CN101239180B - Complexes for target killing tumor cell, preparation and application thereof - Google Patents
Complexes for target killing tumor cell, preparation and application thereof Download PDFInfo
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- CN101239180B CN101239180B CN2008100343294A CN200810034329A CN101239180B CN 101239180 B CN101239180 B CN 101239180B CN 2008100343294 A CN2008100343294 A CN 2008100343294A CN 200810034329 A CN200810034329 A CN 200810034329A CN 101239180 B CN101239180 B CN 101239180B
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Abstract
The present invention provides a compound for target killing tumour cell, a method for preparing the same and an appliance, which relates to nano-technology and life science field. The nanometer compound is composed of carbon nanotube less than 50nm -1mum, ricin protein A-chain, anti-HFR-2 targeting protein. The short carbon nanotube combines with ricin protein A-chain and anti-HER-2 targeting protein respectively. The compound has high killing rate by acting on human breast cancer cell and normal cell. The preparing method is simple, and the prepared compound has better biocompatibility and better target for tumour cell, and provides new thinking for clinic research of target killing tumour cell.
Description
Technical field
The present invention relates to nanotechnology and life science, a kind of complex, method for preparing and application of target killing tumor cell are provided especially.
Background technology
Because the selection permeability of cell membrane, a lot of biomolecule particularly macromole of one type in protein just can not initiatively pass cell membrane, so to seek new biomacromolecule carrier be pressing for of drug conveying, gene and protein for treatment field.
CNT has been showed huge using value to people with its comprehensive excellent properties.In recent years, the biomolecule functionalization of CNT further is applied to living things system alive for CNT opportunity is provided.At present, be that the exploratory development of purpose increases rapidly with the biological applications, become a new research focus gradually.French scientist Pantarotto (Pantarotto D in 2003; Briand J, Prato M, Bianco A.Chem Commun; 2004; 1 (1): 16-17) utilize the confocal fluorescent microscope to find that fluorescently-labeled SWCN (SWNT) itself can get into Cytoplasm and nucleus, when the concentration of SWNT was lower than 10 μ M, the pair cell of CNT own can not produce tangible toxic action.Simultaneously, they find that also with little peptide section on the SWNT covalent bond SWNT can be delivered into cell with little peptide section.Subsequently, people such as Dai (Kam N W S, Dai H.J Am Chem Soc, 2005,127 (16): 6021-6026; Kam N W S, Jessop T C, WenderP A; Dai H.J Am Chem Soc, 2004,126 (22): research 6850-6851) shows that SWNT portability molecular weight gets into the mammalian cell of animal smaller or equal to numerous protein of 80kDa; Like BSA, Streptavidin and cytochrome C etc.Recently domestic also have patented invention that antisense oligonucleotide is assembled into to process pharmaceutical carrier (number of patent application: 200710037582.0, publication number CN101015696) on the multi-walled carbon nano-tubes.
The research that toxin protein is applied to clinical cancer therapy is of long duration.As far back as Lin (Lin JY in 1970; Tserng K Y, Chen C C, Tung T C.Nature; 1970; 227:292-293) etc. the discovery toxalbumin (Ricin) of from castor bean, extracting has very strong antitumor action, and they reach the effect of curing the mouse ascites tumor after 5 days through the Ricin at mouse peritoneal injection high dose.Ricin is a kind of of ribosome inactivating protein, is made up of A chain (RTA) that links to each other through a disulfide bond and B chain (RTB), and wherein RTA is the toxicity chain, can make the big subunit inactivation of ribosome 60S, and it is synthetic to suppress intracellular protein, finally causes cell death.And RTB is a carrier, can combine with the surface of cell membrane binding site, and the contrary secretory pathway of mediation A chain warp gets into Cytoplasm and arrives endoplasmic reticulum, and in this generation disulfide bonds two chains is separated, and makes the A chain be folded into activated state again.Natural Ricin since RTA and RTB combine; Toxicity is quite big; As long as 1 milligram Ricin just is enough to poison with poison an adult, just be enough to cell killing as long as get into several Ricin molecules in the kytoplasm, so high toxicity has limited the use of people to Ricin.Along with the development of technique for gene engineering, people have carried out the trial of a lot of distinct methods according to its toxic action mechanism.Domestic patent (number of patent application: 200410018033.5; Publication number: CN1569234) report utilizes gene recombination technology specific expressed Ricin B catenin in tumor cell; While purification ricin A chain; Then with the RTA intravenous injection, thereby in tumor tissues, be formed with active toxin, killing tumor cell.
Summary of the invention
The purpose of this invention is to provide a kind of nano-complex that can be used for target killing breast cancer tumour cell.
Another object of the present invention provides the method for preparing of above-mentioned nano-complex.
The invention provides a kind of nano-complex of target killing tumor cell; This nano-complex is made up of CNT, Ricin protein A chain and the anti-HER-2 targeting proteins of length 5nm～1 μ m, and carbon nanometer short tube links to each other with Ricin protein A chain and anti-HER-2 targeting proteins respectively.
The present invention selects the functional chain of Ricin---and the A chain is as the element of killing tumor cell.Ricin (the proteic NCBI of the Ricin number of landing P02879) is a kind of of ribosome inactivating protein, is made up of A chain (RTA) that links to each other through a disulfide bond and B chain (RTB), and wherein RTA is the toxicity chain, and RTB carries the A chain to get into cell, avirulence own.Among the present invention purified RTA by technique for gene engineering obtain (number of patent application: 200410018033.5, publication number: CN1569234).
Human epidermal growth factor acceptor 2 (Her-2) human epidermal growth factor acceptor 2 (Her-2) are a kind of known oncogenes, and cross expressing of it shifted 25% patient with breast cancer's tumor.Anti-HER-2 targeting proteins (Herceptin; Anti-HER-2) be one type of ability and the material of tumor cell surface molecular specific effect, can discern monoclonal antibody or the part of a large amount of cytokines that exist of tumor cell surface of the specific antigen of tumor associated antigen and cell surface.Anti-HER-2 targeting proteins can obtain through conventional monoclonal antibody preparation method, also can buy from sigma company.
The present invention obtains purified RTA (number of patent application: 200410018033.5 with technique for gene engineering; Publication number: CN1569234); Utilize CNT and RTA that block, that bio-toxicity is little to process MWNT-RTA then; Substitute the B chain by CNT the RTA chain is carried into cell, avoided expressing the loaded down with trivial details of B chain, practiced thrift cost.Finding that CNT can carry RTA and get into cell; And can highly keep the biological activity of toxin protein, after spectrum ground causes killing and wounding of many cell line, the present invention also adopt can selectively acting in the targeting antibodies Herceptin (anti-HER-2) of tumor cell as targeting agent; It is processed the MWNT-RTA-anti-HER-2 nano-complex jointly with MWNT-RTA; Because HER-2 antibody selectively acting in the HER-2 of human breast carcinoma apparent height receptor, has high affinity, has height targeting property; Therefore it works to cancerous cell, and less to Normocellular effect.Research shows that the mechanism of action of Herceptin treatment breast carcinoma mainly is through combining with the HER-2 receptor-specific, influencing the transmission of growth signals.
On the other hand, the invention provides the method for preparing of above-mentioned complex, this method specifically may further comprise the steps successively:
(1) with the CNT purification and block short tube into length 5nm～1 μ m;
(2) in the dispersion liquid of the CNT of step (1) gained, add Ricin protein A chain and anti-HER-2 targeting proteins, the three stirs and is combined into complex and gets final product; The concentration range of CNT is 0.006～0.1mg/mL in the mixed liquor, and the concentration of Ricin protein A chain is 0.05～1.0 μ mol/L, and the concentration range of anti-HER-2 targeting proteins is 0.25～2.0 μ mol/L.
In the step of method for preparing of the present invention (1), CNT can block through the nitration mixture ultrasonic method.
In the step of method for preparing of the present invention (1), CNT can place the mixed liquor of concentrated nitric acid and concentrated sulphuric acid to carry out ultrasonic blocking, and wherein the volume ratio of concentrated nitric acid and concentrated sulphuric acid is (0.5-1.5): (2-4).
In the step of method for preparing of the present invention (1), CNT can place nitric acid and vitriolic mixed liquor to carry out ultrasonic blocking, and supersonic frequency is 40-80KHz (KHz), and ultrasonic time is 7-9 hour.
In the step of method for preparing of the present invention (2), the dispersion liquid of CNT is that the CNT of step (1) gained is dispersed in gained liquid in the phosphate buffer (for example, cultured cell PBS commonly used).
In the step of method for preparing of the present invention (2), CNT, Ricin protein A chain and anti-HER-2 targeting proteins can combine through the mode that ice bath mixes.
Among the present invention, CNT is SWCN or multi-walled carbon nano-tubes.
Said CNT can be commercial SWCN (SWNT) or multi-walled carbon nano-tubes (MWNT), and the specific surface area of producing like Nanometer Port Co., Ltd., Shenzhen is the multi-walled carbon nano-tubes of 40-300 meters squared per gram greater than the SWCN or the specific surface area of 600 meters squared per gram.Because SWNT research is more relatively, among the present invention, CNT adopts MWNT as object of study.Among the present invention, can adopt the conventional method purifying carbon nano-tube, for example use dense HNO
3The backflow purification is to remove impurity.
The present invention selects the carrier of CNT as complex discrepancy cell for use, and the CNT that blocks has numerous advantages: good water dispersibility; Huge specific surface area has bigger load capacity to albumen; Can be as required to the carbon nano tube surface functionalization; Have excellent biological compatibility, little to living things system toxicity.
The present invention also provides the application of said complex, is about to this complex and is used for killing tumor cell.
For example; The above-mentioned carbon mano-tube composite that has functional protein is joined (human breast cancer cell in the MDA-MB-453 cell of exponential phase; Available from Shanghai Chinese Academy of Sciences cell bank, cell numbering: TCHu35), continue to cultivate 22 hours; Detect this complex to breast cancer cell and the Normocellular ratio that kills and wounds with flow cytometer, find that it is 2: 1 to cancerous cell and the Normocellular ratio of killing and wounding.
The present invention adopts the carrier of CNT as the RTA chain of killer cell, makes RTA keep the biological activity of height, utilize this simply, method is carried into cell still first with " bullet " of toxin protein effectively.The present invention has avoided loaded down with trivial details and expensive in toxin protein self carrier (RTB) the expression process, the new approaches that provide toxin protein to use.In addition, through combining HER-2 antibody on the CNT, realized optionally killing and wounding human breast cancer cell at high proportion and the imagination less to normal cell killing provides new method for target tumor kills and wounds.The carrier systems based on carboxylated multi-walled carbon nano-tubes of this place invention is expected new foundation to medicine conveying and cancer targeted therapy to be provided.
Description of drawings
Figs further describes the present invention, but is not that the present invention is limited.
Fig. 1 is the flow cytometer detection figure that CNT-RTA-anti-HER-2 complex acts on breast cancer cell.Abscissa is a fluorescence intensity, and vertical coordinate is a cell quantity.
Fig. 2 is that CNT-RTA-anti-HER-2 complex acts on the Normocellular flow cytometer detection of CHO figure.Abscissa is a fluorescence intensity, and vertical coordinate is a cell quantity.Compared to Figure 1, CNT-RTA-anti-HER-2 complex ability killing tumor cell is described, and less to normal primary cellular defect.
Fig. 3 is for after different material hatches, Hela, and HL7702, COS7, the mortality rate flow cytometer of Mcf7 cell is figure as a result.For the allogenic cell strain, each post is representative successively from left to right: ghost, hatched independent MWNT, and hatched independent RTA, hatched the cell of MWNT-RTA.
Fig. 4 difference is hatched the streaming detection figure that thing kills and wounds human breast cancer cell MDA-MB-453 and normal cell CHO.Cell mortality after wherein the following material of each post value representative is hatched: 1 blank cell; 2 independent MWNT; 3 independent RTA; 4 independent anti-HER-2 albumen; The 5RTA-anti-HER-2 complex; The 6MWNT-anti-HER-2 complex; The 7MWNT-RTA complex; The 8MWNT-RTA-anti-HER-2 complex.
The specific embodiment
Below in conjunction with the practical implementation example the present invention is done further elaboration, these embodiments only are used to explain the present invention, and non-limiting scope of the present invention.
The preparation of embodiment one MWNT-RTA complex
(1) purification of MWNT
Get 1g MWNT and place round-bottomed flask, add the dense HNO of 16ml
3, 140 ℃ of oil baths refluxed 4 hours, and centrifugalize discards upper strata liquid, and repeated washing is centrifugal till upper strata clear liquid pH～6, and using the aperture is the filtering with microporous membrane of 0.45 μ m, obtains black solid.
(2) block the preparation of MWNT
The MWNT that purification is crossed further volume ratio be in 3: 1 concentrated sulphuric acid and the concentrated nitric acid 40 ℃ blocked the carbon pipe in ultrasonic 8 hours down, make the two ends of pipe and part tube wall a large amount of carboxyls occur.It is the microporous filter membrane sucking filtration of 0.45 μ m that the carbon pipe dispersion liquid that blocks uses diameter; Extremely filtrate near till the neutrality with a large amount of distilled water washs; The filtrating that obtains then with the centrifugal 10min of 9000rpm to remove the bulky grain carbon pipe in the solution; Can obtain the MWNT aqueous dispersion liquid of black homogeneous like this, with the oven dry down of this dispersion liquid infrared lamp, obtain concentration with PBS ultra-sonic dispersion again and be about 0.1mg/mL MWNT dispersion liquid at last.
(3) preparation of MWNT-RTA
250 μ L MWNT (0.05mg/mL) and 50 μ L RTA (2 μ M) are added to 200 μ L PBS (pH=7.4) linings, and ice bath stirred 2-3 hour, and at this moment toxin protein can be adsorbed on carbon nano tube surface owing to static, hydrophobic and other weak interactions.
Embodiment two MWNT-RTA complex are to the wide spectrum lethal effect of numerous cell lines
(1) cultivation of cell
Used cell is all preserved by the Zhong Jiang of life sciences institute of Fudan University professor laboratory among the present invention; Human liver cell HL7702 wherein; Human cervical carcinoma Hela cell and the used culture fluid of African green monkey kidney cell COS-7 are that volume fraction is the RPMI 1640 of 10% hyclone; Human breast cancer cell MCF-7 uses and to contain the DMEM culture fluid that volume fraction is penicillin one streptomycin of 10% hyclone and 1%.Used cell is cultivated in the saturated humidity incubator that 37 ℃ of volume fractions are 5%CO2, and the exponential phase cell is selected in experiment for use.
(2) mensuration of cell mortality
The MWNT-RTA complex that 150 μ L are processed is added in the above-mentioned cell, continues to cultivate 22 hours.Cultured cell is removed culture fluid, and the digestion of pancreatin room temperature after the dyeing of propidium iodide (PI, 50 μ g/mL) lucifuge, detects with flow cytometer rapidly.The numerical value that detection obtains is the ratio of representing the dead cell in the cell of measuring, and the excitation wavelength of PI is 488nm, and detecting wavelength is 620nm.Blank cell is all selected in all experiments for use, and the cell that cell independent and that MWNT is hatched reaches separately and RTA is hatched is as control experiment.As shown in Figure 3, as far as the cell strain of all investigations, all obvious experimental result of the mortality rate of the cell that the MWNT-RTA complex was hatched greater than each matched group.This result shows that MWNT is a kind of broad-spectrum, novelty, effectively carries the carrier that toxin protein RTA gets into cell, can also keep to high strength the biological activity of toxin protein simultaneously.
Embodiment three MWNT-RTA-anti-HER-2 complex are to the targeting killing effect of human breast cancer cell MDA-MB-453
250 μ L MWNT (0.05mg/mL), 50 μ L RTA (2 μ M) and 50 μ L anti-HER-2 albumen (1 μ M) are added to 150 μ L PBS (pH=7.4) linings; Ice bath stirred 2-3 hour, and at this moment toxin protein and targeting proteins can be adsorbed on carbon nano tube surface because of static, hydrophobic and other weak interactions.
The culture fluid of human breast cancer cell MDA-MB-453 and Chinese hamster ovary cell CHO all is the DMEM culture fluid.Each MWNT-RTA-anti-HER-2 complex that 150 μ L are processed is added to respectively in breast cancer cell and the normal cell, continues to cultivate 22 hours.The method of cultured cell according to embodiment 2 (2) detected with flow cytometer rapidly.Post 7 among Fig. 4 is dead data of the two strain cells that cause of MWNT-RTA; Wherein the ratio of cancerous cell and Normocellular mortality rate is 1: 1; And MWNT-RTA-anti-HER-2 (post 8) killing and wounding than being 2: 1 to this two strains cell; Be this targeted molecular of HER-2 antibody in the MWNT-RTA combination, can kill and wound cancerous cell more in high proportion, better choice property is arranged.
Claims (7)
1. the nano-complex of a target killing tumor cell; It is characterized in that; This nano-complex is made up of CNT, Ricin protein A chain and the anti-HER-2 targeting proteins of length 5nm～1 μ m, is adsorbing Ricin protein A chain and anti-HER-2 targeting proteins on the carbon nanometer short tube respectively; Described tumor cell is a breast cancer cell.
2. the method for preparing of complex as claimed in claim 1 is characterized in that this method may further comprise the steps successively:
(1) with the CNT purification and block short tube into length 5nm～1 μ m;
(2) in the dispersion liquid of the CNT of step (1) gained, add Ricin protein A chain and anti-HER-2 targeting proteins, the three stirs and is combined into complex and gets final product; The concentration range of CNT is 0.006～0.1mg/mL in the mixed liquor, and the concentration of Ricin protein A chain is 0.05～1.0 μ mol/L, and the concentration range of anti-HER-2 targeting proteins is 0.25～2.0 μ mol/L; Described dispersion liquid is obtained with PBS ultra-sonic dispersion again by the CNT of step (1) gained.
3. like the said method of claim 2, it is characterized in that in the step (1), CNT blocks through the nitration mixture ultrasonic method.
4. like the said method of claim 3, it is characterized in that CNT places the mixed liquor of concentrated nitric acid and concentrated sulphuric acid to carry out ultrasonic blocking, wherein the volume ratio of concentrated nitric acid and concentrated sulphuric acid is 0.5-1.5: 2-4.
5. like the said method of claim 3, it is characterized in that CNT places the mixed liquor of concentrated nitric acid and concentrated sulphuric acid to carry out ultrasonic blocking, supersonic frequency is the 40-80K hertz, and ultrasonic time is 7-9 hour.
6. like the said method of claim 2, it is characterized in that CNT is SWCN or multi-walled carbon nano-tubes.
7. complex kills and wounds the application in the medicament of breast cancer cell in preparation according to claim 1.
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| CN105561316A (en) * | 2015-12-30 | 2016-05-11 | 西安医学院 | Preparation method of carbon nanotube-dimethyldiguanide/polyethylene glycol composite material |
| CN110129332A (en) * | 2019-04-26 | 2019-08-16 | 浙江大学 | A kind of method of silkworm expression recombination ricin (WA) albumin A chain |
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