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

Abstract

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). 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. Key words: Breast neoplasms; Tumor microenvironment; Hydrogen-ion concentration; Membrane proteins; Fluorescence; Tumor cells, cultured; Mice, nude