Aaron P. Esser-Kahn

Programmable Microcapsules from Self-Immolative Polymers

For the autonomous repair of damaged materials, microcapsules are needed that release their contents in response to a variety of physical and chemical phenomena, not just by direct mechanical rupture. Herein we report a general route to programmable microcapsules. This method creates core-shell microcapsules with polymeric shell walls composed of self-immolative polymer networks. The polymers in these networks undergo a head-to-tail depolymerization upon removal of the triggering end group, leading to breakdown of the shell wall and subsequent release of the capsules liquid interior. We report microcapsules with shell walls bearing both Boc and Fmoc triggering groups. The capsules release their contents only under conditions known to remove these triggering groups; otherwise, they retain their contents under a variety of conditions. In support of the proposed release mechanism, the capsule shell walls were observed to undergo physical cracking upon exposure to the triggering conditions.

In vivo characterization of the physicochemical properties of polymer-linked TLR agonists that enhance vaccine immunogenicity

The efficacy of vaccine adjuvants such as Toll-like receptor agonists (TLRa) can be improved through formulation and delivery approaches. Here, we attached small molecule TLR-7/8a to polymer scaffolds (polymer–TLR-7/8a) and evaluated how different physicochemical properties of the TLR-7/8a and polymer carrier influenced the location, magnitude and duration of innate immune activation in vivo. Particle formation by polymer–TLR-7/8a was the most important factor for restricting adjuvant distribution and prolonging activity in draining lymph nodes. The improved pharmacokinetic profile by particulate polymer–TLR-7/8a was also associated with reduced morbidity and enhanced vaccine immunogenicity for inducing antibodies and T cell immunity. We extended these findings to the development of a modular approach in which protein antigens are site-specifically linked to temperature-responsive polymer–TLR-7/8a adjuvants that self-assemble into immunogenic particles at physiologic temperatures in vivo. Our findings provide a chemical and structural basis for optimizing adjuvant design to elicit broad-based antibody and T cell responses with protein antigens.

Metallothionein-Cross-Linked Hydrogels for the Selective Removal of Heavy Metals from Water

The diverse functional repertoire of proteins promises to yield new materials with unprecedented capabilities, so long as versatile chemical methods are available to integrate biomolecules with synthetic components. As a demonstration of this potential, we have used site-selective strategies to cross-link polymer chains using the N- and C-termini of a metallothionein derived from a pea plant. This arrangement directly relates the swelling volume of the polymer to the folded state of the protein. The material retains the proteins ability to remove heavy metal ions from contaminated water samples, and can be regenerated through the subsequent addition of inexpensive chelators. The change in hydrogel volume that occurs as metal ions are bound allows the detection of contaminants through simple visual inspection. The utility of this bulk property change is demonstrated in the construction of a low-cost device that can report heavy metal contamination with no external power requirements. Most importantly, the generality of the protein modification chemistry allows the immediate generation of new hybrid materials from a wide range of protein sequences.

Protein‐Cross‐Linked Polymeric Materials through Site‐Selective Bioconjugation

Well-defined hybrid materials constructed from proteins and polymers offer significant opportunities for the construction of sensors, actuators, and drug-delivery systems. The enabling concept underlying these materials is the fusion of the specific biological function of proteins with the bulk properties and processability of synthetic polymers. Recent reports have capitalized on this concept to produce materials that undergo a dynamic change based on a number of different inputs. Examples include changes in response to ions, peptides, antigenand carbohydrate-binding interactions, cell surface receptors, and temperature. In a particularly welldefined example, Murphy et al. demonstrated the covalent attachment of polymers to two specific protein sites, effectively generating a biomolecular cross-link. Ligand-induced conformational changes in the protein then afforded a significant change in volume. While these pioneering studies promise great opportunities, they have relied on coupling techniques that are difficult to generalize to all proteins and polymers. Site-selective bioconjugation is at the heart of such materials, and utilizing modern methods for protein activation should provide access to a wider range of materials. In particular, it could be advantageous to attach at two sites, which requires the challenge of modifying proteins selectively at two locations. Ideally, the method for accessing these hybrids would not rely on the primary sequence and would be applicable to a wide array of proteins. We therefore targeted the protein termini because they provide two site-specific, chemically distinct modifications that are independent of any one protein and yet common to all. This attachment strategy also represents an optimal way to relate the folded state of the protein to the properties of the polymer backbone. Herein, we report a method for the construction of protein–polymer hybrid materials utilizing orthogonal chemical reactions to link polymer chains to the two termini of a protein. We also report the initial characterization of a new material, a fluorescent hybrid hydrogel, constructed using this strategy. To develop this methodology, we selected a protein with a unique set of properties to allow facile characterization of the resulting gels. Enhanced green fluorescent protein (eGFP) is a 26.6-kD protein with an internal chromophore that is sensitive to many factors that affect the protein8s structure. When incorporated into a material through termini crosslinking, the fluorescence and size properties of the material should depend on the protein8s folded state. The application of different environmental stimuli was expected to change the protein, and thus the bulk material properties (Figure 1).

Incorporation of Antifreeze Proteins into Polymer Coatings Using Site-Selective Bioconjugation

The diverse functional repertoire of proteins promises to yield new materials with unprecedented capabilities, so long as versatile chemical methods are available to introduce synthetic components at specific sites on biomolecule surfaces. As a demonstration of this potential, we have used site-selective strategies to attach antifreeze proteins found in Arctic fish and insects to polymer chains. This multivalent arrangement increases the thermal hysteresis activity of the proteins and leads to materials that can be cast into thin films. The polymer-protein conjugates retain the ability of the proteins to slow ice growth in subzero water and can inhibit ice formation after attachment to glass surfaces. These inexpensive materials may prove useful as coatings for device components that must function at low temperature without ice buildup. The polymer attachment also allows higher thermal hysteresis values to be achieved while using less protein, thus lowering the cost of these additives for biomedical applications.

Chemical treatment of poly(lactic acid) fibers to enhance the rate of thermal depolymerization.

When heated, poly(lactic acid) (PLA) fibers depolymerize in a controlled manner, making them potentially useful as sacrificial fibers for microchannel fabrication. Catalysts that increase PLA depolymerization rates are explored and methods to incorporate them into commercially available PLA fibers by a solvent mixture impregnating technique are tested. In the present study, the most active catalysts are identified that are capable of lowering the depolymerization temperature of modified PLA fibers by ca. 100 °C as compared to unmodified ones. Lower depolymerization temperatures allow PLA fibers to be removed from a fully cured epoxy thermoset resin without causing significant thermal damage to the epoxy. For 500 μm diameter PLA fibers, the optimized treatment involves soaking the fibers for 24 h in a solvent mixture containing 60% trifluoroethanol (TFE) and 40% H(2)O dispersed with 10 wt % tin(II) oxalate and subsequent air-drying of the fibers. PLA fibers treated with this procedure are completely removed when heated to 180 °C in vacuo for 20 h. The time evolution of catalytic depolymerization of PLA fiber is investigated by gel permeation chromatography (GPC). Channels fabricated by vaporization of sacrificial components (VaSC) are subsequently characterized by scanning electron microscopy (SEM) and X-ray microtomography (Micro CT) to show the presence of residual catalysts.

Directing the immune system with chemical compounds.

Agonists of immune cell receptors direct innate and adaptive immunity. These agonists range in size and complexity from small molecules to large macromolecules. Here, agonists of a class of immune cell receptors known as the Toll-like receptors (TLRs) are highlighted focusing on the distinctive molecular moieties that pertain to receptor binding and activation. How the structure and combined chemical signals translate into a variety of immune responses remain major questions in the field. In this structure-focused review, we outline potential areas where the tools of chemical biology could help decipher the emerging molecular codes that direct immune stimulation.

Covalently Coupled Immunostimulant Heterodimers

We report increased stimulation of dendritic cells via heterodimers of immunostimulants formed at a discrete molecular distance. Many vaccines present spatially organized agonists to immune cell receptors. These receptors cluster suggesting that signaling is increased by spatial organization and receptor proximity, but this has not been directly tested for multiple, unique receptors. In this study we probe the spatial aspect of immune cell activation using heterodimers of two covalently attached immunostimulants.

Stimulation of innate immune cells by light-activated TLR7/8 agonists

The innate immune response is controlled, in part, by the synergistic interaction of multiple Toll-like receptors (TLRs). This multi-receptor cooperation is responsible for the potent activity of many vaccines, but few tools have been developed to understand the spatio-temporal elements of TLR synergies. In this Communication, we present photo-controlled agonists of TLR7/8. By strategically protecting the active agonist moiety based on an agonist-bound crystal structure, TLR activity is suppressed and then regained upon exposure to light. We confirmed NF-κB production upon light exposure in a model macrophage cell line. Primary cell activity was confirmed by examining cytokine and cell surface marker production in bone-marrow-derived dendritic cells. Finally, we used light to activate dendritic cell sub-populations within a larger population.

Covalent modification of cell surfaces with TLR agonists improves & directs immune stimulation.

We present a primary example of a cell surface modified with a synergistic combination of agonists to tune immune stimulation. A model cell line, Lewis Lung Carcinoma, was covalently modified with CpG-oligonucleotides and lipoteichoic acid, both Toll-like receptor (TLR) agonists. The immune-stimulating constructs provided greater stimulation of NF-κB in a model cell line and bone marrow-derived dendritic cells than the components unconjugated in solution.