Triblock polymer cross-linked micelle with autofluorescence property as well as preparation method and application of triblock polymer cross-linked micelle

Abstract

The invention discloses a triblock polymer cross-linked micelle with an autofluorescence property as well as a preparation method and application of the triblock polymer cross-linked micelle. The polymer is PPMAx-b-POPMAy-b-PPEGMAz, x is equal to 15 to 35, y is equal to 15 to 35, and z is equal to 5 to 20. Monomer molecules of the polymer are easy to synthesize, polymer blocks are easy to regulate and control, and the polymer can be self-assembled to form stable polymer micelles. The polymer micelle structure has rich sub-fluorophores, the structural rigidity of the polymer micelle is further enhanced through a cross-linking strategy, non-radiative transition of the sub-fluorophores is inhibited, then conversion from a non-light-emitting state to a light-emitting state is promoted, and autofluorescence emission of the polymer cross-linked micelle is achieved. The polymer cross-linked micelle disclosed by the invention has pH response performance, responds to breakage of cross-linked bonds in a tumor microenvironment, realizes conversion of fluorescence from existence to absence, and is expected to realize visual tracking of in-vivo operation of the polymer micelle.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical polymer materials, and particularly relates to a triblock polymer cross-linked micelle with spontaneous fluorescence properties, a preparation method thereof, and an application thereof. Background Art

[0002] In recent years, theranostic nanocarriers that combine diagnostic imaging and therapeutic capabilities have received increasing attention due to their effective cancer treatment effects. Polymer micelles have become one of the suitable materials for theranostic nanocarriers due to their good biocompatibility, chemical modifiability, and high drug loading capacity. In order to obtain imaging performance, the use of fluorescent probe-labeled polymer micelles for intracellular tracing has become an important method. However, the incorporation of these traditional fluorescent probes into polymer micelles is mainly achieved through physical encapsulation or chemical linkage of conjugated fluorophores, and most reported methods involve many lengthy operating procedures. In addition, the leakage of coupled fluorescent probes also seriously hinders their further application and clinical translation. Therefore, a micelle delivery platform that utilizes the spontaneous fluorescence properties of polymers without coupling external fluorescent probes will have a broader application prospect.

[0003] Currently, some researchers have utilized the property that non-conjugated structure polymers can exhibit fluorescence in the aggregated state to prepare many self-fluorescent polymers. However, there are few studies on the spontaneous fluorescence properties of polymer micelles. This is because the concentration of polymer micelles is low and the structural rigidity is insufficient, resulting in weak or even non-emission of the aggregated fluorescence of the polymer, which ultimately greatly limits the application potential of self-fluorescent polymer micelles in actual tumor theranostics. Summary of the Invention

[0004] In order to overcome the problems in the above-mentioned prior art, such as the complex preparation of polymer micelles, insufficient structural rigidity, and inability to effectively achieve self-luminescence properties, the primary object of the present invention is to provide a triblock polymer cross-linked micelle with spontaneous fluorescence properties. The structure of the triblock polymer is poly(2-hydroxy-3-phenoxypropyl methacrylate)-b-poly(2-oxopropyl methacrylate)-b-poly(methacrylic acid monomethoxy polyethylene glycol ester) (PPMA-b-POPMA-b-PPEGMA). The triblock polymer cross-linked micelle with spontaneous fluorescence properties is obtained by covalently cross-linking the triblock polymer under the action of a catalyst and a cross-linking agent.

[0005] Another object of the present invention is to provide a method for preparing the above-mentioned triblock polymer cross-linked micelles with spontaneous fluorescence properties. The process of this preparation method is as follows: First, a hydrophobic block monomer (2-hydroxy-3-phenoxypropyl methacrylate, PMA) with a lipid group / hydroxyl-rich electron group is prepared through a ring-opening reaction, and a cross-linking site block monomer (2-oxopropyl methacrylate, OPMA) with a lipid group / carbonyl-rich electron group is prepared through an addition reaction method. Then, the electron transfer activation regenerative atom transfer radical polymerization (ARGET ATRP) method is used to polymerize the hydrophobic block monomer 2-hydroxy-3-phenoxypropyl methacrylate (PMA), the cross-linking site block monomer 2-oxopropyl methacrylate (OPMA), and the hydrophilic block monomer methoxypolyethylene glycol methacrylate (PEGMA) in sequence using a small molecule initiator to obtain the triblock polymer PPMA-b-POPMA-b-PPEGMA. After dissolving the triblock polymer in a solvent, it is dialyzed to obtain polymer micelles, and further a catalyst 2-amino-5-methoxybenzoic acid and a cross-linking agent dithiodihydrazide are added for cross-linking reaction to obtain the spontaneously fluorescent polymer cross-linked micelles. The cross-linking to form covalent bonds improves the structural rigidity of the polymer micelles, thereby promoting the aggregation of a large number of electron-rich groups in the polymer, effectively inhibiting the decay of non-radiative pathways, making the polymer micelles exhibit spontaneous fluorescence characteristics, and thus realizing fluorescence imaging. The preparation of the spontaneously fluorescent polymer micelles of the present invention is simple and fast, has good biocompatibility, provides a new candidate system for the development of spontaneously fluorescent nanocarriers, and has good application prospects.

[0006] Another object of the present invention is to provide the application of the above-mentioned triblock polymer cross-linked micelles with spontaneous fluorescence properties in the preparation of tumor imaging and drug delivery products.

[0007] The object of the present invention is achieved through the following technical solutions:

[0008] A method for preparing triblock polymer cross-linked micelles with spontaneous fluorescence properties, comprising the following steps:

[0009] Dissolve the triblock polymer in an organic solvent, adjust the pH to 6-7, stir for 12-24 h, then dialyze in a phosphate buffer solution, and then add a catalyst and a cross-linking agent. After cross-linking reaction at room temperature, dialyze with deionized water to obtain triblock polymer cross-linked micelles with spontaneous fluorescence properties based on covalent bond cross-linking;

[0010] The triblock polymer is poly(2-hydroxy-3-phenoxypropyl methacrylate)-b-poly(2-oxopropyl methacrylate)-b-poly(methoxypolyethylene glycol methacrylate) (PPMA x -b-POPMA y -b-PPEGMA z) wherein the degree of polymerization x of 2-hydroxy-3-phenoxypropyl polymethacrylate is 15 to 35, the degree of polymerization y of 2-oxopropyl polymethacrylate is 15 to 35, the degree of polymerization z of monomethoxypolyethylene glycol polymethacrylate is 5 to 20, and the number of structural units n of the ethylene glycol group in the side chain of the hydrophilic block monomer monomethoxypolyethylene glycol methacrylate (PEGMA) is 5 to 20.

[0011] Preferably, the number-average molecular weight of the triblock polymer is: 8277 to 23010 g / mol.

[0012] Preferably, the organic solvent is dimethyl sulfoxide (DMSO); the mass-volume ratio of the triblock polymer to the solvent is 0.5 to 1 mg: 1 mL.

[0013] Preferably, the concentration of the phosphate buffer solution is 30 to 50 mmol / L and the pH is 6.5.

[0014] Preferably, the concentration of the blank polymer micelle solution obtained by dialysis in the phosphate buffer solution is 0.5 to 1 mg / mL.

[0015] Preferably, the dialysis time in the phosphate buffer solution is 24 to 48 h.

[0016] Preferably, the catalyst is 2-amino-5-methoxybenzoic acid; the molar ratio of the catalyst dosage to the volume of the blank polymer micelle solution (i.e., the triblock polymer phosphate mixed solution) obtained after dialysis is 8 to 12 mmol: 1 L.

[0017] Preferably, the crosslinking agent is at least one of dithiodihydrazide, adipic dihydrazide, and 1,2-bis(2-aminoethoxy)ethane; the equivalent molar ratio of the crosslinking agent to 2-oxopropyl methacrylate in the triblock polymer is 1.25 to 2.25: 1.

[0018] Preferably, the crosslinking reaction time is 24 to 36 h.

[0019] Preferably, the dialysis with deionized water means transferring the reaction product into a dialysis bag with a molecular weight cut-off MWCO = 3500 kDa, and then dialyzing in deionized water for 24 h, changing the dialysis fluid every two hours for the first 12 h, and then changing the dialysis fluid every 6 h.

[0020] The triblock polymer is obtained by the following method:

[0021] (1) Preparation of hydrophobic monomer 2-hydroxy-3-phenoxypropyl methacrylate (PMA): Under an inert atmosphere, phenol, tetraethylammonium bromide, and glycidyl methacrylate are mixed evenly in a solvent, followed by heating for reaction and purification to obtain 2-hydroxy-3-phenoxypropyl methacrylate;

[0022] (2) Preparation of crosslinking site block monomer 2-oxopropyl methacrylate (OPMA): Chloroacetone and triethylamine are mixed evenly and cooled. Under ice bath conditions, methacrylic acid is added dropwise while stirring vigorously, followed by heating for reaction and purification to obtain 2-oxopropyl methacrylate;

[0023] (3) Under an inert gas atmosphere, 2-hydroxy-3-phenoxypropyl methacrylate (PMA), ligand, catalyst, reducing agent, and solvent are mixed evenly, then a small molecule initiator is added, and heating reaction is carried out. After the complete conversion of 2-hydroxy-3-phenoxypropyl methacrylate (PMA), 2-oxopropyl methacrylate (OPMA) is added to continue the reaction. After complete conversion, methoxypolyethylene glycol methacrylate (PEGMA) is added to continue the reaction, and then purification is carried out to obtain a triblock polymer (PPMA x -b-POPMA y -b-PPEGMA z ).

[0024] Preferably, the molar ratio of phenol, tetraethylammonium bromide, and glycidyl methacrylate in step (1) is 1:0.090 - 0.13:1.20 - 1.50.

[0025] Preferably, the solvent in step (1) is at least one of dichloromethane and dimethylformamide.

[0026] Preferably, the molar amount of phenol and the volume ratio of the solvent in step (1) is 4 - 6 mmol:3 mL.

[0027] Preferably, the heating reaction temperature in step (1) is 60 - 80 °C, and the time is 36 - 48 h.

[0028] Preferably, the purification in step (1) refers to gradient elution by silica gel column chromatography (the mobile phase is hexane:ethyl acetate volume ratio 20:1, 15:1, 10:1).

[0029] Preferably, the molar ratio of chloroacetone, triethylamine, and methacrylic acid in step (2) is 1:1.15 - 1.55:1.105 - 1.135.

[0030] Preferably, the cooling temperature in step (2) is 0 °C; the heating reaction temperature is 40 - 45 °C, and the time is 2 - 6 h.

[0031] Preferably, the dropping rate of methacrylic acid in step (2) is 1-5 mL / min to keep the system temperature not higher than 30 °C.

[0032] Preferably, the purification in step (2) means first filtering to remove the obtained ammonium salt precipitate, dissolving the product in water, extracting the organic phase with chloroform, and then performing vacuum distillation to obtain the purified product.

[0033] Preferably, the molar ratio of 2-hydroxy-3-phenoxypropyl methacrylate (PMA), 2-oxopropyl methacrylate (OPMA), methoxypolyethylene glycol methacrylate (PEGMA), ligand, catalyst, reducing agent and small molecule initiator in step (3) is: 15-35:15-35:5-20:0.38-0.95:0.018-0.062:0.181-0.89:0.36-1.15.

[0034] Preferably, the ligand in step (3) is 1,1,4,7,10,10-hexamethyltriethylenetetramine (HMTETA); the catalyst is copper bromide (CuBr 2 ); the reducing agent is stannous octanoate (Sn(Oct) 2 ); the small molecule initiator is ethyl 2-bromoisobutyrate (EBriB).

[0035] Preferably, the solvent in step (3) is anisole.

[0036] Preferably, the temperature of the heating reaction in step (3) is 50-70 °C; the heating reaction time of 2-hydroxy-3-phenoxypropyl methacrylate (PMA) is 6-12 h; the heating reaction time of 2-oxopropyl methacrylate (OPMA) is 24-72 h; the heating reaction time of methoxypolyethylene glycol methacrylate (PEGMA) is 96-168 h.

[0037] Preferably, the purification method in step (3) is: after the conversion of methoxypolyethylene glycol methacrylate (PEGMA) is completed, cool, add tetrahydrofuran (THF) to the product mixture to terminate the reaction, then pass through a neutral alumina chromatography column to remove the catalyst, spin evaporate and concentrate, and then dropwise add to 8-12 times the volume of ice-cold n-hexane and ice-cold methanol for precipitation. The spin evaporation-precipitation is repeated 1-5 times, and the purified product is obtained after vacuum drying.

[0038] Preferably, the inert atmosphere in steps (1) and (3) is at least one of nitrogen, argon and helium.

[0039] The present invention provides a triblock polymer cross-linked micelle with spontaneous fluorescence properties prepared by the above preparation method.

[0040] The present invention provides the application of the above-mentioned triblock polymer cross-linked micelles with spontaneous fluorescence properties in the preparation of tumor imaging and drug delivery products.

[0041] The polymer cross-linked micelles prepared by the present invention have spontaneous fluorescence characteristics (spontaneously emit fluorescence). Under the conditions of tumor acidic microenvironment (pH 5.0) and high concentration of glutathione GSH (10 mmol / L), the pH- and GSH-responsive cross-linking bonds can be broken, resulting in the disappearance of spontaneous fluorescence, thereby realizing the on-off regulation of fluorescence, which is convenient for dynamic monitoring in the processes of tumor imaging and drug delivery.

[0042] The mechanism of the spontaneous fluorescence of the polymer cross-linked micelles of the present invention is as follows:

[0043] The hydrophobic block, cross-linking site block and hydrophilic block of the triblock polymer of the present invention all have rich sub-fluorophores such as lipid groups. The corresponding monomers are polymerized in sequence by the ARGET ATRP method to promote the aggregation of sub-fluorophores to obtain a spontaneously fluorescent polymer. After dissolving it in a solvent and dialysis, and cross-linking at room temperature, a spontaneously fluorescent polymer cross-linked micelle with a hydrophobic block PPMA as the core, a cross-linked structure of POPMA as the middle layer and a hydrophilic layer PPEGMA as the shell is obtained. The electron-donating effect of the β-hydroxy group in the benzene ring structure of the hydrophobic block PMA enhances the π-π conjugation of the system. The keto carbonyl group of the cross-linking site block OPMA can carry out cross-linking reactions with various cross-linking agents at room temperature, which are all beneficial to improving the structural rigidity of the polymer micelles and inhibiting the vibration and rotation of sub-fluorophores, thereby inhibiting the non-radiative transition of sub-fluorophores and promoting the transition from the non-luminescent state to the luminescent state, enhancing the fluorescence emission. The polymer hydrophilic block PEGMA has excellent hydrophilicity and biocompatibility, improves the anti-protein adsorption performance, and can effectively enhance the stability of the polymer micelles, thereby improving the fluorescence efficiency. When the polymer cross-linked micelles are delivered to the tumor acidic (pH 5.0), high GSH concentration (10 mmol / L) microenvironment, the pH- and GSH-responsive cross-linking bonds are broken, realizing a transition of fluorescence from on to off, which helps to realize the visualization tracking of the polymer micelles in vivo operation.

[0044] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0045] (1) The preparation process flow of the spontaneously fluorescent polymer and its cross-linked micelles of the present invention is simple and the conditions are mild, and the degree of polymerization of the triblock polymer can be precisely regulated.

[0046] (2) The polymer cross-linked micelle system constructed by the present invention utilizes the structural characteristics of the polymer and the chemical bond cross-linking strategy, and can exhibit spontaneous fluorescence properties without the coupling of external fluorescent probes. This design can avoid the common problems of modification instability and probe leakage in the process of in vivo drug delivery to the greatest extent, thereby reducing the negative impact of these factors on the drug delivery effect.

[0047] (3) The cross-linking bonds constructed in the present invention are hydrazone bonds or imine bond structures that respond to pH and GSH. In a normal physiological environment, the polymer cross-linked micelles exhibit fluorescence properties. When they enter tumor cells, they can respond to the acidic environment and high GSH concentration environment in the microenvironment to break the cross-linking bonds, and the fluorescence disappears, which is beneficial for dynamically and visually tracking and observing the operation of the polymer micelles delivering drugs in vivo. It is a potential fluorescence imaging material. Description of the Drawings

[0048] Figure 1 They are the synthesis reaction formulas of monomers PMA and OPMA in Example 1 and the synthesis reaction formula of triblock polymer PPMA-b-POPMA-b-PPEGMA in Example 2.

[0049] Figure 2 They are the 1H NMR spectra of monomer PMA in Example 1.

[0050] Figure 3 They are the 1H NMR spectra of monomer OPMA in Example 1.

[0051] Figure 4 They are the 1H NMR spectra and GPC elution curves of triblock polymer PPMA-b-POPMA-b-PPEGMA in Example 2.

[0052] Figure 5 They are the CMC diagrams of triblock polymer PPMA-b-POPMA-b-PPEGMA in Example 4.

[0053] Figure 6 They are the DLS of self-fluorescent polymer cross-linked micelles in Example 5, where POP-NCMs represent blank non-cross-linked micelles and POP-SCMs represent self-fluorescent polymer cross-linked micelles.

[0054] Figure 7 They are the infrared spectra of self-fluorescent polymer cross-linked micelles (POP-SCMs) and blank non-cross-linked micelles (POP-NCMs) in Example 5.

[0055] Figure 8 They are the TEM diagrams of self-fluorescent polymer cross-linked micelles in Example 5.

[0056] Figure 9 They are the ultraviolet absorption spectra (A) and fluorescence emission spectra (B) of self-fluorescent polymer cross-linked micelles (POP-SCMs) and blank non-cross-linked micelles (POP-NCMs) in Example 5. The small figure is the optical photograph under ultraviolet lamp irradiation.

[0057] Figure 10It is the fluorescence emission spectrogram of the self - fluorescent polymer cross - linked micelles in Example 6 under simulated different physiological environments.

[0058] Figure 11 It is the cytotoxicity experiment diagram of the self - fluorescent polymer cross - linked micelles in Example 7. Detailed implementation manners

[0059] The present invention will be further described in detail below in conjunction with examples and drawings, but the implementation manners of the present invention are not limited thereto.

[0060] In the examples of the present invention, those without specific conditions are carried out according to conventional conditions or the conditions recommended by the manufacturer. Raw materials, reagents, etc. without indicating the manufacturer can be obtained as conventional products through commercial purchase.

[0061] Example 1: Preparation of polymer monomers:

[0062] (1) Hydrophobic monomer (PMA)

[0063] Add a magnetic stir bar to a 250 mL dry three - necked flask and connect a condenser reflux device. Purge with argon for 10 minutes, then seal the three - necked flask. Use a syringe to inject 18.8 g of phenol dissolved in 75 mL of dimethylformamide into the three - necked flask. Stir at 60 °C for 10 minutes, then slowly add 8.4 g of tetraethylammonium bromide and 37 g of glycidyl methacrylate dissolved in 75 mL of dimethylformamide to the reaction. Slowly heat to 70 °C and react for 48 h. After the reaction, extract with dichloromethane to obtain a crude product solution. Rotavaporize to remove the solvent. The obtained crude product is eluted by gradient elution on a silica gel column (mobile phase: n - hexane: ethyl acetate 20:1, 15:1, 10:1), and then rotavaporize again to obtain a pure amber - colored oil. The synthesis reaction formula is shown in Figure 1 (A). Use nuclear magnetic resonance hydrogen spectrum ( 1 1H NMR) to characterize and analyze the product structure, and the results are as shown in Figure 2 shown.

[0064] (2) Keto - functionalized monomer (OPMA)

[0065] Connect a thermometer and a dropping tube to a 200 mL dry four - necked flask equipped with a magnetic stir bar. Add 36 mL of chloroacetone and 45 mL of triethylamine, mix well, and then place it in an ice bath and cool to 0 °C. While stirring vigorously, drop in 40.5 mL of methacrylic acid, control the dropping rate at 2 mL / min, and keep the temperature not higher than 30 °C. Continue to stir for 1 h. Then continue to react at 45 °C for 2 h. After the reaction, filter to remove the ammonium salt precipitate obtained, extract the organic phase with dichloromethane, and rotavaporize to remove the solvent to obtain a pure amber - colored oil. The synthesis reaction formula is shown in Figure 1 (B). Use nuclear magnetic resonance hydrogen spectrum ( 1The product structure was characterized and analyzed by 1H NMR, and the results are as Figure 3 shown.

[0066] Example 2: Preparation of triblock polymer (PPMA x -b-POPMA y -b-PPEGMA z )(x:y:z = 15:25:14, n = 9)

[0067] A magnetic stir bar and catalyst CuBr 2 (0.00754 g) were added to a 150 mL dry eggplant-shaped flask. After sealing the reaction flask, it was evacuated and purged with argon three times. Then, the solvent anisole (8 mL), monomer PMA (2 g), and ligand HMTETA (0.092 mL) were added using a syringe, and the mixture was stirred vigorously for 15 min to dissolve the catalyst CuBr 2 ; Reducing agent Sn(Oct) 2 (0.1096 mL) was dissolved in 2 mL of anisole, injected into the eggplant-shaped flask, and stirred for another 10 min. After adding the small molecule initiator EBriB (0.08146 mL), the reaction mixture was transferred to an oil bath at 60 °C and reacted under a nitrogen atmosphere for 8 h, and then a nitrogen balloon was added; Monomer OPMA (2.0058 g) and anisole (2 mL) were added and the reaction continued for 48 h. Monomer PEGMA (3.75 g, n = 9) was added to the reaction and the reaction continued at 70 °C for 144 h. Ice-cold n-hexane was added, and then the mixture was stirred vigorously until precipitation occurred. The precipitate was redissolved in tetrahydrofuran, passed through a column by wet method, precipitated twice with ice-cold n-hexane and twice with ice-cold methanol, and rotary evaporated four times, and then dried under vacuum at 40 °C for 72 h to obtain a yellow paste-like solid product. The synthetic reaction formula is shown in Figure 1 (C). Using 1 1H NMR and GPC to analyze the composition and structure of the product, Mn = 14.9 kDa, The results are as Figure 4 shown.

[0068] Example 3: Preparation of triblock polymer (PPMA x -b-POPMA y -b-PPEGMA z )(x:y:z = 15:20:5, n = 20)

[0069] A magnetic stir bar and catalyst CuBr 2 (0.00504 g) were added to a 150 mL dry eggplant-shaped flask. After sealing the reaction flask, it was evacuated and purged with argon three times. Then, the solvent anisole (8 mL), monomer PMA (2 g), and ligand HMTETA (0.0614 mL) were added using a syringe, and the mixture was stirred vigorously for 15 min to dissolve the catalyst CuBr 2Dissolve; dissolve the reducing agent Sn(Oct) with 2 mL of anisole 2 (0.0731 mL), inject it into a round-bottom flask and stir for 10 min. After adding the small molecule initiator EBriB (0.0564 mL), transfer it to an oil bath at 60 °C and start the reaction for 6 h under a nitrogen atmosphere, then add a nitrogen balloon; add the monomer OPMA (1.60 g), anisole (2 mL) and the reducing agent Sn(Oct) 2 (0.015 mL) and continue the reaction for 36 h. Add the monomer PEGMA (2.68 g, n = 20) to the reaction and continue the reaction at 65 °C for 96 h. Add ice-cold n-hexane, then stir vigorously until precipitation occurs. Redissolve it in tetrahydrofuran, perform column chromatography by wet method, precipitate twice with ice-cold n-hexane and twice with ice-cold methanol, evaporate by rotary evaporation 4 times, and dry under vacuum at 40 °C for 72 h to obtain the product as a yellow oily liquid.

[0070] Example 4: Critical micelle concentration CMC value of triblock polymer

[0071] Use fluorescence spectroscopy to measure the CMC values of micelles formed by different polymers. Dissolve the polymer obtained in Example 2 in a small amount of acetone, then disperse it in deionized water to prepare a polymer stock solution (0.1 mg / mL), and stir at room temperature for 24 h to completely volatilize the acetone. Dilute the polymer solution to a range of 0.0003 - 0.1 mg / mL to obtain a series of solutions with different concentrations. Add 0.1 mL of pyrene / acetone solution (12×10 -7 mol / L) to a 10 mL volumetric flask, volatilize the acetone at room temperature, then add the polymer solution, and let it stand for 24 h to equilibrate so that the concentration of pyrene in the solution is 6×10 -7 mol / L. Set the emission wavelength of the fluorescence spectrometer to 373 nm and scan the fluorescence spectra of different solutions in the range of 300 - 350 nm. Plot a curve with the ratio I 339 / I 335 of the excitation spectral intensities at 339 nm and 335 nm and the logarithm of the concentration. The abscissa corresponding to the inflection point of the curve is the CMC value of the polymer. The critical micelle concentration of PPMA-b-POPMA-b-PPEGMA is 2.09 mg / mL, as Figure 5 shown.

[0072] Example 5: Preparation of self-fluorescent polymer cross-linked micelles

[0073] The dialysis method was used to prepare self - fluorescent polymer - crosslinked micelles. Weigh 15 mg of the triblock polymer obtained in Example 2 and dissolve it in 30 mL of DMSO. Sonicate for 1 h to fully dissolve it and stir overnight. Transfer it into a dialysis bag (MWCO = 3.5 kDa) and dialyze it with a phosphate solution (50 mmol / L, pH 6.5) for 24 h. Change the dialysis fluid every two hours for the first 12 h, and then change the dialysis fluid every 6 h to obtain a blank non - crosslinked micelle solution. Subsequently, transfer 30 mL of the blank non - crosslinked micelle solution (0.5 mg / mL) to a flask, add 10 mmol / L catalyst 2 - amino - 5 - methoxybenzoic acid (54.6 mg, 0.31 mmol) and dithiodihydrazide (4.15 mg, 0.017 mmol). After stirring and reacting for 24 h, dialyze the micelle solution with deionized water for another 12 h, changing the dialysis fluid every two hours. After dialysis, freeze - dry it at - 40 °C to obtain a white powder, which is the self - fluorescent polymer - crosslinked micelle.

[0074] The particle size, cross - linking degree and morphology of the cross - linked micelles were characterized by dynamic light scattering (DLS), infrared spectroscopy and transmission electron microscopy (TEM). The hydrodynamic diameter of the self - fluorescent polymer - crosslinked micelles was 252.93 nm, and the PDI was 0.23, as Figure 6 shown. The disappearance of the characteristic peaks in the infrared spectrum proved the occurrence of the cross - linking process, as Figure 7 shown. The TEM image showed that the cross - linked micelles presented a relatively uniform spherical morphology, as Figure 8 shown.

[0075] The self - fluorescent properties of the polymer micelles were measured by ultraviolet spectroscopy and fluorescence spectroscopy, as Figure 9 shown. The ultraviolet absorption spectrum of the polymer showed that a new characteristic peak appeared at 330 nm for the polymer - crosslinked micelles, while the fluorescence spectrum showed that the cross - linked micelles had a blue fluorescence emission wavelength at 429 nm.

[0076] Example 6: Fluorescence switch imaging performance test of self - fluorescent polymer - crosslinked micelles

[0077] Take 1 mg of the self - fluorescent polymer - crosslinked micelles (obtained in Example 5) and dissolve them in 2 mL of PBS solution (10 mmol / L, pH 7.4), acetate buffer (0.2 mmol / L, pH 5.0) and acetate buffer (0.2 mmol / L, pH 5.0) containing GSH (10 mmol / L) respectively. Put them into a 37 °C constant - temperature mixer and react for 2 h. Test the fluorescence changes of the polymer micelles by a fluorescence spectrometer. The results are shown in Figure 10 .

[0078] Example 7: Cytotoxicity experiment

[0079] The B16 cells were passaged and cultured in DMEM medium supplemented with 10% FBS, 1% penicillin and streptomycin at 37 °C and 5% CO 2 concentration. Then, the cells were seeded in a 96-well plate at a density of 5000 cells per well and incubated for 24 h. After aspirating the old medium and washing, 10 μL of the self-fluorescent polymer crosslinked micelle solution with concentrations ranging from 25 to 1000 μg / mL and 150 μL of the medium were added to the wells and incubation was continued for 24 h. Then, the liquid was aspirated and the wells were washed three times. Next, 150 μL of the medium containing 10 μL of the CCK-8 reagent was added and allowed to act for 1 h. The ultraviolet absorption value of the well plate at a wavelength of 450 nm was measured, and the cell viability at different material concentrations was calculated, as Figure 11 shown.

[0080] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A method for preparing triblock polymer cross-linked micelles with autofluorescence properties, characterized in that: The following steps are involved: The triblock polymer is dissolved in an organic solvent, the pH is adjusted to 6-7, and the mixture is stirred for 12-24 hours, and then dialyzed in a phosphate buffer solution, and then a catalyst and a cross-linking agent are added. After the cross-linking reaction at room temperature, the mixture is dialyzed with deionized water to obtain triblock polymer cross-linked micelles with autofluorescence properties based on covalent cross-linking. The triblock polymer is polymethacrylate-2-hydroxy-3-phenoxypropyl-b-polymethacrylate-2-oxopropyl-b-polymethacrylate monomethoxypolyethylene glycol, wherein the polymerization degree x of polymethacrylate-2-hydroxy-3-phenoxypropyl is 15 to 35, the polymerization degree y of polymethacrylate-2-oxopropyl is 15 to 35, the polymerization degree z of polymethacrylate monomethoxypolyethylene glycol is 5 to 20, and the number n of side chain ethylene glycol group structural units of the hydrophilic block monomer methacrylate monomethoxypolyethylene glycol is 5 to 20.

2. The preparation method according to claim 1, characterized in that: The cross-linking agent is at least one of dithiodihydrazide, adipic acid dihydrazide and 1,2-bis(2-aminoethoxy)ethane; And / or, the molar ratio of the crosslinking agent to 2-oxypropyl methacrylate in the triblock polymer is 1.25-2.25:

1.

3. The preparation method according to claim 1 or 2, characterized in that: The catalyst is 2-amino-5-methoxybenzoic acid; and / or, the molar amount of the catalyst used and the volume ratio of the blank polymer micelle solution obtained after dialysis is 8-12 mmol:1 L; And / or, the concentration of the blank polymer micelle solution obtained after dialysis in a phosphate buffer solution is 0.5-1 mg / mL.

4. The preparation method according to claim 1 or 2, characterized in that: The cross-linking reaction time is 24 to 36 hours.

5. The preparation method according to claim 1 or 2, characterized in that: The organic solvent is dimethyl sulfoxide; and / or, the mass volume ratio of the triblock polymer to the solvent is 0.5 to 1 mg: 1 mL; And / or, the concentration of the phosphate buffer solution is 30-50 mmol / L, and the pH is 6.5; And / or, the dialysis time in the phosphate buffer solution is 24 to 48 hours.

6. The preparation method according to claim 1 or 2, characterized in that: The triblock polymer is obtained by the following method: (1) preparing a hydrophobic monomer 2-hydroxy-3-phenoxypropyl methacrylate: in an inert atmosphere, phenol, tetraethylammonium bromide and glycidyl methacrylate are uniformly mixed in a solvent, heated for reaction, and purified to obtain 2-hydroxy-3-phenoxypropyl methacrylate; (2) Preparation of cross-linking site block monomer 2-oxypropyl methacrylate: Chloroacetone and triethylamine were mixed evenly and cooled, and methacrylic acid was added dropwise while stirring in an ice bath, and then heated for reaction and purified to obtain 2-oxypropyl methacrylate; (3) Under an inert gas atmosphere, 2-hydroxy-3-phenoxypropyl methacrylate, a ligand, a catalyst, a reducing agent and a solvent are uniformly mixed, and a small molecule initiator is added to carry out a heating reaction. After the 2-hydroxy-3-phenoxypropyl methacrylate is completely converted, 2-oxypropyl methacrylate is added to continue the reaction. After the conversion is complete, monomethoxy polyethylene glycol methacrylate is added to continue the reaction, and the reaction is purified to obtain a triblock polymer.

7. The preparation method according to claim 6, characterized in that: The molar ratio of phenol, tetraethylammonium bromide and glycidyl methacrylate in step (1) is 1:0.09-0.13:1.25-1.50; And / or, the heating reaction temperature in step (1) is 60-80° C. and the time is 36-48 h; And / or, the molar ratio of chloroacetone, triethylamine and methacrylic acid in step (2) is 1:1.15-1.55:1.105-1.135; And / or, the heating reaction temperature in step (2) is 40-45° C. and the time is 2-6 hours; And / or, in step (3), the molar ratio of 2-hydroxy-3-phenoxypropyl methacrylate, 2-oxopropyl methacrylate, monomethoxy polyethylene glycol methacrylate, ligand, catalyst, reducing agent and small molecule initiator is: 15-35: 15-35: 5-20: 0.38-0.95: 0.018-0.062: 0.181-0.89: 0.36-1.15; And / or, the temperature of the heating reaction in step (3) is 50-70°C; the heating reaction time of the 2-hydroxy-3-phenoxypropyl methacrylate is 6-12h; the heating reaction time of the 2-oxopropyl methacrylate is 24-72h; the heating reaction time of the monomethoxypolyethylene glycol methacrylate is 96-168h.

8. The preparation method according to claim 6, characterized in that: The ligand in step (3) is 1,1,4,7,10,10-hexamethyltriethylenetetramine; And / or, the catalyst in step (3) is copper bromide; And / or, the reducing agent in step (3) is stannous octoate; And / or, the small molecule initiator in step (3) is ethyl bromoisobutyrate; And / or, the dripping rate of the methacrylic acid in step (2) is 1 to 5 mL / min; And / or, the inert atmosphere in steps (1) and (3) is at least one of nitrogen, argon and helium.

9. A triblock polymer cross-linked micelle with autofluorescence property obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the triblock polymer cross-linked micelle with autofluorescence property as claimed in claim 9 in the preparation of tumor imaging and drug delivery products.

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