Suppression of tumor development by inducing ferroptosis may provide a potential remedy for triple-negative breast cancer (TNBC), which is sensitive to intracellular oxidative imbalance. Recently, artemisinin (ART) and its derivatives have been investigated as potential anticancer agents for the treatment of highly aggressive cancers via the induction of ferroptosis by iron-mediated cleavage of the endoperoxide bridge. Poor water solubility and insufficient availability of intracellular ferrous ions, limit the further application of ART in antitumor therapy. In this study, ferrous-supply nanocarrier was constructed based on tannic acid (TA) and ferrous ion (Fe2+)-coated on the zeolitic imidazolate framework-8(ZIF) with ART encapsulated (TA-Fe/[email protected]). via supramolecular coordination. Following the internalization of the as-prepared nano-system in cells, acidic degradation of the vehicles would facilitate the release of ART and accumulation of Fe2+. The results revealed that the newly developed nano-drug system displayed a high level of intracellular ROS generation, and markedly enhanced ferroptosis. This work provides a novel approach to enhance the potency of ferroptotic nanomedicine and new directions for TBNC therapy.
Nano Express
Fe(II) and tannic acid-cloake MOF as carrier of artemisinin for supply of ferrous ions to enhance treatment of triple-negative breast cancer
https://doi.org/10.21203/rs.3.rs-77012/v1
This work is licensed under a CC BY 4.0 License
Journal Publication
published 23 Feb, 2021
You are reading this latest preprint version
Artemisinin
Ferroptosis
Triple-negative breast cancer
Metal organic frameworks
Reactive oxygen species
Ferroptosis, a newly discovered subtype of cell death, can result in accumulation of iron-dependent lipid hydroperoxides (LPO), leading to damage of the cell structure and integrity.[1-3] Emerging evidence implied that activation of ferroptosis by several small molecules is an effective approach for tumor suppression in various experimental cancer models, and created high expectations for the potential of ferroptosis as a novel anti-cancer therapy.[4-6] Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer lacking targeted therapies, and often associated with tumor recurrence, distant metastasis, and resistance to therapy.[7] Previous studies have pointed out that xCT cystine/glutamate antiporter is highly expressed in numerous TNBC cells, playing an important role in maintaining the glutathione (GSH) levels and redox balance. [8] Reduction of the intracellular GSH content can render TNBC cells sensitive to ferroptosis, thereby killing tumor cells.[8] Notably, ferroptosis can also bypass the resistance of TNBC to routine programmed apoptosis.[9] Therefore, strategies or drugs based on inducing ferroptosis may have therapeutic potential for the clinical treatment of refractory TNBC.
Artemisinin (ART), a sesquiterpene lactone that contains a peroxide group, was isolated from the traditional Chinese plant Artemisia annua and has demonstrated desirable antitumor activity in multiple cancer cell lines.[10,11] Increasing evidence displayed that cancer cells contain significantly more intracellular iron pool than normal cells, while iron-mediated cleavage of the endoperoxide bridge allows ART to selectively cause cell death in multiple cancer cell lines.[12,13] The iron ion-depended antitumor activity has attracted increasing attention on ART-regulated ferroptosis, an iron-dependent cell death.[13] Mechanistically, ART can induce lysosomal degradation of ferritin in an autophagy-independent manner, increasing the cellular levels of ferrous ion and sensitizing cells to ferroptosis.[11]
However, whether ART induces ferroptosis in TNBC remains unclear. In addition, a series of factors, such as poor water solubility and insufficient availability of intracellular ferrous ions, limit the further application of ART in antitumor therapy.[13] ART prodrug nanocomplexes are expected to be successfully used as a prospective nano-drug delivery system for ART-based anti-tumor drugs.[14-16] In recent years, metal organic frameworks (MOFs) have been prominently used in the field of biomaterials. As a representative of MOF-type materials, the zeolitic imidazolate framework (ZIF-8) is widely used in the development of nanomedicines with characteristics of pH-responsiveness, high drug loading, and good biocompatibility.[17-19] Furthermore, the ability to incorporate an adjustable surface on MOF permits the control of surface properties and its endowment with multifunctionalities.[20,21] The supramolecular assembly of a metal-phenolic coordination coat on the MOF surface has recently attracted interest owing to the desirable properties, such as stimuli-responsive disassembly, colloidal stability, and biocompatibility.[22,23] The metal-phenolic coordination materials on the MOF could be an ideal vehicle for delivering the hydrophobic ART.
Inspired by this, we developed ferrous ion -tannic acid coordination cloaked ZIF-8 nano-system encapsulating ART (TA-Fe/[email protected]) for regulating ferroptosis in TNBC cells, which is demonstrated in Fig. 1. ZIF-8 was selected as a nanocarrier to encapsulate ART owing to its high drug loading, good biocompatibility and pH- responsive release. The ferrous ion -TA coordination coat was immobilized onto the surface of [email protected] for the purpose of dispersion stability and supply of ferrous ions (Fe2+). Following the internalization of the as-prepared nanosystem in cells, acidic degradation of the vehicles would facilitate the release of ART and accumulation of Fe2+. The upregulation of Fe2+ levels in cells would decompose ART into radicals through cleavage of the iron-mediated endoperoxide bridge, markedly enhancing the effects of ferroptosis. Conclusively, the discovery of TA-Fe/[email protected] ferroptosis may offer new perspectives for the development of novel treatments against TNBC.
Reagents
Artemisinin (99%), 2-methylimidazole (98%), zinc nitrate (ZnNO3; 98%), Ta (98%), ferrous sulfate (FeSO4; 98%), and anhydrous methanol were provided by Aladdin-Reagent Co. Ltd. (Shanghai, China).
Fabrication of TA-Fe/[email protected] nanoparticles
For the preparation of Art nanoparticles, 200 mg of Art was dissolved in 1 mL of anhydrous methanol, and 2 g of 2-methylimidazole (the solvent was 8 mL of absolute methanol) was slowly added to the obtained Art solution. Under magnetic stirring, 0.2 g of zinc nitrate (the solvent was 1 mL of absolute methanol) was slowly added. Finally, the volume of the solution was adjusted to 15 mL and stirred for 10 min to obtain a light white solution. After centrifugation at 10,000 rpm, the sample was washed thrice with methanol. The supernatant from the first centrifugation was maintained to measure the content of Art, while the precipitant was freeze dried to obtain solid state for further use.
For the preparation of TA-Fe/[email protected] nanoparticles, 40 mg of solid Art nanoparticles was dissolved in water (1 mL). Ta (80 mg) (Ta solvent was 2 mL of deionized water) was slowly added to the nanoparticle solution and the volume of the solution was adjusted to 76 mL. After stirring for 20 min, 20 mg of FeSO4 (the solvent was 4 mL of deionized water) was slowly added to the solution. After repeated stirring for 30 min, a dark purple solution was obtained. The solution was centrifuged at 10,000 rpm, washed thrice with water, and the precipitant was freeze dried to obtain solid statefor further use TA-Fe/[email protected]
Transmission electron microscopy (TEM) and dynamic light scattering analysis of TA-Fe/[email protected] nanoparticles
TEM (JEM-1230; JEOL,Tokyo, Japan) was used to determine the morphological and elemental composition of each part of the nanoparticle. Dynamic light scattering and zeta potential (DLS; Zetasizer Nano system Malvern Instruments, Malvern, United Kingdom) were used to evaluate the particle size and electrical stability of the nanoparticles.
Fourier transform infrared spectroscopy (FTIR) and thermogravimetric analysis of TA-Fe/[email protected] nanoparticles
Fourier transform infrared spectroscopy (VERTEX 70; Bruker, Bremen, Germany) and thermogravimetric analysis were used to analyze the composition of the constituents of the nanoparticles.
Measurement of encapsulation efficiency and loading capacity
High-performance liquid chromatography (HPLC) (Agilent 1200; Agilent Technologies, Santa Clara, CA.) was used to measure the amount of Art in the supernatant. The drug loading and encapsulation rates of ART can be calculated as follows:
In vitro release and pH-response of TA-Fe/[email protected] nanoparticles
The treated dialysis membrane wrapped with 2 mg of nanoparticles was placed in 50 mL of phosphate-buffered saline (PBS) with pH of 7.4 and 5.0, respectively, and shaken continuously at 37°C. The solution outside the dialysis membrane was sampled at 15 min, 30 min, 45 min, 1 h, 2 h, 4 h, 6 h, 8 h, and 10 h after the initiation of the experiment. The contents of Art in the buffer solution were measured using HPLC.
Cell culture
MDA‐MB‐231 and L929 cell lines were acquired from the American Type Culture Collection (American TypeCulture Collection, Manassas, VA, USA). Cells were cultured at 37°C and 5% CO2 humidity in RPMI-1640 medium (Solarbio, Beijing, China), which was supplemented with 10% fetal bovine serum (Cyclone,Utah, USA), 100 µg/mL of sodium pyruvate, penicillin, and streptomycin (Solarbio Beijing, China).
Cellular toxicity test in vitro
Cell viability was determined using 3-(4, 5-Dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) assay (Beyotime Biotechnology, Nanjing, China). MDA-MB-231 cells and L929 cells were cultured in standard cell media in 96-well plates (5,000 cells per well) and incubated in 5% CO2 at 37°C for 24 h. The fluid in the well was discarded, and 100 μL per well of the serum-free medium with PBS and different concentrations of ART, TA-Fe/ZIF, TA-Fe/[email protected], deferoxamine (MedChemExpress, Shanghai, China), N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone (Z-VAD-FMK; MedChemExpress), and ferrostatin-1 (MedChemExpress) were added to the 96-well plates. After 48 h, 10 μL of MTT (5 mg/mL) was added and incubated for 4 h. Finally, an automatic enzyme marker (BioTek Instruments Inc., USA) was used to measure the absorbance in each well. Results were expressed as the percentage of cell viability.
Calcein-acetoxymethyl (Calcein-AM) staining assay
MDA-MB-231 cells were cultured in 24-well plates (2×104 cells per well) and incubated for 24 h. Subsequently, the cells were treated with different concentrations of TA-Fe/[email protected] nanoparticles for 24 h. After discarding the medium, the cells were stained with Calcein-AM (Beyotime Biotechnology, Shanghai, China) in the dark at 4°C for 20 min and observed under a fluorescence inverted microscope (Olympus, Tokyo, Japan).
Flow cytometry for apoptosis
MDA-MB-231 cells were placed in a six-well plate at a density of 2.5×105 cells per well under the same conditions. After treatment with PBS, ART, TA-Fe/ZIF, and TA-Fe/[email protected] were applied for 24 h. Subsequently, the cells were centrifuged and collected from the six-well plate. After propidium iodide and Annexin-V double staining (Annexin V‐FITC Kit; Beckman Coulter, Marseille, France), flow cytometry was used to detect.
In vitro reactive oxygen species (ROS) determination assay
The determination of ROS content in cells was performed using a ROS fluorescence probe (dichloro-dihydro-fluorescein diacetate [DCFH-DA]; Beyotime Biotechnology, Shanghai, China). MDA-MB-231cells were cultured into six-well plates (2.5×105 cells per well) and incubated in 5% CO2 at 37°C for 24 h. The fluid from the wells was discarded, and cells were treated as follows: blank control group (serum-free medium), positive control group and experimental group (different concentrations of nanoparticles). Following incubation for 8 h at 37°C, 0.1% DCFH-DA was added to each well and the cells were incubated for 30 min. Cells unresponsive to DCFH-DA were removed with PBS and observed under a fluorescence inverted microscope (Olympus, Tokyo, Japan).
Malondialdehyde (MDA) and GSH content determination
The MDA assay kit (TBA method; Jiancheng Bioengineering, Nanjing, China) and GSH assay kit (Beyotime Biotechnology, Shanghai, China) were used to measure the intracellular levels of MDA and GSH. After treatment with PBS, ART, TA-Fe/ZIF and TA-Fe/[email protected], MDA-MB-231 cells were collected and counted. The intracellular content of MDA and GSH was determined according to the instructions provided in the kits.
Western blot analysis
The MDA-MB-231 cells which treated with different nanoparticles were lysed with RIPA lysis buffer. After the protein concentration was determined, the proteins of different samples were separated using 10% SDS-PAGE gel and transferred to nitrocellulose membrane. The nitrocellulose membrane which loaded with sample proteins was blocked by 0.5% BSA protein solution for 1 h, and the nitrocellulose membrane and the primary antibodies were incubated for 24 h at 4°C. We rinsed the primary antibodies from the nitrocellulose membrane with TBST and continued to place it with the corresponding secondary antibodies at ordinary temperature for 2 hours. After washing off the secondary antibodies on the in cellulose membrane, the nitrocellulose membrane was used a chemiluminescent solution and observed under the gel imaging system.
Statistical analysis
All experiments were repeated at least thrice. All data were statistically analyzed using the SPSS version 22.0 software (IBM Corp., Armonk, USA). The results were expressed as the mean ± standard deviation. P-values <0.05 denoted statistical significance.
Characterizations of [email protected] nanoparticles
First of all, the [email protected] nanoparticles were synthesized at room temperature from methanol, zinc acetate, ART, and 2-methylimidazole according to literature procedures.[24] Stable metal-polyphenol supramolecular films were then rapidly formed around the [email protected] templates by vortexing TA and ferrous ions. The encapsulation efficiency, which was measured by HPLC using the supernatant from the first centrifugation was 66.7%. The supernatant was obtained the TA-Fe/[email protected] nanoparticles and the calculated drug load of Art was 11.4%. According to FTIR spectroscopy (Fig. 2A), characteristic absorption peaks of ART, namely the carbonyl bond at 1,738 cm−1 and the peroxy bridge at 724 cm−1[25], was observed in TA-Fe/[email protected] nanoparticles, indicating that ART was successfully encapsulated in the nanoparticles. Next, the results of the thermogravimetric analysis revealed that Art completely abolished when the temperature was increased to approximately 400°C (Fig. 2B). Compared with the TA-Fe/ZIF nanoparticles, it was found that ART accounts for 7.1% of the total weight of the TA-Fe/[email protected] nanoparticles, which is basically consistent with the results of the HPLC analysis.
The results of TEM showed that ZIF-8 and [email protected] exhibited a similar uniform hexagon configuration and the particle size distribution was determined at approximately 100 nm (Fig. 3A and 3B). TA-Fe/[email protected] nanoparticles exhibited a spherical configuration and the particle size distribution was 150 nm. Compared with complete [email protected], TA-Fe/[email protected] coated with Fe2+ and TA demonstrated an obvious conventional core-shell structure, and the size of the TA-Fe membrane was approximately 30 nm (Fig. 3A). Moreover, we performed area-elemental mapping on the analysis of the formed nanoparticles. The area-elemental mapping confirmed that the periphery of TA-Fe/[email protected] nanoparticles were encircled by Fe element to determine whether [email protected] had been successfully cloaked with Fe2+and TA (Fig. 3B). By increasing the amount of added Fe2+, we found that the hydrodynamic diameter of TA-Fe/[email protected] nanoparticles was increased (Fig. 1s). To further confirm the coating, we measured the zeta potential of various nanoparticles. The formation TA-Fe layer on [email protected] particles shifted the surface zeta from +21 mV potential to –19.5 mV due to the acidic nature of TA. (Fig. 2s).
Release and cytotoxicity of TA-Fe/[email protected] in vitro
Next, in order to study whether TA-Fe/[email protected] had pH-responsive decomposition behavior, we examined the release of ART from nanoparticles under neutral and acidic conditions. As shown in Fig. 4A, in the acidic PBS solution, the nanoparticles can rapidly release approximately 58% of ART within 1 h. However, under neutral conditions, the nanoparticles can only slowly release a minute amount of ART. In addition, during the treatment of the external solution of the dialysis membrane with external solution of the dialysis membrane with sodium hydroxide, the solution of the pH=5.0 group reacted with sodium hydroxide to produce a considerable white precipitate. However, this phenomenon was not observed in the pH=7.4 group.
According to our hypothesis, the white precipitate is a zinc hydroxide precipitate formed by the dissociation of zinc ions and alkali from the nanoparticles under acidic conditions. Collectively, this evidence successfully shows that our nanoparticles have the ability to dissociate under acidic conditions.
In the design of TA-Fe/ [email protected] nanosystem, the TA-Fe and ZIF structures are ablated to release the encased ART and Fe2+ under the acidic conditions of the tumor microenvironment. The upregulation of Fe2+ levels in cells would decompose Art into radicals through cleavage of the iron-mediated endoperoxide bridge, markedly enhancing the effects of ferroptosis. Accordingly, MTT assays were conducted to study the cytotoxicity of nanosystem to MDA-MB-231 cells and L929 cells. Compared with ART, TA-Fe/ [email protected] nanoparticles showed greater cytotoxicity to MDA-MB-231 cells (Fig. 4B) and low cytotoxicity to normal cells (Fig. 4C). TA-Fe/ [email protected] nanoparticles inhibited the activity of MDA-MB-231 cells by 53.9%, while Art at the same concentration inhibited only 24.1% of the whole cells. The experimental results of calcein-AM staining confirmed that the quantity of viable MDA-MB-231 cells gradually descended with the increasing concentration of TA-Fe/ [email protected] nanoparticles. (Fig. 4D)
TA-Fe/[email protected] enhanced ROS generation in MDA-MB-231 cells
ART is known to exert its anticancer activity via generation of ROS produced by iron-mediated cleavage of the endoperoxide bridge.[26,27] Therefore, we have examined the efficiency of TA-Fe/ [email protected] to induce ROS generation by the 2’,7’-dichlorodihydrofluorescein diacetate (DCHF-DA) probe. As shown in Fig. 5, the slightly stronger fluorescence signal was observed in cells treated with ART and TA-Fe/ZIF compared with untreated groups, indicating the ART and Fe2+ ions could induce ROS generation. By contrast, strong fluorescence signal exposure to TA-Fe/ [email protected] nano-drug is found in cells as an evidence of desired ROS yield. The ROS generation by endoperoxide of ART was mediated Fe2+. The as-prepared nanosystem in cells would be degradated and make accumulation of Fe2+, remarkably enhanced ROS generation.
The anticancer effect of ART and its derivatives has been attributed to their ability to induce apoptosis by various cellular processes, ranging from DNA damage response, the lysosome-mediated catabolic process macroautophagy, and oxidative stress.[13,26,28] And it was also reported that the iron-mediated cleavage of the endoperoxide bridge in ART could affect the intracellular oxidative balance to ferroptosis in many types of cancer cells.[11] This induction of ferroptosis based on oxidative imbalance can be further amplified with the addition of Fe2+. While Fe2+ activates ART, it can also react with intracellular hydrogen peroxide to generate hydroxyl free radicals through the Fenton reaction, which enhances the apoptosis-inducing effect of ART in tumor cells.
TA-Fe/[email protected] induced apoptosis and ferroptosis in MDA-MB-231 cells
Meanwhile, to investigate that cell death and role of Fe2+, we utilized iron chelator deferoxamine, apoptosis inhibitor Z-VAD-FMK, and ferroptosis inhibitor ferrostatin-1 to rescue these cells. As predicted, deferoxamine, a scavenger of Fe2+, could obviously block cell death, suggesting the important role of Fe2+. Moreover, the survival rate of MDA-MB-231 cells was significantly improved by apoptosis and ferroptosis inhibitor from 31.4% to 56.3% and 76.0%, respectively (Fig. 6A), highlighting the potential importance of apoptosis and ferroptosis in TA-Fe/[email protected] nanoparticles mediated cell death. In addition, we employed Annexin V- FITC based assay through flow cytometry, to quantitatively determine the degree of apoptosis. The results state that TA-Fe/[email protected] nanoparticles can induce apoptosis in 21.8% of MDA-MB-231 cells (Fig. 3s). This percentage was higher than that recorded for ART, implied that apoptosis is involved in the TA-Fe/[email protected] nanoparticles mediated, but it is not the main cause.
A common feature of ferroptosis is endogenous lipid peroxidation.[29] MDA, a prouduct of lipid peroxidation[30], was investigated to assess the degree of ferroptosis. Results showed TA-Fe/[email protected] nanoparticles produced approximately 2.5 times higher MDA levels than control group (Fig. 6B). This was presumed due to the presence nanocarrier-enriched Fe2+ and the corresponding elevation of lipid radicals levels. As one of the major antioxidant components in the cells, intracellular GSH is decreased accompanied with ferroptosis.[31] Considering the import role of GSH in ferroptosis, we evaluated intracellular GSH level after treatment with TA-Fe/[email protected] nanoparticles. GSH was decreased significantly compared to vehicle-treated cells (Fig. 6C). This provided strong evidence for the depletion of GSH and oxidation imbalance, which was centered on the Fe2+ mediated activation of ART by TA-Fe/[email protected] nanoparticles. Next, western blotting was performed to understand the impact of TA-Fe/[email protected] nanoparticles. on GPX4 activity.[32] We observed that TA-Fe/[email protected] nanoparticles. caused a more significantly inhibition of GPX4 activity than did ART (Fig. 6D). These data also agreed well with the highest extent of GSH depletion. Although we have successfully demonstrated that TA-Fe/[email protected] nanoparticles can induce apoptosis/ferroptosis in TNBC cell lines, the antitumor effects of ART nanoparticles in tumor-bearing mice and the performance of ART nanoparticles in the field of metal imaging should be further investigated.
In summary, we designed metal (Fe2+)-phenolic (TA) coordination -coated on metal-organic framework MOF-wrapped ART nanoparticles to target TNCB cells through the synergism of apoptosis/ferroptosis. This method may provide new ideas and options for anti-tumor treatments, which are currently ineffective through traditional approaches.
MOF: Metal organic frameworks; TNBC: triple-negative breast cancer
ART: artemisinin; TA: tannic acid; ZIF: zeolitic imidazolate framework-8
TA-Fe/[email protected]: tannic acid and ferrous ion-coated on the zeolitic imidazolate framework-8 with artemisinin encapsulated;
ROS: Reactive oxygen species; LPO: lipid hydroperoxides; GSH: glutathione;
TEM:Transmission electron microscopy;
FTIR: Fourier transform infrared spectroscopy;
HPLC: High-performance liquid chromatography; PBS :phosphate-buffered saline;
MTT: 3-(4, 5-Dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide;
DFO: deferoxamine; Fer-1:ferrostatin-1;
Z-VAD-FMK: N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone;
Calcein-AM: Calcein-acetoxymethyl;
DCFH-DA: dichloro-dihydro-fluorescein diacetate; MDA: Malondialdehyde;
BSA：bovine serum albumin; TBST：Tris Buffered saline Tween;
SDS-PAGE: polyacrylamide gel electrophoresis’ GPX4: glutathione peroxidase 4
Authors’ Contributions:
Z. Li and X. Wu performed the experiments and drafted the manuscript. W. Wang prepared and characterized the nanoparticles. C.Gai performed the statistical design of experiments. This research was carried out under the instruction of W. Zhang, W. Li and D. Ding. All authors helped to correct and polish the manuscript and read and approved the final manuscript.
Funding
The present study was supported by the Projects of Medical and Health Technology Development Program in Shandong province (Grant No. 2019WS590) and Project of Shandong Province Higher Educational Science and Technology Program (Grant No. J18KA279).
Competing Interests
The authors declare that they have no competing interests.
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