CN106924185B - A kind of preparation method for the multivesicular liposome being loaded with vesica - Google Patents
A kind of preparation method for the multivesicular liposome being loaded with vesica Download PDFInfo
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Abstract
The present invention relates to a kind of preparation methods of multivesicular liposome for being loaded with vesica.The preparation method is using nanoscaled vesicle and osmotic pressure regulator as inner aqueous phase, to be oily phase with the organic solvent of good biocompatibility lipid, inner aqueous phase is distributed to formation w/o type colostrum in oily phase, colostrum is distributed to formation W/0/W type emulsion in the outer aqueous phase containing osmotic pressure regulator and coemulsifier again, emulsion is transferred in identical outer aqueous phase, by rotary evaporation or it is passed through nitrogen removing organic solvent, much capsule liposome turbid liquor.The multivesicular liposome preparation not only can guarantee the encapsulation rate of load drug, and the bioactivity of large biological molecule also can be improved, and reduce its immunogenicity, reduces burst effect, guarantees the lasting slow release of drug.
Description
Technical field
The present invention relates to a kind of preparation methods of multivesicular liposome for being loaded with vesica, belong to pharmaceutical technology field.
Background technique
Multivesicular liposome (Multivesicular, MVLs) is to develop a kind of more mature sustained-release preparation at present, in
Nineteen eighty-three uses DepoFoam technology (Depo Foam by Skyepharma PLC company, BritainTM) successfully develop.It is by multiple
Aqueous compartments are constituted in the form of non-concentric, are separated by between each chamber with lipid bilayer, and neutral lipid is as fixation
Attachment is distributed at the interface point of adjacent chamber, so as to form firm topological structure.Multivesicular liposome is as hydrophily
The carrier of drug, is greatly improved the encapsulation rate of water soluble drug, while reducing leakage, during release, with drug containing
Cell gradually rupture and discharge drug, but complete chondritic is still able to maintain original state, to reach sustained release purpose.
Currently, the report of multivesicular liposome embedding medicinal is still confined to grinding for conventional water-soluble drug or polypeptide
Study carefully, such as: the preparation of Zhang Miao, Yu Yueling, Tang Xing cytarabine multivesicular liposome and vitro release investigate [J], and 2009,26
(1): the research [D] of the striking tyroserleutide multivesicular liposome of 1-5. Tao Yu, 2007.More multivesicular liposome research reports are addressed upper
In road, release time is generally 3-5 days drug in vitro, and slow-release time is shorter.Burst drug release phenomenon is still more apparent, this is to Mr. Yu
A little therapeutic windows require in stringent disease treatment that there is also major hidden dangers.For biopharmaceutical macromolecular drug, how as far as possible
Guarantee that drug activity before and after preparation is not lost, is a hot issue to reach good therapeutic effect.But large biological molecule
Self structure is complex, it is easy to be subjected to during preparation, preservation or release due to internal external environment chemical degradation or
Physical change, so that the preparation of loss of activity, especially multivesicular liposome is using W/O/W multi-emulsion method, in oil-water interfaces, albumen
The easy recurring structure of polypeptide drug destroys, and loses some biological activity so as to cause it.
Therefore, it is necessary to research and develop a kind of novel sustained-release preparation, it is made to improve entrapment efficiency, solve burst drug release and
Guarantee that there is preferable advantage in terms of pharmaceutical activity.
Summary of the invention
The present invention can solve phenomenon of burst release and encapsulation rate height in view of the shortcomings of the prior art, providing one kind, protect in preparation process
Demonstrate,prove the preparation method of the multivesicular liposome for being loaded with vesica of drug excellent activity.
The technical scheme to solve the above technical problems is that
A kind of preparation method for the multivesicular liposome being loaded with vesica, steps are as follows:
(1) preparation of three-phase solution
Be solution A respectively: preparation is interior containing the vesica and sucrose for embedding small-molecule drug and/or biopharmaceutical macromolecular drug
Water phase；Wherein, the solution A mass concentration containing sucrose is 2-10%；
Solution B: lipid phase of the preparation containing lipid and organic solvent；Wherein, the lipid includes dioleoyl phosphatidyl
Choline, dipalmitoylphosphatidylglycerol, cholesterol and olein；The dioleyl phosphatidyl choline accounts for the total matter of lipid
The percentage of amount is 20-60%, and the percentage that the olein accounts for lipid gross mass is 5-20%, two palmityl phosphatide
The percentage that acyl glycerol accounts for lipid gross mass is 5-20%, and the percentage that the cholesterol accounts for lipid gross mass is 10-50%；
Solution C: outer aqueous phase of the preparation containing glucose and lysine；Wherein, the solution C is containing glucose mass concentration
5-15%, mass concentration containing lysine are 10-80mmol/L；
(2) preparation of W/O colostrum
Merge, mix the solution A and the solution B, high shear agitation obtains W/O colostrum；Wherein, solution A and molten
The volume ratio of liquid B is 1: 0.5-1: 20；
(3) preparation of W/O/W multivesicular liposome
W/O colostrum is distributed in solution C, high shear agitation, forms W/O/W type emulsion；W/O/W type emulsion redisperse
Into solution C, rotary evaporation or nitrogen are blown at 30-40 DEG C, remove organic solvent；W/O/W type emulsion is centrifuged, precipitating is taken to add
Physiological saline disperses again, obtains multivesicular liposome；Wherein, the volume ratio of W/O colostrum and solution C is 1: 1-1: 10.
Preferably, the vesica is micella or nanoparticle.
Preferably, solution A described in step (2) and the solution B high shear agitation conditions are as follows: shear velocity 3000-
15000rpm, time 2-15min.
Preferably, the colostrum is distributed in the solution C in step (3), condition are as follows: shear velocity 3000-
12000rpm, time 10-100 second.
The utility model has the advantages that the present invention contains vesica using inner aqueous phase, oil mutually contains lipid components, forms multivesicular liposome cell packet
Composite construction containing vesica.The preparation method mainly solves the problems, such as following two aspect: (1) solving fat-soluble medicine and be difficult to make
Fat-soluble medicine first can be prepared into vesica, multivesicular liposome is then made again by the technical issues of at multivesicular liposome；(2) it solves
It has determined the common situation of burst drug release, has guaranteed drug bioactivity in vivo and in vitro, extend the half-life period of drug, reach slow release
Purpose.The multivesicular liposome not only can guarantee the encapsulation rate of load drug, and the biology that large biological molecule also can be improved is living
Property, its immunogenicity is reduced, burst effect is reduced, guarantees the lasting slow release of drug.
Detailed description of the invention
Some specific embodiments of the present invention is described in detail by way of example and not limitation with reference to the accompanying drawings hereinafter.
Identical appended drawing reference denotes same or similar part or part in attached drawing.It should be appreciated by those skilled in the art that these
What attached drawing was not necessarily drawn to scale.In attached drawing:
Fig. 1 is the gelatin nanparticles transmission electron microscope photo for being loaded with Exenatide in the embodiment of the present invention one；
Fig. 2 is that the optical microscopy for being loaded with Exenatide gelatin nanparticles multivesicular liposome in the embodiment of the present invention one shines
Piece；
Fig. 3 is that the release in vitro for being loaded with Exenatide gelatin nanparticles multivesicular liposome in the embodiment of the present invention one is bent
Line；
Fig. 4 is the microscope photo for being loaded with Exenatide multivesicular liposome in comparative example one of the present invention；
Fig. 5 is the In-vitro release curves for being loaded with Exenatide multivesicular liposome in comparative example one of the present invention.
Specific embodiment
The present invention provides be used to prepare can with lag characteristic h substance such as drug, proteins and peptides or activity at
The new formulation method divided, that is, provide a kind of preparation method of multivesicular liposome for being loaded with vesica.Steps are as follows:
(1) preparation of three-phase solution
Be solution A respectively: preparation is interior containing the vesica and sucrose for embedding small-molecule drug and/or biopharmaceutical macromolecular drug
Water phase；
Wherein, the solution A mass concentration containing sucrose is 2-10% (w/v)；
Solution B: lipid phase of the preparation containing lipid and organic solvent；
Wherein, the lipid includes dioleyl phosphatidyl choline, dipalmitoylphosphatidylglycerol, cholesterol and three oleic acid
Glyceride；The percentage that the dioleyl phosphatidyl choline accounts for lipid gross mass is 20-60%, and the olein accounts for
The percentage of lipid gross mass is 5-20%, and the percentage that dipalmitoylphosphatidylglycerol accounts for lipid gross mass is 5-20%, institute
Stating cholesterol and accounting for the percentage of lipid gross mass is 10-50%；
Solution C: outer aqueous phase of the preparation containing glucose and lysine；
Wherein, the solution C is 5-15% (w/v) containing glucose mass concentration, and mass concentration containing lysine is 10-
80mmol/L(w/v)；
(2) preparation of W/O colostrum
Merge, mix the solution A and the solution B, high shear agitation obtains W/O colostrum；
Wherein, the volume ratio of solution A and solution B is 1: 0.5-1:20；
The solution A and the solution B high shear agitation conditions are as follows: shear velocity 3000-15000r pm, time 2-
15min；
(3) preparation of W/O/W multivesicular liposome
W/O colostrum is distributed in solution C, high shear agitation, shear velocity 3000-12000rpm, time 10-
100 seconds, form W/O/W type emulsion；W/O/W type emulsion redisperse is into solution C, and rotary evaporation or nitrogen are blown at 30-40 DEG C, removal
Organic solvent；W/O/W type emulsion is centrifuged, takes precipitating plus physiological saline to disperse again, obtains multivesicular liposome；
Wherein, the volume ratio of W/O colostrum and solution C is 1: 1-1: 10.
Small-molecule drug of the present invention, is selected from antibiotics, and chemotherapeutic agent treats choroidal neovascularization
Drug such as dexamethasone.
The biopharmaceutical macromolecular drug is selected from therapeutic peptide or protein or monoclonal antibody or nucleic acid, as shellfish cuts down pearl list
Resist, Lucentis, VEGF Trap, siRNA and Plasmid DNA.The compound multivesicular liposome being prepared into using the present invention, lipid material
Material has preferable biocompatibility, will not cause to stimulate to skin, muscle and local organization, and biopharmaceutical macromolecular drug is wrapped by bilayer
It wraps up in, guarantees that activity is not lost, and each cell gradually ruptures release vesica, vesica discharges drug one by one again, to reach sustained release
Purpose.Its emphasis solves burst drug release situation, guarantees pharmaceutical activity, drug is allow to keep slow release in vivo.
The vesica is micella or nanoparticle.
The micella is made of block copolymer, as in segment include poly lactic-co-glycolic acid, hyaluronic acid, preferably
MPEG-PLGA micella.
The nanoparticle is selected from chitosan nanoparticle, gelatin nanparticles, the sodium hyaluronate grain of rice, polylactic acid-glycolic
The nanoparticle formed after guanidine-acetic acid nanoparticle and the modification transformation of their materials.
The organic solvent is one of chloroform, ether, methanol or acetone or a variety of.
It is to be appreciated that in the present invention, the dioleyl phosphatidyl choline is to form multivesicular liposome main
Ingredient, only has the phosphatide containing bilayer, and drug can be just encapsulated in wherein.
In the present invention, the olein is otherwise the key factor to form multivesicular liposome can only obtain
Conventional liposome；Olein is filled in the gap between multivesicular liposome capsule and capsule, and it is each small to play stable connection
The effect of room；The glyceryl ester of different chain length influences the in-vitro release rate of drug.
In the present invention, the dipalmitoylphosphatidylglycerol, effect is that enhancing is formed by between multivesicular liposome
Stability, the repulsive force mainly due to neutral phospholipid since van der Waals interaction is also easy to produce agglomeration effect, between identical charges
The agglutination of particle can more preferably be prevented.Its concentration in lipid phase is 1-10mg/ml, bear electricity phosphatide better effect.By reality
Comparison discovery is tested, the multivesicular liposome containing dipalmitoylphosphatidylglycerol is not than containing on spherical rounding and degree of scatter
There is more apparent advantage, the former is sticked together, poor dispersion.
In the present invention, the cholesterol can improve the mobility of immobilized artificial membrane as membrane stabilizer, reduce the leakage of drug
Rate, dosage is excessive or the very few stability and encapsulation rate that can all influence multivesicular liposome, and the concentration of cholesterol is in 5- in lipid phase
20mg/ml, if cholesterol is not added in experiment discovery, obtained multivesicular liposome is just crushed quickly, and slow release effect is not achieved.
In the present invention, the osmotic pressure regulator in the inner aqueous phase is sucrose, the osmotic pressure regulator in the outer aqueous phase
For glucose.The presence of the inner aqueous phase osmotic pressure regulator, outer aqueous phase osmotic pressure regulator is outer aqueous phase pressure in guaranteeing
Difference in a certain range, increases the stability of multivesicular liposome, reduces drug leakage occurs as far as possible.
A kind of preparation method of multivesicular liposome for being loaded with vesica of the present invention is specifically described below by embodiment.
Embodiment one
It is loaded with the multivesicular liposome of the gelatin nanparticles of Exenatide
(1) solution A: the inner aqueous phase of 1 part of gelatin nanparticles for being loaded with Exenatide and osmotic pressure regulator is prepared, specially
It disperses the nanoparticle prepared in the water phase dissolved with osmotic pressure regulator；
Preparation solution B: contain 200 parts of lipid organic solvent；
Prepare solution C: the outer aqueous phase containing osmotic pressure regulator and coemulsifier；
(2) merge, mixed solution A and solution B, 12000rpm high shear agitation obtain W/O colostrum；
Wherein, the volume ratio of solution A and solution B is 1:0.5-1:20；
Solution A mass concentration containing sucrose is 2-10% (w/v)；
Mass concentration containing dipalmitoylphosphatidylglycerol is 0.01-1% (w/v) in solution B；
(3) W/O colostrum is distributed in solution C, shear velocity 5000rpm, the time 10 seconds, forms W/O/W type emulsion；
Wherein, the volume ratio of W/O colostrum and solution C is 1:1-1:10 progress；
Solution C is 5-15% (w/v) containing glucose mass concentration, and mass concentration containing lysine is 10-80mmol/L (w/
v)；
(4) by W/O/W type emulsion redisperse into solution C, rotary evaporation or nitrogen are blown at 30-40 DEG C, remove organic solvent；
Wherein, the volume ratio of W/O/W type emulsion and solution C is 1:1-1: 100；
(5) W/O/W type emulsion is subjected to 2000rpm centrifugation, takes precipitating plus physiological saline to disperse again, obtains more capsule lipids
Body suspension.
Fig. 1 is the gelatin nanparticles transmission electron microscope photo for being loaded with Exenatide, it will be seen from figure 1 that being loaded with Exenatide
Gelatin nanparticles form rounding.Fig. 2 is the optical microscope photograph for being loaded with the multivesicular liposome of Exenatide gelatin nanparticles,
Figure it is seen that the particle size of multivesicular liposome is uniform, spherical rounding.Fig. 3 is to be loaded with Exenatide gelatin nanparticles
The In-vitro release curves of multivesicular liposome.
Embodiment two
It is loaded with the multivesicular liposome of the mPEG-PLGA micella of huperzine
(1) prepare solution A: 1 part is loaded with the mPEG-PLGA micella of huperzine and the inner aqueous phase of osmotic pressure regulator, tool
Body is to disperse the nanoparticle prepared in the water phase dissolved with osmotic pressure regulator；
Solution B: contain 200 parts of lipid organic solvent；
Solution C: the outer aqueous phase containing osmotic pressure regulator and coemulsifier；
(2) merge, mixed solution A and solution B, 12000rpm high shear agitation obtain W/O colostrum；
Wherein, the volume ratio of solution A and solution B is 1:0.5-1:20；
Solution A mass concentration containing sucrose is 2-10% (w/v)；
Mass concentration containing dipalmitoylphosphatidylglycerol is 0.01-1% (w/v) in solution B；
(3) W/O colostrum is distributed in solution C, shear velocity 5000rpm, the time 10 seconds, forms W/O/W type emulsion；
Wherein, the volume ratio of W/O colostrum and solution C is 1: 1-1: 10；
Solution C is 5-15% (w/v) containing glucose mass concentration, and mass concentration containing lysine is 10-80mmol/L (w/
v)；
(4) by W/O/W type emulsion redisperse into solution C, rotary evaporation or nitrogen are blown at 30-40 DEG C, remove organic solvent；
Wherein, the volume ratio of W/O/W type emulsion and solution C is 1: 1-1: 100；
(5) W/O/W type emulsion is subjected to 2000rpm centrifugation, takes precipitating plus physiological saline to disperse again, obtains more capsule lipids
Body suspension.
Wherein the nanoparticle, micella can be replaced liposome.
Comparative example one
Exenatide multivesicular liposome
(1) solution A: the inner aqueous phase containing 1 part of Exenatide and osmotic pressure regulator is prepared, specially by drug dissolved with infiltration
In the water phase for pressing regulator；
Solution B: contain 10 parts of lipid organic solvent；
Solution C: the outer aqueous phase containing osmotic pressure regulator and coemulsifier
(2) merge, mixed solution A and solution B, 12000rpm high shear agitation obtain W/O colostrum；
Wherein, the volume ratio of solution A and solution B is 1: 0.5-1: 20；
Solution A mass concentration containing sucrose is 2-10% (w/v)；
Mass concentration containing dipalmitoylphosphatidylglycerol is 0.01-1% (w/v) in solution B；
(3) W/O colostrum is distributed in solution C, shear velocity 5000rpm, the time 10 seconds, forms W/O/W type emulsion；
Wherein, the volume ratio of W/O colostrum and solution C is 1: 1-1: 10 progress；
Solution C is 5-15% (w/v) containing glucose mass concentration, and mass concentration containing lysine is 10-80mmol/L (w/
v)；
(4) by W/O/W type emulsion redisperse into solution C, rotary evaporation or nitrogen are blown at 30-40 DEG C, remove organic solvent；
Wherein, the volume ratio of W/O/W type emulsion and solution C is 1: 1-1: 100；
(5) W/O/W type emulsion is subjected to 2000rpm centrifugation, takes precipitating plus physiological saline to disperse again, obtains more capsule lipids
Body suspension.
Its microscope photo is shown in that Fig. 4, In-vitro release curves are shown in Fig. 5；The results show that Exenatide multivesicular liposome spherical shape compared with
Rounding.
By the In-vitro release curves of embodiment one and comparative example one it is found that in embodiment the variation of drug Accumulation dissolution it is slow
Slowly, there is apparent slow release effect, the sustainable release of drug 15 days；In comparative example, drug release, which has, to be significantly released now
As, and drug release is rapider, sustainable 7 days or so.
Comparative example two
Bovine serum albumin(BSA) multivesicular liposome
(1) solution A: the inner aqueous phase containing 1 part of bovine serum albumin bletilla osmotic pressure regulator is prepared, it is specially that drug is molten
Have in the water phase of osmotic pressure regulator；
Solution B: contain 10 parts of lipid organic solvent；
Solution C: the outer aqueous phase containing osmotic pressure regulator and coemulsifier；
(2) merge, mixed solution A and solution B, 12000rpm high shear agitation obtain W/O colostrum；
Wherein, the volume ratio of solution A and solution B is 1: 0.5-1: 20；
Solution A mass concentration containing sucrose is 2-10% (w/v)；
Mass concentration containing dipalmitoylphosphatidylglycerol is 0.01-1% (w/v) in solution B；
(3) W/O colostrum is distributed in solution C, shear velocity 5000rpm, the time 10 seconds, forms W/O/W type emulsion；
Wherein, the volume ratio of W/O colostrum and solution C is 1: 1-1: 10 progress；
Solution C is 5-15% (w/v) containing glucose mass concentration, and mass concentration containing lysine is 10-80mmol/L (w/
v)；
(4) by W/O/W type emulsion redisperse into solution C, rotary evaporation or nitrogen are blown at 30-40 DEG C, remove organic solvent；
Wherein, the volume ratio of W/O/W type emulsion and solution C is 1: 1-1: 100；
(5) W/O/W type emulsion is subjected to 2000rpm centrifugation, takes precipitating plus physiological saline to disperse again, obtains more capsule lipids
Body suspension.
The multivesicular liposome for being loaded with vesica for vitreum, oral, intramuscular injection, subcutaneous, intrathecal, abdominal cavity, Epidural cavity,
A variety of injecting pathways such as articular cavity.
A kind of multivesicular liposome for being loaded with vesica of the present invention is widely used in operation and clinical treatment, therapeutic domain include
But be not limited to, ophthalmology disease (such as senile macular degeneration, glaucoma, inflammation of eye section and vitreoretinal diseases), tumour,
Hepatopathy and the nervous system disease etc..
The present invention can be summarized with others without prejudice to the concrete form of spirit or essential characteristics of the invention.Therefore, nothing
By from the point of view of which point, the embodiment above of the invention can only all be considered the description of the invention and cannot limit this hair
Bright, claims indicate the scope of the present invention, and above-mentioned explanation does not point out the scope of the present invention, therefore, with this
Any change in the comparable meaning and scope of claims of invention is all considered as including in claim of the invention
In the range of book.
Claims (6)
1. a kind of preparation method for the multivesicular liposome for being loaded with vesica, which is characterized in that steps are as follows:
(1) preparation of three-phase solution
Be solution A respectively: preparing is in the vesica and sucrose of Exenatide or huperzine drug containing embedding active component
Water phase；Wherein, the solution A mass concentration containing sucrose is 2-10%；
Solution B: lipid phase of the preparation containing lipid and organic solvent；Wherein, the lipid includes dioleyl phosphatidyl choline,
Dipalmitoylphosphatidylglycerol, cholesterol and olein；The dioleyl phosphatidyl choline accounts for lipid gross mass
Percentage is 20-60%, and the percentage that the olein accounts for lipid gross mass is 5-20%, and two palmityl phosphatidyls are sweet
The percentage that oil accounts for lipid gross mass is 5-20%, and the percentage that the cholesterol accounts for lipid gross mass is 10-50%；
Solution C: outer aqueous phase of the preparation containing glucose and lysine；Wherein, the solution C is 5- containing glucose mass concentration
15%, mass concentration containing lysine is 10-80mmol/L；
(2) preparation of W/O colostrum
Merge, mix the solution A and the solution B, high shear agitation obtains W/O colostrum；Wherein, solution A and solution B
Volume ratio is 1:0.5-1:20；
(3) preparation of W/O/W multivesicular liposome
W/O colostrum is distributed in solution C, high shear agitation, forms W/O/W type emulsion；W/O/W type emulsion redisperse is to molten
In liquid C, rotary evaporation or nitrogen are blown at 30-40 DEG C, remove organic solvent；W/O/W type emulsion is centrifuged, precipitating plus physiology are taken
Salt water disperses again, obtains multivesicular liposome；Wherein, the volume ratio of W/O colostrum and solution C is 1:1-1:10.
2. being loaded with the preparation method of the multivesicular liposome of vesica as described in claim 1, which is characterized in that the vesica is glue
Beam or nanoparticle.
3. being loaded with the preparation method of the multivesicular liposome of vesica as claimed in claim 2, which is characterized in that the inner aqueous phase is
The inner aqueous phase of gelatin nanparticles and sucrose containing embedding Exenatide.
4. being loaded with the preparation method of the multivesicular liposome of vesica as claimed in claim 2, which is characterized in that the inner aqueous phase is
The inner aqueous phase of mPEG-PLGA micella and sucrose containing embedding huperzine.
5. the preparation method of the multivesicular liposome for being loaded with vesica as described in claim 3 or 4, which is characterized in that in step (2)
The solution A and the solution B high shear agitation conditions are as follows: shear velocity 3000-15000rpm, time 2-15min.
6. being loaded with the preparation method of the multivesicular liposome of vesica as claimed in claim 5, which is characterized in that will in step (3)
The colostrum is distributed in the solution C, condition are as follows: shear velocity 3000-12000rpm, time 10-100 second.
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| CN110179964A (en) * | 2019-03-07 | 2019-08-30 | 中国农业科学院农产品加工研究所 | A kind of preparation method of functional peanut small peptide carrier |
| CN109985236B (en) * | 2019-04-17 | 2023-04-18 | 烟台大学 | Multivesicular liposome gel for overcoming burst release and maintaining antibody activity and preparation method thereof |
| CN110812254A (en) * | 2019-07-08 | 2020-02-21 | 圣菲之美（湖北）生物科技有限公司 | Phospholipase-coated multivesicular liposome and preparation method and application thereof |
| CN115067460A (en) * | 2022-06-16 | 2022-09-20 | 南京林业大学 | Red date pigment nano-particles and preparation method thereof |
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