CN106310257A - Monodispersed fluorescent magnetic nanometer probe and the preparation and application thereof - Google Patents

Monodispersed fluorescent magnetic nanometer probe and the preparation and application thereof Download PDF

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CN106310257A
CN106310257A CN201610735466.5A CN201610735466A CN106310257A CN 106310257 A CN106310257 A CN 106310257A CN 201610735466 A CN201610735466 A CN 201610735466A CN 106310257 A CN106310257 A CN 106310257A
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magnetic nano
probe
npo
photosensitizer
peg
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CN106310257B (en
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高国
尹婷
张倩
黄鹏
崔大祥
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Shanghai Jiaotong University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
    • A61K41/0071—PDT with porphyrins having exactly 20 ring atoms, i.e. based on the non-expanded tetrapyrrolic ring system, e.g. bacteriochlorin, chlorin-e6, or phthalocyanines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
    • 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
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/10—Organic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/10—Organic compounds
    • A61K49/12—Macromolecular compounds
    • A61K49/124—Macromolecular compounds dendrimers, dendrons, hyperbranched compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/10—Organic compounds
    • A61K49/14—Peptides, e.g. proteins
    • 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
    • A61K49/10—Organic compounds
    • A61K49/14—Peptides, e.g. proteins
    • A61K49/16—Antibodies; Immunoglobulins; Fragments thereof
    • 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
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01G—COMPOUNDS CONTAINING METALS NOT COVERED BY SUBCLASSES C01D OR C01F
    • C01G49/00—Compounds of iron
    • C01G49/02—Oxides; Hydroxides
    • C01G49/08—Ferroso-ferric oxide (Fe3O4)
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
    • C01P2004/00—Particle morphology
    • C01P2004/60—Particles characterised by their size
    • C01P2004/64—Nanometer sized, i.e. from 1-100 nanometer

Abstract

The invention relates to a monodispersed fluorescent magnetic nanometer probe and the preparation and application thereof. The magnetic nanometer probe consists of Fe304 magnetic nanometer particles of load photosensitizer, target molecules modified by PEG and hyperbranched polymer NPO; Molar ratio of Fe304 magnetic nanometer particles of load photosensitizer, target molecules modified by PEG and hyperbranched polymer NPO is (0.0005-1):(1-10):(0.5-2); Fe304 magnetic nanometer particles of load photosensitizer consist of photosensitizer and Fe304 carrier; mass ratio of the photosensitizer and Fe304 carrier is (1-5):(4-20); The nanometer probe is made through the solvothermal method and can be used for making the imaging agent of targeted imaging of cancer cells or drugs for targeted treatment of cancers. Compared with the prior art, the invention has the strengths of simple preparation techniques, high efficiency of targeted treatment, good biologic compatibility, and can improve the efficacy and increase the surface size effect of hydrophobic antineoplastic drugs, and strengthen imaging and treatment.

Description

A kind of single dispersing fluorescence magnetic nano-probe and preparation and application
Technical field
The present invention relates to nano-probe technical field, be specifically related to a kind of single dispersing fluorescence magnetic nano-probe and preparation and Application.
Background technology
Along with the increase of M & M, cancer has become as the dead first cause of Chinese population and main public affairs Health problem altogether.Statistical data shows, China has 4292 in 2015, and 000 example cancer new cases, 2814,000 example cancers are dead Dying, pulmonary carcinoma becomes most common cancer, is also the first cause of cancer mortality.Gastric cancer, the esophageal carcinoma and hepatocarcinoma are also common cancers, Cancer mortality reason is stood out.The position do not specified due to cancer, it can occur any part at internal organs, cancer The symptom of disease and precancerous lesion has invisible and without specificity, and incubation period is longer, once finds reach an advanced stage or shift, Patient is caused to cure and dead.Therefore, early warning and internal cancerous cell spike are most important to patient's incubation period.
Along with the development of Medical Technology in modern times, nano material application in medical science have been enter into one fast-developing time Phase.The especially research at tumor subject is constantly deepened, at image checking, target administration, optical dynamic therapy and photo-thermal therapy etc. Field is with a wide range of applications.Therefore develop diagnosis-treatment integrated multi-functional nano-probe and become a research heat Point.
Nuclear magnetic resonance can be to partes corporis humani position multi-angle, multiple plane imaging, can the most objective more specifically positioning and qualitative disease Stove, diagnosis, early warning and the clinical follow of systemic disease each to whole body all have the biggest value.Hurtless measure relative to other Imaging technique, such as: nuclear medicine, optical imagery, ultra sonic imaging etc., has high spatial resolution, without radiological survey X, soft tissue The advantages such as good contrast, are highly suitable to be applied for the research of molecular imaging.But MR is imaged in lumpectomy procedure along with non- The minimizing of cut-out, the degree of accuracy of its detection is gradually lowered, and check fee is expensive, sweep time is longer, allows patient be difficult to Tolerance.
Excited electronic state photosensitizer can not only produce cytotoxic effect, moreover it is possible to send glimmering when being changed into ground state process Light, forms optical imagery.Therefore photosensitizer can be as photodynamic therapy medication, again can be as image-forming contrast medium.Photosensitizer is glimmering Light detection is the sensitiveest a kind of technology, it is possible to identify the fine structure of tissue, and degree of accuracy will not be along with non-ablated part Minimizing and reduce.But not there is the feature such as high spatial resolution, soft tissue contrast.
Optical dynamic therapy refers under photosensitizer participates in, and under the effect of light, makes organism cell or biomolecule occur Function or metamorphosis, cause cell injury and necrosis.Photodynamic therapy is the new technique of an oncotherapy, owing to it is unique Advantage and good compatibility, new fresh combatants in tumor diagnosis and therapy will be become, at the Comprehensive Treatment of tumor In play a significant role.
At present, there is researcher the advantage that Magnetic Resonance Imaging images with photosensitizer fluorescence to be combined and design fluorescence magnetic nanometer Probe, but its probe performance not formed the probe nuclei of series different-grain diameter contrasts, if the probe of different-grain diameter is in disease The difference that the difference of the distribution in born of the same parents, the difference being distributed in animal body, normal tissue (such as: liver, spleen etc.) damage of attenuating and The aspects such as the holdup time difference of tumor locus are studied.In order to find fluorescence magnetic nano-probe and cell delicate effect machine Reason, the infringement of reduction fluorescence magnetic nano-probe normal tissue (such as: liver, spleen etc.), increased in the holdup time of tumor locus, Improve oncotherapy effect, need the fluorescence magnetic nano-probe of different-grain diameter is compared, fully excavate it and cure at biology The application potential in field.
Summary of the invention
Defect that the purpose of the present invention is contemplated to overcome above-mentioned prior art to exist and provide one to prepare simply, targeting Single dispersing fluorescence magnetic nano-probe that efficiency is high and preparation and application.
The purpose of the present invention can be achieved through the following technical solutions: a kind of single dispersing fluorescence magnetic nano-probe, institute State magnetic Nano probe and include loading the Fe of photosensitizer3O4Magnetic nano-particle, PEG modify target molecule and hyperbranched big point Sub-NPO, the Fe of described load photosensitizer3O4The target molecule of magnetic nano-particle, PEG modification and rubbing of hyperbranched macromolecular NPO Your ratio is (0.0005～1): (1～10): (0.5～2), the Fe of described load photosensitizer3O4Magnetic nano-particle includes photosensitizer And Fe3O4Carrier, described photosensitizer and Fe3O4The mass ratio of carrier is (1～5): (4～20).
The ferroferric oxide magnetic nano-particles that the present invention is little with size, even particle size distribution, magnetic property are excellent is as probe Core, through amphiphilic polymer modified, obtains the carboxylated ferroferric oxide magnetic nano-particles that dispersibility is excellent；Again through PEGization Target molecule and one end be the other branched end of amino be that the hyperbranched macromolecular of hydroxyl is modified, and load photosensitizer drug, i.e. structure Become that preparation technology is simple, targeting efficiency height, good biocompatibility, the curative effect of raising hydrophobic anticancer drug and surface size are imitated Answer the fluorescence magnetic nano-probe of Enhanced Imaging and optical dynamic therapy.
Compare and traditional method, use the ferroferric oxide magnetic nano-particles of series different-grain diameter, pass through covalent coupling By the target molecule of PEGization and one end be the other branched end of amino be that the hyperbranched macromolecular of hydroxyl is attached to magnetic nano particle sublist Face, improves the biocompatibility of magnetic nano-particle and functional.The pharmaceutical carrier obtained (is called for short: Fe3O4@P-NPO/PEG- Glc) shell that in, PEG and hyperbranched macromolecular NPO is constituted stops reunion and the protein adsorption of magnetic nanoparticle, increases magnetic Property nano-particle blood circulation time and the internalization efficiency of internal target cell.Cancer cell surfaces is rich in target molecule receptor, by target Molecule is coupled on nano-probe, can combine the target spot of cancer cell surfaces efficiently, directionally enter cell, spike cancer exactly Cell or the position of tumor, contribute to imaging diagnosis and treatment of cancer.
Described photosensitizer is selected from chlorin e 6, indocyanine green, cyanine 5.5 and amycin one or more；
Described Fe3O4Carrier particle diameter be 3～15nm.
Described target molecule selected from antibody (such as: CD44, CD45, Her2 etc.), antibody fragment, somatomedin (such as: VEGF), In ring-type RGD, octreotide, AP peptide, tLyp-1 peptide, hyaluronic acid HA or 4-aminophenyl β-D-pyranglucoside One or more.These target molecules have selectively targeted effect, have high expressed site on target cell membrane surface, can make Probe medicament identifies efficiently and kills target cell.
The molecular weight of described hyperbranched macromolecular NPO is more than or equal to 5000, and its one end is amino, branched end additionally For hydroxyl.The amino of hyperbranched macromolecular NPO one end and Fe3O4After the carboxyl covalent coupling of@P-COOH nanoparticle surface, shape Become amido link.The hydroxyl of the most branched end promotes water solublity and the biocompatibility of nano-probe.
A kind of preparation method of single dispersing fluorescence magnetic nano-probe as above, including following step:
(1) ferric acetyl acetonade, oleic acid, oleyl amine, hexadecane diol and diphenyl ether are mixed, at N2The lower reaction of protection, reaction Being cooled to room temperature after end, the magnetic nano-particle being filtrated to get is dissolved in chloroform, obtains chloroformic solution, stand-by；
(2) in the chloroformic solution of step (1) gained, add amphipathic nature polyalcohol, evaporate solvent after mix homogeneously, obtain Solid be dissolved in SBB12 buffer solution, this SBB12 buffer solution is that 50mM boric acid solution NaOH solution is adjusted after pH to 12 Solution, the SBB12 buffer solution dissolved with solid is concentrated and centrifugal obtains Fe3O4@P-COOH nanoparticle dispersion liquid；
(3) by Fe3O4Activated carboxylic in@P-COOH nanoparticle dispersion liquid, is subsequently adding the target molecule that PEG modifies, instead Should add hyperbranched macromolecular NPO after a period of time, reaction obtains the Fe of band target molecule and hyperbranched macromolecular3O4@P-NPO/ PEG-TM dispersion liquid；
(4) by step (3) resulting tape target molecule and the Fe of hyperbranched macromolecular3O4@P-NPO/PEG-TM dispersion liquid is with photosensitive Agent solution mixing, oscillating reactions, be then centrifuged for purification, obtain described single dispersing fluorescence magnetic nano-probe.
The mol ratio of step (1) described ferric acetyl acetonade, oleic acid, oleyl amine and hexadecane diol is (0.5～4): (1.5～ 12): (1.5～12): (2.5～20), the volume of described diphenyl ether and the ratio of the mole of described ferric acetyl acetonade are (5～40) ML:(0.5～4) mol；
In two stages, the reaction temperature of first stage is 150～250 DEG C in described reaction, and the response time is 1～3h, the The reaction temperature of two-stage is 280～350 DEG C, and the response time is 0.5～1.5h.
Substep sets reaction temperature so that reacting controlled, product is more uniform.
The synthesis step of step (2) described amphipathic nature polyalcohol is: by poly-(isobutene .-alt-maleic anhydride), lauryl amine With (0.05～1): the mixed in molar ratio of (0.01～2), it is dissolved in oxolane, after ultrasonic 10～20s, is heated to 65 DEG C and stirs Mix 8～12h, then oxolane is evaporated, and be dissolved in chloroform, obtain the amphipathic nature polyalcohol that concentration is 0.1M；Using should Amphipathic nature polyalcohol, can make the Fe of synthesis in oil phase3O4Magnetic nano-particle proceeds to aqueous phase, and makes nanoparticle surface contain There is a large amount of carboxyl, ready for next step synthesis of probe；
Magnetic nano-particle in described chloroformic solution is 1:(5-100 with the mol ratio of addition amphipathic nature polyalcohol)；
The Fe being concentrated to give3O4The concentration of@P-COOH nanoparticle dispersion liquid is 3～20 μMs
One end be the other branched end of amino be that the synthesis step of the hyperbranched macromolecular of hydroxyl is: N-BOC ethylenediamine and contracting Water glycerol is (0.5～1) according to mol ratio: (0.5～1) is mixed in the three-neck flask containing 5～20mL dioxane and utilizes Magnet rotor stirs, and mixture reacts 2-5h at 90 DEG C；After reaction terminates, add 100～200mgNaH (60% mineral oil) anti- Answer 10～30min；Being added dropwise over the 5～20mL dioxane containing 2.1～8.4mL (+)-2,3-Epoxy-1-propanols, mixture reacts at 90 DEG C Overnight, after reaction terminates, pouring out organic facies, surplus materials is dissolved in 5～10mL distilled water, and with bag filter, (molecular retention amount is 3K) dialysing 24～48h, lyophilization obtains transparence gum-like product；Drip two concentrated hydrochloric acid, then the PBS solution adding pH=7.4 is joined Make 0.5～1g/mL solution, must arrive one end be the other branched end of amino be the hyperbranched macromolecular NPO of hydroxyl, standby.
The technique of step (3) described activation is as follows:
At Fe3O4@P-COOH nanoparticle dispersion liquid add EDC/NHS solution, the mole of the EDC/NHS of addition with Fe3O4In@P-COOH nanoparticle dispersion liquid, the ratio of the mole of carboxyl is 1:(1～1.5), then 5～30rpm/min Mixing 20～30min is rotated under rotating speed；
The preparation of PEGization target molecule: add in SBB 9 (being the boric acid solution of 9 with the NaOH titration pH) buffer of PEG EDC/NHS so that it is meeting with the mol ratio of carboxyl on PEG molecule is 1:1.5, at XH-1T type multitube adjustable rotary blender Upper rotation 30min, rotating speed is 30 revs/min；It is 1:(1～3 by the mol ratio of PEG Yu target molecule) add target molecule, 30～40 At DEG C vibrate 1～3h or 20～30 DEG C on rotator rotate 12～24h.
Add PEG modify the post-reacted condition of target molecule be at 30～40 DEG C vibrate 1～3h or 20～30 DEG C at rotation Turn and on device, rotate 12～24h；
Add the post-reacted condition of hyperbranched macromolecular NPO be at 30～40 DEG C vibrate 1～3h or 20～30 DEG C at rotation Turn and on device, rotate 12～24h.Select the reaction condition of above-mentioned gentleness, be beneficial to reaction fully, and do not result in Fe3O4@P-COOH Nanoparticle dispersion liquid is reunited.
The concentration of step (4) described photosensitizing agent solution is 2-5mg/mL；
The addition quality of the photosensitizer added and Fe3O4The ratio of the mole of@P-COOH nanoparticle dispersion liquid is: (2.5- 5) mg:(0.5-10) mmol；
The temperature of described reaction is 30～40 DEG C, and the time is 24～48h, and the speed of vibration is 80～200rpm.
The application of a kind of single dispersing fluorescence magnetic nano-probe as above, described nano-probe is used for preparing targeting As the preparation of cancerous cell or for preparing the medicine of targeted therapy of cancer.
The shell that PEG and hyperbranched macromolecular NPO is constituted stops reunion and the protein adsorption of magnetic nanoparticle, increases The internalization efficiency of magnetic nanoparticle blood circulation time and internal target cell.Cancer cell surfaces, will rich in target molecule receptor Target molecule is coupled on nano-probe, can combine the target spot of cancer cell surfaces efficiently, directionally enter cell, exactly spike Cancerous cell or the position of tumor, contribute to imaging diagnosis and treatment of cancer.
Compared with prior art, beneficial effects of the present invention is embodied in following several respects:
(1) standby simple, fast, low cost, only several frequently seen reagent need to be used to can be prepared by: to pass through amphipathic nature polyalcohol The magnetic nano-particle modified and the hyperbranched macromolecular that the target molecule of PEGization and the other branched end of amino of one end are hydroxyls NPO covalent coupling, can directly prepare the nano-probe that single dispersing biocompatibility is excellent；
(2) the nano-probe average-size prepared is 3.5-20nm, and even particle size distribution；
(3) the nano-probe targeting efficiency prepared is high, improve water solublity and the intake of hydrophobic anticancer drug；
(4) surface size effect strengthens bimodal imaging and optical dynamic therapy effect.
Accompanying drawing explanation
Fig. 1 is the transmission electron microscope picture of the fluorescence magnetic nano-probe of the present invention；
Fig. 2 is the fluorescence spectrum figure of the fluorescence magnetic nano-probe of the present invention；
Fig. 3 is the fluorescent images in animal body of the fluorescence magnetic nano-probe of the present invention；
Fig. 4 is that the fluorescence magnetic nano-probe of the present invention becomes at the nuclear-magnetism of tumor-bearing mice group with blank group tumor locus As photo；
Fig. 5 is the fluorescence magnetic nano-probe treatment curve to tumor-bearing mice tumor of the present invention；
Fig. 6 is the tumor-bearing mice body weight change curve after fluorescence magnetic nano-probe is treated of the present invention.
Detailed description of the invention
Elaborating embodiments of the invention below, the present embodiment is carried out under premised on technical solution of the present invention Implement, give detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following enforcement Example.
Embodiment 1
The preparation of a kind of single dispersing fluorescence magnetic nano-probe, comprises the following steps:
(1) preparation of ferroferric oxide magnetic nano-particles:
By mol ratio it is: ferric acetyl acetonade (III), oleic acid, oleyl amine, hexadecane diol and the 5mL of 0.5:1.5:1.5:2.5 Diphenyl ether is mixed in three-neck flask and utilizes magnet rotor to stir, and mixture is heated to 150 DEG C and persistently adds under nitrogen protection Hot 1h, is subsequently heated to 280 DEG C, and in this thermotonus 0.5h, reaction is cooled to room temperature after terminating, and gained dark solution utilizes Dehydrated alcohol is centrifuged repeatedly 3 times (centrifugal speed 2000rpm, 5min) of precipitation, and final gained precipitation is resuspended in chloroform.
(2) preparation of carboxylated ferroferric oxide magnetic nano-particles:
Take 1mmol poly-(isobutene .-alt-maleic anhydride), 0.2mmol lauryl amine is dissolved in equipped with 10mL oxolane In round-bottomed flask, after ultrasonic 20s, being heated to 65 DEG C and be stirred overnight, after oxolane is evaporated by next day, polymer is dissolved in surely 5mL chloroform obtains the amphiphilic polymers of 0.1M；
Step (1) gained magnetic nano-particle and amphipathic nature polyalcohol are mixed in round-bottomed flask according to mol ratio 1:5, Utilizing Rotary Evaporators to be evaporated by solvent lentamente, the solid obtained is dissolved in the SBB 12 of 3mL under magnetic stirring and buffers In solution, final gained dispersion liquid uses super filter tube to concentrate, and utilizes Ultracentrifuge to remove the most coated polymer, obtains Carboxylated ferroferric oxide magnetic nano-particles, i.e. obtains the Fe of 3 μMs3O4@P-COOH nanoparticle dispersion liquid；
(3) coupling PEGization target molecule and one end be the other branched end of amino be the hyperbranched macromolecular of hydroxyl:
EDC/NHS is added so that it is meet and carboxylic on Fe3O4@P-COOH in Fe3O4@P-COOH nanoparticle dispersion liquid The mol ratio of base is 1:1, rotates 20min on XH-1T type multitube adjustable rotary blender, and rotating speed is 5 revs/min；
PEGization target molecule and one end are that amino additionally holds is that hyperbranched macromolecular NPO and the Fe3O4@P-COOH of hydroxyl receives The process conditions of rice corpuscles dispersion liquid hybrid reaction are: rotate 12h at vibrate at 30 DEG C 1h or 20 DEG C on rotator, its In, the molecular weight of NPO is more than or equal to 5000.
The preparation of PEGization target molecule: add EDC/NHS in SBB 9 buffer of PEG so that it is meet and on PEG molecule The mol ratio of carboxyl is 1:1, rotates 20min on XH-1T type multitube adjustable rotary blender, and rotating speed is 5 revs/min；By PEG It is that 1:1 adds target molecule with the mol ratio of target molecule, on rotator, at vibrate at 30 DEG C 1h or 20 DEG C, rotates 12h.
One end be the other branched end of amino be the preparation of the hyperbranched macromolecular NPO of hydroxyl: N-BOC ethylenediamine with shrink sweet Oil is that 0.5:0.5 is mixed in the three-neck flask containing 5mL dioxane and utilizes magnet rotor to stir according to mol ratio, and mixture exists 2h is reacted at 90 DEG C；After reaction terminates, add 100mgNaH (60% mineral oil) reaction 10min；It is added dropwise over shrinking containing 2.1mL The 5mL dioxane of glycerol, mixture reacts overnight at 90 DEG C, after reaction terminates, pours out organic facies, and surplus materials is dissolved in In 5mL distilled water, with bag filter (molecular retention amount is 3K) dialysis 24h, lyophilization obtains transparence gum-like product；Drip two Concentrated hydrochloric acid, then the PBS solution adding pH=7.4 is configured to 0.5g/mL solution, must arrive one end be the other branched end of amino be hydroxyl Hyperbranched macromolecular NPO, standby.
(4) load photosensitizer drug chlorin e 6:
By the mass ratio of 2.5g:10mmol by photosensitizer drug and Fe3O4@P-NPO/PEG-Glc support dispersion is in 1mL's In deionized water, stirring with the speed of 200rpm and make it fully mix at 30 DEG C, mixing time is 24h；With the 100K of 15mL Super filter tube is centrifuged, and removes the medicine not carried, and then constant volume is to original volume, is centrifuged in triplicate, is gone by the medicine on unsupported Except completely, obtain Fe3O4@P-NPO/PEG-Glc@Ce6。
After testing, the magnetic Nano probe obtained includes the Fe loading photosensitizer3O4The target that magnetic nano-particle, PEG modify Molecule and hyperbranched macromolecular NPO, the Fe of load photosensitizer3O4The target molecule of magnetic nano-particle, PEG modification and over-expense The mol ratio changing macromole NPO is 0.0005:1:0.5, the Fe of load photosensitizer3O4Magnetic nano-particle include photosensitizer and Fe3O4Carrier, photosensitizer and Fe3O4The mass ratio of carrier is 1:20.
Wherein Fe3O4Carrier particle diameter be 3nm.
To above-mentioned prepared Fe3O4@P-NPO/PEG-Glc@Ce6 detects, and by gas optical photograph, fluorescence is received Rice probe stands more than 1 year, is still uniformly dispersed, and has the strongest fluorescence；A, b, c of Fig. 1 is respectively the present embodiment and prepares Fluorescence magnetic nano-probe transmission electron microscope picture under different resolution, it is known that, fluorescence magnetic nano-probe has excellent Dispersibility, after covalent coupling, agglomeration does not occurs；The fluorescence of the fluorescence magnetic nano-probe that Fig. 2 the present embodiment prepares Spectrogram, it is known that, at probe carrier Fe3O4After@P-NPO/PEG-Glc wraps up photosensitizer, there is the strongest fluorescence intensity, can For internal fluorescence imaging；And due to dimensional effect, the particle diameter of probe nuclei directly affects the fluorescence intensity of drug loading and probe.
By above-mentioned prepared fluorescence magnetic nano-probe tail vein injection in tumor-bearing mice body, carry out targeting bimodal one-tenth Picture and optical dynamic therapy, after injection in different time, observe probe distribution situation in nude mouse, and NMR (Nuclear Magnetic Resonance) imaging becomes with fluorescence Positioning altogether as carrying out tumor, spike more accurately contributes to imaging diagnosis and treatment of cancer, and Fig. 3 shows that fluorescence magnetic nanometer is visited Pin is at its internal fluorescent images, and A is the fluorescent images before injection, and B is the fluorescent images after injecting 3 days, Understanding, probe can the most postoperative spike tumor exactly；Fig. 4 shows blank group (tail vein injection 150 microlitre PBS Solution tumor-bearing mice after 3 days) and experimental group (tail vein injection fluorescence magnetic nano-probe (by 5mg/kg be coated with fluorescent drug Dosage injection) tumor-bearing mice after 3 days) the NMR (Nuclear Magnetic Resonance) imaging photo of tumor locus, it is known that, fluorescence magnetic nano-probe has good Good contrasting effects, and the holdup time be 3 days, observing for preoperative and postoperative provides information accurately.And and fluorescence imaging (Fig. 3) combine, altogether positioning tumor position and spike lesion boundary；Fig. 5 shows that fluorescence magnetic nano-probe is to tumor-bearing mice The treatment curve of tumor, it is known that, fluorescence magnetic nano-probe can be effectively targeted to tumor and suppress the growth of tumor；Fig. 6 shows Show the tumor-bearing mice body weight change curve after fluorescence magnetic nano-probe is treated, it is known that, over the course for the treatment of, the weight of nude mice Amount is held essentially constant, and shows that probe is the least to the side effect of mouse.
Embodiment 2
The preparation of a kind of single dispersing fluorescence magnetic nano-probe, comprises the following steps:
(1) preparation of ferroferric oxide magnetic nano-particles:
By mol ratio it is: ferric acetyl acetonade (III), oleic acid, oleyl amine, hexadecane diol and the 40mL hexichol of 4:12:12:20 Ether (or benzyl ether) is mixed in three-neck flask and utilizes magnet rotor to stir, and mixture is heated to 250 DEG C also under nitrogen protection Continuous heating 3h, is subsequently heated to 350 DEG C, and in this thermotonus 1.5h, reaction is cooled to room temperature after terminating, and gained black is molten Liquid utilizes dehydrated alcohol to be centrifuged repeatedly 3 times (centrifugal speed 3500rpm, 20min) of precipitation, and final gained precipitation is resuspended in chloroform In.
(2) preparation of carboxylated ferroferric oxide magnetic nano-particles:
Take 0.05mmol poly-(isobutene .-alt-maleic anhydride), 0.01mmol lauryl amine is dissolved in equipped with 25mL tetrahydrochysene furan In the round-bottomed flask muttered, after ultrasonic 20s, being heated to 65 DEG C and be stirred overnight, after oxolane is evaporated by next day, polymer is fixed It is dissolved in the amphiphilic polymers obtaining 0.1M in 5mL chloroform；
Step (1) gained magnetic nano-particle and amphipathic nature polyalcohol are mixed in round-bottomed flask according to mol ratio 1:100 In, utilize Rotary Evaporators to be evaporated by solvent lentamente, the solid obtained is dissolved in the SBB 12 of 15mL under magnetic stirring In buffer solution, final gained dispersion liquid uses super filter tube to concentrate, and utilizes Ultracentrifuge to remove the most coated polymer, Obtain carboxylated ferroferric oxide magnetic nano-particles, i.e. obtain the Fe3O4@P-COOH nanoparticle dispersion that concentration is 20 μMs Liquid；
(3) coupling PEGization target molecule and one end be the other branched end of amino be the hyperbranched macromolecular of hydroxyl:
EDC/NHS is added so that it is meet and carboxylic on Fe3O4@P-COOH in Fe3O4@P-COOH nanoparticle dispersion liquid The mol ratio of base is 1:1.5, rotates 30min on XH-1T type multitube adjustable rotary blender, and rotating speed is 30 revs/min；
PEGization target molecule and one end are that amino additionally holds is hyperbranched macromolecular NPO and Fe of hydroxyl3O4@P-COOH nanometer The process conditions of particle dispersion hybrid reaction are: rotate 24h at vibrate at 40 DEG C 3h or 30 DEG C on rotator.
The preparation of PEGization target molecule: add EDC/NHS in SBB 9 buffer of PEG so that it is meet and on PEG molecule The mol ratio of carboxyl is 1:1.5, rotates 30min on XH-1T type multitube adjustable rotary blender, and rotating speed is 30 revs/min；Press The mol ratio of PEG and target molecule is that 1:1.5 adds target molecule, on rotator rotates 24h at vibrate 3h or 30 DEG C at 40 DEG C.
One end be the other branched end of amino be the preparation of the hyperbranched macromolecular NPO of hydroxyl: N-BOC ethylenediamine with shrink sweet Oil is that 1:1 is mixed in the three-neck flask containing 20mL dioxane and utilizes magnet rotor to stir according to mol ratio, and mixture is 90 5h is reacted at DEG C；After reaction terminates, add 200mgNaH (60% mineral oil) reaction 30min；It is added dropwise over shrinking sweet containing 8.4mL The 20mL dioxane of oil, mixture reacts overnight at 90 DEG C, after reaction terminates, pours out organic facies, and surplus materials is dissolved in In 10mL distilled water, with bag filter (molecular retention amount is 3K) dialysis 48h, lyophilization obtains transparence gum-like product；Drip two Drip concentrated hydrochloric acid, then the PBS solution adding pH=7.4 be configured to 1g/mL solution, must arrive one end be the other branched end of amino be hydroxyl Hyperbranched macromolecular NPO, standby.
(4) load photosensitizer drug indocyanine green ICG:
By the mass ratio of 5mg:0.5mmol by photosensitizer drug and Fe3O4@P-NPO/PEG-Glc support dispersion is in 1mL's In deionized water, stirring with the speed of 80rpm and make it fully mix at 40 DEG C, mixing time is 48h；Surpass with the 100K of 15mL Chimney filter is centrifuged, and removes the medicine not carried, and then constant volume is to original volume, is centrifuged in triplicate, is removed by the medicine on unsupported Completely, Fe is obtained3O4@P-NPO/PEG-Glc@ICG。
After testing, the magnetic Nano probe obtained includes the Fe loading photosensitizer3O4The target that magnetic nano-particle, PEG modify Molecule and hyperbranched macromolecular NPO, the Fe of load photosensitizer ICG3O4Target molecule that magnetic nano-particle, PEG modify and super The mol ratio of branched macromolecules NPO is 1:10:2, the Fe of load photosensitizer3O4Magnetic nano-particle includes photosensitizer and Fe3O4Carry Body, photosensitizer and Fe3O4The mass ratio of carrier is 5:4.
Wherein Fe3O4Carrier particle diameter be 15nm.
Embodiment 3
The preparation of a kind of single dispersing fluorescence magnetic nano-probe, comprises the following steps:
(1) preparation of ferroferric oxide magnetic nano-particles:
By mol ratio it is: ferric acetyl acetonade (III), oleic acid, oleyl amine, hexadecane diol and the 30mL hexichol of 2:10:10:15 Ether (or benzyl ether) is mixed in three-neck flask and utilizes magnet rotor to stir, and mixture is heated to 200 DEG C also under nitrogen protection Continuous heating 2h, is subsequently heated to 300 DEG C, and in this thermotonus 1h, reaction is cooled to room temperature, gained dark solution after terminating Utilizing dehydrated alcohol to be centrifuged repeatedly 3 times (centrifugal speed 3000rpm, 10min) of precipitation, final gained precipitation is resuspended in chloroform.
(2) preparation of carboxylated ferroferric oxide magnetic nano-particles:
Step (1) gained magnetic nano-particle and amphipathic nature polyalcohol are mixed in round-bottomed flask according to mol ratio 1:50 In, utilize Rotary Evaporators to be evaporated by solvent lentamente, the solid obtained is dissolved in the SBB 12 of 10mL under magnetic stirring In buffer solution, final gained dispersion liquid uses super filter tube to concentrate, and utilizes Ultracentrifuge to remove the most coated polymer, Obtain carboxylated ferroferric oxide magnetic nano-particles, i.e. obtain Fe3O4@P-COOH nanoparticle dispersion liquid；
(3) coupling PEGization target molecule and one end be the other branched end of amino be the hyperbranched macromolecular of hydroxyl:
EDC/NHS is added so that it is meet and carboxylic on Fe3O4@P-COOH in Fe3O4@P-COOH nanoparticle dispersion liquid The mol ratio of base is 1:1.2, rotates 25min on XH-1T type multitube adjustable rotary blender, and rotating speed is 20 revs/min；
PEGization target molecule and one end are that amino additionally holds is that hyperbranched macromolecular NPO and the Fe3O4@P-COOH of hydroxyl receives The process conditions of rice corpuscles dispersion liquid hybrid reaction are: rotate 20h under vibrate at 37 DEG C 2h or room temperature on rotator.
The preparation of PEGization target molecule: add EDC/NHS in SBB 9 buffer of PEG so that it is meet and on PEG molecule The mol ratio of carboxyl is 1:1.2, rotates 25min on XH-1T type multitube adjustable rotary blender, and rotating speed is 20 revs/min；Press PEG is that 1:2 adds glucose with the mol ratio of glucose, rotates 20h at 37 DEG C under vibrate 2h or room temperature on rotator.
One end be the other branched end of amino be the preparation of the hyperbranched macromolecular NPO of hydroxyl: N-BOC ethylenediamine with shrink sweet Oil is that 0.8:0.8 is mixed in the three-neck flask containing 12mL dioxane and utilizes magnet rotor to stir according to mol ratio, mixture 3.5h is reacted at 90 DEG C；After reaction terminates, add 150mgNaH (60% mineral oil) reaction 20min；It is added dropwise over contracting containing 5mL The 12mL dioxane of water glycerol, mixture reacts overnight at 90 DEG C, after reaction terminates, pours out organic facies, and surplus materials is molten In 5-10mL distilled water, with bag filter (molecular retention amount is 3K) dialysis 36h, lyophilization obtains transparence gum-like product； Dripping two concentrated hydrochloric acid, then the PBS solution adding pH=7.4 is configured to 0.8g/mL solution, must arrive one end is that the other branched end of amino is The hyperbranched macromolecular NPO of hydroxyl, standby.
(4) load photosensitizer drug DOX:
By the mass ratio of 3:12 by photosensitizer drug and Fe3O4@P-NPO/PEG-Glc support dispersion is in the deionized water of 1mL In, stirring with the speed of 150rpm and make it fully mix, mixing time is 36h；It is centrifuged with the 100K super filter tube of 15mL, removes not Medicine in load, the parameter of refrigerated centrifuger is set to: 4 DEG C, 3000rpm, 30min, and then constant volume is to original volume, in triplicate from The heart, removes the medicine on unsupported completely, obtains Fe3O4@P-NPO/PEG-Glc@DOX。
Embodiment 4
The preparation of a kind of single dispersing fluorescence magnetic nano-probe, comprises the following steps:
(1) preparation of ferroferric oxide magnetic nano-particles:
By mol ratio it is: the ferric acetyl acetonade (III) of 1:3:3:5, oleic acid, oleyl amine, hexadecane diol and diphenyl ether (or two Benzyl oxide) it is mixed in three-neck flask and utilizes magnet rotor to stir, mixture is heated to 180 DEG C and continuous heating under nitrogen protection 1.5h, is subsequently heated to 300 DEG C, and in this thermotonus 1h, reaction is cooled to room temperature after terminating, and gained dark solution utilizes nothing Water-ethanol is centrifuged repeatedly 3 times (centrifugal speed 2800rpm, 10min) of precipitation, and final gained precipitation is resuspended in chloroform.
(2) preparation of carboxylated ferroferric oxide magnetic nano-particles:
Step (1) gained magnetic nano-particle and amphipathic nature polyalcohol are mixed in round-bottomed flask according to mol ratio 1:20 In, utilize Rotary Evaporators to be evaporated by solvent lentamente, the solid obtained is dissolved in the SBB 12 of 8mL under magnetic stirring and delays In dissolved liquid, final gained dispersion liquid uses super filter tube to concentrate, and utilizes Ultracentrifuge to remove the most coated polymer, To carboxylated ferroferric oxide magnetic nano-particles, i.e. obtain Fe3O4@P-COOH nanoparticle dispersion liquid；
(3) coupling PEGization target molecule and one end be the other branched end of amino be the hyperbranched macromolecular of hydroxyl:
EDC/NHS is added so that it is meet and carboxylic on Fe3O4@P-COOH in Fe3O4@P-COOH nanoparticle dispersion liquid The mol ratio of base is 1:1, rotates 25min on XH-1T type multitube adjustable rotary blender, and rotating speed is 10 revs/min；
PEGization target molecule and one end are that amino additionally holds is that hyperbranched macromolecular NPO and the Fe3O4@P-COOH of hydroxyl receives The process conditions of rice corpuscles dispersion liquid hybrid reaction are: rotate 15h under vibrate at 37 DEG C 1.5h or room temperature on rotator.
The preparation of PEGization target molecule: add EDC/NHS in SBB 9 buffer of PEG so that it is meet and on PEG molecule The mol ratio of carboxyl is 1:1, rotates 25min on XH-1T type multitube adjustable rotary blender, and rotating speed is 10 revs/min；Press The mol ratio of PEG and hyaluronic acid HA is that 1:1 adds hyaluronic acid HA, under vibrate at 37 DEG C 1.5h or room temperature on rotator Rotate 20h.
One end be the other branched end of amino be the preparation of the hyperbranched macromolecular NPO of hydroxyl: N-BOC ethylenediamine with shrink sweet Oil is that 0.5:1 is mixed in the three-neck flask containing 5-20mL dioxane and utilizes magnet rotor to stir according to mol ratio, mixture 3h is reacted at 90 DEG C；After reaction terminates, add 120mgNaH (60% mineral oil) reaction 15min；It is added dropwise over shrinking containing 4mL The 8mL dioxane of glycerol, mixture reacts overnight at 90 DEG C, after reaction terminates, pours out organic facies, and surplus materials is dissolved in 5- In 10mL distilled water, with bag filter (molecular retention amount is 3K) dialysis 24h, lyophilization obtains transparence gum-like product；Drip two Drip concentrated hydrochloric acid, then the PBS solution adding pH=7.4 be configured to 0.5g/mL solution, must arrive one end be the other branched end of amino be hydroxyl Hyperbranched macromolecular NPO, standby.
(4) load photosensitizer drug cyanine 5.5 (Cy5.5)/Ce6:
By the mass ratio of 3mg:7mmol by photosensitizer drug and Fe3O4@P-NPO/PEG-HA support dispersion in 1mL go from In sub-water, stirring with the speed of 300rpm and make it fully mix, mixing time is 24h；It is centrifuged with the 100K super filter tube of 15mL, goes Except the medicine not carried, the parameter of refrigerated centrifuger is set to: 4 DEG C, 3500rpm, 20min, and then constant volume is to original volume, repeats three Secondary centrifugal, the medicine on unsupported is removed completely, obtains Fe3O4@P-NPO/[email protected]/Ce6。
Embodiment 5
The preparation of a kind of single dispersing fluorescence magnetic nano-probe, comprises the following steps:
(1) preparation of ferroferric oxide magnetic nano-particles:
By mol ratio it is: ferric acetyl acetonade (III), oleic acid, oleyl amine, hexadecane diol and the diphenyl ether of 3:10:10:15 (or benzyl ether) is mixed in three-neck flask and utilizes magnet rotor to stir, and mixture is heated to 230 DEG C and holds under nitrogen protection Continuous heating 2.5h, is subsequently heated to 300 DEG C, and in this thermotonus 1.2h, reaction is cooled to room temperature after terminating, and gained black is molten Liquid utilizes dehydrated alcohol to be centrifuged repeatedly 3 times (centrifugal speed 3000rpm, 15min) of precipitation, and final gained precipitation is resuspended in chloroform In.
(2) preparation of carboxylated ferroferric oxide magnetic nano-particles:
Step (1) gained magnetic nano-particle and amphipathic nature polyalcohol are mixed in round-bottomed flask according to mol ratio 1:30 In, utilize Rotary Evaporators to be evaporated by solvent lentamente, the solid obtained is dissolved in the SBB 12 of 12mL under magnetic stirring In buffer solution, final gained dispersion liquid uses super filter tube to concentrate, and utilizes Ultracentrifuge to remove the most coated polymer, Obtain carboxylated ferroferric oxide magnetic nano-particles, i.e. obtain Fe3O4@P-COOH nanoparticle dispersion liquid；
(3) coupling PEGization target molecule and one end be the other branched end of amino be the hyperbranched macromolecular of hydroxyl:
EDC/NHS is added so that it is meet and carboxylic on Fe3O4@P-COOH in Fe3O4@P-COOH nanoparticle dispersion liquid The mol ratio of base is 1:1.3, rotates 30min on XH-1T type multitube adjustable rotary blender, and rotating speed is 20 revs/min；
PEGization target molecule and one end are that amino additionally holds is that hyperbranched macromolecular NPO and the Fe3O4@P-COOH of hydroxyl receives The process conditions of rice corpuscles dispersion liquid hybrid reaction are: rotate 20h under vibrate at 37 DEG C 2h or room temperature on rotator.
The preparation of PEGization target molecule: add EDC/NHS in SBB 9 buffer of PEG so that it is meet and on PEG molecule The mol ratio of carboxyl is 1:1.2, rotates 30min on XH-1T type multitube adjustable rotary blender, and rotating speed is 20 revs/min；Press The mol ratio of PEG Yu tLyp-1 peptide is that 1:1.3 adds tLyp-1 peptide, is rotating under vibrate 2h or room temperature at 37 DEG C 20h is rotated on device.
One end be the other branched end of amino be the preparation of the hyperbranched macromolecular NPO of hydroxyl: N-BOC ethylenediamine with shrink sweet Oil is that 1:0.5 is mixed in the three-neck flask containing 15mL dioxane and utilizes magnet rotor to stir according to mol ratio, and mixture exists 4h is reacted at 90 DEG C；After reaction terminates, add 180mgNaH (60% mineral oil) reaction 25min；It is added dropwise over shrinking sweet containing 6mL The 20mL dioxane of oil, mixture reacts overnight at 90 DEG C, after reaction terminates, pours out organic facies, and surplus materials is dissolved in 8mL In distilled water, with bag filter (molecular retention amount is 3K) dialysis 40h, lyophilization obtains transparence gum-like product；Drip two dense Hydrochloric acid, then the PBS solution adding pH=7.4 is configured to 1g/mL solution, must arrive one end be the other branched end of amino be the over-expense of hydroxyl Change macromole NPO, standby.
(4) load photosensitizer drug Ce6:
By the mass ratio of 4:18 by photosensitizer drug and Fe3O4@P-NPO/PEG-tLyp-1 support dispersion in 1mL go from In sub-water, stirring with the speed of 100rpm and make it fully mix, mixing time is 40h；It is centrifuged with the 100K super filter tube of 15mL, goes Except the medicine not carried, the parameter of refrigerated centrifuger is set to: 4 DEG C, 3200rpm, 35min, and then constant volume is to original volume, repeats three Secondary centrifugal, the medicine on unsupported is removed completely, obtains Fe3O4@P-NPO/PEG-tLyp-1@Ce6。
Embodiment 6
The preparation of a kind of single dispersing fluorescence magnetic nano-probe, comprises the following steps:
(1) preparation of ferroferric oxide magnetic nano-particles:
By mol ratio it is: ferric acetyl acetonade (III), oleic acid, oleyl amine, hexadecane diol and the hexichol of 0.5:1.5:12:20 Ether (or benzyl ether) is mixed in three-neck flask and utilizes magnet rotor to stir, and mixture is heated to 250 DEG C also under nitrogen protection Continuous heating 1h, is subsequently heated to 280 DEG C, and in this thermotonus 0.5h, reaction is cooled to room temperature after terminating, and gained black is molten Liquid utilizes dehydrated alcohol to be centrifuged repeatedly 3 times (centrifugal speed 2000rpm, 5min) of precipitation, and final gained precipitation is resuspended in chloroform.
(2) preparation of carboxylated ferroferric oxide magnetic nano-particles:
Step (1) gained magnetic nano-particle and amphipathic nature polyalcohol are mixed in round-bottomed flask according to mol ratio 1:45 In, utilize Rotary Evaporators to be evaporated by solvent lentamente, the solid obtained is dissolved in the SBB 12 of 10mL under magnetic stirring In buffer solution, final gained dispersion liquid uses super filter tube to concentrate, and utilizes Ultracentrifuge to remove the most coated polymer, Obtain carboxylated ferroferric oxide magnetic nano-particles, i.e. obtain Fe3O4@P-COOH nanoparticle dispersion liquid；
(3) coupling PEGization target molecule and one end be the other branched end of amino be the hyperbranched macromolecular of hydroxyl:
EDC/NHS is added so that it is meet and carboxylic on Fe3O4@P-COOH in Fe3O4@P-COOH nanoparticle dispersion liquid The mol ratio of base is 1:1, rotates 20min on XH-1T type multitube adjustable rotary blender, and rotating speed is 5 revs/min；
PEGization target molecule and one end are that amino additionally holds is that hyperbranched macromolecular NPO and the Fe3O4@P-COOH of hydroxyl receives The process conditions of rice corpuscles dispersion liquid hybrid reaction are: rotate 12h under vibrate at 37 DEG C 1h or room temperature on rotator.
The preparation of PEGization target molecule: add EDC/NHS in SBB 9 buffer of PEG so that it is meet and on PEG molecule The mol ratio of carboxyl is 1:1, rotates 30min on XH-1T type multitube adjustable rotary blender, and rotating speed is 30 revs/min；Press The mol ratio of PEG Yu AP peptide is that 1:1 adds AP peptide, on rotator rotates under vibrate 1h or room temperature at 37 DEG C 12h。
One end be the other branched end of amino be the preparation of the hyperbranched macromolecular NPO of hydroxyl: N-BOC ethylenediamine with shrink sweet Oil is that 0.5:0.5 is mixed in the three-neck flask containing 5mL dioxane and utilizes magnet rotor to stir according to mol ratio, and mixture exists 2h is reacted at 90 DEG C；After reaction terminates, add 100mgNaH (60% mineral oil) reaction 10min；It is added dropwise over shrinking containing 2.1mL The 5mL dioxane of glycerol, mixture reacts overnight at 90 DEG C, after reaction terminates, pours out organic facies, and surplus materials is dissolved in In 5mL distilled water, with bag filter (molecular retention amount is 3K) dialysis 24h, lyophilization obtains transparence gum-like product；Drip two Concentrated hydrochloric acid, then the PBS solution adding pH=7.4 is configured to 0.5g/mL solution, must arrive one end be the other branched end of amino be hydroxyl Hyperbranched macromolecular NPO, standby.
(4) load photosensitizer drug Ce6 and Cy5.5:
By the mass ratio of 3mg:6mmol by photosensitizer drug and Fe3O4@P-NPO/PEG-AP support dispersion in 1mL go from In sub-water, stirring with the speed of 200rpm and make it fully mix, mixing time is 24h；It is centrifuged with the 100K super filter tube of 15mL, goes Except the medicine not carried, the parameter of refrigerated centrifuger is set to: 4 DEG C, 2000rpm, 20min, and then constant volume is to original volume, repeats three Secondary centrifugal, the medicine on unsupported is removed completely, obtains Fe3O4@P-NPO/PEG-AP@Ce6/Cy5.5。
Embodiment 7
The preparation of a kind of single dispersing fluorescence magnetic nano-probe, comprises the following steps:
(1) preparation of ferroferric oxide magnetic nano-particles:
By mol ratio it is: ferric acetyl acetonade (III), oleic acid, oleyl amine, hexadecane diol and the diphenyl ether of 2:10:12:15 (or benzyl ether) is mixed in three-neck flask and utilizes magnet rotor to stir, and mixture is heated to 200 DEG C and holds under nitrogen protection Continuous heating 1.5h, is subsequently heated to 300 DEG C, and in this thermotonus 1h, reaction is cooled to room temperature, gained dark solution after terminating Utilizing dehydrated alcohol to be centrifuged repeatedly 3 times (centrifugal speed 3500rpm, 20min) of precipitation, final gained precipitation is resuspended in chloroform.
(2) preparation of carboxylated ferroferric oxide magnetic nano-particles:
Step (1) gained magnetic nano-particle and amphipathic nature polyalcohol are mixed in round-bottomed flask according to mol ratio 1:60 In, utilize Rotary Evaporators to be evaporated by solvent lentamente, the solid obtained is dissolved in the SBB 12 of 10mL under magnetic stirring In buffer solution, final gained dispersion liquid uses super filter tube to concentrate, and utilizes Ultracentrifuge to remove the most coated polymer, Obtain carboxylated ferroferric oxide magnetic nano-particles, i.e. obtain Fe3O4@P-COOH nanoparticle dispersion liquid；
(3) coupling PEGization target molecule and one end be the other branched end of amino be the hyperbranched macromolecular of hydroxyl:
EDC/NHS is added so that it is meet and carboxylic on Fe3O4@P-COOH in Fe3O4@P-COOH nanoparticle dispersion liquid The mol ratio of base is 1:1.5, rotates 30min on XH-1T type multitube adjustable rotary blender, and rotating speed is 30 revs/min；
PEGization target molecule and one end are that amino additionally holds is that hyperbranched macromolecular NPO and the Fe3O4@P-COOH of hydroxyl receives The process conditions of rice corpuscles dispersion liquid hybrid reaction are: rotate 24h under vibrate at 37 DEG C 3h or room temperature on rotator.
The preparation of PEGization target molecule: add EDC/NHS in SBB 9 buffer of PEG so that it is meet and on PEG molecule The mol ratio of carboxyl is 1:1.5, rotates 30min on XH-1T type multitube adjustable rotary blender, and rotating speed is 30 revs/min；Press The mol ratio of PEG and ring-type RGD is that 1:3 adds ring-type RGD, rotates 24h at 37 DEG C under vibrate 3h or room temperature on rotator.
One end be the other branched end of amino be the preparation of the hyperbranched macromolecular NPO of hydroxyl: N-BOC ethylenediamine with shrink sweet Oil is that 1:0.5 is mixed in the three-neck flask containing 20mL dioxane and utilizes magnet rotor to stir according to mol ratio, and mixture exists 5h is reacted at 90 DEG C；After reaction terminates, add 200mgNaH (60% mineral oil) reaction 30min；It is added dropwise over shrinking sweet containing 6mL The 5-20mL dioxane of oil, mixture reacts overnight at 90 DEG C, after reaction terminates, pours out organic facies, and surplus materials is dissolved in In 10mL distilled water, with bag filter (molecular retention amount is 3K) dialysis 48h, lyophilization obtains transparence gum-like product；Drip two Drip concentrated hydrochloric acid, then the PBS solution adding pH=7.4 be configured to 1g/mL solution, must arrive one end be the other branched end of amino be hydroxyl Hyperbranched macromolecular NPO, standby.
(4) load photosensitizer drug Ce6 and amycin DOX:
By the mass ratio of 5mg:7mmol by photosensitizer drug and Fe3O4@P-NPO/PEG-RGD support dispersion going in 1mL In ionized water, stirring with the speed of 400rpm and make it fully mix, mixing time is 36h；It is centrifuged with the 100K super filter tube of 15mL, Removing the medicine not carried, the parameter of refrigerated centrifuger is set to: 4 DEG C, 3000rpm, 30min, and then constant volume is to original volume, repeats It is centrifuged for three times, the medicine on unsupported is removed completely, obtains Fe3O4@P-NPO/PEG-RGD@Ce6/DOX。
Embodiment 8
The preparation of a kind of single dispersing fluorescence magnetic nano-probe, comprises the following steps:
(1) preparation of ferroferric oxide magnetic nano-particles:
By mol ratio it is: ferric acetyl acetonade (III), oleic acid, oleyl amine, hexadecane diol and the diphenyl ether of 4:1.5:3:10 mixes Together in three-neck flask and utilize magnet rotor to stir, mixture is heated to 250 DEG C and continuous heating 3h under nitrogen protection, subsequently It is heated to 350 DEG C, and in this thermotonus 1h, reaction is cooled to room temperature after terminating, and gained dark solution utilizes dehydrated alcohol anti- Multiple centrifugation 3 times (centrifugal speed 3500rpm, 20min), final gained precipitation is resuspended in chloroform.
(2) preparation of carboxylated ferroferric oxide magnetic nano-particles:
Step (1) gained magnetic nano-particle and amphipathic nature polyalcohol are mixed in round-bottomed flask according to mol ratio 1:80 In, utilize Rotary Evaporators to be evaporated by solvent lentamente, the solid obtained is dissolved in the SBB 12 of 15mL under magnetic stirring In buffer solution, final gained dispersion liquid uses super filter tube to concentrate, and utilizes Ultracentrifuge to remove the most coated polymer, Obtain carboxylated ferroferric oxide magnetic nano-particles, i.e. obtain Fe3O4@P-COOH nanoparticle dispersion liquid；
(3) coupling PEGization target molecule and one end be the other branched end of amino be the hyperbranched macromolecular of hydroxyl:
EDC/NHS is added so that it is meet and carboxylic on Fe3O4@P-COOH in Fe3O4@P-COOH nanoparticle dispersion liquid The mol ratio of base is 1:1, rotates 30min on XH-1T type multitube adjustable rotary blender, and rotating speed is 20 revs/min；
PEGization target molecule and one end are that amino additionally holds is that hyperbranched macromolecular NPO and the Fe3O4@P-COOH of hydroxyl receives The process conditions of rice corpuscles dispersion liquid hybrid reaction are: rotate 20h under vibrate at 37 DEG C 2h or room temperature on rotator.
The preparation of PEGization target molecule: add EDC/NHS in SBB 9 buffer of PEG so that it is meet and on PEG molecule The mol ratio of carboxyl is 1:1, rotates 30min on XH-1T type multitube adjustable rotary blender, and rotating speed is 20 revs/min；Press PEG is that 1:1 adds somatomedin with the mol ratio of somatomedin, on rotator rotates under vibrate 3h or room temperature at 37 DEG C 24h。
One end be the other branched end of amino be the preparation of the hyperbranched macromolecular NPO of hydroxyl: N-BOC ethylenediamine with shrink sweet Oil is that 0.5:1 is mixed in the three-neck flask containing 10mL dioxane and utilizes magnet rotor to stir according to mol ratio, and mixture exists 5h is reacted at 90 DEG C；After reaction terminates, add 150mgNaH (60% mineral oil) reaction 20min；It is added dropwise over shrinking sweet containing 8mL The 20mL dioxane of oil, mixture reacts overnight at 90 DEG C, after reaction terminates, pours out organic facies, and surplus materials is dissolved in In 10mL distilled water, with bag filter (molecular retention amount is 3K) dialysis 24h, lyophilization obtains transparence gum-like product；Drip two Drip concentrated hydrochloric acid, then the PBS solution adding pH=7.4 be configured to 0.8g/mL solution, must arrive one end be the other branched end of amino be hydroxyl Hyperbranched macromolecular NPO, standby.
(4) load photosensitizer drug DOX and Cy5.5:
By the mass ratio of 2.5mg:6mmol by photosensitizer drug and Fe3O4@P-NPO/PEG-growth factor carrier is scattered in In the deionized water of 1mL, stirring with the speed of 500rpm and make it fully mix, mixing time is 48h；With the 100K ultrafiltration of 15mL Pipe is centrifugal, removes the medicine not carried, and the parameter of refrigerated centrifuger is set to: 4 DEG C, 3500rpm, 20min, then constant volume is to substance Long-pending, it is centrifuged in triplicate, the medicine on unsupported is removed completely, obtains Fe3O4@P-NPO/PEG-somatomedin@DOX/ Cy5.5。
Embodiment 9
The preparation of a kind of single dispersing fluorescence magnetic nano-probe, comprises the following steps:
(1) preparation of ferroferric oxide magnetic nano-particles:
By mol ratio it is: ferric acetyl acetonade (III), oleic acid, oleyl amine, hexadecane diol and the diphenyl ether of 4:1.5:5:18 mixes Together in three-neck flask and utilize magnet rotor to stir, mixture is heated to 200 DEG C and continuous heating 1.5h under nitrogen protection, with Post-heating is to 300 DEG C, and in this thermotonus 1h, reaction is cooled to room temperature after terminating, and gained dark solution utilizes dehydrated alcohol Being centrifuged repeatedly 3 times (centrifugal speed 2000rpm, 20min) of precipitation, final gained precipitation is resuspended in chloroform.
(2) preparation of carboxylated ferroferric oxide magnetic nano-particles:
Step (1) gained magnetic nano-particle and amphipathic nature polyalcohol are mixed in round-bottomed flask according to mol ratio 1:60 In, utilize Rotary Evaporators to be evaporated by solvent lentamente, the solid obtained is dissolved in the SBB 12 of 10mL under magnetic stirring In buffer solution, final gained dispersion liquid uses super filter tube to concentrate, and utilizes Ultracentrifuge to remove the most coated polymer, Obtain carboxylated ferroferric oxide magnetic nano-particles, i.e. obtain Fe3O4@P-COOH nanoparticle dispersion liquid；
(3) coupling PEGization target molecule and one end be the other branched end of amino be the hyperbranched macromolecular of hydroxyl:
EDC/NHS is added so that it is meet and carboxylic on Fe3O4@P-COOH in Fe3O4@P-COOH nanoparticle dispersion liquid The mol ratio of base is 1:1.5, rotates 25min on XH-1T type multitube adjustable rotary blender, and rotating speed is 20 revs/min；
PEGization target molecule and one end are that amino additionally holds is that hyperbranched macromolecular NPO and the Fe3O4@P-COOH of hydroxyl receives The process conditions of rice corpuscles dispersion liquid hybrid reaction are: rotate 24h under vibrate at 37 DEG C 3h or room temperature on rotator.
The preparation of PEGization target molecule: add EDC/NHS in SBB 9 buffer of PEG so that it is meet and on PEG molecule The mol ratio of carboxyl is 1:1.5, rotates 30min on XH-1T type multitube adjustable rotary blender, and rotating speed is 5 revs/min；Press PEG is that 1:1.5 adds octreotide with the mol ratio of octreotide, rotates 20h at 37 DEG C under vibrate 2h or room temperature on rotator.
One end be the other branched end of amino be the preparation of the hyperbranched macromolecular NPO of hydroxyl: N-BOC ethylenediamine with shrink sweet Oil is that 1:1 is mixed in the three-neck flask containing 15mL dioxane and utilizes magnet rotor to stir according to mol ratio, and mixture is 90 3h is reacted at DEG C；After reaction terminates, add 150mgNaH (60% mineral oil) reaction 25min；It is added dropwise over containing 5mL (+)-2,3-Epoxy-1-propanol 5-20mL dioxane, mixture reacts overnight at 90 DEG C, reaction terminate after, pour out organic facies, surplus materials is dissolved in 8mL In distilled water, with bag filter (molecular retention amount is 3K) dialysis 24-48h, lyophilization obtains transparence gum-like product；Drip two Concentrated hydrochloric acid, then the PBS solution adding pH=7.4 is configured to 1g/mL solution, must arrive one end be the other branched end of amino be the super of hydroxyl Branched macromolecules NPO, standby.
(4) load photosensitizer drug ICG and Cy5.5:
By the mass ratio of 3mg:10mmol by photosensitizer drug and Fe3O4@P-NPO/PEG-octreotide support dispersion is in 1mL Deionized water in, stir with the speed of 300rpm and make it fully mix, mixing time is 40h；With the 100K super filter tube of 15mL Centrifugal, remove the medicine not carried, the parameter of refrigerated centrifuger is set to: 4 DEG C, 3500rpm, 25min, then constant volume is to substance Long-pending, it is centrifuged in triplicate, the medicine on unsupported is removed completely, obtains Fe3O4@P-NPO/PEG-octreotide@ICG/Cy5.5.
Embodiment 10
The preparation of a kind of single dispersing fluorescence magnetic nano-probe, comprises the following steps:
(1) preparation of ferroferric oxide magnetic nano-particles:
By mol ratio it is: ferric acetyl acetonade (III), oleic acid, oleyl amine, hexadecane diol and the diphenyl ether of 2:10:10:15 mixes Together in three-neck flask and utilize magnet rotor to stir, mixture is heated to 250 DEG C and continuous heating 2h under nitrogen protection, subsequently It is heated to 350 DEG C, and in this thermotonus 1h, reaction is cooled to room temperature after terminating, and gained dark solution utilizes dehydrated alcohol anti- Multiple centrifugation 3 times (centrifugal speed 3500rpm, 20min), final gained precipitation is resuspended in chloroform.
(2) preparation of carboxylated ferroferric oxide magnetic nano-particles:
Step (1) gained magnetic nano-particle and amphipathic nature polyalcohol are mixed in round-bottomed flask according to mol ratio 1:70 In, utilize Rotary Evaporators to be evaporated by solvent lentamente, the solid obtained is dissolved in the SBB 12 of 10mL under magnetic stirring In buffer solution, final gained dispersion liquid uses super filter tube to concentrate, and utilizes Ultracentrifuge to remove the most coated polymer, Obtain carboxylated ferroferric oxide magnetic nano-particles, i.e. obtain Fe3O4@P-COOH nanoparticle dispersion liquid；
(3) coupling PEGization target molecule and one end be the other branched end of amino be the hyperbranched macromolecular of hydroxyl:
EDC/NHS is added so that it is meet and carboxylic on Fe3O4@P-COOH in Fe3O4@P-COOH nanoparticle dispersion liquid The mol ratio of base is 1:1.2, rotates 25min on XH-1T type multitube adjustable rotary blender, and rotating speed is 10 revs/min；
PEGization target molecule and one end are that amino additionally holds is that hyperbranched macromolecular NPO and the Fe3O4@P-COOH of hydroxyl receives The process conditions of rice corpuscles dispersion liquid hybrid reaction are: rotate 20h under vibrate at 37 DEG C 2h or room temperature on rotator.
The preparation of PEGization target molecule: add EDC/NHS in SBB 9 buffer of PEG so that it is meet and on PEG molecule The mol ratio of carboxyl is 1:1.2, rotates 25min on XH-1T type multitube adjustable rotary blender, and rotating speed is 20 revs/min；Press PEG is that 1:3 adds antibody with the mol ratio of antibody, rotates 22h at 37 DEG C under vibrate 2h or room temperature on rotator.
One end be the other branched end of amino be the preparation of the hyperbranched macromolecular NPO of hydroxyl: N-BOC ethylenediamine with shrink sweet Oil is that 1:1 is mixed in the three-neck flask containing 15mL dioxane and utilizes magnet rotor to stir according to mol ratio, and mixture is 90 2h is reacted at DEG C；After reaction terminates, add 200mgNaH (60% mineral oil) reaction 30min；It is added dropwise over containing 7mL (+)-2,3-Epoxy-1-propanol 15mL dioxane, mixture reacts overnight at 90 DEG C, reaction terminate after, pour out organic facies, surplus materials be dissolved in 6mL steam In distilled water, with bag filter (molecular retention amount is 3K) dialysis 48h, lyophilization obtains transparence gum-like product；Drip two dense salt Acid, then the PBS solution adding pH=7.4 is configured to 1g/mL solution, must arrive one end be the other branched end of amino be the hyperbranched of hydroxyl Macromole NPO, standby.
(4) load photosensitizer drug Ce6, DOX and Cy5.5:
By the mass ratio of 5:20 by photosensitizer drug and Fe3O4@P-NPO/PEG-Glc support dispersion is in the deionized water of 1mL In, stirring with the speed of 500rpm and make it fully mix, mixing time is 48h；It is centrifuged with the 100K super filter tube of 15mL, removes not Medicine in load, the parameter of refrigerated centrifuger is set to: 4 DEG C, 3500rpm, 30min, and then constant volume is to original volume, in triplicate from The heart, removes the medicine on unsupported completely, obtains Fe3O4@P-NPO/PEG-antibody@Ce6/DOX/Cy5.5.
The above-mentioned description to embodiment is to be understood that for ease of those skilled in the art and use invention. These embodiments obviously easily can be made various amendment by person skilled in the art, and described herein typically Principle is applied in other embodiments without through performing creative labour.Therefore, the invention is not restricted to above-described embodiment, ability Field technique personnel should be the present invention's according to the announcement of the present invention, the improvement made without departing from scope and amendment Within protection domain.

Claims (10)

1. a single dispersing fluorescence magnetic nano-probe, it is characterised in that described magnetic Nano probe includes loading photosensitizer Fe3O4The target molecule of magnetic nano-particle, PEG modification and hyperbranched macromolecular NPO, the Fe of described load photosensitizer3O4Magnetic Target molecule and the mol ratio of hyperbranched macromolecular NPO that nanoparticle, PEG modify are (0.0005～1): (1～10): (0.5 ～2), the Fe of described load photosensitizer3O4Magnetic nano-particle includes photosensitizer and Fe3O4Carrier, described photosensitizer and Fe3O4Carry The mass ratio of body is (1～5): (4～20).
A kind of single dispersing fluorescence magnetic nano-probe the most according to claim 1, it is characterised in that described photosensitizer choosing In chlorin e 6, indocyanine green, cyanine 5.5 and amycin one or more；
Described Fe3O4Carrier particle diameter be 3～15nm.
A kind of single dispersing fluorescence magnetic nano-probe the most according to claim 1, it is characterised in that described target molecule choosing From antibody, antibody fragment, somatomedin, ring-type RGD, octreotide, AP peptide, tLyp-1 peptide, hyaluronic acid HA or Portugal One or more in grape sugar.
A kind of single dispersing fluorescence magnetic nano-probe the most according to claim 1, it is characterised in that described hyperbranched greatly The molecular weight of molecule NPO is more than or equal to 5000, and its one end is amino, and branched end additionally is hydroxyl.
5. a preparation method for the single dispersing fluorescence magnetic nano-probe as described in Claims 1 to 4 is arbitrary, its feature exists Following step is included in, the method:
(1) ferric acetyl acetonade, oleic acid, oleyl amine, hexadecane diol and diphenyl ether are mixed, at N2The lower reaction of protection, after reaction terminates Being cooled to room temperature, the magnetic nano-particle being filtrated to get is dissolved in chloroform, obtains chloroformic solution, stand-by；
(2) adding amphipathic nature polyalcohol in the chloroformic solution of step (1) gained, evaporate solvent after mix homogeneously, obtain consolidates Body is dissolved in SBB12 buffer solution, and concentration is centrifugal obtains Fe3O4@P-COOH nanoparticle dispersion liquid；
(3) by Fe3O4Activated carboxylic in@P-COOH nanoparticle dispersion liquid, is subsequently adding the target molecule that PEG modifies, and reacts one Adding hyperbranched macromolecular NPO after the section time, reaction obtains the Fe of band target molecule and hyperbranched macromolecular3O4@P-NPO/PEG-TM Dispersion liquid；
(4) by step (3) resulting tape target molecule and the Fe of hyperbranched macromolecular3O4@P-NPO/PEG-TM dispersion liquid is molten with photosensitizer Liquid mixing, oscillating reactions, be then centrifuged for purification, obtain described single dispersing fluorescence magnetic nano-probe.
The preparation method of a kind of single dispersing fluorescence magnetic nano-probe the most according to claim 5, it is characterised in that step (1) mol ratio of described ferric acetyl acetonade, oleic acid, oleyl amine and hexadecane diol is (0.5～4): (1.5～12): (1.5～ 12): (2.5～20), the volume of described diphenyl ether and the ratio of the mole of described ferric acetyl acetonade be (5～40) mL:(0.5～ 4)mol；
In two stages, the reaction temperature of first stage is 150～250 DEG C in described reaction, and the response time is 1～3h, second-order The reaction temperature of section is 280～350 DEG C, and the response time is 0.5～1.5h.
The preparation method of a kind of single dispersing fluorescence magnetic nano-probe the most according to claim 5, it is characterised in that step (2) synthesis step of described amphipathic nature polyalcohol is: by poly-(isobutene .-alt-maleic anhydride), lauryl amine with (0.05～1): The mixed in molar ratio of (0.01～2), is dissolved in oxolane, after ultrasonic 10～20s, is heated to 65 DEG C and stirs 8～12h, so After oxolane is evaporated, and be dissolved in chloroform, obtain the amphipathic nature polyalcohol that concentration is 0.1M；
Magnetic nano-particle in described chloroformic solution is 1:(5-100 with the mol ratio of addition amphipathic nature polyalcohol) it is concentrated to give Fe3O4The concentration of@P-COOH nanoparticle dispersion liquid is 3～20 μMs.
The preparation method of a kind of single dispersing fluorescence magnetic nano-probe the most according to claim 5, it is characterised in that step (3) technique of described activation is as follows:
At Fe3O4@P-COOH nanoparticle dispersion liquid adds EDC/NHS solution, the mole of the EDC/NHS of addition and Fe3O4@P- In COOH nanoparticle dispersion liquid, the ratio of the mole of carboxyl is 1:(1～1.5), then 5～30rpm/min rotating speed backspin Turn mixing 20～30min；
Add PEG modify the post-reacted condition of target molecule be at 30～40 DEG C vibrate 1～3h or 20～30 DEG C at rotator Upper rotation 12～24h；
Add the post-reacted condition of hyperbranched macromolecular NPO be at 30～40 DEG C vibrate 1～3h or 20～30 DEG C at rotator Upper rotation 12～24h.
The preparation method of a kind of single dispersing fluorescence magnetic nano-probe the most according to claim 5, it is characterised in that step (4) concentration of described photosensitizing agent solution is 2～5mg/mL；
The addition quality of the photosensitizer added and Fe3O4The ratio of the mole of@P-COOH nanoparticle dispersion liquid is: (2.5-5) Mg:(0.5-10) mmol；
The temperature of described reaction is 30～40 DEG C, and the time is 24～48h, and the speed of vibration is 80～200rpm.
10. the application of the single dispersing fluorescence magnetic nano-probe as described in Claims 1 to 4 is arbitrary, it is characterised in that institute State nano-probe for preparing the preparation of targeted imaging cancerous cell or for preparing the medicine of targeted therapy of cancer.
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CN106941209B (en) * 2017-01-13 2019-04-09 合肥工业大学 A kind of preparation method for the monodisperse magnetic nano material that Terahertz and infrared optical device performance can be improved
CN106680486A (en) * 2017-01-24 2017-05-17 广东顺德工业设计研究院(广东顺德创新设计研究院) Preparation method of immunomagnetic beads
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