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Transmission electron micrograph showing a prostate cancer cell immediately after exposure to ultrasound. The image has been color enhanced to show the spot where the cell membrane has been removed. Image courtesy of Robyn Schlicher, Robert Apkarian, and Mark Baran.

Ultrasound-Mediated Therapy - Are We There Yet?: March 27, 2007

Everyone is familiar with the sonogram pictures that pregnant women carry with them; now scientists are developing innovative ways to adapt noninvasive ultrasound technology to deliver drugs and genes to specific organs and tissues in the body. Some traditional methods of drug delivery are often not suitable for large molecules such as proteins and DNA, underscoring the need for improved drug delivery strategies. Ultrasound holds considerable clinical promise in this realm.

Understanding the Mechanism

Scientists have known for some time now that exposure of cells to ultrasound at higher intensity levels and different frequencies than those used for diagnostic purposes can drive molecules into living cells, thereby increasing the effects of drugs and the expression of genes. The mechanism by which ultrasound increases permeability of the protective outer membrane of cells, however, was uncertain until Dr. Mark Prausnitz, Professor of Chemical and Biomedical Engineering at Georgia Institute of Technology, and his team directed their research toward this phenomenon. With the aid of various microscopy techniques, the researchers showed that ultrasound can cause bubbles to oscillate and violently collapse – a process known as cavitation – in a cell suspension, producing a shock wave which, in turn, causes fluid to move and open up holes in the cell membranes. The holes allow macromolecules like proteins to enter the cell before the holes close in a matter of minutes by an internal cellular patching process.

Prausnitz and his team have seen that the extent to which the cellular membrane is disrupted can vary depending on the ultrasound parameters used. Through study of the physical, acoustic, and biological conditions in which molecules are driven into cells, they found that the impact of ultrasound can also kill cells. “The cell has its plasma membrane to regulate what is inside the cell, so when you disrupt the membrane all sorts of molecules can go inside the cell and that regulation is disrupted. The cell can be highly stressed by that, and therefore actively tries to repair the membrane. If the membrane breach is not that big, the membrane is able to reseal itself. But if it’s too big, we see evidence of various biochemical pathways of programmed cell death that kick into gear due to that stress,” says Prausnitz.

A major challenge that researchers face is determining the optimal ultrasound qualities that will open holes in cell membranes without killing the cells, so that drug delivery can be maximized while cell viability is maintained. The Prausnitz team is focusing its research on determining the mechanisms by which cellular bioeffects of ultrasound occur. “If we can learn more about how cells respond to plasma membrane disruptions, we will have a valuable tool in designing a controllable therapeutic ultrasound system,” says Joshua Hutcheson, a graduate student in Prausnitz’s lab.

Clinical Possibilities

Prausnitz perceives ultrasound-mediated drug delivery as having a potentially broad range of applications within therapeutic medicine, as this process is independent of both cell and drug type. He also envisions other applications in which the goal might not necessarily be to get molecules into cells, but rather to penetrate deeper into a multicellular tissue to sensitize it. “Ultrasound might be able to open up the permeability of the tissue as a whole and in that way drive drugs more generally into that tissue,” Prausnitz explains. The team is studying the effect of ultrasound on the uptake and viability of cells in explanted carotid arteries from pigs as a three-dimensional tissue model.

Widespread research activity is also under way to understand the bioeffects of ultrasound in the context of gene therapy. “It appears that ultrasound is doing other things to the cell [in addition to enhancing DNA delivery] that might further enhance the transfection efficiency of a cell,” says Prausnitz.

Brightfield (above) and confocal/fluorescence (below) microscopy of prostate cancer cells before (left), immediately after (center), and long after (right) expsoure to ultrasound. Holes in the cell membranes are highlighted with an arrow. Image courtesy of Robyn Schlicher.

Challenges to Overcome

Previous applications of ultrasound as a diagnostic tool and as a direct interventional approach for the pulverization of kidney stones and tumor ablation, among other uses, have contributed to the current standing of medical ultrasonics as a well-developed, noninvasive technology. The therapeutic application of ultrasound to enhance efficacy of drugs and gene-based therapy, however, is still in early stages of development. “If the ultrasonic parameters can be well controlled, drugs could be targeted to a specific region in the body. For example, a tumor site could be targeted so that drugs are preferentially delivered to the tumor cells. This could minimize the side-effects commonly associated with traditional chemotherapeutic treatments,” explains Hutcheson. Thus, noninvasively focused ultrasound has the potential to improve the delivery of drugs and genes to targeted tissues, minimizing side effects, lowering drug dosages, and increasing efficacy.

Prausnitz believes that a number of challenges need to be overcome before ultrasound-mediated therapy can be used in humans. Researchers need to determine the optimum cavitational activity, as well as other physical and chemical parameters, within the body for each given application. To control the impact of ultrasound on cells, researchers first need to better understand the pathways that mediate cell death so that maximum cell viability can be preserved. Further studies are needed to determine whether other cellular and physiological pathways are affected by ultrasound. There is still a long way to go to fully validate initial demonstrations that the ultrasound bioeffects that are effective and desirable in vitro – enhanced transfection and increased sensitivity to chemotherapeutics and other agents – are reproducible in vivo.

Evidence from other research fields suggests that cell membranes are continually ripped open and repaired inside the body without long-term effects. Mechanical impacts such as the beating of the heart, the motility of the gut, and the movement of muscles rip open cells in our bodies, and a similar mechanism of membrane repair as that shown by the Prausnitz team was demonstrated earlier with these mechanical stresses. While these data would suggest that cells may similarly withstand the effects of ultrasound, thorough studies are needed to corroborate this hypothesis and address other safety concerns.

Despite these obstacles, Prausnitz and the ultrasound research community remain optimistic that the day will come when ultrasound-mediated therapy will be widely applied in humans.

This research is funded in part by the National Institute of Biomedical Imaging and Bioengineering and CytoDome.

References:

Hallow DH, Mahajan AD, Prausnitz MR. Ultrasonically targeted delivery into endothelial and smooth muscle cells in ex vivo arteries. Journal of Controlled Release, Jan 12, 2007 (Epub ahead of print). http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=17291619&query_hl=1&itool=pubmed_docsum.

Campbell P, Prausnitz MR. Future directions for therapeutic ultrasound. Ultrasound in Medicine and Biology, in press.

Schlicher RK, Radhakrishna H, Tolentino TP, Apkarian RP, Zarnitsyn V, Prausnitz MR. Mechanism of intracellular delivery by acoustic cavitation. Ultrasound in Medicine and Biology, 32:915-924, 2006.

Zarnitsyn VG, Prausnitz MR. Physical parameters influencing optimization of ultrasound-mediated DNA transfection. Ultrasound in Medicine and Biology, 30: 527-538, 2004.

Keyhani K, Guzman HR, Parsons A, Lewis TN, Prausnitz MR. Intracellular drug delivery using low-frequency ultrasound: Quantification of molecular uptake and cell viability. Pharmaceutical Research, 18:1514-1520, 2001.

Dr. Mark R. Prausnitz