An Autonomous Tumor-targeted Nanoprodrug For Reactive Oxygen Species-activatable Dual-cytochrome C/doxorubicin Antitumor Therapy

In recent years,nanotechnology provides a new way to deliver chemotherapy drugs to tumor tissues with high efficiency and specificity.Protein therapy is a new treatment method which shows a wide application prospect in cancer treatment.However,clinical application of protein therapy has a series of persistent problems.For example,protein is highly unstable and easily deactivation under physiological conditions.It is affected by many factors including chemical factors,changes in pH,temperature,enzyme degradation,etc..The denatured proteins can result in some immune responses and/or adverse effects on the body.How to transport the active proteins to target site is a difficult problem that scientists need to solve.Therefore,a combination of chemotherapy and protein therapy will have important clinical implications.The main contents of this study include: yolk-shell mesoporous silica nanoparticles were synthesized by hexadecyl trimethyl ammonium bromide(CTAB)as template through hydrothermal method.Reactive oxygen species(ROS)-activated YMSN-NBC-Cyt c-NBC-LA nanocomposite particles were prepared though amination and construction of boronic ester-modified Cyt c on it surface by functionalizing again with lactobionic acid.The key feature in this design is that by taking advantage of the boronic ester linkage,the cytotoxicity of Cyt c capped on the nanoparticle could be temporarily deactivated during blood transportation and rapidly restored upon exposure to the ROS-rich microenvironment within liver cancer cells,thereby simultaneously achieving the protein therapy and stimuli-responsive doxorubicin release.(1)The morphology,structure and preparation of nanocomposite particles were characterized by transmission electron microscope,scanning electron microscope,surface area porosity analyzer,fourier transform infrared spectrum,thermogravimetric analysis and Zeta potential analyzer.The loading and controlling behavior of drug/protein was studied in vitro.It could provide an ideal platform for ROS-trigger drug delivery system.(2)The anticancer efficiency of YMSN-NBC-Cyt c-NBC-LA nanocomposite nanoparticles were studied in vitro.Anticancer efficiency in vitro was investigated through cytotoxicity experiment(MTT),endocytosis experiment of nanoparticles(confocal microscope and flow cytometry instrument),targeting experiment(confocal microscope and flow cytometry instrument)and apoptosis experiment(confocal microscope and flow cytometry and western blot).(3)The anticancer efficiency of YMSN-NBC-Cyt c-NBC-LA nanocomposite nanoparticles were studied in vivo.Anticancer efficiency in vivo was evaluated by One-step TUNEL detection kit apoptosis staining,hematoxylin and eosin staining(H&E)about morphological changes and apoptosis of tumor tissue.The results showed that the Cyt c/DOX nano-drug delivery system with reactive oxygen response was successfully prepared,which could effectively protect the bioactivity of Cyt c.Both in vitro and in vivo biological evaluation showed that the system had good biological compatibility in normal cells and strong tumor suppression effect on the HepG2 cells.It could provide certain reference basis for the protein therapy in cancer.