CN102249345B - Preparation method of Exendin-4 coupling superparamagnetic iron oxide nano particle - Google Patents
Preparation method of Exendin-4 coupling superparamagnetic iron oxide nano particle Download PDFInfo
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Abstract
The invention discloses a preparation method of an Exendin-4 coupling superparamagnetism iron oxide nanometer particle. A high temperature hydrolysis of metal alkoxide chelate is employed to prepare an SPIO (superparamagnetic iron oxide) nano particle; an APS ((3-aminopropyl) trimethoxysilane) is employed to carry out silanization modification on the SPIO, and PEG (polyethylene glycol) is employed to carry out a surface coating on an APS-SPIO particle to obtain a PEG-SPIO; an NHS/EDC (N-Hydroxysuccinimide / N-ethyl-N-(3-(dimethylamino)propyl)carbodiimide) method is further employed to couple the Exendin-4 on the PEG-SPIO, so as to obtain a nano-material SPIO-exendin-4 with beta cell targeting. The SPIO-exendin-4 prepared by the method of the invention can be applied to targeting imaging of insulinome and can increase mark efficiency of the SPIO to a beta cell.
Description
Technical field
The invention belongs to biological and medical technical field, relate to materials chemistry, be specially the Superparamagnetic Iron Oxide nano particle (Polyethylene glycol Coated Superparamagnetic Iron Oxide PEG-SPIO) of modifying by synthesizing polyethylene glycol, and with the exendin-4 coupling on it, prepare and have the cell targeted Superparamagnetic Iron Oxide nano particle SPIO-exendin4 of beta, use the targeted imaging that this contrast medium is realized the insulinoma of glucagon-like-peptide-1 (GLP-1) acceptor high expression level.The invention still further relates to this material to the external tag application of beta cell.
Background technology
Insulinoma is the endocrine tumors that derives from the beta cell, although its sickness rate lower (1 ~ 4/106), but modal endocrine tumor of pancreas.Insulinoma is more distributes, and is confined to the real property in the pancreas, optimum little tumour (diameter is many less than 2.0cm).Operation is insulinoma standard care mode, can reach 75 ~ 98% curative ratio, then is the prerequisite of accurate excision to the location (comprising primary tumor and metastasis) of insulinoma.B ultrasonic is adopted in the clinic operation prelocalization that insulinoma is commonly used usually, CT, MRI etc., the Enhanced CT diagnostic sensitivity can reach 57 ~ 94%, and Contrast-enhanced MRI then has 30 ~ 98% susceptibility.But with interior insulinoma, both susceptibility then reduces greatly for 1cm, for be dispersed in, the location of multiple minimal disease is then more difficult; And, have 10 ~ 27% to be invisible insulinoma.But the specific probe that develops a class targeted imaging insulinoma then has realistic meaning to location small and the concealment insulinoma.
The GLP-1 acceptor all has the expression (for 2 times of somatostatin receptor 2 expression levels) of higher level on the insulinoma more than 90%, and lower in the pancreatic exocrine gland expression, this is to realize that insulinoma (being different from pancreatic exocrine gland) video picture provides good target spot.At present, adopt in the world
111The exendin-4(GLP-1 analogue of In mark) wish realizes the SPECT imaging of insulin human's knurl; Also there is the investigator to adopt
68The analogue of the another kind of GLP-1 of Ga mark exendin-3(), use PET at mouse islets element knurl model and carry out tumor imaging.Although SPECT and PET susceptibility are high, spatial resolution is low, and simultaneously, also there is certain influence in radionuclide to the experimenter.Mr (MRI) has outstanding spatial resolution, and without wound, does not relate to radionuclide imaging, thereby uses MRI and realize that the special video picture of insulinoma has better prospect.
The employing nucleic of the past international literature report (as
111In) mark exendin-4 realizes the targeted imaging of insulinoma, but the defectives such as radionuclide imaging Existential Space resolving power is low and the contrast medium internal metabolism is fast, transformation period weak point.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of GLP-1 analogue exendin-4 coupling, but the preparation method of the Superparamagnetic Iron Oxide nano particle (SPIO-exendin4) of target GLP-1 acceptor, adopt the SPIO-exendin4 of the method preparation can be used for the targeted imaging of insulinoma, and can improve SPIO to the labeling effciency of beta cell.
In order to solve the problems of the technologies described above, the invention provides the preparation method of a kind of SPIO-exendin4, adopt pyrohydrolysis metal alkoxide inner complex process to prepare Superparamagnetic Iron Oxide nano particle (SPIO), adopt two carboxy polyethylene glycol that described particle is carried out finishing, prepare PEG-SPIO, on this basis, GLP-1 analogue exendin-4 is coupled to PEG-SPIO and makes this product.
The present invention adopts pyrohydrolysis metal alkoxide inner complex process to prepare the SPIO nano particle, SPIO is carried out silylation modification to application APS and PEG carries out pan coating to the APS-SPIO particle, obtain PEG-SPIO, further adopt the NHS/EDC method that exendin-4 is coupled on the PEG-SPIO, thereby obtain having the cell targeted nano material SPIO-exendin4 of beta.
Concrete steps of the present invention are as follows:
1), under the protection of rare gas element, in room temperature with FeCl
36H
2O and FeCl
24H
2O is dissolved in the Polyethylene Glycol-600 (PEG600), obtains brown solution; FeCl
36H
2O and FeCl
24H
2The mol ratio of O is 2:1, FeCl
36H
2The concentration of O in brown solution is 0.01～0.1M;
2), under the protection of rare gas element, the brown solution of step 1) gained is warming up to 120～140 ℃, then add deionized water and 2, two (ethamine) (2 of 2'-(ethylene dioxy), 2-(ethylenedioxy) bis (ethylamine) EDEA), 120～140 ℃ of insulations 30～60 minutes, get black suspension; Deionized water and FeCl
36H
2The mol ratio of O is 30～10:1,2,2'-(ethylene dioxy) two (ethamine) and FeCl
36H
2The mol ratio of O is 200～400:1;
3), with step 2) black suspension of gained is cooled to room temperature, it is centrifugal to add toluene, removes supernatant liquor, obtains brown ferric oxide nanometer particle (SPIO is pulverulent solids); The volume ratio of toluene and black suspension is 1～3:1;
4), under protection of inert gas, the ferric oxide nanometer particle of step 3) gained is scattered in the toluene, the concentration that makes ferric oxide nanometer particle is 0.005～0.05M; Then in situation about stirring, add 3-(trimethoxysilyl)-1-propylamine ((3-aminopropyl) trimethoxysilane APS) and butyl (tetra) titanate, be heated to 70～90 ℃, back flow reaction 10～16 hours; 3-(trimethoxysilyl)-1-propylamine (APS) is 5～30:1 with the mol ratio of ferric oxide nanometer particle, and the mol ratio of butyl (tetra) titanate and ferric oxide nanometer particle is 1～5:1;
5), the solution with step 4) reaction end gained is cooled to room temperature and centrifugation, clean (3～5 times with toluene, purpose is to remove remaining unreacted 3-(trimethoxysilyl)-1-propylamine (APS) or butyl (tetra) titanate), obtain the SPIO that APS-SPIO(APS modifies, be brown ceramic powder);
APS-SPIO is scattered in the toluene, and the concentration that makes APS-SPIO is 0.005～0.05M; Then in room temperature under ultrasonic and mechanical stirring, add two carboxyl PEG(poly (ethylene glycol) bis (carboxymethyl) ether, MW～600) and dicyclohexylcarbodiimide (N, N-dicyclohexyl carbodiimide, DCC), react after 3～5 hours, the mol ratio of the described couple of carboxyl PEG and APS-SPIO is 1～5:1, and dicyclohexylcarbodiimide (DCC) is 5～20:1 with the mol ratio of APS-SPIO;
Above-mentioned reaction adds 2,2'-(ethylene dioxy) two (ethamine) (EDEA) after finishing again, continues reaction 3～5 hours in room temperature under ultrasonic and churned mechanically condition; Described 2,2'-(ethylene dioxy) two (ethamine) is 1～5:1 with the mol ratio of APS-SPIO (EDEA);
6), the product of step 5) gained is centrifugal, with ethanol and washed with de-ionized water, obtain PEG-SPIO, and be scattered in (Phosphate Buffered Saline PBS buffered soln) in the phosphate buffer soln, obtain the PBS solution of PEG-SPIO; The Fe concentration of element is 3 ~ 7 mg/ml (that is, the Fe element in the SPIO is as the standard test concentration) in the PBS solution of PEG-SPIO;
7), with the 1-(3-dimethylamino-propyl) of mol ratio 1:2.5-3-ethyl-carbodiimide hydrochloride (N-ethyl-N-(3-(dimethylamino) propyl) carbodiimide, EDC) with N-hydroxy-succinamide (N-Hydroxysuccinimide, NHS) mix, get mixture;
The PBS solution of getting (part) PEG-SPIO of step 6) gained adds the exendin-4 aqueous solution and said mixture, stirring reaction is 8 ~ 12 hours under normal temperature, thereby makes exendin-4 be attached to the PEG-SPIO surface by the carboxyl on surface and the amino on PEG-SPIO surface by the reaction of shrinking; By ultrafiltration, with EDC and NHS and byproduct of reaction removal that may be remaining, the SPIO-exendin4 that finally obtains; The mass ratio of exendin-4 and Fe element is 0.5～1.5:1, and the mol ratio of PEG-SPIO and EDC is 1:2～4.
Improvement as the preparation method of the Superparamagnetic Iron Oxide nano particle of Exendin-4 coupling of the present invention: step 2) under agitation condition, carry out.
Improvement as the preparation method of the Superparamagnetic Iron Oxide nano particle of Exendin-4 coupling of the present invention: rare gas element is Ar.
The present invention also discloses the purposes such as the Superparamagnetic Iron Oxide nano particle of the Exendin-4 coupling of above-mentioned method preparation simultaneously: be used for the mark of pancreas islet beta cell and the targeted imaging of insulinoma.
In sum, the present invention adopts 3-(trimethoxysilyl)-1-propylamine (APS) that the SPIO for preparing is carried out silylation modification and two carboxy polyethylene glycol (PEG) are carried out pan coating to the APS-SPIO particle, it is even to obtain particle diameter, good dispersity, the nano particle PEG-SPIO with better magnetic.Further adopt the NHS/EDC method that GLP-1 analogue exendin-4 is coupled on the PEG-SPIO, obtain SPIO-exendin4.
This SPIO-exendin4 nano particle (mark of original position pancreas islet and mark of islet cell transplantation thing etc. in the mark of viable cell and fixed cell, pancreas) in the experiment of external series of markings show to the beta cell than high specific.By setting up the insulinoma model, tail vein injection SPIO-exendin4, MRI scanning knurl body can be realized the targeted imaging to insulinoma.
The sign of the SPIO-exendin4 that employing the present invention makes (Fig. 1～Fig. 4).
TEM scanning prompting granular core diameter is about 5 ~ 10nm, and distribution of sizes is concentrated, favorable dispersity in the aqueous solution.Nanometer laser particle size analyzer analytical results prompting grain size comprises SPIO core and outside decorative layer, and its mean sizes is 25nm.Particle surface is wrapped with the thick PEG decorative layer in about 20nm left and right sides, the sterically hindered effect balance that produces act on magnetic and the Van der Waals'attractive force of particle surface, this provides good colloidal stability and biocompatibility for the PVP-SPIO nano particle.In order to confirm that further modifier successfully is coated on the surface of nano particle, we utilize FTIR that the finishing group of SPIO nano particle is characterized.The charateristic avsorption band of PEG and APS as we can clearly see from the figure.Fig. 4 be PEG-SPIO under room temperature (300K), the magnetic hysteresis loop when foreign field is 2K.As can be seen from the figure PEG-SPIO nano particle sample shows superparamagnetism, does not have residual magnetism and coercive force, still has higher saturation magnetization behind the coating decoration layer, reaches 27emu/g.
In external series of markings experiment, prove that its beta cell to high expression level GLP-1 acceptor has higher specificity by the SPIO-exendin4 nano particle.The non-targeted PEG-SPIO of 100ug/ml concentration of iron and target SPIO-exendin4 are hatched row prussian blue staining behind the 12h to the INS-1 cell, find that SPIO-exendin4 is than the more mark beta cell of PEG-SPIO.INS-1 cell after the SPIO-exendin4 of 200ug/ml concentration of iron and PEG-SPIO is fixing with 4% Paraformaldehyde 96 hatches 1,2, row prussian blue staining behind the 4h, found that non-targeted PEG-SPIO can not mark beta cell, but and SPIO-exendin4 significant notation INS-1 cell, and labeled cell quantity increases along with the prolongation of mark time.After the beta cell section that enters subrenal capsule by PEG-SPIO and SPIO-exendin4 homologous transplantation with the 200ug/ml concentration of iron is hatched 2h, but prussian blue staining results suggest SPIO-exendin4 significant notation beta cellular transplant.After the PEG-SPIO of 200ug/ml concentration of iron and SPIO-exendin4 hatched 2h with the pancreas section, but prussian blue staining results suggest SPIO-exendin4 significant notation original position pancreas islet, then can not mark to exocrine gland.By setting up the insulinoma model of nude mice bearing tumor, tail vein PEG-SPIO and SPIO-exendin4 (3mg Fe/kg), 24h after the injection, give MRI scanning, on the T2 weighting picture (T2-weighted image), SPIO-exendin4 injection group mouse tumour presents obvious negativity and strengthens, and prussian blue staining is presented at the deposition that the Fe particle is arranged in the SPIO-exendin4 injection group tumour, shows that insulinoma can be by the SPIO-exendin4 targeted imaging.
Still there are not at present document and research report that exendin-4 is coupled to the upper synthetic targeted contrast agent of SPIO.SPIO is a kind of novel MRI contrast medium, on its basis can the different material of coupling, such as antibody, small molecules or peptide section etc., have different purposes with formation, the contrast medium of different targetings; And the MRI imaging has the advantages such as higher spatial resolution than radionuclide imaging.The synthetic the first step of SPIO-exendin4 is synthetic PEG-SPIO, then on the basis of PEG-SPIO, further the exendin-4 coupling on it, is made the SPIO-exendin4 with target GLP-1 acceptor.
This SPIO-exendin4 is used for the effect of insulinoma targeted imaging: at experiment in vitro, SPIO-exendin4 is than non-targeted PEG-SPIO, it has higher labeling effciency to insulinoma clone, illustrates that SPIO-exendin4 can more enter in the cell by joining receptors bind.SPIO-exendin4 also can show islet cells group in pancreas section difference.In vivo in the experiment, but the SPIO-exendin4 target be gathered in the insulinoma, and make insulinoma obtain showing at MRI, non-targeted PEG-SPIO then can not show insulinoma.
The present invention has following characteristics: 1, synthetic SPIO-exendin4 has good physico-chemical property and magnetism characteristic; 2, in contrast to non-targeted PEG-SPIO, the SPIO-exendin4 of target all has preferably labeling effciency to viable cell and fixing beta cell; But 3, original position pancreas islet in the SPIO-exendin4 significant notation beta cellular transplant of preparation and the pancreas, and have higher specificity; 4, SPIO-exendin4 has good targeted imaging effect to insulinoma in vivo.
In sum, SPIO-exendin4 nano particle of the present invention is used for the targeted imaging of the upper insulinoma of MRI, for target, the specific diagnostic (comprising primary tumor and metastasis) of the insulinoma of GLP-1 acceptor high expression level provide effective means clinically.Simultaneously, SPIO-exendin4 has higher cell marking efficient to the beta cell, provides good material for better realizing pancreas islet beta cell marking and MRI spike.
Description of drawings
Below in conjunction with accompanying drawing the specific embodiment of the present invention is described in further detail.
The transmission electron microscope photo of Fig. 1 PEG-SPIO nano particle.
The nanometer laser sreen analysis collection of illustrative plates of Fig. 2 PEG-SPIO nano particle.
The fourier infrared absorption spectrum of Fig. 3 PEG-SPIO nano particle.
The vibrating sample magnetometer room temperature magnetism collection of illustrative plates of Fig. 4 PEG-SPIO nano particle.
Fig. 5 is the synthesis strategy figure of Exendin-4 structure and SPIO-exendin4; A, the Exendin-4 molecular structure; B, the molecular formula of Exendin-4; C adopts the NHS/EDC method to synthesize the strategy of SPIO-exendin4.
Fig. 6-the 1st, prussian blue staining figure behind the INS-1 cell 12h of the PEG-SPIO of 100ug/ml concentration of iron and PEG-SPIO-Exendin4 mark equivalent amount;
Left figure is prussian blue staining figure behind the PEG-SPIO mark INS-1 cell 12h of 100ug/ml concentration of iron;
Middle figure is prussian blue staining figure behind the SPIO-exendin4 mark INS-1 cell 12h of 100ug/ml concentration of iron;
Right figure is the amplification of middle figure, and clear showed cell is by the SPIO-exendin4 mark;
Results suggest is with respect to common PE G-SPIO, and the contrast medium that contains target is labeled cell more.
Fig. 6-the 2nd, INS-1 and former generation pancreas islet fixing after, the mark situation map of contrast medium; A, INS-1 cell after Paraformaldehyde 96 is fixing, the PEG-SPIO under the 200ug/ml concentration of iron and PEG-SPIO-Exendin4 labeled cell 1h, 2h and 4h discard contrast medium and clean three times the row prussian blue staining with PBS; The result shows that common PE G-SPIO is fixing INS-1 cell on the energy mark, and the SPIO of coupling exendin-4 is then with the prolongation of incubation time, and the labeled cell number increases gradually.B, former generation pancreas islet section gives 200ug/ml SPIO-Exendin4 and hatches 2h after fixing, and PBS washes three afterwards capable prussian blue stainings, but the fixing islet cells of the same mark of prompting SPIO-Exendin4.
Fig. 7 is PEG-SPIO, and SPIO-exendin4 respectively marking transfer enters the beta cell section prussian blue staining photo of subrenal capsule;
A, the INS-1 Transplanted cells enters the HE colored graph of subrenal capsule; B, C, prussian blue staining behind the PEG-SPIO mark Islet allografts of the 200ug/ml concentration of iron section 2h, the result shows that PEG-SPIO fails mark Islet allografts (C figure is the amplification of B figure); D, behind the SPIO-Exendin4 mark 2h of equal concentration of iron, prussian blue staining shows that uriniferous tubules is not labeled, and E, F, the islet cell transplantation thing can be by SPIO-Exendin4 mark (F figure be the amplification of square frame part among the E figure).
Fig. 8 is PEG-SPIO, the beta cell prussian blue staining photo in the section of SPIO-exendin4 difference mark pancreas;
A contains the pancreas section HE dyeing of pancreas islet; B, C, D, behind the SPIO-Exendin4 mark original position pancreas islet 2h of 200ug/ml concentration of iron, prussian blue staining shows pancreas islet by the SPIO-Exendin4 mark, exocrine gland then is not labeled (C, D figure are that the step by step amplification of pancreas islet in the B figure shows) on every side; E, F, G shows another routine pancreas islet by the SPIO-exendin4 significant notation, exocrine gland then is not labeled (F, G are that the step by step amplification of pancreas islet in the E figure shows) on every side, has proved that SPIO-Exendin4 exists targeting.
Fig. 9 is PEG-SPIO, and SPIO-exendin4 is injected into respectively in the insulinoma tumor bearing nude mice body, MRI scanning imagery T2WI image;
24h MRI scanned T2WI figure after left figure, middle figure, right figure were respectively not injection of contrast medium group, the rear 24h of PEG-SPIO injection and SPIO-exendin4 injection.
The result shows: the insulinoma mice with tumor passes through respectively tail vein injection PEG-SPIO and SPIO-exendin4 (3mg Fe/kg), MRI scanning imagery behind the 24h (T2 map).The result shows that SPIO-exendin4 can be gathered in insulinoma zone (showing as negativity strengthens), and PEG-SPIO is then without targeting.
Figure 10 is PEG-SPIO, and SPIO-exendin4 injects not on the same group insulinoma section prussian blue staining photo, is used for prussian blue staining checking SPIO-exendin4 to the targeting of insulinoma.
A, nude mice insulinoma lotus knurl model is set up, the visible thick supply blood vessel of tumour; B, C, the insulinoma mice with tumor cuts the prussian blue staining of tumour row by tail vein injection PEG-SPIO (3mg Fe/kg) behind 24 h, and the result shows tumor region iron-free particle deposition (C figure is the amplification of part in the B picture frame); D, E, F, the insulinoma mice with tumor is by tail vein injection SPIO-exendin4 (3mg Fe/kg), cut the prussian blue staining of tumour row behind the 24h, the result shows that SPIO-exendin4 can be gathered in tumor locus (D figure and F figure are respectively the amplification of part in the E figure inside casing).
Embodiment
Further specify the present invention below in conjunction with embodiment.
The preparation method of the Superparamagnetic Iron Oxide nano particle of embodiment 1, a kind of exendin-4 coupling (SPIO-exendin4 nano particle), carry out following steps successively:
1), under the protection of rare gas element Ar, in room temperature with FeCl
36H
2O and FeCl
24H
2O is dissolved in the Polyethylene Glycol-600 (PEG600) according to the mol ratio of 2:1, obtains brown solution; FeCl
36H
2The volumetric molar concentration of O in this brown solution is 0.05M.
2), under the protection of rare gas element Ar, with above-mentioned steps 1) brown solution of gained is warming up to 125 ℃, adds a certain amount of deionized water and EDEA, 125 ℃ of insulations 30 minutes, gets black suspension.Wherein deionized water and FeCl
36H
2The mol ratio of O is 20:1, EDEA and FeCl
36H
2The mol ratio of O is 300:1.
3), with above-mentioned steps 2) black suspension of gained is cooled to room temperature, it is centrifugal to add toluene 5000rpm, the volume ratio of toluene and black suspension is 1:1; Remove supernatant liquor, obtain brown ferric oxide nanometer particle (SPIO) (for pulverulent solids).
4), under rare gas element Ar protection, with above-mentioned steps 3) ferric oxide nanometer particle that obtains is scattered in the toluene, is 0.01M thereby make the volumetric molar concentration of ferric oxide nanometer particle; Then under the condition that stirs, add a certain amount of APS and butyl (tetra) titanate, be heated to 80 ℃, back flow reaction 10 hours.The mol ratio of APS and ferric oxide nanometer particle is 20:1, and the mol ratio of butyl (tetra) titanate and ferric oxide nanometer particle is 5:1.
5), with above-mentioned steps 4) the reaction solution that finishes gained is cooled to room temperature 5000rpm centrifugation, cleans 3～5 times with toluene and removes remaining unreacted APS or butyl (tetra) titanate, obtains the SPIO(APS-SPIO that APS modifies) brown ceramic powder.The consumption of toluene is identical with the reaction soln volume during each the cleaning.
The APS-SPIO that obtains is scattered in the toluene, is 0.01M thereby make the concentration of APS-SPIO.Ultrasonic in the supersonic cleaning machine of 30KHz at room temperature, and mechanical stirring, add two carboxyl PEG and DCC, react after 3 hours, add again EDEA, under ultrasonic and mechanical stirring, continue at room temperature reaction 5 hours.Wherein the mol ratio of two carboxyl PEG and APS-SPIO is 3:1, and the mol ratio of DCC and APS-SPIO is 10:1; The mol ratio of EDEA and APS-SPIO is 3:1.
6), the product 5000rpm of step 5) gained is centrifugal, use successively ethanol and washed with de-ionized water, obtain PEG-SPIO.
PEG-SPIO is scattered in (Phosphate Buffered Saline PBS buffered soln) in the phosphate buffer soln, get the PBS solution of PEG-SPIO, its concentration is about every milliliter of 6 milligrams of PEG-SPIO, be that the Fe concentration of element is about 6 milligrams every milliliter (concentration of its accurate Fe element generally records with atomic absorption spectrum, and following concentration all represents with the Fe concentration of element).
7), the EDC of mol ratio 1:2.5 is mixed with NHS, get mixture.
Get the PBS solution that 1 milliliter of Fe concentration of element is 6 milligrams every milliliter PEG-SPIO, add a certain amount of exendin-4 aqueous solution and said mixture (be the EDC of mol ratio 1:2.5 mix with NHS and get), stirring reaction is 10 hours under the normal temperature, the concentration of exendin-4 is 1 milligram every milliliter in the exendin-4 aqueous solution, the mass ratio of exendin-4 and Fe element is 1:1, and the mol ratio of PEG-SPIO and EDC is 1:2.Exendin-4 is attached to the PEG-SPIO surface by the carboxyl on surface and the amino on PEG-SPIO surface by the reaction of shrinking, and by the super filter tube ultrafiltration of 3kDa, EDC and NHS and byproduct of reaction removal with may be remaining obtain final SPIO-exendin4.
The preparation method of the Superparamagnetic Iron Oxide nano particle of embodiment 2, a kind of exendin-4 coupling (SPIO-exendin4 nano particle), carry out following steps successively:
1), under the protection of rare gas element, in room temperature with FeCl
36H
2O and FeCl
24H
2O is dissolved in the Polyethylene Glycol-600 (PEG600), obtains brown solution; FeCl
36H
2O and FeCl
24H
2The mol ratio of O is 2:1, FeCl
36H
2The concentration of O in brown solution is 0.05M.
2), under the protection of rare gas element, the brown solution of step 1) gained is warming up to 125 ℃, add a certain amount of deionized water and EDEA, 125 ℃ of insulations 30 minutes, get black suspension.Wherein deionized water and FeCl
36H
2The mol ratio of O is 20:1, EDEA and FeCl
36H
2The mol ratio of O is 300:1.
3), with step 2) black suspension of gained is cooled to room temperature, it is centrifugal to add toluene 5000rpm, removes supernatant liquor, obtains brown iron oxide nano particle (SPIO) pulverulent solids.Wherein the volume ratio of toluene and reaction soln is 1:1.
4), under protection of inert gas, the ferric oxide nanometer particle that step 3) is obtained is scattered in the toluene, the concentration that makes ferric oxide nanometer particle is 0.01M; then in situation about stirring; add a certain amount of APS and butyl (tetra) titanate, be heated to 80 ℃, back flow reaction 12 hours.The mol ratio of APS and ferric oxide nanometer particle is 20:1, and the mol ratio of butyl (tetra) titanate and ferric oxide nanometer particle is 1:1;
5), solution that above-mentioned reaction is finished is cooled to room temperature and 5000rpm centrifugation, cleans 3～5 times with toluene and removes remaining unreacted APS or butyl (tetra) titanate, the consumption of toluene is identical with the reaction soln volume when cleaning at every turn.Obtain the SPIO(APS-SPIO that APS modifies) brown ceramic powder.
The APS-SPIO that obtains is scattered in the toluene, and making the concentration of APS-SPIO is 0.01M, in room temperature under ultrasonic and mechanical stirring, add two carboxyl PEG and DCC, react after 4 hours, add EDEA, in room temperature, under ultrasonic and mechanical stirring, continue reaction 4 hours.Wherein the mol ratio of two carboxyl PEG and APS-SPIO is 3:1, and the mol ratio of EDEA and APS-SPIO is 3:1, and the mol ratio of DCC and APS-SPIO is 10:1.
6) the product 5000rpm that, step 5) is obtained is centrifugal, clean successively with ethanol and deionized water, obtain PEG-SPIO, and be scattered in (Phosphate Buffered Saline PBS buffered soln) in the phosphate buffer soln, obtain the PBS solution of PEG-SPIO; Its concentration is about every milliliter of 6 milligrams of PEG-SPIO, that is, the Fe concentration of element is 6 mg/ml in the PBS solution of PEG-SPIO.
7), get the PBS solution that 1 milliliter of Fe concentration of element is 6 milligrams every milliliter PEG-SPIO, add the EDC of a certain amount of exendin-4 aqueous solution and mol ratio 1:2.5 and the mixture of NHS, the concentration of exendin-4 is 1 milligram every milliliter in the exendin-4 aqueous solution, stirring reaction is 10 hours under the normal temperature, exendin-4 is attached to the PEG-SPIO surface by the carboxyl on surface and the amino on PEG-SPIO surface by the reaction of shrinking, super filter tube ultrafiltration by 3kDa, EDC and NHS and byproduct of reaction removal with may be remaining obtain final SPIO-exendin4.Wherein, the mass ratio of exendin-4 and Fe element is 1:1, and the mol ratio of PEG-SPIO and EDC is 1:2.
The preparation method of the Superparamagnetic Iron Oxide nano particle of embodiment 3, a kind of exendin-4 coupling (SPIO-exendin4 nano particle), carry out following steps successively:
1), under the protection of rare gas element, in room temperature with FeCl
36H
2O and FeCl
24H
2O is dissolved in the Polyethylene Glycol-600 (PEG600), obtains brown solution; FeCl
36H
2O and FeCl
24H
2The mol ratio of O is 2:1, FeCl
36H
2The concentration of O in brown solution is 0.1M.
2), under the protection of rare gas element, above-mentioned brown solution is warming up to 135 ℃, add a certain amount of deionized water and EDEA, 135 ℃ the insulation 40 minutes, get black suspension; Deionized water and FeCl
36H
2The mol ratio of O is 15:1, EDEA and FeCl
36H
2The mol ratio of O is 200:1.
3), black suspension that reaction is obtained is cooled to room temperature, it is centrifugal to add toluene 5000rpm, removes supernatant liquor, obtains brown iron oxide nano particle (SPIO) pulverulent solids.The volume ratio of toluene and black suspension is 3:1.
4), under protection of inert gas, the ferric oxide nanometer particle that obtains is scattered in the toluene, the concentration that makes ferric oxide nanometer particle is 0.01M; Then in situation about stirring, add a certain amount of APS and butyl (tetra) titanate, be heated to 80 ℃, back flow reaction 12 hours.The mol ratio of APS and ferric oxide nanometer particle is 10:1, and the mol ratio of butyl (tetra) titanate and ferric oxide nanometer particle is 3:1.
5), with above-mentioned steps 4) solution that finishes of reaction is cooled to room temperature and 5000rpm centrifugation, clean 3～5 times with toluene and remove remaining unreacted APS or butyl (tetra) titanate, obtain the SPIO(APS-SPIO that APS modifies) brown ceramic powder, the consumption of toluene is identical with the reaction soln volume when cleaning at every turn.
The APS-SPIO that obtains is scattered in the toluene, and the concentration that makes APS-SPIO is 0.02M; Then in room temperature under ultrasonic and mechanical stirring, add two carboxyl PEG and DCC, react after 5 hours, add again EDEA, in room temperature, under ultrasonic and mechanical stirring, continue to react 3 hours.Wherein the mol ratio of two carboxyl PEG and APS-SPIO is 4:1, and the mol ratio of EDEA and APS-SPIO is 4:1, and the mol ratio of DCC and APS-SPIO is 10:1.
6), the product 5000rpm that obtains is centrifugal, use successively ethanol and washed with de-ionized water, obtain PEG-SPIO, and be scattered in (Phosphate Buffered Saline PBS buffered soln) in the phosphate buffer soln, obtain the PBS solution of PEG-SPIO; Its concentration is about every milliliter of 4 milligrams of PEG-SPIO, and namely the Fe concentration of element is about 4 milligrams every milliliter.
7), the above-mentioned Fe concentration of element of getting 2 milliliters is the PBS solution of 4 milligrams every milliliter PEG-SPIO, add the EDC of a certain amount of exendin-4 aqueous solution and mol ratio 1:2.5 and the mixture of NHS, the concentration of exendin-4 is 1 milligram every milliliter in the exendin-4 aqueous solution, stirring reaction is 10 hours under the normal temperature, exendin-4 is attached to the PEG-SPIO surface by the carboxyl on surface and the amino on PEG-SPIO surface by the reaction of shrinking, super filter tube ultrafiltration by 3kDa, EDC and NHS and byproduct of reaction removal with may be remaining obtain final SPIO-exendin4.Wherein, the mass ratio of exendin-4 and Fe element is 0.5:1, and the mol ratio of PEG-SPIO and EDC is 1:3.
Embodiment 4, SPIO-exendin4 alloy granular solids foreign labeling rat insulin oncocyte are INS-1 cell (one)
(1) with rat INS-1 cell with 8 * 10
4Individual kind enters in 24 orifice plates, growth 24h.
(2) PEG-SPIO and the SPIO-exendin4 nano particle with preparation dilutes with the RPMI-1640 substratum, and Fe concentration is 100ug/ml.
(3) in containing 24 orifice plates of INS-1 cell, add the substratum 500ul that above-mentioned each group contains nano particle, hatch the capable prussian blue staining of 12h.
The result is: prussian blue staining demonstration PEG-SPIO is hatched 12h, and it is lower that the INS-1 cell is labeled efficient; And but SPIO-exendin4 is at the next significant notation INS-1 of 100ug/ml Fe concentration cell.Specifically shown in Fig. 6-1.
Confirm that from the above results targeting SPIO-exendin4 has higher beta cell marking efficient than PEG-SPIO.
Embodiment 5, SPIO-exendin4 alloy granular solids foreign labeling rat insulin oncocyte are INS-1 cell (two)
(1) with rat INS-1 cell with 8 * 10
4Individual kind enters in 24 orifice plates, behind the growth 24h, with the fixing 20min of 4% Paraformaldehyde 96 room temperature;
(2) PEG-SPIO and the SPIO-exendin4 nano particle with preparation dilutes with the RPMI-1640 substratum, and concentration of iron is 200ug/ml.
(3) in 24 orifice plates that contain fixing INS-1 cell, add respectively above-mentioned each group and contain the substratum 500ul of nano particle, hatch row prussian blue staining behind 1,2, the 4h.
The result is: no matter incubation time length, PEG-SPIO all can not mark on fixing beta cell, but and the fixing beta cell of SPIO-exendin4 significant notation and along with the prolongation of time, is labeled cell number and increases gradually.Specifically shown in 6-2.
Can learn from the above results: SPIO-exendin4 not only has than protrude mark efficient the beta cell of living, and still can realize preferably by joining receptors bind mark to fixing beta cell.
Original position pancreas islet in embodiment 6, SPIO-exendin4 mark beta cellular transplant and the pancreas
(1) collects 2 * 10
6Individual INS-1 cell, and it is implanted into C57BL/6 mouse subrenal capsule, taking out beta cellular transplant and pancreas after the week, 4% Paraformaldehyde 96 is fixed, and gradient ethanol dewaters step by step, paraffin embedding, section (bed thickness 4um).
(2) PEG-SPIO and the SPIO-exendin4 nano particle with preparation dilutes with the RPMI-1640 substratum, and concentration of iron is 200ug/ml.
(3) above-mentioned mixed solution is hatched 2h with pancreas section and beta cellular transplant after, the row prussian blue staining.
The result is: PEG-SPIO can not mark pancreas islet in beta cellular transplant in the section and the pancreas; But SPIO-exendin4 is pancreas islet in mark beta cellular transplant and the pancreas then, and beta cellular transplant perienchyma and the pancreatic exocrine gland such as mark uriniferous tubules not.Concrete such as Fig. 7, shown in Figure 8.
Can learn from the above results: the SPIO-exendin4 of preparation can realize good mark to pancreas islet in beta cellular transplant and the pancreas, and shows the specificity to the beta cell.
Embodiment 7, SPIO-exendin4 are to target MRI video picture in the body of insulinoma
(1) collects 2 * 10
7Individual INS-1 cell, be injected into 4 the week age nude mice oxter, set up insulinoma nude mice lotus knurl model.
(2) nude mice that will build the knurl success is weighed, and the vetanarcol of abdominal injection 50mg/kg are with anesthetized mice.On the clinical MRI of 3.0 Tesla, scan with the mouse coil.TR/TE=200/8?ms，Number?of?averages=32，FOV=3.2×3.2?cm
2,?matrix?size=256×256,?resolution=0.125×0.125?mm
2,?slice?thickness=0.5?mm。
(3) not on the same group mice with tumor enter PEG-SPIO or SPIO-exendin4 (3mg Fe/kg) by tail vein injection, carry out MRI scanning behind the 24h, parameter is substantially with (2).
With not on the same group the tumour of tumor bearing nude mice carry out paraffin section, H﹠amp; E dyeing and prussian blue staining.
The result is: insulinoma shows as high signal in MRI T2 weighting, 24h after the PEG-SPIO injection, insulinoma signal be without obvious change, and after the SPIO-exendin4 injection, insulinoma produces clear signal and changes, and presents negativity and strengthens (being the color blackening).The prussian blue staining result shows, has no obvious iron particle deposition in the PEG-SPIO injection group insulinoma, and in SPIO-exendin4 injection group, visible more iron particle aggregation in the tumour.Concrete such as Fig. 9, shown in Figure 10.
Can learn from the above results: but SPIO-exendin4 target insulinoma realizes the targeted imaging of insulinoma on MRI.
At last, it is also to be noted that what more than enumerate only is several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
Claims (3)
1.Exendin-4 the preparation method of the Superparamagnetic Iron Oxide nano particle of coupling is characterized in that carrying out successively following steps:
1), under the protection of rare gas element, in room temperature with FeCl
36H
2O and FeCl
24H
2O is dissolved in the Polyethylene Glycol-600, obtains brown solution; Described FeCl
36H
2O and FeCl
24H
2The mol ratio of O is 2:1, FeCl
36H
2The concentration of O in brown solution is 0.01～0.1M;
2), under the protection of rare gas element, the brown solution of step 1) gained is warming up to 120～140 ℃, then add deionized water and 2,2'-(ethylene dioxy) two (ethamine), 120～140 ℃ of insulations 30～60 minutes, get black suspension; Described deionized water and FeCl
36H
2The mol ratio of O is 30～10:1,2,2'-(ethylene dioxy) two (ethamine) and FeCl
36H
2The mol ratio of O is 200～400:1;
3), with step 2) black suspension of gained is cooled to room temperature, it is centrifugal to add toluene, removes supernatant liquor, obtains brown ferric oxide nanometer particle; The volume ratio of described toluene and black suspension is 1～3:1;
4), under protection of inert gas, the ferric oxide nanometer particle of step 3) gained is scattered in the toluene, the concentration that makes ferric oxide nanometer particle is 0.005～0.05M; Then in situation about stirring, add 3-(trimethoxysilyl)-1-propylamine and butyl (tetra) titanate, be heated to 70～90 ℃, back flow reaction 10～16 hours; The mol ratio of 3-(trimethoxysilyl)-1-propylamine and ferric oxide nanometer particle is 5～30:1, and the mol ratio of butyl (tetra) titanate and ferric oxide nanometer particle is 1～5:1;
5), solution that step 4) reaction is finished gained is cooled to room temperature and centrifugation, cleans with toluene, obtains APS-SPIO;
Described APS-SPIO is scattered in the toluene, and the concentration that makes APS-SPIO is 0.005～0.05M; Then in room temperature under ultrasonic and mechanical stirring, add two carboxyl PEG and dicyclohexylcarbodiimide, react after 3～5 hours, the mol ratio of the described couple of carboxyl PEG and APS-SPIO is 1～5:1, and the mol ratio of dicyclohexylcarbodiimide and APS-SPIO is 5～20:1;
Above-mentioned reaction adds 2,2'-(ethylene dioxy) two (ethamine) after finishing again, continues reaction 3～5 hours in room temperature under ultrasonic and churned mechanically condition; Described 2,2'-(ethylene dioxy) two (ethamine) is 1～5:1 with the mol ratio of APS-SPIO;
Described APS-SPIO is the SPIO that APS modifies, and APS is 3-(trimethoxysilyl)-1-propylamine, and SPIO is the Superparamagnetic Iron Oxide nano particle;
6), the product of step 5) gained is centrifugal, with ethanol and washed with de-ionized water, obtain PEG-SPIO, and be scattered in the phosphate buffer soln, obtain the PBS solution of PEG-SPIO; The Fe concentration of element is 3 ~ 7 mg/ml in the PBS solution of described PEG-SPIO;
Described PEG-SPIO is the Superparamagnetic Iron Oxide nano particle of modifying by synthesizing polyethylene glycol;
7), the 1-(3-dimethylamino-propyl) of mol ratio 1:2.5-3-ethyl-carbodiimide hydrochloride is mixed with N-hydroxy-succinamide, get mixture;
The PBS solution of PEG-SPIO of getting the part of step 6) gained adds the exendin-4 aqueous solution and said mixture, stirring reaction is 8 ~ 12 hours under normal temperature, thereby makes exendin-4 be attached to the PEG-SPIO surface by the carboxyl on surface and the amino on PEG-SPIO surface by the reaction of shrinking; Again by ultrafiltration, the SPIO-exendin4 that finally obtains; The mass ratio of described exendin-4 and Fe element is 0.5～1.5:1, and the mol ratio of PEG-SPIO and EDC is 1:2～4;
Described EDC is 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride;
Described SPIO-exendin4 is the Superparamagnetic Iron Oxide nano particle of exendin4 coupling.
2. the preparation method of the Superparamagnetic Iron Oxide nano particle of Exendin-4 coupling according to claim 1 is characterized in that: described step 2) carry out under agitation condition.
3. the preparation method of the Superparamagnetic Iron Oxide nano particle of Exendin-4 coupling according to claim 1 and 2, it is characterized in that: described rare gas element is Ar.
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| CN1524796A (en) * | 2003-02-28 | 2004-09-01 | 中国科学院过程工程研究所 | Method for synthesizing ferric oxide nanometer powder by hydrothermal back extraction |
| US20040213731A1 (en) * | 2003-01-06 | 2004-10-28 | Jun Xu | Iron oxide based materials for use as ion intercalation hosts in rechargeable lithium batteries |
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| US20040213731A1 (en) * | 2003-01-06 | 2004-10-28 | Jun Xu | Iron oxide based materials for use as ion intercalation hosts in rechargeable lithium batteries |
| CN1524796A (en) * | 2003-02-28 | 2004-09-01 | 中国科学院过程工程研究所 | Method for synthesizing ferric oxide nanometer powder by hydrothermal back extraction |
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