Tris (hydroxymethyl) aminomethane (Tris, 99.9%, CAS: 77-86-1) was purchased from Damas-beta. Dopamine hydrochloride (98%, AR, CAS: 62-31-7) was purchased from Titan (Shanghai, China). TMB (AR, 97%, CAS:108-67-8), dimethyl sulfoxide (DMSO, GC, 99.8%, CAS:67-68-5), glutathione (GSH, CAS: 70-18-8), 5,5’-dithiobis (2-nitrobenzoic acid) (DTNB, CAS: 69-78-3), and methylene blue (MB, CAS: 61-73-4) were purchased from Aladdin Reagent (Shanghai, China). H₂O₂ (30%, CAS: 7722-84-1) and KMnO₄ (CAS: 7722-64-7) were supplied by Chuandong Chemical Co., Ltd. (Chongqing, China). Phosphate buffer saline (PBS, Item No.: BF-0011, Beyotime, Shanghai, China), Pluronic^® F-127, 5-hydroxyfluorescein resinamide (5-FAM, CAS: 76823-03-5), 2’7’-dichlorodihydrofluorescein diacetate (DCFH-DA, CAS: 4091-99-0), Cell Counting Kit-8 (CCK-8, Item No.: C0039), GSH detection kit (Item No.: S0053), H₂O₂ detection kit (Item No.: S0038), Hoechst 33342 (Item No.: C1025) were purchased from Beyotime (Shanghai, China). Annexin V-fluorescein isothiocyanate/propidium iodide (V-FITC/PI) apoptosis detection kit (Item No.: C1062M) was obtained from Solarbio (Beijing, China). Deionized (DI) water (CAS: 7732-18-5) was used in all preparation processes from the purification system (Synergy, Millipore, MA). Other materials include: TMB (CAS:108-67-8, Aladdin Reagent, Shanghai, China); CPT (CAS: 7689-03-4, Aladdin Reagent, Shanghai, China); potassium permanganate solution (CAS: 7722-64-7, Aladdin Reagent, Shanghai, China); portable dissolved oxygen meter (ST300D, OHAUS, Changzhou, China); dialysis bags (Item No.: MD1444, Yunaye Reagent, Shanghai, China); (UV-Vis)-NIR spectrophotometer (UV-1800, Shimadzu, Japan); well plates (Item No.: 11510, Titan, Shanghai, China); flow cytometry (NovoCyte TM 2060R, USA).

For the preparation of MPDA nanoparticles, 0.36 g of Pluronic^® F-127 and 834 μL of TMB were dissolved in a mixture of H₂O (65 mL) and ethanol (60 mL) under stirring. After 30 min, 90 mg of Tris and 60 mg of dopamine hydrochloride were added successively into the mixture. The reaction was performed for 24 h at 30°C and the product pellet was then isolated by centrifugation (8,000 rpm) and washed with ethanol five times.

For CPT loading, 1 mg/mL MPDA aqueous solution and 0.5 mg/mL CPT in DMSO solution were stirred for 24 h under dark conditions, and the product pellets were separated by centrifugation and washed twice with DI water. For MnO₂ coating, 1 mg/mL MPDA-CPT aqueous solution and 0.1 mg/mL potassium permanganate solution were reacted in a water bath at 40°C for 3 h. The product pellets were separated by centrifugation and washed with DI water. The products were dispersed in an aqueous and stored at 4°C. For comparison, MPDA-MnO₂ without CPT loading was also prepared using the same method.

A series of aqueous solutions of MPDA-CPT-MnO₂ nanocomposites in a concentration gradient (0, 100, 200, 300, and 400 ppm) were irradiated with an 808 nm laser (5 min, 1.0 W/cm²) to record the temperature values. The aqueous solution of MPDA-CPT-MnO₂ nanocomposites and MPDA nanoparticles (400 ppm) was irradiated with the same laser (10 min, 1.0 W/cm²) and cooled for 20 min. Their photothermal stability was also tested by six repeated cycles of laser on/off process.

To verify the O₂ production capacity, H₂O₂ (100 mmol/L) was added to an aqueous solution of MPDA-CPT-MnO₂ nanocomposites (0.2 mg/mL). The concentration of O₂ generated was determined by a portable dissolved oxygen meter.

MPDA-CPT and MPDA-CPT-MnO₂ nanocomposites (0.5 mg/mL, 2 mL) were packed into dialysis bags (molecular weight = 8,000–14,000) and immersed in the PBS (pH 6.4, 100 mL) at 37°C under the protection from light. At various time points, the dialysate (1 mL) was taken and the absorption at 369 nm was measured by spectrophotometer (UV-1800, Shimadzu). An equal volume of fresh buffer was replenished to maintain the total volume unchanged.

The solutions of MPDA-CPT-MnO₂ nanocomposites with concentrations of 0, 50, 100, 200, 300, and 400 ppm were added to the GSH PBS solution (1 mmol/L, pH 7.4). After reacted for 6 h, 0.9 mL of supernatant was collected and mixed with DTNB solution (50 μL, 5 mg/mL), which can act as the GSH probe [47]. After reacting for 30 min, the absorbance of the supernatant at 412 nm was measured by an ultraviolet-visible (UV-Vis)-NIR spectrophotometer.

The mixture of H₂O₂ (20 mmol/L), MB (0.1 mg/mL), and sodium bicarbonate (100 mmol/L, CAS: 144-55-8) was prepared. The solutions of MPDA-CPT-MnO₂ nanocomposites with final concentrations at 0, 20, 40, 60, 80, and 100 ppm were added to the above mixtures and reacted for 1 h. Then, the mixture (1 mL) was centrifuged to obtain the supernatant for UV-Vis-NIR measurement to determine the absorbance at 664 nm.

4T1 cells were inoculated in 12-well plates (1.0 × 10⁵ per well) and incubated for 12 h. After co-incubation of the cells with the obtained MPDA-CPT-MnO₂ nanocomposites for 1, 3, and 6 h, the cells were washed three times with PBS. The cell uptake behavior was analyzed by flow cytometry (NovoCyte, ACEA). In addition, fluorescent images were captured by confocal laser scanning microscopy [(CLSM) 780, CarlZeiss, Germany].

4T1 cells were inoculated in 96-well plates (1.5 × 10⁴ per well) and incubated for 12 h. The cells were then co-incubated with different products for 24 h. Afterward, the cells were washed 3 times with PBS and incubated with 100 μL of medium containing 10 μL of CCK-8 reagent for 30 min. Finally, cell viability was assessed and recorded by a microplate reader (SPARK 10M, Tecan). To study the PTT effect, the cells in corresponding groups were treated with laser irradiation (808 nm, 5 min, 1.0 W/cm²) and the cell viability was measured using the same method.

4T1 cells were inoculated in 6-well plates (5.0 × 10⁵ per well) and incubated for 12 h. The cells were then co-incubated with PBS, MPDA, MPDA-MnO₂, and MPDA-CPT-MnO₂ for 6 h. The concentration of all the nanocomposites was kept at 200 ppm. After that, the cells were collected and washed with PBS. The intracellular H₂O₂ and GSH levels were tested using the H₂O₂ detection kit and GSH detection kit, respectively.

4T1 cells were inoculated in 12-well plates (1.0 × 10⁵ per well) and incubated for 12 h. The cells were then co-incubated with different products (200 ppm) for 6 h. Afterward, the cells were washed 3 times with PBS and stained with DCFH-DA as a reactive oxygen species (ROS) probe for 30 min. Finally, fluorescent images were collected by CLSM.

4T1 cells were inoculated in 12-well plates (1.0 × 10⁵ per well) and incubated for 12 h. The cells were then co-incubated with different products (200 ppm) for 24 h. Afterward, the cells were washed 3 times with PBS. The cells were treated according to the apoptosis kit instructions and finally processed by flow cytometry.

Transmission electron microscopy (TEM) image revealed that MPDA showed a regular porous structure with a uniform diameter of around 200 nm (Figure 2a). After CPT loading and MnO₂ coating, the porous structures were invisible, indicating the success of surface coating (Figure 2b). Energy dispersive spectrometer (EDS) mapping revealed the co-existence of C, O, and Mn elements in the formed nanocomposites (Figure 2c). The X-ray photoelectron spectroscopy (XPS) spectrum of Mn 2p displayed two peaks at 652.78 eV and 641.08 eV, corresponding to the Mn(IV) 2p_1/2 and Mn(IV) 2p_2/3 spin-orbit peaks of MnO₂, respectively (Figure 2d). The content of Mn was measured by inductively coupled plasma-optical emission spectrometry (ICP-OES) to be 9.64% in the final MPDA-CPT-MnO₂ nanocomposites. The Fourier transform infrared (FTIR) spectra of the obtained products demonstrated the successful loading of CPT and coating of the MnO₂ shell on the MPDA surface (Figure 2e) [48, 49]. The UV-Vis spectrum of MPDA showed no obvious peak (Figure 2f). After CPT loading, a typical peak at 369 nm was detected, indicating the loading of the CPT drug. The loading content was calculated to be 21.4% according to its standard curve. The mean hydrodynamic diameter of MPDA was tested to be 210.5 nm with a polydispersity of 11.7, which increased to around 270.1 nm for the final MPDA-CPT-MnO₂ nanocomposites (Figure 2g). In addition, the zeta potential of the products kept similar around –20.0 mV (Figure 2h). The formed MPDA-CPT-MnO₂ nanocomposites could disperse well in DI water, PBS (pH 7.4), and fetal bovine serum (FBS) during 48 h, indicating their good stability under physiological conditions (Figure 2i).

The solutions of MPDA-CPT-MnO₂ nanocomposites displayed elevated temperature curves during 5 min laser irradiation, showing a positive correlation with the nanocomposite concentration (Figure 3a). For 400 ppm condition, the solution temperature increased from 25.1°C to 45.2°C after 5 min laser irradiation. The photothermal reproducibility of MPDA-CPT-MnO₂ nanocomposites was assessed under 808 nm laser irradiation (Figure 3b). The photothermal conversion efficiencies of MPDA-CPT-MnO₂ nanocomposites and MPDA nanoparticles were calculated by linear fitting calculations to be 31.5% and 32.3%, respectively (Figure 3c–f).

As shown in Figure 4a, MPDA-CPT-MnO₂ nanocomposites can react with H₂O₂ to produce O₂, which can further alleviate the drug resistance of tumor cells. Then, the loaded CPT was released from the nanocomposites (Figure 4b). When compared to the un-coated nanocomposites (MPDA-CPT), the release of CPT from MPDA-CPT-MnO₂ nanocomposites was postponed. The production of •OH was detected using MB as a probe. The characteristic absorption peak of MB was found to gradually disappear with the increase of nanocomposite concentration, and meanwhile, the color changed from blue to colorless (Figure 4c). DTNB was used as the probe to detect the depletion of GSH (Figure 4d). The characteristic peak was detected to decrease with the increase of nanocomposite concentration, demonstrating the redox reaction-caused GSH depletion.

First, the cellular internalization ability of MPDA-CPT-MnO₂ nanocomposites was characterized. The nanocomposites were fluorescence-labeled using 5-FAM and co-cultured with 4T1 cells. It was found that the fluorescence signal gradually increased with the nanocomposite concentration and incubation time (Figure 5a–c). As shown in the CLSM images, the cells at 6 h showed the strongest green fluorescence signal (Figure 5d).

As shown in Figure 6a, the cells in the MPDA group displayed similar viability data (0–200 ppm) to that of the control group, revealing the good cytocompatibility of the formed MPDA nanoparticles. A slight influence on cell viability was observed when the concentration of MPDA nanoparticles reached 300 ppm and above. The cells in the MPDA-MnO₂ and MPDA-CPT groups decreased due to the therapeutic effect of CDT and CT, respectively. When treated with MPDA-CPT-MnO₂ nanocomposites, the cell viability decreased significantly, which was further decreased under laser irradiation. At 300 ppm and 400 ppm conditions, the cell viability data in the MPDA-CPT-MnO₂ plus laser group displayed great significant difference to all other groups.

Then, the therapeutic mechanism was explored. The H₂O₂ levels in cells after treatments were shown in Figure 6b. Then, DCFH-DA was employed as a fluorescent indicator to measure the intracellular ROS level. The fluorescence images showed that 4T1 cells cultured with MPDA-MnO₂ nanocomposites exhibited higher green fluorescence than that of the PBS group, demonstrating the production of ROS (Figure 6c). It was detected that the GSH level decreased significantly in the presence of MnO₂ (Figure 6d).

In addition, the apoptosis percentages after different treatments were tested using an apoptosis kit (Figure 7). It was found that the apoptosis percentages in the MPDA-MnO₂ and MPDA-CPT groups were 11.35% and 26.34%, respectively, which increased to 29.93% after MPDA-CPT-MnO₂ nanocomposite treatment. The highest apoptosis percentage was detected to be 38.54% in the MPDA-CPT-MnO₂ plus laser group.