Enhanced Phototherapy by Nanoparticle-Enzyme via Generation and

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Enhanced Phototherapy by Nanoparticle-Enzyme via Generation and Photolysis of Hydrogen Peroxide Kaiwen Chang, Zhihe Liu, Xiaofeng Fang, Haobin Chen, Xiaoju Men, Ye Yuan, Kai Sun, Xuanjun Zhang, Zhen Yuan, and Changfeng Wu Nano Lett., Just Accepted Manuscript • Publication Date (Web): 14 Jun 2017 Downloaded from http://pubs.acs.org on June 15, 2017

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Enhanced Phototherapy by Nanoparticle-Enzyme via Generation and Photolysis of Hydrogen Peroxide Kaiwen Chang,1,2 Zhihe Liu,3 Xiaofeng Fang,3 Haobin Chen,3 Xiaoju Men,3 Ye Yuan,3 Kai Sun,3 Xuanjun Zhang,2 Zhen Yuan,2 and Changfeng Wu 1,* 1

Department of Biomedical Engineering, Southern University of Science and Technology, Shenzhen, Guangdong 518055, China 2

3

Faculty of Health Sciences, University of Macau, Taipa, Macau SAR, China

State Key Laboratory on Integrated Optoelectronics, College of Electronic Science and Engineering, Jilin University, Changchun, Jilin 130012, China

* Corresponding author: [email protected]

KEYWORDS Polymer dot, Glucose oxidase, Hydrogen peroxide, Hydroxyl radical, Enzyme-enhanced phototherapy

ACS Paragon Plus Environment

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ABSTRACT

Light has been widely used for cancer therapeutics such as photodynamic therapy (PDT) and photothermal therapy (PTT). This paper describes a strategy called enzyme-enhanced phototherapy (EEPT) for cancer treatment. We constructed a nanoparticle platform by covalent conjugation of glucose oxidase (GOx) to small polymer dots (Pdots), which could be persistently immobilized into tumor.

While the malignant tumors have high glucose uptake, the GOx

efficiently catalyzes the glucose oxidation with simultaneous generation of H2O2. Under light irradiation, the in-situ generated H2O2 was photolyzed to produce hydroxyl radical (•OH), the most reactive oxygen species (ROS), for killing cancer cells. In vitro assays indicated that the cancer cells were destroyed by using a nanoparticle concentration at 0.2 µg/mL and a light dose of ~120 J/cm2, indicating the significantly enhanced efficiency of the EEPT method when compared to typical PDT that requires a photosensitizer of >10 µg/mL for effective cell killing under the same light dose. Furthermore, remarkable inhibition of tumor growth was observed in xenograft-bearing mice, indicating the promise of the EEPT approach for cancer therapeutics.


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Hydrogen peroxide (H2O2) is a key signaling molecule in living organisms that can activate diverse pathways to stimulate cell growth, proliferation, and differentiation.1-2

However,

aberrant generation of H2O2 are closely correlated with many diseases including cancer,3-4 neurodegenerative disorders,5 and cardiovascular diseases.6 In this context, H2O2 has attracted considerable interests from researchers with respect to the chemical mechanisms underlying the development and progression of disease.7 In particular, accumulating evidence reveals that H2O2 plays a significant role in cancer initiation and progression.8-9

For example, cancer cells

commonly have high levels of H2O2, and the increase in cellular H2O2 has been connected with several key alternations of cancer, including DNA damage, cell proliferation, apoptosis resistance, and metastasis.10-15 On the other hand, there is compelling evidence that excessive amount of H2O2 can start toxic and lethal chain reactions, which may be an efficient way of killing cancer cells.8,11 High dose of ascorbate has been shown to generate H2O2-dependent cytotoxicity toward a variety of cancer cells without adversely affecting normal cells.16-17 Further in vivo studies confirmed that the high dose parenteral ascorbate could exert local prooxidant effects, selectively increase the H2O2 formation within interstitial fluids of tumors, and thus significantly reduce the tumor growth in xenograft-bearing mice.18-19 These findings have significant implications for development of novel cancer therapeutic strategies by mediating the in vivo generation of hydrogen peroxide. It is well known that H2O2 is a typical product in a number of enzymatic reactions. Previous studies have demonstrated that the overexpression of superoxide dismutases in tumor cells can reduce tumor cell growth, metastasis, and other malignant features of cancer cells, because these enzymes catalyze the conversion of superoxide to H2O2.20-21 More recently, Qu and coauthors demonstrated that carbon nitride nanomaterials could catalyze the reduction of molecular oxygen


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to the superoxide anion or hydrogen peroxide to the hydroxyl radical for enhanced cancer therapy.22-23 Huang and coauthors reported a strategy involving glucose oxidase to generate H2O2 and NO for synergistic cancer therapy.24 Malignant tumors have much higher glucose uptake as compared to normal tissues, thus enhancing the H2O2 generation in presence of glucose oxidase (GOx). As a nonpolar molecule, H2O2 is able to diffuse across membranes and enter into different cellular organelles. The efficient generation of H2O2 could cause oxidative damage to tumor cells, yielding apparent antitumor effect. Here, we demonstrate a strategy called enzyme-enhanced phototherapy (EEPT) by in vivo generation and photolysis of H2O2 for cancer treatment. We show that GOx conjugated polymer dots (Pdot-GOx) could be persistently immobilized into tumor for in-situ generation of H2O2. Upon light irradiation, each H2O2 was split into two hydroxyl radicals, the most reactive ROS for killing cancer cells. Hydroxyl radical (•OH) has the strong ability to damage biomacromolecules including nucleic acids, proteins, lipids, and carbohydrates.25-27 In vitro and in vivo studies indicated that the Pdot-GOx at low concentrations effectively killed cancer cells and inhibited the tumor growth in xenograft-bearing mice. Besides the well-known PDT and PTT approaches, this study provides a new phototherapy modality for treatment of malignant tumors. Polymer dots (Pdots) are small and bright nanoparticles that have been demonstrated for a wide variety of biological imaging and sensing applications. 28-42 Recently, an oxygen-sensitive Pdots conjugated with GOx enzyme was used as transducer for detection of blood glucose level.38 We use fluorescent Pdots for GOx immobilization and simultaneous imaging of the enzyme retention in tumor. First, deep-red emitting Pdots were prepared by using precipitation method described previously.43 A light-harvesting polymer poly[(9,9-dioctylfluorenyl-2,7-diyl)alt-co-(1,4-benzo-{2,1’,3}-thiadiazole)] (PFBT) as the donor and a deep-red emitting polymer


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poly(9,9-dioctylfluorene)-co-(4,7-di-2-thienyl-2,1,3-benzothiadiazole)

(PF-5DTBT)

as

the

acceptor were used to prepare the Pdots with efficient deep-red emission (Scheme 1). A small amount of functional polymer poly(styrene-co-maleic anhydride) (PSMA) was condensed with the semiconducting polymer to form carboxyl Pdots, which were further covalently linked to the GOx

enzyme

through

the

bioconjugation

reaction

catalyzed

by

1-ethyl-3-(3-

dimethylaminopropyl) carbodiimide (EDC). The Pdot-GOx nanoparticles showed a spherical morphology with narrow size distribution, as observed from transmission electron microscopy (TEM) (Figure S1a). The average sizes of Pdots and Pdot-GOx were determined to ~18 nm and ~22 nm, respectively, as measured by dynamic light scattering (DLS, Figure S1b and S1c). The size increase of Pdot-GOx is due to the GOx conjugation on the Pdot surface. Meanwhile, the zeta potential of Pdot-GOx was apparently decreased when compared to that of the carboxyl Pdots (Figure S1d). These results clearly indicate the successful formation of Pdot-GOx bioconjugates. On the basis of the steric hindrance, we estimated that each Pdot (~18 nm diameter) had 5-10 enzyme molecules, with an average weight ratio of ~50 wt% relative to the Pdot. The Pdots before and after GOx conjugation exhibited similar absorption and emission bands (Figure S1e), indicating that the GOx enzyme does not affect the optical properties of the Pdots. GOx can selectively catalyze the conversion of D-glucose into gluconic acid, with simultaneous generation of H2O2. Each H2O2 can be split to produce two hydroxyl radicals under light irradiation. Previous report has shown that the use of blue light (450-490 nm) in combination with H2O2 yielded synergic antibacterial effect.44 Therefore, we choose a blue light source (blue LED array, center wavelength of 460 nm with a full width at half maximum of 20 nm) for in vitro and in vivo experiments. We use an assay involving terephthalic acid (TA) to


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detect the •OH radicals generated by the photolysis of H2O2.45 The oxidation of TA by •OH produces 2-hydroxyterephthalic acid (TAOH), as shown in Figure 1a. Thus, the fluorescence intensity of TAOH is dependent the •OH concentration generated from H2O2 in the aqueous solution. For the •OH detection, the fluorescence spectra were monitored at ten-minute intervals under continuous light irradiation for 60 minutes (460 nm, 100 mW/cm2).

The emission

intensity of TAOH at 426 nm was plotted as a function of the irradiation time. As seen from Figure 1b, the TA molecule (6 mM) mixed with the Pdot-GOx nanoparticles (10 µg/mL) and glucose (5 mM) in the aqueous solutions show remarkably increased emission from TAOH upon the light irradiation, indicating that •OH was efficiently generated in the system. However, the fluorescence spectra of the mixture solution consisting of pure TA and Pdot-GOx nanoparticles showed negligible changes in absence of the light irradiation, indicating that the light irradiation is necessary for •OH generation (Figure 1c). Meanwhile, the fluorescence spectra of the pure TA and glucose solutions showed negligible changes with or without light irradiation (Figure 1d and e). Furthermore, the fluorescence spectra of the pure TA showed negligible changes with light irradiation (Figure S2a), indicating that the TA molecules were not converted to TAOH by light irradiation alone. These results confirmed that the Pdot-GOx nanoparticles in presence of glucose under light irradiation resulted in the generation of •OH radicals. We directly compared the generation rate of •OH by the Pdot-GOx nanoparticles with the aqueous H2O2 solution (0.4 mM) under the same irradiation condition. The emission intensity of the solution of TA and H2O2 showed negligible change in dark condition (Figure S2b). However, the emission intensity of TA and H2O2 upon light irradiation were increased remarkably (Figure S2c), indicating efficient •OH generation from the H2O2 solution. Figure 1f showed the fluorescence intensity of TAOH as a function of irradiation time for the Pdot-GOx


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nanoparticles and other control experiments.

The •OH generation rate by the Pdot-GOx

nanoparticles (10 µg/mL) in presence of glucose (5 mM) was comparable to that of the aqueous H2O2 solution (0.4 mM) under the same light irradiation (Figure S2d), revealing the high efficiency of •OH generation by the Pdot-GOx system. To further confirm that •OH radicals are indeed involved in the process, we examined the effect of dimethyl sulfoxide (DMSO) as a •OH scavenger on the fluorescence change of TAOH. A small amount of DMSO was added to the aqueous solution of H2O2 and TA, followed by a time-dependent light exposure. As shown by the fluorescence spectra in Figure S3, the presence of DMSO (~0.6 M) significantly inhibited the fluorescence of TAOH when compared to the assays in absence of DMSO. Similarly, with the addition of DMSO (~0.6 M) to the aqueous solution of Pdot-GOx, glucose, and TA, the emission intensity of TAOH were apparently decreased as compared with the results in absence of DMSO (Figure S4). These results further confirmed the oxidation of TA to TAOH by hydroxyl radicals and the light-induced generation of hydroxyl radicals by Pdot-GOx nanoparticles. The cytotoxicity of the Pdot-GOx nanoparticles was evaluated by monitoring the metabolic viability of MCF-7 cells after incubation with Pdot-GOx at different concentrations. MCF-7 cells were incubated with Pdot-GOx in absence of light irradiation as the control group. Figure 2a showed the cell viability after incubation with Pdot-GOx in the dark without light illumination. The cell viability showed negligible decrease when incubated with Pdot-GOx nanoparticles (< 0.1 µg/mL), while apparent decrease was observed with increasing the PdotGOx concentrations (>0.1 µg/mL). However, the cell viability remains unchanged for the Pdots without GOx conjugation, indicating that the cytotoxicity was attributable to the GOx that generated excess amount of H2O2.

In another experimental group, the MCF-7 cells were


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incubated with Pdot-GOx and then exposed to light illumination for 20 min (460 nm, 100 mW/cm2). As shown in Figure 2b, the cellular viabilities were decreased significantly as compared to the viability values in absence of light irradiation. It is worth noting that the PdotGOx nanoparticles exhibited a much higher efficiency for cell killing than the free GOx enzyme at the same concentration. This is likely due to the combined factors such as the Pdot-GOx internalization and subcellular distributions. As the diffusion of •OH radicals is very limited because of the very high reactivity, the effective cell uptake of Pdot-GOx and H2O2 diffusion are essential for intracellular generation of •OH radicals, which ensure the Pdot-GOx to execute their therapeutic functions. MCF-7 cancer cells were significantly destroyed by using the Pdot-GOx nanoparticles at 0.2 µg/mL concentration and a light dose of ~120 J/cm2, indicating the high efficiency of the EEPT method as compared to traditional PDT. Because the photosensitizer dose and light irradiation conditions in PDT are variable in different reports,47-50 we performed a side-by-side treatment comparison by using Pdot-GOx and conventional photosensitizers.

Table S1 presents the

concentrations of several common photosensitizers and light doses for effective PDT in recent reports. We chose Chlorin e6 (Ce6) and Rose Bengal (RB) to investigate their photodynamic effect by MTT under the same light dose as used for Pdot-GOx. Under the light dose of 120 J/cm2, effective cell damage was observed for the Ce6 at 12.5 µg/mL and RB at 50 µg/mL (Figure S5). It is worth noting that the Pdot-GOx concentration (~0.2 µg/mL) in EEPT method is much lower than those of the common photosensitizers (>10 µg/mL), indicating the significantly enhanced therapeutic effect by using Pdot-GOx when comparing with the traditional photosensitizers in clinical PDT.


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To elucidate the EEPT mechanism, we used flow cytometry and a fluorescent probe (dichlorofluorescein diacetate, DCFH-DA) to detect intracellular H2O2 levels. As seen from the data (Figure S6), the cells exposed to Pdots alone showed very minor increases in H2O2 level as compared to the control group and no significant difference was observed for the Pdots at different concentrations. The cells treated with free GOx showed minor increases in H2O2 level with increasing the GOx concentration. In contrast, the cells treated with Pdot-GOx showed higher H2O2 level as compared to the Pdots and free GOx, indicating that the Pdot-GOx enhanced intracellular generation of H2O2. Cellular staining was further performed to investigate the cytotoxic effect induced by the Pdot-GOx nanoparticles and light irradiation. Acridine orange/ethidium bromide (AO/EB) staining allows for the discrimination among live, apoptotic and necrotic cells.

Live cells (green), early apoptotic cells (yellow-orange), and late

apoptotic/necrotic cells (red) can be simultaneously identified by the confocal laser scanning microscopy. The AO/EB staining results revealed apparent cell death after treatment with PdotGOx nanoparticles and light irradiation as compared to the control groups in absence of either Pdot-GOx nanoparticles or light irradiation (Figure 2c). We also evaluated the PDT effect of the two photosensitizers Ce6 and RB by AO&EB cell staining (Figure S7 and S8). As clearly seen, the number of dead cells increased gradually with increasing the concentrations of the photosensitizers.

However, their concentrations required for effective cell killing are

significantly higher than that of the Pdot-GOx nanoparticles. Thus, the cell staining results together with the cell viability assays indicated the enhanced phototherapeutic effect of the EEPT method by Pdot-GOx through the in situ enzymatic generation and photolysis of H2O2. Fluorescence imaging was performed to monitor the tumor retention effect of the Pdot-GOx nanoparticles in xenograft-bearing mice. The fluorescent images were recorded at different time


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intervals after intratumoral injection of Pdot-GOx (150 µL, 100 µg/mL). The fluorescence signal remained relatively constant from day 1 to day 28 post injection (Figure 3a), indicating the long-term retention effect of the Pdot-GOx in tumor.

Meanwhile, the distribution

examinations on different organs indicated the Pdot-GOx nanoparticles were predominantly trapped in the tumor and did not enter into the clearance organs such as the liver, kidney, and spleen (Figure S9).

This observation was consistent with our recent results using Pdot

transducer for subcutaneous glucose measurements.38 We recognize the great promise of local therapy as described in a recent landmark paper,51 because conventional cancer therapies involve the systemic delivery of anticancer agents that neither discriminate between cancer and normal cells nor eliminate the risk of cancer recurrence. In this context, the long-term retention effect offered by Pdots is particularly useful for in vivo enzyme immobilization in tumor and subsequent phototherapy treatment. The in vivo EEPT efficacy of Pdot-GOx was evaluated by measuring tumor growth rate and body weights of the mice. Figure 3b shows representative pictures of the tumor-bearing mice after different treatments. As seen from these pictures, the mice receiving Pdot-GOx and light treatment showed the most effective tumor inhibition as compared to all other control groups. The tumor sizes in the mice receiving the EEPT treatment were decreased markedly, as directly seen from Figure 3c. These observations further confirmed the therapeutic efficacy of PdotGOx under the light irradiation. Particularly, the mice receiving Pdot-GOx and light irradiation exhibited much smaller tumor sizes than those receiving free GOx and light treatment. This comparison unambiguously revealed the important role of the enzyme immobilization by Pdots, as free enzyme molecules may easily diffuse and migrate away from the tumor. The in vivo therapeutic effect by using the Pdot-GOx is also significantly enhanced when comparing the


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treatments using free enzyme and bare Pdots. The Pdot-GOx clearly inhibited the tumor growth in xenograft-bearing mice while both the results using free enzyme and bare Pdots exhibited negligible therapeutic effect. This comparison highlighted the significant role of nanoparticles in the EEPT treatment. No significant body weight change was observed with the prolonged time (Figure 3d), suggesting that the EEPT treatment did not induce adverse effects on the mice as compared to the control groups. Figure 3e showed the tumor growth curves in the xenograft-bearing mice after different treatments.

Again, these curves highlighted the remarkably enhanced EEPT

efficacy by photolysis of H2O2 as compared to the mice receiving Pdot-GOx in absence of light irradiation. Because the toxicity of •OH is much higher than that of H2O2, the EEPT method induced more severe oxidative damage to the tumor by generating the extremely reactive •OH through photolysis. As the Pdot-GOx nanoparticles were primarily trapped in the tumors, the potential toxicity of Pdot-GOx to the major organs was not expected. Indeed, hematoxylin and eosin (H&E) stained tissue sections did not show noticeable damage or inflammatory lesions in all the major organs of the mice 30 days after EEPT treatment (Figure 3f). In summary, we developed the enzyme-enhanced phototherapy for cancer treatment by using the Pdot-GOx nanoparticles. The strategy used the GOx enzyme that efficiently catalyzed the oxidation of glucose to gluconic acid with simultaneous production of H2O2. Meanwhile, the Pdot-GOx nanoparticles were persistently immobilized into tumor, where the high glucose H2O2 could be photolyzed to generate two •OH radicals, which effectively killed cancer cells and inhibited tumor growth. It is worth noting the mechanism of EEPT approach is different from the well-known PDT and PTT. PDT relies on photosensitizers that absorb light and sensitize surrounding O2 to generate singlet oxygen for cancer cell killing.52 PTT directly converts light


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irradiation to heat, leading to local temperature increase to damage cancer cells. In contrast, EEPT uses the nanoparticle-enzyme complex, combined with the high glucose uptake in tumor, to generate and split H2O2 into •OH radicals for killing cancer cells. Our results have shown that the drug dose (Pdot-GOx), which were much less than those in PDT, effectively inhibited or eradicated tumor in mouse models. A light radiation with a shorter wavelength is more efficient for the generation of •OH radicals. However, phototherapy using short-wavelength light can damage normal tissues and is constrained by the light penetration depth. Combined with photon upconversion technology, it is possible to use near-infrared (NIR) light excitation for EEPT because upconversion nanoparticles can convert low-energy NIR photons to UV and visible photons. With further optimization, EEPT is likely to become a promising therapeutic method for treatment of malignant tumors.

ASSOCIATED CONTENT Supporting Information. Supporting Information contains additional Materials, experimental details, particle size and Zeta potential of Pdots and Pdot-GOx, spectroscopic characterizations of Pdots and Pdot-GOx, detection of hydroxyl radical (•OH) generated by aqueous H2O2 solution, effect of DMSO on hydroxyl radical generation, determination of intracellular H2O2 level, MTT results of common photosensitizers, AO&EB cell staining results of common photosensitizers, in vivo distribution of Pdot-GOx in various organs, and a summary of traditional photosensitizers for photodynamic therapy.


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AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGMENT We gratefully acknowledge the financial support from the Key Program of National Natural Science Foundation of China (NSFC 61335001) and Thousand Young Talents Program.

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(28) Pu, K.; Shuhendler, A. J.; Jokerst, J. V.; Mei, J.; Gambhir, S. S.; Bao, Z.; Rao, J. Nat. Nanotechnol. 2014, 9, 233-239. (29) Pu, K.; Mei, J.; Jokerst, J. V.; Hong, G.; Antaris, A. L.; Chattopadhyay, N.; Shuhendler, A. J.; Kurosawa, T.; Zhou, Y.; Gambhir, S. S. Adv. Mater. 2015, 27, 5184-5190. (30) Zhen, X.; Zhang, C.; Xie, C.; Miao, Q.; Lim, K. L.; Pu, K. ACS Nano 2016, 10, 64006409. (31) Miao, Q.; Lyu, Y.; Ding, D.; Pu, K. Adv. Mater. 2016, 28, 3662-3668. (32) Shi, H.; Ma, X.; Zhao, Q.; Liu, B.; Qu, Q.; An, Z.; Zhao, Y.; Huang, W. Adv. Funct. Mater. 2014, 24, 4823-4830. (33) Palner, M.; Pu, K.; Shao, S.; Rao, J. Angew. Chem. Int. Ed. 2015, 127, 11639-11642. (34) Wu, W.; Feng, G.; Xu, S.; Liu, B. Macromolecules 2016, 49, 5017-5025. (35) Liu, H. Y.; Wu, P. J.; Kuo, S. Y.; Chen, C. P.; Chang, E. H.; Wu, C. Y.; Chan, Y. H. J. Am. Chem. Soc. 2015, 137, 10420-10429. (36) Chan, Y. H.; Wu, P. J. Part. Part. Sys. Char. 2015, 32, 11-28. (37) Zhao, Q.; Zhou, X.; Cao, T.; Zhang, K. Y.; Yang, L.; Liu, S.; Liang, H.; Yang, H.; Li, F.; Huang, W. Chem. Sci. 2015, 6, 1825-1831. (38) Sun, K.; Tang, Y.; Li, Q.; Yin, S.; Qin, W.; Yu, J.; Chiu, D. T.; Liu, Y.; Yuan, Z.; Zhang, X.; Wu, C. ACS Nano 2016, 10, 6769-6781. (39) Wu, C.; Chiu, D. T. Angew. Chem. Int. Ed. 2013, 52, 3086-3109.


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(40) Li, Q.; Sun, K.; Chang, K.; Yu, J.; Chiu, D. T.; Wu, C.; Qin, W. Anal. Chem. 2013, 85, 9087-9091. (41) Lyu, Y; Xie, C.; Chechetka, S.; Miyako, E; Pu K. J. Am. Chem. Soc. 2016, 138, 90949052. (42) Lyu, Y; Zhen, X; Miao, Y; Pu, K. ACS Nano 2017, 11, 358-367. (43) Wu, C.; Hansen, S. J.; Hou, Q.; Yu, J.; Zeigler, M.; Jin, Y.; Burnham, D. R.; McNeill, J. D.; Olson, J. M.; Chiu, D. T. Angew. Chem. Int. Ed. 2011, 50, 3430-3434. (44) Feuerstein, O.; Moreinos, D.; Steinberg, D. J. Antimicrob. Chemother. 2006, 57, 872-876. (45) Soh, N. Anal. Bioanal. Chem. 2006, 386, 532-543. (46) Gomes, A.; Eduarda, F.; Lima, J. L. J. Biochem. Bioph. Meth. 2005, 65, 45-80. (47) Allison, R. R.; Gordon, H. D; Rosa, C; Hu, X. H; Childs, JH. C; and Sibata, H. C. Photodiagn. Photodyn. 2004, 1, 27-42. (48) Liang, R.; Tian, R.; Ma, L.; Zhang, L.; Hu, Y.; Wang, J.; Wei, M.; Yan, D.; Evans, D. G.; Duan, X. Adv. Funct. Mater. 2014, 24, 3144-3151. (49) Demidova, T. N.; M. R. Hamblin. Int. J. Immunopath. Ph. 2004, 17, 245-254. (50) Bachor, R.; Shea, C.R.; Gillies, R.; Hasan, T. Proc. Natl. Acad. Sci. USA 1991, 88, 15801584. (51) Conde, J.; Oliva, N.; Zhang, Y.; Artzi, N. Nat. Mater. 2016, 15, 1128-1138.


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(52) Henderson, B. W.; Dougherty, T. J. How Does Photodynamic Therapy Work? Photochem. Photobiol. 1992, 55, 145-157.


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Scheme 1. Schematic illustration of Pdot-GOx nanoparticles for in vivo generation and photolysis of hydrogen peroxide.


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Figure 1. Detection of hydroxyl radical (•OH) generated by Pdot-GOx nanoparticles. Oxidation of terephalic acid (TA) to 2-hydroxy- terephalic acid (TAOH) by •OH.

a)

Non-

fluorescent TA was converted to fluorescent TAOH. b) Fluorescence spectra of TAOH induced by Pdot-GOx and glucose under 460 nm light irradiation for different times (0-60 min). c)


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Fluorescence spectra of TAOH from the same solution in (b) in absence of light irradiation. d) Fluorescence spectra of TAOH from the same solution in (b) in absence of Pdot-GOx. e) Fluorescence spectra of the solution of TA and glucose alone. f) Fluorescence intensity of TAOH at 426 nm as a function of irradiation time for the Pdot-GOx and different control treatments. F0 and F were the fluorescence intensities of the system without or with treatment, respectively. The error bars represent the standard deviations of three separate measurements.


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Figure 2. a) MTT cell viability values of MCF-7 cells incubated with Pdot-GOx and other controls for 6 h. b) MTT cell viability values of MCF-7cells incubated with Pdot-GOx for 6 h and treated with light irradiation (460 nm, 100 mW/cm2) for 20 min. c) Fluorescent images of MCF-7 cells with AO/EB staining after different treatment. The control showed the cell staining in absence of Pdot-GOx. The other three panels showed the cell staining with different times of light irradiation. Green, yellow-orange and red fluorescence indicated live cells, early apoptotic cells and late apoptotic/necrotic cells, respectively.


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Figure 3. a) Fluorescence imaging of tumor-bearing mice at different time intervals after intratumoral injection of Pdot-GOx. b) Representative pictures of the the mice on Day 1, Day 14, and Day 28 post treatment. From top to bottom: i) GOx, ii) GOx + Light, iii) Pdot-GOx, iv) Pdot-GOx + Light. c) Representative pictures of the tumors collected from the mice after the EEPT and the control treatment. The tumors collected from the 1-6 groups were showed from top to bottom, respectively. d) Time-dependent changes of the body weight of the mice after various treatments.

e) Tumor growth curves in the xenograft-bearing mice after different

treatments. Tumor volumes were normalized to their initial sizes. Error bars represent the standard deviations of five mice per group. f) Histological H&E staining of various organs from the mice of the EEPT group.


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TOC Graphics


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Enhanced Phototherapy by Nanoparticle-Enzyme via Generation and

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