Patterns were analyzed through the use of Jade 6.0 software program. five HANPs acquired different physicochemical properties, including morphology, size, particular surface (SSA), crystallinity, etc. With the in vitro cell research, it was discovered that the materials factors played essential assignments in the anti-melanoma aftereffect of HANPs. Among the as-prepared five HANPs, HA-A with granular form, smaller sized size, higher SSA, and lower crystallinity exhibited greatest influence on inhibiting the viability of A375 cells. On the focus of 200 g/mL, HA-A led to TRV130 (Oliceridine) the cheapest cell viability (34.90%) in day 3. All of the HANPs could induce the apoptosis of A375 cells, as well as the fairly higher apoptosis rates of the cells were found in HA-A (20.10%) and HA-B (19.41%) at day 3. However, all the HANPs showed no inhibitory effect on the viability of the normal human epidermal fibroblasts. The preliminary in vivo evaluation showed that both HA-A and HA-C could delay the formation and growth velocity of melanoma tissue significantly. Likely, HA-A exhibited better effect on inhibiting the growth of melanoma tissue than HA-C. The inhibition rate of HA-A for tumor tissue growth reached 49.1% at day 23. Conclusion The current study confirmed the anti-melanoma effect of HANPs and provided a new idea for the clinical treatment of melanoma. Keywords: hydroxyapatite, nanoparticles, melanoma cells, fibroblasts, viability, apoptosis, tumor, suppression Introduction As the largest organ and outer shell of human body, skin mainly protects tissues and organs in the body from the attack of physical factor, chemical substance, mechanical stress, and pathogenic microorganism.1,2 In the epidermal layer of skin, there are five layers from inside to outside, in which the melanocytes in the basal layer are susceptible to lesions and then transform into melanoma.3,4 In recent years, melanoma took around the increasing incidence rate and can also be found in mucosa, choroid, and other tissues.5C9 So far, the general clinical treatment is still surgical resection, accompanied by chemotherapy and immunotherapy. However, melanoma has the characteristics of rapid proliferation, local invasion, TRV130 (Oliceridine) long-distance migration, and strong resistance to currently clinical therapies.1,10 Except the thin primary skin melanoma (<1 mm), the clinical surgery for metastatic melanoma and deep primary malignant melanoma (>4 mm) still have a very high recurrence rate and mortality.11,12 Therefore, new strategies for improving the clinical treatment effect of melanoma are TRV130 (Oliceridine) quite necessary. Hydroxyapatite (HA) is usually a major inorganic component of human bone and teeth, and exhibits excellent biocompatibility, bioactivity, osteoconduction, and even osteoinduction in biomedical application.13C15 In 1990s, Aoki et al and Kano et al first reported the in vitro anti-tumor effect of HA nanoparticles (HANPs).16,17 They occasionally found that HANPs without loading doxorubicin still had the inhibitory effect on the proliferation for Ca-9 tumor cells. After that, the anti-tumor effects of HANPs were widely regarded and investigated. A large number of reports indicated that HANPs could inhibit the proliferation of various tumor cells, such as hepatoma cells,18C20 osteosarcoma cells,21C23 lung cancer cells,24,25 and gastric cancer cells26C28 to some extent. Moreover, HANPs showed little or no inhibitory effect on the normal tissue cells, including osteoblasts,23 hepatocytes,18 lung fibroblasts,25 etc. This was undoubtedly hopeful to overcome the drawbacks of some anti-tumor drugs, which could kill cancer cells as well as normal tissue cells. In previous studies, Li et al reported that HANPs had certain anti-melanoma effect.29 They found that for HANPs, the size had stronger influence around the proliferation of A875 melanoma cells than the morphology. However, the involved mechanism has not been well revealed. Besides, the correlation between the material factors of HANPs and proliferation inhibition or apoptosis of melanoma cells need be further investigated. Hence, in the present study, we prepared five different HANPs by TRV130 (Oliceridine) wet chemical method combining with polymer template and different post-treatments, and investigated their anti-melanoma effects by in vitro and in vivo experiments. Besides, human fibroblasts were chosen as the control to investigate their impacts on normal tissue cells. The influences of various material factors around the anti-melanoma effects of the HANPs were studied systematically and discussed. Materials and methods Reagents Ca(NO3)24H2O, (NH4)2HPO4, and NH3H2O were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). PEG2000 was purchased from the Aladdin (Shanghai, China). Human melanoma cells (A375) and human epidermal fibroblasts (HSF) were purchased from iCell Bioscience Inc (Shanghai, China). FBS, DMEM medium, penicillinCstreptomycin answer, PBS, and Trypsin 0.25% EDTA were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Fluorescein diacetate and propidium iodide (PI) were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). DAPI was purchased from Beyotime (Shanghai, China). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Laboratories (Kumanoto, Japan). Fluorescein Dysf isothiocyanate (FITC) Annexin V Apoptosis Detection Kit I.