The Preparation Of Multifunctional Polysaccharide-Based Gene Carriers Via Ring-Opening Reaction

Gene therapy has been attracting extremely attention of the researchers around the world for the treatment of congenital and acquired diseases caused by genetic abnormalities. One of the key factors in gene therapy is the construction of safe and high efficiency gene vectors. Comparing to viral gene vectors, cationic gene vectors have the advantages of high efficiency, non-immunogenic, easily modified structure and large-scale preparation. Along with the diverse claim of MRI/CT imaging, fluorescence imaging, antibacterial, drug delivery and photothermal therapy in clinical application, the construction of multifunctional gene vectors have been becoming more and more significant. Pullulan is a microbial polysaccharide produced by aureobasidium pullulans with the characteristic of good biocompatibility and liver-targetability. Gentamicin, tobramycin and neomycin belong to aminoglycoside antibiotics and produced by the actinomycetes. They have good biocompatibility and antibacterial properties. According to the various superiorities, pullulan and aminoglycoside antibiotics were used for the preparation of low toxicity, efficient multifunctional gene vectors.1. The ATRP initiation sites were firstly introduced onto pullulan backbone to prepare initiator Pullulan-Br. The polymerization of glycidyl methacrylate were initiated by Pullulan-Br to produce PuPGMA. Cationic PuPGEA were prepared by reacting PuPGMA with excess ethanolamine. The cationic nanoparticles (PuPGEA-GdL or PuPGEA-GdW) with MRI functions were produced accordingly by assembling PuPGEA with aminophenylboronic acid-modified Gd-DTPA (GdL) or GdW10O369- (GdW) via the corresponding etherification or electrostatic interaction. The properties of the PuPGEA-GdL and PuPGEA-GdW nanoparticles including cytotoxicity, gene transfection, cellular uptake, and in vitro and in vivo MRI were characterized in details. Such kinds of cationic nanoparticles exhibited lower cell cytotoxicity than 25 kDa PEI and good performances in gene transfection in liver cells. PuPGEA-GdW demonstrated much better MRI abilities. The present design of PuPGEA-based cationic nanoparticles with the liver cell-targeting polysaccharides and MRI contrast agents would shed light on the exploration of multifunctional gene delivery systems.2. The non-cleavable hyperbranched polymers (HP) or cleavable hyperbranched polymers (SS-HP) were prepared according by reacting gentamicin, tobramycin and neomycin with ethylene glycol diglycidyl ether or hydroxyethyl disulfide diglycidyl ether. In order to increase their water-solubility, the reaction systems were end-capped by the use of ethylenediamine. The properties of the HP and SS-HP including cytotoxicity, gene transfection, cellular uptake, hemolysis assay and antibacterial activity were characterized in details. The results indicated as follows:hyperbranched polymer SS-HPN had better gene transfection efficiency and lower cytotocity than that of HPN with the fact that disulfide bond of the SS-HP could be biointerrupted by intracellular GSH. Such kinds of hyperbranched polymer exhibited low cytotoxicity, good biocompatibility and excellent antibacterial activity. Additionally, the neomycin-based SS-HPN and HPN possessed the most efficient gene transfection efficiency and lowest cell toxicity. p53 mediate by HPN and SS-HPN exhibited good anti-tumor activity in C6 glioma model.