(M1130-12-81) Detection of Voriconazole Nanoaggregates in Rat Lung by Mass Spectrometry Imaging after Dry Powder Insufflation

Purpose: Thin film freezing (TFF) is a technology utilizing ultra-rapid freezing that can prevent particle growth during the freezing process, being able to produce nanoaggregates of high potency voriconazole dry powder. The nanoaggregates are expected to have advantages in distribution and absorption in the lungs when delivered as a dry powder through the pulmonary route. The objective of this study is to develop a method to visualize and quantitate voriconazole deposited in rat lungs after delivering high potency voriconazole nanoaggregates made by TFF and administered by dry powder insufflation. Specifically, Mass Spectrometry Imaging (MSI) was used to map the spatial distribution of voriconazole molecules from lung samples of rats.

Methods: The dry powder of voriconazole nanoaggregates were prepared by TFF. Voriconazole/Leucine (95/5 w/w) were dissolved in water/acetonitrile (50/50 v/v), and the solution was rapidly frozen by TFF technology, and the frozen sample was dried using a shelf lyophilizer (SP Scientific, Gardner, NY). Voriconazole nanoaggregates were sieved (#120) and the powder was insufflated into the lungs (10 mg/kg) using DP-4 dry powder insufflator (Penn-Century Inc. Philadelphia, PA) connected to the AP-1 air pump (Penn-Century Inc. Philadelphia, PA). Lung sample was collected post-mortem at 15 min after insufflation. The airways of left lung were rinsed with PBS, followed by 2% w/v gelatin solution. The left lung was then inflated with 2% w/v gelatin solution before freezing it by the vapor of liquid nitrogen. The left lung was sectioned at 12 microns thickness and collected onto a glass microscope slide. A serial section was collected for hematoxylin and eosin (H&E) staining. The section was coated with 2,5-dihydroxybenzoic acid (40 mg/mL) in 70% v/v acetonitrile and water with 0.1% TFA solution using an HTX M5 Robotic Sprayer (HTX Technologies, LLC, Chapel Hill, NC). The image was collected in positive ion mode at 100 μm resolution using Bruker timsTOF flex MALDI QTOF mass spectrometer (Bruker, Billerica, MA). The images were visualized using SCiLS^TM Lab software. After imaging, the matrix was removed, and the section was stained with H&E.

Results: Voriconazole was readily detected in the rat lung by MALDI-MSI in positive ion mode at a resolution of 100 μm without any background interference. The highest levels of voriconazole were detected in airways (tertiary bronchus and bronchioles) and in the lower lung. The H&E staining image showed bronchi and bronchioles. Non-lung tissue was not detected in the section after H&E staining, presenting that all voriconazole signals detected are from the voriconazole absorbed in the lung tissue.

Conclusion: The dry powder of voriconazole nanoaggregates made by TFF was efficiently insufflated into rat lungs, and inflating the lungs with 2% gelatin solution limited exogenous variations, leading to successful visualization and measurement of voriconazole levels in lung tissue by Mass Spectrometry Imaging. Voriconazole was detected throughout the lung section after insufflation, indicating that the dry powder insufflation and administration of voriconazole nanoaggregates were spatially distributed throughout the lungs and absorbed into the lung tissue of the deep lung very well.

References: Moon C, Sahakijpijarn S, Koleng JJ, Williams III RO. Processing design space is critical for voriconazole nanoaggregates for dry powder inhalation produced by thin film freezing. J Drug Deliv Sci Technol. 2019;54:101295.
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Scott AJ, Chandler CE, Ellis SR, Heeren RMA, Ernst RK. Maintenance of Deep Lung Architecture and Automated Airway Segmentation for 3D Mass Spectrometry Imaging. Sci Rep. 2019;9(1):20160.

Acknowledgements:

Funding: This research was funded by TFF Pharmaceuticals, Inc. through a sponsored research agreement at The University of Texas at Austin. Mass Spectrometry Imaging facility at The University of Texas at Austin was funded by Cancer Prevention & Research Institute of Texas. Conflicts of Interest: Williams and Moon report a relationship with TFF Pharmaceuticals, Inc. that includes consulting or advisory, equity or stocks.

Figure 1. MSI of voriconazole in section, left lung of rat (left), and H&E staining of the section, post-MSI (right)