CN102539760A - Folic-acid-ligand-modified ferric oxide nano-particle with in vitro tumor targeting function, as well as preparation method and in vitro evaluation method thereof - Google Patents
Folic-acid-ligand-modified ferric oxide nano-particle with in vitro tumor targeting function, as well as preparation method and in vitro evaluation method thereof Download PDFInfo
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Abstract
The invention discloses a folic-acid-ligand-modified ferric oxide nano-particle with an in vitro tumor targeting function, as well as a preparation method and an in vitro evaluation method thereof. The folic-acid-ligand-modified ferric oxide nano-particle with the in vitro tumor targeting function, prepared by the method disclosed by the invention, has the targeting function on in vitro tumor cells, and the mechanism of the folic-acid-ligand-modified ferric oxide nano-particle is from high expression of some malignant cells of human onto folic acid receptors. Based on detection of a test, the content of iron in human oral epidermoid cancer cells of a folic acid-dextran-ferric oxide nano-particle group highly expressed by the folic acid receptors is higher than that of iron in a dextran-ferric oxide nano-particle group which is not modified by the folic acid. Through the research on folic acid-dextran-ferric oxide nano-particles, the success rate of tumor early diagnosis is effectively improved.
Description
Technical field
The present invention relates to a kind of preparation method of ferric oxide nanometer particle, specifically have external cancer target effect through folic acid ligand modified ferric oxide nanometer particle and its preparation method and in-vitro evaluation method.
Background technology
Annual nearly 2,500,000 the newly-increased cancer patients of China, wherein great majority have got into the cancer middle and advanced stage when making a definite diagnosis.If realize the effective treatment to tumour, important a bit is the early diagnosis that can realize tumour through various technology.Therefore, check that technological quality is directly connected to the quality of treatment of cancer effect.
Tumour nanometer technology and Nano medication research are at the early-stage, need utilize the knowledge and the research means in fields such as nanometer technology, biology, chemistry, physics, medical science, pharmacy and public health.Nano medication is the international scientific forward position, also is and human health and the closely-related important social concern of life, has been full of the opportunity of innovation.
Along with research in nanotechnology deeply with the raising of people to the nanometer control ability, nanometer technology is that engineering science and technology is studied and technological change has brought breathtaking chance.With regard to medical science, nanometer technology is expected to make a breakthrough in the fields such as drug therapy of cancer, cardiovascular disease, diabetes, spinal cord injury etc.Nanometer technology will be to producing new change in the biomedical research, and new method and medicine will be provided for prevention, detection, the early diagnosis and therapy of disease.The treatment mechanism that Nano medication is brand-new might be to obtain the key that Nano medication research breaks through, but difficulty be also bigger from changing the pattern and the approach of human diseases prevention and treatment in idea.
The quick progress of nano science is for the development of biological medicine has brought new opportunity.How through development with utilize nanometer technology to remove prevention, detection, early diagnosis and therapy human diseases, security how to guarantee Nano medication be pendulum in various countries the problem that is rich in challenge in face of the scientist.
Cancer is 21 century one of the challenging medical problem of tool.Although for capturing cancer, the Chinese and foreign department scholar has carried out the unremitting effort of many decades, prevention, the early diagnosis and therapy of most cancers are still not fully up to expectations so far.The mortality ratio of cancer is high in the world wide, becomes human main healthy " killer ".Nanometer technology is that the early diagnosis and therapy of tumour provides new thinking and method.Scientific discovery, some human malignant cells have the high expressed for folacin receptor.A kind ofly has the ligand modified targeting agent of folic acid or medicine just can detect and treat cancer well if can prepare.
Though a lot of patents possibly all mention how preparing tri-iron tetroxide ferric oxide nanometer particle etc.; But the method for preparing glucosan-iron oxide nano-granule or folic acid-glucosan-iron oxide nano-granule is because the preparation technology characteristics of seeing service property just itself; And; Each patent inventor or producer painstakingly maintain secrecy for process detail, and the information that openly provides is limited, make the many preparation methods that mention in many documents be difficult to repeat enforcement with experimental program; Even perhaps can repeat to implement, states such as prepared nano particle or its suspension are also unstable.
Summary of the invention
Technical matters to be solved by this invention is to above-mentioned prior art present situation; And core is provided is iron oxide; Its shell is mainly the ferric oxide nanometer particle of folic acid, effectively tumor cell have external cancer target effect through folic acid ligand modified ferric oxide nanometer particle and its preparation method and a kind of in-vitro evaluation method.
The present invention solves the problems of the technologies described above the technical scheme that is adopted:
Have external cancer target effect through the ligand modified ferric oxide nanometer particle of folic acid, wherein, ferric oxide nanometer particle is kernel with the iron oxide, covalent modification is carried out with folic acid in the surface of this ferric oxide nanometer particle.
For optimizing technique scheme, the measure of taking also comprises:
The surface adsorption of ferric oxide nanometer particle has the activating agent particle, and described activating agent particle is a dextran molecule; This ferric oxide nanometer particle is folic acid-glucosan-iron oxide nano-granule.
Preparation wherein, may further comprise the steps through the method for the ligand modified ferric oxide nanometer particle of folic acid:
Step 1, synthetic Fe
3O
4Particle suspension;
Step 2, will be the dextran solution and the Fe of carrier fluid with the deionized water
3O
4Particle suspension mixes the back heating, refluxes, the dialysis of cooling back, centrifugal filtration, glucosan that must be stable-iron oxide nano-granule suspending liquid;
Step 3, get the glucosan-iron oxide nano-granule suspending liquid of step 2 preparation; The absolute ethyl alcohol that adds half volume, centrifugal treating, supernatant inclines; Taking precipitate; And sediment cleaned with absolute ethyl alcohol, dimethyl sulfoxide respectively, again through the ultrasonic dispersion of anhydrous dimethyl sulfoxide, the anhydrous dimethyl sulfoxide solution of glucosan-iron oxide nano-granule;
Step 4, anhydrous dimethyl sulfoxide added to folic acid solution and stir it is fully dissolved after; Add dicyclohexylcarbodiimide, N-hydroxy-succinamide and triethylamine solution again; Lucifuge; Fill nitrogen, after the liquid reaction to be mixed, filter away sediment; Contain in the diethyl ether solution of acetone filtering back solution adding; Fully stir, elimination solution promptly gets the folic acid active ester with the excess vacuum drying;
Step 5, the folic acid active ester is added the anhydrous dimethyl sulfoxide solution of glucosan-iron oxide nano-granule, make the dissolving of folic acid active ester, fill nitrogen; Lucifuge, heating, and add triethylamine as catalyzer; After treating that solution stops reaction, solution is carried out magnetic resolution, and separator is cleaned with absolute ethyl alcohol and distilled water respectively; Vacuum drying promptly gets folic acid-glucosan-iron oxide nano-granule.
Fe in the step 1
3O
4The preparation method of particle suspension is mixed with Fe for adopting
3+And Fe
2+Iron salt solutions, under the oxygen free condition, add aqueous slkali, letting nitrogen in and deoxidizing is handled, and stirs fast and heats gradually, treats its reaction back elimination supernatant liquid, obtains Fe
3O
4Sediment is with Fe
3O
4Sediment adds the ultrasonic dispersion of a small amount of distilled water, and diafiltration gets Fe
3O
4Particle suspension.
Fe
3+And Fe
2+Iron salt solutions in the mol ratio of trivalent iron salt and divalent iron salt be between the 2:1 to 3:2, and pH value is between 2 to 4, adds in the alkali lye process; PH value progressively is changed to 9 to 11; The highlyest must not surpass 12, stir fast and heat gradually to 55 ℃, the highest must not be above 60 ℃.
Glucosan in the step 2 in the dextran solution and the mass ratio that adds between the iron of dextran solution are 5:1, and mixed solution to be heated to 100 ℃ of return times be 1 hour.
Solution dropwise joins in the diethyl ether solution that contains 30% acetone below 5 ℃ under well-beaten situation after the filtration in the step 4.
In-vitro evaluation is through the method for the tumour cell folacin receptor target property of the ligand modified ferric oxide nanometer particle of folic acid; It is characterized in that: identical oral cavity epidermal carcinoma cell line is divided into two groups of A, B; With folic acid-glucosan-iron oxide nano-granule and the hatching of A group cell line; Glucosan-iron oxide nano-granule and the hatching of B group cell line adopt luxuriant and rich with fragrance Lip river piperazine method to measure the tumor-selective of iron content, the quantitative and qualitative investigation folic acid-glucosan of Prussian blue iron decoration method-iron oxide nano-granule and glucosan-iron oxide nano-granule in the cell then.
The method that luxuriant and rich with fragrance Lip river piperazine method is measured iron content in the cell is: human mouth epidermal carcinoma cell suspension is inoculated in 12 orifice plates with every hole 2*105; Every pore volume 1ml; Inoculate two plates; Behind the hatching 24h, fully wash twice, add respectively with the glucosan-iron oxide nano-granule of nanoparticle concentration and the full training solution of folic acid-glucosan-iron oxide nano-granule at two plates with PBS solution; And respectively do three and answer holes; And respectively with the culture medium solution that do not add human mouth epidermal carcinoma cell but added the isoconcentration nanoparticle as contrast, the hole that does not add nanoparticle simultaneously to have only human mouth epidermal carcinoma cell as control, do not add human mouth epidermal carcinoma cell and nanoparticle the hole as blank, the result is proofreaied and correct.Cell fully washs three times with PBS solution after placing 37 ℃ of incubators to continue to cultivate 24h, then human mouth epidermal carcinoma cell is suspended in the 0.01MHCl solution of 500 μ l, in each hole, adds by equal-volume 4.5%KMnO
4Solution and 2%HCl and facing the reagent A that the time spent mixes; Reacted 2 hours down in 60 ℃, to be cooled to room temperature, add the reagent B that 50 μ l are made up of the luxuriant and rich with fragrance Lip river of 6.5mM piperazine, 13.1mM zinc-copper reagent, 2M vitamin C, 5M ammonium acetate; React after 10 minutes, with ELIASA in 490nm wavelength reading.
Prussian blue iron decoration method is: A group cell line and the B group cell line that will hatch 24 hours add prussian blue staining respectively, observe the color depth of two groups of cell lines.
Compared with prior art, of the present invention have external cancer target effect through the ligand modified ferric oxide nanometer particle of folic acid, wherein, ferric oxide nanometer particle is kernel with the iron oxide, covalent modification is carried out with folic acid in the surface of this ferric oxide nanometer particle.The mechanism of tumor cell in vitro targeting comes from the high expressed of some human malignant cells for folacin receptor.Detect through overtesting; Iron content is higher than the glucosan-iron oxide nano-granule group without modified with folic acid in the human mouth epidermal carcinoma cell of the folic acid-glucosan of folacin receptor high expressed-iron oxide nano-granule group, and visible folic acid-glucosan-iron oxide nano-granule has more intense tumor-targeting.If realize the effective treatment to tumour, important a bit is the early diagnosis that can realize tumour through various technology.Therefore, check that technological quality is directly connected to the quality of treatment of cancer effect.The research and development of folic acid-glucosan-iron oxide nano-granule will effectively promote the success ratio of early diagnosis of tumor.
Embodiment
Below embodiments of the invention are described in further detail.
Ferric oxide nanometer particle of the present invention adopts chemical coprecipitation to prepare.Employing is mixed with Fe
3+And Fe
2+Iron salt solutions (as: FeCl
2, FeCl
3, FeSO
4, Fe
2(SO4)
3, ferric citrate, ferrous oxalate, ferric phosphate, ferrous fumarate, ferric ammonium sulfate, ferrous sulfate amine, ferric citrate amine and nitrate, hydrochloride etc.), the mol ratio of its trivalent iron salt and divalent iron salt is between the 2:1 to 3:2, under 25 ℃, oxygen free condition; Add aqueous slkali (ammoniacal liquor, NaOH; Optium concentration is 0.5mol/L), the PH initial value is 3.0, adds in the alkali lye process; Progressively be changed to 9 to 11, the highlyest must not surpass 12; (if can not reach oxygen-free environment, under the aerobic situation, the particle of preparing can change logical nitrogen, and its reaction equation is: Fe
3O
4+ 0.25O
2+ 4.5H
2O → 3Fe (OH)
3, this has just caused the instability of the physicochemical property of the particle for preparing.So, for preventing the generation of this phenomenon, in preparing process, take the method for logical nitrogen to remove the oxygen in the solution, can also reach the effect of the particle diameter that reduces ferric oxide nanometer particle simultaneously), stir fast and heat gradually to 55 ℃, the highest must not be above 60 ℃.Reaction time is 30 minutes, precipitates fully and prepares (black precipitate).Chemical equation is: Fe
2++ 2Fe
3++ 8OH
-→ Fe
3O
4↓+4H
2O leaves standstill, the supernatant liquid that inclines, wash 4 times after, add the ultrasonic dispersion of a small amount of distilled water 10 minutes, diafiltration gets Fe
3O
4Particle suspension (a kind of colloidal solution).
But this liquid such as long-time placement (for example several days) can be separated, and produce coagulation or coazevation phenomenon.So need a kind of carrier fluid that contains certain surfactant, make Fe
3O
4The surface adsorption surfactant, stable existence is in carrier fluid.
2, the preparation of glucosan-iron oxide nano-granule
Because nontoxic in human body, the no antigen of glucosan, degradable have good biocompatibility and separating property of biosoluble, are a kind of desirable surfactants; In addition; Activity hydroxy on the dextran molecule can connect the polyfunctional molecule of biologically active, like folic acid through chemical modification; Group or molecule according to link are different, and the ferric oxide nanometer particle of preparing can be expanded as different purposes such as target medicine carrier, molecular image labels.
Therefore, we select glucosan (according to the ratio of glucosan and iron content mass ratio 5:1, iron content are at the Fe of above-mentioned preparation
3O
4Particle suspension adopts colorimetric method for determining) as coating material, be carrier fluid with the deionized water, with the Fe of above-mentioned preparation
3O
4Be heated to 100 ℃ after particle suspension mixes and refluxed 1 hour, the dialysis of cooling back, centrifugal filtration promptly gets stable glucosan-iron oxide nano-granule suspending liquid.
Record the Fe that this method makes with the laser light scattering particle size analyzer
3O
4The ferric oxide nanometer particle particle diameter is (wavelength of LASER Light Source is 663nm, and probe temperature is 25 ℃) about 18-20nm, and behind the surface adsorption glucosan, its whole particle diameter is about about 35nm.
3, carry out covalent modification with folic acid
Get the glucosan-iron oxide nano-granule suspending liquid of above-mentioned preparation, add the absolute ethyl alcohol of half volume, high speed centrifugation; Supernatant inclines; Get deposition and respectively give a baby a bath on the third day after its birth time, the ultrasonic dispersion of anhydrous dimethyl sulfoxide, anhydrous dimethyl sulfoxide solution that must glucosan-iron oxide nano-granule with absolute ethyl alcohol, dimethyl sulfoxide.
Other takes by weighing folic acid 300mg, adds the 10ml anhydrous dimethyl sulfoxide, after stirring is fully dissolved it, adds dicyclohexylcarbodiimide (DDC) 99mg; N-hydroxy-succinamide (NHS) 78mg adds 500 μ l triethylamine solutions, lucifuge; Fill the nitrogen reaction and spend the night, remove by filter the accessory substance of reaction, after partial solvent is removed in decompression distillation; Dropwise under well-beaten situation, join in the ice-cold diethyl ether solution that contains 30% acetone, filter, vacuum drying promptly gets product folic acid active ester.
The folic acid active ester is added the anhydrous dimethyl sulfoxide solution of above-mentioned glucosan-iron oxide nano-granule, and fully vortex makes the dissolving of folic acid active ester, fills nitrogen, and lucifuge is heated to 50 ℃, adds triethylamine as catalyzer, reacts 4 hours.After stopping reaction, magnetic resolution is respectively given a baby a bath on the third day after its birth time with absolute ethyl alcohol and distilled water, and vacuum drying promptly gets product: folic acid-glucosan-iron oxide nano-granule, its particle diameter is about 50nm.
4, the external verification method of folacin receptor cancer target effect.
Folacin receptor tumor-targeting for in-vitro evaluation folic acid-glucosan-iron oxide nano-granule; With without the glucosan-iron oxide nano-granule of modified with folic acid as contrast; With the human mouth epidermal carcinoma cell (KB cell) of cell surface folacin receptor high expressed as model (recover through from liquid nitrogen container, taking out, go down to posterity, frozen, prepare nutrient solution and process single cell suspension); With folic acid-glucosan-iron oxide nano-granule or glucosan-iron oxide nano-granule and this cell line hatching 24h, luxuriant and rich with fragrance Lip river piperazine method is measured iron content, quantitative and qualitative its tumor-selective of investigation of Prussian blue iron decoration method in the cell respectively.
Luxuriant and rich with fragrance Lip river piperazine method is measured iron content in the cell: the KB cell suspension that makes is inoculated in 12 orifice plates with every hole 2*105; Every pore volume 1ml inoculates two plates, behind the hatching 24h; Fully wash twice with PBS solution; Add respectively with the glucosan-iron oxide nano-granule of nanoparticle concentration and the full training solution of folic acid-glucosan-iron oxide nano-granule at two plates, and respectively do three multiple holes, and respectively not add cell but the culture medium solution that has added the isoconcentration nanoparticle as contrast; The hole that does not add simultaneously nanoparticle to have only cell as control, do not add cell and nanoparticle the hole as blank, the result is proofreaied and correct.Cell places 37 ℃ of incubator (5%CO
2) continue to cultivate 24h after, fully wash three times with PBS solution, be suspended in the 0.01MHCl solution of 500 μ l, in each hole, add reagent A (equal-volume 4.5%KMnO
4Solution and 2%HCl, face the time spent and mix); Reacted 2 hours down in 60 ℃; To be cooled to room temperature, add 50 μ l reagent B (the luxuriant and rich with fragrance Lip river of 6.5mM piperazine, 13.1mM zinc-copper reagent, 2M vitamin C, 5M ammonium acetate) reaction after 10 minutes, with ELIASA in 490nm wavelength reading; Find folic acid-glucosan-iron oxide nano-granule group, iron content is apparently higher than glucosan-iron oxide nano-granule group in the cell.
The prussian blue staining method is investigated: the KB cell with contain folic acid-glucosan-iron oxide nano-granule nutrient culture media hatching 24h; Prussian blue staining; Cell is dyed sapphirine, and after containing glucosan-iron oxide nano-granule nutrient culture media and KB cell hatching 24h, it is light blue to have only a little cell to be dyed.The result is consistent with luxuriant and rich with fragrance Lip river piperazine method mensuration result.
In sum, this patent discloses a kind of preparation method based on the ligand modified ferric oxide nanometer particle of folic acid with tumor cell in vitro targeting.The mechanism of its tumor cell in vitro targeting comes from the high expressed of some human malignant cells for folacin receptor.In this example; Iron content is higher than without folic acid finishing group in the KB cell of the folic acid-glucosan of folacin receptor high expressed-iron oxide nano-granule group; Folic acid-glucosan-iron oxide nano-granule gets into the KB cell through the endocytosis mechanism of folacin receptor regulation and control, and visible folic acid-glucosan-iron oxide nano-granule has more intense tumor-targeting.
Most preferred embodiment of the present invention is illustrated, and various variations or the remodeling made by those of ordinary skills can not depart from the scope of the present invention.
Claims (10)
1. have external cancer target effect through the ligand modified ferric oxide nanometer particle of folic acid, it is characterized in that: ferric oxide nanometer particle is kernel with the iron oxide, and covalent modification is carried out with folic acid in the surface of this ferric oxide nanometer particle.
2. according to claim 1 have external cancer target effect through the ligand modified ferric oxide nanometer particle of folic acid, it is characterized in that: the surface of described ferric oxide nanometer particle is with the activating agent particle, and described activating agent particle is a dextran molecule; This ferric oxide nanometer particle is folic acid-glucosan-iron oxide nano-granule.
3. prepare the method through the ligand modified ferric oxide nanometer particle of folic acid as claimed in claim 1, it is characterized in that: may further comprise the steps:
Step 1, synthetic Fe
3O
4Particle suspension;
Step 2, will be the dextran solution and the Fe of carrier fluid with the deionized water
3O
4Particle suspension mixes the back heating, refluxes, the dialysis of cooling back, centrifugal filtration, glucosan that must be stable-iron oxide nano-granule suspending liquid;
Step 3, get the glucosan-iron oxide nano-granule suspending liquid of step 2 preparation; The absolute ethyl alcohol that adds half volume, centrifugal treating, supernatant inclines; Taking precipitate; And sediment cleaned with absolute ethyl alcohol, dimethyl sulfoxide respectively, again through the ultrasonic dispersion of anhydrous dimethyl sulfoxide, the anhydrous dimethyl sulfoxide solution of glucosan-iron oxide nano-granule;
Step 4, anhydrous dimethyl sulfoxide added to folic acid solution and stir it is fully dissolved after; Add dicyclohexylcarbodiimide, N-hydroxy-succinamide and triethylamine solution again; Lucifuge; Fill nitrogen, after the liquid reaction to be mixed, filter away sediment; Contain in the diethyl ether solution of acetone filtering back solution adding; Fully stir, elimination solution promptly gets the folic acid active ester with the excess vacuum drying;
Step 5, the folic acid active ester is added the anhydrous dimethyl sulfoxide solution of glucosan-iron oxide nano-granule, make the dissolving of folic acid active ester, fill nitrogen; Lucifuge, heating, and add triethylamine as catalyzer; After treating that solution stops reaction, solution is carried out magnetic resolution, and separator is cleaned with absolute ethyl alcohol and distilled water respectively; Vacuum drying promptly gets ferric oxide nanometer particle, and this ferric oxide nanometer particle is folic acid-glucosan-iron oxide nano-granule.
4. preparation method according to claim 3 is characterized in that: the Fe in the described step 1
3O
4The preparation method of particle suspension is mixed with Fe for adopting
3+And Fe
2+Iron salt solutions, under the oxygen free condition, add aqueous slkali, letting nitrogen in and deoxidizing is handled, and stirs fast and heats gradually, treats its reaction back elimination supernatant liquid, obtains Fe
3O
4Sediment is with Fe
3O
4Sediment adds the ultrasonic dispersion of a small amount of distilled water, and diafiltration gets Fe
3O
4Particle suspension.
5. preparation method according to claim 4 is characterized in that: described Fe
3+And Fe
2+Iron salt solutions in the mol ratio of trivalent iron salt and divalent iron salt be between the 2:1 to 3:2, and pH value is between 2 to 4, adds in the alkali lye process; PH value progressively is changed to 9 to 11; The highlyest must not surpass 12, stir fast and heat gradually to 55 ℃, the highest must not be above 60 ℃.
6. preparation method according to claim 3 is characterized in that: the glucosan in the described step 2 in the dextran solution and to add mass ratio between the iron of dextran solution be that to be heated to 100 ℃ of return times be 1 hour for 5:1 and mixed solution.
7. preparation method according to claim 3 is characterized in that: solution dropwise joins in the diethyl ether solution that contains 30% acetone below 5 ℃ under well-beaten situation after the filtration in the described step 4.
8. the in-vitro evaluation method through the ligand modified ferric oxide nanometer particle of folic acid as claimed in claim 1; It is characterized in that: identical oral cavity epidermal carcinoma cell line is divided into two groups of A, B; With folic acid-glucosan-iron oxide nano-granule and the hatching of A group cell line; Glucosan-iron oxide nano-granule and the hatching of B group cell line adopt luxuriant and rich with fragrance Lip river piperazine method to measure the tumor-selective of iron content, the quantitative and qualitative investigation folic acid-glucosan of Prussian blue iron decoration method-iron oxide nano-granule and glucosan-iron oxide nano-granule in the cell then.
9. in-vitro evaluation method according to claim 8; It is characterized in that: the method that described luxuriant and rich with fragrance Lip river piperazine method is measured iron content in the cell is: human mouth epidermal carcinoma cell suspension is inoculated in 12 orifice plates with every hole 2*105; Every pore volume 1ml inoculates two plates, behind the hatching 24h; Fully wash twice with PBS solution; Add respectively with the glucosan-iron oxide nano-granule of nanoparticle concentration and the full training solution of folic acid-glucosan-iron oxide nano-granule at two plates, and respectively do three multiple holes, and respectively not add human mouth epidermal carcinoma cell but the culture medium solution that has added the isoconcentration nanoparticle as contrast; The hole that does not add simultaneously nanoparticle to have only human mouth epidermal carcinoma cell as control, do not add human mouth epidermal carcinoma cell and nanoparticle the hole as blank, the result is proofreaied and correct; Cell fully washs three times with PBS solution after placing 37 ℃ of incubators to continue to cultivate 24h, then human mouth epidermal carcinoma cell is suspended in the 0.01MHCl solution of 500 μ l, in each hole, adds by equal-volume 4.5%KMnO
4Solution and 2%HCl and facing the reagent A that the time spent mixes; Reacted 2 hours down in 60 ℃, to be cooled to room temperature, add the reagent B that 50 μ l are made up of the luxuriant and rich with fragrance Lip river of 6.5mM piperazine, 13.1mM zinc-copper reagent, 2M vitamin C, 5M ammonium acetate; React after 10 minutes, with ELIASA in 490nm wavelength reading.
10. in-vitro evaluation method according to claim 8 is characterized in that: described Prussian blue iron decoration method is: A group cell line and the B group cell line that will hatch 24 hours add prussian blue staining respectively, observe the color depth of two groups of cell lines.
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| 范彩霞: "新型肿瘤靶向的叶酸－羧甲基壳聚糖－超顺磁氧化铁纳米粒的合成及评价", 《中国博士学位论文全文库》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102818826A (en) * | 2012-07-21 | 2012-12-12 | 上海师范大学 | Electrochemical cell-based biosensor based on nanometer Ag@BSA biomimetic interface and preparation method thereof |
| CN102818826B (en) * | 2012-07-21 | 2014-09-17 | 上海师范大学 | Electrochemical cell-based biosensor based on nanometer Ag@BSA biomimetic interface and preparation method thereof |
| CN103558170A (en) * | 2013-11-11 | 2014-02-05 | 福建医科大学 | Folic acid-porous platinum-graphene oxide composite nano material as well as application thereof in detecting tumor cells |
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