Elastin-like Polypeptides Research Articles

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.

One-Pot Synthesis of Multiple Stimuli-Responsive Magnetic Nanomaterials Based on the Biomineralization of Elastin-like Polypeptides.

Synthesis of multiple stimuli-responsive magnetic nanomaterials in a green way remains as a big challenge currently. Herein, temperature-responsive elastin-like polypeptides (ELPs) were designed to involve in the biomimetic mineralization and successfully prepared magnetic nanoparticles (MNPs) (named ELPs-MNPs) with multiple responsiveness (temperature, magnetic, and biomimetic silicification responsiveness) in one pot. ELPs-MNPs were identified as cubic nanomaterials with an average size of about 32 nm and in line with the classic ferromagnetic behavior. Interestingly, ELPs-MNPs show clearly lower critical solution temperature phase behavior with a transition temperature of 36 °C. Moreover, ELPs-MNPs can spontaneously trigger the biosilicification of tetramethyl orthosilicate (TMOS) to entrap themselves into silicon oxide as proved by the Fourier transform infrared spectra (FTIR) and elemental mapping of transmission electron microscopy (TEM), with an average size of about 62 nm. The possible role of ELPs in the biomimetic preparation of the multiple stimuli-responsive MNPs was also addressed. The proposed novel and simple one-pot strategy to synthesize multifunctional nanomaterials with higher effectiveness is the first report for preparing MNPs with multiple stimuli response. This strategy conforms to the concept of green chemistry and will pave a new way for the design of smart biomaterials, which may have great potentials for different fields.

Advances in Drug Delivery and Theranostics

Advances in Drug Delivery and Theranostics

Self-assembling systems comprising intrinsically disordered protein polymers like elastin-like recombinamers.

Despite lacking cooperatively folded structures under native conditions, numerous intrinsically disordered proteins (IDPs) nevertheless have great functional importance. These IDPs are hybrids containing both ordered and intrinsically disordered protein regions (IDPRs), the structure of which is highly flexible in this unfolded state. The conformational flexibility of these disordered systems favors transitions between disordered and ordered states triggered by intrinsic and extrinsic factors, folding into different dynamic molecular assemblies to enable proper protein functions. Indeed, prokaryotic enzymes present less disorder than eukaryotic enzymes, thus showing that this disorder is related to functional and structural complexity. Protein-based polymers that mimic these IDPs include the so-called elastin-like polypeptides (ELPs), which are inspired by the composition of natural elastin. Elastin-like recombinamers (ELRs) are ELPs produced using recombinant techniques and which can therefore be tailored for a specific application. One of the most widely used and studied characteristic structures in this field is the pentapeptide (VPGXG)n . The structural disorder in ELRs probably arises due to the high content of proline and glycine in the ELR backbone, because both these amino acids help to keep the polypeptide structure of elastomers disordered and hydrated. Moreover, the recombinant nature of these systems means that different sequences can be designed, including bioactive domains, to obtain specific structures for each application. Some of these structures, along with their applications as IDPs that self-assemble into functional vesicles or micelles from diblock copolymer ELRs, will be studied in the following sections. The incorporation of additional order- and disorder-promoting peptide/protein domains, such as α-helical coils or β-strands, in the ELR sequence, and their influence on self-assembly, will also be reviewed. In addition, chemically cross-linked systems with controllable order-disorder balance, and their role in biomineralization, will be discussed. Finally, we will review different multivalent IDPs-based coatings and films for different biomedical applications, such as spatially controlled cell adhesion, osseointegration, or biomaterial-associated infection (BAI).

Novel Protein Therapeutics Created Using the Elastin-Like Polypeptide Platform.

Elastin-like polypeptides (ELPs) are bioengineered proteins that have a unique physical property, a thermally triggered inverse phase transition, that can be exploited for drug delivery. ELP-fusion proteins can be used as soluble biologics, thermally targeted drug carriers, self-assembling nanoparticles, and slow-release drug depots. Because of their unique physical characteristics and versatility for delivery of nearly any type of therapeutic, ELP-based drug delivery systems represent a promising platform for biologics development.

3D-printed scaffold composites for the stimuli-induced local delivery of bioactive adjuncts.

Polysaccharide scaffolds have been successfully employed to reconstruct environments that sustain skin tissue regeneration after injuries. Three-dimensional (3D) advanced additive manufacturing technologies allow creating scaffolds with controlled and reproducible macro- and micro-structure that improve the quality of the restored tissue to favor spontaneous repair. However, when persistent inflammation occurs, the physiological tissue healing capacity is reduced, like in the presence of pathologies like diabetes, vascular diseases, chronic infection, and others. In these circumstances, the bioavailability of therapeutic adjuncts like the growth factors in addition to the standard treatments represents undoubtedly a promising strategy to accelerate the healing of skin lesions. Precisely designed polysaccharide scaffolds obtained by 3D printing represent a robust platform that can be further implemented with the controlled delivery of bioactive adjuncts. Human elastin-like polypeptides (HELPs) are stimuli-responsive biopolymers. Their structure allows the integration of domains endowed with biological functionality, making them attractive compounds to prepare composites with smart properties. In the present study, 3D-printed alginate and chitosan scaffolds were combined with the HELP components. The HELP biopolymer was fused to the epidermal growth factor (EGF) as the bioactive domain. Different constructs were prepared and the stimuli-responsive behavior as well as the biological activity were evaluated, suggesting that these smart bioactive composites are suitable to realize multifunctional dressings that sustain the local release of therapeutic adjuncts.

Concentration-Independent Multivalent Targeting of Cancer Cells by Genetically Encoded Core-Crosslinked Elastin/Resilin-like Polypeptide Micelles.

Valency is a fundamental principle to control macromolecular interactions and is used to target specific cell types by multivalent ligand-receptor interactions using self-assembled nanoparticle carriers. At the concentrations encountered in solid tumors upon systemic administration, these nanoparticles are, however, likely to show critical micelle concentration (CMC)-dependent disassembly and thus loss of function. To overcome this limitation, core-crosslinkable micelles of genetically encoded resilin-/elastin-like diblock polypeptides were recombinantly synthesized. The amphiphilic constructs were covalently photo-crosslinked through the genetically encoded unnatural amino acid para-azidophenylalanine in their hydrophobic block and they carried different anticancer ligands on their hydrophilic block: the wild-type tenth human fibronectin type III domain, a GRGDSPAS peptide-both targeting αvβ3 integrin-and an engineered variant of the third fibronectin type III domain of tenascin C that is a death receptor 5 agonist. Although uncrosslinked micelles lost most of their targeting ability below their CMC, the crosslinked analogues remained active at concentrations up to 1000-fold lower than the CMC, with binding affinities that are comparable to antibodies.

Proline Isomerization Regulates the Phase Behavior of Elastin-Like Polypeptides in Water.

Responsiveness of polypeptides and polymers in aqueous solution plays an important role in biomedical applications and in designing advanced functional materials. Elastin-like polypeptides (ELPs) are a well-known class of synthetic intrinsically disordered proteins (IDPs), which exhibit a lower critical solution temperature (LCST) in pure water and in aqueous solutions. Here, we compare the influence of cis/trans proline isomerization on the phase behavior of single ELPs in pure water. Our results reveal that proline isomerization tunes the conformational behavior of ELPs while keeping the transition temperature unchanged. We find that the presence of the cis isomers facilitates compact structures by preventing peptide–water hydrogen bonding while promoting intramolecular interactions. In other words, the LCST transition of ELPs with all proline residues in the cis state occurs with almost no noticeable conformational change.

Microphase Separation of Resilin-like and Elastin-like Diblock Copolypeptides in Concentrated Solutions.

Diblock copolymers are valued for their ability to form thin films with nanoscale features that typically reflect those of their microphase-separated structures in concentrated solution. Here, we show that such self-assembled structures can be easily formed with diblock copolymers composed of thermally responsive polypeptides, such as resilin-like polypeptides (RLP) and elastin-like polypeptides (ELP), by exploiting the inverse temperature transition behavior of ELPs in aqueous media. Specifically, we examine the self-assembly of a series of RLP-b-ELP diblock copolypeptides in concentrated aqueous solution (30 and 50 wt %) by small-angle X-ray scattering (SAXS). By systematically varying RLP block length and temperature (10-45 °C), we observed microphase separation into hexagonally packed cylinders and lamellae. By analyzing the observed order-order transitions (OOT) and order-disorder transitions (ODT), we determined that self-assembly in this system is primarily driven by polymer-solvent interactions. While these thermally responsive polymers showed clear ODTs and OOTs at certain temperatures, temperature only had a weak effect on the spacing of the resulting nanostructures. In contrast, we found that nanostructure spacing was far more sensitive to RLP block length. Finally, we used atomic force microscopy (AFM) to demonstrate that spin casting RLP-b-ELP diblock copolypeptides also produce nanostructured thin films with spacings that correlate with those in concentrated solution.

Insights into Structure and Aggregation Behavior of Elastin-like Polypeptide Coacervates: All-Atom Molecular Dynamics Simulations.

The stimuli-responsive character of elastin-like polypeptides (ELP) has led to their use in a wide range of applications. The temperature-triggered aggregation, or LCST behavior, of ELPs is a complex and multistep phenomenon, which proposed to include the structural transitions, loss of hydrophobic hydration, expulsion of water molecules and physical association of chains. Thus, the origin and detailed mechanism of LCST in ELPs is difficult to elucidate. Here, to gain insights into structure and dynamics of coacervates, we performed all-atom molecular dynamics simulations of 27 90-mer ELPs in explicit water at 350 K. Two sequences, poly(VGPVG)18 and poly(VPGVG)18, were examined due to their experimentally observed differences in thermal hysteresis albeit identical overall composition but different arrangement of amino acids. The simulation results indicate that surface hydrophobicity of poly(VGPVG) aggregate is less than that of the poly(VPGVG) aggregate, and there are marked changes in torsion angles and the propensities of secondary structural motifs during the aggregation process. Moreover, there are significant differences between structure of a single polypeptide in water and structure within the aggregate. Overall, the aggregation process is driven by the formation of peptide-peptide interactions whereas the average hydration of peptides remains almost the same between dissolved and aggregated states. Even though the aggregation is driven by the hydrophobic interactions, ELP coacervate has no hydrophobic core and contains many water molecules. Overall, our findings provide an insight into the sequence-dependent structure of coacervates and molecular behavior of individual peptides during aggregation.

Structure-Property Relationships of Elastin-like Polypeptides: A Review of Experimental and Computational Studies.

Elastin is a structural protein with outstanding mechanical properties (e.g., elasticity and resilience) and biologically relevant functions (e.g., triggering responses like cell adhesion or chemotaxis). It is formed from its precursor tropoelastin, a 60-72 kDa water-soluble and temperature-responsive protein that coacervates at physiological temperature, undergoing a phenomenon termed lower critical solution temperature (LCST). Inspired by this behavior, many scientists and engineers are developing recombinantly produced elastin-inspired biopolymers, usually termed elastin-like polypeptides (ELPs). These ELPs are generally comprised of repetitive motifs with the sequence VPGXG, which corresponds to repeats of a small part of the tropoelastin sequence, X being any amino acid except proline. ELPs display LCST and mechanical properties similar to tropoelastin, which renders them promising candidates for the development of elastic and stimuli-responsive protein-based materials. Unveiling the structure-property relationships of ELPs can aid in the development of these materials by establishing the connections between the ELP amino acid sequence and the macroscopic properties of the materials. Here we present a review of the structure-property relationships of ELPs and ELP-based materials, with a focus on LCST and mechanical properties and how experimental and computational studies have aided in their understanding.

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.

Review of Applications and Future Prospects of Stimuli-Responsive Hydrogel Based on Thermo-Responsive Biopolymers in Drug Delivery Systems.

Some of thermo-responsive polysaccharides, namely, cellulose, xyloglucan, and chitosan, and protein-like gelatin or elastin-like polypeptides can exhibit temperature dependent sol–gel transitions. Due to their biodegradability, biocompatibility, and non-toxicity, such biomaterials are becoming popular for drug delivery and tissue engineering applications. This paper aims to review the properties of sol–gel transition, mechanical strength, drug release (bioavailability of drugs), and cytotoxicity of stimuli-responsive hydrogel made of thermo-responsive biopolymers in drug delivery systems. One of the major applications of such thermos-responsive biopolymers is on textile-based transdermal therapy where the formulation, mechanical, and drug release properties and the cytotoxicity of thermo-responsive hydrogel in drug delivery systems of traditional Chinese medicine have been fully reviewed. Textile-based transdermal therapy, a non-invasive method to treat skin-related disease, can overcome the poor bioavailability of drugs from conventional non-invasive administration. This study also discusses the future prospects of stimuli-responsive hydrogels made of thermo-responsive biopolymers for non-invasive treatment of skin-related disease via textile-based transdermal therapy.

Genetically Encoded Elastin-Like Polypeptides for Drug Delivery.

Elastin-like polypeptides (ELPs) are thermally responsive biopolymers that consist of a repeated amino acid motif derived from human tropoelastin. These peptides exhibit temperature-dependent phase behavior that can be harnessed to produce stimuli-responsive biomaterials, such as nanoparticles or injectable drug delivery depots. As ELPs are genetically encoded, the properties of ELP-based biomaterials can be controlled with a precision that is unattainable with synthetic polymers. Unique ELP architectures, such as spherical or rod-like micelles or injectable coacervates, can be designed by manipulating the ELP amino acid sequence and length. ELPs can be loaded with drugs to create controlled, intelligent drug delivery systems. ELPs are biodegradable, nonimmunogenic, and tolerant of therapeutic additives. These qualities make ELPs exquisitely well-suited to address current challenges in drug delivery and have spurred the development of ELP-based therapeutics to treat diseases-such as cancer and diabetes-and to promote wound healing. This review focuses on the use of ELPs in drug delivery systems.

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.

Self-Assembly Behavior of Elastin-like Polypeptide Diblock Copolymers Containing a Charged Moiety.

Elastin-like polypeptides (ELPs) are stimulus-responsive protein-based biopolymers, and some ELP block copolymers can assemble into spherical nanoparticles with thermosensitivity. In this study, two different ELP diblock copolymers, each composed of a hydrophobic and a charged moiety, were synthesized, and the dependence of their physical properties on pH, temperature, and salt concentration was investigated. A series of analyses revealed that hydrophobic core micelles could be generated in response to a change in their surroundings and that micelles did not self-aggregate, a phenomenon due to the repulsive forces between like-charged molecules on the surface. We also demonstrated that self-assembly behavior was closely dependent on the character of the charged amino acid and the specific anion in solution.

Genetically Encoding Light‐Responsive Protein‐Polymers Using Translation Machinery for the Multi‐Site Incorporation of Photo‐Switchable Unnatural Amino Acids

AbstractA general and versatile technology to engineer light‐responsive protein‐based biomaterials can enable the manipulation and interrogation of proteins, pathways, and cells, and it will assist the design of “smart” light‐responsive biomaterials. This study reports the evolution of chromosomal aminoacyl‐tRNA synthetases (aaRSs) for azobenzene‐bearing unnatural amino acids (uAAs) with up to ≈40‐fold increased protein production and improved fidelity, as compared with a previously described aaRS. The evolved translation systems enable efficient and accurate incorporation of up to 10 instances of the various light‐responsive uAAs in elastin‐like polypeptides (ELPs). Azobenzene‐containing ELPs are capable of isothermal, reversible, light‐mediated soluble‐to‐insoluble phase transition, with up to a 12 °C difference in the ELP transition temperature upon cis‐to‐trans azobenzene isomerization. Furthermore, the incorporation of azobenzene‐uAAs in ELP diblock‐copolymers enables the creation of light‐responsive self‐assembled nanostructures. Finally, light‐responsive resilin‐inspired polymers are also generated by multi‐site azobenzene‐incorporation. The translation machinery evolved in this study can be used for the multi‐site incorporation of azobenzene moieties at the polypeptide level and constitute a universal methodology for the design of light‐responsive proteins and additional families of protein‐based biomaterials with customized and tunable light‐responsive behavior.

Refining the Design of Diblock Elastin-Like Polypeptides for Self-Assembly into Nanoparticles.

Diblock copolymers based-on elastin-like polypeptide (ELP) have the potential to undergo specific phase transitions when thermally stimulated. This ability is especially suitable to form carriers, micellar structures for instance, for delivering active cargo molecules. Here, we report the design and study of an ELP diblock library based on ELP-[M1V3-i]-[I-j]. First, ELP-[M1V3-i]-[I-j] (i = 20, 40, 60; j = 20, 90) that showed a similar self-assembly propensity (unimer-to-aggregate transition) as their related monoblocks ELP-[M1V3-i] and ELP-[I-j]. By selectively oxidizing methionines of ELP-[M1V3-i] within the different diblocks structures, we have been able to access a thermal phase transition with three distinct regimes (unimers, micelles, aggregates) characteristic of well-defined ELP diblocks.

Manipulating the solution environment to control the surface roughness of elastin-based polymer coatings

Elastin-like polypeptides (ELP) have been used as a genetically-engineered, biocompatible substitute for elastin. Cell culture coatings prepared using ELP conjugated to low molecular weight polyethyleneimine (PEI) entices cells to form three-dimensional cellular aggregates that mimic their in vivo counterparts. This study seeks to control the deposition of the ELP and ELP-PEI molecules to control the roughness of the final coatings. The two polymers were coated onto three different substrates (glass, polystyrene, tissue-culture polystyrene) and the solution environment was altered by changing the polymer concentration (0.5, 1.0, 1.5 mg/mL) and/or salt concentration (None, 0.2 M phosphate buffered saline) for a total of 36 conditions. Atomic force microscopy (AFM) was used to measure the average roughness (Ra) of the samples and found that ELP coated samples had a higher Ra than their ELP-PEI counterparts. The coatings were tested for stability by performing cell culture media changes every three days for 11 days. AFM showed that the average roughness of the tested samples increased with each media change. To address this, the surfaces were crosslinked using hexamethyl diisocyanate, which minimized the change in surface roughness even when subjected to an intense sonication process. This study provides parameters to achieve elastin-based coatings with controlled roughness that can be used to support stable, long-term in vitro cell culture.