A Thermo-Sensitive NIPA-Based Co-Polymer and Monosize Polycationic Nanoparticle for Non-viral Gene Transfer to Smooth Muscle Cells

Abstract

Primary smooth muscle cells (SMC) isolated from the aorta of fetal calf were transfected with a green fluorescent protein (GFP)-encoding plasmid DNA, which was carried by a water-soluble and temperature-sensitive N-isopropylacrylamide-based (NIPAAm-based)-co-polymer, either poly(N-isopropylacrylamide-co-2-methacryloamidohistidine) (poly(NIPAAm-co-MAH)) or monosized PEGylated nanoparticle poly(styrene/poly(ethylene glycol) ethyl ether methacrylate/N-(3-(dimethylamino)propyl) methacrylamide) (poly(St/PEG-EEM/DMAPM)). Poly(NIPAAm-co-MAH) co-polymer was synthesized by solution polymerization of n-isopropylacrylamide (NIPAAm) and 2-methacrylamidohistidine (MAH). Monosized cationic nanoparticles were produced by emulsifier-free emulsion polymerization of styrene, PEG ethyl ether methacrylate and N-[3-(dimethyl-amino) propyl] methacrylamide, in the presence of a cationic initiator, 2,2-azobis (2-methylpropionamidine) dihydrochloride. The structure of poly(St/PEG-EEM/DMAPM) and poly(NIPAAm-co-MAH) was confirmed by1 H-NMR and FT-IR spectroscopy. Particle size/size distribution and surface charges of both carriers were measured by Zeta Sizer. The LCST behavior of poly(NIPAAm-co-MAH) co-polymer was followed spectrophotometrically. Poly(St/PEG-EEM/DMAPM) nanoparticles, with an average size of 78 nm and zeta potential of 54.4 mV, and an average size of 200 nm with a zeta potential of 54.2 mV, and poly(NIPAAm-co-MAH) were used in the transfection studies. The cytotoxicity of the vectors was tested using the MTT method. According to conditions for the transfection study (polymer/cell ratio and polymer–cell incubation period), cell loss was only 4 and 15% with poly(St/PEG-EEM/DMAPM) sized 78 and 200 nm, respectively. Poly(NIPAAm-co-MAH) cytotoxicity was insignificant. Poly(NIPAAm-co-MAH) uptake efficiency in SMCs was around 85%, but gene expression efficiency were low compared to poly(St/PEG-EEM/DMAPM)/pEGFP-N2 conjugates because of the low zeta potential of the co-polymer. Polymer uptake efficiencies of the nanoparticles were 90–95%. GFP expression efficiency was 68 and 64% after transfection with pEGFP-N2 conjugate with 78 and 200 nm sized poly(St/PEG-EEM/DMAPM) nanoparticles.