X-ray-responsive dissolving microneedles mediate STING pathway activation to potentiate cutaneous melanoma radio-immunotherapy

Abstract

Background: Radiotherapy (RT) often activates the cyclic GMP-AMP synthase (cGAS) stimulator of interferon response cGAMP interactor (STING) signaling pathway and induces systemic immunotherapy effects by triggering immunogenic cell death (ICD) in various solid tumors. However, RT-induced ICD usually falls short in eradicating distant tumors because of moderate anti-tumor immune responses. Methods: In this study, Mn-ZIF-8 nanoparticles and microneedles were prepared, and their physical and chemical properties were characterized. Subsequently, in vitro experiments using B16 and A375 cutaneous melanoma cell lines were conducted to investigate the radiosensitivity characteristics of Mn-ZIF-8 and its mechanism for enhancing RT efficacy. Moreover, mouse models bearing primary and distant B16 cutaneous melanoma were established to clarify the immunomodulatory effect and antitumor efficacy of Mn-ZIF-8 microneedles when combined with RT and immunotherapy. Results: A percutaneous delivery method based on soluble microneedles (MNs) with Mn²⁺-loaded, X-ray-responsive zeolite imidazolate frame-8 (ZIF-8) was designed. This microneedle-based drug delivery system, combined with RT, promoted the radiosensitivity of cutaneous melanoma and reinforces ICD by augmenting STING pathway activation. Furthermore, after X-ray irradiation, Mn-ZIF-8 MNs continuously released Mn²⁺ in the tumor to enhance cGAS-STING activation. This promoted dendritic cell maturation and antigen presentation, and potentiated a T cell mediated immune response. Thus, the local and systemic immune effects induced by RT were amplified when combined with immune checkpoint inhibitors. Conclusion: The microneedle patches with X-ray-responsive, rapid dissolution and controlled release abilities have the potential to enhance the radioimmunotherapy efficacy for cutaneous melanoma.

Keywords: Mn-ZIF-8; dissolving microneedles; melanoma; radio-immunotherapy; radiosensitization.

Scheme 1

Schematic Illustrations. (i) Schematic illustration of the preparation process of Mn-ZIF-8 and (ii) the proposed mechanism of Mn-ZIF-8-mediated radiosensitization and STING pathway-dependent antitumor immunity for enhanced the radioimmunotherapy efficacy.

Figure 1

Construction and characterization of Mn-ZIF-8. (A) TEM images of Mn(20%)-ZIF-8 at different magnifications. (B) Zeta potentials and (C, n = 3 per group) Hydrodynamic diameters of ZIF-8 and Mn-ZIF-8. (D) Elemental mapping images of Mn(20%)-ZIF-8. (E) The XRD patterns of ZIF-8 and Mn-ZIF-8. (F) The XPS survey spectra of Mn(20%)-ZIF-8. (G) The XPS spectra of Mn 2p. The blue line represents the fitted baseline, the purple and green lines correspond to the fitted peaks for Mn⁴⁺ and Mn²⁺, and the red line shows the final fitted curve. (H) The cumulative release profile of Mn²⁺ from Mn-ZIF-8 MNs under different pH conditions. The data are presented as the mean ± SD.

Figure 2

Effect of Mn-ZIF-8 on melanoma cell proliferation and dsDNA damage. (A) Effect of Mn-ZIF-8 at different concentrations and its components in combination with radiotherapy (6 Gy) on the viability of B16 cells (n = 6 per group). (B) Effect of PBS, ZIF-8, and Mn‑ZIF-8 in combination, with or without radiotherapy (B16, 6 Gy; A375, 4Gy), on the proliferation of melanoma cells, as assessed using the CCK8 assay (n = 5 per group). (C) Effect of PBS, ZIF-8, and Mn-ZIF-8 in combination, with or without different doses of radiotherapy (2, 4, and 6 Gy), on the proliferation of melanoma cells, as assessed using a colony formation assay (B16, n = 4 per group; A375, n = 3 per group). (D) Effect of PBS, ZIF-8, and Mn-ZIF-8 in combination, with radiotherapy (2Gy), on the dsDNA breaks in melanoma cells, as evaluated using immunofluorescence staining of γ-H₂AX. Scale bar = 10 μm. The data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 3

Mn-ZIF-8 enhanced ICD and the activation of the STING pathway induced by RT in vitro. (A) and (B) Quantification of CRT fluorescence intensities and representative fluorescence images of melanoma cells subjected to various treatments (n = 5 per group). (C) and (D) Quantification of HMGB1 fluorescence intensities and representative fluorescence images of melanoma cells subjected to various treatments (n = 5 per group). Scale bar = 10 μm. (F) Western blotting analysis of the activation of cGAS-STING in melanoma cells subjected to various treatments. ELISA analysis of the secretion of (E) ATP and (G) IFN-β from melanoma cells subjected to with various treatments (n = 3 per group). The data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 4

Construction and characterization of MNs loaded with Mn-ZIF-8. (A) Photograph of the Mn(20%)-ZIF-8 MNs array. (B) SEM images of Mn(20%)-ZIF-8 and ZIF-8 MNs. (C) Main view and top view of elemental mapping images of Mn(20%)-ZIF-8 MNs. (D) Solubility of Mn(20%)-ZIF-8 MNs. (E) The force-displacement curves of Mn(20%)-ZIF-8 and ZIF-8 MNs. Arrow: Fracture point of MNs. (F) Mechanical strength of the force per individual needle (N per needle) (n = 3 per group). (G) H&E staining of the rat skin punctured with Mn(20%)-ZIF-8 MNs. The data are presented as the mean ± SD.

Figure 5

Antitumor effects of Mn-ZIF-8 MNs in a B16 melanoma xenograft mouse model. (A) Experimental timeline for the treatment of B16 tumor-bearing C57BL/6J mice. (B) Body weight curve of mice during treatment. (C) Photograph of B16 tumors isolated from the mice on day 18. (D) Tumor growth curve of mice subjected to different treatments. (E) and (F) and (G) Individual tumor growth curves of mice after different treatments. (H) Weights of tumors isolated from the mice on day 18. (I) H&E staining of tumor tissues and IHC images showing Ki67, CD4⁺ T, and CD8⁺ T cell infiltration, Foxp3 and GZMB expression after the indicated treatments. (J) Quantitative analysis of mature DCs (CD80⁺ CD86⁺ in CD11c⁺ cells) in inguinal lymph nodes adjacent to tumors after treatment. (K-M) Percentages of tumor-infiltrating CD4⁺ T, CD8⁺ T, and Treg cells. (N) and (O) Percentages of spleen-infiltrating CD4⁺ T and CD8⁺ T cells. The data are presented as the mean ± SD; n = 5 per group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 6

Mn-ZIF-8 MNs combined with RT plus ICB elicited systemic antitumor immunity. (A) Experimental timeline for the treatment of bilateral B16 tumor-bearing C57BL/6J mice. (B) Photograph of primary (left) and metastatic (right) B16 tumors isolated from the mice on day 16. (C) and (D) Weights of primary tumors and distant tumors isolated from the mice on day 16. (E) and (F) Primary tumors and distant tumor growth curves of the mice after different treatments. (G) Quantitative analysis of mature DCs (CD80⁺ CD86⁺ in CD11c⁺ cells) in inguinal lymph nodes adjacent to primary tumors after treatment. (H) Percentages of primary tumor-infiltrating CD4⁺ T, CD8⁺ T, and Treg cells. (I) Percentages of distant tumor-infiltrating CD4⁺ T, CD8⁺ T, and Treg cells. (J) H&E staining of bilateral tumor tissues and IHC images showing Ki67, CD4⁺ T, and CD8⁺ T cell infiltration, and Foxp3 and GZMB expression after the indicated treatments. The data are presented as the mean ± SD; n = 5 per group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.