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.

The rapid degradation of magnesium (Mg)-based implants in physiological environment limits its clinical applications, and alloying treatment is an effective way to regulate the degradation rate of Mg-based materials. In the present study, three Mg alloys, including Mg-0.8Ca (denoted as ZQ), Mg-0.8Ca-5Zn-1.5Ag (denoted as ZQ71) and Mg-0.8Ca-5Zn-2.5Ag (denoted as ZQ63), were fabricated by alloying with calcium (Ca), zinc (Zn) and silver (Ag). The results obtained from electrochemical corrosion tests and in vitro degradation evaluation demonstrated that the three Mg alloys exhibited distinct corrosion resistance, and ZQ71 exhibited the lowest degradation rate in vitro among them. After addition of Zn and Ag, the antibacterial potential of Mg alloys was also enhanced. The in vitro cell experiments showed that all the three Mg alloys had good biocompatibility. After implantation in a rat femoral defect, ZQ71 showed significantly higher osteogenic activity and bone substitution rate than ZQ63 and ZQ, due to its higher corrosion resistance as well as the stimulatory effects of the released metallic ions. In addition, the average daily degradation rate of each Mg alloy in vivo was significantly higher than that in vitro, as could be due to the implantation site located in the highly vascularized trabecular region. Importantly, the correlations between the in vitro and in vivo degradation parameters of the Mg alloys were systematically analyzed to find out the potential predictors of the in vivo degradation performance of the materials. The current work not only evaluated the clinical potential of the three biodegradable Mg alloys as bone grafts but also provided a feasible approach for predicting the in vivo degradation behavior of biodegradable materials.