Light-triggered multifunctional nanoplatform for efficient cancer photo-immunotherapy

Abstract

Cancer immunotherapy is limited by the immune escape of tumor cells and adverse effects. Photo-immunotherapy, the combination of immunotherapy and phototherapy (such as photodynamic therapy (PDT) and photothermal therapy (PTT)), can improve the effectiveness of immunotherapy in cancer treatment. Here, we first explored mesoporous hexagonal core-shell zinc porphyrin-silica nanoparticles (MPSNs), which are composed of a zinc porphyrin core and a mesoporous silica shell, and exhibit high laser-triggered photodynamic and photothermal activity, as well as outstanding drug loading capacity. In other words, MPSNs can be used not only as excellent photosensitizers for photo-immunotherapy, but also as an ideal drug carrier to achieve more efficient synergy. After loading with R837 (imiquimod, a toll-like receptor-7 agonist), MPSNs@R837 will elicit high-efficiency immunogenic cell death via PDT and PTT, and promote dendritic cell maturation after the PH-responsive release of R837, thereby, inducing tumor-specific immune responses. When combined with a programmed death ligand-1 checkpoint blockade, the photo-immunotherapy system markedly restrains primary tumors and metastatic tumors with negligible systemic toxicity. Therefore, the therapeutic strategy of integrating PTT, PDT and checkpoint blockade, shows great potential for suppressing cancer metastasis.

Keywords: Dendritic cell; Hexahedron zinc porphyrin mesoporous nanoparticles; Immune response; PD-L1 checkpoint blockade; Photo-immunotherapy.

Scheme 1

A schematic of the synthetic procedure for core–shell zinc porphyrin nanoplatform (MPSNs@R837) and the schematic illustration of MPSNs@R837 for combined phototherapy (PDT and PTT) and checkpoint blockade to enhance synergistic antitumor immunity

Fig. 1

Characterization of MPSNs. A and B TEM images of MPSNs. C Elemental mapping of C, N, O, Zn, and Si of MPSNs. D Zeta potential. E UV/Vis spectra of MPSNs, MPSNs@R837, ZnP and free R837. F The fluorescence spectra of MPSNs, MPSNs@R837, and ZnP upon irradiation of 450 nm light, G upon irradiation of 780 nm light. H The fluorescence lifetime of ZnP and MPSNs in water. I R837 release from MPSNs@R837 at different pH values (pH 7.4 or 5.0)

Fig. 2

Photothermal heating curves of MPSNs aqueous solution (A) with different concentrations and (B) with different laser power densities. C Photothermal effect of the MPSNs solution (100 μg/mL) under irradiation of 808 nm laser (0.6 W/cm²) for 600 s min and left to cool down then, inset: Linear time data versus negative natural logarithm of the temperature driving force which is obtained from the cooling stage. D Temperature variations of the MPSNs under irradiation (808 nm, 0.6 W/cm²⁾ for five light on/off cycles (600 s of irradiation for each cycle). E Photothermal images of MPSNs in solution under laser irradiation

Fig. 3

A CLSM images of 4T1 treated with MPSNs for different time (scale bar = 20 μm). B Mean fluorescence intensities of 4T1 cells by flow cytometry. C CLSM images of intracellular reactive oxygen species (ROS) (scale bar = 20 μm). D Mean fluorescence intensities. All data are mean ± SD (n = 3), statistical significances were calculated via Student’s t test, *p < 0.05, **p < 0.01

Fig. 4

An Immunofluorescence observation of CRT (green fluorescence) exposure on the 4T1 cells surface after incubation with PBS, MPSNs and MPSNs@R837 with or without laser irradiation (808 nm, 0.6 W/cm²) (scale bar = 25 μm). B Mean fluorescence intensities of 4T1 determined by flow cytometry. C HMGB1 and D ATP levels of 4T1 cells after 24 h. All data are mean ± SD (n = 3), statistical significances were calculated via Student’s t test, *p < 0.05, **p < 0.01

Fig. 5

Maturation and secretion analysis of DCs after treated with laser-treated 4T1 cells in the presence of free R837, MPSNs and MPSNs@R837. A The design of the transwell system experiment, 4T1 cells were placed in the upper chamber and DCs were incubated in the lower chamber. B The expression level of DC maturation (CD11c + CD80 + CD86 +) was determined by flow cytometry after different treatments. C Percentage of DC maturation. D the secretion of IL-12 and (E) TNF-α in DCs suspensions. All data are mean ± SD (n = 3), statistical significances were calculated via Student’s t test, *p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Fig. 6

A The distribution of MPSNs@R837 (red fluorescence) in tumor sections (scale bar = 200 μm). B The fluorescence intensity as described in A determined by flow cytometry. C Thermographic images and (D) tumor temperature changes of 4T1 tumor-bearing mice at different time points under laser irradiation 12 h after injection of saline, MPSNs and MPSNs@R837 (808 nm, 0.6 W/cm²). E The ROS level in (green fluorescence) in tumor sections (scale bar = 100 μm). F The fluorescence intensity as described in e. All data are mean ± SD (n = 5), statistical significances were calculated via Student’s t test, ****p < 0.0001

Fig. 7

MPSNs@R837-mediated PDT and PTT in vivo: A Schematic of treatment schedule in 4T1 orthotropic mammary tumor model. Mice were randomly divided into 6 groups: (1) saline-only, (2) free R837, (3) MPSNs, (4) MPSNs@R837, (5) MPSNs (+), (6) MPSNs@R837 (+). B In vivo apoptosis and/or necrosis of the tumor induced by different treatment as shown by H&E staining (scale bar = 100 μm) and TUNEL assay (scale bar = 50 μm). C Tumor volume D Tumor weight. E Tumor picture. F and (G) DC maturation in the tumor-draining lymph nodes induced by different treatment on mice. H–J cytokine levels of TNF-α, INF-γ and IL-12 in sera from mice. Data are mean ± SD (n = 5), statistical significances were calculated via Student’s t test, *p < 0.05, **p < 0.01

Fig. 8

MPSNs@R837-mediated photo-immunotherapy in vivo: A Schematic of treatment schedule in 4T1 orthotopic mammary tumor model. Mice were randomly divided into 4 groups: (1) saline, (2) Anti-PD-L1, (3) MPSNs@R837 (+), (4) MPSNs@R837 (+) plus Anti-PD-L1. B Tumor volume (C) Tumor weight. D Number of pulmonary metastatic nodules. E Ratio of CD8 + /CD4 + T cells and (F) CTL infiltration in primary. G Survival time. H Lung tissues with metastatic nodules from 4T1 tumor-bearing mice in each group over 21 days. I Representative metastatic lung images of each group in Fig. 8. H. J Tumor images. Data are mean ± SD (n = 5), statistical significances were calculated via Student’s t test, *p < 0.05, **p < 0.01

Fig. 9

The abscopal effect of MPSNs@R837-mediated photo-immunotherapy: A Schematic of treatment schedule a 4T1 bilateral tumor model. Mice were randomly divided into 4 groups: (1) saline, (2) Anti-PD-L1, (3) MPSNs@R837 (+), (4) MPSNs@R837 (+) plus Anti-PD-L1. B Volume and (C) Weight of primary tumors. D Volume and E Weight of distant tumors. F Ratio of CD8 + /CD4 + T cells and (G) CTL infiltration in distant tumor. Data are mean ± SD (n = 5), statistical significances were calculated via Student’s t test, *p < 0.05, **p < 0.01