New Potential Drug Carriers for Tumors

Experiments by Duke biomedical engineers and cancer researchers suggest that genetically engineered biomolecules that precipitate from solution at slightly elevated temperatures eventually may provide a novel way to deliver drugs or radiation therapy to solid tumors. These studies, using laboratory mice implanted with human tumors, are apparently the first demonstration in living animals that such a "thermal phase transition by a freely circulating polymer can be engineered to occur at a specified temperature within a complex physiological system," the investigators reported in the Feb. 15 issue of the research journal Cancer Research. "The idea was to create a carrier for drugs that would be thermally responsive, so that one could then take advantage of all the work done in clinical hyperthermia -- a large part of it done at Duke," lead author Ashutosh Chilkoti, an assistant professor of biomedical engineering at Duke's Pratt School of Engineering, said in an interview. Hyperthermia is a heat treatment technique used in cancer therapy. Chilkoti's ultimate hope is that these large molecules could be linked with anti-cancer drugs so that both would precipitate out at tumor sites undergoing such already-available heat treatments. He has a grant from the Whitaker Foundation to investigate this potential of the biopolymers, known as "elastin-like polypeptides" or ELPs. One of his Duke Medical Center co-authors, Mark Dewhirst, participated under National Institutes of Health funding; the other, Michael Zalutsky, was supported by the U.S. Department of Energy. Polymers are large chain-like molecules. Polypeptides are polymers made up of amino acids, the building blocks of proteins, but have less elaborate structures than proteins. The ELPs Chilkoti's group makes in the laboratory are thus analogous to but less complicated than elastins, elastic proteins that are constituents of connective tissue in animals. His laboratory previously discovered that ELPs come out of solution in water, essentially undergoing a "phase change," when the the polymer's temperatures are raised just a few degrees. In a subsequent 1999 article in the research journal Nature Biotechnology, Chilkoti and graduate student Dan Meyer described how ELP's could be attached to dissolved proteins so that the proteins can themselves be separated from solution, and thus "purified," when the linked ELPs are warmed. To make the ELPs for the Nature Biotechnology study, the Pratt School researchers designed and made artificial genes that encode for ELPs, fused them with blueprint genes for the two proteins, then introduced the combination into colonies of the bacterium Escherichia coli. The bacteria then proceeded to make ELP-protein combinations. In the new work, Meyer used synthetic genes and Escherichia coli to tailor make ELPs that were "tuned" to be soluble at 98.6 Fahrenheit degrees -- approximately normal body temperature -- but go through a phase transition at 107 degrees, Chilkoti said. Such fine tuning can be achieved either by adjusting either the polypeptide's composition or its molecular weight. "Both of those are completely under our control," he added. Dewhirst, a professor of radiation oncology who directs the Comprehensive Cancer Center's Hyperthermia Program, and associates then introduced human tumors to specially bred mice lacking immune-system defenses to such "foreign" tissue. Working with Dewhirst were Garheng Kong, a former graduate student of Dewhirst, and Zalutsky, a professor of radiology. In one technique, pieces of human ovarian tumor were implanted into skin folds in the backs of one group of mice so that tumor-area fluid circulation could then be observed with microscopes through special glass "window chambers." These window chambers were then warmed with 107-degree warm water to simulate hyperthermia treatments used in cancer therapy. And special versions of the temperature-tuned ELPs were introduced into the animals' bloodstreams. Those versions were tagged with fluorescent chemicals that glow under ultraviolet light whenever the biomolecules precipitate to form clumps of particles. A duplicate experiment, but using ELPs that do not precipitate at 107-degree water temperatures, was also run for comparison. Documenting the results with a digital camera, the investigators "could see particles forming in the blood vessels in the tumors, and only in the tumors that were heated with the polymer that was thermally responsive," Chilkoti said. Meanwhile, a team in Zalutsky's lab tagged ELPs with a radioactive chemical for injection into a different type of tumor -- that of the human brain. These "glioma" tumors were implanted into the forelegs of another group of the same kind of mice. Researchers then immersed some of those animals' legs in 107-degree water baths and injected the radioactive ELPs. Other animals received radioactive ELPs but no water baths. After a period, the animals' were checked for how much radioactivity was localizing in their tumors. The results would suggest how much EPLs were concentrating there, and whether those changes were heat-triggered. "We got the most localization in the heated tumor with thermally responsive elastins," Chilkoti reported. The results of Dewhirst's window chamber studies and Zalutsky's radioactive tracer experiments were "complementary," Chilkoti said. As their Cancer Research report concluded, there was approximately "a two-fold increase in tumor localization compared to the same polypeptide without hyperthermia." Chilkoti cautioned that "this is nowhere close to any kind of clinical application." He and Meyer must still engineer other ELPs that deliver radioactive or non-radioactive drugs to tumor sites. They also hope to deliver them at higher concentrations than the current ELPs. Pinpoint localization at the tumor site is essential, he said. "You don't want to get too much drug into the rest of the body, because anti-cancer drugs can be terribly toxic."