title: 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 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.
authors.affiliation: Advanced Technology Education, Research and Application Center, Mersin University, 33343 Mersin, Turkey. melisalacin@yahoo.com
authors.forename: Nelisa Türkoğlu
authors.initials: NT
authors.lastname: Laçin
authors.forename: Güldem Guven
authors.initials: GG
authors.lastname: Utkan
authors.forename: Tülin
authors.initials: T
authors.lastname: Kutsal
authors.forename: Erhan
authors.initials: E
authors.lastname: Pişkin
mesh_terms: D000178:Acrylamides / Q000737:chemistry*; D000818:Animals; D002417:Cattle; D004337:Drug Carriers / Q000737:chemistry* / Q000633:toxicity; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000378:metabolism*; D053758:Nanoparticles / Q000737:chemistry*; D010316:Particle Size; D011092:Polyethylene Glycols / Q000737:chemistry; D011108:Polymers / Q000737:chemistry* / Q000633:toxicity; D013499:Surface Properties; D013696:Temperature; D014162:Transfection / Q000379:methods*
chemical_list: D000178:Acrylamides; D004337:Drug Carriers; D011108:Polymers; D011092:Polyethylene Glycols; C067295:N-isopropylacrylamide; C045985:methacrylamide