CN112851575A

CN112851575A - Ratio type lysosome pH probe with aggregation-induced emission property and application

Info

Publication number: CN112851575A
Application number: CN201911101156.8A
Authority: CN
Inventors: 唐本忠; 石秀娟; 牛广乐; 郭子健
Original assignee: Hong Kong University of Science and Technology HKUST; HKUST Shenzhen Research Institute
Current assignee: Hong Kong University of Science and Technology HKUST; HKUST Shenzhen Research Institute
Priority date: 2019-11-12
Filing date: 2019-11-12
Publication date: 2021-05-28
Anticipated expiration: 2039-11-12
Also published as: CN112851575B

Abstract

The present invention relates to a simple method for the preparation of luminophores (AIEgens) that test the pH of lysosomes and have aggregation-induced emission (AIE). The present invention relates to a ratiometric pH probe that can test for pH in the maximum range of pH between 3 and 6.5. The AIE luminophores provided by the invention can quantitatively test the pH of lysosomes in cells in vitro, and can also test the pH of lysosomes in living small fish. The AIE luminophor provided by the invention can also track the detailed change process of the average lysosome pH and the distribution condition of the lysosome pH in the regeneration process of the small fish tail fin for a long time, and provides a simple, convenient and effective method for observing the regeneration process.

Description

Ratio type lysosome pH probe with aggregation-induced emission property and application

Technical Field

The invention relates to a simple method for preparing a luminophore (AIEgenes) for testing the lysosome pH and having aggregation-induced emission (AIE), to the use of said luminophore as a pH probe of the ratiometric type, and to the use of said luminophore for long-term tracking of the lysosome pH changes during the regeneration of the tail fins of small fish.

Background

Tissue regeneration refers to the process by which an organism undergoes self-renewal and self-repair following injury to regrow a damaged or lost body part. Although humans have some regenerative capacity, for example, skin, fingertips, endometrium and liver can regenerate naturally, this capacity is dwarfed by the regenerative capacity of vertebrates such as lizards, zebrafish, xenopus laevis, etc. In order to promote the development of regenerative medicine, researchers have made many efforts in studying biological processes associated with tissue regeneration using vertebrate transgenic models. Among them, zebrafish and medaka are vertebrate models that are widely used because they can regenerate many organs, for example, the central nervous system, heart, kidney, and especially their tail fins can have unlimited regeneration potential after amputation. And they are excellent transgenic models that can be used to understand the mode of occurrence and the cause of tissue regeneration. In addition, they are easy to raise in the laboratory and have a transparent body in the early stages of life, so that in vivo tissues can be easily imaged in real time without sacrificing the subject.

To study the biological processes in tissue regeneration, the most popular approach for biologists is to use transgenic fluorescent fish with fluorescent protein-tagged proteins. Although transgenic technology of fluorescent protein fusion offers the possibility to study protein kinetics in living cells. However, this technique is complex, time consuming, expensive and prone to photobleaching. Furthermore, the macromolecular size of the fluorescent protein may affect the function of the fusion protein of interest. Another popular method of investigation is fluorescence immunohistochemical staining. It requires the fixation and permeabilization of the cells, which may lead to some drawbacks, such as protein extraction or translocation, blocking of protein epitopes and inability to image in real time, thus possibly leading to information on some biological processes that are erroneous or missing. Therefore, a simple method is urgently required. The method allows direct observation of the tissue regeneration process in real time to further validate the biological changes that are generally accepted but not fully revealed in the caudal fin regeneration process.

Lysosomes are "waste disposal systems" for cells. As they are responsible for intracellular degradation of substances (e.g., damaged organelles, used proteins, DNA, phospholipids, etc.) and release metabolites and ions to maintain cellular homeostasis. Therefore, they are involved in many important cellular processes and disease pathogenesis. Among the most important functions of lysosomes are their involvement in autophagy, a process mediated by the lysosome in the autophagy of eukaryotic cells, which is essential for tissue regeneration. Lysosomal pH is an important parameter indicative of lysosomal hydrolase activity and lysosomal functional status. To perform the normal function of lysosomes, the pH of the lysosome must be maintained within the acidic range of pH 4-5. This is because most lysosomal hydrolases of all classes have optimal activity at acidic pH values. In some cases, if the pH of the lysosome is not considered, erroneous conclusions may be drawn about the biological phenomenon. For example, an increase in the fluorescent protein-labeled LC3-II (tubulin light chain 3) protein recruited to the autophagosome membrane does not necessarily imply an increase in autophagy flux, as this may be due to blockade of lysosomal alkalinization leading to degradation of autophagosomes. Therefore, tracking lysosomal pH changes in real time can lead to a more comprehensive understanding of events in the tissue regeneration process, thereby promoting the development of regenerative medicine.

Probes suitable for use in tracking lysosomal pH during tissue regeneration need to meet the following requirements: (1) good light stability; (2) long-term tracking ability; (3) targeting lysosomes; (4) is sensitive to pH; (5) quantitatively measuring the pH; (6) in vivo non-invasive imaging.

Intensity-based fluorescent lysosomal pH probes are affected by many factors, including variations and maldistribution of probe concentration, temperature, environmental polarity, and fluctuations in excitation light. In contrast, ratiometric probes can overcome systematic errors and quantify more accurately. Conventional ACQ probes have a problem of fluorescence quenching at high concentrations or in an aggregated state due to strong pi-pi stacking, and thus, in biological applications, conventional ACQ probes are often suggested to be used at low concentrations. But this presents other problems such as being easily photobleached and not suitable for long-term tracking applications. In addition, their smaller stokes shift leads to strong self-absorption. The more AIE material compared to ACQ material, the brighter it is. Research over recent decades has demonstrated that AIEgens have high light stability, large stokes shift, excellent long-term trackability and good biocompatibility. Although research on tissue regeneration and lysosomes has been of great interest, there has been no study for monitoring lysosomal pH changes in real time during tissue regeneration in vivo. Therefore, there is an urgent need to develop ratiometric AIEgen probes for long-term follow-up of lysosomal pH changes during tissue regeneration in vivo.

Disclosure of Invention

In one embodiment, the present invention provides a luminophore exhibiting aggregation-induced emission, the luminophore comprising the following structure:

r1 and R2 are each independently

Any one of them.

In one embodiment, the present application is directed to a aggregation-inducing luminophore (AIEgen) comprising: piperazine groups target lysosomes, pyridine groups act as pH-responsive groups, and AIEgen exhibits aggregation-induced emission properties.

Drawings

FIG. 1 shows CSPP¹H NMR spectrum;

FIG. 2 shows CSPP¹³C NMR spectrum;

FIG. 3 shows a high resolution mass spectrometry (MALDI-TOF) plot of CSMPP;

FIG. 4a shows a single crystal structure of CSMCPP; FIG. 4b shows CSMCPP as a function of water volume fraction (f) in an acetonitrile/water mixed solvent_w) A PL spectrum resulting from the change in (c); FIG. 4c shows α_AIEAnd f_wAlpha is I/I₀I and I₀Are respectively represented inHaving f_wIn a mixed solvent of acetonitrile/water and in pure acetonitrile at 509nm, the concentration of CSMPP is 10. mu.M, the excitation wavelength is 365nm, and the inset in FIG. 4c is under a hand-held UV lamp at 365nm, f_w0% and f_wFluorescence photograph of 90% CSMPP solution, the molecular structure of CSMPP is inserted in fig. 4 c;

FIG. 5a shows the PL spectra of CSMPP in glycerol/ethylene glycol mixed solvents of different volume fractions of glycerol; FIG. 5b shows I/I₀Plot of volume fraction of glycerol, I and I₀Represents the PL intensity at 494nm in a glycerol/ethylene glycol mixed solvent with a certain glycerol volume fraction and in pure ethylene glycol, respectively, the concentration of CSMPP is 10 μ M, and the excitation wavelength is 365 nm;

FIG. 6 shows intermolecular interactions in a CSMP single crystal structure;

FIG. 7 shows the reaction in DMSO-d₆Before and after addition of excess deuterated hydrochloric acid solution (2.5 equivalents) to the solution, CSPP¹H NMR spectrum;

FIG. 8 shows electron cloud profiles of HOMO and LUMO based on Frontier molecular orbitals optimized by M062X/6-31G (d, p) levels before and after CSMCP acidification;

FIG. 9a shows fluorescence emission spectra of CSMPP (10. mu.M) in buffer solution (containing 2% DMSO) at pH 2.60-6.80; FIG. 9b shows the fluorescence intensity ratio (I)₆₁₅/I₅₀₃) Graph with pH; the fluorescence pictures inserted in FIG. 9b are CSPP fluorescence pictures in different pH solutions under excitation of a hand-held 365nm UV lamp; FIG. 9c shows the ratio I₆₁₅/I₅₀₃)，R_min、R_maxA pH calibration curve is fitted with the relationship between the pH and the pH; FIG. 9d shows the reversibility of CSMCPP response to pH 7 and pH 3, showing PL intensity ratio (I)₆₁₅/I₅₀₃) Changes with the number of reciprocating cycles of pH; fig. 9e shows the interference of probe signals by different chemical species in buffers at pH 6.8 and pH 3.0, chemical species: (1) blank; (2) ca²⁺；(3)Mg²⁺；(4)Cd²⁺；(5)Co²⁺；(6)Fe²⁺；(7)Fe³⁺；(8) Ni²⁺；(9)Cu²⁺；(10)Al³⁺；(11)Zn²⁺；(12)Ag⁺；(13)Mn² ⁺；(14)Pb²⁺；(15) K⁺；(16)Na⁺；(17)I^-；(18)HPO₄ ^2-；(19)H₂PO₄ ^-；(20)Ac^-；(21)NO₃ ^-； (22)CO₃ ^2-；(23)ClO^-；(24)HS^-；(25)SCN^-；(26)S₂O₃ ^2-；(27)H₂O₂(ii) a (28) Tryptophan; (29) (ii) glutamine; (30) aspartic acid; (31) glycine; (32) leucine; (33) valine; (34) (ii) cysteine; (35) homocysteine; (36) glutathione; (37) arginine; (38) serine; (39) glucose; and (3) testing conditions are as follows: the probe concentration was 10. mu.M; the concentration of the reagent 1-14 is 0.2 mM; the concentration of the reagent 15-39 is 1 mM; all anions of the cation being Cl^-But NO^3-For Ag⁺、Mn²⁺And Pb²⁺An anion of (a); all anions have a cation of Na⁺，λ_exThe fluorescence emission at wavelengths of 503nm and 615nm was recorded at 365 nm;

FIG. 10 shows a plot of fluorescence intensity at 615nm as a function of pH; when PL intensity was changed by half, the corresponding pH represented pK_aThe pK calculated therefrom_a4.75 +/-0.02;

FIG. 11 shows the absorption spectrum after normalization of CSMP in buffers at pH 6.8 and pH 2.6, and the corresponding structural formula of CSMP at pH 6.8 and pH 2.6;

FIG. 12 shows confocal images of laser staining of HeLa cells with 200nM LysoTracker Red (LTR) for 10 min followed by co-staining with 2 μ M CSPP for 10 min; FIG. 12a is a fluorescence image of CSMCPP; FIG. 12b is a fluorescent picture of LTR; FIG. 12c is a picture of a and b merged; FIG. 12d is a picture with a and b merged with a bright field; FIG. 12e is the signal distribution of the green channel of CSMPP and the red channel of LTR; FIG. 12f is a fluorescence intensity distribution curve extracted from the white arrow in FIG. 12 c; and (3) testing conditions are as follows: for CSMCP, lambda_ex＝405nm，λ_em580nm ═ 450-; for LTR, λ_ex＝560 nm，λ _em650 and 700 nm. Scale bar is 10 μm;

FIG. 13 shows confocal images of ARPE-19 cells stained with 240nM LysoTracker Blue (LTB) for 10 min, then co-stained with 1.5. mu.M CSMPP for 10 min; FIG. 13a is a fluorescent photograph of CSMCP; FIG. 13b is a fluorescence image of LTB; FIG. 13c is a picture with a and b merged; FIG. 13d is a picture with a and b merged with a bright field; FIG. 13e is a signal distribution plot of the red channel of CSMCPP and the green channel of LTB; FIG. 13f is a fluorescence intensity distribution curve extracted from the white arrow in FIG. 13 c; and (3) testing conditions are as follows: for CSMCP, lambda_ex＝405nm，λ_em480 nm. For LTB, λ_ex＝ 405nm，λ_em400-; scale bar is 10 μm;

figure 14 shows cell motility diagrams of (a) HeLa and (b) ARPE-19 cells after 24h incubation with different concentrations of CSMPP, giving data as mean ± SD (n ═ 4);

FIG. 15 shows (a) laser confocal images, λ, of the whole body of a medaka juvenile fish after being fed with 5 μ M CSMPP for 4 hours_ex＝405nm，λ_em468-; (b) after CSMCPP (5 mu M) and deep red LysoTracker (LTDR, 200nM) are used for feeding the medaka juvenile fish together, laser confocal images of tail fins of the medaka juvenile fish are sequentially fluorescence images of CSMCPP and LTDR, merged images of two channels and a bright field, merged images of two channels, amplified images cut from the merged images of the two channels and signal distribution maps of the two channels; and (3) testing conditions are as follows: for CSMCP, lambda_ex＝405nm，λ_em468 and 630 nm; for LTDR, λ_ex＝633nm，λ_em647 and 713 nm; the scale bar is 50 μm. The scale bar of the magnified image was 10 μm;

fig. 16 shows the biocompatibility of CSMPP for medaka juvenile fish: (a) survival measured after exposing juvenile fish to different concentrations of CSMPP for 96 hours; (b) mean heart beat of young fish at different times after feeding 5 μ M CSPP for 4 hours; each concentration treatment consisted of three replicate samples, each sample containing 10 individual juvenile fish, the heartbeat of 15 juvenile fish being recorded each time, the data being mean ± SD;

FIG. 17 shows (a) CSMPP, LTR and LThe change in fluorescence signal of TG-stained HeLa cells after continuous scanning with CLSM was mean ± SD (n ═ 4); (b) fluorescence pictures of HeLa cells stained for 12 min with 2 μ M CSMPP at the 1 st and 100 th scans; (c) fluorescence pictures of HeLa cells stained with 500nM LTR for 6 min and (d) with 500nM LTG for 5 min at 1 st and 50 th scans; for CSPP: lambda [ alpha ]_ex＝405nm；λ _em470 and 650 nm; for LTR: lambda [ alpha ]_ex＝561nm，λ _em565 and 650 nm; for LTG: lambda [ alpha ]_ex＝ 488nm，λ_em495 and 580nm, and the scale bar is 20 μm;

FIG. 18 shows (a) a standard curve of HeLa cells photographed using CLSM with 63 fold oil lens and in which probes were tested; first staining the cells with 3. mu.M CSPP for 1h at 37 ℃ and then equilibrating the cells in pH buffer containing 12. mu.M nigericin and 5. mu.M monensin for 8 min at 25 ℃ to capture the fluorescence images of the green channel (470-560nm) and the red channel (560-700nm) of HeLa cells in different pH buffers; ratio images were acquired by dividing the red channel by the green channel using Image J software, with a scale bar of 20 μm; (b) e_m-red/E_m-greenThe relation between the average ratio and the pH value, and the average value +/-SD is obtained from the three pictures; in addition, according to the average ratio, R_min、R_maxAnd the relationship between the pH value and the pH value is fitted to obtain a calibration curve;

fig. 19 shows CLSM pictures after HeLa cells were stimulated with chemicals, including bright field, green channel, red channel, merged two-channel pictures, and distribution pictures of lysosomal pH; pictures are sequentially normal lysosomes without irritant effects; 50nM of effected lysosomes incubated with broomcorn A1 for 30 min at 37 ℃; 0.1mM H₂O₂Lysosomes after incubation at 37 ℃ for 30 minutes; lysosomes after incubation with 40 μ M Tamoxifen (TMX) for 15 minutes at 37 ℃; lysosomes after 8 min incubation with 1.75mM and 10mM acetic acid (HAc) at 37 ℃; analyzing the confocal fluorescence images of the red and green channels by Image J software to obtain ratio images; the scale bar is 20 μm, the excitation wavelength is 405nm, and the green channel of 470-560nm and the red channel of 560-700nm are collectedA fluorescent picture;

FIG. 20 shows (a) standard curves of zebrafish cells photographed using a CLSM with a 40-fold oil mirror and in which probes were tested; cells were first stained with 3. mu.M CSPP for 1h at 37 ℃ and then equilibrated at 25 ℃ for 8 min in pH buffer containing 12. mu.M nigericin and 5. mu.M monensin, lambda_ex405 nm; green channel lambda_em416 and 555 nm; red channel lambda_em557 + 704 nm; ratio images were acquired by dividing the red channel by the green channel using Image J software, scale bar 50 μm; (b) e_m-red/E_m-greenThe relation between the average ratio and the pH value, and the average value +/-SD is obtained from the three pictures; in addition, according to the average ratio, R_min、R_maxAnd the relationship between pH was fitted to a calibration curve.

Fig. 21 shows laser confocal pictures of medaka juvenile tail fins before and after amputation at different times (12, 24, 48, 96 and 120 hpa), including brightfield, green channel, red channel, merged two-channel pictures, and distribution pictures of lysosomal pH; lambda [ alpha ]_ex405 nm; green channel lambda_em416 and 555 nm; red channel lambda_em557 + 704 nm; analysis of E using Image J software_m-red/E_m-greenThe ratio image of (1); hours after amputation abbreviated hpa; the white dotted line represents the amputation plane; the light blue dotted line represents the small fin profile with a scale bar of 50 μm.

Fig. 22 shows the average pH change of lysosomes in medaka juvenile fish during tail fin regeneration; as a control to the amputated medaka juvenile fish, the non-amputated medaka juvenile fish takes the same time as it takes after being fed CSMPP; the times shown here are named according to the time after amputation; "0 h" refers to the time after CSMP feeding until amputation;

fig. 23 shows laser confocal pictures of tail fins of non-amputated medaka juvenile fish at different times after being fed with 5 μ M CSMPP 4h, including brightfield, green channel, red channel, merged two-channel pictures, and distribution pictures of lysosomal pH; lambda [ alpha ]_ex405 nm; green channel lambda_em416 and 555 nm; red channel lambda_em557 + 704 nm; analysis of E using Image J software_m-red/E_m-greenScale bar of 50 μm.

Detailed Description

The present invention provides an AIEgen probe for ratiometric lysosomal pH, wherein a piperazine group is used for lysosomal targeting. The large changes in intramolecular charge transfer before and after protonation of the pyridine group allowed us to quantitatively test lysosomes for pH using a ratiometric method. In this application, one non-limiting example of an AIEgen design, in particular, is CSMPP. And one non-limiting application is to monitor the regeneration process of the tail fin of medaka juvenile fish by testing the lysosomal pH value of the tail fin near the amputation plane.

In one embodiment, the AIEgen of the present invention is a CSMPP having the structure:

in embodiments of the methods of synthesis of AIEgens according to the present application, the AIEgens can be ratiometric pH probes, can specifically target lysosomes in cells, have good photostability, good pH responsiveness, low cytotoxicity, low fish toxicity, and can track lysosomal pH changes during fin regeneration for long periods of time.

Synthesis of Compound CSPP

The starting material was prepared by Suzuki coupling followed by one-step Knoevenagel condensation to form CSMPP. Wherein the α -cyanobenzene is the core backbone that confers the characteristics of AIE. The synthetic route of CSMCPP is shown below.

Synthesis of Compound 3: to a 100mL two-necked round bottom flask equipped with a condenser were added 2- (4-bromophenyl) acetonitrile (compound 30, 0.50g, 2.55mmol), 4-pyridylboronic acid (compound 40; 0.31g, 2.55mmol), potassium carbonate (3.52g, 25.5mmol) and Pd(PPh₃)₄(35mg, 0.03mmol), 50mL of THF and 10mL of water were added to dissolve under nitrogen. The mixture was stirred and heated to reflux overnight. After cooling to room temperature, the mixture was extracted three times with Dichloromethane (DCM). The organic phase was collected, washed with water and dried over anhydrous sodium sulfate. After evaporation of the solvent, the crude product was purified by silica gel column chromatography using DCM/ethyl acetate (v/v ═ 99:1) as eluent to give the product as a white solid. Yield: 81 percent.¹H NMR(400MHz, CDCl₃),δ(ppm):8.69(d,2H,J＝6.0Hz),7.67(d,2H,J＝8.4Hz), 7.51-7.46(m,4H),3.83(s,2H).¹³C NMR(100MHz,CDCl₃),δ(ppm): 149.7,146.7,137.5,130.2,128.1,127.1,120.9,116.9,22.9.HRMS (MALDI-TOF):m/z 194.0914(M⁺Theoretical value 194.0844).

Synthesis of Compound 5: 3(0.1g, 0.515mmol) and 4(0.105g, 0.515mmol) were placed in a 50mL round bottom flask and dissolved with 4mL ethanol. Sodium hydroxide (20.6mg, 0.515mmol) was dissolved in 1mL ethanol and then slowly added to the mixture. After stirring for 2 hours at room temperature, the pale yellow precipitate was filtered off, washed with cold ethanol and dried under reduced pressure. Yield of the product: 80 percent. Crystals of CSPP were obtained by slowly volatilizing a nearly saturated chloroform solution thereof.¹H NMR(400MHz, CDCl₃),δ(ppm):8.76-8.60(m,2H),7.90-7.83(m,2H),7.74(d,J＝ 8.6Hz,2H),7.68(d,J＝8.5Hz,2H),7.55-7.49(m,2H),7.47(s, 1H),7.01-6.84(m,2H),3.45-3.29(m,4H),2.56(t,J＝5.1Hz,4H), 2.35(s,3H).¹³C NMR(400MHz,CDCl₃),δ(ppm):δ152.47, 150.33,147.25,142.52,137.73,136.05,131.32,127.46,126.19, 123.69,121.36,118.82,114.36,105.32,54.73,47.36,46.13.HRMS (MALDI-TOF):m/z 381.2085(M⁺Theoretical value 380.2001).

AIE Properties of CSPP

The photophysical properties of CSMPP were first studied. In the good solvent acetonitrile, CSMPP has very weak emission with a maximum emission wavelength of 509nm, at which the absolute fluorescence quantum yield is 0.8%. With the water content (f) in the mixed solvent of acetonitrile and water_w) Is gradually increased at f_wBelow 80%, CSMP fluorescence emission is very weak and essentially unchangedTo reduce to 80% f_wThereafter, the fluorescence increases dramatically due to the restriction of intramolecular movement by the aggregates produced (FIGS. 4b and 4 c). At 90% f_wMaximum fluorescence emission at time of about 0% f _w7 times of the time. Thus, the change in fluorescence after gradually increasing the poor solvent water indicates that CSPP has AIE properties. In addition, the absolute fluorescence quantum yield of CSPP powder reaches 25.4%, and the emission peak is at 525 nm.

Similarly, under high viscosity conditions, intramolecular motion may also be restricted to excite the radiation channel. As shown in FIG. 5, the CSPP fluorescence emission gradually increased with increasing glycerol content in the glycerol and ethylene glycol mixture. And the quantum yield of CSPP reaches 10.5% in the solution containing 95% glycerol.

To further understand the AIE properties of CSMPP, its single crystal structure was studied (fig. 4 a). The molecule has a slightly distorted conformation with dihedral angles of 10.95 ° and 11.08 °. Therefore, the benzene ring adjacent to the acrylonitrile group can freely rotate in a good solvent, thereby consuming energy of the exciton in a non-radiative manner. When the molecules are in an aggregated state or in a high viscosity solution, the process of restricting intramolecular movement can be activated, thereby enabling the molecules to have a strong light-emitting ability. It was found from the alignment of the crystals that the course of RIM is caused by the intermolecular interactions of C-H. And J-aggregates were observed in the stacking of single crystals (FIG. 6), without strong pi-pi stacking effect.

Mechanism research of pH responsiveness of CSPP

To investigate the pH responsiveness of CSMP, this study tested DMSO-d in CSMP₆Before and after adding deuterated hydrochloric acid to the solution¹H NMR spectrum. The results are shown in FIG. 7, where H in the methyl group on the piperazinyl group and H next to the pyridyl group undergo significant low field shift, indicating that both N-methylpiperazinyl and pyridyl can be protonated. Due to pK of N-methylpiperidine and pyridine_a10.08 and 5.23 respectively, CSMPP has two groups of weak bases enabling it to target lysosomes with acidic pH.

To understand the photophysical properties of CSPP before and after acidification, the study was based on CSPPThe Frontier molecular orbits of the ground state were calculated by the Density Functional Theory (DFT). The results are shown in FIG. 8, CSMCPP-H⁺(N-methyl-piperazinyl is protonated) and CSMCPP-2H⁺(both the N-methyl-piperazinyl and the pyridine group are protonated), the electron clouds in the Highest Occupied Molecular Orbital (HOMO) of the three are located substantially around the cyanophenylacetylene. CSMCPP and CSMCPP-H⁺Is also predominantly located around the cyanophenylacetylene. While CSMCPP-2H⁺The electron cloud in LUMO of (a) is localized to the pyridylphenyl site. In addition, CSPP-H compares to neutral CSPP molecules⁺The HOMO-LUMO energy gap variation of (a) is negligible. However, CSMCPP-2H⁺The HOMO-LUMO gap (4.64eV) is smaller than that of CSPP (5.47 eV). This indicates that intramolecular charge transfer becomes more pronounced after the pyridyl group is protonated, which will cause a red shift in absorption and emission. This is the basis for the CSMP to have ratiometric pH testing properties.

Properties of CSPP Rate-type pH test

PL profiles of CSPP were measured in buffers of varying pH in the range of pH 2.60-6.80. As a result, as shown in FIG. 9, when the pH was decreased from pH 6.80 to pH 2.60, the fluorescence at the maximum emission peak 503nm gradually decreased, while a new emission peak appeared at 615nm, and the fluorescence gradually increased. An isoluminescent point was observed at 560nm, indicating that the conversion between the two luminescent substances occurred in the system. Meanwhile, when the pH was decreased from 6.80 to pH 3.45, the fluorescence image of the probe solution showed a distinct color change, i.e., from green to yellow and then red (inset in fig. 9 b). Further, the ratio of fluorescence emission at two wavelengths (I)₆₁₅/I₅₀₃) The curve with pH shows an inverse "S" shape and can be well fitted by the DoseResp function of Origin software (fig. 9 b). According to the following formula

After calculation, log [ (R-R) is obtained_min)/(R_max-R)]Linear relationship with pH (fig. 9 c). This indicates that the probe mayFor ratiometric pH measurements. In addition, the absolute pK exhibited by the probe_aAt 4.75 ± 0.02 (fig. 10), just in the acidity window of the lysosome (pH 4.5-5.5), indicating that the probe is able to measure the pH of the lysosome.

After protonation, the absorption of CSPP was also red-shifted, with the maximum absorption peak changing from 353nm at pH 6.8 to 383nm at pH 2.6 (FIG. 11). Thus, the enhancement of intramolecular charge transfer after CSMCP protonation causes a significant red shift in the absorption spectrum (30nm) and emission spectrum (112 nm).

pH response reversibility and specificity of CSMP probes

The pH reversibility of CSMPP was checked by alternating the pH of the solution between pH 7 and pH 3 (fig. 9 d). As a result, it was found that the probe can emit fluorescence normally even after 5 cycles of the alkaline-acidic pH. Since the selectivity of the assay is crucial for pH measurements both in vivo and in vitro, the present invention investigates the interference of different chemical species on the binding of the probe to protons. As a result, as shown in FIG. 9e, the fluorescence ratios I of the probe solutions at pH 6.8 and pH 3.0, to which 38 kinds of chemicals possibly existing in the living system were added, respectively₆₁₅/I₅₀₃The changes are all minor. The 38 chemical species include metal ions (K)⁺,Na⁺,Ca²⁺,Mg²⁺,Cd²⁺,Co²⁺,Fe²⁺,Fe³⁺,Ni²⁺,Cu²⁺, Al³⁺,Zn²⁺,Ag⁺,Mn²⁺,Pb²⁺) Common anions (HPO)₄ ^2-,H₂PO₄ ^-,Ac^-, NO₃ ^-,I^-,CO₃ ^2-) Active oxygen species (ClO)^-,H₂O₂) Reactive sulfides (HS)^-, SCN^-,S₂O₃ ^2-Cysteine, homocysteine, glutathione), some natural amino acids and glucose. Thus, the probe can be used for specific pH tests without interference from certain common chemicals in living systems.

Targeting of CSPP to lysosomes in cells and cytotoxicity thereof

It is well known that HeLa cells are the most widely used cell model, while ARPE-19 cells (a human retinal pigment epithelium) are commonly used as an in vitro model of age-related macular degeneration. The present invention investigated the lysosome specific staining of CSMPP in vitro using HeLa cells and ARPE-19 cells and photographed with a Confocal Laser Scanning Microscope (CLSM). Results of co-staining HeLa cells with LysoTracker Red (LTR) and CSMPP showed that their signals overlapped well and the Pearson correlation coefficient was 0.92 (FIG. 12). Co-staining of ARPE-19 cells with LysoTracker Blue (LTB) and CSMPP also showed a higher Pearson correlation coefficient (0.90) (FIG. 13). Thus, CSPP stains acidic organelles as well as LysoTracker excellently.

The cytotoxicity of CSMPP on HeLa cells and ARPE-19 cells was investigated by the MTT method. The MTT results showed that when the CSMPP concentration did not exceed 10 μ M, the cell viability of HeLa cells remained above 91% (fig. 14a) and that of ARPE-19 cells remained above 80% (fig. 14 b). Thus, CSMPP has low cytotoxicity at staining concentrations below 10 μ M.

Targeting of CSMPP to lysosomes in vivo and its biocompatibility

The invention researches the lysosome specific imaging of the tail fin of the medaka juvenile fish. After feeding the young fish with CSMPP for 4 hours, the whole body of the young fish was illuminated with fluorescence (fig. 15 a). Most importantly, its tail fin is also illuminated. We here focused on studying specific imaging of lysosomes on the tail fin. After feeding young fish with CSMPP and LysoTracker deep (LTDR), images of the tail fin were captured using confocal laser microscopy (fig. 15 b). The results show that CSMP and LTDR overlap well with a Pearson correlation coefficient of 0.8 (FIG. 15 b). Thus, CSMPP targets lysosomes in the tail fin of juvenile fish as well as LTDR.

The biocompatibility of CSMPP to medaka juvenile fish is evaluated by adopting two methods. The first method exposes medaka juvenile fish to different concentrations of CSMPP (0-10. mu.M) for 96h, and the survival rate of the juvenile fish is recorded (FIG. 16 a). The second method monitors the change in the heart rate of a medaka juvenile fish after exposure to 5 μ M CSMPP for 4 hours later within 96 hours (fig. 16 b). The results show that the survival rate of young fish is over 80% when the CSMPP concentration does not exceed 10. mu.M (FIG. 16 a). The heart rate of the young fish remained almost unchanged for 96 hours after the young fish stopped exposure to CSMPP (fig. 16 b). Therefore, these results indicate that CSMPP has better biocompatibility with medaka juvenile fish.

Photostability of CSPP

For applications where imaging is to be tracked over a long period of time, photostability is a requirement for fluorescence imaging. The present invention investigated the photostability of CSMPP by scanning stained HeLa cells 100 times consecutively (fig. 17). For comparison, the light stability of LTR and LysoTracker Green (LTG) was also tested. The results show that the fluorescence signals of LTR and LTG are reduced to almost zero after 50 scans (FIGS. 17a and 17 b). Whereas the fluorescence signal of CSMPP remained above 80% even after 100 consecutive scans (fig. 17a and 17b), indicating that CSMPP has good photostability.

CSMP test for pH of lysosomes in cells following chemical stimulation

Before monitoring lysosomal pH changes in living cells, a pH calibration curve for CSMPP in HeLa cells was established. By means of H⁺/K⁺Antiporter nigericin and H⁺/Na⁺The pH standard curve was tested by reverse transport of monensin to homogenize the pH inside and outside the cell. Fig. 18 shows fluorescence ratio images at different pH values and pH calibration curves. To evaluate the performance of CSMPP to measure lysosomal pH, the present invention used some common chemical stimuli to adjust lysosomal pH and calculated the lysosomal pH based on the pH calibration curve (fig. 19). It is known that bafilomycin A1 can inhibit vacuole type H⁺ATPase (proton pump) to prevent protonation of lysosomes, thereby raising the pH of the lysosome. When CSMPP-stained HeLa cells were incubated with 50nM of bafilomycin a1 for 30 minutes, the lysosomal pH rose to 5.65 (fig. 19), while the normal lysosomal pH was 5.06 (fig. 19). Acetic acid is a weak organic acid that can diffuse freely into cells. After incubating HeLa cells with 10mM acetic acid for 8 min, the pH of the lysosome was reduced to 3.97 (fig. 19). Furthermore, it showed concentration-dependent cellular acidification in HeLa cells with 1.75mM acetic acidAfter 8 minutes of incubation, the pH of its lysosome was reduced to 4.45 (fig. 19). H₂O₂Is a reactive oxygen species that can exert oxidative stress on cells and damage vacuole type H⁺ATPase, leading to the basification of lysosomes. With 0.1mM H₂O₂After 30 minutes of treatment of HeLa cells, the lysosome pH rose to 5.55 (fig. 19). It is well known that tamoxifen can alkalify the pH of lysosomes, which rises to 6.14 after HeLa cells are incubated with 40 μ M tamoxifen at 37 ℃ for 15 minutes (fig. 19). Thus, these results indicate that CSMPP probes can sensitively monitor and quantify lysosomal pH in living cells.

Tracking of CSMCPP on lysosome pH in medaka juvenile fish tail fin regeneration process

Since the photographing conditions of the tail fin of the juvenile fish are different from those of the HeLa cell, the pH standard curve needs to be re-established. The pH standard curve was tested using CLSM with a 40x oil lens and zebrafish cells under the same conditions as tail fin photographing conditions. By means of H⁺/K⁺Antiporter nigericin and H⁺/Na⁺Backward transport of monensin to homogenize the pH inside and outside the cells, fluorescence ratio images of fish cells at different pH were obtained (E)_m-red/E_m-green) And a pH calibration curve. As shown in FIG. 20, the ratio of the mean fluorescence intensities of the two emission channels excited at 405nm (E) increases with increasing buffer pH_m-red/E_m-green) Exhibits an inverse "S" curve over a pH range of 2.5 to 7.0, from which R can be obtained_minAnd R_max(FIG. 20 b). In addition, log [ (R-R)_min)/(R_max-R)]Linear relationship with pH between 3.0 and 6.5 (fig. 20b), which will be used to calculate lysosomal pH of the tail fin.

In the process of regeneration of the tail fin of the medaka juvenile fish, the change of the lysosome pH is tracked by using the CSMPP probe. The juvenile fish were fed with 5 μ M CSMPP for 4 hours and then their tail fins were truncated. Tail fin regeneration was followed using CSLM and tail fins of the same juvenile fish were photographed 12, 24, 48, 96 and 120 hours before and after amputation. Here several hours after amputation, abbreviated hpa. As a result, as shown in fig. 21, at 12hpa, the end of the tail fin, which was originally round, became a clean cut surface after being cut by a sharp scalpel. Then, the tail fin gradually regenerates. At 120hpa, the end of the tail fin became almost rounded and the tail fin reached almost the original length, indicating that the amputated tail fin is about to fully regenerate as it did the original tail fin. During regeneration, the lysosomal pH of the tail fin varies greatly. As shown in figure 22, the average lysosomal pH gradually decreased after amputation, reaching a minimum pH at 24-48hpa, which was reported to be the stage of blast formation. At 24hpa, the average lysosomal pH value decreased from the original pH of 5.1 to pH 4.6, for a total decrease of about 0.5. This indicates that lysosomes are most active at this stage. Regeneration at this stage relies primarily on autophagy to remove damaged tissue and cellular debris resulting from the injury. Thus, to aid autophagic degradation, lysosomal acidification of the cell is upregulated. Furthermore, at 24 and 48hpa (fig. 21), the pH distribution results for lysosomes indicate that lysosomes (including regenerated tissue) around the plane of the amputation are more acidic than lysosomes away from the plane of the amputation. Then, the average pH of the lysosomes was elevated after 48hpa and returned to near normal levels after 120hpa (fig. 22). Meanwhile, the pH of lysosomes in the tail fin of medaka juvenile fish hardly changed during the five-day follow-up without amputation (fig. 22 and 23).

In addition, autophagy was found to be activated, and V-ATPase expression and lysosomal acidification were upregulated during tissue regeneration. Our studies not only provided good support for previous studies on lysosome acidification during regeneration, but also showed a detailed course of changes in mean lysosomal pH and distribution of lysosomal pH during tail fin regeneration by a simpler method.

Given only as a non-limiting example herein, it is believed that the lysosomal pH probes designed by this invention can be used in a variety of biological tracking applications.

The implementation of the invention can achieve the following beneficial effects:

1. the preparation of the lysosome probe only needs two steps, and the method is simple.

2. The ratiometric pH probes of the invention can test for pH in the maximum range of pH between 3 and 6.5.

3. The lysosomal probes of the invention can not only target lysosomes, but also test lysosomal pH by the ratiometric method. Only one excitation light is needed to receive the fluorescence emission of two wave bands. The exciting light of 405nm is the light source of common microscope, and has wide application range. And the emission wavelength change before and after probe protonation is 112 nm. This large variation in emission wavelength allows flexibility in the choice of the range of the receive band relative to conventional dyes with small variations in emission shift.

4. The probe is a luminophore (AIEgenes) with aggregation-induced emission (AIE), so that the probe has the advantages of common AIEgenes, such as no quenching of aggregation fluorescence, good light stability, suitability for long-term tracking application and large Stokes shift.

5. The probe of the invention can not only quantitatively test the pH of lysosomes in vitro cells, but also test the pH of lysosomes in living small fish.

6. The probe can track the detailed change process of the average pH of the lysosome and the distribution condition of the pH of the lysosome in the regeneration process of the small fish tail fin for a long time, and provides a simple, convenient and effective method for observing the regeneration process.

7. The lysosomal pH probes designed by the present invention can be used in a variety of other biological tracking applications.

While the present invention has been described with reference to the embodiments shown in the drawings, the present invention is not limited to the embodiments, which are illustrative and not restrictive, and many modifications may be made by those skilled in the art without departing from the spirit and the scope of the present invention as defined in the appended claims.

Claims

1. An AIE emitter, wherein the AIE emitter comprises the structure:

wherein R is₁And R₂Are respectively as

Any one of them.

2. The AIE luminophore of claim 1, wherein said AIE luminophore exhibits pH responsiveness.

3. Use of the AIE luminophore of claim 1 for ratiometric lysosomal pH probes.

4. Use of the AIE luminophore of claim 1 for specific targeting to lysosomes in cells and small fish tail fin lysosomes.

5. Use of the AIE luminophore of claim 1 for long term tracking of lysosomal pH changes during fin regeneration in small fish.

6. Use of the AIE luminophore of claim 1 for long term tracking of the regeneration process of hyaline organisms.

7. A method for preparing the AIE luminophore of claim 1, wherein the AIE luminophore is synthesized by the following route:

the preparation method comprises the following steps:

preparation of starting material 3 by Suzuki coupling:

to a 100mL two-necked round bottom flask equipped with a condenser was added 2- (4-bromophenyl) acetonitrile, 4-pyridylboronic acid, potassium carbonate, and Pd (PPh)₃)₄Adding 50mL of THF and 10mL of water for dissolving under the protection of nitrogen; the mixture was stirred and heated to reflux overnight; is cooled toAfter room temperature, the mixture was extracted three times with dichloromethane; the organic phase was collected, washed with water and dried over anhydrous sodium sulfate; after evaporation of the solvent, the crude product was purified by silica gel column chromatography using DCM/ethyl acetate as eluent to give product 3 as a white solid;

forming the AIE luminophore by Knoevenagel condensation comprising:

placing compounds 3 and 4 in a 50mL round bottom flask, and dissolving with ethanol; sodium hydroxide was dissolved in 1mL of ethanol and then slowly added to the mixture; after stirring for 2 hours at room temperature, the pale yellow precipitate was filtered off, washed with cold ethanol and dried under reduced pressure; the AIE luminophores were obtained by slowly volatilizing a nearly saturated chloroform solution thereof.