Janus nanocarrier-based co-delivery of doxorubicin and berberine weakens chemotherapy-exacerbated hepatocellular carcinoma recurrence

Abstract

Despite the rapid progress which has been made in hepatocellular carcinoma (HCC) chemotherapeutics, recurrence of liver cancer still remains a barrier to achieve satisfying prognosis. Herein, we aimed to decipher the role of berberine (BER) in chemotherapy-exacerbated HCC repopulation via developing a nanocarrier co-deliveries doxorubicin (DOX) and BER to achieve a synergic effect in HCC treatment. The underlying fact of chemotherapy that promotes HCC repopulation was firstly examined and corroborated by clinical samples and murine repopulation model. Then, hyaluronic acid (HA)-conjugated Janus nanocarrier (HA-MSN@DB) was developed to load DOX and BER simultaneously. The HCC targeting efficiency, pH-controlled drug-release and anti-cancer property of HA-MSN@DB were assessed in CD44-overexpressed HCCs and normal liver cells. Magnet resonance imaging, bio-distribution, biocompatibility, tumor and recurrence inhibition studies were performed in H22 tumor-bearing mice. BER significantly reduced doxorubicin (DOX)-triggered HCC repopulation in vitro and in vivo through inhibiting Caspase-3-iPLA2-COX-2 pathway. The delivery of HA-MSN@DB into HCCs through CD44 receptor-mediated targeting effect was demonstrated. The controlled release of DOX and BER in response to acidic tumor microenvironment was validated. Importantly, HA-MSN@DB drastically enhanced the antitumor activity of DOX and suppressed DOX-exacerbated HCC repopulation in vitro and in vivo. Furthermore, HA-MSN@DB exhibited enhanced tumor accumulation and biocompatibility. Our findings revealed the pivotal role of BER in overcoming chemotherapy-exacerbated HCC repopulation through Caspase-3-iPLA2-COX-2 pathway, thereby providing a promising and stable nanocarrier integrating DOX and BER for effective HCC chemotherapy without repopulation.

Statement of significance

In this work, we have first demonstrated the fact that berberine (Ber) reduces chemotherapy-exacerbated HCC recurrence and studied its mechanism by the aid of a doxorubicin-induced mice HCC relapse model. We then developed a promising strategy that simultaneously inhibits HCC and its recurrence with an HCC-targeted co-delivery nanocarrier HA-MSN@DB and revealed that such an inhibition was related with the suppression of Caspase-3-iPLA2-COX-2 pathway by berberine.

Introduction

Hepatocellular carcinoma (HCC) leads to nearly millions of deaths worldwide each year with an increasing mortality rate [1,2]. Advances in chemotherapy partially alleviates the clinical outcomes of HCC patients [3], [4], [5]. Unfortunately, more than ninety percent of patients suffer from recurrence after treatment, which majorly accounts for the morbidity [6], [7], [8], [9]. This fundamental challenge urges clinicians to re-evaluate the application of current cell death-based strategies and to investigate the molecular mechanisms in depth [10,11]. To enhance chemotherapeutic agent-triggered cancer cell apoptosis and to caspase elicits repair, our group and others have recently reported that caspase-3-dependent cleavage of cytosolic calcium-independent phospholipase A2 (iPLA2) during chemotherapy triggers the release of arachidonic acid (AA) and prostaglandin E2 (PGE2). The activation of the AA metabolic pathway leads to the repopulation of surviving cancer cells, ultimately promoting recurrence [12]. Therefore, we speculated that the balance between apoptotic cell death and caspase-driven tumor cell survival and repopulation could be used for effective tumor killing and death-related recurrence inhibition [12], [13], [14]. Among several important targets involved in the ``Phoenix Rising'' signaling pathway [15], [16], [17], [18], iPLA2 acts as a key link between caspase activation and the AA metabolic pathway. We previously demonstrated that berberine (BER), which is a competitive inhibitor of PLA2 [19], [20], [21], inhibits chemotherapy-induced repopulation of ovarian cancer cells by suppressing the AA metabolic pathway and FAK phosphorylation [22]. However, the role of the ``Phoenix Rising'' signaling pathway in chemotherapy-exacerbated HCC recurrence and the effects of BER on this event remain elusive. Therefore, we hypothesized that the combination of a chemotherapeutic agent and the ``Phoenix Rising'' signaling pathway inhibitor might inhibit cell death-induced HCC recurrence, thereby promoting effective chemotherapeutic outcomes.

To achieve synergic effect on chemotherapy-exacerbated HCC recurrence with minimal side effects, drug codelivery system is highly desirable for imaging-guided HCC-targeted and controlled delivery. Chemotherapeutic agent doxorubicin (DOX) and the ``Phoenix Rising'' signaling pathway inhibitor BER were exploited in this co-delivery system. Magnetic mesoporous silica nanoparticles (M-MSNs) have attracted considerable attention as promising carriers due to their good biocompatibility, high drug loading, surface modification versatility, and magnetic properties for magnetic resonance imaging (MRI) contrast [23], [24], [25], [26]. Our group has recently used Janus-type magnetic mesoporous silica nanoparticles (Janus M-MSNs) with an architectural advantage of minimal interfering [23]. Continuous efforts have demonstrated that our drug delivery systems exhibited stronger magnetic properties, a higher intracellular internalization and more tumor accumulation in comparison to traditional core-shell structures such as M-MSNs [27], which could further benefit magnetically targeted chemotherapy and MRI-guided, hyperthermia-enhanced gene therapy of HCC [28], [29], [30]. Furthermore, due to the high expression level of the CD44 receptor on HCC cells surface [31,32], hyaluronic acid (HA), a biodegradable, biocompatible, and nonimmunogenic linear polysaccharide, was employed as a bifunctional ligand to prolong blood circulation and improve the accumulation of nanoparticles in HCC foci [33], [34], [35]. In such a scenario, HA acts as a targeting component due to its high affinity for CD44 receptor, which facilitating the CD44 receptor-mediated endocytosis of HA-modified nanocarriers in HCC cells. Therefore, HA-conjugated Janus M-MSNs with MR imaging and HCC-targeting behavior can be exploited as an ideal co-delivery system for efficient and safe HCC therapy and reducing chemotherapy-exacerbated recurrence [36], [37], [38].

In this study, we first validate our hypothesis of chemotherapy promotes HCC recurrence in clinical samples and a murine repopulation model. Then, we aimed to explore the crucial role of the activated caspase-3-iPLA2-COX-2 pathway in the HCC cell repopulation model. We illustrated BER significantly reverses doxorubicin (DOX)-induced HCC repopulation both in vitro and in vivo. To improve the drug delivery efficiency of DOX and BER and to reduce the side effects of DOX, the co-delivery system of HA-conjugated Janus M-MSNs (HA-MSN@DB) was developed. HCC cell-targeting, pH-sensitive drug release and anticancer properties of HA-MSN@DB were assessed in CD44-overexpressing HCC cells and in normal liver cells. MR imaging, biodistribution, biocompatibility, tumor inhibition and recurrence studies were subsequently performed in H22 tumor-bearing mice. The ``Phoenix Rising'' signaling pathway during DOX-induced HCC repopulation was analyzed in vivo. Our findings prove the co-delivery of DOX and BER via HA-conjugated Janus M-MSNs, which is a promising, efficient and safe HCC chemotherapeutic strategy with decreased recurrence.