PEG-Polyester-Poly(2-dimethylaminoethyl Methacrylate) Triblock Copolymer Nanoparticles For Gene Delivery

Gene therapy has been confirmed as a promising strategy to cure various inherited or acquired diseases, such as cancers, heart cerebrovascular disease, nervous system disease and viral infection. Development of safe and efficient gene delivery system is crucial for successful gene therapy. Viral vectors are widely applied in gene delivery studies because of their evolutionarily high transfection efficiency in clinic trials. However, safety concerns on the random recombination immunogenicity, limited nucleic acid loading capacity and high cost hamper their wide clinical application. Therefore, much attention has been shifted to non-viral vectors for their potential advantages such as low immunogenic response, easy structure modification, capability to carry large inserts and facile manufacturing. Recently, self-assembly nanoparticles (NPs) of biodegradable and amphiphilic cationic copolymers have emerged as efficient candidates for non-viral gene delivery. These amphiphilic cationic copolymers can self-assemble into stable, size-controlled and unique NPs with a hydrophobic core and a hydrophilic, positive charged shell. The NPs provide a strong gene-binding ability, high gene transfection efficiency in various cell lines and relatively low cytotoxicity in vitro and in vivo. Furthermore, the NPs can protect gene from degradation by nucleases and escape the reticuloendothelial system (RES) in vivo. But, comparing with viral vectors, the transfection efficiency and targeted delivery ability in vivo of NPs are still need to improve. In this paper, we prepared several biodegradable and amphiphilic cationic copolymers based on PEG, polyester and poly (2-dimethylaminoethyl methacrylate) (PDMAEMA), and investigated their properties as DNA delivery vectors.Firstly, a kind of biodegradable and amphiphilic copolymer, methoxy poly(ethylene glycol)-b-poly(ε-caprolactone)-b-poly(2-dimethylaminoethyl methacrylate) (mPEG-b-PCL-b-PDMAEMA), was synthesized by combination of ring opening polymerization (ROP) and atom transfer radical polymerization (ATRP) in our lab. We studied the properties of mPEG-b-PCL-b-PDMAEMA NPs to co-deliver hydrophobic drug and DNA. mPEG-b-PCL-b-PDMAEMA can self-assemble into NPs with hydrophobic core and hydrophilic cationic shell, so hydrophobic drug paclitaxel can be entrapped into the core and DNA can be condensed on the shell simultaneously. mPEG-b-PCL-b-PDMAEMA has obviously pH dependent sensitivity. The drug release rate of paclitaxel-loaded NPs in pH 5.0 release medium is higher than that in pH 7.2 release medium. The average size of NPs/pDNA complexes was about 200 nm. mPEG-b-PCL-b-PDMAEMA NPs with or without paclitaxel are both able to complex with pDNA completely when N/P ratio is equal to or above 3, and the combinatorial delivery of paclitaxel and pDNA have equivalent transfection efficiency compared with blank NPs/pDNA complexes when N/P ratio is equal to or above 15, which indicate that the payload of hydrophobic drug does not influence pDNA condensation and transfection efficiency. In addition, gene transfection efficiency was enhanced by the addition of 5% serum. Besides, confocal microscopic measurements indicated that whether or not carrying paclitaxel, the NPs/pDNA complexes can be efficiently internalized into 293T cells and escape from the endosome after transfection for 2 h. The in vivo distribution in the lungs, lives, pancreas and tumors confirmed that mPEG-b-PCL-b-PDMAEMA NPs had much better siRNA delivery efficiency than PEI. These results suggest that mPEG-b-PCL-b-PDMAEMA NPs may be a promising vector to deliver anti-cancer drugs and pDNA simultaneously for achieving the synergistic/combined effect on cancer therapies. While, the stronger crystallinity and slower biodegaradation rate of PCL define mPEG-b-PCL-b-PDMAEMA NPs only for long-acting administration.Further, to overcome the problems of crystallinity and biodegaradation rate of mPEG-b-PCL-b-PDMAEMA NPs, we synthesized methoxy poly(ethylene glycol)-b-poly(D,L-lactide)-b-poly(2-dimethylaminoethyl methacrylate) (mPEG-b-PDLLA-b-PDMAEMA) by ROP and ATRP. The prepared copolymers can self-assemble into spherical core-shell NPs with ultralow critical association concentration (CAC) of 0.025 mg/mL. The NPs can completely condense the pDNA into spherical complexes when the N/P ratio is equal to or above 3. BSA challenging results shown the mPEG-PDLLA-PDMAEMA NPs can effectively protect the DNA against protein. MTT assay results indicate that mPEG-b-PDLLA-b-PDMAEMA NPs/pDNA complexes exhibit obviously lower cytotoxicity compared with commercial gene transfection reagent Lipofectamine 2000/pDNA complexes. Subsequently, in vitro gene transfection studies in HeLa cells without serum show that mPEG-b-PDLLA-b-PDMAEMA NPs/pDNA complexes exhibit higher transfection efficiency than Lipofectamine 2000. Furthermore, the NPs still display equivalent gene transfection efficiency compared to Lipofectamine 2000 when N/P ratio is above 15 in DMEM with 10% serum. Confocal microscopic measurements indicate that the NPs/pDNA complexes can be efficiently internalized into HeLa cells after transfection for 3 h. These results suggest the mPEG-b-PDLLA-b-PDMAEMA NPs may be a potential candidate as gene carriers for human gene therapy.Finally, we investigated the role and influence of hydrophobic modifying in gene delivery. We synthesized a series of mPEG-b-PCL-b-PDMAEMA (PECLD) and mPEG-b-PDLLA-b-PDMAEMA (PEDLD) copolymers with different hydrophobic length. With increasing the hydrophobic length in PECLD and PEDLD, the CAC values decreased. Moreover, the CAC values of PEDLD are greater than that of PECLD when the hydrophobic length in PECLD and PEDLD are the same. The average size and zeta potential of PECLD NPs and NPs/pDNA complexes are both increased with increasing the hydrophobic length in PECLD. The same phenomenon is observed for PEDLD NPs and NPs/pDNA complexes. In contrast, the average size and zeta potential of PEDLD NPs and NPs/pDNA complexes are both higher than those of PECLD NPs and NPs/pDNA complexes. The increasing of hydrophobic length in copolymers does not influence pDNA condensation of PECLD NPs and PEDLD NPs. In vitro gene transfection was performed in HepG2 cells. The gene transfection efficiency of PECLD and PEDLD NPs are both increased with increasing the hydrophobic length in copolymers. It was worth noticed that the PEDLD NPs exhibits obviously higher gene transfection efficiency than PECLD NPs, and even higher than commercial gene transfection reagent Lipofectamine 2000. CCK-8 assay results indicate that both PECLD and PEDLD NPs are low toxic to HepG2 cells when N/P ratio is 15 and 10, respectively. These results suggest that the hydrophobic modifying by using PCL and PDLLA play an important role in gene delivery.