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. 2014 Apr 11;7(4):2956-2977.
doi: 10.3390/ma7042956.

Influence of Layer-by-Layer Polyelectrolyte Deposition and EDC/NHS Activated Heparin Immobilization onto Silk Fibroin Fabric

M Fazley Elahi  1 , Guoping Guan  2 , Lu Wang  3 , Martin W King  4   5
Affiliations

Influence of Layer-by-Layer Polyelectrolyte Deposition and EDC/NHS Activated Heparin Immobilization onto Silk Fibroin Fabric

M Fazley Elahi et al. Materials (Basel). .

Abstract

To enhance the hemocompatibility of silk fibroin fabric as biomedical material, polyelectrolytes architectures have been assembled through the layer-by-layer (LbL) technique on silk fibroin fabric (SFF). In particular, 1.5 and 2.5 bilayer of oppositely charged polyelectrolytes were assembled onto SFF using poly(allylamine hydrochloride) (PAH) as polycationic polymer and poly(acrylic acid) (PAA) as polyanionic polymer with PAH topmost. Low molecular weight heparin (LMWH) activated with 1-ethyl-3-(dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) was then immobilized on its surface. Alcian Blue staining, toluidine blue assay and X-ray photoelectron spectroscopy (XPS) confirmed the presence of heparin on modified SFF surfaces. The surface morphology of the modified silk fibroin fabric surfaces was characterized by scanning electron microscopy (SEM) and atomic force microscopy (AFM), and obtained increased roughness. Negligible hemolytic effect and a higher concentration of free hemoglobin by a kinetic clotting time test ensured the improved biological performance of the modified fibroin fabric. Overall, the deposition of 2.5 bilayer was found effective in terms of biological and surface properties of the modified fibroin fabric compared to 1.5 bilayer self-assembly technique. Therefore, this novel approach to surface modification may demonstrate long term patency in future in vivo animal trials of small diameter silk fibroin vascular grafts.

Keywords: EDC/NHS; hemocompatibility; heparin; layer-by-layer; low molecular weight heparin (LMWH); poly(acrylic acid); poly(allylamine hydrochloride); silk fibroin fabric.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1.
Figure 1.
SEM micrographs of untreated and modified fibroin fabrics: (a1, a2) SFF; (b1, b2) SFF-1 and (c1, c2) SFF-2. Magnification: top row 5000×; bottom row 10,000×.
Figure 2.
Figure 2.
Atomic force microscopy (AFM) images of untreated and surface modified fibroin fabrics: (a) SFF; (b) SFF-1 and (c) SFF-2.
Figure 3.
Figure 3.
FTIR spectra of poly(acrylic acid) (PAA); poly(allylamine hydrochloride) (PAH); 1-ethyl-3-(dimethylaminopropyl) carbodiimide hydrochloride (EDC); N-hydroxysuccinimide (NHS); low molecular weight heparin (LMWH); SFF, SFF-1 and SFF-2.
Figure 4.
Figure 4.
Optical microscopy images of Alcian blue stained untreated and surface modified fibroin fabrics. Magnification: ×40. (a) SFF; (b) SFF-1 and (c) SFF-2.
Figure 5.
Figure 5.
Release percentage of heparin from surface modified silk fibroin fabrics.
Figure 6.
Figure 6.
XPS spectra of (a) SFF (b) SFF-1 and (c) SFF-2 and (d) relative elemental contents.
Figure 7.
Figure 7.
XPS spectra of S 2p peaks: (a) SFF-1 and (b) SFF-2.
Figure 8.
Figure 8.
XPS spectra of C 1s peaks (a) SFF; (b) SFF-1; (c) SFF-2 and (d) relative elemental contents.
Figure 9.
Figure 9.
XPS spectra of O 1s peaks (a) SFF; (b) SFF-1; (c) SFF-2 and (d) relative elemental contents.
Figure 10.
Figure 10.
Mean hemolytic assay results of untreated (SFF) and modified fibroin fabric samples (SFF-1 and SFF-2) compared to exposure to the PBS solution negative control and the water positive control. Mean data for each sample (n = 3) are presented. Error bar = 1× standard deviation. Statistical differences indicated with (***) for p < 0.001.
Figure 11.
Figure 11.
SEM photomicrographs of untreated and surface modified fibroin fabrics after exposure to whole blood: (a1, a2) Untreated SFF sample showing cell attachment; (b1, b2) SFF-1 and (c1, c2) SFF-2 surface treated samples show limited and no cell attachment respectively. Magnification: top row 5000×; bottom row 500×.
Figure 12.
Figure 12.
Rate of coagulation assay of untreated and surface modified silk fibroin fabrics at different time intervals. Data are shown here as mean (n = 3). Error bar = 1 × standard deviation.
Figure 13.
Figure 13.
Chemical structures of the materials used: (a) silk fibroin; (b) LMWH with two typical GAG repeats with multiple sulfate groups; (c) PAH; (d) PAA; (e) EDC and (f) NHS.
Figure 14.
Figure 14.
Schematic illustration of the deposition of poly(allylamine hydrochloride) (PAH) and poly(acrylic acid) (PAA) to create 1.5 and 2.5 polyelectrolytes bilayer on silk fibroin fabric (SFF) surfaces followed by EDC/NHS activated heparin immobilization.
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