CN107952081B - PH controlled-release target medicament nano transport agent and its preparation method and application - Google Patents

PH controlled-release target medicament nano transport agent and its preparation method and application Download PDF

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CN107952081B
CN107952081B CN201711130408.0A CN201711130408A CN107952081B CN 107952081 B CN107952081 B CN 107952081B CN 201711130408 A CN201711130408 A CN 201711130408A CN 107952081 B CN107952081 B CN 107952081B
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msio
nanosphere
preparation
dnm
water
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CN107952081A (en
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赵平
柳敏超
刘冰
张陆勇
孙翔玉
林慧超
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Guangdong Pharmaceutical University
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Guangdong Pharmaceutical University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/02—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
    • A61K49/1818—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles
    • A61K49/1821—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles
    • A61K49/1824—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles
    • A61K49/1827—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle
    • A61K49/1833—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle having a (super)(para)magnetic core coated or functionalised with a small organic molecule

Abstract

The present invention provides a kind of pH controlled-release target medicament nano transport agent and its preparation method and application, and the pH controlled-release target medicament nano transport agent is with Fe3O4For core, mSiO2For shell, in Fe3O4@mSiO2Modification-NH on surface2, receptor targeted folic acid (FA), immobilized daunomycin (DNM) are then connected by amide reaction, finally use CaCO3It is blocked, realizes that pH value responds controlled release, achieve the effect that in blood circulation " zero release ", to reduce drug to the toxic side effect of human body.PH controlled-release target medicament nano transport agent combination mSiO of the present invention2Good biocompatibility, high-specific surface area, surface are easy to the advantages that modifying and wrap up Fe3O4Nanosphere, passes through amino (- NH2) receptor targeted folic acid is grafted after modification, using multiple targeting caused by magnetic targeted and receptor target, the anti-cancer properties of targeted drug are improved, hence it is evident that be better than single targeting, by, in the stability of physiological pH, improving the performance of targeted drug controlled release system using calcium carbonate.

Description

PH controlled-release target medicament nano transport agent and its preparation method and application
Technical field
The invention belongs to target administration technical fields, are related to a kind of medicament nano transport agent, and in particular to a kind of pH control Release targeted drug nanometer transport agent and its preparation method and application.
Background technique
In recent years, with the fast development of nanometer biotechnology, intelligent Organic-inorganic composite nanosphere by The extensive concern of scientists and research, nano material is more and more in the detection of major disease and treatment, biomarker, change It learns in the numerous areas such as catalysis and separation and has embodied its big advantage.Wherein mesoporous silicon dioxide nano microballoon is sub (MSNs) It is exactly the important representative of one type, is confirmed in scientific research and application in the past few years, while also achieving non- Often significant progress.Since mesoporous silicon oxide has a good biocompatibility, high-specific surface area, aperture and hole hold it is adjustable, Duct uniformly, surface be easy to the advantages that modifying, be highly suitable as the carrier of anti-tumor drug, be conducive to improve drug effect while again Can reduce drug toxic side effect (Small, 2010,6,1185-1190, Chem.Mater., 2007,17,4570-4573, Chem.Commun.,2010,46,3019–3921)。
For multifunctional nano-carrier, the constituent in conjunction with two or more is needed to examine for biomedicine The platform that disconnected, treating cancer diseased region provides.Magnetic nano ferroferric oxide nanosphere is that have magnetic hypermnesia raw The good feature of object compatibility makes it in targeted drug conveying, thermotherapy, cell separation, all many-sided tools such as magnetic resonance imaging (MRI) There are many potential application values.But single magnetic Nano microsphere is easy to reunite, and is not easy to load Drug is easy to be degraded in biosystem when it is directly exposed to, at the same simple mesoporous silicon oxide cultivate in vitro it is several Hour also can quickly discharge the limitation that medicine drug makes it face practicability very much, overcome these disadvantages, can be with Fe3O4Nanometer Microballoon is as core, mSiO2As hull shape at core-shell structure can not only overcome single Fe3O4Nanosphere and mSiO2 The limitation of nanosphere, and can be improved it in the performance of targeted drug controlled release system.(ACS nano.2010；4: 529-39.,Chem Phys Chem,2006,7(2):400-406,Journal of the American Chemical Society,2010,132(31):10623-10625)。
Folic acid is a kind of coenzyme nontoxic, stable, that immunogenicity is weak, inexpensive, and folic acid and folacin receptor compatibility are high, and With specificity, folacin receptor is expressed in many malignant cell excessive surfaces, and is then hardly expressed in normal cell Or only a small amount of table E, scientific research personnel link together drug, protein, polymer etc. with folic acid, utilize the targeting of folic acid It identifies to effect, makes drug targeting to tumor tissues are concentrated on, specific treatment is carried out to tumour, improve the treatment effect of drug Fruit.(Biomaterials,2016,82:178-193,Journal of controlled release,2016,232:161- 174,Rsc Advances,2016,6(42):35658-35667)。
Daunomycin (DNM) is first generation anthracene nucleus antineoplastic antibiotic, for various types of acute leukemias (including Granulocytic, lymphatic and monocarpotic cellularity and grain-monocarpotic cellularity), erythroleukemia, the white blood of chronic granulocytic Disease, malignant lymphoma, it can also be used to neuroblast disease, Ewing's sarcoma and nephroblastoma etc..But alopecia, bone can be caused Marrow inhibits and the toxicities such as cardiac toxic.Normal cell and cancer cell cannot be distinguished in presently commercially available daunomycin preparation, lacks Few targeting, causes it to have great damage to normal cell in the treatment.Patient will endure the various pairs of drug after chemotherapy Reaction, wherein cardiac toxic is irreversible.
Fe has been previously reported in inventor2+Adriamycin is combined (J with Superparamagnetic Iron Oxide nanosphere sub (SPION) coordination Biomater Appl.2016；Sub (SPION) immobilized porphin of paramagnetic iron oxide nanosphere 31:261-72) and with carboxyl Quinoline (Rsc Adv.2016；6:103137-48), the release rate under tumour pH environment reaches 85% or more, and in cellular water There is good antitumous effect on flat.It is well known that more satisfactory controlled drug transmits system in antitumor clinical application System does not require nothing more than itself with good biocompatibility and has higher load factor to drug, it is necessary to which satisfaction is followed in blood " zero release " of drug molecule when ring, but (Chemistry of but can quickly and be efficiently discharged after reaching lesions position Materials.2013,25:3030-3037,CS Applied Materials&Interfaces.2013,5:1566-1574, Macromolecules.2013,46:9169-9180), however the Fe of core-shell structure3O4@SiO2Targeting is single, follows in blood It is difficult to achieve the effect that " zero release " when ring, even if " intelligent " the drug release system now based on mesoporous silicon oxide System, mainly pH value response medicine controlled release system (Int.J.Pharm.2011,421:388~396), enzyme response medicine controlled release system Unite (Langmuir.2014,30:243~249), photoresponse controlled release delivery systems (Chem.Int.Ed.2013,52:4375~ 4379) and temperature-responsive controlled release delivery systems (Trans.2014,43:18056~18065 Dalton) etc. have reached so-called The effect of " zero release ", but their organic coating agent higher costs for largely using and synthetic method are complicated, therefore, find A kind of simple organic coating agent of at low cost and synthetic method has important research in conjunction with targeted drug nano-carrier control delivery And application value.
Summary of the invention
In view of the deficiencies of the prior art, the present invention provides a kind of pH controlled-release target medicament nano transport agent and its preparation side Method and application, it is intended to Fe3O4For core, with mSiO2For shell, in Fe3O4@mSiO2Modification-NH on surface2, then anti-by amide Receptor targeted folic acid (FA), immobilized daunomycin (DNM) should be connected, CaCO is finally used3It is blocked, realizes pH value response control It releases, achievees the effect that in blood circulation " zero release ", to reduce drug to the toxic side effect of human body.
To solve the above problems, on the one hand, the invention reside in a kind of pH controlled-release target medicament nano transport agent is provided, wrapping Include a nanometer transport agent, the anti-tumor drug being immobilized on nanometer transport agent and pH controlled release agent.
Further, the nanometer transport agent includes the Fe positioned at core3O4Nanosphere is coated on nanosphere table The mSiO in face2、Fe3O4@mSiO2- the NH of nanosphere surface modification2Be grafted on-NH2On folic acid.
Further, the nanometer transport agent: the mass ratio for the anti-tumor drug being immobilized on nanometer transport agent is 12.81%:1.
Further, the anti-tumor drug is anthracycline antibiotic or porphyrin.
Further, the anthracycline antibiotic is adriamycin, daunomycin, idarubicin, mitoxantrone or table Ah mould Element.
Further, the porphyrin is TMPYP, protoporphyrin, haematoporphyrin or hematoporphyrin monomethyl ether.
Further, the pH controlled release agent is mSiO2、CaCO3。
Further, the diameter of the pH controlled-release target medicament nano transport agent is 10~25nm, the diameter 10 of core~ 20nm, shell with a thickness of 3~10nm.
In an embodiment of the present invention, Fe3O4Nanosphere surface coats mSiO2It is denoted as: Fe3O4@mSiO2Nanosphere； Nanometer transport agent is denoted as: Fe3O4@mSiO2- FA nanosphere；- the NH of surface modification2Fe3O4@mSiO2Nanosphere note It does: Fe3O4@mSiO2-NH2Nanosphere；The nanometer immobilized antineoplastic objects system of transport agent is denoted as: Fe3O4@mSiO2-FA- DNM nanosphere；PH controlled-release target medicament nano transport agent is denoted as: Fe3O4@mSiO2-FA-DNM-CaCO3Nanosphere.
On the other hand, the invention reside in a kind of preparation method of pH controlled-release target medicament nano transport agent is provided, specifically Include the following steps:
Step 1: preparation Fe3O4Nanosphere: in atmosphere of inert gases, to FeCl3.6H2O and FeCl2.7H2O mixture Middle addition deoxygenates deionized water, heats after stirring and dissolving, keeps pH value in reaction 10~11 after rapidly joining ammonium hydroxide, reaction terminates Magnetic-particle is obtained using magnet adsorbing separation afterwards, it is spare with deionized water supersound washing until pH value is neutrality；
Step 2: preparation Fe3O4@mSiO2Nanosphere:
The Fe for 2a) taking step 1 to prepare3O4Nanosphere is placed in dilute hydrochloric acid, is washed with water after ultrasonic disperse to pH value and is Property；Take the Fe of processing3O4Nanosphere is added in water/ethyl alcohol mixed system, and ammonium hydroxide is added, and is slowly dripped under fast stirring Add tetraethyl orthosilicate (TEOS), reacts to obtain magnetic fluid；
It is dispersed in after 2b) alternately washing magnetic fluid deionized water and ethyl alcohol containing cetyl trimethylammonium bromide (CTAB) in deionized water/ethyl alcohol mixed system, ammonium hydroxide is then added and carries out ultrasonic disperse, is slowly added dropwise under stirring at room temperature Magnetic Nano microsphere, magnetic Nano microsphere water and second are isolated in tetraethyl orthosilicate reaction under the action of externally-applied magnetic field After alcohol washing, for use；
It 2c) takes magnetic Nano microsphere to be dispersed in ammonium nitrate-alcohol solution, after ultrasonic disperse is uniform, is heated to reflux Reaction is alternately washed with second alcohol and water, repeats step 2c after washing) twice to get Fe3O4@mSiO2Nanosphere；
Step 3: preparation Fe3O4@mSiO2-NH2Nanosphere: step 2 gained Fe is taken3O4@mSiO2Nanosphere is added to In water/ethyl alcohol mixed system, ammonium hydroxide is added and stirs evenly, 3- aminopropyl triethoxysilane (APTES) is then added and carries out machine Tool stirring is alternately washed after the reaction was completed with second alcohol and water, with magnet absorptive collection to get Fe3O4@mSiO2-NH2Nanometer is micro- Ball；
Step 4: preparation Fe3O4@mSiO2- FA nanosphere: step 3 gained Fe is taken3O4@mSiO2-NH2Nanosphere dispersion It is slowly added drop-wise to after DMSO in the folic acid solution of activation, centrifuge separation, which is collected, after being stirred overnight is made Fe3O4@mSiO2- FA receives Meter Wei Qiu；
Step 5: preparation Fe3O4@mSiO2- FA-DNM nanosphere: step 4 gained Fe is added in DNM solution3O4@mSiO2- Immobilized in FA nanosphere, then bottom material is collected in immobilized rear centrifuge separation, and Fe is made afterwards wash with distilled water3O4@mSiO2- FA-DNM nanosphere；
Step 6: preparation Fe3O4@mSiO2-FA-DNM-CaCO3Nanosphere: to Fe made from step 53O4@mSiO2-FA- Calcium chloride solution is added in DNM nanosphere, after being put in shaking table shaking uniformly, sodium carbonate liquor is added when shaking up, continues to shake It is even to be washed after removing supernatant using magnet absorption settle and separate to fully reacting, obtain Fe3O4@mSiO2-FA-DNM-CaCO3It receives Meter Wei Qiu.
Further, atmosphere of inert gases refers in nitrogen atmosphere in the step 1.
Further, heating refers to and is heated to 80 DEG C after stirring and dissolving in the step 1, and mixing speed control is existed 6.5krp/min。
Further, ammonium hydroxide is the ammonium hydroxide newly prepared, specific process for preparation in the step 1 are as follows: takes the concentration of 1 times of volume The ammonium hydroxide of 4.16 times of volumes is diluted to for 25% concentrated ammonia liquor.
Further, ammonium hydroxide is rapidly joined in the step 1 keeps reaction pH the reaction was continued after 10~11 30min.
It further, is 3~4 times with the number of deionized water supersound washing in the step 1.
Further, the step 2a) in dilute hydrochloric acid concentration be 0.1mol/L.
Further, the step 2a) in the ultrasonic disperse time be 10min.
Further, the step 2a) in water/ethyl alcohol mixed system be water/ethyl alcohol mixed system that volume ratio is 3/7.
Further, the step 2a) in ammonia concn be 25~28%.
Further, the step 2a) in react magnetic fluid reaction time be for 24 hours.
Further, the step 2a) in Fe3O4Nanosphere is 25:7 with the mass ratio of TEOS reacted.
Further, the step 2b) in deionized water/ethyl alcohol mixed system be deionized water/second that volume ratio is 1/1 Alcohol mixed system.
Further, the step 2b) in ammonia concn be 28%.
Further, the step 2b) in ultrasonic disperse time be 10min.
Further, the step 2b) in react time be 6h.
Further, the step 2c) in ammonium nitrate ethanol solution concentration be 10mg/mL.
Further, the step 2c) in the temperature that is heated to reflux be 80 DEG C, mixing speed control exists in reflux course 200rpm/min, the time of back flow reaction are 6h.
Further, water/ethyl alcohol mixed system is water/ethyl alcohol mixed system that volume ratio is 1/15 in the step 3.
Further, the concentration of ammonium hydroxide is 28% in the step 3, and the time of the stirring is 30min.
Further, the churned mechanically time is 4h in the step 3.
Further, the folic acid solution activated in the step 4 the preparation method is as follows: folic acid be dissolved in DMSO after addition EDC.HCl and sulfo-NHS, atmosphere of inert gases are stirred at room temperature, the folic acid solution activated.
Further, atmosphere of inert gases, which is stirred at room temperature, in the preparation method of the folic acid solution activated in the step 4 is 3h is stirred at room temperature in finger in nitrogen atmosphere.
Further, the time being stirred overnight in the step 4 is 14~18h.
Further, Fe in the step 43O4@mSiO2-NH2The mass ratio of nanosphere and folic acid is 1:4.
Further, the concentration of DNM solution is 0.14~0.86mg/ml in the step 5.Preferably 0.65mg/ml.
Further, it is immobilized on shaking table and carries out in the step 5, the speed of shaking table is 180rp/min, and the immobilized time is 24h。
Further, distilled water wash number is 2~3 times in the step 5.
Further, calcium chloride solution concentration is 8mmol/L in the step 6.
Further, sodium carbonate liquor concentration is 8mmol/L in the step 6.
Further, being put in the shaking table shaking time in the step 6 after addition calcium chloride solution is 10min or more.
Further, addition 1~5 is taken to drip sodium carbonate liquor when sodium carbonate liquor being added when shaking up in the step 6 Primary speed is shaken up to carry out.
Further, continuing to shake up in the step 6 to the time of fully reacting is 1h or more.
Further, it is 2~3 times that washing times after supernatant are removed in the step 6.
Room temperature of the present invention refers to room temperature, specifically can be 20~40 DEG C.
On the other hand, the present invention provides a kind of pH controlled-release target medicament nano transport agent and prevents and treats cancer in preparation Purposes in disease medicament.
Compared with prior art, the invention has the following beneficial effects:
The present invention provides a kind of pH controlled-release target medicament nano transport agent and its preparation method and application, the pH controlled release Targeted drug nanometer transport agent is with Fe3O4For core, mSiO2For shell, in Fe3O4@mSiO2Modification-NH on surface2, then pass through Amide reaction connects receptor targeted folic acid (FA), immobilized daunomycin (DNM), finally uses CaCO3It is blocked, realizes that pH value is rung Controlled release is answered, achievees the effect that in blood circulation " zero release ", to reduce drug to the toxic side effect of human body.PH control of the present invention Release targeted drug nanometer transport agent combination mSiO2Good biocompatibility, high-specific surface area, surface are easy to the advantages that modifying Wrap up Fe3O4Nanosphere, passes through amino (- NH2) receptor targeted folic acid is grafted after modification, utilize magnetic targeted and receptor target Generated multiple targeting, improves the anti-cancer properties of targeted drug, hence it is evident that is better than single targeting, by utilizing calcium carbonate In the stability of physiological pH, the performance of targeted drug controlled release system is improved.Specific manifestation is as follows:
Firstly, by by Fe3O4Magnetic targeted and folacin receptor targeting combine, improve anticancer drug to positive folic acid by The targeting ability of body cell (such as breast cancer, oophoroma, lung cancer, kidney, carcinoma of endometrium), while Superparamagnetic Iron Oxide is made For a kind of novel MRI contrast agent, can constantly be monitored.
Secondly, CaCO3As the main inorganic composition of skeleton, have source wide, cheaply.In physiological pH environment It is lower to keep complete, and dissolved under (such as: pH5-6, inflammation part pH3-5 in lysosome) under the lower environment of some pH value, And lysate (Ca2+,CO3 2-) nontoxic, realize that effective inhibition drug discharges in advance in blood circulation, in tumor locus (lesions position) efficiently and the purpose that quickly discharges.
Third, the present invention prepared by pH controlled-release target medicament nano transport agent good water solubility, partial size 100nm with Under, preferably, drugloading rate is high for dispersibility, discharged under tumor environment medication amount it is big and can last very long, and normally giving birth to It is very small to manage release medication amount under environment.
Of the invention preparation method is simple, and the mild raw material source of finance of preparation condition is wide, and product is easy to get, reliably, Post-processing is simple, suitable for mass production.
Detailed description of the invention
The synthetic schemes of Fig. 1 pH controlled-release target medicament nano transport agent.
Fig. 2 pH controlled-release target medicament nano transport agent mechanism of action figure；
In figure, tomur cell cancer cell, nucleus core, Endosome/lysosome inner body/lysosome, Folate Receptors folacin receptor.
Fig. 3 infrared spectroscopy map；
In figure, transmittance transmitance, wavenumber wavelength, (a) Fe3O4@mSiO2Nanosphere, (b) Fe3O4@mSiO2- FA nanosphere, (c) Fe3O4@mSiO2-NH2Nanosphere, (d) FA.
Fig. 4 Fe3O4@mSiO2- FA-DNM nanosphere electron microscope.
Fig. 5 Fe3O4@mSiO2-FA-DNM-CaCO3Nanosphere electron microscope.
Fig. 6 Fe3O4@mSiO2The aperture figure of-FA；
Pore size in figure: aperture, cumulative accumulation, dv/dw indicate the distribution of hole differential, and STP indicates that pore volume is seperated Product.
Fig. 7 sustained release figure；
In figure, a and c respectively represent Fe3O4@mSiO2- FA-DNM nanosphere is under conditions of pH=5.6 and pH=7.4 Elution profiles；B and d respectively represent Fe3O4@mSiO2-FA-DNM-CaCO3Item of the nanosphere in pH=5.6 and pH=7.4 Elution profiles under part；Cumulative release cumulative release, Time time.
Fig. 8 biocompatibility figure；
Wherein, figure a is Fe3O4@mSiO2-CaCO3Biocompatibility figure of the nanosphere to Hela cell；Figure b is Fe3O4@ mSiO2-FA-CaCO3Biocompatibility figure of the nanosphere to Hela cell；
Figure c is Fe3O4@mSiO2-CaCO3Biocompatibility figure of the nanosphere to A549 cell；Figure d is Fe3O4@ mSiO2-FA-CaCO3Biocompatibility figure of the nanosphere to A549 cell；
In figure, cell viability cell activity, concentration concentration.
Fig. 9 a dissociates DNM, Fe3O4@mSiO2-DNM-CaCO3、Fe3O4@mSiO2-FA-DNM-CaCO3To Hela cell MTT figure；
Fig. 9 b Fe3O4@mSiO2-FA-DNM-CaCO3It is right under folic acid culture medium and culture medium condition without folic acid The MTT of Hela cell schemes；
Fig. 9 c dissociates DNM, Fe3O4@mSiO2-DNM-CaCO3、Fe3O4@mSiO2-FA-DNM-CaCO3(the training without folic acid Support base under the conditions of), Fe3O4@mSiO2-FA-DNM-CaCO3The IC50 of (under the culture medium condition containing folic acid) schemes；
In figure, cell viability cell activity, concentration concentration, IC50 503nhibiting concentration.
The immobilized rate figure of the immobilized daunomycin of Figure 10 embodiment 2；
In figure: the immobilized rate of Loading Capacity, concentration concentration.
Figure 11 FA, Fe3O4@mSiO2With Fe3O4@mSiO2The ultraviolet phenogram of-FA；
In figure, absorbance absorbance, wavelength wavelength.
Figure 12 Fe3O4@mSiO2、Fe3O4@mSiO2-NH2With Fe3O4@mSiO2- FA charge pattern；
In figure, zata potential potential.
The free DNM solution of Figure 13 Hela cellular uptake and the fluorescence intensity level for loading DNM nanosphere solution；
In figure, MFI fluorescence intensity.
The free DNM solution of Figure 14 A549 cellular uptake and the fluorescence intensity level for loading DNM nanosphere solution；
In figure, MFI fluorescence intensity.
Specific embodiment
Below with reference to embodiment, the present invention is further illustrated, and however, it is not limited to this.
Embodiment 1pH controlled-release target medicament nano transport agent Fe3O4@mSiO2-FA-DNM-CaCO3The system of nanosphere It is standby
Specific preparation process can refer to Fig. 1, and process is as follows:
Step 1:Fe3O4The preparation of nanosphere
1.99g FeCl is weighed first3.6H2O and 0.99g FeCl2.7H2O(Fe2+: Fe3+Molar ratio is about 2:1, Fe2+ It is slightly excessive, because of Fe2+It is oxidized easily) in the round-bottomed flask of 250mL, 100ml deoxidation is added after being passed through nitrogen for a moment Ionized water, stirring and dissolving are heated to 80 DEG C heating in oil bath pan, speed of agitator 6.5krp/min is rapidly added new preparation Ammonium hydroxide (taking 25% concentrated ammonia liquor 12mL, be diluted to 50mL), test pH range with pH test paper, keep pH value in reaction 10~ 11；Liquid becomes black at once, the reaction was continued 30min；To utilize magnet adsorbing separation to adsorb magnetic-particle after reaction To drag, washed repeatedly 3-4 times with deionized water ultrasound, until pH is neutrality, it is spare.
Step 2: preparation Fe3O4@mSiO2Nanosphere
A. the Fe for taking 200mg step 1 to prepare3O4Nanosphere is in the dilute hydrochloric acid of the 0.1M of 50ml~100ml, ultrasound point After dissipating 10min, it is neutral that 4~5 times, which are washed to pH value,；
B. Fe step a handled3O4Nanosphere is added to the 500ml round-bottomed flask containing 60ml water and 140ml ethyl alcohol In, 2ml ammonium hydroxide (concentration is 25~28%) is then added, the tetraethyl orthosilicate of 0.08ml is then slowly added dropwise under fast stirring (TEOS), reaction obtains magnetic fluid for 24 hours；
C. magnetic fluid made from step b is taken, is alternately washed with 3ml deionized water and 3ml ethyl alcohol, removes extra reactant And impurity；
D. by the magnetic fluid after washing be dispersed in the 70mL containing 350mg cetyl trimethylammonium bromide (CTAB) go from In the mixed solution of sub- water and 70ml ethyl alcohol, the concentrated ammonia liquor that 1.5ml concentration is 28% is then added, carries out ultrasonic disperse 10min, 0.2ml tetraethyl orthosilicate (TEOS) reaction 6h is slowly added dropwise under the stirring of room temperature, after reaction in the effect of externally-applied magnetic field Under isolate magnetic Nano microsphere, alternately washed with water and ethyl alcohol for 3~4 times repeatedly, for use；
E. it takes magnetic Nano microsphere made from 100mg step d, is dispersed in (concentration in the ammonium nitrate-alcohol solution of 50mL For 10mg/ml), after ultrasonic disperse is uniform, 80 DEG C are heated to reflux, and mixing speed control reacts 6h, then in 200rpm/min With alternately washing 3 times of 5ml ethyl alcohol and 5ml water, repeats step e and test 2 times to get Fe3O4@mSiO2Nanosphere, infrared figure Spectrum is as shown in (a) in Fig. 3, and ultraviolet phenogram is as shown in figure 11, and charge pattern is as shown in figure 12.
Step 3: preparation Fe3O4@mSiO2-NH2Nanosphere
Take 80mg step 2 gained Fe3O4@mSiO2Nanosphere is added to the ethanol solution containing 10ml water and 150ml In round-bottomed flask, the ammonium hydroxide that 2ml concentration is 28% is added, stirs 30min, 1ml 3- aminopropyl triethoxysilane is then added (APTES), then mechanical stirring 4h is alternately washed 3 times with 3ml ethyl alcohol and 3ml water, with magnet absorptive collection to get Fe3O4@ mSiO2-NH2In nanosphere, infared spectrum such as Fig. 3 shown in (c), charge pattern is as shown in figure 12.
Step 4: preparation Fe3O4@mSiO2- FA nanosphere
0.1g folic acid is dissolved into the DMSO of 30ml, EDC.HCl the and 0.1151g sulfo- of 0.0575g is then added NHS (N- hydroxy thiosuccinimide), is passed through nitrogen, closed under nitrogen atmosphere that 3h is stirred at room temperature, and sufficiently activates to folic acid The folic acid solution that must be activated；
Take 25mg step 3 gained Fe3O4@SiO2-NH2Nanosphere is dispersed in 10ml DMSO, is then slowly added drop-wise to In the folic acid solution of activation, mixed solution is stirred overnight (14~18h), centrifuge separation, which is collected, is made Fe3O4@mSiO2- FA receives In meter Wei Qiu, infared spectrum such as Fig. 3 shown in (b), aperture figure is as shown in fig. 6, its ultraviolet phenogram is as shown in figure 11, electricity Lotus figure is as shown in figure 12.
Step 5: preparation Fe3O4@mSiO2- FA-DNM nanosphere, it may be assumed that immobilized daunomycin (DNM)
Take 10mg step 4 gained Fe3O4@mSiO25ml DNM solution (concentration is added in test tube in-FA nanosphere 0.65mg/ml), it is placed on shaking table (180rp/min) immobilized 24 hours, is then centrifuged for supernatant, measure the concentration of supernatant, count Calculate the amount of immobilized drug.Immobilized rate is 12.81% in the present embodiment；Magnetic fluid supernatant after will be immobilized refunds returnable bottle, Then it is cleaned magnetic fluid 2~3 times with 5ml distilled water, Fe is made3O4@mSiO2- FA-DNM nanosphere, electron microscope such as Fig. 4 institute Show.
Step 6: preparation Fe3O4@mSiO2-FA-DNM-CaCO3Nanosphere, it may be assumed that carry out CaCO3It blocks
A prepares the CaCl of 8mmol/L respectively2The Na of aqueous solution and 8mmol/L2CO3Aqueous solution (weighs calcium chloride 0.0178g, sodium carbonate 0.0170g are separately added into 20ml water and are configured to 20ml solution to obtain the final product).
B. Fe is made in step 53O4@mSiO25ml calcium chloride solution is added in-FA-DNM nanosphere, and it is equal to be put in shaking table shaking It is even, at least shake 10 minutes or more；
C. the sodium carbonate liquor of 5ml is added into step b test tube, Bian Shoudong shakes up side addition and (takes every 1~5 drop of addition Shake up primary mode) until complete, then test tube is put in shaking table and continues to shake up to fully reacting, at least react 1 hour；
D. after fully reacting, take out test tube, using magnet adsorb settle and separate, go supernatant (after closure, the face of supernatant Color is purple under normal circumstances), it is then cleaned 2~3 times with 5ml distilled water, obtains Fe3O4@mSiO2-FA-DNM-CaCO3Nanometer is micro- Ball, diameter be 10~25nm, 10~20nm of diameter of core, shell with a thickness of 3~10nm, electron microscope is as shown in Figure 5.
The experiment of the amount of 2 immobilized daunomycin (DNM) of embodiment
Take the resulting Fe of 1 step 4 of 10mg embodiment3O4@mSiO2It is molten that 5ml DNM is added in test tube in-FA nanosphere Liquid, the DNM solution concentration are respectively 0.14,0.33,0.49,0.65,0.86mg/ml, and it is immobilized to be placed on shaking table (180rp/min) 24 hours, it is then centrifuged for supernatant, measures the concentration of supernatant, calculates immobilized rate, finds out best immobilized concentration.It is specific real Test that the results are shown in Figure 10.
From fig. 10 it can be seen that immobilized rate also accordingly increases, and in concentration with the increase of DNM solution concentration Immobilized rate, which is increased speed, when 0.86mg/ml has tended to parallel, and subsequent take in DNM solution concentration is the progress of 0.65mg/ml when It is immobilized.
The sustained release experiment of embodiment 3
By Fe3O4@mSiO2- FA-DNM nanosphere and Fe3O4@mSiO2-FA-DNM-CaCO3Nanosphere is respectively placed in It fills in the test tube of PBS buffer solution of 10ml, the pH of the PBS buffer solution is respectively 5.6 and 7.4；Test tube is put into 37 3ml release liquid is taken out in persistent oscillation (180rmp/min) on DEG C constant-temperature table within the specific time (1,2,4,6h ... ..), then It is separately added into the fresh PBS buffer solution of 3ml, measurement is accumulative under the conditions of the PBS buffer solution of pH=5.6 and pH=7.4 Burst size.Concrete outcome is as shown in Figure 7.
As can be seen from Figure 7, Fe3O4@mSiO2- FA-DNM nanosphere and Fe3O4@mSiO2-FA-DNM-CaCO3Nanosphere It is more under the conditions of the PBS buffer solution of Cumulative release amount ratio pH=7.4 under the conditions of the PBS buffer solution of pH=5.6；Identical Under the conditions of PBS buffer solution, Fe3O4@mSiO2The Cumulative release amount ratio Fe of-FA-DNM nanosphere3O4@mSiO2-FA-DNM- CaCO3Nanosphere is more, it is seen that CaCO3Plugging effect.
4 cell biological compatibility experiments of embodiment
Fe3O4@mSiO2-CaCO3Nanosphere and Fe3O4@mSiO2-FA-CaCO3The preparation process of nanosphere is with reference to real Apply the preparation process of example 1.
Fe3O4@mSiO2-CaCO3Nanosphere and Fe3O4@mSiO2-FA-CaCO3The cytotoxicity of nanosphere passes through body Outer MTT experiment detection, specific experiment process are as follows: being first inoculated with Hela cell and A549 cell with the density in 5000/hole In 96 well culture plates, using the DMEM culture medium containing 10% fetal calf serum (FBS) in CO2It is cultivated for 24 hours, so for 37 DEG C in incubator It moves back except culture medium, adds 200 μ g/ml and contain Fe respectively3O4@mSiO2-CaCO3Nanosphere and Fe3O4@mSiO2-FA-CaCO3 The culture medium of nanosphere.By being prepared with DMEM culture medium serial dilution, concentration is the nanosphere solution of various concentration 12.5,25,50,100,200μg/ml.After cell and nanosphere culture 48h, cell is detected at 490nm by mtt assay Survival rate.Specific experiment result is as shown in Figure 8.
From figure 8, it is seen that Fe3O4@mSiO2-CaCO3When nanosphere solution concentration reaches 200ug/ml, do not have to cell Show toxicity, Fe3O4@mSiO2-FA-CaCO3When nanosphere concentration reaches 200ug/ml, Hela cell survival rate reaches 78%, higher cell survival rate is showed, and the survival rate of A549 cell is even more to have reached 95% or more, illustrates nanosphere To the good biocompatibility of Hela cell and A549 cell.
5 hemolytic test of embodiment
Fresh anticoagulant people's blood 4ml is taken, 5ml physiological saline is added and is diluted, sample to be tested is divided into experimental group, positive control Group and negative control group, wherein positive controls deionized water, negative control group physiological saline, every group set three it is parallel Experiment, the Fe for being 0.125mg/ml and 0.25mg/ml by 1ml brand-new concentration3O4@mSiO2The nanosphere suspension of-FA is put into 30min in 37 DEG C of water-baths, then 37 DEG C of water-baths are put under taking 1m dilute blood to be added in nanosphere suspension and shake Middle 60min, 1000*g are centrifuged 5min, then remove supernatant, and ultraviolet absorptivity is surveyed at 545nm, calculates the haemolysis of material, as a result As shown in table 1.
Table 1Fe3O4@mSiO2- FA nanosphere hemolytic experiment result
According to formula hemolysis rate (%)=(sample to be tested absorbance mean value-negative control absorbance)/(positive control extinction Spend mean value-negative control group) * 100%, 0.125mg/m, 0.25mg/m Fe of calculating3O4@mSiO2The haemolysis of-FA nanosphere Rate is respectively 1.97%, 4.21%, meets the requirement of hemolysis rate < 5%, meets the requirement of hemolysis rate < 5% of medical material.
The experiment of 6 cellular uptake of embodiment
Experimental procedure: using the cellular uptake situation of Flow Cytometry observation medicament-carrying nano-microsphere.Logarithmic growth phase Uterine cancer cells (Hela) and lung carcinoma cell (A549), with containing EDTA pancreatin digestion after, take out the pancreatin of EDTA, addition contains The culture medium piping and druming of serum is uniformly dispersed, and cell suspending liquid (1.0 × 10 is made5A/ml), take 1.5ml to be inoculated in 6 orifice plates cell In culture plate.Culture plate is placed in containing 5%CO237 DEG C of incubator in be incubated for for 24 hours, make cell adherent growth.Cell liquid is removed, It is separately added into free DNM solution, Fe3O4@mSiO2-DNM-CaCO3Nanosphere, Fe3O4@mSiO2-FA-DNM-CaCO3Nanometer Culture medium is sucked out after being incubated for 4h by microspheres solution, the final concentration of 0.5ug/ml of DNM, PBS solution washing is added three times, to remove Remove free drug molecule and nanosphere.The pancreatin of 0.5ml is added in each hole, and 1ml is added containing serum free culture system after digesting 1min Base terminates digestion, after piping and druming uniformly, cell suspending liquid is centrifuged 3min at 2000rmp, removes supernatant, PBS cleaning 3 is added Time, then it is centrifugated into cell suspending liquid, as measuring in streaming pipe, cell count is 1000.
Experimental analysis: as shown in Figure 13~14, the case where DNM is ingested is observed using Flow Cytometry and is analyzed. Through to Fe3O4@mSiO2-DNM-CaCO3Nanosphere, Fe3O4@mSiO2-DNM-FA-CaCO3The DNM analysis of nanosphere load, Know the Fe through modified with folic acid3O4@mSiO2-DNM-FA-CaCO3Nanosphere is in the Hela cell that folate receptor-positive is expressed The intake of DNM is apparently higher than the Fe of not modified with folic acid3O4@mSiO2-DNM-CaCO3Nanosphere；And correspondingly, Fe3O4@ mSiO2-DNM-FA-CaCO3Nanosphere and Fe3O4@mSiO2-DNM-CaCO3Nanosphere is expressed in folacin receptor feminine gender The not apparent difference of the intake of DNM in A549 cell, this illustrates Fe3O4@mSiO2-DNM-FA-CaCO3Nanosphere pair The Hela cell of positive receptor has targeting.
The measurement of 7 cell survival rate of embodiment
Free DNM, Fe are measured by MTT experiment3O4@mSiO2-DNM-CaCO3Nanosphere and Fe3O4@mSiO2-FA- DNM-CaCO3Nanosphere is to the antiproliferative effect of Hela cell, in order to detect the nanosphere of modified with folic acid to Hela cell Targeting, cultivated provided with the culture medium of folic acid containing 1mmol/L and without folic acid culture medium.The mechanism of action as shown in Fig. 2, Specific experiment process is as follows: Hela cell is inoculated in 96 well culture plates first with the density in 5000/hole, with containing 10% tire ox The DMEM culture of serum is based on 37 DEG C, 5%CO2Under the conditions of cultivate for 24 hours, then in every hole be added various concentration free DNM, Fe3O4-mSiO2-DNM-CaCO3Nanosphere and Fe3O4-mSiO2-FA-DNM-CaCO3Nanosphere, the final concentration difference of DNM For 0.5 μ g/ml, 0.25 μ g/ml, 0.125 μ g/ml, 0.06 μ g/ml, 0.03 μ g/ml, for 24 hours, each concentration setting 5 right for culture According to hole.After drug-treated, cell and MTT (5mg/mL) cultivate 4h, are dissolved in after first a ceremonial jade-ladle, used in libation (Formazan) precipitating of formation 150 μ L DMSO, finally measure its absorbance value with microplate reader under 490nm wavelength.Concrete outcome is as shown in Fig. 9 a~9c.
It can be seen that the increase of the concentration with DNM, Fe from Fig. 9 a3O4@mSiO2-FA-DNM-CaCO3Nanosphere phase Than in Fe3O4@mSiO2-DNM-CaCO3Nanosphere and free DNM show stronger tumor inhibitory effect, illustrate Fe3O4@ mSiO2-FA-DNM-CaCO3Nanosphere has Targeting Effect.
In order to further prove the Targeting Effect of folic acid, in the item containing 1mmol folic acid culture medium and without folic acid culture medium Under part, to Fe3O4@mSiO2-FA-DNM-CaCO3It is poor that the anti-tumor activity of nanosphere adjust, and Fig. 9 b, which can be seen that, to be free of Fe under the culture medium condition of folic acid3O4@mSiO2-FA-DNM-CaCO3The anti-tumor activity of nanosphere is than anti-swollen containing folic acid Tumor activity is more significant, because folacin receptor has competitiveness above the culture medium Hela cell containing folic acid, illustrates Fe3O4@ mSiO2-FA-DNM-CaCO3Nanosphere have the effect of folate-targeted and preferably it is antitumor.
Free DNM, Fe3O4@mSiO2-DNM-CaCO3Nanosphere, Fe3O4@mSiO2-FA-DNM-CaCO3Nanosphere (under the culture medium condition without folic acid), Fe3O4@mSiO2-FA-DNM-CaCO3Nanosphere (the culture medium condition containing folic acid Under) MTT experiment IC50 value as is shown in fig. 9 c, it is seen that without containing folic acid culture medium in Fe3O4@mSiO2-FA-DNM- CaCO3The IC50 value of nanosphere is smaller, has better antitumous effect.
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any to repair Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.

Claims (6)

1. a kind of preparation method of pH controlled-release target medicament nano transport agent, which is characterized in that specifically comprise the following steps:
Step 1: preparation Fe3O4Nanosphere: in atmosphere of inert gases, to FeCl3·6H2O and FeCl2·7H2In O mixture Deoxidation deionized water is added, is heated after stirring and dissolving, keeps pH value in reaction 10~11 after rapidly joining ammonium hydroxide, after reaction Magnetic-particle is obtained using magnet adsorbing separation, it is spare with deionized water supersound washing until pH value is neutrality；
Step 2: preparation Fe3O4@mSiO2Nanosphere:
The Fe for 2a) taking step 1 to prepare3O4Nanosphere is placed in dilute hydrochloric acid, and being washed with water after ultrasonic disperse to pH value is neutrality；It takes The Fe of processing3O4Nanosphere is added in water/ethyl alcohol mixed system, and ammonium hydroxide is added, silicic acid is slowly added dropwise under fast stirring Tetra-ethyl ester reacts to obtain magnetic fluid；
Be dispersed in after 2b) alternately washing magnetic fluid deionized water and ethyl alcohol containing cetyl trimethylammonium bromide go from In sub- water/ethyl alcohol mixed system, ammonium hydroxide is then added and carries out ultrasonic disperse, it is anti-that tetraethyl orthosilicate is slowly added dropwise under stirring at room temperature It answers, magnetic Nano microsphere is isolated under the action of externally-applied magnetic field, after magnetic Nano microsphere water and ethanol washing, to With；
Magnetic Nano microsphere 2c) is taken to be dispersed in ammonium nitrate-alcohol solution, after ultrasonic disperse is uniform, heating reflux reaction, Alternately washed with second alcohol and water, repeat step 2c after washing) twice to get Fe3O4@mSiO2Nanosphere；
Step 3: preparation Fe3O4@mSiO2-NH2Nanosphere: step 2 gained Fe is taken3O4@mSiO2Nanosphere is added to water/second In alcohol mixed system, ammonium hydroxide is added and stirs evenly, 3- aminopropyl triethoxysilane is then added and carries out mechanical stirring, has reacted Cheng Houyong second alcohol and water alternately washs, with magnet absorptive collection to get Fe3O4@mSiO2-NH2Nanosphere；
Step 4: preparation Fe3O4@mSiO2- FA nanosphere: step 3 gained Fe is taken3O4@mSiO2-NH2Nanosphere is dispersed in It is slowly added drop-wise to after DMSO in the folic acid solution of activation, centrifuge separation, which is collected, after being stirred overnight is made Fe3O4@mSiO2- FA nanometers Microballoon；
Step 5: preparation Fe3O4@mSiO2- FA-DNM nanosphere: step 4 gained Fe is added in DNM solution3O4@mSiO2- FA receives Immobilized in meter Wei Qiu, then bottom material is collected in immobilized rear centrifuge separation, and Fe is made afterwards wash with distilled water3O4@mSiO2-FA- DNM nanosphere；
Step 6: preparation Fe3O4@mSiO2-FA-DNM-CaCO3Nanosphere: to Fe made from step 53O4@mSiO2- FA-DNM receives Calcium chloride solution is added in meter Wei Qiu, after being put in shaking table shaking uniformly, sodium carbonate liquor is added when shaking up, continues to shake up to anti- Should settle and separate be adsorbed using magnet, is washed after removing supernatant, obtains Fe completely3O4@mSiO2-FA-DNM-CaCO3Nanosphere.
2. preparation method according to claim 1, which is characterized in that atmosphere of inert gases refers in nitrogen in the step 1 During atmosphere is enclosed；
Heating refers to and is heated to 80 DEG C after stirring and dissolving in the step 1, and mixing speed is controlled in 6.5krp/min；
Ammonium hydroxide is the ammonium hydroxide newly prepared, specific process for preparation in the step 1 are as follows: takes the dense ammonia that the concentration of 1 times of volume is 25% Water is diluted to the ammonium hydroxide of 4.16 times of volumes；
Ammonium hydroxide is rapidly joined in the step 1 keeps reaction pH the reaction was continued after 10~11 30min；
The number that deionized water supersound washing is used in the step 1 is 3~4 times.
3. preparation method according to claim 1, which is characterized in that the step 2a) in the concentration of dilute hydrochloric acid be 0.1mol/L；
The step 2a) in the ultrasonic disperse time be 10min；
The step 2a) in water/ethyl alcohol mixed system be water/ethyl alcohol mixed system that volume ratio is 3/7；
The step 2a) in ammonia concn be 25~28%；
The step 2a) in react magnetic fluid reaction time be for 24 hours；
The step 2a) in Fe3O4Nanosphere is 25:7 with the mass ratio of TEOS reacted；
The step 2b) in deionized water/ethyl alcohol mixed system be deionized water/ethyl alcohol mixed system that volume ratio is 1/1；
The step 2b) in ammonia concn be 28%；
The step 2b) in ultrasonic disperse time be 10min；
The step 2b) in react time be 6h；
The step 2c) in ammonium nitrate ethanol solution concentration be 10mg/mL；
The step 2c) in the temperature that is heated to reflux be 80 DEG C, mixing speed control is in 200rpm/min, reflux in reflux course The time of reaction is 6h.
4. preparation method according to claim 1, which is characterized in that water/ethyl alcohol mixed system is volume in the step 3 Than the water for 1/15/ethyl alcohol mixed system；
The concentration of ammonium hydroxide is 28% in the step 3, and the time of the stirring is 30min；
The churned mechanically time is 4h in the step 3.
5. preparation method according to claim 1, which is characterized in that the preparation of the folic acid solution activated in the step 4 Method is as follows: EDCHCl and sulfo-NHS is added after being dissolved in DMSO in folic acid, and atmosphere of inert gases is stirred at room temperature, is lived The folic acid solution of change；
The atmosphere of inert gases, which is stirred at room temperature, to be referred to 3h is stirred at room temperature in nitrogen atmosphere；
The time being stirred overnight in the step 4 is 14~18h；
Fe in the step 43O4@mSiO2-NH2The mass ratio of nanosphere and folic acid is 1:4.
6. preparation method according to claim 1, which is characterized in that in the step 5 concentration of DNM solution be 0.14~ 0.86mg/ml；
It is immobilized on shaking table and carries out in the step 5, the speed of shaking table is 180rp/min, and the immobilized time is for 24 hours；
Distilled water wash number is 2~3 times in the step 5；
Calcium chloride solution concentration is 8mmol/L in the step 6；
Sodium carbonate liquor concentration is 8mmol/L in the step 6；
It takes addition 1~5 to drip sodium carbonate liquor when sodium carbonate liquor is added in the step 6 when shaking up and shakes up primary speed It carries out；
It is 2~3 times that the number washed after supernatant is removed in the step 6.
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