CN103463648A - Surface modified ferric oxide nucleus-gadolinium oxide shell composite nano particle, as well as preparation method and application of particle - Google Patents
Surface modified ferric oxide nucleus-gadolinium oxide shell composite nano particle, as well as preparation method and application of particle Download PDFInfo
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Abstract
A ferric oxide nucleus-gadolinium oxide shell composite nano particle with the surface modified by polyethylene glycol is 50-300nm in average particle size, and uses ferric oxide as a nucleus and gadolinium oxide as a shell; the surface of a composite nano porous microsphere is modified by polyethylene glycol; the particle is hydrophilic, has better stability in a wide pH (Potential of Hydrogen) scope, and is biocompatible; the surface of the nano particle is regular; a plurality of ducts are formed in a shell wall; and the nano particle has a T1 and T2 dual-mode magnetic resonance imaging function. The ferric oxide nucleus-gadolinium oxide shell composite nano particle with the surface modified by polyethylene glycol has an obvious dual-mode imaging effect; a preparation method is simple, green and environment-friendly; raw materials are cheap; the particle can be inoculated with a target molecule to detect a tumor more accurately; and the particle has high application values and wide application prospects in the fields of materials, biology and medicine. The invention discloses the preparation method of the particle.
Description
Technical field
The present invention relates to a kind of multifunctional nuclear shell structure inorganic nano contrast agent, specifically a kind of have a medicine carrying function, core is ferrum oxide, shell is Gadolinia., but the hollow-core construction carrying medicament, have the inorganic nano-particle of T1, T2 double-mode magnetic resonance imaging function and in the application of biomedical sector.
Background technology
In the modern medical service diagnosis, means for disease examination are more and more, diagnosis to disease, not only want position, scope and the character of clear and definite pathological changes, disease is made to location, the quantitative Morphologic Diagnosis qualitatively that reaches, also to react its functional variation, even will make comprehensively objectively analysis, estimate peripheral reference and the systemic conditions of pathological changes.Therefore, the modern medicine imaging examination plays vital effect, and has penetrated into the every field of disease treatment.
Cancer is a kind of individual cells or the out of control disease of a plurality of Growth of Cells, and the cell of propagation forms lump and is called tumor.Primary tumor is often because invading blood vessel or organ threat to life.Yet, cause that dead common situations is that primary tumor is diffused into other positions of health by the cell metastasis, the chance that losing perform the operation etc. treats.Therefore, early diagnosis is particularly crucial.At present, along with the development of the popular body scan instrument of the economy based on high-resolution such as mr imaging techniques, the effective examination project that can carry out how dissimilar solid tumor.
Although magnetic resonance imager has obtained great exploitation, but the examination for some atomic little focuses, and no matter be from economics point or ethics angle, examination must be sensitive, accurate as much as possible, a lot of MR diagnosises must rely on the auxiliary of magnetic resonance contrast agent.The magnetic resonance contrast agent of present clinical use has following characteristics:
1, the contrast agent of clinical use has: luxuriant and rich with fragrance power magnetic Feridex is the iron oxide magnetic nano particle, by the T2 weighted magnetic resonance imaging pattern that reduces the signal radiography, is scanned, and when localized magnetization is larger, can make the scalloping distortion.Another kind of tie up the coordination compound that fine Magnevist is gadolinium for mug(unit of measure), the T1 weighted magnetic resonance imaging pattern by the enhancing signal radiography is scanned, clear picture, but accretion rate is too fast, can not fully detect.
2, the T1 weighting looks like to be suitable for observing normal anatomical structures, and T2 weighting picture is better for observing pathological tissues, fat (comprising skeleton) T1, and T2 all presents the white shadow of high signal; Brain and muscle are the ghost of msp signal; Liquid, aqueous T1 is the low signal shadow, and T2 is high signal; Cortical bone, ligament, calcification T1, T2 all is low signal.
For the clinical These characteristics with nuclear magnetic resonance and contrast agent, further improve the effect of magnetic resonance contrast agent, especially improve sensitivity and accuracy rate that tumor tissues is detected, can also carry therapeutant and enter tumor locus, play the double effects of diagnosis and treatment simultaneously.A kind of contrast agent that collects T1 and T2 double-mode magnetic resonance imaging function of plan invention, simultaneously can carrying medicament carry out the targeted therapy of tumor.
Nanoparticle is combined together to form to the concern that composite nano materials more and more causes everybody, be mainly because composite nanoparticle with the character of various one-component nano materials and can realize multi-functional application, and many-sided information is provided, make diagnosis and treatment more accurate.For example, magnetic-fluorescent nano particles, noble metal-magnetic nano-particle, Graphene-magnetic nano-particle, the composites such as CNT-magnetic nano-particle, make single particle have fluorescence and magnetic and have a wide range of applications at chemistry, biological, material science.
Use for reference the theory of Nanocomposites, the object of the invention is to by preparing ferrum oxide (Fe
3o
4) be core, Gadolinia. (Gd
2o
3) be the core-shell structure nanometer particle of shell, but form the method for pore structure carrying medicament by calcining, make compound nano material there is double-mode magnetic resonance radiography and targeted drug transportation function simultaneously.By the EPR effect, tumor tissues is realized to specific recognition, by T1, two kinds of imaging patterns of T2, lesion locations or tumor are carried out to double check, and therapeutant is sent into to tumor tissues, realize the double-mode magnetic resonance imaging detection of tumor tissues and the purpose of targeted therapy.
The invention provides a kind of hud typed T1 of ferrum oxide-Gadolinia. and T2 double-mode magnetic resonance contrast agent with meso-hole structure.The double mode targeted contrast agent of T1, T2 has dual magnetic performance, can to lesion locations or tumor, be detected by T1, two kinds of imaging patterns of T2.Double-mode detects pathological changes or tumor can reduce the detection fault rate greatly, determines more accurately lesion locations.Can change its accretion rate by regulating the size of contrast agent nano-particles size simultaneously, obtain the suitable form time, guarantee to scan fully, detect.
Summary of the invention
The purpose of this invention is to provide a kind of ferrum oxide is core, nucleocapsid structure porous, inorganic nanometer double-mode magnetic resonance contrast agent that Gadolinia. is the shell composition and preparation method thereof.And rely on the EPR effect of tumor to realize the passive target transmission by the physicochemical property (particle diameter, pattern, surface nature) of regulating composite nanoparticle, or realize initiatively targeting radiography and detection by grafting targeting group (small peptide, antibody etc.).
The object of the present invention is achieved like this:
The polyethyleneglycol modified ferrum oxide core in a kind of surface-Gadolinia. shell composite nanometer particle, it is that ferrum oxide that a kind of mean diameter is 50-300nm is the composite nanoparticle that composite Nano porous microsphere finishing that core-Gadolinia. is shell has Polyethylene Glycol, it is hydrophilic, there is stability preferably in the pH scope widely, there is biocompatibility, the nanoparticle surface rule, have a lot of ducts on shell wall, have the function of T1, the imaging of T2 double-mode magnetic resonance.
Above-mentioned composite nanoparticle, the number-average molecular weight of the Polyethylene Glycol of described modification is 200-20000.
The preparation method of the polyethyleneglycol modified ferrum oxide core in a kind of surface-Gadolinia. shell composite nanometer particle, it comprises the steps:
(1) preparation of ferric oxide nano core and finishing:
(1) use hydro-thermal method, solvent thermal decomposition method or coprecipitation prepare the ferric oxide nano microgranule, and mean diameter is 5～20nm.(Chem.Mater., 2003,15,1367; Angew.Chem., Int.Ed., 2005,44,123; Or J.Mater.Chem., 2012,22,3311)
(2) be coated with the preparation of the ferric oxide nano microgranule of chitosan:
Method one: get 50 μ L～200 μ L γ-Fe
2o
3(or Fe
3o
4) to add containing number-average molecular weight be 500 to nanometer solution (4wt%), the chitosan that deacetylation is 90%, in the 20mL aqueous solution of (CS10～50mg) and ethylenediaminetetraacetic acid (EDTA2～10mg), under stirring condition, slowly drip a certain amount of ethanol in system, system adds the amino on 20 μ L～100 μ L glutaraldehyde (25%) cross-linked chitosan surfaces when clear becomes colloid solution, and room temperature reaction spends the night, and obtains crosslinked CS-EDTA-γ-Fe
2o
3(or Fe
3o
4) composite nano-microsphere, crosslinked CS-EDTA-γ-Fe by above-mentioned magnetic polymer microsphere
2o
3(or Fe
3o
4) emulsion of composite nano-microsphere on supercentrifuge centrifugal 30 minutes, rotating speed 12000 turns, and takes out the precipitation of lower floor, with after clear water washing three times, be dispersed in intermediate water, then press the above-mentioned steps centrifugal treating twice, finally by the sample redispersion of gained in intermediate water, obtain pure CS-γ-Fe
2o
3(or Fe
3o
4) solution of composite nano-microsphere;
Method two: take refining 10-30 ten thousand molecular weight, chitosan 0.25 gram of deacetylation (80-95%), be dissolved in acrylic acid aqueous solution that 50ml contains amount of calculation, wherein: in the amino in chitosan and acrylic acid, the ratio of carboxyl is 1:1, under normal temperature condition, stir, until chitosan dissolves fully, probably need 15 minutes, then get 200 μ L γ-Fe
2o
3(or Fe
3o
4) nanometer solution (4wt%) adds above-mentioned mixed solution, strong agitation 10 minutes, load onto condensing unit, connects condensed water, is rapidly heated 70 ℃, and add a small amount of initiator: potassium peroxydisulfate remains on 70 ℃ of reactions.The variation of observe system color in course of reaction.After the system color becomes milky, continue reaction 2 hours, to guarantee that the acrylic acid in system reacts completely, then the glutaraldehyde cross-linking 2 hours that adds 0.5ml, the emulsion of gained is poured one into can only see through molecular weight in the bag filter that is less than 10,000 materials, room temperature, in the phosphate buffer that is 4.5 at pH value, dialysis is 48 hours, and to remove wherein unreacted acrylic molecules and to maintain a stable pH value, room temperature is preserved.
(2) preparation of ferrum oxide core-Gadolinia. shell composite nanometer particle:
The above-mentioned ferric oxide nano particles that is coated with chitosan of 0.05g is dispersed in the 50mL deionized water, then by the Gd (NO of 14.30mL0.2M
3)
3with the 12mL0.5Mol/L aqueous solution of urea, add in above-mentioned ferric oxide nano particles solution, after the ultrasonic 20min of mixture formed, transfer in a rustless steel high-pressure reactor in the tetrafluoroethene village (capacity is 50mL), high-pressure reactor is heated to 180 ℃ and react 18h, then cool to room temperature, separating by magnetic the magnetic product obtained alternately washs 4 times with ethanol and distilled water, then at 60 ℃ of lower vacuum dryings, spend the night, the dry powder obtained is calcined 2 hours under 700 ℃ of argon atmosphers, finally forming ferrum oxide is core, the composite nano particle that Gadolinia. is shell;
Method two, 2mL, the Gd (NO of 0.5Mol/L
3)
3aqueous solution adds (10mL ethanol and 10mL water) in the mixed solvent of the second alcohol and water in the 50mL round-bottomed flask to form limpid solution, add 0.6g carbamide in above-mentioned solution under strong agitation, after 5min, under ultrasonication to the ferric oxide nano microgranule (0.10g) that adds above-mentioned chitosan parcel in above-mentioned mixed solution, powerful ultrasonic 15min, then, the flask of mixed solution will be housed under strong agitation at 90 ℃ of heating 6h, by alternately washing of second alcohol and water for the isolated product of magnetic, at 60 ℃ of lower vacuum dryings, spend the night, the dry powder obtained is at 800 ℃ of calcining 2h, obtain ferrum oxide core-Gadolinia. shell nanoparticle,
(3) prepare polyethyleneglycol modified ferrum oxide core-Gadolinia. shell composite nanometer particle.
Under nitrogen protection, add 25mL anhydrous tetrahydro furan (THF) and 2～10g monomethyl Polyethylene Glycol (m-PEG, Mw=200～20000) in round-bottomed flask, then, by 4mL3-isocyanide propyl-triethoxysilicane ((C
2h
5o)
3si (CH
2)
3nCO, 3-Isocyanatopropyltriethoxysilane, IPTS) and 0.5mL dibutyl tin laurate (DBTL) add in above-mentioned m-PEG solution, at room temperature, continue to stir after two days, obtain silanization m-PEG, it is precipitated out from tetrahydrofuran solution by adding (just) hexane, 60 ℃ of vacuum dryings spend the night, and obtain triethoxysilicane alkanisation methyl Polyethylene Glycol (trialkoxysilylated m-PEG).
By silanization m-PEG grafting in the composite nano-granule sub-surface: during the composite nanoparticle that by 100mg prepared by above-mentioned silanization m-PEG and 20mg step (two) is sneaked into the 50mL deionized water, after the ultrasonic 6h of room temperature, centrifugally wash away unreacted raw material, obtain the polyethyleneglycol modified ferrum oxide core in surface-Gadolinia. shell composite nanometer particle.
The application of the polyethyleneglycol modified ferrum oxide core in above-mentioned surface-Gadolinia. shell composite nanometer particle in the biological contrast agent of preparation.
The polyethyleneglycol modified ferrum oxide core in the above-mentioned surface-application of Gadolinia. shell composite nanometer particle in preparing the drug-loaded biological contrast agent.
Main points of the present invention are: at first prepare the controlled ferric oxide nano particles of particle diameter, ferric oxide nano microgranule and chitosan macromole are assembled in solution, form the ferric oxide nano particles of chitosan parcel, shell by hydro-thermal method at chitosan Surface Creation one deck Gadolinia., prepare the ferrum oxide of porous-Gadolinia. inorganic composite nano microgranule by the method for high-temperature calcination, finally, again at its finishing Polyethylene Glycol, prepare T1 weighted sum T2 weighting double-mode magnetic resonance imaging porous, inorganic nanoparticle.The size control of the polyethyleneglycol modified ferrum oxide core by surface-Gadolinia. shell composite nanometer particle is realized passive target imaging effect, further grafting targeting group also, tumor cell and tissue are realized to specific recognition, realize the application of targeting radiography and treatment, there is good biology and medical value.
The polyethyleneglycol modified ferrum oxide core in surface of the present invention-Gadolinia. shell composite nanometer particle Double-mode imaging successful, preparation method is simple, environmental protection, raw material is cheap, can also detect more accurately tumor by the grafting targeted molecular, the present invention will have great using value and wide application prospect at material, biology, field of medicaments.
Advantage of the present invention is:
1, experimentation carries out fully in aqueous phase system, does not use any organic solvent and surfactant, environmental protection;
2, the present invention has the function of T1, the imaging of T2 double-mode magnetic resonance, not only can provide the information of tumor-localizing, can also present function of organization's information, makes diagnosing tumor accurate.Magnetic resonance radiography reinforced effects at tumor locus is obvious;
3, obtain the longer blood pond half-life by the physical and chemical parameter (particle diameter, surface nature) of regulating nanoparticle, thereby obtain the desirable form phase, make diagnosing tumor fully, thoroughly; Be easy in the tumor locus enrichment as nanoparticle, promote radiography to strengthen simultaneously;
4, the present invention is loose structure inorganic nano carrier, but therapeutic effect is played at load treatment reagent target tumor position;
5, the invention provides the ferrum oxide that a kind of mean diameter is 50～300nm-Gadolinia. composite Nano porous microsphere, it is hydrophilic, has stability preferably, biocompatibility in the pH scope widely.The microsphere surface rule, have a lot of ducts on shell wall, for absorption and the release of medicine provides passage, and the mechanical strength of inorganic nano-particle is high, not yielding.In fields such as biological medicine and clinical medicine, be with a wide range of applications.
The accompanying drawing explanation
Figure l is the preparation technology figure of the polyethyleneglycol modified ferrum oxide core in surface-Gadolinia. shell composite nanometer particle.
Fig. 2 is the transmission electron micrograph of the polyethyleneglycol modified ferrum oxide core in surface-Gadolinia. shell composite nanometer particle.(A) embodiment 1; (B) embodiment 2.
Fig. 3 is the T1 weighted imaging figure of the polyethyleneglycol modified ferrum oxide core in the surface of embodiment 1 preparation-Gadolinia. shell composite nanometer particle.(A) after before the injection, (B) injects 1 hour
Fig. 4 is the T2 weighted imaging figure of the polyethyleneglycol modified ferrum oxide core in the surface of embodiment 1 preparation-Gadolinia. shell composite nanometer particle.(A) after before the injection, (B) injects 1 hour
The specific embodiment
Describe technology contents of the present invention in detail below by embodiment, but the present invention is not so limited.
The preparation of the polyethyleneglycol modified ferrum oxide core of EXAMPLE l, a kind of surface-Gadolinia. shell composite nanometer particle
Press Chem.Mater., 2003,15,1367; Angew.Chem., Int.Ed., 2005,44,123; Or J.Mater.Chem., 2012,22,3311. methods of putting down in writing prepare γ-Fe
2o
3(or Fe
3o
4) nanometer solution.
Get 100 μ L γ-Fe
2o
3(or Fe
3o
4) nanometer solution (4wt%) adds chitosan-containing (CS) (number-average molecular weight 500, deacetylation 90%) in the 20mL aqueous solution of 10mg and EDTA2mg, under stirring condition, slowly drip a certain amount of ethanol in system, system adds the amino on 20 μ L glutaraldehyde (25%) cross-linked chitosan surfaces when clear becomes colloid solution, room temperature reaction spends the night, and obtains crosslinked CS-EDTA-γ-Fe
2o
3(or Fe
3o
4) composite nano-microsphere.By the emulsion of magnetic polymer microsphere on supercentrifuge centrifugal 30 minutes, rotating speed 12000 turned.Take out the precipitation of lower floor, with after clear water washing three times, be dispersed in intermediate water, then press the above-mentioned steps centrifugal treating twice.Finally by the sample redispersion of gained in intermediate water, obtain pure CS-γ-Fe
2o
3(or Fe
3o
4) solution of composite nano-microsphere.
0.05g being coated with the ferric oxide nano particles of chitosan is dispersed in the 50mL deionized water.Then by the Gd (NO of 14.30mL0.2mol/L
3)
3, and the 12mL0.5mol/L aqueous solution of urea adds in above-mentioned ferric oxide nano particles dispersion liquid, after the ultrasonic 20min of the mixture of formation, transfers in a rustless steel high-pressure reactor in the tetrafluoroethene village (capacity is 50mL).High-pressure reactor is heated to 180 ℃ and react 18h, then cool to room temperature.Separate by magnetic the magnetic product of tool obtained and alternately wash 4 times with ethanol and distilled water, then spend the night at lower 60 ℃ of vacuum dryings.Above-mentioned dry powder is calcined 2 hours under 700 ℃ of argon atmosphers.Finally forming ferrum oxide is core, the porous nano microgranule that Gadolinia. is shell.
Under nitrogen protection, 25mL anhydrous tetrahydro furan (THF) adds 2g monomethyl Polyethylene Glycol (m-PEG is housed, Mw=200) in round-bottomed flask, then, by 3-isocyanide propyl-triethoxysilicane ((C2H5O) 3Si (CH2) 3NCO, 3-Isocyanatopropyltriethoxysilane, IPTS) 4mL and 0.5mL dibutyl tin laurate (DBTL) add above-mentioned m-PEG solution, at room temperature, continue to stir after two days, obtain silanization m-PEG, by adding (just) hexane that product is precipitated out from tetrahydrofuran solution, the 60oC vacuum drying spends the night.Obtain triethoxysilicane alkanisation methyl Polyethylene Glycol (trialkoxysilylated m-PEG).By silanization m-PEG grafting in the composite nano-granule sub-surface: during by 100mg, the above-mentioned ferrum oxide of above-mentioned silanization m-PEG and 20mg-Gadolinia. composite nano particle is sneaked into the 50mL deionized water.After the ultrasonic 6h of room temperature, centrifugally wash away unreacted raw material, obtain the polyethyleneglycol modified ferrum oxide core in surface-Gadolinia. shell composite nanometer particle GF1.It is kept to 10mL, in the HEPES buffer solution of the pH=7.3 of 0.05mol/L (HEPES:N-(2-ethoxy) piperazine-N'-2-ethane sulfonic acid).
As shown in Figure 2 (A) shows, the mean diameter of GF1 is 50nm to the transmission electron microscope photo of GF1.
The preparation of the polyethyleneglycol modified ferrum oxide core of embodiment 2, a kind of surface-Gadolinia. shell composite nanometer particle
Take refining after number-average molecular weight 300,000, the chitosan 0.25g of deacetylation 90%, be dissolved in acrylic acid aqueous solution that 50ml contains amount of calculation.Wherein: in the amino in chitosan and acrylic acid, the ratio of carboxyl is 1:1.After 15 minutes, load onto condensing unit, connect condensed water.Get 200 μ L γ-Fe2O3(or Fe3O4) nanometer solution (4wt%) adds above-mentioned mixed solution, strong agitation 10 minutes, the 70oC that is rapidly heated, add a small amount of initiator: potassium peroxydisulfate, remain on 70oC reaction 2h, react completely to guarantee the acrylic acid in system.Then the crosslinked 2h of glutaraldehyde (25wt%) that adds 0.5ml.The emulsion of gained is poured one into can only see through molecular weight in the bag filter that is less than 10,000 materials, room temperature, and the 48h that dialyses in the phosphate buffer that is 4.5 at pH value, to remove wherein unreacted acrylic molecules and to maintain a stable pH value.Room temperature is preserved.
By 2mL, 0.5M Gd (NO3) 3 aqueous solutions add (10mL ethanol and 10mL water) in the mixed solvent of the second alcohol and water in the 50mL round-bottomed flask to form limpid solution.Add 0.6g carbamide in above-mentioned solution under strong agitation.After 5min, under ultrasonication to the ferric oxide nano microgranule (0.10g) that adds the chitosan parcel in above-mentioned mixed solution, powerful ultrasonic 15min.The flask that then, mixed solution will be housed under strong agitation heats 6h at 90oC.Product after separating by magnetic alternately washs 4 times with the second alcohol and water, and under 60oC, vacuum drying spends the night.Above-mentioned dry powder is calcined 2h at 800oC, obtains ferrum oxide core-Gadolinia. shell porous nano microgranule.
Under nitrogen protection; 25mL anhydrous tetrahydro furan (THF) adds 10g monomethyl Polyethylene Glycol (m-PEG is housed; Mw=20000) in round-bottomed flask; then; 3-isocyanide propyl-triethoxysilicane ((C2H5O) 3Si (CH2) 3NCO; 3-Isocyanatopropyltriethoxysilane; IPTS) 4mL and 0.5mL dibutyl tin laurate (DBTL) add m-PEG solution; at room temperature; continue to stir after two days; obtain silanization m-PEG, be precipitated out from tetrahydrofuran solution by (just) hexane, the 60oC vacuum drying spends the night.Obtain triethoxysilicane alkanisation methyl Polyethylene Glycol (trialkoxysilylated m-PEG).By silanization m-PEG grafting in the composite nano-granule sub-surface: during the composite nanoparticle of the above-mentioned preparation of 100mg silanization m-PEG and20mg is sneaked into the 50mL deionized water.After the ultrasonic 6h of room temperature, centrifugally wash away unreacted raw material, obtain the polyethyleneglycol modified ferrum oxide core in surface-Gadolinia. shell composite nanometer particle GF2.It is kept to 10mL, in the HEPES buffer solution of the pH=7.3 of 0.05M.
The GF2 transmission electron microscope photo is as shown in Fig. 2 (B), and the mean diameter of GF2 is 300nm.
The preparation of the ferrum oxide core of embodiment 3, load doxorubicin hydrochloride-Gadolinia. shell composite nano particle
The doxorubicin hydrochloride of getting the polyethyleneglycol modified ferrum oxide core in the surface of lyophilizing-Gadolinia. shell composite nanometer particle powder 15mg and 1.5mg is mixed in the 20mL deionized water, stirring at room 48h, centrifugal, wash three times, remove supernatant liquid and obtain the polyethyleneglycol modified ferrum oxide core in the surface of load doxorubicin hydrochloride-Gadolinia. shell composite nanometer particle.In above-mentioned medicine carrying composite nanoparticle, drug loading is 11.2%, and medicine carrying efficiency is 95.4%.
Embodiment 4, gadolinium ion release test
Surface is polyethyleneglycol modified, and ferrum oxide core-the Gadolinia. shell composite nanometer particle is ultrasonic is dispersed in deionized water, form the suspension of 1.1mMol/L, preserve after 8 days for 37 ℃, centrifugal (13000 turn/30min) gets supernatant, by inductively coupled plasma spectroscopic assay Gd3+ ion concentration wherein.The test result of ICP shows that the amount of Gd3+ in supernatant is 0.086 μ g/5mg composite nanoparticle.
The toy T1 weighted magnetic resonance imaging of the composite nanoparticle that embodiment 5, embodiment 1 make
Mouse H22 hepatoma cell line conservation, in the ICR mouse peritoneal, is got ascites after ascites on the 7th～9 generates, and counts and adjust cell number to 4～6 * 106/mL under microscope, is inoculated in respectively ICR mice (oxter, left side, every 0.2ml).7-8 days after cell seeding, selecting the mice of gross tumor volume 300mm3 left and right is experimental model, zoopery has obtained the approval of regional ethics organizing committee.MRI carries out on 3T Siemens Magnetom Trio, uses T1 weighting sequence (TR=40ms, TE=2.96ms, flip angle=60o, 256 * 256 matrixes, slice thickness=1.0mm, FOV6 * 6, NEX6.0, echo 1/1).0.5h, 1h before the polyethyleneglycol modified ferrum oxide core on intravenous injection surface-Gadolinia. shell composite nanometer particle, after quiet notes, 1.5h and 2h implement scanning.Embodiment 1 (0.2mg/mL) intravenous injection of PBS solution (150 μ L) enters in mouse body, gathers the crown and horizontal tangent plane radiography of MRI of each mouse, and mouse keeps at least normal body temperature of 120min.
After 1 hour, tumor site can see that obvious signal brightens, illustrate and reached Targeting Effect, (A) before borderline tumor is in injection of contrast medium, rear (B) signal have obviously and brighten, the polyethyleneglycol modified ferrum oxide core in proof surface-Gadolinia. shell composite nanometer particle has reached the effect (as Fig. 3 (A), (B) shown in) of T1 nuclear magnetic resonance effect and target tumor tissue.The highest positive signal of liver is also to appear at about 2 hours, and the kidney signal intensity is little, shows that the polyethyleneglycol modified ferrum oxide core in this surface-Gadolinia. shell composite nanometer particle is mainly by liver metabolism.
The toy T2 weighted magnetic resonance imaging of the composite nanoparticle that embodiment 6, embodiment 1 make
By the test of the magnetic resonance imager of 3.0T and obtain, parameter is: the TR/TE=4000/89 millisecond, select 2.2 millimeters of layer thicknesses, bandwidth BW=220 hertz/pixel.Fov=80 * 80 millimeter.Fig. 4 shows: the polyethyleneglycol modified ferrum oxide core in the surface of embodiment 1 preparation-Gadolinia. shell composite nanometer particle injects in the tumor nude mouse by the tail vein, and each normal structure liver and spleen have obviously and brighten at the forward and backward signal of injection of contrast medium.In the time of 30 minutes, tumor site can see that obvious signal is dimmed, illustrate and reached Targeting Effect, at the borderline tumor place, before the polyethyleneglycol modified ferrum oxide core in injection surface-Gadolinia. shell composite nanometer particle, (A), rear (B) signal have obviously dimmed, and As time goes on, signal strengthens more obvious, after lasting till 2 hours, signal falls into a decline, and proves that the polyethyleneglycol modified ferrum oxide core in surface-Gadolinia. shell composite nanometer particle has reached the effect of T2 nuclear magnetic resonance effect and target tumor tissue.
Claims (8)
1. the polyethyleneglycol modified ferrum oxide core in a surface-Gadolinia. shell composite nanometer particle, it is characterized in that: it is that ferrum oxide that a kind of mean diameter is 50-300nm is the composite nanoparticle that composite Nano porous microsphere finishing that core-Gadolinia. is shell has Polyethylene Glycol, it is hydrophilic, there is stability preferably in the pH scope widely, there is biocompatibility, the nanoparticle surface rule, have a lot of ducts on shell wall, have the function of T1, the imaging of T2 double-mode magnetic resonance.
2. composite nanoparticle according to claim 1, it is characterized in that: the number-average molecular weight of the Polyethylene Glycol of described modification is 200-20000.
3. a method for preparing composite nanoparticle claimed in claim 1, is characterized in that it comprises the steps:
(1) preparation of ferric oxide nano core and finishing:
(1) use hydro-thermal method, solvent thermal decomposition method or coprecipitation prepare the ferric oxide nano microgranule, and mean diameter is 5～20nm.
(2) be coated with the preparation of the ferric oxide nano microgranule of chitosan:
Get γ-Fe that 50 μ L～200 μ L concentration are 4wt%
2o
3or Fe
3o
4nanometer solution adds chitosan-containing, and (number-average molecular weight is 500, deacetylation is 90%) in the 20mL aqueous solution of 10～50mg and ethylenediaminetetraacetic acid 2～10mg, under stirring condition, slowly drip ethanol in system, system adds the amino on 20 μ L～glutaraldehyde cross-linking chitosan surface that 100 μ L mass percentage concentration are 25% when clear becomes colloid solution, room temperature reaction spends the night, and obtains crosslinked CS-EDTA-γ-Fe
2o
3or Fe
3o
4composite nano-microsphere, crosslinked CS-EDTA-γ-Fe by above-mentioned magnetic polymer microsphere
2o
3(or Fe
3o
4) emulsion of composite nano-microsphere on supercentrifuge centrifugal 30 minutes, rotating speed 12000 turns, and takes out the precipitation of lower floor, with after clear water washing three times, be dispersed in intermediate water, then press the above-mentioned steps centrifugal treating twice, finally by the sample redispersion of gained in intermediate water, obtain pure CS-γ-Fe
2o
3(or Fe
3o
4) solution of composite nano-microsphere;
(2) preparation of ferrum oxide core-Gadolinia. shell composite nanometer particle:
The ferric oxide nano particles that 0.05g is coated with to chitosan is dispersed in the 50mL deionized water, then by the Gd (NO of 14.30mL0.2M
3)
3with the 12mL0.5Mol/L aqueous solution of urea, add in above-mentioned iron oxide solution, after the ultrasonic 20min of mixture formed, transfer in a rustless steel high-pressure reactor in the tetrafluoroethene village, high-pressure reactor is heated to 180 ℃ and react 18h, then cool to room temperature, separating by magnetic the magnetic product of tool obtained alternately washs 4 times with ethanol and distilled water, then at lower 60 ℃ of vacuum dryings, spend the night, the dry powder obtained is calcined 2 hours under 700 ℃ of argon atmosphers, and finally forming ferrum oxide is the composite nano particle that core, Gadolinia. are shell;
(3) preparation of polyethyleneglycol modified ferrum oxide core-Gadolinia. shell composite nanometer particle
Under nitrogen protection, add 25mL anhydrous tetrahydro furan and 10g monomethyl Polyethylene Glycol in round-bottomed flask, then, 4mL3-isocyanide propyl-triethoxysilicane and 0.5mL dibutyl tin laurate are added in above-mentioned monomethyl polyglycol solution, at room temperature, continue to stir after two days, obtain the monomethyl Polyethylene Glycol of silanization, by adding hexane from tetrahydrofuran solution, it to be precipitated out, 60 ℃ of vacuum dryings spend the night, and obtain triethoxysilicane alkanisation methyl Polyethylene Glycol;
By the monomethyl Polyethylene Glycol grafting of silanization in the composite nano-granule sub-surface: during the composite nanoparticle that by 100mg prepared by above-mentioned silanization monomethyl Polyethylene Glycol and 20mg step (two) is sneaked into the 50mL deionized water, after the ultrasonic 6h of room temperature, centrifugally wash away unreacted raw material, obtain the polyethyleneglycol modified ferrum oxide core in surface-Gadolinia. shell composite nanometer particle.
4. preparation method according to claim 3, it is characterized in that: the described monomethyl Polyethylene Glycol of step (three) is the monomethyl Polyethylene Glycol that number-average molecular weight is 200-20000.
5. preparation method according to claim 3 is characterized in that: the described preparation that is coated with the ferric oxide nano microgranule of chitosan of step () they (2) substitutes with following methods:
Taking refining number-average molecular weight is 10-30 ten thousand molecular weight, chitosan 0.25 gram that deacetylation is 80-95%, be dissolved in acrylic acid aqueous solution that 50ml contains amount of calculation, wherein: in the amino in chitosan and acrylic acid, the ratio of carboxyl is 1:1, under normal temperature condition, stir, until chitosan dissolves fully, probably need 15 minutes, then get γ-Fe that 200 μ L mass percentage concentration are 4%
2o
3or Fe
3o
4nanometer solution adds above-mentioned mixed solution, strong agitation 10 minutes, load onto condensing unit, connect condensed water, be rapidly heated 70 ℃, add initiator: potassium peroxydisulfate, remain on 70 ℃ of reactions, after the reaction system color becomes milky, continue reaction 2 hours, to guarantee that the acrylic acid in system reacts completely, then the glutaraldehyde cross-linking 2 hours that adds 0.5ml, the emulsion of gained is poured one into can only be less than 10 through molecular weight, in the bag filter of 000 material, room temperature, in the phosphate buffer that is 4.5 at pH value, dialysis is 48 hours, to remove wherein unreacted acrylic molecules and to maintain a stable pH value, room temperature is preserved.
6. preparation method according to claim 3 is characterized in that: the preparation of the described ferrum oxide core of step (two)-Gadolinia. shell composite nanometer particle substitutes with following methods:
By 2mL, the Gd (NO of 0.5Mol/L
3)
3aqueous solution joins in the mixed solvent that 10mL ethanol and 10mL water forms and forms limpid solution, add 0.6g carbamide in above-mentioned solution under strong agitation, after 5min, under ultrasonication to the ferric oxide nano microgranule that adds 0.10g chitosan parcel in above-mentioned mixed solution, powerful ultrasonic 15min, then, the flask of mixed solution will be housed under strong agitation at 90 ℃ of heating 6h, by alternately washing of second alcohol and water for the magnetic product of the isolated tool of magnetic, at 60 ℃ of lower vacuum dryings, spend the night, the dry powder obtained is at 800 ℃ of calcining 2h, obtain ferrum oxide core-Gadolinia. shell nanoparticle.
7. the application of the polyethyleneglycol modified ferrum oxide core in surface claimed in claim 1-Gadolinia. shell composite nanometer particle in the biological contrast agent of preparation.
8. the polyethyleneglycol modified ferrum oxide core in the surface claimed in claim 1-application of Gadolinia. shell composite nanometer particle in preparing the drug-loaded biological contrast agent.
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