Synthesis and Characterization of Bismuth-based Nanoparticles for Targeted Computed Tomography Imaging

The cardiac conduction system (CCS) is responsible for rhythmic and coordinated contraction of the heart by rapidly transmitting electrical impulses through fast-conducting fibers. Conduction abnormalities can manifest as life-threatening arrhythmias. Catheter ablation remains the standard treatment for restoring normal cardiac rhythm, relying heavily on accurate localization of the arrhythmogenic substrate. Intraprocedural visualization of CCS abnormalities would be game changing in precisely guiding ablation to the region of interest and minimizing collateral damage of healthy tissue. Although current imaging modalities such as computed tomography (CT) combined with iodinated contrast agents (ICAs) provide high-resolution anatomical detail, they lack the specificity needed to delineate the CCS for targeted intervention. The use of ICAs present several limitations, including nephrotoxicity, short half-life, and risk of hypersensitivity reactions in patients. Bismuth-based nanoparticles (BiNps) offer a promising alternative to ICAs due to bismuth’s higher atomic number (Z=83) compared to iodine (Z=53), resulting in greater X-ray attenuation. Their nanoscale size enables BiNps to evade renal filtration, thereby prolonging circulation time and enhancing the imaging window. Unlike ICAs, BiNps can be functionalized with antibodies targeting specific proteins, such as Contactin-2 (Cntn2), which is preferentially expressed on CCS cells. The aim of this work is to synthesize high-payload BiNps with strong radiopaque properties that can be conjugated with Cntn2 antibodies, enabling targeted binding to Cntn2-expressing cells. BiNps were synthesized by dissolving bismuth (III) nitrate pentahydrate in 1,2-propanediol, using α-D(+) glucose as a surfactant and borane morpholine as a reducing agent. The synthesized BiNPs were subsequently PEGylated using a layer-by-layer (LbL) approach, employing poly(allylamine hydrochloride) (PAH) and poly(acrylic acid) (PAA) as polyelectrolytes, followed by the addition of amino-PEG-acid (NH₂-PEG-COOH). PEGylated particles were further conjugated with Cntn2 antibodies using NHS/EDC chemistry. Dynamic Light Scattering (DLS) and Zeta Potential analyses revealed an average hydrodynamic diameter of 107.8 ± 1.9 nm and a surface charge of -26.6 ± 1.7 mV for the BiNps. Ultraviolet-Visible (UV-vis) spectroscopy showed a broad and stable absorption between 350–400 nm over 20 weeks. Transmission Electron Microscopy (TEM) confirmed the crystalline structure, with a bismuth core-size of 65.6 ± 9.6 nm. MicroCT analysis confirmed significantly higher X-ray attenuation compared to commercial ICAs. The success of the LbL coating was confirmed at each step by DLS analysis, which showed a progressive increase in hydrodynamic diameter. Zeta potential measurements revealed corresponding changes in surface charge, confirming a negative value for the PEGylated particles, which was maintained following Cntn2 conjugation. In conclusion, BiNps demonstrated sustained colloidal stability and superior X-ray attenuation compared to commercial ICAs, reinforcing their potential as a nanoparticle-based alternative for X-ray imaging. Furthermore, BiNps were successfully PEGylated and conjugated with Cntn2 antibodies to enable targeted visualization. Their enhanced physicochemical properties and targeting capability highlight their suitability for visualizing the CCS and advancing precision imaging in electrophysiological interventions.