A Probe Design Strategy Based on Cholesterol‐Induced Differential Cellular Uptake for Cancer Cells Specific Penetration and Imaging

Abstract Cancer metastasis and postoperative recurrence continue to pose significant challenges in the treatment of advanced‐stage cancer, highlighting the urgent necessity for early diagnosis and precise intraoperative identification of lesions. In this study, we synthesized a series of molecules derived from the traditional nucleic acid probe thiazole orange (TO), by incorporating alkyl chains of varying lengths and hydrophilic functional groups. Among them, the specific lipophilicity of TO‐Ac (a derivative of TO) enables its penetration into cancer cells, where it binds to nucleic acids and generates intense fluorescence, while being effectively excluded by normal cell membranes. The differential cholesterol content in cell plasma membranes was confirmed to influence the selective uptake of molecules in cancer cells. Accordingly, we established a quantitative design criterion (LogP = 1.48–1.62) that predicts cancer cell specificity based on oil‐water partition coefficients. Finally, TO‐Ac was successfully applied for detection of early‐stage colon cancer, enabling complete tumor resection via fluorescence imaging while identifying lesions that would otherwise remain undetected. This passive targeting strategy fundamentally challenges the conventional paradigm of ligand‐modified probes, offering a transformative approach for the development of universal tumor identification systems.