Rationally Designed Dynamic Protein Hydrogels with Reversibly Tunable Mechanical Properties

Protein hydrogels have attracted considerable interest due to their potential applications in biomedical engineering. Creating protein hydrogels with dynamic mechanical properties is challenging. Here, the engineering of a novel, rationally designed protein‐hydrogel is reported that translates molecular level protein folding‐unfolding conformational changes into macroscopic reversibly tunable mechanical properties based on a redox controlled protein folding‐unfolding switch. This novel protein folding switch is constructed from a designed mutually exclusive protein. Via oxidation and reduction of an engineered disulfide bond, the protein folding switch can switch its conformation between folded and unfolded states, leading to a drastic change of protein's effective chain length and mechanical compliance. This redox‐responsive protein can be readily photochemically crosslinked into solid hydrogels, in which molecular level conformational changes (folding‐unfolding) can result in significant macroscopic changes in hydrogel's physical and mechanical properties due to the change of the effective chain length between two crosslinking points in the protein hydrogel network. It is found that when reduced, the hydrogel swells and is mechanically compliant; when oxidized, it swells to a less extent and becomes resilient and stiffer, exhibiting an up to fivefold increase in its Young's modulus. The changes of the mechanical and physical properties of this hydrogel are fully reversible and can be cycled using redox potential. This novel protein hydrogel with dynamic mechanical and physical properties could find numerous applications in material sciences and tissue engineering.     

Photogenerated Aldehydes for Protein Patterns on Hydrogels and Guidance of Cell Behavior

The immobilization of proteins to hydrogels is important and plays a significant role to provide suitable biomimetic material as extracellular matrix for cell behavior mediation. This study describes a novel and universal strategy for photopatterning unmodified proteins on hydrogels. The methodology creates photogenerated aldehyde regions within a protein‐resistant hydrogel and then conjugates unmodified proteins by mild imine ligation with spatial, temporal, and dosage control. The relatively stable aldehyde intermediate enables the facile and highly efficient covalent immobilization of proteins by a postfunctionalization methodology and the sequential protein patterns provide an easy access to control the identity and dynamic change of proteins presented to cells on demand, thus mediating cell behaviors. This approach provides important opportunities for understanding and controlling cell behavior mediated by proteins, and opens up new avenues for hydrogels in tissue engineering and biotechnology applications.     

Biomimetic Design of Protein Nanomaterials for Hydrophobic Molecular Transport

Biomaterials such as self‐assembling biological complexes have a variety of applications in materials science and nanotechnology. The functionality of protein‐based materials, however, is often limited by the absence or locations of specific chemical conjugation sites. Here a new strategy is developed for loading organic molecules into the hollow cavity of a protein nanoparticle that relies only on non‐covalent interactions, and its applicability in drug delivery is demonstrated in breast cancer cells. Based on a biomimetic model that incorporates multiple phenylalanines to create a generalized binding site, the anti‐tumor compound doxorubicin is retained and delivered by redesigning a caged protein scaffold. Using structural modeling and protein engineering, variants of the E2 subunit of pyruvate dehydrogenase with varying levels of drug‐carrying capabilities are obtained. An increasing number of introduced phenylalanines within the scaffold cavity generally results in greater drug loading capacity. Drug loading levels greater than conventional nanoparticle delivery systems are achieved. The universal strategy can be used to design de novo hydrophobic binding domains within protein‐based scaffolds for molecular encapsulation and transport and increases the ability to attach guest molecules to this class of materials.     

Reinforcement of Shear Thinning Protein Hydrogels by Responsive Block Copolymer Self‐Assembly

Shear thinning hydrogels are promising materials that exhibit rapid self‐healing following the cessation of shear, making them attractive for applications including injectable biomaterials. Here, self‐assembly is demonstrated as a strategy to introduce a reinforcing network within shear thinning artificially engineered protein gels, enabling a responsive transition from an injectable state at low temperatures with a low yield stress to a stiffened state at physiological temperatures with resistance to shear thinning, higher toughness, and reduced erosion rates and creep compliance. Protein‐polymer triblock copolymers capable of the responsive self‐assembly of two orthogonal networks are synthesized. Midblock association forms a shear‐thinning network, while endblock aggregation at elevated temperatures introduces a second, independent physical network into the protein hydrogel. These reversible crosslinks introduce extremely long relaxation times and lead to a five‐fold increase in the elastic modulus, significantly larger than is expected from transient network theory. Thermoresponsive reinforcement reduces the high temperature creep compliance by over four orders of magnitude, decreases the erosion rate by at least a factor of five, and increases the yield stress by up to a factor of seven. Combined with the demonstrated potential of shear thinning artificial protein hydrogels for various uses, this reinforcement mechanism broadens the range of applications that can be addressed with shear‐thinning physical gels.     

Growth‐Factor Free Multicomponent Nanocomposite Hydrogels That Stimulate Bone Formation

Synthetic osteo‐promoting materials that are able to stimulate and accelerate bone formation without the addition of exogenous cells or growth factors represent a major opportunity for an aging world population. A co‐assembling system that integrates hyaluronic acid tyramine ( HA‐Tyr ), bioactive peptide amphiphiles ( GHK‐Cu²⁺ ), and Laponite ( Lap ) to engineer hydrogels with physical, mechanical, and biomolecular signals that can be tuned to enhance bone regeneration is reported. The central design element of the multicomponent hydrogels is the integration of self‐assembly and enzyme‐mediated oxidative coupling to optimize structure and mechanical properties in combination with the incorporation of an osteo‐ and angio‐promoting segments to facilitate signaling. Spectroscopic techniques are used to confirm the interplay of orthogonal covalent and supramolecular interactions in multicomponent hydrogel formation. Furthermore, physico‐mechanical characterizations reveal that the multicomponent hydrogels exhibit improved compressive strength, stress relaxation profile, low swelling ratio, and retarded enzymatic degradation compared to the single component hydrogels. Applicability is validated in vitro using human mesenchymal stem cells and human umbilical vein endothelial cells, and in vivo using a rabbit maxillary sinus floor reconstruction model. Animals treated with the HA‐Tyr‐HA‐Tyr‐GHK‐Cu²⁺ hydrogels exhibit significantly enhanced bone formation relative to controls including the commercially available Bio‐Oss.     

Affinity‐Based Protein Surface Pattern Formation by Ligand Self‐Selection from Mixed Protein Solutions

Photolithographically prepared surface patterns of two affinity ligands (biotin and chloroalkane) specific for two proteins (streptavidin and HaloTag, respectively) are used to spontaneously form high‐fidelity surface patterns of the two proteins from their mixed solution. High affinity protein‐surface self‐selection onto patterned ligands on surfaces exhibiting low non‐specific adsorption rapidly yields the patterned protein surfaces. Fluorescence images after protein immobilization show high specificity of the target proteins to their respective surface patterned ligands. Time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS) imaging further supports the chemical specificity of streptavidin and HaloTag for their surface patterned ligands from mixed protein solutions. However, ToF‐SIMS did detect some non‐specific adsorption of bovine serum albumin, a masking protein present in excess in the adsorbing solutions, on the patterned surfaces. Protein amino acid composition, surface coverage, density, and orientation are important parameters that determine the relative ToF‐SIMS fragmentation pattern yields. ToF‐SIMS amino acid‐derived ion fragment yields summed to produce surface images can reliably determine which patterned surface regions contain bound proteins, but do not readily discriminate between different co‐planar protein regions. Principal component analysis (PCA) of these ToF‐SIMS data, however, improves discrimination of ions specific to each protein, facilitating surface protein pattern identification and image contrast.     

Micropatterned Protein for Cell Adhesion through Phototriggered Charge Change in a Polyvinylpyrrolidone Hydrogel

Regulated immobilization of proteins on hydrogels allows for the creation of highly controlled microenvironments to meet the special requirements of cell biology and tissue engineering devices. Light is an ideal stimulus to regulate immobilization because it can be controlled in time, space, and intensity. Here, a photoresponsive hydrogel that enables the patterning of proteins by a combination of electrostatic adsorption and photoregulated charge change on a hydrogel is developed. It is based on a photosensitive cationic monomer ( CLA ), a coumarin caged lysine betaine zwitterion, incorporated into a polyvinylpyrrolidone ( PVP ) hydrogel, which can controllably change the charge from an adhesive positive state to an anti‐adhesive zwitterion state upon irradiation at 365 nm. With this strategy, the immobilization of proteins is regulated and cell adhesion is programmed on hydrogels on demand. This approach should open up new avenues for hydrogels in biomedical applications.     

‘Mechanical Engineering’ of Elastomeric Proteins: Toward Designing New Protein Building Blocks for Biomaterials

Elastomeric proteins are subject to stretching force under biological settings and play important roles in regulating the mechanical properties of a wide range of biological machinery. Elastomeric proteins also underlie the superb mechanical properties of many protein‐based biomaterials. The developments of single molecule force spectroscopy have enabled the direct characterization of the mechanical properties of elastomeric proteins at the single molecule level and led to the new burgeoning field of research: single protein mechanics and engineering. Combined single molecule atomic force microscopy and protein engineering efforts are well under way to understand molecular determinants for the mechanical stability of elastomeric proteins and to develop methodologies to tune the mechanical properties of proteins in a rational and systematic fashion, which will lead to the ‘mechanical engineering’ of elastomeric proteins. Here the current status of these experimental efforts is discussed and the successes and challenges in constructing novel proteins with tailored nanomechanical proteins highlighted. The prospect of employing such engineered artificial elastomeric proteins as building blocks for the construction of biomaterials for applications ranging from material sciences to biomedical engineering are also discussed.

     

Highly Efficient Self‐Healable and Dual Responsive Cellulose‐Based Hydrogels for Controlled Release and 3D Cell Culture

To face the increasing demand of self‐healing hydrogels with biocompatibility and high performances, a new class of cellulose‐based self‐healing hydrogels are constructed through dynamic covalent acylhydrazone linkages. The carboxyethyl cellulose‐graft‐dithiodipropionate dihydrazide and dibenzaldehyde‐terminated poly(ethylene glycol) are synthesized, and then the hydrogels are formed from their mixed solutions under 4‐amino‐DL‐phenylalanine (4a‐Phe) catalysis. The chemical structure, as well as microscopic morphologies, gelation times, mechanical and self‐healing performances of the hydrogels are investigated with ¹H NMR, Fourier transform infrared spectroscopy, atomic force microscopy, rheological and compression measurements. Their gelation times can be controlled by varying the total polymer concentration or 4a‐Phe content. The resulted hydrogels exhibit excellent self‐healing ability with a high healing efficiency (≈96%) and good mechanical properties. Moreover, the hydrogels display pH/redox dual responsive sol‐gel transition behaviors, and are applied successfully to the controlled release of doxorubicin. Importantly, benefitting from the excellent biocompatibility and the reversibly cross‐linked networks, the hydrogels can function as suitable 3D culture scaffolds for L929 cells, leading to the encapsulated cells maintaining a high viability and proliferative capacity. Therefore, the cellulose‐based self‐healing hydrogels show potential applications in drug delivery and 3D cell culture for tissue engineering.     

Complementary Co‐assembling Peptides: From In Silico Studies to In Vivo Application

Self‐assembling biomaterials offer an unprecedented chance of successfully facing most of the challenges of various biomedical fields, and, in particular, of tissue engineering. Nonetheless co‐assembling peptides (CAPs), taking advantage of the theory and empirical findings developed for self‐assembling peptides, could provide an even better control over cell cultures, drug delivery, and transplantation therapies. This study follows a “full” bottom‐up approach to develop new CAPs for neural tissue engineering applications. After molecular aggregation analysis via coarse‐grained simulations, LKLK12, LDLD12, and the functionalized KLPGWSG‐LDLD12 CAPs are synthesized and characterized assessing their co‐assembled secondary structures, the biomechanical properties of the obtained hydrogels, and the morphological features of the assembled nanofibers. The biological influence on viability and differentiation of human and murine neural stem cells are tested in vitro and neuroregenerative potentials in complete spinal cord transections are verified in vivo. Upon mixing of CAPs, the spontaneous formation of double layers of β‐sheets with a high degree of integration of the two CAP species is demonstrated. The formation of entangled nanofibrous structures give rise to shear‐thinning hydrogels. The in vitro results are comparable to a standard state‐of‐the‐art cell culture substrate and nervous regeneration in vivo is enhanced.     

Simple and Efficient Targeted Intracellular Protein Delivery with Self‐Assembled Nanovehicles for Effective Cancer Therapy

Protein therapy offers promising prospects for the treatment of various important diseases, thus it is highly desirable to develop a robust carrier that can deliver active proteins into cells. The development of a novel protein delivery platform based on the self‐assembly of multiarmed amphiphilic cyclodextrins (CDEH) is reported. CDEH can self‐assemble into nanoparticles in aqueous solution and achieve superior encapsulation of protein (loading capacity > 30% w/w) simply by mixing with protein solution without introducing any subsequent cumbersome steps that may inactivate proteins. More importantly, CDEH nanovehicles can be easily further modified with various targeting groups based on host–guest complexation. Using saporin as a therapeutic protein, AS1411‐aptamer‐modified CDEH nanovehicles can preferentially accumulate in tumors and efficiently inhibit tumor growth in a MDA‐MB‐231 xenograft mouse model. Moreover, folate‐targeted CDEH nanovehicles can also deliver Cas9 protein and Plk1‐targeting sgRNA into Hela cells, leading to 47.1% gene deletion and 64.1% Plk1 protein reduction in HeLa tumor tissue, thereby effectively suppressing the tumor progression. All these results indicate the potential of targeted CDEH nanovehicles in intracellular protein delivery for improving protein therapeutics.     

Self‐Assembling Peptides as Cell‐Interactive Scaffolds

Cell and tissue engineering therapies for regenerative medicine as well as cell‐based assays require an understanding of the interactions between cells with the surrounding microenvironment at the nanoscale. Engineering a cell‐interactive scaffold therefore entails control over the nanostructure of the biomaterial. Peptides that are able to self‐assemble into 3D scaffolds have emerged as interesting biomaterials for directing cell behavior, with desirable properties such as the capability of tuning the nanostructure by modulating the amino acid composition. Here, an overview of the development of self‐assembling peptide hydrogels as functional cell scaffolds is presented, highlighting recent work on incorporating features such as bioactive ligands, growth factor delivery, controlled degradation, and formulation into microgels for defined cell microenvironments.     

Self‐Assembling Doxorubicin Silk Hydrogels for the Focal Treatment of Primary Breast Cancer

Standard care for early stage breast cancer includes tumor resection and local radiotherapy to achieve long‐term remission. Systemic chemotherapy provides only low locoregional control of the disease; to address this, self‐assembling silk hydrogels that can retain and then deliver doxorubicin locally are described. Self‐assembling silk hydrogels show no swelling, are readily loaded with doxorubicin under aqueous conditions, and release drug over 4 weeks in amounts that can be fine‐tuned by varying the silk content. Following successful in vitro studies, locally injected silk hydrogels loaded with doxorubicin show excellent antitumor response in mice, outperforming the equivalent amount of doxorubicin delivered intravenously. In addition to reducing primary tumor growth, doxorubicin‐loaded silk hydrogels reduce metastatic spread and are well tolerated in vivo. Thus, silk hydrogels are well suited for the local delivery of chemotherapy and provide a promising approach to improve locoregional control of breast cancer.     

Biological Assemblies Provide Novel Templates for the Synthesis of Biocomposites and Facilitate Cell Adhesion

Mechanical mismatch and the lack of interactions between implants and the natural tissue environment are major drawbacks in bone tissue engineering. Biomaterials mimicking the self‐assembly process and the composition of the bone matrix should provide new routes for fabricating biomaterials possessing novel osteoconductive and osteoinductive properties for bone repair. In the present study, bioinspired strategies are employed to design de novo self‐assembled chimeric protein hydrogels comprising leucine zipper motifs flanking a dentin matrix protein 1 domain, which is characterized as a mineralization nucleator. Results show that this chimeric protein could function as a hydroxyapatite nucleator in pseudo‐physiological buffer with the formation of highly oriented apatites similar to biogenic bone mineral. It could also function as an inductive substrate for osteoblast adhesion, promote cell surface integrin presentation and clustering, and modulate the formation of focal contacts. Such biomimetic “bottom‐up” construction with dual osteoconductive and osteoinductive properties should open new avenues for bone tissue engineering.     

Enzyme‐Catalyzed Formation of Supramolecular Hydrogels as Promising Vaccine Adjuvants

Promising vaccine adjuvants of self‐assembling peptide hydrogels for protein ovalbumin (OVA) are introduced in this study. The hydrogels are formed by the enzyme of phosphatase, and the vaccine adjuvant potency of both l ‐ and d ‐peptide hydrogels is evaluated. The results indicate that, compared with the clinically used alum adjuvant, both l ‐ and d ‐peptide hydrogels can increase the IgG production of OVA for about 1.3 and 3.8 times, respectively. Both gels can enhance antigen uptake and induce dendritic cell maturation, and promote and prolong accumulation of antigen in lymph node, as well as evoke germinal center formation. However, the d ‐peptide hydrogel with OVA exhibits a slightly more efficient accumulation of OVA in the lymph nodes and seems preventing tumor growth more significantly than its l ‐counterpart. With the good biocompatibility and degradability of peptide hydrogels, the hydrogels described in this study have big potential for the production of protein vaccines for immunotherapy against different diseases.     

Covalently Adaptable Elastin‐Like Protein–Hyaluronic Acid (ELP–HA) Hybrid Hydrogels with Secondary Thermoresponsive Crosslinking for Injectable Stem Cell Delivery

Shear‐thinning, self‐healing hydrogels are promising vehicles for therapeutic cargo delivery due to their ability to be injected using minimally invasive surgical procedures. An injectable hydrogel using a novel combination of dynamic covalent crosslinking with thermoresponsive engineered proteins is presented. Ex situ at room temperature, rapid gelation occurs through dynamic covalent hydrazone bonds by simply mixing two components: hydrazine‐modified elastin‐like protein (ELP) and aldehyde‐modified hyaluronic acid. This hydrogel provides significant mechanical protection to encapsulated human mesenchymal stem cells during syringe needle injection and rapidly recovers after injection to retain the cells homogeneously within a 3D environment. In situ, the ELP undergoes a thermal phase transition, as confirmed by coherent anti‐Stokes Raman scattering microscopy observation of dense ELP thermal aggregates. The formation of the secondary network reinforces the hydrogel and results in a tenfold slower erosion rate compared to a control hydrogel without secondary thermal crosslinking. This improved structural integrity enables cell culture for three weeks postinjection, and encapsulated cells maintain their ability to differentiate into multiple lineages, including chondrogenic, adipogenic, and osteogenic cell types. Together, these data demonstrate the promising potential of ELP–HA hydrogels for injectable stem cell transplantation and tissue regeneration.     

Guest‐Molecule‐Directed Assembly of Mesostructured Nanocomposite Polymer/Organoclay Hydrogels

Given the urgent need for soft materials with high functional value, hydrogels based on the integrative assembly of organic polymers and nanoscale inorganic building blocks—so‐called nanocomposite polymer hydrogels—offer a generic approach to swollen hybrid networks with tuneable and synergistic properties. Here, we report a new approach to assembling nanocomposite polymer hydrogels with multiple levels of structural complexity and enhanced functionality by using nanoscale integration of mesostructured inorganic building blocks capable of sequestering and releasing drugs (ibuprofen, aspirin, naproxen) and enzymes (glucose oxidase). The viscoelastic materials are produced by noncovalent crosslinking of poly(vinylpyrrolidone) in the presence of low amounts (1–5 wt%) of an exfoliated synthetic organoclay that undergoes in situ guest‐molecule‐directed self‐assembly. The hydrogels can be moulded into shape‐persistent, free‐standing objects that are stable between pH values of 4 to 11 and self‐heal when damaged. Significantly, the mesostructured nanocomposite polymer hydrogels, which can be reversibly dried and reconstituted in the form of highly swollen materials, exhibit sustained drug release and can be recharged and reused. The results provide important guidelines for developing new multifunctional nanocomposite polymer hydrogels based on the concerted self‐assembly of inorganic building blocks with mesostructured interiors.     

Hexahistidine‐Tagged Single‐Walled Carbon Nanotubes (His6‐tagSWNTs): A Multifunctional Hard Template for Hierarchical Directed Self‐Assembly and Nanocomposite Construction

While a hexahistidine affinity tag can be introduced at protein termini or internal sites by standard molecular biology procedures for purification, immobilization, or labeling of proteins, here the versatility of this concept is exploited for the chemical preparation of novel hexahistidine‐tagged single‐walled carbon nanotubes (His₆‐tagSWNTs), a novel hard template useful for solubilizing, assembling, processing, and interfacing SWNTs in aqueous conditions. Water‐soluble and exfoliated His₆‐tagSWNTs are prepared and fully characterized. This functional molecular module is able to interact via robust physisorption (π?π stacking) with the sidewall of SWNTs and combines the versatility of small, water‐soluble reporters (His₆) for hierarchical directed self‐assembly (HDSA) and construction of nanocomposites. It is demonstrated that metal coordination bonds with Ni(II) can be used for the supramolecular self assembly of His₆‐tagSWNTs, generating complex reticulated networks and architectures. The His₆‐tagSWNTs hard template nanohybrid is further utilized for directed self‐assembly with silica nanoparticles. The versatility of the novel hybrids opens a new era for the rational design, smart (bio)functionalization, processing, interfacing, and self assembling of carbon nanotubes for the construction of multicomposites and more complex systems with controllable spatial organization and programmable properties for a wide range of applications in biology, nanoelectronics, and catalysis.