Nanofiber Gels Research Articles

Survival, migration and differentiation of mouse tau-GFP embryonic stem cells transplanted into the rat auditory nerve

Stem cells have been investigated as treatment for a variety of diagnoses such as Parkinson's disease, Alzheimer's disease and spinal cord injuries. Here, we investigated the possibility of using stem cells as a replacement therapy for lesions of the auditory nerve (AN). We transplanted tau-GFP mouse embryonic stem cells into the AN either by the internal auditory meatus or via the modiolus in rats that had been previously deafened by application of β-bungarotoxin to the round window niche. We investigated the effect of brain derived neurotrophic factor (BDNF) on cell transplant survival and differentiation. Additionally chondroitinase ABC (ChABC), a digestive enzyme that cleaves the core chondroitin sulfate proteoglycans, was used in order to promote possible migration of cells and axons through the transitional zone. A bioactive isoleucine–lysine–valine–alanine–valine (IKVAV) peptide amphiphile (PA) nanofiber gel was applied around the cell injection site. This nanofiber gel has been shown to promote neural differentiation and other similar gels have been used to encapsulate and release proteins. Three weeks after injection, transplanted cells were found in the scala tympani, the modiolus, the AN trunk and the brain stem. As compared to cell transplantation and gel only, BDNF content in the PA gel increased cell survival and neuronal differentiation. In the animals treated with ChABC we observed extensive migration of cells through the transitional zone to or from the CNS.

Amphiphilic Schiff Base Organogels: Metal‐Ion‐Mediated Chiral Twists and Chiral Recognition

New amphiphilic gelators that contained both Schiff base and L-glutamide moieties, abbreviated as o-SLG and p-SLG, were synthesized and their self-assembly in various organic solvents in the absence and presence of metal ions was investigated. Gelation test revealed that o-SLG formed a thermotropic gel in many organic solvents, whilst p-SLG did not. When metal ions, such as Cu(2+), Zn(2+), Mg(2+), Ni(2+), were added, different behaviors were observed. The addition of Cu(2+) induced p-SLG to from an organogel. In the case of o-SLG, the addition of Cu(2+) and Mg(2+) ions maintained the gelating ability of the compound, whilst Zn(2+) and Ni(2+) ions destroyed the gel. In addition, the introduction of Cu(2+) ions caused the nanofiber gel to perform a chiral twist, whilst the Mg(2+) ions enhanced the fluorescence of the gel. More interestingly, the Mg(2+)-ion-mediated organogel showed differences in the fluorescence quenching by D- and L-tartaric acid, thus showing a chiral recognition ability.

Nanostructure-templated control of drug release from peptide amphiphile nanofiber gels.

High aspect ratio peptide nanofibers have potential as biodegradable vehicles for drug delivery. We report here the synthesis of four self-assembling peptide amphiphiles (PAs) containing a lysine ε-amine-derivatized hydrazide that was systematically placed at different positions along the backbone of the peptide sequence C(16)V(2)A(2)E(2) (where C(16) = palmitic acid). Hydrazones were formed from each hydrazide by condensation with the solvatochromic dye 6-propionyl-2-dimethylaminonaphthalene (Prodan), which is typically used to probe cell membranes. All four compounds were found to self-assemble into nanofibers, and Prodan release was measured from filamentous gels prepared by screening PA charges with divalent cations. Near zero-order release kinetics were observed for all nanofibers, but release half-lives differed depending on the position of the fluorophore in the PA sequence. Dye release kinetics were rationalized through the use of cryogenic transmission electron microscopy, small-angle X-ray scattering, fluorescence spectroscopy, fluorescence anisotropy, circular dichroism, and partition coefficient calculations. Relative release rates were found to correlate directly with fluorophore mobility, which varied inversely with packing density, degree of order in the hydrophobic PA core, and the β-sheet character of the peptide.

A Peptide-Based Material for Therapeutic Carbon Monoxide Delivery.

We report on the preparation of the first material for therapeutic delivery of CO. A peptide amphiphile was synthesized with a covalently attached ruthenium tricarbonyl. Self-assembled nanofiber gels containing this peptide spontaneously released CO with prolonged release kinetics compared to soluble CO donors. Oxidatively stressed cardiomyocytes had improved viability when treated with this peptide, demonstrating its potential as a biodegradable gel for localized therapeutic CO delivery.

A Pendulum-Like Motion of Nanofiber Gel Actuator Synchronized with External Periodic pH Oscillation

In this study, we succeeded in manufacturing a novel nanofiber hydrogel actuator that caused a bending and stretching motion synchronized with external pH oscillation, based on a bromate/sulfite/ferrocyanide reaction. The novel nanofiber gel actuator was composed of electrospun nanofibers synthesized by copolymerizing acrylic acid and hydrophobic butyl methacrylate as a solubility control site. By changing the electrospinning flow rate, the nanofiber gel actuator introduced an anisotropic internal structure into the gel. Therefore, the unsymmetrical motion of the nanofiber actuator was generated.

Drug release from hydrazone-containing peptide amphiphiles

Hydrolytically-labile hydrazones in peptide amphiphiles were studied as degradable tethers for release of the drug nabumetone from nanofiber gels. On-resin addition of the novel compound tri-Boc-hydrazido adipic acid to a lysine ε-amine allowed for precise placement of a hydrazide in a peptide sequence.

Self-assembling nanostructures to deliver angiogenic factors to pancreatic islets

Supramolecular self-assembly of nanoscale filaments offers a vehicle to signal cells within dense cell aggregates such as pancreatic islets. We previously developed a heparin-binding peptide amphiphile (HBPA) that self-assembles into nanofiber gels at concentrations of 1% by weight when mixed with heparin and activates heparin-binding, angiogenic growth factors. We report here on the use of these molecules at concentrations 100 times lower to drive delivery of the nanofibers into the dense islet interior. Using fluorescent markers, HBPA molecules, heparin, and FGF2 were shown to be present in and on the surface of murine islets. The intraislet nanofibers were found to be necessary to retain FGF2 within the islet for 48 h and to increase cell viability significantly for at least 7 days in culture. Furthermore, enhanced insulin secretion was observed with the nanofibers for 3 days in culture. Delivery of FGF2 and VEGF in conjunction with the HBPA/heparin nanofibers also induced a significant amount of islet endothelial cell sprouting from the islets into a peptide amphiphile 3-D matrix. We believe the infiltration of bioactive nanofibers in the interior of islets as an artificial ECM can improve cell viability and function in vitro and enhance their vascularization in the presence of growth factors such as FGF2 and VEGF. The approach described here may have significant impact on islet transplantation to treat type 1 diabetes.

Bone regeneration mediated by biomimetic mineralization of a nanofiber matrix

Rapid bone regeneration within a three-dimensional defect without the use of bone grafts, exogenous growth factors, or cells remains a major challenge. We report here on the use of self-assembling peptide nanostructured gels to promote bone regeneration that have the capacity to mineralize in biomimetic fashion. The main molecular design was the use of phosphoserine residues in the sequence of a peptide amphiphile known to nucleate hydroxyapatite crystals on the surfaces of nanofibers. We tested the system in a rat femoral critical-size defect by placing pre-assembled nanofiber gels in a 5 mm gap and analyzed bone formation with micro-computed tomography and histology. We found within 4 weeks significantly higher bone formation relative to controls lacking phosphorylated residues and comparable bone formation to that observed in animals treated with a clinically used allogenic bone matrix.

Tuning supramolecular rigidity of peptide fibers through molecular structure.

We synthesized a series of peptide amphiphiles (PAs) with systematically modified amino acid sequences to control the mechanical properties of the nanofiber gels they form by self-assembly. By manipulating the number and position of valines and alanines in the peptide sequence, we found that valines increase the stiffness of the gel, while additional alanines decrease the mechanical properties. Vitreous ice cryo-transmission electron microscopy shows that all PA molecules investigated here form nanofibers 8-10 nm in diameter and several micrometers in length. We found through Fourier transform IR experiments a strong correlation between gel stiffness and hydrogen bond alignment along the long axis of the fiber. Molecules that form supramolecular structures with the highest mechanical stiffness were found by circular dichroism to self-assemble into beta-sheets with the least amount of twisting and disorder, a result that is consistent with IR experiments. Molecular control of mechanical stiffness in three-dimensional artificial peptide amphiphile matrices offers a chemical strategy to control biological phenomena such as stem cell differentiation and cell morphology.

Effect of IKVAV peptide nanofiber on proliferation, adhesion and differentiation into neurocytes of bone marrow stromal cells

This study examined the effect of IKVAV peptide nanofiber on proliferation, adhesion and differentiation into neurocytes of bone marrow stromal cells (BMSCs). IKVAV Peptide-amphiphile was synthesized and purified. Then, hydrogen chloride was added to the diluted aqueous solutions of PA to induce spontaneous formation of nanofiber in vitro. The resultant samples was observed under transmission electron microscope. BMSCs were cultured with IKVAV peptide nanofiber. The effect of IKVAV nanofiber on the proliferation, adhesion and induction differentiation of BMSCs was observed by inverted microscopy, calcein-AM/PI staining, cell counting and immunofluorescence staining. The results demonstrated that IKVAV peptide-amphiphile could self-assemble to form nanofiber gel. BMSCs cultured in combination with IKVAV peptide nanofiber gel grew well and the percentage of live cells was over 90%. IKVAV peptide nanofiber gel exerted no influence on the proliferation of BMSCs and could promote the adhesion of BMSCs and raise the ratio of neurons when BMSCs were induced to differentiate into neurocytes. It is concluded that BMSCs could proliferate and adhere well and yield more neurons during when induced to differente into neurocytes on IKVAV peptide nanofiber gel.

Synergistic effects of tethered growth factors and adhesion ligands on DNA synthesis and function of primary hepatocytes cultured on soft synthetic hydrogels

The composition, presentation, and spatial orientation of extracellular matrix molecules and growth factors are key regulators of cell behavior. Here, we used self-assembling peptide nanofiber gels as a modular scaffold to investigate how fibronectin-derived adhesion ligands and different modes of epidermal growth factor (EGF) presentation synergistically regulate multiple facets of primary rat hepatocyte behavior in the context of a soft gel. In the presence of soluble EGF, inclusion of dimeric RGD and the heparin binding domain from fibronectin (HB) increased hepatocyte aggregation, spreading, and metabolic function compared to unmodified gels or gels modified with a single motif, but unlike rigid substrates, gels failed to induce DNA synthesis. Tethered EGF dramatically stimulated cell aggregation and spreading under all adhesive ligand conditions and also preserved metabolic function. Surprisingly, tethered EGF elicited DNA synthesis on gels with RGD and HB. Phenotypic differences between soluble and tethered EGF stimulation of cells on peptide gels are correlated with differences in expression and phosphorylation the EGF receptor and its heterodimerization partner ErbB2, and activation of the downstream signaling node ERK1/2. These modular matrices reveal new facets of hepatocellular biology in culture and may be more broadly useful in culture of other soft tissues.

Fibronectin-mimetic peptide-amphiphile nanofiber gels support increased cell adhesion and promote ECM production

The design of nanofiber scaffolds has been a key objective in tissue engineering as they structurally mimic the natural extracellular matrix (ECM) found in tissues. The continuous cross talk between cells and the surrounding matrix ligands plays a crucial role during development and wound healing which leads to tissue regeneration and repair. In an attempt to provide a nanofiber scaffold with a ligand that can promote cell adhesion and ECM production, we propose the use of our peptide-amphiphile nanofibers as a potential scaffold for tissue engineering. A fibronectin-mimetic peptide sequence containing both the cell-binding GRGDSP domain and the synergistic PHSRN domain has been recently developed in our group. It has been shown to be a specific ligand for the α5β1 integrin, and has been synthesized to a C16 single-tailed peptide-amphiphile, PR_g (with a peptide headgroup of GGGSSPHSRN(SG)5RGDSP). The PR_g peptide-amphiphiles self-assemble into nanofibers in an aqueous environment and form hydrogels. In this study, the PR_g hydrogels were characterized with cryo-scanning electron microscopy (cryo-SEM) and rheology measurements, and their surfaces were evaluated in terms of sustaining long term human umbilical vein endothelial cell (HUVEC) adhesion and proliferation, cytoskeleton formation, and secretion of extracellular fibronectin. PR_g gels were compared to polyethylene glycol (PEG) gels, PEG gels functionalized with fibronectin as well as commercially available peptide hydrogels (PuraMatrix). Our results show that the PR_g hydrogels outperform the other gels evaluated in this study and therefore hold promise as scaffolds for tissue engineering.

Biocompatibility of FGL peptide self-assembly nanofibers with neural stem cells in vitro

To observe the biocompatibility of self-assembled FGL peptide nano-fibers scaffold with neural stem cells (NSCs). FGL peptide-amphiphile (FGL-PA) was synthesized by solid-phase peptide synthesis technique and thereafter It was analyzed and determined by high-performance liquid chromatography (HPLC) and mass spectrometry (MS). The diluted hydrochloric acid was added into FGL-PA solution to reduce the pH value and accordingly induce self-assembly. The morphological features of the assembled material were studied by transmission electron microscope (TEM). NSCs were cultured and different concentrations of FGL-PA assembled material were added with the terminal concentrations of 0, 50, 100, 200, 400 mg/L, respectively. CCK-8 kit was used to test the effect of FGL assembled material on proliferation of NSCs. NSCs were added into differentiation mediums (control group: DMEM/F12 medium containing 2% B27 supplement and 10% FBS; experimental group: DMEM/F12 medium containing 2% B27 supplement, 10% FBS and 100 mg/L FGL-PA, respectively). Immunofluorescence was applied to test the effect of FGL-PA assembled material on differentiation of NSCs. FGL-PA could be self-assembled to form a gel. TEM showed the self-assembled gel was nano-fibers with diameter of 10-20 nm and length of hundreds nanometers. After NSCs were incubated for 48 hours with different concentrations of FGL-PA assembled material, the result of CCK-8 assay showed that FGL-PA with concentrations of 50, 100 or 200 mg/L could promote the proliferation of NSCs and absorbance of them was increased (P < 0.05). Immunofluorescence analysis notified that the differentiation ratio of neurons from NSCs in control group and experimental group were 46.35% +/- 1.27% and 72.85% +/- 1.35%, respectively, when NSCs were induced to differentiation for 14 days, showing significant difference between 2 groups (P < 0.05). FGL-PA can self-assemble to nano-fiber gel, which has good biocompatibility and neural bioactivity.

Tunable Mechanics of Peptide Nanofiber Gels

The mechanical properties of self-assembled fibrillar networks are influenced by the specific intermolecular interactions that modulate fiber entanglements. We investigate how changing these interactions influences the mechanics of self-assembled nanofiber gels composed of peptide amphiphile (PA) molecules. PAs developed in our laboratory self-assemble into gels of nanofibers after neutralization or salt-mediated screening of the charged residues in their peptide segment. We report here on the gelation, stiffness, and response to deformation of gels formed from a negatively charged PA and HCl or CaCl(2). Scanning electron microscopy of these gels demonstrates a similar morphology, whereas the oscillatory rheological measurements indicate that the calcium-mediated ionic bridges in CaCl(2)-PA gels form stronger intra- and interfiber cross-links than the hydrogen bonds formed by the protonated carboxylic acid residues in HCl-PA gels. As a result, CaCl(2)-PA gels can withstand higher strains than HCl-PA gels. After exposure to a series of strain sweeps with increasing strain amplitude HCl- and CaCl(2)-PA gels both recover 42% of their original stiffness. In contrast, after sustained deformation at 100% strain, HCl-PA gels recover nearly 90% of their original stiffness after 10 min, while the CaCl(2)-PA gels only recover 35%. This result suggests that the hydrogen bonds formed by the protonated acids in the HCl-PA gels allow the gel to relax quickly to its initial state, while the strong calcium cross-links in the CaCl(2)-PA gels lock in the deformed structure and inhibit the gel's ability to recover. We also show that the rheological scaling behaviors of HCl- and CaCl(2)-PA gels are consistent with that of uncross- and cross-linked semiflexible biopolymer networks, respectively. The ability to modify how self-assembled fibrillar networks respond to deformations is important in developing self-assembled gels that can resist and recover from the large deformations that these gels encounter while serving as synthetic cell scaffolds in vivo.

Modulating the Gelation Properties of Self-Assembling Peptide Amphiphiles

Peptide amphiphiles (PAs) are self-assembling molecules that form interwoven nanofiber gel networks. They have gained a lot of attention because of their excellent biocompatibility, adaptable peptide structure that allows for specific biochemical functionality, and nanofibrous assembly that mimics natural tissue formation. However, variations in molecule length, charge, and intermolecular bonding between different bioactive PAs cause contrasting mechanical properties. This potentially limits cell-delivery therapies because scaffold durability is needed to withstand the rigors of clinician handling and transport to wound implant sites. Additionally, the mechanical properties have critical influence on cellular behavior, as the elasticity and stiffness of biomaterials have been shown to affect cell spreading, migration, contraction, and differentiation. Several different PAs have been synthesized, each endowed with specific cellular adhesive ligands for directed biological response. We have investigated mechanical means for modulating and stabilizing the gelation properties of PA hydrogels in a controlled manner. A more stable, biologically inert PA (PA-S) was synthesized and combined with each of the bioactive PAs. Molar ratio (M(r) = PA/PA-S) combinations of 3:1, 1:1, and 1:3 were tested. All PA composites were characterized by observed nanostructure and rheological analysis measuring viscoelasticity. It was found that the PAs could be combined to successfully control and stabilize the gelation properties, allowing for a mechanically tunable scaffold with increased durability. Thus, the biological functionality and natural degradability of PAs can be provided in a more physiologically relevant microenvironment using our composite approach to modulate the mechanical properties, thereby improving the vast potential for cell encapsulation and other tissue engineering applications.

Dynamic in vivo biocompatibility of angiogenic peptide amphiphile nanofibers

Biomaterials that promote angiogenesis have great potential in regenerative medicine for rapid revascularization of damaged tissue, survival of transplanted cells, and healing of chronic wounds. Supramolecular nanofibers formed by self-assembly of a heparin-binding peptide amphiphile and heparan sulfate-like glycosaminoglycans were evaluated here using a dorsal skinfold chamber model to dynamically monitor the interaction between the nanofiber gel and the microcirculation, representing a novel application of this model. We paired this model with a conventional subcutaneous implantation model for static histological assessment of the interactions between the gel and host tissue. In the static analysis, the heparan sulfate-containing nanofiber gels were found to persist in the tissue for up to 30 days and revealed excellent biocompatibility. Strikingly, as the nanofiber gel biodegraded, we observed the formation of a de novo vascularized connective tissue. In the dynamic experiments using the dorsal skinfold chamber, the material again demonstrated good biocompatibility, with minimal dilation of the microcirculation and only a few adherent leukocytes, monitored through intravital fluorescence microscopy. The new application of the dorsal skinfold model corroborated our findings from the traditional static histology, demonstrating the potential use of this technique to dynamically evaluate the biocompatibility of materials. The observed biocompatibility and development of new vascularized tissue using both techniques demonstrates the potential of these angiogenesis-promoting materials for a host of regenerative strategies.

Development of bioactive peptide amphiphiles for therapeutic cell delivery

There is great clinical interest in cell-based therapies for ischemic tissue repair in cardiovascular disease. However, the regenerative potential of these therapies is limited due to poor cell viability and minimal retention following application. We report here the development of bioactive peptide amphiphile nanofibers displaying the fibronectin-derived RGDS cell adhesion epitope as a scaffold for therapeutic delivery of bone marrow derived stem and progenitor cells. When grown on flat substrates, a binary peptide amphiphile system consisting of 10wt.% RGDS-containing molecules and 90wt.% negatively charged diluent molecules was found to promote optimal cell adhesion. This binary system enhanced adhesion 1.4-fold relative to substrates composed of only the non-bioactive diluent. Additionally, no enhancement was found upon scrambling the epitope and adhesion was no longer enhanced upon adding soluble RGDS to the cell media, indicating RGDS-specific adhesion. When encapsulated within self-assembled scaffolds of the binary RGDS nanofibers in vitro, cells were found to be viable and proliferative, increasing in number by 5.5 times after only 5days, an effect again lost upon adding soluble RGDS. Cells encapsulated within a non-bioactive scaffold and those within a binary scaffold with scrambled epitope showed minimal viability and no proliferation. Cells encapsulated within this RGDS nanofiber gel also increase in endothelial character, evident by a decrease in the expression of CD34 paired with an increase in the expression of endothelial-specific markers VE-Cadherin, VEGFR2 and eNOS after 5days. In an in vivo study, nanofibers and luciferase-expressing cells were co-injected subcutaneously in a mouse model. The binary RGDS material supported these cells in vivo, evident by a 3.2-fold increase in bioluminescent signal attributable to viable cells; this suggests the material has an anti-apoptotic and/or proliferative effect on the transplanted bone marrow cells. We conclude that the binary RGDS-presenting nanofibers developed here demonstrate enhanced viability, proliferation and adhesion of associated bone marrow derived stem and progenitor cells. This study suggests potential for this material as a scaffold to overcome current limitations of stem cell therapies for ischemic diseases.

Fabrication of Luminescent CdS Nanoparticles on Short‐Peptide‐Based Hydrogel Nanofibers: Tuning of Optoelectronic Properties

The pH-induced self-assembly of three synthetic tripeptides in water medium is used to immobilize luminescent CdS nanoparticles. These peptides form a nanofibrillar network structure upon gelation in aqueous medium at basic pH values (pH 11.0-13.0), and the fabrication of CdS nanoparticles on the gel nanofiber confers the luminescent property to these gels. Atomic force microscopy, field-emission scanning electron microscopy, and high-resolution transmission electron microscopy clearly reveal the presence of CdS nanoparticles in a well-defined array on the gel nanofibers. This is a convenient way to make organic nanofiber-inorganic nanoparticle hybrid nanocomposite systems. The size of the CdS nanoparticles remains almost same before and after deposition on the gel nanofiber. Photoluminescence (PL) measurement of the CdS nanoparticles upon deposition on the gel nanofibers shows a significant blue shift in the emission spectrum of the nanoparticles, and there is a considerable change in the PL gap energy of the CdS nanoparticles after immobilization on different gel nanofibrils. This finding suggests that the optoelectronic properties of CdS nanoparticles can be tuned upon deposition on gel nanofibers without changing the size of the nanoparticles.

Growth of rat dorsal root ganglion neurons on a novel self-assembling scaffold containing IKVAV sequence

The potential benefits of self-assembly in synthesizing materials for the treatment of both peripheral and central nervous system disorders are tremendous. In this study, we synthesized peptide-amphiphile (PA) molecules containing IKVAV sequence and induced self-assembly of the PA solutions in vitro to form nanofiber gels. Then, we tested the characterization of gels by transmission electron microscopy and demonstrated the biocompatibility of this gel towards rat dorsal root ganglion neurons. The nanofiber gel was formed by self-assembly of IKVAV PA molecules, which was triggered by metal ions. The fibers were 7–8 nm in diameter and with lengths of hundreds of nanometers. Gels were shown to be non-toxic to neurons and able to promote neurons adhesion and neurite sprouting. The results indicated that the self-assembling scaffold containing IKVAV sequence had excellent biocompatibility with adult sensory neurons and could be useful in nerve tissue engineering.

Self-assembling peptide amphiphile-based nanofiber gel for bioresponsive cisplatin delivery.

The aim of this study is to develop a bioresponsive cisplatin (CDDP) delivery system with a self-assembling peptide amphiphile (PA) comprising a cell-adhesive matrix metalloproteinase-2 (MMP-2)-sensitive GTAGLIGQRGDS and a fatty acid. A biomimetic CDDP-PA gel was spontaneously formed upon incubating a mixture of CDDP and the PA for 5 h at 37 degrees C. CDDP-PA gel formation was confirmed by rheological analysis. The structure of self-assembled CDDP-PA nanofibers inside the gel was determined by transmission electron microscopy (TEM). Bioresponsive drug release from the biomimetic gel was demonstrated by in vitro MMP-2-triggered CDDP release. The MMP-2-sensitive CDDP release was dependent on the enzyme concentration in the medium. Enzymatic degradation of the CDDP-PA gel was confirmed by TEM images of the gel degraded in an MMP-2 containing medium. The MMP-2-triggered CDDP release as well as the presentation of RGDS in the gel would potentially provide a spatially and temporally controlled delivery system for targeted anticancer drug delivery.