Focused Ultrasound-Enhanced Delivery of Intranasally Administered Anti-Programmed Cell Death-Ligand 1 Antibody to an Intracranial Murine Glioma Model

Abstract

Immune checkpoint inhibitors have great potential for the treatment of gliomas; however, their therapeutic efficacy has been partially limited by their inability to efficiently cross the blood-brain barrier (BBB). The objective of this study was to evaluate the capability of focused-ultrasound-mediated intranasal brain drug delivery (FUSIN) in achieving the locally enhanced delivery of anti-programmed cell death-ligand 1 antibody (aPD-L1) to the brain. Both non-tumor mice and mice transcranially implanted with GL261 glioma cells at the brainstem were used in this study. aPD-L1 was labeled with a near-infrared fluorescence dye (IRDye 800CW) and administered to mice through the nasal route to the brain, followed by focused ultrasound sonication in the presence of systemically injected microbubbles. FUSIN enhanced the accumulation of aPD-L1 at the FUS-targeted brainstem by an average of 4.03- and 3.74-fold compared with intranasal (IN) administration alone in the non-tumor mice and glioma mice, respectively. Immunohistochemistry staining found that aPD-L1 was mainly located within the perivascular spaces after IN delivery, while FUSIN further enhanced the penetration depth and delivery efficiency of aPD-L1 to the brain parenchyma. The delivered aPD-L1 was found to be colocalized with the tumor cells after FUSIN delivery to the brainstem glioma. These findings suggest that FUSIN is a promising technique to enhance the delivery of immune checkpoint inhibitors to gliomas.

Keywords: blood-brain barrier; brain drug delivery; brainstem glioma; focused ultrasound; immune checkpoint inhibitor; intranasal delivery.

Figure 1

Analysis of 800CW-aPD-L1 conjugates. (A) Schematic illustration of 800CW conjugated to aPD-L1 through NHS ester reaction. (B) Absorbance spectrum of 800CW-aPD-L1 measured by the UV spectrophotometer. (C) SDS-PAGE of reduced aPD-L1 and 800CW-aPD-L1 by both Coomassie blue staining and fluorescence imaging. (D) Flow cytometry analysis of binding affinity of aPD-L1 and 800CW-aPD-L1 to GL261 cells with high surface PD-L1 expression (black: unstained control, red: 800CW-aPD-L1, green: aPD-L1).

Figure 2

Focused-ultrasound-mediated intranasal (FUSIN) delivery of 800CW-aPD-L1 to the brainstem. (A) Fluorescence images of representative ex vivo mouse brain slices. The olfactory bulb (Olf) was in the top slice. The targeted location of the FUS was located at the right side of the brainstem. The dashed box highlights the brain slices containing the brainstem. (B) Fluorescence quantification of the 800CW-aPD-L1 delivery efficiency to the brainstem. (C) The relationship between the fluorescence intensity measured by ex vivo fluorescence imaging and the aPD-L1 concentrations measured by ELISA (*: p < 0.05).

Figure 3

Fluorescence images of the brainstem obtained from non-tumor mice after intranasal (IN) (A) and FUSIN (B) delivery. After IN delivery, immunofluorescence staining shows the aPD-L1 distributed along with the perivascular spaces, defined by the space between the AQP4-stained astrocyte and the lectin-stained blood vessel. FUSIN enhanced the accumulation of aPD-L1, and the delivered aPD-L1 penetrated deep into the brain parenchyma.

Figure 4

FUSIN delivery of 800CW-aPD-L1 to the brainstem glioma. (A) Fluorescence images of representative ex vivo mouse brainstem slices. The white arrow indicates the tumor location. (B) Spatial distribution of FUSIN-delivered 800CW-aPD-L1. Left panel: aPD-L1 distribution in a coronal section of the brainstem after FUSIN delivery imaged at 2×. Right panel: higher magnification view (60×) of the tumor showing the colocalization of aPD-L1 with the tumor cells. (C) Fluorescence quantification of the 800CW-aPD-L1 delivery efficiency to the brainstem glioma (** p < 0.01).