CN107952081A - PH controlled-release target medicament nano transport agents and its preparation method and application - Google Patents

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

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CN107952081A
CN107952081A CN201711130408.0A CN201711130408A CN107952081A CN 107952081 A CN107952081 A CN 107952081A CN 201711130408 A CN201711130408 A CN 201711130408A CN 107952081 A CN107952081 A CN 107952081A
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msio
nanoparticle
dnm
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CN107952081B (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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    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • 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
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    • 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

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Abstract

The present invention provides a kind of pH controlled-release targets medicament nano transport agent and its preparation method and application, and the pH controlled-release targets medicament nano transport agent is with Fe3O4For core, mSiO2For shell, in Fe3O4@mSiO2NH is modified on surface2, receptor targeted folic acid (FA), immobilized daunomycin (DNM) are then connected by acid amides reaction, finally use CaCO3Blocked, realize that pH value responds controlled release, reach in blood circulation the effect of " zero release ", so as to reduce toxic side effect of the medicine to human body.PH controlled-release targets medicament nano transport agent combination mSiO of the present invention2The advantages that good biocompatibility, high-specific surface area, surface are easy to modification wraps up Fe3O4Nanoparticle, 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 using calcium carbonate in the stability of physiological pH, the performance of raising targeted drug controlled release system.

Description

PH controlled-release target medicament nano transport agents and its preparation method and application
Technical field
The invention belongs to target administration technical field, is related to a kind of medicament nano transport agent, and in particular to a kind of pH controls Release targeted drug nanometer transport agent and its preparation method and application.
Background technology
In recent years, with the fast development of nanometer biotechnology, intelligent Organic-inorganic composite nanoparticle is subject to The extensive concern of scientists and research, nano material is more and more in the detection and treatment, biomarker, change of major disease Learn in the numerous areas such as catalysis and separation and 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 achieve non- Often significant progress.Due to mesoporous silicon oxide have good biocompatibility, high-specific surface area, aperture and pore volume it is adjustable, Duct uniformly, surface the advantages that being easy to modification, be highly suitable as the carrier of antitumor drug, be conducive to improve drug effect while again Can reduce medicine toxic side effect (Small, 2010,6,1185-1190, Chem.Mater., 2007,17,4570-4573, Chem.Commun.,2010,46,3019–3921)。
, it is necessary to be examined with reference to the constituent of two or more for biomedicine for multifunctional nano-carrier The platform that disconnected, treating cancer diseased region provides.Magnetic nano ferroferric oxide nanoparticle is that there is magnetic hypermnesia to give birth to The characteristics of thing compatibility is good, makes it in targeted drug conveying, thermotherapy, all many-sided tools such as cell separation, magnetic resonance imaging (MRI) There are many potential application values.But single magnetic Nano microsphere is easy to reunite, and it is not easy to load Medicine, is easily degraded when it is directly exposed in biosystem, at the same simple mesoporous silicon oxide cultivate in vitro it is several Hour also can quickly discharge the limitation that medicine medicine makes it face practicality very much, overcome these shortcomings, can be with Fe3O4Nanometer Microballoon is used as core, mSiO2As hull shape into core shell structure can not only overcome single Fe3O4Nanoparticle and mSiO2 The limitation of nanoparticle, and its performance in targeted drug controlled release system can be improved.(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 is high with folacin receptor compatibility, 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 medicine, protein, polymer etc. with folic acid, utilize the targeting of folic acid Identify to effect, drug targeting is carried out specific treatment to tumour to tumor tissues are concentrated on, improve the treatment effect of medicine 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 The toxicity such as marrow suppression and cardiac toxic.Presently commercially available daunomycin preparation cannot be distinguished by normal cell and cancer cell, lack Few targeting, causes it to have great infringement to normal cell in the treatment.Patient will endure the various pairs of medicine after chemotherapy Reaction, wherein cardiac toxic is irreversible.
Inventor has been previously reported by Fe2+- adriamycin combines (J with Superparamagnetic Iron Oxide nanoparticle (SPION) coordination Biomater Appl.2016；31:Sub (SPION) immobilized porphin of paramagnetic iron oxide nanoparticle 261-72) and with carboxyl Quinoline (Rsc Adv.2016；6:103137-48), the release rate under tumour pH environment reaches more than 85%, and in cellular water There is good antitumous effect on flat.It is well known that in antitumor clinical practice, more satisfactory controlled drug transmission system System, which does not require nothing more than, itself to be had good biocompatibility and has higher load factor to medicine, it is necessary to which satisfaction is followed in blood " zero release " of drug molecule during ring, but but can quickly and efficiently discharge (Chemistry of 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, is followed in blood It is difficult to the effect for reaching " zero release " during ring, even if " intelligent " the insoluble 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~ And temperature-responsive controlled release delivery systems (Dalton Trans.2014,43 4379):18056~18065) etc. reached so-called The effect of " zero release ", but their the organic coating agent costs that largely use are higher and synthetic method is complicated, therefore, find A kind of cost is low and the simple organic coating agent of synthetic method, has important research with reference to targeted drug nano-carrier control delivery And application value.
The content of the invention
In view of the deficiencies of the prior art, the present invention provides a kind of pH controlled-release targets 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, it is then anti-by acid amides Receptor targeted folic acid (FA), immobilized daunomycin (DNM) should be connected, finally uses CaCO3Blocked, realize pH value response control Release, reach in blood circulation the effect of " zero release ", so as to reduce toxic side effect of the medicine to human body.
To solve the above problems, on the one hand, the invention reside in provide a kind of pH controlled-release targets medicament nano transport agent, bag Include a nanometer transport agent, the antitumor drug being immobilized on nanometer transport agent and pH controlled release agents.
Further, the nanometer transport agent includes the Fe positioned at core3O4Nanoparticle, be coated on nanoparticle table The mSiO in face2、Fe3O4@mSiO2- the NH of nanoparticle surface modification2Be grafted on-NH2On folic acid.
Further, the nanometer transport agent：The mass ratio for the antitumor drug being immobilized on nanometer transport agent is 12.81%:1.
Further, the antitumor 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 agents are mSiO2、CaCO3。
Further, a diameter of 10~25nm of the pH controlled-release targets medicament nano transport agent, the diameter 10 of core~ 20nm, the thickness of shell is 3~10nm.
In an embodiment of the present invention, Fe3O4Nanoparticle surface coats mSiO2It is denoted as：Fe3O4@mSiO2Nanoparticle； Nanometer transport agent is denoted as：Fe3O4@mSiO2- FA nanoparticles；- the NH of surface modification2Fe3O4@mSiO2Nanoparticle is remembered Do：Fe3O4@mSiO2-NH2Nanoparticle；The nanometer immobilized antineoplastic objects system of transport agent is denoted as：Fe3O4@mSiO2-FA- DNM nanoparticles；PH controlled-release target medicament nano transport agents are denoted as：Fe3O4@mSiO2-FA-DNM-CaCO3Nanoparticle.
On the other hand, the invention reside in a kind of preparation method of pH controlled-release targets medicament nano transport agent is provided, specifically Include the following steps：
Step 1：Prepare Fe3O4Nanoparticle：In atmosphere of inert gases, to FeCl3.6H2O and FeCl2.7H2O mixtures It is middle to add deoxidation deionized water, heat after stirring and dissolving, rapidly join and pH value in reaction is kept after ammonium hydroxide reaction terminates 10~11 The isolated magnetic-particle of magnet adsorption is utilized afterwards, it is spare with deionized water supersound washing until pH value is neutral；
Step 2：Prepare Fe3O4@mSiO2Nanoparticle：
The Fe for 2a) taking step 1 to prepare3O4Nanoparticle is placed in dilute hydrochloric acid, in being washed with water after ultrasonic disperse and being to pH value Property；Take the Fe of processing3O4Nanoparticle is added in water/ethanol mixed system, adds ammonium hydroxide, is slowly dripped under fast stirring Add tetraethyl orthosilicate (TEOS), react to obtain magnetic fluid；
It is dispersed in after 2b) magnetic fluid is alternately washed with deionized water and ethanol containing cetyl trimethylammonium bromide (CTAB) in deionized water/ethanol mixed system, then add ammonium hydroxide and carry out ultrasonic disperse, be slowly added dropwise under stirring at room temperature Tetraethyl orthosilicate reacts, and magnetic Nano microsphere, magnetic Nano microsphere water and second are isolated under the action of externally-applied magnetic field It is stand-by after alcohol washing；
2c) take magnetic Nano microsphere to be dispersed in ammonium nitrate-alcohol solution, after ultrasonic disperse is uniform, be heated to reflux Reaction, is alternately washed with second alcohol and water, step 2c is repeated after washing) twice, up to Fe3O4@mSiO2Nanoparticle；
Step 3：Prepare Fe3O4@mSiO2-NH2Nanoparticle：Take step 2 gained Fe3O4@mSiO2Nanoparticle is added to In water/ethanol mixed system, add ammonium hydroxide and stir evenly, then add 3- aminopropyl triethoxysilanes (APTES) and carry out machine Tool stirs, and is alternately washed, is collected with magnet adsorption, up to Fe with second alcohol and water after the completion of reaction3O4@mSiO2-NH2Nanometer is micro- Ball；
Step 4：Prepare Fe3O4@mSiO2- FA nanoparticles：Take step 3 gained Fe3O4@mSiO2-NH2Nanoparticle disperses Slowly it is added drop-wise to after DMSO in the folic acid solution of activation, is centrifuged after being stirred overnight and collect obtained Fe3O4@mSiO2- FA receives Meter Wei Qiu；
Step 5：Prepare Fe3O4@mSiO2- FA-DNM nanoparticles：DNM solution is added into step 4 gained Fe3O4@mSiO2- Immobilized in FA nanoparticles, then immobilized rear centrifuge collects bottom material, and Fe is made after being cleaned with distilled water3O4@mSiO2- FA-DNM nanoparticles；
Step 6：Prepare Fe3O4@mSiO2-FA-DNM-CaCO3Nanoparticle：To Fe made from step 53O4@mSiO2-FA- Calcium chloride solution is added in DNM nanoparticles, after being put in shaking table shaking uniformly, sodium carbonate liquor is added when shaking up, continues to shake Even extremely the reaction was complete, using magnet adsorption standing separation, is washed after removing supernatant, obtains Fe3O4@mSiO2-FA-DNM-CaCO3Receive Meter Wei Qiu.
Further, atmosphere of inert gases refers in nitrogen atmosphere in the step 1.
Further, heating refers to be 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 in the step 1, and specific process for preparation is：Take 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 and keeps reaction pH the reaction was continued after 10~11 30min.
Further, it is 3~4 times with the number of deionized water supersound washing in the step 1.
Further, the step 2a) in the concentration of dilute hydrochloric acid be 0.1mol/L.
Further, the step 2a) in the ultrasonic disperse time be 10min.
Further, the step 2a) in water/ethanol mixed system be water/ethanol mixed system that volume ratio is 3/7.
Further, the step 2a) in ammonia concn be 25~28%.
Further, the step 2a) in react reaction time of magnetic fluid is 24h.
Further, the step 2a) in Fe3O4The mass ratio of the reaction of nanoparticle and TEOS is 25:7.
Further, the step 2b) in deionized water/ethanol 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 time of ultrasonic disperse be 10min.
Further, the step 2b) in reaction time be 6h.
Further, the step 2c) in the concentration of ammonium nitrate ethanol solution be 10mg/mL.
Further, the step 2c) in the temperature that is heated to reflux be 80 DEG C, in reflux course mixing speed control exists 200rpm/min, the time of back flow reaction is 6h.
Further, water/ethanol mixed system is water/ethanol 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 preparation method of the folic acid solution activated in the step 4 is as follows：Addition after folic acid is dissolved in DMSO 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 nanoparticle 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, it is more than 10min to be put in the shaking table shaking time in the step 6 after addition calcium chloride solution.
Further, addition 1~5 is taken to drip sodium carbonate liquor when adding sodium carbonate liquor when shaking up in the step 6 The speed shaken up once carries out.
Further, it is more than 1h to continue to shake up to the time that the reaction was complete in the step 6.
Further, washing times are 2~3 times after supernatant is removed in the step 6.
Room temperature of the present invention refers to room temperature, can be specifically 20~40 DEG C.
On the other hand, the present invention provides a kind of pH controlled-release targets medicament nano transport agent and is preparing prevention and treatment cancer Purposes in disease medicament.
Compared with prior art, the present invention has the advantages that：
The present invention provides a kind of pH controlled-release targets medicament nano transport agent and its preparation method and application, the pH controlled releases Targeted drug nanometer transport agent is with Fe3O4For core, mSiO2For shell, in Fe3O4@mSiO2Modification-NH on surface2, then pass through Acid amides reaction connects receptor targeted folic acid (FA), immobilized daunomycin (DNM), finally uses CaCO3Blocked, realize that pH value is rung Controlled release is answered, reaches in blood circulation the effect of " zero release ", so as to reduce toxic side effect of the medicine to human body.PH controls of the present invention Release targeted drug nanometer transport agent combination mSiO2The advantages that good biocompatibility, high-specific surface area, surface is easy to modification Wrap up Fe3O4Nanoparticle, passes through amino (- NH2) receptor targeted folic acid is grafted after modification, utilize magnetic targeted and receptor target Caused multiple targeting, improves the anti-cancer properties of targeted drug, hence it is evident that be better than single targeting, by using calcium carbonate In the stability of physiological pH, the performance of raising targeted drug controlled release system.Specific manifestation is as follows：
First, by by Fe3O4Magnetic targeted and folacin receptor targeting be combined, improve cancer therapy 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 new MRI contrast agent, can constantly be monitored.
Secondly, CaCO3It is wide with source as the main inorganic composition of skeleton, cheaply.In physiological pH environment It is lower to keep complete, and under the relatively low environment of some pH value (such as：PH5-6, inflammation part pH3-5 in lysosome) under dissolve, And lysate (Ca2+,CO3 2-) nontoxic, realize that the effective medicine that suppresses discharges in advance in blood circulation, in tumor locus (lesions position) efficiently and the purpose that quickly discharges.
3rd, the present invention prepared by pH controlled-release target medicament nano transport agent good water solubilities, particle diameter 100nm with Under, preferably, drugloading rate is high for dispersiveness, 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.
The preparation method of the present invention is simple and practicable, and the gentle 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.
Brief description of the drawings
The synthetic schemes of Fig. 1 pH controlled-release target medicament nano transport agents.
Fig. 2 pH controlled-release target medicament nano transport agent mechanism of action figures；
In figure, tomur cell cancer cells, nucleus cores, Endosome/lysosome inner bodies/lysosome, Folate Receptors folacin receptors.
Fig. 3 infrared spectrum collection of illustrative plates；
In figure, transmittance transmitances, wavenumber wavelength, (a) Fe3O4@mSiO2Nanoparticle, (b) Fe3O4@mSiO2- FA nanoparticles, (c) Fe3O4@mSiO2-NH2Nanoparticle, (d) FA.
Fig. 4 Fe3O4@mSiO2- FA-DNM nanoparticle electron microscopes.
Fig. 5 Fe3O4@mSiO2-FA-DNM-CaCO3Nanoparticle electron microscope.
Fig. 6 Fe3O4@mSiO2The aperture figure of-FA；
Pore size in figure：Aperture, cumulative accumulations, dv/dw represent the distribution of hole differential, and STP represents pore volume split Product.
Fig. 7 sustained release figures；
In figure, a and c represent Fe respectively3O4@mSiO2- FA-DNM nanoparticles are under conditions of pH=5.6 and pH=7.4 Elution profiles；B and d represent Fe respectively3O4@mSiO2-FA-DNM-CaCO3Bar of the nanoparticle in pH=5.6 and pH=7.4 Elution profiles under part；Cumulative release cumulative releases, Time times.
Fig. 8 biocompatibility figures；
Wherein, it is Fe to scheme a3O4@mSiO2-CaCO3Biocompatibility figure of the nanoparticle to Hela cells；Figure b is Fe3O4@ mSiO2-FA-CaCO3Biocompatibility figure of the nanoparticle to Hela cells；
Figure c is Fe3O4@mSiO2-CaCO3Biocompatibility figure of the nanoparticle to A549 cells；Figure d is Fe3O4@ mSiO2-FA-CaCO3Biocompatibility figure of the nanoparticle to A549 cells；
In figure, cell viability cytoactives, concentration concentration.
Fig. 9 a dissociate DNM, Fe3O4@mSiO2-DNM-CaCO3、Fe3O4@mSiO2-FA-DNM-CaCO3To Hela cells MTT schemes；
Fig. 9 b Fe3O4@mSiO2-FA-DNM-CaCO3It is right under folic acid culture medium and culture medium condition without folic acid The MTT figures of Hela cells；
Fig. 9 c dissociate DNM, Fe3O4@mSiO2-DNM-CaCO3、Fe3O4@mSiO2-FA-DNM-CaCO3(be free of the training of folic acid Support base under the conditions of), Fe3O4@mSiO2-FA-DNM-CaCO3The IC50 figures of (under the culture medium condition containing folic acid)；
In figure, cell viability cytoactives, concentration concentration, IC50 503nhibiting concentrations.
The immobilized rate figure of 2 immobilized daunomycin of Figure 10 embodiments；
In figure：The immobilized rates of Loading Capacity, concentration concentration.
Figure 11 FA, Fe3O4@mSiO2With Fe3O4@mSiO2The ultraviolet phenograms of-FA；
In figure, absorbance absorbances, wavelength wavelength.
Figure 12 Fe3O4@mSiO2、Fe3O4@mSiO2-NH2With Fe3O4@mSiO2- FA charge patterns；
In figure, zata potential potentials.
Figure 13 Hela cellular uptakes dissociate DNM solution and load DNM nanoparticle solution fluorescence intensity level；
In figure, MFI fluorescence intensities.
Figure 14 A549 cellular uptakes dissociate DNM solution and load DNM nanoparticle solution fluorescence intensity level；
In figure, MFI fluorescence intensities.
Embodiment
With reference to embodiment, the present invention is further illustrated, but is not limited thereto.
Embodiment 1pH controlled-release target medicament nano transport agents Fe3O4@mSiO2-FA-DNM-CaCO3The system of nanoparticle It is standby
Specific preparation process refers to Fig. 1, and flow is as follows：
Step 1：Fe3O4The preparation of nanoparticle
1.99g FeCl are weighed first3.6H2O and 0.99g FeCl2.7H2O(Fe2+：Fe3+Molar ratio is about 2：1, Fe2+ It is somewhat excessive, because of Fe2+It is oxidized easily) in the round-bottomed flask of 250mL, add 100ml deoxidations after being passed through nitrogen for a moment Ionized water, stirring and dissolving, is heated to 80 DEG C, speed of agitator 6.5krp/min, is rapidly added new preparation heating in oil bath pan Ammonium hydroxide (taking 25% concentrated ammonia liquor 12mL, be diluted to 50mL), test pH scopes with pH test paper, keep pH value in reaction 10~ 11；Liquid becomes black at once, the reaction was continued 30min；Magnetic-particle is adsorbed using magnet adsorption separation after question response To drag, with deionized water ultrasound repeated washing 3-4 times, until pH is neutrality, it is spare.
Step 2：Prepare Fe3O4@mSiO2Nanoparticle
A. the Fe for taking 200mg steps 1 to prepare3O4Nanoparticle is in the dilute hydrochloric acid of the 0.1M of 50ml~100ml, ultrasound point After dissipating 10min, it is neutrality that 4~5 times, which are washed to pH value,；
B. Fe step a handled3O4Nanoparticle is added to the 500ml round-bottomed flasks containing 60ml water and 140ml ethanol 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 24h obtains magnetic fluid；
C. magnetic fluid made from step b is taken, is alternately washed with 3ml deionized waters and 3ml ethanol, removes unnecessary reactant And impurity；
D. by the magnetic fluid after washing be dispersed in the 70mL containing 350mg cetyl trimethylammonium bromides (CTAB) go from In the mixed solution of sub- water and 70ml ethanol, the concentrated ammonia liquor that 1.5ml concentration is 28% is then added, carries out ultrasonic disperse 10min, 0.2ml tetraethyl orthosilicates (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 ethanol for 3~4 times repeatedly, it is stand-by；
E. magnetic Nano microsphere made from 100mg steps d is taken, 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 is controlled in 200rpm/min, reacts 6h, then With alternately washing 3 times of 5ml ethanol and 5ml water, repeat step e is tested 2 times, up to Fe3O4@mSiO2Nanoparticle, its infrared figure In spectrum such as Fig. 3 shown in (a), its ultraviolet phenogram is as shown in figure 11, its charge pattern is as shown in figure 12.
Step 3：Prepare Fe3O4@mSiO2-NH2Nanoparticle
Take 80mg step 2 gained Fe3O4@mSiO2Nanoparticle 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, then adds 1ml 3- aminopropyl triethoxysilanes (APTES), mechanical agitation 4h, then with alternately washing 3 times of 3ml ethanol and 3ml water, is collected, up to Fe with magnet adsorption3O4@ mSiO2-NH2In nanoparticle, its infared spectrum such as Fig. 3 shown in (c), its charge pattern is as shown in figure 12.
Step 4：Prepare Fe3O4@mSiO2- FA nanoparticles
0.1g folic acid is dissolved into the DMSO of 30ml, then adds EDC.HCl the and 0.1151g sulfo- of 0.0575g NHS (N- hydroxy thiosuccinimides), is passed through nitrogen, closed under nitrogen atmosphere that 3h is stirred at room temperature, and treats that folic acid fully activates The folic acid solution that must be activated；
Take 25mg step 3 gained Fe3O4@SiO2-NH2Nanoparticle 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), centrifuges and collects obtained Fe3O4@mSiO2- FA receives In meter Wei Qiu, its infared spectrum such as Fig. 3 shown in (b), for its aperture figure as shown in fig. 6, its ultraviolet phenogram is as shown in figure 11, it is electric Lotus figure is as shown in figure 12.
Step 5：Prepare Fe3O4@mSiO2- FA-DNM nanoparticles, i.e.,：Immobilized daunomycin (DNM)
Take 10mg step 4 gained Fe3O4@mSiO2- FA nanoparticles add 5ml DNM solution (concentration in test tube 0.65mg/ml), be placed on shaking table (180rp/min) immobilized 24 it is small when, be then centrifuged for supernatant, measure the concentration of supernatant, count Calculate the amount of immobilized medicine.Immobilized rate is 12.81% in the present embodiment；Magnetic fluid supernatant after will be immobilized refunds returnable bottle, Then clean magnetic fluid 2~3 times with 5ml distilled water, Fe is made3O4@mSiO2- FA-DNM nanoparticles, its electron microscope such as Fig. 4 institutes Show.
Step 6：Prepare Fe3O4@mSiO2-FA-DNM-CaCO3Nanoparticle, i.e.,：Carry out CaCO3Closure
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@mSiO2- FA-DNM nanoparticles add 5ml calcium chloride solutions, and it is equal to be put in shaking table shaking It is even, at least shake more than 10 minutes；
C. toward the sodium carbonate liquor of addition 5ml in step b test tubes, added when shaking up manually and (take every drop of addition 1~5 Shake up mode once) until complete, then test tube is put in shaking table and continues to shake up to the reaction was complete, at least react 1 hour；
D. after the reaction was complete, take out test tube, using magnet adsorption standing separation, go supernatant (after closure, the face of supernatant Color is in purple under normal circumstances), then cleaned 2~3 times with 5ml distilled water, obtain Fe3O4@mSiO2-FA-DNM-CaCO3Nanometer is micro- Ball, a diameter of 10~25nm, 10~20nm of diameter of core, the thickness of shell is 3~10nm, its electron microscope is as shown in Figure 5.
The experiment of the amount of 2 immobilized daunomycin (DNM) of embodiment
Take the Fe obtained by 1 step 4 of 10mg embodiments3O4@mSiO2It is molten to add 5ml DNM in test tube for-FA nanoparticles Liquid, the DNM solution concentrations are respectively 0.14,0.33,0.49,0.65,0.86mg/ml, it is immobilized to be placed on shaking table (180rp/min) 24 it is small when, be then centrifuged for supernatant, measure the concentration of supernatant, calculate immobilized rate, find out optimal immobilized concentration.It is specific real Test that the results are shown in Figure 10.
From fig. 10 it can be seen that with the increase of DNM solution concentrations, immobilized rate also accordingly increases, and in concentration Immobilized rate, which gathers way, during 0.86mg/ml has tended to parallel, subsequently takes and is carried out when DNM solution concentrations are 0.65mg/ml It is immobilized.
The sustained release experiment of embodiment 3
By Fe3O4@mSiO2- FA-DNM nanoparticles and Fe3O4@mSiO2-FA-DNM-CaCO3Nanoparticle is respectively placed in In the test tube of PBS buffer solutions for filling 10ml, the pH of the PBS buffer solutions is respectively 5.6 and 7.4；Test tube is put into 37 Persistent oscillation (180rmp/min) on DEG C constant-temperature table, the specific time (1,2,4,6h ... ..) in take out 3ml release liquids, then The fresh PBS buffer solutions of 3ml are separately added into, are measured accumulative under the conditions of the PBS buffer solutions 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 nanoparticles and Fe3O4@mSiO2-FA-DNM-CaCO3Nanoparticle It is more under the conditions of Cumulative release amount is than the PBS buffer solutions of pH=7.4 under the conditions of the PBS buffer solutions of pH=5.6；Identical Under the conditions of PBS buffer solutions, Fe3O4@mSiO2The Cumulative release amount of-FA-DNM nanoparticles compares Fe3O4@mSiO2-FA-DNM- CaCO3Nanoparticle is more, it is seen that CaCO3Plugging effect.
4 cell biological compatibility experiments of embodiment
Fe3O4@mSiO2-CaCO3Nanoparticle and Fe3O4@mSiO2-FA-CaCO3The preparation process of nanoparticle is with reference to real Apply the preparation process of example 1.
Fe3O4@mSiO2-CaCO3Nanoparticle and Fe3O4@mSiO2-FA-CaCO3The cytotoxicity of nanoparticle passes through body Outer MTT experiment detection, specific experiment process are as follows：Hela cells and A549 cells are inoculated with the density in 5000/hole first In 96 well culture plates, using the DMEM culture mediums containing 10% hyclone (FBS) in CO237 DEG C of culture 24h in incubator, so After remove culture medium, add 200 μ g/ml and contain Fe respectively3O4@mSiO2-CaCO3Nanoparticle and Fe3O4@mSiO2-FA-CaCO3 The culture medium of nanoparticle.The nanoparticle solution of various concentrations is prepared by using the serial dilution of DMEM culture mediums, and concentration is 12.5、25、50、100、200μg/ml.After cell and nanoparticle culture 48h, cell is detected under 490nm by mtt assay Survival rate.The results are shown in Figure 8 for specific experiment.
From figure 8, it is seen that Fe3O4@mSiO2-CaCO3When nanoparticle solution concentration reaches 200ug/ml, do not have to cell Show toxicity, Fe3O4@mSiO2-FA-CaCO3When nanoparticle concentration reaches 200ug/ml, Hela cell survival rates reach 78%, the higher cell survival rate of performance, and the survival rate of A549 cells is even more to have reached more than 95%, illustrates nanoparticle To the good biocompatibility of Hela cells and A549 cells.
5 hemolytic test of embodiment
Fresh anti-freezing people 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, by the Fe that 1ml brand-news concentration is 0.125mg/ml and 0.25mg/ml3O4@mSiO2The nanoparticle suspension of-FA is put into 30min in 37 DEG C of water-baths, then it is put into 37 DEG C of water-baths under taking 1m dilute bloods to be added in nanoparticle suspension and shake Middle 60min, 1000*g centrifuge 5min, then remove supernatant, and ultraviolet absorptivity is surveyed at 545nm, calculate the haemolysis of material, as a result As shown in table 1.
Table 1Fe3O4@mSiO2- FA nanoparticle hemolytic experiment results
According to formula hemolysis rate (%)=(sample to be tested absorbance average-negative control absorbance)/(positive control extinction Spend average-negative control group) * 100%, 0.125mg/m, 0.25mg/m Fe of calculating3O4@mSiO2The haemolysis of-FA nanoparticles Rate is respectively 1.97%, 4.21%, meets hemolysis rate<5% requirement, meets the hemolysis rate of medical material<5% requirement.
6 cellular uptake of embodiment is tested
Experimental procedure：Using the cellular uptake situation of Flow Cytometry observation medicament-carrying nano-microsphere.Take the logarithm growth period Uterine cancer cells (Hela) and lung carcinoma cell (A549), after the pancreatin digestion containing EDTA, 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 plate cells In culture plate.Culture plate is placed in containing 5%CO237 DEG C of incubator in be incubated 24h, grow cell attachment.Remove cell liquid, It is separately added into free DNM solution, Fe3O4@mSiO2-DNM-CaCO3Nanoparticle, Fe3O4@mSiO2-FA-DNM-CaCO3Nanometer Microspheres solution, the final concentration of 0.5ug/ml of DNM, be incubated 4h after, culture medium is suctioned out, add PBS solution washing three times, with except Remove free drug molecule and nanoparticle.Each hole adds the pancreatin of 0.5ml, and addition 1ml contains serum free culture system after digesting 1min Base terminates digestion, after piping and druming uniformly, cell suspending liquid is centrifuged 3min under 2000rmp, removes supernatant, adds PBS cleaning 3 Time, then cell suspending liquid is centrifugated into, as being measured in streaming pipe, cell count is 1000.
Experimental analysis：As shown in Figure 13~14, analyzed using Flow Cytometry observation DNM situations about being ingested. Through to Fe3O4@mSiO2-DNM-CaCO3Nanoparticle, Fe3O4@mSiO2-DNM-FA-CaCO3The DNM analyses of nanoparticle load, Understand the Fe through modified with folic acid3O4@mSiO2-DNM-FA-CaCO3Nanoparticle is in the Hela cells that folate receptor-positive is expressed Fe of the intake of DNM apparently higher than no modified with folic acid3O4@mSiO2-DNM-CaCO3Nanoparticle；And correspondingly, Fe3O4@ mSiO2-DNM-FA-CaCO3Nanoparticle and Fe3O4@mSiO2-DNM-CaCO3Nanoparticle is reached in folacin receptor radiolucent table The intake of DNM does not have apparent difference in A549 cells, this explanation Fe3O4@mSiO2-DNM-FA-CaCO3Nanoparticle pair The Hela cells of positive acceptor have targeting.
The measure of 7 cell survival rate of embodiment
Free DNM, Fe are measured by MTT experiment3O4@mSiO2-DNM-CaCO3Nanoparticle and Fe3O4@mSiO2-FA- DNM-CaCO3Nanoparticle is to the antiproliferative effect of Hela cells, in order to detect the nanoparticle of modified with folic acid to Hela cells Targeting, cultivated there is provided 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 cells are inoculated in 96 well culture plates with the density in 5000/hole first, with containing 10% tire ox The DMEM cultures of serum are based on 37 DEG C, 5%CO2Under the conditions of cultivate 24h, then per add in hole various concentrations free DNM, Fe3O4-mSiO2-DNM-CaCO3Nanoparticle and Fe3O4-mSiO2-FA-DNM-CaCO3Nanoparticle, 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, 24h is cultivated, each concentration setting 5 is right According to hole.After drug-treated, cell cultivates 4h with MTT (5mg/mL), is dissolved in after first a ceremonial jade-ladle, used in libation (Formazan) precipitation of formation 150 μ L DMSO, finally measure its absorbance 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-CaCO3Nanoparticle phase Than in Fe3O4@mSiO2-DNM-CaCO3Nanoparticle and free DNM show stronger tumor inhibitory effect, illustrate Fe3O4@ mSiO2-FA-DNM-CaCO3Nanoparticle has Targeting Effect.
In order to further prove the Targeting Effect of folic acid, in the bar containing 1mmol folic acid culture medium and without folic acid culture medium Under part, to Fe3O4@mSiO2-FA-DNM-CaCO3The antitumor activity of nanoparticle carry out adjust it is poor, Fig. 9 b can be seen that without Fe under the culture medium condition of folic acid3O4@mSiO2-FA-DNM-CaCO3The antitumor activity of nanoparticle is than anti-swollen containing folic acid Tumor activity is more notable, because folacin receptor has competitiveness above the culture medium Hela cells containing folic acid, illustrates Fe3O4@ mSiO2-FA-DNM-CaCO3Nanoparticle has folate-targeted and preferably antitumor effect.
Free DNM, Fe3O4@mSiO2-DNM-CaCO3Nanoparticle, Fe3O4@mSiO2-FA-DNM-CaCO3Nanoparticle (under the culture medium condition without folic acid), Fe3O4@mSiO2-FA-DNM-CaCO3Nanoparticle (the culture medium condition containing folic acid Under) MTT experiment IC50 values as is shown in fig. 9 c, it is seen that the Fe in the culture medium for do not contain folic acid3O4@mSiO2-FA-DNM- CaCO3The IC50 value smallers of nanoparticle, have more preferable antitumous effect.
The foregoing is only a preferred embodiment of the present invention, is not intended to limit the invention, for the skill of this area For art personnel, the invention may be variously modified and varied.Within the spirit and principles of the invention, that is made any repaiies Change, equivalent substitution, improvement etc., should all be included in the protection scope of the present invention.

Claims (10)

1. a kind of pH controlled-release targets medicament nano transport agent, it is characterised in that including nanometer transport agent, be immobilized on nanometer fortune Antitumor drug and pH controlled release agents on transmission carrier；
The nanometer transport agent includes the Fe positioned at core3O4Nanoparticle, the mSiO for being coated on nanoparticle surface2、 Fe3O4@mSiO2- the NH of nanoparticle surface modification2Be grafted on-NH2On folic acid.
2. pH controlled-release targets medicament nano transport agent according to claim 1, it is characterised in that the antitumor drug For anthracycline antibiotic or porphyrin；
The anthracycline antibiotic is adriamycin, daunomycin, idarubicin, mitoxantrone or Epi-ADM；
The porphyrin is TMPYP, protoporphyrin, haematoporphyrin or hematoporphyrin monomethyl ether；
The pH controlled release agents are mSiO2、CaCO3。
3. pH controlled-release targets medicament nano transport agent according to claim 1, it is characterised in that the pH controlled-release targets A diameter of 10~25nm of medicament nano transport agent, 10~20nm of diameter of core, the thickness of shell is 3~10nm.
4. a kind of preparation method of pH controlled-release targets medicament nano transport agent, it is characterised in that specifically comprise the following steps：
Step 1：Prepare Fe3O4Nanoparticle：In atmosphere of inert gases, to FeCl3.6H2O and FeCl2.7H2Add in O mixtures Enter and deoxygenate deionized water, heated after stirring and dissolving, rapidly join and pH value in reaction is kept after ammonium hydroxide 10~11, it is sharp after reaction It is spare with deionized water supersound washing until pH value is neutral with the isolated magnetic-particle of magnet adsorption；
Step 2：Prepare Fe3O4@mSiO2Nanoparticle：
The Fe for 2a) taking step 1 to prepare3O4Nanoparticle is placed in dilute hydrochloric acid, and it is neutrality to be washed with water after ultrasonic disperse to pH value；Take The Fe of processing3O4Nanoparticle is added in water/ethanol mixed system, adds ammonium hydroxide, silicic acid is slowly added dropwise under fast stirring Tetra-ethyl ester, reacts to obtain magnetic fluid；
Be dispersed in after 2b) magnetic fluid is alternately washed with deionized water and ethanol containing cetyl trimethylammonium bromide go from In sub- water/ethanol mixed system, then add ammonium hydroxide and carry out ultrasonic disperse, it is anti-that tetraethyl orthosilicate is slowly added dropwise under stirring at room temperature Should, magnetic Nano microsphere is isolated under the action of externally-applied magnetic field, after magnetic Nano microsphere is washed with water and ethanol, is treated 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, step 2c repeated after washing) twice, up to Fe3O4@mSiO2Nanoparticle；
Step 3：Prepare Fe3O4@mSiO2-NH2Nanoparticle：Take step 2 gained Fe3O4@mSiO2Nanoparticle is added to water/second In alcohol mixed system, add ammonium hydroxide and stir evenly, then add 3- aminopropyl triethoxysilanes and carry out mechanical agitation, reacted Cheng Houyong second alcohol and water alternately washs, and is collected with magnet adsorption, up to Fe3O4@mSiO2-NH2Nanoparticle；
Step 4：Prepare Fe3O4@mSiO2- FA nanoparticles：Take step 3 gained Fe3O4@mSiO2-NH2Nanoparticle is dispersed in Slowly it is added drop-wise to after DMSO in the folic acid solution of activation, is centrifuged after being stirred overnight and collect obtained Fe3O4@mSiO2- FA nanometers Microballoon；
Step 5：Prepare Fe3O4@mSiO2- FA-DNM nanoparticles：DNM solution is added into step 4 gained Fe3O4@mSiO2- FA receives Immobilized in meter Wei Qiu, then immobilized rear centrifuge collects bottom material, and Fe is made after being cleaned with distilled water3O4@mSiO2-FA- DNM nanoparticles；
Step 6：Prepare Fe3O4@mSiO2-FA-DNM-CaCO3Nanoparticle：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 be complete, using magnet adsorption standing separation, washed after removing supernatant, obtain Fe3O4@mSiO2-FA-DNM-CaCO3Nanoparticle.
5. preparation method according to claim 4, it is characterised in that atmosphere of inert gases refers in nitrogen in the step 1 During atmosphere is enclosed；
Heating refers to be 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 in the step 1, and specific process for preparation is：The dense ammonia that the concentration for taking 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 and 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.
6. preparation method according to claim 4, it is characterised 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/ethanol mixed system be water/ethanol mixed system that volume ratio is 3/7；
The step 2a) in ammonia concn be 25~28%；
The step 2a) in react reaction time of magnetic fluid is 24h；
The step 2a) in Fe3O4The mass ratio of the reaction of nanoparticle and TEOS is 25:7；
The step 2b) in deionized water/ethanol mixed system be deionized water/ethanol mixed system that volume ratio is 1/1；
The step 2b) in ammonia concn be 28%；
The step 2b) in time of ultrasonic disperse be 10min；
The step 2b) in reaction time be 6h；
The step 2c) in the concentration of ammonium nitrate ethanol solution 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.
7. preparation method according to claim 4, it is characterised in that water/ethanol mixed system is volume in the step 3 Than the water for 1/15/ethanol 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.
8. preparation method according to claim 4, it is characterised in that the preparation of the folic acid solution activated in the step 4 Method is as follows：Folic acid adds EDC.HCl and sulfo-NHS after being dissolved in DMSO, and atmosphere of inert gases is stirred at room temperature, is activated Folic acid solution；
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 nanoparticle and folic acid is 1:4.
9. preparation method according to claim 4, it is characterised in that in the step 5 concentration of DNM solution for 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 24h；
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；
Take addition 1~5 to drip sodium carbonate liquor when adding sodium carbonate liquor when shaking up in the step 6 and shake up speed once Carry out；
The number for going after supernatant to wash in the step 6 is 2~3 times.
10. a kind of any pH controlled-release target medicament nano transport agents of claims 1 to 3 are preparing prevention and treatment cancer Purposes in disease medicine.
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CN109453376A (en) * 2018-11-08 2019-03-12 东南大学 A kind of preparation method of composite magnetic nanometric photosensitizer
CN112957469A (en) * 2021-02-26 2021-06-15 广东药科大学 PH-responsive magnetic nano core-shell drug-loading system and construction method and application thereof
CN114099470A (en) * 2021-12-09 2022-03-01 济南优科医疗技术有限公司 Method for uniformly and slowly releasing liquid magnetic induction medium for treating tumors

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