Thermoresponsive Poly(N-isopropylacrylamide)/Poly-L-Arginine Conjugate Transgene Vector

In this work, a thermoresponsive poly(N-isopropylacrylamide)/poly-L-arginine bioconjugate(PNIPArg) was prepared. The interaction between PNIPArg and plasmid DNA, and temperature tunable PNIPArg mediated gene delivery into C2C12 cells were investigated. The main work is summarized as follows:1. A thermoresponsive bioconjugate poly(N-isopropylacrylamide)/poly-L-arginine was synthesized by radical polymerization and EDC coupling. The formation of PNIPArg was confirmed by FTIR and 13C NMR. The lower critical solution temperature(LCST) of PNIPArg aqueous solution determined by turbidimetry was around 35.2℃.2. The physichemical properties of PNIPArg/DNA complexes were examined by a variety of methods. The influence of temperature sensitiveness on the formation, particle size and stability of complexes was investigated. The results of transmission electron microscope (TEM) showed that the PNIPArg/DNA complexes appeared uniform nanospheres with size about 50-100nm. Circular dichroism (CD) verified that the conformation of plasmid DNA remained almost unchanged in the complex, which became denser at a temperature above LCST. Agarose gel electrophoresis and DNase I assay revealed that the formation and stability of the complexes were improved by increasing temperature.3. The transfection efficiency of poly-L-arginine and PNIPArg was evaluated in C2C12 cells using two different reporter systems, PGL3 encoding for luciferase and pEGP-C1 encoding for green fluorescent protein (GFP). The results indicated that PNIPArg was able to transfer PGL3 and pEGP-C1 successfully. A controllable gene transfection and expression could be realized by selecting a variable culture temperature route.