BIOFORGE - Artículos de revista

Bioorthogonal mussel-inspired elastin-like nanocoatings for indwelling devices

2025-01-01T00:00:00Z

Medical devices such as vascular grafts, stents, and catheters are crucial for patient treatment but often suffer suboptimal integration with host tissues due to the nature of their surfaces. The materials commonly used, including metals and synthetic polymers, frequently lead to undesired immune responses and device failure. In this context, coating their surfaces with designer proteins has arisen as a promising strategy to improve the device’s biointegration. Here, we present a bioinspired method for coating biomaterial surfaces with protein-engineered polymers designed to mimic tailored functions from the native extracellular matrix (ECM). Combining mussel-inspired catechol chemistry with bioorthogonal click chemistry, we developed a modular grafting method for the surface functionalization of metallic and polymeric implants using a bifunctional peptide containing azide and DOPA (3,4-dihydroxyphenylalanine) groups. This simple dip-coating process enabled the fabrication of bioactive elastin-like coatings with precise peptide presentation. The results reveal enhanced bioactivity and cytocompatibility, as evidenced by improved endothelial cell adhesion, proliferation, and heparin-binding capacity on coated surfaces. The versatility and effectiveness of this bioorthogonal coating method suggest significant potential for creating implant surfaces tailored to diverse clinical applications.

Characterization and release modelling in ELR-based nanocomposite hydrogel loaded with polylactic acid for the implementation of a biomedical device

2025-01-01T00:00:00Z

Cardiac tissues are difficult to regenerate due to the low proliferative capacity of cardiomyocytes. A new ther- apeutic strategy for cardiac regenerative medicine could include a device capable of ensuring cell grafting, stimulating cardiac tissue regeneration, and serving as an appropriate scaffold for the controlled and sustained release of lactate over time as an inducer of cardiomyocyte proliferation. An effective source of lactate could consist of the lactic acid polymer (PLA) itself, which generates free lactic acid during its degradation. In this work, we have developed a nanocomposite hydrogel for lactate release based on a biocompatible and biode- gradable matrix formed by elastin-like recombinamers cross-linked via click chemistry. Polylactic acid particles were encapsulated in the matrix after these particles had been partially degraded to lactic acid through oxygen plasma treatment. In the first 48 h, an early and modulated release of free lactic acid from plasma-treated PLA degradation is observed, and over longer periods, a sustained release of lactic acid produced by the hydrolytic degradation of PLA under physiological conditions occurs. Lactate is available from the very beginning (“early release”), addressing the drawback of the slow degradation (by hydrolysis) of polylactic acid. Therefore, a biomedical device has been designed and implemented, formed by an ELR polymeric matrix as an analogue of cardiac tissue, acting as a device for early, controlled, and sustained lactate release, with dosing at concentrations similar to those previously studied as suitable for promoting cardiomyocyte proliferation, showing promise for its use in the regeneration of infarcted cardiac tissue.

Biocompatible elastin-like click gels: design, synthesis and characterization

2015-01-01T00:00:00Z

Elastin-like recombinamer click gels (ELR-CGs) for biomedical applications, such as drug delivery or tissue engineering, have been developed by taking advantage of the click reaction (CuAAC) in the absence of traditional crosslinking agents. ELRs are functionalized with alkyne and azide groups using conventional chemical techniques to introduce the reactivity required to carry out the 1,3-dipolar cycloaddition under mild biocompatible conditions, with no toxic by-products and in short reaction times. Hydrogels with moduli in the range 1,000–10,000 Pa have been synthesized, characterized, and tested in vitro against several cell types. The cells embedded into ELR-CGs possessed high viability and proliferation rate. The mechanical properties, porosity and swelling of the resulting ELR-CGs can easily be tuned by adjusting the ELR concentration. We also show that it is possible to replicate different patterns on the hydrogel surface, thus allowing the use of this type of hydrogel to improve applications that require cell guidance or even differentiation depending on the surface topography.

“In-situ” formation of elastin-like recombinamer hydrogels with tunable viscoelasticity through efficient one-pot process

2024-01-01T00:00:00Z

Despite the remarkable progress in the generation of recombinant elastin-like (ELR) hydrogels, further improvements are still required to enhance and control their viscoelasticity, as well as limit the use of expensive chemical reagents, time-consuming processes and several purification steps. To alleviate this issue, the reactivity of carboxylic groups from glutamic (E) acid distributed along the hydrophilic block of an amphiphilic ELR (coded as E50I60) with amine groups has been studied through a one-pot amidation reaction in aqueous solutions, for the first time. By means of this approach, immediate conjugation of E50I60 with molecules containing amine groups has been performed with a high yield, as demonstrated by the 1H NMR and MALDI-TOF spectroscopies. This has resulted in the preparation of viscoelastic irreversible hydrogels through the “in-situ” cross-linking of E50I60 with another ELR (coded as VKV24) containing amine groups from lysines (K). The rheology analysis demonstrated that the gelation process takes place following a dual mechanism dependent on the ELR concentration: physical cross-linking of I60 block through the hydrophobic interactions, and covalent cross-linking of E50I60 with VKV24 through the amidation reaction. While the chemical network formed between the hydrophilic E50 block and VKV24 ELR preserves the elasticity of ELR hydrogels, the self-assembly of the I60 block through the hydrophobic interactions provides a tunable physical network. The presented investigation serves as a basis for generating ELR hydrogels with tunable viscoelastic properties promising for tissue regeneration, through an ‘‘in-situ”, rapid, scalable, economically and feasible one-pot method.

Advanced technologies for marine drugs large scale supply

2024-01-01T00:00:00Z

Marine organisms represent a source of unique chemical entities with valuable biomedical potentialities, broad diversity and complexity. It is essential to ensure a reliable and sustainable supply of marine natural products (MNPs) for their translation into commercial drugs and other valuable products. From a structural point of view and with few exceptions, MNPs of pharmaceutical importance derive from the so-called secondary metabolism of marine organisms. When production strategies relied on marine macroorganism, harvesting or culturing coupled with extraction procedures re-main sometimes the only alternative to produce these compounds at industrial scale. Their supply can be often implemented with laboratory scale cultures for bacterial, fungal or microalgal sources. However, a diverse approach, combining traditional methods with a modern synthetic biology and biosynthesis strategies, must be considered for invertebrate MNPs, as they are usually naturally accumulated in only very small quantities. This review offers a comprehensive examination of various production strategies for MNPs, addressing the challenges related to supply, synthesis, and scalability. It also underscores recent biotechnological advancements that are likely to transform the current industrial-scale manufacturing methods for pharmaceuticals derived from marine sources.

Structural characterization of a homopolysaccharide produced by Weissella cibaria FMy 2-21-1 and its potential application as a green corrosion inhibiting film

2024-01-01T00:00:00Z

Steel corrosion is a global issue that affects safety and the economy. Currently, the homopolysaccharide (HoPS) structure of a novel lactic acid bacterium (LAB) is under study, as well as its application as a green corrosion inhibitor. Weissella cibaria FMy 2-21-1 is a LAB strain capable of producing HoPS in sucrose enriched media. The isolated and purified HoPS was characterized by different spectroscopic analyses as a linear α-1,6 dextran adopting a random coil conformation, with high molecular weight and extended size in water. The polysaccharide showed a semi-crystalline organization, which is a requirement for film formation. Its biocoating showed a grainy network structure, with a slightly lesser hydrophobic role in the aqueous environment than in the ionic one. The electrochemical measurements of the steel-HoPS coating showed that the biopolymer layer acts as an anodic-type corrosion inhibitor, with high resistance to corrosion by water and with chloride ions which prevent pitting, a corrosion process typical of bare steel. Few reports have cited the application of LAB HoPS as corrosive coating inhibitors. This work is the first to explore the influence of a structurally characterized dextran from Weissella cibaria strain as a potential steel corrosion inhibitor in ionic environments.

CD44-targeted nanoparticles for co-delivery of docetaxel and an Akt inhibitor against colorectal cancer

2024-01-01T00:00:00Z

New strategies to develop drug-loaded nanocarriers with improved therapeutic efficacy are needed for cancer treatment. Herein we report a novel drug-delivery nanosystem comprising encapsulation of the chemotherapeutic drug docetaxel (DTX) and recombinant fusion of a small peptide inhibitor of Akt kinase within an elastinlike recombinamer (ELR) vehicle. This combined approach is also precisely targeted to colorectal cancer cells by means of a chemically conjugated DNA aptamer specific for the CD44 tumor marker. This 53 nm dual-approach nanosystem was found to selectively affect cell viability (2.5 % survival) and proliferation of colorectal cancer cells in vitro compared to endothelial cells (50 % survival), and to trigger both apoptosis- and necrosis-mediated cell death. Our findings also show that the nanohybrid particles remain stable under physiological conditions, trigger sustained drug release and possess an adequate pharmacokinetic profile after systemic intravenous administration. In vivo assays showed that these dual-approach nanohybrids significantly reduced the number of tumor polyps along the colorectal tract in a murine colorectal cancer model. Furthermore, systemic administration of advanced nanohybrids induced tissue recovery by improving the morphology of gastrointestinal crypts and the tissue architecture. Taken together, these findings indicate that our strategy of an advanced dualapproach nanosystem allows us to achieve successful controlled release of chemotherapeutics in cancer cells and may have a promising potential for colorectal cancer treatment.

Bioglues based on an elastin-like recombinamer: effect of tannic acid as an additive on tissue adhesion and cytocompatibility

2023-01-01T00:00:00Z

More than 260 million surgical procedures are performed worldwide each year. Although sutures and staples are widely used to reconnect tissues, they can cause further damage and increase the risk of infection. Bioadhesives have been proposed as an alternative to reconnect tissues. However, clinical adhesives that combine strong adhesion with cytocompatibility have yet to be developed. In this study, we explored the production of adhesives based on protein-engineered polymers bioinspired by the sequence of elastin (i.e., elastin-like recombinamers, ELRs). We hypothesized that the combination of polyphenols (i.e., tannic acid, TA) and ELRs would produce an adhesive coacervate (ELR+TA), as reported for other protein polymers such as silk fibroin (SF). Notably, the adhesion of ELR alone surpassed that of ELR+TA. Indeed, ELR alone achieved adhesive strengths of 88.8 ± 33.2 kPa and 17.0 ± 2.0 kPa on porcine bone and skin tissues, respectively. This surprising result led us to explore a multicomponent bioadhesive to encompass the complementary roles of elastin (mimicked here by ELR) and silk fibroin (SF), and subsequently mirror more closely the multicomponent nature of the extracellular matrix. Tensile testing showed that ELR+SF achieved an adhesive strength of 123.3 ± 60.2 kPa on porcine bone and excellent cytocompatibility. To express this in a more visual and intuitive way, a small surface of only 2.5 cm2 was able to lift at least 2 kg of weight. This opens the door for further studies focusing on the ability of protein-engineered polymers to adhere to biological tissues without further chemical modification for applications in tissue engineering.

Production of elastin-like recombinamer-based nanoparticles for docetaxel encapsulation and use as smart drug-delivery systems using a supercritical anti-solvent process

2021-01-01T00:00:00Z

This study presents a new groundbreaking methodology for integrating innovative concepts to develop novel drug-delivery strategies. This methodology combines genetically engineered elastin-like recombinamers (ELRs) with supercritical fluid (SCF) techniques to encapsulate a poorly water-soluble drug in a one-step process. The chemotherapeutic agent docetaxel (DTX) is encapsulated with a block copolymer ELR containing the RGD peptide, a specific target sequence for cancer cells, using the supercritical anti-solvent (SAS) technique in a high process yield of up to 70%. SEM studies show spherical microparticles of 10 mm after encapsulation. After dispersion under physiological conditions, microparticles disaggregate into stable monodisperse nanoparticles of 40 nm size and - 30 mV z-potential. This protects the drug, as confirmed by NMR analysis, thereby increasing the water solubility of DTX up to fifty orders of magnitude. The delivery process is governed by the Fick diffusion mechanism and indicates that the presence of DTX on the particles surface is practically negligible. Cellular assays showed that, due to the presence of the cancer target sequence RGD, breast cancer cells were more affected than human endothelial cells, thus meaning that the strategy developed in this work opens the way to new controlled release systems more precise than non-selective chemotherapeutic drugs.

Elastin-like recombinamer-based devices releasing Kv1.3 blockers for the prevention of intimal hyperplasia: An in vitro and in vivo study

2020-01-01T00:00:00Z

Coronary artery disease (CAD) is the most common cardiovascular disorder. Vascular surgery strategies for coronary revascularization (either percutaneous or open) show a high rate of failure because of restenosis of the vessel, due to phenotypic switch of vascular smooth muscle cells (VSMCs) leading to proliferation and migration. We have previously reported that the inhibition of Kv1.3 channel function with selective blockers represents an effective strategy for the prevention of restenosis in human vessels used for coronary angioplasty procedures. However, delivery systems for controlled release of these drugs have not been investigated. Here we tested the efficacy of several formulations of elastin like recombinamers (ELRs) hydrogels to deliver the Kv1.3 blocker PAP-1 in various restenosis models. The dose and time course of PAP-1 release from ELRs click hydrogels was able to inhibit human VSMC proliferation in vitro as well as remodeling of human vessels in organ culture and restenosis in in vivo models. We conclude that this combination of active compound and advanced delivery method could improve the outcomes of vascular surgery in patients. STATEMENT OF SIGNIFICANCE: Vascular surgery strategies for coronary revascularization show a high rate of failure, because of occlusion (restenosis) of the vessel, due to vascular smooth muscle cells proliferation and migration. We have previously reported that blockers of Kv1.3 channels represent an effective anti-restenosis therapy, but delivery systems for their controlled release have not being explored. Here we tested the efficacy of several formulations of elastin like recombinamers (ELRs) hydrogels to deliver the Kv1.3 blocker PAP-1 in various restenosis models, both in vivo and in vitro, and also in human vessels. We demonstrated that combination of active compound and advanced delivery method could improve the outcomes of vascular surgery in patients.

Metronomic anti-cancer therapy: A multimodal therapy governed by the tumor microenvironment

2021-01-01T00:00:00Z

The concept of cancer as a systemic disease, and the therapeutic implications of this, has gained special relevance. This concept encompasses the interactions between tumor and stromal cells and their microenvironment in the complex setting of primary tumors and metastases. These factors determine cellular co-evolution in time and space, contribute to tumor progression, and could counteract therapeutic effects. Additionally, cancer therapies can induce cellular and molecular responses in the tumor and host that allow them to escape therapy and promote tumor progression. In this study, we describe the vascular network, tumor-infiltrated immune cells, and cancer-associated fibroblasts as sources of heterogeneity and plasticity in the tumor microenvironment, and their influence on cancer progression. We also discuss tumor and host responses to the chemotherapy regimen, at the maximum tolerated dose, mainly targeting cancer cells, and a multimodal metronomic chemotherapy approach targeting both cancer cells and their microenvironment. In a combination therapy context, metronomic chemotherapy exhibits antimetastatic efficacy with low toxicity but is not exempt from resistance mechanisms. As such, a better understanding of the interactions between the components of the tumor microenvironment could improve the selection of drug combinations and schedules, as well as the use of nano-therapeutic agents against certain malignancies.

Protease‐sensitive, VEGF‐mimetic peptide, and IKVAV laminin‐derived peptide sequences within elastin‐like recombinamer scaffolds provide spatiotemporally synchronized guidance of angiogenesis and neurogenesis

2022-01-01T00:00:00Z

Spatiotemporal control of vascularization and innervation is a desiredhallmark in advanced tissue regeneration. For this purpose, we design a 3Dmodel scaffold, based on elastin-like recombinamer (ELR) hydrogels. Thiscontains two interior and well-defined areas, small cylinders, withdifferentiated bioactivities with respect to the bulk. Both are constructed on aprotease sensitive ELR with a fast-proteolyzed domain, but one bears aVEGF-mimetic peptide (QK) and the other a laminin-derived pentapeptide(IKVAV), to promote angiogenesis and neurogenesis, respectively. The outerbulk is based on a slow proteolytic sequence and RGD cell adhesion domains.In vitro studies show the effect of QK and IKVAV peptides on the promotion ofendothelial cell and axon spreading, respectively. The subcutaneousimplantation of the final 3D scaffold demonstrates the ability tospatiotemporally control angiogenesis and neurogenesis in vivo. Specifically,the inner small cylinder containing the QK peptide promotes fastendothelialization, whereas the one with IKVAV peptide promotes fastneurogenesis. Both, vascularization and innervation take place in advance ofthe bulk scaffold infiltration. This scaffold shows that it is possible to inducevascularization and innervation in predetermined areas of the scaffold wellahead to the bulk infiltration. That significantly increases the efficiency of theregenerative activity.

Fast and reversible crosslinking of a silk elastin-like polymer

2022-01-01T00:00:00Z

Elastin-like polymers (ELPs) and their chimeric subfamily the silk elastin-like polymers (SELPs) exhibit a lower critical solvation temperature (LCST) behavior in water which has been extensively studied from theoretical, computational and experimental perspectives. The inclusion of silk domains in the backbone of the ELPs effects the molecular dynamics of the elastin-like domains in response to increased temperature above its transition temperature and confers gelation ability. This response has been studied in terms of initial and long-term changes in structures, however, intermediate transition states have been less investigated. Moreover, little is known about the effects of reversible hydration on the elastin versus silk domains in the physical crosslinks. We used spectroscopic techniques to analyze initial, intermediate and long-term states of the crosslinks in SELPs. A combination of thermoanalytical and rheological measurements demonstrated that the fast reversible rehydration of the elastin motifs adjacent to the relatively small silk domains was capable of breaking the silk physical crosslinks. This feature can be exploited to tailor the dynamics of these types of crosslinks in SELPs.

Mineralizing coating on 3D printed scaffolds for the promotion of osseointegration

2022-01-01T00:00:00Z

Design and fabrication of implants that can perform better than autologous bone grafts remain an unmet challenge for the hard tissue regeneration in craniomaxillofacial applications. Here, we report an integrated approach combining additive manufacturing with supramolecular chemistry to develop acellular mineralizing 3D printed scaffolds for hard tissue regeneration. Our approach relies on an elastin-like recombinamer (ELR) coating designed to trigger and guide the growth of ordered apatite on the surface of 3D printed nylon scaffolds. Three test samples including a) uncoated nylon scaffolds (referred to as “Uncoated”), b) ELR coated scaffolds (referred to as “ELR only”), and c) ELR coated and in vitro mineralized scaffolds (referred to as “Pre- mineralized”) were prepared and tested for in vitro and in vivo performance. All test samples supported normal human immortalized mesenchymal stem cell adhesion, growth, and differentiation with enhanced cell proliferation observed in the “Pre- mineralized” samples. Using a rabbit calvarial in vivo model, ‘Pre-mineralized’ scaffolds also exhibited higher bone ingrowth into scaffold pores and cavities with higher tissue- implant integration. However, the coated scaffolds (“ELR only” and “Pre-mineralized”) did not exhibit significantly more new bone formation compared to “Uncoated” scaffolds. Overall, the mineralizing coating offers an opportunity to enhance integration of 3D printed bone implants. However, there is a need to further decipher and tune their immunologic response to develop truly osteoinductive/conductive surfaces.

Mesenchymal stromal cells combined with elastin-like recombinamers increase angiogenesis in vivo after hindlimb ischemia

2022-01-01T00:00:00Z

Hindlimb ischemia is an unmet medical need, especially for those patients unable to undergo vascular surgery. Cellular therapy, mainly through mesenchymal stromal cell (MSC) administration, may be a potentially attractive approach in this setting. In the current work, we aimed to assess the potential of the combination of MSCs with a proangiogenic elastin-like recombinamer (ELR)–based hydrogel in a hindlimb ischemia murine model. Human bone marrow MSCs were isolated from four healthy donors, while ELR biomaterials were genetically engineered. Hindlimb ischemia was induced through ligation of the right femoral artery, and mice were intramuscularly injected with ELR biomaterial, 0.5 × 106 MSCs or the combination, and also compared to untreated animals. Tissue perfusion was monitored using laser Doppler perfusion imaging. Histological analysis of hindlimbs was performed after hematoxylin and eosin staining. Immunofluorescence with anti–human mitochondria antibody was used for human MSC detection, and the biomaterial was detected by elastin staining. To analyze the capillary density, immunostaining with an anti–CD31 antibody was performed. Our results show that the injection of MSCs significantly improves tissue reperfusion from day 7 (p = 0.0044) to day 21 (p = 0.0216), similar to the infusion of MSC + ELR (p = 0.0038, p = 0.0014), without significant differences between both groups. After histological evaluation, ELR hydrogels induced minimal inflammation in the injection sites, showing biocompatibility. MSCs persisted with the biomaterial after 21 days, both in vitro and in vivo. Finally, we observed a higher blood vessel density when mice were treated with MSCs compared to control (p<0.0001), but this effect was maximized and significantly different to the remaining experimental conditions when mice were treated with the combination of MSCs and the ELR biomaterial (p < 0.0001). In summary, the combination of an ELR-based hydrogel with MSCs may improve the angiogenic effects of both strategies on revascularization of ischemic tissues.

Protein-Based Films Functionalized with a Truncated Antimicrobial Peptide Sequence Display Broad Antimicrobial Activity

2021-01-01T00:00:00Z

The increasing bacterial resistance to antibiotics is driving strong demand for new antimicrobial biomaterials. This work describes the fabrication of free-standing films exhibiting antimicrobial properties by combining, in the same polypeptide chain, an elastin-like recombinamer comprising 200 repetitions of the pentamer VPAVG (A200) and an 18-amino-acid truncated variant of the antimicrobial peptide BMAP-28, termed BMAP-18. The fusion protein BMAP-18A200 was overexpressed and conveniently purified by a simplified and scalable nonchromatographic process. Free-standing films of BMAP-18A200 demonstrated to be stable without requiring cross-linking agents and displayed high antimicrobial activity against skin pathogens including Gram-negative and Gram-positive bacteria as well as unicellular and filamentous fungi. The antimicrobial activity of the films was mediated by direct contact of cells with the film surface, resulting in compromised structural integrity of microbial cells. Furthermore, the BMAP-18A200 films showed no cytotoxicity on normal human cell lines (skin fibroblasts and keratinocytes). All of these results highlight the potential of these biotechnological multifunctional polymers as new drug-free materials to prevent and treat microbial infections.