CN106421823A - Preparation method of amphoteric ion modified ultra-fine iron oxide particles - Google Patents

Preparation method of amphoteric ion modified ultra-fine iron oxide particles Download PDF

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CN106421823A
CN106421823A CN201610954284.7A CN201610954284A CN106421823A CN 106421823 A CN106421823 A CN 106421823A CN 201610954284 A CN201610954284 A CN 201610954284A CN 106421823 A CN106421823 A CN 106421823A
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peg
preparation
oxide particles
amphion
extra small
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史向阳
马丹
罗宇
王悍
陈静文
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Shanghai First Peoples Hospital
Donghua University
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Shanghai First Peoples Hospital
Donghua University
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    • 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
    • A61K49/1836—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 the small organic molecule being a carboxylic acid having less than 8 carbon atoms in the main chain
    • 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
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • 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
    • A61K49/1851—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 an organic macromolecular compound, i.e. oligomeric, polymeric, dendrimeric organic molecule
    • A61K49/1857—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 an organic macromolecular compound, i.e. oligomeric, polymeric, dendrimeric organic molecule the organic macromolecular compound being obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. PLGA
    • A61K49/186—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 an organic macromolecular compound, i.e. oligomeric, polymeric, dendrimeric organic molecule the organic macromolecular compound being obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. PLGA the organic macromolecular compound being polyethyleneglycol [PEG]

Abstract

The invention relates to a preparation method of amphoteric ion modified ultra-fine iron oxide particles. The preparation method comprises the following steps: dissolving trivalent ferric salt in solvents, adding sodium citrate, stirring, adding anhydrous sodium acetate, stirring, reacting for 3 to 4 hours at the temperature of 190 to 200 DEG C, cooling, carrying out centrifugation and drying to obtain ultra-fine Fe3O4 nanoparticles, then dispersing the nanoparticles in ultrapure water, performing ultrasound, and EDC and NHS activation, dropwise adding to the ultrapure aqueous solution of Mal-PEG-NH2, reacting for 60 to 72 hours to obtain an aqueous solution of Fe3O4-PEG-Mal, and then dropwise adding the ultrapure aqueous solution of L-cysteine, reacting for 60 to 72 hours, dialyzing and freeze-drying to obtain the amphoteric ion modified ultra-fine iron oxide particles. The method disclosed by the invention is simple, and the prepared nanoparticles are long in half-life periods of blood in mouse bodies, and can realize blood pool angiography in animal levels and enhance imaging for subcutaneous transplanted tumors T1 of HeLa cells, so that the preparation method of the mphoteric ion modified ultra-fine iron oxide particles has potentials of industrialization and commercialization.

Description

A kind of preparation method of the extra small ferric oxide particles that amphion is modified
Technical field
The invention belongs to the preparation field of magnetic resonance imaging (MRI) contrast preparation, modify particularly to a kind of amphion The preparation method of extra small ferric oxide particles.
Background technology
All the time, malignant tumour all be harm human life number one killer, have the death rate high, refractory treat and dislike The features such as change rapid.Therefore, the early diagnosis of tumour and specific treatment are particularly important.At present, the detection means of tumour Mainly have：Ultrasonic imaging, CT imaging, nuclear medicine (PET or SPECT) imaging and magnetic resonance imaging (MRI).With magnetic resonance skill The development of art, its sweep time is gradually shortened, and resolution ratio gradually steps up, and the detection for small lesion is also more accurate, and this also makes Obtaining mr imaging technique becomes New Type of Diseases detection means developed in recent years.In order to improve the spirit of MRI imaging diagnosis Sensitivity and specificity are it is necessary to select suitable MRI contrast agent.Conventional MRI contrast agent is broadly divided into two classes：One class is T1Plus The MRI contrast agent of power, a class is T2The MRI contrast agent of weighting.So far have lot of documents report and utilize magnetic ferric oxide nano Particle is applied to the diagnosis of cancer as MRI negative contrast medium.However, in blood of human body, calcium ion enrichment region, metal ion Deposition and human tissue injury position are in T2Also occur signal to weaken phenomenon in imaging process and obtain negative contrastographic picture, this Often disturb clinical diagnosis.Therefore, the more desirable exploitation of clinical medicine circle has the T of signal enhancing effect1Contrast preparation.And it is current It is applied to small molecule T of clinical MRI1All there is unsurmountable defect in contrast preparation, as too short in blood circulation time, adhesion protein Matter and be easy to assemble etc..Especially also there is renal toxicity under gadolinium base contrast preparation finite concentration.By contrast, metal or metal oxygen Compound nano particle more has security.
As biological internal MRI opaque contrast medium, extra small Fe3O4Nano particle must have good water-soluble, stable Property, biocompatibility and higher T1Relaxation rate.Sodium citrate is a kind of small carboxylic acid molecules' salt, has three carboxyls, extensive Be applied to the crystal growth inhibitor in nano material building-up process and stabilizer, be not only provided that impact nano-particle colloid The Charge repulsion of stability, also makes the carboxyl functional group of negative electrical charge on nano grain surface band, is the surface of nano particle Multi-functional modification provides feasibility.
Retrieval domestic and foreign literature, does not still find the extra small iron oxide MRI sun modified with regard to amphion Cys Property contrast preparation and its relevant report of internal MRI diagnostic application.
Content of the invention
The technical problem to be solved is a kind of preparation of the extra small ferric oxide particles providing amphion to modify Method, the method process is simple, gently it is easy to operate, cost is relatively low for reaction condition；The Fe of preparation3O4When nano particle can be long Between be stably dispersed in the aqueous solution, be not in agglomeration.
The preparation method of the extra small ferric oxide particles that a kind of amphion of the present invention is modified, including：
(1) trivalent iron salt is dissolved in solvent, adds sodium citrate Na3Cit, stirring and dissolving, it is subsequently adding acetic anhydride Sodium, stirring and dissolving, 190～200 DEG C of solvent thermal reactions 3～4 hours, it is cooled to room temperature, centrifugation, it is dried, obtain extra small four oxidations Three iron nano-particles；Wherein, sodium citrate is stabilizer, and anhydrous sodium acetate is as reaction promoter, anion surfactant；
(2) the extra small ferroferric oxide nano granules in step (1) are dispersed in ultra-pure water, ultrasonic, through EDC and NHS activates, by the Fe after activation3O4Solution is added dropwise to Mal-PEG-NH2Ultra-pure water solution in, room temperature reaction 60～72 Hour, obtain Fe3O4The aqueous solution of-PEG-Mal；
(3) Cys are dissolved in ultra-pure water, are added dropwise to the Fe in step (2)3O4- PEG-Mal's is water-soluble In liquid, react 60～72 hours under room temperature, dialysis, freeze-drying, obtain the extra small ferric oxide particles Fe of amphion modification3O4- PEG-(L-Cysteine).
In described step (1), trivalent iron salt, the ratio of solvent, sodium citrate and anhydrous sodium acetate are 1.081～1.09g： 38～40mL：0.47～0.50g：1.312～1.33g.
Described trivalent iron salt is ferric chloride hexahydrate, and solvent is diglycol DEG.
After adding sodium citrate in described step (1), the condition of stirring is：Lower 80 DEG C of air atmosphere stirs 1～2 hour.
In described step (1), the container of solvent thermal reaction is the autoclave of 50ml.
In described step (1), the condition of centrifugation is：8500～9000rpm is centrifuged 10～15 minutes, abandons supernatant, with anhydrous Ethanol back dissolving, 8500～9000rpm is centrifuged 10～15 minutes, repeats 2～3 times.
In described step (1), dry condition is：60～65 DEG C of drying.
Fe in described step (2)3O4, EDC and NHS mass ratio be 160～168：134～144：65～70.
Fe in described step (2)3O4And Mal-PEG-NH2Mass ratio 55～60：9～12.
PEG one end in described step (2) is NH2(amino) other end is Mal (dimaleoyl imino)；Mean molecule quantity For 2000.
Fe in described step (3)3O4The mol ratio of-PEG-Mal and Cys is 1.5～2：1～1.5.
In described step (3), the condition of dialysis is：Dialysed 2～3 days with the bag filter that molecular cut off is 8000～14000 (every time distilled water 1.5～2L used by dialysis, change altogether water 6～9 times).
In described step (3), the particle diameter of the extra small ferric oxide particles that amphion is modified is 2～3nm.
The present invention adopts amphion Cys (L-Cysteine) to extra small Fe3O4Nano particle is modified, and imitates The amphion of raw membrane structure forms the hydrated sheath of protectiveness in nano grain surface, can give nano-particle good dividing Scattered stability and biocompatibility.Cys, as a kind of natural small molecule amino acid, make through bisexual ion purification Inorganic nano-particle after surface modification hardly increases size, and gives its excellent high salt tolerance concentration, anti acid alkali performance, anti-albumen Matter absorption and anti-macrophage endocytosis.These characteristics compensate for magnetic Fe just3O4Nano particle is easy to adhesion protein, assembles The shortcoming short with circulation time in vivo, make nano particle can long-acting circulation in vivo, be progressively enriched in lesions position.This Plant special nano-interface biomedical sector is had great importance.
Present invention employs similar method (a step solvent-thermal method) and synthesize and there is the extra small of good colloidal stability Fe3O4Nano particle.Subsequently, in Fe3O4NH is passed through on the surface of nano particle2- PEG-Mal modifies amphion Cys, Obtain and there is excellent anti acid alkali performance, the nano particle of anti-protein absorption and anti-macrophage phagocytic, make extra small Fe3O4Receive The blood circulation time of rice grain greatly extends, and can apply to T1Enhanced blood pool MRI and the diagnosis of tumour MRI.
The present invention is easy to operation, and the cost of raw material is low；It is steady that the nano particle of preparation has good water solubility, colloid Qualitative and biocompatibility.With traditional polymer (NH2- mPEG) Fe that modifies3O4Magnetic nanoparticle, as comparison, is compared Under, bisexual ion purification modify after Fe3O4Nano particle shows excellent anti-macrophage phagocytic, anti-protein adsorption performance, Blood circulatory half-life increases, it is possible to achieve the imaging of magnetic resonance blood pool and tumor imaging.The Cys of the method preparation are repaiied The extra small ferric oxide nanometer particle of decorations has potential using value in MRI molecular image diagnostic field.
The present invention use NMR spectrum (1H NMR), infrared (FTIR), thermogravimetric analysis (TGA), inductive etc. from The methods such as daughter atom emission spectrometry (ICP-OES), Zeta electric potential and hydration particle diameter (DLS) characterize the magnetic Nano of preparation The physics of grain and chemical property.The impedance albumen subsequently passing through the nano particle that ultraviolet colorimetric evaluation Cys are modified is inhaled Attached ability, and the T of nano particle is measured using MRI imager1Imaging performance and r1Relaxation rate, then passes through hemolytic experiment, CCK- The blood compatibility of 8 methods and fluorescence microscope method evaluation nano particle and cytotoxicity, recycle prussian blue staining method Evaluate the ability of nano particle impedance macrophage (RAW 264.7) endocytosis with ICP-OES.Finally, inspection is tested using internal MRI Survey the blood pool radiography of extra small ferric oxide nanometer particle and the diagnosing tumor effect that Cys are modified.
Beneficial effect
(1) present invention adopts the stable extra small Fe of the water-soluble good sodium citrate of a simple step solvent-thermal method preparation3O4 Nano particle, then modifies amphion Cys, the Fe obtaining in nano grain surface3O4-PEG-(L-Cysteine) Nano particle is applied to MR imaging opaque contrast medium；This method operating procedure is simple, and reaction condition benign environment close friend is it is easy to operate Separate, there is the business-like prospect of enforcement；
(2) Fe of present invention preparation3O4Nano particle can be stably dispersed in for a long time in water be not in reunite or Deposited phenomenon；Sodium citrate increased Fe3O4The stability of nano particle, modifies amphion Cys, in Fe3O4Receive The hydrated sheath of rice grain surface formation protectiveness, the imparting anti-non-specific adsorption of nano particle, the performance of anti-macrophage phagocytic, Extend blood circulatory half-life；These advantages make Fe prepared by the present invention3O4- PEG- (L-Cysteine) nano particle can It is effective as MR imaging opaque contrast medium, the blood vessel in enhancing animal body, the contrast of tumor locus imaging.
Brief description
Fig. 1 is the proton nmr spectra spectrogram of the PEG- (L-Cysteine) of embodiment 1 preparation；
Fig. 2 is Fe in embodiment 13O4(a)、Fe3O4-mPEG(b)、Fe3O4The infrared light of-PEG- (L-Cysteine) (c) Spectrogram；
Fig. 3 is Fe in embodiment 23O4(a)、Fe3O4-mPEG(b)、Fe3O4The thermogravimetric of-PEG- (L-Cysteine) (c) is divided Analysis figure；
Fig. 4 is Fe in embodiment 23O4-mPEG、Fe3O4The ultraviolet colorimetric method of-PEG- (L-Cysteine) nano particle is divided Analysis figure；
Fig. 5 is Fe in embodiment 23O4-mPEG、Fe3O4The T of-PEG- (L-Cysteine) nano particle1Relaxation time is reciprocal Linear relationship chart with Fe concentration；
Fig. 6 is Fe in embodiment 23O4- PEG- (L-Cysteine) (a) and control group material Fe3O4- mPEG (b) nanometer Grain MR T for 0.05-0.8mM in concentration of iron1Weighted imaging；
Fig. 7 is Fe in embodiment 33O4- PEG- (L-Cysteine) and control group material Fe3O4- mPEG in concentration of iron is Hemolytic experiment result under 10-200 μ g/mL；Wherein, (a) is hemolysis rate column analysis chart, and (b) is the haemolysis picture after centrifugation；
Fig. 8 is that in embodiment 4, CCK-8 method records L929 cell through PBS (comparison), Fe3O4-PEG-(L- ) and Fe Cysteine3O4- mPEG process 24 hours under the conditions of concentration of iron 10-100 μ g/mL after cell viability；
Fig. 9 be in embodiment 4 L929 cell through PBS (comparison a), Fe3O4- mPEG (b-e) and Fe3O4-PEG- (L-Cysteine) (f-i) to process 24 under the conditions of concentration of iron 10 (b, f), 25 (c, g), 50 (d, h) and 100 (e, i) μ g/mL little When after Fluorescence microscopy Cells shape appearance figure；
Figure 10 be in embodiment 5 RAW264.7 cell through PBS (a, e), Fe3O4- mPEG (b-d) and Fe3O4- After PEG- (L-Cysteine) (f-h) is processed 4 hours under the conditions of concentration of iron 25 (b, f), 50 (c, g) and 100 (d, h) μ g/mL Result after prussian blue staining；The scale of in figure is 100 μm；
Figure 11 be in embodiment 5 RAW264.7 cell through PBS, Fe3O4- mPEG and Fe3O4-PEG-(L- Cysteine collect cell after) processing 4 hours when Fe concentration is 5-100 μ g/mL, surveyed using ICP-OES after chloroazotic acid digestion Surely obtain each intracellular iron content result；
Figure 12 is the Fe that in embodiment 6, tail vein injection prepares3O4- PEG- (L-Cysteine) nano particle and right According to material Fe3O4- mPEG (Fe concentration be 0.1M, 450 μ L) afterwards in different time points rat blood Fe concentration matching drug metabolism Figure；
Figure 13 is that in embodiment 7, tail vein injection prepares (a) Fe3O4- PEG- (L-Cysteine) nano particle and (b) control group material Fe3O4- mPEG (Fe concentration is 0.1M, 450 μ L) different time points rat aorta T afterwards1Weighting MR imaging Picture；
Figure 14 is the Fe that in embodiment 7, tail vein injection prepares3O4- PEG- (L-Cysteine) nano particle and right According to group material Fe3O4- mPEG (Fe concentration is 0.1M, 450 μ L) different time points rat aorta MRI snr value change post afterwards Shape figure；
Figure 15 is that in embodiment 8, tail vein injection prepares (a) Fe3O4- PEG- (L-Cysteine) nano particle and (b) control group material Fe3O4The T of-mPEG (Fe concentration be 0.1M, 150 μ L) different time points nude mouse tumor afterwards1Weighting MR imaging Picture；
Figure 16 is the Fe that in embodiment 8, tail vein injection prepares3O4- PEG- (L-Cysteine) nano particle and right According to group material Fe3O4The MRI snr value of different time points nude mouse tumor changes post to-mPEG (Fe concentration is 0.1M, 150 μ L) afterwards Shape figure；
Figure 17 is the Fe that in embodiment 8, tail vein injection prepares3O4- PEG- (L-Cysteine) nano particle 4,8 He After 12 hours, distribution results in each internal organs in nude mice body for the nano particle；
Figure 18 is preparation method schematic diagram of the present invention.
Specific embodiment
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention Rather than restriction the scope of the present invention.In addition, it is to be understood that after having read the content of present invention instruction, people in the art Member can make various changes or modifications to the present invention, and these equivalent form of values equally fall within the application appended claims and limited Scope.
Embodiment 1
1.09g ferric chloride hexahydrate is dissolved in 40mL diglycol (also known as diethylene glycol (DEG), DEG), then will 0.47g sodium citrate (Na3Cit) it is dissolved in above-mentioned solution, and in the lower 80 DEG C of stirrings of air atmosphere 1 hour, treat that sodium citrate is complete Again the anhydrous sodium acetate of 1.312g is added in above-mentioned solution after CL, continues stirring until sodium acetate powder and be completely dissolved, Then solution is transferred in the autoclave of 50mL, reacts 4 hours in 200 DEG C；After reaction terminates, naturally cool to room Temperature, product is transferred to 8500rpm in 50mL centrifuge tube and is centrifuged 15 minutes, abandon supernatant, use absolute ethyl alcohol back dissolving, 8500rpm Centrifugation 15 minutes, repeats 3 times, then in 60 DEG C of drying, sediment is obtained extra small ferroferric oxide nano granules, i.e. table The stable extra small ferric oxide nanometer particle of face sodium citrate.
Respectively by Mal-PEG-NH2(10mg) and Fe3O4Nano particle (56mg) is scattered in ultra-pure water (5ml) and ultrasonic 10 minutes, activate Fe using EDC (144mg) and NHS (70mg)3O4Solution 3 hours, by the Fe after activation3O4Solution is added dropwise over To Mal-PEG-NH2In (aqueous solution), react 72 hours under room temperature, obtain Fe3O4The aqueous solution of-PEG-Mal；Again by L- half Guang Propylhomoserin (2mg) is dissolved in ultra-pure water, is then added dropwise over Fe3O4In the solution of-PEG-Mal, reaction 72 hours under room temperature, instead Being dialysed 3 days with the bag filter that molecular cut off is 8000-14000 after should terminating, (distilled water 2L used by dialysis, changes water 9 altogether every time Secondary), then vacuum freeze drying, obtain product Fe3O4-PEG-(L-Cysteine).In order to estimate Cys at PEG end The modification amount of base, passes through1H NMR detects NH2- PEG- (L-Cysteine) in deuterated water hydrogen spectrum (as Fig. 1) it is known that, NH2- The spectral peak that PEG- (L-Cysteine) occurs in 2-4ppm proves that Cys can be connected to by the method being stirred at room temperature On PEG, and calculated by integrated peak areas, L-Cysteine can efficiently be coupled at Mal-PEG-NH2On：Each 0.9 Cys molecule is connected on PEG.
The Fe preparing3O4Nano grain surface has substantial amounts of sodium citrate, has higher negative electrical charge, and this makes to receive Produce mutual repulsive interaction between rice grain and there is good colloidal stability.Surface potential and hydration particle size determination result As shown in table 1：The stable Fe of the sodium citrate that obtained with solvent structure3O4Surface potential and hydration particle diameter be respectively- 30.2mV and 30.9nm.
Table 1.Fe3O4、Fe3O4- mPEG and Fe3O4The potential of-PEG- (L-Cysteine) and hydrodynamic diameter
Embodiment 2
The Fe of Example 1 preparation respectively3O4、Fe3O4- PEG- (L-Cysteine) nano particle and comparative example 1 are prepared into The Fe arriving3O4- mPEG nano particle 2mg is dissolved in ultra-pure water, obtains nano particle suspension, ultrasonic uniformly, survey surface potential and Hydration particle diameter.Test result indicate that (table 1), the Fe preparing3O4、Fe3O4- PEG- (L-Cysteine) and Fe3O4- mPEG receives The surface potential of rice grain is respectively -39.7, -15.7 and -16.4mV；Hydration particle diameter is respectively 30.9,116.2 and 93.6nm. Draw from experimental result, extra small ferric oxide nanometer particle is modifying NH2- PEG- (L-Cysteine) and mPEG-NH2Surface afterwards Potential raises, and hydrodynamic diameter increases, and is mainly caused by the surface modification of PEG or PEG/L-Cysteine.Surface potential With hydration particle diameter change explanation mPEG and PEG- (L-Cysteine) modified to ferric oxide nanometer particle surface.
Weigh the bi-material that embodiment 1 prepares respectively：Fe3O4、Fe3O4- PEG- (L-Cysteine) and comparative example The control group material Fe that 1 obtains3O4- mPEG 2mg carries out examination of infrared spectrum (as shown in Figure 2) and thermogravimetric analysis (as Fig. 3 institute Show).By parsing infared spectrum (as Fig. 2), 3436cm in curve a, b, c-1The peak at place is the flexible of OH on the hydrone of absorption Vibration peak, 2900cm-1And 2850cm-1Neighbouring characteristic absorption peak belongs to the stretching vibration of methylene on sodium citrate.Simultaneously In 1396-1642cm-1(stretching vibration of C=O) and 1064cm-1The peak at (stretching vibration of C-O-C) place, all in Fe3O4、 Fe3O4- mPEG and Fe3O4Embodied on-PEG- (L-Cysteine) sample.And 1433cm in Fig. 2 c-1Neighbouring strong characteristic peak Belong to Fe3O4S-C key on-PEG- (L-Cysteine)；466-601cm-1The characteristic absorption peak of upper appearance is Fe3O4Upper Fe- The stretching vibration (Fig. 2 a, b, c) of O.Infrared spectrogram result shows that there are sodium citrate, L- half Guang ammonia in extra small ferroso-ferric oxide surface Acid and the presence of PEG.Additionally, TGA test result shows, Fe3O4Weight loss be 60.8% (Fig. 3 a), Fe3O4- mPEG and Fe3O4The weightlessness of-PEG- (L-Cysteine) is 52.8% (Fig. 3 b) and 50.7% (Fig. 3 c) respectively, goes out PEG by this quantitative analysis It is connected to Fe with Cys3O4Nano particle ratio is respectively 8% and 2.1%.
Fe by colorimetrically analysing embodiment 1 preparation3O4Prepared by-PEG- (L-Cysteine) nano particle and comparative example 1 Fe3O4The anti-protein adsorption performance (Fig. 4) of-mPEG nano particle, under conditions of 37 DEG C, by Fe concentration be respectively 50,100, 500th, the Fe of 1000 μ g/mL3O4- PEG- (L-Cysteine) and Fe3O4- mPEG nano particle BSA (the ox blood with 10% respectively Albumin) it is incubated 2 hours altogether, subsequently centrifugation (8000rpm, 5 minutes) separates.Use ultraviolet-visible spectrophotometry test protein The stoste and centrifuged supernatant protein absorbance at 280nm, the reduced value of absorbance represents the adsorbance of protein. As can be seen from the figure with the rising of concentration of iron (50-1000 μ g/mL), Fe3O4- PEG- (L-Cysteine) and Fe3O4- MPEG nano particle increases to the absorption of protein, under same concentrations, Fe3O4- PEG- (L-Cysteine) nano particle adsorbs Protein be considerably less than control group.Result explanation prepares Fe3O4- PEG- (L-Cysteine) nano particle has well Anti- protein adsorption performance.
r1Relaxation rate reflects Fe3O4Nano-particle, as the efficiency of MRI contrast agent, is that the longitudinal direction of unit molar concentration iron is relaxed The Henan time, can be by the T under variable concentrations1The Fitting Calculation reciprocal in relaxation time obtains.Fe by preparation3O4-PEG-(L- Cysteine) (embodiment 1) nano particle and control material Fe3O4- mPEG (comparative example 1) is recorded molten by ICP-OES method of testing The concentration of Fe element in liquid, then prepare, with ultra-pure water, the aqueous solution 2mL that Fe concentration is followed successively by 0.1,0.2,0.4,0.8 and 1.6mM, Measure the T under different Fe concentration1Relaxation time (as shown in figure 5, Fe3O4- PEG- (L-Cysteine) nano particle and control group Material Fe3O4The T of-mPEG1The relaxation time Linear Fit Chart with Fe concentration reciprocal) and T1Weighted imaging (as shown in Figure 6).Relax Henan rate test result shows Fe3O4- PEG- (L-Cysteine) and Fe3O4The T of-mPEG nano particle1Relaxation time, inverse was in Fe Concentration is that in the range of 0.1-1.6mM, the increase with concentration of iron has good linear relationship.And by can be calculated Fe3O4- PEG- (L-Cysteine) nano particle and control material Fe3O4The r of-mPEG1Relaxation rate is respectively 1.2mM-1s-1And 0.9mM- 1s-1.Therefore, the Fe of preparation3O4- PEG- (L-Cysteine) nano particle and control material Fe3O4- mPEG nano particle As the excellent T in the diagnosis of MRI molecular imaging1Signal enhancing contrast preparation.As can be seen from Figure 6 with Fe concentration (0.05- Raising MRI signal 0.8mM) gradually strengthens, and in good gradient relation, it is good that test result illustrates that this material has MRI imaging capability.
Embodiment 3
Due to the method for administration of contrast preparation be in most cases enter via intravenous injection mode internal.Therefore, radiography Agent will certainly be with blood directly contact, and the intervention of contrast preparation can or can not produce haemolysis or other ill symptomses become as scientific research One of key factor that worker has to take into account that.Nano particle in order to ensure present invention preparation can be safely used for giving birth in vivo Thing imaging diagnosis, have rated the Fe preparing3O4- PEG- (L-Cysteine) nano particle and control material Fe3O4-mPEG Blood compatibility.Concentration of iron according to the bi-material measuring in embodiment 2 calculates and weighs Fe3O4-PEG-(L- Cysteine) nano particle (embodiment 1) and control material Fe3O4Two kinds of each 1mg of ferro element total amount in-mPEG (comparative example 1) Nano particle, be scattered in respectively be configured in PBS 1mg/mL concentration be mother liquor, then with PBS successively compound concentration be 10 μ g/ ML, 20 μ g/mL, the nano granule suspension of 50 μ g/mL, 100 μ g/mL and 200 μ g/mL.Take appropriate people's new blood, first Supernatant is removed in centrifugation (2000rpm, 5 minutes), then washs erythrocyte 5 times with PBS, and the red blood cell collecting health is used in combination PBS dilutes 10 times.Again by Fe3O4- PEG- (L-Cysteine) and Fe3O4- mPEG nano material (10-200 μ g/mL) is thin with red After 1 hour, 10000rpm is centrifuged 1 minute born of the same parents' mixing standing, takes pictures (Fig. 7 b) and survey the ultraviolet absorption value of supernatant.This process with , as positive control, PBS is as negative control for ultra-pure water.Fe is shown in Fig. 73O4- PEG- (L-Cysteine) and Fe3O4- MPEG given concentration of iron be 10 μ g/mL, the hemolytic test knot under 20 μ g/mL, 50 μ g/mL, 100 μ g/mL and 200 μ g/mL Really.By measuring the hemolytic of the absorbance quantitative assessment nano material of supernatant liquor.As Fig. 7 a shows, reach in concentration During 200 μ g/mL, Fe3O4- PEG- (L-Cysteine) and Fe3O4The hemolysis rate of-mPEG is both less than 5%, and preparation these are described Nano material has good blood compatibility, thus can be safely used for biological internal MR imaging.
Embodiment 4
The Fe being prepared for model cell evaluation with L929 cell3O4- PEG- (L-Cysteine) nano particle and control material Fe3O4The impact to the cell survival for-mPEG.Weigh the Fe of corresponding weight3O4- PEG- (L-Cysteine) nano particle (embodiment 1) and control material Fe3O4- mPEG (comparative example 1) dry powder (content of bi-material ferro element is 1mg), is dispersed in aseptic PBS It is configured to the PBS solution of 1mg/mL, and overnight sterilized with ultraviolet irradiation.Then prepared dense in superclean bench with aseptic PBS Spend the Fe for 10,25,50 and 100 μ g/mL3O4- PEG- (L-Cysteine) and Fe3O4- mPEG nano granule suspension.L929 Cell seeding after 96 orifice plates respectively with Fe3O4- PEG- (L-Cysteine) and Fe3O4- mPEG nano particle (concentration be 10, 25th, 50 and 100 μ g/mL) co-culture 24 hours at 37 DEG C.Then, add 20 μ L CCK-8 in cultivation plate hole, continue 37 After cultivating 4 hours at DEG C, discard nutrient solution, and add 100 μ L DMSO, vibration measures light absorption value after 20 minutes at 450nm, On the basis of the absorption value of buffer solution PBS group, the absorption value after the material process of variable concentrations is calculated L929 by comparison The survival rate (as Fig. 8) of cell.(PBS processes groups of cells), Fe compared with control group3O4- PEG- (L-Cysteine) and Fe3O4- MPEG does not have significant difference to the survival rate of L929 cell in the range of experimental concentration 0～100 μ g/mL, and cell survival rate is all More than 80%.This absolutely proves the Fe of synthesis3O4- PEG- (L-Cysteine) and Fe3O4- mPEG is respectively provided with good cell Compatibility, may apply to biological internal MRI image checking.Meanwhile, dyeed using Calcein-AM fluorometric reagent and pass through glimmering Light microscope observation verifies whether material has an impact to the pattern of cell further.As shown in figure 9, the Fe of variable concentrations3O4- PEG- (L-Cysteine) and Fe3O4- mPEG nano material (10,25,50 and 100 μ g/mL) process 24 hours after cellular morphology Cell after processing with PBS is compared, and does not change significantly, further illustrates the good cell compatibility of the material of synthesis.
Embodiment 5
It should can escape when being used for biological internal by the ferric oxide nanometer particle that amphion Cys are modified The phagocytosis of macrophage, thus extending circulation time in vivo, is progressively enriched in focus, improves blood pool imaging and tumor imaging effect Really.Detect cell after nano particle and RAW 264.7 co-culture of cells 4 hours of variable concentrations by prussian blue staining method Prussian blue staining (as Figure 10) is evaluating Fe3O4The macrophage endocytosis effect of-PEG- (L-Cysteine) nano particle.RAW 264.7 cells are with 2 × 105Individual/hole is planted in 12 orifice plates, after incubated overnight more respectively with Fe in embodiment 13O4-PEG-(L- Cysteine the Fe) and in comparative example 13O4- mPEG nano particle (Fe concentration is 25,50 and 100 μ g/mL) is trained at 37 DEG C altogether Support 4 hours, and with the cell of PBS process as a control group.After co-cultivation, cell PBS washes three times, then is digested simultaneously with pancreatin Centrifugation, abandons supernatant, cell is suspended in 1mL PBS.Phase contrast microscope Taking Pictures recording is used, according to thin after prussian blue staining Born of the same parents dye blueness the depth come qualitative analysis cell swallow nano particle number.In Fig. 10 with the raising of Fe concentration, with The dyed rear blueness of cell that nano particle co-cultures gradually is deepened, and illustrates that macrophage to the phagocytosis of two kinds of nano particles is in all Concentration dependent, and under identical Fe concentration, compared with control group, Fe3O4The cell that-PEG- (L-Cysteine) organizes is blue Substantially more shallow.This explanation Cys imparts the performance of the higher anti-macrophage phagocytic of nano particle after modifying.Additionally, ICP-OES technology quantitative analysis each cell average is also adopted to swallow Fe3O4- PEG- (L-Cysteine) and Fe3O4-mPEG The quality of nano particle, the processing mode of cell is consistent with prussian blue staining.When the nano particle of variable concentrations is common with cell After culture 24 hours, with PBS washed cell 3 times, then pancreatin digestion, count, finally use chloroazotic acid vitellophag, use ICP-OES Test the total amount that every hole swallows ferro element, then divided by cell number, draw each cell phagocytosis iron content.As shown in figure 11, exist Under identical Fe concentration, cell is to Fe3O4The endocytosis amount of-PEG- (L-Cysteine) nano particle is considerably less than to Fe3O4-mPEG The endocytosis amount of nano particle, further demonstrates Fe3O4- PEG- (L-Cysteine) has certain biological pollution resistance.These knots Fruit explanation magnetic Fe3O4Nano particle can carry out bisexual ion purification modification by Cys, thus greatly reducing huge biting carefully The non-specific endocytosis to nano particle for the born of the same parents.
Embodiment 6
Fe by preparation3O4- PEG- (L-Cysteine) (embodiment 1) and control material Fe3O4- mPEG (comparative example 1) presses It is configured to the 450 μ L PBS dispersion liquids that Fe concentration is 0.1M, inject metabolic half life in evaluating blood by rat tail vein (as Figure 12).Different time points (0,0.5,1,2,4,8,12,24,48 and 72 hours) after injection, quiet by rat eye socket By ICP-OES, the blood sampling of arteries and veins hole simultaneously detects that in blood, Fe constituent content is come the matching drug metabolism half-life.As Figure 12 shows, not Same time point, injects Fe3O4In the rat blood of-PEG- (L-Cysteine) nano particle, the content of Fe is all higher than control group.Warp Over-fitting obtains, Fe3O4Half-life (6.2 hours) in rat body for-PEG- (L-Cysteine) nano particle is much larger than Fe3O4Half-life (2.3 hours) in the rat body for-mPEG, and the superparamag-netic iron oxide stable much larger than citric acid Blood circulatory half-life (0.6-1.3 hour) (C.Corot et al./Advanced Drug Delivery Reviews 58(2006)1471–1504).With traditional polymer-modified compared with, amphion Cys can greatly prolong magnetic Nano particle circulation time in vivo.These results illustrate, the Fe of present invention preparation3O4- PEG- (L-Cysteine) nanometer The blood circulatory half-life of grain is longer, has potential using value, can be applicable to internal blood pool radiography and tumor imaging.
Embodiment 7
Fe by preparation3O4- PEG- (L-Cysteine) (embodiment 1) and control material Fe3O4- mPEG (comparative example 1) presses It is configured to the 450 μ L PBS dispersion liquids that Fe concentration is 0.1M.MR is evaluated by the dispersion liquid that rat tail vein injects nano particle Blood pool contrasting effects (as Figure 13), compared with before injection, tail vein injection Fe3O4- PEG- (L-Cysteine) nano particle Rat aorta substantially brightens, and the contrast of MR imaging persistently strengthens 4.5 hours.And in identical time point, with injection Fe3O4The rat of-mPEG is compared, and injects Fe3O4The rat aorta of-PEG- (L-Cysteine) nano particle is substantially brighter.Say Bright Fe3O4- PEG- (L-Cysteine) nano particle can maintain longer circulation time in the blood vessel.Figure 14 is corresponding injection The rat aorta position MRI signal to noise ratio of time point, in identical time point, injects the Fe of preparation in embodiment 13O4-PEG- (L-Cysteine) signal to noise ratio at the rat aorta position of nano particle, apparently higher than control group, demonstrates above-mentioned conclusion again. These results indicate that the nano particle going out Cys modification has good blood pool contrasting effects, and the imaging of MR blood pool is Detection atherosclerotic, the major way of the disease such as kidney failure and miocardial infarction, therefore, the Fe of preparation3O4-PEG-(L- Cysteine) nano particle has potential using value as mr angiography agent.
Embodiment 8
Fe by preparation3O4- PEG- (L-Cysteine) (embodiment 1) and control material Fe3O4- mPEG (comparative example 1) joins Put the 150 μ L PBS dispersion liquids that Fe concentration is 0.1M.2×106Individual HeLa cell is inoculated in nude mice body, treats that tumour is straight after three weeks When footpath reaches 0.6-1cm, by tail vein injection Fe3O4- PEG- (L-Cysteine) and Fe3O4- mPEG nano particle PBS is molten Liquid is evaluating the MR imaging effect (as Figure 15) of tumor locus.With injection before nude mice compared with, after injection 30 minutes little to 3 When interior, inject Fe3O4The nude mouse tumor position of-PEG- (L-Cysteine) nano particle, progressively brightens and 1.5 little after injection When, reach the brightest, and injects Fe3O4Reaching for 45 minutes after injection of-mPEG is the brightest.Figure 16 is the swollen of corresponding injection time point Knurl position MRI signal to noise ratio, injects Fe3O4The nude mouse tumor position signal of-PEG- (L-Cysteine) nano particle progressively strengthens, After injecting 1.5 hours, signal to noise ratio reaches peak, then slowly declines, and injects Fe3O4The nude mice of-mPEG nano particle is swollen Knurl position signal to noise ratio declines rapidly after reaching peak value, and the nano particle that these results show Cys modification has Enhanced MRI diagnosing tumor effect, can be successfully applied to internal MRI diagnosing tumor.
In order to study biological tissue's distribution situation of nano particle, will be through tail vein injection Fe3O4-PEG-(L- Cysteine nude mice) is put to death and dissects, and the taking-up heart, liver, spleen, lung, kidney, tumour are weighed, and are cut into the fragment of 2 × 2mm, Ran Houyong Chloroazotic acid digests 24 hours, then measures the iron content of each sample with ICP-OES, finally calculates in each vitals The content (Figure 17) of iron.As can be seen from Figure in the Fe of injection present invention preparation3O4(Fe concentration is-PEG- (L-Cysteine) 0.1M, 150 μ L) after nano particle, in liver, spleen and blood, the content of iron all substantially increases before relatively injecting, and in other organs, Such as：The heart, lung, kidney and tumour, the content of iron is less.It should also be noted that growth over time, Fe element is first in blood In liquid, content is higher, then assembles in kidney and spleen, is finally all gradually reduced in each organ.These results not only prove Fe3O4- PEG- (L-Cysteine) nano particle has longer blood circulation time, and receiving of present invention preparation is described Rice grain can in mouse body normal metabolite clearance.
Comparative example 1
Surface modification polymer is conventional magnetic nanoparticle method of modifying, and the nanoparticles stable after modification increases Plus.In order to compare the performance of the nano particle that amphion Cys are modified with traditional polymer, according in embodiment 1 Method and steps synthesis obtains Fe3O4Nano particle.Then by the Fe preparing3O4Nano particle is dispersed in DMSO solvent And so that it is completely dispersed in ultrasonic 10 minutes, respectively the DMSO solution of EDC and NHS is added to above-mentioned Fe3O4In (DMSO solution), Stirring reaction 3 hours, by the Fe after activation3O4Solution is added dropwise to obtain mPEG-NH2Reaction 72 hours in (DMSO solution), Obtain product Fe3O4- mPEG, reacts 3 days (first three use of the bag filter dialysis being 8000～14000 with molecular cut off after terminating PBS is dialysed, every time PBS or distilled water 2L used by dialysis, changes PBS or water 9 times altogether), then Vacuum freeze drying, obtains final product Fe3O4-PEG.Product Fe3O4The sign detailed in Example 2 of-mPEG.

Claims (10)

1. the preparation method of the extra small ferric oxide particles that a kind of amphion is modified, including：
(1) trivalent iron salt is dissolved in solvent, adds sodium citrate, stirring and dissolving, be subsequently adding anhydrous sodium acetate, stirring is molten Solution, 190～200 DEG C of solvent thermal reactions 3～4 hours, cooling, centrifugation, it is dried, obtain extra small ferroferric oxide nano granules；
(2) the extra small ferroferric oxide nano granules in step (1) are dispersed in ultra-pure water, ultrasonic, live through EDC and NHS Change, by the Fe after activation3O4Solution is added dropwise to Mal-PEG-NH2Ultra-pure water solution in, room temperature reaction 60～72 hours, Obtain Fe3O4The aqueous solution of-PEG-Mal；
(3) Cys are dissolved in ultra-pure water, are added dropwise to the Fe in step (2)3O4The aqueous solution of-PEG-Mal In, react 60～72 hours under room temperature, dialysis, freeze-drying, obtain the extra small ferric oxide particles Fe of amphion modification3O4- PEG-(L-Cysteine).
2. the preparation method of the extra small ferric oxide particles that a kind of amphion according to claim 1 is modified, its feature exists In in described step (1), trivalent iron salt, the ratio of solvent, sodium citrate and anhydrous sodium acetate are 1.081～1.09g：38～ 40mL：0.47～0.50g：1.312～1.33g.
3. the preparation method of the extra small ferric oxide particles that a kind of amphion according to claim 1 and 2 is modified, its feature It is, described trivalent iron salt is ferric chloride hexahydrate, solvent is diglycol DEG.
4. the preparation method of the extra small ferric oxide particles that a kind of amphion according to claim 1 is modified, its feature exists In in described step (1), the condition of centrifugation is：8500～9000rpm is centrifuged 10～15 minutes, abandons supernatant, uses absolute ethyl alcohol Back dissolving, 8500～9000rpm is centrifuged 10～15 minutes, repeats 2～3 times.
5. the preparation method of the extra small ferric oxide particles that a kind of amphion according to claim 1 is modified, its feature exists In Fe in described step (2)3O4, EDC and NHS mass ratio be 160～168：134～144：65～70.
6. the preparation method of the extra small ferric oxide particles that a kind of amphion according to claim 1 is modified, its feature exists In Fe in described step (2)3O4And Mal-PEG-NH2Mass ratio 55～60：9～12.
7. the preparation method of the extra small ferric oxide particles that a kind of amphion according to claim 1 is modified, its feature exists In the PEG one end in described step (2) is amino, and the other end is dimaleoyl imino；Mean molecule quantity is 2000.
8. the preparation method of the extra small ferric oxide particles that a kind of amphion according to claim 1 is modified, its feature exists In Fe in described step (3)3O4The mol ratio of-PEG-Mal and Cys is 1.5～2：1～1.5.
9. the preparation method of the extra small ferric oxide particles that a kind of amphion according to claim 1 is modified, its feature exists In in described step (3), the condition of dialysis is：Dialysed 2～3 days with the bag filter that molecular cut off is 8000～14000.
10. the preparation method of the extra small ferric oxide particles that a kind of amphion according to claim 1 is modified, its feature exists In in described step (3), the particle diameter of the extra small ferric oxide particles that amphion is modified is 2～3nm.
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CN107987916A (en) * 2017-11-28 2018-05-04 青岛大学 A kind of preparation method of the rheomagnetic energy material with shear shinning
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* Cited by examiner, † Cited by third party
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CN105664158A (en) * 2016-01-07 2016-06-15 复旦大学 Photoluminescence-photothermal nano composite structural material and preparation method and application thereof
CN107987916A (en) * 2017-11-28 2018-05-04 青岛大学 A kind of preparation method of the rheomagnetic energy material with shear shinning
CN110013559A (en) * 2019-05-14 2019-07-16 东华大学 A kind of extra small ferrum nano material of double-metal hydroxide-of HA targeting and its preparation and application
CN110013559B (en) * 2019-05-14 2021-08-31 东华大学 HA-targeted bimetallic hydroxide-ultra-small iron nano material and preparation and application thereof
CN111358954A (en) * 2020-03-24 2020-07-03 中国人民解放军总医院 Composition with function of targeted adjustment of macrophage polarization and preparation method and application thereof
CN112133553A (en) * 2020-10-30 2020-12-25 福州大学 Continuous preparation method of water-based magnetofluid
CN112133553B (en) * 2020-10-30 2021-12-21 福州大学 Continuous preparation method of water-based magnetofluid

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