Gelatin-based nanoparticles as DNA transfection vectors

明胶纳米颗粒作为 DNA 转染载体

• 批准号: RGPIN-2016-04009
• 负责人: Wettig, Shawn
• 金额: $ 2.04万
• 依托单位: University of Waterloo
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=614778
• 关键词: Gelatin; based; nanoparticles; DNA; transfection

The most commonly applied method for facilitating the transfer of DNA into cells is through the use of viral vectors, which are the most efficient vectors currently being studied, accounting for more than 65% of ongoing clinical trials. Viral vectors have several potential disadvantages for application as a gene therapy; most significantly, very real safety concerns exist because viral capsids have the potential to generate a severe immune response, cause mutagenisis , as has been demonstrated in a number of animal experiments and human trials, where patient death (resulting from adenoviral vector) has been reported. An alternative approach is to use self-assembling nanoparticles that are capable of binding DNA and delivering it into the cells. These non-viral systems are typically based upon positively charged lipids or polymers, and are generally non-immunogenic and have low toxicities; however, they also suffer from low efficiency in terms of DNA delivery.Our work is focused on the design of new non-viral delivery systems based upon neutral polymers that are non-toxic, are easily cleared from the body, and are biodegradable in the environment. This proposal focuses on the application of naturally occurring polymers, in this case gelatin, to the formation of nanoparticle vectors for the delivery of DNA. Naturally occurring gelatin will be converted to a cationic form, allowing for efficient packaging of DNA. The gelatin/DNA complexes will then be formulated with other components to enhance the overall expression of the protein of interest, in this case the enhanced green fluorescent protein which is used as a reporter gene for analysis purposes. In particular, we will explore the impact of the cationic gelatin nanoparticles on lipid membranes, utilizing lipid monolayers as a model of cellular membranes implicated in transfection (i.e. plasma membrane, endosomal membrane, etc.). The structure of model membranes will be examined using Langmuir isotherm methods coupled with Brewster Angle Microscopy (BAM), Infra-red reflected absorbance spectroscopy (IRRAS) and Atomic Force Microscopy (AFM). From these studies we hope to determine relationships between nanoparticle composition and the impact on membrane structure, allowing for better prediction of which nanoparticle formulations are more likely to be successful as DNA transfection vectors.

促进DNA转移到细胞中最常用的方法是通过使用病毒载体，这是目前正在研究的最有效的载体，占正在进行的临床试验的65%以上。病毒载体对于作为基因疗法的应用具有几个潜在的缺点;最重要的是，存在非常真实的安全性问题，因为病毒衣壳具有产生严重免疫应答、引起诱变的潜力，如在许多动物实验和人体试验中已经证明的，其中已经报道了患者死亡（由腺病毒载体引起）。另一种方法是使用能够结合DNA并将其递送到细胞中的自组装纳米颗粒。这些非病毒系统通常基于带正电荷的脂质或聚合物，并且通常是非免疫原性的并且具有低毒性;然而，它们也遭受在DNA递送方面的低效率。我们的工作集中于基于中性聚合物的新的非病毒递送系统的设计，所述中性聚合物是无毒的，容易从身体中清除，并且在环境中是可生物降解的。该提案的重点是应用天然存在的聚合物，在这种情况下，明胶，形成纳米粒子载体的DNA的交付。天然存在的明胶将被转化为阳离子形式，允许DNA的有效包装。然后将明胶/DNA复合物与其它组分一起配制，以增强感兴趣的蛋白质的总体表达，在这种情况下，增强的绿色荧光蛋白用作报告基因用于分析目的。特别是，我们将探索阳离子明胶纳米颗粒对脂质膜的影响，利用脂质单层作为涉及转染的细胞膜（即质膜，内体膜等）的模型。模型膜的结构将使用朗缪尔等温线方法结合布鲁斯特角显微镜（BAM）、红外反射吸收光谱（IRRAS）和原子力显微镜（AFM）来检查。从这些研究中，我们希望确定纳米颗粒组成与对膜结构的影响之间的关系，从而更好地预测哪种纳米颗粒制剂更有可能作为DNA转染载体成功。