Abstract
Purpose. The purpose of the current study was to develop a new method to examine the diffusion in fresh unfixed human skin on-line.
Methods. Full thickness skin samples were cut perpendicular to the skin surface (cutting plane facing upwards) with a new cutting device forming part of the final diffusion cell. The donor solution contained 0.1 mg/ml Bodipy FL C5 (moderately lipophilic) dissolved in citric acid buffer, pH 5.0, and the acceptor phase consisted of phosphate-buffered saline, pH 7.4. Images were taken with confocal laser scanning microscopy (CLSM) every 10 min for 8 h.
Results. This new method enabled for the first time visualization of concentration profiles in different skin layers simultaneously as a function of time. For this model penetrant, Bodipy FL C5 showed that the lower stratum corneum layer constitutes the greatest barrier to diffusion. Furthermore, there is preferred partitioning of this probe in epidermis vs. either stratum corneum or dermis.
Conclusions. The on-line diffusion cell in combination with CLSM is a promising tool to study diffusion processes of dyes in fresh unfixed skin on-line. The method has the potential to access deeper skin layers as well as to visualize diffusion processes in cells.
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references
P. Corcuff and G. E. Pierard. Skin imaging: State of the art at the dawn of the year 2000. Skin Bioeng. 26:1–11 (1998).
E. Touitou, V. M. Meidan, and E. Horwitz. Methods for quantitative determination of drug localized in the skin. J. Control. Rel. 56:7–21 (1998).
F. Lund and T. Jogestrand. Video fluorescein imaging of the skin: description of an overviewing technique for functional evaluation of regional cutaneous blood perfusion in occlusive arterial disease of the limbs. Clin. Physiol. 17:619–633 (1997).
R. H. Bull, D. O. Bates, and P. S. Mortimer. Intravital video-capillaroscopy for the study of the microcirculation in psoriasis. Br. J. Dermatol. 126:436–445 (1992).
J. L. Morris. Cotransmission from sympathetic vasoconstrictor neurons to small cutaneous arteries in vivo. Am. J. Physiol. Heart Circ. Physiol. 46:H58-H64 (1999).
T. Salmon, R. A. Walker, and N. K. Pryer. Advances in microscopy-part III; video-enhanced differential interference contrast light microscopy. Biotechniques 7:624–633 (1989).
A. W. B. Stanton, H. S. Patel, J. R. Levick, and P. S. Mortimer. Increased dermal lymphatic density in the human leg compared with the forearm. Microvasc. Res. 57:320–328 (1999).
S. Richard, B. Querleux, J. Bittoun, I. Idy-Peretti, O. Jolivet, E. Cermakova, and J. L. Leveque. In vivo proton relaxation times analysis of the skin layers by magnetic resonance imaging. J. Invest. Dermatol. 97:120–125 (1991).
H. K. Song, F. W. Wehrli, and J. F. Ma. In vivo MR microscopy of the human skin. Magn. Reson. Med. 37:185–191 (1997).
M. Szayna and W. Kuhn. In vivo and in vitro investigations of hydration effects of beauty care products by high-field MRI and NMR microscopy. Eur. Acad. Dermatol. Venereol. 11:122–128 (1998).
T. Herrling, J. Fuchs, and N. Groth. Kinetic measurements using EPR imaging with a modulated field gradient. J. Magn. Reson. 154:6–14 (2002).
D. H. Turnbull, B. G. Starkoski, K. A. Harasiewicz, J. L. Semple, L. From, A. K. Gupta, D. N. Sauder, and F. S. Foster. 40–100 MHz B-SCAN ultrasound backscatter microscope for skin imaging. Ultrasound Med. Biol. 21:79–88 (1995).
P. J. Caspers, G. W. Lucassen, R. Wolthuis, H. A. Bruining, and G. J. Puppels. In vitro and in vivo Raman spectroscopy of human skin. Biospectroscopy 4:S31-S39 (1998).
P. J. Caspers, G. W. Lucassen, E. A. Carter, H. A. Bruining, and G. J. Puppels. In vivo confocal Raman microspectroscopy of the skin: noninvasive determination of molecular concentration profiles. J. Invest. Dermatol. 116:434–442 (2001).
D. Aghassi, R. R. Anderson, and S. Gonzalez. Time-sequence histologic imaging of laser-treated cherry angiomas with in vivo confocal microscopy. J. Am. Acad. Dermatol. 43:37–41 (2000).
C. Bertrand and P. Corcuff. In vivo spatio-temporal visualization of the human skin by real-time confocal microscopy. Scanning 16:150–154 (1994).
P. Corcuff, C. Bertrand, and J. L. Leveque. Morphometry of human epidermis in vivo by real-time confocal microscopy. Arch. Dermatol. Res. 285:475–481 (1993).
C. Cullander. Light microscopy of living tissue: the state and future of the art. J. Invest. Dermatol. Symp. Proc. 3:166–171 (1998).
B. S. Grewal, A. Naik, W. J. Irwin, G. Gooris, G. J. de-Grauw, H. G. Gerritsen, and J. A. Bouwstra. Transdermal macromolecular delivery: Real-time visualization of iontophoretic and chemically enhanced transport using two-photon excitation microscopy. Pharm. Res. 17:788–795 (2000).
A. J. Hoogstraate, C. Cullander, J. F. Nagelkerke, F. Spies, J. Verhoef, A. H. G. J. Schrijvers, H. E. Junginger, and H. E. Bodde. A novel in-situ model for continuous observation of transient drug concentration gradients across buccal epithelium at the microscopical level. J. Control. Rel. 39:71–78 (1996).
M. Rajadhyaksha, S. Gonzalez, J. M. Zavislan, R. R. Anderson, and R. H. Webb. In vivo confocal scanning laser microscopy of human skin II: advances in instrumentation and comparison with histology. J. Invest. Dermatol. 113:293–303 (1999).
M. E. M. J. Meuwissen, J. Janssen, C. Cullander, H. E. Junginger, and J. A. Bouwstra. A cross-section device to improve visualization of fluorescent probe penetration into the skin by confocal laser scanning microscopy. Pharm. Res. 15:352–356 (1998).
Y. Y. Grams and J. A. Bouwstra. A new method to determine the distribution of a fluorophore in scalp skin with focus on hair follicles. Pharm. Res. 19:350–354 (2002).
J. Karolin, L. B. A. Johansson, L. Strandberg, and T. Ny. Fluorescence and absorption spectroscopic properties of Dipyrrometheneboron difluoride (BODIPY) derivatives in liquids, lipid membranes, and proteins. J. Am. Chem. Soc. 116:7801–7806 (1994).
H. Okamoto, F. Yamashita, K. Saito, and M. Hashida. Analysis of drug penetration through the skin by the two-layer skin model. Pharm. Res. 6:931–937 (1989).
R. J. Scheuplein and L. W. Ross. Mechanism of percutaneous absorption. V. J. Invest. Dermatol. 62:353–360 (1974).
F. Yamashita, H. Bando, Y. Koyama, S. Kitagawa, Y. Takakura, and M. Hashida. In vivo and in vitro analysis of skin penetration enhancement based on a two-layer diffusion model with polar and nonpolar routes in the stratum corneum. Pharm. Res. 11:185–191 (1994).
H. Schaefer and T. E. Redelmeier. Skin Barrier: Principles of Percutaneous Absorption, Karger, Basel, 1996.
B. Yu, C. Y. Dong, P. T. So, D. Blankschtein, and R. Langer. In vitro visualization and quantification of oleic acid induced changes in transdermal transport using two-photon fluorescence microscopy. J. Invest. Dermatol. 117:16–25 (2001).
Y. Y. Grams and J. A. Bouwstra. Penetration and distribution of three lipophilic probes in vitro in human skin focusing on the hair follicle. J. Control. Rel. 83:253–262 (2002).
Y. Y. Grams, S. Alaruikka, L. Lashley, J. Caussin, L. Whitehead, and J. A. Bouwstra. Permeant lipophilicity and vehicle composition influence accumulation of dyes in hair follicles of human skin. Eur. J. Pharm. Sci. 18:329–336 (2003).
Y. H. Kim, A. H. Ghanem, and W. I. Higuchi. Model studies of epidermal permeability. Semin. Dermatol. 11:145–156 (1992).
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Grams, Y.Y., Whitehead, L., Cornwell, P. et al. On-Line Visualization of Dye Diffusion in Fresh Unfixed Human Skin. Pharm Res 21, 851–859 (2004). https://doi.org/10.1023/B:PHAM.0000026439.63969.30
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DOI: https://doi.org/10.1023/B:PHAM.0000026439.63969.30