Characteristics of functionalized nano-hydroxyapatite and internalization by human epithelial cell

Abstract

Hydroxyapatite is the main inorganic component of biological bone and tooth enamel, and synthetic hydroxyapatite has been widely used as biomaterials. In this study, a facile method has been developed for the fabrication of arginine-functionalized and europium-doped hydroxyapatite nanoparticles (Arg-Eu-HAP). The synthesized nanoparticles characterized by transmission electron microscopy, X-ray diffractometry, Fourier transform infrared, and Zeta potential analyzer. Its biological properties with DNA binding, cell toxicity, cell binding and intracellular distribution were tested by agarose gel electrophoresis assay, flow cytometry, and fluorescence microscope and laser scanning confocal microscope. The synthesized Arg-Eu-HAP could effectively bind DNA without any cytotoxicity and be internalized into the cytoplasm and perinuclear of human lung epithelial cells.

Figure 1

TEM images of Arg-Eu-HAP crystal synthesized by hydrothermal method. (a) Without amino acid; (b) with arginine

Figure 2

XRD patterns of nanoparticles and Eu-doped nanoparticles.

Figure 3

FTIR spectra of arginine-functionalized nanoparticles: (a) without amino acid; (b) with arginine.

Figure 4

Luminescence excitation (a) and emission (b) spectrum of europium-doped HAP.

Figure 5

Zeta potential curve of Arg-Eu-HAP at pH of 7.5.

Figure 6

Agarose gel electrophoresis of Arg-Eu-HAP/DNA complexes (w/w ratio). M, marker; L1, positive control; L2, 101:; L3, 301:; L4, 501:; L5, 701:; L6, negative control.

Figure 7

Cell viability assay. Cell viability assay showing the effect of varying concentrations of nanoparticles on growth inhibition of human lung epithelial (A549) cancer cells cultured in vitro. Results are reported as mean. There is no statistically significant difference between test groups and control groups (p < 0.05).

Figure 8

Green emission and Laser scanning confocal microscope images. (a) Green emission of the internalized Arg-Eu-HAP into the cells under fluorescence microscopy. Arg-Eu-HAP (green) were evident in the cytoplasm, nucleus were counterstained with DAPI dye (blue). Representative images of four different experiments are shown (magnification ×40). (b) Laser scanning confocal microscope images (magnification ×60, insert magnification ×252). No fluorescent light in the control cells can be detected.