A method for preparing a polymer-gadolinium hybrid contrast agent and its use

Abstract

The application discloses a preparation method and application of a polymer-gadolinium hybrid contrast agent, and utilizes a reversible addition-fragmentation chain transfer polymerization method to prepare an ultraviolet light response fluorine-containing polymer, and the polymer is connected through an amidation reaction of an aminated macrocyclic polydentate ligand and NHS and chelation of a paramagnetic ion to obtain a polymer-gadolinium hybrid probe. 1 H and 19 F dual-mode MRI. The probe has good biocompatibility, simple preparation and high repeatability, and provides design ideas, theoretical guidance and technical support for preparation of an intelligent probe capable of controllable activation and multi-modal and multi-functional imaging.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of nuclear magnetic resonance imaging technology, and particularly relates to a preparation method of a polymer-gadolinium hybrid contrast agent and application thereof. BACKGROUND

[0002] Magnetic resonance imaging (MRI) is widely used in the medical field due to its non-invasiveness, deep tissue penetration, and excellent spatial and tissue resolution. In order to better highlight the anatomical and pathological features of the tissue of interest, a contrast agent is usually used during imaging to improve the sensitivity of MRI scanning by enhancing the image contrast. Paramagnetic or superparamagnetic metal ion-based compounds, such as gadolinium chelates and oxidized metal nanoparticles, have achieved great success. Although metal contrast agents have been successful in the clinic, they still have some inherent drawbacks. Basically, these agents change the relaxation properties of the surrounding water protons, and quantitative analysis is difficult. In addition, the large amount of water present in tissues will cause significant background signal interference, sometimes making it difficult to identify the target tissue. At the same time, there are certain potential safety issues. 19 F nuclei show excellent nuclear magnetic resonance sensitivity because their good magnetism is very close to 1 H, and there is basically no detectable background signal in the human body. Therefore, signal intensity is a direct result of the presence of fluorinated probes and the anatomical localization of fluorine-containing particles or cells. When 19 F MRI images are superimposed on 1 H density images, fluorine-containing particles or cells can be accurately identified. 19 F MRI shows great potential in biological multi-modal imaging and therapy.

[0003] The dipole-dipole interaction between unpaired electrons in paramagnetic molecules and atomic nuclei causes the atomic nucleus to relax at a faster rate, which is known as the PRE effect. The PRE effect causes the nuclear magnetic signal of adjacent atomic nuclei to become broadened or even disappear, and its size is proportional to the negative sixth power of the distance (r) between the paramagnetic center and the atomic nucleus. Various macrocyclic complexes with fluorine-containing functional groups have been studied as potential 19 F MRI contrast agents. These studies use the PRE effect of paramagnetic ions to improve the relaxation properties of 19 F nuclei to shorten the required acquisition time. This type of activatable nanoprobes usually contains paramagnetic or superparamagnetic signal particles, 19 F moieties, and a responsive linking structure between them. Due to the presence of paramagnetic particles and self-assembly leading to 19 F aggregation, their molecular mobility is significantly reduced, and the transverse relaxation of 19 F nuclei is enhanced, resulting in weak 19F MRI signal, when a specific stimulus occurs, in response to the structure breaks, fluorine and paramagnetic particles are separated, resulting in PRE effect significantly weakened, thus producing a strong 19 F MRI signal. Develop a method for 1 H / 19 F MRI multifunctional imaging contrast agent, not only can realize real-time in vivo visualization and accurate disease diagnosis, but also can be combined with nano drug delivery system to realize diagnosis and treatment integration. SUMMARY

[0004] The purpose of the present application is to provide a preparation method of polymer-gadolinium hybrid contrast agent and its application, to solve the following technical problems:

[0005] (1) how to be used for T1-weight 1 H and ultraviolet activated 19 F dual-mode MRI polymer-gadolinium hybrid contrast agent and a preparation method thereof;

[0006] (2) how to provide the application of ultraviolet activated dual-mode probe in magnetic resonance imaging. The purpose of the present application can be realized by the following technical scheme:

[0007] A preparation method of polymer-gadolinium hybrid contrast agent, comprising the following steps:

[0008] Step one, dissolve succinimidyl benzoate-4-hydroxymethyl-3-nitrobenzoate and triethylamine in anhydrous dichloromethane, and add acryloyl chloride, stir at room temperature for 4h to obtain ultraviolet responsive monomer succinimidyl benzoate-o-nitrobenzyl acrylate, namely FNAS;

[0009] Step two, OEGA, FNAS, TFEA, PABTC, AIBN are added to the reaction tube in a molar feed ratio of 20:10:10:1:0.1 and solvent, purged with nitrogen for 20 minutes, then stirred at 70℃ for 24 hours to obtain polymer PF;

[0010] Step three, dissolve PF, DO3AtBu-NH2 and TEA in DMF, degas by nitrogen bubbling, and stir overnight at room temperature to obtain grafted polymer;

[0011] Step four, add the grafted polymer to TFA and stir overnight at room temperature, then add GdCl3·6H2O to the solution; increase the pH value to 6 by adding alkaline solution, and stir at 50℃ for 24 hours to obtain polymer-gadolinium hybrid PF-Gd.

[0012] Preferably, the structure of the polymer-gadolinium hybrid PF-Gd is shown as formula 1:

[0013]

[0014] wherein x, y, z, m are integers between 1-20, including but not limited to 1, 3, 10, 15, 20.

[0015] Preferably, the structure of the FNAS compound is:

[0016]

[0017] Preferably, the structure of the PF polymer is:

[0018]

[0019] Preferably, the structure of the grafted polymer is:

[0020]

[0021] Preferably, DO3AtBu-NH2 refers to a compound with the structure:

[0022] Preferably, the solvent in step two includes one of N,N-dimethylformamide, 1,4-dioxane, dimethyl sulfoxide.

[0023] The present application also proposes a polymer-gadolinium hybrid contrast agent, which is obtained by the preparation method mentioned above and is applied to magnetic resonance imaging.

[0024] The beneficial effects of the present application: the present application is about a kind of for T1-weight 1 H and ultraviolet activatable 19 F dual-mode MRI polymer-gadolinium hybrid contrast agent, the present application uses reversible addition fragmentation chain transfer polymerization method to prepare ultraviolet light response fluoropolymer, and undergoes amidation reaction connection and paramagnetic ion chelation of amination macrocyclic polydentate ligand and NHS, to obtain polymer-gadolinium hybrid probe. This probe can be used for ultraviolet light control PRE effect mediated T1-weight 1 H and 19 F dual-mode MRI. And this probe has good biocompatibility, simple preparation, high repeatability, provides design ideas, theoretical guidance and technical support for the preparation of controllable activation, multi-modal multi-functional imaging of intelligent probe.

[0025] Of course, any product implementing the present application does not necessarily need to achieve all the advantages described above at the same time. BRIEF DESCRIPTION OF DRAWINGS

[0026] ​In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the description of the embodiments. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without any creative effort based on these drawings.

[0027] Figure 1 The preparation method of the polymer-gadolinium hybrid contrast agent of the present application is shown in the following steps.

[0028] Figure 2 The photolysis UV absorption spectrum of the light-responsive fluoropolymer (left) and the polymer-gadolinium (right) hybrid of the present application is shown in the following figure.

[0029] Figure 3 The nuclear magnetic resonance fluorography of the fluorine-gadolinium in different states of the present application is shown in the following figure.

[0030] Figure 4 The magnetic resonance imaging of the fluorine-gadolinium in different states of the present application is shown in the following figure. 1 H / 19 F

[0031] Figure 5 The cytotoxicity of the hybrid in different concentrations (left) and the fluorescence microscope picture under the concentration of 150 μΜ (right) of the present application is shown in the following figure. DETAILED DESCRIPTION

[0032] The technical solutions of the embodiments of the present application will be described clearly and completely in the following with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only some embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without any creative effort are within the scope of protection of the present application.

[0033] Please refer to Figures 1-2 The present application is a preparation method of a polymer-gadolinium hybrid contrast agent and its application, which is used for T1-weighted 1 H and ultraviolet-activated 19 F dual-mode MRI polymer-gadolinium hybrid contrast agent and its preparation method, which provides the application of the ultraviolet-activated dual-mode probe in magnetic resonance imaging.

[0034] In the first aspect, the present application discloses an ultraviolet-activated polymer-gadolinium hybrid, and the structure of the hybrid is shown in formula 1:

[0035]

[0036] wherein x, y, z, and m are integers between 1 and 20, including but not limited to 1, 3, 10, 15, and 20.

[0037] In the most preferred embodiment of the present application, x = 3, y = 5, z = 10, m = 2.

[0038] In a second aspect, the present application provides a method for preparing an ultraviolet-activated polymer-gadolinium hybrid, the method comprising the following steps:

[0039] (1) Dissolve the succinimidyl benzoate-4-hydroxymethyl-3-nitrobenzoate and triethylamine in anhydrous dichloromethane (20 ml), and add acryloyl chloride, stir at room temperature for 4 h to obtain the ultraviolet-responsive monomer succinimidyl benzoate-o-nitrobenzyl acrylate (FNAS).

[0040] The FNAS of the present application refers to a compound with the structure of formula 2:

[0041]

[0042] (2) Add OEGA, FNAS, TFEA, PABTC, AIBN to the reaction tube in a molar feed ratio of 20:10:10:1:0.1 and solvent, purge with nitrogen for 20 minutes, then stir at 70°C for 24 hours to obtain the polymer PF.

[0043] The PF of the present application refers to a polymer with the following structure:

[0044]

[0045] The solvent in the above preparation method includes one of N,N-dimethylformamide, 1,4-dioxane, dimethyl sulfoxide.

[0046] (3) Dissolve the PF, DO3AtBu-NH2 and TEA in DMF, degas by nitrogen bubbling, and stir overnight at room temperature to obtain the grafted polymer. The structure of the grafted polymer is shown as follows:

[0047]

[0048] The DO3AtBu-NH2 of the present application refers to a compound with the structure of .

[0049] (4) Add the grafted polymer to TFA and stir overnight at room temperature, then add GdCl3·6H2O to the solution; increase the pH value to 6 by adding an alkaline solution, and stir at 50°C for 24 hours to obtain the polymer-gadolinium hybrid PF-Gd.

[0050] In this example, the preparation of the ultraviolet-activated polymer-gadolinium hybrid.

[0051] Example 1-1 Preparation of ultraviolet light-responsive monomer FNAS:

[0052] Dissolve 2,5-dioxopyrrolidinyl-4-(hydroxymethyl)-3-nitrobenzoate (0.60 g, 2.04 mmol) and TEA (0.268 g, 2.65 mmol) in anhydrous dichloromethane. Stir the solution at 0 °C for 30 min; then, add a solution of acryloyl chloride (0.241 g, 2.66 mmol) in dichloromethane (10 mL); stir the resulting solution at 0 °C for 30 min, then move to room temperature overnight, remove the dichloromethane by rotary evaporation, then add anhydrous tetrahydrofuran. After filtration and concentration, the crude product is purified by silica gel chromatography (n-hexane / ethyl acetate (v / v = 2:1 to 1 / 1)) to give the photoresponsive monomer FNAS as a white solid.

[0053] Example 1-2 Synthesis of linear random copolymer P(FNAS-co-TFEA-co-OEGA) PF:

[0054] The random copolymer PF is prepared using RAFT polymerization method, OEGA (0.690 g, 1.44 mmol), TFEA (0.110 g, 0.72 mmol), FNAS (0.250 g, 0.72 mmol), PABTC (0.017 g, 0.072 mmol), AIBN (1.180 mg, 0.007 mmol) and dioxane (0.7 mL) are added into a reaction tube. The molar feed ratio [OEGA]:[TFEA]:[FNAS]:[PABTC]:[AIBN] = 20:10:10:1:0.1. Remove oxygen by vacuum freeze-thaw method, purged with nitrogen for 20 min, then stir at 70 °C for 24 h. After cooling to room temperature, the solution is slowly dropped into stirred cold ether to precipitate the yellow oil PF.

[0055] Example 1-3 Grafting of macrocycle DO3AtBu-NH2 to PF:

[0056] Dissolve P(FNAS-co-TFEA-co-OEGA) (0.107 g, 0.016 mmol), DO3AtBu-NH2 (0.050 g, 0.081 mmol) and TEA (0.002 g, 0.019 mmol) in DMF (1 mL). Bubble the solution with nitrogen to degas and stir at room temperature overnight. Precipitate the solution in cold ether three times and dry in vacuum at 40 °C.

[0057] Example 1-4 Chelation of gadolinium ions:

[0058] Graft polymer (0.155 g, 0.017 mmol) was added to TFA (2 mL) and stirred at room temperature overnight. TFA was removed by reduced pressure and the mixture was dissolved in water. To the solution was added GdCl3-6H2O (0.052 g, 0.139 mmol). The pH value was raised to 6 by adding KOH solution. The solution was stirred at 50 °C for 24 hours. Free Gd was removed by adding 2 molar equivalents of gadolinium DOTA to the solution. The mixture was dialyzed to remove small molecules. The dialyzed solution was freeze-dried to obtain a light yellow solid polymer-gadolinium hybrid P[((FNAS-D03A(Gd 3+ )-co-TFEA-co-OEGA)]PF-Gd. 3+

[0059] Effect validation

[0060] In the following experiments, the present application will validate the photolysis performance, relaxation performance, 19 F NMR and T1-weight 1 H / 19 F MRI performance, biocompatibility and cellular uptake ability of the ultraviolet activated polymer-gadolinium hybrid bimodal probe.

[0061] (1) Ultraviolet decomposition test

[0062] Solutions of 0.1 mg / mL polymer and hybrid were prepared in DMSO and exposed to UV irradiation (0.210 W / cm 2 , 365 nm) in a quartz cuvette for a specific time (0-3000 s); UV-Vis spectra were obtained using a HITACHI U-4100. The hybrid and polymer showed similar photolysis behavior, as shown in Figure 2 . The absorbance of the UV responsive group corresponding to the spectral peak at 310 nm decreased within 1 h and tended to be balanced, indicating that the photolysis behavior had ended, and good photodecomposition properties were the basis for achieving activatable magnetic resonance imaging.

[0063] (2) 19 F NMR test

[0064] F NMR spectra of the same mass concentration (20 mg / mL) of polymer PF and hybrid PF-Gd and PF-Gd after photolysis were recorded on a Bruker AVANCE NEO 400 instrument in PBS buffer / D2O (v / v = 9 / 1) solution. The results show that the random copolymer PF shows a clear single peak, and the signal of the hybrid polymer PF-Gd is almost invisible. After 1 hour of intervention by ultraviolet light, 19 19 ​​F NMR recovery. This indicates the specificity of the mixed PF-Gd as UV-activatable hybrid probes.

[0065] (3) Relaxation properties test

[0066] 19 The relaxation properties (T1and T2) of the F atoms were determined at 298 K on a Bruker 565 MHz spectrometer equipped with a CPBBFO probe. All measurements used a 90° pulse of 19 μs, a relaxation delay of 1 s, and an acquisition time of 0.144 s. Data were collected using a spectral width of 7 kHz and 128 scans. Two parameters were measured for each sample at 25 °C. T1measurements used an inversion recovery sequence and T2measurements used a CPMG sequence.

[0067] Table 1. Different states of polymer and hybrid 19 F NMR relaxation times

[0068]

[0069] Table 1. Results show that in PF-Gd, 19 The relaxation times T1and T2of the F nuclei are both drastically shortened. After 1 hour of intervention with UV light, both T1and T2are recovered to similar levels as before. This is all related to the PRE effect between the F atoms and the Gd 19 3+ The PRE effect between the F atoms and the Gd 19 The change in T2corresponds to the change in F NMR, and the excellent UV-controllable activation relaxation properties indicate the potential of PF-Gd as UV-activatable probes.

[0070] (4) T1-wei ght 1 H / 19 F MRI performance test

[0071] Hybrid solution MRI was tested using a 9.4 T MRI scanner (Biospec 94 / 30USR Bruker) with a rat body 1 H / 19 F volume coil combined 19 F META coil. 1 H MRI was collected using a 2D FLASH sequence: TE = 2.3 ms, TR = 100 ms, FA = 30.0, FOV = 76 x 76 mm, and MTX = 256 x 256. F MRI was collected using a 2D FLASH sequence: TR = 1000 ms, TE = 1.3247 ms, FOV = 30 x 30 mm, MTX = 60 x 60. 19

[0072] Results are shown in Figures 3-4 ​​As shown, no imageable signal was detected in the hybrid. Similar to the unmodified fluoropolymer, the hybrid PF-Gd exhibited a bright appearance after 1 hour of UV irradiation. 19 fMRI hotspot signal. Compared with fluoropolymers, PF-Gd shows stronger photoactivated signal after fluoropolymerization. 19 Low F NMR and MRI signal intensity, accompanied by relatively low 19 f MRI SNR. 19 The image quality of fMRI is limited by the inherent magnetic field inhomogeneity of the instrument, which corresponds to 19 Incomplete recovery of F NMR signal and T2. However 19 Activation phenomenon of fMRI signal and 19 The F NMR results were consistent and provided sufficient contrast. This evidence suggests that the hybrid PF-Gd is a viable UV-activated [method / approach]. 19 fMRI probe. In Figure 5 In the middle, the PF-Gd solution before and after photoactivation 1 H MRI ( Figure 5 The right side also showed a bright signal, and a slight increase in intensity was observed after UV activation. This demonstrates its T1-weighted signal strength. 1 Application capabilities in H MRI.

[0073] (5) Biocompatibility and cellular uptake capacity

[0074] Mouse breast cancer cells (4T1) were routinely cultured in a humid environment of 37°C and 5% CO2. CCK-8 staining was used to detect the cytotoxicity of the hybrids against 4T1 cells. During the logarithmic growth phase, seven concentrations of the drug were prepared in six wells, with 15,000 cells per well. 100 μL of culture medium was seeded into each well of a 96-well plate, and the plates were then incubated in a CO2 incubator. When the cells adhered to the cell wall and grew to a suitable density, drug solutions of different concentrations were prepared using culture medium under dark conditions. The original cell culture medium was discarded, and 100 μL of hybridization solution was added to each well. The cells were cultured in a cell culture incubator for 24 h. Then, 100 μL of CCK-8 solution was added to each well of the 96-well plate, and the plates were incubated for 20 min. The absorbance of each well was measured using an enzyme-linked immunosorbent assay (ELISA) monitor (490 nm). Figure 5 Left-handed medullomyeloid breast cancer (4T1) cells showed a survival rate exceeding 80% after 24 hours of incubation with different concentrations of hybridization probes, demonstrating their good biocompatibility. Furthermore, after 2 hours of incubation at 150 μM... Figure 5 The overlapping of the blue-green fluorescence on the right demonstrates the ability of hybridized PF-Gd to enter the nucleus of cancer cells.

[0075] The above merely illustrates and describes the concept of the present application, and those skilled in the art can make various modifications or supplements to the described specific embodiments or adopt similar ways to replace, as long as the modifications or supplements do not deviate from the concept of the present application or exceed the scope defined by the present claims, and the modifications or supplements shall fall within the protection scope of the present application.

Claims

1. A method for the preparation of a polymeric-gadolinium hybrid contrast agent, characterized in that, The method comprises the following steps: Step one, oxalylamino benzoate-4-hydroxymethyl-3-nitrobenzoate and triethylamine are dissolved in anhydrous dichloromethane, and acryloyl chloride is added, and stirring is carried out at room temperature for 4h to obtain an ultraviolet responsive monomer oxalylamino benzoate-benzyl o-nitroacrylate, namely FNAS; Step two, OEGA, FNAS, TFEA, PABTC, AIBN are added into a reaction tube in a molar feeding ratio of 20:10:10:1:0.1 and a solvent, and then purged with nitrogen for 20 minutes, and then stirred at 70 DEG C for 24 hours to obtain a polymer PF; the structural molecular formula of the polymer PF is: ； wherein x, y and z are integers between 1 and 20; Step three, PF, DO3AtBu-NH2 and TEA are dissolved in DMF, degassed by nitrogen bubbling, and stirred at room temperature overnight to obtain a grafted polymer; Step four, the grafted polymer is added into TFA and stirred at room temperature overnight, then GdCl3·6H2O is added into the solution; the pH value is increased to 6 by adding an alkaline solution, and then stirred at 50 DEG C for 24 hours to obtain a polymer-gadolinium hybrid PF-Gd.

2. The method for preparing a polymer-gadolinium hybrid contrast agent according to claim 1, characterized in that, The structural molecular formula of the polymer-gadolinium hybrid PF-Gd is: ； wherein x, y, z and m are integers between 1 and 20.

3. The method for preparing a polymer-gadolinium hybrid contrast agent according to claim 1, characterized in that, The structural molecular formula of the grafted polymer is: ； wherein x, y, z and m are integers between 1 and 20.

4. The method for preparing a polymer-gadolinium hybrid contrast agent according to claim 3, characterized in that, The DO3AtBu-NH2 refers to a compound having the structure of the following compound.

5. The method for preparing a polymer-gadolinium hybrid contrast agent according to claim 1, characterized in that, The solvent in step two comprises one of N,N-dimethylformamide, 1,4-dioxane and dimethyl sulfoxide.

6. A polymeric-gadolinium hybrid contrast agent produced by the method of any one of claims 1 to 5, characterized in that, The contrast agent is applied to magnetic resonance imaging.

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