Surface modification of a polyhedral oligomeric silsesquioxane poly(carbonate-urea) urethane (POSS-PCU) nanocomposite polymer as a stent coating for enhanced capture of endothelial progenitor cells

Abstract

An unmet need exists for the development of next-generation multifunctional nanocomposite materials for biomedical applications, particularly in the field of cardiovascular regenerative biology. Herein, we describe the preparation and characterization of a novel polyhedral oligomeric silsesquioxane poly(carbonate-urea) urethane (POSS-PCU) nanocomposite polymer with covalently attached anti-CD34 antibodies to enhance capture of circulating endothelial progenitor cells (EPC). This material may be used as a new coating for bare metal stents used after balloon angioplasty to improve re-endothelialization. Biophysical characterization techniques were used to assess POSS-PCU and its subsequent functionalization with anti-CD34 antibodies. Results indicated successful covalent attachment of anti-CD34 antibodies on the surface of POSS-PCU leading to an increased propensity for EPC capture, whilst maintaining in vitro biocompatibility and hemocompatibility. POSS-PCU has already been used in 3 first-in-man studies, as a bypass graft, lacrimal duct and a bioartificial trachea. We therefore postulate that its superior biocompatibility and unique biophysical properties would render it an ideal candidate for coating medical devices, with stents as a prime example. Taken together, anti-CD34 functionalized POSS-PCU could form the basis of a nano-inspired polymer platform for the next generation stent coatings.

Figure 1

A clinical-grade biofunctionalized polymer for coating stents. (A) POSS-PCU nanocomposute polymer can be used to coat bare metal stents, and further functionalized with endothelial progenitor cell (EPC)-specific antibodies for enhanced endothelialization. (B) A schematic diagram of an anti-CD34 antibody. The Fab region binds to EPCs, while the Fc region is immobilized onto POSS-PCU.

Figure 2

Detection of amine groups on anti-CD34 antibody. OPA assay showed the presence of amine groups on POSS-PCU. This value was somewhat less that pure anti-CD34, indicating a certain about of loss during functionalization.

Figure 3

Detecting chemical groups via FTIR. FTIR spectra revealed that incorporation of NH2-FS did not alter the spectral read-outs. Amide I band was detected in anti-CD34 antibodies.

Figure 4

SEM images of POSS-PCU. Pure POSS-PCU films displayed a flake-like surface. Immobilization with anti-CD34 antibodies causes the surface to adopt a more ridge-like appearance, possibly due to protein aggregations. Scale bar represents 20 μm.

Figure 5

AFM images of POSS-PCU. Pure POSS-PCU films displayed a topography with “spikes”. Anti-CD34 antibody immobilization changes the topography to a more ridge-like appearance. This is largely consistent with SEM images. Scale bar represents 1 μm.

Figure 6

Raman spectroscopy. Raman intensity at the POSS regions were especially strong. Similar Raman shifts were seen in both POSS-PCU and POSS-PCU-CD34 samples due to the strong POSS signatures in the polymer. The spectral difference between POSS and PCU were used to create Raman integration maps.

Figure 7

Comparison of Raman integration maps. Optical images and Raman maps revealed a modified surface after anti-CD34 antibody immobilization. POSS-PCU-CD34 had a granite-like appearance on both optical and Raman integration. Detection of POSS and PCU –rich regions also revealed a chemically heterogeneous surface. Scale bar represents 5 μm.

Figure 8

Raman integration maps of POSS-PCU-CD34. Raman AFM shows a cobblestone-like appearance, with phase AFM revealing a textured-surface topography. Antibody-quantum dot regions were tracked using Raman, with integrated maps showing it to be highly dispersed. Scale bar represents 5 μm.

Figure 9

Detection of antibody engraftment via XPS. Atomic composition of POSS-PCU-CD34 showed a higher percentage of N compared to POSS-PCU, indicating presence of antibodies on the surface.

Figure 10

Reduction of water contact angle. Anti-CD34 antibody immobilization on the surface of POSS-PCU renders the surface less hydrophobic, compared to POSS-PCU. This is due to the high energy polar groups of proteins present in antibodies.

Figure 11

Assessment of hemocompatibility via TEG. TEG revealed that cuvettes coated with POSS-PCU and CD34-POSS-PCU did not significantly deviate from uncoated cuvettes. This indicates that polymer coatings did not acutely affect blood coagulation kinetics.

Figure 12

EPC staining with anti-CD34 and VEGFR₂. Scale bar represents 40 μm. Compared to POSS-PCU and POSS-PCU-IgG, POSS-PCU-CD34 displayed a higher density of cell adherence which were positive for CD34 and VEGFR₂.

Figure 13

EPC staining with CD31 and vWF. Compared to POSS-PCU and POSS-PCU-IgG, POSS-PCU-CD34 displayed a higher degree of adherent cells that were positive for CD31 and VWF. Scale bar represents 40 μm.

Figure 14

Culturing HUVECs on POSS-PCU-CD34. Growth and proliferation of HUVECs were observed on POSS-PCU-CD34 even after being exposed to physiological flow conditions. Scale bar represents 40 μm.

Figure 15

Assessment of biocompatibility. alamarBlue showed that EPCs and HUVECs grew and proliferated well on both POSS-PCU and POSS-PCU-CD34 films.

Figure 16

Stability under physiological flow conditions. POSS-PCU and CD34-POSS-PCU coated stents were placed in a flow circuit, calibrated to mimic physiological flow conditions, for 28 days. Confocal microscopy using fluorescent QDs on retrieved films showed the presence of anti-CD34 antibodies on the surface even after being exposed to dynamic flow conditions.