IL-13 modification liposome containing gadolinium chelate compound target magnetic resonance imaging contrast agent and its
Preparation method and application
Technical field
The invention belongs to medical imaging technology fields, and in particular to the targeting of liposome containing gadolinium chelate compound of IL-13 modification
Magnetic resonance imaging contrast agent and its preparation method and application.
Background technique
Currently, the technology for diagnosing tumor mainly includes that computed tomography (CT), positron emission emission computer are disconnected
Layer scanning (PET), optical imagery and ultrasonic imaging etc..Because magnetic resonance imaging (MRI) does not have ionising radiation, and can be by comparison
Agent carries out the characteristics such as accurate positioning, quantitative analysis to deep tissue, it has also become clinically common detection and diagnosis means
One of.
Intestinal cancer, also referred to as colorectal cancer, occur in colon or rectum, and it is the most normal to belong to the third that diagnose in the U.S.
See cancer, and belongs to the third-largest cancer of Cancer death.Its harmfulness is, when advanced stage intestinal cancer liver metastasis, part
Hepatectomy only has 25% survival rate, and recurrence rate but has 75%, and most of this 75% recurrence rate eventually leads to death, this 75%
In recurrence rate, there are about 50% transfers can be transferred to the tissue other than liver.There is IL-13 alpha-2 receptor high in pernicious intestinal cancer metastatic cells
Degree expression, and in the transfer process of intestinal cancer adhesion, migrate, live in, metastatic shifting grow during play an important role,
And advanced stage intestinal cancer prognosis is caused to be difficult to ideal.
Adrenocortical carcinoma is relatively rare, but it is a kind of temporarily pernicious swollen without effective treatment method with invasion
Tumor.Result of study shows that adrenocortical carcinoma is expressed with IL-13 alpha-2 receptor height, and IL-13 α 2, which is played, adjusts pernicious adrenal gland
Cortical carcinoma tumour cell lives in, regenerates breeding.The expression of adrenocortical carcinoma IL-13 alpha-2 receptor height is detection and chemotherapy adrenal gland
The novel targeting target of one of cortical carcinoma.
Glioma is a kind of primary malignant tumor for being grown in central nervous system, is had to brain tissue very strong
Invasion, and astrocytoma, mesoglioma, ependymoma, medulloblastoma and pleomorphism glioblast can be subdivided into
Tumor.The conventional treatment regime of glioma is additional radiotherapy or chemotherapy after operation, unfortunately be the pre- of conventional treatment regime
It is very poor afterwards, the average survival time rate only about 14 months.The main reason for causing such low survival rate first is that we lack one kind at present
Sensitive early detection means, conventional detection means can not detect the lesion less than 2mm, when the cancer of the brain is less than 2mm, conventional detection
Means failure, this is by magnetic resonance imaging contrast agent (if Magnevist Solution is containing gadolinium chelate compound) is clinically widely applied at present
What mechanism was determined.Magnetic resonance Magnevist Solution is only able to detect that the cancer greater than 2mm or more, often already belongs to tumour evening
Phase.Advanced tumor blood-brain barrier is impaired, and Magnevist Solution can enter the growth site of tumour by impaired blood-brain barrier
And this is largely accumulated on, so that Nuclear Magnetic Resonance is captured signal intensity caused by Magnevist Solution.And Magnevist Solution
Blood-brain barrier is relatively complete when the early lesion being limited in that when tumour is less than 2mm, and Magnevist Solution is limited to blood-brain barrier
To which tumor focus position can not be accumulated on, thus the variation of Magnetic resonance imaging desired signal can not be provided, cause nuclear magnetic resonance
Instrument can not detect infantile tumour and wellability lesion.Meanwhile conventional contrast agents cannot generate specific binding with tumour cell,
Belong to non-target contrast medium, lesions position and normal tissue can not be accurately distinguished, causes lesion during operation can not be complete
Excision, then recurrence frequency is raw.Therefore, the wellability lesion diagnosis of tumour early stage, especially less than 2mm or less cancer lesions position
Effectively detection all plays key effect to raising oncotherapy effect or tumor eradication.
But conventional MRI imaging (MRI) can not detect early stage brain tumor using contrast medium.Therefore, it develops
It is a kind of can through blood-brain barrier and in conjunction with tumor tissue specificity, the novel contrast medium of early stage invasive tumor tissue can be detected
With very important clinical meaning.
Many tumours express interleukin-13 alpha-2 receptor (IL-13R α 2) in its cell surface height, brain as escribed above
Liver cancer that glioma and rectum metastasis of cancer are gone etc. all there is IL-13 α 2 highly to express.
In order to improve the targeting of gadolinium (Gd) contrast agent, researcher is highly expressed using tumor cell surface IL-13R α 2
Characteristic, allow tumor cell surface IL-13R α 2 to combine with the ligand IL-13 for being loaded with drug.As the IL- on tumour cell
After the 13R α 2 and ligand IL-13 for being loaded with drug is specifically bound, the contained carrier gadolinium of liposome is delivered to receptor
The tumour cell position that IL-13R α 2 is highly expressed, to have the function that target.
Chinese patent 201110312974.X discloses a kind of based on biotinylated mAb and Streptavidin knot
Close the targeting glioma mr contrast agent for containing two kinds of compounds of sterically stabilized immunoliposomes of gadolinium;The monoclonal antibody
Detection for Endoglin (CD105), for glioma new vessels.It is injected in two steps when the contrast medium use, that is, needs elder generation
Biotinylated mAb is given, then gives Streptavidin and combines the sterically stabilized immunoliposomes for containing gadolinium.
Chinese patent 201410252588.X discloses a kind of by aptamer analog and the liposome structure for containing gadolinium
At magnetic resonance imaging contrast agent, the aptamer analog is selected from mercapto-modified, amido modified or site and modifies
GBI-10, AS1411, A10, MUC1, sgc8, sgc3, sgd3, KH1C1, KD05, S6, AGR0100, A30 and STRG2 in
It is one or more, it is used for early detection tumour cell, wherein GBI-10 to can be used for the diagnosis of glioma.But in lipid system
It was squeezed out film up to 20 times during standby.
Chinese patent 201010286508.4 discloses one kind with polypeptide as targeting head base, and dendrimer material is base
Plinth macromolecule carrier, surface connect small molecule magnetic resonance imaging contrast agent, cancer target magnetic resonance imaging contrast agent are made, i.e., sharp
With the 7 peptide modified macromolecule material of peptide T that can specifically bind tumor cell surface Tf receptor, magnetic resonance imaging contrast agent is connected
Cancer target magnetic resonance contrast agent is made.
Chinese patent 200910196477.0 discloses the gadolinium contrast medium of a kind of nano liposomes and target polypeptide modification,
Reach tumour identification and contrast agent delays, target polypeptide modification is selected from GD10 peptide molecule, F56 polypeptide, K237 peptide molecule.Institute
For the contrast medium partial size stated in 20~200nm, size distribution is wide.
Chinese patent 201080026113.9 discloses a kind of pulullan polysaccharide and DTPA (diethylene-triamine pentaacetic acid two
Acid anhydride) in the pulullan polysaccharide-DTPA conjugant that is formed by ester bond gadolinium coordination be incorporated into the gadolinium contrast agent that DTPA formed, be used for
Liver MRI image.
Summary of the invention
In order to improve detection accuracy, the above problem in the prior art is solved, the present invention provides a kind of IL-13 modifications
Liposome containing gadolinium chelate compound targets magnetic resonance imaging contrast agent.
The liposome containing gadolinium chelate compound of IL-13 modification provided by the invention targets magnetic resonance imaging contrast agent, to encapsulate gadolinium
The liposome of chelate, and surface of liposome is modified by IL-13.
Interleukins IL-13 has modified surface of liposome, and liposome encloses gadolinium chelate compound.Interleukins
IL-13 has height homogenieity, and the interleukin-13 R alpha-2 receptor that can be over-expressed with lesion is specifically bound.
For example, there are about 16000~22000 IL-13R alpha-2 receptor knots for each malignant brain tumor cell for malignant brain tumor
Coincidence point, therefore the magnetic resonance imaging contrast agent has very strong parent to the cancer cell of height expression interleukin-13 R alpha-2 receptor
And power.
Preferably, in the targeting magnetic resonance imaging contrast agent of liposome containing gadolinium chelate compound of above-mentioned IL-13 modification, the gadolinium chela
Closing object is Magnevist Solution or gadoterlc acid meglumine saltlniection.
Magnevist Solution (Gd-DATP) also known as the double meglumines of diethylene triamine pentaacetic acid gadolinium, are presently the most and are widely used
Contrast medium.Gadoterlc acid meglumine saltlniection (Gd-DOTA) is also more commonly used gadolinium contrast medium, is equally applicable to the present invention.
Preferably, in the targeting magnetic resonance imaging contrast agent of liposome containing gadolinium chelate compound of above-mentioned IL-13 modification, the lipid
The raw material for preparing of body includes dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylethanolamine-polyethylene glycol
(DSPE-PEG), cholesterol (CHOL) and distearoylphosphatidylethanolamine-polyethylene glycol-maleimide cross-linking agent
(DSPE-PEG-Mal).Multilamelar liposome can be made in these types of material mixing, can effectively encapsulate gadolinium chelate compound, and pass through
Blood-brain barrier reaches lesion.
Preferably, in the targeting magnetic resonance imaging contrast agent of liposome containing gadolinium chelate compound of above-mentioned IL-13 modification, two palmityls
Phosphatidyl choline, distearoylphosphatidylethanolamine-polyethylene glycol, cholesterol and distearoylphosphatidylethanolamine-are poly-
Ethylene glycol-maleimide cross-linking agent molar ratio is 10:5:5:0.5.The liposome of the material preparation of this consumption proportion, packet
Envelope rate height, high yield rate, and be used to inhibit brain colloid carcinoma curative effect ideal.
The present invention also provides the liposome containing gadolinium chelate compound of more than one any IL-13 modifications target magnetic resonance at
As the preparation method of contrast medium, steps are as follows:
(1) dipalmitoylphosphatidylcholine, distearoylphosphatidylethanolamine-polyethylene glycol, cholesterol and two are hard
Fatty acyl group phosphatidyl-ethanolamine-polyethylene glycol-maleimide cross-linking agent is dissolved in the methanol chloroform mixed solvent of volume ratio 1:1
In, solvent forms film by volatilization under 40 DEG C of rotatory vacuum reduced pressures, film dry 10~20min in nitrogen, then
It is mothballed 14~16h in 4 DEG C of driers;Gadolinium chelate compound is added into the container containing liposome membrane, in ultrasonic wave
Under assistance, liposome membrane wraps up gadolinium chelate compound to form the multilamelar liposome containing gadolinium chelate compound;The multilamelar liposome is -80
By freeze thawing four times under the conditions of DEG C, polycarbonate membrane four times of aperture 200nm are then extruded through by nitrogen, then worn by nitrogen extruding
Polycarbonate membrane four times for crossing aperture 100nm;
(2) IL-13 is dissolved in the phosphate buffer containing EDTA, the 2- imido hydrogen chlorine of final concentration 1.4mM is added
Change mercaptan aqueous solution, then stirring keeps IL-13 Thiolation under the conditions of 4 DEG C;G25 will be passed through by Thiolation IL-13 solution
Cross-linked dextran gel column is purified, and purified product is concentrated to 1mL, then is added into step (1) products therefrom,
12h is stirred under the conditions of 4 DEG C, obtains the targeting magnetic resonance imaging contrast agent of liposome containing gadolinium chelate compound of IL-13 modification.
That the Thiolation process of IL-13 protein generates is Thiolation IL-13 and not by the Thiolation IL- freely to dissociate
13 mixture does not remove free not Thiolation IL-13, and crossing G25 cross-linked dextran gel column is essential operation.
Preferably, the preparation method of the targeting magnetic resonance imaging contrast agent of liposome containing gadolinium chelate compound of above-mentioned IL-13 modification,
Further include purification step (3): the liposome containing gadolinium chelate compound for the IL-13 modification that step (2) obtains targets magnetic resonance imaging contrast
Agent crosses agarose CL-2B pillar first to remove unbonded Thiolation IL-13, then dialyses in 4 DEG C of phosphate buffers
To exchange solvent;A phosphate buffer is changed after dialysis 4h, total dialysis time is 16~20h;Then, under the conditions of 4 DEG C from
The heart, which is concentrated, targets magnetic resonance imaging contrast agent to get the IL-13 of the purifying liposome containing gadolinium chelate compound modified.
The targeting magnetic resonance imaging contrast agent of liposome containing gadolinium chelate compound of the above-mentioned IL-13 modification of the present invention is in detection tumour
In application, 2 expression quantity of IL-13 α of the tumour is higher than Healthy People normal value (such as glioma, pernicious intestinal cancer transfer, kidney
Upper gland cortical carcinoma).
Preferably, the tumour is glioma, intestinal cancer and/or adrenocortical carcinoma.
Compared with prior art, the invention has the following advantages:
The targeting magnetic resonance imaging contrast agent of liposome containing gadolinium chelate compound of 1.IL-13 modification can assist contrast medium to pass through blood
Brain barrier;
The nuclear magnetic resonance image that the targeting magnetic resonance imaging contrast agent of liposome containing gadolinium chelate compound of 2.IL-13 modification can will contain
Contrast medium is transmitted to lesion and generates specific binding with cancer cell, has targeting;
The targeting magnetic resonance imaging contrast agent of liposome containing gadolinium chelate compound of 3.IL-13 modification can detecte swollen less than 2mm
Tumor lesion can be used for the early detection of tumour.
Detailed description of the invention
Fig. 1 is IL-13-Liposomes-Gd-DTPA stereoscan photograph.
Fig. 2 is that nude mice is injected intravenously IL-13-Liposomes-Gd-DTPA MRI detection effect.
Fig. 3 is that nude mice is injected intravenously Gd-DTPA MRI detection effect.
Fig. 4 is that nude mice is injected intravenously IL-13-Liposomes-Gd-DTPA Brain slices MRI detection effect.
Fig. 5 is that nude mice is injected intravenously Gd-DTPA Brain slices MRI detection effect.
Specific embodiment
The present invention is further explained in the light of specific embodiments, manages those skilled in the art preferably
The solution present invention can be simultaneously practiced, but illustrated embodiment is not as a limitation of the invention.
The preparation of 1 IL-13-liposomes-Gd-DTPA of embodiment.
By dipalmitoylphosphatidylcholine 80mg, distearoylphosphatidylethanolamine-polyethylene glycol 13mg, cholesterol
20mg and distearoylphosphatidylethanolamine-polyethylene glycol-maleimide cross-linking agent 10mg (molar ratio 10:5:5:
0.5) 40mL methanol chloroform (the two volume ratio is 1:1) in the mixed solvent, solvent under 40 DEG C of rotatory vacuum reduced pressures are dissolved in
Film is formed by volatilization, then film dry 10~20min in nitrogen is mothballed 14~16h in 4 DEG C of driers.Gadolinium
The Portugal Pen Suan amine is added into the container containing liposome membrane, under the assistance of ultrasonic wave, liposome membrane by magnetic resonance at
As contrast medium is wrapped up to form the multilamelar liposome containing magnetic resonance imaging contrast agent.The liposome is under the conditions of -80 DEG C by freeze thawing
Four times, polycarbonate membrane four times of aperture 200nm are then extruded through by nitrogen, then be extruded through aperture 100nm's by nitrogen
Polycarbonate membrane four times.
IL-13 protein 2mg is dissolved in the phosphate-buffered that 6mL contains 0.67mM disodium ethylene diamine tetraacetate (EDTA)
In liquid, the 2 Iminothiolane hydrochloride aqueous solution that ultimate density is 1.4mM is added, then stirs 30min under the conditions of 4 DEG C,
Keep IL-13 protein Thiolation.IL-13 protein after Thiolation is by G-25 cross-linked dextran gel column to be isolated from by swimming
From IL-13, obtain bright sulfur alcoholization IL-13, bright sulfur alcoholization IL-13 eluent be concentrated into 1mL.Thiolation IL-13 is added
Enter in the liposome containing Magnevist Solution, stir 12h under the conditions of 4 DEG C, obtains liposomes vs' agent of IL-13 modification.
Liposomes vs' agent of prepared IL-13 modification crosses agarose CL-2B pillar first to remove unbonded mercaptan
Then the IL-13 of change dialyses in 4 DEG C of phosphate buffers to exchange solvent.A phosphate buffer is changed after dialysis 4h, always
Dialysis time is 16~20h.Then, IL-13-liposomes-Gd- is named as to 8.0mL in 4 DEG C of centrifugal concentratings
DTPA。
Prepared IL-13-liposomes-Gd-DTPA is spherical in shape, and size dimension is 80~140nm, and average-size is
131nm, liposome polydispersity index (PDI) are 0.1~0.2nm, see Fig. 1.
2 IL-13-liposomes-Gd-DTPA of embodiment may pass through blood-brain barrier
Magnetic resonance imaging contrast agent IL-13- is targeted for the IL-13 of the proof liposome containing gadolinium chelate compound modified
Liposomes-Gd-DTPA may pass through blood-brain barrier, and it is that model is tested that we, which select normal nude mice without the cancer of the brain,.Knot
Fruit shows that after magnevist contrast medium (Magnevist) Gd-DTPA 30min is given in the nude mice injection of the no cancer of the brain, nude mice brain hangs down
Body signal noise ratio (signal-to-noise ratio) improves 25%, on the contrary, brain parenchym signal-to-noise ratio but remains unchanged.Show magnevist contrast medium
The blood-brain barrier of nude mice living can not be passed through to reach brain parenchym region.In contrast, IL-13- is given in the nude mice injection of the no cancer of the brain
After liposomes-Gd-DTPA 30min, not only the signal-to-noise ratio at nude mice pituitary position increases 20%, and at the same time, brain is real
Matter signal-to-noise ratio also increases 15%.This is because IL-13-liposomes-Gd-DTPA may pass through living body nude mouse blood brain
Barrier, and penetrate into brain parenchyma region.Liposome, which is proved, as pharmaceutical carrier can assist contrast medium to pass through complete blood brain
Barrier reaches brain parenchym brain tissue.
The targeting of 3 IL-13-liposomes-Gd-DTPA of embodiment
With the liposome mr contrast agent (FITC-IL-13- of fluorescein isothiocynate cyano label IL-13 modification
Liposomes-Gd-DTPA) and without IL-13 liposome contrast agent (FITC-liposomes-Gd-DTPA), so as in nerve
The effect that cell absorbs contrast medium is observed in glioma U251 and GSC T3691 cell cytosol, we have done cell absorption experiment.
Tissue of biological cells the result shows that, observe that cell absorbs more IL- in colloid cancer stem cell GSC T3691 endochylema
The FITC-IL-13-liposomes-Gd-DTPA of 13 modifications.Its prompt the IL-13R α 2 in IL-13 and GSC T3691 cell by
Body has the compatibility of height, can farthest promote suction of the specific tumors cell to IL-13-liposomes-Gd-DTPA
It receives.
The effect of 4 IL-13-lipsomes-Gd-DTPA of embodiment detection tumour cell
To prove that immature glioma can be detected in the IL-13-liposomes-Gd-DTPA of IL-13 modification, carry out thus
Related Experimental Study, normal nude mice are vaccinated glioma U251 cell, and after four weeks, nude mice intracerebral grows mature and early stage
Glioma, in this, as intracerebral glioma animal model.It gives containing the injection magnevist comparison of intracerebral glioma nude mice
Agent (Magnevist) Gd-DTPA obtains image by weighting 7T Nuclear Magnetic Resonance, sees Fig. 3.Give same nude mice note afterwards for 24 hours
IL-13-liposomes-Gd-DTPA is penetrated, image is obtained by weighting 7T Nuclear Magnetic Resonance, sees Fig. 2.The results show that IL-13-
Liposomes-Gd-DTPA can detecte other than nude mice maturity period lesion, and the wellability glioma of minimal disease can also be shown
It shows and, and magnevist contrast medium then only shows the tumour of brain position maturation, because blood-brain barrier has gone to pot at this time,
Therefore mr contrast agent can enter brain tissue to show the brain tissue by lesion destruction rather than tumor cell tissue itself.
In order to further determine testing result, to intravenous injection Gd-DTPA (Magnevist) and IL-13-liposomes-
Nude mice after Gd-DTPA carries out brain deeper time slice detection.After profound level slice is the results show that be injected intravenously Gd-DTPA,
Nude mice minimal disease can not be shown, see Fig. 5;It is injected intravenously the same brain position of nude mice of IL-13-liposomes-Gd-DTPA
The immature cancer of the brain of wellability is clearly shown, sees Fig. 4.Therefore, highly sensitive IL-13-liposomes-Gd-DTPA contrast medium is used
It is most important in the early detection of tumour, early treatment.
Embodiment described above is only to absolutely prove preferred embodiment that is of the invention and being lifted, protection model of the invention
It encloses without being limited thereto.Those skilled in the art's made equivalent substitute or transformation on the basis of the present invention, in the present invention
Protection scope within.Protection scope of the present invention is subject to claims.