Session: Polymer Division Student Poster Session: Celebrating the Diversity of Macromolecules:

Due to high cytotoxicity, endosomal degradation, and aggregation on cell surfaces encountered with gene delivery particles condensed by unmodified branched PEI, chemical modification has been a common strategy to enhance performance. In this work, we have synthesized a series of succinylated (zPEI, 2-35%) and acetylated (acPEI, 10-50%) branched polyethyleneimine (bPEI) polymers to better understand how modulating PEI charge affects both the DNA packaging inside the polyplexes as well as the gene delivery efficacy in vitro. Acetylation decreases the number of protonated amines on the bPEI reducing the PEI charge density, while succinylation introduces COO^- groups to produce zwitterion-like PEI (zPEI). Both acPEI/DNA and zPEI/DNA are capable of condensing DNA and form similar colloidal nanoparticles that are observed to have comparable polyplex diameters as measured by nanoparticle tracking analysis (NTA) and ζ-potential for all but the highest degrees of modification studied. While acPEI and zPEI have been shown improvement under serum-free conditions, we show that only zPEI shows greatly enhanced transfection efficacy in vitro in the presence of serum. Using small-angle X-ray scattering (SAXS), we also investigated the internal structure of DNA condensed with zPEI and acPEI polymers and compared to unmodified bPEI/DNA. As the degree of acetylation increases, the packaging density decreases in a linear fashion. Succinylated PEI is also observed to decrease packaging density at low degrees of modification, but, surprisingly, leads to increased packaging density at higher degrees of modification. Polyplexes in cells have been shown to experience a decrease in pH as particles are transported along the endosomal-lysosomal pathway. The interior of lysosomes (pH ~4.5-5.0) is considerably more acidic than the cytosol (pH~7.2). To mimic these cellular conditions, we also examined the DNA packaging of our polyplexes as a function of pH. For all three systems, tighter DNA packaging results when samples are condensed at pH 4 compared to pH 7.5. Both PEI modifications result in higher pH sensitivity with zPEI/DNA having the largest difference in packaging density as a function of pH. Lastly, we show that condensing DNA at pH 7.5 and then moving to pH 4 also results in tighter DNA packaging over the course of several hours. Together, our results give insight into how modifying PEI alters polymer-DNA interactions and its connection to enhanced gene delivery in vitro.

Jason Derouchey

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