本研究開發裝載PLGA微粒之玻尿酸微針系統，用以長期治療雌激素缺乏所引起的骨質疏鬆症。本研究分別以單層及雙層乳化的方式，將親脂性vitamin D3(cholecalciferol)與親水性含氮雙磷酸鹽(alendronate)包覆進PLGA微粒，再以250 kDa玻尿酸將PLGA微粒裝填於微針前端，後端支撐軸則由混和型玻尿酸(7kDa：250 kDa = 1：1)製成，藉由具機械強度的支撐軸將包有微粒的微針尖端穿刺進皮膚，同時利用玻尿酸的快溶性使微粒鑲嵌於皮膚內進行藥物長效釋放。本研究製備的兩種PLGA微粒其外觀呈球狀且具良好分散性，粒徑皆約為5~20 µm，單層乳化法的vitamin D3包覆效率為40.7 ± 2.3%，而雙層乳化法的alendronate包覆效率則為11.1 ± 0.8%，偏低的包覆效率可歸因alendronate的極親水性；由微粒體外釋放實驗結果證明，包覆alendronate的PLGA微粒可持續釋放一個月；本研究開發之玻尿酸微針可完整穿刺小鼠背部，穿刺深度約為800 µm；骨質疏鬆症療效測試分成六組，分別為未去除卵巢組(Sham)、去除卵巢未治療(OVX)、去除卵巢口服治療(Oral)、去除卵巢高劑量微針治療(MN-H)、去除卵巢低劑量微針治療(MN-L)以及去除卵巢低劑量微針不含維生素D3治療(MN-L-ND)，由微電腦斷層掃描儀(µ-CT)所計算出的骨密度來看，MN-H擁有最佳的治療效果(P<0.05)，證明此微針系統與口服相比可以較低的藥物劑量(1/40倍)達到更好的治療效果；而MN-L治療效果優於MN-L-ND (P<0.05)，顯示有維生素D3的輔助治療下，可增加抑制骨質流失的能力。本研究開發出的玻尿酸微針系統可成功將含藥微粒鑲嵌於皮膚內，相較於一般口服治療，其較高的生物利用率及較短治療頻率可提升骨鬆治療效果及使用方便性，期盼未來能取代口服藥物並應用於臨床治療上。

This study developed a hyaluronic acid microneedles (HA MNs) system containing drug-loaded PLGA microparticles (MPs) for long-term treatment of postmenopausal osteoporosis. A single and double emulsion methods were used to encapsulate the hydrophobic drug, vitamin D3 (cholecalciferol; D3), and hydrophilic drug, nitrogen-containing bisphosphonate (alendronate; ALN) into PLGA MPs, respectively. The MNs, composed of 250 kDa HA, D3-loaded, and ALN-loaded PLGA MPs, were combined with a HA (7 kDa：250 kDa =1：1) supporting array patch. The supporting array can provide mechanical strength for MN insertion. Due to the highly hydrophilic property of HA, the MNs can be quickly dissolved by skin interstitial fluid, thus leaving the MPs in the skin for sustained drug release. The prepared MPs were spherical and the loading efficiency of D3 and ALN were 40.7 ± 2.3 and 11.1 ± 0.8%, respectively. The in vitro drug release study showed that ALN can be sustained release from the MPs for 1 month. The MNs can completely pierce into the back skin of mice at a depth of approximately 800 μm. Six groups were evaluated in the anti-osteoporosis therapy study: non-ovariectomized mice (Sham), ovariectomized mice without treatment (OVX), ovariectomized mice with oral treatment (Oral), ovariectomized mice with high-dose MN treatment (MN-H), ovariectomized mice with low-dose MN treatment (MN-L) and ovariectomized mice with low-dose MN treatment without adding D3 (MN-L-ND). The µ-CT results show that the MN-H group had the highest BMD value (P < 0.05) which is equivalent to the Sham group (P  0.05). The therapeutic effect of MN-L was superior than that of MN-L-ND (P < 0.05), demonstrating that the vitamin D3 adjuvant can increase the anti-bone resorptive ability. These results show that the proposed HA MN system can efficiently deliver the drug-loaded MPs into the skin for prolonged drug release. Compare to the oral treatment, this MNs system has higher bioavailability to increase the anti-osteoporosis efficacy and requires less treatment frequency to enhance convenience. This MN system has potential to replace oral treatment and be applied for clinical treatment of osteoporosis.

1. Feng, X., Chemical and biochemical basis of cell-bone matrix interaction in health and disease. Current chemical biology, 3(2): p. 189-196, 2009.
2. Mow, V.C. and R. Huiskes, Basic orthopaedic biomechanics & mechano-biology. Lippincott Williams & Wilkins, 2005.
3. Steele, D.G. and C.A. Bramblett, The anatomy and biology of the human skeleton. Texas A&M University Press, 1988.
4. Marieb, E.N. and K. Hoehn, Human anatomy & physiology. Pearson Education, 2007.
5. Tortora, G.J. and B.H. Derrickson, Principles of anatomy and physiology. John Wiley & Sons, 2008.
6. Noble, B.S., The osteocyte lineage. Archives of biochemistry and biophysics, 473(2): p. 106-111, 2008.
7. Bourne, G.H., The biochemistry and physiology of bone. Elsevier, 2014.
8. Marquis, M.E., et al., Bone cells-biomaterials interactions. Front Biosci (Landmark Ed), 14: p. 1023-67, 2009.
9. Yabe, H. and H. Hanaoka, Investigation of the origin of the osteoclast by use of transplantation on chick chorioallantoic membrane. Clinical orthopaedics and related research, 197: p. 255-265, 1985.
10. Boyle, W.J., W.S. Simonet, and D.L. Lacey, Osteoclast differentiation and activation. Nature, 423(6937): p. 337-342, 2003.
11. Raggatt, L.J. and N.C. Partridge, Cellular and molecular mechanisms of bone remodeling. Journal of Biological Chemistry, 285(33): p. 25103-25108, 2010.
12. Rachner, T.D., S. Khosla, and L.C. Hofbauer, Osteoporosis: now and the future. The Lancet, 377(9773): p. 1276-1287, 2011.
13. Iqbal, M.M., Osteoporosis: epidemiology, diagnosis, and treatment. Southern medical journal, 93(1): p. 2-18, 2000.
14. Osteoporosis in men, D.o.H.a.H. Services., Editor. 2005.
15. Legrand, E., et al., Trabecular bone microarchitecture, bone mineral density, and vertebral fractures in male osteoporosis. Journal of Bone and Mineral Research, 15(1): p. 13-19, 2000.
16. Orimo, H., The mechanism of mineralization and the role of alkaline phosphatase in health and disease. Journal of Nippon Medical School, 77(1): p. 4-12, 2010.
17. Lee, N.K., et al., Endocrine regulation of energy metabolism by the skeleton. Cell, 130(3): p. 456-469, 2007.
18. Karlström, E., Functional aspects of prothrombin in bone. Institutionen för laboratoriemedicin/Department of Laboratory Medicine, 2010.
19. Marx, R.E., J.E. Cillo, and J.J. Ulloa, Oral bisphosphonate-induced osteonecrosis: risk factors, prediction of risk using serum CTX testing, prevention, and treatment. Journal of Oral and Maxillofacial Surgery, 65(12): p. 2397-2410, 2007.
20. Rosen, H., et al., Serum CTX: a new marker of bone resorption that shows treatment effect more often than other markers because of low coefficient of variability and large changes with bisphosphonate therapy. Calcified tissue international, 66(2): p. 100-103, 2000.
21. Rodan, G.A. and T.J. Martin, Therapeutic approaches to bone diseases. Science, 289(5484): p. 1508-1514, 2000.
22. Muñoz-Torres, M., G. Alonso, and M. Raya, Calcitonin therapy in osteoporosis. Treatments in endocrinology, 3(2): p. 117-132, 2003.
23. McClung, M., Role of RANKL inhibition in osteoporosis. Arthritis research & therapy, 9(1): p. S3, 2007.
24. Ho, Y., et al., Effects of alendronate on bone density in men with primary and secondary osteoporosis. Osteoporosis international, 11(2): p. 98-101, 2000.
25. Ruggiero, S.L., et al., Osteonecrosis of the jaws associated with the use of bisphosphonates: a review of 63 cases. Journal of oral and maxillofacial surgery, 62(5): p. 527-534, 2004.
26. Marx, R.E., Pamidronate (Aredia) and zoledronate (Zometa) induced avascular necrosis of the jaws: a growing epidemic. Journal of Oral and Maxillofacial Surgery, 61(9): p. 1115-1117, 2003.
27. Colditz, G.A., et al., The use of estrogens and progestins and the risk of breast cancer in postmenopausal women. New England Journal of Medicine, 332(24): p. 1589-1593, 1995.
28. Jiao, Y., X. Pang, and G. Zhai, Advances in hyaluronic acid-based drug delivery systems. Current drug targets, 17(6): p. 720-730, 2016.
29. Burdick, J.A. and G.D. Prestwich, Hyaluronic acid hydrogels for biomedical applications. Advanced materials, 23(12), 2011.
30. Brown, M.B. and S.A. Jones, Hyaluronic acid: a unique topical vehicle for the localized delivery of drugs to the skin. Journal of the European Academy of Dermatology and Venereology, 19(3): p. 308-318, 2005.
31. Gentile, P., et al., An overview of poly (lactic-co-glycolic) acid (PLGA)-based biomaterials for bone tissue engineering. International journal of molecular sciences, 15(3): p. 3640-3659, 2014.
32. Makadia, H.K. and S.J. Siegel, Poly lactic-co-glycolic acid (PLGA) as biodegradable controlled drug delivery carrier. Polymers, 3(3): p. 1377-1397, 2011.
33. Esmaeili, F., F. Atyabi, and R. Dinarvand, Preparation and characterization of estradiol-loaded PLGA nanoparticles using homogenization-solvent diffusion method. DARU Journal of Pharmaceutical Sciences, 16(4): p. 196-202, 2015.
34. Plotkin, L.I., et al., Prevention of osteocyte and osteoblast apoptosis by bisphosphonates and calcitonin. Journal of Clinical Investigation, 104(10): p. 1363, 1999.
35. Hayata, K., et al., Bisphosphonates modulate RANKL and OPG expression in human osteoblasts. Harvard Orthop J, 2005.
36. Thorat V, S.R., Bisphosphonates: Benefits, Basics, Potential Dental Side Effects, and Management. Int J Prev Clin Dent Res, 4(1): p. 64-68, 2017.
37. Holick, M.F., Sunlight and vitamin D for bone health and prevention of autoimmune diseases, cancers, and cardiovascular disease. The American journal of clinical nutrition, 80(6): p. 1678S-1688S, 2004.
38. Crissey, S.D., et al., Serum concentrations of lipids, vitamin D metabolites, retinol, retinyl esters, tocopherols and selected carotenoids in twelve captive wild felid species at four zoos. The Journal of nutrition, 133(1): p. 160-166, 2003.
39. Hume, E.M., N.S. Lucas, and H.H. Smith, On the absorption of vitamin D from the skin. Biochemical Journal, 21(2): p. 362, 1927.
40. Narula, N. and J.K. Marshall, Management of inflammatory bowel disease with vitamin D: beyond bone health. Journal of Crohn's and Colitis, 6(4): p. 397-404, 2012.
41. Recker, R., et al., Alendronate with and without cholecalciferol for osteoporosis: results of a 15‐week randomized controlled trial. Current medical research and opinion, 22(9): p. 1745-1755, 2006.
42. Binkley, N., et al., Alendronate/vitamin D3 70 mg/2800 IU with and without additional 2800 IU vitamin D3 for osteoporosis: Results from the 24-week extension of a 15-week randomized, controlled trial. Bone, 44(4): p. 639-647, 2009.
43. Makras, P., et al., Cost-effective osteoporosis treatment thresholds in Greece. Osteoporosis international, 26(7): p. 1949-1957, 2015.
44. Jansen, J.P., et al., Cost-effectiveness of a fixed dose combination of alendronate and cholecalciferol in the treatment and prevention of osteoporosis in the United Kingdom and The Netherlands. Current medical research and opinion, 24(3): p. 671-684, 2008.
45. Rashid, M., et al., Microparticles as controlled drug delivery carrier for the treatment of ulcerative colitis: A brief review. Saudi Pharmaceutical Journal, 24(4): p. 458-472, 2016.
46. Freiberg, S. and X. Zhu, Polymer microspheres for controlled drug release. International journal of pharmaceutics, 282(1): p. 1-18, 2004.
47. Fan, J.-B., et al., Nanoporous microspheres: from controllable synthesis to healthcare applications. Journal of Materials Chemistry B, 1(17): p. 2222-2235, 2013.
48. Park, J.-H., M.G. Allen, and M.R. Prausnitz, Biodegradable polymer microneedles: fabrication, mechanics and transdermal drug delivery. Journal of Controlled Release, 104(1): p. 51-66, 2005.
49. van der Maaden, K., W. Jiskoot, and J. Bouwstra, Microneedle technologies for (trans) dermal drug and vaccine delivery. Journal of controlled release, 161(2): p. 645-655, 2012.
50. Prausnitz, M.R., Microneedles for transdermal drug delivery. Advanced drug delivery reviews, 56(5): p. 581-587, 2004.
51. O'mahony, C., Structural characterization and in-vivo reliability evaluation of silicon microneedles. Biomedical microdevices, 16(3): p. 333, 2014.
52. Ryu, T.-K., et al., Bone-targeted delivery of nanodiamond-based drug carriers conjugated with alendronate for potential osteoporosis treatment. Journal of Controlled Release, 232: p. 152-160, 2016.
53. Bae, J. and J.W. Park, Preparation of an injectable depot system for long-term delivery of alendronate and evaluation of its anti-osteoporotic effect in an ovariectomized rat model. International journal of pharmaceutics, 480(1): p. 37-47, 2015.
54. Cohen-Sela, E., et al., A new double emulsion solvent diffusion technique for encapsulating hydrophilic molecules in PLGA nanoparticles. Journal of controlled release, 133(2): p. 90-95, 2009.
55. Rosca, I.D., F. Watari, and M. Uo, Microparticle formation and its mechanism in single and double emulsion solvent evaporation. Journal of Controlled Release, 99(2): p. 271-280, 2004.
56. Bile, J., et al., The parameters influencing the morphology of poly (ɛ-caprolactone) microspheres and the resulting release of encapsulated drugs. International journal of pharmaceutics, 494(1): p. 152-166, 2015.
57. Al Deeb, S.K., I.I. Hamdan, and S.M. Al Najjar, Spectroscopic and HPLC methods for the determination of alendronate in tablets and urine. Talanta, 64(3): p. 695-702, 2004.
58. Laperre, K., et al., Development of micro-CT protocols for in vivo follow-up of mouse bone architecture without major radiation side effects. Bone, 49(4): p. 613-622, 2011.
59. Wu, Y., S. Adeeb, and M.R. Doschak, Using micro-CT derived bone microarchitecture to analyze bone stiffness–a case study on osteoporosis rat bone. Frontiers in endocrinology, 6, 2015.
60. Miladi, K., et al., Drug carriers in osteoporosis: preparation, drug encapsulation and applications. International journal of pharmaceutics, 445(1): p. 181-195, 2013.
61. Corrigan, O.I. and X. Li, Quantifying drug release from PLGA nanoparticulates. European Journal of Pharmaceutical Sciences, 37(3): p. 477-485, 2009.
62. Arifin, D.Y., L.Y. Lee, and C.-H. Wang, Mathematical modeling and simulation of drug release from microspheres: implications to drug delivery systems. Advanced drug delivery reviews, 58(12): p. 1274-1325, 2006.