Preparation method and application of polymer-gadolinium hybrid contrast agent

Abstract

The invention discloses a preparation method and application of a polymer-gadolinium hybrid contrast agent, an ultraviolet response fluorine-containing polymer is prepared by using a reversible addition fragmentation chain transfer polymerization method, and a polymer-gadolinium hybrid probe is obtained through amidation reaction connection of an aminated macrocyclic polydentate ligand and NHS and chelation of paramagnetic ions. The probe can be used for dual-mode MRI (Magnetic Resonance Imaging) of T1-wei ght 1H and 19F. The probe is good in biocompatibility, simple to prepare and high in repeatability, and a design thought, theoretical guidance and technical support are provided for preparing an intelligent probe with controllable activation, multi-mode and multifunctional imaging.

Description

Technical Field

[0001] The present invention relates to the field of nuclear magnetic resonance imaging technology, and in particular to a preparation method and application of a polymer-gadolinium hybrid contrast agent. Background Art

[0002] Magnetic resonance imaging (MRI) is widely used in the medical field due to its non-invasive nature, deep tissue penetration, and excellent spatial and tissue resolution. In order to better highlight the anatomical and pathological features of the tissue of interest, contrast agents are usually required during imaging to improve the sensitivity of MRI scans by enhancing image contrast. Paramagnetic or superparamagnetic metal ion-based compounds, such as gadolinium chelates and metal oxide nanoparticles, have achieved great success. Despite their clinical success, metal contrast agents still have some inherent disadvantages. Basically, these agents change the relaxation properties of surrounding water protons, and quantitative analysis is difficult. In addition, the large amount of water commonly present in tissues can 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 NMR sensitivity because their good magnetic properties are very close to 1 H, there is essentially no detectable background signal in the human body. Therefore, the signal intensity is a direct result of the presence of the fluorinated probe and the anatomical localization of the fluorinated particles or cells. 19 F MRI images and 1 When H density images are superimposed, fluorine-containing particles or cells can be accurately identified. 19 F MRI shows great potential in biological multimodal imaging and therapy.

[0003] The dipole-dipole interaction between unpaired electrons and nuclei in paramagnetic molecules causes the nuclear relaxation rate to accelerate, a phenomenon called the PRE effect. The PRE effect causes the nuclear magnetic signal spectrum of adjacent nuclei to become wider or even disappear, and its size is proportional to the negative sixth power of the distance (r) between the paramagnetic center and the nucleus. Various macrocyclic complexes with fluorinated functional groups have been studied as potential 19 F MRI contrast agents. These studies use the PRE effect of paramagnetic ions to improve 19 The relaxation properties of F nuclei can be exploited to shorten the acquisition time required. This type of activatable nanoprobe usually contains paramagnetic or superparamagnetic signal particles, 19 Part F and the responsive connection structure between them. Due to the presence of paramagnetic particles and self-assembly 19 F aggregation, which significantly reduces their molecular mobility and enhances 19 The transverse relaxation of the F nuclei leads to a weak 19F MRI signals, when a specific stimulus occurs, the response structure breaks and fluorine separates from the paramagnetic particles, resulting in a significant weakening of the PRE effect, thus producing a strong 19 F MRI signal. Developed for 1 H / 19 The multifunctional imaging contrast agent of F MRI can not only realize real-time visualization and accurate disease diagnosis in vivo, but also can be combined with nano-drug delivery system to realize integrated diagnosis and treatment. Summary of the invention

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

[0005] (1) How to use T1-weight 1 H and UV activatable 19 F dual-mode MRI polymer-gadolinium hybrid contrast agent and preparation method thereof;

[0006] (2) How to provide an application of UV-activated dual-mode probe in magnetic resonance imaging. The purpose of the present invention can be achieved by the following technical solutions:

[0007] A method for preparing a polymer-gadolinium hybrid contrast agent comprises the following steps:

[0008] Step 1: dissolving succinimidylbenzoate-4-hydroxymethyl-3-nitrobenzene and triethylamine in anhydrous dichloromethane, adding acryloyl chloride, and stirring at room temperature for 4 hours to obtain a UV-responsive monomer succinimidylbenzoate-o-nitroacrylate benzyl ester, i.e., FNAS;

[0009] Step 2: Add OEGA, FNAS, TFEA, PABTC, AIBN and solvent into a reaction tube at a molar feed ratio of 20:10:10:1:0.1, purge with nitrogen for 20 minutes, and then stir at 70° C. for 24 hours to obtain polymer PF;

[0010] Step 3, PF, DO3AtBu-NH2 and TEA were dissolved in DMF, degassed by nitrogen bubbling, and stirred at room temperature overnight to obtain a grafted polymer;

[0011] Step 4: Add TFA to the grafted polymer and stir overnight at room temperature, then add GdCl3·6H2O to the solution; raise the pH value to 6 by adding an alkaline solution, and stir at 50°C for 24 hours to obtain a polymer-gadolinium hybrid PF-Gd.

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

[0013]

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

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

[0016]

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

[0018]

[0019] Preferably, the grafted polymer structure is:

[0020]

[0021] Preferably, DO3AtBu-NH2 refers to a structure of of compounds.

[0022] Preferably, the solvent in step 2 includes one of N,N-dimethylformamide, 1,4-dioxane and dimethyl sulfoxide.

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

[0024] Beneficial effects of the invention: The invention relates to a method for T1-weight 1 H and UV activatable 19 F dual-mode MRI polymer-gadolinium hybrid contrast agent, the present invention uses a reversible addition-fragmentation chain transfer polymerization method to prepare a UV-responsive fluorinated polymer, and undergoes an amidation reaction of an amino macrocyclic multidentate ligand with NHS and chelation of paramagnetic ions to obtain a polymer-gadolinium hybrid probe. This probe can be used for UV-controlled PRE effect-mediated T1-weight 1 H and 19 F dual-mode MRI. This probe has good biocompatibility, simple preparation, and high repeatability, providing design ideas, theoretical guidance, and technical support for the preparation of intelligent probes with controllable activation and multi-modal and multi-functional imaging.

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

[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for describing the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.

[0027] Figure 1 A step diagram of a method for preparing a polymer-gadolinium hybrid contrast agent of the present invention;

[0028] Figure 2 The photo-cleavage ultraviolet absorption spectra of the photoresponsive fluorine-containing polymer (left) and the polymer-gadolinium (right) hybrid of the present invention;

[0029] Figure 3 The nuclear magnetic resonance fluorine spectra of fluorine-gadolinium in different states of the present invention;

[0030] Figure 4 The fluorine-gadolinium of the present invention is in different states 1 H / 19 F Magnetic resonance imaging;

[0031] Figure 5 The cytotoxicity of the hybrids of the present invention at different concentrations (left) and fluorescence microscopy images at a concentration of 150 μM (right). DETAILED DESCRIPTION

[0032] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] See also Figure 1-2 As shown, the present invention is a preparation method and application of a polymer-gadolinium hybrid contrast agent for T1-weight 1 H and UV activatable 19 F dual-mode MRI polymer-gadolinium hybrid contrast agent and its preparation method provide the application of ultraviolet activated dual-mode probe in magnetic resonance imaging.

[0034] In a first aspect, the present invention discloses a UV-activatable polymer-gadolinium hybrid, the structure of which 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 invention, x=3, y=5, z=10, and m=2.

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

[0039] (1) Succinimidylbenzoate-4-hydroxymethyl-3-nitrobenzene and triethylamine were dissolved in anhydrous dichloromethane (20 ml), and acryloyl chloride was added, and the mixture was stirred at room temperature for 4 hours to obtain a UV-responsive monomer succinimidylbenzoate-benzyl o-nitroacrylate (FNAS).

[0040] The FNAS of the present invention refers to a compound having a structure of Formula 2:

[0041]

[0042] (2) OEGA, FNAS, TFEA, PABTC, AI BN and solvent were added into a reaction tube at a molar feed ratio of 20:10:10:1:0.1, purged with nitrogen for 20 minutes, and then stirred at 70° C. for 24 hours to obtain polymer PF.

[0043] The PF of the present invention refers to a polymer having the following structure:

[0044]

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

[0046] (3) PF, DO3AtBu-NH2 and TEA were dissolved in DMF, degassed by nitrogen bubbling, and stirred at room temperature overnight to obtain a grafted polymer. The structure of the grafted polymer is shown below:

[0047]

[0048] DO3AtBu-NH2 of the present invention refers to a structure of of compounds.

[0049] (4) The grafted polymer was added to TFA and stirred at room temperature overnight, and then GdCl3·6H2O was added to the solution; the pH value was raised to 6 by adding an alkaline solution, and the mixture was stirred at 50°C for 24 hours to obtain a polymer-gadolinium hybrid PF-Gd.

[0050] In this example, a UV-activated polymer-gadolinium hybrid was prepared.

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

[0052] 2,5-Dioxopyrrolidinyl-4-(hydroxymethyl)-3-nitrobenzoate (0.60 g, 2.04 mmol) and TEA (0.268 g, 2.65 mmol) were dissolved in anhydrous dichloromethane. The solution was stirred at 0°C for 30 minutes; then, acryloyl chloride solution (0.241 g, 2.66 mmol) was added to dichloromethane (10 mL); the resulting solution was stirred at 0°C for 30 minutes, then moved to room temperature overnight, dichloromethane was removed by rotary evaporation, and then anhydrous tetrahydrofuran was added. After filtration and concentration, the crude product was purified by silica gel chromatography (n-hexane / ethyl acetate (v / v=2:1 to 1 / 1)) to obtain a white solid photoresponsive monomer FNAS.

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

[0054] The random copolymer PF was prepared by RAFT polymerization. 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), AI BN (1.180 mg, 0.007 mmol) and dioxane (0.7 mL) were added to the reaction tube. The molar feed ratio [OEGA]: [TFEA]: [FNAS]: [PABTC]: [AI BN] = 20: 10: 10: 1: 0.1. The oxygen was removed by vacuum freeze-thaw method, purged with nitrogen for 20 minutes, and then stirred at 70 ° C for 24 hours. After cooling to room temperature, the solution was slowly dripped into stirred cold ether to precipitate a yellow oily substance PF.

[0055] Example 1-3 Grafting the macrocyclic compound DO3AtBu-NH2 onto PF:

[0056] 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) were dissolved in DMF (1 mL). The solution was degassed by nitrogen bubbling and stirred overnight at room temperature. The solution was precipitated three times in cold ether and dried in a vacuum at 40 °C.

[0057] Example 1-4 Chelation of Gadolinium Ions:

[0058] The grafted polymer (0.155 g, 0.017 mmol) was added to TFA (2 mL) and stirred at room temperature overnight. TFA was removed by decompression and the mixture was dissolved in water. GdCl3·6H2O (0.052 g, 0.139 mmol) was added to the solution. The pH was raised to 6 by adding KOH solution. The solution was stirred at 50 °C for 24 hours. Free Gd was removed by adding DOTA in a molar amount of 2 times that of gadolinium. 3+ The mixture was dialyzed to remove small molecules. The dialyzed solution was freeze-dried to obtain a pale yellow solid polymer-gadolinium hybrid P[((FNAS-DO3A(Gd 3+ ))-co-TFEA-co-OEGA)]PF-Gd.

[0059] Effect verification

[0060] In the following experiments, the present invention will verify the photolysis performance, relaxation performance, 19 F NMR and T1-weight 1 H / 19 F MRI performance, biocompatibility and cellular uptake ability.

[0061] (1) UV decomposition test

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

[0063] (2) 19 F NMR testing

[0064] The photolysis of polymer PF and hybrid PF-Gd at the same mass concentration (20 mg / mL) dissolved in PBS buffer / D2O (v / v=9 / 1) and PF-Gd after photolysis were recorded on a Bruker AVANCENEO 400 instrument. 19 F NMR spectrum. The results show that the random copolymer PF shows a distinct single peak, and the signal of the hybrid polymer PF-Gd is almost invisible. After 1 hour of UV intervention, 19F NMR was recovered, which demonstrated the specificity of the mixed PF-Gd as a UV-activatable hybrid probe.

[0065] (3) Relaxation performance test

[0066] 19 The relaxation properties (T1 and T2) of F atoms were determined at 298 K using a Bruker 565 MHz spectrometer equipped with a CPBBFO probe. All measurements used a 19 μs 90° pulse, a relaxation delay of 1 s, and an acquisition time of 0.144 s. Data were collected using a 7 kHz spectrum width and 128 scans. Both parameters were measured for each sample at 25 °C. T1 measurements used an inversion recovery sequence, and T2 measurements used a CPMG sequence.

[0067] Table 1 Polymers and hybrids in different states 19 F NMR relaxation time

[0068]

[0069] The results in Table 1 show that in PF-Gd, 19 The relaxation times T1 and T2 of the F nuclei were both shortened dramatically. After one hour of UV light intervention, both T1 and T2 returned to levels similar to before. 19 F atom and Gd 3+ The change in T2 corresponds to the PRE effect between 19 The changes in F NMR and the excellent UV-controlled activation relaxation properties indicate the potential of PF-Gd as a UV-activated probe.

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

[0071] Hybrid solution MRI was performed using a 9.4T MRI scanner (Biospec 94 / 30USR Bruker) in rats. 1 H / 19 F volume coil combination 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 × 76 mm, and MTX = 256 × 256. 19 FMRI: TR=1000ms, TE=1.3247ms, FOV=30×30mm, MTX=60×60.

[0072] The results are as follows Figure 3-4As shown, the hybrid was scanned without imageable signals. Like the unmodified fluoropolymer, the hybrid PF-Gd showed bright fluorescence after UV irradiation for 1 h. 19 F MRI hot spot signals. Compared with fluorinated polymers, PF-Gd has a 19 F NMR and MRI signal intensities were low, accompanied by relatively low 19 F MRI SNR. 19 The image quality of F MRI is limited by the inherent magnetic field inhomogeneity of the instrument, which corresponds to 19 Incomplete recovery of F NMR signal and T2. 19 The activation phenomenon of F MRI signal is related to 19 The results of F NMR are consistent and provide sufficient contrast. These evidences indicate that the hybrid PF-Gd is a feasible UV-activated 19 F MRI probe. Figure 5 In the PF-Gd solution before and after photoactivation 1 H MRI( Figure 5 Right) also shows a bright signal, and a slight increase in intensity is observed after UV activation. 1 H MRI application capabilities.

[0073] (5) Biocompatibility and cellular uptake

[0074] Mouse breast cancer cells (4T1) were routinely cultured in a humid environment of 5% CO2 at 37°C. CCK-8 staining was used to detect the cytotoxicity of hybrids to 4T1. In the logarithmic growth phase, 7 concentrations and 6 wells were designed, 15,000 cells were placed in each well, 100 μL of culture medium was inoculated on a 96-well plate, and finally cultured in a carbon dioxide incubator. When the cells adhered to the cell wall and grew to a suitable density, different concentrations of drug solutions were prepared using culture medium under dark conditions. The original cell culture medium was discarded, 100 μL of hybridization solution was added to each well, the cells were cultured in a cell culture incubator for 24 h, 100 μL of CCK-8 solution was added to each well of the 96-well plate, and the culture plate was incubated in an incubator for 20 min. The light absorption value of each well was measured by an enzyme-linked immunosorbent monitor (490 nm). Figure 5 Mouse breast cancer (4T1) cells in the left middle showed a survival rate of more than 80% after incubation with different concentrations of hybridization probe for 24 hours, demonstrating its good biocompatibility. Figure 5 The overlay of the right cyan fluorescence shows the ability of the hybridized PF-Gd to enter the nucleus of cancer cells.

[0075] The above contents are merely examples and explanations of the concept of the present invention. The technicians in this technical field may make various modifications or additions to the specific embodiments described or replace them in a similar manner. As long as they do not deviate from the concept of the invention or exceed the scope defined by the claims, they should all fall within the protection scope of the present invention.

Claims

1. A method for preparing a polymer-gadolinium hybrid contrast agent, characterized in that: The method comprises the following steps: Step 1: dissolving succinimidylbenzoate-4-hydroxymethyl-3-nitrobenzene and triethylamine in anhydrous dichloromethane, adding acryloyl chloride, and stirring at room temperature for 4 hours to obtain a UV-responsive monomer succinimidylbenzoate-o-nitroacrylate benzyl ester, i.e., FNAS; Step 2: Add OEGA, FNAS, TFEA, PABTC, AIBN and solvent into a reaction tube at a molar feed ratio of 20:10:10:1:0.1, purge with nitrogen for 20 minutes, and then stir at 70° C. for 24 hours to obtain polymer PF; Step 3, PF, DO3AtBu-NH2 and TEA were dissolved in DMF, degassed by nitrogen bubbling, and stirred at room temperature overnight to obtain a grafted polymer; Step 4: Add TFA to the grafted polymer and stir overnight at room temperature, then add GdCl3·6H2O to the solution; raise the pH value to 6 by adding an alkaline solution, and stir at 50°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 formula of the polymer-gadolinium hybrid PF-Gd is: Wherein x, y, z, and m are integers between 1 and 20, including but not limited to 1, 3, 10, 15, and 20.

3. The method for preparing a polymer-gadolinium hybrid contrast agent according to claim 1, characterized in that: The structural formula of the FNAS compound is:

4. The method for preparing a polymer-gadolinium hybrid contrast agent according to claim 1, characterized in that: The structural formula of the PF polymer is:

5. The method for preparing a polymer-gadolinium hybrid contrast agent according to claim 1, characterized in that: The grafted polymer structural formula is:

6. The method for preparing a polymer-gadolinium hybrid contrast agent according to claim 5, characterized in that: The DO3AtBu-NH2 is a structure of compounds.

7. The method for preparing a polymer-gadolinium hybrid contrast agent according to claim 1, characterized in that: The solvent in step 2 includes one of N,N-dimethylformamide, 1,4-dioxane and dimethyl sulfoxide.

8. A polymer-gadolinium hybrid contrast agent, which is prepared by the preparation method of the polymer-gadolinium hybrid contrast agent according to any one of claims 1 to 7, characterized in that: This contrast agent is used in magnetic resonance imaging.

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