Modulation Of Dendrimers Gene Delivery Efficiency Via Hydrogen Bonding Strategy And Its Biomedical Applications

Dendrimer is a polymeric gene vector characterized by surface primary amines and internal tertiary amines. Surface primary amines can condense nucleic acid into nano-sized polyplexes. Internal tertiary amines with pH buffering ability enable successful endosomal escape. However, dendrimers only exhibit low gene delivery efficiency and serious cytotoxicity caused by large amounts of primary amines. Many researches show that surface functionalization is capable of improving the transfection efficiency or reducing the cytotoxicity of dendrimers. These surface functionalizations mainly work through modulation of physicochemical properties of dendrimers, for example, amino acids for surface charge density and endosomal escape, fluorination for self-assembly, lipid for hydrophilic-hydrophobic balance, and glycosylation and protein for cell targeting. But, transfection efficiency and biocompatibility of these surface-functionalized dendrimers still cannot meet the need of clinical therapy. Neotype dendrimer based gene vectors with high efficiency, low cytotoxicity and potential for therapeutical use become the focus of research.This thesis proposes a new strategy for surface functionalization. A serious dendrimers are modified with 2,4-diamino-1,3,5-triazine (DAT) moieties. These hydrogen bonding (H-bond) moieties are able to modulate transfection efficiency of surface-engineered dendrimers through H-bond interactions. DAT moieties (1) form multiple hydrogen bonds with nucleobases of DNA strand. By tailoring the electrostatic and H-bond interactions, DAT-functionalized dendrimers are able to condense DNA and form polyplexes with a favored size for gene delivery; (b) form supramolecular gene vector through H-bond assembly between DAT-functionalized low generation (G3) dendrimer and cyanuric acid (CyA). The detailed methods and results are summarized as follows:(1) Dendrimers with H-bond donors/acceptors are synthesized by surface modification with DAT moieties. DAT is able to form multiple hydrogen bonds with nucleobases of DNA strand. Meanwhile, the rest of primary amines bind DNA by electrostatic interactions. The synergistic effect between H-bond and electrostatic interactions results in well-characterized polyplexes, which are stable in water and prevent DNA from degradation. Through the enhanced cellular uptake of polyplexes, DAT-functionalized dendrimers show very high gene transfection efficiency in several cell lines, such as HEK293, COS-7, CHO and HeLa cells, even better than commercial reagents, such as bPEI 25KD and Lipofectamine 2000. DAT-functionalized dendrimers also show excellent serum resistance, good cell penetration, decreased cytotoxicity and low hemolysis. These outcomes lay the foundation in the field of clinical therapy. Furthermore, dendrimers functionalized with other H-bond ligand, such as nucleobase derivatives, and other cationic polymers functionalized with DAT moieties show greatly increased gene delivery efficiency, indicating that this H-bond strategy can be widely applied in the design of neotype gene vectors.(2) High gene delivery efficiency of DAT-functionalized dendrimers have been confirmed in the previous section. Here, these materials are used as siRNA vectors for gene therapy. Oncoprotein mouse double minute 2 (MDM2) protein is amplied in non-small cell lung cancer cell line PC-9. MDM2 can be considered as RNA interference target for gene therapy. The interference of MDM2 will lead to suppression of MDM2 expression, activation of p53 pathway and induction of PC-9 apoptosis. The transfection polyplexes formed between DAT-functionalized dendrimers and MDM2 siRNA are able to efficiently silence the expression of MDM2 protein and induce the apoptosis of PC-9 in vitro. The interference effect is even better than that of commercial transfection reagent, Lipofectamine 2000. Moreover, the delivery of MDM2 siRNA by DAT-functionalized dendrimers is able to effectively inhibit the growth of PC-9 subcutaneous tumor in a nude mice model. These resluts indicates that DAT-functionalized dendrimers are gene vectors for not only DNA but also siRNA with high efficiency.(3) There is a paradox for traditional cationic polymers that high transfection efficiency of polymers with large molecular weight are accompanied by serious cytotoxicity. Cationic vectors covalently assembling through biodegradable cross-linkers (such as disulfide linkers) have high efficiency and low cytotoxicity. However, this solution is faced with instability and over crosslinking, which put forward higher demand to synthesis, storage and consistency from batch to batch. To solve this problem, a H-bond assembly strategy is adopted to construct gene vector in a non-covalent way. DAT-functionalized low generation (G3) dendrimers and CyA can form a supramolecular assembly through multiple H-bond recognition. The H-bond interactions between DAT and CyA are able to help with condensing DNA and forming compact polyplexes; to increase the stability of polyplexes; to protect polyplexes with high stability against DNase; and to enhance the cellular uptake of polyplexes. The supramolecular polyplexes show high gene delivery efficiency in HEK293 and COS-7 cells, which are comparable to bPEI 25KD. The introduced CyA molecules do not cause extra cytotoxicity. In addition, the unimproved cellular uptake and the loss of transfection efficiency through the mono H-bond recognition between CyA and 4,6-dimethoxy-1,3,5-triazine (DMT) indicate that the multiple H-bond recognition is essential to the construction of stable supramolecular gene vector with high transfection efficiency. This H-bond assembly strategy with high feasibility breaks up the paradox of cationic polymers and achieves the goal of high efficiency and low cytotoxicity using low generation dendrimers. More importantly, this strategy offers a promising solution to the development of novel gene vectors.In summary, high gene delivery efficiency and low cytotoxicity can be achieved through multiple H-bond recognition, which is able to modulate the formation of polyplexes between surface-engineered dendrimers and nucleic acids or to modulate the assembly of surface-engineered dendrimers. This strategy can be considered as a new surface modification on dendrimer to enhance gene delivery efficiency. These surface-engineered dendrimers can be used for gene therapy with high efficiency.