A new way to image metallic nanoparticles in real time using multi-photon luminescence

Objectives: Metallic nanoparticles have great potential as both imaging and therapeutic agents, however their distribution and extravasation potential are often difficult to assess in-vivo. Intravital microscopy (IVM) is a powerful tool that offers real-time imaging of cellular and subcellular processes in living preclinical animal models and can provide important quantitative and dynamic insights to enable clinical translation of new imaging/therapeutic agents. Most IVM procedures use fluorescent based labels to track various molecular processes. A limitation to this technique is the need to chemically modify the sample of interest, which may disrupt its natural biological or physiological function. Here we demonstrate a new technique to capture real-time videos of label-free gold nanoparticles in living mice with single particle sensitivity. With the emergence of newer multi-photon based IVM platforms, we have discovered that metallic nanoparticles produce an intrinsic luminescence effect that is easily detected and recorded in real-time without the need for exogenous fluorophore labeling (Figure 1). Several preclinical studies have shown great promise for the use of various metallic-based nanoparticles for the treatment and imaging of various disease processes, but without fully understanding their pharmacokinetics, biodistribution and blood clearance post administration, their likelihood of clinical translation is improbable. Previously, in-vivo tracking and identification of metallic nanoparticles has been difficult without extrinsic labeling using fluorophores or radiolabels.

Methods: Here, we present a label-free single particle sensitive approach to examining gold nanoparticle (AuNP) pharmacokinetics in living mice in real time, utilizing intravital multi-photon microscopy. This method employs a near-infrared femtosecond pulsed laser system to visualize intrinsic AuNP luminescent signal produced by multiphoton absorption induced luminescence (MAIL). Multi-channel imaging allows for simultaneous visualization of the vasculature using another label-free technique known as second-harmonic generation microscopy. This highlights vascular features in real-time without the need to administer exogenous vessel illuminating dyes. Mice were given a 100 ul intravenous dose of silica coated gold nanoparticles (~140 nm) ranging in concentration between 1 nM and 3.5 nM. The ear vasculature was imaged at 220 frames per second with multiphoton IVM.

Results: Nanoparticle blood distribution was assayed from 30 seconds pre-injection to 30 min post AuNP injection and indicated rapid blood clearance (<30 min) for all nanoparticle concentrations with no evidence of passive extravasation. AuNPs exhibited no photobleaching over the imaging sessions along with 4.88x (p<0.001) higher intensity compared to the background vessel autofluorescence. Further, pre-injection concentrations were correlated with in-vivo particle counts and blood elimination profiles, demonstrating effective sensitivity for pharmacokinetic analysis. Conclusion: With this information, researchers can finally get a real-time glimpse at the fate and dynamic behavior of individual metallic-based nanoparticles, marking an important step towards clinical translation.