Delivery of a STING Agonist Using Lipid Nanoparticles Inhibits Pancreatic Cancer Growth

Abstract

Introduction: The tumor microenvironment (TME) of pancreatic cancer is highly immunosuppressive and characterized by a large number of cancer-associated fibroblasts, myeloid-derived suppressor cells, and regulatory T cells. Stimulator of interferon genes (STING) is an endoplasmic reticulum receptor that plays a critical role in immunity. STING agonists have demonstrated the ability to inflame the TME, reduce tumor burden, and confer anti-tumor activity in mouse models. 2'3' cyclic guanosine monophosphate adenosine monophosphate (2'3'-cGAMP) is a high-affinity endogenous ligand of STING. However, delivering cGAMP to antigen-presenting cells and tumor cells within the cytosol remains challenging due to membrane impermeability and poor stability.

Methods: In this study, we encapsulated 2'3'-cGAMP in a lipid nanoparticle (cGAMP-LNP) designed for efficient cellular delivery. We assessed the properties of the nanoparticles using a series of in-vitro studies designed to evaluate their cellular uptake, cytosolic release, and minimal cytotoxicity. Furthermore, we examined the nanoparticle's anti-tumor effect in a syngeneic mouse model of pancreatic cancer.

Results: The lipid platform significantly increased the cellular uptake of 2'3'-cGAMP. cGAMP-LNP exhibited promising antitumor activity in the syngeneic mouse model of pancreatic cancer.

Discussion: The LNP platform shows promise for delivering exogenous 2'3'-cGAMP or its derivatives in cancer therapy.

Keywords: 2’3’-cGAMP; cold tumor; cytosolic delivery; innate immunotherapy; lipid nanoparticle.

Figure 1

Schematics of cGAMP-LNP and its anti-tumor mechanism. (a) Schematics of the cGAMP-LNP (created using Biorender.com). (b) Anti-tumor mechanism of cGAMP-LNP.

Figure 2

Characterization of cGAMP-LNPs. (a) Analysis of particle size using dynamic light scattering, (b) Zeta potential distribution, (c) Morphology was elucidated using TEM.

Figure 3

In-vitro activity of cGAMP-LNP. (a) IRF-inducible SEAP reporter activity of cGAMP LNP, blank LNP, and free 2’3’-cGAMP in THP-1 ISG reporter cell line. Poly (dA: DT)-Lyovec serves as a positive control. NC denotes negative control. (b) EC₅₀ of the cGAMP-LNP. (c) EC₅₀ of free 2’3’-cGAMP. All data are presented as mean ± SD (n=3).

Figure 4

Cellular uptake, endosomolytic and cytotoxic activity of cGAMP-LNP. (a) Cellular uptake of cGAMP LNP and free cGAMP in DC2.4 cells. The DC2.4 cells were treated for 2 h with cGAMP-LNP or free cGAMP at cGAMP concentrations of 0.5 or 0.25 μg/mL, respectively. (b) The LNP induces pH- and concentration- dependent hemolysis of human RBCs. (c) Cytotoxicity of cGAMP-LNP in human pancreatic duct epithelial (HPDE) cells. All data are presented as mean ± SD (n=3).

Figure 5

Anti-tumor activity of cGAMP-LNP in a mouse pancreatic cancer model. (a) C57BL/6 mice was implanted with a mixture of 1×10⁶ Panc-02 and NIH-3T3 cells (2:1 ratio) subcutaneously on right flank. (b) Normalized body weight. (c) Tumor volume. Body weight was presented as mean ± SD (n=8). Tumor volumes were presented as mean ± SEM (n=8). **p<=0.005.

Figure 6

In-vivo toxicological evaluation of cGAMP-LNP. (a) Biochemical analysis levels of albumin, alkaline phosphatase (ALP), alanine transferase (ALT), aspartate aminotransferase (AST), creatinine, glutamate dehydrogenase (GLDH), total protein, glucose, and urea nitrogen in the plasma. (b) H&E staining of the heart, kidney, liver, lung, spleen, and tumor. The scale bar is 200 μm. (c) Inflammation score (scale of 0–5) of the lungs, liver, and heart. Kidney and spleen were reported to have no inflammation. The data were presented as mean ± SD (n=8).