Читать книгу Handbook of Aggregation-Induced Emission, Volume 2 - Группа авторов - Страница 46

Specific subcellular organelle imaging is of great significance as visualization of organelles and their morphology or functional changes is essential for the study of biological processes such as metabolism and diseases at the cellular level, as well as clinical diagnosis, drug development, or medical intervention. Among numerous advanced imaging technologies, fluorescence imaging has been recognized as one of the most powerful tools in biological systems due to the high sensitivity and in situ and real‐time observation, noninvasive testing, and cost‐effective performance. In the past decades, AIE fluorophores have achieved great progresses in specific cell imaging [54]. Despite the significant advantage as “light‐up bioprobe” of most common AIEgen conjugates [55], due to the ESIPT characteristic, probes based on SSB show another unique benefit in bioimaging such as no self‐absorption, large Stokes shift, high contrast ratio, and excellent photobleaching resistance. Furthermore, because the 3, 4, and 5 positions on the benzene ring of the salicylaldehyde molecule are easily functionalized by chemical modification, live‐cell SSB‐based fluorescent probes for specific localization of mitochondria, lysosomes, lipid droplets (LDs), and other organelles can be rationally designed by chemical modification with the target‐reactive functional groups. In general, since fluorescence probes based on SSB generally have a relatively high positive electric potential, most SSB probes prefer to accumulating and lighting up mitochondria through charge interaction due to the high negative mitochondrial membrane potential (MMP). Thus, SSB‐based bioprobes are widely applied in imaging ions as well as biomolecules in mitochondria such as H⁺, S²⁻, and esterase [38, 47, 52]. Moreover, some researches have also reported the application potential of SSB probes in imaging‐guided selective cancer cell recognition and therapy [56, 57].

Figure 3.21 (a) Schematic of deprotonation processes of compound 34. (b) pH‐dependence of the emission spectra of 34 (60 μM) in phosphate buffer and ratiometric calibration curve of I₅₁₆/I₅₅₉. (c) Confocal fluorescence images of H⁺ in HepG2 cells after incubation with 34 (60 μM) and nigericin.

Source: Reprinted from Ref. [49] (Copyright 2011 Royal Society of Chemistry).

Figure 3.22 (a) Chemical structures of some SSB probes with ratiometric pH‐responsive fluorescence. (b) Color changes (top: under room light; bottom: under a 360‐nm UV light) of compound 35‐based test papers at different pH values.

Source: Reprinted with permission from Ref. [50] (Copyright 2015 Royal Society of Chemistry).

(c) Photograph of 36 in water/ethanol (fw = 99%, v/v) with different pH values under a 360‐nm UV light. (d) Confocal fluorescence images of HeLa cells incubated with 36 (50 μmol/l) and different pH buffers.

Source: Panels (c) and (d) are adapted with permission from Ref. [52] (Copyright 2018 American Chemical Society).

The mitochondrion is one of the most important organelles in eukaryotic cells. Mitochondrial metabolism provides the energy required for the metabolism of the entire cell; meanwhile, they are involved in processes such as cell differentiation, cell communication, and cell apoptosis [58]. Most of SSB‐based bioprobes accumulate in mitochondria after passing through cytoplasm membrane may be because of an ~1.3 mV zeta‐potential of SSB derivatives [47] that makes it liable to be attracted by the ~−180 mV extreme negative potential of mitochondria. Modified by mitochondrial targeted groups, triphenylphosphonium (TPP) and pyridinium, Liu and coworkers developed SSB‐based probes 39 and 40, which perform excellent mitochondrial imaging specifically [56, 59]. Moreover, such a modification often gives SSB fluorophores some additional attractive functions. For example, as illustrated in Figure 3.23b, as TPP is sensitive to negative charge, owing to the more negative mitochondrial membrane potential of cancer cells than normal cells, 39 can selectively accumulate in cancer cell mitochondria and light up its fluorescence. Furthermore, this probe also shows high cytotoxicity toward HeLa cells as it could trigger mitochondrial dysfunctions. Thus, chemotherapy with 40 is specifically targeted to mitochondria of cancer cells and monitored by itself. Images of HeLa cells costained with MitoTracker/LysoTracker shown in Figure 3.23c demonstrate that 39 could accumulate on mitochondria with high efficiency, while for normal NIH‐3T3 cell lines, the mitochondria and lysosomes were all lit up without specificity. After incubating tetramethylrhodamine ethyl ester (TMRE), a mitochondrial membrane potential indicator, with 39 pretreated HeLa cells, the fluorescence intensity of the cells decreased, indicating that 39 can cause a reduction in the mitochondrial membrane potential. The reactive oxygen species (ROS) indicator, dihydroethidium (DHE), also clearly indicates that 39 may cause ROS production and induce apoptosis. Further research showed that 39 showed high cytotoxicity to HeLa cells even at low concentrations (0.5–1.0 μM). However, at a probe concentration of 8 μM, the viability of NIH‐3T3 cells was still higher than 80%. This treatment selectivity is related to the higher affinity of 39 for cancerous mitochondria. Due to its great selectivity for imaging and eliminating carcinoma cells' mitochondria, 39 was further self‐assembled together with the lysosome‐targeted photosensitizer AlPcSNa₄ into NPs for mitochondria and lysosome‐targeted therapeutics, with synergistic chemo‐photodynamic therapy (PDT) functions associated with self‐monitoring by dual‐light‐up fluorescence [57]. With such fascinating characteristics, 39 is expected to have promising applications in imaging‐guided precise cancer therapy. Additionally, 40 was also found to have interesting property in differentiating brown adipose cells [59]. Other researches [60, 61] also found that 40 could form a 1 : 1 host–guest complex specially with γ‐cyclodextrin (γ‐CD), which makes the fluorescence intensity on mitochondria in bioimaging enhanced about 30‐folds due to the restriction of intramolecular rotation of 40 in γ‐CD. 40 was also applied as fluorescence lighting‐up probe for the detection of protein aggregates.

Figure 3.23 (a) Chemical structures of typical mitochondrial targeting SSB probes 39 and 40. (b) Schematic representation of intracellular tracking and the therapeutic effect of 39 in cancer cells. (c) Confocal microscopic images of HeLa cells and NIH‐3T3 cells after incubation with 39. The cells were costained with MitoTracker or LysoTracker.

Source: Reprinted from Ref. [56] (Copyright 2014 John Wiley and Sons).

By modifying with various targeting groups, a number of SSB‐based bioprobes for other cellular organelle‐specific imaging such as lysosome [62], cell membrane, and LDs were reported [63]. Besides, a series of SSB derivatives were also designed as photosensitizers for imaging and killing both gram‐positive and ‐negative bacteria over mammalian cells [64, 65].

Morpholine is widely used as a lysosome targeting ligand. SSB‐based bioprobes 41 and 42 thus show high affinity to lysosomes. By substituting hydroxyl groups of salicylaldehyde with acetyl groups, the ESIPT of 41 was blocked and the fluorescence was quenched. After incubating with live cells, the probes accumulated in lysosomes and subsequently the protective acetyl groups were hydrolyzed by esterase, making them a specific fluorescent probe for lysosome esterase imaging [62]. A similar lysosome‐specific SSB bioprobe 42 was developed according to the same principle and applied to monitor the autophagy process [66]. Figure 3.24B shows that the fluorescence of 42 in HeLa cells colocalized finely with commercial lysosome dye with Pearson's coefficient as 0.9. Figure 3.24C shows that even if excess 42 was removed before rapamycin treatment, the newly formed lysosomes were lit up. The results strengthen that the fusion of the autophagic region with the original lysosome occurred during autophagy, demonstrating the satisfactory application of 42 in visualization of the lysosome and lysosome‐involved autophagy process.

LDs are subcellular organelles surrounded by a phospholipid monolayer and contain diverse neutral lipids such as triacylglycerol and cholesteryl esters. Some reports demonstrate that LD is a dynamically complex organelle involved in various physiological processes, and its metabolic balance and stability play a key role in living organisms. The abnormality of LD activities or numbers is a critical signal of various diseases, such as fatty liver diseases, type II diabetes, and inflammatory myopathy. Monitoring the location and distribution of LDs is therefore of great importance for early diagnosis of related diseases. Figure 3.24D displays two hydrophobic SSB compounds 43 and 44 applied in specific LD imaging for both live and fixed cells [67]. 43 and 44 emit yellow and orange fluorescence, respectively. Due to their ESIPT characteristics, the Stokes shift of 43 and 44 is as large as ~200 nm, which is superior to commercial BODIPY dyes for LD staining. As shown in Figure 3.24E, after incubation with 43 or 44, the LDs in A549 cells were lit up with high resolution. The overlap rates of 43 and 44 with commercial LD dyes BODIPY were as high as 0.98 and 0.97, respectively, indicating their high LD‐targeting affinity. No significant inhibition of HeLa cell growth was observed in media with high concentrations of up to 10 μM 43 and 44, indicating that these two probes have excellent biocompatibility.

Figure 3.24 (A) Chemical structures of 41 and 42. (B) Confocal images of HeLa cells stained with 10 μM 42 and containing with 50 nM LysoTracker Red. (C) Fluorescence images of 42‐stained HeLa cells before and after rapamycin treatment for different periods of time.

Source: Panels (b) and (c) are adapted with permission from Ref. [66] (Copyright 2016 John Wiley and Sons).

(D) Chemical structure of 43 and 44. (E) CLSM images of A549 cells incubated with 43 and 44, respectively.

Source: Reprinted from Ref. [67] (Copyright 2016 American Chemical Society).

(F) Chemical structure of 45. (G) Bright‐field and fluorescent images of E‐coli incubated with 45.

Source: Reprinted from Ref. [65] (Copyright 2016 John Wiley and Sons).

A few numbers of SSB probes were also applied in bacterial imaging. For example, 45 is an amphiphilic SSB designed for light‐up detection of anionic surfactants. Due to the positive polarity of the quaternary ammonium salt, the probe can also be used for wash‐free imaging of bacteria enveloped by a negatively charged outer membrane. As Figure 3.24G shows, 45 performs high affinity to Escherichia coli and imaging with excellent contrast ratio.

Liu's group [63] also reported another SSB probe with “AIE + ESIPT” characteristic, which is based on a zinc‐coordinated salicylaldehyde hydrazine backbone for apoptotic cell membranes, LDs, and bacterial imaging. As Figure 3.25b shows, lipophilic 46 displays favorable affinity to LDs in both live and apoptotic HeLa cells. However, after complexation reaction with zinc perchlorate hexahydrate (Figure 3.25a), 47 becomes strongly hydrophilic even with exceptional water solubility, thus exhibiting a weak emission in water and high emission in water–THF mixtures with THF fractions higher than 80%. 46 and 47 displayed large Stokes shifts of 215 and 190 nm, respectively, with no overlap between the absorption and emission spectra. As illustrated in Figure 3.25a, since early apoptosis is characterized by partially exposed phosphatidylserine (PS) on the cell surface, positively charged probes can bind to PS to enhance fluorescence emission. For late‐stage apoptotic cells, the membrane integrity is completely compromised; 47 was found to light up nuclei specifically. As Figure 3.25c shows, 47 only stained the cytoplasm membrane of cells in early‐stage apoptosis induced by 1 μM staurosporine, while no obvious fluorescence signal was observed for healthy cells. The simple manipulation of the presence of metal ions can bring great changes to the properties of the probe and therefore lead to the alteration of the probe function for different applications. Another research also studied the photosensitization property of 47 and found its attractive performance in selective imaging and photodynamic extirpating of both Gram‐positive and Gram‐negative bacteria over mammalian cells [64]. As Figure 3.25d and e displays, due to the electrostatic interaction between the positively charged probe and the bacterial membrane, which has a more negative potential, 47 only emitted a strong green fluorescence upon incubating with bacteria (gram‐positive Bacillus subtilis or gram‐negative E. coli), yet showed nearly no signal with mammalian cells Jurkat T or K562. The probe was found to kill Gram‐positive bacteria due to depolarization of the bacterial membrane even in the dark. For Gram‐negative bacteria, 47 could generate ROS after white light irradiation for selective photodynamic killing.