Blood–brain barrier best breached by small molecules

Focused ultrasound paired with microbubbles can get drugs into the brain, but no intensity of sonication allows large molecules to penetrate very far

Original source: Physics World

Focused ultrasound (FUS) can be used to help drugs pass from the bloodstream into the brain, but the technique’s effectiveness depends on the ultrasound pressure and the size of the drug molecules. Michael Valdez and colleagues at the University of Arizona measured how thoroughly differently sized molecules diffused into mouse brains under a range of ultrasound intensities, and found that the largest molecules could not be delivered under any safe FUS regime. The results set a limit on the types of drugs that might one day be used to treat neurological conditions like Alzheimer’s and Parkinson’s disease (Ultrasound Med. Biol. 10.1016/j.ultrasmedbio.2019.08.024).

Usually, the brain is isolated from substances circulating in the bloodstream by the blood–brain barrier (BBB), a semipermeable layer of cells that permits only certain molecules to pass. This restricts the range of drugs that can be used in the brain to small, hydrophobic molecules (such as alcohol and caffeine), other small drugs like psychotropics and some antibiotics. Extending that range would open the door to new therapeutic possibilities, says Theodore Trouard, who led the team. “The ability to temporarily and safely open the BBB to allow drugs into the brain would help address a number of neurological diseases for which there is currently no effective treatment.”

Previous research has shown that such opening can be achieved by focusing an ultrasound beam in the brain while gas microbubbles circulate in the blood. The microbubbles – perfluorocarbon-filled lipid shells about 1 µm across – are inert while they move around the body, but rapidly expand and contract in the local pressure fluctuations caused by the ultrasound field. Mechanical forces exerted by this phenomenon create temporary gaps in the layer of cells that make up the BBB, giving larger molecules a chance to breach the brain’s defences.

More intense ultrasound fields produce a greater effect than weaker fields, and smaller molecules are more likely to diffuse into the brain than larger molecules. To quantify the relationship, Valdez and colleagues injected mice with dextran solutions containing molecules of three different weights: 3, 70 and 500 kilodaltons. They then administered a solution of microbubbles and subjected each mouse to FUS at one of three intensity levels.

When the procedure was complete, the researchers injected a fixing agent to preserve the distribution of the dextran molecules, then removed and sliced the mice’s brains for study. The method echoes past investigations conducted along similar lines, but in the earlier studies, each animal was given just one size of molecule, meaning inherent physiological differences between individuals made the results less certain. This time, Valdez and colleagues labelled each size of dextran molecule with a specific fluorescent marker, so that the distribution of all three could be measured simultaneously in each individual mouse.

Examining the brain slices using fluorescence microscopy, the researchers found that higher ultrasound pressures allowed the dextran to perfuse larger volumes of brain tissue. They noted, however, that the largest (500 kDa) molecules failed to penetrate the brain whatever the ultrasound intensity, and even the intermediate-weight (70 kDa) molecules only spread over small volumes.

Localized “hotspots” on the fluorescence images suggested that, though these dextran molecules had managed to breach the cells of the BBB, their size prevented them from diffusing further into the brain parenchyma. If large molecules like antibodies and other therapeutic proteins are restricted to the parts of the brain immediately next to where they exit the blood vessel, clinicians will need to get around the BBB at multiple sites simultaneously in order to access sufficient volumes of brain tissue.

Along with the three varieties of dextran, Valdez and colleagues also injected each mouse with a gadolinium-based MRI contrast agent. In some trials of the microbubble–FUS technique conducted until now, researchers have used this contrast as an easily detectable proxy for the therapeutic compound, confirming with MRI the volume perfused by the drug. Trouard’s team, however, showed that the small molecular weight of the contrast agent means that it diffuses into the brain much more readily than larger drug molecules, making it only a surrogate indicator of a procedure’s success.

Next, says Trouard, the researchers will investigate whether the BBB can be breached by a specific antibody that targets a protein associated with Parkinson’s disease, and whether it has any effect on the condition’s symptoms in mice.