Bioactive Peptide Amphiphiles Research Articles

Growth‐Factor Free Multicomponent Nanocomposite Hydrogels That Stimulate Bone Formation

AbstractSynthetic osteo‐promoting materials that are able to stimulate and accelerate bone formation without the addition of exogenous cells or growth factors represent a major opportunity for an aging world population. A co‐assembling system that integrates hyaluronic acid tyramine (HA‐Tyr), bioactive peptide amphiphiles (GHK‐Cu2+), and Laponite (Lap) to engineer hydrogels with physical, mechanical, and biomolecular signals that can be tuned to enhance bone regeneration is reported. The central design element of the multicomponent hydrogels is the integration of self‐assembly and enzyme‐mediated oxidative coupling to optimize structure and mechanical properties in combination with the incorporation of an osteo‐ and angio‐promoting segments to facilitate signaling. Spectroscopic techniques are used to confirm the interplay of orthogonal covalent and supramolecular interactions in multicomponent hydrogel formation. Furthermore, physico‐mechanical characterizations reveal that the multicomponent hydrogels exhibit improved compressive strength, stress relaxation profile, low swelling ratio, and retarded enzymatic degradation compared to the single component hydrogels. Applicability is validated in vitro using human mesenchymal stem cells and human umbilical vein endothelial cells, and in vivo using a rabbit maxillary sinus floor reconstruction model. Animals treated with the HA‐Tyr‐HA‐Tyr‐GHK‐Cu2+ hydrogels exhibit significantly enhanced bone formation relative to controls including the commercially available Bio‐Oss.

Reprint of: 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.

Editor's Comment on: Development of bioactive peptide amphiphiles for therapeutic cell delivery

Editor's Comment on: Development of bioactive peptide amphiphiles for therapeutic cell delivery

Self-assembly of a dual functional bioactive peptide amphiphile incorporating both matrix metalloprotease substrate and cell adhesion motifs.

We describe a bioactive lipopeptide that combines the capacity to promote the adhesion and subsequent self-detachment of live cells, using template-cell-environment feedback interactions. This self-assembling peptide amphiphile comprises a diene-containing hexadecyl lipid chain (C16e) linked to a matrix metalloprotease-cleavable sequence, Thr-Pro-Gly-Pro-Gln-Gly-Ile-Ala-Gly-Gln, and contiguous with a cell-attachment and signalling motif, Arg-Gly-Asp-Ser. Biophysical characterisation revealed that the PA self-assembles into 3 nm diameter spherical micelles above a critical aggregation concentration (cac). In addition, when used in solution at 5-150 nM (well below the cac), the PA is capable of forming film coatings that provide a stable surface for human corneal fibroblasts to attach and grow. Furthermore, these coatings were demonstrated to be sensitive to metalloproteases expressed endogenously by the attached cells, and consequently to elicit the controlled detachment of cells without compromising their viability. As such, this material constitutes a novel class of multi-functional coating for both fundamental and clinical applications in tissue engineering.

Abstract 263: Enhance Cd34+ Cell Therapy For Limb Ischemia Using Novel Bioactive Peptide Amphiphiles Nanofibers

Background: CD34+ cells have shown unprecedented promise for ischemic tissue repair in recent clinical trials; however, their recruitment, retention, and survival in the injured tissue remain one of the major barriers to the success of this novel therapeutic strategy. Methods and Results: In this study, we have developed a self-assembling bioactive peptide amphiphile (PA) nanofiber that displays the integrin-recognized cell adhesion epitope RGDS and tested this novel scaffold material for therapeutic delivery, retention, and functional support of human CD34+ cells in a mouse model of critical hind-limb ischemia (CLI). In culture, PA nanofibers, but not non-bioactive nanostructures or nanostructures with scrambled DGRS epitope, significantly enhanced the adhesion and survival of human CD34+ cells under a number of cellular stresses. In CLI nude mice, intramuscular injection of human CD34+ cells contained in the RGDS nanostructures led to a significantly better blood perfusion, motor ability, limb salvage, and treadmill endurance when compared with those of CD34+ cells contained in DGRS nanostructures, cells alone or RGDS alone. Histology analyses revealed a better viability and retention of CD34+ cells in the limb tissue of the RGDS nanostructures/CD34+ cells group than in the RGDS nanostructures alone or cells alone group. In consistent with the improved functional recovery, the density of capillaries in the ischemic limb of RGDS nanostructures/CD34+ cells group was significantly greater than in other groups. Conclusions: Our data demonstrate that integrin-specific binding epitope RGDS nanostructure enhances viability, adhesion, and angiogenic capability of human CD34+ cells. This pre-clinical study provides a rationale for further investigation of combining RGDS PAs with human CD34+ cells to enhance the effectiveness of cell therapy for ischemic limb disease.

Abstract 19043: Novel Bioactive Peptide Amphiphiles Nanofibers for Enhancement of Human Cd34+ Cell Mediated Ischemic Tissue Repair

Background: CD34+ cells have shown unprecedented promise for ischemic tissue repair in recent clinical trials; however, their recruitment, retention, and survival in the injured tissue remain one of the major barriers to the success of this novel therapeutic strategy. Methods and Results: In this study, we have developed a self-assembling bioactive peptide amphiphile (PA) nanofiber that displays the integrin-recognized cell adhesion epitope RGDS and then tested this novel scaffold material for therapeutic delivery, retention, and functional support of human CD34+ cells in a mouse model of critical hind-limb ischemia (CLI). In culture, PA nanofibers, but not non-bioactive nanostructures or nanostructures with scrambled DGRS epitope, significantly enhanced the adhesion and survival of human CD34+ cells under a number of cellular stresses. In CLI nude mice, intramuscular injection of human CD34+ cells contained in the RGDS nanostructures led to a significantly better blood perfusion, motor ability, limb salvage, and treadmill endurance when compared with those of CD34+ cells contained in DGRS nanostructures, cells alone or RGDS alone. Histology analyses revealed a higher viability and retention of CD34+ cells in the limb tissue of the RGDS nanostructures/CD34+ cells group than in the RGDS nanostructures alone or cells alone group. In consistent with the improved functional recovery, the density of capillaries in the ischemic limb of RGDS nanostructures/CD34+ cells group was significantly greater than in other groups. Conclusions: Our data demonstrate that integrin-specific binding epitope RGDS nanostructure enhances viability, adhesion, and angiogenic capability of human CD34+ cells. This pre-clinical study provides a rationale for further investigation of combining RGDS PAs with human CD34+ cells to enhance the effectiveness of cell therapy for ischemic limb disease.

Bioactive films produced from self-assembling peptide amphiphiles as versatile substrates for tuning cell adhesion and tissue architecture in serum-free conditions.

The development of versatile bioactive surfaces able to emulate in vivo conditions is of enormous importance to the future of cell and tissue therapy. Tuning cell behaviour on two-dimensional surfaces so that the cells perform as if they were in a natural three-dimensional tissue represents a significant challenge, but one that must be met if the early promise of cell and tissue therapy is to be fully realised. Due to the inherent complexities involved in the manufacture of biomimetic three-dimensional substrates, the scaling up of engineered tissue-based therapies may be simpler if based upon proven two-dimensional culture systems. In this work, we developed new coating materials composed of the self-assembling peptide amphiphiles (PAs) C16G3RGD (RGD) and C16G3RGDS (RGDS) shown to control cell adhesion and tissue architecture while avoiding the use of serum. When mixed with the C16ETTES diluent PA at 13 : 87 (mol mol-1) ratio at 1.25 × 10-3 M, the bioactive PAs were shown to support optimal adhesion, maximal proliferation, and prolonged viability of human corneal stromal fibroblasts (hCSFs), while improving the cell phenotype. These PAs also provided stable adhesive coatings on highly-hydrophobic surfaces composed of striated polytetrafluoroethylene (PTFE), significantly enhancing proliferation of aligned cells and increasing the complexity of the produced tissue. The thickness and structure of this highly-organised tissue were similar to those observed in vivo, comprising aligned newly-deposited extracellular matrix. As such, the developed coatings can constitute a versatile biomaterial for applications in cell biology, tissue engineering, and regenerative medicine requiring serum-free conditions.

A hybrid biomimetic nanomatrix composed of electrospun polycaprolactone and bioactive peptide amphiphiles for cardiovascular implants

Current cardiovascular therapies are limited by the loss of endothelium, restenosis and thrombosis. The goal of this study was to develop a biomimetic hybrid nanomatrix that combined the unique properties of electrospun polycaprolactone (ePCL) nanofibers with self-assembled peptide amphiphiles (PAs). ePCL nanofibers have interconnected nanoporous structures, but are hampered by a lack of surface bioactivity to control cellular behavior. It has been hypothesized that PAs could self-assemble onto the surface of ePCL nanofibers and endow them with the characteristic properties of native endothelium. The PAs, which comprised hydrophobic alkyl tails attached to functional hydrophilic peptide sequences, contained enzyme-mediated degradable sites coupled to either endothelial cell-adhesive ligands (YIGSR) or polylysine (KKKKK) nitric oxide (NO) donors. Two different PAs (PA–YIGSR and PA–KKKKK) were successfully synthesized and mixed in a 90:10 (YK) ratio to obtain PA–YK. PA–YK was reacted with pure NO to develop PA–YK–NO, which was then self-assembled onto ePCL nanofibers to generate a hybrid nanomatrix, ePCL–PA–YK–NO. Uniform coating of self-assembled PA nanofibers on ePCL was confirmed by transmission electron microscopy. Successful NO release from ePCL–PA–YK–NO was observed. ePCL–YK and ePCL–PA–YK–NO showed significantly increased adhesion of human umbilical vein endothelial cells (HUVECs). ePCL–PA–YK–NO also showed significantly increased proliferation of HUVECs and reduced smooth muscle cell proliferation. ePCL–PA–YK–NO also displayed significantly reduced platelet adhesion compared with ePCL, ePCL–PA–YK and a collagen control. These results indicate that this hybrid nanomatrix has great potential application in cardiovascular implants.

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.

Light‐Triggered Bioactivity in Three Dimensions

Photocleavage of a 2-nitrobenzyl group in a bioactive peptide amphiphile containing the Arg-Gly-Asp (RGD) epitope triggers a sol-to-gel transition as nanospheres are converted to nanofibers (see picture). This morphological change enhances the bioactivity of the nanostructures surrounding cells in three dimensions.

Covalent functionalization of NiTi surfaces with bioactive peptide amphiphile nanofibers

Surface modification enables the creation of bioactive implants using traditional material substrates without altering the mechanical properties of the bulk material. For applications such as bone plates and stents, it is desirable to modify the surface of metal alloy substrates to facilitate cellular attachment, proliferation, and possibly differentiation. In this work we present a general strategy for altering the surface chemistry of nickel–titanium (NiTi) shape memory alloy in order to covalently attach self-assembled peptide amphiphile (PA) nanofibers with bioactive functions. Bioactivity in the systems studied here includes biological adhesion and proliferation of osteoblast and endothelial cell types. The optimized surface treatment creates a uniform TiO 2 layer with low levels of Ni on the NiTi surface, which is subsequently covered with an aminopropylsilane coating using a novel, lower temperature vapor deposition method. This method produces an aminated surface suitable for covalent attachment of PA molecules containing terminal carboxylic acid groups. The functionalized NiTi surfaces have been characterized by X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectroscopy (ToF-SIMS), and atomic force microscopy (AFM). These techniques offer evidence that the treated metal surfaces consist primarily of TiO 2 with very little Ni, and also confirm the presence of the aminopropylsilane overlayer. Self-assembled PA nanofibers presenting the biological peptide adhesion sequence Arg-Gly-Asp-Ser are capable of covalently anchoring to the treated substrate, as demonstrated by spectrofluorimetry and AFM techniques. Cell culture and scanning electron microscopy (SEM) demonstrate cellular adhesion, spreading, and proliferation on these functionalized metal surfaces. Furthermore, these experiments demonstrate that covalent attachment is crucial for creating robust PA nanofiber coatings, leading to confluent cell monolayers.

Conducting Polymers Confined Within Bioactive Peptide Amphiphile Nanostructures

Conducting polymers within bioactive peptide amphiphile (PA) nanostructures are synthesized (see image). The approach relies on the ability of hydrophobic volumes within self-assembled structures to partition organic molecules from aqueous environments. Electron microscopy and electrochemical measurements indicate confinement of conducting polymers within the self-assembled cylindrical PA nanostructures. PEDOT: poly(3,4-ethylenedioxythiophene).

Self‐Assembly and Applications of Biomimetic and Bioactive Peptide‐Amphiphiles

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.

Self-assembly and applications of biomimetic and bioactive peptide-amphiphiles.

Peptide-amphiphiles are amphiphilic structures with a hydrophilic peptide headgroup that incorporates a bioactive sequence and has the potential to form distinct structures, and a hydrophobic tail that serves to align the headgroup, drive self-assembly, and induce secondary and tertiary conformations. In this paper we review the different self-assembled structures of peptide-amphiphiles that range from micelles and nanofibers, to patterned membranes. We also describe several examples where peptide-amphiphiles have found applications as soft bioactive materials for model studies of bioadhesion and characterization of different cellular phenomena, as well as scaffolds for tissue engineering, regenerative medicine, and targeted drug delivery.