The enhancing effect of DNA tetrahedron-based lactoferrin as an assembled adjuvant in immunotherapy

• The advancement of adjuvants for immunostimulatory cancer treatments has faced considerable obstacles. Engineered immune adjuvants represent one of the approaches to tackle the inadequacy of adjuvants. This study, engineering nanoparticle-based adjuvants, particularly DNA Tetrahedron-Aptamer-Lactoferrin (TAL), significantly enhances the immunostimulatory properties of lactoferrin, promoting effective antigen presentation and macrophage activation through RIG-I-like receptor (RLR) signaling. The implications of RLR pathway activation extend beyond macrophages, potentially informing therapeutic strategies aimed at enhancing immune responses in various contexts, including cancer immunotherapy and infectious diseases. • In vivo studies demonstrate that TAL not only improves the immune response but also works synergistically with Doxorubicin (DOX) in breast cancer models. This synergy facilitates cytotoxic T lymphocyte differentiation and amplifies the therapeutic effects of DOX while allowing for a reduction in its dosage. Furthermore, the activation of the RLR signaling pathway by TAL marks a notable advancement in our understanding of the molecular interactions that drive immune activation. The upregulation of critical genes such as Rig-I, TBK1, and Irf7 suggests that TAL not only stimulates macrophage activation but also triggers essential antiviral and pro-inflammatory signaling cascades. This process is crucial for orchestrating innate immune responses , which may, in turn, influence adaptive immunity . • This research addresses vital gaps in the current understanding and application of immune adjuvants and underscores the promise of intentionally engineering weak immunostimulatory agents into stable structures to develop effective adjuvants, thereby paving the way for innovative therapeutic strategies for cancer treatment in clinical settings. Cancer immunotherapy faces significant challenges in enhancing immune responses, necessitating the development of innovative adjuvants. This study investigates the immune-stimulatory properties of Lactoferrin (LF) when assembled into a DNA tetrahedron structure (TAL) with stable spatial structure as an engineered adjuvant. This approach effectively addressed the limited immunostimulatory ability of lactoferrin in its free state, demonstrating the crucial role of DNA tetrahedron-based assembly of lactoferrin in enhancing adjuvant stimulation of antigen-presenting cells (APCs). TAL promoted antigen presentation, leading to a heightened inflammatory response in macrophage activation through the activation of the RIG-I-like receptor signaling pathway. In vivo studies further demonstrated that the assembled TAL enhanced immune response and exhibited substantial efficacy in enhancing synergy with Doxorubicin (DOX) in the context of cancer immunotherapy for breast cancer models. This synergy facilitated the differentiation of cytotoxic T lymphocytes and significantly amplified the therapeutic effect of DOX, allowing for a reduction in the required DOX dosage. Our findings suggest that TAL holds promise as a novel immune adjuvant in cancer immunotherapy, offering valuable insights for the development of neoadjuvants derived from biological nanomaterials. This study underscores the potential of strategically engineering weak immunostimulatory agents into stable structures to develop effective adjuvants, paving the way for innovative therapeutic strategies of adjuvant in clinical oncology.