August 19, 2010
DURHAM, N.C. – The same process that crumbles kidney stones and pits ship propeller blades may hold the key to successfully injecting drugs directly into individual cells without harming them.
A new technique that harnesses the power of mighty microscopic bubbles, developed by Duke University engineers, can open for a blink of the eye a few hundred nanometer-sized entry into individual cells.
Scientists have long known that miniscule bubbles in a liquid possess a great deal of useful energy, and when they collapse – a process known as inertial cavitation – for the briefest of moments, the potential energy stored in the bubble can be focused and released in a number of ways, such as shock waves or heat (with temperatures equal to that of the surface of a star produced inside a collapsing bubble) or tiny high-speed spurts of liquid that can either destroy cells by bursting them open or change acutely membrane permeability without killing the cells. The inception and dynamics of cavitation bubbles induced by ultrasound, however, are random and difficult to control.
The Duke engineers have now developed a method utilizing focused laser beams to generate microbubbles with precise control of their inception time, size, and location. They have demonstrated in a new set of experiments that collapsing two of these microscopic bubbles in tandem, one right after the other, creates a “microjet” capable of tearing a tiny pore on the surface of a cell placed nearby. This pore formation is transient to allow drugs to enter the cell while the cell membrane will seal itself fast enough to keep the contents of the cell spilling out.
Pei Zhong, associate professor of mechanical engineering and materials science at Duke’s Pratt School of Engineering, said this method could become a valuable technique to not only safely introduce drugs into individual cells, but also genetic materials, such as genes and siRNAs, into cells to help treat patients with cancers, heart disease, and hereditary disorders.
“The controlled creation of a second bubble in close proximity as the first bubble collapses produces a unique bubble-bubble interaction that forms a potent microjet of liquid, which creates a pore on the cell membrane without destroying the cell,” Zhong, who along with Pratt research scientist Georgii Sankin, published the results of their experiments in the journal Physical Review Letters this month. Their paper was selected as one of the highlighted articles in the Editors’ Suggestion. “This microjet is significantly forceful with more focused energy than its counterpart created by a single bubble near a boundary.”
In addition to developing this novel one-two bubble punch approach, the Duke researchers also for the first time captured the entire process of bubble-cell interaction and subsequent drug uptake with high-speed cameras.
For their experiments, the scientists grew breast cancer cells in a microfluidic channel, in which the liquid solution was colored by blue dye and then fired two precisely aligned laser beams into the liquid next to the cell. The microjet is generated by the mutual interaction between the collapsing first bubble and the expanding second bubble. Zhong used a child swinging as an analogy. When someone pushes an already swinging child ready to come down, it gives that child an extra boost to swing higher and faster. That is in essence what the expansion of the second bubble causes – transfer of energy and momentum to the first bubble ready to collapse.
“When we looked at the cells immediately after the laser exposure, we saw that the blue dye had entered the cell through a pinpoint rupture at the microjet impact site and gradually diffused into the cell, but the rest of the cell membrane remained intact,” Zhong said.
The size of the pore varies from a few hundred nanometers to a few microns, sufficient to allow drugs, DNA or other macromolecules to be infused into cells without damaging them, Zhong said. He envisions a time when cells, such as immune or stem cells, could be removed from a patient, new genetic materials inserted, and then reinjected back to the patient, such as in ex vivo therapies.
In an editorial accompanying this issue of the journal, Claus-Dieter Ohl, from the Nanyang Technical University and the Institute of High Performance Computing, both in Singapore, wrote that the Duke experiments were “. . . to my knowledge the most complete experiment that characterizes the flow, measures the uptake on single cells, and obtains a one-to-one correlation between the observed jet and the measured membrane pore.
“The Duke team’s method qualifies as a new technique for drug delivery to single cells, and if engineered further may become a tool for biologists for particularly delicate cell lines and perhaps stem cells,” Ohl concluded.
Zhong, who also researches ways to improve minimally invasive or non-invasive treatments of various diseases, such as cancers and urinary tract stones, also said that their new methodology and experimental technique can provide deeper insights into the mechanisms of cavitation-generated bioeffects at the cellular level, which is key for ensuring the success of therapeutic ultrasound applications in clinic medicine.
“Understanding this process should help us design better medical devices,” he said.
The research was support by the National Institutes of Health. Duke graduate student Fang Yuan was also a member of the team.