SWNTs were taken up by macrophages both in cell culture and in ligated murine carotid arteries, allowing both Cy5.5-based fluorescence imaging and intrinsic NIR imaging. Furthermore, combining SWNTs with laser light exposure caused thermal ablation of macrophages in vitro and in freshly excised ligated carotids. To the best of our knowledge, this study is the first to show that carbon nanotubes allow imaging and thermal ablation of vascular macrophages.
SWNTs have a number of valuable properties for molecular and cellular imaging and therapy. Their nanosize has made them amenable to cellular binding and/or uptake, and their functionality can be enhanced with the attachment of NIR fluorophores and/or targeting ligands (eg, RGD, folate).6,22–24
We found high uptake of nontargeted SWNTs in vascular macrophages in this study, but further enhancement of macrophage uptake with specific targeting ligands may be beneficial.19,25,26
SWNTs can be used for molecular and cellular imaging by attaching a fluorophore6,27
or through their recently described intrinsic photoluminescent properties.16,28–30
In this study, we showed that either approach can be used. The high performance of the intrinsic NIR imaging capability of SWNTs shown in vivo in cancer10
holds promise for in vivo vascular imaging. More data are needed on in vivo sensitivity of intrinsic SWNT fluorescence for imaging vascular inflammation, as this can be challenging in deeper vessels and larger species. FMT is a useful modality for noninvasive whole-body imaging in mice, but anatomic registration is challenging. Hybrid imaging protocols, such as FMT-CT, may improve anatomic localization of signals in vivo.21,31
Heating of SWNTs in response to laser light excitation has shown promise for photothermal ablation of cancer cells,6,8,10
with strong optical absorbance demonstrated in the NIR range6
and specific testing for heating and nontoxicity at 808 nm.6
NIR light is known to penetrate up to 10 cm through certain tissues, even using low (FDA Class 1) microwatt lasers.32
SWNTs are potentially advantageous for photothermal ablation, as they have shown tumor elimination with 10-fold-lower injected doses and lower laser power compared with gold nanorods.10
In this study, side-by-side external laser light exposure of diseased carotid specimens with and without SWNTs showed heating and macrophage apoptosis only in the presence of SWNTs. SWNTs alone did not induce apoptosis, nor did laser exposure alone. We have not observed significant acute or chronic toxicity of these SWNTs with regard to clinical and laboratory parameters, histology, or survival in mice followed 3 to 5 months after injection,10,33
but longer-term studies are needed. Although quantitative analysis of carotid temperature change was not performed, the goal was not to ablate the entire vessel, only the SWNT-containing macrophages. The colocalization of apoptosis and macrophage staining was supportive of achieving this goal, particularly as monitoring the temperature of individual macrophages within the specimen was not feasible.
This macrophage photothermal ablation approach to treat atherosclerosis has both promise and challenges. One advantage is that it requires a double hit—cellular uptake of SWNTs and local laser light excitation—which provides a high degree of specificity. Macrophage-specific uptake and ablation could be further enhanced with targeted SWNTs.19,20,23–26
Alternative therapeutic approaches, such as toxin-loaded targeted nanoparticles, may be prone to uptake in other organs with resulting collateral damage (particularly liver and other components of the reticuloendothelial system). Similarly, direct thermal ablation (eg, focused ultrasound) is difficult to target to specific cells. Of course, clinical translation of this approach in vivo has multiple challenges, including the temperature modulating effect of circulating blood and the need for an intravascular laser light delivery system for deeper vessels, which has been used previously.11,12,15
Furthermore, although reduction of plaque macrophage burden and plaque area has been shown with phototoxic agents,11
thermal macrophage ablation with SWNTs could potentially lead to an increase in vascular inflammation. There may also be heterogeneity of the photothermal response, because of the complexity of human plaque tissue, variable macrophage uptake of SWNTs, and the heat-dissipating effect of flowing blood near the lumen. In vivo animal testing and serial monitoring of disease response will be needed to determine the potential for clinical translation.
In conclusion, we have demonstrated that carbon nanotubes are capable of both fluorescence imaging and photothermal ablation of vascular macrophages. Further development and testing of an in vivo vascular photothermal ablation system are warranted. Thus, carbon nanotubes are promising “theranostic” agents for vascular inflammation and atherosclerosis.