Zaheer Atif, Wheat Thomas E, Frangioni John V
Beth Israel Deaconess Medical Center, Boston, MA, USA.
Mol Imaging. 2002 Oct;1(4):354-64. doi: 10.1162/15353500200221302.
The detection of human malignancies by near-infrared (NIR) fluorescence will require the conjugation of cancer-specific ligands to NIR fluorophores that have optimal photoproperties and pharmacokinetics. IRDye78, a tetra-sulfonated heptamethine indocyanine NIR fluorophore, meets most of the criteria for an in vivo imaging agent, and is available as an N-hydroxysuccinimide ester for conjugation to low-molecular-weight ligands. However, IRDye78 has a high charge-to-mass ratio, complicating purification of conjugates. It also has a potentially labile linkage between fluorophore and ligand. We have developed an ion-pairing purification strategy for IRDye78 that can be performed with a standard C18 column under neutral conditions, thus preserving the stability of fluorophore, ligand, and conjugate. By employing parallel evaporative light scatter and absorbance detectors, all reactants and products are identified, and conjugate purity is maximized. We describe reversible and irreversible conversions of IRDye78 that can occur during sample purification, and describe methods for preserving conjugate stability. Using seven ligands, spanning several classes of small molecules and peptides (neutral, charged, and/or hydrophobic), we illustrate the robustness of these methods, and confirm that IRDye78 conjugates so purified retain bioactivity and permit NIR fluorescence imaging of specific targets.
通过近红外（NIR）荧光检测人类恶性肿瘤，需要将癌症特异性配体与具有最佳光性质和药代动力学的近红外荧光团偶联。IRDye78是一种四磺化七甲川吲哚菁近红外荧光团，满足作为体内成像剂的大部分标准，并且可以作为N-羟基琥珀酰亚胺酯用于与低分子量配体偶联。然而，IRDye78具有高的电荷质量比，使得偶联物的纯化变得复杂。它在荧光团和配体之间还具有潜在不稳定的连接。我们已经开发了一种用于IRDye78的离子对纯化策略，该策略可以在中性条件下使用标准C18柱进行，从而保持荧光团、配体和偶联物的稳定性。通过使用平行蒸发光散射和吸光度检测器，可以识别所有反应物和产物，并使偶联物纯度最大化。我们描述了在样品纯化过程中可能发生的IRDye78的可逆和不可逆转化，并描述了保持偶联物稳定性的方法。使用七种配体，涵盖几类小分子和肽（中性、带电和/或疏水），我们展示了这些方法的稳健性，并证实如此纯化的IRDye78偶联物保留生物活性并允许对特定靶标进行近红外荧光成像。
