We acknowledge support by the Estate of Dr Clem Jones AO, the National Health and Medical Research Council of Australia [GNT1145580, GNT1176326], the Metal Foundation, and the State Government of Queensland (DSITI, Department of Science, Information Technology and Innovation).

All animal experiments were approved by the animal ethics committee of the University of Queensland.
1. In-house microbubble preparation
<ol>
	<li>Weigh out a 9:1 molar ratio of 1,2-distearoyl-sn-glycero-3-phosphocholine and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000] (ammonium salt). 0.5 mg of lipid mixture is required per 1 mL of microbubble solution. Alternatively, lipids can be bought already in chloroform, if using pre-dissolved lipids proceed to s...

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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+and+transient+blood-brain+barrier+opening+in+the+hippocampus+of+Alzheimer's+double+transgenic+mice+using+focused+ultrasound.">Noninvasive and transient blood-brain barrier opening in the hippocampus of Alzheimer's double transgenic mice using focused ultrasound.</a> Ultrasonic Imaging. 30 (3), 189-200 (2008).</li><li>Lipsman, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood-brain+barrier+opening+in+Alzheimer's+disease+using+MR-guided+focused+ultrasound.">Blood-brain barrier opening in Alzheimer's disease using MR-guided focused ultrasound.</a> Nature Communications. 9 (1), 2336 (2018).</li><li>Pandit, R., Chen, L., G&#246;tz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+blood-brain+barrier:+physiology+and+strategies+for+drug+delivery.">The blood-brain barrier: physiology and strategies for drug delivery.</a> Advanced Drug Delivery Reviews. (19), 30238 (2019).</li><li>Golde, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open+questions+for+Alzheimer's+disease+immunotherapy.">Open questions for Alzheimer's disease immunotherapy.</a> Alzheimers Research &amp; Therapy. 6 (1), 3 (2014).</li><li>Nisbet, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+effects+of+scanning+ultrasound+and+a+tau-specific+single+chain+antibody+in+a+tau+transgenic+mouse+model.">Combined effects of scanning ultrasound and a tau-specific single chain antibody in a tau transgenic mouse model.</a> Brain. 140 (5), 1220-1230 (2017).</li><li>Janowicz, P. W., Leinenga, G., G&#246;tz, J., Nisbet, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound-mediated+blood-brain+barrier+opening+enhances+delivery+of+therapeutically+relevant+formats+of+a+tau-specific+antibody.">Ultrasound-mediated blood-brain barrier opening enhances delivery of therapeutically relevant formats of a tau-specific antibody.</a> Scientific Reports. 9 (1), 9255 (2019).</li><li>Leinenga, G., G&#246;tz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scanning+ultrasound+removes+amyloid-beta+and+restores+memory+in+an+Alzheimer's+disease+mouse+model.">Scanning ultrasound removes amyloid-beta and restores memory in an Alzheimer's disease mouse model.</a> Science Translational Medicine. 7 (278), 233 (2015).</li><li>Burgess, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+delivery+of+neural+stem+cells+to+the+brain+using+MRI-guided+focused+ultrasound+to+disrupt+the+blood-brain+barrier.">Targeted delivery of neural stem cells to the brain using MRI-guided focused ultrasound to disrupt the blood-brain barrier.</a> PLoS One. 6 (11), 27877 (2011).</li><li>Chen, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focused+ultrasound-enhanced+intranasal+brain+delivery+of+brain-derived+neurotrophic+factor.">Focused ultrasound-enhanced intranasal brain delivery of brain-derived neurotrophic factor.</a> Scientific Reports. 6, 28599 (2016).</li><li>Leinenga, G., Langton, C., Nisbet, R., G&#246;tz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound+treatment+of+neurological+diseases+-+current+and+emerging+applications.">Ultrasound treatment of neurological diseases - current and emerging applications.</a> Nature Reviews Neurology. 12 (3), 161-174 (2016).</li><li>G&#246;tz, J., Halliday, G., Nisbet, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Pathogenesis+of+the+Tauopathies.">Molecular Pathogenesis of the Tauopathies.</a> Annual Reviews of Pathology. 14, 239-261 (2019).</li><li>Pandit, R., Leinenga, G., G&#246;tz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repeated+ultrasound+treatment+of+tau+transgenic+mice+clears+neuronal+tau+by+autophagy+and+improves+behavioral+functions.">Repeated ultrasound treatment of tau transgenic mice clears neuronal tau by autophagy and improves behavioral functions.</a> Theranostics. 9 (13), 3754-3767 (2019).</li><li>Karakatsani, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unilateral+Focused+Ultrasound-Induced+Blood-Brain+Barrier+Opening+Reduces+Phosphorylated+Tau+from+The+rTg4510+Mouse+Model.">Unilateral Focused Ultrasound-Induced Blood-Brain Barrier Opening Reduces Phosphorylated Tau from The rTg4510 Mouse Model.</a> Theranostics. 9 (18), 5396-5411 (2019).</li><li>Valdez, M. A., Fernandez, E., Matsunaga, T., Erickson, R. P., Trouard, T. 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Fluorescently-labeled antibodies can be delivered to the brain using focused ultrasound together with microbubbles applied in a scanning mode. Antibody delivery, microglial morphology and lysosomal enlargement can be detected by fluorescence microscopy following scanning ultrasound. Microglia can take up into their lysosomes antibodies and antigens that the antibodies have bound to in an Fc-receptor-mediated process4.
There are a number of critical steps to achieve repe...

Therapeutic ultrasound is an emerging technology aimed at treating brain diseases in a noninvasive manner, in part by facilitating access of therapeutic agents to the brain1,2,3. As only a small fraction of therapeutic antibodies targeting brain diseases are taken up by and retained in the brain4, therapeutic ultrasound offers the possibility to increase their uptake and target engagement5,6.
In our laboratory, we are developing therapeutic approaches for neurodegenerative diseases in which an antibody in various formats is delivered across the blood-brain barrier (BBB) using microbubbles. To achieve this, ultrasound is applied through the skull into the brain in multiple spots using a scanning mode we refer to as scanning ultrasound (SUS)7. The mechanical interaction between the ultrasound energy, the intravenously injected microbubbles and the brain vasculature transiently separates the tight junctions of the BBB in a given sonication volume, allowing antibodies and other cargoes including therapeutic agents to effectively cross this barrier7,8,9. Moreover, ultrasound has been shown to facilitate the uptake of antibodies from the interstitial brain into brain cells, such as neurons, where the antibody distributes throughout the cell body and even into neuritic processes5,10.
Alzheimer&#39;s disease is characterized by an amyloid-&#946; and tau pathology11, and a host of animal models is available to dissect pathogenic mechanisms and validate therapeutic strategies. A&#160;SUS approach, by which ultrasound is applied in a sequential pattern across the entire brain, when repeated over several treatment sessions, can reduce amyloid plaque pathology in the brains of amyloid-&#946;-depositing amyloid precursor protein (APP) mutant mice and activate microglia which take up the amyloid, leading to improvement in cognitive function7. BBB opening with ultrasound and microbubbles also reduces tau pathology in pR5, K3 and rTg4510 tau transgenic mice5,12,13. Importantly, whilst microglia remove extracellular protein deposits, one of the underlying clearance mechanisms for intraneuronal pathologies induced by SUS is the activation of neuronal autophagy12.
Here, we outline an experimental process, by which fluorescently labeled antibodies are prepared, and then mixed with in-house lipid-based microbubbles, followed by retroorbital injection into anesthetized mice. Retroorbital injection is an alternative to tail vein injection which we have found to be equally efficacious and simpler to repeatedly perform. This is immediately followed by applying SUS to the brain. To determine the therapeutic antibody uptake, mice are sacrificed and the increased antibody concentration in the brain is then quantified. As a proxy of the change in brain homeostasis, microglial phagocytic activity is determined by histology and volumetric 3D reconstruction.
The generated data suggest that ultrasound delivery of antibodies is a potentially attractive approach to treat neurodegenerative diseases. The protocol can be similarly applied to other drug candidates, as well as model cargos such as fluorescently labeled dextrans of defined sizes14.

<table>
	<tbody>
		<tr>
			<td>1,2-distearoyl-sn-glycero-3-phosphocholine</td>
			<td>Avanti</td>
			<td>850365C</td>
			<td></td>
		</tr>
		<tr>
			<td>1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000]</td>
			<td>Avanti</td>
			<td>880128C</td>
			<td></td>
		</tr>
		<tr>
			<td>AlexaFluor 647 antibody labeling kit</td>
			<td>Thermo Fisher</td>
			<td>A20186</td>
			<td></td>
		</tr>
		<tr>
			<td>CD68 antibody</td>
			<td>AbD Serotec</td>
			<td>MCA1957GA</td>
			<td>Use 1:1000 dilution</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Sigma-Aldrich</td>
			<td>372978</td>
			<td></td>
		</tr>
		<tr>
			<td>Coulter Counter (Multisizer 4e)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG, Alexa Fluor 488</td>
			<td>Thermo FIsher</td>
			<td>A-11008</td>
			<td>Use 1:500 dilution</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG, Alexa Fluor 488</td>
			<td>Thermo Fisher</td>
			<td>A-11077</td>
			<td>Use 1:500 dilution</td>
		</tr>
		<tr>
			<td>head holder (model SG-4N, Narishige Japan)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Iba1 antibody</td>
			<td>Wako</td>
			<td>019-19741</td>
			<td>Use 1:1000 dilution</td>
		</tr>
		<tr>
			<td>Image analysis software</td>
			<td>Beckman Coulter</td>
			<td>#8547008</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflow flow solution</td>
			<td>Beckman Coulter</td>
			<td>B43905</td>
			<td></td>
		</tr>
		<tr>
			<td>Near infrared imaging system Odyssey Fc</td>
			<td>Licor</td>
			<td>2800-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Octafluoropropane</td>
			<td>Arcadophta</td>
			<td>0229NC</td>
			<td></td>
		</tr>
		<tr>
			<td>Propylene Glycol</td>
			<td>Sigma-Aldrich</td>
			<td>P4347</td>
			<td></td>
		</tr>
		<tr>
			<td>TIPS (Therapy Imaging Probe System)</td>
			<td>Philips Research</td>
			<td>TIPS_007</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Bitplane</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

delivery of antibodies into the brain using focused scanning ultrasound

Only a small fraction of therapeutic antibodies targeting brain diseases are taken up by the brain. Focused ultrasound offers a possibility to increase uptake of antibodies and engagement through transient opening of the blood-brain barrier (BBB). In our laboratory, we are developing therapeutic approaches for neurodegenerative diseases in which an antibody in various formats is delivered across the BBB using microbubbles, concomitant with focused ultrasound application through the skull targeting multiple spots, an approach we refer to as scanning ultrasound (SUS). The mechanical effects of microbubbles and ultrasound on blood vessels increases paracellular transport across the BBB by transiently separating tight junctions and enhances vesicle- mediated transcytosis, allowing antibodies and therapeutic agents to effectively cross. Moreover, ultrasound also facilitates the uptake of antibodies from the interstitial brain into brain cells such as neurons where the antibody distributes throughout the cell body and even into neuritic processes. In our studies, fluorescently labeled antibodies are prepared, mixed with in-house prepared lipid-based microbubbles and injected into mice immediately before SUS is applied to the brain. The increased antibody concentration in the brain is then quantified. To account for alterations in normal brain homeostasis, microglial phagocytosis can be used as a cellular marker. The generated data suggest that ultrasound delivery of antibodies is an attractive approach to treat neurodegenerative diseases.

Using this protocol fluorescently-labeled antibodies are delivered to the brain and can be detected, along with microglia activation. The conclusion that can be drawn is the use of focused ultrasound and microbubbles markedly enhances brain uptake of antibodies and can deliver antibodies to the whole brain or hemisphere of a mouse when used in a scanning mode. Figure 1 shows the TIPS ultrasound application device (different components labeled) that is used to open the BBB. <strong class="xfi...

Watch this Scientific Journal Video about Delivery of Antibodies into the Brain Using Focused Scanning Ultrasound at JoVE.com

Delivery of Antibodies into the Brain Using Focused Scanning Ultrasound

Presented here is a protocol to transiently open the blood-brain barrier (BBB) either focally or throughout a mouse brain to deliver fluorescently-labeled antibodies and activate microglia. Also presented is a method to detect the delivery of antibodies and microglia activation by histology.

Only a small fraction of therapeutic antibodies targeting brain diseases are taken up by the brain. Focused ultrasound offers a possibility to increase ...

Only a small fraction of therapeutic antibodies are taken up by the brain. Using our method, antibodies are delivered into the brain by transiently opening the blood-brain barrier. This technique allows us to deliver antibodies into the mouse brain in a noninvasive manner.To perform quality control of the micro bubbles using a cell counter, remove the micro bubble solution from the amalgamator and use a 19 gauge needle to pierce the septum of the micro bubble solution vial. Use a one milliliter syringe equipped with a 19 gauge needle to dilute 100 microliters of the micro bubbles in five milliliters of filtered flow solution and pipette 100 microliters of the diluted micro bubble solution in 10 milliliters of filtered flow solution in a cuvette. Lock the cuvette in place on the cell counter platform and make sure that a 30 micron aperture will be used for the sample acquisition.In the software, load the standard operating method and select edit standard operating method and concentration. Enter 5, 000 times for the dilution and click apply and OK.Click edit info to select a suitable filename. Select preview and verify that the micro bubble sample concentration is less than 10%Select start and OK to begin the sample acquisition.After the measurement, rinse the aperture of the cell counter with filtered flow solution. Sonicate the cuvette of diluted micro bubble solution for 30 seconds in a water bath. Measure the sonicated micro bubble solution as demonstrated and click edit info to label the data as blank.After measuring the blank, subtract the final readout from the initial readout to exclude any particles that are not micro bubbles. To label the sections with fluorescent anti-mouse IgG antibody, label one milligram of mouse IgG antibody with Alexa Flour 647 in 0.1 molar sodium bicarbonate buffer according to the manufacturer's instructions for 15 minutes at room temperature. At the end of the incubation, load the fluorescently labeled antibody solution onto a spin column and purify the antibody by centrifugation.Then use a spectrophotometer to measure the protein concentration. To set up the focused scanning ultrasound system, add a five millimeter space to the water bolus to position the ultrasound focus nine millimeters below the bottom of the water bolus. Fill the water bolus with approximately 300 milliliters of degassed deionized water and place the annular array into the filled water bolus.Use a dental mirror to check that there are no air bubbles on the surface and launch the application software. In the waveform menu, select set waveform duty cycle and set the pulse repetition frequency to 10 Hertz, the duty cycle to 10%the focus to 80 millimeters, the center frequency to one megahertz, the amplitude to 0.65 megapascals, and the mechanical index to 0.65. Press set to define the waveform and store the waveform in memory.After selecting the treatment plan, open the scan tab in the motion controller window and enter start, stop, and increment values for motion in the X dimension and start, stop, and increment values for motion in the Y direction. To define the actions for the treatment sites, click event and open the script editing window. Select a list of actions that will be executed in the order selected at each treatment site and set the movement type to raster grid.In the events tab, select add actions and move the move synchronously start trigger ARB wait and stop trigger ARB actions to the script panel. Then click wait action and select a wait time of 6, 000 milliseconds. To prepare an animal for the analysis, after confirming a lack of response to toe pinch in an anesthetized mouse, use a permanent marker to label the center of the head, then glue plastic wrap to the bottom of a small weigh boat with the bottom cutoff and fill the weigh boat with ultrasound gel.For focused ultrasound treatment, invert the vial of micro bubbles and gently load one microliter of the solution per gram of the mouse's body weight into a 29 gauge insulin syringe. Add 100 microliters of fluorescently labeled antibody to the syringe and gently invert and roll the syringe between thumb and index finger to mix the antibody and the micro bubbles within the syringe. Carefully and slowly retro-orbitally inject 150 microliters of the micro bubble and antibody solution into the mouse and set a timer for two minutes.Apply ophthalmic ointment, then place the mouse in the head holder and fix its nose in the holder. Place the small ultrasound gel-filled weigh boat on top of the head and lower the water bolus until it sits on top of the ultrasound gel within the weigh boat. Then use the joystick to visually align the transducer focus within the center of the head.When the timer goes off, in the motion tab of the software, select reset origin and select a complete scan. When the treatment is complete, apply ophthalmic ointment to the animal's eyes and place the mouse in a warmed recovery chamber. Here, representative results from the culture counter measurements of the size and concentration that can be obtained when the micro bubbles are produced correctly are shown.Antibody uptake by the brain can be easily visualized in the whole brain or tissue sections using an infrared scanner or fluorescent microscopy of the sections. As demonstrated in these images, representative staining for microglia markers can be used to determine whether microglia become more phagocytic following the delivery of the antibody. It is important to ensure the welfare and safety of the animals especially when performing the retro-orbital injections and applying the transducer above the heads of the mice.This method allows the application of various scanning patterns to target different regions of the brain, such as the hippocampus, cortex, or individual brain hemispheres. This technique can be used to develop new therapies and drugs that penetrate the blood-brain barrier and to study the mechanism of the blood-brain barrier formation and opening.

delivery-antibodies-into-brain-using-focused-scanning

Research

JoVE Journal

Neuroscience

2.9K Views.  The University of Queensland. Only a small fraction of therapeutic antibodies are taken up by the brain. Using our method, antibodies are delivered into the brain by transiently opening the blood-brain barrier. This technique allows us to deliver antibodies into the mouse brain in a noninvasive manner.To perform quality control of the micro bubbles using a cell counter, remove the micro bubble solution from the amalgamator and use a 19 gauge needle to pierce the septum of the micro bubble solution vial. Use a one m...