European Biophysics Journal

, Volume 36, Issue 8, pp 1049–1058

Follow-up of drug permeation through excised human skin with confocal Raman microspectroscopy

  • Ali Tfayli
  • Olivier Piot
  • Franck Pitre
  • Michel Manfait
Original Paper

Abstract

Skin is a multilayered organ which covers and protects the surface of human body by providing a barrier function against exogenous agents. Meanwhile, the efficacy of several topically applicated drugs is directly related to their penetration through the skin barrier. Several techniques are commonly used to evaluate the rate, the speed and the depth of penetration of these drugs, but few of them can provide real-time results. Therefore, the use of nondestructive and structurally informative techniques permits a real breakthrough in the investigations on skin penetration at a microscopic scale. Confocal Raman microspectroscopy is a nondestructive and rapid technique which allows information to be obtained from deep layers under the skin surface, giving the possibility of a real-time tracking of the drug in the skin layers. The specific Raman signature of the drug enables its identification in the skin. In this study, we try to follow the penetration of Metronidazole, a drug produced by Galderma as a therapeutic agent for Rosacea treatment, through the skin. The first step was the spectral characterization of Metronidazole in the skin. Then micro-axial profiles were conducted to follow the penetration of the drug in the superficial layers, on excised human skin specimens. For more accurate information, transverse sections were cut from the skin and spectral images were conducted, giving information down to several millimeters deep. Moreover, the collected spectra permit us to follow the structural modifications, induced by the Metronidazole on the skin, by studying the changes in the spectral signature of the skin constituents.

Keywords

Confocal Raman microspectroscopy Drug penetration Metronidazole Skin 

References

  1. Akhtar W, Edwards HG (1997) Fourier-transform Raman spectroscopy of mammalian and avian keratotic biopolymers. Spectrochim Acta A Mol Biomol Spectrosc 53A:81–90CrossRefADSGoogle Scholar
  2. Baldwin KJ, Batchelder DN (2001) Confocal Raman microspectroscopy through a planar interface. Appl Spectrosc 55:517–524(8)CrossRefADSGoogle Scholar
  3. Barry BW (1987) Mode of action of penetration enhancers in human skin. J Controll Release 6:85–97CrossRefGoogle Scholar
  4. Barry BW, Edwards HGM, Williams AC (1992) Fourier transform Raman and infrared vibrational study of human skin: assignment of spectral bands. Raman Spectrosc 23:641–645CrossRefADSGoogle Scholar
  5. Bouwstra JA, de Vries MA, Gooris GS, Bras W, Brussee J, Ponec M (1991) Thermodynamic and structural aspects of the skin barrier. J Controll Release 15:209–219CrossRefGoogle Scholar
  6. Bruneel JL, Lassègues JC, Sourisseau C (2002) In-depth analyses by confocal Raman microspectrometry: experimental features and modeling of the refraction effects. J Raman Spectrosc 33:815–828CrossRefADSGoogle Scholar
  7. Caspers PJ, Lucassen GW, Carter EA, Bruining HA, Puppels GJ (2001) In vivo confocal Raman microspectroscopy of the skin: noninvasive determination of molecular concentration profiles. J Invest Dermatol 116:434–442CrossRefGoogle Scholar
  8. Caspers PJ, Williams AC, Carter EA, Edwards HG, Barry BW, Bruining HA, Puppels GJ (2002) Monitoring the penetration enhancer dimethyl sulfoxide in human stratum corneum in vivo by confocal Raman spectroscopy. Pharm Res 19:1577–1580CrossRefGoogle Scholar
  9. Caspers PJ, Lucassen GW, Puppels GJ (2003) Combined in vivo confocal Raman spectroscopy and confocal microscopy of human skin. Biophys J 85:572–580CrossRefGoogle Scholar
  10. Chrit L, Hadjur C, Morel S, Sockalingum G, Lebourdon G, Leroy F, Manfait M (2005) In vivo chemical investigation of human skin using a confocal Raman fiber optic microprobe. J Biomed Opt 10:44007CrossRefGoogle Scholar
  11. Cohen AF, Tiemstra JD (2002) Diagnosis and treatment of Rosacea. JABFP 15:214–217Google Scholar
  12. EC (2002) Health consumer protection directorate, Directorate E, Guidance document on dermal absorption, vol Sanco/222/2000 rev.6. European commissionGoogle Scholar
  13. Everall NJ (2000a) Confocal Raman microscopy: why the depth resolution and spatial accuracy can be much worse than you think. J Appl Spectrosc 50Google Scholar
  14. Everall NJ (2000b) Modeling and measuring the effect of refraction on the depth resolution of Confocal Raman Microscopy. J Appl Spectrosc 54Google Scholar
  15. Everall NJ (2004a) Depth profiling with confocal Raman Microscopy, Part I. Spectroscopy 19. http://www.spectroscopyonline.com
  16. Everall NJ (2004b) Depth profiling with confocal Raman Microscopy, Part II. Spectroscopy 19. http://www.spectroscopyonline.com
  17. Frost RL (2004) Raman spectroscopy of natural oxalates. Anal Chim Acta 517:207–214CrossRefGoogle Scholar
  18. Fujiwara A, Hinokitani T, Goto K, Arai T (2005) Partial ablation of porcine stratum corneum by argon-fluoride excimer laser to enhance transdermal drug permeability. Lasers Med Sci 19:210–217CrossRefGoogle Scholar
  19. Gniadecka M, Faurskov Nielsen O, Christensen DH, Wulf HC (1998) Structure of water, proteins, and lipids in intact human skin, hair, and nail. J Invest Dermatol 110:393–398CrossRefGoogle Scholar
  20. Grams YY, Whitehead L, Cornwell P, Bouwstra JA (2004a) On-line visualization of dye diffusion in fresh unfixed human skin. Pharm Res 21:851–859CrossRefGoogle Scholar
  21. Grams YY, Whitehead L, Cornwell P, Bouwstra JA (2004b) Time and depth resolved visualisation of the diffusion of a lipophilic dye into the hair follicle of fresh unfixed human scalp skin. J Controll Release 98:367–378CrossRefGoogle Scholar
  22. Hofland HE, Bouwstra JA, Bodde HE, Spies F, Junginger HE (1995) Interactions between liposomes and human stratum corneum in vitro: freeze fracture electron microscopical visualization and small angle X-ray scattering studies. Br J Dermatol 132:853–866CrossRefGoogle Scholar
  23. Kalia YN, Pirot F, Potts RO, Guy RH (1998) Ion mobility across human stratum corneum in vivo. J Pharm Sci 87:1508–1511CrossRefGoogle Scholar
  24. Khan GM, Frum Y, Sarheed O, Eccleston GM, Meidan VM (2005) Assessment of drug permeability distributions in two different model skins. Int J Pharm 303:81–87CrossRefGoogle Scholar
  25. koningsten JA (1971) Introduction of the theory of the Raman effect. D. Reidel publishingGoogle Scholar
  26. Lippert JL, Peticolas WL (1971) Laser Raman investigation of the effect of cholesterol on conformational changes in dipalmitoyl lecithin multilayers. Proc Natl Acad Sci USA 68:1572–1576CrossRefADSGoogle Scholar
  27. Mendelsohn R, Flach CR, Moore DJ (2006) Determination of molecular conformation and permeation in skin via IR spectroscopy, microscopy, and imaging. Biochim Biophys Acta 1758:923–933CrossRefGoogle Scholar
  28. Neubert R, Rettig W, Wartewig S, Wegener M, Wienhold A (1997) Structure of stratum corneum lipids characterized by FT-Raman spectroscopy and DSC. II. Mixtures of ceramides and saturated fatty acids. Chem Phys Lipids 89:3–14CrossRefGoogle Scholar
  29. OECD (2004a) Test guideline 428: skin absorption: in vitro method. Paris, FranceGoogle Scholar
  30. Parker F (1983) Applications of infrared, Raman and Raman Resonance spectroscopy in biochemistry, New YorkGoogle Scholar
  31. Piot O (2000) Caractérisation par microspectroscopie Raman des espèces moléculaires responsables de la cohésion des grains de blé tendre Pharmacy, vol Doctor. University of Reims Champagne - Ardenne, ReimsGoogle Scholar
  32. Piot O, Autran JC, Manfait M (2000) Spatial distribution of protein and phenolic constituents in wheat grain as probed by confocal Raman microspectroscopy. J Cereal Sci 32:57–71CrossRefGoogle Scholar
  33. Potts RO, Guzek DB, Harris RR, McKie JE (1985) A noninvasive, in vivo technique to quantitatively measure water concentration of the stratum corneum using attenuated total-reflectance infrared spectroscopy. Arch Dermatol Res 277:489–495CrossRefGoogle Scholar
  34. Rawlings AV, Scott IR, Harding CR, Bowser PA (1994) Stratum corneum moisturization at the molecular level. J Invest Dermatol 103:731–741CrossRefGoogle Scholar
  35. Schulte A, Bradley L, Williams C (1995) Equilibrium composition of retinal isomers in dark-adapted bacteriorhodopsin and effect of high pressure probed by near-infrared Raman Spectroscopy. Appl Spectrosc 49:80–83CrossRefADSGoogle Scholar
  36. Sett P, Chattopadhyay S, Mallick PK (2000) Raman excitation profiles and excited state molecular configurations of three isomeric phenyl pyridines. Spectrochim Acta A Mol Biomol Spectrosc 56:855–875CrossRefADSGoogle Scholar
  37. Sieg A, Crowther J, Blenkiron P, Marcott C, Matts PJ (2006) Confocal Raman microspectroscopy—measuring the effects of topical moisturisers on stratum corneum water gradients in vivo. In: Mahadevan-Jansen A, Petrich WH (eds) The international society of optical engineeringGoogle Scholar
  38. Song Y, Xiao C, Mendelsohn R, Zheng T, Strekowski L, Michniak B (2005) Investigation of iminosulfuranes as novel transdermal penetration enhancers: enhancement activity and cytotoxicity. Pharm Res 22:1918–1925CrossRefGoogle Scholar
  39. Tsuboi M, Ezaki Y, Aida M, Suzuki M, Yimit A, Ushizawa K, Ueda T (1998) Raman scattering tensors of tyrosine. Biospectroscopy 4:61–71CrossRefGoogle Scholar
  40. van de Sandt JJM, van Burgsteden JA, Cage S, Carmichael PL, Dick I, Kenyon S, Korinth G, Larese F, Limasset JC, Maas WJM, Montomoli L, Nielsen JB, Payan JP, Robinson E, Sartorelli P, Schaller KH, Wilkinson SC, Williams FM (2004) In vitro predictions of skin absorption of caffeine, testosterone, and benzoic acid: a multi-centre comparison study. Regul Toxicol Pharmacol 39:271–281CrossRefGoogle Scholar
  41. Veiro JA, Cummins PG (1994) Imaging of skin epidermis from various origins using confocal laser scanning microscopy. Clin Lab Invest 189:16–22Google Scholar
  42. Wagner H, Kostka K-H, Lehr C-M, Schaefer UF (2001) Interrelation of permeation and penetration parameters obtained from in vitro experiments with human skin and skin equivalents. J Controll Release 75:283–295CrossRefGoogle Scholar
  43. Wegener M, Neubert R, Rettig W, Wartewig S (1997) Structure of stratum corneum lipids characterized by FT-Raman spectroscopy and DSC. III. Mixtures of ceramides and cholesterol. Chem Phys Lipids 88:73–82CrossRefGoogle Scholar
  44. Weigmann H-J, Jacobi U, Antoniou C, Tsikrikas GN, Wendel V, Rapp C, Gers-Barlag H, Sterry W, Lademann J (2005) Determination of penetration profiles of topically applied substances by means of tape stripping and optical spectroscopy: UV filter substance in sunscreens. J Biomed Opt 10:014009–014017CrossRefGoogle Scholar
  45. Xiao C, Flach CR, Marcott C, Mendelsohn R (2004) Uncertainties in depth determination and comparison of multivariate with univariate analysis in confocal Raman studies of a laminated polymer and skin. Appl Spectrosc 58:382–389CrossRefADSGoogle Scholar
  46. Xiao C, Moore DJ, Flach CR, Mendelsohn R (2005a) Permeation of dimyristoylphosphatidylcholine into skin—structural and spatial information from IR and Raman microscopic imaging. Vib Spectrosc 38:151–158CrossRefGoogle Scholar
  47. Xiao C, Moore DJ, Rerek ME, Flach CR, Mendelsohn R (2005b) Feasibility of tracking phospholipid permeation into skin using infrared and Raman microscopic imaging. J Invest Dermatol 124:622–632CrossRefGoogle Scholar
  48. Yu B, Kim KH, So PT, Blankschtein D, Langer R (2003) Visualization of oleic acid-induced transdermal diffusion pathways using two-photon fluorescence microscopy. J Invest Dermatol 120:448–455CrossRefGoogle Scholar
  49. Zip C (2006) An update on the role of topical metronidazole in rosacea. Skin Therapy Lett 11:1–4ADSGoogle Scholar

Copyright information

© EBSA 2007

Authors and Affiliations

  • Ali Tfayli
    • 1
    • 3
  • Olivier Piot
    • 1
  • Franck Pitre
    • 2
  • Michel Manfait
    • 1
  1. 1.MeDIAN Unit, CNRS UMR 6142, Faculty of PharmacyUniversity of Reims ChampagneArdenneFrance
  2. 2.Department of Pharmaceutical Development, Formulation laboratoryGalderma R & DSophia, AntipolisFrance
  3. 3.Champagne ArdenneFrance

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