Anion-induced thermoresponsiveness in cationic polycysteine and DNA binding

Abstract

Cationic polypeptide based smart biomaterials offer immense potential for biomedical applications including the field of drug delivery and the capability of spontaneously binding to biologically active components such as DNA. This study unfolds the synthesis of an L-cysteine-based water-soluble cationic polypeptide, an investigation of its thermoresponsive behaviour in the presence of added anions and its binding ability with DNA. Ring opening polymerization (ROP) of the newly designed bromo-functionalized L-cysteine N-carboxyanhydride (NCA) monomer results in the formation of polycysteine with a pendant Br atom. Subsequent nucleophilic substitution of the Br atom with triphenylphosphine gives cationic polycysteine (P[Cys-PPh₃]⁺[Br⁻]). An aqueous solution of P[Cys-PPh₃]⁺[Br⁻] is responsive to different Hofmeister series anions (BF₄⁻, I⁻, ClO₄⁻ and SCN⁻) showing a clear transformation from transparent to a cloudy suspension due to the formation of water-insoluble polypeptide/anion aggregates. The cloudy suspension of P[Cys-PPh₃]⁺[Br⁻]/anion aggregates in water becomes transparent upon heating and reappears on cooling, revealing an upper critical solution temperature (UCST)-type thermoresponsivity with a tunable cloud point with respect to the concentrations of both the cationic polypeptide and the added anions. The MTT assay result shows low cytotoxicity of the cationic polypeptide against human cells indicating its biocompatible nature. The polyplexation of cationic P[Cys-PPh₃]⁺[Br⁻] with calf-thymus DNA (ctDNA) was monitored by fluorescence spectroscopy, gel electrophoresis and circular dichroism spectroscopy. The effect of ionic strength on the polyplexation was also monitored by fluorescence spectroscopy.