| Gene therapy has emerged as a new approach treating genetic disorders by delivering therapeutic genes to targeted diseased tissues, and is known highly effective for treatments of many diseases. Nonviral gene vectors including cationic lipids, polymers, dendrimers and peptides are particularly attractive in terms of safety, low immunogenicity, biocompatibility, and the potential for large-scale manufacture. However, their applications are bottlenecked by low transfection efficiency compared with viral vectors. Cationic polymers are safe nonviral gene vectors with great potential for large-scale applications, and widely used to condense the large macromolecules into cationic polymer/DNA complexes (polyplexes) nanoparticles, protecting them from degradation and facilitating their cellular internalization. However, once inside cells, unzipping the cationic polymer/DNA complexes is against the strong electrostatic interaction and thus intracellular release of free DNA for transcription is the main barrier to efficient DNA transfection.In this thesis, we first designed reactive oxygen species (ROS)-labile charge-reversal polymer-based fusogenic lipidic polyplexes (FLPPs), successfully overcoming these problems. One key design is the charge-reversal moiety, a quaternary ammonium with a benzylboronic acid. Upon oxidation of the boronic acid group by intracellular ROS (e.g., H2O2), which are known to be elevated in cancerous cells and capable of oxidizing benzylboronic acid/esters, the quaternary ammonium releases p-hydroxylmethylenephenol (HMP) and becomes a tertiary amine, which self-catalyzes further hydrolysis of the polyacrylic ester into poly(acrylic acid). The second key design is the fusogenic lipid coating. Different from the most lipopolyplexes which are internalized by endocytosis and thus may be trapped in lysosomes causing DNA degradation, a pegylated lipid layer was fine tuned and coated to the charge-reversal polyplexes so that lipidic polyplexes nanoparticles were able to fuse with the cell membrane, ejecting the polyplexes into the cytosol. Such viropexis-mimicking process avoids the endosomal trapping and DNA degradation. The most importantly, the CRGKD-targeting FLPPs were stable and long circulating in the bloodstream and actively target tumors, and successfully delivered reporter genes and suicidal TRAIL gene to tumor xenografts, producing significantly better tumor growth inhibition than doxorubicin.In the second part of this thesis we developed two cationic lipidic polyplex gene delivery systems with excellent serum-resistant ability using the ROS-labile charge-reversal polymer for intraperitoneal cancer gene therapy. The lipid coating served as a barrier to prevent polyplexes from premature dissociation in the presence of high concentration serum proteins, promoted the cellular uptake and tumor-penetrating. Furthermore, the cationic liposomes could induce intracellular generation of ROS, which in turn promoted the ROS-responsive charge-reversal and the dissociation and release of DNA. The lipopolyplexes successfully delivered TRAIL gene to intraperitoneal A549 tumors, producing significantly better tumor growth inhibition than B-PDEAEA/TRAIL, Lipo 2000/TRAIL and PEI/TRAIL polyplexes. The therapeutic efficacy was even comparable to the cytotoxin drug paclitaxel. The delivery system also showed significantly better anti-metastatic activities than PEI/TRAIL in lung metastatic 4T1-Luc-GFP model.Using the same concept, in the third part of this thesis we synthesized a series of ROS-labile charge-reversal cationic polymers, B-PDEAEMA, B-PEI and B-PAMAM. All these polymers with the charge-reversal moiety, a quaternary ammonium with a benzylboronic acid, showed excellent gene transfection efficiency. The CRGKD modified fusogenic lipidic polyplexes based on B-PDEAEMA (RGDK-FLPPM) successfully delivered TRAIL gene to SW480 tumor xenografts, producing significantly better tumor growth inhibition than 5-fluorouracil. |
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