Design, Synthesis, And Properties Of Phenylborinic Acid-modified Polymers For Use As Gene Vectors

By integrating normal or therapeutic genes into target cells to regulate andmodulate cell functions and responses, gene therapy holds enormous potential fortreating a broad range of genetic diseases, including infectious diseases, cancers,gene-related disorders, HIV, and Parkinson’ diseases. A gene therapy system includestwo parts which are target gene and gene delivery system. Nowadays, a major hurdlefor clinical applications is that there is no safe and efficient delivery system for geneticdrugs. Generally, there are two different categories of gene transferring methods: genetransfer mediated by viruses and non-viral gene delivery. Non-viral vectors haveattracted much attention because of their safety in clinical trials, excellentbiocompatibility, high DNA-packaging capacity, low cytotoxicity and so on. All ofthese promote researchers to develop non-viral vectors with low toxicity and high genetransfection efficiency. Because of the excellent properties, polymeric gene carriershave attracted significant attention. However, their relatively low transfectionefficiency remains as an obstacle for further development. So, it is important to designand synthesize polymeric carriers to mimic the essential properties of viruses such ascell recognizing and targeting.Boronic acid compounds have versatile applications in the biomedical field, suchas cell identification and saccharide sensor because they could form reversible cyclicboronates with multi-hydroxyl carbohydrates. As it is well known that there are manydifferent carbohydrates on the cell surface, we design and synthesize a series ofphenylboronic-modified polymers and investigate their properties for use a genevectors in this dissertation.Chapter1presents a detailed review on the progress in polymer-based genedelivery and the applications of phenylboronic acids in the biomedical filed. In the nextfour chapters, a series of polymeric carriers modified with phenylboronic acid groupswere designed and synthesized, and physicochemical and biological characteristics ofthe resulting polymers were evaluated.Commercially available Pluronic copolymers have been used as pharmaceutical excipients for a long time. In chapter2, we synthesized a block copolymer(Pluronic-PG-BA) modified with phenylboronic acid groups as a nonviral and neutralgene vector. Pluronic-PG-BA was prepared via the reaction of polyglycidol-b-poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide)-b-polyglycidol(Pluronic-PG) with2-(N,N-dimethylaminomethyl)-5-aminomethyl phenylboronic acidusing phosgene as a coupling reagent. This boronic acid-modified non-cationic polymershowed excellent biocompability and lower cytoxicity than25KDa PEI. It boundplasmid pGL3effectively, formed submicron polymer/DNA complex particles, andgreatly facilitated the cell uptake of the plasmid. Efficiency of the polymer as a genevector was evaluated in vitro by transfection of pGL3to HeLa, COS-7, and HepG2cells. Pluronic-PG-BA enhanced the transfection efficiency by100to1000timescompared with Pluronic-PG. Notably, the presence of serum did not affect thetransfection efficiency significantly.Polyethylenimine (PEI) has been widely used for nonviral transfection in vitro andin vivo. In chapter3, two derivatives (PEI1800-o-BA and PEI1800-p-BA) of PEI1800modified with different types of phenylboronic acid groups were designed,synthesized and investigated for use as gene vectors. PEI1800-o-BA and PEI1800-p-BAwere prepared by the reaction of1800Da PEI with4-(bromomethyl)phenylboronricacid or2-(bromomethyl)phenylboronric acid, respectively. The two polymers couldbind DNA well when the N/P ratio was1. They showed much lower cytoxicity than25KDa PEI. MTT assay indicated that PEI1800-p-BA had lower cytoxicity thanPEI1800-o-BA in HeLa cells, and higher cytoxicity in HepG2cells. Efficiency of thePEI1800-o-BA and PEI1800-p-BA as gene vectors was evaluated in vitro by transfectionof pGL3to HeLa and HepG2cells. Their efficiency was about one order ofmagnitude higher than that of25KDa PEI. The transfection efficiency of PEI1800-o-BAwas much higher than that of PEI1800-p-BA in HeLa cells but lower in HepG2cells,showing a cell selectivity.The transfection efficiency of low molecular weight PEI could be enhanced byincorporated long-chain alkyl groups. In chapter4, a series of phenylboronicacid-modified dodecanyl PEI800(Dod_x-PEI800-BA_y) were synthesized and their characteristics for gene delivery were studied. The results of agarose gel electrophoresisshowed that Dod_x-PEI800-BA_ycould bind DNA better with the increasing contents ofdodecanyl and phenylboronic acid groups. Particle size and zeta potential ofDod_x-PEI800-BA_y/pGL3polyplexes were measured by DLS. The results showed thatparticle sizes ranged from several dozens to300nm and the zeta potentials owner in therange of20-25mV. MTT assay indicated that these polymers had lower cytoxicity than25KDa PEI and the cytoxicity would be enhanced by the increasing of the dodecanylgroups. Efficiency of Dod_x-PEI800-BA_yas gene vectors was evaluated in vitro bytransfection of pGL3to HeLa and HepG2cells. The resluts showed that incorporationof phenylboronic acid groups could enhance the efficiency of transfection.In chapter5, a series of degradable polyethylenimine modified with4-aminomethylphenylboric acid groups (BA_y-PEI800-SSX) were designed, synthesized andcharacterized. BA_y-PEI800-SSXshowed lower cytoxicity in HeLa cells than25KDa PEI.They were able to condense negatively charged DNA based on the electrostasticinteraction to form polymer/DNA polyplexes. The particle sizes of the polyplexesmeasured by DSL were about400nm in150mM NaCl, and the zeta potentials wereabout25mV. BA_y-PEI800-SSXcould deliver foreign pDNA containing luciferase geneinto HeLa and HepG2cells effectively, about one order higher than25KDa PEI ingene expression. Among the polymers with various amount of disulfide cross-linkingbonds and phenylboronic acid groups, BA0.25-PEI800-SS1.63had the highest efficiency.