The Immune-Enhancing Properties of Hwanglyeonhaedok-Tang-Mediated Biosynthesized Gold Nanoparticles in Macrophages and Splenocytes

Background: Despite great advances in the field of immunotherapy, there is still a need for novel and effective immunostimulants to overcome challenges, such as instability and autoinflammatory toxicity, associated with conventional immunostimulants. Nanotechnology provides the possibility to overcome these challenges. The well-known classical Chinese formula, Hwanglyeonhaedok-tang (HHT) has been widely used to treat immune-related diseases in clinical practice. Methods: We developed novel gold nanoparticles (AuNPs) utilizing one-pot synthesis with the herbal formula-HHT. The optimal conditions for HHT-AuNP biosynthesis were established, and physicochemical properties of the optimized HHT-AuNPs were identified using various spectrometric and microscopic techniques. Bio-TEM analysis revealed that HHT-AuNPs were highly engulfed within RAW264.7 cells without inducing cytotoxicity. The effect of HHT-AuNPs on immunostimulatory activity was evaluated in innate and adaptive immune cells (RAW264.7 macrophages and ICR mice splenocytes) using qRT-PCR, immunoblotting, and ELISA. Results: The HHT-AuNPs remarkably increased the nitric oxide (NO) and immune-related cytokines production by activating the mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) signaling pathways in RAW264.7 cells. Furthermore, HHT-AuNPs exerted immunostimulatory effects on mouse splenocytes by priming T/B-cells and macrophages. Discussion: The present study is the first to demonstrate that HHT-AuNPs could be utilized as immunostimulators to activate both innate and adaptive immune systems. These results provide a foundation for the application of traditional Chinese medicinal formulae in the field of nanomedicine.