BBR-RHE Co-Loaded Nanoemulsion Potentiates Anti-PD-L1 Efficacy via Lipid Metabolic Reprogramming in Triple-Negative Breast Cancer

Abstract Triple-negative breast cancer (TNBC) therapy is limited by the poor efficacy of anti-programmed death-ligand 1 (aPD-L1) monotherapy, with dysregulated lipid metabolism in the tumor microenvironment (TME) driving immunosuppression. Herein, a nanoemulsion co-loaded with berberine (BBR) and rhein (RHE) (BBR-RHE-NE), two lipid-modulating bioactives from Dahuang Huanglian Xiexin Decoction, was fabricated to remodel the immunosuppressive TME and boost aPD-L1 efficacy in TNBC. BBR-RHE-NE was characterized with a small droplet size (185.8 ± 0.44 nm), narrow polydispersity (PDI: 0.12 ± 0.02), and good stability. In orthotopic 4T1Fluc TNBC mouse models, BBR-RHE-NE monotherapy inhibited tumor growth, and its combination with aPD-L1 exerted robust synergistic anti-tumor activity. Flow cytometry revealed that BBR-RHE-NE, as a monotherapy or in combination with aPD-L1, enhanced effector immune cell infiltration and reduced immunosuppressive subsets in both tumor tissues and spleens. Lipidomic analysis, Oil Red O staining, and enzymatic assays confirmed BBR-RHE-NE reduced intratumoral lipid droplet accumulation and downregulated intratumoral triacylglycerols and fatty acids (FAs) levels both in vitro and in vivo. Mechanistically, RNA sequencing and functional validation experiments demonstrated that BBR-RHE-NE potentiated aPD-L1 efficacy via PPARγ/FABP4-mediated lipid metabolic remodeling, a process that promoted dendritic cell maturation and was attenuated by the PPARγ agonist GW1929. Moreover, BBR-RHE-NE exhibited excellent biocompatibility with no detectable organ toxicity. Collectively, these findings demonstrate that BBR-RHE-NE effectively remodels the immunosuppressive TME through lipid metabolic reprogramming, offering a promising nanomedicine-based combinatorial strategy to enhance aPD-L1 immunotherapy for TNBC.