CN103191526B

CN103191526B - The heating ablation effect in anti ovary tumour of the CNT of functional modification

Info

Publication number: CN103191526B
Application number: CN201210001297.4A
Authority: CN
Inventors: 汪宏波; 张晓莉
Original assignee: Individual
Current assignee: Individual
Priority date: 2012-01-05
Filing date: 2012-01-05
Publication date: 2016-12-14
Anticipated expiration: 2032-01-05
Also published as: CN103191526A

Abstract

The present invention relates to tumor-targeting function carbon nano tube and preparation thereof.Multidrug resistance gene and coded cell membrane P glycoprotein (P gp) thereof are the big obstacles affecting chemotherapy in ovarian cancer effect.The most do not kill the effective ways of drug resistant cancer cells at present.CNT (CNTs) has the stronger feature absorbing heat energy, and energy quick release of energy in near-infrared region, forms local heat effect.The present invention designs with P gp as target spot, by modifying, increases the accumulation of CNT in drug resistant cancer cells, utilizes near infrared ray light radiation, form the intracellular condition of high temperature and make cytoclasis, to reach to kill cancerous cell.

Description

The heating ablation effect in anti ovary tumour of the CNT of functional modification

Technical field

The present invention relates to preparation and the application of heating ablation effect thereof of functionalized carbon nano-tube, the CNT of functional modification is connected to specific antibody can Identify many cells of resistant tumors, utilize CNT at the extinction thermal effect of near-infrared region, kill tumor cell.It is that a kind of targeting non-intruding is controlled Treat the new method of tumor.

Background technology

Ovarian cancer is as the primary tumor of harm whole world WomanHealth, the treatment one of the research treatment of oophoroma multidrug resistance, especially advanced ovarian cancer It it is directly the problem of Chinese scholars concern.Multidrug resistance (MDR) is the big obstacle affecting chemotherapy of tumors effect.Estimate according to American Cancer Society, Annual about 490,000 example malignant tumor patients are dead, wherein more than 90% drug resistance (1) that there is tumours of chemotherapeutic agents.The proposition of multidrug resistance is early At 20 century 70s, along with the further investigation to MDR mechanism, find that the formation of MDR is the complex process that a polygenes participates in, As multidrug resistance gene (MDR1) and coded cell membrane P glycoprotein (P-gp) thereof express increase, the increased activity of glutathione transferase, DNA The reduction of topoisomerase II activity, multidrug-associated protein increase etc.；Wherein with multidrug resistance gene (MDR1) and coded cell membrane P thereof Glycoprotein (P-gp) expresses the drug resistance the most most commonly seen (2) increasing mediation.Past more than 20 year, Chinese scholars sought various reverse The method of MDR1, such as Antisense OligodeoxynucleotideTechnique Technique, ribozyme technology etc., achieves certain experimental studies results and clinical experience, but at the beginning of these Step research is still in the exploratory stage, and multidrug resistance is still the major reason causing chemotherapy of tumors failed.At present, the five year survival rate of Advanced Ovarian tumor is only It is 15%～30%.Therefore, constantly study the new effective way overcoming MDR1, improve patient's cure rate and survival rate is not only current cancer and is controlled Treat important topic in the urgent need to address, it may have great clinical practice and economic worth.

2. the extinction thermal effect of CNT

CNT (CNTs) since being found (3) for 1991, with structure and the calorifics of excellence, electricity and the mechanical property such as bigger ratio of its uniqueness Surface area, good conductivity of heat, electric conductivity and higher mechanical strength cause extensive concern, become the study hotspot of field of nanometer material technology.In recent years CNTs applies and is increasingly becoming new focus (4-7) in the biomedicine particularly research on pharmaceutical carrier.CNTs has stronger in near-infrared region Absorb the feature of heat energy, and energy quick release of energy, form local heat effect.Kam etc. (8) report CNTs can rapid release soon under near infrared light Put unnecessary energy, form the intracellular condition of high temperature and make cytoclasis.Except near infrared light, CNT can be with radio wave absorbing and release Heat thus tumor cell is destroyed.It is thin in different carcinoma that Gannon etc. (9) have studied the heat effect that the absorption of radio wave causes by carbon pipe Effect in born of the same parents, and inject modified CNTs to the liver neoplasm of animal, with radio wave, carbon pipe is heated, has successfully killed tumor cell, And neighbouring healthy cell has been resulted only in minimal amount of injury.The result of the external living cells of CNTs and living animal experiment shows, carbon pipe is to normally The most significantly affecting of cell and tissue, this makes CNTs apply on human body in the future and is possibly realized (7).

3. by modifying raising CNT cell-penetrating ability.

CNT does not dissolves in organic solvent and water, thus limits its application in pharmaceutical carrier field.Functional modification CNT then can improve it Dissolubility, and its biocompatibility can be improved, make CNT be applied in pharmaceutical carrier field.Whether biology can be carried for CNT (10) employing Fluorescein isothiocyanate (isothi ocyanato fluorescein, FITC) labellings such as bioactive molecule enters this problem of cell, Pantarotto Derivatization modifies SWCN and peptide coupling SWCN, has investigated carbon nano-tube modified (the functi onalized carbon of both Nanotubes, f-CNTs) cell-penetrating ability, find that they all can primarily enter Cytoplasm by permeates cell membranes: CNT1, and peptide modified CNT2 Nucleus can be entered.Cellular uptake nano material also has size critical point.Fagan etc. (11), with DNA coated single-walled carbon nano tube, are prepared for not Affect the stable dispersion solution system of SWCN Electronic Structure, investigate the size difference of cellular uptake CNT.Research shows, cell The length of picked-up CNT is had selection, and (180 ± 17) nm is picked-up separation.They guess that different cellular uptake CNT there may also be difference The length range of choice.Ito etc. (12) use CNT as erythropoietin (erythr poietin, EPO) oral formulations carrier, find that short carbon is received Mitron can deliver more EPO to target cell.Liu etc. (13) utilize PEG to modify CNT, prepare water miscible CNT and carry paclitaxel, with purple China fir alcohol is compared, and CNT carrier band improves penetrance and the retentive period in blood circulation, and 4T1 breast cancer cell is had good inhibiting effect, And normal cell is not had obvious toxic-side effects.Kostarelos etc. (14) research shows that the carbon nano-tube modified sphere of penetration to different cells is wider, he Also find that cell can optionally absorb the CNT of different base group modification, the CNT of i.e. different base group modifications can penetrate different cells. The most up to the present, in terms of application nano material research in Diagnosis of Ovarian Cancer is concentrated mainly on function vector, the most not about CNT The report that extinction thermal effect is studied in ovarian cancer.

Summary of the invention

The present invention designs with P-gp as target spot, specifically increases the accumulation of CNTs in drug-resistant ovarian carcinoma cell, recycles near infrared ray light radiation, to reach To the Research Thinking killing ovarian cancer cell.This research fits to a series of chemical graft process, is connected on CNTs by Polyethylene Glycol (PEG) biotin Form DSPE-PEG (2000)-biotin-CNTs (B-CNTs), on the one hand can increase the water solublity of CNTs, on the other hand connect more Anti-P-gp monoclonal antibody (mAb), eventually form mAb-CNTs complex.For drug-resistant ovarian carcinoma cell Expression of Plant Height P-gp specific with These cells combine, and make CNT optionally orient and accumulate in tumor tissues, application CNTs extinction thermal effect and the cytoclasis that arrives.

For achieving the above object, the technical solution used in the present invention is:

Connect cell membrane P glycoprotein specific antibodies (anti-P-gp monoclonal antibody) specific recognition tumor cell on the carbon nanotubes, utilize CNT closely The extinction thermal effect in infrared light district so that the heating ablation of tumor cell.

The preparation process of described functional modification CNT is:

The preparation of 1.CNTs and purification.

Multi-walled carbon nano-tubes (self-control, caliber about 25nm).Use the method purification cNTs of nitration mixture oxidation.Will add by the unpurified CNT of 0.3g In the concentrated sulphuric acid that 24mL volume ratio is 3: 1 and concentrated nitric acid mixed liquor, sonic oscillation 8 hours, then by sucking filtration and repeatedly rinse with deionized water, Making filtrate pH value to neutral, the CNT obtained by sucking filtration is placed in the calorstat of 80 DEG C drying, is ground into powder, i.e. after being dried completely Obtain carbon nanotube-sample after purification.

The modification of 2.CNTs, utilizes DSPE-PEG (2000)-biotin to modify, and forms B-CNTS.

1. according to proportioning, CNTs Yu DSPE-PEG (2000)-biotin solution is joined in dry flask, ultrasonic disperse. mixture is stirred at room temperature, Product is centrifugal after repeatedly washing. obtain B-CNTS.

The modification of the most anti-P-gb monoclonal antibody, utilizes neutralite avidin (NA) to modify, forms anti-P-gp-NA (mAb-NA).

Business-like P-gb monoclonal antibody and neutralite avidin are purified.The monoclonal antibody of 10mg and Traut reactant liquor co-culture 1 hour, add 10mg NA, finally terminates, by running gel purification with MBS liquid.

4. prepare anti-Pgp-NA-B-CNTs (mAb-CNTs) complex.

B-CNTs and mAb-NA is carried out 1: 2 (wt/wt) mixing, shakes 35min under room temperature, be then centrifuged.Precipitate PBS dissolves, To mAb-CNTs complex.

The invention have the advantages that

1 utilizes the killing to drug-resistant ovarian carcinoma cell of the CNT extinction thermal effect, has no relevant report both at home and abroad.

CNT is modified by 2 by biotin-avidin interconnection function, will be greatly improved the binding ability of CNT and specific monoclonal antibody, and it is right to improve The magnetic target therapy effect of drug-resistant ovarian carcinoma cell.

3 present invention utilize CNT to absorb near-infrared laser thermal effect by experimental verification can play lethal effect to ovarian cancer tumor cell.And Prove that the CNT of non-irradiating laser is inconspicuous to the toxic action of cell.

Accompanying drawing illustrates: Fig. 1-5 is the ovarian cellular apoptosis experimental result of flow cytomery.

Fig. 1 is that blank does not adds CNT, does not irradiates near infrared ray；

Fig. 2 carbon nanotube concentration is 0, and near infrared ray irradiates, and power is 30J/cm2, and irradiation time is 600ms；

Fig. 3 carbon nanotube concentration is 50ug/ml, and near infrared ray irradiates, and power is 30J/cm2, and irradiation time is 600ms；

Fig. 4 is negative control, and carbon nanotube concentration is 80ug/ml, non-irradiating laser；

Fig. 5 carbon nanotube concentration is 80ug/ml, and irradiating laser power is 30J/cm2, and irradiation time is 600ms.

Subordinate list 1

	Fig. 1	Fig. 2	Fig. 3	Fig. 4	Fig. 5
						Q1	0.4%	0.8%	1.1%	0.7%	1.7%
Q2	3.3%	6.8%	10.9%	7.9%	20.8%
						Q3	94.5%	91.2%	81.2%	85.7%	60.7%
Q4	1.7%	1.3%	6.8%	5.7%	16.9%

Subordinate list 1 illustrates:

Q1 is necrocytosis rate, and Q2 is early apoptosis rate, and Q3 is living cell rate, and Q4 is late apoptic rate.

Experimental result is as shown in accompanying drawing subordinate list, after adding CNT and irradiating near-infrared laser, the apoptosis of ovarian cancer tumor cell is served promotion work With, and test result indicate that the increase along with carbon nanotube concentration, apoptosis rate increases the most therewith.

Statistical analysis: carry out single factor analysis with S P S S11.5 software, the comparison chi-square criterion of rate, level of significance α= 0.05.Result shows that process group has significant difference (P ＜ 0.05) with the early apoptosis rate of matched group with total apoptosis rate, illustrates near Infrared irradiation CNT can promote the apoptosis of tumor cell.

Specific implementation method:

1 Ovarian Cancer Cells is cultivated.

Use the independent drug-resistant cell strain to taxol resistance induced as usage by A2780, SKOV3 cell by increasing concen-trations and short time A2780/PTX, SKOV3/PTX.

1. A2780, the SKOV3 ovarian cancer cell that the phase of taking the logarithm grows, adds the culture fluid containing PTX, and continuous action is after 48 hours, with Hank ' S Balanced salt solution washed once, and changes the fresh culture continued growth without PTX, sees that most cells is dead, survivaling cell poor growth.Treat thin Born of the same parents' density passes on once when reaching 70%-80%, cultivates to exponential phase and again gives pastille culture medium, then steps up the drug level in culture fluid, Persister inductive dose is respectively 2ng/ml 2 times, 5ng/ml 2 times, 10ng/ml 3 times, 15ng/ml 3 times, 20ng/ml 3 times, 30ng/ml 3 times, 40ng/ml 3 times, 50ng/ml 3 times.Finally set up persister A2780/PTX, SKOV3/PTX.

2. make mdr cell continued growth, then recover after cell cryopreservation February, repeatedly pass in without the culture medium of PTX and still keep resistance to February more than The property of medicine.

3. mtt assay detection drug resistance multiple: mdr cell being inoculated in 96 orifice plates, adds the chemotherapeutic of variable concentrations, abandon supernatant after 48 hours, every hole adds Entering the MTT 20ul of 5mg/ml, abandon supernatant after continuing to cultivate 4 hours, every hole adds the DMSO of 200ul, lucifuge concussion 10min, makes crystal Fully dissolve.Detect 490nmOD value with microplate reader, calculate cell survival rate.By Logistic models fitting dose-effect relationship, ask half suppression dense Degree.

4. the expression of the mRNA of application round pcr mensuration MDR1.

5. application SABC measures the expression of relevant p-gp glycoprotein.

2 external Ovarian Cancer Cells co-culture with mAb-CNTs.

Ovarian carcinoma drug-resistant cell is divided into six groups, is established as matched group, CNTs group, B-CNTS group, mAb group, mAb-NA group, mAb-CNTs respectively Group, co-cultures 24 hours, utilizes MTT, the impact of technique study cell proliferation, apoptotic state and the cell cycles such as fluidic cell. 7.1064nm near infrared light (NIR) irradiates taxol resistance Ovarian Cancer Cells, measures the change of mdr cell.

With reference to step 6, ovarian carcinoma drug-resistant cell is divided into six groups, then through different capacity, different time length infrared radiation, deposits with mtt assay detection cell Motility rate.After using 1064nm wavelength near infrared light under the different capacity of 60J/cm2 and 120/cm2, observation of cell shape under inverted microscope State changes, the technique study apoptotic state such as mtt assay, fluidic cell and the impact of cell cycle.

According to test result indicate that above, CNT provides new direction to the development of the non-invasive therapy of ovarian tumor.

Claims

1. the preparation method of the CNT of functional modification, it is characterised in that:

(1) preparation of .CNTs and purification；

Self-control multi-walled carbon nano-tubes, caliber about 25nm, use the method purification cNTs of nitration mixture oxidation, will join in the concentrated sulphuric acid and concentrated nitric acid mixed liquor that 24mL volume ratio is 3: 1 by the unpurified CNT of 0.3g, sonic oscillation 8 hours, then by sucking filtration and repeatedly rinse with deionized water, make filtrate pH value to neutral, the CNT obtained by sucking filtration is placed in the calorstat of 80 DEG C drying, is ground into powder, i.e. obtains carbon nanotube-sample after purification after being dried completely；

(2) modification of .CNTs, utilizes DSPE-PEG (2000)-biotin to modify, and forms B-CNTS；

Joining in dry flask according to proportioning by CNTs Yu DSPE-PEG (2000)-biotin solution, ultrasonic disperse, mixture is stirred at room temperature, and product is centrifugal after repeatedly washing, and obtains B-CNTS；

(3). the modification of anti-P-gb monoclonal antibody, utilize neutralite avidin (NA) to modify, form anti-P-gp-NA (mAb-NA)；

Business-like P-gb monoclonal antibody and neutralite avidin are purified, and the monoclonal antibody of 10mg and Traut reactant liquor co-culture 1 hour, add 10mg NA, finally terminate, by running gel purification with MBS liquid；

(4). prepare anti-Pgp-NA-B-CNTs (mAb-CNTs) complex；

B-CNTs and mAb-NA carrying out 1: 2 (wt/wt) mixing, shakes 35min, be then centrifuged under room temperature, precipitate PBS dissolves, and obtains mAb-CNTs complex.