CN100457191C - Magnetic nano radioactive medicine and its preparing method - Google Patents
Magnetic nano radioactive medicine and its preparing method Download PDFInfo
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- CN100457191C CN100457191C CNB2004100020302A CN200410002030A CN100457191C CN 100457191 C CN100457191 C CN 100457191C CN B2004100020302 A CNB2004100020302 A CN B2004100020302A CN 200410002030 A CN200410002030 A CN 200410002030A CN 100457191 C CN100457191 C CN 100457191C
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Abstract
The present invention provides a magnetic nano radioactive drug and its preparation method. Said method includes the following steps: 1. making magnetic nano microparticle undergo the process of surface chemical modification; 2. fixing and carrying ligand; and 3. making radioactive nuclide marking treatment of the ligand. Said invented magnetic nano radioactive drug has good stability underto the physiological condition. Said invention can make the nano characteristics of magnetic nano microparticle, magnetic property and therapeutic property of radioactive nuclide be combined together, and can use external magnetic field to make radioactive drug be quickly concentrated and collected on focal region so as to raise therapeutic effect and can reduce toxic side effect of killing normal tissue cell.
Description
Technical field
The present invention relates to field of medicaments, particularly a kind of magnetic Nano radiopharmaceutical and preparation method thereof.
Background technology
Radiopharmaceutical therapy is present general gradually a kind of scheme for the treatment of tumor, and interior X-ray therapy is otherwise known as.This therapy is to have radiolabeled tumour-specific medicine to be expelled to knub position, and the radiation that radiosiotope sends (α or beta particle, Auger electron and interior conversion electrons) is killed or killing tumor cell.Up to now, the quantity of the radionuclide that has been found that is above 2000 kinds, yet the radionuclide that really can be used in treatment is few, and the radionuclide of treatment usefulness must satisfy: 1. must be according to tumor character, size and may be to the extent of injury of adjacent organs and tissue and ray type and energy that careful selection suits; 2. Shi Yi half-life, to keep the time of one section continuous action, make ray pass to the enough energy of focus cell, the nucleic of half-life several days to tens of days scopes generally selected in short its death; 3. the radiation treatment medicine generally requires high specific activity (specific activity is meant the radioactivity of certain radioactive substance of unit mass), should be near carrier-free; 4. radionuclide should not concentrate in key tissues such as brain and heart, otherwise will produce radiation damage; 5. generally require the content of radionuclide to be not less than 90%; 6. nucleic should be easy to produce and preparation, and supply is convenient, and supply frequency height is cheap.Common medical treatment radionuclide is as shown in the table:
Wherein
188Re, its β
- Max=2.12MeV (79%), 1.96MeV (20%); γ=155keV (15%); Effectively treat range X
90=2.1mm; Half-life T
1/2=16.9h is a kind of attractive treatment radionuclide, can be by reactor-produced W-188 parent (half-life T
1/2=69d) decay and get, therefore can be easily as requested from
188W/
188The drip washing of Re generator obtains (D.Z.Yin et al., Nuclear Techniques 1998,21:51; F.F.J.Knapp, A.L.Beets, S.Guhlke, Anticancer Research 1997,17:1783).The metal carbonyl that proposes Re, Tc since people such as Jaouen in 1993 since using aspect the nuclear medicine, fac-[M (OH
2)
3(CO)
3]
+(M=
99mTc,
186/188Re) labelling that is used for biomolecule has obtained extensive studies (Schibli R.andSchubiger P.A.Current use and future potential of organometallicradiopharmaceuticals.Eur J Nucl Med, 2002,29:1529-1542).It has many advantages, and is better such as water solublity, good stability in air and aqueous solution (pH 2～12).Its ligand H in addition
2The O molecule is easy to be replaced by monodentate, multiple tooth double function ligand (containing N-N-N, N-N-O, N-N-S, P-P, keys such as P-P-O and S-S), forms the carbonyl chelate.This chelate is easy to and biomolecule combinations such as albumen, monoclonal antibody, polypeptide, aminoacid, sugar.Fac-[
188Re (H
2O)
3(CO)
3]
+Can be used as the radioactive label precursor of polylactic acid microsphere, magnetic microsphere, magnetic nanometer particles, monoclonal antibody, protein, polypeptide or saccharide.
The various method that developed during the decade in the past is used for radiopharmaceuticals is transported to tumor cell more specifically, and a kind of method wherein is to the monoclonal antibody of patient infusion tool cancer immunity reaction and relative cancer specific (be marked with emission α-or the radionuclide of beta particle).After the injection, the radiation treatment medicine little by little gathers tumor locus, and normal structure is but much less usually.Radiopharmaceutical is just more optionally transported like this, has reduced the internal radiation to health tissues.Yet this method is often because medicine was transported to the relatively little drug level of tumor locus and the radiation breakdown of normal structure is restricted before the tumor locus optimum concentrates.
On the other hand, because special superparamagnetism and nano-meter characteristic, magnetic nanometer particles has broad application prospects at aspects such as giant magnetoresistance, magnetic fluid and magnetic recording, soft magnetism, permanent magnetism, magnetic cooling, giant magnetic impedance material and magnetic-optic devices, magnetic detectors.Based on its nanoscale and super paramagnetic characteristic, the application of magnetic nanometer particles in biomedical sector is especially noticeable.Magnetic nanometer particles has superparamagnetism and colloidal nano microgranule character, is treating (Dailey JP et al.J Magn MagnMater, 1999,194:140-148 such as retina shedding; Rutnakonituk M et al.Polymer, 2002,43:2337-2348), cell separation (Shinkai M.J Biosci and Bioeng, 2002,94 (6): 606-613; Molday RS and Mackenzie D.J Immunol Methods, 1982,52 (3): 353-367; RoathS.J Magn Magn Mater, 1993,122 (1-3): 329-334; Xu Z and Gu Y.WO03/005029 A2, Canada, 2003), the superthermal therapy of tumor (Shinkai M.J Biosci and Bioeng, 2002,94 (6): 606-613; Yanase M, et al.Jpn.J.Cancer Res., 1986,8 (12): 877-880; Jordan A et al.J Magn Magn Mater, 2001,225:118-126; Hilger I et al.AcademicRadiology, 2002,9 (2): 198-202; Jordan A et al.J Magn Magn Mater, 1999,194:185-196), magnetic resonance video picture (MRI) diagnosis differentiates gain reagent (Hasegawa M et al.Jpn.J.Appl.Phys., 1998,37 (3A): 1029-1032; Kim DK et al.J Magn Magn Mater, 2001,225:256-261; Shinkai M.J Biosci and Bioeng, 2002,94 (6): 606-613; Papisov MI et al.J Magn Magn Mater, 1993,122 (1-3): 383-386; Kim DK et al.J J Magn MagnMater, 2001,225 (1-2): 30-36; Babes L et al.J Colloid and Interface Sci, 1999,212:474-482) and the magnetic oriented carrier (Shinkai M.J Biosci and Bioeng, 2002,94 (6): 606-613 that are used for medicine location or radiation therapy; Xu Z and Gu Y.WO 03/005029 A2, Canada, 2003; Widder KJ, et al.Proc.Soc.Exp.Bio.Med, 1978,58:141-146; Widder KJ, Flouret G and Senyei AE.J Pharm Sci, 1979,68:79-82; Leach JH.Magnetic Targeted Drug Delivery.Thesis of Master of Science in ElectricalEngineering, Virginia Polytechnic Institute and State University, Blacksburg, VA, 2003) etc. the aspect has extensively and important use value.The nanoparticle granularity is little, facilitates penetration of interstice and is absorbed by cell, can also pass through blood brain barrier, and can reduce or avoid the absorption of reticuloendothelial system by the blood capillary of human body minimum.Magnetic nanometer particles is because granularity is little, and suspension stability is good, and specific surface is big, the surface activity height, and high adsorption capacity can be made liposome, but also coated high molecular material perhaps carries out chemical modification, is used to carry medicine or other biological material.Because of its superparamagnetism, in magnetic field, can finely respond, it is almost nil to remove magnetic field remanent magnetism, so can be used for fast enriching, separation and the purification of biological substance, medicine is in the intravital targeting transportation of people and location or the like.In addition, magnetic nanometer particles also can be used as MRI resolution reagent; In superthermal therapy, utilize magnetic nanometer particles under alternating magnetic field, to generate heat, make the tumor tissues that has absorbed magnetic nanometer particles to be rapidly heated and reach 45 ℃～47 ℃, directly the kill tumor cell.
Be used for biomedical sector, particularly being applied to the nano magnetic material that human body medicine transports mainly is that magnetic property is good, and chemical property is stable under physiological condition, good biocompatibility, the Fe that toxic and side effects is little
3O
4, γ-Fe
2O
3Magnetic nanometer particles that modify or unmodified is existing commercially available at present (as the nanomag of German Micromod Partikeltechnologie company
-D), but fetch long price.
If consider that therefore application and cost need, magnetic nanometer particles also can be made by oneself.Adopt silica gel coated magnetic nanoparticle, can form hydrophilic clad, improve the dispersibility of magnetic nanometer particles on the magnetic nanometer particles surface; The silica gel clad is inertia and has biocompatibility, can improve the oxidation resistance of magnetic nanometer particles, reduces toxicity simultaneously; Silica gel is easy to modify in addition, and the silanol base is easy to and dressing agent reaction such as silane coupler.Nineteen ninety Philipse has just developed a kind of method (Albert P.Philipse with silica gel coated ferroferric oxide nanoparticle, Michel P.B.van Bruggen and ChellapahPathmamanoharan.Magnetic Silica Dispersions:Preparation and Stability ofSurface-Modified Silica Particles with a Magnetic Core.Langmuir, 1994,10:92-99).Silane coupler is a kind of bifunctional reagent: an end and silanol base or magnetite nano microparticle surfaces hydroxyl reaction, the other end then are can be directly or indirectly and biomolecule and the bonded organo-functional group of chemicals.Kobayashi in 1991 and Matsunaga modify the ferroferric oxide magnetic nano microgranule with containing amino silane coupler, and be used for immobilization (the Kobayashi H and Matsunaga T.Amino-silane modified superparamagnetic particles with surface-immobilizedenzyme.Journal of Colloid and Interface Science of enzyme, 1991,141 (2): 505-511).
The preparation of relevant magnetic nanometer particles at present both at home and abroad, modification and a lot of in related article, the patent of the application of biomedical sector are not given unnecessary details herein one by one.Our major concern be to use the magnetic nanometer particles that carries radionuclide, be used for radiopharmaceutic location, thereby improve radiopharmaceutic targeting, curative effect, reduce toxic and side effects.
External mainly is the Urs O. of U.S. Cleveland Clinic Foundation
Seminar, they have studied magnetic microsphere and have carried
90Y,
186/188Re,
111In,
67Ga,
153Radionuclides such as Sm (
UO, Sweeney SM, Beresford BA, Humm JL, Macklis RM.Effective targeting ofmagnetic radioactive
90Y-microspheres to tumor cells by an externally appliedmagnetic field.Preliminary in vitro and in vivo results.Nuclear Medicine andBiology-International Journal of Radiation Applications and InstrumentationPart B 1995,22:147-155;
UO, Pauer GJ, and Macklis RM.Treatment ofmouse tumors with radioactive magnetic microspheres:Model for intracavitaryradiotherapy.Proceedings International Symposium on Controlled Release ofBioactive Materials 1995,22:89-90;
UO, Pauer GJ, Roberts W K andHumm J L, Magnetically targeted microspheres for intracavitary and intraspinalY-90 radiotherapy Presented at International Conf.on Scientific and ClinicalApplications of Magnetic Carriers (Rostock, German);
UO, Pauer GJ, Failing S, and Tapolsky G.Radiolabeling of magnetic particles with rhenium-188for cancer therapy.Journal of Magnetism and Magnetic Materials 2001,225:73-78;
UO, in:R.Arshady (Ed.), 2001, Radiolabeled and MagneticParticulates in Medicine and Biology, MML series Vol.3, Citus Books, London, Chapter 18; Yu JF,
UO, Dong Y, Sands MJ, Li YH, Failing S, Leakakos T, Tapolsky G.Radiolabeling of magnetic targeted carriers with several therapeuticand imaging radioisotopes.European Cells and Materials 2002,3 Suppl.2:16-18), the magnetic microsphere of its use has polylactic acid-magnetic iron ore microsphere, iron-carbon alloy microsphere (MTCs is by the exploitation of U.S. FeRx company).
At present, the magnetic microsphere of all radioisotope labelings also is not applied to human body.This is because magnetic microsphere is with all microsphere supported the same, thereby is difficult to limit by biomembrane their application (Rusetskii et al., 1985; Gallo and Hassan, 1988).And just because of this, work of all relevant magnetic microspheres all are the carriers that concentrates on the cancer therapy drug of delivery high dose in the interventional therapy.The MTC-DOX that is used for liver cancer treatment as the exploitation of U.S. FeRx company
By the authentication of U.S. FDA, the I/II clinical trial phase that carries out in the U.S. is near completion.
Magnetic nanometer particles is different from magnetic microsphere, its nano-scale can be so that microgranule can pass interstice and capillary wall, therefore can be by the method for intravenous injection or intra-arterial injection, with the immobilized magnetic nanometer particles that radionuclide or chemotherapeutics are arranged, be directed to focus under the action of a magnetic field fast adding.Domestic have the Hu Jifan of Shandong University and colleague thereof to study the lift-launch radioactivity
125The magnetic nanometer particles of I-bovine serum albumin (the Chinese invention patent application number is 01114912.4).But its be with
125I-BSA and nano magnetic material (Fe
3O
4) and the ultrasonic simply mixing of dispersant (as polyvinyl pyrrolidone), its stable ambigendi locus.
Tumor tissues is because protein synthesis speeds up, and amino acid whose picked-up speed also increases, so labeled amino acid can carry out tumor imaging or treatment.L-histidine (L-Histidine) is a human indispensable amino acid, the imidazole radicals that contains can and fac-[
188Re (CO)
3(H
2O)
3]
+Ligand exchange reaction takes place, and forms stable coordination compound.Cefotaxime acetic acid [(Z)-2-methoxyimino-2-(2-aminothiazol-4-yl)-acetic acid] and derivant thereof are the medicine intermediates of the important semi-synthetic cephalosporins of preparation, has broad-spectrum antibacterial action, their thiazolyl is the heterocycle that contains N-and S-atom, might can with fac-[
188Re (CO)
3(H
2O)
3]
+Ligand exchange reaction takes place, and forms stable coordination compound.If with aglucon,, just might guarantee the fixed radionuclide of magnetic nanometer particles stablizing under physiological condition better as immobilized magnetic nanometer particles surfaces of arriving such as histidine with the chemical bond mode.
Summary of the invention
The objective of the invention is provides a kind of magnetic Nano radiopharmaceutical in order to solve above-mentioned problem; Another object of the present invention provides the radiopharmaceutic preparation method of this magnetic Nano.
The objective of the invention is to realize by following technical proposals: the radiopharmaceutic preparation method of a kind of magnetic Nano, it includes following steps:
1) magnetic nanometer particles is carried out surface chemical modification;
2) immobilized aglucon;
3) aglucon is carried out radioisotope labeling.
Wherein, the silica gel that said surface chemical modification includes magnetic nanometer particles in the said method step 1) coats and two processes of silane coupler modification.
Said silane coupler can be silane couplers such as amino or epoxy radicals.
Best amino silicane coupling agent is N-β-(aminoethyl)-γ-An Bingjisanjiayangjiguiwan.
This method step 1) magnetic nanometer particles in then generally selects for use magnetic property good, and chemical property is stable under physiological condition, good biocompatibility, the Fe that toxic and side effects is little
3O
4Or γ-Fe
2O
3
Said biomolecule refers generally to protein, monoclonal antibody or polypeptide etc.
Said medicine can be a chemotherapeutics, as methotrexate, amycin, daunorubicin etc.; Antibiotic or its intermediate are as cefotaxime acetic acid.
In addition, said radionuclide can be in the said method step 3)
188/186Re,
90Y,
111In or
125/131I etc.
Fe in the inventive method step 1)
3O
4Or γ-Fe
2O
3Magnetic nanometer particles can be bought acquisition; If or consider that application and cost need, and also can make by oneself.Fe wherein
3O
4The preparation of magnetic nanometer particles can be adopted the partial reduction sedimentation method (Qu S, Yang H, Ren D, et al.Magnetite Nanoparticles Preparedby Precipitation from Partial Reduced Ferric Chloride Aqueous Solutions.JColloid Interface Sci, 1999,215:190-192): because preparation ferriferrous oxide nano microgranule is alkalization precipitation Fe usually
3+-Fe
2+Mixed solution, but Fe
2+Raw material is easy to be become Fe by dioxygen oxidation in air and aqueous solution
3+, so products therefrom often purity and magnetic property are poor, the partial reduction sedimentation method can address this problem well.But do not make blanketing with inert gas, FeCl in the above-mentioned document
3With Na
2SO
3Mol ratio be 3: 1, react wayward, product purity is lower; And method of the present invention makes blanketing with inert gas, FeCl
3With Na
2SO
3Mol ratio be adjusted to theoretical than promptly 6: 1, and reaction finish after at 60～80 ℃ of about 30min of ageing, can improve the magnetic performance and the purity of magnetic nanometer particles greatly.Its preparation process is: at FeCl
3In the aqueous solution, inert gas shielding, stirring drip Na down
2SO
3Aqueous solution is with part Fe
3+Be reduced into Fe
2+Drip alkaline solution subsequently.Above-mentioned reaction equation is:
6Fe
3++SO
3 2-+18OH
-→2Fe
3O
4↓+SO
4 2-+9H
2O
Wherein:
FeCl
3Concentration is 10
-3～1mol/L;
FeCl
3With Na
2SO
3Mol ratio be 6: 1～3: 1;
Noble gas refers to nitrogen or argon or their mist;
Alkaline solution refers to NaOH, KOH, ammonia, NR
4 +OH
-Deng aqueous solution;
In the preferred scheme of the present invention, FeCl
3Concentration is 10
-2Mol/L, FeCl
3With Na
2SO
3Mol ratio be 6: 1.
The ferroferric oxide magnetic nano microgranule that above-mentioned reaction generates separates with the Nd-Fe-B permanent magnet, and uses distilled water wash, is dispersed at last in the alcohol-water (its volume ratio is 7: 3～9: 1) so that follow-up modification.The magnetic nanometer particles particle diameter of gained is 7～27nm, mean diameter～15nm; Through XRD analysis is pure inverse spinel magnetic iron ore structure.
Concrete preparation process of the present invention is:
1) surface of above-mentioned magnetic nanometer particles is modified:
A) silica gel coats: reference literature (WO 01/37721 A2), in ferroferric oxide magnetic nano microgranule-alcohol-water dispersion, stir an amount of tetraethyl orthosilicate ester (TEOS) and the ammonia of adding down, room temperature to 40 ℃ heating in water bath 12～24 hours.Magnetic nanometer particles that the silica gel that makes coats such as preceding separation, washing are scattered in the organic solvent such as methanol/ethanol/DMF, so that further modify at last.The about 20nm of magnetic nanometer particles particle diameter of silica obtained parcel; Analyze through vibrating specimen magnetometer (VSM), saturation magnetic moment is about 61emu/g.
B) silane coupler is modified: reference literature (Kobayashi H and Matsunaga T.Amino-silane modified superparamagnetic particles with surface-immobilizedenzyme.Journal of Colloid and Interface Science, 1991,141 (2): 505-511), in the magnetic nanometer particles system that dispersive silica gel coats with organic solvent, add an amount of (amino, epoxy radicals etc.) silane coupler, room temperature to 100 ℃ reaction 2～24 hours.Reaction finishes, and with corresponding organic solvent-water washing, transfers water washing gradually to.It is standby to be dispersed in the middle preservation of suitable buffer solution (as the PBS of pH 7.4) at last.With amino silicane coupling agent N-β-(aminoethyl)-γ-An Bingjisanjiayangjiguiwan (adopting Nanjing Shuguang Chemical General Plant's product, trade name SG-Si900) is example, and modification is shown in following reaction equation:
2) immobilized (immobilization):
List of references (Jiang Zhonghua, Zhang Jinhui chief editor, biomolecule mobilization technology and application, Chemical Industry Press, 1998.7 front pages, Beijing) according to required immobilized aglucon, is selected good magnetic nanometer particles of suitable modification and immobilized condition.With protein is representative, can select amino-magnetic nanoparticle (being the magnetic nanometer particles that amino silicane coupling agent is modified), under the neutrallty condition, with glutaraldehyde as cross linker, by the Michael additive reaction, just proteinaceous solid can be downloaded to the magnetic nanometer particles surface.
3) radioisotope labeling (radionuclide labeling): (Li Yongjian, Sun Qi smoke the chief editor to list of references, radiopharmaceutical and labelled compound, Science Press, 1996.2 front page, Beijing), do not have difference with the radioactive label of biomolecule such as common protein, polypeptide, just can adopt externally-applied magnetic field (Nd-Fe-B permanent magnet) during separation and purification.
By above-mentioned disclosed technical scheme as seen, the present invention is in step 1), and can make by oneself and obtain mean diameter is the ferroferric oxide magnetic nano microgranule of 15nm, or buys Fe
3O
4Or γ-Fe
2O
3Magnetic nanometer particles; It is carried out silica gel with TEOS basic hydrolysis coat, the silanol radical reaction of reuse silane coupler and magnetic nanometer particles and carried out surface chemical modification makes the surface that organic active functional group be arranged; In step 2), can select suitable magnetic nanometer particles and immobilized condition according to aglucon, aglucon is immobilized to the magnetic nanometer particles surface; At last, in step 3), can be according to the method for labelled protein, polypeptide etc. in the past, the magnetic nanometer particles of immobilized aglucon is carried out the labelling of radionuclide.
The present invention is first with radionuclide
188Re is fixed to the magnetic nanometer particles surface by chemical bond.At first by
188Re-ReO
4 -Synthesizing radioactive predecessor fac-[
188Re (CO)
3(H
2O)
3]
+, hydrone part and immobilized aglucon to the magnetic nanometer particles surface then carry out ligand exchange as the imidazole radicals of histidine, thereby are fixed on the magnetic nanometer particles surface.Because the kinetic inertness of carbonyl-complexes is this
188Very stable of Re (I) chemistry, if with the chemical bond mode with aglucon, immobilized as histidine to the magnetic nanometer particles surface, just can guarantee the fixed radionuclide of magnetic nanometer particles stablizing under physiological condition better.By the magnetic Nano radiopharmaceutical of method preparation of the present invention, its 72 hours radioactivity retention rates generally can reach more than 80%.
The present invention combines the treatment characteristic of nano-meter characteristic, magnetic property and the radionuclide of magnetic nanometer particles, use externally-applied magnetic field, can radiopharmaceutical is dense poly-at lesions position fast, thus curative effect improved, reduce toxic and side effects such as killing and wounding normal tissue cell.
Description of drawings
Fig. 1 (1) records Fe for HITACHI-600 type transmission electron microscope (TEM)
3O
4Magnetic nanometer particles photo; The magnetic nanometer particles TEM photo that Fig. 1 (2) coats for silica gel.
Fig. 2 at room temperature uses Cu target K α line for D/max 2250 type x-ray diffractometers
X-ray powder polycrystalline diffraction (XRD) pattern of the magnetic nanometer particles that the silica gel that records for X-ray light source coats.
Fig. 3 is LEO 1530 VP scanning electron microscope (SEM) photos of the magnetic nanometer particles of silica gel coating.
The magnetic nanometer particles EG﹠amp that Fig. 4 coats for silica gel; The hysteresis curve that G Princeton Research 155 type vibrating specimen magnetometers (VSM) are measured.
Fig. 5 is
188Immobilized vitro stability (in the calf serum, the 37 ℃) curve chart that the magnetic nanometer particles of histidine and cefotaxime acetic acid is arranged of Re labelling, wherein MN-His is the magnetic nanometer particles of immobilized histidine; MN-(1) is the magnetic nanometer particles of immobilized cefotaxime acetic acid.
Fig. 6 is
188Immobilized vitro stability (in the calf serum, the 37 ℃) curve chart that the magnetic nanometer particles of BSA is arranged of Re labelling, wherein MN-BSA is the magnetic nanometer particles of immobilized BSA.
The specific embodiment
Below will be described further the specific embodiment of the invention, but embodiment does not limit protection scope of the present invention by embodiment.Agents useful for same among the embodiment, 2-N-morpholino ethane sulfonic acid (MES) and BH
3NH
3Available from Fluka; SG-Si900 is available from Nanjing Shuguang Chemical General Plant; 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide hydrochloride (EDC) is available from ACROS; N; N '-carbonyl dimidazoles (CDI) is available from the biochemical (Shanghai) Co., Ltd. of gill; all the other are available from Shanghai chemical reagents corporation, except that L-histidine and 25% glutaraldehyde are that all the other are analytical pure the biochemical reagents.The Nd-Fe-B permanent magnet is available from Yaolong Non-ferrous Metal Co. Ltd., Shanghai, and Surface field intensity is 1Tesla.Fresh, DNAcarrier free
188Re-ReO
4 -From
188W/
188The Re generator (Ke Xing pharmaceutcal corporation, Ltd in Shanghai produces, its
188The W parent is provided by Oak Ridge laboratory (ORNL)) get with normal saline drip washing.
Embodiment 1
The preparation of step 1 ferroferric oxide magnetic nano microgranule: at first get 2mol/L FeCl
3Solution 12.0ml (in advance with 2mol/L hydrochloric acid preparation) and adds about 100ml redistilled water and dilutes in 500-ml three neck round-bottomed flasks; Logical nitrogen, stirring slowly drip freshly prepared 0.08mol/L Na down
2SO
3Solution 50ml, can be observed solution colour change into gradually from yellow orange red, until change into orange-yellow till; Under 60～80 ℃ of heating in water bath, logical nitrogen, the high degree of agitation, slowly drip weak ammonia (V
28% ammonia/ V
Heavy Steam water=1:, generate a large amount of black precipitates 9) to pH to 8～10; Continue to keep about 70 ℃ of water-bath 15～30min; Stop to heat below the postcooling to 45 ℃, isolate black precipitate with Magnet, and with the redistilled water washing to neutral.Gained Fe
3O
4The TEM photo of magnetic nanometer particles shown in Fig. 1 (1), its particle diameter 7～27nm.
Step 3 silane coupler is modified: the magnetic nanometer particles of above-mentioned silica obtained coating is dispersed in the 100ml methanol, guarantees that system is moisture at 1～4% (percentage by weight); Add 5ml SG-Si900, ultrasonic about 5min mixing, 60 ℃ of water-baths, stirring are reacted more than 12 hours down.After reaction finishes, isolate magnetic nanometer particles with Magnet, with the methanol wash number all over removing excessive silane coupler, then with the redistilled water washing, be dispersed at last preserve among redistilled water or the 0.1mol/L PBS (pH 7.4) standby.
Step 4 histidine immobilized: (abandoning supernatant is with PBS (pH 7.4) washing of 0.1mol/L 1～2 time for solid content＞20mg/ml), magnetic field separation to get the magnetic nanometer particles that 1ml SG-Si900 modifies; Add 2.5% glutaraldehyde solution (25% glutaraldehyde water solution gets with the PBS dilution of 0.1mol/L, pH 7.4) 1ml, ultrasonic mixing, 4 ℃～40 ℃ were reacted 2～24 hours; Reaction finishes, magnetic field separation, and with 0.1mol/L PBS (pH 7.4) washing 3 times, the L-histidine solution that adds the freshly prepared 0.2mol/L of 1ml then (is used pH 7.0, the PBS of 0.1mol/L; 0.15mol/L NaCl; 0.005mol/L EDTA solution preparation), ultrasonic mixing, room temperature reaction 24 hours, the back that finishes is with 0.1mol/L PBS (pH 7.4) washing 3 times; Add 1ml again and contain 0.5mg/ml NaBH
40.1mol/L Na
2B
4O
7(pH9.2), mixing, 4 ℃ of reaction 30min are to eliminate superfluous aldehyde radical and two key.Use 0.1mol/L PBS (pH 7.4) washing 3 times at last, and be dispersed in 1ml 0.5mol/L MES (pH 6.6) buffer solution.Above-mentioned reaction equation is as follows:
Step 5
188The Re labelling the is immobilized magnetic nanometer particles of histidine: we are with fac-[
188Re (H
2O)
3(CO)
3]
+Be the radioactive label precursor, the complex reaction that utilizes the imidazole radicals of histidine and Re (I) coordination center is (with H
2Ligand exchange takes place in O), thus will
188The Re labelling is to the magnetic nanometer particles surface.Take by weighing 5mg NH
3BH
3In 10ml dried and clean ampoule, add rubber closure and aluminium lid, sealing, about 15 minutes of logical high-purity CO gas; Add 6 μ l 85%H with syringe
3PO
4With the fresh normal saline drip washing of 1ml
188Re-ReO
4 -(from
188W/
188The drip washing of Re generator and get) mixed solution, 60～80 ℃ of about 20min of water-bath obtain fac-[
188Re (H
2O)
3(CO)
3]
+The radioactive label precursor needn't separation and purification, can be directly used in the labelling magnetic nanometer particles.In containing the solution of labelled precursor, add the dispersive immobilized magnetic nanometer particles of histidine of MES (pH 6.6) buffer solution of isopyknic 0.5mol/L (solid content＞20mg/ml), 60～80 ℃ of about 30～50min of water-bath.(pH6.6 0.5mol/L MES under optimal conditions; 70 ℃ of reaction temperatures; Response time 40min), we obtain 91.4 ± 0.3% higher mark rate (mark rate of precursor is 85.7 ± 6.1%), the good magnetic nanometer particles of labelling has good stable in calf serum, in 37 ℃, 3 days, as shown in Figure 5, its 24 hours radioactivity retention rates are 89.3 ± 0.3%; The radioactivity retention rate was 86.5 ± 0.3% in 48 hours; The radioactivity retention rate was 81.6 ± 2.2% in 72 hours.
Fixing of step 4 cefotaxime acetic acid: the magnetic nanometer particles that 1ml SG-Si900 modifies (solid content＞20mg/ml), behind the magnetic field separation, PBS (pH 7.4) washing with 0.1mol/L, ultra-sonic dispersion contains in MES (pH 6.3) buffer solution of the 0.1mol/L of 0.2mol/L cefotaxime acetic acid at 1ml then, the carboxyl that adds 500mg EDC activation cefotaxime acetic acid is to start reaction, room temperature reaction is more than 12 hours, back 0.1mol/L PBS (pH 7.4) washing finishes, be dispersed at last in the 0.5mol/LMES buffer solution of 1ml pH about 6.6, be used for next step
188The Re labelling.Above-mentioned reaction equation is as follows:
Step 5
188The Re labelling the is immobilized magnetic nanometer particles of cefotaxime acetic acid: equally with fac-[
188Re (H
2O)
3(CO)
3]
+Be the radioactive label precursor, the complex reaction that utilizes the thiazolyl of cefotaxime acetic acid and Re (I) coordination center is (with H
2Ligand exchange takes place in O), thus will
188The Re labelling is to the magnetic nanometer particles surface.Method is identical with step 5 among the embodiment 1, and its mark rate is 78.4 ± 1.4%, stability as shown in Figure 5: its 24 hours radioactivity retention rates are 77.2 ± 1.2%; The radioactivity retention rate was 73.5 ± 3.5% in 48 hours; The radioactivity retention rate was 72.1 ± 4.7% in 72 hours.
Embodiment 3
Adopt the immobilized magnetic nanometer particles that bovine serum albumin (BSA) arranged of self-control, utilize classical Sn (II) reducing process, in the magnetic nanometer particles surface markers
188Re.
Step 3 silane coupler is modified: at first synthetic hemisuccinic acid N-β-(aminoethyl)-γ-An Bingjisanjiayangjiguiwan; 4.5mmol N-β-(aminoethyl)-γ-An Bingjisanjiayangjiguiwan and 4.5mmol succinic anhydrides (being succinic anhydride) are dissolved in the 1ml dry DMF; Dropwise 35 0 μ l diisopropylamine; argon shield, stirring at room reaction 24 hours.In the 20ml dry DMF, add the Fe that about 500mg hemisuccinic acid N-β-(aminoethyl)-γ-An Bingjisanjiayangjiguiwan and 50mg silica gel coat
3O
4Magnetic nanometer particles, stirring at room reaction 24 hours, dry then with the DMF washing, obtain the magnetic nanometer particles that terminal carboxyl group is contained on the surface.Above-mentioned course of reaction is as follows:
Step 4 bovine serum albumin (BSA) immobilized: at first with the carboxyl on CDI activation magnetic nanometer particles surface; form activatory acylimidazole intermediate; when the BSA that contains primary amino radical existed, imidazole radicals was substituted, and formed stable amido link between carrier and the BSA aglucon.The magnetic nanometer particles that 50mg hemisuccinic acid N-β-(aminoethyl)-γ-An Bingjisanjiayangjiguiwan is modified is scattered in the 1ml anhydrous propanone, add the reaction of 100mg CDI room temperature shake activated in 1 hour, centrifugal then or magnetic field separation, with anhydrous propanone washing several times, the distilled water that reuse is ice-cold is finished 2 washings fast, adding 1ml immediately contains the 0.1mol/L pH 8.5 sodium carbonate buffer solution of 20mg/ml BSA and is uniformly dispersed, 4 ℃ of jolting reactions are more than 24 hours, wash with 0.1mol/L pH 8.5 sodium carbonate buffer solution, to remove free BSA, be dispersed among 1ml 0.1mol/L pH 7.4 PBS 4 ℃ of preservations at last.Above-mentioned course of reaction is as follows:
Step 5
188Re directly marks the magnetic nanometer particles of immobilized BSA: logical N in the 0.1mol/L of freshly prepared 10ml citric acid solution
2(g) 15min adds the SnCl of 80mg then
22H
2O and 80mg vitamin C (Vc) dissolving continue logical N
2(g) preserve labelling usefulness fully.Normally, labeling process is as follows: the magnetic nanometer particles of the immobilized BSA of 25mg (MN-BSA) is dissolved with SnCl with 500 μ l
2Mix mutually with the citric acid solution of Vc, add 25 μ l fresh drip washing then
188Re-ReO
4 -(about 1mCi), boiling water bath reaction 30min, magnetic field or centrifugalize are removed free with the normal saline washing
188Re-ReO
4 -, be dispersed in again at last among the PBS of 0.1mol/L, pH 7.4 of 500 μ l.Under optimal conditions, mark rate can reach 92%, and the good magnetic nanometer particles of labelling has good stable in calf serum, in 37 ℃, 3 days, and as shown in Figure 6: its 24 hours radioactivity retention rates are 100.1 ± 0.3%; The radioactivity retention rate was 95.9 ± 1.5% in 48 hours; The radioactivity retention rate was 81.0 ± 8.1% in 72 hours.
Certainly, the magnetic nanometer particles in the various embodiments described above also can be selected γ-Fe for use
2O
3, can directly buy magnetic nanometer particles that obtain modifying or unmodified (as the nanomag of German Micromod Partikeltechnologie company
-D); Immobilized aglucon also can be biomolecule such as other protein, monoclonal antibody, polypeptide, aminoacid, and chemotherapy, antibiotic etc or its intermediate; Radionuclide also can be selected for use
90Y,
111In or
125/131/123I etc. do not give unnecessary details one by one at this.
Claims (7)
1, the radiopharmaceutic preparation method of a kind of magnetic Nano, it includes following steps:
1) magnetic nanometer particles being carried out surperficial silica gel coats and the silane coupler chemical modification;
2) immobilized aglucon histidine, cefotaxime acetic acid or bovine serum albumin;
3) aglucon is carried out radionuclide
188Re or
186The Re labelling.
2, the method for claim 1 is characterized in that this silane coupler is amino or epoxy silane coupling.
3, method as claimed in claim 2 is characterized in that this amino silicane coupling agent is N-β-(aminoethyl)-γ-An Bingjisanjiayangjiguiwan.
4, the method for claim 1 is characterized in that this method step 1) in magnetic nanometer particles be Fe
3O
4Or γ-Fe
2O
3
5, method as claimed in claim 4 is characterized in that this Fe
3O
4In the preparation process of magnetic nanometer particles inert gas shielding is arranged.
6, method as claimed in claim 5 is characterized in that this noble gas is nitrogen or argon or their mist.
7, the magnetic Nano radiopharmaceutical for preparing as the described method of the arbitrary claim of claim 1-6.
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| CN1325732A (en) * | 2001-04-28 | 2001-12-12 | 山东大学 | Nm-class magnetic material carrying radioisotope and its preparing process |
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