CN101744764A - Blank and topotecan hydrochloride containing polycystin liposome and preparation method thereof - Google Patents
Blank and topotecan hydrochloride containing polycystin liposome and preparation method thereof Download PDFInfo
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- CN101744764A CN101744764A CN200810204206A CN200810204206A CN101744764A CN 101744764 A CN101744764 A CN 101744764A CN 200810204206 A CN200810204206 A CN 200810204206A CN 200810204206 A CN200810204206 A CN 200810204206A CN 101744764 A CN101744764 A CN 101744764A
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Abstract
The invention discloses a blank polycystin liposome and a topotecan hydrochloride containing polycystin liposome and a preparation method thereof; the blank polycystin liposome comprises the following ingredients by mass parts: 1 part of lipid ingredient and 0.1-50 parts of ion gradient regulator, wherein the lipid ingredient comprises amphipathy lipid and neutral lipid which have 5-36:1 molar ratio. The topotecan hydrochloride containing polycystin liposome comprises drug active ingredient-topotecan hydrochloride and the blank liposome which is used as a carrier; in the invention, the blank polycystin liposome which is encapsulated with the encapsulate is prepared firstly, and then the topotecan hydrochloride is led to penetrate through a phospholipid membrane to be loaded in liposome internal water phase by adopting a transmembrane gradient active medicine carrying method, so as to obtain the topotecan hydrochloride containing polycystin liposome. In the invention, the utilization rate of the raw materials is high, the medicine carrying concentration is high, and the polycystin liposome has good slow release function in in-vivo and in-vitro experiments and has better anti-tumor effect.
Description
Technical field
The present invention relates to the slow releasing preparation field, particularly a kind of blank multivesicular liposome and a kind of topotecan hydrochloride multivesicular liposome with and preparation method thereof.
Background technology
Topotecan (Topotecan, TPT) develop by Smithkline Beecham company early than nineteen ninety, be semisynthetic camptothecine soluble derivative, and be used for the treatment of the drug-fast ovarian cancer of progressivity and ratified to be used for the treatment of the lobule cell lung cancer in succession by the FDA approval in 1996.Topotecan counterweight recurrence disease or drug-fast neurocytoma, nonsmall-cell lung cancer, small cell lung cancer, ovarian cancer, breast carcinoma, colon cancer and esophageal carcinoma all have curative effect preferably.
Topotecan is injection clinically.Because the topotecan hydrochloride biological half-life is short, clinical practice needs multiple dosing, and patient's poor compliance, especially drug administration by injection may bring the infection of injection site etc., and therefore developing its slow releasing preparation is urgent clinical needs.But above-mentioned dosage form can't reach the effect of slow release.
Liposome is a kind of targeted drug carrier, belongs to a kind of novel form of targeting drug delivery system.It can be embedded in diameter with drug powder or solution is in the nano level microgranule, this microgranule has the class cellularity, enter the autoimmune function that is mainly activated body in the human body by reticuloendothelial system phagocytic, and the interior distribution of the body that changes encapsulated medicine, drug main will be put aside in histoorgans such as liver, spleen, lung and bone marrow, thereby improve the therapeutic index of medicine, reduce the toxicity of the therapeutic dose and the reduction medicine of medicine.
The preparation of existing conventional liposome comprises two kinds of active loading method and passive medicine carrying methods.Active loading method wherein is also referred to as remote control and seals the loading technology, the drug selectivity ground of some special nature can be spread, and is enriched to water in the liposome.Its ultimate principle is: (1) liposome phospholipid double sublayer can selectivity with the water-soluble substances inside and outside the liposome, comprise that various ions such as the proton of positive charge and potassium/sodium separate effectively, form the inside and outside relatively independent environment of liposome; (2) lipophilic weak acid or alkaline compound are fat-soluble at the nonionic state, can embed phospholipid double sublayer, and enter water in the liposome according to ion gradient through immobilized artificial membrane.Nichols and Deamer have proposed the notion of ion gradient very early, they observe in studying in early days, if in the inside and outside formation pH gradient of liposome, and make in the liposome water, can make catecholamine high efficiency separation and gathering in the tart liposome for acid.On the contrary, when water in the liposome is when alkalescence, the liposome interior that lipophilic faintly acid chemical compound will alcaliotropism is assembled.
Multivesicular liposome is based on the slow releasing pharmaceutical transmission system of liposome, is used to part, zone and system's drug delivery.Multivesicular liposome also is called as multivesicular liposomes or multilamelar liposome, it is the liposome particles that contains a plurality of non-concentric chamber, shape is similar to liposome particles, this particulate structure is different from the multilamelar liposome that contains a plurality of concentric chamber, is different from the unilamelar liposome that only contains single interior water chamber again.Many water soluble drugs can be encapsulated in the multivesicular liposome, by in the animal sheath, subcutaneous, abdominal cavity and epidural administration, human body Intraventricular, sheath are interior, subcutaneous, epidural administration has all demonstrated slow releasing function.
People such as Kim.S find and have studied multivesicular liposome at first in nineteen eighty-three.They are filmogen with phospholipid, cholesterol and neutral lipid by the multiple emulsion solvent evaporation method, are organic solvent with chloroform and ether, have successfully prepared multivesicular liposome, and the microstructure of multivesicular liposome that relied on optical microscope and electron microscopic observation.In preparation process, at first drug solution is formed water-in-oil emulsion with lipid under mechanicals efforts, again with this emulsion and outer water are mixed must emulsion, nitrogen dries up and removes organic solvent and make the multivesicular liposome suspension then.
CN ZL95196186.1 also discloses a kind of preparation of multivesicular liposome of active agents sustained release.Because of its non-concentrically ringed topological structure, make multivesicular liposome form medicine " bank " in the injection site, along with the continuous metabolism of phospholipid bilayer, the medicine that is encapsulated in the vesicle progressively is released into blood or diseased region, and performance well postpones release action.By regulating parameter and the prescription ratio in the preparation process, easily the control drug release time is between several days to several weeks.The particulate mean diameter of multivesicular liposome by multi-emulsion method preparation between 5～50 μ m, envelop rate 20%～90%, but the preparation method utilization ratio of drug of this multivesicular liposome is low, less stable.
The preparation method of existing multivesicular liposome all belongs to above-mentioned passive medicine carrying method, rather than active loading method.This preparation method raw material availability is lower, the multivesicular liposome less stable.
At present, disclosing topotecan has conventional liposome injection dosage form, has reached the purpose of directed conveying medicine substantially.But its slow release effect is undesirable, can not reach the effect of topotecan sustained-release medication in the body substantially.At present, though Cheng Gong the multivesicular liposome preparation of having developed some drugs,, because different medicines has the different physicochemical properties and the mechanism of action, the prescription of its multivesicular liposome and preparation technology generally can not be suitable for other medicine.Therefore, for this specific medicine of topotecan hydrochloride, reach the purpose of sustained release profile in vivo test targeting transmission, also exist following problem: must be at its requirements for clinical application, search out specific prescription and preparation technology, just can prepare its multivesicular liposome preparation.
Summary of the invention
Therefore, the problem to be solved in the present invention just provide a kind of blank multivesicular liposome and a kind of topotecan hydrochloride multivesicular liposome with and preparation method thereof.
The inventor is through extensive studies and test, find: also can prepare multivesicular liposome with the transmembrane gradient active loading method pleasantly surprisedly, and topotecan hydrochloride utilization rate height in this method, the multivesicular liposome good stability, be a kind of good method for preparing the topotecan hydrochloride multivesicular liposome, thereby finished the present invention.
Therefore, the present invention addresses the above problem one of technical scheme of being adopted: a kind of blank multivesicular liposome, each component that comprises following mass parts: 0.1～50 part of 1 part of lipid components and ion gradient regulator comprise in the lipid components that wherein mol ratio is 5～36: 1 amphipathic lipids and neutral lipid.
According to the present invention, described blank multivesicular liposome is meant the multivesicular liposome that does not contain the medicine active component, and it can be used as the initiatively carrier of the medicine multivesicular liposome of medicine carrying.
Among the present invention, described " amphipathic lipids " is meant to have hydrophilic and lipoid compositions two kinds of character of oleophylic, mainly is meant phospholipid, is the Main Ingredients and Appearance that constitutes liposome.Amphipathic lipids preferable for being selected from lecithin, soybean phospholipid, hydrogenated soya phosphatide, dipalmitoyl phosphatidyl choline, stearylamine, dimyristoyl phosphatidyl choline, dioleoyl phospholipid phatidylcholine, PHOSPHATIDYL ETHANOLAMINE, DOPE, cephalin, sphingomyelins, two palmityl Phosphatidylserine, phosphatidyl glycerol, phosphatidylinositols, phosphatidic acid and the two palmityl phosphatidyl glycerols one or more.
Among the present invention, described " neutral lipid " is meant the lipophile lipoid, and itself can not form liposome, needs to constitute liposome with amphipathic lipids.Neutral lipid is one of deciding factor that forms the multivesicular liposome topological structure.It mainly plays the support effect in liposome, be distributed in non-concentric aqueous chamber phospholipid bilayer node place, supports the topological structure of multivesicular liposome.Neutral lipid preferable for being selected from three oils and fatss, three hot fat, soybean oil, pig/tallow, tocopherol and the Squalene one or more.
Among the present invention, described " ion gradient regulator " is meant the material that is used to form the inside and outside ion gradient of liposome.The ion gradient regulator is preferable is selected from ammonium sulfate, copper sulfate, manganese sulfate, magnesium sulfate, calcium acetate, citric acid and the manganous chloride one or more.
Blank multivesicular liposome of the present invention, preferable, comprise each component of following mass parts: 0.5～20 part of 1 part of lipid components and ion gradient regulator wherein comprise mol ratio 10～20: 1 amphipathic lipids and neutral lipid in the lipid components.
Blank multivesicular liposome of the present invention, in the described lipide component, the preferable sterin that can further include, what described sterin was preferable is cholesterol.Sterin can be regulated the flowability of liposome rete by changing the neutral lipid phase transition temperature, thereby improves the stability of liposome and the rate of release of vivo and vitro.Therefore, sterin is considered to membrane stabilizer usually.The consumption of sterin has remarkable influence to the stability and the release behavior of multivesicular liposome.Among the present invention, preferable, the mol ratio of the neutral lipid in described sterin and the lipid components is 0.7～65: 1, and preferable is 2～8: 1, and described percentage ratio is its molar percentage shared in lipid components.
Blank multivesicular liposome of the present invention also further comprises osmotic pressure regulator.Among the present invention, described " osmotic pressure regulator " is meant the material that is used for the regulator solution osmotic pressure, can be material commonly used in the state of the art, as trehalose, succinate, cyclodextrin, arginine, galactose, mannose, maltose, mannitol, glycine, lysine, citrate, sorbitol and glucosan etc., preferable be selected from glucose, sucrose, mannitol and the sodium chloride one or more.What the content of osmotic pressure regulator was preferable is 0.08～15 part (is the situation of 1 mass parts with respect to lipid components).
The present invention addresses the above problem two of the technical scheme that adopted: a kind of preparation method of aforesaid blank multivesicular liposome comprises the following steps:
1. lipide component is dissolved in the organic solvent as the lipid phase, this lipid components comprises amphipathic lipids and neutral lipid;
2. the ion gradient regulator is dissolved in the water, as interior water, water in described is added the lipid phase, mixing and emulsifying makes Water-In-Oil (W/O) type colostrum;
3. outer water is added described water-in-oil type Ruzhong just, mix forming W/O/W (W/O/W) type emulsion;
4. remove the organic solvent in the emulsion that 3. step obtain, make blank multivesicular liposome suspension first product;
5. wash described blank multivesicular liposome suspension first product with isosmotic solution, redispersion makes blank multivesicular liposome finished product in isosmotic solution.
Among the present invention, 1. for lipide component is dissolved in the organic solvent as the lipid phase, this lipid components comprises amphipathic lipids and neutral lipid to step.Wherein, described organic solvent can be can lipin dissolving any solvent of composition, preferable be more preferred from chloroform for being selected from ether, hydrocarbon, halogenated hydrocarbons, halogen ether and the ester one or more, perhaps be the mixture of chloroform and ether.This lipid mutually in, preferable, the concentration of amphipathic lipids is 5～120mM, the mol ratio of amphipathic lipids and neutral lipid is 5～36: 1.
Among the present invention, step as interior water, adds lipid phase with water in described 2. for the ion gradient regulator is dissolved in the water, and mixing and emulsifying makes Water-In-Oil (W/O) type colostrum.
Wherein, described ion gradient regulator is selected from one or more in ammonium sulfate, copper sulfate, manganese sulfate, magnesium sulfate, calcium acetate, citric acid and the manganous chloride.The ion gradient regulator the concentration of interior aqueous phase preferable be 10～1000mM, that better is 50～500mM.The ion gradient regulator of aqueous phase has produced ion gradient inside and outside making the blank multivesicular liposome of gained of the present invention in the present invention.Prepare in the technology of conventional liposome at active loading method, ion gradient commonly used comprises pH gradient, ammonium sulphate gradient and ion gradient-cell plasma carrier.The most basic motive power that ammonium sulphate gradient and other ion gradient methods can be written into medicine liposome interior can be summed up as the effect of pH gradient, and only the production method of pH gradient has direct and indirect difference.The ion gradient that the present invention was suitable for also comprises as above three kinds, sets up by the 2. described ion gradient regulator of step.After the inside and outside ion gradient of blank multivesicular liposome was set up, the power that just can utilize this ion gradient to produce initiatively was written into the active constituents of medicine topotecan hydrochloride in this blank multivesicular liposome, thereby makes the topotecan hydrochloride multivesicular liposome.
Interior aqueous phase can not contain osmotic pressure regulator, because ion gradient regulator itself just can cause osmotic pressure, when the ion gradient modifier concentration is big inadequately, just needs osmotic pressure regulator to regulate osmotic pressure.Osmotic pressure regulator can be any material that is used for the regulator solution osmotic pressure commonly used in this area.The osmotic pressure of water equaled the osmotic pressure of human plasma in the effect of osmotic pressure regulator was to regulate, and can guarantee like this to keep stable after multivesicular liposome of the present invention is injected into human body, and the adjusting medicine was released into intravital speed from multivesicular liposome.Osmotic pressure regulator can be 1～1000mM in the concentration of interior aqueous phase, and that preferable is 50～200mM.
The method that 2. this step prepares Water-In-Oil (W/O) type colostrum is the ordinary skill in the art, and employing is selected from mechanical agitation, ultrasonic and spray-dired method is carried out emulsifying.Wherein preferable, described in water and lipid volume ratio mutually preferable be 1: 1～1: 4, better is 1: 1～1: 2.
Among the present invention, step is mixed formation W/O/W (W/O/W) type emulsion 3. for outer water being added described water-in-oil type Ruzhong just.Wherein, preferable, described outer aqueous phase comprises coemulsifier and osmotic pressure regulator.Coemulsifier can be for being selected from lysine, glycine and the histidine one or more.Aqueous phase outside, that coemulsifier concentration is preferable is 20～80mM, that better is 30～60mM.Osmotic pressure regulator also can be any material that is used for the regulator solution osmotic pressure commonly used in this area.Osmotic pressure regulator makes the osmotic pressure of the outer water of blank multivesicular liposome of the present invention keep equating with interior water, is the osmotic pressure of human plasma.Aqueous phase outside this, the concentration of osmotic pressure regulator can be 0.01～50% (w/v), that better is 0.1～10% (w/v).This described method for preparing W/O/W (W/O/W) type emulsion is the ordinary skill in the art, and employing is selected from mechanical agitation, ultrasonic and spray-dired method is carried out emulsifying.What the volume ratio of described water-in-oil type colostrum and outer water was preferable is 1: 2.5～1: 10, and better is 1: 3～1: 5.
Among the present invention, 4. step for removing the organic solvent in the emulsion that 3. step obtain, makes blank multivesicular liposome suspension first product.Wherein, the method for removing organic solvent is the removal of solvents method of the routine of this area, and preferable for air-flow dries up, rotates reduction vaporization or spray drying, wherein air-flow dries up gases used nitrogen, helium, argon, oxygen, hydrogen and the carbon dioxide of being selected from of method.
Among the present invention, step is 5. for to wash described blank multivesicular liposome suspension first product with isosmotic solution, and redispersion makes blank multivesicular liposome finished product in isosmotic solution.Among the present invention, term " isosmotic solution " is meant the solution that osmotic pressure is identical with human plasma, and preferable is normal saline.Wherein Xi Di purpose is to remove non-encapsulated ion gradient regulator and osmotic pressure regulator constant gradient formation material, makes the enough big gradient difference of the inside and outside formation of liposome, so that more effectively load medicine.
The present invention addresses the above problem three of the technical scheme that adopted: a kind of topotecan hydrochloride multivesicular liposome comprises that active constituents of medicine topotecan hydrochloride and aforesaid blank multivesicular liposome are as carrier.According to the present invention, in the described topotecan hydrochloride multivesicular liposome, medicine fat was than preferably 1: 0.1～1: 250, and better is 1: 3～1: 20.Among the present invention, described " medicine fat ratio " is meant the mass ratio of the lipid components in active constituents of medicine topotecan hydrochloride and the multivesicular liposome.
The present invention addresses the above problem four of the technical scheme that adopted: a kind of preparation method of the multivesicular liposome of topotecan hydrochloride as mentioned above may further comprise the steps:
A. topotecan hydrochloride and osmotic pressure regulator are dissolved in the water;
B. step a gained solution is added in the blank as mentioned above multivesicular liposome finished product, mix homogeneously is hatched at the phase transition temperature that is higher than lipid, makes topotecan hydrochloride multivesicular liposome suspension first product;
C. wash described topotecan hydrochloride multivesicular liposome suspension first product with isosmotic solution, redispersion makes topotecan hydrochloride multivesicular liposome finished product in isosmotic solution.
Among the present invention, step a is for to be dissolved in the water topotecan hydrochloride and osmotic pressure regulator.Wherein, that the concentration of topotecan hydrochloride is preferable is 0.01～100mg/ml, and that better is 0.1～50mg/ml, and that better is 1～10mg/ml.The osmotic pressure that topotecan hydrochloride produces is very little, needs to add osmotic pressure regulator and makes the osmotic pressure of this aqueous solution equal the osmotic pressure of the interior water of blank multivesicular liposome.The concentration of osmotic pressure regulator can be 1～1000mM, is preferably 10～500mM, and that better is 20～50mM.
Among the present invention, step b adds step a gained solution in the blank as mentioned above multivesicular liposome finished product, and mix homogeneously is hatched at the phase transition temperature that is higher than lipid, makes topotecan hydrochloride multivesicular liposome suspension first product.Wherein, medicine fat was than preferably 1: 0.1～1: 250, and better is 1: 3～1: 20.Among the present invention, the temperature when term " phase transition temperature of lipid " is meant phase co-conversion between lipid gel state and the liquid crystal state.Hatch at the phase transition temperature that is higher than lipid, the immobilized artificial membrane permeability is strengthened, it is general for health easier film that penetrates under the driving force of ion gradient that hydrochloric acid is opened up, and is gathered in the interior aqueous phase of multivesicular liposome.Preferable, be 1～240 minute in the time that the phase transition temperature that is higher than lipid is hatched, more preferably 5～30 minutes.
Among the present invention, step c is for to wash described topotecan hydrochloride multivesicular liposome suspension first product with isosmotic solution, and redispersion makes topotecan hydrochloride multivesicular liposome finished product in isosmotic solution.Wherein, can be the method for this area routine with the method for isosmotic solution washing, available isosmotic solution is as wash solution, adopts the method removal free drug wherein of centrifugal, dialysis or membrane filtration, suspends with isosmotic solution then.What isosmotic solution was preferable is normal saline.
Topotecan hydrochloride multivesicular liposome of the present invention can be used for preparing antitumor drug, especially for the medicine of the anti-neurocytoma of preparation, nonsmall-cell lung cancer, small cell lung cancer, ovarian cancer, breast carcinoma, colon cancer and esophageal carcinoma.
The route of administration of topotecan hydrochloride multivesicular liposome of the present invention is the same with conventional liposome, as subcutaneous injection, epidural injection or intrathecal injection etc.
Raw material that the present invention is used and reagent are all commercially available to be got.
Because different encapsulated chemical compounds has the different physicochemical properties and the mechanism of action, topotecan hydrochloride multivesicular liposome of the present invention prepares utilization ratio of drug and release rate has than big-difference, therefore at concrete medicine and release request thereof, can on basis of the present invention, improve, obtain specific prescription and technology.All based on the present invention's blank multivesicular liposome, hydrochloric acid open up general for health multivesicular liposome or their preparation method all within the scope of the present invention.
Than prior art, beneficial effect of the present invention is as follows: the present invention at first prepares the blank multivesicular liposome that is encapsulated with the gradient regulator, with the transmembrane gradient active loading method topotecan hydrochloride is seen through immobilized artificial membrane then and enter water in the liposome, obtained being loaded with the multivesicular liposome with slow release effect of active constituents of medicine topotecan hydrochloride.The raw material availability height of the active substance that the present invention seals---topotecan hydrochloride, waste that can less greatly medicine, thus reduce cost.Topotecan hydrochloride multivesicular liposome medicine carrying concentration height of the present invention, utilization ratio of drug height, good stability, in vivo, all show the good slow release effect in the in vitro tests, thereby reduce medication number of times and medication total amount, reduce untoward reaction, inevitable existing preparation has better antitumous effect.
Description of drawings
Below in conjunction with the description of drawings the features and advantages of the present invention.
Curve when Fig. 1 is the medicine of topotecan hydrochloride multivesicular liposome release in vitro of the present invention.
The specific embodiment
Further specify the present invention with embodiment below, but the present invention is not limited.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer.
The weight of the material that is obtained by the mol ratio conversion among the embodiment feeds intake, because phospholipid mostly is mixture, so get its about molecular weight when calculating.The molecular weight of the material that relates to is as follows: lecithin 750, hydrogenated soya phosphatide 790, phosphatidic acid 424.5, stearylamine 269.5, cholesterol 387, glycerol trioleate 885, three caprylins 470, phosphatidyl glycerol 740, two palmityl phosphatidyl glycerols 745, dioleoyl phospholipid phatidylcholine 786, DOPE 744, phosphatidylinositols 859, two Palmic acid acyl Phosphatidylserine 733, dimyristoyl phosphatidyl choline 678, PHOSPHATIDYL ETHANOLAMINE 744, sphingomyelins 731.
The production firm of used main agents is as follows among the embodiment: lecithin, Degussa; Two Palmic acid Phosphatidylserine, Degussa; Phosphatidyl glycerol, Degussa; Two palmityl phosphatidyl glycerols, Degussa; The dioleoyl phospholipid phatidylcholine, Degussa; Topotecan hydrochloride, Yunyang, Jiangsu pharmaceutcal corporation, Ltd of group; Citric acid, Shanghai chemical reagents corporation of Chinese Medicine group; Cholesterol, Chemical Reagent Co., Ltd., Sinopharm Group; Triglyceride, Guangzhou Chemical Reagent Factory.
The utilization ratio of drug assay method is:
Precision is measured in liposome turbid liquor 1mL to the 5mL measuring bottle, adds normal saline to scale, shakes up, and gets diluent.Precision is measured in diluent 1mL to the 5mL measuring bottle, adds Triton X-100 (10wt%) to scale, ultrasonic 15 minutes, measures total dose; It is an amount of that other gets diluent, centrifugal 10 minutes separation of supernatant of 600 * g, and precision is measured in supernatant 1mL to the 5mL measuring bottle, adds Triton X-100 (10wt%) to scale, ultrasonic 15 minutes, measures free dose.Calculate utilization ratio of drug by following formula:
Utilization ratio of drug (%)=(total dose-free dose)/total dose * 100.
The method of testing of medicine-cholesterol ratio is:
According to the preparation of the step in above-mentioned " utilization ratio of drug assay method " sample, measure total dose, free dose, T-CHOL amount, free cholesterol amount.Calculate medicine-cholesterol ratio by following formula:
Medicine-cholesterol ratio (%)=[(total medication amount-free dose) * phospholipid molecule amount]/[(T-CHOL amount-free cholesterol amount) * cholesterol molecular weight].
The Zeta potential assay method is:
It is an amount of to get the multivesicular liposome suspension, adds the normal saline dilution, measures with ZW380 type particle size analyzer (PCS, photon correlation spectroscopy technology).
Embodiment 1
Step 1: in clean glass container, add 1mL and contain 19.8mM (14.85mg) lecithin, 4.2mM (3.13mg) two palmityl phosphatidyl glycerols, the chloroformic solution of 30mM (11.61mg) cholesterol and 3.75mM (3.32mg) glycerol trioleate.This solution is called the lipid phase.
Step 2: the interior water 1mL that will be dissolved with the aqueous solution of 120mM (15.86mg) ammonium sulfate adds in the above-mentioned glass container that fills the lipid phase, and with 10, the speed of 000rpm stirred 10 minutes with high-speed shearing machine, the w/o type colostrum.
Step 3: with 5mL contain 5% (w/v) (250mg) solution of glucose and 40mM (29.36mg) lysine place on the colostrum layer, mixed for 10 seconds with the speed of 7500rpm then, W/O/W type emulsion.
Step 4: emulsion is transferred to fills 7mL and contain 5% (w/v) (350mg) in the 250mL conical flask of glucose and 40mM (40.93mg) lysine, 37 ℃ of waters bath with thermostatic control, the nitrogen current that makes 8L/min is by the suspension in the conical flask, slowly volatilize chloroform after 15 minutes, get blank multivesicular liposome first product.
Step 5: add the normal saline of 5 times of amounts in conical flask, 600 * g separated liposome in centrifugal 10 minutes then.Abandon supernatant, and with the precipitate redispersion in the 5mL normal saline, centrifugation again, so cycling is 3 times, the enrichment precipitation must contain the blank multivesicular liposome of ammonium sulfate.
Step 6: with blank multivesicular liposome precipitation redispersion in 5mL topotecan hydrochloride (1mg/ml)-glucose (5%, w/v) in the solution, mix homogeneously, room temperature (25 ℃) left standstill 2 hours, the topotecan hydrochloride multivesicular liposome.
The particle diameter that records the topotecan hydrochloride multivesicular liposome is 5～50 μ m; Utilization ratio of drug is 95.32%; Medicine-cholesterol ratio is 30.55%; The zeta current potential is negative 47.8mV.
Embodiment 2
Step 1: in clean glass container, add 1mL and contain 5mM (3.96mg) hydrogenated soya phosphatide, 0.5mM (0.38mg) two Palmic acid Phosphatidylserine, the chloroform-diethyl ether solution of 10mM (3.87mg) cholesterol and 0.155mM (0.073mg) tricaprylin.This solution is called the lipid phase.
Step 2: will be dissolved with 10mM manganese sulfate (0.845mg) and 6% (w/v) (600mg) in the aqueous solution of sucrose water 0.5mL add above-mentioned filling in the lipid glass container mutually, with high-speed shearing machine with 10, the speed of 000rpm stirred 10 minutes, got the w/o type colostrum.
Step 3: with 7.5mL contain 0.5% (w/v) (37.5mg) solution of mannitol and 80mM (87.71mg) lysine place on the colostrum layer, mixed for 40 seconds with the speed of 4500rpm then, W/O/W type emulsion.
Step 4: emulsion is transferred to fills 9mL and contain 0.5% (w/v) (45mg) in the 250mL conical flask of mannitol and 80mM (105.26mg) lysine, 37 ℃ of waters bath with thermostatic control, the nitrogen current that makes 8L/min is by the suspension in the conical flask, slowly volatilize chloroform-ether after 15 minutes, get blank multivesicular liposome first product.
Step 5: with embodiment 1.
Step 6: blank multivesicular liposome precipitation redispersion is contained+A23187 (calcium channel A23187) in 5mL, molecular weight is 523.6, concentration is 0.2mg/ml), topotecan hydrochloride (0.1mg/ml) and mannitol (0.5%, w/v) in the aqueous solution, mix homogeneously, hatched 5 minutes for 40 ℃, get the topotecan hydrochloride multivesicular liposome.
The particle diameter that records the topotecan hydrochloride multivesicular liposome is 5～50 μ m; Utilization ratio of drug is 91.48%; Medicine-cholesterol ratio is 27.31%; The zeta current potential is negative 55.4mV.
Embodiment 3
Step 1: in clean glass container, add 1mL and contain 100mM (75mg) lecithin, 20mM (8.49mg) phosphatidic acid, the dichloromethane solution of 10mM (3.87mg) cholesterol and 13mM (11.51mg) glycerol trioleate.This solution is called the lipid phase.
Step 2: will be dissolved with water 1mL in 1000mM (192.1mg) the Fructus Citri Limoniae aqueous acid and add in the above-mentioned glass container that fills the lipid phase, with 10, the speed of 000rpm stirred 10 minutes with high-speed shearing machine, the w/o type colostrum.
Step 3: with 20mL contain 10% (w/v) (2000mg) solution of mannitol and 20mM (58.48mg) lysine place on the colostrum layer, mixed for 20 seconds with the speed of 7500rpm then, W/O/W type emulsion.
Step 4: emulsion is transferred to fills 22mL and contain 10% (w/v) (2200mg) in the 1000mL conical flask of mannitol and 20mM (64.32mg) lysine, 37 ℃ of waters bath with thermostatic control, the nitrogen current that makes 8L/min is by the suspension in the conical flask, slowly volatilize dichloromethane after 15 minutes, get blank multivesicular liposome first product.
Step 5: with embodiment 1.
Step 6: (10%, w/v) in the aqueous solution, mix homogeneously was hatched 120 minutes for 4 ℃, got the topotecan hydrochloride multivesicular liposome in 5mL topotecan hydrochloride (50mg/ml)-mannitol with blank multivesicular liposome precipitation redispersion.
The particle diameter that records the topotecan hydrochloride multivesicular liposome is 5～50 μ m; Utilization ratio of drug is 97.01%; Medicine-cholesterol ratio is 12.84%; The zeta current potential is negative 45.5mV.
Embodiment 4
Step 1: in clean glass container, add 0.5mL and contain 25mM (9.83mg) dipalmitoyl phosphatidyl choline, 10mM (8.59mg) phosphatidylinositols, chloroform-ether (1: 1) solution of 5mM (0.97mg) cholesterol, 1.2mM (0.53mg) glycerol trioleate and 1.2mM (0.28mg) tricaprylin.This solution is called the lipid phase.
Step 2: will be dissolved with water 0.5mL in the aqueous solution of 350mM (23.12mg) ammonium sulfate and add in the above-mentioned glass container that fills the lipid phase, with 10, the speed of 000rpm stirred 10 minutes with high-speed shearing machine, the w/o type colostrum.
Step 3: with 2.5mL contain 8% (w/v) (200mg) solution of sucrose and 60mM (21.48mg) lysine place on the colostrum layer, mixed for 10 seconds with the speed of 7500rpm then, W/O/W type emulsion.
Step 4: emulsion is transferred to fills 2.5mL and contain 8% (w/v) (200mg) in the 250mL conical flask of sucrose and 60mM (21.48mg) lysine, 37 ℃ of waters bath with thermostatic control, the nitrogen current that makes 8L/min is by the suspension in the conical flask, slowly volatilize chloroform-ether organic solvent after 15 minutes, get blank multivesicular liposome first product.
Step 5: with embodiment 1.
Step 6: (8%, w/v) in the aqueous solution, mix homogeneously was hatched 30 minutes for 60 ℃, got the topotecan hydrochloride multivesicular liposome in 5mL topotecan hydrochloride (25mg/ml)-sucrose with blank multivesicular liposome precipitation redispersion.
The particle diameter that records the topotecan hydrochloride multivesicular liposome is 5～50 μ m; Utilization ratio of drug is 82.99%; Medicine-cholesterol ratio is 21.57%; The zeta current potential is negative 50.2mV.
Embodiment 5
Step 1: in clean glass container, add 2mL and contain 50mM (75mg) soybean phospholipid, 5mM (2.7mg) stearylamine, the chloroformic solution of 50mM (38.7mg) cholesterol and 8.4mM (7.9mg) tricaprylin.This solution is called the lipid phase.
Step 2: will be dissolved with 150mM (18.7mg) copper sulfate and add above-mentioned filling in the lipid glass container mutually with the interior water 0.5mL of glucose 2% (w/v) aqueous solution (10mg), with high-speed shearing machine with 13, the speed of 000rpm stirred 10 minutes, got the w/o type colostrum.
Step 3: with 20mL contain 0.9% (w/v) (180mg) solution of sodium chloride and 40mM (116.96mg) lysine place on the colostrum layer, mixed for 10 seconds with the speed of 7500rpm then, W/O/W type emulsion.
Step 4: emulsion is transferred to fills 20mL and contain 0.9% (w/v) (180mg) in the 1000mL conical flask of sodium chloride and 40mM (116.95mg) lysine, 37 ℃ of waters bath with thermostatic control, the nitrogen current that makes 8L/min is by the suspension in the conical flask, slowly volatilize the chloroform organic solvent after 15 minutes, get blank multivesicular liposome first product.
Step 5: with embodiment 1.
Step 6: (0.9%, w/v) in the aqueous solution, mix homogeneously was hatched 10 minutes for 30 ℃, got the topotecan hydrochloride multivesicular liposome in 5mL topotecan hydrochloride (10mg/ml)-sodium chloride with blank multivesicular liposome precipitation redispersion.
The particle diameter that records the topotecan hydrochloride multivesicular liposome is 5～50 μ m; Utilization ratio of drug is 66.05%; Medicine-cholesterol ratio is negative 44.3mV for the 10.28%zeta current potential.
Embodiment 6
Step 1: in clean glass container, add 0.5mL and contain 5mM (1.97mg) dioleoyl phospholipid phatidylcholine, 1mM (0.73mg) two palmityl Phosphatidylserine, chloroform-ether (1: 1) solution of 0.625mM (0.12mg) cholesterol and 0.27mM (0.16mg) soybean oil.This solution is called the lipid phase.
Step 2: will be dissolved with water 0.5mL in the aqueous solution of 600mM (37.77mg) manganous chloride and add in the above-mentioned glass container that fills the lipid phase, with 13, the speed of 000rpm stirred 10 minutes with high-speed shearing machine, the w/o type colostrum.
Step 3: with 5mL contain 3.5% (w/v) (175mg) solution of glucose and 60mM (43.86mg) lysine place on the colostrum layer, mixed for 10 seconds with the speed of 7500rpm then, W/O/W type emulsion.
Step 4: emulsion is transferred to fills 5mL and contain 3.5% (w/v) (175mg) in the 250mL conical flask of glucose and 60mM (43.86mg) lysine, 37 ℃ of waters bath with thermostatic control, the nitrogen current that makes 8L/min is by the suspension in the conical flask, slowly volatilize chloroform-ether organic solvent after 15 minutes, get blank multivesicular liposome first product.
Step 5: with embodiment 1.
Step 6: blank multivesicular liposome precipitation redispersion is contained+A23187 (0.2mg/ml), topotecan hydrochloride (25mg/ml)-glucose (3.5% in 5mL, w/v) in the aqueous solution, mix homogeneously was hatched 60 minutes for 55 ℃, got the topotecan hydrochloride multivesicular liposome.
The particle diameter that records the topotecan hydrochloride multivesicular liposome is 5～50 μ m; Utilization ratio of drug is 87.38%; Medicine-cholesterol ratio is 23.61%; The zeta current potential is negative 52.8mV.
Embodiment 7
Step 1: in clean glass container, add 1mL and contain 20mM (15mg) lecithin, the chloroformic solution of 20mM (7.74mg) cholesterol and 2.4mM (1.45mg) soybean oil.This solution is called the lipid phase.
Step 2: will be dissolved with water 1mL in the aqueous solution of 125mM (16.52mg) ammonium sulfate and add in the above-mentioned glass container that fills the lipid phase, with 10, the speed of 000rpm stirred 10 minutes with high-speed shearing machine, the w/o type colostrum.
Step 3: the solution that 5mL is contained 5.4% (w/v) glucose (270mg) and 40mM (29.24mg) lysine places on the colostrum layer, and then with 10, the speed of 000rpm mixed for 10 seconds, gets W/O/W type emulsion.
Step 4: emulsion is transferred to fills 5mL and contain 5.4% (w/v) (270mg) in the 250mL conical flask of glucose and 40mM (29.24mg) lysine solution, 37 ℃ of waters bath with thermostatic control, the nitrogen current that makes 8L/min is by the suspension in the conical flask, slowly volatilize chloroform after 15 minutes, get blank multivesicular liposome first product.
Step 5: with embodiment 1.
Step 6: blank multivesicular liposome precipitation redispersion is contained in topotecan hydrochloride (15mg/ml) normal saline solution in 5mL, and mix homogeneously was hatched 5 minutes for 25 ℃, got the topotecan hydrochloride multivesicular liposome.
The particle diameter that records the topotecan hydrochloride multivesicular liposome is that 5～50 μ m utilizations ratio of drug are 83.58%; Medicine-cholesterol ratio is 18.95%; The zeta current potential is negative 32.1mV.
Embodiment 8
Step 1: in clean glass container, add 1mL and contain 3mM (2mg) dimyristoyl phosphatidyl choline, 2mM (1.49mg) PHOSPHATIDYL ETHANOLAMINE, the cyclohexane solution of 9mM (7.9mg) cholesterol and 1mM (0.89mg) glycerol trioleate.This solution is called the lipid phase.
Step 2: will be dissolved with 250mM (4.4mg) calcium acetate and 8.5% (w/v) (85mg) in the aqueous solution of sucrose water 1mL add above-mentioned filling in the lipid glass container mutually, with 10, the speed of 000rpm stirred 10 minutes with high-speed shearing machine, the w/o type colostrum.
Step 3: with embodiment 1.
Step 4: with embodiment 1.
Step 5: with embodiment 1.
Step 6: (5.4%, w/v) in the aqueous solution, mix homogeneously was hatched 120 minutes for 4 ℃, got the topotecan hydrochloride multivesicular liposome in 5mL topotecan hydrochloride (0.01mg/ml)-glucose with blank multivesicular liposome precipitation redispersion.
The particle diameter that records the topotecan hydrochloride multivesicular liposome is 5～50 μ m; Utilization ratio of drug is 56.57%; Medicine-cholesterol ratio is 10.53%; The zeta current potential is negative 34.5mV.
Embodiment 9
Step 1: in clean glass container, add 1mL and contain 45mM (33.5mg) DOPE, 40mM (30mg) cephalin, the chloro methyl ether solution of 9mM (3.5mg) cholesterol and 5mM (4.4mg) glycerol trioleate.This solution is called the lipid phase.
Step 2: will be dissolved with 400mM (48mg) magnesium sulfate and 6.2% (w/v) (62mg) in the aqueous solution of sucrose water 1mL add above-mentioned filling in the lipid glass container mutually, with 10, the speed of 000rpm stirred 10 minutes with high-speed shearing machine, the w/o type colostrum.
Step 3: with embodiment 1.
Step 4: with embodiment 1.
Step 5: with embodiment 1.
Step 6: (5.4%, w/v) in the aqueous solution, mix homogeneously was hatched 120 minutes for 4 ℃, got the topotecan hydrochloride multivesicular liposome in 5mL topotecan hydrochloride (100mg/ml)-glucose with blank multivesicular liposome precipitation redispersion.
The particle diameter that records the topotecan hydrochloride multivesicular liposome is 5～50 μ m; Utilization ratio of drug is 53.21%; Medicine-cholesterol ratio is 11.13%; The zeta current potential is negative 41mV.
Embodiment 10
Step 1: in clean glass container, add 1mL and contain 20mM (14.6mg) sphingomyelins, 10mM (7.4mg) phosphatidyl glycerol, the ethyl acetate solution of 50mM (19.3mg) cholesterol and 2mM (1.77mg) glycerol trioleate.This solution is called the lipid phase.
Step 2: the interior water 1mL that will be dissolved with the aqueous solution of 120mM (15.86mg) ammonium sulfate adds in the above-mentioned glass container that fills the lipid phase, and with 10, the speed of 000rpm stirred 10 minutes with high-speed shearing machine, the w/o type colostrum.
Step 3: with embodiment 1.
Step 4: with embodiment 1.
Step 6: (5.4%, w/v) in the aqueous solution, mix homogeneously was hatched 120 minutes for 4 ℃, got the topotecan hydrochloride multivesicular liposome in 5mL topotecan hydrochloride (1mg/ml)-glucose with blank multivesicular liposome precipitation redispersion.
The particle diameter that records the topotecan hydrochloride multivesicular liposome is 5～50 μ m; Utilization ratio of drug is 81.09%; Medicine-cholesterol ratio is 16.52%; The zeta current potential is negative 44mV.
Comparative Examples 1 active loading method prepares the topotecan hydrochloride conventional liposome
In eggplant-shape bottle, add and contain 400mM lecithin, the chloroformic solution 10ml of 40mM cholesterol.55 ℃ of constant temperature revolve and steam the chloroform of removing wherein, make into immobilized artificial membrane.The ammonium sulfate 10ml that slowly adds 55 ℃ 250mM, vortex vibration 30 minutes comes off immobilized artificial membrane fully.55 ℃ of thermostatic ultrasonics 30 minutes, prepare blank liposome then.The gained blank liposome successively by 0.8,0.45 and 0.22 μ m microporous filter membrane, is carried out granulate.With the dialysis of the liposome behind the granulate, remove the free sulfuric acid ammonium, dialysis solution is a normal saline.(5%, w/v) aqueous solution places 55 ℃ of waters bath with thermostatic control hatchings 5 minutes, can obtain the conventional liposome of topotecan hydrochloride to add topotecan hydrochloride (1mg/ml)-glucose of 1ml again.
The particle diameter that records the topotecan hydrochloride conventional liposome is 100～150nm; Utilization ratio of drug is 91.24%; Medicine-cholesterol ratio is 18.31%; The zeta current potential is negative 41.8mV.
Experimental example 1
(1) release in vitro of topotecan hydrochloride multivesicular liposome is measured
Precision is measured the topotecan hydrochloride multivesicular liposome suspension 5ml of embodiment 1, dilute with the 20ml normal saline, the gained suspension is placed 37 ℃ of constant temperature shaking tables (rotating speed is 15rpm), take out the sample of equivalent at preset time point, with 600 * g centrifugalize supernatant and precipitation, measure the amount of topotecan hydrochloride in the supernatant in accordance with the law.The drug release percentage ratio of each time point calculates with following formula:
Topotecan hydrochloride original vol * 100 of sealing in the amount/multivesicular liposomes of free topotecan hydrochloride in drug release percentage ratio (%)=supernatant
(2) release in vitro of topotecan hydrochloride conventional liposome is measured
Precision is measured the topotecan hydrochloride conventional liposome 1ml of Comparative Examples 1 to (molecular cut off is 1 through pretreated bag filter, 4000) in, place the beaker that the 50mL normal saline is housed, 37 ℃ of constant temperature stir, timing sampling 1mL normal saline dialysis liquid, replenish the blank normal saline of equivalent simultaneously, in accordance with the law the amount of topotecan hydrochloride in the working sample solution.And calculate by as above method and to discharge percentage ratio.
The result: conventional liposome drug release about 6 hours surpasses 90%, and multivesicular liposome is when the 3rd day (72 hours), and drug release just reaches more than 90%, and the topotecan hydrochloride of sealing discharges (as shown in Figure 1) fully substantially.Illustrate that topotecan hydrochloride multivesicular liposome of the present invention compares with conventional liposome, have tangible slow release effect.
Claims (28)
1. blank multivesicular liposome, comprise each component of following mass parts: 0.1～50 part of 1 part of lipid components and ion gradient regulator comprise in the lipid components that wherein mol ratio is 5～36: 1 amphipathic lipids and neutral lipid.
2. blank multivesicular liposome as claimed in claim 1, it is characterized in that described amphipathic lipids is to be selected from lecithin, soybean phospholipid, hydrogenated soya phosphatide, dipalmitoyl phosphatidyl choline, stearylamine, dimyristoyl phosphatidyl choline, dioleoyl phospholipid phatidylcholine, PHOSPHATIDYL ETHANOLAMINE, DOPE, cephalin, sphingomyelins, two palmityl Phosphatidylserine, phosphatidyl glycerol, phosphatidylinositols, phosphatidic acid and the two palmityl phosphatidyl glycerols one or more.
3. blank multivesicular liposome as claimed in claim 1 is characterized in that, described neutral lipid is to be selected from three oils and fatss, three hot fat and the soybean oil one or more.
4. blank multivesicular liposome as claimed in claim 1 is characterized in that, described ion gradient regulator is selected from one or more in ammonium sulfate, copper sulfate, manganese sulfate, magnesium sulfate, calcium acetate, citric acid and the manganous chloride.
5. blank multivesicular liposome as claimed in claim 1, it is characterized in that, each component that comprises following mass parts: 0.5～20 part of 1 part of lipid components and ion gradient regulator wherein comprise mol ratio 10～20: 1 amphipathic lipids and neutral lipid in the lipid components.
6. blank multivesicular liposome as claimed in claim 1 is characterized in that, in the described lipide component, also further comprises sterin.
7. blank multivesicular liposome as claimed in claim 6 is characterized in that, described sterin is a cholesterol.
8. blank multivesicular liposome as claimed in claim 6 is characterized in that, the mol ratio of the neutral lipid in described sterin and the lipid components is 0.7～65: 1.
9. blank multivesicular liposome as claimed in claim 1 is characterized in that, also comprises osmotic pressure regulator.
10. blank multivesicular liposome as claimed in claim 9 is characterized in that, the content of osmotic pressure regulator is 0.08～15 part.
11. blank multivesicular liposome as claimed in claim 9, it is characterized in that described osmotic pressure regulator is selected from one or more in glucose, sucrose, mannitol, sodium chloride, trehalose, succinate, cyclodextrin, arginine, galactose, mannose, maltose, mannitol, glycine, lysine, citrate, sorbitol and the glucosan.
12. the preparation method as each described blank multivesicular liposome of claim 1～11 is characterized in that, comprises the following steps:
1. lipide component is dissolved in the organic solvent as the lipid phase, this lipid components comprises amphipathic lipids and neutral lipid;
2. the ion gradient regulator is dissolved in the water, as interior water, water in described is added the lipid phase, mixing and emulsifying makes Water-In-Oil (W/O) type colostrum;
3. outer water is added described water-in-oil type Ruzhong just, mix forming W/O/W (W/O/W) type emulsion;
4. remove the organic solvent in the emulsion that 3. step obtain, make blank multivesicular liposome suspension first product;
5. wash described blank multivesicular liposome suspension first product with isosmotic solution, redispersion makes blank multivesicular liposome finished product in isosmotic solution.
13. preparation method as claimed in claim 12 is characterized in that, the 1. described organic solvent of step is to be selected from ether, hydrocarbon, halogenated hydrocarbons, halogen ether and the ester one or more.
14. preparation method as claimed in claim 12 is characterized in that, the 1. described lipid of step mutually in, the concentration of amphipathic lipids is 5～120mM, the mol ratio of amphipathic lipids and neutral lipid is 5～36: 1.
15. preparation method as claimed in claim 12 is characterized in that, the 2. described ion gradient regulator of step is selected from one or more in ammonium sulfate, copper sulfate, manganese sulfate, magnesium sulfate, calcium acetate, citric acid and the manganous chloride.
16. preparation method as claimed in claim 12 is characterized in that, the 2. described ion gradient regulator of step is 10～1000mM in the concentration of interior aqueous phase.
17. preparation method as claimed in claim 12 is characterized in that, aqueous phase also added osmotic pressure regulator in step was 2. described, just water in the institute was added the lipid phase then, and mixing and emulsifying makes Water-In-Oil (W/O) type colostrum.
18. preparation method as claimed in claim 12 is characterized in that, what water and lipid volume ratio mutually were preferable in step was 2. described is 1: 1～1: 4.
19. preparation method as claimed in claim 12 is characterized in that, the 3. described outer aqueous phase of step comprises coemulsifier and osmotic pressure regulator, and coemulsifier is selected from one or more in lysine, glycine and the histidine.
20. preparation method as claimed in claim 19 is characterized in that, the coemulsifier concentration of described outer aqueous phase is 20～80mM.
21. preparation method as claimed in claim 12 is characterized in that, what the volume ratio of 3. described water-in-oil type colostrum of step and outer water was preferable is 1: 2.5～1: 10.
22. preparation method as claimed in claim 12 is characterized in that, the 5. described isosmotic solution of step is a normal saline.
23. a topotecan hydrochloride multivesicular liposome is characterized in that, comprise the active constituents of medicine topotecan hydrochloride and as each described blank multivesicular liposome of claim 1～11 as carrier.
24. topotecan hydrochloride multivesicular liposome as claimed in claim 23 is characterized in that, its Chinese medicine fat ratio is 1: 0.1～1: 250.
25. the preparation method as topotecan hydrochloride multivesicular liposome as described in the claim 23 is characterized in that, may further comprise the steps:
A. topotecan hydrochloride and osmotic pressure regulator are dissolved in the water;
B. step a gained solution is added as in each described blank multivesicular liposome finished product of claim 1～11, mix homogeneously is hatched at the phase transition temperature that is higher than lipid, makes topotecan hydrochloride multivesicular liposome suspension first product;
C. wash described topotecan hydrochloride multivesicular liposome suspension first product with isosmotic solution, redispersion makes topotecan hydrochloride multivesicular liposome finished product in isosmotic solution.
26. preparation method as claimed in claim 25 is characterized in that, the concentration of the topotecan hydrochloride described in the step a is 0.01～100mg/ml.
27. preparation method as claimed in claim 25 is characterized in that, step b Chinese medicine fat ratio is 1: 0.1～1: 250.
28. preparation method as claimed in claim 25 is characterized in that, the isosmotic solution described in the step c is a normal saline.
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