CN106924185A - A kind of preparation method of the multivesicular liposome for being loaded with vesica - Google Patents
A kind of preparation method of the multivesicular liposome for being loaded with vesica Download PDFInfo
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Abstract
The present invention relates to a kind of preparation method of the multivesicular liposome for being loaded with vesica.The preparation method with nanoscaled vesicle and osmotic pressure regulator be interior water phase, it is oil phase with the organic solvent with good biocompatibility lipid, w/o type colostrum will be formed in interior Aqueous dispersions to oil phase, colostrum is distributed to formation W/0/W type emulsions in the outer water phase containing osmotic pressure regulator and coemulsifier again, emulsion is transferred in identical outer water phase, by rotary evaporation or it is passed through nitrogen and removes organic solvent, much capsule liposome turbid liquor.Described multivesicular liposome preparation can not only ensure to load the envelop rate of medicine, can also improve the bioactivity of large biological molecule, reduce its immunogenicity, reduce burst effect, it is ensured that medicine continues slow release.
Description
Technical field
The present invention relates to a kind of preparation method of the multivesicular liposome for being loaded with vesica, belong to pharmaceutical technology field.
Background technology
Multivesicular liposome (Multivesicular, MVLs) is a kind of sustained-release preparation of the more maturation of development at present, in
Nineteen eighty-three uses DepoFoam technology (Depo Foam by Skyepharma PLC companies of BritainTM) successfully develop.It is by multiple
Aqueous compartments are constituted in the form of non-concentric, are separated by with lipid bilayer between each chamber, and neutral lipid is used as fixation
Attachment is distributed at the interface point of adjacent chamber, so as to firm topological structure can be formed.Multivesicular liposome is used as hydrophily
The carrier of medicine, its envelop rate for being greatly improved water soluble drug, while seepage is reduced, during release, with pastille
Cell gradually rupture and discharge medicine, but complete chondritic remains to maintain the original state, to reach sustained release purpose.
At present, the report of multivesicular liposome embedding medicinal is still confined to grinding for conventional water-soluble medicine or polypeptide
Study carefully, such as：The preparation of Zhang Miao, Yu Yueling, Tang Xing cytarabine multivesicular liposomes and vitro release investigate [J], and 2009,26
(1):The research [D] of the striking tyroserleutides multivesicular liposomes of 1-5. Tao Yu, 2007.Many multivesicular liposome research reports are addressed upper
In road, release time is generally 3-5 days medicine in vitro, and slow-release time is shorter.Burst drug release phenomenon is still more apparent, and this is for certain
Also there is major hidden danger in the strict disease treatment of a little therapeutic window requirements.For biopharmaceutical macromolecular drug, how as far as possible
Ensure that medicine activity before and after preparing is not lost, be a hot issue to reach good therapeutic effect.But large biological molecule
Self structure is complex, it is easy to prepare, preserve or release during undergo due to internal external environment chemical degradation or
Physical change, so that prepared by loss of activity, especially multivesicular liposome is using W/O/W multi-emulsion methods, in oil-water interfaces, albumen
The easy recurring structure destruction of polypeptide drug, so as to cause it to lose some biological activity.
Therefore, it is necessary to research and develop a kind of new sustained-release preparation, make it improving entrapment efficiency, solve burst drug release and
Ensure that pharmaceutical activity aspect has preferable advantage.
The content of the invention
The present invention can solve phenomenon of burst release and envelop rate is high in view of the shortcomings of the prior art, providing one kind, be protected in preparation process
Demonstrate,prove the preparation method of the multivesicular liposome for being loaded with vesica of medicine excellent activity.
The technical scheme that the present invention solves above-mentioned technical problem is as follows：
A kind of preparation method of the multivesicular liposome for being loaded with vesica, step is as follows：
(1) preparation of three-phase solution
It is respectively solution A：Prepare and contain the interior of 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：Prepare the lipid phase containing lipid and organic solvent；Wherein, the lipid includes dioleoyl phosphatidyl
Choline, DPPG, 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 glycerine accounts for lipid gross mass is 5-20%, and the percentage that the cholesterol accounts for lipid gross mass is 10-50%；
Solution C：Prepare the outer water phase containing glucose and lysine；Wherein, the solution C concentration containing glucose quality is
5-15%, mass concentration containing lysine is 10-80mmol/L；
(2) preparation of W/O colostrums
Merge, mix the solution A and the solution B, high shear agitation obtains W/O colostrums；Wherein, solution A and molten
The volume ratio of liquid B is 1: 0.5-1: 20；
(3) preparation of W/O/W multivesicular liposomes
W/O colostrums are distributed in solution C, high shear agitation, form W/O/W type emulsions；W/O/W type emulsion redisperses
To in solution C, rotary evaporation or nitrogen blow at 30-40 DEG C, remove organic solvent；W/O/W type emulsions are centrifuged, precipitation are taken and is added
Physiological saline disperses again, obtains multivesicular liposome；Wherein, the volume ratio of W/O colostrums 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：Shear rate 3000-
15000rpm, time 2-15min.
Preferably, the colostrum is distributed in the solution C in step (3), condition is：Shear rate is 3000-
12000rpm, time 10-100 seconds.
Beneficial effect：The present invention mutually contains vesica using interior water, and oil phase contains lipid components, forms multivesicular liposome cell bag
Composite construction containing vesica.The preparation method mainly solves the problems, such as following two aspect：(1) fat-soluble medicine is solved to be difficult to make
Into the technical problem of multivesicular liposome, fat-soluble medicine first can be prepared into vesica, multivesicular liposome is then made again；(2) solve
Determine the common situation of burst drug release, it is ensured that medicine bioactivity in vivo and in vitro, extended the half-life period of medicine, reached slow release
Purpose.Described multivesicular liposome can not only ensure to load the envelop rate of medicine, and the biology that can also improve large biological molecule is living
Property, reduce its immunogenicity, reduce burst effect, it is ensured that medicine continues slow release.
Brief description of the drawings
Describe some specific embodiments of the invention in detail by way of example, and not by way of limitation with reference to the accompanying drawings hereinafter.
Identical reference denotes same or similar part or part in accompanying drawing.It should be appreciated by those skilled in the art that these
What accompanying drawing was not necessarily drawn to scale.In accompanying 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 light microscope 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 microphotograph for being loaded with Exenatide multivesicular liposome in comparative example of the present invention；
Fig. 5 is the In-vitro release curves for being loaded with Exenatide multivesicular liposome in comparative example of the present invention.
Specific embodiment
The invention provides for prepare can with lag characteristic h substance such as medicine, proteins and peptides or activity into
A kind of new formulation method divided, i.e., there is provided preparation method of the multivesicular liposome for being loaded with vesica.Step is as follows：
(1) preparation of three-phase solution
It is respectively solution A：Prepare and contain the interior of 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：Prepare the lipid phase containing lipid and organic solvent；
Wherein, the lipid includes dioleyl phosphatidyl choline, DPPG, cholesterol and three oleic acid
Glyceride；The percentage that the dioleyl phosphatidyl choline accounts for lipid gross mass is 20-60%, and the olein is accounted for
The percentage of lipid gross mass is 5-20%, and the percentage that DPPG accounts for lipid gross mass is 5-20%, institute
State cholesterol account for lipid gross mass percentage be 10-50%；
Solution C：Prepare the outer water phase containing glucose and lysine；
Wherein, the solution C concentration containing glucose quality is 5-15% (w/v), and mass concentration containing lysine is 10-
80mmol/L(w/v)；
(2) preparation of W/O colostrums
Merge, mix the solution A and the solution B, high shear agitation obtains W/O colostrums；
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：Shear rate 3000-15000r pm, time 2-
15min；
(3) preparation of W/O/W multivesicular liposomes
W/O colostrums are distributed in solution C, high shear agitation, shear rate is 3000-12000rpm, time 10-
100 seconds, form W/O/W type emulsions；To in solution C, rotary evaporation or nitrogen blow W/O/W type emulsion redisperses at 30-40 DEG C, removal
Organic solvent；W/O/W type emulsions are centrifuged, precipitation are taken plus physiological saline is disperseed again, obtained multivesicular liposome；
Wherein, the volume ratio of W/O colostrums and solution C is 1: 1-1: 10.
Small-molecule drug of the present invention, selected from antibiotics, chemotherapeutic agent treats CNV
Medicine such as dexamethasone.
The biopharmaceutical macromolecular drug is selected from therapeutic peptide or protein or monoclonal antibody or nucleic acid, and such as shellfish cuts down pearl list
It is anti-, Lucentis, VEGF Trap, siRNA and DNA.The compound multivesicular liposome being prepared into using the present invention, its lipid material
Material has preferable biocompatibility, and skin, muscle and local organization will not be caused to stimulate, and biopharmaceutical macromolecular drug is wrapped by bilayer
Wrap up in, it is ensured that activity is not lost, and each cell gradually ruptures release vesica, and vesica discharges medicine one by one again, to reach sustained release
Purpose.Its emphasis solves burst drug release situation, it is ensured that pharmaceutical activity, medicine is kept slow release in vivo.
The vesica is micella or nanoparticle.
The micella is made up of block copolymer, such as includes poly lactic-co-glycolic acid, hyaluronic acid in fragment, preferably
MPEG-PLGA micellas.
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 or more in chloroform, ether, methyl alcohol or acetone.
It is to be appreciated that in the present invention, the dioleyl phosphatidyl choline, it is to form multivesicular liposome main
Composition, only with the phosphatide containing bilayer, medicine can just be encapsulated in wherein.
In the present invention, the olein, it is the key factor to form multivesicular liposome, otherwise, be can only obtain
Conventional liposome；Olein is filled in the space between multivesicular liposome capsule and capsule, and each is small to play stable connection
The effect of room；The glyceryl ester of different chain length, influences the in-vitro release rate of medicine.
In the present invention, the DPPG, its effect is between the formed multivesicular liposome of enhancing
Stability, mainly due to neutral phospholipid because van der Waals interaction is also easy to produce agglomeration effect, the repulsive force between identical charges
The aggegation of particle can more preferably be prevented.Its concentration in lipid phase is 1-10mg/ml, and bear electricity phosphatide is better.By reality
Test contrast and find that the multivesicular liposome containing DPPG 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 seepage of medicine
Rate, consumption is excessive or the very few stability and envelop rate that can all influence multivesicular liposome, and the concentration of cholesterol is in 5- in lipid phase
20mg/ml, if experiment finds to be not added with cholesterol, obtained multivesicular liposome is just crushed quickly, does not reach slow release effect.
In the present invention, the osmotic pressure regulator in the interior water phase is sucrose, the osmotic pressure regulator in the outer water phase
It is glucose.Described interior water phase osmotic pressure regulator, the presence of outer water phase osmotic pressure regulator, is to ensure inside and outside water phase pressure
Difference within the specific limits, increases the stability of multivesicular liposome, reduces as far as possible and medicine leakage occurs.
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 is prepared：The interior water phase of 1 part of gelatin nanparticles for being loaded with Exenatide and osmotic pressure regulator, specially
The nanoparticle that will be prepared is scattered in the water phase dissolved with osmotic pressure regulator；
Prepare solution B：Contain 200 parts of lipid organic solvent；
Prepare solution C：Outer water phase containing osmotic pressure regulator and coemulsifier；
(2) merging, mixed solution A and solution B, 12000rpm high shear agitations obtain W/O colostrums；
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 DPPG is 0.01-1% (w/v) in solution B；
(3) W/O colostrums are distributed in solution C, shear rate is 5000rpm, 10 seconds time, forms W/O/W type emulsions；
Wherein, the volume ratio of W/O colostrums and solution C is 1:1-1:10 are carried out；
Solution C concentration containing glucose quality is 5-15% (w/v), and mass concentration containing lysine is 10-80mmol/L (w/
v)；
(4) by W/O/W type emulsion redisperses to solution C, rotary evaporation or nitrogen blow at 30-40 DEG C, remove organic solvent；
Wherein, W/O/W types emulsion and the volume ratio of solution C are 1:1-1∶100；
(5) W/O/W types emulsion is carried out into 2000rpm centrifugations, takes precipitation plus physiological saline disperses again, obtain many 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 of the multivesicular liposome for being loaded with Exenatide gelatin nanparticles,
Figure it is seen that the particle size of multivesicular liposome is homogeneous, 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 micellas of huperzine
(1) solution A is prepared：The interior water phase of 1 part of mPEG-PLGA micella for being loaded with huperzine and osmotic pressure regulator, tool
Body is that the nanoparticle that will be prepared is scattered in the water phase dissolved with osmotic pressure regulator；
Solution B：Contain 200 parts of lipid organic solvent；
Solution C：Outer water phase containing osmotic pressure regulator and coemulsifier；
(2) merging, mixed solution A and solution B, 12000rpm high shear agitations obtain W/O colostrums；
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 DPPG is 0.01-1% (w/v) in solution B；
(3) W/O colostrums are distributed in solution C, shear rate is 5000rpm, 10 seconds time, forms W/O/W type emulsions；
Wherein, the volume ratio of W/O colostrums and solution C is 1: 1-1: 10；
Solution C concentration containing glucose quality is 5-15% (w/v), and mass concentration containing lysine is 10-80mmol/L (w/
v)；
(4) by W/O/W type emulsion redisperses to solution C, rotary evaporation or nitrogen blow at 30-40 DEG C, remove organic solvent；
Wherein, W/O/W types emulsion and the volume ratio of solution C are 1: 1-1: 100；
(5) W/O/W types emulsion is carried out into 2000rpm centrifugations, takes precipitation plus physiological saline disperses again, obtain many capsule lipids
Body suspension.
Wherein described nanoparticle, micella can be replaced liposome.
Comparative example one
Exenatide multivesicular liposome
(1) solution A is prepared：Interior water phase containing 1 part of Exenatide and osmotic pressure regulator, specially by medicine dissolved with infiltration
In the water phase of pressure conditioning agent；
Solution B：Contain 10 parts of lipid organic solvent；
Solution C：Outer water phase containing osmotic pressure regulator and coemulsifier
(2) merging, mixed solution A and solution B, 12000rpm high shear agitations obtain W/O colostrums；
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 DPPG is 0.01-1% (w/v) in solution B；
(3) W/O colostrums are distributed in solution C, shear rate is 5000rpm, 10 seconds time, forms W/O/W type emulsions；
Wherein, the volume ratio of W/O colostrums and solution C is carried out for 1: 1-1: 10；
Solution C concentration containing glucose quality is 5-15% (w/v), and mass concentration containing lysine is 10-80mmol/L (w/
v)；
(4) by W/O/W type emulsion redisperses to solution C, rotary evaporation or nitrogen blow at 30-40 DEG C, remove organic solvent；
Wherein, W/O/W types emulsion and the volume ratio of solution C are 1: 1-1: 100；
(5) W/O/W types emulsion is carried out into 2000rpm centrifugations, takes precipitation plus physiological saline disperses again, obtain many capsule lipids
Body suspension.
Its microphotograph is shown in Fig. 4, and In-vitro release curves are shown in Fig. 5；Result shows, Exenatide multivesicular liposome it is spherical compared with
Rounding.
By embodiment one and the In-vitro release curves of comparative example one, the change of medicine Accumulation dissolution is slow in embodiment
Slowly, with obvious slow release effect, the sustainable release of medicine 15 days；In comparative example, insoluble drug release has significantly prominent releasing now
As, and insoluble drug release is rapider, sustainable 7 days or so.
Comparative example two
Bovine serum albumin(BSA) multivesicular liposome
(1) solution A is prepared：It is specially that medicine is molten containing 1 part of interior water phase of bovine serum albumin bletilla osmotic pressure regulator
In having a water phase of osmotic pressure regulator；
Solution B：Contain 10 parts of lipid organic solvent；
Solution C：Outer water phase containing osmotic pressure regulator and coemulsifier；
(2) merging, mixed solution A and solution B, 12000rpm high shear agitations obtain W/O colostrums；
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 DPPG is 0.01-1% (w/v) in solution B；
(3) W/O colostrums are distributed in solution C, shear rate is 5000rpm, 10 seconds time, forms W/O/W type emulsions；
Wherein, the volume ratio of W/O colostrums and solution C is carried out for 1: 1-1: 10；
Solution C concentration containing glucose quality is 5-15% (w/v), and mass concentration containing lysine is 10-80mmol/L (w/
v)；
(4) by W/O/W type emulsion redisperses to solution C, rotary evaporation or nitrogen blow at 30-40 DEG C, remove organic solvent；
Wherein, W/O/W types emulsion and the volume ratio of solution C are 1: 1-1: 100；
(5) W/O/W types emulsion is carried out into 2000rpm centrifugations, takes precipitation plus physiological saline disperses again, obtain many capsule lipids
Body suspension.
The described multivesicular liposome for being loaded with vesica be used for vitreum, oral, intramuscular injection, subcutaneous, intrathecal, abdominal cavity, Epidural cavity,
Various 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, and its therapeutic domain includes
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 can not limit this hair
Bright, claims indicate the scope of the present invention, and the scope of the present invention is not pointed out in above-mentioned explanation, therefore, with this
Any change in claims of invention suitable implication and scope, is all considered as being included in claim of the invention
In the range of book.
Claims (6)
1. a kind of preparation method of the multivesicular liposome for being loaded with vesica, it is characterised in that step is as follows：
(1) preparation of three-phase solution
It is respectively solution A：Prepare vesica and the interior water of sucrose containing embedding small-molecule drug and/or biopharmaceutical macromolecular drug
Phase；Wherein, the solution A mass concentration containing sucrose is 2-10%；
Solution B：Prepare the lipid phase containing lipid and organic solvent；Wherein, the lipid includes dioleyl phosphatidyl choline,
DPPG, 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：Prepare the outer water phase containing glucose and lysine；Wherein, the solution C concentration containing glucose quality is 5-
15%, mass concentration containing lysine is 10-80mmol/L；
(2) preparation of W/O colostrums
Merge, mix the solution A and the solution B, high shear agitation obtains W/O colostrums；Wherein, solution A and solution B
Volume ratio is 1: 0.5-1:20；
(3) preparation of W/O/W multivesicular liposomes
W/O colostrums are distributed in solution C, high shear agitation, form W/O/W type emulsions；W/O/W type emulsion redisperses are to molten
In liquid C, rotary evaporation or nitrogen blow at 30-40 DEG C, remove organic solvent；W/O/W type emulsions are centrifuged, precipitation plus physiology is taken
Salt solution disperses again, obtains multivesicular liposome；Wherein, the volume ratio of W/O colostrums and solution C is 1: 1-1: 10.
2. the preparation method of the multivesicular liposome of vesica is loaded with as claimed in claim 1, it is characterised in that the vesica is glue
Beam or nanoparticle.
3. the preparation method of the as claimed in claim 2 multivesicular liposome for being loaded with vesica, it is characterised in that the interior water is mutually
The interior water phase of gelatin nanparticles and sucrose containing embedding Exenatide.
4. the preparation method of the as claimed in claim 2 multivesicular liposome for being loaded with vesica, it is characterised in that the interior water is mutually
The interior water phase of mPEG-PLGA micellas 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, it is characterised in that in step (2)
The solution A and the solution B high shear agitation conditions are：Shear rate 3000-15000rpm, time 2-15min.
6. the preparation method of the multivesicular liposome of vesica is loaded with as claimed in claim 5, it is characterised in that will in step (3)
The colostrum is distributed in the solution C, and condition is：Shear rate is 3000-12000rpm, time 10-100 seconds.
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| CN110339166A (en) * | 2018-04-04 | 2019-10-18 | 沈阳药科大学 | A kind of Liraglutide multivesicular liposome and its preparation method and application |
| 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 |
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