Site-specific mutated albumin for covalently targeting NIR-II dye enables noninvasive imaging of blood-testis barrier integrity

<h2>Summary</h2> Assessing the integrity of the blood-testis barrier (BTB) is critical, as it serves to protect developing sperm cells by creating a protective environment against toxins and pathogens. Here, we optimize a near-infrared (NIR) dye for <i>in situ</i> binding of endogenous albumin or constructing NIR-II fluorescent proteins (FPs) based on exogenous albumin, affording non-invasive <i>in vivo</i> imaging of BTB disruption with tunable time windows. To further enhance the targeting capability of FPs, we employ a site-directed mutagenesis (SDM) strategy to develop the mutant albumin with increased binding efficiency to CO-1080 at room temperature, which serves as a specialized exogenous albumin for accelerating the imaging window of BTB disruption. Given the common drawbacks of traditional modalities for assessing BTB integrity, non-invasive and real-time NIR-II imaging is an effective method for investigating the testicular microenvironment as well as understanding testicular physiology and pathology.