Application of a fluoroborate dipyrrole fluorophore in endoplasmic reticulum super-resolution imaging

Abstract

The present invention discloses the use of a fluoroboron dipyrrole compound in preparing an endoplasmic reticulum imaging fluorescent probe. The fluoroboron dipyrrole compound has the following structure: wherein R 1 and R 2 The same or different, selected from H or C1-C10 alkyl. The fluorescent probe of the present invention can sense the change of hydrophobicity in the endoplasmic reticulum membrane according to the "retention and release" mechanism. When the endoplasmic reticulum is damaged, the hydrophobicity of the fluorescent probe is enhanced and the fluorescence is released. The fluorescent probe of the present invention can accumulate in the endoplasmic reticulum and specifically mark it. Combined with SIM technology, it has a nano-imaging effect that is superior to commercial endoplasmic reticulum dyes.

Description

Technical Field

[0001] The invention belongs to the field of organelle super-resolution imaging, and in particular relates to a fluoroboron dipyrrole fluorescent probe which can be used to trace the local structure and hydrophobicity changes of endoplasmic reticulum in living cells. Background Art

[0002] The endoplasmic reticulum (ER) is the largest organelle in the cell and is a continuous intracellular network composed of lamellar and tubular membrane structures. Given the important role of ER in calcium homeostasis, lipid and protein synthesis, cell-to-cell interactions, and innate immunity, dynamically changing the shape and size of ER to adapt to fluctuations in the ER microenvironment is crucial for cellular homeostasis. Autophagy is a highly conserved cellular process that occurs in eukaryotic cells. It destroys organelles, proteins, and macromolecules in the cytoplasm to clear damaged structures (e.g., misfolded proteins), restore components, or generate energy. ER autophagy is associated with a variety of human diseases, such as esophageal squamous cell carcinoma, colorectal cancer, allergic rhinitis, vascular diseases, viral infections, cancer, and Alzheimer's disease (Hübner, CA & Dikic, I. ER-phagy and human diseases. Cell Death. Differ. 27, 833-842 (2020).). Selective autophagy (endoplasmic reticulum autophagy) can remove excess or damaged ER. Therefore, selective recognition of ER engulfed by autophagosomes and transported to specific sites for lysosomal degradation is of great significance for the protein quality control of ER.

[0003] Although probes have been developed for visualizing the endoplasmic reticulum, most of them are used for conventional microscopic imaging, and the limited resolution restricts the observation of detailed local changes in the ER (Zhu, Z., Wang, Q., Liao, H., Liu, M., Liu, Z., Zhang, Y. & Zhu, W. Trapping endoplasmic reticulum with amphiphilic acid-active sensor via specific interaction of atp-sensitive potassium (k atp).Natl.Sci.Rev.8,nwaa198(2021);Chen,J.,Han,G.,Liu,Z.,Wang,H.,Wang,D.,Zhao,J.,Liu,B.,Zhang,R.&Zhang,Z.Recovery mechanism of endoplasmic reticulum revealed by fluorescencelifetime imaging in live cells.Anal.Chem.94,5173-5180(2022).). To address this shortcoming, the researchers used structured illumination microscopy (SIM) to overcome the Abbe diffraction limit, thereby performing imaging with a resolution of approximately 100nm. In previous work, a series of probes for super-resolution SIM imaging were developed to observe subcellular dynamics and local changes by detecting the morphology of subcellular organelles (Chen, Q., Jin, C., Shao, X., Guan, R., Tian, ​​Z., Wang, C., Liu, F., Ling, P., Guan, J., Ji, L., Wang, F., Chao, H. & Diao, J. Super-resolution tracking of mitochondrial dynamics with an iridium (iii) luminophore. Small 14, 1802166 (2018); Fang, H., Yao, S., Chen, Q., Liu, C., Cai, Y., Geng, S., Bai, Y., Tian, ​​Z., Zacharias, AL, Takebe, T., Chen, Y., Guo, Z., He, W. & Diao, J. De novo-designed near-infrared nanoaggregates for super-resolution monitoring of lysosomes in cells, in whole organoids, and in vivo.ACS Nano 13, 14426-14436 (2019).). In addition, a method for characterizing the endoplasmic reticulum based on the morphological characteristics of organelles revealed by super-resolution imaging has been developed (Fang, H., Geng, S., Hao, M., Chen, Q., Liu, M., Liu, C., Tian, ​​Z., Wang, C., Takebe, T., Guan, J., Chen, Y., Guo, Z., He, W. & Diao, J. Simultaneous zn 2+tracking in multiple organelles using super-resolutionmorphology-correlated organelle identification in living cells.Nat.Commun.12,109(2021).). However, due to the dynamic nature of the ER, only reporting the size and shape of the organelle is not sufficient to quantitatively detect and analyze stress-induced subtle ER autophagy. Therefore, a probe for monitoring changes in local ER autophagy levels is urgently needed. Summary of the invention

[0004] In order to overcome the limitation of using only morphological detection of ER autophagy, the present invention establishes a "reserve-release" mechanism for constructing an environmentally sensitive, light-stable, low-toxic SIM probe ER-BDP. Due to changes in hydrophobicity, changes in ER-BDP fluorescence intensity can indicate stress-induced ER damage. Therefore, by correlating the extent and location of such damage with the distribution of ER-BDP fluorescence intensity revealed by nanoscopy, the present invention can perform local intensity-correlated detection of ER autophagy at the sub-organelle level.

[0005] The specific technical solutions of the present invention are as follows:

[0006] Application of a fluoroboron dipyrrole compound in the preparation of an endoplasmic reticulum imaging fluorescent probe, wherein the fluoroboron dipyrrole compound has the following structure:

[0007]

[0008] Where R 1 and R 2 The same or different, selected from H or C1-C10 alkyl, preferably H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl.

[0009] A preferred structure of the fluoroboron dipyrrole compound is:

[0010]

[0011] The fluorescent probe of the present invention is a hydrophobicity-responsive fluorescent probe.

[0012] In the application described in the present invention, the probe is used for super-resolution imaging of the endoplasmic reticulum, imaging the local fine structure of the endoplasmic reticulum at a resolution level below 200nm.

[0013] Under the existing technology, the difficulty of super-resolution imaging is the need to overcome the influence of photobleaching and fluorescence background interference. The present invention proposes a "retention-release" mechanism, in which the probe remains in an aggregated state under normal conditions and "retains" its fluorescence signal. After stimulation, the fluorescence will be "released" through the deaggregation process. Based on the above technical concept, fluoroborate dipyrrole (BODIPY), which is easy to form π-π stacking, is selected as the fluorescent mother core. This molecule is prone to aggregation quenching (ACQ) in aqueous solution, and its deaggregation in a hydrophobic environment releases the fluorescent signal. The fluorescent mother core is further connected to the toluenesulfonamide group through a benzene ring, which can react with K in the endoplasmic reticulum. + The structure has the ability to target the endoplasmic reticulum by binding to the channel. In addition, the fluorescent probe also has intramolecular hydrogen bonds, which can enhance the aggregation ability of the probe and thus improve the "retention-release" effect.

[0014] The fluorescent probe of the present invention has low background noise and photobleaching ability against aggregation quenching. After being targeted to the endoplasmic reticulum, when the endoplasmic reticulum is damaged, the surrounding hydrophobicity is enhanced to cause the fluorescent probe to deaggregate and release fluorescence.

[0015] Furthermore, the fluorescent probe of the present invention can be used to prepare an imaging agent for studying the molecular mechanism of endoplasmic reticulum autophagy-related diseases, wherein the endoplasmic reticulum-related diseases are allergic rhinitis, vascular diseases, viral infections, Alzheimer's disease, cancer, etc. Another object of the present invention is to provide a method for quantitative analysis of endoplasmic reticulum autophagy fluorescence, using a fluoroborane dipyrrole compound as an endoplasmic reticulum imaging fluorescent probe, and expressing the change in fluorescence intensity during endoplasmic reticulum autophagy in the form of a numerical value, wherein the numerical value = (FF Low ) / F low , F represents the fluorescence intensity of the endoplasmic reticulum damaged area stained by the fluorescent probe, F Low The fluorescence intensity of the normal area of ​​the endoplasmic reticulum stained with a fluorescent probe is represented, and this value is used to quantify the degree of endoplasmic reticulum autophagy, which is of great significance for discovering new functions of endoplasmic reticulum autophagy and developing new cell regulation strategies related to autophagy.

[0016] Beneficial Effects

[0017] The present invention discloses the application of a fluoroboron dipyrrole compound ER-BDP in the preparation of an endoplasmic reticulum imaging fluorescent probe, in particular, endoplasmic reticulum super-resolution imaging. The probe can sense changes in hydrophobicity in the endoplasmic reticulum membrane according to a "retention and release" mechanism. When the endoplasmic reticulum is damaged, the hydrophobicity of the fluorescent probe is enhanced and fluorescence is released. ER-BDP can accumulate in the endoplasmic reticulum and specifically mark it. Combined with SIM (Structured Illumination Microscopy, structured light illumination microscopy) technology, it has a nano-imaging effect that is superior to commercial endoplasmic reticulum dyes. ER-BDP can also detect changes in local hydrophobicity caused by endoplasmic reticulum autophagy under different conditions. Considering the hydrophobic response of ER-BDP, the present invention provides a quantitative analysis method for real-time monitoring of changes in endoplasmic reticulum autophagy. By using an analysis method combined with super-resolution imaging, endoplasmic reticulum autophagy can be visualized at the nanoscale. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 . The fluorescent probe ER-BDP of the present invention 1 H NMR spectra (CDCl 3 ,400MHz).

[0019] Figure 2 . The fluorescent probe ER-BDP of the present invention 13 C NMR spectra (CDCl 3 ,101MHz).

[0020] Figure 3 . The fluorescent probe ER-BDP ([M] + )’s MALDI-TOF-MS image.

[0021] Figure 4 . Absorption (a, c) and fluorescence spectra (b, d) of the fluorescent probe ER-BDP described in the present invention in different solvents (a, b) or dioxane / water binary system (c, d).

[0022] Figure 5 . Fluorescence spectra (a) and fluorescence intensity comparison diagram (b) of the fluorescent probe ER-BDP of the present invention in PBS, urea solution of unfolded protein reagent, and solution of β-lac or BSA incubated with urea.

[0023] Figure 6 .This is the fluorescence response of the fluorescent probe ER-BDP described in the present invention to the lipid mimetic DOPC.

[0024] Figure 7 . The fluorescence response of the fluorescent probe ER-BDP described in the present invention to biological species (a), pH (b), polarity (c) and viscosity (d).

[0025] Figure 8 .This is the effect of the fluorescent probe ER-BDP of the present invention on the proliferation of different cell lines after incubating cells for 24 hours at different concentrations.

[0026] Fig. 9 . This is the co-staining of the fluorescent probe ER-BDP described in the present invention with commercial mitochondrial dyes and commercial lysosomal dyes.

[0027] Fig.10 . The co-localization of the fluorescent probe ER-BDP described in the present invention and the commercial ER plasmid dye (Cell light ER-RFP) (af) and the resolution comparison of super-resolution imaging and confocal imaging of the endoplasmic reticulum (gl).

[0028] Fig.11 .Comparison of the anti-photobleaching performance of the fluorescent probe ER-BDP (ah) described in the present invention and the commercial ER small molecule dye (ip).

[0029] Fig.12 . The fluorescent probe ER-BDP described in the present invention super-resolution traces the local structural changes of the endoplasmic reticulum after untreated (a) and treated with tunicamycin (TM) (b). (c) Schematic diagram of TM-induced endoplasmic reticulum autophagy.

[0030] Fig.13 . The fluorescent probe ER-BDP described in the present invention super-resolution dynamic tracking of endoplasmic reticulum autophagy and fluorescence changes after TM treatment for different time periods (ad), as well as the endoplasmic reticulum autophagy quantification results (e and f). DETAILED DESCRIPTION

[0031] The following is a detailed description of an embodiment of the present invention. This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and a specific operation process are given, but the protection scope of the present invention is not limited to the following examples.

[0032] Example 1: Preparation of fluorescent probe ER-BDP:

[0033] Reference: Aydin D.,Viswanathan,G.,Zehra Topal,S.,Looi,CY,Wong,WF,Min Yi Tan,G.,Zorlu,Y.,Gürek,AG,Lee,HB&Dumoulin,F.Antimicrobialactivity of a quaternized bodipy against staphylococcusstrains.Org.Biomol.Chem.14,2665-2670(2016). Preparation of NO 2 -BDP.

[0034] Ramkumar,K.,Samanta,S.,Kyani,A.,Yang,S.,Tamura,S.,Ziemke,E.,Stuckey,JA,Li,S.,Chinnaswamy,K.,Otake,H.,Debnath,B.,Yarovenko,V.,Sebolt-Leopold,JS,Ljungman,M.&Neamati,N.Mechanistic evaluation and transcriptionalsignature of a glutathione s-transferase omega 1inhibitor.Nat.Commun.7,13084(2016). Preparation of NH 2 -BDP.

[0035] At 0°C, NH 2 -BDP (3.3mmol, 1.12g) and p-toluenesulfonyl chloride (3.3mmol, 627mg) were dissolved in 20ml of dichloromethane, and then three drops of pyridine were added to the reaction solution. The reaction was stirred for 2h. After the reaction was completed, the solvent was removed by rotary evaporation. Silica gel preparative chromatography with dichloromethane / petroleum ether (2:1, v:v) as the mobile phase was used to purify the orange solid ER-BDP (1.34g, 82.3%). And then used 1 H NMR, 13 Its structure was characterized by C NMR and HR-MS. Figure 1-Figure 3 shown.

[0036]

[0037] Example 2: In vitro spectral characterization of ER-BDP

[0038] In order to determine the spectral characteristics of ER-BDP, this example measured its Figure 4 a and 4b). From the absorption spectrum, it can be seen that the maximum absorption wavelength of ER-BDP in organic solvents is 490nm, while the maximum absorption wavelength in water is red-shifted to 500nm, accompanied by the formation of a wider peak, showing obvious aggregation behavior. Similarly, the fluorescence intensity of ER-BDP shows aggregation-induced quenching in water and is significantly increased in organic solvents, indicating that the probe depolymerizes in lipophilic solvents and responds to hydrophobicity.

[0039] This example also examines the effect of ER-BDP in the presence of water and dioxane ( Figure 4 Figure 4 shows the absorption and fluorescence spectra of ER-BDP in the binary solvent system (c and 4d). As the dioxane content increases, the absorbance of ER-BDP at 500 nm gradually decreases, with an absorption peak at 492 nm. In contrast, the fluorescence intensity of ER-BDP at 512 nm increases ("release") with increasing dioxane content, and when the dioxane content reaches 20%, the fluorescence intensity increases by about 160 times compared to the baseline. However, due to the complete depolymerization of ER-BDP, the fluorescence intensity does not increase as the hydrophobicity exceeds 20%.

[0040] Example 3: Fluorescence response of ER-BDP to unfolded proteins

[0041] Endoplasmic reticulum autophagy is usually accompanied by the production of unfolded proteins and the accumulation of lipids, both of which lead to an increase in local hydrophobicity. Since urea as a denaturant can induce protein unfolding, this example uses bovine serum albumin (BSA) and β-lactoglobulin (β-lac) as model proteins and urea as an unfolding agent to investigate the effect of unfolded proteins on hydrophobicity. When BSA or β-lactoglobulin was treated with 6M urea, the fluorescence intensity of ER-BDP increased with more unfolded proteins ( Figure 5 ). To simulate lipid accumulation and membrane environment, we used phospholipid-1,2-diol-octyl-glycerol-3-phosphocholine (DOPC). As the DOPC concentration and incubation time increased, the fluorescence of the probe also increased, indicating that the probe can sense lipid accumulation and distribute in the membrane ( Figure 6 a and 6b).

[0042] In order to exclude the influence of biological species and cell microenvironment, this example also studied the effects of ER-BDP on different biologically rich metal ions, reactive oxygen species, biological thiols, and solutions with different pH, polarity, and viscosity ( Figure 7 ). Among them, the probe is almost not interfered by these factors except hydrophobicity. All these evidences show that the probe has excellent hydrophobicity-specific sensing ability.

[0043] Example 4: Cytocompatibility of ER-BDP

[0044] Prior to the intracellular experiments, the cytotoxicity of the probe was tested in this example. After incubating cancer cell lines (HeLa and HepG2 cells) and non-cancer cell lines (AT3, HMC, and L929 cells) with different concentrations of ER-BDP for 24 h, CCK-8 was used to detect cell proliferation. Increasing the probe concentration from 0 μM to 30 μM had little effect on cell proliferation ( Figure 8 ). Even when the concentration reached 50 μM, the cell viability remained at 80%, indicating that ER-BDP is almost non-toxic to cells and can be used in cell experiments.

[0045] Example 5: Labeling of the endoplasmic reticulum by ER-BDP

[0046] To further investigate the subcellular localization of ER-BDP, commercial dyes targeting different subcellular organelles were used in the colocalization experiments. TM Red, LTR, 0.1 μM), commercial mitochondrial dyes (MitoTracker TM After incubating HeLa cells with 10 μM ER-BDP and DeepRed, MTDR, 0.5 μM, SIM imaging was performed. The Pearson correlation coefficients (PCC) between ER-BDP and lysosomes and mitochondria were 0.111 and 0.208, respectively, with a low degree of overlap ( Fig. 9 ER-BDP and commercial ER plasmid dye cell light ER-RFP (10 4 After co-incubation with 1 μL of 1 μL per cell, ER-BDP showed good overlap with the commercial ER dye, with a PCC of 0.798 ( Fig.10 a-10f). Therefore, ER-BDP is mainly distributed on the ER membrane and shows good co-localization with ER.

[0047] To illustrate the advantages of SIM super-resolution over confocal imaging, we further compared the two imaging techniques after ER-BDP incubation ( Fig.10 g-10l). The images in SIM mode clearly show the continuous reticular morphology of ER, while the images in confocal mode cannot show the continuous reticular morphology of ER ( Fig.10 h and 10k). Considering the distribution of fluorescence intensity (i.e. Fig.10 h and 10k), the minimum half-maximum width was determined to be 100nm in SIM mode, indicating the possibility of local imaging at the nanoscale. A heat map was further drawn based on the fluorescence intensity in the magnified image ( Fig.10 i and 10l). The results showed that it was difficult to distinguish the changes in fluorescence intensity and morphology in the confocal heat map, but the SIM heat map clearly showed the ER structure and local distribution of fluorescence intensity.

[0048] Example 6: Photostability of ER-BDP

[0049] Light-stable fluorescent probes with minimal photobleaching properties are the best probes for super-resolution imaging because they can withstand long-term laser irradiation and can be used for long-term dynamic monitoring in cells. In this example, HeLa cells co-stained with the probe of the present invention and the commercial ER small molecule dye ER-TR were irradiated with 488nm and 561nm continuous lasers to observe the change of fluorescence intensity over time. As the exposure time increases, the fluorescence of the ER-BDP channel decreases slowly, while the fluorescence of the ER-TR channel decreases sharply ( Fig.11 ). When the cells were continuously exposed for 3 min, the fluorescence intensity of ER-BDP decreased by about 20%, while the fluorescence intensity of ER-TR decreased by about 90%, indicating that ER-BDP has excellent anti-photobleaching properties compared with commercial ER small molecule dyes. The fluorescent probe constructed based on the "retention-release" mechanism of the present invention has good photostability.

[0050] Example 7: ER-BDP super-resolution tracing of endoplasmic reticulum autophagy

[0051] Tunicamycin (TM) activates autophagy by inhibiting synthetic glycosylation, which leads to protein misfolding, lipid accumulation and ER fragmentation. In order to maintain intracellular homeostasis, cells selectively engulf excess products and damage the ER. In order to further study endoplasmic reticulum autophagy, this example uses a commercial autophagy dye DAPRed to perform super-resolution microscopy co-staining on HeLa cells and TM-treated cells. In untreated cells, the ER presents a complete network structure, and the DAPRed channel has almost no fluorescent signal, indicating that the cells do not undergo autophagy ( Fig.12 a). In cells treated with TM, ER appears as bright fragments ( Fig.12 b, left column) appears, and the DAPRed channel shows strong punctate fluorescence ( Fig.12 b, middle column). There is good overlap between the channels ( Fig.12 b, right column, PCC = 0.617), indicating that the ER autophagosome engulfs hydrophobic unfolded proteins and lipids, so ER autophagy can be observed using ER-BDP super-resolution imaging ( Fig.12 c).

[0052] Example 8: ER-BDP is used to dynamically track changes in local endoplasmic reticulum autophagy

[0053] Due to its excellent photostability, ER-BDP can be used for long-term dynamic tracking. We co-stained cells with ER-BDP and DAPRed, stimulated cells with TM, and observed intracellular changes with super-resolution imaging. Before adding TM, the green channel showed a complete ER structure, and the red channel had almost no fluorescence signal ( Fig.13 a and 13b, 0min). After adding TM, the ER structure in the green channel began to be destroyed ( Fig.13 a, 5-30min), local fluorescence signal is gradually released ( Fig.13 d, 5-30min). At the same time, red fluorescent spots are formed at those sites where the fluorescence is enhanced ( Fig.13 b and 13c, 15min), and gradually showed a donut-shaped structure ( Fig.13 b, 30 min), indicating the formation of autophagosomes and the accumulation of unfolded proteins and lipids. The results showed that the hydrophobicity of cells increased during ER autophagy. In order to quantitatively evaluate the relationship between ER autophagy and TM treatment time, Fig.13 The calculation formula shown in Figure 5a shows the change in fluorescence intensity in the form of a numerical value, and uses this value to quantify the degree of ER autophagy. First, the quantitative changes in ER in cells treated with TM at different times were calculated, and it was found that its value increased with the increase in TM stimulation time, indicating that the severity of ER autophagy increased ( Fig.13 f). This not only provides support for new detection methods for studying ER autophagy, but also provides a basis for further studying ER-related pathological processes.

Claims

1. Application of a fluoroboron dipyrrole compound in the preparation of an endoplasmic reticulum imaging fluorescent probe, wherein the fluoroboron dipyrrole compound has the following structure: ， Where R 1 and R 2 The same or different, selected from H or C1-C10 alkyl.

2. The use according to claim 1, Features R 1 and R 2 are the same or different and are selected from hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl and tert-butyl.

3. The use according to claim 1, Features The structure of the fluoroboron dipyrrole compound is: 。 4. The use according to any one of claims 1 to 3, Features The application is endoplasmic reticulum super-resolution imaging, which images the local fine structure of the endoplasmic reticulum at a resolution level below 200 nm.

5. The use according to claim 4, Features The fluorescent probe is an imaging agent for studying the molecular mechanism of endoplasmic reticulum autophagy-related diseases.

6. The use according to claim 5, Features The endoplasmic reticulum autophagy-related diseases are allergic rhinitis, vascular disease, viral infection, Alzheimer's disease, and cancer.

7. A fluorescence quantitative analysis method for endoplasmic reticulum autophagy, Features Fluoroboron dipyrrole compounds were used as endoplasmic reticulum imaging fluorescent probes to express the changes in fluorescence intensity during endoplasmic reticulum autophagy in numerical form. The numerical value = (FF Low ) / F low , F represents the fluorescence intensity of the damaged endoplasmic reticulum area stained by the fluorescent probe, F Low represents the fluorescence intensity of the normal region of the endoplasmic reticulum stained by the fluorescent probe, and this value is used to quantify the extent of endoplasmic reticulum autophagy. The fluoroboron dipyrrole compound has the following structure: , Where R 1 and R 2 The same or different, selected from H or C1-C10 alkyl.

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