Antibiotic-loaded nanoemulsions, methods of making and using the same

Abstract

The application provides a kind of antibiotic-loaded nanoemulsion and its preparation method and application, it is related to the technical field of medical adjuvant, antibiotic is mixed with medicinal adjuvant soybean oil, dipalmitoyl phosphatidylcholine and cholesterol, and volatile organic solvent is used to dissolve and form oil phase;Deoxycholic acid sodium and tween are dissolved in sterile liquid to form water phase;Oil phase and water phase are mixed, and are treated by ultrasonic treatment, rotary evaporation treatment and high pressure homogenization treatment, to obtain antibiotic-loaded nanoemulsion.The application adjusts the type of inhalable medicinal adjuvant, the addition ratio of inhalable medicinal adjuvant, the ratio of oil phase and water phase, and the ultrasonic power and homogenization pressure and other factors, so that the prepared nanoemulsion can efficiently load antibiotic, and has good aerosol inhalation performance, better drug stability, the nanoemulsion can be retained in mouse lung tissue for a long time, and the lung drug delivery efficiency is significantly improved.

Description

Technical Field

[0001] This application relates to the field of medical excipients technology, and in particular to an antibiotic-loaded nanoemulsion, its preparation method, and its application. Background Technology

[0002] Currently, there is a severe shortage of inhaled antibiotics for clinical use. In China, only tobramycin inhalation solution is approved for clinical application, and several antibiotics, such as rifampin and polymyxin, have been used off-label via inhalation. Therefore, there is an urgent need to develop antibiotic formulations specifically for inhalation.

[0003] Current antibiotic inhalation methods primarily utilize solution-based antibiotics, with the only commercially available inhaled nano-formulation being amikacin liposome suspension. However, solution-based inhaled antibiotics suffer from drawbacks such as rapid blood entry, low pulmonary retention, relatively high dosage, and insufficient antibacterial efficacy.

[0004] Therefore, there is an urgent need for a new inhaled nano-formulation loaded with antibiotics. Summary of the Invention

[0005] This application provides a nanoemulsion loaded with antibiotics, its preparation method, and its application, in order to solve the problems of existing inhaled antibiotics, such as rapid blood entry, low pulmonary retention, large dosage, and insufficient antibacterial efficiency.

[0006] In a first aspect of this application, a method for preparing an antibiotic-loaded nanoemulsion is provided, the method comprising the following steps:

[0007] Step 1: Mix antibiotics, soybean oil, dipalmitoylphosphatidylcholine and cholesterol evenly to obtain a mixture, and dissolve the mixture in a volatile organic solvent to prepare an oil phase. In the oil phase, add 0.1 to 20 mg of antibiotics, 0.1 to 100 mg of soybean oil, 1 to 100 mg of dipalmitoylphosphatidylcholine and 0.1 to 3 mg of cholesterol to each 1 ml of volatile organic solvent.

[0008] Step 2: Add the sodium organic acid salt and Tween to the sterile liquid and stir until the sodium organic acid salt and Tween dissolve to prepare an aqueous phase. In the aqueous phase, add 0.1 to 4 mg of the sodium organic acid salt and 0.1 to 5 mg of Tween to every 1 ml of sterile liquid.

[0009] Step 3: After the oil phase and the aqueous phase are mixed evenly, the mixture is ultrasonically treated to obtain a first emulsion. The volatile organic solvent in the first emulsion is removed by rotary evaporation to obtain a second emulsion. The mixing volume ratio of the volatile organic solvent in the oil phase to the sterile liquid in the aqueous phase is 1 to 8:1.

[0010] Step 4: The second emulsion is subjected to high-pressure homogenization to obtain an antibiotic-loaded nanoemulsion.

[0011] Optionally, in step 1, the volatile organic solvent is at least one of dichloromethane, ethyl acetate, diethyl ether, and chloroform.

[0012] Optionally, in step 1, the antibiotic is at least one of rifampin, polymyxin, and vancomycin.

[0013] Optionally, in step 1, the sterile liquid is at least one of water, physiological saline, deionized water, phosphate buffer, deoxycholic acid, citrate buffer, and Tween solution.

[0014] Optionally, in step 2, the organic acid sodium salt is at least one of sodium citrate and sodium deoxycholate.

[0015] Optionally, in step 3, the power of the ultrasonic treatment is 30 watts to 400 watts, and the duration of the ultrasonic treatment is 20 seconds to 20 minutes.

[0016] Optionally, in step 3, the temperature for rotary evaporation to remove the volatile organic solvents from the first emulsion is 37°C to 60°C, and the time for rotary evaporation to remove the volatile organic solvents from the first emulsion is 5 minutes to 10 minutes.

[0017] Optionally, in step 4, the pressure of the high-pressure homogenization process is 200 bar to 1000 bar, and the time of the high-pressure homogenization process is 2 minutes to 20 minutes.

[0018] In a second aspect of this application, an antibiotic-loaded nanoemulsion is provided, which is prepared by any one of the preparation methods described in the first aspect above.

[0019] In a third aspect of this application, an antibiotic-loaded nanoemulsion as described in the second aspect above is provided for use in the preparation of an inhaled antibiotic drug.

[0020] This application includes the following advantages: This application proposes an antibiotic-loaded nanoemulsion, its preparation method, and its application. The method includes the following steps: mixing antibiotics, soybean oil, dipalmitoylphosphatidylcholine, and cholesterol evenly to obtain a mixture, and dissolving the mixture in a volatile organic solvent to prepare an oil phase. In the oil phase, 0.1 to 20 mg of antibiotics, 0.1 to 100 mg of soybean oil, 1 to 100 mg of dipalmitoylphosphatidylcholine, and 0.1 to 3 mg of cholesterol are added to every 1 ml of volatile organic solvent; sodium salt of organic acid and Tween are added... In a sterile liquid, the sodium salt of the organic acid and the Tween are stirred until dissolved to prepare an aqueous phase. In the aqueous phase, 0.1 to 4 mg of the sodium salt of the organic acid and 0.1 to 5 mg of Tween are added to every 1 mL of sterile liquid. After the oil phase and the aqueous phase are mixed evenly, the mixture is ultrasonically treated to obtain a first emulsion. The volatile organic solvent in the first emulsion is removed by rotary evaporation to obtain a second emulsion. The mixing volume ratio of the volatile organic solvent in the oil phase to the sterile liquid in the aqueous phase is 1 to 8:1. The second emulsion is subjected to high-pressure homogenization to obtain an antibiotic-loaded nanoemulsion.

[0021] This application achieves a nanoemulsion by specifically adjusting the type and proportion of inhalable pharmaceutical excipients, the ratio of oil and water phases, and the interaction of various factors such as ultrasonic power and homogenization pressure. This allows the prepared nanoemulsion to efficiently load antibiotics and exhibits good nebulization performance and drug stability. Compared with inhaled antibiotic solution formulations, the nanoemulsion can remain in mouse lung tissue for a longer period of time, significantly improving the efficiency of drug delivery to the lungs. Attached Figure Description

[0022] To more clearly illustrate the technical solutions of the embodiments of this application, the drawings used in the description of the embodiments of this application will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 This is a transmission electron microscope (TEM) image of a rifampicin-loaded nanoemulsion provided in an embodiment of this application.

[0024] Figure 2 This is a transmission electron microscope (TEM) image of a polymyxin-loaded nanoemulsion provided in an embodiment of this application.

[0025] Figure 3 This is a transmission electron microscope (TEM) image of a vancomycin-loaded nanoemulsion provided in an embodiment of this application.

[0026] Figure 4This application provides a quantitative detection image of lung tissue in mice 48 hours after inhalation of rifampicin nanoemulsion. Detailed Implementation

[0027] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0028] Currently, there is a severe shortage of inhaled antibiotics for clinical use. In China, only tobramycin inhalation solution is approved for clinical application, and several antibiotics, such as rifampin and polymyxin, have been used off-label via inhalation. Therefore, there is an urgent need to develop antibiotic formulations specifically for inhalation.

[0029] Current antibiotic inhalation methods primarily utilize solution-based antibiotics, with the only commercially available inhaled nano-formulation being amikacin liposome suspension. However, solution-based inhaled antibiotics suffer from drawbacks such as rapid blood entry, low pulmonary retention, relatively high dosage, and insufficient antibacterial efficacy.

[0030] Based on this, this application proposes a method for preparing antibiotic-loaded nanoemulsions, which utilizes various inhalable pharmaceutical excipients, such as soybean oil, dipalmitoylphosphatidylcholine (DPPC), cholesterol, phospholipids, etc., to prepare antibiotic-loaded nanoemulsions.

[0031] The first aspect of this application provides a method for preparing an antibiotic-loaded nanoemulsion, the method comprising the following steps:

[0032] Step 1: Mix antibiotics, soybean oil, dipalmitoylphosphatidylcholine and cholesterol evenly to obtain a mixture, and dissolve the mixture in a volatile organic solvent to prepare an oil phase. In the oil phase, add 0.1 to 20 mg of antibiotics, 0.1 to 100 mg of soybean oil, 1 to 100 mg of dipalmitoylphosphatidylcholine and 0.1 to 3 mg of cholesterol to each 1 ml of volatile organic solvent.

[0033] Step 2: Add the sodium organic acid salt and Tween to the sterile liquid and stir until the sodium organic acid salt and Tween dissolve to prepare an aqueous phase. In the aqueous phase, add 0.1 to 4 mg of the sodium organic acid salt and 0.1 to 5 mg of Tween to every 1 ml of sterile liquid.

[0034] Step 3: After the oil phase and the aqueous phase are mixed evenly, the mixture is ultrasonically treated to obtain a first emulsion. The volatile organic solvent in the first emulsion is removed by rotary evaporation to obtain a second emulsion. The mixing volume ratio of the volatile organic solvent in the oil phase to the sterile liquid in the aqueous phase is 1 to 8:1.

[0035] Step 4: The second emulsion is subjected to high-pressure homogenization to obtain an antibiotic-loaded nanoemulsion.

[0036] In specific implementation step 1, to prepare a highly efficient antibiotic-loaded nanoemulsion, the preparation method proposed in this application first prepares an oil phase containing antibiotics. Lipid-soluble antibiotics are added to the oil phase to promote the binding of antibiotics with the oil phase, enhancing the stability and solubility of the antibiotics, thereby achieving highly efficient antibiotic loading in the nanoemulsion. Soybean oil in the oil phase has good solubility and can effectively dissolve lipid-soluble substances, achieving highly efficient antibiotic loading. Simultaneously, soybean oil can also act as a carrier for antibiotics, facilitating their delivery and release after nebulization and inhalation. Dipalmitoylphosphatidylcholine, as an emulsifier, has good emulsifying properties and can form a stable interfacial film between the oil and aqueous phases, thereby preventing oil droplet aggregation and promoting nanoemulsion formation. It also enhances the physical stability of the nanoemulsion, preventing stratification or demulsification during storage and use. Cholesterol can improve the biocompatibility of the nanoemulsion, enabling it to better interact with cell membranes in vivo and improving drug delivery. Cellular uptake further influences the metabolic pathways and clearance rates of nanoemulsions in vivo, thereby regulating the distribution and residence time of antibiotics and improving drug bioavailability and therapeutic efficacy. Volatile organic solvents can regulate the physical properties of the oil phase, such as viscosity and surface tension. Simultaneously, during emulsification, the volatility of the solvent can affect the stability of the emulsion and droplet formation, helping to form smaller, more uniform droplets and improving the stability and performance of the nanoemulsion. Furthermore, the interaction between soybean oil and the emulsifiers dipalmitoylphosphatidylcholine and cholesterol can enhance the stability of the nanoemulsion and reduce antibiotic leakage and degradation. Soybean oil, dipalmitoylphosphatidylcholine, and cholesterol each play important roles in the oil phase of the nanoemulsion, jointly promoting its formation, stability, and performance enhancement.

[0037] In one optional embodiment of this application, the volatile organic solvent used in step 1 above can be at least one of dichloromethane, ethyl acetate, diethyl ether, and chloroform. Because dichloromethane has strong dissolving power, is easily volatile, has stable physicochemical properties, and a wide range of applications, the volatile organic solvent is preferably dichloromethane.

[0038] In one optional embodiment of this application, the Tween used in step 1 is at least one of Tween 20 and Tween 80, preferably, the Tween used in step 1 is Tween 80.

[0039] In one optional embodiment of this application, the antibiotic used in step 1 is preferably a lipid-soluble antibiotic, i.e. a hydrophobic antibiotic, which may be selected from at least one of rifampin, polymyxin and vancomycin, or other hydrophobic antibiotics with equivalent efficacy.

[0040] In specific implementation step 2, an aqueous phase is prepared by adding sodium organic acid salt and Tween to a sterile liquid. The sodium organic acid salt in the aqueous phase regulates the pH value, maintaining the nanoemulsion within a suitable range to ensure emulsion stability and the effectiveness of active ingredients. Simultaneously, the sodium organic acid salt acts as a co-solvent, increasing the solubility of active ingredients in the aqueous phase, making them easier to emulsify and disperse in the nanoemulsion. Furthermore, as an electrolyte, the sodium organic acid salt affects the double-layer structure and charge distribution in the emulsion system, thereby influencing the emulsion's stability and particle size distribution. An appropriate amount of electrolyte contributes to the formation of a more stable nanoemulsion. Tween possesses excellent emulsifying properties, significantly reducing the interfacial tension between the oil and aqueous phases, promoting their mixing and emulsification, and forming stable nanoemulsions. Tween can adsorb onto the surfaces of oil and water droplets, forming a protective film to prevent aggregation and stratification, thereby improving the stability of the nanoemulsion. Furthermore, Tween can increase the solubility of antibiotics or active ingredients in the aqueous phase, making them easier to emulsify and disperse in the nanoemulsion, thus increasing their loading capacity. Therefore, sodium organic acid salts and Tween each play important roles in the aqueous phase of nanoemulsions, jointly promoting their formation, stability, and performance improvement.

[0041] In one optional embodiment of this application, the sterile liquid used in step 2 above may be at least one of water, physiological saline, deionized water, phosphate buffer, deoxycholic acid, citrate buffer and Tween aqueous solution, wherein each 1 ml of sterile liquid in the Tween aqueous solution contains 0.1 to 5 mg of Tween.

[0042] In one optional embodiment of this application, the sodium organic acid salt used in step 2 is at least one of sodium citrate and sodium deoxycholate.

[0043] In specific implementation step 3, after obtaining the oil phase and aqueous phase containing antibiotics respectively, the oil phase and aqueous phase containing antibiotics are mixed. To ensure thorough mixing of the oil and aqueous phases, considering that ultrasonic treatment has an effective emulsifying effect, can promote mixing uniformity, improve emulsification efficiency, and improve the stability of the emulsion, ultrasonic treatment is used to mix them, resulting in the first emulsion. Considering environmental protection and safety, after mixing, volatile organic solvents are removed by rotary evaporation, resulting in the second emulsion. At this point, the particles of the second emulsion mixed by ultrasonic treatment are relatively large and the particle size distribution is not very uniform. After obtaining the second emulsion, it needs to be further homogenized.

[0044] In one optional embodiment of this application, in step 3, the volume ratio of the volatile organic solvent in the oil phase to the sterile liquid in the aqueous phase is 1 to 8:1.

[0045] In a preferred embodiment of this application, the volume ratio of the volatile organic solvent in the oil phase to the sterile liquid in the aqueous phase is preferably 2 to 6:1.

[0046] In one optional embodiment of this application, the ultrasonic treatment power in step 3 is 30 watts to 400 watts. Since the ultrasonic treatment power is a key factor affecting the nanoemulsion formation effect, preferably, the ultrasonic treatment power in step 3 is 100 watts to 400 watts. Furthermore, to ensure uniform mixing of the oil and water phases before ultrasonic treatment, a vortex treatment is used to mix the oil and water phases uniformly, and the vortex treatment time is 1 minute to 10 minutes.

[0047] In one optional embodiment of this application, the duration of the ultrasonic treatment in step 3 is 20 seconds to 20 minutes.

[0048] In one optional embodiment of this application, in step 3, the temperature for removing the volatile organic solvent from the first emulsion by rotary evaporation is 37 degrees Celsius to 60 degrees Celsius, and the time for removing the volatile organic solvent from the first emulsion by rotary evaporation is 5 minutes to 10 minutes.

[0049] In specific implementation step 4, to further improve the stability and homogeneity of the second emulsion and obtain a nanoemulsion with more uniform and smaller particle size, the second emulsion obtained in step 3 is subjected to high-pressure homogenization to obtain an antibiotic-loaded nanoemulsion. This further high-pressure homogenization breaks down large particles that may not have been fully dispersed during ultrasonic treatment, resulting in higher homogeneity of the emulsion. This significantly improves the uniformity of the emulsion particle size and allows for the preparation of an emulsion with nano-sized particles. The smaller particle size distribution helps reduce emulsion stratification and sedimentation, improving the stability of the nanoemulsion. Simultaneously, high pressure kills microorganisms, improving the safety and extending the shelf life of the nanoemulsion while retaining the active ingredients and enhancing its functionality. Furthermore, compared to solvent-based nanoemulsions, the antibiotic-loaded nanoemulsion prepared in this application provides better nebulization during inhalation and allows for greater retention of the nanoemulsion in the lungs, increasing the inhaled antibiotic dose and the effective concentration of antibiotics in lung lesions.

[0050] In one optional embodiment of this application, in step 4, the pressure of the high-pressure homogenization process is 200 bar to 1000 bar. Since the homogenization pressure is a key factor affecting the nanoemulsion formation effect, preferably, in step 4, the homogenization pressure is 500 bar to 800 bar.

[0051] In one optional embodiment of this application, the high-pressure homogenization process in step 4 takes 2 to 20 minutes.

[0052] The method for preparing antibiotic-loaded nanoemulsions provided in the first aspect of this application involves first mixing antibiotics with inhalable pharmaceutical excipients soybean oil, DPPC, and cholesterol, and then dissolving them in a volatile organic solvent to form an oil phase. Sodium deoxycholate and Tween are then dissolved in a sterile liquid to form an aqueous phase. The oil and aqueous phases are then mixed and subjected to ultrasonic treatment, rotary evaporation, and high-pressure homogenization to obtain an antibiotic-loaded nanoemulsion. By specifically adjusting the type and proportion of inhalable pharmaceutical excipients, the ratio of oil to aqueous phases, and various factors such as ultrasonic power, homogenization pressure, and time, the prepared nanoemulsion can efficiently load antibiotics and exhibits good nebulization performance and high drug stability. Compared with inhaled antibiotic solutions, antibiotic nanoemulsions improve the penetration and retention of antibiotics in lung tissue, further enhancing their ability to combat respiratory infections. This method has a simple preparation process, employs high-pressure homogenization, and can be mass-produced.

[0053] Based on the same application concept, a second aspect of the embodiments of this application proposes an antibiotic-loaded nanoemulsion. The antibiotic-loaded nanoemulsion is prepared by the preparation method described in any one of the first aspects above.

[0054] The antibiotic-loaded nanoemulsions provided in this application, as inhalable antibiotic drugs, can enhance the stability and solubility of antibiotics and increase the effective concentration of antibiotics in lung lesions. In contrast, solution-type antibiotics have poor permeability to lung tissue mucus and are easily recognized and cleared by alveolar macrophages due to their positive charge, resulting in faster elimination. Compared to solution-type antibiotics, nanomaterials can be rationally engineered, such as through size control, surface modification, and stimulus-responsive functionalization, thereby generating a unique bactericidal mechanism with bacteria, improving anti-pathogen activity, and facilitating widespread application.

[0055] Based on the same application concept, in the third aspect of the embodiments of this application, the application of an antibiotic-loaded nanoemulsion as described in the second aspect above in the preparation of an inhaled antibiotic drug is proposed.

[0056] To clearly illustrate the antibiotic-loaded nanoemulsion, its preparation method, and its application proposed in this application, detailed descriptions will be provided below in conjunction with specific examples.

[0057] Example 1: A method for preparing an antibiotic-loaded nanoemulsion

[0058] Example 1A: Preparation method of rifampicin-loaded nanoemulsion

[0059] When the loaded antibiotic is rifampin, the preparation method of the rifampin-loaded nanoemulsion includes the following steps:

[0060] Step 1: Mix 3 mg of rifampicin, 20 mg of soybean oil, 10 mg of DPPC and 3 mg of cholesterol evenly to obtain a mixture; dissolve the mixture in 1.5 mL of dichloromethane to prepare the oil phase.

[0061] Step 2: Add 3 mg of sodium deoxycholate and 5 mg of Tween 80 to 1 mL of sterile water and stir until the sodium deoxycholate and Tween 80 dissolve to prepare an aqueous phase.

[0062] Step 3: Mix the volatile organic solvent in the oil phase and the sterile liquid in the aqueous phase at a volume ratio of 1.5:1, vortex mix for 3 minutes, and then sonicate using a cell disruptor at a power of 200 watts for 8 minutes. After sonication, the first emulsion is obtained. The volatile organic solvent in the first emulsion is removed by rotary evaporation to obtain the second emulsion. The rotary evaporation temperature is 45 degrees Celsius and the rotary evaporation time is 5 minutes.

[0063] Step 4: The second emulsion is subjected to high-pressure homogenization using a high-pressure homogenizer. The high-pressure homogenization pressure is 800 bar, the high-pressure homogenization temperature is 4 degrees Celsius, and the high-pressure homogenization time is 10 minutes to obtain a rifampicin-loaded nanoemulsion.

[0064] Example 1B: Preparation method of rifampicin-loaded nanoemulsion

[0065] When the loaded antibiotic is rifampin, the preparation method of the rifampin-loaded nanoemulsion includes the following steps:

[0066] Step 1: Mix 160 mg of rifampin, 800 mg of soybean oil, 800 mg of DPPC, and 24 mg of cholesterol evenly to obtain a mixture; dissolve the mixture in chloroform and bring the volume to 8 mL to obtain an oil phase containing 20 mg / mL rifampin, 100 mg / mL soybean oil, 100 mg / mL DPPC, and 3 mg / mL cholesterol.

[0067] Step 2: Add 4 mg of sodium deoxycholate and 5 mg of Tween to 1 mL of sterile water and stir until sodium deoxycholate and Tween dissolve to prepare an aqueous phase containing 5 mg / mL Tween and 4 mg / mL sodium deoxycholate.

[0068] Step 3: Mix the mixture in the oil phase and the sterile liquid in the aqueous phase at a volume ratio of 8:1, vortex mix for 10 minutes, and then sonicate using a cell disruptor at a power of 400 watts for 20 minutes. After sonication, the first emulsion is obtained. The volatile organic solvents in the first emulsion are removed by rotary evaporation to obtain the second emulsion. The rotary evaporation temperature is 60 degrees Celsius and the rotary evaporation time is 10 minutes.

[0069] Step 4: The second emulsion is homogenized under high pressure using a high-pressure homogenizer. The high-pressure homogenization pressure is 1000 bar, the high-pressure homogenization temperature is 4 degrees Celsius, and the high-pressure homogenization time is 20 minutes to obtain a rifampicin-loaded nanoemulsion.

[0070] Example 1C: Preparation method of rifampicin-loaded nanoemulsion

[0071] When the loaded antibiotic is rifampin, the preparation method of the rifampin-loaded nanoemulsion includes the following steps:

[0072] Step 1: Mix 5 mg of rifampicin, 5 mg of soybean oil, 50 mg of DPPC, and 5 mg of cholesterol evenly to obtain a mixture; dissolve the mixture in 50 mL of chloroform to prepare an oil phase containing 0.1 mg / mL rifampicin, 0.1 mg / mL soybean oil, 1 mg / mL DPPC, and 0.1 mg / mL cholesterol.

[0073] Step 2: Add 5 mg of sodium deoxycholate and 5 mg of Tween to 50 mL of sterile water and stir until sodium deoxycholate and Tween dissolve to prepare an aqueous phase containing 0.1 mg / mL Tween and 0.1 mg / mL sodium deoxycholate.

[0074] Step 3: Mix the volatile organic solvent in the oil phase and the sterile liquid in the aqueous phase at a volume ratio of 1:1, vortex mix for 1 minute, and then sonicate using a cell disruptor at a power of 30 watts for 20 seconds. After sonication, the first emulsion is obtained. The volatile organic solvent in the first emulsion is removed by rotary evaporation to obtain the second emulsion. The rotary evaporation temperature is 37 degrees Celsius and the rotary evaporation time is 5 minutes.

[0075] Step 4: The second emulsion is homogenized under high pressure using a high-pressure homogenizer. The high-pressure homogenization pressure is 200 bar, the high-pressure homogenization temperature is 4 degrees Celsius, and the high-pressure homogenization time is 2 minutes to obtain a rifampicin-loaded nanoemulsion.

[0076] Example 1D: Preparation method of polymyxin-loaded nanoemulsion

[0077] When the loaded antibiotic is polymyxin, the preparation method of the polymyxin-loaded nanoemulsion in this embodiment includes the following steps:

[0078] Step 1: Mix 3 mg of polymyxin, 20 mg of soybean oil, 10 mg of DPPC and 3 mg of cholesterol evenly to obtain a mixture; dissolve the mixture in 1.5 mL of dichloromethane to prepare the oil phase.

[0079] Step 2: Add 3 mg of sodium deoxycholate and 5 mg of Tween 80 to 1 mL of sterile water and stir until the sodium deoxycholate and Tween dissolve to prepare an aqueous phase.

[0080] Step 3: Mix the volatile organic solvent in the oil phase and the sterile liquid in the aqueous phase at a volume ratio of 1.5:1, vortex mix for 3 minutes, and then sonicate using a cell disruptor at a power of 200 watts for 8 minutes. After sonication, the first emulsion is obtained. The volatile organic solvent in the first emulsion is removed by rotary evaporation to obtain the second emulsion. The rotary evaporation temperature is 45 degrees Celsius and the rotary evaporation time is 5 minutes.

[0081] Step 4: The second emulsion is homogenized under high pressure using a high-pressure homogenizer. The high-pressure homogenization pressure is 800 bar, the high-pressure homogenization temperature is 4 degrees Celsius, and the high-pressure homogenization time is 10 minutes to obtain a nanoemulsion loaded with polymyxin.

[0082] Example 1E: Preparation method of vancomycin-loaded nanoemulsion

[0083] When the loaded antibiotic is vancomycin, the preparation method of the vancomycin-loaded nanoemulsion in this embodiment includes the following steps:

[0084] Step 1: Mix 3 mg of vancomycin, 20 mg of soybean oil, 10 mg of DPPC, and 3 mg of cholesterol evenly to obtain a mixture; dissolve the mixture in 1.5 mL of dichloromethane to prepare the oil phase;

[0085] Step 2: Add 3 mg of sodium deoxycholate and 5 mg of Tween 80 to 1 mL of sterile water and stir until the sodium deoxycholate and Tween dissolve to prepare an aqueous phase.

[0086] Step 3: Mix the volatile organic solvent in the oil phase and the sterile liquid in the aqueous phase at a volume ratio of 1.5:1, vortex mix for 3 minutes, and then sonicate using a cell disruptor at a power of 200 watts for 8 minutes. After sonication, the first emulsion is obtained. The volatile organic solvent in the first emulsion is removed by rotary evaporation to obtain the second emulsion. The rotary evaporation temperature is 45 degrees Celsius and the rotary evaporation time is 5 minutes.

[0087] Step 4: The second emulsion is homogenized under high pressure using a high-pressure homogenizer. The high-pressure homogenization pressure is 800 bar, the high-pressure homogenization temperature is 4 degrees Celsius, and the high-pressure homogenization time is 10 minutes to obtain a vancomycin-loaded nanoemulsion.

[0088] To illustrate the preparation method proposed in this application, by specifically adjusting the type of inhalable pharmaceutical excipients, the addition ratio of inhalable pharmaceutical excipients, the ratio of oil phase and water phase, and various factors such as ultrasonic power, homogenization pressure and time, the prepared nanoemulsion can efficiently load antibiotics and has good nebulization inhalation performance and high drug stability. The following comparative examples are listed for comparison with the antibiotic-loaded nanoemulsions prepared by the preparation method proposed in Example 1 (Examples 1A-Examples 1E).

[0089] Comparative Example 1A: Preparation method of rifampicin-loaded nanoemulsion

[0090] Compared to Example 1A, no soybean oil was added in Comparative Example 1A, but the rest of the operations were the same as in Example 1A. The specific operations are as follows:

[0091] Step 1: Mix 3 mg of rifampin, 10 mg of DPPC and 3 mg of cholesterol evenly to obtain a mixture; dissolve the mixture in 1.5 mL of dichloromethane to prepare the oil phase.

[0092] Step 2: Add 3 mg of sodium deoxycholate and 5 mg of Tween 80 to 1 mL of sterile water and stir until the sodium deoxycholate and Tween dissolve to prepare an aqueous phase.

[0093] Step 3: Mix the volatile organic solvent in the oil phase and the sterile liquid in the aqueous phase at a volume ratio of 1.5:1, vortex mix for 3 minutes, and then sonicate using a cell disruptor at a power of 200 watts for 8 minutes. After sonication, the first emulsion is obtained. The volatile organic solvent in the first emulsion is removed by rotary evaporation to obtain the second emulsion. The rotary evaporation temperature is 45 degrees Celsius and the rotary evaporation time is 5 minutes.

[0094] Step 4: The second emulsion is homogenized under high pressure using a high-pressure homogenizer. The high-pressure homogenization pressure is 800 bar, the high-pressure homogenization temperature is 4 degrees Celsius, and the high-pressure homogenization time is 10 minutes to obtain a rifampicin-loaded nanoemulsion.

[0095] Comparative Example 1B: Preparation method of rifampicin-loaded nanoemulsion

[0096] Compared to Example 1A, Comparative Example 1B does not include DPPC, but the rest of the operations are the same as in Example 1A. The specific operations are as follows:

[0097] Step 1: Mix 3 mg of rifampin, 20 mg of soybean oil and 3 mg of cholesterol evenly to obtain a mixture; dissolve the mixture in 1.5 mL of dichloromethane to prepare the oil phase.

[0098] Step 2: Add 3 mg of sodium deoxycholate and 5 mg of Tween 80 to 1 mL of sterile water and stir until the sodium deoxycholate and Tween dissolve to prepare an aqueous phase.

[0099] Step 3: Mix the volatile organic solvent in the oil phase and the sterile liquid in the aqueous phase at a volume ratio of 1.5:1, vortex mix for 3 minutes, and then sonicate using a cell disruptor at a power of 200 watts for 8 minutes. After sonication, the first emulsion is obtained. The volatile organic solvent in the first emulsion is removed by rotary evaporation to obtain the second emulsion. The rotary evaporation temperature is 45 degrees Celsius and the rotary evaporation time is 5 minutes.

[0100] Step 4: The second emulsion is homogenized under high pressure using a high-pressure homogenizer. The high-pressure homogenization pressure is 800 bar, the high-pressure homogenization temperature is 4 degrees Celsius, and the high-pressure homogenization time is 10 minutes to obtain a rifampicin-loaded nanoemulsion.

[0101] Comparative Example 1C: Preparation method of rifampicin-loaded nanoemulsion

[0102] Compared to Example 1A, Comparative Example 1C did not contain cholesterol, but all other operations were the same as in Example 1A. The specific operations are as follows:

[0103] Step 1: Mix 3 mg of rifampin, 20 mg of soybean oil and 10 mg of DPPC evenly to obtain a mixture; dissolve the mixture in 1.5 mL of dichloromethane to prepare the oil phase.

[0104] Step 2: Add 3 mg of sodium deoxycholate and 5 mg of Tween 80 to 1 mL of sterile water and stir until the sodium deoxycholate and Tween dissolve to prepare an aqueous phase.

[0105] Step 3: Mix the volatile organic solvent in the oil phase and the sterile liquid in the aqueous phase at a volume ratio of 1.5:1, vortex for 3 minutes, and then sonicate using a cell disruptor at a power of 200 watts for 8 minutes. After sonication, the first emulsion is obtained. The volatile organic solvent in the first emulsion is removed by rotary evaporation to obtain the second emulsion. The rotary evaporation temperature is 45 degrees Celsius and the rotary evaporation time is 5 minutes.

[0106] Step 4: The second emulsion is homogenized under high pressure using a high-pressure homogenizer. The high-pressure homogenization pressure is 800 bar, the high-pressure homogenization temperature is 4 degrees Celsius, and the high-pressure homogenization time is 10 minutes to obtain a rifampicin-loaded nanoemulsion.

[0107] Comparative Example 1D: Preparation method of rifampicin-loaded nanoemulsion

[0108] Compared to Example 1A, Comparative Example 1D changes the ultrasonic power, but the rest of the operations are the same as in Example 1A. The specific operations are as follows:

[0109] Step 1: Mix 3 mg of rifampicin, 20 mg of soybean oil, 10 mg of DPPC and 3 mg of cholesterol evenly to obtain a mixture; dissolve the mixture in 1.5 mL of dichloromethane to prepare the oil phase.

[0110] Step 2: Add 3 mg of sodium deoxycholate and 5 mg of Tween 80 to 1 mL of sterile water and stir until the sodium deoxycholate and Tween dissolve to prepare an aqueous phase.

[0111] Step 3: Mix the volatile organic solvent in the oil phase and the sterile liquid in the aqueous phase at a volume ratio of 1.5:1, vortex mix for 3 minutes, and then sonicate using a cell disruptor at a power of 450 watts for 8 minutes. After sonication, the first emulsion is obtained. The volatile organic solvent in the first emulsion is removed by rotary evaporation to obtain the second emulsion. The rotary evaporation temperature is 45 degrees Celsius and the rotary evaporation time is 5 minutes.

[0112] Step 4: The second emulsion is homogenized under high pressure using a high-pressure homogenizer. The pressure of the high-pressure homogenization is 800 bar, the temperature is 4 degrees Celsius, and the homogenization time is 10 minutes to obtain a rifampicin-loaded nanoemulsion.

[0113] Comparative Example 1E: Preparation method of rifampicin-loaded nanoemulsion

[0114] Compared to Example 1A, Example 1E differs from Example 1A in that the homogenization pressure is changed, while the rest of the operations are the same as in Example 1A. The specific operations are as follows:

[0115] Step 1: Mix 3 mg of rifampicin, 20 mg of soybean oil, 10 mg of DPPC and 3 mg of cholesterol evenly to obtain a mixture; dissolve the mixture in 1.5 mL of dichloromethane to prepare the oil phase.

[0116] Step 2: Add 3 mg of sodium deoxycholate and 5 mg of Tween 80 to 1 mL of sterile water and stir until the sodium deoxycholate and Tween dissolve to prepare an aqueous phase.

[0117] Step 3: Mix the volatile organic solvent in the oil phase and the sterile liquid in the aqueous phase at a volume ratio of 1.5:1, vortex mix for 3 minutes, and then sonicate using a cell disruptor at a power of 200 watts for 8 minutes. After sonication, the first emulsion is obtained. The volatile organic solvent in the first emulsion is removed by rotary evaporation to obtain the second emulsion. The rotary evaporation temperature is 45 degrees Celsius and the rotary evaporation time is 5 minutes.

[0118] Step 4: The second emulsion is subjected to high-pressure homogenization using a high-pressure homogenizer. The high-pressure homogenization pressure is 150 bar, the high-pressure homogenization temperature is 4 degrees Celsius, and the high-pressure homogenization time is 10 minutes to obtain a rifampicin-loaded nanoemulsion.

[0119] Comparative Example 1F: Preparation method of rifampicin-loaded nanoemulsion

[0120] Compared to Example 1A, Example 1F changed the volume ratio of volatile organic solvent in the oil phase to sterile liquid in the aqueous phase to 0.5:1. All other operations were the same as in Example 1A, as detailed below:

[0121] Step 1: Mix 3 mg of rifampicin, 20 mg of soybean oil, 10 mg of DPPC and 3 mg of cholesterol evenly to obtain a mixture; dissolve the mixture in 1.5 mL of dichloromethane to prepare the oil phase.

[0122] Step 2: Add 3 mg of sodium deoxycholate and 5 mg of Tween 80 to 1 mL of sterile water and stir until the sodium deoxycholate and Tween dissolve to prepare an aqueous phase.

[0123] Step 3: Mix the volatile organic solvent in the oil phase and the sterile liquid in the aqueous phase at a volume ratio of 5:1, vortex mix for 3 minutes, and then sonicate using a cell disruptor at a power of 200 watts for 8 minutes. After sonication, the first emulsion is obtained. The volatile organic solvent in the first emulsion is removed by rotary evaporation to obtain the second emulsion. The rotary evaporation temperature is 45 degrees Celsius and the rotary evaporation time is 5 minutes.

[0124] Step 4: The second emulsion is homogenized under high pressure using a high-pressure homogenizer. The pressure of high-pressure homogenization is 800 bar, the temperature is 4 degrees Celsius, and the time is 10 minutes to obtain a rifampicin-loaded nanoemulsion.

[0125] Comparative Example 1G: Preparation method of rifampicin-loaded nanoemulsion

[0126] Compared to Example 1A, Comparative Example 1G changed the volume ratio of volatile organic solvent in the oil phase to sterile liquid in the aqueous phase to 10:1. All other operations were the same as in Example 1A, as detailed below:

[0127] Step 1: Mix 3 mg of rifampicin, 20 mg of soybean oil, 10 mg of DPPC and 3 mg of cholesterol evenly to obtain a mixture; dissolve the mixture in 1.5 mL of dichloromethane to prepare the oil phase.

[0128] Step 2: Add 3 mg of sodium deoxycholate and 5 mg of Tween 80 to 1 mL of sterile water and stir until the sodium deoxycholate and Tween dissolve to prepare an aqueous phase.

[0129] Step 3: Mix the volatile organic solvent in the oil phase and the sterile liquid in the aqueous phase at a volume ratio of 10:1, vortex mix for 3 minutes, and then sonicate using a cell disruptor at a power of 200 watts for 8 minutes. After sonication, the first emulsion is obtained. The volatile organic solvent in the first emulsion is removed by rotary evaporation to obtain the second emulsion. The rotary evaporation temperature is 45 degrees Celsius and the rotary evaporation time is 5 minutes.

[0130] Step 4: The second emulsion is homogenized under high pressure using a high-pressure homogenizer. The pressure of high-pressure homogenization is 800 bar, the temperature is 4 degrees Celsius, and the time is 10 minutes to obtain a rifampicin-loaded nanoemulsion.

[0131] Example 2: Performance characterization of rifampicin-loaded nanoemulsions

[0132] First, the morphology of the antibiotic-loaded nanoemulsions prepared in Examples 1A, 1D, and 1E was characterized. The characterization results are shown in [reference needed]. Figures 1 to 3 The morphology of the prepared antibiotic-loaded nanoemulsion was characterized, and the characterization results are shown in [reference needed]. Figure 1 , Figure 1 This is a transmission electron microscope (TEM) image of a rifampicin-loaded nanoemulsion provided in an embodiment of this application. Figure 2 This is a transmission electron microscope (TEM) image of a polymyxin-loaded nanoemulsion provided in an embodiment of this application. Figure 3 This is a transmission electron microscope (TEM) image of a vancomycin-loaded nanoemulsion provided in this embodiment. The nanoemulsions loaded with the antibiotics rifampin, polymyxin, and vancomycin exhibit uniform morphology, all appearing as spherical shapes. The experimental method used in this embodiment to characterize the performance of the rifampin-loaded nanoemulsion is the same as commonly used techniques in the art, and will not be described further here.

[0133] Then, the performance of the antibiotic-loaded nanoemulsions prepared in Examples 1A-1E and Comparative Examples 1A-1G was characterized. The encapsulation efficiency, drug loading, particle size, PDI (Polymer Dispersion Index), potential and stability were tested respectively. The test results are shown in Table 1.

[0134] Table 1. Performance characterization results of antibiotic-loaded nanoemulsions prepared in Examples 1A-1E and Comparative Examples 1A-1G.

[0135]

[0136] As shown in Table 1, the antibiotic-loaded nanoemulsions prepared using the method described in this application, as inhalation-type nanoemulsions, exhibit high antibiotic loading rates, good nebulization performance, and good stability. The ratios of soybean oil and DPPC, as well as sodium deoxycholate and Tween 80 in the antibiotic-loaded nanoemulsions, affect the encapsulation efficiency and drug loading. Higher ultrasonic power can reduce the particle size of the nanoemulsions, but excessively high ultrasonic power can negatively impact the physical stability of the nanoemulsions, leading to precipitation during storage. Sodium deoxycholate significantly promotes the emulsification uniformity of the nanoemulsions, contributing to the formation of emulsions with uniform particle size; without sodium deoxycholate, nanoemulsion formation is difficult. Insufficient ultrasonic power results in incomplete emulsification of the nanoemulsions, hindering emulsion formation.

[0137] Therefore, in the preparation method of this application, through the reasonable design and proportioning of various parameters, the optimal parameter ratio is that in Example 1A. Specifically, the volume ratio of volatile organic solvent in the oil phase to sterile liquid in the aqueous phase is 1.5:1, the vortexing time is 3 minutes, the ultrasonic treatment power is 200 watts, the ultrasonic treatment time is 8 minutes, the rotary evaporation temperature is 45 degrees Celsius, the rotary evaporation time is 5 minutes, the homogenization pressure is 800 bar, and the homogenization time is 10 minutes. Through the above preparation method, a nanoemulsion with good stability at 4 degrees Celsius for 6 months, a minimum dispersion index of 0.124, and high encapsulation efficiency and drug loading (encapsulation efficiency up to 99% and drug loading up to 10%) can be obtained.

[0138] Example 3: Characterization of drug delivery efficiency and drug retention in antibiotic-loaded nanoemulsions

[0139] A nanoemulsion labeled with the fluorescent dye DID, namely 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine, was prepared using the method of Example 1A. The nanoemulsion loaded with rifampicin and labeled with DID (hereinafter referred to as rifampicin nanoemulsion) was used as an inhaled nanoemulsion, and the distribution of the nanoemulsion in mice was observed.

[0140] The specific operation is as follows: DID-labeled rifampicin nanoemulsion was prepared according to the preparation method of Example 1A. Intravenous injection of free DID and nebulized inhalation of free DID were used as control groups. Mice were given an equal amount of DID-labeled nanoemulsion by a mouse micro-nebulization inhalation device. 48 hours after the mice were given the drug via nebulization, the lung tissue of the mice was removed. The fluorescence content in the lung tissue of each group of mice was observed by a small animal in vivo imaging instrument to analyze the drug delivery efficiency and drug retention in the lung tissue.

[0141] The above experimental results are referenced. Figure 4 , Figure 4 This is a quantitative detection image of lung tissue in mice 48 hours after inhalation of a rifampicin nanoemulsion, as provided in an embodiment of this application. Figure 4The DID represents the distribution of the drug (i.e., rifampicin nanoemulsion) in mice. In the mouse model, after the rifampicin nanoemulsion was nebulized in mice, the fluorescence signal in the lung tissue of the nanoemulsion group (i.e., DID-labeled nanoemulsion) was significantly higher than that of the intravenous injection group and the nebulized inhalation group with free DID. The exposure of DID-labeled rifampicin nanoemulsion after nebulization was significantly higher than that of inhalation (nebulized inhalation DID) and intravenous injection (intravenous injection DID) after 48 hours, indicating that nebulized inhalation of rifampicin nanoemulsion can significantly improve the efficiency of drug delivery to the lungs and has the advantage of increasing the amount of drug retained in the lung tissue and the exposure time.

[0142] The above provides a detailed description of an antibiotic-loaded nanoemulsion, its preparation method, and its application. Specific examples have been used to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the method and core ideas of this application. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the ideas of this application. Therefore, the content of this specification should not be construed as a limitation of this application.

Claims

1. A method for preparing an antibiotic-loaded nebulized inhalation nanoemulsion, characterized in that, The method includes the following steps: Step 1: Mix 3 mg of antibiotic, 20 mg of soybean oil, 10 mg of dipalmitoylphosphatidylcholine, and 3 mg of cholesterol evenly to obtain a mixture; dissolve the mixture in 1.5 mL of volatile organic solvent to prepare the oil phase; Step 2: Add 3 mg of sodium deoxycholate and 5 mg of Tween 80 to 1 mL of sterile liquid and stir until sodium deoxycholate and Tween 80 dissolve to prepare an aqueous phase; Step 3: Mix the volatile organic solvent in the oil phase and the sterile liquid in the aqueous phase at a volume ratio of 1.5:

1. After mixing, sonicate the mixture at a power of 200 watts for 8 minutes. After sonication, the first emulsion is obtained. Rotary evaporation is used to remove the volatile organic solvent from the first emulsion to obtain the second emulsion. Step 4: The second emulsion is subjected to high-pressure homogenization to obtain an antibiotic-loaded nebulized inhalation nanoemulsion. The high-pressure homogenization pressure is 800 bar and the high-pressure homogenization time is 10 minutes. In step 1, the antibiotic is rifampin, polymyxin, or vancomycin; The sterile liquid is water, physiological saline, or deionized water; The volatile organic solvent is dichloromethane.

2. The method for preparing the antibiotic-loaded nebulized inhalation nanoemulsion according to claim 1, characterized in that, In step 3, the temperature for removing the volatile organic solvent from the first emulsion by rotary evaporation is 37°C to 60°C, and the time for removing the volatile organic solvent from the first emulsion by rotary evaporation is 5 minutes to 10 minutes.

3. A nanoemulsion for nebulized inhalation administration loaded with antibiotics, characterized in that, The antibiotic-loaded nebulized inhalation nanoemulsion is prepared by the preparation method according to any one of claims 1-2.

4. The use of the antibiotic-loaded nebulized inhalation nanoemulsion as described in claim 3 in the preparation of inhaled antibiotic drugs.

Images