Heat-Triggered Liposomes Carry Drugs to Eradicate Tumors in Mice

DURHAM, N.C. - Human tumors implanted into mice regressed completely within about 12 days when treated with heat-triggered, sub-microscopic drug carriers called "liposomes," and most of those tumors didn't regrow during 60-day trials at Duke University and Duke Comprehensive Cancer Center, researchers reported Tuesday.

The finding raises the possibility of treating cancers by injecting such liposomes into cancer patients and applying heat only at the region of a tumor to selectively release cancer-killing drugs.

The studies, published in the March 1 issue of the journal Cancer Research Advances in Brief, tested a new heat-triggered variety of liposomes - artificially engineered waxy capsules just two molecules thick composed of "lipids" materials similar to those that surround every cell in the body.

Small enough to travel through the bloodstream to deliver drugs to designated targets, liposomes measure only about 100 billionths of a meter in diameter, or about one-hundredths the size of a red blood cell.

"Anything larger tends to be taken up by the spleen," said David Needham, the professor of mechanical engineering and materials science at Duke's Pratt School of Engineering who is the new liposome's inventor. "Anything smaller gets lost in the liver."

Unlike previous liposome versions, which the studies also tested for comparison, the new Duke-engineered variety can release an exceptional 45 percent of its stored drug in one 20-second burst when tumor temperatures are raised to five degrees Celsius. higher than normal human body temperatures, according to the Cancer Research report.

Such temperatures of 42 C. are easily reachable by clinical devices that focus heat onto sites inside the body as part of existing cancer therapies. The promising results in mice, whose grafted human squamous cell carcinoma tumors were warmed to 42 C by water baths as the liposomes were injected, thus raise the possibility of using remote heat sources to trigger precision releases of anti-cancer drugs in future human patients.

These liposomes can quickly dump their cargo because their special membrane chemistry causes parts of their molecular structures to begin "melting" when heat increases to the critical temperature. "It's like a soccer ball with stitches," Needham said in an interview. "When its stitches become leaky, the drug that is trapped inside will come out, and in this invention will come out rapidly."

However, just as melting ice can refreeze, these leaky liposomes are also able to reseal themselves when the temperature falls again, as it would when the liposomes leaves the artificially heated tumor site. That resealing provides a safety factor against delivering toxic drugs to the wrong sites.

"After it goes through it seals up," Needham said. "Then when it comes around again to the tumor it releases again."

The critical temperature that produces this phase change is also lower than temperatures needed to trigger previous versions of heat-sensitive liposomes, a potential benefit for therapy.

Needham's liposomes have received patent protection, and Duke has licensed the rights for commercial development to Celsion Corp. of Columbia, Md. Celsion, which makes microwave devices that deliver heat for medical treatment, co-funded the initial mouse study at Duke's Comprehensive Cancer Center and will also support further research at Duke and elsewhere using the new liposomes. Other support for the initial study came from the National Institutes of Health.

The Cancer Research Advances in Brief study describes contrasting effects on tumor growth of administering the chemotherapy drug doxorubicin alone, giving no drug at all, or three different kinds of liposomes containing equivalent doses of drug. The first liposome variety was not designed to react to heat. A second was an older heat-sensitive liposome that releases its drug cargo at slightly higher temperatures and at much slower rates than the new one. The third was Needham's new version.

Each different preparation was injected into the bloodstreams of mice that previously had human squamous (scaly or plate-like) cell carcinoma tumors implanted into their legs as so-called "xenografts." These special mice were genetically altered to lack thymus glands. Because the mice had no thymuses, they could manufacture no immune-system T-cells to mount an immune system attack on the implanted "foreign" cancer tissues originally derived from a human tumor.

Mice that received no liposomes nor heating at their xenograft sites -- the experimental "controls" -- showed a steady and rapid tumor growth that reached five times the initial tumor volume within 10 days. And the effect of injecting only doxorubicin, a commonly used and potent anti-cancer drug, was "practically nothing" in all instances, said Mark Dewhirst, a Duke professor of radiation oncology who co-authored the Cancer Research Advances in Brief study with Needham, engineering postdoctoral researcher Gopal Anyarambhatla and engineering graduate student Garheng Kong.

Dewhirst said doxorubicin alone had practically no effect on tumor growth because it is known to quickly exit the bloodstream and enter all tissues. "It's gone in about two minutes," he added. "So you can't get a therapeutic gain. It also goes to the bone marrow, the heart, all the normal tissues that just can't tolerate any more drug. It's also going to the tumor, but not in adequate amounts."

In contrast, injections of the different types of drug-filled liposomes into xenografts warmed to 42 C all resulted in some delay of tumor growth, even with liposomes not engineered to react to heat.

In explanation, Dewhirst and Needham said that blood vessels supplying tumors seem to become inherently leakier when heated, in this case letting statistically more liposomes out of circulation to reach the warmed-up tumor sites. While they are in the vicinity, all liposomes will eventually release some drug, whether they are primed for heat release or not, the researchers said. Moreover, heat alone is known to retard tumor cell growth and is itself used for cancer therapy.

However, in all cases "growth delay was highly dependent on how much drug got in," noted Dewhirst, who was able to compare the amounts of drug in different tumor tissue samples because the doxorubicin preparations were fluorescent.

Drug amounts released from the new Duke-engineered liposome was "much higher," he said, than from the other two types.

Study results showed that injecting non-heat sensitive liposomes delayed tumor growth, compared to controls, an average of about 12 days in the first of two trials, and about 14 days on average in the second trial. But all the tumors eventually grew to five times their initial volume, the maximum assessed in the study.

Injecting the older type of heat-sensitive liposome resulted in one remission in the first trial, which involved 12 animals, and one in the second, which studied 11 different mice. "Remission" means that the tumor disappeared and did not return over the 60-day length of each trial. Compared to the controls, tumor growth was only delayed in the other mice treated with the older temperature- triggered liposome by 15 days in one trial and 16 in the other.

In comparison, treating tumors with the new Duke-engineered liposome resulted in remissions in six of nine mice in the first trial, and all 11 animals in the second. "It's pretty unusual to see something work that well," said Dewhirst. "It's not unheard of, but it's certainly unusual."

Dewhirst cautioned that more work is needed. Tumors in this study were relatively small, so the work needs to be repeated studying larger ones, he said. Heating tumors in a water bath also produces more uniform results that would be seen in a clinic. And these tumors were also transplanted from one species to another.

In one followup, the Duke researchers said they will team up with others at the North Carolina State University College of Veterinary Medicine in Raleigh to test how well the new liposomes work with pet dogs being treated as patients for soft tissue sarcomas, another form of cancer.

Preliminary toxicity studies of the new liposomes also are being planned as a prelude to investigatory drug trials in human patients, Dewhirst added.

Localized heat delivery - called hyperthermia - has been used for the past two decades to treat certain cancers, particularly in combination with radiation therapy. The combination has been widely used because the two treatments together have been found to work better than either does alone, said Dewhirst, who supervises a Duke program in hyperthermia.

The problem with both, he said, is that hyperthermia and radiation work best together at slightly higher temperatures than can be achieved in their current clinical applications.