| In recent years, there is a growing interest in nanoparticles (1~100nm) due to their physical and chemical characteristics. Nano-technology is one of the backbones that help to develop new medical treatment techniques, and much research work has been conducted focusing on the aspects of the prevention and treatment of tumors. The treatment of hepatocellular carcinoma with hydroxyapatite (HAP) nanoparticles has aroused much attention.In this study, four kinds of HAP nanoparticles with different sizes were prepared by the homogeneous precipitation and sol-gel method, and their size distribution, crystallization degree, components, and morphology were characterized by laser granularity instrument, X-ray diffraction, FT-IR spectrum, transmission electron microscope (TEM), and electron diffraction. The measured average particle sizes of the different HAP nanoparticles were 59.9nm, 170.7nm, 288.9nm, and 421.1nm. They also showed the characteristic absorbability peak of HAP. As the results revealed, increasing particle sizes leaded to higher crystallization degrees and X-radial diffraction peaks while smaller particle sizes resulted in clearer electron diffraction annuli.The prepared HAP nanoparticles were used for the treatment of hepatocellular carcinoma. The inhibition effect was determined in vitro for four different average particle sizes and three different concentrations by MTT assays. Furthermore, growth curve and time of double proliferation (T_D) tests, colony formation rate measurements, and morphology observations were conducted. The results showed that HAP nanoparticles with an average size of 59.9nm most effectively inhibited the proliferation of hepatocellular carcinoma cells with an inhibition rate of more than 70 percent, which was significantly higher than the effect of 170.7nm size HAP nanoparticles(P<0.01). The inhibition proliferation was free cytotoxicity-mediated However, the inhibition rate for hepatocytes was comparatively low (P<0.01). For the treatment of hepatocellular carcinoma cells with 59.9nm size HAP nanoparticles, the growth curve ascended slowly, the time of double proliferation prolonged, and cloneformed rarely, which were statistically different from the untreated group (P<0.05). However, there was no distinct effect on the hepatocytes (P>0.05).The components of HAP nanoparticles in cells were studied by the use of TEM and electron diffraction methods. The HAP nanoparticles were found entering the cells and locating in the endoplasmic reticulum. The changes of the cell endoplasmic reticulum treated with HAP nanoparticles were further examined with laser scanning confocal microscope. The results showed that the endoplasmic reticulum content in hepatocellular carcinoma cells was lower than that of the hepatocytes (P<0.01). After the treatment with HAP nanoparticles, the content of endoplasmic reticulum in hepatocellular carcinoma cells decreased (P<0.05), while that of hepatocytes did not change (P>0.05).The cause of the hepatocellular carcinoma cell death was paraptosis rather than apoptosis by agarose gel electrophoresis and flow cytometry. Further investigation of the activity of caspase-9 and the expression of mRNA of c-myc and p53 indicated that HAP particles arrest hepatocellular carcinoma cells at Gi phase by activating the caspase-9, inhibiting the expression of c-myc, and up-regulating the expression of p53.In short, HAP nanoparticles significantly inhibited the proliferation of hepatocellular carcinoma cells in vitro in comparison with that of hepatocytes (P<0.01). The inhibition mechanism can be described as the following: First, the HAP nanoparticle enter the cells and accumulate in the cell endoplasmic reticulum, where they affect the activity of caspase-9. By regulating the mRNA expression of c-myc, p53 , the proliferation of hepatocellular carcinoma cells is arrested at G| phase. As a result, the proliferation of hepatocellular carcinoma cells is inhibited, inducing them to paraptosis.
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