Research from the College of Engineering and Applied Science that uses acoustic radiation forces to shape the internal structure of suspended droplets has been published in .

The paper, titled “Acoustically Manipulating Internal Structure of Disk-in-Sphere Endoskeletal Droplets,” is a collaborative work completed by researchers in the Paul M. Rady Department of Mechanical Engineering, the Biomedical Engineering Program and the Materials Science and Engineering Program.

Their work could boost health and drug advancements by giving researchers a better understanding of primary and secondary radiation forces in multiphase colloidal systems – such as emulsions, foams, membranes and gels. Those forces are currently being studied for cell separation for disease diagnoses and drug delivery systems for cancer treatments.

First author Gazendra Shakya, a PhD graduate who worked in the labs of Professors Mark Borden and Xiaoyun Ding, shared how the group came to the more thorough understanding of radiation forces and how the research could benefit future studies.

Can you explain what endoskeletal droplets are and how you used them in this research?

Endoskeletal droplets are tiny liquid droplets that are suspended in an aqueous or water medium. The ones we used are 10 micrometers in diameter, a similar size to biological cells. For comparison, a typical human hair is 100 micrometers in diameter.

The interesting thing about these liquid droplets are that they have a solid skeleton embedded inside them, hence the name endoskeletal droplets. There are different types of endoskeletal droplets but the ones we study in this project have a disk-shaped solid inside the liquid droplet.

What are the real-world impacts from this research? How will the collaborative work benefit society?

The radiation forces are very important in any colloidal system that deals with acoustic waves. For example, these forces are being studied currently for cell separation for disease diagnosis or optimizing drug delivery systems for cancer treatments. But these forces and their behaviors in multiphase colloidal systems have not yet been fully understood. With this current paper, we have gotten a better understanding of the primary and secondary radiation forces.

Moreover, this study demonstrated the possibility of manipulating internal structures of droplets and cells. This can pave the way to manipulating internal organelles in a cell, which is very challenging for current techniques, but could be helpful to understand the communication and function of intracellular organelles.   

Is exploring the internal structure of a droplet this small challenging?

It is very challenging because the internal structure adds a lot of complexity. With that added internal phase, which has different physical and chemical properties, it is very hard to properly explore the behavior of the internal structure.

I think the geometry also played an important part. We have discovered the ideal droplet at the internal phase is not spherical. Instead, it is cylindrical like a flattened disk and free to rotate or move around inside the droplet. This was a major advantage for us because we could now visualize how different forces were affecting the internal structure as it moved and rotated in response. 

How did you use radiation forces to manipulate the structure within the droplet?

Currently, we are using acoustic frequencies which are in the MHz range and hence inaudible. Any particle in an acoustic field experiences a force called the acoustic radiation force. There are two types of radiation forces: the primary radiation force and the secondary radiation force. These two forces have different implications on suspended particles. Since we have particles with two different acoustic properties in a single droplet – liquid and solid properties – they both are affected in different ways.

The liquid droplet is pushed to a specific direction by the primary radiation force and the attractive force from the secondary radiation creates clusters. As for the solid disks inside the droplets, the primary radiation force pushes the disks to the top of the cluster and forces them to be parallel to the substrate, whereas the secondary radiation force pushes it to the edges of the cluster and makes them perpendicular. By manipulating the magnitude of these forces, which can be done by either changing the frequency or the cluster size, we could manipulate the internal structure of the disks.

The other authors on this paper include previous postdoctoral scholar Tao Yang, postdoctoral associate Yu Gao, PhD researcher Apresio K. Fajrial, and professors Baowen Li,Massimo Ruzzene, Mark A. Borden and Xiaoyun Ding.

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Professor Xiaoyun Ding recently earned a $1.8 million grant from the National Institutes of Health (National Institute of General Medical Sciences) to help improve cancer-fighting tools and cut patient costs, exploring ways to streamline delivery of lifesaving treatments into immune cells.

“This funding has the perfect timing to help expand our research,” said Ding.

With current immunotherapy treatments, when disease-fighting white blood cells called T-cells tire and can no longer seek out and destroy cancer cells, modified forms of those cells can be introduced to help the body treat itself. But it’s an imprecise and costly procedure.

Started July 1, the five-year award supports the Ding Lab’s efforts to learn how to efficiently and precisely disrupt cell membranes so that treatment can be introduced to improve cell function, as well as how to repair the membranes faster. In particular, the researchers will attempt to standardize a process that can be automated for millions of cells in a second.

In previous research, Ding’s lab found that they could use microfluidic technology to disrupt the membrane, deliver molecules into the cell and control the dose. They also showed it’s possible to deliver DNA molecules into the cell, a process not achievable in other ways through membrane disruption.

The results are not yet precise enough for commercial use — the mechanisms of cell membrane disruption and cell response to such disruption are not entirely clear — so the team will investigate the process behind controlling doses. They’ll learn whether the procedure becomes more efficient when they monitor the size and number of “pores” at the cell membrane of a cell, for instance.

Conducting interdisciplinary research at the frontiers of biology, medicine, physics and micro/nano engineering, Ding’s group will work to develop technological devices that can disrupt cell membranes to deliver controllable doses to cells through a certain number of pores—treating mainly targeted cell areas and killing fewer healthy cells.

The team will also attempt to control the cell membrane disruption and how quickly it recovers from rupture after treatment is delivered. Because an individual cell will die if its membrane is disrupted too much, the researchers plan to learn what level of disruption they can achieve safely and when they should stop. They’d also like to glean ways to help cells recover to create a stronger rupture that can be repaired later, said Ding.

His lab achieved promising earlier findings in their investigation of cell behavior, including a 2020 discovery that allowed them to diagnose sickle cell disease with greater sensitivity and precision in less than one minute.

Speedy processing could also improve treatment costs for cancer and other diseases.

The principal investigator on this NIH grant, Ding started his academic studies in microelectronic and mechanical engineering.

Focusing on technology development, he looked for applications for the work. Then he realized that was not the best approach, recognizing that drug delivery is a broad category and depends on each disease and its applications. So as a postdoc pursuing chemical and biomedical engineering learning in immunotherapy, he absorbed medical problems first, then developed strategies to deliver treatment for them.

“I want to deliver treatment at high efficiency and with low costs. That’s the goal. I’m working in problem-driven research,” Ding said.

Ding’s lab also does research on Lab-on-a-Chip technologies for micro/nano manipulation, cell mechanics and fast diagnosis.

The goal of the is to increase the efficiency of National Institute of General Medical Sciences funding by providing investigators with greater stability and flexibility, thereby enhancing scientific productivity and the chances for important breakthroughs. This grant is believed to be the first MIRA granted to anyone in the Paul M. Rady Department of Mechanical Engineering.

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Diseases of the blood, like sickle cell disease, have traditionally taken a full day, tedious lab work and expensive equipment to diagnose, but researchers at the University of Colorado Boulder and the University of Colorado Anschutz have developed a way to diagnose these conditions with greater sensitivity and precision in only one minute. Their technology is smaller than a quarter and requires only a small droplet of blood to assess protein interactions, dysfunction or mutations.

Assistant Professor Xiaoyun Ding of the Paul M. Rady Department of Mechanical Engineering and Associate Professor Michael Stowell of the Department of Molecular, Cellular and Developmental Biology at CU Boulder, along with of the Departments of Biochemistry and Molecular Genetics and Medicine, Division of Hematology at CU Anschutz, co-authored a   

This project began when Ding realized that a technology developed in his lab could increase the speed of cell protein analysis performed by Stowell and his research group. As Stowell and D’Alessandro got involved, new applications emerged, including disease diagnosis.

“In Africa, sickle cell disease is the cause of death in 5% of children under 5 years old for lack of early diagnosis,” said D’Alessandro. “This common, life‐threatening genetic disorder is most prevalent in poor regions of the world where newborn screening and diagnosis are rare.”

Sickle cell disease affects hemoglobin, the molecule in red blood cells that delivers oxygen to cells throughout the body. In some areas of the world where malaria is endemic, variants of hemoglobin have evolved that can cause red blood cells to assume a crescent, or sickle, shape.

“Almost all life activities involve proteins,” said Ding. “We thought if we could measure the protein thermal stability change, we could detect these diseases that affect protein stability.”

Proteins have a specific solubility at a specific temperature. When one bonds to another or when the protein is mutated, the solubility changes. By measuring solubility at different temperatures, researchers can tell whether the protein has been mutating.

Before recent developments, Stowell and his group, including researcher Kerri Ball, used Thermal Shift Assays to assess protein stability under varying conditions. Now, with the new technology, an Acousto Thermal Shift Assay, they can do the same but faster and with greater sensitivity.

The ATSA utilizes high-amplitude sound waves, or ultrasound, to heat a protein sample while concentrating the proteins that don’t dissolve. Device components include a channel where the sample is deposited and two electrodes on each side to generate the wave that applies acoustic heating and concentration.

For both a traditional TSA and ATSA, samples are collected and heated from 40 to 70 degrees Celsius. The traditional TSA measures how much of the protein has dissolved at set points over the course of the temperature increase, while the ATSA measures data continuously, recording how much of the protein has dissolved at every fraction of change in degrees Celsius.

“Our method is seven to 34 times more sensitive,” said Ding. “The ATSA can distinguish the sickle cell protein from normal protein, while the traditional TSA method cannot.”

Another benefit of the ATSA is cost reduction in terms of human labor and equipment.

“The traditional methods for thermal profiling require specialized equipment such as calorimeters, polymerase chain reaction machines, and plate readers that require at least some technical expertise to operate,” said Ball. “These instruments are also not very portable, requiring samples to be transported to the instruments for analysis.”

Ball said the ATSA requires only a power source, a microscope and a camera as simple as the one on your smart phone. Because the protein is concentrated, there is also no need to apply a florescent dye as is sometimes required to highlight protein changes in a traditional TSA.

Ding said it is thanks to his collaborators, experts in biochemistry and hematology, that they now know the full impact of the technology.

“This technique is an exciting development, because it represents a new and promising point-of-care platform for a rapid and highly sensitive diagnostic tool of sickle cell disease, and maybe for other hemoglobinopathies such as beta thalassemia too,” said D’Alessandro.

By working with the engineering team, Ball said she and her research group created tools to accomplish their goals in new and better ways.

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