Polyplex-Stability Enhancement by Hydrophobic Modification of PAM-DET Dendrimer and Dermal Gene Delivery Application of PAMAM-Arg Dendrimer

Abstract

Various medical and pharmacological studies have been conducted to identify nano-scale therapeutic methods, and nano-sized therapy via gene-delivery systems has gained attention over the last 25 years. In this regard, gene-delivery carriers that show remarkable properties and outstanding gene-delivery potential have been studied, and several forms of these carriers have been developed, including liposomes, nano-particles, and gene-polymer complexes. Dendrimers, a group of nonviral gene-delivery carriers, are polymers with highly branched structures, globular shapes, well-defined molecular weights, and functionalized end groups. A number of modifications and functionalizations of dendrimers have been attempted by many researchers in order to solve the general problem associated with these structures, i.e., the high cytotoxicity of polymers such as the 25-kDa poly(ethylenimine) (PEI) and polyamidoamine dendrimer (PAMAM). As a result of these attempts, PAMAM-R and PAM-DET, which are respectively arginine- and diethyltriamine (DETA)-modified PAMAM dendrimers, were synthesized by our group and have shown high transfection efficiency and low cytotoxicity. PAMAM-R is a noble gene delivery carrier due to its in vivo application, the wound healing of diabetic mice. It had good ability to deliver the gene which can express growth factors into the cell of wound mouse-skin tissue. From inspiration of former successes like this, I attempted to apply PAMAM-R to in vivo experiment for dermal system and think this application is another appropriate approach for gene therapy. From the results, I knew which substructure in dermal tissue of hairless mouse PAMAM-R G4 can specifically deliver a target gene. PAM-DET is also a well-developed carrier which accelerates the collapse membrane of endosom. But the unstable polyplex which is a complex of PAM-DET with a gene in aqueous solution with salt concentration could cause bad gene delivery efficiency. So, I tried to modify PAM-DET to sustain the stability of its polyplex for better transfaction effect. My main idea of the modification of PAM-DET was based on the conjugation of several moieties which are both hydrophobic and biocompatible on the primary amine of PAM-DET surface. The mechanisms of the conjugations were mainly the formation of amide or carbamate bond, and the linkage using of ring-opening reagent, Trauts reagent. In the final analysis of stability and the results from transfection of the polyplex, the modificated PAM-EDTs showed their enhanced stability at the state of the polyplex with a target gene in hydrophilic environment. In the first part, I tried to conjugate the nontoxic and hydrophobic groups deoxycholate (DC), dimethyl-beta-cyclodextrin (DMβCD), and dexamethasone (DX) with PAM-DET and synthesized PAM-DET-DC PAM-DET-DMβCD, and PAM-DET-DX. In comparison with PAM-DET, all hydrophobic group-conjugated PAM-DET dendrimers showed polyplexes (gene-polymer complexes) with better and longer size-stability in aqueous solutions with high ion strengths. The modified PAM-DET gene carriers showed higher transfection efficiency than PAM-DET and cytotoxicity levels equivalent to that shown by PAM-DET in HeLa and U87MG Cell lines. In second part, I attempted to deliver a green fluorescent protein (GFP) gene into animal dermal tissue by using its polyplex with PAMAM-R, a gene-delivery carrier that has shown a high transfection efficiency and low cytotoxicity in our studies. Using green fluorescence images and a western blot assay, I showed the potential capability of PAMAM-R to act as a gene-delivery carrier to sub-organisms in the skin.