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.

Glioblastoma (GBM) remains one of the most aggressive and deadly types of cancer with an estimated 5-year survival rate of 5.5%.¹ It is also the most common type of primary malignant brain tumor with 12,760 cases projected in the United States in 2018.¹ Management of GBM presents many unique obstacles owing to the relative importance of CNS tissue, necessitating the sparing of tissue during surgical resection. Furthermore, protection of the tumor by the blood–brain barrier (BBB) as well as tumor invasiveness, heterogeneity, and drug resistance limits the effectiveness of systemic therapies.^{2 – 4} In the face of these complex challenges, the role of molecular classification of GBM is increasingly important for diagnosing specific disease phenotypes, choosing the best treatment options, and monitoring treatment response.^3,5

The blood-brain barrier (BBB) is the major obstacle for brain drug delivery and limits the treatment options for central nervous system diseases. To circumvent the BBB, we introduce focused ultrasound-mediated intranasal brain drug delivery (FUSIN). FUSIN utilizes the nasal route for direct nose-to-brain drug administration, bypassing the BBB and minimizing systemic exposure to the major organs, such as heart, lung, liver, and kidney [1]. It also uses transcranial ultrasound energy focused at a targeted brain region to induce microbubble cavitation, enhancing the transport of intranasally administered agents at the FUS-targeted brain location. FUSIN is unique because it can achieve noninvasive and localized brain drug delivery with minimized systemic toxicity to other major organs. The goal of this paper is to provide a detailed protocol for FUSIN delivery to the mouse brain. • FUSIN delivery utilizes the nose-to-brain pathway for brain drug delivery. • FUSIN utilizes FUS combined with microbubble to significantly enhance the delivery efficiency of intranasally administered drugs to the FUS targeted brain regions. • FUSIN achieves efficient brain delivery with minimized systemic exposure in the major organs.

Sonogenetics, which uses ultrasound to noninvasively control cells genetically modified with ultrasound-sensitive ion channels, can be a powerful tool for investigating intact brain circuits. Here we show that TRPV1 is an ultrasound-sensitive ion channel that can modify the activity of TRPV1-expressing cells in vitro when exposing to focused ultrasound. We also show that focused ultrasound exposure at the mouse brain in vivo can selectively activate neurons that are genetically modified to express TRPV1. We demonstrate that precise manipulation of neural activity via TRPV1-based sonogenetics can be achieved by spatiotemporal control of ultrasound-induced heating. The focused ultrasound exposure is safe based on our inspection of neuronal integrity, apoptosis, and inflammation markers. This sonogenetic tool enables noninvasive, cell-type specific, spatiotemporally controlled modulation of mammalian brain activity.

Nanomedicine is the next-generation technology for cancer therapy. Liposomes are one of such nanomedicine that have well-established clinical applications. However, their large sizes make it challenging to deliver them to the brain due to the existence of the blood-brain barrier (BBB). Previous studies have demonstrated the potential of integrating focused ultrasound (FUS)-induced BBB opening with liposomes for brain drug delivery. However, no study has characterized the spatial distributions of the liposomes and released drugs in the brain parenchyma at a microscopic level after the BBB was disrupted by the FUS. In this study, we manufactured liposomes with a fluorescent dye embedded in the lipid layer and chemotherapeutic drug doxorubicin encapsulated in the core. These liposomes were intravenously injected into the mouse followed by FUS sonication of a targeted brain location in the presence of microbubbles. The mouse brains were coronally sectioned and imaged using fluorescence microscopy. Preliminary results confirmed the successful delivery of doxorubicin to the brain. However, the liposome distribution as indicated by the dye embedded in the lipid shell was not spatially correlated with the distribution of the doxorubicin. Further studies are needed to better understand the mechanism of FUS-enabled liposome delivery.

Intranasal route provides therapeutics direct access to the brain through the nose-to-brain pathway, bypassing the BBB and minimizing systemic exposure. However, nasal delivery is limited by its low delivery efficiency and non-localized delivery. Focused ultrasound-enhanced intranasal (FUSIN) delivery is a new technique for noninvasive and localized delivery of therapeutic agents to the brain. Previous studies have shown its feasibility in the delivery of dextrans and brain-derived neurotrophic factor to the brain. The goal of this study was to demonstrate the application of FUSIN for the delivery of temozolomide (TMZ), a first-line drug for treating glioma tumors, for the treatment of glioblastoma in an orthotopic mouse model. U87 glioblastoma cells were trancranially implanted to nude mice. The tumor mice were divided into four groups: (1) FUSIN + TMZ (n = 7); (2) IN TMZ (n = 6); (3) Oral TMZ (n = 6); and (4) IN vehicle (n = 6), and were treated once each week for 4 weeks. Mice were weighed once a week. A body weight loss reaching 20% was selected as the endpoint criteria for the study. Mice in FUSIN group showed statistically significant higher survival rate when compared with mice in all other groups. This pilot study suggested that FUSIN can potentially be an effective technique for the treatment of brain diseases.