Engineered Human Serum Albumin for Mapping Drug Fluctuations: Generatation of Pseudo-Fluorescent Proteins by Covalently Coupling Dyes for Probing Tamoxifen in Vitro, in Cellulo, and in Vivo

Covalent coupling of human serum albumin (HSA) and fluorescent dyes would confer superior performance of the protein for in vivo fluorescence detection and imaging. Here, a series of small molecule probes was developed to achieve HSA-specific enhanced responses. The representative probe, HSAP-6 (IC50 = 11.4 μM, LOD = 30.4 nM), demonstrated specific covalent labeling with lysine-588 in HSA, resulting in the formation of a pseudo-fluorescent protein denoted as PFP-1 (IC50 > 280 μM), characterized by unique fluorescent properties (λex/λem = 460 nm/550–750 nm). Exploiting the distinct drug-binding domains enriched in HSA, we investigated the interaction between drugs, exemplified by tamoxifen, and the pseudo-fluorescent protein PFP-1. Notably, tamoxifen significantly enhanced the fluorescence intensity of PFP-1. Leveraging tamoxifen as a model drug, we achieved real-time, in situ tracing of drug fluctuation and distribution within living cells using PFP-1. Additionally, employing zebrafish and mouse models, we observed PFP-1’s potential to probe tamoxifen distribution in vivo. Furthermore, PFP-1 facilitated quantitative analysis of drug concentrations in human urine, offering promising prospects for drug dosing guidance in clinical settings. This research introduces a novel tool for sensing and imaging tamoxifen drugs in vitro, in cellulo, and in vivo.