Gelatin-based nanoparticles as DNA transfection vectors

促进DNA转移到细胞中最常用的方法是使用病毒载体，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。作为一种基因治疗，病毒载体有几个潜在的缺点；最重要的是，存在非常真实的安全问题，因为病毒衣壳有可能产生严重的免疫反应，导致突变，正如一些动物实验和人体试验所证明的那样，其中已有患者死亡(由腺病毒载体引起)的报道。另一种方法是使用能够结合DNA并将其输送到细胞内的自组装纳米颗粒。这些非病毒系统通常基于带正电荷的脂类或聚合物，通常不具免疫原性，毒性低；然而，它们在DNA传递方面也存在效率低下的问题。**我们的工作重点是设计基于中性聚合物的新的非病毒传递系统，这些聚合物无毒，容易从体内清除，并且在环境中可生物降解。这项建议侧重于应用自然产生的聚合物，在这种情况下，明胶，以形成纳米颗粒载体，以输送DNA。自然生成的明胶将被转化为阳离子形式，从而实现DNA的高效包装。然后，明胶/DNA复合体将与其他成分一起配制，以增强目的蛋白的整体表达，在这种情况下，增强的绿色荧光蛋白被用作分析目的的报告基因。特别是，我们将探索阳离子明胶纳米颗粒对脂膜的影响，利用脂单分子层作为与转染有关的细胞膜(即质膜、内膜等)的模型。模型膜的结构将用朗缪尔等温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)进行检测。通过这些研究，我们希望确定纳米颗粒组成和对膜结构的影响之间的关系，从而能够更好地预测哪些纳米颗粒制剂更有可能成功地作为DNA转染载体。*******

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. *******