Targeted Ferroptosis-Immunotherapy Synergy: Enhanced Antiglioma Efficacy with Hybrid Nanovesicles Comprising NK Cell-Derived Exosomes and RSL3-Loaded Liposomes

Abstract

Ferroptosis therapy and immunotherapy have been widely used in cancer treatment. However, nonselective induction of ferroptosis in tumors is prone to immunosuppression, limiting the therapeutic effect of ferroptosis cancer treatment. To address this issue, this study reports a customized hybrid nanovesicle composed of NK cell-derived extracellular versicles and RSL3-loaded liposomes (hNRVs), aiming to establish a positive cycle between ferroptosis therapy and immunotherapy. Thanks to the enhanced permeability and retention effect and the tumor homing characteristics of NK exosomes, our data indicate that hNRVs can actively accumulate in tumors and enhance cellular uptake. FASL, IFN-γ, and RSL3 are released into the tumor microenvironment, where FASL derived from NK cells effectively lyses tumor cells. RSL3 downregulates the expression of GPX4 in the tumor, leading to the accumulation of LPO and ROS, and promotes ferroptosis in tumor cells. The accumulation of IFN-γ and TNF-α stimulates the maturation of dendritic cells and effectively induces the inactivation of GPX4, promoting lipid peroxidation, making them sensitive to ferroptosis and indirectly promoting the occurrence of ferroptosis. This study highlights the role of the customized hNRV platform in enhancing the effectiveness of synergistic treatment with selective delivery of ferroptosis inducers and immune activation against glioma without causing additional side effects on healthy organs.

Keywords: biomimetic hybrid nanovesicles; enhanced ferroptosis therapy; immunotherapy; natural killer cell; targeted delivery.

Figure 1

Schematic illustration of the action mechanism of hNRVs for synergistic ferroptosis-immunotherapy. After intravenous injection into glioma-bearing mice, hNRVs transmigrate to the brain across the BBB and BBTB and penetrate the infiltrating glioma cells. They actively accumulate in tumors, releasing immune activators and ferroptosis inducers, thereby exerting a synergetic ferroptosis-immunotherapy effect.

Figure 2

Characterization of hNRVs. (A) Fabrication of hNRVs. (B) Representative TEM images of NK-EVs, RLPs, and hNRVs. (C) Western blotting of NK extracellular vesicle-specific protein on NK-EVs, RLPs, and hNRVs. (D) Hydrodynamic size distribution and PDI (E) of different formulations. (F) Validation of hybridization via FRET analysis and CLSM images (G). (H) Cumulative release of RLPs and hNRVs in different release media (n = 3). Data were presented as means ± SD; *p < 0.05, **p < 0.01, and ***p < 0.001; ns, not significant.

Figure 3

Assessment of cellular uptake behavior and cytotoxicity of hNRVs. (A) Flow cytometry of uptake efficiency of hNRVs by different cells. (B) Representative CLSM images and quantification of the cellular uptake of hNRVs in cells treated with PBS, filipin, cytochalasin D, colchicine, and chlorpromazine. DiI: red; DAPI: blue. (C) In vitro BBB and BBTB model penetration analysis. (D) Cell apoptosis induced by various treatments examined by flow cytometry. The cells were stained with annexin V-FITC and PI. (E) Viability of RAW264.7 and DC2.4 cells in response to different concentrations of hNRVs. Data were presented as means ± SD; *p < 0.05, **p < 0.01, and ***p < 0.001; ns, not significant.

Figure 4

hNRV-induced ferroptosis and activation of BMDCs in vitro. (A) ROS and LPO imaging in C6 cells. (B) GPX4 level and GSH level (C) in C6 cells post hNRV incubation. (D) BMDC maturation as detected by FC with staining of anti-CD80 and anti-CD86. (E) TNF-α and IL-6 (F) levels in the supernatants of BMDCs with different treatments. (G) In vitro hemocompatibility of hNRVs. Data were presented as means ± SD; *p < 0.05, **p < 0.01, and ***p < 0.001; ns, not significant.

Figure 5

In vivo glioma targeting and biodistribution of hNRVs. (A) Living images of glioma-bearing mice with different treatments. (B) Fluorescence imaging in different organs and brain tissues (C). (D) Ratio of fluorescence intensity in brain tissues and different organs. (E) Pharmacokinetics behavior of RLPs and hNRVs in glioma-bearing mice. (F) Immunofluorescence staining of hNRVs in glioma-bearing brains at 12 h after administration. Data were presented as means ± SD; *p < 0.05, **p < 0.01, and ***p < 0.001; ns, not significant.

Figure 6

In vivo antiglioma efficacy of hNRVs. (A) Schematic illustration of the establishment of the orthotopic glioma-bearing mouse model and dose regimen. (B) Luciferase luminescence levels of mice following the indicated different treatments. (C) Body weight changes of mice in different treatment groups. (D) Survival rate of mice treated with different formulations. (E) H&E and TUNEL staining of tumor tissues after treatment with different formulations. Data were presented as means ± SD.

Figure 7

Synergistic mechanism of ferroptosis and immunotherapy induced by hNRVs in vivo. (A) Schematic illustration of animal experiment design on the synergistic effect of ferroptosis and immunotherapy. (B) Fluorescence distributions of the treated tumors stained with DCFH-DA and BODIPY C11-581/591. (C) Relative GPX4 and GSH (D) levels of the cells in the treated tumors. (E) Proportion of CD80⁺/CD86⁺ reflecting the ratio of mature DCs in the mouse spleen. (F) Immunofluorescence staining of anti-CD4 (green) and anti-CD8 (red) in the lymph node. Data were presented as means ± SD; *p < 0.05, **p < 0.01, and ***p < 0.001; ns, not significant.