Name: Visualisierung und Quantifizierung pharmazeutischer Verbindungen in der Haut mittels kohärenter Raman-Streubildgebung (Video) | JoVE Journal
Uploaded: 2021-11-24T13:00:00.000+00:00
Duration: 11 min 7 s
Description: Lernen Sie die kohärente Raman-Streubildgebung zur Visualisierung und Quantifizierung pharmazeutischer Verbindungen in menschlicher und Mäusehaut. Diese Methode ermöglicht präzise pharmakokinetische Analysen für die gezielte Arzneimittelentwicklung.

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The authors would like to thank Dr. Fotis Iliopoulos and Daniel Greenfield of the Evans&#39; Group for their discussion and proofreading of this manuscript. In addition, the authors would like to acknowledge support from LEO Pharma. Figure 2 was created with BioRender.com.

<ol><li>Finnin, B., Walters, K. A., Franz, T. J. In vitro skin permeation methodology. In Transdermal and topical drug delivery: principles and methodology. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aFinnin%2C+B+%22Transdermal+and+topical+drug+delivery%3A+principles+and+practice%22">Transdermal and topical drug delivery: principles and practice</a>. Benson, H. E., Watkinson, A. C. , John Wiley & Sons. Hoboken NJ USA. 85-108 (2012).</li>
<li>Shin, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((On+the+road+to+development+of+an+in+vitro+permeation+test+%28IVPT%29+model+to+compare+heat+effects+on+transdermal+delivery+systems%3A+exploratory+studies+with+nicotine+and+fentanyl%5BTitle%5D)+AND+%22Pharmaceutical+Research%22%5BJournal%5D)">On the road to development of an in vitro permeation test (IVPT) model to compare heat effects on transdermal delivery systems: exploratory studies with nicotine and fentanyl.</a> Pharmaceutical Research. 34 (9), 1817-1830 (2017).</li>
<li>Hossain, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Preparation%2C+characterisation%2C+and+topical+delivery+of+terbinafine%5BTitle%5D)+AND+%22Pharmaceutics%22%5BJournal%5D)">Preparation, characterisation, and topical delivery of terbinafine.</a> Pharmaceutics. 11 (10), 548(2019).</li>
<li>Santos, L. L., Swofford, N. J., Santiago, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+permeation+test+%28IVPT%29+for+pharmacokinetic+assessment+of+topical+dermatological+formulations%5BTitle%5D)+AND+%22Current+Protocols+in+Pharmacology%22%5BJournal%5D)">In vitro permeation test (IVPT) for pharmacokinetic assessment of topical dermatological formulations.</a> Current Protocols in Pharmacology. 91 (1), 79(2020).</li>
<li>Iliopoulos, F., Caspers, P. J., Puppels, G. J., Lane, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Franz+cell+diffusion+testing+andquantitative+confocal+Raman+spectroscopy%3A+In+vitro-in+vivo+correlation%5BTitle%5D)+AND+%22Pharmaceutics%22%5BJournal%5D)">Franz cell diffusion testing andquantitative confocal Raman spectroscopy: In vitro-in vivo correlation.</a> Pharmaceutics. 12 (9), 887(2020).</li>
<li>Cordery, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Topical+bioavailability+of+diclofenac+from+locally-acting%2C+dermatological+formulations%5BTitle%5D)+AND+%22International+Journal+of+Pharmaceutics%22%5BJournal%5D)">Topical bioavailability of diclofenac from locally-acting, dermatological formulations.</a> International Journal of Pharmaceutics. 529 (1-2), 55-64 (2017).</li>
<li>Pensado, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stratum+corneum+sampling+to+assess+bioequivalence+between+topicalacyclovir+products%5BTitle%5D)+AND+%22Pharmaceutical+Research%22%5BJournal%5D)">Stratum corneum sampling to assess bioequivalence between topicalacyclovir products.</a> Pharmaceutical Research. 36 (12), 1-16 (2019).</li>
<li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dermal+delivery+of+niacinamide-in+vivo+studies%5BTitle%5D)+AND+%22Pharmaceutics%22%5BJournal%5D)">Dermal delivery of niacinamide-in vivo studies.</a> Pharmaceutics. 13 (5), 726(2021).</li>
<li>Bodenlenz, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Open+flow+microperfusion+as+a+dermal+pharmacokinetic+approach+to+evaluate+topical+bioequivalence%5BTitle%5D)+AND+%22Clinical+Pharmacokinetics%22%5BJournal%5D)">Open flow microperfusion as a dermal pharmacokinetic approach to evaluate topical bioequivalence.</a> Clinical Pharmacokinetics. 56 (1), 91-98 (2017).</li>
<li>Eirefelt, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluating+dermal+pharmacokinetics+and+pharmacodymanic+effect+of+soft+topical+PDE4+inhibitors%3AOpen+flow+microperfusion+and+skin+biopsies%5BTitle%5D)+AND+%22Pharmaceutical+Research%22%5BJournal%5D)">Evaluating dermal pharmacokinetics and pharmacodymanic effect of soft topical PDE4 inhibitors:Open flow microperfusion and skin biopsies.</a> Pharmaceutical Research. 37 (12), 1-12 (2020).</li>
<li>Stagni, G., O'Donnell, D., Liu, Y. J., Kellogg, J. D. L., Shepherd, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Iontophoretic+current+and+intradermal+microdialysis+recovery+in+humans%5BTitle%5D)+AND+%22Journal+of+Pharmacological+and+Toxicological+Methods%22%5BJournal%5D)">Iontophoretic current and intradermal microdialysis recovery in humans.</a> Journal of Pharmacological and Toxicological Methods. 41 (1), 49-54 (1999).</li>
<li>Garcia Ortiz, P., Hansen, S. H., Shah, V. P., Menne, T., Benfeldt, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Impact+of+adultatopic+dermatitis+on+topical+drug+penetration%3A+assessment+by+cutaneous+microdialysis+and+tape+stripping%5BTitle%5D)+AND+%22Acta+Dermato-Venereologica%22%5BJournal%5D)">Impact of adultatopic dermatitis on topical drug penetration: assessment by cutaneous microdialysis and tape stripping.</a> Acta Dermato-Venereologica. 89 (1), 33-38 (2009).</li>
<li>Joshi, A., Patel, H., Joshi, A., Stagni, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pharmacokinetic+applications+of+cutaneous+microdialysis%3A+Continuous%2Bintermittent+vs+continuous-only+sampling%5BTitle%5D)+AND+%22Journal+of+Pharmacological+and+Toxicological+Methods%22%5BJournal%5D)">Pharmacokinetic applications of cutaneous microdialysis: Continuous+intermittent vs continuous-only sampling.</a> Journal of Pharmacological and Toxicological Methods. 83, 16-20 (2017).</li>
<li>Kuzma, B. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+local+bioavailability+of+metronidazole+from+topical+formulations+using+dermal+microdialysis%3A+Preliminary+study+in+a+Yucatan+mini-pig+model%5BTitle%5D)+AND+%22European+Journal+of+Pharmaceutical+Sciences%22%5BJournal%5D)">Evaluation of local bioavailability of metronidazole from topical formulations using dermal microdialysis: Preliminary study in a Yucatan mini-pig model.</a> European Journal of Pharmaceutical Sciences. 159, 105741(2021).</li>
<li>Begley, R., Harvey, A., Byer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((L.Coherent+anti-Stokes+Raman+spectroscopy%5BTitle%5D)+AND+%22Applied+Physics+Letters%22%5BJournal%5D)">L.Coherent anti-Stokes Raman spectroscopy.</a> Applied Physics Letters. 25 (7), 387-390 (1974).</li>
<li>Evans, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chemical+imaging+of+tissue+in+vivo+with+video-rate+coherent+anti-Stokes+Raman+scattering+microscopy%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Chemical imaging of tissue in vivo with video-rate coherent anti-Stokes Raman scattering microscopy.</a> Proceedings of the National Academy of Sciences of the United States of America. 102 (46), 16807-16812 (2005).</li>
<li>Hill, A. H., Manifold, B., Fu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tissue+imaging+depth+limit+of+stimulated+Raman+scattering+microscopy%5BTitle%5D)+AND+%22Biomedical+Optics+Express%22%5BJournal%5D)">Tissue imaging depth limit of stimulated Raman scattering microscopy.</a> Biomedical Optics Express. 11 (2), 762-774 (2020).</li>
<li>Feizpour, A., Marstrand, T., Bastholm, L., Eirefelt, S., Evans, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Label-free+quantification+of+pharmacokinetics+in+skin+with+stimulated+Raman+scattering+microscopy+and+deep+learning%5BTitle%5D)+AND+%22Journal+of+Investigative+Dermatology%22%5BJournal%5D)">Label-free quantification of pharmacokinetics in skin with stimulated Raman scattering microscopy and deep learning.</a> Journal of Investigative Dermatology. 141 (2), 395-403 (2021).</li>
<li>Ghosh, B., Reddy, L. H., Kulkarni, R. V., Khanam, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+skin+permeability+of+drugs+in+mice+and+human+cadaver+skin%5BTitle%5D)+AND+%22Indian+Journal+of+Experimental+Biology%22%5BJournal%5D)">Comparison of skin permeability of drugs in mice and human cadaver skin.</a> Indian Journal of Experimental Biology. 38 (1), 42-45 (2000).</li>
<li>Nielsen, J. B., Plasencia, I., Sørensen, J. A., Bagatolli, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Storage+conditions+of+skin+affect+tissue+structure+and+subsequent+in+vitro+percutaneous+penetration%5BTitle%5D)+AND+%22Skin+Pharmacology+and+Physiology%22%5BJournal%5D)">Storage conditions of skin affect tissue structure and subsequent in vitro percutaneous penetration.</a> Skin Pharmacology and Physiology. 24 (2), 93-102 (2011).</li>
<li>Barbero, A. M., Frasch, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+frozen+human+epidermis+storage+duration+and+cryoprotectant+on+barrier+function+using+two+model+compounds%5BTitle%5D)+AND+%22Skin+Pharmacology+and+Physiology%22%5BJournal%5D)">Effect of frozen human epidermis storage duration and cryoprotectant on barrier function using two model compounds.</a> Skin Pharmacology and Physiology. 29 (1), 31-40 (2016).</li>
<li>Babu, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+influence+of+various+methods+of+cold+storage+of+skin+on+the+permeation+of+melatonin+and+nimesulide%5BTitle%5D)+AND+%22Journal+of+Controlled+Release%22%5BJournal%5D)">The influence of various methods of cold storage of skin on the permeation of melatonin and nimesulide.</a> Journal of Controlled Release. 86 (1), 49-57 (2003).</li>
<li>Skelly, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((FDA+and+AAPS+report+of+the+workshop+on+principles+and+practices+of+in+vitro+percutaneous+penetration+studies%3A+relevance+to+bioavailability+and+bioequivalence%5BTitle%5D)+AND+%22Pharmaceutical+Research%22%5BJournal%5D)">FDA and AAPS report of the workshop on principles and practices of in vitro percutaneous penetration studies: relevance to bioavailability and bioequivalence.</a> Pharmaceutical Research. 4 (3), 265-267 (1987).</li>
<li>OECD. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Guidance+document+for+the+conduct+of+skin+absorption+studies%5BTitle%5D)+AND+%22OECD%22%5BJournal%5D)">Guidance document for the conduct of skin absorption studies.</a> OECD. , (2004).</li>
<li>OECD. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Test+no.+428%3A+Skin+absorption%3A+In+vitro+method%5BTitle%5D)+AND+%22OECD%22%5BJournal%5D)">Test no. 428: Skin absorption: In vitro method.</a> OECD. , (2004).</li>
<li>Saar, B. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Video-rate+molecular+imaging+in+vivo+with+stimulated+Raman+scattering%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Video-rate molecular imaging in vivo with stimulated Raman scattering.</a> Science. 330 (6009), 1368-1370 (2010).</li>
<li>Saar, B. G., Contreras-Rojas, L. R., Xie, X. S., Guy, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imaging+drug+delivery+to+skin+with+stimulated+Raman+scattering+microscopy%5BTitle%5D)+AND+%22Molecular+Pharmaceutics%22%5BJournal%5D)">Imaging drug delivery to skin with stimulated Raman scattering microscopy.</a> Molecular Pharmaceutics. 8 (3), 969-975 (2011).</li>
<li>Pence, I. J., Kuzma, B. A., Brinkmann, M., Hellwig, T., Evans, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multi-windowsparse+spectral+sampling+stimulated+Raman+scattering+microscopy%5BTitle%5D)+AND+%22Biomedical+Optics+Express%22%5BJournal%5D)">Multi-windowsparse spectral sampling stimulated Raman scattering microscopy.</a> Biomedical Optics Express. 12 (10), 6095-6114 (2021).</li>
<li>Herkenne, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+methods+for+the+assessment+of+topical+drug+bioavailability%5BTitle%5D)+AND+%22Pharmaceutical+Research%22%5BJournal%5D)">In vivo methods for the assessment of topical drug bioavailability.</a> Pharmaceutical Research. 25 (1), 87-103 (2008).</li>
<li>Alfonso-Garcıa, A., Mittal, R., Lee, E. S., Potma, E. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biological+imaging+with+coherent+Raman+scattering+microscopy%3A+a+tutorial%5BTitle%5D)+AND+%22Journal+of+Biomedical+Optics%22%5BJournal%5D)">Biological imaging with coherent Raman scattering microscopy: a tutorial.</a> Journal of Biomedical Optics. 19 (7), 071407(2014).</li>
<li>Osseiran, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Longitudinal+monitoring+of+cancer+cell+subpopulations+in+monolayers%2C+3D+spheroids%2C+and+xenografts+using+the+photoconvertible+dye+DiR%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Longitudinal monitoring of cancer cell subpopulations in monolayers, 3D spheroids, and xenografts using the photoconvertible dye DiR.</a> Scientific Reports. 9 (1), 1-10 (2019).</li>
<li>Evennett, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Kohler+illumination%3A+a+simple+interpretation%5BTitle%5D)+AND+%22Proceedings+of+the+Royal+Microscopical+Society%22%5BJournal%5D)">Kohler illumination: a simple interpretation.</a> Proceedings of the Royal Microscopical Society. 28 (4), 189-192 (1983).</li>
<li>Sanderson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fundamentals+of+microscopy%5BTitle%5D)+AND+%22Current+Protocols+in+Mouse+Biology%22%5BJournal%5D)">Fundamentals of microscopy.</a> Current Protocols in Mouse Biology. 10 (2), 76(2020).</li>
<li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fiji%3A+an+open-source+platform+for+biological-image+analysis%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li>
<li>Hunter, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Matplotlib%3A+A+2D+graphics+environment%5BTitle%5D)+AND+%22Computing+in+Science+%26+Engineering%22%5BJournal%5D)">Matplotlib: A 2D graphics environment.</a> Computing in Science & Engineering. 9 (3), 90-95 (2007).</li>
<li>Wickham, H. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aWickham%2C+H+%22ggplot2%3A+Elegant+Graphics+for+Data+Analysis%22">ggplot2: Elegant Graphics for Data Analysis</a>. , Springer-Verlag. New York. (2016).</li>
<li>Kim, H., Han, S., Cho, Y. S., Yoon, S. K., Bae, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+R+packages%3A%27Non-Compart%27+and+%27ncar%27+for+noncompartmental+analysis+%28NCA%29%5BTitle%5D)+AND+%22Translational+and+Clinical+Pharmacology%22%5BJournal%5D)">Development of R packages:'Non-Compart' and 'ncar' for noncompartmental analysis (NCA).</a> Translational and Clinical Pharmacology. 26 (1), 10-15 (2018).</li>
</ol>

CLE is an inventor on patents for CARS microscopy that have been licensed to multiple microscope manufacturers. All other authors have no conflicts of interest to disclose.

The evaluation of topical BA/BE is an area of research the requires a multifaceted approach as no single method can fully characterize in vivo cPK. This protocol presents a methodology for the evaluation of a topical drug product&#39;s BA/BE based on coherent Raman imaging. One of the first points that might be overlooked is how thin the skin samples must be, especially for quantitative transmission SRS imaging. If the skin is too thick (i.e., light cannot readily pass through), there is little to no si...

Methodologies to assess cPK after topical drug product application have expanded from classical in vitro permeation testing (IVPT) studies1,2,3,4,5 and tape-stripping6,7,8 to additional methodologies such as open-flow microperfusion or dermal microdialysis9,10,11,12,13,14. There are potentially various local sites of therapeutic action depending on the disease of interest. Hence, there may be a corresponding number of methodologies to assess the rate and extent to which an API gets to the intended local site of action. While each of the aforementioned methodologies has its advantages, the major disadvantage is the lack of microscale cPK information (i.e., the inability to visualize where the API goes and how it permeates).
One noninvasive methodology of interest to estimate topical BA and BE is CRI, which can be broken down into two imaging modalities: CARS and SRS microscopy. These coherent Raman methods enable chemically specific imaging of molecules via nonlinear Raman effects. In CRI, two laser pulse trains are focused and scanned within a sample; the difference in energy between the laser frequencies is set to target vibrational modes specific to the chemical structures of interest. As CRI processes are nonlinear, a signal is only generated at the microscope focus, allowing for three-dimensional pharmacokinetic tomographic imaging of the tissue. In the context of cPK, CARS has been used to obtain tissue structural information, such as the location of lipid-rich skin structures15. In contrast, SRS has been utilized to quantify molecular concentration as its signal is linear with concentration. For ex vivo skin specimens, it is advantageous to carry out CARS in the epi-direction16 and SRS in transmission mode17. Therefore, tissue samples that are thin will allow for SRS signal detection and quantification.
As a model tissue, the nude mouse ear presents several advantages with minor drawbacks. One advantage is that the tissue is already ~200-300 &#181;m in thickness and does not require further sample preparation. In addition, several skin stratifications are seen by axially focusing through one field of view (e.g., stratum corneum, sebaceous glands (SGs), adipocytes, and subcutaneous fat)16,18. This allows for preliminary preclinical estimation of cutaneous permeation pathways and topical BA estimates before moving to human skin samples. However, the nude mouse model presents limitations such as difficulty in extrapolation to in vivo scenarios due to differences in skin structure19. While the nude mouse ear is an excellent model to obtain preliminary results, the human skin model is the gold standard. Although there have been various commentaries on the suitability and applicability of frozen human skin to accurately recapitulate in vivo permeation kinetics20,21,22, the use of frozen human skin is an accepted method for the evaluation of in vitro&#160;API permeation kinetics23,24,25. This protocol visualizes various skin layers in mouse and human skin while quantifying API concentrations within lipid-rich and lipid-poor structures.
While CRI has been utilized across numerous fields to specifically visualize compounds within tissues, there have been limited efforts investigating the cPK of topically applied drug products. To evaluate the topical BA/BE of topical products using CRI, it is necessary to first have a standardized protocol in place to make accurate comparisons. Previous efforts using CRI for drug delivery to the skin have demonstrated variability within the data. As this is a relatively new application of CRI, establishing a protocol is critical to obtain reliable results18,26,27. This approach only targets one specific wavenumber in the biological silent region of the Raman spectrum. However, most APIs and inactive ingredients have Raman shifts within the fingerprint region. This has previously posed challenges due to the inherent signal arising from the tissue in the fingerprint region. Recent laser and computational advances have removed this barrier, which can also be utilized in combination with the approach presented here28. This approach presented here allows for the quantification of an API, which has a Raman shift in the silent region (2,000-2,300 cm-1). This is not limited to the physiochemical properties of the drug, which might be the case for some previously mentioned cPK monitoring methodologies29.
The protocol must reduce sample-to-sample variability in skin thickness for various preparations, as thick human skin samples will produce minimal signal after drug product application due to light scattering by the thick sample. A goal of this manuscript is to present a tissue preparation methodology that assures reproducible imaging standards. In addition, the CRI system is setup as described to reduce potential sources of error as well as minimize signal-to-noise.&#160;However, this paper will not discuss the guiding principles and technical merits of the CRI microscope as this has been previously covered30. Finally, the extensive data analysis procedure is explored to allow for interpretation of the results to determine an experiment&#39;s success or failure.

<table><tbody><tr><td>&lt;strong&gt;Tissue Preparation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Autoclavable Biohazard Bags</td><td>FisherBrand</td><td>22-044562</td><td>As refered to in text: biohazard bags&lt;br/&gt; https://www.fishersci.com/shop/products/fisherbrand-polyethylene-biohazard-autoclave-bags-without-sterilization-indicator-8/22044562?searchHijack=true&amp;amp;searchTerm= 22044562&amp;amp;searchType=RAPID&amp;amp; matchedCatNo=22044562</td></tr><tr><td>Cell Culture Buffers: Dulbecco&#039;s Phosphate-Buffered Salt Solution 1x</td><td>Corning</td><td>MT21030CV</td><td>As refered to in text: PBS&lt;br/&gt; https://www.fishersci.com/shop/products/corning-cellgro-cell-culture-buffers-dulbecco-s-phosphate-buffered-salt-solution-1x-8/MT21030CV?searchHijack=true&amp;amp;searchTerm= 21-030-cv&amp;amp;searchType= RAPID&amp;amp;matchedCatNo=21-030-cv</td></tr><tr><td>Disposable Scalpels</td><td>Exel International</td><td>14-840-00</td><td>As refered to in text: scalpel&lt;br/&gt; https://www.fishersci.com/shop/products/exel-international-disposable-scalpels-3/1484000?keyword=true</td></tr><tr><td>High Precision 45&amp;deg; Angle Broad Point Tweezers/Forceps</td><td>Fisherbrand</td><td>12-000-132</td><td>As refered to in text: forceps&lt;br/&gt; https://www.fishersci.com/shop/products/high-precision-45-angle-broad-point-tweezers-forceps/12000132#?keyword=</td></tr><tr><td>Kimwipes Delicate Task Wipers, 1-Ply</td><td>Kimberly-Clark Professional Kimtech Science</td><td>06-666</td><td>As refered to in text: task wiper&lt;br/&gt; https://www.fishersci.com/shop/products/kimberly-clark-kimtech-science-kimwipes-delicate-task-wipers-7/06666</td></tr><tr><td>Parafilm M Laboratory Wrapping Film</td><td>Bemis</td><td>13-374-12</td><td>As refered to in text: parafilm&lt;br/&gt; https://www.fishersci.com/shop/products/curwood-parafilm-m-laboratory-wrapping-film-4/1337412</td></tr><tr><td>Petri Dish (35 mm x 10 mm)</td><td>Fisherbrand</td><td>FB0875711YZ</td><td>As refered to in text: small petri dish&lt;br/&gt; https://www.fishersci.com/shop/products/fisherbrand-petri-dishes-specialty-6/FB0875711YZ?keyword=true</td></tr><tr><td>Petri Dish (60 mm x 15 mm)</td><td>Fisherbrand</td><td>FB0875713A</td><td>As refered to in text: large petri dish&lt;br/&gt; https://www.fishersci.com/shop/products/fisherbrand-petri-dishes-clear-lid-12/FB0875713A?keyword=true</td></tr><tr><td>Surgical Scissors</td><td>Roboz</td><td>NC9411473</td><td>As refered to in text: scissors&lt;br/&gt; https://www.fishersci.com/shop/products/scissors-327/NC9411473?searchHijack=true&amp;amp;searchTerm= RS-5915SC&amp;amp;searchType=RAPID&amp;amp; matchedCatNo=RS-5915SC</td></tr><tr><td>&lt;strong&gt;Laser/microscope&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>650/60 nm BrightLine single-band bandpass filter</td><td>Semrock</td><td></td><td>As refered to in text: CARS filter - CH2 vibrations (645nm/60nm filter)</td></tr><tr><td>Control box IX2-UCB</td><td>Olympus</td><td></td><td>As refered to in text: Control Box</td></tr><tr><td>D700/30m</td><td>Chroma</td><td></td><td>As refered to in text: CARS filter - deuterated band&lt;br/&gt; https://www.chroma.com/products/parts/d700-30m</td></tr><tr><td>DeepSee Insight</td><td>Spectra-Physics</td><td></td><td>As refered to in text: Laser&lt;br/&gt; https://www.spectra-physics.com/f/insight-x3-tunable-laser</td></tr><tr><td>Digital Handheld Optical Power and Energy Meter Console</td><td>ThorLabs</td><td>PM100D</td><td>As refered to in text: power meter&lt;br/&gt; https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=3341</td></tr><tr><td>Fluoview Software</td><td>Olympus</td><td></td><td>As refered to in text: Microscope Control software</td></tr><tr><td>Frosted Microscope Slides</td><td>FisherBrand</td><td></td><td>As refered to in text: microscope slides&lt;br/&gt; https://www.fishersci.com/shop/products/fisherbrand-frosted-microscope-slides-4/22265446</td></tr><tr><td>FV1000</td><td>Olympus</td><td></td><td>As refered to in text: Microscope</td></tr><tr><td>Incubation Chamber</td><td>Tokai Hit</td><td>GM-800</td><td>As refered to in text: incubation chamber</td></tr><tr><td>Integrating Sphere Photodiode Power Sensor</td><td>ThorLabs</td><td>S142C</td><td>As refered to in text: photodiode&lt;br/&gt; https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=3341</td></tr><tr><td>Power supply FV31-PSU</td><td>Olympus</td><td></td><td>As refered to in text: Power Supply</td></tr><tr><td>Precision 4063, 80MHz Dual Channel Function Generator</td><td>BK Precision</td><td></td><td>As refered to in text: function generator</td></tr><tr><td>ProScan &amp;ndash; Precision Microscope Automation</td><td>Prior Scientific Instruments</td><td></td><td>As refered to in text: stage controller&lt;br/&gt; https://www.prior.com/microscope-automation/inverted-microscope-systems/proscan-linear-stage-highest-precision-microscope-automation</td></tr><tr><td>SecureSeal Imaging Spacers</td><td>Grace Biolabs</td><td>654004</td><td>As refered to in text: spacer&lt;br/&gt; https://gracebio.com/product/secureseal-imaging-spacers-654004/</td></tr><tr><td>SRS Detection Kit</td><td>APE</td><td></td><td>As refered to in text: SRS detector</td></tr><tr><td>UPLSAPO 20X NA:0.75</td><td>Olympus</td><td></td><td>As refered to in text: 20X Objective&lt;br/&gt; https://www.olympus-lifescience.com/en/objectives/uplsapo/</td></tr><tr><td>&lt;strong&gt;Lipid/Drug Imaging&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;35 mm Dish, No. 0 Uncoated Coverslip, 14 mm Glass Diameter</td><td>MatTek Corporation</td><td>NC9711297</td><td>As refered to in text: Glass bottom dish&lt;br/&gt; https://www.fishersci.com/shop/products/glass-bottom-mircrowell-dish/nc9711297</td></tr><tr><td>Cotton-tipped applicators</td><td>FisherBrand</td><td></td><td>As refered to in text: Cotton-tipped applicator</td></tr><tr><td>Distriman Postive Displacement Pipette</td><td>Gilson</td><td></td><td>As refered to in text: Postive Displacement Pipette&lt;br/&gt; https://www.fishersci.com/shop/products/gilson-distriman-positive-displacement-repetitive-pipette/F164001G#?keyword=</td></tr><tr><td>Distriman Postive Displacement Pipette Tips</td><td>Gilson</td><td></td><td>As refered to in text: Tips for pipette&lt;br/&gt; https://www.fishersci.com/shop/products/gilson-distritip-syringes-6/f164100g?keyword=true</td></tr><tr><td>&lt;strong&gt;Data Analysis&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>FIJI</td><td>Open-source</td><td></td><td>As refered to in text: FIJI/ImageJ&lt;br/&gt; https://imagej.net/software/fiji/</td></tr><tr><td>Jupyter-Lab</td><td>open-source</td><td></td><td>As refered to in text: JupyterLab&lt;br/&gt; https://jupyter.org/</td></tr><tr><td>Rstudio</td><td>Open-source</td><td></td><td>As refered to in text: Rstudio&lt;br/&gt; https://www.rstudio.com/</td></tr></tbody></table>

visualizing and quantifying pharmaceutical compounds within skin using coherent raman scattering imaging

Visualizing and Quantifying Pharmaceutical Compounds within Skin using Coherent Raman Scattering Imaging

Cutaneous pharmacokinetics (cPK) after topical formulation application has been a research area of particular interest for regulatory and drug development scientists to mechanistically understand topical bioavailability (BA). Semi-invasive techniques, such as tape-stripping, dermal microdialysis, or dermal open-flow microperfusion, all quantify macroscale cPK. While these techniques have provided vast cPK knowledge, the community lacks a mechanistic understanding of active pharmaceutical ingredient (API) penetration and permeation at the cellular level. 
One noninvasive approach to address microscale cPK is coherent Raman scattering imaging (CRI), which selectively targets intrinsic molecular vibrations without the need for extrinsic labels or chemical modification. CRI has two main methods-coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS)-that enable sensitive and selective quantification of APIs or inactive ingredients. CARS is typically utilized to derive structural skin information or visualize chemical contrast. In contrast, the SRS signal, which is linear with molecular concentration, is used to quantify APIs or inactive ingredients within skin stratifications. 
Although mouse tissue has commonly been utilized for cPK with CRI, topical BA and bioequivalence (BE) must ultimately be assessed in human tissue before regulatory approval. This paper presents a methodology to prepare and image ex vivo skin to be used in quantitative pharmacokinetic CRI studies in the evaluation of topical BA and BE. This methodology enables reliable and reproducible API quantification within human and mouse skin over time. The concentrations within lipid-rich and lipid-poor compartments, as well as total API concentration over time are quantified; these are utilized for estimates of micro- and macroscale BA and, potentially, BE.

Imaging is considered successful if the tissue has not significantly moved in either axial (&lt;10 μm) or lateral direction upon the completion of the experiment (Figure 4). This is an immediate indication if the SRS measurement for the API of interest is not representative of the initial depth, for which quantification is layer-specific. This is mitigated by imaging z-stacks for each XY position of interest, with the trade-off being the temporal resolution. If frozen skin is used in these s...

A coherent Raman scattering imaging methodology to visualize and quantify pharmaceutical compounds within the skin is described. This paper describes skin tissue preparation (human and mouse) and topical formulation application, image acquisition to quantify spatiotemporal concentration profiles, and preliminary pharmacokinetic analysis to assess topical drug delivery.

Cutaneous pharmacokinetics (cPK) after topical formulation application has been a research area of particular interest for regulatory and drug development ...

This methodology is significant in that it allows for reproducible and accurate method to quantify topically-applied drug products using coherent Raman imaging. Other methodologies provide bulky cutaneous pharmacokinetic information, while this methodology can provide both bulk and microscale cutaneous pharmacokinetic information such as the route of permeation of a drug. The visualization and quantification and permeation pathways will allow the development of drug products with a specific pathway in mind to reach the target site.Tissue preparation will prove to be the most challenging part of this methodology. The tissue should be sufficiently thin so that one may be able to read through the skin. To begin the preparation of a nude mouse ear skin tissue, acquire euthanized nude mice and remove the ears of the mouse using forceps and microsurgical scissors.Then place one ear in a large Petri dish of size 60 millimeters by 15 millimeters. Next, rinse the mouse ear with PBS twice and pat the ear dry each time with the task wiper. If used within 24 hours, place the ear in PBS in a small, 35 millimeters by 10 millimeters Petri dish at two to eight degrees Celsius.To use after 24 hours

visualizing-quantifying-pharmaceutical-compounds-within-skin-using

Research

JoVE Journal

Medicine

A combination of three single wavelength short-pulsed lasers is used to generate coherent anti-Stokes Raman scattering (CARS) and doubly-resonant CARS (DR-CARS). The difference between these signals provides enhanced sensitivity for otherwise difficult to detect coherent Raman signals, enabling imaging of weak Raman scatterers.

Differential Imaging of Biological Structures with Doubly-resonant Coherent Anti-stokes Raman Scattering (CARS)

Coherent anti-Stokes Raman scattering (CARS) microscopy is combined with an intrinsic flow-through dissolution setup to allow in situ and real-time visualization of the surface of pharmaceutical tablets undergoing dissolution. Using this custom-built setup, it is possible to correlate CARS videos with drug dissolution profiles recorded using inline UV absorption spectroscopy.

Coherent anti-Stokes Raman Scattering (CARS) Microscopy Visualizes Pharmaceutical Tablets During Dissolution

Coherent anti-Stokes Raman scattering (CARS) microscopy based on inherent vibration of molecule bonds permits label-free chemically selective live cell imaging. This work presents the implementation of a complementary microscopy technique on a standard multiphoton laser scanning microscope based on a femtosecond Ti:sapphire laser and an OPO laser.

Implementation of a Coherent Anti-Stokes Raman Scattering (CARS) System on a Ti:Sapphire and OPO Laser Based Standard Laser Scanning Microscope

In this manuscript, the implementation of a stimulated Raman scattering (SRS) microscope, obtained by the integration of an SRS experimental set-up with a laser scanning microscope, is described. The SRS microscope is based on two femtosecond (fs) laser sources, a Ti-Sapphire (Ti:Sa) and synchronized optical parametric oscillator (OPO).

Implementation of a Nonlinear Microscope Based on Stimulated Raman Scattering

Here, we present a protocol for collection of confocal Raman spectra from human subjects in clinical studies combined with chemometric approaches for spectral outlier removal and the subsequent extraction of key features.

Resolving Water, Proteins, and Lipids from In Vivo Confocal Raman Spectra of Stratum Corneum through a Chemometric Approach

Stimulated Raman scattering (SRS) microscopy allows selective, label-free imaging of specific chemical moieties and it has been effectively employed to image lipid molecules in vivo. Here, we provide a brief introduction to the principle of SRS microscopy and describe methods for its use in imaging lipid storage in Caenorhabditis elegans.

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

This paper directly compares the resolution, sensitivity, and imaging contrasts of stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS) integrated into the same microscope platform. The results show that CARS has a better spatial resolution, SRS gives better contrasts and spectral resolution, and both methods have similar sensitivity.

Direct Comparison of Hyperspectral Stimulated Raman Scattering and Coherent Anti-Stokes Raman Scattering Microscopy for Chemical Imaging

We present a protocol to acquire chemical images with broadband stimulated Raman scattering (SRS) microscopy. Based on an SRS microscope that operates with differential multichannel-lock-in detection, the protocol describes the sample preparation, adjustment of the SRS apparatus, and chemometrics to disentangle different constituents of chemically heterogeneous samples.

Multiplex Chemical Imaging Based on Broadband Stimulated Raman Scattering Microscopy

This study presents a method for correlative imaging of drugs in soft tissues using nonlinear optical spectroscopy and mass spectrometry imaging. Combining high-resolution skin imaging with sensitive drug detection offers a valuable approach to study drug distribution in the skin, critical for the development of superior topical products.

Correlative Optical Spectroscopy and Mass Spectrometry Imaging Methodology to Visualise Drug Distribution in a Soft Tissue Section

Utilizing a multimodal platform combining label-free optical imaging modalities, we have developed a protocol for visualizing and quantifying cellular dynamics and metabolism. Through imaging via multiphoton fluorescence, second harmonic generation, and stimulated Raman scattering microscopy, we can generate a holistic overview of the cellular and molecular environment.

Multimodal Optical Imaging Platform for Studying Cellular Metabolism

Lernen Sie die kohärente Raman-Streubildgebung zur Visualisierung und Quantifizierung pharmazeutischer Verbindungen in menschlicher und Mäusehaut. Diese Methode ermöglicht präzise pharmakokinetische Analysen für die gezielte Arzneimittelentwicklung.

Visualizing and Quantifying Pharmaceutical Compounds within Skin using Coherent Raman Scattering Imaging

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