Plasma-assisted heparin conjugation on electrospun poly(L-lactide) fibrous scaffolds

Abstract

Heparin conjugation on poly(L-lactide) fibrous scaffolds fabricated by electrospinning was accomplished by surface functionalization with amine (-NH2) groups using a sequential treatment with Ar-NH3 and H2 plasmas. The density of the incorporated -NH2 groups was determined by combining a chemical derivatization method with X-ray photoelectron spectroscopy. The time of Ar-NH3 plasma treatment significantly affected the N/C, -NH2 /N, and -NH2 /C fractions, whereas the plasma power, Ar-NH3 gas composition, and time of H2 plasma treatment only influenced the -NH2 /N and -NH2 /C fractions. Scaffold surface functionalization by -NH2 groups significantly increased the amount of covalently bonded heparin compared to a hydrolysis method. The function of immobilized heparin was confirmed by the decrease of platelet attachment during the exposure of the scaffolds to blood from Sprague-Dawley rats. In vitro experiments with bovine aorta endothelial cells demonstrated that heparin conjugation enhanced cell infiltration through the fibrous scaffolds, regardless of the amount of covalently immobilized heparin.

Keywords: amine groups; conjugation; endothelial cells; heparin; infiltration; plasma treatment; platelets; surface functionalization.

Fig. 1

Schematic of the chemical derivatization process of the –NH₂ group with TFBA on PLLA fibrous scaffolds subjected to Ar-NH₃/H₂ plasma treatment and formula for computing the NH₂/C fraction.

Fig. 2

Effect of plasma power (left column) and plasma treatment time (right column) on the surface chemical characteristics of PLLA fibrous scaffolds subjected to Ar-NH₃/H₂ plasma treatment: (a,d) N/C, (b,e) NH₂/N, and (c,f) NH₂/C fraction. (Plasma parameters: (a)–(c) Ar-NH₃ plasma (50–200 W power, 30 vol% Ar, 0.5 Torr pressure, 100 sccm gas flow rate, 5 min treatment time); H₂ plasma (10 W power, 50 sccm H₂ gas flow rate, 0.5 Torr pressure, 0.5 min treatment time) and (d)–(f) Ar-NH₃ plasma (100 W power, 30 vol% Ar, 0.5 Torr pressure, 100 sccm gas flow rate, 2–10 min treatment time); H₂ plasma (10 W power, 50 sccm H₂ gas flow rate, 0.5 Torr pressure, 0.5 min treatment time)).

Fig. 3

Effect of Ar-NH₃ plasma gas composition (left column) and H₂ plasma post-treatment time (right column) on the surface chemical characteristics of PLLA fibrous scaffolds subjected to Ar-NH₃/H₂ plasma treatment: (a,d) N/C, (b,e) NH₂/N, and (c,f) NH₂/C fraction. Plasma parameters are: (a)–(c) Ar-NH₃ plasma (50 W power, 10–50 vol% Ar, 0.5 Torr pressure, 100 sccm gas flow rate, 5 min treatment time); H₂ plasma (10 W power, 50 sccm H₂ gas flow rate, 0.5 Torr pressure, 0.5 min treatment time) and (d)–(f) Ar-NH₃ plasma (50 W power, 30 vol% Ar, 0.5 Torr pressure, 100 sccm gas flow rate, 5 min treatment time); H₂ plasma (10 W power, 50 sccm H₂ gas flow rate, 0.5 Torr pressure, 10–60 s treatment time).

Fig. 4

Heparin conjugation density on untreated, plasma-treated (Ar or Ar-NH₃/H₂), and PEG-conjugated (by the NaOH hydrolysis method) PLLA fibrous scaffolds. Plasma parameters are: Ar-NH₃ plasma (50 W power, 30 vol% Ar, 0.5 Torr pressure, 100 sccm gas flow rate, 5 min treatment time); H₂ plasma (10 W power, 50 sccm H₂ gas flow rate, 0.5 Torr pressure, 30 s treatment time).

Fig. 5

SEM images showing the attachment of platelets from whole blood of Sprague-Dawley rats on PLLA fibrous scaffolds without (first row) and with (second row) heparin conjugation (by the NaOH hydrolysis method): (a,d) untreated (using the NaOH hydrolysis method for heparin conjugation in d), (b,e) Ar plasma-treated (controls), and (c,f) Ar-NH₃/H₂ plasma-treated scaffolds. (UT = untreated, Ar = Ar plasma treated, Ar-NH₃ = Ar-NH₃/H₂ plasma treated, Hep = heparin conjugated).

Fig. 6

Cross-sectional fluorescence images showing through-thickness infiltration of BAECs into PLLA fibrous scaffolds without (left column) and with (right column) heparin conjugation: (a,d) untreated (using the NaOH hydrolysis method for heparin conjugation in d), (b,c) Ar plasma-treated (controls), and (c,f) Ar-NH₃/H₂ plasma-treated scaffolds. Cells were seeded at 100% confluency at the top scaffold surface with serum medium for 5 days. Top and bottom scaffold surfaces are distinguished by dashed lines. DAPI staining for nuclei is shown in blue color. (UT = untreated, Ar = Ar plasma treated, Ar-NH₃ = Ar-NH₃/H₂ plasma treated, Hep = heparin conjugated).