ELECTRICAL CREATION OF AQUEOUS PATHWAYS IN SKIN

我们建议研究“高电压”脉冲(约20 到800伏的皮肤)导致极大地增强了带电的传输 分子穿过人体皮肤的主要屏障角质层。 目标。我们的目标是测试这一假说 通路是在角质层中创建的，涉及到 使用一系列带电荷和不带电荷的荧光分子来测量 在体外条件下这些分子的通量和存储量。 意义重大。富脂角质层(SC；最外层，死层 人类皮肤)由死亡的角质细胞组成，周围有 多个平行的双层膜，并保护身体免受 脱水、有毒分子进入和感染。然而，SC也 为经皮给药和分析物提取提供了障碍。 高压脉冲有望在水中形成新的通道 角质层，从而增加了分子的运输量 以及对经皮给药的控制程度。特别的 人们感兴趣的是带电分子(例如，多肽)的运输 目前不能大量交付。恰恰相反， 从样品中提取分析物(例如葡萄糖)的免费过程 皮下组织液对于“非侵入性”将有很大的价值 临床化学“化验。以前的工作。我们已经证明 大量的带电分子可以穿过角质层，如果 施加一系列短的“高电压”脉冲。有限动物 对“高压”脉冲的研究和先前的医学用途共同表明 这种微不足道的损害在某些脉冲条件下会发生，但对于 更大和/或更长的脉冲会造成组织损伤。其他使用单元格的工作 悬浮液，以及电穿孔传输的理论模型， 提示通过细胞内瞬时水孔的电泳法 双层膜，以及在地层的多层双层中 角质层可能是电荷运输的主要机制 分子通过电穿孔双层膜。方法：研究方法。在In下 在体外条件下，我们将同时对 每种皮肤制剂：[1]第一个荧光分子的流量，[2] 第二个荧光分子的通量，以及无源电子 属性(如皮肤阻抗或电阻)测量。这种方法 解决皮肤准备可变性的重要问题，同时 通过使用荧光分子提供关键的定量信息 具有不同的大小、电荷和水/脂的溶解度。这些实验 将被设计用来测试一般假设的不同版本 通路是通过“高压”脉冲产生的，而这种电泳法 (也可能是电渗透和扩散)通过这些途径可以 解释了巨大的流量增加。一个配套的理论 建模工作将提供明确的预测，从而直接 对“水通道创造”假说的检验。

We propose study of the mechanism by which "high voltage" pulses (about 20 to 800 V across the skin) cause greatly enhanced transport of charged molecules across the main barrier of the human skin, the stratum corneum. AIMS. Our goals are directed towards testing the hypothesis that aqueous pathways are created in the stratum corneum, and involve experiments which use a series of charged and uncharged fluorescent molecules to measure fluxes and stored amounts of these molecules under in vitro conditions. SIGNIFICANCE. The lipid-rich stratum corneum (SC; outermost, dead layer of the human skin) is comprised of dead corneocytes surrounded by multiple, parallel bilayer membranes, and protects the body from dehydration, entry of toxic molecules and infection. However the SC also presents a barrier for transdermal drug delivery and analyte extraction. High voltage pulsing is expected to create new aqueous pathways across the stratum corneum, thereby increasing both the amount of molecular transport and degree of control for transdermal drug delivery. Of particular interest is the transport of charged molecules (e.g., peptides) which presently cannot be delivered in large amounts. The opposite, complimentary process of extracting analytes (e.g. glucose) from the subcutaneous interstitial fluid would be of great value for "non-invasive clinical chemistry" assays. PREVIOUS WORK. We have shown that significant fluxes of charged molecules can cross the stratum corneum if a series of short "high voltage" pulses are applied. Limited animal studies and prior medical use of "high voltage" pulses together suggest that insignificant damage occurs for some pulsing conditions, but that for larger and/or longer pulses there is tissue damage. Other work with cell suspensions, and with theoretical models for electroporative transport, suggests that electrophoresis through transient aqueous pores in cellular bilayer membranes, and in the multilamellar bilayers of the stratum corneum, may be the dominant mechanism for the transport of charged molecules through electroporated bilayer membranes. METHODS. Under in vitro conditions, we will carry out three simultaneous measurements on each skin preparation: [1] the flux of a first fluorescent molecule, [2] the flux of a second fluorescent molecule, and [3] a passive electrical property (e.g. skin impedance or resistance) measurement. This approach addresses the important issue of skin preparation variability, while providing critical quantitative information by using fluorescent molecules with different size, charge and water/lipid solubility. These experiments will be designed to test versions of the general hypothesis that aqueous pathways are created by "high voltage" pulsing, and that electrophoresis (and possibly electro-osmosis and diffusion) through these pathways can account for the tremendous flux increases. A companion theoretical modeling effort will provide explicit predictions that allow direct testing of the "aqueous pathway creation" hypothesis.