key: cord-0910621-3u1b4orl
authors: Tian, Zhimin; Zhao, Junlong; Zhao, Shoujie; Li, Huicheng; Guo, Zhixiong; Liang, Zechen; Li, Jiayuan; Qu, Yongquan; Chen, Dongfeng; Liu, Lei
title: Phytic acid-modified CeO(2) as Ca(2+) inhibitor for a security reversal of tumor drug resistance
date: 2022-01-29
journal: Nano Res
DOI: 10.1007/s12274-022-4069-0
sha: 42af4208bdd6018c520a8bd440e2adfcd4d108dd
doc_id: 910621
cord_uid: 3u1b4orl

Ca(2+) plays critical roles in the development of diseases, whereas existing various Ca regulation methods have been greatly restricted in their clinical applications due to their high toxicity and inefficiency. To solve this issue, with the help of Ca overexpressed tumor drug resistance model, the phytic acid (PA)-modified CeO(2) nano-inhibitors have been rationally designed as an unprecedentedly safe and efficient Ca(2+) inhibitor to successfully reverse tumor drug resistance through Ca(2+) negative regulation strategy. Using doxorubicin (Dox) as a model chemotherapeutic drug, the Ca(2+) nano-inhibitors efficiently deprived intracellular excessive free Ca(2+), suppressed P-glycoprotein (P-gp) expression and significantly enhanced intracellular drug accumulation in Dox-resistant tumor cells. This Ca(2+) negative regulation strategy improved the intratumoral Dox concentration by a factor of 12.4 and nearly eradicated tumors without obvious adverse effects. Besides, nanocerias as pH-regulated nanozyme greatly alleviated the adverse effects of chemotherapeutic drug on normal cells/organs and substantially improved survivals of mice. We anticipate that this safe and effective Ca(2+) negative regulation strategy has potentials to conquer the pitfalls of traditional Ca inhibitors, improve therapeutic efficacy of common chemotherapeutic drugs and serves as a facile and effective treatment platform of other Ca(2+) associated diseases. [Image: see text] ELECTRONIC SUPPLEMENTARY MATERIAL: Supplementary material (further details of the XRD pattern of CeO(2), TEM images, XPS spectra, cellular uptake study, cytotoxicity data, apoptosis study, biodistribution, and biosecurity of nanocerias in vivo, etc.) is available in the online version of this article at 10.1007/s12274-022-4069-0.

As a ubiquitous intracellular second messenger, Ca 2+ has been wellestablished to play critical biological roles in the metabolic homoeostasis, signaling pathway, and development [1, 2] . Hence, numerous diseases (e.g., neurodegenerative disorders, cardiovascular disease, hypertension, diabetes, and tumors) are generally accompanied with the abnormal expression of Ca 2+ signaling [3] [4] [5] [6] . Besides, a variety of viruses including SARS-CoV-2 can modulate intracellular Ca 2+ expression to facilitate virus infection and replication inside host cells [7] . In particular, the intracellular Ca 2+ levels are crucial in mediating tumorigenesis as well as tumor progression [4, 6, 8] . Thereafter, various Ca regulation methods such as Ca 2+ antagonist, hormones, and Casupplemental agents have been developed with the expectation to realize the successful disease treatments. However, the clinical applications of those Ca regulation methods have been greatly restricted due to their high toxicity, poor reversal efficacy, and nonspecific action [9] [10] [11] [12] . Therefore, it is urgent to develop safe, efficient, and facile Ca 2+ regulation strategy to satisfy practical demands.

With the rapid development of nanoscience and nanotechnology in modern medicine, nanomaterials have shown their great potentials to improve human living standards as well as disease therapy [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] . Specifically, nanomaterials have been designed to greatly overload the intracellular Ca 2+ levels for tumor therapy [22] [23] [24] . However, since the abnormally high levels of Ca 2+ might induce severe side effects including renal injury and cardiac arrest, the intracellular Ca concentration must be dedicatedly balanced within a narrow window to maintain normal Ca homeostasis [25, 26] . Thus, an attractive Ca regulatory strategy should mitigate Ca 2+ -related cytotoxicity to guarantee the high safety of treatments. Considering the powerful and dual roles of Ca 2+ in living organisms, to design a negative regulation strategy of Ca regulation by decreasing the intracellular Ca 2+ level in a controllable manner is of great value in the treatments of various Ca-related diseases as well as tumors.

It has been well documented that excessive Ca 2+ can promote the intracellular Ca 2+ -involved proliferation and provide a survival pathway for cancer cells after drug treatments, thereby decreasing the efficacy of chemotherapeutic drugs. Thus, higher cellular Ca 2+ level is recognized as an essential characteristic of drug-resistant tumor cells, which is a major obstacle for the long-term efficacy of chemotherapeutic drugs [4, 8, [27] [28] [29] . Generally, high doses and/or various combinations of chemotherapeutic drugs are used to achieve effective therapy. However, such a temporary relief, regardless of the negative consequences of chemotherapeutic drugs, is inadvisable. Despite the intensive efforts to overcome drug resistance, only limited achievements have been realized. Herein, we design an unprecedentedly safe and efficient nanoscale platform to effectively remove the excessively expressed intracellular Ca 2+ for reversal of tumor drug resistance and demonstrate the high feasibility of the intracellular Ca 2+ negative regulation strategy for disease treatments. Both in vitro cellular and in vivo animal evaluations highlight that this strategy provides a facile, safe, and efficient platform for the reversal of tumor drug resistance, showing its great potentials for clinical applications.

Extensive examination of literatures emphasizes that high level of the intracellular free Ca 2+ is believed to activate drug-transporting proteins (such as P-glycoprotein, P-gp) to efflux chemotherapeutic drugs out of tumor cells, ultimately reducing the efficacy of chemotherapy and leading to multidrug resistance. Previous studies have suggested that a rise in cytoplasmic Ca 2+ level stimulates P-gp overexpression in tumor cells [27] [28] [29] . Thus, suppression of P-gp expression by regulating intracellular Ca 2+ level offers an opportunity to overcome tumor drug resistance. The blockage of Ca 2+ channels with Ca 2+ antagonists has been demonstrated to exert beneficial effects in cancer cells by downregulating P-gp expression. However, the toxicity of those Ca blockers limits their potential clinical use [9, 10] .

Inspired by the above cognitions, here, the phytic acid (PA)modified porous nanorods of CeO 2 (CePA) have been rationally designed and experimentally demonstrated as an unprecedentedly safe and efficient platform to effectively enable the intracellular Ca 2+ negative regulation through cleating between PA and Ca 2+ , and successfully reverse tumor drug resistance (Scheme 1). Doxorubicin (Dox), which is severely limited in clinical therapy due to serious drug resistance, was selected as a model chemotherapeutic drug [30] [31] [32] . PA, an Food and Drug Administration (FDA)-approved natural product in many plant tissues (e.g., rice bran and seeds) as a tonic, food additive, antioxidant, preservative, and stabilizer, was chosen as a safe Ca nano-inhibitor for down-regulating the intracellular Ca 2+ level due to the outstanding coordination ability between Ca 2+ and PA [33] [34] [35] .

Moreover, Dox as a commonly used chemotherapy drug can induce serious cardiotoxicity and hepatotoxicity due to the reactive oxygen species (ROS)-mediated oxidative damage and subsequent apoptosis of normal cells [30] [31] [32] . Therefore, reducing the side effects of Dox on normal cells is also highly expected. Due to high antioxidant capability of CeO 2 nanozyme for the degradation of ROS [36] [37] [38] [39] [40] [41] [42] [43] , the porous nanoceria (CeO 2 ) was used as an adjuvant medicine to alleviate Dox-induced oxidative stress in normal cells [32] . Therefore, the CePA nanozymes are expected to serve as a highly efficient Ca 2+ nano-inhibitor, reducing Ca 2+ levels in drug-resistant tumor cells, inhibiting P-gp overexpression, and increasing intracellular accumulation of drugs.

Hydrothermally synthesized CeO 2 showed a porous rod-like morphology with fluorite phase, as revealed from transmission electron microscopy (TEM) and X-ray diffraction (XRD) pattern ( Fig. S1 in the Electronic Supplementary Material (ESM)) [44] . Afterwards, CePA hybrids were synthesized by immersing CeO 2 into aqueous PA solution (2 wt.%) for 12 h and subsequent centrifugal separation. TEM and XRD characterizations indicated the preserved morphological features and phases of CeO 2 during treatment ( Fig. 1(a) and Fig. S1 (b) in the ESM). To probe the surface states of CePA, Fourier transform infrared (FT-IR) spectroscopy and TEM elemental mapping were performed. Compared with CeO 2 , the FT-IR spectrum of CePA showed two new emerging absorption peaks at 1,230 and 1,651 cm −1 due to the P=O stretching mode of PO 4 3− and the increased peak at 3,510 cm −1 due to the hydroxyl of PA, confirming the successful modification of CeO 2 by PA ( Fig. 1(b) ). In comparision with a near neutral potential (2.6 mV) of CeO 2 with the abundant interfacial hydroxyl, the zeta potential of CePA decreased to −41.2 mV, also supporting the successful conjugation of the negatively charged PA on CeO 2 ( Fig. 1(c) ). TEM elemental mapping images further proved the formation CePA hybrids due to the homogeneous distribution of Ce, O, and P elements in CePA ( Fig. 1(d) ). The loadings of PA in the CePA was ~ 4.7 wt.% determined by the inductively coupled plasma mass spectrometry (ICP-MS) through the quantification of the P element.

Afterwards, the Ca 2+ coordination capability of CeO 2 and CePA was evaluated in tris-HCl buffer solution (50 mM, pH 7.4, and 50 μM Ca 2+ ) by a Ca 2+ probe, o-cresolphthalein complexone. CeO 2 showed the negligible Ca 2+ coordination capability. Impressively, CePA showed significant Ca 2+ coordination activity (> 70%, Fig. 1 (e)), enabling CePA as a potential Ca 2+ nanoinhibitor for in vitro and in vivo investigations. The Ca 2+ spectral absorption further revealed the outstanding Ca 2+ coordination capability of CePA in a dose-dependent performance, as evidenced by a decrease of spectral absorption at 570 nm ( Fig. 1(f) ) [45] . The corresponding solution colors from dark violet to light purple with the increased doses of CePA indicate the promises as cytoplasmic Ca 2+ nano-inhibitor for reversing tumor drug resistance ( Fig. 1(g) ).

Scheme 1 Synthetic procedures of CePA and mechanism for drug resistance reversal via Ca 2+ negative regulation. PA-modified porous nanorods of CeO 2 were demonstrated as an efficient nano-inhibitor to overcome drug resistance through coordination between PA and Ca 2+ . After the nano-inhibitor enters the body, it reaches the tumor tissue mainly through the blood vessels, effectively scavenging Ca 2+ , significantly suppresses P-gp expression and significantly enhances the intracellular Dox accumulation in drug-resistant tumor cells, thus achieving highly effective chemotherapy.

Due to the severe Dox-induced oxidative stress and the high antioxidant ability of nanoceria as nanozyme to scavenge druginduced ROS [32, 42] , herein, we anticipated that nanocerias would suppress or even prevent adverse effects of Dox on normal organs. Then, the ROS scavenging activity of CeO 2 and CePA was evaluated by calibration from the standard catalase and superoxide dismutase (SOD) assay kits. CePA showed better H 2 O 2 elimination than CeO 2 at the same dose, indicating its higher catalase-like performance ( Fig. S2 (a) in the ESM). For SOD-mimic activity, both CeO 2 and CePA exhibited enhanced performance with the increase of catalyst concentration ( Fig. S2 (b) in the ESM). Consistent with the catalase-like results, the SOD-like activity of CePA was higher than that of CeO 2 at each concentration, demonstrating the higher antioxidant activity of CePA over CeO 2 . The ratio of Ce 3+ /Ce 4+ plays a pivotal role in their cpability of ROS scavenging [32, [39] [40] [41] [42] . Herein, X-ray photoelectron spectra (XPS) of CeO 2 and CePA suggested their similar surface Ce 3+ fractions ( Fig. S3 in the ESM), indicating that the surface defect concentration was not the primary factor for the difference in their antioxidative capabilities. Considering PA as a natural antioxidant [33, 34] , the conjugation of PA on CeO 2 might contribute to the increased antioxidant performance of CePA. Meanwhile, the cytotoxicity experiments showed that both CeO 2 and CePA delivered a better protective capability than free PA (Fig. S4 in the ESM). Importantly, the ROS scavenging efficiency of both CeO 2 and CePA was pH dependent, and their total free radical scavenging activity was significantly higher in normal physiological environments than in weakly acidic conditions (Fig.  S5 in the ESM). The aberrant glycolytic metabolism of the tumor microenvironment (TME) causes pH of tumor tissues to be less than the normal physiological value. Hence, nanocerias showed their high potential to selectively alleviate the adverse drug effects on normal organs during chemotherapy.

Before evaluating their capability for the reversal of drug resistance, the biosafety of CeO 2 and CePA was studied using normal human liver cells (LX-2) and Dox-resistant human hepatic carcinoma cells (HepG2/ADR). No obvious cytotoxicity was observed in either cell lines, suggesting their high biocompatibility 

Next, we evaluated the influence of the CeO 2 and CePA on Dox sensitivity in the Dox-resistant tumor cells (HepG2/ADR). As expected, Dox showed limited toxicity in HepG2/ADR cells, even at a high concentration of 30 μM ( Fig. S9 (a) in the ESM). Notably, CePA significantly enhanced the antitumor activity of the Dox in the HepG2/ADR cells, demonstrating its high capability to overcome the Dox-induced drug resistance ( Fig. 2(a) ). When the CePA dose was > 50 μg/mL, the antagonizing Dox-resistance activity of CePA reached saturation ( Fig. S9 (b) in the ESM). Hence, 50 μg/mL CePA was used to investigate the reversal of Dox resistance, except where otherwise indicated. While, CeO 2 barely enhanced the therapeutic efficacy of Dox in HepG2/ADR cells under same conditions, suggesting that the surface conjugation of PA on CeO 2 played critical roles in the enhanced antitumoral efficacy of Dox in drug-resistant HepG2/ADR cells.

Thereafter, the flow cytometry and fluorescence images further demonstrated that the intracellular Dox concentration was 

Considering the side effects of Dox, we further analyzed the capability of nanocerias against the chemotherapy-induced cell injury on normal LX-2 cells. CeO 2 and CePA significantly improved viability of LX-2 cells treated by Dox ( Fig. 2(g) ).

Importantly, the cytoprotective effects of nanocerias were strongly depended on the environmental pH, delivering the outstanding protection at pH 7.4 but poor cytoprotections at pH 6.5 ( Fig. 2(h) ). Further analysis of ROS and apoptosis also confirmed the above results (Figs. S13 and S14 in the ESM). Overall, CePA with high antioxidant activity is expected to suppress the side effects during tumor chemotherapy in normal cells and enhance chemotherapeutic activity in drug-resistant tumors.

Then, the mechanism of CePA against the drug resistance was explored. As previously reported [27] [28] [29] , the increased intracellular level of free Ca 2+ is closely related to tumor drug resistance. Based on the capability of CePA as Ca 2+ nano-inhibitor resulting in intracellular Ca deficiency (Figs. 1(e)-1(g)) and the reversal of tumor Dox resistance (Figs. 2(a)-2(f) and Figs. S9-S11 in the ESM), we evaluated the capability of CePA for the deprivation of free intracellular Ca 2+ in the Dox-resistant Data are presented as means ± SD, ***P < 0.001 and ****P < 0.0001 (unpaired Student's two-sided t-test analysis).

HepG2/ADR cells. CeO 2 showed a barely interferred intracellular Ca 2+ level (Figs. 3(a) and 3(b)). Impressively, CePA treatment in the HepG2/ADR cells for 6 h dramatically lowered the intracellular free Ca 2+ level by 45% (Figs. 3(a) and 3(b)), indicating the capability of CePA as Ca 2+ nano-inhibitor for regulating the intracellular Ca 2+ level. Both western blotting and immunofluorescence staining analysis confirmed that CePA efficiently captured the intracellular Ca 2+ and then markedly decreased the P-gp expression (Figs. 3(c) and 3(e)).

To further confirm CePA as a Ca 2+ nano-inhibitor against the tumor drug resistance, CALP2, an agonist of Ca 2+ , was used to prevent the activity of CePA. The experimental results showed that the CALP2 pretreatment significantly attenuated the capability of CePA against tumor drug resistance, further demonstrating the CePA-enabled reversal of tumor drug resistance through the Ca 2+ negative regulation strategy (Fig. S15 in the ESM). Based on above analysis and mechanism investigations, CePA as Ca 2+ nano-inhibitor efficiently captured the intracellular Ca 2+ in the HepG2/ADR cells, significantly decreased the P-gp expression and greatly increased drug accumulation within the resistant tumor cells, ultimately reversing the drug resistance.

Encouraged by the in vitro results, the HepG2/ADR tumorbearing model in BALB/c nude mice was established to evaluate the potentials of CePA for reversal of Dox resistance in vivo. The biocompatibility of CeO 2 and CePA as the primary consideration was initially assessed. Primarily, main blood markers and histological analyses of the main organs showed no obvious abnormalities between the CeO 2 /CePA-injected group and control group (Figs. S16 and S17 in the ESM). Meanwhile, after intravenous injection of CeO 2 and CePA, the biodistribution of naocerias in major organs decreased gradually overtime ( Fig.  S18(a) in the ESM), further suggesting that the nanocerias could be effectively removed out of the body. We then proved that the nanoceria could be excreted via urine (Fig. S18(b) in the ESM) [46] . All results demonstrated the high in vivo biocompatibility of CeO 2 and CePA. Conversely, 94% and 76% of Ce was retained in tumors after CeO 2 and CePA injection for 7 days, demonstrating tumor accumulation of CeO 2 and CePA. Such intratumor permeability and retention of two candidates facilitated their effective delivery into tumors for overcoming tumor drug resistance.

We then evaluated the overall therapeutic performance of CeO 2 and CePA against Dox-resistant tumors in HepG2/ADR tumorbearing BALB/c nude mice. The tumor-bearing mice were randomly divided into six groups: control, Dox, CeO 2 , CePA, CeO 2 + Dox, and CePA + Dox. The tumor volumes in the control and Dox groups were increased by factors of 12.1 and 10.8 on the 14 th day, respectively (Fig. 4(a) ), indicating the strong Doxresistance developed in the tumor cells. Conversely, the combination of CePA and Dox completely restricted tumor growth ( Fig. 4(a) ), indicating the excellent efficacy of CePA for reversing chemotherapy resistance. CeO 2 and CePA groups also showed certain inhibitory activity against tumor growth, which could be attributed to the peroxidase-like activity of CeO 2 and CePA to convert the intratumoral H 2 O 2 into toxic ·OH in TME [47, 48] . The digital images and average weight of tumors confirmed the tumor growth profiles (Figs. 4(b) and 4(c) ). Significant weight loss (29.7%) of the tumor-bearing mice treated by Dox was observed due to the main adverse effect of chemotherapy ( Fig. 4(d) ). Notably, CeO 2 and CePA induced no significant weight change throughout the treatments, further verifying their biosafety and their potential protective effects against Dox-induced adverse effects in vivo.

The mechanism of CePA for restoring Dox-sensitivity was further revealed by histological analysis [49, 50] . Hematoxylin and eosin (HE) staining showed that compact tumor tissues were found for the Dulbecco's Phosphate Buffered Saline (DPBS, control)-and Dox-treated tumor-bearing mice (Fig. 4(e) ). In contrast, large areas of apoptosis and necrosis in tumor tissues and more vacancies were observed after the combined CePA + Dox treatment, further suggesting that CePA was able to reverse tumor drug resistance, consistent with the tumor inhibition results (Figs. 4(a)-4(c) ).

Similar results were observed in ROS, TUNEL, and caspase-3 staining images (Fig. 4(f) and Fig. S19 in the ESM). CePA + Dox combination treatment significantly strengthened the intratumoral ROS production by Dox (Fig. 4(f) ), consistent with the CePAinduced increase of Dox level in tumor issues ( Fig. 4(g) ). Significant green fluorescence was observed in mice treated with CePA + Dox, suggesting the widespread DNA strand fragmentation and tumor cell apoptosis ( Fig. S19(a) in the ESM). The apoptosis areas in the CePA + Dox group were significantly larger than those in other groups. Overlapping of the blue (nucleus) and green fluorescence again confirmed DNA (Fig. 4) . P-gp expression of various groups was also monitored by immunofluorescent staining (Fig. 4(g) ). CeO 2 exhibited no significant influence on the P-gp expression. Thus, the intratumoral Dox (red fluorescence) levels in the Dox-treated and CeO 2 + Dox treated groups were negligible. Comparatively, the expression of the intratumoral P-gp (green fluorescence) was markedly downregulated upon exposure to CePA, leading to the significantly enhanced the intratumoral Dox level of the CePA + Dox group. Then, the concentrations of Dox in tumor tissues after the CeO 2 and CePA treatments for 24 h were further identified by high-performance liquid chromatography. CePA markedly enhanced the tumor accumulation of Dox, which was 12.4-fold higher than that of the Dox-treated tumor tissues (Fig. 4(h) ). Therefore, CePA reversed Dox resistance of tumor cells by downregulating P-gp expression, decreasing the efflux pump for Dox and enhancing intratumoral Dox retention. To verify the contribution of Ca 2+ to tumor drug resistance, variation in intratumoral free Ca 2+ concentration was measured for the control, CeO 2 , and CePA groups. The intratumoral free Ca 2+ level in the CePA group showed a significant decrease of 61.5% after 24 h treatment ( Fig. 4(i) ), consistent with the decrease of P-gp expression (Fig. 4(h) ). Thus, the results again demonstrated that CePA as Ca 2+ nano-inhibitor overcame tumor Dox resistance and restored sensitivity by inducing Ca deficiency in tumor cells, triggering P-gp downregulation and increasing intratumoral Dox retention.

Meanwhile, Dox can induce serious toxic adverse effects in major organs due to ROS-mediated DNA damage and apoptosis of normal cells, especially cardio-and hepatotoxicity [30] [31] [32] 51] . Besides the capability of CePA for reversing drug resistance, the protective benefits of CeO 2 and CePA against Dox-induced cardiac injury and liver damage in mice were evaluated. The body weight of the Dox-treated mice significantly decreased (~ 32%) ( Fig. 4(d) ), compared with the DPBS-treated mice, indicating that Dox caused severe systemic injury. More importantly, 60% of mice treated by Dox alone died within 28 days, whereas all mice pretreated with CeO 2 and CePA survived up after Dox treatment during same period (Fig. 5(a) ). Besides, the plasma levels of lactate dehydrogenase (LDH, specific marker of cardiac injury), creatine kinase isoenzyme myocardial band (CK-MB, specific marker of cardiac injury), alanine aminotransferase (ALT, specific marker of liver injury), and aspartate aminotransferase (AST, specific marker of liver injury) of the Dox group were markedly higher than those in the control group (Figs. 5(b)-5(d) ). Conversely, the levels of these serum markers in the CeO 2 + Dox and CePA + Dox groups showed bare changes compared with the control group (Figs. 5(b)-5(d)), further indicating that CeO 2 and CePA significantly inhibited Dox-induced heart and liver injury.

HE-stained cardiac tissues indicated that the Dox group mice exhibited myofibrillar loss (Fig. 5(e) ), vacuolization and swelling. Meanwhile, the hepatic tissues taken from the Dox group showed severe pathological alterations (blood vessel congestion and necrosis) (Fig. 5(f) ). As expected, no obvious myocardial damages and liver injury were found in the CeO 2 + Dox and CePA + Dox groups. Similar to the cytoprotective results ( Fig. 2(g) and Figs. S13 and S14 in the ESM), CeO 2 and CePA also effectively degraded the Dox-induced oxidative stress in the heart and liver 

In a summary, a facile and efficient negative regulation strategy of intracellular Ca 2+ has been dedicatedly designed and successfully developed to overcome tumor drug resistance while preventing from Dox-induced organ damage. PA in the nano-inhibitor, as a Ca 2+ coordination agent, efficiently deprives the intracellular excessive free Ca 2+ , and significantly suppresses expression and activity of P-gp to avoid drug efflux. The high safety of the nanoinhibitor CePA is illustrated by no obvious systemic damage as well as relief of chemotherapy-induced oxidative stress in the major organs, due to its high antioxidant capability. The synergistic effect of the Dox and CePA treatment improves the efficacy of the commonly chemotherapeutic agent and decreases the side effects. This negative regulation strategy of intracellular Ca 2+ with features of high safety and efficacy not only brings new insights on the tumor drug resistance, and also provides an alternative methodology for other Ca related diseases, such as Ca poisoning, atherosclerosis and neurodegenerative diseases, even COVID-19.

Cerium (III) nitrate hexahydrate, Na hydroxide, PA (50%), Ca chloride, Triton X-100, and hydrogen peroxide (3%) were obtained from Sigma-Aldrich Co. (USA). Dulbecco's modified eagle medium, penicillin-streptomycin (100×), fetal bovine serum (FBS), DPBS, 4,6-diamidino-2-phenylindole (DAPI), dihydroethidium (DHE), alamarBlue, and 2',7'-dichlorofluorescein diacetate (DCFH-DA) were purchased from Thermo Fisher. The Dox was purchased from Meilunbio. The primary antibody (Anti-P-gp; ab170904; Abcam biotechnology) was used for western blot and immunofluorescence staining. Ultrapure water (18.2 MΩ/cm), purified by a Milli-Q system was used throughout the experiments.

CeO 2 was synthesized through a two-step hydrothermal method according to our previous report [44] . For a typical preparation of PA-modified CeO 2 , 50 mg of CeO 2 were dispersed in 100 mL of 2.0 wt.% aqueous solution of PA. The mixed solutions were ultrasonicated for 1 h and then vigorously stirred at ambient temperature for 12 h. The hybrids were collected by centrifugation at 12,000 rpm for 10 min, repeatedly washed with ultrapure water for three times and dried at 50 °C. Then, the CePA hybrids were dispersed in H 2 O with a concentration of 5 mg/mL by ultrasonication for 20 min.

TEM and energy dispersive spectroscopy (EDS) mapping were examined on a JEM-2100F filed emission electron microscope with an acceleration voltage of 200 kV. XRD patterns of samples were determined using a Shimadzu X-ray diffractometer (Model 6000) with Cu Kα radiation at a scanning rate of 5°/min in the 2θ range from 10° to 90°. XPS spectra were performed on Thermo Electron Model K-Alpha. Surface charge analysis of samples were detected by Zetasizer Nano ZS 90 (Malvern Instruments). The organ and tissue distributions of cerium were measured by ICP-MS, Thermo. FT-IR spectroscopy were performed on Thermo FT-IR spectrophotometer (NICOLET 6700). Fluorescence imaging photos were taken through confocal laser scanning microscopy (Nikon A1). The SOD activity of nanoceria were determined by hydroxylamine assay-developed from xanthine oxidase assay (SOD assay kits, Beyotime, China). The levels of H 2 O 2 were analyzed by a hydrogen peroxide assay kit (Beyotime, China).

LX-2, HepG2, and HepG2/ADR cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS and 1% antibiotics (penicillin-streptomycin) in a humidified atmosphere at 37 °C (5% CO 2 and 95% air).

LX-2 cells were seeded into 96-well plates, adjusted to a density of 2 × 10 3 per well and incubated in 5% CO 2 at 37 °C for 12 h. Subsequently, LX-2 cells were treated with CeO 2 and CePA at various concentrations (0.1-1,000 μg/mL) for 24 h. After removal of cellular DMEM medium, the LX-2 cells were treated with 100 μL of 10% alamarBlue at 37 °C for 2 h. Finally, the cell survival rates were determined by the standard alamarBlue assay. Each experiment was repeated at least for three times independently.

The determination of ROS levels was via fluorescent imaging. Typically, the LX-2 and HepG2/ADR cells were planted in a confocal dish with a density of 

HepG2/ADR cells were cultured in 6-well plates at a density of 6 × 10 5 cells/well. Then, the cells were treated with DMEM (control group), CeO 2 , and CePA for 6 h, respectively. Subsequently, the HepG2/ADR cells were washed by cold fresh DPBS for three times, collected and then lysed (RIPA Lysis Buffer). The total proteins were distilled through the addition of the total protein extraction kit and the protein concentrations were tested by BCA protein assay kit (Beyotime, China). Afterwards, total proteins were loaded in the wells of 10% Na dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride (PVdF) membranes. The membranes were incubated with P-gp antibodies (Abcam, dilution 1:1,000) and β-actin (Invitrogen, dilution 1:2,000) for 4 h. Then, the membranes were blocked by 5% non-fat milk (Bio-Rad) overnight at 4 °C, then washed for three times with Western washing buffer (TBS-T), and incubated with horseradish peroxidase-conjugated secondary antibody (Invitrogen, dilution 1:5,000) for 1 h at room temperature. After washing for three times with TBS-T buffer, the protein blots could be analyzed using the chemiluminescence system.

To confirm HepG2/ADR cells were resistant to Dox, the HepG2/ADR cells (2 × 10 3 cells/well) were cultured in 100 μL of medium (96-well plates) at 37 °C for 12 h. Then, the fresh 100 μL of medium containing different concentrations of Dox (0-30 μM) was added. After the incubation for 6 h, the alamarBlue method was performed to evaluate cell viabilities. Furthermore, the viability of non-resistant liver tumor cells (HepG2) and LX-2 cells with Dox treatment at different concentrations was further evaluated by the same method. 

HepG2/ADR cells were seeded in the confocal dishes at a density of 10 5 cells/dish for 12 h. Subsequently, the CeO 2 (50 μg/mL) and CePA (50 μg/mL) were added and incubated with cells for 6 h. Dox (20 μM) was then added and co-incubated for another 6 h at 37 °C. Afterwards, the HepG2/ADR cells were fixed with 4% formaldehyde for 30 min at 4 °C, permeabilized with Triton X-100 (1%, DPBS) and washed with DPBS for three times. Finally, the HepG2/ADR cells were incubated in the dark with TUNEL solution for 1 h at 37 °C. A confocal laser scanning microscopy (Nikon A1) was utilized to capture HepG2/ADR cells images.

All male BALB/c nude mice were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China).

All animal experiments were conducted in compliance with the Institutional Animal Experiment Administration Committee of the Fourth Military Medical University. The mice were bred at the animal facilities of Xi'an Jiaotong University under the specificpathogen-free conditions in the closed-top cages. The male BALB/c nude mice were eight weeks of age (18-22 g) and housed in an environmentally controlled room (temperature 22 ± 1 °C; relative humidity 55 ± 5 °C). They were given food and water ad libitum. BALB/c nude mice were subcutaneously injected into the groin with 2 × 10 6 HepG2/ADR cells to establish the tumor mouse model. When the tumor volume reached approxima100-150 mm 3 , the mice were divided randomly into six groups (each group included 4 mice) and subjected to various treatments: (a) control group (0.2 mL of DPBS injection by intravenous), (b) Dox, (c) CeO 2 , (d) CePA, (e) CeO 2 + Dox, and (f) CePA + Dox at 5 mg/kg Dox and 5 mg/kg. CeO 2 /CePA via tail vein injection on the 1 st and 8 th day during the period of therapy (14 days), respectively. During the course of treatments, the mice body weights and tumor volumes in all groups were monitored every two days. Tumor volumes were calculated as follows: V = (width × width × length)/2. The relative tumor volumes were compared with that of the first day. After 14 days of treatment, all the mice were sacrificed with CO 2 . The main organs (heart, liver, spleen, lung, and kidney) and tumors of all mice were collected for histology analysis, TUNEL assay, and immunofluorescent staining.rescent staining.

Tumor-bearing BALB/c nude mice were injected with CeO 2 and CePA in a dosage of 5 mg/kg via tail vein injection. On the 1 st day and the 7 th day of the treatments, the mice humanely were sacrificed. The main organs (heart, liver, spleen, lung, and kidney) and tumor tissues were dissected, weighed, and homogenized. Subsequently, the main organs, tumor tissues, and urine samples were digested in aqua regia for 24 h. Afterwards, the solutions were diluted with water and filtered using 0.22 μm membrane. Finally, the Ce element content was determined by ICP-MS, which was repeatedly for three times.

Tumor-bearing BALB/c nude mice were intravenously injected with CeO 2 (5 mg/kg) and CePA (5 mg/kg), respectively. Blood samples were collected at 24 h post injection. The routine blood tests were carried out by an automatic hematology analyzer (BC-2800Vet, Mindray).

Tumor-bearing BALB/c nude mice were intravenously injected with CeO 2 (5 mg/kg) and CePA (5 mg/kg), respectively. After the treatments, the mice were sacrificed and the blood samples were collected for the cardiac and hepatic function analysis. The levels of alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase and creatine kinase isoenzyme myocardial band were determined by the Biochemical Auto Analyzer (Chemray800, Rayto).

The main organs and the separated solid tumors were washed with cold DPBS, then fixed with 4% formaldehyde at 4 °C for 24 h and then embedded in paraffin and sliced. The slices were stained with HE and the images were obtained by a light microscope. The apoptosis in heart, liver, and tumor tissue was also detected by TUNE staining (Beyotime). DHE staining was performed to test the ROS levels in heart, liver, and tumor tissues. In addition, the heart, liver and tumor tissues were stained with anti-caspase-3 antibody (Invitrogen, dilution 1:200) for immunohistochemistry analysis according to the following procedure. In order to prevent from nonspecific binding, the sections were blocked at 4 °C for 12 h in a blocking buffer containing 5% non-fat milk (Bio-Rad). Afterwards, the slices were incubated with the secondary antibody-FITC (1:50 dilution, Invitrogen) for 1 h at room temperature. After counterstained with DAPI, the slices were acquired with a confocal microscope (Nikon A1). Meantime, the apoptosis in tumor tissue was also detected by TUNE immunofluorescence staining (Beyotime Biotechnology).

All data were expressed in this article as mean result ± standard deviation (SD). Statistical evaluation was performed using unpaired Student's two-sided t test analysis with Prism. Asterisks indicate significant differences (**P < 0.005, ***P < 0.001, and ****P < 0.0001).

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