Elsevier

Biomaterials

Volume 268, January 2021, 120537
Biomaterials

Activatable nanomedicine for overcoming hypoxia-induced resistance to chemotherapy and inhibiting tumor growth by inducing collaborative apoptosis and ferroptosis in solid tumors

https://doi.org/10.1016/j.biomaterials.2020.120537Get rights and content

Abstract

Hypoxia has been firmly correlated to the drug resistance of solid tumors. Alleviation of hypoxia by tumor reoxygenation is expected to sensitize the chemotherapy toward solid tumors. Alternatively, ferroptosis provides a therapeutic strategy to overcome apoptotic resistance and multidrug resistance of solid tumors, collaboratively strengthening the chemotherapy toward hypoxic tumors. Herein, an ultrasound (US)-activatable nanomedicine was developed for overcoming hypoxia-induced resistance to chemotherapy and efficiently inhibiting tumor growth by inducing sensitized apoptosis and collaborative ferroptosis of tumor cells. This nanomedicine was constructed by integrating ferrate and doxorubicin into biocompatible hollow mesoporous silica nanoplatforms, followed by assembling a solid-liquid phase-change material of n-heneicosane. The US-induced mild hyperthermia initiates the phase change of n-heneicosane, enabling US-activated co-release of ferrate and doxorubicin. Results reveal that the released ferrate effectively reacts with water as well as the over-expressed hydrogen peroxide and glutathione in tumor cells, achieving tumor-microenvironment-independent reoxygenation and glutathione-depletion in tumors. The reoxygenation down-regulates expressions of hypoxia-inducible factor 1α and multidrug resistance gene/transporter P-glycoprotein in tumor cells, sensitizing the apoptosis-based doxorubicin chemotherapy. More importantly, exogenous iron metabolism from the nanomedicine initiates intracellular Fenton reactions, leading to reactive oxygen species overproduction and iron-dependent ferroptotic death of tumor cells. Furthermore, the glutathione-depletion inactivates the glutathione peroxidase 4 (GPX4, a critical regulatory target in ferroptosis), inhibiting the reduction of lipid peroxides and reinforcing the ferroptotic cell death. The sensitized chemotherapy together with the iron-dependent ferroptosis of tumor cells play a synergistic role in boosting the growth suppression of hypoxic osteosarcoma in vivo. Additionally, the nanomedicine acts as a nanoprobe for in vivo photoacoustic imaging and glutathione tracking, showing great potential as theranostic agents for hypoxic solid tumors treatment.

Introduction

While chemotherapy has been successful and encouraging, its efficiency has always been limited by the multidrug resistance (MDR) of tumors and severe side effects on normal tissues [[1], [2], [3]]. Hypoxia, a characteristic feature of most solid tumors, has been firmly related to the angiogenesis, metastasis, and drug resistance of tumors [[4], [5], [6]]. It has been proved that the efficacy of chemotherapy often relays on the interaction of chemotherapeutic drugs with oxygen, which produces reactive oxygen species (ROS) and ultimately induces death of neoplastic cells [2,7,8]. Besides, hypoxia has been involved in the regulation of gene/protein expressions related to drug resistance and decreases drug delivery to tumor tissues [9]. Ameliorating tumor hypoxia through reoxygenation thus offers a promising way for the improvement of both the harsh tumor microenvironment (TME) and the efficacy of chemotherapy against solid tumors [[10], [11], [12], [13], [14]].

On the other hand, studies have indicated that chemotherapy principally induces caspase-dependent apoptotic cell death [1,15,16]. However, the apoptosis-based chemotherapy is ineffective for certain malignancies due to the apoptosis evasion and anti-apoptosis induced by the overexpression of apoptotic inhibitors and MDR of tumors [17,18]. In this regard, ferroptosis, a new form of programmed cell death defined by oxidative and iron-dependent accumulation of excessive lipid hydroperoxides and depletion of plasma membrane polyunsaturated fatty acids [19,20], provides a new therapeutic strategy to overcome apoptotic resistance and MDR of tumors due to its morphological, biochemical, and genetical distinction from apoptosis, classic necrosis, autophagy and other forms of cell death [[21], [22], [23]].

On this ground, an activatable nanomedicine was developed for ameliorating tumor hypoxia and efficiently inhibiting tumor growth by inducing collaborative apoptosis and ferroptosis in solid tumors. Oxidative ferrate (Fe(VI)) species, which are highly reactive to water, especially in acidic/neutral media (Scheme 1) [24], were integrated into biocompatible hollow mesoporous silica (HMS)-based nanoplatforms (designated as Fe(VI)@HMS) and used as a powerful oxygen generator for tumor reoxygenation.

The nanoplatforms-based tumor reoxygenation was expected to alleviate tumor hypoxia and promote the sensitivity of tumor cells to chemotherapeutics, such as doxorubicin (DOX). Specifically, the reoxygenation in tumor cells would facilitate the oxygen-involved redox cycling, which activate the DOX to generate active superoxide radical (O2•-) (Scheme 1) [[25], [26], [27]]. The O2•- could be further catalyzed by cellular superoxide dismutase (SOD) to produce hydrogen peroxide (H2O2) [28]. These H2O2, together with the over-expressed H2O2 in tumor cells, may also be used as the substrate for Fe(VI)-mediated redox reactions (Scheme 1), leading to more significant tumor reoxygenation. The sequence of these catalytic reactions would be damaging, since a relatively small amount of DOX is sufficient for the generation of numerous toxic O2•- and H2O2, even under hypoxic conditions, which is expected to promote the efficiency of DOX toward hypoxic and chemotherapy-resistant malignancies.

More importantly, the generated H2O2 may also serve as the substrate for iron-involved Fenton reactions [29,30]. Previous studies have demonstrated that exogenous iron metabolism in cells plays an important role in ferroptosis processes [31]. Overloading iron could induce ferroptotic cell death through converting low toxic H2O2 in tumor cells to highly active O2•- and hydroxyl radical (HO•) [32]. The cellular metabolism of Fe(VI)-based nanoplatforms is thus speculated to promote the ROS level in cells and lead to ferroptotic death of tumor cells. Apart from the iron metabolism, glutathione peroxidase 4 (GPX4) is another critical regulatory target in ferroptosis [33]. Inactivation of GPX4 inhibits the reduction of ROS and lipid peroxides, leading to ROS overproduction and consequently irreversible cell death [34,35]. The oxidative Fe(VI) species in the nanoplatform may convert the over-expressed reductive glutathione (GSH) in tumor cells to glutathiol (GSSG). Since GSH is an essential intracellular antioxidant that serves as the co-substrate of GPX4 to protect cells from oxidative damage [36], the depletion of GSH would thus inactivate the activity of GPX4 and potentiate ferroptotic cell death (Scheme 1).

Herein, we established a low-dose ultrasound (US) responsive nanomedicine by integrating DOX with the Fe(VI)-nanoplatform, followed by incorporation of n-heneicosane (HE) and polyethylene glycol (PEG) chains (designated as DOX-Fe(VI)@HMS-HE-PEG, abbreviated as DFHHP) (Scheme 1B). HE is a solid-liquid phase-change material with a low melting point of ca. 40 °C [37], which may response to the mild hyperthermia induced by US and achieve US-responsive cargo release. When DFHHP nanoparticles were administered intravenously, the nanoparticles could extravasate into tumors based on the enhanced permeability and retention (EPR) effect of leaky tumor vasculatures [38]. Once inside tumor cells, the US-responsive DFHHP nanomedicine is expected to alleviate the hypoxic TME by tumor reoxygenation, which may sensitize the DOX-mediated apoptosis of solid tumors. More importantly, efficient ferroptotic cell death could be simultaneously achieved via exogenous iron metabolism and systemic GPX4 inactivation. The sensitized DOX-based chemotherapy together with the iron-based ferroptosis of tumor cells play a synergistic role in elevating the ROS levels and boosting the growth inhibition of tumors, especially chemotherapy-resistant hypoxic tumors (Scheme 1). Notably, the US irradiation is tumor-site targeted, enabling tumor-specific modulation of microenvironment and more remarkable synergistic anticancer outcome with minimal side effects, which is much desirable and promising for further clinical translation.

Section snippets

Construction and characterization of DFHHP

Silica-based nanomaterials have been extensively used for cargo delivery due to their superior microstructure (i.e., high ratio of surface area/pore volume, tunable pore size and specific hollow mesoporous structure) and abundant surface chemistry [39]. In this work, hollow mesoporous silica (HMS) nanospheres were fabricated according to literatures [40]. The constructed HMS was ca. 100 nm in diameter (Fig. 1A and B). The huge cavity can be clearly seen in the transmission electron microscopy

Conclusion

In conclusion, a US-activatable DFHHP nanomedicine has been developed to overcome the chemoresistance and suppress tumor growths by inducing collaborative apoptosis and ferroptosis of tumor cells. The Fe(VI) species and DOX were integrated into biocompatible HMS nanoplatforms, followed by assembling of a solid-liquid phase-change compound of HE. The activity and release of Fe(VI) species and DOX can be intelligently controlled by the US-induced mild hyperthermia. The Fe(VI) in the DFHHP reacted

Author contribution

J. Fu and Y. Zhu designed the experiments for this study; J. Fu, T. Li, Y. Yang, L. Jiang, W. Wang and L. Fu carried out the experiments and participated in data acquisition and analysis. J. Fu, Y. Zhu and Y. Hao wrote and edited the manuscript. All the authors reviewed and acknowledged the manuscript.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

We acknowledge the financial support from the National Natural Science Foundation of China (Nos. 81801821, 81972058, and 51872313), China Postdoctoral Science Foundation (No. 2018M630448), and Shanghai Municipal Key Clinical Specialty (No. shslczdzk06701).

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