Preparation And Properties Of Cationic Poly(Amino Acid)s For Gene Delivery

Gene therapy is a way of therapy using therapeutic genes as drugs. It is achieved by inserting a functional gene into the targeted cells in order to cure a disease or to repair a dysfunction caused by a genetic defect. A complete gene therapy system includes two parts that are therapeutic gene and gene delivery system. Nowadays, the main problem limiting the clinical applications is lacking of safe and efficient delivery systems for genetic drugs. Gene transfer systems (vectors) are classified into two types:viral vectors and non-viral vectors. There are some drawbacks limiting the applications of viral vectors. Acute immune response may be caused by viral vectors; the production of viral vectors in large quantities is very difficult and expensive; the size of gene that can be delivered by the virus is limited. Non-viral vectors have attracted much attention because of they are non-immunogenic, biocompatible, practicable for large production. Among the non-viral vectors, cationic polymers are very promising and have been most intensively studied. However, relatively high cytotoxicity and low transfection efficiency in comparison with virul vectors remain the main problems that limit their further development. It is an important challenge to design safe and efficient polymeric carriers based on overcoming the extra-and intra-cellular barriers during the transfection process.Chapter1of this thesis presents a detailed review of DNA delivery systems, progresses achieved in cationic polymer-based gene delivery, and the obstacles in polymer gene delivery. In the next four chapters, a series of poly(amino acids)s were designed and synthesized, and their properties for gene delivery were evaluated,In chapter2, multi-arm star polymers PEI-P(Orn)n are prepared by grafting polyornithine arms onto branched polyethylenimine (PEI) with an Mw of600via the ring-opening polymerization of N-carboxyanhydride of benzyloxycarbonyl ornithine. The cytotoxicity of PEI-P(Orn)n increases with the increase of molecular weight. The star polymers could bind DNA at a very low polymer/DNA ratio. The particle sizes of the polyplexes was from400to160nm, and zeta potentials trended to reach a plateau of34mV. PEI-P(Orn),, have high gene transfection efficiency comparable with25KDa PEI in serum-free medium. More importantly, in contrast to the dramatically lowered efficiency of25KDa PEI in the presence of serum, the efficiency of PEI-P(Orn)42and PEI-P(Orn)71can be retained in the medium containing10%serum.Arginine-rich peptides have an ability to penetrate the cellular membrane. Homopolymers of arginine have better cellular uptake than polyhistidines and polylysines. The cell-penetrating properties of arginine-rich pepteides is not only because of the cationic nature of arginine, which provides the positive charge needed for interaction with cell membrane, but it is more attributable to the guanidino head group of arginine. In chapter3, the amino side groups on the polyornithine arms are partially guanidinated that transforms the ornithine units to arginine units. Thus, the guanidinated products G-PEI-P(Orn)n contain multiple poly(ornithine-co-arginine) arms. The cytotoxicity could be remarkably lowered by guinidination of the amino groups. G-PEI-P(Orn)71can transfect pGL3to293T cells and HeLa cells with efficiency of almost the same magnitude of25KDa PEI in serum-free medium. More importantly, the efficiency of G-PEI-P(Orn)71can be retained or even enhanced in the medium containing10%serum. Among all these polymers, the34%G-PEI-P(Orn)71can achieve high transfection efficiency which is2orders more efficient than25KDa PEI at a polyrner/DNA (w/w) ratio of20in the medium containing10%serum.A promising way to realizing the low cytotoxicity of the polyplexes for gene delivery is the use of PEG-modified cationic polymers. In chapter4, an amino-terminated PEG was used as a macro molecular initiator in synthesis of PEG-conjugated poly(ornithine-co-arginine). The polymer had a lower cytotoxicity compared to25KDa PEI. The particle size of the polyplexes with DNA was about400nm, and zeta potential trended to reach a plateau of20mV. PEG-conjugated poly(ornithine-co-arginine) has gene transfection efficiency comparable with25KDa PEI at an appropriate polymer/DNA (w/w) ratio in serum-free medium. The polymer can achieve high transfection efficiency which is10times more efficient than25KDa PEI in the medium containing10%serumRecent years disulfide-linked bioreducible polymers have attracted much attention for gene transfectba They show excellent stability in extracellular environment and degradability in cytoplasm, which is beneficial for unpacking of DNA. In chapter4, we synthesized a low molecular weight PEG-conjugated poly(ornithine-co-cysteine) by co-polymerization of s-benzyloxycarbonyl ornithine N-carboxyanhydride (ZOrn-NCA) and S-benzyloxycarbonyl cysteine N-carboxyanhydride (ZCys-NCA). Their cytotoxity is remarkably lower than that of25KDa PEI. The polyplexes with DNA have particle size from200to600nm, and zeta potentials up to32mV. The polymers have gene transfection efficiency comparable with25KDa PEI in serum-free medium, and the efficiency can be retained in the medium containing10%serum.