CN101480494B - Magnetic resonance contrast agent based on humanized antibody for diagnosing tumor - Google Patents
Magnetic resonance contrast agent based on humanized antibody for diagnosing tumor Download PDFInfo
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Abstract
The invention belongs to the diagnosis field, and particularly discloses a magnetic resonance contrast agent for tumor diagnosis and a preparation method thereof. The magnetic resonance contrast agent SM-USPIO used as a humanized antibody is obtained by crosslinking a humanized SM5-1 antibody and an ultra-small super paramagnetic iron oxide particle USPIO. The SM-USPIO has the advantage of strong specificity.
Description
Technical field
The invention belongs to diagnostic field, more specifically, the invention discloses a kind of mr contrast agent that is used for diagnosing tumor and preparation method thereof.
Background technology
Along with the development of monoclonal antibody technique, prepared a lot of monoclonal antibodies to the tumor correlating markings, antibody plays more and more important effect in oncotherapy.Make Antybody therapy bring into play more effective effect, the molecule parting of necessary clear and definite patient's tumor.Especially for the patient who is not suitable for performing the operation, the responsive non-invasive diagnosis means of research are used for tumor molecular diagnosis particular importance.
(Magnetic Resonance Imaging is a kind of repeatedly used non-invasive diagnostic techniques MRI) to mr imaging technique, has good spatial resolution and contrast.Conventional MRI is still not enough for the resolution capability of tumor tissues and normal structure; Therefore must study new formation method; The molecular probe that will have the cancer target performance is crosslinked in suitable MRI imaging contrast; Promptly possibly carry out the molecular image diagnosis to tumor through MRI, this requires to select the suitable efficient contrast medium and the tumor probe of high specific.
Nanoparticle is widely used in biomedicine; Ultra-fine superparamagnetic iron oxide particle (ultrasmall superparamagnetic iron oxide particle; USPIO) as the existing history that surpasses 10 years of the research of MRI imaging contrast; Compare with the agent of traditional M RI imaging contrast gadolinium, USPIO has hypotoxicity, the advantage of hypersensitivity.Recently, people also are optimized through structure and composition to USPIO, prepare than the stronger USPIO of traditional USPIO magnetic.USPIO is as the MRI imaging contrast; Be used to the imaging of gastrointestinal tract, liver, spleen; And discriminating good, the malignant lymph node enlargement; But in these were used, USPIO was engulfed by the monokaryon macrophage system through passive target and brings into play potentiation, can not be used for the molecule parting of tumor.
The cancer target probe that is used to develop cancer target MRI contrast medium at present is less, mainly is the not high receptor quasi-molecule of several kinds of antibody and other specificity, like lutropin, folic acid etc.Distinct issues are that targeting property is not high, poor specificity.
Summary of the invention:
In order to address the above problem, inventor of the present invention is cross-linked to humanized SM5-1 monoclonal antibody on the nanoparticle USPIO of preparation, prepares the MRI contrast medium with targeting property, called after SM-USPIO.MRI through flow cytometry, Laser Scanning Confocal Microscope observation and cultured cell in vitro confirms can combine with the tumor cell specific of SM5-1 antigen positive through crosslinked antibody at external SM-USPIO, and can be by the tumor cell endocytosis; Behind the injection SM-USPIO, MRI result shows that the T2 signal of tumor tissues obviously reduces, so SM-USPIO can be used for the in-vivo diagnostic of SM5-1 antigen positive tumor in the tumor-bearing mice body.
More specifically, the invention discloses:
1. mr contrast agent SM-USPIO based on humanized antibody is for humanization SM5-1 antibody and superparamagnetic iron oxide particle USPIO is crosslinked gets.
2. the method for preparing of above-mentioned 1 described mr contrast agent SM-USPIO may further comprise the steps:
Step a: preparation superparamagnetic iron oxide particle USPIO,
Step b: the preparation humanized antibody,
Step c: preparation mr contrast agent SM-USPIO.
3. the method for preparing of above-mentioned 2 described mr contrast agent SM-USPIO, wherein the preparation of superparamagnetic iron oxide particle USPIO obtains through coprecipitation method among the step a:
Steps A: glucosan is dissolved in the water, adds iron salt, stir;
Step B: under the condition of heating, polyvinyl alcohol is dissolved in the water;
Step C: mix in the solution that the solution adding steps A that step B is obtained obtains, to 10-12, continue to stir post-heating and obtain deposition with adjusting PH with base;
Step D: the collecting precipitation thing also carries out post processing to it.
4. the method for preparing of above-mentioned 3 described mr contrast agent SM-USPIO, the iron salt that wherein uses in the steps A is FeCl
36H
2O and FeSO
47H
2O, wherein ferric iron and ferrous mol ratio are to be 3: 2.
5. the method for preparing of above-mentioned 3 described mr contrast agent SM-USPIO, wherein the alkali described in the step C is ammonia.
6. the method for preparing of above-mentioned 3 described mr contrast agent SM-USPIO is wherein utilized magnetic field collecting precipitation thing among the step D.
7. the method for preparing of above-mentioned 3 described mr contrast agent SM-USPIO, wherein post-processing step comprise utilize washing, with the resuspended deposition of citric acid solution, ultrasonic, in deionized water the unnecessary citric acid of dialysis removal.
8. the method for preparing of above-mentioned 2 described mr contrast agent SM-USPIO; Wherein the step c step for preparing mr contrast agent SM-USPIO comprises UPSIO is carried out activation, separates the particle after the activation, and particle is mixed with the PBS that contains antibody; Stir, handle and get through the magnetic post.
9. above-mentioned 1 the purposes of mr contrast agent SM-USPIO aspect diagnosing tumor.
10. above-mentioned 9 purposes, wherein tumor is the conjugated protein male tumor of SM5-1.
Inventor of the present invention is crosslinked with the superparamagnetic iron oxide particle (USPIO) of humanized SM5-1 antibody and coprecipitation acquisition, has obtained a kind of targeting MRI contrast medium of nanoscale, called after SM-USPIO.Its average aquation diameter is 25.3nm, and this nanoscale has guaranteed interior long cycle period of its body and targeting efficient.
Inventor of the present invention utilizes several different methods to detect the SM-USPIO specific binding capacity.The flow cytometry result shows, compares with free humanization SM5-1 monoclonal antibody, and SM-USPIO has similar and the bonded ability of the conjugated protein positive human HCC of SM5-1 ch-hep-3, and the result of reference substance H-USPIO is negative.In different cell strains, the average fluorescent strength after SM-USPIO handles and the protein-bonded expression positive correlation of SM5-1 of each cell; But the result of Laser Scanning Confocal Microscope shows the SM-USPIO specificity and is incorporated into the conjugated protein male tumor cell of SM5-1, and can be by this cell endocytic.MRI detect to confirm that the positive tumor cell that SM-USPIO handled has tangible T2 signal weakening, adds humanization SM5-1 monoclonal antibody and does not have this effect and contrast contrast medium H-USPIO and blank contrast medium USPIO.Above results suggest SM-USPIO can get into and accumulate the conjugated protein positive human HCC in SM5-1 through antibody-mediated endocytosis; The T2 signal weakening that occurs tumor region in the tumor-bearing mice behind the injection SM-USPIO, and
125The SM-USPIO of I labelling also obviously is gathered in the tumor cell, and therefore, SM-USPIO can get into and accumulate in tumor by local equally through antibody-mediated endocytosis in vivo, the effect of performance MRI radiography contrast medium.
Above-mentioned result of experiment shows, the USPIO of crosslinked humanization SM5-1 monoclonal antibody can be used for the diagnosis of the conjugated protein male tumor of SM5-1.
Thereby other have tumor high specific targeting antibodies also can carry out the crosslinked diagnosis that is applied to tumor by method disclosed by the invention and USPIO, and these tumor high specific targeting antibodies include but not limited to the anti-Her-2 monoclonal antibody of high-affinity, high-affinity monoclonal antibody against EGFR etc.
Description of drawings
Fig. 1: photo that the SM-USPIO transmission electron microscope detects and laser light scattering particle size analyzer are measured the particle diameter of SM-USPIO; The photo that Fig. 1-1:SM-USPIO transmission electron microscope detects; Show that the particle core footpath is between 8-10nm; Fig. 1-2: the laser light scattering particle size analyzer is measured the particle diameter of SM-USPIO, shows that its hydration radius is 25.3 ± 2.1nm.
Fig. 2: the flow cytometry result, the flow cytometry result of Fig. 2-1:ch-hep-3 cell, wherein dark dotted line is represented the PBS processed group; Fine line is the H-USPIO processed group, and heavy line is the SM-USPIO processed group, and light dotted line is the free antibodies processed group; The flow cytometry result of Fig. 2-2:SM-USPIO and three kinds of different cells binding abilities; Fig. 2-2-1 is the ch-hep-1 cell, and Fig. 2-2-2 is the ch-hep-6 cell, and Fig. 2-2-3 is the ch-hep-3 cell; The cell that on behalf of PBS, wherein light solid line handle, dark solid line is represented SM-USPIO.
Fig. 3: the conjugated protein endocytosis result of Laser Scanning Confocal Microscope inspection SM5-1, wherein A is the cell that PBS handles, and B is the cell that SM-USPIO handles, and C is the cell that H-USPIO handles; First classifies the fluorescence visual field as, and second classifies as and differ the visual field, and the 3rd classifies the two stack as.
Fig. 4: the nuclear magnetic resonance experimental result is illustrated as its sagittal plain t2 weighted image.From left to right be respectively ch-hep-1, ch-hep-6 and ch-hep-3, every kind of cell each layer from top to bottom is respectively the sample of handling through PBS, USPIO, USPIO+ free antibodies, SM-USPIO and H-USPIO.
Fig. 5: the in-vivo imaging experimental result, figure is its t2 weighted image.The animal of first behavior injection SM-USPIO, the animal of second behavior injection H-USPIO, first classifies the image before the injection as, and second classifies the back 8 hours image of injection as, and the 3rd classifies the back 72 hours image of injection as.
Fig. 6: distribution experiment result in the body, Fig. 6-1 are the internal organs increased radioactivity result of 8 hours execution treated animals, and Fig. 6-2 is the internal organs increased radioactivity result of 72 hours execution treated animals, and Fig. 6-3 does
125The SM-USPIO of I labelling with
125The clearance curve of the H-USPIO of I labelling in blood.
The specific embodiment
Following examples, experimental example only further specify the present invention, should not be construed as limitation of the present invention.
Materials and methods:
Cell, antibody and other materials:
SMMC-7721 ch-hep-1, ch-hep-3 and ch-hep-6 are set up by the The 2nd Army Medical College institute of oncology and provide; The protein-bonded expression of these cell lines SM5-1 separately is different, ch-hep-3 high expressed wherein, and the ch-hep-6 medium level is expressed; And ch-hep-1 is negative; Its open source information is referring to Zhao J, Wang H, Wei L; Et al.The cytotoxic effect of E1B 55-kDa mutant adenoviruson human hepatocellular carcinoma cell lines, Cancer Gene Ther 2001; 8:333-341; Humanized SM5-1 antibody prepares Li B by literature method; Wang H; Zhang D, et al.Construction and characterization of a high-affinity humanized SM5-1monoclonal antibody.Biochem Biophys Res Commun 2007; 357:951-6.The goat anti-human igg of FITC labelling (H+L) available from Zymed company (San Francisco, CA).Humanized anti-Her2 antibody is according to Chinese invention patent application number 01132225.X, and denomination of invention is disclosed method preparation in " Humanized anti-HER 2 monoclonal antibody and method for making thereof and pharmaceutical composition ".The organic solvent of using in the experiment is the HPLC level, and other reagent are the highest level that can buy.
Laboratory mice is that the Balb/c nude mice (18-22 gram) in age in 6-8 week available from the The 2nd Army Medical College Experimental Animal Center, is raised in this center by the SPF rank.All zooperies all obtain the approval of management of laboratory animal committee of The 2nd Army Medical College.
Transmission electron microscope: JEM-100CX, Japanese JEOL company;
Laser light scattering particle size analyzer: Zeta Sizer 3000HS, Malvern Instruments, UK;
Flow cytometer: Becton-Dickinson, San Jose, CA;
Laser Scanning Confocal Microscope: Zeiss CLSM 510, German Zeiss company;
4.7T Bruker Biospec
AvanceToy magnetic resonance imaging system: Bruker Biosciences, Germany;
Gamma counter: HPGe ADCAMTM918 type, U.S. ORTEC company.
USPIO prepares through coprecipitation method in containing the solution of glucosan, and it comprises the core of ferrum oxide and the low-molecular-weight glucosan of outer parcel.1 gram glucosan (molecular weight 40kDa) is dissolved in the 15ml deionized water, adds FeCl
36H
2O 24.35mg and FeSO
47H
2O 16.68mg stirs under nitrogen protection.Polyvinyl alcohol (Polyvinyl Alcohol; Molecular weight 1750 ± 50) under 80 ℃ condition, prepares saturated solution in the adding deionized water; Get 10ml and mix, under nitrogen protection and lasting condition of stirring, drip 7% ammonia adjusting pH to 10-12 with above-mentioned solution.After black iron oxide particle occurs, continue to stir 10 minutes, be heated to 85 ℃ afterwards and kept 60 minutes; Through magnetic field results precipitate, the deionized water wash number is all over to remove the soluble iron ion.With citric acid solution (1 gram citric acid is dissolved in the 30ml deionized water) the resuspended deposition of 30ml, ultrasonic 20 minutes of 700w, the product that obtains dialyse 48 hours to remove unnecessary citric acid in the 1L deionized water; Finally, to be adjusted to 0.25mol/L by concentration of iron subsequent use for the USPIO of acquisition.
Humanized SM5-1 antibody is crosslinked as follows on the humanized SM5-1 antibody of preparation; Antibody is dissolved in PBS (the 5mmol/L phosphate of pH 7.4 by the concentration of 5mg/ml; 0.9% sodium chloride) in; EDC (1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide Hydrochloride, 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides) and NHS (N-hydroxysulfosuccinimide, N-hydroxy-succinamide) all are dissolved in PBS (the 5mmol/L phosphate of pH 6.0 by the concentration of 50mg/ml; 0.9% sodium chloride) in; Get 1ml USPIO suspension, mix with above-mentioned EDC/NHS solution 2.5ml, stirring at room 30 minutes is with the activation particle; The PD-10 desalting column is removed unnecessary activator on the reactant liquor afterwards; Particle after the separation activation, particle is scattered among the PBS of 3ml, adds the antibody of the above-mentioned preparation of 300 μ l immediately; Stirring at room 2 hours; Particle through the magnetic post (Miltenyi Biotech GmbH, Germany) (by specification operation is no more than the restriction of magnetic column flow rate) and deionization washing multipass are to remove unconjugated antibody; Combined the USPIO (called after SM-USPIO) of humanized SM5-1 antibody to be resuspended among the 10mmol/L PBS of pH7.4 at last, and it is subsequent use to be adjusted to 0.25mol/L by concentration of iron once more.(Pierce, Rockford IL) measure the protein concentration of magnetic post stream in putting on clearly and obtain the concentration of unconjugated SM5-1 humanized antibody, and can therefore calculate the crosslinked amount of antibody on cross-linking efficiency and the particle of antibody through the MicroBCA protein determination kit; Identical method is crosslinked humanization anti-Her2 antibody on USPIO, obtains particle called after H-USPIO.
The particle diameter of nanoparticle and structure obtain through transmission electron microscope and laser light scattering particle size analyzer.
TEM results shows that the ferrum nuclear diameter of SM-USPIO is 8-10nm (Fig. 1-1), the laser light scattering particle size analyzer show its hydration radius be 25.3 ± 2.1nm (98% between 17nm～50nm) (Fig. 1-2).It is 14mg Fe/ml that SM-USPIO is adjusted to concentration, and promptly the concentration of iron of 0.25mmol/ml is preserved, and AC is 10 ± 2 μ g/mg Fe, and the crosslinking rate of antibody is 60%; During preservation, the mannitol of aqueous citric acid solution and 60mg/ml that has added 4.16mg/ml in the system is as stabilizing agent; The result proves that SM-USPIO can stablize June at ambient temperature.
The radioactive label experiment of experimental example 1 targeting USPIO
Through chlorine ammonia-T method (Hunter WM, Greenwood FC (1962) .Preparation of iodine,
131I-labelled human growth hormone of high specific activity.Nature 194:495-496) uses
125I-Na carries out labelling to SM-USPIO and H-USPIO, and (Miltenyi Biotech GmbH, Germany) separation is removed free still to adopt the magnetic post behind the labelling
125I-Na, SM-USPIO behind the labelling and H-USPIO are used for the metabolism experiment.
The activity experiment that combines of experimental example 2 Flow cytometry SM-USPIO and the conjugated protein high expressing cell of SM5-1
1 * 10
6The cell of/ml (ch-hep-3) was hatched 1 hour for 4 ℃ with the PBS of 0.1ml; The humanization SM5-1, SM-USPIO or the H-USPIO that contain 10 μ g/ml respectively (all calculate with the antibody amount; Comprise free be combined on the particle); Hatched 30 minutes for 4 ℃ with the goat anti-human igg (H+L) of FITC labelling behind the cell washing, cell washs the back once more with machine testing on the FACScan flow cytometer.
The result shows and sees Fig. 2-1; The result shows with there was no significant difference between the cell of H-USPIO processing and the PBS processed group; The ch-hep-3 cell of SM-USPIO and humanization SM5-1 antibody treatment shows very high fluorescence intensity; And PBS and H-USPIO processed group are all negative, and SM-USPIO is similar with free antibodies with the protein-bonded binding ability of SM5-1 in prompting, and this results suggest humanization SM5-1 antibodies has still kept itself and the protein-bonded high-affinity of SM5-1 behind the USPIO.
Afterwards; We are again in the different last binding ability that detects SM-USPIO of human liver cancer cell ch-hep-1, ch-hep-3, ch-hep-6 (method is the same) of three strain SM5-1 binding protein expression levels; The result is shown in Fig. 2-2, and the result shows that the average fluorescent strength of the cell that SM-USPIO handles and the SM5-1 binding protein expression level of cell match.The SM5-1 binding protein expression level of Ch-hep-3 is the highest, and its average fluorescent strength is also the highest; The SM5-1 binding protein expression of ch-hep-1 is negative, and its average fluorescent strength is negative; The SM5-1 binding protein expression level of chhep-6 is medium, and its average fluorescent strength is between between the two.This result shows that SM-USPIO combines to have good specificity with target cell.
Experimental example 3 utilizes Laser Scanning Confocal Microscope to detect the conjugated protein endocytosis SM-USPIO experiment of SM5-1
For whether checking Ch-hep-3 cell can pass through the conjugated protein endocytosis SM-USPIO of SM5-1, we have carried out the Laser Scanning Confocal Microscope observation to it: Ch-hep-3 cell (1 * 10
5) hatched altogether 1 hour for 37 ℃ with each 0.1ml of SM-USPIO, H-USPIO or PBS (in the antibody amount) of 10 μ g/ml; The PBS washing; Fix 10 minutes for 4 ℃ with 4% paraformaldehyde; Carried out rupture of membranes in 5 minutes with the 0.1%TritonX-100 incubated at room afterwards and handle, subsequently with 1% bovine serum albumin sealing 30 minutes; At room temperature hatched 2 hours with the sheep anti-mouse igg of FITC labelling again, PBS washing, cell is observed and is taken pictures through fixing, mounting and through the Zeiss510 laser scanning co-focusing microscope.
The result is as shown in Figure 3; The result shows simple two anti-the hatching with the FITC labelling of ch-hep-3 cell, and cell does not have obvious background fluorescence and occurs, but two resist hatch before; Cell and SM-USPIO were hatched 1 hour at 37 ℃ altogether; Tangible fluorescence then appears in cell cytosol, the cell that H-USPIO handles then with the negative cells zero difference, this explanation SM-USPIO can merge by cell internalizing with the ch-hep-3 cell node specifically.
Experimental example 4 nuclear magnetic resonances experiment
For external imaging, 1 * 10
5Cell (be respectively ch-hep-1; Ch-hep-3; Ch-hep-6) under 37 ℃ of conditions, hatched 1 hour with following composition: SM-USPIO, H-USPIO, USPIO, USPIO add humanization SM5-1 antibody and PBS, and cell is fixed with 4% paraformaldehyde after the trypsinization through three washings; 1000rpm made it in centrifuge tube, to become a cell mass in centrifugal 5 minutes, added one deck 1% agarose in the above.Same a kind of cell harvesting of different condition is in the different aspects of same pipe.Nuclear magnetic resonance is at 4.7T BrukerBiospec
AvanceCarry out on the toy magnetic resonance imaging system, adopt the spiral echo sequence.Vow the dress bit image for the T2 weighting, adopt following imaging parameters: matrix=128 * 128 μ m, FOV=45 * 45mm, sectionthickness=1mm, TE=45ms, TR=3000ms, number of average=1.
Experimental result is as shown in Figure 4, demonstration be the sagittal t2 weighted image of cell mass in the agarose.Hatched 1 hour with the SM-USPIO (calculating by iron content) of 50 μ g/ml before the cell harvesting, matched group comprises that PBS, USPIO, H-USPIO and USPIO add humanization SM5-1 antibody, and its concentration is all calculated by iron content or antibody amount, and is consistent with SM-USPIO dosage.On the image of ch-hep-3 cell pipe, the cell mass that SM-USPIO handles shows tangible low signal, and other four cell masses all show as high signal.On the image of ch-hep-1 cell pipe, 5 cell masses show identical high signal.On the image of ch-hep-3 cell pipe, the signal of the cell mass that SM-USPIO handles is lower slightly than the signal of other four cell masses, but the corresponding ch-hep-3 cell that is far from is obvious.In three kinds of cells; The decline of signal does not all appear in the cell that USPIO adds humanization SM5-1 antibody treatment; This explanation is in short relatively incubation time, and cell can not initiatively be engulfed free USPIO, and free humanization SM5-1 antibody can not engulfed USPIO by mediated cell yet.Therefore, the USPIO that has only humanization SM5-1 antibody crosslinked just can be combined and engulfs by the conjugated protein positive cells of SM5-1 specifically.
The experiment of experimental example 5 in-vivo imagings
What use in the in-vivo imaging experiment is the lotus tumor Balb/c nude mice of inoculation human liver cell hepatocarcinoma ch-hep-3.Balb/c nude mice subcutaneous vaccination 2 * 10
6The ch-hep-3 cell, inoculate back 25 days after the about 5mm of diameter of tumor.The MRI contrast medium SM-USPIO or the H-USPIO of tail vein injection preparation this moment, dosage is 200mg Fe/kg.Animal is with the barbital sodium intraperitoneal anesthesia before the MRI inspection, and radiography reaches injection respectively before injection carried out in back 2 hours, 8 hours, 24 hours and 72 hours, used 4.7T Bruker Biospec equally
AvanceThe toy magnetic resonance imaging system, parameter is following: TR=3000ms, TE-52ms, number of average=2, FOV=3.5 * 3.5cm, matrix=128 * 128 μ m.Three of every treated animals.
Experimental result sees that Fig. 5 result shows that the signal difference of tumor tissues and near muscular tissue is little, injects back 8 hours, and descending appears in the signal of tumor region, injects back 72 hours, and the signal of tumor region obviously descends, and tumor border is clear.The experiment contrast group is employed in the H-USPIO of injection same dose in the identical model; The result shows before and after the injection; The signal of tumor region does not have significant change, and prompting H-USPIO in this model does not have targeting potentiation, and SM-USPIO has tangible targeting potentiation.
The experiment of experimental example 6 biodistribution
125The biodistribution of I labelling SM-USPIO is also carried out on the lotus tumor Balb/c nude mice of inoculation human liver cell hepatocarcinoma ch-hep-3.Inoculate back 25 days, 12 animals are divided into 2 groups at random, and one group through tail vein 1.0 μ Ci's
125The SM-USPIO of I labelling, the same dosage of another group injection
125The H-USPIO of I labelling.Every treated animal was put to death 3 of animals in back 8 hours and 72 hours in injection respectively, got tumor tissues and each normal structure, comprised lung, the heart, liver, spleen, pancreas, stomach, colon and kidney, weighed and measured radioactivity.Each animal of putting to death in 72 hours is got the hematometry radioactivity injection back 5 minutes, 1 hour, 4 hours, 8 hours, 24 hours and 72 hours through cutting tail respectively.Radioactivity is measured through gamma counter.The radioactivity of each organ is represented with the percent of ID.
Statistical analysis: adopt the t check, with the standard of P＜0.05 as significant difference.Result of the test is seen Fig. 6.
Fig. 6 show with
125The H USPIO of I labelling compares,
125The SM-USPIO of I labelling injected back 8 hours and 72 hours tumor by local accumulation showed increased (P＜0.05) (Fig. 6-1,6-2).The accumulation of tumor by local is progressively, with time correlation, injects after back 72 hours and can reach 8.9 ± 2.3%ID g
-1(ID pointed injection dosage, ID g
-1Refer to average every gram tissue injection dosage), be higher than the liver of same time and the summary accumulation level of spleen far away.On the contrary,
125The H-USPIO of I labelling is obviously low in the accumulation of tumor by local, and injects back 72 hours level and be lower than the back 8 hours level of injection.
125The SM-USPIO of I labelling with
125The distribution of the H-USPIO of I labelling in normal structure is consistent,, inject back 72 hours level and all be lower than the back 8 hours level (Fig. 6-1,6-2) of injection.
125The SM-USPIO of I labelling with
125The clearance curve of the H-USPIO of I labelling in blood consistent (Fig. 6-3).For comparing
125The SM-USPIO of I labelling with
125The cumulative difference of the H-USPIO of I labelling specificity in tumor tissues, we have calculated radioactivity ratio in same temporal lesion tissue and the blood.Injected back 72 hours,
125The tumor of the SM-USPIO treated animal of I labelling/blood ratio is 4.93 ± 0.79, and
125The tumor of the H-USPIO treated animal of I labelling/blood ratio is 1.26 ± 0.47, and the former is much larger than the latter (P＜0.01).
Claims (10)
1. mr contrast agent SM-USPIO based on humanized antibody is for humanization SM5-1 antibody and superparamagnetic iron oxide particle USPIO is crosslinked gets.
2. the method for preparing of the described mr contrast agent SM-USPIO of claim 1 may further comprise the steps:
Step a: preparation superparamagnetic iron oxide particle USPIO,
Step b: the preparation humanized antibody,
Step c: preparation mr contrast agent SM-USPIO.
3. the method for preparing of the described mr contrast agent SM-USPIO of claim 2, wherein the preparation of superparamagnetic iron oxide particle USPIO obtains through coprecipitation method among the step a, may further comprise the steps:
Steps A: glucosan is dissolved in the water, adds iron salt, stir;
Step B: under the condition of heating, polyvinyl alcohol is dissolved in the water;
Step C: mix in the solution that the solution adding steps A that step B is obtained obtains, to 10-12, continue to stir post-heating and obtain deposition with adjusting PH with base;
Step D: the collecting precipitation thing also carries out post processing to it.
4. the method for preparing of the described mr contrast agent SM-USPIO of claim 3, the iron salt that wherein uses in the steps A is FeCl
36H
2O and FeSO
47H
2O, wherein ferric iron and ferrous mol ratio are to be 3: 2.
5. the method for preparing of the described mr contrast agent SM-USPIO of claim 3, wherein the alkali described in the step C is ammonia.
6. the method for preparing of the described mr contrast agent SM-USPIO of claim 3 is wherein utilized magnetic field collecting precipitation thing among the step D.
7. the method for preparing of the described mr contrast agent SM-USPIO of claim 3, wherein post-processing step comprise utilize washing, the resuspended deposition of citric acid solution, ultrasonic after, the unnecessary citric acid of removal of in deionized water, dialysing.
8. the method for preparing of the described mr contrast agent SM-USPIO of claim 2; Wherein the step c step for preparing mr contrast agent SM-USPIO comprises UPSIO is carried out activation, separates the particle after the activation, and particle is mixed with the PBS that contains antibody; Stir, handle and get through the magnetic post.
9. the described SM-USPIO of claim 1 is used for the purposes of the mr contrast agent of diagnosing tumor in preparation.
10. the described purposes of claim 9, wherein tumor is the conjugated protein male tumor of SM5-1.
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| Geng Kou et al..Development of SM5-1-conjugated ultrasmall superparamagnetic iron oxide nanoparticles for hepatoma detection.《Biochemical and Biophysical Research Communications》.2008,第374卷(第2期),192–197. * |
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