Figure 1.
Serum half life of nanoparticles.
(A and B) Standard curve generated by quantifying intensity of known concentrations of NP-chitosan-Cy5.5 or NP-CTX-chitosan-Cy5.5. (C and D) Measured fluorescence intensity of nanoparticles in serum. Each data point represents the mean fluorescence intensity integrated above the baseline. This baseline subtraction avoids systematic errors from underlying autofluorescence. Error bars represent standard errors of the mean. The curve indicates an exponential decay curve fit to the data (n = 3 mice per time point).
Figure 2.
Ex vivo NIRF imaging of each organ.
Fluorescence image of whole organs in non-injected (top) or NP-CTX-Chitosan-Cy5.5 injected (bottom) animals. Images were acquired six hours post injection. (A) Mice were injected 200 µl of 1 mg/ml. Six hours after injection, the animals were euthanized and the organs were collected. Ex vivo fluorescence images of whole organ was obtained using the Xenogen imaging system. The spectrum gradient bar corresponds to the fluorescence intensity unit p/sec/cm2/sr. (B) Fluorescence image of 12-micron sections obtained using the Odyssey imaging system. The spectrum gradient bar corresponds to relative fluorescent level. (Top row: brain, heart, lung, liver, and kidney. Bottom row: pancreas, spleen, small intestine, colon, gonad, muscle, and bone marrow) Bone marrow is only shown with the Odyssey scanner.
Figure 3.
Comparison of H&E staining and high-resolution NIRF fluorescence imaging.
(A) In spleen, NIRF signal was observed in large cells within the white pulp (bar, 500 µm). (B) In liver, the pattern was less obvious, but clearly heterogeneous (bar, 500 µm). (C) In heart, the muscle walls of the atria and ventricle showed no signal above background but the walls of the aorta showed significant signal (bar, 50 µm). (D) In kidney, high NIRF signal was found in the renal cortex (bar, 1 mm). All images were taken of tissues that were harvested 24 hours post nanoparticle injection.
Figure 4.
Biodistribution of NP-chitosan-Cy5.5 and NP-CTX-chitosan-Cy5.5 nanoparticles.
Relative fluorescence intensity was determined using the Odyssey scanner. Bars represent tissue from animals that were not injected with nanoparticles (blue), or injected with nanoparticles and harvested 6 hours (red), 24 hours (green), or 48 hours (purple) after injection. Bars represent the average of 3 animals for each nanoparticle at each time point. The error bars are standard deviation from the mean. (A) Ex vivo biodistribution of NP-chitosan-Cy5.5 non-targeted nanoparticle. (B) Ex vivo biodistribution of NP-CTX-chitosan-Cy5.5 targeted nanoparticle.
Figure 5.
Co-localization of iron oxide nanoparticle core and Cy5 signal in liver.
No iron uptake or Cy5 fluorescent signal was detected in non-injected control tissue (top row). Co-localization of Prussian blue/hemotoxylin staining and Cy5 signal from the same tissue section suggest that the nanoparticles remain stable in vivo (bottom row) (bar, 50 µm).
Figure 6.
Rapid and easy method to generate key pharmacokinetic data in the early stages of nanoparticle development.
Our approach minimizes the number of mice required to evaluate nanoparticle pharmacokinetic properties. Serum half-life, whole organ biodistribution, biodistribution at cellular level up to 12 µm resolution, histological and in vivo stability analysis can be performed from the same set of mice that are used to assess nanoparticle targeting to cancer cells or other targets.