The GNRs and SiGNRs were characterized via
TEM and absorbance spectroscopy (). The GNRs had a peak resonance at 665 nm with average dimensions of 42.17 ± 5.11 nm by 14.90 ± 0.58 nm as measured by TEM and ImageJ analysis (). After silica coating (), the dimensions increased to 82.99 ± 3.86 by 64.20 ± 3.48 nm width with an additional 11 nm in red-shift of the plasmon resonance to 676 nm (). This 20 nm shell thickness was previously reported to be optimal for PA imaging.35
DLS indicated that the GNRs had a charge of 14.7 mV and the SiGNRs were 7.8 mV in 1:1 PBS/water.38
The PA signal of GNRs and SiGNRs at 1.4 nM was also calculated and the silica coating produced a 4-fold increase in PA signal (). Previous reports suggest that silica coating provides a 3-fold increase in PA signal.35
The 4-fold increase seen here is likely due to a closer matching of the SiGNR peak (676 nm) with the excitation pulse (680 nm) relative to the uncoated GNRs (665 nm).
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 (more ...)
The PA scanner consisted of three separate components including a light-tight imaging chamber, an excitation source, and a PC-based processing console (Supporting Information, Figures S.1 and S.2
). The imaging conditions (gain, power, and dynamic range) of the PA instrument for this contrast agent were empirically optimized. For additional details of these descriptors, please see the caption of Supporting Information, Figure S.3
. The laser power was monitored with an external power meter as well as internal power sampling. At 680 nm, the average power detected 1 cm away from the transducer was 9.5 mJ (6.9–12.9 mJ) with root-mean-square variation of 10.1% for 500 pulses. Supporting Information, Figure S.3
presents an experiment in which other parameters were sequentially modulated and the resulting signal from the contrast agent was plotted along with the signal-to-background ratio. Optimal conditions were achieved with a gain of 50 dB, 80% power, a persistence of four frames (no persistence was used for real time imaging), and 20 dB of dynamic range. These conditions were used for the remainder of the experiments. The spatial resolution was probed by imaging a test pattern printed on transparency film. Spacing of 340 μ
m was easily resolved while spacing of 58 μ
m could not be resolved (Supporting Information, Figure S.4
). There was a linear relationship (R2
> 0.99) between concentration (up to 0.7 nM) and PA signal of the SiGNRs in an agarose phantom with a LOD of 0.03 nM SiGNRs (Supporting Information, Figure S.5A,B
). No decrease in PA signal intensity was observed for the SiGNRs over 60 days.
The capacity of SiGNRs to label MSCs was studied next. Previously, silica has facilitated endocytosis into a variety of cell types, including MSCs.39,40
To choose the appropriate starting concentration and incubation time of the SiGNRs, we used the MTT cell toxicity assays and centered the study near 0.05 nM SiGNRs, which has previously shown efficacy for cellular labeling with gold core/silica shell nanoparticles ().41
Both SiGNR concentration () and the incubation time () were studied. The results indicate that 0.07 nM of SiGNRs (1.5 × 106
SiGNRs/MSC) at 3 h of incubation time gave no statistically significant change in MSC metabolic activity relative to the negative control (p
> 0.05). To confirm SiGNR endocytosis, TEM images of fixed cells were acquired, and accumulation of SiGNRs inside MSC vesicles was noted ( and Supporting Information, Figure S.6
). The silica coat was not entirely clear because the electron density of silica is approximately the same as the 400 nm section of resin.
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 (more ...)
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 (more ...)
The capacity of GNR- and SiGNR-labeled MSCs to generate a PA signal was studied relative to nano-particle free-MSCs, all at 50 000 cells (in 15 μ
L) in an agarose phantom (Supporting Information, Figure S.7
). Maximum intensity projections were created to analyze the data with ROI analysis. A 2.8-fold increase was measured for the GNR-labeled MSCs relative to unlabeled MSCs. The signal of GNR-MSCs was 7.6-fold lower than the same number of MSCs with SiGNRs (Supporting Information, Figure S.7C
). Theoretically, this increase should have been 20-fold (5 times more contrast with 4 times more signal). This lower observed signal is likely due to optical attenuation and scatter that occurs inside the MSC. The effect of incubation time on PA signal of MSCs was also measured for the 3, 6, and 20 h time points at 0.07 nM. The 6 h time point had the same (p
= 0.33) PA intensity as the 3 h incubation sample, but the 20 h sample was reduced. The PA signal of the 20 h sample was 34% of the 3 h sample. To determine the ex vivo
LOD, we immobilized decreasing numbers of SiGNR-loaded MSCs into a phantom and collected PA images. The LOD above the water blank was 5000 cells (Supporting Information, Figure S.7B
ICP analysis determined the amount of gold loaded into the cells. First, an increasing number of GNRs and SiGNRs were analyzed for their gold content and that signal was plotted in Supporting Information, Figure S.8A
. This calibration plot was used with the gold content of dissolved MSCs to determine the number of SiGNRs present in the total sample as well as the quantity on a per-cell basis. We calculated 102 000 ± 1000 SiGNRs per MSC; the gold signal from SiGNR-loaded MSCs was 5-fold higher than that from GNR-loaded MSCs (Supporting Information, Figure S.8C
We performed additional studies to determine whether the SiGNRs altered the normal behavior of MSCs beyond gross toxicity assays like the MTT metabolic tests. First, to determine whether this loading changes the normal proliferation of MSCs, 3000 MSCs with and without SiGNR loading were seeded in a 96 well plate and monitored sequentially with MTT. There was no significant (p = 0.35) difference between the growth of the two cell populations (). The doubling time for both populations was 3 days.
Next, we used differentiation reagents to determine whether the SiGNRs impacted the pluripotency of the MSCs.42
This work sought to answer two questions: (1) Can SiGNR-loaded MSCs still differentiate?43
and (2) Does the presence of SiGNRs induce any unintended differentiation? We were especially concerned that the SiGNRs might unintentionally transform MSCs into osteogenic cells as silicon-based structures have previously been show to induce such differentiation.44
Fortunately, SiGNR-loaded cells were still easily transformed into osteogenic and adipogenic cell lines (). There was 5-fold more osteogenic signal (as determined by A402) in the induced () cells than noninduced cells (). Adipogenic induction produced many lipid-containing vacuoles in both the control () and SiGNR containing cells () (see photographs of the culture plates in Supporting Information, Figure S.9
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) (more ...)
A final study analyzed the secretome of SiGNR-loaded and control MSCs.45
Of the 31 analyzed proteins, 26 had levels in cell culture media that were measurable by the bead-based Luminex assay (Supporting Information, Table S.1
We compared the levels in the labeled MSCs to unlabeled MSCs and found that only interleukin-6 (IL-6) had expression patterns increased or decreased more than 2-fold.
The utility of SiGNRs in living systems was first probed by implanting decreasing concentrations of SiGNRs (80 μ
L of 0.7, 0.35, and 0.175 nM in 50% matrigel) subcutaneously and performing PA imaging. The LOD for the SiGNR contrast agent in vivo
(subcutaneous) was 0.05 nM and linear at R2
= 0.93 (Supporting Information, Figure S.5B,D
). The next step was to inject SiGNR-labeled MSCs. presents representative sequences of intramuscular cell implantation (, right) including positive (, left) and negative controls (, middle). Images of hind limb muscle and images, during, and after injection are shown. For video of real-time injection of the SiGNR-labeled MSCs presented in the right of , please see Supporting Information, Video 1
, with speed increased 8-fold and Video 2 in real time. The positive control is 3 nM SiGNRs only, the negative control is PBS, and the cell implantation is 800 000 cells. Importantly, the B-mode image shows the implant in all three examples (). The red dashed circle highlights the injection site. For , there is clearly an i.m. bolus injection, but no PA signal. In contrast, with SiGNR-MSCs shows a bolus and PA signal. Spectral analysis of the therapy site was performed before and after injection ().
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), (more ...)
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 (more ...)
The difference pre- and postinjection at the injection site was 670% increase for positive control; no increase in PA signal was observed for the negative (vehicle) control. Decreasing numbers of SiGNR-labeled MSCs (8 × 105
to 1 × 105
) were delivered into a mouse hind limb muscle in three replicate mice at each different cell number ( and Supporting Information, Figure S.10
) The lowest value imaged was 100 000 cells and the calculated in vivo
LOD of MSCs in mouse hind limb muscle is 90 000 cells. One animal with 100 000 cells was monitored longitudinally and a PA image recorded daily. The implanted cell bolus could be monitored for 4 days after injection (see Supporting Information, Figure S.10
To validate the imaging data we performed histological analysis in which treated muscle tissue was removed after injection (cells in this example were labeled prior to injection with a green cell tracking fluorophore), fixed, and stained with hematoxylin and eosin. This sample was fist placed in a fluorescence imaging chamber using green fluorescent protein filter cubes. Intense green fluorescence is seen corresponding to the green cell tracking dye in the MSCs (Supporting Information, Figure S.11
) The resulting histology slide shows very clear morphological differences between skeletal muscle (; right) and the delivered cells (; left). The fluorescence of the cell tracking dye is obvious when an adjacent slice is imaged with fluorescence (). Although there was some damage during sample preparation causing the delivered cells to lose adherence to the muscle tissue, this confirms that the increase in imaging signal is due to cells. Interestingly, at 40× magnification, dark spots are present in MSCs, likely due to SiGNRs ().