Construction Of Novel Gene Delivery System Based On Gold Nanoparticles And Its Application And Mechanism Studies In Skin Melanoma Treatment In Vitro And In Vivo

Objective:The present study prepared modified gold nanoparticles and investigated their transdermal, transfection and gene regulation efficiency as multifunctional vehicles when carrying microRNA-221 inhibitor for treating skin melanoma both in vitro and in vivo.Methods:1. HAuCl4 was reduced by NaBH4 in the presence of PEI. Then AuPT was prepared by adding TAT into AuP solution under continuous stirring overnight. 2.Characterization of AuPT including morphology, diameters, zeta potential and grafting ratios of PEI and TAT.3. Prepare AuPT/pDNA complexes and screen their optimal weight ratio (AuPT:pDNA, w/w) by evaluating the diameter, effect of condensing pDNA and transfection efficiency.4. Evaluate the endocytosis efficiency and mechanism of AuPT/pDNA.5. Evaluate the gene regulation ability of vector/Mi221 and migration, proliferation and cell cycle changes of tumor cells after gene therapy treatment.6. To conduct a quantitative investigation on transdermal efficiency, different vector/FITC-pDNA were used to treat mice skin in vitro. Skin samples that treated with vector/FITC-pDNA were fixed to glass cover slips and observed by CLSM at 24h to study their distribution in different skin layers.7. Investigate anti-tumor effect and mechanism of different vector/Mi221 complexes in vivo and their distribution in melanoma tissues.Results:AuPT was uniformly dispersed in water solution, with diameters range from 10 nm-20 nm. AuPT could also condense pDNA, protecting it from degration. When incubating concentration of TAT was 25 ?g/mL and the weight ratio between AuPT with pDNA was 1:8, AuPT/pDNA complexes had smallest diameter and highest transfecting efficiency on B16F10. Gene regulation results showed that the expression level of microRNA-221 was significantly downregulated along with upregulation of p27 and c-Kit genes after transfecting Mi221. This gene regulation blocked cell division, decreased the metastasis ability and inhibited proliferation of B16F10 cells. In vitro transdermal assay proved that TAT could enhance the transdermal efficiency of pDNA. The skin penetration route was through skin affiliated organs such as hair follicles and sweat glands, which was observed under CLSM. Finally, topical delivery of AuPT/Mi221 could notably inhibit the growth of skin melanoma in nude mice. By analyzing tumor sections, the division of tumor cells was restrained and a large number of apoptosis and necrosis in tumor tissues were observed.Conclusion:AuPT was shown a highly efficient carrier for the transdermal delivery of pDNA. Along with the robust gene delivery efficiency of AuPT, transdermal delivery of AuPT/Mi221 provided a novel strategy based on topical gene therapy for skin cancer with great clinical potential to reverse the progression and metastasis of advanced melanoma.