EP1945192A2

EP1945192A2 - Nanoscale particles used as contrasting agents in magnetic resonance imaging

Info

Publication number: EP1945192A2
Application number: EP06806318A
Authority: EP
Inventors: Armin Kuebelbeck; Heike Schilke
Original assignee: Merck Patent GmbH
Current assignee: Merck Patent GmbH
Priority date: 2005-11-10
Filing date: 2006-10-17
Publication date: 2008-07-23
Also published as: DE102005053618A1; KR20080066999A; WO2007054182A3; JP2009514905A; US20080286370A1; WO2007054182A2; CN101340900A

Abstract

The invention relates to a nanoscale particles that are used as contrasting agents in magnetic resonance imaging. Said nanoscale particles are composed of a nucleus with an inert matrix, one or several covalently bonded organic complexing agents in which one or several metal ions with unpaired electrons are bonded, and one or several optional biomolecules that are covalently bonded on the surface of the nuclei. Also disclosed is a method for producing said nanoparticles.

Description

Nanoscale particles as contrast agent for magnetic resonance imaging

The invention relates to nanoscale particles which consist of a core with an inert matrix, one or more covalently bound organic complexing agents in which one or more metal ions are bound with unpaired electrons and optionally one or more biomolecules covalently bound to the surface of the nuclei and a process for their preparation.

Because of its optimal signaling (T1 shortening, strong paramagnetism by 7 unpaired electrons) gadolinium (Gd) is used in MRI (Magnetic Resonance Imaging or Magnetic Resonance Imaging). Due to the 7 unpaired pairs of electrons, the gadolinium induces a strong alternating electromagnetic field, which influences the spin of the neighboring water protons in such a way that their relaxation time is reduced.

Intravenously applied solutions of gadolinium salts are acutely toxic. Amongst others, the smooth and the striated muscles, the function of the mitochondria and the blood coagulation are affected by the toxicity. Therefore, ways have been sought to reduce the toxicity of this metal without compromising its paramagnetic properties - the tendency to migrate into magnetic fields. The best way to achieve this goal, which has also led to the commercial production of Gd-containing contrast agents, is chelation.

For this purpose, complexing agents with very high complex formation constants are used. Examples of these complexing agents are DOTA and DTPA.

DOTA DTPA

1, 4,7,10-tetraazacyclododecane-diethylenetriaminepentaacetic acid

1, 4, 7, 10-tetraacetic acid

The most stable commercial gadolinium chelate complex to date is macrocyclic gadoteric acid (Gd-DOTA;

DOTAREM ^®, Fa. Guerbet). The danger of dissociation and thus of the

The release of toxic gadolinium ions (LD ₅₀ about 0.1 mmol kg ^"1 ) when using gadoterate as an MRI contrast agent is very low, unlike other complexes whose release half-life stability in acid gastric juice (measured in 0.1 -molar HCl solution as a standardized model) in the range of seconds to hours has

Gadoteric acid here has a half-life of over a month. The exchange of gadolinium for endogenous metal ions such as copper or zinc is well below 1%, while it can be up to 35% in other complexes.

Gd is literally enclosed by the organic acid DOTA and lies in the center of the chelate molecule as in a cavity, as shown by X-ray crystallographic studies. The toxicity of gadolinium is thus virtually completely concealed, while its paramagnetic

Properties that make it interesting as an MRI contrast agent to be preserved.

The use of contrast agents in MRI increases the significance of the visualization of organs. Normally, 10 to 15 ml (0.2 ml / kg

Body weight) contrast agent injected intravenously. The Gd-containing contrast agent used leads to a shortening of the relaxation time and thus to a stronger signal in the images produced. Only in special, especially the liver issues are used contrast agents based on the transition elements manganese, iron or copper.

Paramagnetic complexes such as gadoteric acid have hydrophilic

Properties and do not pass the blood-brain barrier. After intravenous injection, a rapid vascular, followed by an interstitial distribution; a preference for a particular organ is not observed. The complexes are excreted unchanged by glomerular filtration through the kidneys within a few hours. Gadoteric acid is 75% eliminated after three hours. The pharmacokinetics described make Gd a contrast medium which is particularly suitable for the diagnosis of shifts in the extracellular fluid, as it occurs in tumors, edema, necrosis and ischemia.

The compatibility of Gd-DOTA is very good. For example, two studies involving more than 5,000 patients have shown that the rate of side effects is between 0.84% and 0.97%. Of 4,169 patients in the larger of the two studies (Caille 1991), 8 had nausea and 5 vomiting, giving a total of 0.31% for these side effects. Heat, headache, discomfort, rash, and unpleasant mouthfeel occurred in less than 0.15% of all patients. The systemic osmolality load is also negligible with gadoteric acid. The only organ that shows an increased accumulation of Gd-DOTA is the kidney, which has probably pharmacokinetic reasons. However, CDU studies with creatinine clearance below 60 ml / min showed no negative impact of Gd-DOTA on vital signs or renal function. Gd-DOTA, unlike Gd-DTPA-BMA, has not given rise to any pseudo-hypocalcaemia. The recommendation at

Renal Insufficiency: Monitoring + take all measures to prevent renal insufficiency (hydration, dose restriction, risk / Benefit assessment). For all these reasons, gadoteric acid is approved as an MRI contrast agent not only for adults, but also for children until infancy.

Nanoscale gadolinium (III) -containing particles have been known for some years and have advantages over individual complexed gadolinium (III) ions, which are significant in use as contrast agents in diagnostics:

- The accumulation of a large number of complexed gadolinium (III) ions on a spherical surface leads to a significantly stronger signal in the nuclear magnetic resonance tomograph compared to individual gadolinium complexes. This makes it possible either to reduce the contrast agent dose or to obtain a stronger signal through the improved signal-to-noise ratio at the same dose.

From C. Platas-Iglesias et al., Chem. Eur. J. 2002, 8, no. 22, 5121-5131 "Zeolite GdNaY nanoparticles with very high relaxivity for application as contrast agents in magnetic resonance imaging" are Gd ³⁺ -supported zeolite NaY nanoparticles, where gadolinium is bound only by Coloumb forces, ie not covalently the pore size of the Y zeolite is only 1.3 nm, the free proton exchange with the surrounding tissue is severely hindered.

WO 00/30688 (Bracco) describes substituted polycarboxylate ligand molecules and corresponding metal complexes such as Gd-DTPA and GD-DOTA derivatives as contrast agents for MRI.

WO 2004/009134 (Bracco) describes Gd-chelate complexes as MRI contrast agents that are trapped by the cell.

WO 96/09840 (Nycomed) describes a diagnostic agent comprising a particulate material whose particles are a diagnostically effective, essentially water-insoluble crystalline material of a metal oxide (iron oxide) and a polyionic coating agent (eg chitosan, hyaluronic acid, chondroitin).

WO 04/083902 (Georgia Tech Research Corp.) describes magnetic

Nanoparticles (e.g., Gd chelates) with a biocompatible coating (e.g., phospholipid-polyethylene glycol) which can carry biomolecules such as nucleic acids, antibodies, etc.

WO 03/082105 (Barnes Jewish Hospital) describes Gd-DTPA-PE and Gd-DTPA-BOA chelate complexes surrounded by a lipid / surfactant coating.

Object of the present invention was to produce new contrast agents that avoid the disadvantages of the aforementioned compounds.

This could now surprisingly be achieved by the use of high-purity silicon dioxide as a carrier of the covalently bound lanthanide complexes (preferably gadolinium complexes). The good

Compatibility of silica in the patient's body is excellent, and thus clearly superior to many other prior art materials. Examples include: Jain, TK; Roy, I.; Dee, TK; Maitra, ANJ Am. Chem. Soc. 1999, 120, 11092-11095, Shimada, M .; Shoji, N. Takahashi, A. Anticancer Res. 1995, 15, 109-115 and LaI, M. Levy, L .; Kim, KS; He ₁ GS Wang, X .; Min, YH; Pakatchi, S .; Prasad, PN Chem. Mater. 2000, 12, 2632-2639.

The present invention thus nanoscale particles consisting of a core with an inert matrix, one or more covalently bonded organic complexing agents in which a metal ion is bound with unpaired electrons and optionally one or more biomolecules covalently bound to the surface of the nuclei.

Another object of the present invention are nanoscale particles consisting of a core with an inert matrix, optionally one or more, covalently bound to the surface of the nuclei biomolecules and one or more, on the surface of the nuclei via a linker covalently bonded organic complexing agent in which a metal ion is bound with unpaired electrons. The core or carrier preferably consists of the inert matrix

Silica, titania, alumina or zirconia. Particularly preferred is silica. The monodisperse silica particles are prepared by known processes (see EP 0216278) by the hydrolysis of tetraalkoxysilanes. The average particle diameter of the monodisperse particles is 10 to 500 nm, preferably 30 to 300 nm. In principle, however, polymers can also be used, for example. Polystyrene latexes are used.

Furthermore, it is possible to coat other nanoparticles in a first step with a thin layer of silicon dioxide. The coating is possible in a simple manner by the SoI-GeI process known to the person skilled in the art. For this purpose, nanoparticles are suspended in an ethanolic-aqueous solution and a silica ester, for example tetraethyl orthosilicate (TEOS) is added. At possibly elevated temperatures, the hydrolysis of the kieselesters is initiated by the addition of aqueous ammonia solution. In this case, the precipitated silica preferably lies on the nanoparticles in the suspension. By the amount of silica ester used, with a known amount and known average diameter of the nanoparticles to be coated, the layer thickness can be set very accurately. By ultrafiltration or centrifuging at particle diameters> 50 nm, the coated nanoparticles can be separated and purified.

As metal ions are preferably paramagnetic ions from the

Group of lanthanides used. Gadolinium (III) ions are particularly preferably used.

As organic complexing agent, compounds from the group of oligo- or polycarboxylates are preferably used. Diethylenetriaminepentaacetic acid (DTPA) or 1, 4,7,10-tetraazacyclodecane-1, 4,7,10-tetraacetic acid (DOTA) are particularly preferably used.

The metal chelate complexes are covalently linked to the surface of the support via a linker, preferably via a silane. A preferred linker is 3-aminopropyltriethoxysilane (APTES). It is particularly advantageous to carry out the metal-chelate complexes via the copper-catalyzed dipolar 1, 3-cycloaddition of azides to alkynes, the so-called Huisgen reaction. This reaction, which is known to the person skilled in the art, leads, under very mild conditions, to stable 1,2,3-triazoles with excellent yields, and makes it possible in a simple manner to build even very complex molecules. Recently, this reaction has therefore gained renewed interest under the term "click chemistry", which is reflected in a large number of publications (see Bräse et al, Angew Chem 2005, 117, 5336; KoIb, Finn and Sharpless, Click Chemistry: Various Chemical Function from a Few Good Reactions, Angew Chem. Int. Ed. 2001, 40, 2004-2021)

Surprisingly, it was found that the above-mentioned Huisgen reaction can also be advantageously used for the functionalization of the surface of nanoparticles. This means that the "click chemistry" is also at Heterogeneous solid-phase reactions can be used on nanoscale particles.

Thus, another object of the present invention is a process for producing nanoscale particles comprising the following

Process steps: a) Preparation of nanoparticles, preferably of silicon dioxide, titanium dioxide, aluminum oxide and / or zirconium dioxide by wet-chemical methods b) Coating of the nanoparticles with a monomolecular layer of a halosilane. c) reaction of the nanoparticles with an azide-containing agent into azide-functionalized nanoparticles d) Preparation of one or more organic complexing agents containing one or more amines and one or more

Polycarboxylic acids, polycarboxylic acid anhydrides, polycarboxylic acid chlorides or polycarboxylic esters e) Loading of one or more complexing agents with metal ions from the group of the lanthanides f) Reaction of the azide groups functionalized nanoparticles

Step c) with the metal ion-loaded organic complexing agent from step e)

It is preferred if, as halosilane, e.g. 3- (chloropropyl) triethoxysilane is used.

As alkynamines, it is possible to use all known alkynamines, preference being given to using propargylamine or 6-aminohexine (1). This is reacted with a polycarboxylic acid suitable for complexing, a polycarboxylic anhydride, a polycarboxylic acid chloride or else also a polycarboxylic acid ester having a good leaving group. The synthesis of a carboxylic acid amide is carried out by known methods. Preferably, the polycarboxylic acids DOTA and DTPA or their derivatives (for example as Li salts) are reacted with a corresponding amine. It is ensured in the reaction that only one carboxylic acid function of the polycarboxylic acid reacts with the amine (1: 1 batch ). The reaction of, for example, DTPA dianhydride with propargylamine is carried out according to the known Schotten-Baumann method.

In addition, covalent biomolecules such as, for example, enzymes, peptides / proteins, receptor ligands or antibodies can be bound to the nanoparticles. By their specific coupling to the target tissue in the body of the patient, the imaging and the resulting diagnosis is facilitated.

Furthermore, the nanoparticles may be coated with dextran or polyethylene glycol to increase biocompatibility.

Another object of the present invention is the use of nanoscale particles as contrast agents for magnetic resonance imaging. The particles according to the invention can be used as contrast agents in magnetic resonance tomography, since the superficially arranged metal ions can interact with the surrounding protons, for example from tissue fluid.

The following examples are intended to illustrate the present invention without limiting it.

Example 1:

Preparation of monodisperse silica particles with a middle

Particle diameter of 250 nm with superficially bound Gd (III)

1.1 Preparation of Monodisperse Silica Particles The preparation of the monodisperse silica particles is carried out as in

EP0216278 B1, by the hydrolysis of tetraalkoxysilanes in aqueous-alcoholic-ammoniacal medium, wherein initially generates a sol of primary particles and then by a continuous, controlled in accordance with the Abreagierens metered addition of tetraalkoxysilane, the resulting SiO 2 particles to the desired particle size brings.

1.2 Functionalization with 3-aminopropyltriethoxysilane (APTES)

10 g of the silica particles prepared in the first step were suspended in 20 ml of 2-propanol. Subsequently, 0.25 ml of APTES was ₁ diluted with 5 ml of 2-propanol was added dropwise and stirred for 2 hours at 80 ⁰ C at reflux. The suspension was by means of a centrifuge at 4000 ^{min -1} 8 times with

Propanol-2 was washed until in the wash solution no APTES - by means of a dot test with ninhydrin - was more evidence.

1.3 Amide Formation with Diethylenetriamine Pentaacetic Acid (DTPA) The second stage of the APTES functionalized silica

Particles were mixed with 25 ml of dimethyl sulfoxide (DMSO) and the propanol-2 distilled off on a rotary evaporator under vacuum. Subsequently, the suspension with 0.58 g of diethylenetriaminepentaacetic acid dianhydride (DTPA-ca) was added and stirred for 2 hours at 150 ⁰ C. After cooling to room temperature, the reaction product was poured onto 200 ml of 0.1 N TRIS buffer (pH 7.0) and washed several times in a centrifuge with deionized water.

1.4 Loading with Gadolinium (IM) ions

The suspension obtained in the third step was treated with 0.486 g of gadolinium (III) chloride anhydrous and stirred for 8 hours at room temperature. Subsequently, the suspension was washed with deionized water with a centrifuge until chloride was no longer detectable in the wash water by means of silver nitrate solution. Thereafter, the reaction product was dried by freeze-drying.

Characterization:

The dried gadolinium-loaded silica particles were dissolved in dilute hydrofluoric acid and the content of gadolinium determined by ICP-MS. 0.13% gadolinium was found in the sample. A sample of the silica particles was again extensively (3x) washed with deionized water and after drying again analyzed by ICP-MS. The content of gadolinium was determined to be 0.14%. The slightly higher content of gadolinium can be explained by the different degrees of drying or limitations in the measuring method. Crucial, however, is that by the multiple washing of the silica particles, the content of gadolinium did not decline. That is, the gadolinium is obviously tightly covalently bound to the surface of the non-porous silica particles. The same result is also obtained in the treatment with 1 N hydrochloric acid. Example 2

Preparation of monodisperse silica particles having an average particle diameter of 90 nm with superficially bound Gd (III)

2.1 Preparation of the particles

As described in Example 4 of EP0216278 B1

2.2 Functionalization of the particles

10 g of the silica particles prepared in the first step were suspended in 20 ml of 2-propanol. Subsequently, 0.50 ml of APTES, diluted with 5 ml of 2-propanol were added dropwise and for 2 hours at 80 ⁰ C on

Reflux condenser stirred.

The suspension was by means of a centrifuge at 4000 ^{min -1} 8 times with

2.3 Amide Formation with Diethylenetriamine Pentaacetic Acid (DTPA)

The silica particles functionalized in the second step with APTES were admixed with 25 ml of dimethylsulfoxide (DMSO) and the propanol-2 was distilled off on a rotary evaporator under reduced pressure.

The suspension of 1, 0 g diethylenetriaminepentaacetic dianhydride (DTPA-ca) was added and stirred for 2 hours at 150 ⁰ C. After cooling to room temperature, the reaction product was poured onto 200 ml of 0.1 N TRIS buffer (pH 7.0) and washed several times in a centrifuge with deionized water.

2.4 Loading with Gadolinium (III) ions

The suspension obtained in the third step was treated with 1, 0 g of gadolinium (III) chloride anhydrous and at 8 hours

Room temperature stirred. Subsequently, the suspension was washed with deionized water with a centrifuge until in Washing water chloride was no longer detectable by means of silver nitrate solution. Thereafter, the reaction product was dried by freeze-drying.

characterization

The dried gadolinium-loaded silica particles were dissolved in dilute hydrofluoric acid and the content of gadolinium determined by ICP-MS.

0.2% gadolinium was found in the sample. The higher Gd content compared to the particles produced in Example 1 can be explained by the higher surface area to volume ratio of the smaller particles. Calculated are about 1200 gadolinium ions at the

Surface of one of the 90 nm particles.

Example 3

Preparation of monodisperse silica particles having an average particle diameter of 250 nm with superficially bound Gd (III)

3.1 Preparation of the silica nanoparticles In a mixture of 41.5 ml of demineralized water and 111 ml of ethanol, 16.7 ml of tetraethylorthosilicate are added at room temperature and a homogeneous solution is achieved by stirring. Then 26 ml 25% by weight of ammonia solution are added to the original, stirred for 15 sec. Intensive and then allowed to stand for 1 h. Approximately 1 min. After addition of the ammonia solution, the progressing condensation to silica nanoparticles can be observed by clouding of the solution. The reaction mixture is not worked up, but fed directly to the next reaction step.

3.2 Reaction with a halosilane

The nanoparticles produced in the first step are coated with a monomolecular layer of a halosilane. To do so δOμl 3- (chloropropyl) - triethoxysilane was added to the reaction mixture from the 1st step and stirred at 8O ⁰ C for 5h. Subsequently, the particles are centrifuged off and washed neutral with deionized water.

3.3 Preparation of the surface-bonded azide The nanoparticles prepared in step 2 and washed are suspended in 50ml of deionized water and treated with 66 mg of sodium azide and stirred for 24 h at 50 ⁰ C. By nucleophilic substitution, the halogen chlorine is replaced by the pseudohalogen azide. The azide-containing nanoparticles are separated from the educts in the centrifuge, washed with demineralized water and stored as an aqueous suspension.

3.4 Preparation of the alkyne (polycarboxylic monoalkynamide) by

Reaction of DTPA dianhydride with propargylamine (Schotten-Baumann method)

0.62 g (1 mmol) of diethylenetriamine-1, 7-tetrakis (f-butyl acetate) -4-acetic acid from Article B-365 from Macrocyclics are mixed with 0.19 g (1 mmol) of 1-ethyl-3- (3 dimethylaminopropyl) carbodiimide hydrochloride (EDC) from Aldrich and 0.15 g (1.5 mmol) of triethylamine from Merck and 2 ml of dimethylformamide (Merck). After 10 minutes of intensive stirring at room temperature 0.06 g (1 mmol) propargylamine are added to the template. The reaction mixture is stirred for a further 8 hours at room temperature. The

Reaction control is carried out by thin-layer chromatography. The reaction mixture is taken up in 10 ml of dichloromethane, 3 times with 20 ml of 0.1 molar hydrochloric acid and extracted twice with 20 ml of _ß saturated aqueous NaHCO 3. Finally, it is extracted by shaking with saturated aqueous sodium chloride solution and dried over anhydrous sodium sulfate. On a rotary evaporator, the dichloromethane is stripped off and the oily residue is dissolved in 4 ml of tetrahydrofuran / ethanol (1: 1 by volume). To the mixture are added 1 ml of water and 0.1 g (4.4 mmol) of lithium hydroxide to cleave the ester protecting groups. The hydrolysis mixture is stirred overnight and concentrated to dryness on a rotary evaporator. The reaction product is taken up in 10 ml of water and 1 molar

Hydrochloric acid adjusted to pH 7.

3.5 Loading the complexing agent with gadolinium (III) ions

The solution of the lithium salt of diethylenetriamine pentaacetic acid 4-propargylamide prepared in step 4 is mixed with 10 ml of 0.1 molar

Gadolinium (III) chloride solution (= 1 mmol) and stirred for 30 minutes.

3.6 Preparation of Nanoparticles Modified with Complexing Agent Molecules (Huisgen Reaction)

The azide-functionalized nanoparticle suspension prepared in step 3 was adjusted to neutral pH using TRIS buffer. The previously calculated amount of polycarboxylic monoalkynamide (from step 5) was added dropwise to the nanoparticle suspension in the presence of 50 mg of copper (I) chloride. To

The reaction was stopped for 16 hours at room temperature. The particles are centrifuged off and washed thoroughly with 0.1 molar hydrochloric acid, finally with demineralized water.

By means of ICP-MS, the gadolinium content of the particles is determined to be 0.3%

Claims

claims

1. Nanoscale particles consisting of:

a core with an inert matrix

- one or more covalently bonded organic complexing agents in which one or more metal ions are bonded with unpaired electrons and - optionally one or more, on the surface of the

Nuclei covalently bound biomolecules.

2. Nanoscale particles consisting of:

a core with an inert matrix, optionally one or more, on the surface of the

Nuclei covalently bound biomolecules and

- One or more, bound to the surface of the cores via a linker covalently bound organic complexing agent in which a metal ion is bound with unpaired electrons.

3. Nanoscale particles according to claim 1 and / or 2, characterized in that the core consists of silica, titania, alumina and / or zirconia.

4. Nanoscale particles according to claim 3, characterized in that the core consists of silica.

5. nanoscale particles according to one or more of claims 1 to 4, characterized in that they have an average particle diameter of 10 to 500 nm, preferably from 30 to 300 nm, and are monodisperse.

6. nanoscale particles according to one or more of claims 1 to 5, characterized in that the metal ion is selected from the group of lanthanides.

7. nanoscale particles according to one or more of claims 1 to 6, characterized in that the metal ion is a gadolinium (III) ion.

8. nanoscale particles according to one or more of claims 1 to 7, characterized in that the organic complexing agent is selected from the group of oligo- or polycarboxylates.

9. nanoscale particles according to claim 8, characterized in that the organic complexing agent diethylenetriaminepentaacetic acid (DTPA) or 1, 4,7,10-tetraaza-cyclododecane-1, 4,7, 10-tetraacetic acid (DOTA) is.

10. nanoscale particles according to one or more of claims 1 to 9, characterized in that are used as covalently bound biomolecules enzymes, peptides / proteins, receptor ligands or antibodies.

11. nanoscale particles according to claim 2, characterized in that a silane is used as the linker.

12. nanoscale particles according to claim 11, characterized in that is used as the linker 3-aminopropyltriethoxysilane (APTES).

13. A process for producing nanoscale particles with the following process steps: a) Producing nanoparticles, preferably from silicon dioxide, titanium dioxide, aluminum oxide and / or zirconium dioxide by wet-chemical methods b) Assignment of the nanoparticles with a monomolecular layer of a halosilane. c) reaction of the nanoparticles with an azide-containing agent to form azide-functionalized nanoparticles d) preparation of one or more organic complexing agents containing one or more amines and one or more polycarboxylic acids, polycarboxylic anhydrides, polycarboxylic acid chlorides or polycarboxylic esters e) loading of one or more complexing agents with metal ions from the group of the lanthanides f) conversion of the azide groups functionalized nanoparticles from step c) with the metal ion-loaded organic complexing agents from step e)

14. The method according to claim 13, characterized in that in step d) a Polycarbonsäuremonoalkinamid of organic complexing agents such as 1, 4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid (DOTA) or diethylenetriaminepentaacetic acid (DTPA) or their Derivatives and a corresponding alkyne amine is produced.

15. The method according to claim 13 and / or 14, characterized in that in step d) as alkyne amine propargylamine or 6-aminohexyne (1) are used.

16. The method according to one or more of claims 13 to 15, characterized in that are used in step e) as metal ions gadolinium (III) ions.

17. Use of the nanoscale particles according to one or more of claims 1 to 12 as a contrast agent for magnetic resonance imaging.