MIT engineers have designed nanoparticle sensors that can diagnose lung diseases. If a disease-associated protein is present in the lungs, the protein cleaves a gaseous molecule from the nanoparticle, and this gas can be detected in the patient’s breath. [Cygny Malvar]

MIT engineers have developed a nanoparticle sensor system that can detect and monitor lung diseases by measuring compounds exhaled in the breath. Initial studies in mice demonstrated use of the technology to detect pneumonia and the genetic disorder alpha-1 antitrypsin deficiency, but the team said the same approach could one day also be used for other diseases, including lung infections such as coronavirus.

“We envision that this technology would allow you to inhale a sensor and then breathe out a volatile gas in about 10 minutes that reports on the status of your lungs and whether the medicines you are taking are working,” said Sangeeta Bhatia, PhD, the John and Dorothy Wilson professor of health sciences and technology and electrical engineering and computer science at MIT.

Bhatia, who is also a member of MIT’s Koch Institute for Integrative Cancer Research and the Institute for Medical Engineering and Science, is the senior author of the team’s published paper in Nature Nanotechnology, which is titled, “Engineering synthetic breath biomarkers for respiratory disease.” The first author of the paper is MIT senior postdoc Leslie Chan, PhD. Other authors are MIT graduate student Melodi Anahtar, MIT Lincoln Lung Laboratory technical staff member Ta-Hsuan Ong, MIT technical assistant Kelsey Hern, and Lincoln Laboratory associate group leader Roderick Kunz.

Our breath contains many different volatile compounds, but exploiting these compounds for clinical diagnostic applications has been a “challenging and slow process,” the authors noted. “… breath is a practical and potentially informative clinical analyte because it can be sampled non-invasively and contains hundreds of trace volatile organic compounds (VOCs) that are produced in the body as metabolites or from environmental exposure,” they wrote. “However, few breath tests are currently used in the clinic to monitor disease due to bottlenecks in biomarker identification.”

One potential approach to overcoming current challenges is to administer a form of nanosensor that will be metabolized by disease-specific molecular processes into detectable volatile products. This strategy is used, for example, in the 13C-urea breath test for Helicobacter pylori detection, and the 13C methacetin breath test for liver fibrosis. To carry out these tests patients ingest isotope-labeled small molecules, which are metabolized by relevant enzymes into 13CO2. “These clinical tests and others in development leverage known enzyme biology to produce breath read-outs, and in the case of 13C breath tests, produce a volatile that is not naturally found in the body, thereby reducing signal-to-noise ratio (SNR),” the investigators further explained.

For several years, Bhatia’s lab has been working on nanoparticle sensors that can be used as synthetic biomarkers. These markers are peptides that are not naturally produced by the body but are released from nanoparticles when they encounter proteins called proteases. The peptides coating the nanoparticles can be customized so that they are cleaved by different proteases that are linked to a variety of diseases. When a peptide is cleaved from the nanoparticle by a protease it is then later excreted in the urine, where it can be detected with a strip of paper similar to a pregnancy test. Bhatia has developed this type of urine test for pneumonia, ovarian cancer, lung cancer, and other diseases.

More recently, she turned her attention to developing biomarkers that could be detected in the breath rather than in the urine. This would allow test results to be obtained more rapidly, and it also avoids the potential difficulty of having to acquire a urine sample from patients who might be dehydrated, for example, Bhatia said. The investigators realized that by chemically modifying the peptides attached to the synthetic nanoparticles, they could enable the particles to release gases called hydrofluoroamines (HFAs) that would be exhaled in the breath. The researchers attached volatile molecules to the end of the peptides in such a way that when the disease-associated protease cleaves the peptides, the volatiles are released into the air as a gas, and breathed out.

Working with Kunz and Ong at Lincoln Laboratory, Bhatia and her team devised a method for detecting the gas from the breath using mass spectrometry. The researchers then tested their nanosensors, which they called volatile-releasing activity-based nanosensors (vABNs), in mouse models of two diseases, bacterial pneumonia caused by Pseudomonas aeruginosa, and the genetic disorder alpha-1 antitrypsin deficiency. Both diseases trigger inflammatory responses that are linked with the production of protease called neutrophil elastase (NE) by immune cells. It is this enzyme that cleaves the volatile compounds from the nanosensors administered to the mice, which are then detected in the breath using mass spectrometry.

“The nanosensors shed volatile reporters upon cleavage by neutrophil elastase, an inflammation-associated protease with elevated activity in lung diseases such as bacterial infection and alpha-1 antitrypsin deficiency,” the team stated. “After intrapulmonary delivery into mouse models with acute lung inflammation, the volatile reporters are released and expelled in breath at levels detectable by mass spectrometry.”

For both pneumonia and alpha-1 antitrypsin deficiency, the researchers showed that they could detect neutrophil elastase activity within about 10 minutes. Additional studies also demonstrated that the sensors could be used to monitor the effectiveness of drug treatment for both the disorders. “Using these nanosensors, we performed serial breath tests to monitor dynamic changes in neutrophil elastase activity during lung infection and to assess the efficacy of a protease inhibitor therapy targeting neutrophil elastase for the treatment of alpha-1 antitrypsin deficiency.”

For their reported tests the researchers used nanoparticles that were injected intratracheally, but they are working on a version that could be inhaled using a device similar to the inhalers used to treat asthma. Bhatia’s lab is also designing new devices for detecting the exhaled sensors that would be easier to use, potentially even allowing patients to use them at home. “Right now we’re using mass spectrometry as a detector, but in the next generation we’ve been thinking about whether we can make a smart mirror, where you breathe on the mirror, or make something that would work like a car breathalyzer,” Bhatia said. Her lab is also working to develop sensors that could detect more than one type of protease at a time. These nanosensors could be designed to reveal the presence of proteases associated with specific pathogens, potentially including SARS-CoV-2.

The authors acknowledged that further safety testing will be needed before their approach could be used in humans, although in their mouse studies there was no sign of lung toxicity. Nevertheless, they commented, “HFAs have not been used in humans and should be further characterized.”

Breath tests are practical diagnostic tools due to the ease and non-invasive nature of breath sampling, the scientists noted. There are a number of commercial sampling tools that could be used to streamline breath detection, and sensitive, miniaturized gas analysis tools are also available that could be used to bring breath analysis to point-of-care use. And while the rate-limiting step in the translation of breath volatiles to clinical diagnostics is the identification of disease-specific breath biomarkers, the team said their studies illustrate how breath biomarkers can be engineered through the combined application of disease biology and stimulus-responsive nanomaterials.

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