A thermo-sensitive NIPA-based co-polymer and monosize polycationic nanoparticle for non-viral gene transfer to smooth muscle cells.

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 by(1) 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.

Synthesis and characterization of nanomagnetite particles and their polymer coated forms.

Superparamagnetic nanoparticles were prepared by coprecipitation of ferrous (Fe(2+)) and ferric (Fe(3+)) aqueous solution by a base. Nanomagnetite particles were coated with poly(St/PEG-EEM/DMAPM) and poly(St/PEG-MA/DMAPM) layer by emulsifier-free emulsion polymerization. Chemical structure of nanoparticles was characterized by both FTIR and (1)H NMR. Particle morphologies were determined by Zeta Sizer, DLS, XRD and SAXS. Structural analysis showed that after polymer coating nanomagnetite particles kept their superparamagnetic property. Besides the synthesized magnetites, polymer coated forms of these particles are more biocompatible, well dispersable and uniform. These properties make them a very strong candidate for bioengineering applications, such as bioseparation, gene transfer.