Fabrication Of Multifunctional Dendrimers By Amino Acid Modification For Gene Delivery

Gene therapy remains a promising strategy in the treatment of hereditary and cancer diseases in recent years. However, the largest obstacle to the development of gene therapy is the lack of efficient and nontoxic gene vectors. Although viral vectors showed higher gene transfection capability, the immunogenicity and safety issues of viral vectors limits their clinical uses. Therefore, the development of effective non-viral gene vectors has attracted more and more attentions during these years.Dendrimers are one of the most widely investigated non-viral vectors due to their unique molecular structures. However, dendrimer-based gene vectors are usually associated relatively low transfection efficiency and high cytotoxicity. To improve the performance of dendrimers in gene transfection, dendrimers have bene modified with several ligands. These surface-engineered dendrimers are usually single-functionalized. It is difficulty to reveal the structuer-function relation of these surface-engineered dendrimers. In addition, there are multiple barriers in the gene transfection process, and single-functionalized dendrimers cannot overcome all the barriers for efficient gene delivery. Hence, there is an urgent need to systematically investigate the functions of modified ligands in gene delivery and to design multifunctional dendrimers for efficient gene delivery.The content contains two parts in this dissertation:(1) The structure and function relationships of amino acids modified dendirmers in gene delivery; (2) Fabrication of multifunctional dendrimers using amino acid modification for efficient gene delivery, and investigate the synergistic effect of different amino acids in gene delivery. The dissertation is comprised of five chapters.In Chapter one, we summarize the history of gene transfection and gene therapy, and gave a briefly introduction of frequently-used transfection methods. The applications of cationic polymers in gene delivery and their transfection mechanisms are described. Specifically, dendrimers and their application in gene delivery and therapy are introduced. Finally, the design concepts and major contents of the thesis are given.In Chapter two,20 kinds of native amino acid modified dendrimers were synthesized and characterized. These amino acid modified dendrimers are divided into four groups, including cationic, anionic, hydrophobic and hydrophilic amino acid modified dendrimers. The relationship between the side group structure of amino acids and the gene transfection efficiency of related materials are systematically investigated. The results indicate that cationic and hydrophobic amino acid modified dendrimers shows higher gene transfection efficiency. Arginine (Arg) and lysine (Lys) modifiction increases the surface cationic charge density of dendrimer, which enhance the complex stability and cellar uptake; Histidine (His) improves pH-buffering capacity of dendrimers, which promotes endosome escape of complexes. Anionic amino acid modification prevents the complex formation between dendrimers and nucleic acid. Hydrophobic amino acid modification (such as Phenylalanine, Phe) modulates the balance between hydrophobicity and hydrophilicity on dendrimer surface, which enhances the internalization of dendrimer/DNA complexes. On the contrary, hydrophilic amino acid modification decreases cellar uptake of the complexes. These results motivate us to design multifunctional dendrimers by modifying different amino acids for efficient gene delivery.In Chapter three, single-, dual-and triple-functionalized dendrimers with Arg, Phe and His were synthesized using an one-pot strategy, and the synergistic effect of amino acids in gene delivery are investigated. The results suggest that a combination of these amino acids on dendrimer generates a synergistic effect in gene delivery, and increases gene transfection efficiency of single-functional dendrimers on a variety of cells. The triple-functionalized dendrimers such as G5-Arg47Phe24His25 and G5-Arg56Phe22His22 show the highest gene transfection efficiency, and even perform a comparable gene transfection efficacy compared to several commercial transfection reagents such as Lipofectamine 2000, JetPEI and Polyfect. The G5-Arg47Phe24HiS25 also has a high transfection efficacy in vivo. The synergistic effect are caused by the three types of amino acids modified on dendrimer surface, whic play different roles in the gene delivery process. Arg is essential in the formation of stable complexes, Phe improves cellular uptake efficacy, and His increases pH-buffering capacity and minimizes cytotoxicity of the cationic dendrimers. In addition, these amino acids modified multifunctional dendrimers also show minimal cytotoxicity on the transfected cells. The combination of different amino acids on dendrimer can achieve efficient gene delivery with minimal toxicity. These results provide a new strategy in the design of efficient polymer gene vectors.In Chapter four, we propose a facile method to fabricate multifunctional dendrimers for efficient gene delivery. Briefly, different functionalized dendrimers (G5-Phe71, G5-His40 and G5-Arg64) are mixed at required molar ratios, and then DNA is added to form multifunctional nanoparticles. The results suggest that the formed multifunctional nanoparticles are consisted of different functionalized dendrimers, and the size of the prepared complexes is around 200 nm which is suitable for gene delivery. The multifunctional nanoparticles show synergistically increased gene transfection efficiency on HeLa cells, and performed a comparable gene delivery capacity with several commercial transfection reagents. The multifunctional nanoparticles also show minimal cytotoxicity on the transfected cells. The composition of these multifunctional dendrimers can be easily tailored by changing the molar ratios without sophisticated synthesis.In chapter 5, we give the conclusions of this dissertation, and propose the other potential application of the multifunctional dendrimers.