CN117298051A - Preparation method of ATP liposome - Google Patents
Preparation method of ATP liposome Download PDFInfo
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- CN117298051A CN117298051A CN202311396836.3A CN202311396836A CN117298051A CN 117298051 A CN117298051 A CN 117298051A CN 202311396836 A CN202311396836 A CN 202311396836A CN 117298051 A CN117298051 A CN 117298051A
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- 239000002502 liposome Substances 0.000 title claims abstract description 112
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- JQWAHKMIYCERGA-UHFFFAOYSA-N (2-nonanoyloxy-3-octadeca-9,12-dienoyloxypropoxy)-[2-(trimethylazaniumyl)ethyl]phosphinate Chemical compound CCCCCCCCC(=O)OC(COP([O-])(=O)CC[N+](C)(C)C)COC(=O)CCCCCCCC=CCC=CCCCCC JQWAHKMIYCERGA-UHFFFAOYSA-N 0.000 claims abstract description 30
- 238000001704 evaporation Methods 0.000 claims abstract description 19
- 150000002632 lipids Chemical class 0.000 claims description 39
- 239000000243 solution Substances 0.000 claims description 34
- 239000007853 buffer solution Substances 0.000 claims description 25
- 239000000725 suspension Substances 0.000 claims description 23
- 238000009210 therapy by ultrasound Methods 0.000 claims description 23
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 claims description 22
- 238000005303 weighing Methods 0.000 claims description 20
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 14
- 238000000034 method Methods 0.000 claims description 14
- 238000003756 stirring Methods 0.000 claims description 13
- 238000004090 dissolution Methods 0.000 claims description 12
- 230000008020 evaporation Effects 0.000 claims description 12
- 239000011259 mixed solution Substances 0.000 claims description 12
- 235000012000 cholesterol Nutrition 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 210000004027 cell Anatomy 0.000 claims description 7
- 238000000265 homogenisation Methods 0.000 claims description 7
- 238000003760 magnetic stirring Methods 0.000 claims description 7
- 235000019441 ethanol Nutrition 0.000 claims description 2
- 230000002441 reversible effect Effects 0.000 abstract description 6
- 235000013305 food Nutrition 0.000 abstract description 3
- 239000003814 drug Substances 0.000 description 27
- 229940079593 drug Drugs 0.000 description 20
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical group CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 description 9
- 239000002245 particle Substances 0.000 description 9
- 238000005259 measurement Methods 0.000 description 8
- 239000000787 lecithin Substances 0.000 description 7
- 229940067606 lecithin Drugs 0.000 description 7
- 235000010445 lecithin Nutrition 0.000 description 7
- 239000012528 membrane Substances 0.000 description 7
- 230000014759 maintenance of location Effects 0.000 description 6
- 239000000872 buffer Substances 0.000 description 5
- 238000005538 encapsulation Methods 0.000 description 5
- 238000003860 storage Methods 0.000 description 5
- 230000000694 effects Effects 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- 238000003917 TEM image Methods 0.000 description 2
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- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 238000002329 infrared spectrum Methods 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000007704 transition Effects 0.000 description 2
- 238000000108 ultra-filtration Methods 0.000 description 2
- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 description 1
- 239000000232 Lipid Bilayer Substances 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000006664 bond formation reaction Methods 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000013329 compounding Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
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- 238000000354 decomposition reaction Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000032798 delamination Effects 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 210000002969 egg yolk Anatomy 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 239000006260 foam Substances 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000009878 intermolecular interaction Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000004080 punching Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 150000004671 saturated fatty acids Chemical class 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000002459 sustained effect Effects 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000012932 thermodynamic analysis Methods 0.000 description 1
- 238000004627 transmission electron microscopy Methods 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
- 150000004670 unsaturated fatty acids Chemical class 0.000 description 1
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 1
Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
- A61K9/1277—Processes for preparing; Proliposomes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
- A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
- A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
- A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- Medicinal Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Oil, Petroleum & Natural Gas (AREA)
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- Manufacturing Of Micro-Capsules (AREA)
Abstract
The invention belongs to the technical field of food industry biology, and relates to a preparation method of ATP liposome. The invention utilizes egg yolk lecithin to prepare the ATP-loaded liposome by a reverse evaporation method, so that the liposome with higher ATP loading capacity can be obtained, and simultaneously, the ATP stability can be better improved.
Description
Technical Field
The invention belongs to the technical field of food industry biology, and particularly relates to a preparation method of ATP liposome.
Background
Egg yolk lecithin liposome is taken as a drug carrier, has bright targeting property and is an important preparation in the latest fourth-generation drug delivery system. The fatty acid composition and the type of the egg yolk lecithin structure have great influence on liposome properties, for example, saturated fatty acid in the lecithin structure can enhance the firmness and impermeability of a liposome membrane, and unsaturated fatty acid in the lecithin structure can enable the liposome to have lower phase transition, good fluidity and low viscosity. However, the high oxidative susceptibility of egg yolk lecithin limits its use.
Reverse evaporation is the most preferred technique for loading hydrophilic drugs into liposomes. For hydrophilic drugs, the inner water core is the only region that can be loaded with the drug. Thus, a large number of aqueous cores can be entrapped during liposome formation, resulting in high entrapment efficiency and thus high drug loading. In the reverse evaporation method, a W/O emulsion is prepared by dissolving a hydrophilic drug in water and a phospholipid in a water-immiscible solvent. The organic solvent was then slowly removed under vacuum and a gel phase formed. Further evaporation of the organic solvent produces a liposome dispersion and highly entraps the aqueous core in the inner core of the liposome. At present, effective research is carried out on a method for preparing liposome by adopting a reverse evaporation method, however, the application of the liposome in the aspect of encapsulating ATP is little in favor, and meanwhile, the application of most of the liposome is limited to the medical field, and the liposome has good application in food engineering.
Disclosure of Invention
The invention provides a preparation method of ATP liposome. The invention utilizes egg yolk lecithin to prepare the ATP-loaded liposome by a reverse evaporation method, so that the liposome with higher ATP loading capacity can be obtained, and simultaneously, the ATP stability can be better improved.
The invention is realized by adopting the following technical scheme:
a method for preparing ATP liposome, comprising the following steps:
s1, weighing a certain amount of egg yolk lecithin, adding the egg yolk lecithin into absolute ethyl alcohol, stirring for proper dissolution, weighing a certain amount of cholesterol at the same time, weighing a certain amount of VE solution, adding the VE solution into the ethanol, and carrying out ultrasonic treatment to fully dissolve the VE solution;
s2, weighing a certain amount of ATP in a 5ml centrifuge tube, adding PBS buffer solution to enable the ATP to be completely dissolved, wherein the pH value of the PBS buffer solution is 6.5, adding the PBS buffer solution into the mixed solution in the step (1), and carrying out ultrasonic treatment for 3min;
s3, pouring the liposome solution obtained in the step S2 into a round-bottomed flask, and evaporating under reduced pressure until a uniform dry lipid film is formed on the surface of the flask;
s4, adding PBS buffer solution into the round-bottomed flask, performing simple ultrasonic treatment on the round-bottomed flask to ensure that a lipid film on the wall of the flask completely falls off into the buffer solution, and performing magnetic stirring to obtain liposome suspension;
and S5, homogenizing the liposome suspension obtained in the step S4 under high pressure by an ultrahigh pressure cell disruption instrument for three times to obtain ATP liposome, and refrigerating at 4 ℃ for later use.
In the step S1 of the scheme, the yolk lecithin is easy to oxidize, so that the weighing process is rapid, and absolute ethyl alcohol is added after the weighing, and then cholesterol is weighed, so that excessive oxidation loss is avoided as much as possible; and the preservative film is used for simple sealing treatment during ultrasonic treatment, so that the splashing of the solution is reduced.
In the step S4 of the scheme, before magnetic stirring, a round-bottom flask is subjected to simple ultrasonic treatment, so that the lipid membrane on the wall of the flask is ensured to completely fall off; sealing with fresh-keeping film to reduce film evaporation and splashing, and punching 2-4 holes with fine needle to remove small amount of steam; the heating temperature must be kept below 35 ℃.
On the basis of the scheme, further, in the step S1, the mass ratio of the egg yolk lecithin to the cholesterol is 4:1, the concentration of the VE solution is 4g/L, and the adding amount is 240mg of egg yolk lecithin corresponding to each 1ml of VE solution.
On the basis of the scheme, the mass ratio of ATP to egg yolk lecithin in the step S2 is 1:5.
On the basis of the above scheme, further, the condition of the reduced pressure evaporation in the step S3 is that the reduced pressure evaporation is performed at a temperature of 35 ℃ for 30 min. The ATP has poor thermal stability, and the lower temperature can reduce the decomposition of ATP in the preparation process and reduce the loss.
On the basis of the scheme, further, the magnetic stirring condition in the step S4 is that the magnetic stirring is carried out for 50 min in a water bath at 35 ℃, the rotating speed is controlled to be 300-500r/min, embedding loss caused by too high rotating speed is prevented, and meanwhile, the generation of foam is reduced.
On the basis of the above scheme, further, the high-pressure homogenizing pressure in the step S5 is set to 258Pa, and the temperature is 23 ℃.
In this scheme, if the homogenization pressure is too high, the liposome structure may be damaged, resulting in outflow of ATP entrapped in the liposome, which reduces the entrapment rate.
The beneficial technical effects of the invention are as follows:
(1) The invention utilizes egg yolk lecithin to prepare the ATP-loaded liposome by a reverse evaporation method, so that the liposome with higher ATP loading capacity can be obtained, and simultaneously, the ATP stability can be better improved. Starting from the existing liposome preparation materials, the invention changes the compounding ratio between the drug and lecithin, and the ATP-loaded liposome prepared by controlling the liposome preparation conditions has good performances in aspects of appearance, encapsulation efficiency and stability.
(2) The invention determines the optimal encapsulation amount of ATP by changing the medicine lipid ratio (ATP: egg yolk lecithin) so as to better encapsulate the ATP and reduce the embedding loss of the ATP. By embedding ATP, the effect of increasing ATP stability is achieved.
Drawings
FIG. 1 is an image of a liposome suspension of different drug to lipid ratios;
FIG. 2 is an image of the average particle size of a liposome suspension of different ratios;
FIG. 3 is a graph of vesicle size distribution for different drug to lipid ratio liposome suspensions;
FIG. 4 is a PDI image of a liposome suspension of different drug to lipid ratios;
FIG. 5 is a Zeta potential image of a liposome suspension of different drug to lipid ratios;
FIG. 6 is an image of encapsulation and loading rates for different drug-to-lipid ratio liposome suspensions;
FIG. 7 is an image of a liposome suspension of different drug to lipid ratios after 12 days of rest;
FIG. 8 is an image of the average particle size of a liposome suspension of different drug to lipid ratios after 12 days of rest;
FIG. 9 is a graph showing vesicle size distribution of different drug to lipid ratio liposome suspensions after 12 days of rest;
FIG. 10 is a PDI image of a liposome suspension of different drug to lipid ratios after 12 days of rest;
FIG. 11 is a Zeta potential image of a liposome suspension of different drug to lipid ratios after 12 days of rest;
FIG. 12 shows ATP retention of liposome suspensions of different drug-lipid ratios after 12 days of rest;
FIG. 13 is a TEM micrograph of blank liposomes at 500 nm;
fig. 14 is a drug to lipid ratio 1: TEM micrograph of liposome at 500 nm;
fig. 15 is a blank liposome and drug to lipid ratio 1:5 thermogravimetric comparison of liposomes;
fig. 16 is a blank liposome and drug to lipid ratio 1:5, comparing images of the fourier infrared spectra of the liposomes;
Detailed Description
The present invention will be further illustrated with reference to the following examples, but the present invention is not limited to the following examples.
Example 1
A process for preparing ATP liposome, comprising the steps of:
(1) Preparing 0.05mol/L PBS buffer solution with pH of 6.5 for dissolving ATP and lipid film, and preparing 4g/L VE solution;
(2) Weighing 120mg of egg yolk lecithin, adding 20ml of absolute ethyl alcohol, stirring for proper dissolution, simultaneously weighing 30mg of cholesterol and 0.5mLVE solution, and performing ultrasonic treatment to fully dissolve;
(3) 40mg of ATP is weighed into a 5mL centrifuge tube, 1.5mL of PBS buffer is added for complete dissolution, and the ratio of the medicine to the lipid is ATP: the mass ratio of the egg yolk lecithin is 1:3;
(4) Adding the mixed solution obtained in the step (3) into the mixed solution obtained in the step (1), and carrying out ultrasonic treatment for 3min to ensure that ATP is uniformly distributed in the lecithin solution;
(5) Pouring the liposome solution obtained in the step (4) into a round-bottomed flask, and performing reduced pressure evaporation for 30 min at 35 ℃ until a uniform dry lipid film is formed on the surface of the flask;
(6) Adding 20mLPBS buffer solution into a round-bottom flask, performing simple ultrasonic treatment on the round-bottom flask to ensure that lipid membranes on the wall of the flask completely fall off into the buffer solution, magnetically stirring for 50 min in a water bath at 35 ℃ and keeping the rotating speed between 300 r/min and 500r/min to obtain liposome suspension;
(7) Injecting the ATP liposome into an ultrahigh pressure cell disruption instrument for high-pressure homogenization, wherein the pressure is set to 258Pa, and the temperature is 23 ℃;
(8) The prepared ATP liposome is marked and put into a refrigerator at 4 ℃ for standby.
Example 2
(1) Preparing 0.05mol/L PBS buffer solution with pH of 6.5 for dissolving ATP and lipid film, and preparing 4g/L VE solution;
(2) Weighing 120mg of egg yolk lecithin, adding 20ml of absolute ethyl alcohol, stirring for proper dissolution, simultaneously weighing 30mg of cholesterol and 0.5mLVE solution, and performing ultrasonic treatment to fully dissolve;
(3) 30mg of ATP is weighed into a 5mL centrifuge tube, 1.5mL of PBS buffer is added for complete dissolution, and the ratio of the drug to the lipid is ATP: the mass ratio of the egg yolk lecithin is 1:4;
(4) Adding the mixed solution obtained in the step (3) into the mixed solution obtained in the step (1), and carrying out ultrasonic treatment for 3min to ensure that ATP is uniformly distributed in the lecithin solution;
(5) Pouring the liposome solution obtained in the step (4) into a round-bottomed flask, and performing reduced pressure evaporation for 30 min at 35 ℃ until a uniform dry lipid film is formed on the surface of the flask;
(6) Adding 20mLPBS buffer solution into a round-bottom flask, performing simple ultrasonic treatment on the round-bottom flask to ensure that lipid membranes on the wall of the flask completely fall off into the buffer solution, magnetically stirring for 50 min in a water bath at 35 ℃ and keeping the rotating speed between 300 r/min and 500r/min to obtain liposome suspension;
(7) Injecting the ATP liposome into an ultrahigh pressure cell disruption instrument for high-pressure homogenization, wherein the pressure is set to 258Pa, and the temperature is 23 ℃;
(8) The prepared ATP liposome is marked and put into a refrigerator at 4 ℃ for standby.
Example 3
(1) Preparing 0.05mol/L PBS buffer solution with pH of 6.5 for dissolving ATP and lipid film, and preparing 4g/L VE solution;
(2) Weighing 120mg of egg yolk lecithin, adding 20ml of absolute ethyl alcohol, stirring for proper dissolution, simultaneously weighing 30mg of cholesterol and 0.5mLVE solution, and performing ultrasonic treatment to fully dissolve;
(3) 24mg of ATP is weighed into a 5mL centrifuge tube, 1.5mL of PBS buffer is added for complete dissolution, and the ratio of the medicine to the lipid is ATP: the mass ratio of the egg yolk lecithin is 1:5;
(4) Adding the mixed solution obtained in the step (3) into the mixed solution obtained in the step (1), and carrying out ultrasonic treatment for 3min to ensure that ATP is uniformly distributed in the lecithin solution;
(5) Pouring the liposome solution obtained in the step (4) into a round-bottomed flask, and performing reduced pressure evaporation for 30 min at 35 ℃ until a uniform dry lipid film is formed on the surface of the flask;
(6) Adding 20mLPBS buffer solution into a round-bottom flask, performing simple ultrasonic treatment on the round-bottom flask to ensure that lipid membranes on the wall of the flask completely fall off into the buffer solution, magnetically stirring for 50 min in a water bath at 35 ℃ and keeping the rotating speed between 300 r/min and 500r/min to obtain liposome suspension;
(7) Injecting the ATP liposome into an ultrahigh pressure cell disruption instrument for high-pressure homogenization, wherein the pressure is set to 258Pa, and the temperature is 23 ℃;
(8) The prepared ATP liposome is marked and put into a refrigerator at 4 ℃ for standby.
Example 4
(1) Preparing 0.05mol/L PBS buffer solution with pH of 6.5 for dissolving ATP and lipid film, and preparing 4g/L VE solution;
(2) Weighing 120mg of egg yolk lecithin, adding 20ml of absolute ethyl alcohol, stirring for proper dissolution, simultaneously weighing 30mg of cholesterol and 0.5mLVE solution, and performing ultrasonic treatment to fully dissolve;
(3) 12mg of ATP is weighed into a 5mL centrifuge tube, 1.5mL of PBS buffer is added for complete dissolution, and the ratio of the medicine to the lipid is ATP: the mass ratio of the egg yolk lecithin is 1:10;
(4) Adding the mixed solution obtained in the step (3) into the mixed solution obtained in the step (1), and carrying out ultrasonic treatment for 3min to ensure that ATP is uniformly distributed in the lecithin solution;
(5) Pouring the liposome solution obtained in the step (4) into a round-bottomed flask, and performing reduced pressure evaporation for 30 min at 35 ℃ until a uniform dry lipid film is formed on the surface of the flask;
(6) Adding 20mLPBS buffer solution into a round-bottom flask, performing simple ultrasonic treatment on the round-bottom flask to ensure that lipid membranes on the wall of the flask completely fall off into the buffer solution, magnetically stirring for 50 min in a water bath at 35 ℃ and keeping the rotating speed between 300 r/min and 500r/min to obtain liposome suspension;
(7) Injecting the ATP liposome into an ultrahigh pressure cell disruption instrument for high-pressure homogenization, wherein the pressure is set to 258Pa, and the temperature is 23 ℃;
(8) The prepared ATP liposome is marked and put into a refrigerator at 4 ℃ for standby.
Example 5
(1) Preparing 0.05mol/L PBS buffer solution with pH of 6.5 for dissolving ATP and lipid film, and preparing 4g/L VE solution;
(2) Weighing 120mg of egg yolk lecithin, adding 20ml of absolute ethyl alcohol, stirring for proper dissolution, simultaneously weighing 30mg of cholesterol and 0.5mLVE solution, and performing ultrasonic treatment to fully dissolve;
(3) 6mg of ATP is weighed into a 5mL centrifuge tube, 1.5mL of PBS buffer is added for complete dissolution, and the ratio of the medicine to the lipid is ATP: the mass ratio of the egg yolk lecithin is 1:20;
(4) Adding the mixed solution obtained in the step (3) into the mixed solution obtained in the step (1), and carrying out ultrasonic treatment for 3min to ensure that ATP is uniformly distributed in the lecithin solution;
(5) Pouring the liposome solution obtained in the step (4) into a round-bottomed flask, and performing reduced pressure evaporation for 30 min at 35 ℃ until a uniform dry lipid film is formed on the surface of the flask;
(6) Adding 20mLPBS buffer solution into a round-bottom flask, performing simple ultrasonic treatment on the round-bottom flask to ensure that lipid membranes on the wall of the flask completely fall off into the buffer solution, magnetically stirring for 50 min in a water bath at 35 ℃ and keeping the rotating speed between 300 r/min and 500r/min to obtain liposome suspension;
(7) Injecting the ATP liposome into an ultrahigh pressure cell disruption instrument for high-pressure homogenization, wherein the pressure is set to 258Pa, and the temperature is 23 ℃;
(8) The prepared ATP liposome is marked and put into a refrigerator at 4 ℃ for standby.
Experimental results:
(1) The ATP liposome suspensions obtained in examples 1-5 were subjected to particle size and potential measurements using a Zeta potential and particle size analyzer (NanoZs 90, UK), three measurements were made for each sample, and the results were averaged.
According to the method, the measurement results of the ATP liposome with different drug-lipid ratios are shown in figures 1-5, and the ATP liposome with five different drug-lipid ratios all have lower particle sizes, wherein the drug-lipid ratio is 1:5 shows the smallest particle size and highest potential.
(2) The encapsulation efficiency and the loading rate of the liposomes prepared in examples 1 to 5 were measured by liquid chromatography. Precisely measuring ATP liposome 0.2 mL, adding 1% Triton x-100 mL, stirring under vortex, clarifying, adjusting volume to 4 mL with mobile phase, and measuring total ATP concentration C by HPLC 0 . Precisely measuring ATP liposome 5mL, adding into ultrafiltration centrifuge tube, centrifuging at 8000 r min-1 for 20 min, precisely measuring solution 0.2 mL in outer tube of ultrafiltration tube, determining free ATP concentration C by HPLC method with mobile phase constant volume of 4 mL 1 . The following formulas were used to calculate EE% and LC% for liposomes:
EE%=(C 0 -C 1 /C 0 )×100%； LC%=(C 0 -C 1 mass of egg yolk lecithin x 100%
The results of EE% and LC% measurements of ATP liposomes for different ratios are shown in FIG. 6, following the procedure described above. The results show that: LC% increases linearly with the addition of ATP, indicating that ATP is efficiently embedded in the lipid bilayer over the load range (5% -45%). Under the condition that the encapsulation efficiency has no obvious difference, the addition amount of the active substance is positively correlated with the embedding amount. Thus, the ratio of the medicine to the fat is 1: 4. 1:5 has the maximum ATP-embedding amount.
(3) The particle size and the potential change measurement method after the liposome suspension was allowed to stand for 12 days were the same as those shown in step (1).
The particle size and potential measurement results of ATP liposomes with different ratios are shown in FIGS. 7-11; after standing for 12 days, the medicine-fat ratio is 1: 3. 1:4 shows a clear sign of delamination. Meanwhile, the particle sizes of ATP liposome with all the medicine-lipid ratios are slightly increased, the PDI variation range is smaller than 0.1, and all the ATP liposome shows good vesicle size stability in a 12-day storage period. The absolute value of the Zeta potential of the liposome is slightly increased due to the fusion and aggregation of part of liposome vesicles, and the ATP-buried liposome has good stability.
(4) The ATP retention rate of the liposomes after 12 days of storage was measured by liquid chromatography, and the measurement formula was as follows: retention rate (%) = Retained amount of ATP/Initial amount of ATP ×100%
The results of the measurement of the retention rate of the liposomes of different drug to ATP according to the method described above are shown in FIG. 12, and the retention rate of ATP in the liposomes is maintained at 50% -65% level after 12 days of storage, which means that the ATP encapsulated in the liposomes is lost to some extent during the storage. The medicine-fat ratio is 1:5 with the highest retention (over 60%) during storage.
(5) Ratio of medicine to fat 1:5, further research and exploration was performed on the liposomes. The ratio of blank liposomes to drug-to-lipid was observed by transmission electron microscopy to be 1:5, wherein the acceleration voltage is set to 120kv. Morphological photographs of the different active packages were recorded by a camera. The microscopic morphology of the ATP liposomes is shown in FIGS. 13-14. The control lipids showed a morphology similar to that of the ATP-encapsulating liposomes, which were uniformly distributed in the image and showed spherical or spheroidic vesicles, with full and clear appearance and no aggregation.
(6) The liposome samples were subjected to thermodynamic analysis using a comprehensive thermal analyzer (U.S., SDT 650). The samples were freeze-dried prior to measurement. Heating 3g of the sample from 20 ℃ to 400 ℃ at a heating rate of 10 ℃/min; the samples were measured using a Fourier transform infrared spectrometer (Nicolet 5700, U.S.A.) in the range of 4000-400 cm-1. Thermogravimetric results are shown in fig. 15, indicating a sustained slow degradation behavior of egg yolk lecithin and cholesterol over the entire temperature range. In addition, the addition of ATP had no significant effect on the thermal stability of the liposomes. The IR spectrum results are shown in FIG. 16, which shows that the liposomes with ATP embedded therein are 1000-1200cm compared with the blank liposomes -1 Where PO 2- The symmetrical and asymmetrical absorption peaks of (2) are obviously red-shifted from 1166cm -1 Up to 1170cm -1 From 1065cm -1 To 1076cm -1 This indicates that ATP is present in the polar region of phosphatidylcholine within the vesicle. This transition not only suggests that intermolecular interactions between ATP and liposomes promote hydrogen bond formation, but is also believed to support evidence that no other characteristic peaks are found in ATP liposomes that support the presence of ATP inside the vesicles, suggesting that liposomes do not have the formation of new chemical bonds upon addition of ATP, further confirming that ATP is bound by physical rather than chemical interactions with egg yolk lecithin compounds.
Claims (6)
1. A method for preparing ATP liposome, which is characterized by comprising the following steps: the preparation method comprises the following preparation steps:
s1, weighing a certain amount of egg yolk lecithin, adding the egg yolk lecithin into absolute ethyl alcohol, stirring for proper dissolution, weighing a certain amount of cholesterol at the same time, weighing a certain amount of VE solution, adding the VE solution into the ethanol, and carrying out ultrasonic treatment to fully dissolve the VE solution;
s2, weighing a certain amount of ATP in a 5ml centrifuge tube, adding PBS buffer solution to enable the ATP to be completely dissolved, wherein the pH value of the PBS buffer solution is 6.5, adding the PBS buffer solution into the mixed solution in the step (1), and carrying out ultrasonic treatment for 3min;
s3, pouring the liposome solution obtained in the step S2 into a round-bottomed flask, and evaporating under reduced pressure until a uniform dry lipid film is formed on the surface of the flask;
s4, adding PBS buffer solution into the round-bottomed flask, performing simple ultrasonic treatment on the round-bottomed flask to ensure that a lipid film on the wall of the flask completely falls off into the buffer solution, and performing magnetic stirring to obtain liposome suspension;
and S5, homogenizing the liposome suspension obtained in the step S4 under high pressure by an ultrahigh pressure cell disruption instrument for three times to obtain ATP liposome, and refrigerating at 4 ℃ for later use.
2. The method for preparing ATP liposome according to claim 1, wherein: in the step S1, the mass ratio of the egg yolk lecithin to the cholesterol is 4:1, the concentration of the VE solution is 4g/L, and the adding amount is 1ml of VE solution corresponding to 240mg of egg yolk lecithin.
3. The method for preparing ATP liposome according to claim 1, wherein: in the step S2, the mass ratio of ATP to egg yolk lecithin is 1:5.
4. The method for preparing ATP liposome according to claim 1, wherein: the conditions for the reduced pressure evaporation in the step S3 are that the reduced pressure evaporation is performed at a temperature of 35 ℃ for 30 min.
5. The method for preparing ATP liposome according to claim 1, wherein: the magnetic stirring condition in the step S4 is that the magnetic stirring is carried out for 50 min in a water bath at 35 ℃, and the rotating speed is controlled to be 300-500r/min.
6. The method for preparing ATP liposome according to claim 1, wherein: the high-pressure homogenization in step S5 was set at 258Pa and at 23 ℃.
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