CN103720657B - The preparation method of a kind of deformable liposome, and the transmutability liposome of preparation - Google Patents
The preparation method of a kind of deformable liposome, and the transmutability liposome of preparation Download PDFInfo
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Abstract
The present invention discloses the preparation method of a kind of deformable liposome, and the deformable liposome of preparation, and this preparation method is: the lipid material and film softening agent that form liposome are dissolved in chloroform/ethanol mixed solution by step 1.；Water-solubility carrier is soluble in water；If material to be encapsulated can be dissolved in water, material to be encapsulated is dissolved in the above-mentioned aqueous solution being dissolved with water-solubility carrier；If material to be encapsulated is water insoluble, then material to be encapsulated also is soluble in above-mentioned chloroform/ethanol mixed solution；Step 2. chloroform/ethanol mixed solution and aqueous solution obtain single phase soln；Step 3., by lyophilised for single phase soln process, removes solvent；Frozen dried product aquation is obtained deformable liposome by step 4..Its particle diameter of deformable liposome prepared by the inventive method can regulate, it is possible to only have the keratodermatitis intercellular substance of its diameter 1/5 ~ 4/5 times through aperture.Present invention process process is simple, easily realizes, and is very easy to amplify, and is suitable for industrialized production.
Description
Technical field
The present invention relates to biological medicine and cosmetic technical field, be specifically related to liposome technology field, particularly relate to one
Plant the preparation method of deformable liposome, and the transmutability liposome prepared by the method.
Background technology
Complete skin is one outstanding protecting wall of human body, for most drug, is one impassable screen
Barrier, wants to improve the percutaneous absorbtion situation of medicine, it is necessary to improve the skin transparent performance to medicine by other means.
Liposome is the Guan Bi capsule balloon-shaped structure that phospholipid bilayer is formed, and liposome has huge as the carrier of medicine
Value.
At present, the preparation method of liposome is broadly divided into four classes:
(1) based on film dispersion method, the phospholipid forming liposome will be used for usual in organic solvent
For chloroform or chloroform and the mixture of methanol, remove solvent the most at reduced pressure and form dry immobilized artificial membrane, by phospholipid
Film waterization can form multilamellar liposome.There is many shortcomings in the method: as used organic solvent that toxicity is the biggest, cannot apply
In industrialized production and when containing aqueous solution aquation, the multilamellar liposome of formation drug level between layers is uneven
One, it is necessary to by multigelation process etc..
(2) based on w/o breast or w/o/w emulsion.Although this kind of preparation method can realize large-scale production, but
Still suffering from some problems, such as removing into the toxicity solvent that breast is needs special technology；Due to the existence of organic solvent, parcel
Protein-based or nucleic acid drug can frequently result in medicine loss of activity.
(3) detergent dialysis methods, is difficult to industrialized production, is not suitable for encapsulating water soluble drug.
(4) based on injection method, such as alcohol injection, can be with large-scale production.If but using Passive loading
Method prepares liposome, and envelop rate ratio usual for water soluble drug is relatively low, and owing to often preparing (60 DEG C of left sides at relatively high temperatures
Right), it will usually cause the medicine of oxidizable, facile hydrolysis and changeableness to lose activity.
Using the normally liquid preparation of Liposomal formulation prepared by said method, its stability cannot solve, mainly show
In terms of following three:
(1) liposome belongs to the disperse system of thermodynamic instability, serious it is also possible that layering.Change due to particle diameter
Change, it is possible to cause the preparation for intravenously administrable to use.
(2) due to the characteristic of phospholipid itself, when phospholipid is present in aqueous phase, it is easy to the phenomenons such as hydrolysis, oxidation occur.Due to
Lysophosphatide can be formed after phospholipid hydrolysis, on the one hand increase the toxicity of preparation, on the other hand the most easily make liposome disintegrate, cause
The seepage of encapsulated medicine.
(3) liposome is such as suspended in aqueous phase, in storage process, medicine can slow seepage, cause entrapment efficiency
Change.If the envelop rate of medicine changes, must the curative effect of preparation to be affected.Certainly, if medicine itself is easy to water
Solve, then the stability of preparation is the most more problematic.
How sterilizing is also a big problem to Liposomal formulation.Due to the characteristic of phospholipid itself, the heating of final preparation is gone out
Bacterium is typically considered unacceptable.Thus requiring that whole production process must use sterile working, this is at some lipid
Preparation process cannot realize.The size controlling of liposome is also critically important, it is thus achieved that small particle liposome (is less than
200nm) it is always the target that scientific research personnel pursues.
SOD superoxide dismutase (Superoxide dismutase SOD) is specificity fast and efficiently in human body
Remove O2 —Enzyme, sufficient SOD can guarantee that body effectively eliminates O2 —To harm, thus play the work of enhancing human body immunity ability
With.Superoxide dismutase as cosmetics additive, play following some effect:
(1) reduce the ionizing radiation damages to skin such as ultraviolet, thus play anti-sunlight function；
(2) effect such as wrinkle resistant, defying age, speckle dispelling；
(3) antiinflammatory performance based on superoxide dismutase, treatment dermatosis will play a role；
(4) for treatment pigmentation, the effect such as acne is obvious.
Summary of the invention
Present invention aims to the deficiencies in the prior art, it is provided that a kind of variable, good stability, can wear
Cross the keratodermatitis intercellular substance of diameter 1/5 ~ 4/5 times, and the preparation method of preparation method simple deformable liposome.
It is a further object to provide a kind of deformable liposome prepared by above-mentioned preparation method.
The above-mentioned purpose of the present invention is achieved by following scheme:
The preparation method of a kind of deformable liposome, this preparation method comprises the steps:
The lipid material and film softening agent that form liposome are dissolved in chloroform/ethanol mixed solution by step 1.；
Water-solubility carrier is soluble in water；
If material to be encapsulated can be dissolved in water, material to be encapsulated is dissolved in the above-mentioned aqueous solution being dissolved with water-solubility carrier；
If material to be encapsulated is water insoluble, then material to be encapsulated also is soluble in above-mentioned chloroform/ethanol mixed solution；
Chloroform/ethanol mixed solution that step 1 is prepared by step 2. and aqueous solution prepared by step 1 obtain single-phase molten
Liquid；
The lyophilised process of single phase soln that step 2 is prepared by step 3., removes solvent；
The frozen dried product aquation that step 3 is prepared by step 4. obtains deformable liposome.
In above-mentioned steps 1, the lipid material of described formation liposome refers to lecithin or phosphatidylcholine.
In above-mentioned steps 1, film softening agent refers to deoxycholic acid.
In above-mentioned steps 1, water-solubility carrier refers to any one in sucrose, lactose or mannitol.
In above-mentioned steps 1, chloroform/ethanol mixed solution is made after referring to mix chloroform and ethanol according to the volume ratio of 2:3
The standby mixed solution obtained；Because the present inventor is found by research, a lot of materials to be encapsulated are all to be insoluble in water and can be dissolved in
The material of organic solvent, and a lot of organic solvent dissolubility in water is bad, single phase soln of application claims refers to clear
Bright uniform solution, therefore, after to the scheme optimization of a large amount of organic solvents and screening test, the inventors discovered that employing body
The chloroform/ethanol mixed solution that long-pending ratio is 2:3 is capable of the present invention, not only can realize most of materials to be encapsulated very well
Ground solute effect, and last and aqueous solution is under the conditions of certain ratio, can be prepared as single phase soln.
In above-mentioned steps 2, the chloroform/ethanol mixed solution of step 1 preparation and the mixed proportion of the aqueous solution of step 1 preparation
For 1:2, this mixed proportion is also that inventor obtains after assay optimization, it is ensured that the acquisition of single phase soln.
In above-mentioned steps 2, in single phase soln, the mass ratio of lipid material and water-solubility carrier is more than or equal to 1:2.5, then may be used
Yield less than the liposome equal to 200nm.
In above-mentioned steps 3, remove the preferred frozen dried of method of solvent, namely lyophilization；Frozen dried concrete
Operation can be to be freezed at liquid nitrogen or cryogenic system by single phase soln, then lyophilizing in freeze dryer, it is also possible to is directly to freeze
Lyophilizing in dry machine.
In above-mentioned steps 4, aquation lyophilized products, to form liposome, can use distilled water or suitably buffer suitably
At a temperature of complete, for acceleration of hydration, it is possible to complete under the effect of mechanical force, as being stirred or vibration etc..
In above-mentioned preparation method, lipid material and the mass ratio of water-solubility carrier
The present invention also provides for a kind of deformable liposome, and this deformable liposome is to be prepared by above-mentioned preparation method
's.
The material to be encapsulated of the present invention can be medicine, it is also possible to be other materials.
Its effect of the water-solubility carrier of the present invention has following two aspects:
(1) addition of water-solubility carrier, is greatly accelerated lyophilized products aquation and forms the speed of liposome；
(2) addition of water-solubility carrier, it is also possible to adjust the particle diameter of whole deformable liposome；According to deformable liposome
Need the skin gap length passed through, the amount of water-solubility carrier can be adjusted, thus change the particle diameter of deformable liposome.
Compared with prior art, the present invention has a following beneficial aspects:
1. the present invention addition by water-solubility carrier, not only can greatly speed up lyophilized products aquation and form liposome
Speed, and the particle diameter of transmutability liposome can be changed by adjusting the consumption of water-solubility carrier, so that the present invention
Deformable liposome can only have the keratodermatitis intercellular substance of its diameter 1/5 ~ 4/5 times through aperture；
2. the present invention is by the selection to organic solvent, not only ensure that material to be encapsulated, film softening agent and lipid material
Can effectively dissolve, and finally also ensure that the acquisition of single phase soln；
3. the present invention uses frozen dried method to remove solvent, the most simple to operate, and the removal effect of solvent is good；
4. present invention process process is simple, easily realizes, and is very easy to amplify, and is suitable for industrialized production.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is further described through, but specific embodiment is not any to the present invention
Limit.
The impact on lyophilized products characteristic of embodiment 1 water-solubility carrier
The present embodiment is divided into experimental group and contrast groups, wherein:
The preparation method of experimental group is as follows:
Lecithin and deoxycholic acid are dissolved in chloroform/ethanol mixed solution (chlorine in this chloroform/ethanol mixed solution by step 1.
Imitative and ethanol volume ratio is 2:3) in；
Mannitol is soluble in water；
Chloroform/ethanol mixed solution and the step 1 of what step 1 was prepared by step 2. be dissolved with lecithin and deoxycholic acid are made
Standby Osmitrol is mixed to get single phase soln according to the volume ratio of 1:2；
The lyophilised process of single phase soln that step 2 is prepared by step 3., removes solvent.
The preparation method of contrast groups is as follows:
Lecithin and deoxycholic acid are dissolved in ethanol/chloroform mixed solution (chlorine in this chloroform/ethanol mixed solution by step 1.
Imitative and ethanol volume ratio is 2:3) in；
What step 1 was prepared by step 2. is dissolved with the chloroform/ethanol mixed solution of lecithin and deoxycholic acid according to 1:2's
Volume ratio and water are mixed to get single phase soln；
The lyophilised process of single phase soln that step 2 is prepared by step 3., removes solvent.
Making discovery from observation, compared with contrast groups, the lyophilized products of experimental group can be with long term storage and be difficult to the moisture absorption；Additionally
Contrast groups and experimental group are all carried out aquation, it was found that experimental group is substantially fast than the hydration rate of contrast groups.
Permissible by above-mentioned experimental result, really the form of lyophilized products and character are had the adding of water-solubility carrier one fixing
Ring, and the speed of lyophilized products aquation formation liposome can be greatly speeded up.
The impact on liposomal particle size of embodiment 2 water-solubility carrier
The present embodiment is divided into three experimental grouies, specific as follows shown:
The preparation method of experimental group A comprises the steps:
Lecithin and deoxycholic acid are dissolved in ethanol/chloroform mixed solution (chlorine in this chloroform/ethanol mixed solution by step 1.
Imitative and ethanol volume ratio is 2:3) in；
Mannitol is soluble in water；
Chloroform/ethanol mixed solution and the step 1 of what step 1 was prepared by step 2. be dissolved with lecithin and deoxycholic acid are made
Standby Osmitrol is mixed to get single phase soln according to the volume ratio of 1:2；Lecithin and the matter of mannitol in single phase soln
Amount ratio is 1:1；
The lyophilised process of single phase soln that step 2 is prepared by step 3., removes solvent.
The frozen dried product aquation that step 3 is prepared by step 4. obtains the deformable lipid that lecithin mass concentration is 1%
Body.
The preparation method of experimental group B comprises the steps:
Lecithin and deoxycholic acid are dissolved in ethanol/chloroform mixed solution (chlorine in this chloroform/ethanol mixed solution by step 1.
Imitative and ethanol volume ratio is 2:3) in；
Mannitol is soluble in water；
Chloroform/ethanol mixed solution that step 1 is prepared by step 2. and Osmitrol prepared by step 1 are according to 1:2
Volume ratio be mixed to get single phase soln；In single phase soln, lecithin is 1:2.5 with the mass ratio of mannitol；
The lyophilised process of single phase soln that step 2 is prepared by step 3., removes solvent.
The frozen dried product aquation that step 3 is prepared by step 4. obtains the deformable lipid that lecithin mass concentration is 1%
Body.
The preparation method of experimental group C comprises the steps:
Lecithin and deoxycholic acid are dissolved in ethanol/chloroform mixed solution (chlorine in this chloroform/ethanol mixed solution by step 1.
Imitative and ethanol volume ratio is 2:3) in；
Mannitol is soluble in water；
Chloroform/ethanol mixed solution that step 1 is prepared by step 2. and Osmitrol prepared by step 1 are according to 1:2
Volume ratio be mixed to get single phase soln；In single phase soln, lecithin is 1:5 with the mass ratio of mannitol；
The lyophilised process of single phase soln that step 2 is prepared by step 3., removes solvent.
The frozen dried product aquation that step 3 is prepared by step 4. obtains the deformable lipid that lecithin mass concentration is 1%
Body.
With ZETASIZER (malvern company) determination experiment group A, the deformable liposome of experimental group B and experimental group C
Particle diameter, the particle diameter of liposome is respectively 316.2nm, 200.7nm, 125.8nm (intensity mean).This explanation is added water-soluble
Property carrier mass mannitol can change liposome and prepare the particle diameter of liquid.
Embodiment 3
With the deformable liposome obtained in transmission electron microscope observation embodiment 2.Negative staining is used to observe, i.e.
By carrier with 1%, the phosphotungstic acid dyeing of pH=7.0, to observe under an electron microscope, observed result finds: embodiment 2 preparation
Carrier very rounding, regular shape is orderly, uniform particle diameter, and dispersibility is preferable, does not has an adhesion, size with use particle instrument
The result measured matches.
Embodiment 4
The present embodiment uses glycoside material as material to be encapsulated, and the process of preparation deformable liposome is as follows:
Lecithin and deoxycholic acid are dissolved in ethanol/chloroform mixed solution (chlorine in this chloroform/ethanol mixed solution by step 1.
Imitative and ethanol volume ratio is 2:3) in；
By soluble in water to glycoside material and sucrose；
Chloroform/ethanol mixed solution that step 1 is prepared by step 2. and aqueous solution prepared by step 1 are according to the volume of 1:2
Ratio is mixed to get single phase soln；
The lyophilised process of single phase soln that step 2 is prepared by step 3., removes solvent.
The frozen dried product aquation that step 3 is prepared by step 4. obtains deformable liposome.
Above-mentioned deformable liposome is detected, result be medicine be 2mg/ml, phospholipid is 3%, and the concentration of sucrose is
6%.The liposome solutions obtained is optics clear liquid, and particle diameter, at about 200nm, with post separation liposome and free medicine, measures
Entrapment efficiency is 50%.
Embodiment 5 SOD liposome transdermal is tested
The present embodiment utilizes skin of rat to measure common SOD liposome and SOD transporter through keratodermatitis
Ability, is specifically divided into contrast groups and experimental group, wherein:
Contrast groups is SOD conventional liposome, and SOD concentration is 2mg/ml, and film regulator is cholesterol；
Experimental group is to be material to be encapsulated with SOD, uses the SOD deformable fat that in embodiment 2 prepared by the method for experimental group A
Plastid, SOD concentration is 2mg/ml.
Contrast groups and experimental group are dripped respectively on isolated rat depilation skin of abdomen, 37 degree of calorstats are placed 12
Hour, measure and below skin, receive the activity of SOD in liquid.
Result of the test shows that SOD deformable liposome can be measured to SOD enzyme activity receiving in liquid, and common SOD
The reception liquid of liposome group has no SOD enzyme activity.
Result of the test shows that SOD deformable lipid physical ability is through rat skin.
Claims (1)
1. a SOD deformable liposome, it is characterised in that described SOD deformable liposome is by following preparation method system
For obtaining, described preparation method comprises the following steps:
Lecithin and deoxycholic acid are dissolved in the chloroform/ethanol mixed solution that volume ratio is 2:3 of chloroform and ethanol by step 1.
In；By soluble in water to mannitol and SOD, obtain aqueous solution；
Prepared by chloroform/ethanol mixed solution and the step 1 of what step 1 was prepared by step 2. be dissolved with lecithin and deoxycholic acid
The aqueous solution being dissolved with mannitol and SOD, be mixed to get single phase soln according to the volume ratio of 1:2；In single phase soln, lecithin
It is 1:1 with the mass ratio of mannitol；
The lyophilised process of single phase soln that step 2 is prepared by step 3., removes solvent；
The frozen dried product aquation that step 3 is prepared by step 4. obtains the SOD deformable lipid that lecithin mass concentration is 1%
Body.
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Citations (3)
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|---|---|---|---|---|
| CN1369263A (en) * | 2002-02-26 | 2002-09-18 | 刘建平 | Flexible vitaminc A acid liposome and its product |
| CN1969830A (en) * | 2006-12-01 | 2007-05-30 | 哈尔滨医科大学 | Itraconazole liposome and preparation process thereof |
| CN101822669A (en) * | 2010-04-23 | 2010-09-08 | 海南美兰史克制药有限公司 | Liposome injection of amoxicillin sodium sulbactam sodium medicinal composition |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100427064C (en) * | 2003-04-16 | 2008-10-22 | 沈阳药科大学 | New method for preparing liposome |
| RU2648842C2 (en) * | 2011-01-04 | 2018-03-28 | Арчивель Фарма, С.Л. | Liposome formulation suitable for treating or preventing tuberculosis |
| RU2602771C2 (en) * | 2012-01-12 | 2016-11-20 | Арчивель Фарма, С.Л. | Mtb-c vaccine against asthma |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1369263A (en) * | 2002-02-26 | 2002-09-18 | 刘建平 | Flexible vitaminc A acid liposome and its product |
| CN1969830A (en) * | 2006-12-01 | 2007-05-30 | 哈尔滨医科大学 | Itraconazole liposome and preparation process thereof |
| CN101822669A (en) * | 2010-04-23 | 2010-09-08 | 海南美兰史克制药有限公司 | Liposome injection of amoxicillin sodium sulbactam sodium medicinal composition |
Non-Patent Citations (2)
| Title |
|---|
| A Novel Method for the Preparation of Liposomes:Freeze Drying of Monophase Solutions;CHUNLEI LI et al.;《JOURNAL OF PHARMACEUTICAL SCIENCES》;20040630;第93卷(第6期);第1403-1414页，尤其第1404页右栏第4-7段，第1407页右栏第2-3段，第1411页左栏第2段 * |
| 双氯芬酸钠柔性脂质体的研究;陈鹰等;《中国药学杂志》;20020731;第37卷(第7期);第513-516页，尤其第513-514页第2.1.1节 * |
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