Cell Membrane Hybrid Lipid Nanovesicles Enhance Innate Immunity for Synergistic Immunotherapy by Promoting Immunogenic Cell Death and cGAS Activation

Abstract

Immunotherapy shows great therapeutic potential for long-term protection against tumor relapse and metastasis. Innate immune sensors, such as cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING), dissolve DNA and induce type I interferon. Through activation of the cGAS/STING pathway, chemotherapy drugs and reversine (REV) may provide synergetic anti-tumor effects. Here, we prepared drug-loaded cell membrane hybrid lipid nanovesicles (LEVs) (designated LEV@DOX@REV) by fusion of cell membranes, phospholipids, doxorubicin (DOX), and REV, to realize accurate delivery to tumors and chemo-immunotherapy. The cell membranes of LEVs confer "homing" abilities. DOX can induce immunogenic cell death as a result of its specific immunomodulatory effects, which promotes the maturation of immune cells and improves the microenvironment of the immune system. REV is proven to efficiently activate cGAS/STING signaling, thereby enhancing the immune system. The antitumor efficacy of LEV@DOX@REV was evaluated in a 4T1 subcutaneous tumor xenograft model, a distant metastatic tumor model, and a liver metastatic tumor model. LEV@DOX@REV facilitated the infiltration of cytotoxic T lymphocytes within tumors, increased the secretion of proinflammatory cytokines, and modified the tumor microenvironment. In conclusion, LEV@DOX@REV displayed favorable antitumor effects and extended the survival of tumor-bearing mice. We therefore successfully developed nanoparticles capable of enhancing immune activation that have potential therapeutic applications for cancer immunotherapy.

Fig. 1.

Schematic diagram of LEV@DOX@REV used for combined therapy that functions via enhancing the cGAS/STING pathway and ICD.

Fig. 2.

Preparation and characterization of LEV@DOX@REV. (A) TEM image of LEV@DOX@REV. (B) DLS was used to measure the hydrodynamic diameter of LEVs before (C) and after (D) drug loading. (E) The zeta potential of LEV@DOX@REV was assessed after 7 days. (F) Western blotting analysis. Samples were run at equal protein concentration and immunostained against membrane markers. (G) The optimal ratio of cell membranes and liposome film membranes in LEVs, as analyzed by FRET. Cell membranes were labeled with DiO and liposome film membranes were labeled with DiI.

Fig. 3.

Confocal fluorescence images of tumor cells after incubation with ICG-labeled nanoparticles (A) or drug-loaded nanoparticles (B). Cell nuclei were stained blue with DAPI, and filamentous actin cytoskeletons were stained green with FITC phalloidin. Scale bar = 10 μm. Cellular uptake was determined by FCM at different time points (C and D) and different concentrations (E and F). (G) Viability of 4T1 cells following various treatments. (H) Viability of LO-2, HEK293T, and 4T1 cells incubated with PBS, EM, Lip, and LEVs.

Fig. 4.

Representative images of PBS or LEV@REV-treated 4T1 cells stained with anti-cGAS (A), anti-STING (B), anti-IRF3 (C), and anti-TBK1 (D) antibodies. Scale bar = 50 μm. (E) Representative FCM plots and (F) quantification of DCs maturation after treatment with PBS, LEV@DOX, LEV@REV, or LEV@DOX@REV for 24 h.

Fig. 5.

(A) WB analysis showing the impact of LEV@DOX@REV on the expression of the indicated proteins. (B) CRT release examined by CLSM (B) and WB (C). Scale bar = 50 μm. (D) The release of ATP from 4T1 cells with different treatments following incubation with or without NAC. (E) The secretion of HMBG1 in 4T1 cells treated with different treatments following incubation with or without NAC. (F) CXCL10 protein was measured by ELISA. (G) The expression of cGAS target genes, including those encoding TNF-α, ISG56, IL-6, and IFN-I, in 4T1 cells with the indicated treatment, was detected using real-time PCR.

Fig. 6.

Biodistribution and antitumor efficacy of LEV@DOX@REV in 4T1 tumor-xenograft mice. (A) In vivo NIR fluorescent images of tumor-bearing mice after injection of ICG-labeled nanoparticles. (B) Ex vivo NIR fluorescent images of the main organs and tumors at 24 and 48 h post-injection. (C) The tumor/muscle ratio of the LEV@DOX@REV group at different time points. (D) Quantitative biodistribution of LEV@DOX@REV in the main organs at 24 and 48 h post-injection.

Fig. 7.

In vivo therapeutic properties of LEV@DOX@REV. The detection of tumor volume (A, n = 8) and survival curves (B, n = 10) after the indicated treatments. (C) Photographs of the tumors extracted from mice after the indicated treatments. (D) H&E, Ki67, TUNEL, and CTL staining of tumors. Scale bar = 50 μm. Representative images (E) showing memory T cells (CD8⁺CD44⁺CD62L^˗) in splenocytes, as measured by FCM and quantitative analysis (n = 4).

Fig. 8.

Immune responses to LEV@DOX@REV-mediated therapy in the tumor-bearing mouse model. Representative FCM plots and quantification analysis of CD80⁺CD86⁺ DCs in the spleen (A), CD3⁺CD8⁺ or CD3⁺CD4⁺ T cells (B), F4/80⁺CD86⁺ macrophages (C), and F4/80⁺CD206⁺ macrophages (D) in 4T1 tumors after different treatments (n = 4).

Fig. 9.

Biosafety evaluation. (A) Changes in the body weight of mice at 19 days. (B) Blood routine parameter data. (C) Blood biochemistry data including liver function markers ALT, AST, and ALP and kidney function markers BUN and CRE. (D) H&E-stained slice images of major organs. Scale bar = 100 μm.

Fig. 10.

Distant tumor growth inhibition effect of LEV@DOX@REV. (A) Schematic of the administration protocol. The detection of tumor volume (B, n = 8), survival curves (C, n = 10), and weight changes (D, n = 5) after the indicated treatments. (E) H&E, Ki67, TUNEL, and CTL staining of tumors. Scale bar = 50 μm.

Fig. 11.

In vivo therapeutic efficacy against 4T1 liver metastasis. (A) Illustration of the treatment schedule for evaluating the anti-metastasis effect. (B) Photographs of excised livers and H&E staining. Scale bar, left: 1 mm, right: 100 μm. (C) Number of liver metastasis nodules after different treatments (n = 5). (D) Survival curves of mice after different treatments during a 40-day observation period (n = 10). (E) Cytokine levels of TNF-α, ISG56, IL-6, and IFN-I in the serum of mice (n = 4).