A de novo strategy to develop NIR precipitating fluorochrome for long-term in situ cell membrane bioimaging

Abstract

Cell membrane-targeted bioimaging is a prerequisite for studying the roles of membrane-associated biomolecules in various physiological and pathological processes. However, long-term in situ bioimaging on the cell membrane with conventional fluorescent probes leads to diffusion into cells from the membrane surface. Therefore, we herein proposed a de novo strategy to construct an antidiffusion probe by integrating a fluorochrome characterized by strong hydrophobicity and low lipophilicity, with an enzyme substrate to meet this challenge. This precipitating fluorochrome HYPQ was designed by conjugating the traditionally strong hydrophobic solid-state fluorochrome 6-chloro-2-(2-hydroxyphenyl) quinazolin-4(3H)-one (HPQ) with a 2-(2-methyl-4H-chromen-4-ylidene) malononitrile group to obtain closer stacking to lower lipophilicity and elongate emission to the far-red to near-infrared wavelength. As proof-of-concept, the membrane-associated enzyme γ-glutamyltranspeptidase (GGT) was selected as a model enzyme to design the antidiffusion probe HYPQG. Then, benefiting from the precipitating and stable signal properties of HYPQ, in situ imaging of GGT on the membrane was successfully realized. Moreover, after HYPQG was activated by GGT, the fluorescence signal on the cell membrane remained unchanged, with incubation time even extending to 6 h, which is significant for in situ monitoring of enzymatic activity. In vivo testing subsequently showed that the tumor region could be accurately defined by this probe after long-term in situ imaging of tumor-bearing mice. The excellent performance of HYPQ indicates that it may be an ideal alternative for constructing universal antidiffusion fluorescent probes, potentially providing an efficient tool for accurate imaging-guided surgery in the future.

Keywords: antidiffusion probe; cell membrane–targeted bioimaging; in vivo bioimaging; long-term in situ imaging; precipitating fluorochrome.

Scheme 1.

Schematic diagram illustrates the difference between conventional cell membrane-targeted bioimaging probes and our de novo anti-diffusion bioimaging probe. Traditional membrane-associated probes easily diffuse into cells, while the anti-diffusion probe releases a strong hydrophobicity and low lipophilicity fluorochrome that precipitates at the reaction sites in a manner that inhibits diffusion into the cell, hence, realizes long-term in situ cell membrane-targeted bioimaging.

Scheme 2.

The design of hydrophobicity and low lipophilicity NIR solid-state fluorochrome. (A) A de novo design strategy for developing solid-state fluorophores. (B) Solid-state fluorescent photographs of those fluorophores in the powder samples. (C) Diffusion experiments were conducted at the interface between water and dichloromethane (amphiphilic environment). All fluorescent photos were obtained under UV lamp excitation at 365 nm.

Fig. 1.

The comparison of solubility of HPQ, HTPQ, HYPQ, AMC (a water-soluble fluorochrome), and DCM-NH₂ (a liposoluble fluorochrome). The absorbance spectra of HPQ, HTPQ, and HYPQ (10 μM) in PBS containing 1% DMSO and 10% glycerol (A) and in dichloromethane (B). The black and red lines indicate the absorbance spectra intensity of these compounds before and after filtration. (C) Diffusion experiments of DCM-NH₂, HYPQ, and AMC were performed at the interface between water and dichloromethane (amphiphilic environment). (D) Agarose gels containing HYPQ and AMC were immersed in PBS (pH = 7.4), and then real-time images were obtained every 10 min for a total of 30 min. HYPQ showed good antidiffusion ability, while AMC diffused rapidly within 30 min. All the fluorescent photos were conducted under UV lamp excitation at 365 nm.

Fig. 2.

The chemical structure and photophysical properties of HYPQG. (A) The response mechanism of HYPQG with GGT that shows turn-on NIR solid-state fluorescence. (B) UV-Vis absorption spectra of HYPQG (10 μM) in DMSO and HYPQ (10 μM) in glycerol: PBS = 1:1. (C) Fluorescence emission spectra of HYPQG (5 μM) with increasing concentration of GGT (0, 1, 3, 5, 10, 15, 20, 30, 40, 60, 80, 90, and 100 U/L). λ_ex/em = 450/650 nm. (D) Particle size distributions of HYPQG (10 μM) after reaction with GGT (150 U/L). (Inset) Photos of HYPQG (10 μM) before (Left) and after (Right) reaction with GGT (150 U/L) under UV lamp at 365-nm excitation. d, diameter. (E) SEM photos of HYPQG after reaction with GGT. HFW, horizontal field width; HV, high voltage. (Scale bar, 200 nm.)

Fig. 3.

In situ imaging of endogenous GGT on the cell membrane with HYPQG and the other five traditional GGT probes. (A) A2780 cells were pretreated with 5 µM HYPQG (a), 5 µM AMCG (b), 5 µM DCMG (c), 5 µM Cv-Glu (d), 5 µM Bcy-GGT (e), or 5 µM Np-Glu (f) for 40 min, respectively, and then cultured with 5 µM Memb-Tracker Green for 10 min (g–l), followed by fluorescence imaging. All overlapping images (m–r) and colocalization images (s–x) of the probe and Memb-Tracker Green, respectively. (B) Three-dimensional reconstructed images after incubating A2780 cells with HYPQG and Memb-Tracker Green. HYPQG: λ_ex = 488 nm, λ_em = 584–676 nm; AMCG: λ_ex = 405 nm, λ_em = 425–475 nm; DCMG: λ_ex = 488 nm, λ_em = 663–738 nm; Cv-Glu: λ_ex = 560 nm, λ_em = 584–676 nm; Bcy-GGT: λ_ex = 640 nm, λ_em = 663–738 nm; Np-Glu: λ_ex = 405 nm, λ_em = 425–475 nm; Memb-Tracker Green: λ_ex = 488 nm, λ_em = 500–550 nm. (Scale bar, 20 μm.)

Fig. 4.

The long-term in situ images of HYPQG, HTPQA, Memb-Tracker Green, and Memb-Tracker Red in live cells. Cells were incubated with HYPQG (A), HTPQA (B), Memb-Tracker Green (C), and Memb-Tracker Red (D) for different times; then fluorescence imaging was carried out. All “0 h” fluorescence images were times when the fluorescence signal reached a plateau for each probe, thus ensuring that the long-term imaging experiments were performed under the same conditions. (Scale bar, 20 μm.)

Fig. 5.

In situ imaging of different expression levels of GGT on the membrane of cancer cells. (A) A2780, OVCAR3, and NIH 3T3 were incubated with HYPQG (5 μM) for 40 min at 37 °C, respectively, followed by fluorescence imaging. (B) Flow cytometric analysis after incubation with HYPQG in A2780, OVCAR3, and NIH 3T3 cancer cells. λ_ex = 488 nm. (C) HepG2 cells were incubated with NaBu (1 mM) for 24 and 48 h and then further incubated with 5 μM HYPQG for 40 min at 37 °C, followed by fluorescence imaging. (D) The average fluorescence intensity found in C; initial signal intensity was defined as 1.0. Statistical significance P values (***P < 0.001) were determined using two-sided Student’s t test (n = 3). F/F_control represents the ratio of the fluorescence intensity of the experimental group to the control group. (E) Western blot was applied to monitor the change of GGT expression, as regulated by NaBu (1 mM) for 24 and 48 h in HepG2 cells. λ_ex = 488 nm, λ_em = 584–676 nm. (Scale bar, 20 μm.)

Fig. 6.

Long-term in situ imaging of GGT in mouse tumors. In vivo real-time imaging of GGT in A2780-bearing nude mice after tumor injection of 20 μM HYPQG (A), DCMG (B), Bcy-GGT (C), and Folate-PEG5000-CY 5.5 (D), respectively. The quantitative analysis was displayed in SI Appendix, Fig. S36A.