Localized Enzyme-Assisted Self-Assembly in the Presence of Hyaluronic Acid for Hybrid Supramolecular Hydrogel Coating

Abstract

Hydrogel coating is highly suitable in biomaterial design. It provides biocompatibility and avoids protein adsorption leading to inflammation and rejection of implants. Moreover, hydrogels can be loaded with biologically active compounds. In this field, hyaluronic acid has been largely studied as an additional component since this polysaccharide is naturally present in extracellular matrix. Strategies to direct hydrogelation processes exclusively from the surface using a fully biocompatible approach are rare. Herein we have applied the concept of localized enzyme-assisted self-assembly to direct supramolecular hydrogels in the presence of HA. Based on electronic and fluorescent confocal microscopy, rheological measurements and cell culture investigations, this work highlights the following aspects: (i) the possibility to control the thickness of peptide-based hydrogels at the micrometer scale (18-41 µm) through the proportion of HA (2, 5 or 10 mg/mL); (ii) the structure of the self-assembled peptide nanofibrous network is affected by the growing amount of HA which induces the collapse of nanofibers leading to large assembled microstructures underpinning the supramolecular hydrogel matrix; (iii) this changing internal architecture induces a decrease of the elastic modulus from 2 to 0.2 kPa when concentration of HA is increasing; (iv) concomitantly, the presence of HA in supramolecular hydrogel coatings is suitable for cell viability and adhesion of NIH 3T3 fibroblasts.

Keywords: coating; enzyme-assisted self-assembly; hyaluronic acid; peptide; supramolecular hydrogel.

Figure 1

(a) Chemical structure of the precursor peptide Fmoc-FFpY transformed in hydrogelator Fmoc-FFY in presence of an enzyme, i.e., AP; (b) Scheme of inverted tube test of a Fmoc-FFY hydrogel formed from Fmoc-FFpY precursor and AP in presence of HA; (c) Inverted tube tests of self-assembled Fmoc-FFY hydrogels formed in Borax buffer solution (pH 9.5) in presence of various HA concentrations.

Figure 2

Fluorescence confocal microscopy images taken after the addition of AP (1 mg/mL) in Fmoc-FFpY (5 mg/mL) borax buffered solution (25 mM, pH 9.5) containing Thioflavine T in (a) absence or in (b) presence of HA (10 mg/mL).

Figure 3

(a) The diagram provides the mean diameter of elementary fibers (ca. 30 fibers/image) determined after analysis of several TEM images (ca 10 images); (b) TEM images of self-assembled Fmoc-FFY nanofibers formed in presence of different HA concentrations ranging from 2, 5 and 10 mg/mL; (c) Elastic modulus (G′) and loss modulus (G″) values measured on hydrogels prepared in presence of 0, 2, 5 and 10 mg/mL of HA.

Figure 4

(a) Sectional cryo-SEM image of supramolecular hydrogel (dark grey part) formed from silica wafer (light grey part) from Fmoc-FFY self-assembled from PEI/(PSS/PAH)₂/AP multilayer; (b) Hydrogel thickness evolution according to the HA concentration present initially in solution with the precursor peptide Fmoc-FFpY and AP; (c) Cryo-SEM images of hydrogels formed from AP-modified multilayer film in presence of various HA concentrations: 0, 2, 5 and 10 mg/mL. Dashed black lines indicate the separation between the glass substrate and the hydrogel.

Figure 4

(a) Sectional cryo-SEM image of supramolecular hydrogel (dark grey part) formed from silica wafer (light grey part) from Fmoc-FFY self-assembled from PEI/(PSS/PAH)₂/AP multilayer; (b) Hydrogel thickness evolution according to the HA concentration present initially in solution with the precursor peptide Fmoc-FFpY and AP; (c) Cryo-SEM images of hydrogels formed from AP-modified multilayer film in presence of various HA concentrations: 0, 2, 5 and 10 mg/mL. Dashed black lines indicate the separation between the glass substrate and the hydrogel.

Figure 5

(a) AFM images (deflection mode, dry state) of the multilayer film PEI/(PSS/PAH)₂ in the following conditions (from left to right): after the adsorption of AP on the top of the film, when the multilayer is brought in contact with Fmoc-FFpY solution (5 mg/mL), after the buildup of the peptide self-assembled architecture from a precursor peptide solution [Fmoc-FFpY] = 5 mg/mL in absence or in presence of HA (2 mg/mL). Scale bar = 2 µm; (b) SEM images of supramolecular hydrogels formed from PEI/(PSS/PAH)₂/AP in presence of Fmoc-FFpY (5 mg/mL) and HA at 0, 2 and 5 mg/mL.

Figure 6

Apoptotic/necrotic/healthy cells detection kit was used to determine the cell viability of NIH 3T3 fibroblasts after 4 h in contact with different hydrogel coatings containing 0, 2, 5 and 10 mg/mL HA. Live cells are stained in blue and death cells in red. Phase contrast microscopy images showing NIH 3T3 fibroblasts cell spreading on the hydrogel coatings.