Synthesis And Characterization Of Novel PH-sensitive Cationic Polymers For Gene Delivery

A new type of homopolymers, poly (N-((2-(2-(dimethylamino) ethoxy)-1,3-dioxolan-4-yl) methyl) methacrylamide (PMAOE), bearing tertiary amines linked byacid-labile ortho ester rings was synthesized and characterized. PMAOE with differentaverage chain length (19,64, and82repeating units) were obtained via traditional radicalpolymerization, and the characterization was studied by1H-NMR, GPC. NMR analysisrevealed that hydrolysis of the ortho ester group in the side-chains of PMAOE followed adistinct exocyclic mechanism and that the rate of hydrolysis was much accelerated at mildlyacidic pH.The polyplexes were prepared by the electrostatic interactions between PMAOE andplasmid DNA encoding green fluorescent protein. Gel retardation assay, DNase I degradationexperiment, heparin displacement experiment, ethidium bromide (EB) exclusion, dynamiclight scattering (DLS) and zeta potential measurements were applied to determine the abilityof DNA condensing and binding. They were found to effectively bind and condense plasmidDNA at neutral pH, forming narrowly dispersed nano-scale polyplexes. The strength of DNAbinding and stability of polyplexes were dependent on the average length of the PMAOEchains. Gel electrophoresis experiment was used to evaluate the pH sensibility of polyplexesand the pH modulation of DNA release. As a result of losing the cationic side-chains andneutralization of residual cationic charges, PMAOE lost its capability of binding andcondensing plasmid DNA, leading to accelerated disruption of polyplexes and the release ofintact plasmid DNA in mildly acidic environment.PMAOE homopolymers had very low cytotoxicity to cultured NIH3T3, HEK-293andCOS-7cells detected by MTT assay. In vitro transfection by the methods of fluorescentmicroscope image and flow cytometry revealed that PMAOE could transfer HEK-293cellsand COS-7cells, and the transfection efficiency was best at the ratio of N/P16.This study demonstrates a unique mechanism of achieving pH-modulated binding andrelease of DNA from cationic polymers, and the excellent cytocompatibility of the polymerssuggest that they may be practically useful biomaterials for gene delivery applications.