Vascular imaging and therapy are important tools in management of cardiovascular diseases. Ultrasound can image and non-invasively deliver therapy. Microbubbles are intravascular ultrasound contrast agents that provide blood pool imaging in real time and targeted (molecular) imaging or drug delivery vehicles with ultrasound activation. Microbubbles have a short half-life;therapeutic payload is small. Red blood cells have been loaded with drugs and reinjected in the bloodstream, including successful clinical trials. They circulate for weeks and have high therapeutic payload, but have poor acoustic contrast and lack a mechanism to trigger drug release. We propose a new ultrasound triggered drug delivery vehicle - acoustically active RBCs (AARBCs) - combining longevity, drug payload and targetability of RBCs with acoustic activation. AARBC is built as RBC with gas precursor perfluorocarbon (PFC) liquid nanodroplet inside. Entrapped PFC absorbs acoustic energy, converts to gas, which triggers drug release from RBC. Particle surface is natural;an early-generation AARBC shows ultrasound contrast half-life in mice ~15 min, longer than microbubbles.
Specific Aims. Aim #1. Demonstrate and optimize acoustically active red blood cells as vascular contrast agents with extended circulation lifetime. Production of AARBCs will be optimized to maximize PFC entrapment and acoustic triggering. The acoustic response of AARBCs will be assessed at clinically relevant ultrasound conditions. AARBCs will be administered to mice to characterize contrast longevity, biodistribution and biocompatibility.
Aim #2. Develop triggered drug delivery vehicles from AARBC. AARBCs will be loaded with calcein, doxorubicin or doxycycline. Contents release and drug delivery will be investigated after insonation, by fluorescence microscopy and via supernatant fluorescence. Therapeutic efficacy will be investigated in a cell culture drug delivery model and in murine vasculature.
Aim #3. Achieve molecular targeting of drug-loaded AARBC. Antibody against VCAM-1 will be attached to AARBCs by noncovalent anchoring and PEG tether. Flow chamber experiments will verify binding of AARBCs to VCAM-1. AARBC targeting to VCAM-1 will be assessed by contrast ultrasound imaging in ApoE -/- mice on high-cholesterol diet, followed by ultrasound-triggered image-guided AARBC release of contents, and a fluorescent drug (doxycycline) detection in the vessel wall.

Public Health Relevance

We propose a novel drug carrier - acoustically active red blood cells (AARBCs) - combining excellent acoustic properties of ultrasound contrast particles with longevity and drug payload of RBCs. These AARBCs would be made from patient's red blood cells, loaded with drug and perfluorocarbon nanodroplets, reinjected into the bloodstream, circulate for extended time, accumulate and provide targeted ultrasound-triggered release of entrapped drug in the area of disease by external focused ultrasound application.

National Institute of Health (NIH)
National Institute of Biomedical Imaging and Bioengineering (NIBIB)
Exploratory/Developmental Grants (R21)
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Medical Imaging Study Section (MEDI)
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Krosnick, Steven
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University of Virginia
Internal Medicine/Medicine
Schools of Medicine
United States
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Rychak, Joshua J; Klibanov, Alexander L (2014) Nucleic acid delivery with microbubbles and ultrasound. Adv Drug Deliv Rev 72:82-93