Enzyme/inorganic nanoparticle dual-loaded animal protein/plant protein composite nanospheres and their synergistic effect in cancer therapy

Abstract

It is a viable strategy to develop a safer and tumor-specific method by considering the tumor microenvironment to optimize the curative effect and reduce the side effects in cancer treatment. In this study, glucose oxidase (GOx) and Fe₃O₄ nanoparticles were successfully loaded inside regenerated silk fibroin/zein (RSF/zein) nanospheres to obtain dual-loaded Fe₃O₄/GOx@RSF/zein nanospheres. The unique structure of the RSF/zein nanospheres reported in our previous work was favorable to loading sufficient amounts of GOx and Fe₃O₄ nanoparticles in the nanospheres. For Fe₃O₄/GOx@RSF/zein nanospheres, GOx depletes endogenous glucose via an enzyme-catalyzed bioreaction, simultaneously generating plenty of H₂O₂in situ. It was further catalyzed through a Fe₃O₄-mediated Fenton reaction to form highly toxic hydroxyl free radicals (˙OH) in the acidic tumor microenvironment. These two successive reactions made up the combination of starvation therapy and chemodynamic therapy during cancer treatment. The catalytic activity of GOx loaded in the RSF/zein nanospheres is similar to that of the pristine enzyme. It was maintained for more than one month due to the protection of the RSF/zein nanospheres. The methylene blue degradation results confirmed the sequential reaction by GOx and Fe₃O₄ from Fe₃O₄/GOx@RSF/zein nanospheres. The in vitro experiments demonstrated that the Fe₃O₄/GOx@RSF/zein nanospheres entered MCF-7 cells and generated ˙OH free radicals. Therefore, these Fe₃O₄/GOx@RSF/zein nanospheres exhibited a considerable synergistic therapeutic effect. They showed more efficient suppression in cancer cell growth than either single-loaded GOx@RSF/zein or Fe₃O₄@RSF/zein nanospheres, achieving the design goal for the nanospheres. Therefore, the Fe₃O₄/GOx@RSF/zein nanospheres cut off the nutrient supply due to the strong glucose dependence of tumor cells and generated highly toxic ˙OH free radicals in tumor cells, effectively enhancing the anticancer effect and minimizing side effects. Therefore, in future clinical applications, the Fe₃O₄/GOx@RSF/zein nanospheres developed in this study have significant potential for combining starvation and chemodynamic therapy.