BIOMOLECULAR ENGINEERING OF MATERIALS

Nature offers an amazing ability to enlist renewable materials to create complex structures that perform precise functions. Examples include, DNA that can store information in a retrievable form, spider silk that is strong yet lightweight, and the water-resistant adhesive that allows mussels to attach to submerged surfaces. Not only do they offer exquisite functional properties, but these natural materials are constructed under mild, physiological conditions and are biodegradable. We are coupling Nature-inspired methods with standard fabrication methods to create next-generation materials that offer superior performance and safety, while avoiding adverse environmental impacts.

Biofabrication: The Use of Biological or Bio-Inspired Materials and Processes

The successful integration of biological components into microfabricated devices will provide "smarter" biosensors for diagnosing disease, detecting contaminants and discovering drugs. Integrating these biological components however will require hybrid fabrication methods that can exert spatial and temporal control during assembly while accommodating the labile nature of the bio-components (i.e., the nucleic acids, proteins, vesicles, or cells). In our work, we are biofabricating by enlisting the unique properties of biological polymers and enzymes.

One example that illustrates our biofabrication approach is the use of the enzyme tyrosinase to conjugate proteins onto the stimuli-responsive aminopolysaccharide chitosan. Responsive protein-chitosan conjugates can be guided to assemble onto micropatterned electrodes in response to localized electrical signals. The fluorescence photomicrograph demonstrates that deposition allows protein assembly to be performed with high spatial selectivity while the deposition conditions are sufficiently mild to preserve the protein's structure. This work is being extended in two ways -- to create fusion tags that facilitate tyrosinase-mediated conjugation, and deposition of chitosan at specific electrode addresses within a fully closed and packaged microfluidic device.

Biomimetic Soft Tissue Adhesive

Biology employs crosslinking reactions to seal wounds -- insects initiate crosslinking using tyrosinase enzymes while humans employ transglutaminases to crosslink blood proteins. For instance, the scheme at the left shows the final stages of the blood coagulation cascade in which Factor XIIIa mediates the crosslinking of fibrin. Both tyrosinase and fibrin based crosslinking routes have been investigated for soft tissue adhesives but they have limitations of either biocompatibility or mechanical strength.

The scheme at the right shows our biomimetic alternative to fibrin sealants. As illustrated, this adhesive is based on the enzymatic crosslinking of a structural protein (i.e., gelatin) to create a strong yet compliant adhesive bond. This biomimetic adhesive is being studied in collaboration with researchers from the United States Food and Drug Administration (FDA) and retinal surgeons from the University of Maryland medical school.

Bio-inspired Vesicle Restraint and Mobilization

Biology routinely uses vesicles for cell-cell communication. As illustrated in the scheme at the upper left, signaling between pre- and post-synaptic cells in the nervous system is mediated by the release of neurotransmitter molecules from vesicles located at the active zone of the presynaptic cell. Fusion between the vesicle and cytoplasmic membranes during exocytosis results in the release of neurotransmitter from the presynaptic cell into the synaptic cleft, while the postsynaptic cell recognizes this signal by the selective binding of the transmitter to its receptor. In addition to the free vesicles located in the active zone, the presynaptic cell also possesses a reserve pool of transmitter-containing vesicles that are tethered to (i.e., "restrained" by) the actin cytoskeleton. The scheme at the upper right shows these restrained vesicles are tethered to actin by the protein synapsin. These restrained vesicles are mobilized (i.e., "recruited") in response to cellular needs by the enzyme-catalyzed phosphorylation of synapsin. Thus, dephosphorylation and phosphorylation of synapsin provide the localized cues to restrain or mobilize these reserve vesicles.

The lower scheme shows our bio-inspired approach for vesicle restraint and mobilization. We use the aminopolysaccharide chitosan as our scaffold in place of the actin cytoskeleton and the vesicles are tethered to the scaffold using n -dodecyl hydrophobes grafted onto the chitosan backbone. These grafted hydrophobes can insert into the vesicle's bilayer to mediate vesicle-chitosan tethering in place of synapsin-mediated vesicle-actin tethering. As suggested, each hydrophobically-modified chitosan (hm-chitosan) chain has multiple hydrophobes on its backbone, and can "inter-connect" multiple vesicles into a three-dimensional vesicle network. This initial vesicle network, which is formed under acidic conditions, offers some of the features of a reserve vesicle pool as the vesicles are tethered to a scaffold. However, this acidic vesicle network is not sufficiently robust to restrain the vesicles.

To restrain the vesicles, we enlist the pH-responsive network-forming properties of chitosan. At low pH, the primary amines of the glucosamine residues are protonated conferring a positive charge to chitosan. Under these acidic conditions (pH less than about 6), chitosan is a water-soluble. With increasing pH, the primary amines become progressively less charged, inter-polymer repulsions are reduced, and chitosan can form inter-polymer associations that lead to the formation of a robust, three-dimensional network. Importantly, chitosan's sol-gel transition occurs at pHs between 6 and 6.5. Thus, a pH increase to near-neutral, or basic conditions serves as the cue for vesicle restraint. As illustrated, vesicles can be mobilized by the enzymatic hydrolysis of the scaffold using chitosanase.

Phenol Reaction Cascades to Confer Mechanical Function

Nature routinely uses phenol reaction cascades for such diverse processes as the curing of the mussel's adhesive protein, the hardening of insect shells (i.e., quinone tanning) and the browning of foods. We are mimicking these processes by initiating the reaction cascade electrochemically (and not enzymatically as nature does). Specifically, we electrochemically initiate the cascade at micropatterned anodes. This allows the reaction cascade to be controlled spatially and temporally. The following figure illustrates that a pattern from a microfabricated silicon wafer can be electrochemically transferred to a flexible polysaccharide film. This pattern-transfer operation allows tailoring of the film's optical, mechanical, and conducting properties.

Utilization of Renewable Resources for Polymeric Materials

Over the last 75 years synthetic polymers have been created for a stunning array of applications. However, there are down-sides. Synthetic polymers are typically; derived from a non-renewable resource (i.e., petroleum), generated using chemistries that are not entirely safe or environmentally-friendly, and incorporated into products that are often poorly-degraded in the environment. At the same time, renewable sources of polymers have become over-abundant and are being "managed" as wastes. Maryland 's example is the food processing wastes generated from its crab-packing industry. We are the academic lead in an industry-university-government partnership that resulted in the commercial "extraction" of the chitosan polysaccharide from these wastes (this partnership was awarded the state's "Outstanding Engineering Achievement"). Currently, we are examining how chitosan can be enzymatically derivatized to create products with commercially-important functional properties.