CN103638532A - Gadolinium oxide targeted magnetic resonance contrast agent - Google Patents

Gadolinium oxide targeted magnetic resonance contrast agent Download PDF

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CN103638532A
CN103638532A CN201310625257.1A CN201310625257A CN103638532A CN 103638532 A CN103638532 A CN 103638532A CN 201310625257 A CN201310625257 A CN 201310625257A CN 103638532 A CN103638532 A CN 103638532A
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peg
contrast agent
oleic acid
nanoparticle
magnetic resonance
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CN103638532B (en
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徐群渊
叶玲
王昊
李帅
肖宁
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Capital Medical University
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Abstract

The invention relates to a gadolinium oxide targeted magnetic resonance contrast agent, which is polyethylene glycolized Gd2O3 modified by folic acid, expressed as FA-PEG-Gd2O3 and shaped like near-spheres under a transmission electron microscope. A preparation method comprises the following steps: preparing gadolinium oxide nano-particles coated by oleic acid through a high-temperature reduction method, treating gadolinium acetylacetonate in an organic phase to ensure homogeneity and high crystallinity, and performing ligand exchange with silane carboxylic acid to obtain the gadolinium oxide nano-particles with high water solubility. Then, a large number of carboxyl groups of silane are further coupled with NH2-PEG-COOH and NH2-PEG-FA to obtain the gadolinium oxide targeted magnetic resonance contrast agent which is used for early diagnosis of brain glioma.

Description

A kind of Gadolinia. targeting magnetic resonance contrast agent
Technical field
The present invention relates to a kind of Gadolinia. targeting magnetic resonance contrast agent.
The invention still further relates to the preparation method of above-mentioned Gadolinia. targeting magnetic resonance contrast agent.
The invention still further relates to the application of above-mentioned Gadolinia. targeting magnetic resonance contrast agent in cerebral glioma early diagnosis.
Background technology
Glioma is human brain major disease, is the modal primary tumor of central nervous system, and therapeutic modality be take therapeutic alliance as main, but poor prognosis, survival rate is low.This is because early diagnosis and the boundary definition of its minimal disease is global problem always, and glioma has wettability growth, and tumor body and normal surrounding tissue boundary are unclear, the difficult feature through the blood brain barrier in brain (BBB) etc. of contrast agent.
Nuclear magnetic resonance (MRI) is a kind of non-invasive diagnostic method, for assessing the function of human anatomic structure, tissue or organ on clinical medicine.MRI technology has its unique advantage, as high-resolution, "dead" radiation, long-acting imaging etc.But while being used for monitoring the focus of microtissue, sensitivity is not high.By adopting paramagnetism micromolecule contrast agent Gd-DTPA or Gd-DOTA etc. can help to improve sensitivity and the contrast of imaging.This is because micromolecule contrast agent can shorten the longitudinal relaxation time (T1) of water proton, and the brightness of image of T1 weighted imaging is improved.Several micromolecule MRI such as Gd-DTPA and Gd-DOTA contrast agent has obtained the approval of U.S. food and drug administration, and is widely used in the diagnosis of clinical tumor.Yet these micromolecule contrast agent exist serious problem, short such as blood halflife, relaxation rate is low, organ or tissue is lacked to targeting specific etc.Therefore cannot diagnose the infantile tumour of minimal disease, this has just limited their application.In order to overcome above-mentioned shortcoming, MRI contrast agent can carry out early diagnosis to glioma, and research worker has made great efforts to develop nanoparticle containing Gd as new compound contrast agent always.Nanoparticle contrast agent more easily enters tumor by EPR effect, and the functional group of further modification can be provided; Meanwhile, gadolinium-contained nano grain has lower toxicity than gadolinium chelate compound, thereby has vast potential for future development.NaGdF 4, Gd 2o 3and GdCO 3deng gadolinium-contained nano grain, be in the news tumor imaging has been had to better effect.In these nanoparticles, with Gd 2o 3for the nanoparticle of core is because of the highest extensive concern that is subject to of its relaxation rate.Gadolinio nanoparticle MRI contrast agent demonstrates unprecedented advantage, as the raising of the enhancing of contrast, sensitivity, the prolongation of diagnosing image time etc.Although gadolinio nanoparticle contrast agent has increased the absorption of tumor region by EPR effect, it is limited that this passive target causes the absorption of nanoparticle.Another method of strengthen taking in is by targeting group is linked on contrast agent as folic acid, antibody or small-molecular peptides, the contrast agent of this targeting can be identified the receptor on tumor tissues surface specifically, thereby increase nanoparticle in the absorption of tumor region, and be increased in the time of staying of tumor, be specially adapted to the diagnosis of minimal disease and tumor boundaries.But the Gd that up to now, not yet has bibliographical information to modify with target body 2o 3nanoparticle is for the radiography research of cerebral glioma.
Summary of the invention
The object of the present invention is to provide a kind of Gadolinia. targeting magnetic resonance contrast agent.
Another object of the present invention is to provide a kind of method of preparing above-mentioned Gadolinia. targeting magnetic resonance contrast agent.
For achieving the above object, Gadolinia. targeting magnetic resonance contrast agent provided by the invention, is the Gd of the Pegylation of modified with folic acid 2o 3, be expressed as FA-PEG-Gd 2o 3, under transmission electron microscope, be shown as subglobose granular;
By following method, obtain:
1) prepare the Gd of oleic acid parcel 2o 3:
Oleic acid and oleyl amine are added in benzyl oxide after being dissolved in acetylacetone,2,4-pentanedione gadolinium on 1: 1 ground in mass ratio, under 80-100 ℃ of vacuum state, stir and remove moisture and air, be then filled with nitrogen, solution is heated to 260-290 ℃; Reaction finishes after cool to room temperature, and by ethanol precipitation, centrifugal rear collecting precipitation obtains the Gd of oleic acid parcel 2o 3, be distributed to normal hexane stand-by;
2) prepare soluble silane carboxylic acid-Gd 2o 3nanoparticle:
The Gd of oleic acid parcel 2o 3join in toluene ultrasonic after mixing with acetic acid, add N-(trimethoxy silicon propyl group) ethylenediamine triacetic acid sodium-salt aqueous solution to stir at 50-70 ℃, collect and washing precipitation, precipitation is dispersed in water, with the dialysis of fiber bag filter, lyophilization obtains soluble silane carboxylic acid-Gd 2o 3nanoparticle, is expressed as TETT-Gd 2o 3powder;
3) prepare FA-PEG-Gd 2o 3:
By TETT-Gd 2o 3be distributed in water, add the activation of N-hydroxy-succinamide and 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides, add NH 2-PEG-FA, the pH value 7-8 of regulator solution, adds NH after stirring at room 2-PEG-COOH is with saturated remaining carboxyl, stirring at room, and reactant liquor is removed unnecessary reactant with fiber bag filter, obtains light green FA-PEG-Gd after lyophilization 2o 3powder.
Described Gadolinia. targeting magnetic resonance contrast agent, wherein, FA-PEG-Gd 2o 3under transmission electron microscope, be shown as the nanoparticle of subsphaeroidal 6nm, hydration particle diameter 130nm.
The method of the above-mentioned Gadolinia. targeting of preparation of the present invention magnetic resonance contrast agent:
1) prepare the Gd of oleic acid parcel 2o 3:
Oleic acid and oleyl amine are added in benzyl oxide after being dissolved in acetylacetone,2,4-pentanedione gadolinium on 1: 1 ground in mass ratio, under 80-100 ℃ of vacuum state, stir and remove moisture and air, be then filled with nitrogen, solution is heated to 260-290 ℃; Reaction finishes after cool to room temperature, and by ethanol precipitation, centrifugal rear collecting precipitation obtains the Gd of oleic acid parcel 2o 3, be distributed to normal hexane stand-by;
2) prepare soluble silane carboxylic acid-Gd 2o 3nanoparticle:
The Gd of oleic acid parcel 2o 3join in toluene ultrasonic after mixing with acetic acid, add N-(trimethoxy silicon propyl group) ethylenediamine triacetic acid sodium-salt aqueous solution to stir at 50-70 ℃, collect and washing precipitation, precipitation is dispersed in water, with the dialysis of fiber bag filter, lyophilization obtains soluble silane carboxylic acid-Gd 2o 3nanoparticle, is expressed as TETT-Gd 2o 3powder;
3) prepare FA-PEG-Gd 2o 3:
By TETT-Gd 2o 3be distributed in water, add the activation of N-hydroxy-succinamide and 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides, add NH 2-PEG-FA, the pH value 7-8 of regulator solution, adds NH after stirring at room 2-PEG-COOH is with saturated remaining carboxyl, stirring at room, and reactant liquor is removed unnecessary reactant with fiber bag filter, obtains FA-PEG-Gd after lyophilization 2o 3powder.
Described method, wherein, in step 1, the additional proportion of each reagent is: the mass ratio of oleic acid and oleyl amine 1: 1, acetylacetone,2,4-pentanedione gadolinium is 2mmol, benzyl oxide is 20mL.
Described method, wherein, in step 2, the additional proportion of each reagent is: the Gd of oleic acid parcel 2o 3for 100mg, acetic acid is 60 μ L, and toluene is 60mL, and N-(trimethoxy silicon propyl group) ethylenediamine triacetic acid sodium-salt aqueous solution is 1.2mL, and dialysis adopts the fiber bag filter that interception is 1000.
Described method, wherein, in step 3, the additional proportion of each reagent is: TETT-Gd 2o 3for 100mg is distributed in 10mL water, N-hydroxy-succinamide is 29mg, and 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides is 23mg, NH 2-PEG-FA is 150mg, NH 2-PEG-COOH is 60mg, and dialysis adopts the fiber bag filter that interception is 3500.
Gadolinia. targeting magnetic resonance contrast agent of the present invention, determines for contrast enhancing and the border of small glioma.PEG is used for increasing circulation time and the water dispersible of nanoparticle in blood.The targeted contrast agent FA-PEG-Gd obtaining 2o 3nanoparticle, by projection Electronic Speculum (TEM), the infrared conversion spectrum of Fourier (FTIR), X-ray diffraction spectrum (XRD) and ICP-AES (ICP-AES) characterize.Result shows, Gd 2o 3the particle diameter of core, between 2 to 4nm, and has highly crystalline.Targeted contrast agent FA-PEG-Gd 2o 3there is height water dispersible and stability.The FA-PEG-Gd of preparation 2o 3relaxivity is 4.48, higher than the Gd ion chelate complex using clinically.Contrast agent toxicity evaluation in vivo and in vitro shows FA-PEG-Gd 2o 3nanoparticle has good bio-compatible.Show FA-PEG-Gd 2o 3the targeted contrast agent that can be used for cerebral glioma MRI.
Accompanying drawing explanation
Fig. 1 is (A) OA-Gd in the present invention 2o 3, (B) TETT-Gd 2o 3, (C) PEG-Gd 2o 3(D) FA-PEG-Gd 2o 3the TEM image of nanoparticle.Illustration is OA-Gd 2o 3the HRTEM of nanoparticle.
Fig. 2 is after synthesizing in the present invention and is heated to 600 ℃ of nanoparticle dry powder OA-Gd after processing 2o 3xRD (X-ray diffracting spectrum)
Fig. 3 is Gadolinia. targeting magnetic resonance contrast agent Fourier infrared (FTIR) spectrogram in the present invention.
Fig. 4 is Gadolinia. targeting magnetic resonance contrast agent relaxation rate r1 and Gd-DTPA in the present invention, PEG-Gd 2o 3and FA-PEG-Gd 2o 3t1 weighting picture.
Fig. 5 is Gadolinia. targeting magnetic resonance contrast agent tail vein injection 15 minutes in the present invention, after 120 minutes and 24 hours, and the nuclear-magnetism image of day part C6 Glioma Model Mus.
Fig. 6 is that in the present invention, Gadolinia. targeting magnetic resonance contrast agent and C6 glioma cell are cultivated after 24 hours altogether, and CCK-8 method is determined C6 glioma cell survival rate.
Fig. 7 is Gadolinia. targeting magnetic resonance contrast agent tail vein injection nanoparticle after 30 days in the present invention, cores respectively, liver, spleen, lung, kidney and brain.The row HE tissue slice that dyes.Be divided into PEG-Gd 2o 3and FA-PEG-Gd 2o 3nanoparticle group.
The specific embodiment
Below in conjunction with accompanying drawing, the present invention is described in further detail.The abbreviation below occurring in explanation is respectively:
EDC (1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride) 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides;
NHS (N-hydroxysuccinimide) N-hydroxy-succinamide;
FA (Folic acid) folic acid;
HE (Haematoxylin Eosin) haematoxylin-Yihong;
The Eagle/F12 culture medium of DMEM/F12 (Dulbeco ' s modified agle ' s medium/F12) improvement;
CCK-8kit (Cell counting kit-8) CCK-8 cell proliferation/toxicity detection kit;
XRD (X-ray diffraction) X-ray diffraction analysis;
T1WI (T1 weight image) T1 weighted imaging;
PEG (Polyethylene glycol) Polyethylene Glycol;
ICP-AES (Inductively coupled plasma atomic emission spectroscopy) ICP-AES;
OA (Oleic acid) oleic acid;
TETT (N-(trimethoxysilypropyl) ethylene diamine triacetic acid, trisodium salt (45%in water)) N-(trimethoxy silicon propyl group) ethylenediamine triacetic acid sodium-salt aqueous solution;
EPR (Enhanced permeability and retention effect) strengthens permeability and reserve effects;
HRTEM (High Resolution Transmission Electron Microscopy) high resolution transmission electron microscope;
MRI (Magnetic Resonance Imaging) nuclear magnetic resonance.
First the present invention adopts folic acid covalent coupling to Gadolinia. magnetic resonance contrast agent, first, the gadolinium sesquioxide nanoparticle of oleic acid parcel is standby by high temperature reduction legal system, acetylacetone,2,4-pentanedione gadolinium is processed and is guaranteed its homogeneity and high-crystallinity in organic facies, by carrying out ligand exchange with silane carboxylic acid, obtain high water soluble gadolinium sesquioxide nanoparticle.Then a large amount of carboxyls of silane further and NH 2-PEG-COOH (PolyethyleneGlycol) and NH 2-PEG-FA (folacin coupled PEG) coupling, obtains the MRI targeted contrast agent of folacin coupled PEGization.
Particularly, Gadolinia. targeting magnetic resonance contrast agent of the present invention, can obtain by following method:
1) prepare the Gd of oleic acid parcel 2o 3:
The oleic acid of 1.6g, the oleyl amine of 1.6g and 2mmol acetylacetone,2,4-pentanedione gadolinium add in 20mL benzyl oxide, extracting air then, and stir 1h under 100 ℃ of vacuum states, to remove moisture and air.Then, be filled with nitrogen, solution is heated to 290 ℃ and maintain 2.5h.Reaction finishes after cool to room temperature, the Gd of oleic acid parcel 2o 3with 20mL ethanol precipitation, centrifugal rear collecting precipitation, precipitation washing with alcohol three times, are distributed to 10mL normal hexane stand-by.
2) prepare water solublity TETT-Gd 2o 3nanoparticle:
The Gd of 100mg oleic acid parcel 2o 3join 60mL toluene with 60 μ L acetic acid, mix latter ultrasonic 30 minutes, then add the TETT of 1.2mL, at 70 ℃, mixture stirs 48h.During this period, in mixture, there is precipitation.Be precipitated as TETT-Gd 2o 3, after collecting, by toluene and methanol wash, carry out purification three times.Precipitation after purification is dispersed in water, and uses the fiber bag filter that interception is 1000, deionized water dialysis 24h.Lyophilization obtains white TETT-Gd 2o 3powder.
3) prepare FA-PEG-Gd 2o 3powder:
By the TETT-Gd of 100mg 2o 3be distributed in 10mL deionized water, add the NHS of 29mg and the EDC of 23mg to activate half an hour, then add the NH of 60mg 2-PEG-FA, the pH value to 8.0 of regulator solution, stirring at room, after 15 minutes, then adds the NH of 150mg 2-PEG-COOH is with saturated remaining carboxyl, stirring at room 24h.Reactant liquor is used the fiber bag filter that interception is 3500, and deionized water dialysis 24h removes unnecessary reactant.After lyophilization, obtain light green FA-PEG-Gd 2o 3powder.
In Gadolinia. targeting magnetic resonance contrast agent by above-mentioned preparation, under FA-PEG-Gd2O3 transmission electron microscope, be shown as the nanoparticle of subsphaeroidal 6nm left and right, hydration particle diameter 130nm.
Below in conjunction with the drawings and specific embodiments, the present invention is elaborated.
Embodiment 1
1) Gd of oleic acid parcel 2o 3synthetic
The Gd of oleic acid parcel 2o 3synthetic standby by known high temperature reduction legal system, its method has been done a little change.The oleic acid of 1.6g, the oleyl amine of 1.6g and 2mmol acetylacetone,2,4-pentanedione gadolinium add in 20mL benzyl oxide, extracting air then, and stir 1h under 100 ℃ of vacuum states, to remove moisture and air.Then, be filled with nitrogen, solution is heated to 290 ℃ and maintain 2.5h.Reaction finishes after cool to room temperature, the Gd of oleic acid parcel 2o 3with 20mL ethanol precipitation, centrifugal rear collecting precipitation, precipitation washing with alcohol three times, are distributed to 10mL normal hexane stand-by.
2) water solublity TETT-Gd 2o 3the preparation of nanoparticle
Water solublity TETT-Gd 2o 3nanoparticle is standby by ligand exchange legal system.The Gd of 100mg oleic acid parcel 2o 3join 60mL toluene with 60 μ L acetic acid, mix latter ultrasonic 30 minutes, then add the TETT of 1.2mL, at 70 ℃ of mixture, stir 48h.During this period, in mixture, there is precipitation.Be precipitated as TETT-Gd 2o 3, after collecting, by toluene and methanol wash, carry out purification three times.Precipitation after purification is dispersed in water, and uses the fiber bag filter that interception is 1000, deionized water dialysis 24h.Lyophilization obtains white TETT-Gd 2o 3powder.
3) PEGization Gd 2o 3synthetic
By the TETT-Gd of 100mg 2o 3be distributed in 10mL deionized water, add the NHS of 29mg and the EDC of 23mg to activate half an hour, add the NH of 210mg 2-PEG-COOH, the pH value to 8.0 of regulator solution, stirring at room 24h.Reactant liquor is used the fiber bag filter that interception is 3500, and deionized water dialysis 24h removes unnecessary reactant.Lyophilization obtains white PEG-Gd 2o 3powder is as experiment contrast sample.
3) PEG-Gd that FA modifies 2o 3synthetic
Another by the TETT-Gd of 100mg 2o 3be distributed in 10mL deionized water, add the NHS of 29mg and the EDC of 23mg to activate half an hour, then add the NH of 60mg 2-PEG-FA, the pH value to 8.0 of regulator solution, stirring at room, after 15 minutes, then adds the NH of 150mg 2-PEG-COOH is with saturated remaining carboxyl, stirring at room 24h.Reactant liquor is used the fiber bag filter that interception is 3500, and deionized water dialysis 24h removes unnecessary reactant.Lyophilization obtains light green FA-PEG-Gd 2o 3powder.
Gadolinia. targeting magnetic resonance contrast agent transmission electron microscope for cerebral glioma early diagnosis prepared by the present invention, X-ray diffraction, the sign that Fourier is infrared is shown in Fig. 1,2,3.
As can be seen from Figure 1,, by acetylacetone,2,4-pentanedione gadolinium 290 ℃ of pyrolytics in benzyl oxide, obtain the Gd of oleic acid parcel 2o 3(OA-Gd 2o 3), its nanoparticle physicochemical property comprises that size, shape and surface nature can be controlled and can repeat and generate to obtain.High resolution transmission electron microscope (HRTEM) image demonstrates just synthetic OA-Gd 2o 3the form of nanoparticle is subsphaeroidal, and its size diameter estimates average 2 to 3nm, and size distribution is even.Clear and definite lattice fringe has further confirmed OA-Gd 2o 3the high-crystallinity of nanoparticle.In addition, the range measurement between lattice fringe edge is 0.33nm, corresponding to (222) lattice plane Gd 2o 3emission in Cubic.High degree of dispersion water solublity Gd 2o 3nanoparticle replaces oleic acid by ligand exchange method and carboxylated silane obtains.Then, NH 2-PEG2000-FA and NH 2-PEG2000-COOH is successively connected to TETT-Gd 2o 3upper, by EDC/NHS coupling technology, produce respectively PEG-Gd 2o 3and FA-PEG-Gd 2o 3nanoparticle.PEG further helps to increase nanoparticle water dispersible, and the while is as the isolation area of glioma targeting part folic acid.Although TETT-Gd 2o 3, PEG-Gd 2o 3and FA-PEG-Gd 2o 3nanoparticle compares OA-Gd 2o 3demonstrate larger particle degree, but these nanoparticles are high degree of dispersion, even at high concentration (20mg.mL -1) in solution, in aqueous solution, keep stable at least 2 months.
As can be seen from Figure 4, OA-Gd 2o 3nanoparticle is 600 ℃ of heating after 3 hours, and XRD detects its crystal formation.All diffraction maximums are blocked 11-0604 and cube Gd according to joint committee to powder diffraction standard (JCPDS) 2o 3crystalline phase coupling.
As can be seen from Figure 3, FA and Gd 2o 3success coupling.
Embodiment 2
The relaxation rate test result of nanoparticle
Gained PEG-Gd will be prepared in embodiment 1 2o 3, FA-PEG-Gd 2o 3on 7TMRI to FA-PEG-Gd 2o 3nanoparticle is evaluated as the performance of a potential magnetic resonance contrast agent.Use commercially available contrast agent magnevist (Gd-DTPA) in contrast.Relaxation rate (r1) is taken from 1/T1 relaxation time (s -1) and Gd ion concentration (mM) slope of a curve.As shown in Figure 4, and the value of Gd-DTPA nanoparticle r1 be estimated as respectively 8.33mM -1s -1, 4.48mM -1s -1and 4.01mM -1s -1.Obviously, PEG-Gd 2o 3, FA-PEG-Gd 2o 3than Gd-DTPA, demonstrate compared with high relaxation rate r1.
Embodiment 3
The in-vivo imaging test of nanoparticle
Gained PEG-Gd will be prepared in embodiment 1 2o 3, FA-PEG-Gd 2o 3nanoparticle is done MRI research as opaque contrast medium to glioma brain tumour.As Fig. 5 shows intravenous injection PEG-Gd 2o 3and FA-PEG-Gd 2o 3nanoparticle records Mus glioma T1 weighting picture at 7T nuclear-magnetism, is respectively nuclear magnetic resonance image record before and 15 minutes, after 120 minutes and 24 hours.The compound of each tail vein injection includes promising isodose Gd ion (0.1mM.Kg -1).This PEG-Gd 2o 3nanoparticle is compared before contrast-medium injection, reveals slight contrast strengthen at glioma region list, and this shows that PEGization helps Gd 2o 3nanoparticle accumulates in glioma region, and reason may be due to long-time circulation time and EPR effect.For FA-PEG-Gd 2o 3, even inject after 24h and strengthen to obvious contrast at glioma regional observation.In addition, gliomatous edge is more clear, is significantly conducive to accurately gliomatous delineation in preoperative diagnosis, art.This likely adheres to its surface due to FA by receptors bind specific and selectivity and glioma cell surface, is coupled to PEG-Gd 2o 3fA can promote more the accumulation of the Donna grain of rice and be retained in tumor locus, the contrast effect that its strengthens has continued for a long time, at least 24 hours.
Embodiment 4
The assessment of nanoparticle in vitro toxicity:
1) cell culture: C6 glioma cell adopts DMEM culture medium (containing 10% hyclone, 100U/ml penicillin, 100U/ml streptomycin), contains 5%CO at 37 ℃ 2moisturizing incubator in cultivate 24 hours.
2) cytotoxicity: C6 cell is made to single cell suspension, inoculation 1 * 10 4individual cells/well, on 96 porocyte culture plates, 37 ℃, is cultivated 24h in the incubator of 5%CO2.Add the PEG-Gd containing preparation in the embodiment 1 of 10% blood serum medium preparation 2o 3, FA-PEG-Gd 2o 3nanoparticle suspension, final concentration is Gd respectively 3+concentration is 5 μ g.mL -1, 2.5 μ g.mL -1, 1.25 μ g.mL -1, every group 6 multiple hole.Under equal conditions, cell is cultivated respectively to 24h.Abandon supernatant, every Kong Jiahan CCK-8 (100 μ L.mL -1) serum-free culture based sols 100 μ L, continue to cultivate 1h.Concussion shakes up 1min, under 450nm, measures absorbance, calculates gained cells survival rate and sees Fig. 6.
From Figure 6 shows that variable concentrations contrast agent (PEG-Gd 2o 3and FA-PEG-Gd 2o 3) impact on C6 glioma cell relative survival rate.Obviously, cytotoxicity is along with Gd concentration raises and raises.Even if but under the highest Gd concentration, under two kinds of contrast agent effects, cell survival rate is still greater than 80%.But along with concentration raises, contrast agent FA-PEG-Gd 2o 3toxicity compare PEG-Gd 2o 3toxicity slightly high.In sum, PEG-Gd 2o 3and FA-PEG-Gd 2o 3) C6 cell is not produced to overt toxicity.
Embodiment 5
Nanoparticle toxicity in vivo is evaluated
12 of healthy clean level ICR white mice, body weight 20.08 ± 2g, in 6 to 8 week age, purchased from animal housing of the Capital University of Medical Sciences, sex is male, and room temperature maintains 22～25 ℃, humidity maintenance 40%～50%, and standard feed and water are all through autoclaving.12 white mice are divided into experimental group and matched group at random.Every group of each 6 mices.Experimental group: tail vein injection PEG-Gd 2o 3nanoparticle, gives Gd 3+concentration is 0.2mmol.kg -1dosage; Matched group: tail vein injection saline.For carrying out histologic analysis, two groups of mices are raised after 30 days and are dissected, organ (brain, heart, spleen, liver, lung and kidney) is according to matched group and experimental group grouping, and be immersed in 4% neutral formalin more than 24 hours, then collected organ envelope is ensconced in paraffin, the 4 μ m that cut into slices are thick, use equally H & E dyeing, tissues observed section under optical microscope.
From not observing and Gd shown in Fig. 7 2o 3relevant tissue injury and any other adverse effect of processing of nanoparticle.The organ structure of experimental mice is normal as matched group small mouse organ structure.Based on above result of study, can illustrate that nanoparticle possesses higher biocompatibility and is expected to more be applied at biomedical aspect.

Claims (7)

1. a Gadolinia. targeting magnetic resonance contrast agent is the Gd of the Pegylation of modified with folic acid 2o 3, be expressed as FA-PEG-Gd 2o 3, under transmission electron microscope, be shown as subglobose granular;
By following method, obtain:
1) prepare the Gd of oleic acid parcel 2o 3:
Oleic acid and oleyl amine are added in benzyl oxide after being dissolved in acetylacetone,2,4-pentanedione gadolinium on 1: 1 ground in mass ratio, under 80-100 ℃ of vacuum state, stir and remove moisture and air, be then filled with nitrogen, solution is heated to 260-290 ℃; Reaction finishes after cool to room temperature, and by ethanol precipitation, centrifugal rear collecting precipitation obtains the Gd of oleic acid parcel 2o 3, be distributed to normal hexane stand-by;
2) prepare soluble silane carboxylic acid-Gd 2o 3nanoparticle:
The Gd of oleic acid parcel 2o 3join in toluene ultrasonic after mixing with acetic acid, add N-(trimethoxy silicon propyl group) ethylenediamine triacetic acid sodium-salt aqueous solution to stir at 50-70 ℃, collect and washing precipitation, precipitation is dispersed in water, with the dialysis of fiber bag filter, lyophilization obtains soluble silane carboxylic acid-Gd 2o 3nanoparticle, is expressed as TETT-Gd 2o 3powder;
3) prepare FA-PEG-Gd 2o 3:
By TETT-Gd 2o 3be distributed in water, add the activation of N-hydroxy-succinamide and 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides, add NH 2-PEG-FA, the pH value 7-8 of regulator solution, adds NH after stirring at room 2-PEG-COOH is with saturated remaining carboxyl, stirring at room, and reactant liquor is removed unnecessary reactant with fiber bag filter, obtains FA-PEG-Gd after lyophilization 2o 3powder.
2. Gadolinia. targeting magnetic resonance contrast agent according to claim 1, wherein, FA-PEG-Gd 2o 3under transmission electron microscope, be shown as the nanoparticle of subsphaeroidal 6nm, hydration particle diameter 130nm.
3. the method for preparing Gadolinia. targeting magnetic resonance contrast agent described in claim 1:
1) prepare the Gd of oleic acid parcel 2o 3:
Oleic acid and oleyl amine are added in benzyl oxide after being dissolved in acetylacetone,2,4-pentanedione gadolinium on 1: 1 ground in mass ratio, under 80-100 ℃ of vacuum state, stir and remove moisture and air, be then filled with nitrogen, solution is heated to 260-290 ℃; Reaction finishes after cool to room temperature, and by ethanol precipitation, centrifugal rear collecting precipitation obtains the Gd of oleic acid parcel 2o 3, be distributed to normal hexane stand-by;
2) prepare soluble silane carboxylic acid-Gd 2o 3nanoparticle:
The Gd of oleic acid parcel 2o 3join in toluene ultrasonic after mixing with acetic acid, add N-(trimethoxy silicon propyl group) ethylenediamine triacetic acid sodium-salt aqueous solution to stir at 50-70 ℃, collect and washing precipitation, precipitation is dispersed in water, with the dialysis of fiber bag filter, lyophilization obtains soluble silane carboxylic acid-Gd 2o 3nanoparticle, is expressed as TETT-Gd 2o 3powder;
3) prepare FA-PEG-Gd 2o 3:
By TETT-Gd 2o 3be distributed in water, add the activation of N-hydroxy-succinamide and 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides, add NH 2-PEG-FA, the pH value 7-8 of regulator solution, adds NH after stirring at room 2-PEG-COOH is with saturated remaining carboxyl, stirring at room, and reactant liquor is removed unnecessary reactant with fiber bag filter, obtains FA-PEG-Gd after lyophilization 2o 3powder.
4. method according to claim 3, wherein, in step 1, the additional proportion of each reagent is: the mass ratio of oleic acid and oleyl amine 1: 1, acetylacetone,2,4-pentanedione gadolinium is 2mmol, benzyl oxide is 20mL.
5. method according to claim 3, wherein, in step 2, the additional proportion of each reagent is: the Gd of oleic acid parcel 2o 3for 100mg, acetic acid is 60 μ L, and toluene is 60mL, and N-(trimethoxy silicon propyl group) ethylenediamine triacetic acid sodium-salt aqueous solution is 1.2mL, and dialysis adopts the fiber bag filter that interception is 1000.
6. method according to claim 3, wherein, in step 3, the additional proportion of each reagent is: TETT-Gd 2o 3for 100mg is distributed in 10mL water, N-hydroxy-succinamide is 29mg, and 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides is 23mg, NH 2-PEG-FA is 150mg, NH 2-PEG-COOH is 60mg, and dialysis adopts the fiber bag filter that interception is 3500.
7. Gadolinia. targeting magnetic resonance contrast agent claimed in claim 1 is for cerebral glioma early diagnosis.
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CN105963715A (en) * 2016-06-27 2016-09-28 四川大学 Double-polypeptide modified europium doped gadolinium oxide nanorod and preparation thereof
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CN115944726A (en) * 2022-12-12 2023-04-11 无锡市南京大学锡山应用生物技术研究所 Chemically modified B 4 C nanoparticles and applications thereof

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