PdCu nanoparticles with multienzymatic activity to treat tumors through the synergistic photothermal and chemodynamic therapy

Combined chemodynamic/photothermal therapy has great potential in tumor treatment. However, the presence of excessive glutathione (GSH) in the tumor microenvironment (TME) can attenuate its therapeutic effect, and other components in the TME have not been fully utilized as well. In this article, we designed a noble metal nanozyme called PdCu@BSA, which can be used for the combined chemodynamic therapy (CDT) and photothermal therapy (PTT) of tumor. In detail, PdCu@BSA has three different types of enzyme-like activities. Its catalase (CAT)-like activity can degrade extra H2O2 in the TME to create O2 and relieve the hypoxic situation. The glutathione oxidase (GSHox)-like activity can consume high level of GSH in the TME to reduce the consumption of reactive oxygen species (ROS). Peroxidase (POD)-like activity catalyzes H2O2 to form strong oxidized ·OH. The above enzyme-like activities enhance the effectiveness of CDT. Besides, PdCu@BSA has good photothermal effect and can be used for PTT when exposed to 1064 nm laser. Therefore, based on multiple enzyme-like activities and photothermal effects, PdCu@BSA can be employed for synergistic tumor therapy, resulting in good therapeutic outcome.


Introduction
Malignant tumors pose a serious threat on human physical and mental health.In 2020, there are approximately 10 million additional cancer deaths worldwide, according to the World Health Organization's International Agency for Research on Cancer [1].Exploring efficient treatment methods has emerged as a current research hotspot because of the high incidence and mortality rate of cancer.Several novel tumor treatment approaches have been developed as a result of the drawbacks of conventional therapies, including photodynamic therapy (PDT) [2,3], photothermal therapy (PTT) [4,5], chemodynamic therapy (CDT) [6,7], immunotherapy [8][9][10][11], and others.Many benefits come with new tumor treatment techniques, including being noninvasive, slightly poisonous, high selectivity and so on [12,13].These techniques also have several disadvantages despite their many benefits, such as PTT being limited by heat shock proteins and light penetration, and CDT being affected by high glutathione (GSH) in the tumor microenvironment (TME) [14][15][16].Therefore, the design of therapeutic system that can overcome various constraints has become a primary priority in the face of new challenges in tumor treatment.
Additionally, they have a variety of enzymatic activities that play an important role in tumor therapy.For instance, peroxidase (POD)-like activity can produce reactive oxygen species (ROS) by Fenton-like reactions to kill cancer cells [31,32].Glutathione oxidase (GSHox)-like activity can strengthen the killing effect of ROS through GSH consumption [16], and catalase (CAT)-like activity can break down excess H 2 O 2 in TME into O 2 [33,34], thus alleviating the hypoxia state of the tumor.These enzyme-like activities work together to achieve CDT and enhance its therapeutic effect.Therefore, by a reasonable design, precious metal nanozymes can be used in tumor treatment through the synergistic PTT and CDT.
Here, precious metal PdCu@BSA nanozymes were designed and synthesized, which could achieve synergistic treatment of PTT and CDT because of their multienzymatic activity and good photothermal conversion capacity.As shown in Scheme 1, PdCu nanozymes were fabricated by hydrothermal method.In order to enable them to be applied in living organisms, albumin (BSA) with good biocompatibility was used to modify PdCu nanozymes.The as-obtained PdCu@BSA nanozymes had three kinds of enzyme-like activities, such as CAT, POD and GSHox, which alleviated the hypoxia of tumors and enhanced the oxidative stress of TME.Furthermore, under 1064 nm laser irradiation, the synergistic effect of PTT and CDT could induce cancer cell apoptosis and had a prominent anticancer effect.

Characterization
TEM images were performed on a FEI Tecnai G2 F20 transmission electron microscope operating at 200 kV.The absorption spectra were measured by an ultraviolet spectrophotometer (U-3900, Japan).The FT-IR spectra of PdCu and PdCu@BSA was measured by a Bruker VERTEX 70v spectrophotometer.The zeta potential and hydrodynamic of PdCu@BSA were measured by a Zetasizer (NZS, Zetasizer Nano ZS).The 1064 nm laser model was LSR-PS-II.The temperature change was monitored by an infrared thermal imager (FLIR E30).The oxygen Scheme 1. Schematic illustrations for the construction of PdCu@BSA nanozymes and PTT/CDT synergetic therapy.concentration was measured by portable oxygen dissolver (JPB-607A).The fluorescence images were pictured through inverted fluorescence microscope (MF53-N, China).Flow cytometry was performed by the BD Accuri C6 Plus.
2.3.The synthesis of PdCu 0.0031 g of PdCl 2 , 0.062 g of Cu(NO 3 ) 2 •3H 2 O, and 0.35 g of CTAB were introduced into a 25 ml of Teflon reactor.Subsequently, 10 ml of oleylamine was added to the reactor and stirred for 30 min.The reactor was then installed and heated at 170 °C for 24 h with magnetic stirring in an oil bath.After cooling down to room temperature, the products were precipitated via centrifugation (10000 rpm, 5 min), washed with ethanol and dispersed in hexane.
2.4.The synthesis of PdCu@BSA 30 mg of BSA was weighed and transferred into a beaker.Subsequently, the BSA was dissolved in 20 ml of deionized water.Then, the homogeneous PdCu chloroform solution that had been subjected to ultrasound treatment was introduced into the BSA solution.The mixture was stirred at room temperature in a fume hood for 24 h.Finally, PdCu@BSA was cleaned three times with deionized water and precipitated by centrifugation (10000 rpm, 5 min).
2.5.Determination of CAT-like activity of PdCu@BSA Three experiments were set up, namely phosphate buffer solution (PBS) group, PBS with 5 mM of H 2 O 2 group, as well as PBS with 5 mM of H 2 O 2 and 200 μg ml −1 of PdCu@BSA group.The portable dissolved oxygen meter was used to measure the amount of O 2 in each buffer solution.Data were recorded at one-minute intervals for a duration of 20 min.

Determination of GSHox-like activity of PdCu@BSA
The reaction between DTNB and GSH can result in the solution to turn yellow, thus enabling us to employ solution-fading comparison for assessing the GSHox-like activity of PdCu@BSA.To achieve this, we prepared GSH and DTNB solution with the same concentrations of 1.5 mg L −1 .Subsequently, PBS solution with different concentrations of PdCu@BSA (0.00, 0.04, 0.08, 0.16, and 0.32 mg ml −1 ) was prepared.Following this step, each tube was added with 0.1 ml of the GSH solution and the mixture solution reacted for 30 min.After that, each tube was supplemented with 0.1 ml of DTNB solution and left undisturbed for a period of 5 min.The solution absorbance at 425 nm was measured by a UV spectrophotometer.

Determination of POD-like activity of PdCu@BSA
Upon reaction with GSH, the PdCu@BSA nanomaterials generated Cu + that could undergo a Fenton-like reaction with H 2 O 2 , while Pd also exhibited POD-like activity.The generated •OH can make the TMB solution blue and the absorption peak of solution will increase.To perform the assay, 5 mg ml −1 of TMB solution was dissolved in DMSO.Then, 20 μl of TMB solution, 150 μl of 3% H 2 O 2 solution, 1.5 ml of PBS solution, and 200 μg ml −1 of PdCu@BSA were introduced into the colorimetric dish.The UV spectrophotometer was employed to monitor the absorption peak at regular intervals of 2 min until the reaction reached equilibrium.

Photothermal effect of PdCu@BSA
The photothermal effect of PdCu@BSA was examined from three angles: concentration, laser power, and photothermal cycle.
Different concentrations: A laser operating at a wavelength of 1064 nm with a power density of 1 W cm −2 was employed.Subsequently, 200 μl of PdCu@BSA solutions with various concentrations (50, 100, 200, and 400 μg ml −1 ) were taken for analysis.The temperature changes were monitored using a thermal imager at intervals of every 30 s over 5 min.
Different powers: 200 μl of the PdCu@BSA solution (400 μg ml −1 ) was added to 1.5 ml centrifuge tube.The solution was exposed to 1064 nm laser with varied powder density (0.25, 0.50, and 1.00 W cm −2 ) for 5 min.The temperature measurement was recorded every 30 s by a thermal imager.
Photothermal cycle: The PdCu@BSA solution (200 μg ml −1 ) was irradiated three times with a 1064 nm laser (1.00 W cm −2 ).In detail, following irradiation for 5 min, the solution was cooled to room temperature before being reheated.This process was repeated three times, and the temperature was recorded every 30 s using a thermal imager.

Cell culture and biocompatibility
The L929 cells and 4T1 cells were cultured in DMEM and RPMI 1640 medium, respectively.L929 cells and 4T1 cells were seeded in two 96-well plates (8000/well), and different concentrations of PdCu@BSA (0, 25, 50, 100, and 200 μg ml −1 ) were added to the plates and cultured for 24 h.The relative survival rate of the cells was detected by standard cellular methyl thiazolyl tetrazolium (MTT) assays.

Cell endocytosis
Firstly, to activate the hydroxyl group, 0.004 g of RhB, 2 ml of NHS solution, and 2 ml of EDC solution were mixed and stirred for 1 h.Then, 2 ml of PdCu@BSA solution was introduced to the mixture and stirred for 24 h in dark area.Finally, PdCu@BSA/RhB was cleaned three times with deionized water and precipitated by centrifugation (10000 rpm, 5 min).
4T1 cells were seeded in a 6-well plate (10 5 /well) and cultured them for 24 h until they reached the desired density.The old medium was removed, then fresh medium containing PdCu@BSA/RhB (200 μg ml −1 ) was added.Subsequently, different cultivation time (0, 2, and 4 h) was set.After three times of PBS washing, the fluorescence intensity in the cells was observed by inverted fluorescence microscope.The procedures for assessing endocytosis via flow cytometry were the same as those described above except that trypsin was employed to digest the cells prior to conducting flow cytometry analysis.

Determination of CAT-like activity of PdCu@BSA in cells
Four experimental groups were established: PBS, PBS+1064 nm, PdCu@BSA and PdCu@BSA+1064 nm.The concentration of PdCu@BSA should be 200 μg ml −1 .The DMSO solution containing 1 mg ml −1 of RDPP was prepared as test reagent.4T1 cells were seeded in a 6-well plate (10 5 /well), and cultured them for 24 h.After the cells were treated with the different groups, the fluorescence intensity of RDPP in cells was observed by inverted fluorescence microscope.

Determination of GSHox-like activity of PdCu@BSA in cells
The experimental groups and concentration of PdCu@BSA were set up in accordance with the above step.4T1 cells were seeded in a 6-well plate (10 5 /well) and cultured for 24 h.Upon the different treatments, the GSH content in cells was detected by GSH and GSSG assay kit.

Detection of ROS
DCFH-DA was used as a probe to detect intracellular ROS production.Consistent with the above steps, four experimental groups were set up. 4T1 cells were inoculated into 6-well plates (10 5 /well) and cultured for 24 h.PdCu@BSA and cells were cocultured for 4 h, and the non-endocytic nanozymes were washed with PBS.Then DCFH-DA probe was added and incubated for 30 min, and 1064 nm laser (1 W cm −2 ) was irradiated for 5 min.Finally, after washing with PBS, fluorescence microscope was used to observe the fluorescence in the cells.

Therapy effect of tumor cells
The therapeutic effects of PdCu@BSA on 4T1 cells were tested.4T1 cells were seeded into 96-well plates (8 × 10 3 /well) and cultured with RPMI 1640 medium containing different concentrations of PdCu@BSA (0, 25, 50, 100, and 200 μg ml −1 ) for 4 h.The remaining nanozymes were washed away with PBS.The treatment groups were irradiated with 1064 nm (1 W cm −2 ) for 5 min and then continued to incubate for 4 h.Finally, the relative survival rate of cells was tested by MTT method.
To visually observe the therapeutic effect of PdCu@BSA nanozymes, 4T1 cells were stained with Calcein-AM/PI.Four experimental groups were established: PBS, PBS+1064 nm, PdCu@BSA and PdCu@BSA+1064 nm.4T1 cells were inoculated into 6-well plates (10 5 /well) and cultured for 24 h.After the cells were treated differently, they were stained by Calcein-AM/PI for 30 min.Finally, the live/dead cells were observed by inverted fluorescence microscope.

Results and discussions
3.1.Synthesis and characterization of PdCu@BSA PdCu nanomaterials were successfully prepared by the previous route with slight variation [35].To confirm the successful preparation of PdCu nanomaterials, the crystallographic information of PdCu nanoparticles was measured by x-ray diffraction (XRD) patterns.As shown in figure 1, the diffraction peaks of PdCu corresponded to the (111), ( 200), (220), and (311) crystal planes (PDF#48-1551).And according to transmission electron microscopy (TME) image and size distribution (figures 2(a) and (b)), the PdCu nanoparticles were uniformly distributed and their average particle size was 23.7 ± 2.6 nm.In order to facilitate the subsequent biological experiments, PdCu nanomaterials were decorated with BSA for the transition of the nanoparticles from the oil phase to the water phase.From figure 2(c), it can be observed visually that after the modification of BSA, PdCu nanomaterials changed from oil phase to water phase, implying that PdCu@BSA nanomaterials had good water solubility.To confirm the modification of BSA on the surface of PdCu nanoparticles, we measured the Fourier transform infrared (FT-IR) spectra of PdCu@BSA and BSA.As shown in figure 2(d), the same characteristic peaks of the peptide bonds (−C═O at 1653 cm -1 and C-N at 1531 cm -1 ) in FT-IR spectrometer demonstrated the successful modification of BSA.Moreover, the XRD pattern of PdCu@BSA (figure 1) showed that the PdCu nanomaterials still maintained good crystalline structure after BSA modification.Most proteins are negatively charged in vivo, and nanocarriers with positive surface charge have a strong adsorption capacity to proteins, which will result in the nanocarriers being easily identified and phagocytosed by cells after entering the body, and then removed from the blood stream, greatly reducing the bioavailability of the drug [36].The carrier surface design with neutral or negative electric properties can extend the cycle time of the carrier in the body [37,38].The zeta potential of PdCu@BSA was −11.3 mV, which meant that PdCu@BSA nanomaterials could be stable in the blood circulation (figure 2(e)).Furthermore, it has been reported that nanoparticle sizes smaller than 200 nm are more favorable for leakage from tumor blood vessels and taken up by cells [39,40].And according to  figure 2(f), the hydrodynamic size of PdCu@BSA was 142 nm, which suggested that the PdCu@BSA nanomaterials were beneficial to uptake by tumor cells.

Enzyme like activity of PdCu@BSA
It was reported that precious metal nanomaterials had enzyme-like activities and had been widely used in therapy and bioimaging [41,42].The enzyme-mimic process of PdCu@BSA nanoparticles was described in figure 3(a).First, the portable dissolved oxygen meter was used to check CAT-like activity of PdCu@BSA.Figure 3(b) demonstrated that the O 2 content in the PBS solution remained essentially unaltered without H 2 O 2 .After the addition of H 2 O 2 , the concentration of O 2 in the PBS solution gradually increased over time, which caused by the breakdown of H 2 O 2 itself.However, when H 2 O 2 and PdCu@BSA nanozymes coexisted, the O 2 levels in the PBS solution increased rapidly with time, which manifested the strong CAT-like activity of PdCu@BSA.
Then, the GSHox-like activity of PdCu@BSA was confirmed through the chromogenic reaction of GSH and DTNB.The DTNB solution would change from colorless to yellow in the presence of GSH, while PdCu@BSA would oxidize GSH to prevent the occurrence of the chromogenic reaction.In the following, the ability of PdCu@BSA nanozymes to consume GSH was investigated.As can be seen in figure 3(c), with the enhancement of the concentrations of PdCu@BSA, the solution of PdCu@BSA gradually faded from yellow to colorless.Additionally, the characteristic peak of DTNB at 412 nm decreased, which further implied the presence of the GSHox-like activity of PdCu@BSA.
Next, the POD-like activity of PdCu@BSA was verified via the oxidizing reaction of TMB.The generated Cu + from the reaction of GSH and PdCu@BSA could undergo a Fenton-like reaction with H 2 O 2 , producing strong oxidized •OH.TMB would be oxidized by •OH, leading to color change of the solution from colorless to blue.As shown in figure 3(d), the solution showed an obvious blue color.And as time increased, the characteristic absorption peaks of the oxTMB augmented at 370 nm and 652 nm, indicating the production of more •OH, thereby verifying the POD-like activity of PdCu@BSA.3.3.Photothermal performance of PdCu@BSA Photothermal materials can absorb light energy and transform it into thermal energy using for PTT. Figure 4(a) demonstrated that as the concentration of PdCu@BSA increased, the absorption became higher in the UV-vis and NIR region, indicating their broad absorption in these regions.Consequently, PdCu@BSA had the potential to be a photothermal agent.Besides, compared to the NIR-I window, the NIR-II window has a deeper depth of tissue penetration, making it more suitable for PTT, so we chose the latter one as the excitation light.According to Lambert-Beer law, the mass extinction coefficient (ε) was calculated as 3.13 L g −1 cm −1 (figure 4   To test the photothermal conversion ability of PdCu@BSA, we investigated the effect on solution temperature of various concentrations of PdCu@BSA and various excitation powers.As seen in figure 4(c), the temperature of the solution elevated with concentration of PdCu@BSA at the excitation power of 1 W cm −2 .After 5 min of exposure, the solution temperature reached 53 °C at the concentration of 400 μg ml −1 .Similarly, the solution temperature had a positive correlation with laser power (figure 4(d)) under the fixed concentration.Then, by controlling the switch of the laser, the temperature change of the solution (200 μg ml −1 ) reached maximum.The solution was cooled to the starting temperature by turning off the laser and the corresponding temperature variation was recorded.As displayed in figure 4(e), the photothermal conversion efficiency (η) of PdCu@BSA nanomaterials was calculated to be 29.92% by linear fitting and calculation.And figure 4(f) illustrated that there was no discernible change of the solution temperature after three alternative switches (on and off) of the laser, suggesting that the stable photothermal performance of PdCu@BSA.In conclusion, PdCu@BSA could be used as photothermal agents for PPT by utilizing NIR-II window excitation light.
3.4.Cytotoxicity and endocytosis of PdCu@BSA First, the cytotoxicity of PdCu@BSA was tested in cancer cells (4T1 cells) and normal cells (L929 cells).Figures 5(a) and (b) displayed that with the highest concentration up to 200 μg ml −1 , the survival rate of normal cells remained above 85%, whereas only 51.55% of the tumor cells survived, proving that PdCu@BSA nanomaterials were non-toxic to normal cells but damaged tumor cells.The reason might be that POD-like activity of PdCu@BSA nanozymes catalyzed excess H 2 O 2 in TME to form strong oxidizing •OH accompanied by  the depletion of reductive GSH due to their GSHox-like activity, thus killing tumor cells and having little influence on normal cells.Then, the endocytosis of PdCu@BSA was investigated.PdCu@BSA featured a fluorescence after attachment to RhB that emitted red fluorescence when excited by green light, which can be determined by flow cytometer and observed by fluorescence microscope to test the endocytosis of PdCu@BSA.As seen in figure 5(c), with time of co-culturing with 4T1 cells increasing, flow cytometry data exhibited a gradual increase in intracellular red fluorescence.And it can be intuitively seen in figure 5(d) that red fluorescence of 4T1 cells heightened with the prolonging of the cultivation period, validating the successful endocytosis of PdCu@BSA by 4T1 cells.

Enzyme-like activity of PdCu@BSA in cells
The CAT-like activity of PdCu@BSA nanomaterials in cells was checked by RDPP probes, whose red fluorescence can be suppressed by O 2 oxidation.As can be observed in figure 6(a), compared to the control group and 1064 nm laser irradiation group, the red fluorescence in the PdCu@BSA group and PdCu@BSA +1064 nm group was almost completely quenched, indicating the formation of extra O 2 in the cells.This demonstrated that besides in solution, PdCu@BSA could also indeed display CAT-like activity in cells.In the following, DCFH-DA was chosen to measure intracellular ROS production.Green fluorescence can be observed via the oxidation of DCFH-DA by ROS.As illustrated in figure 6(b), significant green fluorescence appeared in  the PdCu@BSA group and PdCu@BSA+1064 nm group, exhibiting that a certain amount of ROS was produced, and confirming the ability of PdCu@BSA to perform the POD-like activity within cells.Then, a GSH assay kit was used to determine the GSH concentration in cells.As indicated in figure 7, the GSH content in the PdCu@BSA and PdCu@BSA+1064 nm groups were significantly reduced compared to the control and 1064 nm laser irradiation groups, presenting the GSHox-like activity of PdCu@BSA.

Therapy effect of PdCu@BSA in cells
Thanks to extra H 2 O 2 in the TME, PdCu@BSA nanozymes could yield •OH for CDT through POD-like activity.Moreover, under 1064 nm laser radiation, PdCu@BSA nanozymes realized the synergistic therapy of CDT and PTT, which could effectively kill tumor cells.Therefore, the damage ability of PdCu@BSA nanozymes to tumor cells was studied.As observed in figure 8, under 1064 nm laser irradiation, the survival rate of 4T1 cells decreased as the concentration of PdCu@BSA nanozymes increased.After treating 4T1 cells in different conditions, the calcein-AM/PI fluorescent dyes were used to stain live cells and dead cells respectively.In figure 9(a), both the 1064 nm group and the control group showed strong green fluorescence, indicating that 1064 nm irradiation alone had no effect on tumor cells.The PdCu@BSA group showed little red fluorescence due to CDT, and the PdCu@BSA+1064 nm group displayed almost all red fluorescence, while the green fluorescence essentially disappeared.This suggested that CDT alone had limited ability to kill tumor cells.In sharp contrast, the combination of CDT and PTT had a significant killing ability, demonstrating the therapy efficacy of PdCu@BSA.In addition, the corresponding flow cytometry data were provided to investigate apoptosis.As shown in figure 9(b), compared to the control groups, the PdCu@BSA group and the PdCu@BSA+1064 nm group showed a considerable increase in apoptosis, reaching 66.50% and 88.00% respectively, further demonstrating the superior therapeutic effect of PdCu@BSA.

Conclusion
In summary, PdCu@BSA nanomaterials with multienzymatic activity were successfully designed and constructed for tumors treatment.It was found that the resulting nanozymes possessed three different types of enzyme-like activity, including CAT, POD and GSHox-like activity, which worked together to improve the lethal effect of ROS by relieving the hypoxic environment in TME, generating •OH for CDT, and consuming reducing substances in tumors.In addition, PdCu@BSA exhibited sustained photothermal properties under laser irradiation at 1064 nm and could be employed for PTT due to the significant NIR absorption.Cell experiments confirmed the enzymatic activity of PdCu@BSA and the synergy of CDT and PTT efficiently destroyed tumor cells.

Figure 2 .
Figure 2. (a) TEM image of PdCu.(b) The size distribution of PdCu.(c) Photograph of PdCu being modified by BSA.(d) FT-IR spectra of PdCu@BSA and BSA.The zeta potential (e) and hydrodynamic size (f) of PdCu@BSA.

Figure 3 .
Figure 3. (a) Schematic diagram of multiple enzyme-like activities detection.(b) Changes in O 2 concentration with time and (c) changes in UV-vis absorption spectra of TMB and the photograph of the change in color after adding different concentrations of PdCu@BSA to the H 2 O 2 -containing solution.(d) Changes in UV-vis absorption spectra of DTNB and the photograph of the changes in color after adding different concentrations of PdCu@BSA to the GSH-containing solution.
(b)) on the basis of the UV-to-NIR absorbance spectra in figure 4(a).

Figure 6 .
Figure 6.(a) RDPP fluorescence images and (b) ROS fluorescence images of 4T1 cells after different treatments.

Figure 7 .
Figure 7.The GSH content of 4T1 cells after different treatments.

Figure 8 .
Figure 8.The relative survival rate of 4T1 cells after 1064 nm laser irradiation with different concentrations of PdCu@BSA.

Figure 9 .
Figure 9. (a) Fluorescent images of calcein AM (green) and PI (red) of 4T1 cells after different treatment.(b) Flow cytometry data of 4T1 cells after different treatments.