A kind of CT visualization nanoparticle based on amphiphilic block copolymer and its preparation method and application

Abstract

The application belongs to the technical field of biological detection, and particularly relates to CT visualized nanoparticles based on amphiphilic block copolymer and a preparation method and application thereof. The application first prepares amphiphilic block copolymer with one end hydrophilic and the other end hydrophobic based on polyethylene glycol and organic siloxane monomer through substitution reaction and atom transfer radical polymerization (ATRP), then makes the amphiphilic block copolymer self-assemble to wrap iodine-containing monomer (3-iodopropyl) trimethoxysilane to form nanoparticles in aqueous solution, and finally forms structure-stable iodine-containing nanoparticles by combining with pH adjustment. The self-assembled nanoparticles prepared by the method have a relatively uniform size distribution, and the iodine content and size of the nanoparticles can be controlled by regulating the ratio of the copolymer and the iodine-containing monomer in the assembly process. Meanwhile, the obtained series of iodine-containing nanoparticles have good CT imaging effect and biocompatibility, and have the potential to become CT imaging contrast agents.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological detection, and particularly relates to a CT visualized nanoparticle based on an amphiphilic block copolymer and a preparation method and application thereof. BACKGROUND

[0002] Since the first X-ray photograph was taken by Roentgen, X-ray imaging has become an indispensable part of medical care, in which computed tomography (CT) is a non-invasive tissue imaging method used in various research and clinical environments by taking hundreds of X-ray images in multiple directions to create three-dimensional images of the body. Its imaging principle is mainly to detect X-ray photons passing through the body, and different X-ray attenuation degrees are generated due to different absorption and scattering of photons by different tissues. Compared with other medical imaging methods, CT imaging has the advantages of better display of calcification and bone lesions, shorter scanning time, clear images, clear anatomical relationship, and low price. However, the ability of this technology to distinguish similar density soft tissues is limited, so it is necessary to improve the imaging effect by injecting contrast agents. Due to the high atomic number, high X-ray absorption coefficient, and flexibility and functionality in chemical synthesis, iodine atoms make iodine contrast medium (ICM) the main category of current CT contrast agents.

[0003] The development of ICM has experienced a transition from inorganic molecules to organic molecules, in which water-soluble sodium iodide and potassium iodide are the earliest used ICM. However, inorganic iodine solution shows high toxicity when meeting the required concentration for imaging, which seriously hinders its clinical application. Subsequently, ICM develops towards organic small molecules, and realizes the transition from ionic to non-ionic, and from monomer to dimer. Currently, almost all of the commercially available and clinically approved ICMs are of the organic small molecule type.

[0004] However, the existing organic small molecule type ICM still has a series of shortcomings such as serious nephrotoxicity, short circulation half-life, and easy to cause related immune reactions, so it is urgent to develop the next generation of ICMs that can realize long circulation, low toxicity and non-immunogenicity. Nanoparticle type ICMs can reduce some of the problems of currently approved organic small molecule type ICMs. One of the reasons is that the low osmotic pressure of nanoparticles can reduce the risk of adverse reactions related to high osmotic pressure during use; another reason is that nanoparticles with a particle size greater than 10 nm can escape the clearance of the kidneys, thereby showing a longer circulation time, and the modifiable / adjustable ability of nanoparticles provides enough development space for multi-modal imaging, organ targeting, etc.

[0005] In summary, to further reduce the ICM existing series of problems (nephrotoxicity, short circulating half-life, etc.), the development of a good biocompatibility and CT imaging effect of nanoparticle type ICM has very important practical significance. SUMMARY

[0006] In order to overcome the above-mentioned prior art deficiencies, the present application develops a chemical synthesis method of amphiphilic block copolymer based on polyethylene glycol and organosiloxane monomer, and through the self-assembly of the synthesized block copolymer in aqueous solution and the wrapping of iodine-containing monomer to form nanoparticles, and then adjusting the pH to realize the hydrolysis crosslinking of the iodine-containing monomer and the hydrophobic segment of the copolymer, thereby realizing the stability of the colloidal structure. The prepared iodine-containing nanoparticles have good CT imaging effect and biocompatibility, and have broad application prospect.

[0007] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is:

[0008] The first aspect of the present application provides a preparation method of CT visualized nanoparticles based on amphiphilic block copolymer, which comprises the following steps:

[0009] S1, under the protection of inert gas atmosphere, polyethylene glycol monomethyl ether, triethylamine and 2-bromoisobutyryl bromide are dissolved in an organic solvent, and the reaction is carried out at room temperature for 40-60h. After the reaction, the compound 1 is obtained by washing, dialysis and drying. The structural formula of the compound 1 is as follows:

[0010]

[0011] S2, under anhydrous and anaerobic conditions, compound 1 and 3-(methacryloyloxy) propyl trimethoxysilane are dissolved in an organic solvent, then copper bromide, pentamethyldiethylenetriamine (PMDETA) and cuprous bromide are added, and then the condensation reflux reaction is carried out in the protection of inert gas atmosphere. After the reaction, the metal ions are removed, and then the compound 2 is obtained by washing. The structural formula of the compound 2 is as follows:

[0012]

[0013] S3, compound 2 and iodine-containing monomer (3-iodopropyl) trimethoxysilane are dissolved in an organic solvent, and then added into water under stirring. After dialysis, the nanoparticles are prepared.

[0014] Preferably, in S1, the average molecular weight of the polyethylene glycol monomethyl ether is 4000-8000.

[0015] Preferably, in S1, the molar ratio of the polyethylene glycol monomethyl ether, triethylamine and 2-bromoisobutyryl bromide is 1:3-4:7-9.

[0016] Preferably, in S1, the concentrated reaction product is washed with cold ether, and after concentration, dissolved in water and dialyzed in a dialysis bag with a molecular weight cut-off of 700-2000 Da for 4-7 days.

[0017] Preferably, in S2, the temperature of the condensation reflux reaction is 60-80℃, and the time is 20-30h.

[0018] Preferably, in S2, the organic solvent is a mixture of tetrahydrofuran and ethanol in a volume ratio of 1:1.

[0019] Preferably, in S2, the amount ratio of compound 1, 3-(methacryloyloxy)propyltrimethoxysilane, copper bromide, pentamethyldiethylenetriamine and cuprous bromide is 350-400mg: 500-550μL: 0.4-0.7mg: 20-30μL: 10-20mg.

[0020] Preferably, in S2, the reaction product is washed with anhydrous n-hexane, and the remaining n-hexane is removed under vacuum.

[0021] Preferably, in S3, the stirring speed is 500rpm-1000rpm.

[0022] Preferably, in S3, the amount ratio of compound 2 and iodine-containing monomer is 68mg: 10-200μL.

[0023] Preferably, in S3, the dialysis is first dialyzed for 20-30h in a dialysis bag with a molecular weight cut-off of 700-1500 Da, then dialyzed for 10-15h after adjusting the pH of the dialysis solution to 8-10, and finally dialyzed for 20-30h with water.

[0024] The second aspect of the present application provides a CT visualizing nanoparticle based on an amphiphilic block copolymer prepared by the preparation method of the first aspect.

[0025] The third aspect of the present application provides the use of the CT visualizing nanoparticle based on an amphiphilic block copolymer of the second aspect in the preparation of a CT contrast agent.

[0026] Compared with the prior art, the present application has the following beneficial effects:

[0027] The application discloses a preparation method of iodine-containing nanoparticles based on an amphiphilic block copolymer. First, a single-hydroxyl-terminated polyethylene glycol is subjected to a substitution reaction with 2-bromoisobutyryl bromide under catalysis of triethylamine, and a reaction product is used as an initiator to polymerize 3-(methacryloyloxy)propyl trimethoxysilane containing a double bond through atom transfer radical polymerization (ATRP), so that an amphiphilic block copolymer with a hydrophilic end and a hydrophobic end is prepared. Then, the amphiphilic block copolymer is self-assembled to wrap iodine-containing monomers (3-iodopropyl) trimethoxysilane in an aqueous solution to form nanoparticles, and the internal iodine-containing monomers are subjected to hydrolysis condensation with the hydrophobic segment of the polymer by adjusting the pH, so that finally, iodine-containing nanoparticles with stable structure are formed. The self-assembled nanoparticles prepared through the method have a relatively uniform size distribution, and the iodine content and size of the nanoparticles can be controlled by regulating the proportion of the copolymer and the iodine-containing monomers in the assembly process. Meanwhile, the obtained series of iodine-containing nanoparticles have good CT imaging effect and biocompatibility, and have the potential to become a CT imaging contrast agent. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 A nuclear magnetic resonance hydrogen spectrum of a compound 1 generated by substitution reaction of polyethylene glycol monomethyl ether and 2-bromoisobutyryl bromide;

[0029] Figure 2 A nuclear magnetic resonance hydrogen spectrum of a block copolymer (compound 2) obtained by polymerizing 3-(methacryloyloxy) propyl trimethoxysilane with the compound 1 as an initiator;

[0030] Figure 3 A synthesis line for preparing iodine-containing nanoparticles by self-assembly of the compound 2 and iodine-containing monomers (in the formula, n = 113, m = 28);

[0031] Figure 4 A particle size distribution diagram of iodine-containing nanoparticles obtained in Example 1;

[0032] Figure 5 A particle size distribution diagram of iodine-containing nanoparticles obtained in Example 2;

[0033] Figure 6 A particle size distribution diagram of iodine-containing nanoparticles obtained in Example 3;

[0034] Figure 7 A particle size distribution diagram of iodine-containing nanoparticles obtained in Example 4;

[0035] Figure 8 A particle size distribution diagram of iodine-containing nanoparticles obtained in Example 5;

[0036] Figure 9 A transmission electron microscope image of the iodine-containing nanoparticles obtained in Example 4;

[0037] Figure 10 CT imaging graph of the iodine-containing nanoparticles obtained in Example 1-5;

[0038] Figure 11 CT value vs. concentration graph of the iodine-containing nanoparticles obtained in Example 1-5;

[0039] Figure 12 Cell toxicity evaluation graph of the iodine-containing nanoparticles obtained in Example 2 at different concentrations (MRC-5 cell line, 24 h);

[0040] Figure 13 Cell toxicity evaluation graph of the iodine-containing nanoparticles obtained in Example 3 at different concentrations (MRC-5 cell line, 24 h);

[0041] Figure 14 Cell toxicity evaluation graph of the iodine-containing nanoparticles obtained in Example 4 at different concentrations (MRC-5 cell line, 24 h);

[0042] Figure 15 Cell toxicity evaluation graph of the iodine-containing nanoparticles obtained in Example 5 at different concentrations (MRC-5 cell line, 24 h). DETAILED DESCRIPTION

[0043] The specific embodiments of the present application will be described further in detail below. It should be noted that the description of these embodiments is used to help understand the present application, but does not constitute a limitation on the present application. In addition, the technical features involved in each of the embodiments of the present application described below can be combined with each other as long as there is no conflict between them.

[0044] The experimental methods in the following examples are all conventional methods unless otherwise specified, and the experimental materials used in the following examples are all commercially available unless otherwise specified.

[0045] Examples 1-5: Preparation of iodine-containing nanoparticles based on amphiphilic block copolymer

[0046] (1) Synthesis of Compound 1:

[0047] Under argon atmosphere at 0°C, 5 g of polyethylene glycol monomethyl ether (average molecular weight of 5000, 1 mmol) and 515 μL of triethylamine (3.71 mmol) were added to 30 mL of anhydrous dichloromethane and stirred for 10 min; 1840 g of 2-bromoisobutyryl bromide (8 mmol) was dissolved in 10 mL of anhydrous dichloromethane, which was then added dropwise into the above mixture, and then reacted at room temperature for 48 h under argon protection. After stopping the reaction, the system was concentrated under reduced pressure to 5-10 mL, and the concentrated system was added dropwise into cold ether (the volume ratio of the concentrated system to ether was about 1:15-1:20), washed with cold ether three times, and connected to a vacuum pump to remove the remaining small amount of ether. The obtained solid was dissolved with an appropriate amount of deionized water (the solid to liquid ratio was about 5 g / 100 mL-200 mL), loaded into a dialysis bag with a molecular weight cut-off of 1000 Da, dialyzed for 5 days, and finally freeze-dried to obtain a pure white solid, whose nuclear magnetic resonance hydrogen spectrum is shown in Figure 1 The synthetic route of this step is shown below:

[0048]

[0049] wherein n = 113.

[0050] (2) Synthesis of compound 2:

[0051] Under anhydrous and anaerobic conditions, 379 mg of compound 1 (0.0729 mmol) and 520 μL of 3-(methacryloyloxy)propyltrimethoxysilane (2.1865 mmol) were added to a mixture of 10 mL of tetrahydrofuran and 10 mL of anhydrous ethanol, stirred for 10 min, and then 0.5 mg of copper bromide, 25 μL of pentamethyldiethylenetriamine (PMDETA), and 15 mg of cuprous bromide were added in sequence. After that, the system was condensed and refluxed at 70°C under argon protection for 24 h. After the reaction, the system was exposed to oxygen to terminate the reaction. Finally, the metal ions were removed by basic aluminum oxide, and the system was concentrated under reduced pressure until the original volume was about one quarter. The system was added dropwise into anhydrous n-hexane (the volume ratio of the concentrated system to n-hexane was about 1:15-1:20), washed with anhydrous n-hexane three times, and connected to a vacuum pump to remove the remaining n-hexane. The obtained white solid was compound 2, and its nuclear magnetic resonance hydrogen spectrum is shown in Figure 2 The synthetic route of this step is shown below:

[0052]

[0053] wherein n = 113, m = 28.

[0054] (3) Preparation of iodine-containing nanoparticles by self-assembly:

[0055] 68 mg of compound 2 was weighed and dissolved in 1 mL of tetrahydrofuran, and then different volumes of iodine-containing monomer (3-iodopropyl)trimethoxysilane (see Table 1 for specific proportions) were added slowly dropwise into 6 mL of deionized water under high-speed stirring (500 rpm-1000 rpm). After the dropwise addition was completed, stirring was continued for 2 h, and the mixture was then placed in a dialysis bag with a molecular weight cut-off of 1000 Da and dialyzed against pure water for 24 h. The dialysis solution was then replaced with fresh pure water, and ammonia was added to the outside dialysis solution (i.e. the fresh pure water) to adjust the pH to about 9. Dialysis was continued for 12 h, and then deionized water was used for dialysis for another 24 h to obtain five different nanoparticles. The synthesis route for this step is shown in Figure 3 .

[0056] Table 1 Sample composition, particle size and Zeta potential of nanoparticles formed in Examples 1-5

[0057]

[0058] Experimental Example: Characterization and performance testing of iodine-containing nanoparticles based on amphiphilic block copolymers

[0059] (1) Particle size characterization:

[0060] The five self-assembled iodine-containing nanoparticles (see Table 1 for specific composition) obtained in the examples were measured for hydrated particle size using a nanoparticle size and Zeta potential analyzer, and the results are shown in Table 1 and Figures 4-8 . It can be seen that each group of nanoparticles has a relatively uniform size distribution, and as the proportion of iodine-containing monomer increases, the particle size of the nanoparticles gradually increases, and the Zeta potential of each group is approximately -2.5 mV to -4.5 mV. In particular, the transmission electron microscopy (TEM) image of Example 4 shows that the nanoparticles are spherical in shape, with a particle size of mostly between 16-24 nm, which is similar to the results of dynamic light scattering (DLS) particle size measurement. Figure 9

[0061] (2) CT imaging effect characterization:

[0062] The iodine-containing nanoparticle solutions of Examples 1-5 were freeze-dried and then reconstituted into aqueous solutions with different mass concentrations (1 mg / mL, 2 mg / mL, 4 mg / mL, 8 mg / mL, 16 mg / mL, 32 mg / mL, 64 mg / mL) using deionized water. The solutions were then transferred to a 96-well plate (150 μL per well) and scanned using a CT scanner. The generated data files were processed and analyzed using RadiAnt DICOM Viewer, and the CT imaging graphs are shown in Figure 10 . In addition, a scatter plot was drawn based on the measured CT values and a curve was fitted (see Figure 11 ​It can be seen that except for Example 1, the CT imaging effect of other groups gradually becomes obvious with the increase of the concentration; for the same mass concentration, the CT imaging effect becomes more and more significant due to the increase of the proportion of the iodine-containing monomer from Example 1 to Example 5.

[0063] (3) Cell toxicity characterization:

[0064] The MRC-5 cell line (purchased from Sibion) was used to characterize the cell toxicity of the nanoparticles in Examples 2 to 5 (Example 1 was not subjected to this characterization due to no CT imaging effect) by CCK-8 method. About 5000 cells were plated in each well of a 96-well plate, and the cells were co-incubated with different concentrations of nanoparticles (the final concentration of the nanoparticles was 50 μg / mL-1 mg / mL) for 24 h. After removing the supernatant, 10 μL of CCK-8 reagent and 90 μL of DMEM medium were added to each well for 1.5 h of incubation. Finally, the relative viability of the cells was determined by an enzyme marker (calculated by the absorbance at 450 nm), and the results are shown in Figures 12-15 It can be seen that the cell compatibility of each group shows a certain concentration dependence, and the cell viability is higher than 80% in the set concentration range (50 μg / mL-1 mg / mL), which proves that the material has good cell compatibility.

[0065] As can be seen from the above, the present application synthesizes an amphiphilic block copolymer with one end hydrophilic and the other end hydrophobic based on polyethylene glycol and organosiloxane monomers, and then uses the copolymer to self-assemble and encapsulate iodine-containing monomers to form nanoparticles, which form nanocolloids with structural stability and high X-ray opacity after adjusting the pH. The nanoparticles have good CT imaging effect and biocompatibility, and have the potential to become a CT imaging contrast agent.

[0066] The embodiments of the present application are described in detail above, but the present application is not limited to the described embodiments. For those skilled in the art, various changes, modifications, replacements and variations of these embodiments can be made without departing from the principles and spirits of the present application, and still fall within the protection scope of the present application.

Claims

1. A method for preparing CT visualizable nanoparticles based on amphiphilic block copolymers, characterized in that, The method comprises the following steps: S1. Dissolving polyethylene glycol monomethyl ether, triethylamine and 2-bromoisobutyryl bromide in an organic solvent under an inert gas atmosphere, and reacting at room temperature for 40-60 hours; after the reaction, washing, dialysis and drying are performed to obtain compound 1, the structural formula of which is shown below: In the formula, n = 110-120; S2. Dissolving compound 1 and 3-(methacryloyloxy)propyl trimethoxysilane in an organic solvent under anhydrous and anaerobic conditions, and then adding copper bromide, pentamethyldiethylenetriamine (PMDETA) and cuprous bromide, and then performing a condensation reflux reaction under an inert gas atmosphere; after the reaction, removing metal ions, and then washing to obtain compound 2, the structural formula of which is shown below: In the formula, n = 110-120, and m = 20-30; S3. Dissolving compound 2 and iodine-containing monomer (3-iodopropyl) trimethoxysilane in an organic solvent, and then adding it to water under stirring, and then performing dialysis to obtain nanoparticles.

2. The method for preparing CT visualizing nanoparticles based on amphiphilic block copolymer according to claim 1, characterized in that, In S1, the average molecular weight of the polyethylene glycol monomethyl ether is 4000-8000.

3. The method for preparing CT visualization nanoparticles based on amphiphilic block copolymers according to claim 1, characterized in that, In S1, the molar ratio of the polyethylene glycol monomethyl ether, triethylamine and 2-bromoisobutyryl bromide is 1:3-4:7-9.

4. The method for preparing CT visualization nanoparticles based on amphiphilic block copolymers according to claim 1, characterized in that, In S2, the condensation reflux reaction is performed at a temperature of 60-80°C for 20-30 hours.

5. The method for preparing CT visualization nanoparticles based on amphiphilic block copolymers according to claim 1, characterized in that, In S2, the organic solvent is a mixture of tetrahydrofuran and ethanol in a volume ratio of 1:

1.

6. The method for preparing CT visualization nanoparticles based on amphiphilic block copolymers according to claim 1, characterized in that, In S2, the amount of compound 1, 3-(methacryloyloxy)propyl trimethoxysilane, copper bromide, pentamethyldiethylenetriamine and cuprous bromide is 350-400 mg:500-550 μL:0.4-0.7 mg:20-30 μL:10-20 mg.

7. The method for preparing CT visualization nanoparticles based on amphiphilic block copolymers according to claim 1, characterized in that, In S3, the amount of compound 2 and iodine-containing monomer is 68 mg:10-200 μL.

8. The method for preparing CT visualization nanoparticles based on amphiphilic block copolymers according to claim 1, characterized in that, In S3, the dialysis is performed by using a dialysis bag with a molecular weight cut-off of 700-1500 Da for 20-30 hours, adjusting the pH of the dialysis solution to 8-10 and then dialyzing for another 10-15 hours, and finally dialyzing with water for 20-30 hours.

9. CT visualizing nanoparticles based on amphiphilic block copolymers prepared by the method of any one of claims 1-8.

10. Use of the CT visualizing nanoparticles based on amphiphilic block copolymers of claim 9 in the preparation of a CT contrast agent.

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