CN103083661A - Application of carbon nanotube in preparation of immunostimulant of anti-tumor immunotherapy medicine - Google Patents
Application of carbon nanotube in preparation of immunostimulant of anti-tumor immunotherapy medicine Download PDFInfo
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- CN103083661A CN103083661A CN2013100318113A CN201310031811A CN103083661A CN 103083661 A CN103083661 A CN 103083661A CN 2013100318113 A CN2013100318113 A CN 2013100318113A CN 201310031811 A CN201310031811 A CN 201310031811A CN 103083661 A CN103083661 A CN 103083661A
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Abstract
The invention discloses new application of a carbon nanotube, and mainly relates to application of the carbon nanotube in preparation of an accelerating agent of an anti-tumor immunotherapy medicine. Experiments prove that when the carbon nanotube serves as the accelerating agent, the immunotherapy effect of inactive tumor cell vaccines can be obviously improved, the tumor curative ratio of tumor-bearing mice is obviously increased, and the tumors of the unhealed mice are also effectively controlled. When the completely healed mice are inoculated with the tumor cells again, no tumor grows, which means that the carbon nanotube effectively plays a role of an accelerating agent, improves the immunotherapy efficiency of the tumor vaccines and simultaneously promotes an organism to generate specific immunoreaction, thus a specific tumor antigen recognition and killing system is established in the organism.
Description
The application is application number 200810102322.1, and the applying date is on March 20th, 2008, and denomination of invention is divided an application for " application of CNT in the immunopotentiating agent of preparation anti-tumor immunotherapy medicaments ".
Technical field:
The invention belongs to biomedical sector, relate to the new purposes of CNT, be specifically related to the new application that CNT can be used as the immunopotentiating agent of anti-tumor immunotherapy medicaments.
Background technology:
The biomedical applications potential of CNT receives great concern in recent years, and has attracted in the world numerous laboratorys to carry out correlational study from all angles.A key property of CNT is the multiple barrier that can cross in cell membrane and organism, enters in the interior multiple organ of Cell and organism body.The result of study demonstration of existing bibliographical information, CNT can pass the cell membrane of various kinds of cell, comprises l cell, human cervical carcinoma cell (HeLa), human breast cancer cell (MCF7) and people T-cell lymphoma cell etc.
[1-5]Seminar's separating Morr. cell from the BALB/c mouse spleen is arranged, hatch the different time from the CNT of the FITC labelling of variable concentrations (2 μ g/ml～10 μ g/ml) (after 4h～24h), can observe green fluorescence in cell, show that CNT has entered into cell interior.They have further studied the impact of CNT on primary immune cell function, find that T cell, B cell and macrophage still keep original function under CNT exists
[6]
The above results be CNT as the novel carriers of gene and medicine, the targeted delivery that is applied to atomic type biosensor and medicine is laid a good foundation.For example, CNT can be used as carrier, is used for DNA, albumen and transport of drug.Protein molecular can be connected to by covalency and noncovalent interaction the surface of CNT.Dai seminar
[7]By the non-specific adsorption effect, the fluorescin molecule is connected to the surface of SWCN (SWNT), utilize cell to the endocytosis of CNT, CNT to be entered in cell, simultaneously fluorescent marker is brought in cell, has realized the cell inner mark colour developing.Further studies show that, can follow the trail of and show CNT-albumen composition by the specific optical property of CNT itself, thereby use for CNT a kind of approach that provides as novel carriers in diagnosing image.And for example, DNA molecular can be incorporated into the surface of CNT, and the change of occurred conformation can detect this signal change by near-infrared fluorescent with the change of effects of ion concentration, thereby makes the CNT might be as the DNA vector of intracellular environment Real-Time Monitoring.
Have researcher to propose, CNT can be positioned at cytoplasm, organelle, even enter in nucleus, and the location of CNT in cell is with to be connected to its surperficial functional molecular relevant.For example, the CNT of nucleic acid function can navigate in nucleus.Therefore, CNT can be used as the carrier of SiRNA, DNA, increases or suppress the expression of differential protein in cell.There are some researches show, can be identified by macrophage specifically and engulf through the CNT of chemical modification, this characteristic makes CNT might become a kind of novel carriers system.For example, studies show that
[8], CNT is combined with some polypeptide, may bring into play certain effect in immunotherapy of tumors.Bianco A etc.
[9]Result of study show, CNT can increase the immunizing antigen of foot and mouth disease virus specific polypeptide, can be used as vaccine carrier.This research is coupled to carbon nano tube surface with the B cell epitope polypeptide of Foot-and-mouth disease virus Infectious Cycle by covalent bond, outside polypeptide epitope still is exposed to, can be identified and bring out body internal specific antibody by its specific antibody and produce.In animal body, experiment shows, this complex has caused the antibody mediated immunity response that strengthens, and CNT itself does not cause that specific antibody produces, and there is no cross reaction between polypeptide antibody.In above-mentioned research, CNT is used mainly as the carrier of biomacromolecule.
Summary of the invention:
Purpose of the present invention is to provide the new application of CNT.
The invention provides CNT application in immunopotentiating agent in the preparation anti-tumor immunotherapy medicaments.
Wherein, described CNT is the water-soluble carbon nanometer tube powder, or be concentration at the carbon nano-tube aqueous solutions of 0.01 ~ 0.5mg/ml, the carbon nano-tube aqueous solutions of preferred 0.1～0.4mg/ml.
Described anti-tumor immunotherapy medicaments is polypeptide or the proteantigen of tumor inactivated vaccine, Tumor-assaciated; Be in particular the tumor inactivated vaccine.
A kind of concrete application of CNT of the present invention is to provide a kind of antineoplastic immune pharmaceutical composition, comprises the combination of the anti-tumor immunotherapy medicaments such as the polypeptide of tumor inactivated vaccine, Tumor-assaciated or proteantigen and water-soluble carbon nanometer tube.
Wherein, the portfolio ratio of described tumor inactivated vaccine and water-soluble carbon nanometer tube is 10
4~ 10
6Cell: 0.1mg ~ 20mg water-soluble carbon nanometer tube.
The another kind of concrete of CNT of the present invention used, and is to provide a kind of anti-tumor immunotherapy medicaments test kit, comprises for as the medicine of immunotherapy of tumors related antigen with as CNT and a operation instructions of immunopotentiating agent.
In this test kit, CNT is the water-soluble carbon nanometer tube powder, and described medicine as the immunotherapy of tumors related antigen is the mitomycin for the preparation of the tumor inactivated vaccine, or is DNA vaccination, polypeptide or the proteantigen of Tumor-assaciated.
In test kit, the CNT working concentration is the aqueous solution of 0.1mg/ml ~ 0.4mg/ml.
The present invention is checking by experiment also, as promoter, can significantly improve the immunization therapy effect of killing tumor cells vaccine with CNT, and the tumor cure rate of tumor-bearing mice is significantly improved, and the tumor of not curing mice has also obtained effective inhibition.After the mice of curing fully is vaccinated tumor cell again, do not have tumor to form.Illustrate that CNT brought into play the effect of effective promoter, improved the immunization therapy efficient of tumor vaccine, simultaneously, promote that body has produced specific immunoreation, set up specific tumor antigen identification and kill system in body.
Description of drawings:
Fig. 1 is the oncotherapy efficiency chart of different immunotherapy methods.
Fig. 2 is for using different CNT dosage as the oncotherapy efficiency chart of tumor vaccine immunopotentiating agent.
Fig. 3 is for curing mice inoculated tumour cell experiment result (tumor challenge) figure again.
The specific embodiment:
The anti-tumor immunotherapy medicaments that uses in tumor immunotherapy, no matter be traditional vaccine, killing tumor cells for example, or the vaccine of a new generation, comprise the proteins and peptides of purification, by the vaccine of the vaccine of microorganism separated product preparation, the preparation of DNA recombinant technique, direct synthetic polypeptide vaccine etc., often have the lower problem of therapeutic efficiency.Improve the efficient of vaccine, need to seek safe and effective Novel immune promoter, the immune response of enhancing body to defined antigen.
The present invention proposes CNT as the new approaches of immunopotentiating agent in anti-tumor immunotherapy medicaments, and verify by experiment the effect that its auxiliary tumor vaccine is treated.
In the present invention, CNT used is water miscible carbon nanotube powder or has been made into certain density carbon nano-tube aqueous solutions.Water miscible carbon nanotube powder can adopt following methods to prepare, water miscible carbon nanotube powder or the carbon nano-tube aqueous solutions that also can directly select other method to obtain.
The preparation of water-soluble carbon nanometer tube powder and aqueous solution:
1) preparation of mixed acid: concentrated sulphuric acid and the concentrated nitric acid ratio preparation of 2:1 are by volume formed mixed strong acids solution;
2) take in the mixed acid solution that the 200mg multi-walled carbon nano-tubes adds 400ml, the ultrasonic probe that is 15mm with diameter extend in multi-walled carbon nano-tubes and mixed acid, and supersound process is 2 minutes under 1000w power, obtains atrous solution.
3) will filter (filter membrane aperture be 2 μ m) after atrous solution thin up, water repeatedly rinses and leaches thing, to filtrate be neutrality; Being deposited in of leaching dried below 70 ℃ to constant weight namely obtain the water-soluble carbon nanometer tube powder.
4) with above acquisition water-soluble carbon nanometer tube powder in the desired amount (as 2mg) add in pure water (as 10ml), again processed 2 minutes with the ultrasonic probe that diameter is 6mm, can obtain uniform and stable dark solution, be the carbon nano-tube aqueous solutions of desired concn (as 0.2mg/ml).Adjust the addition of powder and water, can prepare the carbon nano-tube aqueous solutions of any required concentration.
The experiment that CNT is used as immunopotentiating agent in anti-tumor immunotherapy medicaments
1. set up animal model for tumour
At the female BALB/C mice left hind subcutaneous vaccination H22 cell (available from Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences's cell centre) in 6 ~ 8 ages in week, after the week, subcutaneous have entity tumor to generate, and the average tumor size reaches approximately 5 * 5 * 2(mm
3).
2. immunization therapy efficient experiment
Tumor-bearing mice is divided into 4 groups at random, 10 every group, comprises: (1) matched group (Control), (2) tumor vaccine treatment group (TCV), (3) CNT treatment group (CNT), (4) tumor vaccine associating CNT treatment group (TCV+CNT).
Wherein tumor vaccine derives from deactivation H22 tumor cell.The H22 tumor cell line is available from Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences's cell centre.The H22 tumor cell inoculation in female BALB/C mice abdominal cavity, is carried out cells in vivo propagation.Extract ascites from mouse peritoneal, separate obtaining the H22 cell, after processing with mitomycin (available from BJ Union Hospital), obtain the tumor cell of deactivation, be tumor vaccine.The concentration of tumor vaccine is adjusted into 10
5Cell/ml, the 0.25mg/ml aqueous solution of CNT for being made by introduction method of the present invention.When CNT and tumor vaccine coupling, subcutaneous injection is in the different parts of mice respectively.
Above-mentioned 4 groups of mices were carried out twice immunization therapy in the 7th day and the 14th day respectively after the inoculated tumour cell, it is as follows that the ejection preparation situation is respectively organized in each immunity:
(1) matched group: subcutaneous injection deionized water 0.4ml and PBS solution (0.15M phosphate buffer, pH=7.4) 0.1ml.
(2) TCV group: subcutaneous killing tumor cells 0.1ml and the deionized water solution 0.4ml of injecting respectively.
(3) CNT group: subcutaneous PBS solution (0.15M phosphate buffer, pH=7.4) 0.1ml and the carbon nano-tube aqueous solutions 0.4ml of injecting respectively.
(4) CNT+TCV group: subcutaneous killing tumor cells 0.1ml and the carbon nano-tube aqueous solutions 0.4ml of injecting respectively.
In said process, continue to observe the tumor size variation of mice.Immunity for the second time is after 5 days, and in the CNT+TCV group, the tumor of part mice is dwindled gradually, the final disappearance, and the development of CNT group mouse tumor size is slower, and matched group and the development of TCV group mouse tumor size are very fast., put to death tumor and do not cure mice after 15 days in immunity for the second time, measure final tumor size, statistics cure rate and calculating tumor control rate are with each group small mouse treatment situation classification.Tumor disappearance person is for curing, and tumour inhibiting rate is that tumor is suppressed greater than 50%, and tumour inhibiting rate is the tumor deterioration less than 50%.Fig. 1 is seen in statistical result, demonstrates that between CNT+TCV and other group, tumor cure rate has significant difference.
3. CNT is as the dose response studies experiment of immunopotentiating agent
Experimental arrangement is identical with 2, just establishes the CNT+TCV group of five different CNT dosage, is respectively each immunity every mice CNT injection volume 0.025mg, 0.05mg, 0.1mg, 0.2mg or 0.4mg.
The experiment demonstration, suitable CNT dosage (0.1 ~ 0.2mg/ mice) can reach optimum therapeuticing effect.Experimental result is seen Fig. 2.
4. tumor challenge experiment
At subcutaneous injection H22 cell and EMT cell (human breast cancer cell is available from Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences's cell centre), the injection volume of every mice H22 cell is 1.5 * 10 to the tomor rejection mice that survives in the CNT+TCV group in experiment 2 by respectively
6, the injection volume of every mice EMT cell is similarly 1.5 * 10
6Whether observe has tumor to generate; Matched group is set simultaneously, and matched group is the normal female BALB/C mice in 6 ~ 8 ages in week, H22 cell and the EMT cell of every same quantity of injection.
Experimental result: after 1 week, control group mice all grows tumor, and the tomor rejection mice of subcutaneous plantation H22 cell does not have 1 to grow tumor, and the tomor rejection mice of subcutaneous plantation EMT cell has all grown tumor.Experimental result is seen Fig. 3.
This tests demonstration, after the mice of curing fully is vaccinated tumor cell again, can not form new tumor, illustrate that the immunization therapy scheme of tumor vaccine combination CNT can cause the specific immunoreation of body, the growth of specific inhibition H22 tumor suppresses tumor recurrence.
The present invention has verified when adopting traditional killing tumor cells as vaccine, use the carbon nano-tube aqueous solutions preparation as immunopotentiating agent, mice to lotus human liver cancer cell H22 tumor carries out immunization therapy, relatively show with independent use killing tumor cells vaccine therapy comparison therapeutic effect: with CNT as immunopotentiating agent, can significantly improve the therapeutic effect of killing tumor cells vaccine, the tumor cure rate of tumor-bearing mice is significantly improved, and the tumor of not curing mice has also obtained effective inhibition.After the mice of curing fully is vaccinated tumor cell again, do not have tumor to form.Above-mentioned result of study explanation, CNT has been brought into play effective immunopotentiating agent effect, has improved the immunization therapy efficient of tumor vaccine, simultaneously, promotes that body has produced specific immunoreation, has set up specific tumor antigen identification and kill system in body.
5 and above experiment 2 similar, the present invention has also verified the immunization therapy scheme of CNT combination polypeptide and CNT combined protein matter antigen, immunization therapy efficient as a result all is significantly increased.
Make and use the same method, the present invention carries out immunization therapy to the mice of breast cancer cell (EMT, MCF7) and lung carcinoma cell tumor, carry out repeated authentication by above experimental program, found that, the immunization therapy scheme of the corresponding tumor vaccine of CNT combination, the more single tumor vaccine immunization therapy of its immunization therapy efficient all is significantly increased.Illustrate that CNT all has facilitation for different tumor immunotherapies.
According to above experimental result, the present invention can utilize water miscible CNT preparation to form new immunotherapy medicaments compositions.Said composition is that wherein the tumor vaccine portfolio ratio can be 10 with the polypeptide of anti-tumor immunotherapy medicaments such as tumor inactivated vaccine, tumour-specific or proteantigen and the combination of water miscible CNT
4～10
6Cell: 0.1mg ~ 20mg water-soluble carbon nanometer tube.Composition medicine the most simply use for water as solvent, can certainly add other auxiliary agent or pharmacy adjuvant to help medicine to play a role.
The present invention can also utilize water miscible CNT preparation to form the immunotherapy medicaments test kit.This test kit comprises for as the medicine of immunotherapy of tumors specific antigen with as CNT and a operation instructions of immunopotentiating agent, CNT is water miscible carbon nanotube powder, during use, to specifications indication is mixed with finite concentration (aqueous solution as 0.1～0.4mg/ml), then test kit is used in to specifications operating process (operation of reference experiment 2 they (4)).In this test kit, can also be the reagent mitomycin that can obtain the tumor inactivated vaccine as the medicine of immunotherapy of tumors specific antigen, the operations of describing according to experiment 2 during use obtain tumor vaccine, and then are used in conjunction with carbon nano-tube aqueous solutions.
List of references
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Claims (10)
1. CNT application in immunopotentiating agent in the preparation anti-tumor immunotherapy medicaments; Described tumor is to comprise lung carcinoma cell, breast cancer cell but each quasi-cancer cell of not comprising hepatoma carcinoma cell.
2. use according to claim 1, it is characterized in that, described CNT is the water-soluble carbon nanometer tube powder.
3. use according to claim 1, it is characterized in that, described CNT is that concentration is at the carbon nano-tube aqueous solutions of the carbon nano-tube aqueous solutions of 0.01 ~ 0.5mg/ml, particularly 0.1～0.4mg/ml.
4. according to claim 1 and 2 or 3 described application, is characterized in that, described anti-tumor immunotherapy medicaments is polypeptide or the proteantigen of tumor inactivated vaccine, Tumor-assaciated; Be in particular the tumor inactivated vaccine; Described tumor is to comprise lung carcinoma cell, breast cancer cell but each quasi-cancer cell of not comprising hepatoma carcinoma cell.
5. antineoplastic immune pharmaceutical composition comprises the combination of the anti-tumor immunotherapy medicaments such as the polypeptide of tumor inactivated vaccine, Tumor-assaciated or proteantigen and water-soluble carbon nanometer tube; Described tumor is to comprise lung carcinoma cell, breast cancer cell but each quasi-cancer cell of not comprising hepatoma carcinoma cell.
6. pharmaceutical composition according to claim 5, is characterized in that, the portfolio ratio of described tumor inactivated vaccine and water-soluble carbon nanometer tube is 10
4~ 10
6Cell: 0.1mg ~ 20mg water-soluble carbon nanometer tube.
7. anti-tumor immunotherapy medicaments test kit comprises for as the medicine of immunotherapy of tumors related antigen with as CNT and a operation instructions of immunopotentiating agent; Described tumor is to comprise lung carcinoma cell, breast cancer cell but each quasi-cancer cell of not comprising hepatoma carcinoma cell.
8. anti-tumor immunotherapy medicaments test kit according to claim 7, it is characterized in that: CNT is the water-soluble carbon nanometer tube powder.
9. anti-tumor immunotherapy medicaments test kit according to claim 7, it is characterized in that: described CNT is the water-soluble carbon nanometer tube powder, and described medicine as the immunotherapy of tumors related antigen is DNA vaccination, polypeptide or the proteantigen of Tumor-assaciated.
10. anti-tumor immunotherapy medicaments test kit according to claim 7, it is characterized in that: described CNT is the aqueous solution of working concentration 0.1mg/ml ~ 0.4mg/ml.
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| CN105903034A (en) * | 2016-06-24 | 2016-08-31 | 南京农业大学 | Lentinan-modified carbon nano-tube as well as preparation method and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105903034A (en) * | 2016-06-24 | 2016-08-31 | 南京农业大学 | Lentinan-modified carbon nano-tube as well as preparation method and application thereof |
| CN105903034B (en) * | 2016-06-24 | 2018-10-23 | 南京农业大学 | A kind of carbon nanotube and the preparation method and application thereof of lentinan modification |
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Application publication date: 20130508 |