Materials

3-Mercaptopropionic acid, Camptothecin, Floxuridine(FUDR), 4-Dimethylaminopyridine (DMAP), and 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) were obtained from Energy Chemical Pharmaceutical Chemical Co., Ltd.(Shanghai, China); acetone, n-hexane, and sodium chloride were procured from China National Pharmaceutical Group Chemical Reagent Co., Ltd.(Shanghai, China); anhydrous sodium sulfate was sourced from Tianjin Damao Chemical Reagent Factory (Tianjing, China); IR-780 iodide was acquired from Shanghai Aladdin Biochemical Technology Co., Ltd.(Shanghai, China); dichloromethane was obtained from Chengdu Cologne Chemical Co., Ltd.(Chengdu, China); hyaluronic acid (HA) (10 kDa) was purchased from Shandong Liyang Biotechnology Co., Ltd.(Shandong, China); DSPE-PEG2000 was purchased from AVT (Shanghai) Pharmaceutical Co., Ltd. (Shanghai, China)silica gel powder was sourced from Qingdao Yonghai Silica Gel Co., Ltd.(Qingdao, China); DCFH-DA and Sephadex were acquired from Beijing Ruida Henghui Technology Development Co., Ltd.(Beijing, China); and N-Hydroxysuccinimide (NHS) was obtained from Shanghai Macklin Biochemical Co., Ltd(Shanghai, China). Fetal bovine serum (FBS) was purchased from VivaCell (Shanghai, China). Sodium Pyruvate and glutamine were purchased from Procell Co., LTD (Wuhan, China). Penicillin–streptomycin solution and gentamycin were purchased from NCM Biotech (Suzhou, China). Cell Counting Kit-8 (CCK8) was purchased from Yeasen Biotechnology (China). All cell culture plates, dishes, transwell plates, ultrafiltration tubes, centrifuge tubes, RPMI 1640 medium, Dulbecco’s modified Eagle’s minimum essential medium (DMEM) were purchased from NEST Biotechnology (Wuxi, China). 0.22 μm filters and 70 μm cell strainers were provided by Biosharp Bioscience (Anhui, China). Antibodies used for western blot assay including anti-CRT (ab92516) and anti-HGMB1 antibody (ab18256) was obtained from Abcam (USA). ATP ELISA Kit was purchased from Beyotime, Biotechnology Co. (Jiangsu, China, S0027). Antibodies used for flow cytometry were purchased from BD Pharmingen (USA), including Fixable Viability Dye eFluor™ 506 (catalog no.65–0866-14, eBioscience), anti-CD16/32 mAb (catalog no.553141), APC-Cy7 anti-CD45 (catalog no.557659), FITC anti-CD3 (catalog no.553061), APC anti-CD11c (catalog no.227166), BB515 anti-CD11b (catalog no.564454), APC anti- CD206 (catalog no.550889), BB700 anti-IA/IE (catalog no.746197), PE-Cy7 anti-CD86 (catalog no.25–0862-82), PE anti-CD80 (catalog no.12–0801-80), PerCP-Cy5.5 anti-CD4 (catalog no.550954) and PE-Cy7 anti-CD8 (catalog no.552877) antibodies.

Synthesis of CTF and HAIRSynthesis of CTF

The synthesis procedure and characterization of TK-CPT was depicted in Figure S1 to S5. Solid CPT (2.94 g, 8.40 mmol), TK (2.03 g, 8.40 mmol), and DMAP (2.06 g, 16.80 mmol) were suspended in 50 mL of CH2Cl2, and a solution of EDC (3.24 g, 16.80 mmol) dissolved in CH2Cl2 was slowly added dropwise. The reaction was carried out at 30 °C for 24 h. After completion of the reaction, it was quenched with water and extracted with CH2Cl2 three times, followed by washing with 1 M HCl once and saturated brine three times. The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure to yield a yellow solid, which was purified by column chromatography (DCM: MeOH = 100:1 → 80:1) to obtain white solid TK-CPT with a yield of approximately 60%.

To synthesize CTF (Figure S6 to S8), TK-CPT (100.00 mg, 0.18 mmol), EDC (67.50 mg, 0.35 mmol), and DMAP (21.50 mg, 0.18 mmol) were dissolved in 6 mL of DMF under N2 protection, followed by stirring in an ice bath for 1 h. FUDR (86.65 mg, 0.35 mmol) was then added dropwise to the solution, and the reaction was allowed to proceed at room temperature for 24 h. The reaction mixture was quenched with water, extracted with CH2Cl2 twice, washed with saturated brine three times, dried over anhydrous Na2SO4, and concentrated under reduced pressure to obtain a white solid. Purification was performed by silica gel column chromatography (DCM/MeOH: 100:1 → 80:1 → 60:1 → 40:1 → 20:1) to yield CTF with a yield of approximately 65%.

Synthesis of HAIR

The synthesis and characterization of HAIR was performed according to the procedure described in the reference (Figure S9 to S12). HA (30.00 mg, 0.08 mmol) was placed in a dry round-bottom flask and dissolved in formamide. EDC (28.60 mg, 0.15 mmol) and NHS (22.50 mg, 0.15 mmol) were slowly added to the solution, and the reaction mixture was stirred for 10 h to activate the HA. Subsequently, IR780NH2 (56.70 mg, 0.08 mmol) was slowly added dropwise to the solution, and the reaction was continued for an additional 12 h. The final product, HAIR, was obtained by purification using Sephadex LH-20 gel.

Detection of ROS response performance of CTF prodrug

Chromatographic Conditions: A ThermoFisher C18 reverse-phase column (250 × 4.6 mm, 2.5 μm) was used with a column temperature of 40 °C. The mobile phase was methanol/water (60:40, v/v) with a total flow rate of 1.0 mL/min. The fluorescence detector (FLD) settings were λEX = 360 nm and λEM = 580 nm, and the injection volume was 20 μL. A suitable amount of CTF was weighed and diluted with methanol to concentrations of 0.1, 0.25, 0.5, 0.8 and 1.0 μg/mL, with methanol as the blank solvent. Samples were analyzed under the above chromatographic conditions. A standard curve was plotted with concentration as the x-axis and peak area as the y-axis, and linear fitting was performed (Figure S13). Under 808 nm laser irradiation, IR780 can produce ROS that cleaves TK bonds, releasing free CPT and FUDR. Therefore, we used H2O2 to simulate an in vitro ROS environment to evaluate the hydrolysis of the prodrug. The CTF prodrug was placed in a methanol solution containing 50 mM H2O2 and stirred in a water bath at 37 ºC. At predetermined time points (0, 30, 60, 90, 120, 150, and 180 min), 100 μL samples were taken, and the release of free CPT and FUDR was detected using HPLC.

Preparation of HAIR/CTF nanoparticles

The prepared amphiphilic polymer HAIR (5 mg) was dissolved in DMSO solution (10 mg/ml). Subsequently, CTF (1 mg) solution (10 mg/mL) and DSPE-PEG2000 (10 mg) were added and the solituon was thoroughly mixed. Under sonication, the mixture was rapidly added dropwise into 5 mL of deionized water and sonicated for 5 min. Under these conditions, HAIR spontaneously formed nanoparticles in an aqueous solution (HAIR NPs) due to its amphiphilic nature, with CTF encapsulated within the hydrophobic core. Following this, the solution was dialyzed using a dialysis bag (MW cutoff = 3500 Da) against ultrapure water for 24 h to remove the organic solvent DMSO and any unencapsulated free drug. Finally, the solution was centrifuged using a 10 kDa ultrafiltration centrifuge tube at 5000 g for 15 min. The retained fraction was collected using a 200 μL pipette tip and diluted to the desired concentration.

Characterization of HAIR/CTF NPs

Take 100 μL of freshly prepared HAIR/CTF NPs nanosuspension and dilute it with 900 μL of deionized water. After thorough mixing, transfer the mixture to a particle size analyzer to measure the average particle size and polydispersity index (PDI) using a Malvern zeta sizer. Additionally, take 1 mL samples of IR-780, HAIR NPs, and HAIR/CTF NPs and place them in the Malvern zeta potential cell to measure their zeta potentials. Each sample should be measured three times for accuracy. Drop 10 μL of the HAIR/CTF NPs sample onto a 300-mesh copper grid, let it stand for 5 min, and remove excess liquid with filter paper. Negatively stain the sample with 2% phosphotungstic acid for 2 min, allow it to air dry naturally, and observe the morphology of the nanoparticles using a spherical aberration-corrected transmission electron microscope. Dilute free IR-780 and CTF in methanol to appropriate concentrations, using methanol as a blank solvent for baseline correction. Take 700–800 μL of each sample and place them in UV quartz cuvettes for UV–vis scanning in the wavelength range of 200–900 nm. Prepare HAIR/CTF NPs and dilute them to appropriate concentrations with ultrapure water, using ultrapure water as the blank solvent. Take 700–800 μL of each sample and place them in UV quartz cuvettes for UV–vis scanning in the wavelength range of 200–900 nm. Mix freshly prepared HAIR/CTF NPs with H2O, PBS, and 10% FBS in a 1:1 ratio, and measure their particle sizes at 0, 12, 24, 48, 72, and 96 h using a Malvern particle size analyzer.

Take 1 mL of freshly prepared HAIR/CTF NPs and transfer it to a 10 kDa ultrafiltration centrifuge tube. Centrifuge at 5000 g for 15 min to remove unencapsulated free drugs. Calculate the drug concentration in the filtrate. Use the following formula to calculate the encapsulation efficiency (EE) and drug loading efficiency (DLE) of CTF in HAIR/CTF NPs:

$$}\left( \% \right)\, = \,\left( }}}}} \right) \times 00\%$$

$$} - }\left( \% \right)\, = \,\frac}}}} \times 00\%$$

Photothermal and photodynamic property assessment of HAIR/CTF NPs

Accurately weigh 1.00 mg of DPBF and dissolve it in DMSO to prepare a stock solution with a concentration of 1 mg/mL. Take 500 μL of DPBF solution (20 μg/mL) and mix it with 500 μL of HAIR/CTF NPs (IR-780 concentration of 2 μg/mL) to achieve an absorbance value of approximately 1.0 at 412 nm for DPBF and approximately 0.3 for IR-780. Subsequently, irradiate the mixture with an 808 nm laser (1.0 W/cm2) for 200 s and measure the absorbance at 412 nm every 20 s using UV–vis spectroscopy (a total of 10 measurements).

Prepare fresh HAIR/CTF NPs samples and dilute them with deionized water to IR-780 concentrations of 5, 10, 15, 20, and 40 μg/mL, using deionized water as a blank control. Place 1 mL of each sample into a 24-well plate and irradiate with an 808 nm laser (1.0 W/cm2) for 5 min. Record the temperature changes of the solutions using an infrared thermal imaging camera. Place 1 mL of HAIR/CTF NPs sample (15 μg/mL) into a 24-well plate and irradiate with an 808 nm laser at powers of 0.5, 1.0, 1.5, and 2.0 W/cm2 for 5 min. Record the temperature changes using an infrared thermal imaging camera.

Seed 4T1 cells in 24-well plates at a density of 5 × 104 cells per well and incubate for 24 h to allow for cell attachment. After attachment, replace the old medium with the drug-containing medium. The groups include a blank control, Free IR-780, HAIR NPs, HAIR/CTF NPs, and their respective laser irradiation groups: PBS + Laser, Free IR-780 + NIR, HAIR NPs + NIR, and HAIR/CTF NPs + NIR. After 6 h of incubation, remove the medium, wash twice with PBS, and add 250 μL of 10 μM DCFH-DA diluted in serum-free medium to each well. Incubate in a 37 °C cell incubator for 20 min. Wash the cells three times with serum-free medium to thoroughly remove any DCFH-DA that has not entered the cells. Then, irradiate with an 808 nm laser (1 W/cm2) for 2 min. Finally, capture images using a fluorescence microscope.

Light-activated drug release behavior

The behavior of polymer nanoparticles under 808 nm laser irradiation and/or the presence of H2O2 was studied using a modified dialysis method. Briefly, 1 mL of HAIR/CTF NPs was sealed in a dialysis bag (molecular weight cut-off of 3500) and incubated in 15 mL of phosphate-buffered saline (PBS) containing 5 mM H2O2 at 37 °C with gentle shaking (100 rpm). At predetermined time points, the NIR group was exposed to 808 nm laser irradiation at 1 W/cm2. The media from all groups were then collected and replaced with pre-warmed fresh PBS. The CPT content was determined by HPLC.

Cell culture and animals

4T1 cells were obtained from Procell Co., LTD (Wuhan, China) and were cultured in RPMI 1640 medium containing NaHCO3 1.5 g/L, glucose 2.5 g/L and Sodium Pyruvate 0.11 g/L and supplemented with 10% FBS and 1% antibiotics (penicillin and streptomycin). Cells were incubated at 37 °C in humidified air with 5% CO2. Female BalB/C mice (6 weeks old) were obtained from SJA Laboratory Animals Co., LTD (Changsha, China) and housed following the guidelines of the Institutional Animal Care and Use Committee (IACUC). Animal experiments were performed according to the protocols approved by the Institutional Animal Care and Use Committees of the Department of Laboratory Animals of Central South University (No. 20240507).

Cellular uptake and in vivo targeting of HAIR/CTF NPs

4T1 cells were seeded in confocal dishes at a density of 5 × 104 cells per well and incubated for 24 h to allow for cell attachment. After attachment, the cells were treated with Free IR780, Free CTF, and HAIR/CTF NPs (IR780 concentration of 0.25 μg/mL, CTF concentration of 2 μg/mL) and incubated for 1 h and 4 h. The drug-containing medium was then removed, and the cells were washed twice with PBS. Subsequently, 60 μM Lyso-tracker green dye was added and incubated in the dark for 15 min. The medium was then removed, and the cells were washed twice with PBS. Finally, images were captured using a Zeiss confocal microscope. Additionally, 4T1 cells were seeded in 6-well plates at a density of 2 × 105 cells per well and incubated for 24 h to allow for cell attachment. After attachment, each well was treated with 2 mL of Free IR780 and HAIR/CTF NPs (IR780 concentration of 1.25 μg/mL) and incubated for 1, 4, 6, 8, and 10 h. At each time point, the drug-containing medium was removed, and the cells were washed twice with PBS. Each well was then treated with 250 μL of trypsin and incubated at 37 °C for 2 min to detach the cells. The cells were collected, and centrifuged at 800 rpm for 5 min, and the supernatant was carefully removed. The cells were washed once with PBS and then resuspended in 0.5 mL of PBS. Finally, the samples were analyzed by flow cytometry.

To examine the tumor-targeting ability of our nanoparticles, we used IVIS Spectrum Imaging System (PerkinElmer, USA) to observe nanoparticle biodistribution. BALB/C female mice (6 weeks old) were injected with 1 × 107 4T1 cells into the fourth left mammary fat pad to build the orthotopic 4T1 mice model. Free IR780 and HAIR/CTF NPs were then injected intravenously into BALB/C mice with 4T1 tumors (150 mm3) (n = 3). The mice were imaged on the IVIS system at specified time points of 2, 8, 12, and 24 h. After in vivo fluorescent imaging, the mice were sacrificed to collect tumors and major organs (heart, liver, spleen, lung, and kidney) for ex vivo imaging. We performed a quantification analysis of fluorescence intensity using the IVIS Spectrum Imaging Software (PerkinElmer, USA) and GraphPad Prism 9.0.0.d

In vitro cytotoxicity assay

4T1 cells were seeded in 96-well plates at a density of 5 × 103 cells per well and incubated for 24 h to allow for cell attachment. Following this incubation, the medium was replaced with drug-containing media according to the following groups: PBS, CTF, CPT/FUDR mixture, HAIR NPs, HAIR/CTF NPs, and the NIR irradiation groups PBS + NIR, HAIR NPs + NIR, HAIR/CTF NPs + NIR (the concentration of IR780 was 0, 0.0625, 0.125, 0.25, 0.5, 1, 1.25 μg/ml and corresponded CTF was 0, 0.5, 1, 2, 4, 8, 10 μg/ml). After 6 h of incubation, the media was replaced with fresh medium. The laser treatment groups were subjected to 808 nm laser irradiation (1.0 W/cm2) for 2 min and then incubated for an additional 12 h. Then the medium containing the drug was carefully removed after laser irradiation, and the cells were washed once with PBS. Each well was then filled with 10% CCK-8 diluted in serum-free medium, and a blank control group was established. The plates were incubated at 37 °C in the dark for 2 h, and the OD values at 450 nm were measured using a microplate reader.

For the live/dead cell staining assay, cells were seeded in 24-well plates at a density of 5 × 104 cells per well and incubated for 24 h to allow for cell attachment. After this incubation period, the medium was replaced with a drug-containing medium according to the following groups: PBS, HAIR NPs, HAIR/CTF NPs, and the laser irradiation groups PBS + NIR, HAIR NPs + NIR, HAIR/CTF NPs + NIR (the concentration of IR780 and CTF was 0.5 and 4 μg/ml, respectively). After 6 h of incubation, the medium was replaced with fresh medium. The NIR treatment groups received 808 nm laser irradiation (1.0 W/cm2) for 2 min per well, followed by an additional 12 h of incubation. The medium was then carefully removed, and the wells were washed once with PBS. A total of 250 μL of Calcein AM/PI staining solution was added to each well, and the plates were incubated at 37 °C in the dark for 30 min. The staining solution was then removed, and the wells were washed once with PBS. Finally, the staining was observed using a fluorescence microscope.

For the cell apoptosis evaluation, 4T1 cells were seeded in 6-well plates at a density of 3 × 105 cells per well and incubated at 37 °C with 5% CO₂ for 24 h to allow for cell attachment. After attachment, the cells were treated according to the following groups: PBS, HAIR NPs, HAIR/CTF NPs, and the NIR irradiation groups PBS + NIR, HAIR NPs + NIR, HAIR/CTF NPs + NIR (the concentration of IR780 and CTF was 0.5 and 4 μg/ml, respectively). After 6 h of incubation, the medium was replaced with fresh medium. The NIR treatment groups received 808 nm laser irradiation (1.0 W/cm2) for 2 min per well, followed by an additional 12 h of incubation. The supernatant containing floating cells was collected into centrifuge tubes and washed twice with PBS. Each well was then treated with 250 μL of EDTA-free trypsin and incubated at 37 °C for 10 min to detach the cells, which were gently pipetted into the same centrifuge tubes. The cells were centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cells were washed once with PBS. The cells were resuspended in 100 μL of 1 × Binding Buffer, and 5 μL of Annexin V/PE and 5 μL of 7-AAD were added to each tube, followed by gentle mixing. The samples were incubated at room temperature in the dark for 15 min, and then 400 μL of 1 × Binding Buffer was added and mixed thoroughly. The samples were analyzed by flow cytometry within 1 h.

In vitro immune activation investigation of HAIR/CTF NPs

The immune activation ability of HAIR/CTF NPs were evaluated by DCs maturation assay and immunogenetic cell death (ICD) biomarker detection. In DCs maturation assay, male C56BL6j mice (6 to 8 weeks old) were sacrificed to obtain BMDCs from sterile femur and tibia as reported. 4T1 cells were treated with PBS, PBS + NIR, IR780 + NIR, CTF, HAIR + CTF, HAIR/CTF NPs, HAIR/CTF NPs + NIR. Then BMDCs were added into cell culture medium of treated 4T1 cells and co-incubated for 24 h. After incubation, cells were stained by APC anti-CD11c, PE anti-CD80 and PE-Cy7 anti-CD86. Flow cytometry was performed to investigate the activation of BMDCs.

Similarly, 4T1 cells were treated with different formulations as mentioned above. Then cells were rinsed with PBS and lysed with RIPA lysis buffer containing a protease inhibitor cocktail. Cell lysate was centrifuged at 10,000 rpm for 10 min and total protein in supernatant was measured by BCA assay. Cell culture medium was collected for HMGB1 detection. CRT and HMGB1 protein level was determined by western blot assay. And ATP level in cell supernatant was evaluated by ELISA kit.

Antitumor efficacy of HAIR/CTF NPs in both primary and abscopal tumors

4T1 cells were cultured on 100 mm cell dishes in 37 ℃ cell incubator. After digestion and centrifugation, 1 × 106 cells were inoculated into the fourth left mammary fat pad of BalB/C mice (female, 6 weeks old) to establish an orthotopic breast cancer model. The tumor volume (cubic millimeter) was calculated as 1/2 × long diameter × (short diameter)2. When the tumor volume reached 100 mm3, mice were randomly divided into 7 groups (n = 6): PBS, PBS + NIR, CTF, HAIR + CTF, HAIR/CTF NPs, HAIR NPs + NIR, HAIR/CTF NPs + NIR. Each mice were treated with different drugs every two days via intravenous injection in the vein of the tail (the injection dose was 100 μl, and the concentration of CPT, FUDR and HAIR was 5 mg/kg, 5 mg/kg and 2 mg/kg), the near-infrared laser irradiation was conducted 24 h after administration (808 nm, 1 W/cm2, 5 min). Tumor volume and mice weight were monitored at the interval of two days. When the tumor volume reached more than 1000 mm3, mice were sacrificed and tumors, blood samples and major organs (heart, liver, spleen, lung, kidney) were collected for later evaluation. Tumors were weighed and fixed by paraformaldehyde for further H&E, TUNEL and Ki67 staining. Blood samples were used to determine the serum level of aminotransferase (ALT), aspartate aminotransferase (AST), blood urea (Ur), and creatinine (Cr) for safety assessment. Major organs were fixed by paraformaldehyde for H&E staining to observe tissue damage.

To further investigate the distant tumor inhibition effect of our NPs, we implanted abscopal tumors into the right mammary fat pad of the mice when the primary tumor volume reached 100 mm3. the mice were randomly divided into 5 groups: PBS, HAIR + CTF, HAIR/CTF NPs, HAIR NPs + NIR, HAIR/CTF NPs + NIR (n = 3). The primary tumors were administrated via intratumoral injection while no administration was given to distant tumors. After the final intervention, the mice were euthanized for subsequent evaluation. Systemic immune activation was assessed in distant tumor models by collecting blood samples from mice. The samples were centrifuged at 300g for 5 min to isolate all cells. Red blood cells were then lysed using a lysis buffer and the solution was centrifuged again to obtain cell pellets. Buffer was added to resuspend the cell pellets, followed by using Fixable Viability Dye eFluor™ 506 (65–0866-14, eBioscience) and APC-Cy7 anti-CD45 (557,659, BD Biosciences) to stain total immune cells.

Antitumor immunity evaluation of HAIR/CTF NPs

After euthanizing the mice, we collected the tumors and tumor-draining lymph nodes. These tissues were then transferred to six-well plates (NEST, China) containing a buffer solution of DPBS with 5% FBS. Tissue samples were ground into pieces smaller than 1 mm using a syringe piston. They were then washed twice with the buffer and filtered through a 70 μm cell filter (Biosharp, China) to obtain single-cell suspensions. Lysis buffer was added to lyse red blood cells. Afterward, samples were centrifuged under 300g for 5 min to collect cell pellets. The resuspended cell samples were divided into two parts for DCs and T cell analysis after adjusting cell intensity to 1 × 106 cells/ml. After incubation with Fixable Viability Dye eFluor™ 506 (65–0866-14, eBioscience), the samples were further incubated at 4 °C for 5 min with anti-CD16/32 mAb (553,141, BD Biosciences) to block background signals. For DC analysis, cells were stained with the following antibodies: APC-Cy7 anti-CD45 (557,659, BD Biosciences), FITC anti-CD3 (553,061, BD Biosciences), APC anti-CD11c (227,166, BD Biosciences), PerCP-Cy5.5 anti-IA/IE (746,197, BD Biosciences), PE-Cy7 anti-CD86 (25–0862-82, BD Biosciences), and PE anti-CD80 (12–0801-80, BD Biosciences). To determine T cells, the cells were incubated with APC-Cy7 anti-CD45, FITC anti-CD3 antibodies, PerCP-Cy5.5 anti-CD4 (550,954, BD Biosciences), and PE-Cy7 anti-CD8 (552,877, BD Biosciences). Staining and incubation followed the manufacturer’s protocols. After staining, the cells were washed twice and analyzed using flow cytometry (NL3000, Cytek Bioscience, USA).

Ethics declarations

Female BALB/C mice (6 weeks old) and male C57BL6 (8 weeks old) were obtained from SJA Laboratory Animal Co., LTD (Changsha, China) and housed following the guidelines of the Institutional Animal Care and Use Committee (IACUC). Animal experiments were performed according to the protocols approved by the Institutional Animal Care and Use Committees of the Department of Laboratory Animals of Central South University (No. 20240507).

Statistical analysis

Data were presented as mean ± SD. Statistical analysis was performed by GraphPad Prism (9.5.0). T-test was used for comparison between the two groups. One-way ANOVA or Two-way ANOVA using the Tukey’s multiple-comparison test was applied for comparison among three or more groups. The number of replicates is indicated in the legend of each figure. Specific statistical analysis methods were illustrated in figure legends.