A trimethyl chitosan-modified sodium alginate diester multivesicular liposome and its preparation method and application

Abstract

The present invention discloses a trimethyl chitosan-modified sodium alginate multivesicular liposome and a preparation method and application thereof. The preparation method comprises: (1) dissolving a lipid component in an organic solvent as an oil phase; dissolving sodium alginate in an aqueous solution containing an osmotic pressure regulator as an inner aqueous phase; (2) adding the oil phase to the inner aqueous phase, and ultrasonically forming colostrum; (3) adding the colostrum to an outer aqueous phase containing an osmotic pressure regulator and an auxiliary emulsifier, and homogenizing to form a double emulsion; (4) rotary evaporating the double emulsion to remove the organic solvent, centrifuging to remove the free drug, and dispersing the precipitate in a solvent to obtain sodium alginate multivesicular liposome; (5) synthesizing trimethyl chitosan, and uniformly coating to obtain trimethyl chitosan-modified sodium alginate multivesicular liposome. The liposome prepared by the present invention has a high drug encapsulation rate, and the use of trimethyl chitosan modification can further promote oral absorption, improve the oral bioavailability of the drug, reduce the number of drug use times, and improve patient compliance. Furthermore, the trimethyl chitosan-modified sodium diester alginate multivesicular liposomes have uniform particle size and stable morphology, so as to exert anticoagulation and platelet aggregation inhibition effects and be applied to the treatment of thrombosis-related diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to trimethyl chitosan-modified sodium alginate diester multivesicular liposomes and a preparation method and application thereof. Background Art

[0002] Propylene glycol alginate sodium sulfate (PSS) is the first marine polysaccharide drug in my country. It uses alginic acid extracted from brown algae (such as kelp) as the basic raw material. It is first degraded to obtain oligomeric alginic acid, and then chemically modified to introduce propylene glycol ester groups and sulfate ester groups to obtain a polyanionic compound. The specific structural formula is as follows:

[0003]

[0004] Pharmacological effects of sodium alginate diester: It is mainly used for the prevention and treatment of ischemic cerebrovascular diseases such as cerebral thrombosis, cerebral embolism, transient ischemic attack and cardiovascular diseases such as hypertension, hyperlipoproteinemia, coronary heart disease, angina pectoris, etc. (Li Chunxia, ​​Sun Yang, Guan Huashi. Research progress and inspiration of marine drug sodium alginate diester. Life Science, 2012, 24(9):1019-1023). In recent years, studies have shown that PSS also has expanded applications in a variety of diseases such as lowering blood sugar and blood pressure.

[0005] PSS is mainly used in clinical practice in the form of tablets and injections, but its low oral bioavailability and poor patient compliance limit and restrict the clinical applicability of PSS. In order to meet the needs of patients, more and more new PSS dosage forms are being studied. For example, CN103142539B reports a sodium alginate diester controlled-release tablet and its preparation method, but the preparation prepared by this method basically releases the drug completely within 12 hours. For another example, Li Pengli et al. used polylactic-co-glycolic acid copolymer (PLGA) as a carrier material to prepare PSS enteric nanoformulations. The results showed that PSS nanoformulations have a significant hypoglycemic effect (Li Pengli. Preparation of a new oral preparation of marine polysaccharide drug PSS and its pharmacokinetic and pharmacodynamic study [D]. Ocean University of China, 2013.), but this method has the problem of low PSS encapsulation rate.

[0006] Multivesicular liposomes (MVLs) are composed of multiple non-concentric vesicles with a particle size distribution of 1-100 μm. The volume of the internal aqueous chamber is huge, so it is particularly suitable for encapsulating water-soluble drugs. After the drug is loaded into the multivesicle, the entire liposome becomes a drug reservoir, which can slowly release the drug after administration, exerting the advantages of sustained release and long-term effect, and significantly prolonging the half-life of the drug. However, the particle size of multivesicular liposomes is large, and the solution will agglomerate and break during storage and transportation. Most existing patents add charged phospholipids to the formulation composition, but this type of phospholipid is expensive. How to develop a formulation composition and preparation process with low production cost and easy to scale up production is a technical problem that needs to be solved urgently by those skilled in the art. Summary of the invention

[0007] The purpose of the present invention is to provide a trimethyl chitosan-modified sodium alginate diester multivesicular liposome and a preparation method and application thereof. The present invention uses oleic acid instead of expensive charged phospholipids in the prescription, solves the problem of easy aggregation and breakage in the preparation process of multivesicular liposomes, and uses trimethyl chitosan for modification to obtain trimethyl chitosan-modified sodium alginate diester multivesicular liposomes with uniform surface coating. The multivesicular liposomes have high encapsulation rate, good stability, simple preparation process, and are easy to scale up production.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] The present invention provides a trimethyl chitosan-modified sodium alginate diester multivesicular liposome and a preparation method and application thereof, comprising the following steps:

[0010] (1) dissolving a lipid component in an organic solvent as an oil phase; the lipid component includes soybean lecithin, cholesterol, triolein, and oleic acid; dissolving sodium alginate diester in an aqueous solution containing an osmotic pressure regulator as an internal aqueous phase;

[0011] (2) adding the oil phase to the inner water phase and performing ultrasonic dispersion to form colostrum;

[0012] (3) adding the prepared colostrum to an external aqueous phase containing an osmotic pressure regulator and an auxiliary emulsifier, and homogenizing the colostrum using a high-speed disperser to form a double emulsion;

[0013] (4) removing the organic solvent from the prepared double emulsion by rotary evaporation to form a preliminary product of sodium alginate diester multivesicular liposomes, removing free drugs by centrifugation, and dispersing the precipitate in a solvent to obtain sodium alginate diester multivesicular liposomes;

[0014] (5) synthesizing trimethyl chitosan, and adding the prepared sodium alginate diester multivesicular liposomes dropwise into the trimethyl chitosan solution to prepare the trimethyl chitosan-modified sodium alginate diester multivesicular liposomes.

[0015] Furthermore, in step (1), the phospholipid concentration is 10-20 mg / mL, the cholesterol concentration is 5-10 mg / mL, the triolein concentration is 2-10 mg / mL, and the oleic acid concentration is 1.0-4.0 mg / mL.

[0016] Furthermore, the organic solvent in step (1) is preferably a mixture of chloroform and diethyl ether, and the volume ratio of chloroform to diethyl ether is 10:1-1:1, and more preferably the volume ratio is 1:1.

[0017] Furthermore, the osmotic pressure regulator in step (1) is sucrose, and the mass fraction of the sucrose solution is 2%-8%; the concentration of sodium diester alginate is 5-20 mg / mL; the ultrasonic power is 30-200 W, and the ultrasonic time is 2-20 min; and the volume ratio of the internal water phase to the oil phase is 0.5:1-3:1.

[0018] Furthermore, in step (3), the osmotic pressure regulator is glucose, and the glucose mass ratio concentration is 2%-10%; the auxiliary emulsifier is lysine, and the lysine concentration is 10-50 mmol / L; the speed of the homogenizer is 2000-10000 rpm / min, and the shear time is 1-10 min; the volume ratio of colostrum to external water phase is 1:1-1:6.

[0019] Furthermore, the rotary evaporation pressure in step (4) is 0.02-0.1 MPa; the centrifugal speed is 2000-10000prm / min, and the centrifugal time is 2-20 min; and the multivesicular liposome product dispersion system is at least one of physiological saline, external aqueous phase, and PBS solution, preferably the external aqueous phase.

[0020] Furthermore, the preparation method of trimethyl chitosan in step (5) is as follows:

[0021] a. Weigh chitosan and sodium iodide and dissolve them in a solvent. Stir the reaction continuously in a constant temperature oil bath until the chitosan is completely dissolved. Condensation refluxes during the reaction.

[0022] b. Add sodium hydroxide solution, add iodomethane solution under stirring, and react in a constant temperature oil bath; precipitate the reaction solution with anhydrous ethanol, centrifuge, wash the precipitate, and dry under reduced pressure;

[0023] c. The sample after vacuum drying is dissolved, stirred continuously in a constant temperature oil bath, and then NaI, NaOH solution and CH3I solution are added, and the reaction is completed in a constant temperature oil bath; then CH3I solution and NaOH are added, stirred to react completely, and then the reaction solution is precipitated with anhydrous ethanol, centrifuged, the precipitate is washed, and vacuum dried overnight;

[0024] d. The obtained sample was dissolved in a NaCl solution, iodine ions were exchanged with chloride ions, the solution was precipitated with ether, centrifuged, washed and dried under reduced pressure to obtain trimethyl chitosan.

[0025] Furthermore, in step (5), the concentration of trimethyl chitosan is 2-30 mg / mL, the concentration of soybean lecithin in the multivesicular liposomes is 1-10 mg / mL, and the volume ratio of trimethyl chitosan solution to multivesicular liposome suspension is 1:1-5:1, preferably 3:1.

[0026] The invention also provides trimethyl chitosan-modified sodium alginate diester multivesicular liposomes prepared by the preparation method.

[0027] The present invention also provides the use of trimethyl chitosan modified sodium alginate diester multivesicular liposomes in the preparation of medicines for treating thrombosis-related diseases.

[0028] Furthermore, the sodium alginate diester multivesicular liposome has an anticoagulant effect and an inhibitory effect on platelet aggregation in a dose-dependent manner.

[0029] Compared with the prior art, the technical solution of the present invention has the following advantages:

[0030] 1. The liposomes prepared by the present invention have a high drug encapsulation rate. The modification with trimethyl chitosan can further promote oral absorption, improve the oral bioavailability of the drug, reduce the number of drug use times, and improve patient compliance.

[0031] 2. The formulation of the present invention has a simple composition, and the formulation ingredients are easy to obtain and relatively low in price. In addition, the overall preparation process is easy to operate, and has low requirements for production equipment and energy consumption. Both the formulation composition and the preparation process have great cost advantages and are particularly suitable for large-scale industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] These and / or other aspects and advantages of the present invention will become more clear and easier to understand from the following detailed description of the embodiments of the present invention in conjunction with the accompanying drawings, in which:

[0033] Figure 1 The synthetic route of trimethyl chitosan of the present invention is as follows;

[0034] Figure 2 is a hydrogen nuclear magnetic resonance spectrum of the trimethyl chitosan;

[0035] Figure 3 The morphology of the trimethyl chitosan-modified sodium alginate diester multivesicular liposomes before (left) and after (right) coating under an optical microscope (×400);

[0036] Figure 4 The particle size distribution diagram of the trimethyl chitosan-modified sodium alginate diester multivesicular liposomes before (left) and after (right) coating;

[0037] Figure 5 It is a statistical diagram of the in vitro anticoagulation experiment of the trimethyl chitosan-modified sodium alginate diester multivesicular liposomes;

[0038] Figure 6 The trimethyl chitosan-modified sodium alginate diester multivesicular liposomes improve platelet aggregation. DETAILED DESCRIPTION

[0039] In order to make those skilled in the art better understand the present invention, the technical scheme of the present invention is further described in detail below in conjunction with specific examples. It should be pointed out that the following is a preferred embodiment of the present invention, rather than all embodiments. For those skilled in the art, all other embodiments obtained without making innovative premises belong to the scope of protection of the present invention. In the following examples, unless otherwise specified, the experimental methods used are all conventional methods, and the materials, reagents, etc. used can be purchased from biological or chemical reagent companies.

[0040] Example 1: Preparation of sodium alginate multivesicular liposomes

[0041] Step 1: Formation of water-in-oil colostrum (W / O)

[0042] The lipid component is dissolved in an organic solvent to prepare an oil phase. The organic solvent of the oil phase in this example is chloroform-ether (1:1); the lipid component is used as a solute and is composed of: 20 mg / ml soybean lecithin, 10 mg / ml cholesterol, 5 mg / ml triolein and 4 mg / ml oleic acid.

[0043] Sodium alginate diester is dissolved in an aqueous solution containing an osmotic pressure regulator to serve as the internal aqueous phase. In this embodiment, the solvent of the internal aqueous phase is water; the solute composition is as follows: 20 mg / ml sodium alginate diester, 3% (w / v) sucrose.

[0044] 1 ml of the oil phase was injected into 1 ml of the inner water phase using a 1 ml pipette, and a cell disruptor was used to sonicate at 90 W for 3 min, with a working speed of 2 s and an interval of 3 s to form water-in-oil colostrum.

[0045] Step 2: Formation of a water-in-oil-in-water (W / O / W) emulsion

[0046] The colostrum obtained in step 1 was added to 4 mL of the external aqueous phase and homogenized at 8000 rpm for 1 min to form a water-in-oil-in-water emulsion. The solvent of the external aqueous phase was water, and the solute composition was 3% glucose (osmotic pressure regulator) and 40 mmol / L lysine (auxiliary emulsifier).

[0047] Step 3: Remove the organic solvent by rotary evaporation

[0048] The emulsion obtained in step 2 is transferred to a round-bottom flask containing 4-10 mL of the external aqueous phase, and the organic solvent is removed by rotary evaporation. This process is now stable for 10 min under a rotary pressure of 0.3-0.5 MPa, and then pressurized and rotated until the white emulsion becomes transparent to obtain the sodium alginate diester multivesicular liposomes. The prepared PSS multivesicular liposomes are centrifuged to remove free drugs and then dispersed in the external aqueous phase for storage.

[0049] Example 2: Determination of encapsulation efficiency of sodium alginate multivesicular liposomes

[0050] The drug encapsulation efficiency of PSS was determined by high performance liquid chromatography. The raw material PSS was used as the standard, and PSS solutions of different concentrations were prepared. The standard curve was drawn according to the relationship between concentration and peak area.

[0051] The prepared multivesicular liposome precipitate was subjected to reduced pressure rotary evaporation to remove the solvent, 6 mL of anhydrous methanol was added to dissolve the fat-soluble membrane material, magnetic stirring was performed for 4 h, and the precipitate was obtained by centrifugation. The precipitate was washed 3 times with methanol, and then the dried precipitate was dissolved in 4 mL of mobile phase. The peak area of ​​PSS in the precipitate was determined by HPLC, and the PSS content in the precipitate was calculated by substituting it into the linear equation of the standard curve. The PSS encapsulation efficiency calculated according to the following formula was about 90%.

[0052] .

[0053] Example 3: Preparation and characterization of trimethyl chitosan

[0054] 1. Preparation of trimethyl chitosan

[0055] (1) Weigh 1.0 g chitosan and 2.4 g sodium iodide, dissolve in 40.0 mL N-methyl-2-pyrrolidone, place in a 60°C constant temperature oil bath and stir continuously until the chitosan is completely dissolved. Condensation and reflux occur during the reaction.

[0056] (2) Add 5.5 mL of sodium hydroxide solution (15%, W / V) to a round-bottom flask, add 5.8 mL of iodomethane solution while stirring, and react in a constant temperature oil bath at 60 °C for 1 h.

[0057] (3) After the reaction, the reaction solution was precipitated with 100 mL of anhydrous ethanol, centrifuged at 5000 rpm for 10 min, washed with acetone twice, and dried under reduced pressure at 50 °C;

[0058] (4) The dried sample was dissolved in 40.0 mL N-methyl-2-pyrrolidone and stirred continuously in a 60°C constant temperature oil bath for 10 min. Then, 2.4 g NaI, 5.5 mL NaOH solution (15%, W / V), and 3.5 mL CH3I solution were added and reacted in a 60°C constant temperature oil bath for 30 min.

[0059] (5) After the reaction, add 1.0 mL CH3I solution and 0.3 g NaOH, and continue stirring for 1 h. After the reaction, the reaction solution is precipitated with 100 mL anhydrous ethanol, centrifuged at 5000 rpm for 10 min, washed with acetone twice, and dried under reduced pressure at 50 °C overnight.

[0060] (6) The obtained sample was dissolved in 30 mL of NaCl solution (10%, w / v), iodide ions were exchanged with chloride ions, the solution was precipitated with 150 mL of ether, centrifuged at 5000 rpm for 10 min, washed twice with ether, and the precipitate was dried at 45 °C under reduced pressure.

[0061] The specific reaction route of trimethyl chitosan is as follows Figure 1 shown.

[0062] Characterization of di- and trimethyl chitosan

[0063] NMR analysis method: measured on JNM-ECP 600M superconducting nuclear magnetic resonance spectrometer 1 H-NMR spectrum.

[0064] About 20 mg of prepared trimethyl chitosan was weighed, dissolved in 0.6 mL D2O, placed in an NMR tube, and subjected to NMR at 25 °C. 1 H-NMR analysis.

[0065] Analysis of H NMR spectrum: H NMR spectrum of trimethyl chitosan is as follows Figure 2 As shown, the peak of heavy water solvent is calibrated at 4.79ppm, the H signal of C1 is at 4.5~5.5 ppm, the H signal of C3 and C6 is at 3.6~4.4 ppm, the H signal of trimethyl is at 2.9~3.3 ppm, the H signal of dimethyl is at 2.8~3.1 ppm, and the H signal of N-acetyl is at 2.0~2.1 ppm. Therefore, the ratio of the peak area integral value of the H signal is used to represent the relative content of methylation at different positions in the sample, and the calculation method is as follows:

[0066] (1)

[0067] (2)

[0068] (3)

[0069] (4)

[0070] (5)

[0071] According to the above method, the H-NMR spectrum of the prepared trimethyl chitosan was integrated using MestReNova software, and the trimethylation degree of substitution was calculated to be 57.4%, the dimethylation degree of substitution was 34.7%, the -C3-OCH3 degree of substitution was 28.5%, and the -C6-OCH3 degree of substitution was 28.0%.

[0072] Example 4: Trimethyl chitosan coated sodium alginate multivesicular liposomes

[0073] The free drug was removed from the prepared multivesicular liposomes, and 4 mL of external aqueous phase was added to prepare a multivesicular liposome suspension with a phospholipid concentration of 5 mg / mL. A trimethyl chitosan solution with a mass fraction of 1% was prepared with a 3% glucose solution, and the ratio of V (trimethyl chitosan solution): V (multivesicular liposome suspension) was 2:1. The multivesicular liposome suspension was added to the trimethyl chitosan solution at a ratio of 20 μL per drop, stirred at 300 rpm / min for 4 h, and then centrifuged at 2500 rpm / min for 10 min to remove the supernatant, and the precipitate was dispersed in the external aqueous phase to prepare the trimethyl chitosan-modified sodium alginate diester multivesicular liposomes.

[0074] Example 5: Determination of trimethyl chitosan coating efficiency

[0075] The coating efficiency of trimethyl chitosan was determined by the conductivity method. A standard curve was drawn with the concentration of trimethyl chitosan (mg / mL) as the abscissa and the measured conductivity value (μs / cm) as the ordinate.

[0076] Taking the sample with chitosan concentration of 0 mg / mL as the reference, the conductivity value of the centrifugal supernatant solution after coating was measured. The conductivity value was substituted into the equation to calculate the concentration of trimethyl chitosan in the supernatant. The trimethyl chitosan coating efficiency was calculated to be 85%.

[0077] .

[0078] Example 6: Characterization of sodium diester alginate multivesicular liposomes

[0079] 1. Observation of the morphology of PSS multivesicular liposomes

[0080] 20 μL of the PSS multivesicular liposome suspension before and after coating was aspirated onto a glass slide, covered with a coverslip, and the morphology was observed under an optical microscope (X400 times). The results are shown in Figure 3 As shown, PSS multivesicular liposomes are round in shape, with uniform particle size and no aggregation.

[0081] 2. PSS multivesicular liposome particle size statistics

[0082] The particle sizes of 100 to 200 multivesicular liposomes observed under an optical microscope (x400) were counted using ImageJ software, and the particle size distribution statistics were drawn using Origin software. The results are shown in Figure 4 As shown, the particle sizes of multivesicular liposomes before and after coating were 9.97±1.77 μm and 9.24±2.02 μm, respectively.

[0083] 3. Zeta potential determination of PSS multivesicular liposomes

[0084] Take 1 mL of multivesicular liposome suspension and slowly add it to the potential sample cell to avoid the generation of bubbles. Hold the top of the sample cell to avoid touching the measurement area of ​​the sample cell. Point the front of the sample cell toward the inspector and push it into the sample slot. Close the sample cell cover and measure the Zeta potential of the sample to be tested. The Malvern laser particle size analyzer measured the Zeta potential before coating to be -61 mV, and the Zeta potential after TMC coating to be 30 mV.

[0085] Example 7: In vitro anticoagulation test of sodium diester alginate multivesicular liposomes

[0086] PSS multivesicular liposome suspension and blank plasma were prepared into 25 μg / mL, 35 μg / mL, and 50 μg / mL blood samples to be tested. APTT working solution and 0.025 mol / L CaCl2 solution were placed in a 37°C thermostat and heated for 15 min. 100 μL of the blood sample to be tested was taken into the blood coagulation tank, 100 μL APTT working solution was added, mixed and incubated at 37°C for 180 s. 100 μL of 0.025 mol / L CaCl2 solution preheated at 37°C was added, mixed immediately, timed, and the plasma coagulation time was recorded. The external aqueous phase was used as a blank control, and each test product was measured in parallel 3 times. The statistical data of each group are as follows. Figure 5 As shown, from Figure 5 It can be seen that both MVLs@PSS and TMC-MVLs@PSS have good in vitro anticoagulant activity. At a concentration of 25 μg / mL, the APTT prolongation rate is significantly higher than 150%.

[0087] Example 8: Application of trimethyl chitosan-coated sodium alginate multivesicular liposomes in anti-thrombotic treatment

[0088] 1. Construction of zebrafish thrombosis model

[0089] Phenylhydrazine induced thrombosis: Normally developed 3 dpf Tg (CD41: EGFP) (platelet marker) transgenic zebrafish were selected and transferred into 12-well culture plates. They were randomly divided into blank control group, model group and drug-treated group, with 10 zebrafish per well, and 2 mL of zebrafish culture water was transferred into each well. The blank control group was recorded as the Control group, and the model group was recorded as the Model group. The blank group was cultured in normal culture medium without phenylhydrazine, and the model group and drug-treated group were cultured in culture medium with a final concentration of 1 µM phenylhydrazine. The culture temperature was 28°C, and the culture time was 12 h, respectively, to successfully construct a thrombosis model.

[0090] 2. Preliminary evaluation of the antithrombotic activity of PSS multivesicular liposomes

[0091] After successful modeling, the zebrafish were washed 2-3 times with fresh culture medium, and the embryos were treated with 25, 35 and 50 μg / mL PSS multivesicular liposomes for 24 h. Finally, each group was washed 2-3 times with fresh culture medium, and the zebrafish were placed in 1 mL of fresh culture medium for later use. The zebrafish in each group were anesthetized with tricaine at a mass concentration of 0.4‰ for 30 s, placed on a slide coated with 4% carboxymethyl cellulose, and fixed in a prone position. The circulation of free platelets in the body of zebrafish was observed under a Leica M205A stereo microscope, and the number of free platelets in the body was recorded (platelets showed green light). The statistical data of each group are shown in Figure (5). Figure 6 It can be seen that PSS multivesicular liposomes can significantly improve the platelet aggregation state in a dose-dependent manner, and after coating, the platelet aggregation inhibition effect is better. The reason is that trimethyl chitosan plays an absorption-promoting role and improves the bioavailability of PSS.

[0092] The above contents are further detailed descriptions of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art of the present invention, without departing from the concept of the present invention, several simple deductions or substitutions can be made, which should be regarded as falling within the scope of protection of the present invention.

Claims

1. A method for preparing trimethyl chitosan-modified sodium alginate multivesicular liposomes, characterized in that: The following steps are involved: (1) dissolving lipid components in an organic solvent as an oil phase; the lipid components include soybean lecithin, cholesterol, triolein, and oleic acid; dissolving sodium alginate in an aqueous solution containing an osmotic pressure regulator as an inner aqueous phase; the osmotic pressure regulator is sucrose, and the mass fraction of the sucrose solution is 2%-8%; the concentration of sodium alginate is 5-20 mg / mL; the ultrasonic power is 30-200 W, and the ultrasonic time is 2-20 min; the volume ratio of the inner aqueous phase to the oil phase is 0.5:1-3:1; The concentration of soybean lecithin is 10-20 mg / mL, the concentration of cholesterol is 5-10 mg / mL, the concentration of triolein is 2-10 mg / mL, and the concentration of oleic acid is 1-4 mg / mL; the organic solvent is chloroform or a mixture of chloroform and ether, and the volume ratio of chloroform to ether is 10:1-1:1; (2) adding the oil phase to the inner water phase and performing ultrasonic dispersion to form colostrum; (3) adding the prepared colostrum to an external aqueous phase containing an osmotic pressure regulator and an auxiliary emulsifier, and homogenizing with a high-speed disperser to form a double emulsion; the osmotic pressure regulator is glucose, and the glucose mass ratio concentration is 2%-10%; the auxiliary emulsifier is lysine, and the lysine concentration is 10-50mmol / L; the volume ratio of colostrum to external aqueous phase is 1:1-1:6; (4) removing the organic solvent from the prepared double emulsion by rotary evaporation to form a preliminary product of sodium alginate diester multivesicular liposomes, removing free drugs by centrifugation, and dispersing the precipitate in a solvent to obtain sodium alginate diester multivesicular liposomes; The rotary evaporation pressure is 0.02-0.1 MPa; the dispersed solvent is at least one of physiological saline, external water phase, and PBS solution; (5) synthesizing trimethyl chitosan, and adding the prepared sodium alginate diester multivesicular liposomes dropwise into the trimethyl chitosan solution to prepare the trimethyl chitosan-modified sodium alginate diester multivesicular liposomes; The preparation method of the trimethyl chitosan is as follows: a. Weigh chitosan and sodium iodide and dissolve them in a solvent. Stir the reaction continuously in a constant temperature oil bath until the chitosan is completely dissolved. Condensation refluxes during the reaction. b. Add sodium hydroxide solution, add iodomethane solution under stirring, and react in a constant temperature oil bath; precipitate the reaction solution with anhydrous ethanol, centrifuge, wash the precipitate, and dry under reduced pressure; c. The sample after vacuum drying is dissolved, stirred continuously in a constant temperature oil bath, and then NaI, NaOH solution and CH3I solution are added, and the reaction is completed in a constant temperature oil bath; then CH3I solution and NaOH are added, stirred to react completely, and then the reaction solution is precipitated with anhydrous ethanol, centrifuged, the precipitate is washed, and vacuum dried overnight; d. The obtained sample was dissolved in a NaCl solution, iodine ions were exchanged with chloride ions, the solution was precipitated with ether, centrifuged, washed and dried under reduced pressure to obtain trimethyl chitosan; The concentration of trimethyl chitosan in step (5) is 2-30 mg / mL, the concentration of soybean lecithin in the multivesicular liposomes is 1-10 mg / mL, and the volume ratio of trimethyl chitosan solution to multivesicular liposome suspension is 1:1-5:

1.

2. The trimethyl chitosan-modified sodium alginate diester multivesicular liposomes prepared by the preparation method according to claim 1.

3. Use of the sodium alginate diester multivesicular liposomes according to claim 2 in the preparation of a medicament for treating thrombosis-related diseases, characterized in that: The sodium diester alginate multivesicular liposome has an anticoagulant effect and an inhibitory effect on platelet aggregation and is dose-dependent.

Images