Chemicals and reagents. 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC), distearoyl phosphoethanola-mine-PEG2000 (DSPE-PEG2000), 1,2-dioleoyl-3-trimethylam-monium-propane (DOTAP) were purchased from Meryer (Shanghai) Chemical Technology Co., Ltd. Chloroform (CHCl3) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Auranofin(AUR) was purchased from Target Molecule Corp. AptATP, eCpG, aptPD-L and deoxyribonucrenase I were all purchased from Sangon Biotech (Shanghai) Co., LTD. Peptide nucleic acid (PmP) was purchased from Tahepna Biotechnologies Co., Ltd. Adenosine triphosphate (ATP) was purchased from Beijing Solarbio Science & Technology Co., Ltd. Recombinant matrix metalloproteinase-2 (MMP-2) was purchased from MedChemExpress(MCE).
Cell lines and animal. B16F10 and NIH3T3 cell lines were bought from Yeze Shanghai Biological Technology Co., LTD. B16F10-luc cell line was bought from Nanjing Wanmuchun Biotechnology Co., LTD. C57BL/6 (female, 6-week-old) were provided by in the Second Affiliated Hospital of the Army Medical University (Xinqiao Hospital) and all mice were kept in the animal house of Xinqiao Hospital. All characterizations were carried out following the Animal Management Rules of the Ministry of Health of the People's Republic of China.
Synthesis of Lip@AUR. 7.255mgDMPC, 1.516mg DSPE-PEG2000, 1.963mgDOTAP, 2mgAUR were added into a clean 500mL single-neck flask and dissolved by adding 10mL chloroform, stirred and ultrasonicated for 5min. Lipid film was obtained by rotary evaporation at 80rpm and 40℃ in a water bath overnight. The lipid membranes were rehydrated using 10mL sterile PBS and ultrasonicated for 30min. Impurities or aggregates were removed by centrifugation at 3000rpm for 10min. The liposomes were filtered through 0.22µm membrane and repeatedly extruded by an extruder for about 10 times, followed by dialysis with an MWCO of 1000 Da for two days to obtain Lip@AUR.
Construction of aptATP/eCpG/PmP (ACP) assembly. Moderate amount of DEPC water was added to solubilize the synthesized aptATP, eCpG, and PmP powder at 100µM. aptATP, eCpG, PmP solutions were placed in clean 1.5mL EP tubes. aptATP and eCpG samples were heat in 95℃ oil bath for 10min and then mixed in the ratio of aptATP:eCpG = 2:1, followed by further incubation in the oven at 42℃ for 1h. PmP was added to aptATP/eCpG at the ratio of aptATP:PmP = 1:1.5 and heated in oil bath at 80℃-90℃ for 10min. ACP assembly was obtained after incubating in oven at 42℃ for 1h.
Synthesis of Lip@AUR-ACP-aptPD-L1. Firstly, Lip@AUR was refrigerated at -80℃ and then freeze-dried in a freeze dryer to obtain liposome powder. The powder was rehydrated by DEPC water and mixed with ACP assemblies with the molarity ratio of lipid: aptATP = 80:2, and incubated in the oven at 37℃ for 4h. aptPD-L1 powder was resuspended with DEPC water at 100µM, and then aptPD-L1 was added at the molarityratio of lipid: aptATP: aptPD-L1 = 80:2:1. AptPD-L1 was incubated with Lip@AUR-ACP overnight in a 37℃ oven to obtain Lip@AUR-ACP-aptPD-L1. The product was frozen at -80℃ and then freeze-dried to obtain Lip@AUR-ACP-aptPD-L1 powder.
DNA-PAGE analysis regarding aptamer binding and release. The formulation of 20%PAGE solution is as follows: 6.666mL 30% acrylamide, 1mL 10×TBE buffer, 2.3µL DEPC water, 50µL 10%APS, 5µL TEMED. After solidification, the corresponding samples were added to each hole and then electrophoresis was carried out at 140V constant voltage. After electrophoresis, 0.29g NaCl was dissolved in 50mL deionized water and mixed with 5µL GelRed. The gel was soaked in GelRed solution for 30min and then taken out for observation with a gel imaging system.
Loading and releasing of AUR. Firstly, 2%Triton X-100 solution was prepared with PBS, while 1mg Lip@AUR-ACP-aptPD-L1 powder was dissolved in 1mL PBS to afford Lip@AUR-ACP-aptPD-L1 solution. 100µL Lip@AUR-ACP-aptPD-L1 solution was added into 900µL 2%Triton X-100 solution and incubated at 37℃ for 1h to lyse the liposomes and release AUR. The AUR release was detected by fluorescence spectrophotometer and quantified via standard curve calibration.
The release of eCpG. For ease of understanding, Cy5 labeled eCpG was denoted as eCpGCy5, while the molecular complex of eCpGCy5 and aptATP was denoted as ACCy5. After the complementary binding with PmP, the aptamer assembly was denoted as ACCy5P. Finally, the aptamer-based ligands were inserted into liposomal membrane to afford Lip@AUR-ACCy5-aptPD-L1 or Lip@AUR-ACCy5P-aptPD-L1. Lip@AUR-ACCy5-aptPD-L1, Lip@AUR-ACCy5P-aptPD-L1, Lip@AUR-ACCy5P-aptPD-L1 + MMP-2 (5nM) and Lip@AUR-ACCy5P-aptPD-L1 + MMP-2(10nM) groups were treated with ATP and then centrifuged under 5000rpm for 10min to extract the supernatant. The release of eCpGCy5 was measured via fluorescence spectroscopy.
Loading analysis of eCpG and aptPD-L1. The synthesis of fluorescently labeled liposomes was generally the same with those unmarked ones except that the original aptamers were replaced by Cy5-labeled eCpG or FAM-labeled aptPD-L1, leading to the formation of Lip@AUR-ACCy5P-aptPD-L1 or Lip@AUR-ACP-aptPD-L1FAM. 56µL Lip@AUR-ACCy5P-aptPD-L1 (5mg·mL− 1) or Lip@AUR-ACP-aptPD-L1FAM (5mg·mL− 1) aqueous solution was added to 1×DNase 1 buffer solution and then treated with 20U·mL− 1 DNase 1. They were incubated at 37℃ for 15min and transferred to an ultrafiltration tube. After centrifugation at 10,000rpm for 15min, the supernatant was collected and fluorescence intensity of Cy5 or FAM was detected by fluorescence spectrophotometer. ECpGCy5 or aptPD-L1FAM solution with different concentrations were configured to establish the standard curve via a fluorescence spectrophotometer. The aptamer concentration in Lip@AUR-ACCy5P-aptPD-L1 or Lip@AUR-ACP-aptPD-L1FAM was quantified according to the standard curve, and then the load efficiency of eCpGCy5 or aptPD-L1FAM on liposomes was calculated accordingly.
Morphological characterization of Lip@AUR-ACP-aptPD-L1. 5µL of Lip@AUR-ACP-aptPD-L1 solution was dropped on the carbon support film and dried naturally. Then the film was re-dyed with 4% phosphotungstic acid solution for 3 times (10min each time) to observe its morphology with a transmission electron microscope.
Cell culture. Mouse-derived melanoma cell line B16F10 was cultured in 1640 medium containing 10% fetal bovine serum (Gibco), penicillin (100 µg·mL− 1), and streptomycin (100 µg·mL− 1). Mouse embryonic fibroblasts NIH3T3 and B16F10-luc cell lines were cultured in high-glucose DMEM medium containing 10% fetal bovine serum (Gibco), penicillin (100 µg·mL− 1), and streptomycin (100 µg·mL− 1). The cells were cultured in a 37℃ constant temperature incubator containing 5% carbon dioxide.
For cellular related experiments with MMP-2 pretreatment, the MMP-2 concentration was 10nM and the incubation time was 2h.
Extraction of splenocytes from C57BL/6 mice. Scissors, tweezers, sterile 40µm cell filter and other utensils were sterilized for 30min by ultraviolet light on ultra-clean workbench. C57BL/6 mice were sacrificed and treated with 75% alcohol for 10min. The spleen of the mice was dissected on a clean table. The cell strainer was placed into a six-well plate containing RPMI1640 medium, and the spleen was placed in the strainer. The spleen was pulverized with the tip of the suction head of a sterile 5mL syringe, and the strainer was removed after grinding until no obvious spleen tissue was found on the filter. The cells collected from the six-well plate were homogenized and transferred to a centrifuge tube, centrifuged at 2000rpm for 5min. The supernatant was discarded, the red blood cell lysate was added and mixed for 10min, and the lysis was terminated by adding 7 times the volume of PBS. After centrifugation at 2000rpm for 5min, cells were collected.
Effects of different samples on the activity of B16F10 cells or immune cells. Toxicity analysis of Lip@AUR-aptPD-L1 to B16F10 cells. B16F10 cells were inoculated into the 96-well plate with a density of 1×104 cells per well. When the cell confluence reached 80%, B16F10 cells were mixed with splenocytes at a ratio of 1:10 for co-culture. Medium containing different concentrations of Lip@AUR or Lip@AUR-aptPD-L1 was added for incubation for 12h, and the fresh medium was used as blank control (TCPS). 100µL of serum-free fresh medium containing MTT reagent (0.5 mg·mL− 1) was added to each well, and MTT agent was discarded after incubation at 37℃ for 4h in the dark. Then the absorption intensity of the sample was measured at 490 nm by SpectraMax i3x microplate reader using 100µL dimethyl sulfoxide (DMSO) to dissolve emerging crystals.
Toxicity of Lip@AUR-aptPD-L1 to B16F10 cells or immune cells at different IR doses. B16F10 cells were inoculated into the 24-well plate with a density of 5×104 cells per well. When the cell confluence reached 80%, cells were incubated with medium containing 40µg·mL− 1 Lip@AUR-aptPD-L1 or Lip@AUR-aptPD-L1 for 12h and fresh medium was used as blank control (TCPS). After incubation, 500µL serum-free fresh medium containing MTT reagent (0.5 mg·mL− 1) was added to each well, and MTT agent was discarded after incubation at 37℃ for 4h in the dark. Afterwards, 300µL DMSO was added into each well and homogenized, 100µL of the added DMSO was extracted from each well for analysis. The OD values of the sample were measured at the wavelength of 490 nm using SpectraMax i3x microplate reader. After placing splenocytes in the 12-well plate at a concentration of 1×106 per well, the drug was administered in the same way as above for 12h, and then were stained with CCK-8 for 2h and transferred to a clean 96-well plate. The OD values of the samples were measured at the wavelength of 450 nm using a SpectraMax i3x microplate reader.
Toxicity of Lip@AUR-aptPD-L1 + RT on B16F10 cells. B16F10 cells were inoculated into 24-well plates with a density of 5×104 cells per well. When the cell confluence reached 80%, the co-culture system was constructed with the B16F10: splenocyte ratio of 1:10 and incubated with fresh medium containing different concentrations Lip@AUR-aptPD-L1 for 12h. After incubation, IR groups were treated with 4Gy IR. Splenocytes and tumor cells were separated, and 500µL serum-free fresh medium containing MTT reagent (0.5 mg·mL− 1) was added to each well of tumor cells, and the rest treatment was kept the same.
Concentration-dependent toxicity evaluation. B16F10 cells were inoculated into the 24-well plate with a density of 5×104 cells per well. When the cell confluence reached 80%, the co-culture system was established with the B16F10: splenocyte ratio of 1:10. I: Lip, II: Lip-aptPD-L1, III: Lip-ACP-aptPD-L1, IV: Lip@AUR-aptPD-L1, V: Lip@AUR-ACP-aptPD-L1 (40µg·mL− 1) was incubated for 12h with fresh medium as blank control (TCPS). After incubation, IR groups were treated with 4Gy IR. Splenocytes and tumor cells were separated, and 500µL serum-free fresh medium containing MTT reagent (0.5 mg·mL− 1) was added to each well of tumor cells, and the rest treatment was kept the same.
Flow cytometric analysis on the receptor binding effect of aptPD-L1 and eCpG. B16F10 cells were mixed with splenocytes at a ratio of 1:10 and transferred to an EP tube. 170nM aptPD-L1FAM and 360nM eCpGFAM were added and incubated for 30min, followed by the addition of 1µL APC-anti-CD11c and 1µL PE-anti-MHCII antibody. Flow cytometry was used to detect the binding status between aptPD-L1FAM and B16F10 cells or between eCpGFAM and DCs.
Tumor cell targeting and membrane fusion. Here orange-red probe Dil was loaded into the liposome instead of AUR for fluorescence tracking, of which the samples were denoted as Lip@Dil and Lip@Dil-aptPD-L1. B16F10 or NIH3T3 cells were inoculated into confocal dishes at a density of 1×105cells/well. When the cell confluence reached 80%, the co-culture system was established with the B16F10: splenocyte ratio of 1:10. Subsequently, samples were added and treated for 1, 3, 6, 12, 18 h, respectively. For the IR-incorporated groups of B16F10cells, 4Gy IR was applied 12 h after the addition of nanosamples, and the incubation would continue for 4, 8, 16 h. The cell membrane was stained with Cellmask orange for 10min, while cell nucleus was stained with DAPI for 10min after cleaning with PBS. The cell samples were mounted on the glass slides and sealed with glycerin, and the membrane fusion status was detected by laser confocal microscopy.
B16F10 tumor sphere assay for testing targeting effect. 90mg agarose gel was dissolved in 6mL serum-free 1640 medium and sterilized at 115℃ for 30min. 80µL of the melted gel was added into sterile 96-well plates and cooled down naturally for solidification. The B16F10 cells were homogenized in 1640 medium containing 2.5% matrix gel and added into the wells at 5000 cells per well, of which the volume was 100µL per well. The cells were cultured for about 7 days until pellets were formed under an optical microscope. ACCy5P, Lip-ACCy5P or Lip-ACCy5P-aptPD-L1 were added and incubated for 12h, then cells were detached, centrifuged at 700rpm for 5min to remove matrix gel, cleaned with PBS for 3 times, and transferred to a confocal laser confocal dish for detection.
ICP assay for determining AUR uptake. B16F10 cells were inoculated into 6-well plates with an initial cell density of 3×105 cells/well. After the cell confluence reached 80%, fresh medium containing Lip@AUR or Lip@AUR-aptPD-L1 was added, and untreated cells were used as control. After incubation for 1, 3, 6, 12 and 18 h, the cells were digested by trypsin and collected by centrifugation. After 24h of lysis, supernatant was extracted by centrifugation at 1500 rpm for 5min, while pure AUR solution with concentration gradient was configured for establishing the standard curve. The volume of the above samples was maintained at 5mL. Finally, inductively coupled plasma emission spectroscopy was used to determine AUR uptake in each group.
ATP abundance and MMP-2 expression levels in B16F10 cells or tumors. B16F10 cells were inoculated into 12-well plates, and the initial cell density was 1×105 cells/well. When the cell confluence reached 80%, B16F10 cells were treated with Lip@AUR-aptPD-L1 for 12h and irradiated with 4Gy IR. The concentrations of total ATP in 2, 4, 12, 18, and 24h after radiotherapy were detected by ATP assay kit. For the in vivo analysis, mice were treated with PBS, Lip, Lip@AUR or Lip@AUR-aptPD-L1 (2mg·kg− 1), followed by 4Gy IR at 12 h post intravenous injection. The concentrations of ATP and MMP-2 in each tumor were detected by relevant kits.
AUR induced secretion of critical DAMPs. B16F10 cells were inoculated into 12-well plates, and the initial cell density was 1×105 cells/well. When the cell confluence reached 80%, B16F10 cells were treated with Lip@AUR-aptPD-L1 for 12h and irradiated with 4Gy. The cell supernatant was collected after the whole culture was continued for 18h. Then the concentration of ATP was detected by kit, and the secretion of CRT and HMGB1 in supernatant was detected by ELISA.
CLSM and flow cytometry for determining eCpG release in vitro. B16F10 cells were inoculated into the confocal dish, and the initial cell density was 1×105cells/well. When the cell confluence reached 80%, the co-culture system was established with the B16F10: splenocyte ratio of 1:10. B16F10 cells were treated with PBS, Lip-ACCy5-aptPD-L1 and Lip-ACCy5P-aptPD-L1 for 12h and then treated with 4Gy IR. Confocal and flow cytometry were used to analyze the fluorescence retention on cell membrane under -RT + 4h and RT + 4h conditions.
Validation of AND-gate release of eCpG. B16F10 cells were inoculated into 12-well plates, and the initial cell density was 1×105 cells/well. When the cell confluence reached 80%, B16F10 cells were treated with Lip-ACCy5P-aptPD-L1 for 12h and irradiated with 4Gy IR. Supernatant was collected after incubating for another 18h. The B16F10 cell membrane was stained with Cellmask orange for 10min, while cell nucleus was stained with DAPI for 10min after cleaning with PBS. The cell samples were mounted on the glass slides and sealed with glycerin, and the Cy5 fluorescence intensity on the membrane was detected by laser confocal microscopy. The fluorescence intensity of Cy5 in supernatant was measured by a fluorescence spectrophotometer.
Analysis of eCpG-DC binding and stimulation of DC maturation. After incubation for 30min with Cy5-labeled sequences, the fluorescence intensity of Cy5 on DCs was verified by flow cytometry for profiling aptamer binding. Subsequently, B16F10 cells were inoculated into 12-well plates, and the initial cell density was 1×105 cells/well. When the cell confluence reached 80%, the co-culture system was established with the B16F10: splenocyte ratio of 1:10. Lip@AUR-ACP-aptPD-L1 was co-incubated with B16F10 cells and splenocytes for 12h to detect DC maturation without radiation treatment or at 4, 8, 12, 18 and 24h after 4Gy IR treatment. The mutant or blocked sequences were also co-incubated with DCs for 18h, and the stimulation effect of DC maturation was detected by flow cytometry.
Transcriptome sequencing and protein expression evaluation. B16F10 cells were inoculated into a 100mm cell culture dish, and the initial cell density was 2×106cells/well. When the cell confluence reached 80%, the co-culture system was established with the B16F10: splenocyte ratio of 1:10. After B16F10 cells were treated with PBS, Lip-aptPD-L1, Lip@AUR-aptPD-L1, the tumor cells were extracted and sent to Sangon Biotech (Shanghai) Co., LTD for detection. For the WB assay, B16F10 cells were inoculated into 100mm cell culture dish, and the initial cell density was 1×106cells/well. When the cell confluence reached 80%, the co-culture system was established with the B16F10: splenocyte ratio of 1:10. The cells were treated with PBS, Lip, Lip-aptPD-L1, Lip ACP-aptPD-L1, Lip@AUR-aptPD-L1, Lip@AUR-ACP-aptPD-L1 for 12h, and then cultured for 18h after 4Gy IR. The cells were collected and treated with RIPA lysis solution on ice for 30min to extract markers of interest, which was then subjected to WB assay kit for imaging and quantitative analysis.
Evaluation on the impact of VEGF on anti-tumor immunity. B16F10 cells were inoculated into the 12-well plate at the concentration of 1×105 per well. When the cell confluence reached 80%, the cells were treated with PBS, Lip, Lip@AUR, Lip@AUR-aptPD-L1, the upper chamber is placed into 12-well plate. Splenocytes were added into the upper chamber with B16F10: splenocyte ratio of 1:10. After 12h of co-incubation, the IR groups were treated with 4Gy IR. The culture continued for 18h, cells in the upper chamber were discarded and the bottom chamber supernatant was collected. After centrifugation at 2000rpm for 5min, 200µL PBS was added to each tube to resuspend the spleen immune cells. 1µL APC-anti-CD25/FITC-anti-CTLA-4/PE-anti-CD4 or 1µL APC-anti-CD45/FITC-anti-CD11b/PE-anti-GR1 were added into each tube. Finally, the infiltration of Tregs or MDSCs in the bottom chamber was detected by flow cytometry.
Alternatively, the recovered cell samples in the bottom chamber were treated with 1µL APC-anti-CD3/1µL FITC-anti-CD4/1µL PE-anti-CD8a or 1µL APC-anti-CD11c/1µL FITC-anti-CD80/1µL PE-anti-CD86 were added into each tube. Finally, the infiltration of effector T cells or DCs was detected by flow cytometry.
The B16F10 tumor-bearing mouse model was constructed and treated with PBS, Lip, Lip@AUR, Lip@AUR-aptPD-L1 (2mg·kg− 1) for 12h and treated with 4Gy IR. Tumors were collected from each group after treatment and pulverized to collect various cell populations. 200µL PBS was added to each tube to suspend tumor cells. 1µL APC-anti-CD25/1µL FITC-anti-CTLA-4/1µL PE-anti-CD4 or 1µL APC-anti-CD45/1µL FITC-anti-CD11b/1µL PE-anti-GR1 were added into each tube. Finally, the infiltration of Tregs or MDSCs in tumor tissues was detected by flow cytometry.
Evaluation of immune activation effect of nanoparticlesin vitro. Splenocytes of C57BL/6 mice were extracted and DCs were sorted out according to the above method. B16F10 cells were inoculated into 12-well plates with the initial cell density of 1×105cells/well. When the cell confluence reached 80%, mouse DCs were added into 12-well plates and co-cultured with B16F10 cells at a ratio of B16F10: DC = 1:10. After 12 h treatment with PBS, Lip, Lip-aptPD-L1, Lip-ACP-aptPD-L1, Lip@AUR-aptPD-L1, Lip@AUR-ACP-aptPD-L1, the IR groups were treated with 4Gy IR and incubated for another 18h. DCs were collected via centrifugation and supernatant was recovered for later use. DCs was resuspended with 200µL PBS and 1µL APC-anti-CD11c/1µL FITC-anti-CD80/1µL PE-anti-CD86 antibodies or 1µL APC-anti-CD11c/1µL PE-anti-MHCII antibodies. The treatment-induced stimulation effect on DCs maturation in each group was detected by flow cytometry. The supernatant was used to detect the concentrations of cytokines TNF-α and IL-2 by ELISA kit.
After B16F10 cells were inoculated into the 12-well plate in the above way, mouse splenocytes were added into the 12-well plate and co-cultured with B16F10 cells at the B16F10: splenocyte ratio of 1:10. Splenocytes and supernatants were collected after treatment for later use. Here the splenocytes were suspended with 200µL PBS, 1µL APC-anti-CD3/1µL FITC-anti-CD4/1µL PE-anti-CD8a or 1µL APC-anti-CD3/1µL FITC-anti-IFN-γ/1µL PE-anti-CD8a antibodies were added to each tube. Finally, the activation status of T cells in each group was detected by flow cytometry. TNF-α, IL-2, IFN-γ and CXCL10 secretion was detected by ELISA kit.
Detection of tumor cell apoptosis: C57BL/6 mouse splenocytes were extracted by the above method. B16F10 cells were inoculated into the 12-well plate with the initial cell density of 1×105cells/well. When the cell confluence reached 80%, mouse splenocytes were added into the 12-well plate at the B16F10: splenocyte ratio of 1:10, and the supernatant was collected after relevant treatment. Tumor cells were digested by trypsin, then suspended with 200µL FITC bonding solution at 37℃ for 30min, followed by PI dye solution for 10min. After extensive staining, apoptosis of tumor cells under different treatments was detected by flow cytometry.
For the imaging analysis of melanoma cell apoptosis, B16F10 cells were inoculated into confocal dishes with the initial cell density of 1×105cells/well. When the cell confluence reached 80%, mouse splenocytes were added into the 12-well plate at the B16F10: splenocyte ratio of 1:10. After treatment was complete, the cells were washed with PBS for 3 times and splenocytes were immediately drained. The cells were fixed with 4% paraformaldehyde for 30min, blocked with 5% bovine serum albumin solution for 30min after cleaning, and permeabilized with 0.5%Triton X-100 solution for 5min after cleaning with PBS. Then γ-H2AX antibody was added and incubated at 4℃ overnight. The primary antibody was removed, and Cy3-labeled fluorescent secondary antibody was added after purification, followed by the incubation at room temperature for another 2h. The secondary antibody was removed and the cell nuclei were stained with DAPI for 10min after washing with PBS. After cleaning, the cell samples were mounted on glass slides with glycerin and the immunofluorescence of γ-H2AX was detected by confocal laser microscopy.
Blood circulation stability of different samples. B16F10-luc tumor cells (1×106 cells) were injected subcutaneously into 6-week-old mice to establish B16F10-luc tumor mouse model. The mice were cultured continuously until the tumor size reached 100mm3 and the body weight of mice was maintained at 17.5 ± 0.3g. Three groups of mice were randomly selected and intravenously injected AUR, Lip@AUR, Lip@AUR-aptPD-L1(2mg·kg− 1), respectively. Then tail venous blood was collected according to the scheduled time point, and AUR content in samples of each group was detected by HPLC.
ICP-dependent blood distribution analysis. B16F10-luc tumor cells (1×106 cells) were injected subcutaneously into 6-week-old mice to establish B16F10-luc tumor mouse model. The mice were cultured continuously until the tumor size reached 100mm3 and the body weight of mice was maintained at 17.5 ± 0.3g. Three groups of mice were randomly selected and intravenously injected with AUR, Lip@AUR and Lip@AUR-aptPD-L1 (2mg·kg− 1), respectively. The mice in each group were euthanized at predetermined time points to collect major organs and tumors were collected, and the supernatant was collected after grinding and cracking for 24h. The samples were filled to 5mL with deionized water, and the AUR concentration in each tissue was detected by ICP.
Antitumor evaluation of the liposomesin vivo. C57BL/6 mice were used in animal experiments and were kept in the Second Affiliated Hospital of the Army Medical University (Xinqiao Hospital). All animal tests have been reviewed and approved by the Animal Care and Use Committee of Laboratory Animals Administration of Xinqiao Hospital, which strictly followed the national and institutional guidelines. B16F10-luc tumor cells (1×106 cells) were injected subcutaneously into 6-week-old mice to establish B16F10-luc tumor mouse model. The mice were cultured continuously until the tumor size reached 100mm3 and the body weight of mice was maintained at 17.5 ± 0.3g(n = 5). They were randomly divided into 12 groups with 5 animals in each group, which were subjected to intravenous injection of PBS (100µL) containing Lip, Lip-aptPD-L1, Lip-ACP-aptPD-L1, Lip@AUR-aptPD-L1, Lip@AUR-ACP-aptPD-L1 (2mg·kg− 1), and the same volume of fresh PBS was administered as the control group. 12h after injection, the IR groups were treated with 4Gy IR. Treatment was performed once every 5 days for a total of 15 days. Bioluminescence imaging was performed every 5 days, and 20µL (7.5mg·mL− 1) luciferase was injected into the intraperitoneal cavity of mice. After anesthesia with isoflurane, tumor volume of each group was detected by IVIS imaging system. The tumor volume and body weight of mice were recorded by electronic balance and vernier caliper. The volume and size of the tumor were measured every two days, and the longitudinal and transverse diameters of the tumor were measured. The calculation formula was V = 1/2*A*B2 (A was the longitudinal diameter, B was the transverse diameter). After 15 days of treatment, serums of all tumor mice were collected, and tumor tissues and major organs were collected for subsequent analysis. A parallel set of animal models were established, and the survival of mice in each group was observed until the 50th day after the 15-day treatment (n = 6).
At the end of treatment, the tumors in each group were dissected, and the tumors were pulverized after freezing with liquid nitrogen, and then the cells were disintegrated by tip ultrasonication. The grinded tumors were treated with cell lysis solution on ice, and Western blot assay was carried out to detect the expression levels of related proteins in the tumor. Paraffin sections of tumor and heart, liver, spleen, lung and kidney were created for optical imaging after H&E staining. The tumor was dissected and cleaned with PBS, and further cut into thin sections for TUNEL staining, CD4/CD8/IFN-γ immunofluorescence staining, CRT/HMGB1 immunofluorescence staining and γ-H2AX immunofluorescence staining using related assay kits and observed by CLSM.
The tumor was ground and treated with red cell lysate for 15min, followed by the treatment with 1µL APC-anti-CD45, 1µL APC-anti-CD3/1µL FITC-anti-CD4/1µL PE-anti-CD8a antibodies, 1µL APC-anti-CD3/1µL FITC-anti-IFN-γ /1µL PE-anti-CD8a antibodies, 1µL APC-anti-CD11c/1µL FITC-anti-CD80/1µL PE-anti-CD86 antibodies or 1µL APC-anti-CD11c/1µLPE-anti-MHCII antibodies. The tumor cells were incubated and detected by flow cytometry. IFN-γ, TNF-α, CXCL10 and IL-2 levels in collected blood samples were detected using ELISA kits.
Establishment and treatment of bilateral tumor model in C57BL/6 mice. 1×106 B16F10-luc cells were injected subcutaneously into the right flank of C57BL/6 mice to establish B16F10 tumor bearing mice. They were cultured in the same way as above and divided into groups (n = 5), and intravenously injected with PBS, Lip, Lip-aptPD-L1, Lip-ACP-aptPD-L1, Lip@AUR-aptPD-L1, Lip@AUR-ACP-aptPD-L1 (2mg·mL− 1) (100µL). After 15 days of treatment, Secondary tumors were established by subcutaneous injection of 2×106 B16F10-luc cells on the left flank. The growth of distal tumor was monitored from the 18th day, and the treatment ended on the 28th day. Bilateral tumors were dissected for analysis. In addition, a batch of bilateral tumor models were established. After 15 days of treatment, the survival of mice in each group was observed for up to 50 days(n = 6). The primary and distal tumors were dissected, cleaned with PBS, pulverized and treated with erythrocyte lysate for detection. Cells in the primary tumors in each group were labeled with 1µL APC-anti-CD11c/1µL FITC-anti-CD80/1µL PE-anti-CD86 antibodies or 1µL APC-anti-CD3/1µL FITC-anti-CD4/ 1µL PE-anti-CD8a antibodies or 1µL APC-anti-CD62L/1µL FITC-anti-CD44/ 1µL PE-anti-CD8a antibodies, and the infiltration of immune cells was detected by flow cytometry. Distal tumors were also treated similarly.