链霉亲和素修饰的量子点联合生物素标记的叶酸在卵巢癌SKOV3细胞体外成像中的应用

Streptavidin-modified quantum dots combined with biotinylated folic acid in diagnostic imaging of ovarian cancer SKOV3 cells in vitro

目的 :探讨链霉亲和素(streptavidin,SA)修饰的量子点(quantum dots,QDs)联合生物素标记的叶酸(folic acid,FA)在卵巢癌SKOV3细胞体外成像中的应用价值。方法 :采用活泼酯法合成SA-QDs复合物,并用同样的方法以聚酰胺-胺(polyamidoamine,PAMAM)树状分子为载体合成生物素化FA-PAMAM。采用生物素化FA-PAMAM联合SA-QDs荧光探针标记叶酸受体(folate receptor,FR)高表达的SKOV3细胞,同时以采用游离FA预处理的SKOV3细胞和FR低表达的肺腺癌A549细胞为对照,验证该荧光探针靶向SKOV3细胞的特异性。随后,以未用PAMAM为载体[生物素化FA-聚乙二醇(polyethylene glycol,PEG)联合SA-QDs荧光探针]或无生物素-链霉亲和素放大系统的荧光探针(FA-PAMAM-QDs)为对照,验证该探针的荧光信号双重放大效应。结果 :FR高表达的SKOV3细胞可被生物素化FA-PAMAM联合SA-QDs的荧光探针特异性识别,平均荧光强度高达114.92±2.87,显著高于FA预处理后再用该探针进行检测的SKOV3细胞(57.86±7.59)和FR低表达的肺腺癌A549细胞(14.94±0.83)(P值均<0.000 1)。在双重放大效应的验证实验中,生物素化FA-PAMAM联合SA-QDs荧光探针标记的SKOV3细胞的平均荧光强度为120.89±3.80,其荧光强度明显高于生物素化FA-PEG联合SA-QDs荧光探针标记的SKOV3细胞的77.50±2.43和FA-PAMAMQDs荧光探针标记的SKOV3细胞的47.81±1.35(P值均<0.000 1)。结论 :构建获得了一种特异性靶向卵巢癌SKOV3细胞的生物素化FAPAMAM联合SA-QDs荧光探针,该探针在卵巢癌早期诊断方面具有潜在应用价值。

Objective： To evaluate the value of streptavidin （SA）-modified quantum dots （QDs） combined with biotinylated folic acid （FA） in diagnostic imaging of ovarian cancer SKOV3 cells in vitro.Methods： SA was covalently conjugated to QDs by active ester method, and biotinylated FA was synthesized using the carrier polyamidoamine （PAMAM） （FA-PAMAM-biotin） by the same method. The fluorescent probe of SA-QDs combined with FA-PAMAM-biotin could preferentially target folate receptor （FR） of ovarian cancer SKOV3 cells which were identified as FR-positive cells. To verify the targeting specificity of this fluorescent probe, lung adenocarcinoma A549 cells with low expression of FR and the SKOV3 cells pretreated with FA were used as the controls. Simultaneously, the dual fluorescence amplification effect was verified by using the control fluorescent probe not containing the carrier PAMAM [FA-polyethylene glycol （PEG）-biotin combined with SA-QDs] or not mediated by biotin-avidin-system （FA- PAMAM-QDs）.Results： The FR-positive SKOV3 cells specifically identified by using FA-PAMAM-biotin combined with SA-QDs had an average fluorescent intensity up to 114,92±2.87, which was significantly higher than those of the SKOV3 cells pretreated with FA followed by treatment with FA-PAMAM-biotin combined with SA-QDs （57.86±7.59） and the A549 cells treated with FA-PAMAM-biotin combined with SA-QDs （14.94±0.83） （both P 〈 0.000 1）. The fluorescent intensity of SKOV3 cells labeled by FA-PAMAM-biotin combined with SA-QDs （120.89±3.80） was also higher than those of the SKOV3 cells labeled by FA-PEG- biotin combined with SA-QDs （77.50±2.43） and the cells labeled by FA-PAMAM-QDs （47.81±1.35） （both P 〈 0.000 1）.Conclusion： FA-PAMAM-biotin combined with SA-QDs, which specifically targets FR in SKOV3 cells, may become a potential candidate with high specificity and sensitivity in early diagnosis of ovarian cancer.