Layer-by-layer Assembled Redox-sensitive Gold Nanocomplex For Non-viral Gene Delivery

The success of gene therapy is largely dependent on the development of the genedelivery vector. The development of gene carriers for effectively delivering genes intocells has attracted a great deal of attention in recent years. Nanotechnology and itspromise for clinical translation to targeted drug delivery with limited accompanyingtoxicity provide exciting research opportunities that demand multidisciplinaryapproaches.Our work is divided into three parts. Part one entails the design, preparation, andcharacterization of three different gold nanoparticles (AuNPs), they are include PVPprotective AuNPs, DODAB-AuNPs and AuCM. First, AuNPsprovide non-toxiccarriers for drug and gene delivery applications.Recently, many attempts have beenmade to utilize AuNPs as intracellular gene carriers because they have severaladvantages of straightforward synthesis, easy modification of surfaces withthiolatedmolecules, and biocompatibility with cells or tissues. So, it have attractedincreasing attention in a variety of biomedical fields including DNA mismatchdetection, biomolecular sensing, and hyperthermal cancer therapy due to their uniqueand controllable optical properties termed surface plasmon resonance.Second, Cationic lipid is the most promising non-viral carrier nowadays. However,its practical application is still limited due to the low delivery efficiency. Studies haveshown that many kinds of lipoplex underwent a rapid disintegration and the release ofDNA after being exposure to serum or anionic membrane components, which isbelieved to be the main reason for the low delivery efficiency. In light of this view,many efforts have been made toimprove the stability of the complex of cationic lipidwith DNA, such as synthesizing new cationic lipids and searching better liposomeformulation Dimethyldioctadecylammonium bromide (DODAB) is a commerciallyavailable cationic lipid and its interaction with DNA andthe application as genecarriers have been extensively characterized in other works.Third, the AuNPs chemically modified with primary amine groups were provide apositively charged surface could form stable polyelectrolyte complexes throughelectrostatic interactions with negatively charged materials. AuNPs chemicallymodified with primary and quaternary amine moieties were ionically interacted withplasmid DNA, and they exhibited more efficient intracellular delivery than theconventional transfection agents. GSH/glutathione disulfide (GSSG) is the major redox couple in animal cells thatdetermines the anti-oxidative capacity of cells. The significant difference in GSHlevel has rendered GSH-responsive nano-vehicles most appealing for targetedintracellular drug delivery. The second part of the thesis describessynthesis andcharacterization of SS-PEI and POLYTAY polypeptide, which is the most importantcomponent for GSH-responsive nano-vehicles.First, non-toxic low molecular weightpolyethyleneimine (PEI,2000Da) was cross-linked with cystaminebisacrylamide(CBA) to prepare reducible SS-PEI. Second, use of bioactive cationic peptides asgene carriers is limited by instability of their DNA complexes in vivo and by the lossoftheir biological activity due to undesired interactions of their bioactive parts withthe DNA. To overcome the two majorlimitations,biodegradablehigh-molecular-weight form of TAT peptide (POLYTAT) sensitive tocellularredox-potentialgradients was synthesized in this study. The reducible genecarriers may achieve efficient uncoupling of polymer/DNA complexes by thecleavage of disulfide linkages in a reductive intracellular environment.The third part of the thesis, we have focus on the potential use of the layer-by-layer(LbL) technique for preparing thin films around the cysteamine modified goldnanoparticles (AuCM) as a multifunctional and smart gene delivery vehicle, withwhich we have developed a novel chitosan-alginate microgelpreviously. Delivery andprogrammed release of therapeutic materials to specific physiological targets is a keychallenge for molecular and macromolecular therapeutics. To make rapid progress inthe design of nano-platforms for drug delivery and toward their use in the clinic, basicand mechanistic studies must first be tested in vitro and then progress to in vivostudies in animal models, incorporating an understanding of body functioning. Withthese systems, the colloidal gold core imparts stability to the assembly.One of the most important requirements for non-viral gene delivery systems is theability to mediate high levers of gene expression with low cell toxicity. With thissystem, the easily tunable size and monodisperse gold core as a template for theassembly, while the monolayer allows tuning of surface properties like the surfacecharge. Acysteamine-modified gold nanoparticles (AuCM)/pDNA/reducible TATpolypeptide (pTAT)/hyaluronic acid (HA) nanocomplex was successfully developedusing a layer-by-layer (LbL) method for target-specific and controlled responsiveintracellular deliver of different plasmid by HA receptor mediated endocytosis. TheAuCM/pDNA/pTAT/HA nanoparticles had a small size of86.5±4.2nm and a negative zeta potential of-17±4mV, and the layer-by-layer structure of theAuCM/pDNA/pTAT/HA nanoparticles was demonstrated by dynamic light scattering(DLS), transmission electron microscope (TEM), atomic force microscope (AFM)and UV-Vis spectra. It is noteworthy that the AuCM/pDNA/pTAT/HA nanoparticleswere GSH-sensitive, and showed enhanced pDNA transfection efficacy in thepresence of GSH-OEt, with negligible cytotoxicity. Furthermore, theAuCM/pDNA/pTAT/HA nanoparticles showed effective cellular uptake inCD44-overexpressing HepG2cells in a HA/CD44interaction dependent manner. Invivo distribution revealed that the AuCM/pDNA/pTAT/HA nanoparticles showed ahigh accumulation (80%) in the liver.In conclusion, the work demonstrated development of multifunctional gene carrierwhich has incorporated reducible moiety, tumor targeting ligands as well as HA toachieve efficient release of pDNA, and the ability to mediate high levels of geneexpression with low cell toxicity.TheAuCM/pDNA/pTAT/HAnanocomplex may serveas an effective approach for target-specific treatment of various liver diseases.