A Versatile Carbon Monoxide Nanogenerator for Enhanced Tumor

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A Versatile Carbon Monoxide Nanogenerator for Enhanced Tumor Therapy and Anti-Inflammation Shi-Bo Wang, Cheng Zhang, Zhao-Xia Chen, Jing-Jie Ye, SiYuan Peng, Lei Rong, Chuan-Jun Liu, and Xian-Zheng Zhang ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.9b00345 • Publication Date (Web): 02 May 2019 Downloaded from http://pubs.acs.org on May 2, 2019

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A Versatile Carbon Monoxide Nanogenerator for Enhanced Tumor Therapy and Anti-Inflammation

Shi-Bo Wang,†,‡,# Cheng Zhang,†,# Zhao-Xia Chen,† Jing-Jie Ye,† Si-Yuan Peng,† Lei Rong,† Chuan-Jun Liu,† and Xian-Zheng Zhang†,‡,*

†

Key Laboratory of Biomedical Polymers of Ministry of Education & Department of Chemistry, Wuhan University, Wuhan 430072, P. R. China

‡

Institute for Advanced Studies (IAS), Wuhan University, Wuhan 430072, P. R. China

* To whom correspondence should be addressed. E-mail address: [email protected] #

These authors contributed equally to this work.

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ABSTRACT: Carbon monoxide (CO) is regarded as a potential therapeutic agent with multiple beneficial functions for biomedical applications. In this study, a versatile CO nanogenerator (designated as PPOSD) was fabricated and developed for tumor therapy and anti-inflammation. Partially oxidized tin disulfide (SnS2) nanosheets (POS NSs) were decorated with a tumor targeting polymer (polyethylene glycol-cyclo(Asp-D-PheLys-Arg-Gly), PEG-cRGD) and followed by the loading of chemotherapeutic drug doxorubicin (DOX) to prepare polymer@POS@DOX, or PPOSD. After injected intravenously, PPOSD could selectively accumulate in tumor tissue via the cRGDmediated tumor recognition. Upon 561 nm laser irradiation, the POS moiety in PPOSD can photoreduce CO2 to CO, which significantly sensitized the chemotherapeutic effect of DOX. Besides, the POS in PPOSD can also act as a photothermal agent for effective photothermal therapy (PTT) of tumor upon 808 nm laser irradiation. Furthermore, the generated CO can simultaneously decrease the inflammatory reaction caused by PTT. Blood analysis and hematoxylin-eosin staining of major organs showed that no obvious systemic toxicity was induced after the treatment, suggesting good biosafety of PPOSD. This versatile CO nanogenerator will find great potential for both enhanced tumor inhibition and anti-inflammation.

KEYWORDS: carbon monoxide, sensitization, photothermal therapy, tumor inhibition, anti-inflammation


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Chemotherapy that utilizes chemical substances to interfere with the division and reproduction abilities of cells is a widespread modality for tumor treatment due to its noninvasive characterization and relatively high therapeutic efficacy.1 For chemotherapy, anti-tumor drugs can attack tumor cells throughout the whole body or be targeted to specific tumor sites, which show great advantages than other treatment approaches, such as surgery and radiotherapy for which can only treat tumor cells in certain area.2,3 Unfortunately, even after treatment with chemotherapeutic drugs, only part of tumor cells can be killed. The failure of chemotherapy is mostly attributed to the resistance of tumor cells to antitumor drugs.4,5 To deal with this challenge, chemosensitizers, usually small-molecule compounds that make tumor cells more sensitive to chemotherapeutic drugs, have come into being.6,7 However, it is difficult to ensure the chemotherapeutic drugs and chemosensitizers accumulate in the same tumor site after systemic administration, thus the in vivo sensitization effects are always not as good as expected.8 Therefore, the development of effective chemosensitization system is of great importance and in urgent need. Photothermal therapy (PTT), which relies on photothermal agents (PTAs) to locally absorb and convert near-infrared (NIR) irradiation into heat to ablate cells, has recently emerged as a promising treatment modality for various kinds of tumors.9-11 PTT is of great attraction due to its advantages, such as low cost, highly localizing, and minimal invasiveness to surrounding healthy tissues.12 Besides, combining PTT with chemotherapy has proved to be a feasible way to improve the therapeutic efficacy.13,14 However, note that the heating temperature of PTT is usually above 42 °C, the most common cellular death mode caused by PTT is necrosis, which is characterized by damage of cell membrane integrity and abnormal release of intracellular constituents into extracellular milieu, thus triggering inflammatory responses.15 Although


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inflammation is a common defense reaction of tissues to various insults and important for the activation of antitumor immunity to some extent, there is evidence that the inflammatory responses triggered by PTT are associated with a wide variety of adverse reactions, such as tissue injury, tumor regeneration, and therapeutic resistance.16-18 Thus, effectively weaken the inflammation reaction caused by PTT is necessary and highly desirable. Carbon monoxide (CO), a colorless gas that is toxic to humans at high concentrations has been increasingly regarded as a critical signaling molecule with multiple beneficial effects in biomedical applications, such as inhibiting bacterial growth, protecting normal cells, and improving transplantation survival.19-22 Meanwhile, CO could also sensitize tumor cells towards chemotherapy and reduce inflammatory reactions.23,24 However, the practical use of gaseous CO is risky and severely hampered by the strong affinity towards hemoglobin as well as the low bioavailability.25 Even nongaseous forms of CO delivery can be realized by using CO-releasing molecules (CO-RMs), the complex preparation and imprecise control of CO release in physiological conditions are still barriers for their applications. Recently, CO2 photoreduction has been proved to be a feasible way for in vivo CO production.26 By regulating the illumination, the CO2 photoreduction process can be precisely adjusted, which significantly improves the controllability and accuracy of CO generation. Here, partially oxidized tin disulfide (SnS2) nanosheets (POS NSs) based versatile CO nanogenerator, PPOSD, was fabricated for tumor suppression and antiinflammation. POS was chosen because of its effective CO2 photoreduction ability to produce CO under visible light.27 Besides, the large surface-area-to-volume ratio of NSs allows for efficient drug loading.28,29 Additionally, POS with strong absorption in the NIR region made it a promising agent for efficient hyperthermia generation under


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NIR laser irradiation. As illustrated in Scheme 1, POS NSs were functionalized with a tumor targeting polymer (polyethylene glycol-cyclo(Asp-D-Phe-Lys-Arg-Gly), PEGcRGD) to improve their tumor selectivity and biocompatibility and followed by the loading

of

chemotherapeutic

drug

doxorubicine

(DOX)

to

prepare

polymer@POS@DOX, or PPOSD. It was envisioned that PPOSD could target to tumor cells by the affinity between cRGD motif and the overexpressed integrin αvβ3 receptor in tumor cell surface. Then CO could be produced by PPOSD via CO2 photoreduction upon 561 nm laser irradiation, which effectively sensitized tumor cells towards DOX for enhanced chemotherapy. Apart from this, the POS moiety in PPOSD was expected to serve as a PTA for tumor thermal ablation. In addition, the CO generated above was also expected to decrease the inflammatory responses caused by PTT.

RESULTS AND DISCUSSION Preparation and Characterization of PPOSD. POS atomic layers were first prepared by hydro-thermal method according to the literature report.27 Transmission electron microscope (TEM) and scanning electron microscope (SEM) clearly featured the sheet-like morphology of the product (Figure 1A). The powder X-ray diffraction (PXRD) pattern of the product was consistent with the standard data for hexagonal SnS2 (JCPDS No. 23-0677) (Figure S1). Furthermore, the Raman (Figure S2) and S 2p Xray photoelectron spectroscopy (XPS) spectra (Figure S3) confirmed the presence of Sn oxide domains. The content of Sn oxide was quantified to be about 3.3% by XPS, which was nearly the oxidized SnS2 with highest CO formation rate according to the literature.27 Afterwards, POS atomic layers with large size were converted into small pieces by sonication to provide POS NSs. The as-prepared POS NSs showed an average diameter of 105 nm as evidenced by TEM observation (Figure 1B). To improve their


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biocompatibility and tumor selectivity, POS NSs were functionalized with an amphiphilic polymer, cRGD conjugated PEG (SH-PEG-cRGD) through thiol reaction to provide polymer@POS, or PPOS. Finally, chemotherapeutic drug DOX was loaded on PPOS after mixing to obtain PPOSD. During the preparation process, the hydrodynamic size of POS NSs (112 nm) increased to 132 nm and the zeta potential (19.5 mV) reversed to -16.6 mV (Figure 1D), which indicated the successful package of the polymer and the adherence of DOX on POS NSs.28 In addition, Fourier transform infrared spectroscopy (FTIR) spectra (Figure S4) and XPS analyze (Figure S5) also illustrated the successful preparation of PPOSD. The thin two-dimensional flake nanostructure of the as-prepared PPOSD was observed by TEM (Figure 1C). The PXRD patterns revealed the retained crystal structure of POS in PPOSD (Figure S1). Furthermore, according to the characteristic optical absorbance of DOX at 480 nm (Figure 1E), the loading capacity of DOX in PPOSD was calculated to be 8.0 wt%. Importantly, PPOSD kept strong absorption in the NIR region (Figure 1E), which indicated its great potential for photothermal conversion. Meanwhile, the POS content in PPOSD was calculated to be 78% by detecting the Sn element content in PPOSD via Inductively Coupled Plasma-Atomic Emission Spectroscopy (ICP-AES). Further studies showed that PPOSD exhibited great dispersibility in water and physiological solutions (Figure S6). Additionally, no obvious changes in hydrodynamic size distribution and polymer dispersity index (PDI) of PPOSD were detected in 7 days (Figure S7), indicating the good stability of PPOSD in stock solution. In Vitro Drug Release, CO Detection, and Photothermal Conversion. Note that the loaded drugs can be released from NSs in response to acidic stimuli.30 The drug release profile of DOX from PPOSD at different pH values was first evaluated. As observed in Figure S8, the cumulative release of DOX from PPOSD under


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physiological pH (pH 7.4) was only 14% after 48 h, while a much more rapid release rate was detected under pH 5.0 (imitating endosome of tumor cells) and the cumulative release ratio was as high as 78% during the same period. The relatively high stability at normal physiological conditions but strong sensitivity to the acidic conditions of PPOSD can avoid the premature release of DOX during internal circulation and significantly improve the drug delivery efficiency of PPOSD. Afterwards, the CO production ability of PPOSD was studied. Myoglobin assay, a commonly used method for CO detection was first conducted.31 As shown in Figure 1F, deoxymyoglobin (deoxy-Mb) co-incubated with PPOSD alone gave only one maximum absorbance at 557 nm. While after 10 min of 561 nm laser illustration, the absorbance of the solution at 557 nm decreased significantly and the absorbance peaks at 540 nm and 577 nm increased obviously, which was owing to the transformation of deoxy-Mb to carbonmonoxy-myoglobin (MbCO) and suggested the successful generation of CO. Apart from this, the CO generation was also investigated by a previously reported fluorescent CO probe, which together with PdCl2 can give fluorescent turn-on signal changes in the presence of CO.32 As displayed in Figure 1G, with the extension of the irradiation time of 561 nm laser, the fluorescence intensity of the solution at 516 nm had a continuous enhancement, which clearly demonstrated the generation of CO (the initial fluorescence signal before 561 nm laser irradiation comes from DOX in PPOSD). Both of the results above demonstrated the successful production of CO by PPOSD under 561 nm laser illustration. Importantly, even after illustrated with 561 nm laser for 30 min, the temperature of PPOSD solution (50 μg/mL) showed limited temperature rise of less than 5 °C (Figure S9), indicating the negligible thermal effect caused by PPOSD during CO generation, which mainly attributed to the relatively low power density (0.5 W/cm2) of the 561 nm laser used.


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Furthermore, PPOSD with strong absorption in the NIR region was expected to serve as an effective PTA and its NIR-induced hyperthermia was then assessed. As shown in Figure 1H, the temperature of the solution increased by 15 °C only after 8 min of 808 nm laser irradiation at a PPOSD concentration of 50 μg/mL, suggesting the relatively high photothermal conversion ability of PPOSD. Besides, the temperature of PPOSD solution exhibited a rapid increase in a concentration-dependent manner upon 808 nm laser illustration, indicating the heat generation can be finely regulated. The photothermal conversion efficiency of PPOSD was determined to be ≈13.8% according to Roper’s method (Figure S10). Moreover, PPOSD presented great photothermal stability in which no significant change in photothermal effects was observed after five NIR laser irradiation cycles (Figure 1I). The effective photothermal convention and high tolerance to the NIR laser irradiation suggested the great potential of PPOSD for serving as a PTA. Of special note, no detectable CO was generated by PPOSD after 808 nm laser irradiation (Figure S11A), which due to the limitation of the band gap of POS.27 Additionally, controllable experiment that keeping PPOSD at 60 °C in dark for 1 h also showed no CO was produced (Figure S11B), thus excluding the possibility of heat to catalyze PPOSD to generate CO. Intracellular CO Generation and Chemotherapy Sensitization. In view of the excellent CO generation ability of PPOSD in vitro, the intracellular CO production ability of PPOSD was then investigated. Before then, the cell selectivity of PPOSD was studied. As shown in Figure S12, after 4 h co-incubation with PPOSD, integrin αvβ3 positive murine mammary carcinoma (4T1) cells showed a much higher PPOSD uptake than αvβ3 negative human breast adenocarcinoma (MCF-7) cells. The effective recognition between the cRGD sequence in PPOSD and integrin αvβ3 overexpressed on 4T1 tumor cells enabled the specific tumor targeting of PPOSD.33 Afterwards, the


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same fluorescent CO probe used in vitro was utilized for intracellular CO detection. As displayed in Figure 2A, 4T1 cells co-incubated with PPOSD alone exhibited negligible fluorescent signal of CO, while strong green fluorescence of CO signal was observed in cells treated with PPOSD after 561 nm laser irradiation, which directly proved the CO production of PPOSD in living cells. Previous reports showed that mitochondria are the cellular target of CO and the mitochondria activity of cancer cells could be rapidly enhanced by CO.23,34 To verify the CO produced by PPOSD via CO2 photoreduction has the same effect, the reactive oxygen species (ROS) level and mitochondria activity of 4T1 cells were first detected. 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) was utilized for the intracellular ROS detection.35 As observed in Figure 2A, cells treated with PPOSD or PPOS alone showed weak fluorescence signal of ROS. However, apparent green fluorescence was observed in cells treated with PPOSD and PPOS NSs after 561 nm laser irradiation, indicating the effective ROS generation caused by CO in 4T1 cells. Meanwhile, the mitochondrial stress induced by CO was evidenced by MitoTracker Red CMXRos (MITO-RED) staining, in which much less red fluorescent signal of oxidized state of mitochondria was observed.36 Then whether the presence of CO will enhance the cytotoxicity of DOX from PPOSD was evaluated. Before that, cell viability study showed that whether PPOS, 561 nm laser irradiation, or PPOS + 561 nm laser irradiation would not initiate cell death in 4T1 cells, indicating the low cytotoxicity of PPOS, 561 nm laser, and CO (Figure S13). For 4T1 cells treated with PPOSD, detectable cytotoxicity was found and approximate 40% cells were killed when the PPOSD concentration was up to 100 μg/mL. Importantly, when the 561 nm laser irradiation was added, the survival rate of the cells had a dramatic decrease and only less than 35% cancer cells were still alive at the same PPOSD concentration, which


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suggested the generated CO could significantly sensitize tumor cells towards chemotherapy. Additionally, stronger cleavage of caspase-3 and lower expression of growth arrest and DNA-damage-inducible protein (Gadd45α) proved that the presence of CO induced more apoptosis and inhibited DNA repair of tumor cells after chemotherapeutic drugs treatment (Figure 2C, D), which was consistent with the cytotoxicity results. Moreover, compared with PPOSD alone, PPOSD + 561 nm laser irradiation resulted in higher DNA damage in tumor cells as evidenced by comet assay (Figure 2E), which further suggested the chemosensitization effect of CO. Importantly, note that the POS content in PPOSD acted as both drug carrier and CO generator, such design can effectively ensure the chemotherapeutic drug and chemosensitizer accumulate in same tumor cells, thus resulting in highly efficient chemosensitization. Intracellular Photothermal Effect and Anti-Inflammation Behavior of PPOSD. Motivated by the good photothermal conversion performance of PPOSD in vitro, the NIR laser-triggered cell ablation by PPOSD was then examined. As shown in Figure 3A, PPOSD + 808 nm laser irradiation exhibited obvious higher tumor cell inhibition rate than PPOSD alone (Figure 2B). Besides, PPOSD + 561 nm + 808 nm laser irradiation showed the best tumor cell suppression performance and only less than 15% cells were still alive after the treatment at a PPOSD concentration of 100 μg/mL. Considering the negligible cytotoxicity of the lasers irradiation (Figure S14), the better tumor cell inhibition effect of PPOSD + 561 nm + 808 nm laser compared with PPOSD + 808 nm laser alone was mainly attributed to the chemosensitization effect induced by CO. Apart from this, the live/dead cell staining results also confirmed the photothermal effect of PPOSD and the enhanced chemotherapeutic effect caused by CO (Figure 3B). Apart from chemosensitization, the CO produced by PPOSD was also expected to weaken the inflammatory reaction caused by PTT. To verify our conjecture, the anti-


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inflammation effect of CO was evaluated on RAW 264.7 macrophages. Proinflammatory cytokines, such as necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1beta (IL-1β) were chosen as inflammatory markers for the evaluation. 561 nm, 808 nm, or 561 nm + 808 nm laser irradiation were harmless and proved to be unable to induce proinflammatory responses (Figure S15). Moreover, compared with PBS or PPOSD alone, PPOSD + 808 nm laser irradiation induced an obvious increase in the level of TNF-α, IL-6, and IL-1β at 24 h after the treatment, which indicated the pro-inflammatory response caused by PTT. Excitingly, the secretion of the proinflammatory cytokines was obviously suppressed in the presence of CO (PPOSD + 561 nm +808 nm laser irradiation), confirming the successful anti-inflammation ability of CO produced by PPOSD (Figure 3C-E). In Vivo Tumor Targeting, CO Detection, and Thermal Imaging. To evidence the feasibility of PPOSD in vivo, we then evaluated the performance of PPOSD on a 4T1 tumor-bearing mice model. The biodistribution of PPOSD in vivo was first investigated using a small animal imaging system and Cy5.5-modified PPOSD was constructed for in vivo drug tracing. As shown in Figure 4A, after intravenously (i.v.) injected, obvious fluorescence signal of PPOSD in tumor site was detected 2 h later and the fluorescence intensity reached the highest point about 4 h post injection (Figure 4C), indicating the rapid enrichment of PPOSD in tumor area, which mainly attributed to the introduction of cRGD sequence in PPOSD. Besides, the fluorescence signal was still obvious even up to 24 h, suggesting the relative long retention time of PPOSD in tumor site. Furthermore, the high content of Sn element in tumor tissue detected by ICP-AES after 24 h post injection also demonstrated the selective tumor accumulation of PPOSD (Figure 4E). Additionally, pharmacokinetic study showed that PPOSD reduced gradually over time and maintained higher levels than free DOX (Figure S16). The long


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blood retention time was attributed to the introduction of PEG segment and the negatively charged surface of PPOSD (Figure 1D), which also favored the selective tumor accumulation of PPOSD after long term blood circulation.37,38 Afterwards, the CO generation in tumor tissues was evaluated by collecting the tumors from mice after different treatments and detecting the CO content by using a carbon monoxide assay kit. As shown in Figure S17, the treatment of PPOSD or laser irradiation alone induced no significant change of CO content in tumor tissues. Moreover, PPOSD + 808 nm laser irradiation also showed negligible effect on the CO content in tumor tissues. However, for mice treated with PPOSD + 561 nm and PPOSD + 561 nm + 808 nm laser irradiation, the CO contents in tumor tissues were nearly 3 times as much as the control group. The notable increase of CO content in tumor tissues demonstrated the successful production of CO by PPOSD under 561 nm laser irradiation. Encouraged by the selective tumor accumulation of PPOSD and its good photothermal conversion ability in vitro, the in vivo photothermal activity of PPOSD was then evaluated. As displayed in thermal images and time-temperature curves (Figure 4F,G), after irradiated by 808 nm laser, the tumor temperature of mice treated with PPOSD increased rapidly and raised from 34 °C to 44 °C only in 5 min, which was high enough to kill tumor cells. However, for mice treated with PBS, the local temperature of tumors showed limited increase which only less than 4 °C with a plateau temperature of 37 °C during the same irradiation period. In addition, mice treated with PPOSD followed by 561 nm laser irradiation also showed limited temperature rise in tumor region (Figure S18), demonstrating the ignorable thermal effect during CO generation. These inspiring results indicated the effective tumor accumulation and local hyperthermia generation of PPOSD in vivo.


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Antitumor and Anti-inflammation Studies. Motivated by the excellent performance of PPOSD above, we further investigated the feasibility of PPOSD for synergistic tumor therapy and anti-inflammation in vivo. 4T1 tumor-bearing mice were i.v. injected with various samples and laser irradiations were given at 4 h post injection. The tumor volume and body weight of mice were measured every other day to evaluate the therapeutic effect and the systemic toxicity. As observed in Figure 5A, mice treated with PPOS alone showed no significant effect on tumor volume, indicating that PPOS was harmless in vivo. PPOS + 561 nm laser irradiation showed little tumor inhibition effect, which demonstrated the limited antitumor ability of CO alone (Figure S19A). Mice treated with PPOSD showed tumor inhibition to some extent. However, the tumor suppression effect was significantly enhanced for PPOSD after 561 nm laser irradiation (PPOSD + 561 nm), which was mainly owing to the chemosensitization effect by CO. Moreover, PPOSD +808 nm laser irradiation also exhibited much higher tumor suppression effect when compared with PPOSD alone, indicating the good synergistic effect of PTT and chemotherapy. It was worth mentioning that PPOSD + 561 nm + 808 nm laser irradiation showed the best tumor inhibition performance and the inhibition rate was as high as 95% on the 15th day. Moreover, no significant difference of the body weight from different groups was observed (Figure 5B), which indicated the negligible side effects caused by the treatments. Besides, the tumor weights of mice on the 15th day further confirmed the highest tumor inhibition effect of PPOSD +561 nm + 808 nm laser irradiation in vivo (Figure 5C). Additionally, hematoxylin-eosin (H&E) and TdT-mediated dUTP Nick-End Labeling (TUNEL) staining results of tumor tissues also proved the best tumor suppression effect of PPOSD + 561 nm + 808 nm group, in which the treatment of PPOSD + 561 nm + 808 nm led to the highest apoptosis of tumor cells among the other groups (Figure 5D).


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Afterwards, the in vivo anti-inflammatory effect of the treatments was also investigated. The cytokine levels in sera of mice with different treatments were first examined. As displayed in Figure S19D and Figure 5E-G, the laser irradiation alone didn’t affect the cytokine levels in sera of mice, while PPOSD + 808 nm laser irradiation led to a significant increase in the level of TNF-α, IL-6, and IL-1β in mice blood at 24 h after the treatment, indicating the proinflammatory responses caused by PTT. However, for mice treated with PPOSD + 561 nm + 808 nm laser irradiation, the secretion of TNF-α, IL-6, and IL-1β was effectively inhibited and the proinflammatory cytokines in sera remained about the same level as those of PBS group, which demonstrated the outstanding anti-inflammatory effect of CO produced by PPOSD. Apart from this, the tumor tissues of mice at 24 h after various treatments were collected for further examination. As shown in Figure S20, compared with PPOSD + 808 nm, PPOSD + 561 nm + 808 nm induced much lower expression of TNF-α, IL-6, and IL1β in tumor tissues. Moreover, the CO generated by PPOSD also depressed neutrophil infiltration which plays a key role in inflammation and is a major cause of tissue damage in tumor tissues caused by PTT,39 further proved the anti-inflammation effect of CO produced by PPOSD. Finally, the major organs (hearts, livers, spleens, lungs, and kidneys) of mice were collected on the 15th day for H&E staining (Figure S21). No obvious physiological damage was observed, implying the negligible side effects of the treatments. Besides, the blood biochemistry tests of mice were normal on the 15th day (Figure S22), further suggested the low systemic toxicity of PPOSD involved treatments.

CONCLUSIONS In summary, a versatile CO generator, PP OSD (polymer@POS@DOX) was


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constructed for enhanced tumor therapy and anti-inflammation. After i.v. injected, PPOSD could rapidly accumulate in tumor tissue via active tumor targeting by the cRGD sequence in PPOSD. Moreover, CO could be produced by PPOSD through CO2 photoreduction under 561 nm laser illustration to enhance the chemotherapeutic effect of DOX. Meanwhile, the POS moiety in PPOSD could also serve as a PTA for PTT upon 808 nm laser irradiation. Subsequently, the generated CO could also decrease the inflammatory reaction caused by PTT. In vivo studies indicated the excellent tumor suppression performance by the use of PPOSD. Moreover, the treatment of PPOSD showed high biosafety which was evidenced by H&E staining of major organs and blood test. We believe such a versatile CO nanogenerator could be a powerful weapon against tumor and inflammation.

MATERIALS AND METHODS Materials. Sodium dodecyl benzenesulfonate (SDBS), SnCl4·5H2O, and L-cysteine (Cys) were purchased from Aladdin Reagent (Shanghai, China). Thiol-PEG-cRGD (Mw: 2000) was provided by Xi’an Ruixi Biological Technology Co., Ltd. (China). Thiol-PEG-Cy5.5 (Mw: 5000) was purchased from Shanghai ToYongBio Tech.Inc (China). Horse skeletal myoglobin was purchased from J&K SCIENTIFIC Ltd. (China). DCFH-DA was provided by Beyotime (Shanghai, China). Mitotracker Red CMXRos (MITO-RED) was provided by Thermo Fisher (Shanghai, China). 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) was purchased from Invitrogen (USA). DNA damage assay kit and endogenous carbon monoxide assay kit were provided by Nanjing Jiancheng Bioengineering Institute (China). Calcein-AM, and propidium iodide (PI) were obtained from 4A Biotech Co., Ltd. (Beijing, China). All other chemical reagents were analytical grade and used without purification.


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Preparation of POS Atomic Layers. POS atomic layers were first prepared according to the literature report. Briefly, SDBS (1.2 mmol), SnCl4·5H2O (0.5 mmol), and Cys (4 mmol) were dissolved in 60 mL mixed solvent of DI water and ethylene glycol (v/v = 1:1). Then the mixed solution was moved to a Teflon-lined autoclave and maintained at 160 °C for 12 h. The product, POS atomic layers were obtained by centrifugation, washed with DI water and ethanol, and freeze-drying. Preparation of POS NSs. Briefly, the as-made POS atomic layers (10 mg) were dispersed in 30 mL DI water under untrasonication for 4 h in ice-bath. Then the resultant solution was centrifuged at a speed of 3000 rpm to remove the large particles, the suspension that containing POS NSs was stored at 4 °C for further modification and characterization. Preparation of PPOSD. For PPOSD NSs preparation, 2 mg of SH-PEG-cRGD was added to 10 mL of POS NSs (1 mg/mL) aqueous solution. After untrasonication for 10 min, the mixture was stirred at room temperature for 8 h, centrifuged and washed with DI water for 3 times to obtain PPOS NSs. To prepare PPOSD, 5 mL of PPOS (0.2 mg/mL) in PBS was mixed with 1 mL of DOX (0.2 mg/mL) in PBS. Then the mixture was stirred at room temperature overnight. Excess DOX was removed by centrifugation and repeated washed with PBS. The resulting PPOSD was then stored at 4 °C for further use. To prepare Cy5.5-modified PPOSD NSs, SH-PEG-Cy5.5 was mixed with PPOSD (w/w = 1/40) in PBS for 8 h. Then the excess SH-PEG-Cy5.5 was removed by centrifugation and repeated washed with PBS to obtain Cy5.5-modified PPOSD NSs. Myoglobin Assay. A myoglobin solution (0.5 mg/mL) in PBS buffer was degassed by bubbling with N2 and followed by the addition of sodium dithionite (0.1%), resulting in a solution of deoxy-Mb (27 μM). Then 50 μg of PPOSD was added and the mixture


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was irradiated with a 561 nm laser (0.5 W/cm2). UV-vis spectra of the sample were studied. In Vitro CO Detection. 1.0 mL of aqueous solution that containing PPOSD (50 μg), CO probe (5 μM), and PdCl2 (5 μM) was prepared in a quartz cuvette. The fluorescent spectra of the mixture were recorded before and after minutes of 561 nm laser irradiation (0.5 W/cm2). The excitation wavelength for the CO probe was set at 490 nm. In Vitro Photothermal Effect of PPOSD. PPOSD with different concentrations in PBS were irradiated by an 808 nm laser (1 W/cm2). The temperature of the solution was recorded by an IR camera. For photothermal stability test, 1 mL of PPOSD (50 μg/mL) in DI water was irradiated by 808 nm laser (1 W/cm2) for 8 min and then the solution was naturally cooled to RT. The heating and cooling process was repeated for 5 times. Cell Culture. 4T1 cells, MCF-7 cells, and Raw 264.7 macrophages were incubated in DMEM medium containing 10% FBS and 1% antibiotics (penicillin-streptomycin, 10000 U/mL), and cultured in a humidified atmosphere with 5% CO2 at 37 °C. Intracellular CO Detection. 4T1 cells were co-incubated with PPOS or PPOSD (50 μg/mL) for 4 h. Then the culture medium was replaced by fresh medium and cells were irradiated by 561 nm laser (0.5 W/cm2) for 10 min. After that, cells were incubated with fresh medium that containing CO probe (1 μM) and PdCl2 (1 μM) for 30 min at 37 °C, washed with PBS, and imaged by CLSM. Intracellular ROS Detection. 4T1 cells were co-incubated with PPOS or PPOSD (50 μg/mL) for 4 h. Then cells were co-incubated with fresh culture medium that containing DCFH-DA (10 μM) at 37 °C for 20 min, followed by 561 nm laser irradiation (0.5 W/cm2) for 10 min and CLSM imaging. Cytotoxicity Assay, Western Blot, and Comet Assay. 4T1 cells were incubated with


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PPOSD or PPOS with different concentrations for 4 h. After the culture medium was replaced, cells were given 561 nm (0.5 W/cm2, 10 min) and/or 808 nm (1 W/cm2, 10 min) laser irradiation. After incubated at 37 °C for another 24 h, cells were tested by classical MTT assay to assess the cell viability. For western blot and comet assay, cells were treated the similar way as cytotoxicity assay. The difference was that cells were collected after 24 h incubation for protein expression analyze and DNA breaks detection. Cell Cytokines Detection. 4T1 cells were incubated with PPOS or PPOSD (50 μg/mL) for 4 h. After the culture medium was replaced, cells were given 561 nm (0.5 W/cm2, 10 min) laser irradiation. Then the supernatants of 4T1 cells were transferred to the sixwell plate used to culture RAW 264.7 macrophages, and RAW 264.7 macrophages were incubated overnight. Finally, the supernatants of RAW 264.7 macrophages were collected for the measurement of proinflammatory cytokines by ELISA. Animal Model. Female BALB/c mice (about 5 weeks old) were purchased from Wuhan University Animal Biosafety Level III Lab and used under protocols approved by the animal experiment center of Wuhan University. For 4T1 tumor model building, 4T1 cells (1 × 106) in 100 µL PBS were subcutaneously injected to the right back of hind leg of each mouse. In Vivo Tumor Imaging and Biodistribution. When the tumor volume reached about 150 mm3, mice were i.v. injected with 100 µL of Cy5.5-modified PPOSD (2.5 mg/mL) in PBS. Then mice were imaged by IVIS Spectrum (Perkin-Elmer) at preset time points of 0, 2, 4, 8, 12, 24 h post injection. Mice were sacrificed 24 h post injection and major organs (heart, liver, spleen, lung, and kidney) together with tumor tissue were collected for ex vivo imaging to study the biodistribution of the sample. Besides, the tumor tissue and major organs were then harvested, weighed, and stored at -80 °C before ICP-AES


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analysis of Sn element for the biodistribution evaluation of PPOSD. In Vivo Photothermal Imaging. 4T1 tumor-bearing mice were i.v. injected with 100 μL of PBS and PPOSD (6 mg/mL). After 12 h post injection, 808 nm laser irradiation (1 W/cm2) was given at the tumor tissue and the temperature was quantified by an IR camera. Detection of CO in Tumor Tissues. 4T1 tumor-bearing mice were i.v. injected with 100 μL of PBS or PPOSD (6 mg/mL). Then 561 nm laser irradiation (0.5 W/cm2) was given at the tumor tissue for 20 min. Subsequently, mice were sacrificed and the tumor tissues were quickly collected for CO detection by endogenous carbon monoxide assay kit (A101-2). Antitumor Studies. When the tumors reached about 100 mm3, mice were randomly divided into different groups (5 mice in each group) and i.v. injected with PBS, PPOS or PPOSD (100 μL, 6 mg/mL) at the 1st day. Then mice were irradiated with 561 nm (0.5 W/cm2, 20 min) and/or 808 nm (1 W/cm2, 5 min) laser 12 h post injection. The tumor sizes and body weights of mice were measured every other day. The tumor volumes were calculated as: V = (the shortest tumor diameter)2 × (the longest tumor diameter)/2 and the relative tumor volume was defined as V/V0 (V0 is the tumor volume of mice at the 1st day before any treatment). The relative body weight was defined as W/W0 (W0 is the body weight of mice at the 1st day before any treatment). Detection of Cytokines in Sera. Mice were treated the same way as antitumor studies. Of special, serum samples of mice were collected 24 h post irradiation for proinflammatory cytokines analysis by ELISA. Detection of Cytokines and Neutrophil Filtration in Tumor. Mice after various treatments for 24 h were sacrificed and the tumors were collected. The expression of TNF-α, IL-6, IL-1β, and Lymphocyte antigen 6G (Ly6G, a marker for neutrophils) were


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examined by immunofluorescent staining. Biosafety Assessment. Blood biochemistry analysis was performed by collecting the blood samples of mice at the 15th day after the last time of tumor volume and body weight measurement. Then mice were sacrificed and the major organs were collected for H&E staining. Statistical Analysis. Statistical analysis was carried out using SPSS 13.0 software and all data were presented as means ± standard deviation (SD). The statistical significance was obtained through two-tailed Student’s t-test. The differences were considered to be statistically significant for a p value < 0.05 (*p < 0.05, **p < 0.01, ***p < 0.001).

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.XXXXXX. PXRD patterns of various samples; Raman spectrum; S 2p XPS spectrum; FTIR spectra; XPS analysis; pictures of PPOSD solutions; size distribution and zeta potential; drug release profile; temperature changes of PPOSD under 561 nm laser irradiation; heating/cooling experiment data; in vitro CO detection; cellular selectivity; cytotoxicity of PPOS and lasers irradiation; changes of TNF-α, IL-6, and IL-1β levels of RAW 264.7 macrophages; pharmacrokinetic data; CO detection in tumor tissues; temperature change of tumors under 561 nm laser irradiation; tumor inhibition and anti-inflammation data of PPOS and PPOS + 561 nm groups; immunostaining for TNF-α, IL-6, IL-1β, and Lymphocyte antigen 6G in tumor tissues; H&E staining of major organs; blood biochemistry analysis (PDF)


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AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] ORCID Xian-Zheng Zhang: 0000-0001-6242-6005 Author Contributions S.B.W. and C.Z. contributed equally.

ACKNOWLEDGMENTS The CO probe was kindly provided by Dr. Shu-Min Feng (College of Chemistry, Central China Normal University, Wuhan, China). This work was supported by the National Natural Science Foundation of China (51703168, 51833007 and 21674084).


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Scheme 1. Schematic illustration of PPOSD for enhanced tumor inhibition and antiinflammation: (A) cRGD-mediated specific tumor targeting of PPOSD; (B) CO generation via CO2 photoreduction by the POS moiety in PPOSD upon 561 nm laser irradiation; (C) CO induced mitochondrial collapse, DNA repair inhibition, and chemosensitization; (D) photothermal therapy (PTT) of tumor by the POS in PPOSD upon 808 nm laser irradiation; (E) inhibition of PTT-induced inflammatory reaction by the CO generated above.


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Figure 1. A) TEM and SEM (inset) images of POS atomic layers. TEM and HRTEM (inset) images of B) POS NSs and C) PPOSD. D) Hydrodynamic size and zeta potential of POS NSs, PPOS and PPOSD. E) UV-vis-NIR absorption spectra of POS NSs, DOX, and PPOSD in water. F) Myoglobin assay for the detection of CO generation by PPOSD upon 561 nm laser irradiation (0.5 W/cm2, 10 min). G) Fluorescence spectra changes of PPOSD solution together with CO probe and PdCl2 upon 561 nm laser irradiation. The data was recorded every 5 min. H) Heating curves of PPOSD at different concentrations upon 808 nm laser irradiation (1 W/cm2). Inset: thermal images of samples after 5 min of 808 nm laser irradiation. I) Photothermal conversion cycling test of PPOSD (50 μg/mL) under 808 nm laser irradiation (1 W/cm2).


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Figure 2. A) CO probe, DCFH-DA, and Mitotracker Red CMXRos (MITO-RED) staining images of 4T1 cells after various treatments. The scale bars were all 50 μm. B) Cell viability of 4T1 cells treated with various samples with/without 561 nm laser irradiation (0.5 W/cm2, 10 min). C) Evaluation of caspase-3, cleaved caspase-3, and Gadd45α expression in 4T1 cells after various treatments. D) Corresponding results of C). E) Comet assay results of 4T1 cells after various treatments.


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Figure 3. A) Cell viability of PPOSD treated 4T1 cells with 808 nm or 561 nm + 808 nm laser irradiation (808 nm laser: 1 W/cm2, 10 min; 561 nm laser: 0.5 W/cm2, 10 min). B) Live/dead staining images of 4T1 cells after various treatments. Viable cells were stained green with calcein-AM, and dead/late apoptosis cells were stained red with propidium iodide. The scale bar was 100 μm. C) TNF-α, D) IL-6, and E) IL-1β levels of RAW 264.7 macrophages after treatment with media of different 4T1 cell samples, n = 3, *p < 0.05, **p < 0.01 compared to control group.


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Figure 4. A) In vivo fluorescence imaging of Cy5.5-modified PPOSD at various time points after intravenous injection; B) Ex vivo fluorescence images of Cy5.5-modified PPOSD in tumor and major organs 24 h post injection. C) Quantification of the fluorescence intensity at different points. D) Quantification of fluorescence intensity of tumor tissue and major organs. E) Quantitative analysis of Sn element by ICP-AES in tumor and major organs at 24 h post injection. F) In vivo infrared thermal images and G) temperature increase curves of 4T1 tumors after the injection of PBS and PPOSD upon 808 nm laser irradiation (1 W/cm2).


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Figure 5. A) Relative tumor volume and B) relative body weight curves of different groups of mice after various treatments, n = 5. C) Average tumor weights of different groups of mice sacrificed on the 15th day. D) H&E (upper) and TUNEL (lower) staining pictures of tumors from different groups of mice after different treatments. The scale bar was 50 μm. E) TNF-α, F) IL-6, and G) IL-1β levels in sera of mice at 24 h after the treatments, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001 compared to PBS group.


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TOC graphic


A Versatile Carbon Monoxide Nanogenerator for Enhanced Tumor

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