Fabrication Of Bionic Nitric Oxide-driven Nanomotors And Their Application In Cancer Therapy

At present,most of the chemical micro/nanomotors have some problems such as toxic fuel or exhaust gas and waste generated in the process of movement,which limit their application in biomedical field.In this paper,based on the concept of bionics,we propose to design a kind of nitric oxide（NO）-driven nanomotors with good biocompatibility,and apply them to the research of cancer treatment.The main contents are as follows:（1）Based on the poor biocompatibility of chemical micro/nanomotors,inspired by the endogenous biochemical reaction of human body,that is,L-arginine reacts with reactive oxygen species（ROS）to produce NO and L-citrulline,the concept of zero-waste chemical nanomotors was proposed for the first time.A novel zero-waste NO-driven nanomotor was constructed by combining hyperbranched polyamide with L-arginine through weak electrostatic binding,and driven by NO gas generated by ROS reacts with L-arginine.With the consumption of L-arginine of the nanomotor in an aqueous hydrogen peroxide（H₂O₂）solution,the production of NO can reach 120μM.The Nanomotors with different L-arginine loadings can be driven by bubbles to achieve continuous motion in the simulated ROS solution.Then,when the nanomotors were placed in the environment of cancer cells with high ROS concentration,and the continuous autonomous motion behavior of the nanomotors was also observed.On this basis,the possible construction mechanism of nanomotors was investigated,and it was found that the method based on weak electrostatic force combining positively charged amino-rich substances with negatively charged carboxyl groups in L-arginine was universal,which will provide a new idea for the design of biosafety nanomotors that can be truly applied in human body.（2）In order to meet the challenges of poor penetration of therapeutic agents and multidrug resistance（MDR）in cancer chemotherapy,a novel kind of NO-driven nanomotors（HFLA-DOX）was constructed using heparin folate nanoparticles（HF）with cage-like structure and tumor-targeting properties as carrier,loaded with L-arginine and chemotherapeutic drug adriamycin（DOX）.The targeting efficiency of the nanomotors to tumors was improved by the specific recognition of overexpressed folate receptors on the surface of tumor cells by HF,and the nanomotor were successfully applied to cancer chemotherapy.The nanomotors exhibited excellent NO and DOX drug release performance,and showed significant self-driven behavior in both two-dimensional（2D）planar cells and three-dimensional multicellular tumor spheres（3D MTSs）environments.Then,the motion effect of nanomotor in the process of blood circulation to tumor tissues infiltration was systematically evaluated,including the processes of target recognition,vascular penetration,fate of reaching tumor cells（cellular uptake pathway,intracellular distribution,and intercellular transport）,and tumor infiltration.In particular,in this process,the versatility of NO,including NO as the driving force of nanomotors,degradation of cytoplasmic matrix（ECM）,reversal of MDR,and anticancer effect,was fully explored.Based on the synergistic effect of the movement performance and multifunctionality of the nanomotor,a new mode of chemotherapy based on“recognition-penetration-reversal-elimination”was proposed.The DOX resistant subcutaneous breast cancer model in mice confirmed that the HFLA-DOX nanomotors can provide experimental basis for solving the problems of drug penetration barrier and tumor MDR in current tumor treatment methods.（3）In order to solve the problem of poor infiltration of T cells in tumor sites and inhibition of T cell killing activity in cancer immunotherapy,a kind of NO-driven nanomotors was designed,which can move autonomously in the tumor microenvironment and image in vivo.To achieve in vivo imaging,heparin folic acid（HFC）covalently modified with fluorescent molecules（cy5.5）was used as a carrier.The HFCA/DTX/a PD1 nanomotors were then prepared by the aboved carrier electrostatically combining L-arginine and loading the chemotherapeutic drug Docetaxel（DTX）and immune checkpoint inhibitor（a PD1）for realizing the combination of chemotherapy and immunotherapy.The introduction of cy5.5 makes the nanomotors have good fluorescence imaging performance in vitro and in vivo,which is conducive to their tracking observation.By assessing the mean tumor vascular density and the number of functional tumor vessels perfused into the tumor of mice,t was proved that the NO produced by this kind of nanomotors can significantly improve the abnormal tumor vessels.Experimental results showed that the drug-loaded nanomotors with the ability to release NO and motility showed a significant increase in vivo tumor infiltration ability.The infiltration efficiency of T cell in tumor tissue in vivo was increased from 2.1%to 28.2%,and the indicators of T cell function,TNF-αand IFN-γ,were increased to 3.7%and 1.9%,respectively.Finally,the excellent in vivo antitumor performance of the drug-loaded nanomotors were confirmed by subcutaneous melanoma model and intraperitoneal implanted gastric cancer tumor model,respectively.Therefore,a novel immunochemotherapy mechanism based on NO-driven nanomotor loaded with chemotherapy and immune checkpoint drugs is proposed,which is"tumor recognition-self-infiltration-tumor vascular normalization/ECM degradation-T cell infiltration-T cell activation-tumor killing”,which provides a new idea for cancer immunochemotherapy.