Preparation method of fluorine magnetic resonance imaging polymer probe containing polyamino acid skeleton

Abstract

The present invention discloses a method for preparing a fluorine magnetic resonance imaging polymer probe containing a polyamino acid backbone, which belongs to the technical field of application-based material preparation. In the present invention, an organic fluorine-containing small molecule containing a thioether functional group is grafted onto the backbone of polysuccinimide through the ring-opening reaction of amino-lactam, and then the thioether is oxidized to form sulfoxide, forming a hydrophilic probe with high fluorine atom loading. 19 FMRI polymer probe, the polymer probe obtained by the present invention has a high fluorine content (> 20%), the sulfoxide group and the polyamino acid skeleton formed after the aminolysis of polysuccinimide work together to give the probe extremely good water solubility, so that the fluorine atoms inside the probe are highly loaded while maintaining the good activity of the fluorine-containing fragment, which can be used for high-sensitivity 19 F magnetic resonance imaging; the probe also has good biocompatibility, which is conducive to the detection of deep tissue in living bodies and has important application significance in the fields of materials science and biomedicine.

Description

Technical Field

[0001] The present invention relates to the technical field of preparation of applied biomedical materials, and in particular to a method for preparing a fluorine magnetic resonance imaging polymer probe containing a polyamino acid skeleton and high fluorine atom loading. Background Art

[0002] Magnetic resonance imaging is a molecular imaging diagnostic technology that is non-ionizing radiation, non-invasive, has high spatial resolution, and deep imaging penetration. However, a large number of water molecules in the human body are hydrogen MRI ( 1 While HMRI provides rich information, it also gives it a strong background signal, which can easily lead to "false positive" or "false negative" diagnostic results. The accurate diagnosis of hydrogen magnetic resonance imaging places high demands on the experience and diagnostic technology of clinicians. 19 FMRI) in the retention 1 While HMRI offers the advantages of zero background noise, it facilitates quantification and tracing, providing more pathological details for disease diagnosis. Over the past decade, perfluorinated nanoemulsions have attracted widespread attention due to their exceptional sensitivity. However, nanoemulsions still face challenges with instability and difficulty in functionalization.

[0003] at present, 19 FMRI polymer probes have attracted attention due to their strong modifiability, good stability and high biocompatibility. 19 There are two main strategies for preparing polymer probes for fMRI. The first involves copolymerizing fluorinated monomers with hydrophilic monomers. However, the resulting polymers, which are mixed with fluorinated monomers and have irregular structures, are exposed to different chemical environments by the fluorine atoms, and their NMR signals are dispersed, making them unsuitable for MRI applications. (Chin. J. Chem. 2018, 36, 25-30; Biomacromolecules, 2019, 20, 790-800.) The second method involves functionalizing the polymers with fluorinated chain transfer reagents based on perfluoropolyethers. However, these polymers exhibit multiple peaks in the NMR spectrum, reducing the effective utilization of fluorine atoms and, consequently, signal intensity. (ACS Nano, 2018, 12, 9162-9176; ACS Macro Lett. 2022, 11, 1195–1201.) Moreover, due to the strong dipole-dipole interaction between fluorine atoms, the fluorinated polymers prepared by these two strategies usually have a high fluorine loading, which leads to the weakening of fluorine nucleus activity due to the aggregation of fluorine atoms. 19 The reduced sensitivity of F magnetic resonance imaging limits its practical application.

[0004] Therefore, it is necessary to develop a novel nanostructured ...19 The preparation method of FMRI polymer probes is of great significance. Summary of the Invention

[0005] In view of the problems existing in the background technology, the purpose of the present invention is to provide a method for preparing a fluorine magnetic resonance imaging polymer probe containing a polyamino acid skeleton, combining a hydrophilic organic fluorine-containing small molecule with a polyamino acid skeleton to form a high fluorine atom loaded hydrophilic fluorine magnetic resonance imaging ( 19 FMRI) polymer probes.

[0006] The present invention is achieved in that:

[0007] The present invention provides a method for preparing a fluorine magnetic resonance imaging polymer probe containing a polyamino acid skeleton. The specific preparation steps are as follows: a. mixing an organic fluorine-containing small molecule with polysuccinimide for reaction, wherein the organic fluorine-containing small molecule has a thioether functional group and an amino group, and grafting the organic fluorine-containing small molecule onto the polysuccinimide skeleton through an amino-lactam ring-opening reaction to obtain an intermediate product; b. oxidizing the thioether group in the intermediate product to form a sulfoxide group through an oxidation reaction to obtain a fluorine magnetic resonance imaging polymer probe containing a polyamino acid skeleton.

[0008] Specifically, the organic fluorine-containing small molecule is 2-[(2,2,2-trifluoroethyl)thio]ethylamine.

[0009] Specifically, the molecular weight of the polysuccinimide is 500 to 20,000, preferably 2,000 to 15,000. By using polysuccinimide of different molecular weights as raw materials, super hydrophilic ... 19 FMRI polymer probes.

[0010] The step a is specifically as follows: dissolving polysuccinimide and organic fluorine-containing small molecules in a solvent, mixing them evenly, reacting them at 60-120° C. for 3-8 hours, adding a precipitant for precipitation, centrifuging and discarding the supernatant, washing, and drying to obtain a solid intermediate product.

[0011] In the step a, the solvent used is N,N-dimethylformamide (DMF) or dimethyl sulfoxide (DMSO); the precipitant used is a mixed solution of dichloromethane and n-hexane, and the volume ratio of dichloromethane to n-hexane is 1:10 to 1:5; the washing process uses a mixed solution of tetrahydrofuran (THF) and n-hexane for washing, and the volume ratio of tetrahydrofuran to n-hexane is 1:10 to 1:5.

[0012] The step b specifically comprises: dissolving the obtained intermediate product in a solvent, adding an oxidant and reacting at 40-80° C. for 6-96 hours, adding a precipitant, centrifuging, and drying to obtain a fluorine magnetic resonance imaging polymer probe containing a polyamino acid skeleton.

[0013] In the step b, the oxidant used is hydrogen peroxide, the precipitant used is a mixed solution of ethanol and diethyl ether, and the volume ratio of ethanol to diethyl ether is 1:6 to 1:3.

[0014] Furthermore, in the step a, 0.2-0.6 g of polysuccinimide and 265-1200 μL of 2-[(2,2,2-trifluoroethyl)thio]ethylamine are dissolved in 4-12 mL of N,N-dimethylformamide or dimethyl sulfoxide, mixed evenly, and reacted at 60-120° C. for 3-8 h.

[0015] In the step b, the intermediate product obtained in the step a is dissolved in 2-8 mL of acetone, and 200-1200 μL of 30% by mass H 2 O 2 is added thereto, and the mixture is reacted at 40-80° C. for 6-96 hours.

[0016] On the other hand, the present invention also provides a fluorine magnetic resonance imaging polymer probe containing a polyamino acid backbone prepared according to the above method as 19 Application of F magnetic resonance imaging contrast agents.

[0017] Beneficial effects of the present invention:

[0018] (1) In the fluorine magnetic resonance imaging polymer probe containing a polyamino acid backbone prepared by the present invention, polysuccinimide is a polyamino acid analog after aminolysis, which has good hydrophilicity and biocompatibility. Combined with the good hydrophilicity of the sulfoxide group, the probe has good water solubility and high mobility of the fluorine atom. This allows the probe to have a high fluorine content (>20%) while maintaining the good mobility and relaxivity of the fluorine-containing segment, thereby enabling high-sensitivity fluorine magnetic resonance imaging.

[0019] (2) The fluorine magnetic resonance imaging polymer probe containing a polyamino acid backbone prepared by the present invention has a high 19 FMRI signals can be highly sensitive 19 FMRI test; and as the polymer concentration increases, 19 The FMRI signal is linearly enhanced, overcoming the problem of weak imaging signal commonly faced by fluorinated polymer nanoprobes due to increased fluorine atom concentration and limited mobility.

[0020] (3) The fluorine magnetic resonance imaging polymer probe containing a polyamino acid skeleton prepared by the present invention has excellent biocompatibility and stability, and is expected to be applied to deep tissue in vivo. 19 FMRI has a simple preparation method and has great potential in future clinical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0022] Figure 1 The polymer probe prepared in Example 1 at different concentrations 19 FNMR signal and 19 Linear relationship between FNMR signal and probe concentration.

[0023] Figure 2 These are the T1 and T2 relaxation times of the polymer probe prepared in Example 1 at different concentrations.

[0024] Figure 3 This is a graph showing the cytotoxicity results of the polymer probe prepared in Example 1 in MREpic and MB49 cell lines.

[0025] Figure 4 This is a graph showing the hemolysis test results of the polymer probe prepared in Example 1 at different concentrations.

[0026] Figure 5 The polymer probe prepared in Example 1 at different concentrations 1 HMRI and 19 FMRI imaging and 19 Linear relationship between FMRI signal-to-noise ratio and probe concentration. DETAILED DESCRIPTION

[0027] In order to better explain the present invention, the present invention will be described in detail with reference to the embodiments of the present invention, and the main contents of the present invention will be further illustrated in conjunction with specific examples, but the content of the present invention is not limited to the following examples. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in this area or the product specifications are used. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0028] The present invention provides a method for preparing a fluorine magnetic resonance imaging polymer probe containing a polyamino acid skeleton with high fluorine atom loading. By a one-pot synthesis method, an organic fluorine-containing small molecule containing a thioether functional group is grafted onto the skeleton of polysuccinimide through an amino-lactam ring-opening reaction. Subsequently, through an oxidation reaction, the thioether is oxidized to form a sulfoxide, thereby increasing the hydrophilicity, thereby forming a high fluorine atom loading hydrophilic polymer probe. 19 The preparation method of the FMRI polymer probe is simple and specifically includes the following steps:

[0029] a. Dissolve 0.2-0.6 g of polysuccinimide and 265-1200 μL of 2-[(2,2,2-trifluoroethyl)thio]ethylamine in 4-12 mL of DMF or DMSO, mix well, and react at 60-120° C. for 3-8 hours. Precipitate the resulting solution with a dichloromethane / n-hexane mixture (v:v = 1:10 to v:v = 1:5), centrifuge, and discard the supernatant. Wash the precipitate twice with a THF / n-hexane mixture (v:v = 1:10 to v:v = 1:5) and dry to obtain a solid.

[0030] b. The solid obtained in step a was dissolved in 2 to 8 mL of acetone, 200 to 1200 μL of H2O2 (30%, w / w) was added and the reaction was carried out at 40 to 80 ° C for 6 to 96 h. The reaction solution was precipitated using an ethanol / ether mixed solution (v:v = 1:6 to v:v = 1:3), the supernatant was discarded by centrifugation, and the solid was dried to obtain a superhydrophilic solid. The solid was dispersed in deionized water to obtain a superhydrophilic 19 FMRI polymer probes.

[0031] The following examples illustrate the preparation method of the fluorine magnetic resonance imaging polymer probe containing a polyamino acid backbone of the present invention in more detail, and the performance of the prepared polymer probe is tested. However, the embodiments of the present invention are not limited to the following examples.

[0032] Example 1

[0033] a. 0.4 g of polysuccinimide (Mw = 2000) and 530 μL of 2-[(2,2,2-trifluoroethyl)thio]ethylamine were dissolved in 8 mL of DMF, mixed evenly, added to a polytetrafluoroethylene liner, placed in an autoclave, and reacted at 100°C for 5 h; the resulting solution was precipitated with a dichloromethane / n-hexane mixed solution (v:v = 1:10), centrifuged and the supernatant discarded, and the precipitate was washed twice with a THF / n-hexane mixed solution (v:v = 1:5) and dried to obtain a solid intermediate product;

[0034] b. The solid intermediate obtained above was dissolved in 4 mL of acetone, 800 μL of H2O2 (30%, w / w) was added and reacted at 75°C for 12 h. The reaction solution was precipitated with an ethanol / ether mixed solution (v:v = 1:4), the supernatant was discarded by centrifugation, and the solid was dried to obtain a superhydrophilic 19 FMRI polymer probes.

[0035] Example 2

[0036] a. 0.4 g of polysuccinimide (Mw = 8000-10000) and 530 μL of 2-[(2,2,2-trifluoroethyl)thio]ethylamine were dissolved in 8 mL of DMF, mixed evenly, added to a polytetrafluoroethylene liner, placed in an autoclave, and reacted at 100°C for 5 h; the resulting solution was precipitated with a dichloromethane / n-hexane mixed solution (v:v = 1:10), the supernatant was discarded by centrifugation, and the precipitate was washed twice with a THF / n-hexane mixed solution (v:v = 1:5), and dried to obtain a solid to obtain an intermediate product;

[0037] b. The solid intermediate obtained above was dissolved in 4 mL of acetone, 800 μL of H2O2 (30%, w / w) was added and reacted at 75°C for 12 h. The reaction solution was precipitated with an ethanol / ether mixed solution (v:v = 1:4), the supernatant was discarded by centrifugation, and the solid was dried to obtain a superhydrophilic 19 FMRI polymer probes.

[0038] Example 3

[0039] a. 0.4 g of polysuccinimide (Mw = 15000) and 530 μL of 2-[(2,2,2-trifluoroethyl)thio]ethylamine were dissolved in 8 mL of DMF, mixed evenly, added to a polytetrafluoroethylene liner, placed in an autoclave, and reacted at 100°C for 5 h; the resulting solution was precipitated with a dichloromethane / n-hexane mixed solution (v:v = 1:10), centrifuged and the supernatant discarded, and the precipitate was washed twice with a THF / n-hexane mixed solution (v:v = 1:5) and dried to obtain a solid intermediate product;

[0040] b. The solid intermediate obtained above was dissolved in 4 mL of acetone, 800 μL of H2O2 (30%, w / w) was added and reacted at 75°C for 12 h. The reaction solution was precipitated with an ethanol / ether mixed solution (v:v = 1:4), the supernatant was discarded by centrifugation, and the solid was dried to obtain a superhydrophilic 19 FMRI polymer probes.

[0041] Example 4

[0042] a. 0.4 g of polysuccinimide (Mw = 2000) and 530 μL of 2-[(2,2,2-trifluoroethyl)thio]ethylamine were dissolved in 8 mL of DMF, mixed evenly, added to a polytetrafluoroethylene liner, placed in an autoclave, and reacted at 100°C for 5 h; the resulting solution was precipitated with a dichloromethane / n-hexane mixed solution (v:v = 1:10), centrifuged and the supernatant discarded, and the precipitate was washed twice with a THF / n-hexane mixed solution (v:v = 1:5) and dried to obtain a solid intermediate product;

[0043] b. The solid intermediate obtained above was dissolved in 4 mL of acetone, 800 μL of H2O2 (30%, w / w) was added and reacted at 45°C for 64 h. The reaction solution was precipitated with an ethanol / ether mixed solution (v:v=1:4), the supernatant was discarded by centrifugation, and the solid was dried to obtain a superhydrophilic 19 FMRI polymer probes.

[0044] Example 5

[0045] a. 0.4 g of polysuccinimide (Mw = 2000) and 795 μL of 2-[(2,2,2-trifluoroethyl)thio]ethylamine were dissolved in 8 mL of DMF, mixed evenly, added to a polytetrafluoroethylene liner, placed in an autoclave, and reacted at 100°C for 5 h; the resulting solution was precipitated with a dichloromethane / n-hexane mixed solution (v:v = 1:10), centrifuged and the supernatant discarded, and the precipitate was washed twice with a THF / n-hexane mixed solution (v:v = 1:5) and dried to obtain a solid intermediate product;

[0046] b. The solid intermediate obtained above was dissolved in 4 mL of acetone, 800 μL of H2O2 (30%, w / w) was added and reacted at 75°C for 12 h. The reaction solution was precipitated with an ethanol / ether mixed solution (v:v = 1:4), the supernatant was discarded by centrifugation, and the solid was dried to obtain a superhydrophilic 19 FMRI polymer probes.

[0047] <Performance Test>

[0048] (1) 19 FNMR signal testing

[0049] The polymer probe solution prepared in Example 1 was 19 The FNMR spectrum test was conducted using a "single pulse" sequence (Bruker "zg"), using a D2O capillary for lock field, setting the number of scans to 128 times, and the test temperature to 298K. 19 FNMR signal test results can be found in Figure 1 The results show that the polymer probe prepared by the present invention has high 19 FMRI signals can be highly sensitive 19 FMRI test, and with the increase of polymer concentration, its 19 The FMRI signal is linearly enhanced, overcoming the problem of weak imaging signal commonly faced by existing fluorinated polymer nanoprobes due to increased fluorine atom concentration and limited activity.

[0050] (2) Relaxation time test

[0051] 19The FNMR spin-lattice relaxation time (T1) test uses an inversion-recovery (t1ir) pulse sequence, and the specific test parameters are: relaxation delay (D1=10s); number of blank scans (DS=4); and number of scans (NS=4). 19 FNMR spin-spin relaxation time (T2) measurements were performed using a Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence with the following parameters: relaxation delay (D1 = 10 s), number of empty scans (DS = 4), number of scans (NS = 4), D20 = 2.0 ms, and P2 = 0.0352 ms.

[0052] The T1 and T2 relaxation times of polymer probes at different concentrations can be found in Figure 2 The results indicate that the ultra-high hydrophilicity of the sulfoxide groups and the polyamino acid backbone of the polymer probe prepared in this invention increases the mobility of the fluorinated segments within the polymer, resulting in a longer T2 relaxation time. With increasing polymer concentration, the T1 and T2 values ​​gradually decrease. Even at a concentration of 400 mg / mL, the T2 value remains as high as 380 ms, demonstrating that the fluorinated segments of the polymer probe retain good mobility even at high concentrations.

[0053] (3) Cytotoxicity test

[0054] The cytotoxicity results of the polymer probe in MREpic and MB49 cell lines are shown in the figure Figure 3 As shown, the results show that when the concentration of the polymer probe prepared by the present invention is as high as 1.8 mg / mL, the cell survival rate is still maintained at more than 85% after incubation with cells for 48 hours, indicating that it has excellent biocompatibility.

[0055] (4) Hemolysis test

[0056] The polymer probes of different concentrations were incubated with red blood cell suspension for 1 hour and then centrifuged to obtain the supernatant and test the hemolysis rate. Figure 4 As shown, the positive control ultrapure water group has hemolytic activity, the negative control PBS group has no hemolytic activity, and the supernatant of the polymer probe group (5-75 mg / mL) obtained in this patent is almost colorless. When the concentration of the polymer probe is 75 mg / mL, the hemolysis rate is only 4%, indicating that it has good blood compatibility.

[0057] (5) 1 HMRI and 19 FMRI imaging tests

[0058] 19 FMRI uses T1-RARE sequence, and the relevant parameters are set as follows: the matrix size is 100×100, T R and T EThe T2-TurboRARE sequence was used. 1 HMRI, the relevant parameters are set as follows: the matrix size is 256×256, T R and T E The experimental time is 2500ms and 100ms respectively. The field of view (FOV) is set to 50mm×50mm. The total experimental time is 12s800ms.

[0059] Polymer probes at different concentrations 1 HMRI and 19 FMRI imaging and 19 The linear relationship between FMRI signal-to-noise ratio and probe concentration is as follows Figure 5 As shown in the figure, the results show that as the concentration of polymer probes increases, the number of fluorine atoms increases. 19 The higher the signal brightness in FMRI, the higher the concentration of polymer probes. 19 The FMRI signal intensity also has a good linear relationship (y=0.113x-0.596, R 2 =0.996) This indicates that the polymer probe has a good 19 Effects of fMRI imaging.

Claims

1. A method for preparing a fluorine magnetic resonance imaging polymer probe containing a polyamino acid backbone, characterized in that: The specific preparation steps are as follows: a. mixing an organic fluorine-containing small molecule with polysuccinimide for reaction, wherein the organic fluorine-containing small molecule has a thioether functional group and an amino group, and grafting the organic fluorine-containing small molecule onto the polysuccinimide backbone through an amino-lactam ring-opening reaction to obtain an intermediate product; b. oxidizing the thioether group in the intermediate product to form a sulfoxide group through an oxidation reaction to obtain a fluorine magnetic resonance imaging polymer probe containing a polyamino acid backbone; The organic fluorine-containing small molecule is 2-[(2, 2, 2-trifluoroethyl)thio]ethylamine.

2. The method for preparing a fluorine magnetic resonance imaging polymer probe containing a polyamino acid backbone according to claim 1, characterized in that: The molecular weight of the polysuccinimide is 500-20000.

3. The method for preparing a fluorine magnetic resonance imaging polymer probe containing a polyamino acid backbone according to claim 1, wherein: The step a is specifically as follows: dissolving polysuccinimide and an organic fluorine-containing small molecule in a solvent, mixing them evenly, reacting them at 60-120° C. for 3-8 hours, adding a precipitant for precipitation, centrifuging and discarding the supernatant, washing, and drying to obtain a solid intermediate product.

4. The method for preparing a fluorine magnetic resonance imaging polymer probe containing a polyamino acid backbone according to claim 3, characterized in that: In the step a, the precipitant used is a mixed solution of dichloromethane and n-hexane, the volume ratio of dichloromethane to n-hexane is 1:10 to 1:5, and the mixed solution of tetrahydrofuran and n-hexane is used for washing, the volume ratio of tetrahydrofuran to n-hexane is 1:10 to 1:

5.

5. The method for preparing a fluorine magnetic resonance imaging polymer probe containing a polyamino acid backbone according to claim 1, wherein: The step b specifically comprises: dissolving the obtained intermediate product in a solvent, adding an oxidant and reacting at 40-80° C. for 6-96 hours, adding a precipitant, centrifuging, and drying to obtain a fluorine magnetic resonance imaging polymer probe containing a polyamino acid skeleton.

6. The method for preparing a fluorine magnetic resonance imaging polymer probe containing a polyamino acid backbone according to claim 5, characterized in that: In the step b, the oxidant used is hydrogen peroxide, and the precipitant used is a mixed solution of ethanol and diethyl ether, with the volume ratio of ethanol to diethyl ether being 1:6 to 1:

3.

7. The method for preparing a fluorine magnetic resonance imaging polymer probe containing a polyamino acid backbone according to claim 1, characterized in that: In step a, 0.2 to 0.6 g of polysuccinimide and 265 to 1200 μL of 2-[(2, 2, 2-trifluoroethyl)thio]ethylamine are dissolved in 4 to 12 mL of N,N-dimethylformamide or dimethyl sulfoxide, mixed evenly, and reacted.

8. The method for preparing a fluorine magnetic resonance imaging polymer probe containing a polyamino acid backbone according to claim 7, characterized in that: In the step b, the intermediate product obtained in the step a is dissolved in 2 to 8 mL of acetone, and 200 to 1200 μL of 30% by mass H2O2 is added to carry out an oxidation reaction.

9. The fluorine magnetic resonance imaging polymer probe containing a polyamino acid backbone prepared by the method according to any one of claims 1 to 8 is prepared 19 Application of F contrast agents in magnetic resonance imaging.

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