Summary Molecular design strategies in biomedical applications often involve creating modular “fusion” proteins, in which distinct domains within a single molecule can perform multiple functions. We have synthesized a new class of modular peptides that include a biologically active sequence derived from the growth factor BMP-2 and a series of hydroxyapatite-binding sequences inspired by the N-terminal α-helix of osteocalcin. These modular peptides can bind in a sequence-dependent manner to the surface of “bone-like” hydroxyapatite coatings, which are nucleated and grown on a biodegradable polymer surface via a biomimetic process. The BMP2-derived sequence of the modular peptides is biologically active, as measured by its ability to promote osteogenic differentiation of human mesenchymal stem cells. Our study indicates that the modular peptides described here are multifunctional, and the characteristics of this approach suggest that it can potentially be applied to a range of biomaterials for regenerative medicine applications.
The sequence specificity of the binding of a modular peptide growth factor (eBGa3) that contains a BMP2‐derived sequence and a mineral‐binding sequence inspired by osteocalcin to calcium ions of hydroxyapatite (HA) may be attributed in part to evolution of an α‐helical structure in the presence of HA, which allows for registration of γ‐carboxylated glutamic acid residues in the peptide (cyan) with calcium atoms (orange) in the HA crystal lattice.
Objective The objective of this study is to develop a method for selective detection of the calcific (hydroxyapatite) component in human aortic smooth muscle cells in vitro and in calcified cardiovascular tissues ex vivo. This method uses a novel optical molecular imaging contrast dye, Cy-HABP-19, to target calcified cells and tissues. Methods A peptide that mimics the binding affinity of osteocalcin was used to label hydroxyapatite in vitro and ex vivo. Morphological changes in vascular smooth muscle cells were evaluated at an early stage of the mineralization process induced by extrinsic stimuli, osteogenic factors and a magnetic suspension cell culture. Hydroxyapatite components were detected in monolayers of these cells in the presence of osteogenic factors and a magnetic suspension environment. Results Atherosclerotic plaque contains multiple components including lipidic, fibrotic, thrombotic, and calcific materials. Using optical imaging and the Cy-HABP-19 molecular imaging probe, we demonstrated that hydroxyapatite components could be selectively distinguished from various calcium salts in human aortic smooth muscle cells in vitro and in calcified cardiovascular tissues, carotid endarterectomy samples and aortic valves, ex vivo. Conclusion Hydroxyapatite deposits in cardiovascular tissues were selectively detected in the early stage of the calcification process using our Cy-HABP-19 probe. This new probe makes it possible to study the earliest events associated with vascular hydroxyapatite deposition at the cellular and molecular levels. This target-selective molecular imaging probe approach holds high potential for revealing early pathophysiological changes, leading to progression, regression, or stabilization of cardiovascular diseases.
An effective cellular delivery vector with enhanced intracellular retention was developed by conjugating a cell-penetrating peptide (CPP) with a fatty acid chain. The optimized lipopeptide (LP), myristoylated hendecaarginine (C14R11), penetrated cell membrane with high efficiency, and achieved superior metabolic stability and versatility as compared with unmodified oligoarginine CPPs, offering no adverse effect on cell viability and function. Cellular uptake, intracellular localization, cytotoxicity, and release kinetics of oligoarginines and LPs were investigated using flow cytometry analysis, cytotoxicity assay, and confocal microscopy. The cellular uptake efficiency and intracellular metabolic stability of C14R11 LP was further enhanced by replacing the L-arginine residues with D-arginine isomers. The cellular uptake and intracellular metabolic stability of D-form C14R11 (C14dR11) was significantly increased without any noticeable cytotoxicity compared to the unmodified parent hepta-arginine CPP or L-arginine LPs.
Background In vitro cell culture is a widely used technique for investigating a range of processes such as stem cell behavior, regenerative medicine, tissue engineering, and drug discovery. Conventional cell culture is performed in Petri dishes or flasks where cells typically attach to a flat glass or plastic surface as a cell monolayer. However, 2D cell mono-layers do not provide a satisfactory representation of in vivo conditions. A 3D culture could be a much better system for representing the conditions that prevail in vivo. Methods and results To simulate 3D conditions, vascular smooth muscle cells (VSMCs) were loaded with gold–polyvmer–iron oxide hydrogel, enabling levitation of the cells by using spatially varying magnetic fields. These magnetically levitated 3D cultures appeared as freely suspended, clustered cells which proliferated 3–4 times faster than cells in conventional 2D cultures. When the levitated cells were treated with 10 nM lysophosphatidylcholine (LPC), for 3 days, cell clusters exhibited translucent extensions/rods 60–80 µm wide and 200–250 µm long. When 0.5 µg/µl Schnurri-3 was added to the culture containing LPC, these extensions were smaller or absent. When excited with 590–650 nm light, these extensions emitted intrinsic fluorescence at >667 nm. When the 3D cultures were treated with a fluorescent probe specific for calcium hydroxyapatite (FITC-HABP-19), the cell extensions/rods emitted intensely at 518 nm, the λmax for FITC emission. Pellets of cells treated with LPC were more enriched in calcium, phosphate, and glycosaminogly-cans than cells treated with LPC and Schnurri-3. Conclusions In 3D cultures, VSMCs grow more rapidly and form larger calcification clusters than cells in 2D cultures. Transdifferentiation of VSMC into calcifying vascular cells is enhanced by LPC and attenuated by Schnurri-3. General significance The formation of calcified structures in 3D VSMC cultures suggests that similar structures may be formed in vivo.
Natural proteins are often multifunctional, and therefore capable of activating cell surface receptors, and also binding with high affinity and specificity to natural extracellular matrices (ECMs). To achieve these diverse functions, a strategy commonly employed by nature involves creating modular proteins, in which distinct domains within a single protein are designed to enable either cell signaling or ECM binding. For example, modular proteins such as osteocalcin (OCN) and bone sialoprotein (BSP) contain a domain that binds to hydroxyapatite (HA) -the major biomineral component in the ECM of bony tissues -and a distinct domain that interacts with integrin receptors to mediate cell adhesion.[1] Therefore, these proteins are capable of influencing cell behavior in particular locations within an organism by virtue of their non-covalent linkage to a specific ECM material.The mechanisms that enable binding of signaling molecules to ECM in nature can potentially be extended to synthetic biomaterials as well. For example, a recent study indicates that it is possible to mimic nature's modular cell adhesion proteins (e.g. OCN, BSP) by engineering synthetic modular peptide molecules that bind to synthetic HA, yet remain capable of affecting cell adhesion. [2] This modular design approach has been used to promote cell adhesion to HA materials, which are now used in a wide range of common clinical orthopedic applications. However, previous studies have not yet extended this type of modular design strategy in order to non-covalently immobilize growth factors, which are capable of actively regulating stem cell phenotype.We hypothesized that modular peptides inspired by portions of natural proteins could provide a mechanism to decorate common biomedical materials with growth factors. We reasoned that the surface-immobilized peptide growth factor could then promote stem cell differentiation on the material surface. Specifically, we synthesized modular peptide growth factors, which mimic the HA-binding ability of OCN and the ability of bone morphogenetic protein-2 (BMP-2) to promote stem cell differentiation. OCN is a 5.7 kDa protein that binds to calcium in the HA crystal lattice (600 nM dissociation constant).[3] HA-OCN binding can be largely attributed a 9-mer sequence on the N-terminus, which contains three γ-carboxylated glutamic acid (Gla) residues that coordinate with Ca 2+ ions in the HA crystal. [4] Therefore, we reasoned that the N-terminal helix derived from OCN could be used as a linker to attach BMP-2 to a HA surface. BMP-2 is a 26 kDa protein that exerts its effects by stimulating differentiation of progenitor cells toward an osteoblastic lineage. [5,6] Recently Tanihara and coworkers discovered that a 20 amino acid peptide sequence from the "knuckle" epitope of BMP-2 retains the biological activity of the full-length BMP-2 protein.[7-10] Therefore, we hypothesized that a modular peptide containing an OCN-inspired portion and the 20-mer derived from the BMP-2 knuckle epitope could be used to promote substrate-med...
Twinkle, twinkle little bone! A short peptide derived from a natural bone binding protein, osteocalcin, shows excellent selectivity and affinity to hydroxyapatite, the main component of bone. With a fluorescent label, the intravenously injected peptide revealed detailed bone structures in mice, like an X‐ray image (see figure).
Nanoparticles have great potential as nanotherapeutics, delivery vectors, and molecular imaging agents due to their flexible properties. Although intracellular and nuclear delivery of nanoparticles is desirable for therapeutic applications, it remains a challenge. Cell penetrating peptides (CPPs) are a powerful tool for the intracellular delivery of various cargoes. Here we report that functionalization of nanoparticles with a myristoylated oligoarginine CPP promoted cellular uptake without increased toxicity. It was evidenced that the myristoylated CPP is much more effective in transporting nanoparticles than the unmodified CPPs.
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