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ABSTRACT: Cationic amphiphilic drugs have recently been shown to inhibit receptor recycling by disrupting the assembly-disassembly of clathrin at the plasma membrane and endosomes. It is therefore proposed that amphiphilic and cationic polysaccharide macromolecule, when used as gene delivery vectors, may have potential ability to direct the disassembly process of cell membrane organization, and penetrate across the cell membrane into cell and nucleus. In the current study, N-methylene phosphonic chitosan (NMPCS), an amphiphilic macromolecule, was synthesized by incorporating the methylene phosphonic group into the amino groups of chitosan (CS) using formaldehyde as the coupling agent, and characterized with a FTIR spectrometer. NMPCS/DNA or CS/DNA complexes were prepared using a complex coacervation method, and characterized by agarose gel electrophoresis retardation assay and dynamic light scattering (DLS). MTT assay was employed to evaluate the cytotoxicity of the polymers and pGL3-control luciferase plasmid was utilized as a reporter gene to assess the transgenic efficacy of the polymers. It was demonstrated that NMPCS was able to fully entrap the DNA at N/P ratio of 2:1, whereas CS entrapped the DNA completely at N/P ratio of 1:1. DLS showed that the NMPCS/DNA or CS/DNA complexes were of mean diameters ranging from 110 to 180 nm. Neither NMPCS nor CS induced significant loss of cell viability at the concentrations ranging from 1 to 50 microg/ml, whereas PEI at 5 microg/ml started to result in significantly decreased cell viability. The expression of transgene mediated by NMPCS was much higher (more than 100-folds) than that mediated by CS, indicating that NMPCS was a more efficacious gene ferrying vector than CS.
Tianjin UniversityT’ien-ching-shih, Tianjin Shi, China