An aie probe targeting cell membrane and a preparation method and application thereof

Abstract

The application discloses a cell membrane-targeting AIE probe and a preparation method and application thereof, and the cell membrane-targeting AIE probe is TBTCP-QY, and the method comprises the following steps: heating and refluxing reaction of a tetraphenylethene parent structure compound BCN-PY-TPA with 3-bromo-N,N,N-trimethylpropane-1-bromide ammonium in an organic solvent, and obtaining the cell membrane-targeting AIE probe through post-treatment after the reaction is completed. The molecule with the aggregation-induced emission property is organically combined with the long-chain molecule with the positive charge together, the new probe with excellent AIE property is obtained by enhancing the charge intensity, the probe can be accurately positioned on the cell membrane of fungi, and a strong fluorescence signal is emitted. In addition, the probe can realize good photodynamic therapeutic effect on Candida albicans in and out of the body, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, and in particular to an AIE probe targeting the cell membrane, its preparation method, and its application. Background Technology

[0002] Clinically, photodynamic therapy has many advantages, mainly including: (1) no anesthesia required, short treatment time, no hospitalization required, outpatient completion, and high comfort; (2) no surgical wound, can be used as an adjunct to mechanical therapy, and may reduce the use of flap surgery; (3) rapid sterilization in a short time, reducing the development of bacterial resistance. The side effects of photodynamic therapy mainly come from photosensitizers, manifesting as phototoxicity and photosensitivity. Systemic effects include fever and constipation, which are mostly temporary and require no treatment or can be relieved by medication. Local effects manifest as photosensitivity phenomena such as erythema, itching, burning sensation, or blisters. Avoiding sunlight or strong light can improve symptoms.

[0003] Candida albicans, also known as white cephalosporin, is a typical yeast-hyphae symbiotic fungus, commonly colonizing mucosal sites such as the vagina, gastrointestinal tract, and oral cavity, as well as the skin, as the yeast phase. Through long evolutionary processes, the interaction between the fungus and its host (human) has led to complete mutual adaptation, so it generally does not cause disease. However, if host-related factors, such as weakened immunity or dysbiosis, disrupt the original microecological balance between the fungus and host, Candida albicans can transform from normal flora into an opportunistic pathogen, causing infection.

[0004] Therefore, it is important to prepare an AIE photosensitizer with excellent performance that can specifically and efficiently kill Candida albicans in vitro and combat oral and vaginal candidiasis. Summary of the Invention

[0005] The purpose of this invention is to provide an AIE probe that targets the cell membrane, its preparation method, and its application. This AIE probe that targets the cell membrane can be used as a photosensitizer to specifically and efficiently kill Candida albicans in vitro and to combat oral candidiasis and vaginal candidiasis.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect of the present invention, an AIE probe targeting the cell membrane is provided, the AIE probe targeting the cell membrane being named TBTCP-QY, and having the following structural formula:

[0008]

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

[0010] The tetraphenylene parent structure compound BCN-PY-TPA, containing thiophene-cyanothiophene and pyridine, was reacted with 3-bromo-N,N,N-trimethylpropane-1-ammonium bromide in an organic solvent under reflux. After the reaction was completed, post-processing was performed to obtain an AIE probe targeting the cell membrane. The reaction formula is shown below:

[0011]

[0012] Furthermore, the molar ratio of the compound BCN-PY-TPA to 3-bromo-N,N,N-trimethylpropane-1-ammonium bromide is 1:(1-3).

[0013] Furthermore, the temperature of the heating reflux reaction is 95-100℃, and the reaction time is 18-24h.

[0014] Furthermore, the organic solvent includes one of acetonitrile, tetrahydrofurantoluene, N-dimethylformamide, xylene, and trimethylbenzene.

[0015] Furthermore, the cell membrane includes the cell membrane of a cell or the cell membrane of a fungus.

[0016] In a third aspect of the invention, the use of the AIE probe targeting the cell membrane in the preparation of anti-Candida drugs is provided.

[0017] Furthermore, the fungal disease includes at least one of oral candidiasis and vaginal candidiasis.

[0018] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0019] The method for preparing an AIE probe targeting the cell membrane provided by this invention organically combines molecules with aggregation-induced emission properties with positively charged long-chain molecules, thereby enhancing the charge intensity to obtain a novel probe with excellent AIE properties. This probe can accurately locate on the cell membrane of fungi and emit a strong fluorescent signal. Furthermore, it can achieve good photodynamic therapy against Candida albicans in vitro and in vivo, showing broad application prospects: it can target and bind to the cell membrane of Candida albicans, generating a large amount of ROS after light exposure, effectively inactivating Candida albicans. TBTCP-QY has a high molar absorption coefficient and excellent ROS generation efficiency, effectively inactivating Candida albicans; it inhibits biofilm formation by suppressing the expression of hydrolysis, adhesion, and drug resistance genes; it eradicates mature fungal biofilms; and it kills biofilms at concentrations as low as 0.8 μM using PDT. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 The image shows the 1H NMR spectrum of TBTCP-QY.

[0022] Figure 2 This is the 13C NMR spectrum of TBTCP-QY.

[0023] Figure 3 This is the UV absorption spectrum of TBTCP-QY.

[0024] Figure 4 The fluorescence spectrum of TBTCP-QY varies with different toluene / DMSO ratios.

[0025] Figure 5 A schematic diagram illustrating the targeting of the fungal cell membrane by the compound TBTCP-QY;

[0026] Figure 6 This is a schematic diagram illustrating the photodynamic inactivation of Candida albicans by the compound TBTCP-QY.

[0027] Figure 7 This image shows a representative image of tongue tissue during the treatment of oral candidiasis using the compound TBTCP-QY.

[0028] Figure 8 These are representative images of vaginal tissue during the treatment of vaginal candidiasis using the compound TBTCP-QY. Detailed Implementation

[0029] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0030] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0031] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be obtained by purchasing them from the market or by existing methods.

[0032] This invention provides an AIE probe targeting the cell membrane, the general concept of which is as follows:

[0033] According to a typical embodiment of the present invention, an AIE probe targeting the cell membrane is provided. The AIE probe targeting the cell membrane is named TBTCP-QY and has the following structural formula:

[0034]

[0035] According to a typical embodiment of the present invention, a method for preparing an AIE probe targeting the cell membrane is provided, the method comprising:

[0036] The tetraphenylene parent structure compound BCN-PY-TPA, containing thiophene-cyanothiophene and pyridine, was reacted with 3-bromo-N,N,N-trimethylpropane-1-ammonium bromide in an organic solvent under reflux. After the reaction was completed, post-processing was performed to obtain an AIE probe targeting the cell membrane. The reaction formula is shown below:

[0037]

[0038] In a preferred embodiment, the molar ratio of the compound BCN-PY-TPA to 3-bromo-N,N,N-trimethylpropane-1-ammonium bromide is 1:(1-3). This molar ratio range is conducive to a complete and sufficient reaction.

[0039] In a preferred embodiment, the reflux reaction temperature is 95-100°C, and the reaction time is 18-24 hours. Temperatures that are too high or too low are detrimental to the reaction.

[0040] In one preferred embodiment, the organic solvent includes one of acetonitrile, tetrahydrofurantoluene, dimethylformamide, xylene, and trimethylbenzene.

[0041] The cell membrane includes the cell membrane of a cell or the cell membrane of a fungus.

[0042] This invention provides an AIE probe targeting the cell membrane and its preparation method, breaking with conventional approaches in existing technologies. It employs a simple one-step synthesis of the cell membrane-targeting AIE probe TBTCP-QY. By organically combining molecules with aggregation-induced emission properties with positively charged long-chain molecules, and enhancing charge strength, a novel probe with excellent AIE properties is obtained. This probe can specifically and efficiently kill Candida albicans in vitro and combat oral and vaginal candidiasis.

[0043] This probe is constructed using an enhanced D-π-A structure, which reduces the band gap between the singlet and triplet excited states, facilitating cross-linking between systems and improving ROS generation efficiency. TBTCP possesses the inherent properties of a D-π-A amphiphilic structure and a non-planar conformation mimicking intramembrane phospholipid structures, which is beneficial for high ROS generation efficiency and membrane targeting. TBTCP-QY can selectively bind to Candida albicans membranes through hydrophobic and electrostatic interactions, thereby enhancing the photodynamic antifungal effect against planktonic Candida albicans. Furthermore, TBTCP-QY not only inhibits biofilm formation and effectively eradicates mature biofilms but also suppresses drug resistance, demonstrating excellent anti-infective properties in mouse models of oral and vaginal candidiasis infected with Candida albicans.

[0044] The following will provide a detailed description of an AIE probe targeting the cell membrane, its preparation method, and its application, in conjunction with embodiments and experimental data.

[0045] Example 1: Synthesis of TBTCP-QY

[0046] 1. Add BCN-Py-TPA (107.5 mg, 0.2 mmol) and 1.8 mmol of 3-bromo-N,N,N-trimethylpropane-1-ammonium bromide to 15 mL of acetonitrile, then transfer the mixture to a 50 mL round-bottom flask and heat under reflux. Heat the mixture to 95-100 °C for 24 h, monitoring the reaction progress with a silica gel plate.

[0047] 2. After the reaction is complete, the system is cooled to room temperature, the solvent is removed under vacuum, and the crude product is purified by silica gel column chromatography using DCM / MeOH (v / v = 5:1) as the eluent to obtain a dark red solid, which is the AIE probe targeting the cell membrane.

[0048] 3. Characterization by NMR and mass spectrometry: 1H NMR (400MHz, DMSO-d6) δ. 9.05 (d, J = 4.8Hz, 2H), 8.49 (d, J = 5.6Hz, 2H), 8.38 (s, 1H), 8.13 (s, 1H), 7.93 (d, J = 3.6Hz, 1H), 7.62 (d, J = 7.2Hz, 2H), 7.51 (dd, J = 3.2Hz, 10Hz, 2H) ),7.36(t,J=6.0Hz,4H),7.12(t,J=6.0Hz,2H),7.10(d,J=6.4Hz,4H)7.00(d,J=6.4H z,2H),4.62(t,J=5.6Hz,2H),3.40-3.86(m,2H),3.08(s,9H),2.46-2.41(m,2H).13C NMR(150MHz,DMSO-d6)δ.153.0,152.5,151.8,151.1,150.3,148.9,144.7,141.2,140.9,137.8,136.0,135.0,134.7,131 .9,131.3,130.0,129.3,129.1,128.1,127.5,121.5,109.9,67.0,65.0,62.1,29.3.HRMS(ESI):m / z[M-2Br-]+calculated for C40H38N4S2:638.2527; found 638.2563.

[0049] Example 2: Performance Measurement of TBTCP-QY

[0050] 1. The specific steps for measuring the ultraviolet absorption spectrum of the AIE probe (TBTCP-QY) targeting the fungal cell membrane obtained in Example 1 are as follows:

[0051] Accurately weigh a certain amount of AIE probe (TBTCP-QY) and dissolve it in DMSO to prepare a 20 mM stock solution. Sonicate the solution until completely dissolved and place it in a 2 mL centrifuge tube. Accurately pipette 1 mL of DMSO organic solvent into the 2 mL centrifuge tube. Then, accurately measure 1 μL from the 20 mM AIE probe (TBTCP-QY) stock solution and add it to the above solution to prepare a 1 mL probe solution with a probe concentration of 20 μM. Shake thoroughly before measuring its UV absorption wavelength.

[0052] The results are as follows Figure 1-3 As shown, the probe absorption curve in DMSO has a maximum absorption peak at 522 nm.

[0053] 2. Properties of aggregation-induced emission

[0054] 30 μL of the 2 mM AIE probe (TBTCP-QY) stock solution was accurately measured and placed in a 1.5 mL mixture of toluene and DMSO to prepare a mixed solvent solution with different toluene contents, with a concentration of 40 μM. The changes in fluorescence emission and intensity of the probe in the DMSO / Toluene mixed solution system were then tested.

[0055] The results are as follows Figure 4 As shown, the fluorescence intensity of the compound increases with increasing toluene content, indicating that the compound TBTCP-QY has excellent aggregation-induced emission properties.

[0056] Example 3: Cell membrane targeting experiment of TBTCP-QY

[0057] Under normal circumstances, *Candida albicans* was cultured overnight at 28°C in YPD liquid medium, centrifuged at 10000×g for 1 minute, and washed twice with PBS. After diluting with PBS to OD600 = 0.6, it was co-cultured with TBTCP-QY and DAPI at final concentrations of 5 μM and 10 μM, respectively, at 37°C for 15 min, followed by three washes with PBS. Fluorescence images were recorded using a CLSM (EclipseTi-S, Nikon) equipped with a 100x oil immersion objective.

[0058] The results are as follows Figure 5 As shown, TBTCP-QY can be clearly targeted at the cell edge of Candida auris cells, indicating that TBTCP-QY has excellent targeting effects.

[0059] Example 4: TBTCP-QY staining experiment on the viability and inactivation of Candida albicans

[0060] Candida albicans was incubated overnight at 37°C in YPD medium, harvested, and diluted with PBS to an OD600 value of 0.1 before use. After incubation at 37°C with different concentrations of TBTCP-QY (0, 0.4, 0.8 μM) for 15 minutes, Staphylococcus epidermidis was washed twice and treated with / without white light (80 mW / cm²). 2 Irradiate for 30 minutes. Then use for fluorescent staining, staining the culture with the Live & Dead™ assay kit according to the manufacturer's instructions; dead fungi are stained red, while all fungi are stained green. Fluorescence images were recorded using a CLSM (Eclipse Ti-S, Nikon) equipped with a 100x oil immersion objective.

[0061] The results are as follows Figure 6 As shown, in the 0.8 μM TBTCP-QY group, almost all Candida albicans showed red fluorescence, proving that 0.8 μM TBTCP-QY has a good ability to kill Candida albicans.

[0062] Example 5: Animal Experiment

[0063] 1. Animal experiments of TBTCP-QY on a mouse model of oral candidiasis

[0064] like Figure 8 As shown, a mouse oral candidiasis model was established using 12-week-old male C57BL / 6 mice. The mice were randomly divided into different groups (n=5 per group). The mice were fed drinking water containing 2.5 g / L tetracycline for 5 consecutive days to prevent infection by unknown bacteria. One day before inoculation, 250 mg / kg prednisolone was injected intramuscularly to induce immunosuppression. The mice were then randomly divided into 4 groups of 5 mice each. During treatment, the mice were divided into the following four groups: (1) PBS group, the tongues of OC mice were washed with 20 μL PBS every day. (2) CHX group, OC mice were given 0.12% CHX every day. (3) TBTCP-QY+L group, the tongues of mice were injected with 20 μL TBTCP-QY (5 μM) every day and irradiated with white light for 15 min. (4) Control group, the tongues of uninfected Candida albicans mice were washed with 20 μL PBS every day. 72 h after infection, the lesions and recovery of the tongue mucosa were recorded using digital equipment.

[0065] Experimental results are as follows Figure 7 As shown, this demonstrates that TBTTCP-QY exhibits superior curative ability for oral candidiasis compared to chlorhexidine.

[0066] 2. The animal experiments of the AIE probe targeting the fungal cell membrane obtained in Example 1 on a mouse model of vulvovaginal candidiasis and candidiasis are as follows:

[0067] The mouse VVC model was constructed using 6-week-old female BALB / c mice, which were randomly divided into different groups (n=5 per group). The mice were infected with the standard strain SC5314. SC5314 was cultured overnight at 28℃, and the OD600 was 0.25. 20 μL of Candida albicans suspension was dripped into the vagina of the mice and the mice were inverted for 5 min. Treatment of VVC in mice: (1) PBS group: VVC mice were rinsed with 20 μL of PBS every day. (2) Fluconazole group: VVC mice were orally administered 0.3 mg of fluconazole every day. (3) TBTCP-QY+L group: VVC mice were injected with 20 μL of TBTCP-QY (5 μM) into the vagina every day, inverted for 15 min, and then irradiated with white light for 15 min. (4) Control group: The vaginas of uninfected mice were washed with 20 μL of PBS every day. After 72 h of infection, the vaginal mucosal lesions and recovery were recorded using digital equipment.

[0068] Experimental results are as follows Figure 8 As shown, this demonstrates that TBTTCP-QY exhibits superior curative efficacy against vaginal candidiasis compared to fluconazole.

[0069] In summary, TBTCP-QY of the present invention can selectively bind to Candida albicans membranes through hydrophobic and electrostatic interactions, thereby enhancing the photodynamic antifungal effect against planktonic Candida albicans. Furthermore, TBTCP-QY exhibited good photodynamic therapy (PDT) performance in mouse models of oral candidiasis and vulvovaginal candidiasis, suggesting its potential application value in the treatment of oral candidiasis and VVC.

[0070] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0071] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0072] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A cell membrane-targeted AIE probe, characterized in that, The name of the target cell membrane AIE probe is TBTCP-QY, and the structural formula is as follows: 。 2. A preparation method of the cell membrane-targeted AIE probe according to claim 1, characterized in that, The method comprises: The compound BCN-PY-TPA with a tetraphenylethene parent structure of thiophene-cyan-thiophene and pyridine is reacted with brominated (3-bromopropyl) trimethylammonium in an organic solvent by heating reflux, after the reaction is completed, the target cell membrane AIE probe is obtained by post-treatment, and the reaction formula of the reaction is as follows: 。 3. The method for preparing an AIE probe targeting the cell membrane according to claim 2, characterized in that, The molar ratio of the compound BCN-PY-TPA to brominated (3-bromopropyl) trimethylammonium is 1: (1-3).

4. The preparation method of the cell membrane-targeted AIE probe according to claim 2, characterized in that, The heating reflux reaction is carried out at a temperature of 95-100 °C, and the reaction time is 18-24 h.

5. The method of claim 2, wherein the AIE probe is targeted to a cell membrane. The organic solvent comprises one of acetonitrile, tetrahydrofuran, toluene, N, N dimethylformamide, dimethylbenzene and trimethylbenzene.

6. The method of claim 2, wherein the AIE probe is targeted to a cell membrane. The cell membrane is a cell membrane of fungi.

7. The method of claim 2, wherein the AIE probe is targeted to a cell membrane. The post-treatment comprises: after cooling to room temperature, the solvent is removed under vacuum, and the crude product is purified by silica gel column chromatography.

8. The target cell membrane AIE probe in claim 1 is applied to the preparation of an anti-Candida albicans drug.

9. Use according to claim 8, characterized in that, The Candida albicans comprises at least one of oral Candida albicans and vaginal Candida albicans.

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