CN102167813B - Fluorescent tracing nanometer magnetic resonance imaging contrast agent - Google Patents

Abstract

The invention discloses a compound, as well as the preparation and application of the compound. The expression of the compound of the present invention is F ₃ O ₄ -PEG-QDTPAGd, and its structure is shown in the following formula; the compound can be used for magnetic resonance imaging detection, fluorescent tracer with fluorescent agent, nano magnetic resonance imaging, and liver Targeted contrast agents.

Description

A Fluorescent Tracer Nano Magnetic Resonance Imaging Contrast Agent

technical field

The invention relates to a compound that can be used as a contrast enhancing agent, in particular to a fluorescent tracer nano magnetic resonance imaging contrast agent with a fluorescent agent used in magnetic resonance imaging detection.

Background technique

Magnetic resonance imaging (MRI) technology is extremely effective in the early diagnosis of tumors, cardiovascular and cerebrovascular diseases and other malignant lesions, so it is widely used in medical and high-tech scientific research fields. Since the resolution of the instruments used in MRI technology is always limited, it is necessary to use contrast agents in many cases to increase image clarity. However, the development of contrast agents is far from meeting the needs of clinical diagnosis. The Magen microscope, which is still used clinically, is a polycarboxypolyamine-based Gd(III) complex, which was a German product in the 1980s. Existing magen microscopes are non-targeting, and their imaging effects still need to be improved in some occasions. Finding new targeted MRI compounds that have better effects than existing contrast agents is a research hotspot for workers in related fields.

Contents of the invention

The invention provides a compound and a preparation method of the compound. The compound can be used as a contrast agent for magnetic resonance imaging detection, and the contrast agent has a better effect than the existing Magen microscope, and has targeted fluorescence The tracer group and the Gd(III) complex are bonded on the nanometer triiron tetroxide carrier through δ-amino polyethylene glycol aminomethyl-3,4-dihydroxybenzene.

The compound of the present invention, its expression is F ₃ O ₄ -PEG-QDTPAGd, and its structure is shown in the following formula:

Formula 1

In the expression, PEG is δ-aminopolyethylene glycol aminomethyl-3,4-dihydroxybenzene, and QDTPAGd is diethylenetriamine-acetyl (8'-quinoline) amine-acetyl-triacetate gadolinium (III ). In the structural formula, F is an existing fluorophore, for example, the fluorophore F can be an off-the-shelf flavonoid fluorophore, preferably F is an 8-aminoquinoline fluorophore.

The preparation method of the preferred compound of the present invention is: using iron ferric oxide nanoparticles and δ-aminopolyethylene glycol aminomethyl-3,4-dihydroxybenzene (PEG) as raw materials, first 60mg δ-aminopolyethylene Glycolamine methyl-3,4-dihydroxybenzene is dispersed in chloroform, 150mg ferric oxide nanoparticles are dissolved in the solution of chloroform, after the mixture is fully stirred at room temperature, the modified nanoparticles are added petroleum ether to make the product Fe ₃ O ₄ -PEG-NH ₂ precipitated out, centrifuged to separate the product, dissolved with a small amount of chloroform, then added petroleum ether to produce a precipitate and centrifuged again, so repeated three times for purification, and then the modified nanoparticles Disperse in DMF for use in the next reaction; weigh 239mg of diethylenetriaminepentaacetic dianhydride (DTPAA), dissolve it in anhydrous DMF, stir for a while, add 0.8mL of anhydrous triethylamine, DTPAA is completely Dissolve in DMF, then take 69 mg of 8-aminoquinoline dissolved in 10 mL of anhydrous DMF, and then slowly add it to the DTPAA solution within 2 hours, and after fully stirring at room temperature, the mixed solution is rotary evaporated to remove excess solvent to obtain Saturated 8-AQ-DTPAA DMF solution; take an equimolar amount of saturated 8-AQ-DTPAA DMF liquid and Fe ₃ O ₄ -PEG-NH ₂ and mix and stir for a day and night, then add petroleum ether to precipitate the modified nanoparticles After centrifuging to separate the precipitate, dissolve it with a small amount of chloroform, continue to add petroleum ether to produce a precipitate, and centrifuge. After repeating this three times for purification, the modified nanoparticle Fe ₃ O ₄ -PEG-DTPA-8-AQ is obtained. It is dispersed in water for use; take equimolar gadolinium nitrate and add it to the prepared Fe ₃ O ₄ -PEG-DTPA-8-AQ dispersion, stir at room temperature for a day and night, filter the reaction solution, and then add petroleum ether Precipitate F ₃ O ₄ -PEG-QDTPAGd, dissolve the product with a small amount of chloroform after centrifugation, continue to add petroleum ether to precipitate F ₃ O ₄ -PEG-QDTPAGd, and then centrifuge, repeat the purification process three times Finally, the final product nanometer F ₃ O ₄ -PEG-QDTPAGd was dispersed in water.

The compound of the present invention can be used as a fluorescent tracer nano magnetic resonance imaging contrast agent. According to related experiments, the compound of the present invention has liver targeting, and its longitudinal relaxation efficiency is better than that of the existing MRI contrast agent Magen microscope, and the compound of the present invention also has the advantage of fluorescence display.

Description of drawings

Accompanying drawing 1 is the IR spectrogram of δ-aminopolyethylene glycol aminomethyl-3,4-dihydroxybenzene.

Accompanying drawing 2 is the IR spectrogram of Fe ₃ O ₄ -PEG-NH ₂ .

Accompanying drawing 3 is the IR spectrogram of 8-AQ-DTPAA.

Accompanying drawing 4 is the IR spectrogram of Fe ₃ O ₄ -PEG-DTPA-8-AQ.

Accompanying drawing 5 is the UV-vis spectrogram of 8-AQ-DTPAA.

Accompanying drawing 6 is the UV-vis spectrogram of Fe ₃ O ₄ -PEG-DTPA-8-AQ.

Figure 7 is the fluorescence spectrum of Fe ₃ O ₄ -PEG-DTPA-8-AQ (peak 1) and Fe ₃ O ₄ -PEG-NH ₂ (peak 2).

Figure 8 is a transmission electron microscope image of Fe ₃ O ₄ -PEG-QDTPAGd.

Accompanying drawing 9 is the MRI image of mouse liver without contrast agent (control).

Accompanying drawing 10 is the mouse liver MRI image using the compound of the present invention as a contrast agent.

Detailed ways

The preparation and corresponding test conditions of the compounds of the present invention are given below.

1. Compound preparation and characterization

1. Purchase or prepare a certain amount of ferric oxide nanoparticle raw material.

2. Purchase or prepare δ-aminopolyethylene glycol aminomethyl-3,4-dihydroxybenzene.

3. Preparation of PEG-modified magnetic nanoparticles

Weigh 60 mg of δ-aminopolyethylene glycol aminomethyl-3,4-dihydroxybenzene, disperse it in chloroform, and then add a solution of iron ferric oxide nanoparticles dissolved in chloroform into the above reaction system. The mixture was stirred at room temperature for a whole day and night, and then the modified nanoparticles were precipitated by adding petroleum ether, and the product (Fe ₃ O ₄ -PEG-NH ₂ ) was obtained by centrifugation, dissolved with a small amount of chloroform, and further precipitated by adding petroleum ether Precipitation, centrifugation, repeated washing three times in this way, and finally disperse the modified nanoparticles in DMF for use in the next reaction. The synthesis route is as follows:

Formula 2

4. Preparation of Fluorescent Nanoparticles

Weigh 239mg of diethylenetriaminepentaacetic dianhydride (DTPAA), dissolve it in anhydrous DMF, stir for a while, add 0.8mL of anhydrous triethylamine, DTPAA is completely dissolved in DMF, and then take 69mg and dissolve it in 10mL of anhydrous The 8-aminoquinoline (8-AQ) of DMF was slowly added to the DTPAA solution within 2 hours through a constant pressure dropping funnel, and after stirring at room temperature for 1 day and night, the mixed solution was rotary evaporated to obtain a small amount of liquid, which was recorded as Do 8-AQ-DTPAA. Take a small amount of 8-AQ-DTPAA in DMF, mix it with Fe ₃ O ₄ -PEG-NH ₂ and stir for a day and night. Finally, the modified nanoparticles were precipitated by adding petroleum ether, then centrifuged, and the product was dissolved with a small amount of chloroform, and then petroleum ether was added to precipitate and precipitated, centrifuged, and washed three times repeatedly, and then the modified nanoparticles _Fe3 O ₄ -PEG-DTPA-8-AQ is dispersed in water for use. The synthesis route is as follows:

Formula 3

5. Preparation of Target Contrast Agent

Add gadolinium nitrate to the prepared modified nano Fe ₃ O ₄ -PEG-DTPA-8-AQ dispersion, and stir at room temperature for a day and night. Then filter the reaction solution with cotton, then add petroleum ether to precipitate the modified nanoparticles, then centrifuge, dissolve the product with a small amount of chloroform, continue to add petroleum ether to precipitate, centrifuge, and wash repeatedly Three times, finally the modified nanoparticles are dispersed in water, the expression is F ₃ O ₄ -PEG-QDTPAGd, the structure is shown in formula 1, wherein: F is 8-aminoquinoline fluorescent group; F ₃ O ₄ It is nano ferric oxide; PEG is δ-aminopolyethylene glycol aminomethyl-3,4-dihydroxybenzene; QDTPAGd is diethylenetriamine-acetyl (8'-quinoline) amine-acetyl-triacetic acid Gadolinium(III).

2. Characterization of sample composition:

1. Sample appearance: brown oily solid, dispersed in water for 6 months without precipitation.

2. Characteristics of each component of Fe ₃ O ₄ -PEG-QDTPAGd

3. Compound Characterization

Accompanying drawing 1 is the IR spectrum of δ-aminopolyethylene glycol aminomethyl-3,4-dihydroxybenzene, and 3435cm ^-1 is the v (NH) of amino group and the v (OH) of phenolic hydroxyl group, and 1640cm ^-1 is the v (OH) of benzene Ring skeleton vibration peaks, these peaks are the characteristic IR absorption peaks of δ-aminopolyethylene glycol aminomethyl-3,4-dihydroxybenzene.

Accompanying drawing 2 is the IR spectrum of Fe ₃ O ₄ -PEG-NH ₂ , 3433cm ^-1 is the v(NH) of the amino group, 1636cm ^-1 is the skeleton vibration peak of the benzene ring, and 601cm ^-1 is the catechol compound Fe ₃ The v(Fe-O) of O ₄ proves that δ-aminopolyethylene glycol aminomethyl-3,4-dihydroxybenzene has been bonded on the nano Fe ₃ O ₄ .

Accompanying drawing 3 is the IR spectrogram of 8-AQ-DTPAA, 3425cm ^-1 is the v(NH) of amino, 1725cm ^-1 is the v(C=O) of DTPAA, and 1634cm ^-1 is the vibration peak of quinoline ring skeleton. This indicates that 8-aminoquinoline has been attached to DTPAA.

Accompanying drawing 4 is the IR spectrogram of Fe ₃ O ₄ -PEG-DTPA-8-AQ, 3424cm ^-1 is the v(NH) of amino, 1731cm ^-1 is the v(C=O) of DTPAA, 1634cm ^-1 is quinine The skeletal vibration peaks of the line ring and the benzene ring, 616cm ^-1 are v(Fe-O) of catechol and Fe ₃ O ₄ , the above characteristic peaks are consistent with the precursor IR characteristic peaks, which proves that the prepared samples are correct.

Figures 5 and 6 are the UV-vis spectra of 8-AQ-DTPAA and Fe ₃ O ₄ -PEG-DTPA-8-AQ respectively. The UV maximum absorption peaks of 8-AQ-DTPAA are at 280nm and 350nm, and Fe ₃ O 4 The UV maximum absorption peaks of ₄ -PEG-DTPA-8-AQ are at 280nm and 300nm, further proving that the conclusion of IR (accompanying drawings 2 and 3) is correct.

Accompanying drawing 7 is the fluorescence spectrogram of Fe ₃ O ₄ -PEG-DTPA-8-AQ, in the figure 358nm place front 1 is the fluorescence emission peak of Fe ₃ O ₄ -PEG-DTPA-8-AQ (connection F), 450nm Peak 2 at is the fluorescence emission peak of Fe ₃ O ₄ -PEG-NH ₂ (without connecting F), the fluorescence emission intensity of the former is about 60 times stronger than that of the latter, and the latter has almost no fluorescence. It proves that the fluorescence emission intensity of the compound of the present invention is greatly enhanced after being introduced into F.

Accompanying drawing 8 is the TEM image of Fe ₃ O ₄ -PEG-QDTPAGd. It can be seen from the figure that the target product is still uniformly dispersed nanoparticles with a particle size of about 10nm. ICP analysis shows that the molar ratio of Fe:Gd in the product is 1.82:1 .

3. Mouse live imaging comparison experiment

Accompanying drawing 9 is the MRI image of mouse liver without contrast agent (ie control group). Accompanying drawing 10 is the mouse liver MRI image using the compound of the present invention as a contrast agent, wherein the same numbers in Figures 9 and 10 indicate that the same part uses the MRI image of the existing horse root microscopic drug and uses the compound F ₃ O ₄ of the present invention - PEG-QDTPAGd MRI image. The imaging pictures in accompanying drawings 9 and 10 were all completed on the MiniMR-60 animal magnetic resonance imager, so that some imaging sequences were multi-layer spin echo sequences. In the test, the compound of the present invention was subcutaneously administered to the test mice, and the results showed that the images of the test mice treated with the compound of the present invention had obvious improvement only in the liver. Comparing Figures 9 and 10, it can be seen that the clarity of mouse liver images is significantly improved after using F ₃ O ₄ -PEG-QDTPAGd, which proves that the compound of the present invention is an MRI contrast agent targeting the liver, and its effect is better than that of the existing MRI contrast agent Magen, which can be proved by the R ₁ of the compound of the present invention is 7.69 times higher than that of Magen (see the table below).

sample name T ₁ (ms) C (mmol/L) R ₁ (Mm ^-1 s ^-1 ) Magen microscope (control group) 104.11 1.10 8.14 Fe ₃ O ₄ -PEG-QDTPAGd 30.2 0.531 62.58 H ₂ O 3771.84

The above table lists the measured values of longitudinal relaxation time (T ₁ ) of Fe ₃ O ₄ -PEG-QDTPAGd with concentration of 0.531mmol/L and 1.10mmol/L of Magen microscope and pure solvent (H ₂ O), according to T ₁ calculated the relaxation efficiency R ₁ =62.58Mm ^-1 s ^-1 for Fe ₃ O ₄ -PEG-QDTPAGd, and R ₁ =8.14Mm ^-1 s ^-1 for Magen Microscope. It can be seen that the _R1 of the compound of the present invention is 7.69 times higher than that of the Magen Microscope, which proves that its imaging effect is better than that of the existing MRI contrast agent Magen Microscope.

Claims (4)

1. compound, its expression formula is Fe ₃O ₄-PEG-QDTPAGd, structure is as shown in the formula showing:

PEG is amino polyoxamide methyl-3 in the expression formula, and 4-dihydroxy-benzene, QDTPAGd are that diethylenetriamine-acetyl (8 '-quinoline) amine-ethanoyl-nitrilotriacetic closes gadolinium (III), and F is a fluorophor in the structural formula, Fe ₃O ₄Be nano ferriferrous oxide.

2. the described compound of claim 1 is characterized in that F is a 8-quinolylamine fluorophor.

3. the preparation method of the compound of claim 2; It is characterized in that: with ferriferrous oxide nano-particle and δ-amino polyoxamide methyl-3; The 4-dihydroxy-benzene is a raw material, and earlier with 60mg δ-amino polyoxamide methyl-3, the 4-dihydroxy-benzene is scattered in the chloroform; The 150mg ferriferrous oxide nano-particle is dissolved in the solution of chloroform, fully stirs the back under the mixture room temperature and makes product Fe through adding sherwood oil at adorned nanoparticle ₃O ₄-PEG-NH ₂Be precipitated out, spinning goes out product, with a spot of trichloromethane dissolving, adds sherwood oil more therein and produces deposition and recentrifuge separation, carries out purifying three times so repeatedly, will be dispersed among the DMF by the decorated nanometer particle then, uses in order to next step reaction; Weighing 239mg diethylene triamine pentacetic acid (DTPA) dianhydride (DTPAA) is dissolved in dry DMF with it, stirs moments later; Add the 0.8mL anhydrous triethylamine, DTPAA is dissolved in DMF fully, then gets the 8-quinolylamine that 69mg is dissolved in the 10mL dry DMF; In 2 hours, it is slowly joined in the DTPAA solution again; After fully stirring under the room temperature, the mixed solution rotary evaporation is removed unnecessary solvent, obtain the DMF solution of the 8-AQ-DTPAA of saturated solution; Get DMF liquid and the Fe of the saturated 8-AQ-DTPAA of equimolar amount ₃O ₄-PEG-NH ₂Mix and stir diel, add sherwood oil more therein and make by the decorated nanometer particle precipitation, spinning goes out post precipitation again with a spot of trichloromethane dissolving; Continue to add sherwood oil and produce deposition; Spinning, behind the triplicate purifying like this, will be by decorated nanometer particle Fe ₃O ₄-PEG-DTPA-8-AQ is dispersed in the water for use; Equimolar Gadolinium trinitrate is joined the Fe that has prepared ₃O ₄-PEG-DTPA-8-AQ is by in the decorated nanometer particle dispersion, and the stirring at room diel behind the filtering reacting liquid, adds sherwood oil more therein and makes Fe ₃O ₄-PEG-QDTPAGd is precipitated out, and dissolves with a spot of trichloromethane through the spinning after product, continues to add sherwood oil and makes Fe ₃O ₄-PEG-QDTPAGd is precipitated out, and spinning again is so repeatedly after the purification process three times, at last with adorned nanoparticle Fe ₃O ₄-PEG-QDTPAGd is dispersed in the water.

4. claim 1 or the 2 described compounds application that is to prepare the fluorescent tracing nano magnetic resonance imaging contrast agent.

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