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Polymer Prodrug-Based Nanoreactors Activated by Tumor Acidity for Orchestrated Oxidation/Chemo-Therapy Junjie Li, Yafei Li, Yuheng Wang, Wendong Ke, Weijian Chen, Weiping Wang, and Zhishen Ge Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.7b03531 • Publication Date (Web): 04 Oct 2017 Downloaded from http://pubs.acs.org on October 4, 2017

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Polymer Prodrug-Based Nanoreactors Activated by Tumor Acidity for Orchestrated Oxidation/ChemoTherapy Junjie Li,1 Yafei Li,2,3 Yuheng Wang,1 Wendong Ke,1 Weijian Chen,1 Weiping Wang,2,3,* and Zhishen Ge1,* 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

Dr. Li Dak-Sum Research Centre, The University of Hong Kong-Karolinska Institutet

Collaboration in Regenerative Medicine, The University of Hong Kong, Pokfulam, Hong Kong, China

3

Department of Pharmacology & Pharmacy, Li Ka Shing Faculty of Medicine, The University

of Hong Kong, Pokfulam, Hong Kong, China

Corresponding authors: Email: [email protected], [email protected]

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ABSTRACT

Therapeutic nanoreactors have been proposed to treat cancers through in situ transformation of low-toxicity prodrugs into toxic therapeutics in the body. However, the in vivo applications are limited by low tissue-specificity and different tissue distributions between sequentially injected nanoreactors and prodrugs. Herein, we construct a block copolymer prodrug-based polymersome nanoreactor that can achieve novel orchestrated oxidation/chemo-therapy of cancer via specific activation at tumor sites. The block copolymers composed of poly(ethylene glycol) (PEG) and copolymerized monomers of camptothecin (CPT) and piperidine-modified methacrylate (P(CPTMA-co-PEMA)) were optimized to self-assemble into polymersomes in aqueous solution for encapsulation of glucose oxidase (GOD) to obtain GOD-loaded polymersome nanoreactors (GOD@PCPT-NR). GOD@PCPT-NR maintained inactive in normal tissues upon systemic administration. After deposition in tumor tissues, tumor acidity-triggered protonation of PPEMA segments resulted in high permeability of the polymersome membranes and oxidation reaction of diffused glucose and O2 under the catalysis of GOD. The activation of the reaction generated H2O2, improving the oxidative stress in tumors. Simultaneously, high level of H2O2 further activated PCPTMA prodrugs, releasing active CPT drugs. High tumor oxidative stress and released CPT drugs synergistically killed cancer cells and suppressed tumor growth via oxidation/chemo-therapy. Our study provides a new strategy for engineering therapeutic nanoreactors in an orchestrated fashion for cancer therapy.

KEYWORDS Cancer therapy, polymer prodrug, polymersomes, stimuli-responsive nanoparticles, therapeutic nanoreactors

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Nanoreactors refer to the nanoparticles that provide a confined reaction space for active species (e.g. enzymes) to improve chemical reaction efficiency.1-3 Nanoreactors can protect enzymes from the surrounding medium, thus preserving or controlling the activity. Inspired by the natural biosynthetic processes, construction of nanoreactors based on a series of synthesized nanoparticles or supramolecular nanoassemblies has been explored, such as dendrimers, liposomes, polymeric micelles, layer-by-layer or polymerized capsules, polymersomes and so on.2-5 Nanoreactors have showed wide applications in various fields, for example, organic synthesis,6 polymerization,7 nanoparticle synthesis,8 and even artificial organelles.5,9 In the field of nanomedicine, therapeutic nanoreactors have been proposed as nanofactories for converting low-toxicity molecules into toxic therapeutics or detoxifying toxic substances in the body.10-13 In cancer therapy, nanoreactors offer the feasibility for high-efficiency in situ transformation of prodrugs into original drugs, which show distinct advantages of maximizing therapeutic efficacy and minimizing side effects. For example, Kataoka et al.14 demonstrated a polyion vesicular nanoreactor functioning in tumors to transform model free prodrugs into fluorescence products, which can work even after four days post systemic administration of nanoreactors. Just recently, Akiyoshi et al.15 developed enzyme-loaded polymeric vesicles with an intrinsically permeable membrane, which achieved transformation of free doxorubicin prodrugs for inhibiting tumor growth in mice. Weissleder and coworkers16 reported polymeric micelles loading palladium catalysts to treat cancers through in vivo catalysis of prodrug activation. However, the lack of tissue-specificity of nanoreactors unavoidably resulted in production of toxic drugs in normal tissues. Different preferential tissue distributions between nanoreactors and smallmolecule prodrugs also limited the reaction efficiency. It remains a great challenge to prepare the

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tumor site-specific nanoreactors for “on demand” and efficient production of toxic anticancer drugs from low-toxicity prodrugs for tumor cell killing. As one class of special nanoreactors, polymersomes possess many intrinsic advantages and have attracted great attentions in recent years.4,5,17,18 They show higher stability in aqueous solutions as compared with liposomes. The aqueous interior chamber and hydrophobic membranes enable them to load both hydrophilic and hydrophobic molecules, respectively.17,19,20 Hydrophilic catalysts (e.g. enzymes) can be encapsulated into the aqueous interior and protected by the membranes.14,21,22 Polymersome properties, such as particle sizes and membrane properties, can be adjusted facilely by changing polymer properties and self-assembly methods.23 Moreover, it should be noted that incorporation of stimuli-responsive moieties or pore proteins could modulate the membrane permeability via external (e.g. light) or endogenous stimuli (e.g. pH, redox, and glucose).24-32 We propose that tumor microenvironment-responsive polymersome nanoreactors may achieve the tumor-specific in situ production of toxic therapeutics for cancer therapy. Herein, we for the first time devised a block copolymer prodrug-based polymersome nanoreactor loading glucose oxidase (GOD), which can be specifically activated by tumor acidity for novel orchestrated oxidation/chemo-therapy (Figure 1). Oxidation therapy treats cancers by increasing tumor oxidative stress based on the fact that cancer cells frequently possess deficient reactive oxygen species (ROS)-eliminating systems.33,34 GOD as a high-efficiency H2O2-generating catalyst is exploited to produce in situ H2O2 in tumors.35-37 However, the substrate glucose is available in vivo ubiquitously leading to no regulation of ROS generation. In this work, the novel GOD-loaded polymer prodrug nanoreactors display some distinctive features in an orchestrated fashion: (i) the prodrug-based polymersome avoids the issue of

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different tissue distributions between prodrugs and nanoreactors; (ii) incorporation of tumor acidity-responsive segments into the polymersomes allows for specific activation of the nanoreactors via increased membrane permeability under the tumor microenvironment; (iii) activated nanoreactors can produce high level of H2O2 through oxidation of glucose under GOD catalysis to increase tumor oxidative stress; (iv) anticancer drug camptothecin (CPT) linked to the polymers via a H2O2-cleavable linkage can be released by the high concentration of H2O2. Finally, produced H2O2 and released CPT drugs can synergistically kill cancer cells and suppress tumor growth via orchestrated oxidation/chemo-therapy. We initially synthesized the amphiphilic block copolymer via reversible additionfragmentation

chain

transfer

(RAFT)

copolymerization

of

2-(methacryloyloxy)ethyl

camptothecin oxalate (CPTMA) and 2-(piperidin-1-yl)ethyl methacrylate (PEMA) using poly(ethylene glycol) (PEG)-based macroRAFT agent (Scheme S1). PPEMA polymers have been demonstrated to be an ultra pH-sensitive polymer with the transition from hydrophobic neutral polymer to positively charged hydrophilic one.38 The CPT drug-linked segments facilitate self-assembly of the block copolymers in aqueous solution due to their hydrophobicity and π-π stacking interactions.39 For the block copolymer PEG-b-P(CPTMA-co-PEMA), the molar ratios of CPTMA to PEMA were optimized for self-assembly into uniform vesicles that could maintain the vesicular integrity when PPEMA segments transformed from hydrophobic to hydrophilic. Particularly, the initial molar ratio of 2:1 of CPTMA to PEMA was selected to prepare the amphiphilic diblock copolymer. The final block copolymer, PEG113-b-P(CPTMA34-co-PEMA20), was used for the following nanoreactor preparation and investigation. The precise degrees of polymerization were determined by 1H NMR analysis (Figure S1A). GPC characterization indicated a narrow molecular weight (MW) distribution (Mn = 30.3 kDa, Mw/Mn = 1.11) with a

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symmetric peak (Figure S1B), which is favorable for self-assembly into uniform vesicular nanoparticles. The CPT drug loading capacity was determined to be as high as 43.3%. Amphiphilic block copolymers can self-assemble into various morphologies in aqueous solution via optimizing self-assembly methods and polymer properties.40 Here, PEG113-bP(CPTMA34-co-PEMA20) self-assembled into polymersomes via “solvent-switch” method upon addition of phosphate buffered saline (PBS, pH 7.4) into the polymer tetrahydrofuran (THF) solution. Transmission electron microscopy (TEM) images show distinct contrast between the periphery and the center, indicating the characteristic vesicular morphology with the vesicle size of 105 ± 12 nm and shell thickness of 21 ± 3.1 nm (Figure S2). Dynamic light scattering (DLS) results demonstrate the size of 112 nm with the polydispersity index (PDI) of 0.112 indicating a relatively uniform size distribution. In addition to the narrow MW distribution and suitable block length ratio of the block copolymer, the optimized organic solvent (THF) and PBS addition rate (1 mg/mL) also account for the formation of the relatively uniform polymersomes.23,40 The strong hydrophobic and π-π staking interactions between CPT moieties are beneficial for the self-assembly into stable polymersomes.39,41 GOD enzymes were encapsulated into the interior aqueous chambers of the polymersomes during the self-assembly process. The GOD loading capacities were dependent on the feeding GOD concentrations. Unencapsulated GOD was removed by washing with PBS and centrifugation. Specifically, we investigated the preparation of the nanoreactors with the initial GOD concentration range of 0.1-1.0 mg/mL. The maximum loading capacity was determined to be 5.66% at the initial GOD concentration of 1.0 mg/mL, which can guarantee the highefficiency glucose oxidation reaction under the catalysis of GOD. Therefore, we chose the loading capacity of 5.66% of the polymersomes for the following investigation unless otherwise

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stated, which were denoted as GOD@PCPT-NR. The size and nanoparticle morphology maintained constant after encapsulation of GOD compared with those without GOD (Figures 2A and S2). The polymersomes without GOD encapsulation were used as the control and denoted as PCTP-V. Subsequently, we determined the pKa of PPEMA segments in PEG113-b-P(CPTMA34-coPEMA20) to be 6.91 in aqueous solutions via acid-base titration (Figure 2C). Thus, with pH values changed from 7.4 to 6.8, PPEMA segments can transform from hydrophobic to hydrophilic due to positively charged protonation. We further evaluated the pH-responsive behavior of GOD@PCPT-NR. With the solution pH decreased from 7.4 to 6.8, the size of GOD@PCPT-NR merely increased by 11 ± 3.5 nm and the membrane thickness maintained almost constant (Figure 2A, B). No GOD release can be observed during this process via a dialysis method (MW cut-off 106) using FITC-labelled GOD. Compared with the previous reports concerning the stimuli-responsive polymersomes,24,26,28,42 the high stability of GOD@PCPT-NR is presumably ascribed to low content of pH-sensitive polymer (PPEMA) (~ 15 wt%), the random copolymerization of PEMA and CPTMA monomers, as well as the strong hydrophobic and π-π staking interactions between CPT moieties. To evaluate the pH-responsive permeability of GOD@PCPT-NR, a mixture of glucose and 3,3',5,5'-tetramethylbenzidine (TMB), and horseradish peroxidase (HRP) were added into the solution of GOD@PCPT-NR. If glucose molecules can diffuse into polymersomes, GOD can catalyze the oxidation reaction of glucose, which produces H2O2. With H2O2 diffused out of the polymersomes, they can serve as oxidative donors and react with TMB under the catalysis of HRP. Thus, the permeability of nanoreactors can be evaluated by monitoring the characteristic absorbance of oxidized TMB.30,43 As shown in Figures 2D and S3, the characteristic absorbance at 370 nm was observed clearly at

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pH 6.8 and increased rapidly within 2 h. The solution displayed a blue color. In contrast, there was no observed oxidized TMB at pH 7.4. Collectively, the results demonstrate that GOD@PCPT-NR have distinctive membrane permeability with pH changed from 7.4 to 6.8, which selectively allows for free transportation of hydrophilic small molecules (e.g. O2. glucose, and H2O2) at pH 6.8. We quantitatively determined H2O2 production of GOD@PCPT-NR (100 mU/mL of GOD) at pH 6.8 with the glucose concentration of 1 mg/mL through measuring dissolved oxygen (D.O) in the presence of catalase. As shown in Figure 2E, GOD@PCPT-NR generated H2O2 quickly with the concentration increasing to approximately 0.5 mM within 1 h at pH 6.8. This H2O2 production rate was comparable to that of free GOD. In sharp contrast, no H2O2 production by GOD@PCPT-NR can be observed at pH 7.4. The results indicate tumor acidity (~ pH 6.8) can trigger H2O2 production of GOD@PCPT-NR. On the other hand, the produced H2O2 can cleave oxalate groups and release free active CPT drugs.44-46 We detected CPT release profiles of GOD@PCPT-NR with 100 mU/mL GOD in the presence of 1 mg/mL glucose at pH 7.4 and 6.8 (Figure 2F). Within 72 h, less than 10% CPT was released at pH 7.4 likely due to the slow hydrolysis of ester bond linkages between CPT and the polymer backbones. However, almost 80% CPT was released rapidly at pH 6.8, which is ascribed to H2O2-responsive CPT release. With CPT releasing, the polymersomes gradually degraded and lost the integrity (Figure S4). At 24 h, most polymersomes lost the vesicular structure and no vesicle structures can be detected at 48 h. By contrast, the morphology maintained constant at pH 7.4 (Figure S5). As a control without GOD, PCPT-V showed less than 10% CPT release at both pH 6.8 and 7.4 within 72 h (Figure S6). Collectively, GOD@PCPT-NR show specific tumor acidity-activated H2O2 production in

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the presence of glucose and H2O2-triggered release of CPT in a predictable and orchestrated fashion. High concentration of H2O2 (> 25 µM) can kill cancer cells efficiently and simultaneously sensitizes the cancer cells toward anticancer drugs.47,48 Therefore, GOD@PCPT-NR are expected to synergistically kill cancer cells via H2O2 production and CPT release. To evaluate the cytotoxicity of GOD@PCPT-NR, we performed 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and live/dead staining assay against A549 cells in the presence of glucose at pH 6.8. As shown in Figure 3A, PCPT-V showed low cytotoxicity with CPT-equivalent IC50 of 77.45 µM. At CPT-equivalent concentration of 8.3 µM, the percentage of PI-positive cells was less than 10% indicating relatively low-efficiency cell apoptosis (Figure 3B, C). The relatively low cytotoxicity of PCPT-V is likely ascribed to the slow hydrolysis and release of free CPT. Free GOD also induced modest cytotoxicity due to produced H2O2 (Figure S7). In contrast, GOD@PCPT-NR showed significantly higher cytotoxicity with CPT-equivalent IC50 of 1.11 µM at the GOD concentration of 100 mU/mL compared with free CPT of 3.54 µM. Live/dead assay displayed approximately 100% PI-positive cells at CPT-equivalent concentration of 8.3 µM indicating severe cell apoptosis under the treatment of GOD@PCPTNR at pH 6.8. In contrast, GOD@PCPT-NR showed similar cytotoxicity at pH 7.4 as compared with PCPT-V, which were both significantly lower than free CPT (Figure S8). The combination index (CI) of GOD and CPT in GOD@PCPT-NR was determined to be < 0.2 with the fraction affected (fa) lower than 0.8 via Chou-Talalay's isobolographic method (Figure S9),49 which indicates significant synergism of GOD and CPT after integration into GOD@PCPT-NR at pH 6.8.

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To test cellular DNA and cellular membrane damage, γ-H2AX and lipid peroxidation (malondialdehyde, MDA) assay were performed (Figure 3D, E, F). Significantly severe DNA damage and MDA level can be observed for GOD@PCPT-NR-treated cells as compared with PCPT-V, free CPT, and free GOD. Efficient H2O2 production of GOD@PCPT-NR at pH 6.8 in the presence of glucose increased cellular ROS level and high H2O2 level-induced CPT release facilitated the effective cellular internalization of CPT (Figures S10 and S11), which finally caused high-efficiency cancer cell killing. These results also confirmed the synergistic effect of produced H2O2 and released CPT for cancer cell killing. Reasonably, a high concentration of H2O2 could damage cellular membranes and cleave DNA efficiently.50 Released CPT could induce lesions to DNA and block DNA damage repair via inhibition of topoisomerase (DNA) I (TOP1).51 To further investigate the in vivo performance of nanoreactors, we first tested the stability of PCPT-V and GOD@PCPT-NR in serum-containing solution (30%). DLS analysis showed that both samples maintained a constant size in the serum-containing solution at pH 7.4 and 6.8 at least within 48 h (Figure S12). Proper nanoparticle size, PEG surface, and high stability enabled their long circulation in blood and accumulation in tumors. The terminal elimination half-life (T1/2z) of the polymersomes in bloodstream was determined to be 39.1 h and 42.3 h upon intravenous injection of GOD@PCPT-NR and PCPT-V, respectively, via noncompartmental analysis (Figures 4A and S13).52 A549 tumor models (~ 100 mm3) were established and used to investigate the biodistribution of the polymersomes in main organs and tumors. The results showed that the CPT-equivalent concentration in tumors were high up to 81 and 68 µg/g tumor tissue at 24 h post injection of PCPT-V and GOD@PCPT-NR, respectively, at the CPTequivalent dose of 35 mg/kg body weight (Figure S14), which were significantly higher than

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those in other organs. Therefore, the polymersomes can prolong the circulation in blood stream and accumulate in tumors efficiently. Next, H2O2 production in the blood, liver, or tumors was detected by 6'-opentafluorobenzene sulfonyl-2',7'-difluorofluorescein (BES-H2O2) probe. In the blood and liver, negligible H2O2 can be detected for GOD@PCPT-NR indicating the nanoreactors can keep inactive in the blood and liver at pH 7.4 within 48 h (Figures S15 and S16). However, at tumor sites, H2O2 level increased quickly and reached the peak at 24 h post administration of GOD@PCPT-NR. Reasonably, in tumor tissues, the mildly acidic microenvironment (~ pH 6.56.8) allows for the activation of the nanoreactors via protonation of PPEMA segments and increased membrane permeability.53 Simultaneously, the average glucose level inside tumors is several µM/g tumor tissue, which guarantees the catalysis reaction in the nanoreactors.54,55 In contrast, PCPT-V exhibited no H2O2 production in plasma, liver, and tumors due to no GOD loaded inside the polymersomes. High concentration of H2O2 in tumor tissues could cleave the oxalate linkages between CPT drugs and the polymer backbone, releasing free CPT.46 Thus, we further monitored in vivo free CPT release, which can indicate side effects and therapeutic efficacy of GOD@PCPT-NR. In the blood and liver, negligible released free CPT can be detected post intravenous injection of GOD@PCPT-NR (Figure 4A, B) or PCPT-V (Figures S13 and S17), indicating minimal side effects of off-targeted drugs. Intriguingly, in the tumor tissues, both free and conjugated CPT increased continuously within 24 h after intravenous injection of GOD@PCPT-NR (Figure 4C), indicating efficient tumor accumulation and CPT release via cleavage of oxalate linkage. Notably, free CPT can be measured at 2 h post-injection of GOD@PCPT-NR and increased rapidly, indicating the quick release of CPT. Selective CPT release of GOD@PCPT-NR in tumor

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tissues is consistent with the result of tumor-specific H2O2 production under the catalysis of GOD@PCPT-NR. Moreover, by intratumor injection of BES-H2O2 probe at 24 h after intravenous injection of GOD@PCPT-NR or PCPT-V, the tumor sections in GOD@PCPT-NR group showed 10-fold and 7-fold stronger fluorescence intensity of H2O2 (green) and released CPT (blue), respectively, compared with PCPT-V group (Figure 4D). High concentration of H2O2 can be ascribed to the activation of nanoreactors in tumor tissues. The strong CPT fluorescence intensity is presumably attributed to the release from GOD@PCPT-NR because the aggregated state of CPT in polymersomes can quench the fluorescence intensity while free CPT showed enhanced fluorescence (Figure S18).56 These results demonstrate that GOD@PCPT-NR can be selectively activated in tumor tissues to produce massive H2O2 for increasing the oxidative stress and triggering rapid release of CPT drugs. To evaluate the antitumor efficacy, 20 mice bearing A549 tumors were randomly divided into four groups (n = 5) including GOD@PCPT-NR, PCPT-V, CPT and PBS. The mice were treated via intravenous injections of the formulations at a CPT-equivalent dose of 35 mg/kg body weight. At day 52 after the first injection, CPT group showed modest tumor growth suppression with the tumor sizes increasing to 30 times (Figure 5A). The low antitumor efficiency is presumably ascribed to poor water-solubility of CPT, rapid clearance from the body, and low accumulation at tumors. PCPT-V group showed significantly better antitumor efficacy compared with free CPT owing to prolonged blood circulation and tumor accumulation.46,57,58 Intriguingly, GOD@PCPT-NR group displayed potent tumor inhibition capability. The tumors were efficiently ablated after 6 times of injections and almost disappeared in 52 days. The average tumor weight was only one tenth of the PCPT-V group (p < 0.005) at day 52 (Figure 5B). The tumor images at day 52 showed dramatically small tumor tissues in GOD@PCPT-NR group

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(Figure 5C). No body weight loss was observed in GOD@PCPT-NR and PCPT-V groups indicating unnoticeable systemic toxicity (Figures 5D). In contrast, free CPT group exhibited some body weight decrease due to the side effect of free CPT drugs in the body. The excellent therapeutic efficacy of GOD@PCPT-NR as compared with PCPT-V is reasonably attributed to tumor acidity-activated efficient H2O2 production and rapid CPT release triggered by elevated H2O2 level. To evaluate the cancer cell killing capability of GOD@PCPT-NR in tumor tissues, we observed the tumor cell apoptosis and tumor tissue damage results via hematoxylin and eosin (H&E) staining (Figure S19) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) (Figure 5E) at the end of treatment. GOD@PCPT-NR group induced severe coagulative necrosis in a large area. In comparison, CPT-V group and free CPT group showed significantly mild tissue necrosis. Further TUNEL staining images also showed severe cell apoptosis after treatment by GOD@PCPT-NR while significantly less apoptotic cells can be observed in PCPT-V group. Notably, H&E staining results of main organs at the end of treatment with various formulations revealed low toxicity toward main organs whereas CPT group displayed some toxicity to liver and kidney (Figure S20). Therefore, GOD@PCPT-NR achieved both high therapeutic efficacy and low systemic toxicity. In summary, we developed a novel GOD-loaded PCPTMA prodrug-based nanoreactors that can be specifically activated by tumor acidity to in situ produce H2O2 and further trigger the rapid release of CPT. The block copolymer, PEG-b-P(CPTMA-co-PEMA), was synthesized and self-assembled into polymersomes for GOD encapsulation in the aqueous solution. GOD@PCPT-NR showed specific tumor acidity-responsive activation via improved membrane permeability to produce massive H2O2, which further cleaved oxalate linkages for rapid active

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CPT release. The synergistic effect of increased oxidative stress and released CPT drugs was demonstrated to ablate A549 tumors efficiently. GOD@PCPT-NR resemble an in vivo medicine nanofactory to produce toxic therapeutics specifically functioning at tumor sites without further administration of prodrugs. The polymer prodrug nanoreactor strategy not only offers an effective approach for delivery and release of drugs, but also achieves controlled ROS production with GOD, which opens a new avenue toward the in vivo applications of therapeutic nanoreactors particularly for cancer therapy.

ASSOCIATED CONTENT The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/ Polymer synthesis, self-assembly and characterization; TEM images of GOD@PCPT-NR at pH 7.4 or 6.8 in the presence of glucose; CPT release profile of PCPT-V; cytotoxicity of GOD@PCPT-NR, PCPT-V, and free CPT at pH 7.4; cytotoxicity of free GOD at pH 7.4 or 6.8; Combination index (CI) of CPT and GOD for GOD@PCPT-NR; Confocal laser scanning microscope (CLSM) observation of cellular uptake and ROS level after treatment with GOD@PCPT-NR, PCPT-V, and free CPT; Stability in the serum-containing medium; biodistribution of polymersomes in main organs and tumors; H2O2 production in plasma, liver, and tumor tissue; CPT release of PCPT-V in plasma, liver, and tumor; H&E staining images of tumors and main organs. AUTHOR INFORMATION Corresponding Author

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*Email: [email protected]. *Email: [email protected]. Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS We gratefully acknowledge financial support from National Natural Scientific Foundation of China (NNSFC) Project (21674104), the Fundamental Research Funds for the Central Universities (WK3450000002), Dr. Li Dak-Sum Research Fund (Start-up Fund) of The University of Hong Kong, and Seed Fund for Basic Research of The University of Hong Kong. REFERENCES (1) Vriezema, D. M.; Aragones, M. C.; Elemans, J. A. A. W.; Cornelissen, J. J. L. M.; Rowan, A. E.; Nolte, R. J. M. Chem. Rev. 2005, 105, 1445-1489. (2) Renggli, K.; Baumann, P.; Langowska, K.; Onaca, O.; Bruns, N.; Meier, W. Adv. Funct. Mater. 2011, 21, 1241-1259. (3) Gaitzsch, J.; Huang, X.; Voit, B. Chem. Rev. 2016, 116, 1053-1093. (4) Che, H. L.; van Hest, J. C. M. J. Mater. Chem. B 2016, 4, 4632-4647. (5) Tanner, P.; Baumann, P.; Enea, R.; Onaca, O.; Palivan, C.; Meier, W. Acc. Chem. Res. 2011, 44, 1039-1049. (6) Rossbach, B. M.; Leopold, K.; Weberskirch, R. Angew. Chem. Int. Ed. 2006, 45, 1309-1312. (7) McHale, R.; Patterson, J. P.; Zetterlund, P. B.; O'Reilly, R. K. Nat. Chem. 2012, 4, 491-497. (8) Pang, X. C.; Zhao, L.; Han, W.; Xin, X. K.; Lin, Z. Q. Nat. Nanotechnol. 2013, 8, 426-431. (9) Tanner, P.; Balasubramanian, V.; Palivan, C. G. Nano Lett. 2013, 13, 2875-2883.

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Figure 1. (A) Schematic illustration for the preparation of GOD@PCPT-NR via self-assembly in aqueous solutions. (B) Oxidation/chemo-therapy of cancer via tumor acidity-responsive activation, in situ H2O2 production, and active CPT drug release. (C) Molecular mechanism for orchestrated oxidation/chemo-therapy of GOD@PCPT-NR at tumor sites including H2O2 production by the catalysis of GOD, H2O2-triggered cleavage of oxalate bonds, and free CPT release.

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Figure 2. TEM images of GOD@PCPT-NR at (A) pH 7.4 and (B) pH 6.8. (C) Acid-base titration curve of PEG113-b-P(CPTOEMA34-co-PEM20). (D) Absorbance (Abs, 370 nm) of the product (oxidized TMB) after the cascade reactions in GOD@PCPT-NR with the GOD concentration of 100 mU/mL in the presence of glucose (1 mg/mL), HRP (150 mU/mL), and TMB (100 µM) outside the polymersomes at pH 7.4 or pH 6.8. (E) H2O2 production of GOD@PCPT-NR or free GOD at the GOD concentration of 100 mU/mL in the presence of glucose (1 mg/mL) at pH 7.4 or pH 6.8. (F) CPT release from GOD@PCPT-NR (100 mU/mL GOD) in the presence of 1 mg/mL glucose at pH 7.4 or pH 6.8. Mean ± standard deviation (SD), n = 3.

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Figure 3. (A) CPT concentration-dependent cytotoxicity of GOD@PCPT-NR, PCPT-V, and free CPT in the presence of 1 mg/mL glucose at pH 6.8. Mean ± SD, n = 4. (B) Live/Dead assay and (C) PI-positive cells of A549 cells after incubation for 48 h with GOD@PCPT-NR, PCPT-V, free CPT, and free GOD with the CPT-equivalent concentration of 8.3 µM or GOD of 100

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mU/mL at pH 6.8. Mean ± SD, n = 4. (D) γ-H2AX assay and (E) γ-H2AX foci numbers of A549 cells after incubation for 24 h with GOD@PCPT-NR, PCPT-V, free CPT, and free GOD with the CPT-equivalent concentration of 8.3 µM or GOD of 100 mU/mL at pH 6.8. Mean ± SD, n = 25. (F) Lipid peroxidation profile using Lipid Peroxidation MDA assay kit for A549 cells after incubation with PBS, GOD@PCPT-NR, PCPT-V, free CPT, and free GOD with the CPTequivalent concentration of 8.3 µM or GOD of 100 mU/mL at pH 6.8 for 24 h. Mean ± SD, n = 4. **p < 0.01, ***p < 0.005 (t-test).

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Figure 4. Time-dependent polymer-bound CPT and released free CPT concentrations in (A) plasma, (B) liver, and (C) tumor post intravenous injection of GOD@PCPT-NR with the CPTequivalent dose of 35 mg/kg. Mean ± SD, n = 3. (D) Ex vivo fluorescence images and integrated optical density (IOD) of A549 tumor sections at 24 h post intravenous injection of GOD@PCPTNR and PCPT-V. H2O2 was detected by BES-H2O2 probe via intratumor injection for probing. Mean ± SD, n = 5. **p < 0.01 (t-test).

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Figure 5. (A) A549 tumor growth profiles of the mice treated with PBS, CPT, PCPT-V, and GOD@PCPT-NR at the CPT equivalent dose of 35 mg/kg. The arrows indicate the injection time. (B) Average tumor weight at the end of treatment. (C) Tumor images at the end of treatment. (D) Body weight change of the mice treated with PBS, CPT, PCPT-V, and GOD@PCPT-NR. (E) TUNEL staining of tumors from the mice after treatment with PBS, CPT, PCPT-V, and GOD@PCPT-NR. Mean ± SD, n = 5. ***p < 0.005 (t-test).

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