When amateur artist Cristina Zavaleta signed up to take an illustration class with Pixar animators on character design, she had no idea she’d also be embarking on a new scientific study. At the time, Zavaleta’s work as a post-doctoral biomedical researcher in a molecular imaging lab at Stanford involved evaluating contrasting agents, like dyes, used to detect tumors in animals. During her art class, the researcher was struck by the intensity of the colors of gouache, vibrant water-based paints, that her fellow illustrators were using. “They were bringing back these pieces that were just incredible, really rich colors. And I thought, how do you even achieve that color, visually,” says Zavaleta.
That simple question ultimately led Zavaleta, now an assistant professor of biomedical engineering at the University of Southern California, and her colleagues to create a first-of-its-kind library detailing the optical imaging properties of commonly used pigments and dyes, found in everything from tattoos to food coloring. The researchers hope their study will open the doors for the novel use of everyday colorants as imaging agents in medical tests, that may be more effective at early detection of several kinds of cancers.
Currently, only three dyes with fluorescent properties used as optical imaging contrast agents—methylene blue, indocyanine green and fluorescein—are approved for human use by the U.S. Food and Drug Administration (FDA). In diagnostic medicine and in some surgical procedures, imaging contrast agents are materials used to improve internal body pictures produced by X-rays, computed tomography (CT) scans, magnetic resonance imaging (MRI), and ultrasounds. These materials can be ingested or injected and temporarily color targeted parts of the body, like specific cells, organs, blood vessels and tissues, to help clinicians see differences and abnormalities that may indicate disease. Yet, Zavaleta wondered about the significant catalogue of approved food, drug and cosmetic dyes that people routinely encounter in their everyday lives. Are there other imaging agents hiding in plain sight?
“As my art brain was thinking about these paints [from class], I thought to myself, what paints are already being used in humans?” says Zavaleta. “And a lightbulb went off.”
Tattoos. High quality pigments used in tattooing are made from mineral salts and metal chelates, which have been isolated from natural sources and used by humans for thousands of years.
Zavaleta’s next step was to do her homework, as any good researcher would. She contacted Adam Sky, a tattoo artist in the Bay Area whose work she admired. Sky was interested in her research, and gave her samples of some of the inks he was using, which Zavaleta collected in a well plate, a tray with multiple divots, or wells, that can be used as test tubes, she’d brought along, just in case.
“I immediately took them to my microscope over at Stanford, and I did all these different tests on them,” Zavaleta says. “I was amazed at what I was seeing.”
She measured two optical elements of the inks, their fluorescence properties and Raman properties. Fluorescence relates to a dye or pigment’s capacity for absorption and emission of light, while Raman indicates how light scatters. Both are commonly used in imaging techniques in the cancer field. Highly fluorescent agents offer sensitivity in imaging; very small amounts are needed for them to illuminate areas very brightly. Raman imaging, on the other hand, offers specificity by allowing multiplexing, or the ability to look at several processes happening inside the human body at once. These can help show whether cells or tissues are expressing multiple genes, for example, or expressing one more highly that may be associated with a particular cancer, like HER2 and breast cancer or EGFR with lung cancer. Each of the targets has different receptors that will be illuminated by different agents, and depending on their optical properties, some agents will be better than others.
In all, the researchers evaluated the optical properties of 30 approved food, drug and cosmetic coloring dyes and tattoo ink pigments using a spectrophotometer, an instrument that measures the intensity of light after it passes through a sample solution. Seven of the colorants displayed fluorescence properties that were comparable to or exceeded the three FDA-approved clinical dyes. The researchers next measured the Raman signatures, to see how high the colors’ unique signatures of light photon peaks were, with high peaks being indicative of usefulness in terms of multiplexing. Finally, they tested the best-performing dyes and pigments by injecting them as imaging agents in mice with cancerous tumors.
Data from Zavaleta and her colleagues’ study showed that FDA-approved Green 8 dyes used in drugs and cosmetics have significant tumor targeting potential in mice with cervical and colon tumors, and the Orange 16 pigment found in tattoo inks also showed, according to the authors, promising fluorescent properties and tumor targeting potential. This is significant because, as they note in the study, “no single imaging modality currently meets all the clinical needs of high sensitivity, high spatial and temporal resolution, high multiplexing capacity, high depth of penetration, low cost, and high throughput.” In other words, no single imaging agent can provide all the information a doctor might need.
The USC lab where Zavaleta and her colleagues conducted the research uses nano-based imaging contrast agents, or tiny spherical vesicles that are loaded with the dyes or pigments. While nano-based agents are approved for use as a medium in human imaging, they have been controversial in the past because of potential toxicity. Metallic-based nanoparticles like those made from gold and silver have been known to stay inside the body for long periods of time after exposure. This is one of the main reasons the team instead uses liposomal nanoparticles, made up of biodegradable materials with fatty skins similar to human body cells, that are already used in other applications, like drug and nutrient delivery.
“You can think of it as us having all these different batches of nanoparticles, and one has a different tattoo ink [or other dye or pigment] inside of it. And that tattoo ink has a very special barcode that’s associated with it; every ink has a unique fingerprint, yellow different from red, red different from purple,” Zavaleta explains. “So, if we have all these different flavors of nanoparticles that we can now target to different receptors on tumors, we can enhance our ability to distinguish between different [cancers].”
One use for such materials could be gathering real-time information during a test, such as a colonoscopy, where physicians are visually searching for certain kinds of polyps. Enhanced imaging agents have the potential to also reduce the invasiveness of disease detection and diagnosis, such as the number and size of biopsies needed, by providing more information from a smaller sample.
Christian Kurtis, who made the career change from biomedical researcher at the National Institutes of Health to tattoo artist in Rockville, Maryland, spent his post-doctoral period in a cancer research lab at the Uniformed Services University of the Health Sciences. Kurtis says the specificity these kinds of dyes could offer for imaging is key to better treatment.
“The unfortunate problem with malignant [tumors] is that they comprise a [variety] of molecular markers that may not be present on all cell types. The increased metabolic activity of malignancy is the signature most commonly exploited in imaging, and is the reason these liposomal techniques are effective,” says Kurtis. In other words, because cancer cells tend to spread quickly, researchers and physicians are able to track their growth with imaging. Having multiple types of agents that bind to the different markers would be even more helpful. “In my opinion, it will be personalized or individualized medicine that will hold the key to meaningful early diagnosis of disease,” he adds.
Jocelyn Rapelyea, the associate director of breast imaging and the program director of the radiology residency program at the George Washington University Cancer Center, adds that while tools like molecular breast imaging have been around for a while and help to identify problematic cells before they grow into lumps, advancing knowledge is always a positive. What works well for one patient may not for another.
“It’s always exciting to have the ability to be able to identify tumors at a potentially early stage. It's quite interesting how [Zavaleta] came to dyes,” Rapelyea says. “This is obviously a model in mice at this point, but it is promising to see that there could be potential of being able to identify earlier development.”
Zavaleta knows the dyes and pigments her team has catalogued in a library will be subject to the FDA’s rigorous regulatory procedures before they could ever be used as imaging agents in humans. “We're not suggesting in any way that they’re safe,” she says. “We’re saying, ‘Hey, these are dyes that we’re continuously being exposed to on a day-to-day basis. Let’s have a look at them further.’”