WO2023082185A1 - Aggregation-induced emission principle-based autophagy detection molecular probe and manufacturing method therefor and application thereof - Google Patents

Abstract

The present invention relates to the field of molecular probes, specifically to an aggregation-induced emission principle-based autophagy detection molecular probe. The structure of the probe sequentially comprises an LC3 protein targeting peptide capable of targeting LC3 molecules, a cell penetrating peptide capable of improving the water solubility of the whole probe and promoting the probe to enter cells, and aggregation-induced emission tetraphenyl ethylene capable of emitting fluorescence when the binding movement with LC3 is limited. The molecule probe of the present invention has the characteristics of cellular internalization, specific interaction with LC3, aggregation-induced emission, being non-cytotoxic, not leading autophagy effect by itself, etc.; the difference of the autophagy intensity of cells can be visually observed under an optical microscope; the difference of the autophagy intensity of the cells can be analyzed and monitored by means of flow cytometry; a cell subpopulation with different autophagy levels can be separated from normally cultured cells or a primary cell population; and the general heterogeneity of basic autophagy levels in various cell types is disclosed. The present invention further provides a manufacturing method for and application of the molecule probe.

Description

Cell autophagy detection molecular probe based on the principle of aggregation-induced luminescence and its preparation method and application technical field

The invention belongs to the technical field of molecular probes, and in particular relates to a molecular probe for detecting cell autophagy based on the principle of aggregation-induced luminescence and its preparation method and application.

Background technique

Autophagy is a dynamic and conserved degradation pathway characterized by a series of steps including the emergence of cup-shaped phagosomes, cargo sequestration, formation of double-membrane autophagosomes, and fusion of autophagosomes with lysosomes to form autophagosomes and cargo degradation. An important and complex role of autophagy has been well documented in the development and treatment of many human diseases, such as cancer, neurodegenerative diseases, diabetes, autoimmune diseases and cardiovascular diseases. It is generally accepted that low levels of autophagy exist in virtually every mammalian cell to maintain cellular homeostasis and survival and play a crucial role in many physiological processes. However, circumstantial evidence suggests that, for a given cell type, the level of basal autophagy varies widely across cells. Literature has shown that about one-third of aged hematopoietic stem cells exhibit high levels of autophagy to maintain a hypometabolic state and have a strong long-term regenerative potential. Autophagic variation within a cell population determines cell fate through selective degradation of Fap-1. On the other hand, autophagy is often elevated to a high level under physical, chemical or biological stress, and this enhanced autophagy is closely related to many physiological abnormalities and pathological conditions. Isolation of cell subpopulations with different levels of autophagy will greatly facilitate the study of basal or induced autophagy. However, this work has been hampered by the lack of convenient and reliable methods. Various acidophilic dyes, such as acridine orange, monodansylcadaverine (MDC), and Cyto-ID, have shown some ability to monitor autophagy. Cyto-ID in particular has been used for autophagy-based cell sorting, but the specificity of these probes is questionable since how they interact with the autophagy machinery is largely unknown. A better approach is to utilize one of the autophagy-associated (Atg) proteins, of which the Atg8/LC3/GABARAP protein family (collectively known as LC3, short for microtubule-associated protein 1 light chain 3) is the most widely used because lipidated LC3 (LC3-II) was the only protein marker reliably associated with intact autophagosomes. Because LC3 lacks intrinsic fluorescence and is not easy to track, exogenous expression of LC3 fused to a fluorescent protein (usually GFP) is often deployed. The fluorescence of GFP-LC3 is quenched in the acidic environment of autophagosomes, so cells with high autophagic flux will exhibit reduced GFP fluorescence and be separated from the autophagy-low cell subpopulation by FACS sorting. Methods based on this principle have been successfully applied in the exemplary studies described above. In addition to LC3, protein molecules specifically degraded during autophagy, such as p62 and NBR1, can also be used for flow cytometry analysis of autophagy after fusing GFP or HaloTag as a reporter. However, in all of the above cases, the requirement for the foreign reporter severely limits its applicability to difficult-to-transfect cell lines or primary cells. In addition, the differential expression of foreign reporter genes in different cell individuals will also affect the detection effect of autophagy, such as adverse complications caused by overexpression. Isolation of fully viable and functionally active cell subpopulations with varying levels of autophagy based on autophagy-specific markers without the need for expression of exogenous reporter genes would be highly desirable but not currently achievable.

A powerful emerging technology in fluorescent materials is aggregation-induced emission (AIE), which refers to a unique phenomenon that when molecules are dissolved in solution, some molecules with twisted conformation and molecular rotators (or vibrator) fluorides are weakly emissive, but highly fluorescent as aggregates. Restriction of intramolecular motions (RIM), including restriction of intramolecular rotation (Restriction of intramolecular rotation, RIR) and restriction of intramolecular vibration (Restriction of intramolecular vibration, RIV), is considered to be aggregated AIE luminescence The main mechanism of fluorescence enhancement of body (AIEgens). The rapid development of AIE research in recent years has greatly promoted the application of AIEgens in the field of biomedicine, especially in sensing and imaging. It is worth noting that AIE probes with lysosomal targeting and mitochondrial targeting have been used to visually monitor autophagy and mitophagy, respectively, but these probes mainly target specific organelles and are not specific to the autophagic process, Nor is it used for cell sorting based on autophagy strength.

Contents of the invention

In view of this, the present invention aims at the above problems and provides a molecular probe for detecting autophagy based on the principle of aggregation-induced luminescence, as well as its preparation method and application.

The present invention is achieved through the following technical solutions:

An autophagy detection molecular probe (LKT) based on the principle of aggregation-induced luminescence, whose structure sequentially includes LC3 protein targeting peptide, cell-penetrating peptide and tetraphenylethylene (TPE) with aggregation-induced luminescence (AIE) properties.

Further, the LC3 protein targeting peptide is derived from the LC3 interaction region (LIR) of the p62/SQSTM1 protein (p62); the LC3 includes LC3A, LC3B, LC3C, GABARAP (GABA type A receptor-associated protein) and GABARAP L1/2.

Further, the sequence of the LC3 protein targeting peptide is DDDWTHL (abbreviated as L, SEQ ID NO.1); the motif of the cell-penetrating peptide is KKKKKKKKK (abbreviated as K, SEQ ID NO.2); The tetraphenylethylene is a propeller-shaped tetraphenylethylene molecule (1,1,2,2-Tetraphenylethylene, TPE).

The molecular structure of LKT of the present invention (see Figure 1 for the molecular pattern of LKT) is that one end is LC3 targeting peptide L, which can target LC3 family molecules; Fluorescent; the middle part is the K sequence, which can increase the water solubility of the overall probe and promote the probe to enter the cell. The molecular probe LKT for autophagy detection of the present invention will include characteristics such as cell internalization, specific interaction with LC3, aggregation-induced luminescence, no cytotoxicity, and no autophagy effect by itself. When co-incubating with cells, LKT molecules can pass through the cell membrane with the assistance of K, enter the cells, and combine with free LC3. At this time, due to the single-molecule binding state, TPE molecules emit low fluorescence. When cells induce autophagy, a large number of LC3 molecules gather on the autophagosome membrane to form aggregates. At this time, a large number of TPE molecules gather together and emit strong fluorescence. Therefore, the LKT probe can be used for the detection of autophagy.

The preparation method of the autophagy detection molecular probe based on the principle of aggregation-induced luminescence of the present invention comprises:

Weigh the peptide powder (azide-modified peptide LK) containing the LC3 protein targeting peptide and the cell-penetrating peptide sequence and the tetraphenylethylene molecule with a single alkyne group, and dissolve the two in a mixed solution of DMSO and water, well mixed;

A mixed solution containing CuSO ₄ and sodium ascorbate was added, and the resulting reaction solution was stirred at room temperature at 600 rpm for 24 hours, protected from light;

After 24 hours, the solution that has completed the reaction is filtered to obtain a clear liquid; the obtained clear liquid is separated to obtain a liquid containing the product;

The organic phase in the liquid containing the product is removed, and the obtained solution is freeze-dried to obtain a white powder, which is the product LKT.

Further, the optimal molar ratio of the cell penetrating peptide and the LC3 protein targeting peptide is 1:1.

Further, the volume ratio of DMSO and water in the mixed solution of DMSO and water is v/v=8:2.

Further, in the mixed solution, the final concentration of the sodium ascorbate is twice the final concentration of the CuSO ₄ .

The molecular probe for detecting cell autophagy of the present invention is applied in the fields of detection of cell autophagy, sorting of different autophagy levels, and the like.

Further, the application concentration of the autophagy detection molecular probe LKT of the present invention is 5-10 μM, and the optimum is 5 μM.

Further, the application and treatment time of the autophagy detection molecular probe LKT of the present invention is 0-48 hours, preferably 3-24 hours.

Beneficial effects of the present invention:

In view of the fact that the covalent binding of the cytoplasmic LC3 protein to the autophagosome membrane is a characteristic sign of autophagy, the AIE probe with the ability to specifically interact with LC3 is verified and utilized when binding to membrane-bound LC3 and free LC3 , would show sufficient fluorescence intensity differences to enable the sorting of cell subpopulations that promote different levels of autophagy.

The autophagy detection molecular probe (LKT) based on the principle of aggregation-induced luminescence of the present invention is an engineering molecular probe. The LKT molecular structure of the present invention is that one end is LC3 targeting peptide L, which can target LC3 molecules, and the other end is an aggregation-induced light-emitting molecule TPE, which can emit fluorescence when combined with LC3 and the movement is limited, and the middle section is cell penetration Peptide K sequence, which can increase the water solubility of the overall probe and facilitate the entry of the probe into the cell. The difference in the strength of autophagy can be visually observed under an optical microscope; the difference in the strength of autophagy can be monitored by means of flow cytometry; autophagy can be isolated from normal cultured cells or primary cell populations Cell subpopulations with differential levels reveal general heterogeneity in basal autophagy levels across multiple cell types. The molecular probe LKT for autophagy detection of the present invention has the characteristics of cell internalization, specific interaction with LC3, aggregation-induced luminescence, no cytotoxicity, and no autophagy effect by itself.

Specifically, the LKT of the present invention responds to the difference in the level of autophagy and exhibits the characteristics of "no light/weak light-enhanced light emission". When co-incubating with cells, LKT molecules can pass through the cell membrane with the assistance of K, enter the cells, and combine with free LC3. At this time, due to the single-molecule binding state, TPE molecules emit low fluorescence. When cells induce autophagy, a large number of LC3 molecules gather on the autophagosome membrane to form aggregates. At this time, a large number of TPE molecules gather together and emit strong fluorescence to realize the detection of cell autophagy, which is more specific.

When the internalized LKT in cells induces or blocks autophagy, it specifically interacts with LC3 protein to form fluorescent dots and colocalizes with GFP-LC3 dots. It has little toxicity and has no autophagy regulation activity itself. Importantly, the fluorescence enhancement of LKT upon binding to membrane-conjugated LC3, but binding to free LC3 did not show a significant increase in fluorescence. LKT can be achieved by targeting autophagy-specific molecules, does not require exogenous reporter gene expression, and only requires simple incubation with cells to separate living cells and functional cell subpopulations with different levels of autophagy.

Description of drawings

Fig. 1 is a schematic structural diagram of the autophagy detection molecular probe LKT of the present invention.

Fig. 2 is a schematic diagram of the reaction principle for the synthesis of the autophagy detection molecular probe LKT of the present invention.

Figure 3 shows the liquid chromatographic behavior of LKT at two different absorption wavelengths of 280nm and 350nm; the wavelength of Figure 3A is 280nm, and the wavelength of Figure 3B is 350nm.

Figure 4 The mass spectrometry identification spectrum of LKT.

Figure 5 is a diagram of the dynamic light scattering analysis results of LKT.

Figure 6 is a graph of the LKT results verified by UV-Vis spectroscopy.

Fig. 7 is a comparison result of the fluorescence of LKT in water and dry state on the film verified by ultraviolet-visible spectrum analysis.

Fig. 8 is a graph showing the fluorescence comparison results of LKT and aggregated TPE verified by ultraviolet-visible spectroscopy.

Fig. 9 is a graph showing the results of verifying the binding of LKT to LC3 protein by surface plasmon resonance technology.

Figure 10 is a diagram of the photostability analysis results of LKT.

Figure 11 is a fluorescence image of the binding of LKT to LC3 protein.

Figure 12 is the optical imaging of LKT in autophagy.

Figure 13 is LKT combined with flow cytometry for the detection of autophagy

In Figure 14, panels A and B are the results of flow cytometry analysis of MEF (panel A) and THP-1 (panel B) cells after the specified treatment for 24 hours (dose: LKT: 5 μM; 3-MA: 2.5 mM; trehalose: 0.1 M; mean±s.e.m.n=3; **p<0.01, ***p<0.001).

Panels C and D are Western blot results of LC3 in MEF (panel C) or THP-1 (panel D) cells after the indicated treatment for 24 hours (dose: LKT: 5 μM; 3-MA: 2.5 mM; trehalose : 0.1M).

Panel E is a Western blot of LC3 in HeLa cells treated with PBS, rapamycin (Rap, 1 μM) or chloroquine (CQ, 10 μM).

Figure 15 is a Western blot of LC3 in Hela cells treated with PBS or LKT (5 μM) for 24 hours; and the results of cell viability, NS: not significant.

In Fig. 16, panels A and B are the results of the viability of MEF (panel A) and THP-1 (panel B) cells treated with PBS or LKT (5 μM) for 24 hours, NS: not significant.

Panels C and D are Western blots of LC3 in MEF (panel C) and THP-1 (panel D) cells treated with PBS or LKT (5 μM) for 24 hours.

Figure 17 is a graph showing the experimental results of effectively separating cells with different levels of autophagy from cell populations with differential expression of LC3-II by LKT probe staining combined with flow cytometry technology.

Fig. 18 is a graph showing the experimental results of effectively separating cells with different autophagy levels from normal adherent cultured cell populations by LKT probe staining combined with flow cytometry technology.

Figure 19 is a diagram of the fluorescent cell distribution in the experiment of dividing different types of cells into cell subpopulations with different levels of autophagy by combining LKT probe staining with flow cytometry technology.

FIG. 20 is a western blot of LC3 of F-high and F-low cells in FIG. 19 .

Fig. 21 is a diagram showing the results of the sorting experiment of THP-1 cells differentiated by PMA combined with LKT and flow cytometry.

Figure 22 is a Western blot of NLRP3 from treated THP-1 cells.

In Fig. 23, Fig. 23A is a statistical diagram of IL-1β secretion of treated THP-1 cells; Fig. 23B is a statistical diagram of LDH release of treated THP-1 cells.

Fig. 24 is a graph showing the experimental results of sorting DCs incubated with LKT.

Fig. 25 is a graph showing the results of cell migration experiments of treated DCs cells.

Fig. 26 is a graph showing the statistical results of IL-12P70 of treated DCs cells.

Detailed ways

In order to better illustrate the problems to be solved by the technical solutions of the present invention, the technical solutions adopted and the beneficial effects achieved, further elaborations will now be made in conjunction with specific embodiments. It is worth noting that the technical solutions of the present invention include but are not limited to the following embodiments.

If no specific technique or condition is indicated in the embodiment of the present invention, it shall be carried out according to the technique or condition described in the literature in this field or according to the product specification. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products that could be obtained from the market or other channels.

Example 1 The molecular probe for detecting autophagy based on the principle of aggregation-induced luminescence of the present invention and its preparation method.

1.1 Molecular pattern of LKT probe.

The molecular probe LKT for autophagic cell classification has the characteristics of cell internalization, specific interaction with LC3, aggregation-induced luminescence, no cytotoxicity, and no autophagic effect by itself. LKT contains cell penetrating motif K (sequence shown in SEQ ID NO.2: KKKKKKKKK), LC3 interaction region (LIR, abbreviated as L, sequence shown in SEQ ID NO.1) of p62/SQSTM1 protein (p62) : DDDWTHL) and propeller tetraphenylethylene molecules (1,1,2,2-Tetraphenylethylene, TPE). In addition to facilitating cellular uptake, the K motif increases the solubility of LKT in aqueous solutions. The molecular model of LKT is shown in Figure 1.

1.2. Synthesis of LKT:

Weigh the polypeptide powder of the fusion peptide LK (10 mg, 4.2 μmol) containing the LC3 protein targeting peptide and the cell-penetrating peptide sequence and the tetraphenylethylene molecule (4.49 mg, 12.6 μmol) with a single alkyne group, respectively. Dissolve in 0.8mL DMSO and water mixed solution (v/v=8:2), mix well, add 0.2mL CuSO ₄ (0.42mg, 2.1μmol) and sodium ascorbate (0.83mg, 4.2μmol) mixed solution . The reaction solution was stirred at room temperature at 600 rpm for 24 hours, protected from light. The next day, the completed reaction solution was filtered to obtain a clear liquid, which was then separated by high performance liquid chromatography. Agilent 1260 Infinity LC system (California, USA) was used for separation, 0.1% TFA/acetonitrile and 0.1% TFA/H2O were used as two solvents as mobile phases, and the chromatographic column model used was ZORBAX SB-C18, 5mm (Agilent) , the preparation conditions are: flow rate=2mL min-1, 0min: 5% ACN, 8min: 50% ACN, 15min: 50% ACN, 20min: 95% ACN, 25min: 100% ACN. After the preparation is completed, a liquid containing the product is obtained, and the liquid is placed in a rotary evaporator for rotary evaporation to remove the organic phase, and the remaining part is water. The solution is frozen and then freeze-dried to obtain a white powder, which is the product LKT. After weighing, 6.9 mg of the product was obtained, and the calculated yield was 55%. The schematic diagram of the synthesis of LKT is shown in Figure 2.

1.2. Characterization of LKT.

LKT high performance liquid chromatography analysis:

The LKT aqueous solution of 1mg/mL was prepared and analyzed by high performance liquid chromatography (High Performance Liquid Chromatography, HPLC). The absorption wavelength is detected, and the results obtained are shown in Figure 3 (the wavelength of Figure 3A is 280nm, and the wavelength of Figure 3B is 350nm). It can be known that the prepared LKT product has obvious absorption at two wavelengths, and there is no miscellaneous peak, showing The product has high purity.

Mass spectrometric analysis of LKT:

In order to detect whether the product we obtained was pure LKT, it was detected by MS, and the instrument used was a Waters ZQ2000 mass spectrometer (Waters, USA). The 1 mg/mL product was prepared and detected in 50% acetonitrile solution, and the obtained results are shown in FIG. 4 . The results show that there is an obvious peak at 913.19, and the relative intensity of the ion current reaches 100%. By calculation, the theoretical [M+H] ³⁺ is 913.09, and the MS result is 913.19, in line with the expected value.

Dynamic Light Scattering Analysis of LKT:

Dynamic light scattering analysis was performed using a particle size analyzer (Litesizer 500, Austria). Dynamic light scattering analysis of TPE-yne, LKT and LK in DMSO/water (1:199 V/v) mixed solution. The results are shown in Figure 5, LKT did not form particles of any size in water, indicating that it existed as a single molecule, while the aggregation size of TPE was close to 1000nm, which showed that LKT had better water solubility.

Spectral analysis of LKT:

The properties of LKT were verified by UV-Vis spectroscopic analysis (Fig. 6). Consistent with the expected AIE properties, LKT was hypofluorescent in water but became hyperfluorescent when deposited on thin films (as shown in Fig. 7), whereas the aggregated TPE is already highly fluorescent in water (Figure 8).

Surface plasmon resonance technology to verify the binding specificity of LKT and LC3 protein:

The GE BIAcore 8K instrument was operated at a constant temperature of 25 °C, and the CM5 sensor chip (GE Healthcare) was used in this study. Each CM5 sensor chip consists of 8 identical experimental channels, and each channel is divided into two flow cells. In our experimental setup, flow cell 1 (Fc1) was always kept blank as a reference, while flow cell 2 (Fc2) was functionalized with LC3 for interaction studies with LKT. Specifically, the system was first equilibrated with PBS-T buffer (20 mM sodium phosphate, 150 mM sodium chloride and 0.05% Tween 20, pH 7.4). The sensor chip was activated with a mixture of EDC (0.2M) and NHS (0.05M) for 6 minutes. In Fc2, 40 μM LC3 in 10 mM acetate buffer (pH 5.5) was subsequently injected for 7 min while PBS-T buffer was injected in Fc1. Finally, 1 M ethanolamine HCl solution was injected onto Fc1 and Fc2 to block the remaining NHS ester groups. Monitor sensorgrams online to ensure successful immobilization of LC3 on Fc2. As expected (Fig. 9), LKT interacted with purified LC3 protein with a Kd of approximately 0.312 ± 0.051 μΜ as determined by surface plasmon resonance. This interaction is dependent on the LIR motif, since the KT (containing K,TPE) probe, without LIR, loses the ability to bind LC3.

Photostability analysis of LKT:

After the cells were incubated with LKT (5 μM) for 6 h, the photostability of LKT in HeLa/GFP-LC3 cells was measured for 50 cycles with 5% laser power under excitation at 405 nm. As shown in Figure 10, the results showed that LKT also exhibited good photostability after internalization into cells, showing minimal fluorescence weakening after repeated excitation.

Microplate binding of LKT to LC3 protein:

Purified LC3 protein was added to the 96-well plate in PBS and left overnight at 4 °C. After removing the protein solution, each well was washed 5 times with bovine serum albumin (BSA, 0.05% w/v). Add LKT (10 μM) or KT (10 μM) to LC3 protein-coated wells incubated at 37° C. for 1 h. Fluorescent images (Figure 11) were observed and captured by a Nikon Ti-E microscope using the DAPI channel. Precoating LC3 (but not BSA) was able to "pull down" LKT (but not KT) molecules in solution, exhibiting aggregation-induced luminescence. These results confirm that LKT is an AIE probe with relatively high affinity to LC3.

Example 2 Application of molecular probes for autophagy detection based on the principle of aggregation-induced luminescence in the present invention

2.1 LKT for optical observation and imaging of autophagy:

Both induction and blockade of autophagy lead to increased autophagosome accumulation, thereby increasing the binding of LC3 to autophagosomal membranes. In fact, the autophagy inducers rapamycin, trehalose and blocker chloroquine (CQ), but not the inhibitor 3-methyladenine (3-MA), were activated in HeLa cells stably expressing GFP-LC3 in HeLa/ GFP-LC3 resulted in prominent green spot formation (Fig. 12A). Co-treatment of these cells with LKT also produced blue punctate spots (Fig. 12A), which significantly co-localized with GFP-LC3 punctate spots, as indicated by the Pearson correlation coefficient (PCC) values of rapamycin, trehalose, and CQ treatments, respectively. are shown as 0.47, 0.66 and 0.69 (Fig. 12A). Furthermore, the time course of LKT puncta formation was in good agreement with that of GFP-LC3 puncta after CQ treatment (Fig. 12B). These results suggest that LKT has the potential to replace GFP-LC3 as a more convenient reporter for monitoring autophagy occurrence. It should be noted that the LIR motif of LKT is derived from the p62 protein, and in addition to the form LC3B of LC3 in GFP-LC3, it can also interact with different members of the LC3 family, including LC3A, LC3B, LC3C, GABARAP (GABA type A receptor body-associated protein) and GABARAP L1/2. Therefore, in theory, LKT could provide a wider coverage of autophagosome visualization than GFP-LC3.

2.2 LKT combined with flow cytometry for detection of autophagy:

The ability to emit enhanced fluorescence upon binding to liposome-bound LC3 suggests that LKT exhibits higher fluorescence in cells containing more lipidated LC3, both under autophagy induction and blockade. In fact, HeLa cells treated with trehalose, an inducer of autophagosome formation, increased LC3 conversion as expected (Fig. 13A), and also showed higher LKT fluorescence under flow cytometry analysis ( Figure 13B). In contrast, 3-MA, an inhibitor of autophagosome formation, significantly reduced LC3 conversion and LKT fluorescence enhancement induced by trehalose ( FIGS. 13A and 13B ). Similar enhancement of LKT fluorescence by trehalose and decrease of LKT fluorescence by 3-MA were observed in mouse embryonic fibroblasts (MEFs) and non-adherent undifferentiated THP-1 cells (Figures 14A and 14B, respectively), LC3 Western blotting confirmed the autophagy-inducing and autophagy-inhibiting effects of trehalose and 3-MA, see Figure 14C and Figure 14D, respectively. Similar to the results in HeLa cells (see Figure 15), LKT exhibited minimal toxicity and no autophagy modulating activity in MEF and THP-1 cells (Figure 16, A to D). Notably, enhanced fluorescence of LKT, but not the AIE probe LKR, was observed in HeLa/GFP-LC3 cells after trehalose treatment (Fig. 13C), suggesting that AIE properties are critical for the responsiveness of LKT to induction of autophagy very important. Basically consistent with the reported results (Shvets, E., Fass, E. & Elazar, Z. Utilizing flow cytometry to monitor autophagy in living mammalian cells. Autophagy 4,621-628, doi:10.4161/auto.5939 (2008)), in trehalose Upon induction of autophagy, GFP-LC3 showed diminished fluorescence, although only a small change was observed in our case (Fig. 13C), probably because of the variation in GFP-LC3 expression levels in laboratory-maintained HeLa/GFP-LC3 cells. Heterogeneity. Significant increases in LKT fluorescence were also observed after treatment of HeLa cells with CQ and rapamycin, with CQ causing a more profound enhancement than rapamycin (Fig. 13D and Fig. 14E). To rule out the possibility that the enhanced LKT fluorescence was due to autophagy modulator-induced increased internalization of LKT cells, we added CQ to LKT-pretreated HeLa cells after removing LKT in the medium. Six hours after CQ addition, an increase in LKT fluorescence was observed, similar to that observed with LKT and CQ co-treatment (Fig. 13E), and it is strongly believed that the enhanced LKT fluorescence reflects increased LC3 turnover, but not LKT cellular uptake. Taken together, these results suggest that LKT exhibits enhanced fluorescence in cells due to its AIE properties, while LC3 lipidation increases due to autophagy induction or blockade.

2.3 LKT probe staining combined with flow cytometry sorting can obtain subpopulations of living cells with different levels of autophagy.

The same amount of HeLa cells treated with LKT alone for 24 hours and HeLa cells treated with LKT+CQ for 24 hours were mixed and sorted by flow cytometry, as shown in Figure 17, F-high and F-low represent the highest and lowest 25% of LKT fluorescence, respectively Cells in which the concentration of LKT was 5 μM and CQ was 10 μM, the right panel is LC3 western blot of F-high and F-low cells. The LC3-II content of F-high cells was significantly higher than that of F-low cells, indicating that LKT can effectively separate cells with different levels of autophagy from cell populations with differential expression of LC3-II.

An equal amount of HeLa cells treated with LKT alone for 24 hours was subjected to flow cytometry sorting, as shown in Figure 18, F-high and F-low represent the 25% cells with the highest and lowest LKT fluorescence, respectively, where the concentration of LKT is 5 μM, the right figure is LC3 Western blot of F-high and F-low cells. The LC3-II content of F-high cells was significantly higher than that of F-low cells, indicating that LKT effectively separates cells with different levels of autophagy from normal adherent cultured cell populations.

MEF, THP-1 (undifferentiated), MCF-7, and BMDM cells incubated with LKT (5 μM) for 6 hours were subjected to flow sorting. As shown in Figure 19, F-high and F-low represent the 25% cell fragments with the strongest and weakest LKT fluorescence, respectively. Figure 20 is the LC3 western blot of F-high and F-low cells. Indicates that heterogeneity in the level of basal autophagy is ubiquitous in cultured cells, including adherent cultured cells, suspension cultured cells, and primary cells, and that LKT reliably separates different types of cells into cells with different levels of autophagy subgroup.

2.4 LKT probe incubation combined with flow cytometry to separate autophagy-high cells and autophagy-low cell subpopulations revealed a significant effect of basal autophagy levels on NLRP3 inflammasome activation in THP-1 cells.

After incubation with LKT (5 μM) for 6 hours, PMA-differentiated THP-1 cells were sorted by LKT flow cytometry. Referring to Figure 21, F-high and F-low represent the 25% cells with the strongest and weakest fluorescence, respectively. Right panels are LC3 western blots of F-high and F-low cells. The results showed that there were differences in autophagy levels in THP-1 cells differentiated by PMA stimulation.

The F-high and F-low THP-1 cells sorted according to Figure 21 were treated with PBS or LPS (100ng/mL) for 3 hours, and the protein level of NLRP3 was verified by Western blot, see Figure 22, autophagy was found Strong THP-1 cells have higher expression of NLRP3.

Treat F-high and F-low THP-1 cells with PBS and LPS for 3 hours or LPS for 3 hours and add nigericycin to stimulate 0.5 hours, then collect the supernatant and detect IL-1β secretion (Figure 23A) and LDH release ( FIG. 23B ), the release of IL-1β from F-high cells was 3 times that of F-low cells, and the release of LDH was 2.8 times that of F-low cells.

2.5 LKT probe incubation combined with flow cytometry to separate autophagy-high cells and autophagy-low cell subpopulations, revealing the effect of basal autophagy levels on the function of human monocyte-derived dendritic cells.

Human monocyte-derived dendritic cells (Dendritic cells, DCs) on the 6th day after differentiation were sorted after incubation with LKT (5μM) for 6h. Referring to Figure 24, F-high and F-low represent the 25% cells with the highest and lowest LKT fluorescence, respectively. Among them, the right panel is the LC3 western blot of F-high and F-low cells, and the results show that there are also differences in autophagy in these cells.

Human monocyte-derived dendritic cells (Dendritic cells, DCs) on the 6th day after differentiation were sorted after incubation with LKT (5μM) for 6h. F-high and F-low represent the 25% cells with the highest and lowest LKT fluorescence, respectively. Cell migration experiments were performed after 24 hours of LPS stimulation, see Figure 25, the left picture shows the number of cells migrating to the bottom of the well plate under the microscope, and the right picture shows the quantified number of cells, and the cell migration ability of F-high is obvious Cells below F-low.

Human monocyte-derived dendritic cells (Dendritic cells, DCs) on the 6th day after differentiation were sorted after incubation with LKT (5μM) for 6h. F-high and F-low represent the 25% cells with the highest and lowest fluorescence of LKT respectively. They were centrifuged after 24 hours of LPS stimulation, and the cell supernatant was collected. See Figure 26. ELISA was used to detect the concentration of IL-12P70, and the secretion of F-high cells The capacity of IL-12P70 was decreased by 2.7 times.

The above-mentioned embodiments only express several implementation modes of the present invention, and the description thereof is relatively specific and detailed, but should not be construed as limiting the patent scope of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention should be based on the appended claims.

Claims (10)

1. A molecular probe for autophagy detection based on the principle of aggregation-induced luminescence, characterized in that the structure of the molecular probe includes LC3 protein targeting peptide, cell penetrating peptide and tetraphenylethylene in sequence.
2. The autophagy detection molecular probe based on the principle of aggregation-induced luminescence according to claim 1, wherein the LC3 protein targeting peptide is derived from the LC3 interaction region of the p62/SQSTM1 protein; the LC3 includes LC3A, LC3B, LC3C, GABARAP or GABARAP L1/2.
3. The molecular probe for detecting autophagy based on the principle of aggregation-induced luminescence according to claim 1, wherein the sequence of the LC3 protein targeting peptide is shown in SEQ ID NO.1; the cell-penetrating peptide The motif is shown in SEQ ID NO.2; the tetraphenylethylene is a propeller-shaped tetraphenylethylene molecule.
4. The preparation method of the molecular probe for detecting autophagy based on the principle of aggregation-induced luminescence according to any one of claims 1-3, characterized in that, the method for preparing the molecular probe for detecting autophagy based on the principle of aggregation-induced luminescence include:

   Weigh the peptide powder containing the LC3 protein targeting peptide and the cell penetrating peptide sequence and the tetraphenylethylene molecule with a single alkyne group, dissolve the two in a mixed solution of DMSO and water, and mix well;

   A mixed solution containing CuSO ₄ and sodium ascorbate was added, and the resulting reaction solution was stirred at room temperature at 600 rpm for 24 hours, protected from light;

   Filtrating the reacted solution to obtain a clear liquid; performing a separation operation on the obtained clear liquid to obtain a product-containing liquid;

   The organic phase in the liquid containing the product is removed, and the obtained solution is freeze-dried to obtain a white powder, which is the product of a molecular probe for detecting autophagy based on the principle of aggregation-induced luminescence.
5. The method for preparing a molecular probe for autophagy detection based on the principle of aggregation-induced luminescence according to claim 4, wherein the molar ratio of the cell-penetrating peptide and the LC3 protein targeting peptide in the polypeptide powder is 1:1 .
6. The method for preparing molecular probes for autophagy detection based on the principle of aggregation-induced luminescence according to claim 4, wherein the volume ratio of DMSO and water in the mixed solution of DMSO and water is v/v=8:2 .
7. The preparation method of the autophagy detection molecular probe based on the principle of aggregation-induced luminescence according to claim 4, characterized in that, in the mixed solution, the final concentration of the sodium ascorbate is the final concentration of _CuSO4 2 times.
8. The application of the autophagy detection molecular probe based on the principle of aggregation-induced luminescence according to any one of claims 1-3 in the detection of cell autophagy or in the sorting of different autophagy levels.
9. The application of molecular probes for autophagy detection based on the principle of aggregation-induced luminescence according to claim 8, characterized in that the application concentration of the molecular probes is 5-10 μM.
10. The application of the molecular probe for autophagy detection based on the principle of aggregation-induced luminescence according to claim 8, characterized in that the application and processing time of the molecular probe is 0-48 hours.

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