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Abstract Successful nerve repair using bioadhesive hydrogels demands minimizing tissue–material interfacial mechanical mismatch to reduce immune responses and scar tissue formation. Furthermore, it is crucial to maintain the bioelectrical stimulation‐mediated cell‐signaling mechanism to overcome communication barriers within injured nerve tissues. Therefore, engineering bioadhesives for neural tissue regeneration necessitates the integration of electroconductive properties with tissue‐like biomechanics. In this study, we propose a stretchable bioadhesive based on a custom‐designed chemically modified elastin‐like polypeptides (ELPs) and a choline‐based bioionic liquid (Bio‐IL), providing an electroconductive microenvironment to reconnect damaged nerve tissue. The stretchability akin to native neural tissue was achieved by incorporating hydrophobic ELP pockets, and a robust tissue adhesion was obtained due to multi‐mode tissue–material interactions through covalent and noncovalent bonding at the tissue interface. Adhesion tests revealed adhesive strength ~10 times higher than commercially available tissue adhesive, Evicel®. Furthermore, the engineered hydrogel supported in vitro viability and proliferation of human glial cells. We also evaluated the biodegradability and biocompatibility of the engineered bioadhesive in vivo using a rat subcutaneous implantation model, which demonstrated facile tissue infiltration and minimal immune response. The outlined functionalities empower the engineered elastic and electroconductive adhesive hydrogel to effectively enable sutureless surgical sealing of neural injuries and promote tissue regeneration.

(1) Background: This study aimed to enhance the biological properties of hydraulic calcium silicate cements (HCSCs) by incorporating organic and inorganic components, specifically elastin-like polypeptides (ELPs) and bioactive glass (BAG). We focused on the effects of these composites on the viability, migration, and osteogenic differentiation of human periodontal ligament fibroblasts (hPDLFs). (2) Methods: Proroot MTA was supplemented with 1–5 wt% 63S BAG and 10 wt% ELP. The experimental groups contained various combinations of HSCS with ELP and BAG. Cell viability was assessed using an MTT assay, cell migration was evaluated using wound healing and transwell assays, and osteogenic activity was determined through Alizarin Red S staining and a gene expression analysis of osteogenic markers (ALP, RUNX-2, OCN, and Col1A2). (3) Results: The combination of ELP and BAG significantly enhanced the viability of hPDLFs with an optimal BAG concentration of 1–4%. Cell migration assays demonstrated faster migration rates in groups with 2–4% BAG and ELP incorporation. Osteogenic activity was the highest with 2–3% BAG incorporation with ELP, as evidenced by intense Alizarin Red S staining and the upregulation of osteogenic differentiation markers. (4) Conclusions: The incorporation of ELP (organic) and BAG (inorganic) into HCSC significantly enhances the viability, migration, and osteogenic differentiation of hPDLFs. These findings suggest that composite HCSC might support healing in destructed bone lesions in endodontics.

Rare earth elements (REEs), including those in the lanthanide series, are crucial components essential for clean energy transitions, but they originate from geographically limited regions. Exploiting new and diverse supply sources is vital to facilitating a clean energy future. Hence, we explored the recovery of REEs from coal fly ash (FA), a complex, low-grade industrial feedstock that is currently underutilized (leachate concentrations of REEs in FA are < 0.003 mol%). Herein, we demonstrated the thermo-responsive genetically encoded REE-selective elastin-like polypeptides (RELPs) as a recyclable bioengineered protein adsorbent for the selective retrieval of REEs from coal fly ash over multiple cycles. The results showed that RELPs could be efficiently separated using temperature cycling and reused with high stability, as they retained ∼95% of their initial REE binding capacity even after four cycles. Moreover, RELPs selectively recovered high-purity REEs from the simulated solution containing one representative REE in the range of 0.0001–0.005 mol%, resulting in up to a 100,000-fold increase in REE purity. This study offers a sustainable approach to diversifying REE supplies by recovering REEs from low-grade coal fly ash in industrial wastes and provides a scientific basis for the extraction of high-purity REEs for industrial purposes.

Abstract 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.

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.

Abstract 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.

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.

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

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

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

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

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

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

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

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

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

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

BackgroundPreeclampsia is a hypertensive syndrome that complicates 3% to 5% of pregnancies in the United States. Preeclampsia originates from an improperly vascularized and ischemic placenta that releases factors that drive systemic pathophysiology. One of these factors, soluble fms‐like tyrosine kinase‐1, is believed to sequester vascular endothelial growth factor (VEGF), leading to systemic endothelial dysfunction and hypertension. With the goal of targeting soluble fms‐like tyrosine kinase‐1 while simultaneously preventing fetal exposure to VEGF, we fused VEGF to elastin‐like polypeptide, a biopolymer carrier that does not cross the placental barrier (ELP‐VEGF). Methods and ResultsELP‐VEGF restored in vitro endothelial cell tube formation in the presence of plasma from placental ischemic rats. Long‐term administered ELP‐VEGF in pregnant rats accumulated in maternal kidneys, aorta, liver, and placenta, but the protein was undetectable in the pups when administered at therapeutic doses in dams. Long‐term administration of ELP‐VEGF in a placental ischemia rat model achieved dose‐dependent attenuation of hypertension, with blood pressure equal to sham controls at a dose of 5 mg/kg per day. ELP‐VEGF infusion increased total plasma soluble fms‐like tyrosine kinase‐1 levels but dramatically reduced free plasma soluble fms‐like tyrosine kinase‐1 and induced urinary excretion of nitrate/nitrite, indicating enhanced renal nitric oxide signaling. ELP‐VEGF at up to 5 mg/kg per day had no deleterious effect on maternal or fetal body weight. However, dose‐dependent adverse events were observed, including ascites production and neovascular tissue encapsulation around the minipump. ConclusionsELP‐VEGF has the potential to treat the preeclampsia maternal syndrome, but careful dosing and optimization of the delivery route are necessary.

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.

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.