PGMA-based High-efficient Gene Vector

The unceasing development of human society improve people’s material standard of living; however, unhealthy lifestyle and serious destruction of global environment directly or indirectly lead to fast spread of major disease,such as cancer, cardiovascular disease, genetic disease and neurological disease. These diseases are often difficult to cure and severely threaten people’s health and life. As a method of introducing therapeutic genes into targeted cells, gene therapy is hopeful to cure these intractable diseases at root.The key of gene therapy is design and fabrication of safe, efficient gene delivery systems including viral vectors and non-viral vectors. So far, the safety problems of viral vectors have not been solved, and thus, more and more rearchers have focused on non-viral vectors, in which cationic polymers have been significantly developed in recent decades. In this dissertation, we proposed a series of poly(glycidyl methacrylate)-based gene vectors for overcoming the obstacles during the gene delivery mediated with cationic polymers, and in vitro and/or in vivo characterizations were used to vestify their properties, providing important, beneficial information for the development of gene therapy.Starlike cationic polymer gene vectors are appealing because of their good flexibility. The satisfactory chemical modificability and excellent biocompatibility of P-cyclodextrin β-CD) provide favorable conditions for the preparation of starlike polycation. Considering the possible biological functions from piperazine (PP), N-(aminoethyl)piperazine (AEPP), and N-(3-aminopropyl)-2-pyrrolidinone (APP), we combined atom transfer radical polymerization and ring-opening reaction to propose CD-PGPP, CD-PGAEPP,and CD-PGAPP, which contained β-CD cores and were based on poly(glycidyl methacrylate) (PGMA). These three starlike polyamine gene vectors had rich amine groups and nontoxic hydroxyl groups, and they could effectively condense plasmid DNA (pDNA) and exhibit much lower cytotoxicity than positive control PEI. In most cases, CD-PGAPP exhibited better gene transfection performance than CD-PGPP and CD-PGAEPP,particularly in HepG2 cells. More importantly, CD-PGPP, CD-PGAEPP, and CD-PGAPP vectors almost did not cause undesirable hemolysis, indicating that these three polyamine vectors are hopeful for in vivo application.The transgene delivery is a complicated process, filled with kinds of obstacles. The above three polyamine vectors could protect nucleic acid and possibly have good blood compatibility; however, as a whole, their properties had limitation and needed to be improved via modification. The bioavailability of transgene can be promoted via endowing a vector with specific delivery property. Herein, we replaced PP, AEPP, and APP with ethanol amine (EA) for making PGMA functionalized and generating s-PGEA with low toxicity and high flexibility, and further introduced phenylboronic acid (PBA) (good affinity with glycoprotein) to harvest s-PGEA-B. As proved, compared with s-PGEA without PBA groups, s-PGEA-B exhibited stronger ability to condense pDNA, forming smaller complexes. The cellular internalization rate and transfection efficiency mediated by s-PGEA-B/pDNA complexes were higher, especially in HepG2 cells, because PBA groups can specifically bind to carbohydrate sLex on the surface of HepG2 cells. In addition, during transfection, s-PGEA-B simultaneously emitted fluorescence, benefitting the location of vectors. This work provided new idea for the design of multifunctionalized gene delivery system and the hepatoma therap.A gene vector approaching "idealized state" should simultaneously overcome several obstacles during delivery. Therefore, we need to take multiple aspects into consideration for developing gene vectors. The last project was carried out in terms of improving bioavailibility. Herein, we would like to design vector via facilitating escape of transgene. Series of reducible comb-shaped polycation SS-PGEADMs were proposed. They comprised EA/cystamine-functionalized PGMA (SS-PGEA-NH2) backbone and PDMAEMA side chains carrying disulfide groups, having plentiful secondary amine and hydroxyl groups, as well as moderate amounts of disulfide groups.The amount of disulfide and the length of PDMAEMA side chains were controllable. As vestified, in a reducible condition simulating that inside cells,the molecular weight of SS-PGEADMs decreased obviously. In addition,SS-PGEADM/pDNA complexes effectively released pDNA in the presence of heparin (a kind of ionic polysaccharide) and dithiothreitol (DTT), and the release amount was significantly higher than that of SS-PGEA-NH2/pDNA complexes. It was worth noting that the introduction of relatively short PDMAEMA side chains improved the transfection efficiency and increased cytotoxicity at an acceptable degree, owing to the cleavable disulfide groups responsive to reducible stimuli. Such multi-functionalized well-defined comb-shaped vector provides a flexible, pratical method for synthesis of gene/drug vectors.Nucleic acid-based gene therapy is a promising treatment option to cure numerous intractable diseases. For non-viral gene carriers,low-molecular-weight polymeric vectors generally demonstrate poor transfection performance, but benefit their final removals from the body. The last project inspired us through simultaneously introducing short polycation chains and disulfide groups to build up high-molecular-weight vectors. Hence,based on ethylenediamine (ED)-functionalized low-molecular-weight linear PGMA (denoted by PGED), a flexible strategy was proposed to design new well-defined reducible cationic nanogels (denoted by PGED-NGs) with friendly crosslinking reagents for highly efficient nucleic acid delivery.a-Lipoic acid (LA), one natural antioxidant in human body, was readily introduced into ED-functionalized PGMA and crosslinked to produce cationic PGED-NGs with plentiful reducible lipoyl groups. PGED-NGs could effectively complex plasmid DNA (pDNA) and short interfering RNA(siRNA). Compared with pristine PGED, PGED-NGs exhibited much better performance of pDNA transfection. PGED-NGs also could efficiently transport MALAT1 siRNA (siR-M) into hepatoma cells and significantly suppressed the cancer cell proliferation and migration. The present work indicated that reducible cationic nanogels involving LA crosslinking reagents probably developed as competitive candidates for application in gene therapy.The above research supports valuable reference and idea for the future design of gene delivery systems.