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

Elastin-like polypeptides (ELPs) are thermally responsive biopolymers derived from natural elastin. These peptides have a low critical solution temperature phase behavior and can be used to prepare stimuli-responsive biomaterials. Through genetic engineering, biomaterials prepared from ELPs can have unique and customizable properties. By adjusting the amino acid sequence and length of ELPs, nanostructures, such as micelles and nanofibers, can be formed. Correspondingly, ELPs have been used for improving the stability and prolonging drug-release time. Furthermore, ELPs have widespread use in tissue repair due to their biocompatibility and biodegradability. Here, this review summarizes the basic property composition of ELPs and the methods for modulating their phase transition properties, discusses the application of drug delivery system and tissue repair and clarifies the current challenges and future directions of ELPs in applications. [ABSTRACT FROM AUTHOR]

L‐Asparaginase (ASP) is well‐known for its excellent efficacy in treating hematological malignancies. Unfortunately, the intrinsic shortcomings of ASP, namely high immunogenicity, severe toxicity, short half‐life, and poor stability, restrict its clinical usage. Poly(ethylene glycol) conjugation (PEGylation) of ASP is an effective strategy to address these issues, but it is not ideal in clinical applications due to complex chemical synthesis procedures, reduced ASP activity after conjugation, and pre‐existing anti‐PEG antibodies in humans. Herein, the authors genetically engineered an elastin‐like polypeptide (ELP)‐fused ASP (ASP‐ELP), a core‐shell structured tetramer predicted by AlphaFold2, to overcome the limitations of ASP and PEG‐ASP. Notably, the unique thermosensitivity of ASP‐ELP enables the in situ formation of a sustained‐release depot post‐injection with zero‐order release kinetics over a long time. The in vitro and in vivo studies reveal that ASP‐ELP possesses increased activity retention, improved stability, extended half‐life, mitigated immunogenicity, reduced toxicity, and enhanced efficacy compared to ASP and PEG‐ASP. Indeed, ASP‐ELP treatment in leukemia or lymphoma mouse models of cell line‐derived xenograft (CDX) shows potent anti‐cancer effects with significantly prolonged survival. The findings also indicate that artificial intelligence (AI)‐assisted genetic engineering is instructive in designing protein‐polypeptide conjugates and may pave the way to develop next‐generation biologics to enhance cancer treatment. [ABSTRACT FROM AUTHOR]

Elastin-like polypeptides (ELPs) are attractive materials for the green preparation of silica nanoparticles via biomimetic silicification. However, the critical factors affecting the ELP-mediated silicification remain unclear. Herein, the role of tunable amino acid residues of ELPs in silicification was studied using three ELPs (ELPs[V9F-40], ELPs[KV8F-40], and ELPs[K5V4F-40]) and their fusion proteins (ELPs[V9F-40]-SpyCatcher, ELPs[KV8F-40]-SpyCatcher, and ELPs[K5V4F-40]-SpyCatcher) with different contents of lysine residues. Bioinformatics methods were employed for the first time to reveal the key physicochemical parameters correlated with silicification. The specific activity of ELPs was increased with the promotion of lysine content with a high correlation coefficient (R = 0.899). Furthermore, exogenous acidic protein SpyCatcher would hinder the interactions between the silica precursors and ELPs, leading to the significantly decrease in specific activity. The isoelectric point (p I) of ELPs presented the highest correlation to silicification with a coefficient of 0.963. The charges of the ELPs [K5V4F-40] at different pH were calculated based on the sequence or structure. Interestingly, the excellent correlation between charges based on structure and specific activity was obtained. Collectively, the novel methods developed here may pave a new way for rational design of ELPs or other peptides for efficient and green preparation of silica nanomaterials for biomedicine, biocatalysis, and biosensor. [ABSTRACT FROM AUTHOR]

Elastin-like polypeptides (ELPs), usually as a purification-tag, can be easily expressed and rapidly purified. We found ELPs[V9F-40], which did not contain hydroxyl or charged residues with the p I of 5.52, could biomimetically form silica when they were in ordered structure. The specific activity of ELPs[V9F-40] was 70.89 ± 9.53. Besides, the time needed for the completion of biomimetic silicification was about 138 s, which was only 1/3 of that for other reported peptides. The ELPs@silica is mainly spherical and the sizes of them were around 900 nm. However, currently presented mechanisms for peptide-triggered silica biomimetic mineralization could not explain such unique phenomenon. It would pave a new way for mining or designing peptides with such function, which provide a potential green method for the preparation of biomimetic silica particles. It would endow ELPs more functions and expand the application fields of ELPs such as the bioinspired synthesis of peptide biotemplated metal or nonmetal oxide nanoparticles. Unlabelled Image [ABSTRACT FROM AUTHOR]

Bioinspired silicification is an attractive route for achieving unique silica nanocomposites. Herein, a novel, facile and inexpensive route for biosilica synthesis is developed using the stimuli-responsive elastin-like polypeptide (ELP). The ELP is precisely tailored to a silica-mineralizing peptide by programming it with lysine residues. The resulting cationic ELP[KV 8 F-40] is purified in ultrahigh yield using a chromatography-free ITC purification technique based on thermal-responsive property. Excitingly, the specific activity of ELP is 40-fold higher than that of silaffin. Besides, efficient and strong entrapment of ELP is achieved with over 98% of immobilization yield and less than 2% of leakage. These imply that cationic ELP may be used as a bifunctional tag (purification and immobilization) for fusion protein. An enzyme (xylanase) is therefore chosen to genetically fuse to ELP. The ELP-fused xylanase is purified by ELP with high purity (~98%) and enables the rapid (within minutes) self-immobilization. The immobilization yield was greater than 95%, and the immobilized xylanases hardly leaked from the silica matrix, demonstrating high efficiency of the self-immobilization process. The strategy developed here may provide a new opportunity for fabricating functional silica nanocomposites in a feasible and inexpensive pathway, which will have great potentials in the field of biotechnology. • A novel, powerful and versatile platform for rational design of peptide with function of biomineralization was developed. • An ELP with ultrahigh silica mineralization activity was the first stimuli-response peptide with silicification function. • We proposed a novel strategy for enzyme self-immobilization with high immobilization yield and extremely low leakage. • ELP functioned as both purification and immobilization tag, which may have great potentials in enzyme self-immobilization. [ABSTRACT FROM AUTHOR]

Abstract As a potent macrolide immunosuppressant, cyclosporine A (CsA) is used to treat multiple autoimmune diseases, including non-autoimmune and autoimmune-mediated dry eye disease, rheumatoid arthritis and psoriasis. Despite its potency, CsA has poor solubility, poor bioavailability, and can cause serious adverse reactions such as nephrotoxicity and neurotoxicity. To overcome these limitations, we invented a new strategy to carry CsA by fusing its cognate human receptor, cyclophilin A (CypA), to a 73 kDa elastin-like polypeptide (ELP) termed A192 using recombinant protein expression. Derived from human tropoelastin, ELPs are characterized by the ability to phase separate above a temperature that is a function of variables including concentration, molecular weight, and hydrophobicity. The resultant fusion protein, termed CA192, which assembles into a dimeric species in solution, effectively binds and solubilizes CsA with a K d of 189 nM, comparable to that of endogenous CypA with a K d of 35.5 nM. The release profile of CsA from CA192 follows a one phase decay model with a half-life of 957.3 h without a burst release stage. Moreover, CA192-CsA inhibited IL-2 expression induced in Jurkat cells through the calcineurin-NFAT signaling pathway with an IC 50 of 1.2 nM, comparable to that of free CsA with an IC 50 of 0.5 nM. The intravenous pharmacokinetics of CA192 followed a two-compartment model with a mean residence time of 7.3 h. Subcutaneous administration revealed a bioavailability of 30% and a mean residence time of 15.9 h. When given subcutaneously for 2 weeks starting at 14 weeks in male non-obese diabetic (NOD) mice, a model of autoimmune dacryoadenitis used to study Sjögren's syndrome (SS), CA192-CsA (2.5 mg/kg, every other day) significantly (p = 0.014) increased tear production relative to CA192 alone. Moreover, CA192 delivery reduced indications of CsA nephrotoxicity relative to free CsA. CA192 represents a viable new approach to deliver this effective but nephrotoxic agent in a modality that preserves therapeutic efficacy but suppresses drug toxicity. Graphical abstract Unlabelled Image [ABSTRACT FROM AUTHOR]

Abstract Although many protein fusion tags have been developed for recombinant protein production to improve protein yields or facilitate purification, determining the expression and purification of the fusion protein still remain to be a time-consuming and laborious procedure. In this work, we designed a histidine-rich elastin-like polypeptide (HRELP) fusion tag and found that it could be efficiently expressed in E. coli cells and specifically stained with Pauly's reagent in a couple of minutes post SDS-PAGE analysis. Moreover, in Pauly's reagent-stained polyacrylamide gels, only the bands of HRELP fusion proteins were yellow and could be clearly visualized with little background. Furthermore, both HRELPs and HRELP20-BMP2 fusion protein could be purified by a method of pH shift-mediated inverse transition cycling (ITC). In our opinion, the HRELP established in this study may be considered as a multifunctional protein tag which could make its fusion proteins being quickly detected by Pauly staining and simply purified by pH-triggered ITC in addition to having the potential to sustained release its fusion proteins. Highlights • A HRELP fusion tag is designed and highly expressed in E. coli. • HRELP fusion tag can be specifically and quickly stained with Pauly's reagent. • HRELP fusion protein can be detected in both cell lysate and SDS-PAGE. • HRELP fusion protein can be purified by pH-triggered ITC. [ABSTRACT FROM AUTHOR]

Highlights • We expressed three ELP-tagged and two His-tagged PoIFNs in E. col. • We optimized conditions for soluble expression and purification of ELP-tagged PoIFNs. • We compared two ELP fusion orders on PoIFN expression and bioactivity. • We demonstrated the solubility of ELP-fused of PoIFNs under the physical conditions. • We demonstrated that ELP-fusion is a feasible strategy for easy purification and half-life extension of rPoIFNs. Abstract The clinical use of recombinant interferons (rIFNs) is limited by higher purification cost and quick clearance from circulation. Elastin-like polypeptides (ELPs) are a novel tag for recombinant protein purification and half-life extension. In this study, we evaluated the feasibility of ELP fusion for simple purification and half-life extension of recombinant porcine IFNs (rPoIFNs). After construction of five different fusion expression vectors, we optimized the conditions for soluble protein expression and purification. SDS-PAGE analysis showed that, unlike PoIFNα-His and PoIFNγ-His, PoIFNα-ELP, ELP-PoIFNα and PoIFNαγ-ELP were expressed mainly as soluble proteins at 20 ℃. The optimal conditions for the inverse transition cycling (ITC) of three ELP fusion proteins were 2 M NaCl at 28 ℃. After two rounds of ITC, the three ELP fusion proteins were purified to more than 90% purities, which were comparable to that of affinity-purified PoIFNα-His and PoIFNγ-His. Cytopathic effect inhibition assay showed that the five rPoIFNs had potent but different antiviral activities against two different viruses on two different cell types. The plasma solubility assay showed that the three ELP-fused rPoIFNs remained as soluble proteins under the physical conditions. The plasma stability of three ELP-fused rPoIFNs was significantly improved in comparison with that of PoIFN-α. These data suggest that ELP fusion is a feasible strategy to enhance purification and plasma stability of rPoIFNs. [ABSTRACT FROM AUTHOR]

Musculoskeletal infections caused by bacteria such as Staphylococcus aureus and Pseudomonas aeruginosa in children and adults can lead to adverse outcomes including a need for extensive surgical debridement and limb amputation. To enable targeted antimicrobial release in infected tissues, the objective of this study was to design and investigate novel elastin-like polypeptide (ELP)-based thermally sensitive liposomes in vitro. ELP biopolymers can change their phase behaviour at higher temperatures. We hypothesised that ELP-TSL will improve therapeutic efficacy by releasing antimicrobial payloads locally at higher temperatures (≥39 °C). ELP-TSL library were formulated by varying cholesterol and phospholipid composition by the thin film and extrusion method. A broad-spectrum antimicrobial (Ciprofloxacin or Cipro) was encapsulated inside the liposomes by the ammonium sulphate gradient method. Cipro release from ELP-TSLs was assessed in physiological buffers containing ∼25% serum by fluorescence spectroscopy, and efficacy against Staphylococcus aureus and Pseudomonas aeruginosa was assessed by disc diffusion and planktonic assay. Active loading of Cipro achieved an encapsulation efficiency of 40-70% in the ELP-TSL depending upon composition. ELP-TSL Cipro release was near complete at ≥39 °C; however, the release rates could be delayed by cholesterol. Triggered release of Cipro from ELP-TSL at ∼42 °C induced significant killing of S. aureus and P. aeruginosa compared to 37 °C. Our in vitro data suggest that ELP-TSL may potentially improve bacterial wound therapy in patients. [ABSTRACT FROM AUTHOR]

Objective: To design a fusion polypeptide combining functions of self-assembly and purification for immobilizing enzymes.Results: A collagen-like polypeptide (CLP) was fused to an elastin-like polypeptide (ELP) through genetic engineering. CLP-ELP was separately fused to superoxide dismutase (SOD) and D-amino acid oxidase (DAAO). The recombinant enzymes were purified with using reversible phase transition. The interfering effect of H2O2 on the secondary structures of the recombinant enzymes was significantly reduced. The stability of the recombinant enzymes against denaturing by urea was improved. SOD-CLP-ELP exhibited a proteinaceous microporous network, and DAAO-CLP-ELP exhibited micro-clusters. The superoxide anion (•O2−) scavenging ability of SOD-CLP-ELP was 1.5 times that of SOD, and the catalytic efficiency of DAAO-CLP-ELP was 1.7 times that of DAAO.Conclusions: The advantages of the CLP-ELP-fused enzymes have been demonstrated and CLP-ELP can be used to immobilize other enzymes/proteins. [ABSTRACT FROM AUTHOR]

Many promising targeting ligands are hydrophobic peptides, and these ligands often show limited accessibility to receptors, resulting in suboptimal targeting. A systematic study to elucidate the rules for the design of linkers that optimize their presentation on nanoparticles has not been carried out to date. In this study, we recombinantly synthesized an elastin-like polypeptide diblock copolymer (ELPBC) that self-assembles into monodisperse micelles. AHNP and EC1, two hydrophobic ErbB2-targeted peptide ligands, were incorporated at the C-terminus of the ELPBC with an intervening peptide linker. We tested more than 20 designs of peptide linkers, where the linker could be precisely engineered at the gene level to systematically investigate the molecular parameters--sequence, length, and charge--of the peptide linker that optimally assist ligands in targeting the ErbB2 receptor on cancer cells. We found that peptide linkers with a minimal length of 12 hydrophilic amino acids and an overall cationic charge--and that impart a zeta potential of the micelle that is close to neutral--were necessary to enhance the uptake of peptide-modified ELPBC micelles by cancer cells that overexpress the ErbB2 receptor. This work advances our understanding of the optimal presentation of hydrophobic ligands by nanoparticles and suggests design rules for peptide linkers for targeted delivery by polymer micelles, an emerging class of nanoparticle carriers for drugs and imaging agents. [ABSTRACT FROM AUTHOR]

Intracerebral hemorrhage (ICH) frequently results in severe disabilities and high mortality. RGD-containing elastin-like polypeptide (REP), a modified elastin-like polypeptide (ELP), is a thermally responsive biopolymer. REP has high affinity for cells and is known to show non-immunotoxicity, -cytotoxicity, and -inflammatory responses. Here we found that administration of REP in the acute phase of the ICH rat model reduced the hematoma volume, decreased the number of activated microglia, attenuated the expression of von Willebrand Factor (vWF), and prevented the leakage of immunoglobulin G (IgG) into the cerebral parenchyma without any occlusion of intact microvessels. Therefore, the present data suggest that REP treatment could be a novel therapeutic strategy for attenuating the acute phase of ICH. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM PUBLISHER]

Improving drug delivery to the kidney using renal-targeted therapeutics is a promising but underdeveloped area. We aimed to develop a kidney-targeting construct for renal-specific drug delivery. Elastinlike polypeptides (ELPs) are nonimmunogenic protein-based carriers that can stabilize attached small-molecule and peptide therapeutics. We modified ELP at its NH2-terminus with a cyclic, seven-amino acid kidney-targeting peptide (KTP) and at its COOH-terminus with a cysteine residue for tracer conjugation. Comparative in vivo pharmacokinetics and biodistribution in rat and swine models and in vitro cell binding studies using human renal cells were performed. KTP-ELP had a longer plasma half-life than ELP in both animal models and was similarly accumulated in kidneys at levels fivefold higher than untargeted ELP, showing renal levels 15- to over 150-fold higher than in other major organs. Renal fluorescence histology demonstrated high accumulation of KTP-ELP in proximal tubules and vascular endothelium. Furthermore, a 14-day infusion of a high dose of ELP or KTP-ELP did not affect body weight, glomerular filtration rate, or albuminuria, or induce renal tissue damage compared with salinetreated controls. In vitro experiments showed higher binding of KTP-ELP to human podocytes, proximal tubule epithelial, and glomerular microvascular endothelial cells than untargeted ELP. These results show the high renal selectivity of KTP-ELP, support the notion that the construct is not species specific, and demonstrate that it does not induce acute renal toxicity. The plasticity of ELP for attachment of any class of therapeutics unlocks the possibility of applying ELP technology for targeted treatment of renal disease in future studies. [ABSTRACT FROM AUTHOR]

High throughput methods for recombinant protein production using E. coli typically involve the use of affinity tags for simple purification of the protein of interest. One drawback of these techniques is the occasional need for tag removal before study, which can be hard to predict. In this work, we demonstrate two high throughput purification methods for untagged protein targets based on simple and cost-effective self-cleaving intein tags. Two model proteins, E. coli beta-galactosidase (βGal) and superfolder green fluorescent protein (sfGFP), were purified using self-cleaving versions of the conventional chitin-binding domain (CBD) affinity tag and the nonchromatographic elastin-like-polypeptide (ELP) precipitation tag in a 96-well filter plate format. Initial tests with shake flask cultures confirmed that the intein purification scheme could be scaled down, with >90% pure product generated in a single step using both methods. The scheme was then validated in a high throughput expression platform using 24-well plate cultures followed by purification in 96-well plates. For both tags and with both target proteins, the purified product was consistently obtained in a single-step, with low well-to-well and plate-to-plate variability. This simple method thus allows the reproducible production of highly pure untagged recombinant proteins in a convenient microtiter plate format. [ABSTRACT FROM AUTHOR]

Objective Collagen-based scaffolds for guided bone regeneration (GBR) are continuously improved to overcome the mechanical weaknesses of collagen. We have previously demonstrated superior mechanical characteristics of the elastin-like polypeptide (ELP) reinforced collagen composites. The objectives of this research were to evaluate the efficacy of ELP-collagen composites to culture human adipose-derived stem cells (hASCs) and allow them to undergo osteogenic differentiation. We hypothesized that hASCs would show a superior osteogenic differentiation in stiffer ELP-collagen composites compared to the neat collagen hydrogels. Methods Composite specimens were made by varying ELP (0–18 mg/mL) and collagen (2–6 mg/mL) in a 3:1 ratio. Tensile strength, elastic modulus, and toughness were determined by uniaxial tensile testing. hASCs cultured within the composites were characterized by biochemical assays to measure cell viability, protein content, and osteogenic differentiation (alkaline phosphatase activity, osteocalcin, and Alizarin red staining). Scanning electron microscopy and energy dispersive spectroscopy were used for morphological characterization of composites. Results All composites were suitable for hASCs culture with viable cells over the 22-day culture period. The ELP-collagen composite with 18 mg/mL of ELP and 6 mg/mL of collagen had greater tensile strength and elastic modulus combined with higher osteogenic activity in terms of differentiation and subsequent mineralization over a period of 3 weeks compared to other compositions. The extra-cellular matrix deposits composed of calcium and phosphorous were specifically seen in the 18:6 mg/mL ELP-collagen composite. Significance The success of the 18:6 mg/mL ELP-collagen composite to achieve long-term, 3-dimensional culture and osteogenic differentiation indicates its potential as a GBR scaffold. [ABSTRACT FROM AUTHOR]

One crucial issue in stem cell therapy used for tissue repair is often the lack of selective carriers to deliver stem cells to the site of injury where the native extracellular matrix is pathologically damaged or lost. Therefore, it is necessary to develop a biomaterial that is permissive to stem cells and is suitable to replace injured or missing matrix. The major aim of this study is to investigate the potential of an R GD-containing e lastin-like p olypeptide (REP) with the structure TGPG[VGRGD(VGVPG) 6 ] 20 WPC to engraft adipose stem cells (ASC) to full-thickness excisional wounds in mice. We implanted REP into the wound defects via body temperature-induced in situ aggregation. Engrafted REP exhibited a half-life of 2.6 days in the wounds and did not elicit any pathological immune responses. REP itself significantly accelerated wound closure and reepithelialization and upregulated the expression of dermal tissue components. A combined administration of REP and ASC formed a hydrogel-like ASC/REP composite, which provided better neovascularization than the use of ASCs alone and increased the viability of transplanted ASC, improving overall wound healing. In vitro and in vivo mechanistic investigations suggested that REP enhances ASC survival at least in part via the Fak/Src adhesion-induced upregulation of Mek/Erk and PI3K/Akt survival pathways. We conclude that REP is a promising therapeutic agent for the improvement of stem cell-based therapy for enhanced tissue regeneration and repair. [ABSTRACT FROM AUTHOR]

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 I 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 min; second, the high energy β-emissions of 131 I further stabilize the depot by introducing crosslinks within the ELP depot over 24 h. These injectable brachytherapy hydrogels were used to treat two aggressive orthotopic tumor models in athymic nude mice: a human PC-3 M-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 h. The 131 I-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. [ABSTRACT FROM AUTHOR]

Objectives: An easy-to-operate method of using R-ω-transaminase has been developed by fusing it to an elastin-like polypeptide and forming a complex with D-amino acid oxidase. Results: R- ω-Transaminase ( R-ω-TA) was fused to an elastin-like polypeptide (ELP) through genetic engineering of the enzyme. The enzyme was purified through reversible phase transition. For the single-enzyme system, in the reaction media, ELP- R-ω-TA self-assembled and formed enzyme clusters of micrometer size, and the substrate, ( R)-1-phenylethylamine, also formed droplets of micrometer size. Intimate contact of the enzyme clusters and the substrate droplets provided a microenvironment of high substrate concentration close to the enzyme, facilitating the diffusion of substrate molecules into the active sites. For the two-enzyme system, ELP- R- ω-TA and ELP-fusion D-amino acid oxidase assembled to form two-enzyme complexes, forming clusters with a size much larger size than that of single enzymes. The efficiency of the combined enzymes for producing the product was 99.6 %. Conclusions: The two-enzyme complexes significantly improved the catalytic efficiency. Potentially, the two enzymes forming complex clusters can facilitate the immobilization of the two enzymes together through non covalent methods by entrapping in porous supports. [ABSTRACT FROM AUTHOR]

In an attempt to spatiotemporally control both tumor retention and the coverage of anticancer agents, we developed a photoradiation-controlled intratumoral depot (PRCITD) driven by convection enhanced delivery (CED). This intratumoral depot consists of recombinant elastin-like polypeptide (ELP) containing periodic cysteine residues and is conjugated with a photosensitizer, chlorin-e6 (Ce6) at the N-terminus of the ELP. We hypothesized that this cysteine-containing ELP (cELP) can be readily crosslinked through disulfide bonds upon exposure to oxidative agents, specifically the singlet oxygen produced during photodynamic stimulation. Upon intratumoral injection, CED drives the distribution of the soluble polypeptide freely throughout the tumor interstitium. Formation and retention of the depot was monitored using fluorescence molecular tomography imaging. When imaging shows that the polypeptide has distributed throughout the entire tumor, 660-nm light is applied externally at the tumor site. This photo-radiation wavelength excites Ce6 and generates reactive oxygen species (ROS) in the presence of oxygen. The ROS induce in situ disulfide crosslinking of the cysteine thiols, stabilizing the ELP biopolymer into a stable therapeutic depot. Our results demonstrate that this ELP design effectively forms a hydrogel both in vitro and in vivo . These depots exhibit high stability in subcutaneous tumor xenografts in nude mice and significantly improved intratumoral retention compared to controls without crosslinking, as seen by fluorescent imaging and iodine-125 radiotracer studies. The photodynamic therapy provided by the PRCITD was found to cause significant tumor inhibition in a Ce6 dose dependent manner. Additionally, the combination of PDT and intratumoral radionuclide therapy co-delivered by PRCITD provided a greater antitumor effect than either monotherapy alone. These results suggest that the PRCITD could provide a stable platform for delivering synergistic, anti-cancer drug depots. [ABSTRACT FROM AUTHOR]

Efficiently delivering therapeutic peptides or proteins for cancer therapy remains a significant challenge. Elastin-like polypeptides (ELPs), engineered peptides inspired by natural tropoelastin, have been widely used as efficient platforms for drug delivery. Herein, we fabricated a three-in-one hybrid nanosystem based on ELPs-mCherry fusion proteins for targeting delivery of antitumor peptide (PCK3145) to colon adenocarcinoma. GE11 peptide-decorated ELPs nanoparticles could preferentially bind to the EGFR-expressed colon cancers in vitro and in vivo. GE11&PCK-ELPs-mCherry treatment could trigger a 2-fold increase in apoptotic cell death compared with untargeted nanoparticles. In mice bearing colon cancer xenograft, our multifunctional nanoparticles could significantly suppress tumor growth in vivo. Our study showed the great potential of tumor-targeted ELPs nanocarriers in delivering antitumor peptides or proteins for enhanced cancer therapy. [Display omitted] • An engineered three-in-one hybrid nanosystem was developed. • GE11 peptide decoration improved delivery efficiency of antitumor peptides to colon cancer. • Multifunctional nanoparticles showed increased cytotoxicity toward colon cancer. • Hybrid nanoparticles demonstrated enhanced antitumor efficacy in mice. [ABSTRACT FROM AUTHOR]

[Display omitted] Cytokines are a group of pleiotropic proteins which are crucial for various biological processes and useful as therapeutics. However, they usually suffer from the poor stability, extreme short circulation half-life, difficulty in high-yield and large-scale production and side effects, which greatly restricts their applications. Over the past decades, conjugation of cytokines with elastin-like polypeptides (ELPs), a type of promising biomaterials, have showed great potential in solving these challenges due to ELP's thermal responsiveness, excellent biocompatibility and biodegradability, non-immunogenicity, and ease of design and control at the genetic level. This review presents recent progress in the design and production of a variety of ELP conjugated cytokines for extended circulation, enhanced stability, increased soluble protein expression, simplified purification, improved drug delivery, and controlled release. Notably, the unique thermoresponsive properties of cytokine-ELP conjugates make it possible to self-assemble into micelles with drastically extended circulatory half-life for targeted delivery or to in situ form drug depots for topical administration and controlled release. The challenges and issues in the emerging field are further discussed and the future directions are pointed out at the end of this review. [ABSTRACT FROM AUTHOR]

An elastin-like polypeptide (ELP) was fused to d -amino acid oxidases (DAAO). ELP–DAAO exhibited a better solubility in aqueous solutions than DAAO, and its enzymatic activity is about 1.6 times that of DAAO. The stability of the proteins was investigated by interacting with urea at various concentrations. The circular dichroism and fluorescence spectra were measured. The results demonstrated that that ELP–DAAO exhibited a much better stability than DAAO, and ELP–DAAO has retained the α-helix content with a high percentage even at a high urea concentration. The results of this work have demonstrated that the ELP tag can be utilized to purify DAAO, in the meantime the solubility and stability of the enzyme are improved. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A well-defined elastin-like polypeptide (ELP) block copolymer was synthesized via protein engineering for the formation of shell-crosslinked micellar particles. The block copolypeptide consisted of two domains with different transition temperatures ( T t ), with the more hydrophilic block positioned at the N-terminus. At this protein end additional lysines were introduced to facilitate crosslinking. Upon raising the temperature above the T t of the more hydrophobic block particles were formed. Two methods of crosslinking were investigated. Using genipin the lysines were directly used for particle stabilization. To be able to crosslink and functionalize the nanoparticles in an orthogonal and efficient fashion, another crosslinking strategy was developed, making use of the strain promoted azide–alkyne cycloaddition (SPAAC) reaction. For this purpose the block polypeptide was azidated making use of a diazotransfer reaction. Subsequent assembly and crosslinking using a bis-cyclooctyne reagent resulted in the formation of stable nanoparticles. Encapsulation of a hydrophobic dye showed these nanoparticles to have potential as nanocarriers. [ABSTRACT FROM AUTHOR]

Background: Pregnant females are largely overlooked in drug development due to concerns for fetal health. Additionally, pregnancy is often an exclusion criterion in clinical trials, so the safety of many drugs during pregnancy is unknown. Purpose: The goal of this study was to evaluate Elastin-like Polypeptide (ELP), a synthetic protein derived from human elastin, for maternally sequestered drug delivery. ELP is a versatile drug carrier with a long plasma half-life, low immunogenicity, and the ability to be fused to nearly any small molecule or protein-based therapeutic. Methods: We determined the pharmacokinetics, biodistribution, and fetal exposure to the ELP drug carrier using quantitative fluorescence techniques in a rat pregnancy model. Results: After either bolus IV administration or continuous infusion over five days, ELPs accumulated strongly in the kidneys, liver, and placenta, but importantly, little to no ELPs were detectable in the fetus. Within the placenta, ELPs were localized to the chorionic plate and broadly distributed within the labyrinth, but were excluded from the fetal portion of the chorionic villi. Conclusion: These data indicate that ELP does not cross the placenta, and they suggest that this adaptable drug delivery system is a promising platform for prevention of fetal drug exposure. [ABSTRACT FROM AUTHOR]

Purpose: Hepatocellular carcinoma (HCC) suffers high tumour recurrence rate after thermal ablation. Heat shock protein 90 (Hsp90) induced post-ablation is critical for tumour survival and progression. A combination therapy of thermal ablation and polymer conjugated Hsp90 chemotherapy was designed and evaluated for complete tumour eradication of HCC. Materials and methods: A thermo-responsive, elastin-like polypeptide (ELP)-based tri-block biopolymer was developed and conjugated with a potent Hsp90 inhibitor, geldanamycin (GA). The anti-cancer efficacy of conjugates was evaluated in HCC cell cultures with and without hyperthermia (43 °C). The conjugates were also administered twice weekly in a murine HCC model as a single treatment or in combination with single electrocautery as the ablation method. Results: ELP-GA conjugates displayed enhanced cytotoxicity in vitro and effective heat shock inhibition under hyperthermia. The conjugates alone significantly slowed the tumour growth without systemic toxicity. Four doses of thermo-responsive ELP-GA conjugates with concomitant simple electrocautery accomplished significant Hsp90 inhibition and sustained tumour suppression. Conclusion: Hsp90 inhibition plays a key role in preventing the recurrence of HCC, and the combination of ablation with targeted therapy holds great potential to improve prognosis and survival of HCC patients. [ABSTRACT FROM AUTHOR]

L4F, an alpha helical peptide inspired by the lipid-binding domain of the ApoA1 protein, has potential applications in the reduction of inflammation involved with cardiovascular disease as well as an antioxidant effect that inhibits liver fibrosis. In addition to its biological activity, amphipathic peptides such as L4F are likely candidates to direct the molecular assembly of peptide nanostructures. Here we describe the stabilization of the amphipathic L4F peptide through fusion to a high molecular weight protein polymer. Comprised of multiple pentameric repeats, elastin-like polypeptides (ELPs) are biodegradable protein polymers inspired from the human gene for tropoelastin. Dynamic light scattering confirmed that the fusion peptide forms nanoparticles with a hydrodynamic radius of approximately 50 nm, which is unexpectedly above that observed for the free ELP (~ 5.1 nm). To further investigate their morphology, conventional and cryogenic transmission electron microscopy were used to reveal that they are unilamellar vesicles. On average, these vesicles are 49 nm in radius with lamellae 8 nm in thickness. To evaluate their therapeutic potential, the L4F nanoparticles were incubated with hepatic stellate cells. Stellate cell activation leads to hepatic fibrosis; furthermore, their activation is suppressed by anti-oxidant activity of ApoA1 mimetic peptides. Consistent with this observation, L4F nanoparticles were found to suppress hepatic stellate cell activation in vitro . To evaluate the in vivo potential for these nanostructures, their plasma pharmacokinetics were evaluated in rats. Despite the assembly of nanostructures, both free L4F and L4F nanoparticles exhibited similar half-lives of approximately 1 h in plasma. This is the first study reporting the stabilization of peptide-based vesicles using ApoA1 mimetic peptides fused to a protein polymer; furthermore, this platform for peptide-vesicle assembly may have utility in the design of biodegradable nanostructures. [ABSTRACT FROM AUTHOR]

Elastin-like polypeptides (ELP) are thermally responsive polypeptides that are soluble in solutions at 37 °C, but which aggregate above 42 °C. ELP can be used as effective carrier systems of anticancer molecules, because they can be targeted to tumor sites through the application of local hyperthermia. Since molecular size largely influences how successfully therapeutic agents can cross the vasculatures of tumors, it was crucial to determine an optimal molecular size. In this study, we designed and evaluated three ELP macromolecules with varying molecular weights (43, 63, and 122 kDa), with the goal of determining which would optimize the ELP drug delivery system. The N-terminus of the ELP macromolecule was modified with the cell penetrating peptide Bac to enhance intratumoral and intracellular uptake, and it was also confirmed that each polypeptide had the target transition temperature of 37–42 °C and the results of the studies, using tumor-bearing mice, showed that the tumor accumulations increased in the case of all three peptides when local hyperthermia was applied, but that the elimination patterns from these tumors varied according to peptide size. Local hyperthermia was found to produce prolonged retention of all ELP conjugates in tumors except Bac-ELP 43 . In addition, the pharmacokinetic analysis showed that two larger polypeptides with 63 and 122 kDa have increased AUC in comparison with the 43 kDa polypeptide. These results suggest that, when combined with local hyperthermia, the larger ELP conjugates (63 and 122 kDa) have advantages over the smaller Bac-ELP 43 polypeptide in terms of enhanced permeability and higher retention effects. [ABSTRACT FROM AUTHOR]

A ubiquitous approach to studying protein function is to knock down activity (gene deletions, siRNA, small molecule inhibitors, etc.) and to study the cellular effects. Using a new methodology, this article describes how to rapidly and specifically switch off cellular pathways using thermally responsive protein polymers. A small increase in temperature stimulates cytosolic elastin-like polypeptides (ELPs) to assemble microdomains. It is hypothesized that ELPs fused to a key effector in a target macromolecular complex will sequester the complex within these microdomains, which will bring the pathway to a halt. To test this hypothesis, ELPs are fused to clathrin-light chain (CLC), a protein associated with clathrin-mediated endocytosis. Prior to thermal stimulation, the ELP fusion is soluble and clathrin-mediated endocytosis remains 'on'. Increasing the temperature induces the assembly of ELP fusion proteins into organelle-sized microdomains that switches clathrin-mediated endocytosis 'off'. These microdomains can be thermally activated and inactivated within minutes, are reversible, do not require exogenous chemical stimulation, and are specific for components trafficked within the clathrin-mediated endocytosis pathway. This temperature-triggered cell switch system represents a new platform for the temporal manipulation of trafficking mechanisms in normal and disease cell models and has applications for manipulating other intracellular pathways. [ABSTRACT FROM AUTHOR]

The 23 amino acid-long extracellular domain of the influenza virus transmembrane protein M2 (M2e) has remained highly conserved since the 1918 pandemic, and is thus considered a good candidate for development of a universal influenza A vaccine. However, M2e is poorly immunogenic. In this study we assessed the potential of increasing immunogenicity of M2e by constructing a nanoscale-designed protein polymer containing the M2e sequence and an elastin-like polypeptide (ELP) nanodomain consisting of alanine and tyrosine guest residues (ELP(A2YA2)24). The ELP nanodomain was included to increase antigen size, and to exploit the inherent thermal inverse phase transition behavior of ELPs to purify the protein polymer. The ELP(A2YA2)24 + M2e nanodomained molecule was recombinantly synthesized. Characterization of its inverse phase transition behavior demonstrated that attachment of M2e to ELP(A2YA2)24 increased its transition temperature compared to ELP(A2YA2)24. Using a dot blot test we determined that M2e conjugated to ELP is recognizable by M2e-specific antibodies, suggesting that the conjugation process does not adversely affect the immunogenic property of M2e. Further, upon vaccinating mice with ELP(A2YA2)24 + M2e it was found that indeed the nanodomained protein enhanced M2e-specific antibodies in mouse serum compared to free M2e peptide and ELP(A2YA2)24. The immune serum could also recognize M2 expressed on influenza virions. Overall, this data suggests the potential of using molecules containing M2e-ELP nanodomains to develop a universal influenza vaccine. [ABSTRACT FROM AUTHOR]

Highlights: [•] We first found the active xylanase particles on ELPs with considerable higher activity and stability. [•] We evaluated the characterizations of the insoluble active xylanase particles in detail and the results were encouraging. [•] The active xylanase particles on ELPs can be regard as “site-specific immobilized” on the ELPs. [ABSTRACT FROM AUTHOR]

Abstract: Numerous nanocarriers of small molecules depend on either non-specific physical encapsulation or direct covalent linkage. In contrast, this manuscript explores an alternative encapsulation strategy based on high-specificity avidity between a small molecule drug and its cognate protein target fused to the corona of protein polymer nanoparticles. With the new strategy, the drug associates tightly to the carrier and releases slowly, which may decrease toxicity and promote tumor accumulation via the enhanced permeability and retention effect. To test this hypothesis, the drug Rapamycin (Rapa) was selected for its potent anti-proliferative properties, which give it immunosuppressant and anti-tumor activity. Despite its potency, Rapa has low solubility, low oral bioavailability, and rapid systemic clearance, which make it an excellent candidate for nanoparticulate drug delivery. To explore this approach, genetically engineered diblock copolymers were constructed from elastin-like polypeptides (ELPs) that assemble small (<100nm) nanoparticles. ELPs are protein polymers of the sequence (Val-Pro-Gly-Xaa-Gly)n, where the identity of Xaa and n determine their assembly properties. Initially, a screening assay for model drug encapsulation in ELP nanoparticles was developed, which showed that Rose Bengal and Rapa have high non-specific encapsulation in the core of ELP nanoparticles with a sequence where Xaa=Ile or Phe. While excellent at entrapping these drugs, their release was relatively fast (2.2h half-life) compared to their intended mean residence time in the human body. Having determined that Rapa can be non-specifically entrapped in the core of ELP nanoparticles, FK506 binding protein 12 (FKBP), which is the cognate protein target of Rapa, was genetically fused to the surface of these nanoparticles (FSI) to enhance their avidity towards Rapa. The fusion of FKBP to these nanoparticles slowed the terminal half-life of drug release to 57.8h. To determine if this class of drug carriers has potential applications in vivo, FSI/Rapa was administered to mice carrying a human breast cancer model (MDA-MB-468). Compared to free drug, FSI encapsulation significantly decreased gross toxicity and enhanced the anti-cancer activity. In conclusion, protein polymer nanoparticles decorated with the cognate receptor of a high potency, low solubility drug (Rapa) efficiently improved drug loading capacity and its release. This approach has applications to the delivery of Rapa and its analogs; furthermore, this strategy has broader applications in the encapsulation, targeting, and release of other potent small molecules. [Copyright &y& Elsevier]

We developed a novel temperature-sensitive liposome, STL composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (DSPE-PEG-2000), cholesterol, and a fatty acid conjugated elastin-like polypeptide (ELP). The STL had a unilamellar spherical shape with a mean diameter of 160nm. Doxorubicin (DOX) was encapsulated by the STL using an ammonium sulfate gradient method with a lipid to drug ratio of 1:0.2 (w/w), resulting in 95% loading efficiency. The STL exhibited better stability than conventional low temperature sensitive liposome (LTSL-lysolipid-based temperature sensitive liposomes; DPPC:MSPC:DSPE-PEG-2000 =90:10:4) at 37°C in the presence of serum; there was rapid release of doxorubicin in the range of 39–42°C (≥95% release at 42°C within 10s). A confocal microscope revealed that DOX encapsulated in STL (STL-DOX) was taken up much better by cell nuclei at 42°C than at 37°C. The difference in cell viability between 37 and 42°C was 63% relative to STL-DOX and 18% for LTSL-DOX. The pharmacokinetics (PK) and antitumor effect of STL-DOX combined with high-intensity focused ultrasound (HIFU) were studied, and compared with LTSL. An in vivo study demonstrated that STL-DOX is highly stable, with a long circulating property (half life=2.03±0.77h) in HIFU-untreated mice, and resulted in significant tumor regression for 2days after intravenous injection of STL-DOX at 5mg DOX/kg in combination with HIFU. These results are better than conventional LTSL, for which the blood circulation time is short (0.92±0.17h) and inhibition of tumor growth is weak. These results indicate that the properties of stability at 37°C and burst release at 42°C of STL-DOX act synergistically against tumors. [ABSTRACT FROM AUTHOR]

A diverse range of drug delivery vehicles have been developed to specifically target chemotherapeutics to solid tumours while avoiding systemic dose-limiting toxicity. Many of these active targeting strategies display limited efficacy because they rely on subtle differences in expression patterns between pathogenic tissue and healthy tissue. In contrast, drug delivery systems that exploit thermoresponsive behaviour allow a clinician to spatially and temporally control the accumulation and/or release of the toxic agents within tumour tissue by simply applying mild hyperthermia (defined as 39-43 °C) to the desired site. Although thermally sensitive materials comprise a significant portion of the literature on novel drug delivery systems, only a few systems have been methodically tuned to respond within this narrowly defined physiological temperature range in an in vivo environment. This review discusses the materials and strategies developed to control the primary tumour through the combined application of hyperthermia and chemotherapy. [ABSTRACT FROM AUTHOR]

Previously, we reported a non-chromatographic protein purification method exploiting the highly specific interaction between the dockerin and cohesin domains from Clostridium thermocellum and the reversible aggregation property of elastin-like polypeptide (ELP) to provide fast and cost-effective protein purification. However, the bound dockerin-intein tag cannot be completely dissociated from the ELP-cohesin capturing scaffold due to the high binding affinity, resulting in a single-use approach. In order to further reduce the purification cost by recycling the ELP capturing scaffold, a truncated dockerin domain with the calcium-coordinating function partially impaired was employed. We demonstrated that the truncated dockerin domain was sufficient to function as an effective affinity tag, and the target protein was purified directly from cell extracts in a single binding step followed by intein cleavage. The efficient EDTA-mediated dissociation of the bound dockerin-intein tag from the ELP-cohesin capturing scaffold was realized, and the regenerated ELP capturing scaffold was reused in another purification cycle without any decrease in the purification efficiency. This recyclable non-chromatographic based affinity method provides an attractive approach for efficient and cost-effective protein purification. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:968-971, 2013 [ABSTRACT FROM AUTHOR]

Background: Plants are recognized as an efficient and inexpensive system to produce valuable recombinant proteins. Two different strategies have been commonly used for the expression of recombinant proteins in plants: transient expression mediated by Agrobacterium; or stable transformation of the plant genome. However, the use of plants as bioreactors still faces two main limitations: low accumulation levels of some recombinant proteins and lack of efficient purification methods. Elastin-like polypeptide (ELP), hydrophobin I (HFBI) and Zera® are three fusion partners found to increase the accumulation levels of recombinant proteins and induce the formation of protein bodies (PBs) in leaves when targeted to the endoplasmic reticulum (ER) in transient expression assays. In this study the effects of ELP and HFBI fusion tags on recombinant protein accumulation levels and PB formation was examined in stable transgenic Nicotiana tabacum. Results: The accumulation of recombinant protein and PB formation was evaluated in two cultivars of Nicotiana tabacum transformed with green fluorescent protein (GFP) fused to ELP or HFBI, both targeted and retrieved to the ER. The ELP and HFBI tags increased the accumulation of the recombinant protein and induced the formation of PBs in leaves of stable transgenic plants from both cultivars. Furthermore, these tags induced the formation of PBs in a concentration-dependent manner, where a specific level of recombinant protein accumulation was required for PBs to appear. Moreover, agro-infiltration of plants accumulating low levels of recombinant protein with p19, a suppressor of post-transcriptional gene silencing (PTGS), increased accumulation levels in four independent transgenic lines, suggesting that PTGS might have caused the low accumulation levels in these plants. Conclusion: The use of ELP and HFBI tags as fusion partners in stable transgenic plants of tobacco is feasible and promising. In a constitutive environment, these tags increase the accumulation levels of the recombinant protein and induce the formation of PBs regardless of the cultivar used. However, a specific level of recombinant protein accumulation needs to be reached for PBs to form. [ABSTRACT FROM AUTHOR]

Abstract: Although surgical resection with adjuvant chemotherapy and/or radiotherapy are used to treat breast tumors, normal tissue tolerance, development of metastases, and inherent tumor resistance to radiation or chemotherapy can hinder a successful outcome. We have developed a thermally responsive polypeptide, based on the sequence of Elastin-like polypeptide (ELP), that inhibits breast cancer cell proliferation by blocking the activity of the oncogenic protein c-Myc. Following systemic administration, the ELP – delivered c-Myc inhibitory peptide was targeted to tumors using focused hyperthermia, and significantly reduced tumor growth in an orthotopic mouse model of breast cancer. This work provides a new modality for targeted delivery of a specific oncogene inhibitory peptide, and this strategy may be expanded for delivery of other therapeutic peptides or small molecule drugs. [Copyright &y& Elsevier]

Abstract: Objectives: Hepatitis 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. Methods: The 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. Results: Fusion 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. Conclusion: This 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. [Copyright &y& Elsevier]

Abstract: This paper reports the design, physicochemical characterization and in vitro cytotoxicity of a thermally responsive chimeric polypeptide (CP), derived from an elastin-like polypeptide (ELP). The CP self-assembles into ~40nm diameter nanoparticles upon conjugation of multiple copies of doxorubicin (Dox), and displays a nanoparticle-to-aggregate phase transition between 39 and 42°C in media, a temperature range suitable for mild hyperthermia of solid tumors. The CP-Dox nanoparticle is stable upon dilution to low micromolar concentrations, and is cytotoxic at both 37 and 42°C. A thermally responsive nanoparticle formulation of Dox may prove to be broadly useful in hyperthermia targeted chemotherapy of a variety of solid tumors. [Copyright &y& Elsevier]

Abstract: The integration of viral gene delivery with key features of biomaterial scaffolds that modulate viral delivery in a controlled manner offers a promising strategy for numerous tissue engineering applications. In this study adeno-associated virus (AAV), which is widely utilized in human gene therapy as a gene carrier due to its safety and efficient gene delivery capability, was encapsulated within electrospun nanofibrous scaffolds composed of blended mixtures of elastin-like polypeptides (ELP) and poly (ε-caprolactone) (PCL) and was employed to transduce fibroblasts adherent on the scaffolds. Combinatorial interactions between ELP and PCL chains upon physical blending significantly altered the mechanical properties (i.e. wettability, elastic modulus, strain, etc.) of the ELP/PCL composites, thus providing key tools to mediate controlled release of AAV vectors and robust cellular transduction on the fibrous scaffolds. The ability of ELP/PCL composites to manipulate the controlled release of AAV-mediated gene delivery for subsequent high-efficiency cellular transduction will provide tremendous opportunities for a variety of tissue engineering applications. [Copyright &y& Elsevier]

Abstract: Antivascular targeting is a promising strategy for tumor therapy. This strategy has the potential to overcome many of the transport barriers associated with targeting tumor cells in solid tumors, because the tumor vasculature is directly accessible to targeting vehicles in systemic circulation. We report a novel nanoscale delivery system consisting of multivalent polymer micelles to target receptors that are preferentially upregulated in the tumor vasculature and perivascular cells, specifically CD13. To this end we utilized amphiphilic block copolymers, composed of a genetically engineered elastin-like polypeptide (ELP) that self-assemble into monodisperse spherical micelles. These polymer micelles were functionalized by incorporating the NGR tripeptide ligand, which targets the CD13 receptor, on their corona. We examined the self-assembly and in vivo tumor targeting by these NGR-functionalized nanoparticles and show that multivalent presentation of NGR by micelle self-assembly selectively targets the tumor vasculature by targeting CD13. Furthermore, we show greater vascular retention and extravascular accumulation of nanoparticles in tumor tissue compared to normal tissue, although the enhancement is modest. These results suggest that enhanced delivery to solid tumors can be achieved by targeting upregulated receptors in the tumor vasculature with multivalent ligand-presenting nanoparticles, but additional work is required to optimize such systems for multivalent targeting. [Copyright &y& Elsevier]

Abstract: The tumor margins are the barrier to hepatocellular carcinoma (HCC) eradication for tumors>3cm. Indeed, inadequately treated tumor margins commonly result in local and regional HCC recurrence with increased size and mass. Tumor recurrence is a common problem with chemotherapy, radiotherapy, thermal ablation, and/or surgical resection, by the inability to properly treat the tumor core and the tumor margins. Here we present novel thermosensitive biopolymer-drug conjugates for thermo-targeted chemotherapy at hyperthermic isotherms produced by focal, locoregional thermal ablation. The chemotherapeutic target is heat shock protein 90 (HSP90), a key molecular chaperone of several, and potent pro-oncogenic pathways including Akt, Raf-1, and mutated p53 that is upregulated in HCC. To inhibit HSP90, we have chosen geldanamycin (GA), a potent HSP90 inhibitor. GA has gained significant attention for its low IC50~1nM and inhibition of Akt and Raf-1, amongst other critical pro-oncogenic pathways. Despite such evidence, clinical trials of GA have not shown promise due to off-target toxicity and poor formulation design. Here, we propose using diblock elastin-based biopolymers as a Ringsdorf macromolecular GA solubilizer — a new generation containing functional poly(Asp)/(Glu) blocks for facile drug conjugation and an ELP block for thermo-targeting of hyperthermic ablative margins. GA release is controlled by pH-sensitive, covalent hydrazone bonds with the biopolymer backbone to avoid systemic toxicity and off-target effects. The resultant biopolymer-conjugates form stable nanoconstructs and display tunable, acute phase transitions at high temperatures. Drug release kinetics are favorable with or without the presence of serum. Thermo-targeted chemotherapy and synchronous thermal ablation provide a unique opportunity for simultaneous destruction of the HCC ablative margins and tumor core for focal, locoregional control of HCC. [Copyright &y& Elsevier]

Abstract: Self-assembly driven processes can be utilized to produce a variety of nanostructures useful for various in vitro and in vivo applications. Characteristics such as size, stability, biocompatibility, high therapeutic loading and controlled delivery of these nanostructures are particularly crucial in relation to in vivo applications. In this study, we report the fabrication of tunable monodispersed elastin-like polypeptide (ELP) hollow spheres of 100, 300, 500 and 1000nm by exploiting the self-assembly property and net positive charge of ELP. The microbial transglutaminase (mTGase) cross-linking provided robustness and stability to the hollow spheres while maintaining surface functional groups for further modifications. The resulting hollow spheres showed a higher loading efficiency of plasmid DNA (pDNA) by using polyplex (~70μg pDNA/mg of hollow sphere) than that of self-assembled ELP particles and demonstrated controlled release triggered by protease and elastase. Moreover, polyplex-loaded hollow spheres showed better cell viability than polyplex alone and yielded higher luciferase expression by providing protection against endosomal degradation. Overall, the monodispersed, tunable hollow spheres with a capability of post-functionalization can provide an exciting new opportunity for use in a range of therapeutic and diagnostic applications. [Copyright &y& Elsevier]

Abstract: Current treatment of solid tumors is limited by side effects that result from the non-specific delivery of drugs to the tumor site. Alternative targeted therapeutic approaches for localized tumors would significantly reduce systemic toxicity. Peptide therapeutics are a promising new strategy for targeted cancer therapy because of the ease of peptide design and the specificity of peptides for their intracellular molecular targets. However, the utility of peptides is limited by their poor pharmacokinetic parameters and poor tissue and cellular membrane permeability in vivo. This review article summarizes the development of elastin-like polypeptide (ELP) as a potential carrier for thermally targeted delivery of therapeutic peptides (TP), and the use of cell penetrating peptides (CPP) to enhance the intracellular delivery of the ELP-fused TPs. CPP-fused ELPs have been used to deliver a peptide inhibitor of c-Myc function and a peptide mimetic of p21 in several cancer models in vitro, and both polypeptides are currently yielding promising results in in vivo models of breast and brain cancer. [ABSTRACT FROM AUTHOR]

Abstract: Thermally responsive elastin-like polypeptides (ELPs) are a promising class of recombinant biopolymers for the delivery of drugs and imaging agents to solid tumors via systemic or local administration. This article reviews four applications of ELPs to drug delivery, with each delivery mechanism designed to best exploit the relationship between the characteristic transition temperature (T t) of the ELP and body temperature (T b). First, when T t ≫ T b, small hydrophobic drugs can be conjugated to the C-terminus of the ELP to impart the amphiphilicity needed to mediate the self-assembly of nanoparticles. These systemically delivered ELP–drug nanoparticles preferentially localize to the tumor site via the EPR effect, resulting in reduced toxicity and enhanced treatment efficacy. The remaining three approaches take direct advantage of the thermal responsiveness of ELPs. In the second strategy, where T b < T t <42°C, an ELP–drug conjugate can be injected in conjunction with external application of mild hyperthermia to the tumor to induce ELP coacervation and an increase in concentration within the tumor vasculature. The third approach utilizes hydrophilic–hydrophobic ELP block copolymers that have been designed to assemble into nanoparticles in response to hyperthermai due to the independent thermal transition of the hydrophobic block, thus resulting in multivalent ligand display of a ligand for spatially enhanced vascular targeting. In the final strategy, ELPs with T t < T b are conjugated with radiotherapeutics, injtect intioa tumor where they undergo coacervation to form an injectable drug depot for intratumoral delivery. These injectable coacervate ELP-radionuclide depots display a long residence in the tumor and result in inhibition of tumor growth. [ABSTRACT FROM AUTHOR]

Abstract: Polypeptides based on the alternating hydrophobic and cross-linking domain structure of human elastin are capable of undergoing self-assembly to produce polymeric matrices with unique biological and mechanical properties. Here, we test the initial feasibility of using a genipin cross-linked elastin-based material as an acellular plug in the treatment of an osteochondral defect in the rabbit knee. Full-thickness defects in the weight-bearing surface of the medial femoral condyle in 18 New Zealand White rabbits were surgically produced and press fitted with cylindrical pads composed of genipin cross-linked elastin-like polypeptides, with identical wounds in the opposite knee left untreated as controls. The biocompatibility of the material, overall wound healing and regeneration of subchondral tissue was assessed at 2, 4 and 6weeks by histological evaluation, synovial fluid analysis and microcomputerized tomography scanning. Histological analysis revealed the regeneration of subchondral bone at the periphery of the material, with evidence of hyaline-like overgrowth across the apical surface in 11/16 cases. Pads developed tight contacts with host tissue and appeared completely biocompatible, with no evidence of localized immune response or increased inflammation compared to controls. The material was stable to 6weeks, with an aggregate elastic modulus calculated at ∼470kPa when tested under confined compression. Further studies are required to assess material degradation over time and long-term replacement with repair tissue. [Copyright &y& Elsevier]