Synergistic Treatment Of Tumor-targeted Biotherapy And Chemotherapy Based On Site-specific Anchoring Aptamer On DNA Nanotubes

Objective:The DNA nanotubes with multiple modifiable sites were prepared by using staples DNA and single-strand scaffold DNA?M13mp18?.Modified the aptamer C2NPs onto the DNA nanotubes to build a tumor-targeting drug delivery system,which can enhance the anti-tumor effect of DOX and reduce its toxicity and side effect.Adjust the quantity and spacing of C2NP on DNA nanotube to make DNA nanotubes exert bothchemical therapy and biological therapy after drug loading.Methods:The twisted DNA nanotube?T-DNT?and straight DNA nanotube?S-DNT?were synthesized respectively by M13mp18 single-stranded DNA with the corresponding staples via PCR.Different quantities and spacing of C2NPs were immobilized on DNA nanotubes by complementing with the extended oligonucleotides on nanotubes.The nanotubles were characterized by agarose gel electrophoresis,transmission electron microscope?TEM?and atomic force microscope?AFM?.The drug loading and release in vitro were evaluated by measuring the fluorescence intensity of DOX using plate reader.The stability of nanotubles in 10%FBS was examined by agarose gel electrophoresis.The inhibition rate of K299 cells was measured by CCK8.Laser confocal microscopy and flow cytometry were used to investigate the cellular uptake of drug-loaded nanotubes in K299 cells.The effects of drug-loaded nanotubles on cell apoptosis,cell cycle and reactive oxygen species?ROS?level of K299 cells were examined by flow cytometry.The expression of related proteins in cell signaling pathway were studied by Western Blot.Results:In the present study,DNA nanotubes that targeted lymphoma cells were successfully constructed,and the results of in vitro stability and release showed that the T-DNT could improve the stability of DOX and delay its release.The results of cell experiment showed that the DNA nanotubes and buffer solution had no cytotoxicity to K299.The anti-tumor activity was improved significantly comparing with free DOX.After 4 hours of co-culture,the cellular uptake of DOX by K299 cells was 2.37 times as much as that of free DOX.DNA nanotubes modified with 30 C2NP that was 6 nm to each other showed the highest inhibition rate to K299 cells,and after loading with DOX,the nanotubes showed the effect of biological and chemotherapy on K299 cells at the same time.The nanotubes increased the cell apoptosis rate of K299 cells and mainly by blocking S phase and G₂ phase to inhibit the proliferation of K299 cells.At the same time,the level of reactive oxygen species?ROS?in K299cells was increased.Western Blot assay showed that the expression of p53 protein in K299 cells could be induced by the DNA nanotubes.Conclusion:The C2NP modified DNA nanotubes can precisely target lymphoma cells,which can significantly improve the antitumor effect of DOX in vitro and reduce its toxicity and side effects.The C2NP modified DNA nanotubes can exert chemo-therapeutic and bio-therapeutic effects,which greatly reduces the amount of used drug.Targeted origami carriers and controllable distance and number of ligands provide a new strategy for the precise treatment of cancer.