Synthesis and characterization of amine-functionalized graphene as a nitric oxide-generating coating for vascular stents

Abstract

Drug-eluting stents are commonly utilized for the treatment of coronary artery disease, where they maintain vessel patency and prevent restenosis. However, problems with prolonged vascular healing, late thrombosis, and neoatherosclerosis persist; these could potentially be addressed via the local generation of nitric oxide (NO) from endogenous substrates. Herein, we develop amine-functionalized graphene as a NO-generating coating on polylactic acid (PLA)-based bioresorbable stent materials. A novel catalyst was synthesized consisting of polyethyleneimine and polyethylene glycol bonded to graphene oxide (PEI-PEG@GO), with physicochemical characterization using x-ray diffraction, Raman spectroscopy, Fourier transform infrared spectroscopy, and thermogravimetric analysis. In the presence of 10 μM S-nitrosoglutathione (GSNO) or S-nitroso-N-acetylpenicillamine (SNAP), PEI-PEG@GO catalyzed the generation of 62% and 91% of the available NO, respectively. Furthermore, PEI-PEG@GO enhanced and prolonged real-time NO generation from GSNO and SNAP under physiological conditions. The uniform coating of PEI-PEG@GO onto stent material is demonstrated via an optimized simple dip-coating method. The coated PLA maintains good biodegradability under accelerated degradation testing, while the PEI-PEG@GO coating remains largely intact. Finally, the stability of the coating was demonstrated at room temperature over 60 days. In conclusion, the innovative conjugation of polymeric amines with graphene can catalyze the generation of NO from S-nitrosothiols at physiologically relevant concentrations. This approach paves the way for the development of controlled NO-generating coatings on bioresorbable stents in order to improve outcomes in coronary artery disease.

FIG. 1

A schematic representation of the design and synthesis of functionalized GO conjugate (termed as PEI-PEG@GO). First, GO was synthesized by oxidizing graphite following the modified Hummer’s method. GO was functionalized with PEI and PEG by the formation of an amide bond between PEI, PEG, and GO in the presence of EDC. The product was washed 3–4 times with an aqueous solution that contained 10% NaCl in order to remove any unreacted PEI, PEG, and urea.

FIG. 2. Basic characterization of GO, PEI@GO, and PEI-PEG@GO.

(a) Transmission electron microscopy (TEM) images, (b) XRD patterns, (c) Raman spectroscopy, (d) FTIR spectra, (e) thermogravimetric analysis (TGA), and (f) water contact angle measurements.

FIG. 3. Quantification of NO release from GSNO and SNAP with and without different formulations using a NO electrode sensor under physiological conditions.

(a) NO release measurements from GSNO alone (10 μM) and GSNO (10 μM) in the presence of different formulations of GO, PEI, PEG, and PEI-PEG@GO at the concentration of 250 μg/ml (n = 3 independent samples). (b) NO release measurements from SNAP alone (10 μM) and SNAP (10 μM) in the presence of different formulations of GO, PEI, PEG, and PEI-PEG@GO at the concentration of 250 μ/ml (n = 3 independent samples).

FIG. 4. Catalytic generation of nitric oxide (NO) from GSNO and SNAP using PEI, PEG, and PEI-PEG@GO at different concentrations in PBS buffer (pH 7.4) in the presence of EDTA as determined by a chemiluminescence NO analyzer.

(a) Representative NO release profile from PEI and PEG without GSNO and SNAP. (b) Representative NO release profile from PEI@GO and PEI-PEG@GO without GSNO and SNAP. (c) Representative NO release profile from GSNO and PEI-PEG@GO+GSNO. (d) Representative NO release profile from SNAP and PEI-PEG@GO+GSNO. The arrow shows the injection of compounds. Time on the x axis represents the time since the start of the chemiluminescence time-trace.

FIG. 5. Characterization of the coating of PEI-PEG@GO on the stent material (PLA).

(a) Photographs of 3D-printed stents. (b) SEM micrographs of coated and uncoated stents at different magnifications. The inset figure in PEI-PEG@GO coated stent shows the average thickness of coating which is 0.2 μm. (c) Raman spectra of uncoated and coated stents (with PEI-PEG@GO) using different combinations of coating solutions and PEI-PEG@GO. Three random fields of view were chosen with ~18 random points mapping per field view along with ~54 points per stent. The optimized coating solution (PEG:PCL 1:1 and 10 wt. % PEI-PEG@GO) was prepared after screening different ratios of PEG and PCL in THF with PEI-PEG@GO. The weight percentage of PEG, PCL, and PEI-PEG@GO in the coating solution is 47.5%, 47.5%, and 5%, respectively. Each spectrum in this panel represents characterization at a spatially distinct sampling point. (d) FTIR spectra of uncoated and coated stents under different conditions. The characteristic FTIR bands are shown along with region names.

FIG. 6. Accelerated hydrolytic degradation of coated and uncoated stent material at elevated temperature.

(a) Weight loss of stents as a function of time in PBS (pH 7.4) at 60 °C for 60 days. The data are shown as the mean ± SD, n = 3. (b) The stability of amine groups on stents over 60 days under hydrolytic degradation conditions is presented in (a). The stability of amine groups was measured using a standard colorimetric assay based on Orange acid II (OA II). The data are shown as the mean of two independent experiments. (c) SEM micrographs of the surface of the stent material at different time points of the hydrolytic degradation experiments at 60 °C in PBS (pH 7.4).

FIG. 7

The stability of amine groups on stents for time points up to 60 days. The data are shown as mean ± SD, n = 3. One-way ANOVA test was used to calculate statistical significance. RT, room temperature.