Gene Transfer with Three Amphiphilic Glycol Chitosans-the Degree of Polymerisation is the Main Controller of Transfection Efficiency

• Published in: Journal of drug targeting 2004-09, Vol.12 (8), p.527-539
• Main Authors: Uchegbu, Ijeoma F., Sadiq, Lubna, Pardakhty, Abbas, El-Hammadi, Mazen, Gray, Alexander I., Tetley, Laurence, Wang, Wei, Zinselmeyer, Bernd H., Schätzlein, Andreas G.
• Format: Article
• Language: English
• Subjects: Animals; Cell Survival - drug effects; Cell Survival - physiology; Chitosan; Chitosan - chemistry; Chitosan - pharmacology; Female; Gene delivery; Gene Transfer Techniques; Glycol chitosan; Glycol chitosan amphiphiles; Glycols - chemistry; Glycols - pharmacology; In vivo gene delivery; Mice; Mice, Inbred BALB C; Polymers - chemistry; Polymers - pharmacology; Surface-Active Agents - chemistry; Surface-Active Agents - pharmacology; Transfection - methods
• ISSN: 1061-186X; 1029-2330
• DOI: 10.1080/10611860400011943

A number of studies have examined the possibility of delivering genes for the treatment of genetic diseases using various polymers and lipids. We have previously demonstrated the gene transfer ability of amphiphilic polymers (a soluble amine polymer covalently bound to lipid pendant groups). In the current communication we explore the gene transfer activity of amphiphilic glycol chitosans. Glycol chitosan was acid depolymerised to give polymers of various molecular weights. Palmitoyl or hexadecyl and in some cases additional N-methyl quaternary ammonium groups were attached to the polymers. DNA binding was studied by measuring the reduced fluorescence of ethidium bromide and the polyplex particle size and zeta potential. Biological characterisation of the polyplexes involved haemolysis, cytotoxicity and gene transfer assays. For the 22 polymers tested, DNA binding was optimum at a nitrogen to phosphate ratio of 2:1 and above. Polyplexes were 200-500 nm in diameter with a neutral or positive zeta potential. The haemolytic activity of the N-methyl polymers was studied and no haemolysis was detected up to a concentration of 10 mg ml−1. Cytotoxicity studies showed that the biocompatibility of glycol chitosan was adversely affected by a combination of a palmitoyl group and depolymerisation and that biocompatibility was subsequently restored with the introduction of N-methyl groups. In vitro transfection efficiency superior to the cationic lipid formulation N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulphate (DOTAP) was seen with depolymerised glycol chitosan in the A431 cell line only and with the depolymerised N-methyl quaternary ammonium amphiphilic derivatives in both the A431 and A549 cell lines. Degree of polymerisation (DP) was the most important controller of transfection efficiency and transfection resided within polymers with a DP of 73-171. High DP polymers diminished DNA-cell association, the first step in the cellular gene transfer process, thus apparently diminishing cell uptake. In vivo transfection with the N-methyl quaternary ammonium amphiphile was best at a DP of 86 and this glycol chitosan amphiphile gave superior liver and heart gene expression levels when compared to both Exgen 500 (linear polyethylenimine) and Superfect (a polyamidoamine dendrimer).