CN104382854A - Doxorubicin lipid microbubble and preparation method thereof - Google Patents
Doxorubicin lipid microbubble and preparation method thereof Download PDFInfo
- Publication number
- CN104382854A CN104382854A CN201410550470.5A CN201410550470A CN104382854A CN 104382854 A CN104382854 A CN 104382854A CN 201410550470 A CN201410550470 A CN 201410550470A CN 104382854 A CN104382854 A CN 104382854A
- Authority
- CN
- China
- Prior art keywords
- doxorubicin
- albumin
- preparation
- lipid microbubble
- phospholipid
- 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.)
- Pending
Links
- AOJJSUZBOXZQNB-TZSSRYMLSA-N doxorubicine Natural products O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 title claims abstract description 146
- 229960004679 doxorubicin Drugs 0.000 title claims abstract description 88
- 238000002360 preparation method Methods 0.000 title claims abstract description 36
- -1 Doxorubicin lipid Chemical class 0.000 title claims abstract description 30
- 108010088751 Albumins Proteins 0.000 claims abstract description 75
- 102000009027 Albumins Human genes 0.000 claims abstract description 75
- 239000003814 drug Substances 0.000 claims abstract description 42
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 42
- 150000002632 lipids Chemical class 0.000 claims description 31
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 22
- 239000007864 aqueous solution Substances 0.000 claims description 21
- 150000003904 phospholipids Chemical class 0.000 claims description 20
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 14
- 230000001476 alcoholic effect Effects 0.000 claims description 14
- 239000008363 phosphate buffer Substances 0.000 claims description 14
- 239000000243 solution Substances 0.000 claims description 14
- 239000000725 suspension Substances 0.000 claims description 14
- 239000007789 gas Substances 0.000 claims description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 11
- 238000010438 heat treatment Methods 0.000 claims description 11
- 229910052757 nitrogen Inorganic materials 0.000 claims description 11
- 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 description 5
- 229940067606 lecithin Drugs 0.000 claims description 5
- 239000000787 lecithin Substances 0.000 claims description 5
- 235000010445 lecithin Nutrition 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 5
- 229940042880 natural phospholipid Drugs 0.000 claims description 5
- 238000002390 rotary evaporation Methods 0.000 claims description 5
- 238000007711 solidification Methods 0.000 claims description 5
- 230000008023 solidification Effects 0.000 claims description 5
- 238000003756 stirring Methods 0.000 claims description 5
- MEYZYGMYMLNUHJ-UHFFFAOYSA-N tunicamycin Natural products CC(C)CCCCCCCCCC=CC(=O)NC1C(O)C(O)C(CC(O)C2OC(C(O)C2O)N3C=CC(=O)NC3=O)OC1OC4OC(CO)C(O)C(O)C4NC(=O)C MEYZYGMYMLNUHJ-UHFFFAOYSA-N 0.000 claims description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 3
- 102000002322 Egg Proteins Human genes 0.000 claims description 3
- 108010000912 Egg Proteins Proteins 0.000 claims description 3
- 241000287828 Gallus gallus Species 0.000 claims description 3
- 229940098773 bovine serum albumin Drugs 0.000 claims description 3
- 230000009514 concussion Effects 0.000 claims description 3
- 210000004681 ovum Anatomy 0.000 claims description 3
- 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 2
- JZNWSCPGTDBMEW-UHFFFAOYSA-N Glycerophosphorylethanolamin Natural products NCCOP(O)(=O)OCC(O)CO JZNWSCPGTDBMEW-UHFFFAOYSA-N 0.000 claims description 2
- 150000008104 phosphatidylethanolamines Chemical class 0.000 claims description 2
- 229940083466 soybean lecithin Drugs 0.000 claims description 2
- 229940079593 drug Drugs 0.000 abstract description 17
- 230000000694 effects Effects 0.000 abstract description 12
- 230000009471 action Effects 0.000 abstract description 9
- 239000002105 nanoparticle Substances 0.000 abstract description 6
- 238000009826 distribution Methods 0.000 abstract description 5
- 210000004369 blood Anatomy 0.000 abstract description 4
- 239000008280 blood Substances 0.000 abstract description 4
- 238000011068 loading method Methods 0.000 abstract description 4
- 239000012528 membrane Substances 0.000 abstract description 4
- 230000009286 beneficial effect Effects 0.000 abstract description 3
- 230000015556 catabolic process Effects 0.000 abstract description 3
- 238000006731 degradation reaction Methods 0.000 abstract description 3
- 238000006243 chemical reaction Methods 0.000 abstract description 2
- 238000005538 encapsulation Methods 0.000 abstract description 2
- 239000003960 organic solvent Substances 0.000 abstract description 2
- 231100000331 toxic Toxicity 0.000 abstract description 2
- 230000002588 toxic effect Effects 0.000 abstract description 2
- 239000000232 Lipid Bilayer Substances 0.000 abstract 2
- 231100000086 high toxicity Toxicity 0.000 abstract 1
- 230000003902 lesion Effects 0.000 abstract 1
- 230000000361 pesticidal effect Effects 0.000 abstract 1
- 230000002035 prolonged effect Effects 0.000 abstract 1
- 238000000034 method Methods 0.000 description 8
- 230000018109 developmental process Effects 0.000 description 7
- 239000000047 product Substances 0.000 description 6
- 241000283973 Oryctolagus cuniculus Species 0.000 description 5
- 239000003937 drug carrier Substances 0.000 description 5
- 239000002245 particle Substances 0.000 description 5
- 238000001035 drying Methods 0.000 description 4
- 229960004756 ethanol Drugs 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 239000002872 contrast media Substances 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 239000002502 liposome Substances 0.000 description 3
- 238000011587 new zealand white rabbit Methods 0.000 description 3
- 230000008685 targeting Effects 0.000 description 3
- 238000002604 ultrasonography Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 206010019280 Heart failures Diseases 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 230000000857 drug effect Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000004880 explosion Methods 0.000 description 2
- 238000010304 firing Methods 0.000 description 2
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 230000031142 liver development Effects 0.000 description 2
- 238000010297 mechanical methods and process Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000005192 partition Methods 0.000 description 2
- 238000005057 refrigeration Methods 0.000 description 2
- 238000000859 sublimation Methods 0.000 description 2
- 230000008022 sublimation Effects 0.000 description 2
- 238000005092 sublimation method Methods 0.000 description 2
- 238000002525 ultrasonication Methods 0.000 description 2
- MPDGHEJMBKOTSU-YKLVYJNSSA-N 18beta-glycyrrhetic acid Chemical compound C([C@H]1C2=CC(=O)[C@H]34)[C@@](C)(C(O)=O)CC[C@]1(C)CC[C@@]2(C)[C@]4(C)CC[C@@H]1[C@]3(C)CC[C@H](O)C1(C)C MPDGHEJMBKOTSU-YKLVYJNSSA-N 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 206010003210 Arteriosclerosis Diseases 0.000 description 1
- 206010065553 Bone marrow failure Diseases 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 206010048610 Cardiotoxicity Diseases 0.000 description 1
- LTMHDMANZUZIPE-AMTYYWEZSA-N Digoxin Natural products O([C@H]1[C@H](C)O[C@H](O[C@@H]2C[C@@H]3[C@@](C)([C@@H]4[C@H]([C@]5(O)[C@](C)([C@H](O)C4)[C@H](C4=CC(=O)OC4)CC5)CC3)CC2)C[C@@H]1O)[C@H]1O[C@H](C)[C@@H](O[C@H]2O[C@@H](C)[C@H](O)[C@@H](O)C2)[C@@H](O)C1 LTMHDMANZUZIPE-AMTYYWEZSA-N 0.000 description 1
- MPDGHEJMBKOTSU-UHFFFAOYSA-N Glycyrrhetinsaeure Natural products C12C(=O)C=C3C4CC(C)(C(O)=O)CCC4(C)CCC3(C)C1(C)CCC1C2(C)CCC(O)C1(C)C MPDGHEJMBKOTSU-UHFFFAOYSA-N 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 206010033128 Ovarian cancer Diseases 0.000 description 1
- 206010061535 Ovarian neoplasm Diseases 0.000 description 1
- 229910018503 SF6 Inorganic materials 0.000 description 1
- 206010039491 Sarcoma Diseases 0.000 description 1
- 208000033781 Thyroid carcinoma Diseases 0.000 description 1
- 208000024770 Thyroid neoplasm Diseases 0.000 description 1
- 206010047700 Vomiting Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 239000003570 air Substances 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 230000004596 appetite loss Effects 0.000 description 1
- 206010003119 arrhythmia Diseases 0.000 description 1
- 230000006793 arrhythmia Effects 0.000 description 1
- 208000011775 arteriosclerosis disease Diseases 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 238000005422 blasting Methods 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 230000007177 brain activity Effects 0.000 description 1
- 201000008275 breast carcinoma Diseases 0.000 description 1
- 231100000259 cardiotoxicity Toxicity 0.000 description 1
- 230000007681 cardiovascular toxicity Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 229960000935 dehydrated alcohol Drugs 0.000 description 1
- 230000001934 delay Effects 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 238000004807 desolvation Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- LTMHDMANZUZIPE-PUGKRICDSA-N digoxin Chemical compound C1[C@H](O)[C@H](O)[C@@H](C)O[C@H]1O[C@@H]1[C@@H](C)O[C@@H](O[C@@H]2[C@H](O[C@@H](O[C@@H]3C[C@@H]4[C@]([C@@H]5[C@H]([C@]6(CC[C@@H]([C@@]6(C)[C@H](O)C5)C=5COC(=O)C=5)O)CC4)(C)CC3)C[C@@H]2O)C)C[C@@H]1O LTMHDMANZUZIPE-PUGKRICDSA-N 0.000 description 1
- 229960005156 digoxin Drugs 0.000 description 1
- LTMHDMANZUZIPE-UHFFFAOYSA-N digoxine Natural products C1C(O)C(O)C(C)OC1OC1C(C)OC(OC2C(OC(OC3CC4C(C5C(C6(CCC(C6(C)C(O)C5)C=5COC(=O)C=5)O)CC4)(C)CC3)CC2O)C)CC1O LTMHDMANZUZIPE-UHFFFAOYSA-N 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 229960003720 enoxolone Drugs 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 229960000304 folic acid Drugs 0.000 description 1
- 235000019152 folic acid Nutrition 0.000 description 1
- 239000011724 folic acid Substances 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 230000037356 lipid metabolism Effects 0.000 description 1
- 235000021266 loss of appetite Nutrition 0.000 description 1
- 208000019017 loss of appetite Diseases 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 206010025482 malaise Diseases 0.000 description 1
- 230000010358 mechanical oscillation Effects 0.000 description 1
- 210000004165 myocardium Anatomy 0.000 description 1
- 230000007830 nerve conduction Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 238000000879 optical micrograph Methods 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 238000002601 radiography Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 230000002000 scavenging effect Effects 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- WNIFXKPDILJURQ-UHFFFAOYSA-N stearyl glycyrrhizinate Natural products C1CC(O)C(C)(C)C2CCC3(C)C4(C)CCC5(C)CCC(C(=O)OCCCCCCCCCCCCCCCCCC)(C)CC5C4=CC(=O)C3C21C WNIFXKPDILJURQ-UHFFFAOYSA-N 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- SFZCNBIFKDRMGX-UHFFFAOYSA-N sulfur hexafluoride Chemical compound FS(F)(F)(F)(F)F SFZCNBIFKDRMGX-UHFFFAOYSA-N 0.000 description 1
- 229960000909 sulfur hexafluoride Drugs 0.000 description 1
- 230000033772 system development Effects 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 201000002510 thyroid cancer Diseases 0.000 description 1
- 208000013077 thyroid gland carcinoma Diseases 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 208000037816 tissue injury Diseases 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 230000008673 vomiting Effects 0.000 description 1
Landscapes
- Medicinal Preparation (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention provides a doxorubicin lipid microbubble. The doxorubicin lipid microbubble comprises a lipid bilayer membrane, gas, albumin nanoparticles for carrying doxorubicin; and the gas and the albumin nanoparticles for carrying the doxorubicin are wrapped by the lipid bilayer membrane. The doxorubicin lipid microbubble has the relative higher medicine loading capacity and the relative higher encapsulation efficiency; the blood concentration of target positions can be improved; the action time of medicines at the target positions is prolonged; the medicine degradation is reduced; and the medicine stability is improved. In addition, the nanoparticles have the beneficial effects that the selective distribution of the medicines is promoted; the medicines are better transmitted to enter lesion tissues; the pesticide effect can be improved; and the toxic and side effects can be reduced. The invention further discloses a preparation method of the doxorubicin lipid microbubble. The preparation process is simple; industrialization is easily achieved; the preparation process does not relate to chemical reactions; organic solvents with high toxicity are not adopted; the safety performance is high; and the environment friendliness is high.
Description
Technical field
The present invention relates to doxorubicin, be specifically related to doxorubicin lipid microbubble and preparation method thereof.
Background technology
Doxorubicin is spectrum antitumor drug, and clinical being mainly used in treats the solid tumors such as breast carcinoma, thyroid carcinoma, pulmonary carcinoma, ovarian cancer, sarcoma.Even but doxorubicin has bone marrow depression, loss of appetite, vomiting of feeling sick, and can cause Delayed onset severe heart failure, can occur increased heart rate, arrhythmia, conduction block or ejection heart failure, wait cardiac toxicity, thus applies and be greatly restricted.
The appearance of targeting research brings new probability to a large amount of uses of digoxin, and the key of target administration is efficient, low toxicity and has the pharmaceutical carrier of good biocompatibility.Targeting drug delivery system development trend be utilize liposome as pharmaceutical carrier, wherein liposome due to can vivo degradation, nontoxic, without features such as immunosuppressive actions, be more and more subject to people's attention as pharmaceutical carrier.But liposome also exists a lot of limitation, as targeting is not obvious, envelop rate is lower, easily permeates.Especially after arriving target position, release is slow, is often difficult to reach effectively treat concentration.
Along with the development of excusing from death developing technique, it is found that microvesicle can significantly improve excusing from death image to specific mass, the excusing from death microvesicle developing middle gassiness is thus as excusing from death contrast agent.Excusing from death contrast agent, as the two dimension excusing from death video picture and the flow Doppler effect that strengthen the organa parenchymatosums such as cardiac muscle, liver, brain, significantly improves the resolution capability of excusing from death for diseased region form.
Although the research of microvesicle in contrast agent reaches its maturity, microvesicle is studied actually rare as a kind of novel pharmaceutical carrier.Although microvesicle is at target administration and promote that medicine has unique advantage in the absorption of target site, but there is the technical barrier that much cannot overcome at present: if microvesicle is as pharmaceutical carrier, its drug loading is lower, cause the drug level being transported to target position lower, be not enough to produce effective pharmacological action etc. concerning medicine.
Summary of the invention
For the above-mentioned defect existed in prior art, the invention provides a kind of doxorubicin lipid microbubble.
Another object of the present invention is to provide a kind of preparation method of doxorubicin lipid microbubble.
The object of the present invention is achieved like this:
A kind of doxorubicin lipid microbubble, is characterized in that: described lipid microbubble comprises phospholipid bilayer tunic, gas and carries the albumin nano granular of doxorubicin; The albumin nano granular of described gas and year doxorubicin is all wrapped in phospholipid bilayer tunic inside.
Described albumin is bovine serum albumin.
Inventor finds under study for action, the doxorubicin lipid microbubble of preparation, easily occurs that shell is comparatively hard, elasticity relative mistake, weak at ultrasonic cavitation action effect, ultrasonic can not to be smashed, if strengthen ultrasonic energy can cause normal tissue injury again by low-yield.
The preferred natural phospholipid of above-mentioned phospholipid.
When meeting lipid microbubble easily by ultrasonic breaking up, inventor also finds under study for action, in doxorubicin lipid microbubble, the selection of gas has very large being particular about, once control bad just easily appearance: the gas diffusion in microvesicle is very fast, ball wall subsides and loses rapidly sound reflecting, and can not to distribute whole body with blood, thus affect the therapeutic effect of doxorubicin to a certain extent.
Above-mentioned gas is good with nitrogen.
When meeting lipid microbubble easily by ultrasonic breaking up, inventor also finds, if control bad, obtained doxorubicin lipid microbubble efficiency of transmission is in vivo slow, and the drug effect time is relatively slow, thus affects the medication of doxorubicin to a certain extent.
Further, one or more mixing in the preferred Ovum Gallus domesticus Flavus lecithin of natural phospholipid, soybean lecithin and PHOSPHATIDYL ETHANOLAMINE.
Described albumin nano granular comprises surface year doxorubicin albumin nano granular modifying folic acid, enoxolone, and year doxorubicin albumin nano granular that surface is not modified.
The preparation method of above-mentioned doxorubicin lipid microbubble, comprises the preparation of preparation and the parcel year doxorubicin albumin nano granular lipid microbubble carrying doxorubicin albumin nano granular suspension.
Carry the preparation of doxorubicin albumin nano granular suspension:
Prepare the alcoholic solution of albumin aqueous solution and doxorubicin respectively, be then slowly added drop-wise in albumin aqueous solution by doxorubicin alcoholic solution, then add glutaraldehyde, stir solidification, rotary evaporation removing ethanol, obtains medicine carrying albumin nano granular suspension.
Parcel carries the preparation of doxorubicin albumin nano granular lipid microbubble:
Phospholipid, glycerol and phosphate buffer are mixed in proportion, then add albumin nano granular suspension obtained in appropriate step 1 and heat, then insufflation gas vibration, the lipid microbubble of obtained parcel medicine carrying albumin nano granular.
The consumption of above-mentioned doxorubicin is clinical prescription amount.The present invention uses glutaraldehyde for firming agent, and its consumption is determined according to practical situation for those skilled in the art.
Inventor finds in real process, prepares in lipid microbubble process, if control bad in the above-mentioned preparation method of employing, easily make the doxorubicin lipid microbubble easy to leak obtained, poor stability, the holding time is short, thus has a strong impact on the medication effect of doxorubicin lipid microbubble.
Preferably, the concentration of above-mentioned albumin aqueous solution is 0.8% ~ 5%; The concentration of glutaraldehyde is 0.2% ~ 0.3%; The ratio of phospholipid, glycerol and phosphate buffer is 55 ~ 60: 5 ~ 8: 100.
Further, ensureing obtained doxorubicin lipid microbubble not easy to leak, when good stability, inventor also finds under study for action, if control bad, easily occurs the technical problem that obtained lipid microbubble envelop rate is low.
More preferably, the concentration of above-mentioned albumin aqueous solution is 0.8% ~ 5%; The concentration of glutaraldehyde is 0.2% ~ 0.3%; The ratio of phospholipid, glycerol and phosphate buffer is 55 ~ 60: 5 ~ 8: 100; The alcoholic solution of doxorubicin joins and drips speed control built in 0.1 ~ 6mL/min in albumin aqueous solution.
Above-mentioned percentage ratio is weight percentage.The ratio of phospholipid, glycerol and phosphate buffer is mass ratio.
Most preferably, the concentration of above-mentioned albumin aqueous solution is 0.8% ~ 5%; The concentration of glutaraldehyde is 0.25%; The ratio of phospholipid, glycerol and phosphate buffer is 55 ~ 60: 5 ~ 8: 100; The alcoholic solution of doxorubicin joins and drips speed control built in 0.5 ~ 3mL/min in albumin aqueous solution.
On the basis ensureing encapsulation efficiency, inventor finds under study for action, controls concussion time and heat time heating time bad meeting and brings the particle diameter of obtained doxorubicin lipid microbubble uneven, thus affect ratio and the efficiency of lipid microbubble permeation body inner membrance to a certain extent.
Further, the concentration of the preferred albumin aqueous solution of above-mentioned preparation method is 0.8% ~ 5%; The concentration of glutaraldehyde is 0.25%; The ratio of phospholipid, glycerol and phosphate buffer is 55 ~ 60: 5 ~ 8: 100; The alcoholic solution of doxorubicin joins and drips speed control built in 0.5 ~ 3mL/min in albumin aqueous solution; The time of shaking after insufflation gas is 5-300s; Heat time heating time is 2 ~ 3h.
Specifically, a kind of preparation method of doxorubicin lipid microbubble, comprises the steps:
1) preparation of doxorubicin albumin nano granular suspension is carried:
Compound concentration is the alcoholic solution of 0.8% ~ 5% albumin aqueous solution and doxorubicin respectively; then doxorubicin alcoholic solution is added drop-wise in albumin aqueous solution with 0.5 ~ 3mL/min; then the glutaraldehyde that appropriate concentration is 0.25% is added; stir solidification; rotary evaporation removing ethanol, obtains medicine carrying albumin nano granular suspension.
2) parcel carries the preparation of doxorubicin albumin nano granular lipid microbubble:
By phospholipid, glycerol and phosphate buffer in 55 ~ 60: 5 ~ 8: 100 ratio mixing; add albumin nano granular suspension obtained in appropriate step 1 again and heat 2 ~ 3h; then nitrogen Mechanical Method vibration 30s ~ 50s is filled with, the lipid microbubble of obtained parcel medicine carrying albumin nano granular.
Within above-mentioned year, doxorubicin albumin lipid microbubble diameter is 4 ~ 7 microns.
The microvesicle of gained of the present invention can be used for clinical by quiet note, or makes lyophilized injectable powder for clinical.
A preparation method for doxorubicin lipid microbubble lyophilized injectable powder, comprises the steps:
1) preparation of doxorubicin albumin nano granular suspension is carried:
Compound concentration is the alcoholic solution of 0.8% ~ 5% albumin aqueous solution and doxorubicin respectively; then doxorubicin alcoholic solution is added drop-wise in albumin aqueous solution with 0.5 ~ 3mL/min; then the glutaraldehyde that appropriate concentration is 0.25% is added; stir solidification; rotary evaporation removing ethanol, obtains medicine carrying albumin nano granular suspension.
2) parcel carries the preparation of doxorubicin albumin nano granular lipid microbubble:
By phospholipid, glycerol and phosphate buffer in 55 ~ 60: 5 ~ 8: 100 ratio mixing; add albumin nano granular suspension obtained in appropriate step 1 again and heat 2 ~ 3h; then nitrogen Mechanical Method vibration 30s ~ 50s is filled with, the lipid microbubble of obtained parcel medicine carrying albumin nano granular.
3), pre-freeze: point doxorubicin lipid microbubble installed is put on freeze drying box internal partition, in manual interface, if flaggy temperature is 0 DEG C, 1min (unlatching electrical heating), after inlet, treats that goods reach less than 1 DEG C and keep 60min; Open two compressors, if plate temperature-40 DEG C, 1min, make goods keep 60min below-35 DEG C; If plate temperature-11 DEG C, 60min, when goods reach-11 DEG C, goods are made to keep 60min at-11 DEG C; If plate temperature-38 DEG C, 1min, goods are made to keep 60min below-35 DEG C.
4), primary drying (sublimation drying)
To rear cabinet refrigeration, when rear cabinet temperature reaches-40 DEG C, open vacuum pump, open small butterfly valve after 2s, to rear cabinet evacuation.
When rear cabinet vacuum≤90Pa, every valve in opening, when front case vacuum≤12pa, setting conduction oil temperature is 0 DEG C, and it is 8 ± 2pa that finite quantity is revealed, and to goods heating 180min, setting conduction oil temperature is 10 DEG C, and it is 8 ± 2pa that finite quantity is revealed, and takes out white completely to goods.In whole sublimation process, forward and backward case vacuum answers≤30Pa, products temperature≤-20 DEG C, condenser temperature≤-50 DEG C.As occur abnormal conditions should slow down firing rate, stop heating or reduce plate temperature.
5), redrying (parsing-desiccation)
Setting flaggy temperature is 20 DEG C, crosses after-25 ~-20 DEG C of temperature sections until products temperature, opens finite quantity and leaks 20 ± 5 (≤30Pa), after making products temperature rise to rapidly 20 DEG C, closes finite quantity and leaks, freeze-day with constant temperature more than 5 hours.
In whole process, condenser temperature should≤-50 DEG C.The constant temperature stage, forward and backward case vacuum was≤5Pa.
Beneficial effect of the present invention:
The present invention is medicine carrying ultrasonic microbubble drug-supplying system, and enter after in body, body surface adopts ultrasonic radiation, makes microvesicle directional blasting, in vivo target site release medicine; The explosion of microvesicle simultaneously can cause " acoustic horn effect ", strengthens local blood capillary and permeability of cell membrane, promotes drug osmotic, thus realizes the target administration of medicine.
The natural phospholipid energy scavenging free radicals that the present invention selects, delays body aging; Improve lipid metabolism, prevent and treat quiet arteriosclerosis, participate in composition cell membrane, strengthen immunologic function, promote nerve conduction, improve brain activity.
The present invention is adsorbed with by nanoparticle or is connected to compared with the system of microbubble surface, the present invention prepares the lipid microbubble of parcel medicine carrying albumin nano granular first, medicine carrying albumin nano granular is wrapped in microvesicle adipose membrane inner, accurately explosion can be located in vivo under transabdominal ultrasonography irradiation, discharge the albumin nano granular of its parcel, thus improve the target administration efficiency of nanoparticle.
The present invention has relatively high drug loading and envelop rate, the blood drug level of target position can be improved, prolong drug is in the action time of target position, reduce drug degradation, improve medicine stability, and nanoparticle has the selective distribution promoting medicine, be beneficial to transmission medicine and enter pathological tissues, can drug effect be increased and reduce toxic and side effects.
The preparation method that the present invention adopts can significantly improve the drug loading of microvesicle, reduces the degraded of medicine, realizes the slow release of medicine at target position, prolong drug action time, adds the curative effect of medicine; Preparation technology of the present invention is simple, and preparation process does not relate to chemical reaction, and do not adopt the organic solvent that toxicity is large, safety is high, and environment friendly is strong, is easy to industrialization.
Doxorubicin lipid microbubble freeze-dried powder prepared by the present invention is very easy to storage and the transport of medicine, and the holding time extends greatly.
Accompanying drawing illustrates:
Image under the optical microscope (400 times) of year doxorubicin albumin nano granular lipid microbubble of Fig. 1 prepared by the embodiment of the present invention 1.
Fig. 2 is the schematic diagram carrying the lipid microbubble of doxorubicin albumin nano granular and nitrogen prepared by the present invention.
Fig. 3 is the granularmetric analysis figure that bag prepared by the present invention carries the lipid microbubble of doxorubicin albumin nano granular and nitrogen.
Fig. 4 is the external development figure that bag prepared by the present invention carries the lipid microbubble of doxorubicin albumin nano granular and nitrogen.
Fig. 5 is the rabbit liver development figure that bag prepared by the present invention carries the lipid microbubble of doxorubicin albumin nano granular and nitrogen.
Fig. 6 is the release in vitro situation of lipid microbubble in ultrasonic front and back that bag prepared by the present invention carries doxorubicin albumin nano granular and nitrogen.
Detailed description of the invention:
Describe the present invention by the following examples, but the present invention is not limited in following embodiment.
Embodiment 1
1), the preparation of lecithin mixed liquor
By Ovum Gallus domesticus Flavus lecithin: glycerol: phosphate buffer with 6: 58: 100 ratio mix homogeneously, by percentage to the quality.
2), the preparation of doxorubicin albumin nano granular
Desolvation is adopted to prepare doxorubicin albumin nano granular; precision takes 20mg bovine serum albumin and is dissolved in 2mL water; separately taking 50mg doxorubicin is dissolved in 12mL dehydrated alcohol; with the volume flow of 1mL/min, doxorubicin alcoholic solution is added drop-wise in albumin aqueous solution; add the glutaraldehyde that 100mL concentration is 0.25%, lucifuge stirs 4h solidification, in 35 DEG C of rotary evaporation removing ethanol; obtain doxorubicin albumin nano granular suspension, the results are shown in Figure 1.It is 88.48% that centrifuging records its envelop rate.
3) preparation of doxorubicin albumin nano granular lipid microbubble, is carried
The doxorubicin albumin nano granular 1mL getting the obtained lecithin mixed liquor 4mL of step 1 obtained with step 2 is mixed homogeneously, heating cultivation 2 hours, then nitrogen is filled with to saturated rear employing mechanical oscillation method concussion 30s, the obtained lipid microbubble carrying doxorubicin albumin nano granular.By gained microvesicle in optical microphotograph Microscopic observation, the results are shown in Figure 2.
4) granularmetric analysis of medicine-carrying microvesicle
Malvern MS2000 laser particle analyzer is adopted to measure the particle size distribution of medicine-carrying microvesicle.
The doxorubicin lipid microbubble prepared is added to and 800ml is housed in the beaker of the purified water of ultrasonic degas, be placed in MS2000 laser particle analyzer, 2500rpm, ultrasound intensity 15, time 2min dispersion treatment, detect particle size distribution, the grain size distribution of gained microvesicle is shown in Fig. 3.
5), by the obtained doxorubicin albumin nano granular lipid microbubble water pocket that carries wrap up, measure its development effect.Result shows that microvesicle prepared by the present invention has good external development effect (Fig. 4).
6), new zealand white rabbit liver development
With new zealand white rabbit (new zealand white rabbit derives from Medical University Of Chongqing's Experimental Animal Center) for experimental subject, after application Su Mian Xin II type (1mL/Kg) intramuscular anesthesia White Rabbit, dorsal position solid, adopt the method for self cross-reference, before radiography, by year doxorubicin albumin nano granular lipid microbubble of 1mL, inject in rabbit body through rabbit auricular vein, observe the development effect at rabbit liver position.Result shows that microvesicle prepared by the present invention has development effect (see Fig. 5) in good body.
7), external supersonic release
Application Bag filter method, investigates the obtained doxorubicin albumin nano granular lipid microbubble that carries ultrasonic or do not add the doxorubicin of different time points in ultrasonic situation and add up release rate (see Fig. 6).Result shows that, under ultrasonication, the rate of releasing drug of microvesicle improves greatly, and prove that this microvesicle has good ultrasonic response, under ultrasonication, adipose membrane breaks, and discharge the doxorubicin albumin nano granular of its parcel, rate of releasing drug improves greatly.
Comparative example 1
Change the nitrogen in embodiment 1 into air, oxygen or sulfur hexafluoride, all the other are identical with embodiment 1.Obtained lipid microbubble finds under ultrasound condition, and phonoresponse is weak, 24h release insufficient total amount 70%.
Embodiment 2-10, runs according to following parameter, and other are with embodiment 1, detects when described preparation experiment is for adding ultrasonic.
Embodiment of the present invention 8-10 is comparative example, and experimental result shows: the kind of phospholipid, the proportion relation of phospholipid, glycerol and phosphate buffer, and choosing of glutaraldehyde has very important impact to the present invention.
The experimental result display of embodiment of the present invention 1-7: by the cooperation of parameters of the present invention, envelop rate 82.3% ~ 92% can be realized, an obtained year doxorubicin albumin lipid microbubble diameter is 4 ~ 7 microns, in external, body, development effect is good, and 24h drug disposition release rate is 80 ~ 92%, has a good application prospect.
Embodiment 11
Doxorubicin lipid microbubble obtained for embodiment 1 is made freeze-dried powder
Pre-freeze: point doxorubicin lipid microbubble installed is put on freeze drying box internal partition, in manual interface, if flaggy temperature is 0 DEG C, 1min (unlatching electrical heating), after inlet, treats that goods reach less than 1 DEG C and keep 60min; Open two compressors, if plate temperature-40 DEG C, 1min, make goods keep 60min below-35 DEG C; If plate temperature-11 DEG C, 60min, when goods reach-11 DEG C, goods are made to keep 60min at-11 DEG C; If plate temperature-38 DEG C, 1min, goods are made to keep 60min below-35 DEG C.
Primary drying (sublimation drying)
To rear cabinet refrigeration, when rear cabinet temperature reaches-40 DEG C, open vacuum pump, open small butterfly valve after 2s, to rear cabinet evacuation.When rear cabinet vacuum≤90Pa, every valve in opening, when front case vacuum≤12pa, setting conduction oil temperature is 0 DEG C, and limited leakage is 8 ± 2pa, and to goods heating 180min, setting conduction oil temperature is 10 DEG C, and it is 8 ± 2pa that finite quantity is revealed, and takes out white completely to goods.In whole sublimation process, forward and backward case vacuum answers≤30Pa, products temperature≤-20 DEG C, condenser temperature≤-50 DEG C.As occur abnormal conditions should slow down firing rate, stop heating or reduce plate temperature.
Redrying (parsing-desiccation)
Setting flaggy temperature is 20 DEG C, crosses after-25 ~-20 DEG C of temperature sections until products temperature, opens finite quantity and leaks 20 ± 5 (≤30Pa), after making products temperature rise to rapidly 20 DEG C, closes finite quantity and leaks, freeze-day with constant temperature more than 5 hours.
In whole process, condenser temperature should≤-50 DEG C.The constant temperature stage, forward and backward case vacuum was≤5Pa.
Claims (10)
1. a doxorubicin lipid microbubble, is characterized in that: described lipid microbubble comprises phospholipid bilayer tunic, gas and carries the albumin nano granular of doxorubicin; The albumin nano granular of described gas and year doxorubicin is all wrapped in phospholipid bilayer tunic inside; Described albumin is bovine serum albumin.
2. doxorubicin lipid microbubble as claimed in claim 1, is characterized in that: described phospholipid is natural phospholipid.
3. doxorubicin lipid microbubble as claimed in claim 2, is characterized in that: described natural phospholipid is one or more mixing in Ovum Gallus domesticus Flavus lecithin, soybean lecithin and PHOSPHATIDYL ETHANOLAMINE.
4. doxorubicin lipid microbubble as claimed in claim 1, is characterized in that: described gas is nitrogen.
5. the preparation method of doxorubicin lipid microbubble as described in any one of claim 1-4, is characterized in that: comprise the following steps:
1) preparation of doxorubicin albumin nano granular suspension is carried:
Prepare the alcoholic solution of albumin aqueous solution and doxorubicin respectively, be then slowly added drop-wise in albumin aqueous solution by doxorubicin alcoholic solution, then add glutaraldehyde, stir solidification, rotary evaporation removing ethanol, obtains medicine carrying albumin nano granular suspension;
2) parcel carries the preparation of doxorubicin albumin nano granular lipid microbubble:
By phospholipid, glycerol and phosphate buffer in 55 ~ 60: 5 ~ 8: 100 ratio mixing; add appropriate step 1 again) in obtained albumin nano granular suspension heating; then insufflation gas vibration; the lipid microbubble of obtained parcel medicine carrying albumin nano granular, described ratio is weight percentage.
6. preparation method as claimed in claim 5, is characterized in that: the concentration of described albumin aqueous solution is 0.8% ~ 5%; The concentration of glutaraldehyde is 0.2% ~ 0.3%; The ratio of phospholipid, glycerol and phosphate buffer is 55 ~ 60: 5 ~ 8: 100.
7. the preparation method as described in claim 5 or 6, is characterized in that: the concentration of described albumin aqueous solution is 0.8% ~ 5%; The concentration of glutaraldehyde is 0.2% ~ 0.3%; The ratio of phospholipid, glycerol and phosphate buffer is 55 ~ 60: 5 ~ 8: 100; The alcoholic solution of doxorubicin joins and drips speed control built in 0.1 ~ 6mL/min in albumin aqueous solution.
8. the preparation method as described in claim 5 or 6, is characterized in that: the concentration of described albumin aqueous solution is 0.8% ~ 5%; The concentration of glutaraldehyde is 0.25%; The ratio of phospholipid, glycerol and phosphate buffer is 55 ~ 60: 5 ~ 8: 100; The alcoholic solution of doxorubicin joins and drips speed control built in 0.5 ~ 3mL/min in albumin aqueous solution.
9. preparation method as claimed in claim 5, is characterized in that: the time that described gas is filled with rear concussion is 5-300s.
10. preparation method as claimed in claim 5, is characterized in that: described heat time heating time is 2 ~ 3h.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410550470.5A CN104382854A (en) | 2014-10-09 | 2014-10-09 | Doxorubicin lipid microbubble and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410550470.5A CN104382854A (en) | 2014-10-09 | 2014-10-09 | Doxorubicin lipid microbubble and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104382854A true CN104382854A (en) | 2015-03-04 |
Family
ID=52600888
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410550470.5A Pending CN104382854A (en) | 2014-10-09 | 2014-10-09 | Doxorubicin lipid microbubble and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104382854A (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1440816A (en) * | 1996-10-28 | 2003-09-10 | 奈科姆成像有限公司 | Improvement for diagnosis and/or therapeutic agnet and its relative improvement |
| US20040126400A1 (en) * | 2002-05-03 | 2004-07-01 | Iversen Patrick L. | Delivery of therapeutic compounds via microparticles or microbubbles |
| CN101346131A (en) * | 2005-12-27 | 2009-01-14 | Lts罗曼治疗方法有限公司 | Protein-based delivery system for overcoming resistance in tumour cells |
| WO2014065513A1 (en) * | 2012-10-25 | 2014-05-01 | 서강대학교 산학협력단 | Ultrasound contrast medium in which nanoparticles containing drug are combined, and preparation method therefor |
-
2014
- 2014-10-09 CN CN201410550470.5A patent/CN104382854A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1440816A (en) * | 1996-10-28 | 2003-09-10 | 奈科姆成像有限公司 | Improvement for diagnosis and/or therapeutic agnet and its relative improvement |
| US20040126400A1 (en) * | 2002-05-03 | 2004-07-01 | Iversen Patrick L. | Delivery of therapeutic compounds via microparticles or microbubbles |
| CN101346131A (en) * | 2005-12-27 | 2009-01-14 | Lts罗曼治疗方法有限公司 | Protein-based delivery system for overcoming resistance in tumour cells |
| WO2014065513A1 (en) * | 2012-10-25 | 2014-05-01 | 서강대학교 산학협력단 | Ultrasound contrast medium in which nanoparticles containing drug are combined, and preparation method therefor |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| He et al. | Image-guided hydrogen gas delivery for protection from myocardial ischemia–reperfusion injury via microbubbles | |
| Cao et al. | Drug release from phase-changeable nanodroplets triggered by low-intensity focused ultrasound | |
| Li et al. | Safe and efficient 2D molybdenum disulfide platform for cooperative imaging-guided photothermal-selective chemotherapy: A preclinical study | |
| Wu et al. | A tumor treatment strategy based on biodegradable BSA@ ZIF-8 for simultaneously ablating tumors and inhibiting infection | |
| CN110215438B (en) | Preparation method and application of mesoporous silicon nanoparticles carrying anthracyclines and photosensitizers | |
| CN109276542A (en) | A near-infrared responsive carbon dot photothermal therapy reagent and preparation method thereof | |
| Liu et al. | Improving acute cardiac transplantation rejection therapy using ultrasound-targeted FK506-loaded microbubbles in rats | |
| KR101487088B1 (en) | Ultrasound contrast agent with nanoparticles including drug and method for preparing the same | |
| Xu et al. | Microwave-activated nanodroplet vaporization for highly efficient tumor ablation with real-time monitoring performance | |
| Ao et al. | STING agonist-based hydrogel enhances immune activation in synergy with radiofrequency ablation for hepatocellular carcinoma treatment | |
| Semcheddine et al. | Rapid and label-free cancer theranostics via in situ bio-self-assembled DNA–gold nanostructures loaded exosomes | |
| Mao et al. | Tumor microenvironment-activated self-charge-generable metallosupramolecular polymer nanocapsules for photoacoustic imaging-guided targeted synergistic photothermal-chemotherapy | |
| Hou et al. | Cryoablation-activated enhanced nanodoxorubicin release for the therapy of chemoresistant mammary cancer stem-like cells | |
| CN104096245A (en) | Ultrasound lipid microbubble wrapping drug-carrying albumin nanoparticles and preparation method thereof | |
| Li et al. | Remodeling the tumor microenvironment to improve drug permeation and antitumor effects by co-delivering quercetin and doxorubicin | |
| CN105999314A (en) | Ultrasonic contrast agent and preparing method thereof | |
| CN111450252B (en) | Medicine for targeted blocking of tumor blood vessels and preparation method and application thereof | |
| Li et al. | MnO 2/Ce6 microbubble-mediated hypoxia modulation for enhancing sono-photodynamic therapy against triple negative breast cancer | |
| CN115068636B (en) | Bionic co-delivery system based on ginsenoside and ultrasound combined microbubble contrast agent | |
| Shao et al. | Glucose oxidase and MnO2 functionalized liposome for catalytic radiosensitization enhanced synergistic breast cancer therapy | |
| CN104382904A (en) | Vincristine lipid microbubble and preparation method thereof | |
| Zhu et al. | An Fe–Cu bimetallic organic framework as a microwave sensitizer for treating tumors using combined microwave thermotherapy and chemodynamic therapy | |
| CN104337766A (en) | Digoxin lipid microbubble and preparation method thereof | |
| CN113301904B (en) | Surface modified air bag and preparation and application thereof | |
| CN104382854A (en) | Doxorubicin lipid microbubble and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| DD01 | Delivery of document by public notice |
Addressee: Tang Chunlin Document name: the First Notification of an Office Action |
|
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20150304 |
|
| RJ01 | Rejection of invention patent application after publication |