Abstract
BackgroundThe complement system is a key component of innate immunity implicated in the neutralization and clearance of invading pathogens. Dextran coated superparamagnetic iron oxide (SPIO) nanoparticle is a promising magnetic resonance imaging (MRI) contrast agent. However, dextran SPIO has been associated with significant number of complement-related side effects in patients and some agents have been discontinued from clinical use (e.g., Feridex™). In order to improve the safety of these materials, the mechanisms of complement activation by dextran-coated SPIO and the differences between mice and humans need to be fully understood.Methods20 kDa dextran coated SPIO nanoworms (SPIO NW) were synthesized using Molday precipitation procedure. In vitro measurements of C3 deposition on SPIO NW using sera genetically deficient for various components of the classical pathway (CP), lectin pathway (LP) or alternative pathway (AP) components were used to study mechanisms of mouse complement activation. In vitro measurements of fluid phase markers of complement activation C4d and Bb and the terminal pathway marker SC5b-C9 in normal and genetically deficient sera were used to study the mechanisms of human complement activation. Mouse data were analyzed by non-paired t-test, human data were analyzed by ANOVA followed by multiple comparisons with Student-Newman-Keuls test.ResultsIn mouse sera, SPIO NW triggered the complement activation via the LP, whereas the AP contributes via the amplification loop. No involvement of the CP was observed. In human sera the LP together with the direct enhancement of the AP turnover was responsible for the complement activation. In two samples out of six healthy donors there was also a binding of anti-dextran antibodies and C1q, suggesting activation via the CP, but that did not affect the total level of C3 deposition on the particles.ConclusionsThere were important differences and similarities in the complement activation by SPIO NW in mouse versus human sera. Understanding the mechanisms of immune recognition of nanoparticles in mouse and human systems has important preclinical and clinical implications and could help design more efficient and safe nano-formulations.Electronic supplementary materialThe online version of this article (doi:10.1186/s12989-014-0064-2) contains supplementary material, which is available to authorized users.
Highlights
Superparamagnetic iron oxide (SPIO) is one of the most widely cited metal oxide nanoparticle that has been used as magnetic resonance imaging (MRI) contrast agent alone and as a component of multifunctional nanomedicines [1]
The same method was used for preparation of Feridex and other dextran superparamagnetic iron oxide (SPIO) [27,28], with the difference being that for Feridex manufacturing 10 kDa (T-10) dextran was used, whereas we used 20 kDa dextran
To verify the complement activation by SPIO nanoworms (SPIO NW) and Feridex in mouse serum, we incubated the particles in normal mouse serum at the concentration similar to the concentrations used in vivo (100 μg/mL serum, or 4 mg/kg body weight), washed multiple times by ultracentrifugation and analyzed for the presence of complement fragments in serum supernatant and on the purified particles
Summary
Nanoparticle synthesis and characterization SPIO NW was prepared by precipitation of Fe2+ and Fe3+ salts in ammonia in the presence of branched dextran of 20 kDa Mw (Sigma), as described elsewhere in the literature [27,28,47]. SPIO NW (final concentration 0.4 mg iron/ml in 1× PBS) was incubated with different dilutions of mouse sera (normally 10 μl particles and 30 μl serum) and incubated for 10–30 min at either room temperature of 37°C. To measure complement activation in vitro, we determined SPIOand dextran-induced rise of serum complement activation products C4d, Bb and SC5b-9 using respective ELISA kits (Quidel, San Diego) according to the manufacturer’s protocols as described previously [14,18,39,53]. For measurement of complement activation, the reaction was started by adding the required quantity of SPIO NW (or dextran) to undiluted serum in Eppendorf tubes (either in duplicate or triplicate, depending on experiment) in a shaking water bath at 37°C for 30 min, unless stated otherwise. Control incubations contained saline (the same volume as SPIO) for background measurement of complement activation products.
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