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...

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...

This study evaluates the influence of particle size, PEGylation, and surface coating on the quantitative biodistribution of near-infrared-emitting quantum dots (QDs) in mice. Polymer- or peptide-coated 64Cu-labeled QDs 2 or 12 nm in diameter, with or without polyethylene glycol (PEG) of molecular weight 2000, are studied by serial micropositron emission tomography imaging and region-of-interest analysis, as well as transmission electron microscopy and inductively coupled plasma mass spectrometry. PEGylation and peptide coating slow QD uptake into the organs of the reticuloendothelial system (RES), liver and spleen, by a factor of 6–9 and 2–3, respectively. Small particles are in part renally excreted. Peptide-coated particles are cleared from liver faster than physical decay alone would suggest. Renal excretion of small QDs and slowing of RES clearance by PEGylation or peptide surface coating are encouraging steps toward the use of modified QDs for imaging living subjects.

An optimized noninvasive Raman microscope was used to evaluate tumor targeting and localization of single walled carbon nanotubes (SWNTs) in mice. Raman images were acquired in two groups of tumor-bearing mice. The control group received plain-SWNTs, whereas the experimental group received tumor targeting RGD-SWNTs intravenously. Raman imaging commenced over the next 72 h and revealed increased accumulation of RGD-SWNTs in tumor (p < 0.05) as opposed to plain-SWNTs. These results support the development of a new preclinical Raman imager.Raman spectroscopy is a well established bioanalytical tool with many advantages including excellent sensitivity to small structural and chemical changes, minimal sample preparation, high spatial resolution, and resistance to autofluorescence and photobleaching. 1 Although scientists have reported the use of Raman spectroscopy to image biological processes within living cells and excised tissues, 2-7 its inherently weak effect has limited its application to noninvasively assess small animal models. However, with careful system design and appropriate modifications, our laboratory has developed a Raman microscope capable of noninvasive deep tissue imaging. 8 The current paper focuses on the ability of our optimized Raman microscope to effectively localize functionalized single wall nanotubes (SWNTs) in a tumor model. Carbon nanotubes have played a fundamental role in the rapidly developing field of nanotechnology because of their unique properties and high potential in biomedical applications including targeted chemotherapeutics, diagnostic imaging contrast agents, and photoablative therapy agents. 9,10 Absence of acute toxicity, relatively long circulation time, and rapid renal clearance also make SWNTs an attractive diagnostic and therapeutic nanodevice. 9,11 Raman spectroscopy has proven to be a valuable tool for characterizing SWNTs. The inherent Raman peak, referred to as the G-band (~1593 cm −1 ), is associated with the graphite in SWNTs and makes them ideal for high sensitivity detection with our Raman microspectroscopy system. 8These quasi-one-dimensional structures (Supporting Information (SI) Figure 1) Using microPET to evaluate effective targeting, involves the use of additional labeling of radioactive material to SWNTs. Additionally, in the RGD conjugated SWNT study reported by Liu et al., animals were sacrificed at 8 hours for tissue evaluation with Raman imaging preventing longitudinal assessment of tumor targeting. Our optimized system takes advantage of the inherent Raman peak of the graphite structure in SWNTs to noninvasively image internal tissue systems (i.e., liver, tumor models) over an extended period of time in the same mouse without adding radioactive isotopes that could potentially influence experimental therapeutic outcome. In addition, the modification of SWNTs with both a DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) metal chelator and an RGD peptide is difficult to prepare; and the radiolabeling procedure can be costly and ti...

2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) has extensively been used for clinical diagnosis, staging, and therapy monitoring of cancer and other diseases. Nonradioactive glucose analogues enabling the screening of the glucose metabolic rate of tumors are of particular interest for anticancer drug development. A nonradioactive fluorescent deoxyglucose analogue may have many applications for both imaging of tumors and monitoring therapeutic efficacy of drugs in living animals and may eventually translate to clinical applications. We found that a fluorescent 2-deoxyglucose analogue, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG), can be delivered in several tumor cells via the glucose transporters (GLUTs). We therefore conjugated D-glucosamine with a near-infrared (NIR) fluorphor Cy5.5 and tested the feasibility of the Cy5.5-D-glucosamine (Cy5.5-2DG) conjugate for NIR fluorescence imaging of tumors in a preclinical xenograft animal model. Cy5.5-2DG was prepared by conjugating Cy5.5 monofunctional N-hydroxysuccinimide ester (Cy5.5-NHS) and D-glucosamine followed by high-performance liquid chromatography purification. The accumulation of Cy5.5-2DG and Cy5.5-NHS in different tumor cell lines at 37 and 4 degrees C were imaged using a fluorescence microscope. Tumor targeting and retention of Cy5.5-2DG and Cy5.5-NHS in a subcutaneous U87MG glioma and A375M melanoma tumor model were evaluated and quantified by a Xenogen IVIS 200 optical cooled charged-coupled device system. Fluorescence microscopy imaging shows that Cy5.5-2DG and Cy5.5-NHS are taken up and trapped by a variety of tumor cell lines at 37 degrees C incubation, while they exhibit marginal uptake at 4 degrees C. The tumor cell uptake of Cy5.5-2DG cannot be blocked by the 50 mM D-glucose, suggesting that Cy5.5-2DG may not be delivered in tumor cells by GLUTs. U87MG and A375M tumor localization was clearly visualized in living mice with both NIR fluorescent probes. Tumor/muscle contrast was clearly visible as early as 30 min postinjection (pi), and the highest U87MG tumor/muscle ratios of 2.81 +/- 0.10 and 3.34 +/- 0.23 were achieved 24 h pi for Cy5.5-2DG and Cy5.5-NHS, respectively. While as a comparison, the micropositron emission tomography imaging study shows that [18F]FDG preferentially localizes to the U87MG tumor, with resulting tumor/muscle ratios ranging from 3.89 to 4.08 after 30 min to 2 h postadministration of the probe. In conclusion, the NIR fluorescent glucose analogues, Cy5.5-2DG and Cy5.5-NHS, both demonstrate tumor-targeting abilities in cell culture and living mice. More studies are warranted to further explore their application for optical tumor imaging. To develop NIR glucose analogues with the ability to target GLUTs/hexokinase, it is highly important to select NIR dyes with a reasonable molecular size.