使用相干拉曼散射成像对皮肤内的药物化合物进行可视化和定量
Summary
本文介绍了一种相干拉曼散射成像方法，用于可视化和量化皮肤内的药物化合物。本文介绍了皮肤组织制剂（人和小鼠）和局部制剂的应用，用于量化时空浓度曲线的图像采集，以及用于评估局部药物递送的初步药代动力学分析。
Abstract
局部制剂应用后的皮肤药代动力学（cPK）一直是监管和药物开发科学家特别感兴趣的研究领域，以机械方式了解局部生物利用度（BA）。半侵入性技术，如胶带剥离、真皮微透析或真皮开流微灌注，均可量化大尺度 cPK。虽然这些技术提供了大量的cPK知识，但社区缺乏对细胞水平的活性药物成分（API）渗透和渗透的机制理解。
解决微尺度cPK的一种非侵入性方法是相干拉曼散射成像（CRI），其选择性地靶向内在分子振动，而无需外在标记或化学修饰。CRI有两种主要方法 – 相干反斯托克斯拉曼散射（CARS）和刺激拉曼散射（SRS） – 能够对API或非活性成分进行灵敏和选择性定量。CARS通常用于获取结构皮肤信息或可视化化学对比度。相比之下，SRS信号与分子浓度呈线性关系，用于量化皮肤分层中的API或非活性成分。
虽然小鼠组织通常用于CPK与CRI，但在监管批准之前，最终必须在人体组织中评估局部BA和生物等效性（BE）。本文提出了一种制备和成像 离体 皮肤的方法，用于定量药代动力学CRI研究，以评估局部BA和BE。随着时间的推移，该方法可在人类和小鼠皮肤内实现可靠且可重复的API定量。定量富脂区和贫脂区室内的浓度，以及随时间推移的总API浓度;这些用于估计微观和宏观尺度的BA，以及潜在的BE。
Introduction
局部药物产品应用后评估cPK的方法已从经典的体外渗透试验（IVPT）研究1，2，3，4，5和胶带剥离6，7，8扩展到其他方法，如开流微灌注或真皮微透析9，10，11，12，13，14.根据感兴趣的疾病，可能存在各种治疗作用的局部部位。因此，可能有相应数量的方法来评估 API 到达预期本地操作站点的速率和程度。虽然上述每种方法都有其优点，但主要缺点是缺乏微尺度的cPK信息（即无法可视化API的去向及其渗透方式）。
一种用于估计局部BA和BE的非侵入性方法是CRI，它可以分为两种成像方式：CARS和SRS显微镜。这些相干拉曼方法能够通过非线性拉曼效应对分子进行化学特异性成像。在CRI中，两个激光脉冲序列在样品内聚焦和扫描;激光频率之间的能量差被设置为特定于目标化学结构的振动模式。由于CRI过程是非线性的，因此仅在显微镜焦点处产生信号，从而允许对组织进行三维药代动力学断层扫描成像。在cPK的背景下，CARS已被用于获取组织结构信息，例如富含脂质的皮肤结构的位置15。相比之下，SRS已被用于量化分子浓度，因为其信号与浓度呈线性关系。对于 离体 皮肤标本，在外延方向16 和SRS传输模式下进行CARS是有利的。因此，薄的组织样品将允许SRS信号检测和定量。
作为模型组织，裸鼠耳朵具有几个优点，但缺点很小。一个优点是组织厚度已经〜200-300μm，不需要进一步的样品制备。此外，通过一个视场轴向聚焦（例如角质层、皮脂腺 （SG）、脂肪细胞和皮下脂肪）可看到多个皮肤分层 16，18。这允许在移动到人体皮肤样本之前对皮肤渗透途径进行初步临床前估计和局部BA估计。然而，裸鼠模型存在局限性，例如由于皮肤结构19的差异而难以外推到体内场景。虽然裸鼠耳朵是获得初步结果的优秀模型，但人体皮肤模型是黄金标准。尽管关于冷冻人体皮肤对准确概括体内渗透动力学的适用性和适用性已有各种评论20，21，22，但使用冷冻人体皮肤是评估体外API渗透动力学的公认方法23，24，25.该协议可视化小鼠和人体皮肤中的各种皮肤层，同时量化富脂质和贫脂结构中的API浓度。
虽然CRI已被用于许多领域以特异性地可视化组织内的化合物，但研究局部应用药物产品的cPK的努力有限。为了使用CRI评估外用产品的局部BA / BE，有必要首先制定标准化方案以进行准确的比较。以前使用CRI将药物递送到皮肤的努力已经证明了数据中的可变性。由于这是CRI的相对较新的应用，因此建立协议对于获得可靠的结果至关重要18，26，27。这种方法仅针对拉曼光谱生物沉默区域中的一个特定波数。然而，大多数API和非活性成分在指纹区域内都有拉曼位移。由于指纹区域组织中产生的固有信号，这在以前已经提出了挑战。最近的激光和计算进步已经消除了这一障碍，这也可以与这里介绍的方法结合使用28。这里介绍的这种方法允许量化API，其在沉默区域（2，000-2，300 cm-1）具有拉曼位移。这不仅限于药物的物理化学性质，这可能是前面提到的一些cPK监测方法29的情况。
该协议必须减少各种制剂的皮肤厚度的样品间差异，因为由于厚样品的光散射，厚人皮样品在药物产品应用后会产生最小的信号。本手稿的目标是提出一种组织制备方法，以确保可重复的成像标准。此外，CRI系统的设置如前所述，以减少潜在的误差源，并最大限度地减少信噪比。但是，本文不会讨论CRI显微镜的指导原理和技术优点，因为之前已经介绍了30。最后，探索广泛的数据分析程序，以允许解释结果以确定实验的成功或失败。
Protocol
裸鼠耳组织的使用得到了马萨诸塞州总医院机构动物护理和使用委员会（IACUC）的批准，而人体皮肤组织的使用则得到了马萨诸塞州总医院机构审查委员会（IRB）的批准。根据IACUC协议，新鲜安乐死的小鼠是从具有裸鼠菌落的合作者那里获得的。人体组织是通过批准的协议从马萨诸塞州总医院的选择性腹部整形手术中采购的。此外，腹部皮肤以外的特定组织类型是通过身体捐赠机构获得的，也是通…
Representative Results
如果组织在实验完成后没有在轴向（<10μm）或横向方向上显着移动，则认为成像成功（图4）。如果目标 API 的 SRS 测量值不代表初始深度，则立即表明初始深度的定量是层特异性的。通过对每个感兴趣的 XY 位置进行 z 轴成像来缓解这种情况，权衡是时间分辨率。如果在这些研究中使用冷冻皮肤，则与新鲜皮肤相比，API的渗透和渗透速度很快，并且从配方应用到开始成像之间?…
Discussion
局部BA / BE的评估是一个研究领域，需要多方面的方法，因为没有一种方法可以完全表征 体内 cPK。该协议提出了一种基于相干拉曼成像的局部药物产品BA / BE评估方法。可能被忽视的第一点是皮肤样品必须有多薄，特别是对于定量传输SRS成像。如果皮肤太厚（即光 不能轻易通过），SRS检测器测量的信号很少甚至没有，因此它将提供较差的浓度数据。必须注意正确制备这些组织样本，?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者要感谢埃文斯小组的Fotis Iliopoulos博士和Daniel Greenfield博士对本手稿的讨论和校对。此外，作者还希望得到LEO Pharma的支持。 图 2 是使用 BioRender.com 创建的。
Materials
| Tissue Preparation | |||
| Autoclavable Biohazard Bags | FisherBrand | 22-044562 | 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&searchTerm= 22044562&searchType=RAPID& matchedCatNo=22044562 |
| Cell Culture Buffers: Dulbecco's Phosphate-Buffered Salt Solution 1x | Corning | MT21030CV | 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&searchTerm= 21-030-cv&searchType= RAPID&matchedCatNo=21-030-cv |
| Disposable Scalpels | Exel International | 14-840-00 | As refered to in text: scalpel https://www.fishersci.com/shop/products/exel-international-disposable-scalpels-3/1484000?keyword=true |
| High Precision 45° Angle Broad Point Tweezers/Forceps | Fisherbrand | 12-000-132 | As refered to in text: forceps https://www.fishersci.com/shop/products/high-precision-45-angle-broad-point-tweezers-forceps/12000132#?keyword= |
| Kimwipes Delicate Task Wipers, 1-Ply | Kimberly-Clark Professional Kimtech Science | 06-666 | As refered to in text: task wiper https://www.fishersci.com/shop/products/kimberly-clark-kimtech-science-kimwipes-delicate-task-wipers-7/06666 |
| Parafilm M Laboratory Wrapping Film | Bemis | 13-374-12 | As refered to in text: parafilm https://www.fishersci.com/shop/products/curwood-parafilm-m-laboratory-wrapping-film-4/1337412 |
| Petri Dish (35 mm x 10 mm) | Fisherbrand | FB0875711YZ | As refered to in text: small petri dish https://www.fishersci.com/shop/products/fisherbrand-petri-dishes-specialty-6/FB0875711YZ?keyword=true |
| Petri Dish (60 mm x 15 mm) | Fisherbrand | FB0875713A | As refered to in text: large petri dish https://www.fishersci.com/shop/products/fisherbrand-petri-dishes-clear-lid-12/FB0875713A?keyword=true |
| Surgical Scissors | Roboz | NC9411473 | As refered to in text: scissors https://www.fishersci.com/shop/products/scissors-327/NC9411473?searchHijack=true&searchTerm= RS-5915SC&searchType=RAPID& matchedCatNo=RS-5915SC |
| Laser/microscope | |||
| 650/60 nm BrightLine single-band bandpass filter | Semrock | As refered to in text: CARS filter – CH2 vibrations (645nm/60nm filter) | |
| Control box IX2-UCB | Olympus | As refered to in text: Control Box | |
| D700/30m | Chroma | As refered to in text: CARS filter – deuterated band https://www.chroma.com/products/parts/d700-30m |
|
| DeepSee Insight | Spectra-Physics | As refered to in text: Laser https://www.spectra-physics.com/f/insight-x3-tunable-laser |
|
| Digital Handheld Optical Power and Energy Meter Console | ThorLabs | PM100D | As refered to in text: power meter https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=3341 |
| Fluoview Software | Olympus | As refered to in text: Microscope Control software | |
| Frosted Microscope Slides | FisherBrand | As refered to in text: microscope slides https://www.fishersci.com/shop/products/fisherbrand-frosted-microscope-slides-4/22265446 |
|
| FV1000 | Olympus | As refered to in text: Microscope | |
| Incubation Chamber | Tokai Hit | GM-800 | As refered to in text: incubation chamber |
| Integrating Sphere Photodiode Power Sensor | ThorLabs | S142C | As refered to in text: photodiode https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=3341 |
| Power supply FV31-PSU | Olympus | As refered to in text: Power Supply | |
| Precision 4063, 80MHz Dual Channel Function Generator | BK Precision | As refered to in text: function generator | |
| ProScan – Precision Microscope Automation | Prior Scientific Instruments | As refered to in text: stage controller https://www.prior.com/microscope-automation/inverted-microscope-systems/proscan-linear-stage-highest-precision-microscope-automation |
|
| SecureSeal Imaging Spacers | Grace Biolabs | 654004 | As refered to in text: spacer https://gracebio.com/product/secureseal-imaging-spacers-654004/ |
| SRS Detection Kit | APE | As refered to in text: SRS detector | |
| UPLSAPO 20X NA:0.75 | Olympus | As refered to in text: 20X Objective https://www.olympus-lifescience.com/en/objectives/uplsapo/ |
|
| Lipid/Drug Imaging | |||
| 35 mm Dish, No. 0 Uncoated Coverslip, 14 mm Glass Diameter | MatTek Corporation | NC9711297 | As refered to in text: Glass bottom dish https://www.fishersci.com/shop/products/glass-bottom-mircrowell-dish/nc9711297 |
| Cotton-tipped applicators | FisherBrand | As refered to in text: Cotton-tipped applicator | |
| Distriman Postive Displacement Pipette | Gilson | As refered to in text: Postive Displacement Pipette https://www.fishersci.com/shop/products/gilson-distriman-positive-displacement-repetitive-pipette/F164001G#?keyword= |
|
| Distriman Postive Displacement Pipette Tips | Gilson | As refered to in text: Tips for pipette https://www.fishersci.com/shop/products/gilson-distritip-syringes-6/f164100g?keyword=true |
|
| Data Analysis | |||
| FIJI | Open-source | As refered to in text: FIJI/ImageJ https://imagej.net/software/fiji/ |
|
| Jupyter-Lab | open-source | As refered to in text: JupyterLab https://jupyter.org/ |
|
| Rstudio | Open-source | As refered to in text: Rstudio https://www.rstudio.com/ |
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Cite This Article
Kuzma, B. A., Pence, I. J., Ho, A., Evans, C. L. Visualizing and Quantifying Pharmaceutical Compounds within Skin using Coherent Raman Scattering Imaging. J. Vis. Exp. (177), e63264, doi:10.3791/63264 (2021).