Application Of DNA Cross Nanostructures Containing PD-L1 Nucleic Acid Aptamers In Anti-tumor Immunotherapy

Objective:Cancer is a leading cause of mortality in the 21st century.In recent years,cancer immunotherapy has become an area of focus of medical oncology.Blocking the PD-1/PD-L1 pathway can diminish immunosuppression and enhance anti-cancer immunity.In addition to the antibody,PD-1/PD-L1 blockade can also be realized by aptamers,which have good biocompatibility and can be synthesized in quantity economically.For in vivo applications,aptamers need to evade renal clearance and nuclease digestion.To date,no report in the literature has evaluated the application of a DNA nanostructure to improve the efficacy of PD-1/PD-L1 blockade by aptamers.Here we investigated whether DNA nanostructures could be used to enhance the function of PD-L1 aptamers.A Holliday Junction(HJ)is a popular DNA nanostructure that can serve as a carrier to deliver drugs,nucleic acids.HJ is a cross DNA nanostructure formed by self-assembly of four single-stranded DNA(ssDNA)through the principle of base complementary pairing.Four PD-L1 aptamers were linked to the 4 end points of the cross structure of HJ to form a tetravalent DNA nanostructure(Apt-HJ).The PD-L1 aptamer was attached to the DNA nanostructure to enhance its size and make its size higher than the renal clearance threshold,thereby prolonging the circulation time of the PD-L1 aptamer in vivo.Apt-HJ had also been partially phosphorothioate modified to improve the resistance of nuclease.In this study,we evaluated whether the tetravalent Apt-HJ can enhance the anti-tumor efficacy in vivo compared with free PD-L1 aptamer.Method:Using the principle of base complementary pairing,four ssDNAs with PD-L1 aptamer at the 3’ ends were self-assembled to form a tetravalent DNA nanostructure(Apt-HJ).Agarose gel electrophoresis was used to verify the successful construction of Apt-HJ,and dynamic light scattering(DLS)was used to measure the particle size and zeta potential of Apt-HJ.Apt-HJ underwent partial phosphorothioate modification,then serum digestion experiment was performed.Agarose gel electrophoresis was used to evaluate the Apt-HJ’s nuclease resistance.Apt-HJ was labeled with FAM,and flow cytometry was used to determine whether the DNA nanostructure(Apt-HJ)could bind to CT26 colon cancer cells,and the affinity of Apt-HJ to CT26 cells was calculated.The laser scanning confocal microscopy was used to further verify whether Apt-HJ could bind to CT26 cells.In vivo experiment was conducted to study whether Apt-HJ could enhance the anti-tumor effect vs.free PD-L1 aptamer.Results:In this study,a new DNA nanostructure(Apt-HJ)comprising four PD-L1 aptamers and a Holliday Junction was constructed to enhance the target affinity and the in vivo antitumor efficacy.Apt-HJ was made of four ssDNA chains which had the PD-L1 aptamer at their 3’ end and the HJ sequence at their 5’ end.These ssDNA chains were assembled into a tetravalent structure via a self-assembly process.The average diameter of Apt-HJ measured by dynamic light scattering was 13.22nm,which was higher than the renal clearance threshold(about 10nm),while the average diameter of free PD-L1 aptamer was 7.14 nm,which was lower than the renal clearance threshold.The zeta potential of Apt-HJ was-11.9 mV.Serum digestion experiments showed that compared with unmodified Apt-HJ,phosphorothioate-modified Apt-HJ significantly enhanced nuclease resistance and serum stability.Flow cytometry and confocal microscopy showed that,compared with the monovalent free PD-L1 aptamer,the tetravalent Apt-HJ showed a higher affinity for CT26 colon cancer cells.Quantitative measurement of affinity showed that the apparent Kd of Apt-HJ to CT26 cells was 145nM,while the Kd of monovalent free PD-L1 aptamer for CT26 cells was 283nM.This result further proved that Apt-HJ had a stronger affinity with the target tumor cells.In vivo animal experiment showed that,compared with free PD-L1 aptamer,Apt-HJ significantly improved the anti-tumor efficacy of tumor-bearing mice,and did not generate extra side effects.Conclusion:In this study,a novel DNA nanostructure(Apt-HJ)comprising four PD-L1 aptamers and a HJ is constructed.The tetravalent Apt-HJ has a higher affinity for CT26 colon cancer cells and improves the anti-tumor efficacy in vivo.The results show that such multivalent DNA complexes may become a new strategy to further enhance the potential of PD-L1 aptamers in anticancer immunotherapy.