'Nanobees' deal death sting to cancer in mice
This article first appeared in the St. Louis Beacon, Sept. 1, 2009 - Researchers at Washington University in St. Louis have designed a new way to kill cancer cells with a toxin found in bee venom.
The fact that the toxin, melittin, can kill cancer cells is no surprise. As an anti-viral, anti-fungal, anti-bacterial peptide that bees use to defend the hive, melittin has evolved to kill everything. The breakthrough is in the delivery system, said Samuel Wickline, head of the Siteman Center of Cancer Nanotechnology Excellence at Washington University.
"We didn't invent the idea that melittin was a good anti-tumor agent," Wickline said. "People have known that for a long time. They just didn't know how to deliver it. You definitely do not want to just inject it because it will [destroy] all your red blood cells." As with many cancer drugs, the difficulty is in getting the deadly drugs to the cancer cells without killing cells vital to keeping the patient, or in this case the mouse, alive.
The researchers created the delivery vehicle by attaching melittin to nanoparticles, nano-scale spheres made of perfluorocarbon, an inert chemical also used in blood substitutes. The melittin is stable on the nanoparticle, but when this "nanobee" is in close proximity to a cell membrane, the layer of melittin is transferred to the membrane where it can then kill the cell.
"I stayed late in the lab one night, doing that experiment," said Paul Schlesinger, associate professor of cell biology and physiology and a co-author with Wickline on the research published Aug. 10 in the Journal of Clinical Investigation. Schlesinger got a single layer of melittin onto the surface of the nanoparticle and then put the "nanobees" next to liposomes, cell vesicles often used to model cell membranes. "I had liposomes that are kind of like Christmas tree ornaments," said Schlesinger, "They contain a fluorescent dye that's very bright. When the melittin destroys the liposomes, the dye is released and I can detect a large burst of color."
His experiment worked. "You go home and you know this great thing that nobody else in the world knows. And it's your secret for the next six hours until you tell other people about it. That's just fun," he said.
Of course, destroying a model of a cell membrane in a dish is not treating cancer. The next step was to test the nanobees in mice. One mouse breed was implanted with human melanoma tumors and another received human breast cancer cells.
After four or five doses of the melittin-loaded nanoparticles, breast cancer tumor growth slowed almost 25 percent. And the melanoma tumors decreased in size by about 88 percent compared to tumors left untreated. When the graduate student working on the project, Neelesh Soman, showed Schlesinger the first results, with the 88 percent reduction in the size of a melanoma tumor, Schlesinger was pleased.
"I said, 'Wow, that's pretty neat, so how many of the mice died?'" recalled Schlesinger.
All the mice were still alive. And not only were they alive, they appeared healthy. The scientists found no evidence of organ damage and blood cell counts were normal. In addition, they found that melittin attached to nanoparticles remained in the body far longer than free melittin, giving it more time to work on the tumors. Free melittin is degraded and removed by the body fairly quickly, but not before it has done serious damage.
In this study, the researchers found that the nanobees honed in on their tumor targets with two mechanisms. Both rely on the fact that tumors stimulate new blood vessel growth for a supply of oxygen and nutrients. One mechanism takes advantage of the fact that the tumor's new blood vessels are small and leaky and the nanoparticles tend to get stuck in them. "This is called enhanced permeability and retention," said Wickline, "It simply means small things get stuck in blind alleys or cracks and can leak out and stay in the neighborhood."
The second mechanism uses a target that the nanobee seeks out. New blood vessels near tumors express a small protein called an integrin. The nanobees with the targeting agent seek out their specific integrin and attach to it like a lock and key. The researchers point out that different targeting agents could be attached to direct the nanobees to different integrins, providing flexibility in treatment options.
In another encouraging finding, they showed that nanobees were able to target and reduce the growth of precancerous skin lesions by as much as 80 percent. Schlesinger spoke of the possibility of cancer prevention, especially for people with high risk factors. "It could decrease the risk significantly or delay the onset so that, even if they had the predisposition, many fewer people would end up expressing that kind of tumor," he said.
Once at the site, melittin appears to have two ways of killing cells. In the first, melittin stays on the cell surface. "Four or more melittins get together and form a hole in the membrane. A fairly small hole, but a fairly nasty hole," Wickline said. "It allows the contents of the cell to leak out and the cell dies."
The second method is more insidious, taking advantage of the cancer cell's own machinery and composition. Cancer cell membranes have unusually high cholesterol content, which can inhibit melittin's ability to form a pore at the surface.
On the nanobee, melittin sits in a single lipid layer, a layer of fatty material that doesn't mix with water. But it will mix with other lipids like the cancer cell membrane.
The nanobee's lipid monolayer containing melittin attaches to the cancer cell membrane and the two fuse together. The lipids around the nanobee and the cell's lipid layer can then mix and the melittin literally flows from the particle to the cell membrane. Still inactive, the melittin is then transported inside the cell. Once there, it pokes holes in the mitochondria, triggering apoptosis, the process of cell suicide.
But cancer cells are notoriously good at self-defense, devising pathways around roadblocks that cancer treatments attempt to erect. Since melittin's method of killing is mechanical, however, it is thought that cancer cells will have a tougher time adapting to it.
"A cell would have to figure out how to repair massive disruptions of its membrane -- probably in seconds," said Wickline. "Can it do that? I don't know the answer to that, but the hypothesis is that this kind of mechanical disruption would be hard to get around, much harder than inhibiting a single point in a single signaling pathway."
Mice Are Not Men
Before Phase I clinical trials for safety can begin in people, more animal testing is required.
"One must be cognizant of the fact that mice are not men," Wickline said. He speculates that the earliest human safety trials could begin in 18 months. In the meantime, they will test nanobees in rabbits.
"The original concern was that melittin is very toxic," Schlesinger said. "Putting it on the nanobees has apparently taken care of that in mice. But one would look very carefully in other animal studies for any evidence of that toxicity reappearing."
They must also scale up the production of melittin, and the rabbit trial will be good practice. "Rabbits are about 100 times bigger than mice so we need 100 times the melittin," said Schlesinger. Fortunately for bees, the melittin used in these studies is synthetic. "We don't squeeze any bees," said Wickline with a laugh. "It's a 26 amino acid peptide and it's relatively straightforward to make."
With such a small peptide, they also do not anticipate problems with bee allergies. "We won't know for sure until we test it," Wickline said, "but according to the literature on beekeeper stings, most of those reactions are likely not to melittin itself, but to other components of bee venom, which are much larger peptides."
The Cost of Care
As for the cost of high-tech health care, both Wickline and Schlesinger are optimistic about the possible cost of nanobees.
"I don't think it's going to be prohibitively expensive to make these carriers," Wickline said. This particular nanoparticle self-assembles, they point out. "You mix it up in a batch like salad dressing," Wickline said. "It comes together naturally."
The melittin is relatively expensive right now, Schlesinger said, but he attributes that to the fact that no one is making it in large quantities yet. "We're certainly able to make insulin synthetically and that's at least 10 times larger than melittin," he said.
Wickline acknowledges that nanobees would likely be more expensive than a drug a pharmaceutical company makes. Those drugs are typically single small molecules while the nanobee has two components. But Wickline also points out that a local company he co-founded, Kereos Inc. already makes the nanobee carrier - the perfluorocarbon core -- in what's called "good manufacturing practices," meeting standards set by the Food and Drug Administration for safety and quality.
Wickline urges caution and patience, but also hope. Because nanoparticles are both complex and new to the FDA, he thinks the approval process may be longer than for traditional small molecule drugs. But he points out the already toxic effects of current cancer treatments. Nanotherapeutics "are going to protect healthy tissues much more so than the drugs we see today."
Julia Evangelou Strait is a freelance science writer based in St. Louis. She is a 2009 Missouri Health Journalism Fellow and holds a master's degree in biomedical engineering.