双色上转换纳米荧光探针在套细胞淋巴瘤细胞靶向成像中的应用

Application of dual color upconversion nanofluorescence probes in cell targeted imaging of mantle cell lymphoma

目的构建上转换纳米靶向探针,并探究其在特异性标记套细胞淋巴瘤细胞株中的应用价值,为体内套细胞淋巴瘤的靶向诊断提供理论依据和指导。方法采用H_2O_2对Na YF_4:Er^(3+)和Na YF_4:Yb^(3+),Tm^(3+)上转换纳米粒子表面进行氧化处理,使该粒子由最初的疏水性转变为亲水性;在NHS、EDC的作用下,亲水性的Na YF_4:Er^(3+)上转换纳米粒子与CD20单克隆抗体共价结合,Na YF_4:Yb^(3+),Tm^(3+)上转换纳米粒子与CD5单克隆抗体共价结合;CD20抗体-Na YF_4:Er^(3+)纳米粒子轭合物和CD5抗体-Na YF_4:Yb^(3+),Tm^(3+)纳米粒子轭合物的混合悬液与套细胞淋巴瘤细胞株室温下孵育2 h;CD20抗体-Na YF_4:Er^(3+)纳米粒子轭合物和Na YF_4:Yb^(3+),Tm^(3+)纳米粒子的混合悬液与套细胞淋巴瘤细胞株室温下孵育2 h;Na YF_4:Er^(3+)纳米粒子和CD5抗体-Na YF_4:Yb^(3+),Tm^(3+)纳米粒子轭合物的混合悬液与套细胞淋巴瘤细胞株室温下孵育2 h;未偶联有任何抗体的Na YF_4:Er^(3+)和Na YF_4:Yb^(3+),Tm^(3+)上转换纳米粒子的混合液与套细胞淋巴瘤细胞株室温下孵育2 h;使用配备有980 nm近红外激光的尼康Eclipse Ti-S倒置荧光显微镜对各组细胞进行上转换成像,观察细胞表面上转换荧光的强度。结果实验组细胞表面释放出蓝、绿双色的上转换纳米荧光,但通过观察3组对照实验,细胞表面却仅分别释放出单一的绿色荧光、蓝色荧光以及未见任何荧光。结论上转换纳米粒子与CD20或CD5抗体成功共价偶联,可以对套细胞淋巴瘤细胞株进行特异性标记、成像。

Objective To construct the upconversion targeted nanoprobes,and explore their application value in the specific labeling of mantle cell lymphoma cell lines,so as to provide theoretical basis and guidance for the targeted diagnosis of mantle cell lymphoma in vivo.Methods H2O2 was used to oxidize the surfaces of the NaYF4:Er3+,NaYF4:Yb3+and Tm3+upconversion nanoparticles and change their character from hydrophobicity to hydrophilicity.With the help of NHS and EDC,hydrophilic NaYF4:Er3+upconversion nanoparticles were covalently combined with CD20 monoclonal antibody while NaYF4:Yb3+and Tm3+upconversion nanoparticles were covalently combined with CD5 monoclonal antibody respectively.The mixture containing CD20 antibody-NaYF4:Er3+upconversion nanoparticle conjugates and CD5 antibody-NaYF4:Yb3+and Tm3+upconversion nanoparticle conjugates was incubated with mantle cell lymphoma cell lines at room temperature for 2 h.The mixture containing CD20 antibody-NaYF4:Er3+upconversion nanoparticle conjugates and NaYF4:Yb3+and Tm3+upconversion nanoparticles was incubated with mantle cell lymphoma cell lines at room temperature for 2 h.The mixture including NaYF4:Er3+upconversion nanoparticles and CD5 antibody-NaYF4:Yb3+and Tm3+upconversion nanoparticle conjugates was incubated with mantle cell lymphoma cell lines at room temperature for 2 h.The mixture including dual color upconversion nanoparticles without any antibody was incubated with mantle cell lymphoma cell lines at room temperature for 2 h.The cells in all the groups were imaged using a Nikon Eclipse Ti-S inverted fluorescence microscope equipped with 980 nm near infrared laser,and the intensity of fluorescence conversion on the surface of the cells was observed.Results On the surface of the cells in the experimental group,blue and green dual color upconversion fluorescence could be observed;however,through observation of the 3 control groups,only single green fluorescence,blue fluorescence and no fluorescence were released from the cells surface,respectively.Conclusions Upconversion nanoparticles are covalently coupled with CD20 or CD5 antibody,which can be used for specific labeling and imaging of mantle cell lymphoma cell lines.