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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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 step 1.3.</li>
	<li>Dissolve the lipid in a small volume of chloroform in a glass beaker.</li>
	<li>Evaporate the chloroform with an evaporator or a nitrogen stream.</li>
	<li>Rehydrate the dried lipid film with 10 mL of phosphate-buffered saline (PBS) + 10% glycerol + 10% propylene glycol solution that has been filtered through 0.22 &#181;m filter.</li>
	<li>Place the rehydrated lipid solution in a water bath sonicator set to 55 &#730;C (above the melting temperature of the lipids) and sonicate until fully dissolved.</li>
	<li>Aliquot lipid solution into autoclaved 1.5 mL high-pressure liquid chromatography (HPLC) vials and screwed on the septa caps.</li>
	<li>Aspirate all of the air in the vial with a 5 mL syringe equipped with a 27 G needle and create a vacuum in the vial.</li>
	<li>Add octofluoropropane to the vial with the included syringe, drawing up the gas from the canister. Fill the vial with 1-2 mL of octafluoropropane by reading the volume in the syringe.</li>
	<li>Seal each vial with paraffin film and refrigerate.</li>
	<li>On the day of the experiment, bring the vial to room temperature, add 0.5 mL of 0.9% NaCl solution to the vial, then place the vial in an amalgamator and agitate for 45 s (preset time) to produce the microbubbles.</li>
</ol>
2. Microbubble quality control using a coulter counter
<ol>
	<li>Take the microbubble solution out of the amalgamator and vent the gas from the vial by piercing the septa with a 19&#160;G needle.</li>
	<li>Dilute the microbubble solution by performing two-step 1:5,000 serial dilutions by adding 100 &#181;L of microbubble solution into 5 mL of filtered flow solution using a 1 ml syringe with 19 G needle, and then taking 100 &#181;L of 1:50 diluted microbubble solution and pipetting into 10 mL filtered flow solution in a cuvette using a pipette.</li>
	<li>Check that the electrolyte tank has sufficient flow solution and that the waste tank is empty.</li>
	<li>Place the cuvette in the coulter counter platform and lock it into place. Use a 30 &#181;m aperture for sample acquisition.</li>
	<li>In the software, load the standard operating method (SOM), then select&#160;Edit SOM | concentration.&#160;Enter 5000x dilution.</li>
	<li>In the software, choose a suitable file name. For example, microbubble_1_date.</li>
	<li>Load and secure the cuvette into the platform.</li>
	<li>In the software select&#160;Run| Preview&#160;and verify that the sample concentration is less than 10%. If this number is higher than 10%, perform a new dilution of the microbubbles with a higher dilution factor.</li>
	<li>Select &#39;Start&#39; to begin an acquisition of the sample to obtain the initial readout.</li>
	<li>Rinse the aperture of the Coulter counter with filtered flow solution after each measurement. Repeat steps 2.2-2.9 to obtain 3 replicates.</li>
	<li>Place a cuvette with diluted microbubble solution into a sonicator water bath and sonicate for 30 s.</li>
	<li>Measure the solution with sonicated microbubbles and label as blank.</li>
	<li>In the software, subtract the final readout from the initial readout. This subtracts any particles that are not microbubbles and do not contain gas.</li>
	<li>Select&#160;Display Results&#160;in the software, to display the microbubble concentration, size distribution, average size, and volume concentration.</li>
</ol>
3. Fluorescent antibody labeling
<ol>
	<li>Obtain a 1 mg/mL solution of mouse immunoglobulin G (IgG) in PBS without any additives.</li>
	<li>Label 1 mg of mouse IgG with AlexaFluor 647 in 0.1 M sodium bicarbonate buffer by following the manufacturers&#39; instructions located in the kit. This amount of fluorescently labeled IgG is sufficient to perform this procedure on 5-7 adult mice where a 5 mg/kg dose of antibody is administered.</li>
	<li>Add the dye to the solution of IgG in 0.1 M sodium bicarbonate buffer and incubate for 15 min at room temperature.</li>
	<li>Purify the fluorescently labeled antibody by pipetting the antibody solution into a spin column and centrifuging at 1,000&#160;x g&#160;for 5 min. Free dye will remain in the column bed.</li>
	<li>Use a spectrophotometer to measure the protein concentration. Measure the absorbance of the conjugate solution at 280 nm and 650 nm (A280 and A650). Calculate the concentration of protein in the sample using the equation: 
	Protein concentration&#160;(M) = [A280 - (A650 x 0.03)] x&#160;dilution factor&#160;/ 203,000.</li>
	<li>Use a spectrophotometer to calculate the degree of labeling using the equation: 
	moles dye per mole protein&#160;=&#160;A650 x&#160;dilution factor&#160;/ 239 000 x&#160;protein concentration&#160;(M). 
	NOTE: An acceptable degree of labeling is 3-7 moles dye per mole protein and typically obtained degree of labeling is around 6.</li>
</ol>
4. Ultrasound set-up
<ol>
	<li>Using the focused ultrasound system, add the 5 mm spacer to the water bolus to position the ultrasound focus 9 mm below the bottom of the water bolus.</li>
	<li>Fill the water bolus with approximately 300 mL of deionized water that has been degassed with an inline degasser for 20 min (oxygen content should be below 3 ppm). Place the annular array into the filled water bolus and use a dental mirror to check that there are no air bubbles on the surface. If present on the surface, remove the annular array and replace it in the water bolus.</li>
	<li>Launch the application software. In the waveform menu, select&#160;Set waveform duty cycle. Settings are PRF (Hz) 10, duty cycle 10%, focus 80 mm, center frequency 1 MHz, amplitude (MPa peak negative pressure) 0.65 MPa, mechanical index = 0.65. Press&#160;Set&#160;to define the waveform and store it in memory. 
	NOTE: The focused ultrasound system is pre-calibrated by the manufacturer from measurements taken by a calibrated hydrophone.</li>
	<li>In the focused ultrasound system software, define a treatment plan. This requires defining a treatment region consisting of multiple individual treatments sites and defining actions to be taken at each of those treatment sites. In this case, the treatment zone is one hemisphere of the mouse brain.</li>
	<li>In the motion controller window, go to the&#160;Scan&#160;tab and&#160;Enter&#160;start, stop and increment value for motion in the x dimension, and start, stop and increment value for motion in the y direction. Enter values for X: start -4, stop 3.50 and Y: -3.00, stop 3, Increment 1.5, # loops: 1.</li>
	<li>Define the actions for treatment sites. In the motion controller window, select the&#160;Event&#160;button. In the&#160;Script Editing Window, select a list of actions that will be executed in the order selected at each treatment site. Set&#160;Movement Type&#160;to raster grid at the top of the script window. In the events tab select&#160;Add Actions&#160;to move them to the script panel, and add&#160;Move Synchronously,&#160;Start&#160;trigger arb, wait,&#160;Stop&#160;trigger arb. Click on the wait action and select a wait time of 6, 000 ms. 
	NOTE: These settings will make the treat a 6 x 5 grid of treatment spots spaced 1.5 mm apart, with each spot having a treatment duration of 6 s. The total duration to sonicate a mouse brain is approximately 3 min. This size treatment grid is suitable for adult C57/Bl6 mice weighing approximately 30 g. The size of the grid of treatment spots can be adjusted up or down depending on the size of the mouse.</li>
</ol>
5. Animal preparation
<ol>
	<li>Weigh the mouse with a balance accurate to 0.1 g.</li>
	<li>Anesthetize mouse with 90 mg/kg ketamine and 6 mg/kg xylazine intraperitoneally. Test for absence of reflexes with a toe pinch. Alternatively, mice can be anesthetized with isoflurane using an appropriate inhalational anesthetic apparatus with an appropriate face mask. If using isoflurane, the mouse should be placed on a heat pad during the ultrasound to prevent hypothermia.</li>
	<li>Use an electric razor to shave the hair from the head of the animal, then apply hair removal cream with a cotton bud, leave on for 2-3 min or until the hair is wiped away clean with a damp piece of gauze.&#160;Take care that the hair removal cream does not get in the eyes of the mouse.</li>
	<li>Mark the center of the mouse&#39;s head with a permanent marker. The transducer has a hole in its center and the transducer focus and focal spot can be aligned visually.</li>
	<li>Fill a small weigh boat that has previously had the bottom cut off and replaced with plastic wrap glued to the bottom of the weigh boat with ultrasound gel. This serves as an 8 mm spacer and provides good coupling to the head of the mouse and allows visual inspection of the focus of the transducer aligned with the head of the mouse.</li>
</ol>
6. Microbubble preparation
<ol>
	<li>Warm a microbubble vial to room temperature. To activate, add 0.5 mL of 0.9% NaCl solution to the vial and place in an amalgamator to agitate for 45 s to produce the microbubbles.</li>
	<li>Vent the vial by piercing the septa with a 27 G needle.</li>
</ol>
7. Ultrasound treatment
<ol>
	<li>Invert the vial of microbubbles and gently draw up 1 &#181;L/g bodyweight of the solution. To this add solution of fluorescently labeled antibody and mix gently in the syringe. The maximum volume injected is 150 &#181;L. 
	NOTE: In-house prepared microbubbles are approximately 60-fold less concentrated than the clinically used (e.g., Definity microbubbles). Adjust the volume or concentration such that the number of microbubbles injected are similar to those clinically used (ie. Definity 1.2 x 108&#160;microbubbles/kg body weight).</li>
	<li>Inject the microbubble and antibody solution retroorbitally, taking care to inject gently and slowly.&#160;Then, apply ophthalmic ointment to the mouse&#39;s eyes.</li>
	<li>Place the mouse in the head holder (see Table of Materials) and fix the nose of the mouse. Then place the ultrasound gel-filled small weigh boat on top of the head.</li>
	<li>Lower the water bolus until it sits on top of the ultrasound gel in the weigh boat.</li>
	<li>Use the joystick to move the transducer focus to the center of the head. Select&#160;Reset Origin&#160;in the motion tab.</li>
	<li>Select complete scan. Steps 7.3-7.6 will take 2 min.</li>
	<li>For consistency, set a timer to ensure a 2 min delay between injecting microbubbles and selecting complete scan.</li>
	<li>After the treatment is complete, apply ophthalmic ointment to the eyes and place mouse in a warmed recovery chamber. If hypothermia is observed during the procedure, a warming pad can be placed under the mouse to provide supplemental heat during the procedure.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>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>Head holder (model SG-4N, Narishige Japan)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflow flow solution</td>
			<td>Beckman Coulter</td>
			<td>B43905</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>

an ultrasound-assisted delivery of antibodies in a mouse model

Source: Leinenga, G. et al., <a href="https://app.jove.com/v/61372">Delivery of Antibodies into the Brain Using Focused Scanning Ultrasound.</a>&#160;J. Vis. Exp. (2020)
This video demonstrates a procedure of ultrasound-assisted antibody delivery in a mouse model. Retro-orbitally injected microbubbles oscillate under ultrasound, creating a transient disruption in the blood-brain barrier, permitting labeled antibodies to enter the brain.

An Ultrasound-Assisted Delivery of Antibodies in a Mouse Model

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Begin with an anesthetized mouse and shave its head to improve ultrasound wave transmission.
Take a mixture of fluorescently labeled antibodies and microbubbles &#8212; a gas-filled core shell surrounded by a lipid layer. Inject them retro-orbitally into the mouse.
The injected microbubbles and antibodies enter the mouse&#39;s circulation and reach the brain.
Restrain the mouse and place an ultrasound gel-filled container on the shaved area.
Position the ultrasound transducer and transmit a high-frequency focused ultrasound wave.
The ultrasound waves cause microbubbles to expand and contract repeatedly.
This disrupts the tight junctions of the blood vessels in the brain and creates temporary localized openings.
These openings allow the labeled antibodies to exit the blood vessels and enter the mouse&#39;s brain.
Perform fluorescence imaging of the brain and locate fluorescent antibodies, confirming ultrasound-assisted antibody delivery in the mouse model.

an-ultrasound-assisted-delivery-of-antibodies-in-a-mouse-model

Research

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Antibody-Based Imaging and Detection Methods

140 Views. מקור: Leinenga, G. et al., העברת נוגדנים למוח באמצעות אולטרסאונד סריקה ממוקד.J. Vis. Exp. (2020) סרטון זה מדגים הליך של אספקת נוגדנים בסיוע אולטרסאונד במודל עכבר. מיקרו-בועות המוזרקות רטרו מתנודדות תחת אולטרסאונד, יוצרות הפרעה חולפת במחסום הדם-מוח, ומאפשרות לנוגדנים המסומנים להיכנס למוח. 

Video: משלוח נוגדנים בסיוע אולטרסאונד במודל עכבר - Concept

Predicting In Vivo Payloads Delivery using a Blood-brain Tumor-barrier in a Dish

Highly selective by nature, the blood-brain barrier (BBB) is essential for the brain homeostasis in physiological conditions. However, in the context of brain tumors, the molecular selectivity of BBB also shields the neoplastic cells by blocking the delivery of peripherally administered chemotherapies. The development of novel drugs (including nanoparticles) targeting malignant brain tumors ideally requires the use of preclinical animal models to study the drug&#8217;s transcytosis and antitumor efficacy. In order to comply with the 3R principle (refine, reduce, and replace) to reduce the number of laboratory animals in experimental setup and perform the high-throughput screening of a large library of antitumor agents, we developed a reproducible in vitro human and murine mimic of the blood-brain tumor-barrier (BBTB) using three-layered cultures of endothelial cells, astrocytes, and patient-derived glioblastoma spheres. For higher scalability and reproducibility, commercial cell lines or immortalized cells have been used in tailored conditions to allow the formation of a barrier resembling the actual BBB. Here we describe a protocol to obtain a BBTB mimic by culturing endothelial cells in contact with astrocytes at specific cell densities on inserts. This BBTB mimic can be used, for instance, for the quantification and confocal imaging of the nanoparticle passage through the endothelial and astrocytic barriers, in addition to the evaluation of the tumor cell targeting within the same assay. Moreover, we show that the obtained data can be used to predict the behavior of nanoparticles in preclinical animal models. In a broader perspective, this in vitro model could be adapted to other neurodegenerative diseases for the determination of the passage of new therapeutic molecules through the BBB and/or be supplemented with brain organoids to directly evaluate the efficacy of drugs.

Drug targeting to central nervous system tumors is a major challenge. Here we describe a protocol to produce an in vitro mimic of the blood-brain tumor-barrier using murine and/or human cells and discuss their relevance for the predictability of central nervous system tumor targeting in vivo.

Predicting ב Vivo מטענים משלוח באמצעות הגידול-מחסום דם-מוח בקערה

Highly selective by nature, the blood-brain barrier (BBB) is essential for the brain homeostasis in physiological conditions. However, in the context of ...

JoVE Journal

Cancer Research

Assembly and Operation of an Acoustofluidic Device for Enhanced Delivery of Molecular Compounds to Cells

Efficient intracellular delivery of biomolecules is required for a broad range of biomedical research and cell-based therapeutic applications. Ultrasound-mediated sonoporation is an emerging technique for rapid intracellular delivery of biomolecules. Sonoporation occurs when cavitation of gas-filled microbubbles forms transient pores in nearby cell membranes, which enables rapid uptake of biomolecules from the surrounding fluid. Current techniques for in vitro sonoporation of cells in suspension are limited by slow throughput, variability in the ultrasound exposure conditions for each cell, and high cost. To address these limitations, a low-cost acoustofluidic device has been developed which integrates an ultrasound transducer in a PDMS-based fluidic device to induce consistent sonoporation of cells as they flow through the channels in combination with ultrasound contrast agents. The device is fabricated using standard photolithography techniques to produce the PDMS-based fluidic chip. An ultrasound piezo disk transducer is attached to the device and driven by a microcontroller. The assembly can be integrated inside a 3D-printed case for added protection. Cells and microbubbles are pushed through the device using a syringe pump or a peristaltic pump connected to PVC tubing. Enhanced delivery of biomolecules to human T cells and lung cancer cells is demonstrated with this acoustofluidic system. Compared to bulk treatment approaches, this acoustofluidic system increases throughput and reduces variability, which can improve cell processing methods for biomedical research applications and manufacturing of cell-based therapeutics.

This protocol describes the assembly and operation of a low-cost acoustofluidic device for rapid molecular delivery to cells via sonoporation induced by ultrasound contrast agents.

הרכבה ותפעול של מכשיר אצוטופלואידי לאספקה משופרת של תרכובות מולקולריות לתאים

Efficient intracellular delivery of biomolecules is required for a broad range of biomedical research and cell-based therapeutic applications. ...

Bioengineering

A Benchtop Approach to the Location Specific Blood Brain Barrier Opening using Focused Ultrasound in a Rat Model

Stereotaxic surgery is the gold standard for localized drug and gene delivery to the rodent brain. This technique has many advantages over systemic delivery including precise localization to a target brain region and reduction of off target side effects. However, stereotaxic surgery is highly invasive which limits its translational efficacy, requires long recovery times, and provides challenges when targeting multiple brain regions. Focused ultrasound (FUS) can be used in combination with circulating microbubbles to transiently open the blood brain barrier (BBB) in millimeter sized regions. This allows intracranial localization of systemically delivered agents that cannot normally cross the BBB. This technique provides a noninvasive alternative to stereotaxic surgery. However, to date this technique has yet to be widely adopted in neuroscience laboratories due to the limited access to equipment and standardized methods. The overall goal of this protocol is to provide a benchtop approach to FUS BBB opening (BBBO) that is affordable and reproducible and can therefore be easily adopted by any laboratory.

Focused ultrasound with microbubble agents can open the blood brain barrier focally and transiently. This technique has been used to deliver a wide range of agents across the blood brain barrier. This article provides a detailed protocol for the localized delivery to the rodent brain with or without MRI guidance.

גישה ספסל למיקום ספציפי מחסום הדם מוח פתיחת באמצעות אולטרסאונד ממוקד במודל עכברוש

Stereotaxic surgery is the gold standard for localized drug and gene delivery to the rodent brain. This technique has many advantages over systemic ...

An Ultrasound-Assisted Delivery of Antibodies in a Mouse Model

Transcript

An Ultrasound-Assisted Delivery of Antibodies in a Mouse Model