Chitosan-Poly(Acrylic Acid) Nanoparticles Loaded with R848 and MnCl2 Inhibit Melanoma via Regulating Macrophage Polarization and Dendritic Cell Maturation

Abstract

Purpose: Since immune cells in the tumor microenvironment (TME) can affect the development and progression of tumors, strategies modulating immune cells are considered to have an important therapeutic effect. As a TLR7/8 agonist, R848 effectively activates the innate immune cells to exert an anti-tumor effect. Mn²⁺ has been reported to strongly promote the maturation of antigen-presenting cells (APCs), thereby enhancing the cytotoxicity of CD8⁺ T cells. Thus, we tried to investigate whether chitosan-poly(acrylic acid) nanoparticles (CS-PAA NPs) loaded with R848 and MnCl₂ (R-M@CS-PAA NPs) could exert an anti-tumor effect by regulating the function of immune cells.

Methods: R-M@CS-PAA NPs were prepared, and their basic characteristics, anti-tumor effect, and potential mechanisms were explored both in vitro and in vivo.

Results: R-M@CS-PAA NPs easily released MnCl₂ and R848 at low pH. In B16F10 mouse melanoma model, R-M@CS-PAA NPs exerted the most significant anti-melanoma effect compared with the control group and CS-PAA NPs loaded with R848 or MnCl₂ alone. FITC-labeled R-M@CS-PAA NPs were displayed to be accumulated at the tumor site. R-M@CS-PAA NPs significantly increased the infiltration of M1 macrophages and CD8⁺ T cells but reduced the number of suppressive immune cells in the TME. Moreover, in vitro experiments showed that R-M@CS-PAA NPs polarized macrophages into the M1 phenotype to inhibit the proliferation of B16F10 cells. R-M@CS-PAA NPs also enhanced the killing function of CD8⁺ T cells to B16F10 cells. Of note, R-M@CS-PAA NPs not only promoted the maturation of APCs such as dendritic cells and macrophages by STING and NF-кB pathways, but also enhanced the ability of dendritic cells to present ovalbumin to OT-I CD8⁺ T cells to enhance the cytotoxicity of OT-I CD8⁺ T cells to ovalbumin-expressing B16F10 cells.

Conclusion: These data indicate that the administration of R-M@CS-PAA NPs is an effective therapeutic strategy against melanoma.

Keywords: NF-кB; STING; T cell; antigen-presenting cell.

Figure 1

Characteristics of R-M@CS-PAA NPs. (A) The morphologies of CS-PAA NPs, R848@CS-PAA NPs, MnCl₂@CS-PAA NPs, and R-M@CS-PAA NPs were observed by TEM. (B and C) Particle sizes (B) and zeta potentials (C) of R-M@CS-PAA NPs in 1640 medium containing 10% FBS at the indicated time points. (D and E) Release profiles of MnCl₂ (D) and R848 (E) from R-M@CS-PAA NPs. *p < 0.05, **p < 0.01.

Figure 2

R-M@CS-PAA NPs suppressed tumor growth in vivo. (A) Photograph of tumor tissues from different mice groups. (B, C) Tumor weight (B) and tumor volume (C) of tumor tissues from different mice groups. (D, E) Quantitative data (D) and histopathologic photograph (E) of Ki67 staining of tumor tissues from different mice groups. **p < 0.01.

Figure 3

R-M@CS-PAA NPs were accumulated at the tumor site. (A) The distribution of R-M@CS-PAA NPs in vivo was observed by IVIS Lumina XR. (B) The distribution of R-M@CS-PAA NPs in vivo after 3 h of intravenous injection was detected FCM. (C) Quantitative data of (B). (D) The distribution of R-M@CS-PAA NPs in vivo after 12 h of intravenous injection was detected FCM. (E) Quantitative data of (D). (F) The distribution of R-M@CS-PAA NPs in vivo after 24 h of intravenous injection was detected FCM. (G) Quantitative data of (F). *p < 0.05, **p < 0.01.

Figure 4

R-M@CS-PAA NPs increased the number of M1 macrophages. (A) Quantitative data from FCM analysis of MDSCs, M-MDSCs, and G-MDSCs in the tumor tissues from different mouse groups. (B) Quantitative data from FCM analysis of macrophages, M1 macrophages, and M2 macrophages in the tumor tissues from different mice groups. (C-E) B16F10 cells were treated with CS-PAA NPs, R848@CS-PAA NPs, MnCl₂@CS-PAA NPs, and R-M@CS-PAA NPs for 48 h, and the cell viability (C), apoptotic rate (D), and proliferation ability (E) of B16F10 cells were examined by CCK-8, Annexin V/PI staining, and FCM, respectively. (F) RAW 264.7 cells were treated with CS-PAA NPs, R848@CS-PAA NPs, MnCl₂@CS-PAA NPs, and R-M@CS-PAA NPs for 48 h, and the cell viability was examined by CCK-8 assay. (G) RAW 264.7 cells were treated with CS-PAA NPs, R848@CS-PAA NPs, MnCl₂@CS-PAA NPs, and R-M@CS-PAA NPs for 6 h, and the mRNA expression levels of TNF-α, iNOS, IL-6, and IL-1β were examined by qRT-PCR. (H) In the presence of CS-PAA NPs, R848@CS-PAA NPs, MnCl₂@CS-PAA NPs, and R-M@CS-PAA NPs, B16F10 cells were indirectly co-cultured with RAW 264.7 cells for 48 h, and the proliferation of B16F10 cells was evaluated by FCM. *p < 0.05, **p < 0.01.

Figure 5

R-M@CS-PAA NPs affected the distribution of T cells in vivo. (A) Quantitative data of CD4⁺ T, CD8⁺ T, and Treg cells in tumor tissues with indicated treatments. (B-E) Quantitative data of the ratio of CD69⁺ (B), IFN-γ⁺ (C), TNF-α⁺ (D), and granzyme B⁺ (E) CD8⁺ T cells. *p < 0.05, **p < 0.01.

Figure 6

R-M@CS-PAA NPs affected the function of CD8⁺ T cells in vitro. (A) The effects of different NPs on the proliferation of CD8⁺ T cells. (B-D) CD8⁺ T cells were treated with different NPs for 48 h, and the proportion of IFN-γ⁺ (B), granzyme B⁺ (C), and perforin⁺(D) CD8⁺ T cells were examined by FCM. (E) CD8⁺ T cells were treated with different NPs for 48 h, and the expression levels of TNF-α, IFN-β, and IFN-γ in the supernatant were detected by ELISA assay. (F) In the presence of different NPs, B16F10 cells were co-cultured with CD8⁺ T cells for 48 h, and the proliferation of B16F10 cells was examined by FCM. *p < 0.05, **p < 0.01.

Figure 7

R-M@CS-PAA NPs induced the maturation of DCs. (A) BMDCs were stimulated with FITC-labeled R-M@CS-PAA NPs for 6 h, and the percentage of FITC⁺ BMDCs was examined by FCM. (B) BMDCs were treated with different NPs for 24 h, and the viability of BMDCs was detected by CCK-8 assay. (C) BMDCs were treated with different NPs for 24h, and the expression of CD80 was detected by FCM. (D) Quantitative data of (C). (E) BMDCs were treated with different NPs for 24h, and the expression of CD86 was detected by FCM. (F) Quantitative data of (E). *p < 0.05, **p < 0.01.

Figure 8

R-M@CS-PAA NPs induced the maturation of DCs via MAPK and STING pathways. (A, B) BMDCs were treated with different NPs for 24 h, and the expression of MHC I (A) and MHC II (B) was detected by FCM. (C) BMDCs were treated with different NPs for 24 h, and the expression levels of IL-12p70, TNF-α, IFN-β, and IFN-γ in the supernatant were detected by ELISA assay. (D) BMDCs were treated with different NPs for 24 h, and the expression of target genes was detected by WB assay. (E-H) RAW 264.7 cells were treated with different NPs for 24 h, and the expression of CD80 (E), CD86 (F), MHC I (G), and MHC II (H) was detected by FCM. *p < 0.05, **p < 0.01.

Figure 9

R-M@CS-PAA NPs-treated DCs enhanced the cytotoxicity of OT-I CD8⁺ T cells to B16F10-OVA cells. (A) BMDCs were co-cultured with B16F10-OVA cells for 48 h, and the percentage of OVA⁺ BMDCs was detected by FCM. (B-F) In the presence of different NPs, BMDCs, OT-I CD8⁺ T cells, and B16F10-OVA cells were co-cultured for 48 h. The ratio of IFN-γ⁺ (B), TNF-α⁺ (C), granzyme B⁺ (D), and perforin⁺ (E) OT-I CD8⁺ T cells, and the proliferation of B16F10-OVA cells (F) were examined by FCM. (G) The expression of IL-12p70, TNF-α, IFN-β, and IFN-γ in the serum of mice treated with different NPs was detected by ELISA assay. *p < 0.05, **p < 0.01.

Figure 10

Schematic illustration of macrophage polarization and DC maturation induced by R-M@CS-PAA NPs for the treatment of melanoma.