STING-Pathway Inhibiting Nanoparticles (SPINs) as a Platform for Treatment of Inflammatory Diseases

Abstract

Aberrant activation of the cyclic GMP-AMP synthase (cGAS)/Stimulator of Interferon Genes (STING) pathway has been implicated in the development and progression of a myriad of inflammatory diseases including colitis, nonalcoholic steatohepatitis, amyotrophic lateral sclerosis (ALS), and age-related macular degeneration. Thus, STING pathway inhibitors could have therapeutic application in many of these inflammatory conditions. The cGAS inhibitor RU.521 and the STING inhibitor H-151 have shown promise as therapeutics in mouse models of colitis, ALS, and more. However, these agents require frequent high-dose intraperitoneal injections, which may limit translatability. Furthermore, long-term use of systemically administered cGAS/STING inhibitors may leave patients vulnerable to viral infections and cancer. Thus, localized or targeted inhibition of the cGAS/STING pathway may be an attractive, broadly applicable treatment for a variety of STING pathway-driven ailments. Here we describe STING-Pathway Inhibiting Nanoparticles (SPINS)-poly(lactic-co-glycolic acid) (PLGA) nanoparticles loaded with RU.521 and H-151-as a platform for enhanced and sustained inhibition of cGAS/STING signaling. We demonstrate that SPINs are equally or more effective at inhibiting type-I interferon responses induced by cytosolic DNA than free H-151 or RU.521. Additionally, we describe a SPIN formulation in which PLGA is coemulsified with poly(benzoyloxypropyl methacrylamide) (P(HPMA-Bz)), which significantly improves drug loading and allows for tunable release of H-151 over a period of days to over a week by varying P(HPMA-Bz) content. Finally, we find that all SPIN formulations were as potent or more potent in inhibiting cGAS/STING signaling in primary murine macrophages, resulting in decreased expression of inflammatory M1-like macrophage markers. Therefore, our study provides an in vitro proof-of-concept for nanoparticle delivery of STING pathway inhibitors and positions SPINs as a potential platform for slowing or reversing the onset or progression of cGAS/STING-driven inflammatory conditions.

Keywords: PLGA; STING; anti-inflammatory; cGAS; controlled release; inhibitor; nanoparticle.

Figure 1

SPIN-R characterization and efficacy in cell lines. (A) Schematic depicting the oil-in-water fabrication method to produce SPINs. (B) RU.521 structure. (C) SPIN-R size distribution determined by dynamic light scattering. (D) RU.521 release from SPIN-R in pH 7.4 PBS at 37 °C. (E) Experimental timeline for testing SPIN-R and free RU.521 in reporter cell lines used in panels F and G. (F) Dose–response curves of IFN-I production induced by cotreatment with SPIN-R or RU.521 and 1 μg/mL HT DNA in RAW-Dual and (G) THP1-Dual cells (n = 3). (H) Experimental timeline for testing SPIN-R and free RU.521 in reporter cell lines used in panels I and J. (I) Dose–response of IFN-I production induced by pretreatment with SPIN-R or RU.521 prior to addition of 1 μg/mL HT DNA in RAW-Dual and (J) differentiated THP1-Dual cells (n = 3). Dose–response data is represented as mean ± SEM, fit using inhibitor vs response (four-parameter fit) nonlinear fit in GraphPad Prism.

Figure 2

SPIN-H characterization and efficacy in cell lines. (A) H-151 structure. (B) SPIN-H size distribution determined by dynamic light scattering. (C) H-151 release from SPIN-H in pH 7.4 PBS at 37 °C. (D) Experimental timeline for testing SPIN-H and free H-151 in reporter cell lines used in panels E and F. (E, F) Dose–response curves of IFN-I production induced by cotreatment with SPIN-H or H-151 and 1 μg/mL HT DNA in (E) RAW-Dual and (F) THP1-Dual cells (n = 3). (G) Experimental timeline for testing SPIN-R and free RU.521 in reporter cell lines used in panels H and I. (H, I) Dose–response curves of IFN-I production induced by pretreatment with SPIN-H or H-151 followed by 1 μg/mL HT DNA in (H) RAW-Dual and (I) differentiated THP1-Dual cells (n = 3). Dose–response data is represented as mean ± SEM, fit using inhibitor vs response (four-parameter fit) nonlinear fit in GraphPad Prism.

Figure 3

Including P(HPMA-Bz) in SPINs extends drug release without compromising inhibitor capacity. (A) Schematic of increased inhibitor loading with inclusion of benzylated excipient polymer in SPINs. (B) P(HPMA-Bz) synthesis scheme. (C) SPIN-R_10% size distribution determined by dynamic light scattering. (D) RU.521 release from SPIN-R_10% in pH 7.4 PBS at 37 °C. (E, F) Dose–response curves of IFN-I production induced by cotreatment with SPIN-R_10% and 1 μg/mL HT DNA in (E) RAW-Dual and (F) THP1-Dual cells (n = 3). (G) SPIN-H_1–10% size distribution determined by dynamic light scattering. (H) H-151 release from SPIN-H_1–10% in pH 7.4 PBS at 37 °C. (I, J) Dose–response curves IFN-I production induced by cotreatment with SPIN-H_1–10% and 1 μg/mL HT DNA in (I) RAW-Dual and (J) THP1-Dual cells (n = 3). Dose–response data are represented as mean ± SEM, fit using inhibitor vs response (four-parameter fit) nonlinear fit in GraphPad Prism.

Figure 4

SPINs with P(HPMA-Bz) inhibit cGAS/STING signaling in BMDMs. (A, B) IFN-β ELISA of supernatants from BMDMs cotreated with the designated SPIN or control and HT DNA after (A) 6 h and (B) 24 h (n = 3–4). (C–E) Gene expression of (C) Cxcl10, (D) Ifnb1, and (E) Tmem173 in BMDMs cotreated with the designated SPIN or control and HT DNA for 6 h (n = 3–4). (F, G) Surface expression of (F) CD80 and (G) CD86 in BMDMs cotreated with SPIN-RU_10%, free RU.521, or empty NP and HT DNA for 24 h (n = 2–4). (H, I) Surface expression of (H) CD80 and (I) CD86 in BMDMs cotreated with SPIN-H_5%, free H-151, or empty NP and HT DNA for 24 h (n = 3). Data are represented as mean ± SEM *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001 compared to Vehicle + HT DNA by one-way ANOVA.