A Supramolecular Host鈥揋uest Carrier System for Growth Factors Employing VHH Fragments

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• 作者：Jordi Cabanas-Dan茅s ; Emilie Dooms Rodrigues ; Ellie Landman ; Jasper van Weerd ; Clemens van Blitterswijk ; Theo Verrips ; Jurriaan Huskens ; Marcel Karperien ; Pascal Jonkheijm
• 刊名：Journal of the American Chemical Society
• 出版年：2014
• 出版时间：September 10, 2014
• 年：2014
• 卷：136
• 期：36
• 页码：12675-12681
• 全文大小：398K
• ISSN：1520-5126

文摘

A supramolecular strategy is presented for the assembly of growth factors employing His₆-tagged single-domain antibodies (V_HH). A combination of orthogonal supramolecular interactions of 尾-cyclodextrin (尾CD)鈥揳damantyl (Ad) host鈥揼uest and N-nitrilotriacetic acid (NTA)鈥揾istidine (His) interactions was employed to generate reversible and homogeneous layers of growth factors. A single-domain antibody V_HH fragment was identified to bind to the human bone morphogenetic protein-6 (hBMP6) growth factor and could be recombinantly expressed in E. coli. The V_HH fragment was equipped with a C-terminal hexahistidine (His₆) tether to facilitate the assembly on 尾CD surfaces using a linker that contains an Ad group to bind to the 尾CD receptors and an NTA moiety to interact with the His₆-tag upon cocomplexation of Ni²⁺ ions. After exploring the thermodynamic and kinetic stability of the V_HH assemblies on 尾CD surfaces using a variety of experimental techniques including microcontact printing (渭CP), surface plasmon resonance (SPR), microscale thermophoresis (MST), and theoretical models for determining the thermodynamic behavior of the system, hBMP6 was assembled onto the V_HH-functionalized surfaces. After analyzing the immobilized hBMP6 using immunostaining, the biological activity of hBMP6 was demonstrated in cell differentiation experiments. Early osteogenic differentiation was analyzed in terms of alkaline phosphatase (ALP) activity of KS483-4C3 mouse progenitor cells, and the results indicated that the reversibly immobilized growth factors were functionally delivered to the cells. In conclusion, the supramolecular strategy used here offers the necessary affinity, reversibility, and temporal control to promote biological function of the growth factors that were delivered by this strategy.