Photoacoustic imaging of living subjects offers higher spatial resolution and allows deeper tissues to be imaged compared with most optical imaging techniques 1-7 . As many diseases do not exhibit a natural photoacoustic contrast, especially in their early stages, it is necessary to administer a photoacoustic contrast agent. A number of contrast agents for photoacoustic imaging have been suggested previously 8-15 , but most were not shown to target a diseased site in living subjects. Here we show that single-walled carbon nanotubes conjugated with cyclic Arg-Gly-Asp (RGD) peptides can be used as a contrast agent for photoacoustic imaging of tumours. Intravenous administration of these targeted nanotubes to mice bearing tumours showed eight times greater photoacoustic signal in the tumour than mice injected with non-targeted nanotubes. These results were verified ex vivo using Raman microscopy. Photoacoustic imaging of targeted single-walled carbon nanotubes may contribute to non-invasive cancer imaging and monitoring of nanotherapeutics in living subjects 16 .Recently, we reported on the conjugation of cyclic RGD containing peptides to single-walled carbon nanotubes 17 (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-PEG 5000 ). These SWNT-RGD conjugates bind with high affinity to α v β 3 integrin, which is overexpressed in tumour neovasculature, and to other integrins expressed by tumours but with lower *e-mail: sgambhir@stanford.edu. Author contributions A.D. built the photoacoustic instrument, designed and performed the experiments and wrote the paper. C.Z. designed, performed and analysed the Raman experiments. S.K. built the photoacoustic instrument and designed the experiments. S.V. designed and built the photoacoustic instrument. S.B. performed the experiments and helped write the paper. Z.L. synthesized the single-walled carbon nanotube conjugates. J.L. performed the cell uptake studies. B.R.S. helped write the paper. T.M. and O.O. helped design the photoacoustic instrument. Z.C. helped perform the comparison to fluorescence imaging. X.C. provided the RGD peptides, performed the fluorescence imaging of QD-RGD conjugates and helped write the manuscript. H.D. was responsible for single-walled carbon nanotube conjugation synthesis. B.T.K. was responsible for building the photoacoustic instrument. S.S.G. was responsible for experimental design and wrote the paper.Author information Reprints and permission information is available online at http://npg.nature.com/reprintsandpermissions/. Correspondence and requests for materials should be addressed to S.S.G. (Fig. 1a). Our photoacoustic instrument 20 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 pla...

In vivo imaging, which enables us to peer deeply within living subjects, is producing tremendous opportunities both for clinical diagnostics and as a research tool. Contrast material is often required to clearly visualize the functional architecture of physiological structures. Recent advances in nanomaterials are becoming pivotal to generate the high-resolution, high-contrast images needed for accurate, precision diagnostics. Nanomaterials are playing major roles in imaging by delivering large imaging payloads, yielding improved sensitivity, multiplexing capacity, and modularity of design. Indeed, for several imaging modalities, nanomaterials are now not simply ancillary contrast entities, but are instead the original and sole source of image signal that make possible the modality's existence. We address the physicochemical makeup/design of nanomaterials through the lens of the physical properties that produce contrast signal for the cognate imaging modality-we stratify nanomaterials on the basis of their (i) magnetic, (ii) optical, (iii) acoustic, and/or (iv) nuclear properties. We evaluate them for their ability to provide relevant information under preclinical and clinical circumstances, their in vivo safety profiles (which are being incorporated into their chemical design), their modularity in being fused to create multimodal nanomaterials (spanning multiple different physical imaging modalities and therapeutic/theranostic capabilities), their key properties, and critically their likelihood to be clinically translated.

Raman spectroscopy is a newly developed, noninvasive preclinical imaging technique that offers picomolar sensitivity and multiplexing capabilities to the field of molecular imaging. In this study, we demonstrate the ability of Raman spectroscopy to separate the spectral fingerprints of up to 10 different types of surface enhanced Raman scattering (SERS) nanoparticles in a living mouse after s.c. injection. Based on these spectral results, we simultaneously injected the five most intense and spectrally unique SERS nanoparticles i.v. to image their natural accumulation in the liver. All five types of SERS nanoparticles were successfully identified and spectrally separated using our optimized noninvasive Raman imaging system. In addition, we were able to linearly correlate Raman signal with SERS concentration after injecting four spectrally unique SERS nanoparticles either s.c. (R 2 ‫؍‬ 0.998) or i.v. (R 2 ‫؍‬ 0.992). These results show great potential for multiplexed imaging in living subjects in cases in which several targeted SERS probes could offer better detection of multiple biomarkers associated with a specific disease.imaging in vivo ͉ multiplex ͉ SERS ͉ nanoparticles I n recent years, the biomedical research community has come to realize that no single targeting agent is likely to provide sufficient information needed to characterize or detect a specific disease process. As a result, several efforts have been made toward the discovery of multiple biomarkers and targeting ligands in the hope of improving earlier detection and management of specific diseases. The ability to simultaneously detect multiple targets, sensitively and in vivo, is an attractive feat; but it is a task often difficult to accomplish. Thus far, nanoparticles have played an important role in this endeavor; however most nanostructure-based platforms for multiplex detection methods have been tailored for in vitro applications (1-6), leading to little progress in the field of in vivo multiplex imaging.Recently, there has been an overwhelming interest in sensitive imaging of nanoparticles for both diagnostic and therapeutic applications (7-11). As a result, new preclinical imaging modalities optimized for nanoparticle imaging have been developed, further expanding the field of molecular imaging. Thus far, fluorescence and Raman spectroscopy, in conjunction with quantum dots and surface enhanced Raman scattering (SERS) nanoparticles, respectively, have been the predominant imaging modalities to evaluate in vivo multiplex imaging (12)(13)(14). Raman imaging, in particular, has generated quite a bit of interest recently; we have demonstrated its ability to detect picomolar concentrations in vivo along with its unique ability to multiplex using SERS nanoparticles and others have developed novel Raman nanoparticles with the potential to be used in vivo as well (14-17).Both quantum dots and SERS nanoparticles have shown great potential as multiplexed imaging probes ex vivo, whether for cellular imaging or for biosensor applications; however, seve...

In cancer imaging, nanoparticle biodistribution is typically visualised in living subjects using ‘bulk’ imaging modalities such as magnetic resonance imaging, computerized tomography and whole-body fluorescence. As such the nanoparticle influx is observed only macroscopically and the mechanisms by which they target cancer remain elusive. Nanoparticles are assumed to accumulate via several targeting mechanisms, particularly extravasation ie, leakage into tumour. Here we show that, in addition to conventional nanoparticle uptake mechanisms, single-walled carbon nanotubes are almost exclusively taken up by a single immune cell subset, Ly-6Chi monocytes (almost 100% uptake in Ly-6Chi monocytes, below 3% in all other circulating cells), and delivered to the tumour in mice. Next, we demonstrate that a targeting ligand (RGD) conjugated to nanotubes significantly enhances the number of single-walled carbon nanotube-loaded monocytes reaching the tumour (p<0.001, day 7 p.i.). The remarkable selectivity of this tumour targeting mechanism demonstrates an advanced immune-based delivery strategy for enhancing specific tumour delivery with substantial penetration.