(W0930-07-37) Nanoparticle Development for Systemic Delivery of Resiquimod to Reactivate Immunosuppressive Tumor Microenvironment

Purpose: Immunotherapies have proven clinically effective in treating cancer. A subset of immunotherapies seeks to generate tumor-associated neoantigens and activate immune cells in situ, thereby inducing anti-tumor immune responses. However, prolonged inflammation in the tumor microenvironment (TME) induces myelopoiesis and recruits a highly immunosuppressive population called myeloid-derived suppressor cells (MDSCs) to the tumor [1]. MDSCs recruit other immunosuppressive cells such as M2-like macrophages and regulatory T cells (Treg), hamper lymphocyte homing to the tumor, and suppress T cells by producing reactive oxygen species and expressing PD-L1 immune checkpoint receptor [1]. Toll-like receptor 7 (TLR7) agonists, such as resiquimod (R848), suppress and polarize the immunosuppressive MDSCs to pro-inflammatory phenotypes. Therefore, TLR7 agonists have been used to prevent the development of immunosuppressive TME after cancer immunotherapy; however, dose-limiting toxicities have hampered their therapeutic use [2, 3]. There have been efforts to reduce the toxicity using prodrugs, ligand-drug conjugates, or nanoparticles (NPs) [2-5]. However, these systems require intrinsic or extrinsic stimuli to release the drug, or they do not completely eradicate tumors and achieve long-term survival, possibly due to limited biodistribution to tumors or poor drug release control. Therefore, there is a strong unmet need for a carrier of TLR7 agonists for their safe and effective delivery to tumors. The objective of this study is to develop a carrier for the systemic delivery of R848 to reactivate the immunosuppressive tumor microenvironment while avoiding systemic toxicity.

Methods: Resiquimod nanoparticles (R848@NPs) were made by mixing R848, tannic acid (TA), and Fe³⁺. Polyvinyl alcohol (PVA) was used as a surface stabilizer to improve colloidal stability of R848@NP. R848@NPs were incubated in 4% PVA solution overnight to make R848@NP/PVA. Drug loading was quantified by high-performance liquid chromatography (HPLC). The release kinetics of R848@NP and R848@NP/PVA were assessed by dialysis (20 kDa MWCO). Nanoparticles equivalent to 0.2 mg R848 dispersed in 1 mL of phosphate-buffered saline (PBS) were placed in the dialysis device, immersed in 10 mL of PBS, and incubated at 37°C under shaking. At timed intervals, the release media was sampled for HPLC analysis. Bone marrow cells were collected from Balb/c mice and differentiated into dendritic cells. The bone marrow-derived dendritic cells (BMDCs) were stimulated with free R848, blank NP/PVA, R848@NP, or R848@NP/PVA at an equivalent to 1 µg/mL of R848 for 24 hours. The expression of maturation markers CD11c and CD86 was analyzed by flow cytometry. In vivo safety study was tested with Balb/c mice (female, 5- 6 weeks old). Blank NP/PVA was dispersed in 100 µL of 5% dextrose and administered via tail vail injection at 65 mg/kg or 118 kg/kg. Body weight of the mice was monitored for 5 days.

Results: R848@NP/PVA was prepared by mixing TA and R848 to form TA/R848 complexes, followed by cross-linking with Fe³⁺ ions and stabilization with PVA (Fig. 1a). Both TA/R848 and R848@NP appeared to be clusters of smaller particles (Fig. 1b). R848@NP/PVA had a similar look as R848@NP, but the detail was less defined, likely due to the surface coating of PVA (Fig. 1b). The R848@NP preparation was optimized by varying the R848 feed per fixed amounts of TA and Fe³⁺. The loading capacity ranged from 10 wt% to 20 wt% (Fig. 1c). In water, R848@NP/PVA was smaller than R848@NP with a z-average of 163 nm in diameter vs. 200 nm (Fig. 1d). More importantly, the colloidal stability of R848@NP/PVA was better than R848@NP in PBS: R848@NP/PVA was measured to be 340 nm in PBS, whereas R848@NP underwent uncontrollable aggregation, resulting in a z-average greater than 2000 nm (Fig. 1d). PVA coating also delayed the release of R848 from the NP, which is necessary to reduce systemic toxicity. Within 24 hours of incubation in PBS, R848@NP released 60% of the loaded R848, while R848@NP/PVA only released 40% and slowly released part of the rest over five days (Fig. 1e). BMDCs were used to evaluate the activity of R848. Free R848 induced the maturation of BMDCs most, followed by R848@NP/PVA, then R848@NP (Fig. 2a). The relatively high maturation induced by R848@NP/PVA compared to R848@NP may be explained by the colloidal stability of the NPs that facilitates their cellular uptake. To evaluate the in vivo safety of the carrier, blank NP/PVA was administered to Balb/c mice via intravenous injection at 65 mg/kg and 118 mg/kg. The body weight of the treated mice was stable after the injection, suggesting the safety of the carrier at the tested doses (Fig. 2b).

Conclusion: R848@NP/PVA encapsulated R848 with 20 wt% loading capacity, controlled the R848 release, and maintained its TLR7 agonist activity in vitro. The preliminary in vivo safety study showed that blank NP/PVA was safe for intravenous injection at a dose as high as 118 mg/kg. These results suggest that R848@NP/PVA has the potential to attenuate the systemic toxicity related to free R848.

References: 1. Groth, C., et al., Br J Cancer, 2019. 120(1): p. 16-25.
2. Cresswell, G.M., et al., Cancer Res, 2021. 81(3): p. 671-684.
3. Spinetti, T., et al., Oncoimmunology, 2016. 5(11): p. e1230578.
4. Sun, J., et al., Adv Mater, 2023. 35(29): p. e2207733.
5. Chen, P.M., et al., Biomaterials, 2020. 230: p. 119629.

Acknowledgements: The authors acknowledge the supports of Ronald W. Dollens Graduate Scholarship in the Life Sciences and the Purdue University Office of Research NIH Competing Renewal R01 Program.

Figure 1. Characterization of R848@NP/PVA. (a) Schematics of R848@NP/PVA synthesis. (b) TEM images of blank NP, TA/R848 complex, R848@NP, and R848@NP/PVA. S cale bar: 200 nm. (c) Loading capacity and encapsulation efficacy of R848@NP prepared with 0.3 mg – 1.2 mg R848 and 1.2 mg of TA. (d) Colloidal stability of R848@NP and R848@NP/PVA in water and PBS. (e) Release kinetics of R848@NP and R848@NP/PVA in PBS.

Figure 2. In vitro and in vivo assessment of the carrier. (a) Maturation of BMDCs incubated with free R848, blank NP/PVA, R848@NP, or R848@NP/PVA at an equivalent to 1 µg/ml of R848 for 24 hours and the representative dot plot. (b) Body weight of Balb/c mice after IV administration of blank NP/PVA at 65 mg/kg or 118 mg/kg.