The delivery of decoy DNA shows great potential as an effective agent to block detrimental transcription factor signaling, for example, to block activated NF-kappaB in the myocardium. Activated NF-kappaB is suspected to play a major role in various cardiac pathophysiological processes and thus has become a rational target for new treatments. However, problems with current DNA delivery agents such as immunogenicity, toxicity, and low delivery efficiency have prompted new vectors to be designed. This study will investigate twelve novel polymeric DNA delivery vehicles along with three control vectors: chitosan, branched polyethylenimine (PEI), and linear PEI for DNA decoy delivery to an in vitro myocardial cell model, H9c2(2-1) cells. The new polymers have been designed specifically for our long-term goal of nontoxic and efficient delivery of decoy DNA to the myocardial cytoplasm in vivo. Each of the twelve polymers have been synthesized with different carbohydrates for lowering toxicity (L-tartrate, glactarate, or D-glucarate) and different amounts of secondary amines (1-4) to facilitate DNA binding, compaction, and efficient intracellular transport. The objective of this application is to statistically analyze how the polymer chemical structure affects the observed cytotoxicity, cellular uptake, and endosomal release of the DNA decoys with H9c2(2-1) cells. This innovative study will allow the identification of new candidates for in vivo studies based on their chemical structure. Also, this study will provide a statistical analysis method to help direct future synthetic efforts toward the design of both nontoxic and highly efficient delivery vehicles for clinical use. This goal will be accomplished by pursuing the two specific aims:
Aim #1) The viability of a myocardial cell model will be statistically correlated with the number of secondary amines and the type of carbohydrate within each polymeric vector. The working hypothesis of this aim is that cytotoxicity will increase as the number of amines increase and the carbohydrate type plays a role in the observed toxicity. This hypothesis will be tested by performing two common viability assays: a) a modified version of the Lowry Protein assay, and b) the MTT assay after exposing H9c2(2-1) cells to each synthetic polymeric agent and the three control vectors. The results will be statistically analyzed using a fixed effects analysis-of-covariance model.
Aim #2) With a myocardial model, the cellular uptake and endosomal release of DNA will be statistically correlated with the number of secondary amines and the type of carbohydrate within each polymeric vector.
This aim will test the working hypothesis that cytoplasmic DNA delivery efficiency will be enhanced as the number of amine units within the polymer backbone is increased and the carbohydrate type plays a role in the delivery efficiency. This hypothesis will be tested by studying in vitro decoy delivery with each of the twelve synthetic and three control vectors with fluorescently-labeled DNA decoys. The cellular uptake and endosomal release will be studied with a flow cytometry assay and the results statistically correlated to the chemical properties as in Aim #1.
