Peptide Amphiphile Molecules Research Articles

Self-Assembly and Wound Healing Activity of Biomimetic Cycloalkane-Based Lipopeptides.

The self-assembly of lipopeptide (peptide amphiphile) molecules bearing single linear lipid chains has been widely studied, as has their diverse range of bioactivities. Here, we introduce lipopeptides bearing one or two cycloalkane chains (cycloheptadecyl or cyclododecyl) conjugated to the collagen-stimulating pentapeptide KTTKS used in Matrixyl formulations. The self-assembly of all four molecules is probed using fluorescence probe measurements to detect the critical aggregation concentration (CAC), and cryogenic-TEM and small-angle X-ray scattering (SAXS) to image the nanostructure. The peptide conformation is studied using circular dichroism (CD) and FTIR spectroscopies. All the cycloalkane lipopeptides show excellent compatibility with dermal fibroblasts. The compounds bearing one or two cyclododecyl chains (denoted as DKT and DDKT, respectively) show wound healing in diabetic rats, the improvement being markedly enhanced for DDKT. Interestingly, the revival of hair follicles and blood vessels in the dermis were observed, which are the critical markers of effective wound repair. Analysis of H&E-stained tissue images (from a rat model) shows that the rat groups treated with DDKT and DKT displayed a significantly increased amount of regenerated hair follicles, indicating a faster healing process for DDKT compared to the control group. Collagen deposition was also enhanced, especially for DDKT, and by day 20, the DDKT-treated groups had developed a dense collagen network accompanied by a regenerated epidermis. At the same time, the number of blood vessels in DDKT-treated diabetic wounds was significantly higher than in control groups and neovascularization was substantially enhanced, as assayed using α-SMA (a marker for vascular smooth muscle cells) and CD31 (a marker specific to vascular endothelial cells). These results suggest that the lead lipopeptide DDKT exhibits a remarkable pro-vascularization capability and shows great promise for future application as a wound-healing biomaterial.

Supramolecular Motion Enables Chondrogenic Bioactivity of a Cyclic Peptide Mimetic of Transforming Growth Factor-β1.

Transforming growth factor (TGF)-β1 is a multifunctional protein that is essential in many cellular processes that include fibrosis, inflammation, chondrogenesis, and cartilage repair. In particular, cartilage repair is important to avoid physical disability since this tissue does not have the inherent capacity to regenerate beyond full development. We report here on supramolecular coassemblies of two peptide amphiphile molecules, one containing a TGF-β1 mimetic peptide, and another which is one of two constitutional isomers lacking bioactivity. Using human articular chondrocytes, we investigated the bioactivity of the supramolecular copolymers of each isomer displaying either the previously reported linear form of the mimetic peptide or a novel cyclic analogue. Based on fluorescence depolarization and 1H NMR spin-lattice relaxation times, we found that coassemblies containing the cyclic compound and the most dynamic isomer exhibited the highest intracellular TGF-β1 signaling and gene expression of cartilage extracellular matrix components. We conclude that control of supramolecular motion is emerging as an important factor in the binding of synthetic molecules to receptors that can be tuned through chemical structure.

Peptide Amphiphile‐Mediated Assembly and Fusion of Anisotropic Amorphous Particles for Enamel Remineralization

AbstractEnamel remineralization, which attempts to generate a mineral layer structurally similar to native enamel and restore its mechanical properties, remains a significant challenge. Here, rationally designed peptide amphiphile (PA) molecules bind to amorphous calcium phosphate (ACP) particles via electrostatic interactions, imparting anisotropic properties that drive PA‐modified ACP to form spindle‐shaped aggregates. Furthermore, some residual PA molecules may further regulate crystallization and facilitate the formation of well‐aligned hydroxyapatite bundles. Additionally, the involvement of PA can mediate the ordered deposition and fusion of ACP on the enamel surface, and construct the crystalline‐amorphous mineralization front with a continuous structure. This mineralization front ensures epitaxially oriented growth of enamel crystals and achieves a structurally ordered mineral layer similar to that of native enamel, while the mechanical performance is effectively restored. More significantly, the thickness of the newly formed mineral layer can be augmented through cyclic remineralization, benefitting the design of products intended for practical enamel repair.

Systemic peptide amphiphile nanofiber delivery following subcutaneous injection

Peptide amphiphile (PA) nanofibers have been shown to target and deliver drugs when administered via an intravenous (IV) injection. Subcutaneous administration can broaden the applicability of PA nanofibers in the medical field. The ability of PA nanofibers to be absorbed into systemic circulation after subcutaneous administration was investigated. Four PA molecules with different amino acid sequences were designed to understand the effect of nanofiber cohesion and charge on uptake. Solution small-angle X-ray scattering confirmed nanostructure morphology and provided characteristic lengths for co-assemblies. Circular dichroism and solution wide-angle X-ray scattering confirmed PA secondary structure and molecular order. PAs were co-assembled in a 95 %:5 % molar ratio of unlabeled PA to fluorescently labeled PA. Male and female Sprague Dawley rats were injected in the nape of the neck with PA co-assemblies. In vivo normalized abdominal fluorescence was measured 1–72 h after injection. PA nanofibers with a negative charge and low internal order showed the highest amount of systemic absorption at 1, 6, and 24 h. At 24 h after injection, white blood cell count decreased and glucose was elevated. Glucose began to decrease at 48 h. These data indicate that PA nanofibers can be absorbed into the systemic circulation after subcutaneous injection.

Optimization of peptide amphiphile-lipid raft interaction by changing peptide amphiphile lipophilicity.

Various peptide amphiphile (PA) molecules have been developed to promote bone regeneration. Previously we discovered that a peptide amphiphile with a palmitic acid tail (C16) attenuates the signaling threshold of leucine-rich amelogenin peptide (LRAP)-mediated Wnt activation by increasing membrane lipid raft mobility. In the current study, we found that treatment of murine ST2 cells with an inhibitor (Nystatin) or Caveolin-1-specific siRNA abolishes the effect of C16 PA, indicating that Caveolin-mediated endocytosis is required. To determine whether hydrophobicity of the PA tail plays a role in its signaling effect, we modified the length of the tail (C12, C16 and C22) or composition (cholesterol). While shortening the tail (C12) decreased the signaling effect, lengthening the tail (C22) had no prominent effect. On the other hand, the cholesterol PA displayed a similar function as the C16 PA at the same concentration of 0.001% w/v. Interestingly, a higher concentration of C16 PA (0.005%) is cytotoxic while cholesterol PA at the higher concentration (0.005%) is well-tolerated by cells. Use of the cholesterol PA at 0.005% enabled a further reduction of the signaling threshold of LRAP to 0.20nM, compared to 0.25nM at 0.001%. Caveolin-mediated endocytosis is also required for cholesterol PA, as evidenced by Caveolin-1 siRNA knockdown experiments. We further demonstrated that the noted effects of cholesterol PA are also observed in human bone marrow mesenchymal stem cells (BMMSCs). Taken together, these results indicate that the cholesterol PA modulates lipid raft/caveolar dynamics, thereby increasing receptor sensitivity for activation of canonical Wnt signaling. STATEMENT OF SIGNIFICANCE: Cell signaling involves not only the binding of growth factors (or other cytokines) and cognate receptors, but also their clustering on the cell membrane. However, little or no work has been directed thus far toward investigating how biomaterials can serve to enhance growth factor or peptide signaling by increasing diffusion of cell surface receptors within membrane lipid rafts. Therefore, a better understanding of the cellular and molecular mechanism(s) operating at the material-cell membrane interface during cell signaling has the potential to change the paradigm in designing future biomaterials and regenerative medicine therapeutics. In this study, we designed a peptide amphiphile (PA) with a cholesterol tail to enhance canonical Wnt signaling by modulating lipid raft/caveolar dynamics.

Metal-Chelating Self-Assembling Peptide Nanofiber Scaffolds for Modulation of Neuronal Cell Behavior.

Synthetic peptides are promising structural and functional components of bioactive and tissue-engineering scaffolds. Here, we demonstrate the design of self-assembling nanofiber scaffolds based on peptide amphiphile (PA) molecules containing multi-functional histidine residues with trace metal (TM) coordination ability. The self-assembly of PAs and characteristics of PA nanofiber scaffolds along with their interaction with Zn, Cu, and Mn essential microelements were studied. The effects of TM-activated PA scaffolds on mammalian cell behavior, reactive oxygen species (ROS), and glutathione levels were shown. The study reveals the ability of these scaffolds to modulate adhesion, proliferation, and morphological differentiation of neuronal PC-12 cells, suggesting a particular role of Mn(II) in cell-matrix interaction and neuritogenesis. The results provide a proof-of-concept for the development of histidine-functionalized peptide nanofiber scaffolds activated with ROS- and cell-modulating TMs to induce regenerative responses.

Peptide Nanofiber System for Sustained Delivery of Anti-VEGF Proteins to the Eye Vitreous.

Ranibizumab is a recombinant VEGF-A antibody used to treat the wet form of age-related macular degeneration. It is intravitreally administered to ocular compartments, and the treatment requires frequent injections, which may cause complications and patient discomfort. To reduce the number of injections, alternative treatment strategies based on relatively non-invasive ranibizumab delivery are desired for more effective and sustained release in the eye vitreous than the current clinical practice. Here, we present self-assembled hydrogels composed of peptide amphiphile molecules for the sustained release of ranibizumab, enabling local high-dose treatment. Peptide amphiphile molecules self-assemble into biodegradable supramolecular filaments in the presence of electrolytes without the need for a curing agent and enable ease of use due to their injectable nature-a feature provided by shear thinning properties. In this study, the release profile of ranibizumab was evaluated by using different peptide-based hydrogels at varying concentrations for improved treatment of the wet form of age-related macular degeneration. We observed that the slow release of ranibizumab from the hydrogel system follows extended- and sustainable release patterns without any dose dumping. Moreover, the released drug was biologically functional and effective in blocking the angiogenesis of human endothelial cells in a dose-dependent manner. In addition, an in vivo study shows that the drug released from the hydrogel nanofiber system can stay in the rabbit eye's posterior chamber for longer than a control group that received only a drug injection. The tunable physiochemical characteristics, injectable nature, and biodegradable and biocompatible features of the peptide-based hydrogel nanofiber show that this delivery system has promising potential for intravitreal anti-VEGF drug delivery in clinics to treat the wet form age-related macular degeneration.

Peptide-Protein Coassemblies into Hierarchical and Bioactive Tubular Membranes.

Multicomponent self-assembly offers opportunities for the design of complex and functional biomaterials with tunable properties. Here, we demonstrate how minor modifications in the molecular structures of peptide amphiphiles (PAs) and elastin-like recombinamers (ELs) can be used to generate coassembling tubular membranes with distinct structures, properties, and bioactivity. First, by introducing minor modifications in the charge density of PA molecules (PAK2, PAK3, PAK4), different diffusion-reaction processes can be triggered, resulting in distinct membrane microstructures. Second, by combining different types of these PAs prior to their coassembly with ELs, further modifications can be achieved, tuning the structures and properties of the tubular membranes. Finally, by introducing the cell adhesive peptide RGDS in either the PA or EL molecules, it is possible to harness the different diffusion-reaction processes to generate tubular membranes with distinct bioactivities. The study demonstrates the possibility to trigger and achieve minor but crucial differences in coassembling processes and tune material structure and bioactivity. The study demonstrates the possibility to use minor, yet crucial, differences in coassembling processes to tune material structure and bioactivity.

Amyloid‐β Inspired Short Peptide Amphiphile Facilitates Synthesis of Silver Nanoparticles as Potential Antibacterial Agents

An amyloid-β inspired biocompatible short peptide amphiphile (sPA) molecule was used for controlled and targeted delivery of bioactive silver nanoparticles via transforming sPA nanostructures. Such sPA-AgNPs hybrid structures can be further used to develop antibacterial materials to combat emerging bacterial resistance. Due to the excellent antibacterial activity of silver, the growth of clinically relevant bacteria was inhibited in the presence of AgNPs-sPA hybrids. Bacterial tests demonstrated that the high biocompatibility and low cytotoxicity of the designed sPA allow it to work as a model drug delivery agent. It therefore shows great potential in locally addressing bacterial infections. The results of our study suggest that these nanodevices have the potential to trap and then engage in the facile delivery of their chemical payload at the target site, thereby working as potential delivery materials. This system has potential therapeutic value for the treatment of microbiota triggered progression of neurodegenerative diseases.

Peptide Amphiphile Supramolecular Nanofibers Designed to Target Abdominal Aortic Aneurysms.

An abdominal aortic aneurysm (AAA) is a localized dilation of the aorta located in the abdomen that poses a severe risk of death when ruptured. The cause of AAA is not fully understood, but degradation of medial elastin due to elastolytic matrix metalloproteinases is a key step leading to aortic dilation. Current therapeutic interventions are limited to surgical repair to prevent catastrophic rupture. Here, we report the development of injectable supramolecular nanofibers using peptide amphiphile molecules designed to localize to AAA by targeting fragmented elastin, matrix metalloproteinase 2 (MMP-2), and membrane type 1 matrix metalloproteinase. We designed four targeting peptide sequences from X-ray crystallographic data and incorporated them into PA molecules via solid phase peptide synthesis. After coassembling targeted and diluent PAs at different molar ratios, we assessed their ability to form nanofibers using transmission electron microscopy and to localize to AAA in male and female Sprague-Dawley rats using light sheet fluorescence microscopy. We found that three formulations of the PA nanofibers were able to localize to AAA tissue, but the MMP-2 targeting PA substantially outperformed the other nanofibers. Additionally, we demonstrated that the MMP-2 targeting PA nanofibers had an optimal dose of 5 mg (∼12 mg/kg). Our results show that there was not a significant difference in targeting between male and female Sprague-Dawley rats. Given the ability of the MMP-2 targeting PA nanofiber to localize to AAA tissue, future studies will investigate potential diagnostic and targeted drug delivery applications for AAA.

Acid-Base Equilibrium and Dielectric Environment Regulate Charge in Supramolecular Nanofibers.

Peptide amphiphiles are a class of molecules that can self-assemble into a variety of supramolecular structures, including high-aspect-ratio nanofibers. It is challenging to model and predict the charges in these supramolecular nanofibers because the ionization state of the peptides are not fixed but liable to change due to the acid-base equilibrium that is coupled to the structural organization of the peptide amphiphile molecules. Here, we have developed a theoretical model to describe and predict the amount of charge found on self-assembled peptide amphiphiles as a function of pH and ion concentration. In particular, we computed the amount of charge of peptide amphiphiles nanofibers with the sequence C 16 − V 2 A 2 E 2. In our theoretical formulation, we consider charge regulation of the carboxylic acid groups, which involves the acid-base chemical equilibrium of the glutamic acid residues and the possibility of ion condensation. The charge regulation is coupled with the local dielectric environment by allowing for a varying dielectric constant that also includes a position-dependent electrostatic solvation energy for the charged species. We find that the charges on the glutamic acid residues of the peptide amphiphile nanofiber are much lower than the same functional group in aqueous solution. There is a strong coupling between the charging via the acid-base equilibrium and the local dielectric environment. Our model predicts a much lower degree of deprotonation for a position-dependent relative dielectric constant compared to a constant dielectric background. Furthermore, the shape and size of the electrostatic potential as well as the counterion distribution are quantitatively and qualitatively different. These results indicate that an accurate model of peptide amphiphile self-assembly must take into account charge regulation of acidic groups through acid–base equilibria and ion condensation, as well as coupling to the local dielectric environment.

Hydrogen Bonding Stiffens Peptide Amphiphile Supramolecular Filaments by Aza-Glycine Residues

Peptide amphiphiles (PAs) are a class of molecules comprised of short amino acid sequences conjugated to hydrophobic moieties that may exhibit self-assembly in water into supramolecular structures. We investigate here how mechanical properties of hydrogels formed by PA supramolecular nanofibers are affected by hydrogen bond densities within their internal structure by substituting glycine for aza-glycine (azaG) residues. We found that increasing the number of PA molecules that contain azaG up to 5 mol% in PA supramolecular nanofibers increases their persistence length fivefold and decreases their diffusion coefficients as measured by fluorescence recovery after photobleaching. When these PAs are used to create hydrogels, their bulk storage modulus (G′) was found to increase as azaG PA content in the supramolecular assemblies increases up to a value of 10 mol% and beyond this value a decrease was observed, likely due to diminished levels of nanofiber entanglement in the hydrogels as a direct result of increased supramolecular rigidity. Interestingly, we found that the bioactivity of the scaffolds toward dopaminergic neurons derived from induced pluripotent stem cells can be enhanced directly by persistence length independently of storage modulus. We hypothesize that this is due to interactions between the cells and the extracellular environment across different size scales: from filopodia adhering to individual nanofiber bundles to cell adhesion sites that interact with the hydrogel as a bulk substrate. Fine tuning of hydrogen bond density in self-assembling peptide biomaterials such as PAs provides an approach to control nanoscale stiffness as part of an overall strategy to optimize bioactivity in these supramolecular systems. supramolecular biomaterials. Statement of significanceHydrogen bonding is an important driving force for the self-assembly of peptides in both biological and artificial systems. Here, we increase the amount of hydrogen bonding within self-assembled peptide amphiphile (PA) nanofibers by substituting glycine for an aza-glycine (azaG). We show that increasing the molar concentration of azaG increases the internal order of individual nanofibers and increases their persistence length. We also show that these changes are sufficient to increase survival and tyrosine hydroxylase expression in induced pluripotent stem cell-derived dopaminergic neurons cultured in 3D gels made of these materials. Our strategy of tuning the number of hydrogen bonds in a supramolecular assembly provides mechanical customization for 3D cell culture and tissue engineering.

Control of Peptide Amphiphile Supramolecular Nanostructures by Isosteric Replacements.

Supramolecular nanostructures with tunable properties can have applications in medicine, pharmacy, and biotechnology. In this work, we show that the self-assembly behavior of peptide amphiphiles (PAs) can be effectively tuned by replacing the carboxylic acids exposed to the aqueous media with isosteres, functionalities that share key physical or chemical properties with another chemical group. Transmission electron microscopy, atomic force microscopy, and small-angle X-ray scattering studies indicated that the nanostructure's morphologies are responsive to the ionization states of the side chains, which are related to their pKa values. Circular dichroism studies revealed the effect of the isosteres on the internal arrangement of the nanostructures. The interactions between diverse surfaces and the nanostructures and the effect of salt concentration and temperature were assessed to further understand the properties of these self-assembled systems. These results indicate that isosteric replacements allow the pH control of supramolecular morphology by manipulating the pKa of the charged groups located on the nanostructure's surface. Theoretical studies were performed to understand the morphological transitions that the nanostructures underwent in response to pH changes, suggesting that the transitions result from alterations in the Coulomb forces between PA molecules. This work provides a strategy for designing biomaterials that can maintain or change behaviors based on the pH differences found within cells and tissues.

Molecular design of peptide amphiphiles for controlled self-assembly and drug release.

Peptide amphiphile-based supramolecular hydrogels hold great promise in drug delivery applications. To cater for a specific drug dose in a demanding biomedical scenario, sophisticated design of peptide amphiphile (PA) molecules is required to tune their self-assembling behaviours as well as drug releasing profiles. Herein, we designed a series of PAs with various capping groups and C-terminal amino acids to systematically optimize their self-assembling capabilities for controlled drug release. First, we evaluated the influence of N-terminal capping groups to find that the 2-naphthylacetyl moiety (Nap) greatly assisted hydrogelation of PAs. Next, self-assembling behaviours of Nap-capped PAs were compared among three candidates that bore varying hydrophilic moieties at the C-terminus (Nap-C12-VVAAG, Nap-C12-VVAAD and Nap-C12-VVAADD, denoted as 1-G, 1-D, and 1-DD). It was found that 1-G and 1-D co-assembled with doxorubicin (DOX) and calcium ions (Ca2+) at a higher efficiency than 1-DD, for 1-G/Ca2+/DOX and 1-D/Ca2+/DOX hydrogels displayed a dense nanofibrillar network, with lower minimal gelation concentrations and greater storage modulus values. Interestingly, these PA/Ca2+/DOX hydrogels exhibited tunable release rates of DOX in vitro, with fast release of DOX found in 1-DD/Ca2+/DOX and slow release in 1-G/Ca2+/DOX and 1-D/Ca2+/DOX. Further cell experiments demonstrated that 1-G/Ca2+/DOX and 1-D/Ca2+/DOX exhibited higher inhibitory efficacy against HeLa cells, as compared to DOX solution and 1-DD/Ca2+/DOX. Finally, PA/Ca2+/DOX hydrogels displayed a longer retention time of DOX than aqueous DOX solution in animal experiments, and sustained release of DOX from hydrogels was also evidenced by slow and persisting accumulation of DOX in the major organs of hydrogel-treated mice.

Supramolecular self-assembling peptides to deliver bone morphogenetic proteins for skeletal regeneration.

Recombinant human bone morphogenetic proteins (BMPs) have shown clinical success in promoting bone healing, but they are also associated with unwanted side effects. The development of improved BMP carriers that can retain BMP at the defect site and maximize its efficacy would decrease the therapeutic BMP dose and thus improve its safety profile. In this review, we discuss the advantages of using self-assembling peptides, a class of synthetic supramolecular biomaterials, to deliver recombinant BMPs. Peptide amphiphiles (PAs) are a broad class of self-assembling peptides, and the use of PAs for BMP delivery and bone regeneration has been explored extensively over the past decade. Like many self-assembling peptide systems, PAs can be designed to form nanofibrous supramolecular biomaterials in which molecules are held together by non-covalent bonds. Chemical and biological functionality can be added to PA nanofibers, through conjugation of chemical moieties or biological epitopes to PA molecules. For example, PA nanofibers have been designed to bind heparan sulfate, a natural polysaccharide that is known to bind BMPs and potentiate their signal. Alternatively, PA nanofibers have been designed to synthetically mimic the structure and function of heparan sulfate, or to directly bind BMP specifically. In small animal models, these bio-inspired PA materials have shown the capacity to promote bone regeneration using BMP at doses 10-100 times lower than established therapeutic doses. These promising results have motivated further evaluation of PAs in large animal models, where their safety and efficacy must be established before clinical translation. We conclude with a discussion on the possiblity of combining PAs with other materials used in orthopaedic surgery to maximize their utility for clinical translation.

Transforming Growth Factor β-1 Binding by Peptide Amphiphile Hydrogels.

Supramolecular biomaterials are promising systems to bind or deliver therapeutic growth factors given their great structural versatility and tunability of properties by simply mixing molecules. In this work, we have investigated this approach for the growth factor cytokine TGFβ-1, which is potentially important in the regeneration of damaged cartilage or in the prevention of fibrinogenesis of organs and the progression of tumors. Our previous work identified a peptide sequence capable of binding TGFβ-1 and supramolecular peptide amphiphile (PA) nanofiber hydrogels that displayed the sequence were found to enhance regeneration of cartilage in a rabbit model. In this work, we have synthesized novel PA molecules motivated by the tendency of the original bioactive peptide to undergo deamidation during purification procedures, thus interfering with synthesis of molecularly well-defined structures. We report here on novel PA nanofibers that can be purified without deamidation to establish if the chemical reaction affects chondrogenesis. Interestingly, we found that gels formed from nanofibers displaying a fully deamidated sequence by introducing an asparagine to aspartic acid mutation retain 25% more growth factor relative to those displaying the original bioactive peptide even though the individual peptides have similar affinity for the cytokine. We attribute this difference in growth factor retention to bundling of nanofibers displaying the original asparagine-containing sequence, thus masking the growth factor-binding structure. Improved retention of the growth factor resulted in chondrogenesis of cells encapsulated in the gels as indicated by a more than 50% increase in Sox 9 expressing cells at 3 days and a 100% increase in glycosaminoglycan production at 21 days. We have therefore been able to design a more effective bioactive supramolecular biomaterial to bind TGFβ-1, and also demonstrated how bioactive peptide sequences in supramolecular biomaterials can have impact on their structure at larger length scales that change their biological functions.

Development of Optimized Tissue-Factor-Targeted Peptide Amphiphile Nanofibers to Slow Noncompressible Torso Hemorrhage.

Noncompressible torso hemorrhage accounts for a significant portion of preventable trauma deaths. We report here on the development of injectable, targeted supramolecular nanotherapeutics based on peptide amphiphile (PA) molecules that are designed to target tissue factor (TF) and, therefore, selectively localize to sites of injury to slow hemorrhage. Eight TF-targeting sequences were identified, synthesized into PA molecules, coassembled with nontargeted backbone PA at various weight percentages, and characterized via circular dichroism spectroscopy, transmission electron microscopy, and X-ray scattering. Following intravenous injection in a rat liver hemorrhage model, two of these PA nanofiber coassemblies exhibited the most specific localization to the site of injury compared to controls (p < 0.05), as quantified using immunofluorescence imaging of injured liver and uninjured organs. To determine if the nanofibers were targeting TF in vivo, a mouse saphenous vein laser injury model was performed and showed that TF-targeted nanofibers colocalized with fibrin, demonstrating increased levels of nanofiber at TF-rich sites. Thromboelastograms obtained using samples of heparinized rat whole blood containing TF demonstrated that no clots were formed in the absence of TF-targeted nanofibers. Lastly, both PA nanofiber coassemblies decreased blood loss in comparison to sham and backbone nanofiber controls by 35-59% (p < 0.05). These data demonstrate an optimal TF-targeted nanofiber that localizes selectively to sites of injury and TF exposure, and, interestingly, reduces blood loss. This research represents a promising initial phase in the development of a TF-targeted injectable therapeutic to reduce preventable deaths from hemorrhage.

N-Cadherin Mimetic Peptide Nanofiber System Induces Chondrogenic Differentiation of Mesenchymal Stem Cells.

Cadherins are vital for cell-to-cell interactions during tissue growth, migration, and differentiation processes. Both biophysical and biochemical inputs are generated upon cell-to-cell adhesions, which determine the fate of the mesenchymal stem cells (MSCs). The effect of cadherin interactions on the MSC differentiation still remains elusive. Here we combined the N-Cadherin mimetic peptide (HAV-PA) with the self-assembling E-PA and the resultant N-cadherin mimetic peptide nanofibers promoted chondrogenic differentiation of MSCs in conjunction with chondrogenic factors as a synthetic extracellular matrix system. Self-assembly of the precursor peptide amphiphile molecules HAV-PA and E-PA enable the organization of HAV peptide residues in close proximity to the cell interaction site, forming a supramolecular N-cadherin-like system. These bioactive peptide nanofibers not only promoted viability and enhanced adhesion of MSCs but also augmented the expression of cartilage specific matrix components compared to the nonbioactive control nanofibers. Overall, the N-cadherin mimetic peptide nanofiber system facilitated MSC commitment into the chondrogenic lineage presenting an alternative bioactive platform for stem-cell-based cartilage regeneration.

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.

Assembly and Evolution of Gemini-Type Peptide Amphiphile with a Di-Lysine Spacer.

Peptide amphiphiles (PAs) can self-assemble into a variety of supramolecular structures with excellent biofunctions. However, their assembly with time has rarely been observed and reported. Here, we find that a novel gemini-type PA [12-(Lys)2-12], taking two lysine (Lys) groups as the spacer, shows an obvious assembly and evolution process with time. Driven by the strong hydrophobic interaction between the alkyl chains as well as the electrostatic force and hydrogen bonding among the peptide spacers, the 12-(Lys)2-12 molecules first self-assemble into vesicles and then transform into fibrils, ribbons, and belts with time. If replacing the -(Lys)2- spacer with four lysine groups [-(Lys)4-] or two glutamic acid groups [-(Glu)2-], the PA molecules do not show the aggregate growth with time. This indicates that the lysine structure and its length are important structural factors contributing to the dynamic aggregate evolution behavior. More interestingly, this assembly and evolution behavior is highly dependent on 12-(Lys)2-12 concentration. Only in the proper concentration region (0.5-0.7 mM), the self-assembly displays the aggregate growth with time. At lower or higher concentrations, the aggregate growth is largely delayed or inhibited. Moreover, we also find that the aggregate growth of 12-(Lys)2-12 is related to the fibril solubilization temperature ( Tf→s). The faster aggregate growth occurs when the temperature is much lower than Tf→s. This work gains new insights into the evolution of the self-assembling structures of peptide amphiphiles.