AIE molecular material and application thereof in preparation of medicine for treating candida albicans infectious vaginitis

Abstract

The invention belongs to the technical field of biological medicine, and particularly discloses an AIE molecular material and application of the AIE molecular material in preparation of a medicine for treating candida albicans infectious vaginitis. The AIE molecule prepared by the invention can specifically bind and kill candida albicans in an aggregation state, pathogenic bacteria can be completely removed within 30 minutes under 100 mu M, and lactobacillus and vaginal epithelial cells are not damaged. The action mechanism comprises the steps of inducing release of fungal reactive oxygen species (ROS), destroying a cell structure, and remarkably reducing inflammatory factors (IL-6 and IL-1beta) and reducing hypha and neutrophil infiltration through animal experiment verification. And after treatment, the diversity of vaginal microbiota and abundance of lactobacillus are remarkably improved, and the recurrence risk is reduced. The molecule has the functions of fluorescence imaging diagnosis and treatment, is simple and convenient to synthesize, stable and efficient, provides an accurate and safe novel scheme for VVC treatment, and has remarkable clinical application potential.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a precise antifungal material based on aggregation-induced emission (AIE) active cationic amphiphilic molecules and its application. Background Art

[0002] Vulvovaginal candidiasis (VVC) is a highly prevalent gynecological infectious disease caused by Candida albicans. Globally, approximately 75% of women will be infected at least once in their lifetime, and 40%-50% will experience recurrences. In the United States, VVC is second only to bacterial vaginosis in incidence, with a prevalence of 17.6% in non-pregnant women and as high as 30.2% in pregnant women. The high incidence of VVC is associated with multiple factors, including increased vaginal glycogen, decreased pH, and imbalanced vaginal microecology caused by long-term use of antibiotics and immunosuppressants.

[0003] Currently, the main challenge in treating VVC lies in the limitations of existing antifungal drugs. Traditional drugs such as azoles, echinocandins, and polyenes, while effective against fungi, suffer from indiscriminate killing, killing both beneficial and pathogenic bacteria, disrupting the vaginal microbiome. This disruption not only weakens the vagina's defenses but also leads to the emergence of drug-resistant bacteria. Long-term use of these drugs can also cause side effects such as gastrointestinal discomfort, liver and kidney dysfunction, and allergic reactions, prolonging treatment cycles and increasing the financial and psychological burden on patients.

[0004] The causes of fungal resistance are complex, primarily due to prolonged or inappropriate use of antifungal drugs, which allows resistant strains to survive and thrive. The consequences of resistance are severe, making infections difficult to cure, causing recurrence, and even potentially developing into life-threatening systemic infections. Therefore, rapid identification and precise destruction of pathogenic bacteria are crucial for preventing and controlling fungal infections. Summary of the Invention

[0005] In order to solve the above-mentioned problems of the prior art, the present invention discloses an AIE molecular material and its use in the preparation of a drug for the treatment of Candida albicans infectious vaginitis. Three cationic amphiphilic molecules with AIE activity are developed, in which a tetraphenylethylene (TPE) portion is introduced as a hydrophobic fluorescent skeleton and connected to a hydrophilic ammonium head through a linker (di(oxyethyl)). On this basis, the hydrophilic ammonium head is connected to three different alkyl chains (i.e., ethane, butane, and hexane), respectively named TPE-ET, TPE-BU, and TPE-HEX. The AIE molecules prepared by the present invention can specifically bind to and kill Candida albicans in an aggregated state, completely eliminating the pathogen in 30 minutes at 100 μM, and do not damage lactobacilli and vaginal epithelial cells.

[0006] The present invention includes the following technical solutions:

[0007] An AIE molecular material, comprising a tetraphenylethylene (TPE) as a hydrophobic fluorescent backbone, a dioxyethyl linker connected to a hydrophilic ammonium head, and further connected to an alkyl chain substituent;

[0008] The alkyl chain is selected from one or more of ethane, butane or hexane chains.

[0009] Furthermore, the above-mentioned AIE molecular material specifically recognizes Candida albicans and emits blue fluorescence in an aggregated state, and has no recognition or killing effect on Lactobacillus.

[0010] The present invention also discloses a method for preparing the above-mentioned AIE molecular material, comprising the following steps:

[0011] a. Synthesis of 4-(1,2,2-triphenylvinyl)phenol (1): Benzophenone, 4-hydrobenzophenone and zinc powder were reacted in tetrahydrofuran, extracted with hydrochloric acid and purified by silica gel column chromatography to obtain compound (1);

[0012] b. Synthesis of 2-(4-(2-(2-bromoethoxy)ethoxy)phenyl)ethylene-1,1,2-triyl)-triphenylbenzene (2): Compound (1) was reacted with dibromoethyl ether in acetone and purified by silica gel column chromatography to obtain compound (2);

[0013] c. Synthesis of TPE-ET, TPE-BU or TPE-HEX: Compound (2) is reacted with the corresponding alkyl chain amine in ethanol under reflux, and the target AIE molecular material is obtained by solvent removal and precipitation purification.

[0014] Furthermore, in the above preparation method, the alkyl chain amine in step c is ethylamine, butylamine or hexylamine.

[0015] The present invention also discloses a pharmaceutical composition for treating Candida albicans infectious vaginitis, comprising the above-mentioned AIE molecular material and a pharmaceutically acceptable carrier, wherein the concentration of the AIE molecular material in the composition is 1.0×10-3M to 5.0×10-3M.

[0016] Furthermore, the present invention discloses the use of the above-mentioned AIE molecular material in the preparation of a drug for treating Candida albicans infectious vaginitis.

[0017] Furthermore, in the above application, the drug achieves specific sterilization by promoting the release of reactive oxygen species (ROS) of Candida albicans and destroying its cell structure.

[0018] Furthermore, in the above application, the drug can completely kill Candida albicans at a concentration of 50-150 μM for 15-45 minutes without damaging vaginal epithelial cells; in some embodiments, the drug can completely kill Candida albicans at a concentration of 100 μM for 30 minutes without damaging vaginal epithelial cells.

[0019] Furthermore, in the above application, during the pharmaceutical manufacturing process, the therapeutic effect of the drug is evaluated by the following methods:

[0020] a. Establish an animal model of vaginal infection with Candida albicans by inducing pseudoestrus with estrogen and then inoculating the bacterial solution;

[0021] b. After administering the molecule, detecting the bacterial count in vaginal lavage fluid, the level of tissue inflammatory factors, and the diversity of the microbiome;

[0022] c. The relative abundance of Lactobacilli and pathogenic bacteria in the vaginal microbiota was analyzed by 16S rRNA and ITS sequencing.

[0023] Furthermore, the present invention also discloses the application of the above-mentioned AIE molecular material in Candida albicans-specific fluorescence imaging. After the AIE molecular material is co-incubated with Candida albicans, the aggregated luminescence signal is detected by laser confocal microscopy.

[0024] Compared with the prior art, the present invention has the following outstanding beneficial effects:

[0025] This invention belongs to the field of biomedicine and specifically relates to a precision antifungal material based on aggregation-induced emission (AIE)-active cationic amphiphilic molecules and their applications. To address the problem that existing drugs for treating Candida albicans vaginitis (VVC) indiscriminately kill pathogenic and beneficial bacteria, disrupt the vaginal microecological balance, and easily induce drug resistance, this invention proposes a simple, low-cost AIE molecular material (TPE-ET, TPE-BU, TPE-HEX) that combines diagnostic and therapeutic functions. Its beneficial effects are as follows:

[0026] 1. The synthesis process is efficient and convenient: The target molecule can be synthesized through a three-step reaction (TPE skeleton modification, introduction of a brominated linker, and alkyl chain amination). Conventional reagents (such as tetrahydrofuran, acetone, and ethanol) and silica gel column chromatography are used for purification. The process is simple, the yield is high, and it is suitable for large-scale production.

[0027] 2. Specific identification and precise sterilization:

[0028] Targeted killing: The AIE molecular material specifically binds to Candida albicans in an aggregated state and emits blue fluorescence. It can completely kill the pathogen at a concentration of 100 μM for 30 minutes (colony survival rate is 0%), and has no recognition and killing effect on lactobacilli (there is no significant change in clone number and ROS level).

[0029] 3. Clear mechanism of action: By promoting the release of reactive oxygen species (ROS) from Candida albicans and destroying the fungal cell membrane and internal structure (verified by SEM / TEM), it achieves efficient sterilization while avoiding damage to vaginal epithelial cells (co-incubation experiments showed no fluorescent labeling and cytotoxicity).

[0030] 4. Protect the balance of vaginal microecology:

[0031] Preclinical validation: In the VVC mouse model, compared with clotrimazole, the colony count in the vaginal lavage fluid after TPE-ET treatment was reduced by more than 90%, the expression level of inflammatory factors (IL-6, IL-1β) decreased by 60%-80%, and hyphae formation (PAS staining) and neutrophil infiltration (HE staining) were significantly reduced.

[0032] Microecological recovery: Through 16S rRNA and ITS sequencing analysis, the Shannon diversity index of the vaginal microbiome increased by 2.5 times after treatment, and the abundance of Lactobacillus increased by 4 times, which was significantly better than the traditional drug group, effectively preventing dysbacteriosis and disease recurrence.

[0033] 5. Integrated diagnosis and treatment function: The molecule can monitor the aggregated luminescent signals of Candida albicans in real time through laser confocal microscopy, providing visual guidance for the treatment process, and has the dual value of pathogen diagnosis and efficacy evaluation. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Schematic diagram of the preparation process of the AIE molecular material in Example 1;

[0035] Figure 2 Structural identification diagram of TPE-ET in Example 1;

[0036] Figure 3 A preliminary study of the structural formulas and bactericidal effects of TPE-ET, TPE-BU, TPE-HEX, and TPE-ET in Example 2; Figure A: The structural formulas of the three synthesized molecules; B: Plate antibacterial test; C: Bactericidal rate statistics; D: SEM image of Candida albicans under the action of TPE-ET;

[0037] Figure 4 Molecular formula, absorbance, particle size, and zeta potential of TPE-ET in Example 2; A: molecular formula; B: absorbance; C: PL; D: particle size; E: zeta potential;

[0038] Figure 5 Schematic diagram of TPE-ET in Example 2 accumulating on Candida albicans and emitting blue fluorescence; A: PL; B: TPE-ET specific recognition of Candida albicans; C: Fluorescence statistical graph;

[0039] Figure 6 Detection of the ability of TPE-ET to kill Candida albicans in Example 2, A: plate antibacterial test of TPE-ET; B: bactericidal efficiency statistics; C: live and dead fungi test; D: flow cytometry ROS test; E: TEM image;

[0040] Figure 7 The protective effect of TPE-ET on Lactobacillus in Example 2. A: TPE-ET antibacterial test on Lactobacillus plate; B: colony count; C: live and dead bacteria staining; D: flow cytometry ROS assay;

[0041] Figure 8 The effect of TPE-ET on VVC in vivo in Example 3. A: Images of mouse vagina before and 1, 3, 5, and 7 days after TPE-ET treatment; B: Colony plate images of treatment effects; C: PAS staining; D: HE image of inflammatory cells;

[0042] Figure 9 Expression of inflammatory factors in different groups after TPE-ET treatment in Example 3 A: Immunohistochemical expression of IL-4, IL-6, and IL-1β in different groups; B: Elisa statistics of IL-4, IL-6, and IL-1β;

[0043] Figure 10 16S and ITS analysis of changes in vaginal microecology before and after TPE-ET treatment in Example 3 AB: bacterial α diversity analysis; C: fungal α diversity analysis; D: expression of Proteobacteria; E:

[0044] F: expression of Firmicutes; F: expression of Lactobacillus. DETAILED DESCRIPTION

[0045] In this study, three cationic amphiphilic molecules with AIE activity were developed, in which a tetraphenylethylene (TPE) moiety was introduced as a hydrophobic fluorescent backbone and connected to a hydrophilic ammonium head via a linker (di(oxyethyl)). Furthermore, the hydrophilic ammonium head was attached to three different alkyl chains (i.e., ethane, butane, and hexane), and they were named TPE-ET, TPE-BU, and TPE-HEX, respectively.

[0046] A novel AIE material molecule for precise treatment of Candida albicans vaginitis specifically includes the following steps:

[0047] (1) Synthesis of AIE materials:

[0048] Aggregation-induced emission (AIE) is a unique new material originally developed by Chinese scientists. Traditional organic luminescent materials have long suffered from a difficult-to-overcome problem: aggregation-induced luminescence quenching. Luminescent molecules emit efficiently in dilute solutions, but when they aggregate in a solid state in highly concentrated solutions, their efficiency drops significantly, resulting in aggregation-induced quenching, which severely limits the application of traditional luminescent materials in living systems. AIE materials differ from traditional luminescent materials in that the synthesized molecules are non-luminescent in the single-molecule state but begin to emit light when aggregated. This contrasts with the previous phenomenon of single-molecule luminescence and non-luminescence in aggregates. Molecules are non-luminescent when dissolved in a good solvent, but begin to emit light when aggregated with the addition of a poor solvent. Because the luminescence is caused by aggregation, it is called aggregation-induced emission. Based on previous research and the differences in the surface membrane structure of fungal and mammalian cells, our team collaborated with the team of Academician Tang Benzhong, the "father of AIE," to develop the AIE molecule (TPE-ET) required for this study and used it for selective imaging and killing of Candida albicans. In this study, three cationic amphiphilic molecules with AIE activity were developed, in which a tetraphenylethylene (TPE) moiety was introduced as a hydrophobic fluorescent backbone and connected to a hydrophilic ammonium head via a linker (dioxyethyl). Synthesis of 4-(1,2,2-triphenylvinyl)phenol (1): Benzophenone (1.82 g, 10 mmol), 4-hydrobenzophenone (1.98 g, 10 mol) and zinc powder (2.60 g, 40 mmol) were placed in a two-necked round-bottom flask. The flask was evacuated and filled with nitrogen three times. Under nitrogen protection, 70 mL of dry tetrahydrofuran (THF) was added, and then 2.2 mL of TiCl4 (20 mmol) was slowly added with stirring in a dry ice acetone bath. The reaction mixture was heated under reflux overnight under N2 protection. After cooling to room temperature, 50 mL of dilute hydrochloric acid (1 M) was added to the mixture, and the mixture was extracted with dichloromethane (DCM). The combined organic phase was dried over anhydrous sodium sulfate and filtered. After evaporation of the solvent, the crude product was purified by silica gel column chromatography using n-hexane / ethyl acetate (40:1) as eluent.

[0049] Synthesis of (2-(4-(2-(2-bromoethoxy)ethoxy)phenyl)ethylene-1,1,2-triyl)-triphenylbenzene (2): Hydroxylated TPE (1) (1.74 g, 5 mmol), dibromoethyl ether (1.39 g, 6 mmol) and K2CO3 (1.38 g, 10 mmol) were added to a two-necked round-bottom flask. 30 mL of acetone was added under N2 protection. The reaction mixture was heated to reflux overnight. After evaporation of the solvent, the crude product was purified by silica gel column chromatography using n-hexane / DCM (20:1) as eluent.

[0050] Synthesis of TPE-ET, TPE-BU, and TPE-HEX (3): A mixture of (2-(4-(2-(2-bromoethoxy)-ethoxy)phenyl)ethylene-1,1,2-triyl)triphenyl (2) (2 mmol) and the corresponding amine (10 mmol) was stirred and heated under reflux in ethanol (20 mL) for 24 h. The solvent was removed in vacuo, and the crude product was repeatedly dissolved in a small amount of methanol. Excess THF was then added to precipitate a white powder, which was dried to obtain the target compound.

[0051] Prepare 1.0 or 5.0 × 10⁻³ M TPE-ET, TPE-BU, or TPE-HEX stock solutions in DMSO and dilute with deionized water to the appropriate concentrations for later use. These solutions are designated TPE-ET, TPE-BU, and TPE-HEX, respectively. Functional and physical property testing of the AIE-active luminescent materials TPE-ET, TPE-BU, and TPE-HEX: The materials were tested using plate antibacterial assays, PL, particle size, and zeta potential. TPE-ET was found to have the highest bactericidal efficiency and was used for subsequent validation.

[0052] (2) Experimental Verification: Plate antibacterial assay, live and dead yeast staining, flow cytometry ROS assay, SEM, and TEM were used to test TPE-ET's specific recognition and killing effect on Candida albicans. TPE-ET was incubated with Lactobacillus and found to have no killing effect on Lactobacillus. An in vivo VVC animal model was constructed, and the therapeutic effect of TPE-ET on VVC was evaluated using HE, PAS, immunohistochemistry, and ELISA. 16S and ITS sequencing were used to evaluate changes in microorganisms and changes in the diversity of vaginal flora after TPE-ET treatment.

[0053] (3) Result judgment:

[0054] Part 1: When TPE-ET is co-incubated with Candida albicans and normal vaginal epithelial cells, it only recognizes Candida albicans and emits blue fluorescence; it can completely kill Candida albicans at 100μM for 30min and promote the release of ROS from Candida albicans; SEM and TEM electron microscopy results show that TPE-ET can destroy the structure of Candida albicans.

[0055] Part 2: After TPE-ET was co-incubated with Lactobacillus, the plate antibacterial experiment showed that it had no recognition and killing effect on Lactobacillus; it did not promote the release of ROS by Lactobacillus; live and dead bacteria staining showed that TPE-ET did not kill Lactobacillus.

[0056] Part III: TPE-ET has a significant therapeutic effect on VVC mice. Compared with the clinical drug Clotrimazole, after 7 days of treatment, the results of the colony plate experiment showed that TPE-ET was more effective than Clotrimazole. Immunohistochemistry and ELISA experiments showed that the expression level of inflammatory factors was significantly reduced, PAS showed that the hyphae were significantly reduced after TPE-ET treatment, and HE results showed that the degree of inflammatory neutrophil infiltration was significantly reduced.

[0057] Part 4: ITS and 16S sequencing results showed that after TPE-ET treatment, compared with clotrimazole, TPE-ET can increase the richness and diversity of vaginal microbiota, especially Lactobacillus, thereby effectively treating candidal vaginitis and reducing recurrence, showing the potential for treating and preventing vaginal dysbiosis.

[0058] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0059] Example 1

[0060] Synthesis of TPE-ET, HEX, and BU.

[0061] Synthesis of 4-(1,2,2-triphenylvinyl)phenol (1): Benzophenone (1.82 g, 10 mmol), 4-hydrobenzophenone (1.98 g, 10 mol) and zinc powder (2.60 g, 40 mmol) were placed in a double-necked round-bottom flask. The flask was evacuated and filled with nitrogen three times. Under nitrogen protection, 70 mL of dry tetrahydrofuran (THF) was added, and then 2.2 mL of TiCl4 (20 mmol) was slowly added with stirring in a dry ice acetone bath. The reaction mixture was heated under reflux overnight under N2 protection. After cooling to room temperature, 50 mL of dilute hydrochloric acid (1 M) was added to the mixture, and the mixture was extracted with dichloromethane (DCM). The combined organic phases were dried over anhydrous sodium sulfate and filtered. After evaporation of the solvent, the crude product was purified by silica gel column chromatography using n-hexane / ethyl acetate (40:1) as eluent.

[0062] Synthesis of (2-(4-(2-(2-bromoethoxy)ethoxy)phenyl)ethylene-1,1,2-triyl)-triphenylbenzene (2): Hydroxylated TPE (1) (1.74 g, 5 mmol), dibromoethyl ether (1.39 g, 6 mmol) and K2CO3 (1.38 g, 10 mmol) were added to a two-necked round-bottom flask. 30 mL of acetone was added under N2 protection. The reaction mixture was heated to reflux overnight. After evaporation of the solvent, the crude product was purified by silica gel column chromatography using n-hexane / DCM (20:1) as eluent.

[0063] Synthesis of TPE-ET, TPE-BU, and TPE-HEX (3): A mixture of (2-(4-(2-(2-bromoethoxy)-ethoxy)phenyl)ethylene-1,1,2-triyl)triphenyl (2) (2 mmol) and the corresponding amine (10 mmol) was stirred and heated under reflux in ethanol (20 mL) for 24 h. The solvent was removed in vacuo, and the crude product was repeatedly dissolved in a small amount of methanol. Excess THF was then added to precipitate a white powder, which was dried to obtain the target compound.

[0064] Prepare 1.0 or 5.0×10-3M TPE-ET, TPE-BU or TPE-HEX stock solutions in DMSO and dilute with deionized water to the corresponding concentrations for later use. They are named TPE-ET (yield 84%), TPE-BU and TPE-HEX respectively. The reaction flow is as follows: Figure 1 The structure of TPE-ET is shown in Figure 2 shown.

[0065] The 1H NMR and 13C NMR data of TPE-ET are as follows: 1H NMR (400

[0066] MHz,DMSO-d6,δ):7.17–7.06(m,9H),6.96(m,6H),6.86(dd,J=8.8,3.3Hz,2H),6.70(dd,J=8.8,3.3Hz,2H),4.07–4 .01(m,2H),3.88(s,2H),3.79–3.72(m,2H),3.52(m,2H),3.07(d,J=3.2Hz,2H),3.03(d,J=3.2Hz,6H),1.22(m,3H).

[0067] 13C NMR(100MHz,DMSO-d6,δ):156.86,143.49,143.41143.37,140.12,139.74,135.52,131.94,130.67,130.64,1 27.88,127.78,126.49,126.42,126.38,113.71,68.72,66.58,63.93,63.90,61.78,59.69,50.14,7.98,7.95.

[0068] Mass spectrometry data are MALDI-TOF HRMS: calcd. for C34H38NO2+[M-Br]+: 492.2897, found: 492.2881.

[0069] Example 2

[0070] Physical properties and in vitro bactericidal tests.

[0071] (1) The three materials TPE-ET, TPE-BU and TPE-HEX were subjected to flat plate antibacterial tests. The results are as follows: Figure 3 It shows that TPE-ET is the most efficient in killing Candida albicans among the three materials.

[0072] (2) The physical properties of TPE-ET (molecular structure, absorbance, particle size) were explored, and the results were as follows: Figure 4 show.

[0073] (3) To explore whether TPE-ET specifically recognizes Candida albicans, we co-incubated Candida albicans with normal vaginal epithelial cells and photographed them using a laser confocal microscope. The results are shown in Figure 3. Figure 5 It showed that TPE-ET could aggregate on Candida albicans and emit blue fluorescence, but did not recognize the co-incubated normal vaginal epithelial cells VK2 / E6E7.

[0074] (4) In order to explore the in vitro killing effect of TPE-ET on Candida albicans, we performed a plate antibacterial experiment and tested the ability of TPE-ET to kill Candida albicans at different concentrations (5μM, 10μM, 20μM, 50μM, 80μM, 100μM). Figure 6The results shown in the figure showed that Candida albicans could be completely killed at a concentration of 100 μM and an action time of 30 minutes; the Syto9 / PI live-dead staining results showed that compared with low concentration TPE-ET (5 μM), high concentration TPE-ET (100 μM) had a significant killing effect on Candida albicans; the flow cytometry results showed that TPE-ET would promote the release of ROS in Candida albicans, thereby promoting its apoptosis; SEM electron microscopy results showed that the fungal structure of Candida albicans was significantly destroyed after the action of TPE-ET.

[0075] (5) In order to explore whether TPE-ET has a killing effect on lactobacilli, we conducted a plate antibacterial experiment and tested the ability of TPE-ET to kill lactobacilli at different concentrations (5μM, 10μM, 20μM, 50μM, 80μM, 100μM). The results are as follows: Figure 7 As shown in the results, there was no significant difference in the number of Lactobacillus clones before and after incubation with different concentrations of TPE-ET; the Syto9 / PI live-dead staining results showed that TPE-ET had no obvious killing effect on Lactobacillus; the flow cytometry results showed that TPE-ET did not promote the release of ROS in Lactobacillus.

[0076] Example 3

[0077] (1) To evaluate the therapeutic effect of TPE-ET on VVC, we first injected 0.1 ml of 2 mg mL-1 estradiol oil into the inner thigh of 6-week-old female BALB / c mice subcutaneously. The needle was pushed about 5-10 mm outside the skin to minimize leakage at the injection site. Before and three days after estrogen stimulation, the vagina of the mice was irrigated with sterile PBS, and the irrigated fluid was smeared and stained with crystal violet. 20 μl of PBS was irrigated each time, and the total irrigated volume was 100 μl. 10 μl of irrigated fluid was taken from the center of the slide. Preliminary experiments found that the fixation effect of anhydrous ethanol drip fixation and the appropriate extension of the natural drying time of the smear was good. Microscopic observation was performed to see whether there were a large number of vaginal keratinized squamous epithelial cells. The vaginal irrigated fluid smear of estrus mice showed a large number of irregular anucleated keratinized cells, which were distributed throughout the entire field of view and could aggregate into decidual shapes, and there were also a small number of nucleated epithelial cells. Before and after comparison, if a large number of irregular anucleated squamous epithelial cells were found in the irrigated fluid of the mice after hormone stimulation, the mice were in estrus. Vaginal inoculation of Candida albicans can be prepared: If a large number of epithelial cells are not seen, repeat the hormone stimulation operation and perform smear evaluation again until the mouse pseudo-estrus model is successfully established. If the mouse pseudo-estrus model is successfully established, shake the bacterial solution one day before vaginal inoculation and culture it at 30℃ and 220rpm for 16 hours. Collect the bacterial solution in a 15ml centrifuge tube, collect it by centrifugation, wash it, and resuspend it. Vaginal inoculation of mice is performed one day after the mouse pseudo-estrus model is successfully established. During vaginal inoculation, the mouse is briefly anesthetized with isoflurane. After the mouse is anesthetized and paralyzed, hang the mouse upside down and repeatedly move the gavage needle back and forth 5 to 6 times, close to the mouse vaginal wall, to cause slight local damage to the vaginal mucosa. Then prepare a concentration of 2.0×10 9 CFU / mL of Candida albicans suspension was inoculated into the mouse vagina, 20uL per mouse; during vaginal inoculation, the mouse was stabilized, the bacterial solution was sucked up with a pipette, and inserted into the vaginal cavity about 5mm deep. The operation process was as gentle as possible to avoid causing other discomfort to the mouse. After inoculation, in order to prevent the bacterial solution from flowing out, the mouse was placed upside down for 30 minutes; we randomly divided the estrus mice into a model group, a clotrimazole treatment group, and a TPE-ET treatment group. There were five mice in each group, and the blank group did not receive any treatment. After the model was successfully established, TPE-ET (100μΜ, once every other day) and Clotrimazole (100μΜ, once every other day) were treated for 7 days, and then the mice were killed and vaginal tissues were taken. The vaginal lavage fluid was plated for colonies, and the tissue was stained with HE, immunohistochemistry, and PAS. The results are as follows Figure 8 and Figure 9 shown.

[0078] (2) Next, we investigated the improvement of the vaginal microbiome in mice with Candida albicans-induced vaginitis after TPE-ET treatment and clotrimazole treatment. Figure 10As shown, Chao1 and Shannon indices of alpha diversity, as measured by 16S ribosomal RNA gene sequencing and internal transcribed spacer (ITS) amplicon sequencing, revealed that TPE-ET treatment significantly improved fungal and bacterial diversity in mice with candidal vaginitis compared with clotrimazole. Further analysis at the phylum level revealed that TPE-ET treatment significantly reduced the relative abundance of Proteobacteria and increased that of Firmicutes, reshaping a healthier vaginal microbiome. At the genus level, TPE-ET significantly increased the relative abundance of Lactobacilli. In summary, compared with clotrimazole, TPE-ET can increase the richness and diversity of the vaginal microbiota, particularly Lactobacilli, thereby effectively treating candidal vaginitis and reducing recurrence, demonstrating potential for the treatment and prevention of vaginal dysbiosis.

[0079] The above are only a few preferred embodiments of the present invention, and their description is relatively specific and detailed, but it should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art may make various modifications and improvements without departing from the scope of the present invention, and such modifications and improvements are within the scope of protection of the present invention.

Claims

1. An AIE molecular material, characterized in that: The material is composed of tetraphenylethylene (TPE) as a hydrophobic fluorescent skeleton, connected to a hydrophilic ammonium head via a dioxyethyl linker, and further connected to an alkyl chain substituent. The alkyl chain is selected from one or more of ethane, butane or hexane chains.

2. The AIE molecular material according to claim 1, characterized in that The molecule specifically recognizes Candida albicans and emits blue fluorescence in an aggregated state, and has no recognition or killing effect on lactobacilli.

3. The method for preparing the AIE molecular material according to claim 1, wherein: The following steps are involved: a. Synthesis of 4-(1,2,2-triphenylvinyl)phenol (1): Benzophenone, 4-hydrobenzophenone and zinc powder were reacted in tetrahydrofuran, extracted with hydrochloric acid and purified by silica gel column chromatography to obtain compound (1); b. Synthesis of 2-(4-(2-(2-bromoethoxy)ethoxy)phenyl)ethylene-1,1,2-triyl)-triphenylbenzene (2): Compound (1) was reacted with dibromoethyl ether in acetone and purified by silica gel column chromatography to obtain compound (2); c. Synthesis of TPE-ET, TPE-BU or TPE-HEX: Compound (2) is reacted with the corresponding alkyl chain amine in ethanol under reflux, and the target AIE molecular material is obtained by solvent removal and precipitation purification.

4. The preparation method according to claim 3, characterized in that The alkyl chain amine in step c is ethylamine, butylamine or hexylamine.

5. A pharmaceutical composition for treating Candida albicans infectious vaginitis, characterized in that: The composition comprises the AIE molecular material according to claim 1 and a pharmaceutically acceptable carrier, wherein the concentration of the AIE molecular material in the composition is 1.0×10-3M to 5.0×10-3M.

6. Use of the AIE molecular material according to claim 1 in the preparation of a medicament for treating Candida albicans vaginitis.

7. The use according to claim 6, characterized in that The drug achieves specific sterilization by promoting the release of reactive oxygen species (ROS) from Candida albicans and destroying its cell structure.

8. The use according to claim 6, characterized in that The drug can completely kill Candida albicans at a concentration of 50-150 μM and acts for 15-45 minutes without damaging vaginal epithelial cells.

9. The use according to claim 6, characterized in that During the pharmaceutical manufacturing process, the therapeutic effects of drugs are evaluated by the following methods: a. Establish an animal model of vaginal infection with Candida albicans by inducing pseudoestrus with estrogen and then inoculating the bacterial solution; b. After administering the molecule, detecting the bacterial count in vaginal lavage fluid, the level of tissue inflammatory factors, and the diversity of the microbiome; c. The relative abundance of Lactobacilli and pathogenic bacteria in the vaginal microbiota was analyzed by 16S rRNA and ITS sequencing.

10. Use of the AIE molecular material according to claim 1 in Candida albicans specific fluorescence imaging, characterized in that: After the AIE molecular material is co-incubated with Candida albicans, the aggregated luminescence signal is detected by laser confocal microscopy.

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