Skip to main content
SpringerLink
Log in

Cytotoxicity of hydroxyapatite nanoparticles is shape and cell dependent

  • Inorganic compounds
  • Published:
Archives of Toxicology Aims and scope Submit manuscript

Abstract

Nanosized hydroxyapatite (nHA) has been proposed as drug delivery vehicles because of its biocompatibility. While the possible risks of nHA inducing inflammation have been highlighted, the specific influence of varying nHA particle morphology is still unclear. In order to establish this understanding, nHA of four different shapes—needle (nHA-ND), plate (nHA-PL), sphere (nHA-SP) and rod (nHA-RD)—were synthesized. The particle effects with the concentration of 10–300 μg/mL on cytotoxicity, oxygen species generation, production of inflammatory cytokines (TNF-α and IL-6), particle–cell association and cellular uptake were evaluated on BEAS-2B and RAW264.7 cells. Results show that nHA-ND and nHA-PL induced the most significant cell death in BEAS-2B cultures compared to nHA-SP and nHA-RD. Necrosis–apoptosis assay by FITC Annexin V and propidium iodide (PI) staining revealed loss of the majority of BEAS-2B by necrosis. No significant cell death was recorded in RAW264.7 cultures exposed to any of the nHA groups. Correspondingly, no significant differences were observed in TNF-α level for RAW264.7 cells upon incubation with nHA of different shapes. In addition, nHA-RD exhibited a higher degree of particle–cell association and internalization in both BEAS-2B and RAW264.7 cells, compared to nHA-ND. The phenomena suggested that higher particle–cell association and increased cellular uptake of nHA need not result in increased cytotoxicity, indicating the importance of particle shape on cytotoxicity. Specifically, needle- and plate-shaped nHA induced the most significant cell-specific cytotoxicity and IL-6 expression but showed the least particle–cell association. Taken collectively, we demonstrated the shape-dependent effects of nHA on cytotoxicity, inflammatory cytokine expression and particle–cell association.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

Abbreviations

nHA:

Hydroxyapatite nanoparticle

HAF:

FITC-tagged nHA

nHA-RD:

Rod-shaped nHA

nHA-DN:

Needle-shaped nHA

nHA-SP:

Spherical nHA

nHA-PL:

Plate-shaped nHA

FITC:

Fluorescein isothiocyanate

ARS:

Alizarin red S staining

DFDA:

Dihydrofluorescein diacetate

ROS:

Reactive oxygen species

APTES:

3-Aminopropyltriethoxysilane

CTAB:

Cetyltrimethylammonium bromide

dsDNA:

Double-stranded DNA

BET:

Brunauer–Emmett–Teller

CPC:

Cetylpyridinium chloride

CLSM:

Confocal laser scanning microscopy

FT-IR:

Fourier transform infrared spectra

XRD:

X-ray diffraction

TEM:

Transmission electron microscope

PI:

Propidium iodide

TNF-α:

Tumor necrosis factor

IL:

Interleukin

References

  • Albrecht C, Scherbart AM, Berlo DV, Braunbarth CM, Schins RPF, Scheel J (2009) Evaluation of cytotoxic effects and oxidative stress with hydroxyapatite dispersions of different physicochemical properties in rat NR8383 cells and primary macrophages. Toxicol In Vitro 23:520–530

    Article  PubMed  CAS  Google Scholar 

  • Berghe TV, Kalai M, Denecker G, Meenus A, Saelens X, Vandenabeele P (2006) Necrosis is associated with IL-6 production but apoptosis is not. Cell Signal 18:328–335

    Article  Google Scholar 

  • Bisht S, Bhakta G, Mitra S, Maitra A (2005) pDNA loaded calcium phosphate nanoparticles: highly efficient non-viral vector for gene delivery. Int J Pharm 288:157–168

    Article  PubMed  CAS  Google Scholar 

  • Cai Y, Liu Y, Yan W, Hu Q, Tao J, Zhang M, Shi Z, Tang R (2007) Role of hydroxyapatite nanoparticle size in bone cell proliferation. J Mater Chem 17(36):3780–3787

    Article  CAS  Google Scholar 

  • Cheng XG, Kuhn L (2007) Chemotherapy drug delivery from calcium phosphate nanoparticles. Int J Nanomedicine 2:667–674

    PubMed  CAS  Google Scholar 

  • Fan QH, Wang YE, Zhao XX, Loo SCJ, Zuo Y (2011) Adverse biophysical effects of hydroxyapatite nanoparticles on natural pulmonary surfactant. ACS Nano 5(8):6410–6416

    Article  PubMed  CAS  Google Scholar 

  • Fu Q, Rahaman MN, Zhou N, Huang WH, Wang DP, Zhang LY, Li H (2008) In vitro study on different cell response to spherical hydroxyapatite nanoparticles. J Biomater Appl 23:37–50

    Article  CAS  Google Scholar 

  • Gaiser BK, Fernandes TF, Jepson M, Lead JR, Tyler CR, Stone V (2009) Assessing exposure, uptake and toxicity of silver and cerium dioxide nanoparticles from contaminated enviroments. Environ Health 8(suppl 1):S2

    Article  PubMed  Google Scholar 

  • Gorbunoff MJ, Timasheff SN (1984) The interaction of proteins with hydroxyapatite: III. Mechanism. Anal Biochem 136(2):440–445

    Article  PubMed  CAS  Google Scholar 

  • Goyal AK, Khatri K, Mishra N, Mehta A, Vaidya B, Tiwari S, Paliwal R, Paliwal S, Vyas S (2009) Development of self-assembled nanoceramic carrier construct(s) for vaccine delivery. J Biomater Appl 24(1):65–84

    Article  PubMed  CAS  Google Scholar 

  • Grandjean-Laquerriere A, Laquerriere P, Laurent-Maquin D, Guenounou M, Phillips TM (2004) The effect of the physical characteristics of hydroxyapatite particles on human monocytes IL-18 production in vitro. Biomaterials 25:5921–5927

    Article  PubMed  CAS  Google Scholar 

  • Grandjean-Laquerriere A, Laquerriere P, Guenounou M, Laurent-Maquin D, Phillips TM (2005) Importance of the surface area ratio on cytokines production by human monocytes in vitro induced by various hydroxyapatite particles. Biomaterials 26:2361–2369

    Article  PubMed  CAS  Google Scholar 

  • Hamilton RF, Wu N, Porter D, Buford M, Wolfarth M, Holian A (2009) Particle length-dependent titanium dioxide nanomaterials toxicity and bioactivity. Part Fibre Toxicol 6:35

    Article  PubMed  Google Scholar 

  • Han JYL, Loo JSC, Lee J, Ma J (2007) Investigation of the bioactivity and biocompatibility of different glass interface with hydroxyapatite, fluorohydroxyapatite and 58S bioactive glass. BioFactors 30(4):205–216

    Article  PubMed  CAS  Google Scholar 

  • Han JYL, Loo JSC, Phung NT, Ong HT, Russell SJ, Peng KW, Boey F (2008) Controlled size and morphology of EDTMP-doped hydroxyapatite nanoparticles as model for 153Samarium-EDTMP doping. J Mater Sci Mater Med 19(9):2993–3003

    Article  PubMed  CAS  Google Scholar 

  • Hans ML, Lowman AM (2002) Biodegradable nanoparticles for drug delivery and targeting. Curr Opin Solid State Mater Sci 6:319–327

    Article  CAS  Google Scholar 

  • Hauck TS, Ghazani AA, Chan WCW (2008) Assessing the effect of surface chemistry on gold nanorod uptake, toxicity and gene expression in mammalian cells. Small 4(1):153–159

    Article  PubMed  CAS  Google Scholar 

  • Hench LL, Polak JM (2002) Third-generation biomedical materials. Science 295:1014–1017

    Article  PubMed  CAS  Google Scholar 

  • Heng BC, Zhao X, Tan EC, Khamis N, Assodani A, Xiong S et al (2011) Evaluation of the cytotoxic and inflammatory potential of differentially shaped zinc oxide nanoparticles. Arch Toxicol 85(12):1517–1528

    Article  PubMed  CAS  Google Scholar 

  • Hsin YH, Chen CF, Huang S, Shih TS, Lai PS, Chueh PJ (2008) The apoptotic effect of nanosilver is mediated by a ROS-and JNK-dependent mechanism involving the mitochondrial pathway in NIHsTS cells. Toxicol Lett 179:130–139

    Article  PubMed  CAS  Google Scholar 

  • Huang J, Best SM, Bonfield W, Brooks RA, Rushton N, Jayasinghe SN, Edirisinghe MJ (2004) In vitro assessment of the biological response to nano-sized hydroxyapatite. J Mater Sci Mater Med 15:441–445

    Article  PubMed  CAS  Google Scholar 

  • Kida H, Yoshida M, Hoshino S, Inoue K, Yano Y, Yanagita M, Kumagai T, Osaki T, Tachibana I, Saeki Y (2005) Protective effect of IL-6 on alveolar epithelial cell death induced by hydrogen peroxide. Am J Physiol Lung Cell Mol Physiol 288:L342–L349

    Article  PubMed  CAS  Google Scholar 

  • Kopf M, Baumann H, Freer G, Freudenberg M, Lamers M, Kishimoto T, Zinkernagel R, Bluethmann H, Koehler G (1994) Impaired immune and acute-phase responses in interleukin-6-deficient mice. Nature 368:339–342

    Article  PubMed  CAS  Google Scholar 

  • Laquerriere P, Grandjean-Laquerrier A, Jallot E, Balossier G, Frayssinet P, Guenounou M (2003) Importance of hydroxyapatite particles characteristics on cytokines production by human monocytes in vitro. Biomaterials 24:2739–2747

    Article  PubMed  CAS  Google Scholar 

  • Laquerriere P, Grandjean-Laquerriere A, Addadi-Rebbah S, Jallot E, Laurent-Maquin D, Frayssinet P, Guenounous M (2004) MMP-2, MMP-9 and their inhibitors TIMP-2 and TIMP-1 production by human monocytes in vitro in the presence of different forms of hydroxyapatite particles. Biomaterials 25:2515–2524

    Article  PubMed  CAS  Google Scholar 

  • Li B, Guo B, Fan H, Zhang X (2008) Preparation of nano-hydroxyapatite particles with different morphology and their response to highly malignant melanoma cell in vitro. Appl Surf Sci 255(2):357–360

    Article  CAS  Google Scholar 

  • Loo J, Siew Y, Ho S, Boey F, Ma J (2008) Synthesis and hydrothermal treatment of nanostructured hydroxyapatite of controllable sizes. J Mater Sci Mater Med 19:1389–1397

    Article  PubMed  CAS  Google Scholar 

  • Luo D, Saltzman WM (2000) Synthetic DNA delivery systems. Nat Biotechnol 18(1):33–37

    Article  PubMed  CAS  Google Scholar 

  • Matsumoto T, Okazaki M, Inoue M, Yamaguchi S, Kusunose T, Toyonaga T, Hamada Y, Takahashi J (2004) Hydroxyapatite particles as a controlled release carrier of protein. Biomaterials 25:3807–3812

    Article  PubMed  CAS  Google Scholar 

  • McFarland-Mancini MM, Funk HM, Paluch AM, Zhou M, Giridhar PV, Mercer CA, Kozma SC, Krew AF (2010) Differences in wound healing in mice with deficiency of IL-6 versus IL-6 receptor. J Immunol 84(12):7219–7228

    Article  Google Scholar 

  • Motskin M, Wright DM, Muller K, Kyle N, Gard TG, Porter AE, Skepper JN (2009) Hydroxyapatite nano and microparticles: correlation of particle properties with cytotoxicity and biostability. Biomaterials 30:3307–3317

    Article  PubMed  CAS  Google Scholar 

  • Nan A, Bai X, Son SJ, Lee SB, Ghandehar H (2008) Cellular uptake and cytotoxicity of silica nanotubes. Nano Lett 8(8):2150–2154

    Article  PubMed  CAS  Google Scholar 

  • Ng SX, Guo J, Ma J, Loo SCJ (2010) Synthesis of high surface area mesostructured calcium phosphate particles. Acta Biomater 6:3772–3781

    Article  PubMed  CAS  Google Scholar 

  • Ong HT, Loo JSC, Boey FYC, Russell SJ, Ma J, Peng KW (2008) Exploiting the high-affinity phosphonate-hydroxyapatite nanoparticle interaction for delivery of radiation and drugs. J Nanopart Res 10:141–150

    Article  CAS  Google Scholar 

  • Prego C, Garcia M, Torres D, Alonso MJ (2005) Transmucosal macromolecular drug delivery. J Control Release 101(1–3):151–162

    Article  PubMed  CAS  Google Scholar 

  • Rieux A, Fievez V, Garinot M, Schneider YJ, Préat V (2006) Nanoparticles as potential oral delivery systems of proteins and vaccines: a mechanistic approach. J Control Release 116(1):1–27

    Article  PubMed  Google Scholar 

  • Soppimath KS, Aminabhavi TM, Kulkarni AR, Rudzinski WE (2001) Biodegradable polymeric nanoparticles as drug delivery devices. J Control Release 70(1–2):1–20

    Article  PubMed  CAS  Google Scholar 

  • Stanford CM, Jacobson PA, Eanes ED, Lembke LA, Midura RJ (1995) Rapidly forming Apatitic mineral in an osteoblastic cell line (UMR 106–01 BSP). J Biol Chem 270(16):9420–9428

    Article  PubMed  CAS  Google Scholar 

  • Unfried K, Albrecht C, Klotz LO, Mikecz AV, Grether-Beck S, Schins RPF (2007) Cellular responses to nanoparticles: target structures and mechanisms. Nanotoxicology 1:52–71

    Article  CAS  Google Scholar 

  • Veis A (2005) A window on biomineralization. Science 307:1419–1420

    Article  PubMed  CAS  Google Scholar 

  • Victor SP, Kumar TSS (2008) Tailoring calcium-deficient hydroxyapatite nanocarriers for enhanced release of antibiotics. J Biomed Nanotech 4(2):203–209

    CAS  Google Scholar 

  • Xia T, Kovochich M, Brant J, Hotze M, Sempf J, Oberley T, Sioutas C, Yeh JI, Wiesner MR, Nel AE (2006) Comparison of the abilities of ambient and manufactured nanoparticles to induce cellular toxicity according to an oxidative stress paradigm. Nano Lett 6:1794–1807

    Article  PubMed  CAS  Google Scholar 

  • Xia T, Kovochich M, Liong M, Mädler L, Gilbert B, Shi H, Joanne IY, Zink JZ, Nel AE (2008) Comparison of the mechanism of toxicity of zinc oxide and cerium oxide nanoparticles based on dissolution and oxidative stress properties. ACS Nano 2(10):2121–2134

    Article  PubMed  CAS  Google Scholar 

  • Zhao X, Heng BC, Xiong S, Guo J, Tan TT, Chiang FBY, Ng KW, Loo JS (2011) In Vitro assessment of cellular responses to rod-shaped hydroxyapatite nanoparticles of varying lengths and surface areas. Nanotoxicology 5(2):182–194

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

The authors wish to thank Nikon Inc. and the SBIC-Nikon Imaging Centre (NIC@SBIC) of Singapore for their assistance in the confocal laser scanning microscopy studies. This work is possible because of the research fund received from the Ministry of Education—AcRF Tier 1 (NTU), and the National Medical Research Council (NMRC) Exploratory/Developmental Grant (EDG) grant (EDG09may011).

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Authors and Affiliations

  1. School of Materials Science and Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore, 639798, Singapore

    Xinxin Zhao, SuXiu Ng, Boon Chin Heng, Jun Guo, LwinLwin Ma, Kee Woei Ng & Say Chye Joachim Loo

  2. School of Chemical and Biomedical Engineering, Nanyang Technological University, 62 Nanyang Drive, Singapore, 637459, Singapore

    Timothy Thatt Yang Tan

Authors
  1. Xinxin Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. SuXiu Ng
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Boon Chin Heng
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jun Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. LwinLwin Ma
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Timothy Thatt Yang Tan
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Kee Woei Ng
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Say Chye Joachim Loo
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Kee Woei Ng or Say Chye Joachim Loo.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 12 kb)

Supplementary material 2 (PPT 131 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Zhao, X., Ng, S., Heng, B.C. et al. Cytotoxicity of hydroxyapatite nanoparticles is shape and cell dependent. Arch Toxicol 87, 1037–1052 (2013). https://doi.org/10.1007/s00204-012-0827-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00204-012-0827-1

Keywords

Search

Navigation