CN101564540A - Gd-DTPA-Polylysine-McAb junctional complex, preparation method and application thereof - Google Patents
Gd-DTPA-Polylysine-McAb junctional complex, preparation method and application thereof Download PDFInfo
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- CN101564540A CN101564540A CNA2008100364727A CN200810036472A CN101564540A CN 101564540 A CN101564540 A CN 101564540A CN A2008100364727 A CNA2008100364727 A CN A2008100364727A CN 200810036472 A CN200810036472 A CN 200810036472A CN 101564540 A CN101564540 A CN 101564540A
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Abstract
The invention relates to a junctional complex, in particular to a Gd-DTPA-Polylysine-McAb (anti VEGF) juncitonal complex, a preparation method and the application thereof. The Gd-DTPA-Polylysine-McAb (anti VEGF) juncitonal complex (contrast agent) is prepared by the following methods: a, synthesis of DTPA bisanhydride; b, preparation of DTPA-Polylysine; c, preparation of Gd-DTPA-Polylysine; and d, junction of anti VEGF monoclonal antibody and the Gd-DTPA-Polylysine. With the aid of the expansion effect of Polylysine and the specificity of monoclonal antibody, the contrast agent Gd-DTPA-Polylysine-McAb (anti VEGF monoclonal antibody) has the advantages of high immune activity, good targeting ability, high content of Gd<3+>, and the like.
Description
Technical field
The present invention relates to a kind of union body, especially relate to a kind of Gd-DTPA-Polylysine-McAb (anti-VEGF) union body, preparation method and application thereof.
Background technology
At present, the conventional clinically mr contrast agent Gd-DTPA that uses is (by diethylenetriamine pentaacetic acid (DTPA) and Gd
+ 3Chelate), belong to the ECS contrast medium, do not have specificity.It strengthens principle is to contain gadolinium (Gd
+ 3) contrast medium enters arteries and be assigned in the organizational structure that is imbued with the blood confession with blood flow, and be back to intravenous very soon, pass through renal excretion at last.The gadolinium agent can cause that the T1 time of distributed areas tissue obviously shortens, and causes that signal intensity significantly raises on T1WI, promptly so-called the reinforcement.Gd-DTPA is widely used in the MR imaging of a plurality of systems of whole body, for example, and to the diagnosis and differential diagnosis of neoplastic lesions such as breast carcinoma.
Gd-DTPA is non-specific contrast medium, and when being used for nuclear magnetic resonance, its imaging effective time window is very narrow, does not have targeting, is only organized or focus blood influences for degree, and blood is strengthened significantly for the person of enriching, otherwise then reinforcement is lighter.Certain value is arranged in medical diagnosis on disease, still, adopt the nuclear magnetic resonance of non-specific contrast medium can not accurately differentiate the good, pernicious of pathological changes, can not be at a certain gene or cellularity imaging, no targeting is difficult to reach the purpose of early diagnosis disease.
In recent years, MRI has very high soft tissue resolution and good advantages such as spatial resolution because of it, and the MR targeted imaging becomes the research focus of molecular imaging gradually.The monoclonal antibody mr contrast agent is that monoclonal antibody is combined with MR paramagnetism or particles with superparamagnetism, it is specificity that thereby the chelating agen that makes new formation had both had target function, possesses the effect of conventional MR contrast medium again, it can not only combine specifically with some organ, tissue and pathological changes, and can change their MR signal intensity, thereby reach the purpose of targeting diagnosis.
Molecular biology and molecular pathology research disclose, VEGF (vascularendothelial growth factor, VEGF) there is significant difference in the expression in the mammary gland innocent and malignant tumour, be that VEGF is significant high expressed in the breast carcinoma, and be the important judge index of breast carcinoma biological behaviour, as vegf expression whether and the expression degree be remarkable dependency with breast carcinoma metastasis rate and patient's prognosis, i.e. VEGF high expressed person, metastasis rate height and poor prognosis.
Summary of the invention
The objective of the invention is to:
(1) provides a kind of Gd-DTPA-Polylysine-McAb (anti-VEGF) union body;
(2) provide Gd-DTPA-Polylysine-McAb (anti-VEGF) the union body preparation method;
(3) provide Gd-DTPA-Polylysine-McAb (anti-VEGF) union body to use.
The objective of the invention is to realize: will resist VEGF (VEGF) monoclonal antibody to become a kind of specificity contrast medium Gd-DTPA-polylysine-McAb (anti-VEGF monoclonal antibody) of the MR of being used for molecular imaging with DTPA-Gd (Gd-DTPA) chelating by following technical method, lotus human breast carcinoma nude mice model is carried out testing at the body molecular imaging of this MR contrast medium, estimate its imaging results, succeed.
Technical scheme of the present invention is as follows: a kind of Gd-DTPA-Polylysine-McAb (anti-VEGF) union body makes by following method:
Synthesizing of a, DTPA bisgallic acid acid anhydride: Diethylenetriamine five acid, acetic anhydride and pyridine are mixed;
B, DTPA-Polylysine preparation: poly-D-lysine is dissolved in NaH
2CO
3/ Na
2HCO
3In the buffer, stir, and add in the DTPA bisgallic acid anhydride solution that step a obtains;
C, Gd-DTPA-Polylysine preparation: with Gd
2O
3Join among the HCl, heating, dissolving, the HCl of evaporating surplus promptly obtains GdCl
3Solution, with GdCl
3Solution joins in the DTPA-Polylysine solution that step b obtains;
The connection of d, anti-VEGF monoclonal antibody and Gd-DTPA-Polylysine: will resist VEGF monoclonal antibody dissolving NaH
2CO
3/ Na
2HCO
3In the buffer, add the Gd-DTPA-Polylysine that carbodiimides and step c obtain then, stirring reaction, chromatography, the association of collecting anti-VEGF monoclonal antibody and Gd-DTPA-Polylysine.
In step b, also comprise and take out poly-D-lysine, NaH
2CO
3/ Na
2HCO
3, DTPA bisgallic acid acid anhydride mixed solution, remove free DTPA with equilibrium dialysis, and be transformed into PH=5,0.2ml NaAc/HAc buffer.In steps d, select Sepharose CL-4B chromatographic column chromatography for use.
A kind of preparation method of Gd-DTPA-Polylysine-McAb union body: may further comprise the steps:
Synthesizing of a, DTPA bisgallic acid acid anhydride: Diethylenetriamine five acid, acetic anhydride and pyridine are mixed;
B, DTPA-Polylysine preparation: poly-D-lysine is dissolved in NaH
2CO
3/ Na
2HCO
3In the buffer, stir, and add in the DTPA bisgallic acid anhydride solution that step a obtains
C, Gd-DTPA-Polylysine preparation: with Gd
2O
3Join among the HCl, heating, dissolving, the HCl of evaporating surplus promptly obtains GdCl
3Solution, with GdCl
3Solution joins in the DTPA-Polylysine solution that step b obtains;
The connection of d, anti-VEGF monoclonal antibody and Gd-DTPA-Polylysine: will resist the VEGF monoclonal antibody to be dissolved in NaH
2CO
3/ Na
2HCO
3In the buffer, add the Gd-DTPA-Polylysine that carbodiimides and step c obtain then, stirring reaction, chromatography, the association of collecting anti-VEGF monoclonal antibody and Gd-DTPA-Polylysine.
In step b, also comprise and take out poly-D-lysine, NaH
2CO
3/ Na
2HCO
3, DTPA bisgallic acid acid anhydride mixed solution, remove free DTPA with equilibrium dialysis, and be transformed into PH=5,0.2ml NaAc/HAc buffer.In steps d, select Sepharose CL-4B chromatographic column chromatography for use.
A kind of Gd-DTPA-Polylysine-McAb (anti-VEGF) union body is used, and this union body agent is as a comparison used in nuclear magnetic resonance.
Described nuclear magnetic resonance is specificity, the targeting nuclear magnetic resonance of angiogenesis in the tumor of VEGF gene expression in body shows tumor, VEGF mediation.
The disclosed a kind of Gd-DTPA-Polylys ine-McAb of the present invention (anti-VEGF) union body, preparation method and application thereof, its advantage shows: (1) the present invention adopts the chelating of monoclonal antibody and gadolinium agent to prepare novel contrast medium, can carry out the specificity imaging by means of the targeting of antibody, the present invention adopts the monoclonal antibody of VEGF (VEGF), therefore, can carry out imaging to the angiogenesis of VEGF and mediation thereof.(2) early stage research is directly gripped connection altogether with monoclonal antibody and Gd-DTPA mostly, and such Gd-DTPA amount that connects on the one hand that directly connects is lacked, and reinforced effects is undesirable; On the other hand,, between double-stranded or a plurality of protein moleculars, form dimer or polymer easily, so along with the increase of Gd-DTPA linking number, the immunocompetence decline of DTPA-antibody complex, the targeting poor effect of contrast medium because the DTPA cyclic anhydride is twin nuclei.In our invention, we adopt Polylysine (poly-D-lysine) amplification system to obtain good expanding effect.The technology that we utilize macromole poly physical ability and a plurality of Gd-DTPA to connect, make the macromole polymer of Gd-DTPA labelling earlier, and then the macromole polymer of a plurality of Gd-DTPA labellings is attached on the VEGF monoclonal antibody, on an antibody, can connect 28～36 Gd-DTPA like this, and not reduce the immunocompetence of antibody.With the immunocompetence of ELISA method mensuration Gd-DTPA-Polylysine-McAb (anti-VEGF monoclonal antibody), the result obtains the combine situation approaching with the antibody of VEGF.In external detection, show the purity height of Gd-DTPA-Polylysine-McAb (anti-VEGF monoclonal antibody), in the cellulose acetate membrane electrophoresis collection of illustrative plates, have single speckle.In a word, the present invention is by means of the amplification effect of Polylysine (poly-D-lysine) and the specificity of antibody, and this contrast medium Gd-DTPA-Polylysine-McAb (anti-VEGF monoclonal antibody) has immunocompetence height, strong, the Gd of targeting
3+Advantages such as content is many, feasiblely study at body tumor MR targeted imaging, the situation that shows vegf expression, and the tumor-blood-vessel growth situation of VEGF mediation, for the early diagnosis of the malignant tumor that is imbued with the VEGF gene such as breast carcinoma etc., the Differential Diagnosis of innocent and malignant tumour provide a brand-new approach, be that the angiogenesis inhibitor treatment improves new evaluation means.
Description of drawings
The high-pressure liquid phase of Fig. 1: Gd-DTPA-Polylysine (HPLC) chromatogram.
The SDS-PAGE electrophoresis pattern of Fig. 2: Gd-DTPA-Polylysine.
The cellulose acetate membrane electrophoresis collection of illustrative plates of Fig. 3: Gd-DTPA-Polylysine-McAb (anti-VEGF monoclonal antibody) union body.
Fig. 4: the ELISA method is measured the immunocompetence result of immune union body.
Fig. 5: inject before the contrast medium and each time sweep point MR image of injection contrast medium Gd-DTPA-polylysine-McAb (anti-VEGF monoclonal antibody) back.A among the figure wherein: before injecting contrast medium.B among the figure: 5min behind the injection contrast medium, tumor tissues is not seen reinforcement, and liver organization is strengthened, and blood vessel shows in the liver.C among the figure: 1h behind the injection contrast medium, borderline tumor begin to strengthen, and trunk shows clear.D among the figure: 1h behind the injection contrast medium, liver organization is obviously strengthened, and blood vessel shows clear in the liver.E among the figure, F: 2h behind the injection contrast medium, tumor tissues, liver organization are generally strengthened.G among the figure, H: 3h behind the injection contrast medium, even when tumor tissues, liver organization are strengthened than 2h.I among the figure, J: 4h behind the injection contrast medium, the tumor reinforcement reaches the peak, and liver is strengthened not obvious.K among the figure: tumor continues to strengthen, gradually to the center disperse.L among the figure, M: 12h behind the injection contrast medium, tumor center strengthens obviously.N among the figure, O: 24h behind the injection contrast medium, tumor center weaken when strengthening than 12h.
Fig. 6: liver organization, tumor tissues and muscular tissue enhancing rate are relatively.
Fig. 7: tumor reaches pathology figure substantially.A among the figure wherein: tumor profile, tumor tissues flesh of fish sample.B:HE dyeing (40 * 10 times) shows that tumor tissues cancerous cell size and form differs among the figure, and karyokinesis mutually more to be seen, sees downright bad tumor cell.C:VEGF SABC among the figure (40 * 10 times), the tumor cell slurry sees that brown particle shows the VEGF stained positive.
Fig. 8: inject before the contrast medium and each time point scanning MR image of injection contrast medium Gd-DTPA-polylysine-McAb (anti-VEGF monoclonal antibody) back.A among the figure wherein: before injecting contrast medium.B among the figure: 5min behind the injection contrast medium, tumor tissues is not seen reinforcement.C among the figure: 1h behind the injection contrast medium, borderline tumor begin to strengthen.D among the figure: 2h behind the injection contrast medium, tumor tissues is generally strengthened.E among the figure, F: 3h behind the injection contrast medium, 4h, tumor tissues is generally strengthened.G among the figure: 8h behind the injection contrast medium, tumor continues to strengthen, gradually to the center disperse.H among the figure: 12h behind the injection contrast medium, tumor center strengthens obviously.I among the figure: 24h behind the injection contrast medium, tumor center weaken when strengthening than 12h.
Fig. 9: each time point scans MR image after reaching injection contrast medium Gd-DTPA before the injection contrast medium.A among the figure wherein: before injecting contrast medium.B among the figure, C: 5min behind the injection contrast medium, tumor tissues is obviously strengthened.D among the figure, E: inject 1h behind the contrast medium, tumor tissues is strengthened still obvious, but during than 5min slightly a little less than.F among the figure, G, H: 2h behind the injection contrast medium, 3h, the tumor tissues reinforcing degree prolongs in time and weakens.H among the figure: 4h behind the injection contrast medium, tumor is strengthened not obvious, and contrast medium withdraws from substantially.I among the figure: 8h behind the injection contrast medium, contrast medium withdraws from fully.
Figure 10: experimental group and matched group signature tune line chart.
Figure 11: tumor is picture and pathology picture substantially.A among the figure wherein: breast carcinoma nude mice model (MDA-MB-231 cell).B among the figure: tumor section, tumor tissues are flesh of fish shape.C:HE dyeing (every scale is represented 25um) among the figure, tumor cell is not of uniform size, and it is poor to break up.D:VEGF SABC (40 * 10) among the figure sees that brown particle shows stained positive in the tumor cell slurry.
The specific embodiment
Below in conjunction with embodiment the present invention is further described.
Embodiment 1
The preparation of a kind of Gd-DTPA-Polylysine-McAb (anti-VEGF) union body
Synthesizing of I, DTPA bisgallic acid acid anhydride (CaDTPA): 10g Diethylenetriamine five acid (DTPA) are suspended in the mixed liquor of 30ml acetic anhydride and 15ml pyridine, in 60 ℃ of stirring reaction 6h, add an amount of ethanol after the cooling and filter, the ether washing, drying gets white solid DTPA bisgallic acid acid anhydride (CaDTPA) crude product.Behind the recrystallization, measuring the product fusing point is 178～180 ℃ (decomposition), has 1845 in infrared spectrogram, a 1785cm
-1The C=O stretching vibration of cyclic acid anhydride is bimodal.
II, DTPA-Polylysine preparation: claim Polylysine (poly-D-lysine) 10mg, be dissolved in the 0.2mol/L NaH of 3ml
2CO
3/ Na
2HCO
3In the buffer (PH=9.6), ceaselessly stir in ice bath and add DTPA bisgallic acid acid anhydride (CaDTPA) solution, the restir reaction finishes reaction under the room temperature after 16 hours.Take out reaction mixture, remove free DTPA with equilibrium dialysis, and be transformed into PH=5,0.2ml NaAc/HAc buffer.Take out solution in the bag filter, concentrating under reduced pressure is placed on 4 ℃ of refrigerators and preserves, and is used for the next step test.Simultaneously, sampling 0.5ml, and with the Sephadex post (1 * 25cm), 0.01mol/L PBS, the PH7.4 buffer solution elution is analyzed, as seen only macromole eluting peak appearance.
III, Gd-DTPA-Polylysine preparation: Gd
2O
3Add the people in 0.1mol/L HCl, be heated to 60 ℃, sustained response 10min, after the dissolving, the HCl of evaporating surplus promptly obtains GdCl fully
3Solution.The GdCl that adds preparation
3Solution is in the process DTPA-Polylysine solution of concentrating under reduced pressure, and restir reaction was at room temperature removed free Gd with equilibrium dialysis, and changeed the PH system of this solution after 24～30 hours then.Adopt the high-frequency inductor Rhizoma Nelumbinis to close plasma emission spectroscopy method (ICP-AES) measuring samples Gd content, calculate resulting Gd-DTPA-Polylysine per molecule and contain 30～42 Gd ions.
The connection of IV, anti-VEGF monoclonal antibody and Gd-DTPA-Polylysine:, anti-VEGF monoclonal antibody is dissolved in 0.1mol/L NaH because polylysine also has amido to exist
2CO
3/ Na
2HCO
3Buffer (PH=9.6), add carbodiimides (ECD) and Gd-DTPA-Polylysine then, restir reaction at room temperature is after 24～30 hours, (1 * 25cm) removes the antibody that on not connecting with Sepharose CL-4B chromatographic column, collect the association of anti-VEGF monoclonal antibody and Gd-DTPA-Polylysine, freeze concentration is left with after the 0.22um aseptic filtration.
The evaluation of V, contrast medium:
(1) Gd assay: adopt the high-frequency inductor Rhizoma Nelumbinis to close plasma emission spectroscopy method (ICP-AES) and measure Gd content, the Gd content of Gd-DTPA-Polylysine is 30～42 (n=7).The Gd content that is used for anti-VEGF monoclonal antibody of zooperal per molecule and Gd-DTPA-Polylysine union body is 28～36 Gd-DTPA.
(2), macromolecular purity testing: use high performance liquid chromatography (HPLC), SDS electrophoresis method, cellulose acetate membrane electrophoresis and molecular sieve Sepharose CL-4B column chromatography to prove purified macromolecular purity.(a) HPLC: the purity of using high pressure liquid chromatography (HPLC) assay Gd-DTPA-Polylysine, pillar is TSK3000 (Bio-Rad) molecular sieve gel analytical column, use 0.01mol/L, the PBS liquid eluting of PH=7.4, drip washing speed is 1ml/min, the measurement chart drive speed is 2mm/min, and each sample introduction 2～5ul detects eluent with wavelength X=280nm place.Less molecular weight peaks＜2% (Fig. 1) a big peak (＞95%) appears, in the high pressure liquid chromatography diagram of high pressure liquid chromatography (HPLC) evaluation of markers sample.(b) SDS-PAGE electrophoresis: the sample buffer of 20u l two volumes is added in isopyknic sample and the standard solution small test tube, after in boiling water, placing 3～5min, be added to and carry out the SDS-PAGE electrophoresis in the electrophoresis sample cell, constant voltage 100V arrives the bromophenol blue indicator near forward position (approximately 4h), after taking-up is fixing, with Coomassie brilliant blue R-25 dyeing, the decolouring of reuse 7% acetic acid.The SDS-PAGE electrophoresis pattern of Gd-DTPA-Polylysine shows to have single speckle (Fig. 2).(c) cellulose acetate membrane electrophoresis: sample spot (the 4cm place is as initial point) on acetate film, be placed on then to fill and carry out electrophoresis 40min in PH8.6 barbital-veronal buffer, continuous current, every of 5mA/, take out thin film, with Coomassie brilliant blue R-25 dyeing, the decolouring of reuse 7% acetic acid.The cellulose acetate membrane electrophoresis collection of illustrative plates of Gd-DTPA-Polylysine-McAb (anti-VEGF monoclonal antibody) union body, the result shows that Gd-DTPA-Polylysine-McAb (anti-VEGF monoclonal antibody) union body has single speckle (Fig. 3).
(3), immunocompetence measures: measure the immunocompetence of Gd-DTPA-Polylysine bind antibody with the ELISA method, the result obtains the situation that combines approaching with the antibody of VEGF, and (Gd-DTPA)
26The immunocompetence decline a lot (Fig. 4) of-VEGF antibody.Concrete grammar: 1ug/100ulVEGF wraps quilt, coating buffer 0.01mol/L PBS (PH=7.4), and 4 ℃ of placements of spending the night dry.After 37 ℃ of 30min sealings, 200ul/ hole cleaning mixture washs, dries three times with 1%BSA.100ng/ml is first hole, and antibody or the corresponding Gd-DTPA union body of the VEGF that later 5 times of dilutions are not commensurability carry out immune association reaction, places 1h for 37 ℃.Take out the washing of 200ul/ hole cleaning mixture, drying five times.Add sheep anti-mouse igg-HRPO (1: 2000) that the 70ul/ aperture diluted, 37 ℃ of constant temperature were placed down after 60 minutes, washed, dried six times with 200ul/ hole cleaning mixture.Add 100ul/ hole ABTS colour developing liquid, color development 20min or longer time under the room temperature.Use under wavelength X=414nm situation, measure the absorptance A value in every hole.
The experiment of Gd-DTPA-polylysine-McAb (anti-VEGF monoclonal antibody) in lotus human breast carcinoma nude mice model live body MR molecular imaging
Materials and methods
1, the preparation of animal model
This experiment adopts cell suspension method in-situ inoculating human breast cancer cell strain MDA-MB-231 to make breast carcinoma nude mouse model.Nude mouse and breast carcinoma MDA-MB-231 cell strain are available from Chinese Academy of Sciences institute of oncology.12 nude mices are female 5 week no-special pathogen level in age (SPF) BALB/c nude mouses, and quality is 18～20g.Breast carcinoma MDA-MB-231 cell adopts Leibovitz ' s L-15medimu (GIBCO company) to add 15% hyclone (GIBCO company) and cultivates, treat cell to living cells ratio＞95% o'clock by 1 * 10
7/ 0.2ml/ only is inoculated in the fat pad of the second pair of mammary gland in nude mice right side.Observe tumor bulk-growth situation after the inoculation weekly at least three times, inoculation position forms macroscopic tumor after 2 weeks, and diameter of tumor reach 1.5～2.0cm after 4～5 weeks, carry out the MRI imaging research this moment.
2, MRI scanning technique and graphical analysis:
Adopt GE Signa Advantage 1.5T MR instrument, use 3 inches surface coils.Every all earlier unenhanced back of tumor bearing nude mice strengthens, cross-section position of unenhanced employing and crown position SE T1WI (TR/TE400/13ms), FSE T2WI (TR/TE 3000/84.3ms); Enhanced ct scans is selected cross-section position, crown position and sagittal plain SE T1WI (TR/TE 400/13ms) for use.Bed thickness/spacing: 1/0.5mm (cross-section position); 2/1mm (crown position), 2/1mm (sagittal plain), matrix are 256 * 256, FOV 8cm * 8cm.
Adopt specificity MR I contrast medium Gd-DTPA-polylysine-McAb (anti-VEGF monoclonal antibody) during enhancing, injected dose only is 0.2ml/, through the nude mice tail vein injection.Be sweep time after the injection of contrast agent: 5min, 1h, 2h, 3h, 4h, 8h, 12h and 24h.Anesthesia is 2.5% pentobarbital sodium solution 0.05ml, lumbar injection.
Observe tumor soma, liver organization, the contiguous front leg portions muscular tissue enhancing of tumor body performance, and measure signal intensity.Concrete measuring method is: measure the signal intensity of tumor soma, liver organization, the contiguous front leg portions muscular tissue of tumor body on crown position before and after injecting contrast medium, and calculate the enhancing rate, computational methods are: (S
Strengthen-S
Unenhanced)/S
Unenhanced* 100%.。(region of interest, ROI), the interest area is not less than 4mm to select 5 region of interest of the same size during measurement respectively
2, get as far as possible and strengthen obviously and uniform zone, get its meansigma methods.
3, pathologic finding: experiment finishes the back and puts to death the experiment nude mice, gets tumor tissues and does pathologic finding, row conventional organization pathologic finding and SABC inspection.The used I of VEGF SABC is anti-to be the mouse-anti human monoclonal antibodies, and II is anti-to be the sheep anti mouse monoclonal antibody.Concrete grammar: 1. dehydration, section, it is as usual to dewax.2. PBS washed 3 times each 5 minutes.3. drip 3%H
2O
2, room temperature left standstill 20 minutes, distillation washing 3 times each 3 minutes.4. the citrate buffer solution steaming and decocting is 20 minutes, and the cooling back was washed among the 0.01mlPBS 3 times each 5 minutes at PH7.2.5. drip normal goats serum confining liquid, room temperature left standstill 20 minutes, got rid of unnecessary liquid.6. drip I anti-(dilution in 1: 40), 4 ℃ are spent the night.7. 0.01PBS washes 3 times, each 5 minutes.8. it is anti-directly to drip II, and 37 ℃ left standstill 20 minutes.9. 0.01PBS washes 3 times, each 5 minutes.10. the DAB colour developing is 5-10 minute, grasps dye levels at microscopically.Washing, haematoxylin redyeing, washing, mounting.Contain pale brown or brown granular is the VEGF positive with endochylema.
4, statistical procedures: carry out paired t-test respectively before the signal strength values of each time point and the injection after the injection of contrast agent, statistical software is SPSS 13.0, is the significance standard with P＜0.05.
The result
1, strengthen performance: tumor focus is strengthened not obvious when 5min, and borderline tumor begins to strengthen behind the 1h, and reinforcing degree is lower, along with the prolongation reinforcement of time is obvious gradually, and by outer circumferentially center disperse, strengthens during to 4h and peaks.Observe to the 24h tumor and still have slight reinforcement.The nude mice liver organization begins to strengthen when 5min, strengthens during 2h to reach the peak, and the prolongation contrast medium along with the time withdraws from gradually subsequently, and is not obvious to the 4h reinforcement.Muscular tissue is not seen obvious reinforcement all the time.Trunks such as aorta are visible when 5min obviously to be strengthened, and continues about 2h.(see figure 5)
2, tumor tissues, liver organization, each delaying sweep time point signal results of muscular tissue and enhancing rate are relatively.
(1) tumor tissues is put signal value and enhancing rate (seeing Table 1) each sweep time.The signal value that injects each scanning element behind the contrast medium is carried out paired t-test with the signal value that injects before the contrast medium one by one, statistical result showed, when injecting 5min behind the contrast medium signal value of tumor soma with inject contrast medium before do not have significant difference (t=1.16, P=0.182), there were significant differences (P＜0.05) before the signal intensity of 1h to 24h tumor soma and the injection contrast medium behind the injection contrast medium, and promptly tumor is remarkable reinforcement performance.The peak that tumor is strengthened appears at after the injection of contrast agent 4 hours, maximum reinforcement rate 58.17%.
Table 1: each time point signal intensity of tumor tissues and enhancing rate
(2) liver organization is put signal intensity and enhancing rate (seeing Table 2) each sweep time.The signal value that injects each scanning element behind the contrast medium is carried out paired t-test with the signal value that injects before the contrast medium one by one, statistical result showed, 5min behind the injection contrast medium, 1h, there were significant differences (P＜0.05) for 2h, the signal value of the 3h tumor soma signal value preceding with injecting contrast medium; There is not significant difference (P＞0.05) before the signal value of each time point tumor soma and the injection contrast medium behind the injection contrast medium 4h.The peak that tumor is strengthened appears at 2h, maximum reinforcement rate 56.33%.
Table 2: each time point signal value of liver organization and enhancing rate
(3) muscular tissue is put signal intensity and enhancing rate (seeing Table 3) each sweep time, injects before and after the contrast medium muscular tissue and puts signal value comparing difference not statistically significant (P＞0.05) each sweep time.
Table 3: each time point signal value of muscular tissue and enhancing rate
(4) tumor tissues, liver organization and muscular tissue are put the enhancing rate relatively each sweep time: (see figure 6)
3, pathological examination:
Conventional H E dyeing shows that tumor cell arranges in a jumble, and the cancerous cell size and form differs, and karyokinesis mutually more to be seen.See that brown particle shows stained positive in the VEGF SABC tumor cell slurry.(see figure 7)
Embodiment 3
Gd-DTPA-polylysine-McAb (anti-VEGF monoclonal antibody) and the control experiment of Gd-DTPA in breast carcinoma nude mice model MR molecular imaging
At present, breast cancer incidence all is remarkable ascendant trend both at home and abroad, the first place that the many metropolitan female mammary gland cancer morbidities of China have occupied women's malignant tumor, and age of onset has trend ahead of time.The diagnosis of breast carcinoma mainly relies on imaging examination, comprise X line, B ultrasonic and MRI etc., the value maximum of the dynamic enhanced ct scans of many phases of high field intensity MRI wherein, its specificity and sensitivity are all higher, but, when adopting present most widely used Gd-DTPA, mammary gland is good, the magnetic resonance of malignant tumor performance have part overlapped, intersect.Therefore, can't accomplish specific diagnosis, this had both influenced the discriminating of breast carcinoma and benign lesion, also influenced detecting of breast carcinoma of early stage.How specific, early diagnosis breast carcinoma is one of clinical problem of being concerned about most.
This research is carried out comparative study with VEGF targeting MR contrast medium Gd-DTPA-polylysine-McAb (anti-VEGF monoclonal antibody) and conventional contrast medium Gd-DTPA in breast carcinoma (MDA-MB-231 cell strain) nude mice model, estimate the probability that employing Gd-DTPA-polylysine-McAb (anti-VEGF monoclonal antibody) carries out breast carcinoma MR molecular imaging, and this imaging is to the diagnostic effect of breast carcinoma.
Materials and methods
1, the preparation of animal model and laboratory animal grouping
This experiment adopts cell suspension method in-situ inoculating human breast cancer cell strain MDA-MB-231 to make breast carcinoma nude mouse model, totally 12.Manufacture method is the same.
At random 12 nude mices that inoculated the human breast cancer cell strain are divided into experimental group and matched group.Experimental group injection of contrast agent Gd-DTPA-polylysine-McAb (anti-VEGF monoclonal antibody), the matched group injection contains the conventional contrast medium Gd-DTPA of identical Gd ion concentration.Gd-DTPA adds the normal saline dilution with Magnevist Solution injection (magnevist contains the gadopentetic acid two amine 469mg/ml of Portugal, Schering Guangzhou) and forms.Contrast medium injection dosage only is 0.2ml/, through the nude mice tail vein injection.
2, MRI scanning technique and graphical analysis:
Adopt GE Signa Advantage 1.5T MR instrument, use 3 inches surface coils.Every all earlier unenhanced back of nude mice strengthens the cross-section position of unenhanced employing and crown SE T1WI (TR/TE 400/13ms), FSE T2WI (TR/TE 3000/84.3ms); Enhanced ct scans is selected cross-section position and crown position SE T1WI (TR/TE 400/13ms) for use.Bed thickness/spacing: 1/0.5mm (cross-section position); 2/1mm (crown position), matrix is 256 * 256, FOV 8cm * 8cm.Be the sweep time after the injection of contrast agent: 5min, 1h, 2h, 3h, 4h, 8h, 12h and 24h.Anesthesia is 2.5% pentobarbital sodium solution 0.05ml, lumbar injection.
Observe the tumor body and strengthen performance, and measure signal intensity.Concrete measuring method is: respectively at injecting contrast medium fore-and-aft survey tumor body signal intensity (SI
T), the front leg portions muscular tissue signal intensity (SI that nude mice model tumor homonymy is contiguous
M), and calculate its contrast noise ratio (contrast to noiseratio, CNR).Computational methods are: CNR=(SI
T-SI
M)/SD
NoiseEvery nude mice SI
TAnd SI
mMeasurement select 5 region of interest of the same size (ROI) respectively, the interest area is not less than 4mm
2, get during measurement as far as possible and strengthen obviously and uniform zone, get its meansigma methods.
3, pathologic finding:
Test and finish back execution experiment tumor bearing nude mice, get tumor tissues and send pathologic finding, capable respectively conventional organization pathological examination and VEGF SABC.Concrete grammar is the same.
4, statistical procedures
Carry out statistical analysis respectively to before the signal strength values of each timing point after experimental group and matched group baseline, the injection of contrast agent, CNR value and the injection, statistical software is SPSS 13.0, is the significance standard with P＜0.05.
The result
1, strengthen performance:
Experimental group injects behind contrast medium Gd-DTPA-polylysine-McAb (the anti-VEGF monoclonal antibody) 5min scanning and sees that the mice blood vessel obviously strengthens, and tumor is strengthened not obvious; Borderline tumor begins to strengthen behind the 1h, and reinforcing degree is lower, along with the prolongation reinforcement of time is obvious gradually, and by outer circumferentially center disperse, strengthens during to 4h and peaks.Observe to the 24h tumor and still have slight reinforcement (see figure 8).Matched group injects behind the contrast medium Gd-DTPA 5min scanning and sees that lump obviously evenly strengthens, and along with the prolongation reinforcing degree of time weakens gradually, contrast medium withdraws from (see figure 9) substantially behind 4h.
2, baseline is relatively:
Tumor body signal intensity (SI on the unenhanced image of difference determination experiment group and matched group
T) and homonymy muscle of anterior limb meat tissue signal intensity (SI
M) value, calculate the CNR value, check the difference that compares signal between tumor body and tumor body, muscle and the muscle through two sample t, show two group difference not statistically significant: SI
T(t=0.841, P=0.303), SI
S(t=0.262, P=0.199) and the CNR value (t=0.713 P=0.285), illustrates two groups of baseline no significant differences.
3, each spot scan time delay result is relatively:
Experimental group with scan at 8 time points after nude mice of control group is injected different contrast medium respectively, on crown position, measure SI respectively
TAnd SI
M, and calculate the CNR value.Experimentize between group and matched group group relatively and the group of experimental group, matched group in comparison.Adopt variance analysis (population variance is uneven, and variance analysis is carried out in advanced row rank conversion again) and LSD to analyze respectively.The result shows:
(1) compares between experimental group and matched group group: the SI of experimental group and matched group
TDifference has statistical significance (F=31.348, P＜0.001); The SI of experimental group and matched group
MDifference has statistical significance (F=19.094, P＜0.001); Experimental group and matched group CNR difference have statistical significance (F=45.996, P＜0.001), and the reinforced effects between illustrative experiment group and the matched group is variant.(see figure 10)
(2) compare in the experimental group group: experimental group SIT value has significant difference (F=419.39 before and after injection of contrast agent, P＜0.001), by showing SIT its difference not statistically significant (P=0.301) of comparison before the rising of tumor body signal intensity and injection of contrast agent during 5min after injection of contrast agent after analyzing in twos; Postpone behind the 1h comparison to the rising of 24h tumor body signal intensity and the injection of contrast agent, difference all has statistical significance (P＜0.001).Experimental group SIM value before and after the injection of contrast agent no difference of science of statistics (F=1.371, P=0.209).Experimental group CNR has significant difference (F=138.742, P＜0.001) before and after injection of contrast agent; Analyze when being presented at after the injection of contrast agent 5min its difference not statistically significant (P=0.677) of comparison before the CNR and injection of contrast agent in twos; Postponing behind the 1h all has statistical significance (P＜0.001) to 24h CNR value.(specifically seeing Table 4) this shows, after injecting contrast medium Gd-DTPA-Polylysine-McAb (anti-VEGF monoclonal antibody), the tumor body all reaches reinforced effects in 1 to 24h, and muscular tissue is not obviously strengthened, this has shown that not only Gd-DTPA-Polylysine-McAb (anti-VEGF monoclonal antibody) to the good strengthening effect of tumor, has also shown its target function.
Table 4: test each time point scanning result after the thin injection of contrast agent
Annotate: expression experimental group injection of contrast agent (C+) each time point determining value of back or value of calculation compare with unenhanced result (C-) respectively.
(3) compare in the matched group group: significant difference (F=152.037, P＜0.001) is arranged before and after the matched group SIT injection of contrast agent; Its difference of comparison has statistical significance (P5min, 1H, 2H, 3H, 4H＜0.001) before analyzing the rising be shown in 5min, 1h after the injection of contrast agent, 2h, 3h, 4h tumor body signal intensity and injection of contrast agent in twos; Comparing difference not statistically significant (P＞0.05) before the rising of delay 8h to 24h posterior tuberosity body signal intensity and the injection of contrast agent.Matched group SIM value has significant difference (F=26.236, P＜0.001) before and after injection of contrast agent; Analyze in twos be shown in injection of contrast agent 5min, 1h, 2h and 3h and the change of injection of contrast agent front signal have significant difference (P5min, 1H, 2H＜0.001, P3H=0.039); Signal change no difference of science of statistics (P＞0.05) before and after the contrast medium injection behind the delay 4h to 24h.Matched group CNR has significant difference (F=59.596, P＜0.001) before and after injection of contrast agent; Analysis in twos is shown in injection of contrast agent 5min, 1h, 2h, 3h and the change of injection of contrast agent front signal significant difference (P5min, 1h, 2h, 3h＜0.001); Signal change no difference of science of statistics (P＞0.05) before and after the contrast medium injection behind the delay 4h to 24h.(specifically seeing Table 5) is in conjunction with the analysis result of SIT value and CNR value, because CNR value no difference of science of statistics (P=0.155) when 4h, so we think that the signal change of SIT value when 4h is meaningless.In sum, tumor body and muscular tissue all have strengthening effect when injecting 5min, 1h behind the contrast medium Gd DTPA, 2h, 3h, and contrast medium withdraws from behind the 4h.
Table 5: each time point scanning result before and after the matched group injection of contrast agent
Annotate: expression matched group injection of contrast agent (C+) each time point determining value of back or value of calculation compare with unenhanced result (C-) respectively.
3, pathological examination: conventional H E dyeing shows that the tumor cell size and form differs, and karyokinesis mutually more to be seen.See that brown particle shows stained positive in the VEGF SABC tumor cell slurry.(seeing Figure 11).
Claims (8)
1, a kind of Gd-DTPA-Polylysine-McAb union body is characterized in that: make by following method:
Synthesizing of a, DTPA bisgallic acid acid anhydride: Diethylenetriamine five acid, acetic anhydride and pyridine are mixed;
B, DTPA-Polylysine preparation: poly-D-lysine is dissolved in NaH
2CO
3/ Na
2HCO
3In the buffer, stir, and add in the DTPA bisgallic acid anhydride solution that step a obtains;
C, Gd-DTPA-Polylysine preparation: with Gd
2O
3Join among the HCl, heating, dissolving, the HCl of evaporating surplus promptly obtains GdCl
3Solution, with GdCl
3Solution joins in the DTPA-Polylysine solution that step b obtains;
The connection of d, anti-VEGF monoclonal antibody and Gd-DTPA-Polylysine: will resist VEGF monoclonal antibody dissolving NaH
2CO
3/ Na
2HCO
3In the buffer, add the Gd-DTPA-Polylysine that carbodiimides and step c obtain then, stirring reaction, chromatography, the association of collecting anti-VEGF monoclonal antibody and Gd-DTPA-Polylysine.
2, union body according to claim 1 is characterized in that: also comprise in step b and take out poly-D-lysine, NaH
2CO
3/ Na
2HCO
3, DTPA bisgallic acid acid anhydride mixed solution, remove free DTPA with equilibrium dialysis, and be transformed into PH=5,0.2ml NaAc/HAc buffer.
3, union body according to claim 1 is characterized in that: select SepharoseCL-4B chromatographic column chromatography in steps d for use.
4, a kind of preparation method of Gd-DTPA-Polylysine-McAb union body: may further comprise the steps:
Synthesizing of a, DTPA bisgallic acid acid anhydride: Diethylenetriamine five acid, acetic anhydride and pyridine are mixed;
B, DTPA-Polylysine preparation: poly-D-lysine is dissolved in NaH
2CO
3/ Na
2HCO
3In the buffer, stir, and add in the DTPA bisgallic acid anhydride solution that step a obtains;
C, Gd-DTPA-Polylysine preparation: with Gd
2O
3Join among the HCl, heating, dissolving, the HCl of evaporating surplus promptly obtains GdCl
3Solution, with GdCl
3Solution joins in the DTPA-Polylysine solution that step b obtains;
The connection of d, anti-VEGF monoclonal antibody and Gd-DTPA-Polylysine: will resist VEGF monoclonal antibody dissolving NaH
2CO
3/ Na
2HCO
3In the buffer, add the Gd-DTPA-Polylysine that carbodiimides and step c obtain then, stirring reaction, chromatography, the association of collecting anti-VEGF monoclonal antibody and Gd-DTPA-Polylysine.
5, preparation method according to claim 4 is characterized in that: also comprise in step b and take out poly-D-lysine, NaH
2CO
3/ Na
2HCO
3, DTPA bisgallic acid acid anhydride mixed solution, remove free DTPA with equilibrium dialysis, and be transformed into PH=5,0.2ml NaAc/HAc buffer.
6, preparation method according to claim 4 is characterized in that: select Sepharose CL-4B chromatographic column chromatography in steps d for use.
7, use according to the arbitrary described union body of claim 1-3, it is characterized in that: this union body agent is as a comparison used in nuclear magnetic resonance.
8, application according to claim 7 is characterized in that: described nuclear magnetic resonance is specificity, the targeting nuclear magnetic resonance of angiogenesis in the tumor of VEGF gene expression in body shows tumor, VEGF mediation.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101912624A (en) * | 2010-06-28 | 2010-12-15 | 山东大学 | Active targeting polymer nanoparticle magnetic resonance contrast agent and preparation method thereof |
| EP2694117A1 (en) * | 2011-04-06 | 2014-02-12 | Cedars-Sinai Medical Center | Polymalic acid based nanoconjugates for imaging |
| WO2015017815A1 (en) * | 2013-08-01 | 2015-02-05 | Rochester Institute Of Technology | Modular imaging agents containing amino acids and peptides |
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| CN1317037C (en) * | 2005-06-06 | 2007-05-23 | 中国人民解放军第二军医大学 | Magnetic resonance tumour target contrast media and preparing method thereof |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101912624A (en) * | 2010-06-28 | 2010-12-15 | 山东大学 | Active targeting polymer nanoparticle magnetic resonance contrast agent and preparation method thereof |
| CN101912624B (en) * | 2010-06-28 | 2012-11-07 | 山东大学 | Active targeting polymer nanoparticle magnetic resonance contrast agent and preparation method thereof |
| EP2694117A1 (en) * | 2011-04-06 | 2014-02-12 | Cedars-Sinai Medical Center | Polymalic acid based nanoconjugates for imaging |
| CN103582497A (en) * | 2011-04-06 | 2014-02-12 | 西奈医疗中心 | Polymalic acid based nanoconjugates for imaging |
| EP2694117A4 (en) * | 2011-04-06 | 2014-06-11 | Cedars Sinai Medical Center | Polymalic acid based nanoconjugates for imaging |
| US10383958B2 (en) | 2011-04-06 | 2019-08-20 | Cedars-Sinai Medical Center | Polymalic acid based nanoconjugates for imaging |
| WO2015017815A1 (en) * | 2013-08-01 | 2015-02-05 | Rochester Institute Of Technology | Modular imaging agents containing amino acids and peptides |
| US10610608B2 (en) | 2013-08-01 | 2020-04-07 | Rochester Institute Of Technology | Modular imaging agents containing amino acids and peptides |
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