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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−VEGF. The ELP−VEGF fusion protein was modified by adding a kidney-targeting peptide (KTP) to the N-terminus. All control proteins (ELP, KTP−ELP, ELP−VEGF, and KTP−ELP−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−ELP−VEGF was equipotent to ELP−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−ELP−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−ELP−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−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 (40−41 °C) hyperthermia. The CPP-ELP-Dox conjugate consists of a cell penetrating peptide (CPP), which facilitates transcytosis through the blood brain barrier and cell entry, and a 6-maleimidocaproyl hydrazone derivative of doxorubicin at the C-terminus of ELP. The acid-sensitive hydrazone linker ensures release of Dox in the lysosomes/endosomes after cellular uptake of the drug conjugate. We have shown that CPP-ELP-Dox effectively inhibits cell proliferation in three GBM cell lines. Both the free drug and CPP-ELP-Dox conjugate exhibited similar in vitro cytotoxicity, although their subcellular localization was considerably different. The Dox conjugate was mainly dispersed in the cytoplasm, while free drug had partial nuclear accumulation in addition to cytoplasmic distribution. The intracellular Dox concentration was increased in the CPP-ELP-Dox cells compared to that in the cells treated with free Dox, which positively correlates with cytotoxic activity. In summary, our findings demonstrate that CPP-ELP-Dox effectively kills GBM cells. Development of such a drug carrier has the potential to greatly improve current therapeutic approaches for GBM by increasing the specificity and efficacy of treatment and reducing cytotoxicity in normal tissues.

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]

We have recently developed a novel double-hydrophobic elastin-like triblock polypeptide called GPG, designed after the uneven distribution of two different hydrophobic domains found in elastin, an extracellular matrix protein providing elasticity and resilience to tissues. Upon temperature trigger, GPG undergoes a sequential self-assembling process to form flexible beaded nanofibers with high homogeneity and excellent dispersibility in water. Given that GPG might be a potential elastin-mimetic material, we sought to explore the biological activities of this block polypeptide. Besides GPG, several functionalized derivatives were also constructed by fusing functional motifs such as KAAK or KAAKGRGDS at the C-terminal of GPG. Although the added motifs affected the kinetics of fiber formation and β-sheet contents, all three GPGs assembled into beaded nanofibers at the physiological temperature. The resulting GPG nanofibers preserved their beaded structures in cell culture medium; therefore, they were coated on polystyrene substrates to study their cytocompatibility toward mouse embryonic fibroblasts, NIH-3T3. Among the three polypeptides, GPG having the cell-binding motif GRGDS derived from fibronectin showed excellent cell adhesion and cell proliferation properties compared to other conventional materials, suggesting its promising applications as extracellular matrices for mammalian cells. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2475-2484, 2017. [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]

Herein, we demonstrate a new approach for small-caliber vascular reconstruction using a non-porous elastin-like polypeptide hydrogel tubular scaffold, based on the concept of guided vascular regeneration (GVR). The scaffolds are composed of elastin-like polypeptide, (Val-Pro-Gly-Ile-Gly) n, for compliance matching and antithrombogenicity and an Arg-Gly-Asp (RGD) motif for connective tissue regeneration. When the polypeptide was mixed with an aqueous solution of β-[Tris(hydroxymethyl)phosphino]propionic acid at 37°C, the polypeptide hydrogel was rapidly formed. The elastic modulus of the hydrogel was 4.4 kPa. The hydrogel tubular scaffold was formed in a mold and reinforced with poly(lactic acid) nanofibers. When tubular scaffolds with an inner diameter of 1 mm and length of 5 mm were implanted into rat abdominal aortae, connective tissue grew along the scaffold luminal surface from the flanking native tissues, resulting in new blood vessel tissue with a thickness of 200 μm in 1 month. In contrast, rats implanted with control scaffolds without the RGD motif died. These results indicate that the non-porous hydrogel tubular scaffold containing the RGD motif effectively induced rapid tissue regeneration and that GVR is a promising strategy for the regeneration of small-diameter blood vessels. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1746-1755, 2017. [ABSTRACT FROM AUTHOR]

3D culture systems have the ability to mimic the natural microenvironment by allowing better cell-cell interactions. We have prepared an in vitro 3D osteogenic cell culture model using human adipose derived stem cells (hASCs) cultured atop recombinant elastin-like polypeptide (ELP) conjugated to a charged polyelectrolyte, polyethyleneimine (PEI). We demonstrate that hASCs cultured atop the ELP-PEI coated tissue culture polystyrene (TCPS) formed 3D spheroids and exhibited superior differentiation toward osteogenic lineage compared to the traditional two dimensional (2D) monolayer formed atop uncoated TCPS. Live/dead viability assay confirmed >90% live cells at the end of the 3-week culture period. Over the same culture period, higher protein content was observed in 2D monolayer than 3D spheroids, as the 2D environment allowed continued proliferation, while 3D spheroids underwent contact-inhibited growth arrest. The normalized alkaline phosphatase (ALP) activity, which is an indicator for early osteogenic differentiation was higher for 3D spheroids. The normalized osteocalcin (OCN) production, which is an indicator for osteogenic maturation was also higher for 3D spheroids while 2D monolayer had no noticeable OCN production. On day 22, increased Alizarin red uptake by 3D spheroids showed greater mineralization activity than 2D monolayer. Taken together, these results indicate a superior osteogenic differentiation of hASCs in 3D spheroid culture atop ELP-PEI coated TCPS surfaces than the 2D monolayer formed on uncoated TCPS surfaces. Such enhanced osteogenesis in 3D spheroid stem cell culture may serve as an alternative to 2D culture by providing a better microenvironment for the enhanced cellular functions and interactions in bone tissue engineering. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1230-1236, 2017. [ABSTRACT FROM AUTHOR]

With the emergence of soft robotics, there is a growing need to develop actuator systems that are lightweight, mechanically compliant, stimuli-responsive, and readily programmable for precise and intelligent operation. Therefore, "smart" polymeric materials that can precisely change their physicomechanical properties in response to various external stimuli (e.g., pH, temperature, electromagnetic force) are increasingly investigated. Many different types of polymers demonstrating stimuli-responsiveness and shape memory effect have been developed over the years, but their focus has been mostly placed on controlling their mechanical properties. In order to impart complexity in actuation systems, there is a concerted effort to implement additional desired functionalities. For this purpose, elastin-like polypeptide (ELP), a class of genetically-engineered thermoresponsive polypeptides that have been mostly utilized for biomedical applications, is being increasingly investigated for stimuli-responsive actuation. Herein, unique characteristics and biomedical applications of ELP, and recent progress on utilizing ELP for programmable actuation are introduced., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Noh, Son and Cha.)

A well characterized, peptide derivative of bovine lactoferrin, L12, has been shown to possess anticancer properties in multiple cell lines. However, adverse side effects in normal tissues and poor plasma kinetics that hinder the clinical effectiveness of current chemotherapeutics also deter the potential for effective delivery of this L12 peptide. To overcome these limitations, we have developed an Elastin-like polypeptide (ELP) carrier that has the potential to thermally target therapeutic peptides and chemotherapeutics to a tumor site. The coding sequence of ELP was modified with the L12 peptide at the C-terminus and a membrane transduction domain derived from the HIV-1 Tat protein at the N-terminus (Tat-ELP-L12). The thermally responsive Tat-ELP1-L12 is soluble in aqueous solutions at 37°C but aggregates near 41°C, which makes Tat-ELP1-L12 ideal for targeting to solid tumors on application of focused hyperthermia. We observed that under hyperthermia conditions at 42°C, Tat-ELP1-L12 mediated cytotoxicity in MIA PaCa-2 pancreatic adenocarcinoma cells was enhanced by nearly thirty-fold. We investigated the mechanisms of cell death and found evidence of mitochondrial membrane depolarization and caspase activation, which are characteristic of apoptosis, as well as, increased membrane permeability, as shown by LDH release. These results suggest that Tat-ELP1-L12 possesses cytotoxic properties to cancer cells in vitro and may have the potential to provide an effective vehicle to thermally target solid tumors.

Objective: Chronic skin wounds are one of the most devastating complications in diabetic patients due to the formation of advanced glycation end-products (AGEs) resulting from nonenzymatic glycation of proteins and lipids in hyperglycemia. AGEs, upon binding their receptors (RAGEs), trigger proinflammatory signals that impair wound healing in diabetes and contribute to the pathology of chronic skin wounds. Approach: We previously developed a recombinant fusion protein containing the binding domain of RAGE (vRAGE) linked to elastin-like polypeptides (ELPs) that acts as a competitive inhibitor of AGEs, and another ELP fusion protein containing stromal cell-derived factor 1 (SDF1) that promotes revascularization. In this study, we report the effects of protein coacervates incorporating both vRAGE-ELP and SDF1-ELP on wound healing in an in vitro diabetes-mimicking cell culture system, and in in vivo in full-thickness wounds on diabetic mice. Results: The combination of vRAGE-ELP and SDF1-ELP increased cell metabolic activity in AGE-stimulated endothelial cells, promoted in vitro tube formation and accelerated healing in an in vitro cell migration assay. When used in a single topical application on full-thickness excisional skin wounds in diabetic mice, wound closure in the combination groups reached almost 100% on postwounding day 35, compared to 62% and 85% on the same days in animals treated with fibrin gel control and vehicle control consisting of ELP alone. Innovation: To our knowledge, this is the first study that attempts to reverse the AGE-RAGE-mediated signaling as well as to promote cell proliferation and vascularization in one single treatment. Conclusion: The codelivery of vRAGE-ELP and SDF1-ELP has potential for the treatment of diabetic wounds.

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]

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. [ABSTRACT FROM AUTHOR]

Background: Brachytherapy is a targeted type of radiotherapy utilized in the treatment of cancers. Elastin-like polypeptides are a unique class of genetically engineered peptide polymers that have several attractive properties for brachytherapy.Aims: To explore the feasibility and application of brachytherapy for VX2 liver tumor using elastin-like polypeptides with (131)I so as to provide reliable experimental evidence for a new promising treatment of liver cancer.Methods: Elastin-like polypeptide as carrier was labeled with (131)I using the iodogen method. Ten eligible rabbits with VX2 liver tumor were randomly divided into the treatment group (n = 5) and control group (n = 5). The treatment group received brachytherapy using elastin-like polypeptide with (131)I, and in the control group, elastin-like polypeptide was injected into the VX2 liver tumor as a control. Periodic biochemical and imaging surveillances were required to assess treatment efficacy.Results: The stability of elastin-like polypeptide with (131)I in vitro was maintained at over 96.8 % for 96 h. Biochemistry and imaging indicated brachytherapy using elastin-like polypeptide with (131)I for liver tumor can improve liver function and inhibit tumor growth (P < 0.05).Conclusions: Elastin-like polypeptide can be an ideal carrier of (131)I and have high labeling efficiency, radiochemical purity and stability. Brachytherapy using elastin-like polypeptide with (131)I for liver tumor is a useful therapy that possesses high antitumor efficacy advantages. [ABSTRACT FROM AUTHOR]

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. [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]

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 μg) and IL-10 (10 μg)/eNOS (20 μg) and reduced inflammation with IL-10 (10 μg) 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, ELP injectable system mediating non-viral delivery of human IL10-eNOS is a promising therapy towards treating limb ischemia. [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]

ABSTRACT Preparation of hydrogels that possess an effective antibiotic release profile and better mechanical properties compared to the traditionally used collagen hydrogels has the potential to minimize post-surgical infections and support wound healing. Toward this goal, we prepared elastin-like polypeptide (ELP)-collagen composite hydrogels that displayed a significantly higher elastic modulus compared to the collagen hydrogels. We then characterized the release behavior of the collagen and ELP-collagen hydrogels loaded with varying dosages (1-5% w/w) of a commonly used broad spectrum antibiotic, doxycycline hyclate. Both collagen and ELP-collagen hydrogels showed a gradual time dependent doxycycline release over a period of 5 days. The ELP-collagen hydrogels, in general, showed a slower release of the doxycycline compared to the collagen hydrogels. The released doxycycline was found to be effective against four bacterial strains ( Escherichia coli, Pseudomonas aeruginosa, Streptococcus sanguinis, and methicillin-resistant Staphylococcus aureus) in a dose dependent manner. Combined with their improved mechanical properties, the gradual and effective drug release from the biocompatible ELP-collagen hydrogels shown here may be beneficial for drug delivery and tissue engineering applications. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 103A: 782-790, 2015. [ABSTRACT FROM AUTHOR]

Drug delivery systems (DDSs) have recently gained widespread attention for improving drug loading and delivery efficiency in treating many cancers. Elastin-like polypeptides (ELPs) are synthetic peptides derived from a precursor of elastin (tropoelastin), reserving similar structural and physicochemical properties. ELPs have gained a variety of applications in tissue engineering and cancer therapy due to their excellent biocompatibility, complete degradability, temperature-responsive property, controllable sequence and length, and precisely tuned structure and function. ELPs-based drug delivery systems can improve the pharmacokinetics and biodistribution of therapeutic reagents, leading to enhanced antitumor efficacy. In this review, we summarize the recent application of ELPs in cancer treatment, focusing on the delivery of functional peptides, therapeutic proteins, small molecule drugs, and photosensitizers., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Jiang, Guan, He and Guan.)

Glycoprotein therapeutics are currently used by large patient populations and generate significant revenue for the biopharmaceutical industry. These therapeutic proteins are currently purified at industrial scale using individualized processes involving multiple chromatographic steps. In the absence of a viable affinity platform method, the required chromatographic steps are difficult to develop and inevitably lead to significant yield losses. Further, during preclinical development, there is a need for reliable platform technologies capable of performing high-throughput screening for biologic candidates. Although affinity tags can provide a solution to some of these challenges, they require specific affinity resins, and the tag itself can interfere with the target protein characteristics. Fusion protein systems consisting of elastin-like polypeptide (ELP) and self-cleaving split inteins such as Npu DnaE can serve as potential non-chromatographic platform technologies for the single-step purification of tagless glycoproteins expressed in mammalian cells. In this chapter, we demonstrate the use of this technology to obtain highly purified anti-ErbB2 ML39 single-chain variable fragment (scFv) expressed from Expi293F suspension cells., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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]

The development of protein-based carriers for drug delivery has been well studied. We previously constructed a protein-based nanoparticle consisting of genetically engineered elastin-like polypeptides (ELPs) with a fused poly-aspartic acid tail (ELPD). The size of the self-assembled ELPD nanoparticles was regulated by charged repulsion of the poly-aspartic acid chains. In the present study, epidermal growth factor (EGF) was genetically fused to the C-terminus of ELPD to impart an active targeting ability to the ELPD nanoparticles. We examined the nanoparticle formation with EGF as well as its targeting ability. ELPD with fused EGF was found to form nanoparticles that displayed multivalent EGFs on their surface. EGF-displayed nanoparticles loaded with the anti-cancer drug paclitaxel were internalized into cells overexpressing the EGF receptor, and induced cell death. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 102B: 1792-1798, 2014. [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 last several decades have seen intensive research into the molecular mechanisms underlying the symptoms of preeclampsia. While the underlying cause of preeclampsia is believed to be defective placental development and resulting placental ischemia, it is only recently that the links between the ischemic placenta and maternal symptomatic manifestation have been elucidated. Several different pathways have been implicated in the development of the disorder; most notably production of the anti-angiogenic protein sFlt-1, induction of auto-immunity and inflammation, and production of reactive oxygen species. While the molecular mechanisms are becoming clearer, translating that knowledge into effective therapeutics has proven elusive. Here we describe a number of peptide based therapies we have developed to target theses pathways, and which are currently being tested in preclinical models. These therapeutics are based on a synthetic polymeric carrier elastin-like polypeptide (ELP), which can be synthesized in various sequences and sizes to stabilize the therapeutic peptide and avoid crossing the placental interface. This prevents fetal exposure and potential developmental effects. The therapeutics designed will target known pathogenic pathways, and the ELP carrier could prove to be a versatile delivery system for administration of a variety of therapeutics during pregnancy. [ABSTRACT FROM AUTHOR]

Background Pectinases play an important role in plant cell wall deconstruction and have potential in diverse industries such as food, wine, animal feed, textile, paper, fuel, and others. The demand for such enzymes is increasing exponentially, as are the efforts to improve their production and to implement their use in several industrial processes. The goal of this study was to study the potential of producing polygalacturonase I from Aspergillus niger in plants and to investigate the effects of subcellular compartmentalization and protein fusions on its accumulation and activity. Results Polygalacturonase I from Aspergillus niger (AnPGI) was transiently produced in Nicotiana benthamiana by targeting it to five different cellular compartments: apoplast, endoplasmic reticulum (ER), vacuole, chloroplast and cytosol. Accumulation levels of 2.5%, 3.0%, and 1.9% of total soluble protein (TSP) were observed in the apoplast, ER, and vacuole, respectively, and specific activity was significantly higher in vacuole-targeted AnPGI compared to the same enzyme targeted to the ER or apoplast. No accumulation was found for AnPGI when targeted to the chloroplast or cytosol. Analysis of AnPGI fused with elastin-like polypeptide (ELP) revealed a significant increase in the protein accumulation level, especially when targeted to the vacuole where the protein doubles its accumulation to 3.6% of TSP, while the hydrophobin (HFBI) fusion impaired AnPGI accumulation and both tags impaired activity, albeit to different extents. The recombinant protein showed activity against polygalacturonic acid with optimum conditions at pH 5.0 and temperature from 30 to 50°C, depending on its fusion. In vivo analysis of reducing sugar content revealed a higher release of reducing sugars in plant tissue expressing recombinant AnPGI compared to wild type N. benthamiana leaves. Conclusion Our results demonstrate that subcellular compartmentalization of enzymes has an impact on both the target protein accumulation and its activity, especially in the case of proteins that undergo post-translational modifications, and should be taken into consideration when protein production strategies are designed. Using plants to produce heterologous enzymes for the degradation of a key component of the plant cell wall could reduce the cost of biomass pretreatment for the production of cellulosic biofuels. [ABSTRACT FROM AUTHOR]

Abstract: This work describes the effects of elastin-like polypeptide (ELP) with the p21Waf1/Cip1-derived cell cycle inhibitory peptide (p21) on pancreatic tumor cells with gemcitabine. The thermo-responsive property of ELP permits use of a mild, local hyperthermia to target tumors for the transport of chemotherapeutics. In this study, a p21-ELP construct with Bac cell penetrating peptide was designed, and its anticancer activities in pancreatic cancer cell lines was examined. In combination with gemcitabine, the peptide demonstrated enhanced in vitro cytotoxicity as well as tumor growth inhibition in an animal model. Our data suggest that this ELP construct, with gemcitabine, may improve pancreatic cancer therapy. [Copyright &y& Elsevier]

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]

Though two-dimensional systems have yielded some success in deriving morphological and functional markers of hepatocyte culture, they largely fail to capture the three-dimensional organization, long-term viability, and functionality of the hepatic tissue. We have engineered a system for inducing self-assembly of model H35 rat hepatoma spheroids using a copolymer comprised of biocompatible elastin-like polypeptide (ELP) chemically conjugated to positively charged polyethyleneimine (PEI). We have achieved a conjugation ratio of 30 mol %, though our studies analyzing spheroid organization kinetics indicate conjugate ratios of 5 mol % and greater to be optimal for cell culture based on least variability in spheroid sizes and minimum incidence of overgrown aggregates. Furthermore, our ELP-PEI system indicated the potential for influencing ultimate spheroid dimensions, with spheroid size inversely related to polyelectrolyte conjugation. Overall, this study provides a good starting point to investigate functional correlations between spheroid size and functional markers and their future use as an in vitro diagnostic or tissue engineering tool. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 852-861, 2014. [ABSTRACT FROM AUTHOR]

In this work, we expand on our understanding of the thrombogenicity of coatings prepared with three different recombinant elastin-like polypeptides (ELPs). The bulk platelet response of the ELP coatings was characterized following whole blood contact under physiological shear flow (300 s−1) using flow cytometry. Prolonged exposure to shear flow (1-h) indicated that materials coated with the longer ELP coatings (ELP2 and ELP4) had less bulk platelet activation and microparticle formation than materials coated with the shorter ELP1. Quartz crystal microbalance with dissipation (QCM-D) was used to monitor the binding of the platelet membrane receptor GPIIb/IIIa to ELP-adsorbed fibrinogen (Fg) surfaces. Compared to the shorter ELPs, a lower amount of Fg adsorbed to the ELP4 coated material and ELP4 appeared to form a softer, more structurally flexible coating layer. When Fg was adsorbed to the ELP coated surface it demonstrated an altered binding for GPIIb/IIIa that was inhibited in the presence of an AGDV-containing peptide but not an RGD-containing peptide. Conversely, on the shorter ELP coatings, binding of GPIIb/IIIa to an adsorbed Fg layer was partially inhibited in the presence of an RGD-containing peptide. These results indicate that both the quantity and conformational state of Fg varies when adsorbed to surfaces coated with ELPs of varying sequence length, which may be mediating their platelet response. Collectively, the findings reinforce the applicability of the ELPs as potential thromboresistant coatings, especially with the use of the longer polypeptide—ELP4. [ABSTRACT FROM AUTHOR]

Strategic materials design is essential for the development of small-diameter, tissue-engineered vascular grafts. Self-assembled nanofibers of elastin-like polypeptides represent promising vascular graft components as they replicate the organized elastin structure of native blood vessels. Further, the bioactivity of nanofibers can be modified by the addition of functional peptide motifs. In the present study, we describe the development of a novel nanofiber-forming elastin-like polypeptide (ELP) with an arginine-glutamic acid-aspartic acid-valine (REDV) sequence. The biological characteristics of the REDV-modified ELP nanofibers relevant to applications in vascular grafting were compared to ELP without ligands for integrin, ELP with arginine-glycine-aspartic acid (RGD) sequence, collagen and cell culture glass. Among them, REDV-modified ELP nanofibers met the preferred biological properties for vascular graft materials, i.e. (i) inhibition of platelet adhesion and activation, (ii) endothelial cell adhesion and proliferation and (iii) maintenance of smooth muscle cells in a contractile phenotype to prevent cell overgrowth. The results indicate that REDV-modified ELP nanofibers represent promising candidates for the further development of small-diameter vascular grafts., (© The Author(s) 2022. Published by Oxford University Press.)

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., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)