Detailed Description
The following description of the embodiments of the present application will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are some, but not all embodiments of the application. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
At present, the inhaled antibiotics for clinical application are seriously lacking, only tobramycin inhalation solution is approved for clinical application in China, and various antibiotics such as rifampin, polymyxin and the like exist for inhalation administration of medicaments with super specifications. There is therefore a need to develop antibiotic delivery systems that are specifically designed for inhalation.
The existing inhalation antibiotics mainly adopt solution type antibiotics for inhalation, and the nanometer preparation for inhalation on the market only comprises amikacin inhalation liposome suspension. However, the solution type inhaled antibiotic medicine has the defects of quick blood inflow, low lung retention, larger administration dosage and insufficient antibacterial efficiency.
Based on the above, the application provides a preparation method of an antibiotic delivery system, which utilizes various inhalable medicinal auxiliary materials in the prescription, such as soybean oil, phospholipid, glycerol and other materials to prepare the antibiotic delivery system capable of loading antibiotics.
In a first aspect of an embodiment of the present application, there is provided a method of preparing an antibiotic delivery system, the method comprising the steps of:
Step 1, uniformly mixing 1-100 mg of soybean oil and 1-100 mg of medium-chain triglyceride to prepare an oil phase;
step 2, adding 1-10 mg of antibiotics, 1-12 mg of phospholipids and 1-25 mg of glycerol into sterile liquid and dissolving to prepare a water 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 performing ultrasonic treatment to obtain an initial emulsion, wherein each 1 ml of mixed material contains 1-100 mg of soybean oil, 1-100 mg of medium-chain triglyceride, 1-10 mg of antibiotics, 1-12 mg of phospholipids and 1-25 mg of glycerol;
and 4, carrying out high-pressure homogenization treatment on the initial emulsion to obtain an emulsion loaded with antibiotics as an antibiotic delivery system.
In the preparation method provided by the application, oil phase is firstly prepared, oil phase components selected by the application are soybean oil and Medium Chain Triglyceride (MCT), the soybean oil and the medium chain triglyceride are used as the oil phase of the emulsion, the stability of emulsion particles is maintained, a larger specific surface area is formed, the loading of the drug is facilitated, and in addition, positively charged antibiotic molecules can be combined with negatively charged emulsion particles through positive and negative charge adsorption, intermolecular acting force, hydrogen bond and other acting forces, the stability of the drug is improved, and the antibacterial capability of the antibiotic is facilitated to be improved. In addition, unsaturated fatty acid in soybean oil also has a certain antibacterial effect, so that the emulsion prepared from the oil phase and loaded with antibiotics is used as a drug delivery system, and compared with free antibiotics, the antibacterial capability is remarkably improved through multiple combined action mechanisms.
In the specific implementation of the step 2, in the preparation of the water phase, the antibiotic is added into the water phase, and preferably the water-soluble antibiotic, so that the complete dissolution of the antibiotic in the water phase is promoted, and the loading rate of the antibiotic is improved. The components in the water phase are preferably phospholipid and glycerin, wherein the phospholipid is used as a natural surfactant, so that the oil-water interfacial tension is reduced, a firm emulsion film is formed, the absorption of the medicine is promoted, and the insoluble medicine is solubilized. The formed phospholipid complex has physical and chemical properties and biological characteristics which are obviously different from those of the original compound, can increase the solubility and the permeability of a biological film, and has obvious effect in the preparation process of the atomized inhalation emulsion. The glycerol is used as an auxiliary emulsifier, can increase the solubility of the emulsifier, assist the emulsifier to reduce the interfacial tension between oil and water, increase the fluidity of an interfacial film, and adjust the HLB value (Hydrophilic-Lipophilic Balance, hydrophilic and lipophilic balance value) of the emulsifier, so that nano emulsion drops can be spontaneously formed. The addition of glycerol can improve the antibiotic carrying capacity, increase the solubility of antibiotics and enlarge the range of nanoemulsion formation.
Wherein the phospholipid is selected from egg yolk lecithin, dipalmitoyl phosphatidylcholine (DPPC), soybean lecithin, etc., preferably egg yolk lecithin.
In an alternative embodiment of the present application, the sterile liquid used in the step 2 may be any one of water for injection, deionized water, physiological saline, dextrose injection, citrate buffer and phosphate buffer.
In an alternative embodiment of the present application, the antibiotic used in the step 2 may be at least one of sodium polymyxin E mesylate and polymyxin B sulfate, tobramycin and amphotericin B, or an antibiotic and a drug with the same efficacy.
In the specific implementation of the step 3, in order to uniformly mix the oil phase and the water phase, the mixed oil phase and water phase are subjected to ultrasonic treatment, so that the emulsification efficiency can be improved, the use amount of an emulsifying agent is reduced, meanwhile, the stability of emulsion is ensured, nano-scale liquid drops are formed, the release characteristic of antibiotics is improved, and the bioavailability and the curative effect of the antibiotics are improved. The emulsion with different particle diameters is prepared by controlling the ultrasonic process, such as controlling the frequency and time of ultrasonic, and adjusting the dispersion degree of emulsion droplets, so that accurate control is realized, and in the ultrasonic treatment process, the ultra-high temperature cannot be generated, and the applicable antibiotic range is wider, such as being applicable to the loading of heat sensitive antibiotics.
In the step 3, 1ml of the mixed material obtained by mixing the oil phase and the water phase contains 1-100 mg of soybean oil, 1-100 mg of medium chain triglyceride, 1-10 mg of antibiotics, 1-12 mg of phospholipids and 1-25 mg of glycerin.
In an alternative embodiment of the present application, in the step 3, the power of the ultrasonic treatment is 50 watts to 350 watts, which may be 50 watts, 100 watts, 150 watts, 200 watts, 250 watts, 300 watts, 350 watts, or the like. The sonication time is from 1 minute to 20 minutes and may be 1 minute, 3 minutes, 5 minutes, 7 minutes, 10 minutes, 13 minutes, 15 minutes, 20 minutes, etc.
In the specific implementation of the step 4, in order to further improve the quality of the nanoemulsion, such as stability, production efficiency, sensory quality and the like, the application adopts high-pressure homogenization to further treat the initial nanoemulsion after ultrasonic treatment, and the high-pressure homogenization can further reduce the size of emulsion liquid drops in the initial nanoemulsion, so that the emulsion is more uniform, and the physical stability of the emulsion is improved. The method can effectively inactivate microorganisms and enzymes in the emulsion, prolong the shelf life of the nanoemulsion, further improve the rheological property of the emulsion, reduce the size of emulsion liquid drops, improve the emulsifying property of the emulsion, and ensure that the smaller size can enable more medicines to enter the lung and improve the retention of the medicines in the lung by adopting an atomization inhalation mode to administer the nanoemulsion, and can control the characteristics of the emulsion, such as the size and the distribution of the liquid drops, more accurately by adopting high-pressure homogenization. In practical production, when the nano preparation is required to be produced in a large scale, high-pressure homogenization is a good treatment mode.
In an alternative embodiment of the present application, in the step 4, the pressure of the high-pressure homogenizing treatment is 200 bar to 1200 bar, which may be 200 bar, 400 bar, 600 bar, 800 bar, 1000 bar, 1200 bar, etc. The temperature of the high pressure homogenization treatment is 4 ℃ to 25 ℃, and may be 4 ℃, 8 ℃, 12 ℃, 15 ℃, 18 ℃,20 ℃, 23 ℃, 25 ℃, and the like. The time of the high-pressure homogenizing treatment is 2 minutes to 30 minutes, and may 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, or the like.
The particle size of the antibiotic-loaded emulsion prepared by the preparation method is less than 1 micrometer, preferably less than 300 nanometers, and the antibiotic-loaded emulsion with the particle size of less than 100 nanometers is more preferably used as an antibiotic delivery system.
According to the preparation method of the antibiotic drug delivery system provided by the embodiment of the application, various inhalable pharmaceutic adjuvants such as soybean oil, phospholipid, glycerol and the like are purposefully adjusted, and the nano emulsion preparation capable of loading the antibiotics is prepared as the antibiotic drug delivery system, so that the antibiotic drug delivery system can efficiently load the antibiotics, has good atomization inhalation performance and good drug stability, and has a slow release effect. In addition, the application firstly adds the water-soluble antibiotics into the water phase and combines the water-soluble antibiotics with the oil phase, thereby improving the loading efficiency of the antibiotics. Meanwhile, compared with free antibiotics, the antibiotic drug delivery system prepared by the application can obviously improve the antibacterial capability of the antibiotics.
Based on the same application conception, in a second aspect of the embodiment of the present application, an antibiotic delivery system is provided, which is prepared by the preparation method according to any one of the first aspects.
Based on the same application conception, in a third aspect of the embodiment of the present application, there is provided an application of the antibiotic delivery system according to the second aspect to the preparation of an inhaled antibiotic drug. Alternative routes of administration for the antibiotic delivery system include intravenous, subcutaneous, and oral. Preferably, intravenous administration is performed. More preferably by inhalation.
In order to clearly illustrate the antibiotic-loaded emulsion, the preparation method and application thereof, the present application will be described in detail with reference to examples.
Example 1 preparation 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, uniformly mixing 30 mg of soybean oil and 30 mg of medium-chain triglyceride to obtain a mixed material 1 as an oil phase;
step 2, mixing 3.6 mg of egg yolk lecithin, 7.5 mg of glycerol and 3 mg of polymyxin B sulfate to obtain a mixed material 2, dissolving the mixed material 2 by adopting 2 ml of sterile deionized water, and uniformly mixing to obtain a water phase.
And 3, dropwise adding the oil phase in the step 1 into the water phase in the step 2 to obtain a mixed material 3, regulating the volume of the mixed material 3 to 3 milliliters by adopting sterile deionized water, carrying out vortex mixing for 5 minutes, carrying out ice water bath ultrasonic treatment by adopting a cell breaker, wherein the ultrasonic treatment power is 200 watts, and the ultrasonic treatment time is 10 minutes to obtain the initial emulsion. Wherein, in the mixed material 3, the soybean oil accounts for 10 mg/ml, the MCT accounts for 10 mg/ml, the egg yolk lecithin accounts for 1.2 mg/ml, the glycerol accounts for 2.5 mg/ml, and the polymyxin B sulfate accounts for 1 mg/ml.
And 4, carrying out high-pressure homogenization treatment on the initial emulsion by using a high-pressure homogenizer, wherein the pressure of the high-pressure homogenization treatment is 800 bar, the temperature of the high-pressure homogenization treatment is 4 ℃, and the time of the high-pressure homogenization treatment is 10 minutes, so as to obtain the antibiotic delivery system loaded with polymyxin B sulfate.
Example 1B
When the loaded antibiotic is tobramycin, the preparation method of the tobramycin-loaded antibiotic delivery system comprises the following steps:
step 1, uniformly mixing 30 mg of soybean oil and 30 mg of medium-chain triglyceride to obtain a mixed material 1 as an oil phase;
step 2, mixing 3.6 milligram of egg yolk lecithin, 7.5 milligram of glycerol and 3 milligram of tobramycin to obtain a mixed material 2, and uniformly mixing 2 milliliters of sterile deionized water-soluble mixture 2 to obtain a water phase.
And 3, dropwise adding the oil phase in the step 1 into the water phase in the step 2 to obtain a mixed material 3, regulating the volume of the mixed material 3 to 3 milliliters by adopting sterile deionized water, carrying out vortex mixing for 5 minutes, carrying out ice water bath ultrasonic treatment by adopting a cell breaker, wherein the ultrasonic treatment power is 200 watts, and the ultrasonic treatment time is 10 minutes to obtain the initial emulsion. Wherein, in the mixture material 3, the soybean oil accounts for 10 mg/ml, the MCT accounts for 10 mg/ml, the egg yolk lecithin accounts for 1.2 mg/ml, the glycerol accounts for 2.5 mg/ml, and the tobramycin accounts for 1 mg/ml.
And 4, carrying out high-pressure homogenization treatment on the initial emulsion by using a high-pressure homogenizer, wherein the pressure of the high-pressure homogenization treatment is 800 bar, the temperature of the high-pressure homogenization treatment is 4 ℃, and the time of the high-pressure homogenization treatment is 10 minutes, so as to obtain the tobramycin-loaded antibiotic delivery system.
Example 1C
When the loaded antibiotic is amphotericin B, the preparation method of the amphotericin B loaded antibiotic delivery system comprises the following steps:
step 1, uniformly mixing 30 mg of soybean oil and 30 mg of medium-chain triglyceride to obtain a mixed material 1 as an oil phase;
Step 2, mixing 3.6 mg of egg yolk lecithin, 7.5 mg of glycerol and 3 mg of amphotericin B to obtain a mixed material 2, dissolving the mixed material 2 by adopting 2 ml of sterile deionized water, and uniformly mixing to obtain a water phase.
And 3, dropwise adding the oil phase in the step 1 into the water phase in the step 2 to obtain a mixed material 3, regulating the volume of the mixed material 3 to 3 milliliters by adopting sterile deionized water, carrying out vortex mixing for 5 minutes, carrying out ice water bath ultrasonic treatment by adopting a cell breaker, wherein the ultrasonic treatment power is 200 watts, and the ultrasonic treatment time is 10 minutes to obtain the initial emulsion. Wherein, in the mixed material 3, the soybean oil accounts for 10 mg/ml, the MCT accounts for 10 mg/ml, the egg yolk lecithin accounts for 1.2 mg/ml, the glycerol accounts for 2.5 mg/ml, and the amphotericin B accounts for 1 mg/ml.
And 4, carrying out high-pressure homogenization treatment on the initial emulsion by using a high-pressure homogenizer, wherein the pressure of the high-pressure homogenization treatment is 800 bar, the temperature of the high-pressure homogenization treatment is 4 ℃, and the time of the high-pressure homogenization treatment is 10 minutes, so as to obtain the antibiotic delivery system loaded with amphotericin B.
Example 1D
When the loaded antibiotic is polymyxin E sodium mesylate, the preparation method of the antibiotic drug delivery system loaded with the polymyxin E sodium mesylate comprises the following steps:
step 1, uniformly mixing 30 mg of soybean oil and 30 mg of medium-chain triglyceride to obtain a mixed material 1 as an oil phase;
step 2, mixing 3.6 mg of egg yolk lecithin, 7.5 mg of glycerol and 3 mg of polymyxin E sodium methane sulfonate to obtain a mixed material 2, dissolving the mixed material 2 by adopting 2 ml of sterile deionized water, and uniformly mixing to obtain a water phase.
And 3, dropwise adding the oil phase in the step 1 into the water phase in the step 2 to obtain a mixed material 3, regulating the volume of the mixed material 3 to 3 milliliters by adopting sterile deionized water, carrying out vortex mixing for 5 minutes, carrying out ice water bath ultrasonic treatment by adopting a cell breaker, wherein the ultrasonic treatment power is 200 watts, and the ultrasonic treatment time is 10 minutes to obtain the initial emulsion. Wherein, in the mixture 3, the soybean oil accounts for 10 mg/ml, the MCT accounts for 10 mg/ml, the egg yolk lecithin accounts for 1.2 mg/ml, the glycerol accounts for 2.5 mg/ml, and the polymyxin E sodium methanesulfonate accounts for 1 mg/ml.
And 4, carrying out high-pressure homogenization treatment on the initial emulsion by using a high-pressure homogenizer, wherein the pressure of the high-pressure homogenization treatment is 800 bar, the temperature of the high-pressure homogenization treatment is 4 ℃, and the time of the high-pressure homogenization treatment is 10 minutes, so as to obtain the antibiotic drug delivery system loaded with the polymyxin E sodium mesylate.
In order to illustrate the preparation method provided by the embodiment of the application, the types of inhalable pharmaceutical excipients, the adding types and the proportion of the inhalable pharmaceutical excipients, the ratio of the amounts of the oil phase and the water phase, and the ultrasonic power, the homogenizing pressure and the time are adjusted in a targeted manner, and the prepared antibiotic delivery system can efficiently load antibiotics and has good aerosol inhalation performance and higher drug stability, and the following comparative examples are listed below to be compared with the antibiotic delivery system prepared by the preparation method provided by the embodiment 1 (embodiment 1A-embodiment 1D).
Comparative example 1A
Comparative example 1A the yolk lecithin was replaced with soybean lecithin, and the rest of the procedure was the same as in example 1A, except that the specific procedure was as follows:
step 1, uniformly mixing 30 mg of soybean oil and 30 mg of medium-chain triglyceride to obtain a mixed material 1 as an oil phase;
Step 2, mixing 3.6 mg of soybean lecithin, 7.5 mg of glycerol and 3 mg of polymyxin B sulfate to obtain a mixed material 2, dissolving the mixed material 2 by adopting 2 ml of sterile deionized water, and uniformly mixing to obtain a water phase.
And 3, dropwise adding the oil phase in the step 1 into the water phase in the step 2 to obtain a mixed material 3, regulating the volume of the mixed material 3 to 3 milliliters by adopting sterile deionized water, carrying out vortex mixing for 5 minutes, carrying out ice water bath ultrasonic treatment by adopting a cell breaker, wherein the ultrasonic treatment power is 200 watts, and the ultrasonic treatment time is 10 minutes to obtain the initial emulsion. Wherein, in the mixture 3, the soybean oil accounts for 10 mg/ml, the MCT accounts for 10 mg/ml, the soybean lecithin accounts for 1.2 mg/ml, the glycerol accounts for 2.5 mg/ml, and the polymyxin B sulfate accounts for 1 mg/ml.
And 4, carrying out high-pressure homogenization treatment on the initial emulsion by using a high-pressure homogenizer, wherein the pressure of the high-pressure homogenization treatment is 800 bar, the temperature of the high-pressure homogenization treatment is 4 ℃, and the time of the high-pressure homogenization treatment is 10 minutes, so as to obtain the antibiotic delivery system loaded with polymyxin B sulfate.
Comparative example 1B
Comparative example 1B the yolk lecithin was replaced with dipalmitoyl phosphatidylcholine compared to example 1A, and the rest of the procedure was the same as in example 1A, with the following specific procedures:
step 1, uniformly mixing 30 mg of soybean oil and 30 mg of medium-chain triglyceride to obtain a mixed material 1 as an oil phase;
Step 2, mixing 3.6 mg of DPPC, 7.5 mg of glycerol and 3mg of polymyxin B sulfate to obtain a mixed material 2, dissolving the mixed material 2 by adopting 2 ml of sterile deionized water, and uniformly mixing to obtain a water phase.
And 3, dropwise adding the oil phase in the step 1 into the water phase in the step 2 to obtain a mixed material 3, regulating the volume of the mixed material 3 to 3 milliliters by adopting sterile deionized water, carrying out vortex mixing for 5 minutes, carrying out ice water bath ultrasonic treatment by adopting a cell breaker, wherein the ultrasonic treatment power is 200 watts, and the ultrasonic treatment time is 10 minutes to obtain the initial emulsion. Wherein, in the mixture 3, the soybean oil accounts for 10 mg/ml, the MCT accounts for 10 mg/ml, the DPPC accounts for 1.2 mg/ml, the glycerol accounts for 2.5 mg/ml, and the polymyxin B sulfate accounts for 1 mg/ml.
And 4, carrying out high-pressure homogenization treatment on the initial emulsion by using a high-pressure homogenizer, wherein the pressure of the high-pressure homogenization treatment is 800 bar, the temperature of the high-pressure homogenization treatment is 4 ℃, and the time of the high-pressure homogenization treatment is 10 minutes, so as to obtain the antibiotic delivery system loaded with polymyxin B sulfate.
Comparative example 1C
Comparative example 1C compared to example 1A, no soybean oil was added, and the rest of the procedure was the same as in example 1A, with the following specific operations:
Step 1, using 30 mg of medium-chain triglyceride as an oil phase;
Step 2, mixing 3.6 mg of egg yolk lecithin, 7.5 mg of glycerol and 3 mg of polymyxin B sulfate to obtain a mixed material 1, dissolving the mixed material 2 by adopting 2 ml of sterile deionized water, and uniformly mixing to obtain a water phase.
And 3, dropwise adding the oil phase in the step 1 into the water phase in the step 2 to obtain a mixed material 2, regulating the volume of the mixed material 2 to 3 milliliters by adopting sterile deionized water, carrying out vortex mixing for 5 minutes, carrying out ice water bath ultrasonic treatment by adopting a cell breaker, wherein the ultrasonic treatment power is 200 watts, and the ultrasonic treatment time is 10 minutes to obtain the initial emulsion. Wherein, in the mixed material 3, the MCT accounts for 10 mg/ml, the egg yolk lecithin accounts for 1.2 mg/ml, the glycerol accounts for 2.5 mg/ml, and the polymyxin B sulfate accounts for 1 mg/ml.
And 4, carrying out high-pressure homogenization treatment on the initial emulsion by using a high-pressure homogenizer, wherein the pressure of the high-pressure homogenization treatment is 800 bar, the temperature of the high-pressure homogenization treatment is 4 ℃, and the time of the high-pressure homogenization treatment is 10 minutes, so as to obtain the antibiotic delivery system loaded with polymyxin B sulfate.
Comparative example 1D
Comparative example 1D compared to example 1A, no medium chain triglyceride was added, and the rest was the same as in example 1A, with the following specific operations:
step 1, taking 30 mg of soybean oil as an oil phase;
Step 2, mixing 3.6 mg of egg yolk lecithin, 7.5 mg of glycerol and 3 mg of polymyxin B sulfate to obtain a mixed material 1, dissolving the mixed material 2 by adopting 2 ml of sterile deionized water, and uniformly mixing to obtain a water phase.
And 3, dropwise adding the oil phase in the step 1 into the water phase in the step 2 to obtain a mixed material 2, regulating the volume of the mixed material 2 to 3 milliliters by adopting sterile deionized water, carrying out vortex mixing for 5 minutes, carrying out ice water bath ultrasonic treatment by adopting a cell breaker, wherein the ultrasonic treatment power is 200 watts, and the ultrasonic treatment time is 10 minutes to obtain the initial emulsion. Wherein, in the mixed material 3, the soybean oil accounts for 10 mg/ml, the egg yolk lecithin accounts for 1.2 mg/ml, the glycerol accounts for 2.5 mg/ml, and the polymyxin B sulfate accounts for 1 mg/ml.
And 4, carrying out high-pressure homogenization treatment on the initial emulsion by using a high-pressure homogenizer, wherein the pressure of the high-pressure homogenization treatment is 800 bar, the temperature of the high-pressure homogenization treatment is 4 ℃, and the time of the high-pressure homogenization treatment is10 minutes, so as to obtain the antibiotic delivery system loaded with polymyxin B sulfate.
Comparative example 1E
Comparative example 1E was conducted in the same manner as in example 1A except that no egg yolk lecithin was added in comparison with example 1A, and the specific procedures were as follows:
step 1, uniformly mixing 30 mg of soybean oil and 30 mg of medium-chain triglyceride to obtain a mixed material 1 as an oil phase;
Step 2, mixing 7.5 mg of glycerol and 3 mg of polymyxin B sulfate to obtain a mixed material 2, dissolving the mixed material 2 by adopting 2 ml of sterile deionized water, and uniformly mixing to obtain a water phase.
And 3, dropwise adding the oil phase in the step 1 into the water phase in the step 2 to obtain a mixed material 3, regulating the volume of the mixed material 3 to 3 milliliters by adopting sterile deionized water, carrying out vortex mixing for 5 minutes, carrying out ice water bath ultrasonic treatment by adopting a cell breaker, wherein the ultrasonic treatment power is 200 watts, and the ultrasonic treatment time is 10 minutes to obtain the initial emulsion. Wherein, in the mixture 3, the soybean oil accounts for 10 mg/ml, the MCT accounts for 10 mg/ml, the glycerol accounts for 2.5 mg/ml, and the polymyxin B sulfate accounts for 1 mg/ml.
And 4, carrying out high-pressure homogenization treatment on the initial emulsion by using a high-pressure homogenizer, wherein the pressure of the high-pressure homogenization treatment is 800 bar, the temperature of the high-pressure homogenization treatment is 4 ℃, and the time of the high-pressure homogenization treatment is 10 minutes, so as to obtain the antibiotic delivery system loaded with polymyxin B sulfate.
Comparative example 1F
Comparative example 1F compared to example 1A, no glycerol was added, and the rest was the same as in example 1A, with the following specific operations:
step 1, uniformly mixing 30 mg of soybean oil and 30 mg of medium-chain triglyceride to obtain a mixed material 1 as an oil phase;
Step 2, mixing 3.6 mg of egg yolk lecithin and 3 mg of polymyxin B sulfate to obtain a mixed material 2, dissolving the mixed material 2 by adopting 2 ml of sterile deionized water, and uniformly mixing to obtain a water phase.
And 3, dropwise adding the oil phase in the step 1 into the water phase in the step 2 to obtain a mixed material 3, regulating the volume of the mixed material 3 to 3 milliliters by adopting sterile deionized water, carrying out vortex mixing for 5 minutes, carrying out ice water bath ultrasonic treatment by adopting a cell breaker, wherein the ultrasonic treatment power is 200 watts, and the ultrasonic treatment time is 10 minutes to obtain the initial emulsion. Wherein, in the mixed material 3, the soybean oil accounts for 10 mg/ml, the MCT accounts for 10 mg/ml, the egg yolk lecithin accounts for 1.2 mg/ml, and the polymyxin B sulfate accounts for 1 mg/ml.
And 4, carrying out high-pressure homogenization treatment on the initial emulsion by using a high-pressure homogenizer, wherein the pressure of the high-pressure homogenization treatment is 800 bar, the temperature of the high-pressure homogenization treatment is 4 ℃, and the time of the high-pressure homogenization treatment is10 minutes, so as to obtain the antibiotic delivery system loaded with polymyxin B sulfate.
Comparative example 1G
Comparative example 1G the sonication parameters were changed compared to example 1A, the rest of the procedure being the same as example 1A, the specific procedure being as follows:
step 1, uniformly mixing 30 mg of soybean oil and 30 mg of medium-chain triglyceride to obtain a mixed material 1 as an oil phase;
step 2, mixing 3.6 mg of egg yolk lecithin, 7.5 mg of glycerol and 3 mg of polymyxin B sulfate to obtain a mixed material 2, dissolving the mixed material 2 by adopting 2 ml of sterile deionized water, and uniformly mixing to obtain a water phase.
And 3, dropwise adding the oil phase in the step 1 into the water phase in the step 2 to obtain a mixed material 3, regulating the volume of the mixed material 3 to 3 milliliters by adopting sterile deionized water, carrying out vortex mixing for 5 minutes, carrying out ice water bath ultrasonic treatment by adopting a cell breaker, wherein the ultrasonic treatment power is 50 watts, and the ultrasonic treatment time is1 minute, so as to obtain the initial emulsion. Wherein, in the mixed material 3, the soybean oil accounts for 10 mg/ml, the MCT accounts for 10 mg/ml, the egg yolk lecithin accounts for 1.2 mg/ml, the glycerol accounts for 2.5mg/ml, and the polymyxin B sulfate accounts for 1 mg/ml.
And 4, carrying out high-pressure homogenization treatment on the initial emulsion by using a high-pressure homogenizer, wherein the pressure of the high-pressure homogenization treatment is 800 bar, the temperature of the high-pressure homogenization treatment is 4 ℃, and the time of the high-pressure homogenization treatment is 10 minutes, so as to obtain the antibiotic delivery system loaded with polymyxin B sulfate.
Comparative example 1H
Comparative example 1H the high pressure homogeneity parameters were changed compared to example 1A, the rest of the operations being the same as example 1A, the specific operations being as follows:
step 1, uniformly mixing 30 mg of soybean oil and 30 mg of medium-chain triglyceride to obtain a mixed material 1 as an oil phase;
step 2, mixing 3.6 mg of egg yolk lecithin, 7.5 mg of glycerol and 3 mg of polymyxin B sulfate to obtain a mixed material 2, dissolving the mixed material 2 by adopting 2 ml of sterile deionized water, and uniformly mixing to obtain a water phase.
And 3, dropwise adding the oil phase in the step 1 into the water phase in the step 2 to obtain a mixed material 3, regulating the volume of the mixed material 3 to 3 milliliters by adopting sterile deionized water, carrying out vortex mixing for 5 minutes, carrying out ice water bath ultrasonic treatment by adopting a cell breaker, wherein the ultrasonic treatment power is 200 watts, and the ultrasonic treatment time is 10 minutes to obtain the initial emulsion. Wherein, in the mixed material 3, the soybean oil accounts for 10 mg/ml, the MCT accounts for 10 mg/ml, the egg yolk lecithin accounts for 1.2 mg/ml, the glycerol accounts for 2.5 mg/ml, and the polymyxin B sulfate accounts for 1 mg/ml.
And 4, carrying out high-pressure homogenization treatment on the initial emulsion by using a high-pressure homogenizer, wherein the pressure of the high-pressure homogenization treatment is 200 bar, the temperature of the high-pressure homogenization treatment is 4 ℃, and the time of the high-pressure homogenization treatment is 2 minutes, so as to obtain the antibiotic delivery system loaded with polymyxin B sulfate.
Comparative example 1I
Comparative example 1I the ratio of each substance was changed compared to example 1A, and the rest of the operations were the same as in example 1A, and the specific operations were as follows:
step 1, uniformly mixing 30 mg of soybean oil and 30 mg of medium-chain triglyceride to obtain a mixed material 1 as an oil phase;
step 2, mixing 3.6 mg of egg yolk lecithin, 7.5 mg of glycerol and 3 mg of polymyxin B sulfate to obtain a mixed material 2, dissolving the mixed material 2 by adopting 2 ml of sterile deionized water, and uniformly mixing to obtain a water phase.
And 3, mixing the water phase and the oil phase in the step 1 to obtain a mixed material 3, regulating the volume of the mixed material 3 to 30 milliliters by adopting sterile deionized water, carrying out vortex mixing for 5 minutes, carrying out ice water bath ultrasonic treatment by adopting a cell disruption instrument, wherein the ultrasonic treatment power is 200 watts, and the ultrasonic treatment time is 10 minutes, so as to obtain the initial emulsion. Wherein, in the mixture 3, the soybean oil accounts for 1 mg/ml, the MCT accounts for 1 mg/ml, the egg yolk lecithin accounts for 0.12 mg/ml, the glycerol accounts for 0.25 mg/ml, and the polymyxin B sulfate accounts for 0.1 mg/ml.
And 4, carrying out high-pressure homogenization treatment on the initial emulsion by using a high-pressure homogenizer, wherein the pressure of the high-pressure homogenization treatment is 800 bar, the temperature of the high-pressure homogenization treatment is 4 ℃, and the time of the high-pressure homogenization treatment is 10 minutes, so as to obtain the antibiotic delivery system loaded with polymyxin B sulfate.
Comparative example 1J
Comparative example 1J the ratio of each substance was changed compared to example 1A, and the rest of the operations were the same as in example 1A, and the specific operations were as follows:
step 1, uniformly mixing 3 g of soybean oil and 3 g of medium-chain triglyceride to obtain a mixed material 1 serving as an oil phase;
Step 2, mixing 360 mg of egg yolk lecithin, 750 mg of glycerol and 300 mg of polymyxin B sulfate to obtain a mixed material, dissolving the mixed material 2 by adopting 5ml of sterile deionized water, and uniformly mixing to obtain a water phase. Wherein, in the mixed material 3, the soybean oil accounts for 100 mg/ml, the MCT accounts for 100 mg/ml, the egg yolk lecithin accounts for 12 mg/ml, the glycerol accounts for 25 mg/ml, and the polymyxin B sulfate accounts for 10 mg/ml.
And 3, mixing the water phase and the oil phase in the step 1 to obtain a mixed material 3, regulating the volume of the mixed material 3 to 30 milliliters by adopting sterile deionized water, carrying out vortex mixing for 5 minutes, carrying out ice water bath ultrasonic treatment by adopting a cell disruption instrument, wherein the ultrasonic treatment power is 200 watts, and the ultrasonic treatment time is 10 minutes, so as to obtain the initial emulsion.
And 4, carrying out high-pressure homogenization treatment on the initial emulsion by using a high-pressure homogenizer, wherein the pressure of the high-pressure homogenization treatment is 800 bar, the temperature of the high-pressure homogenization treatment is 4 ℃, and the time of the high-pressure homogenization treatment is 10 minutes, so as to obtain the antibiotic delivery system loaded with polymyxin B sulfate.
EXAMPLE 2 characterization of the Performance of antibiotic delivery System
The method comprises the steps of performing morphology characterization on an antibiotic drug delivery system prepared in an embodiment 1A, an embodiment 1B, an embodiment 1C and an embodiment 1D, wherein characterization results refer to fig. 1 to 4, characterization results refer to fig. 1, fig. 1 is a transmission electron microscope morphology graph of the antibiotic drug delivery system loaded with polymyxin B sulfate provided by the embodiment of the application, fig. 2 is a transmission electron microscope morphology graph of the antibiotic drug delivery system loaded with tobramycin provided by the embodiment of the application, fig. 3 is a transmission electron microscope morphology graph of the antibiotic drug delivery system loaded with amphotericin B provided by the embodiment of the application, and fig. 4 is a transmission electron microscope morphology graph of the antibiotic drug delivery system loaded with polymyxin sodium mesylate provided by the embodiment of the application, wherein the morphology of the antibiotic drug delivery system is uniform and spherical. The experimental manner of performance characterization of the polymyxin-loaded nanoemulsion adopted in this embodiment is the same as the technical means commonly used in the art, and will not be described here again.
The antibiotic delivery systems prepared in examples 1A-1D, and comparative examples 1A-1J were then characterized for performance, and the encapsulation efficiency, drug loading, particle size, PDI (Polymer Dispersion Index ), potential, and stability were each tested, and the test results are shown in table 1.
Table 1 characterization of the performance of the antibiotic delivery systems prepared in example 1A-example 1D, and comparative example 1A-comparative example 1J
As can be seen from the results in Table 1, the antibiotic delivery system prepared by the preparation method provided by the application is used as an inhalation type emulsion, has high-efficiency loading rate on the antibiotic, and has good aerosol inhalation performance and good stability. The proportion of soybean oil, MCT and glycerin in the antibiotic drug delivery system, ultrasonic power and time influence the encapsulation efficiency and drug loading, larger ultrasonic power can enable the particle size of the emulsion to be smaller and reach the nanometer level, but excessive ultrasonic power can influence the physical stability of nanoemulsion to cause precipitation and separation of the emulsion in the storage process, and the emulsion with uniform particle size is formed by proper homogenizing pressure and long-time homogenizing time. In addition, it can be seen that the ratio of each substance in the antibiotic delivery system is a key factor for successfully preparing the delivery system, and when the ratio of each substance in the antibiotic delivery system is too small (comparative example 1I), the emulsion is unstable, turbidity and precipitation are easy to generate, and the preparation cannot be successfully performed. At higher levels of each substance in the antibiotic delivery system (comparative example 1J), the emulsion appeared to precipitate slightly after 4 months of storage at 4℃and regained clarity after shaking mixing. Overall, higher ratio excipients are difficult to obtain drug-loaded emulsions with better stability.
Therefore, according to the preparation method, through reasonable design and proportion of each parameter, wherein the optimal parameter proportion is in the embodiment 1A, specifically, in the prepared antibiotic drug 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, polymyxin B accounts for 1 mg/ml, vortex mixing time is 5 minutes, the cell breaker ice water bath ultrasound is adopted, the power of ultrasound treatment is 200 watts, the time of ultrasound treatment is 10 minutes, the pressure of high-pressure homogenization treatment is 800 bars, the temperature of high-pressure homogenization treatment is 4 ℃, the time of high-pressure homogenization treatment is 10 minutes, and the antibiotic drug delivery system loaded with polymyxin B can be prepared, and the antibiotic drug delivery system with good storage stability for 6 months at 4 ℃, the dispersion index is 0.153, and the encapsulation rate can reach more than 99%.
Example 3 characterization of drug delivery efficiency and drug retention of antibiotic delivery System
The preparation method of example 1A was used to prepare a drug delivery system for the antibiotic coated with Cy 5-labeled polymyxin sulfate B, cy 5-labeled polymyxin sulfate B was used as a control group, a mouse micro-aerosol inhalation device was used to quantitatively administer the same amount of polymyxin sulfate B to mice, and after 48 hours of administration, the in vivo fluorescence signal distribution of each group of mice was observed by a small animal in vivo imager to analyze the drug delivery efficiency and the drug retention condition of lung tissues. As can be seen from fig. 5 (the left side of fig. 5 shows the fluorescence signal distribution results of the isolated organs of the mice, and the right side of fig. 5 shows the fluorescence signal quantification results of free polymyxin B sulfate and polymyxin B sulfate delivery systems in the lung tissues of the mice), the polymyxin B sulfate antibiotic delivery system can significantly enhance the accumulation of antibiotics in the lung tissues of the mice and prolong the exposure time of antibiotics in the lung tissues of the mice.
Example 4 investigation of antibacterial Activity of a polymyxin B sulfate-loaded antibiotic delivery System against carbapenem-resistant Acinetobacter baumannii
The antibiotic delivery system carrying polymyxin B sulfate was prepared by the preparation method of example 1A, acinetobacter baumannii with carbapenems was cultured, polymyxin B sulfate of the same concentration was set in two experiments and the antibiotic delivery system carrying polymyxin B sulfate was divided into a Control group (Control), a free polymyxin B sulfate group (PMB group) and a polymyxin B sulfate antibiotic delivery system group (PMB-NEs group), and incubated with Acinetobacter baumannii (bacterial count: 1.0X105 cfu/ml) for 12 h, respectively, and bacterial growth after two groups of drug treatments was observed. As can be seen from the results of fig. 6, the Control group has more acinetobacter baumannii colonies, and the Control group is next to the PMB group, and the PMB-NEs group has no acinetobacter baumannii colonies, so that compared with the Control group (Control) and the PMB group, the PMB-NEs group has the optimal sterilization effect, can achieve the effect of sterile growth, and shows that the antibiotic delivery system loaded with polymyxin B sulfate can remarkably improve the sterilization effect.
The above detailed description of the preparation method and application of the antibiotic delivery system provided by the present application has been provided, and specific examples are provided herein to illustrate the principles and embodiments of the present application, and the above examples are provided to assist in understanding the method and core concept of the present application, and meanwhile, to those skilled in the art, according to the concept of the present application, there are variations in the specific embodiments and application ranges, so that the disclosure should not be construed as limiting the application.