Flordeliza S. Villanueva

Purpose of Review Ultrasound-targeted microbubble cavitation (UTMC) has emerged as a transformative non-invasive technology in diagnostic imaging, therapy, and targeted gene and drug delivery. As UTMC advances toward clinical translation, understanding its impact on fundamental cellular functions is essential. Recent Findings Recent studies on UTMC have provided insights into its effects on biological processes. These include transient membrane opening and resealing (sonoporation), calcium ion (Ca²⁺) influx, generation of free radicals, nitric oxide synthesis, cytoskeletal remodeling, inter-endothelial gap formation, gene expression changes, and neuroinflammation. Summary This review explores the mechanisms underlying UTMC, with a focus on ultrasound-targeted microbubble cavitation and its bioeffects on cellular function. We examine the molecular processes induced by cavitation, including sonoporation and Ca²⁺ influx, and highlight their effects on key biological processes.

The blood‒brain barrier (BBB) acts as a hindrance to drug therapy reaching the brain. With an increasing incidence of neurovascular diseases and brain cancer metastases, there is a need for an ideal in vitro model to develop novel methodologies for enhancing drug delivery to the brain. Here, we established a multicellular human brain spheroid model that mimics the BBB both architecturally and functionally. Within the spheroids, endothelial cells and pericytes localized to the periphery, while neurons, astrocytes, and microglia were distributed throughout. Ultrasound-targeted microbubble cavitation (UTMC) is a novel noninvasive technology for enhancing endothelial drug permeability. We utilized our three-dimensional (3D) model to study the feasibility and mechanisms regulating UTMC-induced hyperpermeability. UTMC caused a significant increase in the penetration of 10 kDa Texas red dextran (TRD) into the spheroids, 100 µm beyond the BBB, without compromising cell viability. This hyperpermeability was dependent on UTMC-induced calcium (Ca²⁺) influx and endothelial nitric oxide synthase (eNOS) activation. Our 3D brain spheroid model, with its intact and functional BBB, offers a valuable platform for studying the bioeffects of UTMC, including effects occurring spatially distant from the endothelial barrier.

Rationale: Microbubble (MB) contrast agents combined with ultrasound targeted microbubble cavitation (UTMC) are a promising platform for site-specific therapeutic oligonucleotide delivery. We investigated UTMC-mediated delivery of siRNA directed against epidermal growth factor receptor (EGFR), to squamous cell carcinoma (SCC) via a novel MB-liposome complex (LPX).

Therapeutic delivery of oligonucleotides, such as RNAs, has the potential to treat heretofore “undruggable” targets in diseases such as cancer. However, systemic intravascular delivery of RNAs to tumor tissue is limited by the vascular endothelium, which provides a barrier to transendothelial passage of large, charged molecules. To address RNA delivery challenges, we and others have developed an approach employing ultrasound targeted microbubble cavitation (UTMC), which utilizes 2–4 μm diameter gas-filled microbubbles that carry RNA on the shell and also serve as an ultrasound imaging agent. As intravenously injected microbubbles travel through the microcirculation, localized ultrasound induces microbubble cavitation, causing localized payload release from the microbubble. In addition to delivering the RNA payload, UTMC increases endothelial permeability, thus increasing local extravasation of the payload, while sparing non-insonified tissue (reduced off-target effects). Microbubbles are non-immunogenic and protect loaded RNA against nuclease digestion. Because UTMC concentrates the RNA at the tumor site, lower systemic RNA doses can be used, resulting in fewer off-target effects. A unique feature is that the microbubble that carries and releases the RNA also permits simultaneous US imaging of microbubble location. This lecture will review these unique features of UTMC for drug delivery and current work in this area.