Pharmaceutical Research

, Volume 25, Issue 2, pp 407–416 | Cite as

Gene Delivery to the Epidermal Cells of Human Skin Explants Using Microfabricated Microneedles and Hydrogel Formulations

  • Marc Pearton
  • Chris Allender
  • Keith Brain
  • Alexander Anstey
  • Chris Gateley
  • Nicolle Wilke
  • Anthony Morrissey
  • James Birchall
Research Paper



Microneedles disrupt the stratum corneum barrier layer of skin creating transient pathways for the enhanced permeation of therapeutics into viable skin regions without stimulating pain receptors or causing vascular damage. The cutaneous delivery of nucleic acids has a number of therapeutic applications; most notably genetic vaccination. Unfortunately non-viral gene expression in skin is generally inefficient and transient. This study investigated the potential for improved delivery of plasmid DNA (pDNA) in skin by combining the microneedle delivery system with sustained release pDNA hydrogel formulations.

Materials and Methods

Microneedles were fabricated by wet etching silicon in potassium hydroxide. Hydrogels based on Carbopol polymers and thermosensitive PLGA-PEG-PLGA triblock copolymers were prepared. Freshly excised human skin was used to characterise microneedle penetration (microscopy and skin water loss), gel residence in microchannels, pDNA diffusion and reporter gene (β-galactosidase) expression.


Following microneedle treatment, channels of approximately 150–200 μm depth increased trans-epidermal water loss in skin. pDNA hydrogels were shown to harbour and gradually release pDNA. Following microneedle-assisted delivery of pDNA hydrogels to human skin expression of the pCMVβ reporter gene was demonstrated in the viable epidermis proximal to microchannels.


pDNA hydrogels can be successfully targeted to the viable epidermis to potentially provide sustained gene expression therein.

Key words

DNA human skin hydrogel microneedles thermosensitive 



The authors acknowledge the BBSRC for financial support of MP.


  1. 1.
    S. Henry, D. V. McAllister, M. G. Allen, and M. R. Prausnitz. Microfabricated microneedles: a novel approach to transdermal drug delivery. J. Pharm. Sci. 87:922–925 (1998).PubMedCrossRefGoogle Scholar
  2. 2.
    F. Chabri, K. Bouris, T. Jones, D. Barrow, A. Hann, C. Allender, K. Brain, and J. Birchall. Microfabricated silicon microneedles for nonviral cutaneous gene delivery. Br. J. Dermatol. 150:869–877 (2004).PubMedCrossRefGoogle Scholar
  3. 3.
    W. Martanto, S. P. Davis, N. R. Holiday, J. Wang, H. S. Gill, and M. R. Prausnitz. Transdermal delivery of insulin using microneedles in vivo. Pharm. Res. 21:947–952 (2004).PubMedCrossRefGoogle Scholar
  4. 4.
    J. A. Matriano, M. Cormier, J. Johnson, W. A. Young, M. Buttery, K. Nyam, and P. E. Daddona. Macroflux® microprojection array patch technology: a new and efficient approach for intracutaneous immunization. Pharm. Res. 19:63–70 (2002).PubMedCrossRefGoogle Scholar
  5. 5.
    R. K. Sivamani, B. Stoeber, G. C. Wu, H. Zhai, D. Liepmann, and H. Maibach. Clinical microneedle injection of methyl nicotinate: stratum corneum penetration. Skin Res. Technol. 11:152–156 (2005).PubMedCrossRefGoogle Scholar
  6. 6.
    J. H. Park, M. G. Allen, and M. R. Prausnitz. Polymer microneedles for controlled-release drug delivery. Pharm. Res. 23:1008–1019 (2006).PubMedCrossRefGoogle Scholar
  7. 7.
    H. S. Gill and M. R. Prausnitz. Coated microneedles for transdermal delivery. J. Control. Release 117:227–237 (2007).PubMedCrossRefGoogle Scholar
  8. 8.
    J. C. Birchall, S. A. Coulman, M. Pearton, C. Allender, K. Brain, A. Anstey, C. Gateley, N. Wilke, and A. Morrissey. Cutaneous DNA delivery and gene expression in ex vivo human skin explants via wet-etch microfabricated microneedles. J. Drug Target. 13:415–421 (2005).PubMedCrossRefGoogle Scholar
  9. 9.
    S. A. Coulman, D. Barrow, A. Anstey, C. Gateley, A. Morrissey, N. Wilke, C. Allender, K. Brain, and J. C. Birchall. Minimally invasive cutaneous delivery of macromolecules and plasmid DNA via microneedles. Curr. Drug Discov. 3:65–75 (2006).CrossRefGoogle Scholar
  10. 10.
    I. R. Williams and T. S. Kupper. Immunity at the surface: homeostatic mechanisims of the skin immune system. Life Sci. 58:1485–1507 (1996).PubMedCrossRefGoogle Scholar
  11. 11.
    E. F. Fynan, R. G. Webster, D. H. Fuller, J. R. Haynes, J. C. Santoro, and H. L. Robinson. DNA vaccines: protective immunizations by parenteral, mucosal and gene-gun innoculations. Proc. Natl. Acad. Sci. U. S. A. 90:11478–11482 (1993).PubMedCrossRefGoogle Scholar
  12. 12.
    S. Kaushik, A. H. Hord, D. D. Denson, D. V. McAllister, S. Smitra, M. G. Allen, and M. R. Prausnitz. Lack of pain associated with microfabricated microneedles. Anesth. Analg. 92:502–504 (2001).PubMedCrossRefGoogle Scholar
  13. 13.
    M. T. S. Lin, L. Pulkkinen, and J. Uitto. Cutaneous gene therapy—principles and prospects. Dermatol. Clin. 18:177–187 (2000).PubMedGoogle Scholar
  14. 14.
    J. A. Mikszta, J. B. Alarcon, J. M. Brittingham, D. E. Sutter, R. J. Pettis, and N. G. Harvey. Improved genetic immunization via micromechanical disruption of skin-barrier function and targetted epidermal delivery. Nat. Med. 8:415–419 (2002).PubMedCrossRefGoogle Scholar
  15. 15.
    D. W. Pack, A. S. Hoffman, S. Pun, and P. S. Stayton. Design and development of polymers for gene delivery. Nat. Rev. Drug Discov. 4:581–593 (2005).PubMedCrossRefGoogle Scholar
  16. 16.
    L. D. Shea, E. Smiley, J. Bonadio, and D. J. Mooney. DNA delivery from polymer matrices for tissue engineering. Nat. Biotechnol. 17:551–554 (1999).PubMedCrossRefGoogle Scholar
  17. 17.
    I. Csoka, E. Csanyi, G. Zapantis, E. Nagy, A. Feher-Kiss, G. Horvath, G. Blazso, and I. Eros. In vitro and in vivo percutaneous absorption of topical dosage forms: case studies. Int. J. Pharm. 291:11–19 (2005).PubMedCrossRefGoogle Scholar
  18. 18.
    P. Mura, G. P. Bettinetti, A. Liguori, and G. Bramanti. Improvement of clonazepam release from a Carbopol hydrogel. Pharm. Acta Helv. 67:282–288 (1992).PubMedGoogle Scholar
  19. 19.
    F. A. Ismail, J. Napaporn, J. A. Hughes, and G. A. Brazeau. In situ gel formulations for gene delivery: release and myotoxicity studies. Pharm. Dev. Technol. 5:391–397 (2000).PubMedCrossRefGoogle Scholar
  20. 20.
    B. Jeong, Y. H. Bae, D. S. Lee, and S. W. Kim. Biodegradable block copolymers as injectable drug-delivery systems. Nature 388:860–862 (1997).PubMedCrossRefGoogle Scholar
  21. 21.
    M. S. Shim, H. T. Lee, W. S. Shim, I. Park, H. Lee, T. Chang, S. W. Kim, and D. S. Lee. Poly(D,L-lactic acid-co-glycolic acid)-b-poly(ethylene glycol)-b-poly (D,L-lactic acid-co-glycolic acid) triblock copolymer and thermoreversible phase transition in water. J. Biomed. Materi. Res. 61:188–196 (2002).CrossRefGoogle Scholar
  22. 22.
    P. Lee, Z. Li, and L. Huang. Thermosensitive hydrogel as a Tgf-β1 gene delivery vehicle enhances diabetic wound healing. Pharm. Res. 20:1995–2000 (2003).PubMedCrossRefGoogle Scholar
  23. 23.
    J. Nieuwenhuis. Synthesis of polylactides and their copolymers. Clin. Mater. 10:59–67 (1992).PubMedCrossRefGoogle Scholar
  24. 24.
    B. Jeong, Y. H. Bae, and S. W. Kim. In situ gelation of PEG-PLGA-PEG triblock copolymer solutions and degredation thereof. J. Biomed. Materi. Res. 50:171–177 (2000).CrossRefGoogle Scholar
  25. 25.
    W. Amass, A. Amass, and B. Tighe. A review of biodegradable polymers: uses, current developments in the synthesis and characterization of biodegradable polyesters, blends of biodegradable polymers and recent advances in biodegradation studies. Polym. Int. 47:89–144 (1998).CrossRefGoogle Scholar
  26. 26.
    D. V. McAllister, P. M. Wang, S. P. Davis, J. H. Park, P. J. Canatella, M. G. Allen, and M. R. Prausnitz. Microfabricated needles for transdermal delivery of macromolecules and nanoparticles: fabrication methods and transport studies. Proc. Natl. Acad. Sci. U. S. A. 100:13755–13760 (2003).PubMedCrossRefGoogle Scholar
  27. 27.
    J. H. Park, M. G. Allen, and M. R. Prausnitz. Biodegradable polymer microneedles: fabrication, mechanics and transdermal drug delivery. J. Control. Release 104:51–66 (2005).PubMedCrossRefGoogle Scholar
  28. 28.
    J. C. Birchall, I. W. Kellaway, and M. Gumbleton. Physical stability and in-vitro gene expression efficiency of nebulised lipid-peptide-DNA complexes. Int. J. Pharm. 197:221–231 (2000).PubMedCrossRefGoogle Scholar
  29. 29.
    N. Wilke, A. Mulcahy, S.-R. Ye, and A. Morrissey. Process optimisation and characterisation of silicon microneedles fabricated by wet etch technology. Microelectron. J. 36:650–656 (2005).CrossRefGoogle Scholar
  30. 30.
    G. M. Zentner, R. Rathi, J. Shih, J. C. McRea, M. Seo, H. Oh, B. G. Rhee, J. Mestecky, Z. Moldoveanu, M. Morgan, and S. Weitman. Biodegradable block copolymers for delivery of proteins and water-insoluble drugs. J. Control. Release 72:203–215 (2001).PubMedCrossRefGoogle Scholar
  31. 31.
    B. Jeong, Y. H. Bae, and S. W. Kim. Biodegradable Thermosensitive Micelles of PEG-PLGA-PEG Triblock Copolymers. Colloids Surf., B Biointerfaces. 16:185–193 (1999).CrossRefGoogle Scholar
  32. 32.
    K. Rengarajan, S. M. Cristol, M. Mehta, and J. M. Nickerson. Quantifying DNA concentrations using fluorometry: A comparision of fluorophores. Mol. Vis. 8:416–421 (2002).PubMedGoogle Scholar
  33. 33.
    S. Chen, R. Pieper, D. C. Webster, and J. Singh. Triblock copolymers: synthesis, characterization, and delivery of a model protein. Int. J. Pharm. 288:207–218 (2005).PubMedCrossRefGoogle Scholar
  34. 34.
    Z. Li, W. Ning, J. Wang, A. Choi, P. Y. Lee, P. Tyagi, and L. Huang. Controlled gene delivery system based on thermosensitive biodegradable hydrogel. Pharm. Res. 20:884–888 (2003).PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Marc Pearton
    • 1
  • Chris Allender
    • 1
  • Keith Brain
    • 1
  • Alexander Anstey
    • 2
  • Chris Gateley
    • 2
  • Nicolle Wilke
    • 3
  • Anthony Morrissey
    • 3
  • James Birchall
    • 1
  1. 1.Gene Delivery Research Group, Welsh School of PharmacyCardiff UniversityCardiffUK
  2. 2.Gwent Healthcare NHS TrustRoyal Gwent HospitalNewport, South WalesUK
  3. 3.Biomedical Microsystems TeamTyndall National InstituteCorkIreland

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