The tiny bubbles that carry signals between cells throughout the body were discovered decades ago, but precisely what they contain and how their cargo affects recipient cells is still largely a mystery.
Though research to date shows that these bubbles, called extracellular vesicles and particles, influence human health and disease, they come in so many different sizes and contain such a huge range of contents that it's difficult to pinpoint their specific functions. Found circulating in biological fluids and embedded in tissues, they are being explored for applications ranging from early disease detection to drug delivery.
In a study published recently in Nature Methods, scientists at The Ohio State University report on a new approach to immobilize extracellular vesicles in a way that mimics their interactions with tissues, a tricky environment compared to bodily fluids because these particles have a specific characteristic: They're sticking to a surface.
"The extracellular vesicles present in tissues are very poorly understood in terms of how the particles actually interact with cells in our body," said senior author Eduardo Reátegui, associate professor of chemical and biomolecular engineering at Ohio State.
The new technique enables label-free immobilization of extracellular vesicles and particles without damaging them, allowing researchers to study them individually or in clumps and observe how they interact with cells.
"What we want to do is not only understand what these vesicles contain, but also identify their tissue of origin, and how they interact with cells. One way to do that is to analyze them without destroying them," said Reátegui, also a member of the Cancer Biology Program in The Ohio State University Comprehensive Cancer Center.
Researchers began by coating a glass surface with a chemical layer, and then used UV light to "etch" a micropattern on that layer, creating tiny spaces with an attractive electrostatic charge to which the proteins on the extracellular vesicles' outer layers would stick. Computer simulations confirmed that electrostatic attractions dominate the interactions between the tiny spaces and the particles.
The researchers found that every extracellular vesicle type used in experiments adhered to the surface only where the light-induced micropattern was produced, without seeping outside those micropatterns.
The team calls the technology Light-Induced Extracellular Vesicle and Particle Adsorption, or LEVA.
This analytical advance opens the door to studying these particles and their complex interactions with cells at a whole new level, such as exploring their contents for disease biomarker discovery or loading them with therapeutic molecules and watching cells respond, to name just a few possibilities.
Previously, Reátegui and colleagues developed a method of using antibodies to immobilize these extracellular vesicles and particles for analysis, which allowed the team to identify molecules inside specific types of particles that were biomarkers for brain cancer or indicators of immunotherapy response. But the method had a limitation: Only EVs with a specific molecule on their surface that would be recognized by the antibody could be isolated for analysis.
With this new technique focused on surface-based extracellular vesicles and particles (EVPs), the UV light degrades regions of a coating to coax particle adsorption to the surface that is dictated by electrostatic interactions rather than any biological signature.
"We started to think about how we can remove that bias in terms of pre-selecting this extracellular vesicle population with antibodies," he said. "And now we can basically immobilize all of them, and have the ability to interrogate them with molecular probes or even cells."
In one application of the study, researchers showed that the new technique can be used to study early stages of inflammation by mimicking the response of immune cells to pathogens such as bacteria or fungi - but instead of using bacteria such as E. coli, they used the extracellular vesicles produced by the bacteria. They found that the EVs emitted by E. coli induced what is known as a neutrophil swarming - the coordinated recruitment and migration of neutrophils toward sites of infection, along the micropatterns where the bacterial EVs were bound.
"This showed that, first, we can generate matrix-bound EVPs in different contexts for easy analysis, and second, this approach allows exploration of EVP interactions in tissue," Reátegui said.
This work was supported by the Ohio State Center for Cancer Engineering-Curing Cancer Through Research in Engineering Sciences; the National Institutes of Health; the Burroughs Wellcome Fund; and Ohio State postdoctoral scholars programs.
Co-authors include Colin Hisey, Xilal Rima, Jacob Doon-Ralls, Chiranth Nagaraj, Sophia Mayone, Kim Truc Nguyen, Sydney Wiggins, Kalpana Deepa Priya Dorayappan, Xin Huang, Karuppaiyah Selvendiran, David Wood, Chunyu Hu, Divya Patel, Andre Palmer and Derek Hansford of Ohio State; Mangesh Hade and Setty Magaña of Nationwide Children's Hospital; and James Higginbotham, Oleg Tutanov, Jeffrey Franklin and Robert Coffey of Vanderbilt University.
