CN101695502A - Lanthanum fullerenol and application in preparing medicaments for inhibiting tumor growth - Google Patents
Lanthanum fullerenol and application in preparing medicaments for inhibiting tumor growth Download PDFInfo
- Publication number
- CN101695502A CN101695502A CN200910210368A CN200910210368A CN101695502A CN 101695502 A CN101695502 A CN 101695502A CN 200910210368 A CN200910210368 A CN 200910210368A CN 200910210368 A CN200910210368 A CN 200910210368A CN 101695502 A CN101695502 A CN 101695502A
- Authority
- CN
- China
- Prior art keywords
- richness
- reined
- lanthanum
- alcohol
- tumor
- 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
Images
Abstract
The invention relates to lanthanum fullerenol and application in preparing medicaments for inhibiting tumor growth, in particular to lanthanum fullerenol nanoparticles with the general formula of M@C2m(OH)x and the application thereof in preparing medicaments for inhibiting tumor growth, wherein M is La rare earth metal, m is equal to 41 or 30, and X is equal to or larger than 10 and is less than 50; and because of the reorganization of adjacent hydroxyl groups, the quantity of O on C82 carbon cage is actually different from the quantity of H, and therefore, the general formula can be written in the form of M@C2mOXHY. Compared with cyclophosphamide, cis-platinum, taxol, and the like which are clinically and widely used, the lanthanum fullerenol M@C2m(OH)x or M@C2mOXHY has the advantages of low consumption and toxicity and high rate of tumor inhibition.
Description
The application is one and divides an application that the application number of original application is 200610152170.7, and the applying date is JIUYUE in 2006 15 days, and denomination of invention is " the metal richness is reined in alcohol and suppressed application in the tumor growth medicine in preparation ".
Technical field
The present invention relates to a kind of novel nano-material and in bio-medical applications.Specifically, relating to general formula is M@C
2m(OH) the metal richness of x is reined in pure nano-particle and the application in preparation inhibition tumor growth medicine thereof, and wherein, M is selected from Gd, rare earth metals such as La, 10≤X＜50.
Background technology
Malignant tumor is the important diseases that threatens human health, has become human main causes of death at present.In China, hepatocarcinoma, pulmonary carcinoma, gastric cancer and breast carcinoma are the highest tumors of sickness rate.The whole world has at least 7,000,000 people to die from cancer every year at present, and wherein China about 1,300,000.Cancer causes huge financial burden also for family and society except the causing death.The continuous discovery of novel anti-tumor medicine and further investigation have made tumor chemical therapy become subject and Internal Medicine-Oncology are learnt to be born.But the research of antineoplastic agent faces serious challenge, and Here it is, and most common solid tumors such as pulmonary carcinoma, hepatocarcinoma, colon cancer and cancer of pancreas etc. also lack active drug, and many antineoplastic agents produce drug resistance in process of clinical application, and side effect is very big.Cyclophosphamide, amycin, cisplatin, paclitaxel etc. produce bone marrow depression, digestive tract vigorous reaction, Toxicity of Kidney or the like toxic and side effects when playing therapeutical effect, seriously limited its clinical using dosage.Therefore, new type antineoplastic medicine research is imperative.
In life science, nano-particle has demonstrated its special advantages and tempting application prospect, as the relevant nano material of the quantum dot of targeted nanometer medicine carrier, disease efficient detection, efficient medical imaging and oncotherapy technology etc., become international front line science problem.
Thunder is reined in alkene C60 and a kind ofly is made of carbon atom, and the spheroid molecule of nanoscale has unique physicochemical properties, in fields such as biomedicine, material science important application prospects is arranged.It is the spherical molecule of strong-hydrophobicity that Friedman equals to simulate the activity that fullerene C60 derivant can suppress HIV virus: C60 in the J.Am.Chem.Soc.115:6506-6509 Theoretical Calculation in 1993, diameter is 0.71nm, and HIV is open-ended cylindric molecule, size is similar to the C60 diameter, its activity site surface also is a strong-hydrophobicity, both might be with covalent bonds, thereby stops the growth of HIV virus.The adduct that contains 14 nucleotide fullerene derivates and DNA can form more stable triple-helix structure, can carry out optionally site cutting to DNA under photocatalysis.
Because fullerene and many derivants thereof are hydrophobic, can't with " target molecule " effect in the human body, make their research and application in biochemical field be very restricted.In recent years, the application of C60 derivant aspect biological quickened and widened in the breakthrough and the success of research synthesizing water-solubility fullerene derivate aspect greatly.In water environment, the fullerene of hydroxyl derivatization is not to exist with independent molecular forms, but by being agglomerated into nanoparticles with macromolecule interaction, these particulate matters have good bioaffinity (Sayes CM et al, Nano Lett, 2004,4 (10): 1881-1887.Dugan LL et al, Proc Natl Acad Sci USA 1997; 94:9434-9439.Mirkova SM et al, NitricOxide 2004; 11:201-207.Chiang LY et al, J Org Chem 1994,59; 3960-3968).Be published in (Sayes CM et al in one piece of paper of in October, 2004 Nano Lett, Nano Lett, 2004,4 (10): 1881-1887), the Colvin of rice university points out that the cytotoxicity height of fullerene depends on the group whether its carbon cage surface is modified and modified.In two kinds of cell lines, the toxicity of different structures can differ 7 orders of magnitude, and with fullerene toxicity maximum, richness is reined in alcohol (C
60(OH)
24) minimum.Richness is reined in alcohol (C
60(OH)
24) LD50＞5,000ppm, and the LD50 of fullerene is 20ppb.People have carried out preliminary research to fullerene and derivant thereof in the intravital distribution of laboratory animal.Nakamura equal 1994 at first synthetic
14A kind of fullerene pyrrole ring derivant of C labelling, and studied it in intravital bio distribution of mice and drug metabolism, to the SD rat, chemical compound is distributed to each organ of mice health very soon by tail vein injection, and 90-95% is enriched in liver.166Ho@C
82(OH)
xIt is wider then to show bio distribution, successively decreases other tissue distribution extremely low (Cagle DW et al., Proc Natl Acad Sci 1999 successively at the content of liver, skeleton, spleen, kidney, lung; 96:5182-5187).
In the past few years, the research that belongs to fullerene covered with gold leaf has obtained remarkable progress in the cage, up to now, and trivalent metal atom (Sc, Y), alkaline earth metal atom (Ca, Sr, Ba), alkali metal atom (Li, Na, K, Cs) and tetravalent metal atom (Zr Hf) waits successfully by the bag cage in fullerene, has formed monatomic, diatomic, three atom metal bag cage things.Physics and the chemical property that belongs to many excellences of fullerene covered with gold leaf makes them might develop into superconduction, Organic Ferromagnet, nonlinear optical material, functional molecular switch in the cage, new material (Bolskar RD et al., J Am Chem Soc 2003 such as nuclear-magnetism contrast agent, biological tracer; 125:5471-5478).
Summary of the invention
We know that tumor tissues is more normally organized and is rich in blood vessel, and the aperture of many nano-scales is arranged on the capillary wall of tumor tissues, and nutrient substance can be penetrated among the tumor tissues by these apertures.Suppose that these blood channels are stopped up by unidimensional nanoparticle just, will suppress the circulation of blood, make tumor can not obtain enough nutrient substance, and then interrupted the growth of tumor tissues.In order to verify this viewpoint, the inventor designs and has prepared a kind of metal richness and reins in alcoholic compound, its molecular diameter is about 1nm, found that, the metal richness is reined in pure nano-particle very strong tumor inhibition effect, when its when to form diameter in solution be the granule of 1-200nm the tumor suppression effect better, its mechanism of action is not to finish by the direct killing effect to tumor cell.
An object of the present invention is to provide a kind of metal richness and rein in alcohol.
Another object of the present invention provides a kind of tumor suppression compositions, wherein contains the effective metal richness of treatment and reins in pure granule and pharmaceutically acceptable carrier.
A further object of the present invention provides a kind of metal richness and reins in the application of alcohol in preparation inhibition tumor growth medicine.
For achieving the above object, the present invention comprises following scheme:
A kind of metal richness is reined in alcohol, and it represents that with general formula in the general formula, M is selected from La, rare earth metals such as Gd; M=41 or 30; 10≤X＜50.
Above-claimed cpd comprises a magnetic central metallic ions M and the nano-sized carbon cage by the C atomic building, and there are many oh groups in its surface, so M@C
2m(OH)
xGood bioaffinity is arranged in vivo.(referring to Fig. 1) simultaneously because the existence of oh group reduces greatly with metal fullerene comparison toxicity.
Because the rearrangement of adjacent hydroxyl, in fact the number of O and the number of H have that some are different on the carbon cage, therefore, also can be write above-mentioned general formula as M@C
2mO
xH
yForm.
General formula of the present invention is M@C
2m(OH)
x(m=41 or 30; 10≤X＜50) metal richness is reined in alcohol, and wherein M is La.
The M@C that general formula of the present invention is
2m(OH)
x(m=41 or 30; 10≤X＜50) the metal richness is reined in alcohol, and wherein M is Gd.
A kind of tumor suppression compositions wherein contains with Tong ShiM @C
2m(OH)
xThe metal richness of expression is reined in alcohol, and in the general formula, M is selected from rare earth metals such as Gd, La; M=41 or 30; 10≤X＜50.
A kind of tumor suppression compositions wherein contains with general formula [M@C
2m(OH)
x]
nThe metal richness of expression is reined in pure nano-particle, and in the general formula, M is selected from rare earth metals such as Gd, La; M=41 or 30; 10≤X＜50; N represents to be agglomerated into this metal richness and reins in the metal richness of pure nano-particle and rein in pure molecular number, 1≤n＜200.
General formula is M@C
2m(OH)
xThe metal richness rein in alcohol can be agglomerated into nanoparticles by macromolecule interaction.In solvent environment, can pass through the size of method control particulate matters such as selective solvent, control concentration and ultrasound wave, form the granule of diameter Distribution scope at 1-200nm.
Above-mentioned composition also can comprise solvent and/or pharmaceutically acceptable carrier.Above-mentioned solvent preferred water, normal saline, Tris-HCl solution or phosphate buffer.Above-mentioned pharmaceutically acceptable carrier is meant the pharmaceutical carrier of pharmaceutical field routine, for example: diluent, excipient, filler, absorption enhancer etc.
Tumor suppression compositions of the present invention, wherein, the metal richness is reined in the concentration of alcohol in solvent and is preferably 1 * 10 in the described compositions
-5～1mmol/L.The metal richness is reined in the solubility property reduction of alcohol when concentration is higher than 1mmol/L, easily is agglomerated into larger particles.The metal richness is reined in pure final concentration 1 * 10
-5Between～the 1mmol/L, pair cell does not have the overt toxicity effect.
Above-mentioned tumor includes but not limited to pulmonary carcinoma, hepatocarcinoma, gastric cancer, the esophageal carcinoma, carcinoma of the colon and rectum, bladder cancer, breast carcinoma, cervical cancer, ovarian cancer, osteosarcoma, angiosarcoma, lymphosarcoma, leukemia, melanoma or skin carcinoma.
A kind of metal richness is reined in the application of alcohol in preparation inhibition tumor growth medicine, and this metal richness is reined in alcohol with Tong ShiM @C
2m(OH)
xExpression, in the general formula, M is selected from Gd, rare earth metals such as La; M=41 or 30; 10≤X＜50.Described tumor includes but not limited to pulmonary carcinoma, hepatocarcinoma, gastric cancer, the esophageal carcinoma, carcinoma of the colon and rectum, bladder cancer, breast carcinoma, cervical cancer, ovarian cancer, osteosarcoma, angiosarcoma, lymphosarcoma, leukemia, melanoma or skin carcinoma.
A kind of metal richness is reined in the application of alcohol in preparation inhibition tumor growth medicine, and this medicine is prepared to various dosage forms, and the pairing dosage of these dosage forms is reined in alcohol in the metal richness and is preferably 5 * 10
-8～1 * 10
-2Mmol/kg/ days.Above-mentioned dosage at the people is the mice dosage 1 * 10 by effect experiment
-6～2 * 10
-1Conversion in mmol/kg/ days gets.
A kind of metal richness is reined in alcohol and is suppressed application in the tumor growth medicine in preparation, and this medicine is prepared to various dosage forms, and the pairing dosage of these dosage forms is reined in alcohol more preferably 5 * 10 in the metal richness
-6～1.25 * 10
-4Mmol/kg/ days.Above-mentioned dosage at the people is the mice dosage 1 * 10 by effect experiment
-4～2.5 * 10
-3Conversion in mmol/kg/ days gets.
Above-mentioned tumor suppression compositions preferably is applied to the patient who needs treatment by modes such as intravenous injection, intraperitoneal injection, oral or topicals.In a preferred embodiment of the invention, above-mentioned tumor suppression compositions is made injection solution.
Advantage of the present invention is: compare with the at present clinical cyclophosphamide that generally uses, cisplatin, paclitaxel etc., the metal richness is reined in pure M@C
2m(OH)
xIt is little to have consumption, and toxicity is low, and the high advantage of tumor control rate.
Description of drawings
Fig. 1 is M@C
2m(OH)
xSchematic arrangement
Fig. 2 is Gd@C
2mHPLC separating resulting spectrogram (5PBB post).
Fig. 3 is Gd@C
82(OH)
22(a) and Gd@C
82(OH)
12The XPS spectrum of C1s electronics (b).
Fig. 4 is [Gd@C in the normal saline solution
82(OH)
22]
nThe high-resolution AFM figure of nano-particle.
Fig. 5 is Gd@
82(OH)
22Low dose group injection back mice H
22The growth curve chart of hepatocarcinoma.
Fig. 6 is Gd@C
82(OH)
22High dose group injection back mice H
22The growth curve chart of hepatocarcinoma.
Fig. 7 is CTX group tumor control rate and dose relationship curve chart.
Fig. 8 is Gd@C
82(OH)
22Group tumor control rate and dose relationship curve chart.
Fig. 9 is Hepar Mus cancer H
22The different treatment group of model tumor tissue pathology section photo.Wherein, A, B are matched group; C, D are Gd@C
82(OH)
22The treatment group; E, F are cyclophosphamide treatment group.
Figure 10 is La@C
82(OH)
18The growth curve chart of injection back Mice Bearing Lewis Lung Cancer.
Figure 11 reins in pure Gd@C for the metal richness
82(OH)
22Influence to human hepatoma HepG2 cell's survival rate.
Figure 12 reins in pure Gd@C for the metal richness
82(OH)
22Induce the percentage ratio of human hepatoma HepG2 cell's apoptosis.
Figure 13 reins in pure Gd@C for the metal richness
82(OH)
26Induce the apoptotic percentage ratio of Hepar Mus cancer Rh35.
Figure 14 reins in pure Gd@C for the metal richness
82(OH)
32Induce the apoptotic percentage ratio of breast carcinoma MCF-7.
The numerical value of abscissa is M@C among Figure 11, Figure 12, Figure 13 and Figure 14
82(OH)
xThe power value of molar concentration.
Figure 15 reins in pure La@C for the metal richness
82(OH)
20Influence to the neuroglial cytoma survival rate.
The specific embodiment
The inventor adopts chemical synthesis to prepare the metal richness and reins in pure Gd@C
82(OH)
x, Gd@C
60(OH)
x, La@C
82(OH)
x, La@C
60(OH)
x, its preparation method is with reference to Chinese invention patent 03146028.3.Gd@C
60Synthetic and separation and purification list of references Robert D.Bolskar etc., J.AM.CHEM.SOC.2003,125,5471-5478.
Adopt the metal richness of the inventive method preparation to rein in pure M@C
2m(OH)
x, its hydroxy number is in 10～50 scope, and when hydroxyl value during with concrete numeric representation, this numerical value refers to full meansigma methods of adding of hydroxy number in this article.The concentration decision that this value is reined in NaOH solution in the alcohol reaction by synthetic metal richness, the concentration that can regulate NaOH solution as required, thus the metal richness that obtains to specify hydroxy number to add full meansigma methods is reined in alcohol.When hydroxy number was lower than 10, it is bad that the metal richness is reined in pure biocompatibility; When hydroxy number is higher than 50, the structural instability of carbon cage.
It is most important accurately to measure rich hydroxy number of reining in alcohol, among the present invention, and the hydroxy number after we utilize Beijing Synchrotron Radiation Facility x-ray photoelectron power spectrum (XPS) and determine the finishing of metal fullerene in conjunction with elemental analysis method.
The XPS experiment is carried out on Chinese Academy of Sciences's Beijing Synchrotron Radiation Facility.On monocrystalline silicon piece, plate high-purity Pt as XPS sample test substrate by the magnetic control ion sputtering.M@C
82And M@C
82(OH)
xDropping obtains the thin film of XPS measuring in high-purity Pt substrate, thin film is put into 8 * 10
-10The ultra-high vacuum system long enough time of the XPS sample preparation chamber of Torr, can adsorbable air plankton on the sample to remove.The C1s photoelectron emissions spectrum that the metal richness is reined in alcohol is used for the hydroxy number that definite sample comprises.Change incident photon energy, the valence band photoelectron spectroscopy of collected specimens.The fractional yield spectrum of collected specimens is to obtain the absorption spectra of sample.The energy resolution of device is about～0.5eV.Sample carries out XPS scanning earlier before image data, to guarantee the sample surfaces cleaning and to determine that instrument is in good running status.
The metal richness is reined in pure M@C
2m(OH)
xThe particle diameter of molecule is about 1nm.The metal richness is reined in pure molecule can be agglomerated into nanoparticles by macromolecule interaction.In solvent environment, can form the granule of diameter Distribution scope by the size of methods such as ultrasound wave control particulate matter at 1-200nm.Related metal richness is reined in alcoholic solution and is and contains the diameter Distribution scope and rein in pure particulate solution in the metal richness of 1-200nm in this paper following examples.
Further specify the present invention below in conjunction with embodiment and Comparative Examples, but therefore do not limit the present invention among the described scope of embodiments.
Gd@C
2m(OH)
xPreparation
First step Gd@C
2mSynthetic
With high-purity (＞99.999%) Gd
2O
3With high purity graphite powder (＞99.999%) by atomic ratio Gd: C=0.5～mix at 3: 100, be pressed into mould, make the graphite-metal mixed electrode; Perhaps be that 6～20mm graphite rod bores sky with diameter, the filling Gadolinia. obtains the graphite-metal mixed electrode.Behind 1000～2000 ℃ of high temperature sinterings, use noble gas arc electric discharge, synthetic metal fullerene Gd@C
2mNoble gas is He or Ar, and pressure is 50～600Torr, and electric current is 80～500A.
The second step Gd@C
82And Gd@C
60Separation and purification
A. adopt high temperature reflux to increase temperature two step of high pressure highly effective extraction method extract and separate carbon nanometer class material.At first with the soot of arc discharge in toluene 100～200 ℃ refluxed 12～24 hours down, again in DMF (N, dinethylformamide) 100～200 ℃ of following High Temperature High Pressure (50～100MPa) extracted 12～24 hours, with extraction carbon nanometer class material.
B. use circulating HPLC (high performance liquid chromatography) two-step method or the required metal fullerene Gd@C of extraction purification of target of extensive use
82And Gd@C
60, obtain purity greater than 99.99% target product Gd@C
82And Gd@C
60Gd@C
82Productive rate be 10% of carbon-point; Gd@C
60Productive rate is 35% of a carbon-point.HPLC separating resulting spectrogram is referring to Fig. 2.(5PBB post)
The 3rd single metal richness is reined in pure Gd@C
82(OH)
22Synthetic
Use the NaOH method, in toluene solution with Gd@C
82With concentration be that 35% NaOH solution reacts, more after filtration, separation, purge process such as ion-exchange chromatography, remove NaOH after, obtain purity greater than 99.99% product Gd@C
82(OH)
22, lyophilization is preserved.
The metal richness is reined in pure Gd@C
82(OH)
12Synthetic
In toluene solution with Gd@C
82With concentration be that 30% NaOH solution reacts, more after filtration, separation, purge process such as ion-exchange chromatography, remove NaOH after, obtain purity greater than 99.99% product Gd@C
82(OH)
12, lyophilization is preserved.
Gd@C
60(OH)
xSynthetic:
With 300mg Gd@C
60Suspension is with after 210mg KH mixes in 20mL THF, continuous stirring 15 minutes, and 1.255g diethyl bromomalonic acid added mixed liquor dropwise.Stir and generated coffee-like solution in 40 minutes later on.Filter then, remove responseless KH.Crude product is separated with oxolane, the rinsing of reuse hexane, low pressure is drained, and refluxes with NaH in toluene then, and finishes reaction with methanol.Use the acidic ion exchange column separation and purification, with the pH value to 7.0 of NaOH regulator solution.Evaporate to dryness obtains Gd@C
60(OH)
xProduct.
The 4th single metal richness is reined in pure granule [Gd@C
82(OH)
22]
nSynthesizing of (1≤n＜200)
The metal richness is reined in pure Gd@C
82(OH)
22Be dissolved in the normal saline, ultrasonic 1 minute, form the particulate matter [Gd@C of diameter Distribution scope at 1-200nm
82(OH)
22]
n, 1＜n＜200.
Measure Gd@C
82(OH)
22And Gd@C
82(OH)
12Hydroxyl value
Gd@C
82(OH)
22(a) and Gd@C
82(OH)
12The XPS spectrum of C1s electronics (b) as shown in Figure 3, the relative intensity between C-C and the C-O key is used to analyze Gd@C
82(OH)
xIn hydroxy number.The Gauss curve fitting of solid line the analysis showed that and has two kinds of different carbon atoms in the molecule among Fig. 2: the peak position is the binding energy that does not connect the carbon atom with sp2 hydridization (C-C) of other group 284.4eV's; The peak that is positioned at 285.3eV is hydroxylated carbon atom (C-OH).
La@C
2m(OH)
xPreparation
Adopt and above-mentioned preparation Gd@C
82Similarly method prepares La@C
82And La@C
60
(1) the metal richness is reined in pure La@C
82(OH)
18Synthetic
Use the NaOH method, in the toluene solution medium with La@C
82React with 28% NaOH solution, again through a series of separation, purge process, remove NaOH after, obtain purity greater than 99.99% La@C
82(OH)
18Product, lyophilization is preserved.
(2) the metal richness is reined in pure La@C
60(OH)
22Synthetic
Use the NaOH method, in the toluene solution medium with La@C
60React with 35% NaOH solution, again through a series of separation, purge process, remove NaOH after, obtain purity greater than 99.99% La@C
60(OH)
22Product, lyophilization is preserved.
The mensuration of nano-particle
The metal richness is reined in pure M@C
2m(OH)
xThe particle diameter of molecule is about 1nm.In solvent environment, the metal richness is reined in pure molecule and is agglomerated into nanoparticles by macromolecule interaction, can form the granule of diameter Distribution scope at 1-200nm by the size of methods such as ultrasound wave control particulate matter.
Adopt synchrotron radiation little angle X-ray diffraction (SR-SAXS) to record [Gd@C in the normal saline solution
82(OH)
22]
nThe particle size range of nano-particle is 1-200nm, mean diameter 22nm.
(Digital Instruments Inc 3A) confirms [Gd@C in the normal saline solution to the high-resolution atomic force microscope for AFM, Nano IIIa SPM
82(OH)
22]
nThe particle size range of nano-particle is 1-200nm, and mean diameter 22.4nm exists, referring to Fig. 4.By two kinds of unanimities as a result of obtaining of method independently.
Contain [Gd@C
82OH
22]
nThe injection solution of (1≤n＜200)
With 500mg Gd@C
82OH
22, being dissolved in the 400ml normal saline, ultrasonic 1 minute of room temperature is packaged in 100 glass tubings, makes injection solution.
Gd@C
82(OH)
mCompositions is to the inhibitory action of rat liver cancer tumor
The animal strain: the female Mus of Kunming kind, body weight are 18 to 22g.
Tumor model: rat liver cancer H
22The tumor strain.
Experiment grouping: A. negative control group: normal saline (saline); B. positive controls: cyclophosphamide (Cyclophosphamide, CTX), a kind of cancer therapy drug that generally uses clinically; C. medicine group: Gd@C
82(OH)
m, every group of 5-7 mice.
Administering mode: lumbar injection (intraperitoneal, i.p.).
CTX and Gd@C
82(OH)
m(m=22 or 26) solution is solvent with 0.9% normal saline.
Dosage design (1 * 10
-6～2 * 10
-3The mmol/kg mice)
This experiment divides two dosage groups:
A. high dose group:
CTX:30mg/kg (0.1mmol/kg) only needs the injections in preceding 7 days in experiment;
Gd@C
82(OH)
22，2×10
-4mmolGd/kg；
Gd@C
82(OH)
26，2×10
-3mmol/kg；
Volume injected is 0.2ml.
Low dose group:
CTX：15mg/kg(0.05mmol/kg)；
Gd@C
82(OH)
22，1×10
-4mmol/kg；
Gd@C
82(OH)
26，1×10
-6mmol/kg；
Volume injected is 0.2ml.
Experimental technique: every mice right hind subcutaneous vaccination rat liver cancer H
22Tumor strain 1 * 10
6Cancerous cell (being scattered in 0.9% normal saline of 100 μ l) is only inoculated after 24 hours beginning administration 0.2ml/; Experimental session was administered once in per 24 hours, every other day measured the hind leg diameter of inoculated tumour, write down its growing state; Observe the response situation of mice simultaneously; When normal saline group mice right hind diameter length arrives the 20mm left and right sides, stop experiment (normal mouse hind leg diameter is 6mm).
Experiment finishes: extract eyeball and get blood, use the anticoagulant of 109mmol/L sodium citrate, the ratio of blood and anticoagulant is 1: 9; Win tumor, weigh; Taking internal organ/organ is also weighed, and calculates organ coefficient; Use formalin fixed internal organs/organ of 10% simultaneously.
Experimental result: Gd@C
82(OH)
mThe anti-hepatocarcinoma H of compositions
22Active as shown in table 1, injection Gd@C
82(OH)
mThe tumor growth that equally can suppress mice behind the nano-particle with injection CTX, and the tumor suppression characteristic of nano material is better than the clinical antitumor drug CTX that generally uses.
High dose group Gd@C
82(OH)
22To the tumor-bearing mice serum total bilirubin, ALT, AST and inosine level influence the table 2 of data referring to nextpage.From reflection hepatocellular damage the most responsive index is that the activity of glutamate pyruvate transaminase ALT and glutamic oxaloacetic transaminase, GOT AST is analyzed injection Gd@C
82(OH)
22Nano material can significantly suppress increasing of two kinds of enzymes of mice with tumor, and near the level of normal body.And cyclophosphamide group ALT level increases on the contrary, illustrates that it increases the damage of animal liver.
According to formula V=4 π r
3/ 3 calculate gross tumor volume, and obtain tumour inhibiting rate; Found that the Gd@C of 0.1 μ mol/kg in the low dose group
82(OH)
22The efficient that suppresses tumor growth is 32.9% (Fig. 5 and Fig. 6), though lower than the tumour inhibiting rate 52.0% of the CTX of 0.05mmol/kg, its dosage is 1/500 of a CTX dose.
In addition, we have also compared the relation between tumour inhibiting rate and the drug use amount, and when the dosage of CTX was improved 0.05mmol/kg, its tumor control rate had improved 15%, and Gd@C
82OH
22When the using dosage of nano material only improved 0.1 μ mol/kg, its tumor control rate just improved 26% (Fig. 7 and Fig. 8).
Hepar Mus cancer H
22The section result of model group tumor tissue pathology:
Below referring to Fig. 9, Gd@C
82(OH)
22Treatment group mouse tumor infiltration scope is littler, and the tumor body is easily peeled off, and tumor weight obviously alleviates.Form tangible tumor around the visible tumor of HE stained and hold band (inflammatory cell, fibroblast and blood capillary).Can activate body's immunity.
Matched group: tumor cell soaks into obviously to skeletal muscle.
The cyclophosphamide group: tumor cell is downright bad in a large number, but can not prevent that tumor cell from soaking into to skeletal muscle.
Table 1 Gd@C
82(OH)
mAnti-hepatocarcinoma H
22The activity data table
(N is a mice quantity, and data are with (mean+SD) expression in the table)
Table 2 high dose group Gd@C
82(OH)
22The tumor-bearing mice serum total bilirubin, ALT, AST and inosine level
Annotate: a and normal saline group be P＜0.01. relatively
Gd@C
82(OH)
mInhibition tumor effect to people source MCF-7 breast carcinoma human breast cancer in nude mice model
The female nude mice of experimental technique: BALB/c (16.0 ± 1.0g) subcutaneous vaccination MCF-7 cell suspension (1 * 10
7Cell) after, raised 20 days, the tumor of subcutaneous generation is taken out, it is standby that the tumor fritter that cuts into 3mm * 3mm * 3mm is implanted the female nude mice of BALB/c again.Inoculate and begin administration after 3 days, give Gd@C respectively
82(OH)
m(m=32 or 12), cyclophosphamide, paclitaxel, normal saline.
Medication:
Gd@C
82(OH)
32μ mol/kg/ days * 13 days 2.5 (calculating) with Gd concentration; Injection concentration is 0.25 μ mol/ml.
Gd@C
82(OH)
121.0 μ mol/kg/ days * 13 days, calculate with Gd concentration; Injection concentration is 0.1 μ mol/ml.
Cyclophosphamide first all 71.6 μ mol/kg/ days * 7 days, 8-12 days physiology saline
Paclitaxel the 0th, 3,6,9,12 days 15.2 μ mol/kg/ days, the injection of not administration sky
Normal saline
Normal saline 0.2ml q.d. * 13
The female nude mice of animal strain: BALB/c,
Gd@C
82(OH)
32Organize 10;
Gd@C
82(OH)
12Organize 10;
10 of cyclophosphamide groups
9 of paclitaxel groups;
8 of normal saline groups
Tumor model: MCF-7 human breast carcinoma
Administering mode: lumbar injection (intraperitoneal injection, i.p.).
CTX and Gd@C
82(OH)
mSolution, the normal saline with 0.9% are solvent.Paclitaxel is commercial liquid preparation.
It is as shown in table 3 to suppress the tumor experiment result, Gd@C
82(OH)
32Group 2.5 μ mol/kg body weight administrations in the time of 13 days tumour inhibiting rate can reach 47%, 7 days results are consistent with the 71.6 μ mol/kg body weight administrations of cyclophosphamide group.Gd@C
82(OH)
12 Group 1 μ mol/kg body weight administration in the time of 13 days tumour inhibiting rate can reach 35.6%.But obvious toxic and side effects appears in cyclophosphamide group mice, as obviously lose weight, become thin, the animal mental status is poor.Paclitaxel is under 15.2 μ mol/kg body weight dosage, and every administration in 2 days, tumour inhibiting rate can reach 82%, but 45% dead mouse illustrates that paclitaxel toxicity is very big.And the metal richness is reined in alcohol group mice tangible toxic reaction do not occur when experiment is finished.
Experimental result shows, compares with at present clinical cyclophosphamide, the paclitaxel that generally uses, and the metal richness that has different hydroxyl values is reined in pure Gd@C
82(OH)
32And Gd@C
82(OH)
12It is little to have consumption, and toxicity is low, and the high advantage of tumor control rate.
Table 3 Gd@C
82(OH)
mSuppress the people source growth of breast cancers tables of data that nude mice is transplanted
(N is a mice quantity)
La@C
82(OH)
18Compositions is to the inhibition tumor effect of Mice Bearing Lewis Lung Cancer model
Experimental technique: C57 pure lines female mice (18.0 ± 1.0g) subcutaneous vaccination Lewis lung cancer cell suspension (1 * 10
7Cell), inoculation begins administration next day, gives La@C respectively
82(OH)
18, cyclophosphamide, normal saline, 10 every group.
Administering mode: lumbar injection (intraperitoneal injection, i.p.).
Medication:
La@C
82(OH)
18 1μmol/kg?q.d.×14
Gave normal saline in cyclophosphamide first all 71.6 μ mol/kg q.d. * 7, the 8-14 days
Normal saline 0.2ml q.d. * 14
CTX and La@C
82(OH)
18Solution is solvent with 0.9% normal saline all.
Press down the tumor experimental result referring to Figure 10, La@C
82(OH)
18Can obviously suppress growth of tumor in 1 μ mol/kg body weight administration in the time of 14 days, the weight unknown of tumor show to increase, with cyclophosphamide 71.6 μ mol/kg body weight administrations 7 days basically identical as a result.But obvious toxic and side effects appears in cyclophosphamide group mice, as obviously lose weight, become thin, the animal mental status is poor.And the metal richness is reined in alcohol group mice tangible toxic reaction do not occur when experiment is finished.
Above-mentioned experimental result shows, compares with the at present clinical cyclophosphamide that generally uses, and the metal richness is reined in pure La@C
82(OH)
18Nano-particle has that consumption is little, and toxicity is low, and the high advantage of tumor control rate.
Gd@C
60(OH)
20Inhibition tumor effect to the Mice Bearing Lewis Lung Cancer model
Experimental technique: C57 pure lines female mice (20.0 ± 1.0g) subcutaneous vaccination Lewis lung cancer cell suspension (1 * 10
7Cell), inoculation begins administration next day, gives respectively and Gd@C
60(OH)
20, normal saline, 10 every group.Gd@C
60(OH)
20Solution, the normal saline with 0.9% are solvent.
Administering mode: lumbar injection (intraperitoneal injection, i.p.).
Medication:
Gd@C
60(OH)
20 0.5μmol/kg?q.d.×18
Normal saline 0.2ml q.d. * 18
Press down the tumor experimental result and show Gd@C
60(OH)
20Can obviously suppress growth of tumor in 0.5 μ mol/kg body weight administration in the time of 18 days, compare with the normal saline matched group, the not obvious increase of the volume of inoculated tumour, tumour inhibiting rate can reach 42%.
Cell toxicity test:
Experimental technique:
(1) the methods analyst metal richness of MTT is reined in the influence of alcohol to variety classes cancerous cell survival rate
Mtt assay: after entering cell as the MTT of one of tetrazole dyestuff, be reduced to insoluble coloured product, utilize the method for light absorption to measure the amount of coloured product, thereby reflect cell activity indirectly by mitochondrial respiratory chain enzyme (as succinate dehydrogenase).
The trophophase cell dissociation of taking the logarithm becomes single cell suspension, and regulating cell concentration is 2 * 10
4/ ml is inoculated in 96 porocyte culture plates, and 100 μ l are inoculated in every hole, is divided into 6 dosage groups, 8 every group multiple holes.Behind the inoculation 24h, change serum-free medium into, every hole 100 μ l according to dosage organize concentration and add medicine respectively, after continuing to cultivate 48h, abandon supernatant, every hole adds 100 μ l MTT buffer and 10 μ l MTT solution (5mg/ml), continues to cultivate 3h, abandon supernatant, add DMSO 150 μ l/ holes, concussion, microplate reader is measured each hole light absorption value of 595nm place automatically.
(2) flow cytometry analysis method: propidium iodide (PI) dyeing detects apoptosis cell
With 5ml concentration is 10
5The cell inoculation of individual/ml and 25cm
2In the culture bottle, change the 5ml serum-free medium behind the 24h into, the metal richness that adds variable concentrations is respectively reined in alcohol, collect cell behind the 24h, comprise the small amounts of cells in the culture medium, collecting good cell washes twice with normal saline, 4 ℃ of 70% ethanol are fixing then, before doing flow cytometer showed, wash 2 times, hanged cell with normal saline at last with normal saline, add RNase A and propidium iodide (PI), final concentration is respectively 25ppm and 50ppm, places 30 minutes in 37 ℃ of water-baths, filters the back with nylon gauze and goes up machine mensuration.PI can not pass through complete cell membrane, but for apoptosis middle and advanced stage cell or dead cell, PI can permeate through cell membranes and make that nucleus is red to be dyed, and DNA analyzes with flow cytometer in conjunction with PI dyeing back, a hypodiploid peak can be before the G1 peak, occurred, apoptosis cell can be detected according to the percentage rate at this peak.
Experimental result is given an example:
[1] the methods analyst metal richness of MTT is reined in the influence of alcohol to human hepatoma HepG2 cell's survival rate
Gd@C
82(OH)
22Final concentration is 10～10
6In the nmol/L scope, experimental result as shown in figure 11, the cytoactive that cytoactive does not add any medicine increases to some extent, but difference is little between each dosage group of medicine, does not also have tangible linear relationship.
[2] the methods analyst metal richness of flow cytometer is reined in pure Gd@C
82(OH)
22Toxicity to the human hepatoma HepG2 cell
The result as shown in figure 12, the apoptosis ratio all between 3.5%～7%, Gd@C
82(OH)
22 Final concentration 10～10
6In the nmol/L scope, apoptosis ratio and matched group basically identical do not have the significance difference between each dosage group of medicine.Gd@C
82(OH)
22Do not induce the HepG2 apoptosis, not influence of the increment of pair cell and growth.
[3] the methods analyst Gd@C of flow cytometer
82(OH)
26Toxicity to Hepar Mus cancer Rh35 cell
The result as shown in figure 13, Gd@C
82(OH)
26 Final concentration 10～10
6In the nmol/L scope, suitable with the cellular control unit apoptosis percentage ratio that does not add any derivant, the ratio of apoptotic cell all between 2.0%～3.5%, illustrates Gd@C
82(OH)
26Do not induce Hepar Mus cancer Rh35 apoptosis.
[4] the methods analyst Gd@C of flow cytometer
82(OH)
32Toxicity to breast carcinoma MCF-7 cell
The result as shown in figure 14, Gd@C
82(OH)
32 Final concentration 10～10
6In the nmol/L scope, suitable with cellular control unit apoptosis percentage ratio, the ratio of apoptotic cell shows Gd@C between 3.0%～9%
82(OH)
32Do not induce breast carcinoma MCF-7 apoptosis, pair cell toxicity is less.
[5] the methods analyst metal richness of MTT is reined in the influence of alcohol to the neuroglial cytoma survival rate
La@C
82(OH)
20Final concentration is 100～10
6In the nmol/L scope, experimental result as shown in figure 15, cell survival rate with do not add the matched group basically identical of any medicine, to the not significantly influence of growth of neuroglial cytoma.
The metal richness of above-mentioned variable concentrations is reined in the cytotoxicity experiment result demonstration of alcohol to kinds of tumor cells, in the concentration range of 10-1000000nmol/ml, the metal richness is reined in the growth not influence of alcohol to the different types of tumors cell strain, cell death inducing not, cell survival rate is consistent with the matched group that does not add medicine simultaneously, illustrate that the metal richness reins in alcohol and do not possess direct cytotoxicity, direct cell killing.
Claims (12)
1. a lanthanum richness is reined in alcohol, it is characterized in that: it is with formula La@C
82(OH) x or La@C
60(OH) x represents, in the formula, and 10≤X＜50.
2. a tumor suppression compositions is characterized in that, it comprises with formula La@C
82(OH) x or La@C
60(OH) the lanthanum richness represented of x is reined in alcohol, in the formula, and 10≤X＜50.
3. a tumor suppression compositions is characterized in that, it comprises with general formula [M@C
2m(OH)
x] the lanthanum richness represented of n reins in pure nano-particle, in the general formula, M is La, m=41 or 30; 10≤x＜50; N represents to be agglomerated into described lanthanum richness and reins in the lanthanum richness of pure nano-particle and rein in pure molecular number, 1≤n＜200.
4. according to claim 2 or 3 described tumor suppression compositionss, it is characterized in that described compositions also comprises solvent and/or pharmaceutically acceptable carrier.
5. tumor suppression compositions according to claim 4 is characterized in that, described solvent is water, normal saline, Tris-HCl solution or phosphate buffer.
6. tumor suppression compositions according to claim 4 is characterized in that, it is 1 * 10 that described lanthanum richness is reined in the concentration of alcohol in solvent
-5～1mmol/L.
7. tumor suppression compositions according to claim 5 is characterized in that, it is 1 * 10 that described lanthanum richness is reined in the concentration of alcohol in solvent
-5～1mmol/L.
8. according to claim 2 or 3 or 5 or 6 or 7 described tumor suppression compositionss, it is characterized in that described tumor is pulmonary carcinoma, hepatocarcinoma, gastric cancer, esophageal carcinoma, carcinoma of the colon and rectum, bladder cancer, breast carcinoma, official's neck cancer, ovarian cancer, osteosarcoma, angiosarcoma, lymphosarcoma, leukemia, melanoma or skin carcinoma.
9. tumor suppression compositions according to claim 4, it is characterized in that described tumor is pulmonary carcinoma, hepatocarcinoma, gastric cancer, esophageal carcinoma, carcinoma of the colon and rectum, bladder cancer, breast carcinoma, official's neck cancer, ovarian cancer, osteosarcoma, angiosarcoma, lymphosarcoma, leukemia, melanoma or skin carcinoma.
10. a lanthanum richness is reined in the application of alcohol in preparation inhibition tumour medicine, it is characterized in that the metal richness is reined in alcohol with formula M
2m(OH)
xExpression, in the general formula, M is La, m=41 or 30; 10≤x＜50.
11. lanthanum richness according to claim 10 is reined in the application of alcohol in preparation inhibition tumour medicine, it is characterized in that described medicine is prepared to various dosage forms, the pairing dosage of these dosage forms is reined in alcohol with the lanthanum richness and is counted 5 * 10
-8～1 * 10
-2Mmol/kg/ days.
12. lanthanum richness according to claim 11 is reined in the application of alcohol in preparation inhibition tumour medicine, it is characterized in that described medicine is prepared to various dosage forms, the pairing administration of these dosage forms is reined in alcohol with the lanthanum richness and is counted 5 * 10
-6～1.25 * 10
-4Mmol/kg/ days.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200910210368 CN101695502B (en) | 2005-09-19 | 2006-09-15 | Lanthanum fullerenol and application in preparing medicaments for inhibiting tumor growth |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2005101034947A CN1739562A (en) | 2005-09-19 | 2005-09-19 | The metal richness is reined in the application of alcohol in suppressing tumor growth |
| CN200510103494.7 | 2005-09-19 | ||
| CN 200910210368 CN101695502B (en) | 2005-09-19 | 2006-09-15 | Lanthanum fullerenol and application in preparing medicaments for inhibiting tumor growth |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200610152170 Division CN1935812B (en) | 2005-09-19 | 2006-09-15 | Metal fullerol and its pharmaceutical use for inhibiting tumour growth |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101695502A true CN101695502A (en) | 2010-04-21 |
| CN101695502B CN101695502B (en) | 2011-11-02 |
Family
ID=42140654
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200910210368 Expired - Fee Related CN101695502B (en) | 2005-09-19 | 2006-09-15 | Lanthanum fullerenol and application in preparing medicaments for inhibiting tumor growth |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101695502B (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102488657A (en) * | 2011-12-23 | 2012-06-13 | 苏州大学 | Fullerenol solid lipid nano-particles, preparation method thereof, and application thereof |
| CN107693540A (en) * | 2017-10-12 | 2018-02-16 | 浙江理工大学 | Metal fullerol and its application as preparation treatment leukemia medicament |
| CN108403717A (en) * | 2018-05-28 | 2018-08-17 | 中国科学院高能物理研究所 | The purposes of Fullerol nano particle and pharmaceutical composition comprising it |
| CN108904532A (en) * | 2018-08-20 | 2018-11-30 | 上海市第人民医院 | A kind of application of the composition of Fullerene C20 nanocrystal joint CaMKII inhibitor |
| CN110292583A (en) * | 2019-06-28 | 2019-10-01 | 中国科学院高能物理研究所 | Fullerol and combinations thereof is preparing the application in antithrombotic reagent |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1208338C (en) * | 2002-10-23 | 2005-06-29 | 赵宇亮 | Contrast enhancement agent of magnetic resonance imaging radiography for metal fullerene as well as its preparing method and usage |
-
2006
- 2006-09-15 CN CN 200910210368 patent/CN101695502B/en not_active Expired - Fee Related
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102488657A (en) * | 2011-12-23 | 2012-06-13 | 苏州大学 | Fullerenol solid lipid nano-particles, preparation method thereof, and application thereof |
| CN107693540A (en) * | 2017-10-12 | 2018-02-16 | 浙江理工大学 | Metal fullerol and its application as preparation treatment leukemia medicament |
| CN108403717A (en) * | 2018-05-28 | 2018-08-17 | 中国科学院高能物理研究所 | The purposes of Fullerol nano particle and pharmaceutical composition comprising it |
| CN108904532A (en) * | 2018-08-20 | 2018-11-30 | 上海市第人民医院 | A kind of application of the composition of Fullerene C20 nanocrystal joint CaMKII inhibitor |
| CN108904532B (en) * | 2018-08-20 | 2021-07-27 | 上海市第一人民医院 | Application of composition of fullerene C60 nanocrystal combined with CaMKII inhibitor |
| CN110292583A (en) * | 2019-06-28 | 2019-10-01 | 中国科学院高能物理研究所 | Fullerol and combinations thereof is preparing the application in antithrombotic reagent |
| CN110292583B (en) * | 2019-06-28 | 2020-06-16 | 中国科学院高能物理研究所 | Application of fullerol and composition thereof in preparation of antithrombotic drugs |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101695502B (en) | 2011-11-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP5043015B2 (en) | Use of metal furol for producing metal furol and tumor (cancer) growth inhibitory drug | |
| CN1935812B (en) | Metal fullerol and its pharmaceutical use for inhibiting tumour growth | |
| CN109602919B (en) | Black phosphorus quantum dot coated by core-shell metal organic framework and preparation method and application thereof | |
| CN102898542B (en) | Water-soluble fullerene and application thereof | |
| CN101397132A (en) | Water-soluble fullerenes derivates, composition and application thereof in preparation of medicament for inhibiting tumor growth and metastasis | |
| CN101695502B (en) | Lanthanum fullerenol and application in preparing medicaments for inhibiting tumor growth | |
| CN112618727A (en) | Preparation for enhancing photodynamic therapy of hypoxic tumor and preparation method and application thereof | |
| WO2021169744A1 (en) | Fluorescent carbon quantum dot, and preparation method therefor and application thereof in preparation of antitumor drug sensitizer | |
| Pang et al. | Aptamer modified MoS2 nanosheets application in targeted photothermal therapy for breast cancer | |
| CN102258788A (en) | Targeted transmission assembly of adriamycin anticancer medicine and preparation method thereof | |
| CN112933229A (en) | Carrier-free self-assembly nanoparticle of IR820 and atovaquone and preparation method and application thereof | |
| CN102250150A (en) | Organic hydridized tetra-core platinum complex and preparation method thereof as well as application technology field of complex in antitumor medicament preparation | |
| CN109369923B (en) | Fe3+/2+-NO donor mixed valence coordination polymer and application thereof | |
| CN111558043B (en) | Preparation method of terpene micromolecule assembled redox response photosensitive drug | |
| CN109289048B (en) | Tumor vascular blocking synergistic phototherapeutic reagent and synthesis method and application thereof | |
| CN115947745B (en) | Albumin-based photothermal conversion nano material and preparation method and application thereof | |
| CN113332429B (en) | Molybdenum-sulfur compound-protein nano-composite and preparation method and application thereof | |
| CN116253636B (en) | Co (cobalt) 4 Application of N/CNE nano material | |
| CN116162077B (en) | Baicalein derivative and preparation method and application thereof | |
| CN113447462B (en) | Application of metformin-derived carbon quantum dots | |
| CN111087376B (en) | Gadolinium-doped rose bengal nanodots and preparation method and application thereof | |
| CN105906540A (en) | Compound and preparation method and application thereof | |
| Tokunaga et al. | Antitumour Effects of 4‐Pyridoxate Diammine Hydroxy Platinum, a Novel Cisplatin Derivative, Against Malignant Gliomas In‐vitro and In‐vivo: A Comparison with Cisplatin | |
| CN117547606A (en) | Ultrasonic response type pro-apoptosis nano-particle, and preparation method and application thereof | |
| CN114404586A (en) | A-D-A type condensed ring conjugated organic micromolecule photosensitizer and application 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 | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20111102 |