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.

Progress and Perspective of Solid-State Organic Fluorophores for Biomedical Applications

“…31 Compared with other HPQ analogs, HYPQ exhibited the highest hydrophobicity and lowest lipophilicity and consequently possessed significantly improved precipitation and antidiffusion abilities in water-and fat-soluble environments. 31 Recently, Zhang et al also reported several novel precipitating SSOFs to extend the scope of HPQbased biomedical research. 32,33 In 2001, Tang et al reported that 1-methyl-1,2,3,4,5pentaphenylsilole exhibited no fluorescence in its dilute solutions but strong fluorescence in its concentrated solutions and solid state.…”

“…31,56 For compound 8 (Cl-HPQ), the intramolecular Hbond interactions (OH•••N: 1.831 Å) improved the intramolecular planarity by preventing rotation, whereas the intermolecular H-bond interactions (NH•••O: 2.101 Å) resulted in the formation of HPQ dimers that further interconnected to form insoluble precipitates with π−π interactions (2.941 Å). 31 An HPQ-based SSOF probe was fabricated through the introduction of a substrate group to the phenolic hydrogen of HPQ to mask the multiple H-bond interactions and quench fluorescent emission (Figure 1, Type 3). Owing to the inherent high hydrophobicity and low lipophilicity of HPQ, the resultant precipitates are durable and can be immobilized in reaction sites, enabling long-term in situ tracking.…”

“…To overcome this, Zhang et al reported a novel strategy for the fabrication of an NIR antidiffusion probe 26 (HYPQG) that could be preferentially converted by GGT to release the precipitating SSOF 27 (HYPQ) with low lipophilicity and high hydrophobicity (Figure 8B). 31 The high solubility fluorophores released by conventional GGT probes readily diffuse into cells, whereas the inherent insolubility of 27 enabled it to generate stable fluorescent precipitates, resulting in the long term bioimaging of cell membranes (Figure 8C). Thus, owing to the favorable photophysical and photochemical properties of HYPQ, the antidiffusion probe design strategies have undoubtedly opened a promising avenue for realizing longterm in situ tracking biomolecules within their environs.…”

Precipitated Fluorophore-Based Molecular Probe for In Situ Imaging of Aminopeptidase N in Living Cells and Tumors

“…Although the short excitation and emission wavelengths of this probe limit its further applications for in vivo imaging, the antidiffusion performance and the long-term imaging capacity of this probe give it a great potential for in situ imaging in living cells. Recently, our group has also reported a near-infrared fluorescent probe with precipitable properties for cell imaging and intravital imaging, and we believe that such probes may have broad prospects in biosensing and biomedical imaging.…”

Peptide-Conjugated Aggregation-Induced Emission Fluorogen: Precise and Firm Cell Membrane Labeling by Multiple Weak Interactions

“…In recent years, many fluorescent probes with the advantages of strong selectivity, ultrasensitivity, and real-time detection have been developed for cell membrane labeling. [5][6][7][8][9][10][11][12][13][14] The labeling mechanisms of these probes mainly relied on the structural characteristics of cell membrane. [15][16][17][18] Firstly, there are many proteins as receptors highly expressed on tumor cell membrane, such as integrins, [19][20][21][22][23][24] aminopeptidase P (XPNPEP2) protein, [25][26] etc.…”