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

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

Layer roughness.

<p>Gold surfaces treated with A) solvent only (10 mM NaOH) and with KOS (oxidized keratin) solution have relatively smooth profiles compared to those with B) KTN (reduced keratin) extract. C) Partial surface adsorption of KTN increased the roughness, Rq, whereas full coverage by overnight KTN incubation led back to a smoother surface. *p < 0.05 compared to each of the other groups.</p

Rates of release of proteins out of bulk KTN gels.

<p>A) Globular proteins diffused out of gels in the following order: negatively-charged albumin, neutral hemoglobin, and positively-charged lysozyme. At the 24-hr time point, the amount of lysozyme released was significantly lower (*p < 0.01) compared to that of albumin and hemoglobin. B) BMP-2 had a slow release profile in PBS (pH 7.4) medium, suggesting tight electrostatic association with the KTN matrix and BMP-2 intermolecular aggregation due to the salting-out effect. KTN bulk degradation was faster relative to the BMP-2 release.</p

KTN-BMP-2 interaction.

<p>Association (a) and dissociation (d) electrostatic interaction profiles of BMP-2 and the KTN monolayer in PBS and in water. BMP-2 analytes were successively flowed, at incrementally increasing concentrations: A) in PBS (pH 7.4) at 0.2, 0.4, 0.9, 1.7, 3.5, and 6.9 μM, B) in PBS (pH 4.5) at 0.03, 0.05, 0.1, 0.2, 0.4, 0.9, and 1.7 μM, and C) in water (pH 7) at 0.03, 0.05, 0.1, 0.2, and 0.4 μM. KTN-BMP-2 electrostatic attraction was strongest in water (KD = 1.1 × 10–7 M). In the presence of PBS salts at physiological pH, binding association was slightly weakened (KD = 3.2 × 10–5 M). Acidification of the PBS eliminated any binding between BMP-2 and KTN.</p

XPS analysis.

<p>A) Wide-scan XPS spectra of gold surfaces treated with 10 mM NaOH solvent, KOS, and KTN. Overnight incubation of KTN led to no detectable Au signals. The inset graph shows that, compared to the solvent group, KOS has very similar concentration levels of carbon, nitrogen, oxygen, sulfur, and gold, while KTN has elevated amounts of protein elements (C, N, and O) but decreased Au. B) Near-scan analysis displays the formation of an amide (O = C-N) peak at 288 eV, corresponding to KTN protein deposition on gold. Unbound and gold-bound KTN thiols were also detected at 163.6 and 162.5 eV, respectively. Partial adsorption of KTN on gold shifted the Au4f peaks to slightly lower energies.</p

KTN bulk release.

<p>KTN gel bulk degradation in vitro at A-B) constant pH = 7.4, and C-D) constant [KCl] = 154 mM for over a period of 28 days at 37°C. Faster degradation occurred at longer time points, lower [KCl], and higher pH levels.</p

Comparison between reduced (KTN) and oxidized (KOS) keratin biomaterials.

<p>KTN can form disulfide linkages to produce a stable scaffold; but KOS cannot, due to the sulfonic acid modification of thiol groups. Consequently, the electrostatic properties are also altered, resulting in more negatively-charged KOS scaffolds, prone to more rapid hydrolytic degradation than KTN scaffolds. 10 mM NaOH solvent was used for wetting and soaking.</p

Interface behavior.

<p>Adsorption kinetics of KTN on gold analyzed through A) SPR and B) QCM-D methods. After establishing baseline readings using 10 mM NaOH solvent, KTN solution was flowed on gold sensor chips until almost full saturation. A) The irreversible adlayer at SPR response (Δn) = 1410 μRIU was retained after solvent, 2% SDS, and solvent washings, and its surface concentration (Γ) = 2.49 mg/m2. B) The normalized changes in dissipation factor (ΔD) for the adsorbate = 0.46 × 10–6 (red curve, using the right y-axis), suggest rigidity. The changes in frequency per overtone (Δf/n) of the adsorbed KTN = -12.08 Hz (green curve, using the left y-axis), correspond to a surface concentration (Γ’) = 2.14 mg/m2. A comparison between Γ (SPR) and Γ’ (QCM-D) showed statistical similarity (p = 0.4273).</p