A lysosome-targeted AIE probe and its preparation method and application

Abstract

The present invention discloses an AIE probe targeting lysosomes, a preparation method and an application thereof. The structural formula of the AIE probe targeting lysosomes is shown below. The present invention organically combines molecules with aggregation-induced emission properties with long-chain molecules with positive charges, and obtains a novel probe with excellent AIE properties by enhancing the charge intensity. The probe can accurately locate the cell membrane and intracellular lysosomes of planktonic bacteria, emit a strong fluorescent signal, and can achieve good photodynamic therapy for planktonic bacteria and biofilms in vivo and in vitro, and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent probes, and in particular to an AIE probe targeting lysosomes and a preparation method and application thereof. Background Art

[0002] Bacterial infection is one of the leading causes of death worldwide and has long posed a serious threat to public health. At the same time, it has also brought a heavy medical burden to human society. There are many factors that cause bacterial infection. For example, high blood sugar in diabetic patients can prolong the repair period of diabetic wound microvascular dysfunction, significantly increasing the risk of bacterial infection. About 15% of diabetic patients worldwide will have a complication of chronic refractory wounds. If not treated in time or improperly treated, they will also bring the risk of lower limb amputation. Therefore, as one of the most serious and most expensive chronic complications of diabetes, effective and economical treatment and care for diabetic refractory wounds remain a difficulty and hot spot in clinical research. Antibiotics are the most commonly used drugs for the treatment of bacterial infections and are widely used in antibacterial therapy. Unfortunately, the abuse of antibiotics has led to the emergence of a large number of drug-resistant pathogens with tenacious vitality, which once again poses a worrying threat to public health. In addition, the development process of new antibiotics is very slow and cannot keep up with the increasing pace of drug-resistant pathogens. In this context, the development of non-resistance antibacterial drugs and strategies is crucial for bacterial infection and wound recovery in diabetic patients.

[0003] Among all the emerging antimicrobial strategies, fluorescence imaging-guided photodynamic therapy (PDT) has attracted great potential due to its powerful bactericidal, non-invasive and high spatiotemporal control advantages. The photosensitizer (PS) that determines the performance of PDT can be used to generate type I or type II reactive oxygen species (ROS) upon light irradiation. Accompanied by the generation of ROS (singlet oxygen, hydroxyl radicals and superoxide anions), PS can eradicate pathogens efficiently and quickly. At the same time, the targeting function of PS on pathogenic microorganisms and in situ ROS generation have a synergistic effect on bactericidal, so it is difficult to develop drug resistance. The effectiveness of conventional PS in antibacterial applications is insufficient because they suffer from aggregation-caused quenching (ACQ) in aqueous media, which leads to a decrease in fluorescence intensity. In contrast, PS characterized by aggregation-induced emission (AIE PS) show weak or no fluorescence signals in the molecular state, but the fluorescence intensity is significantly enhanced after aggregation due to the restriction of intramolecular motion (RIM). Some AIE PS have been shown to have aggregation-induced emission and enhanced ROS generation, giving them great potential for photodetection and PDT applications of pathogenic microorganisms.

[0004] Therefore, it is particularly important to prepare an AIE PS with excellent performance, broad-spectrum antibacterial activity and multifunctional visualization of pathogenic microorganisms, so that it can be used in the treatment of chronic diabetic wound infections and related applications. Summary of the invention

[0005] The purpose of the present invention is to provide an AIE probe targeting lysosomes and a preparation method and application thereof. The AIE probe targeting lysosomes of the present invention can retain the fluorescence emission performance related to the radiation transition by controlling the length of the introduced side chain, and can also increase the DA intensity of the compound by introducing the side chain, thereby inducing the production of active oxygen in bacteria.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] In a first aspect of the present invention, a lysosome-targeted AIE probe is provided, and the lysosome-targeted AIE probe has the following structural formula:

[0008]

[0009] A second aspect of the present invention provides a method for preparing an AIE probe targeting lysosomes, the method comprising:

[0010] The parent structure compound TPE-CHO of bithiophene tetraphenylethylene with an aldehyde group and the compound 3 with a benzothiazole structure are heated to reflux in an organic solvent for reaction. After the reaction is completed, the AIE probe targeting lysosomes is obtained by post-treatment; the reaction formula is as follows:

[0011]

[0012] Furthermore, the molar ratio of the bithiophene tetraphenylethylene parent compound having an aldehyde group to the compound 3 is 1:(1-2).

[0013] Furthermore, the temperature of the heating reflux reaction is 80-85° C., and the reaction time is 8-15 hours.

[0014] Furthermore, the organic solvent is anhydrous methanol.

[0015] Furthermore, the preparation method of the bithiophene tetraphenylethylene parent structure compound TPE-CHO having an aldehyde group comprises:

[0016] Under an inert atmosphere, compound 1 and compound 2 are heated and refluxed in a mixed solution of tetrahydrofuran and water. After the reaction is completed, post-treatment is performed to obtain a parent structure compound TPE-CHO of bithiophene tetraphenylethylene with an aldehyde group; the reaction formula is as follows:

[0017]

[0018] Furthermore, the molar ratio of the compound 1 to the compound 2 is 1:(1-3).

[0019] Furthermore, the temperature of the heating reflux reaction is 80-85° C., and the reaction time is 8-15 hours.

[0020] Furthermore, the volume ratio of tetrahydrofuran to water is (2-3):1.

[0021] In a third aspect of the present invention, provided is the use of the lysosome-targeted AIE probe in the preparation of antibacterial drugs or diabetic wound model components.

[0022] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0023] The lysosome-targeting AIE probe provided by the present invention, as well as its preparation method and application, organically combines molecules with aggregation-induced emission properties with long-chain molecules with positive charges, and obtains a new probe with excellent AIE properties by enhancing the charge intensity. The probe can accurately locate the cell membrane of planktonic bacteria and intracellular lysosomes, emit a strong fluorescence signal, and can achieve good photodynamic therapy effects on planktonic bacteria and biofilms in vivo and in vitro, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings in the following description are some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.

[0025] Figure 1 This is the HRMS spectrum of compound TPE-Z-3.

[0026] Figure 2 Schematic diagram of the ultraviolet absorption and fluorescence spectra of compound TPE-Z-3 in different polar solvents; wherein: (a) is a schematic diagram of the ultraviolet absorption of TPE-Z-3; (b) is a schematic diagram of the fluorescence spectrum of TPE-Z-3 changing with different ratios of toluene / DMSO; (c) is a schematic diagram of the normalized fluorescence spectrum of TPE-Z-3 in different polar solvents.

[0027] Figure 3 Schematic diagram of the biocompatibility induced by compound TPE-Z-3;

[0028] Figure 4 Schematic diagram of compound TPE-Z-3 targeting cell lysosomes;

[0029] Figure 5 Schematic diagram of the photodynamic inactivation of planktonic Staphylococcus epidermidis by compound TPE-Z-3;

[0030] Figure 6 Schematic diagram of membrane targeting of planktonic Staphylococcus epidermidis by compound TPE-Z-3. DETAILED DESCRIPTION

[0031] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0032] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0033] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or obtained by existing methods.

[0034] The embodiment of the present invention provides an AIE probe targeting lysosomes, and the overall idea is as follows:

[0035] According to a typical embodiment of the present invention, a lysosome-targeted AIE probe is provided, and the lysosome-targeted AIE probe has the following structural formula:

[0036]

[0037] The lysosome-targeting AIE probe of the present invention is referred to as compound TPE-Z-3.

[0038] The present invention provides an AIE probe targeting lysosomes, which contains an aggregation-induced emission structure (i.e., a tetraphenylethylene rotor structure). One end of the molecular structure is an electron-withdrawing end and the other end is an electron-donating end. At the same time, the molecule with the aggregation-induced emission structure is combined with a long-chain molecule with a positive charge, thereby further enhancing the charge strength of the compound. The resulting compound can target the cell membrane of bacteria and the lysosome of cells, generate an immune mechanism, and achieve a certain dark toxicity and phototoxicity therapeutic effect on bacteria in vivo and in vitro. In addition, since the AIE molecule is not resistant to photodynamic therapy, it can also have a certain killing ability on clinical drug-resistant bacterial strains, and therefore has broad application prospects.

[0039] According to a typical embodiment of the present invention, a method for preparing an AIE probe targeting lysosomes is provided, the method comprising:

[0040] Step S1, obtaining a parent structure compound TPE-CHO of bithiophene tetraphenylethylene having an aldehyde group; specifically comprising:

[0041] Under an inert atmosphere, compound 1 and compound 2 are heated and refluxed in a mixed solution of tetrahydrofuran and water. After the reaction is completed, post-treatment is performed to obtain a parent structure compound TPE-CHO of bithiophene tetraphenylethylene with an aldehyde group; the reaction formula is as follows:

[0042]

[0043] As an optional implementation, in step S1,

[0044] The molar ratio of the compound 1 to the compound 2 is 1:(1-3).

[0045] The temperature of the heating reflux reaction is 80-85° C., and the reaction time is 8-15 hours.

[0046] The volume ratio of tetrahydrofuran to water is (2-3): 1. The mixed solution of tetrahydrofuran and water has the advantage of efficient Suzuki coupling. The volume ratio of tetrahydrofuran to water is (2-3): 1, which is conducive to providing a better alkaline environment and reaction integrity. If the volume ratio is too small, the alkalinity is too high, resulting in the adverse effect of premature desorption of boric ester. If the volume ratio is too large, the alkalinity is too weak, which is not conducive to the desorption of boric ester groups.

[0047] Step S2, heating the tetraphenylethylene parent structure compound TPE-CHO having an aldehyde group and the compound 3 having a benzothiazole structure under reflux in an organic solvent for reaction, and after the reaction is completed, post-treatment is performed to obtain an AIE probe targeting lysosomes; the reaction formula is as follows:

[0048]

[0049] The molar ratio of the bithiophene tetraphenylethylene parent compound having an aldehyde group to the compound 3 is 1:(1-2).

[0050] The temperature of the heating reflux reaction is 80-85° C., and the reaction time is 8-15 hours.

[0051] The organic solvent is anhydrous methanol or ethanol.

[0052] The following will describe in detail a lysosome-targeted AIE probe of the present application and its preparation method and application in combination with embodiments and experimental data.

[0053] Example 1: Synthesis of a lysosomal-targeted long-chain AIE probe (TPE-Z-3)

[0054] 1. Preparation of bithiophene tetraphenylethylene matrix structure with aldehyde group (TPE-CHO):

[0055] (1) Under nitrogen protection, weigh compound 1 (compound with tetraphenylethylene parent structure) (100 mg, 0.21 mmol), compound 2 (5-bromo-2,2'-bithiophene-5'-carboxaldehyde) (89.32 mg, 0.32 mmol), anhydrous K2CO3 (120.36 mg, 0.87 mmol), add tetrakis(triphenylphosphine)palladium (25.19 mg, 0.02 mmol) in a three-necked flask, use tetrahydrofuran / water (the volume ratio of tetrahydrofuran / water is 3:1) as the reaction solvent, and heat to reflux at 80°C for 12 h. Post-treatment: Return the reaction to room temperature, stop stirring, remove tetrahydrofuran by rotary evaporation, extract with appropriate water and dichloromethane (extract in small amounts and multiple times), and add anhydrous magnesium sulfate to the organic layer to dry. Filter and spin dry. Column chromatography was performed using a developing solvent (volume ratio: petroleum ether: dichloromethane = 10:1 to petroleum ether: dichloromethane = 1:1) to obtain the product TPE-CHO (light yellow solid, yield 92.3%). The chemical structure of TPE-CHO is:

[0056]

[0057] (2) The chemical reaction formula of the bithiophene tetraphenylethylene parent compound (TPE-CHO) having an aldehyde group is:

[0058]

[0059] (3) Characterized by NMR, 1 H NMR (400MHz, CDCl3) δ9.85 (s, 1H), 7.66 (d, J = 4.0Hz, 1H), 7.35 (d, J = 8.3Hz, 2H), 7.29 (s, 1H), 7.23 (d, J = 3.9Hz, 1H), 7.21 (s, 1H), 7.15-7.02 (m, 18H).

[0060] 2. Preparation of lysosome-targeted AIE probe (TPE-Z-3)

[0061] (1) Weigh TPE-CHO (300 mg, 0.57 mmol) in a reaction apparatus, evacuate, and use nitrogen as protective gas; add 3 (3-ethyl-2-methylbenzothiazole iodide) (198 mg, 0.65 mmol) and anhydrous methanol as solvent, heat and stir at 80°C for 10 min to fully dissolve, add a small amount of piperidine (0.5 mL), and heat and reflux at 80°C for 12 h. Post-treatment: Stop stirring, use dichloromethane to dissolve the solid and then mix with silica gel. Use a developing solvent (volume ratio: dichloromethane: methanol = 30:1 to dichloromethane: methanol = 10:1) for column chromatography separation to obtain the product TPE-Z-3 (purple solid, yield 74%). The chemical structure of TPE-Z-3 is:

[0062]

[0063] (2) The chemical reaction formula of the lysosome-targeted AIE probe (TPE-Z-3) is:

[0064]

[0065] (3) Characterized by NMR and mass spectrometry, 1 H NMR (400MHz, DMSO-d6) δ8.50-8.38(m,2H),8.26(d,J=8.5Hz,1H),7.95(d,J=4.1Hz,1H),7.89-7.83(m,1H),7.81-7.74(m,1H),7.67(d,J=15. 4Hz,1H),7.59(dt,J=7.9,4.0Hz,3H),7.53-7.48(m,2H),7.23-7.10(m,9H),7.08-6.96(m,8H),4.91(q,J=7.2Hz,2H),1.46(t,J=7.2Hz,3H). 13 C NMR(100MHz,DMSO-d6)δ170.61,144.53,144.18,143.46,143.12,143.10,142.97,1 41.23,141.11,140.98,139.91,137.83,137.47,134.60,131.66,130.88,130.80,1 30.75,130.69,129.56,128.31,128.17,128.09,128.02,127.91,126.89,126.80,1 26.04,125.61,124.85,124.43,116.48,110.82,54.96,14.20.HRMS(CH3OH):calcd for[TPE-Z-3]m / z=684.18479, found m / z=684.18417.

[0066] Experimental Example 1: Spectral Measurement

[0067] The ultraviolet absorption spectrum, AIE effect spectrum and fluorescence emission spectrum of different solvents of the lysosomal targeted AIE probe (TPE-Z-3) obtained in Example 1 were measured, and the specific steps were as follows:

[0068] (1) Accurately weigh 3 mg of AIE probe (TPE-Z-3) and dissolve it in 184 μL DMSO to prepare a 20 mM mother solution. Ultrasonicate to completely dissolve it and place it in a 2 mL centrifuge tube. Use a pipette to accurately measure 1 mL of DMSO organic solvent and place it in a 2 mL centrifuge tube. Then accurately measure 1 μL from the 20 mM AIE probe (TPE-Z-3) mother solution and place it in the above solution to prepare 1 mL of probe solution. The probe concentration is 20 μM. After fully shaking, test its ultraviolet absorption wavelength. The results are as follows Figure 2 As shown, the absorption curve of the probe in DMSO has one maximum absorption peak at around 520 nm.

[0069] (2) Accurately measure 30 μL from the 2 mM AIE probe (TPE-Z-3) mother solution and place it in a 1.5 mL mixed solvent solution with different toluene contents and a concentration of 40 μM. Test the changes in the fluorescence emission and intensity of the probe in the DMSO / Toluene mixed solution system. Results: As the toluene content increases, the fluorescence intensity of the compound increases, indicating that the compound TPE-Z-3 has good aggregation-induced emission properties.

[0070] The results are as follows Figure 2 As shown in Figure b, the fluorescence emission intensity of the probe is very weak in a pure DMSO solution or when fw is low. When fw>70%, the probe emits strong fluorescence in a mixed solution, with a fluorescence emission peak of 700nm, which is significantly increased compared to a pure DMSO solution. Therefore, the probe can emit strong fluorescence in an aggregated state, showing an excellent AIE effect.

[0071] (3) The AIE probe (TPE-Z-3) was prepared with different organic solvents (DMSO, THF, DMF, DCM, CHCl3) to a concentration of 40 μM, and its fluorescence emission wavelength was tested after being thoroughly shaken.

[0072] Results In different solvents, TPE-Z-3 exhibited different fluorescence emission ranges, indicating that it has a good solvent effect.

[0073] Experimental Example 2: Toxicity Determination

[0074] The toxicity of the lysosome-targeted AIE probe (TPE-Z-3) obtained in Example 1 to MCF-10a cells was determined, and the specific steps were as follows:

[0075] Cell viability was determined by standard WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) cell counting kit-8 assay (CCK-8, C0040, Beyotime). MCF-10a cells were plated at 5 × 10 cells per well.3 The cells were seeded at a density of 100 μL in a 96-well microplate with 100 μL of culture medium. Then, the culture medium was replaced with 100 μL of fresh culture medium containing different concentrations of TPE-Z-3 (0, 0.5, 1, 2, 5, and 10 μM) and incubated at 37°C for 10 min. The cells were irradiated under white light for 30 min (80 mW / cm 2 ), the samples were incubated at 37°C for 5 hours. DMSO was used as a solvent control. Then, 10 μL of 12 mM CCK-8 stock solution mixed with 90 μL PBS was added to each well and incubated for another 2.5 hours. The absorbance was measured at 450 nm using a SpectraMax M2 microplate reader (Molecular Devices). Cell viability (%) was calculated as follows: (OD 450 Test / OD 450 Control)×100%.

[0076] The results are as follows Figure 3 As shown, with the increase of compound concentration, MCF-10a cells showed a high survival rate regardless of illumination or no illumination, indicating that compound TPE-Z-3 has low toxicity to normal cells.

[0077] Experimental Example 3: Cell Targeting Experiment

[0078] Live MCF-10a cells were incubated with 0.5 μΜ Fluoro Lyso TM Green and 0.5 μM AIE probe (TPE-Z-3) prepared in Example 1 were incubated together for 20 min, washed three times with PBS, and then incubated with 0.5 μM DAPI for 5 min, and the cells were imaged using a confocal laser scanning microscope.

[0079] The results are as follows Figure 4 As shown, it indicates that the probe (TPE-Z-3) can be well localized in lysosomes in cells.

[0080] Experimental Example 4: Photodynamic inactivation experiment

[0081] The specific steps of the photodynamic inactivation experiment of planktonic bacteria using the lysosomal targeted AIE probe (TPE-Z-3) obtained in Example 1 are as follows:

[0082] S. epidermidis was incubated in LB medium at 37 °C overnight, harvested and diluted with PBS to OD before use. 600After incubation with different concentrations of TPE-Z-3 (0, 0.05, 0.1, 0.2 μM) at 37°C for 15 min, Staphylococcus epidermidis were washed twice and irradiated with / without white light (80 mW / cm2) for 30 min. Then, for fluorescent staining, the bacteria were detected using the Bacteria Live & Dead ELISA kit according to the manufacturer's instructions. TM The cultures were stained with an assay kit; dead cells were stained red and live cells were stained green. Bacteria were imaged using a confocal laser scanning microscope.

[0083] The results are as follows Figure 5 As shown, with the increase of compound concentration, the red fluorescence of bacteria increased, and under the illumination of 0.2 μM TPE-Z-3, the red fluorescence area of ​​Staphylococcus epidermidis was as high as 90%, indicating that TPE-Z-3 exhibited a high photodynamic sterilization effect on Staphylococcus epidermidis.

[0084] Experimental Example 5: Bacterial membrane targeting experiment

[0085] The lysosome-targeted AIE probe (TPE-Z-3) obtained in Example 1 was subjected to a bacterial membrane targeting experiment, and the specific steps were as follows:

[0086] Generally, Staphylococcus epidermidis was grown in LB liquid medium at 37°C overnight, harvested by centrifugation at 10,000 × g for 1 min, and washed twice with PBS. 600 After the value reached 0.6, Staphylococcus epidermidis was co-cultured with 1.6 μM TPE-Z-3 at 37°C for 15 min and then washed three times with PBS. Fluorescence images were recorded with a CLSM (Eclipse Ti-S, Nikon) equipped with a 100× oil immersion objective and processed using 8NIS Elements AR software (Nikon).

[0087] The results are as follows Figure 6 As shown, the cell membrane of Staphylococcus epidermidis can be clearly stained red by TPE-Z-3, indicating that TPE-Z-3 has a good membrane targeting effect.

[0088] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0089] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0090] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A lysosome-targeted AIE probe, characterized in that: The structural formula of the lysosome-targeted AIE probe is as follows:

2. A method for preparing the lysosome-targeting AIE probe according to claim 1, characterized in that: The method comprises: Under an inert atmosphere, compound 1 and compound 2 are heated and refluxed in a mixed solution of tetrahydrofuran and water. After the reaction is completed, post-treatment is performed to obtain a parent structure compound TPE-CHO of bithiophene tetraphenylethylene having an aldehyde group; wherein the volume ratio of tetrahydrofuran to water is (2-3):1, and the reaction formula is as follows: The bithiophene tetraphenylethylene parent structure compound TPE-CHO having an aldehyde group and the compound 3 having a benzothiazole structure are heated to reflux in an organic solvent for reaction, and after the reaction is completed, a post-treatment is performed to obtain an AIE probe targeting lysosomes; the reaction formula is as follows:

3. The method for preparing the lysosome-targeting AIE probe according to claim 2, characterized in that: The molar ratio of the bithiophene tetraphenylethylene parent structure compound having an aldehyde group to the compound 3 is 1:(1-2).

4. The method for preparing the lysosome-targeting AIE probe according to claim 2, characterized in that: The temperature of the heating reflux reaction is 80-85° C., and the reaction time is 8-15 hours.

5. The method for preparing the lysosome-targeting AIE probe according to claim 2, characterized in that: The organic solvent is anhydrous methanol or ethanol.

6. The method for preparing the lysosome-targeting AIE probe according to claim 2, characterized in that: The molar ratio of the compound 1 to the compound 2 is 1:(1-3).

7. Use of the lysosome-targeting AIE probe according to claim 1 in the preparation of antibacterial drugs or diabetic wound model components.

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