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There is growing concern about the toxicity of colloidal aluminum salts used as adjuvants in subcutaneous allergen immunotherapy (SCIT). Therefore, alternative adjuvants and delivery systems are being explored to replace alum in SCIT. We applied micellar elastin-like polypeptides (ELPs), a type of self-assembling protein, to replace alum as vaccine adjuvant in birch pollen SCIT. ELP and an ELP-Bet v 1 fusion protein were expressed inE. coliand purified by immuno-affinity chromatography and inverse-transition cycling (ITC). Nanoparticles self-assembled from ELP and a 9:1 ELP/ELP-Bet v 1 mixture were characterized by using dynamic light scattering and atomic force microscopy. Allergenicity was assessed by measuring mediator release from rat basophilic leukemia cells transformed with the human FcϵR1 and sensitized with sera derived from human birch pollen allergic patients. Humoral and T-cell immunity were investigated by immunizing naïve mice with the ELP/ELP-Bet v 1 nanoparticles or alum-adsorbed Bet v 1, both containing 36 µg Bet v 1. ELP and ELP/ELP-Bet v 1 self-assembled at 37°C into spherically shaped micelles with a diameter of ~45 nm. ELP conjugation made Bet v 1 hypo-allergenic (10-fold). Compared to alum-adsorbed Bet v 1, ELP/ELP-Bet v 1 nanoparticles induced stronger IgG responses with an earlier onset. Additionally, ELP/ELP-Bet v 1 did not induce Th2 skewing cytokines and IgE. The hypoallergenic character and strong humoral immune response in the absence of a Th2-skewing T-cell response make ELP-based nanoparticles a promising candidate to replace alum in SCIT.

In recent years, antimicrobial peptides (AMPs) have become a promising alternative to the use of conventional and chemically synthesized antibiotics, especially after the emergence of multidrug-resistant organisms. Thus, this review aims to provide an updated overview of the state-of-the-art for producing antimicrobial peptides fused or conjugated with the elastin-like (ELP) peculiar carriers, and that are mostly intended for biomedical application. The elastin-like biopolymers are thermosensitive proteins with unique properties. Due to the flexibility of their modular structure, their features can be tuned and customized to improve the production of the antimicrobial domain while reducing their toxic effects on the host cells. Both fields of research faced a huge rise in interest in the last decade, as witnessed by the increasing number of publications on these topics, and several recombinant fusion proteins made of these two domains have been already described but they still present a limited variability. Here in, the approaches described to recombinantly fuse and chemically conjugate diverse AMPs with ELPs are reviewed, and the nature of the AMPs and the ELPs used, as well as the main features of the expression and production systems are summarized. This article is protected by copyright. All rights reserved.

Glioblastoma (GBM) is a primary brain tumor that carries a dismal prognosis, which is primarily attributed to tumor recurrence after surgery and resistance to chemotherapy. Since the tumor recurrence appears near the site of surgical resection, a concept of immediate and local application of chemotherapeutic after initial tumor removal could lead to improved treatment outcome. With the ultimate goal of developing a locally-applied, injectable drug delivery vehicle for GBM treatment, we created elastin-like polypeptide (ELP) hydrogels. The ELP hydrogels can be engineered to release anti-cancer drugs over an extended period. The purpose of this study was to evaluate the biomechanical properties of ELP hydrogels, to characterize their ability to release doxorubicin over time, and to investigate, in vitro, the anti-proliferative effect of Dox-laden ELP hydrogels on GBM. Here, we present microstructural differences, swelling ratio measurements, drug release characteristics, and in vitro effects of different ELP hydrogel compositions. We found that manipulation of the ELP–collagen ratio allows for tunable drug release, that the released drug is taken up by cells, and that incubation with a small volume of ELP-Dox hydrogel drastically reduced survival and proliferation of GBM cells in vitro. These results underscore the potential of ELP hydrogels as a local delivery strategy to improve prognosis for GBM patients after tumor resection.

Silk fibroin (SF) is considered biocompatible and biodegradable for osteochondral repair. However, it lacks a bioactive domain for cell adhesion, proliferation and differentiation, limiting its therapeutic efficacy. To revamp SF as a biomimicking and bioactive microenvironment to regulate cell behaviours, we engineered an elastin-like polypeptide (ELP, Val-Pro-Gly-Xaa-Gly) to modify SF fibers via simple and green dehydrothermal (DHT) treatment. Our results demonstrated that the ELP successfully bound to SF, and the scaffold was reinforced by the fusion of the silk fiber intersections with ELP (S-ELP-DHT) via the DHT treatment. Both bone mesenchymal stem cells (BMSCs) and chondrocytes exhibited improved spreading and proliferation on the S-ELP-DHT scaffolds. The ex vivo and in vivo experiments further demonstrated enhanced mature bone and cartilage tissue formation using the S-ELP-DHT scaffolds compared to the naked SF scaffolds. These results indicated that a recombinant ELP-modified silk scaffold can mimic three-dimensional (3D) cell microenvironment, and improve bone and cartilage regeneration. We envision that our scaffolds have huge clinical potential for osteochondral repair., Graphical abstract Schematic illustration showing the S-ELP-DHT scaffolds can be fabricated into fibrocartilage or bone mimic by seeding with chondrocytes or BMSCs for osteochondral regeneration.Image 1, Highlights • Elastin-like polypeptide (ELP) modified silk fibroin (SF) scaffold was developed via dehydrothermal treatment (S-ELP-DHT). • The S-ELP-DHT scaffold provided a beneficial cell microenvironment for osteochondral repair. • Greater mature bone and cartilage tissue formation were achieved. • Improved repair efficacy for articular osteochondral defects was confirmed.

Capsids of the cowpea chlorotic mottle virus (CCMV) are great candidates for the development into in vivo catalytic or therapeutic nanocarriers. However, due to their limited intrinsic stability at physiological pH, thus far no methods exist for incorporating cargo into these nanoparticles in cellulo. Here, we employ a stabilized VW1-VW8 ELP-CCMV variant for the development of a co-expression-based cargo-loading approach. Co-expression of the non-functionalized VW1-VW8 ELP-CCMV coat protein with fusion proteins with enhanced green fluorescent protein (mEGFP) and pyrrolysine synthase D (PylD) in E. coli enabled the purification of cargo-loaded capsids from the bacteria directly either via affinity chromatography or PEG-precipitation and subsequent size exclusion chromatography. Microscopy results indicated that the co-expression does not harm the E. coli cells and that proper folding of the mEGFP domain is not hampered by the co-assembly. Our co-expression strategy is thus a suitable approach to produce cargo-loaded CCMV nanoparticles.

The synthesis of ultra-small gold nanoclusters (Au NCs) with sizes down to 2 nm has received increasing interest due to their unique optical and electronic properties. Like many peptide-coated gold nanospheres synthesized before, modified gold nanoclusters with peptide conjugation are potentially significant in biomedical and catalytic fields. Here, we explore whether such small-sized gold nanoclusters can be conjugated with peptides also and characterize them using atomic force microscopy. Using a long and flexible elastin-like polypeptide (ELP)20 as the conjugated peptide, (ELP)20-Au NCs was successfully synthesized via a one-pot synthesis method. The unique optical and electronic properties of gold nanoclusters are still preserved, while a much larger size was obtained as expected due to the peptide conjugation. In addition, a short and rigid peptide (EAAAK)3 was conjugated to the gold nanoclusters. Their Yong’s modulus was characterized using atomic force microscopy (AFM). Moreover, the coated peptide on the nanoclusters was pulled using AFM-based single molecule-force spectroscopy (SMFS), showing expected properties as one of the first force spectroscopy experiments on peptide-coated nanoclusters. Our results pave the way for further modification of nanoclusters based on the conjugated peptides and show a new method to characterize these materials using AFM-SMFS.

The anticancer agent doxorubicin(dox) has been widely used in the treatment of a variety of hematological malignancies and solid tumors. Despite doxorubicin’s efficiency in killing tumor cells, severe damage to healthy tissues, along with cardiotoxicity, limits its clinical use. To overcome these adverse side effects, improve patient safety, and enhance therapeutic efficacy, we have designed a thermally responsive biopolymer doxorubicin carrier that can be specifically targeted to tumor tissue by locally applying mild hyperthermia (41 °C). The developed drug vehicle is composed of the following: a cell penetrating peptide (SynB1) to promote tumor and cellular uptake; thermally responsive Elastin-like polypeptide (ELP); and the (6-maleimidocaproyl) hydrazone derivative of doxorubicin (DOXO-EMCH) containing a pH-sensitive hydrazone linker that releases doxorubicin in the acidic tumor environment. We used the in vivo imaging system, IVIS, to determine biodistribution of doxorubicin-delivered ELP in MDA-MB-231 xenografts in nude mice. Tumor bearing mice were treated with a single IV injection of 10 mg/kg doxorubicin equivalent dose with free doxorubicin, thermally responsive SynB1 ELP 1-DOXO, and a thermally nonresponsive control biopolymer, SynB1 ELP 2-DOXO. Following a 2 h treatment with hyperthermia, tumors showed a 2-fold higher uptake when treated with SynB1 ELP 1-DOXO compared to free doxorubicin. Accumulation of the thermally non-responsive control SynB1 ELP2 –DOXO was comparable to free doxorubicin, indicating that an increase in dox accumulation with ELP is due to aggregation in response to thermal targeting. Higher levels of SynB1 ELP1–DOXO and SynB1 ELP2 –DOXO with respect to free doxorubicin were observed in kidneys. Fluorescence intensity from hearts of animals treated with SynB1 ELP1–DOXO show a 5-fold decrease in accumulation of doxorubicin than the same dose of free doxorubicin. SynB1-ELP1-DOXO biopolymers demonstrated a 6-fold increase in tumor/heart ratio in comparison to free doxorubicin, indicating preferential accumulation of the drug in tumors. These results demonstrate that thermally targeted polymers are a promising therapy to enhance tumor targeting and uptake of anticancer drugs and to minimize free drug toxicity in healthy tissues, representing a great potential for clinical application.

Wound repair is a fascinatingly complex process, with overlapping events in both space and time needed to pave a pathway to successful healing. This additional complexity presents challenges when developing methods for the controlled delivery of therapeutics for wound repair and tissue engineering. Unlike more traditional applications, where biomaterial-based depots increase drug solubility and stability in vivo, enhance circulation times, and improve retention in the target tissue, when aiming to modulate wound healing, there is a desire to enable localised, spatiotemporal control of multiple therapeutics. Furthermore, many therapeutics of interest in the context of wound repair are sensitive biologics (e.g. growth factors), which present unique challenges when designing biomaterial-based delivery systems. Here, we review the diverse approaches taken by the biomaterials community for creating stimuli-responsive materials that are beginning to enable spatiotemporal control over the delivery of therapeutics for applications in tissue engineering and regenerative medicine.

Background and Aim recombinant DNA technique is a powerful and appropriate method for the production of protein biopolymers with specificity in amino acid sequence and spatial chemistry. Elastin-Like Polypeptide (ELP) is a biocompatible, biodegradable and non-immunological biopolymer used in various biotechnology studies. The ELP tag is a cheap, fast and non-chromatographic technique for purifying target proteins. In this study, pET expression vector was designed for the combination of ELP gene sequences and target recombinant protein in order to produce recombinant fusion protein with the ELP tag. Methods & Materials MOD gene was transformed to E. coli-BL21 (DE3) cells after designing and synthesis among the XbaI and XhoI restriction sites in the pET-32a (+) vector of the clone. Then, colonies were isolated based on plasmid size and examined by cutting using restriction enzymes. The final recombinant colonies was verified using polymerase chain reaction method and DNA sequencing. Ethical Considerations The Research Ethics Committee of Arak University of Medical Sciences approved all ethical considerations ofworking on laboratory animals (Code: 92-146-11). Results Replacing the MOD sequence in the pET-32a vector (+) eliminated the components expressing the fusion tags (Thioredoxin, Histidine, and S-tag), the identification site of protease enzyme (tobacco etch virus), and multiple cloning site. In addition, it added specific restriction enzyme identification sequences of ELP gene and target gene. As a result, in the optimized pET-MODvector, 466 nucleotides reduced in size and the secondary structure was improved. Conclusion Considering the improvement of spatial structure and reduction of pET-MOD vector size, as well as the possibility of the fusion of recombinant protein with the ELP tag, it is possible to use this vector for ELPyation of the target protein.

Proliferative Vitreoretinopathy (PVR) is a refractory retinal disease whose primary pathogenesis involves the epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells. At present, there is no effective treatment other than surgery for PVR. The purpose of this study was to investigate the effect of αB crystallin peptide (αBC-P) on EMT in PVR. We have previously shown that this peptide is antiapoptotic and regulates RPE redox status. Subconfluent primary human RPE (hRPE) cells were stimulated by TGFβ2 (10 ng/mL) with or without αBC-P (50 or 75 μg/mL) for 48 h and expression of EMT/mesenchymal to epithelial transition (MET) markers was determined. Mitochondrial ROS (mtROS) generation in hRPE cells treated with TGFβ2 was analyzed. The effect of TGFβ2 and αBC-P on oxidative phosphorylation (OXPHOS) and glycolysis in hRPE was studied. RPE cell migration was also assessed. A PVR-like phenotype was induced by intravitreal dispase injection in C57BL/6J mice. PVR progression and potential therapeutic efficiency of αBC-Elastin-like polypeptides (ELP) was studied using fundus photography, OCT imaging, ERG, and histologic analysis of the retina. αSMA, E-cadherin, Vimentin, Fibronectin and, RPE65, and CTGF were analyzed on Day 28. Additionally, the amount of VEGF-A in retinal cell lysates was measured. The EMT-associated αSMA, Vimentin, SNAIL and SLUG showed a significant upregulation with TGFβ2, and their expression was significantly suppressed by cotreatment with αBC-P. The MET-associated markers, E-cadherin and Sirt1, were significantly downregulated by TGFβ2 and were restored by αBC-P. Incubation of hRPE with TGFβ2 for 24 h showed a marked increase in mitochondrial ROS which was noticeably inhibited by αBC-ELP. We also showed that after TGFβ2 treatment, SMAD4 translocated to mitochondria which was blocked by αBC-ELP. Mitochondrial oxygen consumption rate increased with TGFβ2 treatment for 48 h, and αBC-P co-treatment caused a further increase in OCR. Glycolytic functions of RPE were significantly suppressed with αBC-P (75 μg/mL). In addition, αBC-P significantly inhibited the migration from TGFβ2 treatment in hRPE cells. The formation of proliferative membranes was suppressed in the αBC-ELP-treated group, as evidenced by fundus, OCT, and H&E staining in dispase-induced PVR in mice. Furthermore, ERG showed an improvement in c-wave amplitude. In addition, immunostaining showed significant suppression of αSMA and RPE65 expression. It was also observed that αBC-ELP significantly reduced the expression level of vimentin, fibronectin, and CTGF. Our findings suggest that the antioxidant αBC-P may have therapeutic potential in preventing PVR by reversing the phenotype of EMT/MET and improving the mitochondrial function in RPE cells.

Regenerative medicine using stem cells offers promising strategies for treating a variety of degenerative diseases. Regulation of stem cell behavior and rejuvenate senescence are required for stem cells to be clinically effective. The extracellular matrix (ECM) components have a significant impact on the stem cell’s function and fate mimicking the local environment to maintain cells or generate a distinct phenotype. Here, human elastin-like polypeptide-based ECM-mimic biopolymer was designed by incorporating various cell-adhesion ligands, such as RGD and YIGSR. The significant effects of bioactive fusion ELPs named R-ELP, Y-ELP, and RY-ELP were analyzed for human bone-marrow-derived stem cell adhesion, proliferation, maintenance of stemness properties, and differentiation. Multivalent presentation of variable cell-adhesive ligands on RY-ELP polymers indeed promote efficient cell attachment and proliferation of human fibroblast cells dose-dependently. Similarly, surface modified with RY-ELP promoted strong mesenchymal stem cell (MSCs) attachment with greater focal adhesion (FA) complex formation at 6 h post-incubation. The rate of cell proliferation, migration, population doubling time, and collagen I deposition were significantly enhanced in the presence of RY-ELP compared with other fusion ELPs. Together, the expression of multipotent markers and differentiation capacity of MSCs remained unaffected, clearly demonstrating that stemness properties of MSCs were well preserved when cultured on a RY-ELP-modified surface. Hence, bioactive RY-ELP offers an anchorage support system and effectively induces stimulatory response to support stem cell proliferation.

Elastin-like polypeptides (ELPs) undergo a characteristic phase transition in response to ambient temperature. Therefore, it has been be used as a thermosensitive vector for the delivery of chemotherapy agents since it can be used to target hyperthermic tumors. This novel strategy introduces unprecedented options for treating cancer with fewer concerns about side effects. In this study, the ELP system was further modified with an enzyme-cleavable linker in order to release drugs within tumors. This system consists of an ELP, a matrix metalloproteinase (MMP) substrate, a cell-penetrating peptide (CPP), and a 6-maleimidocaproyl amide derivative of doxorubicin (Dox). This strategy shows up to a 4-fold increase in cell penetration and results in more death in breast cancer cells compared to ELP-Dox. Even in doxorubicin-resistant cells (NCI/ADR and MES-SA/Dx5), ELP-released cell-penetrating doxorubicin demonstrated better membrane penetration, leading to at least twice the killing of resistant cells compared to ELP-Dox and free Dox. MMP-digested CPP-Dox showed better membrane penetration and induced more cancer cell death in vitro. This CPP-complexed Dox released from the ELP killed even Dox-resistant cells more efficiently than both free doxorubicin and non-cleaved ELP-CPP-Dox.

Proteins display an essential role in numerous natural systems due to their structural and biological properties. Given their unique properties, proteins have been thoroughly investigated in the last few decades, offering a myriad of solutions for the development of innovative biomaterials, including films, foams, composites and gels, in particular for biomedical applications such as drug delivery systems, biosensors and scaffolds for tissue regeneration. In this context, this review intends to give a general overview of the potential of protein based materials within the sustainable polymers context, covering aspects ranging from protein types, selection/isolation to properties of protein based (nano) materials and biomedical applications, passing through preparation methodologies of materials.

The autoimmune disorder, Sj&ouml, gren&rsquo, s syndrome (SS), is characterized by lymphocytic infiltration and loss of function of exocrine glands such as the lacrimal gland (LG) and salivary gland. SS-associated changes in the LG are associated with the development of autoimmune-mediated dry eye disease. We have previously reported the accumulation of intercellular adhesion molecule 1 (ICAM-1) in the LG of Non-Obese Diabetic (NOD) mice, a murine model of autoimmune-mediated dry eye in SS, in both LG acinar cells and infiltrating lymphocytes. ICAM-1 initiates T-cell activation and can trigger T-cell migration through binding to lymphocyte function-associated 1 antigen (LFA). To modulate this interaction, this study introduces a new tool, a multivalent biopolymeric nanoparticle assembled from a diblock elastin-like polypeptide (ELP) using the S48I48 (SI) ELP scaffold fused with a mouse ICAM-1 targeting peptide to form IBP-SI. IBP-SI forms a multivalent, monodisperse nanoparticle with a radius of 21.9 nm. Unlike the parent SI, IBP-SI binds mouse ICAM-1 and is internalized by endocytosis into transfected HeLa cells before it accumulates in lysosomes. In vitro assays measuring lymphocyte adhesion to Tumor Necrosis Factor TNF-&alpha, treated bEnd.3 cells, which express high levels of ICAM-1, show that adhesion is inhibited by IBP-SI but not by SI, with IC50 values of 62.7 &mu, M and 81.2 &mu, M, respectively, in two different assay formats. IBP-SI, but not SI, also blocked T-cell proliferation in a mixed lymphocyte reaction by 74% relative to proliferation in an untreated mixed cell reaction. These data suggest that a biopolymeric nanoparticle with affinity for ICAM-1 can disrupt ICAM-1 and LFA interactions in vitro and may have further utility as an in vivo tool or potential therapeutic.

Duck viral hepatitis type I (DVH I) is a lethal disease in ducklings caused by duck hepatitis A virus (DHAV). Although the commercial vaccine is available for vaccination of one-day-old ducklings or breeder ducks, the disease is still prevalent due to the delayed immune response in ducklings and variable maternal antibody levels in breeder duck flocks. To explore the feasibility of duck interferon-α (DuIFN-α) for control of DVH I, DuIFN-α was expressed as an elastin-like polypeptide (ELP) fusion protein (ELP-DuIFN-α) in E. coli and purified by inverse phase transition cycling (ITC). After detection of its cytotoxicity, bioactivity, plasma stability and serum half-life, the protective efficacy of ELP-DuIFN-α against DHAV-1 infection of embryos or ducklings was evaluated using different treatment routes at different infection times. The results show that ELP-DuIFN-α was correctly expressed and purified to more than 90% purity after two cycles of ITC. The purified fusion protein had a specific anti-DHAV-1 activity of 6.0 × 104 IU/mg protein, significantly extended plasma stability and serum half-life without overt cytotoxicity. After allantoic injection with ELP-DuIFN-α pre-infection, co-infection or post-infection with DHAV-1, 5/5, 5/5 or 4/5 embryos survived from the virus challenge. After intramuscular injection or oral administration with ELP-DuIFN-α, 3/5 or 4/5 ducklings survived from co-infection with DHAV-1. After oral administration with ELP-DuIFN-α pre-infection, co-infection or post-infection with DHAV-1, 3/5, 4/5 or 4/5 ducklings survived from the virus challenge, and the relative transcription levels of interferon-stimulated genes were significantly higher than the normal control group and virus challenge control group (p < 0.01). These experimental data suggest that ELP-DuIFN-α can be used as a long-lasting anti-DHAV-1 reagent.

Although doxorubicin (dox), an anthracycline antibiotic, is widely used and effective in treating cancer, its treatment efficiency is limited by low blood plasma solubility, poor pharmacokinetics, and adverse side effects, including irreversible cardiotoxicity. Moreover, cancer cells often develop drug resistance over time, which decreases the efficacy of anti-cancer drugs, including dox. In this study, we examine a macromolecular drug delivery system for its ability to specifically deliver doxorubicin to cancer cells with and without drug resistance. This drug delivery system consists of a multi-part macromolecule, which includes the following: elastin-like polypeptide (ELP), cell penetrating peptide (CPP), a cleavable linker (releasing at low pH), and a derivative of doxorubicin. ELP is thermally responsive and improves drug solubility, while the CPP mediates cellular uptake of macromolecules. We compared cytotoxicity of two doxorubicin derivatives, where one is cleavable (DOXO) and contains a pH-sensitive linker and releases dox in an acidic environment, and the other is non-cleavable (ncDox) doxorubicin. Cytotoxicity, apoptosis, cell cycle distribution and mechanism of action of these constructs were tested and compared between dox-responsive MCF-7 and dox-resistant NCI/ADR cell lines. Dox delivered by the ELP construct is comparably toxic to both sensitive and drug resistant cell lines, compared to unconjugated doxorubicin, and given the pharmacokinetic and targeting benefits conveyed by conjugation to ELP, these biopolymers have potential to overcome dox resistance in vivo.

Modification of protein-based drug carriers with tumor-targeting properties is an important area of research in the field of anticancer drug delivery. To this end, we developed nanoparticles comprised of elastin-like polypeptides (ELPs) with fused poly-aspartic acid chains (ELP-D) displaying DNA aptamers. DNA aptamers were enzymatically conjugated to the surface of the nanoparticles via genetic incorporation of Gene A* protein into the sequence of the ELP-D fusion protein. Gene A* protein, derived from bacteriophage ϕX174, can form covalent complexes with single-stranded DNA via the latter's recognition sequence. Gene A* protein-displaying nanoparticles exhibited the ability to deliver the anticancer drug paclitaxel (PTX), whilst retaining activity of the conjugated Gene A* protein. PTX-loaded protein nanoparticles displaying DNA aptamers known to bind to the MUC1 tumor marker resulted in increased cytotoxicity with MCF-7 breast cancer cells compared to PTX-loaded protein nanoparticles without the DNA aptamer modification.

The cowpea chlorotic mottle virus (CCMV) is a nanoparticle that holds promise for diagnostic and therapeutic applications. The empty virus-like particle, however, is not stable under physiological conditions. Here, we describe a systematic study into the expansion of the assembly properties of a protein based block copolymer of the CCMV capsid protein and an elastin-like polypeptide. By systematically changing the hydrophobicity of the stimulus-responsive elastin-like polypeptide block, assembly of the capsid proteins could be achieved at close to physiological conditions. This strategy may prove to be useful in the development of a physiologically stable CCMV capsid variant. (C) 2017 Elsevier Ltd. All rights reserved.

Notch pathway was found to be activated in most glioblastomas (GBMs), underlining the importance of Notch in formation and recurrence of GBM. In this study, a Notch inhibitory peptide, dominant negative MAML (dnMAML), was conjugated to elastin-like polypeptide (ELP) for tumor targeted delivery. ELP is a thermally responsive polypeptide that can be actively and passively targeted to the tumor site by localized application of hyperthermia. This complex was further modified with the addition of a cell penetrating peptide, SynB1, for improved cellular uptake and blood–brain barrier penetration. The SynB1–ELP1–dnMAML was examined for its cellular uptake, cytotoxicity, apoptosis, cell cycle inhibition and the inhibition of target genes’ expression. SynB1–ELP1–dnMAML inhibited the growth of D54 and U251 cells by inducing apoptosis and cell cycle arrest, especially in the presence of hyperthermia. Hyperthermia increased overall uptake of the polypeptide by the cells and enhanced the resulting pharmacological effects of dnMAML, showing the inhibition of targets of Notch pathway such as Hes-1 and Hey-L. These results confirm that dnMAML is an effective Notch inhibitor and combination with ELP may allow thermal targeting of the SynB1–ELP1–dnMAML complex in cancer cells while avoiding the dangers of systemic Notch inhibition.

Dengue is a viral disease that represents a significant threat to global public health since billions of people are now at risk of infection by this mosquito-borne virus. The implementation of extensive screening tests is indispensable to control this disease, and the Dengue virus non-structural protein 1 (NS1) is a promising antigen for the serological diagnosis of dengue fever. Plant-based systems can be a safe and cost-effective alternative for the production of dengue virus antigens. In this work, two strategies to produce the dengue NS1 protein in Nicotiana benthamiana leaves were evaluated: Targeting NS1 to five different subcellular compartments to assess the best subcellular organelle for the expression and accumulation of NS1, and the addition of elastin-like polypeptide (ELP) or hydrophobin (HFBI) fusion tags to NS1. The transiently expressed proteins in N. benthamiana were quantified by Western blot analysis. The NS1 fused to ELP and targeted to the ER (NS1 ELP-ER) showed the highest yield (445 mg/kg), approximately a forty-fold increase in accumulation levels compared to the non-fused protein (NS1-ER), representing the first example of transient expression of DENV NS1 in plant. We also demonstrated that NS1 ELP-ER was successfully recognized by a monoclonal anti-dengue virus NS1 glycoprotein antibody, and by sera from dengue virus-infected patients. Interestingly, it was found that transient production of NS1-ER and NS1 ELP-ER using vacuum infiltration of whole plants, which is easier to scale up, rather than syringe infiltration of leaves, greatly improved the accumulation of NS1 proteins. The generated plant made NS1, even without extensive purification, showed potential to be used for the development of the NS1 diagnostic tests in resource-limited areas where dengue is endemic.

A new, bifunctional recombinant protein was expressed as the fusion product of human elastin-like polypeptide (HELP) and the bilirubin-binding protein UnaG. The engineered product displays both the HELP-specific property of forming a functional hydrogel matrix and the UnaG-specific capacity of emitting green fluorescence upon ligand binding. The new fusion protein has been proven to be effective at detecting bilirubin in complex environments with high background noise. A cell culture model of the stress response, consisting of bilirubin released in the cell culture medium, was set up to assess the bilirubin-sensing properties of the functional matrix obtained by cross-linking the HELP moiety. Our engineered protein allowed us to monitor cell induction by the release of bilirubin in the culture medium on a nanomolar scale. This study shows that elastin-like protein fusion represents a versatile platform for the development of novel and commercially viable analytical and biosensing devices.

Elastin-like polypeptides (ELPs) are promising candidates for fabricating tissue-engineering scaffolds that mimic the extracellular environment of elastic tissues. We have developed a &ldquo, double-hydrophobic&rdquo, block ELP, GPG, inspired by non-uniform distribution of two different hydrophobic domains in natural elastin. GPG has a block sequence of (VGGVG)5-(VPGXG)25-(VGGVG)5 that self-assembles to form nanofibers in water. Functional derivatives of GPG with appended amino acid motifs can also form nanofibers, a display of the block sequence&rsquo, s robust self-assembling properties. However, how the block length affects fiber formation has never been clarified. This study focuses on the synthesis and characterization of a novel ELP, GPPG, in which the central sequence (VPGVG)25 is repeated twice by a short linker sequence. The self-assembly behavior and the resultant nanostructures of GPG and GPPG were when compared through circular dichroism spectroscopy, atomic force microscopy, and transmission electron microscopy. Dynamic rheology measurements revealed that the nanofiber dispersions of both GPG and GPPG at an extremely low concentration (0.034 wt%) exhibited solid-like behavior with storage modulus G&prime, &gt, loss modulus G&rdquo, over wide range of angular frequencies, which was most probably due to the high aspect ratio of the nanofibers that leads to the flocculation of nanofibers in the dispersion.

The combination of natural polysaccharides and recombinant elastin-like polypeptides (ELPs) into block copolymers is expected to lead to materials with precise stimuli-responsive self-assembly properties and bioactivities. Herein, we report the synthesis of a series of polysaccharide-b-ELP bioconjugates, in which 4 different hydrophilic polysaccharides were coupled to the N-terminal end of an ELP via “click chemistry”. The resulting bioconjugates were characterized by 1D and 2D NMR, SEC and FTIR. Their thermal sensitivity and temperature-triggered self-assembly in aqueous solution were investigated by UV-Vis spectrometry, DLS, SLS, SANS and temperature-controlled liquid AFM. This study demonstrated that polysaccharide-b-ELP bioconjugates can self-assemble into well-defined nanoparticles in aqueous condition above a specific and tunable transition temperature (Tt) and reversibly disassemble below the Tt, which make them particularly promising candidates for the design of controlled drug delivery nanocarriers. Chemoselective functionalizations of the ELP segment at methionine residues using oxaziridine chemistry were additionally applied for further tuning of bioconjugates’ properties.; L’association de polysaccharides naturels et de polypeptides recombinants de type élastine (ELP) dans des structures de copolymères à blocs doit permettre d’accéder à des matériaux possédant des propriétés d’auto-assemblage sous l’action de stimuli et potentiellement bioactifs. Nous avons réalisé et présentons ici la synthèse d'une série de bioconjugués polysaccharide-b-ELP, dans lesquels 4 polysaccharides hydrophiles différents ont été couplés à l'extrémité N-terminale de l’ELP par "chimie click".Ces bioconjugués ont été caractérisés par RMN 1D et 2D, SEC et FTIR. Leur thermo-sensibilité et leur auto-assemblage induit par la température ont été étudiés par spectroscopie UV-Visible, DLS, SLS, SANS et AFM liquide à température contrôlée. Cette étude a démontré que les bioconjugués polysaccharide-b-ELP peuvent s'auto-assembler en milieu aqueux en nanoparticules bien définies au-dessus d'une température de transition spécifique et modulable (Tt) et se désassembler de façon réversible sous la Tt, ce qui les rend particulièrement prometteurs pour la conception de vecteurs de principes actifs à libération contrôlée. La fonctionnalisation chimiosélective des résidus méthionine du segment ELP par chimie de l'oxaziridine a également été exploitée pour moduler les propriétés des bioconjugués.

The combination of natural polysaccharides and recombinant elastin-like polypeptides (ELPs) into block copolymers is expected to lead to materials with precise stimuli-responsive self-assembly properties and bioactivities. Herein, we report the synthesis of a series of polysaccharide-b-ELP bioconjugates, in which 4 different hydrophilic polysaccharides were coupled to the N-terminal end of an ELP via “click chemistry”. The resulting bioconjugates were characterized by 1D and 2D NMR, SEC and FTIR. Their thermal sensitivity and temperature-triggered self-assembly in aqueous solution were investigated by UV-Vis spectrometry, DLS, SLS, SANS and temperature-controlled liquid AFM. This study demonstrated that polysaccharide-b-ELP bioconjugates can self-assemble into well-defined nanoparticles in aqueous condition above a specific and tunable transition temperature (Tt) and reversibly disassemble below the Tt, which make them particularly promising candidates for the design of controlled drug delivery nanocarriers. Chemoselective functionalizations of the ELP segment at methionine residues using oxaziridine chemistry were additionally applied for further tuning of bioconjugates’ properties., L’association de polysaccharides naturels et de polypeptides recombinants de type élastine (ELP) dans des structures de copolymères à blocs doit permettre d’accéder à des matériaux possédant des propriétés d’auto-assemblage sous l’action de stimuli et potentiellement bioactifs. Nous avons réalisé et présentons ici la synthèse d'une série de bioconjugués polysaccharide-b-ELP, dans lesquels 4 polysaccharides hydrophiles différents ont été couplés à l'extrémité N-terminale de l’ELP par "chimie click".Ces bioconjugués ont été caractérisés par RMN 1D et 2D, SEC et FTIR. Leur thermo-sensibilité et leur auto-assemblage induit par la température ont été étudiés par spectroscopie UV-Visible, DLS, SLS, SANS et AFM liquide à température contrôlée. Cette étude a démontré que les bioconjugués polysaccharide-b-ELP peuvent s'auto-assembler en milieu aqueux en nanoparticules bien définies au-dessus d'une température de transition spécifique et modulable (Tt) et se désassembler de façon réversible sous la Tt, ce qui les rend particulièrement prometteurs pour la conception de vecteurs de principes actifs à libération contrôlée. La fonctionnalisation chimiosélective des résidus méthionine du segment ELP par chimie de l'oxaziridine a également été exploitée pour moduler les propriétés des bioconjugués.

Elastin-like polypeptides (ELP) are versatile protein biopolymers used in drug delivery due to their modular nature, allowing fusion of therapeutics and targeting agents. We previously developed an ELP fusion with vascular endothelial growth factor (VEGF) and demonstrated its therapeutic efficacy in translational swine models of renovascular disease and chronic kidney disease. The goal of the current work was to refine renal targeting and reduce off-target tissue deposition of ELP&ndash, VEGF. The ELP&ndash, VEGF fusion protein was modified by adding a kidney-targeting peptide (KTP) to the N-terminus. All control proteins (ELP, KTP&ndash, ELP, ELP&ndash, VEGF, and KTP&ndash, ELP&ndash, VEGF) were also produced to thoroughly assess the effects of each domain on in vitro cell binding and activity and in vivo pharmacokinetics and biodistribution. KTP&ndash, VEGF was equipotent to ELP&ndash, VEGF and free VEGF in vitro in the stimulation of primary glomerular microvascular endothelial cell proliferation, tube formation, and extracellular matrix invasion. The contribution of each region of the KTP&ndash, VEGF protein to the cell binding specificity was assayed in primary human renal endothelial cells, tubular epithelial cells, and podocytes, demonstrating that the VEGF domain induced binding to endothelial cells and the KTP domain increased binding to all renal cell types. The pharmacokinetics and biodistribution of KTP&ndash, VEGF and all control proteins were determined in SKH-1 Elite hairless mice. The addition of KTP to ELP slowed its in vivo clearance and increased its renal deposition. Furthermore, addition of KTP redirected ELP&ndash, VEGF, which was found at high levels in the liver, to the kidney. Intrarenal histology showed similar distribution of all proteins, with high levels in blood vessels and tubules. The VEGF-containing proteins also accumulated in punctate foci in the glomeruli. These studies provide a thorough characterization of the effects of a kidney-targeting peptide and an active cytokine on the biodistribution of these novel biologics. Furthermore, they demonstrate that renal specificity of a proven therapeutic can be improved using a targeting peptide.

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&ndash, 41 &deg, 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.

Jeremy WD McGowan,1 Qingmei Shao,1 Parminder JS Vig,1,2 Gene L Bidwell III1,2 1Department of Neurology, 2Department of Biochemistry, University of Mississippi Medical Center, Jackson, MS, USA Abstract: Bypassing the blood–brain barrier is one of the primary considerations when designing compounds intended to function in the central nervous system (CNS). Intranasal (IN) administration of otherwise blood–brain barrier impermeable molecules can result in high CNS concentrations and low systemic accumulation, indicating that IN administration may be a useful method of delivering therapeutics to the CNS. Elastin-like polypeptide (ELP) is a large, non-immunogenic, highly manipulable biopolymer with extensive evidence supporting its use as a carrier with the ability to improve drug pharmacokinetics and drug targeting. The ability of ELP to reach the CNS via IN administration has been shown previously. Previous studies have also identified the ability of cell penetrating peptides (CPPs) to increase the uptake of molecules in some instances, including via the IN route. Here, we compared and contrasted the biodistribution of ELPs with or without addition of the CPPs Tat or SynB1 via both the IN and intravenous routes. Administration of ELP via the IN route led to significant accumulation in the brain, especially in the olfactory bulbs. When injected intravenously

Cell encapsulation technology is still a challenging issue. Innovative methodologies such as additive manufacturing, and alternative bioprocesses, such as cell therapeutic delivery, where cell encapsulation is a key tool are rapidly gaining importance for their potential in regenerative medicine. Responsive materials such as elastin-based recombinant expression products have features that are particularly attractive for cell encapsulation. They can be designed and tailored to meet desired requirements. Thus, they represent promising candidates for the development of new concept-based materials that can be employed in this field. Areas covered: An overview of the design and employment of elastin-like polypeptides for cell encapsulation is given to outline the state of the art. Special attention is paid to the design of the macromolecule employed as well as to the method of matrix formation and the biological system involved. Expert opinion: As a result of recent progress in regenerative medicine there is a compelling need for materials that provide specific properties and demonstrate defined functional features. Rationally designed materials that may adapt according to applied external stimuli and that are responsive to biological systems, such as elastin-like polypeptides, belong to this class of smart material. A run through the components described to date represents a good starting point for further advancement in this area. Employment of these components in cell encapsulation application will promote its advance toward 'smart cell encapsulation technology'.

Protein bodies (PBs) are highly specialized protein storage organelles in cereal seeds. PB formation in seeds initiates in the endoplasmic reticulum (ER), and depending on the plant species, PBs remain in the ER or find their way out of the ER, bypass the Golgi and end up in protein storage vacuoles (PSVs) (Khan et al., 2012). Protein bodies have been ectopically induced in leaves by producing high amounts of ER-retrieved recombinant proteins, usually by fusing them to protein tags such as Zera®, elastin-like polypeptide (ELP) and hydrophobin-I (HFBI). This article is protected by copyright. All rights reserved.

International audience; Despite significant progress in the field of biomaterials for bone repair, the lack of attention to the vascular and nervous networks within bone implants could be one of the main reasons for the delayed or impaired recovery of bone defects. The design of innovative biomaterials should improve the host capacity of healing to restore a functional tissue, taking into account that the nerve systems closely interact with blood vessels in the bone tissue. The aim of this work is to develop a cell-free and growth factor-free hydrogel capable to promote angiogenesis and innervation. To this end, we have used elastin-like polypeptides (ELPs), poly(ethylene glycol) (PEG) and increasing concentrations of the adhesion peptide IKVAV (25% (w/w) representing 1.7 mM and 50% (w/w) representing 4.1 mM) to formulate and produce hydrogels. When characterized in vitro, hydrogels have fine-tunable rheological properties, microporous structure and are biocompatible. At the biological level, 50% IKVAV composition up-regulated Runx2, Osx, Spp1, Vegfa and Bmp2 in mesenchymal stromal cells and Tek in endothelial cells, and sustained the formation of long neurites in sensory neurons. When implanted subcutaneously in mice, hydrogels induced no signals of major inflammation and the 50% IKVAV composition induced higher vessel density and formation of nervous terminations in the peripheral tissue. This novel composite has important features for tissue engineering, showing higher osteogenic, angiogenic and innervation potential in vitro, being not inflammatory in vivo, and inducing angiogenesis and innervation subcutaneously. STATEMENT OF SIGNIFICANCE: One of the main limitations in the field of tissue engineering remains the sufficient vascularization and innervation during tissue repair. In this scope, the development of advanced biomaterials that can support these processes is of crucial importance. Here, we formulated different compositions of Elastin-like polypeptide-based hydrogels bearing the IKVAV adhesion sequence. These compositions showed controlled mechanical properties, and were degradable in vitro. Additionally, we could identify in vitro a composition capable to promote neurite formation and to modulate endothelial and mesenchymal stromal cells gene expression, in view of angiogenesis and osteogenesis, respectively. When tested in vivo, it showed no signs of major inflammation and induced the formation of a highly vascularized and innervated neotissue. In this sense, our approach represents a potential advance in the development of new strategies to promote tissue regeneration, taking into account both angiogenesis and innervation.

Hydrogel-based biomaterials have been widely used for tissue engineering applications because of their high water content, swellability, and permeability, which facilitate transport and diffusion of essential nutrients, oxygen, and waste across the scaffold. These characteristics make hydrogels suitable for encapsulating cells and creating a cell supportive environment that promotes tissue regeneration when implanted in vivo. This is particularly important in the context of tissues whose intrinsic regenerative capacity is limited, such as cartilage. However, the clinical translation of hydrogels has been limited by their poor mechanical performance, low adhesive strength, uncontrolled degradation rates, and their susceptibility to bacterial colonization. Here, we introduce an elastic, antimicrobial, and adhesive hydrogel comprised of methacrylated hyaluronic acid (MeHA) and an elastin-like polypeptide (ELP), which can be rapidly photo-cross-linked in situ for the regeneration and repair of different tissues. Hybrid hydrogels with a wide range of physical properties were engineered by varying the concentrations of MeHA and ELP. In addition, standard adhesion tests demonstrated that the MeHA/ELP hydrogels exhibited higher adhesive strength to the tissue than commercially available tissue adhesives. MeHA/ELP hydrogels were then rendered antimicrobial through the incorporation of zinc oxide (ZnO) nanoparticles, and were shown to significantly inhibit the growth of methicillin-resistant Staphylococcus aureus (MRSA), as compared to controls. Furthermore, the composite adhesive hydrogels supported in vitro mammalian cellular growth, spreading, and proliferation. In addition, in vivo subcutaneous implantation demonstrated that MeHA/ELP hydrogels did not elicit any significant inflammatory response, and could be efficiently biodegraded while promoting the integration of new autologous tissue. In summary, we demonstrated for the first time that MeHA/ELP-ZnO hydrogel can be used as an adhesive and antimicrobial biomaterial for tissue engineering applications, because of its highly tunable physical characteristics, as well as remarkable adhesive and antimicrobial properties.

The biochemical properties of muscle extracellular matrix are essential for stem cell adhesion, motility, proliferation and myogenic development. Recombinant elastin-like polypeptides are synthetic polypeptides that, besides maintaining some properties of the native protein, can be tailored by fusing bioactive sequences to their C-terminal. Our laboratory synthesized several Human Elastin-Like Polypeptides (HELP) derived from the sequence of human tropoelastin. Here, we developed a novel HELP family member by fusing the elastin-like backbone to the sequence of human Epidermal Growth Factor. We employed this synthetic protein, named HEGF, either alone or in combination with other proteins of the HELP family carrying RGD-integrin binding sites, as adhesion substrate for C2C12 myoblasts and satellite cells primary cultures. Adhesion of myoblasts to HEGF-based substrates induced scattering, decreased adhesion and cytoskeleton assembly; the concomitant presence of the RGD motifs potentiated all these effects. Recombinant substrates induced myoblasts proliferation, differentiation and the development of multinucleated myotubes, thus favoring myoblasts expansion and preserving their myogenic potential. The effects induced by adhesion substrates were inhibited by AG82 (Tyrphostin 25) and herbimycin A, indicating their dependence on the activation of both the EGF receptor and the tyrosine kinase c-src. Finally, HEGF increased the number of muscle stem cells (satellite cells) derived from isolated muscle fibers in culture, thus highlighting its potential as a novel substrate for skeletal muscle regeneration strategies.

Elastin-like polypeptides (ELPs) are a class of stimuli-responsive biopolymers inspired by the intrinsically disordered domains of tropoelastin that are composed of repeats of the VPGXG pentapeptide motif, where X is a “guest residue”. They undergo a reversible, thermally triggered lower critical solution temperature (LCST) phase transition, which has been utilized for a variety of applications including protein purification, affinity capture, immunoassays, and drug delivery. ELPs have been extensively studied as protein polymers and as biomaterials, but their relationship to other disordered proteins has heretofore not been established. The biophysical properties of ELPs that lend them their unique material behavior are similar to the properties of many intrinsically disordered proteins (IDP). Their low sequence complexity, phase behavior, and elastic properties make them an interesting “minimal” artificial IDP, and the study of ELPs can hence provide insights into the behavior of other more complex IDPs. Motivated by this emerging realization of the similarities between ELPs and IDPs, this review discusses the biophysical properties of ELPs, their biomedical utility, and their relationship to other disordered polypeptide sequences.

The phase-transition temperatures of an elastin-like polypeptide (ELP) with the (GVGVP)40 sequence and solvent dipolarity/polarizability, hydrogen-bond donor acidity, and hydrogen-bond acceptor basicity in its aqueous solutions were quantified in the absence and presence of different salts (Na2SO4, NaCl, NaClO4, and NaSCN) and various osmolytes (sucrose, sorbitol, trehalose, and trimethylamine N-oxide (TMAO)). All osmolytes decreased the ELP phase-transition temperature, whereas NaCl and Na2SO4 decreased, and NaSCN and NaClO4 increased it. The determined phase-transition temperatures may be described as a linear combination of the solvent's dipolarity/polarizability and hydrogen-bond donor acidity. The linear relationship established for the phase-transition temperature in the presence of salts differs quantitatively from that in the presence of osmolytes, in agreement with different (direct and indirect) mechanisms of the influence of salts and osmolytes on the ELP phase-transition temperature.

The intervertebral disc (ND) is the most avascular and acellular tissue in the body and therefore prone to degeneration. During IVD degeneration, the balance between anabolic and catabolic processes in the disc is deregulated, amongst others leading to alteration of extracellular matrix production, abnormal enzyme activities and production of pro-inflammatory substances like cytokines. The established treatment strategy for ND degeneration consists of physiotherapy, pain medication by drug therapy and if necessary surgery. This approach, however, has shown limited success. Alternative strategies to increase and prolong the effects of bioactive agents and to reverse the process of IVD degeneration include the use of delivery systems for drugs, proteins, cells and genes. In view of the specific anatomy and physiology of the ND and depending on the strategy of the therapy, different delivery systems have been developed which are reviewed in this article. (C) 2014 Elsevier B.V. All rights reserved.

Intratumoral radiation therapy – ‘brachytherapy’ – is a highly effective treatment for solid tumors, particularly prostate cancer. Current titanium seed implants, however, are permanent and are limited in clinical application to indolent malignancies of low- to intermediate-risk. Attempts to develop polymeric alternatives, however, have been plagued by poor retention and off-target toxicity due to degradation. Herein, we report on a new approach whereby thermally sensitive micelles composed of an elastin-like polypeptide (ELP) are labeled with the radionuclide 131-Iodine to form an in situ hydrogel that is stabilized by two independent mechanisms: first, body heat triggers the radioactive ELP micelles to rapidly phase transition into an insoluble, viscous coacervate in under 2 minutes; second, the high energy β-emissions of 131-Iodine further stabilize the depot by introducing crosslinks within the ELP depot over 24 hours. These injectable brachytherapy hydrogels were used to treat two aggressive orthotopic tumor models in athymic nude mice: a human PC-3M-luc-C6 prostate tumor and a human BxPc3-luc2 pancreatic tumor model. The ELP depots retained greater than 52% and 70% of their radioactivity through 60 days in the prostate and pancreatic tumors with no appreciable radioactive accumulation (≤ 0.1% ID) in off-target tissues after 72 hours. The 131I-ELP depots achieved >95% tumor regression in the prostate tumors (n=8); with a median survival of more than 60 days compared to 12 days for control mice. For the pancreatic tumors, ELP brachytherapy (n=6) induced significant growth inhibition (p = 0.001, ANOVA) and enhanced median survival to 27 days over controls.We then demonstrated that 131I-ELP brachytherapy can work synergistically with paclitaxel chemotherapy to overcome the intrinsic resistance found in pancreatic tumors. Treating tumors with an optimized radioactivity dose of 10.0 µCi/mg and systemically administered paclitaxel nanoparticles achieved complete regression in BxPc3-luc2, MIA PaCa-2, and AsPc-1 tumor models. Moreover, responses occurred irrespective of the paclitaxel dose (between 12.5-50 mg/kg) or the formulation (Abraxane or micelle formulation). A comparative study utilizing an aggressive 5x 5Gy hypofractionated X-ray radiation produced only minor growth inhibition, with or without paclitaxel. The mechanistic underpinnings of this effect were explored in an orthotopic model to reveal the fundamental differences between 131I-ELP therapy and conventional radiotherapy. Continuous dose exposure was found to coordinate much more effectively with the temporal sensitization mechanisms of paclitaxel, as evidenced by TUNEL immunohistochemistry. Stromal collagen and cellular junctional proteins regulating interstitial permeability (Claudin-4, CD31, and VE-Cadherin) were dysregulated after 131I-ELP treatment. Fluorescent analysis of paclitaxel nanoparticles revealed significantly higher paclitaxel accumulation in brachytherapy tumors after treatment (p

The controlled self-assembly of protein cages is vital for the use of these nanocompartments in biomedical and nanotechnological applications. Recently, we showed that by combining different structural peptide elements, it is possible to assemble viral capsid proteins in distinct well-defined morphologies. In this chapter, a triblock copolypeptide is discussed, consisting of a metal ion-coordinating hexahistidine tag, a stimulus-responsive elastin-like polypeptide and a pH-responsive self-assembling viral capsid protein. This protein is able to form two different types of capsids, depending on the assembly pathway that is followed. Here, we focus on the metal ion-induced assembly process and describe the relevant experimental procedures to induce and utilize this assembly behavior.

Background: Despite the availability of a yearly vaccine and antivirals, the incidence of influenza infections remains high. The genome of the influenza virus can mutate rapidly, therefore novel influenza strains that may be resistant to the current vaccine or antivirals frequently enter the population. Because of the long production time necessary to produce a vaccine, new antivirals must be created to combat early stages of influenza outbreaks. The most effective antivirals will target a highly conserved and essential stage of virus replication. The influenza RNA-dependent RNA polymerase is a heterotrimeric complex composed of three subunits: PA, PB1, and PB2. The three components of the polymerase interact through well-defined domains and are essential for viral replication. Previously, influenza replication has been inhibited using a small synthetic peptide that mimics the interaction domain between PA and PB1 and inhibits the formation of the heterotrimeric complex. Problem and Hypothesis: Although the peptide could inhibit influenza replication, synthetic peptides are costly to produce and are not a viable option for large-scale production. This problem can often be overcome by attaching the peptide to a highly soluble carrier protein. We hypothesize that influenza replication can be inhibited by attaching a peptide, that mimics the binding domain between the PA and PB1 subunits, to a human elastin-like polypeptide (ELP) carrier protein. Methods and Results: The peptide and a nuclear localization sequence was genetically linked to a maltose binding protein (MBP) or ELP carrier protein. The MBP construct was purified by affinity chromatography using FPLC. A high yield of the ELP construct was obtained using inverse transition cycling, a method unique to ELPs because of their temperature-dependent solubility. The ELP construct was designed to be soluble at physiological temperature to limit cellular toxicity due to protein aggregation. The cytotoxicity of the ELP construct was assessed by monitoring the growth of A549 cells, a human lung epithelial cell line. The ELP construct did not have any adverse effects on A549 cell growth. Both constructs could localize to cell nuclei using their respective nuclear localization sequences and could also interact with the PA subunit, demonstrating their potential to inhibit influenza replication. Despite this, only the MBP construct was able to inhibit the replication of influenza. The MBP construct could inhibit the replication of both the H1N1 and H3N2 subtypes of influenza, indicating the recombinant protein had cross-strain activity. Conclusion: Linking a small peptide to carrier protein can result in high protein yields, however a carrier protein must be chosen that will maintain the peptides’ therapeutic activity. In this study, a small anti-influenza peptide inhibited influenza replication when attached to an MBP carrier protein, however was not able to inhibit influenza replication when attached to an ELP carrier protein. Although the peptide was ineffective when attached to this particular ELP carrier protein, different ELP proteins of various lengths and compositions may still be effective carrier proteins for an antiviral peptide. Thesis Master of Science (MSc) The influenza virus causes seasonal outbreaks each year and can have life-threatening symptoms in the young and elderly. In addition, it can rapidly mutate through antigenic drift; therefore, a new vaccine is required each year. Pandemic influenza strains can enter the population when the virus undergoes genetic reassortment by antigenic shift. However, it can take a significant amount of time to formulate a vaccine against pandemic influenzas, which means antiviral drugs are often used as the first line of defense. New antivirals to treat influenza must be developed because resistance to the current influenza antivirals has steadily increased. In this work, we developed an antiviral peptide to disrupt a critical interaction in the influenza RNA-dependent RNA polymerase and inhibit virus replication. This peptide was previously conjugated to an E. coli MBP carrier protein, which would likely not be compatible in vivo. This thesis focused on attaching the antiviral peptide to an elastin-like polypeptide protein, which mimics human tropoelastin, and should be non-immunogenic in humans.

The production of pharmaceutical proteins in plants has made much progress in recent years with the development of transient expression systems, transplastomic technology and humanizing glycosylation patterns in plants. However, the first therapeutic proteins approved for administration to humans and animals were made in plant cell suspensions for reasons of containment, rapid scale-up and lack of toxic contaminants. In this study, we have investigated the production of human interleukin-10 (IL-10) in tobacco BY-2 cell suspension and evaluated the effect of an elastin-like polypeptide tag (ELP) and a green fluorescent protein (GFP) tag on IL-10 accumulation. We report the highest accumulation levels of hIL-10 obtained with any stable plant expression system using the ELP fusion strategy. Although IL-10-ELP has cytokine activity, its activity is reduced compared to unfused IL-10, likely caused by interference of ELP with folding of IL-10. Green fluorescent protein has no effect on IL-10 accumulation, but examining the trafficking of IL-10-GFP over the cell culture cycle revealed fluorescence in the vacuole during the stationary phase of the culture growth cycle. Analysis of isolated vacuoles indicated that GFP alone is found in vacuoles, while the full-size fusion remains in the whole-cell extract. This indicates that GFP is cleaved off prior to its trafficking to the vacuole. On the other hand, IL-10-GFP-ELP remains mostly in the ER and accumulates to high levels. Protein bodies were observed at the end of the culture cycle and are thought to arise as a consequence of high levels of accumulation in the ER.

A heme-dependent peroxidase (HDP) catalyzes the ortho-hydroxylation of l-tyrosine to l-3,4-dihydroxyphenylalanine (l-DOPA) in the presence of hydrogen peroxide. l-DOPA can be used for the treatment of Parkinson's disease. In this work, to improve the catalytic efficiency, the heme-dependent peroxidase has been genetically modified with an elastin-like polypeptide (ELP). bicinchoninic acid (BCA) assay demonstrated that HDP-ELP has a higher solubility in aqueous solutions than HDP. Circular dichroism (CD) spectra showed that HDP-ELP has a higher stability than HDP. Enzyme kinetics has been investigated over a range of substrate concentrations. It has been demonstrated that HDP-ELP exhibited a catalytic efficiency 2.4 times that of HDP.

Jeremy WD McGowan,1 Qingmei Shao,1 Parminder JS Vig,1,2 Gene L Bidwell III1,2 1Department of Neurology, 2Department of Biochemistry, University of Mississippi Medical Center, Jackson, MS, USA Abstract: Bypassing the blood–brain barrier is one of the primary considerations when designing compounds intended to function in the central nervous system (CNS). Intranasal (IN) administration of otherwise blood–brain barrier impermeable molecules can result in high CNS concentrations and low systemic accumulation, indicating that IN administration may be a useful method of delivering therapeutics to the CNS. Elastin-like polypeptide (ELP) is a large, non-immunogenic, highly manipulable biopolymer with extensive evidence supporting its use as a carrier with the ability to improve drug pharmacokinetics and drug targeting. The ability of ELP to reach the CNS via IN administration has been shown previously. Previous studies have also identified the ability of cell penetrating peptides (CPPs) to increase the uptake of molecules in some instances, including via the IN route. Here, we compared and contrasted the biodistribution of ELPs with or without addition of the CPPs Tat or SynB1 via both the IN and intravenous routes. Administration of ELP via the IN route led to significant accumulation in the brain, especially in the olfactory bulbs. When injected intravenously

d-amino acid oxidase (DAAO) and catalase (CAT) have been genetically modified by fusing them to an elastin-like polypeptide (ELP). ELP-DAAO and ELP-CAT have been separately immobilized on multi-walled carbon nanotubes (MWNTs). It has been found that the secondary structures of the enzymes have been preserved. ELP-DAAO catalyzed the oxidative deamination of d-alanine, and H2O2 was evolved continuously. When the MWNT-supported enzymes were used together, the generated hydrogen peroxide of ELP-DAAO could be decomposed in situ. The catalytic efficiency of the two immobilized enzymes was more than five times greater than that of free ELP-DAAO when the ratio of immobilized ELP-CAT to immobilized ELP-DAAO was larger than 1:1.

ObjectivesHepatitis B virus (HBV) is a member of the hepadnavirus family. The HBV genome contains four genes designated as S, C, P, and X. The HBV X (HBx) gene encodes for a 16.5-kDa regulatory protein that enhances HBV replication and exerts multifunctional activities. The aim of this study is to describe the rapid and easy purification of HBx using ELP (elastin-like polypeptide) fusion protein.MethodsThe ELP–HBx fusion protein was overexpressed in Escherichia coli. Environmental sensitivity was demonstrated via turbidity and dynamic light scattering as a function of temperature. HBx was purified as an ELP fusion protein. ELPs are biopolymers of the pentapeptide repeat Val-Pro-Gly-Xaa-Gly that undergo an inverse temperature phase transition. ELP follows in temperature and salt consistency, precipitation, and solution repetition (inverse transition cycling) with polypeptide, where it purifies the protein in a simple manner.ResultsFusion proteins underwent supramolecular aggregation at 40 °C in 1 M NaCl and slowly resolubilized at subphysiologic temperatures. ELP domain proteolysis liberated a peptide of comparable size and immunoreactivity to the commercial HBx.ConclusionThis study suggests that HBx can be purified rapidly and easily using inverse transition cycling, and that this method can be applied in determination of HBx 3D structures and HBx stability study.

New strategies to self-assemble biocompatible materials into nanoscale, drug-loaded packages with improved therapeutic efficacy are needed for nanomedicine. To address this need, we developed artificial recombinant chimeric polypeptides (CPs) that spontaneously self-assemble into sub-100 nm size, near monodisperse nanoparticles upon conjugation of diverse hydrophobic molecules, including chemotherapeutics. These CPs consist of a biodegradable polypeptide that is attached to a short Cys-rich segment. Covalent modification of the Cys residues with a structurally diverse set of hydrophobic small molecules, including chemotherapeutics leads to spontaneous formation of nanoparticles over a range of CP compositions and molecular weights. When used to deliver chemotherapeutics to a murine cancer model, CP nanoparticles have a four-fold higher maximum tolerated dose than free drug, and induce nearly complete tumor regression after a single dose. This simple strategy can promote co-assembly of drugs, imaging agents, and targeting moieties into multifunctional nanomedicines.

The design and application of effective drug carriers is a fundamental concern in the delivery of therapeutics for the treatment of cancer and other vexing health problems. Traditionally utilized chemotherapeutics are limited in efficacy due to poor bioavailability as a result of their size and solubility as well as significant deleterious effects to healthy tissue through their inability to preferentially target pathological cells and tissues, especially in treatment of cancer. Thus, a major effort in the development of nanoscopic drug delivery vehicles for cancer treatment has focused on exploiting the inherent differences in tumor physiology and limiting the exposure of drugs to non-tumorous tissue, which is commonly achieved by encapsulation of chemotherapeutics within macromolecular or supramolecular carriers that incorporate targeting ligands and that enable controlled release. The overall aim of this work is to engineer a hybrid nanomaterial system comprised of protein and silica and to characterize its potential as an encapsulating drug carrier. The synthesis of silica, an attractive nanomaterial component because it is both biocompatible as well as structurally and chemically stable, within this system is catalyzed by self-assembled elastin-like polypeptide (ELP) micelles that incorporate of a class of biologically-inspired, silica-promoting peptides, silaffins. Furthermore, this methodology produces near-monodisperse, hybrid inorganic/micellar materials under mild reaction conditions such as temperature, pH and solvent. This work studies this material system along three avenues: 1) proof-of-concept silicification (i.e. the formation and deposition of silica upon organic materials) of ELP micellar templates, 2) encapsulation and pH-triggered release of small, hydrophobic chemotherapeutics, and 3) selective silicification of templates to potentiate retention of peptide targeting ability.

Elastin-like polypeptides (ELP) and hydrophobins (HFBI) are fusion tags which enhance the accumulation levels of recombinant proteins in plants and aid in the non-chromatographic purification of these proteins. An intein is inserted between the target protein and fusion tag to replace the protease cleavage site. In this study, transient expression of intein-GFP fused to ELP or HFBI in Nicotiana benthamiana yielded the full size fusion protein. The ELP-fused proteins were successfully purified using inverse transition cycling (ITC) with 1.0 M NH4(SO4)2 at room temperature. However, the purification using membrane ITC was unsuccessful due to clogged membrane pores. Purification of HFBI-fused proteins through aqueous two-phase system (ATPS) using Triton X-114 was also unsuccessful. The proteins did not separate into the correct phase for recovery. In crude extract, cleavage of the intein at the C-terminus could be induced with a pH change, while DTT was not sufficient for cleavage at the N-terminus.

Critical limb ischemia is a major clinical problem. Despite rigorous treatment regimes, there has been only modest success in reducing the rate of amputations in affected patients. Reduced level of blood flow and enhanced inflammation are the two major pathophysiological changes that occur in the ischemic tissue. The objective of this study was to develop a controlled dual gene delivery system capable of delivering therapeutic plasmid eNOS and IL-10 in a temporal manner. In order to deliver multiple therapeutic genes, an elastin-like polypeptide (ELP) based injectable system was designed. The injectable system was comprised of hollow spheres and an in situ-forming gel scaffold of elastin-like polypeptide capable of carrying gene complexes, with an extended manner release profile. In addition, the ELP based injectable system was used to deliver human eNOS and IL-10 therapeutic genes in vivo. A subcutaneous dose response study showed enhanced blood vessel density in the treatment groups of eNOS (20 rig) and IL-10 (10 mu g)/eNOS (20 mu g) and reduced inflammation with IL-10 (10 mu g) alone. Next, we carried out a hind-limb ischemia model comparing the efficacy of the following interventions; Saline; IL-10, eNOS and IL-10/eNOS. The selected dose of eNOS, exhibited enhanced angiogenesis. IL-10 treatment groups showed reduction in the level of inflammatory cells. Furthermore, we demonstrated that eNOS up-regulated major proangiogenic growth factors such as vascular endothelial growth factors, platelet derived growth factor B, and fibroblast growth factor 1, which may explain the mechanism of this approach. These factors help in formation of a stable vascular network. Thus, ELF injectable system mediating non-viral delivery of human IL10-eNOS is a promising therapy towards treating limb ischemia. (C) 2015 Elsevier Ltd. All rights reserved. Science Foundation Ireland grant no. 07/SRC/B1163 peer-reviewed 2017-10-31

Protein bodies (PBs) are endoplasmic reticulum (ER) derived organelles found in seeds whose function is to accumulate seed storage proteins. It was shown that PB formation is not limited to seeds, and green fluorescent protein (GFP) fused to either elastin-like polypeptide (ELP), hydrophobin-I (HFBI) or Zera® fusion tags induces PBs in leaves of Nicotiana benthamiana. The mechanism by which fusion tags induce PBs is not well understood. To address how PBs form and develop in plant leaves, I studied the factors involved in their formation including recombinant protein concentration, effect of the fusion tags, PB sequestration patterns and destination in vivo. In this study I showed that PB formation is a concentration-dependent mechanism and that proteins accumulating at levels higher than 0.2% of total soluble protein are capable of inducing PBs in vivo. The presence of fusion tags is not necessary for the formation of PBs but affects their distribution pattern and size. ELP-induced PBs are larger than HFBI-induced PBs and the size of both PBs increases over time along with accumulation levels of the recombinant protein. I found that in the process of PB formation, secretory and ER resident molecules are passively sequestered into the lumen of PBs. This property of PBs was proposed as a tool to increase accumulation levels of erythropoietin and human interleukin-10 by co-expression with PB-inducing proteins. To understand whether PBs are terminally-stored cytosolic organelles or if they are connected to the ER and to each other, I developed an EGFP-based photoconversion technique, which was successfully used to visualize the trafficking of proteins targeted to the cytosol, ER, apoplast, and chloroplast in vivo. Study of PBs with this technique suggested that PBs remain associated with the ER and communicate with one another via the ER.

A wide variety of recombinant proteins has been produced in the dicot model plant, Arabidopsis thaliana. Many of these proteins are targeted for secretion by means of an N-terminal endoplasmic reticulum (ER) signal peptide. In addition, they can also be designed for ER retention by adding a C-terminal H/KDEL-tag. Despite extensive knowledge of the protein trafficking pathways, the final protein destination, especially of such H/KDEL-tagged recombinant proteins, is unpredictable. In this respect, glycoproteins are ideal study objects. Microscopy experiments reveal their deposition pattern and characterization of their N-glycans aids in elucidating the trafficking. Here, we combine microscopy and N-glycosylation data generated in Arabidopsis leaves and seeds, and highlight the lack of a decent understanding of heterologous protein trafficking.