A preparation method of a liposome of prodigiosin

Abstract

The application discloses a preparation method of a prodigiosin liposome, and comprises the following steps: S1, lipid phase emulsification: mixing phospholipid and cholesterol according to a mass ratio of 2-5:1 to form a lipid phase with a concentration of 10-30 g / L, and emulsifying the lipid phase in a buffer solution with a volume ratio of 1:(3-5) to form a preliminary emulsion of the lipid phase; S2, mixed emulsion ultrasonic dispersion: dropping a prodigiosin alcohol solution into the preliminary emulsion of the lipid phase to form a mixed emulsion, and sequentially performing emulsification and ultrasonic dispersion to obtain a dispersion liquid; S3, gradient extrusion refinement: gradient extruding the dispersion liquid through filter membranes with pore diameters of 180-260 nm, 140-160 nm and 80-120 nm in sequence to obtain a prodigiosin liposome suspension; and centrifuging and purifying the prodigiosin liposome suspension to obtain the prodigiosin liposome. The prodigiosin liposome prepared by the method has uniform particle size, high encapsulation efficiency and high stability.

Description

Technical Field

[0001] This invention belongs to the field of liposome pharmaceutical formulation technology, specifically relating to a method for preparing leptospirin liposomes. Background Technology

[0002] Lecithin is a natural compound with various biological activities such as antibacterial, antitumor, and anti-inflammatory effects. However, it has several drawbacks. Lecithin is highly hydrophobic, has extremely low solubility in aqueous phase, and poor bioavailability. Moreover, it is chemically unstable, sensitive to light, heat, and oxygen, and is prone to oxidative degradation, leading to loss of activity. In addition, when lecithin is administered directly, it is highly toxic and is easily metabolized rapidly by enzymes in the body, limiting its clinical application.

[0003] Liposomes, as a novel drug carrier, can encapsulate hydrophobic drugs through a lipid bilayer, improving drug solubility and bioavailability while reducing drug toxicity and protecting the drug from environmental degradation. Existing methods for preparing strychnine liposomes mainly include thin-film dispersion, reverse evaporation, and ultrasonic dispersion, but all have significant shortcomings: liposomes prepared by thin-film dispersion have uneven particle size and encapsulation efficiency generally below 70%; reverse evaporation is complex and carries a high risk of organic solvent residue; traditional ultrasonic dispersion lacks targeted protective measures, resulting in severe oxidative degradation of strychnine during preparation, and poor liposome stability, making them prone to fusion or drug leakage during storage, leading to poor quality strychnine liposomes.

[0004] Therefore, the existing technology for preparing squalene liposomes has poor adaptability, resulting in poor batch-to-batch reproducibility and difficulty in meeting the needs of large-scale production. Therefore, there is an urgent need to develop a simple method for preparing squalene liposomes that can effectively inhibit the oxidative degradation of squalene, improve encapsulation efficiency and particle size uniformity. Summary of the Invention

[0005] The purpose of this invention is to solve the above problems and provide a method for preparing lecithin liposomes. The lecithin liposomes prepared by this method have better encapsulation efficiency and particle size uniformity, and are not easily oxidized and degraded, thus exhibiting higher stability.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] This invention provides a method for preparing strychnine liposomes, comprising the following steps:

[0008] S1. Lipid phase emulsification: Phospholipids and cholesterol are mixed in an organic solvent at a mass ratio of (2~5):1 to form a lipid phase, the total concentration of phospholipids and cholesterol in the lipid phase being 10~30 g / L; the lipid phase is emulsified in a buffer solution with a volume ratio of 1:(3~5) to form a preliminary lipid phase emulsion; the emulsification is carried out at a shear rate of 4000~6000 rpm for 10~15 min;

[0009] S2. Ultrasonic dispersing of mixed emulsion: A solution of styraxol is dropped into a preliminary lipid emulsion to form a mixed emulsion, wherein the mass ratio of styraxol to phospholipid is 1:(10~20). The mixed emulsion is first emulsified at a shear rate of 4000~6000 rpm for 3~10 min. Then, the mixed emulsion is ultrasonically dispersed to obtain a dispersion.

[0010] S3. Gradient extrusion refining: The dispersion is subjected to gradient extrusion through filter membranes with pore sizes of 180~260 nm, 140~160 nm and 80~120 nm to obtain a strychnine liposome suspension; centrifugation and purification are then performed to obtain strychnine liposomes.

[0011] Further, the phospholipid mentioned in step S1 is one or more of soybean lecithin, egg yolk lecithin, or hydrogenated soybean lecithin.

[0012] Furthermore, the emulsification temperature in step S1 is 25~30℃, and the emulsification shear rate is 4000~5000 rpm.

[0013] Further, the gradient extrusion refinement in step S3 is to perform gradient extrusion of the dispersion through filter membranes with pore sizes of 180~260 nm, 140~160 nm, 80~120 nm and 40~60 nm in sequence to obtain a strychnine liposome suspension.

[0014] Further, the gradient extrusion refinement in step S3 is to extrude the dispersion sequentially through polycarbonate filter membranes with pore sizes of 200 nm, 150 nm, 100 nm, and 50 nm, or sequentially through polycarbonate filter membranes with pore sizes of 250 nm, 150 nm, and 100 nm; the extrusion pressure is 0.3~0.5 MPa, and the extrusion temperature is 2~8℃.

[0015] Furthermore, the ultrasound in step S2 is performed using a probe-type ultrasound machine with an ultrasound power of 200~300 W, a working time of 2~4 seconds and a pause of 4~6 seconds, and a total ultrasound time of 5~10 min; the system temperature is maintained at 0~10℃ during the ultrasound process.

[0016] Further, the organic solvent in step S1 is a mixture of chloroform and methanol, wherein the volume ratio of chloroform to methanol is 1~2:1.

[0017] Further, the buffer solution mentioned in step S1 is a phosphate buffer solution with a concentration of 0.01~0.05 mol / L and a pH value of 7.2~7.4.

[0018] Furthermore, the lipid phase emulsification in step S1, the ultrasonication of the mixed emulsion in step S2, and the gradient extrusion refining in step S3 are all carried out under the protection of an inert gas; the inert gas is nitrogen or argon.

[0019] Further, the centrifugation in step S3 involves centrifuging the lecithin liposome suspension at 3-5°C and 7000-9000 rpm for 5-20 min to remove free lecithin and impurities; the purification in step S3 is dialysis purification, with a molecular weight cutoff of 10-30 kDa in the dialysis bag, a dialysis time of 8-12 h, and phosphate buffer as the dialysis medium, with the dialysis solution being changed 3-4 times during dialysis.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] (1) The preparation method of the present invention uses a specific emulsification process and combines ultrasonic dispersion and gradient extrusion to prepare liposomes with high encapsulation efficiency and uniform particle size. The liposome particle size is controlled at 50~200 nm and the encapsulation efficiency is greater than 85%, which is significantly better than the traditional film dispersion method. Moreover, the oxidative degradation rate of this liposome is less than 2%, which can effectively retain the drug activity.

[0022] (2) The liposomes prepared by the method of the present invention have excellent stability. After being stored at 4°C in the dark for 6 months, the encapsulation rate of the liposomes is greater than 80%, and there is no obvious fusion or leakage.

[0023] (3) The preparation method of the lecithin liposome of the present invention is safe and controllable, easy to operate, and has good repeatability, making it suitable for large-scale production. Attached Figure Description

[0024] Figure 1 This is a flowchart illustrating the preparation process of the lecithin liposomes of the present invention.

[0025] Figure 2 The zeta potential is the serotonin liposomes prepared in Examples 1-6 and Comparative Examples 1-11.

[0026] Figure 3 The oxidative degradation rate of the strychnine liposomes prepared in Examples 1-6 and Comparative Examples 1-11 is given.

[0027] Figure 4 The PDI of strychnine liposomes prepared in Examples 1-6 and Comparative Examples 1-11 is shown.

[0028] Figure 5 The encapsulation efficiency of the lecithin liposomes prepared in Examples 1-6 and Comparative Examples 1-11 is given.

[0029] Figure 6 The curves show the oxidative degradation rate of the liposomes prepared in Example 1 and Comparative Example 11 after storage at 4°C for 0 to 6 months.

[0030] Figure 7 The in vitro release rate curves of the lecithin liposomes prepared in Example 1 and Comparative Example 11 are shown.

[0031] Figure 8 The effect of the lecithin liposomes prepared in Example 1 and Comparative Example 11 on the activity of human renal clear cell carcinoma 786-O cells. Detailed Implementation

[0032] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following embodiments are commercially available.

[0033] Example 1

[0034] This embodiment provides a method for preparing strychnine liposomes, including the following steps:

[0035] S1. Lipid phase emulsification: Phospholipids and cholesterol are mixed in a mixed organic solvent of chloroform and methanol to form a lipid phase. The lipid phase is then sheared and emulsified in a phosphate buffer solution to form a preliminary lipid phase emulsion.

[0036] S2. Ultrasonic dispersing of mixed emulsion: The erythromycin alcohol solution is dropped into the lipid phase preliminary emulsion to form a mixed emulsion. The mixed emulsion is first sheared and emulsified, and then ultrasonically dispersed to obtain a dispersion.

[0037] S3. Gradient extrusion refining: The dispersion was extruded sequentially through filter membranes with pore sizes of 180~260 nm, 140~160 nm and 80~120 nm to obtain a suspension of strychnine liposomes; centrifugation and dialysis purification were performed to obtain strychnine liposomes.

[0038] The lipid phase emulsification in step S1, the ultrasonication of the mixed emulsion in step S2, and the gradient extrusion refining in step S3 are all carried out under the protection of an inert gas; the inert gas is nitrogen or argon.

[0039] Combined with appendix Figure 1 The specific preparation method of strychnine liposomes in this embodiment is as follows:

[0040] (1) Raw material preparation: 4 g soybean lecithin and 1 g cholesterol were added to 200 mL of chloroform and methanol mixture. The volume ratio of chloroform to methanol in the chloroform and methanol mixture was 1:1. The mixture was magnetically stirred for 30 min until completely dissolved to obtain a lipid phase of 25 mg / mL. 0.3 g styraxin was placed in a brown volumetric flask, 50 mL of anhydrous ethanol was added, and the mixture was stirred in the dark for 15 min until dissolved to prepare a 6 mg / mL styraxin alcohol solution.

[0041] (2) Temperature-controlled emulsification: The above lipid phase was slowly added to 800 mL of phosphate buffer with a pH of 7.4 and a concentration of 0.01 mol / L. Then, it was sheared and emulsified for 12 min at a temperature of 25℃ and a shear rate of 4000 rpm to form a preliminary lipid phase emulsion. The nitrogen cylinder was opened (flow rate 0.2 L / min), and 50 mL of erythromycin alcohol solution was added dropwise to the preliminary lipid phase emulsion at a rate of 1 mL / min. Emulsification was continued for 6 min to obtain a mixed emulsion.

[0042] (3) Directional ultrasonic dispersion: The mixed emulsion is transferred to an ice bath. The probe of the probe-type ultrasonic instrument is inserted 1.5 cm below the liquid surface. The power is set to 250 W. The ultrasonic conditions are 3 seconds on and 5 seconds off, with a total ultrasonic time of 8 minutes. During the ultrasonic process, the system is stirred at a rate of 600 rpm to maintain the system temperature at no more than 10°C. In this embodiment, 10°C is preferred. Nitrogen gas is continuously introduced at a flow rate of 0.2 L / min.

[0043] (4) Gradient extrusion refining: The ultrasonically purified dispersion is fed into a multi-stage extruder at an extrusion temperature of 4°C and a pressure of 0.4 MPa. The multi-stage extruder extrudes the dispersion through polycarbonate filter membranes of 200 nm, 150 nm, 100 nm and 50 nm in sequence. The extrusion is repeated twice to obtain a liposome suspension.

[0044] (5) Post-processing: The liposome suspension was centrifuged at 4℃ and 8000 rpm for 10 min and the supernatant was collected. The supernatant was dialyzed for 10 h using a dialysis bag with a molecular weight cutoff of 10000 Da. The dialysis medium was phosphate buffer with a pH of 7.4. The dialysis medium was changed every 3 h. After dialysis, the product was collected and stored in a brown bottle at 4℃ in the dark.

[0045] Comparative Example 1

[0046] This comparative example provides a method for preparing lecithin liposomes. The preparation steps are the same as in Example 1, except that the amount of soybean lecithin in Example 1 is changed to 8 g, while other parameters remain unchanged. The lecithin liposomes prepared are referred to as Comparative Example 1.

[0047] Example 2

[0048] This embodiment provides a method for preparing lecithin liposomes. The preparation steps are the same as in Example 1, except that the amount of soybean lecithin in Example 1 is changed to 2 g, while other parameters remain unchanged. The lecithin liposomes prepared are referred to as Example 2.

[0049] Comparative Example 2

[0050] This comparative example provides a method for preparing lecithin liposomes. The preparation steps are the same as in Example 1, except that the amount of soybean lecithin in Example 1 is changed to 1 g, while other parameters remain unchanged. The lecithin liposomes prepared are referred to as Comparative Example 2.

[0051] Comparative Example 3

[0052] This comparative example provides a method for preparing strychnine liposomes. The preparation steps are the same as in Example 1, except that the shear rate during lipid emulsification in Example 1 is changed to 2000 rpm, while other parameters remain unchanged. The strychnine liposomes prepared are referred to as Comparative Example 3.

[0053] Comparative Example 4

[0054] This comparative example provides a method for preparing lecithin liposomes. The preparation steps are the same as in Example 1, except that the shear rate during lipid emulsification in Example 1 is changed to 3000 rpm, while other parameters remain unchanged. The lecithin liposomes prepared are referred to as Comparative Example 4.

[0055] Example 3

[0056] This embodiment provides a method for preparing lecithin liposomes. The preparation steps are the same as in Example 1, except that the shear rate during lipid emulsification in Example 1 is changed to 5000 rpm, while other parameters remain unchanged. The lecithin liposomes prepared are referred to as Example 3.

[0057] Example 4

[0058] This embodiment provides a method for preparing lecithin liposomes. The preparation steps are the same as in Example 1, except that the emulsification time in Example 1 is changed to 3 min, while other parameters remain unchanged. The lecithin liposomes prepared are referred to as Example 4.

[0059] Example 5

[0060] This embodiment provides a method for preparing lecithin liposomes. The preparation steps are the same as in Example 1, except that the emulsification time in Example 1 is changed to 9 min, while other parameters remain unchanged. The lecithin liposomes prepared are referred to as Example 5.

[0061] Comparative Example 5

[0062] This comparative example provides a method for preparing lecithin liposomes. The preparation steps are the same as in Example 1, except that the lipid phase shearing emulsification time in Example 1 is changed to 15 min. After the lecithin alcohol solution is dropped into the lipid phase preliminary emulsion, emulsification is not continued, but it is directly ultrasonically dispersed. Other parameters remain unchanged. The lecithin liposomes prepared are referred to as Comparative Example 5.

[0063] Comparative Example 6

[0064] This comparative example provides a method for preparing strychnine liposomes. The preparation steps are the same as in Example 1, except that in Example 1, the lipid phase is not emulsified, and the strychnine alcohol solution is directly added to the lipid phase to form a mixed emulsion, which is then ultrasonically dispersed. Other parameters remain unchanged. The strychnine liposomes prepared are referred to as Comparative Example 6.

[0065] Comparative Example 7

[0066] This comparative example provides a method for preparing strychnine liposomes. The preparation steps are the same as in Example 1, except that there is no gradient extrusion refining step, and the centrifugation and dialysis purification steps are performed directly. The strychnine liposomes prepared are referred to as Comparative Example 7.

[0067] Example 6

[0068] This embodiment provides a method for preparing strychnine liposomes. The preparation steps are the same as in Example 1, except that the dispersion is extruded sequentially through a 250 nm, 150 nm and 100 nm (denoted as 250 nm→150 nm→100 nm) polycarbonate filter membrane using a multi-stage extruder. The resulting strychnine liposomes are referred to as Example 6.

[0069] Comparative Example 8

[0070] This comparative example provides a method for preparing strychnine liposomes. The preparation steps are the same as in Example 1, except that the dispersion is extruded sequentially through 250 nm, 150 nm and 50 nm polycarbonate filter membranes using a multi-stage extruder (denoted as 250 nm→150 nm→50 nm). The resulting strychnine liposomes are referred to as Comparative Example 8.

[0071] Comparative Example 9

[0072] This comparative example provides a method for preparing lecithin liposomes. The preparation steps are the same as in Example 1, except that the dispersion is extruded sequentially through 150 nm, 100 nm and 50 nm polycarbonate filter membranes using a multi-stage extruder (denoted as 150 nm→100 nm→50 nm). The resulting lecithin liposomes are referred to as Comparative Example 9.

[0073] Comparative Example 10

[0074] This comparative example provides a method for preparing strychnine liposomes. The preparation steps are the same as in Example 1, except that ultrasonic dispersion is not performed. Other steps remain unchanged. The strychnine liposomes prepared are referred to as Comparative Example 10.

[0075] Comparative Example 11 Thin Film Dispersion Method

[0076] This comparative example provides a conventional thin-film dispersion method for preparing squalene liposomes. The specific preparation method is as follows: Weigh 4 g of soybean lecithin, 1 g of cholesterol, and 0.3 g of squalene, dissolve them in 400 mL of anhydrous ethanol, and sonicate in an ultrasonic cell disruptor on ice for 10 min. The sonication conditions are 5 s on, 10 s off, and a power of 300 W, resulting in a red, transparent solution. Transfer the solution to a round-bottom flask, place the flask on a rotary evaporator, and evaporate at 40°C for 15 min until a translucent lipid film forms in the flask. Remove the organic phase. Wash the contents with PBS buffer at 50°C, and sonicate in an ultrasonic cell disruptor on ice for 20 min. The sonication conditions are 5 s on, 10 s off, and a power of 300 W, resulting in a squalene liposome solution. Filter the solution through a 0.22 μm filter membrane and store it at 4°C protected from light. This is designated as Comparative Example 11.

[0077] Test Example 1

[0078] 1. Testing Method

[0079] The product performance of Examples 1-6 and Comparative Examples 1-11 was tested using the following methods. The specific test indicators were: testing the Polydispersity Index (PDI) to characterize the uniformity of liposome size distribution, testing the Zeta potential to characterize the stability of liposomes after dispersion, testing the encapsulation rate to characterize the encapsulation degree of levofloxacin, and testing the oxidative degradation rate to characterize the stability of liposomes.

[0080] The specific methods are as follows:

[0081] (1) The PDI of liposomes was determined by dynamic light scattering method. Specifically, 100 μL of lecithin liposomes from each example and comparative example was taken, diluted 10 times with deionized water, placed in the sample cell, and measured at room temperature using a laser particle size analyzer.

[0082] (2) The Zeta potential was determined by laser Doppler electrophoresis. The specific operation was to take 100 μL of lecithin liposomes, dilute them 10 times with deionized water, place them in a gold-plated electrode U-shaped cell, and measure the Zeta potential at room temperature using a laser particle size analyzer.

[0083] (3) The encapsulation efficiency was determined by spectrophotometry. Specifically, 1 mL of styrax erythrin liposomes was diluted 5 times with deionized water, centrifuged at 12,000 rpm for 10 min, and the absorbance of the supernatant was measured at 535 nm using an ELISA reader. The concentration of free drug was calculated according to the standard curve. C 游 Take 1 mL of lecithin liposomes, dilute 5 times with anhydrous ethanol, and sonicate in an ultrasonic cell disruptor on ice for 20 min. The sonication conditions are: 5 s on, 10 s off, 300 W, and centrifugation at 12000 rpm for 10 min. Measure the absorbance at 535 nm using an ELISA reader, and calculate the free drug concentration according to the standard curve. C 总 Calculate the encapsulation ratio ( EE The formula is as follows:

[0084] EE % = 100%.

[0085] (4) The oxidative degradation rate was determined by high performance liquid chromatography. Specifically, a gradient concentration (0.5, 1, 2, 5, 10 μg / mL) of styracin standard solution was prepared with methanol and injected into the sample. The peak area was recorded, and a standard curve was plotted with concentration as the abscissa and peak area as the ordinate to obtain the regression equation. Freshly prepared samples from Examples 1-6 and Comparative Examples 1-11 were demulsified first, and then the demulsified filtrate was injected into the sample. The peak area was recorded and substituted into the regression equation to calculate the remaining concentration of styracin in the sample. The specific standard curve can be plotted according to the range of styracin content determined by the reagent and prepared based on the different gradients of styracin standard solutions.

[0086] 2. Test Results

[0087] (1) The test results of important parameters of strychnine liposomes under different raw material ratios are shown in Table 1 and Figures 2-5 As shown in Table 1 and Figures 2-5 It is known that the mass ratio of phospholipids to cholesterol is 4:1. At this ratio, cholesterol precisely fills the gaps between phospholipid molecules, forming a dense bilayer that ensures both the encapsulation of erythromycin and improves the homogeneity of the system. When the ratio is 8:1, due to insufficient cholesterol content, the lipid bilayer becomes too fluid, easily leading to drug leakage and poor system stability. At a ratio of 2:1, excessive cholesterol increases the rigidity of the lipid membrane, making it difficult to form stable vesicles during emulsification and sonication, and easily resulting in drug release.

[0088] Table 1. Key parameters of pyruvic erythromycin liposomes under different raw material ratios.

[0089]

[0090] (2) The test results of important parameters of erythromycin liposomes under different emulsification shear rates and emulsification times are shown in Tables 2 and 3. Figures 2-5 As shown. (Based on Tables 2 and 3, combined with...) Figures 2-5 It was found that a shear rate of 4000 rpm and a total emulsification time of 18 min yielded a relatively homogeneous mixed emulsion while avoiding disruption of lipid molecular arrangement or localized thermal effects. Under these conditions, the lipid phase was fully broken down into micron-sized droplets, allowing the styraxanthin alcohol solution to disperse uniformly and be encapsulated by the lipid membrane upon droplet introduction. When the shear rate was ≤3000 rpm, the initial emulsion exhibited a heterogeneous, turbid state regardless of the emulsification time, and the PDI of the liposomes was >0.25 after subsequent sonication. The low shear rate failed to break down lipid aggregates, resulting in poor initial emulsion dispersibility and posing a potential challenge for subsequent particle size control. At a shear rate of 5000 rpm, although the initial emulsion dispersibility was good, the encapsulation efficiency dropped to 75.4% after the total emulsification time exceeded 18 min. Excessively high shear rates generated localized thermal effects, leading to partial degradation of styraxanthin (degradation peaks were detected by HPLC), and excessive emulsification easily disrupted the lipid molecular arrangement.

[0091] Table 2. Key parameters of pyruvic erythrin liposomes under different emulsification shear rates.

[0092]

[0093] Table 3. Key parameters of liposomes containing styraxanthin under different emulsification times.

[0094]

[0095] (3) The test results of important parameters of strychnine liposomes under different extrusion conditions are shown in Table 4 and Figures 2-5 As shown in Table 4 and Figures 2-5 It can be seen that when the dispersion passes sequentially through polycarbonate filter membranes with pore sizes of 200 nm, 150 nm, 100 nm, and 50 nm (denoted as 200 nm→150 nm→100 nm→50 nm) and the extrusion pressure is 0.4 MPa, the PDI of the liposomes decreases to 0.11, the average particle size stabilizes at 55 nm, and the Zeta potential is -10.04 mV. The gradual reduction in pore size avoids instantaneous rupture of the liposome membrane, and the 0.4 MPa pressure ensures extrusion efficiency without damaging the vesicle structure. When there is no gradient extrusion or when the dispersion passes sequentially through polycarbonate filter membranes with pore sizes of 250 nm, 150 nm, and 100 nm (denoted as 250 nm→150 nm→100 nm), the PDI of the liposomes is 0.21 and 0.17, respectively, with a wide particle size distribution. However, skipping the intermediate pore size filter membrane for gradient filtration (Comparative Examples 8 and 9) results in some large-particle-size liposomes not being effectively extruded, leading to poor system uniformity.

[0096] Table 4. Key parameters of styrax erythrin liposomes under different gradient extrusion conditions.

[0097]

[0098] (4) The test results of important parameters of erythromycin liposomes under ultrasound conditions are shown in Table 5 and Figures 2-5 As shown in Table 5 and Figures 2-5 As can be seen, under the ultrasonic conditions in Example 1, the dispersion was transparent and homogeneous, with a PDI of 0.11 and a degradation rate of only 2.0% for styraxin. Intermittent ultrasound effectively dissipated heat, and the combination of an ice bath and temperature control ensured both dispersion and protection of the active ingredient. Low power could not achieve nanoscale dispersion, while continuous ultrasound (excessive working time) caused the probe to generate heat, raising the system temperature to above 12°C, resulting in a degradation rate of 12.3% for styraxin. At an ultrasonic power of 300 W, although the particle size was further reduced to 85 nm, the encapsulation efficiency decreased to 72.5%. Excessive power damaged the lipid bilayer structure, leading to drug leakage.

[0099] Table 5. Important parameters of pyrenoidin liposomes with and without ultrasound.

[0100]

[0101] (5) The test results of important parameters of the strychnine liposomes prepared in Example 1 and Comparative Example 11 are shown in Table 6 and Figures 2-5 As shown in Table 6 and Figures 2-5 It is known that the preparation method of the present invention, through a specific emulsification process, combined with ultrasonic dispersion and gradient extrusion, produces liposomes with high encapsulation efficiency and uniform particle size. The liposome particle size is controlled at 50~200 nm (RSD≤5%), and the encapsulation efficiency is ≥85%, which is significantly better than the traditional method. Moreover, the oxidative degradation rate of this liposome is less than 2%, effectively preserving the drug activity.

[0102] Table 6 Key parameters of the strychnine liposomes prepared in Example 1 and Comparative Example 11

[0103]

[0104] Test Example 2

[0105] 1. Testing Method

[0106] (1) The liposomes of strychnine prepared in Example 1 and Comparative Example 11 were selected and stored at 4°C for 0, 1, 2, 3, 4, 5 and 6 months respectively. The oxidative degradation rate was determined by high performance liquid chromatography, and the method was the same as in Part (4) of Test Example 1.

[0107] (2) The liposomes prepared in Example 1 and Comparative Example 11 were selected to test their in vitro release rate. The specific test steps are as follows: The in vitro drug release behavior of the liposomes was investigated using dynamic membrane dialysis. 5 mL of the prepared liposomes were placed in a dialysis bag and then placed in a beaker containing 50 mL of 0.5% Tween-80 solution. The beaker was then placed in a constant temperature magnetic stirrer and stirred at 120 rpm in the dark at 37°C. At 0, 6, 12, 24, 48, 72, 96, and 100 h of stirring, 500 μL of dialysis solution was accurately pipetted, and an equal volume of Tween-80 solution was added to the beaker. The absorbance of the solution at 535 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader. The content of liposomes in the solution was calculated using the standard curve method.

[0108] (3) The liposomes prepared in Example 1 and Comparative Example 11 were selected to test their activity against human clear cell renal cell carcinoma 786-O cells. The specific test steps are as follows: The activity of liposomes against 786-O tumor cells was detected by the thiazolium blue colorimetry (MTT) method. Liposome administration groups were set up (0.01, 1.00, 5.00, 10.00, 20.00, 50.00 μg / mL), data were recorded and IC50 was calculated for comparison.

[0109] 2. Test Results

[0110] (1) The oxidative degradation rate changes of the liposomes prepared in Example 1 and Comparative Example 11 after storage at 4°C for 0-6 months are shown in the figure. Figure 6 As shown, by Figure 6 It can be seen that after 6 months of storage, the oxidative degradation rate of the lecithin liposomes in Example 1 was less than 10%, while the oxidative degradation rate of the lecithin liposomes prepared in Comparative Example 11, i.e., the lecithin liposomes prepared by the thin film dispersion method, was higher than 40%.

[0111] (2) The in vitro release effect of the strychnine liposomes prepared in Example 1 and Comparative Example 11 is as follows: Figure 7 As shown, by Figure 7 It can be seen that after 100 h, the in vitro release rate of the lecithin liposome prepared in Example 1 reached 72.5%, which was significantly lower than that of the lecithin liposome prepared in Comparative Example 1, which had an in vitro release rate of 92.6%. Therefore, it can be seen that the lecithin liposome prepared in Example 1 can exert its effect in vitro for a long time.

[0112] (3) Test results are as follows Figure 8 As shown, by Figure 8It can be seen that the half-inhibitory concentration (IC50) of the liposomes of lecithin in human clear cell renal cell carcinoma 786-O cells in Example 1 was 2.188 μM, which was significantly lower than the half-inhibitory concentration (IC50) of the liposomes of lecithin prepared by the thin-film dispersion method in Comparative Example 11, which was 7.811. Therefore, it can be seen that the liposomes of lecithin in Example 1 significantly retained the drug activity.

[0113] Finally, it should be noted that the above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing strychnine liposomes, characterized in that, Includes the following steps: S1. Lipid phase emulsification: Phospholipids and cholesterol are mixed in an organic solvent at a mass ratio of (2~4):1 to form a lipid phase, the total concentration of phospholipids and cholesterol in the lipid phase being 10~30 g / L; the lipid phase is emulsified in a buffer solution with a volume ratio of 1:(3~5) to form a preliminary lipid phase emulsion; the emulsification is carried out at a shear rate of 4000~5000 rpm for 10~15 min; S2. Ultrasonic dispersing of mixed emulsion: A solution of styraxol is dropped into a lipid phase preliminary emulsion to form a mixed emulsion, wherein the mass ratio of styraxol to phospholipid is 1:(10~20). The mixed emulsion is first emulsified at a shear rate of 4000~6000 rpm for 3~9 min. Then, the mixed emulsion is ultrasonically dispersed to obtain a dispersion. The ultrasound is performed using a probe-type ultrasonic instrument with an ultrasonic power of 200-300 W, a working time of 2-4 seconds and a pause of 4-6 seconds, and a total ultrasound time of 5-10 minutes; the system temperature is maintained at 0-10℃ during the ultrasound process. S3. Gradient extrusion refining: The dispersion is extruded and refined sequentially through polycarbonate filter membranes with pore sizes of 200 nm, 150 nm, 100 nm, and 50 nm, or sequentially through polycarbonate filter membranes with pore sizes of 250 nm, 150 nm, and 100 nm. The extrusion pressure is 0.3~0.5 MPa and the extrusion temperature is 2~8℃ to obtain a strychnine liposome suspension; centrifugation and purification are then performed to obtain strychnine liposomes.

2. The method for preparing strychnine liposomes according to claim 1, characterized in that, The phospholipids mentioned in step S1 are one or more of soybean lecithin, egg yolk lecithin, or hydrogenated soybean lecithin.

3. The method for preparing strychnine liposomes according to claim 1, characterized in that, The emulsification in step S1 is carried out at a temperature of 25~30℃ and an emulsification shear rate of 4000~5000 rpm.

4. The method for preparing strychnine liposomes according to claim 1, characterized in that, The organic solvent in step S1 is a mixture of chloroform and methanol, wherein the volume ratio of chloroform to methanol is 1~2:

1.

5. The method for preparing strychnine liposomes according to claim 1, characterized in that, The buffer solution mentioned in step S1 is a phosphate buffer solution with a concentration of 0.01~0.05 mol / L and a pH value of 7.2~7.

4.

6. The method for preparing strychnine liposomes according to claim 1, characterized in that, The lipid phase emulsification in step S1, the ultrasonication of the mixed emulsion in step S2, and the gradient extrusion refining in step S3 are all carried out under the protection of an inert gas; the inert gas is nitrogen or argon.

7. The method for preparing strychnine liposomes according to claim 1, characterized in that, The centrifugation in step S3 involves centrifuging the lecithin liposome suspension at 3-5℃ and 7000-9000 rpm for 5-20 min to remove free lecithin and impurities. The purification in step S3 is dialysis purification, with a molecular weight cutoff of 10-30 kDa in the dialysis bag, a dialysis time of 8-12 h, and phosphate buffer as the dialysis medium. The dialysis solution is changed 3-4 times during dialysis.

Images