In-situ oxygen-generation nanoplatform with dual amplication effect for combined chemo-photodynamic therapy


 Background

Photodynamic therapy (PDT) is a promising method for cancer treatment because of its advantages such as easy operation, good targeting, minimal side effects, low systemic toxicity and less invasiveness. However, the hypoxic microenvironment within the tumor significantly inhibited the therapeutic effect of PDT. The development of targeted nanoplatform for regulating hypoxia microenvironment is an important method to give full play to the therapeutic effect of PDT.
Methods

In this study, we designed and prepared a novel chemo-photodynamic therapy nanoplatform, which can continuously catalyze the decomposition of H2O2 in tumors to generate oxygen (O2) to enhance the therapeutic effect of PDT, resulting in DNA damage, while releasing MTH1 inhibitors in tumor cells to inhibit the repair process of DNA damage caused by PDT.
Results

In our work, a simple one-step reduction approach was applied to enable platinum nanoparticles (Pt NPs) growth in situ in the nanochannels of mesoporous silica nanoparticles (MSNs). After physical encapsulation of photosensitizer chlorin e6 (Ce6) and MTH1 inhibitor TH588, the drug loading nanoplatform was modified with an arginine-glycine-aspartic acid (RGD) functionalized liposome shell, resulting in the fabrication of multifunctional nanoplatform MSN-Pt@Ce6/TH588@Liposome-RGD (MPCT@Li-R) with dual amplification effect and achieve the purpose of chemo-photodynamic therapy.
Conclusions

Our study provides a new strategy for PDT to ablation tumor cells by damaging the DNA of tumor nucleus and mitochondria, meanwhile inhibiting the repair process after the damage.


Background
Photodynamic therapy (PDT) is a promising method for cancer treatment because of its advantages such as easy operation, good targeting, minimal side effects, low systemic toxicity and less invasiveness.
However, the hypoxic microenvironment within the tumor signi cantly inhibited the therapeutic effect of PDT. The development of targeted nanoplatform for regulating hypoxia microenvironment is an important method to give full play to the therapeutic effect of PDT.

Methods
In this study, we designed and prepared a novel chemo-photodynamic therapy nanoplatform, which can continuously catalyze the decomposition of H 2 O 2 in tumors to generate oxygen (O 2 ) to enhance the therapeutic effect of PDT, resulting in DNA damage, while releasing MTH1 inhibitors in tumor cells to inhibit the repair process of DNA damage caused by PDT.

Results
In our work, a simple one-step reduction approach was applied to enable platinum nanoparticles (Pt NPs) growth in situ in the nanochannels of mesoporous silica nanoparticles (MSNs). After physical encapsulation of photosensitizer chlorin e6 (Ce6) and MTH1 inhibitor TH588, the drug loading nanoplatform was modi ed with an arginine-glycine-aspartic acid (RGD) functionalized liposome shell, resulting in the fabrication of multifunctional nanoplatform MSN-Pt@Ce6/TH588@Liposome-RGD (MPCT@Li-R) with dual ampli cation effect and achieve the purpose of chemo-photodynamic therapy.

Conclusions
Our study provides a new strategy for PDT to ablation tumor cells by damaging the DNA of tumor nucleus and mitochondria, meanwhile inhibiting the repair process after the damage.

Background
Cancer, as a malignant disease that seriously threatens human health, is a major cause of morbidity and mortality worldwide. Considering the high fatality rate and great harmfulness of cancer, it is particularly urgent to develop more reasonable and effective treatment approaches for cancer [1,2]. As a non-invasive treatment strategy, photodynamic therapy (PDT) can be utilized for the treatment of many types and sites of cancer. The good therapeutic effect and the possibility of combining with other therapeutic methods make it become one of the hot spots in the eld of cancer therapy [3]. The principle of PDT is that the photosensitizer that accumulates in tumor cells can be stimulated by a laser with the appropriate wavelength and produces reactive oxygen species (ROS) in the presence of O 2 [4][5][6][7]. Therefore, PDT is the consequence of the interaction of three essential elements: light, photosensitizer and O 2 . However, the hypoxic microenvironment caused by the uncontrolled proliferation of tumor cells severely limits the conversion e ciency of O 2 to ROS [8][9][10]. Moreover, PDT-induced microvascular collapse further impairs the O 2 supply, leading to a further deterioration of the hypoxic environment. Consequently, the vicious cycle formed by local O 2 consumption and O 2 supply disruption severely limits the e ciency of PDT, leading to incomplete ablation of tumors and metastasis [11,12]. Therefore, it is an urgent task for cancer treatment to develop therapeutic methods to overcome tumor hypoxia and improve PDT e ciency.
At present, various strategies have been developed to alleviate hypoxia in tumors. One approach is to construct NPs with an O 2 donor as the functional center for the targeted delivery of O 2 for PDT therapy.
For example, Tang et al. innovatively utilized red blood cell (RBC) as O 2 carrier to achieve the RBCfacilitated PDT therapy, which effectively solved the problem of tumor hypoxia [13] [17][18][19][20]. However, this method also has problems such as narrow pH response range and unstable catalyst and so on. [21,22].
In recent years, Pt NPs have been regarded as the ideal nanoenzymes for catalyzing H 2 O 2 decomposition due to their antioxidant properties, excellent catalytic e ciency, durable catalytic and good biocompatibility [23,24]. More importantly, Pt NPs has excellent stability and will not be decomposed in the tumor slight acidic and H 2 O 2 micro-environment during the catalytic process like other catalysts (MnO 2 ) [24]. However, Pt NPs are usually small in volume (< 10nm), which makes them easy to be cleared by the urinary system, resulting in a short residence time in the body [25]. MSN has many attractive properties, such as high speci c surface area, adjustable pore size, high porosity, excellent biocompatibility and easy surface modi cation, which make it an ideal nanovehicle for loading other drugs or reagents for the purpose of chemotherapy (CHT), photothermal therapy (PTT) or PDT combination therapy [26,27]. It is encouraging that the inner surface of MSN nanochannels modi ed by abundant amino groups (-NH 2 ) can easily coordinate with Pt ions by substituting chloride ligand, and then achieve the purpose of in situ growth of Pt NPs after NaBH 4 reduction reaction [28,29]. In addition, due to the presence of plentiful electronegative -SH on the outer surface of silica and positively charged -NH 2 in the inner wall of mesopore, the negatively charged PtCl 6 2− were driven into the mesopore under electrostatic action.
ROS are the consequence of PDT treatment and simultaneously become the cause of DNA mispairing [30]. High tension ROS can directly lead to DNA damage, or cause the oxidation of bases in the deoxynucleoside triphosphate (dNTP) pool, leading to DNA mispairing, mutations, and cell necrosis [31]. It has been reported that guanine can be oxidized by ROS to generate 8-oxoguanine, which can be further converted to 8-oxo-2'-deoxyguanosine triphosphate (8-oxo-dGTP), leading to nuclear or mitochondrial DNA mispairing [32][33][34]. The MTH1 protein can sanitize the oxidized dNTP pool by converting 8-oxo-dGTP to 8-oxo-dGMP, thus avoiding the integration of these oxidized nucleotides into DNA. MTH1 is unnecessary for normal cells, but it is closely related to the migration and metastasis behavior of tumor cells, so it is of great signi cance for the survival of tumor cells and the reduction of tumor-related damage [35]. Therefore, speci c chemotherapy (CHT) targeting MTH1 is a very promising anticancer approach. Moreover, the therapeutic effect of CHT can be ampli ed by ROS-induced intracellular base oxidation. Predictably, ampli ed PDT can collaborate with MTH1 inhibitors to generate large amounts of 8-oxo-dGTP, resulting in DNA structure destruction and cell death.
Based on current background knowledge, we tactfully and innovatively designed a dual ampli cation therapeutic e cacy nanotherapy platform (MSN-Pt@Ce6/TH588@Li-R) in which MSN was taken as the template, MSN-Pt formed by in-situ growth of Pt NPs in MSN nanochannels was used as the core, after physical encapsulation of Ce6 and TH588, the RGD functionalized liposome shell was modi ed on the periphery to effectively prevent the premature release of Ce6 and TH588 in the blood circulation [36,37]. NPs can reach the tumor site through the active targeting by RGD and the enhanced permeability and retention (EPR) effect. After being internalized through membrane fusion, the liposome layer was destroyed and Ce6 and TH588 were subsequently released into the cytoplasm. Pt NPs exposed to the Hydrogen hexachloroplatinate (IV) hexahydrate (H 2 PtCl 6 ‚6H 2 O, 99%), sodium borohydride(NaBH 4 ) were purchased from Beijing Hongke Chemical Products Co. Tetraethoxysilane (TEOS) and sodium hydroxide (NaOH) were purchased from Aladdin (Shanghai China). TH588 was purchased from Selleckchem (Houston, TX, USA). All other chemical reagents were analytical grade and do not require further puri cation.
Preparation of MSN-NH 2 15g CTAC and 0.6g TEA were dissolved in 150ml water and incubated in a trimethyl silicone bath at 80°C, followed by intensive magnetic stirring at 200 rpm for 1 h until completely dissolved. Subsequently, 10ml of TEOS was added to the reaction system drop wise at the rate of two seconds per drop, the reaction was carried out at 80°C for 2h. After cooling, the precipitation was collected by centrifugation at 12000rpm for 10min, followed by alternately washed with water and ethanol for 3 times. The removal process of the template was further performed by dissolving the reaction product in an ethanol/hydrochloric acid solution and carried out at 70°C overnight. The MSN were centrifuged at 12000rpm for 10min, then washed alternately with ddH 2 O and ethanol for 3 times, and dissolved in water for later use. Finally, 50μL APTS and 100μl glacial acetic acid were added to the MSN aqueous solution and stirred at room temperature for 24h. After centrifugation at 12000rpm for 15min, precipitation was collected and vacuum freeze-dried to obtain MSN-NH 2 .
In situ growth of Pt NPs in nanochannels 150mg MSN-NH 2 was dispersed in 20ml ddH 2 O, and 20mL H 2 PtCl 6 ·6H 2 O aqueous solution was added into the NPs suspension and stirred at room temperature for 1h. NaBH 4 was then added to the reaction system and stirred for another 1h. NPs were centrifuged at 12000rpm for 10min, washed with ddH 2 O for 3 times and dried in vacuum to obtain MSN-Pt.

Synthesis of MSN-Pt@Ce6/TH588
For successful loading of Ce6, MSN-Pt (75mg) was dispersed in phosphate buffered saline (PBS), 5mg Ce6 (dissolved in 750μl DMSO solution) was added and stirred at room temperature for 24h in the dark. The mixture was centrifuged at 12000rpm for 20min, the supernatant was collected, and the precipitation was washed with PBS for 3 times. The loading process of TH588 is similar to that of Ce6, MSN-Pt/Ce6 75mg was dispersed in PBS, 7.5mg TH588 (20mg/mL,375μl DMSO solution) was added and stirred overnight at room temperature. The precipitation and supernatant were collected after centrifugation at 12000rpm for 20min. The absorbance of Ce6 and TH588 in the supernatant was detected by uorescence spectrophotometer to determine the drug loading content and encapsulation e ciencies, respectively.

Fabrication of DSPE-PEG-RGD
In order to synthesize the RGD-modi ed phospholipid (DSPE-PEG-RGD), DSPE-PEG-NHS and RGD (molar ratio 3:1) were co-incubated in DMF solution for 24 h under nitrogen ow at 25°C. The excess RGD and DSPE-PEG-NHS were removed by dialysis (MWCO:3500) for 48 hours to achieve the purpose of separation and puri cation of the products. Finally, the products were freeze-dried and stored at -80°C for later use.

Synthesis of MSN-Pt@Ce6/TH588@Li-R, MSN-Pt@Ce6/TH588@Li
Liposome shell was prepared by thin lm hydration method [38]. Brie y, cholesterol, DPPC, DSPE-PEG-RGD, DSPE-PEG, and DOTAP (molar ratio: 40:50:3:3:6) were placed in a round ask containing 30 mL chloroform. The organic solvent was removed in a rotary evaporator under a vacuum environment of 40°С and a thin lipid lm was obtained at the bottom of the ask. The ask was then dried in a vacuum desiccator for 24 h until the organic solvent completely evaporated. Subsequently, 15ml of deionized water was added to the ask and the liposome sample was obtained by ultrasonic oscillation at 30°C for 15min. The prepared MSN-Pt/Ce6/TH588 and liposome sample were dispersed in 25ml PBS and continuously stirred in the dark for 12h. The mixture was then centrifuged at 12000 rpm for 10 min to collect the precipitate (MSN-Pt/Ce6/TH588@Li-R). Non-RGD-targeted liposome encapsulated MPCT was synthesized in a similar method as described previously without the addition of DSPE-PEG-RGD. All nanoproducts were re-suspended in saline or PBS for later use.

Characterization
The particle size and morphology of NPs at each stage of synthesis process were characterized by transmission electron microscopy (TEM, JEOL, JEM F200) and scanning electron microscopy (SEM, ZEISS, Gemini 300). Size distribution, zeta potential and polydispersity index (PDI) of NPs were monitored by Malvern zeta sizer Nano-ZS90. The UV-vis spectra of the samples were obtained by using a UV-vis spectrophotometer (Perkin-Elmer, Lambda Bio40).

Drug release in vitro
To explore the drug release kinetics and pH responsive release properties of MPCT@Li-R, two kinds of NPs (MPCT, MPCT@Li-R) with equal amounts were immersed in different aqueous solutions (pH=7.4, 5.0) at room temperature, respectively. The supernatant of the samples was extracted at different time points, and the release amount of TH588 and Ce6 was determined by UV-vis spectrophotometry. All the release tests were repeated three times in parallel and the average of the results were taken.

Cytotoxicity assay
The cytotoxicity of MPCT@Li-R NPs was evaluated by Cell Counting Kit-8 (CCK-8) assay. Speci cally, HOS cells were seeded into 96-well plates at a density of 5000 cells per well and co-incubated with fresh medium at 37°C in 5% CO 2 for 24h. Next, the cells were treated with a series of increasing concentrations of MPCT@Li-R (0, 6.25, 12.5, 25, 50, 100, 200 μg/ml) for 24h and 48h. Then, 10μl of CCK8 solution was added to each well, and incubate for 2h. The absorbance values at the 450nm test wave were determined using an enzyme immunoassay analyzer (Thermo Fisher Scienti c, Inc., Waltham, MA, USA). The experiment was performed in quintuplicate.
The PDT-CHT combination treatment e cacy of MPCT@Li-R was monitored by CCK8 assay. Generally, HOS cells were seeded into a 96-well plate (5000 cells per well) and cultured in humidi ed 5%CO 2 at 37°C for 24 h until completely attached. Subsequently, the cells were washed once with PBS, fresh medium containing a range of concentrations of free Ce6, MPC@Li-R or MPCT@Li-R was then added. After coincubation with HOS cells for 6h, the cells were irradiated with 660nm laser (400 mW cm -2 ) for 10 minutes and incubated for another 24h. Finally, the HOS cell viabilities were determined by CCK8 method in vitro.
Calcein AM/PI staining Calcein AM/PI staining was used to further investigate the antitumor effect of MPCT@Li-R in vitro. HOS cells were inoculated in 24-well plates (5×10 4 cells per well) and cultured at 37°C with 5% CO 2 for 24 hours until completely adherent. TH588(250μL, 20μg/ml), Ce6(250μL, 20μg/ml), MPC@Li-R (250μL, equivalent MSN concentration: 20μg/ml), MPCT@Li-R (250μL, equivalent MSN concentration: 20μg/ml) were added to displace the medium. PBS was added as negative control group. After incubation for 4 h, the treatment group was irradiated with a 660nm laser with an energy density of 2.5W cm -2 for 5 min. Finally, Calcein AM/PI detection solution was added and incubated in dark for 30 minutes at 37°C. After incubation, the staining effect was observed under an inverted uorescence microscope.

Hemolysis Assay
To test the blood biocompatibility of MPCT@Li-R, hemolysis assay was performed. Venous blood was extracted from BALB/c mouse and centrifuged at 8000rpm for 5 min. The serum was discarded and 2ml PBS was added to resuspend the red blood cells (RBC). Then, 200µl RBC suspension was added to 800µl PBS, in which the concentration of MPCT@Li-R ranged from 12.5-400 µg/ml. Moreover, 200µl RBC suspension were added to 800µl PBS and deionized water as negative and positive controls, respectively. The mixture was shaken at 37°C for 2 hours and centrifuged at 12000rpm for 3 minutes. Finally, 8 treatment groups were photographed, 100µl supernatant was taken and placed in 96-well plate. The absorbance was measured by a microplate reader (Bio-Rad, Model 550, USA)

Cellular uptake experiments
To measure the e ciency of the cellular nanoparticle uptake, Human osteosarcoma HOS cells were cultured in a 24-well plate at 37°C and 5% CO 2 for 24 h. After the complete application, the cells were washed once with PBS. Then, fresh medium containing different formulations were added to incubate for another 12 hours. The cells were then washed with PBS and stained with Hoechst 33342 for 15 min, followed by cell imaging with an inverted uorescence microscope.
In vitro catalysis and ROS generation experiment MPCT and MPCT@Li-R NPs were dissolved in 4ml 3% H 2 O 2 solution or deionized water, respectively, and co-incubated for 30 minutes followed by detection using HI-2400 dissolved oxygen meter to demonstrate To detect ROS generation in cancer cells, ve groups were set up (control, TH588, Ce6 + laser, MPC@Li-R + laser, MPCT@Li-R + laser). The HOS cells (1×10 5 per well) were inoculated into 6-well plates and cultured for 24 hours until they adhered completely. Then, different formulations (TH588, Ce6, MPC@Li-R, MPCT@Li-R) were added into the corresponding wells according to the treatment of the above 5 groups.
After the cells were incubated at 37°C for another 6 hours, the culture medium was replaced with fresh MEM again. The cells were irradiated with a 660nm, 500mW cm -2 laser for 5 min, and then co-incubated with the ROS probe: 2′,7′-dichlorodihydro uorescein diacetate (DCFH-DA) for 30 min, followed by ROS uorescence imaging. To further evaluate the e cacy of different treatment modalities, terminal deoxynucleotidyl transferasemediated dUTP-digoxigenin nick-end labeling (TUNEL) staining was used to evaluate the apoptotic response of tumor tissues. Hematoxylin and eosin (H&E) and Ki67 staining were used to evaluate the e cacy of chemo-photodynamic combination therapy on tumor tissues. The main organs (heart, liver, spleen, lungs, kidneys) of mice were extracted, embedded in para n and sectioned for H&E staining to study the toxicity of NPs in vivo.

Statistical analysis
The values are expressed as mean ± standard deviation (SD). GraphPad Prism (version 7.0.0.159) was used to conduct the two-tailed Student's t tests for multiple comparisons. P < 0.05 was considered statistically signi cant.

Preparation of MPCT@Li-R NPs
The synthesis process of MPCT@Li-R is illustrated in Scheme 1. MSN-based drug vehicles were rst synthesized and then modi ed with -NH 2 to effectively chelate Pt ions while improving water stability, then Pt ions were reduced by NaBH 4 , so as to achieve the goal of in-situ growth of Pt NPs in the nanochannels. After the adsorption of photosensitizer Ce6 and MTH1 inhibitor TH588 through electrostatic action, the RGD-functionalized liposome shell was introduced to the periphery of the NPs in order to further realize the controllable release and precise targeting of Ce6 and TH588.

Characterization of MPCT@Li-R NPs
The synthesis process of the core-shell MPCT@Li-R was monitored by TEM. As shown in Figure. 1a, b, MSN was spherical, with uniform particle size (about 95nm by TEM measurement) and possessed homogeneous mesoporous structure, in which the black part is the skeleton of MSN and the bright area is the mesoporous channel. After the reduction of NaBH 4 , it can be observed that the bright black dot structure with an average diameter of 3.4 nm is evenly distributed in the mesoporous structure of MSN, indicating the successful grafting of Pt NPs. The morphology of MPCT@Li-R was monitored by TEM, in which the thickness of the liposome shell was about 7nm, and the particle size increased to nearly 105nm compared with MSN-Pt, indicating the successful aggregation of lipid bilayer on the surface of the NPs (Figure. 1c, d). SEM was used to further observe the uniform spherical structure of MPCT@Li-R and the corresponding element distribution (Figure. S1). The polydispersity index (PDI), as an evaluation of the inhomogeneity of size distribution, showed that the PDI of the nal product in deionized water was 0.265, which proved its good dispersion stability. As shown in the Figure. S2, both the intermediate product and the nal product MPCT@Li-R exhibited good colloidal stability in aqueous solution. In addition, the particle sizes of MSN and MPCT@Li-R were measured by dynamic light scattering (DLS), and the results were similar to those measured by TEM ( Figure. 1e, f). Studies have shown that the cell uptake e ciency of MSN is negatively correlated with particle size, and smaller particle size means higher uptake [39]. Fang et al. reported that MSN with a particle size of around 100nm exhibited the best intracellular uptake rate and endosomal escape e ciency, which perfectly matched our results. In addition, energy dispersive spectrometer (EDS, OXFORD, Xplore 30) was used to analyze the composition of different elements in MPCT@Li-R, in which Si, O, Pt, C and other elements can be clearly observed, further indicating the successful preparation of the composite material ( Figure. 1g-i).
For effectively induce the growth of Pt NPs in mesoporous of MSN, the MSN was modi ed with -NH 2 , resulting in the formation of positively charged nanochannels which further promotes the electrostatic bonding of negatively charged PtCl 6 2with -NH 2 . Zeta potential is shown in Figure. Figure. 2c, d. The drug loading capacity and encapsulation rate of TH588 were 8.67% and 94.93%, and that of Ce6 were 9.08% and 99.85%, respectively.
The pH-responsive release of TH588 and Ce6 is an inevitable requirement for PDT-CHT combination therapy after the targeted arrival of NPs to tumor cells. Different pH values (pH 7.4, 5.0) were selected to simulate the pH values of normal physiological environment and tumor acidic environment. As shown in Figure. 2e, explosive drug release of MPCT NPs was observed in PBS solutions at all pH values, the release proportion of TH588 and Ce6 from MPCT was nearly 90% and 85% at 10h, respectively. In contrast, MPCT@Li-R has a completely different TH588 and Ce6 release pro le. As shown in Figure. 2f, MPCT@Li-R is pH-dependent on the release of TH588 and Ce6. For TH588, only 30% was released at pH 7.4 within 30 h, in contrast to more than 75% at pH 5.0. The release behavior of Ce6 was similar to that of TH588, with only 17% release at pH 7.4 and more than 60% release at pH 5.0 within 36 hours. In general, the gatekeeper liposomes can be cleaved in acidic environments and unlock mesoporous channels, eventually leading to the release of TH588 and Ce6. This property provides a strong guarantee for the continuous drug release of NPs in acidic medium after entering tumor cells.

Evaluation of O 2 and ROS generation in vitro
As shown in Figure. 2g, obvious transparent bubbles were attached to the tube wall after MPCT and MPCT@Li-R were co-incubated with H 2 O 2 solution for 30min. The catalytic performance of Pt NPs was found to be excellent by dissolved oxygen meter, and Ce6, TH588, even liposome shell had a negligible in uence on its performance. In addition, the catalase activity of the nal product was further investigated. As shown in Figure. 2h concentration showed that more than half of the H 2 O 2 was decomposed within 30 minutes (Figure. S4).
After repeated addition of H 2 O 2 for several times, its catalytic activity was still excellent, indicating its good catalytic stability ( Figure. 2i).
ROS can ablate tumors by destroying nucleic acids and proteins in tumor cells [40]. In order to investigate whether MPCT@Li-R can produce ROS under the irradiation of 660nm laser, Ce6, TH588, MPC@Li-R and MPCT@Li-R were co-incubated with HOS cells and DCFH-DA probe was used to detect ROS generation. As shown in the Figure. 3a, negligible uorescence was observed in the control group, while relatively obvious green uorescence was observed in the other 5 treatment groups, indicating that CHT, PDT or NPs + laser can effectively produce ROS in tumor cells. Compared with Ce6 + laser, MPC@Li-R + laser exhibited a stronger green uorescence, which may be attributed to the catalase properties of Pt NPs or the targeting ability of RGD peptide. In all treatment groups, MPCT@Li-R + laser (660nm, 500mW cm -2 , 5min) exhibited the highest green uorescence intensity, which demonstrated the unique advantage of CHT-PDT combination therapy with dual amplifying effect in ablation of tumor cells.
Since MTH1 inhibitor TH588 can inhibit the puri cation of 8-oxo-dGTP, resulting in DNA damage. Therefore, immuno uorescence staining was utilized to observe the content of 8-oxo-dGTP in different treatment groups. There is no doubt that the dissipation of MTH1 protein can lead to the accumulation of 8-oxo-dGTP in DNA, as demonstrated by pink uorescence. In addition, the single PDT or MSN-Pt based in situ oxygen-generation promoting PDT process, and the reciprocal effect of TH588 and O 2 -facilitated PDT could signi cantly increase the intracellular content of 8-oxo-dGTP, resulting in oxidative damage to DNA ( Figure. 3b).

Cancer cellular uptake of NPs
To evaluate the uptake capacity of HOS cells to MPCT@Li-R, different formulations (free Ce6, MPCT@Li, MPCT@Li-R) were co-incubated with HOS cells for 12h and uorescein imaging was performed using a uorescence microscope. As shown in Figure. 3c, after being co-incubated with free Ce6, although the blue uorescence of the nucleus was clearly visible, only a small amount of red uorescence was observed in the cytoplasm, which may be due to the poor solubility of Ce6 and its inability to enter HOS cells effectively. In contrast, there was a strong red uorescence signal in the cytoplasm of MPCT@Li-R group, indicating that RGD peptides enhanced the endocytosis of NPs by interacting with integrin receptors.
Antitumor e cacy of NPs in vitro Excellent biocompatibility is the prerequisite for composite to play the therapeutic role. Hemoglobin can be released from the broken red blood cells, generating the red supernatant and resulting in enhanced absorbance at 570nm. In the hemolysis experiment, composites with a series of gradient concentration were co-incubated with red blood cells in PBS solution, as shown in Figure. 4a, the hemolytic activity of MPCT@Li-R was less than 5% even at concentrations up to 400 μg/ml.
To evaluate the cytotoxicity of NPs, CCK8 assay was utilized to analyze the viability of HOS cells. As shown in Figure. S5, the cellular viability of HOS cells remained above 90% even after 48h co-incubation with 200 μg/ml MP@Li-R, which proves their excellent biocompatibility. Subsequently, we further explored the cytotoxicity of different treatment methods. As shown in Figure. 4b, the TH588 group and the Ce6 + laser group showed relatively low cell viability compared with the control group, indicating the killing effect of CHT or PDT on tumor cells. The cellular viability of MPC@Li-R + laser group was signi cantly lower than that of PDT group, which may be attribute to the Pt-related O 2 -enhanced PDT effect or RGD targeting effect. Undoubtedly, the cellular viability of MPCT@Li-R + laser with dual ampli cation effect is the lowest, indicating that it has the best therapeutic effect. Based on the above ndings, in order to explore whether the inhibition of the proliferation of tumor cells by MPCT@Li-R + laser is caused by inducing their apoptosis, we conducted a live (green)/dead (red) staining analysis on the cells. As shown in Figure. 4c, compared with the TH588 group and the Ce6 + laser group, the MPCT@Li-R + laser group exhibited stronger red uorescence signal, which con rmed its unique advantages in killing tumor cells as a multifunctional nanotherapy platform.
Next, Western blot was used to assess the expression of some key marker proteins during combination therapy. As shown in Figure. 4d, e, MTH1 expression can be inhibited by TH588 or MPCT@Li-R. Moreover, MTH1 inhibitor can trigger p53-mediated apoptosis of cancer cells by inducing DNA damage. The expression of p53 protein was upregulated in all treatment groups, especially in the MPCT@Li-R group ( Figure. S6). Bcl-2 can prevent the release of cytochrome c from mitochondria and has an anti-apoptotic effect, while BAX can interact with voltage-dependent ion channels on mitochondria to mediate the release of cytochrome c and have an apoptotic effect. As can be seen from Figure. S6, BAX and Bcl-2 exhibited an opposite trend. In the MPCT@Li-R + laser treatment group, the expression level of BAX was the highest, while the Bcl-2 was the lowest, which con rmed the e cacy of CHT-PDT combination therapy in mediating mitochondrial injury to kill tumor cells.
Fluorescence imaging and biodistribution of NPs in mouse RGD peptide confers tumor targeting ability to MPCT@Li-R NPs through integrin receptor mediated endocytosis [41]. To further explore the appropriate irradiation time after NPs treatment, HOS tumorbearing mice models were further utilized to investigate the tumor accumulation and biological distribution of MPCT@Li-R. Fluorescence images were collected at different time points (1h, 3h, 6h, 12h, 24h) after tail vein injection. As shown in Figure. 5a, a strong uorescence signal appeared in the liver region 1h after injection. With the EPR effect and the active targeting effect of RGD, uorescence signals began to appear in the tumor region 3 h post injection, and reached the peak intensity 6 h post injection. It is noteworthy that we can still detect residual uorescent signals in the tumor area 24h after injection. Moreover, mice were sacri ced 24h post injection, tumor tissues and organs were harvested. Quantitative analysis of uorescence signals in tumors and major organs showed that the uorescence intensity in tumor tissues was signi cantly higher than that in other major organs, con rming the excellent targeting ability, high uptake and retention ability of MPCT@Li-R NPs (Figure. 5b, c).

Antitumor e cacy of NPs in vivo
Encouraged by the excellent targeting, in situ O 2 generation facilitated PDT effect and CHT effect of MPCT@Li-R, antitumor e cacy was further observed in HOS tumor xenograft mouse model. Tumorbearing mice were randomly divided into ve groups (n=3 each group) and received different prescriptions after tumor volume reached about 80-100mm 3 . Based on the results of uorescence imaging and biological distribution in vivo, 6h after intravenous injection was determined as the optimal time window for laser irradiation (660nm, 1 W cm -2 , 5 min). As shown in Figure. 5d, Tumors in the control group showed a faster growth trend and no inhibition trend was observed. TH588 and Ce6 + laser group had certain inhibitory effect on tumor growth. However, in the Ce6 + laser group, tumor growth volume began to accelerate after day 8, demonstrating that hypoxia within the larger solid tumors may impair the e cacy of PDT. Encouragingly, ROS generation based on Pt NPs catalytic properties can effectively inhibit tumor growth, while the addition of MTH1 inhibitor can bring out the best effect of this dualampli cation therapy model. Figure. 5e shows the general image of mice after 14 days of treatment, in which the change of tumor volume is consistent with the trend of Figure. 5d. Moreover, the tumor tissue images and weight further con rmed the changing trend of tumor volume ( Figure. 5f, h).

Biosafety assessment
To assess the biosafety of MPCT@Li-R, body weight changes of mice were recorded every other day during each treatment period. Compared with the PBS treatment group, the other ve treatment groups showed no signi cant weight uctuations ( Figure.

Conclusions
In summary, on the basis of MSN, we successfully realized the in-situ growth of Pt NPs by one-step reduction method, and nally prepared a dual-ampli cation effect nanotherapy platform MPCT@Li-R, which could elevate the ROS level during PDT by alleviating the hypoxia situation within the tumor, and simultaneously inhibited the puri cation process of oxidized nucleotides regulated by MTH1 through the TME responsive release of TH588. Speci cally, NPs with excellent biocompatibility and biodegradability can accumulate at the tumor site through EPR effect or RGD-mediated active targeting effect. Next, the liposome shell depolymerized in the slightly acidic environment of tumor cells to release Ce6 and TH588 to achieve the purpose of TME response release, and the exposed Pt NPs in the inner layer can decompose H 2 O 2 to generate O 2 . Under the dual action of 660nm laser and the generated O 2 , su cient ROS can be produced to cause oxidative damage of DNA, while TH588 can inhibit the scavenging of oxidative bases by MTH1 protein in dNTP pool, resulting in the accumulation of DNA oxidative damage. Both in vivo and in vitro experiments have con rmed that this dual ampli cation mode treatment strategy, which combines the elevating O 2 -promoted PDT and inhibiting DNA oxidative damage repair pathway, exhibits more ideal anti-tumor e cacy than traditional PDT or CHT. In brief, MPCT@Li-R NPs take MTH1 protein as the target and combine with Pt NPs nanoenzyme to form a novel cancer therapeutic nanoplatform.

Availability of data and materials
All data generated or analyzed during this study are included in this manuscript.
Ethics approval and consent to participate All animal experiments were performed according to protocols approved by the Experimental Animal Ethics Committee of Hebei Ex & Invivo Biotechnology Co., Ltd.

Consent for publication
All authors gave their consent for publication.