Littérature scientifique sur le sujet « Elastin-Like Polypeptides (ELP) »

Thermo-responsive elastin-like polypeptides (ELPs) were successfully obtained by inverse transition cycling (ITC) and recursive directional ligation (RDL). Six ELPs displayed thermal properties, depending on their sequence and chain length. It was found that the ELP[KV8F-4 and ELP[KV8F-8 were effective as thermosensitive materials at the body temperature with phase transition temperature from 35 to 45oC.

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

To increase treatment efficiency for glioblastoma, we have developed a system to selectively deliver chemotherapeutic doxorubicin (Dox) to Glioblastoma (GBM) tumors. This carrier is based on elastin-like polypeptide (ELP), which is soluble at physiological temperatures but undergoes a phase transition and accumulates at tumor sites with externally applied, mild (40–41 °C) hyperthermia. The CPP-ELP-Dox conjugate consists of a cell penetrating peptide (CPP), which facilitates transcytosis through the blood brain barrier and cell entry, and a 6-maleimidocaproyl hydrazone derivative of doxorubicin at the C-terminus of ELP. The acid-sensitive hydrazone linker ensures release of Dox in the lysosomes/endosomes after cellular uptake of the drug conjugate. We have shown that CPP-ELP-Dox effectively inhibits cell proliferation in three GBM cell lines. Both the free drug and CPP-ELP-Dox conjugate exhibited similar in vitro cytotoxicity, although their subcellular localization was considerably different. The Dox conjugate was mainly dispersed in the cytoplasm, while free drug had partial nuclear accumulation in addition to cytoplasmic distribution. The intracellular Dox concentration was increased in the CPP-ELP-Dox cells compared to that in the cells treated with free Dox, which positively correlates with cytotoxic activity. In summary, our findings demonstrate that CPP-ELP-Dox effectively kills GBM cells. Development of such a drug carrier has the potential to greatly improve current therapeutic approaches for GBM by increasing the specificity and efficacy of treatment and reducing cytotoxicity in normal tissues.

Elastin-like peptide (ELP) constructs are typically hydrophobic in nature. A rapid organic solvent extraction method is reported that affords highly pure ELP with removal of key contaminants such as nucleic acids and LPS.

Abstract Extracellular matrix (ECM) including fibronectin (FN) and elastin plays a pivotal role in providing a microenvironment to support tissue regeneration in stem cell therapy. To develop a novel biomimetic ECM for stem cell differentiation, we engineered FN type III 9 and 10 domains fused to elastin-like polypeptides (FN-ELPs). The recombinant FN-ELP fusion protein was expressed in Escherichia coli and purified by inverse transition cycling. Human mesenchymal stem cells (hMSCs) cultured on plates coated with FN-ELP had significantly greater adhesion activity and proliferation than cells grown on non-coated plates. FN-ELP induced the osteogenic differentiation by elevating alkaline phosphatase (ALP) and mineralization activity of hMSCs. Furthermore, the osteogenic marker gene expressions of ALP, collagen type I (Col I), osteopontin (OPN), and transcriptional coactivator with a PDZ-binding motif (TAZ) were increased in hMSCs cultured on plates coated with FN-ELP. We reported a novel biomimetic ECM with potential for bone regeneration that promotes the osteogenic differentiation of hMSCs.

Elastin-like polypeptides (ELPs) are well known for their elastic and thermoresponsive behaviors. Their elasticity originates from the formation of a β-spiral which is the consequence of stacking type-II β-turns, formed from individual VPGVG pentapeptide units. Here, the synthesis of ELPs of varying chain lengths [VPGVG, (VPGVG)2, and (VPGVG)4] and their coupling to a mechanoresponsive catalyst are reported. The attached ELP chains can act as “molecular springs,” allowing for an efficient uptake and transmission of an applied force to the mechanophoric bond. This leads to stress-induced activation of the mechanophoric catalyst, in turn transforming mechanical energy into a “click” reaction. Secondary structure analysis via IR and CD spectroscopy revealed that the β–spiral formation of the ELP is not affected by the coupling process and the β–spiral is still intact in the mechanocatalyst after the coupling. Mechanochemical activation of the synthesized catalysts by an external applied force, studied via ultrasonication, showed conversions of the copper(I)-catalyzed alkyne-azide “click” reaction (CuAAC) up to 5.6% with an increasing chain length of the peptide, proving the potential to incorporate this chemistry into biomaterial engineering.

Contact lenses are widely prescribed for vision correction, and as such they are an attractive platform for drug delivery to the anterior segment of the eye. This manuscript explores a novel strategy to drive the reversible adsorption of peptide-based therapeutics using commercially available contact lenses. To accomplish this, thermo-sensitive elastin-like polypeptides (ELPs) alone or tagged with a candidate ocular therapeutic were characterized. For the first time, this manuscript demonstrates that Proclear CompatiblesTM contact lenses are a suitable platform for ELP adsorption. Two rhodamine-labelled ELPs, V96 (thermo-sensitive) and S96 (thermo-insensitive), were employed to test temperature-dependent association to the contact lenses. During long-term release into solution, ELP coacervation significantly modulated the release profile whereby more than 80% of loaded V96 retained with a terminal half-life of ~4 months, which was only 1–4 days under solubilizing conditions. A selected ocular therapeutic candidate lacritin-V96 fusion (LV96), either free or lens-bound LV96, was successfully transferred to HCE-T cells. These data suggest that ELPs may be useful to control loading or release from certain formulations of contact lenses and present a potential for this platform to deliver a biologically active peptide to the ocular surface via contact lenses.

Physically crosslinked hydrogels with thixotropic properties attract considerable attention in the biomedical research field because their self-healing nature is useful in cell encapsulation, as injectable gels, and as bioinks for three-dimensional (3D) bioprinting. Here, we report the formation of thixotropic hydrogels containing nanofibers of double-hydrophobic elastin-like polypeptides (ELPs). The hydrogels are obtained with the double-hydrophobic ELPs at 0.5 wt%, the concentration of which is an order of magnitude lower than those for previously reported ELP hydrogels. Although the kinetics of hydrogel formation is slower for the double-hydrophobic ELP with a cell-binding sequence, the storage moduli G′ of mature hydrogels are similar regardless of the presence of a cell-binding sequence. Reversible gel–sol transitions are demonstrated in step-strain rheological measurements. The degree of recovery of the storage modulus G′ after the removal of high shear stress is improved by chemical crosslinking of nanofibers when intermolecular crosslinking is successful. This work would provide deeper insight into the structure–property relationships of the self-assembling polypeptides and a better design strategy for hydrogels with desired viscoelastic properties.

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

Antigene e anticorpo sono i due reagenti chiave di un saggio immunodiagnostico. L’indagine di nuove tecniche e il miglioramento di processi quali la purificazione e la marcatura sito-specifica di antigeni e anticorpi possono promuovere lo sviluppo di nuovi reagenti più efficienti capaci di migliorare la performance dei saggi immunodiagnostici. La prima parte di questa tesi è stata focalizzata sull’esplorazione di tecniche biotecnologiche innovative nella produzione di antigeni al fine di migliorare i saggi per la rilevazione degli anticorpi IgM e IgG specifici per il virus Epstein-Barr. Il virus EBV è causa della mononucleosi infettiva ed è associato ad un crescente numero di tumori; per questa motivo è importante sviluppare saggi diagnostici per la rilevazione di EBV ad alta specificità e sensibilità. La proteina VCA p18 è uno degli antigeni più importanti per la diagnosi di EBV. I saggi attuali Diasorin LIAISON EBV VCA IgM and IgG si basano su un singolo antigene corrispondente alla regione C-terminale immunodominante della proteina p18, immobilizzata su fase solida. I vari metodi esplorati in questa tesi hanno permesso di ottenere diverse varianti dell’antigene p18 con lo scopo di migliorare le prestazioni dei saggi EBV VCA IgM e IgG a diversi livelli: 1_produzione dell’antigene p18; 2_immobilizzazione dell’antigene p18 su fase solida; 3_formato di saggio. 1_La lunghezza della regione C-terminale della proteina p18 (57aa), risulta essere considerevole per il processo sintetico ma, allo stesso tempo, troppo piccola per essere prodotta in modo efficiente per via ricombinante. Per superare questo problema, abbiamo esplorato il sistema Elastin Like Polypeptides (ELP)-Inteina basato sull’uso di una proteina capace di effettuare auto-cleavage (inteina) e un tag responsivo alla temperatura (ELP). Questa tecnica si è rivelata un eccellente sistema per la produzione del peptide p18. 2_Lo sviluppo di diverse varianti dell’antigene p18 ha permesso di esplorare varie tecniche per l’immobilizzazione dello stesso antigene su fase solida: coating covalente diretto, attraverso il complesso streptavidina-biotina e attraverso l’uso dei peptidi leucine zipper (o velcro). L’immobilizzazione del peptide p18 su fase solida attraverso questi vari metodi è avvenuta con successo e l’attività immunochimica dell’antigene, immobilizzato con queste tecniche innovative, è comparabile o migliore rispetto a quella del peptide sintetico utilizzato nei saggi attuali. 3_Nonostante il saggio Diasorin LIAISON EBV VCA IgM abbia una buona performance analitica, al fine di ottenere un aumento di specificità, è stato esplorato un nuovo tipo di formato di saggio. Sfortunatamente i risultati indicano che questo diverso tipo di formato raggiunge un livello di specificità minore rispetto a quello del saggio attuale. La seconda parte di questa tesi è stata focalizzata sull’esplorazione di un metodo innovativo per la marcatura sito specifica degli anticorpi. Uno degli approcci più promettenti è basato sulla generazione di gruppi tiolo liberi attraverso la riduzione parziale e selettiva dei ponti disolfuro inter-catena presenti a livello della “hinge region” e la loro reazione con molecole marcanti caratterizzate dal possedere gruppi funzionali reattivi verso i gruppi sulfidrilici. Questa tecnologia è stata utilizzata per la biotinilazione di due diversi anticorpi usati attualmente per il rilevamento della proteina virale p24 di HIV e per l’antigene FGF23. I risultati suggeriscono che la biotinilazione sito-specifica rispetto a quella classica random promuove un miglioramento dell'attività immunochimica degli anticorpi con una conseguente ottimizzazione della performance dei saggi immunodiagnostici.
Antigen and antibody are the two key reagents for an immunodiagnostic assay. Investigation of new techniques and improvement of processes such as purification and site-specific labeling of antigen and antibody molecules can promote the development of new more powerful bioreagents able of improving the performance of immunodiagnostic assays. The first part of this thesis aimed to explore innovative biotechnology techniques in antigen production for the improvement of immunoassays that allow the detection of antibodies directed against the Epstein-Barr virus. EBV virus is the causative agent of infectious mononucleosis and it is considered to be associated with a still increasing number of tumors; for this reason it is important to develop diagnostic assays for EBV detection with high specificity and sensitivity. The viral capsid protein VCA p18 is one of the most important antigens for the diagnosis of EBV. The current Diasorin LIAISON EBV VCA IgM and IgG assays rely on a single antigen, consisting in a synthetic peptide corresponding to the immunodominant C-terminal portion of the p18 protein, which is immobilized on solid phase (indirect format). The several methods explored in this thesis have allowed to obtain different variants of the p18 antigen with the aim to improve the performance of DiaSorin LIAISON EBV VCA IgM and IgG assays at different levels: 1_production of p18 antigen; 2_immobilization of p18 antigen on solid phase; 3_immunoassay format. 1_ The length of the immunodominant C-terminal portion of the p18 protein (57aa) appears to be considerable for the synthetic route but, at the same time, too small to be effectively produced in a recombinant fashion. To overcome this problem, we explored the Elastin Like Polypeptides (ELP)-Intein system, a method based on the use of a self-cleavable protein (the intein) and a temperature responsive tag (ELP). This technique has proved to be an excellent system for the preparation of the p18 peptide. 2_The development of different variants of p18 antigen has enabled to explore different techniques for the immobilization of the same antigen on solid phase: direct covalent coating, through streptavidin-biotin complex and through an innovative procedure based on the use of leucine zipper (or “velcro”) peptides. The immobilization of the p18 peptide on solid phase through these different methods occurred successfully and the immunochemical activity of the antigen, immobilized with these innovative techniques, is comparable or better than that of the synthetic peptide used in the current immunoassays. 3_Despite the DiaSorin LIAISON EBV VCA IgM immonoassay has a good analytical performance, in order to obtain an increase of specificity, a new assay format was explored. Unfortunately, the results indicate that this different type of format reaches a lower level of specificity than that of current assay. The second part of this thesis aimed to explore an innovative method for the site-specific labeling of antibodies. One the most promising approach is based on the generation of free thiol groups by selective partial reduction of the interchain disulfide bridges present at the level of the “hinge region” and their reaction to labels carrying sulfhydryl-reactive chemical groups. This technology was used for the biotinylation of two different antibodies currently used in immunoassays for the HIV p24 viral protein and for FGF23 antigen detection. The results suggest that the site-specific biotinylation compared to the random traditional biotinylation promotes a great improvement of antibodies immunochemical activity with a consequent optimization of immunodiagnostic assays performance.

The specific manner in which ions associate with a peptide surface is a subject of much research. The models currently proposed tend to rely either on computational results from overly simplified systems, or on observations of bulk solution behavior not applicable to peptide solvation. Herein we demonstrate a new platform for directly measuring specific ion interactions with peptides and use a pair of highly conserved model peptides to investigate specific mechanisms by which ions interact with a peptide surface. A system for investigation of charge selective ion-peptide interactions using a conical glass nanopore was designed. The glass nanopore was coated using layer-by-layer depositions of poly(diallyldimethylammonium) chloride and sodium poly(styrenesulfonate) to control the size and charge selectivity of the nanopore. The tip of this nanopore probe was encapsulated in a 5% agarose gel to prevent peptide fouling. This probe was then used to measure the partitioning of cations to or from the surface of two model peptides: nonpolar V5-120 and positively charged KV6-112 elastin-like polypeptide (ELP). Partitioning was measured by clamping the current through the pore at zero amps and measuring the resulting potential across the nanopore. This potential was used to determine the bulk concentration of electrolyte in a 1 mg/mL peptide in 0.1 M electrolyte solution. Measurements were made with a patch clamp using chloride salts with the cations potassium, lithium, cesium, ammonium, and guanidinium at both room temperature and in an ice bath to ensure that the peptides were in their unfolded state and thus that all possible binding sites were exposed to the solution. All salts were observed to partition to the peptide surface with much less affinity than water, resulting in an increase in the bulk electrolyte concentration with the exception of ammonium, which showed a greater affinity than water for the KV6-112 ELP in the ice bath measurements. These results demonstrate that cations do not favorably partition to nonpolar or cationic peptide surfaces.