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Indocyanine green–enhanced, cyclic Arg-Gly-Asp–conjugated, PEGylated single-walled carbon nanotubes

, PhD
National Center for Biotechnology Information, NLM, NIH
Corresponding author.

Created: ; Last Update: July 12, 2010.

Chemical name:Indocyanine green–enhanced, cyclic Arg-Gly-Asp–conjugated, PEGylated single-walled carbon nanotubes
Abbreviated name:SWNT-ICG-RGD
Agent Category:Nanoparticles
Target:Integrin αvβ3
Target Category:Receptor
Method of detection:Ultrasound, Photoacoustic Imaging (PAI)
Source of signal / contrast:Indocyanine green (ICG)
  • Checkbox In vitro
  • Checkbox Rodents
No structure is available.



Photoacoustic imaging (PAI) is a hybrid imaging modality based on the photoacoustic effect where absorption of electromagnetic energy (non-ionizing laser or radio-frequency pulses) is converted into ultrasound waves that are detected outside the subject of interest (1-4). It takes advantage of sensitive optical absorption contrast and low acoustic scattering (two to three orders of magnitude weaker for ultrasound than for optical scattering) in soft tissue. The PAI signal from the photoacoustic effect is proportional to the local optical absorption and thus contains functional and molecular information about the target of interest (3, 5). In addition, PAI can achieve better than submillimeter resolution in centimeters of depth (4). At the same time, PAI inherits some limitations from both acoustic and electromagnetic radiation, such as strong ultrasonic wave aberration from hard tissues, contact measurement, and light attenuation from light scattering (1, 6).

Currently, two types of PAI systems, photoacoustic tomography and photoacoustic microscopy, have been developed for angiogenesis visualization, tumor detection, blood oxygenation mapping, and functional brain imaging (5, 7-10). However, some pathological lesions fail to generate sufficient photoacoustic contrast and require an exogenous agent to enhance the absorption contrast for detection (1, 11). Nanoparticles have been synthesized as PAI contrast agents on the basis of either their surface plasmon resonance or the near-infrared fluorescent dyes they can carry (1, 6, 11). The near-infrared optical window allows light to penetrate up to several centimeters into biological tissues, and the optical properties of tissues within this region are closely related to the tissue's molecular constituents. Challenges remain with respect to producing sufficient photoacoustic signal with low concentrations of contrast agents (2, 4).

de la Zerda et al. developed a targeted nanoparticle probe by conjugating cyclic Arg-Gly-Asp (RGD) peptides to PEGylated single-walled carbon nanotubes (SWNTs) for αvβ3 integrin targeting and attaching indocyanine green (ICG) dye to the surface of nanotubes for absorption contrast enhancement (referred to as SWNT-ICG-RGD) (1). The ultrahigh surface area of nanotubes allows highly efficient loading of ICG on the nanotube surface. SWNT-ICG-RGD have been characterized in vitro and tested in animal tumor models (1).



Synthesis of the cyclic RGD peptide–conjugated and PEGylated SWNTs (RGD-PEG-SWNTs or SWNT-RGD) has been described previously by Liu et al., and relevant data obtained from RGD-PEG-SWNTs have been introduced in the MICAD chapter on RGD-PEG-SWNTs (12). de la Zerda et al. attached ICG dye to the surface of RGD-PEG-SWNTs with overnight incubation to generate ICG-enhanced RGD-PEG-SWNTs (SWNT-ICG-RGD) (1). Unbound ICG molecules were removed with filtration through 100-kDa centrifuge filters. The SWNTs used in this study were 50–300 nm in length and 1–2 nm in diameter. The molar concentrations of SWNTs used in the synthesis were calculated on the basis of an average molecular weight of 170 kDa per SWNT (150 nm in length and 1.2 nm in diameter). Control nanoparticles (SWNT-ICG-RAD) were also synthesized with a mutated nontargeted peptide, RAD, instead of RGD. The purity, ICG labeling efficiency, and yield of the nanoparticles were not described in detail.

In Vitro Studies: Testing in Cells and Tissues


The stability of SWNT-ICG-RGD (peak optical absorbance: 780 nm) in serum was evaluated after incubation of the SWNT-ICG-RGD with 10% serum and after exposure to a 780-nm laser at a power density of 8 mJ/cm2 (the power density used in animal experiments) (1). SWNT-ICG-RGD showed steady optical absorption in serum over 24 h of incubation. The absorbance standard deviation in serum through the entire 24 h was within 2% of the average, and the maximum deviation from average was <7%. Exposure of SWNT-ICG-RGD to laser light resulted in a 30% loss of the optical absorbance after 60 min of irradiation, exhibiting good resistance to photobleaching.

In vitro targeting capability of SWNT-ICG-RGD was studied after incubation of the nanoparticles with cultured U87MG (human glioblastoma) cells for 30 min to 4 h. Cells exposed to SWNT-ICG-RGD had greater absorbance than cells exposed to control SWNT-ICG-RAD (P < 0.05 for all time points). At 1 h and 2 h of incubation, the optical absorption was approximately two-fold higher for cells exposed to SWNT-ICG-RGD than for cells exposed to SWNT-ICG-RAD, suggesting specific binding of SWNT-ICG-RGD to the αvβ3 receptor. Blocking studies were not performed (1).

The photoacoustic signal linearity as a function of agent concentrations was tested with a non-scattering and non-absorbing agar-phantom (1). SWNT-ICG-RGD particles were embedded 2–3 mm deep in the agar phantom. The photoacoustic signal produced by the particles was closely correlated with the particle concentration (R2 = 0.983).

Comparison between plain RGD-PEG-SWNTs and ICG-enhanced SWNT-ICG-RGD was performed after incubation of the nanoparticles with cultured U87MG cells (expressing αvβ3 integrin) for 2 h. Cells were collected and embedded in a clear agarose phantom. PAI showed that cells exposed to SWNT-ICG-RGD were detected at concentrations 20 times lower than the concentrations of cells exposed to plain RGD-PEG-SWNTs (P < 0.0001), which was consistent with the optical absorbance of SWNT-ICG-RGD being ~20 times higher than plain RGD-PEG-SWNTs (1, 12).

Animal Studies



Nanoparticle sensitivity in mice was tested after subcutaneous injection of SWNT-ICG-RAD (instead of SWNT-ICG-RGD) mixed with matrigel into the lower back of mice (n = 3) (1). SWNT-ICG-RAD had similar optical spectrum as SWNT-ICG-RGD. SWNT-ICG-RAD produced significant photoacoustic contrast, while matrigel alone did not. There was a linear correlation between the SWNT-ICG-RAD concentration and the corresponding photoacoustic signal (R2 = 0.97). SWNT-ICG-RAD at 170 pmol generated a photoacoustic signal equivalent to that of the tissue background (i.e., signal/background ratio = 1).

PAI of tumors was performed after tail vein injection of either SWNT-ICG-RGD (targeted) or SWNT-ICG-RAD (untargeted control) to mouse bearing U87MG tumor xenografts (n = 4 mice/group). Mice injected with SWNT-ICG-RGD showed significantly higher photoacoustic signal in the tumor compared with the control group injected with SWNT-ICG-RAD (P < 0.001). At 2 h after injection, the photoacoustic signal in the tumor was >100% higher for mice injected with SWNT-ICG-RGD than for mice injected with SWNT-ICG-RAD (1). No blocking studies were carried out.

Other Non-Primate Mammals


No references are currently available.

Non-Human Primates


No references are currently available.

Human Studies


No references are currently available.


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