Elastin-like Peptides Research Articles

Quantification of the effects of hydrophobicity and mass loading on the effective coverage of surface-immobilized elastin-like peptides

Elastin-like peptides (ELPs) immobilized to solid surfaces have recently gained attention for use in electrochemical applications in sensing as well as bioenabled electrode assembly. Key to the success of these applications is understanding how ELPs impact the access and electron transfer of reacting species to the solid surface (effective surface coverage). In this study, short ELPs with varying hydrophobicity and sequence length were designed for gold attachment, and the effect on the ability of a redox probe to access a gold surface was characterized by cyclic voltammetry. A quantitative model describing the relationship between ELP effective surface coverage as a function of mean hydrophobicity and mass loading was elucidated based on the results, showing the ability to precisely control surface properties by tuning the ELP sequence. This model will be useful for the design of surface-bound ELP sequences that exhibit desired effective surface coverage for electrochemical as well as biomaterial applications.

Histidine-based hydrogels via singlet-oxygen photooxidation.

The formation of hydrogels by photosensitized oxidation and crosslinking of histidine-derived polymers is demonstrated for the first time. The photooxidation of pendant His mediated by singlet oxygen was used to promote covalent coupling by its dimerization. As a proof-of-concept, two systems were studied: (i) chondroitin sulfate (CS) functionalized with His, and (ii) an elastin-like peptide (ELP) containing His produced by recombinant techniques. Both materials were crosslinked by irradiation at 425 nm in the presence of Zn-porphyrin derivatives yielding His-based hydrogels. The molecular structure and physicochemical properties of ELP-His and other 5 ELPs with photooxidizable amino acids were studied in silica by computer simulation. A correlation between the protein conformation and its elastic properties is discussed. CS-His hydrogels demonstrate larger storage moduli than ELPs with other amino acids. The obtained results show the potential use of photooxidation to create a new type of His-based hydrogels.

Recombination and Purification of Elastin-Like Polypeptides.

Elastin, as an extracellular matrix protein, has inherent advantages for biomedical applications. For example, it is highly extensible and biocompatible, biodegradable, and has no immunogenicity. However, directly extracting elastin from biological tissues remains challenging because they usually coexist with other proteins such as collagen. Therefore, an effective strategy to produce elastin is to transfer the elastin's target gene into other expression hosts and synthesize the resultant polypeptides using chemical biology methods. The polypeptides and proteins produced using these methods are usually referred to as elastin-like peptides (ELPs), which have received intensive interests in drug delivery and release, tissue engineering, implanted devices, and so on. Therefore, this chapter introduces the detailed protocol for the preparation of ELPs using genetic recombination, including DNA recombination, expression, and purification. The methods presented here are expected to provide methodological guidance for preparation and application of ELP materials.

Fine structural tuning of the assembly of ECM peptide conjugates via slight sequence modifications.

The self-assembly of nanostructures from conjugates of elastin-like peptides and collagen-like peptides (ELP-CLP) has been studied as means to produce thermoresponsive, collagen-binding drug delivery vehicles. Motivated by our previous work in which ELP-CLP conjugates successfully self-assembled into vesicles and platelet-like nanostructures, here, we extend our library of ELP-CLP bioconjugates to a series of tryptophan/phenylalanine-containing ELPs and GPO-based CLPs [W2F x -b-(GPO) y ] with various domain lengths to determine the impact of these modifications on the thermoresponsiveness and morphology. The lower transition temperature of the conjugates with longer ELP or CLP domains enables the formation of well-defined nanoparticles near physiological temperature. Moreover, the morphological transition from vesicles to platelet-like nanostructures occurred when the ratio of the lengths of ELP/CLP decreased. Given the previously demonstrated ability of many ELP-CLP bioconjugates to bind to both hydrophobic drugs and collagen-containing materials, our results suggest new opportunities for designing specific thermoresponsive nanostructures for targeted biological applications.

Arg-Gly-Asp-modified elastin-like polypeptide regulates cell proliferation and cell cycle proteins via the phosphorylation of Erk and Akt in pancreatic β-cell

ObjectiveEnhancement of β-cell proliferation plays an important role in maintaining β-cell mass and function, and in improving pancreatic β-cell survival before transplantation. Extracellular matrix (ECM) components increase the adhesion and proliferation of β-cells, and the RGD-modified elastin-like polypeptide (RGD-ELP, REP) has been described as a bioactive matrix. In this study, we investigated whether REP could enhance β-cell adhesion and proliferation and elucidated the signaling pathways involved. MethodsWe investigated the effect of REP on cell adhesion, proliferation and insulin secretion via assays using Rin-m and rat islets. Crystal violet, CCK-8, and BrdU assay, FACS, western blot, real time q-PCR analyses and insulin ELISA were examined. To explain the associated mechanisms, phosphorylation of Akt and extracellular signal-regulated kinase (Erk) were measured. ResultsREP more increased the adhesion, proliferation and survival of Rin-m cells compared to elastin-like poly peptide (ELP) without RGD-motif. The enhancement of β-cell proliferation by REP was associated with increased cyclin D1, cyclin D2 and cdk6, and decreased p27 levels. When β-cells were cultured on REP, Erk and the phosphatidylinositol 3-kinase (PI3-kinase) downstream effector, Akt was stimulated. Treatment with the Erk pathway inhibitor and PI3-kinase inhibitor decreased REP-induced β-cell adhesion and proliferation, and regulated REP-induced cell cycle proteins. Additionally, REP increased the mRNA and protein levels of insulin and its transcription factor, PDX-1, and insulin secretion. ConclusionsOur results demonstrate that the up-regulation of the PI3K/Akt and Erk signaling pathways and the regulation of cell cycle proteins by REP could serve as effective strategies for improving pancreatic β-cell adhesion and proliferation.

Effect of Crosslinker Topology on Enzymatic Degradation of Hydrogels

Despite the widespread use of hydrogels in biomedical applications, little is known regarding the effect of crosslinker topology on hydrogel degradation. Dendritic and linear elastin-like peptides (ELPs) were used as crosslinkers for hyaluronic acid (HA) hydrogels, and their enzymatic degradation was studied using trypsin. Rheological studies revealed that hydrogels crosslinked with ELP dendrimers (HA_denELPs) degraded more slowly than those crosslinked with the otherwise equivalent linear ELPs (i.e., both molecules have the same number of pentamers and peripheral lysine residues). The origin of this phenomenon was evaluated using solution studies in which various dendritic and linear ELPs were treated with trypsin. Apart from the expected steric hindrances due to the dendritic topology, we identified the dual directionality of the peptide sequences (generated by a central branching lysine residue) and the likelihood of cleaving a productive crosslinking point as two additional contributors to the lesser degradability of HA_denELPs. Overall, these results highlight how the molecular design of crosslinker topology represents a novel strategy to tune the degradation rate of hydrogels.

Peptide-Controlled Assembly of Anion Exchange Ionomer Thin Films on Electrode Surfaces for Promoting Microphase Separation and Ionic Conductivity

In electrochemical devices such as water electrolyzers and fuel cells, the network and morphology of the conductive ionomers within electrodes are critical for ion delivery and mass transport, significantly influencing the device performance. Most studies investigate these materials as bulk polymer electrolyte membranes and comparatively little attention has been given to their behavior on electrode surfaces as thin films. Anion exchange membrane fuel cells and water electrolyzers (AEMFCs and AEMWEs) would allow the use of non-precious group metals and enable low-cost systems, yet, even less is known about thin films of anion exchange ionomer (AEI). Protein engineering is emerging as a powerful nanomanufacturing tool to control the organization of components on electrodes. In this work, we propose that it can be applied to ionomer control, demonstrating that sequence defined elastin-like peptides assembled on electrode surfaces, and/or solvent vapor annealing processing, alters the microstructure configuration of assembled thin films of anion exchange ionomer. It is observed that peptides form a uniform monolayer on the metal surface, and moderately sized microphase separated ionic domains of the AEI are obtained either by modifying the electrode with peptides or solvent vapor annealing, which correlate with an increased in-plane ionic conductivity of the thin film. Interestingly, the use of peptide-modified electrodes in conjunction with solvent vapor annealing yields excessively large ionic grains that correlates with compromised ionic conductivity. Overall, we show that sequence defined peptides could serve as a tool for controlling electrode architecture, and judicious use of peptides, or solvent vapor annealing, have the potential to control the microstructure of thin AEI films and lead to structure-function understanding.

Tissue scaffolds functionalized with therapeutic elastin-like biopolymer particles.

Tissue engineering scaffolds are intended to provide mechanical and biological support for cells to migrate, engraft and ultimately regenerate the tissue. Development of scaffolds with sustained delivery of growth factors and chemokines would enhance the therapeutic benefits, especially in wound healing. In this study, we incorporated our previously designed therapeutic particles, composed of fusion of elastin-like peptides (ELPs) as the drug delivery platform to keratinocyte growth factor (KGF), into a tissue scaffold, alloderm. The results demonstrated that sustained KGF-ELP release was achieved and the bioactivity of the released therapeutic particles was shown via cell proliferation assay, as well as a mouse pouch model in vivo, where higher cellular infiltration and vascularization were observed in scaffolds functionalized with KGF-ELPs.

Target-Specific Covalent Immobilization of Enzymes from Cell Lysate on SiO2 Nanoparticles for Biomass Saccharification

Herein, we presented a novel strategy for the preparation of peptide/SiO2 nanoparticles enabling enzyme covalent immobilization by combining elastin-like peptide (ELP)-based biomimetic silicificati...

Protease-Resistant Growth Factor Formulations for the Healing of Chronic Wounds.

Objective: Chronic wounds are long-term nonhealing wounds that are refractory to treatment. These wounds can present elevated protease levels, leading to rapid degradation of native and exogenously added growth factors. This work focused on developing a protease-resistant growth factor formulation for treatment of chronic wounds presented with high protease activity. Approach: This study developed protease-resistant growth factor formulations comprising elastin-like peptides (ELPs) fused with a known protease inhibitor peptide or growth factor. The ELP component of the fusion proteins allows assembly of heterogeneous nanoparticles (NPs) putting the inhibitor in close proximity to the growth factor to be protected. Results: We show successful preservation of growth factor activity in high human neutrophil elastase (HNE) environment and in human chronic wound fluid derived from patients. We further show that these NPs result in enhanced collagen remodeling and resolution of inflammation in a full thickness wound supplemented with HNE in genetically diabetic mice. Innovation: Development of heterogeneous NPs that put the protease inhibitor in close proximity of the growth factor. Moreover, the modular nature of the NPs allows for protection of multiple growth factors by the same inhibitor without changing the amino acid sequence of the growth factor. Conclusion: Our results indicate that the developed NPs hold tremendous promise in chronic wound healing therapy and may further help the translation of growth factor therapies to clinic. The customizable template for the NP design allows for multifaceted use across several fields in research and medicine.

Targeted Elastin-like Fusion Proteins for Enhanced Imaging and Treatment of Non-Muscle Invasive Bladder Cancer

Background and Hypothesis: A recent study estimates 17,670 individuals will die from bladder cancer in the United States in 2019. With current imaging and treatment technology, bladder cancer is associated with high recurrence rates making it the highest lifetime treatment cost per patient. High expense is primarily due to the profound heterogeneity associated with bladder cancer necessitating repeated intervention. Traditional treatment of non-muscle invasive bladder (NMIBC) cancer following intravesical tumor resection often includes chemotherapy or immunostimulatory therapy via Bacillus Calmette-Guerin (BCG) infusions. Both lack specificity for cancerous tissue, leading to sub-optimal prognosis and unnecessary discomfort for patients. Furthermore, bladder cancer detection is limited in its precision. Methods such as CT/MRI urography and cystoscopies are unable to accurately detect tumors under 2 centimeters in diameter. Blue light cystoscopy, a gold standard detection method, is unable to accurately differentiate tumors from inflamed tissue. This leads to issues detecting aggressive carcinoma in situ. The goal of this project is to enhance tumor detection by fusing various targeting ligands to elastin-like peptides (ELP).
 Experimental Design or Project Methods: Using synthetic biology approaches, a library of ELP fusions were made and purified using a simplified organic extraction method. (Biomaterials Science 2018, 6 (4), 863-876) This resulted in exceptionally pure protein within a matter of hours. Contrast agents were covalently attached, and performance testing was done in murine bladder cancer cells.
 Results: Novel ELP fusions were successfully produced and sequences verified. Protein expression and purification yielded active targeted constructs which showed enhanced binding efficiency over non-targeted constructs. These ELP fusion proteins are effective transporters for rapid and preferential cargo entry into bladder cancer tissue.
 Conclusion and Potential Impact: Chemotherapeutic, immunostimulatory, and tumor imaging cargo are all bright candidates for targeted ELP fusion technology. Targeted ELP delivery may be a superior tool key to improving tumor visualization and treatment, leading to improved comfort and prognosis in patients with NMIBC.

RGD-containing elastin-like polypeptide improves islet transplantation outcomes in diabetic mice

Successful islet transplantation critically depends on the isolation of healthy islets. However, the islet isolation procedure itself contributes to islet death due to the destruction of intra- and peri-islet extracellular matrices (ECMs) during digestion. We investigated whether an RGD-containing elastin-like polypeptide (REP) could function as a self-assembling matrix to replenish ECMs and protects islets from cell death. Immediately following isolation, islets were coated with REP coacervate particles via isothermal adsorption of an REP solution followed by thermal gelation. REP-coated islets displayed increased viability and insulin secretory capacity in pretransplant culture compared to untreated islets. Co-transplantation of REP-treated islets and REP beneath the renal sub-capsule in streptozotocin-induced diabetic mice restored normoglycemia and serum insulin levels. Mice that received co-transplants maintained normoglycemia for a longer period of time than those receiving untreated islets without REP. Moreover, co-transplantation sites exhibited enhanced β-cell proliferation and vascularization. Thus, the REP-based coacervation strategy improve the survival, function and therapeutic potential of transplanted islets. Statement of Significance1). An artificial matrix polypeptide comprised of thermoresponsive elastin-like peptides and integrin-stimulatory RGD ligands (REP) to reconstitute damaged or lost matrices.2). Through body temperature-induced coacervation, REP reconstitutes intra-islet environment and enhances islet viability and insulin secretion by activating the pro-survival and insulin signaling pathways.3). REP-coated islets were transplanted together with the matrix polypeptide under the kidney sub-capsule of mice, it develops a new peri-insular environment, which protects the islet grafts from immune rejection thus extending islet longevity.4). Our data suggest that in situ self-assembly of biomimetic islet environments become a new platform allowing for improved islet transplantation at extrahepatic sites.

Self-Assembly of Stable Nanoscale Platelets from Designed Elastin-like Peptide-Collagen-like Peptide Bioconjugates.

The self-assembly of nanostructures from elastin-like (poly)peptide (ELP) containing block copolymers has been a subject of intense investigation over decades. However, short synthetic ELPs have rarely been used due to their high inverse transition temperature; the use of short ELPs has largely been limited to polymer conjugates. Motivated by our previous work which successfully overcame this barrier by simply conjugating short ELPs with a triple-helix-forming collagen-like peptide, in this study, we further extend the ELP library to a series of ELPs equipped with aromatic residues and having sequences as short as four pentapeptide motifs. The resulting elastin-like peptide-collagen-like peptide (ELP-CLP) bioconjugates unexpectedly self-assembled into nanosized platelets likely by forming a bilayer structure. Given the previously demonstrated ability of many other CLP conjugates to target collagens and the potential for encapsulation of hydrophobic drugs in collapsed ELPs, these ELP-CLP nanoplatelets may offer similar opportunities for targeted delivery in biomedical and other arenas.

Effect of Peptide Sequence on the LCST-Like Transition of Elastin-Like Peptides and Elastin-Like Peptide-Collagen-Like Peptide Conjugates: Simulations and Experiments.

Elastin-like polypeptides (ELPs) are thermoresponsive biopolymers that undergo an LCST-like phase transition in aqueous solutions. The temperature of this LCST-like transition, Tt , can be tuned by varying the number of repeat units in the ELP, sequence and composition of the repeat units, the solution conditions, and via conjugation to other biomacromolecules. In this study, we show how and why the choice of guest (X) residue in the VPGXG pentad repeat tunes the Tt of short ELPs, (VPGXG)4, in the free state and when conjugated to collagen-like peptides (CLPs). In experiments, the (VPGWG)4 chain (in short, WWWW) has a Tt < 278 K, while (VPGFG)4 or FFFF has a Tt > 353 K in both free ELP and ELP-CLP systems. The Tt for the FWWF ELP sequence decreases from being >353 K for free ELP to <278 K for the corresponding ELP-CLP system. The decrease in Tt upon conjugation to CLP has been shown to be due to the crowding of ELP chains that decreases the entropic loss upon ELP aggregation. Even though the net hydrophobicity of ELP has been reasoned to drive the Tt , the origins of lower Tt of WWWW compared to FFFF are unclear, as there is disagreement in hydrophobicity scales in how phenylalanine (F) compares to tryptophan (W). Motivated by these experimental observations, we use a combination of atomistic and coarse-grained (CG) molecular dynamics simulations. Atomistic simulations of free and tethered ELPs show that WWWW are more prone to acquire β-turn structures than FFFF at lower temperatures. Also, the atomistically informed CG simulations show that the increased local stiffness in W than F due to the bulkier side chain in W compared to F, alone does not cause the shift in the transition of WWWW versus FFFF. The experimentally observed lower Tt of WWWW than FFFF is achieved in CG simulations only when the CG model incorporates both the atomistically informed local stiffness and stronger effective attractions localized at the W position versus the F position. The effective interactions localized at the guest residue in the CG model is guided by our atomistically observed increased propensity for β-turn structure in WWWW versus FFFF and by past experimental work of Urry et al. quantifying hydrophobic differences through enthalpy of association for W versus F.

Solvent Effects on the Self-Assembly and Mechanical Properties of Elastin-Like Peptides

Solvent Effects on the Self-Assembly and Mechanical Properties of Elastin-Like Peptides

Molecular Models of Human Elastin and Elastin Biomaterials

The elastin polymer, assembled from its molecular precursor tropoelastin, is the dominant component of elastic fibers, which confer elasticity and structural integrity to skin, lung, connective and vascular tissue. Historically, elastin's dynamic nature has precluded traditional approaches such as X-ray crystallography to understand its detailed features. Here, I describe recent work using atomistic and coarse-grained models for predicting elastin's molecular structure, mechanical properties and mechanisms, as well as dynamics. We use the models to probe the function of key molecular regions, investigate disease etiology and explore implications for hierarchical assembly. From the materials perspective, elastin-based materials display tunable thermal sensitivity, presenting opportunities to mimic and control these responsive features for biomedical applications. We characterize the temperature response spectrum of elastin-like peptides to design synthetic polymers with tunable switching, resolving effects of peptide chemistry, chain length, and solvent environment. We also study a chimera silk-elastin-like protein polymer that combines silk's strength with elastin's extensibility and responsive features to identify temperature transition effects on molecular-scale mechanics. We analyze the associated free-energy landscape with the Bell-Evans model to interpret temperature-induced phase transitions. Such a feedback loop between simulation and experiment for predictive biomaterial design may enable new applications in drug delivery and tissue engineering.

Self-Assembly of Short Elastin-like Amphiphilic Peptides: Effects of Temperature, Molecular Hydrophobicity and Charge Distribution.

A novel type of self-assembling peptides has been developed by introducing the basic elastomeric β-turn units of elastin protein into the amphiphilic peptide molecules. The self-assembly behaviors of such peptides are affected by the overall molecular hydrophobicity, charge distribution and temperature. The molecules with higher hydrophobicity exhibit better self-assembling capability to form long fibrillar nanostructures. For some peptides, the temperature increase can not only promote the self-assembly process but also change the self-assembly routes. The self-assembly of the peptides with two charges centralized on one terminal show higher dependence on temperature than the peptides with two charges distributed separately on the two terminals. The study probes into the self-assembly behaviors of short elastin-like peptides and is of great help for developing novel self-assembling peptides with thermo sensitivity.

Temperature induced conformational changes in the elastin-like peptide GVG(VPGVG)3.

Elastin-like peptides are biopolymers that display LCST behaviour in solution quite similar to other synthetic polymers like polyethylene oxide. Here we study the structure of the peptide GVG(VPGVG)3 in a temperature range of 25 °C to 70 °C with small angle neutron scattering. The LCST for this peptide is outside the experimental range of temperatures. Molecular conformation is well described within the model of a random coil but increasing temperature leads to significant changes. The peptide displays a combination of conformational change and aggregation that show up in the scattering at low and intermediate scattering vector q. The aggregate size is determined from an integral measure of the scattered intensity. It increases with temperature and concentration. For low concentration we find a size variation with temperature that may be related to the collapse of conformation at the inverse temperature transition (ITT).

Hydrogels composed of hyaluronic acid and dendritic ELPs: hierarchical structure and physical properties.

Hydrogels that mimic the native extracellular matrix were prepared from hyaluronic acid (HA) and amine-terminated dendritic elastin-like peptides (denELPs) of generations 1, 2, and 3 (G1, 2, and 3) as crosslinking units. The physical properties of the hydrogels were investigated by rheology, scanning electron microscopy, swelling tests, small-angle X-ray scattering (SAXS), and model drug loading and release assays. Hydrogel properties depended on the generation number of the denELP, which contained structural segments based on the repeating GLPGL pentamer. Hydrogels with higher generation denELPs (G2 and 3) showed similar properties, but those prepared from G1 denELPs were rheologically weaker, had a larger mesh size, absorbed less model drug, and released the drug more quickly. Interestingly, most of the HA_denELP hydrogels studied here remained transparent upon gelation, but after lyophilization and addition of water retained opaque, "solid-like" regions for up to 4 d during rehydration. This rehydration process was carefully evaluated through time-course SAXS studies, and the phenomenon was attributed to the formation of pre-coacervates in the gel-forming step, which slowly swelled in water during rehydration. These findings provide important insights into the behavior of ELP-based hydrogels, in which physical crosslinking of the ELP domains can be controlled to tune mechanical properties, highlighting the potential of HA_denELP hydrogels as biomaterials.

Towards synthetic cells using peptide-based reaction compartments

Membrane compartmentalization and growth are central aspects of living cells, and are thus encoded in every cell’s genome. For the creation of artificial cellular systems, genetic information and production of membrane building blocks will need to be coupled in a similar manner. However, natural biochemical reaction networks and membrane building blocks are notoriously difficult to implement in vitro. Here, we utilized amphiphilic elastin-like peptides (ELP) to create self-assembled vesicular structures of about 200 nm diameter. In order to genetically encode the growth of these vesicles, we encapsulate a cell-free transcription-translation system together with the DNA template inside the peptide vesicles. We show in vesiculo production of a functioning fluorescent RNA aptamer and a fluorescent protein. Furthermore, we implement in situ expression of the membrane peptide itself and finally demonstrate autonomous vesicle growth due to the incorporation of this ELP into the membrane.