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Rationale: Elastin-like polypeptides (ELPs) are elastic and thermoresponsive biopolymers composed of VPGXG repeats (X can be any amino acid except proline), used in biomedical applications, for example, tissue engineering and drug delivery. As different variants of ELP are mostly produced fermentatively, there is a need for the development of analysis methods that allow for absolute protein quantification in both complex matrices and purified samples and MW determination of the final products., Methods: ELPs were intracellularly expressed in Escherichia coli quantified after cell lysis and enzymatic digestion using a proline-specific protease ProAlanase (Promega) at acidic conditions. Resulting peptides were separated by liquid chromatography, and mass spectrometry analysis was conducted by electrospray ionization high-resolution mass spectrometry using an Orbitrap mass spectrometer. The addition of a stable isotopically labeled internal standard enabled quantification in complex matrices. Prior to intact mass analysis, ELPs were purified from fermentation broth by inverse temperature cycling. Intact protein analysis was performed using reversed-phase liquid chromatography, and mass spectrometry analysis was conducted by electrospray ionization high-resolution mass spectrometry using a time-of-flight mass spectrometer., Results: Absolute quantification of ELPs was achieved by utilizing ELP-specific properties, that is, proline-rich, soluble at low pH and low temperature. The repetitive nature of ELPs allows for sensitivity increase and use of higher dilution factors to minimize the matrix effects. Despite the lack of amino acids with charged side chains (Arg, His, Lys, Asp, and Glu) in ELP, we demonstrated successful intact protein analysis using reversed-phase LC coupled to electrospray ionization TOF MS. Moreover, truncated protein forms could be chromatographically separated and characterized as well as N-terminal modifications., Conclusions: Both methods combined enabled quantitative and qualitative characterization of fermentatively produced ELPs., (© 2024 John Wiley & Sons Ltd.)

There is the rapid growth in application of Brillouin scattering spectroscopy to biomedical objects in order to characterize their mechanoelastic properties in this way. However, the possibilities and limitations of the method when applied to tissues have not yet been clarified. Here, applicability of Brillouin spectroscopy for testing the elastic response of medically relevant tissues of bovine jugular vein and pericardium was considered. Parameters of the Brillouin peak were studied for samples untreated, diepoxide-fixed, and preserved after treatment in alcohol solutions. It was found that diepoxide cross-linking resulted to a slight tendency to increase the Brillouin position for hydrated tissues. The variations in the position and width of the Brillouin peaks, associated with local fluctuations in water concentration, were reduced after diepoxide treatment in the case of the pericardium, but not in the case of the vein wall. To obtain more information about the elastic response of the protein scaffold without the participation of water, dried samples were also studied. Brillouin spectra of the dried pericardium and vein wall revealed a significant increase in the Brillouin peak position (elastic modulus) after conservation in alcohol. In the case of the vein wall, this effect was found for both collagen and elastin-related peaks, which were identified in the Brillouin spectrum. This result corresponds to a denser packing of fibrous proteins after preservation in alcohol solutions. The ability of Brillouin spectroscopy to independently characterize the effect of treatment on the instantaneous elastic modulus of various tissue components is also attractive for its application in the development of new materials for bioimplants. A comparison of the Brillouin longitudinal and Young's elastic moduli determined for the hydrated samples of the vein and pericardium showed that there is no clear correspondence between these material parameters. The usefulness of using both experimental methods to obtain new information about the elastic response of the material is discussed., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Elastin-like peptides (ELPs) exhibit lower critical solution temperature (LCST)-type behavior, being soluble at low temperatures and insoluble at high temperatures. While the properties of linear, long-chain ELPs are well-studied, short-chain ELPs, especially those with branched architectures, have been less explored. Herein, to obtain further insights into multimeric short ELPs, we investigated the temperature-responsive properties of branched molecules composed of a repeating pentapeptide unit of short ELPs, Phe-Pro-Gly-Val-Gly, as side components and oligo(Glu) as a backbone structure. In turbidimetry experiments, the branched ELPs showed LCST-like behavior similar to conventional ELPs and upper critical solution temperature (UCST)-like behavior, which are rarely observed in ELPs. In addition, the morphological aspects and mechanisms underlying the temperature-responsiveness were investigated. We observed that spherical aggregates formed, and the branched ELPs underwent structural changes through the self-assembly process. This study demonstrates the unique temperature-responsiveness of branched short ELPs, providing new insights into the future development and use of ELPs with tailored properties.

A column-free immunoaffinity purification (CFIP) technique for sample preparation of aflatoxin B 1 (AFB 1 ) was developed using an AFB 1 -specific nanobody (named G8) and an elastin-like polypeptide (ELP). The reversible phase transition between liquid and solid in response to temperature changes was exhibited by the ELP which was derived from human elastin. The G8 was tagged with ELPs of various lengths (20, 40, 60, and 80 repeat units) at the C-terminus using recursive directional ligation (RDL). Coding sequences were then subcloned into pET30a at the multiple cloning sites. Bioactive recombinant proteins were produced by expressing them as inclusion bodies in Escherichia coli BL21 (DE3), then dissolved and refolded. Analysis by indirect competitive enzyme-linked immunosorbent assay (icELISA) and transition temperature (T t ) measurement confirmed that the refolded G8-ELPs preserved the ability to recognize AFB 1 as well as phase transition when the temperature rose above T t . To establish the optimal conditions for cleaning AFB 1 , the effects of various parameters on recovery were investigated. The recovery in ELISA tests was 95 ± 3.67% under the optimized CFIP workflow. Furthermore, the CFIP-prepared samples were applied for high-performance liquid chromatography (HPLC) detection. The recovery in the CFIP-HPLC test ranged from 54 ± 1.86% to 98 ± 3.58% for maize, rice, soy sauce, and vegetable oil samples. To the best of our knowledge, this is the first report combining the function of both nanobody and ELP to develop a cleanup technique for small molecules in a complex matrix. The CFIP for the sample pretreatment was easy to use and inexpensive. In contrast to conventional immunosensitivity materials, the reagent utilized in the CFIP was entirely biosynthesized without any chemical coupling reactions. This suggests that the nanobody-ELP may serve as a useful dual-functional reagent for the development of sample cleaning or purification methods., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

The proliferation of nano-plastic particles (NPs) poses severe environmental hazards, urgently requiring effective biodegradation methods. Herein, a novel method was developed for degrading nano-PET (polyethylene terephthalate) using immobilized cutinases. Nano-PET particles were prepared using a straightforward method, and biocompatible elastin-like polypeptide-magnetic nanoparticles (ELPs-MNPs) were obtained as magnetic cores via biomimetic mineralization. Using one-pot synthesis with the cost-effective precursor tetraethoxysilane (TEOS), silica-coated magnetically immobilized ELPs-tagged cutinase (ET-C@SiO 2 @MNPs) were produced. ET-C@SiO 2 @MNPs showed rapid magnetic separation within 30 s, simplifying recovery and reuse. ET-C@SiO 2 @MNPs retained 86 % of their initial activity after 11 cycles and exhibited superior hydrolytic capabilities for nano-PET, producing 0.515 mM TPA after 2 h of hydrolysis, which was 96.6 % that of free enzymes. Leveraging ELPs biomimetic mineralization, this approach offers a sustainable and eco-friendly solution for PET-nanoplastic degradation, highlighting the potential of ET-C@SiO 2 @MNPs in effective nanoplastic waste management and contributing to environmental protection and sustainable development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

It appeared certain that elastin condensates retain liquid-like properties. However, a recent experimental study demonstrated that their aggregate states might depend on the length of hydrophobic domains. To gain microscopic insight into this behavior, we employ atomistic modeling to assess the conformational properties of hydrophobic elastin-like polypeptides (ELPs). We find that short ELPs always remain in coil-like conformations, while the longer ones prefer globule states. While the former engages in intrapeptide hydrogen bonds temporarily, retaining their liquid-like properties, the latter forms hundreds of nanosecond-long intrapeptide hydrogen bonds attributed to ordered secondary structure motifs. Our work demonstrates that the sequence length modulates the material properties of elastin condensates.

Recombinant elastin-like polypeptides (ELPs) have emerged as an attractive nanoplatform for drug delivery due to their tunable genetically encoded sequence, biocompatibility, and stimuli-responsive self-assembly behaviors. Here, we designed and biosynthesized an HER2 (human epidermal growth factor receptor 2)-targeted affibody-ELP fusion protein (Z-ELP), which was subsequently conjugated with monomethyl auristatin E (MMAE) to build a protein-drug conjugate (Z-ELP-M). Due to its thermal response, Z-ELP-M can immediately self-assemble into a nanomicelle at physiological temperature. Benefiting from its active targeting and nanomorphology, Z-ELP-M exhibits enhanced cellular internalization and deep tumor penetration in vitro . Moreover, Z-ELP-M shows excellent tumor targeting and superior antitumor efficacy in HER2-positive ovarian cancer, demonstrating a relative tumor growth inhibition of 104.6%. These findings suggest that an affibody-functionalized elastin-like peptide-drug conjugate nanomicelle is an efficient strategy to improve antitumor efficacy and biosafety in cancer therapy.

Pathological calcification of elastin, a key connective tissue protein in the medial layers of blood vessels, starts with the binding of calcium ions. This Mini-Review focuses on understanding how calcium ions interact with elastin to initiate calcification at a molecular level, and emphasizes water's critical role in mediating this interaction. In the past decade, great strides have been made in understanding and modeling ion-specific hydration and its effects on biomolecule interactions. However, these advances have been largely absent from our understanding of elastin calcification. Historically, charge-neutral backbone carbonyls and negatively charged carboxyl groups have been proposed as elastin's calcium binding sites. Recently, tropoelastin's only four carboxyl groups have been identified as binding sites from classical molecular dynamics (MD). While carboxyl groups have a much higher affinity for binding calcium ions than backbone carbonyls, conflicting evidence persists for both functional group's importance in elastin calcification. This can be attributed to the fact that divalent ions strongly polarize water, leading to a hydration shell that shields electrostatic forces. The hydration shell surrounding both a calcium ion and either of the proposed binding sites must be displaced to enable binding. Providing our own extended X-ray absorption fine structure (EXAFS) data and complementary simulations, we discuss the potential structures of calcium binding in elastin and review prior knowledge regarding the relative importance of the two proposed binding sites.

Natural evolution endows some insects and marine organisms with a special class of protein-based elastic tissues that possess energy feedback characteristics, providing them with the foundation for jumping and flying, and protecting them from the damage caused by movements or waves. However, the design and fabrication of such protein-based elastomeric materials that can function in human society through biomimetic strategies still remains challenging. Recombinant proteins designed by synthetic biology can mimic the advantageous structures in natural proteins and can be biosynthesized without the requirements for harsh conditions such as high temperatures and cytotoxic agents, which provides a great opportunity to prepare protein-based elastomeric materials. In this review, starting from the design of protein molecules, we highlight an overview of the synthesis of elastomeric materials based on recombinant resilin, recombinant elastin-like proteins and other recombinant folded proteins, etc. , and then demonstrate their application progress in the fields of biomedicine and high technology. Finally, the challenges and prospects for the future development of protein-based elastomeric materials are envisioned to provide insights into the design and synthesis of the next generation of protein-based elastomeric materials.

Elastin, a fibrous extracellular matrix (ECM) protein, is the main component of elastic fibers that are involved in tissues' elasticity and resilience, enabling them to undergo reversible extensibility and to endure repetitive mechanical stress. After wounding, it is challenging to regenerate elastic fibers and biomaterials developed thus far have struggled to induce its biosynthesis. This review provides a comprehensive summary of elastic fibers synthesis at the cellular level and its implications for biomaterial formulation, with a particular focus on dermal substitutes. The review delves into the intricate process of elastogenesis by cells and investigates potential triggers for elastogenesis encompassing elastin-related compounds, ECM components, and other molecules for their potential role in inducing elastin formation. Understanding of the elastogenic processes is essential for developing biomaterials that trigger not only the synthesis of the elastin protein, but also the formation of a functional and branched elastic fiber network., (© 2024 The Author(s). Advanced Healthcare Materials published by Wiley‐VCH GmbH.)

Protein hydrogels with tailored stimuli-responsive features and tunable stiffness have garnered considerable attention due to the growing demand for biomedical soft robotics. However, integrating multiple responsive features toward intelligent yet biocompatible actuators remains challenging. Here, a facile approach that synergistically combines genetic and chemical engineering for the design of protein hydrogel actuators with programmable complex spatial deformation is reported. Genetically engineered silk-elastin-like proteins (SELPs) are encoded with stimuli-responsive motifs and enzymatic crosslinking sites via simulation-guided genetic engineering strategies. Chemical modifications of the recombinant proteins are also used as secondary control points to tailor material properties, responsive features, and anisotropy in SELP hydrogels. As a proof-of-concept example, diazonium coupling chemistry is exploited to incorporate sulfanilic acid groups onto the tyrosine residues in the elastin domains of SELPs to achieve patterned SELP hydrogels. These hydrogels can be programmed to perform various actuations, including controllable bending, buckling, and complex deformation under external stimuli, such as temperature, ionic strength, or pH. With the inspiration of genetic and chemical engineering in natural organisms, this work offers a predictable, tunable, and environmentally sustainable approach for the fabrication of programmed intelligent soft actuators, with implications for a variety of biomedical materials and biorobotics needs., (© 2024 Wiley‐VCH GmbH.)

Plants are often seen as a potent tool in the recombinant protein production industry. However, unlike bacterial expression, it is not a popular method due to the low yield and difficulty of protein extraction and purification. Therefore, developing a new high efficient and easy to purify platform is crucial. One of the best approaches to make extraction easier is to utilize the Extensin Signal peptide (EXT) to translocate the recombinant protein to the outside of the cell, along with incorporating an Elastin-like polypeptide tag (ELP) to enhance purification and accumulation rates. In this research, we transiently expressed Shigella dysenteriae's IpaDSTxB fused to both NtEXT and ELP in both Nicotiana tabacum and Medicago sativa. Our results demonstrated that N. tabacum, with an average yield of 6.39 ng/μg TSP, outperforms M. sativa, which had an average yield of 3.58 ng/μg TSP. On the other hand, analyzing NtEXT signal peptide indicated that merging EXT to the constructs facilitates translocation of IpaDSTxB to the apoplast by 78.4% and 65.9% in N. tabacum and M. sativa, respectively. Conversely, the mean level for constructs without EXT was below 25% for both plants. Furthermore, investigation into the orientation of ELP showed that merging it to the C-terminal of IpaDSTxB leads to a higher accumulation rate in both N. tabacum and M. sativa by 1.39 and 1.28 times, respectively. It also facilitates purification rate by over 70% in comparison to 20% of the 6His tag. The results show a highly efficient and easy to purify platform for the expression of heterologous proteins in plant., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Protein and peptide materials have attracted great interest in recent years, especially for biological applications, in light of their possibility to easily encode bioactivity whilst maintaining cytocompatibility and biodegradability. Heterologous recombinant expression to produce antimicrobial peptides is increasingly considered a convenient alternative for the transition from conventional methods to more sustainable production systems. The human elastin-like polypeptide (HELP) has proven to be a valuable fusion carrier, and due to its cutting-edge properties, biomimetic materials with antimicrobial capacity have been successfully developed. In this work, we have taken advantage of this platform to produce a difficult-to-synthesise sequence as that of the human β-defensin 1 (hBD1), an amphipathic cationic peptide with structural folding constraints relevant to its bioactivity. In the design of the gene, highly specific endoproteinases recognition sites were introduced to release the active forms of hBD1. After the expression and purification of the new fusion construct, its biological activity was evaluated. It was found that both the fusion biopolymer and the released active forms can inhibit the growth of Escherichia coli in redox environments. Remarkably, 2D and 3D materials derived from the biopolymer showed a strong cell adhesion-promoting activity. These results suggest that HELP represents a multitasking platform that not only facilitates the production of bioactive domains and derived materials but could also pave the way for the development of new approaches to study biological interactions at the molecular level.

Nucleic acid (NA)-based therapies are revolutionizing biomedical research through their ability to control cellular functions at the genetic level. This work demonstrates a versatile elastin-like polypeptide (ELP) carrier system using a layer-by-layer (LbL) formulation approach that delivers NA cargos ranging in size from siRNA to plasmids. The components of the system can be reconfigured to modulate the biochemical and biophysical characteristics of the carrier for engaging the unique features of the biological target. We show the physical characterization and biological performance of L bL E LP n ucleic acid n anoparticles (LENNs) in murine and human bladder tumor cell lines. Targeting bladder tumors is difficult owing to the constant influx of urine into the bladder, leading to low contact times (typically <2 h) for therapeutic agents delivered via intravesical instillation. LENN complexes bind to bladder tumor cells within 30 min and become rapidly internalized to release their NA cargo within 60 min. Our data show that a readily adaptable NA-delivery system has been created that is flexible in its targeting ability, cargo size, and disassembly kinetics. This approach provides an alternative path to either lipid nanoparticle formulations that suffer from inefficiency and physicochemical instability or viral vectors that are plagued by manufacturing and immune rejection challenges. This agile ELP-based nanocarrier provides an alternative route for nucleic acid delivery using a biomanufacturable, biodegradable, biocompatible, and highly tunable vehicle capable of targeting cells via engagement with overexpressed cell surface receptors.

We present a straightforward strategy for constructing giant, multicompartmentalized vesicles using recombinant fusion proteins. Our method leverages the self-assembly of globule-zipper-elastin-like polypeptide fusion protein complexes in aqueous conditions, eliminating the need for organic solvents and chemical conjugation. By employing the thin-film rehydration method, we have successfully encapsulated a diverse range of bioactive macromolecules and engineered organelle-like compartments─ranging from soluble proteins and coacervate droplets to vesicles─within these protein-assembled giant vesicles. This approach also facilitates the integration of water-soluble block copolymers, enhancing the structural stability and functional versatility of the vesicles. Our results suggest that these multicompartment giant protein vesicles not only mimic the complex architecture of living cells but also support biochemically distinct reactions regulated by functionally folded proteins, providing a robust model for studying cellular processes and designing microreactor systems. This work highlights the transformative potential of self-assembling recombinant fusion proteins in artificial cell design.

A generic model of elastin-like polypeptides (ELP) is derived that includes proline isomerization (ProI). As a case study, conformational transition of a -[valine-proline-glycine-valine-glycine]- sequence is investigated in aqueous ethanol mixtures. While the non-bonded interactions are based on the Lennard-Jones (LJ) parameters, the effect of ProI is incorporated by tuning the intramolecular 3- and 4-body interactions known from the underlying all-atom simulations into the generic model. One of the key advantages of such a minimalistic model is that it readily decouples the effects of geometry and the monomer-solvent interactions due to the presence of ProI, thus gives a clearer microscopic picture that is otherwise rather nontrivial within the all-atom setups. These results are consistent with the available all-atom and experimental data. The model derived here may pave the way to investigate large scale self-assembly of ELPs or biomimetic polymers in general., (© 2024 The Author(s). Macromolecular Rapid Communications published by Wiley‐VCH GmbH.)

Metastasis stands as the primary contributor to mortality associated with tumors. Chemotherapy and immunotherapy are frequently utilized in the management of metastatic solid tumors. Nevertheless, these therapeutic modalities are linked to serious adverse effects and limited effectiveness in preventing metastasis. Here, we report a novel therapeutic strategy named starvation-immunotherapy, wherein an immune checkpoint inhibitor is combined with an ultra-long-acting L-asparaginase that is a fusion protein comprising L-asparaginase (ASNase) and an elastin-like polypeptide (ELP), termed ASNase-ELP. ASNase-ELP's thermosensitivity enables it to generate an in-situ depot following an intratumoral injection, yielding increased dose tolerance, improved pharmacokinetics, sustained release, optimized biodistribution, and augmented tumor retention compared to free ASNase. As a result, in murine models of oral cancer, melanoma, and cervical cancer, the antitumor efficacy of ASNase-ELP by selectively and sustainably depleting L-asparagine essential for tumor cell survival was substantially superior to that of ASNase or Cisplatin, a first-line anti-solid tumor medicine, without any observable adverse effects. Furthermore, the combination of ASNase-ELP and an immune checkpoint inhibitor was more effective than either therapy alone in impeding melanoma metastasis. Overall, the synergistic strategy of starvation-immunotherapy holds excellent promise in reshaping the therapeutic landscape of refractory metastatic tumors and offering a new alternative for next-generation oncology treatments., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Affinity precipitation is a powerful separation method in that it combines the binding selectivity of affinity chromatography with precipitation of captured biomolecules via phase separation triggered by small changes in the environment, e.g., pH, ionic strength, temperature, light, etc. Elastin-like polypeptides (ELPs) are thermally responsive biopolymers composed of pentapeptide repeats VPGVG that undergo reversible phase separation, where they aggregate when temperature and/or salt concentration are increased. Here we describe the generation of an ELP fusion to a soluble streptavidin mutant that enables rapid purification of any Strep-tag II fusion protein of interest. This heterobifunctional protein takes advantage of the native tetrameric structure of streptavidin, leading to binding-induced multivalent crosslinking upon protein capture. The efficient biotin-mediated dissociation of the bound Strep-tag II fusion protein from the streptavidin-ELP capturing scaffold allows for mild elution conditions. We also show that this platform is particularly effective in the purification of a virus-like particle (VLP)-like E2 protein nanoparticle, likely because the high valency of the protein particle causes binding-induced crosslinking and precipitation. Considering the importance of VLP for gene therapy applications, we believe this is a particularly exciting advance. We demonstrated this feasibility by the efficient purification of a VLP-like E2 protein nanoparticle as a surrogate., (© 2024 Wiley Periodicals LLC.)

Current biological research requires simple protein bioseparation methods capable of purifying target proteins in a single step with high yields and purities. Conventional affinity tag-based approaches require specific affinity resins and expensive proteolytic enzymes for tag removal. Purification strategies based on self-cleaving aggregating tags have been previously developed to address these problems. However, these methods often utilize C-terminal cleaving contiguous inteins which suffer from premature cleavage, resulting in significant product loss during protein expression. In this work, we evaluate two novel mutants of the Mtu RecA ΔI-CM mini-intein obtained through yeast surface display for improved protein purification. When used with the elastin-like-polypeptide (ELP) precipitation tag, the novel mutants - ΔI-12 and ΔI-29 resulted in significantly higher precursor content, product purity and process yield compared to the original Mtu RecA ΔI-CM mini-intein. Product purities ranging from 68 % to 94 % were obtained in a single step for three model proteins - green fluorescent protein (GFP), maltose binding protein (MBP) and beta-galactosidase (beta-gal). Further, high cleaving efficiency was achieved after 5 h under most conditions. Overall, we have developed improved self-cleaving precipitation tags which can be used for purifying a wide range of proteins cheaply at laboratory scale., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Large bone defects are a significant health problem today with various origins, including extensive trauma, tumours, or congenital musculoskeletal disorders. Tissue engineering, and in particular bone tissue engineering, aims to respond to this demand. As such, we propose a specific model based on Elastin-Like Recombinamers-based click-chemistry hydrogels given their high biocompatibility and their potent on bone regeneration effect conferred by different bioactive sequences. In this work we demonstrate, using biochemistry, histology, histomorphometry and imaging techniques, the biocompatibility of our matrix and its potent effect on bone regeneration in a model of bone parietal lesion in female New Zealand rabbits., (© 2024. The Author(s).)

Elastin-like polypeptides (ELPs) are a promising material platform for engineering stimuli-responsive biomaterials, as ELPs undergo phase separation above a tunable transition temperature. ELPs with phase behavior that is isothermally regulated by biological stimuli remain attractive for applications in biological systems. Herein, we report protease-driven phase separation of ELPs. Protease-responsive "cleavable" ELPs comprise a hydrophobic ELP block connected to a hydrophilic ELP block by a protease cleavage site linker. The hydrophilic ELP block acts as a solubility tag for the hydrophobic ELP block, creating a temperature window in which the cleavable ELP reactant is soluble and the proteolytically generated hydrophobic ELP block is insoluble. Within this temperature window, isothermal, protease-driven phase separation occurs when a critical concentration of hydrophobic cleavage product accumulates. Furthermore, protease-driven phase separation is generalizable to four compatible protease-cleavable ELP pairings. This work presents exciting opportunities to regulate ELP phase behavior in biological systems using proteases.

Confronted with the profound threat of cardiovascular diseases to health, vascular tissue engineering presents potential beyond the limitations of autologous and allogeneic grafts, offering a promising solution. This study undertakes an initial exploration into the impact of a natural active protein, elastin, on vascular cell behavior, by incorporating with polycaprolactone to prepare fibrous tissue engineering scaffold. The results reveal that elastin serves to foster endothelial cell adhesion and proliferation, suppress smooth muscle cell proliferation, and induce macrophage polarization. Furthermore, the incorporation of elastin contributes to heightened scaffold strength, compliance, and elongation, concomitantly lowering the elastic modulus. Subsequently, a bilayer oriented polycaprolactone (PCL) scaffold infused with elastin is proposed. This design draws inspiration from the cellular arrangement of native blood vessels, leveraging oriented fibers to guide cell orientation. The resulting fiber scaffold exhibits commendable mechanical properties and cell infiltration capacity, imparting valuable insights for the rapid endothelialization of vascular scaffolds., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Due to the limitations of the current skin wound treatments, it is highly valuable to have a wound healing formulation that mimics the extracellular matrix (ECM) and mechanical properties of natural skin tissue. Here, a novel biomimetic hydrogel formulation has been developed based on a mixture of Agarose-Collagen Type I (AC) combined with skin ECM-related components: Dermatan sulfate (DS), Hyaluronic acid (HA), and Elastin (EL) for its application in skin tissue engineering (TE). Different formulations were designed by combining AC hydrogels with DS, HA, and EL. Cell viability, hemocompatibility, physicochemical, mechanical, and wound healing properties were investigated. Finally, a bilayered hydrogel loaded with fibroblasts and mesenchymal stromal cells was developed using the Ag-Col I-DS-HA-EL (ACDHE) formulation. The ACDHE hydrogel displayed the best in vitro results and acceptable physicochemical properties. Also, it behaved mechanically close to human native skin and exhibited good cytocompatibility. Environmental scanning electron microscopy (ESEM) analysis revealed a porous microstructure that allows the maintenance of cell growth and ECM-like structure production. These findings demonstrate the potential of the ACDHE hydrogel formulation for applications such as an injectable hydrogel or a bioink to create cell-laden structures for skin TE., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Patricia Galvez-Martin reports a relationship with Bioibérica SA that includes: employment. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

The objective for this study is to advance the development of a specialized biomaterial that can effectively facilitate the regeneration of adipose tissue. In prior studies, the assessment of collagen (Col), elastin (Ela), and fibrin (Fib) unary scaffolds has been conducted. However, it is important to note that native adipose tissue is comprised of a diverse array of extracellular matrix (ECM) constituents. To mimic this behavior, binary compositions of collagen, elastin, and fibrin are fabricated in a 1:1 ratio, resulting in the formation of Col/Ela, Col/Fib, and Ela/Fib composites through a customized fabrication procedure. The physical properties of these scaffolds are comprehensively analyzed using a range of material characterization techniques. Additionally, the biological properties of the scaffolds are investigated by examining the survival, proliferation, and phenotype of adipose-derived stem cells. Subsequently, the aforementioned binary scaffolds are implanted into a rodent model for 28 days. the explants are analysed through X-ray microtomography, histology, and immunohistochemistry. The findings of the study demonstrate that the utilization of binary combinations of Col/Ela, Col/Fib, and Ela/Fib has a discernible impact on the physical and biological characteristics of the scaffolds. Nevertheless, Ela/Fib exhibits characteristics that make it a suitable candidate for adipogenesis due to its notable upregulation of caveolin-1 expression in both acellular and cellular cohorts. The combination of two natural polymers in this cell-material interaction has significantly enhanced the comprehension of adipogenesis., (© 2024 The Author(s). Macromolecular Bioscience published by Wiley‐VCH GmbH.)

While it is known that calcium phosphate (CaP) minerals deposit in elastin-rich medial layers of arteries during medial calcification, their nucleation and growth sites are still debated. Neutral carbonyl groups and carboxylate groups are possible candidates. Also, while it is known that elastin degradation leads to calcification, it is unclear whether this is due to formation of new carboxylate groups or elastin fragmentation. In this work, we disentangle effects of carboxylate groups and particle size on elastin calcification; in doing so, we shed light on CaP mineralization sites on elastin. We find carboxylate groups accelerate calcification only in early stages; they mainly function as Ca 2+ ion chelation sites but not calcification sites. Their presence promotes formation (likely on Ca 2+ ions adsorbed on nearby carbonyl groups) of CaP minerals with high calcium-to-phosphate ratio as intermediate phases. Larger elastin particles calcify slower but reach similar amounts of CaP minerals in late stages; they promote direct formation of hydroxyapatite and CaP minerals with low calcium-to-phosphate ratio as intermediate phases. This work provides new perspectives on how carboxylate groups and elastin particle size influence calcification; these parameters can be tuned to study the mechanism of medial calcification and design drugs to inhibit the process., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

There is an emerging strong demand for smart environmentally responsive protein-based biomaterials with improved adhesion properties, especially underwater adhesion for potential environmental and medical applications. Based on the fusion of elastin-like polypeptides (ELPs), SpyCatcher and SpyTag modules, biosynthetic barnacle-derived protein was genetically engineered and self-assembled with an enhanced adhesion ability and temperature response. The water resistance ability of the synthetic protein biopolymer with a network structure increased to 98.8 from 58.5% of the original Cp19k, and the nonaqueous adhesion strength enhanced to 1.26 from 0.68 MPa of Cp19k. The biopolymer showed an improved adhesion ability toward hydrophilic and hydrophobic surfaces as well as diatomite powders. The combination of functional module ELPs and SpyTag/SpyCatcher could endow the biosynthetic protein with temperature response, an insoluble form above 42 °C and a soluble form at 4 °C. The combinational advantages including temperature response and adhesion performance make the self-assembled protein an excellent candidate in surgical adhesion, underwater repair, and surface modification of various coatings. Distinct from the traditional approach of utilizing solely ELPs, the integration of short ELPs with Spy partners exhibited a synergistic enhancement in the temperature response. The synergistic effects of two functional modules provide a technical method and insight for designing smart self-assembled protein-based biopolymers.

A major component of the extracellular matrix (ECM), laminins, modulates cells via diverse receptors. Their fragments have emerging utility as components of "ECM-mimetics" optimized to promote cell-based therapies. Recently, we reported that a bioactive laminin peptide known as A99 enhanced cell binding and spreading via fusion to an elastin-like polypeptide (ELP). The ELP "handle" serves as a rapid, noncovalent strategy to concentrate bioactive peptide mixtures onto a surface. We now report that this strategy can be further generalized across an expanded panel of additional laminin-derived elastin-like polypeptides (LELPs). A99 (AGTFALRGDNPQG), A2G80 (VQLRNGFPYFSY), AG73 (RKRLQVQLSIRT), and EF1m (LQLQEGRLHFMFD) all promote cell spreading while showing morphologically distinct F-actin formation. Equimolar mixtures of A99:A2G80-LELPs have synergistic effects on adhesion and spreading. Finally, three of these ECM-mimetics promote the neurite outgrowth of PC-12 cells. The evidence presented here demonstrates the potential of ELPs to deposit ECM-mimetics with applications in regenerative medicine, cell therapy, and tissue engineering.

Despite the current high interest, there is limited information on diffusion data for intrinsically disordered proteins (IDPs). This study investigates the effect of crowding on the diffusion behaviour of an elastin-like peptide (ELP), by combined pulse field gradient (PFG) and static field gradient (SFG) NMR techniques. We interpret our findings in terms of highly dynamic chain assemblies with weak interactions, resulting in ELP diffusion that is primarily governed by the viscous flow of the solvent. The diffusion behaviour of the peptide appears to resemble that of globular proteins rather than flexible linear polymers over a wide concentration range., (© 2024 The Authors. ChemPhysChem published by Wiley-VCH GmbH.)

Nanogels offer hope for precise drug delivery, while addressing drug delivery hurdles is vital for effective prostate cancer (PCa) management. We developed an injectable elastin nanogels (ENG) for efficient drug delivery system to overcome castration-resistant prostate cancer (CRPC) by delivering Decursin, a small molecule inhibitor that blocks Wnt/βcatenin pathways for PCa. The ENG exhibited favourable characteristics such as biocompatibility, flexibility, and low toxicity. In this study, size, shape, surface charge, chemical composition, thermal stability, and other properties of ENG were used to confirm the successful synthesis and incorporation of Decursin (DEC) into elastin nanogels (ENG) for prostate cancer therapy. In vitro studies demonstrated sustained release of DEC from the ENG over 120 h, with a pH-dependent release pattern. DU145 cell line induces moderate cytotoxicity of DEC-ENG indicates that nanomedicine has an impact on cell viability and helps strike a balance between therapeutics efficacy and safety while the EPR effect enables targeted drug delivery to prostate tumor sites compared to free DEC. Morphological analysis further supported the effectiveness of DEC-ENG in inducing cell death. Overall, these findings highlight the promising role of ENG-encapsulated decursin as a targeted drug delivery system for CRPC., (© 2024. The Author(s).)

Efforts to engineer high-performance protein-based materials inspired by nature have mostly focused on altering naturally occurring sequences to confer the desired functionalities, whereas de novo design lags significantly behind and calls for unconventional innovative approaches. Here, using partially disordered elastin-like polypeptides (ELPs) as initial building blocks this work shows that de novo engineering of protein materials can be accelerated through hybrid biomimetic design, which this work achieves by integrating computational modeling, deep neural network, and recombinant DNA technology. This generalizable approach involves incorporating a series of de novo-designed sequences with α-helical conformation and genetically encoding them into biologically inspired intrinsically disordered repeating motifs. The new ELP variants maintain structural conformation and showed tunable supramolecular self-assembly out of thermal equilibrium with phase behavior in vitro. This work illustrates the effective translation of the predicted molecular designs in structural and functional materials. The proposed methodology can be applied to a broad range of partially disordered biomacromolecules and potentially pave the way toward the discovery of novel structural proteins., (© 2024 The Authors. Advanced Materials published by Wiley‐VCH GmbH.)

Despite progress in bone tissue engineering, reconstruction of large bone defects remains an important clinical challenge. Here, a biomaterial designed to recruit bone cells, endothelial cells, and neuronal fibers within the same matrix is developed, enabling bone tissue regeneration. The bioactive matrix is based on modified elastin-like polypeptides (ELPs) grafted with laminin-derived adhesion peptides IKVAV and YIGSR, and the SNA15 peptide for retention of hydroxyapatite (HA) particles. The composite matrix shows suitable porosity, interconnectivity, biocompatibility for endothelial cells, and the ability to support neurites outgrowth by sensory neurons. Subcutaneous implantation leads to the formation of osteoid tissue, characterized by the presence of bone cells, vascular networks, and neuronal structures, while minimizing inflammation. Using a rat femoral condyle defect model, longitudinal micro-CT analysis is performed, which demonstrates a significant increase in the volume of mineralized tissue when using the ELP-based matrix compared to empty defects and a commercially available control (Collapat). Furthermore, visible blood vessel networks and nerve fibers are observed within the lesions after a period of two weeks. By incorporating multiple key components that support cell growth, mineralization, and tissue integration, this ELP-based composite matrix provides a holistic and versatile solution to enhance bone tissue regeneration., (© 2024 The Author(s). Advanced Healthcare Materials published by Wiley‐VCH GmbH.)

Biological water-responsive (WR) materials are abundant in nature, and they are used as mechanical actuators for seed dispersal by many plants such as wheat awns and pinecones. WR biomaterials are of interest for applications as high-energy actuators, which can be useful in soft robotics or for capturing energy from natural water evaporation. Recent work on WR silk proteins has shown that β-sheet nanocrystalline domains with high stiffness correlate with the high WR actuation energy density, but the fundamental mechanisms to drive water responsiveness in proteins remain poorly understood. Here, we design, synthesize, and study protein block copolymers consisting of two α-helical domains derived from cartilage oligomeric matrix protein coiled-coil (C) flanking an elastin-like peptide domain (E), namely, CEC. We use these protein materials to create WR actuators with energy densities that outperform mammalian muscle. To elucidate the effect of structure on WR actuation, CEC was compared to a variant, CEC L44A , in which a point mutation disrupts the α-helical structure of the C domain. Surprisingly, CEC L44A outperformed CEC, showing higher energy density and less susceptibility to degradation after repeated cycling. We show that CEC L44A exhibits a higher degree of intermolecular interactions and is stiffer than CEC at high relative humidity (RH), allowing for less energy dissipation during water responsiveness. These results suggest that strong intermolecular interactions and the resulting, relatively steady protein structure are important for water responsiveness.

Elastin-like polymers are a class of stimuli-responsive protein polymers that hold immense promise in applications such as drug delivery, hydrogels, and biosensors. Yet, understanding the intricate interplay of factors influencing their stimuli-responsive behavior remains a challenging frontier. Using temperature-controlled dynamic light scattering and zeta potential measurements, we investigate the interactions between buffer, pH, salt, water, and protein using an elastin-like polymer containing ionizable lysine residues. We observed the elevation of transition temperature in the presence of the common buffering agent HEPES at low concentrations, suggesting a "salting-in" effect of HEPES as a cosolute through weak association with the protein. Our findings motivate a more comprehensive investigation of the influence of buffer and other cosolute molecules on elastin-like polymer behavior.

Elastic fibers provide critical elasticity to the arteries, lungs, and other organs. Elastic fiber assembly is a process where soluble tropoelastin is coacervated into liquid droplets, cross-linked, and deposited onto and into microfibrils. While much progress has been made in understanding the biology of this process, questions remain regarding the timing of interactions during assembly. Furthermore, it is unclear to what extent fibrous templates are needed to guide coacervate droplets into the correct architecture. The organization and shaping of coacervate droplets onto a fiber template have never been previously modeled or employed as a strategy for shaping elastin fiber materials. Using an in vitro system consisting of elastin-like polypeptides (ELPs), genipin cross-linker, electrospun polylactic- co -glycolic acid (PLGA) fibers, and tannic acid surface coatings for fibers, we explored ELP coacervation, cross-linking, and deposition onto fiber templates. We demonstrate that integration of coacervate droplets into a fibrous template is primarily influenced by two factors: (1) the balance of coacervation and cross-linking and (2) the surface energy of the fiber templates. The success of this integration affects the mechanical properties of the final fiber network. Our resulting membrane materials exhibit highly tunable morphologies and a range of elastic moduli (0.8-1.6 MPa) comparable to native elastic fibers.

Biological condensates are known to retain a large fraction of water to remain in a liquid and reversible state. Local solvation contributions from water hydrating hydrophilic and hydrophobic protein surfaces were proposed to play a prominent role for the formation of condensates through liquid-liquid phase separation (LLPS). However, although the total free energy is accessible by calorimetry, the partial solvent contributions to the free energy changes upon LLPS remained experimentally inaccessible so far. Here, we show that the recently developed THz calorimetry approach allows to quantify local hydration enthalpy and entropy changes upon LLPS of α-elastin in real time, directly from experimental THz spectroscopy data. We find that hydrophobic solvation dominates the entropic solvation term, whereas hydrophilic solvation mainly contributes to the enthalpy. Both terms are in the order of hundreds of kJ/mol, which is more than one order of magnitude larger than the total free energy changes at play during LLPS. However, since we show that entropy/enthalpy mostly compensates, a small entropy/enthalpy imbalance is sufficient to tune LLPS. Theoretically, a balance was proposed before. Here we present experimental evidence based on our spectroscopic approach. We finally show that LLPS can be steered by inducing small changes of solvation entropy/enthalpy compensation via concentration or temperature in α-elastin., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

While direct cell transplantation holds great promise in treating many debilitating diseases, poor cell survival and engraftment following injection have limited effective clinical translation. Though injectable biomaterials offer protection against membrane-damaging extensional flow and supply a supportive 3D environment in vivo that ultimately improves cell retention and therapeutic costs, most are created from synthetic or naturally harvested polymers that are immunogenic and/or chemically ill-defined. This work presents a shear-thinning and self-healing telechelic recombinant protein-based hydrogel designed around XTEN - a well-expressible, non-immunogenic, and intrinsically disordered polypeptide previously evolved as a genetically encoded alternative to PEGylation to "eXTENd" the in vivo half-life of fused protein therapeutics. By flanking XTEN with self-associating coil domains derived from cartilage oligomeric matrix protein, single-component physically crosslinked hydrogels exhibiting rapid shear thinning and self-healing through homopentameric coiled-coil bundling are formed. Individual and combined point mutations that variably stabilize coil association enables a straightforward method to genetically program material viscoelasticity and biodegradability. Finally, these materials protect and sustain viability of encapsulated human fibroblasts, hepatocytes, embryonic kidney (HEK), and embryonic stem-cell-derived cardiomyocytes (hESC-CMs) through culture, injection, and transcutaneous implantation in mice. These injectable XTEN-based hydrogels show promise for both in vitro cell culture and in vivo cell transplantation applications., (© 2024 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

Engineered protein fibers are promising biomaterials with diverse applications due to their tunable protein structure and outstanding mechanical properties. However, it remains challenging at the molecular level to achieve satisfied mechanical properties and environmental tolerance simultaneously, especially under extreme acid conditions. Herein, the construction of artificial fibers comprising chimeric proteins made of rigid amyloid peptide and flexible cationic elastin-like protein (ELP) module is reported. The amyloid peptide readily assembles into highly organized β-sheet structures that can be further strengthened by the coordination of Cu 2+ , while the flexible ELP module allows the formation of imine-based crosslinking networks. These double networks synergistically enhance the mechanical properties of the fibers, leading to a high tensile strength and toughness, overwhelming many reported recombinant spidroin fibers. Notably, the coordination of Cu 2+ with serine residues could stabilize β-sheet structures in the fibers under acidic conditions, which makes the fibers robust against acid, thus enabling their successful utilization in gastric perforation suturing. This work highlights the customization of double networks at the molecular level to create tailored high-performance protein fibers for various application scenarios., (© 2024 Wiley‐VCH GmbH.)

The unique biophysical and biochemical properties of intrinsically disordered proteins (IDPs) and their recombinant derivatives, intrinsically disordered protein polymers (IDPPs) offer opportunities for producing multistimuli-responsive materials; their sequence-encoded disorder and tendency for phase separation facilitate the development of multifunctional materials. This review highlights the strategies for enhancing the structural diversity of elastin-like polypeptides (ELPs) and resilin-like polypeptides (RLPs), and their self-assembled structures via genetic fusion to ordered motifs such as helical or beta sheet domains. In particular, this review describes approaches that harness the synergistic interplay between order-promoting and thermoresponsive building blocks to design hybrid biomaterials, resulting in well-structured, stimuli-responsive supramolecular materials ordered on the nanoscale., (© 2024 Wiley-VCH GmbH.)

Background: The exogenous delivery of miRNA to mimic and restore miRNA-34a activity in various cancer models holds significant promise in cancer treatment. Nevertheless, its effectiveness is often impeded by challenges, including a short half-life, propensity for off-target accumulation, susceptibility to inactivation by blood-based enzymes, concerns regarding patient safety, and the substantial cost associated with scaling up. As a means of overcoming these barriers, we propose the development of miRNA-loaded Tat-A86 nanoparticles by virtue of Tat-A86's ability to shield the loaded agent from external environmental factors, reducing degradation and inactivation, while enhancing circulation time and targeted accumulation., Results: Genetically engineered Tat-A86, featuring 16 copies of the interleukin-4 receptor (IL-4R)-binding peptide (AP1), Tat for tumor penetration, and an elastin-like polypeptide (ELP) for presenting target ligands and ensuring stability, served as the basis for this delivery system. Comparative groups, including Tat-E60 and A86, were employed to discern differences in binding and penetration. The designed ELP-based nanoparticle Tat-A86 effectively condensed miRNA, forming stable nanocomplexes under physiological conditions. The miRNA/Tat-A86 formulation bound specifically to tumor cells and facilitated stable miRNA delivery into them, effectively inhibiting tumor growth. The efficacy of miRNA/Tat-A86 was further evaluated using three-dimensional spheroids of lewis lung carcinoma (LLC) as in vitro model and LLC tumor-bearing mice as an in vivo model. It was found that miRNA/Tat-A86 facilitates effective cell killing by markedly improving miRNA penetration, leading to a substantial reduction in the size of LLC spheroids. Compared to other controls, Tat-A86 demonstrated superior efficacy in suppressing the growth of 3D cellular aggregates. Moreover, at equivalent doses, miRNA-34a delivered by Tat-A86 inhibited the growth of LLC cells in allograft mice., Conclusions: Overall, these studies demonstrate that Tat-A86 nanoparticles can deliver miRNA systemically, overcoming the basic hurdles impeding miRNA delivery by facilitating both miRNA uptake and stability, ultimately leading to improved therapeutic effects., (© 2024. The Author(s).)

Glaucoma, the second most common cause of blindness worldwide, requires the development of new and effective treatments. This study introduces a novel controlled-release system utilizing elastin-like recombinamers (ELR) and the Supercritical Antisolvent (SAS) technique with supercritical CO2. Acetazolamide (AZM), a class IV drug with limited solubility and permeability, is successfully encapsulated in an amphiphilic ELR at three different ELR:AZM ratios, yielding up to 62 %. Scanning electron microscopy (SEM) reveals spherical microparticles that disintegrate into monodisperse nanoparticles measuring approximately 42 nm under physiological conditions. The nanoparticles, as observed via Transmission Electron Microscopy (TEM) and Atomic Force Microscopy (AFM), do not exhibit aggregates, a fact confirmed by the zeta potential displaying a value of -33 mV over a period of 30 days. Transcorneal permeation tests demonstrate a 10 % higher permeation level compared to the control solution, which increases to 30 % after 2 h. Ocular irritation tests demonstrate no adverse effects or damage. Intraocular pressure (IOP) tests conducted on hypertensive rabbits indicate greater effectiveness for all three analyzed formulations, suggesting enhanced drug bioavailability during treatment. Consequently, the combination of recombinant biopolymers and high-pressure techniques represents a promising approach for advancing glaucoma therapy, emphasizing its potential clinical significance., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Stabilization against the dilution-dependent disassembly of self-assembled nanoparticles is a requirement for in vivo application. Herein, we propose a simple and biocompatible cross-linking reaction for the stabilization of a series of nanoparticles formed by the self-assembly of amphiphilic HA- b -ELP block copolymers, through the alkylation of methionine residues from the ELP block with diglycidyl ether compounds. The core-cross-linked nanoparticles retain their colloidal properties, with a spherical core-shell morphology, while maintaining thermoresponsive behavior. As such, instead of a reversible disassembly when non-cross-linked, a reversible swelling of nanoparticles' core and increase of hydrodynamic diameter are observed with lowering of the temperature.

Metal-coordination bonds, a highly tunable class of dynamic noncovalent interactions, are pivotal to the function of a variety of protein-based natural materials and have emerged as binding motifs to produce strong, tough, and self-healing bioinspired materials. While natural proteins use clusters of metal-coordination bonds, synthetic materials frequently employ individual bonds, resulting in mechanically weak materials. To overcome this current limitation, we rationally designed a series of elastin-like polypeptide templates with the capability of forming an increasing number of intermolecular histidine-Ni 2+ metal-coordination bonds. Using single-molecule force spectroscopy and steered molecular dynamics simulations, we show that templates with three histidine residues exhibit heterogeneous rupture pathways, including the simultaneous rupture of at least two bonds with more-than-additive rupture forces. The methodology and insights developed improve our understanding of the molecular interactions that stabilize metal-coordinated proteins and provide a general route for the design of new strong, metal-coordinated materials with a broad spectrum of dissipative time scales.

Vesicles are self-assembled structures comprised of a membrane-like exterior surrounding a hollow lumen with applications in drug delivery, artificial cells, and micro-bioreactors. Lipid or polymer vesicles are the most common and are made of lipids or polymers, respectively. They are highly useful structures for many applications but it can be challenging to decorate them with proteins or encapsulate proteins in them, owing to the use of organic solvent in their formation and the large size of proteins relative to lipid or polymer molecules. By utilization of recombinant fusion proteins to make vesicles, specific protein domains can be directly incorporated while also imparting tunability and stability. Protein vesicle assembly relies on the design and use of self-assembling amphiphilic proteins. A specific protein vesicle platform made in purely aqueous conditions of a globular, functional protein fused to a glutamate-rich leucine zipper (Z E ) and a thermoresponsive elastin-like polypeptide (ELP) fused to an arginine-rich leucine zipper (Z R ) is discussed here. The hydrophobic conformational change of the ELP above its transition temperature drives assembly, and strong Z E /Z R binding enables incorporation of the desired functional protein. Mixing the soluble proteins on ice induces zipper binding, and then warming above the ELP transition temperature ( T t ) triggers the transition to and growth of protein-rich coacervates and, finally, reorganization of proteins into vesicles. Vesicle size is tunable based on salt concentration, rate of heating, protein concentration, size of the globular protein, molar ratio of the proteins, and the ELP sequence. Increasing the salt concentration decreases vesicle size by decreasing the T t , resulting in a shorter coacervation transition stage. Likewise, directly changing the heating rate also changes this time and increasing protein concentration increases coalescence. Increasing globular protein size decreases the size of the vesicle due to steric hindrance. By changing the ELP sequence, which consists of (VPGXG) n , through the guest residue (X) or number of repeats ( n ), T t is changed, affecting size. Additionally, the chemical nature of X variation has endowed vesicles with stimuli responsiveness and stability at physiological conditions.Protein vesicles have been used for biocatalysis, biomacromolecular drug delivery, and vaccine applications. Photo-cross-linkable vesicles were used to deliver small molecule cargo to cancer cells in vitro and antigen to immune cells in vivo . pH-responsive vesicles effectively delivered functional protein cargo, including cytochrome C, to the cytosol of cancer cells in vitro , using hydrophobic ion pairing to improve cargo distribution in the vesicles and release. The globular protein used to make the vesicles can be varied to achieve different functions. For example, enzyme vesicles exhibit biocatalysis, and antigen vesicles induce antibody and cellular immune responses after vaccination in mice. Collectively, the development and engineering of the protein vesicle platform has employed amphiphilic self-assembly strategies and rational protein engineering to control physical, chemical, and biological properties for biotechnology and nanomedicine applications.

The tegument protein pp150 of Human Cytomegalovirus (HCMV) is known to be essential for the final stages of virus maturation and mediates its functions by interacting with capsid proteins. Our laboratory has previously identified the critical regions in pp150 important for pp150-capsid interactions and designed peptides similar in sequence to these regions, with a goal to competitively inhibit capsid maturation. Treatment with a specific peptide (PepCR2 or P10) targeted to pp150 conserved region 2 led to a significant reduction in murine CMV (MCMV) growth in cell culture, paving the way for in vivo testing in a mouse model of CMV infection. However, the general pharmacokinetic parameters of peptides, including rapid degradation and limited tissue and cell membrane permeability, pose a challenge to their successful use in vivo. Therefore, we designed a biopolymer-stabilized elastin-like polypeptide (ELP) fusion construct (ELP-P10) to enhance the bioavailability of P10. Antiviral efficacy and cytotoxic effects of ELP-P10 were studied in cell culture, and pharmacokinetics, biodistribution, and antiviral efficacy were studied in a mouse model of CMV infection. ELP-P10 maintained significant antiviral activity in cell culture, and this conjugation significantly enhanced P10 bioavailability in mouse tissues. The fluorescently labeled ELP-P10 accumulated to higher levels in mouse liver and kidneys as compared to the unconjugated P10. Moreover, viral titers from vital organs of MCMV-infected mice indicated a significant reduction of virus load upon ELP-P10 treatment. Therefore, ELP-P10 has the potential to be developed into an effective antiviral against CMV infection., (© 2024. The Author(s).)

Extraction of nucleic acids (NAs) is critical for many methods in molecular biology and bioanalytical chemistry. NA extraction has been extensively studied and optimized for a wide range of applications and its importance to society has significantly increased. The COVID-19 pandemic highlighted the importance of early and efficient NA testing, for which NA extraction is a critical analytical step prior to the detection by methods like polymerase chain reaction. This study explores simple, new approaches to extraction using engineered smart nanomaterials, namely NA-binding, intrinsically disordered proteins (IDPs), that undergo triggered liquid-liquid phase separation (LLPS). Two types of NA-binding IDPs are studied, both based on genetically engineered elastin-like polypeptides (ELPs), model IDPs that exhibit a lower critical solution temperature in water and can be designed to exhibit LLPS at desired temperatures in a variety of biological solutions. We show that ELP fusion proteins with natural NA-binding domains can be used to extract DNA and RNA from physiologically relevant solutions. We further show that LLPS of pH responsive ELPs that incorporate histidine in their sequences can be used for both binding, extraction and release of NAs from biological solutions, and can be used to detect SARS-CoV-2 RNA in samples from COVID-positive patients., (© 2024. The Author(s).)

This paper outlines a process undertaken by a physician to design a peptide aimed at impacting the extracellular matrix. From a position of very little expertise, a new peptide was designed with amino acid constituents based on the structural proteins collagen and elastin. Sequencing was also considered, given the periodic repetition observed in these proteins, and a peptide with reasonable molecular weight and physical characteristics was designed using available software. The sequence of events concerning intellectual property, functionality investigation, and eventual use of the peptide in new formulations is detailed. This may be of interest to physicians who consider this exercise out of the scope of the usual practice. J Drugs Dermatol. 2024;23(5):347-352.&nbsp; &nbsp; doi:10.36849/JDD.7921.

The increased remodeling of the extracellular matrix (ECM) in pulmonary fibrosis (PF) generates bioactive ECM fragments called matricryptins, which include elastin-derived peptides (EDPs). The interaction between EDPs and their receptors, including elastin-binding protein (EBP), plays a crucial role in exacerbating fibrosis. Here, we present L XJ-02 for the first time, a novel ultralong-acting inhibitor that disrupts the EDPs/EBP peptide-protein interaction, promoting macrophages to secrete matrix metalloproteinase-12 (MMP-12), and showing great promise as a stable peptide. MMP-12 has traditionally been implicated in promoting inflammation and fibrosis in various acute and chronic diseases. However, we reveal a novel role of L XJ-02 that activates the macrophage-MMP-12 axis to increase MMP-12 expression and degrade ECM components like elastin. This leads to the preventing of PF while also improving EDP-EBP interaction. L XJ-02 effectively reverses PF in mouse models with minimal side effects, holding great promise as an excellent therapeutic agent for lung fibrosis.

Thermoresponsive elastin-like peptides (ELPs) have been extensively investigated in biotechnology and medicine, but little attention has been paid to the process by which coacervation causes ELP-decorated particles to aggregate. Using gold nanoparticles (AuNPs) functionalized with a cysteine-terminated 96-repeat of the VPGVG sequence (V96-Cys), we show that the size of the clusters that reversibly form above the ELP transition temperature can be finely controlled in the 250 to 930 nm range by specifying the concentration of free V96-Cys in solution and using AuNPs of different sizes. We further find that the localized surface plasmon resonance peak of the embedded AuNPs progressively red-shifts with cluster size, likely due to an increase in particle-particle contacts. We exploit this fine control over size to homogeneously load precise amounts of the dye Nile Red and the antibiotic Tetracycline into clusters of different hydrodynamic diameters and deliver cargos near-quantitatively by deconstructing the aggregates below the ELP transition temperature. Beyond establishing a key role for free ELPs in the agglomeration of ELP-functionalized particles, our results provide a path for the thermally controlled delivery of precise quantities of molecular cargo. This capability might prove useful in combination photothermal therapies and theranostic applications, and to trigger spatially and temporally uniform responses from biological, electronic, or optical systems.

Elastin-collagen nanovesicles (ECnV) have emerged as a promising platform for drug delivery due to their tunable physicochemical properties and biocompatibility. The potential of nine distinct ECnVs to serve as drug-delivery vehicles was investigated in this study, and it was demonstrated that various small-molecule cargo (e.g., dexamethasone, methotrexate, doxorubicin) can be encapsulated in and released from a set of ECnVs, with extents of loading and rates of release dictated by the composition of the elastin domain of the ECnV and the type of cargo. Elastin-like peptides (ELPs) and collagen-like peptides (CLPs) of various compositions were produced; the secondary structure of the corresponding peptides was determined using CD, and the morphology and average hydrodynamic diameter (∼100 nm) of the ECnVs were determined using TEM and DLS. It was observed that hydrophobic drugs exhibited slower release kinetics than hydrophilic drugs, but higher drug loading was achieved for the more hydrophilic Dox. The collagen-binding ability of the ECnVs was demonstrated through a 2D collagen-binding assay, suggesting the potential for longer retention times in collagen-enriched tissues or matrices. Sustained release of drugs for up to 7 days was observed and, taken together with the collagen-binding data, demonstrates the potential of this set of ECnVs as a versatile drug delivery vehicle for longer-term drug release of a variety of cargo. This study provides important insights into the drug delivery potential of ECnVs and offers useful information for future development of ECnV-based drug delivery systems for the treatment of various diseases.

Plants can produce complex pharmaceutical and technical proteins. Spider silk proteins are one example of the latter and can be used, for example, as compounds for high-performance textiles or wound dressings. If genetically fused to elastin-like polypeptides (ELPs), the silk proteins can be reversibly precipitated from clarified plant extracts at moderate temperatures of ~ 30 °C together with salt concentrations > 1.5 M, which simplifies purification and thus reduces costs. However, the technologies developed around this mechanism rely on a repeated cycling between soluble and aggregated state to remove plant host cell impurities, which increase process time and buffer consumption. Additionally, ELPs are difficult to detect using conventional staining methods, which hinders the analysis of unit operation performance and process development. Here, we have first developed a surface plasmon resonance (SPR) spectroscopy-based assay to quantity ELP fusion proteins. Then we tested different filters to prepare clarified plant extract with > 50% recovery of spider silk ELP fusion proteins. Finally, we established a membrane-based purification method that does not require cycling between soluble and aggregated ELP state but operates similar to an ultrafiltration/diafiltration device. Using a data-driven design of experiments (DoE) approach to characterize the system of reversible ELP precipitation we found that membranes with pore sizes up to 1.2 µm and concentrations of 2-3 M sodium chloride facilitate step a recovery close to 100% and purities of > 90%. The system can thus be useful for the purification of ELP-tagged proteins produced in plants and other hosts., (© 2024. The Author(s).)