The bulk black phosphorus (BP) was purchased from Nanjing Two-dimensional Nanotechnology Co., LTD (Nanjing, China). 1-Methyl-2-pyrrolidinone (NMP) and tris-(2-carboxyethyl)-phosphine hydrochloride (TCEP) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Zinc acetate was obtained from Aladdin (Los Angeles, CA, USA). Aptamer (APT) AS1411 (5’-/SH/GGTGGTGGTGGTTGTGGTGTGGTGGTGG-3’) was customized by Guangzhou Ruibo Biotechnology Co., LTD (Guangzhou, China). Doxorubicin (DOX) was purchased from APExBIO (Houston, USA). PC-3 cells were kindly provided by the Stem Cell Bank, Chinese Academy of Sciences (Shanghai, China). Cell Counting Kit-8 (CCK-8) was provided by Dojindo (Japan). Dulbecco’s minimum essential medium (DMEM)/F12, penicillin-streptomycin liquid, and fetal bovine serum (FBS) were purchased from GIBCO (Grand Island, NY, USA). Alexa Fluor^® 488 Anti-Calreticulin antibody was purchased from Abcam (Cambridge, England). Anti-HMGB1 was obtained from Cell Signaling Technology (Boston, USA). Human HMGB1 ELISA Kit was obtained from Arigo Biolaboratories (Taiwan, China). ATP content detection kit was obtained from Solarbio (Beijing, China). Dendritic cells (DCs) were obtained from AllCells (San Francisco, USA). FITC anti-human CD11c Antibody, Alexa Fluor^® 647 anti-human CD80 Antibody, PE anti-human CD86 Antibody, and PerCP/Cyanine5.5 anti-human HLA-DR Antibody were provided by Biolegend (California, USA).

Black phosphorus nanosheets were obtained using an optimized liquid stripping method and step-by-step centrifugal screening for a suitable size.

First, 8 mg black phosphorus crystal powder was added to 24 mL NMP. Then, the mixture was subjected to ultrasonic treatment (800 W, on/off cycle time was 4 s/6 s) using a probe for 12 h in an ice bath, and the resultant brown suspension was centrifuged at 4000 rpm for 15 min. The supernatant was collected and centrifuged at 12000 RPM for 12 min to collect the sediment and obtain BP NSs, which were stored at 4°C.

About 1 mg Apt-SH was dissolved into 1 mL Tris buffer (tris: 10 mM, pH = 7.4). Then, 2 mg NH₂-PEG-Mal and 50 μg TCEP was added and stirred in the dark for 5 h to obtain NH₂-PEG-Apt. Then, 2 mg BP NSs were added to the solution. After ultrasonic treatment in an ice bath for 20 min and stirring for 8 h, centrifugation was performed at 10000 rpm for 15 min to obtain BP-P-APT, and it was washed twice with deionized water.

About 7 mg zinc acetate powder was added to 5 ml BP-P-APT suspension. The solution was subjected to ultrasound in an ice bath for 2 min, stirred for 1 hour, and centrifuged at 10000 rpm for 15 min, and the precipitate was washed with deionized water.

About 2 mg BP-P-Apt-Zn/NSs was mixed in 2 mL DOX solution (1.5 mg/mL). The solution was stirred in the dark for 8 h. The precipitate was obtained by centrifugation at 10000 rpm for 15 min. It was then washed with deionized water and freeze-dried.

Temperature change and stability of black phosphorus were analyzed under different concentrations and laser powers using an infrared thermal imager (Ti400+, Fluke, USA). The concentrations of BP NSs and BP-P-Apt-Zn/Dox used were 50, 100, and 200 µg/mL, and they were irradiated for 10 min with an 808-nm laser at 0.5, 1, and 2 W/cm² (Beijing Blueprint Photoelectric Technology Co., Ltd) to observe the temperature change. The irradiation was repeated 5 times for 10 min each time.

About 1 × 10⁶ cells were inoculated in 6-well plates and anti-slip slide cell crawl plates were placed at the bottom of the wells. Complete medium(89%DMEM/F12+10%FBS+1% penicillin-streptomycin liquid), DOX, BP-P-Zn/DOX, and BP-P-Apt-Zn/DOX (DOX: 3 µg/mL) were added to separate wells. About 4 h later, the cells were washed with PBS thrice, fixed with 4% formaldehyde for 25 min, washed with PBS thrice again, stained with DAPI for 10 min, and observed using a fluorescence inverted microscope (Inverted fluorescence imaging microscope, Olympus, IX73 + DP80+ fluoview, Tokyo, Japan).

Human prostate cancer PC-3 cells were cultured in a T25 culture bottle containing 10% FBS, 1% penicillin-streptomycin liquid, and 89% DMEM/F12. The culture was maintained at 5% CO₂ and 37°C. The medium was changed every other day.

To evaluate the toxicity of zinc ion in PC-3 cells, PC-3 cells in the logarithmic phase were inoculated in 96-well plates (5 × 10⁴ cells per well) and then incubated with a medium containing zinc ions in different concentrations (1, 2.5, 5, 10, 20, and 30 µg/mL), separately. After 24 h, the cell survival rate was calculated using the CCK-8 assay.

The same method was used to evaluate the toxicity of DOX in PC-3 cells. The concentrations of DOX used were 0.125, 0.25, 0.5, 1, 2.5, 5, and 10 µg/mL.

To evaluate the killing effect of Apt and Zinc ions on PC-3, PC-3 cells were incubated with BP-P, BP-P-Apt, and BP-P-Apt-Zn at concentrations of 1, 10, 25, 50, 75, and 100 µg/mL for 24 h separately, and the cell survival rate was detected using the method mentioned above. The toxicity of BP-P-Apt-Zn/DOX and BP-P-Apt-Zn/DOX + laser (DOX: 0.25µg/mL;Zn:2.5µg/mL) in PC-3 cells in the photothermal treatment group was analyzed using the same method. After 5 h of drug treatment, the cells were irradiated with a laser at 808 nm and 1 W/CM² for 12 min and then incubated. It is worth noting that because the absorbance of black phosphorus at 450 nm interferes with the OD value of the cells, 5 groups of drug-only media were used for normalization.

Western blotting was performed to detect the expression of HMGB1 in the supernatant of PC-3 cells after 24 h of drug treatment (DOX: 3 µg/mL, BP-P: 40 µg/mL, BP-P+ laser: 40 µg/mL, BP-P-Apt-Zn/DOX: 40 µg/mL, and BP-P-Apt-Zn/DOX + laser: 40 µg/mL). The procedure was as follows: β-actin was used as the control protein. The supernatant was collected after 24 h of drug treatment and centrifuged at 12000 rpm for 15 min to remove cell debris. The samples were then mixed with SDS-PAGE protein loading buffer (Beyotime, China) and boiled for 5 min. About 20 µL of the samples were subjected to SDS-PAGE gel electrophoresis and then transferred to nitrocellulose membranes. Nitrocellulose membranes were incubated with rabbit monoclonal antibody (1:1000, CST, Boston, USA) and gently shaken at 4°C overnight. After 3 washes, the membranes were incubated with anti-Rabbit IgG and HRP-Linked Antibodies (1:2000, CST, Boston, USA) for 1 h at room temperature. After three washes, the HMGB1 protein bands were detected using Bio-RAD (California, USA), and the protein concentration was analyzed using ImageJ.

The HMGB1 protein level in the supernatant was detected according to the instructions provided with the Human HMGB1 ELISA Kit (Arigo Biolaboratories, Taiwan, China). This assay employs the sandwich enzyme immunoassay technique for the detection of Human HMGB1 in cell culture supernatant samples. The specific steps were performed according to the protocol provided by the manufacturer.

PC-3 cells were inoculated in 6-well plates at a density of 1 × 10⁵ per well. After 24 h, the drugs (DOX: 3 µg/mL, BP-P:40 µg/mL, BP-P+laser:40µg/mL, BP-P-Apt-Zn/DOX: 40 µg/mL, BP-P-Apt-Zn/DOX + laser: 40 µg/mL) were added. After 24 h, the cells were rinsed thrice, fixed with 4% paraformaldehyde for 20 min, rinsed thrice again, sealed with 10% bovine serum for 30 min, and incubated with Alexa Fluor^® 488 anti-calreticulin antibody (1:1000, Abcam, Cambridge, England) overnight, in the dark, at 4°C. The cells were then washed thrice, their nuclei were stained with DAPI, and the cells were observed using a fluorescence inverted microscope (Inverted fluorescence imaging microscope, Olympus, IX73 + DP80 + fluoview, Tokyo, Japan). The whole process was performed on ice.

PC-3 cells (1 × 10⁵) were inoculated in 6-well plates. The next day, drugs were added and incubated for 24 h (DOX: 3 µg/mL, BP-P:40 µg/mL BP-P+laser:40µg/mL, BP-P-Apt-Zn/DOX: 40 µg/mL, BP-P-Apt-Zn/DOX +laser: 40 µg/mL). After washing the cells thrice, they were digested with trypsin and washed again thrice with PBS containing 10% FBS. The precipitate was collected and incubated with Alexa Fluor^® 488 anti-calreticulin antibody (1:100, Abcam, Cambridge, England), in the dark for 30 min at 4°C. The samples were then washed thrice with PBS containing 10% FBS, centrifuged at 400 g for 5 min, resuspended in 500 µl PBS containing 10% FBS, and placed on ice. Each sample was analyzed using a flow cytometer (BD FASC Aria III, Becton Dickinson, California, USA) to determine the concentration of CRT on the cell membrane.

According to the manufacturer’s instructions, the concentration of ATP in the cell culture medium after different treatments was determined using UV spectrophotometry. Briefly, ATP was extracted from the sample. Due to the characteristic absorption peak of NADPH at 340 nm and the content of NADPH being proportional to the content of ATP, NADPH content was used to calculate ATP content.

Human DCs were provided by AllCells (San Francisco, USA). After resuscitating the DCs, they were co-incubated with PC-3 cells treated with drugs for 24 h. Briefly, DCs were inoculated in 6-well plates with a density of 2 × 10⁵ cells per well, and then, drug-treated PC-3 cells with a density of 2 × 10⁵ per well were added and incubated for 30 h.

About 30 h later, the cells were centrifuged at 1200 rpm for 5 min thrice, and resuspended in PBS containing 10% FBS. They were incubated with FITC anti-human CD11c Antibody, Alexa Fluor^® 647 anti-human CD80 Antibody, PE anti-human CD86 Antibody, and PerCP/Cyanine5.5 anti-human HLA-DR Antibody (Biolegend, California, USA) at 1:100 (by volume) on ice for 30 min in the dark. After centrifugation thrice, they were suspended in 500 µl PBS and the matured DCs were detected using a flow cytometer (BD FASC Aria III, Becton Dickinson, California, USA).

The detailed synthesis process BP-P-Apt-Zn/DOX is shown in Scheme 1 . First, the diameter of black phosphorus powder was changed to about 200 nm using an ultrasonic probe, Apt was loaded on BP nanosheets using electrostatic adsorption, then, the zinc ions were modified on BP-P-Apt using charge coupling, and finally, DOX was modified.

Figures 1A, B show the Transmission Electron Microscopy (TEM) photos before and after modification of BP NSs. As shown in Figure 1A , BP NSs were transparent and thin, indicating that the liquid stripping method had stripped off the black phosphorus and the stripping effect was good. BP NSs were about 100-200 nm in diameter. Figure 1B shows the appearance after modification using Zn, Apt, and DOX. It can be seen that the surface is covered with a transparent substance and the size is changed to 200-300 nm, indicating that the drug was successfully loaded. This was consistent with dynamic light scattering (DLS) results ( Figure 1C ).

As shown in Figure 1D , the initial Zeta potential of unmodified BP was about −17.8 mV, and after loading NH₂-PEG-Apt, the Zeta potential changed to −38.7 mV (due to a large amount of PEG). With the coupling of Zn²⁺, the Zeta potential changed to −23.3 mV, and finally, when DOX was loaded, the Zeta potential changed to 16.7 mV.

The crystal structure of BP NSs was characterized using X-ray diffraction. As shown in Figure 2A , compared to the standard PDF card JCPDS#73-1358, it was found that all the peaks of the prepared material were consistent with those of ortho-crystalline black phosphorus in the pure phase, and the characteristic peaks were not offset. The results showed that the lattice structure of BP was not damaged during ultrasonic stripping.

The surface group structure of BP-P-Apt-Zn/DOX was characterized using FTIR. As shown in Figure 2B , multiple peaks with high intensity appeared on the FTIR spectrum. At 3423.75 cm^-1, multiple absorption peaks of hydrogen bond association formed by Apt, DOX, PEG, and NH₂, including -OH stretching vibration absorption peak and -NH₂ stretching vibration absorption peak. The stretching vibration absorption peak of C-H was at 2923.93 cm^-1. The stretching vibration absorption peak of P = O was at 1632.42 cm^-1. The stretching vibration absorption peaks of the PO4 group were at 1102.21 cm^-1, 1025.73 cm^-1, and 945.95 cm^-1. The bending vibration absorption peak of the PO₄ group was at 568.78 cm^-1. PEG molecules chemically bind to BP by forming P-O-C bonds, which also led to the successful modification of BP surface with PEG.

To further identify the structure of BP-P-Apt-Zn/DOX, Scanning Electron Microscope (SEM) was used to analyze the nanostructure and characterize the element distribution in the nanostructure. The high-angle annular dark-field (HAADF) SEM image in Figure 2C shows the BP-P-Apt-Zn/DOX, and element mapping analysis was performed using the image. Figure 2C show SEM-mapping elements of P, Zn, N, and O, respectively. The SEM element mapping analysis revealed that the compositional distributions of the four elements (P, Zn, N, and O) in the BP-P-Apt-Zn/DOX are uniform, revealing the BP-P-Apt-Zn/DOX structure. DOX:C₂₇H₂₉NO₁₁, PEG: HO(CH₂CH₂O)NH.

The photothermal properties of BP NSs and BP-P-Apt-Zn/DOX were observed at different 808-nm laser powers and concentrations. The process was monitored using an infrared thermal imager. Figures 3A–C respectively show the temperature change curve of BP-NSs at the concentration of 50, 100 and 200μg/mL.After five cycles of irradiation with an 808-nm laser, the temperature changes in BP NSs (24.73°C ~ 25.09°C) ( Figure 3D ).

Figure 4A shows the temperature changes at three powers when the concentration of BP-P-Apt-Zn /DOX is 50μg/mL. As shown in Figure 4B , when the concentration of BP-P-Apt-Zn/DOX is 100μg/mL and the power is 1W/cm2, the irradiation temperature can be increased by 28.03°C.When the concentration of BP-P-Apt-Zn/DOX was 200μg/mL, the temperature could be increased by 39.3°C ( Figure 4C ).It shows that the whole nanosystem has good photothermal conversion efficiency, and the increase of temperature is related to the concentration. Under the different powers, BP-P-Apt-Zn/ DOX at 100μg/mL could still reach 20.1°C at a power of 0.5 W/cm2 ( Figure 4B ).When the power is 2W/cm2, BP-P-Apt-Zn/DOX can reach 49.1°C ( Figure 4B ), and the increase of temperature is positively correlated with the power. As shown in Figure 4D , after five cycles of irradiation with an 808-nm laser, the temperature changes in BP-P-Apt-Zn/DOX (27.5°C ~ 28.5°C) were not significant, indicating good photostability.

To prove that the addition of Apt can improve the targeting efficiency of drugs, we used a fluorescence inverted microscope (Inverted fluorescence imaging microscope, Olympus, IX73 + DP80 + fluoview, Tokyo, Japan) to observe the uptake of DOX, BP-P-Zn/DOX, and BP-P-Apt-Zn/DOX by PC-3 cells. As shown in Figure 5A , compared with BP-P-Zn/DOX, the BP-P-Apt-Zn/DOX group showed stronger fluorescence, indicating that Apt-modified BP-P-Zn/DOX could be taken up in greater amounts by PC-3 cells. It is noteworthy that the fluorescence intensity of the only doxorubicin group was relatively high, probably due to the presence of the nuclear pore complex (37), which enables DOX to enter and exit cells efficiently. However, when only DOX is used, the long cycle capacity and biocompatibility of the nanodrug delivery platform is not achieved.

We first explored the toxicity of Zn²⁺ and DOX alone in PC-3 cells. As shown in Figure 5B , the toxicity of DOX in PC-3 cells was time- and dose-dependent, with a survival rate of 33.7% at 72 h at a concentration of 5 μg/mL.

As shown in Figure 5C , the cytotoxicity of Zn²⁺ in PC-3 was also time- and dose-dependent. After 48 h of treatment, Zn²⁺ showed good cell destruction. When the concentration was only 5 µg/mL, the survival rate was 43.8% at 48 hours.

As shown in the Figure 5D , we analyzed the lethality of the addition of various components to the whole nanosystem towards PC-3 cells. It was observed that BP showed no obvious lethality towards PC-3 cells, the addition of Apt reduced the survival rate of cells, and BP-P-Apt-Zn had a strong killing effect.

However, the BP-P-Apt-Zn/DOX irradiation group had a stronger destruction effect than the non-irradiation group. When the concentration of BP-P-Apt-Zn/DOX was 50µg/mL, the survival rate of cells in the laser group was 32.6%, while that in the unlaser group was 41%.

The transfer of CRT to the cell membrane was observed using the immunofluorescence assay. There was no expression of CRT on the membranes of the cells in the BP-P and control groups ( Figure 6D ), indicating that BP alone had no effect on the expression of CRT on the membrane. Weak green fluorescence of CRT was observed on the membranes in the DOX group, and an increased green fluorescence of BP-PEG was observed in the laser-irradiated group compared to the non-irradiated group. The fluorescence intensity in the BP-P-Apt-Zn/DOX + laser group was slightly higher than that in the BP-P-Apt-Zn/DOX group, but the difference was not significant. The combined application of BP, laser, and DOX played the most significant role in the transfer of CRT to the cell membrane.

The average fluorescence intensity in CRT-positive cells was quantitatively analyzed using flow cytometry. As shown in Figure 7A , the CRT fluorescence intensity in the BP-Apt-Zn-Dox + laser group was about three times that in the control group. The laser-irradiated group also showed an excellent CRT fluorescence intensity ( Figure 7A ), which was consistent with the analysis of immunofluorescence. As can be seen in Figure 7A , the DOX group showed stronger fluorescence of CRT compared with the control group.

The release of ATP was measured using UV spectrophotometry. As shown in Figure 7B , the release of ATP in the extracellular fluid of PC-3 cells treated with DOX increased 5.5 times compared to that in the untreated group, while the release of ATP in the DOX group combined with the whole nanomedicine system increased 9.8 times, which was approximately consistent with the results of CRT and HMGB1. Similarly, the laser-irradiated group also showed significant enhancement.

To explore whether the nanoparticle drug platform-induced apoptosis of PC-3 cells can promote the maturation of anthropogenic DC cells to further induce an immune response, drug-treated PC-3 cells were co-incubated with anthropogenic DCs for 24 h. The maturation of DCs before and after drug treatment and in different drug groups was observed using an inverted microscope. As shown in Figure 7C , immature human DCs in their original state are round and have no dendrite structure. However, several DC suspension cells with the dendritic structure were observed in the DOX, laser, and all nanodrug groups, suggesting that the laser effect and the whole nanodrug platform could stimulate DC maturation.

To further verify the maturation of DCs, flow cytometry was used to analyze the expression of related markers in dendritic cells after co-incubation. We first treated the cells with FITC anti-human CD11c to label the DCs, and then analyzed their CD80 and CD86 co-expression and HLA-DR expression. As shown in Figure 8A , the expression of CD80 and CD86 in the DOX group was significantly higher than that in the untreated group. There were no significant changes observed in the BP-P and BP-P+laser groups, while the expression of CD80 and CD86 in BP-P-Apt-Zn/DOX +laser and non-laser groups significantly increased by 62.2% and 60.3%, respectively. The expression of HLA-DR is shown in Figure 8B . Its expression in DOX and laser groups increased significantly, while there was no significant difference between the BP-P and untreated groups. Figures 8C, D shows the results of quantitative analysis of CD80 and CD86 are co-expressed and HLA-DR expression in dendritic cells. Flow cytometry analysis results were consistent with the microscopy results.