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. 2019 Oct 7;16(10):4274-4281.
doi: 10.1021/acs.molpharmaceut.9b00632. Epub 2019 Sep 26.

Feraheme (Ferumoxytol) Is Recognized by Proinflammatory and Anti-inflammatory Macrophages via Scavenger Receptor Type AI/II

Guankui Wang  1 , Natalie J Serkova  1   2 , Ernest V Groman  1 , Robert I Scheinman  1 , Dmitri Simberg  1
Affiliations

Feraheme (Ferumoxytol) Is Recognized by Proinflammatory and Anti-inflammatory Macrophages via Scavenger Receptor Type AI/II

Guankui Wang et al. Mol Pharm. .

Abstract

Feraheme (ferumoxytol), a negatively charged, carboxymethyl dextran-coated ultrasmall superparamagnetic iron oxide nanoparticle (USPIO, 30 nm, -16 mV), is clinically approved as an iron supplement and is used off-label for magnetic resonance imaging (MRI) of macrophage-rich lesions, but the mechanism of recognition is not known. We investigated mechanisms of uptake of Feraheme by various types of macrophages in vitro and in vivo. The uptake by mouse peritoneal macrophages was not inhibited in complement-deficient serum. In contrast, the uptake of larger and less charged SPIO nanoworms (60 nm, -5 mV; 120 nm, -5 mV, respectively) was completely inhibited in complement deficient serum, which could be attributed to more C3 molecules bound per nanoparticle than Feraheme. The uptake of Feraheme in vitro was blocked by scavenger receptor (SR) inhibitor polyinosinic acid (PIA) and by antibody against scavenger receptor type A I/II (SR-AI/II). Antibodies against other SRs including MARCO, CD14, SR-BI, and CD11b had no effect on Feraheme uptake. Intraperitoneally administered PIA inhibited the peritoneal macrophage uptake of Feraheme in vivo. Nonmacrophage cells transfected with SR-AI plasmid efficiently internalized Feraheme but not noncharged ultrasmall SPIO of the same size (26 nm, -6 mV), suggesting that the anionic carboxymethyl groups of Feraheme are responsible for the SR-AI recognition. The uptake by nondifferentiated bone marrow derived macrophages (BMDM) and by BMDM differentiated into M1 (proinflammatory) and M2 (anti-inflammatory) types was efficiently inhibited by PIA and anti-SR-AI/II antibody. Interestingly, all BMDM types expressed similar levels of SR-AI/II. In conclusion, Feraheme is efficiently recognized via SR-AI/II but not via complement by different macrophage types. The recognition by the common phagocytic receptor has implications for specificity of imaging of macrophage subtypes.

Keywords: bone marrow; iron oxide; macrophage; nanoworms; scavenger receptor; uptake.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1.
Figure 1.
Role of the complement in the uptake of different iron oxide nanoparticles: (A) TEM images of particles prepared for the study. Size bar, 50 nm. (B) Uptake by peritoneal macrophages (Prussian blue) in wild type or C3 knockout sera. (C) Prussian blue quantification shows inhibition of uptake of small and large SPIO NWs, but not of Feraheme, in C3 KO serum. Repeated four times. (D) Measurement of mouse C3 per nanoparticle (in arbitrary units) shows much less deposition on Feraheme and ultrasmall SPIO than on SPIO NWs. EDTA (10 mM) is the inhibitor of all complement pathways. Two-sided t test. N = 3, repeated three times. (E) Smaller nanoparticles accommodate fewer C3b molecules and theoretically cannot accommodate the bulky complement convertase (C3Bb-properdin). Nanoparticle–protein real size models of C3b and C3 convertase were described before. Size bar, 50 μm for all images.
Figure 2.
Figure 2.
Uptake of Feraheme by peritoneal macrophages is mediated by scavenger receptors in vitro and in vivo. (A) Scavenger receptors are a large group of phagocytic receptors recognizing molecular patterns, mostly negatively charged. (B–C) Prussian blue staining and image quantification show blockage of in vitro Feraheme uptake by polyinosinic acid (PIA), a well established SR inhibitor. Repeated three times. (D,E) Blockade of in vivo Feraheme uptake by PIA following intrapertioneal injection. Two-sided t test. N = 3, repeated three times. Size bar, 50 μm for all images.
Figure 3.
Figure 3.
SR-AI/II recognizes Feraheme in vitro. (A,B) Prussian blue and image quantification show that SR-AI/II antibody almost completely abolished the uptake by peritoneal macrophages. Other blocking antibodies did not have any effect on the uptake. N = 3, repeated two times. (C) Immunostaining of PM with anti-SR-AI/II antibody. (D) Uptake of Feraheme by CHO cells transfected with SR-AI plasmid. Repeated three times. Size bar, 50 μm for all images.
Figure 4.
Figure 4.
Expression of SR-AI/II by bone marrow derived macrophages. (A) BMDM differentiation scheme. M1 macrophages are rounded, expressing iNOS and not Arg1, whereas M2 macrophages are elongated, expressing Arg1 and not iNOS. (B) Flow cytometry of BMDM stained for CD11b and SR-AI/II. Upper row, nonstained cells. (C) Confocal microscopy of BMDM stained for CD11b and SR-AI/II.
Figure 5.
Figure 5.
SR-AI/II mediates uptake of Feraheme by BMDM. (A) Representative Prussian blue images and (B) image quantification shows inhibition by PIA and anti-SR-AI/II antibody, regardless of the serum type. WT vs SR-AI/II Ab: p-value 0.012 for M0, M1, M2; two-tailed t test, n = 5, repeated three times. Size bar 50 μm for all images.
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