Recently, we reported on the conjugation of cyclic RGD containing peptides to single-walled carbon nanotubes17
(SWNT–RGD) that is stable in serum. The single-walled carbon nanotubes, which were 1–2 nm in diameter and 50–300 nm in length were coupled to the RGD peptides through polyethylene glycol-5000 grafted phospholipid (PL–PEG5000
). These SWNT–RGD conjugates bind with high affinity to αv
integrin, which is overexpressed in tumour neovasculature, and to other integrins expressed by tumours but with lower affinity18,19
. We also synthesized non-targeted single-walled carbon nanotubes (that is, plain single-walled carbon nanotubes) by conjugating them solely to PL–PEG5000
(). Our photoacoustic instrument20
used a single-element focused transducer to raster scan the object under study, which was illuminated through a fibre head (see Methods and Supplementary Information, Fig. S1
). In a phantom study we measured the photoacoustic signal of plain single-walled carbon nanotubes and SWNT–RGD at wavelengths of 690–800 nm (; shorter wavelengths are less desirable as the depth of penetration through the tissues is reduced21
). These photoacoustic spectra suggest that 690 nm is the preferable wavelength, because the photoacoustic signal of the single-walled carbon nanotubes is highest at that wavelength. Furthermore, the ratio of single-walled carbon nanotubes to haemoglobin signal is higher at this wavelength when compared with other wavelengths. Importantly, the photoacoustic signal of single-walled carbon nanotubes was found to be unaffected by the RGD peptide conjugation. This finding was validated through measurements of the optical absorbance of the two single-walled carbon nanotubes conjugates (see Supplementary Information, Fig. S2
). In a separate non-absorbing and non-scattering phantom study, we also validated that the photoacoustic signal produced by single-walled carbon nanotubes is in linear relationship with their concentration () with R2
Characterization of the photoacoustic properties of single-walled carbon nanotubes
We then subcutaneously injected the lower back of a mouse with 30 μl of mixtures of single-walled carbon nanotubes and matrigel at concentrations between 50 and 600 nM (n
= 3 for each concentration). Matrigel alone produced no photoacoustic signal (data not shown). Upon injection, the matrigel solidified, fixing the single-walled carbon nanotubes in place. Three-dimensional (3D) ultrasound and photoacoustic images of the inclusions were then acquired (). The ultrasound images showed the mouse anatomy (for example, skin and inclusion edges), and the photoacoustic images revealed the single-walled carbon nanotubes contrast in the mouse. The photoacoustic signal from each inclusion was quantified using a 3D region of interest drawn over the inclusion. We observed a linear correlation (R2
= 0.9929) between the single-walled carbon nanotubes concentration and the corresponding photoacoustic signal (). Importantly, this linear relation can only be expected in special cases where the dye concentration does not perturb the tissue light distribution significantly. We concluded that the photoacoustic signal produced by tissues (background) was equivalent to the photoacoustic signal produced by 50 nM of single-walled carbon nanotubes (that is, a signal-to-background ratio of 1). This experimental result correlates well with the theoretical analysis (see Supplementary Information
), which predicts a background signal equal to 7–70 nM of single-walled carbon nanotubes, depending on the location of the nanotubes in the body.
Photoacoustic detection of single-walled carbon nanotubes in living mice
We then injected two groups of mice bearing U87MG tumour xenografts (~100 mm3
) through the tail-vein (IV) with either 200 μl of plain single-walled carbon nanotubes (n
= 4) or SWNT–RGD (n
= 4) at a concentration of 1.2 μM. Three-dimensional ultrasound and photoacoustic images of the tumour and its surroundings were acquired before and up to 4 h after injection. We found that mice injected with SWNT–RGD showed a significant increase of photoacoustic signal in the tumour compared with control mice injected with plain single-walled carbon nanotubes (). The images from the different time points were aligned with one another using simple vertical translations to account for small vertical movements in the transducer positioning. This alignment allowed quantification of the photoacoustic signal at all time points using a single region of interest. We then calculated a subtraction image between the photoacoustic image taken at 4 h post-injection and the photoacoustic image taken before injection. The subtraction image better visualizes the real distribution of the single-walled carbon nanotubes as it removes, to a large extent, the background signal. For example, in the mouse injected with plain single-walled carbon nanotubes (), a high photoacoustic signal, likely produced by a large blood vessel, was seen in the pre-injection and post-injection images. However, the subtraction image showed a much lower signal from this area, reflecting the likely low concentration of plain single-walled carbon nanotubes there. We calculated the photoacoustic signal by drawing a 3D region of interest around the tumour (tumour boundaries were clearly visualized in the ultrasound images). The photoacoustic signal increase was quantified as a function of time (). Although SWNT–RGD led to a consistently higher photoacoustic signal, plain single-walled carbon nanotubes led only to a temporary increase in the photoacoustic signal of the tumour (P
< 0.001 when comparing entire time-curves, and P
< 0.05 when comparing the signals at each time point independently). The temporary photoacoustic signal observed for plain single-walled carbon nanotubes is likely caused by circulating nanotubes that are eventually cleared from the bloodstream. Conversely, SWNT–RGD bind to the tumour vasculature, creating a consistent photoacoustic signal from the tumour. On average, at 4 h post-injection, the SWNT–RGD resulted in ~8 times greater increase in photoacoustic signal compared with plain single-walled carbon nanotubes. The percentage injected dose per gram of tissue was calculated to be ~14 %ID g−1
(see Supplementary Information
Single-walled carbon nanotube targets tumour in living mice
We further validated our photoacoustic results using a Raman microscope, as an independent method for detection of single-walled carbon nanotubes. At the conclusion of the photoacoustic study, 4 h post-injection, the mice were sacrificed; the tumours were surgically removed and scanned ex vivo under a Raman microscope. The two-dimensional Raman images of the excised tumours were found to match the photoacoustic images (). The mean Raman signal from the tumours was calculated from the Raman images. Similarly to the photoacoustic results, the Raman signal from the tumours was ~4 times higher in mice injected with SWNT–RGD than in mice injected with plain single-walled carbon nanotubes ().
Validation of the in vivo photoacoustic images by Raman ex vivo microscopy
Unlike photoacoustic imaging, optical imaging suffers from relatively poor spatial resolution as well as exponentially degraded sensitivity as tissue depth increases22
. We showed the superiority of our photoacoustic strategy by comparing it with fluorescence imaging of tumour-targeted quantum dots. The quantum dots were conjugated to RGD peptides23
(QD–RGD) and imaged 6 h post-injection using a fluorescence imaging instrument (). Although the quantum dot and single-walled carbon nanotube conjugates might have different biodistributions, the photoacoustic images of single-walled carbon nanotubes from the tumour illustrated the depth-information and the greater spatial resolution achieved by photoacoustic imaging compared with fluorescence imaging (). The smeared signal from the tumour in the fluorescence image is due to light scattering. However, the photoacoustic images showed the 3D distribution of SWNT–RGD in the tumour with high spatial resolution. Similar results were also observed in a phantom study (see Supplementary Information, Fig. S4
Comparison between photoacoustic imaging using single-walled carbon nanotubes and fluorescence imaging using quantum dots
We have demonstrated that single-walled carbon nanotubes can be exploited as photoacoustic contrast agents to non-invasively image tumours. Intravenous injection of targeted single-walled carbon nanotubes in mice led to 8 times higher photoacoustic signal in the tumour compared with mice injected with non-targeted single-walled carbon nanotubes. Our photoacoustic images were verified using Raman microscopy on the surgically removed tumours. Furthermore, our results agreed with a previous study17
where radiolabelled SWNT–RGD were monitored using small animal positron emission tomography (microPET). In that study SWNT–RGD were found to accumulate ~3–5 times more in tumours than plain single-walled carbon nanotubes. That study also showed that the SWNT–RGD did not accumulate in the tissue surrounding the tumour.
Most previous work on photoacoustic contrast agents in vivo
is limited to non-targeted agents such as gold nanocages used for highlighting the blood vessels in a rat’s brain11
. A recent preliminary study13
showed that an indocyanine green derivative (IRDye-800-c(KRGDf)) may be applicable for photoacoustic spectroscopic imaging of U87MG tumours; however, the study was carried out on a single mouse and statistical validation of the agent has yet to be shown. Various gold nanoparticles have been previously suggested, primarily for their high absorption characteristics and the ability to control their spectra, which allows multiplexing studies9
. However, their main limitation is their relatively large size, which will lead to their rapid clearance by the reticuloendothelial system (RES) upon intravenous injection. It is possible that single-walled carbon nanotubes, due to their unique high aspect ratio (~1:100) and high surface area to volume ratio, are capable of minimizing RES uptake while having an increased affinity for molecular targets due to multivalency effects17
. A concentration of 50 nM of single-walled carbon nanotubes was found to produce a photoacoustic signal equivalent to mouse tissues (background); however, the minimum detectable concentration of single-walled carbon nanotubes is likely to be less than 50 nM. This is because photoacoustic images were acquired before and after the administration of the contrast agent, thus making it possible to separate the contrast agent signal from the background signal. Further background reduction can be achieved by performing photoacoustic spectral imaging, improving hardware/reconstruction software, or by enhancing the single-walled carbon nanotubes’ photoacoustic signal. With respect to acquisition time, our current instrument acquires a single photoacoustic image in ~20–30 minutes for a tumour ~100 mm3
in size. However, by using lasers with higher repetition rates, scan duration can be greatly reduced.
We are currently investigating the potential of single-walled carbon nanotubes to extravasate out of the leaky vasculature of tumours. Single-walled carbon nanotube extravasation is of particular interest, because upon exiting the vasculature, the nanotubes would have access to many more molecular targets that exist only on the cancer cell’s membranes. Future work should optimize the particles’ extravasation as well as bring new technologies to help quantify the degree of nanotube extravasation. Moreover, future studies can monitor various nanotherapeutic applications such as drug-eluting single-walled carbon nanotubes using photoacoustic imaging. Such nanotherapeutic and cancer imaging applications would gain further clinical interest as single-walled carbon nanotubes continue to show no toxic effects24
. Although single-walled carbon nanotubes have the capability to efficiently bind to molecular targets, their high photoacoustic signal allows for high-resolution 3D photoacoustic images with substantial depth of penetration. None of the other molecular imaging modalities compares with the precise depth information and submillimetre resolution at nanomolar sensitivity that is achieved by photoacoustic imaging. We expect this work to stimulate further studies of biologically relevant problems using photoacoustic molecular imaging.