Eye drops based on amphotericin B liposome and preparation method thereof

Abstract

The invention relates to the technical field of biological medicine, and discloses eye drops based on amphotericin B lipidosome and a preparation method of the eye drops. Comprising the following steps: mixing amphotericin B with hydrogenated soybean phosphatidylcholine, distearoyl phosphatidyl glycerol, cholesterol and methanol, and sequentially carrying out ultrasonic treatment, drying, hydration, homogenization, filtration and freeze drying to obtain the amphotericin B liposome, the amphotericin B liposome, alpha-tocopherol, glucose, hydroxypropyl methyl cellulose, polyaminopropyl biguanide and water are mixed to obtain the eye drops containing the amphotericin B liposome. The hydrogenated soybean phosphatidylcholine, the distearoyl phosphatidyl glycerol and the cholesterol are controlled to form a stable liposome skeleton, so that the liposome can be formed in a relatively stable form, and the bearing capacity of the amphotericin B is improved. When the eye drops are prepared, polyaminopropyl biguanide is adopted as an antiseptic and antibacterial component, so that the integrity of liposome and the health of the ocular surface are maintained while the sterility is ensured.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to an amphotericin B liposome-based eye drop and a preparation method thereof. BACKGROUND

[0002] Fungal keratitis is an eye disease with a high rate of blindness caused by fungal infection. Common pathogenic bacteria include Candida, Aspergillus and Fusarium. The disease has insidious onset and dangerous condition. If not treated in time or improperly, it can lead to corneal ulcer, perforation and even blindness. At present, the treatment of fungal keratitis is a serious challenge worldwide. The main difficulty lies in the lack of efficient, safe and easy-to-administer antifungal drugs for the eye.

[0003] Amphotericin B (AmB) is a polyene broad-spectrum antifungal drug. It is considered as the "gold standard" drug for the treatment of deep fungal infections, especially ocular fungal infections, due to its broad-spectrum antibacterial spectrum and resistance to drug resistance. However, amphotericin B itself has strong hydrophobicity and instability, and is easy to aggregate and degrade in aqueous solution. More importantly, its molecular toxicity is significant, and it has strong irritation and toxicity to ocular surface tissues, especially corneal epithelial cells, which can cause severe conjunctival hyperemia, corneal epithelial shedding, burning sensation and pain, and patients have poor tolerance. In order to overcome the toxicity and stability problems of amphotericin B, researchers have developed amphotericin B liposomes. As a new drug delivery system, liposomes can encapsulate drugs in phospholipid bilayers, simulate biological membrane structures, significantly reduce the toxicity of free drugs, and improve the stability and biocompatibility of drugs. At present, amphotericin B liposome injection has been widely used in the treatment of systemic fungal infections in clinical practice, and its superior safety has been proven.

[0004] However, the successful application of amphotericin B liposome to eye local administration still faces many technical barriers. First, the liposome is a thermodynamically unstable system. After being prepared into eye drops, it is prone to aggregation, fusion, phospholipid oxidation and hydrolysis, or drug leakage due to long-term storage, changes in environmental factors (such as temperature and light), and the influence of other ingredients in the prescription, resulting in increased particle size and decreased encapsulation efficiency, which not only affects the drug efficacy, but also may block the lacrimal duct. Second, in order to meet the specific physicochemical properties and microbiological requirements of eye drops, many excipients such as buffers, osmotic pressure regulators, thickening agents and preservatives are usually added. Many common excipients, especially isotonicity regulators containing electrolytes (such as sodium chloride) and cationic preservatives (such as benzalkonium chloride), can destroy the phospholipid bilayer structure of the liposome, leading to instantaneous collapse of the liposome and complete leakage of the drug, so that the advantages of the liposome are lost. Further, the residence time of conventional eye drops on the ocular surface is short, and the bioavailability is low (usually less than 5%). For liposomes, how to prolong the residence time on the corneal surface without affecting its stability by adding safe adhesive materials is the key to improve the efficacy, but this also puts higher requirements on the complexity of the prescription. Finally, amphotericin B liposome eye drops cannot be terminally heat sterilized and must use aseptic production process. The entire preparation process, including the formation of liposomes, particle size homogenization and filtration sterilization, needs to be carried out under strict conditions, and the process is complex, the cost is high, and the shelf life of the preparation is difficult to guarantee.

[0005] In summary, although amphotericin B liposome is theoretically considered to be an ideal dosage form for the treatment of fungal keratitis, the existing technology has not yet provided an amphotericin B liposome eye drop preparation with high physical stability, good ocular tolerance, feasible preparation process and suitable for long-term storage. Therefore, there is an urgent need in the art to develop a completely new technical solution that can solve all the above technical problems. SUMMARY

[0006] Therefore, the present application provides an amphotericin B liposome-based eye drop and a preparation method thereof, which aims to solve the problems of poor drug solubility, poor liposome stability and complex preservative sterilization operation in the current technology when amphotericin B is prepared into eye drops.

[0007] The present application provides a preparation method of an amphotericin B liposome-based eye drop, comprising the following steps: 1) mixing amphotericin B with hydrogenated soy phosphatidylcholine, distearoyl phosphatidylglycerol, cholesterol and methanol, and then sequentially performing ultrasonic treatment, drying, hydration, homogenization, filtration and freeze-drying to obtain amphotericin B liposomes; 2) mixing amphotericin B liposome, alpha-tocopherol, glucose, hydroxypropyl methyl cellulose, polyaminopropyl biguanide with water to obtain an eye drop containing amphotericin B liposome.

[0008] Preferably, the molar ratio of amphotericin B to hydrogenated soybean phosphatidylcholine, distearoyl phosphatidylglycerol, cholesterol in step 1) is 0.3-0.4:2-2.2:1-1.2:0.8-1; The concentration of amphotericin B in methanol is 8-12 mg / mL.

[0009] Preferably, the power of the ultrasonic in step 1) is 40-60 W, and the ultrasonic time is 3-8 min.

[0010] Preferably, the drying method in step 1) is low-pressure evaporation of methanol, the drying pressure is 0.01-0.02 MPa, and the drying temperature is 30-40℃.

[0011] Preferably, the hydration and homogenization in step 1) are mixing the remaining solid after drying with water and ultrasonic. The mass ratio of water to the remaining solid after drying is 1:80-120; The power of the ultrasonic is 20-30 W, and the ultrasonic time is 2-5 min.

[0012] Preferably, the filtration in step 1) is filtration with a 0.22 µm filter membrane under a pressure of 0.1-0.3 MPa.

[0013] Preferably, the mass fraction of amphotericin B liposome in the eye drop containing amphotericin B liposome in step 2) is 0.25%±0.005%; The mass fraction of alpha-tocopherol in the eye drop containing amphotericin B liposome is 0.01%±0.005%; The mass fraction of glucose in the eye drop containing amphotericin B liposome is 5.0%±0.005% The mass fraction of hydroxypropyl methyl cellulose in the eye drop containing amphotericin B liposome is 0.8%±0.005% The mass fraction of polyaminopropyl biguanide in the eye drop containing amphotericin B liposome is 0.1%±0.005%.

[0014] The application also provides an eye drop based on amphotericin B liposome prepared by the above preparation method.

[0015] Compared with the prior art, the application has the following beneficial effects: (1) Hydrogenated soybean phosphatidylcholine + distearoyl phosphatidylglycerol + cholesterol constitute a stable liposome skeleton. Among them, hydrogenated soybean phosphatidylcholine has a high phase transition temperature, which can enhance the rigidity and stability of the lipid bilayer membrane, and reduce drug leakage. Distearoyl phosphatidylglycerol as a negatively charged phospholipid, effectively prevents the aggregation and fusion of liposomes during storage through electrostatic repulsion, ensuring uniform particle size. The addition of cholesterol can fill the gap between phospholipid molecules, adjust the membrane fluidity, further enhance the membrane density, and significantly improve the physical stability and encapsulation efficiency. By controlling the molar ratio of the three, the liposome can form in a relatively stable form, and the carrying capacity of amphotericin B is improved.

[0016] (2) Glucose is used as an osmotic pressure regulator, which avoids the problems of liposome destruction and drug leakage caused by the use of electrolytes such as sodium chloride.

[0017] (3) Hydroxypropyl methylcellulose can not only increase the viscosity of the drug solution and prolong the residence time on the eye surface, thereby increasing the contact between the drug and the cornea and improving the bioavailability, but also can play a lubricating and moisturizing role, relieve eye dryness and discomfort, and further improve the drug experience.

[0018] (4) Compared with traditional cationic preservatives, polyaminopropyl biguanide does not cause damage to the liposome, and has lower irritation, which can ensure the sterility of multi-dose packaging while maximizing the integrity of the liposome and the health of the eye surface. As an antioxidant, alpha-tocopherol can effectively prevent the oxidation and degradation of phospholipids and amphotericin B during storage, thereby prolonging the effective shelf life of the eye drops. DETAILED DESCRIPTION

[0019] The various illustrative embodiments of the present application will now be described in detail in connection with the accompanying drawings. This description is not intended to be a limitation on the application, but rather a description of certain aspects, features, and embodiments of the application. It is understood that the scope of the application is not limited to the aspects, features, and embodiments described herein, but include all aspects, features and embodiments within the scope of the appended claims.

[0020] 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 also specifically disclosed. Each smaller range within any stated range or within any stated range of intermediate values, and any other stated value or range of values, is also included within the scope of the present application. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0021] 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 documents mentioned herein are incorporated by reference to disclose and describe in full the methods and / or materials useful in connection to the documents. In case of conflict between the content of the specification and that of any document incorporated herein by reference, the content of the specification prevails.

[0022] Many modifications and variations of the present application described in the specification are possible without departing from the scope or spirit of the application. Other implementations of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application. The specification and examples are illustrative only.

[0023] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having" and the like are open-ended terms that are intended to permit but not limit the inclusion of elements or the number of elements, as well as the possibility that one or more other elements can be added or otherwise included.

[0024] The present application provides a preparation method of amphotericin B liposome-based eye drops, comprising the following steps: 1) mixing amphotericin B with hydrogenated soybean phosphatidylcholine, distearoyl phosphatidylglycerol, cholesterol and methanol, and then sequentially performing ultrasonic treatment, drying, hydration, homogenization, filtration and freeze-drying to obtain amphotericin B liposomes; 2) mixing amphotericin B liposomes, α-tocopherol, glucose, hydroxypropyl methyl cellulose and polyaminopropyl biguanide with water to obtain amphotericin B liposome-containing eye drops.

[0025] In the present application, the molar ratio of amphotericin B to hydrogenated soybean phosphatidylcholine, distearoyl phosphatidylglycerol and cholesterol in step 1) is 0.3-0.4:2-2.2:1-1.2:0.8-1, preferably 0.32-0.38:2.05-2.15:1.05-1.15:0.85-0.95, further preferably 0.34-0.36:2.08-2.12:1.08-1.12:0.88-0.92, and more preferably 0.35:2.1:1.1:0.9. The addition ratio of hydrogenated soybean phosphatidylcholine, distearoyl phosphatidylglycerol and cholesterol affects the drug loading capacity, membrane stability and electrostatic repulsion of the liposomes. By adjusting the ratio of the three, high encapsulation efficiency and long-term stability are ensured.

[0026] In the present application, the concentration of amphotericin B in methanol is 8-12 mg / mL, preferably 8.5-11.5 mg / mL, further preferably 9-11 mg / mL, and more preferably 9.5-10.5 mg / mL. This concentration can ensure complete dissolution of the drug, does not affect film formation, and can control the amount of methanol, avoiding a too long drying process.

[0027] In the present application, the power of the ultrasound in step 1) is 40-60 W, preferably 43-58 W, further preferably 45-55 W, and more preferably 50 W. This power is sufficient to fully disperse and mix the lipids and the drug, while avoiding excessive power that can cause drug degradation or oxidation of phospholipids. The ultrasound time is 3-8 min, preferably 3.5-7.5 min, further preferably 4-7 min, and more preferably 5-6 min. Prolonged ultrasound time can cause the temperature of the system to rise, and amphotericin B is heat-sensitive, so prolonged ultrasound time can cause thermal decomposition.

[0028] In the present application, the drying method in step 1) is low-pressure evaporation of methanol, and the drying pressure is 0.01-0.02 MPa. The drying temperature is 30-40℃, preferably 33-38℃, and further preferably 35℃.

[0029] In the present application, the hydration and homogenization in step 1) are mixing the remaining solids after drying with water and ultrasonic treatment.

[0030] In the present application, the mass ratio of water to the remaining solids after drying is 1:80-120, preferably 1:85-115, further preferably 1:90-110, and more preferably 1:95-100. This ratio can form a liposome initial suspension with an appropriate concentration, laying the foundation for subsequent dilution into eye drops. A concentration that is too high or too low is not conducive to hydration.

[0031] In the present application, the power of the ultrasound is 20-30 W, preferably 22-28 W, further preferably 24-26 W, and more preferably 25 W. The ultrasound time is 2-5 min, which can be 2 min, 3 min, 4 min, or 5 min. A moderate power is sufficient to hydrate and break the lipid membrane into small unilamellar / oligolamellar liposomes, avoiding high power that can damage the structure of the liposomes.

[0032] In the present application, the filtration in step 1) is performed using a 0.22 µm filter membrane under a pressure of 0.1-0.3 MPa.

[0033] In the present application, the mass fraction of amphotericin B liposomes in the eye drops containing amphotericin B liposomes in step 2) is 0.25%±0.005%.

[0034] In the present application, the mass fraction of α-tocopherol in the eye drops containing amphotericin B liposomes is 0.01%±0.005%; In the present application, the mass fraction of glucose in the eye drops containing amphotericin B liposomes is 5.0%±0.005% In the present application, the mass fraction of hydroxypropyl methyl cellulose in the eye drops containing amphotericin B liposomes is 0.8%±0.005%, which provides the best viscosity and residence time, and too low effect is insufficient, and too high is poor comfort and easy to blur vision.

[0035] In the present application, the mass fraction of polyaminopropyl biguanide in the eye drops containing amphotericin B liposomes is 0.1%±0.005%.

[0036] The present application also provides an amphotericin B liposome-based eye drops prepared by the above preparation method.

[0037] Example 1 1. Preparation of liposomes: According to the molar ratio of 0.35:2.1:1.1:0.9, amphotericin B 16.5 mg, hydrogenated soybean phosphatidylcholine 100.0 mg, distearoyl phosphatidylglycerol 42.5 mg and cholesterol 19.5 mg were accurately weighed and placed in a 100 mL round-bottom flask. 2 mL of methanol was added to the flask, and it was placed in a 10°C water bath and ultrasonicated at a power of 40W for 7 minutes.

[0038] The flask was connected to a rotary evaporator, and rotary evaporation was carried out at a water bath temperature of 35°C and a pressure of 0.015 MPa until a uniform lipid film formed on the wall of the flask. Vacuum was continued for 30 minutes to completely remove residual solvent.

[0039] According to the water to solid mass ratio of 1:100, sterile water for injection at room temperature (25°C) was added to the flask and manually shaken to fully hydrate and detach the lipid film, obtaining a milky white suspension. The suspension was placed in an ice water bath and treated with a probe ultrasonic instrument at a power of 25W for 4 minutes to obtain a translucent, uniform particle size liposome suspension. The liposome suspension was filtered and sterilized with a 0.22µm microporous filter membrane at a pressure of 0.2 MPa.

[0040] The filtered liposome suspension was freeze-dried to obtain white sponge-like amphotericin B liposome solids.

[0041] 2. Preparation of eye drops: Amphotericin B liposome solids prepared above 10 mg, α-tocopherol 0.4 mg, glucose 200 mg, hydroxypropyl methyl cellulose 16 mg and polyaminopropyl biguanide 2 mg were accurately weighed.

[0042] Dissolve the above components with an appropriate amount of sterile water for injection, and make up to 40 g, fully stir to completely dissolve, mix evenly.

[0043] The mass fraction of amphotericin B liposome, α-tocopherol, glucose, hydroxypropyl methyl cellulose and polyaminopropyl biguanide in the obtained eye drops is 0.025%, 0.01%, 5.0%, 0.8% and 0.005%, respectively.

[0044] Example 2 1. Preparation of liposome: According to the molar ratio of 0.36:2.1:1.1:0.95, accurately weigh amphotericin B 17.01 mg, hydrogenated soybean phosphatidylcholine 100.0 mg, distearoyl phosphatidylglycerol 42.5 mg and cholesterol 20.58 mg, and place them in a 100 mL round-bottom flask. Add 2 mL of methanol to the flask, and place it in a 10°C water bath for ultrasonic treatment at a power of 45 W for 6 min.

[0045] Connect the flask to a rotary evaporator, and rotate to evaporate at a water bath temperature of 35°C and a pressure of 0.015 MPa until a uniform lipid film forms on the wall of the flask. Continue vacuuming for 30 min to completely remove the residual solvent.

[0046] According to the water to solid mass ratio of 1:95, add sterile water for injection at room temperature (25°C) to the flask, and manually shake to fully hydrate and detach the lipid film to obtain a milky white suspension. Place the suspension in an ice water bath, and use a probe ultrasonic instrument to treat it at a power of 24 W for 5 min to obtain a translucent, uniform particle size liposome suspension. Filter the liposome suspension through a 0.22 µm microporous filter membrane at a pressure of 0.2 MPa to sterilize.

[0047] Freeze-dry the filtered liposome suspension to obtain white sponge-like amphotericin B liposome solids.

[0048] 2. Preparation of eye drops: Accurately weigh 10 mg of the amphotericin B liposome solids prepared above, 0.4 mg of α-tocopherol, 200 mg of glucose, 16 mg of hydroxypropyl methyl cellulose and 2 mg of polyaminopropyl biguanide.

[0049] Dissolve the above components with an appropriate amount of sterile water for injection, and make up to 40 g, fully stir to completely dissolve, mix evenly.

[0050] The mass fraction of amphotericin B liposome, α-tocopherol, glucose, hydroxypropyl methyl cellulose and polyaminopropyl biguanide in the obtained eye drops is 0.025%, 0.01%, 5.0%, 0.8% and 0.005%, respectively.

[0051] Example 3 1. Preparation of liposome: According to the molar ratio of 0.36:2.1:1.1:0.95, accurately weigh amphotericin B 16.03 mg, hydrogenated soybean phosphatidylcholine 16.03 mg, distearoyl phosphatidylglycerol 43.27 mg and cholesterol 19.07 mg, and place them in a 100 mL round-bottom flask. Add 2 mL of methanol to the flask, and place it in a 10°C water bath for ultrasonic treatment at a power of 55 W for 4 min.

[0052] Connect the flask to a rotary evaporator, and rotate it at a 35°C water bath and a pressure of 0.015 MPa until a uniform lipid film forms on the wall of the flask. Continue vacuuming for 30 min to completely remove the residual solvent.

[0053] Add sterile water for injection at room temperature (25°C) to the flask at a water to solid mass ratio of 1:105, and manually shake to fully hydrate and detach the lipid film to obtain a milky white suspension. Place the suspension in an ice water bath, and use a probe ultrasonic instrument to treat it with ultrasonic waves at a power of 26 W for 3 min to obtain a translucent, uniform particle size liposome suspension. Filter the liposome suspension with a 0.22 µm microporous filter membrane at a pressure of 0.2 MPa to sterilize it.

[0054] Freeze-dry the filtered liposome suspension to obtain white sponge-like amphotericin B liposome solids.

[0055] 2. Preparation of eye drops: Accurately weigh 10 mg of the amphotericin B liposome solids prepared above, 0.4 mg of α-tocopherol, 200 mg of glucose, 16 mg of hydroxypropyl methylcellulose and 2 mg of polyaminopropyl biguanide.

[0056] Dissolve the above components with an appropriate amount of sterile water for injection, and make up to 40 g. Fully stir to completely dissolve and uniformly mix.

[0057] In the obtained eye drops, the mass fractions of amphotericin B liposomes, α-tocopherol, glucose, hydroxypropyl methylcellulose and polyaminopropyl biguanide are 0.025%, 0.01%, 5.0%, 0.8% and 0.005%, respectively.

[0058] Example 4 1. Preparation of liposome: According to the molar ratio of 0.36:2.1:1.1:0.95, accurately weigh amphotericin B 17.01 mg, hydrogenated soybean phosphatidylcholine 100.0 mg, distearoyl phosphatidylglycerol 42.5 mg and cholesterol 20.58 mg, and place them in a 100 mL round-bottom flask. Add 2 mL of methanol to the flask, and place it in a 10°C water bath for ultrasonic treatment at a power of 40 W for 7 min.

[0059] The flask was connected to a rotary evaporator and rotary evaporation was carried out at 35°C water bath, 0.015 MPa pressure until a uniform lipid film was formed on the wall of the flask, and vacuum was continued for 30 minutes to completely remove residual solvent.

[0060] According to the water to solid mass ratio of 1:100, sterile water at room temperature (25°C) was added to the flask, and the lipid film was manually shaken to fully hydrate and fall off to obtain a milky white suspension. The suspension was placed in an ice water bath, and a probe ultrasonic instrument was used for ultrasonic treatment at 25 W power for 4 minutes to obtain a translucent, uniform particle size liposome suspension. The liposome suspension was filtered with a 0.22 μm microporous filter membrane under a pressure of 0.2 MPa to sterilize.

[0061] The filtered liposome suspension was freeze-dried to obtain white sponge-like amphotericin B liposome solids.

[0062] 2. Preparation of eye drops: 10 mg of amphotericin B liposome solids prepared above, 0.4 mg of α-tocopherol, 200 mg of glucose, 16 mg of hydroxypropyl methyl cellulose, and 2 mg of polyaminopropyl biguanide were accurately weighed.

[0063] The above components were dissolved with an appropriate amount of sterile water for injection, and the volume was adjusted to 40 g. After stirring, the components were completely dissolved and uniformly mixed.

[0064] In the obtained eye drops, the mass fractions of amphotericin B liposomes, α-tocopherol, glucose, hydroxypropyl methyl cellulose, and polyaminopropyl biguanide were 0.025%, 0.01%, 5.0%, 0.8%, and 0.005%, respectively.

[0065] Comparative Example 1 1. Preparation of liposomes: 16.5 mg of amphotericin B, 100.0 mg of hydrogenated soybean phosphatidylcholine, and 19.5 mg of cholesterol were accurately weighed and placed in a 100 mL round-bottom flask. The molar ratio of the feed was 0.35:2.1:0.9. 2 mL of methanol was added to the flask, and the flask was placed in a 10°C water bath and ultrasonicated at 40 W power for 7 min.

[0066] The flask was connected to a rotary evaporator and rotary evaporation was carried out at 35°C water bath, 0.015 MPa pressure until a uniform lipid film was formed on the wall of the flask, and vacuum was continued for 30 minutes to completely remove residual solvent.

[0067] The lipid film was fully hydrated and detached by adding sterile water for injection at room temperature (25℃) to the flask in a water to solid mass ratio of 1:100 with manual shaking to obtain a milky white suspension. The suspension was placed in an ice water bath and treated with a probe sonicator at a power of 25W for 4 minutes to obtain a translucent, uniform particle size liposome suspension. The liposome suspension was filtered with a 0.22μm microporous filter membrane at a pressure of 0.2MPa to obtain a sterile liposome suspension.

[0068] The filtered liposome suspension was freeze-dried to obtain white sponge-like amphotericin B liposome solids.

[0069] 2. Preparation of eye drops: 10mg of the amphotericin B liposome solids prepared above, 0.4mg of α-tocopherol, 200mg of glucose, 16mg of hydroxypropyl methyl cellulose and 2mg of benzalkonium chloride were accurately weighed.

[0070] The above components were dissolved with an appropriate amount of sterile water for injection, and the volume was adjusted to 40g. The mixture was thoroughly stirred until it was completely dissolved and uniformly mixed.

[0071] In the obtained eye drops, the mass fractions of amphotericin B liposomes, α-tocopherol, glucose, hydroxypropyl methyl cellulose and polyaminopropyl biguanide were 0.025%, 0.01%, 5.0%, 0.8% and 0.005%, respectively.

[0072] Comparative Example 2 1. Preparation of liposomes: 16.5mg of amphotericin B, 100.0mg of hydrogenated soybean phosphatidylcholine, 42.5mg of distearoyl phosphatidylglycerol and 19.5mg of cholesterol were accurately weighed and placed in a 100mL round-bottom flask. The molar ratio of the feed was 0.35:2.1:1.1:0.9. 2mL of methanol was added to the flask, which was placed in a 10℃ water bath and sonicated at a power of 40W for 7min.

[0073] The flask was connected to a rotary evaporator, and rotary evaporation was carried out at a water bath temperature of 35℃ and a pressure of 0.015MPa until a uniform lipid film formed on the wall of the flask. Vacuum was then applied for 30min to completely remove the residual solvent.

[0074] The lipid film was fully hydrated and detached by adding sterile water for injection at room temperature (25℃) to the flask in a water to solid mass ratio of 1:100 with manual shaking to obtain a milky white suspension. The suspension was placed in an ice water bath and treated with a probe sonicator at a power of 25W for 4 minutes to obtain a translucent, uniform particle size liposome suspension. The liposome suspension was filtered with a 0.22μm microporous filter membrane at a pressure of 0.2MPa to obtain a sterile liposome suspension.

[0075] 2. Preparation of eye drops: The liposome suspension prepared in the above step, 0.4 mg of α-tocopherol, 200 mg of glucose, 16 mg of hydroxypropyl methyl cellulose and 2 mg of polyaminopropyl biguanide were mixed.

[0076] The above components were dissolved with an appropriate amount of sterile water for injection, and the volume was made to 40 g. After stirring thoroughly, the solution was completely dissolved and uniformly mixed.

[0077] In the obtained eye drops, the mass fractions of amphotericin B liposome, α-tocopherol, glucose, hydroxypropyl methyl cellulose and polyaminopropyl biguanide were 0.025%, 0.01%, 5.0%, 0.8% and 0.005%, respectively.

[0078] The products obtained in Examples 1-4 and Comparative Examples 1-2 were subjected to the following tests: 1. Physicochemical property characterization of liposomes Particle size, polydispersity index and Zeta potential: determined using a dynamic light scattering nanoparticle size analyzer, and the results are shown in Table 1.

[0079] Physicochemical stability test: the product was stored in the dark (5°C) for 30 days, and whether there was flocculation was observed.

[0080] Table 1 Particle size, polydispersity index (PDI) and Zeta potential of the products obtained in Examples 1-4 and Comparative Examples 1-2

[0081] Examples 1-4 had small particle size, narrow distribution (PDI < 0.2) and high negative Zeta potential, and the system was stable. Comparative Example 1 had increased particle size, increased PDI and smaller absolute value of Zeta potential due to the absence of charge repulsion. The product obtained in Comparative Example 2 had a high initial Zeta potential, and after one month of storage, the liposomes aggregated, and the average particle size was 153.1 ± 2.8 nm, which was significantly increased.

[0082] Encapsulation efficiency test: free drug was separated by dextran gel column method, amphotericin B content was determined by HPLC, encapsulation efficiency (EE%) was calculated, and the encapsulation efficiency was tested again after the eye drops were stored in the dark at 5°C for 30 days.

[0083] The test results are shown in Table 2.

[0084] Table 2 Encapsulation efficiency of the products obtained in Examples 1-4 and Comparative Examples 1-2

[0085] All examples showed high initial encapsulation efficiency (>91%), which directly proved that the optimized lipid formulation (precise molar ratio of hydrogenated soy phosphatidylcholine, distearoyl phosphatidylglycerol and cholesterol) of the present application could form a dense and stable lipid bilayer membrane, thereby effectively encapsulating amphotericin B therein. The initial encapsulation efficiency of Comparative Example 1 was significantly lower: this was mainly due to the lack of distearoyl phosphatidylglycerol in the formulation, resulting in an incomplete or defective lipid membrane structure that could not effectively encapsulate the drug; at the same time, the addition of benzalkonium chloride would destroy the formed liposome membrane, leading to leakage of the encapsulated drug. The unstable liposome structure after long-term storage was more easily destroyed, leading to a large amount of drug leakage and a further significant decrease in encapsulation efficiency.

[0086] 2. In vitro release behavior study The dialysis bag method was used to perform the release experiment in simulated tear fluid (pH 7.4) at 37°C, and the release was determined at different time points. The specific test results are shown in Table 3.

[0087] Table 3 In vitro cumulative release (%) of products obtained from Examples 1-4 and Comparative Example 1

[0088] All examples showed the expected sustained-release characteristics. The drug was slowly and continuously released within 24 hours, without a sharp burst phenomenon. This ideal release behavior benefited from the complete and dense lipid bilayer membrane. The diffusion of the drug from the inside of the liposome to the release medium needs to overcome the membrane barrier, which effectively delays the release rate. Comparative Example 1 showed a typical drug burst phenomenon. Within the initial 0.5 hours, its cumulative release was as high as 45.3%, much higher than any of the examples (<20%), and the release degree was more than 90% within 4 hours.

[0089] 3. Sterility test: According to Chinese Pharmacopoeia General 1101, the test results are shown in Table 4.

[0090] Table 4 Sterility test results of products obtained from Examples 1-4 and Comparative Example 1

[0091] The aerobic bacteria test result of Comparative Example 1 was positive, which was due to two reasons: first, the benzalkonium chloride used in its formulation had limited killing effect on some gram-negative bacteria; second, the destroyed liposome provided nutrients for microorganisms, leading to microbial contamination. This illustrates the major defect of the comparative formulation from the core level of drug safety. The eye drops prepared by Examples 1-4 passed the sterility test.

[0092] 4. Anti-bacterial efficacy test: According to the Chinese Pharmacopoeia General 1121, a certain amount of challenge bacteria was inoculated into the sample, and the decrease of the number of colonies was observed within a specified time. The test results are shown in Table 5.

[0093] Table 5 Anti-bacterial efficacy test results of the product obtained in Example 4 and Comparative Example 1 (logarithmic value of the decrease of the number of colonies lg N / N0)

[0094] The eye drops prepared in Example 4 met the requirements of the Chinese Pharmacopoeia for the anti-bacterial efficacy of multi-dose preparations for all types of challenge bacteria. It quickly killed bacteria and effectively inhibited and eventually killed fungi (Candida and Aspergillus). This proves that the preservative system with 0.005% polyaminopropyl biguanide as the core, when coexisting with the stable liposomes of the present application, can provide broad-spectrum, high-efficiency and long-lasting anti-bacterial protection. The eye drops prepared in Comparative Example 1 had acceptable effect on Staphylococcus aureus, but the killing effect on Pseudomonas aeruginosa was significantly insufficient and did not meet the pharmacopoeia standard. This exposed the limitations of the anti-bacterial spectrum of benzalkonium chloride. The killing effect on Candida albicans and Aspergillus niger was extremely poor, and the number of colonies hardly decreased. This is mainly because benzalkonium chloride itself has weak anti-fungal ability, and after destroying the liposomes, the released amphotericin B can resist fungi, but in a liquid mixed system, it can interact with preservatives or other ingredients, and cannot effectively exert its anti-bacterial efficacy, resulting in the collapse of the defense ability of the whole system against fungi.

[0095] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application and 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 still be modified or replaced by equivalents without departing from the spirit and scope of the present application, any modification or equivalent replacement thereof should be covered within the protection scope of the claims of the present application.

Claims

1. A method for preparing an amphotericin B liposome-based eye drop, characterized by, The method comprises the following steps: 1) mixing amphotericin B with hydrogenated soybean phosphatidylcholine, distearoyl phosphatidylglycerol, cholesterol and methanol, and then sequentially performing ultrasonic treatment, drying, hydration, homogenization, filtration and freeze-drying to obtain amphotericin B liposomes; 2) mixing the amphotericin B liposomes, alpha-tocopherol, glucose, hydroxypropyl methyl cellulose, polyaminopropyl biguanide and water to obtain an eye drop containing amphotericin B liposomes.

2. The method for preparing an eye drop based on amphotericin B liposomes according to claim 1, characterized in that, The molar ratio of amphotericin B to hydrogenated soybean phosphatidylcholine, distearoyl phosphatidylglycerol and cholesterol in step 1) is 0.3-0.4:2-2.2:1-1.2:0.8-1. The concentration of amphotericin B in methanol is 8-12 mg / mL.

3. The method for preparing an eye drop based on amphotericin B liposomes according to claim 1, characterized in that, The power of the ultrasonic treatment in step 1) is 40-60 W, and the ultrasonic treatment time is 3-8 min.

4. A method for preparing an eye drop based on amphotericin B liposomes according to claim 2 or 3, characterized in that, The drying method in step 1) is low-pressure evaporation of methanol, the drying pressure is 0.01-0.02 MPa, and the drying temperature is 30-40℃.

5. The method for preparing an eye drop based on amphotericin B liposomes according to claim 4, characterized in that, The hydration and homogenization in step 1) are performed by mixing the remaining solid after drying with water and performing ultrasonic treatment. The mass ratio of water to the remaining solid after drying is 1:80-120. The power of the ultrasonic treatment is 20-30 W, and the ultrasonic treatment time is 2-5 min.

6. The method for preparing an eye drop based on amphotericin B liposomes according to claim 5, characterized in that, The filtration in step 1) is performed using a 0.22 µm filter membrane under a pressure of 0.1-0.3 MPa.

7. The method for preparing an eye drop based on amphotericin B liposomes according to claim 6, characterized in that, The mass fraction of amphotericin B liposomes in the eye drop containing amphotericin B liposomes in step 2) is 0.25%±0.005%. The mass fraction of alpha-tocopherol in the eye drop containing amphotericin B liposomes is 0.01%±0.005%. The mass fraction of glucose in the eye drop containing amphotericin B liposomes is 5.0%±0.005%. The mass fraction of hydroxypropyl methyl cellulose in the eye drop containing amphotericin B liposomes is 0.8%±0.005%. The mass fraction of polyaminopropyl biguanide in the eye drop containing amphotericin B liposomes is 0.1%±0.005%.

8. An eye drop based on amphotericin B liposomes prepared by the method of any one of claims 1-7.

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