Micron-scale lactoferrin proteinosomes potentiate immune activation and chemo-immunotherapy in breast cancer.

Efficient uptake by antigen-presenting cells (APCs) is critical for the immunostimulatory performance of protein-based adjuvants, yet soluble proteins often suffer from poor cellular internalization and transient immune activation. Here, we report a hierarchical micron-scale assembly strategy to construct lactoferrin proteinosomes (LPs) via interfacial self-assembly. The resulting spherical LPs, with an average diameter of ∼2 μm, exhibit robust structural stability while retaining the native secondary structure of lactoferrin. Compared with monomeric lactoferrin, LPs markedly enhance internalization by macrophages and dendritic cells (DCs). LPs promote pro-inflammatory macrophage activation, evidenced by upregulated CD80, CD86 and MHC-II expression and elevated secretion of IL-1β, TNF-α, IL-6, and nitric oxide. Transcriptomic profiling further revealed an inflammatory gene-expression signature associated with multiple innate immune signaling pathways in LP-treated macrophages. Moreover, LPs promote DC phenotypic maturation in vitro and enhance DC activation in lymph nodes in vivo, accompanied by a favorable biosafety profile and elevated systemic levels of immunostimulatory cytokines. In a 4T1 breast tumor model, combined administration of LPs and doxorubicin significantly improves therapeutic outcomes by remodeling the tumor immune microenvironment, as indicated by increased infiltration of macrophages, DCs, and T cells. Collectively, this work demonstrates the potential of hierarchical micron-scale assembly for enhancing the immunoadjuvant activity of proteins in combined cancer therapy.