Repeated three times. is an extensive effort to interrogate the phenotype of disease-associated macrophages via different imaging modalities with the hope that profiling of macrophages can help guide treatments and understand disease biology.4 A notable example is cancer where the inflammatory state of tumor associated Foretinib (GSK1363089, XL880) macrophages (TAMs) plays a critical role in therapy response.5 Additionally, macrophage reprogramming is an emerging therapeutic strategy.6 Of all imaging modalities, MRI appears to be the most versatile due to its excellent anatomical and spatial resolution, tissue contrast, and decreasing cost. Superparamagnetic iron oxides (SPIOs) are nanoparticle-based MRI contrast agents that consist of magnetite-maghemite (Fe3O4 and for 10 min at 4 C in a Beckman Optima TLX ultracentrifuge equipped with a TLA-100.3 Foretinib (GSK1363089, XL880) rotor. The pellets were resuspended in PBS, and 2 (0.02 (transfected with the plasmids) using QIAprep Miniprep Foretinib (GSK1363089, XL880) kit (Catalog# 27104, QIAGEN). CHO-K1 cells were transfected with the plasmid using Lipofectamine 3000 according to the manufacturers instructions. The details of the plasmids were described in full detail in our previous publication.26 Flow Cytometry. Macrophages were first incubated with rat antimouse CD16/32 antibody (as the mouse Fc-gamma blocker) and then stained with FITC antimouse/human CD11b and goat antimouse SR-AI (detected with AlexaFluor 647 chicken antigoat secondary antibody) in 1% BSA and 0.1% sodium azide in 1 PBS. Cells were resuspended at ~0.5 million/mL and 20,000 events were acquired with Guava EasyCyte HT flow cytometer (Merck KGaA). The data were analyzed and plotted using FlowJo version 10. Uptake of Nanoparticle and Cell Staining. Mouse PMs and BMDM were plated in 96-well plates. PMs and three types of BMDM including M0, M1, and M2 were incubated with Feraheme at 0.1 mg/mL in the presence of wild type (WT) mouse serum with or without PIA 20 Uptake of Nanoparticle and Histology. The animal studies were approved by Institutional Animal Care and Use Committee (IACUC) of the University of Colorado. Wild-type BALB/c mice were bred in house. For uptake of Feraheme by PMs, PBS (25 test. P values 0.05 were considered as statistically significant. RESULTS Scavenger Receptor SR-AI/II, but Not Complement, Mediates Recognition of Feraheme by Mouse Peritoneal Macrophages. We prepared small (60 nm) SPIO NWs, large (120 nm) SPIO NWs, and USPIO (30 nm). TEM images of all particles used in the study are shown in Figure 1A. In order to understand the role of the complement in the uptake of Feraheme, we isolated nonactivated peritoneal macrophages (PMs) from BALB/c mice and exposed them to 0.1 mg Fe/mL Feraheme, 60 nm (small) S-SPIO NW, and 120 nm (large) L-SPIO NW for 3C6 h in the presence of wild type (WT) mouse sera or sera deficient for complement C3. Prussian blue staining (Figure 1B) and colorimetric image quantification (Figure 1C) showed a high level of uptake of all nanoparticle types. Open in a separate window Figure 1. Role of the complement in the uptake of different iron oxide nanoparticles: (A) TEM images of Foretinib (GSK1363089, XL880) 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 Mctp1 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 test. = 3, repeated three times. (E) Smaller nanoparticles accommodate fewer C3b molecules and theoretically cannot accommodate the bulky complement convertase (C3Bb-properdin). NanoparticleCprotein real size models of C3b and C3 convertase were described before.23 Size bar, 50 and Feraheme uptake by polyinosinic acid (PIA), a well established SR inhibitor. Repeated three times. (D,E) Blockade of Feraheme uptake by PIA following intrapertioneal injection. Two-sided test. = 3, repeated three times. Size bar, 50 = 3, repeated two times. (C) Immunostaining of PM with anti-SR-AI/II antibody. (D) Uptake.