CN112138144B - Liposome containing active biological factor and preparation method thereof - Google Patents
Liposome containing active biological factor and preparation method thereof Download PDFInfo
- Publication number
- CN112138144B CN112138144B CN201910571937.7A CN201910571937A CN112138144B CN 112138144 B CN112138144 B CN 112138144B CN 201910571937 A CN201910571937 A CN 201910571937A CN 112138144 B CN112138144 B CN 112138144B
- Authority
- CN
- China
- Prior art keywords
- liposome
- fgf
- active biological
- cholesterol
- biological factor
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 239000002502 liposome Substances 0.000 title claims abstract description 71
- 239000003181 biological factor Substances 0.000 title claims abstract description 33
- 238000002360 preparation method Methods 0.000 title abstract description 10
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 claims abstract description 50
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 claims abstract description 50
- 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 abstract description 42
- 235000012000 cholesterol Nutrition 0.000 claims abstract description 21
- MWRBNPKJOOWZPW-CLFAGFIQSA-N dioleoyl phosphatidylethanolamine Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC(COP(O)(=O)OCCN)OC(=O)CCCCCCC\C=C/CCCCCCCC MWRBNPKJOOWZPW-CLFAGFIQSA-N 0.000 claims abstract description 16
- 238000000034 method Methods 0.000 claims abstract description 14
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound 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 claims abstract description 11
- 229940067606 lecithin Drugs 0.000 claims abstract description 11
- 239000000787 lecithin Substances 0.000 claims abstract description 11
- 235000010445 lecithin Nutrition 0.000 claims abstract description 11
- JZNWSCPGTDBMEW-UHFFFAOYSA-N Glycerophosphorylethanolamin Natural products NCCOP(O)(=O)OCC(O)CO JZNWSCPGTDBMEW-UHFFFAOYSA-N 0.000 claims abstract description 7
- 150000008104 phosphatidylethanolamines Chemical class 0.000 claims abstract description 7
- 239000002994 raw material Substances 0.000 claims abstract description 7
- 239000006185 dispersion Substances 0.000 claims abstract description 4
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 30
- JLPULHDHAOZNQI-ZTIMHPMXSA-N 1-hexadecanoyl-2-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCC\C=C/C\C=C/CCCCC JLPULHDHAOZNQI-ZTIMHPMXSA-N 0.000 claims description 15
- 229940083466 soybean lecithin Drugs 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 229930006000 Sucrose Natural products 0.000 claims description 5
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 5
- 239000000839 emulsion Substances 0.000 claims description 5
- 230000000887 hydrating effect Effects 0.000 claims description 5
- 150000002632 lipids Chemical class 0.000 claims description 5
- 239000012982 microporous membrane Substances 0.000 claims description 5
- 238000002390 rotary evaporation Methods 0.000 claims description 5
- 238000007873 sieving Methods 0.000 claims description 5
- 239000005720 sucrose Substances 0.000 claims description 5
- 238000005303 weighing Methods 0.000 claims description 5
- 244000061458 Solanum melongena Species 0.000 claims 1
- 235000002597 Solanum melongena Nutrition 0.000 claims 1
- 239000010408 film Substances 0.000 claims 1
- 239000010409 thin film Substances 0.000 claims 1
- 238000005538 encapsulation Methods 0.000 abstract description 15
- 230000004071 biological effect Effects 0.000 abstract description 10
- 102000018233 Fibroblast Growth Factor Human genes 0.000 description 9
- 108050007372 Fibroblast Growth Factor Proteins 0.000 description 9
- 239000003814 drug Substances 0.000 description 9
- 229940079593 drug Drugs 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- 238000003860 storage Methods 0.000 description 7
- 238000005516 engineering process Methods 0.000 description 5
- 229940126864 fibroblast growth factor Drugs 0.000 description 5
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 229960002897 heparin Drugs 0.000 description 4
- 229920000669 heparin Polymers 0.000 description 4
- 238000001727 in vivo Methods 0.000 description 4
- 230000007774 longterm Effects 0.000 description 4
- 230000008439 repair process Effects 0.000 description 4
- 230000009286 beneficial effect Effects 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000011550 stock solution Substances 0.000 description 3
- 108090000695 Cytokines Proteins 0.000 description 2
- 102000004127 Cytokines Human genes 0.000 description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 102000003971 Fibroblast Growth Factor 1 Human genes 0.000 description 2
- 108090000386 Fibroblast Growth Factor 1 Proteins 0.000 description 2
- 238000003917 TEM image Methods 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 230000006978 adaptation Effects 0.000 description 2
- 239000012876 carrier material Substances 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 239000000017 hydrogel Substances 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000000693 micelle Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000035699 permeability Effects 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 230000001817 pituitary effect Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 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 description 1
- KILNVBDSWZSGLL-KXQOOQHDSA-N 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCCCCCCCCC KILNVBDSWZSGLL-KXQOOQHDSA-N 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 102000009024 Epidermal Growth Factor Human genes 0.000 description 1
- 101800003838 Epidermal growth factor Proteins 0.000 description 1
- 102000003973 Fibroblast growth factor 21 Human genes 0.000 description 1
- 108090000376 Fibroblast growth factor 21 Proteins 0.000 description 1
- 101100281008 Homo sapiens FGF2 gene Proteins 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 108010025020 Nerve Growth Factor Proteins 0.000 description 1
- 102000015336 Nerve Growth Factor Human genes 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 208000026137 Soft tissue injury Diseases 0.000 description 1
- ATBOMIWRCZXYSZ-XZBBILGWSA-N [1-[2,3-dihydroxypropoxy(hydroxy)phosphoryl]oxy-3-hexadecanoyloxypropan-2-yl] (9e,12e)-octadeca-9,12-dienoate Chemical compound CCCCCCCCCCCCCCCC(=O)OCC(COP(O)(=O)OCC(O)CO)OC(=O)CCCCCCC\C=C\C\C=C\CCCCC ATBOMIWRCZXYSZ-XZBBILGWSA-N 0.000 description 1
- 210000004100 adrenal gland Anatomy 0.000 description 1
- AWUCVROLDVIAJX-UHFFFAOYSA-N alpha-glycerophosphate Natural products OCC(O)COP(O)(O)=O AWUCVROLDVIAJX-UHFFFAOYSA-N 0.000 description 1
- 230000033115 angiogenesis Effects 0.000 description 1
- 239000003012 bilayer membrane Substances 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 210000005013 brain tissue Anatomy 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 210000004246 corpus luteum Anatomy 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 229940116977 epidermal growth factor Drugs 0.000 description 1
- 238000002270 exclusion chromatography Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 102000003684 fibroblast growth factor 13 Human genes 0.000 description 1
- 108090000047 fibroblast growth factor 13 Proteins 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 210000002216 heart Anatomy 0.000 description 1
- 229940099578 hydrogenated soybean lecithin Drugs 0.000 description 1
- 210000003016 hypothalamus Anatomy 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 239000004005 microsphere Substances 0.000 description 1
- 230000011278 mitosis Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000002077 nanosphere Substances 0.000 description 1
- 229940053128 nerve growth factor Drugs 0.000 description 1
- 210000000944 nerve tissue Anatomy 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 210000001525 retina Anatomy 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000001541 thymus gland Anatomy 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 208000037816 tissue injury Diseases 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000014621 translational initiation Effects 0.000 description 1
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 description 1
- 210000003556 vascular endothelial cell Anatomy 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/1825—Fibroblast growth factor [FGF]
-
- 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
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Epidemiology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Dispersion Chemistry (AREA)
- Medicinal Preparation (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention aims to provide a liposome containing active biological factors and a preparation method thereof, wherein the raw materials of the liposome are lecithin, cholesterol, phosphatidylethanolamine and the active biological factors, the active biological factors are preferably FGF-2, and the phosphatidylethanolamine is preferably dioleoylphosphatidylethanolamine; the liposome is prepared by a film dispersion method, has the advantages of high encapsulation efficiency, good stability and good biological activity, is suitable to be used as a carrier of active biological factors, and has great application and popularization values.
Description
Technical Field
The invention belongs to the field of active biological factors, and particularly relates to a liposome containing active biological factors and a preparation method thereof.
Background
Fibroblast growth factor is an important cytokine which has various physiological functions and plays an important role in life activities. The fibroblast growth factor family is currently known to comprise 23 members, with 30-80% homology between family members. Research on fibroblast growth factors has been focused on FGF-1 (a-FGF, acidic fibroblast growth factor), FGF-2 (b-FGF, basic fibroblast growth factor), FGF-13, FGF-21, etc.
FGF-2, namely basic fibroblast growth factor, is originally separated from bovine pituitary and brain tissue extracts and can promote division and proliferation of 2T3 cells, and subsequent researches show that FGF-2 is widely distributed in vivo, can be separated from pituitary, brain, hypothalamus, retina, adrenal gland, thymus, corpus luteum, kidney, heart, liver and placenta, and can also be expressed in various tumor tissues. The human FGF-2 gene is a single copy gene, is positioned on the short arm of chromosome 4, its total length is greater than 40kb, and its 5 'end and 3' end have non-coding sequence which can be used for regulating gene transcription, and its mRNA length is about 6802bp, and because of the difference of translation initiation sites it can produce a low Mr FGF-2 and 4 high Mr FGF-2.
FGF-2 is a multifunctional cytokine, such as it can promote mitosis of cells, induce angiogenesis, promote repair of damaged vascular endothelial cells, promote repair of soft tissue injury, promote repair of cartilage and bone tissue injury, promote repair and regeneration of nerve tissue, etc. In conclusion, the fibroblast growth factor, especially the basic fibroblast growth factor, has great clinical value and can be used for treating or assisting in treating various clinical diseases.
The half-life of exogenous active biomolecules such as fibroblast growth factors in vivo is short, for example, the half-life of FGF-2 in vivo is only 3-10min, and the effect of systemic or local administration cannot meet the clinical application requirement.
The existing fibroblast growth factor administration technology mainly focuses on the following aspects: heparin-regulated FGF drug release technology, namely heparin is combined to different carrier materials by a covalent grafting method to form a new material of a heparin-polymer compound, and then the FGF is entrapped or free heparin is directly mixed into the carrier materials in a non-covalent form, so that the release process of the drug FGF is influenced; FGF microspheres and nanospheres; an FGF liposome; FGF micelles; FGF hydrogels, and the like. Compared with liposome, heparin-controlled FGF drug release technology, FGF micelle, FGF hydrogel and other drug delivery technologies, the preparation steps are multiple, the technology is complex, the cost is high and the like, and the liposome is simple in preparation steps, low in cost and wide in clinical popularization prospect.
The liposome is prepared from phospholipid, cholesterol and other amphiphilic substances, a bilayer membrane forms one or more layers of hollow ultramicro spherical carrier dosage forms, the inside of the liposome is a closed vesicle with a water phase, and the liposome serving as a drug carrier can delay the release of drugs in vivo. However, the existing active biological factors, such as fibroblast growth factors, especially FGF-2 liposomes have the defects of low encapsulation efficiency, poor stability and the like, and the demand of further improvement exists.
Disclosure of Invention
In order to overcome the defects of low encapsulation efficiency and stability of the liposome of the active biological factor and easy reduction of biological activity in the prior art, the invention provides the liposome containing the active biological factor and the preparation method thereof.
Specifically, the invention provides a liposome containing an active biological factor, raw materials for preparing the liposome are lecithin, cholesterol, phosphatidylethanolamine and the active biological factor, and preferably the phosphatidylethanolamine is dioleoylphosphatidylethanolamine.
Further, the lecithin is one or more of soybean lecithin, hydrogenated soybean lecithin, egg yolk lecithin and dipalmitoyl phosphatidylcholine, and is preferably soybean lecithin.
Further, the active biological factor is fibroblast growth factor, nerve growth factor, epidermal growth factor, insulin, preferably fibroblast growth factor, more preferably FGF-2.
Furthermore, the ratio of lecithin, cholesterol and dioleoyl phosphatidylethanolamine in the liposome is (by weight ratio) 10-20; the ratio is preferably 20.
Furthermore, the ratio of the lecithin, the cholesterol, the dioleoyl phosphatidylethanolamine and the active biological factor in the liposome is 10-20: 0.1 (weight ratio).
The invention also provides a preparation method of the liposome, and particularly relates to a method for preparing the liposome by a film dispersion method.
Further weighing required amount of lecithin, cholesterol, dioleoylphosphatidylethanolamine and active biological factors, putting into a eggplant-shaped bottle, and adding chloroform to completely dissolve; the chloroform in the solution is pumped out by a rotary evaporator through rotary evaporation, so that a layer of uniform lipid film is formed at the bottom and the inner wall of the eggplant-shaped bottle; place in a vacuum desiccator overnight to thoroughly remove the chloroform; adding 10% sucrose water solution, hydrating at room temperature for 30min to form semitransparent emulsion, extruding at room temperature, sieving with 0.22 μm microporous membrane for 5-10 times, and grading to obtain active biological factor liposome.
Furthermore, in the raw materials, the ratio of lecithin, cholesterol and dioleoyl phosphatidylethanolamine is (by weight ratio) 10-20; the ratio is preferably 20.
Furthermore, in the raw materials, the ratio of lecithin, cholesterol, dioleoyl phosphatidylethanolamine and active biological factors is (10-20): 0.1 (weight ratio).
Most further, the dosage of each raw material is as follows: 20mg of soybean lecithin, 5mg of cholesterol, 1.5mg of dioleoyl phosphatidylethanolamine and 100 mu g of active biological factor.
Advantageous effects
The liposome has high encapsulation efficiency and good stability, can well keep the activity of the active biological factor, and is suitable for long-term storage of the active biological factor; and the preparation method is used for identification, so that the industrial production and application of the active biological factor are facilitated.
Drawings
FIG. 1: transmission electron micrograph of FGF-2 liposome of the invention.
Detailed Description
The present invention is described in more detail below to facilitate an understanding of the present invention.
It should be understood that the terms or words used in the specification and claims should not be construed as having meanings defined in dictionaries, but should be interpreted as having meanings that are consistent with their meanings in the context of the present invention on the basis of the following principles: the concept of terms may be defined appropriately by the inventor for the best explanation of the invention.
The experimental procedures, for which specific conditions are not noted in the following examples, are generally carried out according to the routine experimental procedures in the art or according to the conditions recommended by the manufacturers.
Example 1: preparation of FGF-2 liposome by film dispersion method
The invention surprisingly discovers that the addition of phosphatidylethanolamine, particularly dioleoylphosphatidylethanolamine, into the liposome prepared from soybean lecithin and cholesterol can greatly improve the entrapment rate of the liposome to active biological factors and reduce the leakage rate of the liposome, and the stability of the liposome is also obviously improved.
Furthermore, preliminary experiments show that the liposome prepared by mixing soybean lecithin, cholesterol and dioleoyl phosphatidylethanolamine in a ratio of 10-20 (weight ratio) to 3-6 (weight ratio) has good shape, good stability, good encapsulation efficiency and other parameters, and is suitable for being used as a carrier of active biological factors.
Therefore, in this example, the liposome is prepared according to the above formulation, and the specific process parameters are as follows:
weighing 20mg of soybean lecithin, 5mg of cholesterol, 1.5mg of dioleoyl phosphatidylethanolamine and 100 mu g of FGF-2, putting into a solanaceous bottle, and adding chloroform to completely dissolve the materials; extracting chloroform from the solution by rotary evaporation by using a rotary evaporator, thereby forming a uniform lipid film on the bottom and the inner wall of the eggplant-shaped bottle; place in a vacuum desiccator overnight to thoroughly remove the chloroform; adding 10% sucrose water solution, hydrating at room temperature for 30min to form semitransparent emulsion, extruding at room temperature, sieving with 0.22 μm microporous membrane for 5-10 times, and grading to obtain FGF-2 liposome.
The transmission electron micrograph of the obtained liposome is shown in fig. 1.
The liposome prepared by the method is relatively complete and spheroidal, mainly has a single-chamber structure, has an average particle size of about 100.0nm, is mainly distributed between 65.45 and 136.55nm, has a zeta potential of-45.49 mV, namely an absolute value of the zeta potential is between 30 and 60mV, and is relatively stable.
Comparative example 1:
FGF-2 liposomes were prepared according to the following procedure: weighing 20mg of soybean lecithin, 5mg of cholesterol and 100 mu g of FGF-2, putting the soybean lecithin, the 5mg of cholesterol and the 100 mu g of FGF-2 into an eggplant-shaped bottle, and adding chloroform to completely dissolve the soybean lecithin, the cholesterol and the FGF-2; extracting chloroform from the solution by rotary evaporation by using a rotary evaporator, thereby forming a uniform lipid film on the bottom and the inner wall of the eggplant-shaped bottle; place in a vacuum desiccator overnight to thoroughly remove the chloroform; adding 10% sucrose water solution, hydrating at room temperature for 30min to form semitransparent emulsion, extruding at room temperature, sieving with 0.22 μm microporous membrane for 5-10 times, and grading to obtain FGF-2 liposome.
Comparative example 2:
FGF-2 liposomes were prepared according to the following steps: weighing 20mg of soybean lecithin, 5mg of cholesterol, 2mg of phosphatidyl glycerol and 100 mu g of FGF-2, putting the mixture into an eggplant-shaped bottle, and adding chloroform to completely dissolve the mixture; the chloroform in the solution is pumped out by a rotary evaporator through rotary evaporation, so that a layer of uniform lipid film is formed at the bottom and the inner wall of the eggplant-shaped bottle; the vacuum desiccator was placed overnight to thoroughly remove the chloroform; adding 10% sucrose water solution, hydrating at room temperature for 30min to form semitransparent emulsion, extruding at room temperature, sieving with 0.22 μm microporous membrane for 5-10 times, and grading to obtain FGF-2 liposome.
Test example:
1. determination of encapsulation efficiency
Placing 2mL of FGF-2 liposome in a 10mL quantitative bottle, diluting to the constant volume with dimethyl sulfoxide, diluting by 10 times with 95% ethanol, shaking up, quantifying by using HPLC, wherein the encapsulation efficiency calculation formula is as follows: encapsulation ratio (%) = (amount of FGF-2 encapsulated in liposome/total amount of FGF-2) × 100%.
2. Determination of the stability
Measuring a physical stability parameter KE by adopting a centrifugal spectrophotometry, sucking 1mL of FGF-2 liposome sample, placing the sample in a centrifuge tube for 10min at 3500rpm/min, centrifuging the sample, taking 0.3mL of liposome before centrifugation and supernatant after centrifugation, respectively adding phosphate buffer (pH 6.8) to dilute the sample to 5.0mL, measuring the absorbance of the sample at the wavelength of 500nm by taking the phosphate buffer as a blank, respectively measuring the absorbance values A0 and A before and after liposome centrifugation, calculating a stability parameter KE value, wherein the KE value is (A0-A)/A0 x 100%, and the smaller the KE value, the more stable the liposome is.
3. Determination of the leakage Rate
Separating FGF-2 liposome from free FGF-2 by Sephadex G-50 molecular exclusion chromatography, storing the suspension of the drug-loaded liposome at 4 deg.C, 25 deg.C and 37 deg.C respectively, and measuring the drug leakage rate by HPLC method, wherein the leakage rate (%) = (the amount of drug in liposome before placement-the amount of drug in liposome after placement)/the amount of drug in liposome is multiplied by 100%.
4. Determination of biological Activity
The biological activity of FGF-2 liposome is determined by adopting an MTT method, FGF-2 liposome or FGF-2 stock solution is diluted to 0.1mg/L by DMEM culture solution containing 0.5% serum, 100 microliter of the diluted FGF-2 stock solution is precisely absorbed and added into the 1 st hole, 4 times of dilution is carried out in sequence, 3 multiple holes are arranged at each dilution degree, the initial dose of the FGF-2 stock solution (standard substance) is 100U, blank liposome matrix control is arranged, the A value is determined at the position of 570nm by taking 630nm as a reference wavelength on an enzyme labeling instrument, and the biological activity of the FGF-2 liposome is calculated.
The results were as follows:
first, the encapsulation efficiency of liposomes at 4 ℃ storage conditions was specifically measured, and three samples were measured, and the change with time of the encapsulation efficiency of FGF-2 liposomes prepared in example 1 of the present invention and comparative examples 1-2 is shown in Table 1.
TABLE 1 variation of FGF-2 liposome encapsulation efficiency preserved at 4 ℃
As can be seen from the encapsulation efficiency results in Table 1, the FGF-2 liposome prepared in example 1 of the present invention achieves better encapsulation efficiency, and the encapsulation efficiency can be maintained at a higher level when stored at a low temperature of 4 ℃ for a long time, which is beneficial to the preservation of FGF-2.
Next, the stability of liposomes at 4 ℃ storage conditions was specifically measured, and three samples were measured, and the stability of FGF-2 liposomes prepared in example 1 of the present invention and comparative examples 1-2 as a function of time is shown in Table 2.
TABLE 2 variation of FGF-2 liposome stability when stored at 4 ℃
As can be seen from the stability results in table 2, the FGF-2 liposome prepared in example 1 of the present invention has better stability under low-temperature storage, and can be maintained at a higher level for a long period of time even when stored at low temperature for a long period of time, which is beneficial to the long-term storage of FGF-2.
The leakage rate and biological activity were measured in triplicate for a period of time at 4 ℃ and the results are shown in table 3:
table 3: changes in the leakage and biological Activity of FGF-2 liposomes when stored at 4 deg.C
As can be seen from the results of the changes in the permeability and biological activity of the liposomes shown in Table 3, the FGF-2 liposomes prepared in example 1 of the present invention have a lower permeability and a better biological activity under the condition of long-term low-temperature storage, which is advantageous for the long-term storage of FGF-2.
In summary, it is clear from the experimental data given in the above experimental examples that the liposome prepared from lecithin, cholesterol, dioleoylphosphatidylethanolamine and the active biological factor can obtain the best encapsulation efficiency and stability, and can maintain the biological activity of the active biological factor, thereby being beneficial to the industrial application of the active biological factor.
The above description is only of the preferred embodiments of the present invention, and it should be noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the invention, and such modifications and adaptations are intended to be within the scope of the invention.
Claims (3)
1. A liposome comprising an active biological factor, wherein: raw materials for preparing the liposome comprise lecithin, cholesterol, phosphatidylethanolamine and active biological factors; the lecithin is soybean lecithin, and the phosphatidylethanolamine is dioleoyl phosphatidylethanolamine; the weight ratio of the soybean lecithin, the cholesterol, the dioleoyl phosphatidylethanolamine and the active biological factor in the liposome is 20.
2. A method for preparing the liposome of claim 1, wherein: preparing liposome by thin film dispersion method, weighing desired amount of soybean lecithin, cholesterol, dioleoylphosphatidylethanolamine, and active biological factor, placing into a bottle shaped like a eggplant, and adding chloroform to dissolve completely; the chloroform in the solution is pumped out by a rotary evaporator through rotary evaporation, so that a layer of uniform lipid film is formed at the bottom and the inner wall of the eggplant-shaped bottle; the vacuum desiccator was placed overnight to thoroughly remove the chloroform; adding 10% sucrose water solution, hydrating at room temperature for 30min to form semitransparent emulsion, extruding at room temperature, sieving with 0.22 μm microporous membrane for 5-10 times, and grading to obtain active biological factor liposome; the active biological factor is FGF-2.
3. The method of claim 2, wherein: wherein the dosage of each raw material is as follows: 20mg of soybean lecithin, 5mg of cholesterol, 1.5mg of dioleoyl phosphatidylethanolamine and 100. Mu.g of FGF-2.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910571937.7A CN112138144B (en) | 2019-06-28 | 2019-06-28 | Liposome containing active biological factor and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910571937.7A CN112138144B (en) | 2019-06-28 | 2019-06-28 | Liposome containing active biological factor and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112138144A CN112138144A (en) | 2020-12-29 |
| CN112138144B true CN112138144B (en) | 2023-02-24 |
Family
ID=73869053
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910571937.7A Active CN112138144B (en) | 2019-06-28 | 2019-06-28 | Liposome containing active biological factor and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112138144B (en) |
Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1160582A (en) * | 1996-12-27 | 1997-10-01 | 暨南大学生物工程研究所 | External-use composite containing cell growth factor |
| EP1663152A2 (en) * | 2003-09-05 | 2006-06-07 | Boehringer Ingelheim Pharma GmbH & Co.KG | Process for the production of homogeneous liposomes and lipoplexes |
| KR20070002232A (en) * | 2005-06-30 | 2007-01-05 | 김진석 | Immunoliposomal Sensitive to WH |
| CN101199837A (en) * | 2007-11-30 | 2008-06-18 | 何荫良 | Humanized cell factor hair agent and preparing method thereof |
| CN101234089A (en) * | 2008-03-06 | 2008-08-06 | 四川大学 | Basic fibroblast growth factor liposome vaccine and its preparation method and application |
| CN102512370A (en) * | 2011-12-28 | 2012-06-27 | 广州赛莱拉生物科技有限公司 | HSCGF liposome as well as preparation and application thereof |
| CN102626388A (en) * | 2012-04-19 | 2012-08-08 | 海南永田药物研究院有限公司 | Liposome solid preparation of ozagrel |
| CN103520007A (en) * | 2013-10-15 | 2014-01-22 | 天博医药技术（苏州）有限公司 | Skin active factor flexible nano-liposome and preparation method and application thereof |
| CN105543161A (en) * | 2014-10-31 | 2016-05-04 | 陕西艾美雅生物科技有限公司 | Composite bioactive factor liposome and preparation method thereof |
| CN106265518A (en) * | 2016-08-31 | 2017-01-04 | 河南佰柯生物科技有限公司 | Fgf liposome and preparation method thereof |
| CN106309368A (en) * | 2015-06-24 | 2017-01-11 | 陕西艾美雅生物科技有限公司 | Composite bioactive factor liposome and preparation method thereof |
-
2019
- 2019-06-28 CN CN201910571937.7A patent/CN112138144B/en active Active
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1160582A (en) * | 1996-12-27 | 1997-10-01 | 暨南大学生物工程研究所 | External-use composite containing cell growth factor |
| EP1663152A2 (en) * | 2003-09-05 | 2006-06-07 | Boehringer Ingelheim Pharma GmbH & Co.KG | Process for the production of homogeneous liposomes and lipoplexes |
| KR20070002232A (en) * | 2005-06-30 | 2007-01-05 | 김진석 | Immunoliposomal Sensitive to WH |
| CN101199837A (en) * | 2007-11-30 | 2008-06-18 | 何荫良 | Humanized cell factor hair agent and preparing method thereof |
| CN101234089A (en) * | 2008-03-06 | 2008-08-06 | 四川大学 | Basic fibroblast growth factor liposome vaccine and its preparation method and application |
| CN102512370A (en) * | 2011-12-28 | 2012-06-27 | 广州赛莱拉生物科技有限公司 | HSCGF liposome as well as preparation and application thereof |
| CN102626388A (en) * | 2012-04-19 | 2012-08-08 | 海南永田药物研究院有限公司 | Liposome solid preparation of ozagrel |
| CN103520007A (en) * | 2013-10-15 | 2014-01-22 | 天博医药技术（苏州）有限公司 | Skin active factor flexible nano-liposome and preparation method and application thereof |
| CN105543161A (en) * | 2014-10-31 | 2016-05-04 | 陕西艾美雅生物科技有限公司 | Composite bioactive factor liposome and preparation method thereof |
| CN106309368A (en) * | 2015-06-24 | 2017-01-11 | 陕西艾美雅生物科技有限公司 | Composite bioactive factor liposome and preparation method thereof |
| CN106265518A (en) * | 2016-08-31 | 2017-01-04 | 河南佰柯生物科技有限公司 | Fgf liposome and preparation method thereof |
Non-Patent Citations (5)
| Title |
|---|
| 两种脂质体制备方法对碱性成纤维细胞生长因子活性的影响;陈妍等;《中国生物制品学杂志》;20071231(第1期);第60-61页 * |
| 受脂质体作为药物载体的研究进展;陈建明等;《药学实践杂志》;20011231;第19卷(第4期);第214-218页 * |
| 生长因子脂质体的研究现状;王伟等;《中国新药杂志》;20070521;第16卷(第7期);第516-518页 * |
| 碱性成纤维细胞生长因子脂质体的制备和评价;黄亚东等;《中国药学杂志》;20080731;第43卷(第13期);第992-997页 * |
| 酶联免疫法测定人碱性成纤维细胞生长因子脂质体的包封率;黄亚东等;《分析化学》;20080623;第36卷(第4期);第521-524页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112138144A (en) | 2020-12-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20070166368A1 (en) | Protein-Stabilized Liposomal Formulations of Pharmaceutical Agents | |
| EP0914161A2 (en) | Chitosan - related compositions and methods for delivery of nucleic acids into a cell | |
| Li et al. | Drug depot-anchoring hydrogel: A self-assembling scaffold for localized drug release and enhanced stem cell differentiation | |
| Wang et al. | Pharmacokinetic and pharmacodynamic study of a phospholipid-based phase separation gel for once a month administration of octreotide | |
| CN114469889A (en) | Bionic drug-loaded nanoparticle for inflammation region after specific targeting pulsed electric field ablation and preparation method thereof | |
| Zhang et al. | Replacing cholesterol with asiatic acid to prolong circulation and enhance anti-metastatic effects of non-PEGylated liposomes | |
| CN104069120A (en) | Double-drug loaded thixotropic hydrogel for tumor local treatment and preparation method thereof | |
| CN112138144B (en) | Liposome containing active biological factor and preparation method thereof | |
| CN106924185A (en) | A kind of preparation method of the multivesicular liposome for being loaded with vesica | |
| CN105213313B (en) | A kind of Decitabine long circulating liposome lyophilized preparation and preparation method thereof | |
| CN103040910B (en) | Cervus and cucumis polypeptide liposome injection | |
| CN107334739B (en) | Fibroblast growth factor liposome freeze-dried powder for preventing and treating alopecia and preparation method thereof | |
| Kadlecová et al. | Liposomes affect protein release and stability of ITA-Modified PLGA–PEG-PLGA hydrogel carriers for controlled drug delivery | |
| CN109908085A (en) | A drug carrier for respiratory administration, its preparation method and its application in the preparation of a drug for treating heart disease | |
| CN108159401B (en) | Apelin liposome and preparation method thereof | |
| WO2024222862A1 (en) | Lyophilized tangeretin nano-liposome formulation and method for preparing same | |
| CN104473874B (en) | A kind of preparation method of cordycepin proliposome | |
| CN104146961B (en) | A kind of film evaporation method prepares the method for carrying cell factor and SPP1 microballoon | |
| Zhang et al. | Nanomicelle formulation modifies the pharmacokinetic profiles and cardiac toxicity of daunorubicin | |
| CN116173233A (en) | Nano-preparation, preparation and application of PD-1 targeting loaded curcumin in kidney diseases | |
| CN115708809A (en) | Novel lipid nanoparticle for delivering nucleic acid drug and preparation method thereof | |
| EP1811962A2 (en) | Manufacturing process for liposomal preparations | |
| CN101642432A (en) | Latamoxef sodium proliposome preparation | |
| CN113244193A (en) | Preparation method and application of protein nanoparticles for delivering sterol and derivatives thereof | |
| CN115737565B (en) | Schisandrin B liposome and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |