Statistical analysis is explained in Methods. Next, we tested the role of immunoglobulins in C3 opsonisation of clinically approved liposomal nanomedicines. binding to all nanoparticle types. We therefore show that natural antibodies represent a link between biomolecule corona and C3 opsonisation, and may determine individual complement responses to nanomedicines. Keywords:complement, immunoglobulin, nanoparticle, biomolecule corona, opsonisation, SPIO, liposome, iron oxide, Doxil Significant advances in development of engineered nanomaterials have resulted in several nanopharmaceuticals for clinical use and many more in clinical development stage.1Nevertheless, there are still important issues that need to be resolved, including non-specific clearance of functional nanoparticles by different immune cell types and life threatening infusion reactions that occur in some patients receiving nanopharmaceuticals.2Better understanding of immune mechanisms that lead to the clearance and toxicities of nanomaterials in general, and of clinically approved nanopharmaceuticals in particular, should be one of the primary tasks in nanomedicine research and development. Plasma protein adsorption has long been debated to be an important factor affecting functionality of intravenously injected nanomedicines.3,4,5Recent studies have demonstrated the role of absorbed proteins in nanoparticle targeting,6immune responses,7and intracellular toxicity.8,9Among the most interesting immunological constituents of protein corona are components of the complement system. Upon activation, all three pathways Loganic acid (alternative, classical, and lectin) of the complement system converge at point where the third complement protein (C3) is usually cleaved through assembly of C3 convertases, which generate a common set of effector molecules including C3a and C3b.2,10The latter fragment covalently bind to activating surfaces thereby facilitating their recognition by leukocytes and macrophages; a process termed opsonisation. C3b is usually further involved in surface assembly of C5 convertase that in turn cleaves the fifth complement protein (C5) into C5a and C5b, thereby triggering activation of the terminal pathway of the complement system. Both C3a and C5a are potent anaphylatoxins, which activate mast cells and other immune cells, and therefore induce proinflammatory reactions. While the role of complement in infusion reactions to nanomedicine is being debated,11there is usually a concern that uncontrolled complement activation may affect the disease outcome and nanomedicine performance in general.12,13,14 Since opsonisation with C3 is the central event in complement cascade and immune recognition, mechanisms of C3 deposition on nanomedicines are of special interest for biological performance.2Earlier we found that C3b preferentially binds to the biomolecule corona on superparamagnetic iron oxide nanoworms (SPIO NWs) in both plasma and sera. Moreover, C3 deposition was significantly enhanced in the presence of protein corona, underlying the dependence of the complement activation on other, less characterised corona components.15SPIO is an important magnetic resonance imaging contrast agent and a component of multifunctional theranostic nanomedicines for imaging and treatment.16,17Furthermore, we demonstrated a significant between-subject variability in levels of C3 deposition on SPIO NWs, as well as clinically approved nanomedicines including intravenous iron supplement Feraheme, PEGylated liposomal doxorubicin LipoDoxand non-PEGylated liposomal irinotecan Onivyde.18We hypothesise that complement activation efficiency by these nanomaterials is determined by differences in biomolecule corona composition. Among many plasma proteins, natural antibodies such as IgG and IgM are known to bind to foreign and self-antigens, and modulate complement activation through the three complement pathways.19,20,21,22Here we demonstrate that binding of only a few immunoglobulin molecules, and specifically IgG, determines the efficiency of C3 deposition Loganic acid around the above-mentioned nanomaterials in plasma and sera of healthy donors and in plasma of cancer patients, regardless of the complement activation pathway. Moreover, we demonstrate that the Loganic acid presence of the biomolecule corona enhances IgG binding to all tested nanomaterials. These results establish natural antibodies MAPKAP1 as a critical factor that determines the efficiency of complement activation in different subjects and by different nanomaterials and can promote development of clinically approved nanoparticles with improved safety profile. == 1. Immunoglobulins promote C3 deposition on nanomedicines == We prepared ~110 nm, 20kDa dextran-coated SPIO NWs (Fig. 1aandSupplementary Table 1) as described previously.15,23In order.