Soluble sCR1 (SCR1C10 of CD35) was generously provided by Alexion Pharmaceuticals, Inc. we demonstrate here that complement therapeutics can be used to improve targeting selectivity. INTRODUCTION Antibody-decorated superparamagnetic iron oxide (SPIO) nanoparticles hold an important niche in biomedical research and nanomedicine. On the CORO2A one hand, they have been successfully utilized for magnetic isolation of a variety of targets, including immune cells and circulating tumor cells from blood and cell suspensions, as well as molecular targets in pulldown assays.1,2 Immunomagnetic isolation is frequently used in clinical procedures and assays, for example, in the production of Chimeric Antigen Receptor (CAR)-T cells3 and for analysis of circulating tumor cells (CTCs).4 On the other hand, due to high magnetic resonance imaging contrast properties,2,5 there is a considerable interest in using iron oxide particles targeted to disease markers for molecular imaging.6 Dextran-coated SPIO has been used clinically in patients as an MRI contrast agent and iron supplement, in part due to the scalability and low cost of synthesis.2 Cross-linking the dextran coat with epichlorohydrin leads to the formation of three-dimensional hydrogel coated iron oxide (termed CLIO) with important chemical and biochemical properties: (a) cross-linked hydrogel improves NP stability in plasma and prolongs circulation time in mice;7,8 and (b) residual epoxy groups can be used for further functionalization p32 Inhibitor M36 of CLIO with amines, fluorophores, peptides, and antibodies. CLIO has been functionalized with radioisotopes, fluorophores, and sensor modules, which targeted antibodies for diagnostic and theranostic applications in cancer, inflammation, diabetes, and atherosclerosis.6,9C11 While CLIO represents an impressive and highly versatile platform for in vivo imaging, and notwithstanding the success in preclinical mouse studies, there is a gap in the fundamental understanding of how surface functionalization of CLIO with antibodies and imaging molecules affects immune recognition in humans. Targeting specificity of NPs is usually validated by comparing antibody conjugated and control p32 Inhibitor M36 formulations, and using the cells with and without the targeting marker.12C14 However, another level of specificity, which is often overlooked for targeted nanoparticles, is the level of uptake by immune cells.15,16 It is highly important to reduce nonspecific recognition of targeted nanoparticles by immune cells to improve target/background ratio p32 Inhibitor M36 and specificity,15,17 but the strategies to avoid immune uptake are mostly limited to modifications of surface chemistry. Complement is the critical arm of serum innate immunity responsible for neutralization of foreign pathogens. The exposure of foreign surfaces to serum results in a rapid generation of C3 and C5 convertases that promote opsonization through covalent attachment of C3b and the formation of anaphylatoxins (e.g., C3a and C5a).18 C3b and its cleavage products iC3b, C3dg, and C 3d promote recognition by complement receptors on neutrophils, monocytes, eosinophils, lymphocytes, erythrocytes, and resident tissue macrophages.19C21 Complement is one of p32 Inhibitor M36 the factors negatively affecting the hemocompatibility of nano- and biomaterials. Many reports have shown complement activation by nanoassemblies including carbon nanotubes,22,23 micelles,24 liposomes,25 polymeric nanospheres,26,27 gold NPs,28 and SPIO.29 Here, we used previously described elongated CLIO nanoworms (CLIO NWs)7 to understand the involvement of complement in immune recognition of targeted iron oxides in humans. The easiest way to test hemocompatibility in different human subjects is to use donated anticoagulated blood. Lepirudin (recombinant hirudin) is the selective inhibitor of thrombin and to our knowledge is one of the few anticoagulants that does not interfere with complement activation, as compared to EDTA or citrate.30 CLIO NWs were modified with p32 Inhibitor M36 antibodies against tumor cell marker Her2/neu (over 20% of breast cancers31) and EpCAM (epithelial marker on circulating tumor cells32) and were rigorously characterized to correlate the composition to the biological outcome. Our results suggest that the alternative pathway plays the predominant role in the complement C3 opsonization regardless of surface functionalization and that addition of specific complement inhibitors can dramatically improve targeting selectivity in human blood. This study introduces a novel notion of using complement inhibitors for improving stealth properties and the selectivity of targeted nanomaterials. RESULTS CLIO NWs Show High Targeting Efficiency in BSA and Plasma. We used dextran SPIO NWs (62 nm, ?5 mV, Table 1)33,34 to synthesize cross-linked CLIO NWs (51 nm, ?5 mV, Table 1) by a modified harsh cross-linking method7,8 (see Methods). The cross-linked CLIO NWs were aminated by reacting the residual epoxy groups of epichlorohydrin with ammonia (56 nm, +15.