coated Hydroxyapatite Nanoparticles as Anticancer Drug Delivery ...

5 downloads 0 Views 298KB Size Report
Jan 15, 2015 - College of Pharmacy and Research Institute of Pharmaceutical Sciences, ... Keywords: Hydroxyapatite, 5-Fluorouracil, Surface, Drug delivery.
Communication DOI: 10.1002/bkcs.10085

J. Yoon et al.

BULLETIN OF THE KOREAN CHEMICAL SOCIETY

5-Fluorouracil-coated Hydroxyapatite Nanoparticles as Anticancer Drug Delivery Carriers Jiseol Yoon,† Daehyun Kim,‡ Adrian Siregar,† Bo Young Lee,† Ki-Young Kwon,‡,* June-Ho Byun,§,* and Dong Kyun Woo†,* †

College of Pharmacy and Research Institute of Pharmaceutical Sciences, Gyeongsang National University, Jinju 660-701, Korea. *E-mail: [email protected] ‡ Department of Chemistry, RINS, Research Institute of Pharmaceutical Sciences, Gyeongsang National University, Jinju 660-701, Korea. *E-mail: [email protected] § Department of Oral and Maxillofacial Surgery, Institute of Health Science, Gyeongsang National University School of Medicine, Jinju 660-702, Korea. *E-mail: [email protected] Received October 21, 2014, Accepted November 12, 2014, Published online January 15, 2015 Keywords: Hydroxyapatite, 5-Fluorouracil, Surface, Drug delivery

As a main inorganic component, hydroxyapatite (Ca10(PO4)6(OH)2)1 can be found in bones and teeth within the human body. Because of its excellent biocompatibility, hydroxyapatite has attracted much attention on tissue engineering and regenerative medicine.2,3 For example, hydroxyapatites have been applied as bone substitute materials and scaffolds for bone tissue. In addition, hydroxyapatites have been utilized as drug delivery carriers since they have multiple advantages: (1) low cost and simple manufacture, (2) biosafety, and (3) high affinity to chemical drugs as well as biomolecules including proteins and DNA.4,5 Because of the above-mentioned benefits for biomedical applications, nano-scale hydroxyapatites have been prepared by various ways.6–8 However, most current procedures for hydroxyapatite synthesis include ammonia (or urea) addition, which can result in the fluctuation of pH during the synthesis. In order to handle this pH issue, recently we attempted to replace ammonia with NaOH, a strong base, and optimized NaOH concentrations for nanocrytaline hydroxyapatite synthesis.9 In this study, we examine these NaOH-driven hydroxyapatite nanoparticles (about 100–150 nm) as 5-fluorouracil (5-FU, an anticancer drug) delivery carriers for cancer cell treatment. 5-FU is a pyrimidine analog that inhibits DNA synthesis and is used widely in the treatment of various cancers including gastrointestinal cancers.10,11 Hydroxyapatites were produced by a hydrothermal method as described.9 Briefly, calcium nitrate and sodium phosphate dibasic, calcium and phosphate sources, respectively, were slowly dissolved in distilled water by stirring, and then NaOH (final concentration 0.71 M) was added. This reaction mixture was then autoclaved (200  C for 24 h). After cooling the mixture, white precipitates were obtained. These precipitates were then washed with distilled water and centrifuged repeatedly (at least five times). Finally, the precipitates were freeze-dried at −65  C. In the present study, we loaded 5-FU onto the surface of hydroxyapatites synthesized as mentioned above by adding a 5-FU solution (0.09 g 5-FU in 9 mL of H2O) to 1 g of the Bull. Korean Chem. Soc. 2015, Vol. 36, 445–446

dried hydroxyapatites. Then, we measured the efficiencies of 5-FU adsorption and desorption with respect to hydroxyapatite surface using spectrophotometry (at 265 nm) based on a standard curve generated from dilutes of a 5-FU solution of known concentration. As shown in Figure 1, by subtracting the absorbance of unattached 5-FU molecules from that corresponding to the total 5-FU used, we found that approximately 7% of 5-FU applied can be coated on hydroxyapatites

Figure 1. Adsorption and desorption of 5-FU on the surface of hydroxyapatites.

© 2015 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Wiley Online Library

445

BULLETIN OF THE KOREAN CHEMICAL SOCIETY

Communication

Next, using the MTT assay,12 which is a colorimetric assay for assessing cell viability, we investigated pharmacological anticancer effects of 5-FU that was detached from the surface of hydroxyapatites. Here, we used two different gastric cancer cell lines, SNU-48413 and MKN-28.14 As shown in Figure 2, we observed a dose-dependent cytotoxic effect of detached 5FU from hydroxyapatites. In addition to this colorimetric assay, we also confirmed that the number of cancer cells decreased by 5-FU treatment relative to untreated controls from microscopic cell culture images (Figure 2(b) vs. (c), and (e) vs. (f )). Together, these results clearly show that 5FU released from hydroxyapatites retains its anticancer effect. In summary, we prepared hydroxyapatites using a modified hydrothermal method utilizing NaOH. These NaOH-driven hydroxyapatites were then tested as carrier molecules delivering anticancer drugs. Our results demonstrated the possibility of using hydroxyapatite nanoparticles for chemotherapeutic application on gastric cancer cells. Furthermore, given that hydroxyapatite provides a scaffold for bone regeneration, these results highlight a potential use of hydroxyapatite in therapies aimed at osteomyelitis and osteosarcoma. Acknowledgments. This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2012R1A1A1008721 and NRF2014R1A1A2A16055714) and by a grant (12182MFDS666) from Ministry of Food and Drug safety, Republic of Korea, in 2014. References 1. 2. 3. 4. 5. 6. 7. Figure 2. Cytotoxic effects of 5-FU released from hydroxyapatites on gastric cancer cell lines. (a, d) 5-FU dose-dependent decreases in viabilities of SNU-484 and MKN-28; black bar indicates 5-FU control while gray and white bars indicate 5-FU released from two different batches of hydroxyapatites. (b, d) Representative cell images corresponding to no treatment. (c, e) Representative cell images corresponding to 100 μM of 5-FU treatment (∗∗∗p < 0.001).

(Figure 1(a)). It appears that most of the attached 5-FU molecules were released from the hydroxyapatite surface shortly after adding H2O to the 5-FU/hydroxyapatite complex. In an aqueous solution (e.g., phosphate buffered saline), approximately 90% of the attached 5-FU molecules were released from the surface of hydroxyapatites within 1 h (Figure 1(b)).

Bull. Korean Chem. Soc. 2015, Vol. 36, 445–446

8. 9. 10. 11. 12. 13.

14.

M. I. Kay, R. A. Young, A. S. Posner, Nature 1964, 204, 1050. S. Bose, S. Tarafder, Acta Biomater. 2012, 8, 1401. H. Zhou, J. Lee, Acta Biomater. 2011, 7, 2769. M. Gorbunoff, J. Anal. Biochem. 1984, 136, 425. Y. Mizushima, T. Ikoma, J. Tanaka, K. Hoshi, T. Ishihara, Y. Ogawa, A. Ueno, J. Control. Release 2006, 10, 260. B. Jokic, M. Mitric, V. Radmilovic, S. Drmanic, R. Petrovic, D. Janackovic, Ceram. Int. 2011, 37, 167. A. Cuneyt Tas¸, F. Korkusuz, M. Timucin, N. Akkas¸, J. Mater. Sci. Mater. Med. 1997, 8, 91. S. Bose, S. K. Saha, J. Am. Ceram. Soc. 2003, 86, 1055. Y. Y. Kim, D. H. Kim, S. H. Lee, D. K. Woo, J. H. Byun, K. Y. Kwon, Bull. Korean Chem. Soc. 2014, 35, 8. R. J. Langenbach, P. V. Dancenberg, C. Heidelberger, Biochem. Biophys. Res. Commun. 1972, 48, 1565. D. B. Longley, D. P. Harkin, P. G. Johnston, Nat. Rev. Cancer 2003, 3, 330. T. Mosmann, J. Immunol. Methods 1983, 65, 55. J. G. Park, H. K. Yang, W. H. Kim, J. K. Chung, M. S. Kang, J. H. Lee, J. H. Oh, H. S. Park, K. S. Yeo, S. H. Kang, S. Y. Song, Y. K. Kang, Y. J. Bang, Y. I. Kim, J. P. Kim, Int. J. Cancer 1997, 70, 443. T. Motoyama, H. Hojo, H. Watanabe, Acta Pathol. Jpn. 1986, 36, 65.

© 2015 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.bkcs.wiley-vch.de

446