Using Pulsed-Field Gradient Spin-Echo NMR to Determine Permeation Mechanisms in Human Stratum Corneum

该奖项是根据2009年美国复苏和再投资法案（公法111-5）资助的。这项研究的目的是研究亲脂化学物质是如何穿透人体皮肤的最外层，也就是角质层的。角质层是一层很薄（20到40微米厚）的复合膜，含有被称为角质层的扁平死皮细胞层，被脂质（油性）分子包围。角质层控制着许多化学物质通过皮肤进入人体的速度。化学物质可以通过两种不同的途径穿透角质层：(1)仅通过脂质层，或(2)串联通过角质层和脂质层。在皮肤文献中流行的观点是，化学物质的渗透仅限于脂质层，尽管目前还没有直接的证据。此外，大多数支持转运只通过脂质层的论点同样适用于可渗透角质层的情况。这项研究的结果将提供直接的证据来回答这个问题：化学物质能否通过角质层穿透角质层？选择用于研究的模型化学物质2-（三氟甲基）苯腈是为了模拟能够穿透皮肤对人体产生治疗或毒性作用的化学物质。该分子在角质层中的扩散将使用一种称为脉冲场梯度自旋回波核磁共振（NMR）的方法进行研究。这种方法在短时间内（即毫秒到秒）测量平移分子运动（即扩散）。它还提供了关于短距离（即0.1-100微米）的限制性边界的信息，这将用于区分通过角质层和脂质层的扩散。虽然这种方法已广泛用于研究胶体和固态工程材料，但其在生物膜研究中的应用受到限制。在这个项目中产生的新数据将提高我们对角质层化学渗透机制的理解，包括它如何随着含水量的变化而变化。如果角质层内的扩散被证明是人体皮肤吸收亲脂性化学物质的重要途径，那么皮肤科学的范式将发生转变，这将改变现有的经皮药物输送策略、皮肤病治疗以及预测有毒化学物质通过皮肤吸收的方法。使用脉冲场梯度自旋回波核磁共振来研究角质层等复合介质中的扩散的科学挑战出现在许多其他材料中，包括生物和非生物，这里使用的技术也将适用于这些系统。角质层扩散的建议研究将用于培养研究生和本科生研究人员在非均质膜中化学传输的基础知识，以及将脉冲场梯度自旋回波核磁共振技术应用于包含多相的生物或工程系统中的材料。将积极征聘妇女和其他代表性不足的少数民族参加该项目，该项目旨在通过研究环境中独特的经验培养强大的沟通和组织/管理技能。研究小组将与科罗拉多矿业学院现有的一个成功的外展项目合作，为小学、初中和高中的学生和教师开发屏障膜主题的教育模块。

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).0854343BungeThe goal of this research is to study how lipophilic chemicals penetrate through the outermost layer of human skin, which is called the stratum corneum. The stratum corneum is a thin (20 to 40 um thick), composite membrane containing layers of flattened, dead skin cells called corneocytes surrounded by lipid (oily) molecules that are also organized into layers. The stratum corneum controls the rate at which many chemicals enter the body through the skin. Chemicals can penetrate the stratum corneum by two different paths: (1) through only the lipid layers, or (2) through the corneocytes and lipid layers in series. The prevailing opinion in the skin literature is that chemical penetration is restricted to the lipid layers alone, although no direct proof for this presently exists. Moreover, most arguments offered to support the claim that transport is through the lipid layers exclusively are equally applicable to the case of permeable corneocytes. The results of this research will provide direct evidence to answer the question: Can chemicals penetrate the stratum corneum through the corneocytes? The model chemical chosen for study, 2-(trifluoromethyl) benzonitrile, was selected to simulate chemicals that can penetrate the skin to produce either therapeutic or toxic effects in humans. The diffusion of this molecule in the stratum corneum will be studied using a method called pulsed-field gradient spin-echo nuclear magnetic resonance (NMR). This method measures translational molecular motion (i.e., diffusion) over short periods of time (i.e., millisecond to seconds). It also provides information about restrictive boundaries over short distances (i.e., 0.1-100 um), which will be used to distinguish diffusion through corneocytes and the lipid layers. Although this method has been used widely to study colloids and solid-state engineered materials, its use in the study of biological membranes has been limited. The new data generated in this project will improve our understanding of chemical permeation mechanisms in the stratum corneum, including how it changes with variations in water content. If diffusion within the corneocytes is shown to be a significant route for the absorption of lipophilic chemicals in human skin, the paradigm of skin science will shift, which will change existing strategies for transdermal drug delivery, treatment of skin diseases, and methods for predicting absorption of toxic chemicals through skin. The scientific challenges of using pulsed-field gradient spin-echo nuclear magnetic resonance to study diffusion in a composite medium like the stratum corneum arise in many other materials, both biological and non-biological, and the techniques used here will be applicable to these systems as well. The proposed studies of diffusion in the stratum corneum will be used to train graduate and undergraduate student researchers in the fundamentals of chemical transport in heterogeneous membranes and in applications of the pulsed-field gradient spin-echo nuclear magnetic resonance technique to materials from either biological or engineered systems that contain multiple phases. Women and other underrepresented minorities will be actively recruited into the project, which is designed to develop strong communication and organizational/management skills through experiences that are uniquely available in a research environment. In collaboration with an existing and successful outreach program at the Colorado School of Mines, the research team will develop educational modules on the topic of barrier membranes for elementary, middle and high school students and teachers.