Leung Kam
National Center for Biotechnology Information, NLM, NIH
Optical fluorescence imaging is increasingly used to monitor biological functions of specific targets (1-3). 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 a 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 complement to radionuclide imaging in small animals. Folic acid is a water-soluble B vitamin (4) that is essential for methylation and DNA synthesis. The primary pathway for folate entry into cells is through the facilitated transporter, which has a low affinity for folate (Michaelis constant () = 1–5 μM). Some cells in the choroid plexus, kidney, lung, thyroid, spleen, placenta, and thymus also possess a higher-affinity receptor (dissociation constant () = 0.5 nM) that allows folate uptake receptor-mediated endocytosis. Some human epithelial tumor cells were found to overexpress folate-binding protein (5). More than 90% of human ovarian and endometrial cancers express the high-affinity receptor, which is absent in the respective normal tissues. Breast, colorectal, renal, and lung carcinomas also overexpress the folate receptor but at lower frequencies (20%–50%). Activated macrophages, but not resting macrophages, have also been found to have folate receptor (6). Several folate-based conjugates have been studied in tumor imaging [PubMed]. Lin et al. (7) reported the synthesis of four new, water-soluble, NIR cyanine fluorophores that have superior chemical stability and optical properties. One of the NIR dyes (NIR2) was conjugated to amino-derivatized folic acid to form NIR2-folate, which has an excitation maximum at 665 nm and an emission maximum at 686 nm. NIR2-folate is being developed as an optical imaging agent for detection of folate receptors . Among the various NIR agents, only indocyanine green (ICG), with absorption at 780 nm and emission at 820 nm, is approved by the United States Food and Drug Administration for clinical applications in angiography, blood flow evaluation, and liver function assessment (8). It is also under evaluation in several clinical trials for other applications. Liu et al. (9) conjugated a hydrophobic derivative of ICG (ICG-Der-01) to the ɣ-carboxyl group of folic acid using PEG-bis-amine (4 kDa) as a linker to form folate-PEG-ICG-Der-01 (fPI-01) for NIR imaging of tumors in mice.
光学荧光成像越来越多地用于监测特定靶点的生物学功能(1 - 3)。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。与可见荧光检测相比，近红外荧光团由于散射减少，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，可提供高量子产率。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补充。叶酸是一种水溶性B族维生素(4)，对甲基化和DNA合成至关重要。叶酸进入细胞的主要途径是通过易化转运体，其对叶酸的亲和力较低(米氏常数( ) = 1 - 5 μM)。脉络丛、肾脏、肺、甲状腺、脾脏、胎盘和胸腺中的一些细胞也具有高亲和力受体(解离常数( ) = 0.5 nM)，可通过受体介导的内吞作用摄取叶酸。发现一些人类上皮肿瘤细胞过表达叶酸结合蛋白(5)。超过90%的人类卵巢癌和子宫内膜癌表达高亲和力受体，而相应的正常组织中不存在该受体。乳腺癌、结直肠癌、肾癌和肺癌也过表达叶酸受体，但频率较低(20% - 50%)。还发现活化的巨噬细胞而非静止的巨噬细胞具有叶酸受体(6)。几种基于叶酸的缀合物已在肿瘤成像中进行了研究[PubMed]。Lin等人(7)报道了四种新型水溶性近红外花菁荧光团的合成，它们具有优异的化学稳定性和光学性质。其中一种近红外染料(NIR2)与氨基衍生化的叶酸偶联形成NIR2 - 叶酸，其最大激发波长为665 nm，最大发射波长为686 nm。NIR2 - 叶酸正在被开发为一种用于检测叶酸受体的光学成像剂。在各种近红外试剂中，只有吲哚菁绿(ICG)，其吸收波长为780 nm，发射波长为820 nm，被美国食品药品监督管理局批准用于血管造影、血流评估和肝功能评估的临床应用(8)。它也正在多个临床试验中用于其他应用的评估。Liu等人(9)使用聚乙二醇双胺(4 kDa)作为连接剂，将ICG的疏水衍生物(ICG - Der - 01)与叶酸的γ - 羧基偶联，形成叶酸 - 聚乙二醇 - ICG - Der - 01(fPI - 01)，用于小鼠肿瘤的近红外成像。
