Multiplexed Breath Biopsy of Lung Disease by Controlled Chemical Signal Activation

Introduction:: Breath testing is a fast, non-invasive diagnostic method that can link specific volatile organic compounds (VOCs) in exhaled breath to medical conditions. Complementary to blood and urine assays in clinical disease diagnosis, breath is an informative analyte that can provide real-time information of changes in the body’s metabolome affected by disease activity. However, few breath tests are currently used in the clinic to monitor disease due to bottlenecks in biomarker identification and differentiation of endogenous VOCs in complex breath signatures. One approach to overcome this challenge is the in vivo administration of orthogonal exogenous agents with controlled properties that can be expelled from breath upon being metabolized by disease-specific molecular processes. Exogenous agents have been used in clinical breath tests for gastrointestinal and liver diseases and are more recently developed for respiratory disease.^1-3 As the direct source of breath, the lungs are particularly suited for enzymatic activity-based breath tests. The multiplexed capabilities of designing reliable in vivo probes that release detectable and distinguishable exogenous VOCs are still in the early stages. Here, we report engineered breath biomarkers by protease-sensing nanoparticles for multiplexed detection of dysregulated activities of pulmonary host proteases in response to disease and produce volatile reporters. We show that the nature of proteolytic hydrolysis generating peptide fragments can be engineered to activate and release different types (amine and alcohol) of volatile reporters. Using viral infection and Alk-mutant lung cancer mouse models, we show that multiplexed volatile-releasing activity-based nanosensors (vABNs) can detect and dynamically monitor the diseases.


Materials and Methods:: Engineered breath biomarkers formulated as protease-sensing nanoparticles are prepared by conjugation of volatile modified peptide substrates onto a polyethylene glycol (PEG) nanocarrier. The substrates are synthesized by CPC Scientific by solid phase peptide synthesis, of which amine- and alcohol-based volatile reporters are attached to the peptide C-termini. These volatile-releasing activity-based nanosensors (vABNs) are delivered into the lungs of mouse models via intratracheal instillation. A viral infection influenza A/PR/8/34 [H1N1] [PR8]) and an ALK⁺ non-small cell lung cancer mouse models are used, as described in previous works.^4,5 At 10, 30, and 60 minutes after vABN administration, breath is collected by placing mice in an in-house apparatus consisting of a 100 ml syringe and a stopcock valve with needle. The syringe is sealed for 2 minutes to allow breath to accumulate in the syringe headspace, followed by displacing ~10 ml of headspace into the exetainers. The reporter signals in breath samples are measured by a proton transfer reaction–mass spectrometer (Ionicon). To create suitable multiplexed vABNs, we generate viable peptide substrates that are responsible for disease-related proteases nominated from literature and substrates that had been previously optimized. Prior to generating in vivo vABNs, we create Förster resonance energy transfer (FRET)-paired and volatile-conjugated peptide panels to screen against select recombinant proteases and in bronchiolar lavage fluids collected from the mouse models, which preserve protease activities analogous to the in vivo lung environments. We analyze both volatile release in the headspace and desired cleaved peptide fragments in solution by mass spectrometry.


Results, Conclusions, and Discussions:: To multiplex, we develop vABNs to have two types of volatile release mechanisms in response to protease cleavage to emit amine- and alcohol-based volatile reporters. The mechanisms essentially leverage the formation of peptide fragments upon protease cleavage; in the former, amine-based reporters attached to the peptide C-termini via amide bonds are subsequently released. In the latter, the exposed N^α group spontaneously induced an intramolecular reaction that acts to release alcohol reporters as part of the reaction products. Such intramolecular reactions activated by specific hydrolytic sites of protease cleavage can be temporally controlled by designed peptide sequences and conformational stability. Using mouse models of viral infection and lung cancer, we show that the two types and up to five of vABNs with different substrate-VOC pairs can function orthogonally in a single pool and noninvasively detect and dynamically monitor the in vivo activities of select host proteases in response to disease progression and treatment response. After intrapulmonary delivery into the mouse models, the activated volatile reporters are expelled in breath and can be simultaneously detected by mass spectrometry with high sensitivity and as early as 10 minutes after vABN administration. This multiplexing approach of synthetic biomarkers for monitoring diseases in breath has the potential to offer new methods for disease classification and should be broadly amenable to additional pathophysiological processes and point-of-care diagnostics.


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