Multimodal Nanocarrier Probes Reveal Superior Biodistribution Quantification by Isotopic Analysis over Fluorescence.

Absolute measurements of biodistribution are essential for understanding and optimizing the function of nanomaterials for in vivo diagnostic and therapeutic applications. Biodistribution analysis by optical imaging is desirable due to its low cost, wide accessibility, and high-throughput nature, but it is substantially less accurate than isotopic and chemical techniques. In this work, we developed multimodal probes for optical and nuclear imaging to analyze the quantitative limits of optical contrast in the red and near-infrared spectra for polysaccharide nanocarriers targeting macrophage cells. Probes incorporating three zwitterionic fluorophores together with radioactive copper distributed diffusely to optically dissimilar tissues that were either white (visceral adipose tissue) or dark red (liver and spleen) in obese rodents. We used in vivo positron emission tomography/computed tomography (PET/CT) imaging, in vivo hyperspectral tomographic fluorescence imaging, and ex vivo optical and isotopic analyses to determine correlations between optical and nuclear signals. PET imaging strongly correlated with standardized ex vivo methods for all tissue types, whereas no fluorescence signals exhibited substantial accuracy in quantification or localization in vivo. Optical imaging of resected tissues was most accurate in the 700 nm wavelength window, but only in white tissues. This work suggests that fluorescence can be used to measure diffuse probe distribution in white tissues over time or across animals, but not red tissues and not deep in the body. This work also highlights the importance of choosing validated experimental protocols and describes how optical measurements are impacted by fluorophore class and spectral properties, tissue properties, and imaging workflow.