Modulation of Electroporative Field Thresolds Using Cationic Peptides

描述（由申请人提供）：我们提出了一种新的电刺激方法，用于药物和基因递送，涉及宏观和纳米尺度的刺激。这种方法将提供优于传统电穿孔的改善的细胞活力，并广泛应用于医学科学。它还将为体外直接分化患者源性干细胞的独特方法奠定基础，并为申请人提供必要的背景和资源，以领导未来在基因和药物输送，再生医学以及其他医学领域的研究，其中可以利用电能。我们相信，阳离子肽与细胞质膜（PM）的共定位将使我们能够减少和明确控制电穿孔所需的电场强度。我们假设阳离子肽与细胞PM的共定位将增加电穿孔PM所需的跨膜电压，从而降低外部施加的电场的所需强度。通过改变PM周围的阳离子肽的电荷密度和浓度，我们将能够明确地控制电穿孔场阈值。研究将分三个阶段进行。首先，我们将开发各种正电荷密度的阳离子肽，并表征其短期和长期毒性以及对干细胞多能性的影响（目的1）。然后，我们将使用荧光质膜完整性指示剂确定阳离子肽的存在和电荷密度对电穿孔效率的影响（目的2）。最后，我们将表征阳离子肽电荷密度、肽胞外浓度和报告基因有效电转染所需的电场强度之间的关系（目的3）。虽然这项技术在药物和基因递送方面具有广泛的应用前景，但我们打算将这项技术应用于再生医学。如果我们的结果证实了我们的假设，那么最终我们设想在肽图案化的聚（乙二醇）（PEG）水凝胶中实施所提出的技术，以便在适合组织再生的环境中对基因表达和细胞分化提供空间和顺序控制。完成这项研究后，我们将开发出一种强大的工具，用于指导从患者源性干细胞再生异质功能组织所需的复杂事件序列。这种工程化组织将解决与移植治疗相关的许多问题，包括但不限于组织和器官的可用性和免疫排斥。再生组织的移植将在治疗烧伤受害者、骨折、失明、耳聋、心脏和血管损伤、肝脏和肾脏损伤以及器官衰竭方面直接有助于公共卫生。

DESCRIPTION (provided by applicant): We propose a novel electro-stimulatory approach for drug and gene delivery involving stimuli at both the macro- and nano-scales. This approach will provide improved cell viability over traditional electroporation and be widely applicable in the medical sciences. It will also lay the groundwork for a unique approach to direct differentiation of patient-derived stem cells in vitro and provide the applicant with the background and resources necessary to lead future investigations in gene and drug delivery, regenerative medicine, and other areas of medical science where the use of electrical energy may be exploited. We believe that the co-localization of cationic peptides with the plasma membrane (PM) of cells will allow us to reduce and explicitly control the electric field strength required for electroporation. We hypothesize that co-localization of cationic peptides with the PM of cells will add to the transmembrane voltage required to electroporate the PM, thus reducing the required intensity of an externally applied electric field. By varying charge density and concentration of cationic peptides about the PM, we will be able to explicitly control the electroporative field threshold. The research will proceed in three stages. First, we will develop cationic peptides of various positive charge densities and characterize their short- and long-term toxicity as well their influence on stem cell multi-potency (aim 1). We will then determine the effect of cationic peptide presence and charge density on electroporative efficiency using a fluorescent plasma membrane integrity indicator (aim 2). Finally, we will characterize the relationship between cationic peptide charge density, peptide extracellular concentration and the electric field strength required for effective electrotransfection of a reporter gene (aim 3). While this technology has wide application possibilities in drug and gene delivery, we intend to implement this technique in regenerative medicine. If our results confirm our hypothesis, then ultimately we envision implementing the proposed technique in peptide-patterned polyethylene glycol) (PEG) hydrogels in order to provide both spatial and sequential control over genetic expression and cell differentiation in environments appropriate for tissue regeneration. Upon completion of this research, we will have developed a powerful tool for directing the complex sequence of events required in regenerating heterogeneous, functional tissues from patient-derived stem cells. Such engineered tissues will solve many of the problems associated with transplant therapy, including, but not limited to, tissue and organ availability and immunorejection. Transplantation of regenerated tissues will directly contribute to public health in treating burn victims, broken bones, blindness, deafness, heart and vascular damage, liver and kidney damage, and organ failure.