Preparation And Application Of Dendrimer-based Gene And Drug Carrier With High Efficiency And Low Cytotoxicity

Dendrimers are a class of artificial macromolecules with tree-like structures. These polymers have unique properties such as monodispersity, well-defined nanoscale size and surface functionality, excellent aqueous solubility and biocompatibility. Due to these properties, dendrimers have gained increasing attentions in many fields. Especially in biomedical applications, dendrimers are widely used as gene and drug carriers. However, the relatively low delivery efficacy and severe cytotoxicity of dendrimer-based carriers prevent these polymers from clinical applications. How to prepare dendrimer-based gene or drug carriers with high efficacy and excellent biocompatibility still remains a great challenge in recent years.The aim of this thesis is to develop high efficient and low cytotoxic gene and drug carriers based on dendrimers. The strategies are described as follows:(1) crosslinking low generation dendrimers using redox-sensitive bifunctional crosslinking agents to prepare biodegradable nanoaggregates,(2) fluorination on the surface of low generation dendrimers,(3) using a hydrogen-bond modulation strategy to improve the performance of dendrimers in gene delivery,(4) conjugation of dendrimers with different cyclodextrins to improve their biocompatibility and drug loading efficacy. The detailed methods and results are summarized as follows:(1) Biodegradable nanoaggregates were synthesized by crosslinking low generation dendrimer with disulfide containing bifunctional crosslinking agents and used as gene carriers. The nanoaggregtes can efficiently condense nucleic acids, forming polyplexes around200nm. The polyplexes can be efficiently internalized by cells and the prepared gene materials are degradable after cellular uptake. These materials show superior gene transfection efficacy to low generation and high generation dendrimers, and comparable efficacy to branched polyethylenimine with a molecular weight of25kDa (bPEI25kD) in different cell lines. Furthermore, the cross-linked gene materials exhibit better biocompatibility than high generation dendrimer and bPEI25kD. In some cell lines, the cytotoxicities of crosslinked materials are even lower than low generation dendrimers. Further studies found that the introduction of thiol groups on the surface of low generation dendrimer also improve its transfection efficacy. Besides dendrimers, such a crosslinking strategy can be used to improve the efficacy of other cationic polymers.(2) The surface of low generation dendrimers were modified with fluorous compounds using a facile strategy and the fluorinated low generation dendrimers were used as gene vectors. These fluorinated materials show much higher gene transfection efficacy than unmodified low generation and high generation dendrimers as well as some commercial gene transfection reagents, and is comparable to the most efficient commercial transfection reagent Lipofectamine2000in several cell lines. These materials exhibited much higher transfection efficacy than Lipofectamine2000in a multicellular spheroid model. Even with a low concentration of nucleic acid, the fluorinated materials showed higher transfection efficacy and penetration ability in the multicellular spheroids than Lipofectamine2000with a high concentration of nucleic acid. In addition, the optimized nitrogen to phosphorous (N/P) ratio for the fluorinated materials in gene transfection were significantly reduced with increasing numbers of fluorous ligands modified on the surface of dendrimers. The molecular weights of these fluorinated materials are far below the commonly used polymeric gene vectors. Moreover, these fluorinated materials are able to self-assemble into micelles with sizes of100-200nm in the water, which behave like lipid-based gene vectors. The cytotoxicities of these fluorinated low generation dendrimers are much lower than fluorinated high generation dendrimers as well as bPEI25kD.(3) A series of nucleobase derivatives were conjugated to the surface of cationic dendrimers and the yielding surface-engineered dendrimers were used as gene vectors. The synthesized materials can effectively bind nucleic acid and form stable polyplexes. The gene transfection efficacy of dendrimer is significantly improved after nucleobase modification and is comparable to several commercial transfection reagents. Moreover, these materials exhibit reproducible gene transfection when delivering different nucleic acids. The transfection efficacy of the synthesized materials is correlated with the hydrogen bond formation ability of the modified nucleobase derivatives. The materials that are able to form complementary hydrogen bond with nucleobases in nucleic acid show higher transfection efficacy. In addition, modification of dendrimer surface with nucleobase derivatives does not cause additional cytotoxicity to dendrimer-based gene delivery systems and even reduced the cytotoxicity of unmodified dendrimers. Thus, hydrogen bond modulation can be developed as a new strategy in the preparation of high efficient and low cytotoxic gene vectors.(4) α-, β-and γ-cyclodextrins were conjugated to dendrimer surface and the yielding conjugates were used as drug carriers. The localizations of five guest molecules within the dendrimer-cyclodextrin conjugates were analyzed using a two-dimensional nuclear magnetic resonance technique. The location of guest molecules in dendrimer or cyclodextrin depends on the size, charge Property, hydrophobicity of the guest and the cavity size of the cyclodextrin. Conjugation of cyclodextrins on dendrimers can improve the drug loading capacity, biocompatibility, and cellular uptake ability of dendrimer, and makes more drugs suitable for the dendrimer-based drug delivery systems. The results provide a new strategy in the design of biomaterials for combination therapy.In summary, several new strategies were proposed to improve the efficacy and biocompatibility of cationic dendrimers in this thesis. These studies extend the biomedical applications of dendrimers. We will further evaluate the in vivo efficacy and therapeutic index of the prepared gene and drug carriers in different animal models. Hope these results can promote the clinical applications of dendrimers in a near future.