Fluorescence-magnetic resonance imaging polystyrene microsphere as well as preparation method and application thereof

Abstract

The invention discloses a fluorescent-magnetic resonance imaging polystyrene microsphere as well as a preparation method and application thereof. A styrene group is introduced into a fluorescent dye and a gadolinium agent, and styrene is copolymerized with a fluorescent dye monomer containing a double-bond structure and a magnetic monomer by adopting an emulsion polymerization method, so that the prepared polystyrene microsphere has the capabilities of fluorescence imaging and magnetic resonance imaging at the same time. The prepared polystyrene microspheres have the advantages of being adjustable in size, good in monodispersity in emulsion and the like by adjusting and controlling conditions such as the content of the surfactant, and can be further used for in-vivo imaging of organisms. Included are enhanced marker stability, superior imaging performance, and broad biocompatibility. The multifunctional microsphere is expected to play an important role in biomedical research, for example, the multifunctional microsphere is used for disease diagnosis, tracking of a drug delivery system, research of biological distribution and metabolism and the like, and the multifunctional microsphere shows huge potential in the field of biomedical imaging.

Description

Technical Field

[0001] The present invention relates to the field of polystyrene composite microspheres, and particularly to a fluorescence-magnetic resonance imaging polystyrene microsphere, a preparation method thereof, and an application thereof. Background Art

[0002] As an intuitive and visual technology, bioimaging has been widely studied and applied in fields such as basic biology and medicine, and can help diagnose diseases and provide biological information required for clinical stage research. Currently, the main methods of bioimaging include fluorescence imaging, magnetic resonance imaging, X-ray computed tomography, positron emission tomography, single photon emission computed tomography, etc. Among them, fluorescence imaging, as a real-time dynamic imaging method, has the advantages of high sensitivity, high specificity, multi-modal and multi-spectral capabilities, simple operation, and no radioactive pollution, but there are problems such as limited light penetration depth and biocompatibility of fluorophores. Magnetic resonance imaging, as another non-invasive imaging method, has the advantages of high image clarity and good soft tissue resolution, and is suitable for deep tissue imaging such as nervous system, musculoskeletal, and cardiovascular imaging, but there are problems of long scanning time and inability to perform dynamic imaging. The two complement each other in the field of bioimaging. Currently, the development of fluorophores and contrast agents and how to effectively combine the two have become hot research topics.

[0003] Compared with commonly used fluorophores such as organic dyes and quantum dots, and contrast agents such as magnetic nanoparticles, functionalized polystyrene microspheres have the advantages of stable morphological structure, narrow particle size distribution, good monodispersity, and high luminous efficiency. At the same time, they have good biocompatibility and can load a variety of markers, and have good development prospects in the fields of bioimaging, immunoassay, etc. In addition to surface modification for the functionalization of polystyrene, fluorescent or magnetic materials are usually introduced into the interior of polystyrene to achieve the function of bioimaging.

[0004] In current research, there are many reports on single-labeled polystyrene microspheres. CN118116684A discloses a manganese-zinc ferrite nanoparticle, a magnetic polystyrene composite microsphere prepared therefrom, and an application thereof, and prepares a nanoscale magnetic polystyrene composite microsphere with a core-shell structure, improving its magnetic properties, and is suitable for fields such as nuclear magnetic resonance for studying contrast performance and immunoassay. CN116284978B discloses a fluorescent microsphere, a preparation method thereof, and an application thereof. The fluorescent microsphere is composed of an amino-containing polystyrene microsphere and an acridine-based fluorescent dye connected through an amino group, and has the ability of fluorescence emission, and can increase the fluorescence intensity during the immunoassay process.

[0005] However, although the above research has demonstrated the potential of polystyrene microspheres in a single imaging mode, integrating the two functions of fluorescence and magnetic resonance imaging into a single microsphere system still faces significant challenges. Existing physical embedding methods are difficult to ensure the stability and uniform distribution of fluorescent dyes and gadolinium agents within the microspheres, leading to an increased risk of marker leakage, which affects the imaging effect and reliability. In addition, the lack of effective fixation means at the molecular level makes it more difficult to precisely control the particle size and monodispersity of the microspheres. Therefore, there is an urgent need for an innovative method to solve these problems in order to achieve the preparation of highly efficient and stable dual-mode imaging polystyrene microspheres. This not only requires overcoming the limitations of existing technologies but also exploring new synthesis strategies and technical means to meet the growing demand for biological imaging. Summary of the Invention

[0006] To solve the above technical problems, the object of the present invention is to provide a fluorescence-magnetic resonance imaging polystyrene microsphere, its preparation method and application. The polystyrene microsphere of the present invention combines polystyrene, a fluorescent dye and a gadolinium agent, and has two imaging methods of fluorescence imaging and magnetic resonance imaging. It can enter cells and organisms and observe fluorescence signals and magnetic resonance signals to study its distribution and metabolism in vitro and in vivo.

[0007] The present invention provides a fluorescence-magnetic resonance imaging polystyrene microsphere, which comprises the following components by weight percentage: 97-99 wt% of polystyrene, 0.1-1.5 wt% of a fluorescent dye, and 0.1-1.5 wt% of a gadolinium agent. The preparation method is an emulsion polymerization method of styrene in the presence of a surfactant. Further preferably, polystyrene: 98-99.8 wt%, fluorescent dye: 0.5-1 wt%, gadolinium agent: 0.5-1 wt%. In the most preferred case, polystyrene: 98.5 wt%; fluorescent dye I: 0.75 wt%; gadolinium agent II: 0.75 wt%.

[0008] The fluorescent dye is a fluorescent molecule that can provide efficient fluorescence signals and has the ability of chemical bonding; preferably a rhodamine derivative, and most preferably the fluorescent dye I described in Example 1;

[0009]

[0010] The gadolinium agent is a gadolinium-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid derivative, and most preferably the gadolinium agent II described in Example 2;

[0011]

[0012] The surfactant is at least one of sodium dodecyl sulfate, cetyltrimethylammonium chloride and polyvinyl alcohol.

[0013] The preparation method of the fluorescence-magnetic resonance imaging polystyrene microspheres is as follows:

[0014] Take a fluorescent dye, a gadolinium agent, a surfactant and water, stir at room temperature for 5 - 60 minutes under inert gas protection; raise the reaction temperature to 60 - 95 °C for heating and stirring, add styrene, and stir thoroughly for 10 - 120 minutes; add an aqueous solution of potassium persulfate as an initiator for a full reaction; perform gradient centrifugation on the upper-layer emulsion and then dialysis to obtain a fluorescence-magnetic resonance imaging polystyrene microsphere emulsion;

[0015] The mass ratio of the added fluorescent dye: gadolinium agent: surfactant: styrene: initiator: water is: 1 - 10:1 - 10:1 - 10:250 - 750:3000 - 4000:1 - 10; the more preferred mass ratio is 1 - 5:1 - 5:1 - 5:400 - 600:3200 - 3800:1 - 5, and the most preferred mass ratio is 1:1:1.2:200:4500:1.

[0016] In a further preferred case: the stirring time is more preferably 10 - 40 minutes, and most preferably 30 minutes;

[0017] In a further preferred case: the inert gas protection condition is more preferably nitrogen to displace the air in the system, the number of displacements is 1 - 5 times, 1 - 5 minutes each time, most preferably 3 times, 2 minutes each time.

[0018] In a further preferred case: the temperature of heating and stirring is more preferably 70 - 90 °C, and most preferably 80 °C;

[0019] In a further preferred case: the time of heating and stirring is more preferably 30 - 90 minutes, and most preferably 60 minutes;

[0020] In a further preferred case: the concentration of the aqueous potassium persulfate solution is 1 - 20 mg / ml; more preferably 2 - 15 mg / ml, and most preferably 5 mg / ml;

[0021] In a further preferred case: the reaction time after adding the initiator is 1 - 24 hours; more preferably 2 - 18 hours, and most preferably 12 hours.

[0022] In a further preferred case: the gradient centrifugation is to centrifuge at 5000 - 12000 r / min (more preferably 6000, 8000, 10000 r / min) for 3 - 10 minutes (more preferably 5 minutes);

[0023] In a further preferred case: the dialysis is to dialyze with ultrapure water for 24 - 72 hours (more preferably 48 hours).

[0024] The preparation method of the fluorescent dye is as follows:

[0025] S1: Under the condition of an ice-water bath, dissolve N,N'-dimethylethylenediamine and compound Y-1 in anhydrous dichloromethane and stir well. Raise the reaction temperature to room temperature and react fully under stirring conditions. Remove the solvent, purify, and obtain compound Y-2;

[0026] The molar ratio of the said compound Y-1 to N,N'-dimethylethylenediamine is 1:3 - 5; more preferably 1:4;

[0027] The stirring time is 2 - 6 hours; more preferably 4 hours;

[0028] The solvent is removed by rotary evaporation under reduced pressure

[0029] Purification is carried out using silica gel column chromatography; the volume ratio of the eluent dichloromethane:methanol is 10 - 20:1, more preferably 15:1;

[0030] S2: Under the condition of an ice-water bath, dissolve compound Y-3 and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate in mixed solvent I and stir well; then dropwise add N,N-diisopropylethylamine and continue to stir and react fully in the ice-water bath; finally, add compound Y-2 and react fully at room temperature. Remove the solvent, slurry, filter, dry, and purify to obtain the target product fluorescent dye, and fluorescent dye I is obtained for Example 1;

[0031] The molar ratio of compound Y-3 to O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate is 1:1.5;

[0032] The molar ratio of compound Y-3 to N,N-diisopropylethylamine is 1:3 - 5; more preferably 1:4;

[0033] The said mixed solvent I is a mixture of N,N-dimethylformamide and anhydrous dichloromethane in a volume ratio of 1:1 - 10;

[0034] The molar ratio of compound Y-3 to compound Y-2 is 1:1 - 1.2; more preferably 1:1.1;

[0035] The purification in S2 is carried out using a medium-pressure liquid preparation chromatograph, and the volume ratio of the eluent methanol:water is 2:1 - 4:1, more preferably 3:2.

[0036]

[0037] The preparation method of the gadolinium-based contrast agent is as follows:

[0038] S1: Under the condition of an ice - water bath, dissolve compound Y - 4 and O-(7 - azabenzotriazol - 1 - yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate in anhydrous dichloromethane, and stir well; then dropwise add N,N - diisopropylethylamine, and continue stirring in the ice - water bath; finally, add compound Y - 2, raise the reaction temperature to room temperature and react fully. After the reaction is completed, remove the solvent and purify to obtain compound Y - 5;

[0039] The molar ratio of compound Y - 4 to O-(7 - azabenzotriazol - 1 - yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate is 1:1.5;

[0040] The molar ratio of compound Y - 4 to N,N - diisopropylethylamine is 1:3 - 5; more preferably 1:4;

[0041] The molar ratio of compound Y - 4 to compound Y - 2 is 1:1 - 1.2; more preferably 1:1.1;

[0042] The purification is carried out using silica gel column chromatography, and the volume ratio of the eluent dichloromethane:ethyl acetate is 5:1 - 10:1, more preferably 8:1.

[0043] S2: Dissolve compound Y - 5 in mixed solvent II, and react fully under stirring conditions, then remove the solvent to obtain compound Y - 6;

[0044] The mixed solvent II is obtained by mixing anhydrous dichloromethane and trifluoroacetic acid in a volume ratio of 1:0.8 - 1.2, more preferably 1:1;

[0045] S3: Dissolve compound Y - 6 and gadolinium chloride in water, and stir at room temperature overnight to obtain the target compound II;

[0046] The molar ratio of compound Y - 6 to gadolinium chloride is 1:1 - 1.5; more preferably 1:1.2;

[0047]

[0048] The application of the above - mentioned fluorescence - magnetic resonance imaging polystyrene microspheres in fluorescence imaging and nuclear magnetic resonance imaging of cells and organisms in vivo. For example: The microspheres are used for fluorescence imaging of various types of cells (including but not limited to cancer cell lines, primary cells, stem cells, etc.) and nuclear magnetic resonance imaging of various organisms (including but not limited to mammals, fish, insects, etc.). Among them, the microspheres can enter the organism through any suitable administration route (such as intravenous injection, subcutaneous injection, oral administration, etc.), and imaging detection can be carried out at any time point after injection (such as within several hours to several days).

[0049] Compared with the prior art, the present invention has the following technical effects:

[0050] Provided is a fluorescent-magnetic resonance imaging polystyrene microsphere and its application. By introducing styrene groups with polymerizable double bond structures into the original structures of fluorescent dyes and gadolinium agents through chemical reactions, emulsion polymerization occurs with styrene monomers under the action of surfactants and initiators to obtain polystyrene microspheres. Fluorescent labeling and magnetic labeling of polystyrene microspheres are achieved through chemical bonding. While realizing fluorescent-magnetic resonance imaging, it largely avoids the problem of marker leakage caused by instability in physical embedding and other methods. And by controlling reaction time, temperature, surfactant content, etc., the prepared polystyrene microspheres have characteristics such as adjustable particle size and excellent monodispersity. It can be used for fluorescent-magnetic resonance imaging of cells and organisms and is expected to be further expanded.

[0051] Based on the above technical principles and innovative designs, the technical advantages of the present invention are further elaborated from aspects such as marker stability, particle size regulation, imaging performance, and application prospects, supported by experimental data and charts.

[0052] Enhanced marker stability: Through chemical bonding, fluorescent dyes and gadolinium agents are tightly bound to polystyrene microspheres, avoiding the common problem of marker leakage in physical embedding methods. This is verified by the laser confocal microscopy images of Example 3 ( Figure 8 ) and magnetic resonance imaging images ( Figure 9 ), showing the stability and high efficiency of the microspheres in fluorescent and magnetic resonance imaging.

[0053] Adjustable particle size and good monodispersity: By controlling reaction conditions (such as surfactant content, reaction temperature, and time), the microspheres prepared in the present invention have the advantage of adjustable particle size and show excellent monodispersity in the emulsion state. Figures 3 - 6 The particle size distribution diagram of

[0054] shows that the microsphere particle size follows a normal distribution and the particle size change is controllable under different conditions, demonstrating the uniformity and repeatability of the microspheres. Figure 8 Figure 9 and show that the microspheres exhibit obvious signals in both fluorescent and magnetic resonance imaging, indicating their excellent imaging performance. Especially in the magnetic resonance imaging images, as the microsphere concentration increases, the image brightness significantly enhances, indicating that the binding of gadolinium agents to the microspheres effectively improves the imaging contrast.

[0055]

[0055] Wide application: The microspheres prepared by the present invention can be used for fluorescence and magnetic resonance imaging of a variety of cells (such as cancer cell lines, primary cells, stem cells, etc.) and organisms (such as mammals, fish, insects, etc.), and can enter the organism through a variety of administration routes (such as intravenous injection, subcutaneous injection, oral administration, etc.), having broad application prospects. Description of the drawings

[0056] Figure 1 Is the high-resolution mass spectrum of Compound I; It can be seen from the figure that there is a molecular ion peak with the highest abundance of 615.3537, proving that Compound I was successfully synthesized in Example 1 and has a high purity;

[0057] Figure 2 Is the high-resolution mass spectrum of Compound Y-4; It can be seen from the figure that there is a molecular ion peak with the highest abundance of 781.5290, proving that Compound Y-4 was successfully synthesized in Example 2 and has a high purity;

[0058] Figure 3 Is the particle size distribution diagram of the polystyrene microspheres prepared in Example 3; It can be analyzed from the figure that the particle size of the prepared microspheres shows a normal distribution, proving that the microspheres were successfully synthesized in Example 3 and the particle sizes are concentratedly distributed;

[0059] Figure 4 Is the mass-particle size diagram of sodium dodecyl sulfate in Example 4; It can be seen from the figure that within the experimental range, as the mass of sodium dodecyl sulfate added increases, the particle size of the microspheres gradually decreases;

[0060] Figure 5 Is the reaction temperature-particle size diagram in Example 5; It can be seen from the figure that at 70 - 90 °C, the temperature has no significant effect on the particle size of the microspheres;

[0061] Figure 6 Is the reaction time-particle size diagram in Example 6; It can be seen from the figure that when the reaction time is 2 - 12 hours, the temperature has no significant effect on the particle size of the microspheres;

[0062] Figure 7 Is the scanning electron microscope image of the polystyrene microspheres; It can be analyzed from the figure that the microspheres prepared in Example 3 have a highly uniform size and a smooth surface morphology;

[0063] Figure 8 Is the confocal laser scanning microscope image of the polystyrene microspheres; It can be observed from the figure that there is an obvious fluorescence signal in Example 3, proving that the microspheres prepared in Example 3 have the ability of fluorescence imaging;

[0064] Figure 9 Is the magnetic resonance imaging image of the polystyrene microspheres; It can be observed from the figure that there is an obvious magnetic resonance imaging signal in Example 3, proving that the microspheres prepared in Example 3 have the ability of magnetic resonance imaging.

[0065] Figure 10 This is the cytotoxicity experiment graph of the prepared polystyrene microspheres. It can be analyzed from the graph that Example 3 does not have obvious cytotoxicity, indicating its good biosafety.

[0066] Figure 11 This is the in-vivo fluorescence imaging graph of the prepared polystyrene microspheres in zebrafish. It can be analyzed from the graph that obvious fluorescence signals can be observed in zebrafish after Example 3 is ingested by zebrafish, indicating that it can be applied to in-vivo fluorescence imaging of organisms.

[0067] Figure 12 This is the in-vivo nuclear magnetic resonance imaging graph of the prepared polystyrene microspheres in mice. It can be analyzed from the graph that changes in liver imaging contrast can be observed after Example 3 enters the mice, indicating that it can be applied to in-vivo magnetic resonance imaging of organisms. Detailed implementation manners

[0068] The following combines the accompanying drawings and examples to further describe in detail the specific implementation manners of the present invention. The following examples are used to illustrate the present invention, but do not limit the scope of the present invention.

[0069] In the specific implementation process of the present invention, in addition to the key steps described in detail, a variety of conventional chemical reagents and standard operation procedures are also used. For example, all solvents such as dichloromethane, N,N-dimethylformamide (DMF), methanol, etc. are purchased from commercial suppliers and their purity is ensured. In addition, common chemical reaction conditions such as reaction systems under nitrogen protection, rotary evaporation under reduced pressure, silica gel column chromatography, etc. are all standard operations in the field of organic synthesis. The selection of these conventional steps and reagents is based on their wide applicability and reliability, and does not constitute a key impact on understanding the core technology of the present invention, so they are not described in detail.

[0070] Example 1

[0071] Synthesis of fluorescent dye Ⅰ

[0072] Under an ice-water bath condition, add 100 ml of anhydrous dichloromethane and 2.6 ml (20 mmol) of N,N'-dimethylethylenediamine to a 500 ml single-necked flask and stir magnetically. Dissolve 0.7 ml (5 mmol) of Y-1 in 50 ml of anhydrous dichloromethane and add it dropwise to the reaction solution. Raise the reaction temperature to room temperature and stir magnetically for 4 hours. Remove the solvent by rotary evaporation under reduced pressure, and perform purification using silica gel column chromatography (eluent V DCM :V MeOH = 15:1), and dry under vacuum to obtain the product as a slightly yellow solid Y-3 (0.72 g, 3.56 mmol), with a yield of 71%.

[0073] Dissolve Y-2 (500 mg, 1.16 mmol) and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (661 mg, 1.74 mmol) in a mixed solvent of 2 ml of N,N-dimethylformamide (DMF) and 8 ml of anhydrous dichloromethane, and stir magnetically in an ice-water bath for 30 min. Then, add N,N-diisopropylethylamine (DIPEA) (0.6 ml, 3.48 mmol) dropwise, and stir magnetically in an ice-water bath for 30 min. Finally, add Y-3 (260 mg, 1.27 mmol), raise the reaction temperature to room temperature, and stir the reaction overnight. After the reaction is completed, remove dichloromethane by rotary evaporation under reduced pressure. Dropwise add the remaining solution into 50 ml of methyl tert-butyl ether, stir for one hour, filter, and dry to obtain a dark red solid. Dissolve the product in a small amount of methanol, and purify the product using a medium-pressure liquid chromatography preparative instrument (V MeOH :V H2O = 3:2). Remove methanol by rotary evaporation under reduced pressure, and freeze-dry to obtain the product - fluorescent dye I, which is a dark red solid (390 mg, 0.63 mmol), and the yield is 54.4%.

[0074] The reaction formula of Example 1 is as follows:

[0075]

[0076] Example 2

[0077] Synthesis of Gadolinium Agent II

[0078] Dissolve compound Y-4 (500 mg, 0.87 mmol) and HATU (498 mg, 0.96 mmol) in 20 ml of anhydrous dichloromethane, and stir magnetically in an ice-water bath for 30 min. Then, add DIPEA (0.46 ml, 2.62 mmol) dropwise, and stir magnetically in an ice-water bath for 30 min. Finally, add compound Y-2 (260 mg, 1.27 mmol), raise the reaction temperature to room temperature, and stir the reaction overnight. After the reaction is completed, remove dichloromethane by rotary evaporation under reduced pressure. Add 30 ml of ethyl acetate, wash with 30 ml of 1M dilute hydrochloric acid, saturated sodium carbonate solution, and water respectively. Collect the organic phase, add anhydrous sodium sulfate for drying, remove ethyl acetate by rotary evaporation under reduced pressure, and purify using silica gel column chromatography (eluent V DCM :V EA = 8:1), and dry under vacuum to obtain the product, a slightly yellow solid Y-5 (456 mg, 0.60 mmol), with a yield of 68.8%.

[0079] Dissolve Y-5 (456 mg, 0.60 mmol) in a mixed solution of 10 ml of anhydrous dichloromethane and trifluoroacetic acid (V DCM :VTFA (ratio = 1:1), and the reaction was magnetically stirred at room temperature for 4 hours. After the reaction was completed, the solvent was removed by rotary evaporation under reduced pressure and dried in vacuo to obtain product Y-6 as a light yellow solid (325 mg, 0.55 mmol) with a yield of 91.6%.

[0080] Y-6 (200 mg, 0.34 mmol) and gadolinium chloride (100 mg, 0.38 mmol) were dissolved in 20 ml of ultrapure water. After ultrasonic treatment for 10 min, the mixture was magnetically stirred overnight to obtain an aqueous solution of the target compound, gadolinium agent II, for standby.

[0081] The reaction formula of Example 2 is as follows:

[0082]

[0083] Example 3

[0084] Synthesis of Fluorescent-Magnetic Polystyrene Microspheres

[0085] Step 1: Add 5 mg of fluorescent dye I, 5 mg of gadolinium agent II, 6 mg of sodium dodecyl sulfate, and 8 mL of ultrapure water to a three-necked flask. After ultrasonic treatment for 5 min to assist dissolution, stir at room temperature for 30 minutes. Assemble a spherical condenser, and use a diaphragm pump and a double-tube to perform nitrogen replacement. Evacuate the air in the system for 2 min each time.

[0086] Step 2: Place the three-necked flask in an oil bath at 80 °C and heat with stirring. Use a syringe to dropwise add 1 g of styrene to the reaction system under nitrogen protection and stir for 1 hour.

[0087] Step 3: Quickly add 1 mL of 5 mg / mL aqueous potassium persulfate solution to the reaction system as an initiator and continue the reaction for 12 h.

[0088] Step 4: After the reaction is completed, centrifuge the reaction solution at different gradients. Centrifuge at 6000, 8000, and 10000 r / min for 5 minutes respectively to remove the precipitate. The upper emulsion is dialyzed with ultrapure water for 48 hours to obtain a microsphere solution for standby at room temperature.

[0089] Example 4

[0090] Except for changing the mass of sodium dodecyl sulfate added in Step 1, other parameters are the same as in Example 3.

[0091] The mass of sodium dodecyl sulfate added in Step 1 was changed to 2, 3, 4, 5, 10, 15, and 20 mg respectively.

[0092] Example 5

[0093] Except for changing the reaction temperature in Step 2, other parameters are the same as in Example 3.

[0094] In Step 2, the reaction temperatures were set at 70, 75, 80, 85, and 90 °C respectively.

[0095] Example 6

[0096] Except for changing the reaction time in Step 3, other parameters were the same as in Example 3;

[0097] In Step 3, the reaction times were set at 2, 4, 6, 8, 10, and 12 hours respectively.

[0098] Effect data: Tests, results, and analysis

[0099] (1) Compound structure identification

[0100] The structures of Compound Ⅰ in Example 1 and Compound Y-4 obtained in Example 2 were characterized and identified using high-resolution mass spectrometry and 1H nuclear magnetic resonance spectroscopy. The results showed that the compounds were successfully synthesized and had high purity.

[0101] The identification data of Compound Ⅰ in Example 1 are as follows

[0102] 1H NMR(400MHz,DMSO-d6)δ7.93(s,1H),7.71(d,J=7.5Hz,1H),7.55–7.13(m,5H),6.69(dd,J=17.8,9.4Hz,1H),6.54(d,J=26.5Hz,4H),6.37(d,J=9.8Hz,1H),5.79(t,J=15.7Hz,1H),5.22(d,J=11.0Hz,1H),3.67(s,2H),2.94(d,J=10.3Hz,12H),2.71(s,2H),2.04–1.77(m,2H).

[0103] The identification data of Compound Y-4 in Example 2 are as follows

[0104] 1H NMR(400MHz,Chloroform-d)δ7.35(d,J=7.9Hz,2H),7.26(s,2H),6.69(dd,J=17.6,10.9Hz,1H),5.73(d,J=17.6Hz,1H),5.24(dd,J=10.9,3.4Hz,1H),2.91(s,3H),2.81(d,J=2.6Hz,2H),2.36(s,2H),2.27(s,3H),1.50–1.38(m,27H).

[0105] (2) Fluorescence-magnetic resonance imaging polystyrene microsphere property test

[0106] The particle sizes of Examples 3-6 were tested using a particle size analyzer, and the test results are as follows Figures 3 - 6 ; and the morphology of the fluorescence-magnetic resonance imaging polystyrene microspheres prepared in Example 3 above was observed using a scanning electron microscope, and the results are as follows Figure 7 ; the results show that the microspheres have a highly uniform size distribution, a smooth surface morphology, and good monodispersity. The fluorescence imaging ability of the microspheres prepared in Example 3 was observed using a laser confocal microscope, and the results are as follows Figure 8 ; it shows that it can generate obvious fluorescence signals under excitation, confirming the efficient loading and stable existence of the fluorescent dye in the microspheres prepared by the present invention. The nuclear magnetic resonance imaging ability of the microspheres prepared in Example 3 was tested using a nuclear magnetic resonance imaging instrument, and the results are as follows Figure 9 ; as the concentration of the microspheres increased, the image became significantly brighter. The test results further confirmed the full binding of the gadolinium agent to the microspheres, indicating that the present invention significantly improved the contrast of nuclear magnetic resonance imaging, which helps to improve the resolution performance and observation effect. The cytotoxicity of the microspheres prepared in Example 3 in 4T1 cells was tested, and the results are as follows Figure 10 ; it shows that the cell survival rate did not decrease significantly at different concentrations, indicating its good biosafety. The imaging effect of the microspheres prepared in Example 3 in zebrafish was observed under a laser confocal microscope, indicating its good in vivo fluorescence imaging ability. The microspheres prepared in Example 3 were used to intragastrically administer to mice, and after half an hour, a significant change in the magnetic resonance imaging contrast of the mouse liver could be observed, indicating its in vivo magnetic resonance imaging ability. These results fully demonstrate the effectiveness of the chemical bonding strategy proposed by the present invention in improving the stability and imaging performance of the markers.

[0107] It should be understood that the above embodiments are only for more clearly explaining the technical solutions of the present invention, rather than limiting the scope of its protection. Those of ordinary skill in the art can make various modifications and variations to the above embodiments without departing from the spirit and basic principles of the present invention, but these modifications and variations still fall within the protection scope of the claims of the present invention and their equivalent replacements. The protection scope of the present invention shall be subject to the appended claims, rather than being limited to the specific details of the above embodiments.

Claims

1. A fluorescence-magnetic resonance imaging polystyrene microsphere, a preparation method thereof and an application thereof, characterized in that: It comprises the following components by weight percentage: 97 - 99 wt% of polystyrene, 0.1 - 1.5 wt% of a fluorescent dye, 0.1 - 1.5 wt% of a gadolinium agent, and the preparation method is the emulsion polymerization method of styrene in the presence of a surfactant.

2. The fluorescent-magnetic resonance imaging polystyrene microspheres according to claim 1, wherein: The fluorescent dye is a fluorescent molecule that can provide a high - efficiency fluorescent signal and has the ability of chemical bonding.

3. The fluorescent-magnetic resonance imaging polystyrene microspheres according to claim 1, wherein: The gadolinium agent is a gadolinium - 1,4,7,10 - tetraazacyclododecane - 1,4,7,10 - tetraacetic acid derivative.

4. The fluorescent-magnetic resonance imaging polystyrene microspheres according to claim 1, characterized in that: The surfactant is at least one of sodium dodecyl sulfate, cetyltrimethylammonium chloride, and polyvinyl alcohol.

5. The preparation method of the fluorescence-magnetic resonance imaging polystyrene microspheres according to claim 1, wherein: It includes the following steps: Take the fluorescent dye, gadolinium agent, surfactant, and water, stir at room temperature for 5 - 60 minutes under inert gas protection; raise the reaction temperature to 60 - 95 °C for heating and stirring, add styrene, and stir thoroughly for 10 - 120 minutes; add an aqueous solution of potassium persulfate as an initiator and react fully; subject the upper - layer emulsion to gradient centrifugation and then dialysis to obtain a fluorescent - magnetic resonance imaging polystyrene microsphere emulsion. The mass ratio of the fluorescent dye: gadolinium agent: surfactant: styrene: initiator: water added is: 1 - 10:1 - 10:1 - 10:250 - 750:3000 - 4000:1 - 10.

6. The method according to claim 1, wherein: The concentration of the aqueous potassium persulfate solution is 1 - 20 mg / ml.

7. The method according to claim 1, characterized in that: The preparation method of the fluorescent dye is as follows: S1: Under ice - water bath conditions, dissolve N,N'-dimethyl ethylenediamine and compound Y - 1 in anhydrous dichloromethane and stir thoroughly. Raise the reaction temperature to room temperature and react fully under stirring conditions. Remove the solvent, purify, and obtain compound Y - 2. The molar ratio of compound Y - 1 to N,N'-dimethyl ethylenediamine is 1:3 - 5. S2: Under ice - water bath conditions, dissolve compound Y - 3 and O-(7 - azabenzotriazol - 1 - yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate in mixed solvent I and stir thoroughly; then drop - wise add N,N - diisopropylethylamine and continue to stir fully in the ice - water bath for reaction; finally, add compound Y - 2 and react fully at room temperature. Remove the solvent, slurry, filter, dry, purify, and obtain the target product fluorescent dye. The molar ratio of compound Y - 3 to O-(7 - azabenzotriazol - 1 - yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate is 1:1.

5. The molar ratio of compound Y - 3 to N,N - diisopropylethylamine is 1:3 - 5. The mixed solvent I is a mixture of N,N - dimethylformamide and anhydrous dichloromethane in a volume ratio of 1:1 - 10. The molar ratio of compound Y - 3 to compound Y - 2 is 1:1 - 1.

2.

8. The method according to claim 1, wherein: The preparation method of the gadolinium agent is as follows: S1: Under the condition of an ice-water bath, dissolve compound Y-4 and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate in anhydrous dichloromethane, and stir well; then add N,N-diisopropylethylamine dropwise, and continue to stir in the ice-water bath; finally, add compound Y-2, raise the reaction temperature to room temperature and react fully. After the reaction is completed, remove the solvent and purify to obtain compound Y-5; The molar ratio of compound Y-4 to O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate is 1:1.5; The molar ratio of compound Y-4 to N,N-diisopropylethylamine is 1:3 - 5; The molar ratio of compound Y-4 to compound Y-2 is 1:1 - 1.2; S2: Dissolve compound Y-5 in mixed solvent II, and react fully under stirring conditions, then remove the solvent to obtain compound Y-6; The said mixed solvent II is obtained by mixing anhydrous dichloromethane and trifluoroacetic acid in a volume ratio of 1:0.8 - 1.2; S3: Dissolve compound Y-6 and gadolinium chloride in water, and stir at room temperature overnight to obtain the target compound II; The molar ratio of compound Y-6 to gadolinium chloride is 1:1 - 1.

5.

9. Use of the fluorescent-magnetic resonance imaging polystyrene microspheres according to claim 1 in fluorescence imaging and nuclear magnetic resonance imaging in cells and organisms.

10. The application according to claim 9, wherein: The said microspheres enter the organism through any suitable administration route and are subjected to imaging detection at any subsequent time point.

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