Figures & data
Figure 1 Schematic illustration showing immunotherapy for tumor ablation and metastasis inhibition. After the laser irradiation, immunogenic cell death (ICD) was induced from polyethylene glycol- polycaprolactone-2-[2-[2-Chloro-3-[(1,3-dihydro-3,3-dimethyl-1-propyl-2H-indol-2-ylidene)ethylidene]-1-cycloxen-1-yl]-ethenyl]-3,3-dimethy-1-propyl-1H-indolium iodide-tirapazamine nanoparticles (PEG-PCL-IR780-TPZ NPs)-based synergic phototherapy and hypoxia-activated chemotherapy. Damage-associated molecular patterns (DAMPs) including adenosine triphosphate (ATP), high-motility group box 1 (HMGB1), and calreticulin (CRT) were produced as endogenous potentiators and subsequently promoted dendritic cells (DCs) maturation. Eventually, the naïve T-cells were recruited by mature DCs and cytotoxic T lymphocytes (CTLs) including CD8+T, CD4+T, and natural killer (NK) cells were elicited, which played an indispensable in ablating primary tumor and controlling tumor metastasis.
![Figure 1 Schematic illustration showing immunotherapy for tumor ablation and metastasis inhibition. After the laser irradiation, immunogenic cell death (ICD) was induced from polyethylene glycol- polycaprolactone-2-[2-[2-Chloro-3-[(1,3-dihydro-3,3-dimethyl-1-propyl-2H-indol-2-ylidene)ethylidene]-1-cycloxen-1-yl]-ethenyl]-3,3-dimethy-1-propyl-1H-indolium iodide-tirapazamine nanoparticles (PEG-PCL-IR780-TPZ NPs)-based synergic phototherapy and hypoxia-activated chemotherapy. Damage-associated molecular patterns (DAMPs) including adenosine triphosphate (ATP), high-motility group box 1 (HMGB1), and calreticulin (CRT) were produced as endogenous potentiators and subsequently promoted dendritic cells (DCs) maturation. Eventually, the naïve T-cells were recruited by mature DCs and cytotoxic T lymphocytes (CTLs) including CD8+T, CD4+T, and natural killer (NK) cells were elicited, which played an indispensable in ablating primary tumor and controlling tumor metastasis.](/cms/asset/8c673811-27c1-4c9a-93c4-3d73ea745a0d/dijn_a_12190906_f0001_c.jpg)
Figure 2 (A) Transmission electron microscopy (TEM) photograph of PEG-PCL NPs. (B) TEM photograph of PEG-PCL-IR780-TPZ NPs. (C) Dynamic light scattering (DLS) of PEG-PCL NPs and PEG-PCL-IR780-TPZ NPs. (D) UV-vis-NIR absorption spectra of PEG-PCL (deionized water as solvent), IR780 (dichloromethane as solvent), TPZ (dichloromethane as solvent), and PEG-PCL-IR780-TPZ NPs (deionized water as solvent). (E) Stability of PEG-PCL-IR780-TPZ NPs in fetal bovine serum (FBS) or phosphate buffer saline (PBS) was monitored by the DLS technology. (F) The release profile of polyethylene glycol- polycaprolactone-2-[2-[2-Chloro-3-[(1,3-dihydro-3,3-dimethyl-1-propyl-2H-indol-2-ylidene)ethylidene]-1-cycloxen-1-yl]-ethenyl]-3,3-dimethy-1-propyl-1H-indolium iodide-tirapazamine nanoparticles (PEG-PCL-IR780-TPZ NPs) under an 808 nm laser irradiation (1 W/cm2).
![Figure 2 (A) Transmission electron microscopy (TEM) photograph of PEG-PCL NPs. (B) TEM photograph of PEG-PCL-IR780-TPZ NPs. (C) Dynamic light scattering (DLS) of PEG-PCL NPs and PEG-PCL-IR780-TPZ NPs. (D) UV-vis-NIR absorption spectra of PEG-PCL (deionized water as solvent), IR780 (dichloromethane as solvent), TPZ (dichloromethane as solvent), and PEG-PCL-IR780-TPZ NPs (deionized water as solvent). (E) Stability of PEG-PCL-IR780-TPZ NPs in fetal bovine serum (FBS) or phosphate buffer saline (PBS) was monitored by the DLS technology. (F) The release profile of polyethylene glycol- polycaprolactone-2-[2-[2-Chloro-3-[(1,3-dihydro-3,3-dimethyl-1-propyl-2H-indol-2-ylidene)ethylidene]-1-cycloxen-1-yl]-ethenyl]-3,3-dimethy-1-propyl-1H-indolium iodide-tirapazamine nanoparticles (PEG-PCL-IR780-TPZ NPs) under an 808 nm laser irradiation (1 W/cm2).](/cms/asset/5469709b-b7b1-41b4-bac5-87427e8d351c/dijn_a_12190906_f0002_c.jpg)
Figure 3 (A) The temperature rising profiles of PEG-PCL-IR780-TPZ NPs (the concentration of IR780 was 200 μg/mL) subjected to the irradiation of an 808 nm laser with different power densities for 10 mins. (B) The temperature rising profiles of PEG-PCL-IR780-TPZ NPs with different concentrations of IR780 irradiated with an 808 nm laser (1 W/cm2, 10 mins). (C) In vitro temperature elevation photographs of PEG-PCL-IR780-TPZ NPs irradiated with an 808 nm laser (the concentration of IR780, 200 μg/mL, 1 W/cm2, 10 mins). (D) In vitro reactive oxygen species (ROS)/hypoxia detection of 4T1 cells subjected to various treatments. Scale bar: 20 μm.
Abbreviations: PEG-PCL-IR780-TPZ NPs, polyethylene glycol- polycaprolactone-2-[2-[2-Chloro-3-[(1,3-dihydro-3,3-dimethyl-1-propyl-2H-indol-2-ylidene)ethylidene]-1-cycloxen-1-yl]-ethenyl]-3,3-dimethy-1-propyl-1H-indolium iodide-tirapazamine nanoparticles; PBS, phosphate buffer saline.
![Figure 3 (A) The temperature rising profiles of PEG-PCL-IR780-TPZ NPs (the concentration of IR780 was 200 μg/mL) subjected to the irradiation of an 808 nm laser with different power densities for 10 mins. (B) The temperature rising profiles of PEG-PCL-IR780-TPZ NPs with different concentrations of IR780 irradiated with an 808 nm laser (1 W/cm2, 10 mins). (C) In vitro temperature elevation photographs of PEG-PCL-IR780-TPZ NPs irradiated with an 808 nm laser (the concentration of IR780, 200 μg/mL, 1 W/cm2, 10 mins). (D) In vitro reactive oxygen species (ROS)/hypoxia detection of 4T1 cells subjected to various treatments. Scale bar: 20 μm.Abbreviations: PEG-PCL-IR780-TPZ NPs, polyethylene glycol- polycaprolactone-2-[2-[2-Chloro-3-[(1,3-dihydro-3,3-dimethyl-1-propyl-2H-indol-2-ylidene)ethylidene]-1-cycloxen-1-yl]-ethenyl]-3,3-dimethy-1-propyl-1H-indolium iodide-tirapazamine nanoparticles; PBS, phosphate buffer saline.](/cms/asset/fd88b1ed-6116-4996-8e14-cb0442055213/dijn_a_12190906_f0003_c.jpg)
Figure 4 (A) The cell viability of 4T1 cells incubated with different particles without a laser irradiation. (B) The cell viability of 4T1 cells incubated with different particles with a laser irradiation at the power density of 1 W/cm2 for 5 mins. *p<0.05, **p<0.01. (C) Live/dead examination of 4T1 cells subjected to different treatments was conducted using calcein-acetoxymethyl ester/propidium iodide (Calcein-AM/PI) double staining and observing by confocal laser scanning microscopy (CLSM). Scale bar: 50 μm.
Abbreviations: PEG-PCL-IR780-TPZ NPs, polyethylene glycol-polycaprolactone-2-[2-[2-Chloro-3-[(1,3-dihydro-3,3-dimethyl-1-propyl-2H-indol-2-ylidene)ethylidene]-1-cycloxen-1-yl]-ethenyl]-3,3-dimethy-1-propyl-1H-indolium iodide-tirapazamine nanoparticles.
![Figure 4 (A) The cell viability of 4T1 cells incubated with different particles without a laser irradiation. (B) The cell viability of 4T1 cells incubated with different particles with a laser irradiation at the power density of 1 W/cm2 for 5 mins. *p<0.05, **p<0.01. (C) Live/dead examination of 4T1 cells subjected to different treatments was conducted using calcein-acetoxymethyl ester/propidium iodide (Calcein-AM/PI) double staining and observing by confocal laser scanning microscopy (CLSM). Scale bar: 50 μm.Abbreviations: PEG-PCL-IR780-TPZ NPs, polyethylene glycol-polycaprolactone-2-[2-[2-Chloro-3-[(1,3-dihydro-3,3-dimethyl-1-propyl-2H-indol-2-ylidene)ethylidene]-1-cycloxen-1-yl]-ethenyl]-3,3-dimethy-1-propyl-1H-indolium iodide-tirapazamine nanoparticles.](/cms/asset/e6950a27-653e-4e30-ae00-9f73c4d9fb23/dijn_a_12190906_f0004_c.jpg)
Figure 5 (A) The immunogenic cancer cell death (ICD) of 4T1 cells subjected to different treatments was assessed by immunofluorescence staining. Scale bars: 50 μm. (B) The release of ATP from dying 4T1 cells subjected to different treatments. (C) The release of HMGB1 from dying 4T1 cells subjected to different treatments. (D) The evaluation of biomarkers (CD83+CD86+) expression on mature DCs membrane after different treatments. (E) The expression level of interleukin 12 (IL-12) from mature DCs after different treatments, *p<0.05, **p<0.01 (n=3).
Abbreviations: DC, dendritic cells; HMGB1, high-motility group box 1; ATP, adenosine triphosphate; PEG-PCL-IR780-TPZ NPs, polyethylene glycol- polycaprolactone-2-[2-[2-Chloro-3-[(1,3-dihydro-3,3-dimethyl-1-propyl-2H-indol-2-ylidene)ethylidene]-1-cycloxen-1-yl]-ethenyl]-3,3-dimethy-1-propyl-1H-indolium iodide-tirapazamine nanoparticles.
![Figure 5 (A) The immunogenic cancer cell death (ICD) of 4T1 cells subjected to different treatments was assessed by immunofluorescence staining. Scale bars: 50 μm. (B) The release of ATP from dying 4T1 cells subjected to different treatments. (C) The release of HMGB1 from dying 4T1 cells subjected to different treatments. (D) The evaluation of biomarkers (CD83+CD86+) expression on mature DCs membrane after different treatments. (E) The expression level of interleukin 12 (IL-12) from mature DCs after different treatments, *p<0.05, **p<0.01 (n=3).Abbreviations: DC, dendritic cells; HMGB1, high-motility group box 1; ATP, adenosine triphosphate; PEG-PCL-IR780-TPZ NPs, polyethylene glycol- polycaprolactone-2-[2-[2-Chloro-3-[(1,3-dihydro-3,3-dimethyl-1-propyl-2H-indol-2-ylidene)ethylidene]-1-cycloxen-1-yl]-ethenyl]-3,3-dimethy-1-propyl-1H-indolium iodide-tirapazamine nanoparticles.](/cms/asset/a9a34590-70fa-4120-9dad-e70080212c4e/dijn_a_12190906_f0005_c.jpg)
Figure 6 (A) In vivo infrared (IR) imaging of 4T1-tumor-bearing mice intravenously injected with PBS or PEG-PCL-IR780-TPZ NPs and irradiated with an 808 nm laser for 10 mins at the power density of 1 W/cm2. (B) Heating curve of 4T1-tumor-bearing mice intravenously injected with PBS or PEG-PCL-IR780-TPZ NPs and irradiated with an 808 nm laser for 10 mins at the power density of 1 W/cm2.
Abbreviations: PBS, phosphate buffer saline; PEG-PCL-IR780-TPZ NPs, polyethylene glycol-polycaprolactone-2-[2-[2-Chloro-3-[(1,3-dihydro-3,3-dimethyl-1-propyl-2H-indol-2-ylidene)ethylidene]-1-cycloxen-1-yl]-ethenyl]-3,3-dimethy-1-propyl-1H-indolium iodide-tirapazamine nanoparticles.
![Figure 6 (A) In vivo infrared (IR) imaging of 4T1-tumor-bearing mice intravenously injected with PBS or PEG-PCL-IR780-TPZ NPs and irradiated with an 808 nm laser for 10 mins at the power density of 1 W/cm2. (B) Heating curve of 4T1-tumor-bearing mice intravenously injected with PBS or PEG-PCL-IR780-TPZ NPs and irradiated with an 808 nm laser for 10 mins at the power density of 1 W/cm2.Abbreviations: PBS, phosphate buffer saline; PEG-PCL-IR780-TPZ NPs, polyethylene glycol-polycaprolactone-2-[2-[2-Chloro-3-[(1,3-dihydro-3,3-dimethyl-1-propyl-2H-indol-2-ylidene)ethylidene]-1-cycloxen-1-yl]-ethenyl]-3,3-dimethy-1-propyl-1H-indolium iodide-tirapazamine nanoparticles.](/cms/asset/3c5f3e4e-077e-4554-a380-38d5e02a9d98/dijn_a_12190906_f0006_c.jpg)
Figure 7 (A) Tumor volume growth curves of 4T1-tumor-bearing mice subjected to various treatments (*p<0.05, **p<0.01, n=6). (B) Mice weight of 4T1-tumor-bearing mice subjected to various treatments. (C) Representative images of 4T1-tumor-bearing mice subjected to various treatments. (D) Representative images of tumor tissues separated from 4T1-tumor-bearing mice subjected to various treatments. (E) Hematoxylin-eosin (H&E) staining for tumor tissue slices of different groups. (F) Ki67 staining for tumor tissue slices of different groups. Blue arrows indicate the positive expression of proliferation marker Ki67.
Abbreviations: PEG-PCL-IR780-TPZ NPs, polyethylene glycol-polycaprolactone-2-[2-[2-Chloro-3-[(1,3-dihydro-3,3-dimethyl-1-propyl-2H-indol-2-ylidene)ethylidene]-1-cycloxen-1-yl]-ethenyl]-3,3-dimethy-1-propyl-1H-indolium iodide-tirapazamine nanoparticles.
![Figure 7 (A) Tumor volume growth curves of 4T1-tumor-bearing mice subjected to various treatments (*p<0.05, **p<0.01, n=6). (B) Mice weight of 4T1-tumor-bearing mice subjected to various treatments. (C) Representative images of 4T1-tumor-bearing mice subjected to various treatments. (D) Representative images of tumor tissues separated from 4T1-tumor-bearing mice subjected to various treatments. (E) Hematoxylin-eosin (H&E) staining for tumor tissue slices of different groups. (F) Ki67 staining for tumor tissue slices of different groups. Blue arrows indicate the positive expression of proliferation marker Ki67.Abbreviations: PEG-PCL-IR780-TPZ NPs, polyethylene glycol-polycaprolactone-2-[2-[2-Chloro-3-[(1,3-dihydro-3,3-dimethyl-1-propyl-2H-indol-2-ylidene)ethylidene]-1-cycloxen-1-yl]-ethenyl]-3,3-dimethy-1-propyl-1H-indolium iodide-tirapazamine nanoparticles.](/cms/asset/e2a0e4b8-7d3b-4f59-9024-b4e2d7b445b3/dijn_a_12190906_f0007_c.jpg)
Figure 8 (A) Positron Emission Tomography (PET) images of mice recorded after treating with various treatments. (PBS, IR780+ laser, PEG-PCL-IR780+ laser, and PEG-PCL-IR780-TPZ + laser) (B) Hypoxia-inducible factor-1α (HIF-1α) staining for tumor tissues of different treatment groups. Scale bars: 50 μm. (C) Platelet endothelial cell adhesion molecule-1 (CD31) staining for tumor tissues of different treatment groups. The red arrows represented blood vessel. Scale bars: 50 μm.
Abbreviations: PEG-PCL-IR780-TPZ NPs, polyethylene glycol- polycaprolactone-2-[2-[2-Chloro-3-[(1,3-dihydro-3,3-dimethyl-1-propyl-2H-indol-2-ylidene)ethylidene]-1-cycloxen-1-yl]-ethenyl]-3,3-dimethy-1-propyl-1H-indolium iodide-tirapazamine nanoparticles; PBS, phosphate buffer saline.
![Figure 8 (A) Positron Emission Tomography (PET) images of mice recorded after treating with various treatments. (PBS, IR780+ laser, PEG-PCL-IR780+ laser, and PEG-PCL-IR780-TPZ + laser) (B) Hypoxia-inducible factor-1α (HIF-1α) staining for tumor tissues of different treatment groups. Scale bars: 50 μm. (C) Platelet endothelial cell adhesion molecule-1 (CD31) staining for tumor tissues of different treatment groups. The red arrows represented blood vessel. Scale bars: 50 μm.Abbreviations: PEG-PCL-IR780-TPZ NPs, polyethylene glycol- polycaprolactone-2-[2-[2-Chloro-3-[(1,3-dihydro-3,3-dimethyl-1-propyl-2H-indol-2-ylidene)ethylidene]-1-cycloxen-1-yl]-ethenyl]-3,3-dimethy-1-propyl-1H-indolium iodide-tirapazamine nanoparticles; PBS, phosphate buffer saline.](/cms/asset/37876b1b-c99c-48b3-b0e3-774575330184/dijn_a_12190906_f0008_c.jpg)
Figure 9 (A) Representative lung images showed the metastatic lesions in different treatment groups. The blue circles represented metastatic lesions on lung. Scale bars: 0.5 cm. (B) H&E staining for lung tissues of different treatment groups. Blue arrows indicate apoptosis and necrosis. Scale bars: 200 μm. (C) Ex vivo immunofluorescence indicated the level of CRT expression in primary tumor tissues. The green area indicated CRT while the blue area indicated cell nuclei. Scale bars: 50 μm. (D) The activated CD8+ T-cell infiltrated into tumor tissues after treating with different treatments. The red arrow represented the CD8+ T-cells. Scale bars: 100 μm.
Abbreviations: PEG-PCL-IR780-TPZ NPs, polyethylene glycol- polycaprolactone-2-[2-[2-Chloro-3-[(1,3-dihydro-3,3-dimethyl-1-propyl-2H-indol-2-ylidene)ethylidene]-1-cycloxen-1-yl]-ethenyl]-3,3-dimethy-1-propyl-1H-indolium iodide-tirapazamine nanoparticles; CRT, calreticulin.
![Figure 9 (A) Representative lung images showed the metastatic lesions in different treatment groups. The blue circles represented metastatic lesions on lung. Scale bars: 0.5 cm. (B) H&E staining for lung tissues of different treatment groups. Blue arrows indicate apoptosis and necrosis. Scale bars: 200 μm. (C) Ex vivo immunofluorescence indicated the level of CRT expression in primary tumor tissues. The green area indicated CRT while the blue area indicated cell nuclei. Scale bars: 50 μm. (D) The activated CD8+ T-cell infiltrated into tumor tissues after treating with different treatments. The red arrow represented the CD8+ T-cells. Scale bars: 100 μm.Abbreviations: PEG-PCL-IR780-TPZ NPs, polyethylene glycol- polycaprolactone-2-[2-[2-Chloro-3-[(1,3-dihydro-3,3-dimethyl-1-propyl-2H-indol-2-ylidene)ethylidene]-1-cycloxen-1-yl]-ethenyl]-3,3-dimethy-1-propyl-1H-indolium iodide-tirapazamine nanoparticles; CRT, calreticulin.](/cms/asset/2747fc05-bb57-4636-b516-1eaa667c6177/dijn_a_12190906_f0009_c.jpg)
Figure 10 Long-term safety study. (A) Weight ratio of major tissues (heart, liver, spleen, lung, kidney) to mice body. (B) The relevant blood analysis. (C) H&E staining as a pathologic analysis for major organs (n=3). Scale bar: 50 μm.
Abbreviations: PEG-PCL-IR780-TPZ NPs, polyethylene glycol- polycaprolactone-2-[2-[2-Chloro-3-[(1,3-dihydro-3,3-dimethyl-1-propyl-2H-indol-2-ylidene)ethylidene]-1-cycloxen-1-yl]-ethenyl]-3,3-dimethy-1-propyl-1H-indolium iodide-tirapazamine nanoparticles; RBC, red blood cell; WBC, white blood cell; HGB, hemoglobin concentration; MCH, mean corpuscular hemoglobin content; MCHC, mean corpuscular hemoglobin concentration; PLT, platelets.
![Figure 10 Long-term safety study. (A) Weight ratio of major tissues (heart, liver, spleen, lung, kidney) to mice body. (B) The relevant blood analysis. (C) H&E staining as a pathologic analysis for major organs (n=3). Scale bar: 50 μm.Abbreviations: PEG-PCL-IR780-TPZ NPs, polyethylene glycol- polycaprolactone-2-[2-[2-Chloro-3-[(1,3-dihydro-3,3-dimethyl-1-propyl-2H-indol-2-ylidene)ethylidene]-1-cycloxen-1-yl]-ethenyl]-3,3-dimethy-1-propyl-1H-indolium iodide-tirapazamine nanoparticles; RBC, red blood cell; WBC, white blood cell; HGB, hemoglobin concentration; MCH, mean corpuscular hemoglobin content; MCHC, mean corpuscular hemoglobin concentration; PLT, platelets.](/cms/asset/5da9ef74-6fa7-4934-876e-a43e94eb404a/dijn_a_12190906_f0010_c.jpg)