Archives of Dermatological Research

, Volume 310, Issue 8, pp 657–664 | Cite as

Caffeine delivery in porcine skin: a confocal Raman study

  • Cristina AlonsoEmail author
  • V. Carrer
  • C. Barba
  • L. Coderch
Original Paper


Confocal Raman microscopy is a novel optical method for studies of pro-drug and drug delivery. This method is a promising technique that enables non-destructive measurement of the permeation profile through skin layers. Peaks of compounds are usually normalised to skin peaks (amino-acid and amide I) for semi-quantitative evaluation. The present study seeks to optimise a methodology for complete quantitative measurement of the amount of an active compound at different depths. Caffeine was used as a tracer to evaluate compound’s skin penetration using confocal Raman microscopy. A semi-quantitative depth profile of caffeine was obtained with normalisation of the Raman intensities. These ratios of Raman intensities were correlated with the caffeine concentration using an external calibration curve. The calibration curve was carried out with porcine skin incubated in different concentrations of caffeine; afterwards, each skin sample was analysed by confocal Raman microscopy and HPLC to determine the relation between the Raman signal intensity and the caffeine concentration per skin mass and to create a depth profile. These correlation curves allow the full quantification of the caffeine in skin from Raman intensity ratios at different depths.


Caffeine Percutaneous penetration Confocal Raman microscopy Quantitative evaluation 



The authors are grateful to Montserrat Rigol Muxart and Núria Solanes Batlló of the Department of Cardiology (Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS) Hospital Clínic, Universitat de Barcelona, Spain) for supplying the porcine skin biopsies. We thank Anna Quintana of Almirall for her collaboration and contribution in this project.


This work was partially funded by the Spanish Ministry of Economy and Competitiveness, project code RTC-2014-1901-1.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. Animal handling was approved by the Institutional Review Board and Ethics Committee of Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS) Hospital Clínic, Universitat de Barcelona, Barcelona, Spain. The management of the Landrace Large White pigs used in this study conforms to the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health (Eighth Edition. Washington, DC: The National Academies Press, 2011).

Informed consent

This study did not require formal informed consent.


  1. 1.
    Alonso C, Martí M, Barba C, Carrer V, Rubio L, Coderch L (2017) Skin permeation and antioxidant efficacy of topically applied resveratrol. Arch Dermatol Res. PubMedCrossRefGoogle Scholar
  2. 2.
    Alonso C, Ramon E, Lozano C, Parra JL, Torres JL, Coderch L (2004) Percutaneous absorption of flavan-3-ol conjugates from plant procyanidins. Drugs Exp Clin Res 30:1–10PubMedGoogle Scholar
  3. 3.
    Amr S, Brown MB, Martin GP, Forbes B (2001) Activation of clindamycin phosphate by human skin. J Appl Microbiol 90:550–554. CrossRefPubMedGoogle Scholar
  4. 4.
    Baena JR, Lendl B (2004) Raman spectroscopy in chemical bioanalysis. Curr Opin Chem Biol 8:534–539. CrossRefPubMedGoogle Scholar
  5. 5.
    Bouwstra J, Gooris G, Ponec M (2002) The lipid organisation of the skin barrier: liquid and crystalline domains coexist in lamellar phases. J Biol Phys 28:211–223. CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Bronaugh RL, Stewart RF (1985) Methods for in vitro percutaneous absorption studies IV: the flow-through diffusion cell. J Pharm Sci 74:64–67. CrossRefPubMedGoogle Scholar
  7. 7.
    Bronaugh RL, Stewart RF, Congdon ER (1982) Methods for in vitro percutaneous absorption studies. II. Animal models for human skin. Toxi App Pharm 62:481–488. CrossRefGoogle Scholar
  8. 8.
    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 Investig Dermatol 116:434–442. CrossRefPubMedGoogle Scholar
  9. 9.
    Choe C, Lademann J, Darvin ME (2015) Analysis of human and porcine skin in vivo/ex vivo for penetration of selected oils by confocal raman microscopy. Skin Pharmacol Phys 28:318–330. CrossRefGoogle Scholar
  10. 10.
    Das RS, Agrawal YK (2011) Raman spectroscopy: recent advancements, techniques and applications. Vib Spectrosc 57:163–176. CrossRefGoogle Scholar
  11. 11.
    Dick IP, Scott RC (1992) Pig ear skin as an in-vitro model for human skin permeability. J Pharm Pharmacol 44:640–645CrossRefPubMedGoogle Scholar
  12. 12.
    Dinc E, Palabiyik IM, Ustundag O, Yurtsever F, Onur F (2002) Simultaneous spectrophotometric determination of chlorphenoxamine hydrochloride and caffeine in a pharmaceutical preparation using first derivative of the ratio spectra and chemometric methods. J Pharm Biomed Anal 28:591–600CrossRefPubMedGoogle Scholar
  13. 13.
    Forslind B (1994) A domain mosaic model of the skin barrier. Acta Derm Venereol 74:1–6PubMedGoogle Scholar
  14. 14.
    Franz TJ (1975) Percutaneous absorption. On the relevance of in vitro data. J Investig Dermatol 64:190–195. CrossRefPubMedGoogle Scholar
  15. 15.
    Franzen L, Anderski J, Windbergs M (2015) Quantitative detection of caffeine in human skin by confocal Raman spectroscopy—a systematic in vitro validation study. Eur J Pharm Biopharm 95(Part A):110–116. CrossRefPubMedGoogle Scholar
  16. 16.
    Franzen L, Selzer D, Fluhr JW, Schaefer UF, Windbergs M (2013) Towards drug quantification in human skin with confocal Raman microscopy. Eur J Pharm Biopharm 84:437–444. CrossRefPubMedGoogle Scholar
  17. 17.
    Franzen L, Windbergs M (2014) Accessing Raman spectral variability in human stratum corneum for quantitative in vitro depth profiling. J Raman Spectros 45:82–88. CrossRefGoogle Scholar
  18. 18.
    Hadgraft J (2001) Skin, the final frontier. Int J Pharm 224:1–18. CrossRefPubMedGoogle Scholar
  19. 19.
    Holmgaard R, Benfeldt E, Nielsen JB (2014) Percutaneous penetration—methodological considerations. Basic Clin Pharmacol Toxicol 115:101–109. CrossRefPubMedGoogle Scholar
  20. 20.
    Jacobi U, Kaiser M, Toll R, Mangelsdorf S, Audring H, Otberg N, Sterry W, Lademann J (2007) Porcine ear skin: an in vitro model for human skin. Skin Res Tech 13:19–24. CrossRefGoogle Scholar
  21. 21.
    Manca ML, Cencetti C, Matricardi P, Castangia I, Zaru M, Sales OD, Nacher A, Valenti D, Maccioni AM, Fadda AM, Manconi M (2016) Glycerosomes: use of hydrogenated soy phosphatidylcholine mixture and its effect on vesicle features and diclofenac skin penetration. Int J Pharm 511:198–204. CrossRefPubMedGoogle Scholar
  22. 22.
    Manca ML, Manconi M, Nacher A, Carbone C, Valenti D, Maccioni AM, Sinico C, Fadda AM (2014) Development of novel diolein–niosomes for cutaneous delivery of tretinoin: influence of formulation and in vitro assessment. Int J Pharm 477:176–186. CrossRefPubMedGoogle Scholar
  23. 23.
    Mateus R, Abdalghafor H, Oliveira G, Hadgraft J, Lane ME (2013) A new paradigm in dermatopharmacokinetics—confocal Raman spectroscopy. Int J Pharm 444:106–108. CrossRefPubMedGoogle Scholar
  24. 24.
    Mohammed D, Matts PJ, Hadgraft J, Lane ME (2014) In vitro-in vivo correlation in skin permeation. Pharm Res 31:394–400. CrossRefPubMedGoogle Scholar
  25. 25.
    Mujica Ascencio S, Choe C, Meinke MC, Müller RH, Maksimov GV, Wigger-Alberti W, Lademann J, Darvin ME (2016) Confocal Raman microscopy and multivariate statistical analysis for determination of different penetration abilities of caffeine and propylene glycol applied simultaneously in a mixture on porcine skin ex vivo. Eur J Pharm Biopharm 104:51–58. CrossRefPubMedGoogle Scholar
  26. 26.
    Nakagawa N, Matsumoto M, Sakai S (2010) In vivo measurement of the water content in the dermis by confocal Raman spectroscopy. Skin Res Tech 16:137–141. CrossRefGoogle Scholar
  27. 27.
    Norlen L (2001) Skin barrier formation: the membrane folding model. J Investig Dermatol 117:823–829. CrossRefPubMedGoogle Scholar
  28. 28.
    Norlen L (2001) Skin barrier structure and function: the single gel phase model. J Investig Dermatol 117:830–836. CrossRefPubMedGoogle Scholar
  29. 29.
    OECD (2004) Test No. 428: skin absorption: in vitro method. OECD Publishing, ParisGoogle Scholar
  30. 30.
    OECD (2011) Guidance notes on dermal absorption. Series on testing and assessment (No. 156). OECD Publishing, ParisGoogle Scholar
  31. 31.
    Prusakiewicz JJ, Ackermann C, Voorman R (2006) Comparison of skin esterase activities from different species. Pharm Res 23:1517–1524. CrossRefPubMedGoogle Scholar
  32. 32.
    Ramon E, Alonso C, Coderch L, De la Maza A, Lopez O, Parra JL, Notario I (2005) Liposomes as alternative vehicles for sun filter formulations. Drug Delivery 12:83–88. CrossRefPubMedGoogle Scholar
  33. 33.
    Redoules D, Perie J, Viode C, Mavon A, Fournier D, Daunes S, Casas C, Lougarre A, De Viguerie N (2005) Slow internal release of bioactive compounds under the effect of skin enzymes. J Investig Dermatol 125:270–277. CrossRefPubMedGoogle Scholar
  34. 34.
    Rougier A, Dupuis D, Lotte C, Rouguet R, Shaefer H (1983) In vivo correlation between stratum corneum reservoir function and percutaneous absorption. J Investig Derrmatol 81:275–278. CrossRefGoogle Scholar
  35. 35.
    Rubio L, Alonso C, López O, Rodríguez G, Coderch L, Notario J, de la Maza A, Parra JL (2011) Barrier function of intact and impaired skin: percutaneous penetration of caffeine and salicylic acid. Int J Dermatol 50:881–889. CrossRefPubMedGoogle Scholar
  36. 36.
    Rubio L, Alonso C, Coderch L, Parra JL, Martí M, Cebrián J, Navarro JA, Lis M, Valldeperas J (2010) Skin delivery of caffeine contained in biofunctional textiles. Tex Res J 80:1214–1221. CrossRefGoogle Scholar
  37. 37.
    Sato K, Sugibayashi K, Morimoto Y (1991) Species differences in percutaneous absorption of nicorandil. J Pharm Sci 80:104–107CrossRefPubMedGoogle Scholar
  38. 38.
    Sieg A, Crowther J, Blenkiron P, Marcott C, Matts PJ (2006) Confocal Raman microspectroscopy: measuring the effects of topical moisturizers on stratum corneum water gradient in vivo. In: SPIE BiOS, 2006, Biomedical Vibrational Spectroscopy III: Adv Res Ind, p 7.
  39. 39.
    Simon GA, Maibach HI (2000) The pig as an experimental animal model of percutaneous permeation in man: qualitative and quantitative observations—an overview. Skin Pharmacol Appl Skin Physiol 13:229–234. CrossRefPubMedGoogle Scholar
  40. 40.
    Tfaili S, Gobinet C, Josse G, Angiboust J-F, Manfait M, Piot O (2012) Confocal Raman microspectroscopy for skin characterization: a comparative study between human skin and pig skin. Analyst 137:3673–3682. CrossRefPubMedGoogle Scholar
  41. 41.
    Tfaili S, Josse G, Angiboust JF, Manfait M, Piot O (2014) Monitoring caffeine and resveratrol cutaneous permeation by confocal Raman microspectroscopy. J Biophotonics 7:676–681. CrossRefPubMedGoogle Scholar
  42. 42.
    Tfayli A, Piot O, Pitre F, Manfait M (2007) Follow-up of drug permeation through excised human skin with confocal Raman microspectroscopy. Eur Biophys J36:1049–1058. CrossRefGoogle Scholar
  43. 43.
    Thiele JJ (2001) Oxidative targets in the stratum corneum. A new basis for antioxidative strategies. Skin Pharmacol Appl Skin Physiol 14(Suppl 1):87–91. CrossRefPubMedGoogle Scholar
  44. 44.
    Tippavajhala VK, de Oliveira Mendes T, Martin AA (2017) In Vivo Human Skin Penetration Study of Sunscreens by Confocal Raman Spectroscopy. AAPS PharmSciTech. PubMedCrossRefGoogle Scholar
  45. 45.
    Trommer H, Neubert RH (2006) Overcoming the stratum corneum: the modulation of skin penetration. A review. Skin Pharmacol Physiol 19:106–121. CrossRefPubMedGoogle Scholar
  46. 46.
    Wertz PW (2000) Lipids and barrier function of the skin. Acta dermato venereologica Supplementum 208:7–11. CrossRefPubMedGoogle Scholar
  47. 47.
    Wester RC, Maibach HI (2001) In vivo methods for percutaneous absorption measurements. J Toxicol Cutan Ocul Toxicol 20:411–422. CrossRefGoogle Scholar
  48. 48.
    Zhang G, Moore DJ, Sloan KB, Flach CR, Mendelsohn R (2007) Imaging the prodrug-to-drug transformation of a 5-fluorouracil derivative in skin by confocal Raman microscopy. J Investig Dermatol 127:1205–1209. CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Cristina Alonso
    • 1
    Email author
  • V. Carrer
    • 1
  • C. Barba
    • 1
  • L. Coderch
    • 1
  1. 1.Department of Chemicals and Surfactants TechnologyAdvanced Chemical Institute of Catalonia, (IQAC-CSIC)BarcelonaSpain

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