Enkhzaya Davaa

Enhanced siRNA delivery using cationic liposomes with new polyarginine-conjugated PEG-lipid.

Gene therapy based on small interfering RNA (siRNA) has emerged as an exciting new therapeutic approach. However, insufficient cellular uptake and poor stability have limited its usefulness. Here, we report efficient delivery of siRNA via the use of cationic liposomes that contain a new PEG-lipid. The new lipid, poly-l-arginine-conjugated polyethylene glycol (PLR-PEG), was synthesized. To confirm the synthesis of the amino acid-conjugated PEG-lipid, (1)H NMR and gel permeation chromatography (GPC) were performed. Cationic liposomes as non-viral vectors were formulated using the cationic lipids 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-dioleoyl-sn-glycero-3-phosphoethanolaminepropane (DOPE), cholesterol (Chol) and PLR-PEG. Physicochemical properties of cationic liposomes were investigated. A GFP siRNA was used as a model siRNA to test the efficiency of cationic liposome-mediated siRNA delivery. The liposomes could enhance delivery efficiency and decrease cytotoxicity at an optimized lipid composition. The new cationic liposome formulation using a new PEG-lipid (PLR-PEG) showed not only enhanced intracellular delivery of siRNA but also decreased cytotoxicity in H4II-E and HepG2 cell lines. The GFP siRNA delivered by new cationic liposomes using PLR-PEG was effective in reducing the GFP protein expression levels of the gene. These results suggest that the new cationic liposomes could be used for efficient delivery of siRNA therapeutics.

Encapsulation enhancement and stabilization of insulin in cationic liposomes

The purpose of this study was to enhance encapsulation efficiency and sustained-release delivery for parenteral administration of a protein drug. To reduce the administration frequency of protein drugs, it is necessary to develop sustained delivery systems. In this study, protein drug-loaded cationic liposomes were formulated with dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), dioleoyl-3-trimethylammonium-propane (DOTAP), and cholesterol (CH) at a molar ratio of DOPE/DOTAP/CH of 2/1.5/2. Five mol% of distearoylphosphatidyl ethanolamine polyethylene glycol (DSPE-PEG) was added prior to encapsulation of the drug into liposomes. Insulin was chosen as a model protein drug and encapsulation efficiency was evaluated in various liposomes with and without DSPE-PEG. Scanning electron microscopy was used to examine the insulin-loaded cationic liposomes. Structural analysis was performed using spectropolarimetry. Additionally, the stability and cytotoxicity of insulin-loaded cationic liposomes were evaluated. Liposomes coated with DSPE-PEG showed higher insulin encapsulation efficiency than did those without DSPE-PEG, but not significantly. Moreover, among the liposomes coated with DSPE-PEG, those hydrated with 10% sucrose showed higher encapsulation efficiency than did liposomes hydrated in either phosphate-buffered saline or 5% dextrose. In vitro release of insulin was prolonged by cationic liposomes. Our findings suggest that cationic liposomes may be a potential sustained-release delivery system for parenteral administration of protein and peptide drugs to prolong efficacy and improve bioavailability.

A thermosensitive vaginal gel formulation with HPγCD for the pH-dependent release and solubilization of amphotericin B

To achieve better therapeutic efficacy and patient compliance in the treatment for Candida vaginitis, the antifungal agent amphotericin B (AmB) was formulated in a vaginal gel using Pluronic-based multiblock copolymers (MBCP-2). To increase its aqueous solubility, the drug was incorporated as its inclusion complex with hydroxypropyl-gamma-cyclodextrin (HPgammaCD). The formation of the AmB inclusion complex was characterized using different techniques including XRD, FT-IR spectrophotometry, DSC, and SEM. The sol-gel transition diagrams were determined by the inversion method at temperature intervals of 2 degrees C. Moreover, a histopathology study was performed to determine whether vaginal tissue damage was caused by repeated doses. The inclusion complex between AmB and HPgammaCD was completely formed, and the aqueous solubility of AmB was improved by the formation of the inclusion complex. The sol-gel transition diagrams showed that the aqueous solutions of MBCP-2 gelled at body temperature, and the gelation temperature of the polymer solutions was dependent on polymer concentration. In vitro drug release results indicated that MBCP-2 exhibited a sustained release of AmB in pH 7.4 and pH 9.0 buffers, whereas at pH 5.0, it presented a constant release that was completed within 3 days. There was no visible sign of inflammation or necrosis in vaginal tissues after repetitive intravaginal application. In conclusion, the thermosensitive vaginal gel might be useful in the delivery of an antifungal agent for local treatment.

A new vaginal delivery system of amphotericin B: a dispersion of cationic liposomes in a thermosensitive gel

Amphotericin B (AmB) is used in the treatment of fungal infections; however, its clinical use is limited by its toxic side effects. In this study, AmB-loaded cationic liposome gels were formulated with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), and cholesterol (CH) at a molar ratio of DOPE:DOTAP:CH = 4:5:1 in thermosensitive gel composed of poloxamer 407 (P407) and poloxamer 188 (P188). To enhance the solubility of AmB, 6 mol% of distearoyl phosphatidyl ethanolamine–polyethylene glycol was added prior to encapsulation of the drug into liposomes. Scanning electron microscopy was used to observe the AmB encapsulated cationic liposome gels. In vitro release, stability and cytotoxicity of AmB in cationic liposome gels were evaluated. The particle size and zeta potential of AmB-loaded liposomes were in the range of 400–500 nm and 40–60 mV, respectively. The thermosensitive gel at the ratio of P407:P188 = 15:15 (w/w) gelled at 37°C, approximating body temperature. Encapsulation efficiency of AmB was ∼50–60%, which was influenced by the ratio of AmB to lipid. Moreover, AmB-loaded cationic liposome gels were more stable and less toxic than free AmB. From these results, cationic liposome gel formulations may be useful for vaginal delivery of AmB.

Thermosensitive and Mucoadhesive Sol-Gel Composites of Paclitaxel/Dimethyl-β-Cyclodextrin for Buccal Delivery

The purpose of this study was to develop a buccal paclitaxel delivery system using the thermosensitive polymer Pluronic F127 (PF127) and the mucoadhesive polymer polyethylene oxide (PEO). The anticancer agent paclitaxel is usually used to treat ovarian, breast, and non-small-cell lung cancer. To improve its aqueous solubility, paclitaxel was incorporated into an inclusion complex with (2,6-di-O-methyl)-β-cyclodextrin (DMβCD). The formation of the paclitaxel inclusion complex was evaluated using various techniques, including x-ray diffractometry (XRD), Fourier-transform infrared (FT-IR) spectrophotometry, differential scanning calorimetry (DSC), and scanning electron microscopy (SEM). Hydrogels were prepared using a cold method. Concentrations of 18, 20, and 23% (w/v) PF127 were dissolved in distilled water including paclitaxel and stored overnight in a refrigerator at 4°C. PEO was added at concentrations of 0.1, 0.2, 0.4, 0.8, and 1% (w/v). Each formulation included paclitaxel (0.5 mg/mL). The sol-gel transition temperature of the hydrogels was measured using the tube-inverting method. Drug release from the hydrogels was measured using a Franz diffusion cell containing pH 7.4 phosphate-buffered solution (PBS) buffer at 37°C. The cytotoxicity of each formulation was measured using the MTT assay with a human oral cancer cell (KB cell). The sol-gel transition temperature of the hydrogel decreased when PF127 was present and varied according to the presence of mucoadhesive polymers. The in vitro release was sustained and the release rate was slowed by the addition of the mucoadhesive polymer. The cytotoxicity of the blank formulation was low, although the drug-loaded hydrogel showed acceptable cytotoxicity. The results of our study suggest that the combination of a PF 127-based mucoadhesive hydrogel formulation and inclusion complexes improves the in vitro release and cytotoxic effect of paclitaxel.

Topical treatment of the buccal mucosa and wounded skin in rats with a triamcinolone acetonide-loaded hydrogel prepared using an electron beam.

In this study, a triamcinolone acetonide-loaded hydrogel was prepared by electron beam irradiation and evaluated for use as a buccal mucoadhesive drug delivery system. A poloxamer was modified to have vinyl end groups for preparation of the hydrogel via an irradiation cross-linking reaction. Carbopol was introduced to improve the mucoadhesive properties of the hydrogel. The in vitro release of triamcinolone acetonide from the hydrogel was examined at 37 °C. To investigate the topical therapeutic effect of triamcinolone acetonide on wounded rat skin and buccal mucosa, the appearance and histological changes were evaluated for 15 days after treatment with saline, triamcinolone acetonide solution, triamcinolone acetonide hydrogel, and blank hydrogel, respectively. Triamcinolone acetonide was released constantly from the gel formulation at 37 °C and reach 100% at about 48 h. After 15 days, in the skin of the group treated with the triamcinolone acetonide-loaded hydrogel, the wound was almost completely free of crust and a number of skin appendages, including hair follicles, had formed at the margins of the tissue. Moreover, the inflammatory response in the buccal mucosa was milder than that in the other groups, and the wound surface was completely covered with regenerating, hyperkeratotic, thickened epithelial cells. Our results indicate that the triamcinolone-acetonide hydrogel showed sustained drug release behavior, while causing no significant histopathological changes in buccal and skin tissues. Therefore, this hydrogel system may be a powerful means of drug delivery for buccal administration with controlled release and no tissue irritation.

Preparation and irradiation of Pluronic F127-based thermoreversible and mucoadhesive hydrogel for local delivery of naproxen.

To improve physical properties and modulate the mucoadhesive hydrogel formulation via cross-linking by radiation, hydrogels were prepared using thermoreversible polymer Pluronic F127 (PF127) and mucoadhesive polymer carbopol 934P (C934P). As a model drug, naproxen was loaded in the hydrogel formulation. Sol-gel transition temperatures of hydrogels were measured by the tube-inversion method. The mucoadhesive potential of each formulation was determined by measuring the force required to detach the formulation from oral mucosal tissue. To strengthen the mechanical properties, the formulations were irradiated using an electronic beam. Drug release from the hydrogels and the cytotoxicity of each formulation were investigated. Sol-gel transition temperatures of the formulations were decreased by the addition of carbopol and were close to body temperature. The mucoadhesive force of the PF127 formulation was increased by addition of carbopol. In vitro release was sustained and the release rate was reduced by the addition of carbopol. After irradiation, the mucoadhesive force was increased about five-fold especially in the case of PF127 23% (9.7 kPa) and in vitro release was not sustained further. In conclusion, the use of a PF127 formulation incorporating a mucoadhesive polymer could effectively and safely improve oral residence time and absorption of naproxen. Irradiated formulations showed permanent cross-linking and improved properties.

Combined delivery of the adiponectin gene and rosiglitazone using cationic lipid emulsions

For the combined delivery of an insulin-sensitizing adipokine; i.e., the ADN gene, and the potent PPARγ agonist rosiglitazone, cationic lipid emulsions were formulated using the cationic lipid DOTAP, helper lipid DOPE, castor oil, Tween 20 and Tween 80. The effect of drug loading on the physicochemical characteristics of the cationic emulsion/DNA complexes was investigated. Complex formation between the cationic emulsion and negatively charged plasmid DNA was confirmed and protection from DNase was observed. The in vitro transfection efficiency and cytotoxicity were evaluated in HepG2 cells. The particle sizes of the cationic emulsion/DNA complex were in the range 230-540 nm and those of the rosiglitazone-loaded cationic emulsion/DNA complex were in the range 220-340 nm. Gel retardation of the complexes was observed when the complexation weight ratios of the cationic lipid to plasmid DNA exceeded 4:1 for both the drug-free and rosiglitazone-loaded complexes. Both complexes stabilized plasmid DNA against DNase. The ADN expression level increased dose-dependently when cells were transfected with the cationic emulsion/DNA complexes. The rosiglitazone-loaded cationic emulsion/DNA complexes showed higher cellular uptake in HepG2 cells depending on the rosiglitazone loading, but not depending on the type of plasmid DNA type such as pVAX/ADN, pCAG/ADN, or pVAX. The drug-loaded cationic emulsion/plasmid DNA complexes were less cytotoxic than free rosiglitazone. Therefore, a cationic emulsion could potentially serve as a co-delivery system for rosiglitazone and the adiponectin gene.

Effect of Drug Loading on the Physicochemical Properties and Stability of Cationic Lipid-based Plasmid DNA Complexes

Recently, co-delivery of drug and gene has been attempted for higher therapeutic effects of anticancer agents. In this study, cationic liposomes were prepared using 1,2-dioleoyl-3-trimethylammoniopropane (DOTAP) as a cationic lipid to investigate the effect of drug loading on the physicochemical characteristics of cationic liposomes/DNA complexes. The complex formation between cationic liposomes and negatively charged plasmid DNA was confirmed and the protection from DNase was observed. Particle size of complexes was reduced not by drug loading, but by the increased ratio of cationic lipid to plasmid DNA. Meanwhile, zeta potential of complex was increased by the addition of cationic liposomes to complexes and the effect of drug loading on the zeta potential was not much higher than on particle size. Gel retardation of complexes was indicated when the complexation weight ratios of cationic lipid to plasmid DNA were higher than 24:1 for drug free complexes and 20:1 for drug loaded ones, respectively. Agarose gel retardation showed the similar complexation between plasmid DNA and drug free liposomes or drug loaded liposomes. Both complexes protected plasmid DNA from DNase independent of complexation temperature. From the results, drug loading may affect not the complex formation of cationic liposomes and plasmid DNA, but the particle size of complex.

Preliminary study to determine the optimal conditions for the simultaneous complexation of siRNA and plasmid DNA

We investigated the complexation ratio of siRNA and plasmid DNA combined with Lipofectamine for the effective delivery of genetic materials. First, the amount of Lipofectamine was varied with a fixed amount of DNA or siRNA to determine in which proportions they would form an optimal combination. Finally, to investigate the effect of DNA or siRNA on the co-complexation of both DNA and siRNA, the co-complex of DNA and siRNA was prepared at the various ratios with a fixed amount of DNA. All complexation was confirmed by gel retardation of DNA or siRNA on agarose gels. The effects of siRNA complexes on mRNA expression from plasmid DNA were explored post-transfection, while the influence of plasmid DNA complexes on the transfection of siRNA was determined in GFP-expressing H4IIE cells. The complex between DNA and Lipofectamine was formed at a weight ratio of 0.8:1, whereas the light band of siRNA/Lipofectamine disappeared at a weight ratio of 4:1. When the amounts of DNA, siRNA, and the mixture were fixed, the optimal ratio of nucleic acids and Lipofectamine in our composition was 110:80:350 (ng). Confocal images and flow cytometry showed that inhibition of GFP expression by siRNA was not interfered with by co-complexed plasmid DNA. Moreover, mRNA expression of adiponectin was not hampered by the addition of siRNA; rather, it was increased. Thus, co-complexation of siRNA and plasmid DNA may have a synergistic effect on delivery of the therapeutic gene and siRNA.