Photothermal Therapy Nanomaterials Boosting Transformation of Fe(III)

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Biological and Medical Applications of Materials and Interfaces

Photothermal Therapy Nanomaterials Boosting Transformation of Fe(III) into Fe(II) in Tumor Cells for Highly Improving Chemodynamic Therapy Xuan Nie, Lei Xia, Hai-Li Wang, Guang Chen, Bin Wu, TianYou Zeng, Chun-Yan Hong, Long-Hai Wang, and Ye-Zi You ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b11291 • Publication Date (Web): 08 Aug 2019 Downloaded from pubs.acs.org on August 9, 2019

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ACS Applied Materials & Interfaces

Photothermal

Therapy

Nanomaterials

Boosting

Transformation of Fe(III) into Fe(II) in Tumor Cells for Highly Improving Chemodynamic Therapy Xuan Nie, Lei Xia, Hai-Li Wang, Guang Chen, Bin Wu, Tian-You Zeng, Chun-Yan Hong*, Long-Hai Wang*, and Ye-Zi You* Hefei National Laboratory for Physical Sciences at the Microscale, CAS Key Laboratory of Soft Matter Chemistry, Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei 230026, P. R. China. *Corresponding authors:

Chun-Yan Hong: [email protected]

Long-Hai Wang: [email protected]

Ye-Zi You: [email protected]

ACS Paragon Plus Environment

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Abstract

Chemodynamic therapy based-on Fe2+ catalyzed Fenton reaction holds great promise in cancer treatment. However, low produced hydroxyl radicals in tumor cells constitute its severe challenges due to that Fe2+ with high catalytic activity could be easily oxidized into Fe3+ with low catalytic activity, greatly lowering Fenton reaction efficacy. Here, we codeliver CuS with iron-containing prodrug into tumor cells. In tumor cells, the overproduced esterase could cleave phenolic ester bond in prodrug to release Fe2+, activating Fenton reaction to produce hydroxyl radical. Meanwhile, CuS could act as a nanocatalyst for continuously catalyzing the regeneration of high active Fe2+ from low active Fe3+ to produce enough hydroxyl radical to efficiently kill tumor cells as well as a photothermal therapy agent for generating hyperthermia for thermal ablation of tumor cells upon NIR irradiation. The results have exhibited that the approach of photothermal therapy nanomaterials boosting transformation of Fe3+ into Fe2+ in tumor cells can highly improve Fenton reaction for efficient chemodynamic therapy. This strategy was demonstrated to


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have an excellent anti-tumor activity both in vitro and in vivo, which provides an innovative perspective to Fenton reaction based chemodynamic therapy.

Key words: photothermal therapy, chemodynamic therapy, Fenton reaction, Fe (II) regeneration, combined cancer therapy.


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Introduction Cancer is still a major threat to human health as its complexity and versatility over the past few years.1 Some new cancer therapies based on nanotechnology have gained the gratifying achievements with nanomedicine.2 Reactive oxygen species (ROS), including hydrogen peroxide, superoxide anion, singlet oxygen and hydroxyl radical etc., are widely used in cancer therapy,3-8 which can kill the tumor cells by oxiding the biomolecules such as lipids, proteins and nucleic acid.9-11 In the tumor cells, H2O2 is overproduced, but H2O2 has mild oxidation capability (standard redox potential E(H2O2/H2O)= 1.78 V),12 which can not kill tumor cells effectively. ROS including hydroxyl radical (•OH) (E(•OH/H2O)= 2.80 V)13 and singlet oxygen (1O2)(E(O2/H2O) = 2.17 V)14 have high oxidation capability, which can kill tumor cells effectively. Therefore, transforming the mild active molecules into highly active molecules in cancer cells has been recognized as an effective method for cancer treatment including photodynamic and chemodynamic therapy.15-18 However, it is very difficult for photodynamic therapy to produce enough ROS in the tumor’s hypoxia environment.19-21 Chemodnamic therapy based Fenton reaction could produce hydroxyl radical to kill cancer cells without oxygen. But at present time, the produced hydroxyl radicals are not enough for highly efficiently killing cancer cells due to that Fe2+ could be easily oxidized into Fe3+ in tumor cell, which greatly lowers Fenton reaction efficacy due to that Fe2+ has high activity in catalyzing Fenton reaction, but Fe3+ has low activity.22 If a large amount of Fe2+ was used, there would be serious health problems to the body.23 Therefore, the most facile method is continuously and effciently transforming low active Fe3+ into high active Fe2+ in tumor cells for producing enough hydroxyl radical to efficiently kill tumor cells.24, 25


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Herein, we construct a nanoparticle contianing CuS and iron-containing prodrug coated with temperature sensitive functional polymer. Irradiating tumor tissues by NIR light, CuS nanoparticles could generate much heat for triggering the phase transition of the temperature sensitive polymer to release the prodrug as well as thermal ablation of cancer cells. In tumor cells, the overproduced esterase could cleave the phenolic ester bond in prodrug to release Fe2+ to activate Fenton reaction, forming hydroxyl radical and Fe3+. On the other hand, CuS could boost the regeneration of Fe2+ from Fe3+ to maintain a high level of Fe2+ in tumor cells. Therefore, enough hydroxyl radical produced via Fenton reaction catalyzed by high level of Fe2+ can efficiently killing tumor cells (scheme 1). This ingenious combined therapy strategy and tumor microenvironment-responsive nano-delivery system can achieve efficient chemodynamic and photothermal combinated treatment on tumors with ostentatious superadditive therapeutic effects. Simultaneously, it could minimize the damage to normal tissues.


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Scheme 1. (a) Schematic of typical Fe(II) catalyzed Fenton reaction. (b) Schematic of the photothermal therapy nanomaterials boosting transformation of Fe(III) into Fe(II) in tumor cells for highly improving chemodynamic therapy.


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Results and Discussion The prepared poly(ethylene glycol)-b-poly(acrylamide-co-acrylonitrile) (PEG-b-P(AAm-co-AN)) has a upper critical solution temperature (UCST) of 45 oC (Figure 1a),26 which could assemble into nanoparticles under the physiological temperature in solution. CuS and Fe-containing prodrug were loaded into PEG-b-P(AAm-co-AN) to form the functional composite nanoparticles (denoted as CuS-Fe@polymer). Once coated by the functional polymers, CuS-Fe@polymer nanoparticles could be well dispersed in PBS for several days, but the bare CuS nanopartices aggregated easily only in one day (Figure 1b). TEM and DLS results have shown that the size of CuS-Fe@polymer nanoparticles is about 100 nm with a narrrow size distribution (Figure 1c and Figure 1d), which is suitable for endocytosis. Further, to investigate the structure of the CuS-Fe@polymer as shown in Figure 1e, The EDX mapping experiment (Figure 1f) was carried out, and the results have suggested that Fe containing prodrug and CuS was inside the CuS-Fe@polymer nanoparticles with a temperature sensitive polymer shell on surface. UV-Vis-NIR absorption results have also shown that the absorption in nera-infrared region of the CuS-Fe@polymer nanoparticles was similar to that of bare CuS as shown in Figure 1g, indicating that CuS-Fe@polymer nanoparticles could produce heat under the NIR light like CuS.


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Figure 1. (a) The temperature-transmittance plot of PEG-b-P(AAm-co-AN) in PBS (4.0 mg/mL, and the heating rate at 1.0 oC/min). (b) The pictures of bare CuS and CuS-Fe@polymer nanoparticles in the PBS. The left is the bare CuS and the right is CuS-Fe@polymer nanoparticles. (c) The TEM pictures of CuS-Fe@polymer. The scale in the picture is 50 nm. (d) The size distribution of CuS-Fe@polymer nanoparticles via DLS. (e) The schematic of CuS-Fe@polymer nanoparticles. (f) The EDX mapping pictures of CuS-Fe@polymer nanoparticles. (g) UV-Vis-NIR spectra of bare CuS and CuS-Fe@polymer nanoparticles.

Under the irradiation of NIR light in the presence of CuS-Fe@polymer nanoparticles for 5 min, the temperature of the mixture could be easily rasied by 25 oC as shown in Figure 2a and Figure 2b. At high temperature, the shell of CuS-Fe@polymer nanoparticles became soluble, releasing the CuS nanoparticles and Fe-containing prodrug. Meanwhile, esterase, which is


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commonly overexpressed in cancer cells,27 could cleave the the ester bond in prodrug, giving Fe2+ to initiate the Fenton reaction.28 To identify the release of the prodrug from the CuS-Fe@polymer nanoparticles under 808 nm light treatment, 2,2’-bipyridine was used as a probe to react with Fe2+, forming red complex, which has a absorption at 519 nm. It is very clear that, after CuSFe@polymer nanoparticles were irradiated by 808 nm light for 5 min, the prodrug was hydrolyzed to release all Fe2+ in 100 min (Figure 2c). While the nanoparticles without irridiation showed low release of Fe2+ (Figure S6), indicating that CuS-Fe@polymer nanoparticles could controll the release of Fe2+ in the tumor microenvirenment with the assistance of 808 nm light. Generally, Fe2+ could effectively catalyze Fenton reaction to produce hydroxyl radicals. However, Fe2+ could be easily oxidized into Fe3+ in tumor cells, but Fe3+ could not effectively catalyze Fenton reaction to produce •OH. Therefore, the transformation of Fe2+ into Fe3+in tumor cells will highly lower Fenton reaction and the production of •OH. Subsequently, we investigated the production •OH in enhanced Fenton reaction by CuS-Fe@polymer nanoparticles. Rhodamine B (RhB), a common probe to detect the •OH produced from H2O2, could be degraded by •OH to form colorless substance. Thus, the RhB degradation experiments were carried out to identify whether CuS could boost transformation of Fe3+ to Fe2+ for catalyzing the Fenton reaction to produce enough •OH. H2O2 could hardly degrade the RhB as shown in Figure 2d, while •OH produced by typical Fenton reaction group could degrade RhB. Compared with the group of H2O2, the absorption at 553 nm decreased in Fe2+ based typical Fenton reaction groups, indicating that •OH has higher oxidization ability than H2O2, and 40% RhB was degraded. However, •OH produced via enhanced Fenton reaction by CuS-Fe@polymer nanoparticles could degrede 100% RhB. It is because that CuS nanoparticles could booste the transformation from low active Fe3+ to high active Fe2+, thereby highly enhancing Fenton reaction to yield more •OH. 5,’5 -Dimethyl-1-pyrrolidine-N-oxide


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(DMPO) can serve as a spin trap to detect the •OH directly by electron paramagnetic resonance (EPR) assay. As shown in Figure 2e, there are stronger •OH signal appeared in the present of CuS nanoparticles, which is consistent with above results. Furthermore, we have investigated the RhB degradation experiments of CuS-Fe@polymer nanoparticle. The results also showed that CuS nanoparticle could enhanced the efficacy of Fenton reaction as shown in Figure S7. Based on above results, it is obvious that CuS-Fe@polymer nanoparticles could produce enough heat under NIR irradiation, and the produced heat could trigger polymer shell to become soluble to release the prodrug and CuS. Subsequently, in the presence of esterase, Fe2+ was released from prodrug, which catalyzed Fenton reaction to form Fe3+. Then, the CuS nanoparticles can effectively transform Fe3+ into Fe2+, resulting in the enhanced Fenton reaction and the production of more •OH. To indentify the tranformation of Fe3+ to Fe2+, 1,10-phenanthrolinen was used as a probe to detect the formation of Fe2+ with a absorption at 511 nm. CuS nanoparticles was added into FeCl3 solution, subsequently, 1,10-phenanthrolinen was added to the mixture. As shown in Figure 2f, a strong peak at 511 nm appeared in the UV absorption spectrum, which suggested that the CuS could reduce Fe3+ to Fe2+. According the references,29,30 there are lots of Cu+ on the surface of the CuS nanoparticles (Figure S8), and Cu+ on the surface of CuS nanoparticles could make Fe2+ get reborn from Fe3+ to catalyze the Fenton reaction. It was found that 2.5 times •OH were produced acccording to the RhB degradation experiments, which will highly enhance the efficacy of chemodynamic cancer therapy.


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Figure 2. (a) The photothermal plots of CuS-Fe@polymer at different light (808 nm) power. The concentration is 60 µg/mL. (b) The photothermal plots of CuS-Fe@polymer with the 980 nm light treatment. (c) The plot of Fe2+ release from CuS-Fe@polymer under NIR irradiation (1.0 W/cm2, 5 min). Fe2+ was detected by 2,2’-bipyridine in 100 mM Tris-HCl, and the absorption of 519 nm reflects the released mount of Fe2+, [bipyridine] = 300 µM, [esterase] = 200 U/mL. (d) The experiments on RhB degradation by CuS enhanced Fenton reaction. The absorption of 553 nm reflects the mount of remained RhB. [RhB] = 10 µg/mL, [FeSO4 •7H2O] = 20 µg/mL, [H2O2] = 1.5 mM, [CuS] = 20 µg/mL. (e) EPR spectra of •OH at the room temperature. 5,’5 -Dimethyl-1pyrrolidine-N-oxide (DMPO, 0.14 M) served as a spin trap. (f) Fe2+ concentration detection in the dark: UV spectra of 1,10-phenanthrolinen in the present of FeCl3, CuS and FeCl3 and CuS. The absorption peak at 511 nm is ascribed to the complex of ferrous ions. [FeCl3] = 1.0 mg/mL, [CuS] = 20 μg/mL, [phenanthrolinen] = 0.5 mg/mL.


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High concentration of ROS could cause the oxidation of intracellular biological molecules, resulting in cell death.31-33 To investigate CuS-Fe@polymer nanoparticles could still work well in a complex tumor environment and indentify that the produced •OH could destroy the cancer cell, •OH production in cancer cell was evaluated by the fluorescence probe DCFH-DA, whose green fluorescence strength showed a positive correlation with the ROS level in cell. After treated with 808 nm light, the CuS-Fe@polymer group had the highest fluorescence intensity among all the groups as shown in Figure 3a, indicating that CuS-Fe@polymer nanoparticles could produce lots of •OH as expected under the NIR treatment. As for CuS-Fe@polymer without 808 nm light treatment group, very slightly fluorescence appeared, which resulted from that the temperature sensitive polymer shell protected the Fe-containing prodrug from being hydrolyzed by esterase under the physiological temperature, and there was little Fe2+ to initiate the Fenton reaction and produce little ROS. To evaluate DNA damage caused by ROS from enhanced Fenton reaction by CuS-Fe@polymer nanoparticles, the single cell gel electrophoresis experiments were carried out. If DNA were damaged by ROS, comet-like shapes would appear as shown in Figure 3b.34 The longer the comets were, the more DNA were damaged.35,36 There are much longer comets appeared with CuS-Fe@polymer/NIR, indicating that CuS-Fe@polymer nanoparticles could cause severe DNA damage under NIR light irradiation. Next, the cytotoxicity in Hela cells was investigated by Calcein-AM (green) and PI (red) double stained assay. After incubation with Fe@polymer, CuS@polymer and CuS-Fe@polymer nanoparticles at concentration of 80 µg/mL for 4 h, Hela cells were subjected to 808 NIR light irradiation for 5 min, and then cultured normally for next 12 h in the dark. As shown in Figure 3c, homogeneous red fluorescence appeared in the group of CuSFe@polymer/NIR while green fluorescence appeared in all other groups, indicating that almost all cells have died in CuS-Fe@polymer/NIR group while there were only ignorable cells died for all


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other groups. Interestingly, a clear dividing line between living and dead cells in the group of CuSFe@polymer/NIR was observed because CuS-Fe@polymer nanoparticles could not destroy the cells in the areas without NIR light. Without light irradiation, there was little Fe2+ could be released due to that the polymer shell protected Fe2+ prodrug from esterase cleaving the ester bond (Figure S9). Redox imbalance is an important role for cell death, and GSH is a major antioxidant that provides reducing equivalents for the glutathione peroxidase (GPx) catalyzed reduction of lipid hydroperoxides to their corresponding alcohols.37,38 So we quantitatively evaluated GSH level after treatment by the method of Ellman’s reagent. It was clear that GSH in cells has decreased by 50% for those cells treated with CuS-Fe@polymer nanoparticles under the 808 nm NIR light compared with the normal cells (Figure 3d). We also evaluated the cytotoxicity of CuS-Fe@polymer nanoparticles in cancer and normal cells by (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. CuS-Fe@polymer/NIR group showed an very high cytotoxicity in Hela cells, which was much higher than that of single therapy of chemodynamic (Fe@polymer) or photothermal (CuS@polymer with NIR) therapy as shown in Figure 3e. Under NIR irradiation, the CuS-Fe@polymer nanoparticles with 60 µg/mL could significantly reduce the vialbility of the Hela cells (below 10 % cells could survive after the treatment). As for CuS@polymer and Fe@polymer groups, even at 80 µg/mL, there were still many cells survived after the teratment. Furthermore, if the cells received no NIR tratment, the CuS-Fe@polymer nanoparticles exhibited little cytotoxicity to cells (>90% viability at 80 µg/mL), indicating that NIR light can be used as a switch in combination therapy, and we can selectively kill the cancer cells by the external NIR light. The same results could be obtained in other cancer cell lines including PC-3 cells and 4T1 cells ( Figure S10 and Figure S11). On the other hand, the MTT experiments have shown that the CuSFe@polymer/NIR has a slight cytotoxicity to the normal cells (NIH3T3 cells, Figure 3f) even at


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high concentration (80 µg/mL). This results could be explained by that there is very little esterase and H2O2 in normal cells, and after the NIR treatment, Fe2+ could not be released from the CuSFe@polymer nanoparticles to initiate the Fenton reaction. Furthermore, the H2O2 in normal cells is not as high as cancer cell, thus CuS-Fe@polymer/NIR could not produce enough ROS. All the above results have exhibited that CuS-Fe@polymer nanoparticles have excellent ostentatious superadditive therapeutic effects. The success of the combinated therapy is inseparable from external NIR light and internal tumor microinvironment including esterase and H2O2, otherwise the therapeutic effect will be greatly reduced.


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Figure 3. (a) Intracellluar ROS image Hela cells after treated with Fe@polymer, CuS@polymer/NIR, CuS-Fe@polymer, CuS-Fe@polymer/NIR. The concentration of each sample is 80 μg/mL, 808 nm light for 5 min at 0.5 W/cm2. The cells are treated with DCFH-DA before imaging by inverted fluorescence microscope. (b) DNA damages after Hela cells receiving the different treatments (the condition is the same to the ROS detection experiments) by single cell gel electrophoresis assay. The nuclear DNA was strained by GelRed, U=20 V,I=100 mA, 30 min.


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(c) The fluorescence images of Calcein-AM and PI double-stained cell with different treatments, 808 nm light, 0.75W/cm2 for 5 min. (d) The GSH level in Hela cell after the different treatments, 0.5 W/cm2 for 5 min. The GSH level was determined by Ellman’s regent (5,’5-dithio-bis (2nitrobenzoic acid, DTNB)). (e), f) Cytotoxicity of Fe@polymer, CuS@polymer/NIR, CuSFe@polymer and CuS-Fe@polymer/NIR in Hela cells and NIH3T3 cells. 808nm NIR light, 0.75 W/cm2 for 5 min.

A 4T1 tumor-bearing mouse model was built to examine the in vivo performances of CuSFe@polymer nanoparticles. When tumor grew into approximately 100 mm3, the tumor-bearing mice were administered intravenously CuS-Fe@polymer nanoparticles. After 12 h, the mice were sacrificed, and the tumor and the main organs were harvested for imaging. The biodistribution of the CuS-Fe@polymer nanopaticles (CuS was marked by FITC) was evaluated in small animal imager with average fluorescence intensity. As shown in Figure 4a, it is clear that most of CuSFe@polymer nanoparticles were mainly accumulated in the tumor tissue by the enhanced permeability and retention (EPR) effect, and the amount of which was approximate 7 times higher than that in liver and 20 times higher than that in kidney. The high accumulation of CuSFe@polymer nanoparticles in tumor tissue also could be proved by inductively coupled plasma atomic emission spectrometry (ICP-AES), as shown in Figure S12. The concentration of Cu in tumor is much higher than other organs. To evaluate the anti-tumor ability of CuS-Fe@polymer nanoparticles in vivo, the tumor mice were administered intravenously 100 µL of PBS, Fe@polymer, CuS@polymer and CuS-Fe@polymer. After 12 h, the mice received the NIR (808 nm) irradiation for 5 min. The temperature of the tumor area in the mouse treated with CuSFe@polymer nanoparticles increased to 49.8 oC (Figure S13), which is enough to trigger the release of prodrug. From the tendency of relative body weights as shown in Figure 4b, there was


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no obvious weight change of the mice in all groups, indicating that the CuS-Fe@polymer nanoparticles have not induced the system toxicity. Furthermore, tumor volume of all groups was monitored to evaluate the anti-tumor ability every two days. CuS-Fe@polymer/NIR exhibited remarkable anti-tumor in vivo compared with other groups. Figure 4c showed that the tumor volume change in the treatment. The tumors in the group of PBS, Fe@polymer and CuSFe@polymer grew rapidly, but the tumor growth in the group of CuS@polymer was inhibited to some extent since the NIR light could generate the photothermal effects in the animal experiment. The tumors of CuS-Fe@polymer/NIR groups were obviously suppressed both in volume (Figure 4d) and in weight (Figure 4e). 80% tumor of mice were completely suppressed (Figure 4f) in the group of CuS-Fe@polymer/808 nm light. However, there was no recovered mice appeared in other groups. Based on these results, we can see that CuS-Fe@polymer nanoparticles have showed an excellent anti-tumor ability in vivo via CuS induced photothermal therapy and enhanced chemodynamic therapy induced by CuS boosting the transformation of Fe3+ into Fe2+. Additionally, hematoxylin and eosin (H&E) staining was carried out to evaluate the biosafety of the CuSFe@polymer nanoparticles. The Figure 4g has shown that CuS-Fe@polymer nanoparticles have very limited pathological abnormalities for the main organs (heart, liver, spleen, lung, kidney) with no obvious phsiological morphology changes. Therefore, the photothermal therapy nanomaterials enhanced Fenton reaction have an advanced tumor inhibition effect with good biocompaibility.


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Figure 4. (a) The biodistribution after the mice was administered intravenously 100 μL CuS (FITC)-Fe@polymer, 2.0 mg/Kg. (b) The body weight changes during the treatment. (c) The tumor volume changes in the treatment. (d), (e) The picture of tumors and the tumor weights at the end of treatment. The numbers of 1, 2, 3, 4, 5, 6 respectively represent the group of PBS, CuS@polymer/808,

Fe@polymer,

CuS-Fe@polymer,

CuS-Fe@polymer

/808,

CuS-

Fe@polymer/980, and the power of NIR light is 1.2W/cm2. The absent of the date means that the


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tumor disappeared after receiving the treatment. (f) The tumor picture of mice in the CuSFe@polymer/808 group. (g) H&E-stained slices of the PBS groups and CuS-Fe@polymer/NIR groups mice after treatment, 1 means the group of PBS and the 5 means the group of CuSFe@polymer/808.

Conclusions In conclusion, we have developed a new combined cancer therapy integrated chemodynamic therapy via CuS boosting Fenton reaction with CuS induced photothermal therapy. Under NIR light irradiation, CuS not only can generate heat to trigger the prodrug releasing and directly kill the cancer cell, but also can boost the conversion of Fe3+ into Fe2+ for highly enhancing Fenton reaction, producing enough •OH to kill cancer cells. CuS-Fe@polymer nanoparticles worked well only in the tumor microenvironment with a very limited cytotoxicity to normal tissue as its high tumor accumulation and multi-trigger in tumor microenvironment. CuS-Fe@polymer nanoparticles were an excellent candidate for cancer therapy, and this novel combined therapy strategy with superadditive therapeutic effects will provide a new perspective in the cancer therapy. Experimental Section Materials Copper bromide (CuBr2, 98%), sodium sulfide nonahydrate (Na2S·9H2O, 98%), polyethylene glycol monomethyl ether (PEG-OH, Mn = 4000 g/mol,) triethylamine, 4-dimethylaminopyridine (DMAP, 99%), N,N-dicyclohexylcarbodiimide (DCC, 98%), acrylamide (AAm, 98%), acrylonitrile (AN, 97%), and azobisisobutyronitrile (AIBN, 99%), tetrabutylammonium bromide (98%), 2-(2’-cyanopropyl) dithiobenzoate( CPDB, 97%), acetoxbenzyl alcogol(98%), esterase (from porcine live, ≥150 units/mg protein), were purchased from Sigma-Aldrich. Sodium azide


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(NaN3, 98%), ferrocenecarboxylic acid (97%), acetyl chloride (AcCl, 98%), sodium citrate (99%), rhodamine B (RhB, 98%), H2O2 (30%), FeSO4·7H2O (98%), 5,5’-dithiobis(2-nitrobenzoic acid) (DNTB, 98%) were brought from Sinopharm Chemical Reagent Co., Ltd. Fluorescein was purchased from Thermo Fisher Scientific. General Characterization The temperature-transmittance plot and UV-Vis-NIR spectra was characterized by UV-3600 produced by Shimadzu. The size of nanoparticles were characterized by NanoBrook 90Plus PALS. Transmission electron microscopy (TEM) images were observed on a Hitachi Model H-7650 transmission electron microscope with an accelerating voltage of 100 kV. The EDX mapping results were from JEM-2100F transmission electron microscopy. 1H NMR spectra were recorded on a Bruker AC-400FT spectrometer (300 MHz). The concentration of Cu in main organs were characterized by Optima 7300 DV. The NIR light was generated from MDL-III-808-2W infrared laser. The EPR results were recorded from JEOL JES-FA200 ESR spectrometer. All fluorescent pictures were taken from Olympus microscope IX71. MTT results were recorded on Thermo Scientific Varioskan Flash. The biodistributions of nanoparticles were recorded on IVIS spectrum. The infrared pictures were collected from Fluke Ti401 PRO infrared camera. Synthesis of the macro RAFT agent PEG4K-CPDB PEG-OH (2.0 g, 0.5 mmol), DMAP (0.030 g, 0.25 mmol), and 2-(2’-cyanopropyl) dithiobenzoate (CPDB, 0.560 g, 2.0 mmol) were dissolved into 60 mL of dichloromethane. After stirring in an ice-water bath under nitrogen atmosphere for half an hour, a solution of DCC (0.412 g, 2.0 mmol) in dichloromethane (15.0 mL) was added dropwise into above solution. The reaction mixture was stirred for 48 h at room temperature, then the resultant mixture was filtered to remove the solid byproduct. After removing most of the solvent via rotary evaporation, the concentrated


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residual solution was added dropwise into a large excess of ice diethyl ether to obtain pink precipitate. The precipitate was collected by filtration and dried under vacuum at room temperature overnight, and the desired PEG-CPDB was obtained as pink powder (1.812 g, 85%). Synthesis of the temperature sensitive polymer The synthesis of PEG-b-P(AAm-co-AN) with an AN feed content of 27 mol % was carried as follows. A 25 mL round-bottom flask was charged with AAm (3.114 g, 43.8 mmol), AN (0.859 g, 16.2 mmol), AIBN (1.960 mg, 0.012 mmol), PEG-CPDB (0.249 g, 0.06 mmol) and dimethyl sulfoxide (DMSO, 14.0 mL). The mixture was degassed and then placed into an oil bath preheated to 70 oC. After polymerization of 48 h, the polymer was purified by precipitation from a DMSO solution into methanol for three times. Finally, the precipitate was dried under vacuum at room temperature overnight until the weight of precipitate keeps constant. Mn NMR is 57000 g/mol. Synthesis of the CuS nanoparticles. CuS nanoparticles were synthesized in mild condition.39 Copper bromide (CuBr2, 0.028 g) and sodium citrate (0.020 g) were added into deionized (DI) water (40.0 mL). The mixture was stirred at room temperature for 1 h before the addition of a Na2S·9H2O (0.024 g in 50 μL water) solution. Next, the solution was stirred for another 10 min and transferred to a 90 °C water bath. The reaction was maintained for 15 min until the solution turned to green and subsequently was cooled down with ice. Synthesis of the ferrocenoyl azide The procedure is according to the reference.26 Ferrocenecarboxylic acid (2.0 g, 8.7 mmol) and oxalyl chloride (1.5 mL, 17.4 mmol) were added into anhydrous CH2Cl2 (20.0 mL) at 0 °C, subsequently, a drop of dimethylformamide (DMF) was added to the solution. The solution was allowed to warm to room temperature and left to react for 3 h before removing the solvent under


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reduced pressure. The residue was then dissolved in anhydrous CH2Cl2 (20.0 mL) and cooled to 0 °C. Tetrabutylammonium bromide (0.030 g, 0.09 mmol) was then added followed by a solution of sodium azide (0.850 g, 13.1 mmol, in water, 4.0 mL) solution. The reaction mixture was then allowed to warm to room temperature and left to stir for 16 h. The reaction was then quenched with water (20.0 mL) and the organics was separated. The aqueous layer was then extracted twice with CH2Cl2 (20.0 mL). The combined organics were then dried over anhydrous Na2SO4 and concentrated under reduced pressure to afford the crude product. Purification via silica gel column chromatography (petroleum/CH2Cl2 =1:1 v/v) gave the titled compound as a crystalline orange solid (1.932 g, 87%). Synthesis of the 4-(hydroxymethyl) phenyl acetate Under N2 atmosphere, 4-hydroxybenzyl alcohol (2.000 g, 16.1 mmol) was dissolved in anhydrous THF (30.0 mL), and the mixture was cooled to 0 °C with an ice-water bath, subsequently, TEA (2.23 mL, 16.1 mmol) was added. Then acetyl chloride (1.26 mL, 17.7 mmol) was added dropwise within 25 min. The resulting reaction mixture was stirred for 2 h. Thereafter, the newly formed precipitate was removed by filtration and filtrate was evaporated. The crude was diluted with CH2Cl2, washed twice with 5% NaHCO3 aqueous solution and deionized water. The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under vacuum (2.143 g, 80%). Synthesis of the Fe-containing prodrug To an oven-dried carousel tube charged purged with argon was added ferrocenoyl azide (0.255 g, 1.0 mmol). The solids were dissolved in anhydrous toluene, and to this stirring solution was added the para-acetoxbenzyl alcogol (0.332 g, 2.0 mmol). The reaction mixture was then heated to reflux and stirred for 2 h. After cooled to room temperature, the reaction mixture was


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transferred to a round-bottom flask and concentrated under reduced pressure to afford the crude product. Purification via gel column chromatography (hexane/EtOAc = 1:9, v/v, Rf = 0.15) gave the titled compound as a yellow solid (0.325 g, 82.6%). Preparation the drug-load nanoparticles Drug-load nanoparticles were prepared via one-pot method. CuS nanoparticles were first dispersed in water (8 mg), subsequently, PEG-b-P(AAm-co-AN) (20 mg) and Fe2+ prodrug (5 mg) dissolved in DMSO (1 mL). Then, the above solution was added to the CuS dispersion (15 mL), followed by ultrasonic treatment. After the mixture was purified by dialysis (MWCO, 11000) in PBS, the precipitates (unloaded prodrug and CuS nanoparticles) were removed by centrifugation (3000 r/min). Fe2+ release with the esterase A solution of a ferrocene loaded nanoparticles (0.2 mg/mL) was dispersed in 100 mM TrisHCl buffer (pH= 7.4). The buffer contains 300 μM 2’2-bipyridine, which can coordinate Fe ion, forming red complex in solution. The experiment was divided into two groups, one of which receive no irradiation treatment before hydrolysis, and the other received 808 nm light irradiation for 5 min. Subsequently, the esterase (200 U/mL) was added to the system, the solution was incubated at 37 oC for different time. Before reading the absorption at 519 nm at different time by microplate reader, precipitates should be removed by centrifugation. In order to deal with the data conveniently, the absorption value was normalized in the experiment. The standard curve of Fe2+ was constructed by the FeSO4, and the amount of Fe2+ release from the prodrug was determined by standard curve. We calculated the ratio of the prodrug and CuS in the CuS-Fe@polymer nanoparticles (CuS/Fe/polymer = 7:1:10, w/w) according to this method. Transformation of Fe3+/Fe2+ and •OH production in vitro


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Fe2+ regeneration: The experiment has shown that CuS can accelerate the transformation of Fe3+ to Fe2+. In brief, the FeCl3 (1.0 mg/mL), CuS (20 μg/mL), and the 1,10-phenanthrolinen (0.5 mg/mL) were placed in dark environment for 3 min. Fe2+ could produce the red complex with phenanthrolinen. The absorbance of 511 nm could confirm the transformation of Fe3+ to Fe2+. RhB degradation: Solution of 10 μg/mL RhB, 1.5 mM H2O2, 20 μg/mL FeSO4·7H2O and 40 μg/mL CuS were prepared. After the mixture was incubated for 30 min at room temperature, •OH induced RhB degradation was measured by the change in absorbance at 554 nm by UV spectrophotometry. As for CuS-Fe@polymer, CuS-Fe@polymer (50 μg/mL CuS, 7.1μg/mL Fe prodrug, 71 ug/mL polymer) were incubated in 37 oC for 60 min with 150 U/mL esterase (100 mM Tris-HCl, pH=7.4). Next, the mixture receive 808 nm light (1.0 W/cm2) for 5 min (if needed). Then the 1.5 mM H2O2 and 7.5 μg/mL RhB were added to mixture. After the mixture was incubated for 30 min at room temperature, •OH induced RhB degradation was measured by the change in absorbance at 554 nm by UV spectrophotometry. EPR experiment: Room-temperature EPR spectra of •OH detection experiments were obtained using a JEOL JES-FA200 ESR spectrometer (300 K, 9.063 GHz, X-band). Microwave power employed was 1 mW. Modulation frequency and modulation amplitude were 100 kHz and 0.35mT, respectively. The concentration of each substance is the same to RhB degradation experiment. 1.0 mmol H2O2, 0.14 M DMPO, 20.0 μg/mL FeSO4·7H2O, and 40.0 μg/mL CuS. ROS production in Hela cell: Hela cells were seeded at a density of 50000 cells per well into 24well plate and incubated overnight before use. Before various treatments, the cell medium was replaced with fresh medium, then cultured normally for another 12 h in the dark. Then, the cells were harvested and washed with PBS three times. ROS probe (DCFH-DA) was added at the final


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concentration of 10 µM and incubated for 20 min. Then, the cells were washed three times with ice-cold PBS and imaged with fluorescent microscope (Olympus IX71). GSH concentration in vitro Hela cells were seeded in 24-well plates (50000 cell per well) and incubated for 24 h before use. Before various treatments, the cell medium was replaced with fresh medium, then cultured normally for more 12 h in the dark. The cells were harvested and washed with PBS three times. Subsequently, after the cells were lysed with 100 μL of Triton-X-100 lysis buffer on ice, the lysis buffer was collected by centrifugation to remove the precipitate, and 50 μL of the lysis buffer was mixed with 50 μL of DTNB solution (400 μM). After incubated for 30 min at 37 oC, the UV absorbance of the mixture was measured at 412 nm by microplate reader to determine the content of cellular GSH. The percentage content of GSH was acquired based on the comparison to the GSH content of untreated cells. Single cell gel electrophoresis experiment DNA damage was evaluated through single cell gel electrophoresis experiment. In brief, Hela cells were seeded in 24-well plates (50000 cells per well) incubated overnight before use. Before various treatments (PBS, CuS@polymer/NIR, Fe@polymer, CuS-Fe, CuS-Fe@polymer/NIR, 0.5 W/cm2, 5 min), the cell medium was replaced with fresh medium, then cultured normally for 12 h in the dark. The cells were harvested and then washed with PBS three times. Then the Hela cells were pelleted and re-suspended in ice cold PBS. Aliquot of 20 μL of cell resuspension were mixed with prewarmed 20 μL 2.0 % low melting point gel, followed by loading onto slide whose surface was functionalized to intact the gel. A coverslip was then applied to the slide and maintained for 10 min at room temperature. Then, the coverslip was removed and slide was coated with another 1.0 % low melting point agarose (40 μL). After solidification for 30 min at 4 °C, the slide was immersed in prechilled lysis buffer for 1 h at 4 °C, followed by rinsing in PBS. Subsequently, the


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slide was incubated in alkaline electrophoresis buffer (1 mM ethylenediaminetetraacetic acid (EDTA), 300 mM NaOH) for 30 min for DNA unwinding, followed by electrophoresis at 25 V for 30 min. After electrophoresis, the slide was rinsed twice in Tris-HCl buffer (0.4 mM, pH 7.5). DNA was visualized by GelRed and observed under fluorescent microscope. In vitro cytotoxicity based on MTT method The cytotoxicity of chemodynamic and photothermal was detected with HeLa cells by MTT assay. In brief, HeLa cells were seeded into 96-well plates (1×104 cells per well) for 24 h before use. Various samples were added to each well and incubated for 4 h, and the cell medium was replaced with fresh medium. After various treatments (PBS, CuS@polymer/NIR, Fe@polymer, CuS-Fe@polymer, CuS-Fe@polymer/NIR, 0.75 W/cm2, 5 min) of different samples. The cells were incubated for more 12 h. Thereafter, the culture medium discarded and washed with PBS, subsequently, MTT solution (100 µL, 0.5 mg mL-1) was added to the plate. After 4 h incubation, the medium was replaced with DMSO (100 µL) and shaken for 10 min to dissolve blue Formosan. The absorbance at 492 nm of each well was measured by microplate reader. Cytotoxicity of each group was acquired based on the comparison to the untreated cells. Biodistribution of the nanoparticles in mice After tumor in the BALB/c mice grew to 100 mm3, three of which were administered intravenously with 100 μL CuS(FITC)-Fe@polymer, 12 h later, the mice were put into small animal imager to investigate the biodistribution of the nanoparticles in tumor, heart, liver, spleen, lung and kidney. The biodistribution of the CuS-Fe@polymer was evaluated by the average fluorescence intensity. As for Cu distribution in main organs. After 12 h post-injection, the animals were sacrificed, and the heart, liver, spleen, lung, kidney were collected. The samples were freezedried, and then digested by concentrated nitric acid (HNO3, 67%) for one day (80 oC). The clearly


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yellow solutions were heated (120 °C) to remove the nitric acid and water, then nitric acid (5%, final concentration) and distilled water were added with 5 ml of the final volume. The resulting solutions were prepared for inductively coupled plasma atomic emission spectrometry test. Tumor inhibition in vivo animal models All animal experiments were performed under the University of Science and Technology of China animal Center (USTCACUC1801043). 30 female BALB/c mice bearing 4T1 tumor (about 100 mm3) were randomly divided into 6 groups. when the experiment started, the mice were administered intravenously with 100 μL PBS, 100 μL CuS@polymer, 100 μL Fe@polymer, 100 μL CuS-Fe@polymer (for 3 groups, one received no laser irradiation, one received 808 nm light for 5 min at 1.2 W/cm2, one received 980 nm light for 5 min at 1.2 W/cm2). The dose of each experiment group was 2.0 mg/mL). After the mice were administered intravenously 12 h, the mice received the NIR irradiation for 5 min. The individual tumor volume (V) was calculated according to the following formula: V = (L × W2)/2, where length L is the longer dimension of tumor and width W is the shorter dimension perpendicular to length. At the end of treatment, the tumors were excised and sectioned into 10 μm thick slices with a cryostat for H&E staining according to the manufacture’s protocols. ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. Detailed synthesis route and methods, characterization of temperature sensitive polymer and the Fe-containing prodrug. The MTT results in PC3 cells and 4T1 cells.


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AUTHOR INFORMATION

Corresponding Authors

Chun-Yan Hong: [email protected]

Long-Hai Wang: [email protected]

Ye-Zi You: [email protected]

Author Contributions

The manuscript was written through contributions of all authors.

Notes

The authors declare no competing financial interest.

Acknowledgements

This work was financially supported by the National Natural Science Funds for Distinguished Young Scholars (21525420 and 51625305) and the National Natural Science Foundation of China (51273187, 21374107, and 21474097).


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TOC:


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Scheme 1. (a) Schematic of typical Fe(II) catalyzed Fenton reaction. (b) Schematic of the photothermal therapy nanomaterials boosting transformation of Fe(III) into Fe(II) in tumor cells for highly improving chemodynamic therapy.



Figure 1. (a) The temperature-transmittance plot of PEG-b-P(AAm-co-AN) in PBS (4.0 mg/mL, and the heating rate at 1.0 oC/min). (b) The pictures of bare CuS and CuS-Fe@polymer nanoparticles in the PBS. The left is the bare CuS and the right is CuS-Fe@polymer nanoparticles. (c) The TEM pictures of CuSFe@polymer. The scale in the picture is 50 nm. (d) The size distribution of CuS-Fe@polymer nanoparticles via DLS. (e) The schematic of CuS-Fe@polymer nanoparticles. (f) The EDX mapping pictures of CuSFe@polymer nanoparticles. (g) UV-Vis-NIR spectra of bare CuS and CuS-Fe@polymer nanoparticles.


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Figure 2. (a) The photothermal plots of CuS-Fe@polymer at different light (808 nm) power. The concentration is 60 µg/mL. (b) The photothermal plots of CuS-Fe@polymer with the 980 nm light treatment. (c) The plot of Fe2+ release from CuS-Fe@polymer under NIR irradiation (1.0 W/cm2, 5 min). Fe2+ was detected by 2,2’-bipyridine in 100 mM Tris-HCl, and the absorption of 519 nm reflects the released mount of Fe2+, [bipyridine] = 300 µM, [esterase] = 200 U/mL. (d) The experiments on RhB degradation by CuS enhanced Fenton reaction. The absorption of 553 nm reflects the mount of remained RhB. [RhB] = 10 µg/mL, [FeSO4 •7H2O] = 20 µg/mL, [H2O2] = 1.5 mM, [CuS] = 20 µg/mL. (e) EPR spectra of •OH at the room temperature. 5,’5 -Dimethyl-1-pyrrolidine-N-oxide (DMPO, 0.14 M) served as a spin trap. (f) Fe2+ concentration detection in the dark: UV spectra of 1,10-phenanthrolinen in the present of FeCl3, CuS and FeCl3 and CuS. The absorption peak at 511 nm is ascribed to the complex of ferrous ions. [FeCl3] = 1.0 mg/mL, [CuS] = 20 μg/mL, [phenanthrolinen] = 0.5 mg/mL. 156x96mm (300 x 300 DPI)



Figure 3. (a) Intracellluar ROS image Hela cells after treated with Fe@polymer, CuS@polymer/NIR, CuSFe@polymer, CuS-Fe@polymer/NIR. The concentration of each sample is 80 μg/mL, 808 nm light for 5 min at 0.5 W/cm2. The cells are treated with DCFH-DA before imaging by inverted fluorescence microscope. (b) DNA damages after Hela cells receiving the different treatments (the condition is the same to the ROS detection experiments) by single cell gel electrophoresis assay. The nuclear DNA was strained by GelRed, U=20 V,I=100 mA, 30 min. (c) The fluorescence images of Calcein-AM and PI double-stained cell with different treatments, 808 nm light, 0.75W/cm2 for 5 min. (d) The GSH level in Hela cell after the different treatments, 0.5 W/cm2 for 5 min. The GSH level was determined by Ellman’s regent (5,’5-dithio-bis (2nitrobenzoic acid, DTNB)). (e), f) Cytotoxicity of Fe@polymer, CuS@polymer/NIR, CuS-Fe@polymer and CuS-Fe@polymer/NIR in Hela cells and NIH3T3 cells. 808nm NIR light, 0.75 W/cm2 for 5 min. 156x157mm (300 x 300 DPI)


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Figure 4. (a) The biodistribution after the mice was administered intravenously 100 μL CuS (FITC)Fe@polymer, 2.0 mg/Kg. (b) The body weight changes during the treatment. (c) The tumor volume changes in the treatment. (d), (e) The picture of tumors and the tumor weights at the end of treatment. The numbers of 1, 2, 3, 4, 5, 6 respectively represent the group of PBS, CuS@polymer/808, Fe@polymer, CuSFe@polymer, CuS-Fe@polymer /808, CuS-Fe@polymer/980, and the power of NIR light is 1.2W/cm2. The absent of the date means that the tumor disappeared after receiving the treatment. (f) The tumor picture of mice in the CuS-Fe@polymer/808 group. (g) H&E-stained slices of the PBS groups and CuSFe@polymer/NIR groups mice after treatment, 1 means the group of PBS and the 5 means the group of CuS-Fe@polymer/808


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