Synthetically tractable click hydrogels for three-dimensional cell culture formed using tetrazine-norbornene chemistry.

The implementation of bio-orthogonal click chemistries is a topic of growing importance in the field of biomaterials, as it is enabling the development of increasingly complex hydrogel materials capable of providing dynamic, cell-instructive microenvironments. Here, we introduce the tetrazine-norbornene inverse electron demand Diels-Alder reaction as a new cross-linking chemistry for the formation of cell laden hydrogels. The fast reaction rate and irreversible nature of this click reaction allowed for hydrogel formation within minutes when a multifunctional PEG-tetrazine macromer was reacted with a dinorbornene peptide. In addition, the cytocompatibility of the polymerization led to high postencapsulation viability of human mesenchymal stem cells, and the specificity of the tetrazine-norbornene reaction was exploited for sequential modification of the network via thiol-ene photochemistry. These advantages, combined with the synthetic accessibility of the tetrazine molecule compared to other bio-orthogonal click reagents, make this cross-linking chemistry an interesting and powerful new tool for the development of cell-instructive hydrogels for tissue engineering applications.

Wavelength-controlled photocleavage for the orthogonal and sequential release of multiple proteins.

On the right wavelength: Photolabile molecular units that undergo photocleavage under light of different wavelengths can be used for the independent release of different dyes/proteins from a single, preloaded storage hydrogel. The controlled release of each protein allowed them to be delivered sequentially and at experimenter-determined times.

Supramolecular displacement-mediated activation of a silent fluorescence probe for label-free ligand screening.

We report a new approach for the rapid screening of analyte binding affinities for a target protein. We demonstrate that a molecular probe, with a pro-fluorophore substrate and ligand moieties, can be hindered from enzymatic access when bound to the target protein. When analytes displace the probe from the protein's binding pocket, a fluorescence profile is generated. This profile is used to discriminate analytes based on their relative binding affinities.

Accessing lipophilic ligands in dendrimer-based amphiphilic supramolecular assemblies for protein-induced disassembly.

Supramolecular nanoassemblies that respond to the presence of proteins are of great interest, as aberrations in protein concentrations represent the primary imbalances found in a diseased state. We present here a molecular design, syntheses, and study of facially amphiphilic dendrimers that respond to the presence of the protein, immunoglobulin G. It is of particular interest that the ligand functionality, utilized for causing the binding-induced disassembly, be lipophilic. Demonstration of binding with lipophilic ligands greatly expands the repertoire of binding-induced disassembly, since this covers a rather large class of ligand moieties designed for proteins and these provide specific insights into the mechanistic pathways that are available for the binding-induced disassembly process. Here, we describe the details of the binding induced disassembly, including the change in size of the assembly in response to proteins, concurrent release of noncovalently encapsulated guest molecules, and the specificity of the disassembly process.

Photocontrolled nanoparticles for on-demand release of proteins.

We describe here light-regulated swelling and degradation features of polymeric nanoparticles that are produced using an inverse microemulsion polymerization method. We demonstrate the phototriggered release characteristics of the nanoparticles by sequestering protein molecules and releasing them using light as a trigger. Furthermore, the intracellular translocation of the nanoparticles, along with its fluorescent protein payload, was achieved using a cell-penetrating peptide-based surface modification. We expect that the noncovalent encapsulation of proteins using nanoparticles and their photo triggered release using an external light would provide opportunities for achieving intracellular release of molecular therapeutics for on-demand requirements.

Guest-release control in enzyme-sensitive, amphiphilic-dendrimer-based nanoparticles through photochemical crosslinking.

Stimuli sensitive, facially amphiphilic dendrimers have been synthesized and their enzyme-responsive nature has been determined with dual fluorescence responses of both covalently conjugated and non-covalently bound reporter units. These dual responses are correlated to ascertain the effect of enzymatic action on micellar aggregates and the consequential guest release. The release of the guest molecule is conveniently tuned by stabilizing the micellar aggregates through photochemical crosslinking of hydrophobic coumarin units. This photo-crosslinking is also utilized as a tool to investigate the mode of enzyme-substrate interaction in the context of aggregate-monomer equilibrium.

Disassembly of dendritic micellar containers due to protein binding.

Disassembling a supramolecular assembly and releasing the contents of the assembly in response to a stimulus are important goals of supramolecular chemistry. When proteins are used as the stimulus, the biological relevance of the supramolecular event dramatically increases. Although there have been efforts in which such disassembly has been achieved using enzymatic action, such events based on ligand-receptor interactions have been very limited. Here we demonstrate protein-binding-induced disassembly of dendrimer-based amphiphilic nanocontainers. We show that this disassembly is selective to the targeted protein and that the disassembly event causes a release of the sequestered guest molecules. We propose that the disassembly is caused by alteration of the hydrophilic-lipophilic balance caused by the protein binding.

Bioorthogonal click chemistries enable simultaneous spatial patterning of multiple proteins to probe synergistic protein effects on fibroblast function.

Three biorthogonal click reactions, a photoinitiated thiol-yne reaction, an azide-alkyne cycloaddition, and a methyltetrazine-transcyclooctene Diels Alder, were used to independently control the presentation of several bioactive proteins to valvular interstitial cells (VICs) in hydrogel scaffolds. Tethered fibroblast growth factor (FGF-2) was found to suppress myofibroblast activation (from 48 ± 7% to 17 ± 6%) and promote proliferation (from 10 ± 2% to 54 ± 3%) at a concentration of 10 ng/mL. In the presence of the pro-fibrotic cytokine transforming growth factor-beta (TGF-ß1), FGF-2 could protect the VIC fibroblast phenotype, even at much higher concentrations of TGF-ß1 than that of FGF-2. With respect to the fibrocalcific VIC phenotype, TGF-ß1 and bone-morphogenic protein-2 (BMP-2) were found to synergistically promote calcific nodule formation (a five-fold increase in nodules compared to TGF-ß1 or BMP-2 alone). Exploiting the orthogonal click reactions, FGF-2, TGF-ß1 and BMP-2 combinations were patterned into distinct regions on a hydrogel to control VIC activation and nodule formation. Cellular crosstalk between separate regions of the same scaffold was affected by the size of each region as well as the interfacial area between different regions. Collectively, these results demonstrate the versatility and robustness of a photoinitiated thiol-yne reaction to template pendant functionalities that allow for the bioconjugation of multiple proteins. This approach maintains protein bioactivity, providing an in vitro platform capable of achieving a better understanding of the complex mechanisms involved in tissue fibrosis.

Enzyme-triggered disassembly of dendrimer-based amphiphilic nanocontainers.

We demonstrate a new enzyme-induced disassembly of amphiphilic nanocontainers based on dendrimers. Disassembly and the ensuing release of noncovalently bound guest molecules are of great interest because of their implications in areas such as drug delivery and sensing. Achieving these with a protein as the stimulus is of even greater importance, because proteins are the primary indicators of biological imbalances. We achieved disassembly of the nanocontainers by disturbing the hydrophilic-lipophilic balance in the amphiphilic dendrimer building blocks.

Site-specific installation and study of electroactive units in every layer of dendrons.

Whereas encapsulation of functional groups at the core of dendrimers is well-understood, very little is known about their intermediate layers or even the periphery. Here we report on a systematic investigation of every layer of dendrimers by incorporating a single ferrocene unit in well-defined locations in dendrons. Site-specific incorporation of the ferrocene unit was achieved by utilizing the dendrimer sequencing methodology. We show here that the redox potential values of ferrocene at intermediate layers were remarkably different from those at the core and the periphery. Although redox potential values were location-dependent, no significant change in the rate of heterogeneous electron transfer (k(0)) was observed with respect to locations. This was attributed to the possibility that free rotation of dendrimer nullifies the distance between the electrode and ferrocene unit. Finally, we also show that no Faradaic current was observed for the amphiphilic assemblies of these dendrons, whereas the same dendron did exhibit significant Faradaic current in nonassembling solvent environments.

Amphiphilic nanoassemblies for the detection of peptides and proteins using fluorescence and mass spectrometry.

Amphiphilic nanostructures provide unique environments for molecules that are incompatible with the solvent to be sequestered within their interior. These internal environments provide opportunities for concentrating an analyte or transducer molecule for detection, and the functional groups within the amphiphiles provide an opportunity for incorporating specificity or selectivity toward analytes. In this review, we discuss ways in which amphiphilic assemblies can be used to detect peptides and proteins with a particular emphasis on facially amphiphilic polymers and dendrimers.

Selective peptide binding using facially amphiphilic dendrimers.

Amphiphilic dendrimers, which contain both hydrophobic and hydrophilic groups in every repeat unit, exhibit environment-dependent assemblies both in hydrophilic solvent, water, and in lipophilic solvent, toluene. Upon investigating the status of these assemblies in a mixture of immiscible solvents, these dendrimers were found to be kinetically trapped in the solvent in which they are initially assembled. This property has been exploited to selectively extract peptides from aqueous solution into an organic phase, where the peptides bind to the interior functionalities of the dendritic inverse micelles. While the corresponding small molecule surfactant does not exhibit any selective binding toward peptides, all dendrons (G1-G3) are capable of this selective binding. We show that the inverse micelle-type assembly itself is crucial for the binding event and that the assembly formed by the G1 dendron has a greater capability for binding compared to the G2 or G3 dendrons. We have also shown that the average apparent pKa of the carboxylic acid functionalities varies with generation, and this could be the reason for the observed differences in binding capacity.

Photoregulated Hydrazone-Based Hydrogel Formation for Biochemically Patterning 3D Cellular Microenvironments.

Photodriven click reactions have emerged as versatile tools for biomaterial synthesis that can recapitulate critical spatial and temporal changes of extracellular matrix (ECM) microenvironments in vitro. In this article, we report on the synthesis of poly(ethylene glycol) (PEG) hydrogels using photodriven step-growth polymerization, where one of the reactive functionalities is formed by a photocleavage reaction. Upon photocleavage, an aldehyde functionality is generated that rapidly reacts with hydrazine-functionalized PEGs; the gelation kinetics and final material modulus are distinctly controlled by variations in the light intensity. This light-driven aldehyde generation is further exploited to install biochemical ligands in the hydrazone-based hydrogels with precise spatial control. We expect that user-directed spatial and temporal control over both biophysical and biochemical gel properties through photochemical reactions and photopatterning, respectively, should provide newfound opportunities to probe and understand dynamic cell-matrix interactions.

Photo-click living strategy for controlled, reversible exchange of biochemical ligands.

A novel addition-fragmentation-chain transfer capable allyl sulfide functionalized PEG hydrogel is reported to allow controlled, reversible exchange of biochemical ligands. The exchange of biochemical ligands is achieved without permanent consumption of reactive functionalities. Demonstrated is the ability to exchange biochemical ligands multiple times using cytocompatible 720 nm two-photon light.

Coumarin-Based Photodegradable Hydrogel: Design, Synthesis, Gelation, and Degradation Kinetics.

The design, synthesis, and characterization of a new class of coumarin-based photodegradable hydrogels are reported. Hydrogel formation was achieved rapidly and efficiently under aqueous conditions using copper-catalyzed click chemistry, which afforded excellent control over the rate of network formation. Rapid photodegradation, to the point of reverse gelation, was observed using both 365 and 405 nm light, and micrometer-scale features were eroded using two-photon irradiation at wavelengths as long as 860 nm.

Bioorthogonal Click Chemistry: An Indispensable Tool to Create Multifaceted Cell Culture Scaffolds.

Over the past decade, bioorthogonal click chemistry has led the field of biomaterial science into a new era of diversity and complexity by its extremely selective, versatile, and biocompatible nature. In this viewpoint, we seek to emphasize recent endeavors of exploiting this versatile chemistry toward the development of poly(ethylene glycol) hydrogels as cell culture scaffolds. In these cell-laden materials, the orthogonality of these reactions has played an effective role in allowing the creation of diverse biochemical patterns in complex biological environments that provide new found opportunities for researchers to delineate and control cellular phenotypes more precisely than ever.