Cu-1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid-quantum dot-vascular endothelial growth factor

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, is a homodimeric glycoprotein weighing 45 kDa (1). The VEGF family consists of six groups: VEGF-A, -B, -C, -D, -E, and the placental growth factor (PIGF) (2). Structurally, VEGFs are related to the platelet-derived growth factors (PDGF), and they all contain the characteristic eight-cysteine residues known as the cysteine knot motif (3). Intrachain and interchain disulfide bonds are formed between these cysteine residues in conserved positions (2). VEGFs bind specifically to three cell-surface receptor tyrosine kinases, including fms-like tyrosine kinase-1 (Flt-1) or VEGF receptor-1 (VEGFR-1), kinase insert domain-containing receptor (KDR) or VEGRF-2, and Flt-4 or VEGFR-3. Each VEGFR contains a 750-amino acid-residue extracellular domain that is organized into seven immunoglobulin-like folds. VEGF and VEGFRs have been implicated in angiogenesis in many solid tumors, including breast cancer, colon cancer, hepatoma, bladder cancer, gastric cancer, and prostate cancer (3). VEGFR-2 (220 kDa) is expressed exclusively in endothelial cells in cell differentiation, tumor vascularization, and metastasis. VEGF-A (the original VEGF) binds to the second and third extracellular immunoglobulin G loop of VEGFR-2 with a dissociation constant of ~100 pM (4). Hypoxia appears to be an important stimulus for producing VEGF in malignant and normal endothelial cells (3). Upon binding to its receptor VEGFR-2, VEGF-A elicits a pronounced angiogenic response, so it is considered as a predominant stimulator of angiogenesis. Human VEGF-A has five different isoforms generated by alternative slicing of a single pre-mRNA species, VEGF-A, VEGF-A, VEGF-A, VEGF-A, and VEGF-A, which comprises 121,145,165, 189, and 206 amino acids, respectively (2). These isoforms differ in their ability to bind to heparin sulfate and extracellular matrix (ECM). Quantum dots (QDs) are semiconductor nanocrystals of 2 to 10 nm in diameter (200–10,000 atoms) that possess a quantum confinement effect (hence the name “quantum dots”) caused by the restriction of electrons and holes in all three dimensions (5, 6). Like classic semiconductors that are composed of two types of atoms from the II/VI or III/V group elements in the periodic table, the nanocrystals have a valence band and a conduction band separated by an energy gap (band gap). Upon excitation, an electron is promoted from the filled valence band to the largely empty conduction band, which creates a positive vacancy “hole” in the valence band. The spatial separation (Bohr radius) of this electron-hole pair (“exciton”) is typically 1 to 10 nm for most semiconductors (6). The quantum confinement arises when one of the dimensions in the nanocrystals becomes comparable to its Bohr radius, these valence/conduction bands are quantized with an energy value that is directly related to the nanocrystal size. Thus, the excitons are confined in a manner similar to a particle-in-the-box problem, leading to a finite band gap and discretization of energy levels. When the electron fills the vacancy in the valence band, light of a certain wavelength is emitted, which corresponds to the respective band gap energy that is a function of nanocrystal size. For instance, the emission wavelength is 550 nm for 3-nm CdSe QDs and 650 nm for 7-nm CdSe QDs (7). The wavelength is also a function of semiconductor compositions, i.e., 5-nm CdTe has an emission wavelength of 700 nm, which is much higher than the 620 nm for 5-nm CdSe (8). QDs are 100 to 1,000 times more stable against photobleaching and are 10 to 100 times brighter than organic dyes. QDs have relatively long fluorescence lifetime (20–50 ns), which allows for time-resolved detection of their emitted fluorescence. For biological applications, QDs are generally encapsulated with biocompatible polymers that can increase their hydrodynamic diameter as much as two-fold (9). When their size is <5 nm, QDs are quickly cleared by renal filtration, whereas larger particles are more likely to be taken up by the reticuloendothelial system before reaching the targeted disease sites. Thus, after systematic administration, non-targeted QDs and some targeted QDs accumulate in substantial quantities in reticuloendothelial system, including the phagocytic cells in the liver, spleen, lymph nodes, and bone marrow (5). QDs can also accumulate in solid tumor tissue through the enhanced permeability and retention (EPR) effect regardless of whether they are conjugated with targeting ligands. As a whole, QDs have been widely used in cell trafficking, vasculature imaging, sentinel lymph node mapping, neural imaging, and targeting imaging (5). Cu-1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid-QD-VEGF factor (Cu-DOTA-QD-VEGF) is a multimodal agent used for imaging VEGFRs with positron emission tomography (PET) and near-infrared (NIFR) optical imaging (10). Cu-DOTA-QD-VEGF consists of three components. An amine-functionalized QD (QD705) is used as a NIFR sensor and as a platform for carrying target-specific ligands. VEGF proteins are attached to the surface of QDs for recognition of VEGFRs. Complexes of the macrocyclic chelating agent DOTA with Cu(II) (Cu-DOTA) are also covalently attached to the surface of QDs. Cu is a positron-emitting radionuclide with an intermediate half-life (12.7 h) that decays by positron (β) with a branching factor of 17.4% and a maximum β energy of 0.653 MeV (11). Cu has been used as a radiotracer in PET imaging and as a radiotherapy agent in cancer treatment. QD705, which is commercially available, comprises a CdTe core shell with a thin layer of ZnS and polyethylene glycol (PEG2000)-attached amine groups (12). The emission wavelength of QD705 (705 nm) is located in the NIRF region (700–900 nm) where the absorbance of all biomolecules reaches a minimum (10). The use of a ZnS shell can increase the quantum yield of CdTe up to 30% to 50% (5). The PEG is used to decrease surface charge, increase colloidal stability of QDs, and reduce non-specific binding of QDs (13). Although QDs are used to examine cellular alteration, their detection is limited by the penetration depth of light. Thus, PET as a highly sensitive and quantitative modality can provide complementary information about tissues in depth. This PET/NIRF dual-modality probe may combine the advantages of QD optical imaging and PET imaging to assess the pharmacokinetics and targeting efficacy of QDs.