Recognition of dextran-superparamagnetic iron oxide nanoparticle conjugates (Feridex) via macrophage scavenger receptor charged domains - PubMed (original) (raw)

. 2012 May 16;23(5):1003-9.

doi: 10.1021/bc200685a. Epub 2012 Apr 26.

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Recognition of dextran-superparamagnetic iron oxide nanoparticle conjugates (Feridex) via macrophage scavenger receptor charged domains

Ying Chao et al. Bioconjug Chem. 2012.

Abstract

Dextran-coated superparamagnetic iron oxide nanoparticles (dextran-SPIO conjugates) offer the attractive possibility of enhancing MRI imaging sensitivity so that small or diffuse lesions can be detected. However, systemically injected SPIOs are rapidly removed by macrophages. We engineered embryonic cells (HEK293T) to express major macrophage scavenger receptor (SR) subtypes including SR-AI, MARCO, and endothelial receptor collectin-12. These SRs possess a positively charged collagen-like (CL) domain and they promoted SPIO uptake, while the charge neutral lipoprotein receptor SR-BI did not. In silico modeling indicated a positive net charge on the CL domain and a net negative charge on the cysteine-rich (CR) domain of MARCO and SR-AI. In vitro experiments revealed that CR domain deletion in SR-AI boosted uptake of SPIO 3-fold, while deletion of MARCO's CR domain abolished this uptake. These data suggest that future studies might productively focus on the validation and further exploration of SR charge fields in SPIO recognition.

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Figures

Fig. 1

Fig. 1. Physical characterization of Feridex (SPIO nanoparticles)

(A) SPIO nanoparticle size (intensity) distribution. Particles were processed as described in Methods; (B) Zeta potential of particles; (C), TEM of SPIO nanoparticle (Feridex), magnification: 60,000×. Size bar = 100 μm. Inset, cropped area showing a representative nanoparticle consisting of a cluster of electron-dense magnetite-maghemite crystals (white arrow on the main image).

Fig. 2

Fig. 2. SR ligand tests confirm SPIO uptake mediated by SRs

SPIO nanoparticle uptake by the macrophages in the presence of various inhibitors (n=3–5). Addition of polyanionic SR ligands fucoidan (10 μg/ml), and dextran sulfate (3 μg/ml) to J774A.1 macrophages prior to SPIO resulted in 65% – 76% inhibition of uptake (non-parametric Mann-Whitney test, p value=0.0003, n=3). The uptake was also inhibited by 1mg/ml gelatin. These data suggest that the mechanism of SPIO nanoparticle uptake is SR-dependent and may be based on electrical charge. Pretreatment of J774A.1 cells with 0.1 mg/ml trypsin reduced the uptake by 80% confirming a protein receptor is needed.

Figure 3

Figure 3. SPIO nanoparticle uptake of transiently expressed macrophage SRs

HEK293T cells were transiently transfected with the same amounts of receptor-coding plasmids and then incubated with 0.1 mg/ml SPIO (Feridex) in 10% FBS-supplemented medium for 2 h. (A) Uptake of SPIO by cells transfected with SRs quantified with QuantiChrom™ Iron Assay Kit (see Methods), p-value 0.0007, non-paired t-test, n=5; Only receptors with charged domains show uptake. (B) Example images of cells were stained with Prussian Blue and Nuclear Fast Red and the images were taken at low magnification (4× objective). A representative experiment out of five is shown. Size bar = 200 μm.

Figure 4

Figure 4. SPIO binding to collagen-coated surface

(A) Schematic representation of SRs based on previously published literature {Taylor, 2005 #37;Ohtani, 2001 #599;Shimaoka, 2004 #544}. SR-AI, MARCO and CL-P1 contain collagen-like domain (stem) in the extracellular part. (B) In order to mimic the binding to cationic collagenous receptors, microplate wells were coated with charged collagen type I, and SPIO nanoparticles were incubated in PBS as described in Methods. The binding was only to collagen-coated wells (open bars) and not to BSA coated wells (solid bars). The binding was inhibited by 10 μg/ml dextran sulfate (striped bars).

Figure 5

Figure 5. Comparison of SPIO uptake by SR-AI and MARCO

(A) Upper panel: SPIO uptake by receptor-transfected HEK293T cells. The experiment was performed as in Fig. 3B. Cropped areas are shown. Size bar=50μm. Lower panel: Staining of the receptors on the cell surface (described in Methods). (B) Western blotting of SR-AI and MARCO after cell fractionation. Both cytoplasmic and membrane fractions are positive for the receptors. Majority of the receptor is in intracellular compartment. HSP90 and Na+, K+ ATPase staining confirms fractionation efficiency. (C) SPIO binding to cells at 4°C shows the difference in binding efficiency between SR-AI and MARCO.

Figure 6

Figure 6. Molecular models of SRs and their electrostatic profiles

Electrostatic profiles of mouse MARCO and mouse SR-AI receptors are shown as equipotential surfaces for the values of +1.8 (blue) or −1.8 (red) charges. Note the higher positive charge density of SR-AI and MARCO CL-domain per unit length versus the overall negative charge of the C-terminal CR-domain (red color). Insert: Ribbon presentation of the 3D models of MARCO and SR-AI receptors used for charge profile reconstruction. Different colors denote the three subunits of each receptor.

Figure 7

Figure 7. Modifications and expression of SR-AI and MARCO

(A) CR DOMAIN of SR-AI was deleted. The charged collagen sequence from SR-AI was fused to C-terminal portion of MARCO's CL DOMAIN. CR DOMAIN of MARCO was deleted. (B) SRs were expressed on HEK293T cells and the levels of expression were verified by western blotting. (C) To demonstrate equal efficiency of transfection, the cells were stained with antibodies against the SRs. High magnification images of MARCO receptor staining in the membrane are provided in Supplemental data.

Fig. 8

Fig. 8. CL-CR domains influence SR mediated SPIO uptake in vitro

The uptake of the constructs shown in Fig. 6 was quantified with an iron assay. (A) SR-AI and SR-AII mediated uptake. Deletion of SR-AI CR DOMAIN increases the SPIO uptake 3-fold (non-parametric Mann-Whitney test, p-value 0.0026, n=3). (B) Binding of SPIO to SR-AI and SR-AII transfected HEK293T cells at 4°C. Cells were grown, transfected in 24 well plates and incubated with 0.3 mg/ml SPIO at 4°C for 15 min. Results of a triplicate measurement is shown. (C) MARCO-mediated uptake. Addition of the charged CL to MARCO increased the uptake two-fold, (non-parametric Mann-Whitney test, p-value 0.013, n=5) but the uptake was still much lower than that of SR-AI cells. Deletion of the CR DOMAIN in MARCO completely blocked SPIO uptake.

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