Proteins and their functional domains, peptides, are extensively applied in the development of biomaterials with enhanced cell attachment, directed differentiation and promoted new tissue formation. However, catastrophic complications associated with use of protein-encapsulated scaffolds in spinal fusion surgeries were frequently reported. The causes of these complications and adverse events are mainly attributed to the lack of locally restriction of proteins in the required sites. Therefore, methods that sequester the proteins or their derivative peptides without interrupting their bioactivities were pursued. These methods did not only help the fabrication of model systems to study the cell behavior under variable biochemical stimuli in academic research, but also have promising potency in protein therapy and other biomedicine applications that require localized distribution of proteins or peptides.
To solve this problem, we designed and synthesized a series of modular peptides that contain a surface-targeting domain and a bioactive domain. In the bioactive domain, versatile moieties coming with specific biomedical functionalities, such as detective tags, or bioactive peptides were served to realize bio-imaging diagnostics or promotion of cell proliferation and direction of differentiation, respectively. The surface-targeting domain should possess super strong binding affinity to the corresponding surface, to sequester the whole molecule from a low concentration solution, and eliminate its diffusion into body fluid which may cause adverse events.
To seek for the ideal candidate that acts as the surface-targeting domain, multivalent binding strategies were applied. In the multivalent binding ligand, several binding units that have binding affinity to corresponding receptors were conjugated in one molecule with a dendron serving as the scaffold. Due to multiple ligand-receptor pairs interacting simultaneously, the binding affinity between the multivalent binding ligand and the corresponding homing surface were dramatically enhanced. The ultimate binding affinity is determined by the structure of the scaffold that linked several binding units together. Specifically, binding valency, which is the number of binding units in one molecule, and length of linkage part, play a fundamental role in the enthalpic and entropic part of the free energy of the binding process, respectively. Therefore a series of multivalent ligands were synthesized to optimize the structure of the multivalent ligand for strongest binding with cost efficiency.
The binding units are versatile and chosen depending on the homing surface. As demonstrations, bone implants surface, generally hydroxyapatite (HA) and titanium oxide (TiO2), were chosen to be the targeting surface due to their wide application in orthopedic and dental implants in clinic treatment.
In the first work, a series of hydroxyapatite (HA)-binding peptide functionalized dendrons as the multivalent binding ligands that bind with HA specifically was designed, synthesized and characterized with quartz crystal microbalance with dissipation (QCM-D). The structure factors, valency and length of flexible linkage, were tuned to optimize the structure which provides the strongest binding with lowest cost. (HA)4-2 showed the strongest binding affinity to HA with a Kd equal to 270 nM, a near 1000-fold enhancement compared with HA-binding peptide, which is the strongest binding motif to HA that has been reported. Bone morphogenetic protein-2 (BMP-2) derived peptide that promotes bone formation, and a generally applied detective tag, biotin, was modified in the focal point of the HA-binding dendrons, respectively, for osteoconductive test and fluorescein imaging study. The biotinylated-(HA)2 gave a more clear image of detailed HA morphology with a 10-fold lower concentration compared with biotinylated-HA molecules, which has potency to be used in HA assay to visualize the biomineral formation.
In the second work, a series of catechol-bearing modular peptides, containing a osteogenic growth peptide as the bioactive domain and a surface-binding dendron domain, was obtained through solid phase synthesis to realize efficient and fast surface modification of bioactive peptides. Their binding affinity to TiO2 was characterized with quartz crystal microbalance with dissipation (QCM-d). The fluorescein-labeled modular peptides were readily synthesized, further adding a detective tag to the surface-targeting bioactive peptides. The multivalent binding effect was studied by tuning the number of catechol groups. The presence of immobilized peptides after simple incubation-and-rinse procedure was proved with X-ray photoelectron spectroscopy and fluorescein microscope. The modular peptide with four catechol groups preserved on the surface beyond two weeks, giving enough time for the bioactive domain to interact with upcoming cells. Impressively, the modular peptides also show a strong adhesion to versatile inorganic surfaces, including ZrO2, CeO2, Fe3O4 and gold. Based on the difference in binding affinity of the modular peptides to surfaces, selective modification was achieved on partially TiO2-coated glass slides. The in vitro cell culture studies demonstrated their bio-safety. These catechol-bearing modular peptides provide a fast and efficient method to functionalize a wide range of inorganic materials with bioactive peptides.
Furthermore, the bioactivity of the osteogenic growth peptide (OGP) (10-14) in the focal point of the dendron was evaluated by culturing MC3T3-E1 on the TiO2 substrates with or without modular peptide. The cell proliferation, osteogenic differentiation and mineralization were studied by proliferation assay, immunochemical staining, enzyme and calcium quantification. Compared with bare titanium oxide substrates, MC3T3-E1 cells on OGP (10-14) modified titanium oxide substrates, exhibited significant difference with about 3-fold higher of alkaline phosphatase (ALP) activity and more than 2-times more of calcium deposition, indication they are promoted to differentiate to osteoblast.
In a summary, dendrons were applied in the construction of multivalent surface-binding ligand bearing a bioactive peptide, to realize efficient and convenient surface modification. Peptide-conjugates synthesized via high efficient “click” reactions were developed. The surface interactions of multivalent binding ligand to bone implants surfaces were studied with multiple techniques to prove the successful immobilization and quantify the binding affinity for estimation of load amount and binding kinetics. The modular peptides were non-toxic, and their bioactivity was preserved in promoting MC3T3 cells osteogenic differentiation and mineralization. These bioactive surface-targeting modular peptides appear promising in the development of translational implants with improved bioactive performance to eliminate complications and shorten healing time for patients.