CN113461609B - Sulfatase-responsive AIE nano probe and preparation method and application thereof - Google Patents

Abstract

The invention discloses a sulfatase-responsive AIE nano-probe and a preparation method and application thereof. The structural formula of the probe is shown in the following formula I. The probe of the present invention is a nano-probe with AIE signal amplification function to monitor sulfatase, which uses a hydrophobic quinoline-malononitrile (DQM) derivative as the core, and a hydrophilic sulfate bond as the sulfatase responsive group. The probe of the invention is simple to prepare and convenient to use, and the sulfatase can be rapidly detected from the change of the fluorescence intensity before and after the response, thereby realizing the purpose of rapid detection. The characteristic of the probe is that it has no fluorescence itself, but it can rapidly react with sulfatase to generate obvious fluorescence signal enhancement, so as to realize the selective and rapid detection of sulfatase. Therefore, the probe of the present invention can be used as an effective tool to detect sulfatase in tumors, and has the potential to be used as an inhaled contrast agent for tumor diagnosis.

Description

A kind of sulfatase-responsive AIE nanoprobe and its preparation method and application

technical field

The invention belongs to the field of biomedicine, and in particular relates to a sulfatase-responsive AIE nanoprobe and a preparation method and application thereof.

Background technique

Sulfatase enzymes are a highly conserved protein family with high structural and functional homology. They can catalyze the hydrolysis of sulfate bonds from a variety of substrates, including glycosaminoglycans, thiolipids, and steroid sulfates. Sulfatase enzymes have been implicated in a variety of pathophysiological conditions, such as hormone-dependent cancers, lysosomal storage diseases, dysplasia, and bacterial pathogenesis. The vast majority of breast tumors overexpress the enzyme, and there are indications that sulfatase plays a role in prostate cancer. Therefore, detection of sulfatase is beneficial for diagnosing cancers with high expression of sulfatase and understanding the pathological activity of sulfatase.

In 2001, Tang Benzhong's team proposed the concept of aggregation-induced emission (AIE) (Aggregation-induced emission of 1-methyl-1,2,3,4,5-pentaphenylsilole), which described an abnormality opposite to the ACQ effect Phenomenon, showing weak or even no fluorescence in solution, but strong fluorescence once aggregated. The main factor for solid-state luminescence of AIE molecules is intramolecular motion constraints, including intramolecular rotational and vibrational constraints. When the molecules are aggregated, the intramolecular motion that originally consumes the energy of the excited state of the molecule is inhibited, so that the non-radiative transition that quenches the fluorescence of the molecule is greatly inhibited, and the molecule mainly releases energy through the radiative transition to emit light. At the same time, AIE molecules generally have a twisted molecular structure, and it is difficult to form π-π stacking during aggregation, thus reducing the attenuation of excited state energy through non-radiative channels. The methods to restrict intramolecular motion mainly include electrostatic attraction, hydrogen bonding, hydrophobic interaction and solubility change. Among them, hydrophobic interactions are the main driving force for protein folding. Proteins are stable in water when the hydrophobic side chains in the protein aggregate inside the protein, rather than being solvated by water. Thus, in aqueous media, amphoteric organic luminescent agents are driven into hydrophobic domains or pores in protein folded structures by hydrophobic interactions in aqueous media. Due to the limited volume of the pores, intramolecular motion is restricted allowing the luminescent agent to form aggregates. AIE-restricted intramolecular motility mechanisms make this method suitable for the design of AIE bioprobes. Based on the outstanding properties of AIE photoluminescent agents, specific enzymatic bioprobes were designed by integrating with the recognition unit, showing many advantages, including low background interference, high signal-to-noise ratio, and excellent photostability.

At present, fluorescent probes have the advantages of high sensitivity, good selection, and non-invasiveness, and are a reliable method for the detection of sulfatase. However, few fluorescent probes have been developed to detect sulfatase activity in vivo due to the limitation of short emission wavelength, small Stokes shift, or aggregation-induced quenching effect. Therefore, deep tissue-penetrating fluorescent probes are urgently needed for in vivo imaging of sulfatase.

SUMMARY OF THE INVENTION

Purpose of the invention: In view of the problems existing in the prior art, the present invention provides a sulfatase-responsive AIE nanoprobe, which can be specifically hydrolyzed by sulfatase, release fluorophores, and realize the duality of fluorescent signals. The secondary enhancement is used for the detection of sulfatase, effectively solving the limitation of the quenching effect caused by the short emission wavelength of the fluorescent probe, the small Stokes shift, or the aggregation.

The present invention also provides the preparation method and application of the sulfatase-responsive AIE nanoprobe.

Technical solution: In order to achieve the above purpose, a sulfatase-responsive AIE nanoprobe of the present invention, abbreviated as DQM-SULF, is composed of a fluorophore and a sulfate, and its structural formula is as follows: Show:

The preparation method of the sulfatase-responsive AIE nanoprobe of the present invention comprises the following steps:

(1) 2-methylquinoline and iodoethane are dissolved in anhydrous acetonitrile, after the reaction mixture is cooled to room temperature in a stirring reaction under the protection of inert gas, the precipitate is vacuum filtered, washed and dried to obtain compound 2;

(2) dissolving compound 2 in ethanol, then adding malononitrile and sodium ethoxide to react, after the reaction is completed, the precipitate is filtered and washed to obtain compound 3;

(3) Dissolve compound 3 and 4-hydroxybenzaldehyde in anhydrous acetonitrile containing piperidine, react under the protection of inert gas, cool the reaction mixture to room temperature, evaporate the solvent under reduced pressure, and purify the residue to obtain the compound DQM-OH;

(4) Add dropwise sodium tert-butoxide dissolved in tetrahydrofuran solution to the tetrahydrofuran solution dissolved with DQM-OH; add trimethylammonium sulfur trioxide copolymer to react; evaporate the solvent from the reaction mixture, and purify the residue to obtain Compound probe DQM-SULF.

The reaction route is as follows:

Preferably, in step (1), 2-methylquinoline and iodoethane are dissolved in anhydrous acetonitrile, and stirred at 85-90° C. for 20-24 h under the protection of nitrogen inert gas. After the reaction, after the reaction mixture was cooled to room temperature, there was a large amount of precipitate at the bottom, which was vacuum filtered with a Buchner funnel; the solid crude product was washed with cold acetonitrile without further purification, and after drying overnight, compound 2 was obtained.

Preferably, in step (2), compound 2 is dissolved in dry ethanol, then malononitrile and sodium ethoxide are added, stirred at 0-4 °C for 0.5-1 h, and then stirred at room temperature for 2-3 h. After the reaction is completed , the precipitate was filtered and washed with cold ethanol several times to obtain compound 3.

Preferably, in step (3), compound 3 and 4-hydroxybenzaldehyde are dissolved in anhydrous acetonitrile containing piperidine, stirred at 85-90° C. overnight under nitrogen protection, and the reaction mixture is cooled to room temperature, and the solvent is reduced After evaporation under pressure, the residue was purified by silica gel column chromatography to obtain compound DQM-OH.

Preferably, in step (4), at 20-22° C., add dropwise sodium tert-butoxide dissolved in the tetrahydrofuran solution to the tetrahydrofuran solution dissolved with DQM-OH, and after 15-20min, add trimethylammonium in solid form Sulfur trioxide copolymer, after 30-40 min, the solvent was evaporated from the reaction mixture, and the residue was purified by silica gel column chromatography to obtain the probe DQM-SULF.

The application of the sulfatase-responsive AIE nanoprobe in the detection of sulfatase responsiveness.

The application of the probe in the present invention in preparing a responsive detection tool for detecting sulfatase.

Preferably, the responsive detection process is as follows: adding sulfatase solutions of different concentrations to the reaction system containing the probes, quickly mixing the reaction solutions and then incubating, and transferring the reaction solutions to British Colorimetric after incubation. The UV absorption and fluorescence emission spectra were measured.

The application of the sulfatase-responsive AIE nanoprobe of the present invention in the imaging of cellular endogenous sulfatase.

Preferably, the imaging process is as follows: taking 4T1 cells in logarithmic growth phase, digesting and centrifuging, preparing a cell suspension, adding the cell suspension to a laser confocal culture dish, and using different concentrations of probes in the co-culture Incubation followed by final cell imaging with confocal microscopy.

Use of the probes of the present invention in the preparation of imaging tools for detecting sulfatase in, for example, tumor cells.

The AIE probe structure proposed in the present invention can hydrolyze the AIE probe into a fluorophore with strong fluorescence in the presence of sulfatase, and then combine with the hydrophobic domain of the sulfatase to further amplify the AIE through hydrophobic interaction fluorescence signal. Catalytic and hydrophobic domains of sulfatase that can turn on and enhance AIE fluorescence to detect sulfatase with high sensitivity and signal-to-noise ratio.

The probe of the invention is a nano-probe with AIE signal amplification function to monitor sulfatase, with a hydrophobic quinoline-malononitrile (DQM) derivative as the core, and a hydrophilic sulfate bond as the sulfate The responsive group of the enzyme, a new nanoprobe for detecting sulfatase was synthesized. After the AIE probe was hydrolyzed by sulfatase, the fluorophore was released, and the secondary enhancement of the fluorescence signal was realized, which can be used to detect intracellular sulfatase. Source sulfatase and intratumorally overexpressed sulfatase were imaged. As an effective tool to detect sulfatase in tumors, the probe has the potential to be applied in tumor diagnosis as an inhaled contrast agent.

The probe prepared by the invention becomes a fluorophore after being hydrolyzed by sulfatase, and then the fluorophore is combined into the hydrophobic cavity of the sulfatase to realize the secondary enhancement of the fluorescence signal, which can effectively overcome the short emission wavelength of the fluorescent probe. , the Stokes shift is small, or the aggregation-induced quenching effect is limited. The probe of the present invention has a hydrophilic group and a hydrophobic group, and the amphiphilic structure enables it to self-assemble into a loose nano-probe in an aqueous solution.

Beneficial effect: Compared with the prior art, the present invention has the following advantages:

(1) The probe is simple to synthesize and easy to use, and the mechanism of its response to sulfatase is based on the hydrolysis of sulfate bond catalyzed by sulfatase to release the fluorophore DQM-OH.

(2) The sulfatase can be quickly detected from the change of the fluorescence intensity before and after the response, and the purpose of the rapid detection is realized.

(3) The characteristic of the probe is that it has no fluorescence itself, but it can rapidly react with sulfatase to generate obvious fluorescence signal enhancement, so as to realize the selective and rapid detection of sulfatase. Therefore, the probe of the present invention can be used as an effective tool to detect sulfatase in tumors, and has the potential to be used as an inhaled contrast agent for tumor diagnosis.

Description of drawings

Fig. 1 is the high-resolution mass spectrum of the probe DQM-SULF;

Figure 2 shows the trend and linear relationship of the fluorescence intensity of the probe DQM-SULF (5μM) in PBS buffer with the concentration of sulfatase (0-50U/mL);

Figure 3 is the fluorescence emission spectrum before and after the reaction of the fluorophore DQM-OH with sulfatase (50U/mL), wherein the upper curve is DQM-OH+SULF;

Figure 4 is the imaging diagram of different concentrations of probe DQM-SULF in 4T1 cells.

Detailed ways

The present invention will be further described below with reference to the accompanying drawings and embodiments.

The experimental methods used in the present invention are all conventional methods unless otherwise specified. Materials, reagents, etc. used in the experiments can be obtained from commercial sources unless otherwise specified. All the following reagents selected in the examples are commercially available analytically pure or chemically pure.

Among them, sulfatase (abbreviated as SULF) is a sulfate derived from Roman snail, purchased from sigma-aldrich. The enzyme is active at 37 ° C and the pH value is 5.0. One unit can hydrolyze 1.0 micromoles of p-nitro groups per hour. Catechol sulfate.

Trimethylammonium sulfur trioxide copolymer was purchased from McLean Reagent Company.

Example 1

Preparation method of sulfatase-responsive AIE nanoprobe:

(1) Synthesis of fluorophore: 2-methylquinoline (2.86 g, 20 mmol) and iodoethane (3.90 g, 25 mmol) were weighed and dissolved in 25 mL of anhydrous acetonitrile, and stirred at 85° C. for 24 h under nitrogen protection. After the reaction was completed, after the reaction mixture was cooled to room temperature, there was a large amount of precipitate at the bottom of the flask, which was filtered with a Buchner funnel under vacuum. The crude product was washed with cold acetonitrile without further purification. After drying overnight, compound 2 was obtained.

(2) Compound 2 (1.72 g, 10 mmol) was weighed and dissolved in 10 mL of dry ethanol, and then malononitrile (1 g, 15 mmol) and sodium ethoxide (1.02 g, 15 mmol) were added. The reaction was stirred at 0 °C for 0.5 h, then at room temperature for an additional 3 h. After the reaction was completed, the precipitate was filtered and washed with cold ethanol three times to obtain compound 3.

(3) Compound 3 (470 mg, 2 mmol) and 4-hydroxybenzaldehyde (244 mg, 2 mmol) were weighed and dissolved in 20 mL of anhydrous acetonitrile containing piperidine (1 mL), and stirred at 85° C. overnight under nitrogen protection. After the reaction mixture was cooled to room temperature, the solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane:methanol=10:1) to obtain compound DQM-OH.

(4) Probe DQM-SULF: at 22°C, to the tetrahydrofuran solution (5 mL) of DQM-OH (509 mg, 1.5 mmol) dissolved in constant stirring was added dropwise tert-butanol dissolved in tetrahydrofuran solution (3 mL) Sodium (144 mg, 1.5 mmol) solution. After 15 min, trimethylammonium sulfur trioxide copolymer (278 mg, 2 mmol) was added in solid form. After continuing the reaction for 30 min, the solvent was evaporated from the reaction mixture under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane:methanol=10:1) to obtain probe DQM-SULF.

¹ H NMR (300MHz, DMSO-d ₆ ) δ 8.90(d, J=8.3Hz, 1H), 8.07(d, J=8.8Hz, 1H), 7.89-7.93(m, 1H), 7.67(d, J=8.5Hz, 2H), 7.57-7.62(m, 1H), 7.26-7.38(m, 2H), 6.99(s, 1H), 6.85(d, J=8.5Hz, 2H), 4.55(q, J) =6.9Hz, 2H), 1.40 (t, J=7.0Hz, 3H). ¹³ C NMR (75MHz, DMSO-d ₆ )δ159.8, 155.6, 152.7, 149.9, 140.4, 139.9, 138.3, 134.2, 130.5, 129.4, 125.6, 121.1, 120.7, 119.5, 118.6, 117.4, 116.2, 107.2, 47.3, 44.7, 14.1. HRMS: m/z calcd for C ₂₂ H ₁₆ N ₃ O ₄ S[MH] ^- :418.0862, found:418.0861.

Figure 1 is a high-resolution mass spectrum of the probe DQM-SULF, indicating that the probe DQM-SULF was successfully synthesized.

Example 2

Weigh 4.2 mg of the probe DQM-SULF prepared in the example and dissolve it in 10 mL of DMSO solution to prepare a standard solution with a concentration of 1 mM; similarly, take 3.4 mg of the fluorophore DQM-OH prepared in the example and dissolve it in 10 mL of DMSO solution , prepared into a standard solution with a concentration of 1mM; the sulfatase was prepared into a standard solution of 100U/mL with PBS buffer solution (10Mm, pH 7.4). Take a 4 mL EP tube and add different final concentrations of sulfatase solution (0-50 U/mL) to the reaction system PBS buffer (10 mM, pH=7.4, 1% DMSO) containing the probe DQM-SULF (5 μM). Incubate for 30 min at 37°C in a constant temperature shaker. Under the excitation of 440 nm wavelength, the slit width was set to 10.0/10.0 nm, and the fluorescence emission spectrum of the solution in the 450-800 nm band was collected. As shown in Fig. 2, the fluorescence intensity curve of the probe DQM-SULF increased with the concentration of sulfatase. In addition, the detection limit is 2.1 U/L and the detection range is 0-50 U/mL, which indicates that the probe has high sensitivity and can be used for the detection of endogenous sulfatase in biological systems.

Example 3

The preparation steps of the probe DQM-SULF solution and the sulfatase solution involved in this example are the same as those in Example 2, and other specific steps are as follows: add the PBS buffer (10mM, pH= 7.4, 1% DMSO), add sulfatase solution with a final concentration of 50 U/mL, and incubate for 30 min at 37°C in a constant temperature shaker. Under the excitation of 440 nm wavelength, the slit width was set to 10.0/10.0 nm, and the fluorescence emission spectrum of the solution in the 450-800 nm band was collected. As shown in Figure 3, the AIE fluorescence signal was amplified in the fluorophore-enzyme response compared to the fluorophore alone. It shows that the probe prepared by the present invention can release fluorophore and realize the secondary enhancement of the fluorescence signal, and effectively solve the limitation of the quenching effect caused by the short emission wavelength of the fluorescent probe, the small Stokes shift, or the aggregation. In addition, using the above method, the enzyme is combined with other compounds with AIE properties such as tetraphenylene, 1-(4-hydroxybenzene)-1,2,2-triphenylene, 1,1,2,3,4,5-hexaphenylene Phenylsilole and other responses did not achieve a secondary enhancement of the fluorescence signal.

Example 4

The preparation steps of the probe DQM-SULF solution involved in this example are the same as those in Example 2, and other specific steps are as follows: Digest and centrifuge 4T1 cells in logarithmic growth phase, and use DMEM medium containing 10% FBS to prepare 5× At a density of 10 ⁴ cells/mL, the cell suspension was added to a laser confocal culture dish, and 100 μL of the cell suspension was added to each dish and placed in a 5% CO ₂ , 37°C incubator overnight to form an adherent monolayer, and when the cells When the density reached 60%-70%, the probe DQM-SULF (0-30μM) solutions of different final concentrations were used to co-incubate for 2h in the incubator, then the mixture was removed, washed three times with PBS, and then washed with 4% paraformaldehyde. fixed. Finally, the cells were imaged with a FV-1000 laser confocal microscope. As shown in Figure 4, as the probe concentration increased, the intracellular fluorescence also increased. Therefore, the probe DQM-SULF can be effectively activated by endogenous sulfatase in living cells, and has good application prospects in biology and medicine.

Example 5

The preparation method of Example 5 is the same as that of Example 1, except that in step (1), stirring is performed at 90° C. for 20 h under the protection of nitrogen inert gas. Step (2) Compound 2 was dissolved in dry ethanol, then malononitrile and sodium ethoxide were added, and the mixture was stirred at 4° C. for 1 h, and then at room temperature for 2 h. Step (3) was stirred at 90°C overnight under nitrogen protection. Step (4) at 20°C, dropwise add sodium tert-butoxide dissolved in tetrahydrofuran solution to the tetrahydrofuran solution dissolved with DQM-OH, after 20min, add trimethylammonium sulfur trioxide copolymer in solid form, 40min After that, the solvent was evaporated from the reaction mixture, and the residue was purified by silica gel column chromatography to obtain the probe DQM-SULF.

Claims (10)

1. An AIE nanoprobe responding to sulfatase, which is characterized in that the structural formula is shown as the following formula I:

2. a preparation method of an AIE nano probe responding to sulfatase is characterized by comprising the following steps:

(1) dissolving 2-methylquinoline and iodoethane in anhydrous acetonitrile, stirring and reacting under the protection of inert gas, cooling a reaction mixture to room temperature, and carrying out vacuum filtration, washing and drying on a precipitate to obtain a compound 2;

(2) dissolving the compound 2 in ethanol, adding malononitrile and sodium ethoxide for reaction, filtering precipitates after the reaction is finished, and washing to obtain a compound 3;

(3) dissolving the compound 3 and 4-hydroxybenzaldehyde in anhydrous acetonitrile containing piperidine, reacting under the protection of inert gas, cooling the reaction mixture to room temperature, evaporating the solvent under reduced pressure, and purifying the residue to obtain a compound DQM-OH;

(4) dropwise adding sodium tert-butoxide dissolved in a tetrahydrofuran solution into the tetrahydrofuran solution dissolved with DQM-OH; adding trimethyl ammonium sulfur trioxide copolymer for reaction; the solvent was evaporated from the reaction mixture and the residue was purified to give the compound probe DQM-SULF;

the reaction route is as follows:

3. the preparation method according to claim 2, wherein in the step (1), 2-methylquinoline and iodoethane are preferably dissolved in anhydrous acetonitrile, and are stirred for 20-24h at 85-90 ℃ under the protection of nitrogen inert gas, after the reaction is finished, the reaction mixture is cooled to room temperature, and a large amount of precipitate is at the bottom part, and is subjected to vacuum filtration by using a Buchner funnel; the solid crude product was washed with acetonitrile without further purification and dried overnight to give compound 2.

4. The preparation method according to claim 2, wherein the compound 2 is dissolved in dry ethanol in the step (2), then malononitrile and sodium ethoxide are added, stirring is carried out at 0-4 ℃ for 0.5-1h, then stirring is carried out at room temperature for 2-3h, and after the reaction is completed, the precipitate is filtered and washed with ethanol to obtain the compound 3.

5. The preparation method according to claim 2, characterized in that in step (3), the compound 3 and 4-hydroxybenzaldehyde are dissolved in anhydrous acetonitrile containing piperidine, the mixture is stirred overnight at 85-90 ℃ under nitrogen protection, after cooling the reaction mixture to room temperature, the solvent is evaporated under reduced pressure, and the residue is purified by silica gel chromatography to obtain the compound DQM-OH.

6. The preparation method according to claim 2, characterized in that the step (4) of adding sodium tert-butoxide dissolved in tetrahydrofuran solution dropwise at 20-22 ℃ to the tetrahydrofuran solution in which DQM-OH is dissolved, after 15-20min, trimethylammonium sulfur trioxide copolymer in solid form is added, after 30-40min, the solvent is evaporated from the reaction mixture, and the residue is purified by silica gel chromatography to give the probe DQM-SULF.

7. Use of the sulfatase-responsive AIE nanoprobe of claim 1 in the detection of sulfatase responsiveness.

8. The application of claim 7, wherein the responsiveness detection procedure is: adding sulfatase solutions with different concentrations into a reaction system containing the probe, quickly mixing the reaction solution, incubating, transferring the incubated reaction solution into an English cuvette, and measuring the ultraviolet absorption and fluorescence emission spectra.

9. Use of the sulfatase-responsive AIE nanoprobe of claim 1 in the imaging of cellular endogenous sulfatase.

10. The use according to claim 9, wherein the imaging process is: digesting and centrifuging 4T1 cells in logarithmic growth phase to prepare cell suspension, adding the cell suspension into a laser confocal culture dish, respectively using probes with different concentrations for co-incubation, and finally using a laser confocal microscope for cell imaging.

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