Leung Kam
National Center for Biotechnology Information, NLM, NIH,
Optical fluorescence imaging is increasingly used to observe biological functions of specific targets (1, 2) in small animals. 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. Vascular endothelial growth factor (VEGF) consists of at least six isoforms with various numbers of amino acids (121, 145, 165, 183, 189, and 206 amino acids) produced through alternative splicing (3). VEGF, VEGF, and VEGF are the forms secreted by most cell types and are active as homodimers linked by disulfide bonds. VEGF does not bind to heparin like the other VEGF species (4). VEGF is a potent angiogenic factor that induces proliferation, sprouting, migration, and tube formation of endothelial cells. There are three high-affinity tyrosine kinase VEGF receptors on endothelial cells (VEGFR-1, Flt-1; VEGFR-2, KDR/Flt-1; and VEGFR-3, Flt-4). Several types of non-endothelial cells such as hematopoietic stem cells, melanoma cells, monocytes, osteoblasts, and pancreatic β cells also express VEGF receptors (3). VEGF receptors were found to be overexpressed in various tumor cells and tumor-associated endothelial cells (5). Inhibition of VEGF receptor function has been shown to inhibit pathological angiogenesis as well as tumor growth and metastasis (6, 7). Radiolabeled VEGF has been developed as a tracer for imaging solid tumors and angiogenesis in humans (8-10). Cys-tag, a fusion tag comprising 15 amino acids, was developed for site-specific conjugation the free sulfhydryl group of Cys. Backer et al. (11) prepared a Cys-tagged vector of VEGF by cloning two single-chain fragments (amino acid sequence 3–112) of VEGF joining head-to-tail to express as scVEGF, which can be labeled as Cu-1,4,7,10-tetraazacyclododecane--tetraacetic acid (DOTA)-scVEGF (Cu-DOTA-scVEGF), Tc-hydrazinonicotinic acid (HYNIC)-scVEGF (Tc-HYNIC-scVEGF), and Cy5.5-scVEGF for imaging VEGFR expression to study tumor angiogenesis (12). Cy5.5 is a NIR fluorescent dye with an absorbance maximum at 675 nm and an emission maximum at 694 nm with a high extinction coefficient of 250,000 Mcm according to measurement. Cy5.5-scVEGF is being developed for NIR fluorescence imaging of VEGFR-2 in tumor vasculature.
光学荧光成像越来越多地用于观察小动物体内特定靶点的生物学功能(1,2)。然而，当使用吸收可见光(350 - 700 nm)的荧光团时，生物分子的固有荧光会带来问题。近红外(NIR)荧光(700 - 1000 nm)检测可避免天然生物分子的背景荧光干扰，使靶组织与背景组织之间具有高对比度。与可见光荧光检测相比，由于散射减少，近红外荧光团具有更宽的动态范围和最小的背景。它们还具有高灵敏度，这源于低红外背景，以及高消光系数，从而提供高量子产率。近红外区域也与固态光学组件兼容，如二极管激光器和硅探测器。近红外荧光成像正成为小动物放射性核素成像的一种非侵入性替代方法。血管内皮生长因子(VEGF)至少由六种异构体组成，通过可变剪接产生不同数量的氨基酸(121、145、165、183、189和206个氨基酸)(3)。VEGF、VEGF和VEGF是大多数细胞类型分泌的形式，以通过二硫键连接的同二聚体形式具有活性。VEGF不像其他VEGF种类那样与肝素结合(4)。VEGF是一种有效的血管生成因子，可诱导内皮细胞的增殖、发芽、迁移和管形成。内皮细胞上有三种高亲和力酪氨酸激酶VEGF受体(VEGFR-1, Flt-1; VEGFR-2, KDR/Flt-1; 和VEGFR-3, Flt-4)。几种非内皮细胞，如造血干细胞、黑色素瘤细胞、单核细胞、成骨细胞和胰腺β细胞也表达VEGF受体(3)。已发现VEGF受体在各种肿瘤细胞和肿瘤相关内皮细胞中过表达(5)。抑制VEGF受体功能已被证明可抑制病理性血管生成以及肿瘤生长和转移(6,7)。放射性标记的VEGF已被开发用作人类实体瘤和血管生成成像的示踪剂(8 - 10)。Cys标签是一种由15个氨基酸组成的融合标签，用于与Cys的游离巯基进行位点特异性偶联。Backer等人(11)通过克隆VEGF的两个单链片段(氨基酸序列3 - 112)，头对尾连接以表达为scVEGF，制备了一种Cys标签化的VEGF载体，其可被标记为铜-1,4,7,10-四氮杂环十二烷-四乙酸(DOTA)-scVEGF (Cu-DOTA-scVEGF)、锝-肼基烟酸(HYNIC)-scVEGF (Tc-HYNIC-scVEGF)和Cy5.5-scVEGF，用于成像VEGFR表达以研究肿瘤血管生成(12)。根据测量，Cy5.5是一种近红外荧光染料，最大吸收波长为675 nm，最大发射波长为694 nm，消光系数高达250,000Mcm。Cy5.5-scVEGF正在被开发用于肿瘤脉管系统中VEGFR-2的近红外荧光成像。
