citation Format: Yukako Soma, Ikumi Sugiyama, Yasuyuki Sadzuka. Advanced properties of Vitamin D3 nanocarrier in cancer chemotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2877.