Complement Inhibitors Block Complement C3 Opsonization and Improve Targeting Selectivity of Nanoparticles in Blood

Abstract

Complement is one of the critical branches of innate immunity that determines the recognition of engineered nanoparticles by immune cells. Antibodytargeted iron oxide nanoparticles are a popular platform for magnetic separations, in vitro diagnostics, and molecular imaging. We used 60 nm cross-linked iron oxide nanoworms (CLIO NWs) modified with antibodies against Her2/neu and EpCAM, which are common markers of blood-borne cancer cells, to understand the role of complement in the selectivity of targeting of tumor cells in whole blood. CLIO NWs showed highly efficient targeting and magnetic isolation of tumor cells spiked in lepirudin-anticoagulated blood, but specificity was low due to high uptake by neutrophils, monocytes, and lymphocytes. Complement C3 opsonization in plasma was predominantly via the alternative pathway regardless of the presence of antibody, PEG, or fluorescent tag, but was higher for antibody-conjugated CLIO NWs. Addition of various soluble inhibitors of complement convertase (compstatin, soluble CD35, and soluble CD55) to whole human blood blocked up to 99% of the uptake of targeted CLIO NWs by leukocytes, which resulted in a more selective magnetic isolation of tumor cells. Using well-characterized nanomaterials, we demonstrate here that complement therapeutics can be used to improve targeting selectivity.

Antibody-Dependent Complement Responses toward SARS-CoV-2 Receptor-Binding Domain Immobilized on “Pseudovirus-like” Nanoparticles

“…To test the role of complement, we used 10 μg/mL sCR1, which potently and specifically inhibits complement-dependent uptake in whole blood. 34 The uptake of nanoparticles in all donors was almost completely inhibited by sCR1 ( Figure 6 C). According to the CD11b staining and forward-side scattering plots ( Figure 6 D,E), CD11b+ cells (mostly granulocytes and monocytes) were the predominant cell type positive for CLIO-RBD, whereas CD11b– cells (mostly lymphocytes) showed much less efficient binding/uptake in both donors with high anti-RBD titers ( Figure 6 D,E).…”

“…The resulting protein was partially in a dimeric form (Figure A), likely due to free cysteines in the sequence . To conjugate RBD to nanoparticles, we used 60 nm aminated cross-linked iron oxide nanoworms (CLIO NWs) that show excellent stability, are amenable to ligand and fluorophore modification, and can be easily purified from serum or blood for subsequent biological assays . The particles were first reacted with ∼100 molecules of Cy5 and ∼2000 molecules of NHS-PEG2000-nitrilotriacetic acid (NTA) and then with NiCl 2 to produce CLIO NW-NTA-Ni 2+ (Figure B, hereafter CLIO-NTA-Ni 2+ ).…”

“…At the same time, in the blood with high anti-RBD IgG titer, there was an increased uptake of CLIO-RBD compared to CLIO-NTA-Ni 2+ (Figure C). To test the role of complement, we used 10 μg/mL sCR1, which potently and specifically inhibits complement-dependent uptake in whole blood . The uptake of nanoparticles in all donors was almost completely inhibited by sCR1 (Figure C).…”

Exploring and Analyzing the Systemic Delivery Barriers for Nanoparticles

“…[ 240,241 ] Inhibiting C3 opsonization through complement convertase inhibitors, like compstatin, soluble CD35, and soluble CD55 have effectively blocked the sequestration of iron oxide nanoparticles by leukocytes. [ 242 ] In another work, soluble CD55 successfully suppressed the C3 opsonization in clinically approved nanomedicines, including Feraheme, LipoDox, and Onivyde. [ 243 ] It is worth noting that these experiments were conducted in vitro using human blood, and further in vivo investigations are essential to more comprehensively assess the safety.…”

The Nano-War Against Complement Proteins

“…The other approach to combating C3 opsonization of nanocarriers has been to infuse complement inhibitors before or along with nanocarriers. For example, SPIONs conjugated to targeting antibodies elicit strong C3b opsonization in whole blood and are therefore taken up rapidly by leukocytes, but multiple C3-convertase inhibitors (compstatin, soluble C35, and various fragments of CD59) blocked C3a and C5a production, and prevented 99% of leukocyte uptake of the SPIONs [34,35]. Additionally, infusion of the natural, soluble complement inhibitor Factor H, dramatically reduced complement activation in whole blood exposed to the clinically used nanocarriers AmBisome (a liposome) and Cremophor EL (a polymeric detergent used to solubilize many chemotherapeutics, which forms micelles) [36].…”