The potency of Toll-like receptor 9 (TLR9) agonist to drive innate immune response was limited due to immune suppression or tolerance during TLR9 signaling activation in immune cells. Herein we addressed this problem by introducing hydroxyapatite nanoparticles (HANPs) to CpG ODN (CpG), a TLR9 agonist. The study revealed that HANPs concentration and duration-dependently reprogramed the immune response by enhancing the secretion of immunostimulatory cytokines (tumor necrosis factor α (TNFα) or IL-6) while reducing the production of immunosuppressive cytokine (IL-10) in macrophages in response to CpG. Next, the enhanced immune response benefited from increased intracellular Ca²⁺ in macrophage by the addition of HANPs. Further, we found exposure to HANPs impacted the mitochondrial function of macrophages in support of the synthesis of adenosine triphosphate (ATP), the production of nicotinamide adenine dinucleotide (NAD), and reactive oxygen species (ROS) in the presence or absence of CpG. In vaccinated mice model, only one vaccination with a mixture of CpG, HANPs, and OVA, a model antigen, allowed the development of a long-lasting balanced humoral immunity in mice without any histopathological change in the local injection site. Therefore, this study revealed that HANPs could modulate the intracellular calcium level, mitochondrial function, and immune response in immune cells, and suggested a potential combination adjuvant of HANPs and TLR9 agonist for vaccine development.

Clathrin-mediated endocytosis plays a critical role for hydroxyapatite nanoparticles (HANPs) to enter tumor cells, induce mitochondrial apoptosis, and inhibit tumor growth. This study was aimed to investigate how the morphology of HANPs impacts the endocytosis of the particles in melanoma cells, and their anti-tumor effect by using in vitro cell experiments and in vivo tumor animal model. Three shapes of HANPs, including granular HANPs (G-HANPs), rod-like HANPs (R-HANPs), and needle-like HANPs (N-HANPs), were successfully prepared by wet chemical method. All the three HANPs could be internalized into A375 melanoma cells as indicated by cellular transmission electron microscopy images. Among these HANPs, only G-HANPs induced morphological change of mitochondria and loss of mitochondrial membrane potential (Δψ_m), and exhibited the greatest intracellular internalization efficiency in the tumor cells. Furthermore, the results of immunofluorescence staining and western blotting indicated that the level of adaptin-2 (AP2) protein was up-regulated by all the HANPs, and highest in G-HANPs treated A375 cells. Moreover, in the tumor-bearing mouse model, we found that tumor growth was delayed by all the three HANPs, of which, G-HANPs delayed tumor growth most efficiently and presented a highest expression level of AP2 protein in tumor tissues. Therefore, this study suggested that the morphology of HANPs regulated their endocytosis efficiency and their effect to inhibit tumor growth. This work facilitates to direct the rational design of nano-materials for tumor therapy.