Complement fixation to surface-conjugated ligands plays a critical role in determining the fate of targeted colloidal particles after intravenous injection. and then mixed with fluorescent antibodies specific for various serum components. We found that complement C3/C3b was the main human serum ZM323881 factor to bind 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 ZM323881 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. 1 Introduction In recent years molecularly targeted contrast-enhanced ultrasound has received increasing attention as a diagnostic imaging modality that allows the detection and evaluation of endothelial biomarkers associated with vascular events underlying specific pathologies [1-7]. For such applications targeted contrast agents are injected intravenously into the bloodstream where they accumulate at targeted sites along the vascular endothelium. When imaged with ultrasound  ZM323881 these bound contrast agents provide an acoustic signal and therefore allow the measurement of specific endothelial receptor expressions that are upregulated. Ultrasound molecular imaging has thus been applied to the assessment of tumor angiogenesis [9-11] thrombosis [12 13 atherosclerosis  and inflammation [15 16 Ultrasound contrast agents are typically gas-filled colloidal particles (microbubbles) with diameters less than 10 μm. The surface comprises amphiphilic phospholipids self-assembled to form a lipid monolayer shell. Microbubbles can provide sensitive acoustic responses when detected using ultrasound because of their compressible gas cores [7 17 Similar to the design of long-circulating liposomes poly(ethylene glycol) (PEG) chains or PEG chain derivatives can be incorporated into the shell of microbubbles in ZM323881 order to form a steric barrier against coalescence and adsorption of macromolecules such as antibodies to the microbubble surface [18 19 These agents owing to their small sizes can pass through the pulmonary vasculature  and have been shown to exhibit contrast persistence longer than 10 min . When administered intravenously microbubbles or other conventional colloidal particles are rapidly removed from the bloodstream by the mononuclear phagocyte system (MPS) . The MPS protects the systemic circulation by distinguishing foreign and endogenous substances and the fast clearance of foreign particles is mediated through endocytosis with recognition of specific cell surface receptors such as complement receptor 1 (CR1) and Fc receptor [23 24 Endocytosis is classified into three categories: receptor-mediated endocytosis (RME) pinocytosis and phagocytosis . Depending on the size of the particle it can be eliminated from the system either through RME and/or pinocytosis (for small compounds) or phagocytosis (for large particles such as microbubbles). Evidence of microbubble phagocytosis has been demonstrated both  and [27 28 Although ZM323881 not required phagocytosis is often triggered by specific receptor recognition and such ligand-receptor interactions typically exist between the cellular receptor specific for the proteins bound to the colloidal particles rather than for the particles themselves. Thus serum protein adsorption is extremely important in determining particle uptake by ZM323881 phagocytes and predicting the fate of colloidal Rabbit Polyclonal to CSTL1. particles after administration. Immunoglobulin G (IgG) and complement components are known as major opsonins for the uptake of large particles such as bacteria viruses and remnants of dead cells. In particular complement activation plays a critical role in the recognition of biocolloids by the immune system . The complement system consisting of over 30 soluble plasma and cell-surface bound proteins is an important effector arm of innate.