CN106727328B - Method for preparing liposome based on ternary complex of medicine-phospholipid-cholesterol - Google Patents
Method for preparing liposome based on ternary complex of medicine-phospholipid-cholesterol Download PDFInfo
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
- A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
- A61K31/404—Indoles, e.g. pindolol
- A61K31/405—Indole-alkanecarboxylic acids; Derivatives thereof, e.g. tryptophan, indomethacin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
Abstract
The invention relates to a method for preparing liposome based on a drug-phospholipid-cholesterol ternary complex, which comprises the following steps: dividing the prescribed amount of phospholipid into a first amount of phospholipid and a second amount of phospholipid; taking a target drug, a first amount of phospholipid and cholesterol to prepare a drug-phospholipid-cholesterol ternary complex; preparing a phospholipid aqueous dispersion solution by taking a second amount of phospholipid; and (3) putting the drug-phospholipid-cholesterol ternary complex obtained in the first step into phospholipid aqueous dispersion, and processing to obtain the drug liposome. The method can reduce the usage amount of the organic solvent, has large single treatment capacity, simple process and high production efficiency, and is easy for industrial amplification.
Description
Technical Field
The invention relates to a preparation method of liposome based on a drug-phospholipid-cholesterol ternary complex, belonging to the technical field of pharmaceutical preparations.
Background
It is reported that at least 40% of the drugs in the development of new drugs at present show low water solubility, which may result in undesirable oral bioavailability, or are difficult to develop into liquid preparations (such as injections, etc.), thereby becoming a bottleneck limiting the development of drugs. Traditional strategies for increasing the solubility of poorly soluble drugs include the formation of soluble salts, pH adjustment, solubilization, and the like. However, even with the above methods, it is sometimes difficult to achieve a concentration suitable for clinical use for some drugs, or there is a potential safety risk due to the use of an excessive amount of excipients. Therefore, researchers have been searching for more and safer methods for improving the solubility of poorly soluble drugs. A new nano drug delivery system, such as liposome, nano particle, submicron emulsion, micelle and the like, becomes an important research direction.
Liposomes are the most common and the earliest commercialized of the nano-delivery systems, the safer delivery system available for injection. The liposome forms a closed capsule structure by a single-layer or multi-layer phospholipid bilayer, so that the liposome can encapsulate water-soluble drugs in an internal water phase and also encapsulate fat-soluble drugs in the phospholipid bilayer, and therefore, the liposome has a wider application range for different classes of drugs. Liposomes have the following advantages over the general pharmaceutical dosage form: (1) liposomes reduce drug toxicity; (2) liposomes protect the encapsulated drug; (3) the liposome has certain cell affinity and histocompatibility.
There are many techniques for preparing liposomes, and the methods are mainly classified into a physical dispersion method, a two-phase dispersion method, and the like according to the difference of the dispersion modes of the bilayer in the aqueous phase. The basic principle of the physical dispersion method is to dissolve lipid material and/or fat-soluble drug in organic solvent, remove the solvent to make the material film, then mix with water, and hydrate it by means of shaking, ultrasound, etc. to form liposome. The two-phase dispersion method comprises mixing oil phase solution and water phase solution thoroughly, stirring, and introducing nitrogen to remove organic solvent to obtain liposome suspension. In the method, the defects of large usage amount of organic solvent, difficult removal, time-consuming preparation steps, low production efficiency and the like generally exist, so that the production cost of the product is high.
The search shows that the Chinese patent application with the application number of CN200810039032.7 and the publication number of CN101292957A, which is named as a teniposide phospholipid complex liposome preparation and a preparation method thereof, comprises the following steps: (1) adding teniposide and phospholipid into an organic solvent, heating and refluxing for reaction until the solution is clear and transparent, wherein the molar ratio of the phospholipid to the teniposide is 1-3: 1; (2) evaporating the solution obtained in the step (1) under reduced pressure to remove the organic solvent, and drying in a vacuum dryer to obtain teniposide phospholipid complex powder; (3) placing teniposide phospholipid composite powder, phospholipid and cholesterol in a container according to a predetermined ratio, adding an organic solvent, heating in a water bath and stirring to make the solution clear and transparent, then decompressing and evaporating to remove the organic solvent, forming a transparent film on the wall of the container, adding a buffer solution for full hydration to obtain a primary liposome; (4) the preparation is prepared by a conventional pharmaceutical method.
The Chinese patent application with the application number of CN201610586554.3 and the application publication number of CN106074390A, named as thalidomide liposome nano preparation and the preparation method and the application thereof, comprises the following steps: placing thalidomide, phospholipid and cholesterol in a round-bottom flask, adding an organic solvent ethanol to dissolve the above substances, performing rotary evaporation under reduced pressure at a water bath temperature of 50 ℃ for 40min, removing the organic solvent, and forming a uniform and transparent film on the wall of the flask; adding normal saline, shaking, and then placing in an oscillator at 37 ℃ and shaking for 30min at a constant speed to form a thalidomide phospholipid complex; and (3) putting 10ml of the thalidomide phospholipid complex into a 25ml beaker, and carrying out ultrasonic treatment for 15min to obtain the thalidomide liposome nano preparation.
However, the inventors have further studied and found that in the prior art represented by the above patent, although a phospholipid and a drug are reacted or a phospholipid and cholesterol are reacted with a drug to form a binary or ternary complex, respectively, a membrane dispersion method is inevitably adopted in the process of preparing liposomes, and has significant disadvantages: the method has the advantages of large usage amount of organic solvent, small single treatment amount and large floor area of production equipment, thereby having low production efficiency and high production cost and being not suitable for industrialization. Therefore, there is a need to develop a liposome preparation method with less organic solvent usage, large single treatment capacity, no need of using production equipment with too large floor space, and improved production efficiency.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: the problems in the prior art are overcome, and the preparation method of the liposome based on the ternary drug-phospholipid-cholesterol complex is provided, so that the use amount of an organic solvent can be reduced, the process is simple, the production efficiency is high, and the industrial amplification is easy.
The technical scheme for solving the technical problems of the invention is as follows:
the preparation method of the liposome based on the ternary complex of the drug, phospholipid and cholesterol is characterized by comprising the following steps:
the method comprises the following steps of firstly, dividing a prescription amount of phospholipid into a first amount of phospholipid and a second amount of phospholipid; putting a target drug, a first amount of phospholipid and cholesterol into a container, adding a reaction solvent to completely dissolve the materials, continuously preserving heat and stirring, evaporating to remove the reaction solvent, and putting the obtained product in vacuum to obtain a drug-phospholipid-cholesterol ternary complex;
secondly, putting the phospholipid with the second dosage into pure water, and performing ultrasonic treatment or shearing by using a high-speed dispersion instrument to obtain phospholipid aqueous dispersion;
thirdly, putting the ternary compound of the drug, phospholipid and cholesterol obtained in the first step into the phospholipid aqueous dispersion liquid obtained in the second step, and processing the ternary compound of the drug, phospholipid and cholesterol by adopting the following first mode or second mode to obtain a drug liposome;
the first mode is as follows: shearing by a high-speed dispersion instrument, and then circularly homogenizing by a high-pressure homogenizer;
the second mode is as follows: firstly, ultrasonic treatment is carried out by a probe, and then filtration treatment is carried out by a microporous filter membrane.
In the preparation method, only a small amount of organic solvent is used in the first step of preparing the ternary complex of the drug-phospholipid-cholesterol, and no organic solvent is used in the other steps, so that the use amount of the organic solvent is greatly reduced. In addition, the liposome is obtained from the first step to the third step, and no film is required in the whole process, so that the equipment volume is small, the single treatment capacity is large, the production process is simple, the production efficiency is high, and the industrial amplification is easy.
The technical scheme of the invention is further perfected as follows:
preferably, in the first step, the first amount is less than the second amount; the mass of the first amount of phospholipid is at least 3 times of that of the target medicament, and the mass ratio of the target medicament to cholesterol is 1 (0.1-10);
in the second step, the mass of the second amount of phospholipid is at least 3 times of that of the drug-phospholipid-cholesterol ternary complex obtained in the first step.
In the preferred scheme, the phospholipid is skillfully divided into a front part, a small part and a rear part, the compounding rate of the drug-phospholipid-cholesterol ternary complex is ensured by specifically limiting the first dosage and the second dosage of the phospholipid, and the encapsulation rate of the liposome is ensured; furthermore, the use of organic solvents can be further reduced with a smaller first amount of phospholipids.
Preferably, in the first step, the temperature range of heat preservation is 30-80 ℃, and the stirring time is 1-8 h; the time for placing in vacuum is 8-16 h.
Preferably, in the first step, the amount of the reaction solvent used is 1 to 1.5 times of the minimum amount capable of completely dissolving the materials; the reaction solvent is one of tetrahydrofuran, acetone and chloroform or the combination thereof.
Preferably, the ternary complex of drug-phospholipid-cholesterol obtained in the first step is in the form of a powdery solid or a spongy semisolid.
Preferably, in the first mode of the third step, the shearing speed is 1000-.
Preferably, in the second mode of the third step, 100-1000W probe is adopted for ultrasonic treatment, the ultrasonic treatment time is 1-60min, and the pore diameter of the microporous filter membrane is 0.22-0.8 μm.
Preferably, the particle size of the drug liposome obtained in the third step is 25-500 nm.
Preferably, the target drug is one of or a combination of a non-steroidal anti-inflammatory drug, an antigen antibody protein, a protein polypeptide drug, a metal ion and a traditional Chinese medicine active ingredient.
Preferably, the phospholipid is one of dimyristoyl phosphatidylcholine, dimyristoyl phosphatidylglycerol, egg yolk lecithin, hydrogenated soybean lecithin, soybean lecithin or a combination thereof.
The invention firstly forms a ternary compound of medicine-phospholipid-cholesterol with the medicine, phospholipid and cholesterol, and then the ternary compound reacts with the phospholipid to prepare the liposome, wherein the formation of the compound of the medicine, the phospholipid and the cholesterol can improve the bad properties of a plurality of medicines, particularly improve the hydrophilic and lipophilic properties of the medicine; the liposome is prepared by taking the ternary drug-phospholipid-cholesterol complex as a base and using a phospholipid aqueous dispersion liquid and a high-pressure homogenization or probe ultrasonic method, so that the use amount of an organic solvent can be greatly reduced, the single treatment capacity is large, the process is simple, the production efficiency is high, and the industrial amplification is easy.
Drawings
FIG. 1 is a transmission electron microscope image of liposomes obtained by high pressure homogenization in example 1 of the present invention.
Detailed Description
The present invention will be described in further detail with reference to examples. The invention is not limited to the examples given.
The preparation method of the liposome based on the ternary complex of the drug, phospholipid and cholesterol, which is specifically implemented by the invention, comprises the following steps:
the method comprises the following steps of firstly, dividing a prescription amount of phospholipid into a first amount of phospholipid and a second amount of phospholipid, wherein the first amount is less than the second amount; putting a target drug, a first amount of phospholipid and cholesterol into a container, adding a reaction solvent to completely dissolve the materials, keeping the temperature at 30-80 ℃, stirring for 1-8h, then evaporating to remove the reaction solvent, and putting the obtained product in vacuum for 8-16h to obtain a powdery solid or spongy semisolid drug-phospholipid-cholesterol ternary complex; the mass of the first amount of phospholipid is at least 3 times of that of the target medicament, and the mass ratio of the target medicament to cholesterol is 1 (0.1-10); the usage amount of the reaction solvent is 1-1.5 times of the minimum usage amount capable of completely dissolving the materials, and the reaction solvent is one of tetrahydrofuran, acetone and chloroform or the combination thereof.
Secondly, putting the phospholipid with the second dosage into pure water, and performing ultrasonic treatment or shearing by using a high-speed dispersion instrument to obtain phospholipid aqueous dispersion; the second amount of phospholipid is at least 3 times of the drug-phospholipid-cholesterol ternary complex obtained in the first step. Wherein, the ultrasonic treatment can adopt common ultrasonic dispersion equipment or probe ultrasonic.
And thirdly, putting the drug-phospholipid-cholesterol ternary complex obtained in the first step into the phospholipid aqueous dispersion obtained in the second step, and treating by adopting the following first mode or second mode to obtain the drug liposome with the particle size of 25-500 nm.
The first mode is as follows: shearing by a high-speed dispersion instrument, and then circularly homogenizing by a high-pressure homogenizer; wherein the shearing speed is 1000-10000rpm, the shearing time is 1-30min, the homogenizing pressure is 100-1000bar, and the homogenizing cycle time is 1-10 times.
The second mode is as follows: firstly, carrying out ultrasonic treatment by using a probe, and then carrying out filtration treatment by using a microporous filter membrane; wherein, 100-1000W probe is adopted for ultrasonic treatment, the ultrasonic treatment time is 1-60min, and the aperture of the microporous filter membrane is 0.22-0.8 μm.
In the above method, the target drug is preferably one or a combination of nonsteroidal anti-inflammatory drug, antigen antibody protein, protein polypeptide drug, metal ion, and Chinese medicinal active ingredient; it should be noted that the target drug may be other drugs besides the above-mentioned limitations, and experiments prove that the method of the present invention has wide general applicability.
The phospholipid is one or combination of dimyristoyl phosphatidylcholine, dimyristoyl phosphatidyl glycerol, egg yolk lecithin, hydrogenated soybean lecithin, and soybean lecithin.
Example 1
The mass volume concentration (g/ml) of each component in the prescription is as follows:
0.8 percent of baicalein
Yolk lecithin 16.08%
1.36 percent of cholesterol
The balance being pure water.
The preparation process comprises the following steps: weighing baicalein, cholesterol and egg yolk lecithin with the mass 3 times of that of the baicalein (namely the mass ratio of the baicalein to the first amount of the egg yolk lecithin is 1:3, and the mass ratio of the baicalein to the cholesterol is 1:1.7), placing the mixture in an eggplant-shaped bottle, adopting tetrahydrofuran as a reaction solvent to completely dissolve the three, keeping the temperature at 30 ℃ and stirring for 1h, evaporating to remove the solvent, keeping the obtained product under vacuum for 12h to obtain the baicalein-egg yolk lecithin-cholesterol ternary compound, taking out, keeping out of the sun, and sealing and storing in a cool place. The essence of this step is to prepare baicalein-egg yolk lecithin-cholesterol ternary complex by solvent method.
Weighing the rest yolk lecithin (namely the second amount of yolk lecithin, wherein the mass of the second amount of yolk lecithin is 3 times of that of the ternary complex obtained in the previous step) in the prescription, ultrasonically dispersing the yolk lecithin in pure water in the prescription, adding the baicalein-yolk lecithin-cholesterol ternary complex, shearing the yolk lecithin-cholesterol ternary complex at a high speed of 5000rpm in a high-speed dispersion instrument for 5min, adding the yolk lecithin-cholesterol ternary complex into a high-pressure homogenizer, circulating for 3 times under the homogenizing pressure of 500bar to obtain the medicinal liposome, or ultrasonically treating the yolk lecithin with a probe, wherein the ultrasonic power of the probe is 550W, the working time is 10min, and filtering the obtained solution with a 0.45-micrometer microporous filter membrane to obtain the medicinal liposome. The mass ratio of the baicalein in the final liposome is as follows: egg yolk lecithin: cholesterol 1:20.1: 1.7.
The transmission electron micrograph of the liposome obtained by the high pressure homogenization method in this example is shown in FIG. 1. As can be seen, the liposome is spherical or nearly spherical in appearance, smooth in edge, free of visible drug crystal particles and about 100nm in particle size. The surface of the nanoparticle shows fingerprint-like characteristics, which indicates that a bilayer structure of the liposome exists.
Example 2
The mass volume concentration (g/ml) of each component in the prescription is as follows:
0.2 percent of quercetin
Yolk lecithin 4.49%
0.4 percent of cholesterol
The balance being pure water.
The preparation process comprises the following steps: weighing quercetin, cholesterol and yolk lecithin (the mass ratio of quercetin to the first amount of yolk lecithin is 1:3.2, and the mass ratio of quercetin to cholesterol is 1:2) 3.2 times of that of quercetin, placing in an eggplant-shaped bottle, dissolving the above three components completely with tetrahydrofuran as reaction solvent, stirring at 55 deg.C for 4 hr, evaporating to remove solvent, maintaining the obtained product under vacuum for 12 hr to obtain quercetin-yolk lecithin-cholesterol ternary complex, taking out, keeping out of the sun, and sealing in the shade.
Weighing the rest yolk lecithin (namely the second amount of yolk lecithin, the mass of the second amount of yolk lecithin is about 3.1 times of the mass of the ternary complex obtained in the previous step) in the prescription, ultrasonically dispersing the yolk lecithin in pure water of the prescription, adding the quercetin-yolk lecithin-cholesterol ternary complex, ultrasonically treating the yolk lecithin-cholesterol ternary complex for 10min by adopting a 550W probe, filtering the obtained solution through a 0.45 mu m microporous filter membrane, and sealing and storing to obtain the medicinal liposome.
Example 3
The mass volume concentration (g/ml) of each component in the prescription is as follows:
indometacin 0.5%
Hydrogenated soybean lecithin 16%
1.0 percent of cholesterol
The balance being pure water.
The preparation process comprises the following steps: weighing indomethacin, cholesterol and hydrogenated soybean lecithin with the mass of 4 times of that of the indomethacin (namely the mass ratio of the indomethacin to the first amount of hydrogenated soybean lecithin is 1:4, and the mass ratio of the indomethacin to the cholesterol is 1:2), placing the indomethacin, the cholesterol and the hydrogenated soybean lecithin in an eggplant-shaped bottle, using chloroform as a reaction solvent to completely dissolve the indomethacin, the cholesterol and the cholesterol, keeping the temperature at 55 ℃ for 4 hours, evaporating to remove the solvent, keeping the obtained product in vacuum for 12 hours to obtain the indomethacin-hydrogenated soybean lecithin-cholesterol ternary complex, taking out, keeping out of the sun, and sealing and storing in a cool place.
Weighing the residual hydrogenated soybean lecithin (namely the second amount of hydrogenated soybean lecithin, the mass of which is 4 times of that of the ternary complex obtained in the previous step) in the prescription, ultrasonically dispersing the hydrogenated soybean lecithin in pure water of the prescription, mixing the dispersed hydrogenated soybean lecithin with the indometacin-hydrogenated soybean lecithin-cholesterol ternary complex, shearing the mixture in a high-speed dispersion instrument at a high speed of 5000rpm for 8min, adding the mixture into a high-pressure homogenizer, and circulating the mixture for 5 times under the homogenization pressure of 500bar to obtain the medicinal liposome.
Example 4
The mass volume concentration (g/ml) of each component in the prescription is as follows:
oroxylin 0.5%
16.2 percent of soybean lecithin
0.9 percent of cholesterol
The balance being pure water.
The preparation process comprises the following steps: weighing oroxylin, cholesterol and soybean lecithin (the mass ratio of oroxylin to the first amount of soybean lecithin is 1:5, and the mass ratio of oroxylin to cholesterol is 1:1.8) with the mass of 5 times of oroxylin according to the formula, placing the oroxylin, cholesterol and soybean lecithin in an eggplant-shaped bottle, adopting acetone as a reaction solvent to completely dissolve the oroxylin, cholesterol and cholesterol, keeping the temperature at 60 ℃ for 3.5h, then evaporating to remove the solvent, keeping the obtained product in vacuum for 12h to obtain the oroxylin-soybean lecithin-cholesterol ternary complex, taking out, keeping out of the sun, and sealing and storing in the shade.
Weighing the rest soybean lecithin (namely the second amount of soybean lecithin, the mass of which is about 3.5 times of the mass of the ternary complex obtained in the previous step) in the prescription, dispersing the soybean lecithin in pure water of the prescription, mixing the soybean lecithin with the ternary complex of oroxylin-soybean lecithin-cholesterol, and carrying out ultrasonic treatment by adopting a probe with the ultrasonic power of 550W for 12 min. The obtained solution is filtered through a 0.45 mu m microporous filter membrane to obtain the medicinal liposome.
Example 5
The Chinese patent application with the application number of CN200810039032.7 and the publication number of CN101292957A, named as a teniposide phospholipid complex liposome preparation and a preparation method thereof mentioned in the background technology is called as a first patent, and the essence of the method is as follows: dissolving the medicine and phospholipid in organic solvent, heating and refluxing, then decompressing and evaporating to remove the organic solvent, then drying in vacuum to obtain medicine-phospholipid binary compound, and preparing the liposome from the medicine-phospholipid binary compound, phospholipid and cholesterol by a film dispersion method.
The application number mentioned in the background art is CN201610586554.3, the application publication number is CN106074390A, and the name is "a thalidomide liposome nano preparation and its preparation method and application", the chinese patent application is called patent two below, and the essence of the method is: the medicine, phospholipid and cholesterol are formed into a film by a film dispersion method, then the film is hydrated by normal saline to obtain a medicine-phospholipid-cholesterol ternary complex, and the ternary complex is subjected to ultrasonic treatment to obtain the liposome.
The method of the invention does not need to adopt a film dispersion method at all, and the essence of the method of the invention is as follows: dissolving the medicine, phospholipid and cholesterol in an organic solvent, stirring at a constant temperature, evaporating to remove the organic solvent to obtain a medicine-phospholipid-cholesterol ternary complex, preparing a phospholipid aqueous dispersion, adding the medicine-phospholipid-cholesterol ternary complex, and preparing the liposome by high-pressure homogenization or probe ultrasound. This completely avoids the disadvantages of the film dispersion method.
In order to compare the actual effects of the methods, a comparative experiment was conducted in this example, which was divided into the method group of the present invention and the thin film dispersion method group, and baicalein was used as the drug.
Specifically, the method comprises the steps of preparing baicalein, phospholipid and cholesterol into a baicalein-phospholipid-cholesterol ternary complex, and then forming a liposome with a phospholipid aqueous dispersion.
The film dispersion method comprises dissolving baicalein, phospholipid and cholesterol in dichloromethane, heating in water bath, rotary evaporating to remove organic solvent to form uniform film, hydrating, and treating with ultrasonic probe to obtain liposome. The particle size, PDI, encapsulation efficiency and appearance of each group were examined and the specific data are shown in the following table:
from the above experimental data:
(1) compared with the film dispersion method used in the first patent and the second patent, the method has the advantages of simple process, less organic solvent consumption and no need of production equipment with large occupied area.
(2) The stability of the liposome prepared by the method is obviously superior to that of a film dispersion method.
Example 6
The invention skillfully divides the phospholipid into a front part, a small part and a rear part, and is verified by the following experiments.
The phospholipid is divided into a front part and a rear part, wherein the front part is used for preparing a drug-phospholipid-cholesterol ternary complex, and the ratio of the phospholipid to the drug in the step is a key parameter and influences the compounding rate of the phospholipid complex; the latter part is used for phospholipid aqueous dispersion, and the mass ratio of phospholipid to ternary complex in the step is also a key process parameter, and influences the encapsulation efficiency of the liposome.
(1) Screening of reaction charge ratio of phospholipid and medicine in preparation of compound
Baicalein is taken as an example. The phospholipid is egg yolk lecithin.
Preparing a baicalein-egg yolk lecithin-cholesterol ternary compound by adopting a solvent method: weighing baicalein, yolk lecithin and cholesterol, and placing the baicalein, the yolk lecithin and the cholesterol in an eggplant-shaped bottle at a mass ratio of 1:1, wherein the mass ratios of the yolk lecithin and the baicalein are respectively 2:1, 2.5:1, 3:1, 3.3:1 and 3.5: 1; adding appropriate amount of reaction solvent tetrahydrofuran to dissolve the three completely, stirring at 30 deg.C for 1 hr, rotary evaporating to remove solvent, vacuum maintaining the obtained product for 12 hr, taking out the product, keeping in the dark, drying, and storing in the shade to obtain baicalein-yolk lecithin-cholesterol ternary complex. And (4) measuring the recombination rate. The results are shown in the following table.
Influence of the ratio (mass ratio) of egg yolk lecithin to baicalein on the compounding rate (n ═ 3)
Therefore, the recombination rate is increased along with the reduction of the mass ratio of the baicalein in the recombination system, and when the mass of the phospholipid is 3 times or more of that of the baicalein, the recombination rate is close to 100 percent.
(2) Screening of the mass ratio of phospholipid to ternary complexes in the preparation of liposomes
Baicalein is taken as an example. The phospholipid is egg yolk lecithin. The prescription comprises baicalein, yolk lecithin, cholesterol and pure water.
Preparing a baicalein-egg yolk lecithin-cholesterol ternary compound by adopting a solvent method: weighing baicalein, yolk lecithin and cholesterol, and placing the mixture in an eggplant-shaped bottle, wherein the mass ratio of the baicalein to the cholesterol is 1:1, and the mass ratio of the yolk lecithin to the baicalein is 3: 1; adding appropriate amount of reaction solvent tetrahydrofuran to dissolve the three completely, stirring at 30 deg.C for 1 hr, rotary evaporating to remove solvent, vacuum maintaining the obtained product for 12 hr, taking out the product, keeping in the dark, drying, and storing in the shade to obtain baicalein-yolk lecithin-cholesterol ternary complex.
Adjusting the mass ratio of the egg yolk lecithin to the baicalein-egg yolk lecithin-cholesterol ternary complex to be 5:5, 8:5, 10:5, 13:5, 15:5, 18:5, 20:5 and 23:5 respectively (the mass ratio of the egg yolk lecithin to the baicalein is 8:1, 11:1, 13:1, 16:1, 18:1, 21:1, 23:1 and 26:1 in a conversion mode), and weighing the egg yolk lecithin and the baicalein-egg yolk lecithin-cholesterol ternary complex respectively according to the baicalein concentration of 80 mg/ml. Dispersing yolk lecithin in pure water to obtain yolk lecithin water dispersion, adding baicalein-yolk lecithin-cholesterol ternary complex, shearing at high speed of 5000rpm in a high-speed dispersion instrument for 5min, adding into a high-pressure homogenizer, and circulating for 3 times under the homogenizing pressure of 500 bar. The encapsulation efficiency, particle size and Zeta potential of the resulting liposomes were measured.
Influence of the ratio of the mass of egg yolk lecithin to the ternary complex on the encapsulation efficiency, particle size and Zeta potential of the liposomes (n ═ 3)
It can be seen from this that: (i) the mass ratio of the egg yolk lecithin to the ternary complex is 5:5-15:5, the encapsulation rate is gradually increased along with the increase of the mass ratio of the egg yolk lecithin to the ternary complex, and when the mass ratio of the egg yolk lecithin to the ternary complex reaches 15:5 (namely the mass ratio of the egg yolk lecithin to the baicalein to the cholesterol is 18:1:1) or more, the encapsulation rate approaches 100%.
(ii) When the mass ratio of the egg yolk lecithin to the ternary complex is 5:5-23:5, the particle size of the liposome is in the range of 102-112 nm.
(iii) When the mass ratio of the egg yolk lecithin to the ternary complex is 5:5-23:5, the Zeta potential of the liposome is lower than-25 mV and is relatively stable.
(iv) The above results were combined to determine that the mass ratio of egg yolk lecithin to the ternary complex should be 15:5, i.e., 3:1 or more.
It should be noted that, in the two screening experiments (1) and (2) in this example, other drugs, other phospholipids, and other prescriptions defined by the technical scheme of the present invention are also used for verification, and the results thereof are consistent with the above results.
In addition to the above embodiments, the present invention may have other embodiments. All technical solutions formed by adopting equivalent substitutions or equivalent transformations fall within the protection scope of the claims of the present invention.
Claims (6)
1. The preparation method of the liposome based on the ternary complex of the drug, phospholipid and cholesterol is characterized by comprising the following steps:
the method comprises the following steps of firstly, dividing a prescription amount of phospholipid into a first amount of phospholipid and a second amount of phospholipid; putting a target drug, a first amount of phospholipid and cholesterol into a container, adding a reaction solvent to completely dissolve the materials, continuously preserving heat and stirring, evaporating to remove the reaction solvent, and putting the obtained product in vacuum to obtain a drug-phospholipid-cholesterol ternary complex; the obtained ternary complex of drug-phospholipid-cholesterol is in the form of powdery solid or spongy semisolid; the using amount of the reaction solvent is 1-1.5 times of the lowest using amount which can completely dissolve the materials;
secondly, putting the phospholipid with the second dosage into pure water, and performing ultrasonic treatment or shearing by using a high-speed dispersion instrument to obtain phospholipid aqueous dispersion;
thirdly, putting the ternary compound of the drug, phospholipid and cholesterol obtained in the first step into the phospholipid aqueous dispersion liquid obtained in the second step, and processing the ternary compound of the drug, phospholipid and cholesterol by adopting the following first mode or second mode to obtain a drug liposome;
the first mode is as follows: shearing by a high-speed dispersion instrument, and then circularly homogenizing by a high-pressure homogenizer;
the second mode is as follows: firstly, carrying out ultrasonic treatment by using a probe, and then carrying out filtration treatment by using a microporous filter membrane;
in the first step, the first amount is less than the second amount; the mass of the first amount of phospholipid is at least 3 times of that of the target medicament, and the mass ratio of the target medicament to cholesterol is 1 (0.1-10);
in the second step, the mass of the second amount of phospholipid is at least 3 times of that of the drug-phospholipid-cholesterol ternary complex obtained in the first step;
the target drug is baicalein;
the phospholipid is one or a combination of dimyristoylphosphatidylcholine, dimyristoylphosphatidylglycerol, egg yolk lecithin, hydrogenated soybean lecithin and soybean lecithin.
2. The method for preparing liposome based on ternary complex of drug-phospholipid-cholesterol as claimed in claim 1, wherein in the first step, the temperature is maintained at 30-80 ℃ and the stirring time is 1-8 h; the time for placing in vacuum is 8-16 h.
3. The method for preparing liposome based on ternary complex of drug-phospholipid-cholesterol as claimed in claim 1, wherein the reaction solvent is one or a combination of tetrahydrofuran, acetone and chloroform.
4. The method as claimed in claim 1, wherein the third step comprises a first mode in which the shear rate is 1000-10000rpm, the shear time is 1-30min, the homogenization pressure is 100-1000bar, and the number of homogenization cycles is 1-10.
5. The method as claimed in claim 1, wherein the second mode of the third step comprises using a 100-1000W ultrasonic probe, the ultrasonic treatment time is 1-60min, and the pore size of the microporous membrane is 0.22-0.8 μm.
6. The method for preparing liposome based on ternary drug-phospholipid-cholesterol complex as claimed in any one of claims 1 to 5, wherein the particle size of the drug liposome obtained in the third step is 25-500 nm.
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