一种自猝灭的半乳糖胺-血清白蛋白-罗丹明X缀合物：一种用临床适用材料合成的“智能”荧光分子成像探针，用于检测腹膜卵巢癌转移灶。

A self-quenched galactosamine-serum albumin-rhodamineX conjugate: a "smart" fluorescent molecular imaging probe synthesized with clinically applicable material for detecting peritoneal ovarian cancer metastases.

作者信息

Hama Yukihiro, Urano Yasuteru, Koyama Yoshinori, Gunn Andrew J, Choyke Peter L, Kobayashi Hisataka

机构信息

Molecular Imaging Program, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland 20892-1088, USA.

出版信息

Clin Cancer Res. 2007 Nov 1;13(21):6335-43. doi: 10.1158/1078-0432.CCR-07-1004.

DOI:10.1158/1078-0432.CCR-07-1004

PMID:17975145

Abstract

PURPOSE

Fluorophore activation after cellular internalization of a targeted fluorescently labeled conjugate is an effective molecular imaging strategy to increase target-to-background ratios. The D-galactose receptor on ovarian cancer cells has been used to target self-quenched avidin-rhodamineX conjugates in which the avidin component binds to D-galactose receptor and the rhodamines are optically activated by dequenching only after cellular internalization. As a nonimmunogenic alternative of avidin, galactosamine-conjugated serum albumin (GmSA) targets the D-galactose receptor with higher binding affinity and has more conjugation sites available for rhodamineX than avidin.

EXPERIMENTAL DESIGN

GmSA was conjugated with 20 rhodamineX molecules (GmSA-20ROX) to create a self-quenching complex, which was compared with a conjugate consisting of GmSA and a single rhodamineX (GmSA-1ROX) in ex vivo chemical activation characteristics, intracellular activation, and in vivo molecular imaging for detecting peritoneal micrometastases of SHIN3 ovarian cancer.

RESULTS

GmSA-20ROX was five times brighter than GmSA-1ROX when incubated with SHIN3 ovarian cancer cells for 3 h. Submillimeter SHIN3 ovarian cancer implants in the peritoneal cavity were clearly visualized in vivo with spectral fluorescence imaging due to the high tumor-to-background ratio. The sensitivity and specificity of GmSA-20ROX for implant detection were determined by colocalization of the rhodamineX emission with red fluorescent protein expressed constitutively in the SHIN3 tumor implants. Among 336 lesions, sensitivity and specificity were 99%/99%, respectively, for GmSA-20ROX, whereas the results for GmSA-1ROX were only 24%/100% (n = 388), respectively, for lesions approximately 0.8 mm or greater in diameter.

CONCLUSION

Self-quenched GmSA-20ROX is more efficient than previous d-galactose-targeted fluorescent conjugates.

摘要

目的

靶向荧光标记偶联物细胞内化后进行荧光团激活是一种提高靶标与背景比值的有效分子成像策略。卵巢癌细胞上的D-半乳糖受体已被用于靶向自猝灭抗生物素蛋白-若丹明X偶联物，其中抗生物素蛋白组分与D-半乳糖受体结合，且若丹明仅在细胞内化后通过去猝灭实现光学激活。作为抗生物素蛋白的一种非免疫原性替代物，半乳糖胺偶联血清白蛋白（GmSA）以更高的结合亲和力靶向D-半乳糖受体，并且比抗生物素蛋白有更多可用于若丹明X的偶联位点。

实验设计

将GmSA与20个若丹明X分子偶联（GmSA-20ROX）以形成自猝灭复合物，在体外化学激活特性、细胞内激活以及用于检测SHIN3卵巢癌腹膜微转移的体内分子成像方面，将其与由GmSA和单个若丹明X组成的偶联物（GmSA-1ROX）进行比较。

结果

与SHIN3卵巢癌细胞孵育3小时后，GmSA-20ROX的亮度比GmSA-1ROX高五倍。由于肿瘤与背景比值高，通过光谱荧光成像在体内可清晰地观察到腹膜腔内亚毫米级的SHIN3卵巢癌植入物。GmSA-20ROX对植入物检测的敏感性和特异性通过若丹明X发射与SHIN3肿瘤植入物中组成型表达的红色荧光蛋白的共定位来确定。在336个病灶中，GmSA-20ROX的敏感性和特异性分别为99%/99%，而对于直径约0.8mm或更大的病灶，GmSA-1ROX的结果分别仅为24%/100%（n = 388）。