Early detection and treatment are often key to cancer survival. Scientists have, over the past decade, created nanoparticles that are able to home in on tumor biomarkers, helping to find early-stage cancer; however, most reported approaches are highly specific and do not allow for the diagnosis of a wide range of cancers simultaneously. Building on previous work, researchers at the University of Texas (UT) Southwestern Medical Center have now developed transistor-like nanoprobes that can illuminate solid tumors with fluorescent light when they detect a universal hallmark of cancer: an acidic microenvironment. In the new study, published in Nature Biomedical Engineering, the nanoprobes were able to detect and image a broad range of tumors in mice—from head and neck to breast to brain tumors—and allow for real-time image-guided tumor surgery.

“We are seeing something that is unique,” says study co-lead author Jinming Gao, a UT Southwestern professor of oncology, pharmacology, and otolaryngology, referring to the nanoprobe’s binary response to cancer pH. “It is the conceptual realization of a transistor kind of pH-threshold sensor that can digitize tumor pH. This binary activity could improve the robustness of technology for the visualization and surgery of tumors.”

In recent years, scientists have developed nanoparticles that target specific cancer biomarkers, particularly cell-surface receptors, such as chlorotoxin, epidermal growth factor receptor (EGFR), and human epidermal growth factor receptor 2 (Her2/neu). Though these techniques could successfully steer patients toward personalized therapy, their specificity does not allow them to detect a broad range of tumors or even all patients with the same cancer (Her2/neu, for instance, is expressed in less than 25% of breast cancer patients). “Once you decide on a specific cell-surface receptor, you are limiting yourself to a specific class of tumors with that histological phenotype,” Gao says.

In previous work, Gao and his colleagues instead focused on cancer pH. Cancer cells are known to take up glucose and convert it to lactic acid, which ultimately makes the microenvironment around the cells more acidic. The pH of blood, for example, is 7.4, while the pH of tumors is typically between 6.5 and 6.9. Gao and his colleagues developed nanoparticles—consisting of an ultra pH-sensitive core, fluorophores, and a targeting molecule that binds to angiogenic cell surface receptors on the tumor vasculature—that could detect tumor pH. But the design was a bit too complicated for clinical translation and the nanoparticles used a dye (Cy5.5) that was not FDA-approved for clinical use.

For the new nanoprobes, the team stripped down the design by removing the unnecessary targeting ligand and replaced the Cy5.5 dye with indocyanine green (ICG), an FDA-approved fluorophore. The new pH-activatable ICG-encoded nanosensor, or PINS, has a sharper pH response and deeper fluorescence penetration in tissues than their team’s previous design—and can be used with clinical cameras. 

To create their nanoprobes, Gao and his team first synthesized block copolymers of poly(ethylene glycol) -b-poly(thylpropylamioethyl methacrylate), or PEG-b-P(EPAx-r-ICGy), using a polymer synthesis method called atom transfer radical polymerization.  They conjugated the ICG dye to the primary amino groups of the copolymer, and then used a solvent evaporation method to create the PINS nanoprobes. The nanoprobes were either concentrated to an aqueous stock solution or freeze-dyed with lyoprotectants for later use. In tests, PINS sharply activated at pH 6.9 and below; at pH above 6.9, however, the nanoprobes did not fluoresce due to hydrophobic micellization (the aggregation of surfactant molecules) combined with quenching induced by homo-fluorescence resonance energy transfer. This activity is similar to a transistor that switches on when voltage exceeds a certain threshold. 

Using mouse models, the researchers tested PINS in about 15 types of malignant tumors, Gao says. Specifically, they tested PINS in head and neck, breast, kidney, pancreatic, and brain tumor models, as well as a model of peritoneal metastasis from colorectal cancer. “They all worked out pretty well,” he says, adding that their tests did not show a negative immune reaction even after 5 repeated injections 10 times the optimal imaging dose.

With the clinically used SPY Elite Fluorescence Imaging System, they used PINS for real-time tumor-acidosis-guided surgery in mice bearing head and neck or breast cancers. The PINS, which were injected 12 to 24 hours before surgery, allowed them to not only resect primary tumors, but also residual tumor nodules smaller than 1 mm³ that were not visible under white light—this additional detection helped improve the mice’s odds of long-term survival compared with white light surgery, they found. “It helps visualization quite a bit,” Gao says. “Just [with] our naked eyes, it’s hard to distinguish the tumor from underneath the normal tissue.”

“The groundbreaking breakthrough brought a new concept in the design of nanosensors to the imaging-probe community, which will not be limited to [designing] pH-activatable probes but might be translated to develop chemical sensors with biological significances,” says Jie Zheng, a nanomaterial and bioimaging researcher at the University of Texas at Dallas, who was not involved in the study. He adds that it will be interesting to see if the researchers can develop nanosensors with other pH thresholds and if the nanoprobe’s targeting efficiency can be further improved. “I think the whole community looks forward to seeing the applications of [these] nanoprobes in clinical trials very soon,” he says.

Gao expects that PINS will find broad applications in detecting, imaging, and surgery of cancers. But, he notes, there are certain situations in which the nanoprobes may not work, such as with benign tumors (which may not be metabolically active enough to reduce pH) or people with metabolic diseases like ketoacidosis, which can lower blood pH—more research is needed to test these situations. “We are pushing forward to use the optical agents for surgical resection and may be starting clinical trials this summer,” he says.

Read the article in Nature Biomedical Engineering.