G A Brazeau1, S Attia, S Poxon, J A Hughes. 1. Department of Pharmaceutics, University of Florida, College of Pharmacy, Gainesville 32610, USA. brazeau@cop.health.ufl.edu
Abstract
PURPOSE: Cationic lipid/DNA complexes have been proposed as a method of in vivo gene delivery via intravenous or intramuscular injection. A concern with using these polycationic molecules is whether they are associated with tissue toxicity at the injection site. Therefore, the objective of these studies was to investigate the myotoxic potential of selected non-viral gene delivery macromolecules (e.g., cationic lipids and polymers) with and without plasmid DNA (pDNA) in vitro. METHODS: Myotoxicity was assessed by the cumulative release of creatine kinase (CK) over 90 minutes from the isolated rodent extensor digitorum longus muscle into a carbogenated balanced salt solution (BBS, pH 7.4, 37 degrees C) following a 15 microL injection of the test formulation. Phenytoin (Dilantin) and normal saline served as positive and negative controls, respectively. RESULTS: The myotoxicity of plasmid DNA (pDNA, approximately 5000bp, 1 mg/ml) was not statistically different from normal saline. However, the myotoxicity of Dilantin was 16-times higher than either normal saline or pDNA (p < 0.05). Cationic liposomes were found to be less myotoxic than polylysine and PAMAM dendrimers. Polylysine's myotoxicity was found to be dependent upon concentration and molecular weight. The myotoxicity of formulations of cationic liposomes(s), lower molecular weight polylysine (25,000) and higher concentration of PAMAM dendrimers with pDNA were found to be statistically less significant than those formulations without pDNA. CONCLUSIONS: The cationic liposomes were less myotoxic compared to the dendrimers and polylysine. Myotoxicity was dependent upon the type of cationic lipid macromolecule, concentration, molecular weight and the presence of pDNA. A possible explanation for this reduced tissue damage in cationic lipids complexed with pDNA is that the formation of complex reduces the overall positive charge of the injectable system resulting in less damage.
PURPOSE: Cationic lipid/DNA complexes have been proposed as a method of in vivo gene delivery via intravenous or intramuscular injection. A concern with using these polycationic molecules is whether they are associated with tissue toxicity at the injection site. Therefore, the objective of these studies was to investigate the myotoxic potential of selected non-viral gene delivery macromolecules (e.g., cationic lipids and polymers) with and without plasmid DNA (pDNA) in vitro. METHODS:Myotoxicity was assessed by the cumulative release of creatine kinase (CK) over 90 minutes from the isolated rodent extensor digitorum longus muscle into a carbogenated balanced salt solution (BBS, pH 7.4, 37 degrees C) following a 15 microL injection of the test formulation. Phenytoin (Dilantin) and normal saline served as positive and negative controls, respectively. RESULTS: The myotoxicity of plasmid DNA (pDNA, approximately 5000bp, 1 mg/ml) was not statistically different from normal saline. However, the myotoxicity of Dilantin was 16-times higher than either normal saline or pDNA (p < 0.05). Cationic liposomes were found to be less myotoxic than polylysine and PAMAM dendrimers. Polylysine's myotoxicity was found to be dependent upon concentration and molecular weight. The myotoxicity of formulations of cationic liposomes(s), lower molecular weight polylysine (25,000) and higher concentration of PAMAM dendrimers with pDNA were found to be statistically less significant than those formulations without pDNA. CONCLUSIONS: The cationic liposomes were less myotoxic compared to the dendrimers and polylysine. Myotoxicity was dependent upon the type of cationic lipid macromolecule, concentration, molecular weight and the presence of pDNA. A possible explanation for this reduced tissue damage in cationic lipids complexed with pDNA is that the formation of complex reduces the overall positive charge of the injectable system resulting in less damage.
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