Differential orientation and conformation of surface-bound keratinocyte growth factor on (hydroxyethyl)methacrylate, (hydroxyethyl)methacrylate/methyl methacrylate, and (hydroxyethyl)methacrylate/methacrylic acid hydrogel copolymers

Abstract

The development of hydrogels for protein delivery requires protein-hydrogel interactions that cause minimal disruption of the protein's biological activity. Biological activity can be influenced by factors such as orientational accessibility for receptor binding and conformational changes, and these factors can be influenced by the hydrogel surface chemistry. (Hydroxyethyl)methacrylate (HEMA) hydrogels are of interest as drug delivery vehicles for keratinocyte growth factor (KGF) which is known to promote re-epithelialization in wound healing. The authors report here the surface characterization of three different HEMA hydrogel copolymers and their effects on the orientation and conformation of surface-bound KGF. In this work, they characterize two copolymers in addition to HEMA alone and report how protein orientation and conformation is affected. The first copolymer incorporates methyl methacrylate (MMA), which is known to promote the adsorption of protein to its surface due to its hydrophobicity. The second copolymer incorporates methacrylic acid (MAA), which is known to promote the diffusion of protein into its surface due to its hydrophilicity. They find that KGF at the surface of the HEMA/MMA copolymer appears to be more orientationally accessible and conformationally active than KGF at the surface of the HEMA/MAA copolymer. They also report that KGF at the surface of the HEMA/MAA copolymer becomes conformationally unfolded, likely due to hydrogen bonding. KGF at the surface of these copolymers can be differentiated by Fourier-transform infrared-attenuated total reflectance spectroscopy and time-of-flight secondary ion mass spectrometry in conjunction with principal component analysis. The differences in KGF orientation and conformation between these copolymers may result in different biological responses in future cell-based experiments.

Fig. 1.

Structures of hydrogel copolymers that were crosslinked for the formation of pores.

Fig. 2.

(a) PC1 confidence limits at the 95% confidence level of the three hydrogel copolymer surfaces and (b) PC1 loadings.

Fig. 3.

(a) FTIR-ATR spectrum of active and heat-denatured KGF in PBS, (b) Fourier self-deconvoluted FTIR-ATR spectrum of active KGF in PBS, and (c) Fourier self-deconvoluted FTIR-ATR spectrum of heat-denatured KGF in PBS.

Fig. 4.

(a) FTIR-ATR spectra of KGF at the copolymer surfaces, (b) Fourier self-deconvoluted spectrum of KGF at the HEMA/MAA surface, (c) Fourier self-deconvoluted spectrum of KGF at the HEMA surface, and (d) Fourier self-deconvoluted spectrum of KGF at the HEMA/MMA surface.

Fig. 5.

(a) Crystal structure of KGF with secondary structural elements labeled created in PyMol: red—reverse turns, orange—extended strands, blue—disordered, pink—heparin binding loop, green—unassigned, (b) crystal structure of KGF with hydrophobic (pink) and hydrophilic amino acids labeled (blue) [PDB ID: 1QQK (Ref. 68)].

Fig. 6.

(a) PC1 confidence limits of protein orientation/conformation on the hydrogel surfaces and (b) PC loadings at the 95% confidence level.

Fig. 7.

Peak intensities of (a) threonine and (b) serine when normalized by the sum of selected peaks.