Three birds with one stone: oxygen self-supply engineering palladium nanocluster/titanium carbide hybrid for single-NIR laser-triggered synergistic photodynamic-photothermal therapy

: As a key branch of the cross-discipline biopho-tonics, phototherapy, including photodynamic therapy (PDT), and photothermal therapy (PTT), is promising in biomedicineandvisiblelight-drivingPDThasbeenapplied to clinical treatment. However, extensive applications of phototherapy are limited by the hypoxic microenvironment, laser penetration depth, and potential complexity for combined PDT/PTT. Thus, NIR-responsive oxygen self-supply nanocomposites functionalized with photosensitizers for achieving simultaneous in-depth PDT/PTT are urgently required. Herein, a multifunctional platform has been fabricated by co-immobilizing monodispersed ultrasmall Pd nanoclusters and a photosensitizer 5,10,15,20-*Corresponding

the continuous development of photosensitive materials and nanotechnology, phototherapy provides a minimally invasive, highly effective, and smart switch-off therapeutic approach for tumor treatment, which generally includes photothermal therapy (PTT) and photodynamic therapy (PDT) [2][3][4][5][6]. PTT is generally based on heat generated from light energy absorbed by photothermal conversion agents, causing irreversible thermal damage to tumor tissues, and thus, leading to tumor ablation. However, PTT usually needs high-power and long-time irradiation to generate enough heat to kill cells, which may decrease the therapeutic effect or cause local overheating. PDT utilizes the reaction between photosensitizer with oxygen (O 2 ) or hydrogen peroxide (H 2 O 2 ) under certain laser irradiation to produce reactive oxygen species (ROS), like singlet oxygen ( 1 O 2 ) and hydroxyl radical (•OH), triggering tumor cell apoptosis through oxidative stress. However, the effect might be restricted because of the hypoxic nature of solid tumors. Moreover, the hypoxic environment would further affect the cellular expression program and lead to therapy resistance. Although Type I PDT, which converts H 2  Currently, various strategies with desired synergistic effect of PTT and PDT have been developed for cancer phototherapy with the potential for clinical applications [7][8][9][10][11]. However, there are still some defects such as, the excitation of photosensitizer used in traditional PDT need the ultraviolet light or visible light, which has limited penetration and damages normal cells when exposed for a long time [12,13]. Therefore, the use of short wavelength excitation in PDT treatment has significant disadvantages. Recently, two-dimensional (2D) materials, including graphene and black phosphorus nanosheets [14][15][16][17][18], were found to exhibit perfect effect of phototherapy due to their unique physicochemical properties and photothermal conversion efficiency under the irradiation of near-infrared (NIR) laser. As the latest emerging 2D materials, MXenes have attracted more attention in batteries, supercapacitors, electrocatalysts, and energy storage because of supper metallic conductivity and surface activity, which was first reported in 2011 [19][20][21][22][23][24]. Typically, MXenes were prepared through HF etching, ultrasonication, and exfoliation process to get ultrathin 2D nanosheets from three-dimension MAX. The present MAX is a layered hexagonal structure with a general formula of M n+1 AX n (n = 1, 2, and 3), where M refers to early transition metal, A represents group 13 and 14 elements, and X stands for carbon or nitrogen [25][26][27]. Owning to excellent features, such as hydrophilicity, biocompatibility, and high NIR absorption, MXenes, like Ti 3 C 2 , Ta 4 C 3 [28], and Nb 2 C [29], have attracted considerable interest in biomedical application, especially tumor therapy.
MXene nanosheets have been used widely in tumor phototherapy because of high photothermal conversion efficiency, which also are the ideal drug delivery platform and less toxic to tissue [30][31][32][33][34]. Recently, some studies prepared MXene quantum dots (QDs) via new methods, such as Ti 2 N QDs [35] and Ti 3 C 2 QDs [36], which exhibited high photothermal conversion efficiency and expressed only desired PTT effect in vitro and in vivo tests. Besides, most of the studies were aimed at achieving effective multifunctional tumor therapy through the modification of MXenes. Utilizing the magnetic resonance imaging capabilities of specific materials, the in-situ growth of superparamagnetic Fe 3 O 4 nanoparticles on the surface of Ti 3 C 2 was reported, which could obviously achieve efficient PTT due to the contrast-enhanced magnetic resonance imaging of tumors [37]. Moreover, some specific materials with the activity of Fenton reaction are applied to modify MXenes through the surface engineering, which have the ability to decompose H 2 O 2 to generate highly toxic •OH, inducing tumor-cell apoptosis, achieving synergistic photothermal-enhanced nanocatalytic tumor therapy [38,39]. Up to now, there are only a few reports on the improvements in the type II PDT efficiency of MXenes over a well-configured system through solving the hypoxia of tumor cells, yielding synergistic therapy of PDT/PTT under irradiation on NIR laser [40,41].
In this study, Pd-Thp-Ti 3 C 2 T x was synthesized with Ti 3 C 2 T x nanosheets as carrier loading the photosensitizer, 5,10,15,20-Tetrakis(4-Aminophenyl)-21H,23H Porphyrin (Thp), and Pd nanoclusters. The nanocomposite exhibited some unique features: (1) Pd-Thp-Ti 3 C 2 T x improved the stability and delivery efficiency of Thp; (2) compared with pure Ti 3 C 2 T x , Pd-Thp-Ti 3 C 2 T x not only keep the high photothermal conversion efficiency, but also catalyzed excessive H 2 O 2 of tumor cells to produce O 2 , resolving the hypoxic problems, hence improving the effect of PDT; (3) the nanocomposite effectually took advantage of the combined effect of PTT and enhanced PDT. Therefore, Pd-Thp-Ti 3 C 2 T x produced effective therapeutic effect against hypoxic tumor. Pd-Thp-Ti 3 C 2 T x nanocomposite could aggregate at tumor sites, as demonstrated by in vivo fluorescence imaging and photothermal imaging. In addition, in vivo toxicity evaluation tests indicated that nanocomposites had favorable biocompatibility and biosafety. Our study not only successfully achieves the synergistic therapy of PTT and enhanced PDT for solid tumor based on Ti 3 C 2 T x -based nanosheets (details are shown in Figure 1), but also provides guidance for the design of nanomaterials for cancer therapy.

Preparation of Thp-Ti 3 C 2 T x and Pd-Thp-Ti 3 C 2 T x
Typically, 300 L of Thp (0.1 mg/mL) was added in the Ti 3 C 2 T x (1 mg/mL) and was sonicated for 30 min, and then the solution was vibrated at 4 • C for 24 h. Finally, the sample was collected by centrifugation, and then the Thp-Ti 3 C 2 T x was immersed into deionized water for 10 min sonication for later use. The Pd-Thp-Ti 3 C 2 T x was also prepared via the same protocol.

Oxygen detection
For the detection of oxygen generation, 150 L sample (1 mg/mL) was added into 3 mL of phosphate buffered saline (PBS, pH = 7) containing H 2 O 2 (30 wt%) about 150 L. The changes of dissolved oxygen concentration were tested by using JPB-607A within 20 min in the dark or under irradiation at 808 nm with 1.0 W/cm 2 .

Singlet oxygen generation
As the singlet oxygen ( 1 O 2 ) probe, 1,3-Diphenylisobenzofuran (DPBF) could react with 1 O 2 resulting the decrease for DPBF absorption at about 410 nm. Typically, 50 L of DPBF (1 mg/mL in ethanol), 50 L sample (1 mg/mL in deionized water) were added in the solution, in which the volume ratio of ethanol and deionized water is 1:1. The solution was irradiated under 808 nm laser with power density of 1.0 W/cm 2 at different time point over 20 min, and then the spectra were recorded. For the study of effect of H 2 O 2 in the experiments, another 3 L of H 2 O 2 was added into solution and collected the corresponding spectra.

Photothermal effect and photostability measurement
A certain number of nanocomposites dissolved in deionized water with different concentration were irradiated under 808 nm laser with certain power density. The temperature changes were monitored by FLIR infrared camera.

Cytotoxicity assay
The cytotoxicity analyses of different samples were performed via cell-counting kit 8 (CCK-8) assay. SW480, A549, MHCC97-L and CAKI cells were seeded in 96-well plates. After 24 h cultivation at 37 • C, the medium was replaced by medium including Pd-Thp-Ti 3 C 2 T x with different concentration (0, 12.5, 25, 50, 100, 200 and 400 ppm) for 24 h at 37 • C, respectively. The mixture of CCK-8 and medium (1:10 of the volume ratio) were added wells staying for 3 h at 37 • C, and then the emitted fluorescence was measured by using a 96-well plate reader at 450 nm and the data were analyzed with Prism 7 software (GraphPad, Inc, La Jolla, CA, USA). The photo-cytotoxicity on SW480 cells were also determined by CCK-8 assay. The previous processes are the same as above with different concentration of samples (0, 12.5, 25, 50, 100 ppm), after culture for 4 h, each well of experimental group was irradiated under 808 nm at 1.0 W/cm 2 for 5 min, and the control group was placed aside but at dark environment. Then all 96-well plates were transferred to humidified incubator for 24 h. The medium was replaced by specific medium including 10% of CCK-8 for 3 h at 37 • C, and then the absorbance at 450 nm was recorded.
SW480 cells were inoculated in 96-well plates and cultured for 4 h in different medium containing nanomaterials (50 ppm). The cells were irradiated at 808 nm with 1.0 W/cm 2 for 5 min and then cultured for 24 h. Finally, the living cells were stained with Calcein-AM and the dead cells were stained with propyl iodide (PI) for 30 min. Images were captured by Echo Laboratories Revolve FL.

In vitro PDT study
For ROS generation measurement, SW480 cells were cultured in medium with different sample (50 ppm) for 4 h at 37 • C with 5% CO 2 . The cells were irradiated under 808 nm laser at 1.0 W/cm 2 for 5 min, and then washed with PBS for 2 times. The medium containing certain amount of 2,7-dichlorodihydro-fluorescein diacetate (DCFH-DA) were added staying for 20 min at 37 • C, and fluorescence images were collected by Echo Laboratories Revolve FL.

In vivo fluorescence imaging
For the fluorescence imaging of Pd-Thp-Ti 3 C 2 T x in vivo, the mice model was established by intravenous injection with 100 μL Pd-Thp-Ti 3 C 2 T x -aminated sulfo-cyanine7 (Cy7) (2 mg/mL). For imaging, the 740 nm was used excitation and 760 nm as emission light was detected, and the in vivo images of mice injected at different time was recorded by a PerkinElmer IVIS Spectrum.

In vivo photothermal imaging and anti-tumor experiments
All female BALB/c mice were raised and purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd. The tumor model was generated by injecting CT26 cells (2 × 10 6 cells per mouse) in the back of each mouse.
When the tumor volume reached about 50-100 mm 3 , the mice were randomly divided into 8 groups (n = 5): (8) Pd-Thp-Ti 3 C 2 T x + Laser. After intravenous injection for 4 h, the tumor was irradiated under 808 nm at 0.75 W/cm 2 for 3 min, and the real-time infrared thermal images and tumor issue temperatures were recorded with an FLIR infrared camera. The reagents were intravenously injected about 100 L (0.5 mg/mL) every other day for a total of 7 times over the treatment of 14 days. Meanwhile, the weight and tumor volume were measured and recorded.
The relative volume was calculated as V/V о , where V о and V represent the tumor volume on the first day and on the day of the measurement. After 14 days, all mice were euthanized, and the structure and states of the cells of tumor were analyzed via H & E staining and an anti-Ki-67 antibody. Meantime, the tumors were detected using the TUNEL method to analyze the fragments during apoptosis. And the major organs were subjected to H & E staining for histological analysis.

Synthesis and characterization of the nanocomposites
As a MXene family member, Ti 3 C 2 T x has been explored for tumor phototherapy because of its extraordinary photothermal conversion efficiency for PTT. However, its metallic conductivity hinders its application in PDT. In the study, a Pd-Thp-Ti 3 C 2 T x nanocomposite was synthesized via a wet-chemistry approach to obtain highly efficient NIR PDT while preserving good PTT performance, releasing the synergistic effects of PTT and enhanced PDT for cancer. Pd was selected for its good performance in selectively catalyzing H 2 O 2 into O 2 , which was further converted to 1 O 2 by Thp at 808 nm. The content of Pd measured about 1.7 wt% using inductively coupled plasma mass spectrometry (ICP-MS), and the loading amount of Thp of Thp-Ti 3 C 2 T x and Pd-Thp-Ti 3 C 2 T x was determined to be about 3 wt% according to the standard curve of free Thp ( Figure S1).
Transmission electron microscopy (TEM) images and DLS result showed that Ti 3 C 2 T x had planer topology with lateral size of approximately 300 nm and thickness of about 5 nm, measured using atomic force microscopy ( Figure 2A-C and Figure S2(A)). Figure 2D and E demonstrated that Pd nanoclusters were uniformly immobilized on Ti 3 C 2 T x surface with a size of about 2 nm. TEM results displayed that Thp-Ti 3 C 2 T x and Pd-Thp-Ti 3 C 2 T x nanosheets had planer topology with lateral size of approximately 300 nm ( Figure 2F and Figure S3). Moreover, Figure S2(B) showed that the lateral size of Pd-Thp-Ti 3 C 2 T x was approximately 300 nm after preparation for 8 days, demonstrating the nanosheets possessed desired structural stability. The observation of the characteristic absorption peak of Thp for Thp-Ti 3 C 2 T x and Pd-Thp-Ti 3 C 2 T x indicated successful Thp loading onto nanocatalysts ( Figure 2G). Moreover, the zeta potential of pure Ti 3 C 2 T x nanosheets was −29.7 mV, which increased to −20.3 mV after Thp modification, and then decreased to −26.1 mV with the coating of Pd nanoclusters ( Figure S4), showing that electrostatic interaction was probably the main factor for successful modification. The chemical composition and valence states of elements in various composites were analyzed using X-ray photoelectron spectroscopy (XPS). As shown in Figure 2H, only the Pd3d peaks were observed in the XPS survey of Pd-Thp-Ti 3 C 2 T x , indicating the presence of Pd nanoparticles on the surface of Ti 3 C 2 T x nanosheets. The fitted peaks of Pd3d indicated that Pd existed in the form of PdO x ( Figure 2I) [42][43][44]. The C1s spectra of pure Ti 3 C 2 T x with four peaks located at 282.04, 284.79, 286.78, and 288.52 eV were assigned to C-Ti-T x , C=C, C-O, and C-F, respectively ( Figure S5(A)) [45][46][47]. Compared with pure Ti 3 C 2 T x , the peak density of C-Ti-T x of Thp-Ti 3 C 2 T x and Pd-Thp-Ti 3 C 2 T x decreased significantly as a result of the Thp and Pd cluster coated on the surface of Ti 3 C 2 T x nanosheets ( Figure S5 Table S1. Furthermore, compared with Ti 3 C 2 T x , the peak of the N1s of Thp-Ti 3 C 2 T x and Pd-Thp-Ti 3 C 2 T x shifted to high binding energy, demonstrating the electron transfer and interaction between Thp and Ti 3 C 2 T x ( Figure S6 and Table S2).

Optical properties of nanocomposites
Recent reports indicate that Ti 3 C 2 T x MXene shows high photothermal conversion property with the photothermal conversion efficiency ( ) up to 102.7% [48]. The photothermal effect of nanocomposites mainly depended on two parameters containing and the extinction coefficient ( ). The UV-vis-NIR profile of Pd-Thp-Ti 3 C 2 T x showed the obvious absorption at NIR-I biowindow around 808 nm ( Figure S7(A) and (B)), and the of Pd-Thp-Ti 3 C 2 T x was calculated to be 6.38 L/g/cm, which was higher than that of state-of-art nanoagents, like graphene oxide (3.6 L/g/cm) [49]. As shown in Figure 3A, the temperatures of Ti 3 C 2 T x , Thp-Ti 3 C 2 T x , and Pd-Thp-Ti 3 C 2 T x, were up to about 66.0 • C under the same irradiation condition, indicating that the modification had no significant influence on the photothermal property of Ti 3 C 2 T x . Meanwhile, the photothermal property of Pd-Thp-Ti 3 C 2 T x at different concentration and laser power density was studied systematically. Figure S7(C) showed that the temperature of Pd-Thp-Ti 3 C 2 T x with 12.5 ppm at 808 nm for 10 min reached 48.9 • C and was elevated to 80.7 • C at 200 ppm. In contrast, the temperature of deionized water after irradiation of the same condition underwent no obvious changes. These results indicated Pd-Thp-Ti 3 C 2 T x had concentration-dependent photothermal features and could serve as the potential photothermalconversion nanoagent. As expected, Pd-Thp-Ti 3 C 2 T x also exhibited power density-dependent photothermal feature ( Figure S7(D)), with the solution temperature reaching up to 41.0 • C at 0.2 W/cm 2 and to 87.8 • C at 2.0 W/cm 2 . In particular, the of Pd-Thp-Ti 3 C 2 T x was measured to be almost 62.1%, higher than that of most nanoagents, such as BP QDs (28.4%), TiN NPs (48.0%), and MoSe 2 nanodots ( Figure 3B and C) [50][51][52]. Notably, the Pd-Thp-Ti 3 C 2 T x showed stable absorbance at 808 nm and photothermal performance (Figures S8 and S9). Therefore, the prepared Pd-Thp-Ti 3 C 2 T x nanosheets revealed perfect photothermal conversion effect, which guaranteed good photothermalhyperthermia performance on the ablation of tumors.
For the nanocomposite, the capability for decomposing H 2 O 2 into O 2 and H 2 O to release hypoxia was investigated quantitatively. As expected, a large number of bubbles were rapidly generated after the addition of Pd-Thp-Ti 3 C 2 T x into the H 2 O 2 solution, but there was almost no generation of O 2 bubbles in the presence of Ti 3 C 2 T x and Thp-Ti 3 C 2 T x , H 2 O and H 2 O 2 solution only after the same time ( Figure S10). In addition, an oxygen probe (JPB-607A) was used to measure the O 2 content of the solution. As displayed in Figure S11, the laser irradiation could decompose a small amount of H 2 O 2 to generate O 2 . In the absence of irradiation, Pd-Thp-Ti 3 C 2 T x showed better catalytic activity for O 2 production than other nanocomposites ( Figure S12). After 808 nm irradiation, Pd-Thp-Ti 3 C 2 T x could catalyze H 2 O 2 to produce more O 2 ( Figure 3D), but the increased O 2 content in Ti 3 C 2 T x and Thp-Ti 3 C 2 T x was nearly similar to the amount of O 2 produced by H 2 O 2 decomposition only under irradiation for 20 min. The results demonstrated that Pd-Thp-Ti 3 C 2 T x could effectively decompose H 2 O 2 to generate O 2 , indicating the potential performance of PDT for hypoxic tumors.
ROS accounts for the efficacy of PDT to kill tumor cells, which can cause irreversible damage to important organelles or the DNA, eventually leading to cell apoptosis and necrosis. Thus, 1,3-Diphenylisobenzofuran (DPBF) was used as 1 O 2 detector to investigate the 1 O 2 -production capacity of these nanocomposites, which can react with 1 O 2 to reduce the absorption peak density to about 410 nm.
First, the absorption peak of DPBF solution without the addition of any nanocomposite did not decrease significantly under laser irradiation, showing that negligible 1 O 2 was generated in the process ( Figure S13). In the presence of Ti 3 C 2 T x , the results showed that there was no evidence of 1 O 2 generation ( Figure S14(A)). To our satisfactory, in the presence of Thp-Ti 3 C 2 T x and Pd-Thp-Ti 3 C 2 T x , the absorption peak of DPBF at 410 nm decreased gradually with the increase in irradiation time (808 nm, 1.0 W/cm 2 ) ( Figure S14(D) and (G)). Compared with Thp-Ti 3 C 2 T x , Pd-Thp-Ti 3 C 2 T x was more efficient in the production of 1 O 2 with the same content of photosensitizer, Thp, showing that Pd modification improved the generation of 1 O2. Meanwhile, the influence of H 2 O 2 was investigated. Following the addition of H 2 O 2 , the 1 O 2 generation efficiency of Thp-Ti 3 C 2 T x and Pd-Thp-Ti 3 C 2 T x improved significantly ( Figure 3E, Figure S14(E), (F) and (H)). In the first 5 min, the formation of 1 O 2 of Pd-Thp-Ti 3 C 2 T x was significantly more than that of Thp-Ti 3 C 2 T x , indicating that Pd-Thp-Ti 3 C 2 T x served as an oxygen self-supply nano-factory through the decomposition of H 2 O 2 into O 2 over Pd nanoclusters, which was continuously transformed into 1 O 2 . In contrast, the absorption peaks for Ti 3 C 2 T x were almost unchanged in the presence of H 2 O 2 ( Figure S14(B) and (C)), meaning that the Ti 3 C 2 T x used in this research could not effectively catalyze H 2 O 2 to generate O 2 at the reaction condition. The remaining TEMP spin-trapping ESR also confirmed that the most 1 O 2 was formed with Pd-Thp-Ti 3 C 2 T x under 808 nm irradiation with 1.0 W/cm 2 ( Figure 3F).
In addition, as an ROS detection probe, 2,7dichlorodihydro-fluorescein diacetate (DCFH-DA) was used to research the intercellular ROS generation in human colon cancer cells, SW480, which could be oxidized by ROS to form fluorescent DCF and further emit bright green fluorescence in cells. In Figure 3G, there was almost no green fluorescence in the group without laser irradiation, treated with Ti 3 C 2 T x and Thp-Ti 3 C 2 T x under irradiation (808 nm, 1.0 W/cm 2 , 5 min). Nevertheless, bright green fluorescence was observed in SW480 cells treated with Pd-Thp-Ti 3 C 2 T x under the same irradiation conditions. The results showed that Pd-Thp-Ti 3 C 2 T x produced more ROS under NIR irradiation resulting in the efficient decomposition of H 2 O 2 into O 2 , which was potent for cancer PDT.

In vitro anticancer performance and cellular uptake of nanocomposites
To study the cellular uptake of nanocomposites, cyanine 5.5 (Cy5.5) was used as fluorescence probe to synthesize Pd-Thp-Ti 3 C 2 T x -Cy5.5. As shown in Figure 4A, the fluorescence intensity was gradually enhanced within 4 h of incubation, revealing that Pd-Thp-Ti 3 C 2 T x could be efficiently endocytosed into SW480 cells. In addition, cell-counting kit 8 (CCK-8) assay was used to measure the cytotoxicity of nanocomposites. Following the cultivation of Pd-Thp-Ti 3 C 2 T x at different concentrations (0, 12.5, 25, 50, 100, 200, and 400 ppm) for 24 h, negligible toxicity to SW480, CAKI, MHCC97-L, and A549 cells was found ( Figure 4B), and the cell abilities were still above 85% even at a high concentration of 400 ppm, indicating the excellent cytocompatibility of Pd-Thp-Ti 3 C 2 T x . Subsequently, the phototoxicity of nanocomposites on SW480 cells was investigated, and no significant decrease in cell viability was found in all the groups without laser irradiation ( Figure 4C). In contrast, after irradiation under 808 nm laser at 1.0 W/cm 2 for 5 min, almost 60% of the cells treated with Pd-Thp-Ti 3 C 2 T x were killed, which was much higher than those treated with Thp-Ti 3 C 2 T x (12%) and Ti 3 C 2 T x (0%) with the same concentration of 12.5 ppm, and the phototherapy efficacy of Pd-Thp-Ti 3 C 2 T x was positively correlated with concentration and laser intensity ( Figure 4D and E). Notably, with the same concentration of Thp, Thp-Ti 3 C 2 T x , and Pd-Thp-Ti 3 C 2 T x showed better performance in killing SW480 cells, which might be due to Thp loading on Ti 3 C 2 T x improving its stability and delivery efficiency ( Figure S15). Meanwhile, SW480 cells incubated with nanocomposites were exposed to 808 nm laser at 1.0 W/cm 2 for 5 min and were then co-stained with Calcein-AM (green fluorescence) and propidium iodide (PI: red fluorescence) which could label the living and dead cells. As shown in Figure 4F, strong green fluorescence was detected in all the groups in the absence of 808 nm irradiation, demonstrating that the viability of cells was not obviously affected. However, after irradiation, no red fluorescence was detected in the control group, and a faint red fluorescence signal was displayed in cells incubated with Ti 3 C 2 T x and Thp-Ti 3 C 2 T x . But SW480 cells incubated with Pd-Thp-Ti 3 C 2 T x emitted stronger red fluorescence, illustrating the efficient phototherapy performance of Pd-Thp-Ti 3 C 2 T x and revealing that Pd-Thp-Ti 3 C 2 T x had negligible cytotoxicity and high phototoxicity. The cell apoptosis pathway reported for most nanomaterials follows the mitochondrial control of apoptosis [53]. To further investigate the mechanism of Pd-Thp-Ti 3 C 2 T x -induced SW480 cell apoptosis, some parameters were examined, including Bak, Bax, Bcl-xL, cytochrome c (Cyt-c), and cleaved caspase 3. Generally, the Bcl-2 family proteins are classified into two kinds according to structure and function, namely anti-apoptosis protein (Bcl-2, Bcl-xL, Bcl-w, etc.) and pro-apoptosis protein (Bax, Bak, Bid, Bim, etc.). As shown in Figure 5A, the expression of Bax and Bak increased in tumor cells treated with Pd-Thp-Ti 3 C 2 T x under 808 nm irradiation, while Bcl-xL was downregulated significantly, and the others had almost no change. Meanwhile, the upregulation of Bax/Bcl-xL and Bak/Bcl-xL revealed that Pd-Thp-Ti 3 C 2 T x -induced PDT/PTT had apoptosis-promoting effect, which could make mitochondria dysfunctional, accelerating the release of Cyt-c from mitochondria into cytoplasm. As expected, Cyt-c in the cytoplasm was found to increase significantly ( Figure 5B), and this could lead to the activation of caspases in the apoptosome as a pivotal effector of apoptosis. Caspase 3 is the main effector caspase that is involved in apoptosis, which could be active to generate cleaved caspase 3, leading to the cleavage of cellar substrates and apoptosis. Just as shown in Figure 5B, the expression of cleaved caspase 3 increased in cells cultured with Pd-Thp-Ti 3 C 2 T x . Meanwhile, MitoTracker Red CMXRos was used to examine changes in the mitochondrial membrane, which is an indicator of mitochondrial damage. As shown in Figure S16, SW480 cells cultured with different samples without laser irradiation emitted obvious red fluorescence. It is worth noting that, after 808 nm laser irradiation, the intensity of red fluorescence decreased significantly, especially for Pd-Thp-Ti 3 C 2 T x ( Figure 5C), which manifests the dysfunction of the mitochondrial membrane. Altogether, the results showed that the PDT and PTT induced by Pd-Thp-Ti 3 C 2 T x had obvious pro-apoptosis effects for SW480 cells through the mitochondria control of apoptosis.

In vivo biosafety of nanocomposites
Prior to the application of the nanocomposites in solid tumor, the biosafety analyses were performed through onetime intravenous injection of different nanocomposites at 20 mg/kg into healthy female BALB/c mice. There were no obvious changes in the body weight of the mice in all the groups within 14 days ( Figure 6A), meaning that Ti 3 C 2 T x , Thp-Ti 3 C 2 T x , and Pd-Thp-Ti 3 C 2 T x had neglectable influence on the growth of the mice. Moreover, standard hematology, including some blood biochemical parameters, was tested to investigate the potential impact of nanocomposites. As shown in Figure 6B, E and F, the aspartate aminotransferase (AST), alkaline phosphatase (ALP), alanine aminotransferase (ALT), urea nitrogen (BUN), and creatinine (CREA) were not different from those of the PBS group, which meant that the nanocomposites had negligible adverse effects on the liver and kidneys. Moreover, albumin (ALB), globulin (GLOB), ALB/GLOB (A/G), lipase (LIPA), amylopsin (AMYL), blood glucose (GLU), calcium ion (CA), and phosphonium ion (PHOS) levels also had no significant changes compared to the PBS group (Figure 6C, D and G-J). These results showed that the nanocomposites had no remarkable toxicity effects on the mice at the given dose. In addition, as shown in Figure 6K, compared with healthy mice of the same age, the results of hematoxylin and eosin (H & E) staining of major organs, including heart, liver, spleen, lung, and kidney, shows that there are no obvious damages of cell or tissue. These data unambiguously demonstrates that Ti 3 C 2 T x -based nanomaterials are biocompatible, with potential for application in cancer phototherapy.

In vivo fluorescence imaging, photothermal imaging, and anticancer activity of nanocomposites
In order to reveal its potential in clinical cancer therapy, the distribution of nanocomposites in mice body after intravenous injection was first investigated. Pd-Thp-Ti 3 C 2 T x -Cy7 was prepared using aminated sulfo-cyanine7 (Cy7) as fluorescent labeled molecule. As Figure 7A displayed, the fluorescent intensity attained the highest value at 24 h post-injection, and then decreased gradually, which was possibly due to the metabolism of the mice body and the biodegradation of the composites. Besides, we verified the in vivo tumor accumulation of the Pd-Thp-Ti 3 C 2 T x -Cy7 and their in vivo targeting ability using an ex vivo investigation of the major organs 72 h post-injection. It was found that there is a certain degree of accumulation in the liver and kidney tissues ( Figure S17), which is an issue encountered by many other nanomedicines. The biodegradation property could guarantee the biosafety of the nanocomposite. These results proved that nanomaterials could aggregate at the tumor site, ensuring subsequent effective in vivo phototherapy. Based on the above results, the in vivo anti-tumor effects of Pd-Thp-Ti 3 C 2 T x were investigated in more detail. To this end, female BALB/c mice with CT26 tumor model were randomly divided into 8 groups (n = 5): (1) PBS, (2) PBS + Laser, (3) Ti 3 C 2 T x , (4) Ti 3 C 2 T x + Laser, (5) Thp-Ti 3 C 2 T x , (6) Thp-Ti 3 C 2 T x + Laser, (7) Pd-Thp-Ti 3 C 2 T x , and (8) Pd-Thp-Ti 3 C 2 T x + Laser. Over the treatment period of 14 days, the reagents were intravenously injected every other day for a total of 7 times. After 4 h via tail intravenous injection every time, the tumor was exposed to 808 nm with 0.75 W/cm 2 for 3 min, and the real-time infrared thermal images were recorded ( Figure 7B). Photothermal imaging is helpful to observe the real-time effect of tumor treatment more intuitively. The tumor surface temperature of groups (4), (6), and (8) increased to 60 • C ( Figure 7C), which was high enough to ablate the tumor cells. However, for the control group without injection of any nanocomposites under the same irradiation condition, the tumor temperature increased from 37 • C to 43 • C, while the slight increase in temperature could not make the death of tumor cells. The results revealed that the Pd-Thp-Ti 3 C 2 T x showed obvious photothermal effect and could be used as photothermal imaging. During the therapeutic period, the body weight of the mice in all the groups showed no obvious changes ( Figure 7D), indicating that the dosage of nanocomposites had no significant effect on healthy growth of the mice. For the tumor volume, the groups without irradiation, PBS + Laser and Ti 3 C 2 T x + Laser, showed fast growth ( Figure 7E), revealing that the PTT of Ti 3 C 2 T x + Laser alone could not restrain the tumor growth. In contrast, the tumor suppression rate of Pd-Thp-Ti 3 C 2 T x + Laser group could be up to 100%, however, the value was only 50% for Thp-Ti 3 C 2 T x + Laser group, which was due to the fact that Pd-Thp-Ti 3 C 2 T x could decompose excess H 2 O 2 of tumor cells into O 2 , improving the PDT efficiency, leading to better synergistic therapy of enhanced PDT and PTT. These results are also supported by the representative tumor images and digital images of all groups on the 14th day ( Figure 7F and S18(A)), revealing the outstanding catalytic activity and photothermal conversion of modified Ti 3 C 2 T x , and achieving desirable effect of enhanced PDT and high-efficiency PTT.
Once the phototherapy treatment was conducted, the tumors were dissected and the pathological evaluation and therapeutic efficacy was performed using H & E, antigen Ki-67 expression, and TUNEL staining. Comparatively, large tumor cells damage and decrease in cell proliferation were recorded for the Thp-Ti 3 C 2 T x + Laser and Pd-Thp-Ti 3 C 2 T x + Laser groups ( Figure 7G). In addition, the Pd-Thp-Ti 3 C 2 T x + Laser group had more severe cell damage. Meanwhile, the Pd-Thp-Ti 3 C 2 T x + Laser group indicated a large amount of cell apoptosis with the nuclei being stained with remarkable cyan. These results aptly confirmed that Pd-Thp-Ti 3 C 2 T x could catalyze H 2 O 2 into O 2 , which relieved the hypoxic environment of the solid tumor, in turn improving the effect of PDT, resulting in efficient apoptosis of tumor cells combined with PTT. For further application of this nanocomposite in vivo, the potential toxicity needs to be analyzed. The H & E staining of major organs showed no obvious toxic side effects ( Figure S18(B)), suggesting that the Pd-Thp-Ti 3 C 2 T x had no noticeable in vivo potential toxicity.

Conclusions
In summary, a single NIR-responsive oxygen self-supply Pd-Thp-Ti 3 C 2 T x 2D nanocomposite was developed using a rationally designed method, which took advantage of the unique photothermal conversion efficiency of Ti 3 C 2 T x and relived the hypoxic microenvironment of solid tumor via catalysis to enhance the PDT effect of photosensitizer.
Compared with most traditional 2D Ti 3 C 2 T x with only PTT effect, this nanocomposite is endowed with admirable catalytic performance and broadens the biomedical applications of such excellent optical materials. Through the modification of Pd nanoclusters, Pd-Thp-Ti 3 C 2 T x had catalytic activity of decomposing excess H 2 O 2 in tumor cells to produce O 2 , releasing in situ oxygen supply for the photosensitizer action. Meanwhile, photosensitizer Thp loaded on the surface of MXene made them more easily prone to endocytosis by the cell to realize enhanced NIR PDT. The Pd-Thp-Ti 3 C 2 T x nanoplatform was successfully employed in tumor in vivo PT imaging. In vitro and in vivo experiments indicated that the Pd-Thp-Ti 3 C 2 T x had high biocompatibility and ignorable toxicity and achieved the combined effect of PTT and enhanced PDT for cancer therapy. Hypoxia restricts different disease therapies, and thus, the oxygen self-supply metal-dopant/MXene nanocomposite and it not only improves the PDT performance by loading photosensitizes, but also shows the potential application of Ti 3 C 2 T x -based nanoplatform in more cancer therapy via surface engineering (e.g., enhance immune therapy by replacing the photosensitizer with immune checkpoint inhibitors).