Gelatin-based nanoparticles as DNA transfection vectors

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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. *******