Multimodal Imaging of Oxidized Low-Density Lipoprotein in Living Systems with Engineered Fluorescent Protein Chromophores

Abstract

Targeted, high-fidelity visualization of oxidized low-density lipoprotein (Ox-LDL) in complex living biological systems remains a longstanding bioanalytical bottleneck for investigating metabolic liver and cardiovascular diseases. This limitation stems from the heterogeneous, poorly defined structure of the Ox-LDL and the lack of molecular probes with adequate target specificity. Herein, we describe a bioinspired molecular engineering strategy to generate a series of probes derived from green fluorescent protein (GFP) chromophores. To the best of our knowledge, the lead candidate 1j (MW = 387.2 Da) is the smallest synthetic probe reported for Ox-LDL targeting. Upon Ox-LDL binding, 1j exhibited a 98-fold fluorescence turn-on response. This effect arose from restricted twisted intramolecular charge transfer (TICT), coupled with specific hydrogen-bonding interactions with apolipoprotein B100 (ApoB100) embedded in Ox-LDL particles. The proposed mechanism was supported by density functional theory (DFT) calculations, molecular docking, and 1H NMR spectroscopy. Probe 1j selectively distinguished high-density lipoprotein (HDL), low-density lipoprotein (LDL), and Ox-LDL in live-cell assays. Quantitative fluorescence-lifetime imaging microscopy (FLIM) further resolved intracellular HDL, LDL, and Ox-LDL. Owing to its ultrasmall molecular size, 1j readily penetrated pathological lesions and accumulated at disease-associated sites. By modular installation of a bioorthogonal reactive handle for pretargeting, we achieved combined near-infrared (NIR) fluorescence and microPET/CT multimodal in vivo imaging in hyperlipidemic mice, enabling high-contrast whole-body detection of endogenous lipoproteins with favorable target-to-background ratios. This work establishes a versatile molecular sensing and imaging platform for the spatiotemporal tracking of lipoproteins in biological matrices. The analytical tools presented here meet measurement needs for metabolic liver and cardiovascular research and open new avenues to study these pathologies.