Supramolecular peptide nanofiber morphology affects mechanotransduction of stem cells

Chirality and morphology are essential factors for protein function and interactions with other biomacromolecules. Extracellular matrix (ECM) proteins are also similar to other proteins in this sense; however, the complexity of the natural ECM makes it difficult to study these factors at the cellular level. The synthetic peptide nanomaterials harbor great promise in mimicking specific ECM molecules as model systems. In this work, we demonstrate that mechanosensory responses of stem cells are directly regulated by the chirality and morphology of ECM-mimetic peptide nanofibers with strictly controlled characteristics. Structural signals presented on l-amino acid containing cylindrical nanofibers (l-VV) favored the formation of integrin β1-based focal adhesion complexes, which increased the osteogenic potential of stem cells through the activation of nuclear YAP. On the other hand, twisted ribbon-like nanofibers (l-FF and d-FF) guided the cells into round shapes and decreased the formation of focal adhesion complexes, which resulted in the confinement of YAP proteins in the cytosol and a corresponding decrease in osteogenic potential. Interestingly, the d-form of twisted-ribbon like nanofibers (d-FF) increased the chondrogenic potential of stem cells more than their l-form (l-FF). Our results provide new insights into the importance and relevance of morphology and chirality of nanomaterials in their interactions with cells and reveal that precise control over the chemical and physical properties of nanostructures can affect stem cell fate even without the incorporation of specific epitopes

A comparison of peptide amphiphile nanofiber macromolecular assembly strategies

Supramolecular peptide nanofibers that are composed of peptide amphiphile molecules have been widely used for many purposes from biomedical applications to energy conversion. The self-assembly mechanisms of these peptide nanofibers also provide convenient models for understanding the self-assembly mechanisms of various biological supramolecular systems; however, the current theoretical models that explain these mechanisms do not sufficiently explain the experimental results. In this study, we present a new way of modeling these nanofibers that better fits with the experimental data. Molecular dynamics simulations were applied to create model fibers using two different layer models and two different tilt angles. Strikingly, the fibers which were modeled to be tilting the peptide amphiphile molecules and/or tilting the plane were found to be more stable and consistent with the experiments

Sulfated GAG mimetic peptide nanofibers enhance chondrogenic differentiation of mesenchymal stem cells in 3D <i>in vitro</i> models

Articular cartilage, which is exposed to continuous repetitive compressive stress, has limited self-healing capacity in the case of trauma. Thus, it is crucial to develop new treatment options for the effective regeneration of the cartilage tissue. Current cellular therapy treatment options are microfracture and autologous chondrocyte implantation; however, these treatments induce the formation of fibrous cartilage, which degenerates over time, rather than functional hyaline cartilage tissue. Tissue engineering studies using biodegradable scaffolds and autologous cells are vital for developing an effective long-term treatment option. 3D scaffolds composed of glycosaminoglycan-like peptide nanofibers are synthetic, bioactive, biocompatible, and biodegradable and trigger cell–cell interactions that enhance chondrogenic differentiation of cells without using any growth factors. We showed differentiation of mesenchymal stem cells into chondrocytes in both 2D and 3D culture, which produce a functional cartilage extracellular matrix, employing bioactive cues integrated into the peptide nanofiber scaffold without adding exogenous growth factors

Mineralized peptide nanofiber gels for enhanced osteogenic differentiation

\u3cp\u3eMineral deposition is observed in both bacterial and eukaryotic organisms through a broad range of mechanisms. Both organic and inorganic components play crucial roles in the formation of mineralized tissues, and acidic proteins are particularly important in this context owing to their ability to stimulate nucleation of minerals. Here, we present negatively-charged self-assembling peptide amphiphile molecules as a template to nucleate calcium phosphate mineralization in a bioactive scaffold environment. Acidic peptide molecules were shown to induce formation of hydroxyapatite like calcium phosphate mineralization, which was characterized by scanning electron microscopy, transmission electron microscopy, thermogravimetric analysis, X-ray diffractometry, oscillatory rheology and atomic force microscopy. The osteoblast-like cells were found to reveal enhanced osteogenic differentiation on pre-mineralized peptide nanofiber networks, suggesting that mineral deposition can be used as a means of enhancing the bioactivity of peptide-based scaffold systems.\u3c/p\u3

Oligonucleotide Delivery with Cell Surface Binding and Cell Penetrating Peptide Amphiphile Nanospheres

A drug delivery system designed specifically for oligonucleotide therapeutics can ameliorate the problems associated with the in vivo delivery of these molecules. The internalization of free oligonudeotides is challenging, and cytotoxicity is the main obstacle for current transfection vehicles. To develop nontoxic delivery vehicles for efficient transfection of oligonudeotides, we designed a self-assembling peptide amphiphile (PA) nanosphere delivery system decorated with cell penetrating peptides (CPPs) containing multiple arginine residues (R-4 and R-8), and a cell surface binding peptide (KRSR), and report the efficiency of this system in delivering G-3129, a Bcl-2 antisense oligonucleotide (AON). PA/AON (peptide amphiphile/antisense oligonucleotide) complexes were characterized with regards to their size and secondary structure, and their cellular internalization efficiencies were evaluated. The effect of the number of arginine residues on the cellular internalization was investigated by both flow cytometry and confocal imaging, and the results revealed that uptake efficiency improved as the number of arginines in the sequence increased. The combined effect of cell penetration and surface binding property on the cellular internalization and its uptake mechanism was also evaluated by mixing R-8-PA and KRSR-PA. R-8 and R-8/KRSR decorated PAs were found to drastically increase the internalization of AONs compared to nonbioactive PA control. Overall, the KRSR-decorated self-assembled PA nanospheres were demonstrated to be noncytotoxic delivery vectors with high transfection rates and may serve as a promising delivery system for AONs

Glycosaminoglycan-Mimetic Signals Direct the Osteo/Chondrogenic Differentiation of Mesenchymal Stem Cells in a Three-Dimensional Peptide Nanofiber Extracellular Matrix Mimetic Environment

Recent efforts in bioactive scaffold development focus strongly on the elucidation of complex cellular responses through the use of synthetic systems. Designing synthetic extracellular matrix (ECM) materials must be based on understanding of cellular behaviors upon interaction with natural and artificial scaffolds. Hence, due to their ability to mimic both the biochemical and mechanical properties of the native tissue environment, supramolecular assemblies of bioactive peptide nanostructures are especially promising for development of bioactive ECM-mimetic scaffolds. In this study, we used glycosaminoglycan (GAG) mimetic peptide nanofiber gel as a three-dimensional (3D) platform to investigate how cell lineage commitment is altered by external factors. We observed that amount of fetal bovine serum (FBS) presented in the cell media had synergistic effects on the ability of GAG-mimetic nanofiber gel to mediate the differentiation of mesenchymal stem cells into osteogenic and chondrogenic lineages. In particular, lower FBS concentration in the culture medium was observed to enhance osteogenic differentiation while higher amount FBS promotes chondrogenic differentiation in tandem with the effects of the GAG-mimetic 3D peptide nanofiber network, even in the absence of externally administered growth factors. We therefore demonstrate that mesenchymal stem cell differentiation can be specifically controlled by the combined influence of growth medium components and a 3D peptide nanofiber environment

Cellular Internalization of Therapeutic Oligonucleotides by Peptide Amphiphile Nanofibers and Nanospheres

Oligonucleotides are promising drug candidates due to the exceptionally high specificity they exhibit toward their target DNA and RNA sequences. However, their poor pharmacokinetic and pharmacodynamic properties, in conjunction with problems associated with their internalization by cells, necessitates their delivery through specialized carrier systems for efficient therapy. Here, we investigate the effects of carrier morphology on the cellular internalization mechanisms of oligonucleotides by using self-assembled fibrous or spherical peptide nanostructures. Size and geometry were both found to be important parameters for the oligonucleotide internalization process; direct penetration was determined to be the major mechanism for the internalization of nanosphere carriers, whereas nanofibers were internalized by clathrin- and dynamin-dependent endocytosis pathways. We further showed that glucose conjugation to carrier nanosystems improved cellular internalization in cancer cells due to the enhanced glucose metabolism associated with oncogenesis, and the internalization of the glucose-conjugated peptide/oligonucleotide complexes was found to be dependent on glucose transporters present on the surface of the cell membrane

Cellular Internalization of Therapeutic Oligonucleotides by Peptide Amphiphile Nanofibers and Nanospheres

Oligonucleotides are promising drug candidates due to the exceptionally high specificity they exhibit toward their target DNA and RNA sequences. However, their poor pharmacokinetic and pharmacodynamic properties, in conjunction with problems associated with their internalization by cells, necessitates their delivery through specialized carrier systems for efficient therapy. Here, we investigate the effects of carrier morphology on the cellular internalization mechanisms of oligonucleotides by using self-assembled fibrous or spherical peptide nanostructures. Size and geometry were both found to be important parameters for the oligonucleotide internalization process; direct penetration was determined to be the major mechanism for the internalization of nanosphere carriers, whereas nanofibers were internalized by clathrin- and dynamin-dependent endocytosis pathways. We further showed that glucose conjugation to carrier nanosystems improved cellular internalization in cancer cells due to the enhanced glucose metabolism associated with oncogenesis, and the internalization of the glucose-conjugated peptide/oligonucleotide complexes was found to be dependent on glucose transporters present on the surface of the cell membrane

Heparin mimetic peptide nanofiber gel promotes regeneration of full thickness burn injury

Burn injuries are one of the most common types of trauma worldwide, and their unique physiology requires the development of specialized therapeutic materials for their treatment. Here, we report the use of synthetic, functional and biodegradable peptide nanofiber gels for the improved healing of burn wounds to alleviate the progressive loss of tissue function at the post-burn wound site. These bioactive nanofiber gels form scaffolds that recapitulate the structure and function of the native extracellular matrix through signaling peptide epitopes, which can trigger angiogenesis through their affinity to basic growth factors. In this study, the angiogenesis-promoting properties of the bioactive scaffolds were utilized for the treatment of a thermal burn model. Following the excision of necrotic tissue, bioactive gels and control solutions were applied topically onto the wound area. The wound healing process was evaluated at 7, 14 and 21 days following injury through histological observations, immunostaining and marker RNA/protein analysis. Bioactive peptide nanofiber-treated burn wounds formed well-organized and collagen-rich granulation tissue layers, produced a greater density of newly formed blood vessels, and exhibited increased re-epithelialization and skin appendage development with minimal crust formation, while non-bioactive peptide nanofibers and the commercial wound dressing 3M (TM) Tegaderm (TM) did not exhibit significant efficiency over sucrose controls. Overall, the heparin-mimetic peptide nanofiber gels increased the rate of repair of burn injuries and can be used as an effective means of facilitating wound healing. (C) 2017 Elsevier Ltd. All rights reserved

Tenascin‑C Mimetic Peptide Nanofibers Direct Stem Cell Differentiation to Osteogenic Lineage

Extracellular matrix contains various signals for cell surface receptors that regulate cell fate through modulation of cellular activities such as proliferation and differentiation. Cues from extracellular matrix components can be used for development of new materials to control the stem cell fate. In this study, we achieved control of stem cell fate toward osteogenic commitment by using a single extracellular matrix element despite the contradictory effect of mechanical stiffness. For this purpose, we mimicked bone extracellular matrix by incorporating functional sequence of fibronectin type III domain from native tenascin-C on self-assembled peptide nanofibers. When rat mesenchymal stem cells (rMSCs) were cultured on these peptide nanofibers, alkaline phosphatase (ALP) activity and alizarin red staining indicated osteogenic differentiation even in the absence of osteogenic supplements. Moreover, expression levels of osteogenic marker genes were significantly enhanced revealed by quantitative real-time polymerase chain reaction (qRT-PCR), which showed the remarkable bioactive role of this nanofiber system on osteogenic differentiation. Overall, these results showed that tenascin-C mimetic peptides significantly enhanced the attachment, proliferation, and osteogenic differentiation of rMSCs even in the absence of any external bioactive factors and regardless of the suitable stiff mechanical properties normally required for osteogenic differentiation. Thus, these peptide nanofibers provide a promising new platform for bone regeneration