Pharmaceutical Research

, Volume 28, Issue 1, pp 7–21 | Cite as

Two-Layered Dissolving Microneedles for Percutaneous Delivery of Peptide/Protein Drugs in Rats

  • Keizo Fukushima
  • Ayaka Ise
  • Hiromi Morita
  • Ryo Hasegawa
  • Yukako Ito
  • Nobuyuki Sugioka
  • Kanji Takada
Research Paper



Feasibility study of two-layered dissolving microneedles for percutaneous delivery of peptide/proteins using recombinant human growth hormone (rhGH) and desmopressin (DDAVP).


Two-layered dissolving microneedles were administered percutaneously to the rat skin. Plasma rhGH and DDAVP concentrations were measured by EIA and LC/MS/MS. In vivo dissolution and diffusion rates of drugs in the skin were studied using tracer dyes, lissamine green B (LG) for rhGH and evans blue (EB) for DDAVP. Diffusion of drugs vertically into the skin was studied using FITC-dextran (MW = 20 kDa)-loaded dissolving microneedles. Stability experiments were performed at −80°C and 4°C.


The absorption half-lives, t 1/2a, of rhGH and DDAVP from dissolving microneedles were 23.7 ± 4.3–28.9 ± 5.2 and 14.4 ± 2.9–14.1 ± 1.1 min; the extents of bioavailability were 72.8 ± 4.2–89.9 ± 10.0% and 90.0 ± 15.4–93.1 ± 10.3%, respectively. LG and EB disappeared from the administered site within 2 h and 3 h after administration. Five green fluorescein spots were detected at 15 s and enlarged transversally at 30 s. FITC-dextran was delivered into the microcapillaries at 5 min and 10 min. The rhGH and DDAVP were stable in dissolving microneedles for one month at −80°C and 4°C.


Results suggest that the two-layered dissolving microneedles are useful as an immediate-release transdermal DDS for peptide/protein drugs.


bioavailability desmopressin (DDAVP) dissolving microneedles recombinant human growth hormone (rhGH) transdermal delivery 



This study was supported by a strategic fund of MEXT (Ministry of Education, Culture, Sports, Science and Technology, MEXT) from 2008 to 2013 for establishing research foundation in private universities of Japan.


  1. 1.
    Walsh G. Biopharmaceuticals. West Sussex: Wiley; 2003.Google Scholar
  2. 2.
    Shibata N, Ito Y, Takada K. Pharmacokinetics. In: Gad SC, editor. Handbook of pharmaceutical biotechnology. MA: Wiley-Interscience; 2007. p. 757–814.CrossRefGoogle Scholar
  3. 3.
    Morishita I, Morishita M, Takayama K, Machida Y, Nagai T. Hypoglycemic effect of novel oral microspheres of insulin with protease inhibitor in normal and diabetic rats. Int J Pharm. 1992;78:9–16.CrossRefGoogle Scholar
  4. 4.
    Yamamoto A, Taniguchi T, Rikyuu K, Tsuji T, Murakami M, Muranishi S. Effects of various protease inhibitors on the intestinal absorption and degradation of insulin in rats. Pharm Res. 1994;11:1496–500.CrossRefPubMedGoogle Scholar
  5. 5.
    Amino Y, Kawada K, Toi K, Kumashiro I, Fukushima K. Phenylalanine derivatives enhancing intestinal absorption of insulin in mice. Chem Pharm Bull. 1988;36:4426–34.PubMedGoogle Scholar
  6. 6.
    Morishita M, Morishita I, Takayama K, Machida Y, Nagai T. Site dependent effect of aprotinin, sodium caprate Na2EDTA and sodium glycocholate on intestinal absorption of insulin. Biol Pharm Bull. 1993;16:68–72.PubMedGoogle Scholar
  7. 7.
    Utoguchi N, Watanabe Y, Shida T, Matsumoto M. Nitric oxide donors enhance rectal absorption of macromolecules in rabbits. Pharm Res. 1998;15:870–6.CrossRefPubMedGoogle Scholar
  8. 8.
    Takeuchi H, Yamamoto H, Niwa T, Hino T, Kawashima Y. Enteral absorption of insulin in rats from mucoadhesive chitosan coated liposomes. Pharm Res. 1996;13:896–901.CrossRefPubMedGoogle Scholar
  9. 9.
    Tozaki H, Komoike J, Tada C, Maruyama T, Terabe A, Suzuki J. Chitosan capsules for colon-specific drug delivery: improvement of insulin absorption from the rat colon. J Pharm Sci. 1997;86:1016–21.CrossRefPubMedGoogle Scholar
  10. 10.
    Matsuzawa A, Morishita M, Takayama K, Nagai T. Absorption of insulin using water-in-oil-in-water emulsion from an enteral loop in rats. Biol Pharm Bull. 1995;18:1718–23.PubMedGoogle Scholar
  11. 11.
    Cunha SA, Grossiord LJ, Puisieux F, Seiller M. Insulin in W/O/W multiple emulsions: biological activity after oral administration in normal and diabetic rats. J Microencapsul. 1997;14:321–33.CrossRefPubMedGoogle Scholar
  12. 12.
    Morishita M, Matsuzawa A, Takayama K, Isowa K, Nagai T. Improving insulin enteral absorption using water-in-oil-in-water emulsion. Int J Pharm. 1998;172:189–98.CrossRefGoogle Scholar
  13. 13.
    Suzuki A, Morishita M, Kajita M, Takayama K, Isowa K, Chiba Y, et al. Enhanced colonic and rectal absorption of insulin using a multiple emulsion containing eicosapentaenoic acid and docosahexaenoic acid. J Pharm Sci. 1998;87:1196–202.CrossRefPubMedGoogle Scholar
  14. 14.
    Takada K. Oral delivery of haematopoietic factors: Progress with gastrointestinal mucoadhesive patches, microdevices and other microfabrication technologies. Am J Drug Deliv. 2006;4:65–77.CrossRefGoogle Scholar
  15. 15.
    Khafagy El-S, Morishita M, Isowa K, Imai J, Takayama K. Effect of cell-penetrating peptides on the nasal absorption of insulin. J Control Release 2009;133:103–8.CrossRefGoogle Scholar
  16. 16.
    Kamei N, Morishita M, Takayama K. Importance of intermolecular interaction on the improvement of intestinal therapeutic peptide/protein absorption using cell-penetrating peptides. J Control Release 2009;136:179–86.CrossRefPubMedGoogle Scholar
  17. 17.
    Barry B, Williams A. Penetration enhancers. Adv Drug Deliv Rev. 2004;56:603–18.CrossRefPubMedGoogle Scholar
  18. 18.
    Cevc G. Lipid vesicles and other colloids as drug carriers on the skin. Adv Drug Deliv Rev. 2004;56:675–711.CrossRefPubMedGoogle Scholar
  19. 19.
    Preat V, Vanbever R. Skin electroporation for transdermal and topical delivery. Adv Drug Deliv Rev. 2004;56:659–74.CrossRefPubMedGoogle Scholar
  20. 20.
    Doukas A. Transdermal delivery with a pressure wave. Adv Drug Deliv Rev. 2004;56:559–79.CrossRefPubMedGoogle Scholar
  21. 21.
    Mitragotri S, Kost J. Low-frequency sonophoresis: a review. Adv Drug Deliv Rev. 2004;56:589–601.CrossRefPubMedGoogle Scholar
  22. 22.
    Cormier M, Johnson B, Ameri M, Nyam K, Libiran L, Zhang DD, et al. Transdermal delivery of desmopressin using a coated microneedle array patch system. J Control Release 2004;97:503–11.PubMedGoogle Scholar
  23. 23.
    Prausnitz RM. Microneedles for transdermal drug delivery. Adv Drug Deliv Rev. 2004;56:581–7.CrossRefPubMedGoogle Scholar
  24. 24.
    Levin G, Gershonowitz A, Sacks H, Stern M, Sherman A, Rudaev S, et al. Transdermal delivery of human growth hormone through RF-microchannels. Pharm Res. 2005;22:550–5.CrossRefPubMedGoogle Scholar
  25. 25.
    Qiu Y, Gao Y, Hu K, Li F. Enhancement of skin permeation of docetaxel: A novel approach combining microneedle and elastic liposomes. J Control Release 2008;129:144–50.CrossRefPubMedGoogle Scholar
  26. 26.
    Wermeling DP, Banks SL, Hudson DA, Gill HS, Gupta J, Prausnitz MR, et al. Microneedles permit transdermal delivery of a skin-impermeant medication to humans. Proc Natl Acad Sci USA. 2008;105:2058–63.CrossRefPubMedGoogle Scholar
  27. 27.
    Donnelly RF, Morrow DIJ, McCarron PA, Woolfson AD, Morrissey A, Juzenas P, et al. Microneedle mediated intradermal delivery of 5-aminolevulinic acid: Potential for enhanced topical photodynamic therapy. J Control Release 2008;129:154–62.CrossRefPubMedGoogle Scholar
  28. 28.
    Ito Y, Hagiwara E, Saeki A, Sugioka N, Takada K. Feasibility of microneedles for percutaneous absorption of insulin. Eur J Pharm Sci. 2006;29:82–8.CrossRefPubMedGoogle Scholar
  29. 29.
    Ito Y, Murakami A, Maeda T, Sugioka N, Takada K. Evaluation of self-dissolving needles containing low molecular weight heparin (LMWH) in rats. Int J Pharm. 2008;349:124–9.CrossRefPubMedGoogle Scholar
  30. 30.
    Ito Y, Ohashi Y, Shiroyama K, Sugioka N, Takada K. Self-dissolving micropiles for the percutaneous absorption of human growth hormone in rats. Biol Pharm Bull. 2008;31:1631–3.CrossRefPubMedGoogle Scholar
  31. 31.
    Ito Y, Yoshimitsu J, Shiroyama K, Sugioka N, Takada K. Self-dissolving microneedles fir the percutaneous absorption of EPO in mice. J Drug Target 2006;14:255–62.CrossRefPubMedGoogle Scholar
  32. 32.
    Ito Y, Saeki A, Shiroyama K, Sugioka N, Takada K. Percutaneous absorption of interferon-α by self-dissolving micropiles. J Drug Target 2008;16:243–9.CrossRefPubMedGoogle Scholar
  33. 33.
    Ito Y, Ohashi Y, Saeki A, Sugioka N, Takada K. Antihyperglycemic effect of insulin from self-dissolving micropiles in dogs. Chem Pharm Bull. 2008;56:243–6.CrossRefPubMedGoogle Scholar
  34. 34.
    Takada K. Microfabrication derived DDS: From batch to individual production. Drug Discov Ther. 2008;2:140–55.Google Scholar
  35. 35.
    Schmitz T, Huck CW, Bernkop-Schnurch A. Characterization of the thiol-disulphide chemistry of desmopressin by LC, μg-LC, LC-ESI-MS and Maldi-Tof. Amino Acids 2006;30:35–42.CrossRefPubMedGoogle Scholar
  36. 36.
    Getie M, Neubert RH. LC-MS determination of desmopressin acetate in human skin samples. J Pharm Biomed Anal. 2004;35:921–7.CrossRefPubMedGoogle Scholar
  37. 37.
    Kluge M, Riedl S, Erhart-Hofmann B, Hartmann J, Waldhauser F. Improved extraction procedure and RIA for determination of arginine8-vasopressin in plasma: role of premeasurement sample treatment and reference values in children. Clin Chem. 1999;45:98–103.PubMedGoogle Scholar
  38. 38.
    Agerso H, Seiding Larsen L, Riis A, Lovgren U, Karlsson MO, Senderovitz T. Pharmacokinetics and renal excretion of desmopressin after intravenous administration to healthy subjects and renally impaired patients. Br J Clin Pharmacol. 2004;58:352–8.CrossRefPubMedGoogle Scholar
  39. 39.
    Gibaldi M, Perrier D. Pharmacokinetics. New York: Marcel Dekker; 2006.Google Scholar
  40. 40.
    Ito Y, Ise A, Sugioka N, Takada K. Molecular weight dependence on bioavailabity of FITC-dextran after administration of self-dissolving micropile to rat skin. Drug Dev Ind Pharm. in press (2010).Google Scholar
  41. 41.
    Monteiro-Riviere AN, Bristol GD, Manning OT, Rogers AR, Riviere EJ. Interspecies and interregional analysis of the comparative histologic thickness and laser Doppler blood flow measurements at five cutaneous sites in nine species. J Invest Dermatol. 1990;95:582–6.CrossRefPubMedGoogle Scholar
  42. 42.
    Bauer J, Bahmer AF, Worl J, Neuhuber W, Schuler G, Fartasch M. A strikingly constant ration exists between Langerhans cells and other epidermal cells in human skin. A stereologic study using the optical dissector method and the confocal laser scanning microscope. J Invest Dermatol. 2001;116:313–8.CrossRefPubMedGoogle Scholar
  43. 43.
    Wermeling PD, Banks LS, Hudson AD, Gill SH, Gupta J, Prausnitz RM, et al. Microneedles permit transdermal delivery of a skin-impermeant medication to human. Proc Natl Acad Sci USA. 2008;105:2058–2063.CrossRefPubMedGoogle Scholar
  44. 44.
    Caspers JP, Lucassen WG, Bruining AH, Puppels JG. Automated depth-scanning confocal Raman microspectrometer for rapid in vivo determination of water concentration profiles in human skin. J Raman Spectrosc. 2000;31:813–8.CrossRefGoogle Scholar
  45. 45.
    Ito Y, Hasegawa R, Fukushima K, Sugioka N, Takada K. Self-dissolving micropile array chip as percutaneous delivery system of protein drug. Biol Pharm Bull. in press (2010).Google Scholar
  46. 46.
    Kolli CS, Banga AK. Charcterization of solid maltose microneedles and their use for transdermal delivery. Pharm Res. 2007;25:104–13.CrossRefPubMedGoogle Scholar
  47. 47.
    Wermeling PD, Banks LS, Hudson AD, Gill SH, Gupta J, Prausnitz RM, et al. Micropiles permit transdermal delivery of a skin-impermeant medication to humans. Proc Natl Acad Sci USA. 2008;105:2058–63.CrossRefPubMedGoogle Scholar
  48. 48.
    Duan H-G, Wei Y-H, Li B-X, Qin H-Y, Wu X-A. Improving the dissolution and oral bioavailability of the poorly water-soluble drug aloe-emodin by solid dispersion with polyethylene glycol 6000. Drug Dev Res. 2009;70:363–9.CrossRefGoogle Scholar
  49. 49.
    Park YJ, Kwon R, Quan ZQ, Oh HD, Kim OJ, Hwang RM, et al. Development of novel ibuprofen-loaded solid dispersion with improved bioavailability using aqueous solution. Arch Pharm Res. 2009;32:767–72.CrossRefPubMedGoogle Scholar
  50. 50.
    Kennedy M, Hu J, Gao P, Li L, Ali-Reynolds A, Chal B, et al. Enhanced bioavailability of a poorly soluble VR1 antagonist using an amorphous solid dispersion approach: a case study. Mol Pharm. 2008;5:981–93.CrossRefPubMedGoogle Scholar
  51. 51.
    Fukushima K, Haraya K, Terasaka S, Ito Y, Sugioka N, Takada K. Long-term pharmacokinetic efficacy and safety of low-dose ritonavir as a booster and atazanavir pharmaceutical formulation based on solid dispersion system in rats. Biol Pharm Bull. 2008;31:1209–14.CrossRefPubMedGoogle Scholar
  52. 52.
    Vasconcelos T, Sarmento B, Costa P. Solid dispersions as strategy to improve oral bioavailability of poor water soluble drugs. Drug Discov Today 2007;12:1068–75.CrossRefPubMedGoogle Scholar
  53. 53.
    Cheng YH, Dyer AM, Jabbal-Gill I, Hinchcliffe M, Nankervis R, Smith A, et al. Intranasal delivery of recombinant human growth hormone (somatropin) in sheep using chitosan-based powder formulations. Eur J Pharm Sci. 2005;26:9–15.CrossRefPubMedGoogle Scholar
  54. 54.
    Moore JA, Pletcher SA, Ross MJ. Absorption enhancement of growth hormone from the gastrointestinal tract of rats. Int J Pharm. 1986;34:35–43.CrossRefGoogle Scholar
  55. 55.
    Li H, Song JW, Park JS, Han K. Polyethylene glycol-coated liposomes for oral delivery of recombinant human epidermal growth factor. Int J Pharm. 2003;258:11–9.CrossRefPubMedGoogle Scholar
  56. 56.
    Lam XM, Duenas ET, Cleland JL. Encapsulation and stabilization of nerve growth factor into poly (lactic-co-glycolic) acid microspheres. J Pharm Sci. 2001;90:1356–65.CrossRefPubMedGoogle Scholar
  57. 57.
    Kim HK, Park TG. Microencapsulation of dissociable human growth hormone aggregates within poly (D, L-lactic-co-glycolic) acid microparticles for sustained release. Int J Pharm. 2001;229:107–16.CrossRefPubMedGoogle Scholar
  58. 58.
    Joukhadar C, Schenk B, Kaehler ST, Kollenz CJ, Bauer P, Muller M, et al. A replicate study design for testing bioequivalence: a case study on two desmopressin nasal spray preparations. Eur J Clin Pharmacol. 2003;59:631–6.CrossRefPubMedGoogle Scholar
  59. 59.
    Steiner IM, Kaehler ST, Sauermann R, Rinosl H, Muller M, Joukhadar C. Plasma pharmacokinetics of desmopressin following sublingual administration: an exploratory dose-escalation study in healthy male volunteers. Int J Clin Pharm Ther. 2006;44:172–9.Google Scholar
  60. 60.
    Cormier M, Johnson B, Ameri M, Nyam K, Libiran L, Zhang D, et al. Transdermal delivery of desmopressin using a coated microneedle array patch system. J Control Release 2004;97:503–11.PubMedGoogle Scholar
  61. 61.
    de Jager MW, Ponec M, Bouwstra JA. The lipid organization in stratum corneum and model systems based on ceramides. In: Touitou E, Barry BW, editors. Enhancement in drug delivery. London: CRC; 2007. p. 217–32.Google Scholar
  62. 62.
    Chabri F, Bouris K, Jones T, Barrow D, Hann A, Allender C, et al. Microfabricated silicon microneedles for nonviral cutancous gene delivery. Br J Dermatol. 2004;150:869–77.CrossRefPubMedGoogle Scholar
  63. 63.
    Gardeniers HJGE, Luttge R, Berenschot EJW, de Boer MJ, Yeshurun SY, Hefetz M, van’t Oever R, van den Berg A. Silicon micromachined hollow microneedles for transdermal liquid transport. J Microelectromech Syst. 2003;12:855–62.CrossRefGoogle Scholar
  64. 64.
    Ito Y, Hagiwara E, Saeki A, Sugioka N, Takada K. Feasibility of microneedles for percutaneous absorption of insulin. Eur J Pharm Sci. 2006;29:82–8.CrossRefPubMedGoogle Scholar
  65. 65.
    Davis PS, Martanto W, Allen GM, Prausnitz RM. Hollow metal micropiles for insulin delivery to diabetic rats. IEEE Trams Biomed Eng. 2005;52:909–15.CrossRefGoogle Scholar
  66. 66.
    Arora A, Prausnitz MR, Mitragotri S. Micro-scale devices for transdermal drug delivery. Int J Pharm. 2008;364:227–36.CrossRefPubMedGoogle Scholar
  67. 67.
    Morawski M, Alpár A, Brückner G, Fiedler A, Jäger C, Gati G, et al. Chondroitin sulfate proteoglycan-based extracellular matrix in chicken (Gallus domesticus) brain. Brain Res. 2009;1275:10–23.CrossRefPubMedGoogle Scholar
  68. 68.
    Faissner A, Clement A, Lochter A, Streit A, Mandl C, Schachner M. Isolation of a neural chondmitin sulfate proteoglycan with neurite outgrowth promoting properties. J Cell Biol. 1994;126:783–99.CrossRefPubMedGoogle Scholar
  69. 69.
    Umehara Y, Yamada S, Nishimura S, Shioi J, Robakis NK, Sugahara K. Chondroitin sulfate of appican, the proteoglycan form of amyloid precursor protein, produced by C6 glioma cells interacts with heparin-binding neuroregulatory factors. FEBS Lett. 2004;557:233–8.CrossRefPubMedGoogle Scholar
  70. 70.
    Wilson TM, Snow DM. Chondroitin sulfate proteoglycan expression pattern in hippocampal development: potential regulation of axon tract formation. J Comp Neurol. 2000;424:532–46.CrossRefPubMedGoogle Scholar
  71. 71.
    Sintov A, Di-Capua N, Rubinstein A. Cross-linked chondroitin sulfate: characterization for drug delivery purposes. Biomaterials 1995;16:473–8.CrossRefPubMedGoogle Scholar
  72. 72.
    Tsai FM, Chiang LY, Wang FL, Huang WG, Wu CP. Oral sustained delivery of diclofenac sodium using calcium chondroitin sulfate matrix. J Biomater Sci Polym Ed. 2005;16:1319–l331.CrossRefPubMedGoogle Scholar
  73. 73.
    Lee JW, Park J-H, Prausnitz MR. Dissolving microneedles for transdermal drug delivery. Biomaterials 2008;29:2113–24.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Keizo Fukushima
    • 1
  • Ayaka Ise
    • 1
  • Hiromi Morita
    • 1
  • Ryo Hasegawa
    • 1
  • Yukako Ito
    • 1
  • Nobuyuki Sugioka
    • 1
  • Kanji Takada
    • 1
  1. 1.Department of PharmacokineticsKyoto Pharmaceutical UniversityKyotoJapan

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