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.

The use of nude mouse ear tissue was approved by Massachusetts General Hospital Institutional Animal Care and Use Committee (IACUC), while the use of human skin tissue was approved by the Massachusetts General Hospital Institutional Review Board (IRB). According to IACUC protocols, freshly euthanized mice were obtained from collaborators with nude mice colonies. Human tissue was procured from elective abdominoplasty procedures at Massachusetts General Hospital via an approved protocol. In addition, specific tissue types ...

<ol>
	<li>Finnin, B., Walters, K. A., Franz, T. J., Benson, H. E., Watkinson, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+skin+permeation+methodology.+In+Transdermal+and+topical+drug+delivery:+principles+and+methodology.">In vitro skin permeation methodology. In Transdermal and topical drug delivery: principles and methodology.</a> Transdermal and topical drug delivery: principles and practice. , 85-108 (2012).</li><li>Shin, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation,+characterisation,+and+topical+delivery+of+terbinafine.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+permeation+test+(IVPT)+for+pharmacokinetic+assessment+of+topical+dermatological+formulations.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Franz+cell+diffusion+testing+andquantitative+confocal+Raman+spectroscopy:+In+vitro-in+vivo+correlation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topical+bioavailability+of+diclofenac+from+locally-acting,+dermatological+formulations.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stratum+corneum+sampling+to+assess+bioequivalence+between+topicalacyclovir+products.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dermal+delivery+of+niacinamide-in+vivo+studies.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open+flow+microperfusion+as+a+dermal+pharmacokinetic+approach+to+evaluate+topical+bioequivalence.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+dermal+pharmacokinetics+and+pharmacodymanic+effect+of+soft+topical+PDE4+inhibitors:Open+flow+microperfusion+and+skin+biopsies.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iontophoretic+current+and+intradermal+microdialysis+recovery+in+humans.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+adultatopic+dermatitis+on+topical+drug+penetration:+assessment+by+cutaneous+microdialysis+and+tape+stripping.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacokinetic+applications+of+cutaneous+microdialysis:+Continuous+intermittent+vs+continuous-only+sampling.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+local+bioavailability+of+metronidazole+from+topical+formulations+using+dermal+microdialysis:+Preliminary+study+in+a+Yucatan+mini-pig+model.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L.Coherent+anti-Stokes+Raman+spectroscopy.">L.Coherent anti-Stokes Raman spectroscopy.</a> Applied Physics Letters. 25 (7), 387-390 (1974).</li><li>Evans, C. 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A., Bagatolli, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Storage+conditions+of+skin+affect+tissue+structure+and+subsequent+in+vitro+percutaneous+penetration.">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. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-windowsparse+spectral+sampling+stimulated+Raman+scattering+microscopy.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+methods+for+the+assessment+of+topical+drug+bioavailability.">In vivo methods for the assessment of topical drug bioavailability.</a> Pharmaceutical Research. 25 (1), 87-103 (2008).</li><li>Alfonso-Garc&#305;a, A., Mittal, R., Lee, E. S., Potma, E. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+imaging+with+coherent+Raman+scattering+microscopy:+a+tutorial.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longitudinal+monitoring+of+cancer+cell+subpopulations+in+monolayers,+3D+spheroids,+and+xenografts+using+the+photoconvertible+dye+DiR.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kohler+illumination:+a+simple+interpretation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fundamentals+of+microscopy.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Hunter, J. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Tissue Preparation</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 
			https://www.fishersci.com/shop/products/fisherbrand-polyethylene-biohazard-autoclave-bags-without-sterilization-indicator-8/22044562?searchHijack=true&#38;searchTerm= 22044562&#38;searchType=RAPID&#38; matchedCatNo=22044562</td>
		</tr>
		<tr>
			<td>Cell Culture Buffers: Dulbecco&#39;s Phosphate-Buffered Salt Solution 1x</td>
			<td>Corning</td>
			<td>MT21030CV</td>
			<td>As refered to in text: PBS 
			https://www.fishersci.com/shop/products/corning-cellgro-cell-culture-buffers-dulbecco-s-phosphate-buffered-salt-solution-1x-8/MT21030CV?searchHijack=true&#38;searchTerm= 21-030-cv&#38;searchType= RAPID&#38;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 
			https://www.fishersci.com/shop/products/exel-international-disposable-scalpels-3/1484000?keyword=true</td>
		</tr>
		<tr>
			<td>High Precision 45&#176; Angle Broad Point Tweezers/Forceps</td>
			<td>Fisherbrand</td>
			<td>12-000-132</td>
			<td>As refered to in text: forceps 
			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 
			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 
			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 
			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 
			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 
			https://www.fishersci.com/shop/products/scissors-327/NC9411473?searchHijack=true&#38;searchTerm= RS-5915SC&#38;searchType=RAPID&#38; matchedCatNo=RS-5915SC</td>
		</tr>
		<tr>
			<td>Laser/microscope</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 
			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 
			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 
			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 
			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 
			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 &#8211; Precision Microscope Automation</td>
			<td>Prior Scientific Instruments</td>
			<td></td>
			<td>As refered to in text: stage controller 
			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 
			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 
			https://www.olympus-lifescience.com/en/objectives/uplsapo/</td>
		</tr>
		<tr>
			<td>Lipid/Drug Imaging</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;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 
			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 
			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 
			https://www.fishersci.com/shop/products/gilson-distritip-syringes-6/f164100g?keyword=true</td>
		</tr>
		<tr>
			<td>Data Analysis</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 
			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 
			https://jupyter.org/</td>
		</tr>
		<tr>
			<td>Rstudio</td>
			<td>Open-source</td>
			<td></td>
			<td>As refered to in text: Rstudio 
			https://www.rstudio.com/</td>
		</tr>
	</tbody>
</table>

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 (&#60;10 &#956;m) 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 t...

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

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 of harvesting, place the ear in a Petri dish without PBS, then cover the dish with parafilm to store in a freezer at minus 20 degrees Celsius. While working under the biological hood, place the human skin tissue in a large Petri dish with the stratum corneum side facing down so that the subcutaneous fat is accessible. Then use forceps and microsurgical scissors to remove the subcutaneous fat.Once subcutaneous fat can no longer be removed with the scissors, use a 10 blade disposable scalpel at a 45-degree angle to the skin to remove the remaining subcutaneous fat while holding the skin still with forceps. When the fat is removed, section the human skin into one centimeter by one centimeter pieces. For lipid imaging, place the anterior portion of the mouse ear facing toward the glass bottom of a 35-millimeter, number zero dish.For the human skin, place the skin with the stratum corneum faced down to allow drug quantification from the superficial to the deeper layers. Once the tissue has been centered on the glass bottom, use a cotton tip applicator to ensure that the skin is flat and has complete contact with the cover slip surface of the glass bottom dish. To prevent any movement while imaging, place a washer on top of the skin and ensure that the tissue is visible through the center hole of the washer for Stimulated Raman Scattering or SRS transmission detection.Pipette the pre-determined dose of the formulation onto the skin, then use a gloved finger to rub the formulation clockwise for 30 seconds. After the allotted time for the formulation to permeate has elapsed, remove the excess formulation before placing the skin with the formulation side facing toward the glass bottom dish. Next, proceed to the experimental setup by turning on the Microscope Control or MC software.Looking through the eyepiece, adjust the axial focus with the adjustment knob to ensure that the tissue is within focus. When done, unblock both the pump and Stokes beams and confirm that ALG1 and ALG2 channels are enabled. Ensure the SRS photodiode is in position above the condenser, then click Focus x2 on the MC software to visualize the skin in the CARS and SRS channels.In the Multi-Area Time Lapse or MATL module, go to the View tab and click on the Registered Point List to have the imaging list appear. Once the stratum corneum is identified, scroll through the axial focus or Z-focus to identify specific tissue stratifications. Specific depths can be added within the skin such as stratum corneum, sebaceous glands, adipocytes, or subcutaneous fat, as determined during live imaging with lipid-tuned contrast.However, if entire depth stacks are to be taken, XY positions can be added, and the relative Z-position can be ignored. In the case of imaging specific depth for each skin stratification, select the Register Point to add it to the MATL queue. For full depth stacks, click Register Point for Each XY Position with Depth Selection in the Acquisition Parameters window.Next, set the number of repeats to one in the MATL module and click Ready. When the Play arrow changes from gray to black, hit Play to begin imaging the preliminary lipid stack. Once the imaging cycle has been completed, block both the pump and Stokes beams and change the wavelength on the laser graphical user interface to the desired wavelength based upon the targeted wave number or Raman vibration.Adjust the manual time delay stage while ensuring that the same powers are used to image the lipid and active pharmaceutical ingredient or API which are established a priori. Once the total number of cycle repeats has been chosen, unblock the pump beam and check if the power matches the desired power using the photodiode before pressing Play to begin automated imaging of the set points. Next, perform data analysis for each skin stratification by importing a sebaceous gland lipid image into Fiji and checking the box labeled Split channels to split the file into the CARS and SRS channels.To open the Region of Interest or ROI Manager, click on Analyze, Tools, and ROI Manager. Using the SRS channel such that C is equal to one, de-marquee the sebaceous gland in the image and add the markings to the ROI Manager by clicking Add T in the ROI Manager. Repeat the process for each sebaceous gland within the image.To mask out the lipid-rich regions, select each ROI and then click More, OR Combine, and Add tabs. To mask out the lipid-poor regions, use the Rectangle Tool in the Fiji menu and draw a square around the entire image. Add the region to the ROI Manager.Click on the newly-added square ROI in addition to the ROI that selects all lipid-rich regions in the ROI Manager. Under More, select XOR to generate a mask of the lipid-poor regions and add it to the ROI Manager. Concatenate the images in numerical order by going to Image, Stacks, Tools, and concatenate tabs in order.Alternatively, import the images by loading one into Fiji and then selecting an option called Group files with similar names on the setup page. While having the concatenated image active, go to the ROI Manager to select the lipid-rich regions and click the More tab before selecting Multi Measure, then wait for the Results window to appear. From the Results window, export the data to a spreadsheet and add a column titled Region to add lipid-rich regions to each row of data.Add lipid poor to regions that were outside the lipids. To visualize the data, save and import the spreadsheet into R package, ggplot2. Then plot the data as a function of the image number versus mean intensity to estimate the concentration-time data.Run non-compartmental analysis or NCA in RStudio on the intensity time data imported from the spreadsheet with the call for one layer. When done, plot and visually compare Jmax and AUCflux-all metrics. Additionally, carry out statistical comparisons across experimental conditions.The representative analysis depicts stratum corneum, sebaceous glands, adipocytes, subcutaneous fat from the nude mouse ear skin, and stratum corneum, papillary dermis, and a sebaceous gland from the human skin. In nude mouse ear skin, the limited tissue movement of sebaceous glands was observed with the same tissue depth of the glands at the time of formulation application and 120 minutes after application. The substantial tissue movement in the skin showed barely-visible sebaceous glands after 120 minutes of drug application, demonstrating the inefficiency of the original protocol.In several studies, the intensity versus time profiles showed high initial concentrations, followed by decreasing concentrations, indicating no further absorption of the drug into the tissue. On the other hand, some studies indicated a rise in the intensities which later declined over the experimental duration, demonstrating that the flux into the tissue had not reached a maximum prior to imaging. NCA analysis of concentration-time profiles of two formulations for the same API indicated that the ethoxy-ethoxyethanol provided extended API permeation compared to that of the gel formulation regardless of the skin layer.The total exposure analysis between the same formulations displayed similar observations in the deeper layers of the skin. The preparation of human skin and proper skin placement on the glass bottom dish after drug application are critical to the experiment's success. The clear solutions here demonstrated the suitability of this methodology.Further research will involve marketed formulations such as creams to comprehend how the formulation impacts API penetration and permeation.

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Research

JoVE Journal

Medicine

2.6K visualizações.  Massachusetts General Hospital, Harvard Medical School. Esta metodologia é significativa na sua vez permite que o método reprodutível e preciso quantifique produtos medicamentos aplicados topicamente usando imagens coerentes de Raman. Outras metodologias fornecem informações farmacocinéticas cutâneas volumosas, enquanto essa metodologia pode fornecer informações farmacocinéticas a granel e microescala, como a rota de permeação de uma droga. As vias de visualização e quantificação e permeação permitirão o desenvolvimento de produ...