Host-guest Assembly As A Novel Strategy To Construct Stimulus-responsive Gene Delivery Systems

Gene therapy as one of the most exciting areas brings great hope to cure many critical diseases. Developing gene vectors with extracellular stability, intracellular responsive disassembly and efficient transfection attract great interest of researchers. Moreover, higher quality of life puts forward higher requirements of early diagnosis of the diseases. Therefore, many researchers aspired to develop multifunctional nanocarriers combining diagnostic detection agents and therapeutic payloads. In this thesis, host-guest assembly were used as novel strategy to develop novel responsive nanoacrriers for DNA delivery. The relationship between the structure of the polyplexes or multifunctional tags and delivery efficiency were further investigated.Problematic challenge between excellent extracellular stability and easy intracellular gene release capability are still major limitation for non-viral gene vectors. In this study, host-guest interactions were exploited to develop light-responsive PEG-detachable gene vector.β-CD modified with PEI (PEI-CD) and azobenzene-conjugated with PEG (Az-PEG) were separately synthesized. It was found that CD grafting-levels showed significant influence on the DNA condensation ability and PEI-CD15 can condensed DNA into small nanoparticles with size below 120 nm. In vitro experiments by human kidney epithelial cells (HEK293T) suggested that CD modification improved the cellular uptake and gene transfection efficiency. Based on CD/Az host-guest interactions, PEI-CD15/Az-PEG was further prepared and could condense DNA ito PEI-CD15/Az-PEG/DNA polyplexes with particle size of around 110 nm. PEG shells significantly decreased the ζ-pontential of polyplexes and equipped the polyplexes with excellent physiological salt stability and competition stability gainst anionic polymers. Gel electrophoresis and ζ-potential mesearuements suggested that light irradiation (λ=365 nm) made Az/CD inclusion complexes disassembly and PEG shell detachment. Furthermore, the 365 nm light irradiation had little influence on the cell viability and significantly improved the gene transfection of PEGylated polyplexes. The reason was ascribed to the light-induced PEG-detachment caused by the photoisomerization of Az, which was believed to facilitate DNA release and the nuclei entry. The supramolecular system offered a novel and straightforward strategy for design of responsive gene vectors.Multifunctional nanoparticles combing diagnostic detection agents and therapeutic payloads will be the trend of future development for their exciting applications. Modular assembly based on host-guest interaction was exploited to construct the multifunctional nanoparticles for targeting, imaging and responsive delivery. Branched polyethylenimine (PEI) was selected as a scaffold polymer and b-CDs were conjugated as host units. Galactose (Gal) was conjugated on azobenzene-modified polyethylene glycol (Az-PEG-Gal). Fluorescein isothiocyanate (FITC) was selected as a fluorescence imaging probe and conjugated on adamantane (AD-FITC). Subsequently, the supramolecular polymer AD-FITC/PEI-CD9/Az-PEG-Gal with multi-functions was easily constructed by host-guest assembly and designated as SPG-Gal, which could condense DNA into nanoparticles with size of below 100 nm and ζ-potential of +6mV. In addition, the polyplexes showed excellent physiological salt stability and competition stability gainst anionic polymers. In vitro experiments by human hepatoma cells (HepG2) showed that the incorporation of FITC can be used to trace the distribiution of the multifunctional nanoparticles. Specific recognition promoted the multifunctional nanoparticles accumulate at the disease site, which facilitated noninvasive bioimaging. The light-responsive CD/Az host-guest interaction facilitated the intracellular DNA release and nuclei entry of DNA, resulting in imporved trasnfection. Therefore, the host-guest assembly acted as a flexible, universal platform to construct multifunctional nanoparticles that physically incorporated targeted imaging and controllable gene delivery into one package.The above PEI-based delivery vector has some disadvantages, such as lack of biological degradation, cell toxicity, etc., which limits its application in clinical treatment. Polysaccharides show great potential of application in non-viral gene vector due to their excellent biocompatibility. Octa-arginine as cell penetrating peptides (CPP) was firstly conjugated with α-cyclodextrin (CDR) and dextran (MW 40 000) was modified with azobenzene (Az-I-Dex) by acid-labile imine bonds. The dextran shell-detachable polyplexes CDR/Az-I-Dex/DNA polyplexes were prepared based on the host-guest interaction. The the molar ratio of CD to Az (C/A) significantly influenced the particle sizes of the polyplexes. It was found that the polpylexes had smaller particle sizes when C/A ratios were at 0.4 and 0.7, respectively. In addition, dextran with high molecular weight improved the stability under physiological salt condition and the imine bonds equipped the polyplexes with pH-sensitivity. In vitro experiment by HEK293T and Cos7 cells suggested that the supramolecular polycation CDR/Az-I-Dex still had over 80% cellular viability even when the concentration of CDR increased to 100μg/mL, indicating excellent biocompatibility. R8 significantly improved the uptake efficiency of the polyplexes. Furthermore, CDR/Az-I-Dex/DNA polyplexes had 6～300 times higher transfection efficiency than CDR/DNA polyplexes, which was comparable to that of high molecular weight polylysine (PLL)-based polyplexes. We belive that the strategy combing Az/CD host-guest interactions and acid-labile imine bonds provides a novel idea for design of non-viral vectors.