Michael A. Daniele

Interpenetrating networks based on gelatin methacrylamide and PEG formed using concurrent thiol click chemistries for hydrogel tissue engineering scaffolds.

The integration of biological extracellular matrix (ECM) components and synthetic materials is a promising pathway to fabricate the next generation of hydrogel-based tissue scaffolds that more accurately emulate the microscale heterogeneity of natural ECM. We report the development of a bio/synthetic interpenetrating network (BioSINx), containing gelatin methacrylamide (GelMA) polymerized within a poly(ethylene glycol) (PEG) framework to form a mechanically robust network capable of supporting both internal cell encapsulation and surface cell adherence. The covalently crosslinked PEG network was formed by thiol-yne coupling, while the bioactive GelMA was integrated using a concurrent thiol-ene coupling reaction. The physical properties (i.e. swelling, modulus) of BioSINx were compared to both PEG networks with physically-incorporated gelatin (BioSINP) and homogenous hydrogels. BioSINx displayed superior physical properties and significantly lower gelatin dissolution. These benefits led to enhanced cytocompatibility for both cell adhesion and encapsulation; furthermore, the increased physical strength provided for the generation of a micro-engineered tissue scaffold. Endothelial cells showed extensive cytoplasmic spreading and the formation of cellular adhesion sites when cultured onto BioSINx; moreover, both encapsulated and adherent cells showed sustained viability and proliferation.

Microfluidic strategies for design and assembly of microfibers and nanofibers with tissue engineering and regenerative medicine applications.

Fiber-based materials provide critical capabilities for biomedical applications. Microfluidic fiber fabrication has recently emerged as a very promising route to the synthesis of polymeric fibers at the micro and nanoscale, providing fine control over fiber shape, size, chemical anisotropy, and biological activity. This Progress Report summarizes advanced microfluidic methods for the fabrication of both microscale and nanoscale fibers and illustrates how different methods are enabling new biomedical applications. Microfluidic fabrication methods and resultant materials are explained from the perspective of their microfluidic device principles, including co-flow, cross-flow, and flow-shaping designs. It is then detailed how the microchannel design and flow parameters influence the variety of synthesis chemistries that can be utilized. Finally, the integration of biomaterials and microfluidic strategies is discussed to manufacture unique fiber-based systems, including cell scaffolds, cell encapsulation, and woven tissue matrices.

Bacterial Nanobioreactors–Directing Enzyme Packaging into Bacterial Outer Membrane Vesicles

All bacteria shed outer membrane vesicles (OMVs) loaded with a diverse array of small molecules, proteins, and genetic cargo. In this study we sought to hijack the bacterial cell export pathway to simultaneously produce, package, and release an active enzyme, phosphotriesterase (PTE). To accomplish this goal the SpyCatcher/SpyTag (SC/ST) bioconjugation system was utilized to produce a PTE-SpyCatcher (PTE-SC) fusion protein and a SpyTagged transmembrane porin protein (OmpA-ST), known to be abundant in OMVs. Under a range of physiological conditions the SpyTag and SpyCatcher domains interact with one another and form a covalent isopeptide bond driving packaging of PTE into forming OMVs. The PTE-SC loaded OMVs are characterized for size distribution, number of vesicles produced, cell viability, packaged PTE enzyme kinetics, OMV loading efficiency, and enzyme stability following iterative cycles of freezing and thawing. The PTE-loaded OMVs exhibit native-like enzyme kinetics when assayed with paraoxon as a substrate. PTE is often toxic to expression cultures and has a tendency to lose activity with improper handling. The coexpression of OmpA-ST with PTE-SC, however, greatly improved the overall PTE production levels by mitigating toxicity through exporting of the PTE-SC and greatly enhanced packaged enzyme stability against iterative cycles of freezing and thawing.

Platinum-Paper Micromotors: An Urchin-like Nanohybrid Catalyst for Green Monopropellant Bubble-Thrusters

Platinum nanourchins supported on microfibrilated cellulose films (MFC) were fabricated and evaluated as hydrogen peroxide catalysts for small-scale, autonomous underwater vehicle (AUV) propulsion systems. The catalytic substrate was synthesized through the reduction of chloroplatinic acid to create a thick film of Pt coral-like microstructures coated with Pt urchin-like nanowires that are arrayed in three dimensions on a two-dimensional MFC film. This organic/inorganic nanohybrid displays high catalytic ability (reduced activation energy of 50-63% over conventional materials and 13-19% for similar Pt nanoparticle-based structures) during hydrogen peroxide (H2O2) decomposition as well as sufficient propulsive thrust (>0.5 N) from reagent grade H2O2 (30% w/w) fuel within a small underwater reaction vessel. The results demonstrate that these layered nanohybrid sheets are robust and catalytically effective for green, H2O2-based micro-AUV propulsion where the storage and handling of highly explosive, toxic fuels are prohibitive due to size-requirements, cost limitations, and close person-to-machine contact.

3D hydrodynamic focusing microfluidics for emerging sensing technologies.

While the physics behind laminar flows has been studied for 200 years, understanding of how to use parallel flows to augment the capabilities of microfluidic systems has been a subject of study primarily over the last decade. The use of one flow to focus another within a microfluidic channel has graduated from a two-dimensional to a three-dimensional process and the design principles are only now becoming established. This review explores the underlying principles for hydrodynamic focusing in three dimensions (3D) using miscible fluids and the application of these principles for creation of biosensors, separation of cells and particles for sample manipulation, and fabrication of materials that could be used for biosensors. Where sufficient information is available, the practicality of devices implementing fluid flows directed in 3D is evaluated and the advantages and limitations of 3D hydrodynamic focusing for the particular application are highlighted.

Sweet Substrate: A Polysaccharide Nanocomposite for Conformal Electronic Decals

A conformal electronic decal based on a polysaccharide circuit board (PCB) is fabricated and characterized. The PCBs are laminates composed of bioderived sugars - nanocellulose and pullulan. The PCB and decal transfer are a bioactive material system for supporting electronic devices capable of conforming to bio-logical surfaces.

Microfluidic fabrication of multiaxial microvessels via hydrodynamic shaping

A microfluidic fiber fabrication device was developed to prepare multiaxial microvessels with defined architecture and material constituency. Hydrodynamic focusing using passive wall structures directed biologically relevant macromer solutions into coaxial flow patterns, which were subsequently solidified via photopolymerization. Solid, coaxial, and triaxial microfibers as well as microtubes were generated from the multiaxial flows composed of both synthetic macromers and biomacromolecules.

Nanoparticles and direct immunosuppression

Targeting the immune system with nanomaterials is an intensely active area of research. Specifically, the capability to induce immunosuppression is a promising complement for drug delivery and regenerative medicine therapies. Many novel strategies for immunosuppression rely on nanoparticles as delivery vehicles for small-molecule immunosuppressive compounds. As a consequence, efforts in understanding the mechanisms in which nanoparticles directly interact with the immune system have been overshadowed. The immunological activity of nanoparticles is dependent on the physiochemical properties of the nanoparticles and its subsequent cellular internalization. As the underlying factors for these reactions are elucidated, more nanoparticles may be engineered and evaluated for inducing immunosuppression and complementing immunosuppressive drugs. This review will briefly summarize the state-of-the-art and developments in understanding how nanoparticles induce immunosuppressive responses, compare the inherent properties of nanomaterials which induce these immunological reactions, and comment on the potential for using nanomaterials to modulate and control the immune system.

Affinity purification of bacterial outer membrane vesicles (OMVs) utilizing a His-tag mutant

To facilitate the rapid purification of bacterial outer membrane vesicles (OMVs), we developed two plasmid constructs that utilize a truncated, transmembrane protein to present an exterior histidine repeat sequence. We chose OmpA, a highly abundant porin protein, as the protein scaffold and utilized the lac promoter to allow for inducible control of the epitope-presenting construct. OMVs containing mutant OmpA-His6 were purified directly from Escherichia coli culture media on an immobilized metal affinity chromatography (IMAC) Ni-NTA resin. This enabling technology can be combined with other molecular tools directed at OMV packaging to facilitate the separation of modified/cargo-loaded OMV from their wt counterparts. In addition to numerous applications in the pharmaceutical and environmental remediation industries, this technology can be utilized to enhance basic research capabilities in the area of elucidating endogenous OMV function.

Electrolyte-Sensing Transistor Decals Enabled by Ultrathin Microbial Nanocellulose.

We report an ultra-thin electronic decal that can simultaneously collect, transmit and interrogate a bio-fluid. The described technology effectively integrates a thin-film organic electrochemical transistor (sensing component) with an ultrathin microbial nanocellulose wicking membrane (sample handling component). As far as we are aware, OECTs have not been integrated in thin, permeable membrane substrates for epidermal electronics. The design of the biocompatible decal allows for the physical isolation of the electronics from the human body while enabling efficient bio-fluid delivery to the transistor via vertical wicking. High currents and ON-OFF ratios were achieved, with sensitivity as low as 1 mg·L−1.