Iris Mironi-Harpaz

Self-Assembled Fmoc-Peptides as a Platform for the Formation of Nanostructures and Hydrogels

Hydrogels are of great interest as a class of materials for tissue engineering, axonal regeneration, and controlled drug delivery, as they offer 3D interwoven scaffolds to support the growth of cells. Herein, we extend the family of the aromatic Fmoc-dipeptides with a library of new Fmoc-peptides, which include natural and synthetic amino acids with an aromatic nature. We describe the self-assembly of these Fmoc-peptides into various structures and characterize their distinctive molecular and physical properties. Moreover, we describe the fabrication of the bioactive RGD sequence into a hydrogel. This unique material offers new opportunities for developing cell-adhesive biomedical hydrogel scaffolds, as well as for establishing strategies to modify surfaces with bioactive materials.

Photopolymerization of cell-encapsulating hydrogels: crosslinking efficiency versus cytotoxicity.

Cell-encapsulating hydrogels used in regenerative medicine are designed to undergo a rapid liquid-to-solid phase transition in the presence of cells and tissues so as to maximize crosslinking and minimize cell toxicity. Light-activated free-radical crosslinking (photopolymerization) is of particular interest in this regard because it can provide rapid reaction rates that result in uniform hydrogel properties with excellent temporal and spatial control features. Among the many initiator systems available for photopolymerization, only a few have been identified as suitable for cell-based hydrogel formation owing to their water solubility, crosslinking properties and non-toxic reaction conditions. In this study, three long-wave ultraviolet (UV) light-activtied photoinitiators (PIs) were comparatively tested in terms of cytotoxicity, crosslinking efficiency and crosslinking kinetics of cell-encapsulating hydrogels. The hydrogels were photopolymerized from poly(ethylene glycol) (PEG) diacrylate or PEG-fibrinogen precursors using Irgacure® PIs I2959, I184 and I651, as well as with a chemical initiator/accelerator (APS/TEMED). The study specifically evaluated the PI type, PI concentration and UV light intensity, and how these affected the mechanical properties of the hydrogel (i.e. maximum storage modulus), the crosslinking reaction times and the reactions cytotoxicity to encapsulated cells. Only two initiators (I2959 and I184) were identified as being suitable for achieving both high cell viability and efficient crosslinking of the cell-encapsulating hydrogels during the photopolymerization reaction. Optimization of PI concentration or irradiation intensity was particularly important for achieving maximum mechanical properties; a sub-optimal choice of PI concentration or irradiation intensity resulted in a substantial reduction in hydrogel modulus. Cytocompatibility may be compromised by unnecessarily prolonging exposure to cytotoxic free radicals or inadvertently enhancing the instantaneous dose of radicals in solution, both of which are dependent on the PI type/concentration and irradiation intensity. In the absence of a radical initiator, the short exposures to long-wave UV light irradiation (up to 5 min, 20 mW cm(-2), 365 nm) did not prove to be cytotoxic to cells. Therefore, it is important to understand the relationship between PIs, light irradiation conditions and crosslinking when attempting to identify a suitable hydrogel formation process for cell encapsulating hydrogels.

The rheological and structural properties of Fmoc-peptide-based hydrogels: the effect of aromatic molecular architecture on self-assembly and physical characteristics.

Biocompatible hydrogels are of high interest as a class of biomaterials for tissue engineering, regenerative medicine, and controlled drug delivery. These materials offer three-dimensional scaffolds to support the growth of cells and development of hierarchical tissue structures. Fmoc-peptides were previously demonstrated as attractive building blocks for biocompatible hydrogels. Here, we further investigate the biophysical properties of Fmoc-peptide-based hydrogels for medical applications. We describe the structural and thermal properties of these Fmoc-peptides, as well as their self-assembly process. Additionally, we study the role of interactions between aromatic moieties in the self-assembly process and on the physical and structural properties of the hydrogels.

Seamless metallic coating and surface adhesion of self-assembled bioinspired nanostructures based on di-(3,4-dihydroxy-L-phenylalanine) peptide motif.

The noncoded aromatic 3,4-dihydroxy-l-phenylalanine (DOPA) amino acid has a pivotal role in the remarkable adhesive properties displayed by marine mussels. These properties have inspired the design of adhesive chemical entities through various synthetic approaches. DOPA-containing bioinspired polymers have a broad functional appeal beyond adhesion due to the diverse chemical interactions presented by the catechol moieties. Here, we harnessed the molecular self-assembly abilities of very short peptide motifs to develop analogous DOPA-containing supramolecular polymers. The DOPA-containing DOPA–DOPA and Fmoc–DOPA–DOPA building blocks were designed by substituting the phenylalanines in the well-studied diphenylalanine self-assembling motif and its 9-fluorenylmethoxycarbonyl (Fmoc)-protected derivative. These peptides self-organized into fibrillar nanoassemblies, displaying high density of catechol functional groups. Furthermore, the Fmoc–DOPA–DOPA peptide was found to act as a low molecular weight hydrogelator, forming self-supporting hydrogel which was rheologically characterized. We studied these assemblies using electron microscopy and explored their applicative potential by examining their ability to spontaneously reduce metal cations into elementary metal. By applying ionic silver to the hydrogel, we observed efficient reduction into silver nanoparticles and the remarkable seamless metallic coating of the assemblies. Similar redox abilities were observed with the DOPA–DOPA assemblies. In an effort to impart adhesiveness to the obtained assemblies, we incorporated lysine (Lys) into the Fmoc–DOPA–DOPA building block. The assemblies of Fmoc–DOPA–DOPA–Lys were capable of gluing together glass surfaces, and their adhesion properties were investigated using atomic force microscopy. Taken together, a class of DOPA-containing self-assembling peptides was designed. These nanoassemblies display unique properties and can serve as multifunctional platforms for various biotechnological applications.

Fluorescent DNA Hydrogels Composed of Nucleic Acid‐Stabilized Silver Nanoclusters

Y-shaped DNA units functionalized with Ag-nanoclusters are crosslinked by nucleic acids to yield fluorescent hydrogels with controlled luminescence properties.

In‐Situ Architectures Designed in 3D Cell‐Laden Hydrogels Using Microscopic Laser Photolithography

DOI: 10.1002/adma.201404185 crosslinking that is compatible with cell-laden 3D culture. The confl uence of a thermal gelation in tandem with a chemical crosslinking by photopolymerization culminates in an irreversibly crosslinked contiguous polymer network at the focal region of the microscope objective lens. The result is a cell-laden hydrogel with dispersed cells cultivated in 3D within a material having small-scale architectures that guide cellular morphogenesis based on material interactions. Cell-compatible LCST hydrogel materials were synthesized using semisynthetic adducts of fi brinogen and functionalized poloxamer (ethylene oxide/propylene oxide block copolymer) ( Figure 1 ). The fi brinogen provides the basic bioactive features essential for in situ 3D cell culture, including adhesion and proteolytic susceptibility. [ 13 ] The functionalized poloxamers provide the reactive groups that are responsible for the mild photochemistry leading to covalent bond formation, as well as for the LCST properties. [ 14 ] The poloxamer Pluronic F127 was chosen because it was able to facilitate a phase transition below 37 °C; the concentrations of constituents and photoinitiator were chosen to allow in situ 3D cell culture with low cytotoxicity (>85% cell viability after 3 days in culture). [ 15 ] Physical crosslinking of the Pluronic F127-fi brinogen adducts (Pluronic–fi brinogen) occurs upon temperature elevation from 20 to 37 °C (Figure 1 and the Supporting Information). Patterned photochemistry of the Pluronic–fi brinogen hydrogels was performed using an inverted microscope setup with a temperature-controlled stage ( Figure 2 a). Illumination from a nanosecond pulse UV (355 nm) laser was used for initiating the free-radical polymerization of the Pluronic–fi brinogen containing photoinitiator (Irgacure 2959, CIBA). With the stageincubator set to a temperature of 37 °C, the positioning of the focal region within the Pluronic–fi brinogen construct enabled pinpoint covalent crosslinking with spatial resolutions that depend mainly on the illuminating beam waist. A variable aperture at the back focal plane of the objective lens was used to control the beam diameter, while widefi eld transmission microscopy using visible light was used to monitor the patterning process. In order to verify covalent reaction, rhodamine-labeled Pluronic F127-acrylate (red) was added to the Pluronic–fi brinogen and co-immobilized in regions of chemical crosslinking (Figure 2 b). Poly(ethylene glycol) (PEG)-coated fl uorescein isothiocyanate (FITC) polystyrene microbeads embedded in the Pluronic–fi brinogen constructs were used to map spatial variations in material properties associated with physical/chemical (Figure 2 b, inner) versus physical (Figure 2 b, outer) crosslinked regions. Comparing the mean square displacements (MSD) of the beads within chemically physically crosslinked regions to those of beads located in physically crosslinked regions (Figure 2 c), we confi rmed a statistically signifi cant increase in the storage component of the complex shear modulus ( n = 20, The ability to apply photochemistry and microscopic patterning techniques for making hydrogel scaffolds enables formation of heterogeneous mechanical structures and chemical gradients that can guide tissue morphogenesis and control stem cell fate. [ 1 ] Microscopy-guided methods to pattern cell-laden hydrogels with micrometer-scale 3D features have been hampered by the complexity of the localized photochemical interactions. This is because cell-compatible hydrogels contain dilute solutions and limited amounts of photoinitiator – resulting in crosslinking reactions on timescales of seconds to minutes. [ 2,3 ]

Time-dependent cellular morphogenesis and matrix stiffening in proteolytically responsive hydrogels

Mesenchymal stromal cells residing in proteolytically responsive hydrogel scaffolds were subjected to changes in mechanical properties associated with their own three-dimensional (3-D) morphogenesis. In order to investigate this relationship the current study documents the transient degradation and restructuring of fibroblasts seeded in hydrogel scaffolds undergoing active cell-mediated reorganization over 7days in culture. A semi-synthetic proteolytically degradable polyethylene glycol-fibrinogen (PF) hydrogel matrix and neonatal human dermal fibroblasts (NHDF) were used. Rheology (in situ and ex situ) measured stiffening of the gels and confocal laser scanning microscopy (CLSM) measured cell morphogenesis within the gels. The assumption that the matrix modulus systematically decreases as cells locally begin to enzymatically disassemble the PF hydrogel to become spindled in the material was not supported by the bulk mechanical property measurements. Instead, the PF hydrogels exhibited cell-mediated stiffening concurrent with their dynamic morphogenesis, as indicated by a four-fold increase in storage modulus after 1week in culture. Fibrin hydrogels, which were used as the control biomaterial, proved similarly adaptive to cell-mediated remodeling only in the presence of the exogenous serine protease inhibitor aprotinin. Acellular and non-viable hydrogels also served as control groups to verify that transient matrix remodeling was entirely associated with cell-mediated events, including collagen deposition, cell-mediated proteolysis, and the formation of multicellular networks within the hydrogel constructs. The fact that cell network formation and collagen deposition both paralleled transient stiffening of the PF hydrogels, further reinforces the notion that cells actively balance between proteolysis and ECM synthesis when remodeling proteolytically responsive hydrogel scaffolds.

Fabrication of PEGylated Fibrinogen: A Versatile Injectable Hydrogel Biomaterial

Hydrogels are one of the most versatile biomaterials in use for tissue engineering and regenerative medicine. They are assembled from either natural or synthetic polymers, and their high water content gives these materials practical advantages in numerous biomedical applications. Semisynthetic hydrogels, such as those that combine synthetic and biological building blocks, have the added advantage of controlled bioactivity and material properties. In myocardial regeneration, injectable hydrogels premised on a semisynthetic design are advantageous both as bioactive bulking agents and as a delivery vehicle for controlled release of bioactive factors and/or cardiomyocytes. A new semisynthetic hydrogel based on PEGylated fibrinogen has been developed to address the many requirements of an injectable biomaterial in cardiac restoration. This chapter highlights the fundamental aspects of making this biomimetic hydrogel matrix for cardiac applications.

A method for preparation of hydrogel microcapsules for stem cell bioprocessing and stem cell therapy

A method for the preparation of suspension culture microcapsules used in the bioprocessing of human mesenchymal stem cells (hMSCs) is reported. The microcapsules are prepared from a semi-synthetic hydrogel comprising Pluronic®F127 conjugated to denatured fibrinogen. The Pluronic-fibrinogen adducts display a lower critical solubility temperature (LCST) at ∼30 °C, thus enabling mild, cell-compatible physical crosslinking of the microcapsules in a warm gelation bath. Cell-laden microgels were prepared from a solution of Pluronic-fibrinogen hydrogel precursor and hMSCs; these were cultivated for up to 15 days in laboratory-scale suspension bioreactors and harvested by reducing the temperature of the microcapsules to disassemble the physical polymer network. The viability, proliferation and cell recovery yields of the hMSCs were shown to be better than photo-chemically crosslinked microcapsules made from a similar material. The cell culture yields, which exceeded 300% after 15 days in suspension culture, were comparable to other microcarrier systems used for the mass production of hMSCs. The simplicity of this methodology, both in terms of the cell inoculation and mild recovery conditions, represent distinct advantages for stem cell bioprocessing with suspension culture bioreactors.

Heart Regeneration: The Bioengineering Approach

The low cell survival following direct cell injection motivated the use of biomaterials in cardiac restoration therapies. Two distinct categories of biomaterial-based cardiac cell therapy were introduced: a tissue engineered cardiac patch and an injectable biomaterial/cell graft. The tissue engineering method attempts to create a tissue analog in vitro, which is to be sutured directly onto the infracted myocardium. The injectable biomaterial approach is designed to locally deliver cell grafts. Generally, cardiac restoration with injectable biomaterials is focused on the use of liquid-to-solid hydrogels as cell carriers that, when combined with cells, should increase the cell survival and improve the overall contractility of the infarcted myocardium. In spite of the enormous potential of cardiac cell therapy in this regard, some recent studies have focused on using only biomaterials alone to stabilize the cardiac wall geometry and prevent cardiac remodeling, without cell therapy.