Real-time intravital microscopy of individual nanoparticle dynamics in liver and tumors of live mice

Abstract

Intravital microscopy is emerging as an important experimental tool for the research and development of multi-functional therapeutic nanoconstructs. The direct visualization of nanoparticle dynamics within live animals provides invaluable insights into the mechanisms that regulate nanotherapeutics transport and cell-particle interactions. Here we present a protocol to image the dynamics of nanoparticles within the liver and tumors of live mice immediately following systemic injection using a high-speed (30-400 fps) confocal or multi-photon laserscanning fluorescence microscope. Techniques for quantifying the real-time accumulation and cellular association of individual particles with a size ranging from several tens of nanometers to micrometers are described, as well as an experimental strategy for labeling Kupffer cells in the liver in vivo. Experimental design considerations and controls are provided, as well as minimum equipment requirements. The entire protocol takes approximately 4-8 hours and yields quantitative information. These techniques can serve to study a wide range of kinetic parameters that drive nanotherapeutics delivery, uptake, and treatment response.

Aptamer photoregulation in vivo

“…Intratumoral photoactivation of A 1411 /OliP was demonstrated by intravital microscopy (38,39) in nude mice with 4T1 tumors (diameters ∼6-8 mm) on their mammary fat pads. Mice were administered A 1411 or A 1411 /OliP (250 nmol/kg) i.v., both labeled with the fluorescent dye Cy3.…”

Cell source determines the immunological impact of biomimetic nanoparticles

“…Mice were anesthetized with isoflorane, surgically prepared to expose the left lobe of the liver [19], and injected intravenously with a 40 kDa FITC-dextran tracer (1 μM, Invitrogen) to delineate the vasculature [20]. Three days prior to imaging, mice received a one-time intravenous injection of DiD-labeled autologous red blood cells (RBCs, ~3% hematocrit) [21].…”

“…Three days prior to imaging, mice received a one-time intravenous injection of DiD-labeled autologous red blood cells (RBCs, ~3% hematocrit) [21]. High-resolution time-lapse images of particle accumulation in the liver were acquired across multiple field-of-views (FOVs) for up to 2 h at 10–30 second intervals (30 fps) following a one-time intravenous injection of 0.5 x 10 8 particles in 50 μl PBS [19]. Particle accumulation dynamics were quantified frame-by-frame using NIS Elements v4.1 [19].…”

“…High-resolution time-lapse images of particle accumulation in the liver were acquired across multiple field-of-views (FOVs) for up to 2 h at 10–30 second intervals (30 fps) following a one-time intravenous injection of 0.5 x 10 8 particles in 50 μl PBS [19]. Particle accumulation dynamics were quantified frame-by-frame using NIS Elements v4.1 [19]. Particle biodistribution was assessed 1 h after particle injection, at which time the tissues harvested, sliced using a vibratome (Leica VT-1000), and imaged using the IVM system.…”

Imaging in animal models

“…Small scale experimental systems are being adapted for use in rodent models and for clinical imaging [38][39][40]. Furthermore, intravital imaging has identified differences in tumour cell movement in animal models in comparison to in vitro models which could provide more information on tumour microenvironment and mechanisms of invasion and metastasis [41,42].…”