荧光标记的低pH插入肽靶向乳腺癌酸性微环境的显像研究

Fluorescence-labeled pH-low insertion peptide imaging of the acid microenvironment of breast tumor

目的利用荧光标记低pH插入肽(pHLIP)显像,分析其在体外不同pH环境下与乳腺癌细胞膜的亲和力及其在乳腺癌荷瘤裸鼠体内的动态分布特征。方法通过红色荧光染料罗丹明B及近红外荧光染料青色素5(Cy5)分别标记pHLIP羟基端(B-pHLIP和Cy5-pHLIP),进行体外和体内荧光显像。分析在体外不同pH值(7.8、7.4、7.0和6.6)环境下B-pHLIP与MDA-MB-231乳腺癌细胞膜结合的荧光强度及其对细胞活性的影响。体内观测Cy5-pHLIP在肿瘤及各组织脏器不同时间点(2 h、24 h、3 d和7 d)的动态荧光分布、荧光强度变化及肿瘤/本底比值(T/NT),并进行离体组织的荧光显像。采用单因素方差分析和最小显著差异t检验分析数据。结果pH值6.6、7.0、7.4和7.8时B-pHLIP在细胞内相对荧光强度分别为(100.00±9.70)%、(69.90±5.50)%、(19.80±1.40)%和(0.40±0.04)%,其中pH值6.6时的相对荧光强度最高,与其他组相比差异有统计学意义(F=230.504,t=5.029~17.669,均P<0.05)。pHLIP对细胞活性没有显著影响。荷瘤裸鼠尾静脉注射Cy5-pHLIP后2 h、24 h、3 d和7 d的T/NT分别为3.42±0.27、3.00±1.23、3.38±0.62、3.51±0.37,差异无统计学意义(F=0.192,P>0.05)。离体荧光分布显示,Cy5-pHLIP在肿瘤组织内有较高浓聚,同时大肠段有大量pHLIP分布。结论pHLIP在细胞外酸性微环境下与肿瘤细胞膜的亲和力显著增加。Cy5-pHLIP能够在体内长效、可视化监测pHLIP的靶向肿瘤分布,但pHLIP在肠道的分布增加了肿瘤显像判读的复杂性。

Objective Fluorescence-labeled pH-low insertion peptide(pHLIP)imaging was used to analyze in vitro acidophilic characteristic of pHLIP and its dynamic distribution in the transplanted breast tumor models.Methods The red fluorescent dye Rhodamine B(B-pHLIP)and near-infrared fluorescent dye Cy5(Cy5-pHLIP)were respectively labeled at the hydroxyl terminal of pHLIP for imaging in vitro and in vivo.The fluorescent intensity in the MDA-MB-231 breast cancer cells and the cell vitality were analyzed under different pH values(7.8,7.4,7.0,6.6).In vivo dynamic fluorescent distribution,fluorescent intensities and tumor/non-tumor(T/NT)ratios in the regions of interest of tumor and normal organs at different time points(2 h,24 h,3 d,7 d)were observed or calculated,and then fluorescence imaging of the isolated tissues was finally performed.One-way analysis of variance and least significant difference t test were used to analyze the data.Results Relative fluorescent intensity of B-pHLIP in the MDA-MB-231 cells at pH 6.6,7.0,7.4 and 7.8 were(100.00±9.70)%,(69.90±5.50)%,(19.80±1.40)%and(0.40±0.04)%,respectively.There were significant differences between pH 6.6 group and other groups(F=230.504,t=5.029-17.669,all P<0.05).pHLIP had no significant toxic effect on MDA-MB-231 cells.After Cy5-pHLIP injection in vivo,the fluorescent intensity of tumor in mice gradually decreased,but the T/NT ratios were stable(3.42±0.27,3.00±1.23,3.38±0.62 and 3.51±0.37 at 2 h,24 h,3 d and 7 d respectively;F=0.192,P>0.05).The ex vivo fluorescence distribution showed that Cy5-pHLIP had a higher uptake in the tumor tissue,and the large intestine also secreted a large amount of pHLIP.Conclusions The affinity between pHLIP and tumor cell membrane is significantly increased in the extracellular acidic microenvironment.Cy5-pHLIP enables long-term and visual monitoring the tumor-targeted distribution of pHLIP in vivo.However,the high accumulation of pHLIP in the large intestine increases the interpretation complexity of tumor imaging.