# Biological and Physical Mechanisms of ultrasound/microbubble-mediated therapeutic gene delivery across the endothelial barrier

> **NIH NIH R01** · UNIVERSITY OF PITTSBURGH AT PITTSBURGH · 2021 · $598,984

## Abstract

RNA-based therapeutics offer a powerful paradigm for treating disease by targeting heretofore “undruggable”
genes, allowing highly specific silencing of pathologic gene expression to heal heretofore hopeless illnesses.
However, a safe and efficient method for targeted delivery of cell-impermeant RNA drugs has remained elusive.
A major hurdle for RNA-based therapies using vascular delivery is to circumvent the endothelial barrier. We have
been developing a unique technology using intravenously injected RNA-loaded microbubbles (MB) which are
triggered to cavitate by ultrasound (US), causing transient permeabilization of the adjacent cell membrane and
endocytosis-independent uptake of the RNA by extravascular target cells. The potential of this site-specific, non-
invasive delivery method is extra-ordinary, more so because the MBs and US transducer also confer capability
for simultaneous real-time image-guided therapy. Despite its pre-clinical proof of concept, fundamental
mechanisms underlying the delivery efficacy of ultrasound-targeted MB cavitation (UTMC) are poorly
understood. Without this knowledge, the potential for UTMC to overcome many of the cellular barriers to bedside
RNA therapeutics will not be realized. Accordingly, this proposal utilizes 2 distinct MB formulations and RNA
payloads to systematically, for the first time, perform studies spanning individual cell signaling pathways, in vivo
MB acoustic behaviors, and three-dimensional tissue interrogation of UTMC effects in vivo, to develop a cohesive
paradigm addressing the mechanisms of UTMC-mediated endothelial hyperpermeability leading to RNA
delivery. We hypothesize that MBs cavitating in the microcirculation mechanically perturb endothelial cells,
leading to signaling events that culminate in endothelial barrier hyperpermeability and enhanced payload uptake.
Using model systems, we propose in vitro studies to interrogate mechanistic pathways, then in vivo studies
investigating UTMC endothelial barrier effects in real time, with 3 Aims: (1) Determine mechanisms by which
UTMC increases endothelial barrier permeability. We will use endothelialized transwells and manipulate
candidate pathways to test the hypothesis that UTMC-induced Ca2+ influx increases endothelial permeability,
and optically measure attendant cellular events (multicolor confocal microscopy), correlating barrier function to
cell response. (2) Determine the relationship between in vivo MB behaviors and transendothelial transport
of siRNA using a custom ultrafast camera to visualize microvascular MB vibrations in vivo, testing the hypothesis
that UTMC causes quantifiable mechanical events, then deriving physical principles governing UTMC-mediated
hyperpermeability (3) Determine extravasation pathways and cellular fate of RNA-loaded MBs during
UTMC in vivo using intravital high-speed multicolor confocal microscopy in cremaster microcirculation. Our
multidisciplinary team unites physics/acoustics with biology/physiology t...

## Key facts

- **NIH application ID:** 10220968
- **Project number:** 5R01EB026966-04
- **Recipient organization:** UNIVERSITY OF PITTSBURGH AT PITTSBURGH
- **Principal Investigator:** Flordeliza S Villanueva
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $598,984
- **Award type:** 5
- **Project period:** 2018-09-20 → 2023-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10220968

## Citation

> US National Institutes of Health, RePORTER application 10220968, Biological and Physical Mechanisms of ultrasound/microbubble-mediated therapeutic gene delivery across the endothelial barrier (5R01EB026966-04). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10220968. Licensed CC0.

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