高尔基体靶向次氯酸响应型近红外荧光探针的设计、合成及细胞成像应用OA

Objective To design and synthesize a novel Golgi apparatus(GA)-targeted near-infrared fluorescent probe,MB-GA,and to investigate its spectral response to hypochlorous acid(HCIO)and its cellular imaging capability,thereby providing a reliable tool for monitoring dynamic changes in HCIO within the GA at the subcellular level.Methods MB-GA was designed and synthesized using methylene blue(MB)as the near-infrared fluorophore,4-(2-aminoethyl)benzenesulfonamide as the GA-targeting moiety,and a lactam group as HCIO responsive switch.The structure of the probe was verified by ¹H NMR and ¹³C NMR spectroscopy,high-resolution mass spectrometry,and density functional theory(DFT)calculations.The spectral response properties of MB-GA were evaluated using ultraviolet-visible absorption spectroscopy and fluorescence spectrophotometry.CCK-8 assay was used to assess the viability of HepG2 and 4T1 cells.Confocal laser scanning microscopy imaging was further applied to verify the GA-targeting ability of MB-GA,as well as its imaging performance in response to exogenous and endogenous HCIO.Results MB-GA features a simple synthetic route with a total yield of 65%.DFT calculations showed that the HOMO-LUMO energy gap of MB-GA was larger than that of MB(4.08 eV vs 2.47 eV),accompanied by a weak oscillator strength(f=0.0 010)and a low molar extinction coefficient(3.0×10³ L·mol⁻¹·cm⁻¹).MB-GA exhibited a rapid response to HCIO(approxi-mately 10 s),with high sensitivity(detection limit of 0.067 μmol/L),excellent selectivity,and good stability over a wide pH range of 4.0-11.0 and good photostability under continuous irradiation.The proliferation-inhibitory effect of MB-GA on HepG2 and 4T1 cells viability was dose-dependent(all P<0.001),and cell viability remained above 50.0%at 10 μmol/L.Confocal imaging showed that MB-GA showed GA-targeting ability,with a Pearson correlation coefficient of 0.6;fluorescence intensity increased significantly after addition of exogenous HCIO or lipopolysaccharide induction,and the fluorescence enhancement in HepG2(50.67±2.08)and 4T1(59.33±3.65)cancer cells was notably higher than that in normal hepatic THLE-3 cells(26.33±2.52).Conclusions This study successfully developed a GA-targeted HClO-responsive fluorescent probe MB-GA.The probe exhibits excellent spectral performance and enables discrimination of HClO-related fluorescence responses between cancer cells and normal cells,suggesting its potential for practical biological applications.