CN108324962A - A kind of preparation method of the ferroferric oxide nano granules of cluster structure - Google Patents
A kind of preparation method of the ferroferric oxide nano granules of cluster structure Download PDFInfo
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- CN108324962A CN108324962A CN201810074527.7A CN201810074527A CN108324962A CN 108324962 A CN108324962 A CN 108324962A CN 201810074527 A CN201810074527 A CN 201810074527A CN 108324962 A CN108324962 A CN 108324962A
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- ferroferric oxide
- oxide nano
- nano granules
- cystamine
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- SZVJSHCCFOBDDC-UHFFFAOYSA-N ferrosoferric oxide Chemical compound O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 title claims abstract description 176
- 239000008187 granular material Substances 0.000 title claims abstract description 37
- 238000002360 preparation method Methods 0.000 title claims abstract description 19
- 239000002105 nanoparticle Substances 0.000 claims abstract description 119
- OOTFVKOQINZBBF-UHFFFAOYSA-N cystamine Chemical compound CCSSCCN OOTFVKOQINZBBF-UHFFFAOYSA-N 0.000 claims abstract description 105
- 229940099500 cystamine Drugs 0.000 claims abstract description 104
- 230000004913 activation Effects 0.000 claims abstract description 9
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims description 158
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical group OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 claims description 58
- 239000002904 solvent Substances 0.000 claims description 23
- 238000006243 chemical reaction Methods 0.000 claims description 22
- YUFRRMZSSPQMOS-UHFFFAOYSA-N 2-(2-aminoethyldisulfanyl)ethanamine;hydron;dichloride Chemical compound Cl.Cl.NCCSSCCN YUFRRMZSSPQMOS-UHFFFAOYSA-N 0.000 claims description 15
- 238000003756 stirring Methods 0.000 claims description 10
- 239000003381 stabilizer Substances 0.000 claims description 8
- 239000001509 sodium citrate Substances 0.000 claims description 7
- 239000006185 dispersion Substances 0.000 claims description 6
- 150000002505 iron Chemical class 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 6
- 239000012498 ultrapure water Substances 0.000 claims description 6
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical group [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 5
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical group O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 5
- 238000002604 ultrasonography Methods 0.000 claims description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 4
- 230000003252 repetitive effect Effects 0.000 claims description 4
- 229940040526 anhydrous sodium acetate Drugs 0.000 claims description 3
- 239000003795 chemical substances by application Substances 0.000 claims description 3
- NGDIAZZSCVVCEW-UHFFFAOYSA-M sodium;butyl sulfate Chemical compound [Na+].CCCCOS([O-])(=O)=O NGDIAZZSCVVCEW-UHFFFAOYSA-M 0.000 claims description 3
- 229910021578 Iron(III) chloride Inorganic materials 0.000 claims description 2
- 238000005119 centrifugation Methods 0.000 claims description 2
- 238000001816 cooling Methods 0.000 claims description 2
- 238000004108 freeze drying Methods 0.000 claims description 2
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 claims description 2
- 230000035484 reaction time Effects 0.000 claims description 2
- 238000003384 imaging method Methods 0.000 abstract description 39
- 206010028980 Neoplasm Diseases 0.000 abstract description 36
- 230000000694 effects Effects 0.000 abstract description 18
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- 241001465754 Metazoa Species 0.000 abstract description 5
- 210000004027 cell Anatomy 0.000 description 41
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 37
- 239000000243 solution Substances 0.000 description 31
- 229910052742 iron Inorganic materials 0.000 description 25
- 238000002347 injection Methods 0.000 description 19
- 239000007924 injection Substances 0.000 description 19
- 238000002595 magnetic resonance imaging Methods 0.000 description 19
- 229960003180 glutathione Drugs 0.000 description 18
- 239000000463 material Substances 0.000 description 16
- 238000000034 method Methods 0.000 description 15
- 238000012360 testing method Methods 0.000 description 14
- 235000013339 cereals Nutrition 0.000 description 12
- 238000011580 nude mouse model Methods 0.000 description 11
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- 239000002616 MRI contrast agent Substances 0.000 description 8
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- 239000002872 contrast media Substances 0.000 description 7
- 238000001727 in vivo Methods 0.000 description 7
- 239000002245 particle Substances 0.000 description 7
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- 235000011083 sodium citrates Nutrition 0.000 description 6
- 239000000725 suspension Substances 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 241000699666 Mus <mouse, genus> Species 0.000 description 5
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- 238000001514 detection method Methods 0.000 description 4
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- 230000002121 endocytic effect Effects 0.000 description 4
- DCYOBGZUOMKFPA-UHFFFAOYSA-N iron(2+);iron(3+);octadecacyanide Chemical compound [Fe+2].[Fe+2].[Fe+2].[Fe+3].[Fe+3].[Fe+3].[Fe+3].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-] DCYOBGZUOMKFPA-UHFFFAOYSA-N 0.000 description 4
- 239000002122 magnetic nanoparticle Substances 0.000 description 4
- 239000002086 nanomaterial Substances 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- 229960003351 prussian blue Drugs 0.000 description 4
- 239000013225 prussian blue Substances 0.000 description 4
- 239000011734 sodium Substances 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 241000581650 Ivesia Species 0.000 description 3
- 108010087230 Sincalide Proteins 0.000 description 3
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- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 3
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- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 2
- 241000699660 Mus musculus Species 0.000 description 2
- 241000209094 Oryza Species 0.000 description 2
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- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 150000001805 chlorine compounds Chemical class 0.000 description 2
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- 238000000338 in vitro Methods 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 238000010253 intravenous injection Methods 0.000 description 2
- UQSXHKLRYXJYBZ-UHFFFAOYSA-N iron oxide Inorganic materials [Fe]=O UQSXHKLRYXJYBZ-UHFFFAOYSA-N 0.000 description 2
- 210000003734 kidney Anatomy 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 210000004072 lung Anatomy 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- NICDRCVJGXLKSF-UHFFFAOYSA-N nitric acid;trihydrochloride Chemical compound Cl.Cl.Cl.O[N+]([O-])=O NICDRCVJGXLKSF-UHFFFAOYSA-N 0.000 description 2
- NDLPOXTZKUMGOV-UHFFFAOYSA-N oxo(oxoferriooxy)iron hydrate Chemical compound O.O=[Fe]O[Fe]=O NDLPOXTZKUMGOV-UHFFFAOYSA-N 0.000 description 2
- 238000004445 quantitative analysis Methods 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 235000009566 rice Nutrition 0.000 description 2
- 239000013049 sediment Substances 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 239000001632 sodium acetate Substances 0.000 description 2
- 235000017281 sodium acetate Nutrition 0.000 description 2
- 210000000952 spleen Anatomy 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 238000007447 staining method Methods 0.000 description 2
- 101100298998 Caenorhabditis elegans pbs-3 gene Proteins 0.000 description 1
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 229910017135 Fe—O Inorganic materials 0.000 description 1
- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 description 1
- RWSXRVCMGQZWBV-PHDIDXHHSA-N L-Glutathione Natural products OC(=O)[C@H](N)CCC(=O)N[C@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-PHDIDXHHSA-N 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
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- 238000002835 absorbance Methods 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 239000012752 auxiliary agent Substances 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 229910001424 calcium ion Inorganic materials 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 238000013043 cell viability test Methods 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
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- 229910052802 copper Inorganic materials 0.000 description 1
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- 238000004132 cross linking Methods 0.000 description 1
- 239000003431 cross linking reagent Substances 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
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- 238000002329 infrared spectrum Methods 0.000 description 1
- WTFXARWRTYJXII-UHFFFAOYSA-N iron(2+);iron(3+);oxygen(2-) Chemical compound [O-2].[O-2].[O-2].[O-2].[Fe+2].[Fe+3].[Fe+3] WTFXARWRTYJXII-UHFFFAOYSA-N 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 238000002385 metal-ion deposition Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 238000009206 nuclear medicine Methods 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Chemical class 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 230000033116 oxidation-reduction process Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 230000032696 parturition Effects 0.000 description 1
- 239000002953 phosphate buffered saline Substances 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
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- 230000001105 regulatory effect Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
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- 238000012549 training Methods 0.000 description 1
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- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical class [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 1
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Classifications
-
- 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
- A61K49/1887—Agglomerates, clusters, i.e. more than one (super)(para)magnetic microparticle or nanoparticle are aggregated or entrapped in the same maxtrix
Landscapes
- Health & Medical Sciences (AREA)
- Nanotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Epidemiology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Radiology & Medical Imaging (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The present invention relates to a kind of preparation methods of the ferroferric oxide nano granules of cluster structure, including:The preparation of extra small ferroferric oxide nano granules, the preparation of the extra small ferroferric oxide nano granules solution after activation, the preparation of the ferroferric oxide nano granules of cluster structure.The present invention is simple, the Fe being prepared3O4/ Cystamine nano particles are with good stability and biocompatibility, sensitive to reductive condition, can realize the T of tumor locus in animal body1‑T2Bimodal MR imaging effects, can be effective as MR image-forming contrast mediums, have industrialization and commercialized application prospect.
Description
Technical field
The invention belongs to the preparation field of magnetic resonance imaging (MRI) contrast agent, more particularly to four oxygen of a kind of cluster structure
Change the preparation method of three iron nano-particles.
Background technology
All the time, malignant tumour is all to endanger the number one killer of human life, has death rate height, refractory treatment and evil
Change the features such as rapid.Therefore, the early diagnosis of tumour and specific treatment are particularly important.Currently, the detection means of tumour
Mainly have:Ultrasonic imaging, CT imagings, nuclear medicine (PET or SPECT) imaging and magnetic resonance imaging (MRI).With magnetic resonance skill
The development of art, sweep time are gradually shortened, and resolution ratio is gradually increased, and also more accurate for the detection of small lesion, 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, it is necessary to select suitable MRI contrast agent.Conventional MRI contrast agent is broadly divided into two classes:One kind is T1Add
The MRI contrast agent of power, one kind are T2The MRI contrast agent of weighting.T2The MRI contrast agent of weighting is to detect the first choice of soft tissue injury
Method has lot of documents report and is applied to cancer using superparamag-netic iron oxide as MRI negative contrast mediums so far
Diagnosis.However, in blood of human body, calcium ion enrichment region, metal ion deposition and human tissue injury position be in T2Imaging
It also will appear signal in the process to weaken phenomenon and obtain negative contrastographic picture, this often interferes clinical diagnosis, at this moment then needs into
As the higher T of resolution ratio1Weighted mri contrast agent.In short, the detection method of single mode often can not provide pinpoint accuracy and complete
The diagnostic result in face.With the development of science and technology, people are not only intended to relaxation rate higher to MRI contrast agent research, also tend to more
Function, multi-modalization, by integrating different diagnostic messages, therapeutic scheme is formulated in preferably guidance.Wherein, T1-T2Bimodal core
Magnetic resonance imaging contrast becomes the extensive concern that an emerging research direction causes people.
Build T1-T2The common methods of bimodal contrast agent have following 3 kinds:(1) by certain T2Contrast agent by with T1Radiography
Agent is combined, and the composite nano materials of acquisition is made to have T1-T2Bimodal MR imaging performances;(2) by magnetic nanoparticle
(such as Fe3O4Nano particle) synthetic method regulated and controled, synthesize the magnetic Nano material with suitable dimension.Studies have shown that
Fe3O4Magnetism and the size of nano particle have prodigious association, grain size<The Fe of 5nm3O4Nano particle (extra small Fe3O4Nanometer
Grain) magnetic moment is greatly reduced, T2Effect is suppressed, therefore may be used as T1Weighting or T1-T2The contrast agent of bimodal MR imagings;(3)
The nano particle of cluster type.With T1The weak magnetic nanoparticle of effect becomes large-sized magnetic enhancement and obtains because forming cluster
Obtain T2Imaging effect.Studies have shown that itself has T1Weight the extra small Fe of MR imaging effects3O4Magnetic nanoparticle forms cluster
After become large-sized, cause its magnetic enhancement, magnetic moment to be consequently increased.Therefore, the extra small Fe of cluster type3O4The T of nano particle1Effect
It is suppressed, and then is converted into T2Image-forming contrast medium.The extra small Fe of cluster type is built by suitable method3O4Nano particle, can also
Realize T1-T2Bimodal imaging effect.For example, Mao etc. prepares the Fe that grain size is 3.5nm, oligosaccharide wraps up3O4Nano particle.
This mono-dispersed nano particle shows good T1Effect, but can form cluster knot under acid tumor microenvironment
The Fe of structure3O4Self-assembly, and it is converted to T2Effect realizes the T of tumor locus1-T2Bimodal MR imaging contrasts (Mao et
al.,ACS Nano.2017,11,4582–4592)。
Domestic and foreign literature is retrieved, still without finding about the extra small ferroferric oxide nano granules of structure cluster type and for giving birth to
T in object1-T2The relevant report of bimodal MRI diagnosis.
Invention content
Technical problem to be solved by the invention is to provide a kind of preparations of the ferroferric oxide nano granules of cluster structure
Method, this method is simple, and reaction condition is mild, easily operated, and cost is relatively low, the Fe for the cluster structure being prepared3O4/
Cystamine nano particles are with good stability and biocompatibility, are easy to by cell endocytic, in vivo can be real
Existing T1-T2The bimodal MR imagings of conversion.
A kind of preparation method of the ferroferric oxide nano granules of cluster structure of the present invention, is as follows:
(1) trivalent iron salt being dissolved in solvent, stabilizer stirring is added, reaction promoter is added, solvent thermal reaction is cooling,
Centrifugation, it is dry, obtain extra small ferroferric oxide nano granules, the wherein ratio of trivalent iron salt, solvent, stabilizer and reaction promoter
For 0.62~0.68g:38~40mL:0.47~0.50g:1.312~1.33g;
(2) in a solvent by the extra small ferroferric oxide nano granules dispersion in step (1), ultrasound, by EDC and NHS
Activation, the extra small ferroferric oxide nano granules solution after being activated, wherein extra small ferroferric oxide nano granules, EDC and
The mass ratio of NHS is 13~16:28~36:17~20, the ratio of extra small ferroferric oxide nano granules and solvent is 28~
32mg:2~4mL;
(3) 2-aminoethyl disulfide dihydrochloride Cystamine dihydrochloride are scattered in solvent, step is added in ultrasound
(2) the extra small ferroferric oxide nano granules solution reaction in after activation is dialysed, and freeze-drying obtains the Fe of cluster structure3O4/
Cystamine nano particles, wherein the molar ratio of the extra small ferroferric oxide nano granules and 2-aminoethyl disulfide dihydrochloride after activation is 3
~5:1~2, the ratio of 2-aminoethyl disulfide dihydrochloride and solvent is 15~19mg:2~4mL.
Trivalent iron salt is anhydrous ferric chloride in the step (1);Solvent is diethylene glycol (DEG) DEG;Stabilizer is sodium citrate;Instead
It is anhydrous sodium acetate to answer auxiliary agent;The condition that stabilizer stirs, which is added, is:1~2h of the lower 80 DEG C of stirrings of air atmosphere.
Solvent thermal reaction temperature is 190~200 DEG C in the step (1), and the solvent thermal reaction time is 3~4h.
Centrifuge in the step (1) the specific steps are:8500~9000rpm centrifuges 10~15min, abandons supernatant, uses
Absolute ethyl alcohol back dissolving, 8500~9000rpm centrifuge 10~15min, repetitive operation 2~3 times.
Solvent thermal reaction carries out in 50mL autoclaves in the step (1);Drying is in 60~65 DEG C of items
It is dried under part.
Solvent is ultra-pure water in the step (2), (3).
Soak time is 2.5~3h in the step (2).
The temperature of the extra small ferroferric oxide nano granules solution reaction in step (2) after activation is added in the step (3)
Degree is room temperature, and the time is 60~72h.
Dialyse in the step (3) the specific steps are:The bag filter dialysis 2 for being 8000~14000 with molecular cut off
~3 days (1.5~2L of dialysis distilled water used every time, change water altogether 6~9 times).
The present invention uses 2-aminoethyl disulfide dihydrochloride (Cystamine dihydrochloride) by extra small Fe3O4Nano particle is handed over
It is unified into cluster structure.By controlling crosslinking agent and Fe3O4Ratio can prepare the cluster type that property is stable, size is controllable
Fe3O4/ Cystamine nano particles, and assign its higher T2Relaxation rate (26.4mM-1s-1).The Fe of cluster structure3O4/
Cystamine nano particles show the characteristic to reducing agent GSH response in vitro, under reductive condition, 2-aminoethyl disulfide dihydrochloride
In S-S can be broken, to make cluster type Fe3O4/ Cystamine nano particles are dispersed into Fe3O4Nano particle obtains higher
T1Relaxation rate (4.3mM-1s-1), realize the T of material1-T2Bimodal MR imaging functions.With individual extra small Fe3O4It compares, rolls into a ball
The Fe of clustering architecture3O4/ Cystamine nano particle hydrodynamic diameter biggers (134.4nm), it is easier to by cell endocytic, and it is right
The reproducibility environment of 4T1 cell interiors is sensitive, and the MR imagings of cellular level may be implemented.In addition to this, the Fe of cluster structure3O4/
Cystamine can be applied to by tail vein injection or intratumor injection in animal body, mice with tumor in-vivo tumour injection location
Fe3O4It can be with T visible in detail after/Cystamine1-T2The conversion process of bimodal MR imagings, passes through tail vein injection
Fe3O4It is observed that the MR imaging signals enhancing of tumor locus after/Cystamine.Therefore, the cluster structure that prepared by the present invention
Fe3O4/ Cystamine nano particles are worth with potential MR diagnostic applications.
The present invention uses infrared (FTIR), thermogravimetric analysis (TGA), inductively coupled plasma atomic emission spectrometry
(ICP-OES), the physics of magnetic nanoparticle prepared by Zeta electric potential and hydration grain size the methods of (DLS) characterization and chemically
Matter.And the T of nano particle is measured by MRI imagers1-T2Bimodal imaging performance is mixed by test material with GSH solution
Its external reduction response is tested in Relaxivity variation afterwards.Then nano particle is evaluated by hemolytic experiment and CCK-8 methods
Blood compatibility and cytotoxicity recycle prussian blue staining method and ICP-OES methods verification 4T1 cells to gulp down nano particle
Bite ability.By to mice with tumor intratumor injection or tail vein injection nano particle, observing the T of material1-T2Bimodal MR imagings are made
Shadow effect.
Advantageous effect
(1) present invention is simple, using the extra small Fe of high relaxation rate3O4Nano particle is made by the crosslinking of 2-aminoethyl disulfide dihydrochloride
With making extra small Fe3O4Nano particle forms stable cluster type Fe3O4/ Cystamine nano particles, the cluster type being prepared
Fe3O4/ Cystamine nano particles are with good stability and biocompatibility, T2Higher (the 26.4mM of relaxation rate-1s-1);
(2) the cluster type Fe being prepared3O4/ Cystamine nano particles show to respond reducing agent GSH in vitro
Characteristic, under reductive condition, the S-S in 2-aminoethyl disulfide dihydrochloride can be broken, to make cluster type Fe3O4/ Cystamine nanometers
Particle is dispersed into Fe3O4Nano particle obtains higher T1Relaxation rate (4.3mM-1s-1), realize the T of material1-T2Bimodal MR at
As function, has and implement commercialized foreground;
(3) Fe for the cluster structure being prepared3O4/ Cystamine can be applied to by intratumor injection in animal body,
Mice with tumor in-vivo tumour injection location Fe3O4It can be with T visible in detail after/Cystamine1-T2The conversion of bimodal MR imagings
Process.After tail vein injection, Fe3O4/ Cystamine nano particles have good internal T1MR imaging capabilities are weighted,
It can realize T in animal body1The MRI of weighting is diagnosed;These advantages make Fe prepared by the present invention3O4/ Cystamine nano particle energy
Enough it is effective as MR image-forming contrast mediums.
Description of the drawings
Fig. 1 is Fe in embodiment 33O4(a) and Fe3O4The infrared spectrogram of/Cystamine (b);
Fig. 2 is Fe in embodiment 33O4(a) and Fe3O4The thermogravimetric analysis figure of/Cystamine (b);
Fig. 3 is Fe in embodiment 43O4The stability test figure of/Cystamine nano particles;
Fig. 4 is Fe in embodiment 53O4(a)、Fe3O4Fe after/Cystamine (b) and glutathione GSH is handled3O4/
The TEM of Cystamine (c and d) nano particle schemes;
Fig. 5 is Fe in embodiment 63O4, Fe3O4/ Cystamine and glutathione GSH treated Fe3O4/Cystamine
(a) T of nano particle1Relaxation rate and (b) T2Relaxation rate;
Fig. 6 is Fe in embodiment 63O4, Fe3O4/ Cystamine and glutathione GSH treated Fe3O4/Cystamine
(a) T of nano particle1Weight MR images and (b) T2Weight MR images;
Fig. 7 is that CCK-8 methods measure 4T1 cells by PBS buffer solution (control), Fe in embodiment 73O4And Fe3O4/
Cystamine nano particles handled 24 hours under the conditions of concentration of iron 5-100 μ g/mL after cell viability test result;
Fig. 8 is Fe in embodiment 83O4Hemolytic experiment of/Cystamine the nano particles in the case where concentration of iron is 10-200 μ g/mL
As a result;
Fig. 9 is that 4T1 cells pass through PBS buffer solution (control a and e), Fe in embodiment 93O4PBS solution (the b- of nano particle
And Fe d)3O4The PBS solution (f-h) of/Cystamine handle 4 hours after after prussian blue staining as a result, wherein (b, f) iron
A concentration of 25 μ g/mL, (c, g) concentration of iron are 50 μ g/mL, and (d, h) concentration of iron is 100 μ g/mL;
Figure 10 is that 4T1 cells pass through PBS buffer solution, Fe in embodiment 93O4The PBS solution and Fe of nano particle3O4/
The PBS solution (within the scope of a concentration of 5-100 μ g/mL of Fe) of Cystamine nano particles collects cell after handling 4 hours, through king
It measures to obtain the iron content result of each cell endocytic using ICP-OES after water digestion;
Figure 11 is mouse intratumor injection Fe in embodiment 103O4PBS solution (the Fe a concentration of 3.2mM, 20 μ of/Cystamine
L) different time points afterwards, the T at tumour1-T2Bimodal MR image built-in testing results;
Figure 12 is to inject Fe respectively to mouse tail vein in embodiment 113O4The PBS solution (a) and Fe of nano particle3O4/
The PBS solution (b) (Fe a concentration of 0.1M, 150 μ L) of Cystamine nano particles different time points (0~120 minute) tumour afterwards
The T at position1It weights MR and is imaged picture;
Figure 13 is to inject Fe respectively to mouse tail vein in embodiment 113O4The PBS solution and Fe of nano particle3O4/
The T at the PBS solution (Fe a concentration of 0.1M, 150 μ L) of Cystamine nano particles different time points nude mouse tumor position afterwards1Add
Weigh the variation of MRI snr values;
Figure 14 is to inject PBS, Fe respectively to mouse tail vein in embodiment 123O4The PBS solution and Fe of nano particle3O4/
For the PBS solution (Fe a concentration of 0.1M, 150 μ L) of Cystamine nano particles after 12 hours, nano particle is each dirty in nude mouse
Distribution results in device.
Specific implementation mode
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In addition, it should also be understood that, after reading the content taught by the present invention, people in the art
Member can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims and limited
Range.
Embodiment 1
(1) 0.65g anhydrous ferric chlorides are dissolved in 40mL diglycols (also known as diethylene glycol (DEG), DEG), are added
0.47g sodium citrates (Na3Cit), in air atmosphere 80 DEG C stirring 1h, add 1.312g after sodium citrate is completely dissolved
Anhydrous sodium acetate powder continues stirring until sodium acetate powder and is completely dissolved, then solution is transferred in 50mL autoclaves,
It is reacted 4 hours in 200 DEG C;After reaction, cooled to room temperature, by product be transferred in 50mL centrifuge tubes 8500rpm from
The heart 15 minutes, abandons supernatant, and with absolute ethyl alcohol back dissolving, 8500rpm is centrifuged 15 minutes, then repetitive operation 3 times exists sediment
60 DEG C of drying, obtain extra small Fe3O4The extra small ferric oxide nanometer particle that nano particle, i.e. surface sodium citrate are stablized.
(2) by extra small Fe in step (1)3O4Nano particle (30mg) is scattered in 3mL ultra-pure waters, and ultrasonic 10min keeps its molten
Solution, EDC (59mg) and NHS (35mg) are dissolved in respectively in 1mL ultra-pure waters, above-mentioned extra small Fe is added to after being completely dissolved3O4Nanometer
It is reacted 3 hours in particle solution, activates Fe3O4The carboxyl of nano grain surface, the extra small ferriferrous oxide nano after being activated
Particle solution.
(3) 2-aminoethyl disulfide dihydrochloride (17mg) is scattered in 3mL ultra-pure waters, ultrasonic 10min makes it dissolve, and then adds dropwise
Enter the extra small ferroferric oxide nano granules solution after activation in step (2), react 72h at room temperature, after reaction with retention
The bag filter that molecular weight is 8000-14000 is dialysed 3 days (dialysis distilled water 2L used every time, change water altogether 9 times), and vacuum refrigeration is dry
It is dry, obtain the Fe of cluster3O4/ Cystamine nano particles.
Embodiment 2
Extra small Fe in Example 1 respectively3O4The Fe of nano particle and cluster3O4The 2mg dissolvings of/Cystamine nano particles
In 2mL ultra-pure waters, nano particle suspension is obtained, ultrasound uniformly, surveys surface potential and hydration grain size, test result such as table 1.It surveys
Test result shows:The extra small Fe being prepared3O4The Fe of nano particle and cluster3O4The surface electricity of/Cystamine nano particles
Gesture is respectively -33.2 and -21.4mV;It is respectively 25.6 and 134.4nm to be hydrated grain size.It is obtained from experimental result, it is monodispersed super
Small ferric oxide nanometer particle surface potential after being cross-linked into cluster increases, and hydrodynamic diameter significantly increases.Surface potential
Variation with hydration grain size illustrates the Fe of cluster3O4/ Cystamine nano particles have been formed.
Table 1
Sample | Potential (mV) | Hydrodynamic diameter (nm) | Polydispersity coefficient |
Fe3O4 | -33.2±1.7 | 25.6±1.6 | 0.53 |
Fe3O4/Cystamine | -21.4±2.6 | 134.4±2.6 | 0.18 |
Embodiment 3
Extra small Fe in Example 1 respectively3O4The Fe of nano particle and cluster3O4/ Cystamine nano particles 5mg is carried out
Examination of infrared spectrum (as shown in Figure 1) and thermogravimetric analysis (as shown in Figure 2).By parsing infared spectrum (such as Fig. 1), 466-
601cm-1The characteristic absorption peak of upper appearance is Fe3O4The stretching vibration of upper Fe-O, 3451cm-1Neighbouring peak is OH on hydrone
Stretching vibration peak, 2931cm-1And 2800cm-1Neighbouring characteristic absorption peak belongs to the stretching vibration of sodium citrate methylene.
Simultaneously in 1396-1642cm-1For the stretching vibration of C=O.And 1736 strong absworption peak belongs to 2-aminoethyl disulfide dihydrochloride in curve b
Amino and Fe3O4The amido bond that the carboxyl on surface generates after combining.551cm-1The characteristic peak at place is remarkably reinforced, and should belong to cystamine
S -- S in dihydrochloride.Infrared spectrogram is the result shows that Fe3O4Nano particle success is connect with 2-aminoethyl disulfide dihydrochloride.This
Outside, TGA test results (as shown in Figure 2) show Fe3O4Weight loss be 35.3%, Fe3O4The weight loss of/Cystamine is
37.7%, thus quantitative analysis goes out 2-aminoethyl disulfide dihydrochloride and is connected to Fe3O4Mass ratio on nano particle is 2.4%.
Embodiment 4
Weigh the Fe prepared in embodiment 13O4/ Cystamine nano particle 2mg, be dissolved in respectively 2mL water, PBS, DMEM and
In FBS, nanoparticulate dispersion hydrodynamic diameter (as shown in Figure 3) after 24 hours is monitored.The experimental results showed that Fe3O4/
Cystamine is dissolved in the solution in different medium and shows clear state, is not occurred after 24 hours stand
The phenomenon that any precipitation or aggregation, and nano particle is dispersed in the hydrodynamic diameter in different medium numerically without apparent
Variation.Illustrate Fe3O4/ Cystamine has good colloidal stability.
Embodiment 5
The Fe for taking the embodiment of the present invention 1 to prepare respectively3O4/ Cystamine and control material (Fe3O4Nano particle) dissolving
It is configured to nano granule suspension in 100 μ L ultra-pure waters.Respectively take 5 μ L Fe3O4/ Cystamine and Fe3O4Nanoparticle suspension
Drop is used for TEM after drying in air and tests (as shown in Figure 4) on copper mesh surface.In order to observe cluster type Fe3O4/
Pattern variation of the Cystamine nano particles under reductive condition, glutathione GSH is dissolved in ultra-pure water and is configured to concentration
For the reducing solution of 10mM, it is then added to Fe3O4In the aqueous solution of/Cystamine nano particles, 5 μ L glutathione is taken to handle
Fe3O4/ Cystamine nano particles are used for TEM after drying in air and test (as shown in Figure 4).TEM the result shows that:It is extra small
Fe3O4It shows monodispersed state and size ratio is more uniform, after the crosslinked action of 2-aminoethyl disulfide dihydrochloride, Fe3O4Group
It is polymerized to Fe3O4/ Cystamine forms cluster structure and becomes large-sized.And under the action of reducing agent GSH, two hydrochloric acid of cystamine
S-S in salt is disconnected, the Fe of cluster3O4/ Cystamine nano particles are dismissed again into monodispersed extra small Fe3O4.Illustrate Fe3O4/
Cystamine nano particles have cluster type structure, and sensitive to Redox Condition, can be in reducing environment by assembling
State is changed into monodisperse status.
Control material (Fe3O4Nano particle) specific preparation method be:
0.65g anhydrous ferric chlorides are dissolved in 40mL diglycols (also known as diethylene glycol (DEG), DEG), 0.47g lemons are added
Lemon acid sodium (Na3Cit), in air atmosphere 80 DEG C stirring 1h, the anhydrous vinegar of 1.312g is added after sodium citrate is completely dissolved
Sour sodium powder end, continues stirring until sodium acetate powder and is completely dissolved, then solution is transferred in 50mL autoclaves, in 200
DEG C reaction 4 hours;After reaction, product is transferred to 8500rpm in 50mL centrifuge tubes and centrifuges 15 points by cooled to room temperature
Clock abandons supernatant, and with absolute ethyl alcohol back dissolving, 8500rpm is centrifuged 15 minutes, then repetitive operation 3 times dries sediment at 60 DEG C
It is dry, obtain extra small Fe3O4Nano particle.
Embodiment 6
Relaxation rate reflects efficiency of the nano particle as MRI contrast agent, can falling by the relaxation time under various concentration
Number the Fitting Calculation obtains.Fe in embodiment 1 is measured by ICP-OES methods of testing3O4Fe in/Cystamine and embodiment 53O4It receives
Fe contents in rice grain are respectively 192 μ g/mg and 234 μ g/mg.Again with ultra-pure water prepare Fe concentration be followed successively by 0.1,0.2,
0.4, each 2mL of the aqueous solution of 0.8 and 1.6mM.In order to test cluster type Fe3O4The reduction response of/Cystamine nano particles,
Glutathione GSH is dissolved in the reducing solution for being configured to a concentration of 10mM in ultra-pure water, is then added to Fe3O4/Cystamine
In the aqueous solution of nano particle, the Fe of glutathione will be added to3O4/ Cystamine nano particles are configured to Fe concentration and are followed successively by
0.1, each 2mL of the aqueous solution of 0.2,0.4,0.8 and 1.6mM.Three kinds of material (Fe are measured respectively3O4/ Cystamine, Fe3O4With add
The Fe of glutathione is added3O4/ Cystamine nano particles) T of nano particle under different Fe concentration1And T2Relaxation time,
Relaxation time inverse and Fe concentration are subjected to linear fit (as shown in Figure 5) and test its T1And T2Weighted imaging figure is (such as Fig. 6 institutes
Show).Relaxation rate test result shows within the scope of a concentration of 0.1-1.6mM of Fe, the T of three kinds of materials1And T2Relaxation time and Fe
Concentration all has good linear relationship.Wherein, Fe3O4T1Relaxation rate is higher, r1For 4.3mM-1s-1.Form cluster
Fe3O4After/Cystamine nano particles, the T of material1Relaxation rate reduces (r1For 1.4mM-1s-1), and under the conditions of GSH,
Fe3O4/ Cystamine dismisses into Fe again3O4, the T of nano particle1Relaxation rate also significantly increases, and reaches and monodisperse Fe3O4It relaxes
The approximate numerical value of Henan rate.On the other hand, Fe3O4T2Relatively low (the r of relaxation rate2For 7.3mM-1s-1), form the Fe of cluster3O4/
After Cystamine nano particles, T2Relaxation rate significantly increases to 26.4mM-1s-1, under the conditions of GSH, Fe3O4/
Cystamine dismisses into Fe again3O4, the T of nano particle2Relaxation rate is also restored to monodisperse Fe3O4The numerical approximation of relaxation rate.
This result explanation, Fe3O4/ Cystamine not only has higher T2Relaxation rate can be converted into single point under reductive condition
Scattered Fe3O4, obtain higher T1Imaging performance.Fe3O4/ Cystamine has good T1-T2Bimodal MR imaging performances.
As can be seen from Figure 6 with the variation of Fe concentration (0.1-1.6mM), MRI signal is in good graded trend, test knot
Fruit illustrates that the material has good MRI imaging capabilities, can be used as the excellent T in the diagnosis of MRI molecular imagings1-T2Bimodal is made
Shadow agent.
Embodiment 7
The Fe prepared as model cell evaluation using 4T1 cells3O4And Fe3O4/ Cystamine nano particles are to cell survival
Influence.The content for weighing ferro element in embodiment 5 is the Fe of 1mg3O4With Fe in embodiment 13O4/ Cystamine nanometers
Grain is dispersed in the PBS solution for being configured to that concentration of iron is 1mg/mL in sterile PBS, ultraviolet irradiation is used in combination to sterilize overnight.Then super
In net workbench the Fe that concentration of iron is 5,10,25,50 and 100 μ g/mL is prepared with sterile PBS3O4And Fe3O4/ Cystamine receives
Rice grain suspension.4T1 cell seedings after 96 orifice plates respectively with Fe3O4And Fe3O4/ Cystamine nano particle (concentration of iron
For 5,10,25,50 and 100 μ g/mL) it is co-cultured 24 hours at 37 DEG C.Then, 20 μ L CCK-8 are added into cultivation plate hole,
After continuation is cultivated 4 hours at 37 DEG C, culture solution is discarded, and 100 μ L DMSO are added, oscillation measures at 450nm after twenty minutes
Light absorption value, on the basis of the absorption value of buffer solution PBS groups, the absorption value after the material processing of various concentration calculates by comparison
To the vigor (such as Fig. 7) of 4T1 cells.Compared with the cell of control group PBS processing, Fe3O4And Fe3O4/ Cystamine nanometers
Grain does not have a significant difference within the scope of 5~100 μ g/mL of experimental concentration to the vigor of 4T1 cells, cell viability all 85% with
On.This absolutely proves Fe prepared by embodiment 53O4The Fe prepared with embodiment 13O4/ Cystamine nano particles have good
Cell compatibility, can be applied to MRI image checkings in organism.
Embodiment 8
Contrast agent will certainly be in direct contact when being injected in vivo with blood, and the intervention of contrast agent can or can not generate haemolysis or
Other ill symptoms become one of an important factor for being had to take into account that for researcher.The nanometer prepared in order to ensure the present invention
Particle can be safely used for vivo biodistribution imaging diagnosis, have rated the Fe being prepared3O4The blood of/Cystamine nano particles
Compatibility.Weigh Fe in embodiment 13O4/ Cystamine nano particles, which are scattered in, is configured to concentration of iron as the molten of 1mg/mL in PBS
Liquid is mother liquor, and it is 10 μ g/mL, 20 μ g/mL, 50 μ g/mL, 100 μ g/mL and 200 μ g/mL then to prepare concentration of iron successively with PBS
Nano granule suspension.Suitable people's new blood is taken, (2000rpm, 5 minutes) is centrifuged first and removes supernatant, then by blood
Red blood cell is washed 5 times with PBS, is collected the red blood cell of health and is diluted 10 times with PBS.Again by the Fe of different concentration of iron3O4/
Cystamine nano granule suspensions are mixed with red blood cell respectively, and after standing 1 hour, 10000rpm is centrifuged 1 minute, is taken pictures simultaneously
Survey the ultraviolet absorption value (as shown in Figure 8) of supernatant.The process is using ultra-pure water as positive control, and PBS is as negative control.Figure
Fe is shown in 83O4The hemolytic test result of/Cystamine nano particles under given concentration of iron.It is clear by measuring upper layer
The hemolytic of the absorbance value quantitative assessment nano material of liquid.From test result as can be seen that reaching 200 μ g/mL in concentration of iron
When, Fe3O4The hemolysis rate of/Cystamine nano particles illustrates Fe prepared by embodiment 1 still less than 5%3O4/ Cystamine has
There is good blood compatibility, thus MR in organism can be can be safely used for and be imaged.
Embodiment 9
The Fe of various concentration is detected by prussian blue staining method3O4And Fe3O4/ Cystamine nano particles and 4T1 are thin
Born of the same parents co-culture 4 hours after endocytosis effect (such as Fig. 9).4T1 cells are with 2 × 105A/hole plantation is in 12 orifice plates, after being incubated overnight
Again respectively with Fe in embodiment 13O4Fe in/Cystamine and embodiment 53O4Nano particle PBS solution (Fe a concentration of 25,
50 and 100 μ g/mL) it is co-cultured 4 hours at 37 DEG C, and as a control group with the cell of PBS processing.Training is discarded after co-cultivation
Base is supported, cell is washed three times with PBS, photographed to record with phase contrast microscope after prussian blue staining, and blue is dyed according to cell
The depth come qualitative analysis cell phagocytosis nano particle number.In fig.9 with the raising of Fe concentration, trained altogether with nano particle
Blue is gradually deepened after foster cell is dyed, illustrates that the phagocytosis of two kinds of nano particles of 4T1 cells pair is in concentration dependent.
Under identical Fe concentration, Fe3O4Treated that cell blue degree is obviously deeper than Fe by/Cystamine3O4Treated for nano particle
Cell illustrates 4T1 cells to Fe3O4The phagocytosis of/Cystamine nano particles is more efficient.In addition, also using ICP-OES technologies
The average each cell of quantitative analysis swallows Fe3O4And Fe3O4The amount of/Cystamine nano particles.When the nanometer of various concentration
After particle (a concentration of 5,10,20,50 and 100 μ g/mL of Fe) PBS solution co-cultures 4 hours with 4T1 cells, cell is washed with PBS
3 times, then pancreatin digestion, counting, finally use chloroazotic acid vitellophag, and the total amount for swallowing ferro element per hole is tested with ICP-OES, is removed
Each cell is calculated with cell number and swallows iron content, and as a control group with the cell of PBS processing.As shown in Figure 10,
Intracellular almost without ferro element, and Fe is handled by PBS3O4Intracellular as concentration improves, the iron of/Cystamine processing
The content of element obviously rises.When concentration of iron is 100 μ g/mL, single 4T1 cells swallow Fe3O4/ Cystamine nano particles
Ferro element amount reaches 6.2pg/cell;And Fe3O4What is handled is intracellular less with the increase phagocytosis amount increase of concentration of iron, when iron is dense
When degree is 100 μ g/mL, single 4T1 cells swallow Fe3O4Nano particle ferro element is only 4.6pg/cell.And comparing two
Phagocytosis amount of the group material under identical concentration of iron can be seen that the Fe of cluster3O4/ Cystamine nano particles are by 4T1 cells
Phagocytosis will be apparently higher than extra small Fe3O4Nano particle.This is the experiment results show that Fe3O4/ Cystamine can more effectively by
4T1 cell endocytics provide reliable foundation to be efficiently applied to internal MR imagings for the material.
Embodiment 10
Fe prepared by embodiment 13O4/ Cystamine is configured to the 20 μ L PBS dispersion liquids of a concentration of 3.2mM of Fe.By 2
×106In a 4T1 cell inoculations to nude mouse, after two weeks when diameter of tumor reaches 0.6-1cm, pass through mouse intratumor injection
Fe3O4The PBS solution of/Cystamine nano particles scans the difference after mice with tumor injection material using NMR imaging instrument
The MR of time point (0,5,20 minute) tumor locus is imaged, and evaluates its T1-T2Bimodal MRI contrasting effects (such as Figure 11).As a result table
It is bright, in injection Fe3O4After/Cystamine, the MR T of mice with tumor tumor locus2Image is obviously dimmed, and after injection
5min reaches best T2Imaging effect.Then due to cluster type Fe3O4/ Cystamine nano particles are gradually dismissed at monodispersed
Fe3O4, T2Effect gradates as T1Effect.By T1Image can be seen that the T at mouse tumor position1Weight MR imaging effects
It gradually increases after injection, and tumour T1Image after injection 20min when reach most bright.This result also demonstrates above-mentioned knot
By illustrating cluster type Fe3O4/ Cystamine nano particles can dismiss into Fe under tumor microenvironment in vivo3O4, realize
By MR T2It is imaged onto T1The effect transfer process of imaging.These are the result shows that Fe3O4/ Cystamine has excellent, convertible
T1-T2Bimodal MRI diagnosing tumor effects, and MRI diagnosing tumors in organism can be successfully applied to.
Embodiment 11
Fe prepared by embodiment 13O4Fe prepared by/Cystamine and embodiment 53O4The iron measured according to ICP-OES
Concentration is configured to the 150 μ L PBS dispersion liquids of a concentration of 0.1M of Fe.By 2 × 106In a 4T1 cell inoculations to nude mouse, two weeks
After when diameter of tumor reaches 0.6-1cm, Fe is injected by tail vein respectively3O4/ Cystamine and Fe3O4Nano particle PBS
Solution evaluates the MR imaging effects (such as Figure 12) of tumor locus.After injection in 0 to 120 minutes, Fe is injected3O4Nude mice it is swollen
The light and shade variation of tumor position is not obvious, and injects Fe3O4The nude mouse tumor of/Cystamine nano particles obviously brightens, and is injecting
Afterwards 30 minutes when reach most bright.Show Fe3O4/ Cystamine nano particles have excellent T in vivo1Weight MR imaging effects
Fruit, imaging effect are better than Fe3O4Nano particle, it may be possible to due to Fe3O4/ Cystamine nano particles can more effectively lead to
Caused by the tumour infiltration retention effect that crossing enhances is enriched with by tumour.Figure 13 is the T of different time points tumor locus after injection1Add
MR imaging snr value variations are weighed, after injection in 0 to 120 minutes, inject Fe3O4Nude mouse tumor MRI signal value variation it is unknown
It is aobvious, and inject Fe3O4The nude mouse tumor MRI signal value of/Cystamine is remarkably reinforced, and signal-to-noise ratio reaches most at 30 minutes
Big value.This is consistent with the result of Figure 12.These results illustrate Fe prepared by embodiment 13O4/ Cystamine nano particles have very
Good internal T1MR imaging capabilities are weighted, can realize T in animal body by intravenous injection1The MRI of weighting is diagnosed.
To the Fe of mouse mainline cluster structure3O4/ Cystamine nano particles and material Fe3O4Nano particle is seen
Examine its T2MR imaging contexts are weighted, find the T that two kinds of materials are not shown2The MR imaging effects of weighting.May be Fe3O4/
Cystamine nano particles are very sensitive to organism oxidation-reduction quality condition, soon divide in reproducibility microenvironment in vivo
Dissipate Fe3O4Nano particle, to lose T2Caused by effect.
Embodiment 12
In order to study biological tissue's distribution situation of nano particle, Fe prepared by embodiment 13O4/ Cystamine and reality
Apply the Fe prepared in example 53O4(control group) is each configured to the 150 μ L PBS dispersion liquids of a concentration of 0.1M of Fe.By 2 × 106It is a
In 4T1 cell inoculations to nude mouse, after three weeks when diameter of tumor reaches 0.6-1cm, Fe is injected by tail vein respectively3O4With
Fe3O4After the PBS solution of/Cystamine nano particles, by 12 hours, nude mice is put to death and dissected, and is noted with tail vein
The nude mice of PBS is penetrated as blank group.It takes out the heart, liver, spleen, lung, kidney, tumour to weigh, is cut into the fragment of 2 × 2mm, then uses chloroazotic acid
It digests 24 hours, then measures the iron content of each sample with ICP-OES, finally calculate iron in each vitals
Content (Figure 14).The Fe prepared as can be seen from Figure in intravenous injection embodiment 13O4/ Cystamine nano particles or control material
Expect Fe3O4Nano particle (Fe a concentration of 0.1M, 150 μ L) after 12 hours, before the content of iron is relatively injected in the liver of mouse and spleen
It obviously increases, and in other organs, such as:The content of the heart, lung, kidney and tumour, iron is less.These results demonstrate embodiment
1 prepare nano particle can in Mice Body normal metabolite clearance.
Claims (8)
1. a kind of preparation method of the ferroferric oxide nano granules of cluster structure, is as follows:
(1) trivalent iron salt being dissolved in solvent, stabilizer stirring is added, reaction promoter is added, solvent thermal reaction is cooling, centrifugation,
It is dry, extra small ferroferric oxide nano granules are obtained, the ratio of wherein trivalent iron salt, solvent, stabilizer and reaction promoter is
0.62~0.68g:38~40mL:0.47~0.50g:1.312~1.33g;
(2) in a solvent by the extra small ferroferric oxide nano granules dispersion in step (1), ultrasound is activated by EDC and NHS,
Extra small ferroferric oxide nano granules solution after being activated, wherein extra small ferroferric oxide nano granules, EDC and NHS
Mass ratio is 13~16:28~36:17~20, the ratio of extra small ferroferric oxide nano granules and solvent is 28~32mg:2~
4mL;
(3) 2-aminoethyl disulfide dihydrochloride Cystamine dihydrochloride are scattered in solvent, ultrasound is added in step (2)
Extra small ferroferric oxide nano granules solution reaction after activation is dialysed, and freeze-drying obtains the Fe of cluster structure3O4/
Cystamine nano particles, wherein the molar ratio of the extra small ferroferric oxide nano granules and 2-aminoethyl disulfide dihydrochloride after activation is 3
~5:1~2, the ratio of 2-aminoethyl disulfide dihydrochloride and solvent is 15~19mg:2~4mL.
2. a kind of preparation method of the ferroferric oxide nano granules of cluster structure described in accordance with the claim 1, feature exist
In trivalent iron salt is anhydrous ferric chloride in the step (1);Solvent is diethylene glycol (DEG) DEG;Stabilizer is sodium citrate;Reaction helps
Agent is anhydrous sodium acetate;The condition that stabilizer stirs, which is added, is:1~2h of the lower 80 DEG C of stirrings of air atmosphere.
3. a kind of preparation method of the ferroferric oxide nano granules of cluster structure described in accordance with the claim 1, feature exist
In solvent thermal reaction temperature is 190~200 DEG C in the step (1), and the solvent thermal reaction time is 3~4h.
4. a kind of preparation method of the ferroferric oxide nano granules of cluster structure described in accordance with the claim 1, feature exist
Centrifuged in, the step (1) the specific steps are:8500~9000rpm centrifuges 10~15min, supernatant is abandoned, with anhydrous second
Alcohol back dissolving, 8500~9000rpm centrifuge 10~15min, repetitive operation 2~3 times.
5. a kind of preparation method of the ferroferric oxide nano granules of cluster structure described in accordance with the claim 1, feature exist
In solvent is ultra-pure water in the step (2), (3).
6. a kind of preparation method of the ferroferric oxide nano granules of cluster structure described in accordance with the claim 1, feature exist
In soak time is 2.5~3h in the step (2).
7. a kind of preparation method of the ferroferric oxide nano granules of cluster structure described in accordance with the claim 1, feature exist
In the temperature that the extra small ferroferric oxide nano granules solution reaction in step (2) after activation is added in the step (3) is room
Temperature, time are 60~72h.
8. a kind of preparation method of the ferroferric oxide nano granules of cluster structure described in accordance with the claim 1, feature exist
Dialyse in, the step (3) the specific steps are:The bag filter for being 8000~14000 with molecular cut off is dialysed 2~3 days.
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Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
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Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102372307A (en) * | 2011-11-21 | 2012-03-14 | 中国科学院苏州纳米技术与纳米仿生研究所 | Method for preparing magnetic hollow cluster from ferroferric oxide nano crystals by one step |
CN102526769A (en) * | 2010-09-28 | 2012-07-04 | 谢达斌 | Double-developer for CT and MRI simultaneously and preparation method thereof |
CN104826139A (en) * | 2015-05-04 | 2015-08-12 | 东华大学 | Method for preparing RGD peptide targeted ultra-small ferriferrous oxide MRI positive nanoprobe |
CN105884942A (en) * | 2016-05-18 | 2016-08-24 | 辽宁大学 | Polyacrylic acid-cystamine dihydrochloride-vitamin E succinate polymer, and preparation method and application thereof |
WO2017210612A1 (en) * | 2016-06-02 | 2017-12-07 | Case Western Reserve University | Stabilized crosslinked nanobubbles for diagnostic and therapeutic applications |
-
2018
- 2018-01-25 CN CN201810074527.7A patent/CN108324962B/en not_active Expired - Fee Related
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102526769A (en) * | 2010-09-28 | 2012-07-04 | 谢达斌 | Double-developer for CT and MRI simultaneously and preparation method thereof |
CN102372307A (en) * | 2011-11-21 | 2012-03-14 | 中国科学院苏州纳米技术与纳米仿生研究所 | Method for preparing magnetic hollow cluster from ferroferric oxide nano crystals by one step |
CN104826139A (en) * | 2015-05-04 | 2015-08-12 | 东华大学 | Method for preparing RGD peptide targeted ultra-small ferriferrous oxide MRI positive nanoprobe |
CN105884942A (en) * | 2016-05-18 | 2016-08-24 | 辽宁大学 | Polyacrylic acid-cystamine dihydrochloride-vitamin E succinate polymer, and preparation method and application thereof |
WO2017210612A1 (en) * | 2016-06-02 | 2017-12-07 | Case Western Reserve University | Stabilized crosslinked nanobubbles for diagnostic and therapeutic applications |
Non-Patent Citations (1)
Title |
---|
RAZIEH MALEKI ET.AL: "Fe3O4–cysteamine hydrochloride magnetic nanoparticles: New,efficient and recoverable nanocatalyst for Knoevenagel condensation reaction", 《APPL ORGANOMETAL CHEM》 * |
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