de Guzman Roche C, Tsuda Shanel M, Ton Minh-Thi N, Zhang Xiao, Esker Alan R, Van Dyke Mark E
School of Biomedical Engineering and Sciences, Virginia Polytechnic Institute and State University, Blacksburg, Virginia, United States of America.
Department of Chemistry, Virginia Polytechnic Institute and State University, Blacksburg, Virginia, United States of America.
PLoS One. 2015 Aug 28;10(8):e0137233. doi: 10.1371/journal.pone.0137233. eCollection 2015.
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.
主要由还原角蛋白（角蛋白）组成的毛发来源的角蛋白生物材料已证明其作为用于组织工程的生物制剂和药物载体的效用。带负电荷的角蛋白与生物大分子之间的静电力可实现有效的药物保留；对角蛋白与带正电荷的生长因子（如骨形态发生蛋白2，BMP-2）的吸引已被用作骨诱导策略。在本研究中，对角蛋白的分子间表面和本体相互作用特性进行了研究。富含硫醇的角蛋白化学吸附到金基底上，形成用于表面等离子体共振分析的不可逆2纳米刚性层。在pH值为中性的水中观察到角蛋白与角蛋白之间的内聚力，平衡解离常数（KD）为1.8×10（-4）M，表明非库仑吸引力（即疏水和范德华力）在起作用。发现BMP-2与角蛋白的结合更强（KD = 1.1×10（-7）M），处于特异性结合范围内。添加盐（磷酸盐缓冲盐水；PBS）缩短了德拜长度或静电场影响，这削弱了角蛋白 - BMP-2结合（KD = 3.2×10（-5）M）。本体角蛋白凝胶中的BMP-2在PBS中释放有限（4周内约10%解离），表明静电分子间吸引力对于将BMP-2保留在角蛋白基质中很重要。当PBS的pH值降低到5.3的角蛋白等电点以下（降至4.5）时，角蛋白与BMP-2之间完全解离。这种现象可归因于较低pH值下角蛋白的质子化，导致正 - 正排斥。因此，角蛋白 - BMP-2结合的动力学高度依赖于pH值和盐浓度，以及BMP-2在不同pH值和摩尔浓度下的溶解度。该研究结果可能有助于我们理解药物从角蛋白生物材料中的释放动力学，并有助于开发更好的、更具临床相关性的用于骨修复和再生的BMP-2共轭系统。
