Application of multi-target nano sulfur antifungal agent to in-vivo and in-vitro efficient killing of pathogenic fungi

Abstract

The invention discloses a multi-target nano sulfur antifungal agent which can efficiently kill pathogenic fungi such as candida albicans and cryptococcus in vivo and in vitro. The sterilization site of the nano sulfur antifungal agent for efficiently killing pathogenic fungi is different from the single or multi-drug combined sterilization site of the existing antifungal drug in the market at present. The nano sulfur antifungal agent performs multi-target sterilization, and main bactericidal action sites relate to synthetic substrates GDP-mannose, UDP-glucose and UDP-N-acetyl glucosamine of mannose glycoprotein on the outer layer of a fungal cell wall, beta-(1, 3) glucan on the middle layer and chitin on the inner layer, and ergocalciferol on a fungal cell membrane and a fungal metabolic process. The novel multi-target sterilization mode is efficient, the problems of drug resistance and cross drug resistance of pathogenic bacteria can be effectively avoided, and the application prospect is wide.

Description

Technical Field

[0001] The invention relates to the field of antibacterial agents, and in particular to the application of a multi-target nano-sulfur antifungal agent in efficiently killing pathogenic fungi in vivo and in vitro. Background Art

[0002] Fungal infections are a serious threat to public health, causing more than 1.5 million deaths each year. Candida albicans and Cryptococcus neoformans are the main pathogenic fungi in clinical practice, which can cause superficial infections such as mucosa, skin, nails, etc. to life-threatening invasive deep infections such as blood infections. Among them, Candida albicans, as the most common pathogenic fungus, can parasitize in multiple parts of the human body, causing millions of superficial infections such as thrush and oropharyngeal candidiasis in individuals with normal and low immune function, and is most associated with severe fungal infections, accounting for more than 90% of cases. In clinical practice, fungal resistance and cross-resistance are receiving increasing attention.

[0003] Fungi are different from pathogens such as bacteria. As eukaryotic organisms, fungi have a highly similar cell structure to mammals, and both have many of the same potential drug targets, making it very difficult to develop drugs that are highly efficient and selective in damaging fungi but safe for mammalian cells. There are three main antifungal drugs in clinic, namely: 1) Echinocandins, which affect the key β-(1,3) glucan synthase in the synthesis of β-(1,3) glucan in fungal cell wall, represented by caspofungin. However, due to the high content of other glucans such as β-(1,6) glucan in Cryptococcus neoformans, the biosynthesis of cryptococcus is not affected by echinocandins, resulting in the natural resistance of cryptococcus to echinocandins; 2) Polyenes, which affect ergosterol on fungal cell membrane, represented by amphotericin B. However, amphotericin B can easily lead to a decrease in glomerular filtration rate and renal blood flow, impair the reabsorption of electrolytes by the proximal and distal tubules, and cause severe nephrotoxicity; 3) Azoles, which affect sterol 14-α-demethylase and thus interfere with the synthesis of fungal ergosterol, represented by ketoconazole. Candida glabrata and Candida krusei have a high resistance rate to azoles. Therefore, it is of great significance to develop new antifungal agents that are highly selective in damaging pathogenic fungi and safe for mammalian cells. Using specific cell membrane / wall structures or components as antibacterial sites is a good killing target for pathogens in both growing and non-growing states. At present, antifungal drugs destroy cell membranes / walls through a single target or a combination of multiple drugs to achieve selective killing of pathogens. However, if the target site of the drug is not changed, it is still easy to form resistance and cross-resistance with existing antibacterial drugs, which is only a matter of time. This not only greatly hinders the clinical application of antibacterial drugs, but also makes people very worried. It is reported that in addition to the inherent resistance of fungi (such as the natural resistance of Cryptococcus neoformans to echinocandin), acquired resistance involves almost all antifungal drugs and major pathogenic fungi. Therefore, the development of new target sites is crucial to solving the resistance and cross-resistance of pathogens.

[0004] Elemental sulfur is the oldest known environmentally friendly fungicide. It not only has inherent biosafety advantages, but is also one of the most abundant elements in the world. As early as 1000 BC, there were records of sulfur repelling pests. To this day, sulfur ointment is still used to treat clinical microbial infections, including rosacea, seborrheic dermatitis, dandruff, and tinea versicolor. However, large particles of elemental sulfur have poor solubility and can only be used as ointments, and cannot be used for in vivo antibacterial treatment, which limits its biomedical application. Moreover, large particles of sulfur (larger than 10nm in size) mostly kill bacteria by destroying structures such as phospholipids in the cell membrane structure. Such bactericidal targets cause indiscriminate damage to mammalian cells while killing bacteria, and cannot achieve the purpose of selective damage. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides a multi-target nano-sulfur antifungal agent that can achieve highly selective damage to pathogenic fungi in vivo and in vitro while being safe for mammalian cells.

[0006] In a first aspect, the nano-sulfur antifungal agent can effectively kill pathogenic fungi such as Candida albicans and Cryptococcus neoformans in vitro, including but not limited to these.

[0007] In a second aspect, the nano-sulfur antifungal agent can effectively kill Candida albicans in vivo and promote the healing of Candida albicans infected wounds in mice.

[0008] On the third aspect, the main bactericidal action sites of the nano-sulfur antifungal agent involve the synthetic substrates GDP-mannose, UDP-glucose and UDP-N-acetylglucosamine of the outer layer mannoprotein, the middle layer β-(1,3) glucan and the inner layer chitin of the fungal cell wall, and ergocalciferol on the fungal cell membrane and the fungal metabolic process.

[0009] The benefits of the present invention are:

[0010] The nano-sulfur prepared by the present invention not only breaks through the problem of water solubility limitation, shows efficient in vitro anti-pathogenic fungi effect, but also can be further applied to the treatment of pathogenic fungal infection in vivo. Compared with large-particle sulfur, the nano-sulfur antifungal agent hardly affects membrane phospholipids, and the main bactericidal sites involve the synthetic substrates GDP-mannose, UDP-glucose and UDP-N-acetylglucosamine of the outer layer mannoprotein, the middle layer β-(1,3) glucan and the inner layer chitin of the fungal cell wall, ergocalciferol on the fungal cell membrane and the fungal metabolic process, thereby achieving selective killing of fungi. This multi-target action is completely different from the single or multi-drug combined drug killing sites of the existing antifungal drugs on the market. Only this nano-sulfur antifungal agent is needed to achieve efficient sterilization in vivo and in vitro, solve the problems of pathogenic fungal resistance and cross-resistance, and is expected to solve practical difficulties and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is a transmission electron microscope characterization image of the nano-sulfur antifungal agent prepared in Example 1.

[0012] Figure 2 This is an X-ray photoelectron spectrum characterization diagram of the nano-sulfur antifungal agent prepared in Example 1.

[0013] Figure 3 This is a graph showing the growth of Candida albicans on a solid culture medium after incubation with PBS, large-particle sulfur, and nano-sulfur antifungal agents in Example 2.

[0014] Figure 4 This is a graph showing the growth of Candida albicans on a solid culture medium after incubation with different concentrations of nano-sulfur antifungal agents in Example 2, with the minimum inhibitory concentration being 0.5 mg / mL.

[0015] Figure 5 This is a graph showing the growth of Cryptococcus neoformans on solid culture medium after incubation with PBS, large-particle sulfur, and nano-sulfur antifungal agents in Example 3.

[0016] Figure 6 It is a diagram showing the in vivo treatment process of Candida albicans wound infection in mice treated with PBS, large particle sulfur, and nano-sulfur antifungal agents in Example 4, as well as the growth of residual fungi in the grinding fluid of the infected wound tissue of each treatment group on a solid culture medium after the treatment.

[0017] Figure 7 It is a cluster analysis heat map of specific differential metabolites in the incubation group of large particle sulfur and Candida albicans, the incubation group of nanosulfur antifungal agent and Candida albicans and the Candida albicans control group in Example 5, and a comparison map of the abundance of fungal cell membrane / wall specific metabolites.

[0018] Figure 8 It is a cluster analysis heat map of the differential metabolites of phospholipids in the incubation group of large particle sulfur and Candida albicans, the incubation group of nanosulfur antifungal agent and Candida albicans, and the Candida albicans control group in Example 5. DETAILED DESCRIPTION

[0019] This section describes in detail the experimental methods used in the experiments of the present invention. The technical means used in the present invention are all methods known to those skilled in the art. The embodiments should be understood to illustrate the present invention, rather than to limit the present invention.

[0020] Example 1 Preparation of Nano-sulfur Antifungal Agent

[0021] 1.5g sublimed sulfur powder, 0.15g sodium polystyrene sulfonate, 4g sodium hydroxide are placed in a round-bottom flask, 50mL ultrapure water is added, and the mixture is placed in a 70℃ oil bath, condensed and refluxed, and magnetically stirred for 12h. When the solution turns orange-yellow, let it cool to room temperature. This is a large-particle sulfur solution. Take 1.5mL of the orange-yellow solution synthesized in the previous step and add 2mL of 4.8% hydrogen peroxide dropwise. A large number of bubbles are generated and heat is released. The solution changes from orange-yellow to colorless. This is a nano-sulfur antifungal agent solution.

[0022] pass Figure 1 Transmission electron microscopy showed that the nanosulfur antifungal agent was evenly dispersed and had a size of 1.9±0.7nm. Figure 2 The X-ray photoelectron spectroscopy (XPS) of S2p shows that 161.97, 163.13 and 163.95 eV are attributed to sulfur atoms, while 168.49 and 169.56 eV are attributed to SO3 2- (2p 3 / 2 ) and SO3 2- (2p 1 / 2 ), indicating that the nanosulfur antifungal agent is composed of a central sulfur atom and oxidized sulfur.

[0023] Example 2 In vitro anti-Candida albicans experiment of nano-sulfur antifungal agent

[0024] Comparison of the antifungal ability of large-particle sulfur and nano-sulfur antifungal agents against Candida albicans

[0025] The strain Candida albicans (C.albicans) was inoculated into a modified Martin liquid medium and then cultured in a 37°C incubator with a rotational motion of 220 rpm. The logarithmic growth phase of C.albicans was measured using a micro-UV spectrophotometer. When the optical density (OD) at 600 nm increased to 0.75, C.albicans grew to 10 per ml. 8 The C.albicans suspension was diluted to 10 with PBS buffer (10 mM, pH 7.4). 6 CFU / mL. Then, 100 μL of C.albicans suspension was mixed with PBS, 3 mg / mL large particle sulfur, and 3 mg / mL nanosulfur antifungal agent, and incubated at 37°C for 20 minutes. After that, 10 μL of the above suspension was evenly spread on Sabor solid medium and cultured in a 37°C incubator for 48-72 hours. Observe and take photos to record the C.albicans colonies on the solid medium.

[0026] Study on the minimum inhibitory concentration of nanosulfur antifungal agent against Candida albicans

[0027] The strain C.albicans was inoculated into modified Martin liquid medium and then cultured in a 37°C incubator with a rotation motion of 220 rpm. The logarithmic growth phase of C.albicans was measured using a micro-volume UV spectrophotometer. When OD 600 When it increases to 0.75, the concentration of C.albicans is 10 8 CFU / mL. Dilute the C.albicans suspension to 10 6 CFU / mL. Then, 100 μL of C.albicans suspension was mixed with 100 μL of PBS buffer and different concentrations (0, 0.0050, 0.010, 0.020, 0.10, 0.20, 0.50, 0.80, 1.2, 1.8 mg / mL) of nanosulfur antifungal agent, and incubated at 37°C for 20 minutes. After that, 10 μL of the above suspension was evenly spread on Sabor solid medium and cultured in a 37°C incubator for 48-72 hours. The C.albicans colonies on the solid medium were observed and photographed.

[0028] from Figure 3 It can be seen that many Candida albicans colonies grew on the solid culture medium of the PBS control group and the large-particle sulfur group, while no colonies grew on the solid culture medium of the nanosulfur antifungal agent group, indicating that at the same concentration, the nanosulfur antifungal agent can more effectively kill Candida albicans than the large-particle sulfur, and the in vitro antibacterial ability of the nanosulfur antifungal agent is much stronger than that of the large-particle sulfur. Figure 4 It can be seen that as the concentration of the nanosulfur antifungal agent increases, the colonies on the solid culture medium gradually decrease. When the concentration of the nanosulfur antifungal agent is greater than or equal to 0.5 mg / mL, no colonies grow on the solid culture medium, indicating that the minimum inhibitory concentration for Candida albicans is 0.5 mg / mL. In summary, the nanosulfur antifungal agent can effectively kill Candida albicans in vitro.

[0029] Example 3 In vitro anti-Cryptococcus neoformans experiment of nano-sulfur antifungal agent

[0030] The strain Cryptococcus neoformans (C.neoformans) was inoculated into modified Martin liquid medium and then cultured in a 37°C incubator with a rotational motion of 220 rpm. The logarithmic growth phase of C.neoformans was measured using a micro-UV spectrophotometer. When OD 600 When the concentration of C. neoformans increases to 0.75, 8CFU / mL. Then the C. neoformans suspension was diluted to 10 6 CFU / mL. Then, 100 μL of C.neoformans suspension was mixed with 100 μL of PBS buffer, 3 mg / mL of large particle sulfur, and 3 mg / mL of nanosulfur antifungal agent, respectively, and incubated at 37°C for 20 minutes. After that, 10 μL of the above suspension was evenly spread on Sabor solid medium and cultured in a 37°C incubator for 48-72 hours, and the fungal colonies on the solid medium were observed and photographed.

[0031] Figure 5 It can be seen that there are a large number of new Cryptococcus colonies on the solid culture medium of the control group PBS and the large-particle sulfur and Candida albicans groups, while no colonies grow in the group adding the nanosulfur antifungal agent, indicating that the nanosulfur antifungal agent can also effectively kill the new Cryptococcus compared with the large-particle sulfur, which shows the extensive and efficient killing effect of the nanosulfur antifungal agent on pathogenic fungi.

[0032] Example 4 Animal Experiment on Antifungal Infection by Nano-sulfur Antifungal Agents

[0033] Female BALB / c mice weighing about 20 g were randomly divided into 3 groups, 3 mice in each group. The back of the mice was shaved with an electric shaver, and then depilatory cream was used to remove the hair. A wound with a diameter of about 5 mm was cut on the epidermis of the mice with surgical scissors. 100 μL of bacterial solution (10 9 CFU / mL), applied to the wound of mice. PBS (10mM, pH 7.4), large-particle sulfur (concentration of 0.5mg / mL), and nano-sulfur antifungal agent (concentration of 0.5mg / mL) were given for treatment, 50μL per group, applied to the wound of mice for 10 minutes, once a day, for 7 consecutive days. Pictures were taken every day to record the recovery of the wounds of mice in each group. After the treatment, the mice were killed. The tissue of the epidermal wound of the mouse was cut with sterile surgical scissors, ground and homogenized, and coated with solid culture medium, and the amount of fungi remaining in the wound was observed and counted.

[0034] Figure 6 a shows that after 5 days of treatment, the infected wounds in the nanosulfur antifungal agent treatment group were significantly smaller than those in the PBS control group and the large-particle sulfur treatment control group. At the end of the treatment on the 7th day, the wounds infected with Candida albicans were almost completely healed, indicating that compared with large-particle sulfur, nanosulfur antifungal agents can more effectively treat and promote the healing of Candida albicans infected wounds in mice. After the treatment, the fungal residues in the abrasive fluid of the infected wound tissue were detected. Figure 6b The results showed that compared with the PBS group and the large-particle sulfur treatment group, there was no growth of Candida albicans colonies on the solid culture medium of the nano-sulfur antifungal group after treatment, indicating that the nano-sulfur antifungal agent has a stronger ability to kill pathogenic fungi in vivo than large-particle sulfur, and can achieve safe and efficient treatment of fungal infections in vivo.

[0035] Example 5 Analysis of multi-target effects on Candida albicans

[0036] The Candida albicans cells incubated with large-particle sulfur and nano-sulfur antifungal agents were centrifuged, collected into 15 mL centrifuge tubes, discarded, and washed three times with pre-cooled 10 mL PBS solution. Resuspended with 1 mL PBS and accurately counted, equal amounts of cells were divided into 1.5 mL centrifuge tubes, centrifuged at 8000g for 5-10 min, supernatant discarded, nitrogen frozen, stored in a -80℃ refrigerator, and metabolite detection was performed at Shanghai Major Biopharmaceutical Technology Co., Ltd. The metabolites of Candida albicans were detected using an ultra-high performance liquid chromatography-tandem Fourier transform mass spectrometry (UHPLC-QExactive HF-X) system (Thermo Fisher Scientific, USA). All data were peak extracted, aligned, and identified using the metabolomics software Proogene QI (Waters Corporation, Milford, USA). Finally, a data matrix containing retention time, peak area, mass-to-charge ratio, and identification information was obtained for post-processing and bioinformatics analysis. All data were analyzed on the online platform of Shanghai Major Biopharmaceutical Technology Co., Ltd. (www.majorbio.com). All quantitative data are expressed as mean ± standard deviation (n = 6). Statistical analysis was performed using GraphPad Prism 8.0. One-way ANOVA was used to calculate significant differences. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 were considered to be significantly different.

[0037] Figure 7a Cluster analysis heat map results of specific differential metabolites of the nanosulfur antifungal agent incubated with Candida albicans group (Control) and the large-particle sulfur incubated with Candida albicans group showed that after incubation of nanosulfur antifungal agent with Candida albicans, the relative expression abundance of GDP-mannose (guanosine diphosphate mannose, GDP-Man), UDP-glucose (uridine diphosphate glucose, UDP-Glc) and UDP-N-acetylglucosamine (uridine diphosphate-N-acetylglucosamine, UDP-GlcNAc) and ergocalciferol, phosphoenol pyruvate (phosphoenol pyruvate, PEP), citric acid, malic acid and reduced nicotinamide adenine dinucleotide (Nicotinamideadenine dinucleotide, NADH) in the four important intermediate substances in the glycolysis EMP pathway and tricarboxylic acid cycle were significantly changed compared with the other two groups. Further quantitative comparison of these metabolites, Figure 7 b The results showed that after Candida albicans was treated with nanosulfur antifungal agents, the relative abundance of GDP-mannose and UDP-N-acetylglucosamine increased significantly, while the relative abundance of UDP-glucose decreased significantly compared with the Candida albicans group and the group incubated with large sulfur particles and Candida albicans, indicating that nanosulfur antifungal agents can act on the synthetic substrates of the fungal cell wall, resulting in the selective destruction of the cell wall integrity. Figure 7 c The results showed that after the nanosulfur antifungal agent acted on Candida albicans, the relative abundance of ergocalciferol was significantly reduced compared with the Candida albicans group and the group incubated with large sulfur particles and Candida albicans, indicating that the nanosulfur antifungal agent can also selectively destroy the integrity of the fungal cell membrane by acting on ergocalciferol, the metabolite of sterols in the fungal cell membrane. Figure 7dThe results showed that after the nanosulfur antifungal agent treated Candida albicans, the relative abundance of phosphoenolpyruvate and citric acid also increased significantly, while the relative abundance of malic acid and NADH decreased significantly, compared with the Candida albicans group and the group incubated with large sulfur particles and Candida albicans, indicating that the nanosulfur antifungal agent can affect the energy metabolism process of fungi and exert a highly efficient bactericidal effect. GDP-mannose, UDP-glucose, UDP-N-acetylglucosamine and ergocalciferol are the synthetic substrates of the outer layer mannoprotein, the middle layer β-(1,3) glucan and the inner layer chitin of the fungal cell wall, and the metabolites of ergosterol on the fungal cell membrane. Phosphoenolpyruvate, citric acid, malic acid and NADH are important intermediates in the fungal energy metabolism glycolysis EMP (Embden-Meyerhof-Parnas) pathway and the tricarboxylic acid cycle pathway. The above results show that the main bactericidal sites of nanosulfur antifungal agents involve the synthetic substrates GDP-mannose, UDP-glucose and UDP-N-acetylglucosamine of the outer layer mannoprotein, the middle layer β-(1,3) glucan and the inner layer chitin of the fungal cell wall, ergocalciferol on the fungal cell membrane and the fungal energy metabolism process. Figure 8Sixty metabolites specifically induced by large particle sulfur were shown, which mainly induced significant changes in the expression levels of Candida albicans lipid metabolites: PS (16:0 / 18:00), PC (14:1 (9Z) / 20:1 (11Z)), PE (15:0 / 16:1 (9Z)), PC (18:3 (6Z, 9Z, 12Z) / 18:3 (6Z, 9Z, 12Z)) [U], PC (14:0 / 18:2 (9Z, 12Z)), PE (18:3 (9Z, 12Z, 15Z) / P-16:0), PS (18:1 (9Z) / 0:0), PC (18:2 (2E, 4E) / 18:2 (2E, 4E)) and PE (18:0 / 0:0). The Human Metabolome Database (HMDB) matched the specific types of the above metabolites and found that PC (14:0 / 18:2 (9Z, 12Z)) and PC (14:1 (9Z) / 20:1 (11Z)) are phosphatidylcholine, PE (15:0 / 16:1 (9Z)) and PE (18:3 (9Z, 12Z, 15Z) / P-16:0) are phosphatidylethanolamine, PS (18:1 (9Z) / 0:0) and PS (16:0 / 18:00) are phosphatidylserine. The lipid composition of fungal and mammalian cell membranes is mainly composed of glycerophospholipids (including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol). Therefore, large-particle sulfur will significantly affect the metabolism of phospholipids. However, there was almost no significant difference in the expression of the above phospholipid metabolites in the nanosulfur antifungal agent incubated with Candida albicans compared with the Candida albicans group. Therefore, compared with large-particle sulfur, nanosulfur antifungal agents have almost no effect on membrane phospholipids, thereby achieving selective killing of fungi.

[0038] In summary, the present invention discloses a nano-sulfur antifungal agent that can effectively kill Candida albicans and Cryptococcus neoformans in vitro, and can also effectively treat Candida albicans infection in mice, showing an efficient in vitro and in vivo killing effect of pathogenic fungi. Compared with large-particle sulfur, this nano-sulfur antifungal agent has almost no effect on membrane phospholipids, and the main bactericidal sites involve the synthetic substrates GDP-mannose, UDP-glucose and UDP-N-acetylglucosamine of the outer layer mannoprotein, the middle layer β-(1,3) glucan and the inner layer chitin of the fungal cell wall, ergocalciferol on the fungal cell membrane and the fungal metabolic process, thereby achieving selective killing of fungi and safety in vivo and in vitro. This multi-target action is efficient and safe, completely different from the single or multi-drug combined bactericidal sites of existing antifungal drugs on the market, and is expected to efficiently solve the problems of pathogenic fungal resistance and cross-resistance, and has broad application prospects.

[0039] The above description is only a specific embodiment of the present invention, and is not intended to limit the present invention. Any person skilled in the art may modify or change the above experimental examples within the technical scope disclosed by the present invention, and the modifications or changes shall still be covered by the claims of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims

1. A multi-target nano-sulfur antifungal agent can effectively kill pathogenic fungi in vivo and in vitro. The bactericidal characteristics of the nano-sulfur antifungal agent are that it can effectively kill pathogenic fungi such as Candida albicans and Cryptococcus neoformans in vitro, including but not limited to these.

2. According to claim 1, the nano-sulfur antifungal agent can effectively kill Candida albicans in vivo and promote the healing of Candida albicans infected wounds in mice, including but not limited to the in vivo treatment and application of such fungi.

3. The main bactericidal sites of the multi-target nano-sulfur antifungal agent according to claims 1 and 2 involve the synthetic substrates GDP-mannose, UDP-glucose and UDP-N-acetylglucosamine of the outer layer mannoprotein, the middle layer β-(1,3) glucan and the inner layer chitin of the fungal cell wall, and ergocalciferol on the fungal cell membrane and the fungal metabolic process.

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