Targeted self-activating Au-Fe₃O₄ composite nanocatalyst for enhanced precise hepatocellular carcinoma therapy via dual nanozyme-catalyzed cascade reactions

Highlights

• A novel strategy of nanocatalytic tumor precision therapy was devised to improve systemic toxicity, off-target, and non-specificity.

• The in situ coupled Au NPs in ^CD44MMSN/AuNPs was synergically catalyzed Fe₃O₄ NPs to produce more plenty hydroxyl radicals (•OH) in tumor microenvironment.

• The ^CD44MMSN/AuNPs was selectively uptaken by HepG2 cells, and performed self-activable ROS-mediated cascade reactions, and thereby resulted in remarkable and specific inhibition of HepG2 cell growth.

• The stradegy represents an insightful paradigm in developing targeted nanozymes with self-activable cascade reactions for achieving nanocatalytic tumor precision therapy.

Abstract

Catalytic nanozyme-based tumor therapy is currently considered as a promising strategy in anti-tumor treatment, but it also has some disadvantages such as systemic toxicity, off-target, and non-specificity, leading to lowered therapeutic precision and efficacy in clinical curability. To overcome these limitations, herein we report a distinct strategy of nanocatalytic tumor precision therapy via tumor cell-specific CD44 targeted nanozyme-catalyzed cascade reactions. Briefly, the composite nanozyme ^CD44MMSN/AuNPs were assembled with two self-activable nanocatalysts including the inner core peroxidase-mimic Fe₃O₄ magnetic nanoparticles (MNPs), and the outer glucose oxidase-mimic AuNPs situated within large aperture mesoporous silicon (MMSN/AuNPs), and then functionalized with cell ligand hyaluronic acid (HA), which can specifically target transmembrane CD44 receptor of HepG2 tumor cells. In terms of catalysis, we found the coupled AuNPs effectively catalyzed glucose to produce H₂O₂, which was inversely catalyzed by Fe₃O₄ NPs and AuNPs to produce more hydroxyl radicals (•OH) in tumor microenvironment (TME). That is, this composite nanozyme performed self-activable reactive oxygen species (ROS)-mediated cascade reactions, and thereby resulted in significantly specific growth inhibition of hepatocellular carcinoma HepG2 cells. The present results also showed that the composite nanozyme ^CD44MMSN/AuNPs could specifically induce a greater number of HepG2 cell apoptosis and death. More importantly, we elucidated the mechanism of the composite nanozyme ^CD44MMSN/AuNPs via inducing HepG2 cell apoptosis and death from subcellular level. Therefore, this study represents an insightful paradigm for achieving nanocatalytic tumor precision therapy through rationally designing tumor cell-targeting inorganic nanozyme with self-activable cascade reactions.

Introduction

Largely attributed to lack of tumor-targeted specificity, chemotherapy as one of commonly used procedures in tumor treatments, usually leads to severe side effects [1,2]. Therapeutic approaches including radiation, ultrasound, and photothermal treatments generally enable positioning the therapeutic sites, but may be harmful to surrounding tissues, or even arouse unwanted tumor invasion and metastasis [3], [4], [5], [6], [7]. To achieve efficient and tumor site-specific treatments, tumor microenvironment (TME) has been extensively investigated recently, in which the tumor cell metabolism, physical environment and vascular formation are significantly different from those in normal tissues [8,9]. TME-responsive drug releasing and diagnostic imaging have been widely developed, however, the introduced toxic antitumor agents would induce undesired cytotoxicity and damage to normal tissues subsequently [10].

Recently, chemodynamic therapy (CDT) has gained increasing research interest due to its special mechanism of producing reactive oxygen species (ROS) [11,12]. During the CDT process, hydroxyl radicals (•OH), a stronger ROS than H₂O₂, can be generated by endogenous H₂O₂ through a metal ion mediated Fenton reaction, and cause a more oxidative damage to tumor cells [13]. Therefore, converting endogenous H₂O₂ to more toxic •OH by catalysts is promising for tumor therapy. Furthermore, this process can avoid disavantages such as nonspecific side effects and low efficiency existing in traditional cancer therapy methods. However, the in vivo level of H₂O₂ is low and the efficiency of catalysts is also not sufficient, both of which have limited the CDT application in cancer therapy. Therefore, it is now urgent and important to develop a nanocatalytic system with prominent catalytic activity for specific and efficient tumor treatments.

Nanozymes integrating functions of natural nanomaterials and enzymes have inspired great research interest because of their tunable catalytic activities, low cost, and high stability and comparable to natural enzymes [14], [15], [16], which allow their wide applications in biomolecular detection [17,18], biosensing [19], disease diagnosis [20,21], antibacterial treatment [22], etc. The potential capability of nanozymes in various tumor treatments has drawn more and more attention recently. These nanocatalytic tumor treatment strategies based non-toxic but can be catalytically activated nano-sized catalysts to produce high-toxic ROS in tumors [23], [24], [25]. Tumor cells need more energy and nutrients than normal cells for growth because of the disorder in their metabolism [26,27]. Most cellular energy in tumor cells is produced via the glycolytic pathway, leading to a higher glucose consumption in tumor cells than in normal cells [28]. Once shutting down of glucose supply happens, tumor growth will be dramatically suppressed, therefore, cancer-starving via consumption of glucose is a promising strategy in cancer therapy [29,30]. Some glucose consumption strategies have been put forward in cancer therapy [31]. Glucose oxidase (GOD) can catalyze glucose to yield gluconic acid and H₂O₂ with O₂, which has been shown promosing in tumor treatment [32]. Nevertheless, delivery of GOD strategies currently are mainly through electrostatic or covalent conjugation GOD with nanocarriers [33], the natural GOD has shortcomings like low operational stability and high cost, which limit its applications in physiological environments. Therefore, developing novel natural GOD alternatives with lower cost and higher stability is urgently desired.

Herein, we endeavored to formulate a novel upgraded stradegy for tumor precision treatment, which is in practice of great use to improve therapeutic efficiency in clinical tumor treatments. In this stradegy, as illustrated in Scheme 1, the acidic TME can specifically initiate the sequentially catalyzed cascade reactions in tumor cells and/or tissues including: (i) the hyaluronic acid (HA)-conjugated nanocatalysts effectively recognize the CD44 receptor-expressing HepG2 cells, making the nanocatalysts more easily to enter tumor cells; (ii) glucose oxidation is catalyzed by the GOD-mimic ultrasmall AuNPs in the silicon pores to produce abundant of H₂O₂ and gluconic acid in tumor cells; (iii) self-accelerated glucose oxidation in the acidic condition generates in situ elevation of the H₂O₂ level; (iv) the elevated H₂O₂ is then catalyzed through the synergistic peroxidase (POD)-like activity between ultrasmall AuNPs and Fe₃O₄ NPs component to release highly toxic •OH, leading to apoptosis of tumor cells. The well-defined ^CD44MMSN/AuNPs can trigger a ROS-mediated apoptosis mechanism in cells, and thus remarkably and specifically suppress tumor growth. Therefore, the fabricated nanocatalysts with dual enzymatic activities represent an insightful paradigm for selective and effective tumor therapy.