Magnetic resonance imaging contrast and the method for making thereof that bone tissue targeting detects that have based on metal fullerene
Technical field
The present invention relates to have the efficient magnetic resonance imaging contrast that bone tissue targeting detects, the invention still further relates to the preparation method of above-mentioned magnetic resonance imaging contrast.
Background technology
Nuclear magnetic resonance (MRI) technology has now developed into a kind of common means in the clinical treatment diagnosis, is diagnosing tumour one of effective method the most.In order to strengthen contrast and the definition between pathological tissues and the normal structure image, need to select suitable contrast medium to show anatomical features.These contrast mediums overwhelming majority utilizes the paramagnetic characteristic of metal ion.Gd
3+Because have over paramagnetism, its complex is the present general and the most effective most widely used class contrast medium.Nearest result of study shows, Gd@C
82(OH)
40MRI radiography efficient be clinical used Gd-DTPA[diethylene triamine pentaacetic acid, DTPA] 20 times of (Mikawa, M., et al., Bioconjugate Chemistry, 2001.12 (4): p.510-514) of chelate contrast agent.This table of discovery Benq can be used for making MRI contrast agent efficiently in the soluble derivative of metal fullerene.Yet,, make this material not have targeting detection sexual function, thereby under identical using dosage, greatly reduce detection sensitivity because the functional group that is woven with affinity with a certain particular group of organism is not modified on carbon cage surface.
Summary of the invention
One of purpose of the present invention is to provide the efficient magnetic resonance imaging contrast that bone tissue targeting detects that has based on metal fullerene.
Another object of the present invention is to provide above-mentioned method for making with efficient magnetic resonance imaging contrast that bone tissue targeting detects based on metal fullerene.
The chemical modification preparation is carried out on the surface of Gd-complex a certain particular organization is had the broad research of the MRI contrast agent of targeting detection, inspire the inventor metal fullerene Gd@C
82Cage carry out biological osseous tissue is had the functional modification of affinity outward, the high-efficiency low-toxicity MRI contrast agent that preparation detects at the osseous tissue targeting.The phospholipid base that the invention has the advantages that finishing has affinity to osseous tissue, can make this material in the enrichment of osseous tissue place, thereby makes osseous tissue that tangible contrast be arranged under the dosage that uses seldom, realizes the early diagnosis of osseous tissue pathological changes.
The magnetic resonance imaging contrast that bone tissue targeting detects that has based on metal fullerene of the present invention is the soluble derivative of the metal fullerene that contains Gd, and the developed by molecule formula of this contrast agent is:
Gd@C
82O
x(OH)
y(C (PO
3Et
2)
2)
10Or
Gd
aM
3-aN@C
80O
x(OH)
y(C (PO
3Et
2)
2)
10Or
Gd
2@C
80O
x(OH)
y(C(PO
3Et
2)
2)
10
Wherein: a=1,2 or 3; M=Sc or Er; X=2~8, y=12~16; Et is an ethyl;
The method of the above-mentioned contrast agent of preparation provided by the invention, be to utilize the metal fullerene that contains Gd to be raw material, direct and tetraethyl Medronate ester generation Bingel additive reaction in the presence of NaH, with OH nucleophilic addition takes place then again, synthesize cage on a large scale and modify the metal fullerene soluble derivative of phospholipid base outward, carry out according to the following steps:
(a) getting 15 to 30 times of tetraethyl Medronate esters to metal fullerene of molar equivalent is dissolved in anhydrous, the non-protonization organic solvent (as anhydrous toluene solution etc.);
(b) preferably at room temperature (as 15~30 ℃), the drips of solution that step (a) is obtained are added in the anhydrous toluene solution of the metal fullerene that contains NaH of stirring, and the mol ratio of wherein said NaH and tetraethyl Medronate ester is 50~100: 1;
Described metal fullerene is Gd@C
82, Gd
aM
3-aN@C
80(in the formula: a=1,2 or 3; M=Sc or Er) or Gd
2@C
80
Described metal fullerene can prepare by following list of references respectively:
Gd@C
82The preparation document is seen Shu, C.Y., et al., Carbon, 2006.44 (3): p.496-500.Gd
aM
3-aN@C
80The preparation document is seen Dorn, H.C., et al., Abstracts of Papers of theAmerican Chemical Society, 1999.218:p.U726-U726.
Gd
2@C
80The preparation document is seen Lu, X., et al., Carbon, 2005.43 (7): p.1546-1549
(c) preferably at room temperature (as 15~30 ℃), the mixture solution that step (b) is obtained stirs 1~3h, dripping deionized water then while stirring produces until no bubble, continue to stir back (preferred 12~24h), leave standstill separatory, the colourless toluene layer of reject concentrates the brown water layer that obtains, preferably with brown water layer with organic solvent washing after reconcentration, described organic solvent is an analytical pure toluene etc.With the polydextran gel phase that fixes, deionized water is done mobile phase, and the brown colour band of collecting pH=6~7 is the magnetic resonance imaging contrast that bone tissue targeting detects that has based on metal fullerene of the present invention.
For improving the purity of metal fullerene, preferably in advance metal fullerene to be purified, the available method commonly used of purifying is as contain the carbon soot of metal fullerene by the preparation of direct-current arc electric discharge, with Carbon bisulfide (CS
2), organic solvent extraction such as dimethyl formamide (DMF), toluene separate to purify in conjunction with high performance liquid chromatography and obtain the toluene solution of highly purified metal fullerene, carry out prepared in reaction magnetic resonance imaging contrast of the present invention with this toluene solution then.
Compare with other prior art, the present invention has following characteristics:
1. the present invention's characteristics of utilizing phospholipid base functional group to have bone are modified the fullerene contrast agent, make it become the targeting contrast agent.
2. novel MRI contrast agent (EMF-TEMDP) the radiography efficiency far based on metal fullerene of the present invention's preparation is better than now clinical used Gd-DTPA.
The present invention ingenious utilize characteristic that tetraethyl Medronate ester has reactive hydrogen in the presence of NaH with the Gd@C of electron deficiency
82These characteristics of Bingel additive reaction take place, and at room temperature make the Gd@C that contains the phospholipid base in a large number through single step reaction
82Derivant, thereby have simple economy, the easy to operate and advantage being convenient to accomplish scale production.
4. the present invention need not add the separation intermediate product, effectively utilizes the direct and intermediate material generation nucleophilic addition of OH in the aqueous solution, prepares the efficient MRI developing agent of good water solubility, has simplified synthesis step to a great extent, has reduced Financial cost.
5. synthetic targeting contrast agent among the present invention owing to the existence of carbon cage, has been protected deleterious Gd effectively
3+Ion has prevented its leakage under physiological condition, thereby greatly reduces its toxicity.
Description of drawings
Fig. 1. the embodiment of the invention 1 is based on metal fullerene Gd@C
82Cage modify the preparation process sketch map of the magnetic resonance imaging contrast of phospholipid base outward.
Fig. 2. the water-soluble metal fullerene Gd@C that is modified with the phospholipid base that the embodiment of the invention 1 is prepared
82Derivant (Gd-TEMDP) structural representation (dispenses OH for simplicity,
-).
The 1/T reciprocal of the longitudinal relaxation time that Fig. 3 A. reference examples 1 usefulness counter-rotating-recovery sequence of the present invention records
1To different Gd concentration mappings.
The 1/T reciprocal of the longitudinal relaxation time that Fig. 3 B. embodiment of the invention 1 counter-rotating-recovery sequence records
1To different Gd concentration mappings.
Fig. 4. Gd@C in the embodiment of the invention 1
82O
2(OH)
16(C (PO
3Et
2)
2)
10(Gd-TEMDP) and the clinical use contrast agent Gd-DTPA in the reference examples 1 under same concentration and the external T of pure water
1(Gd concentration is 4 μ M, 1.5T spin-echo sequence T as comparison diagram in weighting
R/ T
E=800ms/15ms, FOV=17.1 * 22.8cm
2, T=24 ℃).
Fig. 5. the infrared spectrogram of embodiment 1Gd-TEMDP.
Fig. 6. embodiment 1Gd-TEMDP is in heavy water
31The P-NMR spectrum.
Fig. 7. the C1s bonded energy empirical curve and the matched curve of different valence state among the embodiment 1Gd-TEMDP.
Fig. 8. the 01s bonded energy empirical curve and the matched curve of different valence state among the embodiment 1Gd-TEMDP.
The specific embodiment
Reference examples 1
In contrast, (Gd-DTPA is that (German Schering Corp produces the Magnevist Solution injection, and first miraculous cure industry company limited divides packing Magnevist) to the magnevist of used clinical use.Import drugs registration certificate number: H20030138, authentication code: the accurate word J20030062 of traditional Chinese medicines), prepare the aqueous solution of a series of concentration respectively, record longitudinal relaxation time T1 with upset-recovery sequence, then concentration is mapped, obtain characterizing the important parameter R1 value 3.18mM of its water proton relaxation rate size with its inverse
-1s
-1
Embodiment 1
1g NaH is added to Gd@C
82In the anhydrous toluene solution (5mg) (25mL), drip the anhydrous toluene solution (0.5mL) of tetraethyl Medronate ester (10 μ L) while stirring, after stirring 1h under 25 ℃, in mixed liquor, slowly drip deionized water (50mL), continuation is stirred 24h down at 25 ℃, fades fully until toluene layer.Brown water layer is with concentrating under reduced pressure after the analytical pure toluene wash three times, with sephadex G-25 phase that fixes, makes eluant with deionized water, and what the brown component of collecting pH=6~7 was the present invention's preparation contains to become the Gd@C that the bone sexual function is rolled into a ball the phospholipid base
82Soluble derivative
Gd@C
82O
2(OH)
16(C(PO
3Et
2)
2)
10(Gd-TEMDP)。
Fig. 1 has described based on metal fullerene Gd@C
82The cage preparation process of modifying the magnetic resonance imaging contrast of phospholipid base outward.
(a) contain metal fullerene Gd@C by the preparation of direct-current arc electric discharge
82The carbon soot, use CS
2, DMF, organic solvent extraction such as toluene separate purification in conjunction with high performance liquid chromatography and obtain highly purified metal fullerene Gd@C
82Toluene solution (A).
(b) get 15 times of tetraethyl Medronate esters of molar equivalent and be dissolved in (B) in an amount of anhydrous toluene solution to A.
(c) at room temperature, add 50 times of molar equivalents in the A solution, then solution B is added drop-wise in the solution A of stirring and promptly obtains mixture solution C to the NaH of tetraethyl Medronate ester.
(d) with the mixture solution C that obtains at stirring at room 1h, drip deionized water then while stirring lentamente and produce until no bubble, after continuing to stir 24h, leave standstill separatory, the colourless toluene layer of reject concentrates the brown water layer that obtains.With the polydextran gel phase that fixes, deionized water is done mobile phase, and the brown colour band of collecting pH=6~7 is the water-soluble metal fullerene Gd@C that cage of the present invention is modified with the phospholipid base outward
82Derivant (Gd-TEMDP).
The longitudinal relaxation rate R that is newly prepared the MRI contrast agent (Gd-TEMDP) that contains the phospholipid base by Fig. 3 B
1Be 58.0mM
-1s
-1The longitudinal relaxation rate R that obtains by Fig. 3 A and Fig. 3 B
1More as can be known, the radiography efficient of novel type radiographic contrast of our preparation is nearly 20 times of clinical use Gd-DTPA.
Fig. 4 illustrates that the radiography effect of this material obviously is better than clinical used Gd-DTPA.
Fig. 5 FT-IR spectrum is at 3427cm
-1The strong absworption peak at place belongs to for the stretching vibration of O-H, 2979, and 2930cm
-1The weak absworption peak ownership at place is the stretching vibration of C-H.1616,1392,1205 and 1048cm
-1The absworption peak ownership of the point at place is the bending vibration of the stretching vibration of C=C, O-H, the stretching vibration of P=O in the phospholipid base and the stretching vibration of C-OH.At 527cm
-1Weak absorption ownership be C
82The skeletal vibration peak.Disclosed the existence of the functional group of being modified.
Gd-TEMDP is in the heavy water among Fig. 6
31P-NMR spectrum only occurred at δ 16.45ppm place one unimodal, the P that has only a kind of chemical environment in this material is described.
As shown in Figure 7
Peak (C1s) |
Position (eV) |
Area |
Half-peak breadth (eV) |
%GL(%) |
C-C,C=C |
284.713 |
6643.267 |
1.742 |
30 |
C-O,C-P |
286.442 |
4208.675 |
1.735 |
80 |
C=O |
288.154 |
2813.134 |
2.169 |
80 |
As shown in Figure 8
Peak (O1s) |
Position (eV) |
Area |
Half-peak breadth (eV) |
%GL(%) |
P=O,C=O |
531.344 |
3378.000 |
2.010 |
80 |
C-O,P-O |
532.662 |
8453.000 |
2.438 |
80 |
Na(A) |
535.499 |
2029.838 |
4.311 |
80 |
According to the content of the atomic ratio of the C1s in the XPS spectrum of prepared fresh Gd-TEMDP, O1s and P2p and various element different chemical attitudes than inferring that the average mark minor that this material is Gd@C
82O
2(OH)
16(C (PO
3Et
2)
2)
10
Shown in equation 1, the Bingel additive reaction can take place with A in B in the presence of NaH.After adding water, excessive N aH can generate NaOH, and OH and intermediate product nucleophilic addition further take place generate water-soluble substances, thereby cause yellowish green Gd@C
82Toluene solution become colourlessly gradually, and that following water layer becomes is brown.
Embodiment 2
With the TNT type metal fullerene that contains Gd, as Gd through purifying
2ScN@C
80(about 10mg) is mixed with toluene solution, also the Bingel additive reaction can take place with tetraethyl Medronate ester in the presence of NaH, and 30 ℃ are stirred 2h down, and all the other conditions are with embodiment 1.
Embodiment 3
Contain Gd metal fullerene Gd with what have a radiography effect through the another kind of purifying
2@C
80(about 5mg) is mixed with toluene solution, also the Bingel additive reaction can take place with tetraethyl Medronate ester in the presence of NaH, and 23 ℃ are stirred 2h down, and all the other conditions are with embodiment 1.