Preparation method of drug carrier for enhancing antifungal activity of drug and antifungal application of drug carrier

Abstract

The invention discloses a preparation method of a drug carrier for enhancing antifungal activity of a drug, which 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 membrane, then adding PBS (Phosphate Buffer Solution), and carrying out ultrasonic treatment under a low-temperature condition to obtain a drug-loaded liposome; s2, blending the drug-loaded liposome and vesicles, and performing ultrasonic treatment to obtain vesicle fused drug-loaded liposome; and S3, blending the vesicle fused drug-loaded liposome with a polylysine solution, adjusting the pH value, heating and stirring for reaction, and purifying to obtain the polylysine modified vesicle fused drug-loaded liposome. The antifungal activity of existing drugs is improved through a novel drug carrier, more drugs capable of being used for treating fungal infection are found, and the infection problem caused by drug-resistant candida albicans is solved; the composition can reverse fungal drug resistance, has a better curative effect of eradicating Candida albicans, and also can keep the health and balance of the vaginal microenvironment.

Description

Technical Field

[0001] The invention relates to the field of drug carriers, and in particular to a preparation method of a drug carrier for enhancing the antifungal activity of drugs and the antifungal application thereof. Background Art

[0002] In recent decades, the incidence of serious illnesses caused by fungi has increased due to an increase in the number of immunocompromised people, including patients undergoing cancer treatment, organ transplant recipients, people with HIV infection, and the expanding elderly population. Recently, the World Health Organization (WHO) released the first list of fungi of medical concern, which aims to guide research and public health initiatives to improve the global strategy to address fungal infections and antifungal drug resistance. Vulvovaginal candidiasis (VVC) is usually caused by vaginal colonization of the fungus Candida albicans. It is estimated that at least 75% of women of childbearing age will develop the disease worldwide, and about 6%-10% of women will experience more than 4 recurrent infections per year, a condition known as recurrent vulvovaginal candidiasis (RVVC). Commonly used antifungal drugs for the treatment of vulvovaginal candidiasis include azoles (such as clotrimazole, fluconazole, itraconazole, miconazole, and ketoconazole) and polyenes (such as amphotericin B). However, existing treatment options for recurrent vulvovaginal candidiasis often fail to completely kill 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 new antifungal drugs are urgently needed to deal with the difficult-to-cure RVVC and the current situation of clinical fungal resistance. However, the traditional new drug 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" have a definite guarantee for the safety of drugs. It only requires the development of 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 “Anti-Candida albicans Combination Drug” (Application Number: CN202010217155.6) combines dihydroartemisinin and fluconazole. As a non-antifungal drug, dihydroartemisinin has almost no antifungal effect when used alone, but the combination of dihydroartemisinin and fluconazole can enhance the antibacterial activity of fluconazole against drug-resistant Candida albicans and produce 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 (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 treat fungal infections as an antifungal drug alone.

[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 view of the defects and deficiencies in the above-mentioned prior art, the present invention discloses a liposome carrier capable of delivering drugs to the interior of fungi in a targeted manner, successfully enhancing the antifungal activity of the encapsulated drug, and realizing the "new use of old drugs" for fungi using ibuprofen, a drug that cannot be traditionally used to treat fungal infections. The process includes first preparing liposomes (Lipo) encapsulating ibuprofen, 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 biofilms 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 non-steroidal anti-inflammatory drugs 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, realize its "old drug new use", and solve the problem of Candida albicans resistance and the problem that RVVC is difficult to cure. Clinically, fungal infection is difficult to eradicate and easy to recur, and the fungal resistance formed is mainly due to the formation of biofilm. The present invention modifies the outer layer of EV-Lipo with polylysine PLL that can penetrate the biofilm to form PLL-EV@Lipo, and eliminates Candida albicans; at the same time, the commonly used antifungal drugs in clinical practice often achieve a broad-spectrum bactericidal effect, and the vaginal flora is imbalanced after use, thereby leading to low vaginal immunity and causing subsequent infections. 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 antifungal active drugs 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 ultrasonicating 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 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 Candida albicans outer membrane vesicles, Cryptococcus neoformans extracellular vesicles, Candida glabrata outer membrane vesicles, Candida auris outer membrane vesicles or 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 power of the ultrasound is 40-60W; and the ultrasound time is 5-15min.

[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 a drug prepared by the method for preparing the drug carrier for enhancing the antifungal activity of a drug.

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

[0022] The present invention innovatively designs a vesicle fusion drug-loaded liposome and a polylysine-modified vesicle fusion drug-loaded liposome, aiming to improve the antifungal activity of existing drugs through new drug carriers, discover more drugs that can be used to treat fungal infections, and solve the infection problem caused by drug-resistant Candida albicans. Specifically, the present invention prepares Candida albicans vesicle fusion liposomes EV@Lipo encapsulating ibuprofen, and obtains PLL-EV@Lipo after modifying its outer layer with polylysine, aiming to solve the drug resistance problem of Candida albicans through the biomembrane penetration of polylysine, the homologous targeting of EV, and the "new use of old drugs" of ibuprofen. We applied it to in vitro and in vivo antifungal experiments, and the results showed that compared with azole drugs, this nanodrug 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 schematic diagrams 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 activity 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 the mixture of DiO-labeled Lipo and DiI-labeled EV;

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

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

[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 The test results of EV targeting efficiency study and in vitro antibacterial study are shown;

[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 (lg CFU / mL) after treatment with different groups (n=3);

[0041] Figure 3 D shows the counts of colony-forming units (lg 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 treated with EV@Lipo and PLL-EV@Lipo for 30 min (blue represents the 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 the 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 test results of the PLL-EV@Lipo treatment effect on VVC in Balb / c mice and the in vivo biocompatibility study 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 (lg 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 heat map of the relative abundance of vaginal phylum-level taxa in mice in 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 alpha diversity. (*p<0.05, ***p<0.001 and ns indicate not significant). DETAILED DESCRIPTION

[0063] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments 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, Termo Fisher Scientific, USA.

[0072] Dulbecco's 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 Scissorhand Gene Co., Ltd.

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

[0078] The present invention uses Balb / c mice (6-8 weeks, female, weighing 21-23g, SPF grade) as animal experimental subjects. 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 (EV) 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 for 48 h under a shaker. The culture suspension was centrifuged at 4500 g for 10 min to remove Candida albicans cells. Subsequently, the supernatant was ultracentrifuged at 100000 g at 4°C for 1 h. The obtained 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. 200 mg of lecithin, 20 mg of cholesterol and 172 μg of ibuprofen were mixed and dissolved in 5 mL of organic solvent (chloroform: methanol = 3:1, v / v). After complete dissolution, the organic solvent was evaporated using a rotary evaporator at 45 ° C and 60 rpm to form a lipid film. Then 5 mL of PBS was added and the lipid film was hydrated with ultrasound 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 with a power of 45 W and a cycle mode of 3 s on and 2 s off 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 for 2 h at 45° C. and pH 7.0, 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 schematic diagrams 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 activity of EV@Lipo and PLL-EV@Lipo.

[0091] The present invention designs a Candida albicans vesicle-fused drug-loaded liposome EV@Lipo that encapsulates ibuprofen, and obtains PLL-EV@Lipo ( Figure 1 A), enabling ibuprofen to fight Candida albicans alone, repurposing 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 liposomes to the inside of Candida albicans through the homologous targeting of fungal vesicles to Candida albicans, increase the concentration of the drug in the cytoplasm of fungal cells, and enhance the antifungal activity of ibuprofen against planktonic Candida albicans and Candida albicans in biofilms. In addition, the present invention explores the antifungal mechanism of action of ibuprofen: transcriptome analysis revealed that PLL-EV@Lipo and ibuprofen down-regulated 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 contained in it are essential substances for fungi. The vacuole is also crucial for the virulence and pathogenicity of Candida albicans. The loss of its activity also affects hyphal growth and calcium ion channel function. Therefore, the antifungal activity of ibuprofen may originate from its interaction with the vacuole.

[0092] Based on the above, the present invention first invented a Candida albicans vesicle fusion drug-loaded liposome EV@Lipo containing ibuprofen and PLL-EV@Lipo ( 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 the mixture of DiO-labeled Lipo and DiI-labeled EV;

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

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

[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 the 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) It was confirmed 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 EV@Lipo was successfully surface modified with polylysine cations.

[0105] To investigate the release of ibuprofen from PLL-EV@Lipo, we determined the cumulative release over 48 h at pH = 4 ( Figure 2 F). The results show that under pH = 4 (vaginal pH), the release rate of ibuprofen does not have any burst release, and the cumulative release amount is about 70%.

[0106] Test Example 1

[0107] 1. EV targeting efficiency study

[0108] Test method:

[0109] (1) DiI-labeled Candida albicans vesicles: Take 1 mL of 120 μg / mL Candida albicans vesicles, add DiI dye to the vesicles, and incubate 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, incubated at 37°C for 10 min, and then centrifuged 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 Candida albicans vesicles with various samples: 500 μL of stained Candida albicans vesicles and 500 μL of each Candida albicans, Candida glabrata and HaCat cell sample were taken and incubated for 4 h. Then, the mixture was 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 by confocal laser scanning microscopy (CLSM) and flow cytometry was used to count the samples.

[0113] 2. In vitro antibacterial study

[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 the 24h results, the samples (diluted 10 4 The cells were inoculated on agar plates and CFU counted (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 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 5 ). Add 4 groups of samples: PBS, ibuprofen (final concentration 31.25 μg / mL), PLL-EV@Lipo, itraconazole (final concentration 1 μg / mL). Incubate at 30°C for 24 h. Measure OD at 0 h, 12 h, and 24 h. 595 For the 24h results, the samples (diluted 104 The cells were inoculated on agar plates and CFU counted (n=3).

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

[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 (lg CFU / mL) after treatment with different groups (n=3);

[0124] Figure 3 D shows the counts of colony-forming units (lg 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 treated with EV@Lipo and PLL-EV@Lipo for 30 min (blue represents the 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 the 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%, which is attributed to the action of EV. We speculated that if enough ibuprofen enters the fungal cells, the fungi will be killed, so we conducted an in vitro anti-free bacteria experiment ( Figure 3 C) in the EV@Lipo group, the ibuprofen dose was increased from 31.25 μg / mL to 62.5 μg / mL in order to achieve high enrichment of ibuprofen 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 no significant effect on the four clinical isolates ( Figure 3 D) showed that EV@Lipo can eradicate clinical Candida albicans. Taking advantage of the property of polylysine (PLL) to penetrate biofilms, we modified it on the outside of EV@Lipo to form PLL-EV@Lipo, in order to achieve good anti-biofilm effect. We observed the Z-stack images of nanoparticles penetrating biofilms after EV@Lipo and PLL-EV@Lipo treated Candida albicans biofilms for 30 minutes ( 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) The membrane permeability of Candida albicans treated with EV@Lipo and PLL-EV@Lipo was analyzed. It was found that after PLL-EV@Lipo treatment, the membrane permeability of Candida albicans increased, allowing more drugs to enter the fungal cells, indicating that PLL not only promotes drug delivery in biofilms, but also achieves intracellular delivery in fungi. In the biofilm crystal violet quantitative experiment ( 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 against 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, once every 2 days for a total of 3 times. 6 cells / mL of Candida albicans spore suspension dissolved in PBS (inoculation dose of 5×10 4 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 counting. CFU counts exceeding 10 4 It 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 dropping 50 μL of the test substance into the vagina for 2 minutes, and then inverting the mice for 2 minutes, and treating once every 2 days. On the 3rd, 5th and 7th days of treatment, vaginal irrigation was performed with PBS, and the collected irrigation fluid was cultured on YM agar plates 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 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 test results of the PLL-EV@Lipo treatment effect on VVC in Balb / c mice and the in vivo biocompatibility study 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 (lg 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 of 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 decrease. 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 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, and compared with day 0, there was a significant improvement in vaginal redness and no yellow discharge.

[0144] For samples collected on day 7 (end point), vaginal lavage fluid was analyzed using 16S rRNA sequencing to study the abundance and diversity of the vaginal microbial community. At the end point of the experiment, mice were euthanized and vaginal tissue samples were collected for immunohistochemical staining (IL-6, TNF-α) to observe inflammation and PAS staining to assess fungal load. The supernatant of vaginal tissue homogenate was subjected to ELISA for quantitative analysis of IL-6 and TNF-α factors.

[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 heat map of the relative abundance of vaginal phylum-level taxa in mice in 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 alpha 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 indicates that PLL-EV@Lipo effectively eliminated fungal infection. In the immunohistochemistry results ( Figure 5 In B), the PLL-EV@Lipo group and the healthy group showed a lower degree of vaginal inflammation in mice. (Brown represents the area of ​​inflammatory factors IL-6 and TNF-α, while blue represents the area of ​​the cell nucleus) ELISA was used to quantitatively analyze IL-6 and TNF-α, and the results were consistent with the immunohistochemical results ( Figure 5 C to 5D): PLL-EV@Lipo effectively inhibits 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 with itraconazole treatment, we employed 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 present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

[0159] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description 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 may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

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Claims

1. A method for preparing a drug carrier for enhancing the antifungal activity of a drug, characterized in that: The steps include: S1, dissolving lecithin, cholesterol and antifungal active drugs 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 ultrasonicating 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.

2. The method for preparing a drug carrier for enhancing the antifungal activity of a drug according to claim 1, characterized in that: In step S1, the mass ratio of lecithin, cholesterol and antifungal active drug is (5-15):1:(0.001-0.01).

3. The method for preparing a drug carrier for enhancing the antifungal activity of a drug according to claim 1, characterized in that: In step S1, the heating temperature is 40-60°C.

4. The method for preparing a drug carrier for enhancing the antifungal activity of a drug according to claim 1, characterized in that: In step S2, the vesicles are selected from one or more of Candida albicans outer membrane vesicles, Cryptococcus neoformans extracellular vesicles, Candida glabrata outer membrane vesicles, Candida auris outer membrane vesicles or Candida tropicalis outer membrane vesicles.

5. The method for preparing a drug carrier for enhancing the antifungal activity of a drug 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.

6. The method for preparing a drug carrier for enhancing the antifungal activity of a drug according to claim 1, characterized in that: In step S2, the power of the ultrasound is 40-60W; and the ultrasound time is 5-15min.

7. The method for preparing a drug carrier for enhancing the antifungal activity of a drug according to claim 1, characterized in that: In step S3, the pH range is 6-8.

8. The method for preparing a drug carrier for enhancing the antifungal activity of a drug according to claim 1, characterized in that: In step S3, the heating temperature is 40-60°C.

9. Antifungal application of a drug carrier with enhanced antifungal activity prepared by the method for preparing a drug carrier with enhanced antifungal activity of a drug according to any one of claims 1 to 8.

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