Ultrasound and Photoacoustic Imaging of the Brain via Laser-Activated Perfluorocarbon Nanodroplets

Introduction:: Treatments for diseases of the central nervous system, such as neurodegenerative diseases and brain tumors, face incredible challenges including the delivery of drugs to intracerebral targets. An obstacle in this area is the blood-brain barrier (BBB), a size-restricting endothelial filter that prevents the passage of many intravenous drugs into the brain. Recent innovations in focused ultrasound have permitted transient opening of this barrier, but there is still a lack of methods for validating that drugs have reached their target sites. A potential and exciting tool to address these challenges are perfluorocarbon nanodroplets (PFCnDs), nano-scale emulsions capable of dynamic contrast for ultrasound and photoacoustic (US/PA) imaging, blood-brain barrier (BBB) opening, drugs delivery, and more. PFCnDs function by repeatably phase-changing between liquid nanodroplets and gas microbubbles when remotely activated by acoustic or optical energy. Recently, PFCnDs, activated using pulsed laser light, have been demonstrated for use in multiplex imaging, which involves uniquely labelling subpopulations of nanodroplets with distinct optical dyes such that these color-coded groups can be independently activated. In this study, we introduce the combination of PFCnD-based BBB opening and multiplex imaging of PFCnDs within the brain that have extravasated through the opened BBB. We demonstrate our approach in mice using in vivo and ex vivo US/PA imaging of the brain and validate our findings using immunohistology of IgG distribution – an endogenous molecule that can only extravasate into the brain after BBB opening.

Materials and Methods:: Two species of lipid-shelled nanodroplets were synthesized, perfluorohexane nanodroplets (PFHnDs) and perfluoropentane nanodroplets (PFPnDs) loaded with dyes having a peak of 1064nm and 760 nm, respectively (Fig. 1A). The droplet’s absorbance spectrums were analyzed using a spectrophotometer (Evolution 220, Thermo Scientific) (Fig. 1B). PFHnDs are used for BBB opening, because they remain stable after repeated phase-changes, and PFPnDs are used because they vaporize at lower laser energies, making them ideal for US/PA imaging. In mice, PFHnDs were injected and then laser-activated on the right side of the brain for blood brain barrier opening (Fig. 1C). When vaporized, PFHnDs release a portion of 1064 nm dye. Right after BBB opening, PFPnDs were injected. Next, US/PA imaging (Vevo LAZR equipped with LZ-250 and LZ-550 transducers) was performed four hours later, in vivo and then ex vivo to visualize extravasation of PFPnDs (780 nm wavelength) and 1064 nm dye (Fig. 1D). We confirm the location of PFPnDs in in vivo US imaging based on the unique temporal US signal of PFPnDs’ vaporization (Fig. 1D). To validate the US/PA imaging results, we used immunohistology to characterize the spatial distribution of IgG molecules that penetrate the BBB after PFHnD-induced opening by staining with DAPI and anti-IgG (Fig. 1E). Finally, ex vivo US/PA images were acquired and processed with AMIRA (Thermo Scientific) to generate whole brain 3D images (Fig. 1F).

Results, Conclusions, and Discussions:: Results and Discussion: Spectral analysis of the droplets shows that the synthesized PFPnDs and PFHnDs have a peak absorption at 760 nm and 1064 nm, respectively. Therefore, the droplets can independently be activated by two distinct wavelengths (Fig. 1B). In vivo US/PA multiplexed imaging shows US and PA signal below the skull, localized to the treated (right) side of the brain (Fig. 1D). PA imaging with a 760 nm and 1064 nm wavelengths shows spatially overlapping signals corresponding to PFPnDs (blue) and 1064 nm dye released from PFHnDs (red), respectively. Further, with US imaging, there is an echogenic region in the same region that corresponds to the dynamic PFPnDs detected signa (yellow). Next, fluorescence microscopy of brain sections shows minimal fluorescence from IgG seen in the untreated (left) side of the brain, but on treated side, there is high IgG fluorescence in agreement with the US/PA imaging results (Fig. 1E). Next, 3D US/PA multiplexed imaging ex vivo was performed revealing the three-dimensional localization of 760 nm PFPnDs (blue) and 1064 nm dye (red) on the treated side of the brain (purple shows overlap) (Fig. 1F).

Conclusion: By utilizing two populations of color-coded PFCnDs activated at different wavelengths (1064 nm and 780 nm) and composed of different types of perfluorocarbons (PFP and PFH), both BBB opening and in vivo US/PA imaging of the brain were achieved. Together, these results elucidate the utility of multiplexed PFCnDs as tool for US/PA neuroimaging and provide a foundation for future work in diagnostics and image-guided drug delivery.


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