GSH-responsive polymeric micelles-based augmented photoimmunotherapy synergized with PD-1 blockade for eliciting robust antitumor immunity against colon tumor

Abstract

Phototherapy is a promising antitumor modality, which consists of photothermal therapy (PTT) and photodynamic therapy (PDT). However, the efficacy of phototherapy is dramatically hampered by local hypoxia in tumors, overexpression of indoleamine 2,3-dioxygenase (IDO) and programmed cell death ligand-1 (PD-L1) on tumor cells. To address these issues, self-assembled multifunctional polymeric micelles (RIMNA) were developed to co-deliver photosensitizer indocyanine green (ICG), oxygenator MnO₂, IDO inhibitor NLG919, and toll-like receptor 4 agonist monophosphoryl lipid A (MPLA). It is worth noting that RIMNA polymeric micelles had good stability, uniform morphology, superior biocompatibility, and intensified PTT/PDT effect. What's more, RIMNA-mediated IDO inhibition combined with programmed death receptor-1 (PD-1)/PD-L1 blockade considerably improved immunosuppression and promoted immune activation. RIMNA-based photoimmunotherapy synergized with PD-1 antibody could remarkably inhibit primary tumor proliferation, as well as stimulate the immunity to greatly suppress lung metastasis and distant tumor growth. This study offers an efficient method to reinforce the efficacy of phototherapy and alleviate immunosuppression, thereby bringing clinical benefits to cancer treatment.

Keywords: Hypoxic tumor microenvironment; Immune checkpoint inhibitor; Immunotherapy; Indoleamine 2,3-dioxygenase (IDO); Phototherapy; Polymeric micelles.

Scheme 1

Schematic illustration of RIMNA polymeric micelles as amplifier for robust photoimmunotherapy synergized with PD-1 blockade antibody against primary tumors, distant tumors, and lung metastasis

Fig. 1

Characterization and CT26 cell-level evaluation of RIMNA. (A) TEM images of RIMNA (Scale bar = 50–200 nm). (B) The particle size stability of RIMNA stored at 4 °C (n = 3). (C) The particle size distribution of RIMNA in PBS solution (pH 7.4) with or without GSH. (D) The absorption spectra of RIMNA, ICG, and NLG919. (E) Temperature curves and (F) corresponding infrared thermal imageries of RIMNA, RINA, Free ICG, and PBS under NIR laser irradiation. (G) The CLSM images indicated cellular uptake (Scale bar = 20 μm). (H) The CLSM images and (I) representative flow cytometric plots indicated ROS production (Scale bar = 20 μm). The survival rate of CT26 cells incubated with various concentrations of RIMNA, RINA or Free ICG (J) without or (K) with NIR laser irradiation (n = 3)

Fig. 2

PTT-PDT effect in vivo. (A) Schematic diagram of the treatment plan. The temperature change curves of tumor sites at (B) 24 h or (C) 48 h timepoint after administration (n = 3). (D) Representative NIR thermography at 24–48 h timepoint. CLSM images of (E) hypoxia and (F) ROS generation in tumor site after indicated treatments (Scale bar = 50 μm). (G) H&E images of the tumors (Scale bar = 25 μm)

Fig. 3

DCs activation in tumor-draining lymph nodes. Representative flow cytometric plots of (A) CD40, (B) CD80, and (C) CD86 expression on the membrane of DCs from the lymph nodes of CT26 tumor-bearing mice following indicated treatments. Corresponding quantification of (D) CD40, (E) CD80, and (F) CD86 (n = 3). Data were displayed as mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001 vs. RIMNA + Laser group)

Fig. 4

Evaluation of systemic antitumor immune. (A, B) The percentages of CTLs and Ths in the tumors through flow cytometric examination (n = 3). (C, D) The percentages of CTLs and Ths in the spleens through flow cytometric examination (n = 3). (E, F) The percentages of IFN-γ-secreting CD3⁺CD8⁺ T cells and CD3⁺CD4⁺ T cells in the spleens through flow cytometric examination. Data were displayed as mean ± SEM (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001 vs. RIMNA + Laser group)

Fig. 5

Antitumor effects in vivo. (A) Treatment schedule in bilateral CT26 tumor model. Volume changes of (B) primary tumor and (C) distant tumor of mice after indicated treatments. (D) Corresponding survival curves. Data were displayed as mean ± SEM (n = 5, **P < 0.01, ***P < 0.001 vs. RIMNA + aPD-1 + Laser group)

Fig. 6

Anti-metastasis effect. The percentages of (A) CTLs, (B) Ths, (C) CD8⁺CD69⁺ T cells, (D) CD4⁺CD69⁺ T cells, (E) IFN-γ⁺CD8⁺ T cells, and (F) Tregs in splenocytes (n = 3). Quantification of (G) TNF-α and (H) IL-4 released in sera after indicated treatments (n = 3). (I) H&E staining images of lung (Scale bar = 200–50 μm). The percentages of CD44^hiCD62L^hi among (J) CD8⁺ T cells and (K) CD4⁺ T cells in spleens (n = 3). Data were displayed as mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001 vs. RIMNA + aPD-1 + Laser group)