Two ORNL facilities used to develop synthetic cell membranes.
If imitation is the sincerest form of flattery, then Mother Nature may be blushing at researchers' efforts to emulate the molecular activity that occurs at cell membranes—the boundary between living cells and their environment. Utilizing the combined capabilities of the Spallation Neutron Source and the adjacent Center for Nanophase Materials Sciences, a team of scientists is building bio-inspired, biocompatible synthetic cell membranes to help them understand a range of interactions between synthetic materials and biomolecules.
"Our inspiration comes from Mother Nature," says researcher Brad Lokitz. "In the body and in nature, a number of biological processes occur at interfaces and membranes. We are trying to mimic nature in a very basic way to gain some insight into these processes."
In addition to Lokitz, the research effort includes John Ankner, Jamie Messman and Dean Myles of ORNL; Mike Kilbey and Jimmy Mays, who have joint appointments with ORNL and the University of Tennessee; and Juan Pablo Hinestrosa of Clemson University.
The team's bio-inspired membrane starts with a silicon base, to which polymers are attached more or less evenly across the silicon's surface. The polymer strands provide a framework, or scaffold, for the biomolecules and synthetic cell membrane that are added later.
"The design of the substrate is somewhat similar to a hairbrush," says Lokitz. "If you take a hairbrush and turn it over, the base of the brush would be the silicon substrate, and the bristles that extend out would be the polymer."
While the researchers needed several attempts before identifying a process that resulted in a polymer framework with a uniform thickness evenly distributed across the surface, the results were worth the effort. Lokitz says that in order to use neutrons to study the framework, the process had to be very uniform on the molecular level. "The rougher the surface, the less information can be extracted, so the surfaces must be as molecularly smooth as possible. Considerable time was required before we could consistently produce samples that were sufficiently smooth to take to the SNS and obtain good results."
Having recently perfected the process of creating the substrate and attaching the biomolecules, the team has begun experimenting with attaching a synthetic cell membrane to the substrate. "The membrane was created by team members working at the nanoscience center," says project leader John Ankner. "We developed a synthetic membrane composed of biocompatible synthetic polymers that has some of the physical and chemical properties of a cell membrane." Because the substrate is fairly soft, the attached synthetic cell membrane will be more flexible and more elastic than if attached to a hard surface. Also, by attaching biomolecules to the substrate before applying the membrane, Ankner's team can populate the membrane with structures, such as proteins, that are found in real cell membranes.
Proteins are a particular focus of the team's research because they play key roles in a variety of cellular functions, such as immune response, the operation of ion channels and reaction to toxins. By embedding biomolecules within the membrane, the researchers hope to be able to study the structure-function relationships of the various biomolecules—proteins, peptides, cholesterol—that form the cell's environment.
The team is using the SNS's Liquids Reflectometer to study each phase of this bio-inspired structure, including the polymer scaffold, the attached biomolecules and the synthetic cell membrane. One feature of the reflectometer is its ability to investigate the boundaries between hard and soft matter, an ideal capability for examining the bio-inspired surfaces with which Lokitz and his colleagues are working.
Lokitz notes that having the world's most powerful pulsed neutron source next door to one of America's most modern nanocenters provides a rare opportunity to perform synergistic nanoscience research at co-located, state-of-the-art facilities. "We have the ability to create our samples at the nanocenter, walk next door, and test them at a world-class facility. As we run our tests, the feedback is immediate. If we see that we need to do something differently, we can walk down the hall to the nanocenter, tweak our procedure, and then go back to the SNS and continue the testing."
Once a satisfactory scaffold is in place, researchers "functionalize" the process by starting a chemical reaction that allows the biomolecules to bind to the polymers. Researchers then examine the scaffold to understand three things: how the attached biomolecules affect the structure and organization of the polymer scaffold, how being attached to the scaffold affects the stability and structure of the biomolecules and whether the biomolecules can still perform their biological functions when attached to the scaffold.
The Liquids Reflectometer can characterize the thickness, density and orientation of polymers. The instrument is very sensitive to small changes in density or thickness, thus providing researchers with an opportunity to study minute variations in the scaffold, the attached biomolecules or the synthetic cell membrane. "The researcher can observe the surface with just the polymer attached and get an idea of the layer thickness and composition," Lokitz says. He adds that when the team functionalizes the layer by attaching the biomolecules, the instrument is sensitive enough to notice even minute changes.
Anker says the team has reached the point at which they can attempt to attach a synthetic cell membrane to the polymer scaffold. "We are taking the first steps in this process. The membrane analogues represent the holy grail in the neutron reflectivity business."
Ankner concedes that the process of attaching the membrane has proven to be tricky. "We're taking a small three-dimensional cell membrane and attempting to spread it out on a flat surface the size of a hockey puck. This goal has been pursued unsuccessfully in a variety of ways. This is our attempt. Our efforts are promising, so we are pushing ahead."
The near-term goal for the team is to assemble a synthetic cell membrane around various biomolecules that have already been attached to the substrate—thus avoiding the chemically problematic issue of inserting biomolecules into the membrane after it has been created.
"In a real cell," Ankner explains, "when proteins or peptides are created, the cellular components that create them inject them directly into the cell membrane one amino acid at a time. The proteins fold and form under the influence of this unique environment."
This process can't easily be duplicated in a laboratory, so researchers typically are forced to find chemical approaches to embedding biomolecules into synthetic membranes—with less than satisfactory results.
"If we can attach them to the substrate and then assemble the membrane around them," Ankner says, "we avoid that whole problem. That's our ambition."
Understanding how cell membranes and other functional areas of cells work may enable researchers to develop bioinspired systems that apply biological processes to specific tasks in materials and chemical sciences. Lokitz notes that "these functional areas are able to sense and respond to external stimuli; capture, store and convert energy; carry out chemical reactions; and transport a wide range of chemicals."
The ability to engineer and control these cellular features would open up a wide range of possibilities for developing next-generation materials, devices and processes, such as therapeutic agents, drug delivery systems and new methods of diagnosing disease.
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