Binding Interactions of Keratin-Based Hair Fiber Extract to Gold, Keratin, and BMP-2

Abstract

Hair-derived keratin biomaterials composed mostly of reduced keratin proteins (kerateines) have demonstrated their utility as carriers of biologics and drugs for tissue engineering. Electrostatic forces between negatively-charged keratins and biologic macromolecules allow for effective drug retention; attraction to positively-charged growth factors like bone morphogenetic protein 2 (BMP-2) has been used as a strategy for osteoinduction. In this study, the intermolecular surface and bulk interaction properties of kerateines were investigated. Thiol-rich kerateines were chemisorbed onto gold substrates to form an irreversible 2-nm rigid layer for surface plasmon resonance analysis. Kerateine-to-kerateine cohesion was observed in pH-neutral water with an equilibrium dissociation constant (KD) of 1.8 × 10−4 M, indicating that non-coulombic attractive forces (i.e. hydrophobic and van der Waals) were at work. The association of BMP-2 to kerateine was found to be greater (KD = 1.1 × 10−7 M), within the range of specific binding. Addition of salts (phosphate-buffered saline; PBS) shortened the Debye length or the electrostatic field influence which weakened the kerateine-BMP-2 binding (KD = 3.2 × 10−5 M). BMP-2 in bulk kerateine gels provided a limited release in PBS (~ 10% dissociation in 4 weeks), suggesting that electrostatic intermolecular attraction was significant to retain BMP-2 within the keratin matrix. Complete dissociation between kerateine and BMP-2 occurred when the PBS pH was lowered (to 4.5), below the keratin isoelectric point of 5.3. This phenomenon can be attributed to the protonation of keratin at a lower pH, leading to positive-positive repulsion. Therefore, the dynamics of kerateine-BMP-2 binding is highly dependent on pH and salt concentration, as well as on BMP-2 solubility at different pH and molarity. The study findings may contribute to our understanding of the release kinetics of drugs from keratin biomaterials and allow for the development of better, more clinically relevant BMP-2-conjugated systems for bone repair and regeneration.

In Vivo Validation of Spray-Dried Mesoporous Bioactive Glass Microspheres Acting as Prolonged Local Release Systems for BMP-2 to Support Bone Regeneration

“…Furthermore, SD-MBG possess a negative surface charge, due to deprotonated silanols, able to bind positively charged protein molecules through electrostatic interactions. This charge-dependent binding was reported for BMP-2 binding the kerateine [ 68 ] or alginate carriers [ 69 ].…”

PEG-Immobilized Keratin for Protein Drug Sequestration and pH-Mediated Delivery

“…Irgacure ® 2959 (I2959), provided by BASF (Ludwigshafen, Germany), was dissolved at a stock solution of 10% (m/V) in MeOH. Reduced keratin biomaterial (KTN; MW = 98 kDa) was extracted [ 22 ] and obtained from Mark Van Dyke's Lab at the School of Biomedical Engineering and Sciences, Virginia Tech (Blacksburg, VA). Distilled deionized water (at 18.2 MΩ·cm electrical resistivity) was used as the default aqueous solvent, unless otherwise indicated.…”

“…Diacrylates of linear PEG (PEGDA) can be photopolymerized for PEG chain growth to form scaffolds [ 20 , 21 ]. Reduced KTN proteins containing free thiols (–SH) can be gelled by reforming of disulfide bonds (–S–S–) [ 22 , 23 ]. PEGDA and KTN can also be combined and crosslinked via a photopolymerization thiol-ene reaction [ 24 , 25 ].…”

“…We capitalized on these reactions ( Figure 1 ) to form stable KTN-PEG scaffolds. KTN, having a pI of 5.3, is negatively charged in PBS, pH 7.4 ( Table 1 ) [ 22 ]. Moreover, the reactions do not target any of the ionizable amino acid groups [ 9 ] and should not significantly alter the crosslinked keratin pI.…”

Homo‐ and heteropolymer self‐assembly of recombinant trichocytic keratins

“…Collagen, elastin, silk, and keratin provide a few exquisite examples of functional biopolymers that have been implemented for materials design. Among these, keratin biopolymers have some of the most unique properties that make them especially well‐suited for the generation of biomaterials . First, as with silk and collagen proteins, keratin dimers can be reconstituted into functional materials following extraction from natural sources .…”