CN101018568A - Superparamagnetic gadolinium oxide nanoscale particles and compositions comprising such particles - Google Patents

Abstract

Superparamagnetic nanoscale particles are disclosed which are useful for providing a contrast agent with high signal intensity, high relaxivity and high intrinsic magnetism. The disclosed contrast agents will have utility and magnetic resonance imaging (MRI) and associated techniques.

Description

Superparamagnetic gadolinium oxide nanoscale granule and comprise this grains of composition

Invention field

The present invention relates to superparamagnetic gadolinium oxide nano-particle and the purposes in selectivity imaging of tissue and cell or analysis of molecules thereof.

Background of invention

The unique ability of high spatial resolution and differentiation soft tissue makes nuclear magnetic resonance (MRI) become one of most important instrument of medical image diagnosis usefulness.The existence of MRI contrast agent is by changing the relaxation time T of proton ₁And T ₂And influence image.The different hydrogen relaxation times causes the picture contrast among the MRI in different tissues.In MRI, there are two hydrogen-like relaxation time T ₁And T ₂T ₁Be called as longitudinal relaxation time and determine that magnetization turns back to poised state after the disturbance that is caused by magnetic field pulse.T ₂Be called as T2 and determine that the signal phase that causes because of the interaction between magnetic moment moves.In addition, T ₂* (" T ₂Star ") be actual T2, it also comprises the influence that produces by the magnetic field unevenness.Reduce T ₁Influence be that signal increases, and reduce T ₂Influence be that signal descends.Contrast agent can be divided into positive reagent or negative agents, and this depends on that in the presence of contrast agent signal increases still and descends.

All contrast agents influence both relaxation times, but some reagent mainly influence T ₁Perhaps T ₂The several properties of the paramagnetic element of contrast agent influences the contrast of MR image.Most important performance be magnetic moment, electronics relaxation time and in inner or outside coordination ball with the coordinate ability of water.The exchange of the rotation of paramagnetism reagent, diffusion and water also is important mechanism.As the function of sweep parameter, from the spin sequence signal echo can be expressed as:

S(TR，TE)＝ρe ^-TE/T2(1-e ^-TM/T1) (1)

Wherein ρ=spin density, TE=echo time and TR=repetition time.According to equation 1, can find out that the relaxation time influences signal on higher degree.Have competitive relation between two kinds of relaxation times, this has explained the concentration with respect to contrast agent, viewed peak value in signal.Viewed relaxation rate (1/T _i, i=1,2) be directly proportional with the concentration (C) of contrast agent:

1/T _i(observation)=1/T _i(intrinsic)+r _iC (2)

1/T wherein _i(observation) is the relaxation time in the presence of contrast agent, 1/T _i(intrinsic) is inherent organizing the relaxation time, and r _iIt is relaxation constant.

Since its magnetic property, Gd ³⁺Ionic complex (chelate) in clinical MRI usually as contrast agent.Yet the increase of the weak signal strength of this reagent is insufficient to molecular imaging.Along with to better contrast, increasing demand of the better profile of different tissues wishes to have the contrast agent that increases than large-signal intensity day by day.Being chosen to of atherosclerotic plaque or pulmonary infarction similarly is the example of novel MRI applications and has great potential early diagnosis disease widely.In MRI contrast agent of new generation, the biocompatible nano-particle with unique magnetic properties is highly interesting for exploitation.Compare with chelate, superparamagnetic nano particle has the advantageous property of molecular imaging, because for each binding site molecule point, they have higher relaxation property.Therefore, provide the new probability of cells in vivo and molecular MRI by the new method of superparamagnetic nano particle magnetic spike, referring to people such as Jaffer FA, JAMA, 2005; 293:855-862; Gillies RJ.J Cell Biochem.2002; 39:231-238; People such as Dijkhuizen RM, J Cerebral Blood Flow and Metabolism, 2003:23:1383-1402; With people such as Wickline SA, J Cellular Biochemistry, 2002; S39:90-97.Superparamagnetic Iron Oxide (SPIO) granule is developed (people such as Perez, Nature Biotch 20:816 (2002)) because of its novel diagnostic application and molecular imaging.SPIO has very high T ₂Relaxation effect, this makes them be suitable for the T of cell and interaction of molecules ₂Draw.Yet SPIO is because of causing causing the loss of signal to the artefact sensitivity.Show these artefacts with signal hole form in image, described signal hole can not be distinguished mutually with organizing hole.This artefact also can hinder describing of the interior fine structure of tissue.These are major defects of negative contrast medium.

U.S. Patent application 2004/015784 people such as () Haase discloses the granule of being made by Gadolinium monophosphate, and with the water ratio, it demonstrates the improved signal intensity of 100-200%.Yet, there is not coating (capping) method about suggested for particle size control, as if the result use this method may be difficult to obtain fully little granularity of 1-10nm, and this is that the superparamagnetism energy is required.

People such as Morawski are at Magnetic Resonance in Medicine, the another kind of granule that contains gadolinium has been discussed among the 51:480 (2004), wherein advise using clinical MRI equipment, working load that the perfluoroparaffin nano-particle of gadolinium, the isotopic picomole concentration of quantitative molecular in single cell are arranged.Yet, these granules have big relatively size (～250nm) and not demonstrate superparamagnetism.

As the high-resolution imaging technology magnetic granule imaging (MPI) has been proposed recently.This technology is directly utilized the magnetic property of contrast agent itself, but does not have to utilize the remote-effects (latter is the mechanism of the contrast agent of routine) to the proton relaxation time.MPI has the potentiality of high spatial resolution and high sensitivity simultaneously.(Nature, in June, 2005) shows the proof of MPI principle, but the application of untapped reality still.MPI will depend on and detect the magnetic granule with strong intrinsic magnetic in the future, and superparamagnetism will be required feature.

Owing to reason listed above, need have the contrast agent of high signal intensity, high relaxation and high intrinsic magnetic.The invention provides the rare earth nanometer particle of bio-compatible, superparamagnetism, described nano-particle can be used as contrast agent, to satisfy mentioned requirement.

Invention is described

The purpose of this invention is to provide supperparamagnetic particles, in nuclear magnetic resonance was used, when using in the compositions with low concentration active material, described supperparamagnetic particles allowed good contrast to improve.

The present invention also aims to improve the contrast properties of contrast agent, so that allow the imaging of molecular imaging or cell processes.

Another purpose of the present invention provides the biocompatible nano-particle that is suitable for tissue-specific ligand-labeled, so that contrast agent is accumulated at required tissue.

Aim to provide the gadolinio nano-particle prescription that satisfies mentioned requirement in the present invention described in the following part.In general, the present invention relates to the super-paramagnetism nano grade particles, it comprises that particle mean size is lower than 50nm, preferably about 0.1-50nm and the rare-earth oxide of 1-15nm more preferably from about.This granule typically can be included in or several particulate fractions in the mentioned size range.Preferred rare-earth oxide comprises the oxide of gadolinium and dysprosium.Particularly preferably be and contain Gadolinia., especially Gd ₂O ₃Granule.The mentioned granule of the present invention can further comprise the additional materials of fraction, and for example ferrous material is so that improve its characteristic.Obtain being of a size of about 0.5-15nm and median size granule in the synthetic method of the Gadolinia. nano-particle described in the following experimental section for about 4nm.Utilize the size classification, can obtain the part of narrow distribution: 1-3nm, 3-6nm, 6-9nm, 9-15nm.

When material when forming, occurs superparamagnetism by very little microstructure (about 1-15nm).The dipole of material has identical direction, and the magnetic moment of the whole crystallite of gained is calibrated with the external magnetic field.In this case, too low even temperature is lower than Curie or N é el temperature and heat energy, so that do not overcome bonding force between the adjacent atom, but heat energy is high enough to the direction of magnetization that changes whole crystallite.The most important thing is nano-scale particle of the present invention and comprise that this grains of composition demonstrates the superparamagnetism energy.Granule of the present invention preferably has the coating of bio-compatible and/or biospecific.Introduce the normally part of grain processing technique of coating, and in the following experimental section of application, several such methods have been shown.Coating plays offsets particle aggregation one-tenth than big unit and the effect of therefore losing superparamagnetism; Make granule compatible in the biotic environment of selecting; And/or can introduce certain biologic specificity.Suitable coating compounds includes, but not limited to diethylene glycol (DEG), Polyethylene Glycol, citric acid, oleic acid, juniperic acid, the amino hexadecanoic acid of 16-, cetylamine or trioctyl phosphine oxide (TOPO).More preferably, coating comprises diethylene glycol (DEG) and/or citric acid.In preferred embodiments, particulate particle mean size is about 5nm and has the coating that contains diethylene glycol (DEG).According to another specific embodiment, coating for example comprise with amido link or at interval base be connected to Polyethylene Glycol on the folic acid, thereby the granule that provides the specificity to tumor tissues to increase.The invention still further relates to the compositions that contains mentioned supperparamagnetic particles.Said composition typically is used as the contrast agent of nuclear magnetic resonance (MRI).Compositions comprises proper auxiliary agent or excipient, comprising, but being not limited to, pH regulator agent, isotonicity regulator and/or be suitable for using are for example by other reagent to the human body of parenteral administration, specific acceptor site or tissue sample.Suitable is, the concentration range of gadolinium is 0.01-500mM in this compositions, preferably about 0.01-5mM and 0.01-2.5mM more preferably from about.The concentration of reagent to be administered depends on the dosage that application-specific is desired and required to a great extent, has provided wide scope for this reason.Yet, think and utilize the present invention that concentration and the dosage that is provided can significantly descend.

The compositions that contains oxidation gadolinio supperparamagnetic particles of the present invention has the relaxation time T that reduces adjacent proton in rich proton environment ₁And/or T ₂The T that obtains to the compositions that is lower than the ionic complex by gadolinium ₁And/or T ₂The ability of numerical value.In addition, the signal intensity ratio water height at least 500% based on the contrast agent of mentioned compositions has is preferably greater than 700%, and the high signal intensity of signal intensity that obtains than the nanoscale iron oxide particles that by concentration range is 0.1mM-1.5mM is provided.Use commercially available iron-based preparation as a reference, carry out this in (mol metallic atom) relatively in identical concentration range, this will describe in more detail following.

The following description that exemplifies of the present invention shows the granule of nano-scale of the present invention and contains this grains of composition, compares with the iron complex of prior art, can provide high contrast to improve and significantly improved relaxation property.Therefore, nano-particle of the present invention and compositions thereof can be used for the high differential of gadolinium concentration ground Cellular tracking and are used for carrying out method interaction of molecules for deliberation or the cell processes of MRI (nuclear magnetic resonance).In addition, supperparamagnetic particles of the present invention allows development approach to study endovascular speckle with the compositions that comprises them, so that support the early diagnosis atherosclerosis, the cell that diagnosis thromboembolism, spike are implanted and be difficult to up to now or can not diagnosis and treatment is up to the early stage onset mechanism of generally destroying other pathologic conditions that comes true.Especially, think that the present invention helps the formation distinguishing the pathologic conditions of commitment and check selected Therapeutic Method as the aid of measuring therapeutic effect.This will improve the probability of the optimization dosage of the therapeutic agent of being used, and provide need to substitute or replenish select Therapeutic Method early stage indication.

Of the present invention in detail and exemplify explanation

Fig. 1 a-1f shows the different synthetic Gd that is spun on the silicon base ₂O ₃The wide scanning XPS spectrum of nano-particle.

Fig. 2 is the Gd that coats with DEG ₂O ₃The HREM microphotograph of nanocrystal.

Fig. 3 is the Gd that coats with oleic acid ₂O ₃The HREM microphotograph of nanocrystal and visual (222) plane.

Fig. 4 is by the synthetic Gd of burning ₂O ₃The HREM microphotograph of nanocrystal.

Fig. 5 a and 5b show for Gd of the present invention ₂O ₃Nano-particle and Gd-DTPA (Magnevist), 1/T _iRelaxation property to the diagrammatic form of gadolinium concentration.

The signal intensity of first echo of employed spin-echo sequence during Fig. 6 shows and measures from relaxation time of Fig. 5 a and 5b (TE=30ms, TR=500ms).

Fig. 7 show with 0.1,0.3,0.6 and 0.9mM Gd cultivate 8 hours monocytic T1-figure: a) Gd ₂O ₃, b) Gd-DTPA.

Fig. 8 and 9 shows for Gd of the present invention ₂O ₃Nano-particle and Resovist , 1/T _iRelaxation property to the diagrammatic form of concentration.

Figure 10 shows from Gd of the present invention ₂O ₃The signal intensity of first echo of employed spin-echo sequence in the relaxation time measurement of nano-particle and Resovist  (TE=30ms, TR=500ms).

Figure 11 shows the Gd under the concentration of water and 0.1-1.5mM Gd ₂O ₃The comparison of the signal intensity of nano-particle.

Embodiment 1

Gd with diethylene glycol (DEG) coating ₂O ₃Synthesizing of nanocrystal

By front Feldmann C. in Polyol-mediated synthesis ofnanoscale functional materials (mediation of the polyhydric alcohol of nanoscale functional material synthetic); Adv.Funct.Mater.2003; 13:101-107; People such as Bazzi R, Synthesis and luminescent properties of sub-5-nm lanthanideoxides nanoparticles (being lower than the synthetic and luminescent properties of 5nm lanthana nano-particle), Journal of Luminescence.2003; 102-103:445-450; With S  derlind, people such as F., Synthesis and characterization of Gd ₂O ₃(synthetic and sign is passed through the functionalized Gd of organic acid to nanocrystals functionalized by organic acids ₂O ₃Nanocrystal), J.Colloid Interface Sci., the polyhydric alcohol method described in the 288:140-148 (2005), synthesis of nano crystallization Gadolinia..

With Gd (NO ₃) ₃6H ₂O (2mmol), solid NaOH (2.5mmol) and deionized water (several) are dissolved in 15ml diethylene glycol ((HOCH ₂CH ₂) ₂O, DEG) in, and heat this mixture to 140 ℃.When reactant dissolved fully, elevated temperature to 180 ℃ also kept constant 4 hours, thereby obtains luteotestaceous colloid.Dilute this colloid with deionized water, the concentration of regulating Gadolinia. is to predetermined value, for example 2.5mM.By in the Pt crucible that carefully cleaned,, determine concentration by thermogravimetry 700 ℃ of following heated sample 3 hours.Confirmed the Gd that DEG coats by x-ray powder diffraction and transmission electron microscopy as the front ₂O ₃Nanocrystal is the crystal of 1-15nm to a great extent for size range.Prepare these crystal,, can obtain to have the part of narrow distribution: 1-3nm, 3-6nm, 6-9nm, 9-15nm by bonded filter/centrifugalize (from A-filter AB V  stra Fr  lunda, the VIVASPIN filter that the SE place obtains).

Embodiment 2

Gd with other reagent coating ₂O ₃Synthesizing of nanocrystal, perhaps alternate synthetic

Independently dissolve Gd (NO in the flask at two that contain 10ml DEG separately ₃) ₃6H ₂O (2mmol) and NaOH (6mmol).Mix this two kinds of solution, be heated to about 210 ℃, and under agitation under this temperature, kept 30 minutes.In this red-hot solution, be added on the oleic acid (1.6mmol in 5ml) in the DEG, thereby obtain the brown paste.In methanol, after washing and the centrifugal several, collect linen powder.Substitute oleic acid with citric acid, juniperic acid, the amino hexadecanoic acid of 16-or cetylamine respectively.In all cases, the 1.6mmol acid/amine of use in 5ml DEG.

Also can adopt the diverse method that is called combustion method suitably, prepare Gd in an identical manner ₂O ₃Nanocrystal [people such as W.Zhang, " Optical properties ofnanocrystalline Y ₂O ₃: Eu depending on its odd structure (depends on the structure that it is peculiar, nanocrystal Y ₂O ₃: the optical property of Eu) ", J.Colloid andInterface Sc., 262 (2003), 588-593].In flask, mix equal-volume (10ml) Gd (NO ₃) ₃With aminoacid glycine (0.1M separately) and be boiled to almost dry.After further heating 1 or 2 minute, (goo) spontaneous combustion of brown goo also forms white micropowder.

Embodiment 3

Characterize with the sub-spectrum of X-ray photoelectric (XPS)

In order to confirm to form and particulate binding energy, prepared appropriate Gd by research ₂O ₃Nanocrystal uses polyenergetic A1 K α photon (1486.6eV) and CLAM2 analyser, on the VG instrument, and the record XPS spectrum.The power of X-ray gun is 300W.Spectrum is with the normal direction with respect to substrate surface, based on the photoelectron of 30 ° the angles of departure.At the indoor pressure of analysis cavity is 3 * 10 ^-10Mbar and in measuring process, temperature is 297K.Use the VGX900 data analysis software, analyze the peak position.In order to clean silicon (SiO _x) substrate, at first at 80 ℃ of MilliQ water that descend with 6: 1: 1: HCl (37%): H ₂O ₂(28%) mixture washing surface 5-10 minute is then at the 80 ℃ of MilliQ water that descends with 5: 1: 1: NH ₃(25%): H ₂O ₂(28%) mixture washing surface 5-10 minute.After each washing step, with the careful rinsing silicon face of MilliQ water.Gadolinia. nano-particle (the Gd that mixing coats with diethylene glycol ₂O ₃-DEG) with alkaline MilliQ water, and under the speed of 2000rpm at the silicon (SiO of fresh cleaning _x) spin coating in the substrate, be placed on immediately in the XPS instrument then.

Fig. 1 a shows the Gd that is spin-coated on the silicon base ₂O ₃The wide scanning optical spectrum of-DEG nano-particle.Under 1120eV and 1188eV, find the most intensive photoelectron peak value.These two peaks come from Gd (3d respectively _3/2) and Gd (3d _5/2).Peak position and Gd ₂O ₃Oxidation level unanimity [people such as Raiser D: Study of XPS photoemission of some gadolinium compounds (the XPS light emission researchs of some gadolinium compounds).J Electron Spectrosc.1991；57：91-97]。It has verified the oxidation level of sample.The O that finds under 532eV (1s) peak is by from three kinds of different components, i.e. Gd ₂O ₃, coat molecule DEG and silicon (SiO _x) oxygen in the substrate forms.Below coat the coordinate more detailed analysis of molecule and nano-particle.Two peak values under 151eV and 99eV come from Si (2s) and Si (2p) as the contribution of substrate.The Gd of spin coating ₂O ₃-DEG film is thin, thereby is minimized in the charged of sample in the XPS measuring process.The peak of prominence of finding under 978eV comes from O (KLL) Auger line.

Also adopt identical operation, use combustion method respectively, perhaps the Gd that makes with oleic acid or citric acid method for coating (preparation method is according to the disclosed operation in front) with the sub-spectral investigation of x-ray photoelectric ₂O ₃The nano-particle sample.Fig. 1 b shows at silicon (SiO _x) Gd that coats of the oleic acid of spin coating in the substrate ₂O ₃The Gd of nanocrystal (3d) spectrum.Gd (3d) energy level by the division of spin(-)orbit bimodal and respectively 1187.7 and 1220.3eV under Gd (3d _5/2) and Gd (3d _7/2) the peak composition.Wire shaped and peak position and be pressed into the Gd of sheet material ₂O ₃Powder (confirms that sample is by Gd ₂O ₃Form) early stage disclosed data people such as (, J.Electron.Spec.57 (1991) 91-97) D.Raiser concordance fine.The citric acid coated pellet is not identical with the oleic acid coated pellet astoundingly with particulate Gd (3d) spectrum made from combustion method.

The C of oleic acid coated pellet (1s) spectrum demonstrates three different peak values (Fig. 1 c).Main peak under 285eV is owing to the aliphatic carbons in the oleic acid.Peak under about 287 is attributable to hydroxyl carbon and corresponding to the end-blocking carbon in diethylene glycol.Peak under 289.1eV is corresponding to the carboxyl in the oleic acid.The O of oleic acid coated pellet (1s) spectrum demonstrates three peaks (Fig. 1 d).Peak under 531.1eV is corresponding at Gd ₂O ₃Oxygen in the oxide and as desired, the outstanding peak under 532.1eV is to come from SiO _xThe contribution of substrate.Peak under 533eV comes from carbonyl and C-O-C in DEG and the C-OH in oleic end-capping group.The O of citric acid coated pellet (1s) spectrum demonstrates three peaks (Fig. 1 e).531.2 and 532.3eV under the peak correspond respectively to oxygen and the carbonyl in the citric acid (C=O and/or O-C=O) in the Gadolinia..The 3rd peak under 533.9eV relates to the oxygen in ester group (C-O-C=O).The formation of ester may take place in building-up process, and this is because it involves alcohol and carboxylic acid.Come O (1s) spectrum of the synthetic sample of spontaneous combustion to demonstrate three peaks (Fig. 1 f).The same with above (Fig. 1 d and 1e), the peak under 531.2eV is corresponding to the oxygen in the Gadolinia..At the peak of prominence at 532.3 places with under 535.6eV, be interesting than small peak.The former is attributable to carbonyl (C=O and/or O-C=O) and the latter owing to oxygen and the most adjacent nitrogen.The source at these peaks or unreacted reactant (glycin, Gadolinium trinitrate) and/or contain the product of carbonyl and nitrogen.

Embodiment 4

Characterize with transmission electron microscopy (TEM)

The Philips CM20 ultramicroscope that employing is operated under 200kV carries out TEM research.Gd by the DEG path of preparing ₂O ₃Nano-particle is of a size of about 5nm, and this HREM microphotograph from Fig. 3 is found out.Although poor contrast, (222) plane (d ≈ 3.1 dusts) are visual.Fig. 4 shows the HREM microphotograph with the synthetic middle nanocrystal that obtains of oleic acid, and its diameter is about 15nm.There is not contrast (if it is in words there) with clad, but in whole crystal, can sees not broken (222) plane.Fig. 5 shows the TEM image of the nanocrystal that obtains in burning is synthetic.The aggregation of at least three nanocrystals is visual and is of a size of and is less than or equal to about 10nm.Result from TEM demonstrates the acquisition crystalline nanoparticles equably.

Embodiment 5

Sample preparation

Has H ₂The 10mm NMR of O in vitro under 9 kinds of 0.1-2.5mM different Gd concentration, prepares the Gd from embodiment 1 ₂O ₃-DEG sample (particle mean size is that about 5nm and particle size range are about 1-15nm) and Gd-DTPA (Magnevist ).In measurement, test tube is immersed in having in the brinish vessel of 22-23 ℃ (this temperature being the temperature of scanner chamber).

Embodiment 6

Relaxation time measures and nuclear magnetic resonance

Adopt 1.5 T Philips Achieva entire scan instrument, use head coil (headcoil) to measure T ₁And T ₂Relaxation time.Be used for measuring [kleef_mrm_1987] with the blended many echoes SE of the 2D of many echoes IR sequence interleaving (interleave).Change over the picture time parameter, minimize the standard deviation of relaxation time calculating: TE=30ms, TR (SE)=500ms, TI=150ms and TR (IR)=1150ms (the 1st group); TE=50ms, TR (SE)=760ms, TI=370ms and TR (IR)=2290ms (the 2nd group).Other MR parameter is: FOV=23cm, sheet thickness=7mm, number of echoes=4.

Realization is compared with Gd-DTPA, at H ₂The remarkable increase of the proton relaxation degree of gadolinium nano-particle in the O.Table 1 shows Gd ₂O ₃Relaxivity almost be 2 times of numerical value of Gd-DTPA: r ₁(Gd ₂O ₃)/r ₁(Gd-DTPA)=1.89, r ₂(Gd ₂O ₃)/r ₂(Gd-DTPA)=1.94.1/T among Fig. 5 a and the 5b _iThe graph shows that gadolinium concentration is done has good fit linear relationship (r according to equation 2 ₁, r ₂＞0.99, table 1).

Table 1:Gd-DTPA (Magnevist ) and Gd ₂O ₃Relaxation constant (the r of-DEG ₁, r ₂) and good match (r ₁, r ₂)

	r 1	r 1	r 2	r 2
						(mM -1s -1)		(mM -1s -1)
Gd-DTPA	4.86±0.08	0.9983	5.53±0.14	0.9975
					Gd 2O 3-DEG	9.19±0.10	0.9984	10.74±0.27	0.9957

The analysis of signal intensity shows use and measures the data of first echo in the spin echo part of employed sequence in the relaxation time, TE=30ms, and TR=500ms (Fig. 6) compares with Gd-DTPA, at the following Gd of low concentration ₂O ₃Sample higher signal intensity.Higher concentration (＞0.9mM) under, strong T ₂The signal of influence decay nano-particle sample.That is to say that with in this sequence, the signal intensity that occurs the Gd-DTPA of peak value under about 1.2mM is compared, Gd ₂O ₃Sample is issued to signal intensity in lower concentration (0.6mM).

The analysis showed that with Gd-DTPA and compare, at H ₂Gd among the O ₂O ₃Relaxivity significantly increase.These test another interesting feature is tangible T ₁Reduce effect and therefore observed signal increase under low concentration.The concentration range that is lower than 0.6mM in plasma is a maximally related concentration range in the clinical practice.Under the dosage (manufacturer is recommended) of Magnevist 0.1mmol/kg, the injection after 3 minutes, the detection plasma density of Gd is 0.6mM and was 0.24mM (by Medical Product Agency of Sweden, the data that FASS provides) in 60 minutes after injection.

Compare Gd in spin-echo sequence shown in Figure 6 with the Gd-DTPA signal ₂O ₃Signal intensity both raise fast, descend fast again.Can pass through high T ₁Relaxivity explains that (＜rapid signal under 0.6mM) increases at low concentration.Yet, under higher concentration, for Gd ₂O ₃Granule, T ₂The decline effect is more outstanding.Can cause that signal descends more fast by the sensitivity influence that causes because of unevenness in magnetic field, particle position place.

In addition, with above identical condition under the preparation and the test Gd ₂O ₃-DEG sample and Resovist .6 kind different Gd and the Fe concentration of existence between 0.1 to 1.5mM.Resovist  is based on the ferrocarbotran colloidal sol of Superparamagnetic Iron Oxide nano-particle (SPIO).This granule has the hydrodynamic diameter of 60nm on the ferrum nuclear of 4nm.Fig. 8,9 and 10 shows relaxivity and signal intensity.These results show, with Gd ₂O ₃-DEG compares, and Resovist  has higher T ₁And T ₂Relaxivity.When comparison curves, obviously Resovist  has significantly higher T ₂Relaxivity.This means and Gd ₂O ₃-DEG compares (it provides positive contrast), and Resovist provides negative contrast (signal intensity of c.f. in Figure 10).Therefore, granule can make to give based on those of SPIO and have the contrast agent of complementary performance.

As shown in figure 11, in order to compare Gd ₂O ₃With the signal intensity of water, in that (TE=30ms during TR=500ms) relaxation time measures, realizes having the signal intensity of first echo.Test tube is immersed in the saline for measuring water and Gd simultaneously ₂O ₃The signal intensity of sample.

Embodiment 7

Mononuclear cell research

For monocyte experiments, under the situation of adding L-glutamate, Glu and penicillin/streptomycin (Invitrogen), in RPMI 1640 media with 10% hyclone (GIBCO, Invitrogen, Carlsbad, CA USA) cultivates the THP-1 cell.Counting cells also finds that 97% can survive.0.1,0.3,0.6 and the concentration of 0.9mM under, use Gd ₂O ₃-DEG or Gd-DTPA handle cell.Stay the cell in next hole to be untreated.Only adopt the Gd of variable concentrations ₂O ₃-DEG granule, the cell culture medium of preparation control series.The two boards for preparing every block of plate 24 holes as mentioned above; Under 37 ℃, use Gd for one ₂O ₃-DEG and Gd-DTPA inoculation 2 hours and another piece inoculation 8 hours.After cultivating, with cell transfer in the Falcon pipe, and with medium washing 2 times and under 1100rpm centrifugal 8 minutes.Monocyte experiments shows after washing procedure, nano-particle or attached on the cell surface or pass through cell internalizing.After washing, Gd-DTPA is not present in the cell suspending liquid.T ₁And T ₂Relaxation time is at higher Gd ₂O ₃All descend under the concentration and under long incubation time (data not shown goes out).Fig. 7 shows the T1 figure that comes from cultivation in 8 hours.

Make Magnevist (Gd-DTPA), in born of the same parents' external space, keep.In Fig. 4 b, can find out that Gd-DTPA washes out effectively from sample.On the contrary, Gd ₂O ₃After washing, be retained in that (Fig. 4 a) in the cell culture.Show some cell types, for example macrophage can be by the little granule of phagocytosis internalization [people: Magnetically labelledcells can be detected by MR imaging (can pass through MR imaging magnetic labeled cell) such as Weissleder R., J Magn Res Imag.1997; 7:258-263].To studies show that in early days of the THP-1 that cultivates with ferric oxide nanometer particle, cell absorb and dosage/incubation time between linear relationship [people: Application of the static dephasing regime theoryto superparamagnetic iron-oxide loaded cells (application on the cell of static phase difference scheme theory at marked by superparamagnetism iron oxide) such as Bowen CV, Magn Res Med.2002; 48:52-61].These earlier results show that in experiment of the present invention, the Gd-granule may be absorbed by cell equally.

Claims (16)

1. super-paramagnetism nano grade particles, it comprises the Gadolinia. of particle mean size between about 0.5-50 nanometer.

2. the granule of claim 1, it has the coating of bio-compatible and/or biospecific.

3. the granule of claim 2, it has the coating that contains diethylene glycol (DEG) and/or citric acid.

4. the granule of claim 2, it has the coating that contains folic acid.

5. the granule of claim 4, its floating coat comprises the Polyethylene Glycol that is connected on the folic acid.

6. the granule of claim 1, its particle mean size is about 5 nanometers, has the coating that contains diethylene glycol (DEG).

7. compositions, it comprises any one granule of claim 1-6.

8. the compositions of claim 7, its gadolinium concentration is 0.01-500mM, preferred 0.01-2.5mM.

9. claim 7 or 8 compositions, it is suitable for being administered on the body position.

10. the compositions of claim 9, but it comprises the carrier of parenteral administration.

11. any one compositions of claim 7-10, it has the relaxation time T that reduces adjacent proton in rich proton environment ₁And/or T ₂The T that obtains to the compositions that is lower than the ionic complex by gadolinium ₁And/or T ₂The ability of numerical value.

12. any one compositions of claim 7-10, in the concentration range of 0.1mM-1.5mM, it has the high signal intensity of signal intensity that obtains than by nanoscale iron oxide particles.

13. a contrast agent, it comprises any one compositions of claim 7-12.

14. the contrast agent of claim 13, the signal intensity ratio water height at least 500% that it has is preferably greater than 700%.

15. carry out the method for MRI (nuclear magnetic resonance), it comprises uses claim 13 or 14 any one contrast agent interaction of molecules for deliberation or cell processes.

16. claim 1-12 any one supperparamagnetic particles or compositions in the purposes of preparation in the opaque contrast medium.

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