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Résumé

This thesis deals with development of polymeric carriers for many different therapeutic agents such as immune-suppressant drug (everolimus), nitric oxide (NO), and polynucleotide. Polymeric carriers developed in this project were all synthesized by reversible additionfragmentation transfer (RAFT) polymerization, which is one of the living radical polymerization techniques. Synthesis, characterization, and results from in vitro/ex vivo experiments are described. New polymeric amphiphiles were obtained from the polymers synthesized by RAFT polymerization. Monomeric building blocks were derived from N-acryloyl derivatives of the cyclic secondary amines; morpholine, piperidine and azocane. Both homopolymerization and block-copolymerization of N-acryloylmorpholine (AM), N-acryloylpiperidine (AP), and N-acryloylazocane (AH) were carried out. From diblock copolymers in these compositions, block copolymeric amphiphiles were aggregated in nano-size. Details of aggregation behaviour were studied. Everolimus, a hydrophobic immune-suppressant drug was loaded in the micelles aggregated from poly[(N-acryloylmorpholine)-block-(N-acryloylazocane)] (PAM-PAH) diblock copolymer and drug-loaded micelles released the encapsulated drug over three weeks under physiological conditions. New polymeric NO donors were conceived from piperazine derivatives such as piperazine, homopiperazine, and 2,5-dimethylpiperazine. Depending on the hydrophobicity of the grafted ring of polymeric NO donors, we observed that the releasing rate of NO varied. Furthermore, we developed self-assembled micelles releasing NO with remarkably long release time ca. over 3 weeks. By encapsulating diazeniumdiolate (NONOates) in a hydrophobic micelle core, we were able to benefit from its slow hydrolysis rate, creating this unique release pattern. As NO is released, self-assembled micelles, turning into water-soluble polymer, will ultimately be dissociated and secreted out of the body. As demonstrated by ex vivo infusion experiments with rabbit carotid arteries, NO-releasing micelles (~80 nm) readily penetrate the arterial wall at which point their therapeutic aspects could be exploited, namely the delivery of NO to restenotic lesions. A new class of polycation, namely poly(N-acryloylpiperazine) (PAZ) was synthesized by RAFT polymerization and attempted as a non-viral gene delivery system. PAZ was capable to condense plasmid DNA and formed particles with less than 100 nm of diameter. PAZ-DNA polyplexes exhibited less incidence of cytotoxicity than linear PEI (LPEI)-complexed DNA, which is a commercially available synthetic gene delivery vector. In vitro transfection experiments were carried out with various cell lines such as human embryonic kidney (HEK) 293T cell line. The results were analyzed by fluorescent-activated cell sorting (FACS). In most cases, all homo/copolymers showed comparable or better transfection efficiency than LPEI when cells were transfected without serum. However, in the presence of serum, transfection efficiency of PAZ decreased. The details of the results, characterization, and discussion are described herein.

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