Anti-CD34-Grafted Magnetic Nanoparticles Promote Endothelial Progenitor Cell Adhesion on an Iron Stent for Rapid Endothelialization

Abstract

Iron stents, with superior mechanical properties and controllable degradation behavior, have potential for use as feasible substitutes for nondegradable stents in the treatment of coronary artery occlusion. However, corrosion renders the iron surface hard to modify with biological molecules to accelerate endothelialization and solve restenosis. The objective of this study is to demonstrate the feasibility of using endothelial progenitor cells (EPCs) to rapidly adhere onto iron surfaces with the assistance of anti-CD34-modified magnetic nanoparticles. Transmission electron microscopy, Fourier transform infrared spectroscopy, Thermogravimetric analysis, XRD, and anti-CD34 immunofluorescence suggested that anti-CD34 and citric acid were successfully modified onto Fe₃O₄, and Prussian blue staining demonstrated the selectivity of the as-prepared nanoparticles for EPCs. Under an external magnetic field (EMF), numerous nanoparticles or EPCs attached onto the surface of iron pieces, particularly the side of the iron pieces exposed to flow conditions, because iron could be magnetized under the EMF, and the magnetized iron has an edge effect. However, the uniform adhesion of EPCs on the iron stent was completed because of the weakening edge effect, and the sum of adherent EPCs was closely linked with the magnetic field (MF) intensity, which was validated by the complete covering of EPCs on the iron stent upon exposure to a 300 mT EMF within 3 h, whereas almost no cells were observed on the iron stent without an EMF. These results verify that this method can efficiently promote EPC capture and endothelialization of iron stents.

Figure 1

(A) Because of ferromagnetism, magnetization under an EMF converts ferromagnetic domains in iron from a random arrangement to be parallel to the EMF. (B) Schematic diagram of EPCs expressing the CD34 membrane protein and anti-CD34-coated MNPs and EPCs magnetically tagged by anti-CD34-coated MNPs. (C) Superimposition of the EMF and the responsive field of the magnetizing iron enables the iron stent to magnify the surrounding MF intensity and augment the quantity of captured MNP-tagged EPCs for rapid endothelialization.

Figure 2

TEM micrographs of Fe₃O₄, Fe₃O₄@CA, and Fe₃O₄@CA–CD34.

Figure 3

(A) FTIR spectra of Fe₃O₄, Fe₃O₄@CA, and Fe₃O₄@CA–CD34. TGA analysis (B), XRD spectra (C) and magnetic hysteresis loops (D) of different kinds of Fe₃O₄, Fe₃O₄@CA, and Fe₃O₄@CA–BSA (because of its high price, anti-CD34 antibody was replaced by BSA in these analysis).

Figure 4

Bright-field microscopy (A) and immunofluorescence (B) images of Fe₃O₄@CA, and the bright-field microscopy (C) and immunofluorescence (D) images of Fe₃O₄@CA–CD34. EPCs were incubated with Fe₃O₄@CA (E) and Fe₃O₄@CA–CD34 (F), and then cellular labeling was visualized histochemically with prussian blue (PB) staining, which indicated that anti-CD34-coated MNPs have selective affinity to EPCs.

Figure 5

Microphotographs of the EPCs incubated with different concentrations of Fe₃O₄@CA–CD34 [(A) 0 μg/mL; (B) 100 μg/mL; (C) 500 μg/mL] for 3 days. Cell viability of the EPCs incubated with the different concentration of Fe₃O₄@CA–CD34 for 1 and 3 d (D).

Figure 6

Residual concentration of the MNP suspension (Fe₃O₄@CA–CD34) in a circulating closed loop system at different time points (A): 100 mT without iron, (B): 100 mT with iron, (C): 300 mT without iron, and (D): 300 mT with iron (Left). Images of absorbed MNPs around the iron and 316L SS pieces after circulation for 30 min (right).

Figure 7

EPC adhesion onto the surface (A) and the side (C) of iron pieces assisted by anti-CD34-coated MNPs in the absence of an EMF and EPC adhesion onto the surface (B) and the side (D) of iron pieces assisted by anti-CD34-coated MNPs in the presence of a 300 mT EMF under flow conditions in vitro for 1 h.

Figure 8

(A) Stereo-micrograph of the iron stent. (B) Bright-field image of the iron stent; (C) EPC adhesion onto iron stents assisted by Fe₃O₄@CA–CD34 and different EMF (0, 100 and 300 mT) for 1 and 3 h under flow conditions in vitro; cellular labeling was visualized histochemically with rhodamine 123 staining.