Ultrahigh sensitivity carbon nanotube agents for photoacoustic molecular imaging in living mice

Abstract

Photoacoustic imaging is an emerging modality that overcomes to a great extent the resolution and depth limitations of optical imaging while maintaining relatively high-contrast. However, since many diseases will not manifest an endogenous photoacoustic contrast, it is essential to develop exogenous photoacoustic contrast agents that can target diseased tissue(s). Here we present a novel photoacoustic contrast agent, Indocyanine Green dye-enhanced single walled carbon nanotube (SWNT-ICG). We conjugated this contrast agent with cyclic Arg-Gly-Asp (RGD) peptides to molecularly target the alpha(v)beta(3) integrins, which are associated with tumor angiogenesis. Intravenous administration of this tumor-targeted contrast agent to tumor-bearing mice showed significantly higher photoacoustic signal in the tumor than in mice injected with the untargeted contrast agent. The new contrast agent gave a markedly 300 times higher photoacoustic contrast in living tissues than previously reported SWNTs, leading to subnanomolar sensitivities. Finally, we show that the new contrast agent can detect approximately 20 times fewer cancer cells than previously reported SWNTs.

Figure 1. Characterization of the ICG dye-enhanced SWNT

(A) Illustration of a SWNT-ICG particle. ICG molecules (red) are attached to the SWNT surface through non-covalent pi-pi stacking bonds. Polyethylene glycol-5000 (blue) is conjugated to a targeting peptide in one end and to the SWNT surface on the other end through phospholipids. (B) Optical spectra of plain SWNT (black), SWNT-ICG-RGD (blue) and SWNT-ICG-RAD (red). ICG dye-enhanced SWNTs particles showed 20-times higher optical absorption than plain SWNT at the peak absorption wavelength, 780 nm. The similarity of SWNT-ICG-RAD and SWNT-ICG-RGD spectra suggests that the peptide conjugation does not notably perturb the photoacoustic signal. (C) The photoacoustic signal produced by SWNT-ICG was observed to be linearly dependent on the concentration (R² = 0.9833).

Figure 1. Characterization of the ICG dye-enhanced SWNT

(A) Illustration of a SWNT-ICG particle. ICG molecules (red) are attached to the SWNT surface through non-covalent pi-pi stacking bonds. Polyethylene glycol-5000 (blue) is conjugated to a targeting peptide in one end and to the SWNT surface on the other end through phospholipids. (B) Optical spectra of plain SWNT (black), SWNT-ICG-RGD (blue) and SWNT-ICG-RAD (red). ICG dye-enhanced SWNTs particles showed 20-times higher optical absorption than plain SWNT at the peak absorption wavelength, 780 nm. The similarity of SWNT-ICG-RAD and SWNT-ICG-RGD spectra suggests that the peptide conjugation does not notably perturb the photoacoustic signal. (C) The photoacoustic signal produced by SWNT-ICG was observed to be linearly dependent on the concentration (R² = 0.9833).

Figure 2. Photoacoustic detection of SWNT-ICG in living mice

(A) Mice were injected subcutaneously with SWNT-ICG at concentrations of 0.82-200 nM. The images represent ultrasound (gray) and photoacoustic (green) vertical slices through the subcutaneous injections (dotted black line). The skin is visualized in the ultrasound images, while the photoacoustic images show the SWNT-ICG distribution. The white dotted lines on the images illustrate the approximate edges of each inclusion. (B) The photoacoustic signal from each inclusion was calculated using 3D regions of interest and the ‘background’ represents the endogenous signal measured from tissues. The error bars represent standard error (n = 3 mice). Linear regression (R² = 0.97) of the photoacoustic signal curve estimates that a concentration of 170 pM of SWNT-ICG will give the equivalent background signal of tissues.

Figure 3. SWNT-ICG-RGD tumor targeting in living mice

(A) Ultrasound (gray) and photoacoustic (green) images of one vertical slice through the tumor (dotted black line). The ultrasound images show the skin and the tumor boundaries. Subtraction photoacoustic images were calculated as 2 hr post-injection minus pre-injection images. As can be seen in the subtraction images, SWNT-ICG-RGD accumulates in higher amount in the tumor as compared to the control SWNT-ICG-RAD. (B) Mice injected with SWNT-ICG-RGD showed significantly higher photoacoustic signal than mice injected with the untargeted control SWNT-ICG-RAD (p < 0.001). The error bars represent standard error (n = 4 mice)

Figure 4. Comparison of plain SWNT-RGD to SWNT-ICG-RGD

(A) Photoacoustic vertical slice image through an agarose phantom containing increasing number of U87 cancer cells exposed to SWNT-ICG-RGD and plain SWNT-RGD particles. While 1.7×10⁶ cells exposed to SWNT-RGD are barely seen on the image, a clear photoacoustic signal was observed from 1.4×10⁵ cells exposed to SWNT-ICG-RGD. The signal inside the ROI (dotted white boxes) is not homogenous due to possible aggregates of cells. (B) Quantitative analysis of the photoacoustic signals from the phantom (n = 3) showed that SWNT-ICG-RGD can visualize ~20-times less cancer cells than SWNT-RGD can (p < 0.0001). The background line represents the average background signal in the phantom. Linear regression was calculated on the linear regime of both curves.