Poster X16 - Delivery of ultrasound-sensitive, CO-loaded microbubble for repairing the blood brain barrier

Introduction: The endothelial cells (ECs) in the blood brain barrier (BBB) are disrupted with age, genetic predisposition, or stroke, and this can lead to vascular cognitive impairment and dementia (VCID). Current treatments approaches involve opening the BBB to deliver drugs to alleviate the cognitive symptoms, but our unique delivery strategy treats the fundamental cause of the disease by promoting brain capillary EC (BCEC) proliferation and restoration of the BCEC tight junctions. Specifically, we are delivering the gasotransmitter carbon monoxide (CO) from ultrasound-sensitive microbubbles (MBs) to restore the BBB integrity. Previously, we computationally determined that the amount of free, MB-released CO should be able to positively impact the ECs at the BBB. However, not all MBs are expected to rupture with low-frequency ultrasound, and unruptured MBs can potentially be endocytosed by the BCECs at the BBB and release CO while inside the cells. In this study, both experimental and computational analyses are used to determine the unruptured MBs that could reach the BBB for endocytosis.

Materials and

Methods: We prepared perfluoropentane-based MBs that could be used for this strategy through a nanoprecipitation method (1.30±0.40 µm) and loaded them with CO. Then we designed a gelatin-based phantom with a 3 mm diameter embedded straw and applied ultrasound with variable ultrasound probe intensities to experimentally test the effects on the MBs. GFP+ human umbilical ECs (HUVECs) were cultured and imaged with time-lapse, confocal microscopy to investigate the MBs endocytosis rate. One-way ANOVA and Tukey comparisons were performed for experimental data. We also modeled the MBs in silico with particle tracking as their movement was driven by pulsatile blood flow, buoyancy, Brownian motion with drag, and /or ultrasound acoustic pressure. The analysis of the results included the amount of MBs that reach the ECs at the BBB arterial wall after ultrasound activation in the internal carotid artery (ICA). For pulsatile blood flow driven movement, we released 100 MBs per 0.1 s over 6 s. For ultrasound directed MB movement, there was a single initial release of 9 MBs. Frequencies of 3, 7 and 18 MHz were simulated to determine their effects on the MBs.

Results, Conclusions, and Discussions: We experimentally determined MBs movement, velocity, and rupture using an 18 MHz transducer with different intensities. As expected, the normalized number of ruptured MBs systematically increased with increasing percent intensity and was significantly higher for 100% intensity. Further, our analysis showed average and maximum speeds of 10.2 mm/s and 111 mm/s, respectively, caused only by application of ultrasound. This resulted in MBs displacements of 0.1 - 3.5 mm over 30 s with application of 18 MHz ultrasound. The in-silico model for acoustic pressure showed comparable MB displacements (i.e., 0.1 - 0.6 mm) with application of ultrasound. In these simulation, the MBs moved faster within the application region with increasing ultrasound frequency. This MB movement along the radial direction could impact the proximity to the wall and how the MBs are carried along with blood flow. The in-silico model for hemodynamics with pulsatile blood flow yielded an average and maximum of 67.0 and 68.3 mm/s MBs speed which is slightly higher than the experimental ultrasound-induced movement. Pulsatile blood flow also shows that buoyancy and the direction of gravity can have an impact in the percentage of MBs that reach the BBB, but only for the larger MBs (i.e., 3–5µm). Our ongoing computational studies are combining ultrasound-induced and blood flow-induced MBs movement to determine the number of MBs available to be endocytosed by BCECs at the BBB. Our preliminary experimental studies show particles with diameters between 0.82-6.3 µm accumulating within ECs but not the larger agglomerates. Overall, this study shows that CO can reach the BCECs at the BBB through CO-loaded MBs that can be endocytosed. Further, this study shows that linking traditional ultrasound analysis with MB movement may be important. Finally, this approach shows potential as a treatment to restore the BBB, although further research is needed.

Acknowledgements (Optional): This study was supported by a Brevard Foundation Medical Research Grant.