When nanoparticles are intravenously injected into the body, complement proteins deposit on the surface of nanoparticles in a process called opsonization. These proteins prime the particle for removal by immune cells and may contribute toward infusion-related adverse effects such as allergic responses. The ways complement proteins assemble on nanoparticles have remained unclear. Here, we show that dextran-coated superparamagnetic iron oxide core-shell nanoworms incubated in human serum and plasma are rapidly opsonized with the third complement component (C3) via the alternative pathway. Serum and plasma proteins bound to the nanoworms are mostly intercalated into the nanoworm shell. We show that C3 covalently binds to these absorbed proteins rather than the dextran shell and the protein-bound C3 undergoes dynamic exchange in vitro. Surface-bound proteins accelerate the assembly of the complement components of the alternative pathway on the nanoworm surface. When nanoworms pre-coated with human plasma were injected into mice, C3 and other adsorbed proteins undergo rapid loss. Our results provide important insight into dynamics of protein adsorption and complement opsonization of nanomedicines.

Balancing surface functionalization and low immune recognition of nanomedicines is a major challenge. Opsonization with the third component of the complement protein (C3) plays a major role in immune cell recognition of nanomedicines. We used dextran-coated superparamagnetic iron oxide nanoworms (SPIO NWs) to study the effect of surface functionalization on C3 opsonization in mouse serum and subsequent macrophage/leukocyte recognition in vitro as well as on intravenous injection into mice. Previously, we found that in mouse serum, SPIO NWs became opsonized with C3 via complement lectin pathway. Crosslinking the dextran shell with epichlorohydrin significantly decreased C3 opsonization and uptake by mouse peritoneal macrophages. Crosslinked nanoworms (NWs) further functionalized with polyethylene glycol (PEG) or with PEG-antibody (Ab) (~160 IgG molecules/particle) did not show an increase in C3 opsonization and peritoneal macrophage uptake in vitro . Following tail vein injection into mice, plain crosslinked NWs and PEGylated crosslinked NWs showed very low C3 opsonization and mouse leukocyte uptake. However, Ab-decorated crosslinked NWs showed significant C3 opsonization and high level of complement-dependent uptake by leukocytes in mice. Decreasing the number of conjugated Abs to 46 IgG molecules/particle significantly reduced C3 opsonization and leukocyte uptake. Using fresh mouse lepirudin plasma rather than serum showed better correlation with C3 opsonization in vivo . The reason for this difference could be related to the known instability of complement classical pathway in mouse sera. Our data illustrate that fine-tuning in nanoparticle surface functionalization with Abs is required to avoid excessive complement activation and complement-mediated immune uptake in mice, and raise issues with in vitro immunological assays of nanomedicines intended to mimic in vivo conditions.

Accumulation of intravenously injected cytotoxic liposomes in the skin induces serious toxicity. We used single time point and longitudinal intravital microscopy to understand skin accumulation dynamics of non-PEGylated and PEGylated liposomes after systemic injection into mice. Non-PEGylated egg phosphatidylcholine (PC) liposomes showed short circulation half-life (1.3 h) and immediate aggregation in the blood, with some aggregates lodging in skin microvasculature soon after the injection. At 24 h, and more prominently at 48 h postinjection, liposomes appeared in dermal and subdermal cells. PEGylated egg PC liposomes showed long circulation half-life (22 h) and no aggregation in the blood. PEGylated liposomes started to accumulate in the skin microvasculature as soon as 5 min after the injection. Within 3 h postinjection, PEGylated liposomes accumulated in extravascular cells in the dermis and subdermis. Liposomes were present in the skin for at least 7 days postinjection. A regulatory approved PEGylated liposomal doxorubicin (LipoDox) and empty liposomes of the same composition as LipoDox showed similar skin distribution as PEGylated egg PC liposomes, suggesting that this phenomenon is relevant to liposomes of different lipid composition. Decorating liposomes with shorter PEGs (350 or 700) in addition to PEG 2000 did not decrease the deposition. Outside the capillaries, liposomes partially colocalized with CD45−, F4/80+ cells. The accumulation of liposomes was not due to prior neutrophil/platelet binding and transport across endothelium. Moreover, our studies have excluded a role of complement in the skin accumulation of liposomes. Further understanding of mechanisms of this important phenomenon can improve the safety of liposomal nanocarriers.