Nystatin combined low-intensity pulsed ultrasound synergistic antifungal method and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a nystatin and low-intensity pulsed ultrasound synergistic antifungal method and application thereof. The nystatin and ultrasonic combined antifungal method comprises the following steps: under the assistance of ultrasonic waves, applying nystatin, and carrying out antifungal treatment. The application discovers that nystatin combined with low-intensity pulsed ultrasound can significantly improve the antibacterial effect on fungi (specifically candida albicans) in vitro, has an obvious injury effect on cell structures, and can effectively and rapidly remove candida albicans colonized in vagina, improve vaginal mucosa structure and relieve inflammation symptoms in rabbit bodies, so that the anti-inflammatory effect of the rabbit bodies is improved, and the anti-inflammatory effect of the rabbit bodies is improved. The expression level of vaginal epithelial apoptotic cells is reduced, and the treatment effect on high-drug-resistance and high-recurrence-rate infectious diseases induced by fungi (specifically candida albicans) is remarkably improved.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a synergistic antifungal method of nystatin combined with low-intensity pulsed ultrasound and its application. Background Art

[0002] Vulvovaginal candidiasis (VVC) is one of the most common vaginal infections in women of childbearing age, mainly caused by Candida albicans. Global statistics show that about 80% of women will experience VVC at least once in their lifetime, and the disease is highly recurrent, with a recurrence rate of up to 50%, of which 5%-8% will recur and form recurrent vulvovaginal candidiasis (RVVC). If VVC is not treated promptly and effectively, it may lead to a decrease in the local immune function of women, and then cause other gynecological diseases, such as cervicitis, pelvic inflammatory disease, and cervical erosion. At present, oral antifungal drugs or intravaginal antifungal drugs are commonly used in the clinical treatment of VVC, including azole drugs, polyene drugs, echinocandins, acrylamide, and nucleoside analogs. However, traditional antifungal drugs can only prevent the growth of some fungi. For recurrent and refractory VVC, antifungal drugs have limited effect in fungal clearance. In addition, excessive use of antibiotics can damage normal vaginal cells and tissues, leading to a decrease in symbiotic bacteria, causing a series of problems such as vaginal microecological imbalance and pathogen resistance.

[0003] Common clinical symptoms of VVC are vaginal pain, vulvar burning, dyspareunia, external urination difficulties, and increased vaginal mucus. Among them, increased vaginal mucus is an important clinical manifestation, and the viscosity of mucus ranges from watery to cheese-like. The main component of cervicovaginal mucus (CVM) is water, and gel-forming mucin (GFM) accounts for more than 80% of the organic part of mucus. CVM can accommodate a variety of vaginal flora and is the first line of defense for the vagina against harmful substances from the outside world. It is directly involved in reproduction and helps to eliminate pathogens while protecting beneficial lactobacilli, acting as a selective barrier. However, this physical barrier makes it difficult for traditional drugs to penetrate the epithelial layer, and the mucus secreted by the vagina of VVC patients will aggravate this problem. On the other hand, the special wrinkled structure of the vaginal cavity also leads to insufficient permeability and short retention time of traditional drugs.

[0004] In summary, there is an urgent need to develop more effective vaginal drug delivery methods to improve the treatment of vulvovaginal candidiasis. Summary of the invention

[0005] In view of the above-mentioned shortcomings of the prior art, the present invention provides a synergistic antifungal method of combining nystatin with low-intensity pulsed ultrasound and its application to improve the therapeutic effect of vulvovaginal candidiasis.

[0006] To achieve the above objectives, the scheme of this application is as follows:

[0007] In a first aspect, the present invention provides an ultrasound-assisted drug delivery system for antifungal drugs, the ultrasound-assisted drug delivery system comprising an ultrasound device and a drug delivery system, the drug delivery system being loaded with antifungal drugs, the antifungal drugs comprising nystatin (NYS), the ultrasound device being used to emit ultrasound waves, and the drug delivery mechanism being used to assist in drug delivery to a site to be administered with the assistance of the ultrasound waves.

[0008] Optionally, the site to be administered of the drug is cells or vagina.

[0009] Optionally, the fungus is selected from Candida albicans.

[0010] Optionally, the ultrasonic device includes an ultrasonic transducer, and the ultrasonic transducer is a tubular ultrasonic transducer.

[0011] Optionally, the drug delivery mechanism is a syringe.

[0012] In a second aspect, the present invention also provides a method for antifungal treatment using nystatin combined with ultrasound, wherein nystatin is applied with the assistance of ultrasound to perform antifungal treatment.

[0013] Optionally, the fungus is selected from Candida albicans.

[0014] Optionally, the mode of the ultrasonic wave is a pulse mode.

[0015] Optionally, the intensity of the ultrasonic wave is 0.5-1.0 W / cm 2 , preferably 0.8-1.0W / cm 2 .

[0016] Optionally, the duty cycle of the ultrasonic wave is 30%-50%, preferably 40%-50%.

[0017] Optionally, the frequency of the ultrasonic wave is 0.5-1.0 MHz, preferably 0.8-1.0 MHz.

[0018] Optionally, the ultrasonic irradiation time is 3-5 min, preferably 4-5 min.

[0019] Optionally, the drug is administered to cells or vagina.

[0020] Optionally, when the administration site is cells or vagina, the dosage form of nystatin is injection.

[0021] Optionally, when the drug is administered to cells, the injection volume is 100-200 μL.

[0022] Optionally, when the drug is administered to cells, the concentration of nystatin is 8 μg / mL.

[0023] Optionally, when the drug is administered into the vagina, the dosage of the injection is 150-250 μL.

[0024] Optionally, when the administration site of the drug is vagina, the concentration of nystatin is 10 mg / mL.

[0025] Optionally, when the administration site of the drug is cells, the drug is administered once a day for at least 1 consecutive day.

[0026] Optionally, when the administration site of the drug is in vivo tissue, the drug is administered once a day for at least 7 consecutive days.

[0027] Optionally, the ultrasound wave used to provide the ultrasound device to the vagina is a circular ultrasound wave.

[0028] Optionally, the ultrasound waves used to provide the ultrasound device to the cells are planar focused ultrasound waves.

[0029] Optionally, the nystatin combined with ultrasound antifungal method uses the ultrasound-assisted drug delivery system as described above for antifungal treatment, and the nystatin combined with ultrasound antifungal method comprises the following steps:

[0030] The ultrasonic device is used to emit differential sound waves, and with the assistance of the ultrasonic waves, the drug is administered through the drug administration mechanism to carry out anti-fungal treatment.

[0031] In a third aspect, the present invention also provides the use of nystatin combined with ultrasound in the preparation of an antifungal drug or a drug for treating a disease caused by a fungal infection. The drug comprises nystatin, and the drug is antifungal or treats a disease caused by a fungal infection with the assistance of ultrasound.

[0032] Optionally, the drug is administered to the vagina or cells.

[0033] Optionally, the ultrasound waves emitted by the device for providing ultrasound to the vagina are circular ultrasound waves.

[0034] Optionally, the ultrasound waves used to provide the ultrasound device to the cells are planar focused ultrasound waves.

[0035] Optionally, when the drug is administered by in vitro combined ultrasound administration, the drug is administered once a day for at least 1 consecutive day.

[0036] Optionally, when the drug is administered by in vivo combined ultrasound administration, the drug is administered once a day for at least 7 consecutive days.

[0037] Optionally, the fungus is Candida albicans.

[0038] Optionally, the disease caused by fungal infection is a disease caused by vaginal infection by Candida albicans.

[0039] Beneficial effects of the present invention:

[0040] The present application innovatively discovered for the first time that nystatin combined with low-intensity pulsed ultrasound can significantly improve the antibacterial effect against fungi (specifically Candida albicans) in vitro and have a significant damaging effect on the cell structure. At the same time, it can effectively and quickly eliminate Candida albicans colonized in the vagina in rabbits, improve the vaginal mucosal structure, relieve inflammatory symptoms, and reduce the expression level of vaginal epithelial apoptotic cells, significantly improving the therapeutic effect of highly resistant and high recurrence rate infectious diseases induced by fungi (specifically Candida albicans). BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying creative work.

[0042] Figure 1 Figures 1A and 1B show the results of the activity test of macrophages after being treated with different ultrasound parameters; Figure 1B shows the results of the activity test of Lactobacillus crispatus after being treated with different ultrasound parameters; Figure 1C shows the results of the lactic acid content test of Lactobacillus crispatus after being treated with different ultrasound parameters; Figure 1D shows the results of the activity test of macrophages after being treated with different concentrations of drugs (including the minimum inhibitory concentration) combined with ultrasound; Figure 1E shows the results of the activity test of Lactobacillus crispatus after being treated with different concentrations of drugs (including the minimum inhibitory concentration) combined with ultrasound; Figure 1F shows the results of H&E staining after the drug combined with ultrasound treatment of rabbit vulvovaginal candidiasis; Macrophage means macrophage, viability means activity, Lactobacillus means Lactobacillus, Lactic acid means lactic acid, and concentration means concentration. Control means control group, US means ultrasound group, NYS means nystatin group, CPX means cyclohexetine group, CTZ means clotrimazole group, NYS+US means nystatin combined with ultrasound group, CPX+US means cyclohexetine combined group, CTZ+US means combined with clotrimazole group, MIC means minimum inhibitory concentration, the same below;

[0043] Figure 2The antibacterial effect of drug combined with ultrasound on Candida albicans. 2A is 1MIC drug against 3x10 5 CFU / mL Candida albicans antibacterial effect plate count chart, 2B is 10MIC drug against 3x10 8 CFU / mL Candida albicans antibacterial effect plate count diagram, 2C is the laser confocal microscope observation of 10MIC drug against 3x10 8 CFU / mL Candida albicans antibacterial effect diagram (red fluorescence: dead cells, green fluorescence: live cells), 2D is transmission electron microscopy observation of 10MIC drug against 3x10 8 CFU / mL Candida albicans internal structure influence diagram;

[0044] Figure 3 The results of the detection of active oxygen produced by Candida albicans by drug combined with ultrasound treatment. Figure 3 A is the laser confocal observation of the production of active oxygen in Candida albicans after drug combined with ultrasound treatment. Figure 3 Figure B is the flow cytometer observation of the production of reactive oxygen species in Candida albicans after drug combined with ultrasound treatment, and Figure C is the fluorescence quantitative graph of Figure B;

[0045] Figure 4 The effect diagram of different conditions for treating rabbit vulvovaginal candidiasis model. Figure 4 A is the clinical symptom picture of the rabbit vulva and the plate colony picture after treatment under different conditions; Figure 4 B is the plate count of rabbit vaginal wash fluid after treatment under different conditions;

[0046] Figure 5 The results of H&E analysis of rabbit vaginal tissue after treatment under different conditions. The red arrows indicate the hyphae / strains accumulated in the vaginal cavity, and the black arrows indicate inflammatory cells.

[0047] Figure 6 The results of apoptosis detection in rabbit vaginal cells after treatment under different conditions. DETAILED DESCRIPTION

[0048] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0049] In the following description, numerous details are discussed to provide a more thorough explanation of embodiments of the present invention; however, it is apparent to one skilled in the art that embodiments of the present invention may be practiced without these specific details.

[0050] (I) Security Verification

[0051] 1.1.1 Newly grown highly active RAW264.7 macrophages were inoculated at a density of 1×10 4 Cells / well were seeded in a 96-well plate and incubated at 37°C for 12 h. 200 μL of serum-free DEME medium was added to each well and the cells were immediately stimulated with a circular planar transducer (commercially available, with a diameter of 3.0 cm) at 0.5 W / cm 2 , 1.0W / cm 2 and 1.5W / cm 2 The cells were irradiated with an intensity of 50% for 5 minutes in a pulse mode with a duty cycle of 50% and an ultrasonic frequency of 1.0 MHz. A control group was set up, the control group was placed on an ultrasonic probe, and ultrasonic irradiation was not turned on. The irradiation method was that the ultrasonic transducer (commercially available) was in direct contact with the bottom of the culture plate coated with an ultrasonic coupling agent. After irradiation, the 96-well plate was placed in a cell incubator at 37° C. and 5% CO2 for 24 hours. 10% CCK-8 reagent was added to each well, reacted at 37° C. for 2 hours, and the absorbance at 450 nm was tested. Then, the activity of the macrophages was detected by a multifunctional microplate reader (commercially available) according to the absorbance. The results are as follows Figure 1 A As shown in the left figure.

[0052] 1.2.1 Lactobacillus Crispatus was revived from the storage state, and then 1 mL of the revived Lactobacillus Crispatus was inoculated into 100 mL of MRS medium, cultured in a microaerophilic (specifically, a gas atmosphere consisting of 5% oxygen, 10% carbon dioxide and 85% nitrogen, volume ratio) at 37°C until mature, and irradiated with different ultrasonic parameters. Specifically, 200 μL of the bacterial solution was inoculated into a 96-well plate and immediately irradiated with the circular planar transducer at 0.5 W / cm 2 , 1.0W / cm 2 and 1.5W / cm 2 The cells were irradiated with ultrasound for 5 minutes in pulse mode with a duty cycle of 50% and an ultrasound frequency of 1.0 MHz. A control group was set up with the same settings as above. Immediately after treatment, the bacterial solution was cultured at 37°C for 24 hours under microaerobic conditions, and samples were taken and plate counts were performed to detect the activity of the Lactobacillus crispatus strain. The results are as follows: Figure 1 As shown in the left figure of B, a lactic acid detection kit (commercially available) was used to detect the lactic acid concentration. The results are as follows Figure 1 C shown in the left figure.

[0053] 1.3 Newly grown highly active RAW264.7 macrophages were inoculated at a density of 1×10 4Cells / well were seeded in a 96-well plate and incubated at 37°C for 24 h. Then, three antifungal drugs, namely clotrimazole (CTZ), nystatin (NYS), and ciclopirox olamine (CPX), were added at different concentrations (including the minimum inhibitory concentration of each drug, CTZ: 2 μg / mL, NYS: 8 μg / mL, CPX: 0.25 μg / mL), namely CTZ (0.5 μg / mL, 1 μg / mL, 2 μg / mL, and 4 μg / mL), NYS (2 μg / mL, 4 μg / mL), and CPX (0.5 μg / mL, 1 μg / mL, 2 μg / mL, and 4 μg / mL). / mL, 8μg / mL and 16μg / mL), CPX (0.0625μg / mL, 0.125μg / mL, 0.25μg / mL and 0.5μg / mL), 200μL were taken in 96-well plates respectively, and the groups were set as single drug group and ultrasound combined drug group. The same volume of serum-free culture medium was used as the control group, and then ultrasound irradiation was performed (i.e., drug combined with ultrasound treatment group) in pulse mode. The ultrasound frequency was 1.0MHz and the sound intensity was 1.0W / cm 2 , the duty cycle was 50%, and the irradiation time was 5 minutes; the drug treatment group was treated in the same way as the drug combined with ultrasound treatment group under the corresponding drug concentration conditions, except that ultrasound sham irradiation (the flat ultrasound treatment head irradiated the 96-well plate but there was no energy output) was used instead of ultrasound irradiation; after the treatment, the plates were cultured at 37°C for 24 hours, 10% CCK-8 reagent was added to each well, reacted at 37°C for 2 hours, and the activity of macrophages was detected by the above method. The results are as follows Figure 1 As shown in D.

[0054] 1.4 Resuscitate Lactobacillus crispatus from the storage state, then inoculate 1 mL of the resuscitated Lactobacillus crispatus into 100 mL of MRS medium, culture for 24 h at 37 °C in a microaerophilic atmosphere (specifically, a gas atmosphere consisting of 5% oxygen, 10% carbon dioxide, and 85% nitrogen, volume ratio), and resuspend the bacterial solution in PBS buffer to 3 × 10 8 CFU / mL, and three antifungal drugs, namely clotrimazole (CTZ), nystatin (NYS) and ciclopirox olamine (CPX), were added to the bacterial solution at different concentrations, namely CTZ (0.5μg / mL, 1.0μg / mL, 2.0μg / mL and 4.0μg / mL), NYS (4.0μg / mL, 8.0μg / mL, 16.0μg / mL and 32.0μg / mL), CPX (0.0625μg / mL, 0.125μg / mL, 0.25μg / mL and 0.5μg / mL), and 100μL was taken in a 96-well plate and divided into a single drug group and a drug combined with ultrasound group. The same volume of PBS resuspended lactobacilli was used as the control group, and then ultrasonic irradiation (i.e., drug combined with ultrasound treatment group) was performed in pulse mode with an ultrasound frequency of 1.0MHz and an acoustic intensity of 1.0W / cm 2, the duty cycle was 50%, and the irradiation time was 5 minutes; the drug treatment group was treated in the same way as the drug combined with ultrasound treatment group under the corresponding drug concentration conditions, except that ultrasound sham irradiation (the flat ultrasound treatment head irradiated the 96-well plate but there was no energy output) was used instead of ultrasound irradiation; after the treatment, the cells were cultured at 37°C for 24 hours, and the activity of the Lactobacillus crispatus strain was detected by the above method. The results are as follows Figure 1 As shown in E.

[0055] 1.5 First, 200 μL of NYS solution with a concentration of 20 mg / mL, CTZ solution with a concentration of 10 mg / mL, CPX solution with a concentration of 5 mg / mL, or 0.9% saline solution were injected into the rabbit vagina using a syringe. Then, a tubular ring transducer (commercially available, with a diameter of 1.0 cm) was extended into the rabbit vagina for ultrasound irradiation at an intensity of 1.0 W / cm 2 The pulse mode was used, the duty cycle was 50%, and the ultrasound frequency was 1.0 MHz. The rabbits were euthanized 24 hours after irradiation, and the vaginal tissue was separated and sliced. The pathological changes were observed by hematoxylin and eosin (H&E) staining. The results are as follows Figure 1 As shown in F.

[0056] Depend on Figure 1 From A to 1C, it can be seen that when different ultrasound parameters are used for irradiation, the activity of macrophages decreases with the increase of ultrasound intensity and irradiation time, but when the ultrasound parameter is 1.0 W / cm 2 , after irradiation for 5 minutes, the activity of macrophages was still greater than 80%; at the same time, under this ultrasonic irradiation parameter, it can be seen that ultrasound can significantly promote the proliferation of lactobacillus and stimulate the increase of lactic acid content.

[0057] Depend on Figure 1 D to Figure 1 E shows that when the sound intensity is 1.0W / cm 2 Under the same conditions, the cell activity of cells treated with three drugs for 5 minutes was further reduced compared with the single drug, but the macrophage activity remained above 80% after ultrasound combined with the MIC of the three drugs (NYS: 8μg / mL, CPX: 0.25μg / mL, CTZ: 2μg / mL). Figure 1 D); Ultrasound combined with three drugs can promote the proliferation of Lactobacillus. As the concentrations of the three drugs increase, the promoting effect of ultrasound irradiation on the activity of Lactobacillus gradually decreases. Among them, ultrasound combined with CTZ and CPX can promote the proliferation of Lactobacillus crispatus by about 1.2 times. Ultrasound combined with NYS has an inhibitory effect on the activity of Lactobacillus crispatus, but the activity is still higher than 80% when the minimum inhibitory concentration (8μg / mL) of ultrasound combined with NYS is 8μg / mL. Figure 1 E).

[0058] Depend on Figure 1F shows that no obvious tissue and mucosal damage was observed after ultrasound combined with three drugs. Therefore, this application chooses an ultrasound frequency of 1.0 MHz and a sound intensity of 1.0 W / cm 2 The ultrasonic irradiation condition with an irradiation duration of 5 min and a duty cycle of 50% was taken as the safety threshold dose.

[0059] (II) Antibacterial effect of drugs combined with ultrasound on Candida albicans in vitro

[0060] 2.1 Candida albicans (commercially available) was divided into eight groups, namely, Control group, CTZ group, CPX group, NYS group, US group, CTZ+US group, CPX+US group and NYS+US group. Before the experiment, the concentrations of CTZ solution, CPX solution and NYS solution were diluted to 1MIC / 10MIC with PBS buffer for later use;

[0061] Take 100 μL of the solution with a concentration of 3×10 5 CFU / mL or 3×10 8 CFU / mL of Candida albicans were added to a 96-well plate, and three drugs with the concentration of 1MIC / 10MIC were added respectively. The ultrasound group and ultrasound combined with drug group were treated with the above-screened ultrasound parameters (i.e., pulse mode, ultrasound frequency of 1.0 MHz, sound intensity of 1.0 W / cm 2 After irradiation, the 96-well plate was placed in a bacterial incubator at 37°C and 5% CO2 for 24 hours, and then the plate count was performed to judge the antibacterial effect. The results are shown in FIG. Figure 2 As shown in A and B; 10MIC treatment 3×10 8 CFU / mL of Candida albicans was incubated with SYTO9 / PI live and dead cell dye for 30 min after 24 h, and then the cell survival was observed using a laser confocal microscope. The results are as follows Figure 2 As shown in C; 3×10 8 CFU / mL albicans After 24 hours, 1 mL of each sample was transferred to a 2 mL Eppendorf tube, followed by centrifugation at 12000 rpm for 15 min, and then rinsed with sterile PBS buffer. This was repeated 3 times, and finally fixed with 2.5% glutaraldehyde fixative. The effects of different conditions on the internal structure of Candida albicans were observed using a transmission electron microscope. The results are shown in Figure 2 D; the specific ultrasound irradiation methods are as follows:

[0062] The CPX treatment groups were injected with 100 μL of CPX solution with a concentration of 0.25 μg / mL (1 MIC) or 2.5 μg / mL (10 MIC), respectively, and then sham ultrasound irradiation was performed (the flat ultrasound treatment head irradiated the 96-well plate but without energy output);

[0063] The NYS treatment group was injected with 100 μL of NYS solution with a concentration of 8 μg / mL (1 MIC) and 80 μg / mL (10 MIC), respectively, and then subjected to sham ultrasound irradiation (the flat ultrasound treatment head irradiated the 96-well plate but without energy output);

[0064] The CTZ treatment group was injected with 100 μL of NYS solution with a concentration of 2 μg / mL (1 MIC) and 20 μg / mL (10 MIC), respectively, and then subjected to sham ultrasound irradiation (the flat ultrasound treatment head irradiated the 96-well plate but without energy output);

[0065] Ultrasound treatment group (US): 100 μL PBS buffer was injected, followed by ultrasound irradiation in pulse mode with a frequency of 1.0 MHz and an intensity of 1.0 W / cm 2 , the duty cycle is 50%, and the irradiation time is 5 minutes;

[0066] The difference between the CTZ combined with ultrasound (US) treatment group and the CTZ treatment group was that ultrasound irradiation was used instead of sham ultrasound irradiation, and the parameters of ultrasound irradiation were the same as those of the ultrasound treatment group;

[0067] The difference between the CPX combined with ultrasound (US) treatment group and the CPX treatment group was that ultrasound irradiation was used instead of sham ultrasound irradiation, and the parameters of ultrasound irradiation were the same as those in the ultrasound treatment group;

[0068] The difference between the NYS combined with ultrasound (US) treatment group and the NYS treatment group was that ultrasound irradiation was used instead of sham ultrasound irradiation, and the parameters of ultrasound irradiation were the same as those in the ultrasound treatment group;

[0069] Control group: injected with 100 μL PBS buffer, followed by sham ultrasound irradiation (the flat ultrasound treatment head irradiated the 96-well plate but without energy output);

[0070] like Figure 2 As shown in A and 2B, compared with the control group, the survival rate of Candida albicans in the US group alone was slightly reduced, the survival rate of Candida albicans in the drug group alone was further reduced, and the survival rate of Candida albicans after the three drugs combined with US treatment was significantly reduced ( Figure 2 A, B) (P < 0.01), compared with NYS, the survival rate of the NYS combined with US group decreased by about 20%-38%, while compared with CTZ and CPX, the survival rate of the drug combined with US treatment group decreased by only about 13%-17%, among which NYS combined with US had the most obvious antibacterial effect. Figure 2 As shown in C, compared with the single drug, the number of live cells of Candida albicans was significantly reduced and the number of dead cells was significantly increased after the drug combined with US treatment. Among them, the number of red fluorescence in the NYS combined with US group was the largest, and the antibacterial effect was the most significant.

[0071] like Figure 2 As shown in D, the Candida albicans in the control group had a regular oval shape, complete cell wall and plasma membrane, and intact organelle structure; the Candida albicans cells in the ultrasound treatment group had a complete morphology, smooth and complete cell membrane and cell wall structure, and only small vacuoles were seen in the cells; the cell membrane morphology of the Candida albicans cells in the drug-only group was irregular, the nucleus was deformed, and vacuoles were formed; the Candida albicans cells in the drug-combined ultrasound treatment group had severe deformation, obvious cytoplasm dissolution, and more obvious damage to the cell structure, among which the cell membrane and cell wall were severely deformed and ruptured, and the cytoplasm was obviously dissolved in the NYS-combined ultrasound treatment group. The results showed that compared with the drug-only treatment group, the drug-combined ultrasound treatment group had more obvious damage to the cell structure of Candida albicans, among which NYS-combined ultrasound had the most obvious damage to the structure of Candida albicans. Obviously, nystatin combined with ultrasound had the most significant antibacterial effect on Candida albicans.

[0072] comprehensive Figure 2 As a result, the applicant speculated that the synergistic antibacterial effect of NYS+US against Candida albicans may be based on the fact that the ultrasound-induced shear force and acoustic radiation force change the permeability of cells, promote drug penetration, and have a more significant antibacterial effect.

[0073] (III) Detection of reactive oxygen species produced by Candida albicans by drug combined with ultrasound treatment

[0074] In order to further explore the synergistic antibacterial mechanism of drugs combined with ultrasound on Candida albicans, this application studied the effects of different treatment methods on the production of reactive oxygen by Candida albicans. This application conducted the following experiments:

[0075] The total reactive oxygen species (ROS) level in the Candida albicans cells of different groups in (II) was detected by using a reactive oxygen species assay kit. Specifically, after Candida albicans was treated with different methods for 5 h, the Candida albicans suspension was incubated with the fluorescent probe DCFH-DA at a dilution of 1:1000 (10 μM) at 37°C for 30 min, and then the total ROS level inside the Candida albicans cells was observed by flow cytometry and confocal microscopy. The results are shown in Figure 2. Figure 3 shown.

[0076] like Figure 3As shown, the NYS group alone can produce a small amount of ROS, and ultrasound can stimulate NYS to produce a large amount of ROS (P < 0.01), while the other experimental groups and the control group did not see obvious ROS production. The generation of ROS in cells exists in a balance with various antioxidant defenses. Excessive ROS may damage proteins, nucleic acids, lipids, membranes and organelles, thereby leading to cell death. The applicant speculates that the synergistic antibacterial effect of NYS + US on Candida albicans may be based on the large amount of ROS produced by ultrasound-activated NYS. Excessive amounts of this toxic compound will induce more damage and apoptosis of Candida albicans.

[0077] (IV) Evaluation of the therapeutic effect of drugs combined with ultrasound on vulvovaginal candidiasis in rabbits

[0078] In order to verify the therapeutic effect of drugs combined with ultrasound on vulvovaginal candidiasis caused by Candida albicans, the present application conducted the following experiments:

[0079] The rabbit model of vulvovaginal candidiasis infection was established and grouped for treatment. The specific steps were as follows: 200 μL of 3×10 8 CFU / ml of Candida albicans solution was injected once a day for 3-5 consecutive days until inflammatory symptoms appeared. The rabbits with vulvovaginal candidiasis infection that were successfully modeled were randomly divided into 8 groups, with 5 rabbits in each group, namely: Control group, CTZ group, CPX group, NYS group, US group, CTZ+US group, CPX+US group and NYS+US group. The treatment methods of each group were the same as those of the corresponding groups in 1.5; all treatments were carried out for 7 consecutive days, once a day. After the treatment, the vulvar symptoms of the rabbits were observed, and the vaginal wash fluid was collected for plate counts. The results are shown in the figure below. Figure 4 As shown in A and 4B;

[0080] The rabbit vaginal tissue pathology analysis was performed. The specific steps were as follows: after 7 days of treatment, all rabbits were euthanized, the vaginal tissue was separated, and fixed with 4% paraformaldehyde for 24 hours. The vaginal tissue was then cut into 4 μm thick slices and stained with H&E. The changes in the vaginal tissue pathological structure, Candida residues, and inflammatory infiltration in each group after treatment were observed under an inverted microscope. The results are as follows Figure 5 As shown;

[0081] Immunofluorescence was used to observe the apoptosis of rabbit vaginal cells. The specific steps were as follows: after 7 days of treatment, all rabbits were euthanized, vaginal tissues were separated, and paraffin sections were preserved. The terminal deoxynucleotidyl transferase dUTP nickel-end labeling (TUNEL) method was used to detect vaginal apoptotic cells, and the vaginal tissue contour was marked with DAPI staining. The sections were digested with proteinase K, and the one-step TUNEL cell apoptosis detection kit was used for step-by-step staining and incubation according to the product instructions. The distribution of apoptotic cells was observed under a confocal microscope, and the apoptotic cells showed bright green fluorescence. The results are as follows Figure 6shown.

[0082] like Figure 4 As shown in the figure, after 7 days of treatment with different methods, the vulvar and vaginal symptoms of rabbits in each group were observed with the naked eye. The vulva of rabbits in the control group was congested, red and swollen, and the white secretions increased; there was no obvious change in the US group; after drug treatment alone, the swelling of the vulva of rabbits was reduced, and the vaginal wall was still red and swollen, with no obvious secretions; compared with drug treatment alone, there was no significant improvement in the vulvar symptoms of rabbits in the CTZ+US and CPX+US groups after treatment; compared with NYS alone, the vaginal swelling of the NYS+US treatment group was reduced, the color became lighter, and returned to normal mucosal structure. Then, the vaginal fungal colonies of rabbits in each group were quantified. The average fungal load after US treatment alone was (258±20)×10 3 CFU / mL, compared with the control group (245±30)×10 3 There was no significant difference in CFU / mL compared with the control group; after 7 days of NYS+US treatment, the average fungal load was (10±15)×10 3 The CFU / mL was significantly lower than that of the NYS group alone (121±26)×10 3 CFU / mL (P<0.01); while there was no significant difference in fungal load between the CTZ+US and CPX+US groups and the CTZ or CPX groups alone; the above results show that NYS combined with US treatment can effectively eliminate Candida albicans colonized in the vagina, while the treatment effect of CTZ, CPX combined with US is not significantly different from that of single drugs.

[0083] like Figure 5 As shown in the figure, H&E staining analysis showed that in the control group, a large number of Candida albicans yeast cells and hyphae adhered to the mucosa (red arrows) and infiltrated the submucosal layer (black arrows) on the vaginal wall. The number of vaginal epithelial cells decreased. After the 7th day of treatment, there was no significant change in the US group alone. Compared with CTZ and CPX, there was no significant change in the CTZ+US and CPX+US groups after treatment. Some colonies and hyphae remained in the vagina, and inflammatory cells were still present under the mucosa. Compared with NYS, after NYS+US treatment, no obvious colonies and hyphae were found in the vaginal cavity, the vaginal wall structure was intact, and the submucosal inflammatory cells were significantly reduced, with no significant difference from the normal vaginal structure.

[0084] Depend on Figure 6 It can be seen that a large number of apoptotic cells with green fluorescence were distributed on the mucosal surface in the vaginal tissue of the control group. The expression of apoptotic cells in the vagina decreased significantly in the CTZ treatment group and the CPX treatment group after treatment, but there was no significant difference in the treatment effect between the CTZ and CPX treatment groups and the drug combined with ultrasound treatment group. However, compared with the NYS treatment group, the expression of apoptotic cells on the vaginal mucosal surface in the NYS+US treatment group was significantly reduced. This result shows that Candida albicans infection was significantly alleviated and the apoptosis of vaginal epithelial cells was significantly improved.

[0085] The above results show that compared with NYS, the NYS+US group showed the best synergistic antifungal effect in the rabbit VVC model, which can effectively remove vaginal colonization of Candida albicans, relieve inflammatory infiltration, repair vaginal mucosal structure, and reduce vaginal cell apoptosis. The applicant speculates that the synergistic antibacterial mechanism of ultrasound combined with NYS in vivo is that on the one hand, the ultrasound-induced sonoporation effect promotes the penetration of drugs in cells, and ultrasound excites NYS to produce more toxic ROS. On the other hand, it may be that ultrasound interacts with vaginal mucus to produce cavitation effect or other acoustic physical effects, and has a certain effect on mucin, the main component of mucus, which increases the permeability of the mucus barrier, thereby improving the delivery efficiency of NYS in the vagina, and then enhancing the antibacterial effect against Candida albicans.

[0086] In summary, the present application provides a safe and efficient synergistic antifungal treatment strategy of combining ultrasound with antifungal drugs for clinically recurrent and refractory vulvovaginal candidiasis.

[0087] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the technology may modify or change the above embodiments without violating the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by a person of ordinary skill in the art without departing from the spirit and technical ideas disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. An ultrasound-assisted drug delivery system for antifungal drugs, characterized in that: The ultrasound-assisted drug delivery system comprises an ultrasound device and a drug delivery mechanism. The antifungal drug comprises nystatin. The ultrasound device is used to emit ultrasound. The drug delivery mechanism is used to assist in drug delivery to a site to be administered with the assistance of the ultrasound.

2. A method for antifungal treatment with nystatin combined with ultrasound, characterized in that: Nystatin was administered with ultrasound assistance for antifungal treatment.

3. The antifungal method of nystatin combined with ultrasound according to claim 2, characterized in that: The fungus is selected from Candida albicans; And / or, the mode of the ultrasonic wave is a pulse mode; And / or, the intensity of the ultrasonic wave is 0.8-1W / cm 2 ; And / or, the duty cycle of the ultrasonic wave is 30%-50%; And / or, the frequency of the ultrasonic wave is 0.5-1.0 MHz; And / or, the ultrasonic irradiation duration is 3-5 min.

4. The antifungal method of nystatin combined with ultrasound according to claim 3, characterized in that: The drug is administered to cells or vagina; and / or, when the administration site of the drug is a cell, the drug is administered once a day for at least 1 consecutive day; And / or, when the administration site of the drug is in vivo tissue, the drug is administered once a day for at least 7 consecutive days.

5. The antifungal method of nystatin combined with ultrasound according to claim 2, characterized in that: The nystatin combined with ultrasound antifungal method uses the ultrasound-assisted drug delivery system as claimed in claim 1 for antifungal treatment.

6. Use of nystatin combined with ultrasound in the preparation of antifungal drugs or drugs for treating diseases caused by fungal infections, characterized in that: The medicine includes nystatin, and the medicine is antifungal with the assistance of ultrasound, or treats a disease caused by fungal infection.

7. The use according to claim 6, characterized in that The administration site of the drug is vagina or cells.

8. The use according to claim 7, characterized in that The ultrasonic wave used to provide the ultrasonic device to the vagina is a circular ultrasonic wave; And / or, the ultrasound wave used to provide the cells with the ultrasound device is planar focused ultrasound wave.

9. The use according to claim 6, characterized in that The fungus is Candida albicans.

10. The use according to claim 8, characterized in that The disease caused by fungal infection is a disease caused by vaginal infection by Candida albicans.

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