Materials. 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was purchased from AVT Pharmaceutical Tech Co. (Shanghai). DSPE-Hyd-PEG5000-MAL and DSPE-PEG2000-NH2 were obtained from Xi'an Ruixi Biological Technology Co., Ltd. 1-MT was purchased from Sigma–Aldrich (United States). IR780 was obtained from Shanghai Aladdin Bio-Chem Technology Co., Ltd. NGR was purchased from Leon Biological Technology Co. Ltd. (Nanjing, China). MMP2 sensitive/nonsensitive peptides (Mal-sensitive pep-COOH, Mw = 1105) were purchased from Nanjing Peptide Biological Science and Technology Limited Co., Ltd. (Nanjing, China). Murine IL-2, recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF), murine IL-4 and murine IL-15 were purchased from Pepro Tech (United States). Collagenase type IV (MMP2 enzyme) was purchased from Sigma–Aldrich (San Diego, USA). An Annexin V-FITC/PI apoptosis detection kit and ATP assay kit were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). Methylthiazol tetrazolium (MTT) was purchased from Solarbio (Shanghai, China). IFN-γ, TNF-α, TGF-β, IL-10, IL-6 and IL-12 ELISA kits were purchased from MultiSciences (Lianke) Biotech Co., Ltd. The PD-1 antibody, MojoSort™ Mouse NK Cell Isolation Kit protocol, MojoSort™ Buffer (5X) and MojoSort™ Magnet were purchased from Dakewei Co., Ltd. (Shenzhen, China). Anti-CD3-APC, anti-CD49b-PE, anti-CD11c-FITC, anti-CD80-APC and anti-CD86-PE were purchased from BioLegend. All other materials used were of analytical reagent grade.
Cell lines. Mouse malignant melanoma cells (B16F10), mouse colorectal cancer cells (CT26) and human umbilical vein endothelial cells (HUVECs) were obtained from the Chinese Academy of Sciences (China). B16F10 cells, CT26 cells and NK cells isolated from C57BL/6 female mice were incubated in RPMI 1640 media with streptomycin and penicillin (1%) and 10% foetal bovine serum. HUVECs were incubated in DMEM with streptomycin and penicillin (1%) and foetal bovine serum (FBS; 10%). All cells were cultured in a 37 °C incubator with 5% CO2.
Animals. Female C57BL/6 mice (6-8 weeks) were purchased from SPF Biotechnology Co., Ltd. (Beijing). Female BALB/c mice (6-8 weeks) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All relevant animal experiments were performed in compliance with the animal management rules of the Ministry of Health, People's Republic of China and Animal Experiment Ethics Review Board of Shandong University.
Synthesis of functional materials. DSPE-PEG2000-MMP2 sensitive/nonsensitive peptides (pep) were synthesized as shown in Supplementary Fig. 1. Mal-MMP2 sensitive/nonsensitive pep-COOH was dissolved in dimethyl formamide (DMF) and activated with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) in an ice bath for 1 h. Then, DSPE-PEG2000-NH2 was dissolved in DMF, and the two reaction solutions were mixed and stirred for 12 h. The obtained solutions were purified via dialysis against deionized water (MWCO = 3500 Da) for 48 h.
DSPE-Hyd-PEG5000-NGR was synthesized as shown in Supplementary Fig. 3. Briefly, NGR and DSPE-Hyd-PEG5000-Mal with a molar ratio of 1.5:1 were dissolved in PBS, pH 7.4, containing 1 mM EDTA. Then, the solution was stirred at room temperature under nitrogen protection for 12 h. The excess NGR was removed via dialysis against deionized water (MWCO = 3500 Da) for 48 h, followed by lyophilization. The structures of DSPE-Hyd-PEG5000-NGR and DSPE-PEG2000-MMP2-sensitive/nonsensitive pep were verified by 1H NMR.
Thiolation of IL-15 was carried out with Traut’s reagent. First, IL-15 was diluted in PBS, pH 7.8-8.0, containing 5 mM EDTA, a 20-fold molar excess of Traut’s reagent was added, and the solutions were mixed. Then, the solutions were stirred for 1 h at room temperature. Excess Traut’s reagent was removed by using a Thermo Scientific Zeba column. After that, IL-15-SH and DSPE-PEG2000-MMP2-sensitive pep-Mal were dissolved in PBS (pH 7.4 with 1 mM EDTA) and the solution stirred at room temperature under nitrogen protection for 12 h.
Preparation of NIL-IM-Lip. NIL-IM-Lip was prepared by the film dispersion method. Briefly, DPPC (10 mg/mL), cholesterol (1.25 mg/mL), and IR780 (0.2 mg/mL) were dissolved in ethanol and evaporated to form a dried lipid film (40 °C, 30 min). Then, the dried lipid film was hydrated with 1-MT (1.25 mg/mL) containing DSPE-Hyd-PEG5000-NGR (3% mol ratio relative to DPPC) and DSPE-PEG2000-MMP2-sensitive pep-IL-15. DPPC liposomes (Blank-Lip) and liposomes containing IR780 and 1-MT (IM-Lip) were prepared as described above. The large liposomes were extruded five times using a LiposoFast-Basic extruder (100 nm) (Avestin, Ottawa, ON, Canada), and the small liposomes were extruded using a 50 nm membrane.
Characterization of NIL-IM-Lip. A Malvern Zeta Sizer Nano-ZS instrument was selected to detect the particle size, polydispersity index (PDI) and zeta potential of NIL-IM-Lip. Transmission electronic microscopy (TEM) was selected to visualize the morphology of NIL-IM-Lip. The encapsulation efficiency (EE %) and drug loading (DL %) of NIL-IM-Lip were calculated by the following equations:
DL % = W loaded drug/W liposome × 100%
EE % = W loaded drug/W total drug × 100%
where W loaded drug and W liposome represent the weight of drug in the liposome and weight of all added to the liposome. W total drug is the weight of drug added to the liposome.
To further verify the effective loading of IR780 and 1-MT, ultraviolet−visible (UV−vis) spectrophotometric analyses were performed. Briefly, IR780, 1-MT, and NIL-IM-Lip were subjected to scanning from 200 nm to 900 nm.
In vitro drug release. The dialysis bag method was selected to determine the 1-MT release profiles. Briefly, 1.0 mL of free 1-MT or NIL-IM-Lip was added to a dialysis bag, which were then placed into a tube with 10 mL of PBS, pH 7.4. The tubes were incubated at 37 °C with a stirring speed of 100 rpm. The 1-MT+laser and NIL-IM-Lip+laser groups were subjected to 5 min of irradiation with an 808 nm NIR laser at 1.0 W/cm2 twice with a 10 min interval between the two treatments. A sample was then collected, and the volume was replaced with 10 mL of fresh PBS, pH 7.4, at predetermined time points. The concentration of released 1-MT was determined by HPLC. Experiments were performed in triplicate.
MMP2-sensitive enzymatic digestion assay. The enzymatic digestion method was selected to evaluate the properties of the MMP2-sensitive peptide using the MMP2 enzyme. Briefly, 1.0 mg/mL MMP2-sensitive peptide was incubated with MMP2 enzyme (0, 0.5, 1, 5, 10, and 50 μg/mL) in PBS, pH 7.4, for 24 h at 37 °C. Subsequently, the enzymatic cleavage time was explored. MMP2-sensitive peptide (1.0 mg/mL) was incubated with 50 μg/mL MMP2 enzyme at 37 °C for 5, 15, 30, 60 and 90 min. The digested fragments were identified by HPLC.
Stability of the liposomes in different media. The formulations were diluted with 20% plasma and RPMI 1640 medium, and then we measured the particle size of NIL-IM-Lip after incubation for different lengths of time. In addition, the particle size of NIL-IM-Lip was recorded every day for 7 days to evaluate the long-term physical stability.
In vitro photothermal performance. To investigate the appropriate IR780 concentration and laser irradiation power, a series of IR780–concentration and irradiation power–temperature curves were constructed after measuring with a thermocouple needle. Briefly, 1 mL of water and a series of concentrations of I-Lip (0.1, 1, 10, 30 μg/mL) were irradiated with an 808 nm laser at 1.0 W/cm2 for 10 min. In addition, 10 μg/mL I-Lip was irradiated with an 808 nm laser at 0.1, 0.6, 1.0 and 2.0 W/cm2 for 10 min. At predetermined times, the temperature was recorded. Then, 1 mL of water, free IR780 and I-Lip (10 μg/mL) were irradiated with an 808 nm laser at 1.0 W/cm2 for 10 min, and we captured photographs using an infrared imaging device. IR780 and I-Lip were irradiated with an 808 nm laser for four on/off cycles, and the temperature was recorded to evaluate photothermal stability.
In vivo photothermal performance. A female B16F10 tumour-bearing C57BL/6 mouse model was used to evaluate photothermal conversion efficiency. A total of 1×106 B16F10 cells were injected into the right axilla of C57BL/6 mice to establish a xenograft tumour model. After 7 days, the mice were intravenously injected with NS, IR780, I-Lip or N-I-Lip. The mice were then treated with laser irradiation (808 nm) at 1.0 W/cm2 for 10 min 12 h after the administration of the different formulations. The mice were anaesthetized and imaged using an infrared thermal imaging camera.
In vivo NIRF imaging. A female B16F10 tumour-bearing C57BL/6 mouse model was used to evaluate biodistribution. A total of 1×106 B16F10 cells were injected into the right axilla of C57BL/6 mice to establish a xenograft tumour model. After 15 days, the mice were intravenously injected with free IR780, I-Lip or N-I-Lip (1.5 mg/kg). At 1, 2, 4, 8, 12, 24 and 48 h, mice were anaesthetized with gas and images were captured with a real-time IVIS spectrum. Then, the mice were sacrificed, and the tumours, LNs and major organs (including the heart, liver, spleen, lung, and kidneys) were taken out for imaging.
To evaluate the LNs accumulation of the different formulations, female C57BL/6 mice were injected with 1 × 106 B16F10 cells into the right axilla to establish the model. After 15 days, the mice were intravenously injected with a large nanoinducer (N-I-Lip-L) or a small nanoinducer (N-I-Lip-S) (1.5 mg/kg). At different time points (12, 24, 48 h), the mice were sacrificed and the LNs were acquired for in vitro imaging. Then, the effect of cholesterol transport on the LNs accumulation capacity was studied. Nanoinducers consisting of 1/2, 1/8 and 1/16 mass ratios of cholesterol were prepared. After intratumoural injection, the LNs were taken out at 4 h, 8 h, and 12 h for NIRF imaging.
Furthermore, we evaluated LNs accumulation of IL-15. Cy5.5-BSA was selected to imitate IL-15. Similar to the LNs accumulation of N-I-Lip, mice were intravenously injected with free Cy5.5-BSA, MMP2-sensitive liposomes and MMP2-nonsensitive liposomes. At 4 h, 8 h and 12 h, the mice were sacrificed the acquire the LNs for in vitro imaging.
In vitro cytotoxicity. Cytotoxicity in B16F10 cells was investigated by MTT assay. Briefly, 5×103 B16F10 cells per well were seeded in 96-well plates and incubated overnight. Subsequently, different concentrations of Blank-Lip, IR780, 1-MT, IL-15 and NIL-IM-Lip were added to the wells and further incubated for 48 h. The photothermal therapy group was irradiated with a laser (808 nm) at 1.0 W/cm2 for 5 min. Then, 20 μL of MTT (5 mg/mL) was added to each well for another 4 h of incubation. Afterwards, the medium was removed and substituted with 200 μL of DMSO. Cell viability was measured with a microplate reader at 570 nm.
Cell apoptosis analysis. B16F10 cells were seeded in 12-well plates at a density of 2×105/well and incubated overnight. Then, fresh medium containing PBS, IR780, 1-MT, IR780+1-MT, NIL-IM-Lip, IR780+Laser (IR780+L), IR780+1-MT+Laser (IR780+1-MT+L), or NIL-IM-Lip+Laser (NIL-IM-Lip+L) was added. The photothermal therapy group was irradiated with a laser (808 nm) at 1.0 W/cm2 for 5 min. The cells were collected and resuspended in PBS after incubation for 24 h. Apoptosis was analysed by flow cytometry after staining with PI (10 μL) and annexin V-FITC (5 μL).
Cellular uptake analysis. Fluorescein isothiocyanate (FITC) was selected to label liposomes. HUVECs were seeded into 12-well plates and cultured overnight. The cells were then incubated with free FITC, F-Lip or N-F-Lip (27 μg/mL) for 1, 2 and 4 h. The cells were washed with cold PBS three times and fixed with 4% paraformaldehyde at 4 °C for 10 min. After washing with cold PBS three times, the cells were stained with DAPI for 15 min. Finally, images of the cells were captured by fluorescence microscopy. To quantify HUVEC uptake efficiency, the HUVECs were incubated with FITC-loaded liposomes (5 μg/mL) and evaluated by flow cytometry.
Deep tumour penetration assay. The hanging drop method was used to prepare B16F10 3D tumour spheroids. Briefly, 1.5×105 B16F10 cells dispersed in 0.24% methylcellulose RPMI 1640 medium (20 µL) were transferred to the lids of 6-well plates. After one week of incubation, the B16F10 tumour spheroids were added to 12-well plates and cultured with F-Lip-Large (F-Lip-L) and F-Lip-Small (F-Lip-S) (FITC: 20 μg/mL). After incubation for 6 h, the B16F10 tumour spheroids were washed with cold PBS. Finally, the deep tumour penetration ability was determined using an LSM 900 microscope.
In vitro immunogenic cell death induction. B16F10 cells were seeded in 12-well plates and cultured overnight. Then, the cells were incubated with PBS, IR780, 1-MT, IR780+1-MT, NIL-IM-Lip, IR780+L, IR780+1-MT+L, or NIL-IM-Lip+L for 4 h. The IR780+L, IR780+1-MT+L and NIL-IM-Lip+L groups were irradiated with an 808 nm laser at 1.0 W/cm2 for 5 min, followed by incubation for another 4 h to promote CRT exposure. Then, the cells were fixed with 4% paraformaldehyde for 10 min at 4 °C. Finally, the cells were incubated with primary antibodies and an AF488-conjugated secondary antibody for 30 min each and stained with DAPI. Images were captured with an inverted fluorescence microscope. The quantitative expression of CRT was determined by flow cytometry.
For intracellular HMGB1 detection, B16F10 cells were seeded in 12-well plates and cultured overnight. After incubation for 4 h with different formulations, the groups with laser irradiation were treated with an 808 nm laser at 1.0 W/cm2 for 5 min. Following further incubation for another 12 h, the cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 for 10 min. Finally, the cells were incubated with primary antibodies and an AF488-conjugated secondary antibody for 30 min each and stained with DAPI. Images were captured with an inverted fluorescence microscope. In addition, the HMGB1 released into the supernatant and ATP were collected and analysed with an ELISA kit (Solarbio, China) and an ATP assay kit.
In vitro ROS evaluation. A total of 1×105 B16F10 cells per well were seeded in 12-well plates and cultured overnight for ROS evaluation. Subsequently, PBS, IR780, 1-MT, IR780+1-MT, NIL-IM-Lip, IR780+L, IR780+1-MT+L, or NIL-IM-Lip+L were added for another 4 h of incubation. Following incubation with DCF (fluorescent probe for ROS) for 1 h, the IR780+L, IR780+1-MT+L, and NIL-IM-Lip+L groups were irradiated with an 808 nm laser at 1.0 W/cm2 for 5 min. Then, the cells were stained with DAPI for 15 min. Finally, an inverted fluorescence microscope was selected to image the cells. ROS production was quantitatively evaluated by flow cytometry.
In vitro DC maturation. Bone marrow-derived dendritic cells (BMDCs) were extracted from the bones of female C57BL/6 mice. Then, the obtained BMDCs were cultured in 6-well plates with RPMI 1640 medium containing GM-CSF (20 μg/mL) and IL-4 (20 μg/mL). Then, immature DCs were seeded in 12-well plates, and B16F10 cells were seeded in a transwell chamber. B16F10 cells were treated with different formulations and then cocultured with the DCs using a transwell system. Mature DCs were stained with an anti-CD11c-FITC antibody, anti-CD80-PE antibody and anti-CD86-APC antibody for flow cytometry analysis. In addition, the collected culture medium was centrifuged to analyse the amount of TNF-α secreted.
In vitro NK killing ability. Briefly, NK cells were obtained from the spleens of female C57BL/6 mice using the MojoSort™ Mouse NK Cell Isolation Kit under aseptic conditions. Subsequently, the purity of the obtained NK cells was determined by flow cytometry. The in vitro NK killing ability was measured by MTT assay. Briefly, NK cells were cultured with IL-2, IL-15 and NIL-IM-Lip after preincubation with the MMP2 enzyme. After 3 days, NK cells were added to B16F10 cells cultured in 96-well plates (5000 cells per well) overnight at ratios of 10:1 and 20:1. Following 6 h of incubation, MTT solution (5 mg/mL, 20 μL) was added to each well, and the cells were incubated for another 4 h. Finally, 200 μL of DMSO was added to each well, and the absorbance of each well was measured with a microplate reader (Model 680; Bio–Rad, CA, USA).
In vivo antitumour postsurgical recurrence efficacy. B16F10 tumour-bearing female C57BL/6 mice were used to evaluate the antitumour postsurgical recurrence efficacy. A total of 1×106 B16F10 cells were subcutaneously injected into the right axilla of the mice to establish the model. After 10 days, 90% of the tumour was surgically removed, and the wounds were sutured. The mice were randomly divided into 14 groups (n = 5): (1) NS, (2) Blank-Lip, (3) IR780, (4) IR780+L, (5) 1-MT, (6) IL-15, (7) N-I-Lip+L, (8) IR780+1-MT+L, (9) N-IM-Lip+L, (10) IR780+IL-15+L, (11) NIL-I-Lip+L, (12) IR780+1-MT+IL-15+L, (13) IL-IM-Lip+L, and (14) NIL-IM-Lip+L. Moreover, we evaluated the efficacy of NIL-IM-Lip without laser irradiation. The NS and NIL-IM-Lip+L groups were selected as control groups. The dosages of IR780, 1-MT and IL-15 were 1 mg/kg, 2.7 mg/kg and 1 μg/per mouse, respectively. The mice were treated every 4 days, and the tumour volumes and body weights were measured every 2 days. The mice in the photothermal therapy groups were irradiated with a laser (808 nm) at 1.0 W/cm2 for 5 min 12 h after administration of the different formulations. In addition, the mice were imaged at -1, 0, 7 and 14 days. The spleens were taken out to evaluate the memory T cells, which were marked using an anti-CD3-APC antibody, anti-CD8a-PE antibody, anti-CD62L-FITC antibody and anti-CD44-PerCP/Cyanine 5.5 antibody. The tumour volume (V) was calculated as V=W2(width)×L(length)/2.
In vivo antitumour activity. B16F10 tumour-bearing female C57BL/6 mice were used to evaluate antitumour activity. B16F10 cells (8×105) were subcutaneously injected into the right axilla of the mice. After 10 days, the mice were randomly divided into 14 groups (n=6): (1) NS, (2) Blank-Lip, (3) IR780, (4) IR780+L, (5) 1-MT, (6) IL-15, (7) N-I-Lip+L, (8) IR780+1-MT+L, (9) N-IM-Lip+L, (10) IR780+IL-15+L, (11) NIL-I-Lip+L, (12) IR780+1-MT+IL-15+L, (13) IL-IM-Lip+L, and (14) NIL-IM-Lip+L. The dosages of IR780, 1-MT and IL-15 were 1 mg/kg, 2.7 mg/kg and 1 μg/mouse, respectively. The mice in the photothermal therapy groups were irradiated with a laser (808 nm) at 1.0 W/cm2 for 5 min 12 h after administration of the different formulations. The mice used for survival period studies were treated in the same manner. The mice were treated every 4 days. Tumour volumes and body weights were measured every 2 days. On Day 16, the mice were sacrificed, and the tumours were excised and photographed. Similar to the antitumour postsurgical recurrence efficacy assay, NIL-IM-Lip was also evaluated.
Furthermore, the efficacy of NIL-IM-Lip+L combined with a PD-1 mAb was evaluated in the B16F10 model and CT26 model. The B16F10 model was constructed by subcutaneously injecting 8×105 B16F10 cells into the right axilla of female C57BL/6 mice. The CT26 model was constructed by subcutaneously injecting 1×106 CT26 cells into the right axilla of female BALB/c mice. The mice were divided into 3 groups (n = 5): (1) NS, (2) NIL-IM-Lip+L, and (3) NIL-IM-Lip+PD-1+L. The treatment procedure was similar to that describe before. The PD-1 mAb was intraperitoneally injected at a dose of 5 mg/kg.
In vivo immunization study. After evaluating the in vivo antitumour activity, the tumours and LNs were excised and filtered through a copper network. Lymphocytes were separated from the tumour mixture with a 40% Percoll solution. CD3+CD4+ and CD3+CD8+ T cells were stained with an anti-CD3-APC antibody, anti-CD4-FITC antibody and anti-CD8-PE antibody. DCs were stained with an anti-CD11c-PE antibody, anti-CD80-FITC antibody and anti-CD86-PerCP/Cyanine 5.5 antibody. NK cells were stained with an anti-CD3-APC antibody and anti-CD49b-PE antibody. Treg cells were stained with an anti-CD4-PE antibody and anti-FOXP3-AF647 antibody. CTLs were stained with an anti-CD8-PE antibody and anti-IFNγ-APC antibody. The changes in the immune cells were measured by flow cytometry. In addition, the levels of cytokines in the tumours, including IFN-γ, TGF-β, IL-10, IL-12 and IL-6, were examined with ELISA kits.
Furthermore, the excised tumours were cut and filtered through a copper network to obtain a homogenate. Then, the homogenate was incubated with trichloroacetic acid (30%) for 30 min at 50 °C. The concentrations of tryptophan (Trp) and kynurenine (Kyn) were measured by HPLC at 360 and 280 nm after centrifugation.
Immunohistochemical analysis. Tumour tissues and major organs were fixed in 4% formaldehyde and embedded in paraffin. The tumour sections were stained with haematoxylin and eosin (H&E), Ki67, and TUNEL. The major organs were stained with H&E. Furthermore, IDO1 was observed in the tumour sections and LNs. In addition, CRT and HMGB1 in the tumour sections were detected.
NIL-IM-Lip was incubated with a red blood cell (RBC) (2%, v/v) suspension at 37 ±0.5 °C for 3 h. The NS group and distilled water group were used as the negative control and positive control, respectively. The absorbance of haemoglobin was measured with a UV–vis spectrophotometer at 576 nm.
Statistical analysis. All data were presented as the mean ± SD. The statistically significant differences were analyzed by the Student’s t-test (two groups) and a one-way analysis of variance (ANOVA) (multiple groups). The P value was calculated with the help of GraphPad Prism 8.0.1 and Microsoft Excel 2019 software. * P < 0.05, ** P < 0.01, *** P < 0.001, # P < 0.001 were considered statistically significant.