Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide. Although liver biopsy remains the diagnostic gold standard, the procedure is invasive and prone to sampling variability. Conventional imaging techniques, including ultrasound, computed tomography (CT) and magnetic resonance imaging (MRI), also have limited sensitivity for detecting early and mild steatosis.
Fluorescent probes have emerged as a potential alternative for liver fat imaging. However, existing aggregation-induced emission (AIE) probes largely depend on cellular viscosity and tend to label lipid-rich regions nonspecifically. This makes it difficult to distinguish pathological lipid overload from normal lipid storage.
Addressing this limitation, Zhuo Wang, Yulong Jin and colleagues at Beijing University of Chemical Technology, China, have designed a cholesterol-biomimetic AIE probe called TPAS-PyCHO+. The probe exploits the hyperactive clathrin-mediated endocytosis (CME) pathway in steatotic hepatocytes, where cholesterol uptake becomes abnormally elevated under pathological conditions.
TPAS-PyCHO+ incorporates a triphenylamine-thiophene unit as its AIE luminogen. Its positively charged pyridinium salt mimics the polar hydroxyl group of cholesterol, while an intact cholesterol moiety serves as the hydrophobic tail. This molecular structure allows the probe to mimic cholesterol metabolism and promotes its selective internalisation by pathological hepatocytes.
Experiments in HepG2 cells demonstrated a marked difference in fluorescence between normal and steatotic conditions. In normal HepG2 cells, TPAS-PyCHO+ produced almost no fluorescence. Following the induction of steatosis, its fluorescence increased 4.81-fold, outperforming the commercial lipid droplet dye BDP 493/503, which recorded a 3.68-fold increase.
The probe also showed nearly complete colocalisation with BDP 493/503 in steatotic cells, with a Pearson’s correlation coefficient (PCC) of 0.98. In comparison, the cholesterol-free control probe TPAS-Py2+ showed no comparable selectivity, recording a fluorescence increase of just 1.18-fold.
Quantum yield measurements further demonstrated the probe's fluorescence response. Its fluorescence quantum yield (ΦF) increased from 0.095 in dimethyl sulfoxide (DMSO) to 0.403 in 90% glycerol, representing a 4.2-fold enhancement.
Mechanistic investigations identified Niemann-Pick C1-like 1 (NPC1L1)-mediated, clathrin-dependent endocytosis as the primary route through which TPAS-PyCHO+ enters steatotic hepatocytes.
Three CME inhibitors, chlorpromazine, Pitstop 2 and Dynasore, significantly suppressed probe uptake. Researchers also used small interfering RNA (siRNA) to knock down clathrin heavy chain (CHC), which reduced the probe's fluorescence. Western blotting confirmed that CHC protein levels fell to 0.77-fold following the knockdown.
Further experiments identified NPC1L1, rather than the low-density lipoprotein receptor (LDLR), as the primary entry receptor. Ezetimibe, an NPC1L1 inhibitor, significantly reduced probe uptake, while excess unlabeled low-density lipoprotein (LDL), which competes for LDLR, had no effect.
A cholesterol competition assay recorded a half-maximal inhibitory concentration (IC₅₀) of 18.68 µM, with an R² value of 0.9955. The findings confirmed that TPAS-PyCHO+ shares a limited number of cell-surface binding sites with cholesterol.
The researchers subsequently evaluated the probe in methionine-choline-deficient (MCD) diet-induced NAFLD mice. Following tail-vein injection of TPAS-PyCHO+ at a dose of 1 mg/kg, the probe accumulated rapidly in the liver.
Liver fluorescence in NAFLD mice peaked at 40 minutes, reaching 2.28 times the initial intensity. In healthy mice, the corresponding increase was only 1.21 times. At 20 minutes, the liver signal-to-noise ratio (SNR) reached 5.27 in NAFLD mice, compared with 1.56 in healthy mice.
Ex vivo imaging further showed that the probe accumulated almost exclusively in the liver. The control probe TPAS-Py2+ displayed almost no difference between NAFLD and healthy mice, recording SNRs of 1.69 and 1.78, respectively. This demonstrated that the high SNR achieved by TPAS-PyCHO+ resulted from cholesterol-mimetic selective uptake rather than intrinsic fluorescence brightness.
The experiments also indicated that the fluorescence response could reflect changes in the disease condition. NAFLD mice switched to a normal diet for 14 days showed a substantial decline in liver fluorescence, with levels returning to those comparable to healthy controls.
The findings introduce a biomimetic 'uptake-targeted' design approach for imaging metabolic diseases. By actively exploiting the pathological NPC1L1/CME endocytic pathway, TPAS-PyCHO+ achieved high-contrast and selective imaging of NAFLD in both cells and live mice.
The strategy could also be extended to other metabolic diseases characterised by dysregulated endocytic activity, offering a potential direction for developing imaging probes that identify pathological metabolic changes rather than simply detecting lipid accumulation.
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