ANTICANCER RESEARCH 38: 6715-6720 (2018)

doi:10.21873/anticanres.13040

Evaluation of a New Hydroxyapatite Nanoparticle as a Drug

Delivery System to Oral Squamous Cell Carcinoma Cells
TAKU MURATA, TOMOHIKO KUTSUNA, KAZUTO KUROHARA,
KASUMI SHIMIZU, AKIRA TOMEOKU and NAOYA ARAI

Department of Oral and Maxillofacial Surgery, Department of Clinical Sciences, Medical Life Science,
Mie University, Graduate School of Medicine, Mie, Japan

Abstract. Background/Aim: Due to its abilities of substance different properties depending on morphology, size, and
adsorption and intracellular transportation, hydroxyapatite is surface properties, there is currently no consensus on what
a potential carrier in drug delivery systems (DDS). This in nano-HAP characteristics are most suitable for DDS (2, 3).
vitro study investigated whether newly-developed, highly- Therefore, nano-HAP with various structures are produced
dispersive calcined hydroxyapatite nanoparticles with an using various techniques (2, 3).
average grain diameter of 20 nm (nano-SHAP) were suitable Bisphosphonates (BPs) are first-line drugs for osteolytic
as a DDS for the drugs zoledronic acid (ZA), cisplatin, and diseases, including osteoporosis, Paget’s disease, and cancer-
carboplatin. Material and Methods: The effects of drug- induced hypercalcemia (4). BPs act as potent osteoclastic
bearing nano-SHAP on cell proliferation were assessed using bone resorption inhibitors by inhibiting osteoclast activity and
three human oral squamous cell carcinoma cell lines (HSC-4, inducing their apoptosis (4-6). Zoledronic acid (ZA) is a
KOSC, and SAS) and one human breast cancer cell line (MCF- third-generation, nitrogen-containing BP and is potentially a
7). Results: Nano-SHAP alone did not affect proliferation of greater inhibitor of bone resorption (4, 7). ZA is widely used
any cell line until a concentration of 1 μg/ml was reached. to prevent metastatic cancer-induced bone diseases (4, 7).
Although the effective concentration of ZA in ZA-bearing nano- Moreover, the direct antitumor activity of ZA has been
SHAP differed, it inhibited cell proliferation better than ZA reported in many cancer cell types in vitro, including oral
alone. Cisplatin and carboplatin-bearing nano-SHAP had the squamous cell carcinoma (OSCC) cells, demonstrating
same effect as these drugs alone. Conclusion: The nano-SHAP significant growth inhibition and apoptosis induction (4, 7-
system is of potential use as a drug delivery system. 9). However, inhibition of cancer cell proliferation in clinical
research using ZA is difficult because plasma concentrations
Hydroxyapatite (HAP), which has a structural formula of rapidly peak after intravenous administration of the standard
Ca10(PO4)6(OH)2, is the principal inorganic constituent of dose of ZA (4 mg) and decline to 1% of the peak within 24
human bones and teeth (1, 2). HAP is widely used in medical h (4, 8, 10). For ZA to directly inhibit tumor cell proliferation,
applications, including treating bone defects, tissue it is necessary to develop a new method of administration.
engineering systems, and bioactive coatings on metallic This study investigated whether newly developed, highly-
osseous implants (1-3). Hydroxyapatite nanoparticle (nano- dispersive calcined HAP nanoparticles with an average grain
HAP) structures have a large surface area, high loading diameter of 20 nm (nano-SHAP) can be applied as a DDS of
capacity, and are capable of intracellular transportation. As a ZA or two antitumor drugs (cisplatin and carboplatin) using
result, nano-HAP has been explored as a therapeutic molecule three human OSCC cell lines and one breast cancer cell line.
for drug delivery systems (DDS) (2, 3). Since nano-HAP has
Materials and Methods

Cells and reagents. Three human oral squamous cell lines (HSC-4,
Correspondence to: Taku Murata, Department of Oral and KOSC, and SAS) and one human breast cancer cell line (MCF-7)
Maxillofacial Surgery, Department of Clinical Sciences, Medical were kindly provided by Dr. M Noguchi, Department of Oral and
Life Science, Mie University, Graduate School of Medicine, 2-174 Maxillofacial Surgery, Graduate School of Medicine and
Edobashi, Tsu, Mie 514-8507, Japan. Tel: +81 592321111, Fax: +81 Pharmaceutical Sciences, University of Toyama. The cells were
592315207, e-mail: muratat@clin.medic.mie-u.ac.jp cultured and maintained at 37°C and 5% CO2 in a humidified
atmosphere in RPMI 1640 medium (Thermo Fisher Scientific,
Key Words: Hydroxyapatite nanoparticle, drug delivery system, Waltham, MA, USA) supplemented with 5% fetal bovine serum
bisphosphonate, oral squamous cell carcinoma. (FBS; Biowest, Paris, France), 100 U/ml penicillin, and 100 μg/ml

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Figure 1. Effect of highly-dispersive calcined hydroxyapatite nanoparticles (nano-SHAP) on cell proliferation of human oral squamous cell carcinoma
cell lines HSC4 (A), KOSC (B), SAS (C) and human breast cancer cell line MCF-7 (D). The cells were exposed to increasing concentrations of
nano-SHAP (100-1,000 ng/ml) for 72 h, and the effects on cell proliferation were measured using the MTS assay. All experiments were performed
independently three times.

streptomycin (Thermo Fisher Scientific). Highly-dispersive nano- plated at 2,000 cells per well in 96-well plates in RPMI 1640
SHAP with an average grain diameter of 20 nm were purchased medium containing 5% FBS; 24 h after plating, the medium was
from SofSera (Tokyo, Japan). ZA (Zometa®) was obtained from replaced with the same medium containing nano-SHAP alone, ZA-
Novartis Pharmaceuticals (Basel, Switzerland). Cisplatin and bearing nano-SHAP, cisplatin-bearing nano-SHAP or carboplatin-
carboplatin were purchased from Tocris Bioscience (Bristol, UK). bearing nano-SHAP. After 72 h, the cell number was determined
using the assay kit according to the manufacturer’s instructions.
Preparation of drug-bearing nano-SHAP and nano-SHAP alone. Nano- Each sample’s absorbance was measured at 490 nm using a
SHAP was dissolved in distilled water, after which each drug was microplate reader (Promega Corporation), which was then used to
added and suspended. The mixture was kept at room temperature for determine the relative cell count in each well. All experiments were
60 min. Nano-SHAP alone was prepared as follows. Nano-SHAP was performed independently three times.
dissolved in distilled water, and kept at room temperature for 60 min.
Statistical analysis. Statistical analysis was performed using
Cell proliferation assay. Cell proliferation was assessed using the Student’s unpaired t-test or one-way analysis of variance followed
CellTiter 96® Aqueous One Solution Cell Proliferation Assay by Tukey’s multiple comparison test. Statistical significance was
(Promega Corporation, Madison, WI, USA). In brief, the cells were defined as a calculated p-value of less than 0.05.

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Figure 2. Effect of zoledronic acid (Zometa®)-bearing highly-dispersive calcined hydroxyapatite nanoparticles (nano-SHAP) on cell proliferation of human
oral squamous cell carcinoma cell lines HSC4 (A), KOSC (B), SAS (C) and human breast cancer cell line MCF-7 (D). Cells were exposed to increasing
concentrations of ZA or ZA-bearing nano-SHAP (1,000 ng/ml) for 72 h, and the effects on cell proliferation were measured using the MTS assay. All
experiments were performed independently three times. Significantly different at *p<0.05 and **p<0.01 versus the respective value without ZA.

Results the effect of ZA-bearing nano-SHAP. In HSC4 cells, ZA-

bearing nano-SHAP significantly inhibited cell proliferation
Effect of nano-SHAP on cell proliferation. As the effect of at 0.1 μM and 1 μM relative to ZA alone. In SAS cells, ZA-
nano-SHAP on cell proliferation in human OSCC and breast bearing nano-SHAP at >0.1 μM significantly inhibited cell
cancer cells was unclear, the effects of different proliferation. In KOSC and MCF-7 cells, ZA-bearing nano-
concentrations of nano-SHAP on cell proliferation were SHAP at >1 μM significantly inhibited cell proliferation
assessed by the MTS assay. Nano-SHAP alone did not (Figure 2).
inhibit proliferation of any of the four cell lines at final
concentrations ≤1,000 ng/ml (Figure 1). Effect of cisplatin-bearing nano-SHAP on cell proliferation.
Cisplatin is a platinum-containing antitumor agent used to treat
Effect of ZA-bearing nano-SHAP on cell proliferation. a variety of tumors, including SCC and breast cancer. The
Since 1,000 ng/ml nano-SHAP had no significant effect on effect of cisplatin-bearing nano-SHAP on cell proliferation was
cell proliferation, this concentration was used to examine examined. However, no differences were found between the

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Figure 3. Effect of cisplatin-bearing highly-dispersive calcined hydroxyapatite nanoparticles (nano-SHAP) on cell proliferation of human oral
squamous cell carcinoma cell lines HSC4 (A), KOSC (B), SAS (C) and human breast cancer cell line MCF-7 (D). The cells were exposed to
increasing concentrations of cisplatin alone or cisplatin-bearing nano-SHAP (1,000 ng/ml) for 72 h, and the effects on cell proliferation were
measured using the MTS assay. All experiments were performed independently three times.

effects of cisplatin alone and cisplatin-bearing nano-SHAP on Discussion

cell proliferation of any of the four cell lines (Figure 3).
HAP can be loaded with therapeutic molecules as a DDS,
Effect of carboplatin-bearing nano-SHAP on cell proliferation. but is known to have different properties depending on
Carboplatin is a second-generation platinum-containing morphology, size, and surface properties (2, 3). In the
antitumor agent commonly used to treat tumors. The effects present in vitro study, we demonstrated that a newly
of carboplatin and carboplatin-bearing nano-SHAP on cell developed, highly-dispersive calcined nano-SHAP with an
proliferation were examined. However, no differences were average grain diameter of 20 nm might be useful as a drug
found between the effects of carboplatin alone and delivery carrier of ZA, but not of cisplatin or carboplatin, to
carboplatin-bearing nano-SHAP on cell proliferation of any of OSCC cells and breast cancer cells. OSCC can directly
the four cell lines (Figure 4). invade adjacent jaw bone at relatively early stages and

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Figure 4. Effect of carboplatin-bearing highly-dispersive calcined hydroxyapatite nanoparticles (nano-SHAP) on cell proliferation of human oral
squamous cell carcinoma cell lines HSC4 (A), KOSC (B), SAS (C) and human breast cancer cell line MCF-7 (D). The cells were exposed to
increasing concentrations of carboplatin alone or carboplatin-bearing nano-SHAP (1,000 ng/ml) for 72 h, and the effects on cell proliferation were
measured using the MTS assay. All experiments were performed independently three times.

breast cancer often metastasizes to bone. It has been of their backbone P-C-P structure and ability to chelate
reported that ZA, a therapeutic agent for osteoporosis and calcium ions (11). Bone is mainly composed of HAP with a
cancer bone metastasis, directly inhibits tumor cell structural formula of Ca10(PO4)6(OH)2 (1, 2). Therefore, we
proliferation at peak plasma concentrations following investigated the effect of ZA-bearing nano-SHAP on cell
intravenous administration of the standard dose (4 mg). proliferation. As expected, ZA-bearing nano-SHAP
However, because plasma concentrations rapidly decline significantly inhibited cell proliferation relative to ZA alone
after less than 24 h (4, 8, 10), a new approach for inhibiting in OSCC and breast cancer cell lines. HAP is sensitive to
tumor cell proliferation at low concentrations of ZA is pH and can be degraded into calcium and phosphorous
needed. Previously, we reported in vitro synergistic effects elements under weak acidic conditions (12). Therefore, we
of ZA and calcium on the viability of human OSCC and thought that after ZA-bearing nano-SHAP was taken into
human skin keratinocytes (4, 8). It is known that BPs, cells by endocytosis, it would be dissolved in the
including ZA, have a high affinity for bone mineral because intracellular acidic environment and ZA would be released.

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Platinum-based drugs have been used to treat various 3 Mondal S, Dorozhkin SV and Pal U: Recent progress on
cancer types for decades, including ovarian, cervical, head and fabrication and drug delivery applications of nanostructured
neck, and non-small-cell lung cancer. Although platinum- hydroxyapatite. Wires Nanomed NanobioI 10: 32, 2018.
4 Inoue S, Arai N, Tomihara K, Takashina M, Hattori Y and
based drugs exhibit strong anticancer activity, they can have
Noguchi M: Extracellular Ca2+-dependent enhancement of
serious side-effects. Therefore, a DDS should increase drug cytocidal potency of zoledronic acid in human oral cancer cells.
concentration to the tumor while reducing the side-effects. Eur J Pharmacol 761: 44-54, 2015.
Cisplatin is a first-generation platinum agent (13). The specific 5 Fleisch H, Russell RG and Francis MD: Diphosphonates inhibit
properties of the drugs and morphology of nano-SHAP were hydroxyapatite dissolution in vitro and bone resorption in tissue
found to affect the adsorption of drug molecules. Positively culture and in vivo. Science 165: 1262-1264, 1969.
charged cisplatin was strongly adsorbed to nano-SHAP, 6 Hughes DE, Wright KR, Uy HL, Sasaki A, Yoneda T, Roodman
GD, Mundy GR and Boyce BF: Bisphosphonates promote
whereas cisplatin adsorption was favored at the needle-shaped
apoptosis in murine osteoclasts in vitro and in vivo. J Bone
HAP surface (3). However, cisplatin-bearing nano-SHAP did Miner Res 10: 1478-1487, 1995.
not inhibit cell proliferation compared with cisplatin alone in 7 Tamura T, Shomori K, Nakabayashi M, Fujii N, Ryoke K and
any of the cell lines. This may indicate difference in Ito H: Zoledronic acid, a third-generation bisphosphonate,
morphology of nano-SHAP or cell type. Furthermore, inhibits cellular growth and induces apoptosis in oral carcinoma
information on cisplatin concentration in nano-SHAP and the cell lines. Oncol Res 25: 1139-1143, 2011.
release rate of cisplatin from nano-SHAP is lacking. 8 Arai N, Inoue S, Tomihara K, Tsuno H and Noguchi M: In vitro
Carboplatin is a second-generation platinum agent that synergistic effects of zoledronic acid and calcium on viability of
human epithelial cells. Oral Dis 19: 200-205, 2013.
exhibits strong antitumor activity (13). To our knowledge,
9 Sun YY, Chen L, Wu XW and Ding Q: Bifunctional
there is only one in vivo report on carboplatin-loaded bisphosphonate derivatives and platinum complexes with high
hydroxyapatite, which was the spherical type with a mean affinity for bone hydroxyapatite. Bioorg Med Chem Let 27:
diameter of 36.1 μm, and it prolonged the mean survival 1070-1075, 2017.
time in rats with AH130 peritoneal carcinomatosis. It is not 10 Koto K, Murata H, Kimura S, Horie N, Matsui T, Nishigaki Y,
clear why our in vitro results and their in vivo results Ryu K, Sakabe T, Itoi M, Ashihara E, Maekawa T, Fushiki S and
differed, but it may relate to HAP shape and size, cell type, Kubo T: Zoledronic acid inhibits proliferation of human
fibrosarcoma cells with induction of apoptosis, and shows
or loaded condition.
combined effects with other anticancer agents. Oncol Res 24:
In conclusion, our results suggest that nano-SHAP may be 233-239, 2010.
effective as a drug delivery carrier for ZA. Further studies 11 Rogers MJ, Crockett JC, Coxon FP and Monkkonen J:
are required to clarify this possibility. Biochemical and molecular mechanisms of action of
bisphosphonates. Bone 49: 34-41, 2011.
Conflicts of Interest 12 Sun W, Fan JL, Wang SZ, Kang Y, Du JJ and Peng XJ:
Biodegradable drug-loaded hydroxyapatite nanotherapeutic agent
The Authors declare no conflicts of interest. for targeted drug release in tumors. Acs App Mater Inter 10:
7832-7840, 2018.
Acknowledgements
13 Chen X, Wang JG, Fu ZP, Zhu B, Wang J, Guan SW and Hua
ZC: Curcumin activates dna repair pathway in bone marrow to
improve carboplatin-induced myelosuppression. Sci Rep 7: 11,
This work was supported by JSPS KAKENHI Grant Number 2017.
JP25861975.

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