Synthesis of intracellular reduction-sensitive amphiphilic polyethyleneimine and poly(ε-caprolactone) graft copolymer for on-demand release of doxorubicin and p53 plasmid DNA

This study aims to present a new intelligent polymeric nano-system used for combining chemotherapy with non-viral gene therapy against human cancers. An amphiphilic copolymer synthesized through the conjugation of low molecular weight polyethyleneimine (LMw-PEI) and poly(e-caprolactone) (PCL) via a bio-cleavable disulfide linkage was successfully employed for the simultaneous delivery of drug and gene molecules into target cells. Compared to the conventional PCL copolymerization pathway, this paper represents a straightforward and efficient reaction pathway including the activation of PCL-diol hydroxyl end groups, cystamine attachment and LMw-PEI conjugation which are successfully performed at mild conditions as confirmed by FTIR and 1 H NMR. Thermal, morphological characteristics as well as biocompatibility of the copolymer were investigated. The copolymer showed great tendency to form positively charged nanoparticles (∼163.1 nm, +35.3 mV) with hydrophobic core and hydrophilic shell compartments implicating its potential for encapsulation of anti-cancer drug and plasmid DNA, respectively. The gel retardation assay confirmed that the nanoparticles could successfully inhibit the migration of pDNA at ∼5 nanoparticle/pDNA w/w. The in vitro cytotoxicity tests and LDH assay revealed that the cationic amphiphilic copolymer was essentially non-toxic in different carcinoma cell lines in contrast to branched PEI 25K. Moreover, the presence of redox sensitive disulfide linkages provided smart nanoparticles with on-demand release behavior in response to reducing agents such as cytoplasmic glutathione (GSH). Importantly, confocal microscopy images revealed that in contrast to free Dox, the nanoparticles were capable of faster internalizing into the cells and accumulating in the perinuclear region or even in the nucleus. Finally, the co-delivery of Dox and p53-pDNA using the copolymer displayed greater cytotoxic effect compared with the Dox-loaded nanoparticle counterpart as revealed by cell viability and Caspase 3 expression assay. These results suggest the copolymer as a promising candidate for the development of smart delivery systems. Statement of Significance We employed cystamine dihydrochloride as a disulfide linkage for the conjugation of PCL diol and low molecular weight PEI segments through a straightforward and efficient reaction pathway at mild conditions. The new copolymer was essentially non-toxic in different carcinoma cell lines and showed great tendency to form positively charged nanoparticles. Therefore, it can be utilized as a promising platform for simultaneous drug and gene delivery to aggressive cancers. The results of drug and gene co-delivery experiments confirmed the pivotal role of disulfide linkage on the controlled release of both drug and gene molecules in response to glutathione concentration gradient between extracellular and intracellular microenvironments. In addition, the co-delivery of doxorubicin and p53 plasmid DNA using the new copolymer displayed greater cytotoxic effect compared with single agent (i.e. Dox) loaded counterpart, which indicated the significance of rapid dissociation of therapeutic agents from the carrier for synergistic cytotoxic effects on cancer cells.