Interactions Of Biomimetic Hydroxyapatite Nanoparticles With Liver Cancer Cells

In the human body, Hydroxyapatite (Ca₁₀(PO₄)6(OH)₂, HAP) is present in form of biological apatite in 60-70 % of the bone and dental tissue with the characteristics of low crystallinity, calcium deficient, and carbonate impurities. In many living organisms, amorphous calcium phosphates (ACP) were observed to work as a precursor for the formation of biological apatite. Recent studies showed that HAP nanoparticles in form of aqueous sol have a selective inhibition effect on the proliferation of liver cancer cells. However, the exact mechanism for the found anti-cancer effect could not be clearly identified. In this work, the interactions of biomimetic HAP nanoparticles with liver cancer cells were investigated. The correlations between the synthesis process on the one hand and nanoparticle characterstics and cellular effects on the other hand were described. Along with the optimization of the HAP nanoparticle sol concentration and the experimental use in vitro, the pathway of internalization as well as the functional mechanism for the effects of nano HAP sol on the proliferation of liver cancer cells were analysed and determined. The commonly used method to prepare HAP nanoparticles in our institute was based on monocalcium phosphate and calcium hydroxide and extensive ultrasound treatment. In this work, only one minute of ultrasound was applied to obtain nano dispersed ACP sol as biocompatible precursor with about 1 wg% carbonate impurities to imitate the natural formation of biological apatite in form of aqueous nanoparticle sol. The correlations of the parameters of time and temperature with the HAP nanoparticle characteristics were investigated by laser scattering, pH value measurement, XRD, FTIR, and TEM. Until peak temperatures of 50篊, the nano ACP with particle sizes above 130 nm remained stable and no phase transformation or change of the particle shape was observed. When kept for several weeks at room temperature, the carbonated amorphous calcium phosphate precursor transformed into HAP as well as some traces of calcite via slow diffusion controlled crystallization. The storage of ACP sol in the refrigerator at 4篊 dramatically reduced the stability of the sol and leaded to particle agglomerations. When the nano ACP sol was heated to peak temperatures between 50-75篊, the induced heat enabled the dissolution and re-precipitation of the Precursor and the formation of needle shaped HAP nanoparticles with sizes below 90 nm driven by chemical controlled surface reactions. The obtained HAP nanoparticles were low crystalline, calcium deficient (Ca/P＜1.65) with about 1 wg% carbonate impurity mainly of type B where CO₃^2- replaced PO₄^3-. Longer heating or sterilization had the effect of ripening which slowly increases the crystallinity and reduces the calcium deficiency. The heat-induction method was proposed to increase the HAP sol concentration through water evaporation. When heated for 25 minutes at 100篊, stable HAP sol with concentrations of up to 16.8 mg/ml was obtained. Longer heat-induced as well as steam sterilized HAP sols revealed very low calcium deficiency and slightly higher crystallinity. According to the standard procedure, more calcium hydroxide was added compared to stoichiometric HAP composition at Ca/P = 1.67 in order to achieve higher pH values to support the formation of HAP. This leaded to probably about 12% excessive Ca²⁺ ions. Furthermore, by-products such as amorphous and crystalline calcium carbonate were suggested. The experimental study of the interactions between HAP nanoparticle sol and the cell culture suspension revealed that HAP sol immediately became instable in cell culture medium and formed agglomerates of above 500 nm in size. When more than 20 vol% HAP sol with above pH 9 was added to the cell culture medium, the pH value of the cell suspension exceed the upper pH value tolerance of 7.45. The refreshing of medium after 12 hours and 2 days resulted in 17% higher proliferation activity after 5 days. Hence, with longer durations of incubations without refreshing the medium, the in vitro conditions worsened and thereby reduced the reliability of the calculated inhibition rates. The in vitro studies showed that the substitution of 40 vol% cell culture medium by aqueous HAP sol or distilled water both leaded to higher osmotic pressure inside of the cells that typically caused oncrosis. In contrast, the cell proliferation was only sighly affected'by the addition of 20 vol% water. At optimized in vitro conditions with identical nanoparticles and final HAP concentration of 0.56 mg/ml in the cell suspension, 33% stimulation after 2 and 5 days were determined. Alamar Blue confirmed the curve developing of the inhibition rate of HAP nanoparticles but was shifted to lower inhibition rates into the range of stimuation and measured 11% stimulation after 5 days. Increasing final HAP concentrations under optimized conditions from 0.56-1.12 mg/ml slightly stimulated cancer cell growth after 2 days and decreased the cell proliferation after 5 days. Furthermore, increasing crystallinity of the HAP nanoparticles leaded to higher proliferation activity after 2 days of incubation and multible steam sterilization leaded to highly toxic effects. The investigation of the internalization mechanism of HAP nanoparticles in hepatoma cells clearly verified clathrin mediated endocytosis as main entrance pathway of HAP nanoparticles in the liver cancer cells.The TEM analysis of the HAP nanoparticle treated cancer cells under optimized conditions clearly revealed the characteristics of paraptosis as dominant cell death type. The cell size and organells did not show visible changes in response to the HAP treatment. The only effect was found in the high density of vacuoles with various sizes. Not all of these vacuoles contained HAP nanoparticles and the enclosed nano HAP content was very low. Propoably due the similarity and relationship of apoptosis and paraptosis, apoptosic patterns were indicated by the flow cytometry of the HAP treated cancer cells. The cell cycle analysis revealed higher amounts of cells in the G1 and S phase.The short-term stimulation of cell growth was probably caused by ionic HAP sol by-products and early HAP solutes that functioned as additional nutritions. The long-term inhibition effect resulted from risen ion contents in the cytoplasma close or above toxic concentrations and accumulating vacuoles began to destroy the cytoskeleton. These changes induced by the internalized HAP nanoparticle sol induced paraptosis which leaded to further vacuolization within the process of programmed cell death.This work provided the foundation for the optimization of the interactions and effects between biomimetic HAP nanoparticles and liver cancer cells for biomedical applications. The usage of nano HAP as carrier for drug or gene delivery was proposed as very promising application for the treatment of cancer and other deaseases.