Sato Kazuhide, Gorka Alexander P, Nagaya Tadanobu, Michie Megan S, Nakamura Yuko, Nani Roger R, Coble Vince L, Vasalatiy Olga V, Swenson Rolf E, Choyke Peter L, Schnermann Martin J, Kobayashi Hisataka
Molecular Imaging Program, Center for Cancer Research, National Cancer Institute, USA.
Mol Biosyst. 2016 Oct 20;12(10):3046-56. doi: 10.1039/c6mb00371k. Epub 2016 Jul 25.
Near-infrared (NIR) fluorophores show superior in vivo imaging properties than visible-light fluorophores because of the increased light penetration in tissue and lower autofluorescence of these wavelengths. We have recently reported that new NIR cyanine dyes containing a novel C4'-O-alkyl linker exhibit greater chemical stability and excellent optical properties relative to existing C4'-O-aryl variants. In this study, we synthesized two NIR cyanine dyes with the same core structure and charge but different indolenine substituents: FNIR-Z-759 bearing a combination of two sulfonates and two quaternary ammonium cations, and FNIR-G-765 bearing a combination of two sulfonates and two guanidines, resulting in zwitterionic charge with distinct cationic moieties. In this study, we compare the in vitro and in vivo optical imaging properties of monoclonal antibody (mAb) conjugates of FNIR-Z-759 and FNIR-G-765 with panitumumab (pan) at antibody-to-dye ratios of 1 : 2 or 1 : 5. One-to-five conjugation of pan-to-FNIR-G-765 was not successful due to aggregate formation during the conjugation reaction. Conjugates of both dyes to pan (2 : 1) demonstrated similar quenching capacity, stability, and brightness in target cells in vitro. However, FNIR-Z-759 conjugates showed significantly lower accumulation in the mouse liver, resulting in higher tumor-to-liver ratio. Thus, FNIR-Z-759 conjugates appear to have superior in vivo imaging characteristics compared with FNIR-G-765 conjugates, especially in the abdominal region. Moreover, from a chemistry point of view, mAb conjugation with FNIR-Z-759 has an advantage over FNIR-G-765, because it does not form aggregates at high dye-to-mAb ratio. These results suggest that zwitterionic cyanine dyes are a superior class of fluorophores for conjugating with mAbs for fluorescence imaging applications due to improving target-to-background contrast in vivo. However, zwitterionic cyanine dyes should be designed carefully, as small changes to the structure can alter in vivo pharmacokinetics of mAb-dye conjugates.
近红外（NIR）荧光团由于在组织中的光穿透性增强以及这些波长下的自发荧光较低，因此在体内成像特性方面优于可见光荧光团。我们最近报道，含有新型C4'-O-烷基连接基的新型近红外花菁染料相对于现有的C4'-O-芳基变体表现出更高的化学稳定性和优异的光学性质。在本研究中，我们合成了两种具有相同核心结构和电荷但吲哚啉取代基不同的近红外花菁染料：带有两个磺酸盐和两个季铵阳离子组合的FNIR-Z-759，以及带有两个磺酸盐和两个胍组合的FNIR-G-765，从而产生具有不同阳离子部分的两性离子电荷。在本研究中，我们以1 : 2或1 : 5的抗体与染料比例比较了FNIR-Z-759和FNIR-G-765与帕尼单抗（pan）的单克隆抗体（mAb）缀合物的体外和体内光学成像特性。由于在缀合反应过程中形成聚集体，pan与FNIR-G-765的1 : 5缀合未成功。两种染料与pan（2 : 1）的缀合物在体外靶细胞中表现出相似的淬灭能力、稳定性和亮度。然而，FNIR-Z-759缀合物在小鼠肝脏中的积累显著较低，从而导致更高的肿瘤与肝脏比率。因此，与FNIR-G-765缀合物相比，FNIR-Z-759缀合物似乎具有更优异的体内成像特性，尤其是在腹部区域。此外，从化学角度来看，mAb与FNIR-Z-759缀合比与FNIR-G-765缀合具有优势，因为它在高染料与mAb比例下不会形成聚集体。这些结果表明，两性离子花菁染料是一类用于与mAb缀合以进行荧光成像应用的优异荧光团，因为它可改善体内的靶标与背景对比度。然而，两性离子花菁染料的设计应谨慎，因为结构上的微小变化可能会改变mAb-染料缀合物的体内药代动力学。
