CN101444630B - Method for preparing high magnetic resonance sensitivity ferroferric oxide nano-particle with tumor-targeting function - Google Patents
Method for preparing high magnetic resonance sensitivity ferroferric oxide nano-particle with tumor-targeting function Download PDFInfo
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- CN101444630B CN101444630B CN2008102207275A CN200810220727A CN101444630B CN 101444630 B CN101444630 B CN 101444630B CN 2008102207275 A CN2008102207275 A CN 2008102207275A CN 200810220727 A CN200810220727 A CN 200810220727A CN 101444630 B CN101444630 B CN 101444630B
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Abstract
The invention discloses a method for preparing high magnetic resonance sensitivity ferroferric oxide nano-particle with tumor-targeting function. Amphiphilic block polymer of the end capping of tumor-targeted ligand and super-paramagnetic ferroferric oxide are dissolved in tetrahydrofuran, then the obtained solution is added into ionized water or distilled water, the tetrahydrofuran is volatilized, the super-paramagnetic ferroferric in the solution is separated out, washed by the distilled water, then dispersed into the ionized water or the distilled water, and finally centrifugalized, thus the nano-particle is obtained; the tumor-targeted ligand is folic acid, polypeptide or antibody. In the invention, by loading lipophilic ferroferric oxide nano-particle at self-assembled micellar inner core, the tumor-targeted and water-soluble ferroferric oxide nano-particle that is below 50 nm is developed. The tumor-targeting function is realized by coupling the folic acid or antibody at micellar surface. The invention has wide application prospect.
Description
Technical field
The present invention relates to a kind of method for preparing of ferriferrous oxide nano-particle; Especially a kind of have tumor-targeting function, a magnetic resonance video picture method for preparing highly sensitive, the small particle diameter ferriferrous oxide nano-particle, belongs to polymer chemistry and biomedical engineering field.
Background technology
Nuclear magnetic resonance, NMR video picture (MRI) is a kind of very important diagnostic techniques in the clinical medicine, and in recent years, researcher has been paid increasing effort so that improve its resolution and to specific mass.The contrast agent targeting to be transferred to the purpose position be a kind of most important method, so not only can improve contrast effect but also can reduce negative effect and toxicity.Mr techniques is particularly useful for the early stage sign that detects disease.SPIO or superparamagnetism ferroso-ferric oxide (super paramagnetic iron oxide; SPIO); It is the novel contrast medium of magnetic resonance; Because the early diagnosis that its tissue specificity is high, high safety has been used for some cancer abroad as mr contrast agent, in addition can be on molecule and cellular level the intravital situation of non-invasive monitoring.Up to the present, everybody mainly concentrates on ferroso-ferric oxide (Fe for the attention of superparamagnetic nanoparticle
3O
4) and iron sesquioxide (γ-Fe
2O
3).
Magnetic nano-particle prepares through the coprecipitation method in aqueous solution usually in the prior art; This method is suitable for producing in enormous quantities magnetic nanoparticle; But; In building-up process, need the pH value of strict control solution, be difficult to control particle diameter and particle size distribution, especially when the size of particle diameter during less than 20 nanometers.The size ratio of the nanoparticle that high-temperature decomposition obtains is easier to control, and distribution of sizes is single.The magnetic nano-particle that high-temperature decomposition obtains is suitable for biologic applications more; Separate such as being used for magnetic resonance video picture, magnetic cell, the effective absorption that in these are used, wants to reach long circulation or cell requires nanoparticle to have excellent particle size and homogeneous particle diameter distribution of sizes usually.Yet, assemble in order to prevent nanoparticle, the nanoparticle surface of preparation is covered by some oil loving surfactant molecules usually in this way.The long carbochain of these surfactants makes the surface of SPIO particle have the lipophile of height, makes the non-constant of the dissolubility of SPIO in water, and then limits their application aspect biological.And the SPIO with height lipophilic surface is easy to wrapped by the composition in the blood, and then from blood circulation, is discharged very soon.Therefore, carrying out surface modification for the magnetic oxide crystal that is used in biological aspect is very important.Ideal surface modification should have following characteristics: (a) can stable dispersion SPIO under physiological environment; (b) some reactive functional groups are provided so that further modification at particle surface; (c) can restrain the absorption of reticuloendothelial system to nanoparticle.Up to the present, what certain methods was successful is converted into hydrophilic to oil loving SPIO, such as ligand exchange method, bipolarity molecule process, macromolecule stabilizer method.These modifications can avoid the lipophilic surface of SPIO to be exposed to the outside, prevent by the absorption of blood protein, and might prolong the circulation time of SPIO in blood.Current, increasing attention is focused on the surface that utilizes amphipathic polymer to come modification SPIO, because with respect to the micromolecule surfactant, they have better colloidal stability.Such as, with amphipathic gathering-maleic anhydride-alt-1-tetradecene) the butyrous SPIO of load, and then crosslinked with hexamethylene diamine, can obtain hydrophilic SPIO; Also be used to be converted into hydrophilic SPIO to oil loving SPIO by polyethylene glycol oxide and polyoxygenated third rare ABA-type triblock polymer of forming (Pluronic F127).In addition, roll into a ball a bunch kernel that loads to polymer micelle to oil loving magnetic nano-particle and can obtain a kind of overdelicate magnetic resonance video picture spin spin relaxation time (MRI T
2) contrast medium.These methods have been proved to be and can successful preparation have had magnetic nano-particle stable in good monodispersity, the higher aqueous solution.What deserves to be mentioned is that the prepared nanoparticle of reported method all lacks the specificity for lesions position in the above document, and then limited their application in vivo.In fact, SPIO is to obtain with the SPIO of particle diameter less than 50 nanometers as experimental result in the most compellent bodies of video picture probe.Reducing particle diameter so that macrocyclic while, improving MRI sensitivity, and to have the transmission of molecular targeted realization fixed point concurrently be the significant challenge that faces in the mr contrast agent research and development.
Summary of the invention
Technical problem to be solved by this invention is the deficiency that overcomes prior art, and purpose is to provide a kind of method for preparing with high magnetic resonance sensitivity small particle diameter ferriferrous oxide nano-particle of tumor-targeting function.
The present invention realizes above-mentioned purpose through following technical scheme:
A kind of method for preparing with tumor-targeting function high magnetic resonance sensitivity ferroferric oxide nano-particle is that end capped amphipathic block polymer of cancer target part and superparamagnetism ferroso-ferric oxide are dissolved in the oxolane (THF), the solution that obtains is joined in deionized water or the distilled water again; Fling to THF; Superparamagnetism ferroso-ferric oxide in the separation solution is used distilled water wash, is distributed to then in deionized water or the distilled water; Centrifugal, promptly get.
Said tumor targeting part is folic acid, polypeptide, antibody.
Said folic acid or antibody function ratio are 0～100%.
The hydrophilic section of the end capped amphipathic block polymer of said cancer target part is a Polyethylene Glycol, and the oleophylic section is a polyester, like polycaprolactone, and polylactic acid etc.; The molecular weight of Polyethylene Glycol section is 1000～5000Da, and reasonable is 2500～3500Da; The molecular weight of oil loving polyester is 500～2500Da, and reasonable is 800～1500Da.
Said solution is joined in deionized water or the distilled water, preferred version is under ultrasonication, solution to be joined in the deionized water.
The said THF of flinging to can adopt stirring method under the room temperature condition.
SPIO in the said separation solution uses with the SPIO in the Magnet separation solution.
Said centrifugal preferred version is centrifugal 10min under 3000rpm.
Technique scheme of the present invention is to adopt solvent evaporation method; Load to the formed micelle of amphipathic polymer inside to oil loving individual particle ferriferrous oxide nano-particle; Ferriferrous oxide nano-particle of a load in each micelle; Wherein, be in order to remove excessive polymer with distilled water wash; Be distributed to the product that obtains once more in the distilled water, centrifugal then is in order to remove bigger aggregation wherein.
Said method for preparing with tumor-targeting function high magnetic resonance sensitivity ferroferric oxide nano-particle (SPIO) more specifically can be described as:
Pi-allyl-polyethylene glycol-caprolactone (ally-PEG-PCL) mixture and the oil loving ferriferrous oxide nano-particle of 2mg that 20mg are contained 20% folic acid-polyethylene glycol-caprolactone (folate-PEG-PCL) are dissolved among the 1mL THF; Under ultrasonication, slowly be added drop-wise in the 10mL deionized water, stirred 48 hours under the room temperature so that the THF volatilization is complete.Isolate the SPIO in the solution with Magnet then, and with distilled water wash to remove excessive polymer.At last, be distributed to products therefrom once more in the distilled water, to remove wherein bigger aggregation, promptly get the ferriferrous oxide nano-particle of novel tumor target function at the centrifugal 10min of 3000rpm.
The diameter of described oil loving ferriferrous oxide nano-particle is 4～20 nanometers, is preferably 6～10 nanometers.
Described mean diameter with tumor-targeting function high magnetic resonance sensitivity ferroferric oxide nano-particle is 20～80nm, and reasonable is 30～50nm.
Compared with prior art, the present invention has following beneficial effect:
The present invention is through loading to oil loving ferriferrous oxide nano-particle the micellar kernel that polyethylene glycol-caprolactone or polylactic acid self assembly form; Developed a kind of tumor-targeting that has; Water-soluble, the ferriferrous oxide nano-particle of size below 50 nanometers.The present invention loads to the SPIO of individual particle in the micellar nuclear, through being coupled folic acid at micellar surface or cancer target antibody is realized micellar tumor-targeting function.Studied the micellar MRI T of load individual particle SPIO targeting
2Sensitivity and studied its on 1.5T MRI scanner as MRI T
2The potentiality of probe.
The present invention is inner through loading to oil loving ferriferrous oxide nano-particle amphipathic polymer micelle; Can easily obtain hydrophilic ferriferrous oxide nano-particle; And through targeting modification to this load ferriferrous oxide nano-particle; Through in nano-micelle hydrophilic shell mechanism, introduce can with the bonded part of tumor cell surface receptor-specific; Utilize the bonded principle of part and receptor-specific, produce the active target function of nano-micelle, reduce toxic and side effects, prolong life cycle tumor cell; Simultaneously, through nano-micelle high-efficient carrier SPIO and prolongation micelle circulation time in vivo, the nano-micelle of load SPIO is expected as a kind of novel, specificity MRI molecular probe, to promote the targeting diagnosis of MRI to infantile tumour.
And; The nanoparticle of the present invention's preparation is having great importance aspect the diagnosis of tumor; The mr contrast agent conventional with prior art is relative, and it is easy that the mr contrast agent of this method preparation has method for preparing, characteristics such as Nazi's particle magnetic resonance sensitivity height that makes; Method for preparing of the present invention; Use the ligand modified good biocompatibility of cancer target, biodegradable amphipathic nature block polymer to be support material; Preparation can be water-soluble ferriferrous oxide nano-particle, this ferriferrous oxide nano-particle has that particle diameter is little, the highly sensitive and tumor-targeting function of magnetic resonance video picture.
Method for preparing provided by the invention; Obtained water-soluble, as to have tumor-targeting ultra paramagnetic iron oxide nanoparticle through loading to SPIO folic acid-polyethylene glycol-caprolactone (folate-PEG-PCL) or cancer target antibody-micellar kernel of polyethylene glycol-caprolactone (Antibody-PEG-PCL); The micelle (Fa-PEG-PCL-SPIONs) of load individual particle SPIO has less size (40nm) and helps long circulation in the body, also has higher MRI T simultaneously
2Sensitivity, folic acid functionalization can improve cell to the micellar absorption of SPIO; And; The MRI experiment is analysed the polyethylene glycol-caprolactone-ferriferrous oxide nano-particle (PEG-PCL-SPIONs) that shows targetingization with the vitro tissue credit and can in tumor tissues, be assembled in the body, shows that it has the potentiality as the used MRI diagnostic probe of the tumor of receptor transition expression.
Description of drawings
Fig. 1 is the sketch map of synthetic ferriferrous oxide nano-particle;
Fig. 2 is the oil loving SPIONs transmission electron microscope of a 6nm picture among the embodiment 6; Oil loving SPIONs can be dissolved in the normal hexane, and can not be dissolved in the water;
Fig. 3 is SPIO (PEG-PCL-SPIONs) the transmission electron microscope picture of polymer modification amphipathic among the embodiment 6; PEG-PCL-SPIONs can be dissolved in the water, and can not be dissolved in the normal hexane;
Fig. 4 is the electronogram of ferriferrous oxide nano-particle among the embodiment 6; Wherein the 1,2,3,4,5, the 6th, marked six diffraction rings and respective wavelength thereof, can infer in view of the above that it is a ferriferrous oxide nano-particle.
Fig. 5 is the polyethylene glycol-and the micellar particle diameter of lactone of embodiment 6 empty polyethylene glycol-s and lactone and load individual particle ferriferrous oxide nano-particle.
Fig. 6 is 6nm Fe among the embodiment 6
3O
4The B-H loop of nanoparticle; Measuring temperature is 300K and 10K.
Fig. 7 is the B-H loop of PEG-PCL-SPIONs among the embodiment 6, and measuring temperature is 300K and 10K; Can know synthetic Fe by figure
3O
4Particle and PEG-PCL-SPIONs all have superparamagnetism, and saturation magnetization is considerable, can be as the MRI contrast medium.
Fig. 8 is PEG-PCL-SPIONs and WSPIO T among the embodiment 6
2Relaxation rate (1/T
2, s
-1) and concentration of iron between relation (mM) (1.5T, 25 ℃) figure.
The PEG-PCL-SPIONs of modified with folic acid is at external targeting design sketch to HCC among Fig. 9 embodiment 7.
Figure 10 and Figure 11 are T among the embodiment 7
2-weighted mri image (TT/TE; 5000/100ms) the variation of (Figure 10) and back 3.5 hours (Figure 11) mouse tumor locus signal intensitys of injection before the injection; Injection volume is the weight of 5mg of Fe/kg nude mice, and injection system is a tail vein injection, and the diameter of tumor is about 1cm; Among Figure 10 and Figure 11,1 is tumor, and 2 is muscular tissue.
The specific embodiment
Below further specify technical scheme of the present invention through concrete embodiment.
The tumor double-target nano-micelle carrier material folate-PEG-PCL of modified with folic acid or the preparation of folate-PEG-PLA
1) synthetic (ally-PEG-PCL) of pi-allyl-polyethylene glycol-caprolactone
Utilize oxirane (EO) and caprolactone (the synthetic di-block copolymer of the anionic ring-opening polymerization thing of ε-CL).In there-necked flask, add the 0.48g naphthalene, room temperature vacuum drying 12h adds the new THF that steams of about 20mL, and magnetic agitation is even, adds excessive potassium, stirs 0.5h, preparation naphthalene potassium reagent.The propenyl and the naphthalene potassium reagent that in two mouthfuls of bottles, add 0.25mL, magnetic agitation 0.5h adds the THF solution of 1.0g 18-hat-6 ethers of 36 ℃ of vacuum drying 12h in advance, stirs 0.5h.Under cryosel bath condition, feed the exsiccant oxirane of about 25mL, stirring at room reaction 5 days.Under agitation, to the caprolactone of reaction system continuation adding 7.0mL, room temperature reaction 3 days.Product is removed naphthalene with petroleum ether precipitation earlier, the dissolving of reuse dichloromethane, ether sedimentation washing, vacuum drying.
1H-NMR(CDCl
3)：δ＝1.40(m，2H，-COCH
2CH
2CH
2CH
2CH
2O-)，1.65(m，4H，-COCH
2CH
2CH
2CH
2CH
2O-)，2.31(t，2H，-COCH
2CH
2CH
2CH
2CH
2O-)，3.65(s，4H，-CH
2CH
2O-)，4.05(t，2H，-COCH
2CH
2CH
2CH
2CH
2O-)，4.2(s，2H，-CH
2CH
2OCO-)，5.15～5.30(q，1H，CH
2＝CH-)，5.80(m，2H，CH
2＝CH-).
2) synthetic (H of amino-polyethylene glycol-caprolactone
2N-PEG-PCL)
Take by weighing 2.0g Ally-PEG-PCL (Mn=3800Da), be dissolved among the THF of about 8mL, remove by filter insoluble matter.Under ultrasonic emulsification, be added drop-wise in the secondary deionized water of 60mL, stir volatilization and spend the night, remove residual THF with Rotary Evaporators, the micellar aqueous solution of preparation Ally-PEG-PCL.Pipette the micellar solution of 30mL, after half an hour, add the Mercaptamine of 308.3mg and the K of 36.7mg successively with nitrogen bubble
2S
2O
8, 50 ℃ of oil baths were reacted 6 hours down.
After reaction stops, system solution is rotated evaporation and concentration down to 10mL at 30 ℃, use the bag filter dialysis of molecular cut off as 1K, to remove unreacted mercaptan and potassium peroxydisulfate, lyophilizing then.Get white powdered product, yield is 80.3%.
1H-NMR(CDCl
3)：δ＝1.40(m，2H，-COCH
2CH
2CH
2CH
2CH
2O-)，1.65(m，4H，-COCH
2CH
2CH
2CH
2CH
2O-)，2.31(t，2H，-COCH
2CH
2CH
2CH
2CH
2O-)，2.65～2.70(m，4H，-CH
2SCH
2-)，2.94(t，3H，H
2NCH
2CH
2S-)，3.65(s，4H，-CH
2CH
2O-)4.05(t，2H，-COCH
2CH
2CH
2CH
2CH
2O-)，4.2(s，2H，-CH
2CH
2OCO-).
3) synthetic (folate-PEG-PCL) of folic acid-polyethylene glycol-caprolactone
1g folic acid adds in the 50ml two-mouth bottle, behind the vacuum drying 5h, adds the exsiccant dimethyl sulfoxide of 30ml (DMSO); Add N-hydroxy-succinamide (NHS) 0.9g then successively; N, N-dicyclohexylcarbodiimide (DCC) 0.5g, lucifuge reaction 12h; The centrifugal N that reaction generates, the N '-1,3-Dicyclohexylurea (DCU) of removing; Subsequently, join activatory folic acid and be dissolved with 0.4g NH
2In the 5ml DMSO solution of-PEG-PCL (Mn=3800Da), be transferred to 8 to pH value of reaction system with triethylamine then, lucifuge reaction 24h, (72h that in distilled water, dialyses of the molecular weight that dams=1kDa), lyophilization gets yellow powder shape product to the bag filter of usefulness.Be dissolved in powdery product among the THF (3mL), overanxious, remove insoluble matter, consider liquid and under agitation drip in the water.THF is wherein removed in volatilization, and the micellar solution that obtains dialysis five days is so that remove unreacted folic acid and the residual THF (molecular weight that dams=8kDa).The freeze dried powdery product of micellar solution (productive rate >=82%).
1H-NMR(DMSO-d
6)：δ＝1.40(m，2H，-COCH
2CH
2CH
2CH
2CH
2O-)，1.65(m，4H，-COCH
2CH
2CH
2CH
2CH
2O-)，2.31(t，2H，-COCH
2CH
2CH
2CH
2CH
2O-)，2.65～2.70(m，4H，-CH
2SCH
2-)，3.2(t，3H，H
2NCH
2CH
2S-)，3.65(s，4H，-CH
2CH
2O-)，4.05(t，2H，-COCH
2CH
2CH
2CH
2CH
2O-)，4.2(s，2H，-CH
2CH
2OCO-)，4.45(d，2H，C
9-H
2?of?folic?acid)，6.61(d，2H，aromatic?protons?offolic?acid)，7.60(d，2H，aromatic?protons?of?folic?acid)，8.62(s，1H，C
7-H?of?folicacid).
1) Alpha-hydroxy-omega-amino-Polyethylene Glycol (HO-PEG-NH
2) synthetic
In there-necked flask, add the 1g naphthalene, vacuum drying 24h under the room temperature adds the exsiccant THF of 20mL rapidly, and stirring adds fresh metallic potassium 1g rapidly after treating the naphthalene dissolving, and solution becomes blackish green behind the stirring 0.5h; Take by weighing 0.7g (5.73mmol) 18-hat-6 ethers and join in the 100mL there-necked flask, behind 40 ℃ of left and right sides vacuum drying 24h, add the exsiccant THF dissolving of 20mL; Pipette the exsiccant ethanolamine of 0.5mL and place other 100mL there-necked flask; Fill the immigration of naphthalene potassium solution in the there-necked flask of ethanolamine; Stir 0.5h; Reaction color becomes peony, is transferred to the THF solution of naphthalene potassium and ethanolamine rapidly to fill in 18-hat-6 ether there-necked flasks, stirs 0.5h (all operations all carries out under argon shield).
Place the reaction bulb that fills initiator cryosel to bathe; Under argon shield logical 1h through 3
oxirane crossed of molecular sieve drying; The reaction system color gradually becomes glassy yelloe; Viscosity strengthens, and continues reaction 72h under the room temperature; Add hydrochloric acid adjust pH to 4.5, crude product is deposited in the normal hexane, is dissolved in then in the dichloromethane, is deposited in the cold absolute ether, filters back reuse absolute ether washed product, obtains the white powder solid.
2) Alpha-hydroxy-ω-maleimide Polyethylene Glycol (mal-PEG-OH) is synthetic
2g H
2N-PEG-OH (Mn=3600Da) is dissolved into (5mL) in the saturated sodium bicarbonate aqueous solution, is cooled to 0 ℃.Under strong agitation, add N-methoxycarbonyl maleimide (0.1g) to above-mentioned solution.Add the 10mL distilled water behind the 10min, stir 45min to reactant again.Sulphuric acid with 0.5N transfers to 3.0 to the pH value of solution, adds the sodium chloride of 15wt%.Use the dichloromethane extraction reactant, be incorporated into 3 times extracts together, use anhydrous sodium sulfate drying; Filter, remove the part methylene chloride in the filtrating, be precipitated in a large amount of absolute ethers with Rotary Evaporators; Filter, the deposition of collecting vacuum drying at room temperature.Product is the white solid powder, productive rate 90%.
3) synthetic (mal-PEG-PCL) of maleimide-polyethylene glycol-caprolactone
Under the argon shield with 0.2g dimaleoyl imino PEG at 70 ℃ of following vacuum drying 8h, add three Sn (Oct)
2Continue dry 0.5h, add the exsiccant 6-caprolactone of 20mL then, at 105 ℃ of stirring reaction 24h; The cooling back adds the unreacted 6-caprolactone of 5mL dissolve with ethanol, filters, and thick product is dissolved in the 15mL oxolane, is deposited in a large amount of absolute ethers, filters after drying and gets the white powder product, productive rate 95%.
4) synthetic (antibody-PEG-PCL) of antibody-polyethylene glycol-caprolactone
A CD3 antibody 50 μ L are with EDTA solution (10 μ L 0.5M) pretreatment 15min.
B is dissolved in 0.5mL PBS (containing 10 μ L, 0.5M EDTA) to the 60mg mercaptoethylmaine, hatches 15min at 4 ℃.Join then in the solution of the above-mentioned CD3 of containing antibody.Hatch 90min at 37 ℃.Excessive mercaptoethylmaine is removed in centrifugal 3 times of ultrafiltration (containing EDTA with the PBS washing) then.
C mixes (pre-cooling) to the antibody that contains free sulfhydryl groups with PBS (the containing EDTA) solution that contains mal-PEG-PCL (10 μ g) immediately, spends the night 4 ℃ of placements.Centrifugal three times (, not containing EDTA), remove EDTA wherein, in order to avoid influence next step reaction with the PBS washing.
Embodiment 3
Synthesizing of lipophile super-paramagnetic ferriferrous oxide
Under anhydrous and oxygen-free and nitrogen protection condition, add Fe (acac) fast
3(2mmol), 1,2-Hexadecandiol (10mmol), oleic acid (6mmol), oleyl amine (6mmol) and 20mL benzyl oxide, magnetic agitation is even.Under magnetic agitation and nitrogen protection, in sand bath, be warmed up to 200 ℃ rapidly, treat temperature stabilization after, continue heating 2 hours.Then, be warming up to reflux state (outer bath temperature is about 300 ℃) rapidly, continue to reflux 1 hour.After having reacted, reaction system moves apart thermal source, under the nitrogen protection condition, is cooled to room temperature.
Under air atmosphere, magnetic agitation condition, the product of black is joined in the dehydrated alcohol of about 3～4 times of volume ratios, after stirring, centrifugalize 5min under the condition of 10000r/min.The precipitate of black is dissolved in the normal hexane of oleyl amine of the oleic acid that is mixed with about 0.05mL and 0.05mL of 20mL centrifugalize 10min under the condition of 6000r/min.The solution of the upper strata black dehydrated alcohol with about 3～4 times of volume ratios is precipitated, with mixed liquor centrifugalize 10min under the condition of 6000r/min.The precipitate of black is dissolved in the normal hexane of 20mL, deposits in the reagent bottle of the sealing under the argon atmosphere, and places refrigerator to preserve.
Embodiment 4
The mixture of 20mg ally-PEG-PCL and folate-PEG-PCL (containing 20%folate-PEG-PCL), 2mg SPIO are dissolved among the 1mL THF.Under ultrasonication, be added to above-mentioned drips of solution in the 10mL deionized water, stirred 48 hours under the room temperature, wherein THF volatilizees.With the SPIO in the Magnet separation solution, go out excessive polymer with distilled water wash.Be distributed to the product that obtains once more in the distilled water, centrifugal 10min under 3000rpm is so that remove bigger aggregation wherein.The preparation process is as shown in Figure 1, and the fundamental property of prepared ferriferrous oxide nano-particle is as shown in table 1.
The fundamental property of table 1 ferriferrous oxide nano-particle
Annotate: the unit of r1 and r2 value is wrapped up by PEG-PCL by the surface of Fe mM-1s-1. [a] SPIO, and wherein the content of folic acid is that 20%. [b] measure through thermal analysis system. the hydrophilic SPIO. that [c] obtains through the micromolecule surfactant
20mg mal-PEG-PCL, 2mg SPIO are dissolved among the 1mL THF.Under ultrasonication, be added to above-mentioned drips of solution in the 10mL deionized water, stirred 48 hours under the room temperature, wherein THF volatilizees.
1) CD3 antibody 50 μ L are with EDTA solution (10 μ L 0.5M) pretreatment 15min.
2) be dissolved in 0.5mL PBS (containing 10 μ L, 0.5M EDTA) to the 60mg mercaptoethylmaine, hatch 15min at 4 ℃.Join then in the solution of the above-mentioned CD3 of containing antibody.Hatch 90min at 37 ℃.Excessive mercaptoethylmaine is removed in centrifugal 3 times of ultrafiltration (containing EDTA with the PBS washing) then.
3) mix (pre-cooling) to the antibody that contains free sulfhydryl groups with PBS (the containing EDTA) solution that contains mal-PEG-PCL (10 μ g) immediately, spend the night 4 ℃ of placements.Centrifugal three times (, not containing EDTA), remove EDTA wherein, in order to avoid influence next step reaction with the PBS washing.
With the SPIO in the Magnet separation solution, go out excessive polymer with distilled water wash.Be distributed to the product that obtains once more in the distilled water, centrifugal 10min under 3000rpm is so that remove bigger aggregation wherein.
The test of the micellar key property of embodiment 6 load individual particle ferriferrous oxide nano-particles
1) test of micellar size of load individual particle ferriferrous oxide nano-particle and form
The micellar size of gained adopts the dynamic light scattering system to measure, and its form is then observed definite through transmission electron microscope, and test result is seen Fig. 2, Fig. 3, Fig. 4, Fig. 5.
2) test of the micellar magnetic property of load individual particle ferriferrous oxide nano-particle
The micellar alternating temperature susceptibility of gained is measured through gaussmeter, and test result is seen Fig. 6, Fig. 7.
3) mensuration of the magnetic resonance sensitivity of cancer target ferriferrous oxide nano-particle
The MRI signal intensity is relevant with physics and chemical parameters, like proton density, spin-lattice relaxation time T
1, spin spin relaxation time T
2T
1, T
2Parameter controlled imaging to specific strength.The hydrogen proton density changes very for a short time in soft tissue, therefore in diagnosis, uses T
1Weighted imaging, T
2Weighted imaging.
The efficient of MRI contrast medium can be through its vertical and horizontal relaxation rate r
1And r
2Assess.r
1And r
2React contrast medium respectively and influenced T
1And T
2Ability.r
2/ r
1Ratio big more, T
2The efficient of class preparation is high more.
The sensitive intensity of the micellar magnetic resonance of load individual particle ferriferrous oxide nano-particle is shown in Fig. 8 and table 1, and the abscissa among Fig. 8 is an iron concentration, and vertical coordinate is spin spin relaxation time T
2Inverse, the result shows that it is highly sensitive in the ferriferrous oxide nano-particle of micromolecule modification.
Embodiment 7 external and in-vivo imagings
1.5T magnetic resonance imaging system (Philips Inter 1.5T) is adopted in MRI scanning, annular surface coil, axle position and crown bit scan, T
1WI adopts the SE sequence, TE 15ms, TR 500ms, bed thickness 1.5mm, spacing 1mm, matrix 64 * 64, excitation number of times 3.T2WI adopts the TSE sequence, TE 100ms, TR2600ms, bed thickness 1.5mm, spacing 1mm, matrix 64 * 64, excitation number of times 4.Measure the region of interest signal respectively, signal calculated rate of change (Δ SI), Δ SI=(SIL-SIU)/SI * 100%, wherein SIL and SIU are respectively the signal intensity of processing and untreated cell
1) experiment in vitro:
After folic acid targeting and non-folic acid targeted nano micelle hatched 1 hour with human liver cancer cell Bel 7402 respectively altogether,, utilize the T of SPIO through external MRI imaging
2The negativity enhancement effect, observation signal Strength Changes situation is explored and is utilized clinical type MRI to estimate the targeting effect of nano-micelle to Bel 7402 cells.
Folic acid targeting and the non-targeted nano micelle that Fe concentration is respectively 5,10,20,40 and 80 μ g/ml respectively with human liver cancer cell Bel 7402 (cell counting 1 * 10
6Individual) in no folic acid RPMI 1640 culture medium, cultivate 1h altogether; With the digestion of 0.25% trypsinization liquid,, place 1.5ml EP pipe (Ependoff pipe) with washing of PBS liquid and re-suspended cell; Row MRI scanning, simultaneously with Bel 7402 cells of not doing any processing as the blank group.
As shown in Figure 9, folacin receptor mediated polymer nano micelle has targeting effect preferably in the cell in vitro level to human liver cancer cell Bel 7402.
2) zoopery
Set up nude mouse lotus people hepatocarcinoma subcutaneous transplantation tumor animal model,
Utilize the T of SPIO (being Fe)
2Negativity contrast enhancement effect is through measuring T
2The last MRI signal intensity of WI, signal calculated rate of change (Rate of signal intensity variety) and contrast noise ratio (contrastnoise ratio; CNR); Reflection local tumor tissue contains the situation of Fe, explores a kind of new targeting delivery system and utilizes clinical type MRI appearance to its feasibility of monitoring.
Scanner is a Philips Intera 1.5T MR imaging system, annular surface coil (being the C3 coil), row axial plane and coronalplane SE T
2WI and T
1WI scanning.SE T
2WI adopts FSE (FSE) sequence, TR 2600ms, TE 100ms, bed thickness 2.0mm, matrix 256 * 256, the visual field (Field of view, FOV) 120～150mm, excitation number of times 4 times.SE T
1The WI sweep parameter is TR 500ms, TE 15ms, bed thickness 2.0mm, matrix 256 * 256, the visual field (Field of view, FOV) 120～150mm, excitation number of times 2 times.
Nude mouse MRI inspection step is following:
1) with 4.3% chloral hydrate (430mg/kg) nude mouse is carried out intraperitoneal injection of anesthesia.
2) every nude mouse is all put down in advance and sweeps (plain scan), promptly injects nanoparticle scanning before as baseline scan.
3), inject folic acid targeted nano-particle and non-targeted nano-particle respectively through caudal vein by the grouping requirement.ID is 5mg Fe/kg.
4) carry out the MRI scanning of different time points after the injection.Being chosen as after the injection at once of scanning time point (represent, refer to after the injection in the 10min), 1h, 3h, 6h and 24h, totally 5 different scanning time points with st.
5) after MRI scanning is accomplished, data are reached the post processing work station, carry out follow-up MRI graphical analysis.
Like Figure 10, Figure 11, table 2, show the T of the different time points of tumor tissues behind the injection folic acid targeted nano micelle in body MRI imaging results
2WI intensity and change rate signal all have obvious decline, have significant difference.But not the targeting group after injection immediately, the signal of 1h and three time points of 3h also has slight decline; But fall is little than folic acid targeting group; And the persistent period is than targeting group short (signal intensity of non-targeting group injection back 6h promptly returns to put down and sweeps level, and the signal intensity of targeting group injection back 24h still maintains reduced levels).
Nude mice tumor locus magnetic resonance signal intensity over time behind the table 2 injection ferriferrous oxide nano-particle
*P＞0.05
Claims (7)
1. the method for preparing with tumor-targeting function high magnetic resonance sensitivity ferroferric oxide nano-particle is characterized in that end capped amphipathic block polymer of cancer target part and superparamagnetism ferroso-ferric oxide are dissolved in the oxolane, the solution that obtains is joined in deionized water or the distilled water again; Fling to oxolane; Superparamagnetism ferroso-ferric oxide in the separation solution is used distilled water wash, is distributed to then in deionized water or the distilled water; Centrifugal, promptly get; Said cancer target part is folic acid, polypeptide or antibody.
2. method for preparing as claimed in claim 1 is characterized in that the hydrophilic section of the end capped amphipathic block polymer of said cancer target part is a Polyethylene Glycol, and molecular weight is 1000～5000Da, and the oleophylic section is a polyester, and molecular weight is 500～2500Da.
3. method for preparing as claimed in claim 2 is characterized in that said polyester is polycaprolactone or polylactic acid.
4. method for preparing as claimed in claim 1 is characterized in that said solution being joined in deionized water or the distilled water, is under ultrasonication, solution to be joined in deionized water or the distilled water.
5. method for preparing as claimed in claim 1 is characterized in that the said oxolane of flinging to is to stir volatilization at ambient temperature.
6. method for preparing as claimed in claim 1 is characterized in that the superparamagnetism ferroso-ferric oxide in the said separation solution is with the superparamagnetism ferroso-ferric oxide in the Magnet separation solution.
7. method for preparing as claimed in claim 1, it is characterized in that said centrifugal be centrifugal 10min under 3000rpm.
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| CN101818128B (en) * | 2010-04-02 | 2012-06-27 | 川北医学院附属医院 | Superparamagnetic iron oxide labeled human pancreas cancer cell strain as well as labeling method and application thereof |
| CN101940791B (en) * | 2010-08-27 | 2012-08-22 | 天津医科大学 | Dually-targeted pH-sensitive antineoplastic drug carrier material as well as preparation and application thereof |
| CN102657881B (en) * | 2012-05-16 | 2013-11-27 | 上海师范大学 | Preparation method of Fe3O4 nano-magnetic resonance contrast medium material |
| CN103663570A (en) * | 2013-11-15 | 2014-03-26 | 太原理工大学 | Method for preparing ovarian cancer target ferroferric oxide nano-particle at room temperature |
| KR101916288B1 (en) * | 2014-01-06 | 2018-11-07 | 나노코 테크놀로지스 리미티드 | Surface-Modified Nanoparticles |
| CN104027357A (en) * | 2014-06-16 | 2014-09-10 | 上海应用技术学院 | Targeted nano magnetic donkey-hide gelatin as well as preparation method and application thereof |
| CN106399226A (en) * | 2016-08-27 | 2017-02-15 | 上海交通大学 | Preparation method and application method of targeted superparamagnetic nano-probe |
| CN110339373A (en) * | 2018-04-04 | 2019-10-18 | 中国科学院宁波材料技术与工程研究所 | A kind of nano combined micella and its preparation method and application |
| CN111012928A (en) * | 2019-11-26 | 2020-04-17 | 安徽医科大学 | Multifunctional nano-cluster and preparation method thereof |
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