The vexing difficulty in delineating brain tumor margins represents a major obstacle toward better outcome of brain tumor patients. Current imaging methods are often limited by inadequate sensitivity, specificity, and spatial resolution. Here we show that a unique triple-modality Magnetic resonance imaging - Photoacoustic imaging – surface enhanced Raman scattering (SERS) nanoparticle (MPR) can accurately help delineate the margins of brain tumors in living mice both pre- and intra-operatively. The MPRs were detected by all three modalities with at least picomolar sensitivity both in vitro and in living mice. Intravenous injection of MPRs into glioblastoma-bearing mice led to specific MPR accumulation and retention by the tumors, allowing for non-invasive tumor delineation by all three modalities through the intact skull. Raman imaging allowed guidance of intra-operative tumor resection, and histological correlation validated that Raman imaging is accurately delineating brain tumor margins. This novel triple-modality nanoparticle approach holds promise to enable more accurate brain tumor imaging and resection.

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...

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...

Molecular imaging of living subjects continues to rapidly evolve with bioluminescence and fluorescence strategies, in particular being frequently used for small-animal models. This article presents noninvasive deep-tissue molecular images in a living subject with the use of Raman spectroscopy. We describe a strategy for small-animal optical imaging based on Raman spectroscopy and Raman nanoparticles. Surface-enhanced Raman scattering nanoparticles and single-wall carbon nanotubes were used to demonstrate whole-body Raman imaging, nanoparticle pharmacokinetics, multiplexing, and in vivo tumor targeting, using an imaging system adapted for small-animal Raman imaging. The imaging modality reported here holds significant potential as a strategy for biomedical imaging of living subjects.nanotubes ͉ SERS nanoparticles M olecular imaging of living subjects provides the ability to study cellular and molecular processes that have the potential to impact many facets of biomedical research and clinical patient management (1-4). Imaging of small-animal models is currently possible by using positron emission tomography (PET), single photon emission computed tomography, magnetic resonance imaging, computed tomography, optical bioluminescence and fluorescence, high frequency ultrasound, and several other emerging modalities. However, no single modality currently meets the needs of high sensitivity, high spatial and temporal resolution, high multiplexing capacity, low cost, and high-throughput.Fluorescence imaging, in particular, has significant potential for in vivo studies but is limited by several factors (5, 6), including a limited number of fluorescent molecular imaging agents available in the near infra-red (NIR) window with large spectral overlap between them, which restricts the ability to interrogate multiple targets simultaneously (multiplexing). In addition, background autofluorescence emanating from superficial tissue layers restricts the sensitivity and the depth to which fluorescence imaging can be used. Moreover, rapid photobleaching of fluorescent molecules limits their useful lifetime and prevents studies of prolonged duration. Therefore, we have attempted to develop new strategies that may solve some of the limitations of fluorescence imaging in living subjects.Raman spectroscopy can differentiate the spectral fingerprint of many molecules, resulting in very high multiplexing capabilities. Narrow spectral features are easily separated from the broadband autofluorescence, because Raman is a scattering phenomenon as opposed to absorption/emission in fluorescence, and Raman active molecules are more photostable compared with fluorophores, which are rapidly photobleached. Unfortunately, the precise mechanism for photobleaching is not well understood. However, it has been linked to a transition from the excited singlet state to the excited triplet state. Photobleaching is significantly reduced for single molecules adsorbed onto metal particles because of the rapid quenching of excited electrons by the metal surface...

Gold has been used as a therapeutic agent to treat a wide variety of rheumatic diseases including psoriatic arthritis, juvenile arthritis and discoid lupus erythematosus. Although the use of gold has been largely superseded by newer drugs, gold nanoparticles are being used effectively in laboratory based clinical diagnostic methods whilst concurrently showing great promise in vivo either as a diagnostic imaging agent or a therapeutic agent. For these reasons, gold nanoparticles are therefore well placed to enter mainstream clinical practice in the near future. Hence, the present review summarizes the chemistry, pharmacokinetics, bio-distribution, metabolism and toxicity of bulk gold in humans based on decades of clinical observation and experiments in which gold was used to treat patients with rheumatoid arthritis. The beneficial attributes of gold nanoparticles, such as their ease of synthesis, functionalization and shape control are also highlighted demonstrating why gold nanoparticles are an attractive target for further development and optimization. The importance of controlling the size and shape of gold nanoparticles to minimize any potential toxic side effects is also discussed.