Stability Of Liposomes Research Articles

Liposomes containing 3-arylamino-nor-β-lapachone derivative: Development, characterization, and in vitro evaluation of the cytotoxic activity

This study aimed to encapsulate the novel synthetic naphthoquinone ENSJ39 in liposomes, characterize them, and evaluate their in vitro cytotoxic activity in different cancer cell lines. Liposomes were obtained by the dried-lipid film hydration method, and characterizations included thermogravimetric analysis, differential scanning calorimetry, dynamic light scattering technique, and atomic force microscopy. Simulation of storage conditions, in vitro cytotoxicity by MTT, Trypan blue exclusion assay, and evaluation of morphological changes in tumor cell lines were also analyzed. Liposomes containing ENSJ39 (LE39) exhibited an average size of 31 ± 5.81 nm, zeta potential of +53.5 mV, polydispersity index of 0.24, and high encapsulation efficiency (96.58 ± 0.09%). They were thermally stable up to 250 °C and able to preserve the mass loss of the free drug. After four months of storage, they maintained excellent physical-chemical characteristics. The encapsulated drug showed less cytotoxic activity than the free drug only in HCT-116 and SNB-19 cells and caused cytoplasmic vacuolization plus an increase in the number of non-viable HCT-116 cells. ENSJ39 encapsulation method was effective in obtaining stable liposomes that presented substantial cytotoxicity activity against tumor cell lines.

A thermodynamic study of F108 and F127 block copolymer interactions with liposomes at physiological temperature

The interactions of egg yolk phosphatidylcholine liposomes with F108 and F127 triblock copolymers, in the monomer state, were analyzed by isothermal titration calorimetry (ITC) at 37 °C. According to the results, the critical micelle concentration was determined to be 0.4 and 0.04 wt.% for F108 and F127, respectively, by surface tension at 37 °C. According to the results, liposomes/poloxamers were not favoured energetically, since endothermic interactions were observed. However, positive changes in entropy promoted a spontaneous process. F127 had a greater partition coefficient (51.97 ± 1.77 × 104), stronger affinity, than F108 (8.19 ± 0.37 × 104) towards the vesicle lipid bilayer due to its larger hydrophobic block. After the ITC experiments, an increased vesicle size (within about 1–3 nm average) by dynamic light scattering and the formation of bilayer discs by electron microscopy (EM) was observed at low copolymer concentrations (0.57 mol% of F108 and 1.01 mol% of F127). The EM and ITC results confirmed the intimate association of the copolymers with the membrane instead of being simply absorbed onto the bilayer surface. Our results indicate that the temperature of the system (37 °C), the copolymer concentration and hydrophobic chain length are important factors for the interaction of poloxamers with lipid bilayers and the stability of liposomes.

Shell properties and concentration stability of acoustofluidic delivery agents.

This paper investigates the shell elastic properties and the number-concentration stability of a new acoustofluidic delivery agent liposome in comparison to Definity™, a monolayer ultrasonic contrast agent microbubble. The frequency dependent attenuation of an acoustic beam passing through a microbubble suspension was measured to estimate the shell parameters. The excitation voltage was adjusted to ensure constant acoustic pressure at all frequencies. The pressure was kept at the lowest possible magnitude to ensure that effects from nonlinear bubble behaviour which are not considered in the analytical model were minimal. The acoustofluidic delivery agent shell stiffnessSp and frictionSf parameters were determined as (Sp = 0.11N/m, Sf = 0.31 × 10-6Kg/sat25°C) in comparison to the Definity™ monolayer ultrasound contrast agent which were (Sp = 1.53N/m, Sf = 1.51 × 10-6Kg/sat25°C). When the temperature was raised to physiological levels, the friction coefficientSf decreased by 28% for the monolayer microbubbles and by only 9% for the liposomes. The stiffness parameter Sp of the monolayer microbubble decreased by 23% while the stiffness parameter of the liposome increased by a similar margin (27%) when the temperature was raised to 37°C. The size distribution of the bubbles was measured using Tunable Resistive Pulse Sensing (TRPS) for freshly prepared microbubbles and for bubble solutions at 6h and 24h after activation to investigate their number-concentration stability profile. The liposome maintained >80% of their number-concentration for 24h at physiological temperature, while the monolayer microbubbles maintained only 27% of their number-concentration over the same period. These results are important input parameters for the design of effective acoustofluidic delivery systems using the new liposomes.

Effects of Phytosterol Butyrate Ester on the Characteristics of Soybean Phosphatidylcholine Liposomes.

The nutritional and structural properties of phytosterols (PS)/phytosterol esters (PEs) facilitate their use as substitutes for cholesterol in liposome encapsulation systems designed for oral drugs and health products. The purpose of this study was to determine the effect of phytosterol butyrate ester (PBE) on the properties of liposomes. PBE was encapsulated within liposomes (approximately 60 nm) prepared using soybean phosphatidylcholine using the thin-film hydration method. There was no significant change in the average particle diameter and zeta potential of these liposomal vesicles corresponding to the increasing amounts of encapsulated PBE. The incorporation of PBE increased the polydispersity index (PDI) independent of concentration. Additionally, we observed that the storage stability of PBE liposomes with uniform particle size and approximately spherical shape vesicle was better at low concentration. The results of Fourier-transform infrared (FTIR) spectroscopy and Raman spectroscopy showed that PBE was positioned at the water interface, which increased the order of hydrophobic alkyl chains in the lipid membranes. The incorporation of PBE led to an increase in the trans conformation of hydrophobic alkyl chain and consequently, the thermal stability of liposomes, which was confirmed by differential scanning calorimetry (DSC). The results of powder X-ray diffraction (XRD) analysis confirmed that PBE was present in an amorphous form in the liposomes. Additionally, the incorporation of PBE reduced the micropolarity of the lipid membrane. Thus, when preparing liposomes using thin-film hydration, the presence of PBE affected the characteristics of liposomes.

Vesicle Delivery Systems of Biologically Active Compounds: From Liposomes to Cerasomes

Modern pharmaceutics aims at creating new drugs with high bioavailability, biocompatibility and efficiency, as well as minimally toxic effects. One of the ways to reach this goal is to create nanosized particles that carry a small but sufficient dose of a medicinal drug. The diversity of nanoscale delivery systems allows designing therapeutic complexes with necessary characteristics. Liposomes are lipid vesicles with a bilayer membrane possessing the properties required for transportation of various drugs and genes. They consist of natural components, which can minimize cytotoxicity. In addition, the creation of artificial cationic lipids makes it possible to adjust the properties of delivery systems to particular purposes. Cerasomes are organosilicon particles that have been developed to overcome the low stability of liposomes. These are nanoscale spherical aggregates consisting, like liposomes, of a lipid bilayer, but their surface is modified by a silicone polymer network. Cerasomes have all advantages of liposomes. This review presents basic methods for the synthesis of liposome components and cerasome-forming lipids to create stable delivery systems. Different variants of cationic lipid structures and modifications of cerasome-forming lipids are presented. Potential areas of their application are described, including gene transfection, photodynamic therapy, visualization and diagnosis of diseases using magnetic resonance, and tumor treatment.

Specific Targeting of PEGylated Liposomal Doxorubicin (Doxil®) to Tumour Cells Using a Novel TIMP3 Peptide.

Doxorubicin is a cytotoxic anthracycline derivative that has been used as a chemotherapeutic in many different forms of human cancer with some success. However, doxorubicin treatment has several side-effects, the most serious of which is cardiomyopathy, that can be fatal. Doxorubicin encapsulation in PEGylated liposomes (Doxil®) has been shown to increase tumour localisation and decrease cardiotoxicity. Conversely, the stability of such liposomes also leads to increased circulation times and accumulation in the skin, resulting in palmar planter erythrodysesthesia, while also limiting release of the drug at the tumour site. Specific targeting of such liposomes to tumour cells has been attempted using various receptor-specific peptides and antibodies. However, targeting a single epitope limits the likely number of tumour targets and increases the risk of tumour resistance through mutation. In this report, Doxil® was coupled to peptide sequence p700 derived from tissue inhibitor of metalloproteinase 3. This Doxil® -P700 complex results in an approximately 100-fold increase in drug uptake, relative to Doxil® alone, by both mouse and human breast cancer cells and immortalised vascular cells resulting in an increase in cytotoxicity. Using p700 to target liposomes in this way may enable specific delivery of doxorubicin or other drugs to a broad range of cancers.

Physical stability of microliposomes in bene (Pistacia atlantica) oil with different formulations

Bene oil (pistacia atlantica), as a plant source, is rich in phenolic and tocopherol compounds and has significant antioxidant, therapeutic and antimicrobial effects. Encapsulation of hydrophobic compounds in liposome system is an ideal solution for protecting them against destruction during storage. An important advantage of liposomes is the encapsulation of hydrophilic, hydrophobic and amphiphilic compounds by using natural phospholipids, such as lecithin, with beneficial effects. The aim of this study is to encapsulate the bene kernel oil in the form of microliposomes. For this purpose, the effect of composition of liposomes based on lecithin and cholesterol was studied using the Mozafari method. Liposomes are prepared using lecithin and cholesterol in the ratios of 60:0, 50:10, 40:20 and 30:30. Particle size, size distribution, zeta potential and encapsulation efficiency were charac-terised. According to the result, the size of liposomes was dependent on their composition, but the wasaffected significantly affected by adding cholesterol (P < 0.05). Average diameter of the particles was between 4 and 9 µm. Liposome with a ratio of 40:20 had the smallest size. By applying cholesterol, zeta potential increased from 16.4 mV to 32.39 mV, which indicates electrostatic stability of liposomes. In general, with encapsulation efficiency of 84.33%, the ratio of 40:20 is considered as an ideal concentration in the formulation of microliposomes. Based on the results, bene oil-loaded liposomes with a lecithin:cholesterol formulation ratio of :as 40:20 was chosen as an optimal formulation because of its smaller particle size, higher zeta potential and suitable stability, which can be used in trapping, delivering and releasing hydrophilic, adipose-friendly and amphiphilic compounds (dual-friendly).

Physical stability of microliposomes in bene (Pistacia atlantica) oil with different formulations

Bene oil (pistacia atlantica), as a plant source, is rich in phenolic and tocopherol compounds and has significant antioxidant, therapeutic and antimicrobial effects. Encapsulation of hydrophobic compounds in liposome system is an ideal solution for protecting them against destruction during storage. An important advantage of liposomes is the encapsulation of hydrophilic, hydrophobic and amphiphilic compounds by using natural phospholipids, such as lecithin, with beneficial effects. The aim of this study is to encapsulate the bene kernel oil in the form of microliposomes. For this purpose, the effect of composition of liposomes based on lecithin and cholesterol was studied using the Mozafari method. Liposomes are prepared using lecithin and cholesterol in the ratios of 60:0, 50:10, 40:20 and 30:30. Particle size, size distribution, zeta potential and encapsulation efficiency were characterised. According to the result, the size of liposomes was dependent on their composition, but the wasaffected significantly affected by adding cholesterol (P < 0.05). Average diameter of the particles was between 4 and 9 µm. Liposome with a ratio of 40:20 had the smallest size. By applying cholesterol, zeta potential increased from 16.4 mV to 32.39 mV, which indicates electrostatic stability of liposomes. In general, with encapsulation efficiency of 84.33%, the ratio of 40:20 is considered as an ideal concentration in the formulation of microliposomes. Based on the results, bene oil-loaded liposomes with a lecithin: cholesterol formulation ratio of :as 40:20 was chosen as an optimal formulation because of its smaller particle size, higher zeta potential and suitable stability, which can be used in trapping, delivering and releasing hydrophilic, adipose-friendly and amphiphilic compounds (dual-friendly).

Improving Vesicular Integrity and Antioxidant Activity of Novel Mixed Soy Lecithin-Based Liposomes Containing Squalene and Their Stability against UV Light.

In order to improve the membrane lipophilicity and the affinity towards the environment of lipid bilayers, squalene (SQ) could be conjugated to phospholipids in the formation of liposomes. The effect of membrane composition and concentrations on the degradation of liposomes prepared via the extrusion method was investigated. Liposomes were prepared using a mixture of SQ, cholesterol (CH) and Tween80 (TW80). Based on the optimal conditions, liposome batches were prepared in the absence and presence of SQ. Their physicochemical and stability behavior were evaluated as a function of liposome constituent. From the optimization study, the liposomal formulation containing 5% (w/w) mixed soy lecithin (ML), 0.5% (w/w) SQ, 0.3% (w/w) CH and 0.75% (w/w) TW80 had optimal physicochemical properties and displayed a unilamellar structure. Liposome prepared using the optimal formulation had a low particle size (158.31 ± 2.96 nm) and acceptable %increase in the particle size (15.09% ± 3.76%) and %trolox equivalent antioxidant capacity (%TEAC) loss (35.69% ± 0.72%) against UV light treatment (280–320 nm) for 6 h. The interesting outcome of this research was the association of naturally occurring substance SQ for size reduction without the extra input of energy or mechanical procedures, and improvement of vesicle stability and antioxidant activity of ML-based liposome. This study also demonstrated that the presence of SQ in the membrane might increase the acyl chain dynamics and decrease the viscosity of the dispersion, thereby limiting long-term stability of the liposome.

Preparation of nanoliposomes containing HER2/neu (P5+435) peptide and evaluation of their immune responses and anti-tumoral effects as a prophylactic vaccine against breast cancer.

HER2/neu is an immunogenic protein inducing both humoral and cell-mediated immune responses. The antigen-specific cytotoxic T lymphocytes (CTLs) are the main effector immune cells in the anti-tumor immunity. To induce an effective CTL specific response against P5+435 single peptide derived from rat HER2/neu oncogene, we used a liposome delivery vehicle. In vivo enhancement of liposome stability and intracytoplasmic delivery of peptides are the main strategies which elevate the liposome-mediated drug delivery. Liposomes containing high transition temperature phospholipids, such as DSPC, are stable with prolonged in vivo circulation and more accessibility to the immune system. Incorporation of DOPE phospholipid results in the effective delivery of peptide into the cytoplasm via the endocytotic pathway. To this end, the P5+435 peptide was linked to Maleimide-PEG2000-DSPE and coupled on the surface of nanoliposomes containing DSPC: DSPG: Cholesterol with/without DOPE. We observed that mice vaccinated with Lip-DOPE-P5+435 formulation had the highest number of IFN-γ- producing CTLs with the highest cytotoxic activity that consequently led to significantly smallest tumor size and prolonged survival rate in the TUBO mice model. In conclusion, our study indicated that the liposomal form of P5+435 peptide containing DOPE can be regarded as a promising prophylactic anti-cancer vaccine to generate potent antigen-specific immunity.

Impact of Quercetin Encapsulation with Added Phytosterols on Bilayer Membrane and Photothermal-Alteration of Novel Mixed Soy Lecithin-Based Liposome.

This study used highly lipophilic agents with an aim to increase the oxidant inhibitory activity and enhance photothermal stability of a novel mixed soy lecithin (ML)-based liposome by changing the composition of formulation within the membrane. Specifically, the development and optimization of the liposome intended for improving Trolox equivalent antioxidant capacity (TEAC) value and %TEAC loss was carried out by incorporating a natural antioxidant, quercetin (QU). In this context, a focus was set on QU encapsulation in ML-based liposomes and the concentration-dependent solubility of QU was investigated and calculated as encapsulation efficiency (EE). To explore the combined effects of the incorporation of plant sterols on the integrity and entrapment capacity of mixed phospholipid vesicles, conjugation of two types of phytosterols (PSs), namely β-sitosterol (βS) and stigmasterol (ST), to mixed membranes at different ratios was also performed. The EE measurement revealed that QU could be efficiently encapsulated in the stable ML-based liposome using 0.15 and 0.1 g/100 mL of βS and ST, respectively. The aforementioned liposome complex exhibited a considerable TEAC (197.23%) and enhanced TEAC loss (30.81%) when exposed to ultraviolet (UV) light (280–320 nm) over a 6 h duration. It appeared that the presence and type of PSs affect the membrane-integration characteristics as well as photodamage transformation of the ML-based liposome. The association of QU with either βS or ST in the formulation was justified by their synergistic effects on the enhancement of the EE of liposomes. Parallel to this, it was demonstrated that synergistic PS effects could be in effect in the maintenance of membrane order of the ML-based liposome. The findings presented in this study provided useful information for the development and production of stable QU-loaded ML-based liposomes for food and nutraceutical applications and could serve as a potential mixed lipids-based delivery system in the disease management using antioxidant therapy.

A new family of hydroxytyrosol phenolipids for the antioxidant protection of liposomal systems

Hydroxytyrosol (HT) is a well-known olive oil polyphenol for its high antioxidant capacity and important cardio and neuroprotective effects. However, its use in lipidic systems is limited, due to its hydrophilic character. In this study, we approach the particular structure of xanthophylls and synthetize HT esters specially designed for the protection of liposomal systems. These HT esters contain two polyphenolic moieties separated by a lipophilic alkyl spacer of different length (12, 16 or 22 carbons). To evaluate the antioxidant activity of these compounds against the 2,2′-azobis(2-amidinopropane) hydrochloride induced oxidation, soybean phospholipid liposomes were used. Fluorescence quenching studies were used to assess the insertion of the compounds in the liposomes. The synthetized HT derivatives were able to protect liposomes from induced oxidation when added to the suspensions. The rank of activity was severely influenced by the alkyl chain length of the spacer molecule, being the C12 derivative the most active antioxidant, with an increase in the oxidative stability of liposomes of 2.2 times when compared with the control. The incorporation of compounds during liposome preparation improved the antioxidant capacity of all HT derivatives by about 2.8 times, when compared to the control. This is probably due to a similar transmembrane position with both polyphenolic rings located at the phospholipid polar heads. The synthesis of bis-ester derivatives seems to be a promising strategy to fine-tune antioxidant molecules at biomembranes, thus increasing the oxidative stability of liposomal systems by improving the antioxidant activity of hydrophilic phenolic compounds with high free radical scavenging activity.

Comprehensive analysis of liposome formulation parameters and their influence on encapsulation, stability and drug release in glibenclamide liposomes

An understanding of the factors that affect liposome size, drug loading, stability and drug release is critical for the rational design of liposomes with desired pharmacokinetics and biodistribution. This article presents a report on the formulation and characterization of BIIB093 (glibenclamide) liposomes as well as a detailed analysis of the influence of formulation methods and parameters on encapsulation efficiency, liposome size, charge (zeta potential, ZP), polydispersity index (PDI), and drug release. PEGylated liposomes containing BIIB093 were made using ethanol injection and calcium acetate remote loading. The critical formulation parameters investigated include: the effect of lipid chain length, lipid unsaturation, lipid phase transition temperature (Tc) and the amount of cholesterol. Liposomes generated in this study had low average particle size (130 ± 20 nm), PDI (0.15 ± 0.1) and ZP (-2 ± 1 mV). Liposomes made from lipids with long acyl chains showed enhanced drug loading, encapsulation efficiency and drug retention. Similarly, liposomes made from lipids with high degree of unsaturation and low Tc exhibited faster drug release rates. Additionally, increasing the amount of cholesterol in the liposome bilayer improved PDI, decreased drug incorporation and accelerated drug release but had negligible impact on liposome size and ZP. Furthermore, encapsulating the drug in the liposome core enabled sustained drug release.

A Phospholipase-A Activity in Soluble Leishmania Antigens Causes Instability of Liposomes.

This study aimed to investigate the existence of phospholipase-A (PLA) activity in Soluble L. major Antigens (SLA) because of no reports for it so far. Liposomes were used as sensors to evaluate PLA activity. Liposomal SLA consisting of Egg Phosphatidylcholine (EPC) or Sphingomyelin (SM) were prepared by two different methods in different pH or temperatures and characterized by Dynamic Light Scattering (DLS) and Thin Layer Chromatography (TLC). Lipid hydrolysis led to the disruption of EPC liposomal SLA in both methods but the Film Method (FM) produced more stable liposomes than the Detergent Removal Method (DRM). The preparation of EPC liposomal SLA at pH 6 via FM protected liposomes from hydrolysis to some extent for a short time. EPC liposomes but not SM liposomes were disrupted in the presence of SLA. Therefore, a phospholipid without ester bond such as SM should be utilized in liposome formulations containing PLA as an encapsulating protein.

Biofunctionalized Liposomes to Monitor Rheumatoid Arthritis Regression Stimulated by Interleukin-23 Neutralization.

Even after the revolution of rheumatoid arthritis (RA) treatment with biologic agents, this debilitating disease remains a major clinical problem. The outstanding outcomes of the systemic administration of antibodies (Abs) are narrowed by the risk of serious side effects and limited efficacy due to their short half-life. Interleukin-23 (IL-23) is a crucial pro-inflammatory cytokine involved in inflammation that potently enhances the generation of T-helper type-17 (Th17) cells. Hence, in this work, anti-IL-23 Abs are immobilized at the surface of liposomes to increase their therapeutic efficacy, being gold nanoparticles (AuNPs) incorporated to allow monitoring the biodistribution of the liposomes after systemic administration as well as due to their anti-inflammatory and antioxidant effects. A stable monodispersed liposomes' suspension with around 130nm is produced and efficiently biofunctionalized with anti-IL-23 Abs. IL-23 capture and neutralization capacity are confirmed using activated macrophages. Biological assays demonstrate their hemocompatibility and cytocompatibility with human articular chondrocytes, macrophages, and endothelial cells. Moreover, the neutralization of IL-23 by the biofunctionalized liposomes efficiently decreases the production of IL-17A by peripheral blood mononuclear cells of healthy donors and RA patients who are activated to Th17 differentiation. Therefore, the developed formulation may be a promising strategy to treat RA.

Pulse Magnetic Fields Induced Drug Release from Gold Coated Magnetic Nanoparticle Decorated Liposomes

Magnetic nanoparticle-assisted drug release from liposomes is an important way to enhance the functionality/usefulness of liposomes. This work demonstrates an approach how to integrate magnetic nanoparticles with liposomes with the assistance of gold–thiol chemistry. The gold coated magnetic particles cover the thiolated liposomes from the outside, which removes the competition of the drug molecules and the triggering magnetic particles to free the inner space of the liposomes when compared to previous magneto liposome formulations. The liposome consists of dipalmitoyl phosphatidylcholine (DPPC) combined with distearoylphosphatidylcholine (DSPC) in addition to regular cholesterol or cholesterol-PEG-SH. Permeability assays and electron microscopy images show efficient coupling between the liposomes and nanoparticles in the presence of thiol groups without compromising the functionality of the liposomes. The nanoparticles such as gold nanoparticles, gold coated iron oxide nanoparticles and bare iron oxide nanoparticles are added following the model drug encapsulation. The efficient coupling between the gold coated nanoparticles (NPs) and the thiolate liposomes is evidenced by the shift in transition temperature of the thiolated liposomes. The addition of magnetically triggerable nanoparticles externally makes the entire interior of liposomes available for drug loading. The drug release efficiencies of these liposomes/NPs complexes were compared under exposure to pulsed magnetic fields. The results indicate up to 20% of the drug can be released in short time, which is comparable in efficiency to previous studies performed when magnetic NPs were located inside liposomes. Interestingly, the liposomes were found to exhibit variations in release efficiency based on different dilution media which is attributed to an osmotic pressure effect on liposomal stability.

Detection of material-derived differences in the stiffness of egg yolk phosphatidylcholine-containing liposomes using atomic force microscopy

Naturally sourced phospholipids are used in many liposomal pharmaceuticals. The present report describes a method to detect the effects of different egg yolk phosphatidylcholines (EPCs) on liposomal physicochemical properties. Five EPC-containing liposomes were prepared using five different EPCs obtained from different suppliers. There was no significant difference in purity between each EPC. The stiffness of the liposomes was examined via atomic force microscopy (AFM) in relation to the liposomal membrane permeability coefficient of encapsulated calcein after gel filtration, which is indicative of liposomal stability including the release of a hydrophilic drug from a liposome. Although the size of the liposome and the encapsulation efficiency of calcein did not significantly change with the type of EPC used, the liposome stiffness was found to vary depending on the EPC used, and liposomes with a similar stiffness were found to show a similar membrane permeability to calcein. Our results indicate the usefulness of stiffness measurement, using AFM as the analytical method, to detect material-derived differences in EPC-containing liposomes that affect drug release from the liposomes. Because drug release is one of the most important liposomal functions, combining this method with other analytical methods could be useful in selecting material for the development and quality control of EPC-containing liposomes.

Manufacturing drug co-loaded liposomal formulations targeting breast cancer: Influence of preparative method on liposomes characteristics and in vitro toxicity.

Developing more efficient manufacturing methods for nano therapeutic systems is becoming important, not only to better control their physico-chemical characteristics and therapeutic efficacy but also to ensure scale-up is cost-effective. The principle of cross-flow chemistry allows precise control over manufacturing parameters for the fabrication of uniform liposomal formulations, as well as providing reproducible manufacturing scale-up compared to conventional methods. We have herein investigated the use of microfluidics to produce PEGylated DSPC liposomes loaded with doxorubicin and compared their performance against identical formulations prepared by the thin-film method. The isoprenylated coumarin umbelliprenin was selected as a co-therapeutic. Umbelliprenin-loaded and doxorubicin:umbelliprenin co-loaded liposomes were fabricated using the optimised microfluidic set-up. The role of umbelliprenin as lipid bilayer fluidity modulation was characterized, and we investigated its role on liposomes size, size distribution, shape and stability compared to doxorubicin-loaded liposomes. Finally, the toxicity of all liposomal formulations was tested on a panel of human breast cancer cells (MCF-7, MDA-MB 231, BT-474) to identify the most potent formulation by liposomal fabrication method and loaded compound(s). We herein show that the microfluidic system is an alternative method to produce doxorubicin:umbelliprenin co-loaded liposomes, allowing fine control over liposome size (100-250nm), shape, uniformity and doxorubicin drug loading (>80%). Umbelliprenin was shown to confer fluidity to model lipid biomembranes, which helps to explain the more homogeneous size and shape of co-loaded liposomes compared to liposomes without umbelliprenin. The toxicity of doxorubicin:umbelliprenin co-loaded liposomes was lower than that of free doxorubicin, due to the delayed release of doxorubicin from liposomes. An alternative, rapid and easy manufacturing method for the production of liposomes has been established using microfluidics to effectively produce uniform doxorubicin:umbelliprenin co-loaded liposomal formulations with proven cytotoxicity in human breast cancer cell lines in vitro.

Preparation and Characterization of Chitosan-Coated Oleic Acid Liposomes for Intravenous Delivery

Liposome has been studied as a potential carrier for targeting and controlled drug delivery. However, poor stability remains a challenge because it can lead to drug leakage from the vesicles thus reduce the effectiveness towards the target cell. For this aim, the present study incorporated the low molecular weight chitosan (LMWC) into the oleic acid liposome to maintain the stability and prolong the lifetime in the blood circulation. The thin-film hydration method was employed to prepare the oleic acid liposomes prior to coating them with LMWC. The stability of the liposomes was determined by the measurement of particle size and zeta potential for 28 days. The morphology of the liposome was confirmed by observing the shape under transmission electron microscopy (TEM) and it showed almost spherical in shape. The average particle size increased to 201.23 nm and -51.4 mV when 5 mg of LMWC was added to the oleic acid liposome. The increase of particle size and zeta potential of LMWC-coated liposome indicated that polymer-liposome interaction had changed the stability of liposome thus this invention could be useful for delivering active ingredients through intravenous delivery.

Cathepsin B-Responsive Liposomes for Controlled Anticancer Drug Delivery in Hep G2 Cells.

In recent decades, several types of anticancer drugs that inhibit cancer cell growth and cause cell death have been developed for chemotherapeutic application. However, these agents are usually associated with side effects resulting from nonspecific delivery, which may induce cytotoxicity in healthy cells. To reduce the nonspecific delivery issue, nanoparticles have been successfully used for the delivery of anticancer drugs to specific target sites. In this study, a functional polymeric lipid, PEG-GLFG-K(C16)2 (PEG-GLFG, polyethylene glycol-Gly-Leu-Phe-Gly-Lys(C16)2), was synthesized to enable controlled anticancer drug delivery using cathepsin B enzyme-responsive liposomes. The liposomes composed of PEG-GLFG/DOTAP (1,2-dioleoyl-3-trimethylammonium-propane (chloride salt))/DPPC (dipalmitoylphosphatidylcholine)/cholesterol were prepared and characterized at various ratios. The GLFG liposomes formed were stable liposomes and were degraded when acted upon by cathepsin B enzyme. Doxorubicin (Dox) loaded GLFG liposomes (GLFG/Dox) were observed to exert an effective anticancer effect on Hep G2 cells in vitro and inhibit cancer cell proliferation in a zebrafish model.