靶向EGFR可见光量子点用于三阴乳腺癌显像的实验研究

Visible light emitted quantum dots targeting EGFR in triple-negative breast cancer

目的探索可见光量子点(QDs)作为载体靶向表皮生长因子受体(EGFR)用于三阴乳腺癌体外和在体靶向显像的可行性。方法水溶性QDs与西妥昔单克隆抗体(Cetuximab)反应合成探针QD-Cetuximab,检测其形态、粒径、稳定性和发光特性。培养人乳腺癌细胞MDA-MB-468(EGFR+)和MDA-MB-453(EGFR-),与QD-Cetuximab和QDs温育进行细胞毒性实验、细胞显像和荧光定量分析。制备MDA-MB-468荷瘤裸鼠8只,经尾静脉注射100μl QD-Cetuximab和QDs,观察不同时间点的显像结果和探针分布。采用两独立样本t检验分析数据。结果电镜下QD-Cetuximab粒径为(40.34±2.44)nm,动态光散射(DLS)结果示其水合粒径为(57.85±4.69)nm,且结构稳定。QD-Cetuximab浓度≤50 nmol/L时,细胞相对存活率>90%;而当浓度增至100 nmol/L,细胞相对存活率降至(72.52±4.91)%。MDA-MB-468经QD-Cetuximab温育后发出的红色荧光比MDA-MB-468用QDs温育和MDA-MB-453用QD-Cetuximab、QDs温育的均强,标准化后的荧光强度定量分析显示QD-Cetuximab组荧光强度是QDs组的5.1倍(863.36/169.97)。流式细胞分析示MDA-MB-468摄取QD-Cetuximab和QDs均随其浓度增加而增加,但前者明显高于后者(t值:12.25~38.11,均P<0.05),25、50、100和200 nmol/L浓度下QD-Cetuximab组的平均荧光强度与QDs组的比值分别为5.4、6.9、7.4和6.2。QD-Cetuximab和QDs探针在体内都主要聚集在肝,在肝发出荧光的背景下肿瘤发出的荧光不明显,但离体瘤组织可见荧光,QD-Cetuximab组和QDs组的离体瘤组织荧光强度分别为(2.46±0.60)×104和(1.29±0.05)×104(t=3.392,P=0.015)。结论偶联Cetuximab的QDs增加了对表达EGFR的三阴乳腺癌细胞的靶向能力;QD-Cetuximab在三阴乳腺癌裸鼠离体成像效果尚可,但需进一步修饰,减少肝摄取。

Objective To explore the feasibility and conditions of in vitro and in vivo imaging of triple-negative breast cancer using visible light emitted quantum dots(QDs)as the carrier to target epidermal growth factor receptor(EGFR).Methods The water-soluble QDs reacted with Cetuximab to synthesize the probe QD-Cetuximab.The morphology,particle size,stability and luminescence properties of the probe were examined.Human breast cancer cells MDA-MB-468(EGFR+)and MDA-MB-453(EGFR-)were cultured.Cytotoxicity assays,in vitro imaging and fluorescence intensity quantification were performed after cells incubation with QD-Cetuximab and QDs.Eight MDA-MB-468 tumor-bearing mice models were constructed,100μl QD-Cetuximab and QDs were injected through the tail vein.In vivo imaging and probe distribution were obtained at different time points.Independent-sample t test was used to analyze the data.Results QD-Cetuximab had a particle size of(40.34±2.44)nm detected by transmission electron microscope(TEM),a hydrated particle size of(57.85±4.69)nm detected by dynamic light scattering(DLS),and a stable structure.When the concentration of QD-Cetuximab was≤50 nmol/L,the relative survival rate of cells was more than 90%,and when the concentration exceeded 100 nmol/L,the relative survival rate of cells was reduced to(72.52±4.91)%(P<0.05).The red fluorescence of MDA-MB-468 incubated with QD-Cetuximab was stronger than that of MDA-MB-468 incubated with QDs and MDA-MB-453 incubated with QD-Cetuximab or QDs.The confocal fluorescent intensity quantitative determination showed that the ratio of QD-Cetuximab group/QDs group was 5.1(863.36/169.97).Flow cytometry showed that the uptake of QD-Cetuximab and QDs by MDA-MB-468 increased with incremental incubating concentration,and the former was more significantly(t values:12.25-38.11,all P<0.05).When the incubating concentration was 25,50,100,and 200 nmol/L,the quantitative average fluorescent intensity ratio of QD-Cetuximab group/QDs group was 5.4,6.9,7.4 and 6.2,respectively.The QD-Cetuximab and QDs probes mainly accumulated in the liver in vivo.The fluorescence emitted by tumor was not obvious under the high fluorescence of liver as a background.However,the fluorescence was visible in the isolated tumor tissue,and the quantitative fluorescence intensity of experimental group and control group were(2.46±0.60)×104 and(1.29±0.05)×104,respectively(t=3.392,P=0.015).Conclusions Cetuximab can increase the targeting ability of QDs and promote cell uptake.Although the isolated tumor imaging results are acceptable,further modification of QDs should be considered to reduce the liver uptake and improving in vivo fluorescence imaging efficiency.