Elastin-like Recombinamers Research Articles

Self-assembly of Janus Au:Fe3O4 branched nanoparticles. From organized clusters to stimuli-responsive nanogel suprastructures.

Janus nanoparticles offer enormous possibilities through a binary selective functionalization and dual properties. Their self-assembly has attracted strong interest due to their potential as building blocks to obtain molecular colloids, supracrystals and well-organized nanostructures that can lead to new functionalities. However, this self-assembly has been focused on relatively simple symmetrical morphologies, while for complex nanostructures this process has been unexplored. Here, we study the assembly of plasmonic-magnetic Janus nanoparticles with a branched (nanostar) – sphere morphology. The branched morphology enhances their plasmonic properties in the near-infrared region and therefore their applicability, but at the same time constrains their self-assembly capabilities to obtain more organized or functional suprastructures. We describe the self-assembly of these nanoparticles after amphiphilic functionalization. The role of the nanoparticle branching, as well as the size of the polymer-coating, is explored. We show how the use of large molecular weight stabilizing polymers can overcome the anisotropy of the nanoparticles producing a change in the morphology from small clusters to larger quasi-cylindrical nanostructures. Finally, the Janus nanoparticles are functionalized with a thermo-responsive elastin-like recombinamer. These nanoparticles undergo reversible self-assembly in the presence of free polymer giving rise to nanoparticle-stabilized nanogel-like structures with controlled size, providing the possibility to expand their applicability to multi-stimuli controlled self-assembly.

Small Caliber Compliant Vascular Grafts Based on Elastin-Like Recombinamers for in situ Tissue Engineering.

Vascular disease is a leading cause of death worldwide, but surgical options are restricted by the limited availability of autologous vessels, and the suboptimal performance of prosthetic vascular grafts. This is especially evident for coronary artery by-pass grafts, whose small caliber is associated with a high occlusion propensity. Despite the potential of tissue-engineered grafts, compliance mismatch, dilatation, thrombus formation, and the lack of functional elastin are still major limitations leading to graft failure. This calls for advanced materials and fabrication schemes to achieve improved control on the grafts' properties and performance. Here, bioinspired materials and technical textile components are combined to create biohybrid cell-free implants for endogenous tissue regeneration. Clickable elastin-like recombinamers are processed to form an open macroporous 3D architecture to favor cell ingrowth, while being endowed with the non-thrombogenicity and the elastic behavior of the native elastin. The textile components (i.e., warp-knitted and electrospun meshes) are designed to confer suture retention, long-term structural stability, burst strength, and compliance. Notably, by controlling the electrospun layer's thickness, the compliance can be modulated over a wide range of values encompassing those of native vessels. The grafts support cell ingrowth, extracellular matrix deposition and endothelium development in vitro. Overall, the fabrication strategy results in promising off-the-shelf hemocompatible vascular implants for in situ tissue engineering by addressing the known limitations of bioartificial vessel substitutes.

Genetically engineered elastin-like recombinamers with sequence-based molecular stabilization as advanced bioinks for 3D bioprinting

The versatility of 3D bioprinting techniques has demonstrated great potential for the development of artificial engineered tissues that more closely resemble native tissues. Despite this, challenges remain as regards the search for new bioinks that embrace all the complex parameters that this technique demands. In an attempt to develop such an advanced material, a novel smart material based on elastin-like recombinamers (ELRs) has been developed by molecularly programming its sequence to exhibit a sequential three-stage gelation process, thus providing printing attributes.The thermoresponsive behavior of the ELR is exploited for the deposition of well-controlled fibres on a platform heated to 37 °C, and its recombinant nature ensures batch-to-batch reproducibility and its applicability to a desired target tissue by the introduction of selected bioactive amino-acid sequences into its molecular chain.The biocompatible nature of the ELR enables the printing of loaded cells while providing a protective environment as part of the printing process. Thus, HFF-1 cells were found to be able to proliferate within the printed structures upon culture, displaying their natural morphology.The results of this work highlight the applicability and novelty of the bioprinting of biomimetic ELR-based structures for advanced applications.

Layer-by-layer biofabrication of coronary covered stents with clickable elastin-like recombinamers

Coronary artery disease is the leading cause of death around the world. Endovascular stenting is the preferred treatment option to restore blood flow in the coronary arteries due to the lower perioperative morbidity when compared with more invasive treatment options. However, stent failure is still a major clinical problem, and further technological solutions are required to improve the performance of current stents. Here, we developed coronary stents covered with elastin-like recombinamers (ELRs) by exploiting a layer-by-layer technique combined with catalyst-free click chemistry. The resulting ELR-covered stents were intact after an in vitro simulated implantation procedure by balloon dilatation, which evidenced the elastic performance of the membrane. Additionally, the stents were mechanically stable under high flow conditions, which is in agreement with the covalent and stable nature of the click chemistry crosslinking strategy exploited during the ELR-membrane manufacturing and the successful embedding of the stent. Minimal platelet adhesion was detected after blood exposure in a Chandler loop as shown by scanning electron microscopy. The seeding of human endothelial progenitor cells (EPCs) on the ELR-membranes resulted in a confluent endothelial layer. These results prove the potential of this strategy to develop an advanced generation of coronary stents, with a stable and bioactive elastin-like membrane to exclude the atherosclerotic plaque from the blood stream or to seal coronary perforations and aneurysms, while providing a luminal surface with minimal platelet adhesion and favouring endothelialization.

Recombinant AMP/Polypeptide Self-Assembled Monolayers with Synergistic Antimicrobial Properties for Bacterial Strains of Medical Relevance.

Nosocomial infections are one of the most frequent causes of indwelling biomedical device failure. In this regard, the use of antibiofilm nanocoatings based on antimicrobial peptides (AMPs) is a promising alternative to prevent multiresistant biofilm infections. However, the limitations of chemical production impede the large-scale development of advanced antimicrobial materials that improve the properties of AMPs. Herein, we present a multifunctional modular design for the recombinant coproduction of self-assembled monolayers (SAMs) based on AMPs and elastin-like recombinamers (ELRs), which combine the antimicrobial properties of a designer AMP, GL13K, and low-fouling activity of an ELR in a synergistic manner. The inclusion of a grafting domain intended for oriented tethering onto surfaces allowed the recombinant polymers to be covalently immobilized onto model gold surfaces. The antibiofilm properties against two of the bacterial strains most frequently responsible for indwelling medical device-associated infections, namely Staphylococcus epidermidis and Staphylococcus aureus, were then evaluated. GL13K peptide was found to provide antibiofilm properties to the surface, with these being synergistically enhanced by the antifouling effect of the ELR. This new design offers a promising tool for the development of advanced AMP-based nanocoatings for medical devices with powerful and enhanced features.

Hydrophobic Cholesteryl Moieties Trigger Substrate Cell-Membrane Interaction of Elastin-Mimetic Protein Coatings in Vitro.

A cellular coating based on hydrophobic interactions of an elastin-like recombinamer (ELR) with the cell membrane is presented. It is well-documented that biophysical properties such as net charge, hydrophobicity, and protein-driven cell–ligand (integrin binding) interactions influence the interaction of polymers, proteins or peptides with model membranes and biological cells. Most studies to enhance membrane–substrate interactions have focused on the introduction of positively charged groups to foster electrostatic interactions with the negatively charged membrane. Herein, we present an antagonistic approach based on ELRs with varying amounts of hydrophobic cholesteryl groups (ELRCTAs). The ability of the membranes to stabilize cholesteryl groups is hypothesized to assist the coordination of hydrophobic ELRs with the membrane. The main objective was to generate a defined cellular coating of a recombinant protein that allows for total sequence control and less host, or batch-to-batch, variation as a substitute for the existing coatings like alginate, polyelectrolytes, collagens, and fibronectin. We used an in vitro cell-binding assay to quantify cell–substrate interactions, showing enhanced cellular recognition and matrix distribution with an increasing number of cholesteryl groups incorporated. These novel materials and the versatile nature of their protein sequence have great potential as cellular markers, drug carriers, or hydrophobic cell-binding domains.

An elastin-like recombinamer-based bioactive hydrogel embedded with mesenchymal stromal cells as an injectable scaffold for osteochondral repair.

The aim of this study was to evaluate injectable, in situ cross-linkable elastin-like recombinamers (ELRs) for osteochondral repair. Both the ELR-based hydrogel alone and the ELR-based hydrogel embedded with rabbit mesenchymal stromal cells (rMSCs) were tested for the regeneration of critical subchondral defects in 10 New Zealand rabbits. Thus, cylindrical osteochondral defects were filled with an aqueous solution of ELRs and the animals sacrificed at 4 months for histological and gross evaluation of features of biomaterial performance, including integration, cellular infiltration, surrounding matrix quality and the new matrix in the defects. Although both approaches helped cartilage regeneration, the results suggest that the specific composition of the rMSC-containing hydrogel permitted adequate bone regeneration, whereas the ELR-based hydrogel alone led to an excellent regeneration of hyaline cartilage. In conclusion, the ELR cross-linker solution can be easily delivered and forms a stable well-integrated hydrogel that supports infiltration and de novo matrix synthesis.

Use of proteolytic sequences with different cleavage kinetics as a way to generate hydrogels with preprogrammed cell-infiltration patterns imparted over their given 3D spatial structure

Control over biodegradation processes is crucial to generate advanced functional structures with a more interactive and efficient role for biomedical applications. Herein, a simple, high-throughput approach is developed based on a three-dimensional (3D)-structured system that allows a preprogramed spatial-temporal control over cell infiltration and biodegradation. The 3D-structured system is based on elastin-like recombinamers (ELRs) characterized by differences in the kinetics of their peptide cleavage and consists of a three-layer hydrogel disk comprising an internal layer containing a rapidly degrading component, with the external layers containing a slow-degrading ELR. This structure is intended to invert the conventional pattern of cell infiltration, which goes from the outside to the inside of the implant, to allow an anti-natural process in which infiltration takes place first in the internal layer and later progresses to the outer layers. Time-course in vivo studies proved this hypothesis, i.e. that it is possible to drive the infiltration of cells over time in a given 3D-structured implant in a controlled and predesigned way that is able to overcome the natural tendency of conventional cell infiltration. The results obtained herein open up the possibility of applying this concept to more complex systems with multiple biological functions.

Self-Assembling ELR-Based Nanoparticles as Smart Drug-Delivery Systems Modulating Cellular Growth via Akt.

This work investigates the physicochemical properties and in vitro accuracy of a genetically engineered drug-delivery system based on elastin-like block recombinamers. The DNA recombinant techniques allowed us to create this smart complex polymer containing bioactive sequences for internalization, lysosome activation under acidic pH, and blockage of cellular growth by a small peptide inhibitor. The recombinant polymer reversibly self-assembled when the temperature was increased above 15 °C into nanoparticles with a diameter of 72 nm and negative surface charge. Furthermore, smart nanoparticles were shown to enter in the cells via clathrin-dependent endocytosis and properly blocked phosphorylation and consequent activation of Akt kinase. This system provoked apoptosis-mediated cell death in breast and colorectal cancer cells, which possess higher expression levels of Akt, whereas noncancerous cells, such as endothelial cells, fibroblasts, and mesenchymal stem cells, were not affected. Hence, we conclude that the conformational complexity of this smart elastin-like recombinamer leads to achieving successful drug delivery in targeted cells and could be a promising approach as nanocarriers with bioactive peptides to modulate multiple cellular processes involved in different diseases.

Bicyclic RGD peptides with high integrin αvβ3 and α5β1 affinity promote cell adhesion on elastin-like recombinamers

Biomaterial design in tissue engineering aims to identify appropriate cellular microenvironments in which cells can grow and guide new tissue formation. Despite the large diversity of synthetic polymers available for regenerative medicine, most of them fail to fully match the functional properties of their native counterparts. In contrast, the few biological alternatives employed as biomaterials lack the versatility that chemical synthesis can offer. Herein, we studied the HUVEC adhesion and proliferation properties of elastin-like recombinamers (ELRs) that were covalently functionalized with each three high-affinity and selectivity αvβ3- and α5β1-binding bicyclic RGD peptides. Next to the bicycles, ELRs were also functionalized with various integrin-binding benchmark peptides, i.e. knottin-RGD, cyclo-[KRGDf] and GRGDS, allowing for better classification of the obtained results. Covalent functionalization with the RGD peptides, as validated by MALDI-TOF analysis, guarantees flexibility and minimal steric hindrance for interactions with cellular integrins. In addition to the covalently modified RGD-ELRs, we also synthesized another benchmark ELR comprising RGD as part of the backbone. HUVEC adhesion and proliferation analysis using the PicoGreen® assay revealed a higher short-term adhesion and proliferative capacity of cells on ELR surfaces functionalized with high affinity, integrin-binding bicyclic RGD-peptides compared with the ELRs containing RGD in the backbone.

Self-assembly in elastin-like recombinamers: a mechanism to mimic natural complexity.

The topic of self-assembled structures based on elastin-like recombinamers (ELRs, i.e., elastin-like polymers recombinantly bio-produced) has released a noticeable amount of references in the last few years. Most of them are intended for biomedical applications. In this review, a complete revision of the bibliography is carried out. Initially, the self-assembly (SA) concept is considered from a general point of view, and then ELRs are described and characterized based on their intrinsic disorder. A classification of the different self-assembled ELR-based structures is proposed based on their morphologies, paying special attention to their tentative modeling. The impact of the mechanism of SA on these biomaterials is analyzed. Finally, the implications of ELR SA in biological systems are considered.

A transferrin receptor-binding mucoadhesive elastin-like recombinamer: In vitro and in vivo characterization

The development of mucoadhesive materials is of great interest and is also a major challenge. Being adsorption sites, mucosae are suitable targets for drug delivery, but as defensive barriers they are complex biological surfaces to interact with, mainly due to their protective mucus layer. As such, first- and second-generation mucoadhesives focused on material-mucus interactions, whereas the third generation of mucoadhesives introduced structural motifs that are able to interact with the cells beneath the mucus layer. The combination of different prerequisites (water solubility, soft gel formation at body temperature and able to interact with the mucus) in a single molecule is easily achieved using elastin-like recombinamers (ELRs) given their multiple block design. Moreover, we have been able to introduce a short amino-acid sequence known as T7 that is able to bind to transferrin receptors in the epithelial cell layer. The T7 sequence enhances the cell-binding properties of the mucoadhesive ELR (MELR), as demonstrated using a Caco-2 epithelial cell model. In vivo experiments confirmed the mucoadhesive properties found in vitro. Statement of SignificanceThe development of a mucoadhesive material is a major challenge. Mucosae are suitable targets for drug delivery, but as defense barriers, they are complex surfaces to interact with. In this work we report the first ELR that combines different functional blocks, in a single molecule, which provide it with the properties of soft-gel forming at body temperature and being able of efficiently adhering to the mucus layer of mucosas, as well as to the underlying epithelial cell layer, as demonstrated in vitro and in vivo.The rationally designed materials presented in this work sets the basis for developing ELR-based, mucosa-directed drug delivery systems, which could improve patient’s compliance, enhancing drug retention at the mucosal site.

A novel lipase-catalyzed method for preparing ELR-based bioconjugates

Herein we present a novel one-pot method for the chemical modification of elastin-like recombinamers (ELRs) in a mild and efficient manner involving enzymatic catalysis with Candida antarctica lipase B. The introduction of different functionalities into such ELRs could open up new possibilities for the development of advanced biomaterials for regenerative medicine and, specifically, for controlled drug delivery given their additional ability to respond to stimuli other than pH or temperature, such as glucose concentration or electromagnetic radiation.Candida antarctica lipase B immobilized on a macroporous acrylic resin (Novozym 435) was used to enzymatically couple different aminated substrates to a recombinamer containing carboxylic groups along its amino acid chain by way of an amidation reaction. A preliminary study of the kinetics of this amidation in response to different reaction conditions, such as solvent, temperature or reagent ratio, was carried out using a phenylazobenzene derivative (azo-NH2) as a model. The optimal amidation conditions were used to couple other amine reagents, such as phenylboronic acid (FB-NH2) or polyethylene glycol (PEG-NH2), thus allowing us to obtain photoresponsive, glucose-responsive or PEGylated ELRs that could potentially be useful as sensors in devices for controlled drug delivery.

Synthesis and characterization of new recombinant Elastin-Like Recombinamers fused to scFv for the formation of targeted polyplex

Synthesis and characterization of new recombinant Elastin-Like Recombinamers fused to scFv for the formation of targeted polyplex

PHBV wet-spun scaffold coated with ELR-REDV improves vascularization for bone tissue engineering

A bone tissue replacement with relevant anatomical size requires the production of 3D scaffolds, which in turn limits the mass transport of nutrients and oxygen to sustain cell survival. A viable vascular network is required to overcome this problem. However, this cannot be established immediately after the implantation of a scaffold. The aim of this study was to develop a 3D wet-spun bone tissue engineering scaffold, coated with an elastin-like recombinamer (ELR) peptide with an endothelial cell-attracting REDV sequence to promote early vascularization. Scaffolds were produced using biodegradable poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), and an ELR was immobilized onto it after oxygen plasma treatment (PHBV-O2-ELR-REDV). O2 plasma treatment and ELR modification of the PHBV changed the wettability, topography, and composition of the surface. A moderately hydrophilic surface was obtained after oxygen plasma treatment and ELR-REDV coating with a contact angle of 66.63 ± 0.77°. The surface roughness decreased after plasma treatment from 343.4 to 160.0 nm and increased to 280.3 nm after ELR-REDV coating. FTIR-ATR showed amide I and amide II bonds after ELR-REDV coating showing that the coating was successful. Scaffolds were tested in vitro with rabbit bone marrow mesenchymal cells. ELR modification did not cause a significant difference in adhesion or proliferation compared to unmodified controls. On the other hand, ELR-modified scaffolds attracted a higher number of human umbilical vein endothelial cells (HUVECs) due to the REDV sequence. The Alamar Blue test and confocal laser scanning microscopy micrographs showed that HUVEC migration and attachment on PHBV-O2-ELR-REDV scaffolds was around 2.5-fold higher than untreated PHBV scaffolds after 14 d. Plasma-treated scaffolds (PHBV-O2) showed an increase in the number of adhered HUVECs due to increased surface wettability. It can, therefore, be suggested that PHBV-O2-ELR-REDV scaffolds have significant potential to induce early vascularization due to increased attractiveness for endothelial cells. This could alleviate the vascularization problem of 3D implants for bone tissue engineering.

Production of bioactive hepcidin by recombinant DNA tagging with an elastin-like recombinamer

With the lack of new chemical antibiotics and increasing pathogen resistance to those available, new alternatives are being explored. Antimicrobial peptides (AMPs) with a broad range of effects, including antibacterial, antifungal, and antiviral actions, have emerged as one of the options. They can be produced by recombinant DNA technology, but the chromatographic methods used for peptide purification are expensive and time consuming. Here, we describe the design, production, purification and assessment of the antibacterial activity of the human peptide hepcidin, using an elastin-like recombinamer as fusion partner. The recombinant protein Hep-A200 was produced in Escherichia coli and purified by a non-chromatographic procedure, exploiting the thermal properties of the A200 elastin-like recombinamer. Recombinant Hep-A200 was found to retain antibacterial activity against Gram-positive and Gram-negative species.

Tuning the Stiffness of Surfaces by Assembling Genetically Engineered Polypeptides with Tailored Amino Acid Sequence.

We introduce elastin-like recombinamers (ELRs) as polypeptides with precise amino acid positioning to generate polypeptide coatings with tunable rigidity. Two ELRs are used: V84-ELR, a hydrophobic monoblock, and EI-ELR, an amphiphilic diblock. Both were modified with the amine-reactive tetrakis (hydroxymethyl) phosphonium chloride compound. We evaluated the affinity, conformation, and dissipative behavior of ELRs assembled on alkanethiol self-assembled coatings by quartz crystal microbalance with dissipation monitoring, multiparametric surface plasmon resonance, and atomic force microscopy. The thickness of the polypeptide coatings showcases the preferential affinity of ELRs to NH2- and CH3-terminated surfaces. We demonstrate that V84-ELR strongly bonded to the substrate and reorganizes into an extended and more hydrated layer as the adsorbed amount increases, whereas EI-ELR has a less dissipative behavior. The results suggest that ELR adsorption depends on the amino acid sequence and the substrate chemistry, ultimately influencing the stiffness of the polypeptide coatings.

Spatial control and cell adhesion selectivity on model gold surfaces grafted with elastin-like recombinamers

A simple system for cell selectivity and spatially controlled cell adhesion has been developed using model gold surfaces grafted with a combination of two ELRs containing into their backbone cell-adhesion domains such as RGD and REDV. Grafting onto gold was achieved via redox reaction through thiol groups present in amino terminal cysteine tails of the ELRs. The correlation among contact angle, SEM micrographs, AFM, XPS and QCM-D have been carried out.After in-depth adhesion studies, a mixture of 75% ELR-REDV and 25% ELR-RGD was found to exhibit high selectivity for endothelial cells, promoting strong adhesion thereof.Consequently, certain areas of gold surfaces (strips) were cleaned by laser ablation and functionalized with the mixture 75% ELR-REDV – 25% ELR-RGD leading to a spatial segregation of the co-culture made of HUVEC and HFF1 cells. This platform therefore exhibits selective spatial control over cell adhesion associated with the bioactive epitopes (RGD and REDV) contained in the ELR sequence, since each functionalized surface (including strips) have similar topographic and hydrophobic characteristics and mechanical properties are in the same order of magnitude.

PO-429 Elastin-like recombinamers nanoparticles: an effective drug delivery system on colorectal cancer cells

IntroductionWith almost 1,4 million new cases each year worldwide, colorectal cancer kills 228.000 Europeans every year. Although new therapies have been discovered, the use of recombinant elastin-based biomaterials could open a new approach in cancer research as polymeric carriers for drug delivery in order to improve the accuracy of the action and reduce the toxicity of chemotherapeutic agents. In this work, we have developed a new elastin-like recombinamer (ELR) fused to a peptide inhibitor of the protein kinase Akt. This polymer self-assembled into nanoparticles, which showed killing ability on colorectal cancer cells (Caco-2).Material and methodsTaking advantage of the recombinant DNA technology, the constructs were formed by an amphiphilic backbone and several bioactive sequences, with an Akt inhibitor among others. ELR were produced by E. coli fermentation and, after purification process, were characterised and tested in vitro. Cellular viability assays on cancerous cells were carried out in order to study the killing ability of these nanoparticles.Results and discussionsBoth physical and chemical characterisation of this novel ELR showed that it is able to self-assemble into nanoparticles with a diameter of 68 nm, which are an effective way to deliver the Akt inhibitor into the cytoplasm, where it will bind to its protein target Akt and stop the kinase activity. With a transition temperature of 18°C and a high negatively-charged surface, nanoparticles perfectly met all requirements for its use as biomedical tools. Moreover, nanoparticles showed increased killing ability on cancerous cells compared to non-cancerous cells.ConclusionThus, we have designed a new system able to enter into the cancerous cells and inhibit Akt signalling pathway. As an interesting advantage, ELRs have the ability to be modified by adding different molecules, such as aptamers or single chain antibodies, in order to be selectively targeted against cancerous cells. Thus, the action of these nanoparticles will be more accurate so as to achieve a new therapeutic tool for colorectal cancer treatment.

Protein disorder\u2013order interplay to guide the growth of hierarchical mineralized structures

A major goal in materials science is to develop bioinspired functional materials based on the precise control of molecular building blocks across length scales. Here we report a protein-mediated mineralization process that takes advantage of disorder–order interplay using elastin-like recombinamers to program organic–inorganic interactions into hierarchically ordered mineralized structures. The materials comprise elongated apatite nanocrystals that are aligned and organized into microscopic prisms, which grow together into spherulite-like structures hundreds of micrometers in diameter that come together to fill macroscopic areas. The structures can be grown over large uneven surfaces and native tissues as acid-resistant membranes or coatings with tuneable hierarchy, stiffness, and hardness. Our study represents a potential strategy for complex materials design that may open opportunities for hard tissue repair and provide insights into the role of molecular disorder in human physiology and pathology.