Aβ斑块靶向近红外荧光探针的合成及评价

Synthesis and evaluation of near-infrared fluorescent probes for β-amyloid plaques

目的制备近红外荧光探针ZZN-4、ZZN-6,并考察其光学性质及与Aβ1-42聚集体、APP/PS1转基因小鼠脑内Aβ斑块的结合能力。方法以2-喹啉甲醛及4-(二甲氨基)肉桂醛为原料,通过缩合反应制备探针ZZN-4、ZZN-6,并通过1HNMR、13CNMR、MS确认结构。考察所制备探针的紫外吸收及与蛋白结合前、后荧光性质的变化;通过蛋白结合饱和实验测定探针与Aβ1-42聚集体结合的解离常数(Kd);对APP/PS1转基因小鼠脑切片进行体外荧光染色,考察其与Aβ斑块的结合能力。结果ZZN-4、ZZN-6与Aβ1-42聚集体结合后,荧光最大发射波长均发生蓝移(分别为635、629nm),但仍处于近红外区;荧光强度分别增强52.4、15.9倍;其中,ZZN-4与Aβ1-42聚集体结合的Kd为6.89nmol·L^-1。APP/PS1转基因鼠脑切片的荧光染色结果表明:ZZN-4可与Aβ斑块特异性结合。结论荧光探针ZZN-4在体外表现出对Aβ1-42聚集体的高亲和力,并对脑切片Aβ斑块具有较好的靶向性,有潜力成为阿尔兹海默症患者脑内Aβ斑块的近红外荧光成像剂。

OBJECTIVE To synthesize a series of near - infrared ( NIR) fluorescent probes ZZN - 4 and ZZN-6, and to investigate their optical properties, binding ability towards Ap, Aβ1-42 aggregates in vitro, and targeted affinity for Ap plaques in brain sections of APP/PS1 transgenic mouse. METHODS ZZN - 4 or ZZN - 6 was synthesized by reaction with 2 - quinolinecarboxaldehyde and 4-( dimethylamino) cinnamaldehyde as primary reagents, and the structures were confirmed by 1HNMR, i3CNMR and MS. UV absorption spectra and fluorescence property with Aβ1-42 aggregates were measured. In vitro saturation binding assays and fluorescence staining were conducted to evaluate binding ability of the probes towards A[3 plaques. RESULTS Large blue shifts were observed upon binding to Aβ1-42 aggregates,the emission maximum of ZZN -4 and ZZN -6 were still retained in the NIR spectral range ( 635 nm and 629 nm, respectively). ZZN - 4 displays a higher affinity towards Aβ1-42 aggregates with a 52. 4 -fold increase in fluorescence intensity while ZZN - 6 with a 15. 9 -fold increase. The dissociation constant between ZZN - 4 and Aβ1-42 aggregates further confirmed its high affinity ( Kd = 6. 89 nmol . L^-1 ). Fluorescence staining results revealed specific affinity of ZZN -4 towards Ap plaques in sections of APP/PS1 transgenic mouse brain. CONCLUSION Fluorescence probe ZZN -4 displays high affinity for Aβ1-42 aggregates in vitro and specific affinity for Ap plaques in brain sections, which possesses the potential to be NIR fluorescence imaging agent for early diagnosis of Alzheimer's disease.