Mitochondria-targeted antifungal nano sound-sensitive agent as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of antifungal drugs, and discloses a mitochondria-targeted antifungal nano sound-sensitive agent as well as a preparation method and application thereof. The mitochondrial targeting type antifungal nano sound-sensitive agent comprises purpurine 18, tannic acid and copper ions. According to the nanometer sound-sensitive agent, a stable nanometer network structure is constructed through the coordination effect of metal polyphenols, chlorophyll derivatives with mitochondrial affinity are carried, the nanometer sound-sensitive agent can efficiently enter fungal cells and can be selectively enriched in mitochondria, and precise delivery of fungal mitochondria is achieved. Under ultrasonic activation, the nanometer sound-sensitive agent generates active oxygen at a targeting site, mitochondria oxidative damage is induced, then cell walls and membrane structures are destroyed, and therefore efficient fungus killing is achieved, and the nanometer sound-sensitive agent has good clinical transformation prospects.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of antifungal drugs, and particularly relates to a mitochondria-targeting antifungal nano sonosensitizer as well as a preparation method and application thereof. BACKGROUND

[0002] In recent years, the incidence of fungal infections continues to rise, especially invasive candidiasis (IC) caused by drug-resistant Candida albicans, with a mortality rate of 80%, which seriously threatens public health and brings a heavy burden to society. Drug-resistant fungal infections not only increase the suffering of patients, but also significantly prolong hospital stays and increase medical expenses, which brings a heavy burden to the social medical system. Therefore, it is urgent to develop an antifungal drug that is not dependent on antibiotics and precisely targets fungal organelles to achieve efficient and precise fungal killing and overcome the treatment difficulties of drug-resistant fungal infections.

[0003] Sonodynamic therapy (SDT) as a non-antibiotic-dependent anti-infection method has shown good prospects in bacterial infection and tumor treatment in recent years. This technology activates sonosensitizers to produce reactive oxygen species (ROS) through external ultrasound, induces oxidative stress and structural damage in target cells, and thus achieves pathogen clearance. However, the development of sonosensitizers for fungi is still in its infancy and lacks selectivity and targeting, which limits its application in the field of antifungal agents.

[0004] Mitochondria is the core organ of energy metabolism in fungal cells, and its membrane potential, high concentration of reactive oxygen species and unique protein composition determine its high targetability in external drug intervention. Previous studies have shown that interfering with the function of fungal mitochondria can effectively block its survival and proliferation process, and has become a new target for antifungal agents. However, there is still a lack of antifungal sonosensitizers that can precisely deliver and act on fungal mitochondria.

[0005] In the prior art, some photosensitizers or metal oxide-based nanomaterials do not have clear mitochondrial targeting ability in structure, or lack selective recognition of fungal cells, which can easily cause toxicity to host cells. In addition, sonosensitizers often have low uptake efficiency and poor biological stability in fungal cells, making it difficult to achieve the unity of targeted therapy and efficient activation. Therefore, the development of a nano sonosensitizer that can be efficiently delivered to fungal mitochondria, produce reactive oxygen species under ultrasonic excitation and selectively kill fungi is of great significance for breaking through the current bottleneck of antifungal therapy.

[0006] To solve the above problems, the present application aims to provide a mitochondria-targeting antifungal nano-sensitizer for sonodynamic therapy to improve the treatment effect of clinically drug-resistant fungal infection and reduce the risk of drug resistance. SUMMARY

[0007] The present application aims to at least solve one of the technical problems existing in the prior art. To this end, the present application proposes a mitochondria-targeting antifungal nano-sensitizer and its preparation method and application. The nano-sensitizer described in the present application uses metal polyphenol coordination to construct a stable nano-network structure, carries chlorophyll derivatives with mitochondrial affinity, can not only efficiently enter fungal cells, but also selectively enrich in mitochondria, realizing precise delivery to fungal mitochondria. Under the activation of ultrasound, the nano-sensitizer produces reactive oxygen species at the target site, induces mitochondrial oxidative damage, and then destroys the cell wall and membrane structure, thereby realizing efficient fungal killing and having good clinical conversion prospects.

[0008] The first aspect of the present application provides a mitochondria-targeting antifungal nano-sensitizer.

[0009] The mitochondria-targeting antifungal nano-sensitizer comprises purpurin 18, tannic acid and copper ions.

[0010] Preferably, the nano-sensitizer uses tannic acid and Cu 2+ as a carrier and dynamically coordinates purpurin 18.

[0011] Preferably, the preparation raw material of the nano-sensitizer comprises purpurin 18 (Purpurin 18, P18), tannic acid (Tannic Acid, TA) and copper ions (Cu 2+ ).

[0012] The nano-sensitizer described in the present application uses TA and Cu 2+ as a carrier, and forms a metal polyphenol nano-sensitizer (TCP) by dynamically coordinating P18 self-assembly. P18 as the main active ingredient can selectively aggregate in fungal mitochondria; TA provides polyphenol coordination sites, enhances stability and biocompatibility; Cu 2+ not only constitutes the skeleton, but also participates in disturbing metal homeostasis and aggravating mitochondrial oxidative damage under the induction of reactive oxygen species. Through the dual targeting effect of P18 mitochondrial targeting and ultrasound catalytic production of reactive oxygen species in situ damage, high targeting and high killing functions are realized, which can be used as an effective targeted drug for treating fungal infections (including drug-resistant fungal infections).

[0013] It is worth mentioning that the nano-sensitizer and the sonodynamic therapy method thereof have good targeting, ultrasonic response and antifungal activity, and especially show significant effects in the treatment of drug-resistant fungal infection. In order to facilitate subsequent product development and technology identification, the nano-sensitizer mediated sonodynamic therapy platform is named "Sonocure" (including but not limited to spelling forms such as "SonoCure", "Sono-cure", etc.), which is used to refer to the antifungal treatment system developed by the present application, which has mitochondrial targeting characteristics, copper homeostasis interference mechanism and ultrasonic response activity. The naming does not limit the technical scope of the present application, but is only used as an identification name for subsequent product development and clinical transformation. "Sonocure" is a proposed trademark name used to describe the treatment system related to the present application.

[0014] The second aspect of the present application provides a preparation method of a mitochondria-targeting antifungal nano-sensitizer.

[0015] The preparation method of the mitochondria-targeting antifungal nano-sensitizer comprises the following steps:

[0016] (1) Prepare solutions of purpurin 18, tannic acid and copper salt respectively to obtain a purpurin 18 solution, a tannic acid solution and a copper salt solution;

[0017] (2) Mix and react the purpurin 18 solution, the tannic acid solution and the copper salt solution to obtain a mixture containing metal polyphenol self-assembled nanostructures, and separate and purify to obtain the nano-sensitizer.

[0018] Preferably, in step (2), the separation process comprises filtering, centrifuging, sterilizing and filtering the mixture containing metal polyphenol self-assembled nanostructures to obtain the nano-sensitizer.

[0019] Preferably, in step (1), the metal salt comprises copper chloride or copper sulfate, preferably copper chloride.

[0020] Preferably, in step (1), the concentration of the purpurin 18 solution is 1-5 mg / mL, and further preferably 2-3 mg / mL.

[0021] Preferably, in step (1), the concentration of the tannic acid solution is 20-40 mg / mL, and further preferably 25-30 mg / mL.

[0022] Preferably, in step (1), the concentration of the copper salt solution is 3-8 mg / mL, and further preferably 4-5 mg / mL.

[0023] Preferably, in step (1), the solvent used in the process of preparing the solution is a mixed solvent formed by dimethyl sulfoxide and ultrapure water.

[0024] Preferably, the volume ratio of dimethyl sulfoxide to ultrapure water is 4:(12-18), further preferably 4:16.

[0025] Preferably, in step (2), the mass ratio of the tannic acid solution, purpurin 18 solution and copper salt solution is (1000-2000):(660-670):1667, further preferably (1000-2000):667:1667.

[0026] Preferably, in step (2), the temperature of the mixed reaction is 20-30°C, and the time is 40-60 minutes. For example, the temperature of the mixed reaction is 25°C, and the reaction time is 60 minutes.

[0027] Preferably, in step (2), the mixed reaction is carried out under neutral conditions with pH = 7-7.4.

[0028] Preferably, in the separation process, the relative centrifugal force is 2000-3000 rpm, and the time is 10-15 minutes.

[0029] Preferably, in the separation process, a filter membrane made of polyether sulfone (PES) material is used for filtration to improve purity and biological safety.

[0030] Preferably, a preparation method of a mitochondria-targeting antifungal nano-sensitizer comprises the following steps:

[0031] (1) Dissolve P18, TA and copper chloride (CuCl2) in a mixture of dimethyl sulfoxide and ultrapure water to prepare a raw material solution: the final concentration of P18 is 3 mg / mL, the final concentration of TA is 30 mg / mL, and the final concentration of copper chloride is 5 mg / mL;

[0032] (2) Add TA, P18 and copper chloride solutions to a reaction container in sequence, and magnetically stir at 25°C for 60 minutes to form a metal polyphenol self-assembled nanostructure;

[0033] (3) Filter the reaction solution through a 40μm filter membrane, then place it in an ultrafiltration centrifuge tube with a molecular weight cutoff of 3000 Da, wash with ultrapure water and centrifuge (2800 rpm, 25°C, 15 min), repeat 3 times.

[0034] (4) After sterilization and filtration through a 0.22μm needle filter, a stable dispersed nano-sensitizer solution is obtained.

[0035] Preferably, in step (4), ultrasonic-assisted dispersion is used.

[0036] Preferably, in step (4), the nanometer sound sensitizer is finally stored in a light-proof sealed condition, preserved at 4℃, and has a validity period of up to 6 months.

[0037] The third aspect of the present application provides an application of the mitochondrion-targeting antifungal nanometer sound sensitizer.

[0038] A medicine comprising the mitochondrion-targeting antifungal nanometer sound sensitizer or the mitochondrion-targeting antifungal nanometer sound sensitizer prepared by the preparation method.

[0039] Preferably, the medicine is an antifungal medicine.

[0040] Preferably, the medicine is a medicine for treating deep drug-resistant C. albicans infection.

[0041] The mitochondrion-targeting antifungal nanometer sound sensitizer prepared by the present application can generate active oxygen and destroy fungal mitochondria under the activation of ultrasound, induce programmed death, and shows excellent anti-infection effect and tissue safety in in-vivo and in-vitro experiments.

[0042] Compared with the prior art, the present application has the following beneficial effects:

[0043] The present application creatively introduces a mitochondrion-targeting mechanism into the design of an antifungal sound sensitizer, constructs a nanometer carrier through a metal polyphenol network, realizes the accurate delivery and sound-controlled activation of fungal mitochondria, and endows the nanometer sound sensitizer with the ability of selective recognition and structural embedding, so that the nanometer sound sensitizer can selectively recognize and embed into the mitochondria of C. albicans. 2+ The nanometer sound sensitizer can generate oxidative and metal homeostasis interference double strikes, realize non-antibiotic-dependent efficient antifungal treatment, and provide a new treatment scheme and strategy for clinically refractory fungal infection. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 The results of the preparation and physicochemical characterization experiments of the nanometer sound sensitizer in Example 1 and Example 2 are shown in the following table:

[0045] Figure 2 The results of the cell uptake and fungal mitochondrion targeting of the nanometer sound sensitizer TCP in Example 3 are shown in the following figures:

[0046] Figure 3 The results of the in-vitro killing of drug-resistant C. albicans and the removal of biofilm by TCP sonodynamic therapy in Example 4 are shown in the following figures:

[0047] Figure 4 The results of the sonodynamic therapy of TCP for treating deep drug-resistant C. albicans skin infection in Example 5 are shown in the following figures:

[0048] Figure 5H&E staining histopathological images of major organs (heart, liver, spleen, lung, kidney) of mice in control group and TCP sonodynamic group. DETAILED DESCRIPTION

[0049] In order to make the skilled in the art more clearly understand the technical solutions described in the present application, the following examples are listed for illustration. It should be pointed out that the following examples do not constitute a limitation on the scope of protection required by the present application.

[0050] The raw materials, reagents or devices used in the following examples, if not specifically stated, can be obtained from conventional commercial channels, or can be obtained by existing known methods.

[0051] Figure 1 Results of preparation and physicochemical characterization of nano-sonosensitizer in Example 1 and Example 2; wherein (a) is a schematic diagram of synthesis of nano-sonosensitizer TCP; (b) is a transmission electron microscope and element mapping image of TCP; (c) is a particle size distribution graph of TCP; (d) is an element energy spectrum abundance graph of TCP; (e) is a UV-Vis absorption spectrum curve graph of TCP; (f) is a fluorescence emission spectrum curve graph of TCP and P18; (g) is a SOSG fluorescence intensity graph of TCP and P18 respectively after ultrasonic excitation; (h) is a P18 cumulative release curve graph of TCP under different pH conditions. Figure 1 In the above table, "Purpurin 18" means purpurin 18, "Tannic acid" means tannic acid, "Addin sequence" means adding in order, "Stirring" means stirring, "Ultrafiltration" means ultrafiltration, "Purification" means purification, "Concentration" means concentration; "Intensity" means intensity, "PDI" means polydispersity index, "Size" means particle size. "Energy" means energy; "Absorbance" means absorbance, "Wavelength" means wavelength; "Fluointensity" means fluorescence intensity; "Cumulative release" means cumulative release rate, "Time" means time.

[0052] Figure 2These are the results of the cellular uptake and fungal mitochondrial targeting of the nanosonosensitizer TCP in Example 3; (a) to (c) are time series fluorescence signal imaging and fluorescence quantification of C.albicans uptake of the nanosonosensitizer TCP; (d) is a super-resolution three-dimensional fluorescence modeling image of the nanosonosensitizer TCP specifically targeting fungal mitochondria; (e) to (g) are the reverse molecular docking results of P18 in the nanosonosensitizer TCP and the COR1 protein of the mitochondrial cytochrome b (cytochrome b) and ubiquinol-cytochrome-c reductase subunit 8 (ubiquinol-cytochrome-c reductase subunit 8) complex of the international standard model strain of C.albicans. Figure 2 “Cell wall” refers to cell wall, “Mitochondria” refers to mitochondria, “Membrane” refers to membrane, and “Nucleus” refers to nucleus.

[0053] Figure 3 These are the results of TCP sonodynamics in vitro killing of drug-resistant C.albicans and removal of biofilms in Example 4; (a) is the scanning electron microscope and transmission electron microscope images of C.albicans cells treated with the control group and the TCP sonodynamics group; (b) and (c) are the fluorescence images and fluorescence quantification images of intracellular reactive oxygen species in C.albicans treated with the control group and the TCP sonodynamics group; (d) and (e) are the fluorescence images and fluorescence quantification images of mitochondrial membrane potential in C.albicans treated with the control group and the TCP sonodynamics group; (f) and (g) are the fluorescence images and fluorescence quantification images of intracellular copper ions in C.albicans treated with the control group and the TCP sonodynamics group; (h) is a three-dimensional fluorescence live-dead staining image of C.albicans biofilm treated with the control group and the TCP sonodynamics group. Figure 3 "DAPI" in the text indicates 4 ′ ,6-Diamidino-2-phenylindoledilactate (4',6-Diamidino-2-phenylindoledilactate), "DCF" means 2 ′ ,7 ′ -Dichlorofluorescein (2 ′ ,7 ′ -dichlorofluorescein), "JC-1" means 5,5 ′ ,6,6 ′ -Tetrachloro-1,1 ′ ,3,3 ′- Tetraethylbenzimidazolylcarbocyanine iodide (5',6,6'-tetrachloro-1,1',3,3'- tetraethylbenzimidazolylcarbocyanine iodide), "Copperion" means copper ion, "Percentage" means percentage, "aggregates" means JC-1 aggregates, "monomer" means JC-1 monomer.

[0054] Figure 4 Figure 1 is the results chart of Example 5 TCP sonodynamic therapy of drug-resistant C. albicans deep skin infection; (a) and (b) are the general images of the lesions and the quantitative images of the lesion area of the drug-resistant C. albicans deep infection mice treated with the control group and the TCP sonodynamic group; (c) and (d) are the images of the fungal culture colonies and the quantitative images of the colony count of the homogenate of the lesion tissue of the drug-resistant C. albicans deep infection mice treated with the control group and the TCP sonodynamic group on the 14th day; (e) is the PAS staining pathological chart of the lesion tissue of the drug-resistant C. albicans deep infection mice treated with the control group and the TCP sonodynamic group; Figure 4 "Day" means day, "Lesion area" means lesion area.

[0055] Figure 5 Figure 2 is the H&E staining histopathological chart of the main organs (heart, liver, spleen, lung, kidney) of the mice in the control group and the TCP sonodynamic group. Figure 5 "Heart" means heart, "Liver" means liver, "Spleen" means spleen, "Lung" means lung, "Kidney" means kidney.

[0056] Example 1: Preparation of mitochondria-targeted nano-sonosensitizer TCP

[0057] The following nano-sonosensitizers were prepared using P18 as a simulated delivery sonosensitizer:

[0058] (1) Solution preparation: P18 (purpurin 18), TA (tannic acid), and CuCl2 were dissolved in a mixed solvent of DMSO (dimethyl sulfoxide) and ultrapure water in a volume ratio of 4:16 to prepare P18 solution with a concentration of 3 mg / mL, TA solution with a concentration of 30 mg / mL, and CuCl2 solution with a concentration of 5 mg / mL, respectively;

[0059] (2) Assembly reaction: The above TA solution, P18 solution, and CuCl2 solution were added to a reaction container in the order of mass ratio 1000:667:1667, and magnetically stirred at 25°C for 60 minutes to form a reaction solution containing nano-sonosensitizer (TCP);

[0060] (3) Purification: The above reaction solution was preliminarily removed by 40 pm filter screen, and then transferred into an ultrafiltration tube with MWCO 3000, and an appropriate amount of ultrapure water was added and centrifuged (2800 rpm, 15 min) and repeated once. After filtration through a 0.22 pm PES (polyether sulfone) membrane, a stable dispersed TCP solution was obtained;

[0061] (4) Storage: The TCP solution was stored at 4°C in the dark, and the best results were obtained within 1 month.

[0062] Example 2: Physicochemical characterization of TCP

[0063] (1) TCP morphology

[0064] a. The TCP nanoparticle solution prepared in Example 1 was ultrasonically treated at 25°C for 15 min in a light-proof environment to obtain a uniformly dispersed TCP nanosolution 1;

[0065] b. The TCP nanosolution 1 was added dropwise to an ultrathin carbon film copper grid carrier and naturally dried to form a sample film (i.e., a TCP sample), and this step was repeated twice to enhance the stability of signal acquisition;

[0066] c. The morphology of the above TCP sample was observed using a transmission electron microscope (TEM), and its morphology image was recorded for analysis of particle size distribution and structural morphology;

[0067] d. Further analysis of the elemental composition of the above TCP sample was performed using a matching energy dispersive spectrometer (EDS), including but not limited to elements such as C, N, O, Cu, etc., and combined with high-angle annular dark field imaging (HAADF) to collect and record element distribution maps.

[0068] (2) TCP particle size

[0069] a. 500 pL of the TCP nanosolution 1 was diluted with ultrapure water at a volume ratio of 1:9 to prepare a clear dispersion solution to avoid instrument detection errors caused by high concentration;

[0070] b. The average particle size and polydispersity index (PDI) of TCP were measured at 25°C using a dynamic light scattering instrument (DLS), and the detection results were recorded.

[0071] (3) Spectral characteristics of TCP

[0072] a. Take an appropriate amount of TCP nanosolution 1, respectively prepare P18 solution and TA solution with the same molar concentration, use ultrapure water as solvent, ensure that the solution is clear and free of impurities;

[0073] b. Use UV-Vis spectrophotometer to scan the three solutions in the wavelength range of 200-800 nm, record the UV absorption spectrum;

[0074] c. Comparative analysis of the changes of TCP and its precursor components (P18 and TA) in characteristic absorption peak position and spectral profile, to verify the changes of its assembly characteristics and spectral behavior.

[0075] (4) Fluorescence spectral characteristics of TCP

[0076] a. Take 1 mL of TCP nanosolution 1, prepare P18 solution with the same molar concentration, dilute with ultrapure water and mix well, ensure that the solution is clear and free of impurities;

[0077] b. Use fluorescence spectrophotometer to scan the fluorescence emission spectrum of the two solutions in the full wavelength range, record the fluorescence spectrum;

[0078] c. Compare the emission peak position, fluorescence intensity and spectral morphology changes of TCP and free P18, evaluate whether the fluorescence behavior of TCP after self-assembly has blue shift, red shift or quenching, to confirm the influence of the formation of nanostructure on the fluorescence characteristics.

[0079] (5) In vitro production of reactive oxygen species by TCP

[0080] a. Prepare TCP solutions with different mass concentrations (12.5, 25, 50, 75, 100 μg / mL) and P18 solutions with equivalent content, each group is added with a final concentration of 5 μM of singlet oxygen probe SOSG (Singlet Oxygen Sensor Green), and mixed well;

[0081] b. Use ultrasonic irradiation (1.0 W / cm 2 , 1 MHz, duty cycle (duty cycle) 50%, 2 min) to load the mixed solution with probe;

[0082] c. Use fluorescence spectrophotometer (excitation wavelength 488 nm, emission wavelength 525 nm) to detect the fluorescence intensity of each group of solutions; by comparing the fluorescence intensity changes of different groups, evaluate the reactive oxygen generation ability of TCP, and verify that the assembly does not affect the sound triggered oxygen production performance.

[0083] (6) pH-responsive release of TCP

[0084] a. The TCP nanosolution (concentration 25 pg / mL) was loaded into a dialysis bag (molecular weight cut-off 3000 Da), and the dialysis bag was immersed in phosphate buffer at pH 7.4, pH 6.5 and pH 5.5, respectively, to simulate the drug release behavior in different tissue environments;

[0085] b. The entire release system was placed in a constant temperature oscillator at 37°C to simulate the in vivo microenvironment;

[0086] c. At the set time points (0 min, 10 min, 30 min, 1 h, 4 h, 8 h, 12 h, 24 h), 200 pL of solution sample was taken out from the release medium, and an equal volume of fresh phosphate buffer was added to maintain the constant volume of the system;

[0087] d. The fluorescence intensity of P18 in the sample was measured using a multifunctional microplate detector, and the cumulative release amount at different time points was calculated according to the standard curve to draw the release curve and evaluate the release performance.

[0088] The experimental results are shown in Figure 1 , wherein Figure 1 (a) shows the schematic diagram of TCP preparation, showing its assembly structure and material composition; Figure 1 (b) shows the transmission electron microscope image and energy dispersive spectrometer element distribution map of TCP, indicating that its morphology structure is uniform, and each element (C, N, O, Cu) is uniformly distributed; Figure 1 (c) shows the particle size distribution map of TCP and the polydispersity index (PDI) analysis results, showing that the average particle size is 145 ± 6.14 nm, and the PDI is 0.056, indicating that the particle size distribution is concentrated and the dispersity is good; Figure 1 (d) shows the element energy spectrum of TCP, verifying the effective loading of P18 and copper ions embedded; Figure 1 (e) shows the ultraviolet-visible absorption spectrum of TCP, P18 and TA, showing that the characteristic absorption peaks of P18 are located at 412 nm, 547 nm, 642 nm and 699 nm, and the characteristic absorption peak of TA is located at 282 nm, confirming that P18 and TA are effectively loaded in TCP; Figure 1 (f) shows the fluorescence emission spectrum of TCP and P18, and the results show that P18 is successfully loaded into TCP, and TCP shows red-shifted fluorescence characteristics, providing a good application basis for its biological imaging and tracking; Figure 1 (g) shows the detection results of the ability of different concentrations of TCP and P18 to induce the generation of reactive oxygen species under ultrasonic excitation, and both of them show a concentration-dependent ROS generation trend, and there is no difference in fluorescence intensity. Figure 1Figure 6 shows in vitro drug release profiles of TCP under different pH conditions, showing that TCP has acid microenvironment-responsive release characteristics.

[0089] Example 3: Cellular uptake and mitochondrial targeting of TCP

[0090] (1) Polarized structured light super-resolution microscopy (SIM) delayed fluorescence imaging

[0091] a. A suspension of Candida albicans (10 6 CFU / mL) was incubated with mitochondrial fluorescent probe PKMito Red (final concentration 0.1 μM) and cell wall-specific fluorescent probe (final concentration 0.1 μM), respectively, with PKMito Red incubation time of 20 min and cell wall fluorescent probe incubation time of 5 min, and incubation temperature controlled at 30°C.

[0092] b. After fluorescence labeling, the bacterial solution was transferred to a confocal imaging dish, and an appropriate amount of RPMI 1640 medium was added, and the sample was allowed to stand for 10 min to stabilize the structure. Then an appropriate amount of TCP solution was added, and delayed fluorescence imaging was performed under structured light super-resolution microscopy. The excitation wavelength was set to 405 nm, 561 nm and 640 nm, and the image acquisition frequency was 1 frame / min, and continuous imaging was performed for 20 min.

[0093] c. The images collected at each time point were analyzed for fluorescence intensity using Imaris software (three-dimensional / four-dimensional image visualization and analysis software), and the distribution of TCP in mitochondria and the fluorescence change trend were evaluated.

[0094] (2) Three-dimensional fluorescence super-resolution imaging and modeling analysis method of TCP and fungal mitochondria

[0095] a. A suspension of C. albicans in the logarithmic growth phase (concentration 10 6 CFU / mL) was added with mitochondrial fluorescent probe PKMito Red (final concentration 0.5 μM, incubation time 20 min) and cell wall-specific fluorescent probe (final concentration 0.1 μM, incubation time 5 min), respectively, and incubated at 30°C.

[0096] b. After probe loading, TCP solution with a concentration of 25 μg / mL was added for incubation times of 0 h, 0.5 h, 1 h and 2 h, respectively.

[0097] c. After incubation, the reaction solution was discarded and washed twice with RPMI 1640 medium, and then placed under structured light super-resolution microscope for 10 min before Z-stack scanning imaging. The excitation wavelength was set to 405 nm, 561 nm and 640 nm, and the laser intensity was controlled to ensure cell activity and low light toxicity;

[0098] d. The Z-stack images obtained were three-dimensionally modeled using Imaris software (version x64 10.0), and the mitochondrial, cell wall and TCP fluorescence signals were segmented and modeled analyzed by the Surface module, respectively, to obtain their spatiotemporal distribution characteristics.

[0099] e. Another logarithmic growth phase human immortalized epidermal keratinocytes (HaCaT, 10 4 / mL) were uniformly inoculated in a confocal imaging dish, and DMEM culture medium containing 10% fetal bovine serum was added, and the cells were cultured at 37°C and 5% CO2 for 12 h until they adhered to the wall;

[0100] f. Mitochondrial fluorescent probe PK Mito Red (0.1 μM, 20 min), cell membrane fluorescent probe CellMask (0.5 μM, 15 min) and nuclear dye DAPI (0.5 μM, 20 min) were sequentially added for staining, and the staining process was carried out at 37°C;

[0101] g. After staining, 25 μg / mL TCP solution was added for incubation for different times (0 h, 0.5 h, 1 h, 2 h), and then washed twice with serum-free DMEM medium, and then placed for 10 min before SIM imaging. The excitation wavelength was set to 405 nm, 488 nm, 561 nm and 640 nm;

[0102] h. The HaCaT images obtained were three-dimensionally reconstructed using Imaris software, and the mitochondria, cell membrane, nucleus and TCP fluorescence signals were segmented, modeled and visualized analyzed by the Surface function, respectively, to obtain their spatiotemporal distribution characteristics

[0103] (3) Reverse molecular docking simulation of P18 in TCP

[0104] a. Receptor protein structure preparation: The three-dimensional structure files of a total of 13844 proteins were downloaded from protein databases (including RCSB, UniProt, AlphaFold). First, all proteins were pre-processed using AutoDock Tools (version 1.5.6) to remove water molecules, ligands and heteroatoms, and to add hydrogen atoms, assign Gasteiger charges, and generate pdbqt files. For proteins with missing residues, Swiss-Model (protein homology modeling automatic modeling platform) was used for homology modeling to complete the missing residues, and the rationality of the structure was verified by MolProbity (protein structure verification tool) or PROCHECK (protein structure evaluation program). For proteins lacking resolved structures, the predicted structures provided by the AlphaFold database were used, and the conformational rationality was verified by PyMOL (three-dimensional molecular visualization software) and the conformation with higher pLDDT value (predicted Local Distance Difference Test, an index for evaluating the reliability of predicted protein three-dimensional structure) was selected as the final model;

[0105] b. Small molecule ligand structure preparation: The three-dimensional structure of P18 was downloaded from the PubChem database, and Chemoffice2019 was used to complete structure optimization under the MMFF94 force field, with the convergence criterion set to an energy change of less than 0.001 kcal / mol. The optimized small molecule was processed using AutoDock Tools to add hydrogen atoms, assign Gasteiger charges, and generate a pdbqt file;

[0106] c. Reverse molecular docking: AutoDock Vina 1.2.5 was used for high-throughput virtual screening. The docking box center was set at the geometric center of the protein, the docking space covered the entire protein structure, the step size was set to 0.375, and the exhaustiveness (exhaustiveness) was set to 32. The binding conformation obtained by docking was ranked according to the score, and the lower score was selected for subsequent structure analysis. The docking results were visualized using PyMOL and LigPlot+ software to evaluate the interaction mode between the ligand and the protein.

[0107] The results of the cellular uptake and mitochondrial targeting of TCP are shown in Figure 2 . Figure 2In (a) to (c), it was shown that TCP fluorescence signal (magenta) started to appear in C. albicans intracellularly at 7 min of incubation, and the signal was enhanced with time. PK MitoRed fluorescence (red) labeled mitochondria, which showed continuous fluorescence when the membrane potential was intact. With TCP uptake, the mitochondrial fluorescence gradually weakened and almost completely disappeared at 15 min, while the blue fluorescence signal of the cell wall did not change significantly within 20 min. This phenomenon suggests that TCP can be rapidly taken up and enriched in the mitochondrial location, which may affect its membrane potential. Figure 2 In (d), it was shown that the co-localization volume ratio of TCP and mitochondrial fluorescence signal was time-dependent, TCP gradually enriched in C. albicans intracellularly and specifically distributed to mitochondria, which possessed the ability of fungal mitochondrial targeting.

[0108] Figure 2 In (e) to (f), it was shown that P18 could stably bind to complex III of the respiratory chain of C. albicans. It formed hydrogen bonds with Tyr103 and Asn27 residues of cytochrome b, and an additional hydrogen bond with Thr53 residue of ubiquinol-cytochrome-c reductase subunit 8, which cooperatively stabilized its binding site. This binding mode supports the potential of P18 to target and functionally interfere with the mitochondrial respiratory chain complex.

[0109] Example 4: TCP sonodynamic in vitro killing of drug-resistant C. albicans and biofilm removal

[0110] (1) Ultrastructure observation of C. albicans after TCP sonodynamic treatment

[0111] a. Electron microscopy sample preparation: Drug-resistant C. albicans (concentration of 10 6 CFU / mL) was divided into Control (control group, PBS), TCP (25 μg / mL TCP), US (PBS + ultrasound), and TCP-US (25 μg / mL TCP + ultrasound) groups according to the experimental design. The TCP group was incubated for 2 h, and the ultrasound conditions were 1 MHz, 1.0 W / cm 2 , duty cycle 50%, and irradiation for 2 min. After treatment, the bacterial solution was centrifuged at 3500 rpm for 5 min at 4°C, the supernatant was discarded, and the pre-cooled cell fixation solution was added for fixation for 24 h, and the mixture was gently inverted every 8 h;

[0112] b. Scanning Electron Microscope (SEM) observation: After fixation, the samples were dehydrated in a series of ethanol gradient (30%, 50%, 70%, 80%, 90%, 95%, 100%) for 15-20 min each time, and then replaced with isoamyl acetate after anhydrous ethanol treatment. After critical point drying, the samples were attached to a conductive carbon film, sprayed with gold, and vacuumed for observation under a scanning electron microscope to observe the changes in bacterial morphology;

[0113] c. Transmission Electron Microscope (TEM) observation: After washing the fixed samples with PB buffer 3 times, 1% agarose was added for embedding. Further dehydration was performed with ethanol and acetone (2 times with anhydrous ethanol, and then 2 times with 100% acetone for 15 min each time). The samples were then embedded with acetone-epoxy resin in a proportion of acetone: SPI 812 epoxy resin = 1:1 at 37°C for 4 h; acetone: SPI 812 epoxy resin = 1:2 at 37°C overnight; SPI 812 epoxy resin at 37°C for 8 h; and SPI 812 epoxy resin and sample embedding in an embedding plate in a 37°C oven overnight, polymerization in a 60°C oven for 48 h). After polymerization, ultrathin sections (60-80 nm) were cut, stained with uranyl acetate and lead citrate, and observed under a TEM to observe the changes in intracellular ultrastructure and record images.

[0114] (2) Intracellular reactive oxygen species detection of C. albicans

[0115] a. The drug-resistant C. albicans suspension (concentration 10 6 CFU / mL) was divided into Control (PBS), TCP (25 μg / mL TCP), US (PBS + ultrasound), and TCP-US (25 μg / mL TCP + ultrasound) groups according to the experimental design. The spores accounted for more than 90% in each group of bacterial solution, which was inoculated in a confocal dish and cultured to adhere. The TCP group was incubated for 2 h, and the US treatment parameters were 1 MHz, 1.0 W / cm 2 , duty cycle 50%, and irradiation for 2 min;

[0116] b. After the treatment of each group was completed, the supernatant was discarded, and PBS buffer was used for washing 2 times. DCFH-DA probe (final concentration 10 μM) was added, and incubated at 37°C for 30 min in the dark, and gently shaken every 5 min to promote the probe into the cells. After incubation, the supernatant was discarded, and PBS was used for washing 2 times. DAPI was added (dilution ratio 1:1000), and incubated at 37°C for 20 min in the dark, and PBS was used for washing 1 time;

[0117] c. Then, an appropriate amount of fluorescence quenching liquid was added, and the excitation wavelength for confocal imaging was DAPI 405 nm and DCF 488 nm. The fluorescence images and fluorescence intensity signals were recorded.

[0118] (3) C. albicans mitochondrial membrane potential detection

[0119] a. Drug-resistant C. albicans bacterial suspension (concentration of 10 6 CFU / mL) was divided into four groups: Control group (PBS), TCP group (25 μg / mL TCP), US (PBS + ultrasound) and TCP-US (25 μg / mL TCP + ultrasound) group. After adherent culture in the confocal dish, each group was treated according to the above scheme, and the TCP incubation time was 2 h, the ultrasound parameters were 1 MHz, 1.0 W / cm 2 , duty cycle 50%, and irradiation time was 2 min.

[0120] b. After the treatment of each group was completed, the supernatant in the dish was discarded, and the PBS buffer was gently washed twice. Then, the JC-1 probe was preheated to 37°C (final concentration of 0.2 μM), and incubated at 37°C in the dark for 20 min. After incubation, the supernatant was discarded, and the pre-cooled PBS buffer was gently washed twice.

[0121] c. On ice in the dark, the sample was sent to the machine for detection, and the JC-1 monomer and JC-1 aggregates fluorescence signals were recorded, with excitation wavelengths of 488 nm and 525 nm, respectively. The fluorescence image and corresponding fluorescence intensity signal were collected simultaneously.

[0122] (4) C. albicans intracellular copper ion detection

[0123] a. Drug-resistant C. albicans bacterial suspension (concentration of 10 6 CFU / mL) was divided into four groups: Control group (PBS), TCP group (25 μg / mL TCP), US (PBS + ultrasound) and TCP-US (25 μg / mL TCP + ultrasound) group. After adherent culture in the confocal dish, each group was treated according to the above scheme, and the TCP incubation time was 2 h, the ultrasound parameters were 1 MHz, 1.0 W / cm 2 , duty cycle 50%, and irradiation time was 2 min.

[0124] b. Then, the samples were treated according to different grouping schemes, and incubated for another 2 h. After incubation, the culture medium was discarded, and the serum-free RPMI-1640 medium was gently washed twice. Copper ion fluorescent probe CuprosGreen was added to each group with a final concentration of 5 μmol / L, and incubated at 30°C in the dark for 3 h.

[0125] c. After incubation, the confocal laser scanning microscope was used for detection, the fluorescence channel image with excitation wavelength of 488 nm was collected, and the fluorescence signal intensity was recorded to evaluate the intracellular copper ion accumulation.

[0126] (5) TCP sonodynamic antifungal biofilm evaluation in vitro

[0127] a. Preparation of mature C. albicans biofilm: drug-resistant C. albicans colonies in the logarithmic growth phase were selected and prepared into 5-8 x 10 6 / mL bacterial suspension (using 10% fetal bovine serum RPMI 1640 medium). The bacterial suspension was placed in a confocal 6-well plate and incubated at 37°C for 48 h, and the medium was replaced every 24 h. The mature C. albicans was in the form of a milky white film, and obvious foreign matter was attached to the glass bottom of the confocal plate and was not easy to fall off.

[0128] b. TCP sonodynamic in vitro biofilm removal: the prepared mature biofilm was divided into: Control group (PBS), TCP group (25 μg / mL TCP), US group (PBS + ultrasound), and TCP-US group (25 μg / mL TCP + ultrasound), wherein the TCP incubation time was 2 h, and the ultrasound parameters were 1 MHz, 1.0 W / cm 2 , duty cycle 50%, and irradiation for 2 min. After treatment, the supernatant was discarded and washed with PBS twice.

[0129] c. Fungal live / dead staining: 1 mL of sterile water and 3 μL of SYTO 9 + PI premix (SYTO 9: 1.67 mM, PI: 1.67 mM) were added to the biofilms of different groups after treatment, and incubated at room temperature for 30 min in the dark. After light washing with sterile water, Z-stack collection was performed using LSM 880 laser confocal microscope, and the distribution of live / dead fungi in each group of biofilm was observed to evaluate the anti-biofilm ability after TCP sonodynamic treatment.

[0130] The results of TCP sonodynamic killing of drug-resistant C. albicans and biofilm removal are shown in Figure 3 . Figure 3 As shown in (a), the cell morphology of C. albicans changed significantly after TCP sonodynamic treatment. The scanning electron microscope images showed that the cell surface was severely shrunk, ruptured and formed fragments (green labeled fungal spores), indicating that the cell wall and membrane structure integrity was damaged. The transmission electron microscope images further revealed that the cell wall and membrane density of C. albicans was reduced, the structure was blurred, and there was leakage of intracellular material, while the structure of organelles such as mitochondria was unclear, indicating that TCP sonodynamic could damage the cell wall and mitochondria of drug-resistant fungi.

[0131] Figure 3 Fig. 3 shows that in (b) and (c), DCF fluorescence (green) in TCP sonodynamic group was significantly enhanced, indicating that intracellular ROS level was significantly increased. Only DAPI blue fluorescence was observed in PBS, TCP and US groups, and ROS generation was limited, indicating that ultrasound activation was essential for the generation of reactive oxygen species by nano-sonosensitizer. Figure 3 Fig. 3 shows that in (d) and (e), C. albicans JC-1 fluorescence turned from red to green after TCP sonodynamic treatment, indicating that mitochondrial membrane potential was significantly decreased. In the control group, JC-1 remained red fluorescence, indicating that mitochondrial function was not affected. This result further indicates that TCP sonodynamic therapy can effectively destroy mitochondrial membrane potential and cause fungal cell apoptosis. Figure 3 Fig. 3 shows that in (f) and (g), CuprosGreen fluorescence was significantly enhanced in the TCP-US group, indicating that TCP can induce intracellular copper accumulation after ultrasound activation, leading to mitochondrial copper homeostasis disorder and copper-induced death. Figure 3 Fig. 3 shows that in (h), the thickness of the biofilm was significantly reduced in the TCP-US treatment group, and the surface area decreased, indicating that it had good anti-biofilm ability. This group mainly showed red fluorescence (PI staining), indicating that the fungal cell wall in the biofilm was ruptured and the cells were dead; while the PBS, TCP and US groups were mainly green fluorescence (SYTO 9), indicating that the bacteria were still alive and the biofilm structure remained intact.

[0132] Example 5: TCP sonodynamic therapy for drug-resistant C. albicans deep skin infection

[0133] (1) Establishment of drug-resistant C. albicans-induced mouse subcutaneous infection model

[0134] Drug-resistant C. albicans colonies in the logarithmic growth phase were picked and prepared into a 2x10 7 / mL bacterial suspension (using 10% fetal bovine serum RPMI 1640 medium), and cultured at 37°C and 100 rpm for 4h to make ≥90% of the spores under the microscope to be in the budding state. Then centrifuged at 4°C and 3500 rpm for 5 min, resuspended after discarding the supernatant, and adjusted the bacterial solution concentration to 5x10 7 CFU / mL. After the mice were anesthetized, the left hind leg thigh to the midline area was prepared and routinely disinfected, and a microsyringe was used to inject 50μL of bacterial suspension subcutaneously. Within 24h, a red rash and local swelling appeared at the infection site, and 3 mice were randomly selected for skin tissue PAS staining, and positive results confirmed the success of the infection model.

[0135] (2) TCP sonodynamic therapy for drug-resistant C. albicans subcutaneous infection

[0136] The successfully modeled mice were randomly divided into 5 groups: Control group (control group, PBS), TCP group (intralesional multi-point injection, TCP 3 mg / kg), US group (PBS + ultrasound), TCP-US group (intralesional multi-point injection, TCP 3 mg / kg + ultrasound) and ITZ group (oral itraconazole, ITZ 40 mg / kg). PBS or TCP was injected into the lesion at 0, 3, 6 days after modeling, respectively, and low-intensity ultrasound irradiation (1 MHz, 1.0 W / cm 2 , duty cycle 50%, 6 min) was performed after waiting for 2 h. The lesion area was monitored during treatment, and the lesion skin tissue and major organs were collected at the 14th day of treatment for tissue homogenate fungal culture and histopathological analysis (H&E and PAS staining).

[0137] The results of TCP sonodynamic therapy of drug-resistant C. albicans deep skin infection are shown in Figs. 1 Figure 4 and Figure 5 . Figure 4 Figs. 1 (a) and (b) show that the treatment effect of the TCP sonodynamic group is the most significant, and the subcutaneous abscess gradually shrinks with the increase of the number of treatments, and basically disappears at the 14th day; Figure 4 Figs. 1 (c) and (d) show that the fungal infection lesion tissue homogenate of the TCP-US group does not form colonies, indicating that the fungus is effectively eliminated; Figure 4 Fig. 1 (e) shows that the skin tissue structure of the TCP-US group is complete, the epidermis, dermis, muscle and accessory organs are clearly distinguishable; the subcutaneous infection site is basically eliminated, only a small amount of necrotic area is seen, the infiltration of inflammatory cells (mainly neutrophils) is obviously reduced, and no PAS staining positive fungal spore or hypha structure is seen in the field, indicating that TCP sonodynamic therapy can effectively eliminate drug-resistant C. albicans, inhibit the progression of infection, promote skin tissue repair, and cure drug-resistant fungal deep infection.

[0138] Figure 5 It is shown that the main organs (heart, liver, spleen, lung, kidney) of the mice in each treatment group are complete in structure, and no tissue damage or inflammation is seen, indicating that TCP sonodynamic therapy has good in vivo biological safety.

[0139] The embodiments of the application are described in detail above in combination with the drawings, but the application is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the purpose of the application.

Claims

1. A mitochondria-targeted antifungal nanosonosensitizer, characterized in that: Includes rhodamine 18, tannic acid and copper ions.

2. The mitochondria-targeted antifungal nanosonosensitizer according to claim 1, characterized in that: The nano sonosensitizer is composed of tannic acid and Cu2 + It acts as a carrier and dynamically coordinates rhodopsin 18.

3. The mitochondria-targeted antifungal nanosonosensitizer according to claim 1, characterized in that: The raw materials for preparing the nano sonosensitizer include rhodamine 18, tannic acid and copper ions.

4. The method for preparing the mitochondria-targeted antifungal nanosonosensitizer according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) preparing solutions of rhodopsin 18, tannic acid, and copper salt, respectively, to obtain rhodopsin 18 solution, tannic acid solution, and copper salt solution; (2) The rhodamine 18 solution, tannic acid solution, and copper salt solution are mixed and reacted to obtain a mixture containing metal polyphenol self-assembled nanostructures, which is then separated to obtain the mitochondria-targeted antifungal nanosonosensitizer.

5. The preparation method according to claim 4, characterized in that In step (2), the separation process includes: filtering the mixture containing the metal polyphenol self-assembled nanostructure, centrifuging, sterilizing, and filtering to obtain the nanosonosensitizer.

6. The preparation method according to claim 4, characterized in that In step (1), the metal salt includes copper chloride or copper sulfate.

7. The preparation method according to claim 4, characterized in that In step (1), the concentration of the rhodamine 18 solution is 1-5 mg / mL; and / or the concentration of the tannic acid solution is 20-40 mg / mL; and / or the concentration of the copper salt solution is 3-8 mg / mL.

8. The preparation method according to claim 7, characterized in that In step (2), the mass ratio of the tannic acid solution, the rhodamine 18 solution and the copper salt solution is (1000-2000):(660-670):1667.

9. The preparation method according to claim 4, characterized in that In step (2), the temperature of the mixing reaction is 20-30° C., and the time is 40-60 minutes.

10. A drug, characterized in that The invention comprises the mitochondria-targeted antifungal nanosonosensitizer according to any one of claims 1 to 3 or the mitochondria-targeted antifungal nanosonosensitizer prepared by the preparation method according to any one of claims 4 to 9.

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