Biomedical engineers at Vanderbilt are gunning for this lethal disease, joining forces with other Vanderbilt researchers in nanoscience, engineering, molecular biology, physics and medicine.

Their research, partly funded by the U.S. Department of Defense Breast Cancer Research Program, is fighting the disease on several fronts by finding new ways to enter the

fortress of the cancer cell and to penetrate its inner sanctum, without harming healthy cells elsewhere.

Already the team, headed by Associate Professor of Biomedical Engineering and Chemical Engineering Todd D. Giorgio, has developed novel ways to infiltrate the heart of the enemy-the nuclei of the malignant cells--to deliver the knock-out punch of chemotherapy more effectively than conventional methods, with few side effects.

Professor Giorgio and his colleagues are particularly excited about their discovery of five previously unidentified molecules that can penetrate cancer cell nuclei. These molecules are being further tested for their ability to deliver "payloads" such as chemotherapy or gene therapy, and the prospects look promising. In fact, the first one of these new molecules the team tested has already proven its potency by successfully bypassing defenders and depositing the payload in the cancer nuclei.

Professor Giorgio's team has also found ways to detect breast cancer in the very early stages of the disease, using coated gold nanoparticles that bind to breast cancer cells, where they can be easily imaged with current mammogram and other imaging technology. And to target even earlier stages of the disease, Professor Giorgio has found a way to develop an at-home breast cancer test that will be used much like a home pregnancy test.

Search and Destroy

Cancer cells are not particularly hearty nor hard to kill. Household bleach would do the trick. Unfortunately, it would also kill the patient's healthy cells. Plus cancer cells are small and hard to detect until their malignant colony has grown to an advanced size and perhaps spread unpredictably throughout the body.

"We know that cancer cells need room to grow, and one of the things they do in order to invade the surrounding tissue is to release a protein that softens the cells," Professor Giorgio says. "This softening process is called proteolysis, and the protease it produces would be an excellent target for finding and killing cancer cells if not for the fact that healthy cells in the kidneys and other organs also use this process and produce the substance."

Professor Giorgio devised a strategy of combining two different targeting mechanisms that will act together to selectively identify breast cancer cells while not attaching to healthy cells. His idea was to coat a nanoparticle with a substance that would be ignored by healthy cells but attacked by the proteins cancer cells produce in proteolysis. Once the cancer cells remove the protective coating through proteolysis, the second targeting mechanism is revealed-a folate (Vitamin B) molecule highly desired by cancer cells. When the cancer cells gobble up the folate, they also take in the nanoparticle attached to it.

Until recently there were no materials that were small enough to be injected but large enough to attach both the folate and the coating.

Cross-disciplinary Collaboration

A member of the Vanderbilt Institute of Nanoscale Science and Engineering (VINSE), Giorgio became intrigued with the idea of using gold nanoparticles being produced by David Cliffel, Assistant Professor of Chemistry. Gold nanoparticles are not only quite small, but are easily imaged by computed tomography (CT) and mammography.

VINSE is an interdisciplinary center that conducts theoretical and experimental research in science and engineering at the nanoscale level. Professor Cliffel produces gold nanoparticles in conjunction with his research in nanostructured metallic catalysts.

"The gold nanoparticles are a good size for our purposes and are clearly visible using existing imaging technology," Professor Giorgio says.

Because gold images well in CT scans, Professor Giorgio can evaluate the effectiveness of his targeting techniques through testing the new particles on laboratory mice.

"Making something work in vitro is quite a different challenge from succeeding in the body," Professor Giorgio points out. "We needed to explore these new techniques in vivo, and Vanderbilt's capabilities in this area happen to be quite strong."

Vanderbilt is home to the Vanderbilt University Institute of Imaging Science (VUIIS), which has recently opened a laboratory for imaging small animals. The equipment in the new facility provides better image resolution for small animals and thus produces more precise and useful images.

"We wanted to see exactly where these targeting particles are

traveling throughout the body," Professor Giorgio says. "We are able to quantify the success rates of the method and can determine the percentage of particles that successfully attach to the tumor we are targeting."

At-home cancer test

Then Professor Giorgio had another idea. Since the protective coating of the new nanoparticle is broken down by proteolysis into a byproduct molecule that is not ordinarily found in the body, why not test for that byproduct in the urine stream, as in a home pregnancy test?

Professor Giorgio and his associates are developing and testing the optimal composition of the coating, a chain of fatty liposome molecules called polyethylene glycol (PEG) and peptide bonds to link them. These liposome molecules are chemically similar to the membranes of human cells, which enables them to enter cells more easily.

By designing these molecules to include a PEG that will break off only during proteoloysis caused by tumor cells, Professor Giorgio's team can pave the way toward producing a simple urine test that checks for this broken-off PEG that is excreted in the urine.

"The ease and low cost of this approach establishes a new paradigm for breast cancer screening that offers the frequency and practical accessibility of self-examination with sensitivity that exceeds current medical imaging methods," he says.

Panning for Peptides

Getting inside a cancer cell without invading its healthy neighbors is hard enough, but getting into the nucleus to either heal through genetic engineering or to destroy through chemotherapy is quite another challenge.

But an important one. The effectiveness of conventional chemotherapy is limited by its impact on healthy cells and its rather random success at penetrating cell nuclei.

"The nucleus containing the cell's DNA is extremely selective about what molecules are allowed inside," Professor Giorgio says. "We have been searching for peptide ligands that can bind to the nucleus and allow a molecular payload to be transferred into the nucleus."

This task is a bit like finding a needle in a haystack. Peptides, which are molecules made up of several amino acids, vary on the order of millions of varieties. Where to begin?

With all of them, Professor Giorgio decided, thanks to a process called "biopanning."

Biopanning begins with a "library" of viruses that invade bacteria but are harmless to humans. These viruses, called bacteriophages or phages, each consist of a tubular-shaped body with several protein streamers. One of these streamers includes a sequence of seven amino acids that create the peptide bond. Within the library or collection of these phages, purchased from a biotechnology vendor, all possible combinations of these seven amino acid peptides are represented.

"We need seven amino acids to ensure sufficient peptide binding to the surface of the nucleus," Professor Giorgio says. "Each phage in the library has one peptide sequence of seven amino acids, so they're all different."

The phages are poured onto two cell lines of human cells, one line of normal cells and the other of breast cancer cells. After waiting a day for the phages to do their work of entering the cell nuclei, the researchers use a detergent method that breaks open the cells but keeps the nuclei intact. They then put the mixtures into a centrifuge containing high-density fluid to extract the nuclei.

Once they have the nuclei, they put them into a low pH solution that bursts the nuclei and frees the phages. These phages are the successful ones.

Then next step is to put the successful phages into dishes of e. coli bacteria to let the phages replicate. These phages are sent through two human cell lines, as with the first process, harvesting the successful phages and introducing this next batch into e. coli bacteria in order to grow.

After the third round of biopanning, the samples are taken to the Vanderbilt DNA Sequencing Facility to be genetically sequenced. Researchers there know where to look on the phages' genome in order to determine the peptide sequence that codes for the phages' amino acids. These peptide sequences, the recipes for the peptides sequence that can carry genetic or chemotherapy payload into the nuclei, are matched against known peptide sequences.

Hitting Paydirt

Professor Giorgio's team has already test-driven one of the successful, previously unknown, peptides and has found that it can indeed deliver into the nucleus a phage as large as 70 nanometers, which is about the size of the liposome used to deliver gene therapy.

"This is particularly exciting because we can harness the penetration power of bacterial viruses' peptide sequence, without using the virus itself, to pull non-viral liposomes into the nucleus," Professor Giorgio says. "At a time when the FDA is concerned about the use of retroviruses in gene therapy, this discovery can be a highly beneficial method."

This success has captured the attention of another collaborator, Dr. Dennis Hallahan, professor of radiation oncology and chair of the department. Dr. Hallahan had already conducted research into using peptides to bond to tumor-feeding endothelial cells, which are cells that line the blood vessels. In addition to conducting National Institutes of Health with Professor Giorgio, Dr. Hallahan has opened his animal laboratories for Professor Giorgio's use in the breast cancer research.

"He has been invaluable to us in helping us take our ideas and winnow through them to focus on the ones that have a good chance of being accepted at the clinical level," Professor Giorgio says.