The effects of lipoplex formulation variables on the protein corona and comparisons with in vitro transfection...

The use of lipoplexes for the intracellular delivery of nucleic acids typically involves the optimization of several parameters that are known to affect delivery. Researchers commonly vary charge ratio, and often incorporate different amounts of helper lipids (e.g., cholesterol) to optimize formulations for transfection in cell culture and in vivo. The results of such experiments are often interpreted in the context of nuclease resistance and cell association, but effects on the protein corona are usually not considered. While many studies have demonstrated that lipoplex structure and function can be dramatically compromised in the presence of serum, little attention has been paid to the adsorption of specific proteins and how this might be affected by formulation parameters. In this study, we characterize changes in the protein corona that occur as DOTAP-based lipoplexes are formulated with different amounts of cholesterol and prepared at different charge ratios. Our results demonstrate a significant effect of lipid composition on both total protein adsorption as well as the individual proteins from fetal calf serum that are associated with lipoplexes. In addition, we show that PEGylation increases protein adsorption with our formulations; effects that depend on the type of PEG conjugate employed in the lipoplex. Attempts to identify a specific protein responsible for enhancing transfection were unsuccessful.