| This research predicts that copolymers of N-isopropylacrylamide (NIPAAm) and dimethylacrylamide (DMAAm) can be synthesized with good characterization, that this copolymer can be grafted with poly(ethyleneimine) (PEI) with good characterization, and that this graft-copolymer can then be condensed with DNA to form DNA nanoparticles (polyplexes) that will allow not only the regulation of surface charge and solubility of the polyplexes by local hyperthermia, but also enhanced cellular uptake, transfection, and transgene expression at the site of hyperthermia with minimal cytotoxicity. Initially, the copolymer of P(NIPAAm-co-DMAAm) is synthesized, and then characterized by several methods, including light scattering, gel permeation chromatography (GPC), nuclear magnetic resonance (NMR), and titration. Analysis shows that the copolymer achieves the following targets: a lower critical solution temperature (LCST) of approximately 38.5--39.5°C; a molecular weight of less 135 than 10,000 g/mole; good composition; and appropriate pH. The copolymer is then grafted to PEI to form the graft-copolymer PEI-g-P(NIPAAm-co-DMAAm), which is then purified and shows an LCST within the target range of 41.5--42.5°C. The graft demonstrates good condensation with DNA, as evidenced via the ethidium bromide (Et-Br) displacement assay and agarose gel electrophoresis. Dynamic light scattering (DLS) reveals good physicochemical characteristics of the polyplexes, as evidenced by appropriate surface charge (zeta potential) and good nanoparticle size (particle size analyzer); the LCST of the polyplexes, however, cannot be determined. Cytotoxicity results show that the graftcopolymer produces greater cell viability in DU145 cells than PEI. Cellular uptake studies show temperature-controlled effects, but transgene expression studies do not confirm this data. Transfection and transgene expression studies show that PEI complexes (for the most part), compared to graft-copolymer complexes, remain superior at 37°C in this regard; however, at 43°C, virtually no transgene expression occurs with any complex. Overall, especially before hyperthermic xenograft gene delivery, further questions still need to be answered to fully assess the PEI-g-P(NIPAAm-co-DMAAm) polyplexes used in this hyperthermic gene delivery system. |
