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Integrated Nanoparticles to Synergistically Elevate Tumor Oxidative Stress and Suppress Antioxidative Capability for Amplified Oxidation Therapy Wei Yin, Junjie Li, Wendong Ke, Zengshi Zha, and Zhishen Ge ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b08347 • Publication Date (Web): 11 Aug 2017 Downloaded from http://pubs.acs.org on August 13, 2017

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ACS Applied Materials & Interfaces

Integrated Nanoparticles to Synergistically Elevate Tumor Oxidative Stress and Suppress Antioxidative Capability for Amplified Oxidation Therapy Wei Yin,†,1,2 Junjie Li, †,1 Wendong Ke,1 Zengshi Zha,1 and Zhishen Ge*,1

1

CAS Key Laboratory of Soft Matter Chemistry, Department of Polymer Science and

Engineering, University of Science and Technology of China, Hefei 230026, Anhui, China

2

Department of Pharmacology, Xin Hua University of Anhui, Hefei 230088, China

ACS Paragon Plus Environment

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ABSTRACT

The improved antioxidant system of cancer cells renders them well-adaptive to the intrinsic oxidative stress in tumor tissues. On the other hand, cancer cells are more sensitive to elevated tumor oxidative stress as compared with normal cells due to their deficient reactive oxygen species (ROS)-eliminating systems. Oxidation therapy of cancers refers to the strategy of killing cancer cells through selectively increasing the oxidative stress in tumor tissues. In this report, to amplify the oxidation therapy, we develop integrated nanoparticles with the properties to elevate tumor oxidative stress and concurrently suppress the antioxidative capability of cancer cells. The amphiphilic block copolymer micelles of poly(ethylene glycol)-b-poly[2-((((4-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxy)carbonyl)oxy)ethyl

methacrylate]

(PEG-b-

PBEMA) are integrated with palmitoyl ascorbate (PA) to form hybrid micelles (PA-Micelle). PA molecules at pharmacologic concentrations serve as prooxidant to upregulate the hydrogen peroxide (H2O2) level in tumor sites and PBEMA segment exhibits H2O2-triggered release of quinone methide (QM) for glutathione (GSH)-depletion to suppress the antioxidative capability of cancer cells, which synergistically and selectively kill cancer cells for tumor growth suppression. Given that the significantly low side toxicity against normal tissues, this novel integrated nanoparticle design represents a novel class of nanomedicine systems for highefficiency oxidation therapy with the potentials to be translated to clinical applications.

KEYWORDS:

oxidation therapy, polymeric micelle, peroxide-responsive, cancer therapy,

GSH depletion


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INTRODUCTION

Normal cells usually maintain a redox homeostasis.1 Specifically, reactive oxygen species (ROS) are produced and used to regulate many biological functions including modulation of many enzyme activity (e.g. ribonucleotide reductase), mediating inflammation, clearing pathogen and foreign materials; on the other side, cells develop a series of systems to eliminate and balance the ROS level, for example, superoxide dismutases, glutathione peroxide, and catalase. In tumor tissue, cancer cells are usually immersed in the oxidative stress due to deficient ROS-eliminating systems resulting in the alteration of redox balance.2 Under the intrinsic oxidative stress, the cancer cells become adapted well and survive through many pathways such as upregulation of glutathione (GSH) level inside cells.3 Notably, increased ROS level as a hallmark of cancer is associated with cancer cell proliferation, tumor growth, metastasis, and even resistance to chemotherapy or radiotherapy.2-4 However, too high ROS level beyond a certain threshold value will selectively kill cancer cells due to their deficient ROSeliminating systems.5,6 Therefore, selective elevation of tumor ROS level or inhibition of the antioxidative capacity have been explored to selectively kill cancer cells and treat cancers.7,8 Oxidation therapy refers to a unique therapeutic strategy towards cancers through administration of exogenous agents for selective generation of ROS in tumor or inhibition of the antioxidant systems to alter redox balance of cancer cells and kill them.7 A variety of ROSproducing agents in tumor tissues have been explored for oxidation therapy through selectively enhancing the tumoral ROS level, such as xanthine oxidase,9 arsenic trioxide (As2O3),10 D-amino acid oxidase,11 high dose of vitamin C (Vc),12 cinnamaldehyde,13 and piperlongumine.14 Alternatively, for inhibition of antioxidant systems, some therapeutic agents have been tested including heme oxygenase-1 (HO-1) inhibitor (zinc protoporphyrin),15 superoxide dismutase


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(SOD) inhibitor (2-methoxyestradiol and copper chelators),16,17 GSH-depleting agents (isothiocyanates, aulphoraphane, and quinone methide).8 Recently, the combination of increasing the ROS level and decreasing antioxidant capacity was exploited to maximally treat tumors.8,18-20 For example, combination of As2O3 for ROS production and ascorbic acid-mediated GSH depletion exhibited clinical effectiveness to treat refractory multiple myeloma.21 Lee et al.18 reported a small-molecule hybrid prodrug with responsive decomposition into quinone methide (QM) and cinnamaldehyde for combination of cinnamaldehyde-based ROS generation and QMbased GSH depletion to enhance cell death. The well-defined polymeric nanoparticles with long blood circulation and high tumor accumulation have been explored to treat cancer through elevation of ROS level in tumor tissues.22 However, to date, nanomedicines with the combined synergistic functions of elevation of tumor oxidative stress and inhibition of antioxidant capability were rarely reported, which may achieve amplified oxidation therapy. Vc and its derivative palmitoyl ascorbate (PA) usually act as antioxidants at relatively low concentration. However, they function as prooxidants at pharmacologic concentrations in tumor tissues and kill cancer cells through increasing the level of hydrogen peroxide (H2O2) at tumor site.12,23-25 Although antitumor activity of Vc and PA has been observed against a variety of cancer cells and tumor types, and has attracted great attentions in recent years, the modest therapeutic efficacy may limit their clinical translation as an efficient anticancer drug.26-29 Thus, Vc was frequently explored to combine with anticancer drugs to treat tumors. Herein, to increase the therapeutic efficacy of nanomedicine-based oxidation therapy, we design integrated micellar nanoparticles composed of amphiphilic block copolymer poly(ethylene glycol)-b-poly[2-((((4(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxy)carbonyl)-oxy)ethyl

methacrylate]

(PEG-b-PBEMA) and PA molecules (Scheme 1). In tumor tissue, high concentration of PA acts


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as a prooxidant agent to upregulate H2O2 level. Meanwhile, PBEMA segments can release QM responsive to upregulated H2O2 level, which depletes intracellular GSH to weaken the antioxidant capacity of the cancer cells. Finally, the amplified oxidation therapy can be anticipated to selectively kill cancer cells by the synergistic effect of strong oxidative stress.

Scheme 1. Schematic illustration of integrated micellar nanoparticles with tumor-specific H2O2 generation to increase the oxidative stress in tumor tissue and simultaneous GSH depletion to inhibit the antioxidant capability of cancer cells for efficient cancer cell killing. EXPERIMENTAL SECTION Materials. 2-Hydroxyethyl methacrylate (HEMA, ≥ 99%, contains ≤ 50 ppm monomethyl ether hydroquinone as inhibitor) was obtained from Sigma-Aldrich and purified based on a previously


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reported method.30 Azobisisobutyronitrile (AIBN) was purified via recrystallization twice in methanol. Fetal bovine serum (FBS), Dulbecco's modified Eagle's medium (DMEM), and trypsin were purchased from Gibco Company (USA). PA (≥ 99%) from Alfa Aesar was obtained and used as received. PEG-based macroRAFT agent (Mn = 5 kDa, Mn/Mw = 1.02) was synthesized based

on

the

previously

reported

procedures.31

1,1'-Dioctadecyl-3,3,3',3'-

tetramethylindotricarbocyanine iodide (DiR) was purchased from Fanbo Chemicals (Beijing, China). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), propidium iodide (PI, 94%), 2′,7′-dichlorofluorescin diacetate (DCFH-DA, ROS probe), adenosine triphosphate (ATP) Assay Kit, and GSH/GSSG Assay Kit were obtained from Beyotime Institute of Biotechnology (Shanghai, China). DNA Damage Assay Kit (Tris-HCl buffer, low melting point agarose, LMA, normal melting point agarose, NMA) was obtained from Nanjing Jiancheng Bioengineering Institute. Mouse breast cancer cell line 4T1, non-small cell lung cancer cell line A549, mouse embryonal fibroblast cell NIH3T3, and human cervical carcinoma cell line HeLa were obtained from the cell bank belonging to Chinese Academy of Sciences in Shanghai (China). Female BALB/c mice at the age of 5-6 weeks were purchased from Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China). The animal studies were conducted according to the Regulations for the Administration of Affairs Concerning Experimental Animals (Hefei, revised in June 2013). Sample Preparation (Scheme S1) Synthesis of Monomer 2.

The monomer 2 was synthesized from pinacol boronic ester-

containing imidazoyl carbamate 1 and HEMA. 1 (2.5 g, 7.62 mmol) was dispersed in dry CH2Cl2 (50 mL) in a flame-dried 100-mL flask. HEMA (1.0 g, 7.68 mmol) and 4-dimethylaminopyridine (DMAP) (0.8 g, 6.5 mmol) were added to the flask and reacted at 40 °C for 30 h. Subsequently,


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the reaction mixture was concentrated under reduced pressure, affording a yellowish solid which was further purified by chromatography eluting with CH2Cl2/EtOAc (9:1). The final white solid was obtained (1.7 g, yield: 58.4%). 1H NMR (300 MHz, CDCl3, Figure 1A) δ: 1.27 (s, 12H), 1.86 (s, 3H), 4.33 (m, 4H), 5.20 (s, 2H), 5.50 (s, 1H), 6.03 (s, 1H), 7.32 (d, 2H), 7.72 (d, 2H); 13C NMR (75 MHz, CDCl3) δ:18.24, 24.85, 58.60, 62.46, 65.68, 69.64, 83.90, 126.26, 127.28, 127.33, 135.04, 135.76, 137.99, 138.09, 154.93,167.07. ESI-MS Calcd. for (C20H27BO77 + H)+: 391.2; Found: 391.4. Synthesis of PEG-b-PBEMA. Monomer 2 (0.8 g, 2.04 mmol), PEG-based RAFT agent (0.128 g, 0.04 mmol), 2,2′-azobisisobutyronitrile (AIBN) (0.6 mg, 3.64 × 10-3 mmol), and 1,4-dioxane (4 mL) were added into a 10-mL Schenk flask. The mixture was degassed through three cycles of freeze-pump-thaw. The flask was subsequently sealed under vacuum followed by reaction for 12 h in a preheated oil bath at 80 °C. After the reaction was quenched in liquid nitrogen, the reaction solution was precipitated in cold diethyl ether. The dissolution-precipitation cycle was repeated for three times. After drying in a vacuum oven overnight, the final product was obtained as a pale yellow powder (0.676 g, Yield: 68.5%, Mn,GPC = 35400, Mw/Mn = 1.10). The degree of polymerization (DP) of the PBEMA segment was determined to be 78 according to the analysis of 1H NMR (Figure 1B). Thus, the final block polymer was denoted as PEG113-b-PBEMA78. Micelle Preparation. PA was encapsulated into the micelles of the amphiphilic block copolymer, PEG113-b-PBEMA78 via the thin-film hydration method.24 In brief, PEG113-bPBEMA78 block copolymer and various amounts of PA were dissolved in the mixed solvent of methanol/chloroform (2:1, v/v). The solvent was then evaporated and dried under vacuum for 2 h to prepare the film, followed by hydration using phosphate buffered saline (PBS) (pH 7.4, 10 mM) for 2 h at 25 oC. Subsequently, the solutions were filtered by 0.45-µM Millipore filters to


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remove the undissolved substance. The final encapsulation efficiencies and loading contents into the micelles were determined by reversed-phase high performance liquid chromatography (RPHPLC). The polymer concentration was fixed at 1.0 mg/mL (28 µM) loading varying concentrations of PA (0, 0.25, 0.5, 1.0, 2.0, and 4.0 mM). The final encapsulation efficiencies of PA were determined to be higher than 95% in the concentration range that we used. Therefore, the concentrations of PA in PEG-b-PBEMA micelles were approximatively considered as 0, 0.25, 0.5, 1.0, 2.0 and 4.0 mM. The micellar nanoparticles without and with PA was denoted as Micelle and PA-Micelle, respectively. The serum stability of the PA-Micelle at the polymer concentration of 1 mg/mL and PA concentration of 1 mM was evaluated through incubation in DMEM containing 20% FBS and the particle sizes were monitored using DLS. H2O2 Production and QM Release. The H2O2 production from PA in the cell culture medium containing 10% FBS was tested using dissolved oxygen (D.O.) meter under the catalysis of catalase.12 In brief, PA (0.5, 1.0, or 2.0 mM) and PA-Micelle (PA: 1 mM, polymer: 28 µM) were added into DMEM medium containing 10% FBS and incubated at 25 °C. The solution (10 mL) was then token out at predetermined time intervals and added catalase solution (200 µL, 1000 units). O2 sensor was soaked in the collected medium. The H2O2 concentration in the sample was determined by establishing the prior standard calibration regarding O2 production and H2O2 concentrations in the solutions in the range of 10-200 µM. In order to evaluate H2O2-responsive QM release, the micelles were first treated by 0, 50, 100, or 500 µM H2O2 at 37 °C for different times. The amounts of 4-hydroxybenzyl alcohol (HA) release were determined by 1H NMR analysis. On the other hand, PA-Micelle containing 1 mM PA and 0.25 mg/mL polymer was incubated in DMEM containing 10% FBS and HA release was analyzed at varying time points by RP-HPLC.


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To test the GSH depletion, PA-Micelle containing 1 mM PA and 56 µM (2 mg/mL) polymer was incubated in DMEM medium containing 10% FBS in the presence of GSH (1 mM) for 24 h. After lyophilization, the sample was measured by LTQ-Qrbitrap XL mass spectrometer. In Vitro Cytotoxicity. 4T1, HeLa, A549, or NIH3T3 cells were seeded in 96-well plates at a density of 1 × 104 cells each well in DMEM (100 µL) containing 10% FBS at 37 °C under 5% CO2 humidified atmosphere. After 12 h of incubation, the medium was removed, and then fresh DMEM with 10% FBS was added. Various final concentrations of PEG-b-PBEMA polymer (0.028, 0.28, 1.4, 2.8, and 28 µM) with PA concentration fixed at 1.0 mM, or various final concentrations of PA (0, 0.25, 0.5, 1.0, 2.0 mM) with the polymer concentration fixed at 28 µM. PA (1.0 mM) was dissolved in a little dimethyl sulphoxide (DMSO) and added into cell culture medium. To minimize the influence of DMSO, the final DMSO concentration in the medium was controlled to less than 0.1%. After 48 h incubation, for MTT assay, MTT solution (20 µL, 5 mg/mL) was added. After incubation for 4 h, the culture medium was removed. DMSO solution (150 µL) was added and the plates were subsequently placed on the microplate plate oscillator for 10 minutes, the microplate reader was used to determine the final UV absorbance at detection wavelength 490 nm. In Vivo Pharmacokinetics and Distribution. Free PA or PA-Micelle were intravenously injected into the tail vein at the PA-equivalent dose of 15 mg/kg mouse body weight. At different time intervals, 200 µL plasma samples were collected from mouse orbital and charged into heparinized tubes. Then, the samples were suspended in DMSO to extract the drugs. After centrifugation at 10000 ×g for 5 min to collect the supernatant, the content of PA in supernatant was examined by RP-HPLC (mobile phase: acetonitrile/CH3OH (2:8, v/v), 1 mL / min, UV-vis detection-wavelength fixed at 243 nm).


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4T1 cells (3 × 106) dispersed in PBS (200 µL) were subcutaneously injected into the right limb armpits of 5-week-old female BALB/c. When tumor volume reached ~ 200 mm3, DiRloaded PA-Micelle in PBS were intravenously injected into the tail vein at the DiR concentration of 1 mg/kg mouse body weight. At different time intervals (1, 12, 24, 48 h), the mice were anesthetized and an IVIS small-animal imaging system was used to obtain the images to investigate biodistribution of PA-Micelle. Antitumor Efficacy. 4T1 tumor-bearing mice were used for investigation of the antitumor efficacy of the nanoparticles. The tumor growth profiles were observed by detecting tumor size using a vernier caliper. The single tumor volume (V) was determined by the equation: V = (L × W2)/2, where length (L) represents the long dimension of tumors and width (W) represents the short one. 4T1 tumor-bearing mice (tumor size ~ 200 mm3) were divided into four groups containing five mice in each group. Subsequently, various formulations were intravenously injected into the tail vein of the mice at a dose of Micelle (polymer 35 mg/kg mice body weight), PA (63 mg/kg), or PA-Micelle (polymer 35 mg /kg and PA 63 mg /kg) on day 0, 2, and 4. The tumor growth was observed by detecting tumor size. At day 21 post administration, the mice were killed to collect tumors and main organs. The weights of tumors were recorded. Then, the tumors and organs were embedded in paraffin and sectioned into 5-µm slices. The sections were stained with haematoxylin and eosin (H&E) according to manufacturer’s protocols. Statistical Analysis. All data obtained were presented as mean ± standard deviation (s.d.). Furthermore, student’s t-test was engaged to analyze the difference between the study groups. In particular, the obtained p-values (< 0.05) were considered to be statistically significant.


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RESULTS AND DISCUSSION Block Copolymer Synthesis and Self-Assembly. Boronate esters were extensively explored to act as H2O2-responsive moieties in the oxidation-responsive materials.32-37 After treatment by H2O2, boronate esters will release QM, which has been demonstrated to react with GSH.18,19 Given that GSH peptide works as the primary reductive agent inside cancer cells to maintain the redox balance, efficient GSH depletion based on massive QM release from nanoparticles was hypothesized to continuously inhibit the antioxidant ability of cancer cells. To construct welldefined boronate ester-containing polymeric nanoparticles, we design the amphiphilic block copolymer, PEG-b-PBEMA, for self-assembly in aqueous solution (Scheme S1, Supporting Information). Initially, BEMA monomer, 2-((((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2yl)benzyl)oxy)carbonyl)oxy)ethyl methacrylate, was synthesized via the reaction between hydroxyl group of HEMA monomer and imidazoyl carbamate 1. Subsequently, PEG macroRAFT agent was used as the chain transfer agent (CTA) to polymerize BEMA through RAFT polymerization. DPs can be determined by comparing the integrals of PEG signal (a) and boronate ester peaks (e, d, c) (Figure 1A). Gel permeation chromatography (GPC) characterization of PEG-b-PBEMA block copolymer revealed a narrow-dispersity peak without a shoulder in the PEG position indicating efficient initiation of polymerization (Figure 1B). For the following self-assembly and in vivo application, PEG and PBEMA blocks with DPs of 113 and 78, respectively, were optimized for further evaluation.


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Figure 1. (A) 1H NMR spectra recorded for the monomer 2 and block copolymer PEG-bPBEMA in CDCl3. (B) GPC traces recorded for PEG-RAFT agent (Mn=5000, Mw/Mn = 1.02) and block copolymer PEG-b-PBEMA (Mn = 35400, Mw/Mn =1.10). Next, the amphiphilic block copolymer was self-assembled into micelles via nanoprecipitation upon addition of PEG-b-PBEMA solution from THF into PBS buffer, which was denoted as Micelle. PA was encapsulated into the micelles via the thin-film hydration method. According to RP-HPLC characterization, the encapsulation efficiency was high up to 95% which enabled the loading content of the micelle as high as 13% at the initial PEG-b-PBEMA and PA concentration of 28 µM (1 mg/mL) and 1 mM, respectively. PA-loaded PEG-b-PBEMA micelles were denoted as PA-Micelle for the following evaluation. Notably, PA-Micelle displayed transparent and bluish stable solution at polymer concentration of 1 mg/mL and PA concentration of 1 mM (Figure S1). In contrast, sole PA solution showed obvious precipitation in aqueous solution. PA-


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Micelle was further characterized by dynamic laser light scattering (DLS) and transmission electron microscopy (TEM) analysis. DLS results showed the particle diameter was 65 nm (Figure 2A). TEM images demonstrated that the particle sizes were 52 ± 10 nm (Figure 2B). The deviation between DLS and TEM results was attributed to different hydrated state of the observed samples in aqueous solution by DLS and invisible PEG shells under TEM observation. Moreover, serum stability of PA-Micelle were evaluated after incubation for 48 h in DMEM containing 20% FBS at the polymer concentration of 1 mg/mL and PA concentration of 1 mM. After 48 h, DLS results indicated that the average particle sizes maintained constant indicating high stability of PA-Micelle in serum-containing solutions (Figure S2).

Figure 2. (A) and (B) DLS and TEM characterization of PA-Micelle. (C) Time-dependent H2O2 production in DMEM cell culture medium containing 10% serum in the presence of various


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concentrations of PA and PA-Micelle. Mean ± s.d., n = 3. (D) 1H NMR spectrum recorded for H2O2-responsive decomposition of PEG-b-PBEMA and formation of 4-hydroxybenzyl alcohol from released QM after incubation for 12 h in the presence of 100 µM H2O2. (E) Time-dependent QM release based 1H NMR spectra characterization after treatment by 0, 50, 100, 500 µM H2O2 and 1 mM PA-Micelle in DMEM containing 10% FBS. Mean ± s.d., n = 3. H2O2 Production and QM Release. Pharmacologic Vc concentrations can serve as a prooxidant generating H2O2 in serum-containing cell culture medium and tumor tissue.12,29 To validate PAMicelle can produce ROS efficiently, we firstly incubated PA or PA-Micelle with cell culture medium, DMEM containing 10% serum. As shown in Figure 2C, PA-Micelle loading 1 mM PA showed high-efficiency H2O2 generation with over 100 µM H2O2 in the culture medium at 2 h which was slightly lower than that of pure PA molecules. The relatively lower H2O2 generation was likely owing to consumption by PBEMA segments. Notably, H2O2 concentrations in the medium reached a peak at 2 h and gradually fell. If H2O2-scanvenger catalase was added, no H2O2 was measured in the medium due to quick decomposition by catalase. Moreover, we further evaluated H2O2-responsive QM release. 1H NMR analysis revealed that significant HA production after incubation of Micelle with low concentration of 100 µM H2O2 for 24 h (Figure 2D). QM has been confirmed by the previous reports to convert into HA rapidly in aqueous solution in the absence of nucleophiles (e.g. GSH).18,19,32 HA production suggests that H2O2-triggered QM release occurred at 100 µM H2O2. Thus, we determined the amount of QM release through monitoring the amount of produced HA by RP-HPLC. Time-dependent QM release profiles as shown in Figure 2E exhibited H2O2 concentration-dependent manner. In the absence of H2O2, few QM release was observed despite of incubation for 72 h. In the presence of the low concentration of 50 µM H2O2, 23.8% QM release can be achieved after 72 h incubation. At 100 and 500 µM, QM release rate was significantly increased. Moreover, through


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incorporation of PA into the micelles at PA concentration of 1 mM, H2O2 concentration in the cell culture medium can be improved to more than 100 µM (Figure 2C). Therefore, QM rapid release can be easily achieved responsive to improved H2O2 concentration by PA-Micelle. To test whether the reaction between released QM and GSH occurred in aqueous solution, we incubated PA-Micelle in DMEM containing 10% FBS in the presence of 10 mM GSH for 24 h. The reaction mixture was freeze-dried and applied to LTQ-Qrbitrap XL mass spectrometer measurements. From the final results, effective reaction between GSH and QM generated from PEG-b-PBEMA in aqueous solution was confirmed. The signal of produced GSH-QM can be found clearly in the final spectrum (m/z = 412.1) (Figure S3). In Vitro Cytotoxicity. Next, we evaluated the cytotoxicity of PA-Micelle against a series of cancer cells (4T1, HeLa, and A549) and a normal cell line (NIH3T3) (Figure 3). We fixed PA concentration or polymer concentration and changed the corresponding polymer concentrations or PA concentrations, respectively. For cytotoxicity evaluation, PA and polymers were first dissolved in minimal amount of DMSO and then added into the cell culture medium. Apparently, 4T1 cell line is remarkably more sensitive to PA-Micelle compared with HeLa and A549 cells, which may be attributed to the different complex networks of H2O2 cytotoxicity and different functional mutations among various cancer cell lines.38 The results were in good agreement with the previous studies of Vc-based cancer therapy.23 PEG-b-PBEMA or PA alone can cause modest cytotoxicity even at high concentration.12 Cytotoxicity of PEG-b-PBEMA may be attributed by massive GSH depletion while that of PA is owing to the H2O2 generation. The combined PA and Micelle were expected to increase the cytotoxicity synergistically via H2O2 generation and GSH depletion. For 4T1, at PA concentration of 1.0 mM, block copolymer PEGb-PBEMA exhibited half maximal inhibitory concentration (IC50) of 2.5 µM compared with


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140.50 µM without PA. At the polymer concentration of 28 µM, IC50 of PA was determined to be 0.44 mM compared with 0.63 mM without the polymer. Notably, we also measured the cytotoxicity of PA-Micelle against normal cell line NIH3T3. PA-Micelle did not show any cytotoxicity towards NIH3T3 in our studied concentration range. Presumably, normal cells have perfect systems to maintain the redox balance which may avoid the damage of GSH depletion and H2O2 generation toward cells. Therefore, we can reasonably infer that PA-Micelle will cause minimal side toxicity to normal tissues while show significant cytotoxicity towards some cancer cell lines, particularly 4T1.

Figure 3. Cytotoxicity against several cell lines (A: 4T1, B: HeLa, C: A549, D: NIH3T3) as a function of PEG-b-PBEMA polymer concentrations (PA concentration was fixed at 1.0 mM for PA-Micelle) or as a function of PA concentrations (polymer concentration was fixed at 28 µM for PA-Micelle). In Vitro ROS Elevation, GSH Depletion, and DNA Damage. To further investigate the ROS elevation in 4T1 cells, DCFH-DA was used as a fluorescent probe to observe the intracellular ROS, which can stain various ROS inside cells (Figure 4A,B).39 PBS control and Micelle


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showed weak fluorescence intensity which should be attributed to the intrinsic ROS level inside cells. In sharp contrast, PA and PA-Micelle showed significantly higher ROS concentrations (***p < 0.005). The generated H2O2 in the cell culture medium or inside cells by PA resulted in significantly increased ROS concentration inside cells. And the generated QM from PA-Micelle also inhibited the antioxidative capability via GSH depletion, thereby elevating the intracellular ROS concentration. To evaluate the GSH depleting ability of PA-Micelle, GSH concentration in 4T1 cells after treatment with PA-Micelle was further investigated by using GSH/GSSG Assay Kit After incubation with various concentrations of PEG-b-PBEMA at PA concentration of 100 µM for 24 h, the GSH level was quantitatively measured. As shown in Figure 4C, PA-Micelle at the polymer concentration of 1.3 µM and PA concentration of 100 µM can reduce the GSH concentration to lower than 30% of the intrinsic one (0 µM). Notably, pure PA showed weak GSH-depleting ability (Figure S4). Thus, the GSH depletion is presumably attributed to the potent GSH depletion ability of PEG-b-PBEMA polymer. PA-Micelle with ROS elevation and GSH depletion against 4T1 cells has been confirmed to be highly cytotoxic, which caused damage to the cells particularly destroying DNA. ATP concentration analysis also confirmed that PA-Micelle significantly reduced the cellular activity, which showed significantly better therapeutic efficacy towards 4T1 cells as compared with Micelle and PA alone (Figure 4D). To evaluate the cellular damage, we further examined the DNA damage via comet assay (Figure 4E,F). Micelle showed as low as ~ 20% tail DNA indicating low DNA damage while PA showed ~ 50% tail DNA at PA concentration of 1 mM. Intriguingly, PA-Micelle exhibited ~ 80% tail DNA suggesting severe DNA damage. DNA


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damage results revealed that the synergistic effect of GSH depletion and ROS elevation of PAMicelle induced high lesions to 4T1 cells.

Figure 4. (A) and (B) CLSM images of ROS level inside 4T1 cells through green fluorescence after treatment with PBS, Micelle (28 µM ), PA (1 mM polymer), and PA-Micelle (1 mM PA and 28 µM polymer). DCFH-DA probe was used to stain ROS. Intracellular ROS of green fluorescence was quantified and summarized in bar graph. Scale bars represent 25 µm. Mean ± s.d., n = 6, ***p < 0.005 (t-test) (C) GSH depletion in 4T1 cells after treatment by PA-Micelles at PA concentration of 100 µM and various polymer concentrations. Mean ± s.d., n = 4, **p < 0.01 (t-test). (D) ATP detection in 4T1 cells after treatment with PBS (control), Micelle (28 µM), PA


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(1 mM polymer), and PA-Micelle (1 mM PA and 28 µM polymer). Mean ± s.d., n = 3, **p < 0.01 (t-test). (E) Comet assay after treatment by PBS (control), Micelle (28 µM), PA (1 mM polymer), and PA-Micelle (1 mM PA and 28 µM polymer) against 4T1 cells. PI was used stain DNA into red for visualization. (F) Ratio of comet tails according to the images in comet assay. Mean ± s.d., n = 20, *p

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