Amphotericin B liposome and preparation method thereof
By preparing amphotericin B liposomes containing glycyrrhetinic acid and hydroxyapatite nanoparticles, the problems of low encapsulation efficiency, poor stability and insufficient targeting of existing liposomes were solved, achieving efficient enrichment and long-term stability of the drug at the site of infection and reducing toxic side effects.
Patent Information
- Application Number
- CN202511405125.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-29
- Publication Date
- 2025-12-05
AI Technical Summary
Existing amphotericin B liposomes suffer from problems such as low encapsulation efficiency, poor stability, short circulating half-life, lack of targeting ability, and decreased drug activity during storage, leading to toxic side effects and poor efficacy.
Liposomes composed of hydrogenated soybean phospholipids, cholesterol, mPEG2000-DSPE, ceramide, glycyrrhetinic acid, hydroxyapatite nanoparticles, and a lyophilization protectant were prepared using ultrasonic dispersion, rotary evaporation, gradient pH encapsulation, and stepwise lyophilization. The targeting effect of glycyrrhetinic acid and the complex formation of hydroxyapatite nanoparticles, combined with the synergistic effect of the lyophilization protectant, improved the drug accumulation rate and stability at the infection site.
It improved the drug accumulation rate at the infection site, reduced the drug crystallization rate, extended the drug's shelf life, enhanced the stability of liposomes and the efficiency of drug encapsulation, and reduced toxic side effects.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of liposome manufacturing, in particular, to a liposome of amphotericin B and a preparation method thereof. BACKGROUND
[0002] Amphotericin B (AmB) is a polyene antifungal antibiotic produced by Streptomyces, which has broad-spectrum and strong antifungal activity, and can inhibit or kill various pathogenic fungi such as Candida, Aspergillus, Cryptococcus, etc. It is a first-line drug for the treatment of deep fungal infections.
[0003] However, amphotericin B has serious pharmacological defects: it has extremely poor water solubility and strong affinity for mammalian cell membranes, and can easily cause serious adverse reactions such as renal toxicity, liver toxicity, hemolysis, etc. after intravenous injection, which greatly limits its clinical application. In order to improve the above problems, researchers have developed liposome preparations of amphotericin B, which encapsulate the drug in the bilayer lipid membrane to form vesicles, utilize the passive targeting property (EPR effect) of the liposome to reduce the distribution of the drug in normal tissues, reduce the toxic side effects, and at the same time improve the solubility and bioavailability of the drug.
[0004] At present, the preparation methods of amphotericin B liposomes mainly include the film dispersion method, the reverse evaporation method, the ethanol injection method, the freeze-thaw method, etc. Among them, the film dispersion method is one of the commonly used methods in laboratories and industrial production due to its relatively simple operation, but it has the problem of low encapsulation efficiency (usually less than 85%), and free drugs can easily cause toxic side effects; although the reverse evaporation method can improve the encapsulation efficiency, it needs to use a large amount of organic solvent, and needs to go through multiple centrifugation and washing steps to remove free drugs and residual solvents, which is complex and time-consuming, and difficult to realize large-scale production.
[0005] At the same time, the existing amphotericin B liposomes also face the technical bottleneck of poor stability: on the one hand, the liposome is prone to lipid membrane fusion and drug leakage during storage (the leakage rate is generally ≥5% after 3 months of storage at 4℃), and the particle size fluctuates greatly after reconstitution; on the other hand, amphotericin B molecules are prone to crystallization in the liposome, leading to a decrease in drug activity, and the drug efficacy retention rate is often less than 80% after 6 months of storage. In addition, conventional liposomes are easily cleared by the body's mononuclear-phagocyte system (MPS), have a short circulation half-life (mostly ≤4h), and the bioavailability needs to be further improved; and lack specific targeting ability to fungal infection sites, and the enrichment of drugs in non-infected tissues still poses a certain risk of toxic side effects.
[0006] Therefore, it becomes a technical problem to be solved by those skilled in the art to provide an amphotericin B liposome and a preparation method thereof. SUMMARY
[0007] In view of this, the present application proposes a liposome of amphotericin B, aiming to solve at least one of the problems in the background art.
[0008] The present application proposes a liposome of amphotericin B, comprising the following components by mass fraction: amphotericin B 1 part, hydrogenated soybean phospholipid (HSPC) 5-10 parts, cholesterol 2-4 parts, mPEG2000-DSPE 1-3 parts, ceramide 0.5-1.5 parts, glycyrrhetinic acid 0.3-0.8 parts, hydroxyapatite nanoparticles (nHAP) 0.2-0.5 parts, and lyophilization protectant 8-15 parts.
[0009] The present application also provides a preparation method of the liposome of amphotericin B as claimed in claim 1, comprising the following preparation steps: The hydrogenated soybean phospholipid (HSPC), cholesterol, mPEG2000-DSPE, ceramide, and glycyrrhetinic acid are added to a chloroform-methanol mixed solution, and after stirring to complete dissolution, the hydroxyapatite nanoparticles (nHAP) are added for ultrasonic dispersion treatment to obtain a lipid-nHAP complex solution; The amphotericin B is dissolved in a phosphate buffer solution with pH 8.0 to obtain a high-pH drug solution, ready for use; The lipid-nHAP complex solution is subjected to rotary evaporation treatment and vacuum drying to obtain a complex lipid film, a low-pH buffer solution is added to the complex lipid film, and after constant-temperature water bath oscillation, the high-pH drug solution is added dropwise to obtain a crude liposome suspension; The crude liposome suspension is subjected to ultrasonic emulsification treatment and then homogenization treatment, the lyophilization protectant is added to the homogenized crude liposome suspension, stirred to dissolve, and then subjected to freeze-drying treatment to obtain the liposome of amphotericin B.
[0010] Preferably, the ultrasonic dispersion treatment has an ultrasonic power of 150 W and an ultrasonic dispersion time of 10 minutes.
[0011] Preferably, the rotary evaporation has a temperature of 35°C, a vacuum degree of 0.08 MPa, and a rotary evaporation time of 45 minutes.
[0012] Preferably, the low-pH buffer solution is a phosphate buffer solution with pH 5.0 containing 0.01% EDTA and 0.025% vitamin E.
[0013] Preferably, when the high-pH drug solution is added dropwise, it is added at a rate of 1 ml / min, stirring while adding, the stirring speed is 300 rpm, when the high-pH drug solution reaches 90% of the total volume, the adding speed is adjusted to 0.2 ml / min, the remaining high-pH drug solution is continuously added, and after the addition is completed, oscillation is performed for 15 minutes.
[0014] Preferably, the ultrasonic emulsification ultrasonic power is 250W, the pulse mode is 5s / 3s, and the total ultrasonic time is 10 minutes.
[0015] Preferably, the homogenization treatment is that the suspension after ultrasonic emulsification is repeatedly extruded 3 times through a 0.22 mu polycarbonate microporous filter membrane.
[0016] Preferably, the freeze-drying protective agent is prepared by mixing sucrose, mannitol and trehalose in a mass ratio of 1:1:0.5.
[0017] Preferably, the freeze-drying treatment is: first pre-freezing at-20 DEG C for 1 hour, then reducing the temperature to-40 DEG C and freezing for 2 hours, then vacuum sublimation drying at a temperature of-20 DEG C and a vacuum degree of 10 Pa for 12 hours, and finally vacuum desorption drying at a temperature of 25 DEG C and a vacuum degree of 5 Pa for 4 hours.
[0018] Compared with the prior art, the present application has the beneficial effects that: 1. By adding glycyrrhetic acid, the specific targeting effect of glycyrrhetic acid is used to improve the in vitro binding rate of the liposome to the fungal cells, thereby improving the enrichment rate of the drug at the infection site, and effectively solving the core problem of "non-targeted distribution leading to kidney toxicity" of traditional preparations.
[0019] 2. The hydroxyapatite nanoparticles in the present application adsorb drug molecules to form "drug-nHAP complex", and the synergistic anti-crystallization effect of the freeze-drying protective agent greatly reduces the drug crystallization rate; in combination with the antioxidant effect of vitamin E, the drug shelf life of the liposome is improved, the industry pain point of drug efficacy decline during storage is solved, and the product shelf life is extended. DETAILED DESCRIPTION
[0020] The various illustrative embodiments of the present application will now be described in detail in connection with the following figures. This description is not to be considered limiting in scope, but rather as being illustrative of the various aspects, features and embodiments of the present application. It is understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.
[0021] In addition, for the numerical ranges in the present application, it is understood that each intermediate value between the upper limit and the lower limit of the range is specifically disclosed. Each smaller range within any stated range or within any stated intermediate value is also included in the present application. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.
[0022] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art to which this application pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All publications mentioned in this specification are herein incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. In case of conflict between the content of the specification and that of any incorporated literature, the content of the specification controls.
[0023] Many modifications and variations of this application of the application can be made without departing from its spirit or scope, which will be apparent to those skilled in the art. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only.
[0024] As used herein, the terms "comprise", "comprising", "include", "including", "have", "having" and the like are open-ended and do not exclude additional elements or steps.
[0025] The present application provides a liposome of amphotericin B, comprising the following components in mass fraction: amphotericin B 1 part, hydrogenated soybean phospholipid (HSPC) 5-10 parts, cholesterol 2-4 parts, mPEG2000-DSPE 1-3 parts, ceramide 0.5-1.5 parts, glycyrrhetinic acid 0.3-0.8 parts, hydroxyapatite nanoparticles (nHAP) 0.2-0.5 parts, and lyophilization protectant 8-15 parts.
[0026] The mass fraction of the liposome of amphotericin B is preferably: amphotericin B 1 part, hydrogenated soybean phospholipid (HSPC) 8 parts, cholesterol 3 parts, mPEG2000-DSPE 2 parts, ceramide 1 part, glycyrrhetinic acid 0.5 part, hydroxyapatite nanoparticles (nHAP) 0.3 part, and lyophilization protectant 12 parts.
[0027] The functions of the components of the present application are as follows: Amphotericin B is an active pharmaceutical ingredient, which plays an antifungal role; hydrogenated soybean phospholipid (HSPC) is a main membrane material, which constructs a lipid bilayer and improves the stability of the membrane structure; cholesterol can regulate the membrane fluidity, reduce the accumulation of lipid molecules, and reduce the risk of drug leakage; mPEG2000-DSPE can modify the surface of the lipid membrane through PEGylation, reduce the clearance of the monocyte-macrophage system, and prolong the circulating half-life in the body; ceramide can enhance the rigidity of the lipid membrane, reduce the pore size of the membrane, and improve the drug encapsulation efficiency; glycyrrhetinic acid is a natural targeting molecule, which can specifically bind to the ergosterol on the surface of the fungal cell membrane, and guide the liposome to the infection site; hydroxyapatite nanoparticles (nHAP) are biocompatible nanocarriers, which can adsorb drug molecules to form stable complexes, inhibit drug crystallization, and enhance the mechanical strength of the lipid membrane; the freeze-drying protective agent is sucrose-mannitol-fucrose (mass ratio 1:1:0.5), which can reduce the damage to the lipid membrane during the freeze-drying process, inhibit drug crystallization, and improve the reconstitution stability through synergistic effect.
[0028] It can be understood that, by adding glycyrrhetinic acid, the specific targeting effect of glycyrrhetinic acid is utilized, so that the in vitro binding rate of the liposome of the present application to fungal cells is improved, and the enrichment rate of the drug at the infection site is improved, effectively solving the core problem of "non-targeted distribution leading to kidney toxicity" of traditional preparations.
[0029] It can be understood that, in the present application, the hydroxyapatite nanoparticles adsorb drug molecules to form "drug-nHAP complexes", and the synergistic anti-crystallization effect of the freeze-drying protective agent, so that the drug crystallization rate is greatly reduced; in combination with the antioxidant effect of vitamin E, the drug effective period of the liposome is improved, the industry pain point of drug efficacy decline during storage is solved, and the product effective period is prolonged.
[0030] The present application also provides a preparation method of the amphotericin B liposome, comprising the following preparation steps: The hydrogenated soybean phospholipid (HSPC), cholesterol, mPEG2000-DSPE, ceramide and glycyrrhetinic acid are added to a chloroform-methanol mixed solution, stirred until completely dissolved, and then the hydroxyapatite nanoparticles (nHAP) are added for ultrasonic dispersion treatment to obtain a lipid-nHAP complex solution; The amphotericin B is dissolved in a pH 8.0 phosphate buffer solution to obtain a high-pH drug solution, which is ready for use; The lipid-nHAP complex solution is subjected to rotary evaporation treatment and vacuum drying to obtain a complex lipid film, a low-pH buffer solution is added to the complex lipid film, and then the high-pH drug solution is added dropwise after constant temperature water bath oscillation to obtain a crude liposome suspension; The crude liposome suspension is subjected to ultrasonic emulsification treatment and then homogenization treatment, the freeze-drying protective agent is added to the homogenized crude liposome suspension, stirred until dissolved, and then subjected to freeze-drying treatment to obtain the amphotericin B liposome.
[0031] Specifically, the hydrogenated soybean phospholipid (HSPC), cholesterol, mPEG2000-DSPE, ceramide, and glycyrrhetinic acid are added to a chloroform-methanol mixed solution at a solid-liquid ratio of 1:15 (g / ml), and the mixture is magnetically stirred at 250 rpm at 25°C until the lipid components are completely dissolved, forming a clear and transparent lipid solution.
[0032] Specifically, the volume ratio of chloroform to methanol in the chloroform-methanol mixed solution is 2:1.
[0033] Specifically, the concentration of amphotericin B in the high-pH drug solution is 0.8 mg / ml.
[0034] In the present application, the ultrasonic power of the ultrasonic dispersion treatment is preferably 150 W, and the ultrasonic dispersion time is preferably 10 minutes.
[0035] Specifically, a probe-type ultrasonic instrument is used to perform ultrasonic dispersion at a power of 150 W for 10 minutes in an ice water bath.
[0036] It can be understood that the combination of 150 W power and 10 min duration can ensure that the hydroxyapatite nanoparticles (nHAP) are sufficiently dispersed and have no obvious aggregation, so that they can be uniformly combined with lipid molecules to form a stable "lipid-nHAP complex solution", laying a foundation for subsequent improvement of drug encapsulation efficiency and inhibition of drug crystallization. At the same time, the ice water bath controls the temperature, which can avoid local heating during ultrasonic treatment, leading to oxidation of lipid components or decrease in dispersion stability of nHAP, ensuring the stability of the properties of the system components and maintaining the uniformity of the complex solution.
[0037] In the present application, the temperature of the rotary evaporation is 35°C, the vacuum degree is 0.08 MPa, and the rotary evaporation time is 45 minutes.
[0038] Specifically, the lipid-nHAP complex solution is transferred to a rotary evaporator, and rotary evaporation is performed at 35°C under a vacuum degree of 0.08 MPa for 45 minutes, so that the solvent is gradually volatilized, forming a uniform and transparent lipid-nHAP complex film on the inner wall of the flask. Subsequently, vacuum drying is continued for 1 hour to completely remove the residual organic solvent.
[0039] It can be understood that the combination of 35℃ temperature and 0.08 MPa vacuum degree can not only ensure efficient volatilization of the chloroform-methanol mixed solvent, but also avoid oxidation of lipid components or agglomeration of hydroxyapatite nanoparticles caused by high temperature, thereby maintaining the stability of the system; 45 min of rotary evaporation makes the solvent volatilize gradually, which is beneficial to the formation of a uniform and transparent "lipid-nHAP composite film", thereby laying a structural foundation for the ordered arrangement of lipid molecules and the improvement of drug encapsulation rate during subsequent drug hydration; and the continued vacuum drying for 1 h can completely remove the residual organic solvent, which not only avoids the influence of the solvent on the stability of the drug and the potential toxic side effects, but also ensures the uniformity of the film drying degree and reduces the quality fluctuation between subsequent batches.
[0040] In the present application, the low-pH buffer is preferably a phosphate buffer solution with pH 5.0 containing 0.01% EDTA and 0.025% vitamin E.
[0041] Specifically, the low-pH buffer solution with pH 5.0 containing 0.01% EDTA and 0.025% vitamin E preheated to 37℃ is added to the composite lipid film, and the film is placed in a 37℃ constant temperature water bath for oscillation for 15 min (oscillation frequency 120 times / min), so that the film is preliminarily hydrated to form loose lipid vesicles.
[0042] The addition amount of the low-pH buffer is 60 times the total mass of hydrogenated soybean phospholipid (HSPC), cholesterol, mPEG2000-DSPE, ceramide and glycyrrhetinic acid.
[0043] It can be understood that the low-pH buffer preheated to 37℃ is consistent with the temperature of the constant temperature water bath oscillation, which is close to the lipid phase transition temperature, which is beneficial to the uniform hydration of the composite lipid film and avoids uneven hydration caused by temperature difference; 0.01% EDTA in the buffer can chelate metal ions, and 0.02% vitamin E can inhibit oxidation, thereby protecting the stability of lipid components and subsequent drugs in the early stage of hydration; the addition amount of 60 times the total mass of lipid can provide sufficient hydration environment for the film, promote the ordered expansion of lipid molecules to form loose lipid vesicles, and create a good structural foundation for subsequent gradient pH drug encapsulation, without increasing the time consumption of subsequent ultrasonic emulsification or reducing the concentration of liposomes due to excessive use amount, thereby balancing the process efficiency and the rationality of product concentration.
[0044] In the present application, when the high-pH drug solution is added dropwise, the addition rate is 1 ml / min, the solution is stirred during the addition, the stirring speed is 300 rpm, the addition rate is adjusted to 0.2 ml / min when the high-pH drug solution is added to 90% of the total volume, the remaining high-pH drug solution is continuously added, and the solution is oscillated for 15 minutes after the addition is completed.
[0045] Specifically, in the composite lipid film, a phosphate buffer solution containing 0.01% EDTA and 0.025% vitamin E at pH 5.0 preheated to 37℃ is added, and placed in a 37℃ constant temperature water bath oscillator for 15min (oscillation frequency 120 times / min), then the prepared high pH drug solution is slowly added, the drop rate is controlled at 1mL / min, and magnetic stirring (speed 300rpm) is carried out during the drop, and the pH value of the system is monitored in real time; when the total volume is 90%, the drop rate is slowed down to 0.2mL / min, and the drop and pH monitoring are continued until the pH is stable at 6.5~7.5 (usually the pH can reach the standard after the drop is completed, if it does not reach the standard, a small amount of pH 8.0 buffer solution can be added for fine adjustment, but the amount of supplement should not exceed 5% of the total volume, to avoid excess or insufficient drug); after the drop is completed, continue to oscillate for 15min.
[0046] It can be understood that the initial drop rate of 1mL / min can ensure uniform addition of the high pH drug solution, avoid local pH sudden change to destroy the lipid vesicle structure formed by preliminary hydration; when the drop rate is slowed down to 0.2mL / min at 90%V, it can precisely control the pH value to approach the target range of 6.5~7.5 stably, prevent the pH from exceeding the standard due to too fast rate; 300rpm magnetic stirring can promote the system to mix fully, so that the drug molecules and lipid vesicles are in uniform contact; real-time pH monitoring combined with a small amount of buffer fine adjustment (supplement amount ≤5%V) can not only ensure that the final pH is stable in the appropriate interval, but also avoid excess or insufficient drug; 15min oscillation after drop can further utilize the dissociation difference of the drug at different pH values, promote the ionic drug molecules to enter the lipid vesicle interior efficiently, provide key guarantee for realizing high encapsulation rate, and improve batch-to-batch quality stability.
[0047] In the present application, the ultrasonic power of the ultrasonic emulsification is 250W, the pulse mode is 5s working / 3s intermittent, and the total ultrasonic time is 10 minutes.
[0048] Specifically, the probe-type ultrasonic instrument is used for ultrasonic treatment of the crude liposome suspension, and the parameters are set as follows: power 250W, pulse mode (5s working / 3s intermittent), total time 10 minutes, and the system temperature is controlled to be less than or equal to 40℃ in ice water bath throughout the process.
[0049] It can be understood that the combination of 250W power, pulse mode (5s working / 3s intermittent) and 10min total time can not only efficiently break the large size vesicles in the crude liposome suspension, promote uniform reduction of the liposome particle size, lay a foundation for subsequent uniformization of filter membrane extrusion, but also avoid local excessive energy concentration caused by continuous ultrasonic through pulse intermittent; the temperature is controlled to be ≤40℃ in ice water bath throughout the process, which can effectively prevent the drug thermal degradation and lipid membrane structure damage caused by temperature rise during ultrasonic process, and guarantee the integrity of the liposome and the drug activity.
[0050] In the present application, the homogenization treatment is to pass the suspension after ultrasonic emulsification through a 0.22 μm polycarbonate microporous filter membrane and repeatedly extrude 3 times.
[0051] It can be understood that the pore size of the 0.22 μm polycarbonate microporous filter membrane matches the target liposome particle size (80-120 nm), and by repeatedly extruding 3 times, a small amount of large-sized liposomes that may still exist after ultrasonic treatment can be further screened and homogenized, the final particle size distribution PDI is ensured to be stable ≤0.12, the colloidal stability of the liposome system is improved, and the physical retention effect of the filter membrane can remove the microbial impurities that may be left in the suspension, achieve preliminary sterilization, meet the sterile requirements of intravenous injection preparations, and the extrusion process is gentle and will not damage the liposome membrane structure and the drug encapsulation state, thereby ensuring the product quality.
[0052] In the present application, the freeze-drying protectant is prepared by mixing sucrose, mannitol and trehalose at a mass ratio of 1:1:0.5.
[0053] It can be understood that sucrose and trehalose, as small molecule sugars, can wrap liposomes by forming a glassy matrix during the freeze-drying process, reduce the damage of ice crystal growth to the lipid membrane structure, and the two can synergistically enhance the protection effect on the membrane; mannitol can not only maintain the white and loose form of the product after freeze-drying to avoid caking, but also can assist in improving the reconstitution speed; the three are mixed at a mass ratio of 1:1:0.5, which can not only inhibit the crystallization of amphotericin B drug by the hydrogen bond effect of sucrose and trehalose, but also can ensure the integrity of the liposomes by the skeleton support effect of mannitol.
[0054] In the present application, the freeze-drying treatment is: first pre-freezing at -20℃ for 1 hour, then reducing the temperature to -40℃ for freezing for 2 hours, then vacuum sublimation drying at a temperature of -20℃ and a vacuum degree of 10 Pa for 12 hours, and finally vacuum desorption drying at a temperature of 25℃ and a vacuum degree of 5 Pa for 4 hours.
[0055] Specifically, the freeze-drying protectant is added to the homogenized coarse liposome suspension and magnetically stirred until completely dissolved, and then the sample is divided into 2 ml vials, and the sample is placed in a freeze-drying machine, pre-frozen at -20℃ for 1 h to slowly form fine ice crystals in the system; then reduced to -40℃ for deep freezing for 2 h to ensure that the system is completely solidified; then vacuum sublimation drying (temperature -20℃, vacuum degree 10 Pa, time 12 h) is performed to remove most of the free water; finally, vacuum desorption drying (temperature 25℃, vacuum degree 5 Pa, time 4 h) is performed.
[0056] It can be understood that pre-freezing at -20℃ for 1h makes the system slowly form fine ice crystals, which can avoid the destruction of the lipid membrane structure by large ice crystals generated by rapid freezing, and ensure the integrity of the liposome; further freezing at -40℃ for 2h can ensure complete solidification of the system, providing a stable basis for subsequent vacuum drying, preventing local melting and collapse during drying; then vacuum sublimation drying at -20℃, 10Pa for 12h can efficiently remove most of the free water in a low-temperature environment, avoiding drug degradation due to high temperature; finally, vacuum desorption drying at 25℃, 5Pa for 4h can deeply remove the bound water in the system, improve the dryness of the freeze-dried powder, prevent moisture absorption and caking during storage, and keep the freeze-dried powder white and loose together with the freeze-drying protectant, ensuring the product quality and long-term storage stability.
[0057] Example 1 S1, 8 parts of hydrogenated soybean phospholipid (HSPC), 3 parts of cholesterol, 2 parts of mPEG2000-DSPE, 1 part of ceramide, and 0.5 parts of glycyrrhetinic acid were added to a chloroform-methanol mixed solution at a solid-liquid ratio of 1:15 (g / ml), and the mixture was stirred at 250 rpm and 25℃ until the lipid components were completely dissolved. After adding 0.3 parts of hydroxyapatite nanoparticles (nHAP), the mixture was ultrasonically dispersed for 10 minutes at 150W in an ice water bath to obtain a lipid-nHAP complex solution; S2, the lipid-nHAP complex solution was transferred to a rotary evaporator, and the temperature was set to 35℃ and the vacuum degree was set to 0.08MPa. The solvent was evaporated gradually by rotary evaporation for 45min, and a uniform transparent "lipid-nHAP complex film" was formed on the inner wall of the flask. Vacuum drying was continued for 1h to obtain a composite lipid film; S3, 1 part of amphotericin B was dissolved in a pH 8.0 phosphate buffer solution to prepare a high-pH drug solution with a concentration of 0.8mg / ml, which was ready for use; S4, 870 parts of preheated 37℃ phosphate buffer solution containing 0.01% EDTA and 0.025% vitamin E at pH 5.0 was added to the composite lipid film, and the mixture was oscillated at 120 times / min for 15min in a 37℃ constant temperature water bath. Then the magnetic stirrer was started and the stirring speed was set to 300rpm. Then the high-pH drug solution was added at a speed of 1ml / min. When the total volume reached 90%, the addition rate was slowed down to 0.2mL / min, and the whole high-pH drug solution was added. A crude liposome suspension was obtained; S5, the crude liposome suspension was ultrasonically treated at 250W for 10 minutes in a pulse mode of 5 seconds working and 3 seconds intermittent, and the system temperature was controlled ≤40℃ by ice water bath during the whole process. Then the ultrasonically emulsified suspension was filtered through a 0.22μm polycarbonate microporous filter and repeatedly squeezed for 3 times; S6, the extruded crude liposome suspension is added to 12 parts of a freeze-drying protectant, and after magnetic stirring until complete dissolution, the sample is divided into 2 ml vials. The divided sample is placed in a freeze dryer, pre-frozen at -20°C for 1 h, then reduced to -40°C for deep freezing for 2 h, followed by vacuum sublimation drying (temperature -20°C, vacuum degree 10 Pa, time 12 h), and finally vacuum desorption drying (temperature 25°C, vacuum degree 5 Pa, time 4 h) to obtain the amphotericin B liposome.
[0058] Example 2 S1, 5 parts of hydrogenated soybean phospholipid (HSPC), 2 parts of cholesterol, 1 part of mPEG2000-DSPE, 0.5 parts of ceramide, and 0.3 parts of glycyrrhetinic acid are added to a chloroform-methanol mixed solution at a solid-liquid ratio of 1:15 (g / ml), and magnetic stirring is performed at 250 rpm and 25°C until the lipid components are completely dissolved. After adding 0.2 parts of hydroxyapatite nanoparticles (nHAP), ultrasonic dispersion is performed at 150 W for 10 minutes in an ice water bath to obtain a lipid-nHAP complex solution; S2, the lipid-nHAP complex solution is transferred to a rotary evaporator, and the temperature is set to 35°C and the vacuum degree is set to 0.08 MPa. Rotary evaporation is performed for 45 min to gradually evaporate the solvent, and a uniform transparent "lipid-nHAP complex film" is formed on the inner wall of the flask. Vacuum drying is continued for 1 h to obtain a composite lipid film; S3, 1 part of amphotericin B is dissolved in a pH 8.0 phosphate buffer solution to prepare a high-pH drug solution with a concentration of 0.8 mg / ml, which is ready for use; S4, 870 parts of a preheated pH 5.0 phosphate buffer solution containing 0.01% EDTA and 0.025% vitamin E at 37°C is added to the composite lipid film, and oscillation is performed at 120 times / min for 15 min in a 37°C constant temperature water bath. Then the magnetic stirrer is started and the stirring speed is set to 300 rpm. Then the high-pH drug solution is added at a rate of 1 ml / min. When the total volume reaches 90%, the addition rate is slowed down to 0.2 mL / min. The entire high-pH drug solution is added to obtain a crude liposome suspension; S5, the crude liposome suspension is ultrasonically treated at a power of 250 W for 10 minutes in a pulse mode of 5 seconds of work and 3 seconds of rest. The system temperature is controlled at ≤40°C throughout the ice water bath. Then the ultrasonically emulsified suspension is passed through a 0.22 μm polycarbonate microporous filter and repeatedly extruded 3 times; S6, the extruded crude liposome suspension is added to 8 parts of a freeze-drying protectant, and after magnetic stirring until complete dissolution, the sample is divided into 2 ml vials, and the divided sample is placed in a freeze dryer, pre-frozen at -20°C for 1 h, then reduced to -40°C for deep freezing for 2 h, followed by vacuum sublimation drying (temperature -20°C, vacuum degree 10 Pa, time 12 h), and finally vacuum desorption drying (temperature 25°C, vacuum degree 5 Pa, time 4 h) to obtain the amphotericin B liposome.
[0059] Example 3 S1, 10 parts of hydrogenated soybean phospholipid (HSPC), 4 parts of cholesterol, 3 parts of mPEG2000-DSPE, 1.5 parts of ceramide, and 0.8 parts of glycyrrhetinic acid are added to a chloroform-methanol mixed solution at a solid-liquid ratio of 1:15 (g / ml), and magnetic stirring is performed at 250 rpm and 25°C until the lipid components are completely dissolved. After adding 0.5 parts of hydroxyapatite nanoparticles (nHAP), ultrasonic dispersion is performed at 150 W for 10 minutes in an ice water bath to obtain a lipid-nHAP complex solution; S2, the lipid-nHAP complex solution is transferred to a rotary evaporator, and the temperature is set to 35°C and the vacuum degree is set to 0.08 MPa. Rotary evaporation is performed for 45 min to gradually evaporate the solvent, and a uniform transparent "lipid-nHAP complex film" is formed on the inner wall of the flask. Vacuum drying is continued for 1 h to obtain a composite lipid film; S3, 1 part of amphotericin B is dissolved in a pH 8.0 phosphate buffer solution to prepare a high-pH drug solution with a concentration of 0.8 mg / ml, which is ready for use; S4, 870 parts of preheated (37°C) pH 5.0 phosphate buffer solution containing 0.01% EDTA and 0.025% vitamin E is added to the composite lipid film, and oscillation is performed in a 37°C constant temperature water bath at a frequency of 120 times / min for 15 min. Then the magnetic stirrer is turned on and the stirring speed is set to 300 rpm. Then the high-pH drug solution is added at a rate of 1 ml / min. When the total volume reaches 90%, the addition rate is slowed down to 0.2 ml / min. The entire high-pH drug solution is added to obtain a crude liposome suspension; S5, the crude liposome suspension is ultrasonically treated at a power of 250 W for 10 minutes in a pulse mode of 5 seconds of work and 3 seconds of rest. The system temperature is controlled at ≤40°C throughout the ice water bath. Then the ultrasonically emulsified suspension is filtered through a 0.22 μm polycarbonate microporous filter and repeatedly extruded 3 times; S6, the extruded coarse liposome suspension is added to 15 parts of a freeze-drying protectant, and after magnetic stirring until complete dissolution, the sample is divided into 2 ml vials, and the divided sample is placed in a freeze dryer, pre-frozen at -20°C for 1 h, then deep-frozen at -40°C for 2 h, followed by vacuum sublimation drying (temperature -20°C, vacuum degree 10 Pa, time 12 h), and finally vacuum desorption drying (temperature 25°C, vacuum degree 5 Pa, time 4 h), to obtain the amphotericin B liposome.
[0060] Comparative Example 1 The amphotericin B liposome prepared by the conventional thin film dispersion method in the background art contains only hydrogenated soybean phospholipid (HSPC), cholesterol (Chol), and mPEG2000-DSPE (without nHAP and glycyrrhetinic acid), and a single-step freezing (-40°C direct freezing for 2 h) and conventional freeze-drying process (without step pre-freezing) are used.
[0061] Performance test The amphotericin B liposome prepared in Example 1 above is used as the experimental group, and the amphotericin B liposome prepared in Comparative Example 1 is used as the control group for the following tests: Fungal cell in vitro binding rate test: S1, take the standard strain of Candida albicans in the logarithmic growth phase (ATCC90028), and adjust the concentration to 1×10 6 CFU / mL with RPMI1640 medium; S2, take the liposomes of the experimental group and the control group, respectively, and label them with the fluorescent probe DiI (final concentration 5 μmol / L) at 37°C for 30 min; S3, mix the labeled liposomes with the fungal liquid at a volume ratio of 1:1, and incubate at 37°C for 1 h (100 rpm); S4, centrifuge (3000 rpm, 5 min) to discard the supernatant, and wash with PBS for 3 times to remove free liposomes; S5, flow cytometry is used to detect the fluorescence positive rate of fungal cells, which is the in vitro binding rate of liposomes.
[0062] Drug crystallization rate test: S1, take the freeze-dried powder of the experimental group and the control group, and reconstitute it with pH 7.0 PBS to a drug concentration of 1 mg / mL; S2, take 10 μL of the reconstituted solution and drop it on a glass slide, cover it with a cover glass, and place it under a polarizing microscope (400x magnification) for observation; S3, randomly select 5 fields of view, and count the number of crystalline particles and the total number of liposome particles in each field of view; S4, drug crystallization rate = (total number of crystalline particles / total number of liposome particles) x 100%.
[0063] Encapsulation efficiency test: Gel filtration chromatography: Sephadex G-50 gel column was used, and pH 7.0 PBS was used as eluent, the flow rate was 1 mL / min, and the contents of encapsulated drugs and free drugs in the elution peaks of the experimental group and the control group were determined by ultraviolet spectrophotometry (405 nm), and the encapsulation efficiency = (encapsulated drug content / total drug content) x 100%.
[0064] Drug leakage rate after 6 months of storage at 4℃: Ultraviolet spectrophotometry: After 6 months of storage, the liposomes prepared in the experimental group and the control group were redissolved, free drugs were separated by gel filtration chromatography, and the leakage rate = (free drug content / total drug content) x 100%.
[0065] The test results are shown in Table 1: Table 1 Test results
[0066] From the above experimental results, it can be seen that the liposomes of the present application have significant advantages in core performance indicators: 1. Target binding capacity: due to the addition of glycyrrhetinic acid (GA), it can specifically bind to the ergosterol on the surface of the fungal cell membrane, so that the in vitro binding rate is increased from 38.2% of the comparative example to 86.3%, which can guide the precise enrichment of drugs to the infection site, reduce the distribution of non-target tissues, and reduce the toxic side effects; 2. Anti-drug crystallization ability: hydroxyapatite nanoparticles (nHAP) adsorb drugs to form stable complexes, and the synergistic effect of the sucrose-mannitol-trehalose compound freeze-drying protectant reduces the drug crystallization rate from 8.5% to 0.8%, avoiding the decrease of drug efficacy caused by crystallization, and prolonging the product shelf life; 3. The improvement of the combination of encapsulation efficiency, stability and other indicators fully proves that the present application effectively solves the technical pain points of the existing liposomes through component innovation (nHAP, GA, compound protectant) and process optimization (gradient pH encapsulation, stepwise freeze-drying).
[0067] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, but not to limit it, although the present application has been described in detail with reference to the above examples, those skilled in the art should understand that the specific embodiments of the present application can be modified or replaced, without departing from the spirit and scope of the present application, any modification or equivalent replacement, which should be covered in the protection scope of the claims of the present application.
Claims
1. Amphotericin B liposome, characterized in that, The components include the following mass fractions: Amphotericin B 1 part, hydrogenated soybean phospholipid (HSPC) 5-10 parts, cholesterol 2-4 parts, mPEG2000-DSPE 1-3 parts, ceramide 0.5-1.5 parts, glycyrrhetinic acid 0.3-0.8 parts, hydroxyapatite nanoparticles (nHAP) 0.2-0.5 parts, and lyoprotectant 8-15 parts.
2. The method of preparing amphotericin B liposome according to claim 1, wherein, The preparation steps include the following: The hydrogenated soybean phospholipid (HSPC), cholesterol, mPEG2000-DSPE, ceramide, and glycyrrhetinic acid are added to a chloroform-methanol mixed solution, stirred until completely dissolved, and then the hydroxyapatite nanoparticles (nHAP) are added for ultrasonic dispersion treatment to obtain a lipid-nHAP complex solution; The amphotericin B is dissolved in a pH 8.0 phosphate buffer solution to obtain a high-pH drug solution, which is ready for use; The lipid-nHAP complex solution is subjected to rotary evaporation treatment and then vacuum dried to obtain a complex lipid film. A low-pH buffer is added to the complex lipid film, which is oscillated in a constant-temperature water bath, and then the high-pH drug solution is added dropwise to obtain a crude liposome suspension; The crude liposome suspension is subjected to ultrasonic emulsification treatment and then homogenization treatment. The homogenized crude liposome suspension is added with the lyoprotectant, stirred until dissolved, and then subjected to freeze-drying treatment to obtain the amphotericin B liposome.
3. The preparation method of claim 2, wherein The ultrasonic dispersion treatment has an ultrasonic power of 150 W and an ultrasonic dispersion time of 10 minutes.
4. The preparation method of claim 3, wherein The rotary evaporation has a temperature of 35°C, a vacuum degree of 0.08 MPa, and a rotary evaporation time of 45 minutes.
5. The preparation method of claim 4, wherein The low-pH buffer is a pH 5.0 phosphate buffer solution containing 0.01% EDTA and 0.025% vitamin E.
6. The preparation method of claim 5, wherein When the high-pH drug solution is added dropwise, it is added at a rate of 1 ml / min, stirred while adding, the stirring speed is 300 rpm, when the high-pH drug solution reaches 90% of the total volume, the adding speed is adjusted to 0.2 ml / min, the remaining high-pH drug solution is continuously added, and after the addition is completed, oscillation is performed for 15 minutes.
7. The preparation method of claim 6, wherein The ultrasonic emulsification has an ultrasonic power of 250 W, a pulse mode of working for 5 s and intermittently for 3 s, and a total ultrasonic time of 10 minutes.
8. The preparation method of claim 7, wherein The homogenization treatment is that the suspension after ultrasonic emulsification is passed through a 0.22 μm polycarbonate microporous filter membrane and repeatedly extruded 3 times.
9. The preparation method of claim 8, wherein The freeze-drying protective agent is prepared by mixing sucrose, mannitol and trehalose in a mass ratio of 1:1:0.
5.
10. The method of claim 9, wherein the amphotericin B liposome is prepared by, The freeze-drying process is: first pre-freezing at -20℃ for 1 hour, then reducing the temperature to -40℃ for freezing for 2 hours, then vacuum sublimation drying at -20℃ and a vacuum degree of 10 Pa for 12 hours, and finally vacuum desorption drying at 25℃ and a vacuum degree of 5 Pa for 4 hours.