Refining the Design of Diblock Elastin-Like Polypeptides for Self-Assembly into Nanoparticles.
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ABSTRACT:
SUBMITTER: Dai M
PROVIDER: S-EPMC8125372 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundElastin-like polypeptides (ELPs) are a fascinating biomaterial that has undergone copious development for a variety of therapeutic applications including as a nanoscale drug delivery vehicle. A comprehensive understanding of ELP self-assembly is lacking and this knowledge gap impedes the advancement of ELP-based biomaterials into the clinical realm. The systematic examination of leucine-containing ELPs endeavors to expand existing knowledge about fundamental assembly-disassembly behaviours.ResultsIt was observed that these marginally soluble, short ELPs tend to behave consistently with previous observations related to assembly-related ELP phase transitions but deviated in their disassembly. It was found that chain length, concentration and overall sequence hydrophobicity may influence the irreversible formation of sub-micron particles as well as the formation of multi-micron scale, colloidally unstable aggregates. Amino acid composition affected surface charge and packing density of the particles. Particle stability upon dilution was found to vary depending upon chain length and hydrophobicity, with particles composed of longer and/or more hydrophobic ELPs being more resistant to disassembly upon isothermal dilution.ConclusionsTaken together, these results suggest marginally soluble ELPs may self-assemble but not disassemble as expected and that parameters including particle size, zeta potential and dilution resistance would benefit from widespread systematic evaluations. This information has the potential to reveal novel preparation methods capable of expanding the utility of all existing ELP-based biomaterials.
| S-EPMC5816514 | biostudies-literature
Project description:Elastin is a self-assembling protein of the extracellular matrix that provides tissues with elastic extensibility and recoil. The monomeric precursor, tropoelastin, is highly hydrophobic yet remains substantially disordered and flexible in solution, due in large part to a high combined threshold of proline and glycine residues within hydrophobic sequences. In fact, proline-poor elastin-like sequences are known to form amyloid-like fibrils, rich in β-structure, from solution. On this basis, it is clear that hydrophobic elastin sequences are in general optimized to avoid an amyloid fate. However, a small number of hydrophobic domains near the C terminus of tropoelastin are substantially depleted of proline residues. Here we investigated the specific contribution of proline number and spacing to the structure and self-assembly propensities of elastin-like polypeptides. Increasing the spacing between proline residues significantly decreased the ability of polypeptides to reversibly self-associate. Real-time imaging of the assembly process revealed the presence of smaller colloidal droplets that displayed enhanced propensity to cluster into dense networks. Structural characterization showed that these aggregates were enriched in β-structure but unable to bind thioflavin-T. These data strongly support a model where proline-poor regions of the elastin monomer provide a unique contribution to assembly and suggest a role for localized β-sheet in mediating self-assembly interactions.
| S-EPMC3000959 | biostudies-literature
Project description:The self-assembly of synthetic diblock copolymers has been extensively studied experimentally and theoretically. In contrast, self-assembly of polypeptide diblock copolymers has so far been mostly studied experimentally. We discovered that the theory developed for synthetic diblock copolymer does not fully explain the self-assembly of elastin-like polypeptide diblock copolymers, leading us to generalize the theory to make it applicable for these polypeptides. We demonstrated that elastin-like polypeptide diblocks self-assemble into weak micelles with dense cores and almost unstretched coronas, a state not previously observed for synthetic diblock copolymers. Weak micelles form if the surface tension at the core-corona interface is low compared to that expected of a micelle with a dense core. The predictions of the theory of weak micelles for the critical micelle temperature, hydrodynamic radius, and aggregation number of elastin-like polypeptide diblocks are in reasonable agreement with the experimentally measured values. The unique and unprecedented control of amphiphilicity in these recombinant peptide polymers reveals a new micellar state that has not been previously observed in synthetic diblock copolymer systems.
| S-EPMC4822422 | biostudies-literature
Project description:Control over the placement and activity of biomolecules on solid surfaces is a key challenge in bionanotechnology. While covalent approaches excel in performance, physical attachment approaches excel in ease of processing, which is equally important in many applications. We show how the precision of recombinant protein engineering can be harnessed to design and produce protein-based diblock polymers with a silica-binding and highly hydrophilic elastin-like domain that self-assembles on silica surfaces and nanoparticles to form stable polypeptide brushes that can be used as a scaffold for later biofunctionalization. From atomic force microscopy-based single-molecule force spectroscopy, we find that individual silica-binding peptides have high unbinding rates. Nevertheless, from quartz crystal microbalance measurements, we find that the self-assembled polypeptide brushes cannot easily be rinsed off. From atomic force microscopy imaging and bulk dynamic light scattering, we find that the binding to silica induces fibrillar self-assembly of the peptides. Hence, we conclude that the unexpected stability of these self-assembled polypeptide brushes is at least in part due to peptide-peptide interactions of the silica-binding blocks at the silica surface.
| S-EPMC8154268 | biostudies-literature
Project description:Elastin is a major structural component of elastic fibres that provide properties of stretch and recoil to tissues such as arteries, lung and skin. Remarkably, after initial deposition of elastin there is normally no subsequent turnover of this protein over the course of a lifetime. Consequently, elastic fibres must be extremely durable, able to withstand, for example in the human thoracic aorta, billions of cycles of stretch and recoil without mechanical failure. Major defects in the elastin gene (ELN) are associated with a number of disorders including Supravalvular aortic stenosis (SVAS), Williams-Beuren syndrome (WBS) and autosomal dominant cutis laxa (ADCL). Given the low turnover of elastin and the requirement for the long term durability of elastic fibres, we examined the possibility for more subtle polymorphisms in the human elastin gene to impact the assembly and long-term durability of the elastic matrix. Surveys of genetic variation resources identified 118 mutations in human ELN, 17 being non-synonymous. Introduction of two of these variants, G422S and K463R, in elastin-like polypeptides as well as full-length tropoelastin, resulted in changes in both their assembly and mechanical properties. Most notably G422S, which occurs in up to 40% of European populations, was found to enhance some elastomeric properties. These studies reveal that even apparently minor polymorphisms in human ELN can impact the assembly and mechanical properties of the elastic matrix, effects that over the course of a lifetime could result in altered susceptibility to cardiovascular disease.
| S-EPMC3458006 | biostudies-literature
Project description:The biofabrication of structural proteins with controllable properties via amino acid sequence design is interesting for biomedicine and biotechnology, yet a complete framework that connects amino acid sequence to material properties is unavailable, despite great progress to establish design rules for synthesizing peptides and proteins with specific conformations (e.g., unfolded, helical, β-sheets, or β-turns) and intermolecular interactions (e.g., amphipathic peptides or hydrophobic domains). Molecular dynamics (MD) simulations can help in developing such a framework, but the lack of a standardized way of interpreting the outcome of these simulations hinders their predictive value for the design of de novo structural proteins. To address this, we developed a model that unambiguously classifies a library of de novo elastin-like polypeptides (ELPs) with varying numbers and locations of hydrophobic/hydrophilic and physical/chemical-cross-linking blocks according to their thermoresponsiveness at physiological temperature. Our approach does not require long simulation times or advanced sampling methods. Instead, we apply (un)supervised data analysis methods to a data set of molecular properties from relatively short MD simulations (150 ns). We also experimentally investigate hydrogels of those ELPs from the library predicted to be thermoresponsive, revealing several handles to tune their mechanical and structural properties: chain hydrophilicity/hydrophobicity or block distribution control the viscoelasticity and thermoresponsiveness, whereas ELP concentration defines the network permeability. Our findings provide an avenue to accelerate the design of de novo ELPs with bespoke phase behavior and material properties.
| S-EPMC9832484 | biostudies-literature
Project description:Human granulocyte-macrophage colony-stimulating factor (hGMCSF) is crucial in the immune system as it stimulates survival, proliferation, differentiation, and functional activation of myeloid hematopoietic cells. hGMCSF is integral to approved therapies, including monoclonal antibodies against checkpoint inhibitors, chimeric antigen receptors, and prevention of chemotherapy-induced neutropenia. Recombinant hGMCSF can be purified from Escherichia. coli; however, it forms inclusion bodies that require solubilization and refolding. Alternatively, this manuscript describes its fusion with an elastin-like polypeptide (ELP). Previously reported as purification tags and solubility enhancers, ELPs are recombinant polypeptides that undergo reversible temperature-dependent phase separation. This report is the first to show that fusion to an ELP enables direct purification of hGMCSF fusions from the soluble fraction of bacterial lysate. Surprisingly, these ELP-fusions assemble stable, small, spherical nanoparticles that maintain pro-mitotic activity of hGMCSF. These nanoparticles exhibit ELP-mediated phase separation; however, nanoparticle assembly significantly increases the entropic and enthalpic cost of phase separation compared to ELP alone. The attachment of a high molecular weight ELP to a difficult-to-express protein, like hGMCSF, appears to be a useful strategy to stabilize bioactive, protein-based nanoparticles, which may have broad applications in medicine and biology.
| S-EPMC7489497 | biostudies-literature
Project description:Silk-elastin-like protein polymers (SELPs) combine the mechanical and biological properties of silk and elastin. These properties have led to the development of various SELP-based materials for drug delivery. However, SELPs have rarely been developed into nanoparticles, partially due to the complicated fabrication procedures, nor assessed for potential as an anticancer drug delivery system. We have recently constructed a series of SELPs (SE8Y, S2E8Y, and S4E8Y) with various ratios of silk to elastin blocks and described their capacity to form micellar-like nanoparticles upon thermal triggering. In this study, we demonstrate that doxorubicin, a hydrophobic antitumor drug, can efficiently trigger the self-assembly of SE8Y (SELPs with silk to elastin ratio of 1:8) into uniform micellar-like nanoparticles. The drug can be loaded in the SE8Y nanoparticles with an efficiency around 6.5% (65 ng doxorubicin/μg SE8Y), S2E8Y with 6%, and S4E8Y with 4%, respectively. In vitro studies with HeLa cell lines demonstrate that the protein polymers are not cytotoxic (IC50 > 200 μg/mL), while the doxorubicin-loaded SE8Y nanoparticles showed a 1.8-fold higher cytotoxicity than the free drug. Confocal laser scanning microscopy (CLSM) and flow cytometry indicate significant uptake of the SE8Y nanoparticles by the cells and suggest internalization of the nanoparticles through endocytosis. This study provides an all-aqueous, facile method to prepare nanoscale, drug-loaded SELPs packages with potential for tumor cell treatments.
| S-EPMC3983132 | biostudies-other
Project description:BackgroundSafe and effective hemostatic materials are important for reducing mortality resulting from excessive hemorrhage. In this work, new biomaterials with hemostatic effects were created by fusing the gene coding for RADA-16, a self-assembling peptide with the sequence RADARADARADARADA, to the 3'-end of the open reading frame (ORF) encoding elastin-like polypeptides through gene recombination.ResultsThe fusion proteins, termed 36R, 60R and 96R, were solubly over-expressed in Escherichia coli BL21 (DE3) based on genetic manipulation of the high-efficiency prokaryotic expression vector pET28a (+) and bacterial transformation. Western Blot analysis showed that the over-expressed proteins were the target fusion proteins. The target proteins 36R with 94.72% purity, 60R with 96.91% purity and 96R with 96.37% purity were prepared using an inverse phase transition cycle at 65 °C followed by His-tag affinity chromatography. The proliferation results of the mouse fibroblast cell line L929 and hippocampus neuron cell line HT22 indicated that the fusion proteins did not cause obvious cell toxicity. The lyophilized spongy film of the purified 36R, 60R and 96R could stop the hemorrhage of a 2?×?2 mm bleeding wound in the mouse liver after 27.21?±?1.92 s, 18.65?±?1.97 s and 15.85?±?1.21 s, respectively. The hemostasis time was 21.23?±?1.84 s for rat-tail collagen and 14.44?±?1.33 s for RADA-16 lyophilized on gauze. The hemostatic time of three treated groups were all significantly superior to that of the negative control without any hemostasis treatment, which spontaneously stopped bleeding after 37.64?±?1.34 s. Statistical analysis showed that the spongy film with purified 96R exhibited an exciting hemostatic effect that was superior to rat-tail collagen and close to that of RADA-16 lyophilized on gauze.ConclusionsThese results revealed that the fusion proteins achieved by gene recombination technology could serve as a promising hemostatic material.
| S-EPMC5842521 | biostudies-literature
Project description:Recently, catalytic peptides were introduced that mimicked protease activities and showed promising selectivity of products even in organic solvents where protease cannot perform well. However, their catalytic efficiency was extremely low compared to natural enzyme counterparts presumably due to the lack of stable tertiary fold. We hypothesized that assembling these peptides along with simple hydrophobic pockets, mimicking enzyme active sites, could enhance the catalytic activity. Here we fused the sequence of catalytic peptide CP4, capable of protease and esterase-like activities, into a short amyloidogenic peptide fragment of Aβ. When the fused CP4-Aβ construct assembled into antiparallel β-sheets and amyloid fibrils, a 4.0-fold increase in the hydrolysis rate of p-nitrophenyl acetate (p-NPA) compared to neat CP4 peptide was observed. The enhanced catalytic activity of CP4-Aβ assembly could be explained both by pre-organization of a catalytically competent Ser-His-acid triad and hydrophobic stabilization of a bound substrate between the triad and p-NPA, indicating that a design strategy for self-assembled peptides is important to accomplish the desired functionality.
| S-EPMC4846159 | biostudies-literature