Imaging of the nanoparticle’s distribution in animal models

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One of the key issues in CUPIDO is to track where and when the inhaled CaP nanoparticles travel in the body in vivo (biodistribution). As a first approach, the imaging studies, performed by our partner BIOEMTECH, has been focused on the spatio-temporal biodistribution of the unloaded nanoparticles (without the drug) in mice, in particular in lungs, heart, intestine and kidneys.

Nanoparticles have been labelled with technetium-99m (99mTc), the most common medical radioisotope used in clinical practice, that allows to track the signal of the nanoparticles inside the body through real-time, dynamic scintigraphy and/or tomography. Thanks to a new protocol, the incorporation of radioisotope in the nanoparticles is very high; furthermore, the size and physicochemical properties of the CaP nanoparticles aren’t altered. The stability of the technetium-99m attached to the nanoparticles remains high (93-98 %) up to 24h after the labelling and at all tested temperatures. Overall, the results highlight an excellent radiolabeling capacity and stability of the tested nanoparticles and also the ability to remain unaltered, making them appropriate candidates for all the next tests.

The in vivo fate of the nanoparticles is assessed through the company’s molecular screening tools that provide real-time, dynamic scintigraphy (for visualizing Single Photon Emission Tomography (SPECT) isotopes), in combination with x-ray imaging. While the SPECT imaging provides a semi-quantitative picture of the accumulation of the nanoparticles in the organs throughout time, the x-ray imaging provides an anatomical image at very high resolution, that can act as an anatomical map. The combination of the two methods provides a detailed and clear biodistribution of the tracked nanoparticles for up to 24 hours. BIOEMTECH also exploits a micro-SPECT (spatial resolution of 0.6 mm for mouse imaging) and a micro-CT system (spatial resolution 0.05 mm) as this allows a clear visualization and monitoring of internal organ parts and are capable to monitor the accumulation in each heart region and passing through the lung-heart barriers.

All the imaging experiments have been performed so far using 3 administration routes in mice: (i) intravenous (tail vein), which is the most common administration route currently used to deliver pharmaceuticals; (ii) intratracheal, which injects the nanoparticles directly into the trachea and resembles the inhalation envisioned in CUPIDO; and (iii) intravenous (retro-orbital) administration, which is considered a close pathway to reach the heart through the circulatory system.

After the injection, real-time fast dynamic imaging (one image every 2 minutes) is performed during the first hour, when kinetics is expected to be fast. After the first hour, tomographic, static, higher resolution images are collected only at specific times on the 3D micro-SPECT and microCT systems up to 24h after the injection, to clearly visualize all the individual heart parts. Ex vivo biodistribution studies are also performed at selected time points and in case of very low uptake (<1%) of the nanoparticles in the organs. When uptake values are close or below 1% (% of the administered substance reaching a certain organ), imaging operates close to its detection limits and therefore ex vivo studies on each organ can be used to estimate the nanoparticles uptake with greater accuracy.

The evidence collected so far confirmed that the intravenous administration route results in a very limited accumulation of the nanoparticles in the heart and supports the idea that inhalation represents a better option to target the heart, as already suggested by other studies from the CUPIDO Consortium. Currently, another administration route via nebulization has been included and further studies are aiming to characterize the nanoparticles fractions that cross the air-blood barrier and assess their distribution among the organs.