A Yeast Cell Wall Derived Hybrid Hydrogel with Photothermal and Immune Combined Modality Therapy for Enhanced Anti-Melanoma Efficacy

Abstract

Introduction: The effect of traditional treatment for melanoma is quite limited, especially for its recurrence. As the major components of yeast cell wall, chitin and β-glucan exhibit good immune activation effect and are promising candidates for adjuvant. Therefore, melanoma cell membrane (CM) and indocyanine green (ICG) was loaded in a chitin and β-glucan hybrid hydrogel to achieve an enhanced anti-melanoma therapy.

Methods: The novel hybrid hydrogel was prepared, and its physicochemical properties were examined. Its effect towards melanoma prevention and treatment was evaluated via a melanoma-bearing mice model.

Results: The CM-ICG-hybrid hydrogel was successfully prepared with excellent injectability, self-healing, drug loading, rheological, in vitro and in vivo photothermal stability, and retention properties. It also exhibited good cellular and in vivo safety profiles. In the primary melanoma mice model, it quickly ablated the in-situ melanoma, effectively inhibited the tumor growth, increased the survival rate of melanoma-bearing mice, and increased the level of IFN-γ and TNF-α. In the distal secondary melanoma model, it efficiently prevented the reoccurrence of melanoma and activated the memory T cells. In both models, a synergistic effect of photothermal therapy and immune therapy was found. The hydrogel effectively recruited CD3⁺ CD4⁺ T cells and CD3⁺ CD8⁺ T cells, inhibited the proliferation of melanoma cells, and induced the apoptosis of melanoma cells.

Conclusion: The hybrid hydrogel was successfully prepared, and it showed excellent efficacy towards melanoma prevention and treatment due to its efficient tumor ablation and immune activation capability.

Keywords: chitin; hybrid hydrogel; immunotherapy; melanoma cell membrane; photo thermotherapy; β-glucan.

Graphical abstract

Figure 1

The preparation and characterization of the hybrid hydrogels. (A) SDS-PAGE of the protein in the cell membrane of B16F10 cells; (B) the SEM images of (a) β-glucan hydrogel, (b) chitin hydrogel, and (c) hybrid hydrogel; (C) images of the CM-hybrid hydrogel and ICG-CM-hybrid hydrogel; (D) rheological properties of blank hybrid hydrogel and ICG-CM-hybrid hydrogel; (E) the injectability of blank hybrid hydrogel and ICG-CM-hybrid hydrogel; (F) the self-healing performance of blank hybrid hydrogel; (G) the self-healing performance of ICG-CM-hybrid hydrogel.

Figure 2

The photothermal properties of the CM-ICG-hybrid hydrogels. (A and B) In vitro photothermal conversion ability; (C and D) in vivo photothermal conversion ability in the gel; (E) photothermal conversion stability; (F) in vivo retention of the hybrid hydrogels; (G) B16F10 cell viability after different treatments with or without laser; (H) live/dead images of B16F10 cells after different treatments; bar: 100 μm.

Figure 3

The inhibitory effect of different treatment groups on the primary tumor in the melanoma mouse model. (A) Time line; (B) the weight records of mice under different treatments; (C) the average volume size of the melanoma under different treatments; (D) the volume records of the primary melanoma in each group. (E) Flow chart of the 60-day survival test; (F) time line; (G) 60-day survival rate of mice under different treatments (Log-rank (Mantel–Cox) test, ** denotes P <0.01, *** denotes P <0.001, **** denotes P <0.0001).

Figure 4

The pathological changes in the primary melanoma after different treatments. (A) H+E staining of the melanoma (green: tissue necrosis; yellow: bleeding; gray: inflammatory infiltration; blue: pycnosis and nucleolysis; red: sparsely arranged cells); (B and C) ki-67 immunohistochemical staining of the melanoma sections; (D) TUNEL immunohistochemical staining of the melanoma sections. *P<0.05, **P<0.01, ***P<0.001.

Figure 5

The immunoregulatory effects of different treatments. (A) Time line; (B) ELISA analysis of the TNF-α level in serum; (C) ELISA analysis of the IFN-γ level in serum; (D) quantitation of T cells in the spleen analyzed by flow cytometry; (E) flow cytometry analysis of the proportion of T cells in the spleen; (F) the proportion of effector T cells (CD3⁺ CD8⁺ T cells) and helper T cells (CD3⁺ CD4⁺ T cells) in the spleen; (G) quantitation of helper T cells analyzed by flow cytometry; (H) quantitation of effector T cells analyzed by flow cytometry. *P<0.05, **P<0.01, ***P<0.001.

Figure 6

The inhibitory effect of different treatments on distal secondary melanoma in mice. (A) Time line; (B) the occurrence and development of secondary melanoma in mice; (C) images of the distal secondary melanoma; (D) the volume records of the distal second melanoma in each group.

Figure 7

The immunoregulatory effects of different treatments on the distal secondary melanoma. (A) The activation tumor infiltrating T cells in secondary melanoma; (B) the proportion of helper T cells in tumor infiltrating T cells in the secondary melanoma; (C) the proportion of cytotoxic T cells in tumor infiltrating T cells in the secondary melanoma; (D) immune status of the distal secondary melanoma; (E) the helper T cells in the secondary melanoma sections after immunofluorescent staining; (F) the cytotoxic T cells in the secondary melanoma sections after immunofluorescent staining; (G) the detection of central memory T cells and effector memory T cells in the spleen via flow cytometry; (H) the proportion of central memory T cells in the spleen; (I) the proportion of effector memory T cells in the spleen. *P<0.05, **P<0.01, ***P<0.001.