Glucose OxidaseâPolymer Nanogels for Synergistic Cancer-Starving

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Glucose Oxidase-Polymer Nanogels for Synergistic Cancer Starving and Oxidation Therapy Wenguo Zhao, Jin Hu, and Weiping Gao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b06814 • Publication Date (Web): 26 Jun 2017 Downloaded from http://pubs.acs.org on June 28, 2017

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ACS Applied Materials & Interfaces

Glucose Oxidase-Polymer Nanogels for Synergistic Cancer Starving and Oxidation Therapy Wenguo Zhao, Jin Hu, and Weiping Gao * Department of Biomedical Engineering, School of Medicine, Tsinghua University, Beijing, 100084 KEYWORDS: glucose oxidase; nanogel; protein-polymer conjugate; cancer therapy; synergistic therapy

ABSTRACT: Glucose oxidase (GOX) can convert glucose into gluconic acid and hydrogen peroxide (H2O2), which is potentially useful for synergistic cancer starving and oxidation therapy. Herein we demonstrate a glucose-responsive nanomedicine of GOX-polymer nanogels to regulate H2O2 production for synergistic melanoma starving and oxidation therapy. GOX-polymer nanogels showed glucose-responsive H2O2generating activity in vitro, improved stability, and considerably enhanced tumor retention as compared to native GOX. More importantly, they exhibited high anti-melanoma efficacy and no obvious systemic toxicity, whereas native GOX was ineffective and systemically toxic at the same dose. This work paves a way for establishing an endogenous and non-invasive cancer treatment paradigm that is based on intratumoral glucose-responsive H2O2-generating chemical reactions.

1. INTRODUCTION Melanoma is the most dangerous type of skin cancer that develops from melanocytes. The incidence rate in America has increased dramatically over the past 30 years and the number of newly diagnosed cases was estimated to be 76,380 in 20161,2. Melanoma accounts for only 1% of all skin cancer cases, but approximately 75% of skin cancer deaths. Patients with melanoma have poor prognosis and low 5-year survival rate3,4. The widely used therapies of melanoma include surgery, chemotherapy, radiation therapy and immunotherapy1,5. However, surgery cannot accurately identify and radically excise the nidus5; chemotherapy and radiation

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therapies have adverse side effects5; immunotherapy can only slightly activate the immune system and is always used as adjuvant therapy6. Therefore, new therapeutic strategies are urgently needed to achieve increased anti-melanoma efficacy and reduced systemic toxicity. Glucose is a key nutrient and plays an essential role in supplying energy for tumor growth7. Glucose oxidase (GOX) can oxidize glucose into gluconic acid and H2O2 (Figure. 1a)8, which potentially provides an alternative strategy for cancer starving therapy9-12. Additionally, H2O2 can induce cancer cell death at high concentrations through H2O2–dependent activation of apoptosis of tumor cells13-16. Therefore, the delivery of GOX to tumor is expected to consume intratumoral glucose for blocking the energy supply and simultaneously increase the H2O2 level for killing cancer cells, which leads to the emergence of a new paradigm of synergistic cancer starving and oxidation therapy. GOX suffers from poor stability, short in vivo half-life, immunogenicity and systemic toxicity. Particularly, it is problematic to regulate H2O2 production after exogenous administration of GOX in tumorbearing hosts. This is because GOX’s substrates, glucose and oxygen, are ubiquitous in the body, which results in the systemic generation of H2O2 and thus induces severe systemic side effects. To minimize the systemic toxicity of H2O2, injection of antioxidants was required after intratumoral administration of PEGylated GOX to produce H2O217, and alternatively GOX was coupled to polystyrene microspheres to prolong the tumor retention of the H2O2-generating system post intratumoral injection18. However, both strategies did not show significantly improved anti-tumor efficacy. More recently, GOX was attached covalently to hollow mesoporous organosilica nanoparticles containing L-arginine for synergistic cancer starving and nitric oxide therapy19. This strategy showed much better antitumor efficacy than the above two strategies, but could not address the other problems of GOX such as poor stability and immunogenicity. Therefore, new strategies that can solve the problems of GOX are needed for synergistic cancer starving and oxidation therapy. Nanogels have been used as nanocarriers for drug delivery20-22, but they have not been explored for protein therapeutics by in situ formation of protein-polymer conjugates. Herein we introduce a glucoseresponsive nanomedicine of GOX-polymer nanogels to modulate H2O2 production for melanoma starving and


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oxidation therapy via constraining GOX in tumor (Figure. 1b). In this technology, GOX reacted with an aldehyde-functionalized polymer to form GOX-polymer nanogels via the imine or Schiff base formation reaction between the amino groups of GOX and the aldehyde groups of the polymer. GOX is a dimeric protein with eleven free amino groups per monomer, which provides sufficient amino groups for the formation of multiple imine bonds between GOX and the aldehyde-functionalized polymer and thus makes it possible to form GOX-polymer nanogels. GOX-polymer nanogels showed not only retained activity but also improved stability relative to native GOX. More importantly, they exhibited 88-fold higher tumor retention than native GOX after intratumoral injection. As a result, GOX-polymer nanogels displayed not only high anti-melanoma efficacy but also no systemic toxicity, while native GOX at the same dose is not only ineffective

in

melanoma

therapy

but

also

severe


in

systemic

toxicity.


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Figure.1. Schematic diagram of synthesis of GOX-poly(FBMA-co-OEGMA) nanogels (NGs) for synergistic cancer starving and oxidation therapy. (a) Catalytic mechanism of GOX. (b) Synthetic route of NGs and working principle of NGs in tumor. 2. MATERIALS AND METHODS 2.1 Materials

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All the chemical reagents (J&K chemical or Sigma-Aldrich), biological reagents (New England Biolabs), cell culture reagents and media (HyClone) used in this research were purchased and used as received. PBS was self-confected, unless otherwise specified.

Cells were purchased from Chinese

Academy of Medical Sciences. Mice were from Vital River Laboratories. 2.2 Preparation of GOX-polymer nanogels 2.2.1 Synthesis of tert-butyl 3-(2-(2-(3-aminopropoxy)ethoxy)ethoxy) propylcarbamate

O

H2 N

NH2 + (Boc)2O 3

DCM H2 N rt., overnight

O

NHBoc 3

4,7,10-trioxa-1,13-tridecanediamine (5.5 mL) in DCM (40 mL) was allowed to stir at 0 °C, 10 mL of DCM containing 1.1 g of Di-tert-butyl dicarbonate ((Boc)2O) was added dropwise over 30 min. After 16 h reaction at room temperature, the crude product was purified by flash chromatography (DCM : MeOH = 5 : 1, Rf = 0.5), yielded colorless oil (C15H32N2O5, 1.48g, 90.7 %). 1H NMR (400 MHz, CDCl3): δ 5.11 (s, 1H), 3.54-3.65 (m, 12 H), 3.22 (m, 2H), 2.80 (t, 2H), 1.74 (m, 4H), 1.44 (s, 9 H). ESI-mass m/z: 321.1 ([M+H]+). 2.2.2 Synthesis of tert-butyl 3-(2-(2-(3-methacrylamidopropoxy)ethoxy) ethoxy) propylcarbamate NHBoc Cl

+ O

NH2 3

O

TEA, DCM rt., overnight

NHBoc H N

O 3

Ο

Tert-butyl 3-(2-(2-(3-aminopropoxy)ethoxy)ethoxy)propyl carbamate (0.8 g) and triethylamine (385 µL) were dissolved in DCM (20 mL), 5 mL of DCM containing 266 µL of methacryloyl chloride was added dropwise at 0 °C. After 16 h reaction at room temperature, the solution was washed with saturated NaHCO3 solution (20 mL) and saline (20 mL) in sequence. Separation through flash chromatography (DCM : MeOH = 20 : 1, Rf = 0.5) yielded colorless oil (C19H36N2O6, 0.69 g, 71.1 %). 1H


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NMR (400 MHz, CDCl3): δ 6.68 (bs, 1H), 5.72 (s, 1H), 5.31 (s, 1H), 4.97 (bs, 1H), 3.41-3.68 (m, 14H), 3.22 (m, 2H), 1.95 (s, 3H), 1.73-1.86 (m, 4H), 1.44 (s, 9H). ESI-mass m/z: 389.2 ([M+H]+), 411.2 ([M+Na]+). 2.2.3

Synthesis

of

N-(3-(2-(2-(3-aminopropoxy)ethoxy)ethoxy)propyl)

methacrylamide

hydrochloride NHBoc HCl, EA

H N

O

rt., overnight

3

NH3Cl H N

O 3

Ο

Ο

Tert-butyl 3-(2-(2-(3-methacrylamidopropoxy)ethoxy)ethoxy) propylcarbamate (0.69 g) was treated with 4 M HCl/EA (15 mL) at room temperature overnight. The colorless viscous oil product (C14H29ClN2O4, 0.78 g, 100 %) was used directly in the following reaction without further characterization. 2.2.4

Synthesis

of

4-formyl-N-(3-(2-(2-(3-methacrylamidopropoxy)ethoxy)ethoxy)propyl)

benzamide (FBMA)

CHO

NH3Cl H N

O

CHO EDC, HOBT, DIPEA, DCM

+ rt., overnight

3

Ο

COOH

O NH H N

O 3

Ο

4-Formyl benzoic acid (0.3 g), N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC, 375 mg), N-hydroxybenzotriazole (HOBT, 265 mg), and N,N-di-2-propyl N-ethyl amine (DIPEA, 0.7 mL) were dissolved successively in DCM (20 mL) with agitation, followed by 30 min reaction at room temperature. N-(3-(2-(2-(3-aminopropoxy)ethoxy)ethoxy)propyl) methacrylamide hydrochloride (0.78 g in 5 mL of DCM) was added dropwise over 15 min. After 16 h reaction at room temperature, the


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solution was washed with saturated NaHCO3 solution (20 mL) and saline (20 mL) in sequence. Separation via flash chromatography (petroleum ether : ethyl acetate = 1 : 1, Rf = 0.2) yielded colorless oil (C22H32N2O6, 0.67 g, 89.7 %). 1H NMR (400 MHz, CDCl3): δ 10.00 (s, 1H), 7.85-7.94 (d×d, 4H), 7.46 (s, 1H), 6.57 (s, 1H), 5.04 (s, 1H), 5.22 (s, 1H), 3.51-3.59 (m, 10H), 3.45 (m 4H), 3.32 (m, 2H), 1.87 (s, 3H), 1.84 (m, 2H), 1.70 (m, 2H). ESI-mass m/z: 443.6 ([M+Na]+), 459.3 ([M+K]+). 2.2.5 Synthesis of poly(FBMA-co-OEGMA) 4-Cyano-4-(phenylcarbonothioylthio)pentanoic acid (1.85 mg), azodiisobutylnitrile (AIBN, 0.217 mg), FBMA (92.5 mg), and oligo ethylene glycol monomethyl ether methacrylate (OEGMA, 220 µL) were dissolved in 2 mL of N,N-dimethylformamide (DMF) in a reaction tube. The solution was deoxygenated by 3 freeze-thaw cycles under nitrogen atmosphere, and then the reaction tube was sealed, moved into an oil-bath, and kept at 70 ℃ for 16 h. After reaction, the polymer was precipitated in cold (20 ℃) diethyl ether (50 mL) for 3 times and dried under nitrogen atmosphere, yielded viscose red oily poly (FBMA-co-OEGMA). 2.2.6 Synthesis of GOX-polymer nanogels (NGs) Glucose oxidase (5 mg) and poly(FBMA-co-OEGMA) (25 mg) were dissolved in PBS (6 mL) and incubated at 37 oC for 48 h to yield GOX-polymer nanogels. 2.2.7 Synthesis of FITC labeled GOX and NGs GOX (20.0 mg) was treated with fluorescein isothiocyanate (FITC, 1.1 mg, 20 eq.) in 50 mM borate buffer (pH 8.5, 2 mL) for 1 h at 25 ℃. Excess FITC was removed via a Hitrap desalting column (mobile phase: PBS, pH 7.4). The efficiency of GOX-FITC conjugation was quantified by the FITC/GOX molar ratio of the GOX-FITC conjugate. GOX concentration and FITC concentration were quantified by UV absorbance (280 nm) and fluorescence intensity (excitation at 492 nm, emission at 525 nm), respectively.


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FITC labeled NGs were synthesized similarly to the synthesis of NGs using the FITC labeled GOX instead of the native GOX. The ratio of GOX to polymer was 1:5 (w/w), and the final concentraion of the nanogels was 5 mg/mL. 2.3 Analytical instruments and methods The analytical instruments and methods used in this research are similar to those used in the previous work reported by our group23. 2.4 Enzyme activity assay The enzyme activities of GOX and NG were determined using enzyme activity assays. Solutions of horseradish peroxidase (HRP) (0.179 mg/mL), dianisidine (0.14 mg/mL), glucose (306.27 mg/mL), and GOX (9.3×10-4 mg/mL) in PBS were prepared separately. 2.4 mL of dianisidine solution, 0.1 mL of HRP solution, and 0.5 mL of glucose solution were combined and agitated. To each well of a plain bottom 96 well plate 0.2 mL of the above working solution was added. Upon the addition of 3.3 µL of GOX solution to each well, the absorbance of oxidated dianisidine at 460 nm was recorded for 15 minutes. Wells with 0.2 mL of working solution and 3.3µL of PBS served as background. The activity of GOX of NGs was proportional to the slope of the absorbance curve. 2.5 MTT assay MTT assay (Promega) was utilized to measure the cytotoxicity of poly(FBMA-co-OEGMA), GOX or NGs. Each well of a flat bottom 96-well tissue culture plate was filled with 200 µL of culture media containing 3000 cells. After 5 hours of incubation, cells were exposed to serial diluted solutions of poly(FBMA-co-OEGMA), GOX or NGs for 48 hours. The cell proliferation was measured by MTT assay according to the manufacturer’s directions. The cytotoxicity was quantiﬁed by the cells survival rate relative to untreated controls. The data fitting and calculation of half maximal inhibitory concentration


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(IC50) were operated using GraphPad Prism (Version 5.0), and the results were shown as mean ± standard deviation (SD). 2.6 In vivo anti-tumor efficacy The C8161 cells (5 × 106 cells per mouse) were implanted subcutaneously into the left dorsal skin of female BALB/c nude mice of 6 weeks old with body weights of 19 to 22 g (average body weight of 20.1 g). When the average tumor size increased to ca. 100 mm3 (length × width2 × 0.5) after 6 days, the mice were blindly divided into 7 groups (5 mice per group). Each mouse received intratumoral injection of 100 µL of PBS containing different concentrations (1, 10, and 100 mU) of GOX or NGs. The group treated with pure PBS served as a negative control group. The body weight and the tumor size of the mice were recorded until the tumor size was over 800 mm3. Note: Mice with a tumor volume over 800 mm3 were sacrificed. The anti-tumor efficacy study ended on day 16 because all the mice of the GOX group and 4 mice of the PBS group had tumor volumes larger than 800 mm3. 2.7 Pharmocokinetics Tumors were implanted as described above. Mice bearing C8161 tumors about 100 mm3 were blindly divided into 2 groups (3 mice per group). The FITC labelled GOX and NGs at a dosage of 100 mU per mouse were intratumorally injected to group 1 and group 2 mice, respectively. Blood samples were drawn from retro orbita at 1, 5, 15, 30 min, 1, 3, 6, 24, 48, 72, and 120 h, 20-30 µL of blood per mouse at each time point. Plasma was obtained by centrifugation (4000 g, 15 min) of the blood samples for the measurement of GOX concentration by detecting the fluorescence of FITC. To each well of an opaque plain bottom 96-well plate, 190 µL of PBS and 10 µL of the plasma were added sequentially. Plasma obtained from untreated mice served as background. GOX concentration in the plasma was quantified according to the FITC concentration. 2.8 Tumor retention


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Mice bearing C8161 tumors about 100 mm3 were blindly divided into 2 groups (3 mice per group). The FITC labelled GOX and NGs with the same fluorescence intensity at the GOX dose of 100 mU per mouse were intratumorally injected to group 1 and group 2 mice, respectively. The fluorescent images were acquired using Caliper IVIS Lumina II multispectral imaging system and treated with IVIS living imaging software version 4.52. The photons were calculated using the same software. 2.9 Hematological and histological evaluations At 21 days post GOX, NG, or PBS administration at the GOX dose of 100 mU per mouse, the mice (3 mice in each group) were executed, and blood, heart, liver, kidney and tumor were collected. Hemoglobin, red blood cells, white blood cells, and platelet were quantified by routine clinical laboratory techniques. Plasma obtained via centrifugation of the blood samples (4000 g, 15 min) was used to measure the urea, creatinine, alanine aminotransferase, creatine kinase MB isoenzyme, aspartate aminotransferase, and lactate dehydrogenase values by Automatic Biochemical Analyzer (HITACHI). Tissues such as kidney, heart, liver, and tumor were ﬁxed by 10% buffered neutral formalin solution for 24 hours, coated with wax, and sliced. After H&E staining, the slices were observed and images were captured via a Nikon micro imaging system.

3. RESULTS AND DISCUSSION To design an aldehyde-functionalized polymer for reacting with GOX to form GOX-polymer nanogels (Figure. 1b), we chose to copolymerize an aldehyde-functionalized monomer of 4-formyl-N-(3(2-(2-(3-methacrylamidopropoxy)ethoxy)ethoxy) propyl) benzamide (FBMA) with a PEG-based monomer OEGMA to yield poly(FBMA-co-OEGMA) by RAFT polymerization. FBMA was copolymerized with OEGMA to increase the aqueous solubility of polyFBMA, tune the number of aldehyde groups per chain, and control the size of nanogels. The molecular weight of poly(FBMA-co-


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OEGMA) was determined by gel permeation chromatography (GPC) to be 30 kDa with a mass dispersity of 1.15 (Figure. S1). The molar ratio of FBMA to OEGMA was determined by proton nucleic magnetic resonance (1H NMR) to be ca. 1:7, which means that there were ca. 8 aldehyde groups per chain (Figure. S2). Hence the polymer provided a sufficient number of aldehyde groups for reacting with the amino groups of GOX to form GOX-poly(FBMA-co-OEGMA) nanogels (NGs). The yield of NGs with respect to the used quantity of GOX was 92.5 ± 5.5 %, indicating the high efficiency of the reaction. The diameter of NGs was determined to be 802 nm with a dispersity of 0.24 by dynamic light scattering (DLS), which was 102-fold larger than that (7.9 nm) of native GOX (Figure. 2a). Transmission electron microscopy (TEM) further revealed that the morphology of NGs in dry state was approximately spherical with an average diameter of 112 nm (Figure. 2b). These DLS and TEM data indicated that NGs were highly swollen in aqueous solution. Glucose oxidase (GOX) is an enzyme that can catalyze D-glucose to be oxidated to D-gluconic acid. In this procedure, oxygen is consumed, in the meantime hydrogen peroxide is released8. To determine the H2O2-generating activity of NGs, we used the o-dianisidine-peroxidase spectrophotometric method8. Approximately 54% of the activity of native GOX was retained after GOX was conjugated to the polymer to form NGs (Figure. 2c). Native GOX is not stable due to the decomposition of flavin adenine dinucleotide (FAD), the catalytic center of GOX24-27. It quickly lost its activity and only remained 17% of its initial activity after stored at the body temperature of 37 °C for 10 days, whereas under the same storage condition, NGs just slowly lost their activity and remained 55% of their initial activity (Figure. 2d). The data suggested that conjugating GOX to polymers can depress the decomposition of FAD from GOX. These results indicated that NGs not only retained their enzymatic activity but also enhanced the stability of GOX even at the body temperature of 37 °C. Melanoma is the most dangerous type of skin cancer and amenable to local therapy, so we chose to treat melanoma with NGs. At first, we studied the cytotoxicity of NGs against C8161 melanoma cells (Figure. 2e). NGs were highly toxic to the melanoma cells with a half maximal inhibitory concentration


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(IC50) of 24.4 ng/mL, which was almost comparable to native GOX with an IC50 of 17.3 ng/mL. Notably, poly(FBMA-co-OEGMA) was non-toxic to normal cells and tumor cells (Figure. S3). These data indicated that the chemical reaction between GOX and poly(FBMA-co-OEGMA) did not significantly reduce the cytotoxicity of GOX.

Figure.2. Synthesis and characterization of GOX-poly(FBMA-co-OEGMA) nanogels (NGs). (a) DLS curves of NGs, poly(FBMA-co-OEGMA) and GOX. (b) TEM images of NGs. (c) Relative activity of native GOX and NGs at the same GOX concentration of 9.31× 10-4 mg/mL. (d) Change of enzymatic activity of native GOX and NGs stored at 37 °C. (e) Antiproliferative activity of native GOX and NGs. Data are shown as mean ± SD (n = 3). Next, we investigated the in vivo antitumor activity of NGs in a C8161 melanoma mouse model (Figure. 3a-c). Mice with an average tumor size of ca. 100 mm3 were intratumorally injected with NGs at different GOX equivalent doses of 1, 10, and 100 mU per mouse. NGs showed a dose-dependent antitumor activity, as indicated by the fact that increasing the dose enhanced their inhibition in tumor growth. In contrast, GOX was ineffective in inhibiting tumor growth at these doses as compared to PBS,


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which was also observed in previously reported work28,29. Particularly, at 16 d post administration, the average tumor size in the NGs (100 mU) treatment group was 250 mm3, which was 3.5-fold smaller than that (882 mm3) in the GOX (100 mU) treatment group. The significant improvement in inhibiting tumor growth was correlated with the elongation in median survival time (Figure. 3d-f). The median survival times of GOX treated mice were no longer than 15 d, similar to the median survival time of PBS treated mice (14.5 d). In contrast, NGs treated mice exhibited longer median survival times than GOX treated mice at the doses. Particularly, the median survival time (28 d) of 100 mU NGs treated mice was 1.9–fold longer than that (15 d) of 100 mU GOX treated mice. No loss of body weight was observed in all groups (Figure. S4). Collectively, these results indicated that NGs were much more efficient in melanoma therapy than native GOX particularly at the dose of 100 mU per mouse.

Figure.3. Anti-tumor efficacy of NGs in a melanoma mouse model. (a-c) Tumor growth post PBS, GOX, or NGs administration at different GOX doses of 1 mU (a), 10 mU (b), and 100 mU (c). Data are shown as mean ± SD (n = 5). p < 0.05 (GOX group and NGs group versus PBS group). (d-f) Cumulative survival rate of mice treated with 1 mU (d), 10 mU (e), and 100 mU (f) of GOX or NGs.


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To elucidate the mechanism of the improved in vivo antitumor activity of NGs over GOX, we studied the pharmacokinetics and tumor retention of NGs and GOX. To trace GOX in vivo, we labeled it with fluorescein isothiocyanate (FITC) at a labeling molar ratio of FITC/GOX = 1.12. After intratumoral injection of FITC labeled NGs and GOX, blood samples were collected at given times and centrifuged to obtain plasma samples for quantification of GOX concentration in the plasma (Figure. 4a). Upon intratumoral injection of free GOX, a huge amount of GOX was detected in the plasma and then the GOX concentration in the plasma decreased with time. This result suggested that GOX quickly leaked from the tumor region into circulating system. In contrast, the GOX concentration (356 ng/mL) of NGs in the plasma was much lower than that (603 ng/mL) of free GOX at 1 min post intratumoral injection. The plasma GOX concentration of NGs slightly increased with time and then decreased with time. At 3 d post intratumoral injection, the plasma GOX concentration of NGs (150 ng/mL) was 5-fold higher than that (30 ng/mL) of free GOX. These results implied that NGs leaked from the tumor region into circulating system much more slowly than free GOX. The above results were further confirmed by in situ fluorescence imaging of NGs and free GOX in tumors (Figure. 4b). NGs showed a much slower decrease in fluorescent intensity than free GOX, and the fluorescence of NGs (2.95 × 109 photons) was 88-fold higher than that of GOX (3.34 × 107 photons) at 3 h post intratumoral injection (Figure. 4c). These data indicated that NGs possessed much higher tumor retention than GOX. Taken together, the results of pharmacokinetics and tumor retention suggested that GOX could be confined to the tumor region by NGs mainly due to the enlarged size of NGs over native GOX.


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Figure.4. Pharmacokinetics and tumor retention of NGs after intratumoral injection. (a) Change of concentration of GOX in plasma post intratumoral administration of 100 mU of GOX or NGs. Data are shown as mean ± SD (n = 3). p < 0.05 (NGs group versus GOX group). (b) Fluorescent images at different time points post intratumoral administration of 100 mU of FITC labelled GOX (left mouse) or NGs (right mouse) (c) Change of fluorescent radiant intensity post intratumoral administration of 100 mU of FITC labelled GOX or NGs. Data are shown as mean ± SD (n = 3). p < 0.05 (NGs group versus GOX group).

A major problem of GOX is that it can induce serious side effects in vivo17. To evaluate the side effects of NGs, we tested the hematological parameters of the treated mice (Figure. 5a-e). White blood cells (WBC) amounts, urea (CR), creatinine (CR), creatine kinase MB isoenzyme (CK-MB), lactate


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dehydrogenase (LDH) levels of the mice treated with GOX were much higher than those of the mice treated with PBS, which indicated that GOX was harmful to kidney and heart. In contrast, all blood cells amounts and blood chemistry parameter levels of the NGs treated group were comparable to those of the PBS treated group, which indicated that NGs did not cause damage to important organs including kidney, liver, and heart. We further investigated the histological morphology of important organs of tumor, kidney, liver and heart of the treated mice. The images of H&E stained slices of tumor, kidney, liver and heart of the GOX treated mice displayed the damaged rental tubules and broken myocardia (Figure. 5f), indicating that GOX was toxic to kidney and heart but not to tumor. In contrast, NGs did not cause apparent damage to kidney, heart and liver but induced apoptosis of tumor cells. Collectively, these in vivo results demonstrated that NGs could not only substantially enhance the antitumor efficacy of GOX but also considerably reduce its side effects.


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Figure.5. Hematological and histological evaluations after treatment. (a-e) Blood biochemistry analysis post intratumoral administration of 100 mU of GOX or NGs. (a) WBC and RBC, (b) HGB and PLT, (c) UR CR, (d) ALT and AST, (e) LDH and CK-MB. Data are shown as mean ± SD (n = 3). (f) H&E staining of kidney, liver, heart and tumor post intratumoral administration of 100 mU of GOX or NGs. The arrows denote the damage sites.


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4. CONCLUSION In this proof-of-concept study, we have demonstrated a glucose-responsive nanomedicine of GOX-polymer nanogels to regulate H2O2 production for synergistic melanoma starving and oxidation therapy. On the one hand, GOX-polymer nanogels not only retain the bioactivity of GOX but also improve the thermal stability of GOX. On the other hand, GOX-polymer nanogels not only significantly enhance the antitumor activity of GOX but also effectively reduce the side effects of GOX through the mechanism of confining GOX to the tumor region. To our knowledge, this work is the first study to present a successful example for synergistic cancer starving and oxidation therapy. Moreover, this work paves a way for establishing an endogenous and non-invasive treatment paradigm that is based on intratumoral glucose-responsive H2O2-generating chemical reactions. ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS

Publications

website

at

DOI:

10.1021/acsami.XXX.

GPC and 1H NMR graphics of poly (FBMA-co-OEGMA), data of Cytotoxicity of poly(FBMAco-OEGMA) to tumor cells (C8161 cells) and normal cells, and carves of changes of body weight of the mice post administration (Figure. S1-S4).

Corresponding Author *E-mail: [email protected]. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes


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The authors declare no competing financial interest. ACKNOWLEDGMENT This study was financially supported by Grants from National Natural Science Foundation of China (Grant No. 21534006 to W. G.). ABBREVIATIONS GOX, glucose oxidase; NGs, glucose oxidase-polymer nanogels; DCM, dichloromethane; TEA, triethylamine. REFERENCES 1. American Cancer Society. Cancer Facts & Figures 2015. Atlanta: American Cancer Society, 2015. 2. McCourt, C.; Dolan, O.; Gormley, G., Malignant Melanoma: A Pictorial Review. Ulster Med. J. 2014, 83(2): 103-110. 3. Balch, C. M.; Gershenwald, J. E.; Soong, S. J.; Thompson, J. F.; Atkins, M. B.; Byrd, D. R.; Buzaid, A. C.; Cochran, A. J.; Coit, D. G.; Ding, S. L.; Eggemont, A. M.; Flaherty, K. T.; Gimotty, P. A.; Kirkwood, J. M.; McMasters, K. M.; Mihm, M. C.; Morton, D. L.; Ross, M. I.; Sober, A. J.; Sondak, V. K., Final Version of 2009 AJCC Melanoma Staging and Classification. J. Clinn. Oncol. 2009, 27(36): 6199-6206. 4. Arrangoiz, R.; Dorantes, J.; Cordera, F.; Juarez, M. M.; Paquentin, E. M.; de León, E. L., Melanoma Review: Epidemiology, Risk Factors, Diagnosis and Staging. J. Cancer Treat. Re. 2016, 4(1): 1-15. 5. Maverakis, E.; Cornelius, L. A.; Bowen, G. M.; Phan, T.; Patel, F. B.; Fitzmaurice, S.; He, Y.; Burrall, B.; Duong, C.; Kloxin, A. M.; Sultani, H.; Wilken, R.; Martinez, S. R.; Patel, F,. Metastatic Melanoma–A Review of Current and Future Treatment Options. Acta Derm-venereol 2015, 95(5): 516-527.


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Glucose OxidaseâPolymer Nanogels for Synergistic Cancer-Starving

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