CN103585108B - A kind of tylonolide liposome and preparation method thereof - Google Patents
A kind of tylonolide liposome and preparation method thereof Download PDFInfo
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- CN103585108B CN103585108B CN201310570929.3A CN201310570929A CN103585108B CN 103585108 B CN103585108 B CN 103585108B CN 201310570929 A CN201310570929 A CN 201310570929A CN 103585108 B CN103585108 B CN 103585108B
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- tylonolide
- liposome
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- cholesterol
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- XMEGXHDYCSUOJC-KXOLBLKYSA-N 2-[(4r,5s,6s,7r,9r,11e,13e,15r,16r)-16-ethyl-4,6-dihydroxy-15-(hydroxymethyl)-5,9,13-trimethyl-2,10-dioxo-1-oxacyclohexadeca-11,13-dien-7-yl]acetaldehyde Chemical compound CC[C@H]1OC(=O)C[C@@H](O)[C@H](C)[C@@H](O)[C@@H](CC=O)C[C@@H](C)C(=O)\C=C\C(\C)=C\[C@@H]1CO XMEGXHDYCSUOJC-KXOLBLKYSA-N 0.000 title claims abstract description 73
- XMEGXHDYCSUOJC-UHFFFAOYSA-N Tylonolide Natural products CCC1OC(=O)CC(O)C(C)C(O)C(CC=O)CC(C)C(=O)C=CC(=CC1CO)C XMEGXHDYCSUOJC-UHFFFAOYSA-N 0.000 title claims abstract description 73
- 239000002502 liposome Substances 0.000 title claims abstract description 63
- 238000002360 preparation method Methods 0.000 title abstract description 8
- 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 72
- 235000012000 cholesterol Nutrition 0.000 claims abstract description 36
- 239000006185 dispersion Substances 0.000 claims abstract description 9
- 239000000243 solution Substances 0.000 claims description 23
- 239000007853 buffer solution Substances 0.000 claims description 17
- GVJHHUAWPYXKBD-UHFFFAOYSA-N (±)-α-Tocopherol Chemical compound OC1=C(C)C(C)=C2OC(CCCC(C)CCCC(C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-UHFFFAOYSA-N 0.000 claims description 16
- 150000003904 phospholipids Chemical class 0.000 claims description 15
- 238000003756 stirring Methods 0.000 claims description 13
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 12
- 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 10
- 229940083466 soybean lecithin Drugs 0.000 claims description 10
- 229930003427 Vitamin E Natural products 0.000 claims description 8
- WIGCFUFOHFEKBI-UHFFFAOYSA-N gamma-tocopherol Natural products CC(C)CCCC(C)CCCC(C)CCCC1CCC2C(C)C(O)C(C)C(C)C2O1 WIGCFUFOHFEKBI-UHFFFAOYSA-N 0.000 claims description 8
- 235000019165 vitamin E Nutrition 0.000 claims description 8
- 229940046009 vitamin E Drugs 0.000 claims description 8
- 239000011709 vitamin E Substances 0.000 claims description 8
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 6
- 229920000053 polysorbate 80 Polymers 0.000 claims description 6
- 238000002390 rotary evaporation Methods 0.000 claims description 6
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims description 4
- 125000002252 acyl group Chemical group 0.000 claims description 4
- AKHNMLFCWUSKQB-UHFFFAOYSA-L sodium thiosulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=S AKHNMLFCWUSKQB-UHFFFAOYSA-L 0.000 claims description 3
- 235000019345 sodium thiosulphate Nutrition 0.000 claims description 3
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 claims description 2
- 239000003814 drug Substances 0.000 abstract description 28
- 238000000034 method Methods 0.000 abstract description 13
- 239000003995 emulsifying agent Substances 0.000 abstract description 8
- 229940079593 drug Drugs 0.000 abstract description 5
- 238000001704 evaporation Methods 0.000 abstract description 5
- 230000008020 evaporation Effects 0.000 abstract description 5
- 238000002347 injection Methods 0.000 abstract description 5
- 239000007924 injection Substances 0.000 abstract description 5
- 230000000844 anti-bacterial effect Effects 0.000 abstract description 4
- 230000014759 maintenance of location Effects 0.000 abstract description 2
- 238000005728 strengthening Methods 0.000 abstract description 2
- 238000001132 ultrasonic dispersion Methods 0.000 abstract description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 22
- GEHJYWRUCIMESM-UHFFFAOYSA-L sodium sulfite Chemical compound [Na+].[Na+].[O-]S([O-])=O GEHJYWRUCIMESM-UHFFFAOYSA-L 0.000 description 22
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- 239000002245 particle Substances 0.000 description 12
- 238000000108 ultra-filtration Methods 0.000 description 12
- 235000010265 sodium sulphite Nutrition 0.000 description 11
- 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 9
- PORPENFLTBBHSG-MGBGTMOVSA-N 1,2-dihexadecanoyl-sn-glycerol-3-phosphate Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(O)=O)OC(=O)CCCCCCCCCCCCCCC PORPENFLTBBHSG-MGBGTMOVSA-N 0.000 description 9
- 210000003022 colostrum Anatomy 0.000 description 9
- 235000021277 colostrum Nutrition 0.000 description 9
- 239000006227 byproduct Substances 0.000 description 8
- 239000003960 organic solvent Substances 0.000 description 8
- 229920001983 poloxamer Polymers 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- 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 description 7
- 239000003963 antioxidant agent Substances 0.000 description 7
- 230000003078 antioxidant effect Effects 0.000 description 7
- 235000006708 antioxidants Nutrition 0.000 description 7
- 239000000787 lecithin Substances 0.000 description 7
- 235000010445 lecithin Nutrition 0.000 description 7
- 229940067606 lecithin Drugs 0.000 description 7
- TZCPCKNHXULUIY-RGULYWFUSA-N 1,2-distearoyl-sn-glycero-3-phosphoserine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(=O)OC[C@H](N)C(O)=O)OC(=O)CCCCCCCCCCCCCCCCC TZCPCKNHXULUIY-RGULYWFUSA-N 0.000 description 6
- 102000002322 Egg Proteins Human genes 0.000 description 6
- 108010000912 Egg Proteins Proteins 0.000 description 6
- 241000287828 Gallus gallus Species 0.000 description 6
- ZWZWYGMENQVNFU-UHFFFAOYSA-N Glycerophosphorylserin Natural products OC(=O)C(N)COP(O)(=O)OCC(O)CO ZWZWYGMENQVNFU-UHFFFAOYSA-N 0.000 description 6
- 239000007864 aqueous solution Substances 0.000 description 6
- 210000004681 ovum Anatomy 0.000 description 6
- -1 acyl PHOSPHATIDYL ETHANOLAMINE Chemical class 0.000 description 5
- NRJAVPSFFCBXDT-HUESYALOSA-N 1,2-distearoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCCCCCCCCCCC NRJAVPSFFCBXDT-HUESYALOSA-N 0.000 description 4
- CFWRDBDJAOHXSH-SECBINFHSA-N 2-azaniumylethyl [(2r)-2,3-diacetyloxypropyl] phosphate Chemical compound CC(=O)OC[C@@H](OC(C)=O)COP(O)(=O)OCCN CFWRDBDJAOHXSH-SECBINFHSA-N 0.000 description 4
- 241000283690 Bos taurus Species 0.000 description 4
- 230000001476 alcoholic effect Effects 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- YRWWOAFMPXPHEJ-OFBPEYICSA-K sodium L-ascorbic acid 2-phosphate Chemical compound [Na+].[Na+].[Na+].OC[C@H](O)[C@H]1OC(=O)C(OP([O-])([O-])=O)=C1[O-] YRWWOAFMPXPHEJ-OFBPEYICSA-K 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 244000046052 Phaseolus vulgaris Species 0.000 description 3
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- HNDXPZPJZGTJLJ-UEJFNEDBSA-N (4r,5s,6s,7r,9r,11e,13e,15r,16r)-6-[(2r,3r,4s,5s,6r)-4-(dimethylamino)-3,5-dihydroxy-6-methyloxan-2-yl]oxy-16-ethyl-4-hydroxy-5,9,13-trimethyl-7-(2-piperidin-1-ylethyl)-15-(piperidin-1-ylmethyl)-1-oxacyclohexadeca-11,13-diene-2,10-dione Chemical compound O([C@@H]1[C@@H](C)[C@H](O)CC(=O)O[C@@H]([C@H](/C=C(\C)/C=C/C(=O)[C@H](C)C[C@@H]1CCN1CCCCC1)CN1CCCCC1)CC)[C@@H]1O[C@H](C)[C@@H](O)[C@H](N(C)C)[C@H]1O HNDXPZPJZGTJLJ-UEJFNEDBSA-N 0.000 description 2
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- JZNWSCPGTDBMEW-UHFFFAOYSA-N Glycerophosphorylethanolamin Natural products NCCOP(O)(=O)OCC(O)CO JZNWSCPGTDBMEW-UHFFFAOYSA-N 0.000 description 2
- 238000013019 agitation Methods 0.000 description 2
- 239000003513 alkali Substances 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 210000000232 gallbladder Anatomy 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 150000008105 phosphatidylcholines Chemical class 0.000 description 2
- 208000023504 respiratory system disease Diseases 0.000 description 2
- HRZFUMHJMZEROT-UHFFFAOYSA-L sodium disulfite Chemical compound [Na+].[Na+].[O-]S(=O)S([O-])(=O)=O HRZFUMHJMZEROT-UHFFFAOYSA-L 0.000 description 2
- 235000010262 sodium metabisulphite Nutrition 0.000 description 2
- 238000001694 spray drying Methods 0.000 description 2
- FVXDQWZBHIXIEJ-LNDKUQBDSA-N 1,2-di-[(9Z,12Z)-octadecadienoyl]-sn-glycero-3-phosphocholine Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCC\C=C/C\C=C/CCCCC FVXDQWZBHIXIEJ-LNDKUQBDSA-N 0.000 description 1
- 241000606748 Actinobacillus pleuropneumoniae Species 0.000 description 1
- 241000588807 Bordetella Species 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 1
- 241000606807 Glaesserella parasuis Species 0.000 description 1
- 239000000232 Lipid Bilayer Substances 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241001597008 Nomeidae Species 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 241000606856 Pasteurella multocida Species 0.000 description 1
- RVGRUAULSDPKGF-UHFFFAOYSA-N Poloxamer Chemical compound C1CO1.CC1CO1 RVGRUAULSDPKGF-UHFFFAOYSA-N 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 229930194936 Tylosin Natural products 0.000 description 1
- 239000004182 Tylosin Substances 0.000 description 1
- 229930003268 Vitamin C Natural products 0.000 description 1
- SORGEQQSQGNZFI-UHFFFAOYSA-N [azido(phenoxy)phosphoryl]oxybenzene Chemical compound C=1C=CC=CC=1OP(=O)(N=[N+]=[N-])OC1=CC=CC=C1 SORGEQQSQGNZFI-UHFFFAOYSA-N 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- 239000003012 bilayer membrane Substances 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 description 1
- 229960001231 choline Drugs 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- BIABMEZBCHDPBV-UHFFFAOYSA-N dipalmitoyl phosphatidylglycerol Chemical compound CCCCCCCCCCCCCCCC(=O)OCC(COP(O)(=O)OCC(O)CO)OC(=O)CCCCCCCCCCCCCCC BIABMEZBCHDPBV-UHFFFAOYSA-N 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 238000004945 emulsification Methods 0.000 description 1
- 238000005538 encapsulation Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 150000008282 halocarbons Chemical class 0.000 description 1
- 230000002949 hemolytic effect Effects 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000003120 macrolide antibiotic agent Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 210000004877 mucosa Anatomy 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 229940042880 natural phospholipid Drugs 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000003182 parenteral nutrition solution Substances 0.000 description 1
- 229940051027 pasteurella multocida Drugs 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 238000011056 performance test Methods 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000003495 polar organic solvent Substances 0.000 description 1
- 229960000502 poloxamer Drugs 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 239000008347 soybean phospholipid Substances 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 210000000434 stratum corneum Anatomy 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- LSNNMFCWUKXFEE-UHFFFAOYSA-L sulfite Chemical compound [O-]S([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-L 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- UBCKGWBNUIFUST-YHYXMXQVSA-N tetrachlorvinphos Chemical compound COP(=O)(OC)O\C(=C/Cl)C1=CC(Cl)=C(Cl)C=C1Cl UBCKGWBNUIFUST-YHYXMXQVSA-N 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- WBPYTXDJUQJLPQ-VMXQISHHSA-N tylosin Chemical compound O([C@@H]1[C@@H](C)O[C@H]([C@@H]([C@H]1N(C)C)O)O[C@@H]1[C@@H](C)[C@H](O)CC(=O)O[C@@H]([C@H](/C=C(\C)/C=C/C(=O)[C@H](C)C[C@@H]1CC=O)CO[C@H]1[C@@H]([C@H](OC)[C@H](O)[C@@H](C)O1)OC)CC)[C@H]1C[C@@](C)(O)[C@@H](O)[C@H](C)O1 WBPYTXDJUQJLPQ-VMXQISHHSA-N 0.000 description 1
- 229960004059 tylosin Drugs 0.000 description 1
- 235000019375 tylosin Nutrition 0.000 description 1
- 239000000052 vinegar Substances 0.000 description 1
- 235000021419 vinegar Nutrition 0.000 description 1
- 235000019154 vitamin C Nutrition 0.000 description 1
- 239000011718 vitamin C Substances 0.000 description 1
- 229940080752 zuprevo Drugs 0.000 description 1
Landscapes
- Medicinal Preparation (AREA)
Abstract
The invention belongs to technical field of veterinary, open a kind of tylonolide liposome and preparation method thereof.Comprise the component of following weight portion: phosphatidase 5-10 parts, cholesterol 0.5-2 part, tylonolide 1-2 part, emulsifying agent 0.5-100 part.The preparation method that the present invention also provides for above-mentioned tylonolide liposome, such as injection method, ultrasonic dispersion, reverse phase evaporation or film dispersion method.The product cut size of the present invention is between 80～500nm so that tylonolide has light more preferably and ordinary temperature stability.And the liposome of the present invention, the product prepared especially by film dispersion method has good envelop rate；Additionally, the present invention is by wrapping up in the liposome with specific components by medicine tylonolide, liposome and biomembrane have better adhesiveness, medicine not easily runs off, medicine is made to maintain higher concentration in local, improving drug level and the bioavailability of tylonolide, thus effectively strengthening antibacterial effect, and extending the action time of medicine due to the increase of medicine retention amount.
Description
Technical field
The invention belongs to technical field of veterinary, be specifically related to a kind of tylonolide liposome and preparation method thereof.
Background technology
Tylonolide, name of commodity in English: Tylonolide, No. CAS is 328898-40-4, and molecular structure is:
, molecular formula is C41H71N3O8, molecular weight 734.02, fusing point is 192 DEG C, is dissolved in polar organic solvent (such as methanol, acetone etc.), slightly soluble in water.It is the Macrolide semisynthetic antibiotics of the up-to-date animal specific of offshore company's exploitation, for the derivant of tylosin.On March 8th, 2011, veterinary medicine committee of European Union (CVMP) has permitted the market license application of the aseptic parenteral solution (commodity are called Zuprevo) that Intervet company is main component with tylonolide, will in succession ratify listing in European Union member countries subsequently.At present, the high incidence of respiratory system disease and mortality rate are the matters of utmost importance that whole world pig, cattle aquaculture face.Tylonolide is used for preventing and treat pig, cattle by sensitive microbial respiratory infectious disease, and single-dose can provide whole-course treatment.Pig muscle injection, cattle cervical region subcutaneous administrations.Tylonolide is broad spectrum antibiotic, some Gram-positives and gram negative bacteria are respectively provided with antibacterial activity, to cause pig, cattle respiratory system disease pathogen particularly sensitive, such as Actinobacillus pleuropneumoniae, pasteurella multocida, bordetella branchiseptica, haemophilus parasuis and hemolytic Mannheim bacterium.But, this aqueous solubility is poor, and bioavailability is low.
Liposome is by the drug encapsulation of some unstable different oxidations in liposome, and medicine, because being subject to the protection of liposome bilayer membrane, improves the stability of medicine to a great extent.It addition, liposome is as pharmaceutical carrier, oneself increasingly comes into one's own and obtains own extensive use.Utilize the characteristic that liposome and biological cell membrane affinity are strong, antibiotic is wrapped in liposome and can improve antibacterial effect.Interacted with Stratum corneum lipids by the lipoid of liposome, promote passing through of medicine, and reach tissue deep, due to transit dose and the increase of hold-up in mucosa, delayed the release of medicine, extended the pharmaceutically-active time.But the local action time of concrete medicine and effect, and the preparation method of liposome, the composition of liposome etc. is relevant；And for different pharmaceutical active component, especially for tylonolide, the envelop rate improving its liposome is also a difficult problem.
Summary of the invention
It is an object of the invention to provide a kind of tylonolide liposome and preparation method thereof.
For achieving the above object, the technical scheme that the present invention takes is as follows:
A kind of tylonolide liposome, comprises the component of following weight portion: phosphatidase 5-10 parts, cholesterol 0.5-2 part, tylonolide 1-2 part, emulsifying agent 0.5-100 part.
Wherein, described phospholipid can be lecithin, the natural phospholipids such as cephalin, and DLPC, two bean bandit's phosphatidyl cholines, dipalmitoyl phosphatidyl choline, distearoyl phosphatidylcholine, two phosphatidyl cholines, two bean bandit's acyl PHOSPHATIDYL ETHANOLAMINE, two palmityl phosphatidyl ethanol acyls, DPPG, two bean bandit's acyl phosphatidic acid, DPPA, DPPC, one or more in the synthetic phospholipids such as 1-stearic acid-2-palmitoylphosphatidyl choline and Polyethylene Glycol-2-stearyl phosphatidyl ethanolamine, they are respectively provided with two positive electricity water chains, the formation lipid bilayer that energy is spontaneous in water, and constitute a cell closed.Preferably soya lecithin of the present invention, Ovum Gallus domesticus Flavus lecithin, cephalin, DPPC, distearoyl phosphatidylcholine, dipalmitoyl phosphatidyl choline, phosphatidic acid (PA) and one or more in Phosphatidylserine (PS).The use of cholesterol, can affect the mobility of film, permeability and then affect the stability of film.The uniformity of liposome turbid liquor and stability are mainly had certain effect by emulsifying agent of the present invention, and can obtain stable colostrum when using reverse evaporation, the preferred poloxamer of the present invention or tween.The content proportioning of above-mentioned phospholipid, cholesterol, emulsifying agent and medicine is too little can affect the stability of liposome, envelop rate and drug loading；Otherwise then causing wastage of material and increase cost, wherein emulsifier is too much, also can increase toxicity.
The tylonolide liposome of the present invention can also include the fat-soluble antioxidant and water soluble antioxidant that respectively account for phospholipid and cholesterol gross weight 0.1-5%, oxidized during to prevent it from storing at normal temperatures.
Described fat-soluble antioxidant can be selected for but is not limited only to one or more in sodium L-ascorbate-2-phosphate, vitamin E, described water soluble antioxidant can be selected for but is not limited only to sulphite, such as sodium sulfite, sodium sulfite, sodium pyrosulfite or sodium thiosulfate etc..
The preparation method that the present invention also provides for above-mentioned tylonolide liposome, such as injection method, ultrasonic dispersion, reverse phase evaporation or film dispersion method.
Adopt film dispersion method, comprise the steps:
1) phospholipid and cholesterol are dissolved in volatilizable organic solvent, and rotation flashes to film at 30～50 DEG C；
2) emulsifying agent is dissolved in the PBS of pH=6.0～7.3, then tylonolide is dissolved in this buffer；
3) by step 2) solution of gained adds in the film that step 1) prepares, and mix and blend obtains tylonolide liposome turbid liquor.
Adopt injection method, comprise the steps:
1) phospholipid and cholesterol are dissolved in volatilizable organic solvent；
2) emulsifying agent is dissolved in the PBS of pH=6.0～7.3, then tylonolide is dissolved in this buffer；
3) by the solution of step 1) gained stirring and 30～50 DEG C of constant temperatures under, join step 2) prepare solution in, after continue stirring to wave most organic solvent, obtain tylonolide liposome turbid liquor.
Adopt reverse phase evaporation, comprise the steps:
1) phospholipid and cholesterol are dissolved in volatilizable organic solvent；
2) emulsifying agent is dissolved in the buffer of pH=6.0～7.3, then tylonolide is dissolved in this buffer；
3) by step 2) solution of gained joins in the solution of step 1) gained, carries out ultrasonic or use cutter to form it into colostrum；
4) colostrum step 3) obtained is evaporated under reduced pressure and obtains tylonolide liposome turbid liquor to remove organic solvent.
In the above-mentioned various methods of the present invention, volatilizable organic solvent can be volatilizable alcohols, such as methanol, ethanol；Halogenated hydrocarbons, such as chloroform；And one or more in the organic solvent such as acetic acid.Step 2) in PBS adopt conventional phosphate PBS.
As antioxidant need to be added, then in the step 1) of above-mentioned various methods, the fat-soluble antioxidant accounting for phospholipid and cholesterol gross weight 0.1-5% is codissolved in volatilizable organic solvent with phospholipid and cholesterol, and addition accounts for phospholipid and the water soluble antioxidant of cholesterol gross weight 0.1-5% in the tylonolide liposome turbid liquor that step 3) obtains.
The positive effect of the present invention: the product cut size of the present invention is between 80～500nm so that tylonolide has light more preferably and ordinary temperature stability.And the liposome of the present invention, the product prepared especially by film dispersion method has good envelop rate；It addition, the present invention is by wrapping up in the liposome with specific components by medicine tylonolide, liposome and biomembrane have better adhesiveness, and medicine not easily runs off, and make medicine maintain higher concentration in local.Drug level and the bioavailability of tylonolide can being improved by medicine tylonolide being prepared into liposome, thus effectively strengthening antibacterial effect, and extending the action time of medicine due to the increase of medicine retention amount.
Detailed description of the invention
Film dispersion method
Embodiment 1
500mg soybean lecithin, 100mg cholesterol and 11mg sodium L-ascorbate-2-phosphate are codissolved in ethanol, by rotary evaporation film forming in the water-bath of 50 DEG C；300mg PLURONICS F87 is dissolved in the PBS 10ml of pH=6, then tylonolide 100mg is dissolved in this buffer solution；Being added in above-mentioned film by prepared solution, vortex oscillation disperses to obtain liposome turbid liquor；Last addition in liposome turbid liquor accounts for soybean lecithin and the sodium sulfite of cholesterol gross weight 0.1%.
The particle diameter grain size analysis analyzer of obtained tylonolide liposome records between 200～300nm；It is 60% by product ultrafiltration centrifugal determination envelop rate.
Embodiment 2
The vitamin E of 900mg Ovum Gallus domesticus Flavus lecithin, 90mg cholesterol and 6.3mg is codissolved in ethanol, by rotary evaporation film forming in the water-bath of 30 DEG C；100mg PLURONICS F87 is dissolved in the PBS 5ml of pH7.3, tylonolide 180mg is dissolved in this buffer solution；Being added in above-mentioned film by prepared solution, after vortex mixed, magnetic agitation homogenizes 1 hour, obtains liposome turbid liquor again；Last addition in liposome turbid liquor accounts for Ovum Gallus domesticus Flavus lecithin and the sodium sulfite of cholesterol gross weight 0.1%.
Obtained tylonolide liposomal particle size is between 80～200nm；It is 70% by products application ultrafiltration centrifugal determination envelop rate.
Embodiment 3
The vitamin E of the soybean lecithin with synthetic phospholipid acyl serine (weight ratio 20:1) and 0.5mg cholesterol, 0.21mg that are total up to 5mg is dissolved in chloroform, by rotary evaporation film forming in the water-bath of 40 DEG C；100mg Tween 80 is dissolved in the PBS 2ml of pH6.5, then tylonolide 1mg is dissolved in this buffer solution；Being added by prepared solution in above-mentioned film, after vibrating dispersion, stirring homogenizes 1 hour, obtains liposome turbid liquor；In liposome turbid liquor, finally add the sodium thiosulfate accounting for soybean lecithin, synthetic phospholipid acyl serine and cholesterol gross weight 0.1%.
Obtained tylonolide liposomal particle size is between 100-300nm；Products application ultrafiltration centrifuging is recorded envelop rate is 80%.
Embodiment 4
The cephalin being total up to 50mg is dissolved in ethanol with phosphatidic acid (weight ratio 15:1), 10mg cholesterol, 2.3mg vitamin C Petiolus Trachycarpi vinegar, by rotary evaporation film forming in the water-bath of 30 DEG C；200mg PLURONICS F87 is dissolved in the PBS 2ml of pH7.2, tylonolide 5mg is dissolved in this buffer solution；Being added by prepared solution in above-mentioned film, after vibrating dispersion, stirring homogenizes 1 hour to obtain liposome turbid liquor；In liposome turbid liquor, finally add the sodium sulfite accounting for cephalin, phosphatidic acid and cholesterol gross weight 0.1%.
Obtained tylonolide liposomal particle size is between 100～200nm；It is 68% by products application ultrafiltration centrifugal determination envelop rate.
Embodiment 5
1000mg is synthesized dipalmitoyl phosphatidyl choline (DPPC), 100mg cholesterol and 16mg vitamin E and is dissolved in ethanol, by rotary evaporation film forming in the water-bath of 30 DEG C；700mg Tween 80 is dissolved in the PBS 14ml of pH6.8, tylonolide 200mg is dissolved in above-mentioned buffer solution；Being added by prepared solution in above-mentioned film, after vortex mixed, magnetic agitation homogenizes 1 hour to obtain liposome turbid liquor；Last addition in liposome turbid liquor accounts for the sodium sulfite synthesizing dipalmitoyl phosphatidyl choline and cholesterol gross weight 0.1%.
Obtained tylonolide liposomal particle size is between 200～300nm；It is 50% by products application ultrafiltration centrifugal determination envelop rate.
Injection method
Embodiment 6
Take 20mg tylonolide and be dissolved in the 5mlPBS buffer solution (pH7.3) of the PLURONICS F87 containing 250mg, the vitamin E of 80mg soybean lecithin, 10mg cholesterol and 5.4mg is codissolved in 10ml ethanol, buffer solution containing medicine is placed in container, under constant temperature 50 DEG C stirring, at the uniform velocity drip above-mentioned alcoholic solution, stirring 1 hour is continued after adding, by high pressure dispersing emulsification machine 2 times, obtain liposome turbid liquor, add in this suspension and account for soybean lecithin and the sodium sulfite of cholesterol gross weight 0.1%.
Obtained tylonolide liposomal particle size is between 300～500nm；Ultrafiltration centrifugal determination envelop rate is 40%.
Embodiment 7
Take 1mg tylonolide and be dissolved in the 2mlPBS buffer solution (pH7.3) of the Tween 80 containing 50mg, the Ovum Gallus domesticus Flavus lecithin and the phosphatidic acid (weight ratio 18:1) that are total up to 4mg, and 0.5mg cholesterol is codissolved in 10ml ethanol, buffer solution containing medicine is placed in container, under constant temperature 30 DEG C stirring, at the uniform velocity drip above-mentioned alcoholic solution, continue stirring 0.5 hour after adding, obtain liposome turbid liquor.
Obtained tylonolide liposomal particle size is between 300～500nm；It is 50% by products application ultrafiltration centrifugal determination envelop rate.
Embodiment 8
Take 200mg tylonolide and be dissolved in the 10mlPBS buffer solution (pH7.2) of the PLURONICS F87 containing 500mg, the sodium L-ascorbate-2-phosphate of the soybean lecithin and Phosphatidylserine (weight ratio 15:1), 100mg cholesterol and 11.8mg that are total up to 1000mg is codissolved in 10ml methanol, buffer solution containing medicine is placed in container, under constant temperature 50 DEG C stirring, at the uniform velocity drip above-mentioned methanol solution, continue stirring 1.5 hours after adding, obtain liposome turbid liquor；In this suspension, finally add the sodium sulfite accounting for soybean lecithin, Phosphatidylserine and cholesterol gross weight 0.1%.
Obtained tylonolide liposomal particle size is between 200～500nm；Ultrafiltration centrifugal determination envelop rate is 55%.
Embodiment 9
Take 100mg tylonolide and be dissolved in the 8mlPBS buffer solution (pH7) of the Tween 80 containing 250mg, the sodium L-ascorbate-2-phosphate of the synthetic phospholipid (DPPC) and phosphatidic acid (weight ratio 20:1), 80mg cholesterol and 9.2mg that are total up to 800mg is codissolved in 15ml chloroform, buffer solution containing medicine is placed in container, under constant temperature 50 DEG C stirring, at the uniform velocity drip above-mentioned chloroformic solution, continue stirring after adding, obtain liposome turbid liquor；Last add in this suspension account for DPPC, phosphatidic acid and cholesterol gross weight 0.1% sodium sulfite.
Obtained tylonolide liposomal particle size is between 100～500nm；It is 60% by products application ultrafiltration centrifugal determination envelop rate.
Reverse phase evaporation
Embodiment 10
Take 100mg Ovum Gallus domesticus Flavus lecithin and 8mg cholesterol and 5.4mg vitamin E is codissolved in 5ml ethanol, the tylonolide of 10mg is dissolved in the 5mlPBS buffer solution (pH7) of 250mg PLURONICS F87, solution containing tylonolide is joined in alcoholic solution, carrying out ultrasonic making formation colostrum, colostrum obtains liposome turbid liquor through being evaporated under reduced pressure removing ethanol；This liposome turbid liquor last adds the sodium sulfite of Ovum Gallus domesticus Flavus lecithin and cholesterol gross weight 0.1%.
Obtained tylonolide liposomal particle size is between 100～400nm；It is 60% by products application ultrafiltration centrifugal determination envelop rate.
Embodiment 11
Take the two palmityl phospholipid ester gallbladder alkali being total up to 1000mg, the mixture (mol ratio 12:1:1) of phosphatidic acid and Phosphatidylserine, and 100mg cholesterol and 14.3mg vitamin E are codissolved in 12ml ethanol, the tylonolide of 200mg is dissolved in the 12.5mlPBS buffer solution (pH6.8) of 500mg PLURONICS F87, solution containing tylonolide is joined in alcoholic solution, shear with high-speed shearing machine and make formation colostrum, colostrum obtains liposome turbid liquor through being evaporated under reduced pressure to remove ethanol, this liposome turbid liquor adds and accounts for two palmityl phospholipid ester gallbladder alkali, phosphatidic acid, the sodium sulfite of Phosphatidylserine and cholesterol gross weight 0.1%.
Obtained tylonolide liposomal particle size is between 200～400nm；By products application ultrafiltration centrifugation liposome and non-encapsulated free drug, recording envelop rate is 60%.
Embodiment 12
Take 10mg distearoyl phosphatidylcholine and 1mg cholesterol is codissolved in 1ml chloroform, the tylonolide of 2mg is dissolved in the 1mlPBS buffer solution (pH7.3) of 50mg Tween 80, solution containing tylonolide is joined in chloroformic solution, mixed solution carries out high speed shear makes formation colostrum, colostrum obtains liposome turbid liquor through being evaporated under reduced pressure to remove chloroform, adds the sodium pyrosulfite of distearoyl phosphatidylcholine and cholesterol gross weight 0.1% in this liposome turbid liquor.
Obtained tylonolide liposomal particle size is between 100～400nm；Ultrafiltration centrifugal determination envelop rate is 40%.
For being more conducive to storage and transport, the liposome turbid liquor that above-described embodiment finally gives also through techniques such as conventional spray drying or lyophilizations, can obtain its powder further.
Liposome turbid liquor embodiment 1 finally prepared, through conventional spray drying, obtains its powder further, as test object, carries out following performance test:
Test example 1
The aqueous solution of tylonolide liposome (powder) of the present invention of same concentration (16.7mg/ml) same volume (100ml) and the aqueous solution of existing tylonolide are joined in water white transparency volumetric flask, and it is placed under 25 DEG C of environment, each solution concentration is measured after 24 hours respectively by high performance liquid chromatography.Result shows that the content of the tylonolide in tylonolide liposome of the present invention is 14.2mg/ml, and the content of the tylonolide in tylonolide aqueous solution is 8.9mg/ml.
Test example 2
The aqueous solution of tylonolide liposome (powder) of the present invention of same concentration (16.5mg/ml) same volume (100ml) and the aqueous solution of existing tylonolide are joined in water white transparency volumetric flask, opening is placed under the environment that intensity of illumination is 4000Lx, measures each solution concentration after 24 hours respectively by high performance liquid chromatography.Result shows that the content of the tylonolide in tylonolide liposome of the present invention is 12.4mg/ml, and the content of the tylonolide in tylonolide aqueous solution is 6.8mg/ml.
The result of above-mentioned test shows that tylonolide can be effectively improved ordinary temperature stability and the light stability of medicine after liposomal encapsulated.
Claims (1)
1. a tylonolide liposome, it is characterized in that preparing as follows: be dissolved in chloroform by being total up to 5mg, the soybean lecithin of weight ratio 20:1 and the vitamin E of synthetic phospholipid acyl serine and 0.5mg cholesterol, 0.21mg, by rotary evaporation film forming in the water-bath of 40 DEG C；100mg Tween 80 is dissolved in the PBS 2ml of pH6.5, then tylonolide 1mg is dissolved in this buffer solution；Being added by prepared solution in above-mentioned film, after vibrating dispersion, stirring homogenizes 1 hour, obtains liposome turbid liquor；In liposome turbid liquor, finally add the sodium thiosulfate accounting for soybean lecithin, synthetic phospholipid acyl serine and cholesterol gross weight 0.1%.
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