The role of surface charge on the uptake and biocompatibility of hydroxyapatite nanoparticles with osteoblast cells

Abstract

The objective of this study is to evaluate the effect of hydroxyapatite (HAP) nanoparticles with different surface charges on the cellular uptake behavior and in vitro cell viability and proliferation of MC3T3-E1 cell lines (osteoblast). The nanoparticles' surface charge was varied by surface modification with two carboxylic acids: 12-aminododecanoic acid (positive) and dodecanedioic acid (negative). The untreated HAP nanoparticles and dodecanoic acid modified HAP nanoparticles (neutral) were used as the control. X-ray diffraction (XRD) revealed that surface modifications by the three carboxylic acids did not change the crystal structure of HAP nanoparticles; Fourier transform infrared spectroscopy (FT-IR) confirmed the adsorption and binding of the carboxylic acids on the HAP nanoparticles' surfaces; and zeta potential measurement confirmed that the chemicals successfully modified the surface charge of HAP nanoparticles in water based solution. Transmission electron microscopy (TEM) images showed that positively charged, negatively charged and untreated HAP nanoparticles, with similar size and shape, all penetrated into the cells and cells had more uptake of HAP nanoparticles with positive charge compared to those with negative charge, which might be attributed to the attractive or repulsive interaction between the negatively charged cell membrane and positively/negatively charged HAP nanoparticles. The neutral HAP nanoparticles could not penetrate the cell membrane due to their larger size. MTT assay and LDH assay results indicated that as compared with the polystyrene control, greater cell viability and cell proliferation were measured on MC3T3-E1 cells treated with the three kinds of HAP nanoparticles (neutral, positive, and untreated), among which positively charged HAP nanoparticles showed the strongest improvement for cell viability and cell proliferation. In summary, the surface charge of HAP nanoparticles can be modified to influence the cellular uptake of HAP nanoparticles and the different uptake also influences the behavior of cells. These in vitro results may also provide useful information for investigations of HAP nanoparticle applications in gene delivery and intracellular drug delivery.

Figure 1

XRD patterns of (a) 12-aminododecanoic modified HAP nanoparticles, (b) dodecanedioic acid modified HAP nanoparticles, (c) dodecanoic acid modified HAP nanoparticles, and (d) untreated HAP nanoparticles.

Figure 2

The TEM images of HAP nanoparticles: (a) untreated, (b) 12- aminododecanoic acid modified, (c) dodecanedioic acid modified, (d) dodecanoic acid modified.

Figure 3

The FT-IR spectra of HAP nanoparticles treated with (a) 12-aminododecanoic acid, (b) dodecanedioic acid, and (c) dodecanoic acid, and (d) untreated HAP nanoparticles.

Figure 4

Zeta potentials of the HAP nanoparticles: (a) 12-aminododecanoic modified, (b) dodecanedioic acid modified, (c) dodecanoic acid modified, (d) untreated, in PBS solution at pH value of 7.4.

Figure 5

The TEM images of MC3T3-E1 cell lines cultured in medium with different HAP nanoparticles for 3days: (a, b) positively charged HAP nanoparticles, (c, d) negatively charged HAP nanoparticles, (e, f) neutral HAP nanoparticles, (g, h) untreated HAP nanoparticles. The TEM images on the right are the enlargement of rectangle area on the corresponding images on the left side.

Figure 5

The TEM images of MC3T3-E1 cell lines cultured in medium with different HAP nanoparticles for 3days: (a, b) positively charged HAP nanoparticles, (c, d) negatively charged HAP nanoparticles, (e, f) neutral HAP nanoparticles, (g, h) untreated HAP nanoparticles. The TEM images on the right are the enlargement of rectangle area on the corresponding images on the left side.

Figure 6

The proliferation of MC3T3-E1 cell lines on different dosages of untreated HAP nanoparticles by MTT assay. The polystyrene well without hydroxyapatite nanoparticles was used as the control. Data are presented as the average±SD for n=5. *indicate P<0.05 relative to controls.

Figure 7

The proliferation of MC3T3-E1 cell lines on differently charged HAP nanoparticles with dosage 1 mg/ml by MTT assay. The polystyrene well without hydroxyapatite nanoparticles was used as the control. Data are presented as the average±SD for n=5. *indicate P<0.05 relative to controls, # indicate P<0.05 relative to untreated HAP nanoparticles.

Figure 8

The LDH activities in the cell culture medium after 3 or 7 days exposure to the differently charged HAP nanoparticles on the dosage of 1mg/ml. The polystyrene well without hydroxyapatite nanoparticles was used as the control. Data are presented as the average±SD for n=5. *indicate P<0.05 relative to controls, # indicate P<0.05 relative to untreated HAP nanoparticles.

Scheme 1

Reaction routes for surface modification on HAP nanoparticles.