CN107096038A - The preparation method of crosslinking nano medicine based on active reaction type one-step method - Google Patents
The preparation method of crosslinking nano medicine based on active reaction type one-step method Download PDFInfo
- Publication number
- CN107096038A CN107096038A CN201710237726.0A CN201710237726A CN107096038A CN 107096038 A CN107096038 A CN 107096038A CN 201710237726 A CN201710237726 A CN 201710237726A CN 107096038 A CN107096038 A CN 107096038A
- Authority
- CN
- China
- Prior art keywords
- polymer
- medicine
- molecular weight
- crosslinking
- preparation
- 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.)
- Granted
Links
Classifications
-
- 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
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
- A61K31/537—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines spiro-condensed or forming part of bridged ring systems
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicinal Preparation (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a kind of preparation method of the crosslinking nano medicine based on active reaction type one-step method, following steps are specifically included, after the addition solution of the medicine by polymer and with sulfydryl is mixed, dialysis obtains crosslinking nano medicine；The polymer lateral chain contains dithiolane structure.The present invention can obtain the Nano medication of stable circulation by simply preparing, and solves prior art chemical bonding nanometer medicine and prepares complexity, needs the defects such as catalyst；And the product for preparing of the present invention has stable cross-linked structure, it is to avoid the problem of existing chemical bonding nanometer medicine is not crosslinked；On the model of lotus B16F10 melanomas, the therapeutic effect shown, tumor control rate illustrates that the active targeting crosslinking nano medicine has huge potential application in terms of cancer chemotherapy more than 90%.
Description
Technical field
The invention belongs to polymer nanocomposite technical field of pharmaceuticals, and in particular to a kind of friendship based on active reaction type one-step method
The preparation method of the stable Nano medication of connection, the Nano medication of stable circulation is can obtain by simply preparing.
Background technology
Chemotherapy is the treatment most common means of malignant tumour.In in the past few decades, in order to improve chemotherapeutics
Selectivity, reduction toxic side effect, researchers explore various polymer nanoparticle drug carriers.In general, polymer nanocomposite is carried
Body packaging medicine has two ways, and a kind of is the mode of physically trapping, it is another be formed by way of chemical bond polymer-
The conjugate of medicine.Such as, NK105 and Genexol-PM are exactly to wrap up taxol using micella（PTX）Two exemplaries, and
NK911 is the model as chemical bonding nanometer medicine.The Nano medication of physically trapping is easier to occur that stability is poor, drug leakage
The problems such as.But, chemical bonding nanometer medicine often faces the more complicated synthesis step of needs and/or in lesions position functioning efficiency
Low the problem of.
Reversible crosslink type polymer nanocomposite medicine（Such as polymer micelle and polymeric bladder soak medicine）Research shown
The strategy can solve the not enough defect of the physically encapsulation Nano medication stability of a system to a certain extent.Before this not yet
About the report of the cross-linked stable Nano medication of active reaction type one-step method.
The content of the invention
Active reaction type one-step method is based on it is an object of the invention to provide one kind（Referred to as react stowage）Crosslinking receive
The preparation method of rice medicine, by simply prepare can obtain drug substance stable be bonded, the Nano medication of stable circulation, solve
Prior art chemical bonding nanometer medicine prepares complexity, needs the defect such as catalyst and multistep preparation；And production prepared by the present invention
Product have stable cross-linked structure, it is to avoid the problem of existing chemical bonding nanometer medicine is not crosslinked.
To achieve the above object of the invention, the present invention is adopted the following technical scheme that：Crosslinking based on active reaction type one-step method
The preparation method of Nano medication, comprises the following steps, and by polymer, the medicine with sulfydryl adds after being mixed in solution and dialyse and obtain
Crosslinking nano medicine；The polymer lateral chain contains dithiolane structure.
In above-mentioned technical proposal, the solution is water or buffer solution；The polymer is biodegradable polymer；Institute
It is itself medicine with sulfydryl medicine or sulfydryl modification to state the medicine with sulfydryl.
In above-mentioned technical proposal, the polymer is target polymer and/or non-targeted polymer；In the present invention, two sulphur
The sulfur-crosslinked stability that superelevation is shown under physiological environment of sulphur that penta ring structure is provided, and in cytoplasmic reproducibility environment
Can rapid delivery of pharmaceuticals because reduction potential two to three orders of magnitude higher than the circulatory system in tumour cell.
In above-mentioned technical proposal, the non-targeted polymer contained by side chain dithiolane structure carbonate monomer, its
His monomer, hydrophilic high polymers reaction are obtained；In the non-targeted polymer, the total molecular weight of hydrophobic segment is hydrophilic segment molecule
0.3~7 times of amount；The total molecular weight that the side chain contains the carbonate monomer construction unit of dithiolane structure is other monomers
The total molecular weight of construction unit contains the 8% of the carbonate monomer construction unit total molecular weight sum of dithiolane structure with side chain
~60%；
Carbonate monomer, other monomers, hydrophilic high polymers, the target for containing dithiolane structure by side chain with target polymer
Obtained to molecule reaction；In the target polymer, the total molecular weight of hydrophobic segment is 0.3~7 times of hydrophilic segment molecular weight；
It is the total of other monomers construction unit that the side chain, which contains the total molecular weight of the carbonate monomer construction unit of dithiolane structure,
Molecular weight contains the 8%~60% of the carbonate monomer construction unit total molecular weight sum of dithiolane structure with side chain；
Or the target polymer contained by side chain dithiolane structure carbonate monomer, other monomers, hyaluronic acid it is anti-
It should obtain；In the target polymer, the total molecular weight of hydrophobic segment is 0.3~7 times of hydrophilic segment molecular weight；The side chain
The total molecular weight of carbonate monomer construction unit containing dithiolane structure for other monomers construction unit total molecular weight with
Side chain contains the 8%~60% of the carbonate monomer construction unit total molecular weight sum of dithiolane structure.
In above-mentioned technical proposal, the other monomers are trimethylene carbonate（TMC）, lactide（LA）And caprolactone
（CL）；The chemical structural formula for the carbonate monomer that side chain contains dithiolane structure is
In the present invention, carbonate monomer and the other monomers copolymerization of dithiolane structure are contained by side chain, or in conjunction with targeting
Molecule or hydrophilic high polymers, obtain the biodegradable polymer that side chain contains dithiolane structure.Obtained polymer knot
In structure, hydrophobic segment includes the carbonate monomer construction unit and other monomers construction unit that side chain contains dithiolane structure,
Hydrophilic high polymers constitute hydrophilic segment, limit the total molecular weight of hydrophobic segment and the ratio and hydrophobic chain of hydrophilic segment molecular weight
The ratio of different structure unit, can obtain the Nano medication of different structure in section.When the total molecular weight of hydrophobic segment is hydrophilic
0.3~1.5 times of chain segment molecular weight, the total molecular weight that side chain contains the carbonate monomer construction unit of dithiolane structure is it
The total molecular weight and side chain of his monomeric building blocks contain the carbonate monomer structure total molecular weight sum of dithiolane structure
15%~60%, obtain nano-micelle structure；When 1.6~7 times that the total molecular weight of hydrophobic segment is hydrophilic segment molecular weight, side chain
The total molecular weight of carbonate monomer construction unit containing dithiolane structure for other monomers construction unit total molecular weight with
Side chain contains the 8%~30% of the carbonate monomer structure total molecular weight sum of dithiolane structure, obtains nano vesicle structure.
In above-mentioned technical proposal, the targeted molecular is polypeptide（Such as cRGD, cNGQ, CC9）, antibody, folic acid, hyaluronic acid
Deng；Hydrophilic high polymers are polyethylene glycol, including modify unmodified etc..In the presence of hydrophilic high polymers, in the polymer of preparation, parent
The molecular weight of water segment is 3000-10000Da；Such as polymer of the invention can be following structure：
Wherein, R1ForOr；
R2For;
Wherein, R3For
In above-mentioned technical proposal, it is described mixing under air, room temperature carry out.Preparation process of the present invention is extremely simple, obtained production
Thing is the stable crosslinking nano medicines structure for carrying medicine, and medicine forms stable chemical bond with polymer；Solve existing medicine with
Polymer, which is combined, needs complicated or harsh conditions, such as the defect such as sealing, heating, multi-step；The friendship that particularly present invention is obtained
Join Nano medication load medicine highly stable, achieve unexpected technique effect.
The present invention further discloses crosslinking nano medicine and the crosslinking nano medicine prepared by above-mentioned preparation method
Application in antineoplastic is prepared.The targeting that novel nano drug system such as disclosed by the invention is used for melanoma is controlled
Treat, the system loads medicine, and the characteristic with ultrastability and triggering release by active reaction type one-step method.
The crosslinking nano medicine of the present invention is such as that targeted molecular cRGD is modified, medicine is maytansine（DM1）Vesica
Nano medication（cRGD-PS-DM1）, gathered by copolymer polyethylene glycol-b-（The methylene of trimethylene carbonate-co- dithiolanes three
Base carbonic ester）（PEG-P(TMC-DTC)）Gather with the polyethylene glycol-b- that cRGD is modified（Trimethylene carbonate-co- dithiolanes
Trimethylene carbonate）（cRGD-PEG-P(TMC-DTC)）While self assembly by active reaction type one-step method, utilize mercapto
Base-sulphur sulphur exchange reaction loads maytansine DM1, and the sulfur-crosslinked vesica Nano medication for forming stabilization of sulphur being catalyzed on vesica film.
Present invention firstly discloses a kind of preparation method of the crosslinking nano medicine based on active reaction type one-step method, pass through
Simple preparation can obtain DM1 drugloading rates in the Nano medication of stable circulation, crosslinking nano medicine and be up to 15 wt%, in PB
In in 24 hours medicine leakage it is negligible；Chemical bonding nanometer medicine in the prior art is solved to prepare complexity, need catalysis
The defects such as agent；And the product for preparing of the present invention has stable cross-linked structure, it is to avoid existing chemical bonding nanometer medicine is not handed over
The problem of connection.
Product prepared by the present invention being capable of specific recognition αvβ3The tumour cell that integral protein is overexpressed, while reduction pair
The toxicity of normal cell；MTD results of study show that the MTD values of crosslinking nano medicine are 4 mg DM1 equiv./kg, with dissociating
DM1 compared to adding 5 times, it is existing be by can not the MTD values of antibody-maytansine conjugate that connect of break chemical bonds also only
It is 2.7 mg DM1 equiv./kg.So, structure and few medicine leakage of the present invention by its stability superelevation are received
The maximum tolerance of rice medicine in vivo is greatly improved, and achieves unexpected technique effect.
On the model of lotus B16F10 melanomas, product cRGD-PS-DM1 prepared by the present invention is than non-targeted PS-
DM1 shows more preferable therapeutic effect, and especially for this grade malignancy very high tumor model, its tumor control rate exceedes
90%, illustrate that the active targeting crosslinking nano medicine has huge potential application in terms of cancer chemotherapy.
Brief description of the drawings
Fig. 1 is the nmr spectrum of copolymer p EG-P (TMC-DTC) in embodiment one；
Fig. 2 is copolymer cRGD-PEG-P (TMC-DTC) nmr spectrum in embodiment one；
Fig. 3 is the grain size distribution of Nano medication cRGD-PS-DM1 in embodiment one；
Fig. 4 is the electronic transmission electron microscope of Nano medication cRGD-PS-DM1 in embodiment one；
Fig. 5 is the ultraviolet absorpting spectrum of Nano medication cRGD-PS-DM1 in embodiment one；
Fig. 6 is the stability test figure of Nano medication cRGD-PS-DM1 in embodiment one；
The high-efficient liquid phase chromatogram that Fig. 7 is free DM1 and Nano medication cRGD-PS-DM1 in embodiment one；
The external release profile that Fig. 8 is Nano medication cRGD-PS-DM1 in embodiment one；
Fig. 9 is toxicity data figures of the cRGD-PS-DM1 in embodiment one to B16F10 melanoma cells；
Figure 10 is toxicity data figures of the cRGD-PS-DM1 in embodiment one to L929 cells；
Figure 11 is to the cell endocytic result figures of B16F10 melanoma cells in embodiment one；
Figure 12 is maximal tolerance dose result figures of the cRGD-PS-DM1 in embodiment one to mouse；
Figure 13 is treatment results figures of the cRGD-PS-DM1 in embodiment one to lotus melanoma mouse, and wherein A is that tumour growth is bent
Line, B is tumour picture after mouse treatment, and C is tumour inhibiting rate, and D is changes of weight, E curves for survival；
Figure 14 is H＆E dyeing of the cRGD-PS-DM1 to the major organs section after the treatment of lotus melanoma mouse in embodiment one
Figure；
Figure 15 is alkynyl-modified HA, N in embodiment six3- P (TMC-DTC), HA-P (TMC-DTC) nmr spectrum；
Figure 16 is alkynyl-modified HA, N in embodiment six3- P (TMC-DTC), HA-P (TMC-DTC) infrared absorption spectra；
Figure 17 is HA-P (TMC-DTC) vesica sign in embodiment six, and A is particle diameter distribution and electronic transmission electron microscope, and B is stable
Property test chart, C is high-efficient liquid phase chromatogram, D for reduction response test chart.
Embodiment
Embodiment one
CRGD-PS-DM1 crosslinking nanos medicine is by copolymer p EG-P (TMC-DTC) and cRGD-PEG-P (TMC-DTC) (w/w
80/20) DM1 being wrapped up the self assembly while and directly being formed, sulphur sulphur-sulfydryl exchange reaction and sulphur are realized in the invention
It is sulfur-crosslinked to carry out simultaneously.
The present embodiment general reaction thinking is：
Under nitrogen protection, by NHS-PEG-OH（M n=7.5 kg/mol, 75 mg, 10 μm of ol）, TMC（200 mg, 1.6
mmol）And DTC（50 mg, 0.26 mmol）It is dissolved in 1.1 mL anhydrous methylene chlorides, is subsequently added diphenyl phosphate（DPP, 25
Mg, 100 μm of ol）Reacted in closed reactor under the conditions of 30 DEG C after 72 h, terminate, precipitated twice in ice ether, suction filtration is simultaneously
It is dried in vacuum overnight, obtains NHS-PEG-P (TMC-DTC).Yield：84.6%.1H NMR (600 MHz, CDCl3): PEG: δ
3.64; TMC: δ 2.06, 4.24; DTC: δ 3.02, 4.19; NHS: δ 2.52. M n (1H NMR) =31.8 kg/
mol. M n (GPC) = 34.8 kg/mol, M w /M n= 1.14；Then the copolymer NHS-PEG-P (TMC-DTC) obtained
（200 mg, 6.5 μm of ol）With polypeptide cRGD in 5mL DMF（8 mg, 13.2 μm of ol）48h is reacted at room temperature, after dialysis
To product cRGD-PEG-P (TMC-DTC).Yield：89.3%.CRGD grafting rate is by BCA protein reagent boxes（Thermo
scientific）Detection is obtained, and grafting rate is 94%.
PEG-P (TMC-DTC) is by initiator of poly glycol monomethyl ether, bicyclic (4.4.0) the decyl- 5- alkene of the nitrine of 1,5,7- tri-
（TBD）For catalyst, by being obtained with above similar polymerization.Yield：93.2%.M n (1H NMR) =29.8 kg/
mol, M n(GPC) =33.9 kg/mol, M w /M n =1.38。
The chemical structural formula of polymer manufactured in the present embodiment is as follows, characterizes such as table 1：
The PEG-P of table 1 (TMC-DTC) and NHS-PEG-P (TMC-DTC) sign
a1H NMR；b GPC
Copolymer p EG-P (TMC-DTC) and cRGD-PEG-P (TMC-DTC) molecular weight pass through1H NMR are characterized, and see accompanying drawing
1st, accompanying drawing 2.As it can be seen from table 1 PEG-P (TMC-DTC) and NHS-PEG-P (TMC-DTC) number-average molecular weight are with design
Molecular weight all very close to molecular weight distribution is very narrow, embodies good controlling.The hydrophilic section and hydrophobic section of two kinds of copolymers
Ratio be 19.4 wt.% and 30.9 wt.%, be more likely to form vesica.The total molecular weight of hydrophobic segment is respectively in the two
5.16 times of hydrophilic segment molecular weight and 3.24 times.
DM1, PEG-P (TMC-DTC) will be mixed with) and cRGD-PEG-P (TMC-DTC) the lower direct injection of DMF solution stirring
Into phosphate buffer（The mM of PB, pH 7.4,10）, crosslinking nano medicine, referred to as cRGD- are just directly formd after dialysis
PS-DM1, is PS-DM1 without targeted molecular.Usually, by 100 μ L cRGD-PEG-P (TMC-DTC)（2 mg/mL）、
PEG-P(TMC-DTC)（8 mg/mL）And DM1（2 mg/mL）DMF solution be slowly expelled to 900 μ L phosphate buffer
In（PB, 10 mM, pH 7.4）, standing was dialysis medium dialysis 48 with the PB containing 0.5 % Tween 80s after 4 hours at room temperature
Hour（Spectra/Pore, MWCO 14000）.The hydrodynamics size of the Nano medication is distributed very narrow in 100 nm or so
（PDI is 0.11）, see accompanying drawing 3；Transmission electron microscope photo（TEM）（Accompanying drawing 4）It also demonstrate that the vesica knot of the Nano medication
Structure, size is close with DLS results.Ultraviolet absorpting spectrum is shown（Accompanying drawing 5）, dithiolane is 330 after the completion of prepared by Nano medication
Nm characteristic absorption peak disappears, and illustrates to realize self-crosslinking by way of sulphur sulfide linkage is exchanged；Nano medication is in 100 times of dilutions
Higher stability is kept with 10% FBS solution（Accompanying drawing 6）, proved the structure of its cross-linked stable.
The sign of the crosslinking nano medicine of table 2
a DLS room temperatures are determined in PB buffer solutions (mM of pH 7.4,10), 1.0 mg/mL；b HPLC is determined
In order to prove that DM1 is to be connected to by way of disulfide bond on vesica, cRGD-PS-DM1 nanometers of medicines are investigated respectively with HPLC
Thing is in 10 mM dithiothreitol (DTT)s（DTT）Change before and after the processing.As a result show that the Nano medication has higher drugloading rate, DLC
For 15.5 wt.%（Table 2）.Particularly, in HPLC spectrograms（Accompanying drawing 7）, sample cRGD-PS-DM1 do not detect free DM1
Presence, it is to be connected to by way of covalent bond above vesica to illustrate DM1.And pass through dithiothreitol (DTT)（DTT）After processing,
DM1 peak is reappeared, and shows that reducing environment can trigger DM1 release, and can be restored to prototype structure.cRGD-PS-
Release in vitro results of the DM1 in 2-10 mM GSH environment shows that, by the stability of its superelevation, crosslinking nano medicine is in PB
In 24 hours DM1 leakage can be ignored；On the contrary, DM1 quick releases come out in reproducibility environment, as shown in Figure 8, point
The DM1 for not having 73.4%, 60.7% or 49.8% is discharged in 10,5 or 2 mM GSH solution.
Nano medication Anticancer Activity in vitro
The active anticancer of cRGD-PS-DM1 Nano medications is assessed with MTT experiment（Accompanying drawing 9）, cRGD-PS-DM1 Nano medications pair
αvβ3The B16F10 melanoma cells that integral protein is overexpressed have very strong inhibition, its IC50It is worth for 8.7 ng DM1
Equiv./mL, the 1/5 of about non-active targeting control group PS-DM1 and free DM1.To αvβ3The Negative control mice of low expression
Fibroblast L929, cRGD-PS-DM1 and PS-DM1 has close cytotoxicity, and significantly lower than free DM1（Accompanying drawing
10）.The above results show that cRGD-PS-DM1 being capable of specific recognition αvβ3The tumour cell that integral protein is overexpressed, is reduced simultaneously
To the toxicity of normal cell.In order to further study endocytosis behavior of the B16F10 cells to Nano medication, mark on polymer
Cy5 is remembered, has been observed with laser confocal microscope.After being incubated 4 hours, cRGD-PS-Cy5 is in Cy5 intracellular B16F10
Fluorescence intensity is apparently higher than non-targeted control group PS-Cy5（Accompanying drawing 11）, illustrate that cRGD-PS is efficiently entering B16F10 intracellular.
Maximum tolerated dose is studied in Mice Body
DM1 is a kind of toxicity very strong tubulin polymerization inhibitor, its maximum tolerated dose（MTD）It is very low, to liver,
Marrow, lymphoid organ, intestines and stomach or nervous system have very strong toxic side effect；MTD results of study show that cRGD-PS-DM1 receives
The MTD values of rice medicine are 4 mg DM1 equiv./kg（Accompanying drawing 12）, 5 times are added compared with free DM1.It is worth one herein
It is mentioned that, the MTD values of the antibody connected by sulphur sulfide linkage-maytansine conjugate are about 0.9 mg DM1 equiv./kg, existing
Even by can not break chemical bonds connection antibody-maytansine conjugate MTD values also only 2.7 mg DM1
equiv./kg.So, structure and few leakage of the present invention by its stability superelevation so that medicine is in vivo most
Big tolerance is greatly improved, and achieves unexpected technique effect.Internal antitumor activity
Nano medication and free DM1 antitumor activity are have evaluated on the mouse model of lotus B16F10 knurls.In view of nanometer medicine
The difference of thing and free DM1 maximum tolerated doses, have studied the antitumor behavior of different groups under two dosage.Accompanying drawing 13 is nanometer
Medicine and free DM1 antitumous effects figure；The suppression of cRGD-PS-DM1 treatment groups under 0.4 mg DM1 equiv./kg dosage
Knurl effect is considerably better than free DM1 groups（**p < 0.01）, when dosage reaches 1.2 mg DM1 equiv./kg, cRGD-PS-
DM1 treatment groups have substantially completely inhibited the growth of tumour, and in the case where other conditions are consistent, inhibition is obvious
Better than non-active targeted therapy group PS-DM1（Accompanying drawing 13A）；The lump photo taken out after 12 days is it can also be seen that cRGD-
PS-DM1 is under 1.2 mg DM1 equiv./kg dosage, and with best tumor killing effect, gross tumor volume is minimum（Accompanying drawing
13B）；Tumour inhibiting rates of the cRGD-PS-DM1 and PS-DM1 when dosage is 1.2 mg DM1 equiv./kg（TIR）Respectively 93%
With 70%（Accompanying drawing 13C）；Calculated and obtained by tumor quality.Within the whole observation cycle, the body weight of the mouse of each group is all without obvious
Reduction, illustrate with and without cRGD active targeting groups, toxic side effect all very littles of Nano medication（Accompanying drawing 13D）；Simultaneously
The survival rate survivorship curve of mouse shows that cRGD-PS-DM1 significantly improves the survival rate of mouse；CRGD-PS-DM1 treatment groups exist
When dosage is 0.4 mg DM1 equiv./kg and 1.2 mg DM1 equiv./kg, median survival is respectively 19 days and 28 days.
By contrast, PS-DM1 (1.2 mg/kg), free DM1 (0.4 mg/kg) and PBS treatment groups median survival are respectively 18
My god, 14 days and 12 days（Accompanying drawing 13E）.H＆E staining analysis results are shown（Accompanying drawing 14）, each treatment group is to major organs all without table
Reveal obvious toxic side effect.Greatly reduce DM1 systemic toxicity this demonstrates the design of Nano medication, even in compared with
Under the conditions of high dose and multiple dosing, mouse is still kept fit.
The above results all show, on the model of lotus B16F10 melanomas, and cRGD-PS-DM1 is than non-active targeting
PS-DM1 shows more preferable therapeutic effect, and cRGD-PS-DM1 inhibiting rate illustrates the active targeting crosslinking nano more than 90%
Medicine has huge potential application in terms of cancer chemotherapy.
Embodiment two
According to embodiment one, polymer P EG-P (TMC-DTC) and MAL-PEG-P (TMC-DTC) are prepared, prepares and targets from the latter
Molecule is the target polymer Anti-CD19-PEG-P (TMC-DTC) of anti-CD19 antibody, and chemical structural formula is as follows：
Crosslinking nano medicine and targeting crosslinking nano medicine are further prepared, is the vesica knot of particle diameter 90-100 rans
Structure；Maytansine DM1 is loaded, drugloading rate DLC is up to 10-15wt.%；MTD results of study display crosslinking vesica Nano medication and targeting
It is crosslinked above free nearly 4 times of the DM1 of MTD values of vesica Nano medication；It has acute leukemia to mouse subcutaneous transplanting（AML-2）
The tumour inhibiting rate of the mouse of tumour（TIR）Respectively more than 60% and 90%；Mouse survival intermediate value is respectively more than 35 days, 60 days（PBS is 25
My god）, to the toxic side effect very little of the main organs of mouse.
Embodiment three
According to embodiment one, TMC is replaced by LA and prepares polymer P EG-P (LA-DTC) and NHS-PEG-P (LA-DTC), from
The latter prepares the target polymer GE11-PEG-P (LA-DTC) that targeted molecular is EGFR target polypeptides GE11, and chemical structural formula is such as
Under：
Crosslinking nano medicine and targeting crosslinking nano medicine are further prepared, is the vesica knot of particle diameter 50-70 rans
Structure；Load the medicine Ismipur for itself carrying sulfydryl（6MP）, drugloading rate DLC is 5-10 wt.%；MTD results of study are shown
The MTD values of crosslinking vesica Nano medication and targeting crosslinking vesica Nano medication are 6 times of free 6MP；It is crosslinked vesica Nano medication
With tumour inhibiting rate of the targeting crosslinking vesica Nano medication to three cloudy breast cancer MDA-MB-231 hypodermic tumours（TIR）Respectively more than 60%
With 85%；Mouse survival intermediate value is respectively more than 47 days, 58 days（PBS is 30 days）, it is secondary to almost no poison of the main organs of mouse
Effect.
Example IV
According to embodiment one, TMC is replaced by CL, polymer P EG-P (CL-DTC) and NHS-PEG-P (CL-DTC) is prepared, from
The latter prepares the target polymer cNGQ-PEG-P (CL-DTC) that targeted molecular is ring type polypeptide cNGQ, and chemical structural formula is as follows：
Crosslinking nano medicine and targeting crosslinking nano medicine are further prepared, is the micella knot of particle diameter 30-50 rans
Structure；Load the medicine MMAE of sulfydryl modification（Monomethyl auristatin E, the auspicious statin E of methyl Australia, antimitotic agent,
Can be by blocking the Inhibition of polymerization cell division of tubulin）, drugloading rate DLC is up to 18 wt.%；The display of MTD results of study is handed over
The MTD values of connection micelle nano medicine and targeting crosslinking micella Nano medication are 7 times or so of the MMAE of free sulfhydryl groups modification；Crosslinking
Micelle nano medicine has the tumor suppression of the mouse of non-small cell lung cancer A549 hypodermic tumours with targeting crosslinking micella Nano medication to lotus
Rate（TIR）Respectively more than 65% and 95%；Mouse survival intermediate value is respectively more than 48 days, 60 days（PBS is 29 days）.It is crosslinked micelle nano
Medicine with targeting crosslinking micella Nano medication have to lotus non-small cell lung cancer A549 in situ tumors mouse lung bioluminescence and
The decline that the mouse lung bioluminescence of PBS processing is compared is respectively more than 60% and 99%；Mouse survival intermediate value respectively more than 47 days,
68 days（PBS is 20 days）.Almost no toxic side effect of the targeting crosslinking micella Nano medication to the main organs of mouse.
Embodiment five
According to embodiment one, TMC is replaced by CL, polymer P EG-P (CL-DTC) and MAL-PEG-P (CL-DTC) is prepared, from
The latter prepares targeted molecular for folic acid（Use hydrosulphonyl functionalized folic acid）Target polymer FA-PEG-P (CL-DTC), chemistry
Structural formula is as follows：
Crosslinking nano medicine and targeting crosslinking nano medicine are further prepared, is the micella knot of particle diameter 20-40 rans
Structure；The toxic polypeptide medicine LTX-315 (CKKWWKKWDipK-NH2) of sulfydryl modification is loaded, drugloading rate DLC is up to 12 wt.%；
Crosslinking micella Nano medication and targeting crosslinking micella Nano medication have the mouse of non-small cell lung cancer H460 hypodermic tumours to lotus
Tumour inhibiting rate（TIR）Respectively more than 68% and 94%；Mouse survival intermediate value is respectively more than 42 days, 61 days（PBS is 28 days）, targeting crosslinking
Almost no toxic side effect of the micelle nano medicine to the main organs of mouse.
Embodiment six
The hydrophilic section PEG of polymer in embodiment one is replaced by the hyaluronic acid with CD44 active targeting functions（HA）Can be with
Obtain the following polymer of chemical structural formula（HA-P(TMC-DTC)）.
Specifically, HA-P (TMC-DTC) is synthesized by three steps.
Synthesize alkynyl hyaluronic acid（HA-alkynyl）It is to be obtained by HA terminal aldehyde groups and propargylamine amino reduction amination
Arrive.N2It is lower by HA（3.0 g, 0.375 mmol）In the deionized water for being dissolved in 20 mL, propargylamine is then sequentially added
（82.6 mg, 1.5 mmol）And NaCNBH3（94.3 mg, 1.5 mmol）, 48 h are reacted at 60 DEG C, then 40 oC react
48 h.After reaction terminates, dialysed in deionized water（MWCO 3500）48 h.Finally solution is freeze-dried.Yield：85%.HA-
Propargylamine is to form two grades of aldimines by the aldehyde radical of HA ends and the amino of propargylamine（Schiff bases）Afterwards, in sodium cyanoborohydride
Reducing condition under, schiff bases are reduced into secondary amine structure.Nuclear magnetic spectrogram is shown（Accompanying drawing 15A）, except HA signal peak (δ
1.86,3.28-4.02,4.21-4.75) beyond, there is new signal peak to occur at δ 2.64,2.82,3.21, be HA
Terminal aldehyde group forms the methylene proton peak beside secondary amine and the methylene proton peak beside nitrine after being reacted with propargylamine.Compare HA features
Peak (δ 4.21-4.75) understands that alkynyl-functional's degree is 100% with signal peak integral area at δ 2.64,2.82,3.21.
N3- P (TMC-DTC) synthesis such as embodiment one, is that, using nitrine ethanol as initiator, diphenyl phosphate is as urging
Prepared by agent ring-opening polymerisation, yield 92%.Nuclear-magnetism figure（Accompanying drawing 15B）In show PTMC characteristic peak（δ 4.24 and 2.05
ppm）And PDTC characteristic peak（The ppm of δ 4.32 and 3.03）And methylene at the ppm of δ 3.73 of nitrine side.Number-average molecular weight
15.0 kg/mol and 1.9 kg/mol are determined as, it is 1.13 that GPC, which determines molecular weight distribution,.
Click chemistry synthesis HA-P (TMC-DTC).N2The lower HA-alky that will be dissolved in respectively in 5.0 mL DMSO solution
(278 mg, 0.035 mmol) and N3-P(TMC-co- DTC) it is well mixed.The water of copper sulphate (1.45 mg, 5.8 μm of ol)
The aqueous solution of solution and sodium ascorbate (2.38 mg, 12 μm of ol) is added sequentially in double-neck flask.50 DEG C of lucifuge reactions 24
After h, dialysed in DMSO（MWCO 14000）Dialyse 2h in 24 h, the deionized water of being dialysed in 48 h, EDTA aqueous solution, lucifuge freezing
Dry.Yield：84%.Nuclear-magnetism figure（Accompanying drawing 15C）In show the characteristic peak of HA signal peak, PTMC characteristic peak and PDTC, really
Accept HA-P (TMC-DTC) structure.Infrared test（Accompanying drawing 16）Show HA-b-P(TMC-co- DTC) react complete, 2100
cm-1The nitrine and alkynyl peak of left and right are wholly absent, and representation polymer reaction is completely and free from admixture is remained.
Targeting crosslinking vesica Nano medication is further prepared, such as method of embodiment one prepares maytansine hyaluronic acid capsule
Nano medication HA-PS-DM1 is steeped, PB is injected directly under the DMSO solution for being mixed with DM1 and HA-P (TMC-DTC) is stirred（pH
7.4, 10 mM）, be incubated, dialysis after just directly form crosslinking vesica Nano medication.Specifically, being 10 by 100 μ L, concentration
Mg/mL is dissolved in DMSO HA-P (TMC-DTC) solution and DM1 solution（Theoretical drugloading rate is 10% and 20%）It is well mixed and even
900 μ L of speed injection rotate at a high speed（700 rpm）PB（10 mM, pH 7.4）In, 37 DEG C of shaking tables（200 rpm）It is incubated 24 h
Afterwards, dialysed in the PB containing 0.2% Tween 80 48 h, in PB dialyse 2h after obtain.DLS measures the stream of HA-PS-DM1 vesicas
Body dynamics diameter is in 80 ~ 85 nm, PDI<0.1, the result observed under electronic transmission Electronic Speculum is approached with this（Accompanying drawing 17A）.
HA-PS-DM1 vesicas remain in that constant particle diameter and particle diameter distribution in the presence of dilution for many times and hyclone（Accompanying drawing 17B）,
But the quick release under simulation tumour cell reducing environment, solution crosslinking（Figure 17 D）.HPLC results show（Accompanying drawing 17C）, HA-
PS-DM1 vesicas go out peak position compared with free DM1 substantially it is to the rear, have no the appearance at free DM1 peaks, it was demonstrated that all DM1 connect
Branch is mixed wherein on polymer, without free DM1.After the DTT and HA-PS-DM1 that add 10mM are incubated, it goes out peak position
As free DM1, it was demonstrated that cystine linkage is broken under the reducing conditions, release DM1 comes out.HPLC measurements can calculate connecing for DM1
Branch rate is more than 99%, and its drugloading rate DLC is up to 16.7 wt.%（Table 3）；HA-PS-DM1 cross-linked stables, medicine will not be revealed；And 10
After mM GSH processing, the obvious particle diameter of vesica becomes big, and unimodal shape is changed into multiplet, medicine leakage.
The HA-PS-DM1 of table 3 sign
a DLS room temperatures are determined in PB buffer solutions (mM of pH 7.4,10), 1.0 mg/mL；b HPLC is determined
The MTD values of MTD results of study display crosslinking vesica Nano medication are more than 5 times of free DM1；It has human ovarian cancer to lotus
（SKOV-3）The tumour inhibiting rate of the mouse of hypodermic tumour（TIR）More than 94%；Mouse survival intermediate value was more than 74 days（PBS is 28 days）；And
To mouse main organs almost without toxic side effect, DM1 group mouse are dead quickly.
Claims (10)
1. the preparation method of the crosslinking nano medicine based on active reaction type one-step method, it is characterised in that comprise the following steps, will
Polymer, the medicine with sulfydryl, which add to dialyse after mixing in solution, obtains crosslinking nano medicine；The polymer lateral chain contains two
Thiophane structure.
2. the preparation method of the crosslinking nano medicine according to claim 1 based on active reaction type one-step method, its feature exists
In the solution is water or buffer solution；The polymer is degradable polymer.
3. the preparation method of the crosslinking nano medicine according to claim 2 based on active reaction type one-step method, its feature exists
In the polymer is non-targeted polymer and/or target polymer；The non-targeted polymer contains dithiolane by side chain
The carbonate monomer of structure, other monomers, hydrophilic high polymers reaction are obtained；The target polymer contains dithiolane by side chain
The carbonate monomer of structure, other monomers, hydrophilic high polymers, targeted molecular reaction are obtained；Or it is described poly- with targeted molecular
Carbonate monomer that compound contains dithiolane structure by side chain, other monomers, hyaluronic acid reaction are obtained.
4. the preparation method of the crosslinking nano medicine according to claim 3 based on active reaction type one-step method, its feature exists
In in the non-targeted polymer, the total molecular weight of hydrophobic segment is 0.3~7 times of hydrophilic segment molecular weight, and the side chain contains
The total molecular weight for having the carbonate monomer construction unit of dithiolane structure is total molecular weight and the side of other monomers construction unit
Chain contains the 8%~60% of the carbonate monomer construction unit total molecular weight sum of dithiolane structure；In the target polymer,
The total molecular weight of hydrophobic segment is 0.3~7 times of hydrophilic segment molecular weight, and the side chain contains the carbonic ester of dithiolane structure
The total molecular weight of monomeric building blocks contains the carbon of dithiolane structure for the total molecular weight of other monomers construction unit with side chain
The 8%~60% of acid ester monomer construction unit total molecular weight sum.
5. the preparation method of the crosslinking nano medicine according to claim 4 based on active reaction type one-step method, its feature exists
In the other monomers include trimethylene carbonate, lactide, caprolactone；The side chain contains the carbon of dithiolane structure
The chemical structural formula of acid ester monomer is；The targeted molecular includes polypeptide, antibody, folic acid, transparent
Matter acid.
6. the preparation method of the crosslinking nano medicine according to claim 1 based on active reaction type one-step method, its feature exists
In, it is described mixing under air, room temperature carry out；Added again with the medicine with sulfydryl in solution after polymer is dissolved.
7. a kind of antineoplastic, it is characterised in that the preparation method of the antineoplastic comprises the following steps, by polymer
Added with the medicine with sulfydryl in solution, dialysis obtains crosslinking nano medicine after mixing；By the crosslinking nano medicine and medicine
Auxiliary material is mixed to get antineoplastic；The polymer lateral chain contains dithiolane structure.
8. crosslinking prepared by the preparation method of the crosslinking nano medicine according to claim 1 based on active reaction type one-step method
Nano medication.
9. application of the crosslinking nano medicine in antineoplastic is prepared described in claim 8.
10. a kind of preparation method of antineoplastic, it is characterised in that comprise the following steps, by polymer and the medicine with sulfydryl
Thing is added in solution, and dialysis obtains crosslinking nano medicine after mixing；The crosslinking nano medicine is mixed to get with excipient substance
Antineoplastic；The polymer lateral chain contains dithiolane structure.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710237726.0A CN107096038B (en) | 2017-04-12 | 2017-04-12 | Preparation method of cross-linked nano-drug based on active reaction type one-step method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710237726.0A CN107096038B (en) | 2017-04-12 | 2017-04-12 | Preparation method of cross-linked nano-drug based on active reaction type one-step method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107096038A true CN107096038A (en) | 2017-08-29 |
| CN107096038B CN107096038B (en) | 2021-06-18 |
Family
ID=59676229
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710237726.0A Active CN107096038B (en) | 2017-04-12 | 2017-04-12 | Preparation method of cross-linked nano-drug based on active reaction type one-step method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107096038B (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111150833A (en) * | 2020-03-16 | 2020-05-15 | 中国科学院微生物研究所 | Application of LTX-315 in preparation of products for inhibiting coronavirus |
| WO2020109428A1 (en) * | 2018-11-29 | 2020-06-04 | Midatech Ltd | Therapeutic compounds, nanoparticles and uses thereof |
| CN111714457A (en) * | 2020-06-22 | 2020-09-29 | 苏州大学 | Carbonate polymer vesicle carrying small-molecule drugs, and preparation method and application thereof |
| CN113197860A (en) * | 2021-04-28 | 2021-08-03 | 苏州大学 | Polymer vesicle nano STING agonist and preparation method and application thereof |
| CN113244175A (en) * | 2021-05-22 | 2021-08-13 | 苏州大学 | Immune vesicle maytansine conjugate as well as preparation method and application thereof |
| CN113332241A (en) * | 2021-06-04 | 2021-09-03 | 苏州大学 | Small micelle nano-drug and preparation method and application thereof |
| CN113350283A (en) * | 2021-06-04 | 2021-09-07 | 苏州大学 | Chemotherapy-immunization combined medicine and preparation method and application thereof |
| WO2022253344A1 (en) * | 2021-06-04 | 2022-12-08 | 苏州大学 | Polymer vesicle loaded with oncolytic peptide and combined medication of same with vesicle immune adjuvant and pd-1 monoclonal antibodies |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105997880A (en) * | 2016-07-15 | 2016-10-12 | 苏州大学 | Anti-tumor nano medicine based on cross-linking biodegradable polymer vesica and preparation method of anti-tumor nano medicine |
| CN106137968A (en) * | 2016-07-15 | 2016-11-23 | 苏州大学 | Inner membrance reversible crosslink Biodegradable polymer vesicles with positive electricity and preparation method thereof and the application in preparing antitumor drug |
| CN106177975A (en) * | 2016-06-30 | 2016-12-07 | 苏州大学 | Reversible crosslink Biodegradable polymer vesicles with asymmetric membrane structure and preparation method thereof and the application in nucleic acid drug |
-
2017
- 2017-04-12 CN CN201710237726.0A patent/CN107096038B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106177975A (en) * | 2016-06-30 | 2016-12-07 | 苏州大学 | Reversible crosslink Biodegradable polymer vesicles with asymmetric membrane structure and preparation method thereof and the application in nucleic acid drug |
| CN105997880A (en) * | 2016-07-15 | 2016-10-12 | 苏州大学 | Anti-tumor nano medicine based on cross-linking biodegradable polymer vesica and preparation method of anti-tumor nano medicine |
| CN106137968A (en) * | 2016-07-15 | 2016-11-23 | 苏州大学 | Inner membrance reversible crosslink Biodegradable polymer vesicles with positive electricity and preparation method thereof and the application in preparing antitumor drug |
Non-Patent Citations (1)
| Title |
|---|
| LIJUAN ZENG,ET AL: ""Treatment of Malignant Brain Tumor by Tumor‐Triggered Programmed Wormlike Micelles with Precise Targeting and Deep Penetration"", 《ADVANCED FUNCTIONAL MATERIALS》 * |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020109428A1 (en) * | 2018-11-29 | 2020-06-04 | Midatech Ltd | Therapeutic compounds, nanoparticles and uses thereof |
| CN111150833A (en) * | 2020-03-16 | 2020-05-15 | 中国科学院微生物研究所 | Application of LTX-315 in preparation of products for inhibiting coronavirus |
| CN111150833B (en) * | 2020-03-16 | 2022-11-08 | 中国科学院微生物研究所 | Application of LTX-315 in preparation of product for inhibiting coronavirus |
| CN111714457A (en) * | 2020-06-22 | 2020-09-29 | 苏州大学 | Carbonate polymer vesicle carrying small-molecule drugs, and preparation method and application thereof |
| CN113197860A (en) * | 2021-04-28 | 2021-08-03 | 苏州大学 | Polymer vesicle nano STING agonist and preparation method and application thereof |
| CN113244175A (en) * | 2021-05-22 | 2021-08-13 | 苏州大学 | Immune vesicle maytansine conjugate as well as preparation method and application thereof |
| CN113332241A (en) * | 2021-06-04 | 2021-09-03 | 苏州大学 | Small micelle nano-drug and preparation method and application thereof |
| CN113350283A (en) * | 2021-06-04 | 2021-09-07 | 苏州大学 | Chemotherapy-immunization combined medicine and preparation method and application thereof |
| WO2022253344A1 (en) * | 2021-06-04 | 2022-12-08 | 苏州大学 | Polymer vesicle loaded with oncolytic peptide and combined medication of same with vesicle immune adjuvant and pd-1 monoclonal antibodies |
| WO2022253080A1 (en) * | 2021-06-04 | 2022-12-08 | 苏州大学 | Combined chemotherapeutic and immune drug, preparation method therefor and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107096038B (en) | 2021-06-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107096038A (en) | The preparation method of crosslinking nano medicine based on active reaction type one-step method | |
| Ghosh et al. | Polymeric micelles in cancer therapy: State of the art | |
| Zhong et al. | Reversibly crosslinked hyaluronic acid nanoparticles for active targeting and intelligent delivery of doxorubicin to drug resistant CD44+ human breast tumor xenografts | |
| Zhong et al. | Hyaluronic acid-shelled acid-activatable paclitaxel prodrug micelles effectively target and treat CD44-overexpressing human breast tumor xenografts in vivo | |
| Aryal et al. | Doxorubicin conjugated gold nanoparticles as water-soluble and pH-responsive anticancer drug nanocarriers | |
| Wang et al. | Facile fabrication of robust, hyaluronic acid-surfaced and disulfide-crosslinked PLGA nanoparticles for tumor-targeted and reduction-triggered release of docetaxel | |
| Prabaharan et al. | Gold nanoparticles with a monolayer of doxorubicin-conjugated amphiphilic block copolymer for tumor-targeted drug delivery | |
| Li et al. | Supramolecular PEGylated dendritic systems as pH/redox dual-responsive theranostic nanoplatforms for platinum drug delivery and NIR imaging | |
| Li et al. | Dual stable nanomedicines prepared by cisplatin-crosslinked camptothecin prodrug micelles for effective drug delivery | |
| CN102060991B (en) | Amphiphilic prodrug of 7- ethyl-10-hydroxycamptothecin and preparation method thereof | |
| CN106995516B (en) | The nanometer medicine-carried system and preparation method thereof of tumour-specific enrichment | |
| Meng et al. | Self-immolative micellar drug delivery: The linker matters | |
| Wang et al. | Tailor-made gemcitabine prodrug nanoparticles from well-defined drug–polymer amphiphiles prepared by controlled living radical polymerization for cancer chemotherapy | |
| Han et al. | Redox-sensitive micelles for targeted intracellular delivery and combination chemotherapy of paclitaxel and all-trans-retinoid acid | |
| Huang et al. | Fabrication and reduction-sensitive behavior of polyion complex nano-micelles based on PEG-conjugated polymer containing disulfide bonds as a potential carrier of anti-tumor paclitaxel | |
| Liu et al. | Inherently nitric oxide containing polymersomes remotely regulated by NIR for improving multi-modal therapy on drug resistant cancer | |
| Liu et al. | Bio-responsive Bletilla striata polysaccharide-based micelles for enhancing intracellular docetaxel delivery | |
| Zhou et al. | Gold nanocage decorated pH-sensitive micelle for highly effective photothermo-chemotherapy and photoacoustic imaging | |
| Hsieh et al. | Tumor site-specific PEG detachment and active tumor homing of therapeutic PEGylated chitosan/folate-decorated polydopamine nanoparticles to augment antitumor efficacy of photothermal/chemo combination therapy | |
| Jaiswal et al. | Chitosan modified by organo-functionalities as an efficient nanoplatform for anti-cancer drug delivery process | |
| Luo et al. | pH-responsive stearic acid-O-carboxymethyl chitosan assemblies as carriers delivering small molecular drug for chemotherapy | |
| Abandansari et al. | Enhance chemotherapy efficacy and minimize anticancer drug side effects by using reversibly pH-and redox-responsive cross-linked unimolecular micelles | |
| Wan et al. | Polymeric micelles with reduction-responsive function for targeted cancer chemotherapy | |
| Nie et al. | In vitro and in vivo evaluation of stimuli-responsive vesicle from PEGylated hyperbranched PAMAM-doxorubicin conjugate for gastric cancer therapy | |
| Ma et al. | Size-shrinkable and protein kinase Cα-recognizable nanoparticles for deep tumor penetration and cellular internalization |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |