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• W62464013 abstract "Throughout these last years, many synthetic polymers have been developed for non-viral gene delivery systems in attempt to overcome potential shortcomings of viral vectors. However, it is still difficult to achieve highly efficient transfection under physiological condition due to severe interaction between cationic polyplexes and abundant proteins in serum. In this project, we aim to suggest different strategies to enhance transfection efficiency of non-viral polymeric vector, which was already developed in our research group, i.e. poly(N-acryloylpiperazine) (PAZ). As a first strategy, cancer-targeting delivery was attempted with a folic acid (FA)-conjugated poly(N-acryloylpiperazine) (PAZ-FA), because folic acid receptors (FRs) are over-expressed on many types of cancer cells. We hypothesized that folic acid moieties on PAZ-FA would facilitate the attachment and the internalization of polyplexes to cancer cells. As a second strategy, block copolymerization was attempted. In this approach, a diblock copolymer, i.e. poly[(N-acryloylmorpholine)-bl-(N-acryloylpiperazine)] (PAM-PAZ) was synthesized. We hypothesized that a neutral and hydrophilic poly(N-acryloylmorpholine) (PAM) block would reduce electrostatic interaction with proteins in serum which deteriorates transfection efficiency. Synthesized polymers were thoroughly characterized and details are described herein. The physical properties of polyplexes were examined such as size, surface charge by zetapotential and gel retardation assay, protection capability from enzyme, etc. To carry out transfection experiments, two types of cells, i.e. human embryonic kidney (293T) cells and human cervical epitheloid carcinoma (HeLa) cells were employed with green fluorescent protein (GFP)-expressing plasmid DNA. Transfection results were analyzed by fluorescence-activated cell sorting (FACS). As a positive control, linear poly(ethylene imine) (LPEI) was employed in the experiments. In serum-free condition, there was no substantial difference in size and/or zeta potential among PAZ-polyplexes, PAZ-FA polyplexes, PAM-PAZ polyplexes, and LPEI-polyplexes. This reveals that all PAZ derivatives are similarly capable to condense DNA into nano-sized particulate polyplexes as LPEI. PAZ and PAZ-FA showed comparable or even better transfection efficiency than LPEI, whereas PAM-PAZ showed lower transfection efficiency than the other two PAZ derivatives and LPEI. In the presence of 10% of fetal bovine serum (FBS), we observed that, the size of polyplexes of PAZ-derivatives spontaneously increased up to a few microns in an hour or two. This size increment was more severe compared to LPEI-polyplexes, although the surface charge of all polyplexes remained the same. As for transfection, with 10% of FBS in cell media, all PAZ-derivatives showed lower transfection efficiency than LPEI. To get a better insight of serum effect, we prepared a fluorescent-labeled PAZ and examined how serum affects the internalization of polyplexes. In result, we observed that serum prevents large extent of attachment on the extracellular membranes, but there was no difference in the actual amount of internalized polyplexes either with or without serum. Thus, we believe that serum does not influence on the internalization of polyplexes, but other critical pathway may involve an intracellular compartment." @default.
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• W62464013 date "2008-01-01" @default.
• W62464013 modified "2023-09-27" @default.
• W62464013 title "Strategies in non-viral gene delivery: to overcome serum effect" @default.
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