Effect of Hydroxyapatite Nanoparticle Crystallinity and Colloidal Stability on Cytotoxicity

Abstract

Hydroxyapatite nanoparticles (nHA) have gained attention as potential intracellular drug delivery vehicles due to their high binding affinity for various biomolecules and pH-dependent solubility. Yet, the dependence of nHA cytocompatibility on their physicochemical properties remains unclear since numerous studies have revealed starkly contrasting results. These discrepancies may be attributed to differences in size, shape, crystallinity, and aggregation state of nHA, which complicates fundamental understanding of the factors driving nHA cytotoxicity. Here, we hypothesize that nHA cytotoxicity is primarily driven by intracellular calcium levels following the internalization of nHA nanoparticles. By investigating the cytotoxicity of spherical nHA with different crystallinity and dispersity, we find that both lower crystallinity and increased agglomeration of nHA raise cytotoxicity, with nanoparticle agglomeration being the more dominant factor. We show that the internalization of nHA enhances intracellular calcium levels and increases the production of reactive oxygen species (ROS). However, only subtle changes in intracellular calcium are observed, and their physiological relevance remains to be confirmed. In conclusion, we show that nHA agglomeration enhances ROS production and the associated cytotoxicity. These findings provide important guidelines for the future design of nHA-containing formulations for biomedical applications, implying that nHA crystallinity and especially agglomeration should be carefully controlled to optimize biocompatibility and therapeutic efficacy.

Keywords: agglomeration; crystallinity; cytotoxicity; hydroxyapatite nanoparticles; intracellular calcium; reactive oxygen species.

Figure 1

Physicochemical characterization of nHA. (A) Scanning electron microscopy image of nanoparticle morphology, (B) zeta potential distribution, and (C) photographs of suspensions of nHA synthesized with (+)/without (−) citrate as dispersant. (D) X-ray diffractograms of nHA as a function of citrate addition (with (+)/without (−) citrate) and aging time. (E) Calcium release at pH 6. All nanoparticles were synthesized at 40 °C. * indicates peaks characteristic for apatite. Scale bar represents 500 nm.

Figure 2

Cytocompatibility of nHA synthesized with (+)/without (−) citrate at 40 °C and aged for either 10 min, 1 or 5 h. (A) Metabolic activity and (B) DNA content of MC-3T3 cells after exposure to hydroxyapatite for 72 h. Untreated cells were set as 100%. Statistical analysis can be found in Table S1.

Figure 3

Internalization of nHA. Confocal live cell images showing internalization of nHA (green) after 24 h and their localization in lysosomal compartments (magenta). Co-localization of nHA and lysosomal compartments appears as white. (A/C) nHA were synthesized at 40 °C with/without citrate, or (B/D) synthesized at 60 °C with citrate and agglomerated by lyophilization. All nanoparticles were aged for 5 h. Enlarged regions of interest are marked by white squares in the images on the left, and a vacuole-like structure is indicated by the white arrow. Scale bars represent 25 and 10 μm (zoom-in image).

Figure 4

Intracellular calcium levels in MC-3T3 cells exposed to nHA (A/C) synthesized at 40 °C with/without citrate or (B/D) synthesized at 60 °C with citrate and agglomerated by lyophilization. All nanoparticles were aged for 5 h. Calcium levels were determined after 24 h using inductively coupled plasma mass spectrometry (A/B) or microscopically after 4 and 24 h using a calcium-sensitive dye (C/D).

Figure 5

ROS after exposure to nHA. Confocal live cell images of MC-3T3 cells exposed to (A) nHA synthesized at 40 °C with/without citrate aged for 5 h and (B) nHA synthesized at 60 °C with citrate aged for 5 h and agglomerated by lyophilization. Cells were stained for ROS. Images are colored with Fire LUT to indicate ROS intensity. (C,D) Quantification of staining intensity. Scale bar represents 50 μm.