Fe3+ ions chelated with ultrasmall polydopamine nanoparticles

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Fe /Fe ions chelated with ultrasmall polydopamine nanoparticles inducing Ferroptosis for cancer therapy Lu Chen, ZhenJie Lin, LiZhu Liu, XiuMing Zhang, Wei Shi, Dongtao Ge, and Yanan Sun ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.9b00461 • Publication Date (Web): 12 Aug 2019 Downloaded from pubs.acs.org on August 12, 2019

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ACS Biomaterials Science & Engineering

Fe2+/Fe3+ ions chelated with ultrasmall polydopamine nanoparticles inducing Ferroptosis for cancer therapy

Lu Chen, Zhenjie Lin, Lizhu Liu, Xiuming Zhang, Wei Shi, Dongtao Ge, Yanan Sun*

Key Laboratory of Biomedical Engineering of Fujian Province University/Research Center of Biomedical Engineering of Xiamen, Department of Biomaterials, College of Materials, Xiamen University, Xiamen 361005, China

Corresponding Author: Yanan Sun, PhD., Associate Professor Department of Biomaterials/Biomedical Engineering Research Center College of Materials Xiamen University Xiamen 361005 P. R. China Tel: +86-15805912545 Fax: +86-592-2185502 *E-mail: [email protected]

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Abstract

Ferroptosis, a promising mechanism of killing cancer cells, has become a research hotspot in cancer therapy. Besides, polymeric nanomaterials held superiority in improving anti -cancer efficacy, reducing side effect that has been widely accepted. In this work, based on the chelating metal ions property of polypodamine, UPDAPEG@Fe2+/3+ nanoparticles, a novel ferroptosis agent, was rationally designed by chelating iron ions on the ultrasmall polydopamine nanoparticles modified by PEG. The ultrasmall treatment leaded to bigger specific surface area, which could support more reactive sites to chelating a large number of iron ions, which benefited to explore detail mechanism of ferroptosis-induced tumor cell death by iron ions. And the iron ions release with pH-responsible can reach to approximately 70% at pH = 5.0, which take advantage of applying in tumor microenvironment. The in vitro tests showed that the as-prepared NPs exhibit effective anticancer effect towards tumor cells including 4T1 and U87MG cells, yet, ferric ions show stronger ability of killing cancer cells than ferrous ions. Differences between ferrous ions and ferric ions in the ferroptosis pathway were monitored by the change of marker including ROS, GPX4 and LPO, as well as the promoter and inhibiter of ferroptosis pathway. UPDA-PEG@Fe2+ nanoparticles induce ferroptosis more depended on ROS, however, more LPO dependent ferroptosis was induced by UPDA-PEG@Fe3+ nanoparticles. Additionally, the in vivo studies using tumor-bearing balb/c mice demonstrated that the as-prepared NPs could significantly inhibit tumor progression. The UPDA-PEG@Fe2+/3+ nanoparticles reported herein represent iron ions-related nanoparticles for chemotherapy against


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cancer by the ferroptosis pathway.

Keywords: Ultrasmall Polydopamine, Iron ions, Ferroptosis, Cancer therapy

Introduction

Ferroptosis, an iron-dependent form non-apoptotic regulated cell death, is defined by Dr. Brent R. Stockwell in 20121, 2. It is different from other well-known pathways of cell death including apoptosis, necrosis, autophagy in morphological, biochemical and genetical fields. Furthermore, ferroptosis is found closely related to intercellular iron concentration3, 4. The iron-mediated reactive oxygen species (ROS) produced by Fenton reaction is important for inducing ferroptosis5-7. During the ferroptosis, it is initialized through inactivation of glutathione peroxidase 4 (GPX4) activity or glutathione (GSH) depletion, besides that, lipid peroxidation products and reactive oxygen species are consider playing the main role in this process8-11. Cell death is crucial for normal development, homeostasis and prevention of hyper proliferative diseases, such as cancer. Therefore, it is new and promising strategy for cancer therapy that activation of regulating cell death by ferroptosis during tumor development.

Previously, there are lots of iron-based nanomaterials such as iron oxide nanoparticles, iron-doped nanomaterials, iron-based metal-organic networks, and iron-



based nanomaterials-drug composites have been reported to apply as anticancer agents12-17. Most materials related with Fe were applied in cancer chemodynamic therapy by Fenton reaction generating ROS18-21. However, little work has focus on the regulation mechanisms of different iron ions in the cancer therapy, which prevents the iron-based nanomaterials further application in development the strategies for cancer therapy. In addition, little work has focus on the regulation mechanisms of different iron ions in the cancer therapy, which prevents the iron-based nanomaterials further application in development the strategies for cancer therapy. The low iron loading and the tedious releasing route greatly restrict the therapy efficiency of these materials in biomedical usages.

Recently, melanin like polydopamine attract great attention as biomaterials applied in anticancer therapy due to their interesting properties and various biological functions, including high biocompatibility, photothermal property, racical scavenging and strong metal ion chelation22-28. Previous research has conducted that the 98 nm PDA@Fe3+ exhibited the size-dependent Fe3+ loading with 7.2 ug Fe per mg PDA nanoparticles29. By copolymerization strategy, the ferric ion loading can reach to 102.6 ug/mg30, which is still lower than iron binding and storage protein in the body. Thus, how to increase the metal ions loading efficiency with polydopamine to enhance application effect is worth thinking about.

In this report, we synthesized ultrasmall polyethylene glycol-modified polydopamine nanoparticles (UPDA-PEG) in order to increase the surface-to-volume


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ratio and provide more reaction site to increase the iron ions chelation. The UPDAPEG-Fe with high metal loading capacity has controllable pH-sensitive iron ion release, thus reducing the side effect and allowing for their medical application. To further investigate the regulation mechanism of different iron ions in the cancer therapy, we chelated UPDA-PEG with ferrous ions or ferric ions. As is shown in Scheme 1, UPDAPEG nanoparticles can be used as the carrier to transport the iron ions inside the cell, then iron ions were released though pH and H2O2 responsive disassembly and induce cell death by ferroptosis. We found that ferrous ions eager to induce Fenton-like reaction to produce ROS, and ferric ions incline to regulate the intercellular LPO. Herein, we assessed the therapeutic effect of UPDA-PEG@Fe2+ and UPDA-PEG@Fe3+ nanoparticles as a novel nanomedicine for the cancer therapy, and discuss the role of iron ion in cancer therapy by ferroptosis, providing an improved understanding of ferroptotic mechanism-based therapeutic approaches.



Scheme 1. Synthesis and ferroptosis mechanism of UPDA-PEG@Fe2+/3+ nanoparticles

Results and discussion

Synthesis and Characterization of nanoparticles

PDA nanoparticles was synthesized by oxidation and self-polymerization of dopamine in NaOH solution at a temperature of 60 °C for 5 h, which size was 100 nm approximately (Figure S1)31. Then the ultrasmall PDA nanoparticles was obtained through ultrasonication in NaOH solution32. To maintain the stability of UPDA nanoparticles for further metal ion-chelating, they were modified with amino-


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terminated polyethylene glycol (NH2-PEG-NH2) on surface by Michael addition reaction. Furthermore, it could enhance the nanoparticles biocompatibility and prolong the blood circulation time. To verify the stability of UPDA-PEG nanoparticles, they were dispersed into different mediums, and there were no visible sediments after 72 h (Figure S2). The results indicate that UPDA-PEG nanoparticles had excellent stability. Lastly, ferric ions and ferrous ions were chelated with the UPDA-PEG nanoparticles.

TEM images demonstrate the as-prepared UPDA, UPDA-PEG, UPDAPEG@Fe2+ and UPDA-PEG@Fe3+ nanoparticles showed well defined spherical morphology and well-dispersed in water (Figure 1a-d). Based on the TEM, the average diameter of as-prepared nanoparticles was increased by modification of PEG and chelation of iron ions with size of 5.5, 8.4, 12.8 and 13.2 nm for UPDA, UPDA-PEG, UPDA-PEG@Fe2+

and

UPDA-PEG@Fe3+

nanoparticles,

respectively.

The

hydrodynamic diameter measured by the dynamic light scattering (DLS) analysis were 6.9, 14.2, 17.4 and 18.7 nm, with the poly-dispersity index data were 0.169, 0.165, 0.074, 0.121 respectively, which identified with the results of TEM image (Figure S3). As is revealed in Figure S4, the zeta potential results suggeste the surface charge of nanoparticles increased gradually with the ultrasonication disruption reaction and PEGylating from -34.2 mV to -24.3 mV and -10.3 mV. After ultrasmall by ultrasonication, the nanoparticles had less negative functional groups, which induce the surface charger changed. The positive NH2 groups provided by PEG modification decreased the surface potential of UPDA-PEG nanoparticles. Along with chelating iron ions, the surface charge was changed from -10.3 mV to 7.88 mV and 10.1 mV,



respectively, which was due to the iron ions were positive. Moreover, the changes of size and zeta potentials could indirectly demonstrate PEG and iron ions were integrated successfully with UPDA nanoparticles.

PDA nanoparticles have the capability of chelating metal ion, which owes to the catechol-like structures that make rich of phenyls and hydroxyl groups exist on the surface of nanoparticles. As is shown in Figure 1e, the Fourier Transform infrared spectrometer (FTIR) spectrum of the PDA showed two weak typical peaks of indole and indoline structures at ≈ 1624 and 1530 cm-1, which indicates that the oxidative polymerization process of PDAs formation. Besides that, a broad peak around 3320 cm1 could be observed, which relates to the -NH- and -OH- groups in PDA nanoparticles33.

According to the FTIR spectrum, it shows that ultrasmall reaction could barely alter the structure of PDA nanoparticles, which means that UPDA nanoparticles also had the ability of chelating metal ions. After PEG modified on the surface of UPDA nanoparticles, the two typical peak at≈2876 and 1100 cm-1 were observed, relating to the -CH2- groups in PEG and -C-O-C- bond in UPDA-PEG nanoparticles, which confirms the successful modification of PEG. With the chelation of iron ions, the peak at≈2974 cm-1 was disappeared, the peak at≈1097 cm-1 changed from double peaks to single peak and the peak at≈950 cm-1 was observed which indicates the formation of Fe-OH bond, that all means the successful chelation of iron ions. The peak at 210 nm in ultraviolet visible and near infrared spectrophotometer (UV-Vis-NIR) spectrum is related to the catechol groups, which confirms the existence of PDA nanoparticles34. As the chelation of ferric ions with UPDA-PEG nanoparticles, according to the


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previously reported results, the UV-Vis-NIR spectra suggests that the increase at the peak of ≈300 nm with the increase of the reactants quality ratio, which confirms that UPDA-PEG@Fe3+ complexes formed by chelating reaction at the ratio of 1:3 (Figure S5)35. However, the UV-Vis-NIR spectra of UPDA-PEG@Fe2+ complexes reveals there were not obvious change of peak and no difference among the UPDA-PEG@Fe2+ complexes. But compared to the UPDA-PEG@Fe2+ complexes, the absorbance of UPDA-PEG at 300~800 nm slightly declined, which due to the chelation of ferrous ions. It indicates that ferrous ion has no variation of valence states during the reaction period (Figure S6). To explore the specific iron ions valence situation, the valence of iron ions in UPDA-PEG@Fe2+ and UPDA-PEG@Fe3+ nanoparticles were measured by X-ray photoelectron spectrometry (XPS). As is showed in Figure 1f, this result demonstrates the successful loading of iron ions on the surface of UPDA-PEG. Additionally, the central peak centered at ∼ 725.0 eV (Fe 2p2/3) and the shakeup satellite peak at ∼ 711.0 eV (Fe 2p1/2) in the spectra of UPDA-PEG@Fe3+ nanoparticles showes the existence of ferric ions, however, the central peak centered at ∼ 723.0 eV (Fe 2p2/3) and the shakeup satellite peak at ∼ 708.5 eV (Fe 2p1/2) in the spectra of UPDA-PEG@Fe2+ nanoparticles indicates the existence of ferrous ions.

Ultrasmall nanoparticles have more reactive site on the surface that contributed to larger specific surface area. Therefore, UPDA-PEG nanoparticles can chelate more iron ions. To evaluate the chelation maximum, different ratio of iron ions reacted with UPDA-PEG nanoparticles, and then the iron content of products was analyzed by inductively coupled plasma mass spectrometry (ICP-MS). It turned out that the ferrous



ion peak concentration of UPDA-PEG@Fe2+ nanoparticles is 860.75 μg/mg, and the ferric ion peak concentration of UPDA-PEG@Fe3+ nanoparticles is 691 μg/mg (Figure S7), which is far more than 7.2 ug/mg of 98 nm polydopamine nanoparticle doping with ferric ion29. By changing synthetic method with prepolymerization doping strategy, the maximum Fe ions loading could only reach 102.6 ug/mg30. It was worth noting that our iron ions loading concentration were even higher than endogenous iron binding and storage protein in the body, such as hemoglobin (340 μg/mg) and ferritin (560 μg/mg).

As we all know, the endogenous stimulations, such as enzymes, pH and redox reactions, can provide the controllable tumor-targeted drug release. We investigated the iron ion release of UPDA-PEG@Fe2+ and UPDA-PEG@Fe3+ nanoparticles, by simulating mildly acid tumor microenvironment and under oxidative stress associated with the increased generation of ROS including H2O2. The pH-responsive release was tested by dialysis in three buffer solutions with different pH value. The results of the iron ions content in buffer solution measured by ICP-MS demonstrate that the more acidic the solution, the more accumulative release (Figure 1g-h). In the neutral buffer, there were little iron ions release, which means UPDA-PEG@Fe2+ and UPDAPEG@Fe3+ nanoparticles are may steady under normal physiological environment. The release of UPDA-PEG@Fe3+ nanoparticles reached 59.0 % at an acidic condition of pH 5.0, which was 18 % higher than in acid buffer of pH 6.5, and also was almost 11–fold of that in the neutral buffer, while the release of UPDA-PEG@Fe2+ nanoparticles in acid buffer of pH 5.0 was 65.52%, which was also higher compare to that in an acidic condition of pH 6.5 (44.52%) and neutral condition (6.85%). With the addition of


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10mM H2O2 in the acid buffer solution, the release improved to 73.13% for UPDAPEG@Fe2+ nanoparticles and to 67.15% for UPDA-PEG@Fe3+ nanoparticles. The acid triggered release is probably due to hydroxyl protonation on the UPDA part, which destroyed the coordinate bond between UPDA nanoparticles and iron ions partially. In addition, in the presence of H2O2, the Fenton reaction and degradation of UPDA nanoparticles could also facilitate the release of iron ions. The stimulus including pH and H2O2 may enhance the accumulation of free iron ions in cancer cells so that the cancer therapeutic efficacy could be improved. Meanwhile, the controlled-released manner of UPDA-PEG@Fe2+ or UPDA-PEG@Fe3+ nanoparticles are beneficial to attenuate systematic toxicity of iron ions. On the other hand, UPDA-PEG nanoparticles are excellent as carrier, which could chelate diverse kind of metal ions without other additional modification and respond to different stimuli to initiate the release of metal ions.

Considering the iron ions can induce a Fenton reaction and convert less-reactive H2O2 into most harmful •OH to induce the chemodynamic therapy, we use the electron paramagnetic resonance (EPR) spectroscopy to detect the generated 1:2:2:1 hydroxyl radical signals via nanoparticles reacted with 10 mM H2O2. The spin-trapping agents DMPO was used to trap •OH and produce a stable EPR signal of paramagnetic adduct DMPO-OH. According to the signal peak in Figure 1i, it has been suggested that under the existing of H2O2, ferrous ions can generate much more •OH than ferric ions. Note that iron ions loading on UPDA-PEG nanoparticles can reduce the ability of producing •OH28. We reason that UPDA nanoparticles have the strong chelating capability with



iron ions, thereby inhibiting the production of •OH. The signal of •OH could be the result of the iron ions released by reaction between UPDA-PEG@Fe2+ or UPDAPEG@Fe3+ nanoparticles with H2O2. These can explain by the controlled-released result in Figure 1g and 1h.

Figure 1. TEM image of UPDA (a), UPDA-PEG (b), UPDA-PEG@Fe2+ nanoparticles (c), UPDA-PEG@Fe3+ nanoparticles (d). (e) FT-IR spectra of PDA, UPDA, UPDA-PEG, UPDAPEG@Fe2+ and UPDA-PEG@Fe3+ nanoparticles from top to bottom. (f) XPS spectra of UPDAPEG@Fe2+ and UPDA-PEG@Fe3+ nanoparticles. (g) Iron ions release of UPDA-PEG@Fe2+ nanoparticles for 24 h. (h) Iron ions release of UPDA-PEG@Fe3+ nanoparticles for 24 h. (i) •OH detection of UPDA-PEG, UPDA-PEG@Fe2+,UPDA-PEG@Fe3+ nanoparticles, FeCl2 and FeCl3 with H2O2 by EPR


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In Vitro anticancer activity

Iron-based nanomaterials have attracted much attention for generating •OH in Fenton reaction and can induce cell death by chemodynamic therapy13. In this work, we first examined the cytotoxic effect of UPDA-PEG@Fe2+ and UPDA-PEG@Fe3+ nanoparticles by using the MTT assay (Figure 2a-b). The nanoparticles were incubated with breast cancer 4T1 cells, glioblastoma U87MG cells and normal L929 cells at a series of different concentrations for 24 h, respectively, which included 0 / 25/50/100/200/300/400/500 μg/mL, containing 0/0.35/0.7/1.4/2.8/4.2/5.6/7 mM iron ions. PBS, FeCl2, FeCl3 and UPDA-PEG nanoparticles were used as controls with the same concentration series. For UPDA-PEG nanoparticles groups, the cell viability result suggests all cells viability were over 95%, which means that there were no obvious cytotoxic for UPDA-PEG nanoparticles in the analyzed concentration range, and this is consistent with the previously report (Figure S8). Combining with the MTT assay results of FeCl2 and FeCl3 (Figure S9-10), the results show that there was dosedependent cytotoxicity for FeCl2, FeCl3, UPDA-PEG@Fe2+ and UPDA-PEG@Fe3+ nanoparticles groups, and that the toxicity of UPDA-PEG@Fe2+ and UPDAPEG@Fe3+ nanoparticles were significantly higher than corresponding free iron ions in the same concentration treatment.

The free iron ions need to enter cells by active transport, while the uptake method



of the nanoparticles is endocytosis. We assume that the two different ways above mentioned influence the internalization of iron ions. To prove the foregoing viewpoints, the cell uptake of iron ions was detected by ICP-MS. As is shown in Figure 2c, the ICPMS result shows the Fe content of cells treated with UPDA-PEG@Fe2+ and UPDAPEG@Fe3+ nanoparticles were almost 5-fold than the cells treated with FeCl2 or FeCl3, which demonstrates that UPDA-PEG carrier can transport more iron ions into cells.

Besides that, we found that the cytotoxic of UPDA-PEG@Fe3+ nanoparticles were higher than UPDA-PEG@Fe2+ nanoparticles. As is known to all, materials that related with iron ions can generate ROS by Fenton reaction, which can induce cell death through chemodynamic, so we use the ROS fluorescence probe 2,7-dichlorofluorescin diacetate (DCFH-DA) to detect the generation of ROS in U87MG and 4T1 cancer cells treated with PBS, UPDA-PEG nanoparticles, FeCl2, FeCl3, UPDA-PEG@Fe2+ nanoparticles and UPDA-PEG@Fe3+ nanoparticles. As is shown in Fig 2d and Fig S11, ferrous ions could regulate ROS level more remarkable than ferric ions. This is same with the EPR result in Figure 1i. Compared to the PBS treated cells, UPDA-PEG nanoparticles without iron ions can eliminate the production of ROS in cells. According to the previous report, PDA nanoparticles have the ability of scavenging free radical and activity of anti-oxidative effect28. As a consequence, the degree of intracellular ROS induced by UPDA-PEG@Fe2+ and UPDA-PEG@Fe3+ nanoparticles were lower than FeCl2 treated cells. In addition, UPDA-PEG@Fe3+ nanoparticles can generate ROS little less than UPDA-PEG@Fe2+ nanoparticles, which may be related to the interconversion of ferrous ion and ferric ion in cellular. Nevertheless, compared to


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UPDA-PEG@Fe2+ nanoparticles, UPDA-PEG@Fe3+ nanoparticles could induce more cancer cell death in cytotoxicity assay (Fig2a and b), so we consider it must be another cell death pathways related with iron ions beside the ROS-mediated cell death.

Figure 2. (a) Cell cytotoxicity of UPDA-PEG@Fe2+ nanoparticles (b) Cell cytotoxicity of UPDAPEG@Fe3+ nanoparticles (c) Iron ions uptake of FeCl2, FeCl3, UPDA-PEG@Fe2+ and UPDAPEG@Fe3+ nanoparticles in U87MG and 4T1 cell (d) ROS assay of U87MG cells after treatment of various agents at 4 h



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It is well known that ferroptosis is a new iron-dependent cell death form. As is shown in Figure 3, cells treated with UPDA-PEG@Fe2+ and UPDA-PEG@Fe3+ nanoparticles had change of mitochondrial shrinkage, compared to cells with PBS treatment,

which

could

indicate

UPDA-PEG@Fe2+

and

UPDA-PEG@Fe3+

nanoparticles induced ferroptosis to make cells death.

Figure 3. TEM images of mitochondrial in cells treated with PBS, UPDA-PEG@Fe2+ and UPDA-PEG@Fe3+ nanoparticles

To further verify if UPDA-PEG@Fe2+ and UPDA-PEG@Fe3+ nanoparticles could cause cancer cell by the pathway of ferroptosis, different inhibitors target cell death associated pathways were applied to regulate the cell viability of UPDA-PEG@Fe2+ and UPDA-PEG@Fe3+ nanoparticles treated cells (Figure 4a and Figure S12). Ferrostatin-1 (Fer), a small molecule inhibitor of ferroptosis, was demonstrated to obviously rescue the nanoparticles regulated cancer cells from death. Besides, AcDEVE-CHO (inhibitor for apoptosis), 3-methyi-adenine (inhibitor for autophagy) and Necrostatin-1 (inhibitor for necroptosis) could hardly influence the nanoparticles induced cell death. These results show that only adding the Fer inhibitor, the cell


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viability obviously increased, however, the cell viability was almost changeless after adding other inhibitors, which means UPDA-PEG@Fe2+ and UPDA-PEG@Fe3+ nanoparticles induced cancer cells death by ferroptosis. In addition, the Fer inhibitor could inhibit more cancer cells death in UPDA-PEG@Fe3+ nanoparticles group than in UPDA-PEG@Fe2+ nanoparticles group, which means UPDA-PEG@Fe3+ nanoparticles could trigger stronger ferroptosis than UPDA-PEG@Fe2+ nanoparticles.

To further confirm that ferroptosis caused by UPDA-PEG@Fe2+ and UPDAPEG@Fe3+ nanoparticles induced the cells death, various types of inhibitors and promotors related ferroptosis pathway previously reported were used to regulate the pathway. As is shown in Figure 4b and Figure S13, DFO could rescue UPDAPEG@Fe2+ and UPDA-PEG@Fe3+ nanoparticles-treated cells, which is an iron chelating agent. It coincides with the iron-dependent feature of ferroptosis. Moreover, Vitamin C (VC) is the scavenger of cytoplasmic ROS, which showed significantly reduce the cytotoxicity of UPDA-PEG@Fe2+ nanoparticles and UPDA-PEG@Fe3+ nanoparticles, besides that, VC can inhibit more cancer cells death in UPDAPEG@Fe2+ nanoparticles group than in UPDA-PEG@Fe3+ nanoparticles group, which indicates UPDA-PEG@Fe2+ nanoparticles induced ferroptosis more depended on ROS. Vitamin E is the scavenger of cytoplasmic LPO, the result showed more effective ability to alleviate the cytotoxicity of UPDA-PEG@Fe3+ nanoparticles than UPDAPEG@Fe2+ nanoparticles, which means UPDA-PEG@Fe3+ nanoparticles induced ferroptosis more depended on LPO. UPDA-PEG@Fe2+ and UPDA-PEG@Fe3+ nanoparticles-treated cancer cells have suffered from Cystine and glutathione applied



as the inhibitor of ferroptosis. The UPDA-PEG@Fe2+ and UPDA-PEG@Fe3+ nanoparticles-treated cell viability obviously increased with the addition of Cystine as same as glutathione. In contrast, glutamate is the promoter of ferroptosis, which facilitated more cancer cells death suffered from ferroptosis that UPDA-PEG@Fe2+ nanoparticles and UPDA-PEG@Fe3+ nanoparticles induced. Above all, there was no doubt that the pathway of cell death induced by UPDA-PEG@Fe2+ and UPDAPEG@Fe3+ nanoparticles is ferroptosis. Additionally, UPDA-PEG@Fe2+ nanoparticles could induce more ROS-dependent ferroptosis. But the ferric ions and ferrous ions could interconvert in cells, so the detail mechanism of different iron ions function in ferroptosis need further investigate.

Ferroptosis is usually driven by loss of activity of GPX4 and subsequent accumulation of LPO. To further confirm UPDA-PEG@Fe2+ and UPDA-PEG@Fe3+ nanoparticles evoking ferroptosis mechanism, the intercellular GPX4 and LPO content were quantitatively tested. As shown in Figure 4c and S14, UPDA-PEG@Fe2+ and UPDA-PEG@Fe3+ nanoparticles treatment cause remarkable not only the decrease of the GPX4 activity ratio, but also the increase of the intercellular LPO ratio (Figure 4d and Figure S15). Notably, ferric ions influence cellular GPX4 and LPO level more than ferrous ions, and as the result of that UPDA-PEG could transport more iron ions into cell, the ability of influencing GPX4 and LPO of UPDA-PEG@Fe2+ and UPDAPEG@Fe3+ nanoparticles was stronger than corresponding iron ions. Our preliminary result indicates that UPDA-PEG@Fe2+ and UPDA-PEG@Fe3+ nanoparticles could induce cancer cell death by ferroptosis and ferric ions have much stronger ability than


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ferrous ions to induce ferroptosis in cancer cells result.

Figure 4. (a) Viability of U87MG cells treated with UPDA-PEG@Fe2+/3+ nanoparticles ± ferrostatin-1 (Fer, 100 nM), Ac-DEVD-CHO (Apo, 50 μM), Necrostatin-1 (Nec, 490 nM), 3methyladenine (Aut, 60 μM) (b) Viability of U87MG cells treated with UPDA-PEG@Fe2+/3+ nanoparticles ± deferoxamine (DFO, 100 μM), sodium ascorbate (VC, 20 μM), vitamin E (VE, 20 μM),glutamic (Glu,1 mM), cystine (Cys, 1 mM) and glutathione (GSH, 1 mM) (c) GPX4 activity assay of U87MG cells after treatment of various agents at 1, 2 and 4 h (d) LPO content assay of U87MG cells after treatment of various agents at 1, 2 and 4 h



In Vivo Anticancer Therapy

Inspired by the above anticancer result in vitro, we study the anticancer therapeutic application of UPDA-PEG@Fe2+ and UPDA-PEG@Fe3+ nanoparticles in vivo using 4T1 tumor-bearing Balb/c mice. 4T1 tumor-bearing Balb/c mice were randomly divided into six groups: PBS, UPDA-PEG, FeCl2, FeCl3, UPDA-PEG@Fe2+ and UPDA-PEG@Fe3+ nanoparticles by injecting intravenously. The treatment started when the tumor volume reached about 200 mm3, and kinds of agents were injected at a time of point of first, third and fifth day, respectively. As is depicted in Figure 5a-b, UPDA-PEG@Fe2+ and UPDA-PEG@Fe3+ nanoparticles treatment was found to have a higher efficiency in inhibiting tumor growth than FeCl2, FeCl3, UPDA-PEG or PBS treatment, which confirms UPDA-PEG@Fe2+ and UPDA-PEG@Fe3+ nanoparticles are effective for tumor suppression. To demonstrate the side effect, thus the change of body weight was monitored. As is given in Figure 5c, during the treatment period, there were no significant body weight loss for each group of mice. The result showed that the UPDA-PEG@Fe2+ and UPDA-PEG@Fe3+ nanoparticles have a good biocompatibility and did not induce detectable side effects in vivo.

The biodistribution of UPDA-PEG@Fe2+ and UPDA-PEG@Fe3+ nanoparticles in heart, lung, kidney, spleen and tumor over 24 h were investigated after injected intravenously through ICP-MS (Figure 5d) and fluorescence imaging (Figure S16 & Figure S17). The result indicates that UPDA-PEG@Fe2+ and UPDA-PEG@Fe3+ nanoparticles were mainly in liver and spleen, a minority of them in lung and heart, and


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a few in kidney and tumor. The Fe contents in major organs were in the normal range. In addition, to examine if the experimental mice treated with UPDA-PEG@Fe2+ and UPDA-PEG@Fe3+ nanoparticles were in healthy condition, the blood biochemical indexes were tested, and the results show that the blood biochemical indexes of experimental mice was hardly different with control mice treated with physiological saline, which means UPDA-PEG@Fe2+ and UPDA-PEG@Fe3+ nanoparticles could not induce the serious side effect (Table S1).

Figure 5. (a)Tumor volume of mice receiving injections of UPDA-PEG/ UPDA-PEG@Fe2+/3+ nanoparticles at a dose of 10 mg/kg, FeCl3/FeCl2 at a dose of 7 mg/kg (n = 5 for all groups) in 4T1



tumor bearing mice (b) Images of 4T1 tumor after 25 days (c) Body Weight of mice receiving injections of UPDA-PEG/ UPDA-PEG@Fe2+/3+ nanoparticles at a dose of 10 mg/kg, FeCl3/FeCl2 at a dose of 7 mg/kg (n = 5 for all groups) in 4T1 tumor bearing mice (b) Biodistribution of UPDA-PEG@Fe2+ and UPDA-PEG@Fe3+ nanoparticles after treatment 24h

To explore the UPDA-PEG@Fe2+/3+ nanoparticles toxicity, the Fe content of blood were monitored by time for 24h after injected intravenously (Figure S18), the result shows that the Fe content in blood gradually decrease, after 24h, it returned to the normal level, which means the UPDA-PEG@Fe2+/3+ nanoparticles can be metabolized and have no damage in vivo. The pathomorphology analysis for major organs (including heart, liver, lung, kidney, spleen and tumor. was also performed. It could be found from Figure 5 that there were also no apparent damages or changes for these major organs. The tumor tissues of FeCl2, FeCl3, UPDA-PEG@Fe2+ and UPDA-PEG@Fe3+ nanoparticles group had obvious damages, which were more and more cancer cells death in sequence. This result showed that there were outstanding therapeutic effect more or less in tumor treated with ferrous or ferric ions, which was not seen in PBS and UPDA-PEG nanoparticles groups. It indicates that all materials have no damages for the major organs, and based on that, some of these materials, related with iron ion, can kill tumor cells effectively.


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Figure 6. H&E stains imaging of major organs after 25 days

Conclusion

In summary, we fabricated an ultrasmall polydopamine nanoparticles with an ultrahigh chelating quantities of iron ions. This UPDA-PEG@Fe nanoparticle has sensitively pH-dependent iron ions release behavior and chemo-Fenton catalysis with H2O2. The in vitro anticancer research revealed that UPDA-PEG@Fe nanoparticle could induce cell death by ferroptosis pathway. Moreover, the detailed mechanism study suggested that UPDA-PEG@Fe2+ and UPDA-PEG@Fe3+ nanoparticles could greatly promote the synthesis of reactive oxygen species (ROS), suppress the GPX4



activity, produce lots of lipid peroxide (LPO), and thus result in enhancing the oxidative stress induced ferroptosis in different degree within the cancer cells. Additionally, the experimental results indicate ferroptosis induced by UPDA-PEG@Fe2+ nanoparticles depended more on ROS, however, UPDA-PEG@Fe3+ nanoparticles induce LPOdependent ferroptosis. The UPDA-PEG@Fe nanoparticle exhibited excellent tumor inhibition in response the tumor microenvironment and by inducing the ferroptosis. Therefore, we used iron ions chelating nanoparticles to discuss the role of iron ion in ferroptosis induced cancer cell death pathway and provided an improved understanding of ferroptotic mechanism-based therapeutic approaches. This high metal ions loading strategy can be expanded to chelating more different metal ions for applying to other nanomedical research area.

Author information

Corresponding author *E-mail: [email protected]

Notes

There are no conflicts to declare.


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Acknowledgment

This work was supported by the National Nature Science Foundation of China (31870986, 81271689 and 31271009), the Program for New Century Excellent Talents in University, the Program for New Century Excellent Talents in Fujian Province University, and the Fundamental Research Funds for the Central Universities (20720150087).

Supporting Information This material is available free of charge via the Internet at http://pubs.acs.org.

References （1）Dixon, S. J.; M.;

Lemberg, K. M.;

Gleason, C. E.;

Patel, D. N.;

Lamprecht, M. R.; Bauer, A. J.;

Skouta, R.; Zaitsev, E.

Cantley, A. M.; Yang, W. S.;

Morrison, B.; Stockwell, B. R., Ferroptosis: An Iron-Dependent Form of Nonapoptotic Cell Death. Cell 2012, 149 (5), 1060-1072. （2）Stockwell, B. R.;

Angeli, J. P. F.;

Dixon, S. J.;

Fulda, S.;

Jiang, X. J.;

Linkermann, A.;

Pagnussat, G. C.;

Gascon, S.;

Park, J.;

Bayir, H.;

Hatzios, S. K.;

Murphy, M. E.;

Ran, Q.;

Bush, A. I.;

Kagan, V. E.; Noel, K.;

Overholtzer, M.;

Rosenfeld, C. S.;


Conrad, M.;

Oyagi, A.;

Salnikow, K.; Tang, D.


L.;

Torti, F. M.;

Torti, S. V.;

Toyokuni, S.;

Page 26 of 32

Woerpel, K. A.; Zhang, D. D.,

Ferroptosis: A Regulated Cell Death Nexus Linking Metabolism, Redox Biology, and Disease. Cell 2017, 171 (2), 273-285. （3）Yang, W. S.; Stockwell, B. R., Ferrootosis: Death by Lipid Peroxidation. Trends in Cell Biology 2016, 26 (3), 165-176. （4）Yang, W. S.; R.;

SriRamaratnam, R.;

Viswanathan, V. S.;

C. B.;

Brown, L. M.;

Welsch, M. E.;

Shimada, K.; Skouta,

Cheah, J. H.; Clemons, P. A.; Shamji, A. F.;

Girotti, A. W.;

Cornish, V. W.;

Clish,

Schreiber, S. L.;

Stockwell, B. R., Regulation of Ferroptotic Cancer Cell Death by GPX4. Cell 2014, 156 (1-2), 317-331. (5) Bystrom, L. M.;

Guzman, M. L.; Rivella, S., Iron and Reactive Oxygen Species:

Friends or Foes of Cancer Cells? Antioxidants & Redox Signaling 2014, 20 (12), 1917-1924. (6) Doll, S.; Conrad, M., Iron and Ferroptosis: A Still Ill-Defined Liaison. Iubmb Life 2017, 69 (6), 423-434. (7) Brillas, E.;

Sires, I.; Oturan, M. A., Electro-Fenton Process and Related

Electrochemical Technologies Based on Fenton's Reaction Chemistry. Chemical Reviews 2009, 109 (12), 6570-6631. (8) Xie, Y.;

Hou, W.;

Song, X.;

Yu, Y.;

Huang, J.;

Sun, X.;

Kang, R.;

Tang, D., Ferroptosis: process and function. Cell Death and Differentiation 2016, 23 (3), 369-379. (9) Banjac, A.; Perisic, T.;

Sato, H.;

Seiler, A.;

Bannai, S.;


Weiss, N.;

Page 27 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Kolle, P.;

Tschoep, K.; Issels, R. D.;

Daniel, P. T.;

Conrad, M.; Bornkamm, G.

W., The cystine/cysteine cycle: a redox cycle regulating susceptibility versus resistance to cell death. Oncogene 2008, 27 (11), 1618-1628. (10)Dixon, S. J.;

Patel, D.;

Thomas, A. G.;

Welsch, M.;

Skouta, R.;

Gleason, C.; Tatonetti, N.;

Lee, E.;

Hayano, M.;

Slusher, B. S.; Stockwell, B. R.,

Pharmacological inhibition of cystine-glutamate exchange induces endoplasmic reticulum stress and ferroptosis. Elife 2014, 3. (11)Angeli, J. P. F.;

Shah, R.;

Pratt, D. A.; Conrad, M., Ferroptosis Inhibition:

Mechanisms and Opportunities. Trends in Pharmacological Sciences 2017, 38 (5), 489-498. (12)Corot, C.;

Robert, P.;

Idee, J. M.; Port, M., Recent advances in iron oxide

nanocrystal technology for medical imaging. Advanced Drug Delivery Reviews 2006, 58 (14), 1471-1504. (13)Tang, Z. M.;

Liu, Y. Y.; He, M. Y.; Bu, W. B., Chemodynamic Therapy:

Tumour Microenvironment-Mediated Fenton and Fenton-like Reactions. Angewandte Chemie-International Edition 2019, 58 (4), 946-956. (14)Wang, X. G.; M. Z.;

Dong, Z. Y.;

Huo, J. W.;

Cheng, H.;

Wan, S. S.; Chen, W. H.;

Zou,

Deng, H. X.; Zhang, X. Z., A multifunctional metal-organic

framework based tumor targeting drug delivery system for cancer therapy. Nanoscale 2015, 7 (38), 16061-16070. (15)Burk, J.;

Sikk, L.;

Fordsmand, J. J.;

Burk, P.;

Manshian, B. B.;

Soenen, S. J.;

Scott-

Tamm, T.; Tämm, K., Fe-Doped ZnO nanoparticle toxicity:



Page 28 of 32

assessment by a new generation of nanodescriptors. Nanoscale 2018, 10 (46), 2198521993. (16)Huang, G.;

Liu, R.;

Hu, Y.;

Li, S.-H.;

Wu, Y.; Qiu, Y.; Li, J.; Yang,

H.-H., FeOOH-loaded mesoporous silica nanoparticles as a theranostic platform with pH-responsive MRI contrast enhancement and drug release. Science China Chemistry 2018, 61 (7), 806-811. (17)Chen, J.;

Lei, S.;

Zeng, K.;

Wang, M.;

Asif, A.; Ge, X., Catalase-

imprinted Fe3O4/Fe@fibrous SiO2/polydopamine nanoparticles: An integrated nanoplatform of magnetic targeting, magnetic resonance imaging, and dual-mode cancer therapy. Nano Research 2017, 10 (7), 2351-2363. (18)Huang, G.; A.;

Chen, H.;

Dong, Y.;

Luo, X.;

Yu, H.;

Moore, Z.;

Bey, E.

Boothman, D. A.; Gao, J., Superparamagnetic iron oxide nanoparticles:

amplifying ROS stress to improve anticancer drug efficacy. Theranostics 2013, 3 (2), 116-126. (19)Zhang, C.; Yao, H.;

Bu, W.;

Ni, D.;

Zhang, S.;

Li, Q.; Yao, Z.;

Zhang, J.;

Wang, Z.; Shi, J., Synthesis of Iron Nanometallic Glasses and Their

Application in Cancer Therapy by a Localized Fenton Reaction. Angewandte Chemie International Edition 2016, 55 (6), 2101-2106. (20)Ma, P. a.; Li, C.;

Xiao, H.;

Wang, J.;

Yu, C.;

Liu, J.;

Cheng, Z.;

Song, H.;

Zhang, X.;

Gu, Z.; Lin, J., Enhanced Cisplatin Chemotherapy by Iron Oxide

Nanocarrier-Mediated Generation of Highly Toxic Reactive Oxygen Species. Nano Letters 2017, 17 (2), 928-937.


Page 29 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(21)Wu, H.;

Cheng, K.;

He, Y.;

Li, Z.;

Su, H.;

Zhang, X.;

Sun, Y.;

Shi,

W.; Ge, D., Fe3O4-Based Multifunctional Nanospheres for Amplified Magnetic Targeting Photothermal Therapy and Fenton Reaction. ACS Biomaterials Science & Engineering 2019, 5 (2), 1045-1056. (22)Liu, Y. L.;

Ai, K. L.; Lu, L. H., Polydopamine and Its Derivative Materials:

Synthesis and Promising Applications in Energy, Environmental, and Biomedical Fields. Chemical Reviews 2014, 114 (9), 5057-5115. (23)Bettinger, C. J.;

Bruggeman, P. P.;

Misra, A.;

Borenstein, J. T.; Langer, R.,

Biocompatibility of biodegradable semiconducting melanin films for nerve tissue engineering. Biomaterials 2009, 30 (17), 3050-3057. (24)Cui, J. W.;

Yan, Y.;

Such, G. K.;

Liang, K.;

Ochs, C. J.; Postma, A.;

Caruso, F., Immobilization and Intracellular Delivery of an Anticancer Drug Using Mussel-Inspired Polydopamine Capsules. Biomacromolecules 2012, 13 (8), 22252228. (25)Liu, F. Y.;

He, X. X.;

Lei, Z.;

Liu, L.; Zhang, J. P.;

You, H. P.;

Zhang,

H. M.; Wang, Z. X., Facile Preparation of Doxorubicin-Loaded Upconversion@Polydopamine Nanoplatforms for Simultaneous In Vivo Multimodality Imaging and Chemophotothermal Synergistic Therapy. Advanced Healthcare Materials 2015, 4 (4), 559-568. (26)Dreyer, D. R.;

Miller, D. J.;

Freeman, B. D.;

Paul, D. R.; Bielawski, C. W.,

Elucidating the Structure of Poly(dopamine). Langmuir 2012, 28 (15), 6428-6435. (27)Lynge, M. E.; van der Westen, R.; Postma, A.; Stadler, B., Polydopamine-a



Page 30 of 32

nature-inspired polymer coating for biomedical science. Nanoscale 2011, 3 (12), 4916-4928. (28)Liu, Y.;

Ai, K.;

Ji, X.;

Askhatova, D.;

Du, R.; Lu, L.; Shi, J.,

Comprehensive Insights into the Multi-Antioxidative Mechanisms of Melanin Nanoparticles and Their Application To Protect Brain from Injury in Ischemic Stroke. Journal of the American Chemical Society 2017, 139 (2), 856-862. (29)Ju, K.-Y.; Lee, J. W.;

Im, G. H.;

Lee, S.;

Pyo, J.;

Park, S. B.;

Lee, J.

H.; Lee, J.-K., Bio-Inspired, Melanin-Like Nanoparticles as a Highly Efficient Contrast Agent for T-1-Weighted Magnetic Resonance Imaging. Biomacromolecules 2013, 14 (10), 3491-3497. (30)Li, Y.;

Xie, Y.; Wang, Z.;

Andolina, C. M.;

Parent, L. R.;

Zang, N.;

Carniato, F.;

Ditri, T. B.;

Huang, Y.;

Walter, E. D.; Botta, M.;

Rinehart, J. D.; Gianneschi, N. C., Structure and Function of Iron-Loaded Synthetic Melanin. ACS Nano 2016, 10 (11), 10186-10194. (31)Ju, K. Y.; Lee, Y.;

Lee, S.;

Park, S. B.; Lee, J. K., Bioinspired

Polymerization of Dopamine to Generate Melanin-Like Nanoparticles Having an Excellent Free-Radical-Scavenging Property. Biomacromolecules 2011, 12 (3), 625632. (32)Fan, Q. L.; Lu, X. M.;

Cheng, K.;

Xing, L.;

Hu, X.;

Ma, X. W.;

Zhang, R. P.;

Yang, M.;

Huang, W.; Gambhir, S. S.; Cheng, Z., Transferring

Biomarker into Molecular Probe: Melanin Nanoparticle as a Naturally Active Platform for Multimodality Imaging. Journal of the American Chemical Society 2014,


Page 31 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


136 (43), 15185-15194. (33)Zhang, S. X.;

Zhang, Y. Y.;

Bi, G. M.;

Liu, J. S.;

Wang, Z. G.;

Xu, Q.;

Xu, H.; Li, X. Y., Mussel-inspired polydopamine biopolymer decorated with magnetic nanoparticles for multiple pollutants removal. Journal of Hazardous Materials 2014, 270, 27-34. (34)Wang, X. Y.;

Zhang, J. S.;

Wang, Y. T.;

Wang, C. P.;

Xiao, J. R.;

Zhang, Q.; Cheng, Y. Y., Multi-responsive photothermal-chemotherapy with drugloaded melanin-like nanoparticles for synergetic tumor ablation. Biomaterials 2016, 81, 114-124. (35)Sever, M. J.; Wilker, J. J., Visible absorption spectra of metal-catecholate and metal-tironate complexes. Dalton Transactions 2004, 7, 1061-1072.



Fe2+/Fe3+ ions chelated with ultrasmall polydopamine nanoparticles inducing Ferroptosis for cancer therapy Lu Chen, Zhenjie Lin, Lizhu Liu, Xiuming Zhang, Wei Shi, Dongtao Ge, Yanan Sun*

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Fe3+ ions chelated with ultrasmall polydopamine nanoparticles

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