⁶⁴Cu-1,4,7,10-Tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid-polyethylene-glycol-single-walled nanotube-c(RGDyK)

[⁶⁴Cu-DOTA-SWNT-PEG-c(RGDyK)]

Background

[PubMed]

Optical fluorescence imaging is increasingly used to visualize biological functions of specific targets (1, 2). However, the intrinsic fluorescence of biomolecules poses a problem when fluorophores that absorb visible light (350–700 nm) are used. Near-infrared (NIR) fluorescence (700–1,000 nm) detection avoids the background fluorescence interference of natural biomolecules, providing a high contrast between target and background tissues. NIR fluorophores have wider dynamic range and minimal background as a result of reduced scattering compared with visible fluorescence detection. They also have high sensitivity, resulting from low infrared background, and high extinction coefficients, which provide high quantum yields. The NIR region is also compatible with solid-state optical components, such as diode lasers and silicon detectors. NIR fluorescence imaging is becoming a non-invasive alternative to radionuclide imaging in small animals.

Carbon nanotubes are made of carbon fullerene carbon units, which respond to local dielectric changes without photobleaching (3, 4). They can be tuned in a range of wavelengths for NIR absorption, thus providing broad excitation profiles and high absorption coefficients. They can be coated and capped with hydrophilic materials for additional conjugation with biomolecules, such as peptides, antibodies, nucleic acids, and small organic compounds for in vitro and in vivo studies (5). Single-walled carbon nanotubes (SWNTs) have a diameter of 1–5 nm and a length of 200–1,000 nm, and they have been shown to be non-toxic to cells in vitro (6). However, there have been limited in vivo studies of SWNT toxicological and pharmacological profiles in small animals.

Integrins are a family of heterodimeric glycoproteins on cell surfaces that mediate diverse biological events involving cell–cell and cell–matrix interactions (7). Integrins consist of an α and a β subunit and are important for cell adhesion and signal transduction. The α_vβ₃ integrin is the most prominent receptor affecting tumor growth, tumor invasiveness, metastasis, tumor-induced angiogenesis, inflammation, osteoporosis, and rheumatoid arthritis (8-13). Expression of the α_vβ₃ integrin is strong in tumor cells and activated endothelial cells, whereas expression is weak in resting endothelial cells and most normal tissues. Antagonists of α_vβ₃ are being studied as antitumor and antiangiogenic agents and the agonists of α_vβ₃ are being studied as angiogenic agents for coronary angiogenesis (12, 14, 15). A peptide sequence consisting of Arg-Gly-Asp (RGD) has been identified as a recognition motif used by extracellular matrix proteins (vitronectin, fibrinogen, laminin, and collagen) to bind to a variety of integrins, including α_vβ₃. Various ligands have been introduced for imaging of tumors and tumor angiogenesis (16). Liu et al. (17) conjugated ⁶⁴Cu-1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (⁶⁴Cu-DOTA) and c(RGDyK) to phospholipid-polyethylene-glycol (PL-PEG)–coated SWNTs (⁶⁴Cu-DOTA-SWNT-PEG-c(RGDyK)) for imaging α_vβ₃ integrin expression in tumors and their vasculatures.

Synthesis

[PubMed]

Liu et al. (17) performed sonication of SWNTs in a solution of PL-PEG₄₅₀₀-NH₂ for 1 h to obtain SWNT-PEG₄₅₀₀-NH₂, which exhibits a molar extension coefficient of 7.9 × 10⁶ M^-1cm^-1 at 808 nm absorbance. The average length of the SWNTs was ~200 nm. SWNT-PEG₄₅₀₀-NH₂ nanotubes were incubated with DOTA-N-hydroxysulfonosuccinimide (SNHS) for 4 h (pH 7.4) to form SWNT-PEG₄₅₀₀-DOTA; there were ~62 DOTA molecules per SWNT. To prepare conjugates with both DOTA and RGD, DOTA-SNHS and sulphosuccinimidyl 4-N-maleimidomethyl cyclohexane-1-carboxylate were mixed at 1:5 molar ratios and incubated in SWNT-PEG-NH₂ solutions for 2 h (pH 7.4). The resulted SWNTs were reacted overnight with 0.2 mM thiolated c(RGDyK) in the presence of 10 mM Tris(2-carboxyethyl) phosphine hydrochloride (pH 7.4), yielding DOTA-SWNT-PEG₅₄₀₀-RGD with both RGD and DOTA on the SWNTs. There were 36.2 c(RGDyK) molecules per SWNT with a c(RGDyK)/DOTA ratio of 0.59. DOTA-SWNT-PEG₂₀₀₀-RGD was prepared similarly with the use of PL-PEG₂₀₀₀-NH₂. There were 47.4 c(RGDyK) molecules per SWNT with a c(RGDyK)/DOTA ratio of 0.71. ⁶⁴CuCl₂ (74 MBq (2 mCi)) was added to 150 nM SWNT conjugates in 0.1 M NaOAc solution (pH 6.5). The reaction mixture was incubated for 1 h at 40°C. The radiolabeled yield was 60–80% with a specific activity of ~370 MBq/µmol (~10 mCi/µmol). ⁶⁴Cu-SWNT conjugates in mouse serum remained intact at 37°C after 24 h incubation.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Liu et al. (17) performed in vitro inhibition studies of DOTA-SWNT-PEG₅₄₀₀-RGD and DOTA-SWNT-PEG₂₀₀₀-RGD in cultured U87MG human glioblastoma cells with ¹²⁵I-echistatin. The 50% inhibition concentration values for DOTA-SWNT-PEG₅₄₀₀-RGD and DOTA-SWNT-PEG₂₀₀₀-RGD were 11.1 and 4.1 nM, respectively.

Animal Studies

Human Studies

[PubMed]

No publication is currently available.

NIH Support

R21 EB001785, R21 CA102123, P50 CA114747, R24 CA93862, U54 CA93862