CN116115566A - A escin sodium liposome and preparation method thereof - Google Patents

Abstract

The invention relates to the technical field of medicine, specifically, sodium aescinate is used as the main drug, and lecithin, cholesterol and DSPE-PEG2000 are also included, sodium aescinate is wrapped in liposomes, and sodium aescinate is stimulated by sodium aescinate. The defects that are too large to affect clinical use. According to the physical and chemical properties of sodium aescinate itself, the present invention has carried out matching research on the composition of the prescription and the preparation process, and developed a sodium aescinate lipid with minimal irritation, safety, stable quality and simple preparation process in the true sense. The plastid has laid a solid foundation for the further research and application of sodium aescinate.

Description

A escin sodium liposome and preparation method thereof

technical field

The invention relates to the technical field of medicine, in particular to a sodium aescinate liposome and a preparation method thereof.

Background technique

Sodium aescinate is a triterpene saponin sodium salt extracted from the dried ripe fruit of Aesculus Wilsonii Rehd., a plant of the Aesculus family. This product is a white powder or crystalline powder. Soluble, slightly soluble in ethanol. Sodium aescinate is a new generation of anti-inflammatory, anti-osmosis and detumescence drugs. Sodium aescinate can promote the blood vessel wall to increase the secretion of PGE-2, and can remove free radicals in the body, thereby anti-inflammation, anti-exudation, accelerating venous blood flow, promoting lymphatic return, increasing venous tension, improving blood circulation and Microcirculation, and protect the blood vessel wall, its clinical efficacy is remarkable. The drug was successfully developed in my country in the 1980s and used clinically. It mainly treats edema and hematoma in various parts of the human body caused by various reasons; acute and chronic injuries of various tissues, fractures, trauma, various causes such as traumatic brain injury and cerebrovascular disease. Brain dysfunction caused by stroke, brain tumor, cerebrovascular disease, intracranial infection, etc.; local blood circulation disorder caused by various vascular diseases, etc. It has been proved by clinical experiments that sodium aescinate has stable and long-lasting effects, can greatly shorten the course of treatment, relieve pain of patients, avoid water and electrolyte disorders and kidney function damage, and overcome the rebound of mannitol and the side effects of corticosteroids. The curative effect is excellent, and it is a safe, effective and reliable anti-inflammatory and detumescence drug.

Sodium aescinate liniment, gel, tablet, freeze-dried powder injection, etc. have been widely used in the treatment of anti-inflammatory and detumescence. Among them, sodium aescinate liniment and gel are used as external preparations, which have a better curative effect on the treatment of acute closed soft tissue injuries such as sprains, ecchymosis and crushing of tendons, muscles, ligaments and joints. . However, due to the limitations of external preparations, sodium aescinate cannot be used for in-depth treatment of brain dysfunction and various vascular diseases caused by various reasons such as traumatic brain injury, stroke, cerebrovascular disease, brain tumor, intracranial infection, etc. Local blood circulation disorders, etc. At the same time, since sodium aescinate belongs to triterpenoid saponins containing polyester bonds, it has strong irritation and cannot be used on damaged skin and mucous membranes, and may cause local pain and allergic reactions during use. . Oral administration of sodium aescinate tablets has poor absorption and low bioavailability. Large doses will burden the kidneys and cannot be used in comatose patients, which has certain limitations. Sodium aescinate for injection has the advantages of rapid onset of action and long-lasting biological effect. After a single administration, the anti-inflammatory and anti-exudation activities can be maintained for more than 16 hours, which can effectively treat and prevent postoperative edema and reduce the suffering of patients. But because of its strong irritant and the physical and chemical properties of saponins, the preparation has many defects: 1. sodium aescinate is a triterpenoid saponin sodium salt, which itself has a strong irritant to blood vessels, and intravenous injection can cause pain at the injection site. Prolonged medication may cause phlebitis. Strict observation is required during the infusion to prevent the fluid from leaking into surrounding tissues and causing redness and swelling. As reported in the following literature: (Liu Mingjie. Clinical application, adverse reactions and pharmacological studies of sodium aescinate [J]. Shizhen Guoyi Guoyao, 2005 (11): 1158-1160; Wang Qiyun. Clinical application of sodium aescinate for injection and adverse reactions[J]. Clinical Medicine Practice, 2009,18(11):1596-1597.); ②It is easy to cause hemolytic reaction during infusion (Wu Fengzhi et al. Compatibility and infiltration of three kinds of infusions of sodium aescinate for injection pressure analysis[J]. China Pharmaceutical Industry, 2020,29(11):42-43); ③has strong renal function damage, and often causes skin rashes during infusion; it is required to use thicker veins during infusion Note; ④ It is easy to produce foam during preparation, which is inconvenient to prepare. In view of the many problems of sodium aescinate for injection, how to develop a sodium aescinin intravenous administration preparation that is less irritating, safe, easy to use and easy to prepare has become a hot spot in the research of sodium aescinate preparations.

In view of the shortcomings of the existing sodium aescinate preparations, scholars at home and abroad have conducted extensive research on sodium aescinate liposomes or other nano-preparations. We know that nano-preparations have a passive targeting effect on inflammation, which can significantly improve the efficacy and reduce side effects. Sodium aescinate nano-preparation has become a research hotspot in the field of medicine. Patent CN200510089090.7 provides a nano-aescin injection preparation and its preparation method. In this paper, the compatibility of different lipids and emulsifiers is explored. The nano-injection prepared by the compatibility of stearic acid and soybean lecithin has good form, stability, etc., but nano-preparations have defects such as complex preparation process, reasonable proportioning, high processing difficulty, high cost, and difficulty in large-scale promotion. In addition, from the perspective of the formulation process of the preparation, the preparation belongs to the theoretical Solid lipid nanoparticles, the safety of intravenous administration of solid lipid nanoparticles formed by solid lipid stearic acid remains to be further studied. Literature (Li Ziwei, et al. Preparation and quality evaluation of aescin sodium microemulsion injection [J]. Journal of Sichuan University (Medical Science), 2014, 45(01): 147-151; Qin Yao, et al. Aescin Safety and Pharmacodynamics of Sodium Microemulsion Injection [J]. Chinese Modern Applied Pharmacy, 2013, 30(10): 1043-1046.) and patent CN201210338585.9, developed a escin sodium microemulsion, The microemulsion formulation contains a large amount of surfactant, which further increases the incidence of vascular irritation and hemolysis of sodium aescinate. Patents CN200610027444.X and CN201510304196.8 both provide a method for preparing a escin sodium freeze-dried emulsion, but the preparation of the freeze-dried emulsion and the freeze-drying process are complicated, and a large amount of surfactant will be added during the emulsion preparation process, which may cause Lead to more serious vascular irritation and hemolysis, and the water-soluble sodium aescinate is encapsulated in the emulsion, and its encapsulation rate will be relatively low, thereby affecting its in vivo effect. It can be seen that the practicability of preparing sodium aescinate as nano-injection, microemulsion or freeze-dried emulsion and other conventional coating preparations is very different, mainly because sodium aescinate is a water-soluble drug with poor encapsulation and increased microemulsion. The safety hazards of vascular irritation and hemolysis, as well as the complicated preparation process of nano-injection and freeze-dried emulsion are caused by many reasons.

Liposomes were proposed by Bangham in the mid-1960s. In the early 1970s, Gregoriadis first proposed the use of liposomes as β-galactosidase carriers to treat glycogen accumulation diseases. Since then, liposomes have received widespread attention as a drug delivery carrier. The phospholipid bilayer of liposomes is similar to biological membranes, has good biocompatibility and low toxicity, and has been widely used in cancer, fungal treatment and viral infection. For this reason, we plan to use liposome technology to reduce the direct contact between the drug and the blood vessel wall, so as to fundamentally solve the problem of strong irritation and strong hemolysis of sodium aescinate, and achieve high efficiency, low irritation, and low hemolysis. Research purposes.

Through literature search, there have been related reports on aescin sodium liposomes both at home and abroad. Patent CN202011530050.2 has developed a compound preparation of sodium aescinate for reducing swelling and analgesia. The article involves the preparation method of sodium aescinate liposomes. The ratio of sodium aescinate to membrane material is 1:30- 40 (weight ratio), the molar ratio of phospholipids to cholesterol is 5:2. However, its preparation process is complicated, and it is necessary to dissolve the aqueous solution of sodium aescinate and the ethanol solution of the membrane material to form a film. This feature is difficult to realize in process amplification, and the industrial operability is poor. In addition, repeated extrusion is required during the preparation process. 15 times, this process has a certain impact on the stability of the preparation. This patent uses DEPC normal saline for hydration, but DEPC water needs to be autoclaved before being prepared into DEPC normal saline. And this article does not carry out correlation evaluation such as characterization, drug efficacy and safety etc. to the prescription of liposome different ratio, this shows that it does not carry out systematic research on liposome. Patents CN201710325262.9 and CN201710324980.4 both mentioned a preparation method of a escin sodium liposome hydrogel patch, which uses 20-50% ethanol to dissolve the lipid film material and medicine, and then through the thin film dispersion method Preparation of liposomes. We made the same preparation according to the method shown in Example 1 of patent CN201710324980.4. During the preparation process, we found that it is difficult to form a film by adding 30% ethanol, and 70% of the water is almost difficult to volatilize at 45°C. Speed up the rotation appropriately Speed, because the nature of aescin itself will produce a large number of bubbles and suck back, so there is a big loophole in the method of the embodiment of this patent, which needs to be discussed. Patents CN201710212793.7, CN201710212717.6 and CN201710212709.1 disclose an external preparation of aescin liposome gel for improving the poor skin penetration and skin irritation of sodium aescinate, all of which are prepared by ethanol injection method Liposomes, although the preparation method is simple, the technology is feasible, but the liposome particle diameters prepared by the formula and process it adopts are all greater than 300nm or even greater than 500nm, and the particle diameters are relatively large, and the encapsulation efficiency of the liposomes is almost all Less than 90%, which will directly affect the stability of drug-loaded liposomes. We still prepared the Example 1 shown in the patent CN201710212793.7, and the encapsulation efficiency obtained was basically the same as that shown in the patent, but we found that there was a lot of drug loss before and after ultrafiltration, which further shows that the topical aescin liposome has not yet been perfected Liposome formulation process and drug matching research, in order to achieve smaller particle size, higher encapsulation efficiency, and less drug ultrafiltration loss. Literature (De Groot C, Musken M, Muller-Goymann C C.Novel Colloidal Microstructures of beta-Escin and the Liposomal Components Cholesterol and DPPC[J].Planta Med,2018,84(16):1219-1227.) by film dispersion method to prepare sodium aescinate liposomes, and explored the interaction between β-sodium aescinate, cholesterol and DPPC, but this document did not discuss the prescription, process and liposomes of β-escin sodium liposomes. Do a systematic study on the relationship between the encapsulation efficiency of plastids, drug release, drug efficacy in vivo, hemolysis, and vascular irritation. Literature (Li Ziwei. Research on liposome inflammatory lesion delivery system constructed by fMLP modified cholesterol [D]. Chongqing Medical University, 2014.) It provides a fMLP (leukocyte chemotactic peptide) modified aescin sodium lipid Plastid preparation method, in order to improve the targeting of inflammatory lesions of liposomes, the paper adopts thin film dispersion method to prepare liposomes, prescription technology research shows that when cholesterol: phospholipid ratio (molar ratio) is 2:1, 1: 1, 1:2, 1:4, the drug encapsulation efficiency is 54.06%, 70.45%, 79.42%, 72.31%; when the drug lipid ratio (mass ratio) is 1:6, 1:3, 1:1 , the drug encapsulation efficiency was 77.34%, 79.42%, 30.23% respectively, and the encapsulation efficiency of the final optimized formula was (80.1 ± 4.8)%. From this, it can be seen that the encapsulation efficiency of the liposomes made is generally not high, indicating that The formulation process has not yet reached the optimization level, and the thin film dispersion method may cause the residue of the organic solvent chloroform and cause harm to the human body.

It can be seen that, at present, the technology of liposome sodium aescinate for external use and sodium aescinate for intravenous administration is still very immature, and due to the imperfection of formula technology etc., the particle size of the drug-loaded liposome prepared , encapsulation efficiency, stability, vascular irritation, hemolysis and other aspects have not been fundamentally improved.

Contents of the invention

Based on the above dosage form does not fundamentally improve the strong irritant and hemolytic defects of sodium aescinate, the object of the present invention is to provide a safe, efficient and stable quality sodium aescinate liposome. Another object of the present invention is to provide a preparation method of sodium aescinate liposome.

The first aspect of the present invention provides a kind of sodium aescinate liposome, with sodium aescinate as active ingredient, also includes lecithin, cholesterol and DSPE-PEG2000; Wherein sodium aescinate consumption is 0.1-0.5% ( w/v); the dosage of lecithin is 1-10% (w/v); the dosage of cholesterol is 0.5-5% (w/v); the dosage of DSPE-PEG2000 is 0-1% (w/v).

Further, the dosage of lecithin is 2-7% (w/v); the dosage of cholesterol is 1-3% (w/v); the dosage of DSPE-PEG2000 is 0.01-0.3% (w/v).

Further, the sodium aescinate liposome is an injection, preferably a freeze-dried powder injection.

Further, the organic solvent for injection of the injection is selected from one or more of absolute ethanol, propylene glycol, and tert-butanol, preferably absolute ethanol. The amount used is 7-15% g/ml; more preferably 9-13% g/ml.

Further, in the sodium aescinate liposome, the drug lipid ratio is 1:5-1:30 g/g, lecithin:cholesterol is 1:1-5:1 g/g, and the pH value is 2.0-4.8 .

Further, the sodium aescinate liposome is composed of sodium aescinate, lecithin, cholesterol, DSPE-PEG2000, a lyoprotectant and a pH regulator.

Further, the described sodium aescinate liposome is prepared from the following formula:

Furthermore, the described sodium aescinate liposome is prepared from the following formula:

Further, the lecithin is selected from egg yolk lecithin, high-purity egg yolk lecithin, hydrogenated soybean lecithin, distearoylphosphatidylcholine, distearoylphosphatidylglycerol, dipalmitoylphosphatidylcholine , soybean lecithin, phosphatidylserine, dimyristoyl phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin; preferably high-purity egg yolk lecithin.

Further, the lyoprotectant is selected from one or more of sucrose, trehalose, maltose, glucose, lactose, mannitol, sorbitol, xylitol, erythritol, and threonine. Preferably, one or more of sucrose, trehalose and maltose are used in combination.

Further, the pH regulator is selected from citric acid, hydrochloric acid, sodium hydroxide, phosphoric acid, disodium hydrogen phosphate, disodium phosphate, dipotassium hydrogen phosphate, disodium citrate, trisodium citrate One or more in combination; preferably one or more of citric acid and hydrochloric acid in combination. Adjust the pH value to 2.0-4.8, preferably adjust the pH value to 2.5-4.5.

The second aspect of the present invention provides a method for preparing the sodium aescinate liposome as described above, and the preparation method is an injection method. Described preparation method comprises the following steps:

Weigh the prescribed amount of lecithin, cholesterol, DSPE-PEG2000, place in an organic solvent, heat and dissolve at 50-65°C to obtain an organic phase; weigh the prescribed amount of sodium aescinate and dissolve it in an appropriate amount of water for injection, and dissolve it in 50 Keep warm at -65°C, adjust the pH with a pH regulator to obtain an aqueous phase; inject the organic phase into the aqueous phase under stirring conditions, mix well, and dilute to the prescribed amount with water for injection to obtain crude liposomes; Emulsification of the crude plastid can be carried out by ultrasonic probe, or placed in an extruder, extruded several times through the extrusion film, or placed in a high-pressure homogenizer for homogeneous emulsification to obtain liposomes solution. Add the same amount of water for injection or the same amount of water for injection after adjusting the pH and ultra-filter several times to remove the organic solvent; weigh the prescribed amount of lyoprotectant, put it in the above liposome solution, stir to dissolve, pass through a 0.22 μm filter membrane to remove bacterium, aliquoting, freeze-drying, and capping to obtain sodium aescinin liposome freeze-dried powder injection.

Further, in the preparation method, the pH regulator is selected from citric acid, hydrochloric acid, sodium hydroxide, phosphoric acid, disodium hydrogen phosphate, disodium phosphate, dipotassium hydrogen phosphate, disodium citrate, citric acid One or two or more of trisodium are used in combination; preferably one or more of citric acid and hydrochloric acid are used in combination to adjust the pH value to 2.0-4.8, preferably to adjust the pH value to 2.5-4.5.

Further, in the preparation method, the organic solvent for injection is selected from one or more of absolute ethanol, propylene glycol, and tert-butanol, preferably absolute ethanol, and the dosage is 7-15% g g/ml; more preferably 9-13% g/ml.

Further, in the preparation method, the organic solvent for injection can be removed by ultrafiltration after the crude liposome is emulsified, or it can be removed during the freeze-drying process. It is preferably removed by ultrafiltration, and the dialysate can be water for injection or water for injection after pH adjustment, and the pH is 2.0-4.8, preferably 2.5-4.5.

Further, in the preparation method, the injection method may be injecting the oil phase into the water phase, or injecting the water phase into the oil phase.

Further, in the preparation method, the crude liposome is emulsified, and the pore diameter of the extruded membrane is selected from 0.8 μm, 0.6 μm, 0.4 μm, 0.2 μm, 0.1 μm, 0.08 μm, and 0.05 μm. One species, or more than two species are extruded through large orifices to small orifices in sequence. Preferably extruded film is 0.2 μm, 0.1 μm.

The average particle diameter of the sodium aescinate liposome of the present invention is 70-200nm.

The third aspect of the present invention provides an application of the above-mentioned sodium aescinate liposome in the preparation of anti-inflammatory drugs.

The sodium aescinate liposome of the present invention exhibits a better curative effect than sodium aescinate for injection at equal clinical doses.

The present invention has the advantage that:

1. The present invention encapsulates the sodium aescinate in the liposome, which solves the defect that the sodium aescinate is highly irritating and affects clinical use.

2. The sodium aescinate liposome provided by the present invention has high encapsulation efficiency and good stability.

3. The sodium aescinate liposome provided by the present invention has better anti-inflammatory effect.

4. Aiming at the physical and chemical properties of sodium aescinate itself, the present invention has carried out matching research on the composition of the prescription and the preparation process. Through the matched prescription process, it has truly overcome the problems of strong irritation and easy hemolysis of sodium aescinate for injection. To solve the problem, a escin sodium liposome with minimal irritation, safety, stable quality and simple preparation process was developed, which laid the foundation for further research and application of aescin sodium.

Description of drawings

Figure 1. Parallel comparison of hemolytic properties of different embodiments;

Figure 2. Irritation comparison of different examples.

Detailed ways

The specific implementation modes provided by the present invention will be described in detail below in conjunction with the examples. It should be understood that the following examples are only used to illustrate the present invention but not to limit the scope of the present invention. Based on the encapsulation efficiency of liposome after ultrafiltration is about 99%, so, in order to reflect the rationality of liposome formulation process, the encapsulation efficiency described in the following examples all refers to the encapsulation efficiency before ultrafiltration.

Embodiment 1: the criticality of aqueous phase pH to sodium aescinate liposome development

1. Experimental prescription

Table 1 Prescription Design of Experimental Verification Scheme

2. Preparation process

Weigh high-purity egg yolk lecithin (EPCS), cholesterol, and DSPE-PEG2000 of the prescribed amount, place them in 11.00 g of absolute ethanol, heat and dissolve at 65°C to obtain an organic phase; weigh 0.25 g of the prescribed amount of sodium aescinate Dissolve in 82g of water for injection, keep warm at 65°C, adjust the pH with citric acid to obtain the aqueous phase; inject the organic phase into the aqueous phase under stirring conditions, mix well, and dilute to volume with the corresponding pH-adjusted water for injection To 100mL, the crude liposome is obtained; the crude liposome is extruded twice through a 0.2μm extrusion membrane, and then extruded 5 times through a 0.1μm extrusion membrane to obtain a liposome solution; add an equal amount of water for injection Ultrafiltration 4 times to remove the organic solvent; weigh 12g of sucrose, put it in the above liposome solution, stir to dissolve, pass through a 0.22μm filter membrane to sterilize, sub-package, freeze-dry, and cap to obtain aescin sodium lipid Plastid freeze-dried powder for injection.

3. Experimental results

Since the encapsulation efficiency of water-soluble drugs is generally low, we mainly use the encapsulation efficiency as the main index of investigation. We have carried out a parallel comparison of aescin sodium liposomes with a series of aqueous phase pH, and found that the aqueous phase pH Encapsulation efficiency has a more obvious impact. When the pH of the aqueous phase is less than 2.0, the acidity is too strong, and it is not suitable for intravenous administration; when the pH of the aqueous phase is between 2.0-4.8, the encapsulation efficiency is all above 90%, and when the pH of the aqueous phase is greater than 4.8, the encapsulation efficiency will be lower. significantly reduced. The results are shown in Table 2 below.

The influence result of table 2 aqueous phase pH on the druggability of sodium aescinate liposome

group Exterior Encapsulation rate (%) particle size PDI Prescription 1 translucent homogeneous solution 97.84 124.6 0.145 Prescription 2 translucent homogeneous solution 98.31 115.3 0.112 Prescription 3 translucent homogeneous solution 97.06 111.7 0.103 Prescription 4 translucent homogeneous solution 94.53 108.4 0.117 Prescription 5 translucent homogeneous solution 90.27 105.7 0.115 Prescription 6 translucent homogeneous solution 82.66 103.6 0.108

Result analysis: After a large number of experimental verifications, we obtained the above experimental results. We found that the pH of the aqueous phase will directly affect the encapsulation of sodium aescinate, and the encapsulation efficiency of sodium aescinin liposomes is closely related to the pH value of the aqueous phase. Visible, the regulation of aqueous phase pH is crucial for this liposome, will directly influence the druggability of liposome.

Embodiment 2: choose embodiment 1 prescription 3 different dialysis fluids to the evaluation of aescin sodium liposome

1. Preparation process

Prescription 3 in Example 1 was selected to prepare sodium aescinate liposomes, and water for injection and water for injection (pH3.5) after adjusting the pH were respectively used as dialysate to remove the organic solvent. All the other preparation steps are the same as the preparation method of prescription 3 in Example 1.

2. Effects of different dialysates on drug loss before and after ultrafiltration of sodium aescin liposomes and encapsulation efficiency after ultrafiltration

Table 3 Different dialysates on drug loss before and after ultrafiltration of sodium aescin liposomes and encapsulation efficiency after ultrafiltration

dialysate Before ultrafiltration (mg/mL) After ultrafiltration (mg/mL) loss% Encapsulation efficiency% after ultrafiltration Water for Injection 2.35 2.27 3.40 99.21 Water for injection after pH adjustment (pH3.5) 2.35 2.26 3.83 99.46

Result analysis: We analyzed and verified the results of the removal of organic solvents by ultrafiltration of the two dialysates, and found that whether the dialysate was water for injection or water for injection after adjusting the pH, the loss of the drug before and after ultrafiltration of sodium aescinate liposomes and There was no significant effect on the encapsulation efficiency after ultrafiltration, so water for injection or water for injection after adjusting the pH could be selected as the dialysate.

Embodiment 3: the criticality of the amount of organic solvent for injection to the development of sodium aescinate liposome

1. Experimental prescription

Table 4 Prescription Design of Experimental Verification Scheme

2. Preparation process

Weigh high-purity egg yolk lecithin (EPCS), cholesterol, and DSPE-PEG2000 of the prescribed amount, place them in absolute ethanol, heat and dissolve at 55°C to obtain an organic phase; weigh 0.25 g of the prescribed amount of sodium aescinate for injection Dissolve in water, keep warm at 55°C, adjust the pH to 3.5 with citric acid to obtain the aqueous phase; inject the organic phase into the aqueous phase under stirring conditions, mix well, and dilute to the corresponding volume with water for injection after pH adjustment 100mL to obtain the crude liposome; extrude the crude liposome twice through a 0.2μm extrusion membrane, and then extrude through a 0.1μm extrusion membrane 5 times to obtain a liposome solution; add an equal amount of water for injection for ultrafiltration Remove the organic solvent 4 times; weigh 12g of sucrose, place it in the above liposome solution, stir to dissolve, pass through a 0.22μm filter membrane to sterilize, subpackage, freeze-dry, and cap to obtain sodium aescinate liposome Freeze-dried powder.

3. Experimental results

The appearance, encapsulation efficiency and smoothness of the 0.22 μm filter membrane of the above-mentioned liposomes were investigated, and the results are shown in Table 5 below.

The influence result of table 5 injection organic solvent dosage on sodium aescinate liposome

The experimental results show that different dosages of organic solvents for injection have a greater impact on the encapsulation efficiency of sodium aescinate liposomes, which is another important reason why this preparation has a higher encapsulation efficiency. When the content of the organic solvent for injection is small, the cholesterol cannot be completely dissolved even under heating, which affects the druggability of the liposome. And when the content of the organic solvent for injection is large, the encapsulation efficiency of the prepared sodium aescinate liposome is low, which affects the quality, so the appropriate amount of the organic solvent for injection is the key of the present invention.

Example 4: Comparative Evaluation of Aescin Sodium Liposomes Prepared by Different Phospholipids and Cholesterol Ratio

1. Preparation of sodium aescinate liposomes with different ratios of phospholipids and cholesterol

Table 6 Prescription Design of Experimental Verification Scheme

2. Preparation process

Weigh high-purity egg yolk lecithin (EPCS), cholesterol, DSPE-PEG2000 of prescription quantity, place in 11.00g of absolute ethanol, heat and dissolve at 55°C to obtain an organic phase; weigh prescription quantity of sodium aescinate 0.25 g was dissolved in 82g of water for injection, kept at 55°C, and adjusted to pH 3.3 with citric acid to obtain the aqueous phase; the organic phase was injected into the aqueous phase under stirring conditions, mixed well, and injected with the corresponding pH-adjusted Dilute the volume to 100mL with water to obtain the crude liposome; extrude the crude liposome twice through the 0.2μm extrusion membrane, and then extrude through the 0.1μm extrusion membrane 5 times to obtain the liposome solution; add an equal amount Remove the organic solvent by ultrafiltration of water for injection 4 times; weigh 12g of sucrose, place it in the above liposome solution, stir to dissolve, pass through a 0.22μm filter membrane to sterilize, subpackage, freeze-dry, and cap to obtain aescin Sodium liposome lyophilized powder for injection.

The impact result of table 7 different phospholipids and cholesterol ratio on sodium aescinate liposome

group Encapsulation rate (%) particle sizenm PDI Prescription 1 90.36 103.4 0.176 Prescription 2 91.03 107.7 0.183 Prescription 3 97.51 110.1 0.102 Prescription 4 96.78 111.6 0.104

After a series of parallel comparisons of experimental results, it is found that the ratio of lecithin to cholesterol is too small, the extrusion pressure of the preparation will be too high, and the extrusion cannot be smoothly extruded, and when the ratio of lecithin to cholesterol is too large, it will directly affect the sodium aescin lipid The encapsulation efficiency of the body, the encapsulation efficiency decreases with the increase of the ratio of the two, and it will lead to the increase of PDI, which will affect the stability of the preparation.

Example 5: Comparative evaluation of aescin sodium liposomes prepared with different drug-to-lipid ratios

1. Preparation of sodium aescinate liposomes with different lipid ratios

Table 8 Prescription Design of Experimental Verification Scheme

2. Preparation process

Weigh high-purity egg yolk lecithin (EPCS), cholesterol, and DSPE-PEG2000 of the prescribed amount, place them in 11.00 g of absolute ethanol, heat and dissolve at 60°C to obtain an organic phase; weigh 0.25 g of the prescribed amount of sodium aescin Dissolve in 81g of water for injection, keep warm at 60°C, adjust the pH to 3.6 with citric acid to obtain the aqueous phase; inject the organic phase into the aqueous phase under stirring conditions, mix well, and use the corresponding pH-adjusted water for injection Set the volume to 100mL to obtain the crude liposome product; extrude the crude liposome product twice through the 0.2μm extrusion membrane, and then extrude through the 0.1μm extrusion membrane 5 times to obtain the liposome solution; add an equal amount of injection Ultrafiltration with water for 4 times to remove the organic solvent; weigh 12g of sucrose, place it in the above liposome solution, stir to dissolve, pass through a 0.22μm filter membrane to sterilize, subpackage, freeze-dry, and cap to obtain sodium aescinate Liposome lyophilized powder for injection.

3. Drug loss before and after ultrafiltration of sodium aescinate liposomes with different drug lipid ratios

Table 9 Drug loss before and after ultrafiltration of sodium aescinate liposomes compared to different drug lipids

Before ultrafiltration (mg/mL) After ultrafiltration (mg/mL) loss% Medicine fat ratio 1:5 2.46 2.20 10.57 Medicine fat ratio 1:10 2.49 2.38 4.42 Medicine fat ratio 1:15 2.28 2.19 3.94 Medicine fat ratio 1:20 2.39 2.31 3.35 Drug fat ratio 1:30 2.45 2.38 2.86

Result analysis: Different drug-to-lipid ratios have obvious effects on drug loss. When the drug-lipid ratio is relatively large, the drug loss is obvious, resulting in unstable process, and to a certain extent, the lipid ratio is increased. We unexpectedly found that when the drug-lipid ratio is increased to between 1:10 and 1:30, the Drug loss tends to plateau, but on the basis of adding less excipients and reducing economic costs, we prefer a drug-to-lipid ratio of 1:10 to 1:20.

Embodiment 6: Preparation of sodium aescinate liposome

Weigh 3g of high-purity egg yolk lecithin (EPCS), 1.5g of cholesterol, and 0.1g of DSPE-PEG20000 in 11.00g of absolute ethanol, and heat to dissolve at 65°C to obtain an organic phase; weigh the recipe Dissolve 0.25g of sodium aescinate in 82g of water for injection, adjust the pH value to 3.5 with citric acid, keep warm at 55°C to obtain the water phase; inject the organic phase into the water phase under stirring conditions, mix well, and use water for injection Dilute the volume to 100mL to obtain the crude liposome product; pass the crude liposome product through a high-pressure homogenizer, and homogenize three times under pressures of 15000psi, 20000psi and 25000psi respectively to obtain a liposome solution; add an equal amount of water for injection for ultrafiltration Remove the organic solvent 4 times; weigh 12g of sucrose, place it in the above liposome solution, stir to dissolve, pass through a 0.22μm filter membrane to sterilize, subpackage, freeze-dry, and cap to obtain sodium aescinate liposome Freeze-dried powder.

It was determined that the encapsulation efficiency was 96.85%, the particle size was 153.4nm, and the PDI was 0.178.

Embodiment 7: Preparation of sodium aescinate liposome

Weigh 3g of high-purity egg yolk lecithin (EPCS), 1.5g of cholesterol, and 0.1g of DSPE-PEG20000 in 11.00g of absolute ethanol, and heat to dissolve at 65°C to obtain an organic phase; weigh the recipe Dissolve 0.25g of sodium aescinate in 82g of water for injection, adjust the pH value to 3.5 with citric acid, keep warm at 55°C to obtain the water phase; inject the organic phase into the water phase under stirring conditions, mix well, and use water for injection Set the volume to 100mL to obtain the crude liposome product; extrude the crude liposome product twice through the 0.2μm extrusion membrane, and then extrude through the 0.1μm extrusion membrane 5 times to obtain the liposome solution; add an equal amount of injection Ultrafiltration with water for 5 times to remove the organic solvent; weigh 12g of sucrose, put it in the above liposome solution, stir to dissolve, pass through a 0.22μm filter membrane to sterilize, subpackage, freeze-dry, and cap to obtain sodium aescinate Liposome lyophilized powder for injection.

It was determined that the encapsulation rate was 97.05%, the particle size was 110.1 nm, and the PDI was 0.102.

Embodiment 8: Preparation of sodium aescinate liposome

Weigh 3g of high-purity egg yolk lecithin (EPCS), 1.5g of cholesterol, and 0.1g of DSPE-PEG20000 in 11.00g of absolute ethanol, and heat to dissolve at 65°C to obtain an organic phase; weigh the recipe Dissolve 0.20g of sodium aescinate in 84g of water for injection, adjust the pH value to 3.35 with citric acid, keep warm at 55°C to obtain the water phase; inject the organic phase into the water phase under stirring conditions, mix well, and use water for injection Set the volume to 100mL to obtain the crude liposome product; extrude the crude liposome product twice through the 0.2μm extrusion membrane, and then extrude through the 0.1μm extrusion membrane 5 times to obtain the liposome solution; add an equal amount of injection Ultrafiltration with water for 5 times to remove the organic solvent; weigh 10.00 g of sucrose, place it in the above liposome solution, stir to dissolve, pass through a 0.22 μm filter membrane to sterilize, subpackage, freeze-dry, and cap to obtain aescin Sodium liposome lyophilized powder for injection.

It was determined that the encapsulation efficiency was 95.71%, the particle size was 108.4nm, and the PDI was 0.104.

Embodiment 9: Preparation of sodium aescinate liposome

Weigh 3g of high-purity egg yolk lecithin (EPCS), 1.5g of cholesterol, and 0.1g of DSPE-PEG20000 in 10.00g of absolute ethanol, and heat to dissolve at 55°C to obtain an organic phase; Dissolve 0.25g of sodium aescinate in 82g of water for injection, adjust the pH value to 2.0 with citric acid, keep warm at 55°C to obtain the water phase; inject the organic phase into the water phase under stirring conditions, mix well, and use water for injection Set the volume to 100mL to obtain the crude liposome product; pass the crude liposome product through a high-pressure homogenizer, and homogenize three times at 10000psi, 15000psi and 20000psi respectively to obtain a liposome solution; add an equal amount of corresponding pH The final water for injection was ultrafiltered 4 times to remove the organic solvent; 8g of sucrose was weighed, placed in the above-mentioned liposome solution, stirred to dissolve, passed through a 0.22 μm filter membrane to sterilize, sub-packaged, freeze-dried, and capped to obtain seven Sodium leaf saponin liposome lyophilized powder for injection.

It was determined that the encapsulation efficiency was 95.84%, the particle size was 167.3nm, and the PDI was 0.192.

Embodiment 10: Preparation of sodium aescinate liposome

Weigh 3g of high-purity egg yolk lecithin (EPCS), 1.5g of cholesterol, and 0.1g of DSPE-PEG20000 in 13.00g of absolute ethanol, and heat to dissolve at 60°C to obtain an organic phase; weigh the recipe Dissolve 0.25g of sodium aescinate in 80g of water for injection, adjust the pH value to 3.5 with citric acid, keep warm at 60°C to obtain the water phase; inject the organic phase into the water phase under stirring conditions, mix well, and use water for injection Set the volume to 100mL to obtain the crude liposome product; extrude the crude liposome product twice through the 0.2μm extrusion membrane, and then extrude through the 0.1μm extrusion membrane 5 times to obtain the liposome solution; add an equal amount of injection Ultrafiltration with water for 5 times to remove the organic solvent; weigh 15.00g of sucrose, put it in the above liposome solution, stir to dissolve, pass through a 0.22μm filter membrane to sterilize, subpackage, freeze-dry, and cap to obtain aescin Sodium liposome lyophilized powder for injection.

It was determined that the encapsulation efficiency was 92.07%, the particle size was 116.2nm, and the PDI was 0.116.

Embodiment 11: Preparation of sodium aescinate liposome

Weigh 5g of high-purity egg yolk lecithin (EPCS), 2.5g of cholesterol, and 0.3g of DSPE-PEG20000 in the prescription amount, place them in 13.00g of absolute ethanol, and heat to dissolve at 50°C to obtain an organic phase; weigh the recipe amount Dissolve 0.3g of sodium aescinate in 76g of water for injection, adjust the pH value to 3.5 with citric acid, keep warm at 55°C to obtain the water phase; inject the organic phase into the water phase under stirring conditions, mix well, and use water for injection Dilute the volume to 100mL to obtain the crude liposome; pass the crude liposome through a high-pressure homogenizer, and homogenize three times at 10,000 psi, 15,000 psi, and 20,000 psi respectively to obtain a liposome solution; add an equal amount to adjust the pH for injection Ultrafiltration with water for 6 times to remove the organic solvent; weigh 11.00g of sucrose, place it in the above liposome solution, stir to dissolve, pass through a 0.22μm filter membrane to sterilize, subpackage, freeze-dry, and cap to obtain aescin Sodium liposome lyophilized powder for injection.

It was determined that the encapsulation efficiency was 93.11%, the particle size was 127.9nm, and the PDI was 0.188.

Embodiment 12: Preparation of sodium aescinate liposome

Weigh 4.0g of high-purity egg yolk lecithin (EPCS), 1.0g of cholesterol, and 0.1g of DSPE-PEG20000 in 12g of absolute ethanol, and heat at 60°C to dissolve to obtain an organic phase; Dissolve 0.25g of sodium aescinate in 81g of water for injection, adjust the pH value to 2.5 with citric acid, keep warm at 60°C to obtain the water phase; inject the organic phase into the water phase under stirring conditions, mix well, and use water for injection Dilute the volume to 100mL to obtain the crude liposome; extrude the crude liposome twice through a 0.2μm extrusion film, and then extrude through a 0.1μm extrusion film five times to obtain a liposome solution; add an equal amount of phase The corresponding pH-adjusted water for injection was ultrafiltered 5 times to remove the organic solvent; weighed 25g of trehalose, placed it in the above liposome solution, stirred to dissolve, passed through a 0.22μm filter membrane to sterilize, subpackaged, freeze-dried, pressed Cover to get sodium aescin liposome freeze-dried powder injection.

It was determined that the encapsulation efficiency was 90.67%, the particle size was 109.1 nm, and the PDI was 0.186.

Embodiment 13: Preparation of sodium aescinate liposome

Weigh 1.64g of high-purity egg yolk lecithin (EPCS), 1.36g of cholesterol, and 0.01g of DSPE-PEG2000 in the prescribed amount, place them in 11.00g of absolute ethanol, and heat to dissolve at 65°C to obtain an organic phase; weigh the recipe Dissolve 0.2 g of sodium aescinate in 78 g of water for injection, adjust the pH value to 3.38 with citric acid, and keep warm at 55°C to obtain the water phase; inject the organic phase into the water phase under stirring conditions, mix well, and inject Dilute the volume to 100mL with water to obtain crude liposomes; pass the crude liposomes through a high-pressure homogenizer, and homogenize 3 times at 10,000 psi, 15,000 psi, and 20,000 psi respectively to obtain a liposome solution; add an equal amount of corresponding adjustment The water for injection after the pH is ultra-filtered 5 times to remove the organic solvent; weigh 8 g of sucrose, place it in the above-mentioned liposome solution, stir to dissolve, pass through a 0.22 μm filter membrane to sterilize, subpackage, freeze-dry, and press the cap to obtain Aescin sodium liposome lyophilized powder for injection.

It was determined that the encapsulation rate was 94.31%, the particle size was 103.9nm, and the PDI was 0.111.

Embodiment 14: Preparation of sodium aescinate liposome

Weigh 3g of high-purity egg yolk lecithin (EPCS), 1.5g of cholesterol, and 0.1g of DSPE-PEG20000 in 11.00g of propylene glycol, and heat to dissolve at 50°C to obtain an organic phase; Dissolve 0.25 g of sodium saponin in 83 g of water for injection, adjust the pH value to 4.8 with hydrochloric acid and keep warm at 50°C to obtain the water phase; inject the organic phase into the water phase under stirring conditions, mix well, and dilute to 100 mL with water for injection , to obtain the crude liposome; the crude liposome was extruded twice through a 0.2 μm extrusion membrane, and then extruded 5 times through a 0.1 μm extrusion membrane to obtain a liposome solution; an equivalent amount of water for injection was added for ultrafiltration for 4 Remove the organic solvent once; weigh 15.00 g of maltose, place it in the above-mentioned liposome solution, stir to dissolve, pass through a 0.22 μm filter membrane to sterilize, subpackage, freeze-dry, and cap to obtain sodium aescinate liposome Freeze-dried powder.

It was determined that the encapsulation efficiency was 90.27%, the particle size was 123.5nm, and the PDI was 0.168.

Embodiment 15: Preparation of sodium aescinate liposome

Weigh 3g of high-purity egg yolk lecithin (EPCS), 1.5g of cholesterol, and 0.1g of DSPE-PEG20000 in 11.00g of absolute ethanol, and heat to dissolve at 55°C to obtain an organic phase; weigh the recipe Dissolve 0.25g of sodium aescinate in 86g of water for injection, adjust the pH value to 2.5 with citric acid, keep warm at 55°C to obtain the water phase; inject the organic phase into the water phase under stirring conditions, mix well, and use water for injection Set the volume to 100mL to obtain the crude liposome product; pass the crude liposome product through a high-pressure homogenizer, and homogenize it at 12000psi, 18000psi and 22000psi respectively for 3 times to obtain a liposome solution; add an equal amount of water for injection for ultrafiltration for 4 Remove the organic solvent once; take 5g of sucrose, place it in the above-mentioned liposome solution, stir to dissolve, pass through a 0.22 μm filter membrane to sterilize, sub-package, freeze-dry, and press the cap to obtain the sodium aescin liposome jelly Dry powder injection.

It was determined that the encapsulation efficiency was 97.48%, the particle size was 163.2nm, and the PDI was 0.194.

Embodiment 16: Preparation of sodium aescinate liposome

Weigh 3g of high-purity egg yolk lecithin (EPCS), 1.5g of cholesterol, and 0.1g of DSPE-PEG20000 in 11.00g of absolute ethanol, and heat to dissolve at 55°C to obtain an organic phase; weigh the recipe Dissolve 0.25g of sodium aescinate in 82g of water for injection, adjust the pH value to 3.5 with citric acid, keep warm at 55°C to obtain the water phase; inject the organic phase into the water phase under stirring conditions, mix well, and use water for injection Dilute the volume to 100mL to obtain the crude liposome product; pass the crude liposome product through ultrasonic probe (working for 30s, stop for 10s, a total of 8 cycles) to obtain a liposome solution; add an equal amount of water for injection after adjusting the pH for ultrafiltration Remove the organic solvent 5 times; weigh 20g of sucrose, put it in the above liposome solution, stir to dissolve, pass through a 0.22μm filter membrane to sterilize, subpackage, freeze-dry, and cap to obtain sodium aescinate liposome Freeze-dried powder.

It was determined that the encapsulation efficiency was 95.05%, the particle size was 197.4nm, and the PDI was 0.233.

Embodiment 17: Preparation of sodium aescinate liposome

Weigh 3 g of high-purity egg yolk lecithin (EPCS), 1.5 g of cholesterol, and 0.1 g of DSPE-PEG in 9 g of absolute ethanol, and heat to dissolve at 55° C. to obtain an organic phase; Dissolve 0.20 g of leaf saponin sodium in 84 g of water for injection, adjust the pH value to 3.5 with citric acid, keep warm at 55°C to obtain the water phase; inject the organic phase into the water phase under stirring conditions, mix well, and use water for injection to set the pH value to 3.5. When the volume reaches 100mL, the crude liposome product is obtained; the crude liposome product is extruded twice through a 0.2 μm extrusion film, and then extruded five times through a 0.1 μm extrusion film to obtain a liposome solution; The water for injection after pH adjustment was ultrafiltered 3 times to remove the organic solvent; 12 g of sucrose was weighed, placed in the above-mentioned liposome solution, stirred to dissolve, sterilized through a 0.22 μm filter membrane, subpackaged, freeze-dried, and capped. The freeze-dried powder injection of liposome sodium aescinate was obtained.

It was determined that the encapsulation efficiency was 97.11%, the particle size was 125.1 nm, and the PDI was 0.154.

Embodiment 18: Preparation of sodium aescinate liposome

Weigh 3g of high-purity egg yolk lecithin (EPCS), 1.0g of cholesterol, and 0.1g of DSPE-PEG20000 in 11.00g of absolute ethanol, and heat to dissolve at 55°C to obtain an organic phase; Dissolve 0.25g of sodium aescinate in 82g of water for injection, adjust the pH value to 3.5 with citric acid, keep warm at 55°C to obtain the water phase; inject the organic phase into the water phase under stirring conditions, mix well, and use water for injection Set the volume to 100mL to obtain the crude liposome product; extrude the crude liposome product twice through the 0.2μm extrusion membrane, and then extrude through the 0.1μm extrusion membrane 5 times to obtain the liposome solution; add an equal amount of injection Ultrafiltration with water for 4 times to remove the organic solvent; weigh 20g of sucrose, place it in the above liposome solution, stir to dissolve, pass through a 0.22μm filter membrane to sterilize, subpackage, freeze-dry, and cap to obtain sodium aescinate Liposome lyophilized powder for injection.

It was determined that the encapsulation efficiency was 92.34%, the particle size was 107.3nm, and the PDI was 0.126.

Embodiment 19: Preparation of sodium aescinate liposome

Weigh 5g of high-purity egg yolk lecithin (EPCS), 2.5g of cholesterol, and 0.3g of DSPE-PEG20000 in 15.00g of propylene glycol, and heat to dissolve at 65°C to obtain an organic phase; Dissolve 0.25g of sodium saponin in 74g of water for injection, adjust the pH value to 3.5 with citric acid, keep warm at 55°C to obtain the aqueous phase; inject the aqueous phase into the organic phase under stirring conditions, mix well, and use water for injection to make up the volume To 100mL, the liposome crude product is obtained; the liposome crude product is extruded twice through a 0.2 μm extrusion film, and then extruded 5 times through a 0.1 μm extrusion film to obtain a liposome solution; Filter 6 times to remove the organic solvent; weigh 8 g of sucrose, place it in the above liposome solution, stir to dissolve, pass through a 0.22 μm filter membrane to sterilize, subpackage, freeze-dry, and cap to obtain sodium aescinate lipid Body freeze-dried powder injection.

It was determined that the encapsulation efficiency was 92.42%, the particle size was 128.1nm, and the PDI was 0.178.

Embodiment 20: Preparation of sodium aescinate liposome

Weigh 5 g of high-purity egg yolk lecithin (EPCS), 1 g of cholesterol, and 0.1 g of DSPE-PEG 20000 in 13.00 g of tert-butanol, and heat to dissolve at 55° C. to obtain an organic phase; Dissolve 0.20 g of sodium leaf saponin in 78 g of water for injection, adjust the pH value to 2.5 with citric acid, and keep warm at 55°C to obtain the water phase; inject the water phase into the organic phase under stirring conditions, mix well, and set the pH value with water for injection. When the volume reaches 100mL, the crude liposome product is obtained; the crude liposome product is extruded twice through 0.8 μm, and then 5 times through 0.4 μm to obtain a liposome solution; Freeze-dried and capped to obtain the freeze-dried powder of sodium aescinate liposome.

It was determined that the encapsulation efficiency was 90.53%, the particle size was 177.3nm, and the PDI was 0.154.

Example 21: Preparation of Aescin Sodium Liposomes

Weigh 1 g of high-purity egg yolk lecithin (EPCS), 0.5 g of cholesterol, and 0.1 g of DSPE-PEG in 7 g of absolute ethanol, and heat to dissolve at 55 ° C to obtain an organic phase; Dissolve 0.3 g of leaf saponin sodium in 90 g of water for injection, adjust the pH value to 3.5 with hydrochloric acid, keep warm at 55°C to obtain the water phase; inject the organic phase into the water phase under stirring conditions, mix well, and use water for injection to make up the volume to 100mL to obtain the crude liposome product; extrude the crude liposome product twice through a 0.2 μm extrusion membrane, and then extrude through a 0.1 μm extrusion membrane 5 times to obtain a liposome solution; add an equivalent amount of corresponding Ultrafiltration 3 times of water for injection to adjust the pH to remove the organic solvent; weigh 7g of sucrose, place it in the above liposome solution, stir to dissolve, pass through a 0.22μm filter membrane to sterilize, subpackage, freeze-dry, and cap, to obtain Aescin sodium liposome lyophilized powder for injection.

It was determined that the encapsulation efficiency was 93.57%, the particle size was 121.13nm, and the PDI was 0.136.

Example 22: Preparation of Aescin Sodium Liposomes

Weigh 3g of high-purity egg yolk lecithin (EPCS), 1.5g of cholesterol, and 0.1g of DSPE-PEG20000 in the prescription amount, place in 15.00g of absolute ethanol, heat and dissolve at 55°C to obtain an organic phase; weigh the recipe amount Dissolve 0.25 g of sodium aescinate in 78 g of water for injection, adjust the pH value to 3.5 with hydrochloric acid, and keep warm at 55°C to obtain the water phase; inject the organic phase into the water phase under stirring conditions, mix well, and set the pH value to 3.5 with water for injection. When the volume reaches 100mL, the crude liposome product is obtained; the crude liposome product is extruded twice through a 0.2 μm extrusion film, and then extruded five times through a 0.1 μm extrusion film to obtain a liposome solution; add an equal amount of water for injection Ultrafiltration 7 times to remove the organic solvent; weigh 25g of sucrose, put it in the above liposome solution, stir to dissolve, pass through a 0.22μm filter membrane to sterilize, sub-package, freeze-dry, and cap to obtain aescin sodium lipid Plastid freeze-dried powder for injection.

It was determined that the encapsulation rate was 90.91%, the particle size was 113.2nm, and the PDI was 0.118.

Example 23: Preparation of Aescin Sodium Liposomes

Weigh 3g of high-purity egg yolk lecithin (EPCS), 1.5g of cholesterol, and 0.2g of DSPE-PEG20000 in 11.00g of absolute ethanol, and heat to dissolve at 60°C to obtain an organic phase; weigh the recipe Dissolve 0.25g of sodium aescinate in 82g of water for injection, adjust the pH value to 4.8 with citric acid, keep warm at 60°C to obtain the water phase; inject the organic phase into the water phase under stirring conditions, mix well, and use water for injection Set the volume to 100mL to obtain the crude liposome product; extrude the crude liposome product twice through the 0.2μm extrusion membrane, and then extrude through the 0.1μm extrusion membrane 5 times to obtain the liposome solution; add an equal amount of injection Ultrafiltration with water for 4 times to remove the organic solvent; weigh 12g of trehalose, put it in the above liposome solution, stir to dissolve, pass through a 0.22μm filter membrane to sterilize, subpackage, freeze-dry, and cap to obtain aescin Sodium liposome lyophilized powder for injection.

It was determined that the encapsulation efficiency was 90.27%, the particle size was 115.6nm, and the PDI was 0.121.

Example 24: Preparation of Aescin Sodium Liposomes

Weigh 3 g of distearoylphosphatidylcholine (DSPC), 1.5 g of cholesterol, and 0.1 g of DSPE-PEG20000 in the prescribed amount, place them in 14 g of absolute ethanol, and heat to dissolve at 65° C. to obtain an organic phase; Dissolve 0.2g of sodium aescinate in 78g of water for injection, adjust the pH value to 4.8 with citric acid, and heat to 65°C to obtain the aqueous phase; inject the organic phase into the aqueous phase under stirring conditions, and mix well to obtain Liposome crude product; the liposome crude product is extruded 2 times through a 0.2 μm extrusion membrane, and then extruded 6 times through a 0.1 μm extrusion membrane to obtain a liposome solution; add an equal amount of water for injection and ultrafilter 6 times to remove organic Solvent: Weigh 20g of maltose, place it in the above liposome solution, stir to dissolve, use water for injection to make up to 100mL, pass through a 0.22μm filter membrane to sterilize, subpackage, freeze-dry, and cap to obtain aescin Sodium liposome lyophilized powder for injection.

It was determined that the encapsulation efficiency was 90.58%, the particle size was 196.4nm, and the PDI was 0.266.

Example 25: Preparation of Aescin Sodium Liposomes

Weigh 3g of hydrogenated soybean lecithin (HSPC), 1.5g of cholesterol, and 0.1g of DSPE-PEG20000 in 13.00g of absolute ethanol, and heat to dissolve at 55°C to obtain an organic phase; Dissolve 0.2 g of leaf saponin sodium in 78 g of water for injection, adjust the pH value to 4.8 with citric acid, and heat to 55°C to obtain the aqueous phase; inject the organic phase into the aqueous phase under stirring conditions, and mix well to obtain liposomes Crude product; the liposome crude product is extruded 2 times through a 0.2 μm extrusion film, and then extruded 5 times through a 0.1 μm extrusion film to obtain a liposome solution; adding an equal amount of water for injection and ultrafiltration 6 times to remove the organic solvent; Take 10.00g of maltose, put it in the above liposome solution, stir to dissolve, use water for injection to make up to 100mL, pass through a 0.22μm filter membrane to sterilize, subpackage, freeze-dry, and press cap to obtain aescin sodium lipid Plastid freeze-dried powder for injection.

It was determined that the encapsulation efficiency was 91.27%, the particle size was 164.2nm, and the PDI was 0.151.

Example 26: Preparation of Aescin Sodium Liposomes

Weigh 1g of high-purity egg yolk lecithin (EPCS), 0.5g of cholesterol, and 20000g of DSPE-PEG in the prescribed amount, place them in 10.00g of absolute ethanol, and heat to dissolve at 55°C to obtain an organic phase; weigh the prescribed amount of Aescin Dissolve 0.10.00g of sodium saponin in 86g of water for injection, adjust the pH value to 3.5 with hydrochloric acid, keep warm at 55°C to obtain the water phase; inject the organic phase into the water phase under stirring conditions, mix well, and use water for injection to make up the volume to 100mL to obtain the crude liposome; extrude the crude liposome through a 0.1μm extrusion membrane twice, and then through a 0.05μm extrusion 4 times to obtain a liposome solution; add an equal amount of water for injection and ultrafilter 4 times Remove the organic solvent; weigh 12g of trehalose, put it in the above liposome solution, stir to dissolve, pass through a 0.22μm filter membrane to sterilize, subpackage, freeze-dry, and cap to obtain the sodium aescin liposome jelly Dry powder injection.

It was determined that the encapsulation rate was 91.03%, the particle size was 78.72nm, and the PDI was 0.145.

Example 27: Preparation of Aescin Sodium Liposomes

Weigh 10.00g of high-purity egg yolk lecithin (EPCS), 5g of cholesterol, and 1g of DSPE-PEG20001g of the prescription amount, place in 12g of absolute ethanol, heat and dissolve at 60°C to obtain an organic phase; weigh the recipe amount of aescin Dissolve 0.50 g of sodium in 70 g of water for injection, adjust the pH value to 3.4 with hydrochloric acid, keep warm at 60°C to obtain the water phase; inject the organic phase into the water phase under stirring conditions, mix well, and dilute to 100 mL with water for injection , to obtain the crude liposome; the crude liposome was extruded 3 times through a 0.2 μm extrusion film, and then 6 times through a 0.1 μm extrusion film to obtain a liposome solution; The final water for injection was ultrafiltered 5 times to remove the organic solvent; 12 g of sucrose was weighed, placed in the above-mentioned liposome solution, stirred to dissolve, passed through a 0.22 μm filter membrane to sterilize, sub-packaged, freeze-dried, and capped to obtain seven Sodium leaf saponin liposome lyophilized powder for injection.

It was determined that the encapsulation efficiency was 95.43%, the particle size was 147.4nm, and the PDI was 0.122.

Example 28: Preparation of Sodium Aescinate Liposome

Weigh 1.33g of high-purity egg yolk lecithin (EPCS), 0.67g of cholesterol, and 0.01g of DSPE-PEG2000 in the prescribed amount, place them in 7g of absolute ethanol, and heat to dissolve at 50°C to obtain an organic phase; weigh the prescribed amount Dissolve 0.20 g of sodium aescinate in 88 g of water for injection, adjust the pH value to 3.4 with hydrochloric acid, keep warm at 50°C to obtain the water phase; inject the organic phase into the water phase under stirring conditions, mix well, and set the pH value to 3.4 with water for injection. When the volume reaches 100mL, the crude liposome product is obtained; the crude liposome product is extruded twice through the 0.1μm extrusion membrane, and then extruded 5 times through the 0.08μm extrusion film to obtain the liposome solution; The final water for injection was ultrafiltered 3 times to remove the organic solvent; 12g of sucrose was weighed, placed in the above-mentioned liposome solution, stirred to dissolve, passed through a 0.22 μm filter membrane to sterilize, subpackaged, freeze-dried, and capped to obtain seven Sodium leaf saponin liposome lyophilized powder for injection.

It was determined that the encapsulation efficiency was 94.89%, the particle size was 95.51nm, and the PDI was 0.188.

Embodiment 29: Preparation of sodium aescinate liposome

Weigh 1.2g of high-purity egg yolk lecithin (PL-100M), 1g of cholesterol, and 0.2g of DSPE-PEG2000 in the prescribed amount, place them in 11.00g of absolute ethanol, and heat to dissolve at 50°C to obtain an organic phase; Dissolve 0.25g of sodium aescinate in 84g of water for injection, adjust the pH value to 3.37 with hydrochloric acid, keep warm at 55°C to obtain the water phase; inject the organic phase into the water phase under stirring conditions, mix well, and inject Dilute the volume to 100mL with water to obtain the crude liposome; extrude the crude liposome twice through the 0.2μm extrusion membrane, and then extrude through the 0.1μm extrusion membrane 5 times to obtain the liposome solution; add an equal amount Remove the organic solvent by ultrafiltration of water for injection 4 times; weigh 12g of sucrose, place it in the above liposome solution, stir to dissolve, pass through a 0.22μm filter membrane to sterilize, subpackage, freeze-dry, and cap to obtain aescin Sodium liposome lyophilized powder for injection.

It was determined that the encapsulation rate was 92.11%, the particle size was 117.0 nm, and the PDI was 0.168.

Example 30: Preparation of Aescin Sodium Liposomes

Weigh 2 g of high-purity egg yolk lecithin (EPCS), 2 g of cholesterol, and 0.05 g of DSPE-PEG in 10.00 g of tert-butanol, and heat to dissolve at 55° C. to obtain an organic phase; Dissolve 0.30 g of sodium leaf saponin in 83 g of water for injection, adjust the pH value to 3.4 with hydrochloric acid, keep warm at 55°C to obtain the water phase; inject the organic phase into the water phase under stirring conditions, mix well, and use water for injection to make up the volume to 100mL, the crude liposome is obtained; the crude liposome is extruded twice through 0.6μm, and then 5 times through 0.08μm to obtain a liposome solution; After drying and pressing the cap, the sodium aescin liposome freeze-dried powder for injection is obtained.

It was determined that the encapsulation efficiency was 93.47%, the particle size was 86.00nm, and the PDI was 0.104.

Example 31: Preparation of Aescin Sodium Liposomes

Weigh 7g of high-purity egg yolk lecithin (EPCS), 3g of cholesterol, and 0.3g of DSPE-PEG in 11.00g of propylene glycol, and heat to dissolve at 55°C to obtain an organic phase; weigh the prescription amount of aescin Dissolve 0.4g of sodium in 74g of water for injection, adjust the pH value to 4.5 with hydrochloric acid, keep warm at 55°C to obtain the aqueous phase; inject the organic phase into the aqueous phase under stirring conditions, mix well, and dilute to 100mL with water for injection , to obtain crude liposomes; the crude liposomes were extruded twice through a 0.2 μm extrusion film, and then extruded 6 times through a 0.1 μm extrusion film to obtain a liposome solution; The final water for injection was ultrafiltered 6 times to remove the organic solvent; 12g of sucrose was weighed, placed in the above-mentioned liposome solution, stirred to dissolve, passed through a 0.22 μm filter membrane to sterilize, subpackaged, freeze-dried, and capped to obtain seven Sodium leaf saponin liposome lyophilized powder for injection.

It was determined that the encapsulation efficiency was 90.65%, the particle size was 115.7nm, and the PDI was 0.128.

Example 32: Preparation of Aescin Sodium Liposomes

Weigh 3g of high-purity egg yolk lecithin (EPCS), 1.5g of cholesterol, and 0.05g of DSPE-PEG20000 in 11.00g of absolute ethanol, and heat to dissolve at 55°C to obtain an organic phase; weigh the recipe Dissolve 0.25g of sodium aescinate in 82g of water for injection, adjust the pH value to 2.7 with citric acid, keep warm at 55°C to obtain the water phase; inject the organic phase into the water phase under stirring conditions, mix well, and use water for injection Set the volume to 100mL to obtain the crude liposome product; extrude the crude liposome product twice through the 0.2μm extrusion membrane, and then extrude through the 0.1μm extrusion membrane 5 times to obtain the liposome solution; add an equal amount of injection Ultrafiltration with water for 5 times to remove the organic solvent; weigh 15.00g of sucrose, put it in the above liposome solution, stir to dissolve, pass through a 0.22μm filter membrane to sterilize, subpackage, freeze-dry, and cap to obtain aescin Sodium liposome lyophilized powder for injection.

It was determined that the encapsulation rate was 96.19%, the particle size was 95.65.1nm, and the PDI was 0.135.

Example 33: Select the sodium aescinate liposomes for injection prepared in Examples 6, 7, and 15 for pharmacodynamic research

1. Mouse ear swelling model

40 ICR mice were randomly divided into 5 groups (n=8), respectively blank group, injection sodium aescinate group (3.6mg/kg) and injection sodium aescinate liposome dosage group (Example 6, 7, 15). The tail vein was administered for three consecutive days. 30 minutes after the last administration, 30 μL xylene was applied to both sides of the right ear of each mouse to induce inflammation, and the left ear was not applied as a control. Ears were punched with a 7.5 mm diameter hole punch at the same site, weighed with an analytical balance, and the difference in weight (mg) between the left and right ear pieces was used to indicate the degree of inflammatory swelling, and the swelling inhibition rate was calculated.

Mouse ear swelling situation (x ± s) in each group of table 10

group Dose (mg/kg) Ear swelling degree (mg) Swelling inhibition rate (%) model group - 0.0078±0.0029 / Sodium Aescinate for Injection Group 3.6 0.0045±0.0012 42.31 Example 6 3.6 0.0021±0.0011 73.18** Example 7 3.6 0.0023±0.0010 70.51** Example 15 3.6 0.0024±0.0013 69.23**

Note: Compared with the control group *P<0.05, **P<0.01.

2. Rat cotton ball granuloma model

40 SD rats are randomly divided into 5 groups, 8 in every group, respectively blank group, injection sodium aescinate group (1.8mg/kg) and injection sodium aescinate liposome dosage group (embodiment 6 , 7, 15). One day before the first administration, a 35 mg sterile cotton ball was implanted in the groin of each rat, and the administration was continued for 7 days. After 24 hours of the last administration, the cotton ball was taken out together with the surrounding connective tissue, and the fat tissue was removed, and placed in Dry in a drying oven at 60°C for 12 hours and weigh. Subtract the weight of the cotton ball from the weighed weight to obtain the dry weight of the granulation.

Table 11 The situation of rat granuloma in each group (x ± s)

group Dose (mg/kg) Granuloma weight (mg) Inhibition rate(%) model group - 0.0863±0.0093 / Sodium Aescinate for Injection Group 1.8 0.0586±0.0094 32.10%** Example 6 1.8 0.0420±0.0078 51.33%**, ## Example 7 1.8 0.0445±0.0153 48.44%**, # Example 15 1.8 0.0461±0.0122 46.58%**, #

Note: Compared with the control group **P<0.01, compared with the model group ^# P<0.05, ^## P<0.01.

Result analysis: the sodium aescinate liposome for injection and the sodium aescinate for injection prepared by the method of three examples were selected to carry out the mouse ear swelling and the rat cotton ball granuloma model for pharmacodynamic evaluation. The experimental results showed that, The curative effect of the sodium aescinate liposome for injection prepared by randomly selected 3 examples is obviously better than that of sodium aescinate for injection, indicating that the overall curative effect of the liposome prepared by the present invention is greatly improved.

Example 34: Select the sodium aescinate liposomes for injection prepared in Examples 6, 7, and 15 for hemolytic evaluation

1. Hemolytic experimental design

Hemolysis test is used to evaluate the safety of clinical use of sodium aescinate for injection. 20 mL of blood was collected from the rabbit's ear vein, and the fibrinogen was removed with a glass rod to prepare defibrillated blood. Use 10 times the amount of normal saline, shake well, centrifuge at 2500r/min for 5min, and discard the supernatant. According to the above method, the red blood cells deposited in the lower layer were repeatedly washed with normal saline for 2 to 3 times until the supernatant was colorless and transparent. The obtained erythrocytes were diluted with physiological saline according to the volume ratio to form a 2% erythrocyte suspension.

Eight glass test tubes containing 2.5 mL of 2% erythrocyte suspension were numerically labeled. Add different amounts of aescin sodium liposomes for injection (0.5 mg/mL) to test tubes 1-5 (0.1, 0.2, 0.3, 0.4 and 0.5 mL). Tube 6 contained only 2.5 mL of 0.9% saline as a negative control. Tube 7 contained 2.5 mL of distilled water as a positive control. Tube 8 contained 0.5 mL of sodium aescinate for injection (0.5 mg/mL) as a reference control. Add saline solution to 5 mL in each tube. After mixing, incubate at 37°C and observe at 15min, 30min, 45min, 1h, 2h, 3h and 4h.

2. The hemolytic comparison results between different examples and sodium aescinate for injection

Result analysis: as shown in Figure 1, the selected 3 examples did not suffer from hemolysis, red blood cells were deposited at the bottom of the test tube, and the supernatant was transparent, while tube No. 8 had hemolysis, red blood cells ruptured, and the liquid was bright red. This further shows that the liposome prepared by the present invention reduces the occurrence of hemolysis and is safer for clinical use.

Example 35: Select the sodium aescinate liposomes for injection prepared in Examples 6, 7, and 15 for irritation evaluation

1. Stimulus program design

Fifteen rabbits (body weight 1.8-2.0kg) were randomly divided into five groups, and injected sodium aescinate liposome and sodium aescinate for injection respectively through the vein of the right ear margin of the rabbits, and the administration dose was 1.3 mg/kg. The injection speed is 1mL/min. At the same time, the left ear was given an equal volume of normal saline via the marginal ear vein as a control, once a day for three consecutive days. Animal behavior and injection site changes were observed visually during daily dosing. Euthanasia was carried out 24h-48h after the last administration, and the ear vein tissue was taken about 0.5cm-3.0cm from the injection site to the heart on both sides, fixed in 10% paraformaldehyde solution, dehydrated with ethanol gradient, and embedded in paraffin and stained with hematoxylin and ovallobulin. All samples were examined using a BX43-DP21 light microscope (Olympus, Japan, Ltd.) and evaluated for pathological changes.

2. The results of irritation evaluation are analyzed as follows:

As shown in Figure 2, the rabbit ear veins in the blank group had no obvious congestion, the vessel wall structure was intact, and the surrounding tissues had no obvious inflammatory lesions. The stratified squamous epithelium on the skin surface of the auricle was intact, and the subcutaneous appendages such as hair follicles, sebaceous glands, and sweat glands had no obvious lesions. The shape of the elastic cartilage in the center was normal, the interstitial fibrous tissue of the auricle had no obvious lesions, and there was a small amount of chronic inflammatory cell infiltration in the interstitium.

In the group of sodium aescinate for injection, there was obvious congestion and hemorrhage in the ear vein, and the infiltration of inflammatory cells in the tissues around the vessel wall was obvious. The subcutaneous connective tissue was loose, edema, and collagen fibers were broken; many acute inflammatory cell infiltrations appeared in the interstitial fibrous tissue of the auricle.

Aescin sodium liposome group (Example 6, 7, 15) ear vein vascular endothelial cells were basically intact, a small amount of inflammatory cell infiltration and a small amount of bleeding around the blood vessel wall. Attachment of fibrinous material to the vein wall. No obvious degeneration, necrosis, hyperplasia and other lesions were seen in the epidermal tissue of the skin. Deep skin appendages such as hair follicles, sebaceous glands and sweat glands showed no obvious lesions. The shape of elastic cartilage in the center is normal, and normal chondrocytes, cartilage matrix and perichondrium structure can be seen.

Result analysis:

The sodium aescinate liposome for injection of the present invention has higher encapsulation efficiency, small particle size, narrow distribution, and smooth sterilization and filtration. More importantly, the sodium aescinate liposome for injection of the present invention can fundamentally Above all, solve the problems of sodium aescinate with strong hemolytic property, great irritation, and great toxic and side effects.

The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the described embodiments, and those skilled in the art can also make various equivalents without violating the spirit of the present invention. These equivalent modifications or replacements are all included within the scope defined by the claims of the present application.

Claims (10)

1. a sodium aescinate liposome, is characterized in that, taking sodium aescinate as active ingredient, also comprises lecithin, cholesterol and DSPE-PEG2000; Wherein sodium aescinate consumption is 0.1-0.5% (w/ v) The dosage of lecithin is 1-10% (w/v); the dosage of cholesterol is 0.5-5% (w/v); the dosage of DSPE-PEG2000 is 0-1% (w/v). 2. sodium aescinate liposome according to claim 1, is characterized in that, described sodium aescinate liposome is freeze-dried powder injection. 3. sodium aescinate liposome according to claim 2, is characterized in that, described sodium aescinate liposome is formulated by following formula:

4. sodium aescinate liposome according to claim 2, is characterized in that, described sodium aescinate liposome is formulated by following formula:

5. sodium aescin liposome according to claim 1, is characterized in that, described lecithin is selected from egg yolk lecithin, high-purity egg yolk lecithin, hydrogenated soybean lecithin, distearoyl phosphatidyl One or both of choline, distearoylphosphatidylglycerol, dipalmitoylphosphatidylcholine, soybean lecithin, phosphatidylserine, dimyristoylphosphatidylcholine, phosphatidylethanolamine, sphingomyelin The above are used together. 6. sodium aescinate liposome according to claim 1, is characterized in that, described lyoprotectant is selected from sucrose, trehalose, maltose, glucose, lactose, mannitol, sorbitol, xylitol One or more of erythritol, threonine; the pH regulator is selected from citric acid, hydrochloric acid, sodium hydroxide, phosphoric acid, disodium hydrogen phosphate, disodium phosphate, dihydrogen phosphate One or more of potassium, disodium citrate, and trisodium citrate are used in combination. 7. a preparation method of sodium aescinate liposome as described in any one of claims 1-6, is characterized in that, comprises the following steps: Weigh the prescribed amount of lecithin, cholesterol, DSPE-PEG2000, place in an organic solvent, heat and dissolve at 50-65°C to obtain an organic phase; weigh the prescribed amount of sodium aescinate and dissolve it in an appropriate amount of water for injection, and dissolve it in 50 Keep warm at -65°C, adjust the pH with a pH regulator to obtain an aqueous phase; inject the organic phase into the aqueous phase under stirring conditions, mix well, and dilute to the prescribed amount with water for injection to obtain crude liposomes; Emulsification of the crude plastid can be carried out by ultrasonic probe, or placed in an extruder, extruded several times through the extrusion film, or placed in a high-pressure homogenizer for homogeneous emulsification to obtain liposomes solution; add an equal amount of water for injection or an equal amount of water for injection after pH adjustment, and ultra-filter several times to remove the organic solvent; weigh the prescribed amount of lyoprotectant, place it in the above-mentioned liposome solution, stir to dissolve, and filter through 0.22 μm Membrane sterilizing, subpackaging, freeze-drying, and capping to obtain escin sodium liposome freeze-dried powder injection. 8. the preparation method of sodium aescinate liposome according to claim 7, is characterized in that, described organic solvent, is selected from one or more in dehydrated alcohol, propylene glycol, tert-butyl alcohol, The dosage is 7-15% g/ml. 9. the preparation method of sodium aescinate liposome according to claim 7, is characterized in that, described liposome crude product is emulsified, and extrusion membrane pore diameter is selected from 0.8 μm, 0.6 μm, 0.4 μm, 0.2 μm, 0.1μm, 0.08μm, 0.05μm, choose one or more than two kinds of extrusion through large aperture to small aperture in sequence. 10. The application of a escin sodium liposome as described in any one of claims 1-6 in the preparation of anti-inflammatory drugs.

Images