CN101991850A - Preparation technology of nanometer aluminum hydroxide adjuvant - Google Patents
Preparation technology of nanometer aluminum hydroxide adjuvant Download PDFInfo
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- CN101991850A CN101991850A CN 201010533334 CN201010533334A CN101991850A CN 101991850 A CN101991850 A CN 101991850A CN 201010533334 CN201010533334 CN 201010533334 CN 201010533334 A CN201010533334 A CN 201010533334A CN 101991850 A CN101991850 A CN 101991850A
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Abstract
The invention discloses a preparation technology of nanometer aluminum hydroxide adjuvant, comprising the following steps: taking benzalkonium bromide, n-caprylic alcohol and cyclohexane at equal ratio; stirring mixture at high speed, and pouring the mixture into a triangular flask; putting in a stir bar; putting the triangular flask on a magnetic stirrer, adding a proper quantity of AlCl3 solution, and stirring; slowing dropwise adding ammonia water into a separating funnel to regulate dripping rate; causing a reaction system to react for 2 hours at the pH of 10; after the reaction ends, adding acetone for demulsification and centrifugation; abandoning supernatant; respectively adding absolute ethyl alcohol and deionized water; evenly stirring by a glass rod; washing for three times, and decentralizing; collecting decentralized sediment into a beaker; drying for 12 hours in a drying oven to obtain fluffy Al(OH)3 adjuvant. The prepared nanometer aluminum hydroxide adjuvant has small grain diameter, good dispersibility and stability and favourable biocompatibility. In an animal experiment, the anti-tumor effect of nanometer aluminium adjuvant adsorption autovaccine is obviously superior to the curative effect obtained by using common aluminium adjuvant adsorption autovaccine.
Description
Technical field
The present invention relates to a kind of as the preparation technology of the nano-class aluminum adjuvant for the treatment of cancer drug and the anti-tumor therapeutical effect of its associating autologous tumor vaccine.
Background technology
In immunological therapy, adjuvant can come the reaction of enhancing body to vaccine by a lot of modes: the antigenicity that can strengthen poor antigen; Can be used to enhance immunity reaction duration and speed; Adjustablely antigenicly tire, specificity, homotype or differentiation; But irritation cell immunne response; Can strengthen with mucosa immunity-inducing and react; But the immunity of the individuality of enhance immunity system immaturity or aging; Can reduce antigenic dosage or reduce antigenic cost, also can help to overcome the antigenic competition in the combined vaccine.The at present extensive adjuvant that uses has only aluminium compound, wherein aluminium hydroxide most importantly.The vaccine of using aluminium compound now mainly is divided into two kinds, and a kind of is aluminium compound precipitation vaccine; Another kind is an aluminium compound absorption vaccine.Though the safety of ordinary hydrogen aluminum adjuvant is good, because can not the inducing cell immunne response and can not be as the adjuvant of tumour immunity.And very strong material of some adjuvant effects such as lipopolysaccharide etc. are because poor stability can not be applied to human vaccine.Qing He, Leibl H etc. studies show that, when adjuvant-antigenic compound during less than the granule of 1000nm, often cause Th1 type immunne response, and do not induce IgE antibody, and, cause Th2 type immunne response, and induce IgE antibody when adjuvant-antigenic compound during greater than the granule of 3um.This may make vertebrate natural immune system have the antigen of identification various trait with long-term evolution and induce the corresponding immunne response of generation relevant.Volume is less than 1-1.5um
3The adjuvant antigenic compound can cause immunne response based on cellular immunization.Because to spirillum, volume is mostly at 1-1.5 um from antibacterial, mycoplasma, rickettsia, chlamydia for occurring in nature, microorganism
3About, long-term evolution has made the immune system of body have the instinct that tackles big or small material like this.Certainly the adjuvant antigenic compound is best less than 200nm, because the diameter of most of virion is about 100nm, maximum is no more than 300nm, the little 18-30nm that is about, like this, adjuvant-the antigenic compound of similar virus size is easily absorbed by antigen presenting cell, especially by dendritic cell with the mode antigen uptaking that gulps down drink (dendritic cell is effective antigens presenting cell, gulps down the little bulb diameter of drink and is generally less than 150nm) thus cause intensive cellular immunization of body and humoral immunoresponse(HI).
Nanoparticle is meant the generally microgranule of the polymer formation between 10～1000nm of diameter, and it has unique small-size effect and interfacial effect.Maybe can the encode DNA or the RNA of immunogen polypeptide of antigenic substance can be wrapped in inside nanoparticles or be adsorbed on nanoparticle surface, also can combine with nanoparticle by chemical interconnect function, the nanoparticle adjuvant can effectively improve cellular immunization, humoral immunization and mucosa-immune.Usefulness calcium phosphate nano particles such as QingHe add herpes simplex types 2 virus protein (CAP+HSV-2), CAP phosphate buffered solution (PBS), and herpes simplex types 2 virus protein (HSV-2) carries out intravaginal or nasal cavity immunity inoculation to female BALB/C mice respectively.The result shows the mucosa-immune globulin IgA and the IgG of CAP+HSV-2 inducing producing specificity, improves whole body IgG level simultaneously, and has found neutralizing antibody in mice serum.Inoculate back the 45th day first, with herpes simplex types 2 virus attack mice, result of the test shows with CAP+do the adjuvant side reaction slight, can cause that the Th1 type reacts, produce the IgG2a antibody of very high titre, thereby think that it can replace aluminum salt adjuvant in the near future and be applied to human body.Zhong Shigen etc. are prepared into the Ca-NP30 conjugate with calcium nanoparticle and NP30, and the active immunity BALB/c mouse is observed it is attacked by schistosomicide to mice protective effect.The result shows that the calcium nanoparticle can strengthen the protective effect of NP30 to the host, and worm reduction rate obviously improves, and brings up to 57.8% from single with 30.4% of NP30; Serological specificity IgG antibody level is than the remarkable rising of matched group; Foot pads to test and can cause delayed hypersensitivity.This studies show that: the calcium nanoparticle can be used as the adjuvant of schistosomicide anti-idio-typic antibody NP 30 vaccine, and its mechanism of action is with to cause that simultaneously humoral immunization and cellullar immunologic response strengthen relevant.
Based on above achievement in research, we prepare nano-class aluminum adjuvant, observe nano-class aluminum adjuvant absorption autovaccine to mice H
22The treatment curative effect of hepatocarcinoma is studied its possible mechanism of action and toxic and side effects simultaneously, provides reliable experimental evidence with expectation for later clinical practice.This research expectation is by changing the size of aluminium adjuvant, changes the limitation that the conventional aluminium adjuvant can not the active cell immunity, for new thinking has been opened up in the immunization therapy of tumor.
Summary of the invention
The invention provides a kind of preparation technology of the nano-aluminum hydroxide adjuvant as cancer treatment drugs, the nano-aluminum hydroxide adjuvant that is obtained by the present invention can improve the effect of treatment cancer.
The present invention adopts following technical scheme:
A kind of preparation technology of the nano-aluminum hydroxide adjuvant as cancer treatment drugs:
Step 1: its preparation method is to pour triangular flask into after getting benzalkonium bromide that mass ratio is 1:1:1, n-octyl alcohol, cyclohexane extraction high-speed stirred, puts into stirrer, and triangular flask is placed on the magnetic stirring apparatus, adds the AlCl of 1mol/l
3Solution 10mL stir about 20 minutes.Slowly drip 10mL ammonia and in separatory funnel, regulate a speed, keep 30 per minutes, keep reaction system pH〉10 reactions 2 hours, reaction finishes the back and adds acetone breakdown of emulsion centrifugal (R=500rpm), abandoning supernatant, use distilled water, ethanol cyclic washing 6 times then respectively, the precipitation after centrifugal is collected in placed 70 ℃ of drying baker in the beaker dry 12 hours, promptly obtain bulk nanometer Al (OH)
3Adjuvant.
Step 2: mouse peritoneal inoculation H22 hepatoma carcinoma cell, treat that ascites volume produces the back and puts to death, being prepared into concentration under the aseptic condition is 1 * 10
8/ ml cell suspension, with cell suspension through 65 ℃ of 30min water bath processing, will be respectively with concentration be that 2mg/ml nano-class aluminum adjuvant and 4 ℃ of absorption of conventional aluminium assist agent solution 7 days are stand-by.。
Compared with prior art, the present invention has obtained following beneficial effect:
1. adopt the microemulsion method successfully to prepare the nano-aluminum hydroxide adjuvant, the nano-aluminum hydroxide adjuvant of preparation is a white powder, behind the dehydrated alcohol ultra-sonic dispersion, be about 100 nm with the transmission electron microscope observing size, favorable dispersibility, nano-class aluminum adjuvant particle squarish, the electron density height, good dispersion, size are consistent, are to be dispersed in distribution [with reference to Fig. 1].Under the scanning electron microscope prepared material surface is carried out energy spectrum analysis [with reference to Fig. 2], as seen wherein only contain two kinds of compositions of aluminum and oxygen, the quality percentage composition is respectively aluminum and accounts for 80.05 ﹪, and oxygen accounts for 19.95 ﹪, with AL (OH)
3Middle alumina quality percentage composition coincide.
2.The nano-class aluminum adjuvant of the present invention's preparation has excellent biological compatibility, we measure by the mice LD50 of material lixiviating solution cell toxicity test, hemolytic test, material, and genetic toxicity test---methods such as micronucleus test in the body, the biocompatibility of self-control nano-class aluminum adjuvant is estimated.Find the effect of homemade nano-class aluminum adjuvant in vitro tests no cytotoxicity; No haemolysis meets no teratogenesis or mutagenic action; And has wider safety value scope.
3. generally use at present and common aluminium adjuvant refers to aluminum hydroxide adjuvant.It mainly induces humoral immunoresponse(HI), and antibody stimulates to produce the reaction of Th2 type based on the IgG1 class.Though the conventional aluminium adjuvant can stimulate body to produce persistent high antibody horizontal rapidly, it can not the inducing cell immunity.Thereby limited anti-tumor immunotherapy greatly based on cellular immunization.We carry out mice H with the aluminium adjuvant absorption tumor autovaccine and the conventional aluminium adjuvant absorption autovaccine of invention
22Hepatocarcinoma is prevented and is treated, and confirms that nano-class aluminum adjuvant helps vaccine to suppress H significantly
22The quality of hepatocarcinoma and volume [with reference to Fig. 3] strengthen the cell toxicant killing activity, and serum I FN-γ is than the obviously rising (P＜0.05) of all the other groups; Serum il-4 obviously reduces (P＜0.05) than all the other groups.Prompting nano-class aluminum adjuvant absorption autovaccine is to mice H
22Hepatocarcinoma has tangible prevention and therapeutical effect, and inducing with cellular immunization is main anti-tumor immune response.
Description of drawings
Fig. 1. the nano-class aluminum adjuvant transmission electron microscope photo of the present invention's preparation.As seen nano-class aluminum adjuvant particle squarish, the electron density height, good dispersion, size is consistent, is to be dispersed in distribution.Size is about 100nm.
Fig. 2. the energy spectrum analysis figure of nano-class aluminum adjuvant, as seen wherein only contain two kinds of compositions of aluminum and oxygen, the quality percentage composition is respectively aluminum and accounts for 80.05 ﹪, and oxygen accounts for 19.95 ﹪, with AL (OH)
3Middle alumina quality percentage composition coincide.
Fig. 3. mice H
22Hepatocarcinoma is handled the appearance of tumors photo through different immunotherapy methods.A group: negative control group; B group: simple autovaccine group; C group: conventional aluminium adjuvant absorption autovaccine group; D group: nano-class aluminum adjuvant absorption autovaccine group.
The specific embodiment
Embodiment 1:
One, the preparation method of the nano-aluminum hydroxide of conduct treatment cancer drug:
Select suitable microemulsion prescription, under 37 ℃ of constant temperature magnetic agitation
1. benzalkonium bromide and the n-octyl alcohol of getting mass ratio 1:1 shake up in beaker;
2. adding waits the cyclohexane extraction high-speed stirred of mass ratio to pour into after 5 minutes and puts into stirrer in the triangular flask under the room temperature;
3. triangular flask is placed magnetic stirring apparatus adjusted mixing speed to add the 1mol/L AlCl of 10ml
3Solution stirring 10 minutes;
4. accurately measure 10ml ammonia with pipet and in separatory funnel, regulate and drip speed (30 droplets/minute slowly splash into ammonia as far as possible), keep system pH 10 reacted 2 hours;
5. reaction finishes the back and adds acetone breakdown of emulsion centrifugal (R=500rpm) and abandon supernatant to add mass concentration then respectively be 95% dehydrated alcohol and deionized water with Glass rod centrifugal the washing altogether 6 times of washing that stir;
6. the precipitation after centrifugal is collected in the vacuum drying oven that places 70 ℃ in the beaker and directly obtained bulk nanometer Al (OH) in dry 12 hours
3Xerogel.
Two, the preparation of H22 vaccine and vaccine absorption adjuvant
Mouse peritoneal inoculation H22 hepatoma carcinoma cell treats that ascites volume produces the back and puts to death, and being prepared into concentration under the aseptic condition is 1 * 10
8/ ml cell suspension, with cell suspension through 65 ℃ of 30min water bath processing, will be respectively with concentration be that 2mg/ml nano-class aluminum adjuvant and 4 ℃ of absorption of conventional aluminium assist agent solution 7 days are stand-by.
Claims (1)
1. the preparation technology of a nano-aluminum hydroxide adjuvant is characterized in that:
Step 1: its preparation method is to pour triangular flask into after getting benzalkonium bromide that mass ratio is 1:1:1, n-octyl alcohol, cyclohexane extraction high-speed stirred, puts into stirrer, and triangular flask is placed on the magnetic stirring apparatus, adds the AlCl of 1mol/l
3Solution 10mL stir about 20 minutes, slowly drip 10mL ammonia and in separatory funnel, regulate a speed, keep 30 per minutes, keep reaction system pH〉10 reactions 2 hours, reaction finishes the back and adds acetone breakdown of emulsion centrifugal (R=500rpm), and abandoning supernatant is used distilled water, ethanol cyclic washing 6 times then respectively, precipitation after centrifugal is collected in placed 70 ℃ of drying baker in the beaker dry 12 hours, promptly obtain bulk nanometer Al (OH)
3Adjuvant.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103412085A (en) * | 2013-08-27 | 2013-11-27 | 成都生物制品研究所有限责任公司 | Method for detecting adsorption capacity of aluminum hydroxide adjuvant prepared from ammonia water |
| CN103877553A (en) * | 2014-04-04 | 2014-06-25 | 文斌 | Medicine for treating cancers |
| CN103948921A (en) * | 2014-04-23 | 2014-07-30 | 东南大学 | Preparation method of nano aluminum adjuvant/ autologous tumor vaccine |
| CN104042628A (en) * | 2013-03-15 | 2014-09-17 | 复旦大学 | Application of aluminium hydroxide in preparation of medicine for curing liver cancer |
| CN104587464A (en) * | 2015-01-23 | 2015-05-06 | 四川大学 | Vaccine vector based on aluminum hydoxide nano-particles |
| CN108324939A (en) * | 2018-03-06 | 2018-07-27 | 重庆大学 | A kind of preparation method and application of rod-like nano aluminum hydroxide adjuvant |
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| CN1824305A (en) * | 2005-12-29 | 2006-08-30 | 中国人民解放军第三军医大学第三附属医院 | Preparation method of nano-aluminium adjuvant capable of raising vaccine effect and its use |
| CN1824302A (en) * | 2005-12-29 | 2006-08-30 | 中国人民解放军第三军医大学第三附属医院 | Preparation method of rabies vaccine for human and its use |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104042628A (en) * | 2013-03-15 | 2014-09-17 | 复旦大学 | Application of aluminium hydroxide in preparation of medicine for curing liver cancer |
| WO2014139456A1 (en) * | 2013-03-15 | 2014-09-18 | 复旦大学 | Application of aluminum hydroxide in preparation of medicament for treatment of liver cancer |
| CN103412085A (en) * | 2013-08-27 | 2013-11-27 | 成都生物制品研究所有限责任公司 | Method for detecting adsorption capacity of aluminum hydroxide adjuvant prepared from ammonia water |
| CN103412085B (en) * | 2013-08-27 | 2015-10-28 | 成都生物制品研究所有限责任公司 | The detection method of aluminum hydroxide adjuvant adsorptive power prepared by a kind of ammoniacal liquor |
| CN103877553A (en) * | 2014-04-04 | 2014-06-25 | 文斌 | Medicine for treating cancers |
| CN103948921A (en) * | 2014-04-23 | 2014-07-30 | 东南大学 | Preparation method of nano aluminum adjuvant/ autologous tumor vaccine |
| CN104587464A (en) * | 2015-01-23 | 2015-05-06 | 四川大学 | Vaccine vector based on aluminum hydoxide nano-particles |
| CN108324939A (en) * | 2018-03-06 | 2018-07-27 | 重庆大学 | A kind of preparation method and application of rod-like nano aluminum hydroxide adjuvant |
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Application publication date: 20110330 |