α,β-poly(N-2-hydroxyethyl)-d,l-aspartamide (PHEA) was used as starting polymer being a highly water-soluble synthetic polymer, already proposed with success as therapeutic carrier by our group. In this study, PHEA was firstly functionalised with ethylendiamine, obtaining a well defined copolymer with pendant primary amine groups (PHEA-EDA), to which N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) and 3-(carboxypropyl)trimethyl-ammonium chloride (CPTA) were linked in two subsequent steps, allowing the introduction of thiol and cationic groups respectively. Finally DTT treatment lead to the final PHEA-EDA-SH-CPTA thiopolycation, named PESC.
The present work describes the synthesis and characterization of the thiopolycation PESC. 1H NMR spectroscopy detected the derivatization molar degrees in SPDP and CPTA; the formation of DNA complexes (thiopolyplexes), their stability in the presence of polyanions and the ability to release DNA under reductive conditions were studied by agarose gel electrophoresis. DNase II degradation study was carried out to detect the ability of thiopolyplex to stabilize DNA towards enzymatic metabolism. Thiopolyplexes were then characterized by Dynamic Light Scattering (DLS) and Zeta Potential analysis. Finally, in vitro toxicity profile (MTT) and gene transfer efficiency (Luciferase assay) were carried out to evaluate thiopolyplex biocompatibility, safety and efficacy to be used as gene delivery system.