Open Access Thesis
Date of Award
Bioreducible, layer-by-layer (LbL) polyelectrolyte films show great promise for use in biomedical implant and gene-delivery applications. These nanometer-scale thin films can easily be coated onto a variety of implantable surfaces or devices. To achieve proof of concept, HEK-293, NIH/3T3, RAW, and MC3T3 cells were transfected using plasmid DNA with the green fluorescent protein reporter gene (GFP-DNA). In order to optimize transfection, a number of polyelectrolytes and biological components were systematically incorporated into the two similar basic LbL structures. Based on our previous degradation assays, it is shown that cellular interactions in vitro can break down the layers of the thin films on substrates, allowing for DNA and future therapeutics to be released gradually over periods of time, or in bulk, depending on modifications to the structure and composition. Using an efficient in vitro polyplex testing method, different bioreducible polycations were examined in order to select a polyelectrolyte with high transfection. Following polyplex testing, thin films were made using the layer-by-layer dip coating method and cells were cultured on the subsequent films in an in vitro setting. The basic LbL structure comprises alternating polycation, GFP-DNA, and polyethylenimine (PEI) as an effective barrier layer. In vitro testing for transfection was monitored using fluorescence microscopy over 10
days, among other evaluation methods. Through these studies, transfection was achieved. This lays the groundwork for future studies using bioreducible thin films to deliver DNA, small molecules, or other therapeutics to cells.
Muniz, Maria, "Programmable Dna Delivery To Cells Using Bioreducible Layer-By-Layer (lbl) Polyelectrolyte Thin Films" (2015). Wayne State University Theses. 410.