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

Abstract

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.

Questioning the use of PEGylation for drug delivery

“…In fact, several studies have reported that PEGylation increases protein adsorption, suggesting that the binding of specific proteins involved in clearance might be preferentially inhibited by PEG [13,14]. Alternatively, it has been suggested that PEG may preferentially bind proteins that function as “dysopsonins”, i.e., proteins that prevent opsonization [11].…”

“…Consistent with the studies cited above, Figure 1 shows data from our laboratory indicating that PEGylation of lipoplexes dramatically increases the extent of serum protein binding compared to non-pegylated lipoplexes. Furthermore, our recent publication also demonstrates that small proteins (potentially protein fragments) bind to PEGylated nanoparticles that are not adsorbed to particles lacking PEG [14]. While it has been shown that the density/conformation of PEG molecules on the surface affects protein binding [6,11,15], studies demonstrating enhanced serum protein binding and the adsorption of specific proteins after PEGylation raise significant questions about the ability of PEG to prevent/reduce interactions with serum proteins.…”

Nonadditive Effects of Repetitive Administration of Lipoplexes in Immunocompetent Mice

“…In addition to the lipoplex formulation described above, mice were treated with phosphate buffered saline (negative control), lipoplexes formulated with 5% DSPE-PEG2000 (Avanti), CpG-containing plasmid (Valentis, Inc.), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP; Avanti) instead of sphingosine, or Lipofectamine 2000 (Invitrogen, Carlsbad, CA) as previously described [19, 20, 22, 25]. In these studies, a single mouse was treated with each formulation as described above, and blood was collected in Eppendorf tubes 2 h after the second injection of lipoplexes.…”

Therapeutic and diagnostic applications of extracellular vesicles

“…Besides the changes in the composition of the EV itself, the presence or absence of serum proteins will likely also influence the protein corona surrounding the EVs . It is well documented that this corona strongly influences the extra-and intracellular (transfection) behavior of synthetic nanoparticles, including liposomes [56,57]. Given the analogy between EVs and liposomes [58] it is conceivable that a protein corona will also impact the EV interactome and hence biological function.…”