A target-activated AS1411 aptamer probe for in vivo and ex vivo imaging of nucleolin-overexpressing triple-negative breast cancer

Abstract

Fluorescence imaging of breast cancer is often limited by high background signals from conventional “always-on” probes. The nucleolin-targeting AS1411 aptamer provides a promising recognition element for tumors with elevated nucleolin expression, including triple-negative breast cancer (TNBC). This study aimed to develop a target-activated molecular probe to enable high-contrast detection of nucleolin-overexpressing breast tumors. An activatable probe (AAFP-AS1411) based on the AS1411 aptamer was engineered by hybridizing fluorescent AS1411 with a quencher strand to generate a target-responsive “OFF-to-ON” fluorescence response. Its preferential fluorescence response and concentration-dependent behavior were evaluated in vitro using flow cytometry, fluorescence spectroscopy, and confocal microscopy. In vivo tumor-associated fluorescence, ex vivo tissue fluorescence distribution, and preliminary acute safety were evaluated in MDA-MB-231 tumor-bearing mice. AAFP-AS1411 exhibited low background interference and preferential fluorescence activation in MDA-MB-231 TNBC cells compared with MCF-10A cells, with reduced signals following sequence mismatch or competitive blocking. In vivo , preferential tumor-associated fluorescence was observed, with the strongest signal detected at 6 h post-injection. Ex vivo analysis showed higher fluorescence in tumor tissue than in the examined major organs. No evident acute toxicity was observed at the tested dose of 2 nmol per mouse and the 24-h time point based on hematological, biochemical, and histopathological assessments. AAFP-AS1411 enabled preferential, high-contrast fluorescence imaging in the tested MDA-MB-231 TNBC model across in vitro , in vivo , and ex vivo experiments. These findings provide proof-of-concept support for further evaluation of activatable AS1411-based probes for molecular imaging of nucleolin-overexpressing TNBC.