Self-assembled Peptide Amphiphile Nanofibers Research Articles

Self-Assembled Peptide Amphiphile Nanofibers for Controlled Therapeutic Delivery to the Atherosclerotic Niche.

Atherosclerotic plaque remains the leading contributor to cardiovascular disease and requires invasive surgical procedures for its removal. Nanomedicine offers a minimally invasive approach to alleviate plaque burden by targeted therapeutic delivery. However, nanocarriers are limited without the ability to sense and respond to the diseased microenvironment. In this study, targeted self-assembled peptide amphiphile (PA) nanofibers were developed that cleave in response to biochemical cues expressed in atherosclerotic lesions-reactive oxygen species (ROS) and intracellular glutathione-to deliver a liver X receptor agonist (LXR) to enhance macrophage cholesterol efflux. The PAs released LXR in response to physiological levels of ROS and reducing agents and could be co-assembled with plaque-targeting PAs to form nanofibers. The resulting LXR PA nanofibers promoted cholesterol efflux from macrophages in vitro as well as LXR alone and with lower cytotoxicity. Further, the ApoA1-LXR PA nanofibers targeted plaque within an atherosclerotic mouse model in vivo and activated ATP-binding cassette A1 (ABCA1) expression as well as LXR alone with reduced liver toxicity. Taken together, these results demonstrate the potential of self-assembled PA nanofibers for controlled therapeutic delivery to the atherosclerotic niche.

Tailorable Exciton Transport in Doped Peptide-Amphiphile Assemblies.

Light-harvesting biomaterials are an attractive target in photovoltaics, photocatalysis, and artificial photosynthesis. Through peptide self-assembly, complex nanostructures can be engineered to study the role of chromophore organization during light absorption and energy transport. To this end, we demonstrate the one-dimensional transport of excitons along naturally occurring, light-harvesting, Zn-protoporphyrin IX chromophores within self-assembled peptide-amphiphile nanofibers. The internal structure of the nanofibers induces packing of the porphyrins into linear chains. We find that this peptide assembly can enable long-range exciton diffusion, yet it also induces the formation of excimers between adjacent molecules, which serve as exciton traps. Electronic coupling between neighboring porphyrin molecules is confirmed by various spectroscopic methods. The exciton diffusion process is then probed through transient photoluminescence and absorption measurements and fit to a model for one-dimensional hopping. Because excimer formation impedes exciton hopping, increasing the interchromophore spacing allows for improved diffusivity, which we control through porphyrin doping levels. We show that diffusion lengths of over 60 nm are possible at low porphyrin doping, representing an order of magnitude improvement over the highest doping fractions.

Self-assembled peptide amphiphile nanofibers and peg composite hydrogels as tunable ECM mimetic microenvironment.

Natural extracellular matrix (ECM) consists of complex signals interacting with each other to organize cellular behavior and responses. This sophisticated microenvironment can be mimicked by advanced materials presenting essential biochemical and physical properties in a synergistic manner. In this work, we developed a facile fabrication method for a novel nanofibrous self-assembled peptide amphiphile (PA) and poly(ethylene glycol) (PEG) composite hydrogel system with independently tunable biochemical, mechanical, and physical cues without any chemical modification of polymer backbone or additional polymer processing techniques to create synthetic ECM analogues. This approach allows noninteracting modification of multiple niche properties (e.g., bioactive ligands, stiffness, porosity), since no covalent conjugation method was used to modify PEG monomers for incorporation of bioactivity and porosity. Combining the self-assembled PA nanofibers with a chemically cross-linked polymer network simply by facile mixing followed by photopolymerization resulted in the formation of porous bioactive hydrogel systems. The resulting porous network can be functionalized with desired bioactive signaling epitopes by simply altering the amino acid sequence of the self-assembling PA molecule. In addition, the mechanical properties of the composite system can be precisely controlled by changing the PEG concentration. Therefore, nanofibrous self-assembled PA/PEG composite hydrogels reported in this work can provide new opportunities as versatile synthetic mimics of ECM with independently tunable biological and mechanical properties for tissue engineering and regenerative medicine applications. In addition, such systems could provide useful tools for investigation of how complex niche cues influence cellular behavior and tissue formation both in two-dimensional and three-dimensional platforms.

Post-Assembly Functionalization of Supramolecular Nanostructures with Bioactive Peptides and Fluorescent Proteins by Native Chemical Ligation

Post-assembly functionalization of supramolecular nanostructures has the potential to expand the range of their applications. We report here the use of the chemoselective native chemical ligation (NCL) reaction to functionalize self-assembled peptide amphiphile (PA) nanofibers. This strategy can be used to incorporate specific bioactivity on the nanofibers, and as a model, we demonstrate functionalization with the RGDS peptide following self-assembly. Incorporation of bioactivity is verified by the observation of characteristic changes in fibroblast morphology following NCL-mediated attachment of the signal to PA nanofibers. The NCL reaction does not alter the PA nanofiber morphology, and biotinylated RGDS peptide was found to be accessible on the nanofiber surface after ligation for binding with streptavidin-conjugated gold nanoparticles. In order to show that this strategy is not limited to short peptides, we utilized NCL to conjugate yellow fluorescent protein and/or cyan fluorescent protein to self-assembled PA nanofibers. Förster resonance energy transfer and fluorescence anisotropy measurements are consistent with the immobilization of the protein on the PA nanofibers. The change in electrophoretic mobility of the protein upon conjugation with PA molecules confirmed the formation of a covalent linkage. NCL-mediated attachment of bioactive peptides and proteins to self-assembled PA nanofibers allows the independent control of self-assembly and bioactivity while retaining the biodegradable peptide structure of the PA molecule and thus can be useful in tailoring design of biomaterials.

Slow Release and Delivery of Antisense Oligonucleotide Drug by Self-Assembled Peptide Amphiphile Nanofibers

Antisense oligonucleotides provide a promising therapeutic approach for several disorders including cancer. Chemical stability, controlled release, and intracellular delivery are crucial factors determining their efficacy. Gels composed of nanofibrous peptide network have been previously suggested as carriers for controlled delivery of drugs to improve stability and to provide controlled release, but have not been used for oligonucleotide delivery. In this work, a self-assembled peptide nanofibrous system is formed by mixing a cationic peptide amphiphile (PA) with Bcl-2 antisense oligodeoxynucleotide (ODN), G3139, through electrostatic interactions. The self-assembly of PA-ODN gel was characterized by circular dichroism, rheology, atomic force microscopy (AFM) and scanning electron microscopy (SEM). AFM and SEM images revealed establishment of the nanofibrous PA-ODN network. Due to the electrostatic interactions between PA and ODN, ODN release can be controlled by changing PA and ODN concentrations in the PA-ODN gel. Cellular delivery of the ODN by PA-ODN nanofiber complex was observed by using fluorescently labeled ODN molecule. Cells incubated with PA-ODN complex had enhanced cellular uptake compared to cells incubated with naked ODN. Furthermore, Bcl-2 mRNA amounts were lower in MCF-7 human breast cancer cells in the presence of PA-ODN complex compared to naked ODN and mismatch ODN evidenced by quantitative RT-PCR studies. These results suggest that PA molecules can control ODN release, enhance cellular uptake and present a novel efficient approach for gene therapy studies and oligonucleotide based drug delivery.

A hybrid biomimetic scaffold composed of electrospun polycaprolactone nanofibers and self-assembled peptide amphiphile nanofibers

Nanofibrous electrospun poly (ε-caprolactone) (ePCL) scaffolds have inherent structural advantages, but lack of bioactivity has limited their usefulness in biomedical applications. Thus, here we report the development of a hybrid, nanostructured, extracellular matrix (ECM) mimicking scaffold by a combination of ePCL nanofibers and self-assembled peptide amphiphile (PA) nanofibers. The PAs have ECM mimicking characteristics including a cell adhesive ligand (RGDS) and matrix metalloproteinase-2 (MMP-2) mediated degradable sites. Transmission electron microscope imaging verified successful PA self-assembly into nanofibers (diameters of 8–10 nm) using a solvent evaporation method. This evaporation method was then used to successfully coat PAs onto ePCL nanofibers (diameters of 300–400 nm), to develop hybrid, bioactive scaffolds. Scanning electron microscope characterization showed that the PA coatings did not interfere with the porous ePCL nanofiber network. Human mesenchymal stem cells (hMSCs) were seeded onto the hybrid scaffolds to evaluate their bioactivity. Significantly greater attachment and spreading of hMSCs were observed on ePCL nanofibers coated with PA-RGDS as compared to ePCL nanofibers coated with PA-S (no cell adhesive ligand) and uncoated ePCL nanofibers. Overall, this novel strategy presents a new solution to overcome the current bioactivity challenges of electrospun scaffolds and combines the unique characteristics of ePCL nanofibers and self-assembled PA nanofibers to provide an ECM mimicking environment. This has great potential to be applied to many different electrospun scaffolds for various biomedical applications.

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.

Bone Regeneration on a Collagen Sponge Self-Assembled Peptide-Amphiphile Nanofiber Hybrid Scaffold

The objective of this study was to create a novel approach to promote bone induction through sustained release of growth factor from a 3-dimensional (3D) hybrid scaffold. Peptide-amphiphile (PA) was synthesized by standard solid-phase chemistry that ends with the alkylation of the NH2 terminus of the peptide. Collagen sponge was reinforced by incorporation of poly(glycolic acid) (PGA) fiber. A 3D network of nanofibers was formed by mixing basic fibroblast growth factor (bFGF) suspensions with dilute aqueous solutions of PA. A hybrid scaffold was fabricated by combination of self-assembled PA nanofibers and collagen sponge reinforced with incorporation of PGA fibers. The in vitro release profile of bFGF from hybrid scaffold was investigated, and ectopic bone formation induced by the released bFGF was assessed after subcutaneous implantation of hybrid scaffold into the backs of rats. Homogeneous bone formation was histologically observed throughout the hybrid scaffolds, in marked contrast to collagen sponge-incorporated bFGF. The level of alkaline phosphatase activity and osteocalcin content at the implanted sites of hybrid scaffolds were significantly high compared with collagen sponge incorporated with bFGF. The combination of bFGF incorporated in a collagen sponge self-assembled PA nanofiber hybrid scaffold is a promising procedure to improve bone regeneration.

Design of Tissue-engineered Nanoscaffold Through Self-assembly of Peptide Amphiphile

In order to mimic in vivo topography of the native tissue created by extracellular matrix (ECM) components, which make up all soft tissues, the surface features of each biomaterial should be considered as a nanodimensional structure. In this study, an artificial ECM was designed to mimic the nanostructured topography created by ECM components in native tissue. The proliferation and differentiation of mesenchymal stem cells (MSCs) was investigated in a three dimensional (3-D) network of nanofibers formed by the self-assembly of peptide amphiphile (PA) molecules. PA was synthesized by standard solid phase chemistry that ends with the alkylation of the NH2 terminus of the peptide. The sequence of arginine-glycine-aspartic acid (RGD) was included in peptide design as well. A 3-D network of nanofibers was formed by mixing MSC suspensions in a media with dilute aqueous solution of PA. The attachment, proliferation and osteogenic differentiation of MSCs were influenced by the self-assembled PA nanofibers as the cell scaffold and the values were significantly high compared with those in the static culture (2-D tissue culture plate).

Ectopic bone formation in collagen sponge self-assembled peptide–amphiphile nanofibers hybrid scaffold in a perfusion culture bioreactor

The objective of this study was to enhance ectopic bone formation in a three-dimensional (3-D) hybrid scaffold in combination with bioreactor perfusion culture system. The hybrid scaffold consists of two biomaterials, a hydrogel formed through self-assembly of peptide-amphiphile (PA) with cell suspensions in media, and a collagen sponge reinforced with poly(glycolic acid) (PGA) fiber incorporation. PA was synthesized by standard solid-phase chemistry that ends with the alkylation of the NH2 terminus of the peptide. A 3-D network of nanofibers was formed by mixing cell suspensions in media with dilute aqueous solution of PA. Scanning electron microscopy (SEM) observation revealed the formation of fibrous assemblies with an extremely high aspect ratio and high surface areas. Osteogenic differentiation of mesenchymal stem cells (MSC) in the hybrid scaffold was greatly influenced by the perfusion culture method compared with static culture method. When the osteoinduction activity of hybrid scaffold was studied following the implantation into the back subcutis of rats in terms of histological and biochemical examinations, significantly homogeneous bone formation was histologically observed throughout the hybrid scaffolds when perfusion culture was used compared with static culture method. The level of alkaline phosphatase activity and osteocalcin content at the implanted sites of hybrid scaffolds were significantly high for the perfusion group compared with those in static culture method. We conclude that combination of MSC-seeded hybrid scaffold and the perfusion method was promising to enhance in vitro osteogenic differentiation of MSC and in vivo ectopic bone formation.

Osteogenic differentiation of mesenchymal stem cells in self-assembled peptide-amphiphile nanofibers

The proliferation and differentiation of mesenchymal stem cells (MSC) was investigated in a three dimensional (3-D) network of nanofibers formed by self-assembly of peptide-amphiphile (PA) molecules. PA was synthesized by standard solid phase chemistry that ends with the alkylation of the NH 2 terminus of the peptide. The sequence of arginine-glycine-aspartic acid (RGD) was included in peptide design as well. A 3-D network of nonofibers was formed by mixing cell suspensions in media with dilute aqueous solution of PA. Scanning electron microscopy (SEM) observation revealed the formation of fibrous assemblies with an extremely high aspect ratio and high surface areas. When rat MSC were seeded into the PA nanofibers with or without RGD, larger number of cells attached was observed in the PA nanofibers including RGD. When measured to evaluate the osteogenic differentiation of MSC, the alkaline phosphatase (ALP) activity and osteocalcin content became maximum for the PA nanofibers including RGD compared with those without RGD, although both the values were significantly higher compared with those in the static tissue culture plate (2-D culture). We concluded that the attachment, proliferation, and osteogenic differentiation of MSC were influenced by PA nanofibers as the cell scaffold.

Proliferation and differentiation of mesenchymal stem cells using self-assembled peptide amphiphile nanofibers

The objective of this study is to enhance the proliferation and differentiation of mesenchymal stem cells (MSC) in nanodimensional scaffolds. The proliferation and differentiation of MSC was investigated in a three-dimensional network of nanofibers formed by self-assembly of peptide amphiphile (PA) molecules. PA was synthesized by standard solid phase chemistry that ends with the alkylation of the NH2 terminus of the peptide. The sequence of arginine–glycine–aspartic acid was included in the peptide design as well. A three-dimensional network of nanofibers was formed by mixing MSC suspensions in media with dilute aqueous solution of PA. The attachment, proliferation and osteogenic differentiation of MSC were influenced by the self-assembled PA nanofibers as the cell scaffold and the values were significantly high compared with those in the static culture. The alkaline phosphatase activity and osteocalcin content of MSC cultured in the PA nanofibers significantly increased compared with the static culture method. It may be concluded that PA nanofibers enable MSC to positively improve the proliferation and differentiation extent.

Encapsulation of pyrene within self-assembled peptide amphiphile nanofibers

Certain peptide amphiphiles (PAs) in aqueous media are known to form high-aspect-ratio cylindrical nanofibers with hydrophobic cores. Using cholesterol or palmitic acid as the hydrophobe and the biological adhesion epitope RGDS as the hydrophilic segment, we studied the encapsulation of pyrene, a small hydrophobic molecule, within the cores of the self-assembling PA nanofibers. Circular dichroism (CD), transmission electron microscopy (TEM) and fluorescence were used to characterize formation of the supramolecular structures. Pyrene excimer formation observed by fluorescence demonstrated the encapsulation and aggregation of pyrene within the hydrophobic cores. In addition, peptide amphiphiles covalently functionalized with pyrene linked to the hydrophobic portion of the molecule exhibited excimer formation upon self-assembly into nanofibers. Interestingly excimer formation was not observed in similar molecules that formed spherical aggregates rather than cylindrical nanofibers.

Self-Assembled Peptide Amphiphile Nanofibers Conjugated to MRI Contrast Agents

Self-assembled peptide amphiphile nanofibers have been investigated for their potential use as in vivo scaffolds for tissue engineering and drug delivery applications. We report here the synthesis of magnetic resonance (MR) active peptide amphiphile molecules that self-assemble into spherical and fiber-like nanostructures, enhancing T(1) relaxation time. This new class of MR contrast agents can potentially be used to combine high-resolution three-dimensional MR fate mapping of tissue-engineered scaffolds with targeting of specific cellular receptors.