High‐Integrity Nanoformulation of Resiquimod (R848) for Dual Autophagy Activation and PD‐L1 Modulation in Triple‐Negative Breast Cancer

Abstract

Triple-negative breast cancer (TNBC) remains a formidable clinical challenge due to its molecular heterogeneity and resistance to conventional therapies. This study presents a high-integrity nanoemulsion (NE) formulation designed to enhance the delivery and stability of the Toll-like receptor 7/8 (TLR7/8) agonist resiquimod (R848) for immunotherapy. Neutral and negatively charged NEs were developed with and without the reactive lipophilic compound ricinoleic acid. Physicochemical characterization and in vitro studies in RAW 264.7 macrophages and 4T1 TNBC cells demonstrated that R848-loaded NEs exhibit prolonged shelf-life, minimal protein binding, and efficient cellular uptake. Incorporation of ricinoleic acid improved drug retention and delivery, likely through enhanced drug-lipid interactions. Molecular profiling in 4T1 cells revealed modulation of key biomarkers (TLR4/7, Cyclin D1, NF-κB), induction of autophagy (LC3II, p62, Beclin-1), and upregulation of PD-L1 expression. These dual effects, autophagy-mediated antitumor mechanisms and immune checkpoint modulation, highlight the potential of R848-NEs as a synergistic partner in anti-PD-L1 combination therapy, offering a promising strategy for TNBC treatment.