Efficient Gene Delivery Using β-Cyclodextrin-Modified Dendrimer-entrapped Gold Nanoparticles

Gene therapy as a new technology combines modern medicine and molecular biology, carrying exogenous normal gene into the target cells to correct or compensate the diseases caused by genetic defect and abnormality, so as to achieve the therapeutic purpose. However, the main obstacle of gene therapy is lack of safe and efficient carriers. In gene delivery system, viral vectors and nonviral vectors were mainly used to transfer the genetic materials to the objective position in vivo. Viral vectors were used in clinical gene therapy because of its high transfection efficiency, but its further application has been restricted due to its numerous security risks. On the other hand, nonviral vectors were gaining more and more attetion because of its property of low cytotoxicity, nonimmunogenicity, easy operation and high genetic loading, etc.PAMAM dendrimer is one of the most widely and deeply researched dendrimers used for nonviral vectors, it retains the common properties of dendrimers with its own characteristics. Research has shown that with the raising of PAMAM generation, the cytotoxicity and gene transfection efficiency also increases. The PAMAMs of generation one to four have both lower cytotoxicity and transfection efficiency, while the PAMAMs of generation above five have a high cytotoxicity and significantly improved transfection efficiency. The efficiency of Generation 5(G5) PAMAM enhanced when compared with that of low generations despite the fact it possesses higher cytotoxicity. Nevertheless, owing to the inherent characteristics of highly branched structure, abundant surface groups and internal cavity structure of G5 PAMAM, we can further reduce the cytotoxicity and improve its transfection efficiency by surface modification or entraping nanoparticles in its interior. Therefore, G5 PAMAM dendrimer was applied as a gene delivery vector, in spite of its high cytotoxicity and relatively low gene transfection efficiency.In order to improve its property as efficiency gene delivery vector, b-cyclodextrin(β-CD) was grafted to the surface of G5 PAMAM dendrimer and then the dendrimers were used as templates to entrap gold nanoparticles with a dendrimer/Au salt molar ratio of 1:25. In this study, ~1H NMR spectra, UV-vis spectra and transmission electron microscopy(TEM) were utilized to characterize the formed β-CD modified dendrimer-entrapped gold nanoparticles(Au DENPs-β-CD). The DNA compaction ability of the formed Au DENPs-β-CD with different N/P ratios was determined by gel retardation assay, zeta potential measurements and dynamic light scattering. The cytocompatibility of the Au DENPs-β-CD was detected by MTT assay.Then, the gene delivery applications of the obtained Au DENPs-β-CD were tested by using them as an effective vector to condense two different pDNAs encoding luciferase(Luc) and enhanced green fluorescent protein(EGFP) to a model cancer cell line(293T cells), respectively. Besides, two different siRNA, Bcl-2 and VEGF, were also transfected into another model cancer cell line(U87MG cells).Our results showed that the Au DENPs-β-CD can compact the pDNA at an N/P ratio of 0.5, compact the siRNA at an N/P ratio of 2, possesses less cytotoxicity and enables highly efficient gene delivery to cancer cells than Au DENPs without β-CD conjugation. With the excellent pDNA compression capability, low cytotoxicity and high gene transfection efficiency, Au DENPs-β-CD are expected to be used as a novel, safe and effective non-viral gene delivery vector for gene therapy applications.