本研究乃結合聚麩胺酸[poly--glutamic acid, -PGA)]微針及聚乙烯吡咯烷酮(polyvinylpyrrolidone, PVP)和聚乙烯醇(polyvinyl alcohol, PVA)支撐陣列，開發出一快溶型微針貼片，並包覆胰島素以評估其應用於糖尿病治療之可行性。此微針可在穿入皮膚約四分鐘內完全溶解，並成功刺入死豬皮及活鼠皮膚分別約400及500微米之深度，相當於真皮層上方。將微針包覆胰島素後連續應用於糖尿病鼠兩次，間隔二十四小時。由體內藥物動力學結果可知，以皮下注射做為對照組時，微針傳輸胰島素之相對生物可利用率皆可達到9899%，並具有與皮下注射組相似的降血糖效果，顯示微針所釋放的胰島素可完全被體內吸收，並保有其藥理活性。經儲存穩定性測試證實，將胰島素包覆於微針中並放置於25 C 及37 C的溫度下一個月，仍有約90%以上的胰島素存在於微針中。以上結果證實，本研究所開發之聚麩胺酸微針適合包覆蛋白質藥物，可相對無痛、快速且穩定地經皮傳輸胰島素，有潛力取代針劑注射，重覆地應用於糖尿病患者，以提升病人的生活品質。

This study presents a dissolving microneedle system, which is composed of poly--glutamic acid (-PGA) microneedles with a poly(vinyl pyrrolidone)/poly(vinyl alcohol) supporting array, for rapid and efficient transdermal delivery of insulin. The microneedles can be completely dissolved after insertion into the skin for 4 min (and the encapsulated insulin is released rapidly). A histological examination shows that the mechanical strength of microneedles is strong to pierce into porcine cadaver skin to a depth of approximately 400 µm and about 500 µm in rat skin depth. Insulin-loaded microneedles were administered to diabetic rats using a homemade applicator to evaluate the feasibility of using these microneedles for diabetes treatment. Pharmacodynamic and pharmacokinetic results show a similar hypoglycemic effect in rats receiving insulin-loaded microneedles and subcutaneous injection of insulin. The relative pharmacological availability and relative bioavailability of insulin were both 98-99% compared to subcutaneous injection for the two administration, indicating that insulin delivered via γ-PGA microneedles was almost completely absorbed from the skin into systemic circulation. The hypoglycemic effect of insulin-loaded microneedles was almost similar to that of the subcutaneous administration of insulin of the same dose. Storage ability analysis confirms that more than 90% of the insulin remained in the microneedles even after storage at room temperature (25 C) and 37 C for 1 month. These results demonstrate that the proposed γ-PGA microneedles are stable for encapsulating bioactive molecules and have great potential for transdermal delivery of protein drugs in a relatively painless, rapid, and convenient manner.

[1] Poretsky L (2010) Principle of Diabetes Mellitus, 2nd ed., Springer, New York.
[2] Liu S, Jin MN, Quan YS, Kamiyama F, Katsumi H, Sakane T, Yamamoto A (2012) “The development and characteristics of novel microneedle arrays fabricated from hyaluronic acid, and their application in the transdermal delivery of insulin.” Journal of Controlled Release 161(3): 933-941.
[3] Ling MH, Chen MC (2013) “Dissolving polymer microneedle patches for rapid and efficient transdermal delivery of insulin to diabetic rats.” Acta Biomaterialia 9(11): 8952-8961.
[4] Prausnitz MR, Mitragotri S, Langer R (2004) “Current status and future and future potential of transdermal drug delivery.” Nature Reviews Drug Discovery 3(2): 115-124.
[5] Gill HS, Denson DD, Burris BA, Prausnitz MR (2008) “Effect of microneedle design on pain in human volunteers.” The Clinical Journal of Pain 24 (7): 585-594.
[6] Martanto W, Davis SP, Holiday NR, Wang J, Gill HS Prausnitz MR (2004) “Transdermal delivery of insulin using microneedles in vivo”. Pharmaceutical Research 21 (6): 947-952.
[7] Sullivan SP, Murthy N, Prausnitz MR (2008) “Minimally invasive protein delivery with rapidly dissolving polymer microneedles.” Advanced Materials 20 (5): 933-938.
[8] Chen MC, Ling MH, Lai KY, Pramudityo E (2012) “Chitosan microneedle patches for sustained transdermal delivery of macromolecules.” Biomacromolecules 13(12):4022-4031.

[9] Zhang W, Gao J, Zhu Q, Zhang M, Ding X, Wang X, Hou X, Fan W, Ding B, Wu X, Wang X, Gao S (2010) “Penetration and distribution of PLGA nanoparticles in the human skin treated with microneedles.” International Journal of Pharmaceutical 402(1-2): 205-212.
[10] Makhlof A, Tozuka Y, Takeuchi H (2011) “Design and evaluation of novel pH-sensitive chitosan nanoparticles for oral insulin delivery.” European Journal of Pharmaceutical Sciences 42(5):445-451.
[11] Sajeesh S, Sharma CP (2006) “Cyclodextrin-insulin complex encapsulated polymethacrylic based nanoparticles for oral insulin delivery.” International Journal of Pharmaceutical 325(1-2): 147-154.
[12] Lee K, Kim JD, Lee CY, Her S, Jung H (2011) “A high-capacity, hybrid electro-microneedle for in-situ cutaneous gene transfer.” Biomaterials 32(30):7705-7710.
[13] Lee J, Park JW, Prausnitz MR (2008) “Dissolving microneedles for transdermal drug delivery.” Biomaterials 29(13):2113-2124.
[14] Fluhr JW, Elsner P, Berrdesca E, Maibach HI (2005) Bioengineering of the Skin: Water and the Stratum Corneum, 3rd ed., CRC Press, Boca Raton, FL.
[15] Roskos KV, Guy RH (1989) “Assessment of skin barrier function using transepidermal water loss: effect of age.” Pharmaceutical Research 6(11):949-953.
[16] Rosado C, Pinto P, Rodrigues LM (2005) “Modeling TEWL-desorption curves: a new practical approach for the quantitative in vivo assessment of skin barrier.” Experimental Dermatology 14(5):386-390.
[17] Gooma YA, Morrow DI, Garland MJ, Donnelly RF, El-Khordaqui LK, Meidan VM (2010) “Effects of microneedle length, density, insertion time and multiple applications on human skin barrier fuction: Assessments by transepidermal water loss.” Toxicology in Vitro 24:1971-1978.
[18] Chu LY, Choi SO, Prausnitz MR (2010) “Fabrication of dissolving polymer microneedles for controlled drug encapsulation and delivery: Bubble and pedestal microneedles designs.” Journal of Pharmaceutical Sciences 99(10):4228-4238.
[19] Wise LD (2000) Handbook of pharmaceutical controlled release technology. Marcel Dekker, Inc. New York.
[20] Ratner BD, Hoffman AS, Schoen FJ, Lemons JE (1996) Biomaterials Science: An Introduction to Materials in Medicine, Academic Press, New York.
[21] Brown L, Munoz C, Siemer L, Edelman E, Langer R (1986) “Controlled release of insulin from polymer matrices.” Diabetes 35(6):692-697.
[22] Raval A, Parikh J, Engineer C (2010) “Mechanism of controlled release kinetics from medical devices.” Brazilian Journal of Chemical Engineering 27(2): 211-225.
[23] Constantino HR, Langer R, Klibanov AM (1994) “Moisture-induced aggregation of lyophilized insulin.” Pharmaceutical Research 11(1): 21-29.
[24] Brange J (1987) Galenics of insulin: the physico-chemical and pharmaceutical aspects of insulin and insulin preparations, Springer-Verlag, Berlin.
[25] Qiu YQ, Qin GJ, Zhang SH, Wu Y, Xu B, Gao Y (2012) “Novel lyophilized hydrogel patches for convenient and effective administration of microneedle-mediaed insulin delivery.” International Journal of Pharmaceutics 437(1-2):51-56.
[26] Amorij JP, Huckriede A, Wilschut,J, Frijlink HW, Hinrichs WL (2008) “Development of stable influenza vaccine powder formulations: challenges and possibilities.” Pharmaceutical Research 25(6):1256-1273.
[27] Kanthamneni N, Sharma S, Meenach SA, Billet B, Zhao JC, Bachelder EM, Ainslie KM (2012) “Enhanced stability of horseradish peroxidase encapsulated in acetalated dextran microparticles stored outside cold chain conditions.” International Journal of Pharmaceutics 431(1-2):101-110.
[28] Omidian H, Hashemi SA, Sammes PG, Meldrum I (1998) “A model for the swelling of superabsorbent polymers.” Polymer 39(26):6697-6704.
[29] Kabiri K, Omidian H, Hashemi SA, Zohuriaan-Mehr MJ (2003) ‘Synthesis of fast-swelling superabsorbent hydrogels: effect of crosslinker type and concentration on porosity and absorption rate.” European Polymer Journal 39(7):1341-1348.
[30] Hsueh PJ (2004) “Effect of γ-polyglutamic acid on the properties and biocompatibility of polyvinylalcohol hydrogel membrane.” National Taiwan University of Science and Technology, Taipei, Taiwan.
[31] Park JH, Allen MG, Prausnitz MR (2005) “Biodegradable polymer microneedles: Fabrication, mechanics, and transdermal drug delivery.” Journal of Controlled Release 104(1):51-66.
[32] Davis SP, Landis BJ, Adams ZH, Allen MG, Prausnitz MR (2004) “Insertion of microneedles into skin: measurement and prediction of insertion force and needle fracture force.” Journal of Biomechanics 37(8):1155-1163.
[33] Boonma A, Narayan RJ, Lee YS (2013) “Analytical modeling and evaluation of microneedles apparatus with deformable soft tissues for biomedical application.” Computer-Aided Design and Aplications 10(1):139-157.
[34] Donnelly RF, Morrow DI, Singh TR, Migalska K, McCarron PA, O’Mahony C, Woolfson AD (2009) “Processing difficulties and instability of carbohydrate microneedle arrays.” Drug Development and Industrial Pharmcy 35(10):1242-1254.
[35] World Health Organization (WHO) (2014) “About Diabetes”.

[36] Badran MM, Kuntsche J, Fahr A (2009) “Skin penetration enhancement by a microneedle device (Dermaroller®) in vitro: Dependency on needle size and applied formulation.” European Journal of Pharmaceutical Sciences 36(4-5):511-523.
[37] Zhou CP, Liu YL, Wang HL, Zhang PX, Zhang JL (2010) “Transdermal delivery of insulin using microneedle rollers in vivo.” International Journal of Pharmaceutics 392(1-2):127-133.
[38] Verbaan FJ, Bal SM, van den Berg DJ, Dijksman JA, van Hecke M, Verpoorten H, van den Berg A, Luttge R, Bouwstra JA (2008) “Improved piercing of microneedle arrays in dermatomed human skin by an impact insertion method.” Journal of Controlled Release 128(1):80-88.
[39] Adler AI, Stratton IM, Neil H, Andrew W, Yudkin JS, Matthews DR, Cull CA, Wright AD, Turner RC, Holman RR (2000) “Association of systolic blood pressure with macrovascular and microvascular complications of type 2 diabetes.” British Medical Journal 321(7528):412–419.
[40] Centers for Disease Control and Prevention (2014) “National Diabetes Statistics Report: Estimates of Diabetes and Its Burden in the United States” US Department of Health and Human Services. Atlanta, GA.
[41] Alzira MF, Jorge AL (2004) “Insulin or insulin-like studies on unicellular organisms: a review.” Brazilian Archives of Biology and Technology 47(6):973-981.
[42] Le Roith D, Shiloach J, Heffron R, Rubinovitz C, Tanebaum R, Roth J (1985) “Insulin-related material in microbes: similarities and differences from mammalian insulins.” Canadian Journal of Biochemistry and Cell Biology 63(8):839-849.
[43] Glenn GM, Kenney RT, Ellingsworth LR, Frech SA, Hammond SA, Zoeteweij JP (2003) “Transcutaneous immunization and immunostimulant strategies: Capitalizing on the immunocompetence of the skin.” Expert Reviews of Vaccines 2(2):253-267.
[44] Glenn GM, Taylor DN, Li X, Frankel S, Montemarano A, Alving CR (2000) “Transcutaneous immunization: A human vaccine delivery strategy using a patch.” Nature Medicine 6(12):1403-1406.
[45] Prausnitz MR, Langer R (2008) “Transdermal drug delivery.” Nature Biotechnology 26(11):1261-1268.
[46] Roberts MS, Walters KA (2009) Dermal Absorption and Toxicity Assessment, 2nd., Informa Healthcare USA, Inc. New York.
[47] Bal SM, Ding Z, van Riet E, Jiskoot W, Bouwstra JA (2010) “Advances in transcutaneous vaccine delivery: Do all ways lead to Rome?” Journal of Controlled Release 148(3):266-282.
[48] Williams AC, Barry B (2004) “Penetration enhancers.” Advanced Drug Delivery Reviews 56(5):603-618.
[49] Cevc G (2004) “Lipid vesicles and other colloids as drug carriers on the skin.” Advanced Drug Delivery Reviews 56(5):675-711.
[50] Denet AR, Vanbever R, Preat V (2004) “Skin electroporation for transdermal and topical delivery.” Advance Drug Delivery Reviews 56(5):659-674.
[51] Doukas AG, Kollias N (2004) “Transdermal delivery with a pressure wave.” Advance Drug Delivery Reviews 56(5):559-579.
[52] Mitragotri S, Kost J (2004) “Low-frequency sonophoresis: a review.” Advance Drug Delivery Reviews 56(5):589-601.
[53] Ludvigsson J (2006) “Why diabetes incidence increases – a unifying theory.” Annals of the New York Academy of Sciences 1079:374-382.
[54] Siekmeier R, Scheuch G “Inhaled insulin: does it become reality.” Journal of Physiology and Pharmacology 59:81-113.
[55] Prausnitz MR (2004) “Microneedles for transdermal drug delivery.” Advance Drug Delivery Reviews 56(5):581-587.
[56] McAllister DV, Wang PM, Davis SP, Park JH, Canatella PJ, Allen MG, Prausnitz MR (2003) “Microfabricated needles for transdermal delivery of macromolecules and nanoparticles: fabrication methods and transport studies.” Proceedings of the National Academy of Sciences 100(24):13755-13760.
[57] Ito Y, Hagiwara E, Saeki A, Sugioka N, Takada K (2006) “Feasibility of microneedles for percutaneous absorption of insulin.” European Journal of Pharmaceutical Sciences 29(1):82-88.
[58] Ito Y, Yamazaki T, Sugioka N, Takada K (2010) “Self-dissolving micropile array tips for percutaneous administration of insulin.” Journal of Material Science: Materials in Medicine 21(2):835-841.
[59] Ito Y, Yoshimitsu J, Shiroyama K, Sugioka N, Takada K (2006) “Self-dissolving microneedles for the percutaneous absorption of EPO in mice.” Journal of Drug Targeting 14(5):255-261.
[60] Migalska K, Morrow DI, Garland MJ, Thakur R, Woolfson AD, Donelly RF (2011) “Laser-engineered dissolving microneedle arrays for transdermal macromolecular drug delivery.” Pharmaceutical Research 28(8):1919-1930.
[61] Huang MH, Yang MC (2010) “Swelling and biocompatibility of sodium alginate/poly(γ-glutamic acid) hydrogels.” Advanced Polymer Technology 21(8):561-567.
[62] Wang CC, Su CH, Chen JP, Chen CC (2009) “An enhancement on healing effect of wound dressing: Acrylic acid grafted and gamma-polyglutamic acid/chitosan immobilized polypropylene non-woven.” Materials Science and Engineering 29(5):1715-1724.
[63] Yu DG (2007) “Formatian of colloidal silver nanoparticles stabilized by Na+-poly(γ-glutamic acid)-silver nitrate complex via chemical reduction process.” Colloids and Surfaces B: Biointerfaces 59(2):171-178.
[64] Lieske JC, Leonard R, Swift H, Toback FG (1996) “Adhesion of calcium oxalate monohydrate cyrstals to anionic sites on the surface of renal epithelial cells.” American Journal of Physiology 270(1-2):192-199.
[65] Lee W, Lee SH, Ahn DG, Cho H, Sung MH, Han SH, Oh JW (2013) “The antiviral activity of poly-γ-glutamic acid, a polypeptide secreted by Bacillus sp., through induction of CD14-dependent type I interferon responses.” Biomaterials 34(37):9700-9708.
[66] Lee YH, Chang JJ, Yang MC, Chien CT, Lai WF (2012) “Acceleration of wound healing in diabetic rats by layered hydrogel dressing.” Carbohydrate Polymers 88(3):809-819.
[67] Park JS, Choi SH, Choi WY, Yoon MH (2005) “Characteristics of hydrogel prepared from microbial poly(γ-glutamic acid) by chemical crosslinker.” Journal of Agricultural and Food Chemistry 48(4):213-217.
[68] Horie K, Baron R, Fox RB, He M, Kahovec J, Kitayama T, Kubisa P, Marechal E, Mormann W, Stepto RF, Tabak D (2004) “Definitions of terms relating to reactions of polymers and functional polymeric materials.” Pure and Applied Chemistry 76(4):889-906.
[69] Yao B, Yang C, Zhang K, Ni C, Song H, Ni Z, Chen M (2009) “Syntheses and characterization of pH-sensitive hydrogel from poly (γ-glutamic) acid.” Materials Science-Poland 27(1).
[70] Shih IL, Van YT (2008) “The production of poly-( γ-glutamic acid) from microorganisms and its various applications.” Bioresource Technology 79(3):207-225.
[71] Caspers PJ, Lucassen GW, Bruining HA, Puppels GJ (2000) “Automated depth-scanning confocala Raman microspectrometer for rapid in vivo determination of water concentration profiles in human skin.” Journal of Raman Spectroscopy 31(8-9):813-818.
[72] Gok E, Olgaz S (2004) “Binding of fluorescein isothiocyanate to insulin: a fluorimetric labeling study.” Journal of Fluoescence 14(2):203-206.
[73] Hentz NG, Richardson JM, Sportsman JR, Daijo J, Sittampalam GS (1997) “Synthesis and characterization of insulin-fluorescein derivatives for bioanalytical applications.” Analytical Chemistry 69(24):4994-50.

[74] Burtis CA, Ashwood ER (1994) Tietz Textbook of Clinical Chemistry, 2nd ed., W.B. Saunders, Philadelphia, PA.
[75] Vedan (2003) “Stable biodegradable, water absorbing gamma-polyglutamic acid hydrogel.” European Patent 1550469.
[76] Korsmeyer RW, Gurny R, Doelker E, Buri P, Peppas NA (1983) “Mechanisms of solute release from porous hydrophilic polymers.” International Journal of Pharmaceutics 15:25-35.
[77] Kopecek J, Ulbrich K (1983) Biodegradation of Biomedical Polymers, Pergamon Press Ltd., Great Britain.
[78] Magaji V, Johnston JM (2011) “Inpatient Management of Hyperglycemia and Diabetes.” Clinical Diabetes 29(1):3-9.