An ultrasmall particle size 19 F magnetic resonance imaging nanoprobe and its preparation method and application

Abstract

The present invention discloses a <supgt;19< / supgt;F magnetic resonance imaging nanoprobe with ultra-small particle size and its preparation method and application, belonging to the technical field of applied material preparation. In the present invention, poly(succinimide) is grafted with oleylamine, or histamine and oleylamine to obtain an amphiphilic polymer. The perfluoro-15-crown-5 ether is successfully introduced into the interior of the nanoparticles by ultrasonic emulsification method to obtain a <supgt;19< / supgt;F magnetic resonance imaging nanoprobe with ultra-small particle size, with a particle size of less than 5 nm, uniform distribution, good biocompatibility and strong hydrophilicity. Due to its ultra-small size, it has good in-vivo circulation ability and tissue penetration ability. The probe can be stable in aqueous solution for a long time, is easy to store, has a strong <supgt;19< / supgt;F magnetic resonance imaging signal, and is very suitable for <supgt;19< / supgt>F magnetic resonance imaging of tumors. The probe will attract extensive attention in the fields of nanomaterial science and biomedical science, and has important application significance in biomedical fields such as biosensors, cell imaging, and medical diagnosis.

Description

Technical Field

[0001] The present invention belongs to the technical field of the preparation of applied nano-materials, and particularly relates to a new method for preparing a nano-probe for 19F magnetic resonance imaging with an ultra-small particle size, and its application as a contrast agent for 19F magnetic resonance imaging. 19 and its application as a 19 19F magnetic resonance imaging contrast agent. Background Art

[0002] Magnetic Resonance Imaging (MRI) technology has high soft tissue resolution and deep tissue penetration. Currently, 1 1H MRI has been widely used in the imaging examination of screening diseased tissues in clinical medicine. 1 1H MRI is based on the difference in the content of hydrogen atoms and the relaxation time in a living body for imaging. Since there are only slight differences between normal tissues and diseased tissues, it is difficult to determine the lesion site in actual diagnosis. Fluorine atoms also have the advantages of high natural abundance, high sensitivity, and low imaging background interference, and are also a very potential nucleus for magnetic resonance imaging. Compared with 1 1H MRI, fluorine only exists in the form of solid salts in teeth and bones, 19 and 19F MRI has almost no background interference. And its signal has a chemical shift range of about 400 ppm and is very sensitive to changes in chemical bonds and microenvironments.

[0003] However, currently, 19 19F MRI has not been able to 1 be widely used in clinical practice like 1H MRI. Since the content of fluorine atoms in a living body is extremely low, 19 the signals of 19F MRI all come from exogenous fluorine-containing probes. Therefore, synthesizing fluorine-containing probes with good stability and high sensitivity is the key to promoting the 19 development of 19F MRI technology. Currently, the common 19 19F MRI probes mostly use liposome shells to coat perfluorocarbon droplets to form nanoemulsion droplets with a micelle structure. The nanoemulsion droplet-type fluorine-containing probes composed of perfluorocarbon molecules generally have 19 the advantages of high 19F MRI signal intensity and high sensitivity. However, such probes generally have a relatively large size (>50 nm), and the droplets will also undergo Ostwald ripening over time. This will lead to unstable probe structures and coalescence, and the sizes will become more uneven, seriously limiting the imaging effect of tumor enrichment in vivo circulation. Existing studies have shown that ultra-small-sized nano-materials have the property of being cleared by the kidneys, which is of great significance for realizing the clinical application of nano-probes. Summary of the Invention

[0004] In view of the problems existing in the background art, the purpose of the present invention is to provide an ultra-small particle size19 F magnetic resonance imaging nanoprobe and its preparation method. The probe has the characteristics of small size (<5nm), uniform particle size distribution, high biocompatibility, good stability, etc.

[0005] The present invention is realized as follows:

[0006] The present invention provides a super-small particle size 19 F magnetic resonance imaging nanoprobe, which has a nanoparticle structure formed by the assembly of perfluoro-15-crown-5 ether and an amphiphilic polymer. The perfluoro-15-crown-5 ether with good imaging effect is located inside the nanoparticle, and its particle size is less than 5nm; the amphiphilic polymer is poly(succinimide) grafted with oleylamine, or poly(succinimide) grafted with histamine and oleylamine.

[0007] The present invention also provides a preparation method of the above-mentioned super-small particle size 19 F magnetic resonance imaging nanoprobe. By grafting oleylamine, or histamine and oleylamine onto poly(succinimide), an amphiphilic polymer is obtained. Using the ultrasonic emulsification method, perfluoro-15-crown-5 ether is introduced into the interior of the nanoparticle assembled with the amphiphilic polymer to obtain a super-small particle size 19 F magnetic resonance imaging nanoprobe.

[0008] This preparation method successfully introduces perfluoro-15-crown-5 ether with good imaging effect into the interior of the nanoparticle by preparing an amphiphilic polymer, and successfully prepares a nanoprobe with a super-small particle size by adjusting the ratio of the hydrophilic and hydrophobic segments of the amphiphilic polymer. It has 19 strong F NMR signal, super-small size, uniform particle size distribution, high biocompatibility, good stability, etc.

[0009] The specific steps of this preparation method are as follows:

[0010] a. Dissolve 0.5 - 2g of poly(succinimide) in 10 - 20mL of organic solvent A, add 0 - 1g of histamine, stir evenly, dropwise add 0.2 - 2mL of oleylamine, heat to 60 - 120°C, stir constantly at a constant temperature for 5 - 24h, add a precipitating agent, centrifuge, and dissolve the obtained precipitate in N,N-dimethylformamide to obtain an amphiphilic polymer;

[0011] b. Dissolve 1 - 20mg of the amphiphilic polymer and 1 - 20mL of perfluoro-15-crown-5 ether in 0.5 - 2mL of organic solvent B, quickly pour it into 5 - 15mL of an aqueous sodium hydroxide solution with a concentration of 0.12 - 0.20g / L, ultrasonically emulsify until it becomes clear, centrifuge and wash to collect the product to obtain a super-small particle size 19 F magnetic resonance imaging nanoprobe.

[0012] Preferably, in step a, the histamine used is histamine hydrochloride.

[0013] Preferably, in step a, the weight-average molecular weight Mw of the poly(succinimide) used is 3000 - 20000 g / mol.

[0014] Preferably, in step a, the organic solvent A used is N,N-dimethylformamide.

[0015] Preferably, in step a, the precipitating agent used is methanol.

[0016] Preferably, in step b, the organic solvent used is a mixed solution of N,N-dimethylformamide and dichloromethane.

[0017] The present invention also provides the above-mentioned ultrasmall-sized 19 19F magnetic resonance imaging nanoprobe as 19 an application of a 19F magnetic resonance imaging contrast agent.

[0018] Advantages of the present invention:

[0019] (1) The preparation method of the ultrasmall-sized 19 19F magnetic resonance imaging nanoprobe provided by the present invention successfully introduces perfluoro-15-crown-5 ether with good imaging effect into the interior of nanoparticles by preparing an amphiphilic polymer, improving the problems of poor water solubility and high toxicity in vivo of fluorinated organic small molecules. At the same time, the ratio of hydrophilic and hydrophobic segments of the amphiphilic polymer is adjusted, and ultrasmall-sized nanoprobes of different sizes are successfully prepared by the ultrasonic emulsification method.

[0020] (2) The ultrasmall-sized 19 19F magnetic resonance imaging nanoprobe provided by the present invention has the characteristics of ultrasmall size, uniform particle size distribution, high biocompatibility, and good stability while retaining the ultra-strong 19 19F NMR of perfluoro-15-crown-5 ether. Its ultrasmall size enables the probe to effectively escape the capture of the reticuloendothelial system, has a longer blood circulation time, and is more easily metabolized to the outside of the body. This probe will attract wide attention in the fields of nanomaterial science and biomedical science and has important application significance in biomedical fields such as biosensors, cell imaging, and medical diagnosis. Description of the Drawings

[0021] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required to be used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0022] Figure 1 The transmission electron microscope photograph and particle size distribution diagram of the ultrasmall particle size 19 F magnetic resonance imaging nanoprobe prepared in Example 1;

[0023] Figure 2 The ultrasmall particle size 19 F magnetic resonance imaging nanoprobe in aqueous solution 19 F MRI;

[0024] Figure 3 The ultrasmall particle size 19 F magnetic resonance imaging nanoprobe prepared in Example 1; Stability test results;

[0025] Figure 4 The ultrasmall particle size 19 F magnetic resonance imaging nanoprobe prepared in Example 1; MTT assay graph of cytotoxicity at different concentrations;

[0026] Figure 5 The ultrasmall particle size 19 F magnetic resonance imaging nanoprobe prepared in Example 1; Hemolysis test graph at different concentrations. Detailed implementation manners

[0027] To better explain the present invention, it is described in detail with reference to the implementation manners of the present invention, and the main content of the present invention is further clarified in combination with specific examples. However, the content of the present invention is not limited to the following examples. For those not specified in the examples in terms of specific technologies or conditions, they are carried out according to the technologies or conditions described in the literature in this field or according to the product specifications. For the reagents or instruments not specified in terms of the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0028] The preparation method of the ultrasmall particle size 19 F magnetic resonance imaging nanoprobe provided by the present invention. By preparing an amphiphilic polymer, perfluoro-15-crown-5 ether with good imaging effect is successfully introduced into the interior of the nanoparticles, and by adjusting the ratio of the hydrophilic and hydrophobic segments of the amphiphilic polymer, an ultrasmall particle size nanoprobe is successfully prepared. The specific steps are as follows:

[0029] a. Dissolve 0.5 - 2 g of poly(succinimide) in 10 - 20 mL of an organic solvent, add 0 - 1 g of histamine, stir evenly, dropwise add 0.2 - 2 mL of oleylamine, heat to 60 - 120 °C, and stir at a constant temperature for 5 - 24 h to obtain an amphiphilic polymer;

[0030] b. Dissolve 1 - 20 mg of amphiphilic polymer and 1 - 20 mL of perfluoro-15-crown-5 ether in 0.5 - 2 mL of organic solvent, quickly pour it into 5 - 15 mL of sodium hydroxide aqueous solution with a concentration of 0.12 - 0.20 g / L, ultrasonically emulsify until clear, centrifuge and wash to collect the product, obtaining ultrasmall particle size 19 19F magnetic resonance imaging nanoprobe.

[0031] Hereinafter, the preparation method of the ultrasmall particle size 19 19F magnetic resonance imaging nanoprobe of the present invention will be more specifically described through examples, and the performance of the prepared nanoprobe will be tested. However, the embodiments of the present invention are not limited to the following examples.

[0032] Example 1

[0033] a. Dissolve 0.8 g of poly(succinimide) in 10 mL of N,N-dimethylformamide. Add 400 mg of histamine and stir evenly. Dropwise add 0.8 mL of oleylamine, heat to 100 °C, and stir at a constant temperature for 12 h. After cooling to room temperature, precipitate with methanol and centrifuge. Dissolve the obtained precipitate in N,N-dimethylformamide to obtain an amphiphilic polymer.

[0034] b. Dissolve 6 mg of amphiphilic polymer and 10 mL of perfluoro-15-crown-5 ether in 1.5 mL of organic solvent, quickly pour it into 10 mL of sodium hydroxide aqueous solution with a concentration of 0.16 g / L, ultrasonically emulsify until clear. Centrifuge and wash to collect the product, and finally obtain ultrasmall particle size 19 19F magnetic resonance imaging nanoprobe.

[0035] Example 2

[0036] a. Dissolve 0.5 g of poly(succinimide) in 12 mL of N,N-dimethylformamide. Add 0 g of histamine and stir evenly. Dropwise add 0.5 mL of oleylamine, heat to 90 °C, and stir at a constant temperature for 12 h. After cooling to room temperature, precipitate with methanol and centrifuge. Dissolve the obtained precipitate in N,N-dimethylformamide to obtain an amphiphilic polymer.

[0037] b. Dissolve 5 mg of amphiphilic polymer and 5 mL of perfluoro-15-crown-5 ether in 1 mL of organic solvent, quickly pour it into 12 mL of sodium hydroxide aqueous solution with a concentration of 0.12 g / L, ultrasonically emulsify until clear. Centrifuge and wash to collect the product, and finally obtain ultrasmall particle size 19 19F magnetic resonance imaging nanoprobe.

[0038] Example 3

[0039] a. Dissolve 1 g of poly(succinimide) in 15 mL of N,N-dimethylformamide. Add 600 mg of histamine and stir evenly. Dropwise add 0.8 mL of oleylamine, heat to 100 °C, and stir at a constant temperature for 12 h. After cooling to room temperature, precipitate with methanol and centrifuge. Dissolve the obtained precipitate in N,N-dimethylformamide to obtain an amphiphilic polymer.

[0040] b. Dissolve 10 mg of the amphiphilic polymer and 20 mL of perfluoro-15-crown-5 ether in 2 mL of an organic solvent, quickly pour it into 10 mL of an aqueous sodium hydroxide solution with a concentration of 0.20 g / L, and ultrasonically emulsify until it becomes clear. Centrifuge and wash to collect the product, and finally obtain a super-small particle size 19 19F magnetic resonance imaging nanoprobe.

[0041] Example 4

[0042] a. Dissolve 1.2 g of poly(succinimide) in 8 mL of N,N-dimethylformamide. Add 400 mg of histamine and stir evenly. Dropwise add 1 mL of oleylamine, heat to 120 °C, and stir at a constant temperature for 10 h. After cooling to room temperature, precipitate with methanol and centrifuge. Dissolve the obtained precipitate in N,N-dimethylformamide to obtain an amphiphilic polymer.

[0043] b. Dissolve 8 mg of the amphiphilic polymer and 4 mL of perfluoro-15-crown-5 ether in 0.8 mL of an organic solvent, quickly pour it into 10 mL of an aqueous sodium hydroxide solution with a concentration of 0.12 g / L, and ultrasonically emulsify until it becomes clear. Centrifuge and wash to collect the product, and finally obtain a super-small particle size 19 19F magnetic resonance imaging nanoprobe.

[0044] Example 5

[0045] a. Dissolve 1 g of poly(succinimide) in 15 mL of N,N-dimethylformamide. Add 500 mg of histamine and stir evenly. Dropwise add 0.8 mL of oleylamine, heat to 80 °C, and stir at a constant temperature for 24 h. After cooling to room temperature, precipitate with methanol and centrifuge. Dissolve the obtained precipitate in N,N-dimethylformamide to obtain an amphiphilic polymer.

[0046] b. Dissolve 15 mg of the amphiphilic polymer and 10 mL of perfluoro-15-crown-5 ether in 1.2 mL of an organic solvent, quickly pour it into 15 mL of an aqueous sodium hydroxide solution with a concentration of 0.08 g / L, and ultrasonically emulsify until it becomes clear. Centrifuge and wash to collect the product, and finally obtain a super-small particle size 19 19F magnetic resonance imaging nanoprobe.

[0047] Example 6

[0048] a. Dissolve 0.6 g of polysuccinimide in 16 mL of N,N-dimethylformamide. Add 400 mg of histamine and stir until uniform. Add 0.8 mL of oleylamine dropwise, heat to 110°C, and stir at this constant temperature for 8 hours. Cool to room temperature, precipitate with methanol, and centrifuge. The resulting precipitate is dissolved in N,N-dimethylformamide to obtain an amphiphilic polymer.

[0049] b. Dissolve 18 mg of amphiphilic polymer and 6 mL of perfluoro-15-crown-5 ether in 2 mL of organic solvent, quickly pour into 10 mL of 0.16 g / L sodium hydroxide aqueous solution, and ultrasonically emulsify until clear. Centrifuge and wash to collect the product, and finally obtain ultra-small particles. 19 F magnetic resonance imaging nanoprobe.

[0050] <Performance Test>

[0051] (1) 19 F NMR signal testing

[0052] Nanoprobe solution 19 F NMR spectrum, using the "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 F magnetic resonance imaging nanoprobes at different concentrations 19 F NMR signal test results can be found in Figure 2 The results show that the nanoprobes prepared by the present invention have high 19 F MRI signal, which can be performed with high sensitivity 19 F MRI test, and with the increase of nanoprobe concentration, 19 The FMRI signal increased linearly.

[0053] (2) Stability test

[0054] The ultra-small particle size prepared in Example 1 19 The stability of the F magnetic resonance imaging nanoprobe was tested. Figure 3 As shown, after 30 days of storage, 19 The F NMR signal is basically unchanged with the size of the nanoparticles, indicating that the nanoprobe prepared by the present invention overcomes the problems of large size and unstable signal commonly faced by existing fluorine-containing nanoprobes.

[0055] (3) Cytotoxicity test

[0056] Ultra-small particle size prepared in Example 1 19 The cytotoxicity results of the F magnetic resonance imaging nanoprobe in the 4T1 cell line are shown in Figure 2. Figure 4 As shown, the results show that the ultra-small particle size prepared by the present invention 19When the F magnetic resonance imaging nanoprobe is incubated with cells for 24 h at a concentration up to 0.4 mg / mL, the cell viability remains above 90%, indicating its excellent biocompatibility.

[0057] (4) Hemolysis test

[0058] The hemolysis test is a method for measuring the degree of red blood cell lysis caused by the contact between materials and red blood cells in vitro, and is commonly used for in vitro blood compatibility evaluation. If the hemolysis rate exceeds the standard (>10%), it indicates that the material may cause unsafe factors of hemolysis when used in vivo. The supernatant obtained by centrifuging the red blood cell suspension incubated with different concentrations of nanoprobes for 1 h. The results are as Figure 5 shown. The positive control ultrapure water group has hemolytic activity, and the negative control PBS group has no hemolytic activity. The supernatant of the nanoprobe group (40 - 280 mg / mL) obtained in the present invention is almost colorless. When the concentration of the nanoprobe is 280 mg / mL, the hemolysis rate is only 3.8%, indicating its good blood compatibility.

[0059] (5) 19 F MRI imaging test

[0060] 19 F MRI uses the T1-RARE sequence, and the relevant parameter settings are as follows: the matrix size is 100×100, T R and T E are 1500 ms and 4.64 ms respectively, the field of view (FOV) is set to 40 mm×40 mm, and the total experimental time is 1 min 54 s.

[0061] The 19 F MRI imaging diagrams of the nanoprobe at different concentrations and 19 the linear relationship between the Figure 2 F MRI signal-to-noise ratio and the probe concentration are as 19 shown. The results show that as the concentration of the nanoprobe increases, the number of fluorine atoms increases, and the 19 signal brightness in F MRI is higher. At the same time, there is also a good linear relationship between the nanoprobe concentration and the 2 F MRI signal intensity (y = 12.98x + 0.07, R 19 = 0.991), which indicates that the nanoprobe has good 19 F MRI imaging effect.

Claims

1. An ultrasmall particle size 19 F magnetic resonance imaging nanoprobe, characterized in that It has a nanoparticle structure formed by the assembly of perfluoro-15-crown-5 ether and an amphiphilic polymer, and the particle size is less than 5 nm; the amphiphilic polymer is poly(succinimide) grafted with oleylamine, or poly(succinimide) grafted with histamine and oleylamine.

2. An ultrafine particle size 19 Preparation method of an F magnetic resonance imaging nanoprobe, characterized in that By grafting poly(succinimide) with oleylamine, or grafting histamine and oleylamine, an amphiphilic polymer is obtained. Using the ultrasonic emulsification method, perfluoro-15-crown-5 ether is introduced into the interior of the nanoparticles formed by assembling with the amphiphilic polymer, obtaining ultrasmall nanoparticles with a particle size of less than 5 nm. 19 F magnetic resonance imaging nanoprobe.

3. The preparation method of the ultrasmall particle size 19 F magnetic resonance imaging nanoprobe according to claim 2, characterized in that The specific steps are as follows: a. Dissolve 0.5 - 2 g of poly(succinimide) in 10 - 20 mL of organic solvent A, add 0 - 1 g of histamine, stir evenly, dropwise add 0.2 - 2 mL of oleylamine, heat to 60 - 120 °C, stir at a constant temperature for 5 - 24 h, add a precipitating agent, and centrifuge to obtain the amphiphilic polymer; b. Dissolve 1 - 20 mg of amphiphilic polymer and 1 - 20 mL of perfluoro-15-crown-5 ether in 0.5 - 2 mL of organic solvent B, quickly pour it into 5 - 15 mL of an aqueous sodium hydroxide solution with a concentration of 0.12 - 0.20 g / L, ultrasonically emulsify until clear, centrifuge and wash to collect the product, obtaining an ultrasmall particle size 19 F magnetic resonance imaging nanoprobe.

4. The preparation method according to claim 3, characterized in that, In step a, the histamine used is histamine hydrochloride.

5. The preparation method according to claim 3, characterized in that, In step a, the weight-average molecular weight Mw of the poly(succinimide) used is 3000 - 20000 g / mol.

6. The preparation method according to claim 3, wherein In step a, the organic solvent A used is N,N-dimethylformamide.

7. The preparation method according to claim 3, characterized in that, In step a, the precipitating agent used is methanol.

8. The preparation method according to claim 3, characterized in that, In step b, the organic solvent used is a mixed solution of N,N-dimethylformamide and dichloromethane.

9. The ultrasmall particle size according to claim 1 19 F magnetic resonance imaging nanoprobe or the ultrasmall particle size of the F magnetic resonance imaging nanoprobe prepared by the method according to any one of claims 2-8 19 in the preparation of the F magnetic resonance imaging nanoprobe 19 Application in an F magnetic resonance imaging contrast agent.

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