Recombinant Elastin-like Polypeptides Research Articles

Tuning pro-survival effects of human induced pluripotent stem cell-derived exosomes using elastin-like polypeptides

Exosome-based regenerative therapies are potentially easier to manufacture and safer to apply compared to cell-based therapies. However, many questions remain about how to bio-manufacture reproducible and potent exosomes using animal-free reagents. Here we evaluate the hypothesis that designer biomaterial substrates can be used to alter the potency of exosomes secreted by human induced pluripotent stem cells (iPSCs). Two animal-free designer matrices were fabricated based on recombinant elastin-like polypeptides (ELPs): one including a cell-adhesive RGD ligand and a second with a non-adhesive RDG peptide. While iPSCs cultured on these two substrates and Matrigel-coated controls had similar levels of proliferation, the RDG-ELP substrate significantly increased protein expression of stemness markers OCT4 and SOX2 and suppressed spontaneous differentiation compared to those on RGD-ELP. The pro-survival potency of iPSC-derived exosomes was evaluated using three distinct stress tests: serum starvation in murine fibroblasts, hypoxia in human endothelial cells, and hyperosmolarity in canine kidney cells. In all three cases, exosomes produced by iPSCs grown on RDG-ELP substrates had similar pro-survival effects to those produced using iPSCs grown on Matrigel, while use of RGD-ELP substrates led to significantly reduced exosome potency. These data demonstrate that recombinant substrates can be designed for the robust bio-manufacturing of iPSC-derived, pro-survival exosomes.

De Novo Synthesis of Elastin-like Polypeptides (ELPs): An Applied Overview on the Current Experimental Techniques.

Elastin-like polypeptides (ELPs) are protein-based biopolymers genetically produced from polypeptides composed of a repeating pentapeptide sequence V-P-G-X-G. The inherent properties of recombinant ELPs, such as smart nature, controlled sequence complexity, physicochemical properties, and biocompatibility, make these polymers suitable for use in nanobiotechnological applications, as biofunctionalized scaffolds for tissue-engineering purposes and drug delivery. In this work, we report the design and synthesis of two elastomeric self-assembling polypeptides (ELPs) that mimic the endogenous human tropoelastin. Using molecular biology techniques, two artificial genes that encode two ELP concatemers of approximate molecular mass 60 kDa, one of them carrying biotin-binding peptide motifs, were constructed. These motifs could facilitate biofunctionalization of the ELPs through tethering biotinylated factors, such as growth factors. The ELPs were heterologously overexpressed in E. coli and subsequently purified in two steps: a nonchromatographic technique by organic solvent extraction, followed by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. The characterization of the biochemical properties and biocompatibility of ELPs was also performed in this study. The ELP carrying the biotin-binding motifs was tested for its capability to bind biotin, and indeed, it was observed that it can bind biotinylated proteins specifically. Additionally, results concerning the cytotoxicity of the ELPs exhibited excellent compatibility of the ELPs with mammalian cells in vitro. We anticipate that these ELPs can be used as components of a scaffold that mimics the extracellular matrix (ECM) for the regeneration of endogenously highly elastic tissues.

Photooxidation Responsive Elastin-Like Polypeptide Conjugates for Photodynamic Therapy Application.

Stimuli-responsive recombinant elastin-like polypeptides (ELPs) are artificial protein polymers derived from the hydrophobic domain of tropoelastin that have attracted significant interest for drug delivery and tissue engineering applications. In the present study, we have conjugated a photosensitizer (PS) to a hydrophobic methionine-containing ELP scaffold, which upon reaction with singlet oxygen (1O2) is transformed into a hydrophilic sulfoxide derivative facilitating the disassembly of photosensitizer-delivery particles during the photodynamic therapy (PDT) process. A peripherally substituted carboxy-Zn(II)-phthalocyanine derivative (TT1) bearing a carboxyl group directly linked to the Pc-ring, and presenting an absorption maximum around 680 nm, was selected as PS which simultaneously acted as a photooxidation catalyst. A TT1-ELP[M1V3-40] conjugate was prepared from ELP[M1V3-40] modified with an alkyne group at the N-terminal chain end, and from TT1-amide-C3-azide by copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) reaction. This innovative model photooxidation sensitive PS delivery technology offers promising attributes in terms of temperature-controlled particle formation and oxidation-triggered release, narrow molar mass distribution, reproducibility, scalability, non-immunogenicity, biocompatibility, and biodegradability for pharmaceutical applications in an effort to improve the clinical effectiveness of PDT treatments.

Thermosensitive Vesicles from Chemically Encoded Lipid‐Grafted Elastin‐like Polypeptides

AbstractBiomimetic design to afford smart functional biomaterials with exquisite properties represents synthetic challenges and provides unique perspectives. In this context, elastin‐like polypeptides (ELPs) recently became highly attractive building blocks in the development of lipoprotein‐based membranes. In addition to the bioengineered post‐translational modifications of genetically encoded recombinant ELPs developed so far, we report here a simple and versatile method to design biohybrid brush‐like lipid‐grafted‐ELPs using chemical post‐modification reactions. We have explored a combination of methionine alkylation and click chemistry to create a new class of hybrid lipoprotein mimics. Our design allowed the formation of biomimetic vesicles with controlled permeability, correlated to the temperature‐responsiveness of ELPs.

Thermosensitive Vesicles from Chemically Encoded Lipid-Grafted Elastin-like Polypeptides.

Biomimetic design to afford smart functional biomaterials with exquisite properties represents synthetic challenges and provides unique perspectives. In this context, elastin-like polypeptides (ELPs) recently became highly attractive building blocks in the development of lipoprotein-based membranes. In addition to the bioengineered post-translational modifications of genetically encoded recombinant ELPs developed so far, we report here a simple and versatile method to design biohybrid brush-like lipid-grafted-ELPs using chemical post-modification reactions. We have explored a combination of methionine alkylation and click chemistry to create a new class of hybrid lipoprotein mimics. Our design allowed the formation of biomimetic vesicles with controlled permeability, correlated to the temperature-responsiveness of ELPs.

Thermosensitive Hybrid Elastin-like Polypeptide-Based ABC Triblock Hydrogel

We report the synthesis of a well-defined hybrid ABC triblock terpolymer containing a synthetic poly(trimethylene carbonate) (PTMC) block A and a thermosensitive BC diblock of recombinant elastin-like polypeptides (ELPs). The triblock in diluted solution (0.1–0.3% w/v), at low temperatures in ultrapure water, forms micellar structures of 10–60 nm sizes in diameter as characterized by dynamic light scattering (DLS) and liquid atomic force microscopy (AFM). While heated above its transition temperature (Tt), larger particles of 200–300 nm sizes are obtained, consistent with the formation of coacervates. When concentrated, the viscosity of the triblock copolymer solution progressively increases, giving a free-standing gel at 4% w/v formed by a network of micron-sized particles. The formed hydrogels are thermally reversible, and their sol–gel transitions are fast and sharp. The gel formation mechanism appears to interestingly biomimic tropoelastin, the native monomeric form of natural elastin, as demonstrated by optical and cryogenic-scanning electron microscopy (cryo-SEM) imaging.

Emerging Role of Elastin-Like Polypeptides in Regenerative Medicine.

Significance: Wound dressing based on naturally derived polymer provides a useful platform for treatment of skin injuries. Owing to the high mechanical strength and tunable structural and physicochemical properties of human elastin-like polypeptides (ELPs), they may be used as excellent materials for fabricating biocompatible scaffolds and other products for wound management. Recent Advances: Designing recombinant ELPs mimicking natural elastin to fabricate synthetic polymers suitable for human health care has generated significant interest. ELP-based cell-adhesive biopolymers have been used as an alternative for successful sutureless wound closure due to the physicochemical characteristics of the extracellular matrix. Critical Issues: Different systems of ELPs are being developed in the form of scaffolds, films, hydrogels, photo-linkable sheets, and composites linked with various types of growth factors for wound healing application. However, optimizing the quality and safety attributes for specific application needs designing of recombinant ELPs with structural and functional modifications as needed for the intervention. Future Direction: Chronic wounds are difficult to treat as the wound repair process is interrupted by conditions such as excessive inflammation, impaired extracellular matrix formation, and persistent infections. Conventional therapies such as skin substitutes or autologous skin grafts, in many cases, are unable to reestablish tissue homeostasis and proper healing. The development of innovative materials could induce a better regenerative healing response. In this study, we are reviewing different types of elastin-based materials for wound care application and their future prospects in regenerative medicine.

Catechol-Functionalized Elastin-like Polypeptides as Tissue Adhesives.

Adhesives can potentially be used to achieve fast and efficient wound closure; however, current products show poor bonding on wet surfaces, undergo swelling, and lack adequate biocompatibility. We designed a hydrogel adhesive with recombinant elastin-like polypeptides (ELPs), which are flexible proteins that can be customized for biomedical needs. The adhesive proteins are synthesized by chemically modifying the ELPs with dopamine, which contain adhesive catechol moieties. The resulting catechol-functional ELPs or Cat-ELPs can form flexible hydrogels that show stable swelling in aqueous conditions at 37 °C. We demonstrate their flexibility and viscoelastic properties through rheology. We also show the advantage of using customizable recombinant proteins to improve the material biological properties by demonstrating improved fibroblast binding on Cat-ELP by adding ELP with "RGD" peptides. We further confirmed in vivo biocompatibility and biodegradation of Cat-ELP hydrogels by implanting them in mice and monitoring for 10 weeks. Finally, we tested the bonding strength of the adhesives on porcine skin through tensile pull-off and lap-shear testing, in which we found strengths of 37 and 39 kPa, respectively. We demonstrated the tensile bonding strength by suspending a 2 kg mass on a one square inch (6.5 cm2) skin sample. As our adhesives are developed further, our strategy combining recombinant protein engineering and chemical modification will help yield an ideal bioadhesive for wound closure.

Design of Thermoresponsive Elastin-Like Glycopolypeptides for Selective Lectin Binding and Sorting.

Selective lectin binding and sorting was achieved using thermosensitive glycoconjugates derived from recombinant elastin-like polypeptides (ELPs) in simple centrifugation-precipitation assays. A recombinant ELP, (VPGXG)40, containing periodically spaced methionine residues was used to enable chemoselective postsynthetic modification via thioether alkylation using alkyne functional epoxide derivatives. The resulting sulfonium groups were selectively demethylated to give alkyne functionalized homocysteine residues, which were then reacted with azido-functionalized monosaccharides to obtain ELP glycoconjugates with periodic saccharide functionality. These modifications were also found to allow modulation of ELP temperature dependent water solubility. The multivalent ELP glycoconjugates were evaluated for specific recognition, binding and separation of the lectin Ricinus communis agglutinin (RCA120) from a complex protein mixture. RCA120 and ELP glycoconjugate interactions were evaluated using laser scanning confocal microscopy and dynamic light scattering. Due to the thermoresponsive nature of the ELP glycoconjugates, it was found that heating a mixture of galactose-functionalized ELP and RCA120 in complex media selectively yielded a phase separated pellet of ELP-RCA120 complexes. Based on these results, ELP glycoconjugates show promise as designer biopolymers for selective protein binding and sorting.

Heuristics for the Optimal Presentation of Bioactive Peptides on Polypeptide Micelles.

Bioactive peptides describe a very large group of compounds with diverse functions and wide applications, and their multivalent display by nanoparticles can maximize their activities. However, the lack of a universal nanoparticle platform and design rules for their optimal presentation limits the development and application of peptide ligand-decorated nanoparticles. To address this need, we developed a multivalent nanoparticle platform to study the impact of nanoparticle surface hydrophilicity and charge on peptide targeting and internalization by tumor cells. This system consists of micelles of a recombinant elastin-like polypeptide diblock copolymer (ELPBC) that present genetically encoded peptides at the micelle surface without perturbing the size, shape, stability, or peptide valency of the micelle, regardless of the peptide type. We created the largest extant set of 98 combinations of 15 tumor-homing peptides that are presented on the corona of this ELPBC micelle via 8 different peptide linkers that vary in their length and charge and also created control micelles that present the linker only. Analysis of the structure-function relationship of tumor cell targeting by this set of peptide-decorated nanoparticles enabled us to derive heuristics to optimize the delivery of peptides based on their physicochemical properties and to identify a peptide that is likely to be a widely useful ligand for targeting across nanoparticle types. This study shows that ELPBC micelles are a robust and convenient system for the presentation of diverse peptides and provides useful insights into the appropriate presentation of structurally diverse peptide ligands on nanoparticles based on their physicochemical properties.

Nucleic acids complexation with cationic elastin-like polypeptides: Stoichiometry and stability of nano-assemblies

Positively charged elastin-like polypeptides (ELPs) were synthesized for the compaction of genetic material. A recombinant ELP (VPGXG)40 with X = V,M (3:1) was post-modified in two steps to introduce chemoselectively either primary or secondary amine pendant groups at each methionine residue. Positively charged ELPs were characterized by SDS-PAGE, size exclusion chromatography, 1H NMR, potentiometric titrations and dynamic light scattering to assess their purity and determine their degree of functionalization, molecular weight, isoelectric point and thermo-responsive behaviour. Electrostatic complexation between the different ELP derivatives and nucleic acids was studied to determine the stoichiometry of ELPS/nucleic acids complex formation, and to find optimal conditions leading to stable nanoparticles with controlled size and surface potential. The stability of these complexes was investigated in the presence of salts at physiological concentrations and in the presence of surfactant. This study revealed that two regimes of stable nanoparticles in terms of size and charge can be obtained from the electrostatic complexation between the primary amine containing ELP derivative, ELP(-NH2), and plasmid DNA. Resulting complexes were found to be stable to dissociation for charge ratios up to 2.5 under physiological salt concentrations (154 mM NaCl), showing that plasmid DNA was completely condensed by the polycationic ELP and protected against electrolyte-mediated dissociation.

Injectable Polypeptide Hydrogel Depot System for Assessment of the Immune Response-Inducing Efficacy of Sustained Antigen Release Alone.

Vaccines typically contain an antigen, delivery system (vehicle), and adjuvant, all of which contribute to inducing a potent immune response. Consequently, design of new vaccines is difficult, because the contributions and interactions of these components are difficult to distinguish. Here, it is aimed to develop an easy-to-use, non-immunogenic, injectable depot system for sustained antigen release that will be suitable for assessing the efficacy of prolonged antigen exposure per se for inducing an immune response. This should mimic real-life infections. Recombinant elastin-like polypeptides with periodic cysteine residues (cELPs) are selected, which reportedly show little or no immunogenicity, as carriers and tetanus toxoid (Ttd) as an antigen. After subcutaneous injection of the mixture, cELP rapidly forms a disulfide cross-linked hydrogel in situ, within which Ttd is physically incorporated, affording a biodegradable antigen depot. A series of Ttd-containing hydrogels is examined. A single injection induces high levels of tetanus antibody with high avidity for at least 20 weeks in mice. The chain length of cELP proves critical, whereas differences in hydrophobicity has little effect, although hydrophilic cELPs are more rapidly biodegraded. This system's ability to distinguish the contribution of sustained antigen release to antibody induction should be helpful for rational design of next-generation vaccines.

Self-Assembly of Stimuli-Responsive Biohybrid Synthetic- b-Recombinant Block Copolypeptides.

The synthesis and original thermoresponsive behavior of hybrid diblock copolypeptides composed of synthetic and recombinant polypeptides are herein reported. A thermoresponsive recombinant elastin-like polypeptide was used as a macroinitiator to synthesize a range of poly( l-glutamic acid)- block-elastin-like polypeptide (PGlu- b-ELP) diblock copolypeptides with variable PGlu block lengths. Their temperature-triggered self-assembly in water and in phosphate-buffered saline (PBS) was investigated at the macroscopic scale using complementary techniques such as turbidimetry, dynamic and static light scattering, small-angle neutron scattering, and at the molecular scale by 1H NMR and circular dichroism (CD). In deionized water, PGlu- b-ELP copolypeptides showed one transition from free soluble chains below the transition temperature ( Tt) of the ELP block to macroscopic aggregates above the Tt. In contrast, in PBS, four successive regimes were observed upon increasing temperature: below the Tt, copolypeptides were soluble, above the Tt, large aggregates appeared and fell apart into discrete and defined spherical nanoparticles at a temperature named critical micellization temperature (CMT), before finally reaching an equilibrium. During the last regime, neutron scattering experiments revealed that the micelle-like structures underwent a densification step and expelled water from their core. In addition, 1H NMR and CD experiments revealed, in deionized water, the formation of type II β-turns into the ELP block upon temperature increase. These β-turns are known to participate in the intrinsic thermoresponsive behavior of the ELPs. In contrast, in PBS, circular dichroism measurements showed an attenuation of folded structure during the self-assembly phase, leading to less cohesive aggregates able to reorganize into nanoparticles at the CMT.

Epidermal Growth Factor – based adhesion substrates elicit myoblast scattering, proliferation, differentiation and promote satellite cell myogenic activation

The biochemical properties of muscle extracellular matrix are essential for stem cell adhesion, motility, proliferation and myogenic development. Recombinant elastin-like polypeptides are synthetic polypeptides that, besides maintaining some properties of the native protein, can be tailored by fusing bioactive sequences to their C-terminal. Our laboratory synthesized several Human Elastin-Like Polypeptides (HELP) derived from the sequence of human tropoelastin. Here, we developed a novel HELP family member by fusing the elastin-like backbone to the sequence of human Epidermal Growth Factor. We employed this synthetic protein, named HEGF, either alone or in combination with other proteins of the HELP family carrying RGD-integrin binding sites, as adhesion substrate for C2C12 myoblasts and satellite cells primary cultures. Adhesion of myoblasts to HEGF-based substrates induced scattering, decreased adhesion and cytoskeleton assembly; the concomitant presence of the RGD motifs potentiated all these effects. Recombinant substrates induced myoblasts proliferation, differentiation and the development of multinucleated myotubes, thus favoring myoblasts expansion and preserving their myogenic potential. The effects induced by adhesion substrates were inhibited by AG82 (Tyrphostin 25) and herbimycin A, indicating their dependence on the activation of both the EGF receptor and the tyrosine kinase c-src. Finally, HEGF increased the number of muscle stem cells (satellite cells) derived from isolated muscle fibers in culture, thus highlighting its potential as a novel substrate for skeletal muscle regeneration strategies.

Critical factors for the bulk adhesion of engineered elastomeric proteins.

Many protein-based materials, such as soy and mussel adhesive proteins, have been the subject of scientific and commercial interest. Recently, a variety of protein adhesives have been isolated from diverse sources such as insects, frogs and squid ring teeth. Many of these adhesives have similar amino acid compositions to elastomeric proteins such as elastin. Although elastin is widely investigated for a structural biomaterial, little work has been done to assess its adhesive potential. In this study, recombinant elastin-like polypeptides were created to probe the factors affecting adhesion strength. Lap shear adhesion was used to examine the effects of both extrinsic factors (pH, concentration, cross-linker, humidity, cure time and cure temperature) and intrinsic factors (protein sequence, structure and molecular weight). Of the extrinsic factors tested, only humidity, cure time and cure temperature had a significant effect on adhesion strength. As water content was reduced, adhesion strength increased. Of the intrinsic factors tested, amino acid sequence did not significantly affect adhesion strength, but less protein structure and higher molecular weights increased adhesion strength directly. The strengths of proteins in this study (greater than 2 MPa) were comparable to or higher than those of two commercially available protein-based adhesives, hide glue and a fibrin sealant. These results may provide general rules for the design of adhesives from elastomeric proteins.

Translatable High Drug Loading Drug Delivery Systems Based on Biocompatible Polymer Nanocarriers.

Most nanocarriers possess low drug loading, resulting in frequently repeated administration and thereby high cost and increased side effects. Furthermore, the characteristics of nanocarrier materials, especially the drug loading capacity, plays a vital role in the drug delivery efficacy. In this review, we focus on the readily translatable polymeric drug delivery systems with high drug loading, which are comprised of biocompatible polymers such as poly(ethylene glycol), poly( N-vinylpyrrolidone), polyoxazoline, natural proteins like albumin and casein, non-natural proteins such as recombinant elastin-like polypeptides, as well as nucleic acids. At the end of this review, applications of these polymeric nanocarriers on the delivery of proteins and gene drugs are also briefly discussed.

Engineering Elastin-Like Polypeptide-Poly(ethylene glycol) Multiblock Physical Networks.

Hybrids of protein biopolymers and synthetic polymers are a promising new class of soft materials, as the advantages of each component can be complementary. A recombinant elastin-like polypeptide (ELP) was conjugated to poly(ethylene glycol) (PEG) by macromolecular coupling in solution to form multiblock ELP-PEG copolymers. The hydrated copolymer preserved the thermoresponsive properties from the ELP block and formed hydrogels with different transition temperatures depending on salt concentration. Small angle scattering indicates that the copolymer hydrogels form sphere-like aggregates with a "fuzzy" interface, while the films form a fractal network of nanoscale aggregates. The use of solutions with different salt concentrations to prepare the hydrogels was found to influence the transition temperature, the mechanical properties, and the size of the nanoscale structure of the hydrogel without changing the secondary structure of the ELP. The salt variation and the addition of a plasticizer also affected the nanoscale structure and the mechanical characteristics of the films.

In situ depot formation of anti-HIV fusion-inhibitor peptide in recombinant protein polymer hydrogel.

In this paper, we present an anti-HIV peptide delivery system using oxidatively and thermally responsive polypeptides that contain multiple periodic cysteine residues as an injectable biomaterial capable of in situ self-gelation, and we demonstrate its utility as an injectable depot capable of sustained release of anti-HIV peptides. The novelty of this work stems from the platform employed to provide the depot encapsulating the peptide drugs (without chemical conjugation), which consists of rationally designed, genetically engineered polypeptides that enable the release rate of the peptide drugs to be precisely controlled.

Tuning Thermoresponsive Properties of Cationic Elastin-like Polypeptides by Varying Counterions and Side-Chains.

We report the synthesis of methionine-containing recombinant elastin-like polypeptides (ELPs) of different lengths that contain periodically spaced methionine residues. These ELPs were chemoselectively alkylated at all methionine residues to give polycationic derivatives. Some of these samples were found to possess solubility transitions in water, where the temperature of these transitions varied with ELP concentration, nature of the methionine alkylating group, and nature of the sulfonium counterions. These studies show that introduction and controlled spacing of methionine sulfonium residues into ELPs can be used as a means both to tune their solubility transition temperatures in water using a variety of different parameters and to introduce new side-chain functionality.

Phase Behavior and Self-Assembly of Perfectly Sequence-Defined and Monodisperse Multiblock Copolypeptides.

This paper investigates how the properties of multiblock copolypeptides can be tuned by their block architecture, defined by the size and distribution of blocks along the polymer chain. These parameters were explored by the precise, genetically encoded synthesis of recombinant elastin-like polypeptides (ELPs). A family of ELPs was synthesized in which the composition and length were conserved while the block length and distribution were varied, thus creating 11 ELPs with unique block architectures. To our knowledge, these polymers are unprecedented in their intricately and precisely varied architectures. ELPs exhibit lower critical solution temperature (LCST) behavior and micellar self-assembly, both of which impart easily measured physicochemical properties to the copolymers, providing insight into polymer hydrophobicity and self-assembly into higher order structures, as a function of solution temperature. Even subtle variation in block architecture changed the LCST phase behavior and morphology of these ELPs, measured by their temperature-triggered phase transition and nanoscale self-assembly. Size and morphology of polypeptide micelles could be tuned solely by controlling the block architecture, thus demonstrating that when sequence can be precisely controlled, nanoscale self-assembly of polypeptides can be modulated by block architecture.