The role of poly(ethylene glycol) brush architecture in complement activation on targeted microbubble surfaces

Complement fixation to surface-conjugated ligands plays a critical role in determining the fate of targeted colloidal particles after intravenous injection. In the present study, we examined the immunogenicity of targeted microbubbles with various surface architectures and ligand surface densities using a flow cytometry technique. Targeted microbubbles were generated using a post-labeling technique with a physiological targeting ligand, cyclic arginine-glycine-asparagine (RGD), attached to the distal end of the poly(ethylene glycol) (PEG) moieties on the microbubble surface. Microbubbles were incubated in human serum, washed and then mixed with fluorescent antibodies specific for various serum components. We found that complement C3/C3b was the main human serum factor to bind in vitro to the microbubble surface, compared to IgG or albumin. We also investigated the effect of PEG brush architecture on C3/C3b fixation to the microbubble surface. RGD peptide was able to trigger a complement immune response, and complement C3/C3b fixation depended on microbubble size and RGD peptide surface density. When the targeting ligand was attached to shorter PEG chains that were shielded by a PEG overbrush layer (buried-ligand architecture), significantly less complement activation was observed when compared to the more traditional exposed-ligand motif. The extent of this protective role by the PEG chains depended on the overbrush length. Taken together, our results confirm that the buried-ligand architecture may significantly reduce ligand-mediated immunogenicity. More generally, this study illustrates the use of flow cytometry and microbubbles to analyze the surface interactions between complex biological media and surface-engineered biomaterials.