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Spatiotemporally controlled O2 and singlet oxygen self-sufficient nanophotosensitizers enable the in vivo high-yield synthesis of drugs and efficient hypoxic tumor therapy†

Carrying out the in vivo syntheses of drugs toxic to tumors based on the specific features of the tumor microenvironment is critical for ensuring specific antitumor efficacy. However, achieving in situ high-yield synthetic toxic drugs from non-toxic agents and reducing their drug resistance in hypoxic tumors remain challenges. Herein we created a tumor-microenvironment-responsive porous Pt/Pt(iv) methylene blue coordination polymer nanoshuttle (Pt/PtMBCPNS) photosensitizer with spatiotemporally controlled O2 and singlet oxygen (1O2) self-sufficient for the in vivo high-yield synthesis of drugs and efficient hypoxic tumor therapy. After being endocytosed, the nanophotosensitizer as a cascade catalyst was observed to effectively catalyze the conversion of endogenous H2O2 to O2, and was hence found to play a dual role in the enhanced tumor therapy. PtMBCPNSs, upon being irradiated with red light, efficiently converted O2 into 1O2. Subsequently, 1O2 oxidized non-toxic 1,5-dihydroxynaphthalene to form the anticancer agent juglone with a high yield. In addition, O2 was found to be able to improve the hypoxic microenvironment without light irradiation, thus enhancing the antitumor efficacy of the produced drugs and reducing drug resistance. As a result, by enhancing the synergistic effect of the treatment, this nanophotosensitizer significantly inhibited the growth of tumors and avoided damage to normal tissues/organs. Collectively, this work highlights a robust nanoplatform with the spatiotemporally controlled in vivo high-yield synthesis of drugs and generation of O2 to help overcome the current limitations of chemical-based therapies against hypoxic tumors.

UV−vis absorbance measurements were recorded on Shimadzu UV-1750. X-ray photoelectron spectroscopy (XPS) measurements were performed on a PHI-5702 multifunctional spectrometer using Al Kα radiation. The XPS profiles were fitted by the Gaussian-Lorentzian method after background subtraction using Shirley's method. 1 H NMR spectra were recorded on a JNM-ECS400 spectrometer. Oxygen production was measured with an oxygen probe (JPBJ-609L Portable Dissolved Oxygen Meter). Photosynthesis was performed using a xenon lamp (HSX-F/UV 300), equipped with 400−780 nm filters. An optical fiber-coupled 660 nm laser (MIL-N-660-5W) was purchased from Changchun New Industries Optoelectronics Tech Co. Ltd. (CNI). Pt content was determined by inductively coupled plasma atomic emission spectrometry (ICP-AES, Varian VISTA-MPX).

Synthesis of PtMBCPNSs
1 mL of 1.0 mM MB aqueous solution, 10 mg of PVP, and 10 mL of 0.4 mM H 2 PtCl 6 aqueous solution were added to the reaction vessel, respectively. The reaction mixture was irradiated by visible light (420 nm < λ < 780 nm filter) for 1 min in a constant temperature water bath at S3 37 o C. The product of Pt/PtMBCPNSs was collected by centrifugation (10000 rpm) and washed with ethanol for several times until the supernatant was colorless.

Statistical method of size distribution
A statistical software (Nano Measurer 1.2) was used to randomly count 100 nanoparticles.
The exported data was then analyzed and plotted in a professional function drawing software (Origin 9) to obtain the size distributions of nanoparticles.

Calculation of photooxidation yield
The consumption of DHN was monitored by the reduced intensity of the absorption peak at 298 nm, and the concentration of DHN was calculated based on its molar extinction coefficient (ε = 7664 M −1 cm −1 ). 1 The generation of juglone was monitored by the increased intensity of the absorption peak at 419 nm. The molar extinction coefficient (ε = 3500 M −1 cm −1 ) 1 was used to calculate the concentration of juglone, and the yield of juglone was obtained by calculating the final amount of juglone and the initial amount of DHN. 1 The structure of the target product was further proved by 1 H NMR.

The GSH-triggered DHN release
DHN@Pt/PtMBCPNSs (2 mg) were packaged into a dialysis bag (MWCO = 3500), and then immersed within 15 mL PBS or aqueous solution containing GSH (10 mM) at 37 °C in a beaker. At different time points, 2.0 mL solution was collected to determine the concentration of DHN using UV−vis spectra, and then 2.0 mL fresh PBS or aqueous solution containing GSH was added to the beaker to make up for the reduced solution volume. Three independent experiments were carried out to minimize the deviations.

Cell culture
Human cervical carcinoma (HeLa) cells were purchased from the cell bank of the Chinese Academy of Sciences (Shanghai, China). HeLa cells were cultured in DMEM with an atmosphere of 21% O 2 and 5% CO 2 at 37 o C to mimic the normoxic environment. Hypoxic condition was achieved by incubating the cells under a hypoxic incubator with an atmosphere of 1% O 2 , 5% CO 2 , and 94% N 2 . Cell viability was detected by a cell counting kit-8 (CCK-8, Beyotime Biotechnology) test following the manufacturer's instructions.

Cell uptake of DHN@Pt/PtMBCPNSs
HeLa cells were inoculated into 12-well plates at a density of 1 × 10 5 cells per well. After incubating for 24 h, 40 µg of DHN@Pt/PtMBCPNSs were added and the cells were incubated for another 30 min, 1 h, and 2 h. Then the cells were washed three times with PBS to remove free nanomaterials. Finally, the cell uptake of nanomaterials was tracked by a confocal imaging system.

Intracellular O 2 detection
RDPP was used as a probe to detect intracellular O 2 levels. HeLa cells were seeded in confocal dishes for 24 h of incubation in hypoxic microenvironment. After 4 h of treatment with 5 µmol L −1 RDPP for 4 h at 37 o C, the cells were incubated with 40 µg mL −1 PtMBCPNSs, Pt/PtMBCPNSs, and DHN@Pt/PtMBCPNSs for 4 h. After washing with PBS for three times, the fluorescence images of cells were obtained at 455 nm excitation using CLSM.

Cytotoxicity assay
The cytotoxicity against HeLa cells was measured by CCK-8 assay. The cells were cultured in 96-well plates and incubated for 24 h at 37 °C under normoxic or hypoxic condition.

In vivo biodistribution and metabolize evaluation
To study the biodistribution and metabolize in vivo of DHN@Pt/PtMBCPNSs, mice were intravenously injected with DHN@Pt/PtMBCPNSs. Samples including blood, urine, and feces were collected at different time post injection. And mice were sacrificed at different time points. Tumors and major organs (heart, liver, spleen, lung, and kidney) were harvested.
Typically, blood, urine, feces, tumors and major organs were dissolved in chloroazotic acid.
The mixtures were heated and the Pt amounts in the above samples were assessed via ICP-AES measurement.

H&E staining
For evaluating the pathological damages to tumors of different treatments, the tumor tissues in each group were retrieved after the 15 days treatment and processed for H&E staining analysis.

Statistical analysis
The data were presented as means ± standard deviation (SD). The statistical significance between two groups was obtained through two-tailed student t-test (*p < 0.05, **p < 0.01, and ***p < 0.001).          To verify the degradation product, the Pt/PtMBCPNSs were immersed in PBS buffer solution (pH 6.0) containing 10 mM GSH for 24 h, and then the degradation product was collected to confirm species and its catalytic performance. As shown in Figure 2a−c, Pt/PtMBCPNSs could undergo remarkable transformation from the original shuttle-like shape to the final spherical shape in the presence of GSH ( Figure  S14a). The TEM image showed that Pt nanoparticles are uniformly distributed in the spherical Pt/PtMBCP nanoparticles (Pt/PtMBCPNPs) ( Figure S14b). As observed from the HRTEM image (the inset in Figure S14b), the lattice spacing of the nanocrystal was 0.22 nm, which matches the spacing of Pt (111) crystal planes. 2 Furthermore, XRD patterns ( Figure S15) showed that the final Pt/PtMBCPNPs have diffraction peaks similar to Pt/PtMBCPNSs. The above results indicated that the species of degradation product is small size Pt/PtMBCPNPs. The formed small size Pt/PtMBCPNPs were further confirmed by XPS. As shown in Figure S16a, the Pt/PtMBCPNPs also existed the Pt 4f peak, which is consistent with Pt/PtMBCPNSs. In addition, for the high resolution of Pt 4f ( Figure S16b), Pt/PtMBCPNPs showed peaks belonging to Pt 4+ species 3 at 72.2 eV and 75.5 eV as well as peaks belonging to Pt 0 species 4 at 70.1 eV and 73.4 eV. Compared with Pt/PtMBCPNSs, peaks belonging to Pt 2+ species 4 at 71.2 and 74.7 eV were only observed in Pt/PtMBCPNPs, indicating that a small amount of Pt 4+ was reduced to Pt 2+ by GSH. N 1s XPS spectrum in Figure  S16c showed that the N 1s moves from 396.9 eV for MB to 397.2 eV for Pt/PtMBCPNPs, indicating that N atom coordinates with Pt 4+ . 5 Similarly, the S 2p XPS spectrum in Figure S16d showed that S 2p moves from 161.4 and 162.9 eV for MB to 161.5 and 163.0 eV for Pt/PtMBCPNPs, revealing the coordination of S atom with Pt 4+ . 6 Together, these results ultimately showed the degradation product is small size Pt/PtMBCPNPs.             As shown in Figure 5a, Pt/PtMBCPNSs and DHN@Pt/PtMBCPNSs could significantly inhibit the growth of tumor. As reported in the literature, Pt(IV) prodrugs could react with GSH in tumor cells to generate active cytotoxic Pt(II). 7−9 Therefore, we hypothesized that the intracellular GSH could reduce Pt(IV) in Pt/PtMBCPNSs to cytotoxic Pt(II), thus inhibiting the growth of tumor. In order to confirm our hypothesis, the valence state of Pt after the incubation of Pt/PtMBCPNSs with GSH was determined by XPS ( Figure S36). The disappearance of the original peaks at 72.2 and 75.5 eV and the appearance of the new peaks at 71.2 and 74.7 eV over time proved the reduction of Pt from the +4 to +2 valence state by GSH. To further confirm this, the cell viability of HeLa cells treated with Pt/PtMBCPNSs and DHN@Pt/PtMBCPNSs was measured by a cell counting kit-8 (CCK-8) assay. For 24 h treatment with Pt/PtMBCPNSs and DHN@Pt/PtMBCPNSs, the cell viability was higher than 80% without laser irradiation under normoxia and hypoxia condition (Figure 4a, b). Interestingly, after 48 h and 72 h incubation, Pt/PtMBCPNSs and DHN@Pt/PtMBCPNSs presented a relatively high cytotoxicity ( Figure S37a, b). Therefore, Pt/PtMBCPNSs itself can significantly inhibit the growth of tumor. As shown in Figure 5b, c, in vivo experiments showed that there is no significant difference between the therapeutic effects of Pt/PtMBCPNSs + Laser and DHN@Pt/PtMBCPNSs + Laser. This was because that the intracellular GSH could reduce Pt(IV) in Pt/PtMBCPNSs to cytotoxic Pt(II). Moreover, after 48 h and 72 h incubation, Pt/PtMBCPNSs + Laser and DHN@Pt/PtMBCPNSs + Laser presented a relatively close cytotoxicity toward HeLa cells ( Figure S38a, b), further indicating that there is no significant difference between the therapeutic effects of Pt/PtMBCPNSs + Laser and DHN@Pt/PtMBCPNSs + Laser.