靶向整合素αvβ6的光声及荧光成像探针的合成

Production of a dual model probe for photoacoustic imaging and fluorescence imaging targeting integrin αvβ6

【摘要】目的合成靶向整合素αvβ6的光声成像及荧光成像的双功能探针。方法用吲哚氰绿一羟基琥珀酰亚胺（ICG-NHS）与胱氨酸结肽反应生成ICG一结肽探针，用反相高效液相色谱仪（RP-HPLC）分离出探针，质谱仪及光谱仪检测探针的分子量及最大吸收波长；光声成像仪及荧光成像仪检测不同浓度探针的光声信号及荧光信号；评估探针在激光照射下的稳定性；使用整合素αvβ6阳性和阴性的细胞进行细胞摄取实验，从而评估探针对整合素a，B。的靶向性；评估光声成像及荧光成像所能检测到的最少细胞数。结果使用RP—HPLC将ICG-结肽从反应液中分离，保留时间为21．4min，质谱分析测得分子量为4727，荧光标记率为1：1；光谱仪测得最大吸收波长为790nm；探针浓度与其光声信号强度呈线性相关，当探针浓度〈1．5×10^6mol／L时，其浓度与荧光信号呈线性相关，光声成像与荧光成像能从背景中检测到的探针最低浓度分别为0．09×10^6mol／L和0．05×10^6mol／L；对探针进行连续20次激光扫描，每次持续1min，其光声信号未见明显减退；αvβ6阳性A431细胞与αvβ6阴性293T细胞对ICG-结肽探针的摄取在不同浓度及不同孵育时间均存在差异（P〈0．001）。加入5μmol／L未标记的结肽可以降低A431细胞对ICG-结肽探针的摄取（P=0．001）；光声成像和荧光成像可分别检测到低至0．4×10^6个和0．05×10^6个ICG-结肽探针标记的A431细胞。结论靶向整合素αvβ6的光声成像及荧光成像的双功能探针具有良好的靶向性及敏感性，在αvβ6阳性肿瘤早期检测方面有潜在的实用价值。

Objective To develop a probe for photoacoustic imaging and fluorescence imaging targeting integrin αvβ6. Methods The probe was separated by RP-HPLC. Molecular weight and the maximum absorption wavelength of the probe were detected by mass spectrum instrument and optical spectrum instrument. Various concentrations of the probe were detected by photoacoustic imaging and fluorescence imaging. The stability of the probe was evaluated when exposed under laser. Targeting of the probe on integrin αvβ6 was evaluated in cell uptake assay with integrin αvβ6 positive and negative cells. The minimum number of cells that could be detected by photoacoustic imaging and fluorescence imaging was also evaluated. Results The probe ICG-peptide was separated from reaction mixture by RP-HPLC. The probe had a retention time of 21.4 minutes and m/z of 4 727. The labeling ratio of the probe was 1 ： 1. The maximum absorption wavelength of the probe was 790 nm. The photoacoustic signal was linearly dependent on the concentration of the probe. The fluorescence signal was linearly dependent on the concentration of the probe when the concentration was smaller than 1.5 × 10^-6 mol/L. The lowest concentration of the probe that could be detected above the background by photoacoustic imaging and fluorescence imaging was 0.09× 10^-6mol/L and 0.05 × 10^-6 mol/L, respectively. No obvious decrease of the photoacoustic signal was observed after the probe was scanned 20 times （each time lasted for 1 min） by laser. There existeddifferences （ P 〈0.001） in cell uptake of the probe with various concentrations and reaction time between A431 cells （αvβ6 positive） and 293T cells （αvβ6 negative）. Cell uptake was inhibited by the addition of 5 μmol/L unlabeled peptide in A431 cells （ P = 0.001）. The lowest number of the labeled A431 cells detected by photoacoustic imaging and fluorescence imaging was 0.4 × 10^6 and 0.05 × 10^6, respectively. Conclusions The dual functional photoacoustic and fluorescence probe targeting integrin αvβ6 was successfully developed. The targeting and sensitivity of the probe makes it potentially useful in early detection of αvβ6 positive tumors.