A method for the preparation of a pharmaceutical carrier for enhancing the antifungal activity of a drug and its antifungal use

Abstract

The application discloses a preparation method of a drug carrier for enhancing the antifungal activity of a drug, and comprises the following steps: S1, dissolving lecithin, cholesterol and an antifungal active drug in an organic solvent, heating and stirring until the organic solvent is completely volatilized to form a lipid film, then adding PBS, and obtaining drug-loaded liposomes by ultrasonic treatment under low temperature; S2, blending the drug-loaded liposomes and vesicles, and obtaining vesicle fusion drug-loaded liposomes by ultrasonic treatment; and S3, blending the vesicle fusion drug-loaded liposomes and a polylysine solution, adjusting pH, heating and stirring to react, and purifying to obtain polylysine-modified vesicle fusion drug-loaded liposomes. The application can improve the antifungal activity of existing drugs by using a new drug carrier, find more drugs for treating fungal infections, solve the infection problem caused by drug-resistant Candida albicans, reverse the fungal drug resistance, have better curative effect for eradicating Candida albicans, and can also maintain the health and balance of the vaginal microenvironment.

Description

Technical Field

[0001] The present invention relates to the field of drug carriers, in particular to a method for preparing a drug carrier capable of enhancing the antifungal activity of a drug and its antifungal application. Background Art

[0002] In recent decades, the incidence of serious fungal diseases has increased due to the growing number of immunocompromised individuals, including patients undergoing cancer treatment, organ transplant recipients, individuals living with HIV, and the expanding elderly population. The World Health Organization (WHO) recently released its first list of fungi of medical concern, aiming to guide research and public health initiatives and improve global strategies to address fungal infections and antifungal resistance. Vulvovaginal candidiasis (VVC), commonly caused by vaginal colonization by the fungus Candida albicans, is estimated to affect at least 75% of women of reproductive age worldwide, with approximately 6%-10% experiencing more than four recurrent infections per year, a condition known as recurrent vulvovaginal candidiasis (RVVC). Commonly used antifungal drugs for the treatment of VVC include azoles (such as clotrimazole, fluconazole, itraconazole, miconazole, and ketoconazole) and polyenes (such as amphotericin B). However, existing treatment options for recurrent VVC often fail to completely eliminate all pathogens and eradicate the disease. Therefore, how to effectively cure RVVC is a clinical problem.

[0003] Currently, there are only three types of antifungal drugs in clinical practice. Azoles and polyenes have been put into medical use as early as before 1980, and the third type (echinocandin) has been on the market for more than 20 years. However, these drugs can only temporarily suppress Candida albicans vaginitis, and patients often relapse after taking the drugs, which brings about the problem of drug resistance. The research and development of new antifungal drugs has stagnated, and there is an urgent need for new antifungal drugs to deal with the difficult-to-cure RVVC and the current situation of clinical fungal resistance. However, the traditional new drug research and development process has lasted for several years or even decades, and the capital investment is huge, so it is not an ideal means of discovering new antifungal drugs. "New uses for old drugs" can ensure the safety of drugs. It only requires developing new uses for existing drugs, which can save time and money.

[0004] The existing “new uses of old drugs” for antifungal drugs are mostly drug combination strategies. For example, the patent “A combination drug against Candida albicans” (application number: CN202010217155.6) combines dihydroartemisinin and fluconazole. Dihydroartemisinin, as a non-antifungal drug, has almost no antifungal effect when used alone, but the combined use of dihydroartemisinin and fluconazole can enhance the antibacterial activity of fluconazole against drug-resistant Candida albicans, producing a synergistic antifungal effect. In addition, ibuprofen, as a commonly used non-steroidal anti-inflammatory drug in clinical practice, is usually used in combination with azole drugs to reduce fungal resistance to azole drugs. For example, ibuprofen combined with fluconazole has a reversal effect on the fluconazole resistance of clinically isolated Candida albicans. The above-mentioned combination scheme mainly inhibits the efflux effect of fungi on fluconazole by inhibiting the activity of fungal efflux pumps by ibuprofen, thereby reversing the fungal resistance to fluconazole. The above studies show that ibuprofen itself has certain antifungal activity, but it is difficult to use it alone as an antifungal drug to treat fungal infections.

[0005] In summary, it is urgent to develop a new technical solution to solve the problems existing in the existing technology. Summary of the Invention

[0006] In response to the defects and shortcomings in the above-mentioned prior art, the present invention discloses a liposome carrier that can deliver drugs to the interior of fungi in a targeted manner, successfully enhancing the antifungal activity of the encapsulated drugs, and realizing the "new use of old drugs" for fungi using ibuprofen, a drug that traditionally cannot be used to treat fungal infections. The process includes first preparing liposomes (Lipo) encapsulating ibuprofen, and then fusing these liposomes with vesicles (EVs) derived from Candida albicans to form EV@Lipo, and then modifying the outer layer with polylysine (PLL) to form PLL-EV@Lipo. The experimental results show that the drug delivery system achieves the retention of drugs in the biofilm through the targeting effect of vesicles on homologous fungi and polylysine, thereby enhancing the content of ibuprofen in fungal cells, improving antifungal activity, and achieving effective treatment of Candida albicans infection. This scheme is expected to provide an effective strategy for the discovery of antifungal drugs and overcoming fungal resistance, and has good application prospects for fungal infections such as clinically refractory vulvovaginal candidiasis (VVC).

[0007] The present invention aims to develop an antifungal nanomedicine and drug carrier, and to encapsulate antifungal active drugs such as the non-steroidal anti-inflammatory drug ibuprofen through vesicle fusion drug-loaded liposomes to enhance its antibacterial activity against fungi, so that ibuprofen can be used to treat Candida albicans infection, realizing its "old drug new use" and solving the problem of Candida albicans resistance and the problem of RVVC being difficult to cure. Clinically, fungal infections are difficult to eradicate and easily relapse, and the fungal resistance formed is mainly due to the formation of biofilms. The present invention modifies the outer layer of EV-Lipo with polylysine PLL that can penetrate the biofilm to form PLL-EV@Lipo, thereby achieving the elimination of Candida albicans. At the same time, commonly used antifungal drugs in clinical practice often achieve a broad-spectrum bactericidal effect. After use, the vaginal flora is imbalanced, resulting in low vaginal immunity and subsequent infection. The present invention aims to achieve specific antibacterial effects on Candida albicans without destroying the balance of vaginal flora.

[0008] One of the objects of the present invention is to provide a method for preparing a drug carrier for enhancing the antifungal activity of a drug, comprising the following steps:

[0009] S1. Dissolving lecithin, cholesterol, and an antifungal drug in an organic solvent, heating and stirring until the organic solvent is completely evaporated to form a lipid film, then adding PBS and sonicating to obtain drug-loaded liposomes;

[0010] S2, blending the drug-loaded liposomes and vesicles, and sonicating under low temperature conditions to obtain vesicle-fused drug-loaded liposomes;

[0011] S3, blending the vesicle fusion drug-loaded liposomes with a polylysine solution, adjusting the pH, heating and stirring for reaction, and purifying to obtain polylysine-modified vesicle fusion drug-loaded liposomes.

[0012] Furthermore, the antifungal active drug is a drug having weak antifungal activity, preferably ibuprofen.

[0013] Furthermore, in step S1, the mass ratio of the lecithin, cholesterol and antifungal active drug is (5-15):1:(0.001-0.01).

[0014] Furthermore, in step S1, the heating temperature is 40-60°C.

[0015] Furthermore, in step S2, the vesicles are selected from one or more of the group consisting of Candida albicans outer membrane vesicles, Cryptococcus neoformans extracellular vesicles, Candida glabrata outer membrane vesicles, Candida auris outer membrane vesicles, and Candida tropicalis outer membrane vesicles.

[0016] Furthermore, in step S2, the mass ratio of the drug-loaded liposomes to the vesicles is (250-350):1.

[0017] Furthermore, in step S2, the ultrasonic power is 40-60W; and the ultrasonic time is 5-15 minutes.

[0018] Furthermore, in step S3, the pH range is 6-8.

[0019] Furthermore, in step S3, the heating temperature is 40-60°C.

[0020] Another object of the present invention is to provide an antifungal application of the drug carrier for enhancing the antifungal activity of drugs prepared by the method for preparing the drug carrier for enhancing the antifungal activity of drugs.

[0021] The present invention has at least the following beneficial effects:

[0022] This invention innovatively designs a vesicle-fusion drug-loaded liposome and a polylysine-modified vesicle-fusion drug-loaded liposome, aiming to enhance the antifungal activity of existing drugs through novel drug carriers, discover more drugs that can be used to treat fungal infections, and address the infection problem caused by drug-resistant Candida albicans. Specifically, the invention prepares ibuprofen-encapsulated Candida albicans vesicle-fusion liposomes EV@Lipo and modifies their outer layer with polylysine to obtain PLL-EV@Lipo. This aims to address the drug resistance problem of Candida albicans by leveraging the biomembrane penetration of polylysine, the homologous targeting of EVs, and the "new use" of ibuprofen. We applied it to in vitro and in vivo antifungal experiments, and the results showed that compared with azole drugs, this nano-drug delivery system can successfully reverse the fungal resistance of patients with vulvovaginal candidiasis (VVC). Compared with currently commonly used clinical drugs such as azoles (such as clotrimazole, fluconazole, itraconazole, miconazole and ketoconazole), polyenes and echinocandins, it has better efficacy in eradicating Candida albicans, while also maintaining the health and balance of the vaginal microenvironment. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The preparation methods, possible antibacterial mechanisms, and in vivo antibacterial schematics of EV@Lipo and PLL-EV@Lipo are shown;

[0024] in,

[0025] Figure 1 A shows the preparation method of EV@Lipo and PLL-EV@Lipo;

[0026] Figure 1 B shows the possible antibacterial mechanism of EV@Lipo and PLL-EV@Lipo;

[0027] Figure 1 C shows the schematic diagram of the in vivo antibacterial effect of EV@Lipo and PLL-EV@Lipo.

[0028] Figure 2 Fluorescence colocalization images, fluorescence resonance energy transfer spectra, transmission electron microscopy images, zeta potential, dynamic light scattering size, and release curves of EV, Lipo, EV@Lipo, and PLL-EV@Lipo are shown;

[0029] in,

[0030] Figure 2 A shows the fluorescence colocalization image (CLSM) of a mixture of DiO-labeled Lipo and DiI-labeled EVs;

[0031] Figure 2 B shows the fluorescence resonance energy transfer (FRET) spectra of EVs after sonication with DiO- and DiI-labeled Lipo probes;

[0032] Figure 2 C shows transmission electron microscopy (TEM) images of Candida albicans-derived EVs, Lipo, EV@Lipo, and PLL-EV@Lipo.

[0033] Figure 2 D shows the zeta potential of EV, Lipo, EV@Lipo, and PLL-EV@Lipo (n=3);

[0034] Figure 2 E shows the dynamic light scattering size (DLS) of EV, Lipo, EV@Lipo, and PLL-EV@Lipo;

[0035] Figure 2 F shows the release curve of PLL-EV@Lipo within 48 h under pH = 4 condition (n = 3) (****p < 0.0001).

[0036] Figure 3 Shown are the test results of EV targeting efficiency study and in vitro antibacterial study;

[0037] in,

[0038] Figure 3 A shows the CLSM images of Lipo (green), EV@Lipo (green), and PLL-EV@Lipo (green) targeting Candida albicans (red);

[0039] Figure 3 B shows the flow cytometry quantitative analysis of the targeting efficiency of EV@Lipo and Lipo to Candida albicans;

[0040] Figure 3 C shows the count of standard Candida albicans colony-forming units (1 g CFU / mL) after treatment with different groups (n=3);

[0041] Figure 3 D shows the count of colony-forming units (1 g CFU / mL) of clinically isolated Candida albicans after treatment with different groups (n=3);

[0042] Figure 3 E shows the Z-stack image of the penetration of Candida albicans biofilm after EV@Lipo and PLL-EV@Lipo treatment for 30 min (blue represents Candida albicans biofilm stained with DAPI; green represents EV@Lipo or PLL-EV@Lipo stained with DIO);

[0043] Figure 3 F shows the membrane permeability analysis of Candida albicans treated with EV@Lipo and PLL-EV@Lipo by flow cytometry;

[0044] Figure 3 G shows crystal violet-stained biofilm quantification (n=3);

[0045] Figure 3 H shows the Z-stack images of live and dead cell staining of Candida albicans biofilm after treatment in different groups;

[0046] Figure 3 I shows scanning electron microscopy (SEM) images of Candida albicans after treatment with ibuprofen and PLL-EV@Lipo for 3 h (hyphae inhibition and cell membrane rupture) (*p<0.05, **p<0.01, ****p<0.0001).

[0047] Figure 4 The results of the in vivo biocompatibility study and the therapeutic effect of PLL-EV@Lipo on VVC in Balb / c mice are shown.

[0048] in,

[0049] Figure 4 A shows a schematic diagram of VVC and treatment timeline in the Balb / c mouse model;

[0050] Figure 4 B shows the Candida albicans colony counts (1g CFU / mL) in the vaginal lavage fluid of mice 3 days, 5 days, and 7 days after treatment in different groups;

[0051] Figure 4 C shows the changes in vaginal redness, swelling, and discharge after treatment in different groups.

[0052] Figure 5 Shown are the test results of samples collected after PLL-EV@Lipo treatment;

[0053] in,

[0054] Figure 5 A shows PAS staining of vaginal sections of mice in different treatment groups;

[0055] Figure 5 B shows immunohistochemical staining of vaginal sections of mice in different treatment groups;

[0056] Figure 5 C shows the quantitative analysis of IL-6 by ELISA;

[0057] Figure 5 D shows the quantitative analysis of TNF-α by ELISA;

[0058] Figure 5 E shows the relative abundance heat map of vaginal phylum-level taxa in mice of different treatment groups;

[0059] Figure 5 F shows the Simpson index of mouse vaginal α diversity;

[0060] Figure 5 G shows the Shannon index of mouse vaginal α diversity;

[0061] Figure 5 H shows the ACE index of mouse vaginal α diversity;

[0062] Figure 5 I shows the Chao1 index of mouse vaginal α diversity (*p<0.05, ***p<0.001 and ns indicate not significant). DETAILED DESCRIPTION

[0063] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0064] Unless otherwise stated, the raw materials, reactions and post-treatment methods mentioned in the examples are common raw materials on the market and technical methods well known to those skilled in the art.

[0065] The following reagents, instruments or animals are used in the examples of the present invention.

[0066] Ibμprofen was purchased from Shanghai Leyan Company, China.

[0067] Lecithin was purchased from Shanghai MacLean Company, China.

[0068] Cholesterol was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0069] Sodium hydroxide (NaOH), hydrochloric acid (HCl), chloroform (CHCl3), dimethyl sulfoxide (DMSO), propidium iodide (PI), 1,1′-docosanyl-3,3,3′,3′-tetramethylindole cyanide (DiI), 3,3′-docosanyl-indole oxide (DiO), and bicinchoninic acid (BCA) detection kit were purchased from Beyotime Biotechnology Co., Ltd., Shanghai, China.

[0070] IL-6 and TNF-α ELISA kits were purchased from Shenzhen New Life Biotechnology Co., Ltd., China.

[0071] Cell counting kit (CCK-8) and live / dead bacterial viability detection kit (L-7012) were purchased from Invitrogen and Termo Fisher Scientific, USA.

[0072] Dulbecco's membrane medium (DMEM) was purchased from Beyotime Chemical Reagent Company, Shanghai, China.

[0073] Estradiol benzoate injection was purchased from Shanghai Quanyu Biotechnology Animal Drug Co., Ltd., China.

[0074] Candida albicans (ATCC 10231) was purchased from the American Type Culture Collection (ATCC).

[0075] Clinical Candida albicans strains were purchased from clinical patients at Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China.

[0076] PBS, phosphate buffered saline (Biosharp), was purchased from Guangzhou Scissorhands Gene Co., Ltd.

[0077] All aqueous solutions were prepared using deionized water (resistivity 18.2 MΩ·cm; Millipore).

[0078] Balb / c mice (6-8 weeks, female, weighing 21-23 g, SPF grade) were used as animal subjects in the present invention. Healthy female BALB / c mice aged six to eight weeks were purchased from the Experimental Animal Center of Sun Yat-sen University and raised in a specific pathogen-free facility.

[0079] The extracellular vesicles (EVs) of the present invention are extracellular vesicles derived from Candida albicans, and the preparation method thereof comprises the following steps:

[0080] Candida albicans (strain ATCC 10231) was cultured in YM medium at 30°C on a shaker for 48 h. The culture suspension was centrifuged at 4500 g for 10 min to remove the C. albicans cells. The supernatant was then ultracentrifuged at 100,000 g for 1 h at 4°C. The resulting extracellular vesicles were washed three times with PBS and then resuspended in PBS.

[0081] Example

[0082] A method for preparing a drug carrier for enhancing the antifungal activity of a drug comprises the following steps:

[0083] S1. Blend 200 mg of lecithin, 20 mg of cholesterol, and 172 μg of ibuprofen and dissolve in 5 mL of an organic solvent (chloroform:methanol = 3:1, v / v). After complete dissolution, evaporate the organic solvent using a rotary evaporator at 45°C and 60 rpm to form a lipid film. Then, add 5 mL of PBS and hydrate the lipid film with ultrasonication for 30 minutes to obtain drug-loaded liposomes (Lipo).

[0084] S2. 35.7 mg of the Lipo was mixed with 120 μg of extracellular vesicles, and ultrasonicated for 10 min in an ice bath using a 45 W ultrasonic wave with a 3 s on, 2 s off cycle mode to obtain vesicle fusion drug-loaded liposomes (EV-Lipo);

[0085] S3. The EV-Lipo was mixed with 1 mL of polylysine aqueous solution (15 mg / mL), stirred at 45°C and pH 7.0 for 2 h, and then centrifuged to remove unmodified polylysine to obtain polylysine-modified vesicle fusion drug-loaded liposomes (PLL-EV@Lipo).

[0086] Figure 1 The preparation methods, possible antibacterial mechanisms, and in vivo antibacterial schematics of EV@Lipo and PLL-EV@Lipo are shown;

[0087] in,

[0088] Figure 1 A shows the preparation method of EV@Lipo and PLL-EV@Lipo;

[0089] Figure 1 B shows the possible antibacterial mechanism of EV@Lipo and PLL-EV@Lipo;

[0090] Figure 1 C shows the schematic diagram of the in vivo antibacterial effect of EV@Lipo and PLL-EV@Lipo.

[0091] The present invention designs a Candida albicans vesicle fusion drug-loaded liposome EV@Lipo that encapsulates ibuprofen, and obtains PLL-EV@Lipo ( Figure 1 A), which enables ibuprofen to fight Candida albicans alone and repurpose existing drugs for new applications. The PLL-EV@Lipo system can deliver drugs to the inside of the biofilm through the action of polylysine penetrating the biofilm of Candida albicans; EV@Lipo can target the ibuprofen encapsulated in the liposomes to the inside of Candida albicans through the homologous targeting of fungal vesicles to Candida albicans, thereby increasing the concentration of the drug in the cytoplasm of the fungal cells and enhancing the antifungal activity of ibuprofen against planktonic Candida albicans and Candida albicans in the biofilm. In addition, the present invention explores the antifungal mechanism of action of ibuprofen: transcriptome analysis revealed that PLL-EV@Lipo and ibuprofen downregulated genes that control fungal vacuolar function, cation co-transporter activity, and monocarboxylic acid metabolism ( Figure 1 B) The vacuole, as a fungal organelle, has important biological functions. For example, the various enzymes it contains are essential for fungi. The vacuole is also crucial for the virulence and pathogenicity of Candida albicans. Loss of its activity also affects hyphal growth and calcium channel function. Therefore, ibuprofen's antifungal activity may originate from its interaction with the vacuole.

[0092] Based on the above, the present invention invented for the first time a Candida albicans vesicle fusion drug-loaded liposome EV@Lipo containing ibuprofen and PLL-EV@Lipo with polylysine modified outer layer ( Figure 1 A), effectively penetrates the biofilm at the site of fungal infection through the action of polylysine, and then the Candida albicans vesicles deliver antibacterial drugs to the bacterial site, achieving high antifungal efficacy. Experimental studies have shown that PLL-EV@Lipo has good bactericidal and therapeutic effects in the recurrent Candida albicans vaginal infection (RVVC) model in mice ( Figure 1 C), which provides a new treatment method and idea for Candida albicans infection.

[0093] Figure 2 Fluorescence colocalization images, fluorescence resonance energy transfer spectra, transmission electron microscopy images, zeta potential, dynamic light scattering size, and release curves of EV, Lipo, EV@Lipo, and PLL-EV@Lipo are shown;

[0094] in,

[0095] Figure 2 A shows the fluorescence colocalization image (CLSM) of a mixture of DiO-labeled Lipo and DiI-labeled EVs;

[0096] Figure 2B shows the fluorescence resonance energy transfer (FRET) spectra of EVs after sonication with DiO- and DiI-labeled Lipo probes;

[0097] Figure 2 C shows transmission electron microscopy (TEM) images of Candida albicans-derived EVs, Lipo, EV@Lipo, and PLL-EV@Lipo.

[0098] Figure 2 D shows the zeta potential of EV, Lipo, EV@Lipo, and PLL-EV@Lipo (n=3);

[0099] Figure 2 E shows the dynamic light scattering size (DLS) of EV, Lipo, EV@Lipo, and PLL-EV@Lipo;

[0100] Figure 2 F shows the release curve of PLL-EV@Lipo within 48 h under pH = 4 condition (n = 3) (****p < 0.0001).

[0101] CLSM( Figure 2 A) The results show that the EV membrane stained with DiI emits red fluorescence, the liposome membrane stained with DiO emits green fluorescence, and yellow fluorescence is observed in the merged view, indicating that the liposome membrane has successfully fused with the vesicle membrane. To further verify the fusion process, we labeled the liposome membrane with dyes DiO and DiI with Fōrster resonance energy transfer (FRET) properties and then fused them with unlabeled EV membranes. After adding EV membrane, the fluorescence intensity (DiI) at 569nm decreased ( Figure 4 B), The fluorescence signal of DiI dye decays, indicating the fusion of liposome membrane with EV membrane.

[0102] TEM studies ( Figure 2 C) Confirmation that EVs have nanostructures with clear boundaries and a size of 100 nm.

[0103] Dynamic light scattering (DLS) particle size determination results ( Figure 2 E) shows that the sizes of EV, Lipo, EV@Lipo and PLL-EV@Lipo are around 100 nm, which is consistent with the TEM results.

[0104] Zeta potential results ( Figure 2 D) shows that the potential of Lipo is -11.69 mV, EV@Lipo is -15.90 mV, and PLL-EV@Lipo is 16.41 mV, indicating that the surface of EV@Lipo was successfully modified with polylysine cations.

[0105] To investigate the release of ibuprofen from PLL-EV@Lipo, we measured the cumulative release over 48 h at pH = 4 ( Figure 2 F) The results showed that at pH = 4 (vaginal pH), the release rate of ibuprofen did not have any burst release, and the cumulative release was approximately 70%.

[0106] Test Example 1

[0107] 1. EV targeting efficiency study

[0108] Test method:

[0109] (1) DiI-labeled Candida albicans vesicles: 1 mL of 120 μg / mL Candida albicans vesicles was added with DiI dye and incubated at 37°C for 1 h.

[0110] (2) DAPI staining of Candida albicans, Candida glabrata and HaCat cells: Take 100 μL 10 6 CFU / mL of Candida albicans, Candida glabrata and 100μL 10 4 Each sample was stained with DAPI and incubated at 37°C for 10 min, followed by centrifugation at 4500 g and 4°C for 10 min. Finally, the supernatant was removed and the pellet was resuspended in 1 mL of PBS.

[0111] (3) Incubation of stained C. albicans vesicles with various samples: 500 μL of stained C. albicans vesicles and 500 μL of each C. albicans, C. glabrata, and HaCat cell sample were incubated for 4 h. The mixture was then centrifuged at 4500 g and 4°C for 10 min, and the pellet was resuspended in PBS.

[0112] (4) Confocal Observation and Flow Cytometry: The samples were observed using a confocal laser scanning microscope (CLSM) and flow cytometry was used to count the samples.

[0113] 2. In vitro antibacterial studies

[0114] Test method:

[0115] (1) Antibacterial study on free cells: Prepare a 96-well plate containing SDB medium. Add microbial suspension (ATCC 64548, CFU 10 5 ). Add 8 groups of samples: PBS, ibuprofen (final concentration 31.25 μg / mL), PLL-EV@Lipo (final concentration 31.25 μg / mL), itraconazole (final concentration 1 μg / mL). Incubate at 30°C for 24 h. Measure OD at 0 h, 12 h, and 24 h. 595For 24h results, the samples (diluted 10 4 The cells were inoculated on agar plates and CFU counts were performed (n=3).

[0116] (2) Antibacterial study on biofilm: The fungal suspension (OD 1.5) was diluted in YM medium (to reach CFU10 5 ). Add 500 μL of fungal suspension to each well of a 24-well plate. Incubate at 30°C for 48 h. Remove the supernatant. Add 100 μL of PBS, ibuprofen (final concentration 31.25 μg / mL), PLL-EV@Lipo (final concentration 31.25 μg / mL), and itraconazole (final concentration 1 μg / mL) respectively. Incubate for 12 h (n=3). Prepare a 1% crystal violet solution and filter through a 0.45 μm membrane. Remove the supernatant and add 100 μL of crystal violet solution to each well. Incubate for 40 min. Remove the supernatant and dry for 5 min. Add 200 μL of anhydrous ethanol for decolorization. After shaking for 30 min, measure the OD using an enzyme-linked immunosorbent assay (ELISA) reader. 595 value.

[0117] (3) Live / dead staining confocal observation: The fungal suspension (OD 1.5) was diluted in YM medium (to reach CFU = 10 5 ). 1 μL of fungal suspension was added to each confocal culture dish. Incubated at 37°C for 48 h. The supernatant was removed. 100 μL of PBS, ibuprofen (final concentration 31.25 μg / mL), PLL-EV@Lipo, and itraconazole (final concentration 1 μg / mL) were added respectively. Incubated for 12 h (n=3). The supernatant was removed. Stained with BacLight Live / Dead Stain (Invitrogen, USA) for 30 min. Images were captured using a confocal microscope. Incubated with propidium iodide (PI) and SYTO 9 dye, and free dye was removed after centrifugation. The stained fungal suspension was imaged on a microscope slide (SYTO 9 for live cells, excitation: 488 nm, emission: 501 nm; PI for dead cells, excitation: 535 nm, emission: 617 nm).

[0118] (4) Antibacterial study on clinical isolates: Prepare a 96-well plate containing SDB medium. Add microbial suspension (clinical strains 5, 2, 4, 9, CFU = 10) to each well. 5 ). Four groups of samples were added: PBS, ibuprofen (final concentration 31.25 μg / mL), PLL-EV@Lipo, and itraconazole (final concentration 1 μg / mL). Incubated at 30°C for 24 h. OD was measured at 0 h, 12 h, and 24 h. 595 For 24h results, the samples (diluted 104 The cells were inoculated on agar plates and CFU counts were performed (n=3).

[0119] Figure 3 Shown are the test results of EV targeting efficiency study and in vitro antibacterial study;

[0120] in,

[0121] Figure 3 A shows the CLSM images of Lipo (green), EV@Lipo (green), and PLL-EV@Lipo (green) targeting Candida albicans (red);

[0122] Figure 3 B shows the flow cytometry quantitative analysis of the targeting efficiency of EV@Lipo and Lipo to Candida albicans;

[0123] Figure 3 C shows the count of standard Candida albicans colony-forming units (1 g CFU / mL) after treatment with different groups (n=3);

[0124] Figure 3 D shows the count of colony-forming units (1 g CFU / mL) of clinically isolated Candida albicans after treatment with different groups (n=3);

[0125] Figure 3 E shows the Z-stack image of the penetration of Candida albicans biofilm after EV@Lipo and PLL-EV@Lipo treatment for 30 min (blue represents Candida albicans biofilm stained with DAPI; green represents EV@Lipo or PLL-EV@Lipo stained with DIO);

[0126] Figure 3 F shows the membrane permeability analysis of Candida albicans treated with EV@Lipo and PLL-EV@Lipo by flow cytometry;

[0127] Figure 3 G shows crystal violet-stained biofilm quantification (n=3);

[0128] Figure 3 H shows the Z-stack images of live and dead cell staining of Candida albicans biofilm after treatment in different groups;

[0129] Figure 3 I shows scanning electron microscopy (SEM) images of Candida albicans after treatment with ibuprofen and PLL-EV@Lipo for 3 h (hyphae inhibition and cell membrane rupture) (*p<0.05, **p<0.01, ****p<0.0001).

[0130] We verified the targeting of Lipo, EV@Lipo and PLL-EV@Lipo to Candida albicans. CLSM( Figure 3 A) The results showed that compared with Lipo, the merged images of the EV@Lipo group and the PLL-EV@Lipo group showed more yellow areas, indicating that the green-labeled EV@Lipo and PLL-EV@Lipo had homologous targeting ability to the red-labeled Candida albicans; quantitative flow cytometry results ( Figure 3 B) shows that the homologous targeting efficiency of EV@Lip to Candida albicans is 25.8%, while that of Lipo is only 5.96%. This targeting effect is attributed to the role of EV. We speculated that if a sufficient amount of ibuprofen enters the fungal cells, the fungus will be killed. Therefore, we conducted an in vitro anti-free bacteria experiment ( Figure 3 In the EV@Lipo group of C), the ibuprofen dose was increased from 31.25 μg / mL to 62.5 μg / mL in order to achieve high ibuprofen enrichment in fungal cells. The results showed that the doubled dose could kill all fungi, just like PLL-EV@Lipo. In addition, the doubled dose of EV@Lipo had a significant effect on the four clinical isolates ( Figure 3 The antibacterial results of the EV@Lipo assay (D) showed that EV@Lipo was able to eradicate clinical Candida albicans. Taking advantage of the ability of polylysine (PLL) to penetrate biofilms, we modified it onto the outside of EV@Lipo to form PLL-EV@Lipo, hoping to achieve a good anti-biofilm effect. We observed Z-stack images of nanoparticles penetrating the biofilm after 30 minutes of treatment with EV@Lipo and PLL-EV@Lipo on Candida albicans biofilms ( Figure 3 E), the results showed that PLL successfully delivered EV@Lipo into the interior of the C. albicans biofilm.

[0131] Flow cytometry ( Figure 3 F) Membrane permeability analysis of Candida albicans treated with EV@Lipo and PLL-EV@Lipo revealed that PLL-EV@Lipo treatment increased the permeability of Candida albicans membranes, allowing more drugs to enter the fungal cells. This indicates that PLL not only promotes drug delivery within biofilms, but also enables intracellular delivery of fungi. Figure 3 G), PLL-EV@Lipo effectively inhibited the formation of biofilm.

[0132] Z-stack images ( Figure 3 H) Live / dead staining results of C. albicans biofilm showed that PLL-EV@Lipo essentially eradicated the C. albicans biofilm (almost all cells appeared red, indicating dead cells).

[0133] To investigate the phenotypic differences in the antifungal effects of ibuprofen alone and PLL-EV@Lipo on C. albicans, we performed scanning electron microscopy (SEM) imaging ( Figure 3 I). We observed that ibuprofen inhibited hyphae formation, whereas PLL-EV@Lipo not only inhibited hyphae formation but also eradicated C. albicans cells (causing fungal cell atrophy). Therefore, the PLL-EV@Lipo system exhibited excellent in vitro efficacy against C. albicans.

[0134] Test Example 2

[0135] Study on the therapeutic effect and in vivo biocompatibility of PLL-EV@Lipo on vulvovaginal candidiasis (VVC) in Balb / c mice

[0136] Test method:

[0137] Each Balb / c mouse was injected subcutaneously with 50 μL of estradiol benzoate in the hind leg 6 days before inoculation, and injected once every 2 days for a total of 3 times. 6 cells / mL of Candida albicans spore suspension was dissolved in PBS (inoculation dose was 5×10 4 Candida albicans spores) were injected into the vaginal cavity of mice, and then the mice were inverted for 5 minutes. After inoculation, the vaginal mucosa of each mouse was observed daily for signs of inflammation, such as congestion, edema, bleeding, erosion, and increased secretions. On days 3, 5, and 7 after inoculation, vaginal irrigation was performed with PBS, and the irrigation solution was thoroughly mixed. Subsequently, the irrigation solution was incubated on YM agar plates at 30°C for 24 hours for CFU count. CFU counts exceeding 10 4 This shows that the model was successfully established. After the model was successfully established, the mice were divided into different experimental groups (n=6) for treatment. The treatment process involved dripping 50 μL of the test substance into the vagina for 2 minutes, and then inverting the mice for 2 minutes, with treatment every 2 days. On the 3rd, 5th and 7th days of treatment, the vagina was rinsed with PBS, and the collected rinsing fluid was cultured on a YM agar plate at 30°C for 24 hours for CFU counting. Photos were taken to record inflammatory changes such as redness, erosion and increased secretions. On the 7th day of treatment, the mice were killed and vaginal tissue samples were collected for immunohistochemical staining (IL-6, TNF-α) to observe inflammation, and PAS staining was performed to evaluate fungal infection. The supernatant of the vaginal tissue homogenate was subjected to Elisa quantitative analysis of IL-6 and TNF-α factors. In addition, 16S rRNA analysis of the vaginal tissue microenvironment was performed to study α diversity.

[0138] Figure 4 The results of the in vivo biocompatibility study and the therapeutic effect of PLL-EV@Lipo on VVC in Balb / c mice are shown.

[0139] in,

[0140] Figure 4 A shows a schematic diagram of VVC and treatment timeline in the Balb / c mouse model;

[0141] Figure 4 B shows the Candida albicans colony counts (1g CFU / mL) in the vaginal lavage fluid of mice 3 days, 5 days, and 7 days after treatment in different groups;

[0142] Figure 4 C shows the changes in vaginal redness, swelling, and discharge after treatment in different groups.

[0143] To bring this research closer to clinical application, we used EV@Lipo and PLL-EV@Lipo to study the therapeutic effects in a mouse model of Candida albicans vaginitis, which has a high clinical incidence. Figure 4 A shows the process from model establishment to treatment endpoint. Figure 4 B shows the results of vaginal lavage colony counts in different groups of mice (PLL-EV@Lipo, EV@Lipo, Lipo, ibuprofen, itraconazole, PBS) on days 0, 3, 5, and 7. In the EV@Lipo group, log 10 The CFU decreased from 6.9 on day 0 to 5.0 on day 7, a 2 log reduction. 10 In the PLL-EV@Lipo group, log 10 The CFU decreased from 6.8 on day 0 to 4.3 on day 7, a 2.5 log reduction. 10 CFU. Figure 4 C depicts the changes in vaginal redness, swelling, and discharge over time in the different treatment groups. Compared with the other groups, the EV@Lipo and PLL-EV@Lipo groups showed the most significant recovery of vaginal redness on day 7. Compared with day 0, vaginal redness was significantly improved, with no yellow discharge.

[0144] For samples collected on day 7 (endpoint), vaginal lavage fluid was analyzed using 16S rRNA sequencing to investigate the abundance and diversity of the vaginal microbiome. At the end of the experiment, mice were euthanized, and vaginal tissue samples were collected for immunohistochemical staining (IL-6, TNF-α) to visualize inflammation and post-transcriptional scintigraphy (PAS) staining to assess fungal burden. Supernatants from vaginal tissue homogenates were analyzed by ELISA for IL-6 and TNF-α.

[0145] Figure 5 Shown are the test results of samples collected after PLL-EV@Lipo treatment;

[0146] in,

[0147] Figure 5 A shows PAS staining of vaginal sections of mice in different treatment groups;

[0148] Figure 5 B shows immunohistochemical staining of vaginal sections of mice in different treatment groups;

[0149] Figure 5 C shows the quantitative analysis of IL-6 by ELISA;

[0150] Figure 5 D shows the quantitative analysis of TNF-α by ELISA;

[0151] Figure 5 E shows the relative abundance heat map of vaginal phylum-level taxa in mice of different treatment groups;

[0152] Figure 5 F shows the Simpson index of mouse vaginal α diversity;

[0153] Figure 5 G shows the Shannon index of mouse vaginal α diversity;

[0154] Figure 5 H shows the ACE index of mouse vaginal α diversity;

[0155] Figure 5 I shows the Chao1 index of mouse vaginal α diversity (*p<0.05, ***p<0.001 and ns indicate not significant).

[0156] PAS staining results ( Figure 5 A) shows that in the PLL-EV@Lipo group and the healthy group, almost no fungi were observed (purple area), and most of them were cell nuclei (blue area). This shows that PLL-EV@Lipo effectively eliminated fungal infection. In the immunohistochemistry results ( Figure 5 In B), lower levels of vaginal inflammation were observed in the PLL-EV@Lipo group and the healthy group. (Brown represents the areas of inflammatory factors IL-6 and TNF-α, while blue represents the areas of cell nuclei.) ELISA quantitative analysis of IL-6 and TNF-α was consistent with the immunohistochemistry results. Figure 5 C to 5D): PLL-EV@Lipo effectively suppresses inflammation in fungal-infected mice. Patients with candidal vaginitis are associated with dysbiosis, characterized by reduced biodiversity, a lower proportion of Firmicutes, and an increased proportion of Proteobacteria.

[0157] To evaluate whether PLL-EV@Lipo therapy could restore the healthy composition of the vaginal microbiota in mice compared to itraconazole treatment, we used second ribosomal internal transcribed spacer amplicon sequencing and assessed alpha diversity using Simpson, Shannon, ACE, and Chao 1 entropy indices by 16S ribosomal RNA gene sequencing. Phylum-level analysis showed that after PLL-EV@Lipo treatment, the relative abundance of Proteobacteria decreased significantly, while the relative abundance of Firmicutes increased, contributing to the restoration of a more balanced and healthier vaginal microbiome ( Figure 5 E), demonstrating the safety of the vaginal microenvironment treated with PLL-EV@Lipo.

[0158] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations that come within the meaning and range of equivalents of the claims be embraced therein.

[0159] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

[0160] References:

[0161] [1]Lee Y, Puumala E, Robbins N, et al. Antifungal Drug Resistance: Molecular Mechanisms in Candida albicans and Beyond[J]. Chem Rev, 2021, 121(6): 3390-3411.

[0162] [2]Pfaller MA,Diekema D J.Epidemiology of invasive mycoses inNorthAmerica[J].Crit Rev Microbiol,2010,36(1):1-53.

[0163] [3]Hoenigl M,Seidel D,Sprute R,et al.COVID-19-associated fungalinfections[J].Nat Microbiol,2022,7(8):1127-1140.

[0164] [4]Lopes J P,Lionakis M S.Pathogenesis and virulence of Candidaalbicans[J].Virulence,2022,13(1):89-121.

[0165] [5]Denning D W,Kneale M,Sobel J D,et al.Global burden ofrecurrentvulvovaginal candidiasis:a systematic review[J].Lancet Infect Dis,2018,18(11):e339-e347.

[0166] [6]Cooke G,Watson C,Deckx L,et al.Treatment for recurrentvulvovaginal candidiasis(thrush)[J].Cochrane Database Syst Rev,2022,1(1):Cd009151.

[0167] [7]Butts A,Krysan D J.Antifungal drug discovery:something old andsomething new[J].PLoS Pathog,2012,8(9):e1002870.

[0168] [8]Zhang Q,Liu F,Zeng M,et al.Drug repurposing strategies in thedevelopment ofpotential antifungal agents[J].Appl Microbiol Biotechnol,2021,105(13):5259-5279.

[0169] [9]Pushpakom S,Iorio F,Eyers PA,et al.Drug repurposing:progress,challenges andrecommendations[J].Nat Rev Drug Discov,2019,18(1):41-58.

[0170]

[10] Lohse M B,Gulati M,Johnson A D,et al.Development and regulationofsingle-and multi-species Candida albicans biofilms[J].Nat Rev Microbiol,2018,16(1):19-31.

[0171]

[11] Finkel J S,Mitchell A P.Genetic control of Candida albicansbiofilmdevelopment[J].Nat Rev Microbiol,2011,9(2):109-18.

[0172]

[12] Mukherjee P K,Chandra J.Candida biofilm resistance[J].Drug ResistUpdat,2004,7(4-5):301-9.

[0173]

[13] Du X,Yin S,Xu L,et al.Polylysine and cysteine functionalizedchitosannanoparticle as an efficient platform for oral delivery of paclitaxel[J].Carbohydr Polym,2020,229:115484.

[0174]

[14] Shen C,Islam M T,Masuda Y,et al.Transcriptional changes involvedininhibition of biofilm formation byε-polylysine in Salmonella Typhimurium[J].ApplMicrobiol Biotechnol,2020,104(12):5427-5436.

[0175]

[15] Wei L,Wu R,Wang C,et al.Effects ofε-Polylysine on PseudomonasAeruginosaandAspergillus Fumigatus Biofilm In Vitro[J].Med Sci Monit,2017,23:4225-4229.

[0176]

[16] Wei G,Liu Q,Wang X,et al.A probiotic nanozyme hydrogel regulatesvaginalmicroenvironment for Candida vaginitis therapy[J].Sci Adv,2023,9(20):eadg0949.

[0177]

[17] Olsen I.Attenuation ofCandida albicans virulence with focus ondisruption ofitsvacuole functions[J].J Oral Microbiol,2014,6.

[0178]

[18] Lv Q,Yan L,Jiang Y.The Importance of Vacuolar Ion HomeostasisandTrafficking in Hyphal Development and Virulence in Candida albicans[J].Front Microbiol,2021,12:779176.

[0179]

[19] Park M,Do E,Jung W H.Lipolytic enzymes involved in the virulenceofhumanpathogenic fungi[J].Mycobiology,2013,41(2):67-72.

[0180]

[20] Li X,Hou Y,Yue L,et al.Potential Targets for Antifungal DrugDiscovery Basedon Growth and Virulence in Candida albicans[J].AntimicrobAgents Chemother,2015,59(10):5885-91.

Claims

1. A method for preparing polylysine-modified vesicle-fused drug-loaded liposomes, characterized in that: The steps include: S1. Dissolving lecithin, cholesterol, and an antifungal drug in an organic solvent, heating and stirring until the organic solvent is completely evaporated to form a lipid film, then adding PBS and sonicating to obtain drug-loaded liposomes; S2, blending the drug-loaded liposomes and vesicles, and sonicating under low temperature conditions to obtain vesicle-fused drug-loaded liposomes; S3, blending the vesicle fusion drug-loaded liposomes with a polylysine solution, adjusting the pH, heating and stirring the mixture for reaction, and purifying the mixture to obtain polylysine-modified vesicle fusion drug-loaded liposomes; The antifungal active drug is ibuprofen; The vesicles are outer membrane vesicles of Candida albicans.

2. The method for preparing the polylysine-modified vesicle-fused drug-loaded liposome according to claim 1, characterized in that: In step S1, the mass ratio of the lecithin, cholesterol and antifungal active drug is (5-15):1:(0.001-0.01).

3. The method for preparing the polylysine-modified vesicle-fused drug-loaded liposome according to claim 1, characterized in that: In step S1, the heating temperature is 40-60°C.

4. The method for preparing the polylysine-modified vesicle-fused drug-loaded liposome according to claim 1, characterized in that: In step S2, the mass ratio of the drug-loaded liposomes to the vesicles is (250-350):

1.

5. The method for preparing the polylysine-modified vesicle-fused drug-loaded liposome according to claim 1, characterized in that: In step S2, the ultrasonic power is 40-60W; and the ultrasonic time is 5-15 minutes.

6. The method for preparing the polylysine-modified vesicle-fused drug-loaded liposome according to claim 1, characterized in that: In step S3, the pH range is 6-8.

7. The method for preparing the polylysine-modified vesicle-fusion drug-loaded liposome according to claim 1, characterized in that: In step S3, the heating temperature is 40-60°C.

8. Use of the polylysine-modified vesicle-fusion drug-loaded liposomes prepared by the method for preparing the polylysine-modified vesicle-fusion drug-loaded liposomes as described in any one of claims 1 to 7 in the preparation of drugs for treating Candida albicans infection.

Images