Each year, nearly one million Americans undergo angioplasty to widen narrowed arteries. During the procedure, a tube (catheter) is inserted into the diseased artery through a small incision. A balloon at the tip of the tube is subsequently inflated, widening the artery and dislodging any blockage. Angioplasty is often combined with placement of a stent, a wire mesh tube intended to prevent re-narrowing of the blood vessel.
When Vascular Interventions Fail
A major cause of vascular reconstruction failure is intimal hyperplasia, a process in which an injury to the blood vessel (such as a surgical incision) triggers increased growth and migration of vascular smooth muscle cells and precursor cells, resulting in narrowing of the affected artery. Stents that release drugs to curtail cell growth have had clinical success in preventing re-narrowing of blood vessels. More recently, however, researchers have been exploring protein and gene-based therapies that might provide more effective ways to combat the onset of intimal hyperplasia. The investigators have a good idea about what genes are important for controlling intimal hyperplasia. "You can think of intimal hyperplasia as a problem of having too many cells. One way to control cell number is to control growth of the cells. You can also enhance cell death," explains Lynn's collaborator, Bo Liu, Associate Professor at the Department of Surgery, University of Wisconsin - Madison.
Multilayered Polymer Films - A Versatile Approach to Gene Delivery
Lynn and his research team have devised a way to deliver genes to vascular tissue from the surfaces of catheter balloons and stents coated with alternating layers of a biodegradable polymer and genetic material called plasmid DNA.
The assembly of the polymer film is fairly simple, explains Lynn. "By dipping an object in a solution of DNA, which is negatively charged, a thin layer of DNA is adsorbed on the surface. You can then take that and dip it in a solution of a positively-charged polymer, and a thin layer of that will adsorb to the surface. You can do that over and over again to build thin, multilayered structures." Devices of any shape and size can be coated this way, and the film is thin enough not to interfere with their functions. The layers cling to each other through electrostatic interactions.
Any gene of interest can be inserted into a plasmid for delivery to cells. "You might want to deliver multiple agents that act synergistically or at different times," says Lynn. Layer-by-layer assembly allows scientists to put different therapeutic agents into each layer, like the layers of meat, cheese, tomato sauce, and noodles found in lasagna.
The polymer films are engineered to fall apart when they come into contact with water, releasing the DNA. By tweaking the chemical composition of the polymers in the film, researchers can control how the film breaks down. For example, the film can be designed to degrade all at once (like a sugar cube) or gradually (like a bar of soap). In the latter design, the agents that are positioned near the top of the film would be delivered to cells first. "We developed a suite of polymer agents that give us control over release from 1 to 2 days to 2 to 3 months. We are now moving down to hours and minutes and seconds," states Lynn. Fast-degrading films are needed for angioplasty, because a catheter balloon can be left in a blood vessel for a maximum of 20 minutes, as it blocks blood flow.
Polymer films have many potential applications. Ultrathin films can be coated on the surface of microneedles to deliver DNA-based agents through the skin. Working with Mark R. Prausnitz of the Georgia Institute of Technology, David Lynn (University of Wisconsin – Madison) recently designed fast-releasing films for the transdermal delivery of DNA- and protein-based agents via microneedles, which are long enough to penetrate the skin but not long enough to cause pain. When the microneedle penetrates the skin, the DNA is quickly released into the surrounding tissue. The approach could be used for pain-free administration of DNA- and protein-based vaccines.
As the film falls apart, the positively-charged polymers escort the plasmid DNA across the negatively-charged cell membrane. Once inside the cell, the plasmid DNA instructs the cell to begin making a specific protein. When the cell divides, the plasmid DNA is not duplicated, so eventually production of the plasmid-encoded protein stops. Although this could prove advantageous for some applications, the investigators think that therapeutic protein production could be prolonged by using slow-degrading films that deliver a steady supply of fresh plasmid to the cells.
Localized delivery reduces the problem of systemic side effects and dilution of therapeutic agent. "The thin film allows us to zoom into the right neighborhood, to get into the diseased vessel. That's better than giving patients a pill or an injection, where the therapeutic agents would go all over the bloodstream and get to all tissues," says Liu.
What Lies Ahead
Ideally, one would be able to control the growth of only specific cell types. Liu's research team is using Lynn's suite of polymer films and other delivery modes to explore how to introduce therapeutically relevant genes only into desired cell types and how long the gene remains active. "We can construct a plasmid in such a way that, even though the gene is delivered to most cells, only the targeted cell will express that gene," Liu explains.
In Liu's opinion, the thin film technology is a safe and efficient way to deliver gene therapy to the blood vessel walls. According to Lynn, gene-based therapies that target the wide range of cellular mechanisms that underlie re-narrowing of blood vessels could substantially contribute to the success of vascular interventions.
Dr. Lynn's work is supported in part by the National Institute of Biomedical Imaging and Bioengineering and the Alfred P. Sloan Foundation.