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Herein, peptide nucleic acids (PNAs) are employed in the design of a participatory duplex PNA‐peptide crosslinking agent. Biophysical and mechanical studies show that crosslinkers present during peptide assembly leading to hydrogelation participate in the formation of fibrils while simultaneously installing crosslinks into the higher‐order network that constitutes the peptide gel. The addition of 2 mol % crosslinker into the assembling system results in a ~100 % increase in mechanical stiffness without affecting the rate of peptide assembly or the local morphology of fibrils within the gel network. Stiffness enhancement is realized by only affecting change in the elastic component of the viscoelastic gel. A synthesis of the PNA‐peptide duplex crosslinkers is provided that allows facile variation in peptide composition and addresses the notorious hydrophobic content of PNAs. This crosslinking system represents a new tool for modulating the mechanical properties of peptide‐based hydrogels. [ABSTRACT FROM AUTHOR]

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Here, we report the design, synthesis and efficacy of a new class of ultrasound (US)-sensitive self-assembled peptide-based nanoparticle. Peptisomes are prepared via templated assembly of a de novo designed peptide at the interface of fluorinated nanodroplets. Utilizing peptide assembly allows for facile particle synthesis, direct incorporation of bioactive sequences displayed from the particle corona, and the ability to easily encapsulate biologics during particle preparation using a mild solvent exchange procedure. Further, nano-peptisome size can be precisely controlled by simply modulating the starting peptide and fluorinated solvent concentrations during synthesis. Biomolecular cargo encapsulated within the particle core can be directly delivered to the cytoplasm of cells upon US-mediated rupture of the carrier. Thus, nano-peptisomes represent a novel class of US-activated carriers that can shuttle cell-impermeable biomacromolecules into cells with spatial and temporal precision. [ABSTRACT FROM AUTHOR]

Dopamine is a small versatile molecule used for various biotechnological and biomedical applications. This neurotransmitter, in addition to its biological role, can undergo oxidative self-polymerization to yield polydopamine, a robust universal coating material. Herein, we harness dopamine self-polymerization to modulate the viscoelastic mechanical properties of peptide-based gels, expanding their ever-growing application potential. By combining rapid peptide assembly with slower dopamine auto-polymerization, a double network gel is formed, where the fibrillar peptide gel network serves as a scaffold for polydopamine deposition, allowing polydopamine to interpenetrate the gel network as well as establishing crosslinks within the matrix. We have shown that triggering the assembly of a lysine-rich peptide gelator in the presence of dopamine can increase the mechanical rigidity of the resultant gel by a factor of 90 in some cases, while retaining the gel's shear thin-recovery behavior. We further investigate how factors such as polymerization time, dopamine concentration and peptide concentration alter the mechanical properties of the resultant gel. The hybrid peptide–dopamine gel systems were characterized using rheological measurements, circular dichroism spectroscopy and transmission electron microscopy. Overall, triggering peptide gelation in the presence of dopamine represents a simple yet powerful approach to modulate the viscoelastic mechanical properties of peptide-based gels. [ABSTRACT FROM AUTHOR]

Hydrogels formed from self-assembling peptides are finding use in tissue engineering and drug delivery applications. Given the notorious difficulties associated with producing self-assembling peptides by recombinant expression, most are typically prepared by chemical synthesis. Herein, we report the design of a family of self-assembling β-hairpin peptides amenable to efficient production using an optimized bacterial expression system. Ex pressing peptides, EX1, EX2 and EX3 contain identical eight-residue amphiphilic β-strands connected by varying turn sequences that are responsible for ensuring chain reversal and the proper intramolecular folding and consequent self-assembly of the peptide into a hydrogel network under physiological conditions. EX1 was initially used to establish and optimize the bacterial expression system by which all the peptides could be eventually individually expressed. Expression clones were designed to allow exploration of possible fusion partners and investigate both enzymatic and chemical cleavage as means to liberate the target peptide. A systematic analysis of possible expression systems followed by fermentation optimization lead to a system in which all three peptides could be expressed as fusions with BAD-BH3, the BH3 domain of the proapoptotic BAD (Bcl-2 Associated Death) Protein. CNBr cleavage followed by purification afforded 50, 31, and 15 mg/L yields of pure EX1, EX2 and EX3, respectively. CD spectroscopy, TEM, and rheological analysis indicate that these peptides fold and assembled into well-defined fibrils that constitute hydrogels having shear-thin/recovery properties. [ABSTRACT FROM AUTHOR]

Abstract: Controlled biodegradation specific to matrix metalloproteinase-13 was incorporated into the design of self-assembling β-hairpin peptide hydrogels. Degrading Peptides (DP peptides) are a series of five peptides that have varying proteolytic susceptibilities toward MMP-13. These peptides undergo environmentally triggered folding and self-assembly under physiologically relevant conditions (150 mm NaCl, pH 7.6) to form self-supporting hydrogels. In the presence of enzyme, gels prepared from distinct peptides are degraded at rates that differ according to the primary sequence of the single peptide comprising the gel. Material degradation was monitored by oscillatory shear rheology over the course of 14 days, where overall degradation of the gels vary from 5% to 70%. Degradation products were analyzed by HPLC and identified by electrospray-ionization mass spectrometry. This data shows that proteolysis of the parent peptides constituting each gel occurs at the intended sequence location. DP hydrogels show specificity to MMP-13 and are only minimally cleaved by matrix metalloproteinase-3 (MMP-3), another common enzyme present during tissue injury. In vitro migration assays performed with SW1353 cells show that migration rates through each gel differs according to peptide sequence, which is consistent with the proteolysis studies using exogenous MMP-13. [Copyright &y& Elsevier]

DNA that encodes tumor-specific antigens represents potential immunostimulatory agents. However, rapid enzymatic degradation and fragmentation of DNA during administration can result in limited vector expression and, consequently, poor efficacy. These challenges have necessitated the use of novel strategies for DNA delivery. Herein, we study the ability of cationic self-assembling peptide hydrogels to encapsulate plasmid DNA, and enhance its immunostimulatory potential in vivo . The effect of network charge on the gel's ability to retain the DNA was assessed employing three gel-forming peptides that vary systematically in formal charge. The peptide HLT2, having a formal charge of +5 at neutral pH, was optimal in encapsulating microgram quantities of DNA with little effect on its rheological properties, allowing its effective syringe delivery in vivo . The plasmid, DNA(TA), encapsulated within these gels encodes for a melanoma-specific gp100 antigen fused to the alarmin protein adjuvant HMGN1. Implantation of DNA(TA)-loaded HLT2 gels into mice resulted in an acute inflammatory response with the presence of polymorphonuclear cells, which was followed by infiltrating macrophages. These cellular infiltrates aid in the processing of encapsulated DNA, promoting increased lymphoproliferation and producing an enhanced immune response mediated by CD4+/IFNγ+ expressing Th1 cells, and complemented by the formation of gp100-specific antibodies. [ABSTRACT FROM AUTHOR]

Abstract: Hydrogel materials that display inherent activity against bacteria can be used to directly treat accessible wounds to prevent or kill existing infection. Hydrogels composed of self-assembling β-hairpin peptides, having a high content of arginine, were found to be extremely effective at killing both gram-positive and gram-negative bacteria, including multi-drug resistant Pseudomonas aeruginosa. No added antibacterial agents are necessary to realize activity. Using self-assembling peptides for material construction allows facile structure–activity relationships to be determined since changes in peptide sequence at the monomer level are directly transposed to the bulk material''s antibacterial properties. SAR studies show that arginine content largely influences the hydrogel''s antibacterial activity, and influences their bulk rheological properties. These studies culminated in an optimized gel, composed of the peptide PEP6R (VKVRVRVRVDPPTRVRVRVKV). PEP6R gels prepared at 1.5 wt % or higher concentration, demonstrate high potency against bacteria, but are cytocompatible toward human erythrocytes as well as mammalian mesenchymal stem cells. Rheological studies indicate that the gel is moderately stiff and displays shear-thin recovery behavior, allowing its delivery via simple syringe. [Copyright &y& Elsevier]

Abstract: Iterative peptide design was used to generate two peptide-based hydrogels to study the effect of network electrostatics on primary chondrocyte behavior. MAX8 and HLT2 peptides have formal charge states of +7 and +5 per monomer, respectively. These peptides undergo triggered folding and self-assembly to afford hydrogel networks having similar rheological behavior and local network morphologies, yet different electrostatic character. Each gel can be used to directly encapsulate and syringe-deliver cells. The influence of network electrostatics on cell viability after encapsulation and delivery, extracellular matrix deposition, gene expression, and the bulk mechanical properties of the gel-cell constructs as a function of culture time was assessed. The less electropositive HLT2 gel provides a microenvironment more conducive to chondrocyte encapsulation, delivery, and phenotype maintenance. Cell viability was higher for this gel and although a moderate number of cells dedifferentiated to a fibroblast-like phenotype, many retained their chondrocytic behavior. As a result, gel-cell constructs prepared with HLT2, cultured under static in vitro conditions, contained more GAG and type II collagen resulting in mechanically superior constructs. Chondrocytes delivered in the more electropositive MAX8 gel experienced a greater degree of cell death during encapsulation and delivery and the remaining viable cells were less prone to maintain their phenotype. As a result, MAX8 gel-cell constructs had fewer cells, of which a limited number were capable of laying down cartilage-specific ECM. [Copyright &y& Elsevier]

Abstract: Curcumin, a hydrophobic polyphenol, is an extract of turmeric root with antioxidant, anti-inflammatory and anti-tumorigenic properties. Its lack of water solubility and relatively low bioavailability set major limitations for its therapeutic use. In this study, a self-assembling peptide hydrogel is demonstrated to be an effective vehicle for the localized delivery of curcumin over sustained periods of time. The curcumin-hydrogel is prepared in-situ where curcumin encapsulation within the hydrogel network is accomplished concurrently with peptide self-assembly. Physical and in vitro biological studies were used to demonstrate the effectiveness of curcumin-loaded β-hairpin hydrogels as injectable agents for localized curcumin delivery. Notably, rheological characterization of the curcumin-loaded hydrogel before and after shear flow have indicated solid-like properties even at high curcumin payloads. In vitro experiments with a medulloblastoma cell line confirm that the encapsulation of the curcumin within the hydrogel does not have an adverse effect on its bioactivity. Most importantly, the rate of curcumin release and its consequent therapeutic efficacy can be conveniently modulated as a function of the concentration of the MAX8 peptide. [Copyright &y& Elsevier]

Abstract: Hydrogel materials are promising vehicles for the delivery of protein therapeutics. Proteins can impart physical interactions, both steric and electrostatic in nature, that influence their release from a given gel network. Here, model proteins of varying hydrodynamic diameter and charge are directly encapsulated and their release studied from electropositive fibrillar hydrogels prepared from the self-assembling peptide, MAX8. Hydrogelation of MAX8 can be triggered in the presence of proteins for their direct encapsulation with neither effect on protein structure nor the hydrogel’s mechanical properties. Bulk release of the encapsulated proteins from the hydrogels was assessed for a month time period at 37 °C before and after syringe delivery of the loaded gels to determine the influence of the protein structure on release. Release of positively charged and neutral proteins was largely governed by the sterics imposed by the network. Conversely, negatively charged proteins interacted strongly with the positively charged fibrillar network, greatly restricting their release to <10% of the initial protein load. Partition and retention studies indicated that electrostatic interactions dictate the amount of protein available for release. Importantly, when protein encapsulated gels were delivered via syringe, the release profiles of the macromolecules show the similar trends as those observed for non-sheared gels. This study demonstrates that proteins can be directly encapsulated in self assembled MAX8 hydrogels, which can then be syringe delivered to a site where subsequent release is controlled by protein structure. [Copyright &y& Elsevier]

Abstract: A peptide-based hydrogel has been designed that directs the formation of hydroxyapatite. MDG1, a twenty-seven residue peptide, undergoes triggered folding to form an unsymmetrical β-hairpin that self-assembles in response to an increase in solution ionic strength to yield a mechanically rigid, self supporting hydrogel. The C-terminal portion of MDG1 contains a heptapeptide (MLPHHGA) capable of directing the mineralization process. Circular dichroism spectroscopy indicates that the peptide folds and assembles to form a hydrogel network rich in β-sheet secondary structure. Oscillatory rheology indicates that the hydrogel is mechanically rigid (G′ ˜ 2500Pa) before mineralization. In separate experiments, mineralization was induced both biochemically and with cementoblast cells. Mineralization-domain had little effect on the mechanical rigidity of the gel. SEM and EDXS show that MDG1 gels are capable of directing the formation of hydroxapatite. Control hydrogels, prepared by peptides either lacking the mineral-directing portion or reversing its sequence, indicated that the heptapeptide is necessary and its actions are sequence specific. [ABSTRACT FROM AUTHOR]

Abstract: Self-assembling peptide hydrogels are used to directly encapsulate and controllably release model FITC–dextran macromolecules of varying size and hydrodynamic diameters. MAX1 and MAX8 are two peptide sequences with different charge states that have been designed to intramolecularly fold and self assemble into hydrogels at physiological buffer conditions (pH 7.4, 150mm NaCl). When self-assembly is initiated in the presence of dextran or protein probes, these macromolecules are directly encapsulated in the gel. Self-diffusion studies using fluorescence recovery after photobleaching (FRAP) and bulk release studies indicate that macromolecule mobility within, and release out of, these gels can be modulated by varying the hydrogel mesh size. The average mesh size can be modulated by simply varying the concentration of a given peptide used to construct the gel or by altering the peptide sequence. In addition, results suggest that electrostatic interactions between the macromolecules and the peptide network influence mobility and release. Depending on probe size, release half-lives can be varied from 8h to over a month. [Copyright &y& Elsevier]

Abstract: The pro-inflammatory potential of β-hairpin peptide hydrogels (MAX1 and MAX8) was assessed in vitro by measuring the cellular response of J774 mouse peritoneal macrophages cultured on the hydrogel surfaces. An enzyme-linked immunosorbent assay (ELISA) was used to measure the level of TNF-α, a pro-inflammatory cytokine, secreted by cells cultured on the gel surfaces. Both bulk and thin films of gels did not elicit TNF-α secretion from the macrophages. In addition, live/dead assays employing laser scanning confocal microscopy (LSCM) and phase-contrast light micrographs show the hydrogel surfaces are non-cytotoxic toward the macrophages and allow the cells to adopt healthy morphologies. When macrophages were activated with lipopolysaccharide (LPS), a known bacterial pathogen that activates an innate immune response, an increase in the TNF-α titers by two orders of magnitude was observed. On LPS induction, macrophages displayed a decrease in cell density, enlarged nuclei, and an increase in cytoplasmic granularity, all characteristics of activated macrophages indicating that the cells are still capable of reacting to insult. The data presented herein indicate that MAX1 and MAX8 gels do not elicit macrophage activation in vitro and suggest that these materials are excellent candidates for in vivo assessment in appropriate animal models. [Copyright &y& Elsevier]

Abstract: MAX1 is a 20 amino acid peptide that undergoes triggered self-assembly to form a rigid hydrogel. When dissolved in aqueous solutions, this peptide exists in an ensemble of random coil conformations rendering it fully soluble. The addition of an exogenous stimulus results in peptide folding into β-hairpin conformation. This folded structure undergoes rapid assembly into a highly crosslinked hydrogel network. DMEM cell culture media is one stimulus able to initiate folding and consequent self-assembly of MAX1. The cytocompatibility of this gel towards NIH 3T3 murine fibroblasts is demonstrated. Gels were shown to be non-toxic to the fibroblast cells. MAX1 hydrogels also foster the ability of the cells to attach to the hydrogel scaffold in the absence or presence of serum proteins. Additionally MAX1 hydrogels were able to support fibroblast proliferation to confluency with little effect on the rheological properties of the scaffold. MAX1 hydrogels meet the preliminary mechanical and cytocompatibiltiy requirements of a tissue engineering scaffold. [Copyright &y& Elsevier]