Strain-Stiffening Mechanoresponse in Dynamic-Covalent Cellulose Hydrogels.

Mechanical stimuli such as strain, force, and pressure are pervasive within and beyond the human body. Mechanoresponsive hydrogels have been engineered to undergo changes in their physicochemical or mechanical properties in response to such stimuli. Relevant responses can include strain-stiffening, self-healing, strain-dependent stress relaxation, and shear rate-dependent viscosity. These features are a direct result of dynamic bonds or noncovalent/physical interactions within such hydrogels. The contributions of various types of bonds and intermolecular interactions to these behaviors are important to more fully understand the resulting materials and engineer their mechanoresponsive features. Here, strain-stiffening in carboxymethylcellulose hydrogels cross-linked with pendant dynamic-covalent boronate esters using tannic acid is studied and modulated as a function of polymer concentration, temperature, and effective cross-link density. Furthermore, these materials are found to exhibit self-healing and strain-memory, as well as strain-dependent stress relaxation and shear rate-dependent changes in gel viscosity. These features are attributed to the dynamic nature of the boronate ester cross-links, interchain hydrogen bonding and bundling, or a combination of these two intermolecular interactions. This work provides insight into the interplay of such interactions in the context of mechanoresponsive behaviors, particularly informing the design of hydrogels with tunable strain-stiffening. The multiresponsive and tunable nature of this hydrogel system therefore presents a promising platform for a variety of applications.

Iterative Design Reveals Molecular Domain Relationships in Supramolecular Peptide-Drug Conjugates.

Supramolecular peptide-drug conjugates (sPDCs) are prepared by covalent attachment of a drug moiety to a peptide motif programmed for one-dimensional self-assembly, with subsequent physical entanglement of these fibrillar structures enabling formation of nanofibrous hydrogels. This class of prodrug materials presents an attractive platform for mass-efficient and site-specific delivery of therapeutic agents using a discrete, single-component molecular design. However, a continued challenge in sPDC development is elucidating relationships between supramolecular interactions in their drug and peptide domains and the resultant impacts of these domains on assembly outcomes and material properties. Inclusion of a saturated alkyl segment alongside the prodrug in the hydrophobic domain of sPDCs could relieve some of the necessity for ordered, prodrug-produg interactions. Accordingly, nine sPDCs are prepared here to iterate the design variables of amino acid sequence and hydrophobic prodrug-alkyl block design. All molecules spontaneously formed hydrogels under physiological conditions, indicating a less hindered thermodynamic path to self-assembly relative to previous prodrug-only designs. However, material studies on the supramolecular arrangement, formation, and mechanical properties of the resultant sPDC hydrogels as well as their drug release profiles showed complex relationships between the hydrophobic and peptide domains in the formation and function of the resulting assemblies. Together, these results indicate that sPDC material properties are intrinsically linked to holistic molecular design with coupled contributions from their prodrug and peptide domains in directing properties of the emergent materials.

Engineering a Pathway to Glucose-Responsive Therapeutics.

In 2014, the American Diabetes Association instituted a novel funding paradigm to support diabetes research through its Pathway to Stop Diabetes® Program. Pathway took a multifaceted approach to provide key funding to diabetes researchers in advancing a broad spectrum of research programs centered on all aspects of understanding, managing, and treating diabetes. Herein the personal perspective of a 2019 Pathway Accelerator awardee is offered, describing a research program seeking to advance a materials-centered approach to engineering glucose-responsive devices and new delivery tools for better therapeutic outcomes in treating diabetes. This is offered alongside a personal reflection on five years of support from the ADA Pathway Program.

Using a biocatalyzed reaction cycle for transient and pH-dependent host-guest supramolecular hydrogels.

The formation of transient structures plays important roles in biological processes, capturing temporary states of matter through influx of energy or biological reaction networks catalyzed by enzymes. These natural transient structures inspire efforts to mimic this elegant mechanism of structural control in synthetic analogues. Specifically, though traditional supramolecular materials are designed on the basis of equilibrium formation, recent efforts have explored out-of-equilibrium control of these materials using both direct and indirect mechanisms; the preponderance of such works has been in the area of low molecular weight gelators. Here, a transient supramolecular hydrogel is realized through cucurbit[7]uril host-guest physical crosslinking under indirect control from a biocatalyzed network that regulates and oscillates pH. The duration of transient hydrogel formation, and resulting mechanical properties, are tunable according to the dose of enzyme, substrate, or pH stimulus. This tunability enables control over emergent functions, such as the programmable burst release of encapsulated model macromolecular payloads.

Engineering a Pathway to Glucose-Responsive Therapeutics.

In 2014, the American Diabetes Association instituted a novel funding paradigm to support diabetes research through its Pathway to Stop Diabetes program. This program took a multifaceted approach to providing key funding to diabetes researchers to advance a broad spectrum of research programs on all aspects of understanding, managing, and treating diabetes. Here, the personal perspective of a 2019 Pathway Accelerator awardee is offered, describing a research program seeking to advance a materials-centered approach to engineering glucose-responsive devices and new delivery tools for better therapeutic outcomes in treating diabetes. This is offered alongside a personal reflection on 5 years of support from the ADA Pathway Program.

Glucose-Driven Droplet Formation in Complexes of a Supramolecular Peptide and Therapeutic Protein.

Biology achieves remarkable function through processes arising from spontaneous or transient liquid-liquid phase separation (LLPS) of proteins and other biomolecules. While polymeric systems can achieve similar phenomena through simple or complex coacervation, LLPS with supramolecular materials has been less commonly shown. Functional applications for synthetic LLPS systems are an expanding area of emphasis, with particular focus on capturing the transient and dynamic state of these structures for use in biomedicine. Here, a net-cationic supramolecular peptide amphiphile building block with a glucose-binding motif is shown that forms LLPS structures when combined with a net-negatively charged therapeutic protein, dasiglucagon, in the presence of glucose. The droplets that arise are dynamic and coalesce quickly. However, the interface can be stabilized by addition of a 4-arm star PEG. When the stabilized droplets formed in glucose are transferred to a bulk phase containing different glucose concentrations, their stability and lifetime decrease according to bulk glucose concentration. This glucose-dependent formation translates into an accelerated release of dasiglucagon in the absence of glucose; this hormone analogue itself functions therapeutically to correct low blood glucose (hypoglycemia). These droplets also offer function in mitigating the most severe effects of hypoglycemia arising from an insulin overdose through delivery of dasiglucagon in a mouse model of hypoglycemic rescue. Accordingly, this approach to use complexation between a supramolecular peptide amphiphile and a therapeutic protein in the presence of glucose leads to droplets with functional potential to dissipate for the release of the therapeutic material in low blood glucose environments.

Dynamic-Covalent Crosslinking of Benzenetricarboxamide-Phenylboronate Conjugates.

In an effort to augment the function of supramolecular biomaterials, recent efforts have explored the creation of hybrid materials that couple supramolecular and covalent components. Here, the benzenetricarboxamide (BTA) supramolecular polymer motif is modified to present a phenylboronic acid (PBA) in order to promote the crosslinking of 1D BTA stacks by PBA-diol dynamic-covalent bonds through the addition of a multi-arm diol-bearing crosslinker. Interestingly, the combination of these two motifs serves to frustrate the resulting assembly process, yielding hydrogels with worse mechanical properties than those prepared without the multi-arm diol crosslinker. Both systems with and without the crosslinker do, however, respond to the presence of a physiological level of glucose with a reduction in their mechanical integrity; repulsive electrostatic interactions in the BTA stacks occur in both cases upon glucose binding, with added competition from glucose with PBA-diol bonds amplifying glucose response in the hybrid material. Accordingly, the present results point to an unexpected outcome of reduced hydrogel mechanics, yet increased glucose response, when two disparate dynamic motifs of BTA supramolecular polymerization and PBA-diol crosslinking are combined, offering a vision for future preparation of glucose-responsive supramolecular biomaterials.

Glucose-Triggered Gelation of Supramolecular Peptide Nanocoils with Glucose-Binding Motifs.

Peptide self-assembly is a powerful tool to prepare functional materials at the nanoscale. Often, the resulting materials have high aspect-ratio, with intermolecular ß-sheet formation underlying 1D fibrillar structures. Inspired by dynamic structures in nature, peptide self-assembly is increasingly moving toward stimuli-responsive designs wherein assembled structures are formed, altered, or dissipated in response to a specific cue. Here, a peptide bearing a prosthetic glucose-binding phenylboronic acid (PBA) is demonstrated to self-assemble into an uncommon nanocoil morphology. These nanocoils arise from antiparallel ß-sheets, with molecules aligned parallel to the long axis of the coil. The binding of glucose to the PBA motif stabilizes and elongates the nanocoil, driving entanglement and gelation at physiological glucose levels. The glucose-dependent gelation of these materials is then explored for the encapsulation and release of a therapeutic agent, glucagon, that corrects low blood glucose levels. Accordingly, the release of glucagon from the nanocoil hydrogels is inversely related to glucose level. When evaluated in a mouse model of severe acute hypoglycemia, glucagon delivered from glucose-stabilized nanocoil hydrogels demonstrates increased protection compared to delivery of the agent alone or within a control nanocoil hydrogel that is not stabilized by glucose.

Insulin-Dendrimer Nanocomplex for Multi-Day Glucose-Responsive Therapy in Mice and Swine.

The management of diabetes in a manner offering autonomous insulin therapy responsive to glucose-directed need, and moreover with a dosing schedule amenable to facile administration, remains an ongoing goal to improve the standard of care. While basal insulins with reduced dosing frequency, even once-weekly administration, are on the horizon, there is still no approved therapy that offers glucose-responsive insulin function. Herein, a nanoscale complex combining both electrostatic- and dynamic-covalent interactions between a synthetic dendrimer carrier and an insulin analogue modified with a high-affinity glucose-binding motif yields an injectable insulin depot affording both glucose-directed and long-lasting insulin availability. Following a single injection, it is even possible to control blood glucose for at least one week in diabetic swine subjected to daily oral glucose challenges. Measurements of serum insulin concentration in response to challenge show increases in insulin corresponding to elevated blood glucose levels, an uncommon finding even in preclinical work on glucose-responsive insulin. Accordingly, the subcutaneous nanocomplex that results from combining electrostatic- and dynamic-covalent interactions between a modified insulin and a synthetic dendrimer carrier affords a glucose-responsive insulin depot for week-long control following a single routine injection.

High-Affinity Host-Guest Recognition for Efficient Assembly and Enzymatic Responsiveness of DNA Nanostructures.

The combination of multiple orthogonal interactions enables hierarchical complexity in self-assembled nanoscale materials. Here, efficient supramolecular polymerization of DNA origami nanostructures is demonstrated using a multivalent display of small molecule host-guest interactions. Modification of DNA strands with cucurbit[7]uril (CB[7]) and its adamantane guest, yielding a supramolecular complex with an affinity of order 1010 m-1 , directs hierarchical assembly of origami monomers into 1D nanofibers. This affinity regime enables efficient polymerization; a lower-affinity ß-cyclodextrin-adamantane complex does not promote extended structures at a similar valency. Finally, the utility of the high-affinity CB[7]-adamantane interactions is exploited to enable responsive enzymatic actuation of origami nanofibers assembled using peptide linkers. This work demonstrates the power of high-affinity CB[7]-guest recognition as an orthogonal axis to drive self-assembly in DNA nanotechnology.

Hyaluronic Acid Hydrogels with Phototunable Supramolecular Cross-Linking for Spatially Controlled Lymphatic Tube Formation.

The dynamics of the extracellular matrix (ECM) influences stem cell differentiation and morphogenesis into complex lymphatic networks. While dynamic hydrogels with stress relaxation properties have been developed, many require detailed chemical processing to tune viscoelasticity, offering a limited opportunity for in situ and spatiotemporal control. Here, a hyaluronic acid (HA) hydrogel is reported with viscoelasticity that is controlled and spatially tunable using UV light to direct the extent of supramolecular and covalent cross-linking interactions. This is achieved using UV-mediated photodimerization of a supramolecular ternary complex of pendant trans-Brooker's Merocyanine (BM) guests and a cucurbit[8]uril (CB[8]) macrocycle. The UV-mediated conversion of this supramolecular complex to its covalent photodimerized form is catalyzed by CB[8], offering a user-directed route to spatially control hydrogel dynamics in combination with orthogonal photopatterning by UV irradiation through photomasks. This material thus achieves spatial heterogeneity of substrate dynamics, recreating features of native ECM without the need for additional chemical reagents. Moreover, these dynamic hydrogels afford spatial control of substrate mechanics to direct human lymphatic endothelial cells (LECs) to form lymphatic cord-like structures (CLS). Specifically, cells cultured on viscoelastic supramolecular hydrogels have enhanced formation of CLS, arising from increased expression of key lymphatic markers, such as LYVE-1, Podoplanin, and Prox1, compared to static elastic hydrogels prepared from fully covalent cross-linking. Viscoelastic hydrogels promote lymphatic CLS formation through the expression of Nrp2, VEGFR2, and VEGFR3 to enhance the VEGF-C stimulation. Overall, viscoelastic supramolecular hydrogels offer a facile route to spatially control lymphatic CLS formation, providing a tool for future studies of basic lymphatic biology and tissue engineering applications.

Biomimetic strain-stiffening in fully synthetic dynamic-covalent hydrogel networks.

Mechanoresponsiveness is a ubiquitous feature of soft materials in nature; biological tissues exhibit both strain-stiffening and self-healing in order to prevent and repair deformation-induced damage. These features remain challenging to replicate in synthetic and flexible polymeric materials. In recreating both the mechanical and structural features of soft biological tissues, hydrogels have been often explored for a number of biological and biomedical applications. However, synthetic polymeric hydrogels rarely replicate the mechanoresponsive character of natural biological materials, failing to match both strain-stiffening and self-healing functionality. Here, strain-stiffening behavior is realized in fully synthetic ideal network hydrogels prepared from flexible 4-arm polyethylene glycol macromers via dynamic-covalent boronate ester crosslinks. Shear rheology reveals the strain-stiffening response in these networks as a function of polymer concentration, pH, and temperature. Across all three of these variables, hydrogels of lower stiffness exhibit higher degrees of stiffening, as quantified by the stiffening index. The reversibility and self-healing nature of this strain-stiffening response is also evident upon strain-cycling. The mechanism underlying this unusual stiffening response is attributed to a combination of entropic and enthalpic elasticity in these crosslink-dominant networks, contrasting with natural biopolymers that primarily strain-stiffen due to a strain-induced reduction in conformational entropy of entangled fibrillar structures. This work thus offers key insights into crosslink-driven strain-stiffening in dynamic-covalent phenylboronic acid-diol hydrogels as a function of experimental and environmental parameters. Moreover, the biomimetic mechano- and chemoresponsive nature of this simple ideal-network hydrogel offers a promising platform for future applications.

Supramolecular approaches for insulin stabilization without prolonged duration of action.

Aggregation represents a significant challenge for the long-term formulation stability of insulin therapeutics. The supramolecular PEGylation of insulin with conjugates of cucurbit[7]uril and polyethylene glycol (CB[7]‒PEG) has been shown to stabilize insulin formulations by reducing aggregation propensity. Yet prolonged in vivo duration of action, arising from sustained complex formation in the subcutaneous depot, limits the application scope for meal-time insulin uses and could increase hypoglycemic risk several hours after a meal. Supramolecular affinity of CB[7] in binding the B1-Phe residue on insulin is central to supramolecular PEGylation using this approach. Accordingly, here we synthesized N-terminal acid-modified insulin analogs to reduce CB[7] interaction affinity at physiological pH and reduce the duration of action by decreasing the subcutaneous depot effect of the formulation. These insulin analogs show weak to no interaction with CB[7]‒PEG at physiological pH but demonstrate high formulation stability at reduced pH. Accordingly, N-terminal modified analogs have in vitro and in vivo bioactivity comparable to native insulin. Furthermore, in a rat model of diabetes, the acid-modified insulin formulated with CB[7]‒PEG offers a reduced duration of action compared to native insulin formulated with CB[7]‒PEG. This work extends the application of supramolecular PEGylation of insulin to achieve enhanced stability while reducing the risks arising from a subcutaneous depot effect prolonging in vivo duration of action.

Dynamic Covalent Dextran Hydrogels as Injectable, Self-Adjuvating Peptide Vaccine Depots.

Dextran-based hydrogels are promising therapeutic materials for drug delivery, tissue regeneration devices, and cell therapy vectors, due to their high biocompatibility, along with their ability to protect and release active therapeutic agents. This report describes the synthesis, characterization, and application of a new dynamic covalent dextran hydrogel as an injectable depot for peptide vaccines. Dynamic covalent crosslinks based on double Michael addition of thiols to alkynones impart the dextran hydrogel with shear-thinning and self-healing capabilities, enabling hydrogel injection. These injectable, non-toxic hydrogels show adjuvant potential and have predictable sub-millimolar loading and release of the peptide antigen SIINFEKL, which after its release is able to activate T-cells, demonstrating that the hydrogels deliver peptides without modifying their immunogenicity. This work demonstrates the potential of dynamic covalent dextran hydrogels as a sustained-release material for the delivery of peptide vaccines.

Transient and Dissipative Host-Guest Hydrogels Regulated by Consumption of a Reactive Chemical Fuel.

The transient self-assembly of molecules under the direction of a consumable fuel source is fundamental to biological processes such as cellular organization and motility. Such biomolecular assemblies exist in an out-of-equilibrium state, requiring continuous consumption of high energy molecules. At the same time, the creation of bioinspired supramolecular hydrogels has traditionally focused on associations occurring at the thermodynamic equilibrium state. Here, hydrogels are prepared from cucurbit[7]uril host-guest supramolecular interactions through transient physical crosslinking driven by the consumption of a reactive chemical fuel. Upon action from this fuel, the affinity and dynamics of CB[7]-guest recognition are altered. In this way, the lifetime of transient hydrogel formation and the dynamic modulus obtained are governed by fuel consumption, rather than being directed by equilibrium complex formation.

Supramolecular Protein Stabilization with Zwitterionic Polypeptide-Cucurbit[7]uril Conjugates.

Protein aggregation is an obstacle for the development of new biopharmaceuticals, presenting challenges in shipping and storage of vital therapies. Though a variety of materials and methods have been explored, the need remains for a simple material that is biodegradable, nontoxic, and highly efficient at stabilizing protein therapeutics. In this work, we investigated zwitterionic polypeptides prepared using a rapid and scalable polymerization technique and conjugated to a supramolecular macrocycle host, cucurbit[7]uril, for the ability to inhibit aggregation of model protein therapeutics insulin and calcitonin. The polypeptides are based on the natural amino acid methionine, and zwitterion sulfonium modifications were compared to analogous cationic and neutral structures. Each polymer was end-modified with a single cucurbit[7]uril macrocycle to afford supramolecular recognition and binding to terminal aromatic amino acids on proteins. Only conjugates prepared from zwitterionic structures of sufficient chain lengths were efficient inhibitors of insulin aggregation and could also inhibit aggregation of calcitonin. This polypeptide exhibited no cytotoxicity in human cells even at concentrations that were five-fold of the intended therapeutic regime. We explored treatment of the zwitterionic polypeptides with a panel of natural proteases and found steady biodegradation as expected, supporting eventual clearance when used as a protein formulation additive.

Glucose-Responsive Injectable Thermogels via Dynamic-Covalent Cross-Linking of Pluronic Micelles.

Stimuli-responsive hydrogels are an area of active discovery for approaches to deliver therapeutics in response to disease-specific indicators. Glucose-responsive delivery of insulin is of particular interest in better managing diabetes. Accordingly, hydrogels have been explored as platforms that enable both a rate and dose of insulin release aligning with the real-time physiological disease state; materials often include glucose sensing by dynamic-covalent cross-linking between phenylboronic acids (PBAs) and diols, with competition from ambient glucose reducing cross-link density of the material and accelerating release of encapsulated insulin. Yet, these materials historically have challenges with insulin leakage, offer limited glucose-responsive release of the insulin payload, and require unreasonably high injection pressures for syringe administration. Here, a thermogel platform prepared from temperature-induced micelles formed into a network by PBA-Diol cross-linking is optimized using a formulation-centered approach to maximize glucose-responsive insulin delivery. Importantly, the dual-responsive nature of this platform enables a low-viscosity sol at ambient temperature for facile injection, solidifying into a stable viscoelastic hydrogel network once in the body. The final optimized formulation affords acceleration in insulin release in response to glucose and enables single dose blood glucose control in diabetic rodents when subjected to multiple glucose challenges.

Embracing Simplicity in Biomaterials Design.

Biomaterials offer elegant frameworks to uncover mysteries of biology and vital tools to treat diseased or damaged tissues. Complex natural materials in the living world inspire the design of many engineered biomaterial constructs. Yet, complexity in materials design introduces practical, functional, and economic constraints. These challenges point to some virtues for a simplified approach in the design of biomaterials, especially when intended for clinical impact. But what is simplicity, and how can simple synthetic systems interface and intervene with application-specific complexities in the living world? Herein, both the philosophy and inherent benefits of simplicity in biomaterials design are discussed.

Rheological Characterization and Theoretical Modeling Establish Molecular Design Rules for Tailored Dynamically Associating Polymers.

Dynamically associating polymers have long been of interest due to their highly tunable viscoelastic behavior. Many applications leverage this tunability to create materials that have specific rheological properties, but designing such materials is an arduous, iterative process. Current models for dynamically associating polymers are phenomenological, assuming a structure for the relationship between association kinetics and network relaxation. We present the Brachiation model, a molecular-level theory of a polymer network with dynamic associations that is rooted in experimentally controllable design parameters, replacing the iterative experimental process with a predictive model for how experimental modifications to the polymer will impact rheological behavior. We synthesize hyaluronic acid chains modified with supramolecular host-guest motifs to serve as a prototypical dynamic network exhibiting tunable physical properties through control of polymer concentration and association rates. We use dynamic light scattering microrheology to measure the linear viscoelasticity of these polymers across six decades in frequency and fit our theory parameters to the measured data. The parameters are then altered by a magnitude corresponding to changes made to the experimental parameters and used to obtain new rheological predictions that match the experimental results well, demonstrating the ability for this theory to inform the design process of dynamically associating polymeric materials.