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Characterization of the extracellular and intracellular electric fields during electric field-mediated gene delivery.

机译：电场介导的基因传递过程中细胞外和细胞内电场的表征。

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摘要

Electric field-mediated gene delivery is currently plagued by its inefficiency. From local injection to expression of a transgene, several barriers, namely, interstitial transport, cellular uptake, intracellular transport, and nuclear uptake, impede the process. However, in order to determine the limiting steps in the treatment, or to design new treatment strategies, each of the aforementioned barriers must be characterized and understood.; Since diffusion is negligible in the interstitial and intracellular regions, electrophoresis of DNA is the primary mode of transport in these regions. Also, the permeability of the cell membrane is increased in response to the local electric field in tumor tissues. Therefore, since the local electric field ultimately determines the extent of DNA electrophoresis or cellular electroporation, the inefficiency of electric field-mediated gene delivery may, in part, be explained in terms of the properties of the fields around and within the target cells.; In the past, the local electric field that exists in tumors during treatment has been poorly characterized. In fact, in many studies, the local electric field in tumors was assumed to be equal to the applied field. To this end, this work aims to quantitatively assess the fields that exist during electric field-mediated gene delivery on both the macroscopic (i.e. tissue) and microscopic (i.e. cellular) levels.; Experimental and theoretical studies were conducted to characterize the electric field that exists in tumor tissue and around and within tumor cells in response to an applied external electric stimulus. The results show that in ex vivo tumor tissue slices and in tissue phantoms, the macroscopic electric field is relatively uniform, and unaffected by the presence of cells. However, the magnitude of the electric field in tumor tissues was significantly lower than the applied field during both ex vivo and in vivo stimulation. During in vivo stimulation, the ratio of the intratumoral versus the applied field was a sigmoidal function of the applied field. In fact, the field in tumors can be as low as ∼25% of the applied field during in vivo stimulation. During electroporation, the magnitude of the field in tumors increases, but asymptotically reaches only ∼50% of the applied field.; The resistances of the skin and electrode interface and the tumor were also experimentally determined during in vivo stimulation. The resistance of the skin and electrode-tissue interface decreased exponentially when the applied field was increased from 50 V/cm to 400 V/cm, but the resistance at the center of tumors was independent of the applied field.; The microscopic electric field was determined using a 3D model to determine the electric field that exists in both the extracellular and intracellular domains of a 10-mum spherical cell exposed to an applied field of 100 V/cm. Electroporation was modeled as a decrease in cell membrane resistivity over a specified angle. The results showed that the extracellular and membrane fields and the extracellular currents are less sensitive to electroporation, compared to the intracellular field and currents. Also, the extent of electroporation affects the transmembrane potential flanking the pore region, which may have consequences for pore growth. The results also showed that extracellular field redirection occurs only in a small volume when pores are formed in the cell membrane, which may help explain some of the inefficiencies currently seen during electric field-mediated gene delivery. Finally, the percent changes in the total current flow are relatively small compared to the changes in the current flow inside the cell.
机译：电场介导的基因传递目前受到效率低下的困扰。从局部注射到转基因的表达，间质运输，细胞吸收，细胞内运输和核吸收的几个障碍阻碍了这一过程。但是，为了确定治疗中的限制步骤或设计新的治疗策略，必须对每个上述障碍进行特征描述和理解。由于在间隙和细胞内区域的扩散可忽略不计，因此DNA电泳是在这些区域中运输的主要方式。而且，响应于肿瘤组织中的局部电场，细胞膜的渗透性增加。因此，由于局部电场最终决定了DNA电泳或细胞电穿孔的程度，电场介导的基因传递的效率低下可以部分地根据靶细胞周围和靶细胞内电场的性质来解释。过去，在治疗过程中存在于肿瘤中的局部电场的特征很差。实际上，在许多研究中，假定肿瘤中的局部电场等于施加的电场。为此，这项工作旨在从宏观（即组织）和微观（即细胞）水平定量评估电场介导的基因传递过程中存在的电场。进行了实验和理论研究以表征响应于施加的外部电刺激而存在于肿瘤组织中以及肿瘤细胞周围和内部的电场。结果表明，在离体肿瘤组织切片和组织模型中，宏观电场相对均匀，不受细胞存在的影响。然而，在离体和体内刺激期间，肿瘤组织中的电场强度显着低于施加的电场。在体内刺激期间，肿瘤内与外加电场的比率是外加电场的S形函数。实际上，在体内刺激过程中，肿瘤的视野可能低至所施加视野的约25％。在电穿孔过程中，肿瘤的电场强度增加，但渐近仅达到所施加电场的约50％。在体内刺激期间，还通过实验确定了皮肤和电极界面以及肿瘤的电阻。当施加的电场从50 V / cm增加到400 V / cm时，皮肤和电极-组织界面的电阻呈指数下降，但肿瘤中心的电阻与施加的电场无关。使用3D模型确定微观电场，以确定暴露于100 V / cm施加电场的10微米球形细胞的细胞外和细胞内域中都存在的电场。电穿孔被建模为在特定角度下细胞膜电阻率的降低。结果表明，与细胞内电场和电流相比，细胞外和膜电场以及细胞外电流对电穿孔的敏感性较低。而且，电穿孔的程度会影响孔区域两侧的跨膜电位，这可能会影响孔的生长。结果还表明，当在细胞膜上形成孔时，细胞外电场重定向仅在很小的体积内发生，这可能有助于解释当前在电场介导的基因传递过程中发现的一些低效率现象。最后，与电池内部电流的变化相比，总电流的变化百分比相对较小。

著录项

  • 作者

    Mossop, Brian J.;

  • 作者单位

    Duke University.;

  • 授予单位 Duke University.;
  • 学科 Engineering Biomedical.
  • 学位 Ph.D.
  • 年度 2006
  • 页码 140 p.
  • 总页数 140
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 生物医学工程;
  • 关键词

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