Application of combined composition nanoparticles in preparation of medicines for resisting fungal and bacterial infection

Abstract

The invention relates to an application of a combined composition nanoparticle in preparation of a medicine for resisting fungal and bacterial infection. The active components of the combined composition nanoparticle are antibiotics and antibiotic adjuvants, the antibiotic adjuvant is tripterine or a pharmaceutically acceptable salt thereof. Compared with the prior art, the tripterine or the medicinal salt thereof and the antibiotics are prepared into the nanoparticles by utilizing a nanotechnology, so that the solubility of the fat-soluble antibiotics is effectively improved, the biocompatibility of the tripterine or the medicinal salt thereof is improved, and the tripterine or the medicinal salt thereof has an excellent effect in resisting fungal and bacterial infection. The tripterine or the medicinal salt thereof can enhance the antibacterial effect, especially the antifungal effect, of the antibiotics, increase the antibacterial sensitivity of the antibiotics and inhibit the occurrence of fungal or bacterial drug resistance.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to the application of a combination composition of nanoparticles in the preparation of drugs against fungal and bacterial infections. Background Technology

[0002] Bacterial and fungal infections are a major cause of human death, and the discovery of antibiotics has significantly reduced mortality rates among patients with these infections. However, the overuse of antibiotics has led to the rapid development of multidrug-resistant bacteria, posing a long-term threat to human health and the sustainable production and development of food. The development of novel antibiotics is challenging and time-consuming, severely limiting the clinical treatment of drug-resistant infections. The arrival of the post-antibiotic era urges us to develop new treatment options to address the concerning situation of antibiotic resistance. The discovery of antibiotic adjuvants offers hope for addressing the obstacles in the development of new antibiotics and the inadequacy of existing drugs to combat drug-resistant infections. Antibiotic adjuvants, used in combination with antibiotics, enhance their efficacy, offering new hope for restoring the activity of existing antibiotics and inhibiting the development of resistance, while also providing an orthogonal strategy for the discovery of new antibiotics.

[0003] Nanotechnology is playing an increasingly important role in improving drug bioavailability and reducing drug toxicity. The emergence of nanotechnology has provided new strategies for improving the water solubility and bioavailability of hydrophobic drugs. Tripterygium wilfordii, also known as yellow wax vine or water vine, has various effects such as dispelling wind and dampness, promoting blood circulation, reducing swelling and relieving pain, and anti-inflammatory and detoxifying. Celastrol (CST), also known as southern celastrine, is a triterpenoid active ingredient in Tripterygium wilfordii extract, possessing various biological activities. Studies have found that celastrol has anti-tumor, anti-rheumatoid, and anti-Parkinson's disease effects. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the purpose of this invention is to provide an application of combined composition nanoparticles in the preparation of drugs for antifungal and antibacterial infections.

[0005] To achieve this objective, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides the application of a combination composition of nanoparticles in the preparation of drugs for antifungal and antibacterial infections, characterized in that the active component of the combination composition is an antibiotic and an antibiotic adjuvant; the antibiotic adjuvant is triptolide or a pharmaceutically acceptable salt thereof.

[0007] This invention utilizes nanotechnology to prepare nanoparticles from triptolide or its pharmaceutically acceptable salts with antibiotics, effectively improving the solubility of lipid-soluble antibiotics and enhancing the biocompatibility of triptolide or its pharmaceutically acceptable salts. Furthermore, it exhibits excellent efficacy against fungal and bacterial infections. Specifically, triptolide or its pharmaceutically acceptable salts can enhance the antibacterial activity of antibiotics, especially their antifungal activity, increase antibiotic susceptibility, and inhibit the development of fungal or bacterial resistance.

[0008] Preferably, the fungus includes any one or a combination of at least two of the following: Candida albicans, Candida glabrata, Candida tropicalis, Candida krusei, Aspergillus fumigatus, Aspergillus niger, Aspergillus flavus, Aspergillus oryzae, Fusarium, Cryptococcus neoformans, Candida albicans, Mucor, Histoplasma capsulatum, Sarcodactylum, Coccidioides, Cryptococcus gutterus, or Paracoccidioides.

[0009] Preferably, the bacteria include any one or a combination of at least two of the following: Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, Escherichia coli, Salmonella, Shigella, Streptococcus pneumoniae, Staphylococcus aureus, Mycobacterium tuberculosis, or Clostridium difficile.

[0010] The combined composition nanoparticles involved in this invention include the following three forms:

[0011] Firstly, the combined composition nanoparticles include carrier-free nanoparticles formed from antibiotics and antibiotic adjuvants; the antibiotic adjuvant is triptolide or its pharmaceutically acceptable salt.

[0012] Preferably, the antibiotic is selected from any one or a combination of at least two of the following: amphotericin B, nystatin, miconazole, ketoconazole, fluconazole, itraconazole, voriconazole, posaconazole, flucytosine, terbinafine, caspofungin, micafungin, anidoxuridine, griseofulvin, penicillin, amoxicillin, ampicillin, cefadroxil, cephalexin, ceftazidime, imipenem, amikacin, gentamicin, doxycycline, tetracycline, minocycline, erythromycin, clarithromycin, azithromycin, sulfadiazine, ciprofloxacin, levofloxacin, metronidazole, clindamycin, or fosfomycin.

[0013] Preferably, the molar ratio of the antibiotic to the antibiotic adjuvant is 1:(0.001-500), such as 1:0.001, 1:0.01, 1:0.05, 1:0.1, 1:0.5, 1:1, 1:5, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:100, 1:150, 1:200, 1:300, 1:400, 1:500, etc. Other specific values ​​within this range can be selected, and will not be elaborated here.

[0014] In this invention, the above-mentioned carrier-free nanoparticles are prepared by a method comprising the following steps:

[0015] (1) Dissolve triptolide or its pharmaceutically acceptable salt and hydrophobic antibiotic in a good solvent to obtain solution A;

[0016] (2) Add solution A to triptolide or its pharmaceutically acceptable salts and hydrophobic antibiotics in a poor solvent to obtain solution B;

[0017] (3) After stirring solution B, the free drug is separated to obtain carrier-free nanoparticles.

[0018] Preferably, the good solvent includes any one or a combination of at least two of methanol, ethanol, propanol, isopropanol, acetone, tetrahydrofuran, or dimethyl sulfoxide.

[0019] Preferably, the unsuitable solvent includes deionized water and / or phosphate buffer.

[0020] Preferably, the stirring time is 5-60 min, such as 5 min, 10 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, etc. Other specific values ​​within this range can be selected, and will not be elaborated here.

[0021] Preferably, the separation of free drugs is performed by ultrafiltration centrifugation. The conditions for ultrafiltration centrifugation are: rotation speed of 8000-10000g, such as 8000g, 8500g, 9000g, 9500g, 10000g, etc.; and duration of 10-30min, such as 10min, 15min, 20min, 25min, 30min, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0022] Secondly, the nanoparticles of the combined composition include composite liposomes prepared from antibiotics, antibiotic adjuvants, lipids, and cholesterol; the antibiotic adjuvant is triptolide or its pharmaceutically acceptable salt.

[0023] Preferably, the antibiotic is selected from any one or a combination of at least two of the following: amphotericin B, nystatin, miconazole, ketoconazole, fluconazole, itraconazole, voriconazole, posaconazole, flucytosine, terbinafine, caspofungin, micafungin, anisofungin, griseofulvin, penicillin, amoxicillin, ampicillin, cefadroxil, cephalexin, ceftazidime, imipenem, amikacin, gentamicin, doxycycline, tetracycline, minocycline, erythromycin, clarithromycin, azithromycin, sulfadiazine, ciprofloxacin, levofloxacin, metronidazole, clindamycin, or fosfomycin.

[0024] Preferably, the molar ratio of the antibiotic to the antibiotic adjuvant is 1:(0.001-500), such as 1:0.001, 1:0.01, 1:0.05, 1:0.1, 1:0.5, 1:1, 1:5, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:100, 1:150, 1:200, 1:300, 1:400, 1:500, etc. Other specific values ​​within this range can be selected, and will not be elaborated here.

[0025] Preferably, the lipid comprises any one or a combination of at least two of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidic acid, cardiolipin, sphingomyelin, phosphatidylserine, DSPE-PEG, or fluorinated DSPE-PEG.

[0026] In this invention, the above-mentioned composite liposomes can be prepared by a preparation method (first method) including the following steps:

[0027] (1) Mix the first solvent, triptolide or its pharmaceutically acceptable salt, hydrophobic antibiotic, phospholipid, and cholesterol to obtain solution A; mix the second solvent and hydrophilic antibiotic to obtain solution B;

[0028] (2) Remove the first solvent from solution A, mix it with solution B and hydrate to obtain solution C;

[0029] (3) Solution C was subjected to ultrasonication and gradient extrusion to obtain the composite liposomes.

[0030] Preferably, the first solvent comprises any one or a combination of at least two of methanol, ethanol, propanol or chloroform; the second solvent comprises deionized water and / or phosphate buffer.

[0031] Preferably, the hydration time is 10-60 min, such as 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, etc.; the temperature is 15-35℃, such as 15℃, 18℃, 20℃, 22℃, 25℃, 28℃, 30℃, 35℃, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0032] Preferably, the ultrasonic treatment in step (3) is performed at 0-10℃, such as 1℃, 2℃, 3℃, 4℃, 5℃, 7℃, 8℃, 10℃, etc.; the ultrasonic power is 80-250W, such as 80W, 100W, 120W, 150W, 180W, 200W, 220W, 250W, etc.; the ultrasonic time is 3-30min, such as 4min, 8min, 10min, 12min, 15min, 20min, 25min, 30min, etc. Other specific values ​​within the above ranges can be selected, and will not be elaborated here.

[0033] Preferably, the pore sizes of the gradient extruded filter membrane are 400 nm and 200 nm, respectively.

[0034] In this invention, the above-mentioned composite liposomes can be prepared by a preparation method (second method) including the following steps:

[0035] (1) Mix the first solvent, phospholipids, and cholesterol to obtain solution A; mix triptolide or its pharmaceutically acceptable salt, antibiotics, and organic solvent thoroughly, and add it dropwise to the second solvent to obtain solution B;

[0036] (2) Remove the first solvent from solution A, mix it with solution B and hydrate to obtain solution C;

[0037] (3) Solution C was subjected to ultrasonication and gradient extrusion to obtain the composite liposomes.

[0038] Preferably, the first solvent includes any one or a combination of at least two of methanol, ethanol, propanol or chloroform; the organic solvent includes any one or a combination of at least two of methanol, ethanol, DMF or DMSO; and the second solvent includes deionized water and / or phosphate buffer.

[0039] Preferably, the hydration time is 10-30 minutes, such as 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, etc.; the temperature is 15-35°C, such as 15°C, 18°C, 20°C, 22°C, 25°C, 28°C, 30°C, 35°C, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0040] Preferably, the ultrasonic treatment in step (3) is performed at 0-10℃, such as 1℃, 2℃, 3℃, 4℃, 5℃, 7℃, 8℃, 10℃, etc.; the ultrasonic power is 80-250W, such as 80W, 100W, 120W, 150W, 180W, 200W, 220W, 250W, etc.; the ultrasonic time is 3-30min, such as 4min, 8min, 10min, 12min, 15min, 20min, 25min, 30min, etc. Other specific values ​​within the above ranges can be selected, and will not be elaborated here.

[0041] Preferably, the pore sizes of the gradient extruded filter membrane are 400 nm and 200 nm, respectively.

[0042] Third, the combined composition nanoparticles include composite polymer nanoparticles made from antibiotics, antibiotic adjuvants, and pharmaceutically degradable polymers; the antibiotic adjuvant is triptolide or its pharmaceutically acceptable salt.

[0043] Preferably, the antibiotic is selected from any one or a combination of at least two of the following: amphotericin B, nystatin, miconazole, ketoconazole, fluconazole, itraconazole, voriconazole, posaconazole, flucytosine, terbinafine, caspofungin, micafungin, anisofungin, griseofulvin, penicillin, amoxicillin, ampicillin, cefadroxil, cephalexin, ceftazidime, imipenem, amikacin, gentamicin, doxycycline, tetracycline, minocycline, erythromycin, clarithromycin, azithromycin, sulfadiazine, ciprofloxacin, levofloxacin, metronidazole, clindamycin, or fosfomycin.

[0044] Preferably, the molar ratio of the antibiotic to the antibiotic adjuvant is 1:(0.001-500), such as 1:0.001, 1:0.01, 1:0.05, 1:0.1, 1:0.5, 1:1, 1:5, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:100, 1:150, 1:200, 1:300, 1:400, 1:500, etc. Other specific values ​​within this range can be selected, and will not be elaborated here.

[0045] Preferably, the biodegradable pharmaceutical polymer includes any one or a combination of at least two of PLGA, PEG, mPEG-PLGA, or DSPE-PEG.

[0046] In this invention, the composite polymer nanoparticles are prepared by a method comprising the following steps:

[0047] (1) Mix the first solvent and the biodegradable pharmaceutical polymer to obtain solution A; mix triptolide or its pharmaceutically acceptable salt, antibiotic and organic solvent, and add dropwise to the second solvent containing emulsifier to obtain solution B;

[0048] (2) Mix solution A with solution B to obtain solution C;

[0049] (3) Add solution C to a third solvent containing an emulsifier, mix, and obtain solution D;

[0050] (4) Remove the first solvent remaining in solution D to obtain the composite polymer nanoparticles.

[0051] Preferably, the first solvent comprises any one or a combination of at least two of methanol, ethanol, propanol, or dichloromethane; the organic solvent comprises any one or a combination of at least two of methanol, ethanol, DMF, or DMSO; the second solvent comprises deionized water and / or phosphate buffer; and the third solvent comprises deionized water and / or phosphate buffer.

[0052] Preferably, each group of emulsifiers in the second solvent and the third solvent independently includes any one or a combination of at least two of polyvinyl alcohol, Tween-80, or vitamin E polyethylene glycol succinate.

[0053] Preferably, the mass fraction of the emulsifier in the second solvent is 3%-15%, such as 3%, 7%, 8%, 10%, 12%, 14%, 15%, etc.; and the mass fraction of the emulsifier in the third solvent is 30%-60%, such as 30%, 35%, 40%, 45%, 50%, 55%, 60%, etc.

[0054] Compared with the prior art, the present invention has the following beneficial effects:

[0055] This invention creatively discovers that triptolide or its pharmaceutically acceptable salts can be used in combination with antibiotics to exhibit excellent efficacy in antifungal and bacterial infections, particularly in antifungal infections. Preparing antibiotics and triptolide into nanoparticles further enhances the antibacterial sensitivity of antibiotics and inhibits the development of fungal or bacterial resistance. The efficacy in antifungal and bacterial infections is even better when the nanoparticles exist in the form of composite liposomes.

[0056] This antibiotic adjuvant-antibiotic drug platform can be formulated into various dosage forms to meet the needs of different treatment scenarios and different physicochemical characteristics of antibiotics, while also satisfying clinical needs for oral, injection, inhalation, or topical administration.

[0057] Meanwhile, the nanoparticles involved in this invention avoid the introduction of excessive toxic organic reagents and carriers during the preparation process, thereby preventing potential biotoxicity problems; and the preparation method is simple, mild, green and environmentally friendly, low in cost, requires no special reaction equipment, and is easy to promote and apply. Attached Figure Description

[0058] Figure 1 This is a graph showing the hydrated particle size detection results of the carrier-free nanoparticles prepared in Example 1.

[0059] Figure 2 This is a graph showing the potential detection results of the carrier-free nanoparticles prepared in Example 1;

[0060] Figure 3 The figure shows the hydration particle size of the carrier-free nanoparticles prepared in Example 2 at different times in PBS solution or ultrapure water containing 10% FBS.

[0061] Figure 4 The graph shows the potential detection results of the carrier-free nanoparticles prepared in Example 2 in PBS solution or ultrapure water containing 10% FBS at different times.

[0062] Figure 5 This is an in vitro release curve of the carrier-free nanoparticles prepared in Example 2 under neutral or slightly acidic conditions.

[0063] Figure 6 The figure shows the results of the crystal violet biomembrane staining experiment to verify the effects of composite liposomes (preparation example 8), triptolide (CST), and fluconazole (Flu) on the cell membranes of Aspergillus fumigatus and its drug-resistant strains.

[0064] Figure 7 The graph shows the statistical results of the hemolysis rate of the carrier-free nanoparticles prepared in Preparation Example 3 and Preparation Example 4.

[0065] Figure 8 This is a graph showing the hydrated particle size detection results of the composite liposomes prepared in Example 7;

[0066] Figure 9 This is a graph showing the potential detection results of the composite liposomes prepared in Example 7;

[0067] Figure 10 The figure shows the hydration particle size of the composite liposomes prepared in Example 7 at different times in PBS solution or ultrapure water containing 10% FBS.

[0068] Figure 11 The graph shows the potential detection results of the composite liposomes prepared in Example 7 in PBS solution or ultrapure water containing 10% FBS at different times.

[0069] Figure 12This is an in vitro release curve of the composite liposomes prepared in Example 7 under neutral or slightly acidic conditions.

[0070] Figure 13 This is a survival curve of the composite liposomes prepared in Example 9 for the treatment of fungal infections in the larvae of the giant wax moth. Detailed Implementation

[0071] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0072] Preparation Example 1

[0073] This preparation example provides a carrier-free nanoparticle, which is prepared by the following method:

[0074] (1) Dissolve 2 mg of triptolide and 3 mg of fluconazole in 1 mL of dimethyl sulfoxide and mix thoroughly;

[0075] (2) Under stirring conditions, add the above mixture dropwise to 10 mL of deionized water;

[0076] (3) Place the resulting mixture in a magnetic stirrer and stir for 10 min to obtain the initial carrier-free nanoparticles;

[0077] (4) Obtain carrier-free nanoparticles by ultrafiltration centrifugation at 8000g / 15min.

[0078] Preparation Example 2

[0079] This preparation example provides a carrier-free nanoparticle, which is prepared by the following method:

[0080] (1) Dissolve 2 mg of triptolide and 2 mg of fluconazole in 1 mL of methanol and mix thoroughly;

[0081] (2) Under stirring conditions, add the above mixture dropwise to 5 mL of deionized water;

[0082] (3) Place the obtained mixture in a magnetic stirrer and stir for 15 min to obtain the initial carrier-free nanoparticles.

[0083] (4) Obtain carrier-free nanoparticles by ultrafiltration centrifugation at 10000g / 10min.

[0084] Preparation Example 3

[0085] This preparation example provides a carrier-free nanoparticle, which is prepared by the following method:

[0086] (1) Dissolve 2 mg of triptolide and 3 mg of fluconazole in 1.5 mL of methanol and mix thoroughly;

[0087] (2) Under stirring conditions, the above mixture was added dropwise to 6 mL of deionized water;

[0088] (3) The resulting mixture was placed in a magnetic stirrer and stirred for 25 min to obtain the initial carrier-free nanoparticles;

[0089] (4) Obtain carrier-free nanoparticles by ultrafiltration centrifugation at 8000g / 15min.

[0090] Preparation Example 4

[0091] This preparation example provides a carrier-free nanoparticle, which is prepared by the following method:

[0092] (1) Dissolve 2 mg of triptolide and 5 mg of amphotericin B in 2 mL of dimethyl sulfoxide and mix thoroughly;

[0093] (2) Under stirring conditions, the above mixture was added dropwise to 6 mL of deionized water;

[0094] (3) The resulting mixture was placed in a magnetic stirrer and stirred for 25 min to obtain the initial carrier-free nanoparticles;

[0095] (4) Obtain carrier-free nanoparticles by ultrafiltration centrifugation at 8000g / 15min.

[0096] Preparation Example 5

[0097] This preparation example provides a composite liposome, which is prepared by the following method:

[0098] (1) Dissolve 1 mg triptolide, 2 mg amphotericin B, 3.5 mg 1,2-dioleoyl-SN-glycerol-3-phosphoethanolamine, 1.0 mg DSPE-mPEG2000 and 0.28 mg cholesterol in 2 mL of methanol, mix thoroughly in a 10 mL round-bottom flask, and evaporate to form a film in a 30 °C water bath.

[0099] (2) Add 4 mL of deionized water and hydrate for 15 min to obtain the initial complex liposomes.

[0100] (3) The initial composite liposomes were subjected to probe ultrasound (200w, 5min, ultrasound on for 3s, ultrasound off for 3s) and then extruded sequentially through 400nm and 200nm filter membranes to finally obtain composite liposomes.

[0101] Preparation Example 6

[0102] This preparation example provides a composite liposome, which is prepared by the following method:

[0103] (1) Dissolve 1.5 mg triptolide, 1 mg fluconazole, 4.5 mg 1,2-dioleoyl-SN-glycerol-3-phosphoethanolamine, 1.5 mg DSPE-mPEG2000 and 0.35 mg cholesterol in 2 mL of methanol, mix thoroughly in a 10 mL round bottom flask, and evaporate to form a film in a 30 °C water bath.

[0104] (2) Add 4 mL of deionized water and hydrate for 25 min to obtain the initial complex liposomes.

[0105] (3) The initial composite liposomes were subjected to probe ultrasound (200w, 5min, ultrasound on for 3s, ultrasound off for 3s) and then extruded sequentially through 400nm and 200nm filter membranes to finally obtain composite liposomes.

[0106] Preparation Example 7

[0107] This preparation example provides a composite liposome, which is prepared by the following method:

[0108] (1) Dissolve 2 mg triptolide, 2 mg amphotericin B, 3.5 mg DOPE, 1.0 mg DSPE-mPEG2000 and 0.28 mg cholesterol in 2 mL of methanol, place in a 10 mL round bottom flask and mix thoroughly. Then, evaporate the mixture in a 30 °C water bath to form a film.

[0109] (2) Add 4 mL of deionized water and hydrate for 15 min to obtain the initial complex liposomes.

[0110] (3) The initial composite liposomes were subjected to probe ultrasound (200w, 5min, ultrasound on for 3s, ultrasound off for 3s) and then extruded sequentially through 400nm and 200nm filter membranes to finally obtain composite liposomes.

[0111] Preparation Example 8

[0112] This preparation example provides a composite liposome, which is prepared by the following method:

[0113] (1) Dissolve 2 mg of triptolide and 2 mg of amphotericin B in 1 mL of methanol and mix thoroughly;

[0114] (2) Under stirring conditions, add the above mixture dropwise to 5 mL of deionized water;

[0115] (3) Place the obtained mixture in a magnetic stirrer and stir for 15 min to obtain the initial carrier-free nanoparticles.

[0116] (4) Dissolve 15mg DPPC and 2mg cholesterol in 3mL of CHCl3, mix thoroughly, remove CHCl3 by rotary evaporation (45℃, 15min), and then add the carrier-free nanoparticle solution obtained in step (3) and fully hydrate (25℃, 30min).

[0117] (5) Subsequently, composite liposomes were obtained by using probe ultrasound (15 min, 100 w) and gradient extrusion.

[0118] Preparation Example 9

[0119] This preparation example provides a composite liposome, which is prepared by the following method:

[0120] (1) Dissolve 1 mg of triptolide and 4 mg of fluconazole in 0.5 mL of methanol and mix thoroughly;

[0121] (2) Under stirring conditions, add the above mixture dropwise to 2.5 mL of deionized water;

[0122] (3) The resulting mixture was stirred in a magnetic stirrer for 12 min to obtain the initial carrier-free nanoparticles.

[0123] (4) Synthesis of fluorinated DSPE-PEG2000:

[0124] Weigh 35.5 mg of 4,4-trifluorobutyric acid, 71.9 mg of EDC:HCl, 28.8 mg of NHS, and 3.05 mg of DMAP, and dissolve them together in 10 mL of CHCl3 (1 mL). Stir on ice for 4 h to obtain system 1. Then, dissolve 139.5 mg of DSPE-PEG-NH2 in CHCl3 (1 mL) and add it to system 1. Stir at room temperature for 20 h to obtain system 2. After removing the organic solvent from system 2 by vacuum drying, add 5 mL of ultrapure water to obtain system 3. Dialyze system 3 in a dialysis bag for 2 days. After 2 days, freeze-dry the product in the dialysis bag to obtain powdered fluorinated DSPE-PEG2000.

[0125] (5) Dissolve 10 mg DPPC, 5 mg DOPE, 1 mg fluorinated DSPE-PEG2000, and 1 mg cholesterol in 3 mL of CHCl3. After thorough mixing, remove CHCl3 by rotary evaporation (45 °C, 20 min). Then add the carrier-free nanoparticles obtained in step 3 and hydrate thoroughly (25 °C, 15 min). Subsequently, obtain composite liposomes by ultrasonication (15 min, 150 W) and gradient extrusion.

[0126] Preparation Example 10

[0127] This preparation example provides a composite polymer nanoparticle, the preparation method of which is as follows:

[0128] (1) Dissolve 10 mg PLGA in 1 mL of dichloromethane;

[0129] (2) Dissolve 0.5 mg triptolide and 5 mg amphotericin B in dichloromethane and add dropwise to 3 mL of deionized water containing 5% Tween-80;

[0130] (3) Slowly drop the PLGA solution from step (1) into the aqueous solution from step (2), and sonicate it for 2 minutes using a 150W ultrasonic disruptor to obtain the initial emulsion;

[0131] (4) The initial emulsion was slowly added to deionized water containing 10 mL of 50% Tween-80, and ultrasonicated for 3 min with a 150 W ultrasonic disruptor. After mixing thoroughly for 12 h, the double emulsion was obtained.

[0132] (5) Remove dichloromethane from the complex emulsion by rotary evaporation to obtain the composite polymer nanoparticles (AmB-CST NPs).

[0133] Test Example 1

[0134] The hydrated particle size and potential of the carrier-free nanoparticles prepared in Example 1 were determined using a dynamic light scattering instrument (Zetasizer NanoZS). The results are as follows: Figure 1 and Figure 2 As shown, the hydrated particle size of the carrier-free nanoparticles is around 120 nm, the polydispersity index is 0.15, and the surface charge is -47.5 mV.

[0135] Test Example 2

[0136] The carrier-free nanoparticles prepared in Example 2 were added to PBS solution containing 10% FBS or ultrapure water, and the hydration particle size and potential were measured on days 0, 1, 2, 3, 4, 5, 6, 7, and 14. Figure 3 and Figure 4 As shown, this demonstrates that carrier-free nanoparticles possess good physical stability.

[0137] Test Example 3

[0138] The encapsulation efficiency and drug loading of the carrier-free nanoparticles prepared in Example 2 were calculated. The absorption peaks of triptolide, fluconazole, and the carrier-free nanoparticles were scanned across the entire wavelength range using a UV spectrophotometer. The maximum absorption peaks of triptolide, fluconazole, and the carrier-free nanoparticles were 423 nm, 251 nm, and 425 nm, respectively. Fluconazole concentration was determined by liquid chromatography, and triptolide concentration was determined by UV-Vis absorption spectroscopy. The contents of triptolide and fluconazole in the carrier-free nanoparticles were then measured, and the encapsulation efficiency and drug loading were calculated. The encapsulation efficiency of triptolide was 87.8%, and the drug loading was 72.7%. The encapsulation efficiency of fluconazole was 83.6%, and the drug loading was 27.3%.

[0139] Test Example 4

[0140] The in vitro release capacity of the carrier-free nanoparticles prepared in Preparation Example 2 under a slightly acidic environment was evaluated. 1 mL of the carrier-free nanoparticles was added to a dialysis bag with a molecular weight of 3500. The dialysis bag was placed in 50 mL of 0.1 M PBS solution (containing 0.5% Tween 80) at pH 5.5 and pH 7.0, respectively. 1.5 mL of PBS solution was taken at different time points, and the absorbance at 423 nm was measured using UV-Vis absorption spectroscopy to determine the concentration of triptolide. The results are as follows: Figure 5 As shown, carrier-free nanoparticles can be rapidly released in acidic buffer solution.

[0141] Test Example 5

[0142] The antibacterial effects of the products obtained in Preparation Example 3, Preparation Example 4, and Preparation Example 10 were evaluated.

[0143] The method described in Example 1 was used to test the minimum inhibitory concentrations (MICs) of the carrier-free nanoparticles prepared in Example 3 (denoted as Flu-CSTNPs), the carrier-free nanoparticles prepared in Example 4 (denoted as AmB-CST NPs), the composite polymer nanoparticles prepared in Example 10 (denoted as AmB-CST NPs), and free triptolide (CST), fluconazole (Flu), and amphotericin B (AmB) against Aspergillus fumigatus. The concentrations of Flu-CST NPs used were expressed as Flu concentrations, and the concentrations of AmB-CST NPs used were expressed as AmB concentrations. The results are shown in Table 1.

[0144] Table 1

[0145] Group Minimum inhibitory concentration (μg / mL) Flu-CST NPs 0.5 AmB-CST NPs (Preparation Example 4) 0.25 AmB-CST NPs (Preparation Example 10) 0.25 CST 4 Flu 32 AmB 0.5

[0146] As shown in Table 1, compared with antibiotics alone or triptolide alone, carrier-free nanoparticles or composite polymer nanoparticles prepared from antibiotics and triptolide can significantly reduce the minimum inhibitory concentration (MIC) of antibiotics, effectively broaden the therapeutic window of drugs, and improve the safety of treatment.

[0147] In addition, the effects of the composite liposomes (denoted as Flu-CST NPs), triptolide (CST), and fluconazole (Flu) prepared in Example 8 on the cell membranes of Aspergillus fumigatus and its drug-resistant strains were verified by crystal violet biofilm staining experiments:

[0148] (1) Prepare SDA medium, add 50 mL of chloramphenicol aqueous solution (2 mg / mL), adjust the pH to 7.0, bring the volume to 1000 mL, autoclave, and store at 4℃. Activate the test strain of non-drug-resistant Aspergillus fumigatus and its clinically resistant strain on SDA medium plates to induce the formation of conidia and sporangiospores.

[0149] (2) Prepare a bacterial suspension using 0.85% NaCl solution (containing 0.01 mL Tween 20). After allowing the bacterial suspension to stand for 10 min, take the upper homogeneous liquid containing sporangiospores or conidia and hyphal fragments, and adjust the concentration to 1×10⁻⁶ using RPMI 1640 culture medium. 4 CFU / mL.

[0150] (3) Then, 1 mL of bacterial suspension was inoculated into a 24-well plate, and PBS, Flu (final concentration of 0.25 μg / mL), CST (final concentration of 0.75 μg / mL), and Flu-CST NPs (final concentration of 0.25 μg / mL based on Flu) were added respectively, and the plate was incubated at 37°C for 24 h.

[0151] (4) Remove the culture medium and wash three times with PBS. Fix the biofilm adhering to each well with 200 μL methanol at 25°C for 15 min, then dry at 25°C for 30 min. Stain each well with 1 mL of 0.1% crystal violet solution for 20 min. Wash thoroughly to remove unbound crystal violet solution. Then add 1 mL of 95% ethanol solution to each well. Measure A using a microtiter microplate reader. 600 The absorbance value at nm was used to determine the content of the biofilm.

[0152] The results are as follows Figure 6 As shown, compared with Flu and CST, Flu-CST NPs have a more significant effect in inhibiting fungal biofilm formation.

[0153] Test Example 6

[0154] The biosafety of the carrier-free nanoparticles prepared in Preparation Example 3 and Preparation Example 4 was evaluated.

[0155] Fresh blood was extracted from female Balb / c nude mice, gently mixed with PBS, centrifuged at 3000 rpm for 10 min, and washed until the supernatant was colorless. The lower precipitate was diluted with PBS to obtain a red blood cell (RBC) suspension. The carrier-free nanoparticles prepared in Example 3 (denoted as Flu-CST NPs), the carrier-free nanoparticles prepared in Example 4 (denoted as AmB-CST NPs), triptolide (CST), Tween 80, and the RBC suspension were incubated at 37°C for 4 h, followed by centrifugation at 3000 rpm for 10 min. The absorbance of the supernatant at 541 nm was measured using a microplate reader, and the hemolysis rate was calculated. The results are as follows: Figure 7 As shown, the carrier-free nanoparticles involved in this invention have good blood compatibility.

[0156] Test Example 7

[0157] The hydrated particle size and potential of the composite liposomes prepared in Example 5 were determined using a dynamic light scattering instrument (Zetasizer NanoZS). The results are as follows: Figure 8 and Figure 9 As shown, the hydrated particle size of this composite liposome is around 113 nm, the polydispersity index is 0.18, and the surface charge is -32.7 mV.

[0158] Test Example 8

[0159] The composite liposomes prepared in Example 7 were added to PBS solution containing 10% FBS or ultrapure water, and the hydration particle size and potential were measured on days 0, 1, 2, 3, 4, 5, 6, 7, and 14. Figure 10 and Figure 11 As shown, this indicates that the composite liposomes have good physical stability.

[0160] Test Example 9

[0161] The encapsulation efficiency and drug loading rate of the composite liposomes prepared in Preparation Example 7 were calculated. The absorption peaks of triptolide, amphotericin B, and the composite liposomes were scanned across the entire wavelength range using a UV spectrophotometer. The maximum absorption peaks of triptolide, amphotericin B, and the carrier-free nanoparticles were 423 nm, 405 nm, and 425 nm, respectively. The concentration of amphotericin B was determined by liquid chromatography, and the concentration of triptolide was determined by UV-Vis absorption spectroscopy. Subsequently, the contents of triptolide and amphotericin B in the composite liposomes were measured, and the encapsulation efficiency and drug loading rate were calculated. It was found that the encapsulation efficiency of triptolide was 80.8%, and the drug loading rate was 52.3%. The encapsulation efficiency of amphotericin B was 43.4%, and the drug loading rate was 17.9%.

[0162] Test Case 10

[0163] The in vitro release capacity of the composite liposomes prepared in Example 7 under slightly acidic conditions was evaluated. 1 mL of the composite liposomes was added to a dialysis bag with a molecular weight of 3500. The dialysis bag was placed in 50 mL of 0.1 M PBS solution (containing 0.5% Tween 80) at pH 5.5 and pH 7.0, respectively. At different time points, 1.5 mL of PBS solution was collected, and the absorbance at 423 nm was measured using UV-Vis absorption spectroscopy to determine the concentration of triptolide. The results are as follows: Figure 12 As shown, the complex liposomes can be rapidly released in acidic buffer solution.

[0164] Test Example 11

[0165] The antibacterial effects of the products obtained in Preparation Example 6, Preparation Example 7, and Preparation Example 10 were evaluated.

[0166] The minimum inhibitory concentrations (MICs) of the composite liposomes prepared in Example 6 (denoted as Flu-CST Lips), the composite liposomes prepared in Example 7 (denoted as AmB-CST Lips), the composite polymer nanoparticles prepared in Example 10 (denoted as AmB-CST NPs), free triptolide (CST), fluconazole (Flu), and amphotericin B (AmB) against Aspergillus fumigatus were tested using the method described in Example 1. The concentration of Flu-CST Lips was expressed as Flu concentration, and the concentrations of AmB-CST Lips and AmB-CST NPs were expressed as AmB concentration. The results are shown in Table 2.

[0167] Table 2

[0168] Group Minimum inhibitory concentration (μg / mL) Flu-CST Lips 0.25 AmB-CST Lips 0.125 AmB-CST NPs 0.5 CST 4 Flu 32 AmB 0.5

[0169] As shown in Table 2, compared with antibiotics alone or triptolide alone, composite liposomes or composite polymer nanoparticles prepared from antibiotics and triptolide can significantly reduce the minimum inhibitory concentration (MIC) of antibiotics, and the composite liposomes are more effective. This effectively broadens the therapeutic window of the drug and improves the safety of treatment.

[0170] Test Example 12

[0171] Inject 10 μL 2×10 8 After infecting *Hemiberlesia lataniae* larvae with *Fusarium* and *Aspergillus fumigatus* CFU, they were injected with 10 μL PBS, 10 mg / mL CST, 30 mg / mL Flu, and Flu-CST NPs (preparation example 9, actual Flu concentration was 30 mg / mL). They were then placed in a 37°C incubator, and the survival status of the larvae was recorded every 8 hours for 48 hours. Figure 13 As shown, the survival rate of *C. thunbergii* larvae inoculated with *Fusarium* and *Aspergillus fumigatus* was less than 40% within 48 hours in the experimental groups treated with CST or Flu alone, indicating that CST or Flu alone is not effective in treating fungal infections. After combined treatment, the survival rate of *C. thunbergii*-infected *C. thunbergii* larvae reached 90%, and the survival rate of *C. thunbergii*-infected *C. thunbergii* larvae reached 80%, demonstrating that the combined nanoparticle composition involved in this invention can effectively treat fungal infections and significantly improve the survival rate of *C. thunbergii* larvae.

[0172] The applicant declares that the technical solution of this invention is illustrated by the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the products of this invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

[0173] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0174] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. The application of a combination of nanoparticles in the preparation of drugs for antifungal and antibacterial infections, characterized in that, The active components of the combined composition are an antibiotic and an antibiotic adjuvant; the antibiotic adjuvant is triptolide or its pharmaceutically acceptable salt.

2. The application according to claim 1, characterized in that, The fungi include any one or a combination of at least two of the following: Candida albicans, Candida glabrata, Candida tropicalis, Candida krusei, Aspergillus fumigatus, Aspergillus niger, Aspergillus flavus, Aspergillus oryzae, Fusarium, Cryptococcus neoformans, Candida albicans, Mucor, Histoplasma capsulatum, Cetacea, Coccidioides, Cryptococcus greatus, or Paracoccidioides. The bacteria include any one or a combination of at least two of the following: Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, Escherichia coli, Salmonella, Shigella, Streptococcus pneumoniae, Staphylococcus aureus, Mycobacterium tuberculosis, or Clostridium difficile.

3. The application according to claim 1, characterized in that, The combination composition nanoparticles include carrier-free nanoparticles formed from antibiotics and antibiotic adjuvants; the antibiotic adjuvant is triptolide or a pharmaceutically acceptable salt thereof. Preferably, the antibiotic is selected from any one or a combination of at least two of the following: amphotericin B, nystatin, miconazole, ketoconazole, fluconazole, itraconazole, voriconazole, posaconazole, flucytosine, terbinafine, caspofungin, micafungin, anidoxuridine, griseofulvin, penicillin, amoxicillin, ampicillin, cefadroxil, cephalexin, ceftazidime, imipenem, amikacin, gentamicin, doxycycline, tetracycline, minocycline, erythromycin, clarithromycin, azithromycin, sulfadiazine, ciprofloxacin, levofloxacin, metronidazole, clindamycin, or fosfomycin. Preferably, the molar ratio of the antibiotic to the antibiotic adjuvant is 1:(0.001-500).

4. The application according to claim 3, characterized in that, The carrier-free nanoparticles are prepared by a method comprising the following steps: (1) Dissolve triptolide or its pharmaceutically acceptable salt and hydrophobic antibiotic in a good solvent to obtain solution A; (2) Add solution A to triptolide or its pharmaceutically acceptable salts and hydrophobic antibiotics in a poor solvent to obtain solution B; (3) After stirring solution B, the free drug is separated to obtain carrier-free nanoparticles.

5. The application according to claim 4, characterized in that, The good solvent includes any one or a combination of at least two of methanol, ethanol, propanol, isopropanol, acetone, tetrahydrofuran, or dimethyl sulfoxide; Preferably, the unsuitable solvent includes deionized water and / or phosphate buffer; Preferably, the stirring time is 5-60 minutes; Preferably, the separation of free drugs is performed by ultrafiltration centrifugation, with the conditions being a rotation speed of 8000-10000g and a duration of 10-30min.

6. The application according to claim 1, characterized in that, The combined nanoparticles comprise a complex liposome prepared from antibiotics, antibiotic adjuvants, lipids, and cholesterol; the antibiotic adjuvant is triptolide or its pharmaceutically acceptable salt. Preferably, the antibiotic is selected from any one or a combination of at least two of the following: amphotericin B, nystatin, miconazole, ketoconazole, fluconazole, itraconazole, voriconazole, posaconazole, flucytosine, terbinafine, caspofungin, micafungin, anidoxuridine, griseofulvin, penicillin, amoxicillin, ampicillin, cefadroxil, cephalexin, ceftazidime, imipenem, amikacin, gentamicin, doxycycline, tetracycline, minocycline, erythromycin, clarithromycin, azithromycin, sulfadiazine, ciprofloxacin, levofloxacin, metronidazole, clindamycin, or fosfomycin. Preferably, the molar ratio of the antibiotic to the antibiotic adjuvant is 1:(0.001-500); Preferably, the lipid comprises any one or a combination of at least two of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidic acid, cardiolipin, sphingomyelin, phosphatidylserine, DSPE-PEG, or fluorinated DSPE-PEG.

7. The application according to claim 6, characterized in that, The composite liposomes are prepared by a method comprising the following steps: (1) Mix the first solvent, triptolide or its pharmaceutically acceptable salt, hydrophobic antibiotic, phospholipid, and cholesterol to obtain solution A; mix the second solvent and hydrophilic antibiotic to obtain solution B; (2) Remove the first solvent from solution A, mix it with solution B and hydrate to obtain solution C; (3) Solution C was subjected to ultrasonication and gradient extrusion to obtain the composite liposomes; Preferably, the first solvent comprises any one or a combination of at least two of methanol, ethanol, propanol, or chloroform; the second solvent comprises deionized water and / or phosphate buffer. Preferably, the hydration time is 10-60 minutes and the temperature is 15-35°C; Preferably, the pore sizes of the gradient-extruded filter membrane are 400 nm and 200 nm, respectively; Preferably, the ultrasonic treatment in step (3) is performed at 0-10℃, the ultrasonic power is 80-250W, and the ultrasonic time is 3-30min.

8. The application according to claim 6, characterized in that, The composite liposomes are prepared by a method comprising the following steps: (1) Mix the first solvent, phospholipids, and cholesterol to obtain solution A; mix triptolide or its pharmaceutically acceptable salt, antibiotics, and organic solvents, and add them dropwise to the second solvent to obtain solution B; (2) Remove the first solvent from solution A, mix it with solution B and hydrate to obtain solution C; (3) Solution C was subjected to ultrasonication and gradient extrusion to obtain the composite liposomes; Preferably, the first solvent comprises any one or a combination of at least two of methanol, ethanol, propanol, or chloroform; the organic solvent comprises any one or a combination of at least two of methanol, ethanol, DMF, or DMSO; and the second solvent comprises deionized water and / or phosphate buffer. Preferably, the hydration time is 10-30 minutes and the temperature is 15-35°C; Preferably, the ultrasonic treatment in step (3) is performed at 0-10°C, the ultrasonic power is 80-250W, and the ultrasonic time is 3-30min; Preferably, the pore sizes of the gradient extruded filter membrane are 400 nm and 200 nm, respectively.

9. The application according to claim 1, characterized in that, The combined nanoparticle composition comprises composite polymer nanoparticles prepared from antibiotics, antibiotic adjuvants, and pharmaceutically biodegradable polymers; the antibiotic adjuvant is triptolide or its pharmaceutically acceptable salt. Preferably, the antibiotic is selected from any one or a combination of at least two of the following: amphotericin B, nystatin, miconazole, ketoconazole, fluconazole, itraconazole, voriconazole, posaconazole, flucytosine, terbinafine, caspofungin, micafungin, anidoxuridine, griseofulvin, penicillin, amoxicillin, ampicillin, cefadroxil, cephalexin, ceftazidime, imipenem, amikacin, gentamicin, doxycycline, tetracycline, minocycline, erythromycin, clarithromycin, azithromycin, sulfadiazine, ciprofloxacin, levofloxacin, metronidazole, clindamycin, or fosfomycin. Preferably, the molar ratio of the antibiotic to the antibiotic adjuvant is 1:(0.001-500); Preferably, the biodegradable pharmaceutical polymer includes any one or a combination of at least two of PLGA, PEG, mPEG-PLGA, or DSPE-PEG.

10. The application according to claim 9, characterized in that, The composite polymer nanoparticles are prepared by a method comprising the following steps: (1) Mix the first solvent and the biodegradable pharmaceutical polymer to obtain solution A; mix triptolide or its pharmaceutically acceptable salt, antibiotic and organic solvent, and add dropwise to the second solvent containing emulsifier to obtain solution B; (2) Mix solution A with solution B to obtain solution C; (3) Add solution C to a third solvent containing an emulsifier, mix, and obtain solution D; (4) Remove the first solvent remaining in solution D to obtain the composite polymer nanoparticles; Preferably, the first solvent comprises any one or a combination of at least two of methanol, ethanol, propanol, or dichloromethane; the organic solvent comprises any one or a combination of at least two of methanol, ethanol, DMF, or DMSO; the second solvent comprises deionized water and / or phosphate buffer; and the third solvent comprises deionized water and / or phosphate buffer. Preferably, each group of emulsifiers in the second solvent and the third solvent independently includes any one or a combination of at least two of polyvinyl alcohol, Tween-80 or vitamin E polyethylene glycol succinate; Preferably, the mass fraction of the emulsifier in the second solvent is 3%-15%; and the mass fraction of the emulsifier in the third solvent is 30%-60%.

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