An antimicrobial peptide with anti-Candida albicans activity and its application

Abstract

The present invention discloses an antimicrobial peptide with anti-Candida albicans activity and its application, belonging to the field of biomedicine technology. The amino acid sequence of the antimicrobial peptide is NH2-GFKKLKLFKLKPF-COOH. The minimum inhibitory concentration of the antimicrobial peptide is 4.0 μM; the antimicrobial peptide is prepared into nanovesicles with egg yolk lecithin, cholesterol, and DSPE-MPEG2000, and its minimum inhibitory concentration is 2.0 μM. The heptamer oligomerization structure formed by the phenylalanine (Phe)-rich region in the antimicrobial peptide of the present invention can, on the one hand, specifically target ergosterol in the fungal cell membrane and significantly enhance the anti-Candida albicans activity; on the other hand, it can destroy the rigid conformation of the α-helix by inserting proline (Pro) residues, increase the molecular flexibility to penetrate the thick cell wall of the fungus, thereby enhancing its core mechanism of action against Candida albicans, and can also effectively treat fungal vaginitis without developing drug resistance.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to an antimicrobial peptide with anti-Candida albicans activity and application thereof. Background Art

[0002] Fungal infections have become a significant challenge to global public health, posing a significant threat to human health and socioeconomic development. According to the World Health Organization, fungal infections affect approximately 1.5 billion people worldwide annually, resulting in over 1.7 million deaths. Currently, clinical antifungal treatments primarily rely on azoles (e.g., fluconazole), polyenes (e.g., amphotericin B), and echinocandins (e.g., caspofungin). These drugs primarily exert their efficacy by targeting ergosterol or inhibiting cell wall β-glucan synthesis. However, with the long-term use of several drugs, fungal resistance is increasing. Therefore, the search and development of new therapeutic approaches is crucial. In this context, antimicrobial peptides, with their multi-target mechanism of action, high specificity, and safety profile, have become an emerging strategy to overcome the limitations of traditional drugs.

[0003] Candida albicans ( Candida albicans ) is one of the core pathogens in the field of fungal infections and can cause infectious diseases such as vaginitis, esophagitis, oropharyngeal candidiasis, and thrush. In the "List of Priority Fungal Pathogens" that threatens health, published for the first time in 2022 by the WHO, Candida albicans was listed as a severe level group. It is reported that the annual incidence of invasive candidiasis mainly caused by Candida albicans reaches 750,000 cases, and the mortality rate of candidemia is as high as 40%-60%, which seriously endangers public health. With the long-term use of mainstream drugs, Candida albicans has developed corresponding drug resistance mechanisms such as overexpression of efflux pumps, gene mutations, and enzyme mutations, which gradually reduce the pathogen's sensitivity to drugs; and the nephrotoxicity of traditional antifungal drugs has always been a lingering topic; in addition, antifungal drugs may also cause side effects such as gastrointestinal discomfort, liver damage, and allergic reactions, which not only affect patients' treatment compliance, but may also cause other safety issues. Therefore, the development of new antimicrobial peptides is imperative.

[0004] Antimicrobial peptides are a class of peptides with broad-spectrum antimicrobial activity. They can exert their antimicrobial effects by targeting fungal cell membranes, interfering with bacterial cell wall synthesis, and targeting intracellular metabolic pathways. They possess advantages such as strong specificity, high yield, and high purity. As promising antifungal biomaterials, they have demonstrated unprecedented advantages. Despite these advantages, their relatively poor biostability, low permeability, and dose-dependency hinder their further clinical translation and application. Drug delivery systems are systems that deliver drugs to their target sites. Peptide-based drug delivery systems can not only achieve precise targeting, high-efficiency antimicrobial activity, enhanced membrane permeability, and improved biostability, but can also regulate drug metabolic kinetics, improve drug bioavailability, prolong drug retention time, extend half-life, and reduce drug toxicity and side effects. They are an important way to resolve the dilemma of antimicrobial peptides and promote their further clinical translation and application. Summary of the Invention

[0005] In response to the above-mentioned prior art, the present invention aims to provide an antimicrobial peptide with anti-Candida albicans activity and its applications. The heptamer oligomerization structure formed by the phenylalanine (Phe)-rich region in the antimicrobial peptide can specifically target ergosterol in the fungal cell membrane, significantly enhancing anti-Candida albicans activity. Furthermore, the insertion of proline (Pro) residues disrupts the rigid conformation of the α-helix, increasing the molecular flexibility to penetrate the thick fungal cell wall, thereby enhancing its core anti-Candida albicans mechanism of action and addressing the current issue of antibiotic resistance in C. albicans. Furthermore, a microfluidics-based method for preparing antimicrobial peptide nanovesicles is provided. This invention utilizes a peptide-based drug delivery system to actively target the site of infection, inhibit the growth of C. albicans, and effectively address the issue of C. albicans resistance.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] In a first aspect of the present invention, an antimicrobial peptide having anti-Candida albicans activity is provided, wherein the amino acid sequence of the antimicrobial peptide is NH2-GFKKLKLFKLKPF-COOH, as shown in SEQ ID NO: 1.

[0008] The second aspect of the present invention provides a method for preparing an antimicrobial peptide, wherein the method comprises synthesizing the peptide sequentially from the C-terminus to the N-terminus using a solid-phase synthesis method.

[0009] The third aspect of the present invention provides the use of antimicrobial peptides in the preparation of antimicrobial drugs for inhibiting Candida albicans.

[0010] Preferably, the minimum inhibitory concentration of the antimicrobial peptide is 4.0 μM.

[0011] The fourth aspect of the present invention provides the use of antimicrobial peptides in the preparation of nanomedicines for treating vaginitis.

[0012] In a fifth aspect, the present invention provides a nanomedicine for treating vaginitis, wherein the nanomedicine has an antimicrobial peptide as an active ingredient and is in the form of a nanovesicle.

[0013] Preferably, the nanovesicles are prepared by a microfluidic method using antimicrobial peptides, egg yolk lecithin, cholesterol, and DSPE-MPEG2000 as raw materials.

[0014] Preferably, in the nanovesicles, the molar ratio of antimicrobial peptide is 10.14%, the molar ratio of egg yolk lecithin is 50.7%, the molar ratio of cholesterol is 38.89%, and the molar ratio of DSPE-MPEG2000 is 0.27%.

[0015] Preferably, the minimum inhibitory concentration of the nanomedicine is 2.0 μM.

[0016] Beneficial effects of the present invention:

[0017] (1) The novel antimicrobial peptide of the present invention has the advantages of high anti-Candida albicans activity, high yield, and high purity, providing a new strategy for the clinical treatment of candidiasis. When the antimicrobial peptide concentration reaches 4 μM or above, it has a strong antibacterial effect on Candida albicans and has good application prospects in the treatment of candidiasis such as vaginitis caused by Candida albicans.

[0018] (2) The present invention adopts the standard Fmoc solid phase peptide synthesis (SPSS) synthesis technology. The principle is to protect the amino group of the polypeptide main chain by the Fmoc group, protect the amino group of the amino acid side chain by other groups, deprotect the amino group of the main chain on the resin, and then iteratively couple with the next amino acid to form a polypeptide. The order of peptide synthesis is from C-terminus to N-terminus. The heptamer oligomerization structure formed by the phenylalanine (Phe)-rich region in the antimicrobial peptide can, on the one hand, specifically target ergosterol in the fungal cell membrane and significantly enhance the anti-Candida albicans activity; on the other hand, it can destroy the rigid conformation of the α-helix by inserting proline (Pro) residues, increase the molecular flexibility to penetrate the thick cell wall of the fungus, thereby enhancing its core mechanism of action against Candida albicans.

[0019] (3) The nanovesicles of the present invention, prepared by microfluidics, can achieve precise targeting, high-efficiency antibacterial properties, and enhanced membrane permeability. They can also regulate drug metabolic kinetics, improve drug bioavailability, prolong drug retention time, extend half-life, and reduce drug toxicity and side effects. This is an important way to resolve the dilemma of antimicrobial peptides and promote their further clinical application.

[0020] (4) The nanovesicles prepared by the microfluidic method of the present invention have uniform particle size, stable PDI and are all less than 0.1, with good stability. In addition, the reproducibility of different batches reaches more than 98%, with good quality control effect, providing a preparation method and ideas for enterprise preparation research and development and prescription scale-up.

[0021] (5) The antimicrobial peptides of the present invention and the nanovesicles prepared therefrom can not only treat vaginitis caused by Candida albicans infection, but also have good application prospects in treating other diseases caused by Candida albicans, such as esophagitis, oropharyngeal candidiasis, and thrush. This provides a new method for the current treatment of candidiasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 :(a)Chemical formula of antimicrobial peptide,(b)3D structure diagram of antimicrobial peptide;

[0023] Figure 2 : Mass spectrum of antimicrobial peptides;

[0024] Figure 3 : HPLC chromatogram of antimicrobial peptides;

[0025] Figure 4 : TEM characterization of nanovesicles;

[0026] Figure 5 : Dynamic light scattering diagram of nanovesicles, where (a) is potential measurement and (b) is particle size measurement;

[0027] Figure 6 : Hemolytic activity evaluation, where (a) is the hemolytic activity evaluation of nanovesicles, and (b) is the hemolytic activity evaluation of antimicrobial peptides;

[0028] Figure 7 : Antibacterial performance experiments, where (a) is the minimum inhibitory concentration of antimicrobial peptides, (b) is the minimum inhibitory concentration of nanovesicles, and (c) is the minimum fungicidal concentration of nanovesicles.

[0029] Figure 8 : Comparison of vaginal secretions before and after modeling of fungal vaginitis;

[0030] Figure 9 : Results of vaginal tissue flat-plate coating in the model group and drug-administered group after treatment, where (a) is the flat-plate coating image of the model group, and (b) is the flat-plate coating image of the nanovesicle-administered group;

[0031] Figure 10 : Pathological examination of rat vaginal tissue, where (a) is the model group and (b) is the drug-treated group. DETAILED DESCRIPTION

[0032] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0033] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below with reference to specific embodiments.

[0034] The test materials used in the examples of the present invention are all conventional test materials in the art and can be purchased through commercial channels.

[0035] Example 1: Synthesis of antimicrobial peptide with sequence NH2-GFKKLKLFKLKPF-COOH

[0036] Solution A was prepared by dissolving 0.5 g of ninhydrin in 10 mL of anhydrous ethanol, solution B was prepared by dissolving 20 g of phenol in 5 mL of anhydrous ethanol, and solution C was prepared by dissolving 0.1 g of ascorbic acid in 5 mL of anhydrous ethanol. One drop each of solution A, solution B, and solution C was mixed to obtain the Kaiser test reagent.

[0037] 490 mL of DMF and 10 mL of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) were mixed, and 25 g of anhydrous piperazine was added. After mixing, the mixture was ultrasonicated to obtain a deprotecting agent.

[0038] DMF and N-methylmorpholine were mixed in a volume ratio of 19:1 to prepare a coupling agent.

[0039] TFA, triisopropylsilane and water were mixed in a volume ratio of 95:2.5:2.5 to prepare a cleavage solution.

[0040] Weigh 300 mg of Fmoc-Phe-Wang resin (purchased from Shanghai Jizhi Biochemical Technology Co., Ltd.) into a peptide synthesis tube and allow to swell in ultra-dry DMF for 6 hours. Drain the tube using a circulating water vacuum pump, then wash three times with DCM and DMF alternately using a wash bottle and drain to obtain the deprotected resin. Add the deprotected resin to Kaiser detection reagent using a capillary glass tube and incubate in a boiling water bath for 1 minute. Observe the resin for color changes; if the resin changes color, deprotection is complete.

[0041] Take 10 times the mass of Fmoc-Pro-OH amino acid (purchased from Shanghai Jizhi Biochemical Technology Co., Ltd.), add HBTU (benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate) and 8 mL of coupling agent, and activate on a shaker for 10 minutes to obtain the activated amino acid.

[0042] In a peptide synthesis tube, add the activated amino acid to the deprotected resin and shake for 45 minutes. The carboxyl groups of the activated amino acids react with the exposed amino groups on the deprotected resin backbone through a dehydration condensation reaction to produce a coupled resin. After the reaction, drain the liquid from the peptide synthesis tube using a circulating water vacuum pump. Wash the resin three times, alternating between DCM and DMF, using a wash bottle and drain. Then, use a capillary glass tube to add the Kaiser detection reagent and place it in a boiling water bath for 1 minute. Observe the resin color change. If the resin does not change color, the coupling is complete. Repeat the deprotection-coupling process until the last amino acid, Gly (G), is deprotected. Add methanol to shrink the resin. Finally, transfer the resin to a cleavage vial and add 10 mL of cleavage buffer. Cleave the resin in an ice-water bath at 180 rpm for 2.5 hours to cleave the peptide from the resin.

[0043] The cleaved peptide was filtered to remove the resin. Nitrogen was blown through the solution until it became viscous. Ice-cold ether was added to allow precipitation, resulting in a white solid. The solid was then centrifuged and resuspended, then washed three times at 8000 rpm for 5 minutes to obtain a white powder. This was then concentrated in a concentrator for 3 hours to obtain a dry white powder, which was the antimicrobial peptide. Its three-letter sequence is: NH2-Gly-Phe-Lys-Lys-Leu-Lys-Leu-Phe-Lys-Leu-Lys-Pro-Phe-COOH.

[0044] Example 2: Mass spectrometry analysis of antimicrobial peptides

[0045] The antimicrobial peptide prepared in Example 1 was dissolved in acetonitrile by ultrasonication and then characterized by a quadrupole electrostatic field orbital trap Fourier transform mass spectrometer (QE). Figure 2 As shown in the mass spectrum, the single-charged ion peak of the antimicrobial peptide ([M+H] + ) The experimental observation value was 1595.0195, and the theoretical molecular weight was 1594.04, which were highly consistent, indicating that the antimicrobial peptide was successfully synthesized.

[0046] Example 3: Purity Verification of Antimicrobial Peptides

[0047] The purity of the obtained peptide was determined by high performance liquid chromatography.

[0048] Mobile phase preparation: Mobile phase A was water containing 0.1% TFA, and mobile phase B was acetonitrile containing 0.1% TFA.

[0049] Selection of chromatographic column: Select Agilent C18 column.

[0050] Sample loading and procedure: The antimicrobial peptide solution was manually loaded in a 25 μL sample volume at a flow rate of 1 mL / min. A gradient elution was performed: the pump B concentration was linearly increased from 10% to 90% at a rate of 3.20% / min between 0.01 and 25.00 minutes, followed by a rapid increase to 100% at a rate of 10.10% / min between 25.01 and 26.00 minutes. After the experiment, the tubing and column were flushed with methanol for 30 minutes.

[0051] like Figure 3 As shown in the figure, the purity of the antimicrobial peptide synthesis was measured to be 95.2% according to the integrated area peak. The synthesized antimicrobial peptide was qualified and could be used in subsequent experiments.

[0052] Experimental Example 4: Preparation of Nanovesicles by Microfluidics

[0053] The antimicrobial peptide prepared in Example 1, along with egg yolk lecithin, cholesterol, and DSPE-MPEG2000 (all pharmaceutical excipients approved by the Chinese Pharmacopoeia) were dissolved in anhydrous ethanol to prepare a lipid-ethanol solution. The lipid-ethanol solution contained 9.0785 mg / mL of antimicrobial peptide, 21.34 mg / mL of egg yolk lecithin, 8.442 mg / mL of cholesterol, and 0.426 mg / mL of DSPE-MPEG2000 (the molar ratios of antimicrobial peptide, egg yolk lecithin, cholesterol, and DSPE-MPEG2000 were 10.14%, 50.7%, 38.89%, and 0.27%, respectively).

[0054] Use a 3 mL syringe to draw 500 μL of lipid ethanol solution as the lipid ethanol phase. Use a 3 mL syringe to draw 1500 μL of ultrapure water as the aqueous phase.

[0055] A microfluidic preparation instrument (ATS Nanotechnology (Suzhou) Co., Ltd., AE-nano) was used to add the lipid ethanol phase and the aqueous phase into the two flow channels of the device, respectively. The instrument temperature was set to 70°C, the instrument operation time was 29.3 s, and the lipid ethanol phase: aqueous phase (v:v) ratio was set to 1:3. After the operation was completed, the effluent was collected (the first 1 / 4 and the last 1 / 4 of the volume of the effluent were discarded), which was the nanovesicle.

[0056] Comparative Example 1: Preparation of basic vesicles

[0057] Egg yolk lecithin, cholesterol, and DSPE-MPEG2000, all approved pharmaceutical excipients in the Chinese Pharmacopoeia, were dissolved in anhydrous ethanol to prepare a lipid-ethanol solution. The concentration of egg yolk lecithin in the lipid-ethanol solution was 25.613 mg / mL; the concentration of cholesterol was 8.442 mg / mL; and the concentration of DSPE-MPEG2000 was 0.426 mg / mL. Aspirate 500 μL of the lipid-ethanol solution using a 3 mL syringe to prepare the lipid-ethanol phase. Aspirate 1500 μL of ultrapure water using a 3 mL syringe to prepare the aqueous phase.

[0058] A microfluidic preparation instrument (ATS Nanotechnology (Suzhou) Co., Ltd., AE-nano) was used to add the lipid ethanol phase and the aqueous phase into the two flow channels of the device respectively. The instrument temperature was set to 70°C, the instrument running time was 29.3s, and the lipid ethanol phase: aqueous phase (v:v) ratio was set to 1:3. After the run was completed, the effluent was collected (the first 1 / 4 and the last 1 / 4 of the volume of the effluent were discarded), which was the basic vesicle.

[0059] Experimental Example 5: TEM Characterization of Nanovesicles

[0060] 10 μL of the nanovesicles prepared in Experimental Example 4 were spotted onto a carbon support membrane. After incubation at room temperature for 10 minutes, the remaining solution was removed with filter paper. 10 μL of 1% phosphotungstic acid negative stain was added and stained for 5 minutes. After removal of the stain, the membrane was rinsed with 10 μL of ultrapure water (ddH₂O) for 1 minute, dried, and stored at room temperature until further analysis. TEM observation revealed stable and uniform nanovesicles.

[0061] Experimental Example 6: Characterization of Zeta Potential and Particle Size of Nanovesicles

[0062] The surface potential and particle size of the nanovesicles prepared in Example 4 were measured using a nanoparticle size analyzer (Nano-ZS-2019), and the measurement was repeated three times for each group.

[0063] like Figure 5 As shown in (a), the Zeta potential of the nanovesicles is 15.5 mV, and the Zeta potential of the basic vesicles is close to 0. This shows that the antimicrobial peptides successfully participated in the assembly, the nanovesicles were successfully prepared, and they were positively charged. Figure 5 As shown in (b), the particle size of the nanovesicles is 102.6 nm and the PDI is less than 0.2, indicating that the prepared nanovesicles have uniform and stable particle sizes.

[0064] Experimental Example 7: Hemolytic activity evaluation

[0065] Blood Processing: Fresh blood from SD rats (purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., healthy ICR 6-week-old rats, weighing 25-30 g) was used to evaluate the hemolytic activity of the antimicrobial peptides prepared in Example 1 and the nanovesicles prepared in Example 4. Untreated SD rats were enucleated and blood was collected. The blood was then dripped into an anticoagulant tube. Freshly drawn mouse blood was slowly added to normal saline at a 1:1 ratio. The saline-containing blood was centrifuged at 1500 rpm and 4°C for 15 minutes. The supernatant was removed and the tube was washed three times with an equal amount of normal saline until the supernatant was clear.

[0066] Drug preparation: 1 mL of nanovesicle solution and antimicrobial peptide solution were prepared in normal saline at final concentrations of 64, 32, 16, 8, 4, 2, 1, and 0.5 μM, respectively. 490 μL of nanovesicle solution or antimicrobial peptide solution was added to 10 μL of blood cells. The mixture was incubated at 37°C for 3 h, centrifuged at 3000 rpm for 10 min, and photographed. 100 μL of the supernatant from each tube was added to a 96-well plate. This was repeated three times. The absorbance (As) was measured at 570 nm. A negative control group (normal saline, An) and a positive control group (deionized water, Ap) were also set up. The hemolysis rate was calculated using the formula:

[0067] Hemolysis rate (%) = (As-An) / (Ap-An) × 100%.

[0068] like Figure 6 As shown, compared with the positive control group, both antimicrobial peptides and nanovesicles exhibited lower hemolytic activity, and when the concentration was as high as 64 μM, the hemolysis rate was less than 5%.

[0069] Experimental Example 1: Verification of the replacement ratio of antimicrobial peptides in basic vesicles

[0070] The basic vesicles prepared in Comparative Example 1 were used as a control. The effects of replacing egg yolk lecithin with antimicrobial peptides in different ratios on vesicle particle size, polydispersity index (PDI), and stability were verified. Ten parallel experiments were designed, with different replacement ratios of egg yolk lecithin: antimicrobial peptide (mol / mol) = 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, and 1:1, numbered A to J. The molar proportions of each component in the ten experiments are shown in the table below. Following the preparation method of Experimental Example 4, ten groups of nanovesicles, A to J, were prepared. The particle size and PDI of the nanovesicles were measured using dynamic light scattering, with three repeated cycles for each group. The results are shown in Table 1.

[0071] Table 1 Particle size and PDI of nanovesicles

[0072]

[0073]

[0074] According to Table 1, when the ratio of egg yolk lecithin to antimicrobial peptide (mol / mol) is 5:1, the prepared nanovesicles are stable and uniform, and have the best effect as a drug delivery system.

[0075] Test Example 2: Antibacterial Performance Test

[0076] (1) Minimum inhibitory concentration test

[0077] The effectiveness of the antimicrobial peptide prepared in Example 1 and the nanovesicles prepared in Example 4 was evaluated by testing their minimum inhibitory concentration (MIC) against Candida albicans.

[0078] Candida albicans was incubated in SDB medium at 30°C for 28 h. Candida albicans was diluted in RPMI (Roswell Park Memorial Institute) 1640 medium to a final concentration of 5×10 3 CFUmL -1 , as a working suspension (buffered with 3-morpholinopropanesulfonic acid (MOPS) at a final concentration of 0.165 M). In a 96-well plate, the nanovesicles were serially diluted in RPMI (Roswell Park Memorial Institute) 1640 medium to 128, 64, 32, 16, 8, 4, 2, and 1 μM and then mixed with an equal volume (100 μL) of the working suspension; the final concentrations of the nanovesicles in the 96-well plate were 64, 32, 16, 8, 4, 2, 1, and 0.5 μM, respectively.

[0079] Incubate at 30°C for 28 hours. RPMI 1640 medium was used as a blank control, and 100 μL of the working suspension was added to 100 μL of RPMI 1640 medium as a negative control. Each experiment was repeated at least three times. The absorbance of each well at 600 nm was measured using a microplate reader, and the inhibition rate was calculated according to the following formula:

[0080] Antibacterial rate = (A 实验组 -A 空白组 ) / (A 阴性对照组 -A 空白组 )×100%.

[0081] according to Figure 7 (a) shows the MIC of antimicrobial peptides against Candida albicans 90 is 4μM; according to Figure 7 (b) MIC of nanovesicles against Candida albicans 90The new antimicrobial peptide and nanovesicles both have a good antibacterial effect on Candida albicans, and the antibacterial effect of nanovesicles is better than that of antimicrobial peptides. They are expected to be widely used as new antibacterial agents for the prevention and treatment of Candida albicans.

[0082] (2) Minimum fungicidal concentration test of nanovesicles

[0083] Determination of the minimum fungicidal concentration (MFC) of the nanovesicles prepared in Example 4. 100 μL of nanovesicles at concentrations of 32, 16, and 8 μM and 100 μL of the working suspension were added to a 96-well plate, resulting in final nanovesicle concentrations of 16 μM (denoted as 8×MIC), 8 μM (denoted as 4×MIC), and 4 μM (denoted as 2×MIC). A control group consisted of 100 μL of the working suspension added to 100 μL of RPMI 1640 medium. The plates were incubated at 30°C for 28 hours and then removed. 15 μL of bacterial suspension from each well was spread on an SDA (Sabouraud dextrose agar) plate. The plates were incubated inverted at 30°C for 16 hours, and colony growth was observed at 4, 8, and 16 hours. The minimum concentration at which no colony growth occurred was defined as the MFC of the nanovesicles against Candida albicans. The experiment was repeated three times for each drug group.

[0084] according to Figure 7 As shown in (c), the minimum fungicidal concentration of nanovesicles is 8 μM.

[0085] Test Example 3: Animal Model Test

[0086] A Candida albicans-induced fungal vaginitis model in rats was established to evaluate the antibacterial properties of nanocapsules against Candida albicans and the reduction of inflammatory response.

[0087] Establishment of a fungal vaginitis model in rats: 24 female SD rats (purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., 8 weeks old; 180-200g) were randomly divided into a modeling group and a normal group, with 16 rats in the modeling group and 8 rats in the normal group. The normal group received no treatment. The modeling group received intraperitoneal injections of 100 μL of β-estradiol (5 mg / mL) for three consecutive days to promote estrus and facilitate infection. Subsequently, Candida albicans (1×10 8 CFU / mL, 50 μL / day) was injected into the vagina of female mice for in situ infection for seven consecutive days to establish a fungal vaginitis model caused by Candida albicans infection. Typical symptoms, such as a significant increase in yellow vaginal discharge and vulvar redness and swelling, indicated successful modeling of fungal vaginitis. The modeling group was randomly divided into a treatment group and a model group, with 8 mice in each group.

[0088] Administration: After successful modeling, the treatment group received vaginal administration of the nanovesicles prepared in Example 5 for seven consecutive days. The nanovesicle concentration was 4 μM, with 100 μL administered each time. The model group received 0.9% saline. After seven days of treatment, vaginal secretions from female mice in the treatment and model groups were collected with cotton swabs and diluted 1000-fold with PBS. Plates were then plated and colony counted to assess the nanovesicles' ability to inhibit Candida albicans. Hematoxylin-eosin (H&E) staining was used to further assess the level of vaginal inflammation in female mice following nanovesicle treatment.

[0089] The experimental results are as follows Figure 8 As shown in the figure, compared with the normal group, the vaginal secretions of the rats in the modeling group were yellow and significantly increased, indicating that the modeling was successful. Figure 9 As shown in the figure, the number of colonies after administration was significantly less than that in the model group, indicating that the nanovesicles can effectively treat vaginitis in rats. Figure 10 As shown in the results of H&E staining, the vaginal epithelial mucosa in the model group was sloughed, and an increase in small dark blue round inflammatory cells revealed obvious inflammatory cell infiltration. In contrast, the vaginal epithelial mucosa in the drug-treated group was intact, with normal density of multi-layered squamous epithelium, fewer inflammatory cells, and better mucosal integrity.

[0090] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application. Various modifications and variations are possible for those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present application shall be included within the scope of protection of the present application.

Claims

1. An antimicrobial peptide having anti-Candida albicans activity, characterized in that: The amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO: 1, specifically: NH2-GFKKLKLFKLKPF-COOH.

2. The method for preparing the antimicrobial peptide according to claim 1, characterized in that: The preparation method adopts a solid phase synthesis method to synthesize the polypeptide sequentially from the C-terminus to the N-terminus.

3. Use of the antimicrobial peptide according to claim 1 in the preparation of antimicrobial drugs for inhibiting Candida albicans.

4. The use according to claim 3, characterized in that The minimum inhibitory concentration of the antimicrobial peptide is 4.0 μM.

5. Use of the antimicrobial peptide according to claim 1 in the preparation of nanomedicines for treating vaginitis.

6. A nanomedicine for treating vaginitis, characterized in that: The nanomedicine uses the antimicrobial peptide according to claim 1 as an active ingredient, and its dosage form is nanovesicles.

7. The nanomedicine according to claim 6, characterized in that The nanovesicles are prepared by a microfluidic method using the antimicrobial peptide according to claim 1, egg yolk lecithin, cholesterol, and DSPE-MPEG2000 as raw materials.

8. The nanomedicine according to claim 6, characterized in that In the nanovesicles, the molar ratio of antimicrobial peptide is 10.14%, the molar ratio of egg yolk lecithin is 50.7%, the molar ratio of cholesterol is 38.89%, and the molar ratio of DSPE-MPEG2000 is 0.27%.

9. The nanomedicine according to claim 6, characterized in that The minimum inhibitory concentration of the nano drug is 2.0 μM.

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