Poly (2-dimethylamino) Ethyl Methacrylate) Copolymers Designed For Non-viral Gene Delivery

Gene therapy is one of the most promising treatments for various diseases such as cancers, cardiovascular diseases, and genetic disorders. The clinical applications of gene therapy, however, are restricted by shortage of safe, efficient gene delivery technology. In the past decade, polymer-based gene delivery systems have attracted great attention due to many advantages including improved safety, low immune responses, enabling repeated uses, and ease of production. Poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) has been often studied for gene transfection.High molecular weight (Mw>300 kDa) PDMAEMA has show to display high buffer capacity at endosomal pH and mediates efficient transfection in various types of cells. However, PDMAEMA is not readily biodegradable, which may render long-term and/or acute toxicity. In addition, polyplexes of PDMAEMA expose insufficient colloidal and serum stability, which restricts their applications in vivo. In the past years, bioreducible and biodegradable PDMAEMA copolymers have been developed to achieve reduced toxicity and/or enhanced transfection activity. Furthermore, unlike other cationic polymers including PEI and PAMAM, PDMAEMA copolymers can be conveniently prepared with controlled macromolecular structures and compositions by living radical polymerization. In this paper, we adopted reversible addition-fragmentation chain transfer (RAFT) polymerization to prepare several new types of PDMAEMA copolymers for gene delivery.First, biodegradable cationic micelles based on PDMAEMA-PCL-PDMAEMA triblock copolymers were designed for the combinatorial delivery of siRNA and paclitaxel into cancer cells. The cationic micelles revealed lower cytotoxicity than 25 kDa branched PEI. Notably, GFP siRNA complexed with micelle achieved over 70% silencing efficiency at an N/P ratio of 36/1 which is higher than 25kDa bPEI (40% silencing efficiency). In contrast,20 kDa PDMAEMA revealed no silencing effect at an N/P ratio of 36/1.Moreover, the combinatorial delivery of VEGF siRNA and paclitaxel resulted in significantly lower VEGF expression as compared to delivery of VEGF siRNA or paclitaxel alone, reaching a high silencing efficiency of ca.85%. Confocal laser scanning microscope (CLSM) studies revealed that nile red was delivered into MDA-MB-435-GFP cells and that GFP expression was significantly inhibited. These results demonstrated that cationic biodegradable micelles are highly promising for the combinatorial delivery of siRNA and lipophilic anticancer drugs.Second, bioreducible PDMAEMA-SS-PEG-SS-PDMAEMA triblock copolymers were designed, prepared and investigated for gene transfection. Like the non-reducible analogues, PDMAEMA-SS-PEG-SS-PDMAEMA triblock copolymers could effectively condense DNA into small particles with average diameters less than 120 nm and zeta potentials close to neutral (0-+6 mV) at and above an N/P ratio of 3/1. The resulting polyplexes showed excellent colloidal stability against 150 mM NaCl, which contrasts with polyplexes of 20 kDa PDMAEMA homopolymer. In the presence of 10 mM DTT, however, polyplexes of PDMAEMA-SS-PEG-SS-PDMAEMA were rapidly de-shielded and unpacked. Release of DNA in response to 10 mM DTT was further confirmed by gel retardation assays. Notably, in vitro transfection studies showed that reversibly shielded polyplexes afforded up to 28 times higher transfection efficacy as compared to stably shielded control under the same conditions. These results demonstrated that reversibly shielded DNA polyplexes have a great potential in achieving safe and efficient gene transfection.Third, a versatile family of PDMAEMA copolymers containing varying amounts of primary amino side groups were synthesized and investigated for in vitro gene transfection. The copolymer compositions were well controlled by feed ratios. These PDMAEMA copolymers could effectively condense DNA at an N/P ratio of 3/1 to give small sized polyplexes, which were smaller than for PDMAMEA. The polyplexes of these copolymers revealed similar level of cytotoxicity as those of PDMAEMA homopolymer. Interestingly, the in vitro transfection in COS-7 cells in serum free medium demonstrated significantly enhanced (up to 24-fold) transfection efficiencies for PDMAEMA copolymer polyplexes as compared to PDMAEMA control, with following the order of P(DMAEMA/AHMA)> P(DMAEMA/AEMA)> PDMAMEA. It is remarkable to note that in the presence of 10% serum, P(DMAEMA/AEMA) and P(DMAEMA/AHMA) displayed comparable or better transfection activity with respect to 25kDa PEI at its optimal formulation. These results suggest that cationic methacrylate copolymers are highly promising for development of safe and efficient non-viral gene transfer agents.Last, reduction-responsive cationic micelles based on PDMAEMA-SS-PCL-SS-PDMAEMA triblock copolymers were investigated as non-viral gene vectors. PDMAEMA-SS-PCL-PDMAEMA triblock copolymers were prepared by reversible addition-fragmentation chain transfer (RAFT) polymerization of dimethylaminoethyl methacrylate using CPADN-SS-PCL-SS-CPADN as RAFT agent. Both DNA complexation assay and gel retardation assay showed that the cationic micelles could effectively complex DNA at and above N/P ratios of 3/1. These resulting polyplexes showed good colloidal stability against 150 mM NaCl, which contrasts with polyplexes of 12 kDa PDMAEMA homopolymer. Notably, the polyplexes were prone to fast aggregation in the presence of 10 mM DTT, as further confirmed by gel retardation assay. Moreover, the reducible micelles from PDMAEMA-SS-PCL-SS-PDMAEMA revealed a lower cytotoxicity than corresponding non-reducible triblock copolymers.