Photoacoustic imaging of mesenchymal stem cells in living mice via silica-coated gold nanorods

Abstract

Improved imaging modalities are critically needed for optimizing stem cell therapy. Techniques with real-time content to guide and quantitate cell implantation are especially important in applications such as musculoskeletal regenerative medicine. Here, we report the use of silica-coated gold nanorods as a contrast agent for photoacoustic imaging and quantitation of mesenchymal stem cells in rodent muscle tissue. The silica coating increased the uptake of gold into the cell more than 5-fold, yet no toxicity or proliferation changes were observed in cells loaded with this contrast agent. Pluripotency of the cells was retained, and secretome analysis indicated that only IL-6 was disregulated more than 2-fold from a pool of 26 cytokines. The low background of the technique allowed imaging of down to 100,000 cells in vivo. The spatial resolution is 340 μm, and the temporal resolution is 0.2 s, which is at least an order of magnitude below existing cell imaging approaches. This approach has significant advantages over traditional cell imaging techniques like positron emission tomography and magnetic resonance imaging including real time monitoring of stem cell therapy.

Figure 1

Characterization of SiGNR contrast agent. TEM images of GNRs (A) and SiGNRs (C) were obtained and the materials were studied by absorption spectroscopy at 1:30 dilution of stock solution (~5 nM) in water. A slight red shift was noted for the silica-coated agents (B). (D) The backscatter (B-mode) and PA signals were studied and the addition of the silica coat increased the PA signal 4-fold. Panels A and C show some imaging artifacts (white) from heterogeneity in nanoparticle size when imaged in a finite focal depth. This is not indicative of an impurity or other species.

Figure 2

Toxicity and proliferation of SiGNR-labled MSCs. (A) The capacity of the MTT assay to count cells was confirmed with increasing numbers of plated MSCs (“#” indicates cytotoxic positive control; 0.25 mg/mL CTAB). (B) Increasing concentrations of SiGNRs show increasing toxicity to 10 000 MSCs after overnight (~20 h) incubation with SiGNRs. (C) The incubation time of one concentration (0.07 nM) was further optimized with 3, 6, and 20 h of incubation. “Ctrl.” in panel C indicates no SiGNRs. Incubation at 3 h at this concentration produced an insignificant (p > 0.05) decrease in cell metabolism. (D) To study the impact SiGNRs have on MSC growth proliferation, MSCs both loaded and unloaded with SiGNRs were serially monitored. There was no significant change to their growth as probed by MTT assay. Both unlabeled and SiGNR-labeled MSCs showed a doubling time of three days. In panels A–C, error bars represent the standard deviation of three replicate experiments. Error bars in panel D represent standard deviation of six replicate wells.

Figure 3

Confirmation of SiGNRs inside MSCs. (A–E) TEM images of MSCs loaded with SiGNRs were collected at increasing magnifications. The dashed, colored inset in panels A–E correspond to the sequential, higher magnification image in the following panel. Panel E clearly shows nanorods inside a MSC vesicle. Panel F is a portion of the EDS spectra acquired on panel E (dashed inset) that confirms the presence of gold. The presence of Cu is from the Formvar-coated TEM grid and Os is from the OsO₄ stain. The silica coat is not highly visible because the electron density of silica is nearly equivalent to the stained cells. See additional example in Supporting Information, Figure S.6.

Figure 4

Histology images confirm that the osteogenic and adipogenic differentiation capacity of MSCs is unchanged by the presence of SiGNRs. Cells in images on the top row are noninduced controls, while the bottom row was cultured in either osteogenic (left) or adipogenic (right) media. The experiments presented in panels B, F, D, and H were performed on cells loaded with SiGNRs before plating. Panels A, E, C, and G are unloaded control cells. Both loaded and unloaded cells show increased mineral deposition as determined by Alizarin Red S staining in the osteogenic experiments (red color; E, F). Differentiation into adipocytes is similarly unaffected by the presence of the nanoparticles. Black arrows in G and H highlight lipid vacuoles stained by Oil Red O. Importantly, not only is differentiation capacity retained, but the presence of SiGNRs does not induce unintended differentiation (B and D) (see Supporting Information, Figure S.9 for white light photographs of the cell culture plates).

Figure 5

Secretome analysis of labeled cells. The change in secretome cytokine expression levels is shown for 26 different proteins. Cell culture media from SiGNR-loaded MSCs and control MSCs was analyzed for these proteins. The concentration of protein in SiGNR-loaded cells was divided by the concentration in controls cells to produce the metric above. Except for IL-6, no protein had a concentration that changed more than 200% (1-fold change). Black bars indicate a statistically significant (p < 0.05) change in expression; green bars indicate a p-value above 0.05. Please see Supporting Information, Table S.1 for actual valuesand additional statistical content.

Figure 6

In vivo positive and negative controls; labeled MSC injection. This figure presents both B-mode (gray scale) and PA (red) images of the intramuscular injection of a positive control (0.7 nM SiGNRs; left), negative control (0 nM SiGNRs (no cells); middle), and 800 000 SiGNR-labeled MSCs (right) all in 50% matrigel/PBS into hind limb muscle of an athymic mouse. Imaging sequence is as follows: preinjection (A, B, C); needle insertion and position (D, E, F); postinjection (G, H, I); needle removal and final imaging (J, K, L), and contrast enhancement to illustrate increased signal (M, N, O). Pixels increased relative to preinjection image are coded yellow. Note significant signal increase in M and O at injection site relative to A and C (dashed circles highlight injection site). Also, note low signal in negative control (N). Scale bar in M and intensity scale in L/O applies to all images. The “b” in all panels indicates bone and the red dashed circle in J, K, L indicates that the injection bolus can also be seen with B-mode ultrasound. Real time injection imaging of cells between panels F and I may be seen in Supporting Information, Video 1 (at 8× speed) and in real time (Supporting Information, Movie 2).

Figure 7

Validation of imaging data. (A) Spectral analysis of tissue and 800 000 MSCs after i.m. injection. Also shown in green is the normalized spectral analysis of the MSCs in vivo. A broad increase in PA signal is seen, which may be due to aggregation and resonance coupling of the contrast (see 7E and Supporting Information, Figure S.6). This normalized spectrum is more red-shifted versus SiGNRs imaged in a phantom (Supporting Information, Figure S.3D). That experiment suggested an intensity maximum near the absorbance peak shown in Figure 1. (B) The average signal for decreasing numbers of cells and as well as the negative control indicate that the calculated estimate for limit of detection is 90 000 cells. A sampling of 100 000 cells was easily imaged above background injection. Error bars represent the standard error of the background-corrected signal for each group of mice. (C) H&E staining of muscle tissue (right) with adjacent MSCs (left). (D) The fluorescence of a cell tracking dye (green) from an adjacent section illustrates higher signal for MSCs than muscle tissue. (E) Higher magnification of MSCs shows SiGNRs (black) inside the cells (black arrows).