Main

To develop tools that meet the cell tracking needs of the research community, scientists at Carestream Molecular Imaging recently invented a class of fluorescent nanoparticles, approximately 17 nanometers in diameter, that encapsulate hydrophobic analogs of carbocyanine dyes with emissions ranging from green to near-infrared. The nanoparticle used for this study is commercially available from Carestream Molecular Imaging under the name Kodak X-Sight 761 Nanospheres.

Knowledge of the location and amount of cells is often desirable in many types of in vivo experiments. This information may be needed to prove a central hypothesis in microbiology1,2 or tumor biology studies3, or to determine how to appropriately normalize data from reporters on other biological pathways. Often, strong constitutive promoters, such as the cytomegalovirus promoter (CMVp), drive the expression of a genetic reporter to tag cells and thus reflect their cell mass. Although this offers a first approximation, CMVp is often epigenetically silenced in vivo4,5,6. Additionally, it may not be the best control or normalization tool for a variety of important pathways and experiments because the NFκB pathway can regulate the CMVp7,8. Thus, alternative methods are needed to detect cells in a convenient manner. Here we demonstrate the use of Kodak X-Sight 761 Nanospheres for both in vitro and in vivo cell labeling applications.

Design of the nanoparticle

The hydrophobic interior of Kodak X-Sight 761 Nanospheres serves to both protect from the surrounding environment and increase the quantum yield of encapsulated organic fluorophores. This bright latex nanoparticle is coated with polyethylene glycol chains to make the nanoparticles water-soluble and biocompatible. The polyethylene glycol chains are terminated with primary amines to facilitate synthetic access for bioconjugation to various targeting moieties to make optical molecular probes. The amines also impart a special property to the nanoparticle: the ability to penetrate into cells without the need for additional conjugation to protein induction domain peptides such as TAT. Cationic species are well known for their uptake into cells using endocytosis and macropinocytosis9.

Kodak X-Sight 761 Nanospheres are loaded with a dye with spectral properties (excitation peak, 761 nm; emission peak, 789 nm) similar to those of Cy7 and Alexa Fluor 750. This bright and stable nanosphere is optimal for in vivo studies because its excitation and emission wavelength are in the near-infrared region, which is best for optical imaging through tissue.

Labeling and monitoring cells

First, we performed in vitro tests to determine the efficacy of Kodak X-Sight 761 Nanospheres in vitro. We incubated melanoma cells (B16-F10) with a 2 μM solution of Kodak X-Sight 761 Nanospheres in cell culture medium (DMEM) at 37 °C for 4 hours. Cells were then trypsinized, rinsed with DMEM and then washed twice with PBS to remove excess nanoparticles. Subsequently we imaged these cells at ×600 magnification and compared them with unlabeled cells (Fig. 1). Epifluorescence images were acquired using a Cy7 filter set (Chroma). The entire population of melanoma cells appeared to be labeled with the near-infrared fluorescent nanoparticles. Inspection of these cells with higher magnification indicated that the nanoparticles appeared to be localized to vesicular compartments in the cells and brightly stained the perinuclear environment. There was almost no detectable fluorescence emanating from within the nucleus (Fig. 1b). Unlabeled cells did not exhibit any detectable near-infrared autofluorescence (Fig. 1d). These data illustrate the usefulness of near-infrared imaging of Kodak X-Sight 761 Nanospheres for simple cellular and subcellular labeling purposes.

Figure 1: Near-infrared epifluorescence microscopic imaging of melanoma cells labeled with Kodak X-Sight 761 Nanospheres.
figure 1

(a–d) Phase-contrast (a,c) and fluorescence (b,d) images of B16-F10 melanoma cells that were incubated with a 2 μM solution of Kodak X-Sight 761 Nanospheres (a,b) and of unlabeled cells (c,d). Magnification factor was ×600. Color bars range from 0 (dark) to 3,865 in b and 79 in d.

Full size image

To demonstrate the utility of these nanoparticles for cell tracking in live animals, we prelabeled of 5 × 106 melanoma cells with Kodak X-Sight 761 Nanospheres as described above and implanted them subcutaneously in both a nude immunodeficient (Nu/Nu) and a black wild-type (C57BL/6) mouse. (Melanoma tumors grow in both of these strains.) After a 24-hour incubation, we could not detect derived tumors via standard palpation or visual inspection of the mice. (Melanoma cell xenografts usually need up to 10 days to form palpable tumors.) However, the location and relative size of the cell implant site was readily apparent (Fig. 2) upon whole-animal fluorescence imaging using the Kodak In-Vivo Multispectral Imaging System FX (configured for 750 nm excitation, 830 nm emission, 60 s exposure, 4 × 4 binning, f-stop 2.5, 120 mm field of view; black mouse was depilated). To determine how long the nanoparticles could be detected, we reimaged the same mice 10 days later, after palpable tumors had formed. The location of the tumor was readily apparent, though the overall fluorescence intensity at the site had decreased, indicating that intensity may not reflect cell mass at longer time points. Finally, we euthanized the mice, dissected them and imaged the organs ex vivo. The fluorescence was confirmed to originate from the tumor (data not shown), illustrating that Kodak X-Sight 761 Nanospheres can be readily used to delineate the sites of living cancer cell xenografts in living mice.

Figure 2: Visualization of tumors in live mice.
figure 2

B16-F10 melanoma cells were prelabeled with Kodak X-Sight 761 Nanospheres and tumors were imaged at 24 hours (top) and 240 hours (bottom). At 24 h, the tumors were impalpable, yet their location was readily detected from the images.

Full size image

Conclusions

To facilitate noninvasive cell imaging experiments in living mice, we used fluorescent nanoparticles as an alternative to genetically encoded reporters. Cells can be prelabeled with Kodak X-Sight 761 Nanospheres and monitored both in vitro and in vivo. Because these nanoparticles are bright and biocompatible and emit in the near-infrared wavelengths, they should allow investigators to conduct rigorous cell-tracking experiments in live mice.