A Supramolecular Host–Guest Carrier System for Growth Factors Employing V_HH Fragments

Abstract

A supramolecular strategy is presented for the assembly of growth factors employing His6-tagged single-domain antibodies (VHH). A combination of orthogonal supramolecular interactions of β-cyclodextrin (βCD)-adamantyl (Ad) host-guest and N-nitrilotriacetic acid (NTA)-histidine (His) interactions was employed to generate reversible and homogeneous layers of growth factors. A single-domain antibody V(H)H 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(H)H fragment was equipped with a C-terminal hexahistidine (His6) tether to facilitate the assembly on βCD surfaces using a linker that contains an Ad group to bind to the βCD receptors and an NTA moiety to interact with the His6-tag upon cocomplexation of Ni(2+) ions. After exploring the thermodynamic and kinetic stability of the V(H)H assemblies on βCD surfaces using a variety of experimental techniques including microcontact printing (μCP), surface plasmon resonance (SPR), microscale thermophoresis (MST), and theoretical models for determining the thermodynamic behavior of the system, hBMP6 was assembled onto the V(H)H-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.

Supramolecular cyclodextrin-based drug nanocarriers

“…16 One of the most interesting attributes of CDs is their ability to host a variety of lipophilic compounds which can be partially or 55 totally included in the hydrophobic internal cavity; the hydrophilic outer face being exposed to the aqueous medium. 6, 17 The phenomenon of CD inclusion complex formation is a multifaceted process driven by many factors, mainly due to displacement of enthalpy-rich water molecules from the CD 60 cavity (repulsive polar-non polar interactions) by a "guest molecule" with appropriate geometry and physicochemical properties. 18 The predominant interactions engaged in the drug-CD complex include van der Waals and hydrophobic binding, but other such as hydrogen bonding, release of ring strain in CD and 2 | Journal Name, [year], [vol], 00-00…”

Supramolecular Surface Immobilization of Knottin Derivatives for Dynamic Display of High Affinity Binders

“…Their loop-structure would also ensure better solvent-exposure of the incorporated peptide sequences and their size (3 -5 kDa) allows for tighter, oriented packing than antibodies and nanobodies. 21 Since they contain only about 30 amino acids that fold into a tight cage-like structure, non-specific interactions with surfaces and proteins are also substantially reduced compared to larger constructs. However, so far, there are no studies attempting to fabricate biofunctional surfaces using knottins.…”

Supramolecular Protein Immobilization on Lipid Bilayers

“…This immobilization can be reversed by the addition of ethylenediaminetetraacetic acid (EDTA) or imidazole. [22][23][24][25][26] Another reported supramolecular immobilizations trategy is the use of complementary DNA strandst hat are fused to the protein and immobilized on the surface. [27][28][29][30] More recently,s ynthetic supramolecular ligand-receptor pairs have been used for protein immobilization.…”

“…By incorporation of theses ynthetic receptors on as olids upport, proteins containing the correspondingl igand can be immobilized. [20,26,[31][32][33][34][35][36][37][38][39] There are, however, only very limited examples of the use of supramolecular chemistry fort he immobilization of proteins on lipid bilayers. The potentialo f as upramolecular approach forp rotein immobilization on lipid membranes is most impressively illustrated by the membrane incorporation of nitrilotriacetic acids that recruit His 6 -tagged proteins upon addition of nickel.…”