CN103622906B - High drug-loading capacity amphotericin B polymer composite micelle and preparation method thereof - Google Patents
High drug-loading capacity amphotericin B polymer composite micelle and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the field of biotechnology and particularly relates to a high drug-loading capacity amphotericin B polymer composite micelle and a preparation method thereof. According to the invention, polyethylene glycol-phospholipid with clinical application potentials and indissolvable amphotericin B are used to form amphotericin B polyethylene glycol-phospholipid composite micelle; amphotericin B can combine with polyethylene glycol-phospholipid to form the amphotericin B polymer composite micelle with high drug-loading capacity, high encapsulation efficiency and high stability through the electrostatic attraction function and the interaction between carbon chains. The amphotericin B polymer composite micelle is stable in normal saline and plasma and lower in toxicity and infusion reaction, can be diluted by adopting normal saline, and greatly improves the clinical application range and convenience.
Description
Technical field
The invention belongs to pharmaceutical technology field, relate to a kind of high drug load amphotericin B polymer composite micelle and preparation method thereof,
Being specifically related to can the nano combined micellar preparation and preparation method thereof of intravenous amphotericin B.
Background technology
Amphotericin B belongs to many alkenes broad-spectrum antifungal antibiotic, is mainly used in treating deep fungal infection, and has the highest curative effect,
Employ nearly half a century in clinic, be referred to as " goldstandard " of anti-fungal infection.
Amphotericin B antifungal mechanism of action is that the ergosterol on fungal cell membrane is combined, and forms micropore on cell membrane,
Intracellular matter extravasation is made to cause fungus dead.Owing to amphotericin B has hydrophobic polyene structure and hydrophilic polyhydroxy structure,
Its critical aggregation concentration in water the lowest (< 1.0 μ g/ml) as easy as rolling off a log gathering, and respectively with insoluble aggregates, solvable
Property aggregation and three kinds of forms of monomer exist.Wherein, the amphotericin B only existed with monomeric form could optionally act on
Ergosterol on fungal cell membrane, and the specificity of aggregation is relatively low, also can tie with the cholesterol on mammalian cell membrane
Close, cause same damage, therefore its toxicity is bigger.
Owing to amphotericin B water dissolution character is poor, NaTDC is used it to be carried out solubilising, as clinical practice clinically
Normal injection agent.When amphotericin B normal injection agent is administered, drug main will be through renal excretion, and therefore nephrotoxicity is bigger.
Additionally, amphotericin B normal injection agent is unstable in normal saline, potassium ion in erythrocyte can be promoted to ooze out, cause various
Infusion reaction is such as shivered with cold, is felt cold, nausea and vomiting.Tradition amphotericin B injection shows serious when clinical practice
Toxic and side effects, significantly limit its application.Therefore, a kind of novel formulation is developed to reduce the kidney poison of amphotericin B
Property and infusion reaction have important clinical meaning.
In order to reduce the toxic and side effects of amphotericin B, domestic and international researcher is devoted to the exploitation of amphotericin B novel formulation always,
The most most successful preparation is AM Bison.AM Bison in 1991 first at UK & Ireland
Listing, is also first Lipidosome medicine.At present, the liposome of domestic listing mainly has " floating special gram of peace " and " cutting edge of a knife or a sword gram
Pine ".Amphotericin B, by after liposomal encapsulated, is mainly distributed in the more rich organ of the reticuloendothelial cell such as liver, spleen, and
Abundance in heart and kidney reduces, such that it is able to reduce the nephrotoxicity of medicine.But AM Bison " cutting edge of a knife or a sword gram pine "
Prescription still contains a certain amount of surfactant sodium deoxycholate, there is certain hemolytic toxicity, deposit during Clinical practice
In bigger potential safety hazard.Additionally, Liposomal formulation existence and stability is poor, particle diameter instability, drug encapsulation during storing
Rate leakage low, easy and be easily formed the shortcomings such as bigger Particles Blocking blood capillary.
The carrier that Amphipathilic block polymer micelle delivers as a kind of novel hydrophobic medicine, has that medicine carrying scope is wide, structure is steady
Fixed, excellent tissue permeability, internal holdup time length, can effectively carry medicine and arrive the features such as target organ.With liposome
Dosage form is compared, and polymer micelle most clear advantage is that more preferable tissue permeability, and its reason is that polymer micelle has more
Little particle diameter and more soft structure.Amphotericin B is used clinically for the treatment of deep tissue fungal infection, and permeability is more preferable
Micellar preparation than Liposomal formulation, there is more preferable therapeutic effect.
The nano-micelle prepared based on polyglycol derivatization phospholipid has the advantages that 1) good biocompatibility；2) particle diameter
Little, good stability, it is to avoid other particulate delivery systems such as liposomees, it is easy to the shortcoming of gathering；3) medicine body can be changed
Interior distribution, reduces medicine accumulation in Liver and kidney, reduces the toxic and side effects of medicine；4) extend the circulation time in vivo of medicine, increase
The action time adding medicine and the targeting that tumor etc. is organized.
Summary of the invention
The present invention seeks to for current clinical existing amphotericin B preparation such as Amphotericin B for injection and injection both sexes mould
The problems such as the element nephrotoxicity of B liposome, hemolytic toxicity and infusion reaction, it is provided that a kind of high drug load amphotericin B polymer is multiple
Close micelle administration system.
The present invention use polyglycol derivatization phospholipid class as carrier material, by mould with a certain proportion of insoluble drug both sexes
Element B interaction force in aqueous is self-assembly of compound skeleton micelle, thus significantly improves in amphotericin B aqueous solution
Dissolubility.Wherein, carrier material is 0.4～4:1 with the mol ratio of amphotericin B, preferably 0.5:1～3:1.
Wherein said polyglycol derivatization phospholipid is that peg molecule passes through covalent bond and the nitrogenous base base junction on phospholipid molecule
Conjunction forms.
In polyglycol derivatization phospholipid, the fatty acid of phospholipid moiety comprises 10～24 carbon atoms, and fatty acid chain is
Saturated and fractional saturation, preferably lauric acid, myristic acid, Palmic acid, stearic acid, oleic acid or linoleic acid.Described phosphorus
Fat is PHOSPHATIDYL ETHANOLAMINE, phosphatidylcholine, phosphatidylinositols, Phosphatidylserine, diphosphatidylglycerol or haemolysis choline phosphorus
Fat.
Wherein the molecular weight polyethylene glycol scope of polyglycol derivatization phospholipid is 200～20000, preferably
500～10000, more preferably 2000～4000.
Described polyglycol derivatization phospholipid is selected from: Macrogol 2000-PHOSPHATIDYL ETHANOLAMINE, Polyethylene Glycol
4000-PHOSPHATIDYL ETHANOLAMINE, Macrogol 2000-phosphatidylcholine, polyethylene glycol 6000-phosphatidylcholine, Macrogol 2000-
Phosphatidylserine, Macrogol 4000-Phosphatidylserine, Macrogol 2000-phosphatidylinositols, Macrogol 4000-
Phosphatidylinositols, polyethylene glycol 6000-phosphatidylinositols, preferably: Macrogol 2000-PHOSPHATIDYL ETHANOLAMINE, Macrogol 4000-
PHOSPHATIDYL ETHANOLAMINE, Macrogol 2000-phosphatidylinositols.
Polyglycol derivatization phospholipid has the advantages such as safe and nontoxic, good biocompatibility, and it is dense to have relatively low critical micell
Degree (10-6～10-5mol·L-1), its micelle formed remains to keep stable after internal hemodilution.Therefore can answer safely
In the preparation of amphotericin B preparation.
The polyglycol derivatization phospholipid of the present invention and the composite micelle of medicine amphotericin B, can be prepared by the following method: thin
Film dispersion method and lyophilization.
Film dispersion method comprises the following steps:
(1) mixture of amphotericin B and polyglycol derivatization phospholipid is dissolved in organic solvent；
(2) thing mixed above is rotated in a vacuum volatilize, obtain polyglycol derivatization phospholipid with amphotericin B mixture film；
(3) add appropriate amount of deionized water, redissolve and i.e. obtain the micellar solution uniformly clarified.
Lyophilization comprises the following steps:
(1) mixture of amphotericin B and polyglycol derivatization phospholipid is dissolved in organic solvent；
(2) add appropriate freeze drying protectant, remove moisture removal through lyophilization, obtain dry bag and carry hydrophobic antitumor drug amphotericin
The polymer micelle of B；
(3) prepared lyophilizing micelle needed amount is added water, after hydration vibration, be recovered to micellar solution.
Described deionized water includes: distilled water, normal saline, isotonic glucose solution or buffer solution
Described organic solvent is methanol, ethanol, chloroform, acetone, dimethyl sulfoxide one or its mixed solvent.
Freeze drying protectant include glucose, mannitol, sucrose, trehalose, maltose, Macrogol 2000, Macrogol 4000,
Polyethylene glycol 6000 or Pluronic F68.
The polymer micelle composition prepared by said method, high speed homogenization even by high pressure breast or the technique such as ultrasonic can reduce micelle
Particle diameter and particle diameter distribution.This polymer micelle composition also such as lyophilization, can be spray-dried, subtract through certain preparation process
Pressure evaporation etc. is further prepared into suitable formulations, such as injection, oral formulations etc..
The present invention identifies carrier polyglycol derivatization phospholipid by infrared spectrometry and there is interaction with medicine amphotericin B.
Infrared spectrometer is used respectively carrier micelle, polyglycol derivatization phospholipid, amphotericin B and mixture thereof to be scanned,
By investigating the change of the absworption peak peak position of compound characteristic group, confirm the change of compound ambient electron environment, it is determined that
There is interaction between the two.Speculate that mPEG2000-DSPE carbonyl exists hydrogen bond physics with the hydroxyl of amphotericin B
Interact.
The present invention, by measuring particle diameter, has investigated carrier micelle stability in normal saline.The injection two used clinically
Property mycin B and Amphotericin B for injection liposome all can only use glucose solution to be diluted, and can produce through normal saline dilution
Raw precipitation.And the product prepared by the present invention can keep long stability in a large amount of normal saline, improve clinic and make
Scope and convenience.
The present invention has investigated the stability of carrier micelle lyophilized powder by envelop rate, drug loading and particle diameter.Composite micelle is protected in lyophilizing
Protecting under the protective effect of agent, have good stability, medicine is difficult to reveal, and particle is difficult to assemble.
The present invention has investigated the release in vitro situation of amphotericin B composite micelle.Amphotericin B composite micelle has the most slow
Releasing feature, release is steadily without burst effect.Micellar carrier can effectively wrap up amphotericin B, it is to avoid preparation is during blood circulation
Medicine quickly discharges, thus reduces the toxic and side effects of medicine；Can significantly extend drug treating time, reach long-acting.
The present invention by having investigated the hemolysis in vitro toxicity of carrier micelle to the mensuration of hemolysis rate, sub-with the dimethyl of amphotericin B
Sulfolane solution, Amphotericin B for injection are compared with Amphotericin B for injection liposome solutions, amphotericin B polymer compound adhesive
Bundle hemolytic toxicity significantly reduces.Owing to amphotericin B composite micelle can delay Slow release, it is to avoid drug accumulation and produce molten
Blood toxicity；Composite micelle can make amphotericin B discharge with the monomeric form that toxicity is relatively low, reduces hemolytic toxicity.
The present invention determines the MIC of carrier micelle by liquid-based dilution method80Value, has investigated antibacterial activity in vitro.Its in-vitro antibacterial is lived
Property is better than dimethyl sulphoxide solution and the Amphotericin B for injection solution of amphotericin B.The dimethyl sulfoxide of amphotericin B is molten
Liquid and Amphotericin B for injection solution are when being diluted, and part amphotericin B is likely to be formed aggregation, cause it the denseest
Degree reduces；And the dilution-resistant of composite micelle is preferable, drug release is slow, is difficult to assemble, thus valid density is higher.Additionally,
Composite micelle can prevent the polyene radical oxidation of amphotericin B to a certain extent, keeps the activity of medicine.
Accompanying drawing explanation
Fig. 1 is polyglycol derivatization phospholipid acyl ethanolamine1H-NMR collection of illustrative plates
Fig. 2 is amphotericin B1H-NMR collection of illustrative plates
Fig. 3 is amphotericin B and polyglycol derivatization phospholipid acyl ethanolamine mixtures1H-NMR collection of illustrative plates
Fig. 4 is the composite micelle prepared by embodiment 31H-NMR collection of illustrative plates
Fig. 5 is the IR collection of illustrative plates of Macrogol 2000-PHOSPHATIDYL ETHANOLAMINE
Fig. 6 is the IR collection of illustrative plates of amphotericin B
Fig. 7 is the IR collection of illustrative plates of amphotericin B and Macrogol 2000-phosphatidyl ethanol amine blends
Fig. 8 is the IR collection of illustrative plates of the composite micelle prepared by embodiment 3
Fig. 9 is the release profiles in embodiment 1～6 product 72h
The dimethyl sulphoxide solution of Figure 10 amphotericin B, Amphotericin B for injection solution, Amphotericin B for injection liposome solutions
Haemolysis situation with embodiment 3 solution
Detailed description of the invention
According to following embodiment, the present invention be may be better understood.But, as it will be easily appreciated by one skilled in the art that embodiment is retouched
Concrete material proportion, process conditions and the result thereof stated are merely to illustrate the present invention, and should be also without limitation on claims
Invention described in detail by.
Embodiment 1: the composite micelle of Macrogol 2000-PHOSPHATIDYL ETHANOLAMINE and amphotericin B prepares (mol ratio is 0.4:1)
Weigh 8.4mg amphotericin B and 10mg Macrogol 2000-PHOSPHATIDYL ETHANOLAMINE is dissolved in 20ml methanol, at 50 DEG C of water
The rotary evaporation that reduces pressure in bath removes organic solvent, adds distilled water 10ml, aquation 15 minutes, crosses 0.22 μm filtering with microporous membrane,
Obtain composite micelle.Recording its envelop rate is 95.8%, and drug loading is 44.4%, and particle diameter is 57.89nm.
Embodiment 2: the composite micelle of Macrogol 2000-PHOSPHATIDYL ETHANOLAMINE and amphotericin B prepares (mol ratio is 4:1)
Weigh 0.8mg amphotericin B and 10mg Macrogol 2000-PHOSPHATIDYL ETHANOLAMINE is dissolved in 10ml methanol-dimethyl sulfoxide
(1:1), the rotary evaporation that reduces pressure in 50 DEG C of water-baths removes organic solvent, adds phosphate-buffered salt 10ml aquation 20 minutes, mistake
0.22 μm filtering with microporous membrane, adds 20mg mannitol as freeze drying protectant, lyophilization.Redissolve with water for injection, survey
Obtaining its envelop rate is 99.5%, and drug loading is 7.4%, and particle diameter is 60.03nm.
Embodiment 3: the composite micelle of Macrogol 4000-PHOSPHATIDYL ETHANOLAMINE and amphotericin B prepares (mol ratio is 0.5:1)
Weigh 6.6mg amphotericin B and 10mg Macrogol 4000-PHOSPHATIDYL ETHANOLAMINE is dissolved in 18ml methanol, at 60 DEG C of water
The rotary evaporation that reduces pressure in bath removes organic solvent, adds distilled water 10ml aquation 15 minutes, crosses 0.22 μm filtering with microporous membrane,
Obtain composite micelle.Recording its envelop rate is 98.7%, and drug loading is 39.8%, and particle diameter is 62.02nm.
Embodiment 4: the preparation (mol ratio is 3:1) of Macrogol 2000-phosphatidylinositols and amphotericin B composite micelle
Weigh 1.1mg amphotericin B and 10mg Macrogol 2000-phosphatidyl-4 hydramine is dissolved in 18ml methanol, in 60 DEG C of water-baths
Decompression rotary evaporation removes organic solvent, adds distilled water 10ml aquation 15 minutes, crosses 0.22 μm filtering with microporous membrane, i.e.
Obtain composite micelle.Recording its envelop rate is 99.8%, and drug loading is 9.9%, and particle diameter is 63.42nm.
Embodiment 5: the composite micelle of Macrogol 4000-PHOSPHATIDYL ETHANOLAMINE and amphotericin B prepares (mol ratio is 1:1)
Weigh 3.3mg amphotericin B and 10mg Macrogol 2000-PHOSPHATIDYL ETHANOLAMINE be dissolved in 10ml methanol-dimethyl sulfoxide (1:1),
The rotary evaporation that reduces pressure in 50 DEG C of water-baths removes organic solvent, adds phosphate-buffered salt 10ml aquation 20 minutes, crosses 0.22 μm
Filtering with microporous membrane, adds 20mg mannitol as freeze drying protectant, lyophilization.Redissolve with water for injection, record its encapsulating
Rate is 99.5%, and drug loading is 24.8%, and particle diameter is 58.62nm.
Embodiment 6: the composite micelle of Macrogol 4000-PHOSPHATIDYL ETHANOLAMINE and amphotericin B prepares (mol ratio is 2:1)
Weigh 1.7mg amphotericin B and 10mg Macrogol 2000-PHOSPHATIDYL ETHANOLAMINE is dissolved in 10ml methanol, in 50 DEG C of water-baths
Decompression rotary evaporation removes organic solvent, adds distilled water 10ml, aquation 15 minutes, crosses 0.22 μm filtering with microporous membrane, i.e.
Obtain composite micelle.Recording its envelop rate is 99.8%, and drug loading is 14.5%, and particle diameter is 56.89nm.
Embodiment 7: the carrier micelle prepared by embodiment 3 method, after lyophilization, weighs 1.0mg and is dissolved in 0.5ml deuterium-oxide, uses
Mercury Plus400MHz nuclear magnetic resonance chemical analyser is identified, it is thus achieved that nuclear magnetic resonance map (such as Fig. 4).Weigh 1.0mg respectively
Polyglycol derivatization phospholipid acyl ethanolamine, amphotericin B and both mixture (mol:mol1:2) be dissolved in 0.5ml deuterium
For in dimethyl sulfoxide, Mercury Plus400MHz nuclear magnetic resonance chemical analyser is used to identify, it is thus achieved that nuclear magnetic resonance map (as
Fig. 1,2 and 3).As figure shows, after forming carrier micelle, the nuclear-magnetism vibration absorption peak of polymer phospholipid end disappears, and illustrate only
The absworption peak of Polyethylene Glycol end, shows that polyglycol derivatization phospholipid acyl ethanolamine defines stable nucleocapsid structure；Amphotericin B
Nuclear-magnetism vibration absorption peak disappear, show that amphotericin B is loaded in micelle core by bag.
Embodiment 8: the carrier micelle prepared by embodiment 3 method, after lyophilization, uses Brukervector22 type infrared spectrometer
Measure the infrared spectrum of the lyophilized powder of Macrogol 2000-PHOSPHATIDYL ETHANOLAMINE, amphotericin B, embodiment 3 preparation, side respectively
Method is solid sample and potassium bromide powder mixed grinding, carries out tabletting, the most directly tests.Scanning gained infared spectrum is such as
5～8, mPEG2000-DSPE is positioned at 1738cm as seen from the figure-1The absworption peak at place (-C=O) is moved to 1730cm-1, both sexes
Mycin B is positioned at the absworption peak of (-OH) at 3411cm and is moved to 3419cm-1, show to have between the two interaction.
Embodiment 9: composite micelle envelop rate and the assay method of drug loading
The accurate product solution drawing 0.5ml embodiment 1～6, is slowly added on sephadex column, is centrifuged 2 with 3000r/min
Minute, collect first three pipe filtrate, merge, methanol dilution, use ultraviolet spectrophotometer measure, be calculated as follows envelop rate and
Drug loading:
Wherein, W1For the drug quality being wrapped in micelle；W2For dosage, W3For carrier material quality.Record prepared by embodiment 1～6
The envelop rate of product and drug loading such as table 1.In table, data show.
The envelop rate of table 1 example 1～6 product and drug loading
Embodiment 10: amphotericin B composite micelle stability study in normal saline
Product prepared by embodiment 1～6 is diluted with 100ml normal saline respectively, uses Malvern particle size determination instrument to divide
After 0.5,1,2,4,6,12,24 hours, do not measure its particle diameter.Particle size determination result such as table 2.
Table 2. amphotericin B composite micelle stability in normal saline
Embodiment 11: the research of amphotericin B composite micelle freeze-dried powder stability
By the amphotericin B composite micelle prepared by embodiment 3, add freeze drying protectant, place in room temperature after lyophilization,
Redissolved with water for injection respectively at 1,2,4,6,8,10,20,30 days, and measure its envelop rate, drug loading and particle diameter.
Measurement result such as table 3.
The stability of table 3. amphotericin B composite micelle lyophilized powder
Embodiment 12: the investigation of amphotericin B micelle release in vitro
The each 2ml of amphotericin B micelle prepared by Example 1～6, is placed in bag filter (molecular weight 12000～15000), then
Put into the conical flask equipped with 200mL pH10.0PBS release medium.Conical flask is placed in constant temperature oscillator, in 37 DEG C, 100r/min
Condition on carry out release experiment.Respectively at 0.5,1,2,4,6,12,24,36,48,72h take extracellular fluid dialysis, simultaneously
Add the mutually synthermal release medium of equivalent.Use ultraviolet spectrophotometer to measure release amount of medicine, draw release profiles such as Fig. 9.
Result shows, amphotericin B composite micelle has obviously sustained releasing character.
Embodiment 13: the investigation of amphotericin B composite micelle hemolysis in vitro
Taking healthy new zealand rabbit 1, auricular vein takes blood, is placed in the test tube that heparin processes, adds appropriate 0.9% chlorination
Sodium injection washs.2000r/min is centrifuged 10min, abandoning supernatant.Repeat aforesaid operations for several times, until centrifuged supernatant
In water white transparency.Abandoning supernatant, adds 0.9% sodium chloride injection by gained erythrocyte and is diluted, be configured to the erythrocyte of 2%
Suspension, standby.
By the carrier micelle prepared by embodiment 3, Amphotericin B for injection, Amphotericin B for injection liposome and amphotericin B
Free solution use normal saline dilution, compound concentration scope is the series concentration solution of 2～256 μ g/ml.Accurate absorption is above-mentioned molten
Liquid 2.5ml, is placed in test tube, is subsequently adding 2% red blood cell suspension 2.5ml, mixing, after cultivating 3h at 37 DEG C, by sample
Solution is placed in ice bath, terminates hemolytic reaction, is centrifuged 10min, Aspirate supernatant with 2000r/min, measures its extinction at 540nm
Degree, hemolysis rate according to the following formula:
Four kinds of solution haemolysis situation such as Figure 10 when 3h.Micellar preparation hemolysis rate prepared by embodiment 3 is substantially less than other three kinds of solution.
Embodiment 14: the investigation of amphotericin B composite micelle in-vitro antibacterial effect
Use RPMI1640 fluid medium dilution Candida albicans solution, be configured to concentration about 0.5～2.5 × 103CFU/mL connects
Plant bacterium solution.By the free solution RPMI1640 liquid of the micelle prepared by embodiment 3, Amphotericin B for injection and amphotericin B
Body culture medium carries out doubling dilution.Drug solution and placebo solution are separately added in 96 orifice plates, add 0.1mL containing bacterium
Culture fluid.96 orifice plates are placed in 35 DEG C of cultivation 48h, observed result in constant incubator.With enzyme linked immunological monitor at 540nm
Measure the optical density value in the 96 each holes of orifice plate.Calculate MIC value such as table 3.
The In Vitro Anti fungus result of 3 three kinds of solution of table
In sum, amphotericin B composite micelle, compared with existing normal injection agent and liposome, has obvious advantage, main body
Now the most following aspects: 1. drug loading is high, the concentration of the amphotericin B polymer micelle preparation Chinese medicine of the present invention up to
1.2mg/ml, is significantly higher than normal injection, AM Bison；2. the amphotericin B polymer micelle system of the present invention
Agent hemolytic is significantly less than normal injection and AM Bison；3. the amphotericin B polymer micelle preparation of the present invention
In Vitro Anti antifungal effect significantly better than normal injection；4. the amphotericin B polymer micelle preparation slow release effect of the present invention is bright
Show and be better than AM Bison.
Claims (5)
1. a high drug load amphotericin B polymer composite micelle, it is characterized in that, weigh 8.4mg amphotericin B and 10mg Macrogol 2000-PHOSPHATIDYL ETHANOLAMINE is dissolved in 20ml methanol, the rotary evaporation that reduces pressure in 50 DEG C of water-baths removes organic solvent, add distilled water 10ml, aquation 15 minutes, crosses 0.22 μm filtering with microporous membrane, obtains composite micelle.
2. a high drug load amphotericin B polymer composite micelle, it is characterized in that, weigh 6.6mg amphotericin B and 10mg Macrogol 4000-PHOSPHATIDYL ETHANOLAMINE is dissolved in 18ml methanol, the rotary evaporation that reduces pressure in 60 DEG C of water-baths removes organic solvent, add distilled water 10ml aquation 15 minutes, cross 0.22 μm filtering with microporous membrane, obtain composite micelle.
3. a high drug load amphotericin B polymer composite micelle; it is characterized in that; weigh 3.3mg amphotericin B and 10mg Macrogol 2000-PHOSPHATIDYL ETHANOLAMINE is dissolved in 10ml methanol-dimethyl sulfoxide 1:1; the rotary evaporation that reduces pressure in 50 DEG C of water-baths removes organic solvent; add phosphate-buffered salt 10ml aquation 20 minutes; cross 0.22 μm filtering with microporous membrane, add 20mg mannitol as freeze drying protectant, lyophilization.
4. according to the composite micelle described in claim 1-3 any one, it is characterised in that wherein said composite micelle is solution form or lyophilized form.
5. a pharmaceutical composition, comprises the polymer micelle described in claim 1-4 any one and pharmaceutically acceptable carrier.
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