Quantitative Whole Body Biodistribution of Fluorescent-Labeled Agents by Non-Invasive Tomographic Imaging

When small molecules or proteins are injected into live animals, their physical and chemical properties will significantly affect pharmacokinetics, tissue penetration, and the ultimate routes of metabolism and clearance. Fluorescence molecular tomography (FMT) offers the ability to non-invasively image and quantify temporal changes in fluorescence throughout the major organ systems of living animals, in a manner analogous to traditional approaches with radiolabeled agents. This approach is best used with biotherapeutics (therapeutic antibodies, or other large proteins) or large-scaffold drug-delivery vectors, that are minimally affected by low-level fluorophore conjugation. Application to small molecule drugs should take into account the significant impact of fluorophore labeling on size and physicochemical properties, however, the presents studies show that this technique is readily applied to small molecule agents developed for far-red (FR) or near infrared (NIR) imaging. Quantification by non-invasive FMT correlated well with both fluorescence from tissue homogenates as well as with planar (2D) fluorescence reflectance imaging of excised intact organs (r2 = 0.996 and 0.969, respectively). Dynamic FMT imaging (multiple times from 0 to 24 h) performed in live mice after the injection of four different FR/NIR-labeled agents, including immunoglobulin, 20–50 nm nanoparticles, a large vascular imaging agent, and a small molecule integrin antagonist, showed clear differences in the percentage of injected dose per gram of tissue (%ID/g) in liver, kidney, and bladder signal. Nanoparticles and IgG1 favored liver over kidney signal, the small molecule integrin-binding agent favored rapid kidney and bladder clearance, and the vascular agent, showed both liver and kidney clearance. Further assessment of the volume of distribution of these agents by fluorescent volume added information regarding their biodistribution and highlighted the relatively poor extravasation into tissue by IgG1. These studies demonstrate the ability of quantitative FMT imaging of FR/NIR agents to non-invasively visualize and quantify the biodistribution of different agents over time

Development of the Merton Partnership's Website

The project team developed a web-based communications system for the Merton Partnership, a local strategic partnership (LSP) in London, England. We conducted research on the Partnership's past and current issues, interviewed the partners, and interviewed IT professionals and project managers from other LSPs to obtain technical advice on e-government systems. This project provided the Partnership with a web-based tool that enables them to communicate more efficiently

Characteristics of Fluorescent-Labeled Agents.

<p>Characteristics of Fluorescent-Labeled Agents.</p

Non-invasive FMT correlation to post-mortem tissue fluorescence.

<p>Quantitative FMT imaging data from three mice injected with BSA-VT680XL was compared to post-mortem tissue assessment of organ fluorescence. <b>A</b>, Comparison of FMT quantitation to FRI gross tissue imaging results, represented in mean intensity units of counts/energy, was performed. <b>B</b>, As a second approach to validation, an independent comparison to quantitative fluorescence was performed from homogenized and diluted tissue samples, assayed in comparison to a VivoTag680XL standard curve. For this second approach, the %ID/g was calculated from quantified fluorescence and weighed organ tissue. Both comparisons yielded excellent correlation to FMT imaging, and tissue homogenate data also generated good agreement between absolute values of %ID/g.</p

Determining relative fluorescence volume of distribution changes of different imaging agents.

<p>Fluorescence data from the study in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020594#pone-0020594-g006" target="_blank">Fig. 6</a> was further analyzed for kinetic changes in the volume of fluorescence at different concentrations within the body as determined by FMT2500. Fluorescence volume changes for four different concentration ranges were represented in graphical form as normalizations to starting volumes.</p

Establishing FMT image analysis techniques for accurate assessment of specific organ systems.

<p>Nude or BALB/c mice received administration of a variety of NIR fluorescence imaging agents to help to define specific organ regions and the appropriate placement of regions of interest, ROI, for quantification. <b>A</b>, GastroSense 680 was orally gavaged, and mice were imaged immediately to visualize the stomach and to place a 3-dimensional ROI (see front and side views). At 2 h post-gavage, the agent emptied from the stomach and moved into the intestines, allowing clear 3D intestinal ROI placement. <b>B</b>, Free fluorophore (VivoTag 680XL) was injected IV, revealing very rapid appearance in the bladder (left panels) and circulation through the vessels of the brain (right panels). <b>C</b>, BSA, known as a protein that predominantly localizes to the liver, was labeled with a NIR fluorophore (VivoTag 680XL) and injected IV to define the liver region. <b>D</b>, The large NIR vascular agent, AngioSense 680, was injected IV to provide signal defining the heart and carotid/jugular veins. <b>E</b>, Asthma-like inflammation was induced by immunization of BALB/c mice as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020594#pone.0020594-Korideck1" target="_blank">[16]</a>, and ProSense 680 was used to image the pulmonary eosinophilia cathepsin activity, thus defining the lung regions in 3D. <b>F</b>, IV ReninSense 680 was activated by normal kidney renin activity, providing signal defining the kidney regions. The resulting 3D ROIs for each of these organ/tissue regions defined the regions to be analyzed in subsequent whole body biodistribution studies, with a 2X4 grid providing a reference for proper ROI placement. Fluorescence outside of each tissue region’s ROI is omitted for clarity.</p

Validation of FMT biodistribution imaging techniques using NIR-labeled BSA.

<p>Nu/nu mice (n = 3 per group) received either BSA-VT680XL or no injection (Control), and mice were noninvasively imaged by FMT2500 at 24 h post-injection, a time when the agent would no longer be in circulation but would be at maximal levels in the tissue. Organ ROIs defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020594#pone-0020594-g002" target="_blank">Figs. 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020594#pone-0020594-g003" target="_blank">3</a> were applied for proper quantification of fluorescence, and the data was normalized to % injected dose per gram of tissue (assessed at study termination). Organ weights were adjusted appropriately for liver and lungs to account for incomplete capture of the full organs, based on prior studies (not shown). <b>A</b>, Tomographic images of injected and control mice revealing BSA-VT680XL distribution (left panel) and normal chow-related fluorescence in the controls (right panel). Brain fluorescence was collected in separate FMT image acquisitions (not shown). <b>B</b>, Quantification of fluorescence in injected and control mice, revealing predominant liver distribution of labeled BSA, with control mice data suggesting that all of the stomach and intestine signal could be attributed to chow-related fluorescence. <b>C</b>, Collection of specific organs was performed as an independent verification of the fluorescence quantified non-invasively by FMT. <b>D</b>, Fluorescence intensity patterns seen for the different organs, although not objectively quantitative due to the 2D acquisition, were similar to those seen by FMT imaging.</p

Comparison of the kinetics of whole body fluorescence changes for four different NIR-labeled agents.

<p>Four different agents were injected IV into nu/nu mice (n = 3 mice/group), and animals were imaged immediately and at 1, 3, 6, 12, 24, and 100h post injection by FMT2500. <b>A</b>, Whole body fluorescence was quantified at each time point using ROIs to capture the torso and head. Data was normalized as the percentage of the injected dose. <b>B</b>, Plasma pharmacokinetics data was collected in separate sets of animals (as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020594#s4" target="_blank">Materials and Methods</a>) to provide data regarding circulating agent kinetics. Data was normalized as the percentage of the injected dose. <b>C</b>, An approximation of the kinetics of agent accumulation in tissue was established by subtracting the normalized plasma pharmacokinetic profiles from the whole body fluorescence profiles, making the assumption for each agent that the earliest imaging time point represented 100% of the agent in circulation. Corrected results reveal differences in extravasation and whole-body tissue clearance rates for all four agents.</p

Diagrammatic representation of the FMT biodistribution model.

<p>Mice are injected with NIR-labeled agents at time 0 and imaged by FMT immediately, at the time when all of the signal would be attributed to the vasculature. At regular times thereafter, mice are reimaged to assess the dynamic changes in tissue distribution, including kinetics of extravasation into specific organ regions as well as the kinetics of tissue clearance.</p