ABSTRACT
In this work, we analyzed protein interaction, cell toxicity, and biodistribution of liposome formulation for further possible applications as DNA vehicles in gene-therapy protocols. In relation to protein interaction, cationic liposomes showed the lowest protein interaction, but this parameter was incremented with DNA association. On the other hand, noncharged liposomes presented high protein interaction, but DNA association decreased this parameter. Protein interaction of polymeric liposomes did not change with DNA association. Cell toxicity of these three liposome formulations was low, cell death became present at concentrations higher than 0.5 mg/mL, and these concentrations were higher than those usually used in transfection assays. In the case of noncharged and polymeric liposomes, toxicity increased upon interaction with serum proteins. DNA/liposome-mediated tissue distribution was analyzed in Balb-c female mice. Results indicated that noncharged liposomes were able to deliver DNA to liver after intraperitoneal (i.p.) inoculation, while polymeric liposomes were able to deliver DNA to kidney by using the same inoculation route. Cationic liposomes were able to deliver DNA to a wide range of tissues by the i.p. route (e.g., liver, intestine, kidney, and blood). After subcutaneous inoculation, only cationic liposomes were able to deliver DNA to blood, but not the other two formulations within the detection limits of the method.
Subject(s)
DNA/administration & dosage , Liposomes/administration & dosage , Animals , DNA/blood , DNA/metabolism , Female , Injections, Intraperitoneal , Injections, Subcutaneous , Kidney/metabolism , Liposomes/metabolism , Liposomes/toxicity , Liver/metabolism , Mice , Mice, Inbred BALB C , Particle Size , Serum Albumin, Bovine/metabolism , Tissue Distribution , TransfectionABSTRACT
Murine melanoma B16F0 cells were transfected with SA:DPPC:DOPE (2:1:1 molar ratio) liposomes associated with a plasmid encoding murine IL-12. Stearylamine, a cationic lipid, showed a greater transfection efficiency compared to DOTAP-containing liposomes. The lipid:DNA ratio was 2:1 (w/w). Control groups were mock transfected or transfected with an empty plasmid (pNeo). pNeo or IL-12 transfected cells and controls were inoculated intradermically into the dorsal region of the foot or the lateral flank of C57BL6 mice. Results showed that IL-12 expression had a marked effect on in vivo growth of B16 melanoma tumors developed in both anatomic sites, significantly retarding their growth and prolonging host survival.
Subject(s)
Interleukin-12/genetics , Melanoma, Experimental/therapy , Transfection/methods , Amines , Analysis of Variance , Animals , Cell Line, Tumor , Cell Survival , Disease Progression , Fatty Acids, Monounsaturated , Genes, Reporter , Interleukin-12/metabolism , Liposomes , Melanoma, Experimental/metabolism , Melanoma, Experimental/mortality , Melanoma, Experimental/pathology , Mice , Mice, Inbred C57BL , Neoplasm Transplantation , Quaternary Ammonium Compounds , Tumor BurdenABSTRACT
Small unilamellar vesicles associated with plasmid DNA showed maximum association efficiency for a cationic mixture of egg phosphatidylcholine (EPC):1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE):di-1,2-dioleoyl-3-trimethyl ammonium propane (DOTAP) (16:8:1 molar ratio) [65%], followed by neutral lipids EPC:1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE):cholesterol (Chol) (2:2:1 molar ratio) [30%], and a polymerized formulation 1,2-bis(10,12-tricosadiynoyl)sn-glycero-3-phosphocholine (DC8,9PC):DMPE:Chol (2:2:1 molar ratio) [11%]. The hydrophobicity factor (HF) for these formulations followed the trend DC8,9PC:DMPE:CHOL < EPC:DMPE:Chol < EPC:DOPE DOTAP, and DNA association did not alter this trend. Results suggest that the higher the HF value, the more fluid the membrane and the higher the efficiency of DNA association. On the other hand, no differences were observed in cell toxicity with lipids up to 1 mg/ml in VERO cells.
Subject(s)
DNA/genetics , Gene Transfer Techniques , Plasmids/genetics , Unilamellar Liposomes , Animals , Cell Survival , Chlorocebus aethiops , Hydrophobic and Hydrophilic Interactions , Unilamellar Liposomes/chemical synthesis , Unilamellar Liposomes/toxicity , Vero CellsABSTRACT
In order to evaluate liposomes as vehicle for oral vaccines the characterization and stability of polymerized and non-polymerized liposomes were examined. Mixtures of 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3 phosphocholine) (DC8,9PC) with saturated 1,2-dimiristoyl-sn-glycero-3-phosphocholine in molar ratio 1:1 were used. Saturated and non-saturated lipids were combined to give a chemically modified membrane by UV polymerization derived from DC8,9PC. Characterization was carried out by electronic microscopy, differential scanning calorimetry (DSC) and by hydrophobicity factor (HF). The stability towards the digestive tract (including saliva): acidic solutions, bile and pancreatin are compared to buffer pH 7.4, measuring the release of Glucose-6-phosphate or bovine plasma albumin entrapment. The polymerized liposomes showed further augmentation of the HF and the size. DSC showed phase separation and lower Tt if compared to data obtained for DC8,9PC. The HF, as main factor is discussed in relation to in vitro stability, suggesting that polymerized and non-polymerized liposomes would serve effectively as an oral delivery vehicle.
