CN100461310C - Double function magnetic fluid and its preparing method - Google Patents
Double function magnetic fluid and its preparing method Download PDFInfo
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- CN100461310C CN100461310C CNB2005100305142A CN200510030514A CN100461310C CN 100461310 C CN100461310 C CN 100461310C CN B2005100305142 A CNB2005100305142 A CN B2005100305142A CN 200510030514 A CN200510030514 A CN 200510030514A CN 100461310 C CN100461310 C CN 100461310C
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Abstract
This invention relates to a double-function magnetic fluid and its preparation method, in which, said magnetic fluid is composed of magnetic inorganic nm particles, a decoration layer and a guiding peptide, the particles are those of ferro-oxide with the diameter of 5~20nm, the decoration layer is a natural high polymer: starch or glucosan, the guiding peptide is a multi-peptide molecule composed of 12-15 amino acid, the decoration layer covers the surface of the particles and is coupled with the guiding peptide. The preparation method includes: preparation of the magnetic inorganic nm particles, the surface decoration of the particles and the coupling of the guiding peptide and the decoration layer. Said magnetofluid has double functions: magnetic responsibility and bio-affinity.
Description
Technical field
The present invention relates to a kind of double function magnetic fluid and preparation method thereof, belong to the technical field of nanoscale science and technology and biological medicine.
Background technology
Magnetic nanoparticle makes it have broad application prospects at biomedical sector because of its biocompatibility, special magnetic property, as immobilised enzymes, targeted drug, control release, magnetic resonance imaging, DNA and cell separation technology etc., become the focus of current research.
The key of these application technologies is that selection and realization boiomacromolecule with boiomacromolecule of special biological function combine with the coupling of magnetic nanoparticle.What application was more at present is among realizing large biological molecules such as protein, enzyme and antibody and the coupling of magnetic nanoparticle combines and be applied to the research of biological medicine by methods such as physical absorption, chemical bondings.
As an important use field, magnetic control target is meant with the magnetic nanoparticle to be pharmaceutical carrier to drug technique, under the effect of outside magnetic field cancer therapy drug is positioned the tumour target area to improve the drug concentration of this target area, and medicine is discharged from carrier in a controlled manner, thereby reach the purpose of effective chemotherapy tumour.Compare with other targeted drug technology have efficient, low toxicity, the characteristics of high anelasticity.
The very important developing direction of another of oncotherapy is the biological targeting treatment.The polypeptide that utilizes the high specificity that the phage peptide library technology screened be a kind of have strong with the tumour-specific binding ability, molecule is little and targeted molecular with strong penetration capacity.Thereby, be that carrier and drug molecule link with this targeted peptide, can improve the ability of targeted delivery medicine effectively.
Recently, targeted peptide is combined with nano particle begin to draw attention with the research of using in its diagnosis of abundant exploitation, treatment and the target administration in tumour.Akerman etc. have reported to have specific targeted peptide and the coupling research of target in animal body mutually of ZnS/CdSe quantum dot to lung cancer by what the phage peptide library technology screening went out, and its result shows that targeted peptide will have wide practical use aspect diagnosis, treatment and the target administration of disease with combining of nano particle.
Summary of the invention
The purpose of this invention is to provide a kind of double function magnetic fluid.This magnetic fluid is a kind of dispersion with magnetic responsiveness and bioaffinity dual-use function, is suitable for being used for novel two target administration carriers in fields such as cell separation, tumor thermotherapy and magnetic resonance imaging.
For achieving the above object, the present invention adopts following technical scheme.A kind of double function magnetic fluid, it is characterized in that, comprise magnetic inorganic nano particle, decorative layer and targeted peptide, the magnetic inorganic nano particle is that particle diameter is the particle of oxide of the iron of 5～20nm, decorative layer is a natural high polymer: starch or glucan, targeted peptide has specific peptide molecule by what 12～15 amino acid were formed to liver cancer, and decorative layer is coated on the magnetic inorganic nano grain surface, targeted peptide and decorative layer coupling mutually.
Of the present invention being further characterized in that, the oxide of described iron is Fe
3O
4Or γ-Fe
2O
3
Of the present invention being further characterized in that, described targeted peptide carries out mark with green fluorescent protein GFP or fluorescein FAM.
Another object of the present invention provides the method for the described magnetic fluid of preparation.
For achieving the above object, the present invention adopts following technical scheme.A kind of method for preparing double function magnetic fluid is characterized in that it may further comprise the steps:
The preparation of first step magnetic inorganic nano particle
Prepare Fe respectively with deionized water
3+And Fe
2+Mixed solution and NaOH solution, Fe in the mixed solution of molysite
3+The concentration of ion is 0.02～0.05mol/L, Fe
2+The concentration of ion is 0.01～0.04mol/L, and the concentration of NaOH is 0.5～2.0mol/L in the NaOH solution, at N
2Gas shiled or do not have N
2Gas shiled and stirring are added to the NaOH drips of solution in the mixed solution of molysite down, to pH=10～11 of reaction solution, after precipitation is finished, under the room temperature, at N
2The protection of gas continues down to stir 1～3h or at no N
2Under the gas shiled with reaction solution at 60～80 ℃ of ageing 0.5～3h, after separate in magnetic field, successively sediment undergoes washing to reaction solution is neutral with deionized water and ethanol, filter the back in 60～80 ℃ of vacuumizes or in 60～80 ℃ at air drying, grind, getting particle diameter is the magnetic inorganic nano particle of 5～20nm;
The finishing of the second step magnetic inorganic nano particle
The first step is made magnetic inorganic nano-particle product natural high polymer: starch or glucan carry out finishing, taking by weighing 5～12g starch or glucan throws in the 50ml deionized water, 60～80 ℃ of heating make its dissolving, with the magnetic inorganic nanoparticulate dispersed of the first step of 0.8～2.5g preparation in starch or dextran solution, at N
2Gas shiled or do not have N
2Under gas shiled and 60～80 ℃, stir 2～3h, behind starch or the dextran solution volatilization 50wt%, natural cooling, the bulky grain or the aggregation of the sedimentation of centrifugal removal part, adopt Magnetic Isolation post or SephacrylS-300 gel column to separate and remove non magnetic component, getting starch or glucan content that coat, the magnetic inorganic nano particle is the magnetic fluid of 5～15mg/ml;
The coupling of the 3rd step targeted peptide and decorative layer
In PBS, compound concentration is targeted peptide 0.5～2.0ml of 0.5～2.0mg/ml, adding concentration is carbodiimide solution 0.5～2.0ml of 0.5～2.0mg/ml, magnetic fluid 100～200 μ l that add the preparation of second step again, under 18～25 ℃ and the lucifuge, with hunting of frequency 20～26h of 200～400r/min, PBS cleans, and gets double function magnetic fluid.
Method of the present invention is further characterized in that in the 3rd step, targeted peptide is the targeted peptide with green fluorescent protein GFP mark, A54-GFP.
Method of the present invention is further characterized in that in the 3rd step, targeted peptide is the targeted peptide with fluorescein FAM mark, FAM-A54.
The present invention has following beneficial effect:
1, preparation method of the present invention is simple.
2, the product of method of the present invention, double function magnetic fluid have external magnetic field response and biological affine dual-use function, and because the high bioaffinity of targeted peptide, its effect in vivo obviously is better than antibody.
3, the product of method of the present invention, double function magnetic fluid can be used for multiple uses such as target administration, cell separation and magnetic resonance imaging.
Embodiment
Below in conjunction with specific embodiment, the invention will be further elaborated.Below all embodiment all according to the operation of the described operating procedure of foregoing invention content.
Embodiment 1
In the first step, Fe in the mixed solution of molysite
3+And Fe
2+The concentration of ion is respectively 0.036mol/L and 0.024mol/L, and the concentration of NaOH solution is 1mol/L.After precipitation is finished, under the room temperature, at N
2The protection of gas continues to stir 2h down.Filter the back in 60～80 ℃ of vacuumizes.Getting particle diameter is the Fe of 5～20nm
3O
4Nano particle, promptly a kind of magnetic inorganic nano particle.
In second step, the used natural high polymer of finishing is a starch, and consumption is 6g.The magnetic inorganic nano particle 1.5g that the first step is made is scattered in the starch solution N
2Under gas shiled and 60～80 ℃, stir 2～3h.Get starch coats, Fe
3O
4Nano-particle content is the magnetic fluid of 9mg/ml.
In the 3rd step, in PBS, compound concentration is targeted peptide 0.5mg/ml, that use green fluorescent protein GFP mark, i.e. A54-GFP solution.Get above-mentioned solution 2ml, the carbodiimide content that adds 2ml is the solution of 0.5mg/ml, then gets the magnetic fluid 100 μ l that second step made, and adds in the above-mentioned solution.Under 18～25 ℃ and the lucifuge, with the hunting of frequency 24h of 300r/min.Get double function magnetic fluid.
Embodiment 2
In the first step, Fe in the mixed solution of molysite
3+And Fe
2+The concentration of ion is respectively 0.048mol/L and 0.032mol/L, and the concentration of NaOH solution is 1.5mol/L.The precipitation finish after, under the room temperature, with reaction solution at 60～80 ℃ of ageing 2h.Filter the back in 60～80 ℃ of air dryings.Getting particle diameter is γ-Fe of 5～20nm
2O
3Nano particle, promptly a kind of magnetic inorganic nano particle.
In second step, the used natural high polymer of finishing is a starch, and consumption is 10g.The magnetic inorganic nano particle 2.5g that the first step is made is scattered in the starch solution no N
2Under gas shiled and 60～80 ℃, stir 2～3h.Get starch coats, γ-Fe
2O
3Nano-particle content is the magnetic fluid of 15mg/ml.
In the 3rd step, in PBS, compound concentration is targeted peptide 1mg/ml, that use green fluorescent protein GFP mark, i.e. A54-GFP solution.Get above-mentioned solution 1ml, the carbodiimide content that adds 1ml is the solution of 1mg/ml, then gets the magnetic fluid 150 μ l that second step made, and adds in the above-mentioned solution.Under 18～25 ℃ and the lucifuge, with the hunting of frequency 20h of 400r/min.Get double function magnetic fluid.
Embodiment 3
In the first step, Fe in the mixed solution of molysite
3+And Fe
2+The concentration of ion is respectively 0.021mol/L and 0.014mol/L, and the concentration of NaOH solution is 0.8mol/L.After precipitation is finished, under the room temperature, at N
2The protection of gas continues to stir 2h down.Filter the back in 60～80 ℃ of vacuumizes.Getting particle diameter is the Fe of 5～15nm
3O
4Nano particle, promptly a kind of magnetic inorganic nano particle.
In second step, the used natural high polymer of finishing is glucan T-40, and consumption is 12g.The magnetic inorganic nano particle 0.8g that the first step is made is scattered in the glucan T-40 solution, at N
2Under gas shiled and 60～80 ℃, stir 2～3h.Get glucan T-40 coats, Fe
3O
4Nano-particle content is the magnetic fluid of 5mg/ml.
In the 3rd step, in PBS, compound concentration is targeted peptide 2mg/ml, that use green fluorescent protein GFP mark, i.e. A54-GFP solution.Get above-mentioned solution 0.5ml, the carbodiimide content that adds 0.5ml is the solution of 2mg/ml, then gets the magnetic fluid 200 μ l that second step made, and adds in the above-mentioned solution.Under 18～25 ℃ and the lucifuge, with the hunting of frequency 26h of 200r/min.Get double function magnetic fluid.
Embodiment 4
The first step is identical with second step with the first step of embodiment 1 with second step.
In the 3rd step, in PBS, compound concentration is targeted peptide 1mg/ml, that use fluorescein FAM mark, i.e. FAM-A54 solution.Get above-mentioned solution 1ml, the carbodiimide content that adds 1ml is the solution of 1mg/ml, then gets the magnetic fluid 150 μ l that second step made, and adds in the above-mentioned solution.Under 18～25 ℃ and the lucifuge, with the frequency lucifuge vibration 24h of 300r/min.Get double function magnetic fluid.
Embodiment 5
The first step is identical with second step with the first step of embodiment 2 with second step.
In the 3rd step, in PBS, compound concentration is targeted peptide 1mg/ml, that use fluorescein FAM mark, i.e. FAM-A54 solution.Get above-mentioned solution 1ml, the carbodiimide content that adds 1ml is the solution of 1mg/ml, then gets the magnetic fluid 150 μ l that second step made, and adds in the above-mentioned solution.Under 18～25 ℃ and the lucifuge, with the frequency lucifuge vibration 24h of 300r/min.Get double function magnetic fluid.
Claims (7)
1. double function magnetic fluid, it is characterized in that, comprise magnetic inorganic nano particle, decorative layer and targeted peptide, the magnetic inorganic nano particle is that particle diameter is the particle of oxide of the iron of 10～50nm, decorative layer is a natural high polymer: starch or glucan, targeted peptide has specific peptide molecule by what 12～15 amino acid were formed to liver cancer, and decorative layer is coated on the magnetic inorganic nano grain surface, targeted peptide and decorative layer coupling mutually.
2. double function magnetic fluid according to claim 1 is characterized in that the oxide of described iron is Fe
3O
4Or γ-Fe
2O
3
3. double function magnetic fluid according to claim 1 is characterized in that, described targeted peptide carries out mark with green fluorescent protein GFP or fluorescein FAM.
4. prepare the method for the described double function magnetic fluid of claim 1, it is characterized in that, it may further comprise the steps:
The preparation of first step magnetic inorganic nano particle
Prepare Fe respectively with deionized water
3+And Fe
2+Mixed solution and NaOH solution, Fe in the mixed solution of molysite
3+The concentration of ion is 0.02～0.05mol/L, Fe
2+The concentration of ion is 0.01～0.04mol/L, and the concentration of NaOH is 0.5～2.0mol/L in the NaOH solution, at N
2Gas shiled or do not have N
2Gas shiled and stirring are added to the NaOH drips of solution in the mixed solution of molysite down, to pH=10～11 of reaction solution, after precipitation is finished, under the room temperature, at N
2The protection of gas continues down to stir 1～3h or at no N
2Under the gas shiled with reaction solution at 60～80 ℃ of ageing 0.5～3h, after separate in magnetic field, successively sediment undergoes washing to reaction solution is neutral with deionized water and ethanol, filter afterwards in 60～80 ℃ of vacuumizes or in 60～80 ℃ at air drying, grind, get the magnetic inorganic nano particle;
The finishing of the second step magnetic inorganic nano particle
The first step is made magnetic inorganic nano-particle product natural high polymer: starch or glucan carry out finishing, taking by weighing 5～12g starch or glucan throws in the 50ml deionized water, 60～80 ℃ of heating make its dissolving, with the magnetic inorganic nanoparticulate dispersed of the first step of 0.8～2.5g preparation in starch or dextran solution, at N
2Gas shiled or do not have N
2Under gas shiled and 60～80 ℃, stir 2～3h, behind starch or the dextran solution volatilization 50wt%, natural cooling, the bulky grain or the aggregation of the sedimentation of centrifugal removal part, adopt Magnetic Isolation post or SephacrylS-300 gel column to separate and remove non magnetic component, getting starch or glucan content that coat, the magnetic inorganic nano particle is the magnetic fluid of 5～15mg/ml;
The coupling of the 3rd step targeted peptide and decorative layer
In PBS, compound concentration is targeted peptide 0.5～2.0ml of 0.5～2.0mg/ml, adding concentration is carbodiimide solution 0.5～2.0ml of 0.5～2.0mg/ml, magnetic fluid 100～200 μ l that add the preparation of second step again, under 18～25 ℃ and the lucifuge, with hunting of frequency 20～26h of 200～400r/min, PBS cleans, and gets double function magnetic fluid.
5. method according to claim 4 is characterized in that, in the 3rd step, targeted peptide is the targeted peptide with green fluorescent protein GFP mark, A54-GFP.
6. method according to claim 4 is characterized in that, in the 3rd step, targeted peptide is the targeted peptide with fluorescein FAM mark, FAM-A54.
7. the purposes of the described double function magnetic fluid of claim 1 is characterized in that, in the field of cell separation, tumor thermotherapy and magnetic resonance imaging, as the carrier of two target administrations.
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| CN101759882B (en) * | 2008-12-25 | 2012-02-29 | 陕西北美基因股份有限公司 | Sephadex magnetic composite particles and preparation and use thereof |
| CN101984096B (en) * | 2010-11-15 | 2012-05-09 | 北京科技大学 | Method for extracting noble metal ions by using magnetic medium |
| CN102071018B (en) * | 2010-12-24 | 2014-01-29 | 苏州方昇光电装备技术有限公司 | Method for preparing magnetic fluorescent nanocomposite material |
| CN104525092B (en) * | 2014-12-17 | 2016-08-17 | 华中科技大学 | A kind of preparation method of superparamagnetic nano particle and products thereof |
| CN107068320B (en) * | 2017-03-24 | 2018-09-21 | 青岛大学 | A kind of preparation method based on the regulatable cobalt-nickel alloy magnetorheological fluid of surface roughness |
| CN109516503A (en) * | 2019-01-11 | 2019-03-26 | 福州大学 | A kind of preparation method of high stability water-base nano magnetic fluid |
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| CN1647821A (en) * | 2004-09-29 | 2005-08-03 | 中国人民解放军第四军医大学 | Method for preparing specific nano vaccine for stomach cancer |
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