Preparation method of reduction response activated < 19 > F magnetic resonance signal amplification self-degradation nano diagnosis and treatment micelle

Abstract

The invention belongs to the technical fields of medical chemistry, new materials and nanotechnology, and relates to a novel diagnosis and treatment agent in the field of interdisciplinary, in particular to a preparation method of a reduction response activation < 19 > F magnetic resonance signal amplification self-degradation nanometer diagnosis and treatment micelle. Poly (benzyl carbamate) is obtained through condensation polymerization as a basic structure, then chemical modification is performed to obtain a self-degradable fluorine-containing amphiphilic block polymer, hydrophobic drug chlorin (Ce6) is loaded through self-assembly, the self-degradable fluorine-containing amphiphilic block polymer has the characteristic of responding to degradation of glutathione (GSH) at a tumor site, and on the basis of maintaining self-degradation of a hydrophobic chain segment of the nano-micelle, the self-degradable fluorine-containing amphiphilic block polymer can be used for preparing the self-degradable fluorine-containing amphiphilic block polymer. The nano-micelle loaded with Ce6 has the advantages that the nano-micelle loaded with Ce6 is used as a carrier, the characteristic of efficient 19F MR imaging is additionally endowed, under laser irradiation, the nano-micelle loaded with Ce6 can enhance a new scheme of breast cancer photodynamic therapy, the effects of responding to tumor microenvironment self-degradation drug release and 19F MRI signal amplification are achieved, and therefore diagnosis and treatment integration of breast cancer is achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug synthesis, and specifically relates to a reduction response activation 19 Preparation method of self-degradable nano-therapeutic micelles with F magnetic resonance signal amplification. Background Art

[0002] Cancer (malignant tumor) is one of the major diseases that threaten human life and health. Early diagnosis and timely treatment of tumors are effective means to reduce their mortality rate. With the continuous development of modern biomedical imaging technology, clinical practice has put forward higher requirements for imaging technology: not only to obtain more accurate and rich diagnostic information, but also to achieve real-time monitoring of lesions during treatment, accurately distinguish between normal tissue and diseased areas, thereby improving the targeting and effectiveness of treatment, and promoting the process of personalized treatment. In recent years, the development of nano-diagnosis and treatment technology has provided a new platform for precise diagnosis and efficient treatment of major diseases such as cancer. By constructing nano-diagnosis and treatment probes that integrate diagnosis, treatment and efficacy monitoring, new strategies and technical paths can be provided for cancer treatment, showing good clinical application prospects. However, how to construct an integrated nano-probe with high imaging sensitivity, good biocompatibility and therapeutic synergy is still a key scientific issue that needs to be broken through in the current field of nanomedicine.

[0003] Traditional magnetic resonance imaging (MRI) is mainly based on hydrogen proton (1H) imaging, but it has limitations in actual diagnosis. Due to the small difference in T1 / T2 relaxation time between normal tissue and lesion tissue, and the widespread distribution of hydrogen atoms in the human body, it often leads to strong background signals and low contrast in the image, thus affecting the accuracy of diagnosis. At the same time, 1 The nonlinear relationship between contrast agents and imaging signals in H MRI makes quantitative analysis difficult. Therefore, the development of new MR imaging technologies with high contrast, low background, and quantitative performance has become a research hotspot. 19 As an emerging non-hydrogen nuclear magnetic resonance technology, F MRI has many advantages. 19 F has 100% natural abundance and is the only stable fluorine isotope with a spin quantum number of 1 / 2 and a gyromagnetic ratio close to 1 H( 19 F is 40.08MHz / T, 1 H is 42.58MHz / T), and its NMR sensitivity is about 83.4% of 1H, which has a high efficiency in acquiring nuclear magnetic signals. In addition, 19 F atoms have abundant outer electrons and a large chemical shift range (>350ppm), which can realize multispectral imaging. More importantly, the content of fluorine in human tissue is extremely low, far below the detection limit of MRI imaging technology, so 19F MRI has extremely low background signals, which helps to obtain high signal-to-noise ratio and high specificity of "hot spot" imaging. 19 The F MRI imaging signal is linearly related to the contrast agent concentration, which enables precise positioning and quantitative analysis, and is of great significance for early diagnosis of diseases. 19 F nanoprobes must simultaneously meet the following characteristics: high fluorine atom density, good imaging activity of fluorine atoms (long T2 relaxation time) and excellent biocompatibility. However, due to the hydrophobicity and oleophobicity of fluorine atoms, it is difficult to simultaneously take into account high fluorine content and good dispersibility in the design. Therefore, the development of fluorine-containing structures with strong water solubility and good stability has become the key to constructing high-performance probes. 19 The research results of F nanoprobes can be divided into the following categories according to the fluorine source molecules: Perfluorocarbon emulsions 19 F probe, fluorine-containing inorganic compounds 19 F probe, fluorinated organic compounds 19 F probe. Perfluorocarbons (PFCs) are hydrocarbon molecules in which fluorine completely replaces hydrogen atoms. They are the first 19 Fluorine source for F imaging. Typical representatives include perfluoropentane (PFP), perfluorohexane (PFH), perfluoro-15-crown-5-ether (PFCE), perfluorooctyl bromide (PFOB), etc. The commercially available CellSense and V-Sense emulsions have been used in cell tracking imaging studies. Due to the weak activity of fluorine atoms in the solid state, the fluorine T2 relaxation time of inorganic fluorinated materials is short and the imaging signal is low, and there are few reports on nanoprobes using fluorine-containing inorganic compounds as fluorine source materials. Fluorine-containing organic compounds have trifluoro (CF3) methyl compounds as the main functional group and can be divided into small molecules and high molecular polymer types. In contrast, fluorine-containing polymers have gradually become the current mainstream due to their advantages such as high fluorine density, adjustable structure, diverse functions, and good biocompatibility. 19 The mainstream direction of F probe design is to introduce hydrophilic groups to further improve its water solubility and targeting function, which is the core strategy for optimizing this type of material.

[0004] In addition to fluoropolymers, the design of derivatives with phosphorylcholine (CP)-like structures is also of great significance. These structures are inspired by the biomimetic design of cell membrane structures and possess excellent biocompatibility and hydrophilicity. The fluorinated phosphorylcholine (FCP) monomers synthesized by Professor Todd Emrick's team at the University of Massachusetts, USA, address the strong aggregation and poor water solubility issues of traditional fluorinated contrast agents, providing new ideas and molecular structure templates for the development of high-performance MRI probes.

[0005] In recent years, self-immolative polymers (SIPs), as an emerging class of macromolecular materials, have attracted widespread attention due to their unique self-depolymerization properties. Since this type of material was first reported in China, SIPs have shown potential for application in signal amplification, biosensing, drug delivery, etc. due to their ability to rapidly depolymerize into small molecular fragments through quinone methination cascade reactions (including 1,4-elimination, 1,6-elimination) or intramolecular cyclization mechanisms under triggering conditions. At present, the SIP systems that have been studied cover a variety of structural types, such as (thio)carbamates, (thio)carbonates, o-phthalaldehyde derivatives, benzyl ethers / esters, and glyoxylates. However, not all structural types are suitable for the biomedical field, and their bioapplicability needs to comprehensively consider the following three aspects: (1) the water solubility of polymer degradation products; (2) the rate of hydrolysis process and induced trigger reaction; and (3) the biotoxicity of the polymer itself and its degradation products. Among them, polybenzylcarbamate SIPs have been widely used in drug delivery research due to their good biocompatibility and controllable depolymerization properties. Moore et al. prepared microcapsules based on this polymer structure through interfacial polymerization, which can achieve controlled release of contents under external stimulation conditions for drug delivery. Liu et al. constructed block copolymers of polybenzyl carbamate and hydrophilic polymers, assembling them into micelles. The end-capping groups can be activated by stimuli such as visible light and ultraviolet light, triggering depolymerization and thus achieving targeted drug release.

[0006] These studies have laid the foundation for the in-depth application of self-cleaving polymeric micelles in targeted drug delivery. Building on this foundation, researchers have recently combined the response mechanisms of self-cleaving polymers with tumor-specific biomarkers to further expand their applications in cancer diagnosis and treatment. Glutathione (GSH), a widely recognized tumor marker, is present in tumor cells and tumor tissues at concentrations approximately three times and 100 times higher than normal, respectively. Therefore, the development of GSH-responsive smart drug delivery systems has become a hot topic in cancer research in recent years. In this context, self-cleaving polymers (DSIPs) with disulfide linkers have attracted widespread attention due to their excellent reduction sensitivity. These polymers are stable under normal physiological conditions. However, upon entering the tumor microenvironment, where GSH concentrations are high, their disulfide bonds are cleaved by reduction, triggering rapid depolymerization of the polymer backbone, exhibiting a "domino-like" disintegration behavior. This property makes them valuable for the construction of tumor microenvironment-responsive imaging probes, prodrug systems, bioconjugates, and drug delivery nanocarriers. For example, Liu Shiyong's research group designed for the first time a self-cleaving block copolymer containing a disulfide bond linker, and constructed an intelligent drug delivery system that can self-assemble to form a micelle structure, achieving targeted and controlled release of anti-tumor drugs. Although relevant research has made certain progress, the existing fluorine-containing MRI contrast agents still have the following major problems in practical applications: (1) Fluorine source materials generally have poor hydrophilicity and low dispersibility, which affects their activity and distribution in the body; (2) Insufficient fluorine content leads to limited MRI signal intensity and low imaging sensitivity; (3) Fluorine probes lack tumor specificity, which limits their further expansion in precise diagnosis and treatment. At present, there is no public literature or patent report on the integration of self-cleaving polymers with disulfide bond structures (DSIP) and fluorine-containing MRI imaging technology to construct a system that can achieve targeted responsive release and 19 A composite nano-diagnosis and treatment platform with synchronous enhancement of F imaging. Summary of the Invention

[0007] In order to overcome the deficiencies of the prior art, the present invention provides a reduction response activation 19Preparation method of self-degradable nano-diagnostic micelles with F magnetic resonance signal amplification. The present invention first synthesizes a block polymer containing a self-cleaving structural unit and a high-fluorine content side chain. The polymer introduces a choline phosphate (CP) structural unit, which significantly improves the hydrophilicity and dispersibility of the fluorine group and enhances the stability of the polymer in the aqueous phase. This structure also gives the polymer good biocompatibility, and the proportion of fluorine-containing monomers is adjusted by a controllable polymerization method to achieve precise control of the fluorine content. The fluorine-containing block polymer self-assembles into nano-micelles in the aqueous phase through hydrophobic forces and π-π stacking, and loads the photosensitive drug dihydrochlorin (Ce6) in the hydrophobic core of the micelles. The nano-micelles give the system good reduction response characteristics by introducing a disulfide bond connection structure in the polymer main chain. When the nano-micelles enter the tumor microenvironment with a high glutathione (GSH) concentration, the disulfide bond breaks, triggering the self-cleavage depolymerization reaction of the polymer skeleton, releasing Ce6, and achieving precise release of the drug at the tumor site. At the same time, the small molecule fluorinated fragments released after depolymerization can significantly improve the sensitivity and resolution of 19F MRI signals due to their higher molecular activity and longer T2 relaxation time, achieve the "amplification activation" effect of the imaging signal, and realize the precise imaging and positioning of tumors.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is:

[0009] The first aspect of the present invention provides a reduction response activation 19 A method for preparing F magnetic resonance signal amplifying self-degrading nano-diagnostic and therapeutic micelles, the method comprising the following steps:

[0010] (1) Synthesis of small molecule compounds: 4-aminobenzyl alcohol reacts with phenyl chloroformate to prepare compound 1a; 2,2'-dithiodipyridine reacts with β-mercaptoethanol to prepare compound 2a; 3,3,3-trifluoro-1-propanol reacts with 2-chloro-2-oxo-1,3,2-dioxaphosphatol in the presence of anhydrous triethylamine to prepare compound 3a; hexafluoroisopropanol reacts with 2-chloro-2-oxo-1,3,2-dioxaphosphatol in the presence of anhydrous triethylamine to prepare compound 4a;

[0011] The synthetic routes of compound 1a, compound 2a, compound 3a and compound 4a are as follows:

[0012]

[0013] (2) Synthesis and modification of polymers: Compound 1a is subjected to condensation polymerization in the presence of a catalyst, dibutyltin dilaurate, to obtain PB1, which is then reacted with a condensation polymerization terminator, compound 2a, to prepare SIP, namely PB2; PB2 is reacted with glacial acetic acid in the presence of dicyclohexylcarbodiimide and 4-dimethylaminopyridine to prepare SIA, namely PB3; PB3 is reacted with β-mercaptoethanol in the presence of glacial acetic acid to prepare SIM, namely PB4; PB4 is reacted with 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid in the presence of dicyclohexylcarbodiimide and 4- SIR (PB5) was prepared by reaction in the presence of dimethylaminopyridine; PB5 was subjected to reversible addition-fragmentation chain transfer polymerization with polyethylene glycol methyl ether methacrylate and 2-dimethylaminoethyl methacrylate in the presence of azobisisobutyronitrile to prepare SIP-b-DMA-b-PEG (PB6); PB6 was reacted with compound 3a to prepare fluorinated amphiphilic block polymer SIP-b-CPF3-b-PEG (PB7); PB6 was reacted with compound 4a to prepare fluorinated amphiphilic block polymer SIP-b-CPF6-b-PEG (PB8);

[0014] The synthetic routes for the synthesis and modification of the polymer are as follows:

[0015]

[0016] (3) Preparation of nano-diagnostic and therapeutic micelles: The fluorinated amphiphilic block polymer SIP-b-CPF3-b-PEG and dihydrochlorin, i.e., Ce6, were dissolved in DMSO, and then added dropwise to PBS buffer, stirred at room temperature, dialyzed, collected and freeze-dried to obtain nano-diagnostic and therapeutic micelles Ce6@SPTF; the fluorinated amphiphilic block polymer SIP-b-CPF6-b-PEG and dihydrochlorin, i.e., Ce6, were dissolved in DMSO, and then added dropwise to PBS buffer, stirred at room temperature, dialyzed, collected and freeze-dried to obtain nano-diagnostic and therapeutic micelles Ce6@SPHF.

[0017] Preferably, in step (1), the preparation of compound 1a and compound 2a are both carried out under an inert gas environment; in the preparation of compound 1a, the molar ratio of 4-aminobenzyl alcohol to phenyl chloroformate is 1:2; in the preparation of compound 2a, the molar ratio of 2,2'-disulfide dipyridine to β-mercaptoethanol is 3:1.

[0018] Preferably, in step (1), the preparation of compound 3a and compound 4a is carried out under an inert gas environment, and the molar ratio of 3,3,3-trifluoro-1-propanol or hexafluoroisopropanol, 2-chloro-2-oxo-1,3,2-dioxaphospholane, and anhydrous triethylamine is 1.05:1:1.05.

[0019] Preferably, in step (2), the preparation of PB1 and PB2 is carried out under an inert gas environment; the condensation polymerization time in the preparation of PB1 is 24 minutes; and in the preparation of PB2, the molar ratio of compound 1a, dibutyltin dilaurate, and compound 2a is 20:1:15.

[0020] Preferably, in step (2), in the preparation of PB3, the molar ratio of PB2, glacial acetic acid, dicyclohexylcarbodiimide, and 4-dimethylaminopyridine is 1:10:15:1; in the preparation of PB4, the molar ratio of PB3, β-mercaptoethanol, and glacial acetic acid is 1:0.4:0.4; in the preparation of PB5, the molar ratio of PB4, 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid, dicyclohexylcarbodiimide, and 4-dimethylaminopyridine is 1:5:7:0.5.

[0021] Preferably, in step (2), the preparation of PB6 is carried out under an inert gas environment, and the molar ratio of PB5, polyethylene glycol methyl ether methacrylate, 2-dimethylaminoethyl methacrylate, and azobisisobutyronitrile is 1:10:50:0.2.

[0022] Preferably, in step (2), the preparation of PB7 and PB8 is carried out under an inert gas environment; the molar ratio of compound 3a or compound 4a to PB6 is 1.5:1.

[0023] Preferably, in step (3), the mass ratio of the fluorinated amphiphilic block polymer SIP-b-CPF3-b-PEG or SIP-b-CPF6-b-PEG to Ce6 is 2.5-20:1.

[0024] Preferably, in step (3), the molecular weight cut-off of the dialysis is 7000 Da, and the dialysis time is 12 h.

[0025] The second aspect of the present invention provides a reduction response activation prepared by the above preparation method 19 F magnetic resonance signal amplification of self-degradable nanotheranostic micelles.

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

[0027] The present invention provides a reduction response activation 19Preparation method of self-degradable nano-diagnostic and therapeutic micelles with F magnetic resonance signal amplification. First, a self-shearable fluorinated block polymer is prepared. The polymer uses a phosphorylcholine structure to greatly improve the hydrophilicity of the fluorinated group. The design of the polymer makes the fluorine content flexible and controllable, while improving the imaging effect and ensuring strong fluorine atom activity (long T2 relaxation time) and biocompatibility; while self-assembling, the drug dihydrochlorin (Ce6) is loaded through π-π stacking and hydrophobic forces to construct a self-degradable polymer nano-diagnostic and therapeutic micelle that responds to the tumor microenvironment (GSH). On the one hand, Ce6 can achieve photodynamic therapy; on the other hand, the broken fluorinated fragments have enhanced activity and achieve a higher relaxation rate. 19 F MR imaging effects, thereby achieving accurate diagnosis and treatment of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is the compound 1a in Example 1 1 H NMR and 13 C NMR spectrum, where A is 1 H NMR spectrum, B is 13 CNMR spectrum.

[0029] Figure 2 is the compound 2a in Example 1 1 H NMR and 13 C NMR spectrum, where A is 1 H NMR spectrum, B is 13 CNMR spectrum.

[0030] Figure 3 is the compound 3a in Example 1 1 H NMR, 13 C NMR, 19 F NMR, 31 P NMR spectrum, where A is 1 HNMR spectrum, B is 13 C NMR spectrum, C is 19 F NMR spectrum, D is 31 P NMR spectrum.

[0031] Figure 4 is the compound 4a in Example 1 1 H NMR, 13 C NMR, 19 F NMR, 31 P NMR spectrum, where A is 1 HNMR spectrum, B is 13 C NMR spectrum, C is 19 F NMR spectrum, D is 31P NMR spectrum.

[0032] Figure 5 is PB6, PB7, PB8 in Example 2 1 H NMR spectrum.

[0033] Figure 6 These are the GPC spectra of PB2, PB6, and PB6+10 mM GSH and the FTIR spectra of PB2-PB8 in Example 2, where A is the GPC spectra of PB2, PB6, and PB6+10 mM GSH, and B is the FTIR spectra of PB2-PB8.

[0034] Figure 7 These are the particle size distribution diagrams of SPTF, Ce6@SPTF, SPHF, and Ce6@SPHF in Examples 3 and 4, and the transmission electron micrographs of the four nanomicelles and SPTF and SPHF after applying GSH, wherein A is the particle size distribution diagram of SPTF and Ce6@SPTF, B is the particle size distribution diagram of SPHF and Ce6@SPHF, and C is the transmission electron micrograph of the four nanomicelles of SPTF, Ce6@SPTF, SPHF, and Ce6@SPHF, and SPTF and SPHF after applying GSH (scale = 100 nm).

[0035] Figure 8 Comparison chart of T1 and T2 relaxation times and T2 / T1 values ​​of the four nanomicelles SPTF, Ce6@SPTF, SPHF, and Ce6@SPHF in Examples 3 and 4, and the SPTF, Ce6@SPTF, SPHF, and Ce6@SPHF nanomicelles after application of GSH, where A is a comparison chart of T1 and T2 relaxation times, and B is a comparison chart of T2 / T1 values.

[0036] Figure 9 These are the changes in the absorbance value of DPBF at 410 nm after incubation of PBS group, Ce6@SPTF, and Ce6@SPHF for different times, and a comparison graph of the absorbance values ​​of DPBF at different time points in Experimental Example 4, wherein A is a comparison graph of the change in the absorbance value of DPBF at 410 nm after incubation of PBS group for different times, B is a comparison graph of the change in the absorbance value of DPBF at 410 nm after incubation of Ce6@SPTF for different times, C is a comparison graph of the change in the absorbance value of DPBF at 410 nm after incubation of Ce6@SPHF for different times, and D is a comparison graph of the absorbance values ​​of DPBF at different time points after incubation of PBS group, Ce6@SPTF, and Ce6@SPHF.

[0037] Figure 10Comparison of the toxicity of SPTF, Ce6@SPTF, SPHF, and Ce6@SPHF at different concentrations on HUVEC and 4T1 cells after 24 h of co-incubation in Experimental Example 5 (n=3), wherein A is a comparison of the toxicity of SPTF and Ce6@SPTF on 4T1 cells, B is a comparison of the toxicity of SPTF and Ce6@SPTF on HUVEC cells, C is a comparison of the toxicity of SPHF and Ce6@SPHF on 4T1 cells, and D is a comparison of the toxicity of SPHF and Ce6@SPHF on HUVEC cells.

[0038] Figure 11 The figure is a comparison of the survival rates of 4T1 cells in different treatment groups in Experimental Example 6 (n=3), wherein ***p<0.001, ****p<0.0001. DETAILED DESCRIPTION

[0039] The following is a further description of specific embodiments of the present invention. It should be noted that the description of these embodiments is intended to facilitate understanding of the present invention and does not constitute a limitation of the present invention. In addition, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

[0040] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0041] Example 1: Preparation of small molecule compounds

[0042] (a) Synthesis of Compound 1a: Under argon (Ar) protection, 1 g of 4-aminobenzyl alcohol was placed in a 50 mL dry round-bottom flask. 30 mL of a mixed solvent of tetrahydrofuran (THF), saturated sodium bicarbonate (NaHCO₃), and water (volume ratio 2:2:1) was added. 2.08 mL of phenyl chloroformate was slowly added dropwise with stirring. The mixture was stirred at room temperature for 12 h, during which the reaction was monitored by thin-layer chromatography. After completion of the reaction, ethyl acetate was added, and the mixture was washed twice with saturated NaHCO₃ solution, dried over anhydrous sodium sulfate (Na₂SO₄), and concentrated under reduced pressure. The product was purified by crystallization from chloroform and dried under vacuum at room temperature to obtain 3.66 g of white crystals in a 93% yield.

[0043] (b) Synthesis of Compound 2a: 10.5494 g of 2,2'-disulfide dipyridine was weighed into a 100 mL round-bottom flask, 50 mL of anhydrous methanol was added and stirred to dissolve, 1.2448 g of β-mercaptoethanol was slowly added dropwise, and the mixture was stirred at room temperature for 12 h under Ar protection; the mixture was concentrated under reduced pressure, and the crude product was further purified by column chromatography using the following eluent: V 乙酸乙酯 / V 石油醚=1:5, the collected crude product was concentrated and dried in vacuo to obtain 2.7731 g of a light yellow oily product with a yield of 57%.

[0044] (c) Synthesis of Compound 3a: Under Argon protection, a dry 100-mL two-necked flask was charged with 40 mL of anhydrous dichloromethane (DCM) and 2.5214 g of 3,3,3-trifluoro-1-propanol. 3.5 mL of anhydrous triethylamine (TEA) was added in an ice bath. 3.42 g of 2-chloro-2-oxo-1,3,2-dioxaphospholane was dissolved in 15 mL of anhydrous DCM and slowly added dropwise (approximately 5 min). Stirring was continued in an ice bath for 45 min, followed by 12 h of reaction at room temperature. The organic phase was filtered, washed three times with saturated sodium chloride solution, dried over anhydrous NaSO, and concentrated under reduced pressure to afford 2.754 g of the product as a pale yellow oil in a 51.9% yield.

[0045] (d) Synthesis of Compound 4a: Under Argon protection, a dry 100-mL two-necked flask was charged with 40 mL of anhydrous dichloromethane (DCM) and 4.2336 g of hexafluoroisopropanol. 3.5 mL of anhydrous triethylamine (TEA) was added in an ice bath. 3.42 g of 2-chloro-2-oxo-1,3,2-dioxaphospholane was dissolved in 15 mL of anhydrous DCM and slowly added dropwise (approximately 5 min). Stirring was continued in an ice bath for 45 min, followed by reaction at room temperature for 12 h. The mixture was filtered, and the organic phase was washed three times with saturated sodium chloride solution, dried over anhydrous NaSO, and concentrated under reduced pressure to afford 3.5498 g of the product as a pale yellow oil in a 54.0% yield.

[0046] Example 2: Synthesis and modification of polymers

[0047] (a) Synthesis of PB1 and SIP (PB2): 1 g of compound 1a and 0.1298 g of dibutyltin dilaurate (DBTL) were dissolved in 2 mL of anhydrous N,N-dimethylformamide (DMF) and quickly added to a 25 mL anhydrous, oxygen-free reaction tube. The apparatus was evacuated and the reaction tube was placed in an oil bath at 110°C for 24 min under Ar protection. 0.75 g of compound 2a (condensation polymerization terminator) was dissolved in an appropriate amount of anhydrous DMF and quickly added to the polymerization system below the liquid level using a long needle through the rubber stopper on the reaction tube. The reaction was stirred for 2 h to ensure complete termination. After cooling in an ice bath and purging with oxygen, the reaction solution was added dropwise to 20 mL of cold methanol. The reaction was centrifuged at 4°C (3000 rpm for 5 min). The precipitate was collected and separated three times before vacuum drying to obtain SIP (PB2) as a yellow powder.

[0048] (b) Synthesis of SIA (PB3): In a clean, dry 50-mL round-bottom flask, add 2 mL of anhydrous DMF and 0.8001 g of PB2, which were dissolved by sonication. Then, 0.927 g of dicyclohexylcarbodiimide (DCC), 0.18 g of glacial acetic acid, and 0.0366 g of 4-dimethylaminopyridine (DMAP) were added sequentially, and the mixture was stirred at room temperature for 24 h. After the reaction, the white precipitate was removed by filtration, and the resulting solution was added dropwise to a large amount of cold methanol. The mixture was centrifuged, washed three times, and dried in vacuo to yield 0.9056 g of the product as a pale yellow powder.

[0049] (c) Synthesis of SIM(PB4): In a clean, dry 50-mL round-bottom flask, 12 mL of anhydrous DMF, 0.7987 g of PB3, and 0.0064 g of glacial acetic acid were added and sonicated. 0.0172 mL of β-mercaptoethanol was dissolved in 1 mL of anhydrous methanol and added dropwise to the reaction system (to prevent side reactions). Stirring was performed at room temperature for 6 h. After precipitation, the reaction solution was precipitated in cold methanol, washed by centrifugation three times, and dried under vacuum to obtain 0.732 g of the product as a light yellow powder with a yield of approximately 91.64%.

[0050] (d) Synthesis of SIR (PB5): 0.732 g of PB4, 0.653 g of 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid, 0.389 g of DCC, and 0.0165 g of DMAP were dissolved in 5 mL of anhydrous DMF and added to a 25 mL reaction tube. The mixture was stirred at room temperature for 24 h. After the reaction, the precipitate was removed by filtration. The reaction solution was dropwise added to cold methanol and the precipitation was repeated three times. The product was then dried under vacuum to obtain 0.7382 g of a light pink powder with a yield of approximately 86.97%.

[0051] (e) Synthesis of SIP-b-DMA-b-PEG (PB6): This reaction was a typical reversible addition-fragmentation chain transfer (RAFT) polymerization. Under Ar protection, 0.7045 g of PB5, 1.0898 g of polyethylene glycol methyl ether methacrylate, 1.7948 g of 2-dimethylaminoethyl methacrylate, and 0.00753 g of azobisisobutyronitrile (AIBN) were dissolved in 3 mL of anhydrous DMSO and rapidly added to a 25 mL reaction tube. The tube was aspirated with a long needle under Ar for 20 minutes to remove oxygen from the system. The reaction was then incubated at 66°C under Ar protection for 48 hours. After completion of the reaction, the polymerization was terminated by ice bath and oxygen flow. The reaction solution was then added dropwise to 30 mL of cold diethyl ether, yielding a pale yellow viscous precipitate. After repeated precipitation and purification, the product was dried in vacuo to yield 1.8211 g of a yellow solid.

[0052] (f) Synthesis of SIP-b-CPF3-b-PEG(PB7): 0.175 g of compound 3a and 0.2094 g of PB6 were dissolved in 1 mL of anhydrous DMSO and added to a 25 mL reaction tube. The mixture was reacted in an oil bath at 70°C under Ar protection for 110 h. The reaction solution was transferred to a 1000 Da regenerated cellulose membrane dialysis bag and dialyzed against anhydrous ethanol for two days. The crude product was collected, the solvent removed by rotary evaporation, and vacuum dried to yield 0.2169 g of a yellow solid.

[0053] (g) Synthesis of SIP-b-CPF6-b-PEG(PB8): 0.219 g of compound 4a and 0.2094 g of PB6 were dissolved in 1 mL of anhydrous DMSO and added to a 25 mL reaction tube. The mixture was reacted in an oil bath at 70°C under Ar protection for 110 h. The reaction solution was transferred to a 1000 Da regenerated cellulose membrane dialysis bag and dialyzed against anhydrous ethanol for two days. The crude product was collected, the solvent removed by rotary evaporation, and vacuum dried to yield 0.2203 g of a yellow solid.

[0054] Example 3: Preparation of Nanomicelles

[0055] Weigh 50 mg of fluorinated amphiphilic block polymer (SIP-b-CPF3-b-PEG or SIP-b-CPF6-b-PEG), dissolve it with 0.2 mL of DMSO, slowly add it dropwise into PBS buffer (1 mL) with high-speed stirring (500 rpm), stir at room temperature for 30 min, put it into a dialysis bag with MWCO = 7000 Da, dialyze for 12 h, collect and freeze-dry to obtain SIP-b-CPF3-b-PEG nanomicelles (SPTF) and SIP-b-CPF6-b-PEG nanomicelles (SPHF).

[0056] Example 4: Preparation of drug-loaded nanomicelles

[0057] Weigh 50 mg of fluorinated block polymer (SIP-b-CPF3-b-PEG or SIP-b-CPF6-b-PEG) and 5 mg of Ce6, dissolve them in 0.2 mL of DMSO, slowly add them dropwise into PBS buffer (1 mL) with high-speed stirring (500 rpm), stir at room temperature for 30 min, put them into dialysis bags with MWCO = 7000 Da, dialyze for 12 h, collect and freeze-dry to obtain drug-loaded nanomicelles (Ce6@SPTF) and drug-loaded nanomicelles (Ce6@SPHF).

[0058] Experimental Example 1: Characterization of small molecule compounds and polymers

[0059] (1) Characterization of small molecule compounds: 5 mg of compound 1a, compound 2a, compound 3a, and compound 4a were dissolved in 500 μL of deuterated reagent and characterized by nuclear magnetic resonance spectroscopy ( 1H NMR) (scan number set to 16), carbon nuclear magnetic resonance spectrum ( 13 C NMR) (scan number is set to 1024) to characterize the structure of each small molecule compound. Dissolve in methanol and use mass spectrometry (ESI-MS) to characterize the molecular weight of the compound. For compound 3a and compound 4a, 19 F NMR and 31 P NMR characterization. 1 HNMR and 13 C NMR spectrum Figure 1 As shown, where A is 1 H NMR spectrum, B is 13 C NMR spectrum of compound 2a 1 H NMR and 13 C NMR spectrum Figure 2 As shown, where A is 1 H NMR spectrum, B is 13 C NMR spectrum of compound 3a 1 H NMR, 13 C NMR, 19 F NMR, 31 P NMR spectrum is as follows Figure 3 As shown, where A is 1 H NMR spectrum, B is 13 C NMR spectrum, C is 19 F NMR spectrum, D is 31 P NMR spectrum of compound 4a 1 H NMR, 13 C NMR, 19 F NMR, 31 P NMR spectrum is as follows Figure 4 As shown, where A is 1 HNMR spectrum, B is 13 C NMR spectrum, C is 19 F NMR spectrum, D is 31 P NMR spectrum. 1 The peaks of H NMR were assigned, indicating that compounds 1a-4a were successfully synthesized.

[0060] (2) Characterization of polymers: 16 mg of pure polymers (PB6, PB7, PB8) were accurately weighed and dissolved in 500 μL DMSO-d6. The polymers were tested using a 400M nuclear magnetic resonance spectrometer with the number of cycles (NS) set to 32. 1 H NMR spectrum Figure 5As shown; 4 mg of pure polymer (PB2, PB6, PB6 + 10 mM GSH) was weighed and dissolved in 1 mL of HPLC grade DMF solution, filtered with a 0.22 μm nylon filter membrane, and used for GPC analysis. The mobile phase flow rate was 1.0 mL / min, and a DMF phase PLgel gel chromatography column (10 μm MIXED-B, 20 μm MIXED-A, 20 μm MIXED-A) was used. The column temperature was 50°C, and polystyrene was used as the internal standard. The GPC spectra of PB2, PB6, and PB6 + 10 mM GSH were shown as follows. Figure 6 As shown in A, in addition, the FTIR spectra of PB2-PB8 are as follows Figure 6 As shown in B, combined Figure 5 H NMR spectrum of the polymer and Figure 6 From the GPC curve in A, we can judge that the fluorinated amphiphilic block polymers PB7 and PB8 were successfully synthesized. Moreover, compared with PB2, the molecular weight of PB7 and PB8 was significantly improved, proving that the RAFT polymerization was successful. Figure 6 As shown in A, after PB6 responds to GSH, the GPC curve drops significantly and a new peak appears, proving that it can effectively respond to GSH and undergo degradation. Figure 6 FTIR spectrum in B, 700-900 cm -1 The strong absorption peak at 819.01 cm -1 The peak at 1099.45 cm-1 proves the existence of benzene ring structure on polymers PB2-PB8. The unique vibration peaks of PB6, PB7, and PB8 (1099.45 cm-1 -1 ) proved the existence of tertiary amine structure and the RAFT polymerization reaction was successful. 1722.93cm -1 and 3350.36cm -1 The obvious carbon-oxygen double bond peaks appear, corresponding to the large number of carbon-oxygen double bonds in the polymer. The above auxiliary evidence proves that the structure of the synthesized polymer is correct.

[0061] Experimental Example 2: Characterization of basic properties of nanomicelles

[0062] (1) Determination of the particle size of nanomicelles: The particle size and potential of the nanomicelles were detected by dynamic light scattering (DLS) using a Zetasizer nano-laser particle size analyzer (Malvem Instruments). The obtained particle size distribution diagrams of SPTF, Ce6@SPTF, SPHF, and Ce6@SPHF are shown in Figure 2. Figure 7 As shown in the figure, A is the particle size distribution diagram of SPTF and Ce6@SPTF, and B is the particle size distribution diagram of SPHF and Ce6@SPHF. The results show that the particle size of nanomicelles with different fluorine contents is around 70-100nm, which is conducive to achieving long circulation in the body.

[0063] (2) Transmission electron microscopy (TEM) observation of the morphology of nanomicelles: The surface morphology of the nanomicelles was observed by F200 transmission electron microscopy at an accelerating voltage of 200 kV. 10 μL of pure water sample solution was dropped onto a copper grid and naturally air-dried at room temperature. The morphology, particle size and dispersion of the nanoparticles were observed by transmission electron microscopy at a voltage of 200 kV. The transmission electron microscopy images of the four nanomicelles obtained and SPTF and SPHF after applying GSH are shown in Figure 2. Figure 7 As shown in Figure C, the results show that the prepared nanomicelles are all spherical with a particle size of about 70-100 nm. After GSH response, the morphology of the nanomicelles decomposes to a certain extent, indicating its potential for responsive drug release at tumor sites.

[0064] Experimental Example 3: Determination of T1 and T2 relaxation times of nanomicelles

[0065] A 10 mg / mL solution of SPTF, SPHF, Ce6@SPTF, or Ce6@SPHF (SPTF or SPHF: 10 mg / mL) was prepared in a 9:1 PBS / D2O solvent. The spin-lattice relaxation time (T1) and spin-spin relaxation time (T2) of each sample were measured using a Bruker Avance Neo 400. The same measurements were performed after adding 10 mM GSH and incubating overnight at 37°C.

[0066] T1 is measured using a standard inversion-recovery pulse sequence (RARE). The relaxation delay time for each measurement is 1s, the number of scans is 16, and 14 points are scanned. The scanning range refers to the corresponding SPTF and Ce6@SPTF. 19 Chemical shifts of peaks in F NMR.

[0067] T2 was measured using a Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence. The relaxation delay was 2 seconds, and eight scans were performed. For each measurement, the echo time was set from 0 to 50,000 seconds, and 12 points were collected. The decay of the spin echo amplitude was described by a single exponential function, and the number of fitted cycles was used to calculate the T2 relaxation time using the following formula.

[0068] T2 = fitting cycle number × (2D 20 +P2) where D20 is the delay time and P2 is the pulse width.

[0069] Comparison of T1 and T2 relaxation times and T2 / T1 values ​​of the four nanomicelles of SPTF, Ce6@SPTF, SPHF, and Ce6@SPHF obtained and the nanomicelles of SPTF, Ce6@SPTF, SPHF, and Ce6@SPHF after GSH addition. Figure 8As shown, A is a comparison diagram of T1 and T2 relaxation times, and B is a comparison diagram of T2 / T1 values. The results show that the T2 relaxation time of SPTF before the application of GSH is 563.6ms. After the application of GSH, the relaxation time increases to 928.9ms, and the T2 / T1 value increases from 0.292 to 0.481. Similarly, the T2 / T1 values ​​of Ce6@SPTF before and after responding to GSH are 0.168 and 0.303, respectively, which are lower than those of SPTF. This proves that when the fluorine content is low, the presence of hydrophobic Ce6 does have a certain effect on the activity of fluorine atoms, but Ce6@SPTF still shows a certain amplification of T2 / T1 after responding to GSH, which is higher than the current T2 / T1 amplification used for mouse breast cancer. 19 F MRI intravenous injection of polymer nanoprobes, which have been shown to have good 19 F MRI imaging potential. However, the T2 / T1 values ​​of high-fluorine content SPHF and Ce6@SPHF were 0.118 and 0.112, respectively. After adding GSH, T2 / T1 decreased to 0.07 and 0.032, respectively. The reason may be that the aggregation of high-density fluorine atoms after breakage reduced the activity, resulting in stronger dipole-dipole interaction and shortening the T2 relaxation time. Combined with literature reports, it is expected to achieve a T2 relaxation time above a certain level. 19 F MRI imaging. The T2 relaxation time of the nanomicelles prepared by the present invention is greater than 50ms, so they are expected to be used in vivo 19 F MRI.

[0070] Experimental Example 4: Characterization of the Photodynamic Effect of Nanotheranostic Micelles

[0071] The content of reactive oxygen species (ROS) is an important criterion for evaluating the degree of killing of cancer cells by photodynamic therapy. The following tests the in vitro ROS production ability of the nano-diagnostic agents Ce6@SPTF and Ce6@SPHF. 1,3-Diphenylisobenzofuran (DPBF) is a commonly used probe for detecting extracellular ROS levels. When the ROS generated in the system binds to DPBF, the ultraviolet absorbance of DPBF at 410nm is reduced. The changes in the absorbance of DPBF at 410nm after incubation of PBS group, Ce6@SPTF and Ce6@SPHF for different times and the comparison of the absorbance of DPBF at different time points are shown in the figure below. Figure 9As shown, A is a comparison graph of the absorbance changes of DPBF at 410 nm after incubation for different times in the PBS group, B is a comparison graph of the absorbance changes of DPBF at 410 nm after incubation for different times in Ce6@SPTF, C is a comparison graph of the absorbance changes of DPBF at 410 nm after incubation for different times in Ce6@SPHF, and D is a comparison graph of the absorbance of DPBF at different time points incubated with PBS group, Ce6@SPTF, and Ce6@SPHF. The results show that under 635 nm laser irradiation, the absorbance values ​​of DPBF co-incubated with the two probes (Ce6@SPTF and Ce6@SPHF) at 410 nm gradually decrease with the irradiation time, which proves the ROS production ability of Ce6@SPTF and Ce6@SPHF under laser irradiation. The absorbance value of the DPBF solution in the control group at 410 nm remained almost unchanged. This result indicated that Ce6@SPTF and Ce6@SPHF have the ability to generate ROS under external laser irradiation, showing their potential application value in PDT.

[0072] Experimental Example 5: Characterization of Cell Compatibility of Nanomicelles

[0073] Human umbilical vein endothelial cells (HUVEC) were used to verify the biocompatibility of the nanomicelles. The steps are as follows: HUVEC cells were seeded in a 96-well cell culture plate at a density of 2000-2500 cells / well. After incubation for 24 hours, the culture medium was replaced with fresh culture medium containing different concentration gradients of SPTF, SPHF, Ce6@SPTF, and Ce6@SPHF, and a blank control group was set up. After another 24 hours of co-incubation, the cell activity was detected using the MTT method using an enzyme marker. The toxicity comparison of different concentrations of SPTF, Ce6@SPTF, SPHF, and Ce6@SPHF on HUVEC and 4T1 cells is shown in the figure below. Figure 10 As shown, Figure 10 A is a comparison of the toxicity of SPTF and Ce6@SPTF to 4T1 cells. Figure 10 B is a comparison of the toxicity of SPTF and Ce6@SPTF to HUVEC cells. Figure 10 C is a comparison of the toxicity of SPHF and Ce6@SPHF to 4T1 cells. Figure 10 Figure D compares the toxicity of SPHF and Ce6@SPHF on HUVEC cells. The experimental results show that within 24 hours, the survival rate of HUVEC cells co-incubated with Ce6@SPTF was above 75%, indicating that the nanomicelles had no significant cytotoxic effect, further demonstrating their safety. In contrast, nanomicelles containing 21.5% fluorine exhibited a stronger cytotoxic effect on HUVEC, suggesting that their biosafety may be inhibited by high fluorine content. Subsequent therapeutic experiments are considering using nanomicelles with lower fluorine content.

[0074] Experimental Example 6: In vitro cell photodynamic therapy effect of nanotherapeutic micelles Ce6@SPTF

[0075] 4T1 cells, a mouse breast cancer cell line, were selected and seeded in a 96-well cell culture plate at a density of 200 cells / well. After incubation for 24 hours, the culture medium was replaced with fresh culture medium containing different concentration gradients of nanodiagnostic micelles, and a blank control group was set up. After incubation for 6 hours, the cells were irradiated with a 635nm laser (0.5W / cm 2 After irradiation, the cells were placed in an incubator for 24 hours. Cell viability was then detected using an MTT assay using a microplate reader. Figure 11 As shown in the figure, the experimental results show that under the same irradiation dose, Ce6@SPTF nano-diagnostic and therapeutic micelles improve the killing ability of 4T1 tumor cells and have an ideal photodynamic therapy effect.

[0076] The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. It is apparent to those skilled in the art that various changes, modifications, substitutions, and variations of these embodiments may be made without departing from the principles and spirit of the present invention, and the changes still fall within the scope of protection of the present invention.

Claims

1. A reduction response activation 19 The preparation method of F magnetic resonance signal amplifying self-degrading nano-diagnostic micelles is characterized by It can release drugs in response to GSH self-degradation at the tumor site and amplify 19 F MRI signal, the preparation method comprises the following steps: (1) Synthesis of small molecule compounds: 4-aminobenzyl alcohol reacts with phenyl chloroformate to prepare compound 1a; 2,2'-dithiodipyridine reacts with β-mercaptoethanol to prepare compound 2a; 3,3,3-trifluoro-1-propanol reacts with 2-chloro-2-oxo-1,3,2-dioxaphosphatol in the presence of anhydrous triethylamine to prepare compound 3a; hexafluoroisopropanol reacts with 2-chloro-2-oxo-1,3,2-dioxaphosphatol in the presence of anhydrous triethylamine to prepare compound 4a; (2) Synthesis and modification of polymers: Compound 1a is subjected to condensation polymerization in the presence of a catalyst, dibutyltin dilaurate, to obtain PB1, which is then reacted with a condensation polymerization terminator, compound 2a, to prepare SIP, namely PB2; PB2 is reacted with glacial acetic acid in the presence of dicyclohexylcarbodiimide and 4-dimethylaminopyridine to prepare SIA, namely PB3; PB3 is reacted with β-mercaptoethanol in the presence of glacial acetic acid to prepare SIM, namely PB4; PB4 is reacted with 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid in the presence of dicyclohexylcarbodiimide and 4- SIR (PB5) was prepared by reaction in the presence of dimethylaminopyridine; PB5 was subjected to reversible addition-fragmentation chain transfer polymerization with polyethylene glycol methyl ether methacrylate and 2-dimethylaminoethyl methacrylate in the presence of azobisisobutyronitrile to prepare SIP-b-DMA-b-PEG (PB6); PB6 was reacted with compound 3a to prepare fluorinated amphiphilic block polymer SIP-b-CPF3-b-PEG (PB7); PB6 was reacted with compound 4a to prepare fluorinated amphiphilic block polymer SIP-b-CPF6-b-PEG (PB8); (3) Preparation of nano-diagnostic and therapeutic micelles: The fluorinated amphiphilic block polymer SIP-b-CPF3-b-PEG and dihydrochlorin, i.e., Ce6, were dissolved in DMSO, and then added dropwise to PBS buffer, stirred at room temperature, dialyzed, collected and freeze-dried to obtain nano-diagnostic and therapeutic micelles Ce6@SPTF; the fluorinated amphiphilic block polymer SIP-b-CPF6-b-PEG and dihydrochlorin, i.e., Ce6, were dissolved in DMSO, and then added dropwise to PBS buffer, stirred at room temperature, dialyzed, collected and freeze-dried to obtain nano-diagnostic and therapeutic micelles Ce6@SPHF.

2. A reduction response activation according to claim 1 19 The preparation method of self-degradable nano-diagnostic and therapeutic micelles with F magnetic resonance signal amplification is characterized by: In step (1), the preparation of compound 1a and compound 2a are both carried out under an inert gas environment; in the preparation of compound 1a, the molar ratio of 4-aminobenzyl alcohol to phenyl chloroformate is 1:2; in the preparation of compound 2a, the molar ratio of 2,2'-disulfide dipyridine to β-mercaptoethanol is 3:

1.

3. A reduction response activation according to claim 1 19 The preparation method of self-degradable nano-diagnostic and therapeutic micelles with F magnetic resonance signal amplification is characterized by: In step (1), the preparation of compound 3a and compound 4a is carried out under an inert gas environment, and the molar ratio of 3,3,3-trifluoro-1-propanol or hexafluoroisopropanol, 2-chloro-2-oxo-1,3,2-dioxaphosphacyclopentane, and anhydrous triethylamine is 1.05:1:1.

05.

4. A reduction response activation according to claim 1 19 The preparation method of self-degradable nano-diagnostic and therapeutic micelles with F magnetic resonance signal amplification is characterized by: In step (2), the preparation of PB1 and PB2 is carried out under an inert gas environment; the condensation polymerization time in the preparation of PB1 is 24 minutes; in the preparation of PB2, the molar ratio of compound 1a, dibutyltin dilaurate, and compound 2a is 20:1:

15.

5. A reduction response activation according to claim 1 19 The preparation method of self-degradable nano-diagnostic and therapeutic micelles with F magnetic resonance signal amplification is characterized by: In step (2), in the preparation of PB3, the molar ratio of PB2, glacial acetic acid, dicyclohexylcarbodiimide, and 4-dimethylaminopyridine is 1:10:15:1; in the preparation of PB4, the molar ratio of PB3, β-mercaptoethanol, and glacial acetic acid is 1:0.4:0.4; in the preparation of PB5, the molar ratio of PB4, 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid, dicyclohexylcarbodiimide, and 4-dimethylaminopyridine is 1:5:7:0.

5.

6. A reduction response activation according to claim 1 19 The preparation method of self-degradable nano-diagnostic and therapeutic micelles with F magnetic resonance signal amplification is characterized by: In step (2), the preparation of PB6 is carried out under an inert gas environment, and the molar ratio of PB5, polyethylene glycol methyl ether methacrylate, 2-dimethylaminoethyl methacrylate, and azobisisobutyronitrile is 1:10:50:0.

2.

7. A reduction response activation according to claim 1 19 The preparation method of self-degradable nano-diagnostic and therapeutic micelles with F magnetic resonance signal amplification is characterized by: In step (2), the preparation of PB7 and PB8 is carried out under an inert gas environment; the molar ratio of compound 3a or compound 4a to PB6 is 1.5:

1.

8. A reduction response activation according to claim 1 19 The preparation method of self-degradable nano-diagnostic and therapeutic micelles with F magnetic resonance signal amplification is characterized by: In step (3), the mass ratio of the fluorinated amphiphilic block polymer SIP-b-CPF3-b-PEG or SIP-b-CPF6-b-PEG to Ce6 is 2.5-20:

1.

9. A reduction response activation according to claim 1 19 The preparation method of self-degradable nano-diagnostic and therapeutic micelles with F magnetic resonance signal amplification is characterized by: In step (3), the molecular weight cut-off of the dialysis is 7000 Da, and the dialysis time is 12 h.

10. A reduction-responsive activated carbon prepared by the preparation method according to any one of claims 1 to 9 19 F magnetic resonance signal amplification of self-degradable nanotheranostic micelles.

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