Photoacoustic Molecular Imaging using Single Walled Carbon Nanotubes in Living Mice

Photoacoustic molecular imaging is an emerging technology offering non-invasive high resolution imaging of the molecular expressions of a disease using a photoacoustic imaging agent. Here we demonstrate for the first time the utility of single walled carbon nanotubes (SWNTs) as targeted imaging agents in living mice bearing tumor xenografts. SWNTs were conjugated with polyethylene-glycol-5000 connected to Arg-Gly-Asp (RGD) peptide to target the αvβ3 integrin that is associated with tumor angiogenesis. In-vitro, we characterized the photoacoustic spectra of the particles, their signal linearity and tested their uptake by αvβ3expressing cells (U87MG). The photoacoustic signal of SWNTs was found not to be affected by the RGD conjugation to the SWNTs and was also found to be highly linear with concentration (R = 0.9997 for 25-400nM). The cell uptake studies showed that RGD-targeted SWNTs gave 75% higher photoacoustic signal than non-targeted SWNTs when incubated with U87MG cells. In-vivo, we measured the minimal detectable concentration of SWNTs in living mice by subcutaneously injecting SWNTs at increasing concentrations. The lowest detectable concentration of SWNTs in living mice was found to be 50nM. Finally, we administered RGDtargeted and non-targeted SWNTs via the tail-vein to U87MG tumor-bearing mice (n=4 for each group) and measured the signal from the tumor before and up to 4 hours post-injection. At 4 hours post-injection, tumors of mice injected with RGD-targeted SWNTs showed 8 times higher photoacoustic signal compared with mice injected with non-targeted SWNTs. These results were verified ex-vivo using a Raman microscope that is sensitive to the SWNTs Raman signal. Contact information: [email protected] INTRODUCTION Photoacoustic imaging is an emerging imaging modality that overcomes, to a great extent, the resolution and depth limitations of optical imaging but maintains the high-contrast of optics. Photons Plus Ultrasound: Imaging and Sensing 2009, edited by Alexander A. Oraevsky, Lihong V. Wang, Proc. of SPIE Vol. 7177, 717725 · © 2009 SPIE · CCC code: 1605-7422/09/$18 · doi: 10.1117/12.806497 Proc. of SPIE Vol. 7177 717725-1 When a short light pulse is used to illuminate tissues, the light is scattered and absorbed as it propagates through the tissues. The absorbed light is converted into heat, which in return causes the material to locally expand, creating a pressure wave. The pressure wave can then be detected by an ultrasound system placed outside the subject of interest. By measuring the pressure waves from several positions, a full tomographic image can be reconstructed. However, many diseases, especially in their early stages, do not exhibit a natural photoacoustic contrast, therefore administering an external photoacoustic contrast agent is necessary. While a number of contrast agents for photoacoustic imaging have been suggested previously, most were not shown to target a diseased site in living subjects. The ideal molecular imaging agent will have a sufficiently large optical absorption cross section to maximize the agent’s photoacoustic signal, but yet be small enough to escape uptake by the reticuloendothelial system (RES), specifically the liver and the spleen. However, designing such an imaging agent is not trivial since a particle’s absorption cross section and its size are highly correlated. In this study, single walled carbon nanotubes (SWNTs) were shown to have utility as photoacoustic contrast agents. Since SWNTs are essentially folded single layers of graphite, which have strong light absorption characteristics, they may act as photoacoustic contrast agents. SWNTs can be made as small as 1 nm in diameter but yet their length can extend to hundreds of nanometers increasing their absorption cross section and their intrinsic photoacoustic contrast. This unique geometry of SWNTs led to several applications of SWNTS in nanomedicine including drug delivery and photothermal therapy. However, in order to successfully translate these emerging applications into practice, it is essential to non-invasively monitor the physical location of the SWNTS in the subject of interest. To monitor SWNTs in living subjects, previous studies attached reporting molecules (fluorophores or radioactive isotopes) to the nanotube’s surface. Conversely, photoacoustic imaging of SWNTs does not require attaching any additional reporting molecules on the nanotubes and can produce three dimensional images with much higher spatial resolution and high depth penetration. 1. METHODS AND MATERIALS Proc. of SPIE Vol. 7177 717725-2 SWNT conjugates synthesis. A complete description of the synthesis of SWNT-RGD and plain SWNT can be found elsewhere. The SWNTs used in this work were 50-300 nm in length and 1-2 nm in diameter and have an average molar weight of 170 kDa per SWNT (based on 150 nm length and 1.2 nm diameter). were coupled to the RGD peptides through polyethylene glycol5000 grafted phospholipid (PL-PEG5000). These SWNT-RGD conjugates bind with high affinity to αvβ3 integrin that is over-expressed in tumor neovasculature as well as to other integrins expressed by tumors but with lower affinity . We also synthesized non-targeted SWNTs by conjugating them solely to PL-PEG5000 (plain SWNT) (Fig. 1). Figure 1 Illustration of plain SWNT and SWNT-RGD. The phospholipid (PL) binds to the sidewall of the SWNT connecting the polyethylene glycol-5000 (PEG5000) to the SWNT. The RGD allows the SWNT to bind to tumor integrins such as αvβ3. Photoacoustic imaging instrument. Our photoacoustic system is illustrated in Fig. 2. A tunable pulsed laser with a repetition rate of 10 Hz and a pulse width of 5 ns (Nd:YAG SurelightIII-10 connected to Surelite OPO Plus, Continuum) illuminated the object through a fiber optic ring light (50-1353 Ringlight, Fiberoptic Systems Inc.). The average energy density of the laser at 690 nm wavelength was measured to be ~9 mJ/cm at the target site, which is below the ANSI limitation for laser skin exposure. A 5 MHz focused transducer (25.5 mm focal length, 4 MHz bandwidth, F number of 2.0, depth of focus of 6.5 mm, lateral resolution of 600 μm, and axial resolution of 380 μm. A309S-SU-F-24.5-MM-PTF, Panametrics) was used to acquire both pulseecho and photoacoustic images. In addition, high resolution ultrasound images were acquired using a 25 MHz focused transducer (27 mm focal length, 12 MHz bandwidth, F number of 4.2, depth of focus of 7.5 mm, lateral resolution of 250 μm, and axial resolution of 124 μm. V324SU-25.5-MM, Panametrics). A precision xyz-stage (U500, Aerotech Inc.) with minimum step Proc. of SPIE Vol. 7177 717725-3 Nd:YAG laser opo /Digitizing oscillosco . e Si Photodiode Amplifier