A Bioenvironment-responsive Versatile Nanoplatform Enabling Rapid

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A Bioenvironment-responsive Versatile Nanoplatform Enabling Rapid Clearance and Effective Tumor-homing for Oxygen Enhanced Radiotherapy Qiuhong Zhang, Jingwen Chen, Ming Ma, Han Wang, and Hangrong Chen Chem. Mater., Just Accepted Manuscript • Publication Date (Web): 17 Jul 2018 Downloaded from http://pubs.acs.org on July 17, 2018

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Chemistry of Materials

A Bioenvironment-responsive Versatile Nanoplatform Enabling Rapid Clearance and Effective Tumor-homing for Oxygen Enhanced Radiotherapy Qiuhong Zhang,†,‡ Jingwen Chen,§ Ming Ma,† Han Wang,*, §, ∥ and Hangrong Chen*, † †

State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050, P. R. China

‡

University of Chinese Academy of Sciences, 100049 Beijing, P. R. China

§

Department of Radiology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200080, P. R. China

∥

National Engineering Research Center for Nanotechnology, Shanghai 200241, P. R. China

ABSTRACT: Nano-radiosensitizer-augmented cancer radiotherapy (RT) has been widely investigated, whereas the desirable nano-radiosensitizer with the characteristics of rapid clearance, effective tumor-homing and tumor hypoxia relieve is still lacking. Herein, bismuth sulfide-albumin composite nanospheres followed by catalase conjugation (denoted as BSNSsCAT) has been well constructed as a bioenvironment-responsive nano-radiosensitizer platform. The BSNSs-CAT phagocytosed by normal cell demonstrates architecture disintegration into small one upon responding to physiological pH, achieving small size-favored rapid clearance, which largely mitigates the concern of long-term toxicity of BSNSs-CAT in normal tissues. More importantly, benefiting from their large size-favored enhanced permeability and retention effect, BSNSs-CAT exhibits efficient tumor accumulation and keeps architecture stable in mild acidic tumor microenvironment (TME), which much favors to response with H2O2 overproduced TME. As results, the produced intratumor oxygen could overcome tumor hypoxia-associated RT resistance, together with the radio-sensitization effect of bismuth, collectively enhancing the RT efficacy. This research demonstrates a versatile material solution through fully exploring its bioenvironment responsive nature, offering a new strategy for nanomedicine design and application.

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INTRODUCTION Radiotherapy (RT), which utilizes ionizing radiation (X-ray) to ablate tumor,1,2 is of a unique advantage of depth-independent, effectively favoring both superficial and deep-seated tumor regression. Recently, the inorganic nanomaterials containing high Z-elements, such as, rareearth element containing up-conversion nanoparticles,3-5 wolfram oxide nanoparticles6 and gold nanoparticles,7-9 etc. have been widely investigated as nano-radiosensitizers. In comparison with pure RT, during which high energy X-rays are radiated at tumorous region to kill cancer, those nano-radiosensitizers accumulated at tumorous region can interact with Xrays, producing photoelectrons, compton electrons and secondary charged particles to promote RT-induced cancer killing.8 In order to maximize the RT efficacy, the effective tumor-homing of these nano-radiosensitizers through the enhanced permeability and retention (EPR) effect is highly desirable, meanwhile, the rapid clearance to circumvent their long-term toxicity is also considerably expected, both of which, as the growing evidences shown, are closely related to their hydrodynamic size. On one hand, the hydrodynamic size in the range of 30-200 nm is quite beneficial for improved tumor-homing through the EPR effect, but inevitably results in retention in reticuloendothelial systems (RES) such as liver and spleen,10 wherein long-term toxicity would be potentially amplified if without biodegradation and clearance.11-13 On the other hand, the hydrodynamic size below 10 nm will largely facilitate rapid clearance from body by urinary excretion, effectively mitigating long-term toxicity, but is at the expense of low tumor accumulation.13-15 Unfortunately, the reported nano-radiosensitizers either focus on large size-favored tumor-homing or small size-favored rapid clearance, rare efforts devoted to both. In addition, tumor hypoxia that often leads to hypoxia-associated RT resistance is another concerned issue.16-19 Despite some strategies have been explored to mitigate tumor hypoxia,2021,22

the highly expected characteristics of effective tumor-homing and rapid clearance as well

as tumor hypoxia relieve are indispensable in an individual nano-radiosensitizer platform, which, therefore, desperately needed to be solved but still of daunting challenge. As well known, tumor demonstrates a unique metabolic profile, such as generating massive lactic acid and producing excessive amounts of H2O2, exhibiting a typical acidic and H2O2-rich tumor microenvironment (TME), which is quite different from the physiological environment of normal organs.23-30 Inspired by such bioenvironment difference, herein, bismuth sulfidealbumin composite nanospheres followed by catalase conjugation (denoted as BSNSs-CAT) has been well constructed as a bioenvironment-responsive nano-radiosensitizer platform (Scheme 1a), for the first time. Owing to the structural expansion of BSA under physiological 2 ACS Paragon Plus Environment

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pH,31,32 BSNSs-CAT phagocytosed by RES can gradually disintegrate their architecture for small size-favored rapid clearance from normal organs (Scheme 1b). Moreover, the BSNSsCAT demonstrates effective tumor-homing contributed by their large size-favored EPR effect and keeps architecture stable under acidic TME, which not only acts as a nano-radiosensitizer for the enhancement of RT based on the high photoelectric absorption coefficient of bismuth element, but also responds to the excessive H2O2 inside TME for the relief of hypoxia, thus collectively promoting cancer RT efficacy (Scheme 1c).

Scheme 1. (a) Synthetic procedure of BSNSs-CAT. (b, c) Schematic illustrations of BSNSsCAT as bioenvironment-responsive versatile nanoplatform for simultaneously rapid clearance, effective tumor-homing and oxygen enhanced RT.

RESULTS AND DISCUSSION The BSNSs-CAT nanoplatform was facilely synthesized by a two-step procedure. Firstly, BSNSs were synthesized by mixing bismuth nitrate into BSA aqueous solution followed by the addition of thioacetamide, after incubated at 37 °C for 12 h, the BSNSs were obtained. Figure 3 ACS Paragon Plus Environment


1a presents the X-ray diffraction pattern of the BSNSs, whose diffraction peaks can be well indexed to the orthorhombic Bi2S3 (JCPDS No. 17-0320). The transmission electron microscopy (TEM) image at low magnification demonstrates the obtained BSNSs have a uniform size (Figure 1b). Moreover, the TEM image of an individual BSNS (Figure 1c) and its enlarged area (Figure 1d) clearly reveal that the Bi2S3 nanocrystals with the size below 5 nm are embedded into BSA scaffold. The scanning TEM (STEM) images of BSNSs also verify that the Bi2S3 nanocrystals are homogeneously distributed into BSA scaffold (Figure 1e-g and Figure S1), which endows the as-synthesized BSNSs with better biocompatibility without need of additional surface modification.33,34 The chemical composition of BSNSs was confirmed by element mapping as shown in Figure 1h. With respect to the formation of Bi2S3 nanocrystals, it is believed that the thiol-containing amino acids are apt to coordinate with metal ions,35 since the BSA molecule involves 17 disulfide bonds with one free thiol in cysteine residues,36 which is possible for Bi3+ ions binding via thiolate linkage.37,38 In addition, thioacetamide is unstable and slowly hydrolyzed to release S2− into the reaction solution which combines with Bi3+ ions to form Bi2S3 nucleus on the BSA molecules and eventually selfassembled into well-defined BSNSs. Interestingly, such a facile method can also be extended to construct the bismuth sulfide-protein composite nanospheres with other thiol-containing proteins, such as human serum albumin (HSA) and holo-Transferrin (Figure S2), indicating the universality of this synthetic method.

Figure 1. Characterizations of BSNSs. (a) X-ray diffraction pattern of the BSNSs. TEM images of BSNSs at (b) low and (c, d) high magnifications. STEM images of BSNSs using (e) 4 ACS Paragon Plus Environment

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SEM, (f) bright-field and (g) dark-field modes of TEM. (h) Corresponding element mapping of BSNSs. Scale bars: 100 nm. Next, CAT was conjugated onto BSA scaffold of BSNSs through amide reaction. The obtained BSNSs-CAT shows a similar structure to the BSNSs (Figure 2a, b) and exhibits high stability in water, phosphate buffered saline (PBS) and fetal bovine serum (FBS) (Figure S3). As shown in Figure 2c, BSNSs-CAT has an average hydrodynamic size of 126 nm, slightly larger than BSNSs (92 nm) owing to the CAT conjugation, which is beneficial for the large size-favored EPR effect and thereafter effective tumor-homing. The CAT coupling capacity is calculated to be 290 milligram CAT per gram of BSNSs according to bicinchoninic acid protein assay, and its activity, on the basis of Góth method,39 maintains about 46% that of equivalent free CAT molecules due to the inevitable conformation changes, which is a normal phenomenon, similarly as widely reported in other articles.40,41 The oxygen generation capability of BSNSs-CAT toward H2O2 is further evaluated through detecting O2 concentration with dissolved oxygen meter upon addition of BSNSs, BSNSs-CAT and CAT into H2O2 solution (100 µM), respectively. As shown in Figure 2d, it is interesting to find that the prepared BSNSs-CAT generates similar amount of oxygen to free CAT molecules in H2O2 solution.

Figure 2. In vitro oxygen generation and RT. TEM images of BSNSs-CAT at (a) low and (b) high magnifications. (c) Hydrodynamic size of BSNSs and BSNSs-CAT, respectively. (d) Quantitative oxygen generation of BSNSs-CAT, BSNSs and CAT reacted with H2O2 solution (100 µM). (e) Relative cell viabilities of 4T1 cells incubated with various bismuth concentrations of BSNSs or BSNSs-CAT for 36 h in an anaerobic atmosphere. (f) Relative cell 5 ACS Paragon Plus Environment


viabilities of 4T1 cells treated with BSNSs or BSNSs-CAT (CBi = 100 µg mL-1) under series of radiation doses of 0, 4, 6, and 8 Gy. Afterwards, the in vitro RT enhancement effect of BSNSs-CAT was evaluated by a standard cell counting kit-8 (CCK-8) assay. The cytotoxicities of BSNSs and BSNSs-CAT were tested by CCK-8 assay with 4T1 cells, respectively. It is demonstrated that both BSNSs and BSNSsCAT show little effects on cell viability, even at a high bismuth concentration up to100 µg mL1

(Figure 2e). For in vitro RT analysis, 4T1 cells incubated in an anaerobic atmosphere were

irradiated under clinical 220 keV X-ray at the radiation doses of 4, 6, and 8 Gy in the presence of BSNSs or BSNSs-CAT at a bismuth concentration of 100 µg mL-1. As shown in Figure 2f, cells treated with BSNSs exhibit lower cell viability compared with pure RT, indicative of the efficient radio-sensitization effect of BSNSs. Moreover, cells treated with BSNSs-CAT display much enhanced inhibition of cell proliferation after X-ray irradiation than that treated with BSNSs, demonstrating that CAT anchored on BSNSs could further enhance RT by reacting with endogenic H2O2 to increase intracellular oxygen level, which is highly favorable for in vivo cancer RT enhancement. Furthermore, the in vitro pH-responsive size evolution of BSNSs-CAT was investigated. The normal physiological environment with a pH value of 7.2-7.4 as well as the mild acidic tumor environment

23,24,42

were simulated with PBS at 37 °C. As shown in Figure 3a1-a3, the

TEM images of BSNSs-CAT dispersed in the milieu of pH 5.4 over 24 h provide the direct evidence that BSNSs-CAT remains stable under mild acidic tumor environment. Nevertheless, the BSNSs-CAT, when bathed in a milieu of pH 7.4 for 6 h, gradually disintegrates their architecture and releases a small quantity of Bi2S3 nanocrystals embedded in BSNSs-CAT (Figure 3a4), and much more nanocrystals could be released when increasing the bath time to 12 h, just leaving the BSA skeletons (Figure 3a5 and Figure S4). Eventually, the BSNSs-CAT disintegrates into Bi2S3 nanocrystals and protein molecules upon bathing in the milieu of pH 7.4 for 24 h (Figure 3a6 and Figure S5). Furthermore, the hydrodynamic sizes of BSNSs-CAT dispersed in different pHs for different time were analyzed. As shown in Figure 3b, hydrodynamic size below 10 nm could be found for BSNSs-CAT dispersed in simulated physiological pH post 12 h, which could be attributed to the releasing of Bi2S3 nanocrystals based on above TEM results. For comparison, the BSNSs-CAT still keeps large hydrodynamic size when dispersed in mild acidic tumor environment, further indicating that BSNSs-CAT could disintegrate their architecture into small one upon triggering with physiological pH. According to the pH-dependent expansion and reverse expansion of BSA molecules previously reported,31 we speculate that the BSA molecules, which have undergone expansion under 6 ACS Paragon Plus Environment

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strong acid reaction condition, then reversely expand to their native structures when bathed in a normal physiological pH, leading to the architectural disintegration of BSNSs-CAT. In addition, as we know, the isoelectric point of BSA molecules is pH 4.7, in which condition, the BSA molecules are electroneutral and apt to aggregate together. Therefore, under a milieu of pH 5.4 (close to the isoelectric point of pH 4.7), the BSNSs-CAT can remain stable and show no obvious structural disintegration.

Figure 3. In vitro size evolution property. (a) TEM images of BSNSs-CAT dispersed in PBS for 6, 12 and 24 h at a pH value of 5.4 (a1-a3) and 7.4 (a4-a6), respectively. The insets are the high magnifications of selected areas. Scale bars: 50 nm. (b) Hydrodynamic size of BSNSsCAT dispersed in PBS for 6, 12 and 24 h at different pHs. To further evidence the disintegrable property of BSNSs-CAT at the cellular level, BSNSsCAT were incubated with 4T1 cells for 24 h to allow cell uptake. After the incubation, the cells were collected and subjected to the standard sample preparation procedure for TEM characterization. In order to clearly illuminate the architectural disintegration of BSNSs-CAT, 7 ACS Paragon Plus Environment


three different regions of the cell slice were observed with TEM. As demonstrated in Figure 4a, all the TEM images evidence the BSNSs-CAT endocytosed into the cell can completely disintegrate inside, well in agreement with above TEM and hydrodynamic size results for BSNSs-CAT dispersed in simulated physiological pH. The energy-dispersive X-ray analysis of the selected area in Figure 4a3 shows the existence of bismuth element (Figure 4b), indicating the fragments in selected area are corresponding to BSNSs-CAT, which further verifies the intracellular architectural disintegration of BSNSs-CAT. According to the TEM and hydrodynamic size results, Figure 4c schematically illustrates the disintegration process of BSNSs-CAT dispersed in physiological pH.

Figure 4. Intracellular architectural disintegration. (a) TEM images of the disintegrated BSNSs-CAT distributed in three regions of cell slice. (b) Energy-dispersive X-ray analysis of the selected area in region 3. The red ellipses in (b) show the existence of bismuth element. (c) Schematic illustration of the disintegration process of BSNSs-CAT dispersed in physiological pH. To further study the in vivo metabolic behavior of the released Bi2S3 nanocrystals, mice were sacrificed on the 1st, 7th, and 14th day post intravenous (i.v.) injection of BSNSs-CAT, and then 8 ACS Paragon Plus Environment

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the bismuth contents in main organs were analyzed using inductively coupled plasma optical emission spectrometry (ICP-OES). As shown in Figure 5a, the BSNSs-CAT were phagocytosed by the reticuloendothelial systems such as liver and spleen on the 1st day post injection, thus displaying accumulation in liver and spleen, which is the typical phenomenon for all the nanomaterials after systemic administration. But it quickly decreased to rather low level on the 7th day, confirming that BSNSs-CAT can be rapidly excreted out of body. Furthermore, the bismuth contents in feces and urine collected from BSNSs-CAT injected mice were also analyzed using ICP-OES. As shown in Figure 5b, it is found that 34.9% and 10.8% injected dose (ID) were cleared out via urine and feces, respectively within 2 days. More than 80% ID could be excreted out of the body after 7 days, thereinto, 54.7% and 26.2% ID were cleared out via urine and feces, respectively, further confirming BSNSs-CAT can indeed be rapidly excreted, which is mainly contributed by the physiological pH triggered architectural disintegration to facilitate small size-favored rapid clearance. Additionally, owing to such rapid clearance, there are no obvious BSNSs-CAT retention in main organs on the 14th day, effectively circumventing the in vivo long-term toxicity.

Figure 5. In vivo clearance. (a) The time-dependent biodistribution of bismuth element (% injected dose (ID) of Bi per gram of tissues) in main tissues of mice after i.v. injection of BSNSs-CAT. (b) Excretion profiles of bismuth element in mice after i.v. injection of BSNSsCAT. Afterwards, the in vivo biosafety evaluation, including blood biochemistry, complete blood panel, histological and weight analysis for mice receiving i.v. injection with BSNSs-CAT at different bismuth doses based on the weight of a mouse (5, 10 and 15 mg Bi kg-1) for 30 days were conducted. The mice intravenously injected with PBS as control group. As shown in Figure 6a, thanks to the rapid clearance of BSNSs-CAT post injection, the blood parameters for BSNSs-CAT treated mice are found to be comparable to those of PBS treated healthy mice in control group and the difference is not statistically significant. Moreover, the hematoxylin 9 ACS Paragon Plus Environment


and eosin (H&E) stained slices of main organs, including the heart, liver, spleen, lung, and kidney, for BSNSs-CAT treated mice also exhibit no histological lesions in comparison with control group (Figure 6b). In addition, the body weights of mice are not affected by different treatments and have been increasing (Figure S6). All these results indicate that the BSNSsCAT possess high biosafety, which is particularly promising for effective cancer RT.

Figure 6. In vivo biosafety evaluation. (a) Blood biochemistry and complete blood panel analysis of mice after receiving i.v. injection with BSNSs-CAT at different bismuth doses based on the weight of a mouse (5, 10 and 15 mg Bi kg-1) for 30 days. The examined parameters included alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), blood urea nitrogen (BUN), creatinine (CREA), white blood cell (WBC) counts, red blood cell (RBC) counts, lymphocyte (LYM), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelets (PLT). (b) Corresponding H&E stained slices obtained from the heart, liver, spleen, lung, and kidney of mice on the 30th day. Before in vivo RT treatment, the tumor-homing and tumor hypoxia relieve property of BSNSs-CAT, which plays a vital role for desirable RT efficacy, were evaluated through photoacoustic (PA) imaging. PA imaging is a newly developed noninvasive diagnostic technology, which possesses the advantages of both optical imaging and ultrasound (US) imaging, excellent sensitivity, high resolution, and the capability of real-time imaging. For 10 ACS Paragon Plus Environment

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tumor-homing analysis, strong PA signal could be detected after BSNSs-CAT was injected into tumor attributed to the strong near infrared absorption of bismuth sulfide nanoparticles (Figure 7a).43,44 Encouraged by the strong PA signal after intratumor (i.t.) injection with BSNSs-CAT, tumor-bearing mice were further intravenously injected with BSNSs-CAT, and then the 3dimensional (3 D) PA images of tumor were acquired at 2, 4 and 6 h post injection. As shown in Figure 7b, the intensity of PA signal of tumor reaches maximum at 4 h, indicating the effective tumor-homing of BSNSs-CAT through the large size-favored EPR effect. The corresponding tumor uptake of BSNSs-CAT, according to the quantitative analysis with ICPOES, is 0.644% ID, which is higher than the reported tumor uptake values of less than 0.6%ID for a majority of nanomaterials.45 For tumor hypoxia relieve evaluation, the vascular saturated O2 (sO2) within tumor post injection of BSNSs-CAT was investigated using PA imaging through measuring oxygenated and deoxygenated hemoglobin at different characteristic wavelengths of 850 nm and 750 nm, respectively. As demonstrated in Figure 7c, the US imaging depicts the profile of the tumor, and PA imaging exhibits the oxygenated hemoglobin (shown in red) and deoxygenated hemoglobin (shown in blue) in tumor, both of which reveal the sO2 level. In contrast, it is found that the injection of BSNSs-CAT can significantly increase tumor sO2 by about 6.65% (Figure 7d). Furthermore, after sO2 PA imaging, the tumor tissues were collected for staining hypoxia areas with anti-hypoxia inducible factor (HIF)-1α antibody (shown in green). As shown in Figure 7e, micrographs of tumor slices treated with BSNSs-CAT display a remarkable down-expression of HIF-1α. Both the PA sO2 imaging and slices results manifest the decreased hypoxia owing to oxygen generation in tumor contributed by BSNSs-CAT reacting with endogenic H2O2 within TME, which lays a foundation for in vivo oxygen-enhanced RT.



Figure 7. In vivo tumor-homing, tumor oxygenation and RT. (a) 3D US/PA images of 4T1 tumor-bearing mice at different time after receiving i.v. injection of BSNSs-CAT and only i.t. injection. The PA signal of BSNSs-CAT is highlighted in green. (b)The corresponding PA signal intensities of the tumorous area. (c) sO2 PA imaging of tumor post injection of PBS or BSNSs-CAT. Oxygenated hemoglobin is shown in red and deoxygenated hemoglobin is shown in blue. (d) Quantitative comparison of intratumor sO2 levels after different treatments. (e) Micrographs of tumor slices with cell nuclei and hypoxia area stained with DAPI (shown in blue) and HIF-1α antibody (shown in green), respectively. Scale bars: 200 µm. (f) Tumor growth curves of mice for 12 days after various treatments. (n = 5, mean ± s.d., *P < 0.05, **P < 0.01, one-way ANOVA) (g) Digital photos of tumor-bearing mice in control and BSNSsCAT+RT groups on the 12th day. (h) Corresponding body weight of mice for 12 days after various treatments. (i) Micrographs of H&E and TUNEL stained tumor slices collected from different groups of mice on the third day post treatments. Scale bars: 100 µm.


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Furthermore, in vivo RT was conducted with tumor-bearing mice and clinical 220 keV X-ray with a dose of 8 Gy. The mice treated with PBS, BSNSs-CAT, pure RT, BSNSs together with RT, BSNSs-CAT together with RT are denoted as control, BSNSs-CAT, RT, BSNSs+RT, BSNSs-CAT+RT group, respectively. After various treatments, the tumor volume and body weight of mice were recorded every 3 days. As shown in Figure 7f, BSNSs-CAT group presents similar tumor growth curve to control group, indicating BSNSs-CAT has no therapeutic effect on tumor. Owing to the radio-sensitizing effect of bismuth element, both BSNSs+RT and BSNSs-CAT+RT groups show obvious tumor growth inhibition compared with RT alone. It is noteworthy that BSNSs-CAT+RT exhibits the most significant tumorgrowth inhibition, mainly attributed to the extra combining effect of hypoxia relieve resulted from CAT responding to endogenic H2O2 in tumor (Figure 7c-e). Thanks for such synergistic effect of radio-sensitizing and hypoxia relieve, BSNSs-CAT+RT group, compared to control group without any treatment, exhibits considerably significant inhibition of tumor growth and the corresponding photos of mice are shown in Figure 7g. Besides, the body weight of mice in BSNSs-CAT+RT group grow over time, indicating the negligible adverse effect of such therapeutic protocol on the mice (Figure 7h). To further evaluate RT efficacy, three days after conducting various treatments, tumors from different groups were dissected for H&E and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining, which clearly illustrates that BSNSs-CAT+RT cause the most severe damage to cancer cells by inducing tumor cell apoptosis (Figure 7i).

CONCLUSIONS In summary, a bioenvironment-responsive nanoplatform, i.e., BSNSs-CAT, enabling rapid clearance and effective tumor-homing for oxygen enhanced RT, was well constructed. The BSNSs-CAT composed of BSA, Bi2S3 nanocrystals with size below 5 nm, and CAT were synthesized via a very facile method. Among which, BSA molecules coordinate with Bi3+ ions via thiolate linkage during synthesis, providing nucleation sites for Bi2S3 nanocrystals which are embedded into BSA scaffold subsequently. Owing to the structural expansion of BSA molecule upon responding to physiological pH, the BSNSs-CAT can specifically and gradually disintegrate their architecture into small one for rapid clearance from normal organs, largely mitigating the concern of long-term toxicity. Moreover, the BSNSs-CAT exhibits effective tumor-homing contributed by their large size-favored EPR effect and keeps architecture stable under acidic TME, which further favors intratumor oxygen generation by reacting with endogenic H2O2 within TME to overcome tumor hypoxia-associated RT resistance, together with the radio-sensitization of bismuth element, thus collectively enhancing the RT efficacy. 13 ACS Paragon Plus Environment


The design concept demonstrated in our research combines the unique bioenvironment (i.e., physiological pH and TME) difference with rapid clearance and effective tumor-homing derived therapeutic benefits, lays foundation for the novel yet practical nanomedicine design in the future.

EXPERIMENTAL SECTION Chemicals and reagents. Bovine serum albumin (BSA), bismuth nitrate and nitric acid were obtained from Aladdin Industrial Co.. Thioacetamide was obtained from J&K Chemical Co.. Catalase, 1-ethyl-3-(3-dimethly-aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were obtained from Sigma-Aldrich. Phosphate buffered saline (PBS, 10 mM, pH 7.4) was obtained from Shanghai Runcheng Biomedical Co., Ltd.. Deionized water was used in all experiments. All chemicals were used as received without further purification. Preparation of BSNSs-CAT. 10.0 mg of BSA was dissolved in 17.0 mL of deionized water, and 2.0 mL of bismuth nitrate (50.0 mM) nitric acid (2 M) solution was slowly added under vigorous stirring, followed by the addition of 1.0 mL of thioacetamide (100.0 mM). The mixture was allowed to react at 37 °C for 12 h. After centrifugation and washing with deionized water several times, BSNSs were obtained. 6 mg catalase was dissolved in 4 mL PBS followed by the addition of 4.65 mg EDC and 2.79 mg NHS. The mixture was stirred at room temperature for 15 min, then 10 mL BSNSs aqueous (2 mg mL-1) was added to the above solution and stirred for another 12 h at room temperature. After centrifugation and washing with deionized water, BSNSs-CAT were acquired. The CAT coupling capacity was estimated via bicinchoninic acid protein assay. Characterization of BSNSs-CAT. X-ray diffraction measurement was performed on a Rigaku D/MAX-2250 V at Cu Kα (λ = 0.154056 nm) with a scanning rate of 4 ° min-1. TEM and STEM images were obtained with JEM-2100F and Magellan 400, respectively. The hydrodynamic size was measured by dynamic light scattering (DLS) using Malvern Zetesizer (Nano-ZS90). Agilent 700 Series inductively coupled plasma optical emission spectrometry (ICP-OES) was used to determine element concentration. Oxygen Generation Capability of BSNSs-CAT in Vitro. For measurement of catalysis ability of BSNSs-CAT in vitro, the oxygen electrode probe of a dissolved oxygen meter (YSI 550A) was soaked into H2O2 aqueous solution (100 µM) followed by adding BSNSs, BSNSsCAT and CAT, respectively. The oxygen concentration was recorded in real time.


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Cellular Experiments. Murine breast cancer 4T1 cells were cultured in Dulbecco’s modified Eagle

medium

(DMEM)

containing

10%

fetal

bovine

serum

(FBS)

and

1%

penicillin/streptomycin at 37 °C in humidified atmosphere with 5% CO2. Cells with different oxygen tension were obtained by culturing them in aerobic incubator (21% O2) and anaerobic incubator (2% O2), respectively. For in vitro cytotoxicity test, the cells seeded in 96-well plates (100 µL containing 5000 cells per well) were incubated in aerobic incubator. Then the medium was replaced with fresh culture medium containing BSNSs or BSNSs-CAT with different concentrations. After incubation for 36 h in anaerobic incubator, the standard Cell Counting Kit-8 (CCK-8) assay was used to evaluate the cell viabilities relative to the control group. For in vitro radiotherapy study, the cells pre-seeded in 96-well plates (100 µL containing 5000 cells per well) were incubated with BSNSs or BSNSs-CAT (CBi = 100 µg mL-1) in anaerobic incubator for 12 h, and then the cells were subjected to irradiation of X-ray (SARRP, Gulmay Medical Inc., USA) with series of doses of 0, 4, 6, and 8 Gy. After another 24 h incubation in anaerobic incubator, cells viability was evaluated by CCK-8 assay. Architectural disintegration in Vitro and Intracellular. 0.1 mL BSNSs-CAT at bismuth concentration of 2 mg mL-1 were added into vials loaded with 3.9 mL of buffer media at different pH values (7.4 and 5.4) and stirred at 37 °C. At indicated time point of 6, 12 and 24 h, a drop of buffer media was collected for the analysis by TEM and hydrodynamic size. The 4T1 cells were incubated with BSNSs-CAT at bismuth concentration of 100 µg mL-1 for 24 h to guarantee that there was enough time for cell endocytosis, then the cells were collected and subjected to the standard sample preparation procedure for TEM observation. Biodistribution, Clearance and Biocompatibility Assay. To determine the biodistribution, after receiving i.v. injection of 150 µL BSNSs-CAT saline (15 mg Bi kg-1), Kunming mice (n = 3) were sacrificed on the 1st, 7th, and 14th day to collect the major organs (heart, liver, spleen, lung and kidney). The collected organs were wet-weighted and solubilized in aqua regia for the measurement of bismuth concentration by ICP-OES. To study the excretion, Kunming mice (n = 3) after injection of 150 µL BSNSs-CAT saline (15 mg Bi kg-1) were housed in metabolic cages to collect their urine and feces for seven days. The collected urine and feces were digested by aqua regia for the measurement of bismuth concentration by ICP-OES. For biocompatibility assay study, Kunming mice were randomly divided into four groups (n = 5) and each group was intravenously injected with 150 µL saline with different doses of 5, 10, and 15 mg Bi per kilogram weight of a mouse (5, 10 and 15 mg Bi kg-1). The mice were sacrificed on the 30th day post injection, the blood samples were collected for blood biochemistry and complete blood panel analysis, major organs (heart, liver, spleen, lung and kidney) were 15 ACS Paragon Plus Environment


dissected, fixed in 4% paraformaldehyde solution and stained with hematoxylin and eosin (H&E) for histological analysis. PA Imaging. For in vivo PA imaging, 150 µL saline of BSNSs-CAT (15 mg Bi kg-1) were intravenously injected into 4T1 tumor-bearing mice (n = 3). PA imaging was performed at pre-, 2, 4, and 6 h post injection. For the study of oxygen generation within tumor, after receiving injection of 150 µL BSNSs-CAT saline (15 mg Bi kg-1 for a mouse) into tumor-bearing mice (n = 3), the vascular saturated O2 (sO2) within tumor was evaluated using PA imaging through measuring oxygenated and deoxygenated hemoglobin at different characteristic wavelengths of 850 nm and 750 nm, respectively. After the sO2 PA imaging, the tumor tissues were collected for staining hypoxia areas with anti-hypoxia inducible factor (HIF)-1α antibody. Cancer Radiotherapy in Vivo. All animal experiments operations were in accord with the statutory requirements of People’s Republic of China and care regulations approved by the administrative committee of laboratory animals of Shanghai Jiao Tong University. Female BALB/c nude mice with average age of 4 weeks were used in vivo tumor models. The xenograft 4T1 tumor models were established by subcutaneous injection of 2×106 4T1 cells in 100 µL PBS into each mouse. When the tumor approached 7 mm in average diameter, 4T1 tumor-bearing mice were randomly divided into five groups (n = 5): Group 1: PBS; Group 2: BSNSs-CAT; Group 3: pure RT; Group 4: BSNSs+RT; Group 5: BSNSs-CAT+RT. The dose of BSNSs-CAT is 15 mg Bi kg-1 and tumors of group 3, 4, 5 and 6 irradiated under clinical 220 keV X-ray with a dose of 8 Gy. After various treatments, the length and width of the tumor were measured by a digital caliper every 3 days for 12 days. The tumor volume was calculated according to the following formula: volume =width2 × length/2. Tumors from different groups were dissected on third day post treatments, fixed in 4% paraformaldehyde solution, stained with H&E and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL).

ASSOCIATED CONTENT Supporting Information TEM characterization, extension of the synthetic method and stability analysis of BSNSs, TEM images of the disintegrated BSNSs-CAT, and body weight of mice receiving different treatments.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] 16 ACS Paragon Plus Environment

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*E-mail: [email protected] ORCID Hangrong Chen: 0000-0003-0827-1270 Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Grant No. 51772316, 51402329, 81671740), the National Key Research and Development Program of China (Grant No. 2017YFB0702602, 2016YFC0107108), the Key Projects of International Cooperation and Exchanges of NSFC (No.81720108023), the Shanghai Academic / Technology Research Leader Program (17XD1424200), and the Gaofeng Clinical Medicine Grant of Shanghai Municipal Education Commission (20152230).

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A Bioenvironment-responsive Versatile Nanoplatform Enabling Rapid

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