Elastin-like Protein Research Articles

In situ formed depot of elastin-like polypeptide-hirudin fusion protein for long-acting antithrombotic therapy

Thrombosis, induced by abnormal coagulation or fibrinolytic systems, is the most common pathology associated with many life-threatening cardio-cerebrovascular diseases. However, first-line anticoagulant drugs suffer from rapid drug elimination and risk of hemorrhagic complications. Here, we developed an in situ formed depot of elastin-like polypeptide (ELP)-hirudin fusion protein with a prodrug-like feature for long-term antithrombotic therapy. Highly secretory expression of the fusion protein was achieved with the assistance of the Ffu312 tag. Integration of hirudin, ELP, and responsive moiety can customize fusion proteins with properties of adjustable in vivo retention and controllable recovery of drug bioactivity. After subcutaneous injection, the fusion protein can form a reservoir through temperature-induced coacervation of ELP and slowly diffuse into the blood circulation. The biological activity of hirudin is shielded due to the N-terminal modification, while the activated key proteases upon thrombus occurrence trigger the cleavage of fusion protein together with the release of hirudin, which has antithrombotic activity to counteract thrombosis. We substantiated that the optimized fusion protein produced long-term antithrombotic effects without the risk of bleeding in multiple animal thrombosis models.

Modular Protein Fibers with Outstanding High-Strength and Acid-Resistance Performance Mediated by Copper Ion Binding and Imine Networking.

Engineered protein fibers are promising biomaterials with diverse applications due to their tunable protein structure and outstanding mechanical properties. However, it remains challenging at the molecular level to achieve satisfied mechanical properties and environmental tolerance simultaneously, especially under extreme acid conditions. Herein, the construction of artificial fibers comprising chimeric proteins made of rigid amyloid peptide and flexible cationic elastin-like protein (ELP) module is reported. The amyloid peptide readily assembles into highly organized β-sheet structures that can be further strengthened by the coordination of Cu2+, while the flexible ELP module allows the formation of imine-based crosslinking networks. These double networks synergistically enhance the mechanical properties of the fibers, leading to a high tensile strength and toughness, overwhelming many reported recombinant spidroin fibers. Notably, the coordination of Cu2+ with serine residues could stabilize β-sheet structures in the fibers under acidic conditions, which makes the fibers robust against acid, thus enabling their successful utilization in gastric perforation suturing. This work highlights the customization of double networks at the molecular level to create tailored high-performance protein fibers for various application scenarios.

Multiresponsive Nanoscale Self-Assembly of Azurin-Elastin-like Polypeptide Fusion Protein for Enhanced Prostate Cancer Therapy.

A fusion protein composed of a bacterial protein, azurin, having antineoplastic properties and a thermally responsive structural cationic elastin-like protein (ELP), is designed, cloned, expressed, and purified. A simple method of inverse transition cycle (ITC) is employed to purify the fusion protein azurin-ELP diblock copolymer (d-bc). The molecular weight of the azurin-ELP fusion protein is ∼32 kDa. Further, its self-assembly properties are investigated. Interestingly, the engineered azurin-ELP d-bc in response to increasing temperature shows a dual-step phase separation into biofunctional nanostructures. Around the physiological temperature, azurin-ELP d-bc forms stable coacervates, which is dependent on the concentration and time of incubation. These coacervates are formed below the lower critical solubility temperature (LCST) of the ELP block at physiological temperature. Above LCST, i.e., 50-55°C, micelles of size ranging from 25 to 30 nm are formed. The cytotoxicity of azurin-ELP d-bc depends on the size of the coacervates formed and their cellular uptake at physiological temperature. Further, MTT assay of azurin-ELP d-bc in the cross-linked micelles prepared ex situ shows > six times higher killing of LNCaP cells than the unimeric form of azurin-ELP at 5 μM concentration. The flow cytometric results of these micelles at 20 μM concentration show ∼97% LNCaP cells in the apoptotic phase. Thus, azurin-ELP cross-linked micelles have enhanced potential for anticancer therapy due to their higher avidity.

A potential bilayer skin substitute based on electrospun silk-elastin-like protein nanofiber membrane covered with bacterial cellulose

Skin substitutes are designed to promote wound healing by replacing extracellular matrix. Silk-elastin-like protein is a renewable extracellular matrix-like material that integrated the advantages of silk and elastin-like protein. In this study, electrospun silk-elastin-like protein (SELP) nanofiber membrane covered with bacterial cellulose (BC) was created as a potential skin substitute to mimic gradient structure of epidermis and dermis of skin. The two layers were glued together using adhesive SELP containing 3,4-dihydroxyphenylalanine (DOPA) converted from tyrosine by tyrosinase. Skin topical drugs commonly used in clinical practice can penetrate through the SELP/BC barrier, and the rate of penetration is proportional to drug concentration. BC with dense fibrous structure can act as a barrier to preserve the inner SELP layer and prevent bacterial invasion, with a blocking permeation efficiency over 99% against four species of bacteria. Cell experiments demonstrated that the reticular fibers of SELP could provide an appropriate growth environment for skin cells proliferation and adhesion, which is considered to promote tissue repair and regeneration. The promising results support this strategy to fabricate a silk-elastin-like protein-based biomaterial for skin substitutes in the clinical treatment of full skin injuries and ulcers.

An artificial silk elastin-like protein modifies the polarization of human macrophages line THP-1.

A silk elastin-like protein (SELP) is an artificial compound with silk fibroin-like and elastin-like tandem repeats. The objective of this study is to evaluate the influence of SELP on the polarization of human monocytoma cell line (THP-1)-derived macrophages. When the macrophages of inflammation-type (M1) were cultured with different concentrations of SELP solution, the secretion of a pro-inflammatory cytokine, tumor necrotizing factor (TNF) -α was significantly suppressed at the higher concentrations. In addition, the secretion of an anti-inflammation cytokine, interleukin (IL)-10, was significantly enhanced from the macrophage of M0-, M1-, and M2-types. By the incubation with soluble SELP, the morphology of M2-type macrophages changed to be of an extended shape. Following incubation with the sponge of SELP, M0-type macrophages secreted IL-10 with time. It is concluded that the SELP itself in solution has an ability to induce the anti-inflammation of M2-type macrophages.

Design Parameters for Injectable Biopolymeric Hydrogels with Dynamic Covalent Chemistry Crosslinks.

Dynamic covalent chemistry (DCC) crosslinks can form hydrogels with tunable mechanical properties permissive to injectability and self-healing. However, not all hydrogels with transient crosslinks are easily extrudable. For this reason, two additional design parameters must be considered when formulating DCC-crosslinked hydrogels: 1) degree of functionalization (DoF) and 2) polymer molecular weight (MW). To investigate these parameters, hydrogels comprised of two recombinant biopolymers: 1) a hyaluronic acid (HA) modified with benzaldehyde and 2) an elastin-like protein (ELP) modified with hydrazine (ELP-HYD), are formulated. Several hydrogel families are synthesized with distinct HA MW and DoF while keeping the ELP-HYD component constant. The resulting hydrogels have a range of stiffnesses, G' ≈ 10-1000Pa, and extrudability, which is attributed to the combined effects of DCC crosslinks and polymer entanglements. In general, lower MW formulations require lower forces for injectability, regardless of stiffness. Higher DoF formulations exhibit more rapid self-healing. Gel extrusion through a cannula (2 m length, 0.25 mm diameter) demonstrates the potential for minimally invasive delivery for future biomedical applications. In summary, this work highlights additional parameters that influence the injectability and network formation of DCC-crosslinked hydrogels and aims to guide future design of injectable hydrogels.

Elastin-like protein hydrogels with controllable stress relaxation rate and stiffness modulate endothelial cell function.

Mechanical cues from the extracellular matrix (ECM) regulate vascular endothelial cell (EC) morphology and function. Since naturally derived ECMs are viscoelastic, cells respond to viscoelastic matrices that exhibit stress relaxation, in which a cell-applied force results in matrix remodeling. To decouple the effects of stress relaxation rate from substrate stiffness on EC behavior, we engineered elastin-like protein (ELP) hydrogels in which dynamic covalent chemistry (DCC) was used to crosslink hydrazine-modified ELP (ELP-HYD) and aldehyde/benzaldehyde-modified polyethylene glycol (PEG-ALD/PEG-BZA). The reversible DCC crosslinks in ELP-PEG hydrogels create a matrix with independently tunable stiffness and stress relaxation rate. By formulating fast-relaxing or slow-relaxing hydrogels with a range of stiffness (500-3300 Pa), we examined the effect of these mechanical properties on EC spreading, proliferation, vascular sprouting, and vascularization. The results show that both stress relaxation rate and stiffness modulate endothelial spreading on two-dimensional substrates, on which ECs exhibited greater cell spreading on fast-relaxing hydrogels up through 3 days, compared with slow-relaxing hydrogels at the same stiffness. In three-dimensional hydrogels encapsulating ECs and fibroblasts in coculture, the fast-relaxing, low-stiffness hydrogels produced the widest vascular sprouts, a measure of vessel maturity. This finding was validated in a murine subcutaneous implantation model, in which the fast-relaxing, low-stiffness hydrogel produced significantly more vascularization compared with the slow-relaxing, low-stiffness hydrogel. Together, these results suggest that both stress relaxation rate and stiffness modulate endothelial behavior, and that the fast-relaxing, low-stiffness hydrogels supported the highest capillary density in vivo.

Mechanical Properties of Protein-Based Hydrogels Derived from Binary Protein Mixtures-A Feasibility Study.

Hydrogels based on natural polymers such as proteins are considered biocompatible and, therefore, represent an interesting class of materials for application in the field of biomedicine and high-performance materials. However, there is a lack of understanding of the proteins which are able to form hydrogel networks by photoinduced dityrosine crosslinking as well as a profound knowledge of the formed network itself and the mechanisms which are responsible for the resulting mechanical properties of such protein-based hydrogels. In this study, casein, bovine serum albumin, α-amylase, and a hydrophobic elastin-like protein were used to prepare binary protein mixtures with defined concentration ratios. After polymerization, the mechanical properties of the resulting homopolymeric and copolymeric hydrogels were determined using rheological methods depending on the protein shares used. In additional uniaxial compression tests, the fracture strain was shown to be independent of the protein shares, while hydrogel toughness and compressive strength were increased for protein-based hydrogels containing casein.

An Artificial Silk Elastin-Like Protein Modifies the Polarization of Macrophages.

A silk elastin-like protein (SELP) is an artificial compound with silk fibroin-like and elastin-like tandem repeats. The objective of this study is to evaluate the influence of SELP on the polarization of mouse bone marrow-derived macrophages. When the macrophages of inflammation-type (M1) were cultured with different concentrations of SELP solution, the secretion of a pro-inflammatory cytokine, tumor necrotizing factor (TNF)-α, was significantly suppressed at the higher concentrations. In addition, the secretion of an anti-inflammation cytokine, interleukin (IL)-10, was significantly enhanced from the macrophage of an original type (M0). By the incubation with soluble SELP, the morphology of M0- and M1-type macrophages changed to be of a round shape with a large size. Following incubation with the sponge of SELP, the M0-type macrophages secreted IL-10 with time. When injected into an air pouch of mice subcutis which had been prepared by the injection of air, the SELP sponge and 5 wt % of SELP solution induced IL-10 secretion to a significantly high extent compared with the saline injection. Cells isolated from the air pouch 24 h after the injection were stained by the CD206 of a M2 marker. It is concluded that the SELP itself in solution has an ability to induce the anti-inflammation M2-type macrophages.

Abstract 11376: Catheter-Injectable Hydrogel for the Delivery of a Minicircle Encoding SDF-la as Therapy for Myocardial Infarction

Myocardial infarction ( MI ) is a leading cause of cardiovascular disease in the U.S. and abroad. Transient delivery of stromal cell-derived factor 1 ( SDF-1 α ) either through the delivery of protein-encoding genes or the protein itself has demonstrated improved cardiac function in preclinical models. However, the delivery of the therapy suffers from two limitations: (1) poor retention when injected into the contracting myocardium, and (2) rapid degradation of the protein, which requires prolonged activity to be therapeutically effective. To mitigate these limitations, we developed a catheter-injectable gel to deliver SDF-1 α encoding minicircles ( MC ). Design of gels for injection into the myocardium is historically challenging since traditional chemical strategies result in gels that are either too stiff and not catheter injectable or too weak and quickly ejected out of the contracting heart muscle. To address this challenge, our approach is to use dynamic covalent chemical (DCC) bonds, which are mechanically strong yet reversible, resulting in a shear-thinning gel (hence catheter injectable) and self-healing (hence retained in the contracting heart muscle). This biocompatible gel is chemically defined for future FDA approval and is synthesized from completely biodegradable polymers: recombinant elastin-like protein and hyaluronic acid. Rheological characterization identified several gel formulations that were easily extruded through a catheter (2 m long and .25 mm internal diameter) and rapidly reformed into a gel within 5 seconds after extrusion. To demonstrate the potential for clinical translatability, we evaluated the injectability and retention of the gel within the contracting rat myocardium. Our gel significantly improved the retention of a microparticle cargo by 200% compared to delivery in Matrigel. Current work is focused on evaluating the therapeutic efficacy of the gel in a rat myocardial infarct model when delivering a therapeutic cargo of SDF-1a-encoding MC. This biocompatible, catheter-injectable gel has great potential to significantly improve the delivery and retention of therapeutic cargo within mechanically active tissues, significantly improving the prolonged efficacy of MI therapies.

Abstract 15334: Tunable Elastin-Like Protein (Elp)-Based Hydrogels for Cell Transplantation in Vascular Repair

Introduction: Endothelial cells (ECs) can improve blood perfusion in diseased blood vessels; however, direct injection of cells significantly decreases their survival and functionality for angiogenesis. To address these limitations, we study a family of engineered extracellular matrices with tunable biochemical and biomechanical cues for enhanced survival and improved angiogenic behavior of primary human ECs. Hypothesis: We hypothesize that the biomechanical properties of ELP-PEG hydrogels will modulate EC survival and function in vitro. Methods: Engineered hydrogels, termed ELP-PEG, consists of two components of a hydrazine-modified elastin-like protein (ELP-HYD) and an aldehyde- or benzaldehyde-modified, polyethylene glycol (PEG-ALD or PEG-BZA), which interact with each other through hydrazone dynamic covalent chemistry (DCC) bonds to form ELP-PEG hydrogels. Stiffness is controlled by altering the number of PEG-ALD or PEG-BZA crosslinks, and the stress relaxation rate is tuned by varying the PEG-ALD crosslinks vs. PEG-BZA. Stiffness and stress relaxation rates of the hydrogels were assessed by dynamic oscillatory rheology. Afterwards, human umbilical vein endothelial cells (HUVECs) were encapsulated within gels to assess cell viability and spreading using a Live/Dead Cytotoxicity assay and confocal fluorescence imaging. Results: Stress relaxation rate in ELP/PEG-ALD is much slower compared with similar combination in ELP/PEG-BZA. Rheology measurements of the RGD-ELP/PEG-BZA (1.75%/1%) and RGD-ELP/PEG-ALD (1.25%/1%) hydrogels demonstrated a storage modulus of 700Pa. It confirms the tunability of stress relaxation rate with a constant stiffness. Cell viability assay demonstrated that both hydrogels could support high cell viability (>90%) for 7 days. After 7 days, cell spreading increased in RGD-ELP/PEG-BZA hydrogels and tubular networks appeared inside the gels. However, cells did not form elongated morphology in RGD-ELP/PEG-ALD hydrogels, suggesting that stress relaxation rate and mechanical stiffness are key characteristics in modulating endothelial cell behavior. Conclusions: ELP/PEG-ALD/BZA promotes angiogenic behavior of ECs and is a promising candidate for cell delivery in vascular diseases.

Targeted elastin-like polypeptide fusion protein for near-infrared imaging of human and canine urothelial carcinoma.

Cystoscopic visualization of bladder cancer is an essential method for initial bladder cancer detection and diagnosis, transurethral resection, and monitoring for recurrence. We sought to develop a new intravesical imaging agent that is more specific and sensitive using a polypeptide based NIR (near-infrared) probe designed to detect cells bearing epidermal growth factor receptors (EGFR) that are overexpressed in 80% of urothelial carcinoma (UC) cases. The NIR imaging agent consisted of an elastin like polypeptide (ELP) fused with epidermal growth factor (EGF) and conjugated to Cy5.5 to give Cy5.5-N24-EGF as a NIR contrast agent. In addition to evaluation in human cells and tissues, the agent was tested in canine cell lines and tissue samples with naturally occurring invasive UC. Flow cytometry and confocal microscopy were used to test cell-associated fluorescence of the probe in T24 human UC cells, and in K9TCC-SH (high EGFR expression) and K9TCC-Original (low EGF expression) canine cell lines. The probe specifically engages these cells through EGFR within 15 min of incubation and reached saturation within a clinically relevant 1 h timeframe. Furthermore, ex vivo studies with resected canine and human bladder tissues showed minimal signal from normal adjacent tissue and significant NIR fluorescence labeling of tumor tissue, in good agreement with our in vitro findings. Differential expression of EGFR ex vivo was revealed by our probe and confirmed by anti-EGFR immunohistochemical staining. Taken together, our data suggests Cy5.5-ELP-EGF is a NIR probe with improved sensitivity and selectivity towards BC that shows excellent potential for clinical translation.

Genetically Engineered Viral Vectors and Organic-Based Non-Viral Nanocarriers for Drug Delivery Applications.

Conventional drug delivery systems are challenged by concerns related to systemic toxicity, repetitive doses, drug concentrations fluctuation, and adverse effects. Various drug delivery systems are developed to overcome these limitations. Nanomaterials are employed in a variety of biomedical applications such as therapeutics delivery, cancer therapy, and tissue engineering. Physiochemical nanoparticle assembly techniques involve the application of solvents and potentially harmful chemicals, commonly at high temperatures. Genetically engineered organisms have the potential to be used as promising candidates for greener, efficient, and more adaptable platforms for the synthesis and assembly of nanomaterials. Genetically engineered carriers are precisely designed and constructed in shape and size, enabling precise control over drug attachment sites. The high accuracy of these novel advanced materials, biocompatibility, and stimuli-responsiveness, elucidate their emerging application in controlled drug delivery. The current article represents the research progress in developing various genetically engineered carriers. Organic-based nanoparticles including cellulose, collagen, silk-like polymers, elastin-like protein, silk-elastin-like protein, and inorganic-based nanoparticles are discussed in detail. Afterward, viral-based carriers are classified, and their potential for targeted therapeutics delivery is highlighted. Finally, the challenges and prospects of these delivery systems are concluded.

Purification of a Hydrophobic Elastin-Like Protein Toward Scale-Suitable Production of Biomaterials.

Elastin-like proteins (ELPs) are polypeptides with potential applications as renewable bio-based high-performance polymers, which undergo a stimulus-responsive reversible phase transition. The ELP investigated in this manuscript—ELP[V2Y-45]—promises fascinating mechanical properties in biomaterial applications. Purification process scalability and purification performance are important factors for the evaluation of potential industrial-scale production of ELPs. Salt-induced precipitation, inverse transition cycling (ITC), and immobilized metal ion affinity chromatography (IMAC) were assessed as purification protocols for a polyhistidine-tagged hydrophobic ELP showing low-temperature transition behavior. IMAC achieved a purity of 86% and the lowest nucleic acid contamination of all processes. Metal ion leakage did not propagate chemical modifications and could be successfully removed through size-exclusion chromatography. The simplest approach using a high-salt precipitation resulted in a 60% higher target molecule yield compared to both other approaches, with the drawback of a lower purity of 60% and higher nucleic acid contamination. An additional ITC purification led to the highest purity of 88% and high nucleic acid removal. However, expensive temperature-dependent centrifugation steps are required and aggregation effects even at low temperatures have to be considered for the investigated ELP. Therefore, ITC and IMAC are promising downstream processes for biomedical applications with scale-dependent economical costs to be considered, while salt-induced precipitation may be a fast and simple alternative for large-scale bio-based polymer production.

Tuning Polymer Hydrophilicity to Regulate Gel Mechanics and Encapsulated Cell Morphology.

Mechanically tunable hydrogels are attractive platforms for 3D cell culture, as hydrogel stiffness plays an important role in cell behavior. Traditionally, hydrogel stiffness has been controlled through altering either the polymer concentration or the stoichiometry between crosslinker reactive groups. Here, an alternative strategy based upon tuning the hydrophilicity of an elastin-like protein (ELP) is presented. ELPs undergo a phase transition that leads to protein aggregation at increasing temperatures. It is hypothesized that increasing this transition temperature through bioconjugation with azide-containing molecules of increasing hydrophilicity will allow direct control of the resulting gel stiffness by making the crosslinking groups more accessible. These azide-modified ELPs are crosslinked into hydrogels with bicyclononyne-modified hyaluronic acid (HA-BCN) using bioorthogonal, click chemistry, resulting in hydrogels with tunable storage moduli (100-1000Pa). Human mesenchymal stromal cells (hMSCs), human umbilical vein endothelial cells (HUVECs), and human neural progenitor cells (hNPCs) are all observed to alter their cell morphology when encapsulated within hydrogels of varying stiffness. Taken together, the use of protein hydrophilicity as a lever to tune hydrogel mechanical properties is demonstrated. These hydrogels have tunable moduli over a stiffness range relevant to soft tissues, support the viability of encapsulated cells, and modify cell spreading as a consequence of gel stiffness.

Cellular synthesis of protein pretzelanes

Topology has been recognized as a unique dimension in molecular engineering, yet the topological diversity remains largely untapped, especially in macromolecules. Herein, we report the molecular design, cellular synthesis, and detailed characterization of protein pretzelanes with a chemical topology of a bridged Hopf link. The synergy between the intramolecular chain entwining guided by the p53dim (X) domains and the genetically encoded side-chain coupling by SpyTag(A)-SpyCatcher(B) reaction facilitates the direct synthesis of the model protein pretzelane BXA-BXA in Escherichia coli. The approach tolerates the insertion of various proteins-of-interest, such as elastin-like protein (ELP), superfolder green fluorescent protein (GFP) and dihydrofolate reductase (DHFR), at the bridge region between two rings, giving rise to three protein pretzelanes BXA-ELP-BXA, BXA-GFP-BXA, and BXA-DHFR-BXA. Their topology has been verified by combined techniques of MALDI-TOF mass spectrometry, ion mobility-mass spectrometry, site-specific mutation, and orthogonal proteolytic digestion experiments. Not only are the fluorescent properties of GFP and the catalytic properties of DHFR fully retained, the pretzelane topology also renders BXA-DHFR-BXA more thermally resilient than the wild-type DHFR. These results expand the topological diversity of proteins and demonstrate protein stabilization as a potential functional benefit for the pretzelane topology.

Hyaluronan and elastin-like protein (HELP) gels significantly improve microsphere retention in the myocardium.

Heart disease is the leading cause of death globally, and delivery of therapeutic cargo (e.g., particles loaded with proteins, drugs, or genes and cells) through direct injection into the myocardium is a promising clinical intervention. However, retention of deliverables to the contracting myocardium is low, with as much as 60-90% of payload being lost within 24 hr. Commercially-available injectable hydrogels, including Matrigel, have been hypothesized to increase payload retention but have not yielded significant improvements in quantified analyses. Here, we assess a recombinant hydrogel composed of chemically modified hyaluronan and elastin-like protein (HELP) as an alternative injectable carrier to increase cargo retention. HELP is crosslinked using dynamic covalent bonds, and tuning the hyaluronan chemistry significantly alters hydrogel mechanical properties including stiffness, stress relaxation rate, and ease of injectability through a needle or catheter. These materials can be injected even after complete crosslinking, extending the time window for surgical delivery. We show that HELP gels significantly improve in vivo retention of microsphere cargo compared to Matrigel, both 1 day and 7 days post-injection directly into the rat myocardium. These data suggest that HELP gels may assist with the clinical translation of therapeutic cargo designed for delivery into the contracting myocardium by preventing acute cargo loss.

Multifunctional Elastin-Like Polypeptide Fusion Protein Coacervates Inhibit Receptor-Mediated Proinflammatory Signals and Promote Angiogenesis in Mouse Diabetic Wounds.

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.

Hybrid fusion protein as a dual protease inhibitor for the healing of chronic wounds.

Diseases bring about the need for interventions that pinpoint each specific aspect of the illness. Commonly, remission of a complex disease is accomplished by mixing treatments, medications, and therapeutics together in a fashion where they may negatively interact with each other or never arrive at the diseased site as a systemic heterogeneous mixture. Chronic wounds display intricacy as they are very localized and have their own environment where tissue deconstruction due to high levels of numerous proteases outweighs normal tissue reconstruction. This idea leads to the necessity of a protein that contains low diffusivity rates for localized treatment, strength against high concentrations of proteolytic species that lead to degradation of short chain peptides, while encompassing broad inhibitory effects against multiple proteases. Elastin-like peptides are an attractive, thermoresponsive, protein-based drug delivery partner as they contain low diffusivity and serve as a stable architecture for short chain peptide fusion. In this project, a novel elastin-like peptide-based protein has been created to target the inhibition of both human neutrophil elastase and matrix metalloprotease-2. As a biologic, this is unique as it is a protein with specific biological activities against multiple proteases, ultimately displaying the potential to mix and match differing biologically active peptides within one amino acid sequence.

Abstract P180: Tunable Elastin-like Protein-based Hydrogels For Cell Transplantation In Vascular Repair

Introduction: Endothelial cells can improve blood perfusion in diseased blood vessels; however, direct injection of cells significantly decreases their survival and functionality for angiogenesis. To address these limitations, we study a family of engineered extracellular matrices with tunable biochemical and biomechanical cues for enhanced survival and improved angiogenic behavior of ECs. Materials & Methods: Engineered hydrogels, termed ELP-PEG, consists of two components of a hydrazine-modified elastin-like protein (ELP-HYD) and an aldehyde- or benzaldehyde-modified, polyethylene glycol (PEG-ALD or PEG-BZA), which interact with each other through hydrazone dynamic covalent chemistry (DCC) bonds to form ELP-PEG hydrogels. Stiffness is controlled by altering the number of PEG-ALD or PEG-BZA crosslinks, and the stress relaxation rate is tuned by varying the PEG-ALD crosslinks vs. PEG-BZA. Stiffness and stress relaxation rates of the hydrogels were assessed by dynamic oscillatory rheology. Afterward, human umbilical vein endothelial cells (HUVECs) were encapsulated within gels to assess cell viability and spreading using a Live/Dead Cytotoxicity assay and confocal fluorescence imaging. Results and Discussion: Stress relaxation rate in ELP/PEG-ALD is much slower compared with a similar combination in ELP/PEG-BZA. Rheology measurements of the RGD-ELP/PEG (2%/2%) hydrogels demonstrated a storage modulus of 800Pa. It confirms the tunability of the stress relaxation rate with constant stiffness. Cell viability assay demonstrated that both hydrogels could support high cell viability (>90%) for 7 days. After 7 days, cell spreading increased in RGD-ELP/PEG-BZA hydrogels, however, cells did not form elongated morphology in RGD-ELP/PEG-ALD hydrogels, suggesting that stress relaxation rate and mechanical stiffness are key characteristics in modulating endothelial cell behavior. Conclusions: ELP/PEG-ALD/BZA promotes the angiogenic behavior of endothelial cells and is a promising candidate for cell delivery in vascular diseases.