Photolabile moieties have been utilized in applications ranging from peptide synthesis and controlled protein activation to tunable and dynamic materials. The photochromic properties of nitrobenzyl ...(NB) based linkers are readily tuned to respond to cytocompatible light doses and are widely utilized in cell culture and other biological applications. While widely utilized, little is known about how the microenvironment, particularly confined aqueous environments (e.g., hydrogels), affects both the mode and rate of cleavage of NB moieties, leading to unpredictable limitations in control over system properties (e.g., rapid hydrolysis or slow photolysis). To address these challenges, we synthesized and characterized the photolysis and hydrolysis of NB moieties containing different labile bonds (i.e., ester, amide, carbonate, or carbamate) that served as labile crosslinks within step-growth hydrogels. We observed that NB ester bond exhibited significant rates of both photolysis and hydrolysis, whereas, importantly, the NB carbamate bond had superior light responsiveness and resistance to hydrolysis within the hydrogel microenvironment. Exploiting this synergy and orthogonality of photolytic and hydrolytic degradation, we designed concentric cylinder hydrogels loaded with different cargoes (e.g., model protein with different fluorophores) for either combinatorial or sequential release, respectively. Overall, this work provides new facile chemical approaches for tuning the degradability of NB linkers and an innovative strategy for the construction of multimodal degradable hydrogels, which can be utilized to guide the design of not only tunable materials platforms but also controlled synthetic protocols or surface modification strategies.
The acid-catalyzed thiol-ene reaction (ACT) is a unique thiol-X conjugation strategy that produces S,X-acetal conjugates. Unlike the well-known radical-mediated thiol-ene and anion-mediated ...thiol-Michael reactions that produce static thioether bonds, acetals provide unique function for various fields such as drug delivery and protecting group chemistries; however, this reaction is relatively underutilized for creating new and unique materials owing to the unexplored reactivity over a broad set of substrates and potential side reactions. Solution-phase studies using a range of thiol and alkene substrates were conducted to evaluate the ACT reaction as a conjugation strategy. Substrates that efficiently undergo cationic polymerizations, such as those containing vinyl functional groups, were found to be highly reactive to thiols in the presence of catalytic amounts of acid. Additionally, sequential initiation of three separate thiol-X reactions (thiol-Michael, ACT, and thiol-ene) was achieved in a one-pot scheme simply by the addition of the appropriate catalyst demonstrating substrate selectivity. Furthermore, photoinitiation of the ACT reaction was achieved for the first time under 470 nm blue light using a novel photochromic photoacid. Finally, using multifunctional monomers, solid-state polymer networks were formed using the ACT reaction producing acetal crosslinks. The presence of S,X-acetal bonds results in an increased glass transition temperature of 20 °C as compared with the same polymeric film polymerized through the radical thiol-ene mechanism. This investigation demonstrates the broad impact of the ACT reaction and expands upon the diverse thiol-X library of conjugation strategies towards the development of novel materials systems.
Kinetics of the acid-catalyzed thiol-ene (ACT) reaction were explored over a range of thiol and vinyl functional groups. Its applicability in material synthesis was demonstrated in the design of photopolymerized polymer networks.
After a myocardial infarction (MI), the heart undergoes changes including local remodeling that can lead to regional abnormalities in mechanical and electrical properties, ultimately increasing the ...risk of arrhythmias and heart failure. Although these responses have been successfully recapitulated in animal models of MI, local changes in tissue and cell-level mechanics caused by MI remain difficult to study in vivo. Here, we developed an in vitro cardiac microtissue (CMT) injury system that through acute focal injury recapitulates aspects of the regional responses seen following an MI. With a pulsed laser, cell death was induced in the center of the microtissue causing a loss of calcium signaling and a complete loss of contractile function in the injured region and resulting in a 39% reduction in the CMT's overall force production. After 7 days, the injured area remained void of cardiomyocytes (CMs) and showed increased expression of vimentin and fibronectin, two markers for fibrotic remodeling. Interestingly, although the injured region showed minimal recovery, calcium amplitudes in uninjured regions returned to levels comparable with control. Furthermore, overall force production returned to preinjury levels despite the lack of contractile function in the injured region. Instead, uninjured regions exhibited elevated contractile function, compensating for the loss of function in the injured region, drawing parallels to changes in tissue-level mechanics seen in vivo. Overall, this work presents a new in vitro model to study cardiac tissue remodeling and electromechanical changes after injury.
We report an in vitro cardiac injury model that uses a high-powered laser to induce regional cell death and a focal fibrotic response within a human-engineered cardiac microtissue. The model captures the effects of acute injury on tissue response, remodeling, and electromechanical recovery in both the damaged region and surrounding healthy tissue, modeling similar changes to contractile function observed in vivo following myocardial infarction.
The engineering of biological molecules is a key concept in the design of highly functional, sophisticated soft materials. Biomolecules exhibit a wide range of functions and structures, including ...chemical recognition (of enzyme substrates or adhesive ligands
, for instance), exquisite nanostructures (composed of peptides
, proteins
or nucleic acids
), and unusual mechanical properties (such as silk-like strength
, stiffness
, viscoelasticity
and resiliency
). Here we combine the computational design of physical (noncovalent) interactions with pathway-dependent, hierarchical 'click' covalent assembly to produce hybrid synthetic peptide-based polymers. The nanometre-scale monomeric units of these polymers are homotetrameric, α-helical bundles of low-molecular-weight peptides. These bundled monomers, or 'bundlemers', can be designed to provide complete control of the stability, size and spatial display of chemical functionalities. The protein-like structure of the bundle allows precise positioning of covalent linkages between the ends of distinct bundlemers, resulting in polymers with interesting and controllable physical characteristics, such as rigid rods, semiflexible or kinked chains, and thermally responsive hydrogel networks. Chain stiffness can be controlled by varying only the linkage. Furthermore, by controlling the amino acid sequence along the bundlemer periphery, we use specific amino acid side chains, including non-natural 'click' chemistry functionalities, to conjugate moieties into a desired pattern, enabling the creation of a wide variety of hybrid nanomaterials.
Networks formed using photo-initiated copper-catalyzed azide-alkyne cycloaddition (photo-CuAAC) polymerizations exhibit extraordinary toughness and high stability making them an ideal material for ...applications ranging from dental composites to membranes. The current design for photoinitiation of solvent-free CuAAC networks is accomplished under blue light using a Norrish Type II initiating system comprising camphorquinone (CQ) and a tertiary amine (TA). Although TAs are required for rapid polymerization kinetics, their presence in the network can act as a plasticizer and is often malodorous and toxic. Here, a one-component, Type II photoinitiation scheme for the photo-CuAAC polymeriziation is achieved by incorporating the TA coinitiating species into the backbone of the polymer network as a crosslinking moiety. Formulations with the polymer network-incorporated TA exhibited rapid polymerization kinetics, reaching >90% conversion with just 5 min of 470 nm irradiation at 30 mW cm
−2
. Varying the structure of the amine centered trialkynes allowed for tunability of the glass transition temperature from 53 to 82 °C. Moreover, only catalytic amounts of the amine-centered alkyne monomers were required to replace the external TA without adversely affecting the rapid polymerization kinetics or the thermomechanical properties of the network. Development of such monomers further demonstrates the tunability of bulk photo-CuAAC polymer network properties and expands upon their broad range of applications.
A one-component photoinitiation scheme was devised utilizing amine-centered trialkyne monomers for the formation of bulk photo-CuAAC polymer networks. The novel monomers maintain rapid polymerization kinetics and allow for tuning of the
T
g
.
Engineered hydrogels are increasingly used as extracellular matrix (ECM) surrogates for probing cell function in response to ECM remodeling events related to injury or disease (e.g., degradation ...followed by deposition/crosslinking). Inspired by these events, this work establishes an approach for pseudo‐reversible mechanical property modulation in synthetic hydrogels by integrating orthogonal, enzymatically triggered crosslink degradation, and light‐triggered photopolymerization stiffening. Hydrogels are formed by a photo‐initiated thiol–ene reaction between multiarm polyethylene glycol and a dually enzymatically degradable peptide linker, which incorporates a thrombin‐degradable sequence for triggered softening and a matrix metalloproteinase (MMP)‐degradable sequence for cell‐driven remodeling. Hydrogels are stiffened by photopolymerization using a flexible, MMP‐degradable polymer‐peptide conjugate and multiarm macromers, increasing the synthetic matrix crosslink density while retaining degradability. Integration of these tools enables sequential softening and stiffening inspired by matrix remodeling events within loose connective tissues (Young's modulus (E) ≈5 to 1.5 to 6 kPa with >3x ΔE). The cytocompatibility and utility of this approach is examined with breast cancer cells, where cell proliferation shows a dependence on the timing of triggered softening. This work provides innovative tools for 3D dynamic property modulation that are synthetically accessible and cell compatible.
This work establishes an approach for sequential modulation of the mechanical properties of cell‐degradable synthetic matrices inspired by matrix remodeling events and relevant for probing‐related cellular responses during 3D culture. In situ property modulation is enabled by integration of orthogonal enzymatically triggered linker degradation for matrix softening and light‐triggered crosslinking for matrix stiffening.
Synthetic DNA analogues are of great interest for their application in information storage, therapeutics, and nanostructured materials, yet are often limited in scalability. Vinyl sulfonamide click ...nucleic acids (VS-CNAs) have been developed to overcome this limitation using the highly efficient thiol-Michael 'click' reaction. Utilizing all four nucleobases, sequence-defined click nucleic acids (CNAs) were synthesized using a simple and scalabale solution-phase approach. Employing a polyethylene glycol (PEG) support, synthesis of the CNA sequence, GATTACA, was achieved in high yields. CNA crosslinked hydrogels were assembled using multiarm PEG-CNAs resulting in materials that dynamically respond to temperature, strain, and competitive sequences.
A scalable synthetic strategy was developed towards the creation of sequence-defined DNA analogues employing thiol-Michael click chemistry and a soluble polymer support.
Macrocyclization of linear peptides imparts improved stability to enzymatic degradation and increases potency of function. Many successful macrocyclization of peptides both in solution and on-resin ...have been achieved but are limited in scope as they lack selectivity, require long reaction times, or necessitate heat. To overcome these drawbacks a robust and facile strategy was developed employing thiol-Michael click chemistry via an N-methyl vinyl sulfonamide. We demonstrate its balance of reactivity and high stability through FTIR model kinetic studies, reaching 88% conversion over 30 min, and NMR stability studies, revealing no apparent degradation over an 8 day period in basic conditions. Using a commercially available reagent, 2-chloroethane sulfonyl chloride, the cell adhesion peptide, RGDS, was functionalized and macrocyclized on-resin with a relative efficiency of over 95%. The simplistic nature of this process demonstrates the effectiveness of vinyl sulfonamides as a thiol-Michael click acceptor and its applicability to many other bioconjugation applications.
Protein therapeutics have become increasingly popular for the treatment of a variety of diseases owing to their specificity to targets of interest. However, challenges associated with them have ...limited their use for a range of ailments, including the limited options available for local controlled delivery. To address this challenge, degradable hydrogel microparticles, or microgels, loaded with model biocargoes were created with tunable release profiles or triggered burst release using chemistries responsive to endogenous or exogeneous stimuli, respectively. Specifically, microfluidic flow-focusing was utilized to form homogenous microgels with different spontaneous click chemistries that afforded degradation either in response to redox environments for sustained cargo release or light for on-demand cargo release. The resulting microgels were an appropriate size to remain localized within tissues upon injection and were easily passed through a needle relevant for injection, providing means for localized delivery. Release of a model biopolymer was observed over the course of several weeks for redox-responsive formulations or triggered for immediate release from the light-responsive formulation. Overall, we demonstrate the ability of microgels to be formulated with different materials chemistries to achieve various therapeutic release modalities, providing new tools for creation of more complex protein release profiles to improve therapeutic regimens.
Adoptive T‐cell therapies (ATCTs) are increasingly important for the treatment of cancer, where patient immune cells are engineered to target and eradicate diseased cells. The biomanufacturing of ...ATCTs involves a series of time‐intensive, lab‐scale steps, including isolation, activation, genetic modification, and expansion of a patient's T‐cells prior to achieving a final product. Innovative modular technologies are needed to produce cell therapies at improved scale and enhanced efficacy. In this work, well‐defined, bioinspired soft materials are integrated within flow‐based membrane devices for improving the activation and transduction of T‐cells. Hydrogel coated membranes (HCM) functionalized with cell‐activating antibodies are produced as a tunable biomaterial for the activation of primary human T‐cells. T‐cell activation utilizing HCMs lead to highly proliferative T‐cells that express a memory phenotype. Further, transduction efficiency is improved by several folds over static conditions by using a tangential flow filtration (TFF) flow‐cell, commonly used in the production of protein therapeutics, to transduce T‐cells under flow. The combination of HCMs and TFF technology leads to increased cell activation, proliferation, and transduction compared to current industrial biomanufacturing processes. The combined power of biomaterials with scalable flow‐through transduction techniques provides future opportunities for improving the biomanufacturing of ATCTs.
Well‐defined, bioinspired soft materials are integrated within scalable, flow‐based membrane devices for improving the activation and transduction of T‐cells, which are essential steps in the production of adoptive T‐cell therapies. These innovative technologies provide opportunities for improving the manufacturing of cell therapies.
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