Biochemical and Structural Characterization of OvoATh2: A Mononuclear Nonheme Iron Enzyme from Hydrogenimonas thermophila for Ovothiol Biosynthesis.

Ovothiol A and ergothioneine are thiol-histidine derivatives with sulfur substitutions at the δ-carbon or ε-carbon of the l-histidine imidazole ring, respectively. Both ovothiol A and ergothioneine have protective effects on many aging-related diseases, and the sulfur substitution plays a key role in determining their chemical and biological properties, while factors governing sulfur incorporation regioselectivities in ovothiol and ergothioneine biosynthesis in the corresponding enzymes (OvoA, Egt1, or EgtB) are not yet known. In this study, we have successfully obtained the first OvoA crystal structure, which provides critical information to explain their C-S bond formation regioselectivity. Furthermore, OvoATh2 exhibits several additional activities: (1) ergothioneine sulfoxide synthase activity akin to Egt1 in ergothioneine biosynthesis; (2) cysteine dioxygenase activity using l-cysteine and l-histidine analogues as substrates; (3) cysteine dioxygenase activity upon mutation of an active site tyrosine residue (Y406). The structural insights and diverse chemistries demonstrated by OvoATh2 pave the way for future comprehensive structure-function correlation studies.

An S=1 Iron(IV) Intermediate Revealed in a Non-Heme Iron Enzyme-Catalyzed Oxidative C-S Bond Formation.

Ergothioneine (ESH) and ovothiol A (OSHA) are two natural thiol-histidine derivatives. ESH has been implicated as a longevity vitamin and OSHA inhibits the proliferation of hepatocarcinoma. The key biosynthetic step of ESH and OSHA in the aerobic pathways is the O2 -dependent C-S bond formation catalyzed by non-heme iron enzymes (e.g., OvoA in ovothiol biosynthesis), but due to the lack of identification of key reactive intermediate the mechanism of this novel reaction is unresolved. In this study, we report the identification and characterization of a kinetically competent S=1 iron(IV) intermediate supported by a four-histidine ligand environment (three from the protein residues and one from the substrate) in enabling C-S bond formation in OvoA from Methyloversatilis thermotoleran, which represents the first experimentally observed intermediate spin iron(IV) species in non-heme iron enzymes. Results reported in this study thus set the stage to further dissect the mechanism of enzymatic oxidative C-S bond formation in the OSHA biosynthesis pathway. They also afford new opportunities to study the structure-function relationship of high-valent iron intermediates supported by a histidine rich ligand environment.

Identification of a humanized mouse model for functional testing of immune-mediated biomaterial foreign body response.

Biomedical devices comprise a major component of modern medicine, however immune-mediated fibrosis and rejection can limit their function over time. Here, we describe a humanized mouse model that recapitulates fibrosis following biomaterial implantation. Cellular and cytokine responses to multiple biomaterials were evaluated across different implant sites. Human innate immune macrophages were verified as essential to biomaterial rejection in this model and were capable of cross-talk with mouse fibroblasts for collagen matrix deposition. Cytokine and cytokine receptor array analysis confirmed core signaling in the fibrotic cascade. Foreign body giant cell formation, often unobserved in mice, was also prominent. Last, high-resolution microscopy coupled with multiplexed antibody capture digital profiling analysis supplied spatial resolution of rejection responses. This model enables the study of human immune cell-mediated fibrosis and interactions with implanted biomaterials and devices.

A Quantitative Metal-Encoded Conjugate Platform for Targeting Ligand Discovery.

The indiscriminate biodistribution of therapeutics can be a key barrier to their safety and efficacy. Localization of compounds into non-diseased tissues often leads to both toxic and dose-limiting effects. To overcome this barrier, nanomedicine implements targeting agents to localize or selectively uptake drugs at disease sites. However, to date there are only a small number of targeting agents with limited scope for targeting tissues. Small-molecule ligands are particularly attractive as targeting agents due to their relatively low cost, tunability, and ease of conjugation. Currently, there are no systematic approaches to the discovery of new small-molecule targeting ligands. Here, we developed a quantitative metal-encoded conjugate platform to determine the biodistribution of multiple small molecules in vivo. By utilizing lanthanide metal complexes, this platform successfully distinguished known ligands with differential tissue targeting in vivo. This system will facilitate the discovery of small molecules as targeting ligands and can accelerate the identification of novel biological targets for tissue-targeted drug delivery.

Alginate-Based Amphiphilic Block Copolymers as a Drug Codelivery Platform.

Structured nanoassemblies are biomimetic structures that are enabling applications from nanomedicine to catalysis. One approach to achieve these spatially organized architectures is utilizing amphiphilic diblock copolymers with one or two macromolecular backbones that self-assemble in solution. To date, the impact of alternating backbone architectures on self-assembly and drug delivery is still an area of active research limited by the strategies used to synthesize these multiblock polymers. Here, we report self-assembling ABC-type alginate-based triblock copolymers with the backbones of three distinct biomaterials utilizing a facile conjugation approach. This "polymer mosaic" was synthesized by the covalent attachment of alginate with a PLA/PEG diblock copolymer. The combination of alginate, PEG, and PLA domains resulted in an amphiphilic copolymer that self-assembles into nanoparticles with a unique morphology of alginate domain compartmentalization. These particles serve as a versatile platform for co-encapsulation of hydrophilic and hydrophobic small molecules, their spatiotemporal release, and show potential as a drug delivery system for combination therapy.

A Small-Molecule Inhibitor to the Cytokine Interleukin-4.

Interleukin-4 (IL-4) is a multifunctional cytokine and an important regulator of inflammation. When deregulated, IL-4 activity is associated with asthma, allergic inflammation, and multiple types of cancer. While antibody-based inhibitors targeting the soluble cytokine have been evaluated clinically, they failed to achieve their end points in trials. Small-molecule inhibitors are an attractive alternative, but identifying effective chemotypes that inhibit the protein-protein interactions between cytokines and their receptors remains an active area of research. As a result, no small-molecule inhibitors to the soluble IL-4 cytokine have yet been reported. Here, we describe the first IL-4 small-molecule inhibitor identified and characterized through a combination of binding-based approaches and cell-based activity assays. The compound features a nicotinonitrile scaffold with micromolar affinity and potency for the cytokine and disrupts type II IL-4 signaling in cells. Small-molecule inhibitors of these important cell-signaling proteins have implications for numerous immune-related disorders and inform future drug discovery and design efforts for these challenging protein targets.

Domesticating the foreign body response: Recent advances and applications.

The foreign body response is an immunological process that leads to the rejection of implanted devices and presents a fundamental challenge to their performance, durability, and therapeutic utility. Recent advances in materials development and device design are now providing strategies to overcome this immune-mediated reaction. Here, we briefly review our current mechanistic understanding of the foreign body response and highlight new anti-FBR technologies from this decade that have been applied successfully in biomedical applications relevant to implants, devices, and cell-based therapies. Further development of these important technologies promises to enable new therapies, diagnostics, and revolutionize the management of patient care for many intractable diseases.

Targeting Type 1 Diabetes: Selective Approaches for New Therapies.

The clinical onset of type 1 diabetes is characterized by the destruction of the insulin-producing ß cells of the pancreas and is caused by autoantigen-induced inflammation (insulitis) of the islets of Langerhans. The current standard of care for type 1 diabetes mellitus patients allows for management of the disease with exogenous insulin, but patients eventually succumb to many chronic complications such as limb amputation, blindness, and kidney failure. New therapeutic approaches now on the horizon are looking beyond glycemic management and are evaluating new strategies from protecting and regenerating endogenous islets to treating the underlying autoimmunity through selective modulation of key immune cell populations. Currently, there are no effective treatments for the autoimmunity that causes the disease, and strategies that aim to delay or prevent the onset of the disease will play an important role in the future of diabetes research. In this review, we summarize many of the key efforts underway that utilize molecular approaches to selectively modulate this disease and look at new therapeutic paradigms that can transform clinical treatment.

Alginate encapsulation as long-term immune protection of allogeneic pancreatic islet cells transplanted into the omental bursa of macaques.

The transplantation of pancreatic islet cells could restore glycaemic control in patients with type-I diabetes. Microspheres for islet encapsulation have enabled long-term glycaemic control in diabetic rodent models; yet human patients transplanted with equivalent microsphere formulations have experienced only transient islet-graft function, owing to a vigorous foreign-body reaction (FBR), to pericapsular fibrotic overgrowth (PFO) and, in upright bipedal species, to the sedimentation of the microspheres within the peritoneal cavity. Here, we report the results of the testing, in non-human primate (NHP) models, of seven alginate formulations that were efficacious in rodents, including three that led to transient islet-graft function in clinical trials. Although one month post-implantation all formulations elicited significant FBR and PFO, three chemically modified, immune-modulating alginate formulations elicited reduced FBR. In conjunction with a minimally invasive transplantation technique into the bursa omentalis of NHPs, the most promising chemically modified alginate derivative (Z1-Y15) protected viable and glucose-responsive allogeneic islets for 4 months without the need for immunosuppression. Chemically modified alginate formulations may enable the long-term transplantation of islets for the correction of insulin deficiency.

Reduction of measurement noise in a continuous glucose monitor by coating the sensor with a zwitterionic polymer.

Continuous glucose monitors (CGMs), used by patients with diabetes mellitus, can autonomously track fluctuations in blood glucose over time. However, the signal produced by CGMs during the initial recording period following sensor implantation contains substantial noise, requiring frequent recalibration via fingerprick tests. Here, we show that coating the sensor with a zwitterionic polymer, found via a combinatorial-chemistry approach, significantly reduces signal noise and improves CGM performance. We evaluated the polymer-coated sensors in mice as well as in healthy and diabetic non-human primates, and show that the sensors accurately record glucose levels without the need for recalibration. We also show that the polymer-coated sensors significantly abrogated immune responses to the sensor, as indicated by histology, fluorescent whole-body imaging of inflammation-associated protease activity, and gene expression of inflammation markers. The polymer coating may allow CGMs to become standalone measuring devices.

Colony stimulating factor-1 receptor is a central component of the foreign body response to biomaterial implants in rodents and non-human primates.

Host recognition and immune-mediated foreign body response to biomaterials can compromise the performance of implanted medical devices. To identify key cell and cytokine targets, here we perform in-depth systems analysis of innate and adaptive immune system responses to implanted biomaterials in rodents and non-human primates. While macrophages are indispensable to the fibrotic cascade, surprisingly neutrophils and complement are not. Macrophages, via CXCL13, lead to downstream B cell recruitment, which further potentiated fibrosis, as confirmed by B cell knockout and CXCL13 neutralization. Interestingly, colony stimulating factor-1 receptor (CSF1R) is significantly increased following implantation of multiple biomaterial classes: ceramic, polymer and hydrogel. Its inhibition, like macrophage depletion, leads to complete loss of fibrosis, but spares other macrophage functions such as wound healing, reactive oxygen species production and phagocytosis. Our results indicate that targeting CSF1R may allow for a more selective method of fibrosis inhibition, and improve biomaterial biocompatibility without the need for broad immunosuppression.

Microfluidic Fabrication of Colloidal Nanomaterials-Encapsulated Microcapsules for Biomolecular Sensing.

Implantable sensors that detect biomarkers in vivo are critical for early disease diagnostics. Although many colloidal nanomaterials have been developed into optical sensors to detect biomolecules in vitro, their application in vivo as implantable sensors is hindered by potential migration or clearance from the implantation site. One potential solution is incorporating colloidal nanosensors in hydrogel scaffold prior to implantation. However, direct contact between the nanosensors and hydrogel matrix has the potential to disrupt sensor performance. Here, we develop a hollow-microcapsule-based sensing platform that protects colloidal nanosensors from direct contact with hydrogel matrix. Using microfluidics, colloidal nanosensors were encapsulated in polyethylene glycol microcapsules with liquid cores. The microcapsules selectively trap the nanosensors within the core while allowing free diffusion of smaller molecules such as glucose and heparin. Glucose-responsive quantum dots or gold nanorods or heparin-responsive gold nanorods were each encapsulated. Microcapsules loaded with these sensors showed responsive optical signals in the presence of target biomolecules (glucose or heparin). Furthermore, these microcapsules can be immobilized into biocompatible hydrogel as implantable devices for biomolecular sensing. This technique offers new opportunities to extend the utility of colloidal nanosensors from solution-based detection to implantable device-based detection.

A Facile and Versatile Method to Endow Biomaterial Devices with Zwitterionic Surface Coatings.

The surface modification of implantable biomaterials with zwitterionic phosphorylcholine polymer is demonstrated through mussel-mimetic catecholamine polymer thin films. Using this method, the surfaces of alginate hydrogel microspheres and polystyrene microbeads, a model material known to produce robust foreign body responses and fibrosis, are successfully modified to reduce the tissue reaction by reducing the fibrosis in immunocompetent C57BL/6J mice.

Sequence-Defined Oligomers from Hydroxyproline Building Blocks for Parallel Synthesis Applications.

The functionality of natural biopolymers has inspired significant effort to develop sequence-defined synthetic polymers for applications including molecular recognition, self-assembly, and catalysis. Conjugation of synthetic materials to biomacromolecules has played an increasingly important role in drug delivery and biomaterials. We developed a controlled synthesis of novel oligomers from hydroxyproline-based building blocks and conjugated these materials to siRNA. Hydroxyproline-based monomers enable the incorporation of broad structural diversity into defined polymer chains. Using a perfluorocarbon purification handle, we were able to purify diverse oligomers through a single solid-phase extraction method. The efficiency of synthesis was demonstrated by building 14 unique trimers and 4 hexamers from 6 diverse building blocks. We then adapted this method to the parallel synthesis of hundreds of materials in 96-well plates. This strategy provides a platform for the screening of libraries of modified biomolecules.

Combinatorial hydrogel library enables identification of materials that mitigate the foreign body response in primates.

The foreign body response is an immune-mediated reaction that can lead to the failure of implanted medical devices and discomfort for the recipient. There is a critical need for biomaterials that overcome this key challenge in the development of medical devices. Here we use a combinatorial approach for covalent chemical modification to generate a large library of variants of one of the most widely used hydrogel biomaterials, alginate. We evaluated the materials in vivo and identified three triazole-containing analogs that substantially reduce foreign body reactions in both rodents and, for at least 6 months, in non-human primates. The distribution of the triazole modification creates a unique hydrogel surface that inhibits recognition by macrophages and fibrous deposition. In addition to the utility of the compounds reported here, our approach may enable the discovery of other materials that mitigate the foreign body response.

Long-term glycemic control using polymer-encapsulated human stem cell-derived beta cells in immune-competent mice.

The transplantation of glucose-responsive, insulin-producing cells offers the potential for restoring glycemic control in individuals with diabetes. Pancreas transplantation and the infusion of cadaveric islets are currently implemented clinically, but these approaches are limited by the adverse effects of immunosuppressive therapy over the lifetime of the recipient and the limited supply of donor tissue. The latter concern may be addressed by recently described glucose-responsive mature beta cells that are derived from human embryonic stem cells (referred to as SC-ß cells), which may represent an unlimited source of human cells for pancreas replacement therapy. Strategies to address the immunosuppression concerns include immunoisolation of insulin-producing cells with porous biomaterials that function as an immune barrier. However, clinical implementation has been challenging because of host immune responses to the implant materials. Here we report the first long-term glycemic correction of a diabetic, immunocompetent animal model using human SC-ß cells. SC-ß cells were encapsulated with alginate derivatives capable of mitigating foreign-body responses in vivo and implanted into the intraperitoneal space of C57BL/6J mice treated with streptozotocin, which is an animal model for chemically induced type 1 diabetes. These implants induced glycemic correction without any immunosuppression until their removal at 174 d after implantation. Human C-peptide concentrations and in vivo glucose responsiveness demonstrated therapeutically relevant glycemic control. Implants retrieved after 174 d contained viable insulin-producing cells.

High Throughput Layer-by-Layer Films for Extracting Film Forming Parameters and Modulating Film Interactions with Cells.

A high-throughput approach which automates the synthesis of polyelectrolyte-based layer-by-layer films (HT-LbL) to facilitate rapid film generation, systematic film characterization, and rational investigations into their interactions with cells is described. Key parameters, such as polyelectrolyte adsorption time and polyelectrolyte deposition pH, were used to modulate LbL film growth to create LbL films of distinct thicknesses using the widely utilized polyelectrolytes poly(allylamine hydrochloride) (PAH) and poly(acrylic acid) (PAA). We highlight how HT-LbL can be used to rapidly characterize film-forming parameters and robustly create linearly growing films of various molecular architectures. Film thickness and growth rates of HT-LbL films were shown to increase as a function of adsorption time. Subsequently, we investigated the role that polyelectrolyte solution pH (ranging from 2.5 to 9) has in forming molecularly distinct films of weak polyelectrolytes and report the effect this has on modulating cell attachment and spreading. Films synthesized at PAA-pH of 5.5 and PAH-pH 2.5-5.5 exhibited the highest cellular attachment. These results indicate that HT-LbL is a robust method that can shift the paradigm regarding the use of LbL in biomedical applications as it provides a rapid method to synthesize, characterize, and screen the interactions between molecularly distinct LbL films and cells.