Novel lysine-based reducible copolymers for intracellular gene delivery

Abstract

The development of biodegradable gene delivery systems, which have the ability to effectively deliver therapeutic DNA to a target tissue, is paramount to the success of nonviral gene delivery. One approach to developing biodegradable polymers is to introduce disulfide bonds along the backbone of the polymers to ensure release of the DNA in the reductive environment of the cytoplasm, whilst simultaneously reducing the molecular weight of the polymers. There is a crucial need to develop biocompatible and biodegradable polymers, which have low cytotoxicities so as to maintain cell viability and hence increase transfection efficiencies. Therefore, to produce a biocompatible gene delivery system, we have designed and synthesized novel reducible copolymers of the type (AB)n, which consist of repeating units of the natural amino acid, L-lysine and cystamine bisacrylamide (CBA). These novel reducible linear L-lysine copolymers (LLCs) were then modified with ethylenediamine so as to introduce primary amines for efficient DNA condensation. The molecular weight (MW) of the copolymers was found to be ~3.2 kDa with a polydispersity index of ~1.2. Gel retardation assays showed complete condensation of DNA at N/P ratios greater than 20/1 and exceptional LLC/pDNA polyplex stability during incubation with DNase I. To investigate the mechanism of DNA release from the polymer/pDNA complexes, fluorescence spectroscopy studies were performed with 1,4-dithio-DL-threitol (DTT). These data showed a significant reduction in fluorescence intensity following the addition of LLCs to DNA. After the addition of DTT, there was a 95 % increase in fluorescence intensity, which indicated the reduction of the disulfide bonds and the release of the DNA from the complexes. The particle sizes of LLC/pDNA polyplexes were found to be between 100-231 nm with surface charges of 0.8-17 mV respectively. The transfection efficiencies of the polyplexes as determined with a luciferase assay showed that LLC polyplexes produced five times higher transfection efficiencies in HDF cells, three times higher transfection efficiencies in MCF-7 cells, and four times higher transfection efficiencies in MA cells as compared to the optimal PLL control. The LLC/pDNA polyplexes showed significantly lower cytotoxicities as compared to the PLL/pDNA control in HDF, MCF-7, and MA cells at certain N/P ratios. Finally, in an exvivo study, LLCs were used as a nonviral gene carrier system to generate genetically modified stem cells to produce sufficient amounts of the angiogenic cytokine, vascular endothelial growth factor (VEGF165). These genetically modified stem cells were used to promote revascularization of an infarcted region of the heart, which can reduce myocardial damage and scar formation. A myocardial infarction model was generated in SCID mice deficient in T and B cells by permanent ligation of the left anterior descending coronary (LAD) artery. Cardiac hemodynamics, H&E staining and immunohistostaining results from this ex vivo study presented improved cardiac contractility, potential differentiation of hMSCs, new blood vessel formation, and a reduction in infarct size after treatment with the LLC genetically modified stem cells compared to the control animals. In conclusion, these results suggest that these novel LLCs are efficient, reducible and biocompatible polymers for nonviral gene delivery. Moreover, LLCs, as a nonviral gene carrier vector, hold great potential for the treatment of myocardial infarction in conjunction with stem cell therapy. Finally, adoption of novel nano-therapeutics strategies and techniques combining gene and cell therapies together could open the gate towards endless possibilities in the future of therapeutics and medicine.Pharmacological and Pharmaceutical Sciences, Department o