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PH-Responsive Smart Polymers for Gene delivery

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Tutor: SuiMeiHua;ShenYouQing
School: Zhejiang University
Course: Biochemical Engineering
Keywords: Non-viral vector,pH-responsive,Charge-reversal,Gene delivery
CLC: R730.5
Type: Master's thesis
Year:  2013
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Despite the great potential of non-viral delivery systems in gene therapy, their application has been impaired by various impediments. The differences in physiochemical and metabolic characteristics between tumor and normal tissues have inspired the development of smart polymers as gene vectors that could respond specifically to the changes in tumor tissues, such as decreased pH value in extracellular compartments and elevated level of oxidizing reactive oxygen species (ROS) in tumor cells. These smart polymers are expected to target tumor cells more specifically, and thereby reducing the side toxicity of gene delivery systems. Among all these stimuli-responsive polymers, pH-responsive polymers have attracted major attention and hold great promise in gene delivery.In this thesis, we aimed to develop two novel pH-responsive smart polymeric vectors for targeted gene delivery. In the first project, a degradable, charge-reversal polyethyleneimine (PEI) derivative for gene delivery was developed based on the reaction between2-(N,N-dimethylamino)ethyl acrylate (DMAEA) and branched PEI. The obtained PEI-DMAEA exhibited significantly reduced cytotoxicity in several cultured tumor cell lines compared to PEI-25kDa. Moreover, as they hydrolyzed quickly at neutral condition, PEI-DMAEA copolymers were further quaternized using iodomethane with varied degrees of quaternization to adjust the hydrolysis rate. Our data demonstrated that at an N/P ratio of40/1, quaternized PEI-DMAEA copolymers could effectively condense and protect DNA, and the unique positive-to-negative charge-reversal induced by self-catalyzed hydrolysis may efficiently promote the subsequent DNA release. Furthermore, polyanionic compound (pEP), a novel membrane disruptive agent synthesized by2-(ethoxymethyl) acrylic acid and2-(propoxymethyl) acrylic acid, was coated onto the surface of PEI-DMAEA/DNA polyplexes at different EP/DNA weight ratios, in order to promote the lysosomal escape and increase the gene transfection efficacy. The obtained pEP/PEI-DMAEA/DNA nanocomplexes had favorable particle size (150~170nm) and moderate negative surface charges (-20~25mV). More importantly, the cellular uptake of pEP/PEI-DMAEA/DNA nanocomplexes reached about6-fold higher than that of PEI/DNA, and the gene transfection efficiency mediated by these nanocomplexes was about2.0-fold higher than that mediated by PEI/DNA.In the second project, we designed a pH-responsive poly (benzyl 2-amino-6-methacrylamidohexanoate)(pLLB) based on the lysine. Further characterization indicated that the hydrolysis rate of this novel polymer at pH7.4was much higher than that at pH5.0, which was desired for gene delivery. Compared to PEI-25kDa, this pH-responsive polymer exhibited lower cell toxicity and better biocompatibility. Moreover, pLLB could condense and protect DNA to form nanocomplexes with favorable particle size (-150nm), even at low N/P ratios. Finally, the in vitro gene transfection of these nanocomplexes was analyzed by flow cytometry in H1299lung cancer cell line. Our data demonstrated that when co-transfected with chloroquine, these nanocomplexes exihibited gene transfection efficiency as high as40%, which was comparable to PEI.In brief, these two novel types of polymeric gene vectors deserve further investigations and may hold great promise for efficient in vivo gene delivery.
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