CN101537015A - Application of carbon nano-tubes in immunity accelerators for preparing anti-tumor immunotherapy medicaments - Google Patents
Application of carbon nano-tubes in immunity accelerators for preparing anti-tumor immunotherapy medicaments Download PDFInfo
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- CN101537015A CN101537015A CN200810102322A CN200810102322A CN101537015A CN 101537015 A CN101537015 A CN 101537015A CN 200810102322 A CN200810102322 A CN 200810102322A CN 200810102322 A CN200810102322 A CN 200810102322A CN 101537015 A CN101537015 A CN 101537015A
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Abstract
The invention discloses novel application of carbon nano-tubes, mainly application thereof in the preparation of accelerators of anti-tumor immunotherapy medicaments. Experiment verifies that the immunotherapy effects of inactivated tumor cell vaccine can be remarkably improved by taking the carbon nano-tubes as accelerators; the tumor cure rate of tumor-bearing mice is remarkably raised; tumors of mice which are not cured are also effectively inhibited; and no tumor forms after the mice which are completely cured are inoculated with tumor cells again. The Experiment shows that the carbon nano-tubes give effective play to accelerator effects, improve the immunotherapy efficiency of tumor vaccine, simultaneously promote organisms to produce specific immune response, and establish systems for recognizing and killing specific tumor antigens in the organisms.
Description
Technical field:
The invention belongs to biomedical sector, relate to novel application of carbon nano-tubes, 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 numerous in the world laboratorys to carry out correlational study from all angles.A key property of CNT is to cross over cell membrane and biological intravital multiple barrier, enters in the interior multiple organ of cell and organism.The result of study of existing bibliographical information shows that 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 with the CNT of the FITC labelling of variable concentrations (2 μ g/ml～10 μ g/ml) (behind the 4h～24h), in cell, can observe green fluorescence, show that CNT has entered into cell interior.They have further studied the influence of CNT to former generation immune cell function, find that T cell, B cell and macrophage still keep original function in the presence of CNT
[6]
The above results is the novel carriers of CNT as gene and medicine, and 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 the surface of CNT by covalency and noncovalent interaction.Dai seminar
[7]The fluorescin molecule is connected to the surface of SWCN (SWNT) by the non-specific adsorption effect, utilize cell CNT to be entered in the cell to the endocytosis of CNT, simultaneously fluorescent marker is brought in the cell, has realized the cell inner mark colour developing.Further studies show that, can follow the trail of and show CNT-albumen composition, thereby in diagnosing image, use a kind of approach that provides as novel carriers for CNT by the specific optical property of CNT itself.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 real-time dna vector of monitoring of intracellular environment.
Have researcher to propose, CNT can be positioned at cytoplasm, organelle even enter in the nucleus, and the location of CNT in cell is with to be connected its surperficial functional molecular relevant.For example, the CNT of nucleic acid functionization can navigate in the nucleus.Therefore, CNT can be used as the carrier of SiRNA, DNA, increases or suppress the expression of differential protein in the cell.There are some researches show, can be discerned by macrophage specifically and engulf that this characteristic makes CNT might become a kind of novel carriers system through the CNT of chemical modification.For example, studies show that
[8], CNT combines with some polypeptide, may bring into play certain effect in immunotherapy of tumors.Bianco A etc.
[9]Result of study show that 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 infection cell by covalent bond, outside polypeptide epitope still is exposed to, can be discerned and bring out body internal specific antibody by its specific antibody and produce.Experiment shows in the animal body, and this complex has caused enhanced antibody mediated immunity response, and CNT itself does not cause that specific antibody produces, and does not have cross reaction between the 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 the immunopotentiating agent in the preparation anti-tumor immunotherapy medicaments.
Wherein, described CNT is the water-soluble carbon nanometer tube powder, or is the carbon nano-tube aqueous solutions of concentration at 0.01～0.5mg/ml, the carbon nano-tube aqueous solutions of preferred 0.1～0.4mg/ml.
Described anti-tumor immunotherapy medicaments is the polypeptide or the proteantigen of tumor inactivated vaccine, tumour-specific; Be in particular the tumor inactivated vaccine.
A kind of concrete application of CNT of the present invention provides a kind of antineoplastic immune pharmaceutical composition, comprises the combination of anti-tumor immunotherapy medicaments such as the polypeptide of tumor inactivated vaccine, tumour-specific 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 provides a kind of anti-tumor immunotherapy medicaments test kit, comprises being used for as the medicine of immunotherapy of tumors specific antigen with as the 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 specific antigen is the mitomycin that is used to prepare the tumor inactivated vaccine, or is dna vaccination, polypeptide or the proteantigen of tumour-specific.
The CNT working concentration is the aqueous solution of 0.1mg/ml～0.4mg/ml in the test kit.
The present invention is checking by experiment also, as promoter, can significantly improve the immunization therapy effect of deactivation tumour-cell 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 once more by the inoculated tumour cell, there is not 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, in body, set up specific tumor antigen identification and killed system.
Description of drawings:
Fig. 1 is the oncotherapy efficiency chart of different immunotherapy methods.
Fig. 2 is for using the oncotherapy efficiency chart of different CNT dosage as the tumor vaccine immunopotentiating agent.
Fig. 3 is for curing mice inoculated tumour cell experiment result (tumor challenge) figure once more.
The specific embodiment:
The anti-tumor immunotherapy medicaments that in the immunization therapy of tumor, uses, no matter be traditional vaccine, deactivation tumor cell for example, still vaccine of new generation, comprise the albumen of purification and polypeptide, 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 immunopotentiating agent, enhancing body is to determining antigenic immune response.
The present invention proposes the new approaches of CNT, and verify the effect that its auxiliary tumor vaccine is treated by experiment as immunopotentiating agent in the anti-tumor immunotherapy medicaments.
Among the present invention, used CNT 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 method to prepare, water miscible carbon nanotube powder or the carbon nano-tube aqueous solutions that also can directly select for use 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 preparation of 2: 1 by volume ratio of concentrated nitric acid are formed mixed strong acids solution;
2) taking by weighing the 200mg multi-walled carbon nano-tubes and add in the mixed acid solution of 400ml, is that the ultrasonic probe of 15mm extend in multi-walled carbon nano-tubes and the mixed acid with diameter, and supersound process is 2 minutes under 1000w power, obtains atrous solution.
3) with atrous solution thin up after-filtration (the filter membrane aperture is 2 μ m), water washes repeatedly and leaches thing, is neutral to filtrate; Being deposited in of leaching dried below 70 ℃ to constant weight promptly 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 the pure water (as 10ml), with diameter is that the ultrasonic probe of 6mm was handled 2 minutes once more, 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 the 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 about 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 handling 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 making by introduction method of the present invention.When CNT and tumor vaccine coupling, subcutaneous injection is in the different parts of mice respectively.
Twice immunization therapy carried out to above-mentioned 4 groups of mices in the 7th day and the 14th day behind the inoculated tumour cell respectively, and 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 deactivation tumor cell 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 deactivation tumor cell 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 the tumor of part mice is dwindled gradually in the CNT+TCV group, 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, add up cure rate and calculate tumor control rate, with mice treatment situation classification in each group.Tumor disappearance person is for curing, and tumour inhibiting rate is greater than 50% for tumor is suppressed, and tumour inhibiting rate is the tumor deterioration less than 50%.Fig. 1 is seen in statistical result, demonstrates that tumor cure rate has significant difference between CNT+TCV and other group.
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 every mice CNT of each immunity injection volume 0.025mg, 0.05mg, 0.1mg, 0.2mg or 0.4mg.
Experiment shows that 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 tumor healing mice that survives in the CNT+TCV group in the 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, the 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 tumor of subcutaneous plantation H22 cell is cured mice does not have 1 to grow tumor, and the tumor healing 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 once more by the inoculated tumour cell, can not form new tumor, illustrate that the immunization therapy scheme of tumor vaccine combination CNT can cause the specific immunoreation of body, specific inhibition H22 growth of tumor suppresses tumor recurrence.
The present invention has verified when adopting traditional deactivation tumor cell 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, with the treatment of independent use deactivation tumour-cell vaccine relatively therapeutic effect relatively show: with CNT as immunopotentiating agent, can significantly improve the therapeutic effect of deactivation tumour-cell 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 once more by the inoculated tumour cell, there is not 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 CNT combination polypeptide and the antigenic immunization therapy scheme of CNT combined protein matter, 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, CNT makes up the immunization therapy scheme of corresponding tumor vaccine, and the more single tumor vaccine immunization therapy of its immunization therapy efficient all is significantly increased.Illustrate that CNT all has facilitation for the immunization therapy of different tumors.
According to above experimental result, the present invention can utilize water miscible made of carbon nanotubes 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.The simplest use of composition medicine be with water as solvent, can certainly add other auxiliary agent or pharmacy adjuvant and play a role to help medicine.
The present invention can also utilize water miscible made of carbon nanotubes to form the immunotherapy medicaments test kit.Comprise in this test kit and being used for as the medicine of immunotherapy of tumors specific antigen with as the CNT and a operation instructions of immunopotentiating agent, CNT is water miscible carbon nanotube powder, to specifications indication is mixed with finite concentration (aqueous solution as 0.1～0.4mg/ml), test kit is used in operating process to specifications then (operation of reference experiment 2 they (4)) during use.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 with carbon nano-tube aqueous solutions.
List of references
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3.Mitchell?LA，Gao?J，Wal?RV，Gigliotti?A，Burchiel?SW，McDonald?JD.Pulmonaryand?systemic?immune?response?to?inhaled?multiwalled?carbon?nanotubes.ToxicolSci.2007?Nov；100(1)：203-14.
4.Sato?Y，Yokoyama?A，Shibata?K，Akimoto?Y，Ogino?S，Nodasaka?Y，Kohgo?T，Tamura?K，Akasaka?T，Uo?M，Motomiya?K，Jeyadevan?B，Ishiguro?M，HatakeyamaR，Watari?F，Tohji?K.Influence?of?length?on?cytotoxicity?of?multi-walled?carbonnanotubes?against?human?acute?monocytic?leukemia?cell?line?THP-1?in?vitro?andsubcutaneous?tissue?of?rats?in?vivo.Mol?Biosyst.2005?Jul；1(2)：176-82.
5.Tian?F，Cui?D，Schwarz?H，Estrada?GG，Kobayashi?H.Cytotoxicity?of?single-wallcarbon?nanotubes?on?human?fibroblasts?Toxicol?In?Vitro.2006?Oct；20(7)：1202-12
6.Dumortier?H，Lacotte?S，Pastorin?G，Marega?R，Wu?W，Bonifazi?D，Briand?JP，PratoM，Muller?S，Bianco?A.Functionalized?carbon?nanotubes?are?non-cytotoxic?andpreserve?the?functionality?of?primary?immune?cells.Nano?Lett.2006Jul；6(7)：1522-8.
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Claims (10)
1, CNT application in the immunopotentiating agent in the preparation anti-tumor immunotherapy medicaments.
According to the described application of claim 1, it is characterized in that 2, described CNT is the water-soluble carbon nanometer tube powder.
According to the described application of claim 1, it is characterized in that 3, described CNT is the carbon nano-tube aqueous solutions of concentration at 0.01～0.5mg/ml, the particularly carbon nano-tube aqueous solutions of 0.1～0.4mg/ml.
According to claim 1 or 2 or 3 described application, it is characterized in that 4, described anti-tumor immunotherapy medicaments is the polypeptide or the proteantigen of tumor inactivated vaccine, tumour-specific; Be in particular the tumor inactivated vaccine.
5, a kind of antineoplastic immune pharmaceutical composition comprises the combination of anti-tumor immunotherapy medicaments such as the polypeptide of tumor inactivated vaccine, tumour-specific or proteantigen and water-soluble carbon nanometer tube.
According to the described pharmaceutical composition of claim 5, it is characterized in that 6, 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, a kind of anti-tumor immunotherapy medicaments test kit comprises being used for as the medicine of immunotherapy of tumors specific antigen with as the CNT and a operation instructions of immunopotentiating agent.
8, according to the described anti-tumor immunotherapy medicaments test kit of claim 7, it is characterized in that: CNT is the water-soluble carbon nanometer tube powder, and described medicine as the immunotherapy of tumors specific antigen is the mitomycin that is used to obtain the tumor inactivated vaccine.
9, according to the described anti-tumor immunotherapy medicaments test kit of 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 specific antigen is dna vaccination, polypeptide or the proteantigen of tumour-specific.
10, according to the described anti-tumor immunotherapy medicaments test kit of 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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