Pyridinium AIE photosensitizer and antifungal application thereof

Abstract

The invention discloses an AIE pyridinium surfactant and antifungal application thereof.The AIE pyridinium surfactant is obtained by introducing an alkyl chain to an AIE photosensitizer group through a simple quaternization reaction, and the AIE pyridinium surfactant permeates into fungi based on electrostatic and hydrophobic effects, so that efficient antifungal activity is shown; in addition, under the irradiation of white light, active oxygen can be effectively generated, and the antifungal efficiency is further improved.

Description

Technical Field

[0001] The invention relates to a pyridinium salt AIE photosensitizer and antifungal application thereof, belonging to the technical field of biological application materials. Background Art

[0002] Fungal infections are the leading cause of infectious disease-related deaths worldwide, among which Candida is the main cause of invasive fungal infections, but the high evolutionary similarities between fungal and mammalian cells greatly limit the development of antifungal drugs. With the emergence of fungal resistance, there are currently few types of antifungal drugs available in clinical practice, which poses a serious threat to human safety. There is an urgent need to develop antifungal drugs with novel mechanisms of action.

[0003] In recent years, photodynamic therapy (PDT) has been used as a non-antibiotic treatment method with the advantages of non-invasiveness, high spatiotemporal resolution, and low side effects. Due to the aggregation-induced quenching (ACQ) problem of traditional photosensitizers, their singlet oxygen production performance is significantly inhibited when they aggregate in an aqueous environment. In contrast, aggregation-induced emission (AIE) molecules exhibit enhanced fluorescence emission and high singlet oxygen production performance in the aggregated state. Therefore, AIE photosensitizers usually exhibit higher photodynamic efficiency than traditional photosensitizers when aggregated.

[0004] Therefore, a new pyridinium salt AIE photosensitizer compound is needed to solve the above problems. Summary of the invention

[0005] Purpose of the invention: To address the problems existing in the prior art, the present invention provides a compound.

[0006] A compound having a structure as shown in Formula 1:

[0007]

[0008] Wherein, G is an alkyl chain; X - is a counter anion; each R is independently selected from a hydrogen group, a hydroxyl group, an amino group or an alkyl group.

[0009] Furthermore, the alkyl chain G is butane or hexane.

[0010] Furthermore, the counter anion X - Anions have one charge.

[0011] Furthermore, the compound has a structure as shown in Formula 2 or Formula 3:

[0012]

[0013] Beneficial effects: The compound of the present invention introduces a hydrophobic chain into the AIE photosensitizer group to construct a cationic amphiphilic AIE photosensitizer, which achieves effective binding with fungi based on electrostatic and hydrophobic effects, thereby being able to efficiently kill fungi; under white light irradiation, active oxygen can be generated, further improving the killing efficiency of fungi.

[0014] The invention also discloses the use of the compound as a fungicide.

[0015] Beneficial effects: The compounds of the present invention belong to a cationic AIE photosensitizer system and can be used as antifungal agents, which have high killing power against fungi.

[0016] Furthermore, the fungus is Candida albicans. The compound of the present invention has a highly effective antibacterial activity against Candida albicans.

[0017] The invention also discloses that the compound is used as a probe for fungus detection or imaging.

[0018] Beneficial effects: Due to the introduction of the AIE group, the compound of the present invention is endowed with AIE properties, and the visual monitoring of the antibacterial mechanism of this type of photosensitizer is achieved based on its fluorescence properties. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The synthetic route of AIE pyridinium surfactants TPy-Bu and TPy-Hex;

[0020] Figure 2 The UV-visible absorption spectrum and fluorescence spectrum of TPy-Bu with a concentration of 20 μM in PBS solution, where (a) is the UV-visible absorption spectrum and (b) is the fluorescence spectrum with an excitation wavelength of 420 nm;

[0021] Figure 3 is the fluorescence spectra of TPy-Bu with different concentrations in PBS solution, with an excitation wavelength of 420 nm;

[0022] Figure 4 The particle size distribution diagram and potential diagram of TPy-Bu with a concentration of 10 μM forming aggregates in PBS solution, wherein (a) is the particle size distribution diagram; (b) is the potential diagram;

[0023] Figure 5 To detect the ABDA as a probe under white light irradiation (60mW / cm 2) The active oxygen generation performance of TPy-Bu, wherein (a) is the UV-visible absorption spectrum of 50 μM ABDA in PBS solution as a function of illumination time, (b) is the UV-visible absorption spectrum of ABDA as a function of illumination time after the addition of TPy-Bu, and (c) is the absorbance of ABDA at 379 nm as a function of white light irradiation time before and after the addition of TPy-Bu;

[0024] Figure 6 The CLSM images of Candida albicans cultured with 10 μM TPy-Bu for 40 min, where (a) is a fluorescence image; (b) is a bright field image; (c) is an overlay field image;

[0025] Figure 7 The antibacterial properties of TPy-Bu at different concentrations against Candida albicans in the dark or under light and the photos of colonies growing on agar plates, wherein (a) is the antibacterial properties of TPy-Bu at different concentrations against Candida albicans in the dark or under light, and (b) is the photos of colonies growing on agar plates of Candida albicans after being treated with TPy-Bu at different concentrations in the dark or under light;

[0026] Figure 8 For TPy-Bu 1 HNMR spectrum (600 MHz, CDCl 3 );

[0027] Fig. 9 For TPy-Bu 13 CNMR spectrum (600MHz, CDCl 3 );

[0028] Fig.10 MALDI-TOF high-resolution mass spectrum of TPy-Bu;

[0029] Fig.11 The UV-visible absorption spectrum and fluorescence spectrum of TPy-Hex with a concentration of 20 μM in PBS solution, where (a) is the UV-visible absorption spectrum; (b) is the fluorescence spectrum, with an excitation wavelength of 420 nm;

[0030] Fig.12 is the fluorescence spectra of TPy-Hex with different concentrations in PBS solution, with an excitation wavelength of 420 nm;

[0031] Fig.13 The particle size distribution and potential diagram of TPy-Hex aggregates formed in PBS solution with a concentration of 10 μM, (a) is the particle size distribution diagram; (b) is the potential diagram;

[0032] Fig.14 To detect the ABDA as a probe under white light irradiation (60mW / cm 2) TPy-Hex active oxygen generation performance diagram, wherein (a) is a graph showing the change of the UV-visible absorption spectrum of 50 μM ABDA in PBS solution with the illumination time, (b) is a graph showing the change of the UV-visible absorption spectrum of ABDA with the illumination time after adding TPy-Hex, and (c) is a graph showing the change of the absorbance of ABDA at 379 nm with the white light irradiation time before and after adding TPy-Hex;

[0033] Fig.15 The CLSM images of Candida albicans cultured with 10 μM TPy-Hex for 40 min, where (a) is a fluorescence image; (b) is a bright field image; (c) is an overlay field image;

[0034] Fig.16 Figure 2 shows the antibacterial performance of TPy-Hex of different concentrations against Candida albicans in the dark or under light and photos of colonies grown on agar plates, where (a) shows the antibacterial performance of TPy-Hex of different concentrations against Candida albicans in the dark or under light, and (b) shows photos of colonies grown on agar plates of Candida albicans after being treated with TPy-Hex of different concentrations in the dark or under light;

[0035] Fig.17 For TPy-Hex 1 HNMR spectrum (600 MHz, CDCl 3 );

[0036] Fig.18 For TPy-Hex 13 CNMR spectrum (600MHz, CDCl 3 );

[0037] Fig.19 This is the MALDI-TOF high-resolution mass spectrum of TPy-Hex. DETAILED DESCRIPTION

[0038] The preferred embodiments of the present invention will be described below in conjunction with the accompanying drawings to more clearly and completely illustrate the technical solutions of the present invention.

[0039] Example 1

[0040] TPy-Bu was prepared via a facile synthetic route.

[0041] like Figure 1 As shown, under nitrogen protection, compound TPy (100 mg, 0.2 mmol) was dissolved in 7 mL of N,N-dimethylformamide, and then CH 3 (CH 2 ) 3Br (108.79 mg, 0.8 mmol), the reaction mixture was reacted at 70°C for 14 h. After the reaction, the solvent was evaporated and purified by silica gel column chromatography using an eluent (dichloromethane / methanol). After vacuum drying, orange-red powder TPy-Bu was obtained.

[0042] Confirmation of TPy-Bu structure

[0043] like Figure 8 , Fig. 9 and Fig.10 As shown, combined with the hydrogen spectrum, carbon spectrum and high-resolution mass spectrum of TPy-Bu, it can be confirmed that the structure of the molecule is as shown in the following figure:

[0044]

[0045] Characterization of photophysical properties of TPy-Bu

[0046] like Figure 2 As shown in the UV-visible absorption spectrum of part a, the maximum absorption of TPy-Bu is at 426 nm. Figure 2 As shown in part b, the maximum emission wavelength is around 587nm. Figure 3 As shown in the figure, with the increase of TPy-Bu concentration, the fluorescence intensity gradually increases, showing a typical AIE property.

[0047] Characterization of aggregation properties of TPy-Bu

[0048] Dynamic light scattering (DLS) was used to characterize the particle size and potential of TPy-Bu. Figure 4 As shown, the aggregates formed by 10 μM TPy-Bu have a particle size distribution of about 400 nm and a potential value of 13 mV, which is conducive to electrostatic binding with the negatively charged surface of fungi.

[0049] Evaluation of the ability of TPy-Bu to generate singlet oxygen

[0050] The ability of TPy-Bu to generate singlet oxygen was tested using the commercial probe 9,10-anthracenediyl-bis(methylene)dimalonic acid (ABDA). The characteristic absorption peak of ABDA decreased after being oxidized by singlet oxygen. Therefore, the ability of TPy-Bu to generate singlet oxygen was evaluated based on the degree of decrease in the characteristic absorption peak of ABDA. 10 μL of probe ABDA (10 mM) was added to 2 mL of TPy-Bu (20 μM) PBS solution, mixed well, and then heated at 60 mW / cm 2 Irradiate with white light and measure the absorbance value at regular intervals. Figure 5As shown in the figure, under illumination, in the control group without TPy-Bu, the characteristic absorption peak intensity of ABDA remained basically unchanged, but after adding TPy-Bu molecules, the absorbance of ABDA decreased with the illumination time, confirming that TPy-Bu can effectively produce singlet oxygen. 0 and A are the absorbances of ABDA at 379 nm before and after irradiation, respectively. The concentrations of TPy-Bu and ABDA are 10 μM and 50 μM, respectively.

[0051] The interaction of TPy-Bu with fungi

[0052] like Figure 6 As shown, it can be clearly seen that after the addition of TPy-Bu, Candida albicans is illuminated and emits a strong orange-yellow fluorescence, indicating that TPy-Bu has a strong interaction with the fungus. Figure 6 Imaging conditions: For TPy-Bu, the excitation wavelength was 405 nm and the emission wavelength range was 500-700 nm.

[0053] Evaluation of antifungal activity of TPy-Bu

[0054] In this experiment, Candida albicans was used as an example to evaluate the antibacterial effect of TPy-Bu in the dark and under light by the traditional plating method. The monoclonal colony was placed in 10 mL of YPD liquid culture medium and cultured in a shaking incubator at 30°C and 180 rpm for 20-24 hours. A certain volume of bacterial solution was taken, the culture medium was removed by centrifugation (7000 rpm, 2 min), washed twice with PBS, and finally suspended in PBS and the bacterial solution concentration was adjusted to OD 600 =2.0. In the dark room, the bacterial solution was exposed to different concentrations of TPy-Bu at 30°C for 40 min. In the light group, the bacterial solution was exposed to different concentrations of TPy-Bu at 30°C for 10 min, using 60 mW / cm 2 irradiated with white light for 30 min; then diluted 10 4 Times, take 100 μL of the diluted bacterial solution and evenly spread it on the YPD solid medium (solid medium plate size is 90 mm), and conduct three parallel experiments for each concentration, culture at 30°C for 22-24 hours, and count the number of plaques. The bactericidal activity (IR) of TPy-Bu is calculated according to the following formula: Where A is the number of colony forming units (cfu) in the control group, and B is the number of colony forming units (cfu) in the experimental group.

[0055] like Figure 7 As shown in Figure 2, when the concentration of TPy-Bu was 20 μM, the bactericidal efficiency was 58.1%. 2) irradiated for 30 min, the bactericidal efficiency reached 100%, indicating that TPy-Bu has a high killing efficiency against fungi.

[0056] Example 2

[0057] Characterization of photophysical properties of TPy-Hex

[0058] like Fig.11 As shown in the UV-visible absorption spectrum of part a, the maximum absorption of TPy-Hex is at 472nm. Fig.11 As shown in part b, the maximum emission wavelength is around 626nm. Fig.12 As shown, TPy-Hex exhibits typical AIE properties, and its fluorescence intensity gradually increases with the increase of the concentration of TPy-Hex solution.

[0059] Characterization of aggregation properties of TPy-Hex

[0060] Dynamic light scattering (DLS) was used to characterize the particle size and potential of TPy-Hex. Fig.13 As shown, the aggregates formed by 10 μM TPy-Hex have a particle size distribution of about 955 nm and a potential value of 15 mV, which is conducive to electrostatic binding with the negatively charged surface of fungi.

[0061] Evaluation of the ability of TPy-Hex to generate singlet oxygen

[0062] The ability of TPy-Hex to generate singlet oxygen was tested using the commercial probe 9,10-anthracenediyl-bis(methylene)dimalonic acid (ABDA). The characteristic absorption peak of ABDA decreased after being oxidized by singlet oxygen. Therefore, the ability of TPy-Hex to generate singlet oxygen was evaluated based on the degree of decrease in the characteristic absorption peak of ABDA. 10 μL of probe ABDA (10 mM) was added to 2 mL of TPy-Hex (10 μM) PBS solution, mixed well, and then charged with 60 mW / cm 2 Irradiate with white light and measure the absorbance value at regular intervals. Fig.14 As shown, under illumination, in the control group without TPy-Hex, the characteristic absorption peak intensity of ABDA remained basically unchanged, while after adding TPy-Hex molecules, the absorbance of ABDA decreased with the illumination time, confirming that TPy-Hex can produce singlet oxygen. Fig.14 Part C, A 0 A and A are the absorbance of ABDA at 379 nm before and after irradiation, respectively. The concentration of TPy-Hex is 10 μM, and the concentration of ABDA is 50 μM.

[0063] TPy-Hex and fungi

[0064] like Fig.15As shown, it can be clearly seen that after adding TPy-Hex, Candida albicans is illuminated and emits strong orange-yellow fluorescence, indicating that TPy-Hex has a strong interaction with fungi. Fig.15 Imaging conditions: For TPy-Hex, excitation wavelength: 405 nm, emission wavelength range: 500-700 nm.

[0065] Evaluation of antifungal activity of TPy-Hex

[0066] In this experiment, Candida albicans was used as an example to evaluate the antibacterial effect of TPy-Hex in the dark and under light by the traditional plating method. The monoclonal colony was placed in 10 mL of YPD liquid culture medium and cultured in a shaking incubator at 30°C and 180 rpm for 20-24 hours. A certain volume of bacterial solution was taken, the culture medium was removed by centrifugation (7000 rpm, 2 min), washed twice with PBS, and finally suspended in PBS and the bacterial solution concentration was adjusted to OD 600 =2.0. In the dark room, the bacterial solution was exposed to different concentrations of TPy-Hex at 30°C for 40 min. In the light group, the bacterial solution was exposed to different concentrations of TPy-Hex at 30°C for 10 min, using 60 mW / cm 2 irradiated with white light for 30 min; then diluted 10 4 Times, take 100μL of the diluted bacterial solution and evenly spread it on the YPD solid medium (solid medium plate size is 90mm), and conduct three parallel experiments for each concentration, culture at 30℃ for 22-24h, and count the number of plaques. The bactericidal activity (IR) of TPy-Hex is calculated according to the following formula: Among them, A is the number of colony forming units (cfu) in the control group, and B is the number of colony forming units (cfu) in the experimental group.

[0067] like Fig.16 As shown in the figure, when the concentration of TPy-Hex was 20 μM, the sterilization efficiency was 35.4% in the dark, and further in white light (60 mW / cm 2 ) irradiated for 30 minutes, its bactericidal efficiency can be increased to 94.2%. With the increase of concentration, the effect is more significant. When the concentration is 30μM, the activity in the dark increases to 67.4%. After combined with light, the antibacterial activity is as high as 97.8%. When the concentration increases to 50μM, the bactericidal efficiency combined with light can be increased to 100%. It shows that TPy-Hex has a high killing efficiency against fungi.

[0068] like Fig.17 , Fig.18 and Fig.19 As shown, Fig.17 For TPy-Hex 1 HNMR spectrum (600 MHz, CDCl 3 ); Fig.18 For TPy-Hex 13 CNMR spectrum (600MHz, CDCl 3 ); Fig.19 This is the MALDI-TOF high-resolution mass spectrum of TPy-Hex.

Claims

1. A compound, characterized in that It has a structure as shown in Formula 1: Wherein, G is an alkyl chain; X - is a counter anion; each R is independently selected from hydrogen, hydroxyl, amino or alkyl.

2. The compound according to claim 1, characterized in that The alkyl chain G is butane or hexane.

3. The compound according to claim 1, characterized in that Counter anion X - Anions have one charge.

4. The compound according to claim 1, characterized in that The compound has a structure as shown in Formula 2 or Formula 3:

5. Use of the compound according to any one of claims 1 to 4 as a fungicide.

6. The use according to claim 5, characterized in that The fungus is Candida.

7. The compound according to any one of claims 1 to 4 is used as a probe for fungal detection or imaging.

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