Biomimetic "Gemini nanoimmunoregulators" orchestrated for boosted photoimmunotherapy by spatiotemporally modulating PD-L1 and tumor-associated macrophages

Abstract

A novel strategy of not only stimulating the immune cycle but also modulating the immunosuppressive tumor microenvironment is of vital importance to efficient cancer immunotherapy. Here, a new type of spatiotemporal biomimetic "Gemini nanoimmunoregulators" was engineered to activate robust systemic photoimmunotherapy by integrating the triple-punch of amplified immunogenic cell death (ICD), tumor-associated macrophages (TAMs) phenotype reprogramming and programmed cell death ligand 1 (PD-L1) degradation. The "Gemini nanoimmunoregulators" PM@RM-T7 and PR@RM-M2 were constructed by taking the biocompatible mesoporous polydopamine (mPDA) as nanovectors to deliver metformin (Met) and toll-like receptor 7/8 agonist resiquimod (R848) to cancer cells and TAMs by specific biorecognition via wrapping of red blood cell membrane (RM) inlaid with T7 or M2 peptides. mPDA/Met@RM-T7 (abbreviated as PM@RM-T7) was constructed to elicit an amplified in situ ICD effect through the targeted PTT and effectively stimulated the anticancer immunity. Meanwhile, PD-L1 on the remaining cancer cells was degraded by the burst metformin to prevent immune evasion. Subsequently, mPDA/R848@RM-M2 (abbreviated as PR@RM-M2) specifically recognized TAMs and reset the phenotype from M2 to M1 state, thus disrupting the immunosuppressive microenvironment and further boosting the function of cytotoxic T lymphocytes. This pair of sister nanoimmunoregulators cooperatively orchestrated the comprehensive anticancer activity, which remarkably inhibited the growth of primary and distant 4T1 tumors and prevented malignant metastasis. This study highlights the spatiotemporal cooperative modalities using multiple nanomedicines and provides a new paradigm for efficient cancer immunotherapy against metastatic-prone tumors.

Keywords: Amplified immunogenic cell death; Biomimetic immunoregulator; Immunosuppressive tumor microenvironment; Metastasis inhibition; PD-L1 degradation; Spatiotemporal delivery; TAMs phenotype reversion; Targeted photothermal therapy.

Graphical abstract

Scheme 1

Schematic illustration of (A) the construction of the biomimetic “Gemini nanoimmunoregulators” and (B) the synergistic photoimmunotherapy via spatiotemporal targeted PTT, phenotype reversion of TAMs, and PD-L1 inhibition.

Figure 1

Characterization of biomimetic “Gemini nanoimmunoregulators”. TEM images of (A) mPDA, (B) PM@RM-T7, and (C) PR@RM-M2 (scale bar = 100 nm). (D) Fluorescence microscopic images of PF@RM-T7, FITC was doped into mPDA, and DiI was used to label RM (scale bar = 100 μm). (E) SDS-PAGE analysis of mPDA, RM, PM@RM-T7, and PR@RM-M2. (F) Z-average size and surface zeta potential of mPDA, PM, PR, PM@RM-T7, and PR@RM-M2. (G) UV–Vis absorption spectra of different formulations. (H) Hydrodynamic size of PM@RM-T7 and PR@RM-M2 in saline; (I) IR thermal images of mPDA and PM@RM-T7 (at the equivalent concentration of mPDA = 200 μg/mL) under laser irradiation (808 nm, 1.0 W/cm², 10 min). (J) Photothermal curves of PM@RM-T7 in four cycles of laser on/off. (K) Cumulative release of Met at pH 7.4 and (L) pH 5.0 from PM and PM@RM-T7 with or without laser irradiation.

Figure 2

Photoimmune activation and downregulation of PD-L1 by PM@RM-T7 in vitro. (A) Representative CLSM images of cellular uptake of PC@RM-T7 and relevant controls by 4T1 cells after 6 h incubation. Relative cell viability of 4T1 cells treated with PM@RM-T7 and the relevant controls (scale bar = 100 μm) (B) under dark and (C) laser irradiation. (D) Representative flow cytometry plots of 4T1 cells after different treatments. (E) CRT exposure and (F) CLSM images of 4T1 cells with IF staining of CRT (scale bar = 50 μm). Expression of PD-L1 in the 4T1 cells detected by (G) western blotting and (H) IF staining after treatment with PM@RM-T7 and relevant controls with or without laser irradiation (scale bar = 50 μm). Data are presented as mean ± SD (n = 3). ∗P < 0.05, ∗∗P < 0.01 and ∗∗∗∗P < 0.0001 vs. indicated.

Figure 3

Selective recognition and reprogramming ability of PR@RM-M2 on M2 type RAW264.7 cells. (A) Representative CLSM images of M2 type RAW264.7 cells incubated with PC@RM, PC@RM-M2, or PC@RM-T7 for 6 h (scale bar = 100 μm). (B) Representative flow cytometry plots of M1 type RAW264.7 cells (CD86^high) and M2 type RAW264.7 cells (CD206^high) gating on F4/80⁺ after different treatments and (C) the corresponding statistical analysis. (D) TNF-α, IL-6, IFN-γ, and IL-10 secretion by M2 type RAW264.7 cells after different treatments. (E) CLSM images of IF staining of CD86 (green) and CD206 (red) on the surfaces of M2 type RAW264.7 cells after various treatments (scale bar = 50 μm; zoom in: scale bar = 20 μm). (F) Representative CLSM images of the phagocytosis of 4T1 cells (labeled by Hoechst) by M2 type RAW264.7 cells (labeled by DiO) after treatment of R848, PR@RM, and PR@RM-M2 (scale bar = 100 μm; zoom in: scale bar = 20 μm). Data are presented as mean ± SD (n = 3). ∗P < 0.05, ∗∗P < 0.01 and ∗∗∗∗P < 0.0001 vs. indicated. n.s., not significant.

Figure 4

Spatiotemporal targeting behavior and biodistribution of “Gemini nanoimmunoregulators” in vivo. Biofluorescence images of (A) 4T1 tumor-bearing mice, (B) dissected tumors and organs, and (C) semiquantitative analysis of fluorescence signals after intravenous injection of PCy@RM, PCy@RM-M2 and PCy@RM-T7. (D) IF staining results of tumor sections (scale bar = 50 μm) and (E) quantitative analysis of the coincidence of fluorescence signals (Cy5.5 and anti-CD206-FITC) by Image J for groups treated by PCy@RM, PCy@RM-M2 and PCy@RM-T7. Data are presented as mean ± SD (n = 3). ∗P < 0.05 and ∗∗P < 0.01 vs. indicated.

Figure 5

Assessment of synergistic photoimmunotherapeutic effect against 4T1 primary tumors. (A) Therapeutic schedule for 4T1 bilateral tumor-bearing mice. (B) The volumes, (C) weight, and (D) photographs of primary tumors after various treatments. Microscopic images of primary tumor sections stained with (E) HE (scale bar = 200 μm), TUNEL (scale bar = 50 μm), and immunofluorescence staining of (F) CRT, HSP 70, and (G) PD-L1 (scale bar = 100 μm). Data are presented as mean ± SD (n = 3). ∗∗P < 0.01 and ∗∗∗∗P < 0.0001 vs. indicated. n.s., not significant.

Figure 6

TAMs reprogramming and immune activation by “Gemini nanoimmunoregulators”. (A) Representative flow cytometry plots and (B) corresponding percentages of matured DCs (gated by CD80⁺ CD86⁺ CD11c⁺) in lymph nodes. (C) Populations of the M1 type (gated by F4/80⁺ CD86⁺) and M2 type TAMs (gated by F4/80⁺ CD206⁺) in tumors. (D) The ratio of M1/M2 macrophages in tumors. (E) IF staining of CD86 and CD206 markers in the primary tumor section (scale bar = 100 μm). (F) Representative flow cytometry plots of the intratumor infiltration of CD4⁺ and CD8⁺ T cells (gated on CD3⁺ T cells) in the primary tumor after treatments. (G) Levels of TNF-α, IL-6, IFN-γ, and IL-10 in the serum of mice from each group were measured by ELISA. (H) Representative flow cytometry plots of the intratumoral infiltration of Tregs cells (CD4⁺ Foxp3⁺) in the primary tumor after treatments. Data are presented as mean ± SD (n = 3). ∗∗P < 0.01, ∗∗∗P < 001, and ∗∗∗∗P < 0.0001 vs. indicated. n.s., not significant.

Figure 7

Abscopal effect of “Gemini nanoimmunoregulators” and metastasis inhibition. (A) Photographs and (B) growth curves of distal tumors. (C) Microscopic images of distal tumor sections with IF staining of Ki67, CD4, CD8, and Foxp3 (scale bar = 100 μm), and (D) further quantified by Image J. (E) Therapeutic schedule of 4T1 lung metastatic model. (F) Representative photographs of lung tissues with tumor metastasis and HE staining images (scale bar = 2 mm). (G) Microscopic images of lung sections with immunohistochemistry analysis of Ki67 (scale bar = 100 μm) and IF staining of CD4 and CD8 (scale bar = 50 μm).