dioleoyl-phosphatidylethanolamine and 1-2-distearoylphosphatidylethanolamine

Acanthamoeba keratitis is an ophthalmic disease with no specific treatment that specially affects contact lens users. The silencing of serine phosphatase (SP) and glycogen phosphorylase (GP) proteins produced by Acanthamoeba has been shown to significantly reduce the cytopathic effect, although no vehicle was proposed yet to deliver the siRNA sequences to the trophozoites. In this study, PEGylated cationic liposomes were proposed and optimized using Box-Behnken design. The influence of DOTAP:DOPE ratio, DSPE-PEG concentration, and siRNA/DOTAP charge ratio were evaluated over both biological response and physicochemical properties of liposomes. The ratio of DOTAP:DOPE had an effect in the trophozoite activity whereas the charge ratio influenced both size and protease activity. The predicted values were very close to the observed values, yielding a formulation with good activity and toxicity profile, which was used in the following experiments. A murine model of ocular keratitis was treated with siGP + siSP-loaded liposomes, as well as their respective controls, and combined treatment of liposomes and chlorhexidine. After 15 days of eight daily administrations, the liposomal complex combined with chlorhexidine was the only treatment able to reverse the more severe lesions associated with keratitis. There was 60% complete regression in corneal damage, with histological sections demonstrating the presence of an integral epithelium, without lymphocytic infiltrate. The set of results demonstrate the efficacy of a combined therapy based on siRNA with classical drugs for a better prognosis of keratitis caused by Acanthamoeba.

Topics: Acanthamoeba; Acanthamoeba Keratitis; Animals; Chlorhexidine; Cornea; Disease Models, Animal; Drug Administration Schedule; Drug Compounding; Drug Delivery Systems; Drug Therapy, Combination; Factor Analysis, Statistical; Fatty Acids, Monounsaturated; Gene Expression Regulation; Glycogen Phosphorylase; Humans; Liposomes; Phosphatidylethanolamines; Phosphoric Monoester Hydrolases; Polyethylene Glycols; Protozoan Proteins; Quaternary Ammonium Compounds; Rats; Rats, Wistar; RNA, Small Interfering; Trophozoites

pH-sensitive drug carriers that are sensitive to the acidic (pH = ~6.5) microenvironments of tumor tissues have been primarily used as effective drug/gene/siRNA/microRNA carriers for releasing their payloads to tumor cells/tissues. Resistance to various drugs has become a big hurdle in systemic chemotherapy in cancer. Therefore delivery of chemotherapeutic agents and siRNA's targeting anti apoptotic genes possess advantages to overcome the efflux pump mediated and anti apoptosis-related drug resistance. Here, we report the development of nanocarrier system prepared from kojic acid backbone-based cationic amphiphile containing endosomal pH-sensitive imidazole ring. This pH-sensitive liposomal nanocarrier effectively delivers anti-cancer drug (Paclitaxel; PTX) and siRNA (Bcl-2), and significantly inhibits cell proliferation and reduces tumor growth. Tumor inhibition response attributes to the synergistic effect of PTX potency and MDR reversing ability of Bcl-2 siRNA in the tumor supporting that kojic acid based liposomal pH-sensitive nanocarrier as efficient vehicle for systemic co-delivery of drugs and siRNA.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Cell Line, Tumor; Drug Compounding; Drug Delivery Systems; Gene Expression Regulation, Neoplastic; Hydrogen-Ion Concentration; Imidazoles; Liposomes; Melanoma, Experimental; Mice; Nanoparticles; Paclitaxel; Phosphatidylethanolamines; Proto-Oncogene Proteins c-bcl-2; Pyrones; RNA, Small Interfering; Skin Neoplasms; Tumor Burden

Nonviral gene delivery vectors hold great promise for gene therapy due to the safety concerns with viral vectors. However, the application of nonviral vectors is hindered by their low transfection efficiency. Herein, in order to tackle this challenge, we developed a nonviral vector integrating lipids, sleeping beauty transposon system and 8-mer stem cell targeting peptides for safe and efficient gene delivery to hard-to-transfect mesenchymal stem cells (MSCs). The 8-mer MSC-targeting peptides, when synthetically reiterated in three folds and chemically presented on the surface, significantly promoted the resultant lipid-based nanoparticles (LBNs) to deliver VEGF gene into MSCs with a high transfection efficiency (∼52%) and long-lasting gene expression (for longer than 170 h) when compared to nonreiterated peptides. However, the reiterated stem cell targeting peptides do not enable the highly efficient gene transfer to other control cells. This work suggests that the surface presentation of the reiterated stem cell-targeting peptides on the nonviral vectors is a promising method for improving the efficiency of cell-specific nonviral gene transfection in stem cells.

Topics: Amino Acid Sequence; Animals; Cell Survival; Endocytosis; Fatty Acids, Monounsaturated; Gene Expression; Gene Transfer Techniques; Liposomes; Mesenchymal Stem Cells; Molecular Sequence Data; Nanoparticles; Particle Size; Peptides; Phosphatidylethanolamines; Quaternary Ammonium Compounds; Rats; Transgenes; Transposases; Vascular Endothelial Growth Factor A

A "cluster-bomb"-like lipid-dendrimer nanoassembly synergizes the functions of its components and thereby efficiently accomplishes the drug delivery cascade for high efficacy in treating cancer. The nanoassembly successfully circulates in the blood and accumulates in the tumor. Once in the tumor, it releases small dendrimers that act like "bomblets", enabling tumor penetration, cell internalization, and drug release.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Cell Membrane; Cholesterol; Dendrimers; Doxorubicin; Drug Delivery Systems; Female; Humans; Mice; Mice, Nude; Micelles; Nanomedicine; Nanostructures; Neoplasms, Experimental; Phosphatidylethanolamines; Polyethylene Glycols

The aim of this study was to investigate the fusogenic properties of poly(ethylene glycol) (PEG)ylated dioleoylphosphatidylethanolamine/cholesteryl hemisuccinate (DOPE/CHEMS) liposomes. These pH-sensitive liposomes were prepared by incorporating two different PEG lipids: distearoylphosphatidylethanolamine (DSPE)-PEG₂₀₀₀ was mixed with the liposomal lipids using the conventional method, whereas sterol-PEG₁₁₀₀ was inserted into the outer monolayer of preformed vesicles. Both types of PEGylated liposomes were characterized and compared for their entrapment efficiency, zeta potential and size, and were tested in vitro for pH sensitivity by means of proton-induced leakage and membrane fusion activity. To mimic the routes of intracellular delivery, fusion between pH-sensitive liposomes and liposomes designed to simulate the endosomal membrane was studied. Our investigations confirmed that DOPE/CHEMS liposomes were capable of rapidly releasing calcein and of fusing upon acidification. However, after incorporation of DSPE-PEG₂₀₀₀ or sterol-PEG₁₁₀₀ into the membrane, pH sensitivity was significantly reduced; as the mol ratio of PEG-lipid was increased, the ability to fuse was decreased. Comparison between two different PEGylated pH-sensitive liposomes showed that only vesicles containing 0.6 mol% sterol-PEG₁₁₀₀ in the outer monolayer were still capable of fusing with the endosome-like liposomes and showing leakage of calcein at pH 5.5.

Topics: Cholesterol Esters; Hydrogen-Ion Concentration; Kinetics; Liposomes; Membrane Fusion; Phosphatidylethanolamines; Polyethylene Glycols

The efficient delivery of plasmids encoding antigenic determinants into dendritic cells (DCs) that control immune response is a promising strategy for rapid development of new vaccines. In this study, we prepared a series of targeted cationic lipoplex based on two synthetic lipid components, mannose-poly(ethylene glycol, MW3000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (Mannose-PEG3000-DSPE) and O-(2R-1,2-di-O-(1'Z-octadecenyl)-glycerol)-3-N-(bis-2-aminoethyl)-carbamate (BCAT), that were formulated with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) for evaluation as nonviral vectors for transgene expression in DCs. First, we optimized the N/P ratio for maximum transfection and then screened the effects of mannose targeting for further enhancement of transfection levels. Our results indicate that efficient delivery of gWIZ GFP plasmid into DCs was observed for mannose compositions of ∼10%, whereas low transfection efficiencies were observed with nontargeted formulations. Mannose-targeted lipofectamine complexes also showed high GFP expression levels in DCs relative to nontargeted lipofectamine controls. The best transfection performance was observed using 10 mol % Mannose-PEG3000-DSPE, 60 mol % BCAT, and 30 mol % DOPE, indicating that the most efficient delivery into DCs occurs via synergistic interaction between mannose targeting and acid-labile, fusogenic BCAT/DOPE formulations. Our data suggest that mannose-PEG3000-DSPE/BCAT/DOPE formulations may be effective gene delivery vehicles for the development of DC-based vaccines.

Topics: Animals; Carbamates; Cell Proliferation; Cells, Cultured; Dendritic Cells; Flow Cytometry; Gene Transfer Techniques; Genetic Therapy; Green Fluorescent Proteins; Lipids; Macrophages; Mannose; Phosphatidylethanolamines; Plasmids; Swine

Novel sonosensitive doxorubicin-containing liposomes comprising dioleoylphosphatidylethanolamine (DOPE) as the main lipid constituent were developed and characterized in terms of ultrasound-mediated drug release in vitro. The liposome formulation showed high sonosensitivity; where approximately 95% doxorubicin was released from liposomes after 6min of 40kHz US exposure in buffered sucrose solution. This represented a 30% increase in release extent in absolute terms compared to liposomes comprising the saturated lipid analogue distearoylphosphatidylethanolamine (DSPE), and a 9-fold improvement in release extent when compared to standard pegylated liposomal doxorubicin, respectively. Ultrasound release experiments in the presence of serum showed a significantly reduction in sonosensitivity of DSPE-based liposomes, whilst the release properties of DOPE-based liposomes were essentially maintained. Dynamic light scattering measurements and cryo-transmission electron microscopy of DOPE-based liposomes after ultrasound treatment indicated liposome disruption and formation of various lipid structures, corroborating the high release extent. The results point to the potential of DOPE-based liposomes as a new class of drug carriers for ultrasound-mediated drug delivery.

Topics: Antineoplastic Agents; Chemistry, Pharmaceutical; Doxorubicin; Drug Carriers; Liposomes; Models, Chemical; Phosphatidylethanolamines; Polyethylene Glycols; Ultrasonics

A gene transfer vector has been developed utilising anionic liposomes as a carrier of plasmid DNA (pEGlacZ, 7.6 kb) to transfect CD3+ T lymphocytes (Jurkat cells). The plasmid DNA that contained the Escherichia coli beta-galactosidase reporter gene was condensed using poly-l-lysine of molecular mass 20,700 (PLK99) to form a polyplex which was interacted with several anionic liposome formulations to form lipopolyplexes. The liposome formulations where based on dioleoylphosphatidylethanolamine (DOPE) in combination with cholesterol and dioleoylphosphatidylcholine (DOPC) and oleic acid, or dimyristoylphosphatidylethanolamine (DMPE). For targeting to the Jurkat cells distearoylphosphatidylethanolamine (DSPE) linked to poly (ethylene glycol) molecular mass 2,000 and coupled to anti-CD3 antibody was incorporated. The polyplexes and lipopolyplexes were characterised in terms of size, zeta potential, agarose gel electrophoresis and electron microscopy and the permeability of the lipopolyplexes to liposome-encapsulated glucose was determined. The polyplexes consisted of a mixed population of rod-like structures (53-160 nm long and 23-31 nm diameter) and spheres (18-30 nm diameter). The lipopolyplexes retained a permeability barrier although were more permeable to glucose than their component liposomes. The poly-l-lysine condensing agent was still susceptible to pronase digestion suggesting that the polyplex was associated with the outer surface of the liposome. The lipopolyplexes with lipid composition DOPE/cholesterol/OA/DSPE-PEG2000 anti-CD3+ PLK99-plasmid DNA had significant gene transfer activity, as monitored by beta-galactosidase expression, that depended on the charge ratio of the component polyplex and the lipid/DNA weight ratio. The anti-CD3 antibody, the liposomal lipid and pH sensitivity were essential for transfection activity.

Topics: Antibodies; DNA; Genetic Vectors; Humans; Hydrogen-Ion Concentration; Jurkat Cells; Liposomes; Macromolecular Substances; Microscopy, Electron; Phosphatidylethanolamines; Polylysine; Pronase; Transfection