Search Results (40)

Three-dimensional (3D) in vitro adipocyte models provide physiologically relevant platforms for studying adipogenesis and obesity-related metabolic dysfunction. However, long-term adipocyte culture is often hindered by limited cell–matrix adhesion and spheroid detachment. Previously, we demonstrated that elastin-like polypeptide (ELP)–polyethyleneimine (PEI) coatings functionalized with a trivalent RGD motif enhanced spheroid retention during frequent media changes. The present study investigates the long-term functional consequences of RGD incorporation over a 28-day culture period. 3T3-L1 preadipocytes were seeded, differentiated, and matured on ELP-PEI or ELP-(RGD)₃-PEI coatings. Spheroid morphology, triglyceride content, expression of PPAR-γ, adiponectin, HIF-1α genes, and insulin-stimulated glucose uptake were assessed. Both coatings supported initial spheroid formation, but only ELP-PEI maintained the 3D architecture and supported adipogenic maturation and insulin responsiveness. ELP-(RGD)₃-PEI promoted early retention but led to spheroid disassembly by mid-culture; notably, by day 28, cells reaggregated into abnormally large spheroids with impaired metabolic function, likely due to continued proliferation. These findings highlight the critical role of extracellular matrix-mediated cell–cell versus cell–substrate interactions in maintaining 3D culture fidelity. While RGD enhances adhesion, it disrupts spheroid integrity and compromises adipogenic and metabolic maturation. Taken together, ELP-PEI coatings offer a more conducive microenvironment for long-term 3D adipocyte culture and hold promise for modeling obesity-associated metabolic dysfunction in vitro. Full article

Conventional calcium silicate cement (CSC) formulations often exhibit insufficient mechanical strength and low initial stability. This study aimed to develop an organic–inorganic hybrid biomaterial by incorporating an elastin-like polypeptide (ELP) (V125E8) and bioactive glass (BG) (63S) into CSC to improve its mechanical properties and wash-out resistance during the initial setting. Experimental groups included ProRoot MTA (Dentsply Sirona, USA) as a control (0BG), inorganic hybrids containing BG (2% or 5%; 2BG, 5BG), and organic–inorganic hybrids combining BG (2% or 5%; 2BG-L, 5BG-L) with a 10 wt% ELP solution. The compressive strength, microhardness, and wash-out resistance of the specimens were evaluated. The organic–inorganic hybrid groups (2BG-L and 5BG-L) exhibited significantly higher compressive strength and microhardness than the control (0BG) and inorganic-only groups (2BG and 5BG). Additionally, the incorporation of ELP markedly improved wash-out resistance, minimizing material disintegration during the initial setting in aqueous environments. The organic–inorganic hybrid groups (2BG-L and 5BG-L) exhibited significantly higher compressive strength and microhardness than the control (0BG) and inorganic-only groups (2BG and 5BG). Additionally, the incorporation of ELP markedly improved wash-out resistance, minimizing material disintegration during the initial setting in aqueous environments. Full article

To mimic the important features of progressing adiposity, in vitro adipose cell culture models must allow gradual intracellular fat accumulation in the three-dimensional (3D) arrangement of adipose-derived stem cells (ASCs) over a long-term culture period. Previously, elastin-like polypeptide (ELP) and polyethyleneimine (PEI) have been used to culture human adipose-derived stem cells (hASCs) as 3D spheroids and to differentiate them to adipocytes over a relatively long culture period of up to 5 weeks. In this study, to further enhance the spheroid adhesion properties, ELP was fused with Arginine–Glycine–Aspartic Acid (RGD) residues, known for their role as cell-attachment sites. This study aimed to assess whether the addition of RGD to the C-or N-terminus of ELP would impact the spheroid-forming ability of ELP-PEI coatings. ELP-RGD conjugates were produced using genetically modified Escherichia coli to express ELP-(RGD)₃ and (RGD)₃-ELP, followed by chemical conjugation with PEI. SDS gel electrophoresis, FTIR spectroscopy, and turbidimetry analyses revealed that ELP was conjugated with RGD without much alteration in the molecular weight, functional groups present, and transition temperature of ELP. The addition of RGD to ELP also did not affect the chemical conjugation capacity of ELP to PEI. We observed that the ELP-PEI coating formed slightly larger spheroids (61.8 ± 3.2 µm) compared to the ELP-(RGD)₃-PEI and (RGD)₃-ELP-PEI coatings (56.6 ± 3.0 and 53.4 ± 2.4 µm, respectively). Despite the size difference, ELP-(RGD)₃-PEI coatings exhibited superior spheroid retention during media changes, with minimal spheroid loss. DNA assay results confirmed a significant decrease in the DNA concentration (p < 0.05) after the 20 media changes for spheroids cultured on the ELP-PEI coating, indicating spheroid loss. However, there was no significant difference in DNA concentration before and after 20 media changes for spheroids cultured on the ELP-(RGD)₃-PEI and (RGD)₃-ELP-PEI coatings (p > 0.05). These findings suggest that RGD incorporation does not hinder the initial spheroid formation ability of the ELP-PEI coating and enhances spheroid retention under dynamic culture conditions. Full article

Protein purification is a crucial step for various downstream applications like drug development, antibody preparation, and structure determination. The constant pursuit is for methods that are more efficient and cost-effective. We propose a novel approach using an elastin-like polypeptide (ELP) as an aggregation core that serves as an anchor between the beads in a chromatography column. In this method, a chilled sample containing a [target protein type] fusion protein is loaded onto a pre-equilibrated IMAC (immobilized metal affinity chromatography) column with a low-salt buffer. The column is then washed with a warm buffer containing high salt to remove impurities. Here, the key step involves warming the column above the ELP’s transition temperature (Tt), which triggers its aggregation. This aggregation is expected to trap the target protein tightly between the beads. Subsequently, a harsh wash with high salt and high imidazole can be applied to remove even persistent contaminants, achieving high protein purity. Finally, the temperature is lowered, and a cold, low-salt buffer is introduced to reverse the aggregation and elute the purified target protein. This method has the potential to eliminate the need for sophisticated chromatography systems while still achieving high protein purity. Full article

This paper reports the development of a highly crosslinked hyper-branched polyglycerol (HPG) polymer bound to elastin-like proteins (ELPs) to create a membrane that undergoes a distinct closed-to-open permeation transition at 32 °C. The crosslinked HPG forms a robust, mesoporous structure (150–300 nm pores), suitable for selective filtration. The membranes were characterized by FTIR, UV–visible spectroscopy, SEM, and AFM, revealing their structural and morphological properties. Incorporating a synthetic polypeptide introduced thermo-responsive behavior, with the membrane transitioning from impermeable to permeable above the lower critical solution temperature (LCST) of 32 °C. Permeation studies using crystal violet (CV) demonstrated selective transport, where CV permeated only above 32 °C, while water permeated at all temperatures. This hybrid HPG-ELP membrane system, acting as a molecular switch, offers potential for applications in drug delivery, bioseparations, and smart filtration systems, where permeability can be controlled by temperature. Full article

(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. Full article

Elastin-like polypeptides (ELPs) are popular biomaterials due to their reversible, temperature-dependent phase separation and their tunability, which is achievable by evolving procedures in recombinant technology. In particular, recursive direction ligation by plasmid reconstruction (PRe-RDL) is the predominant cloning technique used to generate ELPs of varying lengths. Pre-RDL provides precise control over the number of (VPGXG)_n repeat units in an ELP due to the selection of type IIS restriction enzyme (REs) sites in the reconstructed pET expression plasmid, which is a low-to-medium copy number plasmid. While Pre-RDL can be used to seamlessly repeat essentially any gene sequence and overcome limitations of previous cloning practices, we modified the Pre-RDL technique, where a high copy number plasmid (pBluescript II SK(+)—using a new library of type IIS REs) was used instead of a pET plasmid. The modified technique successfully produced a diblock ELP gene of 240 pentapeptide repeats from 30 pentapeptide “monomers” composed of alanine, tyrosine, and leucine X residues. This study found that the large, GC-rich ELP gene compromised plasmid yields in pBluescript II SK(+) and favored higher plasmid yields in the pET19b expression plasmid. Additionally, the BL21 E. coli strain expression consistently provided a higher transformation efficiency and higher plasmid yield than the high cloning efficiency strain TOP10 E. coli. We hypothesize that the plasmid/high GC gene ratio may play a significant role in these observations, and not the total plasmid size or the total plasmid GC content. While expression of the final gene resulted in a diblock ELP with a phase separation temperature of 34.5 °C, future work will need to investigate RDL techniques in additional plasmids to understand the primary driving factors for improving yields of plasmids with large ELP-encoding genes. Full article

Soybean seeds show great potential as a safe and cost-effective host for the large-scale production of biopharmaceuticals and industrially important macromolecules. However, the yields of desired recombinant proteins in soybean seeds are usually lower than the economic threshold for their potential commercialization. Our previous study demonstrated that polypeptide fusion such as maize γ-zein or elastin-like polypeptide (ELP) could significantly increase the accumulation of foreign proteins. In the present study, a recombination strategy of polypeptide fusions (γ-zein or ELP) and suppression of intrinsic storage proteins (glycinin or conglycinin) via RNA interference was further exploited to improve the yield of the target protein in soybean seeds. Transgenic soybean plants harboring both polypeptide-fused green fluorescent protein (GFP) and glycinin/conglycinin RNAi expression cassettes were generated and confirmed by molecular analysis. The results showed that on both the glycinin and conglycinin suppression backgrounds, the average accumulation levels of recombinant zein-GFP and GFP-ELP proteins were significantly increased as compared to that of their counterparts without such suppressions in our previous study. Moreover, zein-GFP and GFP-ELP accumulation was also remarkably higher than unfused GFP on the glycinin suppression background. However, no significant differences were detected in the glycinin or conglycinin suppression backgrounds for the same polypeptide fusion constructs, though suppression of one of the storage proteins in soybean seeds led to a significant increase in the other. Additionally, the increases in the recombinant protein yield did not affect the total protein content and the protein/oil ratio in soybean seeds. Taken together, the results indicate that both the fusion of the foreign protein with polypeptide tags together with the depletion of endogenous storage proteins contributed to a higher accumulation of the recombinant proteins without affecting the total protein content or the protein/oil ratio in soybean seeds. Full article

Background: Oxidative stress-induced retinal degeneration is among the main contributing factors of serious ocular pathologies that can lead to irreversible blindness. αB-crystallin (cry) is an abundant component of the visual pathway in the vitreous humor, which modulates protein and cellular homeostasis. Within this protein exists a 20 amino acid fragment (mini-cry) with both chaperone and antiapoptotic activity. This study fuses this mini-cry peptide to two temperature-sensitive elastin-like polypeptides (ELP) with the goal of prolonging its activity in the retina. Methods: The biophysical properties and chaperone activity of cry-ELPs were confirmed by mass spectrometry, cloud-point determination, and dynamic light scattering ’DLS’. For the first time, this work compares a simpler ELP architecture, cry-V96, with a previously reported ELP diblock copolymer, cry-SI. Their relative mechanisms of cellular uptake and antiapoptotic potential were tested using retinal pigment epithelial cells (ARPE-19). Oxidative stress was induced with H₂O₂ and comparative internalization of both cry-ELPs was made using 2D and 3D culture models. We also explored the role of lysosomal membrane permeabilization by confocal microscopy. Results: The results indicated successful ELP fusion, cellular association with both 2D and 3D cultures, which were enhanced by oxidative stress. Both constructs suppressed apoptotic signaling (cleaved caspase-3); however, cry-V96 exhibited greater lysosomal escape. Conclusions: ELP architecture is a critical factor to optimize delivery of therapeutic peptides, such as the anti-apoptotic mini-cry peptide; furthermore, the protection of mini-cry via ELPs is enhanced by lysosomal membrane permeabilization. Full article

In the healing of wounds, human-like collagen (hCol) is essential. However, collagen-based composite dressings have poor stability in vivo, which severely limits their current therapeutic potential. Based on the above, we have developed a recombinant fusion protein named hCol-ELP, which consists of hCol and an elastin-like peptide (ELP). Then, we examined the physicochemical and biological properties of hCol-ELP. The results indicated that the stability of the hCol-ELP fusion protein exhibited a more compact and homogeneous lamellar microstructure along with collagen properties, it was found to be significantly superior to the stability of free hCol. The compound hCol-ELP demonstrated a remarkable capacity to induce the proliferation and migration of mouse embryo fibroblast cells (NIH/3T3), as well as enhance collagen synthesis in human skin fibroblasts (HSF) when tested in vitro. In vivo, hCol-ELP demonstrated significant enhancements in healing rate and a reduction in the time required for scab removal, thereby exhibiting a scar-free healing effect. The findings provide a crucial theoretical foundation for the implementation of an hCol-ELP protein dressing in fields associated with the healing of traumatic injuries. Full article

African swine fever (ASF) is one of the most severe diseases caused by the ASF virus (ASFV), causing massive economic losses to the global pig industry. Serological tests are important in ASF epidemiological surveillance, and more antigen targets are needed to meet market demand for ASFV antibody detection. In the present study, ASFV p15 protein was fusion-expressed in Escherichia coli (E. coli) with elastin-like polypeptide (ELP), and the ELP-p15 protein was purified using a simple inverse transition cycling (ITC) process. The ELP tag was cleaved off using tobacco etch virus protease (TEVp), resulting in a tag-free p15 protein. Western blot analysis demonstrated that the p15 protein reacted strongly with ASFV-positive serum. The p15 protein was used as a coating antigen in an indirect ELISA (iELISA) for detecting ASFV antibodies. The p15-iELISA method demonstrated high specificity to ASFV-positive sera, with a maximum detection dilution of 1:1600. Moreover, the method exhibited good reproducibility, with less intra-assay and inter-assay CV values than 10%. Therefore, p15-iELISA offers a novel approach for accurately detecting ASFV antibodies with significant clinical application potential. Full article

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

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. Full article

Elastin-like polypeptides (ELPs) are stimulus-responsive artificially designed proteins synthesized from the core amino acid sequence of human tropoelastin. ELPs have good biocompatibility and biodegradability and do not systemically induce adverse immune responses, making them a suitable module for drug delivery. Design strategies can equip ELPs with the ability to respond to changes in temperature and pH or the capacity to self-assemble into nanoparticles. These unique tunable biophysicochemical properties make ELPs among the most widely studied biopolymers employed in protein purification, drug delivery, tissue engineering and even in tumor therapy. As a module for drug delivery and as a carrier to target tumor cells, the combination of ELPs with therapeutic drugs, antibodies and photo-oxidation molecules has been shown to result in improved pharmacokinetic properties (prolonged half-life, drug targeting, cell penetration and controlled release) while restricting the cytotoxicity of the drug to a confined infected site. In this review, we summarize the latest developments in the application methods of ELP employed in tumor therapy, with a focus on its conjugation with peptide drugs, antibodies and photosensitizers. Full article

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. Full article