Recyclable Endogenous H2S Activation of Self‐Assembled Nanoprobe with Controllable Biodegradation for Synergistically Enhanced Colon Cancer‐Specific Therapy

Abstract Excessive production of hydrogen sulfide (H2S) plays a crucial role in the progress of colon cancer. Construction of tumor‐specific H2S‐activated smart nanoplatform with controllable biodegradation is of great significance for precise and sustainable treatment of colon cancer. Herein, an endogenous H2S triggered Co‐doped polyoxometalate (POM‐Co) cluster with self‐adjustable size, controlled biodegradation, and sustainable cyclic depletion of H2S/glutathione (GSH) is designed for synergistic enhanced tumor‐specific photothermal and chemodynamic therapy. The designed POM‐Co nanocluster holds H2S responsive “turn‐on” photothermal property in colon cancer via self‐assembling to form large‐sized POM‐CoS, enhancing the accumulation at tumor sites. Furthermore, the formed POM‐CoS can gradually biodegrade, resulting in release of Co2+ and Mo6+ for Co(II)‐catalyzed •OH production and Russell mechanism‐enabled 1O2 generation with GSH consumption, respectively. More importantly, the degraded POM‐CoS is reactivated by endogenous H2S for recyclable and sustainable consumption of H2S and GSH, resulting in tumor‐specific photothermal/chemodynamic continuous therapy. Therefore, this study provides an opportunity of designing tumor microenvironment‐driven nanoprobes with controllable biodegradation for precise and sustainable anti‐tumor therapy.


Synthesis of POM-Co cluster
The POM cluster was simply synthesized through the one-pot method modified as previous report. S1 At first, (NH 4 )6Mo 7 O 24 ·4H 2 O (0.4414 g) was dissolved in 10 mL ultrapure water and stirred for 0.5 h at room temperature. Subsequently, 5 mL solution of CoCl 2 ·6H 2 O (35 mg/mL) was added and stirred for another 0.5 h. At last, 2 mL solution of L-ascorbic acid (100 mg/mL) was added dropwisely into the system under stirring. After stirring at room temperature for 2 h, 80 mL ethanol was added to precipitate. Finally, the samples were centrifuged and washed with water and ethanol for three times.

Characterization
The morphology, structure and elemental compositions of the samples were characterized by transmission electron microscopy (TEM, FEI Tecnai F20) operated at 200 kV. The elements in the sample were further measured by the energy dispersive X-ray spectroscopy (EDS) and X-ray photoelectron spectroscopy (XPS, Thermo Fisher Scientific Escalab 250Xi).
The UV-Vis adsorption spectra were recorded using the absorption spectrometer (Lambda750, Perkin-Elmer) at room temperature. The thermal imaging was recorded by Fluke Ti95 infrared camera.

3
The mixed solution of different concentrations of 25,50,100 and 200 μg/mL) and Na 2 S, and the mixed solution of different concentrations of Na 2 S (0, 0.5, 1, 2 and 4 mM) and POM-Co were irradiated with an 808 nm laser (1 W/cm 2 ). Thermal infrared camera (Ti95, Fluke, USA) was used to record the temperature changes and real-time thermal images at the designed time intervals. The photothermal stability test was conducted by irradiating POM-CoS aqueous solution with 808 nm laser and then cooling it for three times. The photothermal conversion efficiency (η) of POM-CoS was calculated by the previous method. S2,S3 The assay of •OH production MB (10 μg/mL), POM-CoS (100 μg/mL) and H 2 O 2 with different concentrations were mixed and reacted for 10 min. MB degradation is detected with characteristic absorption at 665 nm. POM-Co was added in the solution containing OPD (1 mM) and H 2 O 2 (10 mM). The •OH generation was monitored by the absorbance change at 452 nm. S1 For electron spin resonance (ESR) assay, POM-CoS, H 2 O 2 and DMPO were mixed and reacted for 10 min.
EPR spectra were recorded.

The assay of GSH consumption and 1 O 2 production
GSH (1 mM) was mixed with POM-CoS (12.5, 25, 50 and 100 μg/mL) for 2 h, and DTNB (0.5 mM) was added. The degradation of GSH was monitored by the absorbance change at 412 nm. The mixture of GSH (10 mM) and POM-CoS (100 μg/mL) was reacted for 12 h. DPBF (20 μg/mL) and 200 μL H 2 O 2 (10 mM) were added into 400 μL of the mixture. The absorbance at 419 nm was monitored by absorption spectrometer. For ESR assay, the trapping agent TEMP of 1 O 2 was added for 10 min, and then the EPR spectrum was recorded.

In vitro cell experiment
The biocompatibility of POM-Co was evaluated by standard CCK8 assay. CT26 cells were inoculated in 96-well plates for 24 h, and then incubated with different concentrations of POM-Co for 24 h. Finally, the cells were washed with PBS and added with culture medium 4 and CCK8. The cells were further cultured at 37 ℃ for 2 h. The cytotoxicity was assessed by testing the absorbance at 450 nm via CCK8 assay.

Tumor models
8×10 6 CT26 cancer cells were subcutaneously injected into BALB/C mice, after further cultured about four weeks, the tumor-bearing mouse models were obtained for in vivo synergistic antitumor therapy experiments.

In vivo pharmacokinetic and biodistribution assays
For blood circulation assay, 15 μL blood sample was collected at different time points (5 min, 30 min, 1 h, 2 h, 4 h, 8 h, 24 h) after intravenous injection. And the Co content in blood was determined by inductively coupled plasma mass spectrometry (ICP-MS). Similarly, major organs (heart, liver, spleen, lung and kidney) and tumor (n=3) were harvested at time points of 12 h, 24 h, 48 h and 7 day for biodistribution assessment.

In vivo synergistic antitumor therapy
The tumor-bearing BALB/C mice (CT26 cancer cells) were randomly divided into four groups (n=5 per group): Group Ⅰ-only PBS group; Group Ⅱ-solo 808 nm laser irradiation; Group Ⅲ-POM-Co (150 μL, 2 mg/mL, single CDT group); Group Ⅳ-POM-Co solution (150 μL, 2 mg/mL) and 808 nm laser irradiation (PTT + CDT group). Mice from Group Ⅱ and Group Ⅳ were irradiated with 808 nm laser at a power density of 1.5 W/cm 2 for 5 min at 24 h injection. The temperature change during the treatment was monitored by using a thermal camera. The tumor sites were irradiated 5 min by using the 808 nm laser every two days. The mice weights and tumor sizes were measured every 2 days.

Tumor biopsies
For hematoxylin and eosin (H&E) and Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, the tumors of CT26 tumor-bearing mice after different treatments were dissected. And then the tumor slices were stained with H&E and TUNEL for histological test. 5

Blood biochemical analysis.
For in vivo safety evaluation, the Kunming mice were injected with POM nanoprobes (3 mg/mL) for 14 days and control group without treatment. The blood samples were then collected for further biochemistry tests. And the biomarkers of alanine transferase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN) and creatinine were measured.

Histopathological examination
To evaluate the in vivo toxicity, the main organs including heart, liver, spleen, lung and kidney from untreated and treated normal mice (intravenous injection of POM-Co solution) for 7, 15 and 30 days were also collected for H&E staining.

Statistical analysis
The results of statistical analysis were presented as mean ± SD. Statistical significance was calculated by one-way ANOVA analysis. The statistical significance was defined as *p < 0.05; **p < 0.01; ***p < 0.001.                 The half-time (t 1/2 ) was calculated to be ~1.63 h. Figure S18. H&E stained main tissues including heart, liver, spleen, lung and kidney collected from normal mice treated without or with POM-Co clusters for 7, 15 and 30 day.