Interventional Molecular Imaging Laboratory, Department of Radiology, Leiden University Medical Center, Albinusdreef 2, 2300 RC Leiden, The Netherlands.
Laboratory of BioNanoTechnology, Wageningen University & Research, Bornse Weilanden 9, 6708 WG Wageningen, The Netherlands.
Molecules. 2022 Dec 17;27(24):9015. doi: 10.3390/molecules27249015.
Surgically induced nerve damage is a common but debilitating side effect. By developing tracers that specifically target the most abundant protein in peripheral myelin, namely myelin protein zero (P0), we intend to support fluorescence-guided nerve-sparing surgery. To that end, we aimed to develop a dimeric tracer that shows a superior affinity for P0.
Following truncation of homotypic P0 protein-based peptide sequences and fluorescence labeling, the lead compound Cy5-P0 was selected. Using a bifunctional fluorescent dye, the dimeric Cy5-(P0) was created. Assessment of the performance of the mono- and bi-labeled compounds was based on (photo)physical evaluation. This was followed by in vitro assessment in P0 expressing Schwannoma cell cultures by means of fluorescence confocal imaging (specificity, location of binding) and flow cytometry (binding affinity; K).
Dimerization resulted in a 1.5-fold increase in affinity compared to the mono-labeled counterpart (70.3 +/- 10.0 nM vs. 104.9 +/- 16.7 nM; = 0.003) which resulted in a 4-fold increase in staining efficiency in P0 expressing Schwannoma cells. Presence of two targeting vectors also improves a pharmacokinetics of labeled compounds by lowering serum binding and optical stability by preventing dye stacking.
Dimerization of the nerve-targeting peptide P0 proves a valid strategy to improve P0 targeting.
手术引起的神经损伤是一种常见但使人衰弱的副作用。通过开发专门针对周围髓鞘中最丰富的蛋白质，即髓鞘蛋白零（P0）的示踪剂，我们旨在支持荧光引导的神经保护手术。为此，我们旨在开发一种对 P0 具有更高亲和力的二聚体示踪剂。
在截短同源 P0 蛋白基肽序列并进行荧光标记后，选择了先导化合物 Cy5-P0。使用双功能荧光染料，创建了二聚体 Cy5-（P0）。基于（光）物理评估来评估单标记和双标记化合物的性能。然后通过荧光共焦成像（特异性，结合位置）和流式细胞术（结合亲和力；K）在表达 P0 的施万细胞瘤培养物中进行体外评估。
与单标记物相比，二聚化导致亲和力增加了 1.5 倍（70.3 +/- 10.0 nM 对 104.9 +/- 16.7 nM； = 0.003），这导致在表达 P0 的施万细胞瘤中染色效率增加了 4 倍。存在两个靶向载体还通过降低血清结合和通过防止染料堆积来提高光学稳定性，从而改善了标记化合物的药代动力学。
神经靶向肽 P0 的二聚化被证明是提高 P0 靶向性的有效策略。
