CN105963278B - A kind of preparation method for the adriamycin controlled release chitosan nanoparticle answered with pH/ redox double-bang firecracker - Google Patents
A kind of preparation method for the adriamycin controlled release chitosan nanoparticle answered with pH/ redox double-bang firecracker Download PDFInfo
- Publication number
- CN105963278B CN105963278B CN201610512675.3A CN201610512675A CN105963278B CN 105963278 B CN105963278 B CN 105963278B CN 201610512675 A CN201610512675 A CN 201610512675A CN 105963278 B CN105963278 B CN 105963278B
- Authority
- CN
- China
- Prior art keywords
- chitosan
- solution
- mixed liquor
- adriamycin
- thio
- 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
Classifications
-
- 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
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/513—Organic macromolecular compounds; Dendrimers
- A61K9/5161—Polysaccharides, e.g. alginate, chitosan, cellulose derivatives; Cyclodextrin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
-
- 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
- A61K9/0002—Galenical forms characterised by the drug release technique; Application systems commanded by energy
-
- 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
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/5115—Inorganic compounds
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Physics & Mathematics (AREA)
- Optics & Photonics (AREA)
- Biomedical Technology (AREA)
- Nanotechnology (AREA)
- Inorganic Chemistry (AREA)
- Molecular Biology (AREA)
- Medicinal Preparation (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The present invention relates to a kind of preparation methods of adriamycin controlled release chitosan nanoparticle answered with pH/ redox double-bang firecracker.The present invention, which uses, uses thio chitosan, carboxymethyl chitosan, sodium tripolyphosphate, doxorubicin hydrochloride, hydrogen peroxide is raw material, thio chitosan solution is mixed into obtain mixed liquor one with doxorubicin hydrochloride solution, carboxymethyl chitosan solution is mixed to obtain to mixed liquor two with sodium tripolyphosphate solution, under stirring, mixed liquor one is instilled in mixed liquor two, control the dosage of carboxymethyl chitosan and thio chitosan, solution acid alkalinity is adjusted to pH6-8, ultrasound mixes, complete ionomer and polymer agglomerates, and it is oxidation cross-linked that hydrogen peroxide completion is added dropwise in backward system, Reaction Separation product is stirred at room temperature, it is dried to obtain the adriamycin controlled release chitosan nanoparticle answered with pH/ redox double-bang firecracker, can escape tumour cell inner body/lysosome, it can be based on tumour cell unique microenvironment, cytoplasm release is realized in spontaneous regulation, improve Ah Mycin passs drug effect rate and drug effect, has good research and development application background at many aspects such as medicine, medical material.
Description
Technical field
The present invention relates to a kind of preparation sides of adriamycin controlled release chitosan nanoparticle answered with pH/ redox double-bang firecracker
Method.
Background technique
Nano medication can realize that targeting conveying and slow release to chemotherapeutics thus have become a hot topic of research.For medicine
The characteristics of internal targeted delivery of object is spontaneous reply vivo environment and the release of tumor microenvironment regulating medicine, pharmaceutical carrier needs
Have the functional characteristic adaptable with it.Tumour cell has unique environmental characteristics (such as pH, reduction characteristic, temperature, enzyme etc.),
Environment sensitive carrier is by the way that single or multiple environment characteristic response, spontaneous control drug is discharged in correct space-time.On the one hand,
The interior environment of inner body/lysosome is weakly acidic (pH 4.5-6.5), and the pH sensitivity based on groups such as ortho esters, hydrazone or acetals carries
Body, can disintegrate drug release in inner body/lysosome acidic environment.But if drug is in inner body/lysosome retention, acidic environment
And the presence of enzyme will lead to medicines structure and destroy and inactivate, and drug is needed to escape from inner body/lysosome in time.O-shaped carboxymethyl
Chitosan (CMCS) is gone back on strand under the premise of retaining amino of chitosan group while having carboxylic group, become
A kind of ampholytes.As pH < 4.8, protonated amino on CMCS molecular skeleton, pH is in 4.8-6.0, the carboxylic of part
Methyl group deprotonation, CMCS reach its isoelectric point, as pH > 6, the amino of CMCS and carboxyl deprotonation.Therefore CMCS
With pH responsiveness, in inner body/lysosome acidic environment, the adsorbable proton of the carboxymethyl group of deprotonation, in initiation
Body/lysosome Permeation Swelling and disintegrate, realize nanoparticle escape.On the other hand, tumour cell matter GSH-PX activity (GSH) contains
Amount is apparently higher than normal tissue, makes tumour cell matter in high reproducibility.Reduction sensitive carrier based on disulfide bond crosslinking, such as sulfydryl
Change chitosan, can disintegrate in cytoplasm, discharges drug in cytoplasm.PH/ redox double-bang firecracker answers drug-carrying nanometer particle combinable
The unique microenvironment of tumor tissues, spontaneous regulating medicine are discharged in correct space-time.
Summary of the invention
The purpose of the present invention is to provide a kind of adriamycin controlled release chitosan nanoparticles answered with pH/ redox double-bang firecracker
Preparation method, use thio chitosan, carboxymethyl chitosan, sodium tripolyphosphate, doxorubicin hydrochloride, hydrogen peroxide for raw material,
By ionomer, oxidation cross-linked, polymer agglomerates method, load adriamycin chitosan nano that the pH/ redox double-bang firecracker of preparation is answered
The grain of rice can escape tumour cell inner body/lysosome, and cytoplasm release is realized in spontaneous regulation, and that improves adriamycin passs drug effect rate and medicine
Effect.
The specific embodiment of the invention includes the following steps.
(1) thio chitosan is dissolved in dilute acid soln, wherein dilute acid concentration is 1 mM, and thio chitosan concentration is 0.1%
(w/v), the doxorubicin hydrochloride solution that concentration is 1 mg/mL is added into the solution, 30 min are stirred at room temperature, controls hydrochloric acid Ah mould
Plain dosage obtains mixed liquor one.
(2) carboxymethyl chitosan is dissolved in deionized water, carboxymethyl chitosan concentration is 0.1%(w/v), into the solution
Addition concentration is 0.2%(w/v) sodium tripolyphosphate solution, 30 min are stirred at room temperature, control carboxymethyl chitosan and sodium tripolyphosphate
Dosage, obtain mixed liquor two.
(3) under stiring, mixed liquor one is instilled in mixed liquor two, additive amount is that carboxymethyl chitosan and sulfydryl shell are poly-
The mass ratio of sugar is 1.5-2:1, and sodium tripolyphosphate and thio chitosan mass ratio are 0.1-0.5:1, rate of addition be 30 drops/point
Clock adjusts solution acid alkalinity to pH6-8,1 h of reaction is stirred at room temperature, ultrasound mixes, and 200 W of ultrasonic power, 5 min of time open 2
S stops 1 s.
(4) hydrogen peroxide that concentration is 30% is instilled in the system of step 3, hydrogen peroxide and the free mercapto of thio chitosan
The molar ratio of base is 1:1, and 2 h of reaction are stirred at room temperature, and 15000 revs/min of 30 min separation products of centrifugation are dried to obtain solid production
Product, i.e., the adriamycin controlled release chitosan nanoparticle answered with pH/ redox double-bang firecracker.
Thio chitosan used in the present invention is that chitosan and sulfhydryl compound pass through the shapes such as amido bond, ester bond or amidino groups
At conjugate, thio chitosan molecular weight be 150 kDa, degree of substitution range be greater than 150 μm ol/g；Carboxymethyl chitosan
Molecular weight ranges be 150-1500 kDa, deacetylation is greater than 90%；Signified dilute acid soln can be concentration be 1 mM hydrochloric acid or
Person is that concentration is 1 mM acetic acid；Signified carboxymethyl chitosan can be O-CMC, be also possible to N, O- carboxymethyl
Chitosan, carboxymethyl chitosan degree of substitution range are greater than 80%；It is used that product is subjected to partial size and Zeta potential analysis, as a result
Show that the particle diameter distribution for the adriamycin controlled release chitosan nanoparticle that prepared pH/ redox double-bang firecracker is answered is relatively narrow, average grain diameter exists
150-250 nm, Zeta potential are -27 mv of -31-, and the encapsulation rate of doxorubicin hydrochloride is 54.9-70.7%.
The present invention has many advantages, such as easy to operate, and preparing technique process is easy.It is formed by the mercapto for containing doxorubicin hydrochloride
Base chitosan/carboxymethyl chitosan nano controlled release particle is spherical in shape.Significance of the invention is the nanoparticle to be formed to oxygen
Changing reduction and pH has double responsiveness, assigns the material property that the temporal characteristics that carrier passs medicine to tumour cell are adapted, can base
In tumour cell unique microenvironment, destruction of the inner body/lysosome to drug of escaping is realized and is released effectively in cytoplasm, improve drug
Transfer efficiency.There is good research and development application background at many aspects such as medicine, medical material.
Further explanation is made to the present invention below in conjunction with subordinate list, chart and embodiment.
Detailed description of the invention
The transmission electron microscope photo for the adriamycin controlled release chitosan nanoparticle that Fig. 1, pH/ redox double-bang firecracker are answered.
The adriamycin controlled release chitosan nanoparticle that Fig. 2, pH/ redox double-bang firecracker are answered releases the drug to redox responsiveness bent
Line.
The adriamycin controlled release chitosan nanoparticle that Fig. 3, pH/ redox double-bang firecracker are answered is to pH responsiveness drug release profiles.
Specific embodiment
The present invention is made below by embodiment and being further elaborated with, but the present invention is not limited to these embodiments.
Embodiment 1
2 mg thio chitosans are weighed, are dissolved in the hydrochloric acid solution of 2 mL, 1 mM, instill 0.3 mL 1 under agitation
Mg/ml doxorubicin hydrochloride solution, obtains mixed liquor one, and taking 3 mg molecular weight is that the carboxymethyl chitosan of 150 kDa is dissolved in 3 mL
In ionized water, the sodium tripolyphosphate solution for instilling 0.1 mL concentration under agitation as 2%(w/v) obtains mixed liquor two, will mix
Liquid one is added dropwise in mixed liquor two, and rate of addition is 30 drops/minute, adjusts pH value to pH 7.5, at room temperature with hydrochloric acid
It is stirred to react 1 h, ultrasound mixes, and 200 W of ultrasonic power, 5 min of time open 2 s, stop 1 s, 0.05 is added dropwise under agitation
The hydrogen peroxide of mL 30% is stirred to react 2 h, 15000 revs/min of 30 min of centrifugation at room temperature, removes supernatant, and freeze-drying is
The adriamycin controlled release chitosan nanoparticle that must have pH/ redox double-bang firecracker to answer.
Embodiment 2
2 mg thio chitosans are weighed, are dissolved in the hydrochloric acid solution of 2 mL, 1 mM, instill 0.3 mL 1 under agitation
Mg/ml doxorubicin hydrochloride solution, obtains mixed liquor one, and taking 4 mg molecular weight is that the carboxymethyl chitosan of 400 kDa is dissolved in 4 mL
In ionized water, the sodium tripolyphosphate solution for instilling 0.2 mL concentration under agitation as 2%(w/v) obtains mixed liquor two, will mix
Liquid one is added dropwise in mixed liquor two, and rate of addition is 30 drops/minute, adjusts pH value to pH 8.0, at room temperature with hydrochloric acid
It is stirred to react 1 h, ultrasound mixes, and 200 W of ultrasonic power, 5 min of time open 2 s, stop 1 s, 0.05 is added dropwise under agitation
The hydrogen peroxide of mL 30% is stirred to react 2 h, 15000 revs/min of 30 min of centrifugation at room temperature, removes supernatant, and freeze-drying is
The adriamycin controlled release chitosan nanoparticle that must have pH/ redox double-bang firecracker to answer.
Embodiment 3
2 mg thio chitosans are weighed, are dissolved in the hydrochloric acid solution of 2 mL, 1 mM, instill 0.3 mL 1 under agitation
Mg/ml doxorubicin hydrochloride solution, obtains mixed liquor one, and taking 3 mg molecular weight is that the carboxymethyl chitosan of 1500 kDa is dissolved in 3 mL
In ionized water, the sodium tripolyphosphate solution for instilling 0.5 mL concentration under agitation as 2%(w/v) obtains mixed liquor two, will mix
Liquid one is added dropwise in mixed liquor two, and rate of addition is 30 drops/minute, adjusts pH value to pH 6.0, at room temperature with hydrochloric acid
It is stirred to react 1 h, ultrasound mixes, and 200 W of ultrasonic power, 5 min of time open 2 s, stop 1 s, 0.05 is added dropwise under agitation
The hydrogen peroxide of mL 30% is stirred to react 2 h, 15000 revs/min of 30 min of centrifugation at room temperature, removes supernatant, and freeze-drying is
The adriamycin controlled release chitosan nanoparticle that must have pH/ redox double-bang firecracker to answer.
Claims (2)
1. a kind of preparation method for the adriamycin controlled release chitosan nanoparticle answered with pH/ redox double-bang firecracker, it is characterised in that
Thio chitosan solution 0.1%(w/v) mixes to obtain mixed liquor one with the doxorubicin hydrochloride solution of 1 mg/mL, by 0.2% (w/v)
Sodium tripolyphosphate solution mixed liquor two is mixed to obtain with the carboxymethyl chitosan solution of 0.1%(w/v), under stiring, by mixed liquor
One instills in mixed liquor two, wherein the mass ratio of carboxymethyl chitosan and thio chitosan is 1.5-2:1, sodium tripolyphosphate and
Thio chitosan mass ratio is 0.1-0.5:1, adjusts solution acid alkalinity to pH6-8, ultrasound is mixed, is added dropwise into above-mentioned system
The molar ratio of 30% hydrogen peroxide, hydrogen peroxide and thio chitosan free sulfhydryl groups is 1:1, stirs, obtains stable at room temperature
The nanometer disperse system of doxorubicin hydrochloride is contained, separates drying to get the product is arrived.
2. a kind of preparation for the adriamycin controlled release chitosan nanoparticle answered with pH/ redox double-bang firecracker according to right 1
Method, it is characterised in that thio chitosan is the coupling that chitosan and sulfhydryl compound are formed by amido bond, ester bond or amidino groups
Object.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610512675.3A CN105963278B (en) | 2016-07-04 | 2016-07-04 | A kind of preparation method for the adriamycin controlled release chitosan nanoparticle answered with pH/ redox double-bang firecracker |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610512675.3A CN105963278B (en) | 2016-07-04 | 2016-07-04 | A kind of preparation method for the adriamycin controlled release chitosan nanoparticle answered with pH/ redox double-bang firecracker |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105963278A CN105963278A (en) | 2016-09-28 |
| CN105963278B true CN105963278B (en) | 2018-12-04 |
Family
ID=56954278
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610512675.3A Active CN105963278B (en) | 2016-07-04 | 2016-07-04 | A kind of preparation method for the adriamycin controlled release chitosan nanoparticle answered with pH/ redox double-bang firecracker |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105963278B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108324680A (en) * | 2018-03-21 | 2018-07-27 | 武汉理工大学 | As segmented intestine targeted multiple response intelligent aqueous gel and preparation method thereof |
| CN109908105B (en) * | 2018-12-30 | 2021-05-28 | 浙江中医药大学 | Deoxycholic acid modified nano compound and preparation and application thereof |
| CN113143867B (en) * | 2020-12-25 | 2023-09-26 | 武汉理工大学 | CMCS-DSP-IPI549 anti-tumor nano-delivery system and preparation method thereof |
| CN113456611A (en) * | 2021-06-11 | 2021-10-01 | 淮阴工学院 | Double-response rapid controlled release nano-carrier and preparation method of nano-drug formed by nano-carrier |
| CN114715994B (en) * | 2022-01-24 | 2023-06-30 | 南华大学 | PH response type nano iron-based slow release material, preparation method and application |
| CN116919886B (en) | 2023-07-21 | 2024-01-30 | 中国海洋大学 | Injectable photo-thermal hydrogel based on black pigment, and preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101766820A (en) * | 2010-02-23 | 2010-07-07 | 厦门大学 | Novel method for preparation of chitosan nano carrier and functionalization thereof |
| CN103143028A (en) * | 2013-03-26 | 2013-06-12 | 中国药科大学 | Sulfhydrylated amphipathic chitosan polymer carrier as well as preparation method and application thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102203800B (en) * | 2010-01-21 | 2015-09-23 | 计量仪器公司 | Comprise the tag reader terminal of optical filter |
| WO2013069629A1 (en) * | 2011-11-11 | 2013-05-16 | 日本電気株式会社 | Wireless transmission device, failure-information forwarding method, and failure-information notification method |
-
2016
- 2016-07-04 CN CN201610512675.3A patent/CN105963278B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101766820A (en) * | 2010-02-23 | 2010-07-07 | 厦门大学 | Novel method for preparation of chitosan nano carrier and functionalization thereof |
| CN103143028A (en) * | 2013-03-26 | 2013-06-12 | 中国药科大学 | Sulfhydrylated amphipathic chitosan polymer carrier as well as preparation method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| pH/redox responsive core cross-linked nanoparticles from thiolated carboxymethyl chitosan for intro release strudy of methotrexate;Cheng Gao et al;《carbohydrate polymers》;20140513;第111卷;第964页"abstract"，第965页"materials and methods"，第970页"conclusion" * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105963278A (en) | 2016-09-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105963278B (en) | A kind of preparation method for the adriamycin controlled release chitosan nanoparticle answered with pH/ redox double-bang firecracker | |
| Jiang et al. | Natural polymer-based stimuli-responsive hydrogels | |
| Tian et al. | Smart stimuli-responsive chitosan hydrogel for drug delivery: A review | |
| Fan et al. | Development and characterization of soybean protein isolate and fucoidan nanoparticles for curcumin encapsulation | |
| Gan et al. | Modulation of surface charge, particle size and morphological properties of chitosan–TPP nanoparticles intended for gene delivery | |
| US7833765B2 (en) | Galenic formulation for colon-targeted delivery of active ingredients | |
| Li et al. | Intelligent nanogels with self-adaptive responsiveness for improved tumor drug delivery and augmented chemotherapy | |
| Chandran et al. | An electric field responsive drug delivery system based on chitosan–gold nanocomposites for site specific and controlled delivery of 5-fluorouracil | |
| Mohammadpourdounighi et al. | Preparation of chitosan nanoparticles containing Naja naja oxiana snake venom | |
| Cavalu et al. | Preparation, structural characterisation and release study of novel hybrid microspheres entrapping nanoselenium, produced by green synthesis | |
| Liu et al. | Preparation and characterization of α-galactosidase-loaded chitosan nanoparticles for use in foods | |
| Pahwa et al. | Chitosan-based gastroretentive floating drug delivery technology: an updated review | |
| Liu et al. | Preparation and evaluation of lysozyme-loaded nanoparticles coated with poly-γ-glutamic acid and chitosan | |
| Nalini et al. | In vitro cytocompatibility assessment and antibacterial effects of quercetin encapsulated alginate/chitosan nanoparticle | |
| Aminabhavi et al. | Production of chitosan-based hydrogels for biomedical applications | |
| US20080160096A1 (en) | Polymeric nanoparticles by ion-ion interactions | |
| CN105969825A (en) | Enzymatic catalysis crosslinking reduction-responsive hyaluronic acid microgel and preparation method thereof | |
| Wang et al. | Semi-permeable nanocapsules of konjac glucomannan–chitosan for enzyme immobilization | |
| Deng et al. | Tea polyphenol liposomes overcome gastric mucus to treat helicobacter pylori infection and enhance the intestinal microenvironment | |
| Singha et al. | Applications of alginate-based bionanocomposites in drug delivery | |
| Venkatesan et al. | Synthesis and characterization of chitosan tripolyphosphate nanoparticles and its encapsulation efficiency containing Russell's viper snake venom | |
| Zhang et al. | Fabrication and characterization of polydopamine-mediated zein-based nanoparticle for delivery of bioactive molecules | |
| CN108096214A (en) | A kind of magnetotactic bacteria quantum dot microcapsules and preparation method thereof | |
| CN104546725A (en) | Preparation method and application of enzyme-supported chitosan nanoparticle | |
| García-Couce et al. | Dexamethasone‐Loaded Chitosan Beads Coated with a pH‐Dependent Interpolymer Complex for Colon‐Specific Drug Delivery |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |