Construction And Application Of Dual-targeting DNA Nano-drug-loading Complexes Based On Nucleic Acid Aptamer

Objective In this study,we aimed to develop a kind of dual-targeting DNA na no-drug delivery system,which is composed of CD44 aptamer,nucleolin aptamer a nd DNA tetrahedron that could carry doxorubicin(Dox)within its DNA double stra nds via intercalation.Then,the targeting and efficacy of these drug delivery system s were further evaluated on melanoma A375 cells.Methods(1)DNA nano-drug-loaded complexes were prepared by PCR;(2)Constructed nanostructures were incubated with DOX to obtain DNA nano-drug-loaded complexes with loaded DOX;(3)Serum stability test was performed to evaluate the nuclease degradation resistance;(4)Flow cytometry was performed to confirm the targeting of constructed nanostructures on melanoma A375 cells;(5)CCK8 assay was used to evaluate the ability of inhibiting proliferation of melanoma A375 cells;(6)Flow cytometry was used to detect the effect of constructed nanostructures on cell cycle of melanoma A375 cell.Results(1)Five single-targeting and four dual-targeting DNA nano-drug-loading complexes were successfully assembled in this experiment;(2)Nine targeted DNA nano-drug-loading complexes loaded with DOX were successfully constructed;(3)Serum stability experiments showed that the serum stability of these constructed nanastructures was significantly enhanced;(4)Flow cytometry results showed that the four dual-targeting DNA nano-drug-loading complexes exhibited stronger targeting ability on melanoma A375 cells than the single-targeting DNA nano-drug-loading complex and the negative control group.Among them,the combination of AS1411-1 and Aptamer1 is the best;5)CCK8results showed that compared with single-targeting DNA nano-drug-loading complexes and free doxorubicin control group,four dual-targeting DNA nano-drug-loading complexes demonstrated higher cell inhibiton on melanoma A375 cells in a time-and dose-dependent manner.Moreover,the combination of AS1411-1 and Aptamer1 displayed the strongest inhibition ability;(6)Flow cytometry results showed that compared with the free DOX group,the ability of four dual-targeting drug-loaded complexes to induce G1 phase arrest in melanoma A375 cells was significantly enhanced.Conclusion In this study,four dual-targeting DNA nano-drug delivery systems based on CD44 aptamer and nucleolin aptamer have been successfully constructed.In vitro experiments results showed that,compared with the single-targeting drug-loading complexand the negative control group,the constructed four dual-targeting drug-loading complexes have obvious advantage on resisting nuclease degradation,targeting A375 cells,inhibiting the proliferation of A375 cells and inducing G1 phase arrest of A375 cells.These findings provide new methods and techniques for targeted therapy of tumors.