Preparation method and application of antibiotic drug delivery system

Abstract

The invention provides a preparation method and application of an antibiotic drug delivery system, and relates to the technical field of medical excipients, various pharmaceutic adjuvants such as soybean oil, phospholipid and glycerol are specifically adjusted to prepare an emulsion capable of loading antibiotics, and the emulsion is used as the antibiotic drug delivery system, so that the antibiotic drug delivery system can efficiently load the antibiotics, and the antibiotic drug delivery effect is improved. According to the present invention, the antibiotic-loaded emulsion has characteristics of low cost, significant improvement of the sterilization efficiency, good aerosol inhalation performance, good drug stability, and slow release effect of the antibiotic drug delivery system, and compared with the inhalation antibiotic solution dosage form, the antibiotic-loaded emulsion can remain in the mouse lung tissue for a long time after being inhaled and administered by the mouse so as to significantly improve the lung drug delivery efficiency.

Description

Technical Field

[0001] The present application relates to the technical field of medical excipients, and in particular to a preparation method and application of an antibiotic drug delivery system. Background Art

[0002] At present, there is a serious shortage of inhaled antibiotics for clinical use. The only inhaled antibiotic approved for clinical use in China is tobramycin inhalation solution, and many antibiotics such as rifampicin and polymyxin have been used for inhalation beyond the instructions. Therefore, it is urgent to develop antibiotic preparations specifically for inhalation.

[0003] The existing inhaled antibiotics are mainly solution-type inhaled antibiotics, and the only nano-preparation for inhalation on the market is amikacin inhaled liposome suspension. However, solution-type inhaled antibiotics have the disadvantages of rapid blood entry, low lung retention, large dosage, and insufficient antibacterial efficiency.

[0004] Based on this, a new antibiotic delivery system is urgently needed. Summary of the invention

[0005] The present application provides a preparation method and application of an antibiotic drug delivery system to solve the problems of existing inhaled antibiotics such as rapid entry into the blood, low lung retention, large dosage, and insufficient antibacterial efficiency.

[0006] In a first aspect, the present application provides a method for preparing an antibiotic drug delivery system, the method comprising the following steps: Step 1: Evenly mix 1-100 mg of soybean oil and 1-100 mg of medium chain triglyceride to prepare an oil phase; Step 2: adding 1 to 10 mg of antibiotics, 1 to 12 mg of phospholipids and 1 to 25 mg of glycerol into a sterile liquid and dissolving them to prepare an aqueous phase, wherein the antibiotics are water-soluble antibiotics; Step 3: uniformly mixing the oil phase and the water phase to obtain a mixed material, and after ultrasonic treatment, obtaining an initial emulsion, wherein each 1 ml of the mixed material contains 1 to 100 mg of soybean oil, 1 to 100 mg of medium-chain triglycerides, 1 to 10 mg of antibiotics, 1 to 12 mg of phospholipids and 1 to 25 mg of glycerol; Step 4: The initial emulsion is subjected to high pressure homogenization to obtain an antibiotic-loaded emulsion as an antibiotic delivery system.

[0007] Optionally, in step 2, the sterile liquid is at least one of water for injection, deionized water, physiological saline, glucose injection, citrate buffer and phosphate buffer.

[0008] Optionally, in step 2, the antibiotic is at least one of polymyxin E sodium methanesulfonate, polymyxin B sulfate, tobramycin and amphotericin B.

[0009] Optionally, in step 3, the power of the ultrasonic treatment is 50 watts to 350 watts, and the ultrasonic treatment time is 1 minute to 20 minutes.

[0010] Optionally, in step 4, the pressure of the high-pressure homogenization treatment is 200 bar to 1200 bar, the temperature of the high-pressure homogenization treatment is 4° C. to 25° C., and the time of the high-pressure homogenization treatment is 2 minutes to 30 minutes.

[0011] Optionally, the particle size range of the prepared antibiotic-loaded emulsion is less than 1 micron, preferably below 300 nanometers, and most preferably below 100 nanometers.

[0012] In a second aspect, the present application provides an antibiotic drug delivery system, which is prepared by the preparation method described in any one of the above-mentioned first aspects.

[0013] In a third aspect, the present application proposes a use of the antibiotic delivery system described in the second aspect in the preparation of inhaled antibiotic drugs.

[0014] Optionally, the administration route of the antibiotic delivery system includes at least intravenous administration, subcutaneous administration, and oral administration.

[0015] The present application includes the following advantages: The present application proposes a preparation method and application of an antibiotic drug delivery system. The present application specifically adjusts various inhalable pharmaceutical excipients, such as soybean oil, phospholipids, glycerol and other materials, to prepare an emulsion that can load antibiotics, and uses it as an antibiotic drug delivery system, so that the antibiotic drug delivery system can efficiently load antibiotics, and has good atomization inhalation performance, good drug stability, so that the antibiotic drug delivery system has a sustained release effect. Compared with the inhaled antibiotic solution dosage form, the antibiotics in the antibiotic drug delivery system can be retained in the lung tissue of mice for a longer time, significantly improving the efficiency of lung drug delivery. At the same time, compared with free antibiotics, the antibiotic drug delivery system prepared by the present application can significantly improve the antibacterial ability of antibiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the description of the embodiments of the present application will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0017] Figure 1This is a transmission electron micrograph of an antibiotic drug delivery system loaded with polymyxin B sulfate provided in an embodiment of the present application; Figure 2 A transmission electron microscopy image of an antibiotic drug delivery system loaded with tobramycin provided in an embodiment of the present application; Figure 3 Transmission electron microscopy image of an antibiotic drug delivery system loaded with amphotericin B provided in an embodiment of the present application; Figure 4 Transmission electron microscopy image of an antibiotic drug delivery system loaded with polymyxin E sodium methanesulfonate provided in an embodiment of the present application; Figure 5 This is a quantitative detection diagram of lung tissue of mice 48 hours after inhalation of an antibiotic delivery system loaded with polymyxin B sulfate provided in an embodiment of the present application; Figure 6 The diagram is a diagram of the antibacterial efficacy of an antibiotic delivery system loaded with polymyxin B sulfate provided in an embodiment of the present application against carbapenem-resistant Acinetobacter baumannii. DETAILED DESCRIPTION

[0018] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0019] At present, there is a serious shortage of inhaled antibiotics for clinical use. The only inhaled antibiotic approved for clinical use in China is tobramycin inhalation solution, and there are many antibiotics such as rifampicin and polymyxin that have been used for inhalation administration beyond the instructions. Therefore, it is urgent to develop an antibiotic delivery system specifically for inhalation.

[0020] Existing inhaled antibiotics are mainly solution-type antibiotics for inhalation, and the only nano-preparation for inhalation on the market is amikacin inhalation liposome suspension. However, solution-type inhaled antibiotics have the disadvantages of rapid blood entry, low lung retention, large dosage, and insufficient antibacterial efficiency.

[0021] Based on this, the present application proposes a method for preparing an antibiotic drug delivery system, which utilizes various inhalable pharmaceutical excipients in the prescription, such as soybean oil, phospholipids, glycerol and other materials, to prepare an antibiotic drug delivery system that can load antibiotics.

[0022] In a first aspect of an embodiment of the present application, a method for preparing an antibiotic drug delivery system is provided, the method comprising the following steps: Step 1: Evenly mix 1-100 mg of soybean oil and 1-100 mg of medium chain triglyceride to prepare an oil phase; Step 2: adding 1 to 10 mg of antibiotics, 1 to 12 mg of phospholipids and 1 to 25 mg of glycerol into a sterile liquid and dissolving them to prepare an aqueous phase, wherein the antibiotics are water-soluble antibiotics; Step 3: uniformly mixing the oil phase and the water phase to obtain a mixed material, and after ultrasonic treatment, obtaining an initial emulsion, wherein each 1 ml of the mixed material contains 1 to 100 mg of soybean oil, 1 to 100 mg of medium-chain triglycerides, 1 to 10 mg of antibiotics, 1 to 12 mg of phospholipids and 1 to 25 mg of glycerol; Step 4: The initial emulsion is subjected to high pressure homogenization to obtain an antibiotic-loaded emulsion as an antibiotic delivery system.

[0023] When implementing step 1 specifically, in order to prepare an emulsion with high efficiency of loading antibiotics, in the preparation method proposed in the present application, the oil phase is first prepared. The oil phase components selected in the present application are soybean oil and medium-chain triglycerides (MCT). Soybean oil and medium-chain triglycerides are used as the oil phase of the emulsion to maintain the stability of the emulsion particles and form a larger specific surface area, which is helpful for loading drugs. In addition, positively charged antibiotic molecules can be combined with negatively charged emulsion particles through positive and negative charge adsorption, intermolecular forces and hydrogen bonds, etc., to improve the stability of the drug and form antibiotic nanoaggregates, which helps to improve the antibacterial ability of antibiotics. In addition, the unsaturated fatty acids in soybean oil also have a certain antibacterial effect. Therefore, the antibiotic-loaded emulsion prepared by the oil phase as a drug delivery system significantly improves the antibacterial ability through a multi-faceted combined action mechanism compared to free antibiotics.

[0024] When the step 2 is specifically implemented, the present application adds antibiotics to the aqueous phase when preparing the aqueous phase, and preferably water-soluble antibiotics, to promote the complete dissolution of the antibiotics in the aqueous phase and increase the loading rate of the antibiotics. The components in the aqueous phase are preferably phospholipids and glycerol. As natural surfactants, phospholipids reduce the interfacial tension between oil and water, form a firm emulsion film, promote drug absorption, and solubilize poorly soluble drugs. The phospholipid complex formed exhibits significantly different physical and chemical properties and biological properties from the original compound, which can increase solubility and biomembrane permeability, and has a significant effect in the preparation process of aerosol inhalation emulsions. Glycerol, as an emulsifier, can increase the solubility of the emulsifier, assist the emulsifier in reducing the interfacial tension between oil and water, increase the fluidity of the interfacial film, and adjust the HLB value (Hydrophilic-Lipophilic Balance) of the emulsifier, so that nanoemulsion droplets can be formed spontaneously. The addition of glycerol can increase the amount of antibiotics loaded, increase the solubility of antibiotics, and form a larger range of nanoemulsions.

[0025] Among them, the phospholipid can be selected from egg yolk lecithin, dipalmitoylphosphatidylcholine (DPPC), soybean lecithin, etc., preferably egg yolk lecithin.

[0026] In an optional embodiment of the present application, the sterile liquid used in the above step 2 can be any at least one of water for injection, deionized water, physiological saline, glucose injection, citrate buffer and phosphate buffer.

[0027] In an optional embodiment of the present application, the antibiotic used in the above step 2 can be at least one of polymyxin E sodium methanesulfonate and polymyxin B sulfate, tobramycin and amphotericin B, or antibiotics and drugs with equivalent efficacy.

[0028] When implementing step 3, in order to make the oil phase and the water phase mix evenly, the present application performs ultrasonic treatment on the mixed oil phase and the water phase, which can improve the emulsification efficiency, reduce the amount of emulsifier used, and ensure the stability of the emulsion, form nano-scale droplets, and improve the release characteristics of antibiotics, thereby improving the bioavailability and efficacy of antibiotics. By controlling the ultrasonic process, such as controlling the frequency and time of ultrasound, adjusting the dispersion of emulsion droplets, preparing emulsions of different particle sizes, achieving precise control, and during the ultrasonic treatment process, no excessive temperature is generated, and a wider range of applicable antibiotics can be used, such as for the loading of heat-sensitive antibiotics.

[0029] In the above step 3, 1 ml of the mixture obtained by mixing the oil phase and the water phase contains 1-100 mg of soybean oil, 1-100 mg of medium-chain triglycerides, 1-10 mg of antibiotics, 1-12 mg of phospholipids and 1-25 mg of glycerol.

[0030] In an optional embodiment of the present application, in the above step 3, the power of the ultrasonic treatment is 50 watts to 350 watts, which can be 50 watts, 100 watts, 150 watts, 200 watts, 250 watts, 300 watts, 350 watts, etc. The ultrasonic treatment time is 1 minute to 20 minutes, which can be 1 minute, 3 minutes, 5 minutes, 7 minutes, 10 minutes, 13 minutes, 15 minutes, 20 minutes, etc.

[0031] When implementing step 4, in order to further improve the quality of the nanoemulsion, such as stability, production efficiency, sensory quality, etc., the present application uses high-pressure homogenization to further process the initial nanoemulsion after ultrasonic treatment. High-pressure homogenization can further reduce the size of the emulsion droplets in the initial nanoemulsion, making the emulsion more uniform, thereby improving the physical stability of the emulsion. It can also effectively inactivate microorganisms and enzymes in the emulsion and extend the shelf life of the nanoemulsion; it can further improve the rheological properties of the emulsion, reduce the size of the emulsion droplets, and improve the emulsification properties of the emulsion; atomization inhalation administration has certain requirements on the size of the nanoemulsion. The smaller the size, the more drugs can enter the lungs and increase the drug retention in the lungs. The use of high-pressure homogenization can more accurately control the characteristics of the emulsion, such as droplet size and distribution. In actual production, when the above-mentioned nanoformulations need to be mass-produced, high-pressure homogenization is a good treatment method.

[0032] In an optional embodiment of the present application, in the above step 4, the pressure of the high-pressure homogenization treatment is 200 bar to 1200 bar, which can be 200 bar, 400 bar, 600 bar, 800 bar, 1000 bar, 1200 bar, etc. The temperature of the high-pressure homogenization treatment is 4°C to 25°C, which can be 4°C, 8°C, 12°C, 15°C, 18°C, 20°C, 23°C, 25°C, etc. The time of the high-pressure homogenization treatment is 2 minutes to 30 minutes, which can be 2 minutes, 3 minutes, 5 minutes, 7 minutes, 10 minutes, 13 minutes, 15 minutes, 18 minutes, 20 minutes, 23 minutes, 25 minutes, 28 minutes, 30 minutes, etc.

[0033] The antibiotic-loaded emulsion prepared by the above preparation method has a particle size of less than 1 micron, preferably less than 300 nanometers as an antibiotic delivery system, and more preferably less than 100 nanometers as an antibiotic delivery system.

[0034] The preparation method of the antibiotic drug delivery system provided in the first aspect of the embodiment of the present application is to adjust various inhalable pharmaceutical excipients, such as soybean oil, phospholipids, glycerol and other materials in a targeted manner, and prepare a nanoemulsion preparation that can load antibiotics as an antibiotic drug delivery system, so that the antibiotic drug delivery system can efficiently load antibiotics, and has good atomization inhalation performance, good drug stability, so that the antibiotic drug delivery system has a sustained release effect. Compared with the inhaled antibiotic solution dosage form, the antibiotic drug delivery system can make the loaded antibiotics stay in the mouse lung tissue for a longer time, significantly improving the efficiency of lung drug delivery. In addition, the present application first adds water-soluble antibiotics to the aqueous phase, and then combines it with oil, thereby improving the loading efficiency of antibiotics. At the same time, the antibiotic drug delivery system prepared by the present application can significantly improve the antibacterial ability of antibiotics compared with free antibiotics.

[0035] Based on the same application concept, in a second aspect of the embodiment of the present application, an antibiotic drug delivery system is proposed, which is prepared by the preparation method described in any one of the above-mentioned first aspects.

[0036] Based on the same application concept, in the third aspect of the embodiment of the present application, an application of the antibiotic delivery system as described in the second aspect above in the preparation of an inhaled antibiotic drug is proposed. The optional administration routes of the antibiotic delivery system include intravenous administration, subcutaneous administration, and oral administration. Preferably, intravenous administration. More preferably, inhalation administration.

[0037] In order to clearly illustrate the antibiotic-loaded emulsion and its preparation method and application proposed in the present application, a detailed description is given below in conjunction with examples.

[0038] Example 1: Preparation method of an antibiotic delivery system Example 1A When the loaded antibiotic is polymyxin B sulfate, the preparation method of the antibiotic drug delivery system loaded with polymyxin B sulfate comprises the following steps: Step 1: 30 mg of soybean oil and 30 mg of medium-chain triglycerides were mixed evenly to obtain a mixture 1 as an oil phase; Step 2: 3.6 mg of egg yolk lecithin, 7.5 mg of glycerol and 3 mg of polymyxin B sulfate were mixed to obtain a mixture 2; 2 ml of sterile deionized water was used to dissolve the mixture 2, and after mixing evenly, an aqueous phase was prepared.

[0039] Step 3: Add the oil phase in step 1 dropwise to the water phase in step 2 to obtain a mixed material 3, adjust the volume of the mixed material 3 to 3 ml with sterile deionized water, vortex mix for 5 minutes, and use a cell disruptor to perform ice water bath ultrasound, the ultrasound treatment power is 200 watts, and the ultrasound treatment time is 10 minutes to obtain an initial emulsion. In the mixed material 3, the proportion of soybean oil is 10 mg / ml, the proportion of MCT is 10 mg / ml, the proportion of egg yolk lecithin is 1.2 mg / ml, the proportion of glycerol is 2.5 mg / ml, and the proportion of polymyxin B sulfate is 1 mg / ml.

[0040] Step 4: The initial emulsion is subjected to high-pressure homogenization treatment using a high-pressure homogenizer. The pressure of the high-pressure homogenization treatment is 800 bar, the temperature of the high-pressure homogenization treatment is 4° C., and the time of the high-pressure homogenization treatment is 10 minutes to obtain an antibiotic drug delivery system loaded with polymyxin B sulfate.

[0041] Example 1B When the loaded antibiotic is tobramycin, the preparation method of the tobramycin loaded antibiotic drug delivery system comprises the following steps: Step 1: 30 mg of soybean oil and 30 mg of medium-chain triglycerides were mixed evenly to obtain a mixture 1 as an oil phase; Step 2: 3.6 mg of egg yolk lecithin, 7.5 mg of glycerol and 3 mg of tobramycin are mixed to obtain a mixture 2; 2 ml of sterile deionized water is used to dissolve the mixture 2, and after mixing evenly, an aqueous phase is prepared.

[0042] Step 3: Add the oil phase in step 1 dropwise to the water phase in step 2 to obtain a mixed material 3, adjust the volume of the mixed material 3 to 3 ml with sterile deionized water, vortex mix for 5 minutes, and use a cell disruptor to perform ice water bath ultrasound, the ultrasound treatment power is 200 watts, and the ultrasound treatment time is 10 minutes to obtain an initial emulsion. In the mixed material 3, the proportion of soybean oil is 10 mg / ml, the proportion of MCT is 10 mg / ml, the proportion of egg yolk lecithin is 1.2 mg / ml, the proportion of glycerol is 2.5 mg / ml, and the proportion of tobramycin is 1 mg / ml.

[0043] Step 4: The initial emulsion is subjected to high-pressure homogenization treatment using a high-pressure homogenizer. The pressure of the high-pressure homogenization treatment is 800 bar, the temperature of the high-pressure homogenization treatment is 4° C., and the time of the high-pressure homogenization treatment is 10 minutes to obtain an antibiotic drug delivery system loaded with tobramycin.

[0044] Example 1C When the loaded antibiotic is amphotericin B, the preparation method of the antibiotic drug delivery system loaded with amphotericin B comprises the following steps: Step 1: 30 mg of soybean oil and 30 mg of medium-chain triglycerides were mixed evenly to obtain a mixture 1 as an oil phase; Step 2: 3.6 mg of egg yolk lecithin, 7.5 mg of glycerol and 3 mg of amphotericin B were mixed to obtain a mixture 2; 2 ml of sterile deionized water was used to dissolve the mixture 2, and the mixture was mixed evenly to prepare an aqueous phase.

[0045] Step 3: Add the oil phase in step 1 dropwise to the water phase in step 2 to obtain a mixed material 3, adjust the volume of the mixed material 3 to 3 ml with sterile deionized water, vortex mix for 5 minutes, and use a cell disruptor to perform ice water bath ultrasound, the ultrasound treatment power is 200 watts, and the ultrasound treatment time is 10 minutes to obtain an initial emulsion. In the mixed material 3, the proportion of soybean oil is 10 mg / ml, the proportion of MCT is 10 mg / ml, the proportion of egg yolk lecithin is 1.2 mg / ml, the proportion of glycerol is 2.5 mg / ml, and the proportion of amphotericin B is 1 mg / ml.

[0046] Step 4: The initial emulsion is subjected to high-pressure homogenization treatment using a high-pressure homogenizer. The pressure of the high-pressure homogenization treatment is 800 bar, the temperature of the high-pressure homogenization treatment is 4° C., and the time of the high-pressure homogenization treatment is 10 minutes to obtain an antibiotic drug delivery system loaded with amphotericin B.

[0047] Example 1D When the loaded antibiotic is polymyxin E sodium methanesulfonate, the preparation method of the antibiotic drug delivery system loaded with polymyxin E sodium methanesulfonate comprises the following steps: Step 1: 30 mg of soybean oil and 30 mg of medium-chain triglycerides were mixed evenly to obtain a mixture 1 as an oil phase; Step 2: 3.6 mg of egg yolk lecithin, 7.5 mg of glycerol and 3 mg of polymyxin E sodium methanesulfonate were mixed to obtain a mixture 2; 2 ml of sterile deionized water was used to dissolve the mixture 2, and after mixing evenly, an aqueous phase was prepared.

[0048] Step 3: Add the oil phase in step 1 dropwise to the water phase in step 2 to obtain a mixed material 3, adjust the volume of the mixed material 3 to 3 ml with sterile deionized water, vortex mix for 5 minutes, and use a cell disruptor to perform ice water bath ultrasound, the ultrasound treatment power is 200 watts, and the ultrasound treatment time is 10 minutes to obtain an initial emulsion. In the mixed material 3, the proportion of soybean oil is 10 mg / ml, the proportion of MCT is 10 mg / ml, the proportion of egg yolk lecithin is 1.2 mg / ml, the proportion of glycerol is 2.5 mg / ml, and the proportion of polymyxin E sodium methanesulfonate is 1 mg / ml.

[0049] Step 4: The initial emulsion is subjected to high-pressure homogenization treatment using a high-pressure homogenizer. The pressure of the high-pressure homogenization treatment is 800 bar, the temperature of the high-pressure homogenization treatment is 4° C., and the time of the high-pressure homogenization treatment is 10 minutes to obtain an antibiotic drug delivery system loaded with polymyxin E sodium methanesulfonate.

[0050] In order to illustrate the preparation method proposed in the examples of the present application, by targetedly adjusting the types of inhalable pharmaceutical excipients, the types and proportions of the added inhalable pharmaceutical excipients, the ratio of the amounts of the oil phase and the aqueous phase, and the ultrasonic power, homogenization pressure and time, and coordinating with each other, the prepared antibiotic delivery system can efficiently load antibiotics and has good atomization inhalation performance and high drug stability. The following comparative examples are listed below for comparison with the antibiotic delivery systems prepared by the preparation method proposed in Example 1 (Example 1A-Example 1D).

[0051] Comparative Example 1A Comparative Example 1A Compared with Example 1A, the egg yolk lecithin was replaced with soybean lecithin, and the remaining operations were the same as those of Example 1A. The specific operations are as follows: Step 1: 30 mg of soybean oil and 30 mg of medium-chain triglycerides were mixed evenly to obtain a mixture 1 as an oil phase; Step 2: 3.6 mg of soybean lecithin, 7.5 mg of glycerol and 3 mg of polymyxin B sulfate were mixed to obtain a mixture 2; 2 ml of sterile deionized water was used to dissolve the mixture 2, and after mixing evenly, an aqueous phase was prepared.

[0052] Step 3: Add the oil phase in step 1 dropwise to the water phase in step 2 to obtain a mixed material 3, adjust the volume of the mixed material 3 to 3 ml with sterile deionized water, vortex mix for 5 minutes, and use a cell disruptor to perform ice water bath ultrasound, the ultrasound treatment power is 200 watts, and the ultrasound treatment time is 10 minutes to obtain an initial emulsion. In the mixed material 3, the proportion of soybean oil is 10 mg / ml, the proportion of MCT is 10 mg / ml, the proportion of soybean lecithin is 1.2 mg / ml, the proportion of glycerol is 2.5 mg / ml, and the proportion of polymyxin B sulfate is 1 mg / ml.

[0053] Step 4: The initial emulsion is subjected to high-pressure homogenization treatment using a high-pressure homogenizer. The pressure of the high-pressure homogenization treatment is 800 bar, the temperature of the high-pressure homogenization treatment is 4° C., and the time of the high-pressure homogenization treatment is 10 minutes to obtain an antibiotic drug delivery system loaded with polymyxin B sulfate.

[0054] Comparative Example 1B Comparative Example 1B Compared with Example 1A, the egg yolk lecithin was replaced with dipalmitoylphosphatidylcholine, and the remaining operations were the same as those of Example 1A. The specific operations are as follows: Step 1: 30 mg of soybean oil and 30 mg of medium-chain triglycerides were mixed evenly to obtain a mixture 1 as an oil phase; Step 2: 3.6 mg of DPPC, 7.5 mg of glycerol and 3 mg of polymyxin B sulfate were mixed to obtain a mixture 2; 2 ml of sterile deionized water was used to dissolve the mixture 2, and after mixing evenly, an aqueous phase was prepared.

[0055] Step 3: Add the oil phase in step 1 dropwise to the water phase in step 2 to obtain a mixed material 3, adjust the volume of the mixed material 3 to 3 ml with sterile deionized water, vortex mix for 5 minutes, and use a cell disruptor to perform ice water bath ultrasound, the ultrasound treatment power is 200 watts, and the ultrasound treatment time is 10 minutes to obtain an initial emulsion. In the mixed material 3, the proportion of soybean oil is 10 mg / ml, the proportion of MCT is 10 mg / ml, the proportion of DPPC is 1.2 mg / ml, the proportion of glycerol is 2.5 mg / ml, and the proportion of polymyxin B sulfate is 1 mg / ml.

[0056] Step 4: The initial emulsion is subjected to high-pressure homogenization treatment using a high-pressure homogenizer. The pressure of the high-pressure homogenization treatment is 800 bar, the temperature of the high-pressure homogenization treatment is 4° C., and the time of the high-pressure homogenization treatment is 10 minutes to obtain an antibiotic drug delivery system loaded with polymyxin B sulfate.

[0057] Comparative Example 1C Comparative Example 1C is compared with Example 1A, except that soybean oil is not added, and the remaining operations are the same as those of Example 1A. The specific operations are as follows: Step 1: Add 30 mg of medium chain triglycerides as the oil phase; Step 2: 3.6 mg of egg yolk lecithin, 7.5 mg of glycerol and 3 mg of polymyxin B sulfate were mixed to obtain a mixture 1; 2 ml of sterile deionized water was used to dissolve the mixture 2, and after mixing evenly, an aqueous phase was prepared.

[0058] Step 3: Add the oil phase in step 1 dropwise to the water phase in step 2 to obtain a mixed material 2, adjust the volume of the mixed material 2 to 3 ml with sterile deionized water, vortex mix for 5 minutes, and use a cell disruptor to perform ice water bath ultrasound, the ultrasound treatment power is 200 watts, and the ultrasound treatment time is 10 minutes to obtain an initial emulsion. In the mixed material 3, the proportion of MCT is 10 mg / ml, the proportion of egg yolk lecithin is 1.2 mg / ml, the proportion of glycerol is 2.5 mg / ml, and the proportion of polymyxin B sulfate is 1 mg / ml.

[0059] Step 4: The initial emulsion is subjected to high-pressure homogenization treatment using a high-pressure homogenizer. The pressure of the high-pressure homogenization treatment is 800 bar, the temperature of the high-pressure homogenization treatment is 4° C., and the time of the high-pressure homogenization treatment is 10 minutes to obtain an antibiotic drug delivery system loaded with polymyxin B sulfate.

[0060] Comparative Example 1D Compared with Example 1A, Comparative Example 1D did not add medium chain triglycerides, and the remaining operations were the same as those of Example 1A. The specific operations are as follows: Step 1: 30 mg soybean oil was used as the oil phase; Step 2: 3.6 mg of egg yolk lecithin, 7.5 mg of glycerol and 3 mg of polymyxin B sulfate were mixed to obtain a mixture 1; 2 ml of sterile deionized water was used to dissolve the mixture 2, and after mixing evenly, an aqueous phase was prepared.

[0061] Step 3: Add the oil phase in step 1 dropwise to the water phase in step 2 to obtain a mixed material 2, adjust the volume of the mixed material 2 to 3 ml with sterile deionized water, vortex mix for 5 minutes, and use a cell disruptor to perform ice water bath ultrasound, the ultrasound treatment power is 200 watts, and the ultrasound treatment time is 10 minutes to obtain an initial emulsion. In the mixed material 3, the proportion of soybean oil is 10 mg / ml, the proportion of egg yolk lecithin is 1.2 mg / ml, the proportion of glycerol is 2.5 mg / ml, and the proportion of polymyxin B sulfate is 1 mg / ml.

[0062] Step 4: The initial emulsion is subjected to high-pressure homogenization treatment using a high-pressure homogenizer. The pressure of the high-pressure homogenization treatment is 800 bar, the temperature of the high-pressure homogenization treatment is 4° C., and the time of the high-pressure homogenization treatment is 10 minutes to obtain an antibiotic drug delivery system loaded with polymyxin B sulfate.

[0063] Comparative Example 1E Compared with Example 1A, in Comparative Example 1E, egg yolk lecithin was not added, and the remaining operations were the same as those in Example 1A. The specific operations are as follows: Step 1: 30 mg of soybean oil and 30 mg of medium-chain triglycerides were mixed evenly to obtain a mixture 1 as an oil phase; Step 2: 7.5 mg of glycerol and 3 mg of polymyxin B sulfate were mixed to obtain a mixture 2; 2 ml of sterile deionized water was used to dissolve the mixture 2, and after mixing evenly, an aqueous phase was prepared.

[0064] Step 3: Add the oil phase in step 1 dropwise to the water phase in step 2 to obtain a mixed material 3, adjust the volume of the mixed material 3 to 3 ml with sterile deionized water, vortex mix for 5 minutes, and use a cell disruptor to perform ice water bath ultrasound, the ultrasound treatment power is 200 watts, and the ultrasound treatment time is 10 minutes to obtain an initial emulsion. In the mixed material 3, the proportion of soybean oil is 10 mg / ml, the proportion of MCT is 10 mg / ml, the proportion of glycerol is 2.5 mg / ml, and the proportion of polymyxin B sulfate is 1 mg / ml.

[0065] Step 4: The initial emulsion is subjected to high-pressure homogenization treatment using a high-pressure homogenizer. The pressure of the high-pressure homogenization treatment is 800 bar, the temperature of the high-pressure homogenization treatment is 4° C., and the time of the high-pressure homogenization treatment is 10 minutes to obtain an antibiotic drug delivery system loaded with polymyxin B sulfate.

[0066] Comparative Example 1F Comparative Example 1F is compared with Example 1A, except that glycerol is not added, and the remaining operations are the same as those of Example 1A. The specific operations are as follows: Step 1: 30 mg of soybean oil and 30 mg of medium-chain triglycerides were mixed evenly to obtain a mixture 1 as an oil phase; Step 2: 3.6 mg of egg yolk lecithin and 3 mg of polymyxin B sulfate were mixed to obtain a mixture 2; 2 ml of sterile deionized water was used to dissolve the mixture 2, and after mixing evenly, an aqueous phase was prepared.

[0067] Step 3: Add the oil phase in step 1 dropwise to the water phase in step 2 to obtain a mixed material 3, adjust the volume of the mixed material 3 to 3 ml with sterile deionized water, vortex mix for 5 minutes, and use a cell disruptor to perform ice water bath ultrasound, the ultrasound treatment power is 200 watts, and the ultrasound treatment time is 10 minutes to obtain an initial emulsion. In the mixed material 3, the proportion of soybean oil is 10 mg / ml, the proportion of MCT is 10 mg / ml, the proportion of egg yolk lecithin is 1.2 mg / ml, and the proportion of polymyxin B sulfate is 1 mg / ml.

[0068] Step 4: The initial emulsion is subjected to high-pressure homogenization treatment using a high-pressure homogenizer. The pressure of the high-pressure homogenization treatment is 800 bar, the temperature of the high-pressure homogenization treatment is 4° C., and the time of the high-pressure homogenization treatment is 10 minutes to obtain an antibiotic drug delivery system loaded with polymyxin B sulfate.

[0069] Comparative Example 1G Compared with Example 1A, the ultrasonic treatment parameters were changed in Comparative Example 1G, and the remaining operations were the same as those in Example 1A. The specific operations were as follows: Step 1: 30 mg of soybean oil and 30 mg of medium-chain triglycerides were mixed evenly to obtain a mixture 1 as an oil phase; Step 2: 3.6 mg of egg yolk lecithin, 7.5 mg of glycerol and 3 mg of polymyxin B sulfate were mixed to obtain a mixture 2; 2 ml of sterile deionized water was used to dissolve the mixture 2, and after mixing evenly, an aqueous phase was prepared.

[0070] Step 3: Add the oil phase in step 1 dropwise to the water phase in step 2 to obtain a mixed material 3, adjust the volume of the mixed material 3 to 3 ml with sterile deionized water, vortex mix for 5 minutes, and use a cell disruptor to perform ice water bath ultrasound, the ultrasound treatment power is 50 watts, and the ultrasound treatment time is 1 minute to obtain an initial emulsion. In the mixed material 3, the proportion of soybean oil is 10 mg / ml, the proportion of MCT is 10 mg / ml, the proportion of egg yolk lecithin is 1.2 mg / ml, the proportion of glycerol is 2.5 mg / ml, and the proportion of polymyxin B sulfate is 1 mg / ml.

[0071] Step 4: The initial emulsion is subjected to high-pressure homogenization treatment using a high-pressure homogenizer. The pressure of the high-pressure homogenization treatment is 800 bar, the temperature of the high-pressure homogenization treatment is 4° C., and the time of the high-pressure homogenization treatment is 10 minutes to obtain an antibiotic drug delivery system loaded with polymyxin B sulfate.

[0072] Comparative Example 1H Comparative Example 1H is compared with Example 1A, the high pressure homogenization parameters are changed, and the remaining operations are the same as those of Example 1A. The specific operations are as follows: Step 1: 30 mg of soybean oil and 30 mg of medium-chain triglycerides were mixed evenly to obtain a mixture 1 as an oil phase; Step 2: 3.6 mg of egg yolk lecithin, 7.5 mg of glycerol and 3 mg of polymyxin B sulfate were mixed to obtain a mixture 2; 2 ml of sterile deionized water was used to dissolve the mixture 2, and after mixing evenly, an aqueous phase was prepared.

[0073] Step 3: Add the oil phase in step 1 dropwise to the water phase in step 2 to obtain a mixed material 3, adjust the volume of the mixed material 3 to 3 ml with sterile deionized water, vortex mix for 5 minutes, and use a cell disruptor to perform ice water bath ultrasound, the ultrasound treatment power is 200 watts, and the ultrasound treatment time is 10 minutes to obtain an initial emulsion. In the mixed material 3, the proportion of soybean oil is 10 mg / ml, the proportion of MCT is 10 mg / ml, the proportion of egg yolk lecithin is 1.2 mg / ml, the proportion of glycerol is 2.5 mg / ml, and the proportion of polymyxin B sulfate is 1 mg / ml.

[0074] Step 4: The initial emulsion is subjected to high-pressure homogenization treatment using a high-pressure homogenizer. The pressure of the high-pressure homogenization treatment is 200 bar, the temperature of the high-pressure homogenization treatment is 4° C., and the time of the high-pressure homogenization treatment is 2 minutes to obtain an antibiotic drug delivery system loaded with polymyxin B sulfate.

[0075] Comparative Example 1I Comparative Example 1I is compared with Example 1A, the proportion of each substance is changed, and the remaining operations are the same as Example 1A. The specific operations are as follows: Step 1: 30 mg of soybean oil and 30 mg of medium-chain triglycerides were mixed evenly to obtain a mixture 1 as an oil phase; Step 2: 3.6 mg of egg yolk lecithin, 7.5 mg of glycerol and 3 mg of polymyxin B sulfate were mixed to obtain a mixture 2; 2 ml of sterile deionized water was used to dissolve the mixture 2, and after mixing evenly, an aqueous phase was prepared.

[0076] Step 3: Mix the aqueous phase and the oil phase in step 1 to obtain a mixed material 3, adjust the volume of the mixed material 3 to 30 ml with sterile deionized water, vortex mix for 5 minutes, and use a cell disruptor to perform ice water bath ultrasound, the ultrasound treatment power is 200 watts, and the ultrasound treatment time is 10 minutes to obtain an initial emulsion. In the mixed material 3, the proportion of soybean oil is 1 mg / ml, the proportion of MCT is 1 mg / ml, the proportion of egg yolk lecithin is 0.12 mg / ml, the proportion of glycerol is 0.25 mg / ml, and the proportion of polymyxin B sulfate is 0.1 mg / ml.

[0077] Step 4: The initial emulsion is subjected to high-pressure homogenization treatment using a high-pressure homogenizer. The pressure of the high-pressure homogenization treatment is 800 bar, the temperature of the high-pressure homogenization treatment is 4° C., and the time of the high-pressure homogenization treatment is 10 minutes to obtain an antibiotic drug delivery system loaded with polymyxin B sulfate.

[0078] Comparative Example 1J Comparative Example 1J is compared with Example 1A, the proportion of each substance is changed, and the remaining operations are the same as Example 1A. The specific operations are as follows: Step 1: 3 g of soybean oil and 3 g of medium-chain triglycerides were mixed evenly to obtain a mixture 1 as an oil phase; Step 2: 360 mg of egg yolk lecithin, 750 mg of glycerol and 300 mg of polymyxin B sulfate were mixed to obtain a mixed material; 5 ml of sterile deionized water was used to dissolve the mixed material 2, and after mixing evenly, an aqueous phase was prepared. Among them, in the mixed material 3, the proportion of soybean oil was 100 mg / ml, the proportion of MCT was 100 mg / ml, the proportion of egg yolk lecithin was 12 mg / ml, the proportion of glycerol was 25 mg / ml, and the proportion of polymyxin B sulfate was 10 mg / ml.

[0079] Step 3: The aqueous phase and the oil phase in step 1 are mixed to obtain a mixed material 3, the volume of the mixed material 3 is adjusted to 30 ml with sterile deionized water, vortex mixed for 5 minutes, and ultrasonicated in an ice water bath with a cell disruptor at a power of 200 watts and a time of 10 minutes to obtain an initial emulsion.

[0080] Step 4: The initial emulsion is subjected to high-pressure homogenization treatment using a high-pressure homogenizer. The pressure of the high-pressure homogenization treatment is 800 bar, the temperature of the high-pressure homogenization treatment is 4° C., and the time of the high-pressure homogenization treatment is 10 minutes to obtain an antibiotic drug delivery system loaded with polymyxin B sulfate.

[0081] Example 2: Performance Characterization of Antibiotic Delivery System First, the morphology of the antibiotic drug delivery system prepared in Example 1A, Example 1B, Example 1C, and Example 1D was characterized. Figures 1 to 4The prepared antibiotic delivery system was characterized for morphology. The characterization results are shown in Figure 1 , Figure 1 This is a transmission electron microscopy morphology of an antibiotic drug delivery system loaded with polymyxin B sulfate provided in an embodiment of the present application; Figure 2 This is a transmission electron microscopy morphology image of a tobramycin-loaded antibiotic drug delivery system provided in an embodiment of the present application; Figure 3 This is a transmission electron microscopy morphology image of an antibiotic drug delivery system loaded with amphotericin B provided in an embodiment of the present application; Figure 4 This is a transmission electron microscope morphology of an antibiotic drug delivery system loaded with polymyxin sodium methanesulfonate provided in an embodiment of the present application. The morphology of the antibiotic drug delivery system is uniform and all present a spherical shape. The experimental method used in this embodiment to characterize the performance of the nanoemulsion loaded with polymyxin is the same as the commonly used technical means in the field, and will not be repeated here.

[0082] Then, the antibiotic drug delivery systems prepared in Example 1A to Example 1D and Comparative Example 1A to Comparative Example 1J were characterized by testing the encapsulation efficiency, drug loading, particle size, PDI (Polymer Dispersion Index), potential and stability. The test results are shown in Table 1.

[0083] Table 1 Performance characterization results of the antibiotic delivery systems prepared in Example 1A-Example 1D and Comparative Example 1A-Comparative Example 1J

[0084] It can be seen from the results in Table 1 that the antibiotic delivery system prepared by the preparation method of the present application, as an inhalation emulsion, has a high loading rate for antibiotics, good atomization inhalation performance, and good stability. The ratio of soybean oil, MCT and glycerol in the antibiotic delivery system, as well as the ultrasonic power and time, affect the encapsulation rate and drug loading. A larger ultrasonic power can make the particle size of the emulsion smaller, reaching the nanometer level, but too high an ultrasonic power will affect the physical stability of the nanoemulsion, causing the emulsion to precipitate during storage; suitable homogenization pressure and a longer homogenization time are helpful to form an emulsion with uniform particle size. In addition, it can be seen that the proportion of each substance in the antibiotic delivery system is a key factor in whether the delivery system can be successfully prepared. When the proportion of each substance in the antibiotic delivery system is too small (Comparative Example 1I), the emulsion is unstable and prone to turbid precipitation, and cannot be successfully prepared. When the proportion of each substance in the antibiotic delivery system is high (Comparative Example 1J), a small amount of precipitation appears after the emulsion is stored at 4°C for 4 months, and after shaking and mixing, it can be restored to clarity and transparency. In general, it is difficult to obtain a drug-loaded emulsion with better stability when a higher proportion of excipients is used.

[0085] Therefore, in the preparation method of the present application, various parameters are reasonably designed and proportioned, and the optimal parameter ratio is that in Example 1A. Specifically, in the prepared antibiotic delivery system, soybean oil accounts for 10 mg / ml, MCT accounts for 10 mg / ml, egg yolk lecithin accounts for 1.2 mg / ml, glycerol accounts for 2.5 mg / ml, and polymyxin B sulfate accounts for 1 mg / ml. The vortex mixing time is 5 minutes, and a cell disruptor is used for ice water bath ultrasound. The power of the ultrasonic treatment is 200 watts, the time of the ultrasonic treatment is 10 minutes, the pressure of the high-pressure homogenization treatment is 800 bar, the temperature of the high-pressure homogenization treatment is 4°C, and the time of the high-pressure homogenization treatment is 10 minutes. An antibiotic delivery system loaded with polymyxin B sulfate is obtained, and an antibiotic delivery system with good stability, a dispersion index of 0.153, and an encapsulation rate of more than 99% can be obtained when stored at 4 degrees Celsius for 6 months.

[0086] Example 3: Characterization of drug delivery efficiency and drug retention of antibiotic delivery systems The antibiotic drug delivery system containing Cy5-labeled polymyxin B sulfate was prepared by the preparation method of Example 1A. The Cy5-labeled polymyxin B sulfate was used as the control group. The mice were quantitatively given an equal amount of polymyxin B sulfate by inhalation using a mouse micro-atomizer inhalation device. After 48 hours of administration, the distribution of fluorescence signals in each group of mice was observed by a small animal in vivo imaging device to analyze the drug delivery efficiency and drug retention in lung tissue. Figure 5 It can be seen that ( Figure 5 The left side shows the fluorescence signal distribution results of isolated mouse organs. Figure 5 On the right are the quantitative results of the fluorescence signals of free polymyxin B sulfate and polymyxin B sulfate delivery system in mouse lung tissue). The polymyxin B sulfate antibiotic delivery system can significantly enhance the accumulation of antibiotics in mouse lung tissue and prolong the exposure time of antibiotics in mouse lung tissue.

[0087] Example 4: Investigation of the antibacterial activity of the antibiotic delivery system loaded with polymyxin B sulfate against carbapenem-resistant Acinetobacter baumannii The preparation method of Example 1A was used to prepare the antibiotic drug delivery system loaded with polymyxin B sulfate, and carbapenem-resistant Acinetobacter baumannii was cultured. The same concentration of polymyxin B sulfate was set in the two groups of experiments, and the groups were divided into: control group (Control), free polymyxin B sulfate (PMB group), and antibiotic drug delivery system group of polymyxin B sulfate (PMB-NEs group), and Acinetobacter baumannii (bacterial number was 1.0×10 5 cfu / ml) for 12 h, and the bacterial growth after drug treatment in the two groups was observed. Figure 6The results show that the control group has more Acinetobacter baumannii colonies, followed by the PMB group, and the PMB-NEs group has no Acinetobacter baumannii colonies. It can be seen that the PMB-NEs group has the best bactericidal effect compared with the control group (Control) and the PMB group, and can achieve the effect of sterile growth, indicating that the antibiotic delivery system loaded with polymyxin B sulfate can significantly improve the bactericidal effect.

[0088] The preparation method and application of an antibiotic drug delivery system provided by the present application are introduced in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the present application. The description of the above embodiments is only used to help understand the method of the present application and its core idea. At the same time, for those skilled in the art, according to the idea of ​​the present application, there will be changes in the specific implementation methods and application scopes. In summary, the content of this specification should not be understood as limiting the present application.

Claims

1. A method for preparing an antibiotic drug delivery system, characterized in that: The method comprises the following steps: Step 1: 1-100 mg of soybean oil and 1-100 mg of medium chain triglyceride are mixed evenly to prepare an oil phase; Step 2: adding 1 to 10 mg of antibiotics, 1 to 12 mg of phospholipids and 1 to 25 mg of glycerol into a sterile liquid and dissolving them to prepare an aqueous phase, wherein the antibiotics are water-soluble antibiotics; Step 3: uniformly mixing the oil phase and the water phase to obtain a mixed material, and after ultrasonic treatment, obtaining an initial emulsion, wherein each 1 ml of the mixed material contains 1 to 100 mg of soybean oil, 1 to 100 mg of medium-chain triglycerides, 1 to 10 mg of antibiotics, 1 to 12 mg of phospholipids and 1 to 25 mg of glycerol; Step 4: subjecting the initial emulsion to high pressure homogenization to obtain an antibiotic-loaded emulsion as an antibiotic delivery system.

2. The method for preparing the antibiotic drug delivery system according to claim 1, characterized in that: In step 2, the sterile liquid is at least one of water for injection, deionized water, physiological saline, glucose injection, citrate buffer and phosphate buffer.

3. The method for preparing the antibiotic drug delivery system according to claim 1, characterized in that: In step 2, the antibiotic is at least one of polymyxin E sodium methanesulfonate, polymyxin B sulfate, tobramycin and amphotericin B.

4. The method for preparing the antibiotic drug delivery system according to claim 1, characterized in that: In step 3, the power of the ultrasonic treatment is 50 watts to 350 watts, and the ultrasonic treatment time is 1 minute to 20 minutes.

5. The method for preparing the antibiotic drug delivery system according to claim 1, characterized in that: In step 4, the pressure of the high-pressure homogenization treatment is 200 bar to 1200 bar, the temperature of the high-pressure homogenization treatment is 4° C. to 25° C., and the time of the high-pressure homogenization treatment is 2 minutes to 30 minutes.

6. The method for preparing the antibiotic drug delivery system according to claim 1, characterized in that: The particle size range of the prepared antibiotic-loaded emulsion is less than 1 micron.

7. An antibiotic delivery system, characterized in that: The antibiotic drug delivery system is prepared by the preparation method according to any one of claims 1-6.

8. Use of the antibiotic delivery system according to claim 7 in the preparation of inhaled antibiotic drugs.

9. The use according to claim 8, characterized in that: The administration routes of the antibiotic delivery system at least include intravenous administration, subcutaneous administration, and oral administration.

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