Functionalized Manganese Dioxide Nanozyme As Nucleic Acid Drugs Carriers For Synergistic Tumor Therapy

Maligant tumors are serious threat to people’s physical and mental health.Although there are many tumor therpies developed,the treatments of tumors have gradually developed from single therapy to combination of multi-therapy.Nucleic acid drugs can play a role of immunotherapy and gene therapy in tumor therpy,but there are some defects such as low cell uptake rate and easy inactivation when applied.Safe and efficient delivery carriers are the key to nucleic acid drugs to play a therapeutic role.Nanozymes,as nanomaterials with enzyme-like activity,can be easily modified and prepared as the carriers of drugs.In addition,nanozymes can exert the efficacy of chemodynamic therapy by using the conditions and substances in tumor microenvironment.Now there are few reports on the delivery of nucleic acid drugs using nanozyme carriers for tumor therapy.In order to improve the therapeutic effect of tumors,a functional manganese dioxide nanozyme as a drug carrier was constructed in this paper,and CpG oligonucleotides（CpG ODNs）and antisense oligonucleotides（ASOs）were used as drugs.The effect of nucleic acid drug delivery and the synergistic anti-tumor effect of the nanozyme/nucleic acid drugs delivery system were explored.It mainly includes the following three parts:（1）Firstly,highly positive NH₂-glycogen（NH₂-Gly）and bovine serum albumin modified manganese dioxide（BSA-MnO₂）were synthesized,and NH₂-Gly-BSA-MnO₂ nanozyme was prepared by chemical crosslinking of NH₂-Gly and BSA-MnO₂.The nanozyme had high positive and was suitable for electrostatic adsorption of nucleic acid drugs.In vitro enzyme activity experiments showed that NH₂-Gly-BSA-MnO₂ nanozyme had glutathione oxidase（GSH-OXD）activity and peroxidase（POD）activity.In vitro cytotoxicity experiments showed that NH₂-Gly-BSA-MnO₂ nanozyme had good cytocompatibility with normal cells.On the contrary,it could inhibit the proliferation of tumor cells（B16 cells and HepG2 cells）.At the same time,NH₂-Gly-BSA-MnO₂ nanozyme also had good blood compatibility.（2）NH₂-Gly-BSA-MnO₂/CpG ODNs were successfully prepared by electrostatic adsorption,and NH₂-Gly-BSA-MnO₂ had a high loading capacity of CpG ODNs,which was about 95μg/mg.NH₂-Gly-BSA-MnO₂/CpG ODNs were mainly taken up by RAW264.7 cells through clathrin-mediated endocytosis pathway.NH₂-Gly-BSA-MnO₂/CpG ODNs could stimulate immune cells to secrete a large number of cytokines both in vivo and in vitro.It had strong immunostimulating activity.In vitro anti-tumor experiments showed that NH₂-Gly-BSA-MnO₂/CpG ODNs still had GSH-OXD and POD activities,and had good growth inhibition effect on B16 cells and HepG2 cells.NH₂-Gly-BSA-MnO₂ and NH₂-Gly-BSA-MnO₂/CpG ODNs had good antitumor effect in tumor bearing mouse model,indicating great potential for clinical anti-tumor therapy of NH₂-Gly-BSA-MnO₂/CpG ODNs.（3）By loading NH₂-Gly-BSA-MnO₂ with Smyd3-ASO nanozyme through electrostatic interaction,the nucleic acid drug delivery system,NH₂-Gly-BSA-MnO₂/Smyd3-ASO,was successfully prepared.NH₂-Gly-BSA-MnO₂ also had a high loading capacity of Smyd3-ASO,which was about 98μg/mg.The results of cell experiments showed that NH₂-Gly-BSA-MnO₂/Smyd3-ASO,which was mainly taken up by HepG2 cells through macropinocytosis pathway,could effectively inhibit the proliferation of HepG2 cells.These results indicate that nucleic acid drugs delivery system based on NH₂-Gly-BSA-MnO₂ nanozyme can provide a new direction for tumor combination therapy.