Combining Catalyst-Free Click Chemistry with Coaxial Electrospinning to Obtain Long-Term, Water-Stable, Bioactive Elastin-Like Fibers for Tissue Engineering Applications.

Elastic fibers are a fundamental requirement for tissue-engineered equivalents of physiologically elastic native tissues. Here, a simple one-step electrospinning approach is developed, combining i) catalyst-free click chemistry, ii) coaxial electrospinning, and iii) recombinant elastin-like polymers as a relevant class of biomaterials. Water-stable elastin-like fibers are obtained without the use of cross-linking agents, catalysts, or harmful organic solvents. The fibers can be directly exposed to an aqueous environment at physiological temperature and their morphology maintained for at least 3 months. The bioactivity of the fibers is demonstrated with human vascular cells and the potential of the process for vascular tissue engineering is shown by fabricating small-diameter tubular fibrous scaffolds. Moreover, highly porous fluffy 3D constructs are obtained without the use of specially designed collectors or sacrificial materials, further supporting their applicability in the biomedical field. Ultimately, the strategy that is developed here may be applied to other click systems, contributing to expanding their potential in medical technology.

Construction of Thermo-Responsive Elastin-Like Polypeptides (ELPs)-Aggregation-Induced-Emission (AIE) Conjugates for Temperature Sensing.

In this work, an aggregation-induced emission (AIE) molecule (tetraphenylethene derivative, TPE-COOH) was conjugated to elastin-like polypeptides (ELPs40) via an amide bond to form ELPs40-TPE. The successful synthesis of ELPs40-TPE was confirmed by Circular Dichroism spectroscopy, gel electrophoresis, UV-vis absorption, and fluorescence emission spectroscopy. ELPs40-TPE possessed both amphiphilicity and the features of an AIE, and the fluorescence intensity was dependent on the local temperature. The Hela cells imaging indicated that ELPs40-TPE has great potential for bio-imaging applications because of its advantages of high fluorescence intensity, good water-solubility, and remarkable biocompatibility.

Random and oriented electrospun fibers based on a multicomponent, in situ clickable elastin-like recombinamer system for dermal tissue engineering.

Herein we present a system to obtain fibers from clickable elastin-like recombinamers (ELRs) that crosslink in situ during the electrospinning process itself, with no need for any further treatment to stabilize them. These ELR-click fibers are completely stable under in vitro conditions. A wrinkled fiber morphology is obtained. In addition to a random fiber orientation, oriented fibers with a high degree of alignment and coherence can also be obtained by using a rotational electrode. The production of multicomponent fibers means that different functionalities, such as cell-adhesion domains (RGD peptides), can be incorporated into them. In a subsequent study, two main cell lines present in the dermis and epidermis, namely keratinocytes and fibroblasts, were cultured on top of the ELR-click fibers. Adhesion, proliferation, fluorescence, immunostaining and histology studies showed the cytocompatibility of these scaffolds, thus suggesting their possible use for wound dressings in skin tissue engineering applications. STATEMENT OF SIGNIFICANCE: For the first time stable electrospun bioactive fibers are obtained by the in situ mixing of two "clickable" ELR components previously described by Gonzalez et al (Acta Biomaterialia 2014). This work describes an efficient system to prepare fibrous scaffolds based on peptidic polymers by electrospinning without the need of crosslinking agents that could be harmful for cells or living tissues. These bioactive fibers support cell growth due to the inclusion of RGD motifs (Staubli et al. Biomaterials 2017). Finally, the in vitro biocompatibility of the two main cell types found in the outer layers of skin, fibroblasts and keratinocytes, indicates that this system is of great interest to prepare elastic artificial skin substitutes for wound healing applications.

Order, Disorder, and Temperature-Driven Compaction in a Designed Elastin Protein.

Artificial minielastin constructs have been designed that replicate the structure and function of natural elastins in a simpler context, allowing the NMR observation of structure and dynamics of elastin-like proteins with complete residue-specific resolution. We find that the alanine-rich cross-linking domains of elastin have a partially helical structure, but only when capped by proline-rich hydrophobic domains. We also find that the hydrophobic domains, composed of prominent 6-residue repeats VPGVGG and APGVGV found in natural elastins, appear random coil by both NMR chemical shift analysis and circular dichroism. However, these elastin hydrophobic domains exhibit structural bias for a dynamically disordered conformation that is neither helical nor ß sheet with a degree of nonrandom structural bias which is dependent on residue type and position in the sequence. Another nonrandom-coil aspect of hydrophobic domain structure lies in the fact that, in contrast to other intrinsically disordered proteins, these hydrophobic domains retain a relatively condensed conformation whether attached to cross-linking domains or not. Importantly, these domains and the proteins containing them constrict with increasing temperature by up to 30% in volume without becoming more ordered. This property is often observed in nonbiological polymers and suggests that temperature-driven constriction is a new type of protein structural change that is linked to elastin's biological functions of coacervation-driven assembly and elastic recoil.

Elastin-like protein-hyaluronic acid (ELP-HA) hydrogels with decoupled mechanical and biochemical cues for cartilage regeneration.

Hyaluronic acid (HA) is a major component of cartilage extracellular matrix and is an attractive material for use as 3D injectable matrices for cartilage regeneration. While previous studies have shown the promise of HA-based hydrogels to support cell-based cartilage formation, varying HA concentration generally led to simultaneous changes in both biochemical cues and stiffness. How cells respond to the change of biochemical content of HA remains largely unknown. Here we report an adaptable elastin-like protein-hyaluronic acid (ELP-HA) hydrogel platform using dynamic covalent chemistry, which allows variation of HA concentration without affecting matrix stiffness. ELP-HA hydrogels were created through dynamic hydrazone bonds via the reaction between hydrazine-modified ELP (ELP-HYD) and aldehyde-modified HA (HA-ALD). By tuning the stoichiometric ratio of aldehyde groups to hydrazine groups while maintaining ELP-HYD concentration constant, hydrogels with variable HA concentration (1.5%, 3%, or 5%) (w/v) were fabricated with comparable stiffness. To evaluate the effects of HA concentration on cell-based cartilage regeneration, chondrocytes were encapsulated within ELP-HA hydrogels with varying HA concentration. Increasing HA concentration led to a dose-dependent increase in cartilage-marker gene expression and enhanced sGAG deposition while minimizing undesirable fibrocartilage phenotype. The use of adaptable protein hydrogels formed via dynamic covalent chemistry may be broadly applicable as 3D scaffolds with decoupled niche properties to guide other desirable cell fates and tissue repair.

Computational smart polymer design based on elastin protein mutability.

Soluble elastin-like peptides (ELPs) can be engineered into a range of physical forms, from hydrogels and scaffolds to fibers and artificial tissues, finding numerous applications in medicine and engineering as "smart polymers". Elastin-like peptides are attractive candidates as a platform for novel biomaterial design because they exhibit a highly tunable response spectrum, with reversible phase transition capabilities. Here, we report the design of the first virtual library of elastin-like protein models using methods for enhanced sampling to study the effect of peptide chemistry, chain length, and salt concentration on the structural transitions of ELPs, exposing associated molecular mechanisms. We describe the behavior of the local molecular structure under increasing temperatures and the effect of peptide interactions with nearest hydration shell water molecules on peptide mobility and propensity to exhibit structural transitions. Shifts in the magnitude of structural transitions at the single-molecule scale are explained from the perspective of peptide-ion-water interactions in a library of four unique elastin-like peptide systems. Predictions of structural transitions are subsequently validated in experiment. This library is a valuable resource for recombinant protein design and synthesis as it elucidates mechanisms at the single-molecule level, paving a feedback path between simulation and experiment for smart material designs, with applications in biomedicine and diagnostic devices.

Rationally Designed Redox-Sensitive Protein Hydrogels with Tunable Mechanical Properties.

Protein hydrogels are an important class of materials for applications in biotechnology and medicine. The fine-tuning of their sequence, molecular weight, and stereochemistry offers unique opportunities to engineer biofunctionality, biocompatibility, and biodegradability into these materials. Here we report a new family of redox-sensitive protein hydrogels with controllable mechanical properties composed of recombinant silk-elastin-like protein polymers (SELPs). The SELPs were designed and synthesized with different ratios of silk-to-elastin blocks that incorporated periodic cysteine residues. The cysteine-containing SELPs were thermally responsive in solution and rapidly formed hydrogels at body temperature under physiologically relevant, mild oxidative conditions. Upon addition of a low concentration of hydrogen peroxide at 0.05% (w/v), gelation occurred within minutes for the SELPs with a protein concentration of approximately 4% (w/v). The gelation time and mechanical properties of the hydrogels were dependent on the ratio of silk to elastin. These polymer designs also significantly affected redox-sensitive release of a highly polar model drug from the hydrogels in vitro. Furthermore, oxidative gelation was performed at other physiologically relevant temperatures, and this resulted in hydrogels with tunable mechanical properties, thus, providing a secondary level of control over hydrogel stiffness. These newly developed injectable SELP hydrogels with redox-sensitive features and tunable mechanical properties may be potentially useful as biomaterials with broad applications in controlled drug delivery and tissue engineering.

Total synthesis of elastin peptide using high pressure-liquid phase synthesis assisted by a soluble tag strategy.

A highly aggregating elastin peptide was prepared efficiently using a high pressure-liquid phase synthesis approach assisted by a soluble tag strategy. Two standard syringes were connected to each other to construct a reactor. This simple reactor was used to apply high pressure to the highly viscous reaction mixture thereby maintaining its fluidity. The reactions were completely inhibited due to aggregation when conducted in a standard flask reactor, whereas our high pressure approach accelerated the couplings to realize complete conversion within 5-7 min. All steps were conducted at 0.10 M concentration, affording grams of the desired product.

Enhancement predicting accuracy for elastin-like polypeptides temperature transition by back propagation neural network.

Elastin-like polypeptides (ELPs) have been widely used to promote the development of a variety of smart biomaterials. Transition temperature is a key attribute of ELPs central to ELPs researches. Therefore, it is essential to establish predictive models of transition temperature that are both computationally efficient and reliable by employing simple parameters. Back propagation neural network (BPNN), a powerful feed-forward algorithm designed to solve problems with overwhelming complexity, has been successfully used in non-linear predictor model. In this study, two pH-sensitive ELPs were expressed, purified and determined for temperature transition across a range of pH. The pH, concentration and molecular weight (MW) as well as isoelectric point (PI) and pseudo amino acid (PseAA) of these two ELPs were adopted as input parameters. Support vector machine (SVM) and back propagation neural network (BPNN) were performed respectively. We selected Lamda (λ) value by training set and evaluated predictor model by jackknife test that combined with Uniform Design (UD). According to the results of BPNN and SVM, whose mean absolute error (MAE) of BPNN model jackknife test were 4.80 and 4.95 respectively, the predictive ability of BPNN is a minor improvement over SVM. Applying Mackay's data, MAE of BPNN jackknife test was 2.02, while the MAE between experimental and predicted transition temperature was 2.30 in Mackay's predictor model. Compared with Mackay predictor model, the enhancement in the accuracy indicates that the proposed BPNN method could play a complementary role to predict ELPs transition temperature.

Design and synthesis of elastin-like polypeptides for an ideal nerve conduit in peripheral nerve regeneration.

The study involves design and synthesis of three different elastin like polypeptide (ELP) gene monomers namely ELP1, ELP2 and ELP3 that encode for ELP proteins. The formed ELPs were assessed as an ideal nerve conduit for peripheral nerve regeneration. ELP1 was constructed with a small elongated pentapeptide carrying VPGVG sequence to mimic the natural polypeptide ELP. The ELP2 was designed by the incorporation of 4-penta peptide chains to improve the biocompatibility and mechanical strength. Thus, the third position in unique VPGVG was replaced with alanine to VPAVG and in a similar way modified to VPGKG, VPGEG and VPGIG with the substitution of lysine, glutamic acid and isoleucine. In ELP3, fibronectin C5 domain endowed with REDV sequence was introduced to improve the cell attachment. The ELP1, ELP2 and ELP3 proteins expressed by Escherichia coli were purified by inverse transition cycling (ITC). The purified ELPs were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. The Schwann cell (SC) morphology and cell adhesion were assessed by fabrication of ELP membrane cross-linked with glutaraledhyde. The Schwann cell proliferation was measured by WST-1 assay. Immunofluorostaining of Schwann cells was accomplished with SC specific phenotypic marker, S100.

Temperature-sensitive elastin-mimetic dendrimers: Effect of peptide length and dendrimer generation to temperature sensitivity.

Dendrimers are synthetic macromolecules with unique structure, which are a potential scaffold for peptides. Elastin is one of the main components of extracellular matrix and a temperature-sensitive biomacromolecule. Previously, Val-Pro-Gly-Val-Gly peptides have been conjugated to a dendrimer for designing an elastin-mimetic dendrimer. In this study, various elastin-mimetic dendrimers using different length peptides and different dendrimer generations were synthesized to control the temperature dependency. The elastin-mimetic dendrimers formed ß-turn structure by heating, which was similar to the elastin-like peptides. The elastin-mimetic dendrimers exhibited an inverse phase transition, largely depending on the peptide length and slightly depending on the dendrimer generation. The elastin-mimetic dendrimers formed aggregates after the phase transition. The endothermal peak was observed in elastin-mimetic dendrimers with long peptides, but not with short ones. The peptide length and the dendrimer generation are important factors to tune the temperature dependency on the elastin-mimetic dendrimer.

Protein polymer hydrogels by in situ, rapid and reversible self-gelation.

Protein-based biomaterials are an important class of materials for applications in biotechnology and medicine. The exquisite control of their composition, stereochemistry, and chain length offers unique opportunities to engineer biofunctionality, biocompatibility, and biodegradability into these materials. Here, we report the synthesis of a thermally responsive peptide polymer-based hydrogel composed of a recombinant elastin-like polypeptide (ELP) that rapidly forms a reversibly cross-linked hydrogel by the formation of intermolecular disulfide cross-links. To do so, we designed and synthesized ELPs that incorporate periodic cysteine residues (cELPs), and show that cELPs are thermally responsive protein polymers that display rapid gelation under physiologically relevant, mild oxidative conditions. Gelation of cELPs, at concentrations as low as 2.5 wt%, occurs in ≈ 2.5 min upon addition a low concentration of hydrogen peroxide (0.3 wt%). We show the utility of these hydrogels for the sustained release of a model protein in vitro, and demonstrate the ability of this injectable biomaterial to pervade tumors to maximize tumor coverage and retention time upon intratumoral injection. cELPs represent a new class of injectable reversibly cross-linked hydrogels with properties intermediate between ELP coacervates and chemically cross-linked ELP hydrogels that will find useful applications in drug delivery and tissue engineering.

Tuning the properties of elastin mimetic hybrid copolymers via a modular polymerization method.

We have synthesized elastin mimetic hybrid polymers (EMHPs) via the step-growth polymerization of azide-functionalized poly(ethylene glycol) (PEG) and alkyne-terminated peptide (AKAAAKA)(2) (AK2) that is abundant in the cross-linking domains of the natural elastin. The modular nature of our synthesis allows facile adjustment of the peptide sequence to modulate the structural and biological properties of EMHPs. Therefore, EMHPs containing cell-binding domains (CBDs) were constructed from α,ω-azido-PEG and two types of alkyne-terminated AK2 peptides with sequences of DGRGX(AKAAAKA)(2)X (AK2-CBD1) and X(AKAAAKA)(2)XGGRGDSPG (AK2-CBD2, X = propargylglycine) via a step-growth, click coupling reaction. The resultant hybrid copolymers contain an estimated five to seven repeats of PEG and AK2 peptides. The secondary structure of EMHPs is sensitive to the specific sequence of the peptidic building blocks, with CBD-containing EMHPs exhibiting a significant enhancement in the α-helical content as compared with the peptide alone. Elastomeric hydrogels formed by covalent cross-linking of the EMHPs had a compressive modulus of 1.06 ± 0.1 MPa. Neonatal human dermal fibroblasts (NHDFs) were able to adhere to the hydrogels within 1 h and to spread and develop F-actin filaments 24 h postseeding. NHDF proliferation was only observed on hydrogels containing RGDSP domains, demonstrating the importance of integrin engagement for cell growth and the potential use of these EMHPs as tissue engineering scaffolds. These cell-instructive, hybrid polymers are promising candidates as elastomeric scaffolds for tissue engineering.

Cell behavior on a CCN1 functionalized elastin-mimetic protein polymer.

We report the design of an elastin-mimetic triblock copolymer with the ability to guide endothelial cell adhesion, spreading, and migration while maintaining the elastomeric properties of the protein polymer. The V2 ligand sequence from matricellular protein CCN1 (cysteine-rich 61, CYR61) was multimerized and cloned into elastin polymer LysB10, creating LysB10.V2. Cell adhesion studies demonstrated that a LysB10.V2 surface density of at least 40 pmol/cm(2) was required to elicit cell attachment. Peptide blocking studies confirmed V2 specific engagement with integrin receptor α(v)ß(3) (P < 0.05) and we observed the formation of actin stress fiber networks and vinculin clustering, characteristic of focal adhesion assembly. Haptotatic migration assays demonstrated the ability of LysB10.V2 surfaces to stimulate migration of endothelial cells (P < 0.05). Significantly, we illustrated the ability of LysB10.V2 to support a quiescent endothelium. The CCN1 molecule functions to support many key biological processes necessary for tissue repair and thus presents a promising target for bioengineering applications. Collectively, our results demonstrate the potential to harness CCN1 specific function in the design of new scaffold materials for applications in regenerative medicine.

Design and production of a chimeric resilin-, elastin-, and collagen-like engineered polypeptide.

Protein-inspired biomaterials have gained great interest as an alternative to synthetic polymers, in particular, for their potential use as biomedical devices. The potential inspiring models are mainly proteins able to confer mechanical properties to tissues and organs, such as elasticity (elastin, resilin, spider silk) and strength (collagen, silk). The proper combination of repetitive sequences, each of them derived from different proteins, represents a useful tool for obtaining biomaterials with tailored mechanical properties and biological functions. In this report we describe the design, the production, and the preliminary characterization of a chimeric polypeptide, based on sequences derived from the highly resilient proteins resilin and elastin and from collagen-like sequences. The results show that the obtained chimeric recombinant material exhibits promising self-assembling properties. Young's modulus of the fibers was determined by AFM image analysis and lies in the range of 0.1-3 MPa in agreement with the expectations for elastin-like and resilin-like materials.

Synthesis and characterization of macroporous thermosensitive hydrogels from recombinant elastin-like polymers.

Multifunctional bioactive chemically cross-linked elastin-like polymers (ELPs) have been prepared as three-dimensional scaffolds for tissue engineering. The salt-leaching/gas-foaming technique was found suitable to prepare highly porous biodegradable hydrogels based on this novel material type. The porosity can be controlled by the amount of sodium hydrogen carbonate incorporated during the cross-linking reaction, whereas the mean pore size is determined by the salt particle size. The gas-foaming process, which involves immersion in a citric acid solution after the cross-linking, facilitates pore interconnectivity and allows a grooved surface essential for cell colonization. Due to the thermoresponsive nature of the ELPs, their physical properties are strongly influenced by the temperature of the aqueous medium. The feasibility to obtain tridimensional scaffolds for tissue engineering has been studied by testing the adhesion and spreading of endothelial cells into the porous ELP hydrogels. The methods and structures described herein provide a starting point for the design and synthesis of macroporous multifunctional elastin-like hydrogels with potential broad applicability.

Creation of cross-linked electrospun isotypic-elastin fibers controlled cell-differentiation with new cross-linker.

Effective application of elastin materials for vascular grafts in tissue engineering requires these materials to retain the elastic and biological properties of native elastin. To clarify the influence of soluble elastin isotypes on vascular smooth muscle cells (VSMCs), soluble elastin was prepared from insoluble elastin by hydrolysis with oxalic acid. Its fractions were separated and classified into three isotypes. Elastin retaining 2.25 mol% of cross-linked structures exhibited significant differentiation of VSMCs, which adhered to the elastin with contraction phenotypes similar to that of native elastin, causing proliferation to cease. This trend was more strongly demonstrated in cotton-like elastin fibers with a new cross-linker. The results suggest that elastin isotypes could be applied as new effective biomaterials for suppressing intimal hyperplasia in vascular grafts.

Elastin-mimetic protein polymers capable of physical and chemical crosslinking.

We report the synthesis of a new class of recombinant elastin-mimetic triblock copolymer capable of both physical and chemical crosslinking. These investigations were motivated by a desire to capture features unique to both physical and chemical crosslinking schemes so as to exert optimal control over a wide range of potential properties afforded by protein-based multiblock materials. We postulated that by chemically locking a multiblock protein assembly in place, functional responses that are linked to specific domain structures and morphologies may be preserved over a broader range of loading conditions that would otherwise disrupt microphase structure solely stabilized by physical crosslinking. Specifically, elastic modulus was enhanced and creep strain reduced through the addition of chemical crosslinking sites. Additionally, we have demonstrated excellent in vivo biocompatibility of glutaraldehyde treated multiblock systems.

Genetic engineering of self-assembled protein hydrogel based on elastin-like sequences with metal binding functionality.

Recombinant DNA methods have been exploited to enable the creation of protein-based block copolymers with programmable sequences, desired properties, and predictable three-dimensional structures. These advantages over conventional polymer counterparts facilitate the utility of this new class of biomaterials in a wide range of applications. In this project, we exploited the environmental application of protein-based block copolymers based on elastin-like protein (ELP) sequences. Triblock copolymers containing charged and hydrophobic segments were synthesized. Chain lengths of each segment were manipulated in order to maintain a gelation point below room temperature. Polyhistidine sequences were successfully incorporated into the hydrophilic segment without disruption of the self-assembled hydrogel formation. The microscopic structure was further investigated using laser confocal microscopy. The metal binding capability and capacity of resulting hydrogel were studied to demonstrate the functionality of polyhistidine and its environmental application for heavy metal removal. Reversibility of metal binding was demonstrated, indicating the cost-effectiveness of this hydrogel. Significantly, we envision that this versatile strategy of incorporating functional groups within a 3-D protein network provides new possibilities in creation of biomaterials with great control over structure-property relationships.

Efficient synthesis of protein-drug conjugates using a functionalizable recombinant elastin-mimetic polypeptide.

This report describes the efficient conjugation of doxorubicin-glycine-phenylalanine-leucine-glycine (1a) and rhodamine-glycine-phenylalanine-leucine-glycine (1b) units to a monodisperse elastin-mimetic polypeptide (EMM)(7) bearing eight primary amine groups for chemical attachment. The synthetic approach is based on the solid-phase synthesis of 1a and 1b followed by chemical conjugation to the elastin-mimetic polypeptide in the presence of HOBt/PyBob as activating agents to form the polypeptide conjugates 2a and 2b. Conjugation efficiency was 61.2% (4.9 doxorubicin units per polypeptide chain) for 2a and 53.7% (4.3 rhodamine units per polypeptide chain) for 2b, demonstrating the feasibility of using these tailor-made, recombinant polypeptides as potential drug carriers for cancer therapy.