Protein adsorption and cell adhesion on nanoscale bioactive coatings formed from poly(ethylene glycol) and albumin microgels

Abstract

Late-term thrombosis on drug-eluting stents is an emerging problem that might be addressed using extremely thin, biologically active hydrogel coatings. We report a dip-coating strategy to covalently link poly(ethylene glycol) (PEG) to substrates, producing coatings with approximately <100 nm thickness. Gelation of PEG-octavinylsulfone with amines in either bovine serum albumin (BSA) or PEG-octaamine was monitored by dynamic light scattering (DLS), revealing the presence of microgels before macrogelation. NMR also revealed extremely high end-group conversions prior to macrogelation, consistent with the formation of highly crosslinked microgels and deviation from Flory-Stockmayer theory. Before macrogelation, the reacting solutions were diluted and incubated with nucleophile-functionalized surfaces. Using optical waveguide lightmode spectroscopy (OWLS) and quartz crystal microbalance with dissipation (QCM-D), we identified a highly hydrated, protein-resistant layer with a thickness of approximately 75 nm. Atomic force microscopy in buffered water revealed the presence of coalesced spheres of various sizes but with diameters less than about 100 nm. Microgel-coated glass or poly(ethylene terephthalate) exhibited reduced protein adsorption and cell adhesion. Cellular interactions with the surface could be controlled by using different proteins to cap unreacted vinylsulfone groups within the coating.

Figure 1

Crosslinking of bovine serum albumin (BSA) and poly(ethylene glycol)-octavinylsulfone (PEG-OVS, MW 10,000) may lead to microgel formation if the principle of equal end-group reactivity does not apply. Vinylsulfone groups on PEG molecules undergo a Michael-type addition with solvent-exposed and sterically accessible lysines on BSA, forming covalent linkages at neutral pH. If the crosslinking reaction is slowed before the gel point by dilution, microgel-containing solutions can be rapidly reacted with nucleophile-derivatized surfaces, such as thiol-silanized glass.

Figure 2

Evidence for formation of microgels during the crosslinking reaction: a) Dynamic light scattering (DLS) with intensity-weighted (d_PCS) and volume-weighted mean effective diameters of reacting PEG-OVS/BSA solutions (0.4:1 ratio of BSA amine groups to PEG vinylsulfone groups, 44 h gel time). b) NMR was used to measure the kinetics of end-group conversion during the reaction of PEG-OVS with PEG-octaamine (both MW 10,000; 1:1 ratio of PEG-OA to PEG-OVS, 6.5 h gel time). The time scales of both (a) and (b) were normalized relative to their respective gel times and error bars display the standard deviations for 4 separate reactions. c) SDS-PAGE of BSA during the crosslinking reaction with PEG-OVS. d) Analysis of the 2^nd order reaction kinetics for part (c) for two separate experiments (circles: first experiment; squares: second experiment; ordinate units: L/mol·h).

Figure 3

Monte Carlo simulation of the reaction between PEG-OVS and PEG-OA suggested a mechanism for microgel formation: (A–C) The weight fraction of polymer chains with 1–2, 3–9, 10–100, 101–1000 or >1000 mers is shown. The simulation assumed: (A,C) equal reactivity of functional groups regardless of polymer size (Flory-Stockmayer), or (B,C) reactivity scaled as (mol. wt.)^−3/5, an estimate of the effects of steric stabilization. With equal reactivity of end groups, monomers and dimers are present in large amounts even well past the theoretical gel point (p_gel = 0.1429, the end-group conversion at the gel point). For equally reactive end groups, polymer chains with 10–100 monomer units were never more prevalent than monomers and dimers. With steric stabilization considered, large polymer chains were not present until p > 0.23, indicating a delay in gelation well past p_gel. At p= 0.21–0.23, greater than 60% of the mass of polymer was contained in 10–100 mer chains. (D–F) The distribution of polymer sizes just prior to the gel point without (D) and with steric stabilization considered (E, F).

Figure 4

AFM analysis of: (A) MPTS glass, 10 × 10 microns, (B) PEG-OVS/BSA microgel-coated MPTS glass, 10 × 10 microns , (C) PEG-OVS/BSA microgel-coated MPTS glass with BSA capping step, 10 × 10 microns, (D) PEG-OVS/ BSA microgel-coated MPTS glass, 3 × 3 microns with height profiles. The height data scales are 20 nm for (A), 30 nm for (B) and (D), and 120 nm for (C).

Figure 5

OWLS analysis of fibrinogen adsorption to microgel-coated surfaces. Si/Ti/O₂ surfaces of OWLS waveguide chips were oxygen-plasma etched, vapor-silanized with MPTS, and incubated with PEG-OVS/BSA microgels (d_PCS = 100–120 nm). The adsorption of bovine fibrinogen (bFg) from: a) 2.5 mg/mL, or b) 20 mg/mL solutions onto microgel-coated surfaces was monitored. All surfaces were exposed to the same series of solutions flowing at 0.1 mL/min at 37°C: (1) DI water, (2) PBS pH 7.4, (3) 2.5 mg/mL or 20 mg/mL bovine fibrinogen in PBS pH 7.4, (4) wash with PBS, pH 7.4, and (4) wash with DI water. The higher concentration of bovine fibrinogen in part b demonstrated that the microgel layer was thin enough to detect protein above the microgel coating.

Figure 6

QCM-D frequency and dissipation changes illustrating the attachment of microgels to the MPTS-silanized crystal and subsequent resistance to non-specific protein adsorption. The following solutions were flowed over the crystal: (1) 0.5 mL of BSA/PEG-OVS microgels (d_PCS = 100–120 nm) flowed onto the crystal and then incubated for 60 min. (2) 30 mL wash with PBS and incubation with PBS until readings stabilized. (3) 0.5 mL of 100 mg/mL BSA flowed over the crystal and incubated for about 90 minutes. (4) 30 mL wash with PBS and incubation with PBS until readings stabilized. (5) 0.5 mg/mL of 2.5 mg/mL bovine fibrinogen flowed over crystal and incubated for 2 h. (6) 30 mL wash and incubation with PBS until readings stabilized.

Figure 7

Cell adhesion on microgel-coated glass at 10× magnification after a 24 h incubation with endothelial (3.5 × 10⁴ cells/cm²), CHO (2.5 × 10⁵ cells/cm²), or fibroblast (2.5 × 10⁵ cells/cm²) cells. MPTS-silanized glass coverslips were incubated at 37°C with one of the following solutions: PBS for 12 h, 100 mg/mL BSA for 12 h, 100 mg/mL PEG-OVS for 12 h, or PEG-OVS/BSA microgels (d_PCS = 100–120 nm) for 1 h followed by capping with 50 mg/mL BSA for 12 h.

Figure 8

Cell adhesion to microgel-coated glass at 10× magnification after repeated seedings with fibroblasts after: a) 1 day, b) 5 days, and c) 19 days. MPTS surfaces coated with BSA-capped BSA/PEG-OVS microgels were washed and reseeded with fibroblasts every 2 days at 2.5 × 10⁵ cells/cm². Cell spreading was not observed until 19 days, at which time aggregates of fibroblasts spread. Controls consisting of non-silanized glass incubated with BSA-capped BSA/PEG-OVS microgels are displayed in the lower right corner of each image at 10× magnification, showing complete cell spreading.

Figure 9

Cell counting results for CHO and fibroblast adhesion to microgel surfaces. a) Comparison of CHO cell adhesion to MPTS-silanized glass incubated overnight with (in order from left to right): (1) PBS pH 7.4; (2) 50 mg/mL bovine serum albumin (BSA) in PBS; (3) 100 mg/mL PEG-OVS in PBS; (4–5) PEG-OVS/BSA microgels (d_PCS = 100–120 nm), capped with (4) BSA or (5) bovine fibrinogen (bFg), and (6) PEG-OVS/BSA microgels (d_PCS = 100–120 nm), capped with BSA then incubated with 2.5 mg/mL bFg in PBS for 2 h at 37°C. b) Fibroblast adhesion after the same surface treatments described in part a. c) CHO cell adhesion to MPTS-silanized glass reacted with: (in order from top to bottom): (1) 100 mg/mL PEG-OVS in PBS; (2) 20 PEG-OVS layers alternating with DTT applied using a layer-by-layer method; (3–4) PEG-OVS/PEG-OA microgels (d_PCS = 100–120 nm) capped with (3) BSA; (4) BSA then incubated with bFg for 2 h; (5–8) PEG-OVS/BSA microgels (d_PCS = 100–120 nm) that were capped with: (5) PEG-OVS, (6) BSA, (7) BSA, then incubated with bFg for 2 h, or (8) bFg. CHO and fibroblast cells were seeded at a density of 2.5 × 10⁵ cells/cm² and incubated with the surfaces for 24 h at 37°C.

Figure 10

10× magnification of CHO cell adhesion on glass slides for 24 h demonstrating the effects of the crosslinker and final capping step. MPTS-silanized glass was incubated with: a) 2.5 mg/mL bovine fibrinogen in PBS at pH 7.4 for 2 h, b) PEG-OVS/BSA microgels overnight in PBS pH 7.4 at 37°C, c) PEG-OVS/PEG-OA microgels capped overnight with 50 mg/mL BSA in PBS pH 7.4 at 37°C, d) PEG-OVS/BSA microgels capped overnight at 37°C with 2.5 mg/mL bovine fibrinogen in PBS pH 7.4, e) PEG-OVS/BSA microgels capped overnight with 50 mg/mL BSA and subsequently incubated for 2 h at 37°C with a 2.5 mg/mL bovine fibrinogen. f) PEG-OVS/PEG-OA microgels capped overnight with 50 mg/mL BSA and subsequently incubated for 2 h at 37°C with 2.5 mg/mL bovine fibrinogen.

Figure 11

CHO cell adhesion at 24 h to air RFGD-treated PET films that were incubated with: a) PBS, b) BSA, c) PEG-VS, or d) PEG-OVS/BSA microgels. 10× magnification.