Assistant Professor Georgia Institute of Technology, United States
Introduction:: Despite their non-invasiveness and speed, breath tests have limited utility in clinical diagnostics due to a scarcity of breath biomarkers. Thousands of volatile organic compounds (VOCs) are exhaled in breath [1]. However, breath biomarker discovery using conventional -omic approaches have failed to identify disease-specific VOCs [2]. As an alternative to biomarker discovery, we have devised a method to engineer breath biomarkers by leveraging disease-associated protease activity to trigger the release of VOC cargos from activity-based nanosensors. Our nanosensors consist of volatile reporter molecules that are tethered to a nanocarrier through protease-cleavable peptide linkers (Fig. 1). After in vivo delivery, peptide linkers are cleaved by dysregulated proteases in diseased tissues. Liberated reporters are subsequently cleared from the body in breath, quantified via mass spectrometry, and used to indicate the presence or absence of disease. Here, we discuss applications of this platform for respiratory disease and the recent development of multiplexing capabilities.
Materials and Methods:: In initial feasibility studies, we sought to develop breath biomarkers to monitor the activity of neutrophil elastase (NE), a protease with elevated activity in the lungs during inflammatory respiratory diseases. Nanosensors for NE activity were synthesized by conjugating a volatile reporter to the cleavage site of an NE peptide substrate. Peptides were conjugated to 8-arm polyethylene glycol-maleimide via a cysteine residue. Excess peptides were removed via spin filtration. To characterize volatile release from nanosensors, cleavage reactions with NE and other proteases were carried out in glass vials with rubber septa caps for headspace sampling using syringes. Once NE-triggered reporter release was confirmed in vitro, nanosensors were administered via intratracheal instillation into 2 different mouse models for diseases known to have elevated NE activity – lung infection and α1-antitrypsin deficiency. Breath samples were collected by placing each mouse into a sealed chamber for a fixed amount of time after nanosensor delivery, after which, the headspace was displaced into evacuated glass vials for analysis by mass spectrometry.
Results, Conclusions, and Discussions:: Results & Discussion: Intrapulmonary delivery of NE nanosensors resulted in exhaled reporter levels corresponding to pulmonary NE activity. Peak breath signal was observed 10 min after nanosensor administration and was elevated in lung infection models compared to healthy controls. Intrapulmonary delivery of a small molecule NE inhibitor before nanosensor administration reduced breath signal in lung infection models, confirming that breath signal was driven specifically by NE activity. In antibiotic-treated infection models, we showed that nanosensors could be administered at multiple timepoints to monitor the dynamics of NE activity via breath analysis (Fig. 2). Nanosensor-induced breath signal was also used to detect tissue-damaging NE activity and monitor protease inhibitor therapy in mouse models of α1-antitrypsin deficiency. With success of these initial studies, we have since moved on to develop nanosensors for multiplexed protease sensing. More recently, we showed that VOCs of distinct mass can be used to barcode nanosensors for different proteases (Fig. 3). This powerful capability enables us to create breath biomarker signatures for highly specific disease detection.
Conclusion: Breath biomarkers can be rationally engineered through the delivery and controlled release of volatile molecules. Using this approach, we can enable non-invasive detection and monitoring of diverse diseases through simple breath tests.
Acknowledgements (Optional): : This work was supported by a K99/R00 Pathway to Independence Award (NIH EB028311).
References (Optional): : 1. Pauling, L., Robinson, A. B., Teranishi, R. & Cary, P. Quantitative analysis of urine vapor and breath by gas-liquid partition chromatography. Proc. Natl. Acad. Sci.68, 2374–2376 (1971).
2. Gaude, E. et al. Targeted breath analysis: exogenous volatile organic compounds (EVOC) as metabolic pathway-specific probes. J. Breath Res.13, 032001 (2019).
3. Chan, L. W. et al. Engineering synthetic breath biomarkers for respiratory disease. Nat. Nanotechnol.15, 792–800 (2020).
