Scalable Synthesis of Calcium Fluoride Nanoparticles as a Novel Ultrasound Contrast Agent for Imaging Tumor Targeted Delivery of Therapeutics

Ultrasound (US) imaging is one of the most widely used noninvasive diagnostic techniques in clinical practice due to its safety, real-time capability, and cost-effectiveness. However, traditional B-mode imaging provides limited information with low sensitivity. Recently microbubble (MB)-based contrast agents have been employed to enhance contrast signals while also facilitating the imaging of molecular targets in target tissues or pathological locations. Currently available MBs face several limitations, including large size (typically 1-10 μm), limited ability to penetrate beyond blood vessels, poor in vivo stability, susceptibility to gas leakage, and high production costs, which collectively hinder their clinical efficiency and applicability. To address these limitations, we developed calcium fluoride (CaF₂) nanoparticles (NPs) using a scalable synthesis process as a next-generation ultrasound contrast agent via a coprecipitation method using citrate as a stabilizing agent. The synthesized NPs exhibited a uniform size distribution (∼100 nm) with consistent reproducibility during scalable production conditions. Notably, CaF₂ NPs generated substantially better contrast-enhanced ultrasound signals under acidic conditions (pH 5.5) than at neutral pH (7.2). The porous structure of the CaF₂ NPs enabled us to efficiently load doxorubicin with the loading efficiency of 29.3 ± 5% and controlled intracellular delivery in MDA-MB-231 breast cancer cells in vitro. In vivo studies using an orthotopic breast cancer model demonstrated a pronounced pH-responsive ultrasound contrast signal following intravenous administration of CaF₂ NPs (15 mg/kg). Tumor signal intensity increased ∼25-fold compared to controls at 1 h postinjection, with contrast persisting up to 5 h, dramatically outperforming conventional microbubbles that typically dissipate within 10 min. Biosafety analyses confirmed excellent hemocompatibility and no detectable organ toxicity. To the best of our knowledge, this is the first report of CaF₂ NPs functioning as a US contrast agent for in vivo tumor imaging and drug delivery. Overall, we identify CaF₂ NPs as safe, long-lasting, and multifunctional US contrast agents with integrated drug delivery capability, offering a promising platform for advanced ultrasound imaging and theranostic applications.