Cyclodextrin-Based High-Performance Gene Delivery Vectors

Gene therapy refers to the delivery of nucleic acids (as drugs to regulate the targeted gene expression) to cure diseases, which shows much promise in tackling various human key diseases such ascancers.However, the current challenging task in gene therapy field is to develop safe and efficient nucleic acid delivery vectors. Compared with viral vectors, cationic polymers (polycations) as the major type of nonviralgene delivery vectors possess several advantages, such as low host immunogenicity, chanches for mass production, and vestaile design of polymers. Thus, there has been a lively interest in the development of new polycationic gene vectors. Recently, Atom transfer radical polymerization (ATRP) and supramolecular assembly have been widely employed for thepreparation of novel polymers for biomedical applicayions. The functionalizationof biocompatiable polysaccharides including chitosan,dextran and cyclodextrin (CD) has provided a versatileplatform for the design of new gene vectors.Star cationic polymers have recently attracted considerable attention as non-viral gene carriers because of their dense molecular architecture with moderate flexibility. In Chapter 2, the reducible star vectors (CD-SS-PGEAs) consisting of biocompatiable CD core and disulfide-linked low-molecular-weight polycationic arms were proposed for highly efficient gene delivery. A simple two-step method was first adopted to introduce reduction-sensitive disulfide-linked initiation sites of atom transfer radical polymerization (ATRP) onto CD cores. The disulfide-linked poly(glycidyl methacrylate) (PGMA) arms prepared subsequently via ATRP, were functionalized via the ring-opening reaction with ethanolamine (EA) to produce the cationic EA-functionalized PGMA (PGEA) arms with plentiful nonionic hydroxyl and secondary aminogroups. The cationic PGEA arms can be readily cleavable from the CD cores under intracellular redoxstimuli. Such reducible (biocleavable) star CD-SS-PGEA vectors exhibited good pDNA-condensing ability, low cytotoxicity, and efficient gene transfection in differentcell lines.Supramolecular polymers based on non-covalent interactions possess appropriate association strength and have been increasingly explored as nonviral gene vectors in preclinical studies and clinical trials. The easy supramolecular assembly process facilates preparation and optimization of supramolecularcarriers.Among varieties of supramolecular structures, the easily assembled system based on the molecular recognition of CD and adamantane (Ad) is typical and well-demonstrated. In Chapter 3,a series of novel supramolecular comb cationic polymers (1-PGEA-Ad/CD-PGEAs) were synthesized by assembling an Ad-modified linear PGEA (1-PGEA-Ad) backbone with multiple CD-cored PGEA star polymers (CD-PGEAs) via the host-guest interaction. The comb 1-PGEA-Ad/CD-PGEAs exhibited better plasmid DNA-condensing ability, similarly low cytotoxicity, and much higher gene transfection efficiency (at various PGEA nitrogen/DNA phosphate molar ratios) than their counterparts, CD-PGEA and 1-PGEA.Nevertheless, the advantages of novel supramolecular comb vectors in gene delivery can be further improved by different star polymers and backbones, which can be easily modified to respond to external stimuli such as redox reactions for intracellular gene delivery as intelligent structures. In Chapter 4, different polysaccharide-based supramolecular cationic polymers were readily synthesized by assembling multiple CD-cored star cationic polymers with an Ad-functionalized dextran via host-guest interaction in the absence or presence of bioreducible linkages. Compared with nanoplexes of the starting star cationic polymers and pDNA, the supramolecular cationic polymer/pDNA nanoplexes exhibited similarly low cytotoxicity and significantly higher gene transfection efficiency, which is ascribed to improved cellular internalization. Reducible Dex-SS-Ad/CD-SS-pDM demonstrated better gene delivery properties than corresponding Dex-Ad/CD-pDM, owing to the disulfide linkage-induced intracellular bioreducibility. In addition, in vitro anti-tumor results further confirmed that reducible Dex-SS-Ad/CD-SS-pDM2-mediated suicide gene therapy system possess effective anti-tumour ability.Incorporation of poly(poly(ethylene glycol)ethyl ether methacrylate) (PPEGEEMA) to polycations has been reported to improve gene delivery. However, the mechanism requires further research, as well as the appropriate strategy to incorporate PPEGEEMA. In Chapter 5, a series of supramolecular block cationic polymers (CD-SS-pDM/Ad-pPEGs) were realized by assembling bioreducible CD-cored star poly (2-dimethyl amino)ethyl methacrylate with different molecular weight and an Ad-ended linear PPEGEEMA via the host-guest interaction. The supramolecular block carriers exhibited undiminished pDNAcondensing ability and intracellular bioreducibility, compared with the starting star carriers. Meanwhile, the supramolecular block carriers displayed lower cytotoxicity and higher gene transfection efficiency. Furthermore, serum-free transfection assay, cellular internalization assay and in vivo anti-tumor activity analysis demonstrated that assembled PPEGEEMA could enhance the serum-stability of supramolecular block carriers by inhibiting non-specific absorption of proteins, thus improving the cellular uptake rate and gene transfection efficiency.To sum up, a series of novel cyclodextrin-based cationic polymershave been synthesized through ATRP and supramolecular assembly. In vitro and in vivo experiments demonstrated their high performance as new gene vectors.The current work is of great value for the construction of new gene vectors and wider biomedical applications of new cyclodextrin-based systems.