Materials

All the chemicals were used as-received without further purification if not specified otherwise. The bulk black phosphorus (BP) was purchased from Nanjing XFNANO Technology Co., Ltd. 1-Methyl-2-pyrrolidinone (NMP) was bought from Aladdin. CD73 inhibitor AMPCP was purchased from Merck BioInc. Chitosan was purchased from Sigma-Aldrich, Sodium tripolyphosphate (TPP) was purchased from Wokai Biotechnology Co., Ltd, sodium hydroxide was purchased from Sinopharm Chemical Reagent Co., Ltd, and the nucleic acid aptamers AS1411, Lib, 5’Cy5-labeled AS1411 and Lib were all synthesized by Shanghai Biotechnology Co. DSPE-PEG2000-maleamide was purchased from Shanghai Pengsheng Biotechnology Co. Cy5-NHS was purchased from Changsha Hanchen Biotechnology Co. Elisa assay kits of TNF-a, IFN-γ, and IL-2 were purchased from Beyotime, ELISA Kit for adenosine, BUN, CR, ALT, AST detection was purchased from Shanghai Enzyme-linked Biotechnology Co. Primary antibody of Calreticulin(CRT) and High mobility group protein B1(HMGB1) was purchased from Abcam. Cy3-labeled secondary antibody for CRT was purchased from Boster Biological Technology Co., Ltd and Alexa Fluor® 488-labeled secondary antibody for HMGB1 was purchased from Abcam. Ki67, MMP9, CD8, CD86, FOXP3, CD11b, CD206 and iNOS antibodies used for immunohistochemistry and immunofluorescence were purchased from Abcam. For flow cytometry testing, Antibodies of FOXP3-PE, CD8-PerCP-Cy5, CD4-APC, CD45-APC-Cy7, CD25-BV421, CD3-BV510, MHCII-PE, CD11C-PE-Cy7, CD80-APC, CD86-BV421 were purchased from BD Biosciences. Granzyme B-PE- CY7 was purchased from Thermo.

Preparation of BPQDs

According to our previous work [49], 25 mg of bulk BP was ground into powder in a mortar and pestle, 25 mL of NMP was added and conducted probe ultrasound at 1200 W (ultrasonic frequency: 19–25 kHz, On / Off cycle: 2 s / 2 s) in an ice bath for 4 h. Then the sample was ultrasonicated at 800 W for 12 h in an ice bath. The supernatant was centrifuged at 7500 ×g for 5 min and the drawn supernatant was centrifuged at 12,000 ×g for 5 min, then the aspirated supernatant was centrifuged for 30 min at 21,000 ×g, the precipitates were obtained as BPQDs.

Preparation of chitosan nanogel and encapsulation of BPQDs and AMPCP

The preparation of chitosan nanogel and encapsulation BPQDs and AMPCP was similar with the reported method [50,51,52]. Briefly, under rapid stirring, a certain amount of 1 M aqueous sodium hydroxide solution was added to 2 mg/mL chitosan solution, adjusting the pH to 5–6, then TPP (0.4 mg/mL) was dropwise added and the mixture was continuously stirred for 30 min at room temperature. The mixture was centrifuged 10,000 ×g for 10 min, discarding the supernatant, and resuspending the precipitate with ddH2O to obtain the chitosan nanogel solution.

To encapsulate BPQDs and AMPCP in the chitosan nanogel, BPQDs (20 µg/mL), and AMPCP (200 µg/mL) were added together with TPP during the cross-linking process. Following similar subsequent process, the chitosan nanogel containing BPQDs and AMPCP was obtained, and was defined as CBA.

Preparation of EM@CBA

Red blood red cells were separated from fresh whole blood obtained from C57 mice, washed twice with DPBS, followed by hypotonic treatment with 0.25× DPBS under ice bath conditions for 1 h and then centrifugated at 12,000 rpm for 10 min. The supernatant was discarded, and the residue was washed twice with DPBS to obtain red blood red cell membranes. Then the erythrocyte cell membrane nanovesicles (EM) were prepared by sequentially passing the polycarbonate membrane with 800 nm and 400 nm pore sizes. Then equal amounts of EM were mixed with CB or CBA solution and co-extruded 15 times through a 400 nm pore size polycarbonate membrane to obtain EM@CBA.

Preparation of AptEM@CBA

3’-SH modified AS1411 nucleic acid aptamer was mixed with an equal amount of DSPE-PEG-Mal, rotary shaking at room temperature for 2 h. Then the mixture was mixed with EM@CBA, and left in an incubator at 37 oC for 30 min. Afterwards, the mixture was centrifuged at 10,000 ×g for 10 min. Then the supernatant was discarded, and the precipitate was resuspended with ddH2O to obtain AptEM@CBA.

Characterization

The TEM images were acquired on the Tecnai G2 F20 S-Twin transmission electron microscope at an acceleration voltage of 200 kV. UV-NIR absorption spectra were acquired at room temperature on a T2601 Ultraviolet-visible Spectrophotometer (Yoke Instruments) with QS-grade quartz cuvettes. The zeta potentials and dynamic diameter were measured on a NanoBrook 90Plus analyzer (Brookhaven Instruments).

Performance of AptEM@CBA for NIR photothermal conversion

100 µL of different concentrations of AptEM@CBA were added into 96-well plates, and the nanodrug was irradiated with NIR at 808 nm with a power of 1 W cm− 2 while the temperature was recorded at specific time points using an infrared thermography camera. For the evaluation of photothermal stability, the nanodrug with BPQDs concentration of 60 µg/mL was irradiated for 5 min with NIR light of 808 nm at a power of 1 W cm− 2, and was allowed to cool naturally to room temperature, which was defined as one cycle. 5 cycles were repeated and the temperature data was recorded at specific time points.

AMPCP loading capacity

CBA and EM were co-extruded through a 400 nm pore size polycarbonate membrane. The extrudate was subjected to ultrafiltration (MWCO 10 KDa, Millipore) and the filtrate was collected. The concentration of the AMPCP in the filtrate was calculated by the UV-Vis-NIR absorption curve of the filtrate. The encapsulation efficiency (EE) of the AMPCP was defined as follows:

Where Ci denoted the initial AMPCP concentration of the mixed system, and Cf denoted the concentration of the filtrate.

In vitro stability of AptEM@CBA

Equal amount of AptEM@CBA was resuspended with DPBS and DMEM medium respectively with BPQDs concentration of 60 µg/mL, and stored at 4 oC for 7 days. The particle size of the nanodrug was detected and recorded the temperature rise of the nanodrug after irradiated with NIR at a power of 1 W cm− 2 for 5 min every other day to assess its photothermal ability.

Cell culture

The cells used in this experiment, such as LLC (Mouse Lewis lung cancer cells), HACAT (Human immortalized fibroblasts), 293T (Human embryonic kidney cells), and LO2 (Human normal hepatocytes), were all obtained from the cell bank of Chinese Academy of Sciences. LLC were cultured in DMEM basic medium (Gibco, USA) supplemented with 10% FBS (VivaCell, China) and 100 UI mL− 1 penicillin and 100 UI mL− 1 streptomycin (Solarbio, China). HACAT, 293T, LO2 were cultured in RPMI 1640 basic medium (Gibco, USA) with 10% FBS and 100 UI mL− 1 penicillin and 100 UI mL− 1 streptomycin. All cells were cultured in a humidified incubator (Thermo Fisher 311 Direct Heat CO2 Incubator) at 37 oC and 5% CO2.

Red blood cell membrane protein imprinting

Red blood cell membranes from fresh mouse red blood cells, EM@CBA and AptEM@CBA, were lysed with 2% sodium dodecyl sulfate (SDS) solution and then loading buffer was added. After the performed sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), coomassie brilliant blue staining was carried out for 2 H, and photograph was taken after elution.

Aptamer cell targeting and flow cytometry

The 5’-Cy5-labeled AS1411 with the sequence of 5’GGTGGTGGTGGGTTGTGGTGGTGGTGGGTGGG was synthesized by Shanghai Bioengineering Company. To verify the targeting specificity, the 5’-Cy5-labeled Lib with the sequence of 5’ATCCGTTACTCAGAACTAGCNNNNNNNNNNNNNNNATTCGACTCATACATGCATT was also synthesize. Briefly, LLC, 293T, HACAT were digested down with non-enzyme cell detach solution, washed twice with DPBS, and the supernatant was discarded. Then 5’Cy5-labeled AS1411 and Lib aptamer dissolved in binding buffer at a concentration of 300 nM were mixed with the above cell pellets respectively. The mixtures were incubated at 37 oC away from light for 30 min, then washed with washing buffer twice and conducted with flow cytometry by DxP Athena V5-B5-R3 Flow cytometer. The data were processed and analyzed by FlowJo V10.0.7 software.

In vitro cellular targeting of AptEM@CBA

Cy5-AptEM@CBA and Cy5-LibEM@CBA were obtained by modifying EM@CBA with Cy5-labelled AS1411 and Lib at a concentration of 300 nM, respectively. LLC, 293T, HACAT were digested with trypsin-free digest, then the three cell pellets were resuspended with Cy5-AptEM@CBA and Cy5-LibEM@CBA, respectively, and washed twice with washing buffer after 30 min in an ice bath protected from light, and then analysed by flow cytometry.

In vitro cellular uptake of AptEM@CBA

Chitosan was co-mixed with TPP, BP, AMPCP, Cy5 and then co-extruded with EM to obtain EM@CBA-Cy5. EM@CBA-Cy5 was modified with DSPE-PEG2000-AS1411 to obtain AptEM@CBA-Cy5. LLC, 293T, HACAT cells were inoculated in 96-well plates (3 × 104 cells/well) separately and cultured overnight, then co-incubated with AptEM@CBA-Cy5 for 6 h respectively. Afterwards, the medium was discarded and the wells was washed twice with DPBS. Then the nuclei were stained with 50 μm Hoechst for 15 min, and the fluorescence was observed by inverted fluorescence microscope (Nikon DIAPHOT300, Japan).

In vitro cytotoxicity

LLC and LO2 cells were inoculated in 96-well plates (1 × 104 cells/well), cultured overnight, and then co-incubated with different concentrations of AptEM@CBA for 12 h. Then MTT reagent was added to detect cell activity. The cell viability was calculated by using the following formula: Cell viability (%) = (Abs sample/Abs control) ×100%. Where Abs sample denotes the absorbance of the solution for the nanodrug treatment group, and Abs control denotes the absorbance of the solution for the normal cultured cells.

Hemolysis assay

100 µL of whole blood extracted from C57 mice was centrifuged at 400 ×g for 5 min at 4 oC, then the supernatant was discarded and the precipitates were washed twice with DPBS to isolate the blood cells. Then the blood cell clumps were resuspended with DBPS, H2O, different concentrations of CBA and AptEM@CBA. After incubated at 37 oC for 6 h, the mixture was centrifuged at 600 ×g for 5 min. To exclude the effect of nanodrug, the supernatant was further centrifuged at 12,000 ×g for 10 min. Then an identical amount of supernatant was pipetted out to a 96-well plate to measure the absorption at 540 nm.

In vitro calcein AM/PI live-dead staining

LLC cells were inoculated in 96-well plates (3 × 104 cells/well), cultured overnight, and then co-incubated with different concentrations of AptEM@CBA for 2 h. Then the cells were irradiated with NIR at 808 nm for 5 min at 1 W cm− 2 and the incubation was continued for 6 h. After the medium was aspirated, and Calcein AM/PI staining was carried out according to the operation manual. Fluorescence was observed using an inverted fluorescence microscope (Nikon DIAPHOT300, Japan).

In vitro photothermal induced immunogenic cell death (ICD) of tumor cells

LLC cells were inoculated in 96-well plates (3 × 104 cells/well), cultured overnight, and then co-incubated with AptEM@CB(60 µg/mL BPQDs), EM@CBA (60 µg/mL BPQDs, 10 µg/mL AMPCP), AptEM@CBA (60 µg/mL BPQDs, 10 µg/mL AMPCP) separately for 2 h, followed by irradiation with 808 nm near infrared light at 1 W cm− 2 for 5 min. For Calreticulin (CRT) detection: After continuous incubation for 6 h, the medium was aspirated and washed cells twice with DPBS, cells were fixed with − 20 ℃ pre-cooled methanol for 10 min and were blocked with 100 µL of blocking solution at room temperature for 2 h. After aspirated out the blocking solution, 100 µL 1:500 dilution of CRT antibody was incubated with the cells at room temperature for 1 h. Then the cells were incubated with 1:200 dilution of Cy3-labeled secondary antibody (BOSTER, China) at room temperature for 1 h. Next, 50 µM Hoechst was used to stain nuclei and fluorescence was observed using an inverted fluorescence microscope. For the detection of high mobility group protein B1 (HMGB1): After continuous incubation for 6 h, the medium was aspirated and washed cells twice with DPBS, 100 µL of 4% paraformaldehyde was used to fix cells which was followed by incubation with 100 µL of blocking solution for 2 h at room temperature. Then cells were incubated with 100 µL of 1:100 dilution of HMGB1 antibody at 4 ℃ overnight. Afterwards, Alexa Fluor® 488-labeled secondary antibody at 1:500 dilution was incubated with cells at room temperature for 2 h. The nucleus was stained with 50 µM Hoechst for 15 min. After washing twice with DPBS, fluorescence was observed using an inverted fluorescence microscope. As for the detection of released ATP: After continuous incubation for 8 h, the medium was collected and centrifuged at 12,000 g for 10 min. The supernatant was retained and ATP levels were measured with an enhanced ATP Assay Kit (Beyotime, USA) according to the instructions.

In vitro adenosine inhibition assay

LLC cells were inoculated in 96-well plates (3 × 104 cells/well) and cultured overnight, then cells were treated with fresh complete medium, AMPCP (10 µg/mL), AptEM@CB (60 µg/mL BPQDs), AptEM@CB (60 µg/mL BPQDs) + NIR, AptEM@CBA (10 µg/mL AMPCP, 60 µg/mL BPQDs) + NIR, respectively. After irradiation with NIR laser 1.0 W cm− 2 for 5 min, the treated cells were continued to be cultured for 8 h. Then the cell culture supernatant was aspirated to be centrifuged at 12,000 ×g for 10 min, and adenosine Elisa assay was performed according to the instructions.

In vitro facilitation of DCs maturation

Eight-week-old male C57 mice were executed by cervical dislocation, and the limbs were separated with scissors. The muscles of the limbs were stripped, and the femur and tibia were soaked in 75% alcohol for 10 min and then transferred to an ultra-clean bench. A cut was made at each end of the femur and tibia, and the bone marrow cavity was blown and washed with a syringe filled with DPBS. The blowing solution with bone marrow tissue was collected and filtered through a cell sieve with a pore size of 70 μm to obtain a single cell suspension. Then, centrifugation was performed at 400 ×g for 5 min, and the supernatant was discarded. The cell pellet was resuspended with 2 mL erythrocyte lysate, and was left to stand for 3 min. The mixture was centrifuged at 400 ×g for 5 min, and the supernatant was discarded. The cell pellet was resuspended with complete medium of RPMI 1640 supplemented with 10% FBS, 20 ng/mL GM-CSF (PEPROTECH, USA) and 10 ng/mL IL-4 (PEPROTECH, USA) to adjust the cell concentration to 1.5 × 106. The cell suspension was spread into 24-well plates and cultured for 7 days, and the medium was changed every other day to obtain the immature dendritic cells (iDCs). The iDCs were collected and the RPMI 1640 complete medium supplemented with 10% FBS, 20 ng/mL GM-CSF and 10 ng/mL IL-4 was used to adjust the iDCs concentration to 5 × 105/mL, then the iDCs was inoculated into the receiving wells of Transwell 6-well plates. While the LLC cells were spread to the chambers of Transwell 6-well plates and incubated at 37 ℃ for 12 h before different treatments (n = 3): DMEM complete medium group, AptEM@CB(60 µg/mL BPQDs) group, AptEM@CB (60 µg/mL BPQDs) + NIR (1.0 W cm− 2 for 5 min) group, AptEM@CBA (60 µg/mL BPQDs, 10 µg/mL AMPCP) + NIR group (1.0 W cm− 2 for 5 min), and 1 µg/mL lipopolysaccharide( LPS, Solarbio, China) group. Then the chambers were gently placed into the receiving wells and cultured at 37 °C in a cell culture incubator for 24 h. The DCs in the receiving wells were collected and co-incubated with fluorescence labeled antibody against MHCII-PE, CD11C-Pe-Cy7, CD80-APC, CD45-APC-CY7, and CD86-BV421 for flow cytometric analysis.

Construction of bilateral tumor model

All animal experiments related to this study were approved by the Ethics Committee of the Department of Laboratory Animals, Central South University (CSU-2022-0635). Specific pathogen Free (SPF) grade 4-week-old male C57 mice were purchased from Hunan SJA Laboratory Animal Co., and housed in individually ventilated cages in the barrier environment of the Department of Laboratory Animal Science, Central South University. The mice were ensured access to adequate food and water as well as a certain degree of freedom of movement. The primary tumor was inoculated by subcutaneous injection of 100 µL (2 × 106) of logarithmically growing LLC on the right flank of the mice, and the distal tumor was inoculated by subcutaneous injection of 100 µL l (1 × 106) of LLC cells on the contralateral side of the mice 5 days after inoculation of the primary tumor. Tumor volume was calculated based on the following formula: Tumor volume (mm3) = 0.5 × Length × Width2.

In vivo antitumor and distal tumor- inhibitory effects

When the volume of the primary tumor reached ∼ 100 mm3, tumor-bearing C57 mice were randomly divided into five groups (n = 4), and subjected to different treatments. G1 was the DPBS group, G2 was the AptEM@CBA group, G3 was the AptEM@CB + NIR irradiation group, G4 was the EM@CBA + NIR irradiation group, and G5 was the AptEM@CBA + NIR irradiation group. At 4 h after the intraperitoneal injection of all materials (the amount of BPQDs was 40 µg/mouse, and the amount of AMPCP was 200 µg/mouse), the primary tumors were irradiated by NIR 1 W cm− 2 for 10 min. Volume changes of the primary tumor and distal tumor were recorded every other day.

Flow cytometric analysis of immune cells in tumor tissues

At the end of the treatment, mice were executed and tumor tissues on both sides were quickly stripped. Appropriate size of tumor tissue mass was put into 24-well plates and cut into ∼ 1 mm3 pieces with scissors. Then tissue dissociation solution (Absin, China) was added, static at 37 ℃ for 30 min and 2 mL of DPBS-BSA (0.25% BSA) solution was used to terminate the digestion and then 70 μm cell filters were used to obtain the single cell suspension. The single cell suspension was centrifuged at 400 ×g for 5 min in 4 ℃ and the cell pellets were blocked with Mouse BD FC Block followed by antibody incubation. Fluorescence labeled antibodies used to identify T cells and regulatory T(Treg) cells were FOXP3-PE, CD8-PerCP-CY5.5, CD4-APC, CD45-APC-CY7, CD25-BV421, CD3-BV510. Fluorescence labeled antibodies used to characterize DC cells were MHC II-PE, CD11C-Pe-cy7, CD80-APC, CD45-APC-CY7, CD86-BV421. Dxp Athena (Cytek, USA) was used to acquire the flow data which were analyzed by Flowjo-V10 software.

At the end of the treatment, mice were executed and tumor tissues on both sides were quickly stripped. Appropriate size of tumor tissue mass was put into 24-well plates and cut into ∼ 1 mm3 pieces with scissors. Then tissue dissociation solution (Absin, China) was added, static at 37 ℃ for 30 min and 2 mL of DPBS-BSA (0.25% BSA) solution was used to terminate the digestion and then 70 μm cell filters were used to obtain the single cell suspension. The single cell suspension was centrifuged at 400 ×g for 5 min in 4 ℃ and the cell pellets were blocked with Mouse BD FC Block followed by antibody incubation. Fluorescence labeled antibodies used to identify T cells and regulatory T(Treg) cells were FOXP3-PE, CD8-PerCP-CY5.5, CD4-APC, CD45-APC-CY7, CD25-BV421, CD3-BV510. Fluorescence labeled antibodies used to characterize DC cells were MHC II-PE, CD11C-Pe-cy7, CD80-APC, CD45-APC-CY7, CD86-BV421. Dxp Athena (Cytek, USA) was used to acquire the flow data which were analyzed by Flowjo-V10 software.

Detection of serum immunostimulatory cytokines

When the treatment was finished, whole blood of mice was collected into 1.5 mL centrifuge tubes and left to stand at 4 ℃ for 10 min, then centrifuged at 400 ×g for 5 min. The serum was aspirated out, and the levels of immunostimulatory cytokines TNF-ɑ, IFN-γ, and IL-2 in serum were detected by using the ELISA Kit (Beyotime, China) according to the instructions.

Adenosine level in tumour tissue

At the end of the treatment regimen, an appropriate amount of tumour tissue was excised and cut into small pieces of approximately 1mm3, followed by grinding with a mortar and adding the corresponding amount of PBS (mass-volume ratio of 1:4). The homogenate was collected and then centrifuged at 12,000 g for 10 min at 4 oC, then the supernatant was taken and tested for adenosine content using a adenosine Elisa assay.

Tumor tissue immunohistochemistry and immunofluorescence staining

T cell marker CD8, mature DC marker CD86, Treg marker FOXP3, MDSC marker CD11b, M1-like TAMs marker iNOS and M2-like TAMs marker CD206 were detected by immunofluorescence. The primary distal tumors were submerged in 4% paraformaldehyde for 1 day, then placed in liquid paraffin, and then sliced into 4 μm thickness sections using a slicer. The slices were treated with 1% Triton X-100 and 30% H2O2 after baked in an oven at 60 °C for 60 min. Antigen repair was performed with 0.01 M sodium citrate at pH6.0, followed by blocking with 10% goat serum for 30 min. Freshly prepared DAB chromogenic solution was added and then the sections were stained with hematoxylin and sealed with neutral resin for observation. Primary antibodies for CD8, CD86, FOXP3, CD11b, iNOS and CD206 were incubated with sections overnight at 4 °C, and then incubated with fluorescence labeled goat anti-rabbit secondary antibody (Abcam, England) for 1 h at room temperature.

In vivo biosafety

To monitor the effect of treatment on the health of mice, one day before the end of the treatment regime, whole blood of mice was taken in EDTA-2 K anticoagulation tubes by tails cutting. The levels of White blood cells (WBC), red blood cells (RBC), hemoglobin (HGB) and platelets (PLT) were detected by XN-1000-B1 fully automated hemocyte analyzer (Sysmex, Japan). After the mice were sacrificed, the major organs including heart, liver, spleen, lung and kidney were isolated, fixed in 4% paraformaldehyde for 12 h, paraffin embedded and sectioned. After deparaffinization, the sections were stained with hematoxylin and eosin, then dehydrated and hyaluronized, and sealed with neutral resin. The serum was separated and the levels of Blood urea nitrogen (BUN), creatinine (CR), alanine aminotransferase (ALT), and aspartate aminotransaminase (AST) usually refers to were detected by the corresponding ELISA Kit (Shanghai Enzyme-linked Biotechnology Co, China).

Statistical analysis

The experimental results were expressed as mean standard deviation (n = 3 in cell experiments and n = 4 in animal experiments). Two-sided Student’s t-test was used between the two groups of data, and one-way ANOVA and Dunnett’s multiple comparisons test were used to compare the statistical differences between several groups. We considered differences to be statistically significant at the following p-values: *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. All statistical analyses were performed using GraphPad Prism 8.0.1 software.