Delivery of Antibodies into the Brain Using Focused Scanning Ultrasound

Summary

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.

Abstract

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.

Introduction

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 brain^1,2,3. 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 engagement^5,6.

In our laboratory, we are developing therapeutic approaches for neurodegene....

Protocol

All animal experiments were approved by the animal ethics committee of the University of Queensland.

1. In-house microbubble preparation

1. 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.
2. Dissolve the lipid in a small volume of chloroform in a glass beaker.
3. Evaporate the chloroform with an evaporator or a ni....

Results

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.

Discussion

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.......

Materials

List of materials used in this article

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