CN118320125A - A supramolecular self-assembly nano-diagnostic agent based on fluorinated cyclodextrin and porphyrin, and its preparation method and application - Google Patents

Abstract

The invention belongs to the technical field of magnetic resonance imaging and medicinal chemistry, and particularly relates to a supermolecule self-assembled nano diagnosis and treatment agent based on fluorinated cyclodextrin and porphyrin, and a preparation method and application thereof. The invention takes zwitterionic fluorinated beta-cyclodextrin and tetraphenylporphyrin as basic structures, firstly modifies the tetraphenylporphyrin into zinc porphyrin coupled adamantane, then utilizes the host-guest interaction between the adamantane and the beta-cyclodextrin to construct an amphiphilic structure, and the amphiphilic structure is assembled in two stages to form a stable nano structure. The disulfide bond structure in the nano system can intelligently respond to glutathione, the fluorinated cyclodextrin micromolecule released after the system is disintegrated can realize the specific response amplification of ¹⁹ F signals, and the singlet oxygen generated by the porphyrin structure after illumination can be effectively used for photodynamic therapy. Therefore, the nano diagnosis and treatment agent can be applied to the fields of tumor diagnosis and treatment, detection, catalysis and the like, and has important potential application value.

Description

A supramolecular self-assembly nano-diagnostic agent based on fluorinated cyclodextrin and porphyrin and its preparation method and application

Technical Field

The present invention belongs to the technical field of magnetic resonance imaging and medicinal chemistry, and specifically relates to a supramolecular self-assembly nano-diagnostic agent based on fluorinated cyclodextrin and porphyrin, and a preparation method and application thereof.

Background technique

In modern medicine, the diagnosis and treatment of diseases such as tumors and inflammation are particularly important. Among the various existing imaging modes, ^1H magnetic resonance imaging (MRI) has the advantages of being non-invasive and having no penetration depth limitation, and is suitable for soft tissue imaging and is widely used in clinical practice. However, due to the strong background signal interference of water in the tissue, there may be diagnostic errors. Although the use of paramagnetic metal contrast agents can enhance the imaging contrast of lesions, it may cause safety issues such as renal fibrosis and brain deposition, which limits its clinical use. Therefore, the development of "heteronuclear" MRI has received widespread attention and research.

The ¹⁹ F nucleus has excellent magnetic resonance properties, high natural abundance, and a gyromagnetic ratio very close to that of hydrogen. Only a trace amount of fluorine in the human body is fixed in teeth and bones, and there is almost no background signal interference. Therefore, ¹⁹ F is considered to be the most promising imaging nucleus. The ¹⁹ FMRI imaging signal intensity is positively correlated with the fluorine content and T2 relaxation time, so high fluorine content and long T2 relaxation time can meet the needs of high-performance imaging. However, due to the super hydrophobicity of fluorine atoms, their poor mobility in aqueous solution leads to extensive aggregation, resulting in the obstruction of the rotation of fluorine atoms, resulting in a short T2 relaxation time (<100ms), which cannot meet the actual imaging needs. Therefore, the current bottleneck of ¹⁹ FMRI is how to increase the fluorine content while ensuring a longer T2 relaxation time, which is essentially how to overcome the problem of hydrophobic aggregation of fluorine.

At present, some studies have achieved certain results by introducing hydrophilic groups (sulfoxide, short-chain polyethylene glycol, amide groups, etc.) near fluorine atoms to reduce the aggregation of fluorine atoms, but the T2 relaxation time of most of these contrast agents is still less than 100ms, and they cannot be effectively used. There are also studies that use some nano-platforms (such as albumin and gold nanoparticles) to couple fluorine-containing molecules as ¹⁹ F MRI contrast agents, and the T2 relaxation time can reach 200ms, but the low fluorine content (<5%) limits its in vivo imaging application.

In addition to early diagnosis and screening of tumors, treatment is the most important step. Currently, among various emerging therapies, photodynamic therapy has been widely studied due to its non-invasiveness. It kills tumor cells by using photosensitizers, light sources and endogenous molecular oxygen. Among them, porphyrin is the most commonly used type of photosensitizer, which can produce singlet oxygen under light to destroy the cell structure, thereby causing apoptosis of tumor cells. At the same time, porphyrin can have the ability of ^1H MRI by complexing superparamagnetic metal ions (such as Gd ³⁺ , Mn ²⁺ , etc.), but after complexing superparamagnetic metal ions, porphyrin cannot undergo intersystem crossing transition to the triplet state, resulting in its inability to produce singlet oxygen, thereby losing its therapeutic function. Therefore, it is impossible to integrate magnetic resonance imaging and photodynamic therapy to achieve the goal of integrated diagnosis and treatment.

Therefore, it is necessary to develop a diagnostic and treatment system that integrates ¹⁹ F MRI and photodynamic therapy, so that it can be applied not only to tumor diagnosis and treatment, but also to detection and catalysis.

Summary of the invention

In order to overcome the deficiencies of the above-mentioned prior art, the present invention proposes a supramolecular self-assembled nano-diagnostic agent based on fluorinated cyclodextrin and porphyrin and a preparation method thereof. The prepared nano-diagnostic agent belongs to a photodynamic therapy system that can effectively and specifically respond to ¹⁹ F MRI guidance, which can be applied not only to tumor diagnosis and treatment, but also to detection and catalysis and other fields, and has important potential application value.

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

The first aspect of the present invention provides a method for preparing supramolecular self-assembled nanomicelles based on fluorinated cyclodextrin and porphyrin, comprising the following steps:

S1. Preparation of compound 1: benzaldehyde and p-hydroxybenzaldehyde are added to a solvent under an inert gas atmosphere, and the mixture is stirred and reacted in an oil bath at 130-140°C for 3-5 hours, and then pyrrole is added dropwise, and then the mixture is stirred and reacted in an oil bath at 130-140°C for 3-5 hours. After the reaction is completed and the solvent is removed, methanol is added, and the mixture is allowed to stand at (-10)-(-30)°C overnight, and the precipitate is collected. The precipitate is separated by silica gel column chromatography and dried to obtain 1-hydroxytetraphenylporphyrin;

S2, preparation of compound 2: under an inert gas atmosphere, dithiodipyridine is added to a solvent, and mercaptoethanol is added under stirring, and the mixture is stirred at room temperature for 1-3 hours, and then washed, dried, desolventized, and separated by silica gel column chromatography to obtain dithiopyridinol;

S3, preparation of compound 3: under an inert gas atmosphere, compound 2 and 4-dimethylaminopyridine are added to a solvent, the temperature is lowered to 0°C, and then p-nitrophenyl chloroformate is added under stirring, the reaction is stirred at room temperature for 20-30 hours, the solvent is removed, and then separated and dried by silica gel column chromatography to obtain p-nitrophenyl dithiopyridine ester;

S4, preparation of compound 4: Compound 1 is added to a solvent under an inert gas atmosphere, and compound 3 and 4-dimethylaminopyridine (DMAP) are added under stirring, followed by condensation reflux reaction at 45-55° C. in an oil bath for 20-30 hours, and after the reaction, the solvent is removed, and then separated and dried by silica gel column chromatography to obtain dithiopyridyl porphyrin;

S5. Preparation of compound 5: Compound 4 is added to a solvent, and mercaptoethanol is added dropwise under stirring. After stirring and reacting for 1-3 hours at room temperature and in the dark, dithiol porphyrin is obtained by separation and drying through silica gel column chromatography;

S6, preparation of compound 6: under an inert gas atmosphere, compound 5, 4-dimethylaminopyridine, dicyclohexylcarbodiimide and 1-adamantanecarboxylic acid are added to a solvent, stirred at room temperature for 20-30 hours under light-proof conditions, and then the solvent is removed, followed by separation by silica gel column chromatography to obtain porphyrin-coupled adamantane;

S7, preparation of compound 7: compound 6 and anhydrous zinc acetate are added to a solvent under an inert gas atmosphere, stirred and reacted at room temperature for 4-6 hours, the solvent is removed, and then separated by silica gel column chromatography and dried to obtain zinc porphyrin-coupled adamantane;

S8. Preparation of compound 8:

S81, under an inert gas atmosphere, dissolve triphenyl phosphine in a solvent and keep it in an ice bath, separately dissolve N-bromosuccinimide (NBS) in a solvent, then dropwise add the N-bromosuccinimide solution to the triphenyl phosphine solution, and stir the reaction at room temperature for 20-40 minutes;

S82, dissolving β-cyclodextrin in a solvent, and then dropping the mixed solution obtained in S81, heating to 75-85°C after dropping, stirring and reacting for 10-15 hours under an inert gas atmosphere, cooling to room temperature after the reaction, adding methanol and continuing to stir for 20-40 minutes, and after stirring, adjusting the pH of the solution to 8.5-9.5 in an ice bath, continuing to stir for 1-2 hours, diluting the reaction solution with ice water to produce a fine precipitate, and finally removing the supernatant, washing and drying to obtain brominated cyclodextrin;

S9, preparation of compound 9: Compound 8 and thiourea are dissolved in a solvent under an inert gas atmosphere, and heated to 65-75° C. for reaction for 20-30 hours. After the reaction is completed and the solvent is removed, sodium hydroxide is added, and refluxed for 1 hour, acidified to form a white precipitate, which is then filtered, washed and dried to obtain thiolated cyclodextrin;

S10, preparation of compound 10: compound 9, dimethylallylamine and azobisisobutyronitrile are dissolved in a solvent under an inert gas atmosphere, reacted at 65-75° C. for 20-30 hours, cooled to room temperature, and then precipitated and dried to obtain tertiary aminated cyclodextrin;

S11, preparation of compound 11: dissolving 3,3,3-trifluoropropane-1-ol and anhydrous triethylamine in a solvent to prepare solution 1, dissolving cyclic vinyl chlorophosphate in a solvent to prepare solution 2, then adding solution 2 dropwise to solution 1, placing in an ice bath for 20-40 minutes after adding the sample, removing the ice bath and stirring at room temperature to react for 3-5 hours, and after the reaction, washing, drying and removing the solvent to obtain vinyl trifluorophosphate;

S12, preparation of compound 12: dissolving compound 10 and compound 11 in a solvent, heating to 85-95° C., stirring and reacting for 2-4 days, cooling to room temperature, and then precipitating to obtain fluorinated cyclodextrin;

S13. Assembly of nano-diagnostic and therapeutic agents: Compound 12 of S12 and compound 7 of S7 are dissolved in solvents respectively, and then the zinc porphyrin-coupled adamantane solution is slowly added dropwise to the fluorinated cyclodextrin solution under rapid stirring. After the addition, stirring is continued for 1-3 hours, and then the solvent is removed to obtain supramolecular self-assembled nano-micelles based on fluorinated cyclodextrin and porphyrin.

Preferably, in S1, the ratio of benzaldehyde, p-hydroxybenzaldehyde, pyrrole and methanol is 4-6mL: 2-3g: 4-6mL: 90-110mL. The concentration of p-hydroxybenzaldehyde in the solvent is 2-3g/180mL. The solvent includes (but is not limited to) propionic acid.

Preferably, in S2, the ratio of dithiodipyridine to mercaptoethanol is 6-7 g:0.5-1 g. The concentration of dithiodipyridine in the solvent is 6-7 g/20 mL. The washing is first washed once with a 10% NaOH aqueous solution and then washed once with a NaCl aqueous solution. The solvent (including but not limited to) is dry dichloromethane.

Preferably, in S3, the usage ratio of the compound 2, 4-dimethylaminopyridine and p-nitrophenyl chloroformate is 1.5-2.5 g: 260-270 mg: 3-4 g. The concentration of the compound 2 in the solvent is 3-4 g/50 mL. The solvent (including but not limited to) is dry dichloromethane.

Preferably, in S4, the usage ratio of compound 1, compound 3 and 4-dimethylaminopyridine is 620-640 mg: 420-440 mg: 140-150 mg. The concentration of compound 1 in the solvent is 620-640 mg/100 mL. The solvent (including but not limited to) is dry dichloromethane.

Preferably, in S5, the ratio of the compound 4 to mercaptoethanol is 670-690 mg:45-55 mg. The concentration of the compound 4 in the solvent is 670-690 mg/10 mL. The solvent (including but not limited to) is dry dichloromethane.

Preferably, in S6, the usage ratio of the compound 5, 4-dimethylaminopyridine, dicyclohexylcarbodiimide and 1-adamantanecarboxylic acid is 390-410 mg: 10-15 mg: 200-210 mg: 160-170 mg. The concentration of the compound 5 in the solvent is 390-410 mg/10 mL. The solvent (including but not limited to) is dry tetrahydrofuran.

Preferably, in S7, the usage ratio of the compound 6 and anhydrous zinc acetate is 190-210 mg:40-50 mg. The concentration of the compound 6 in the solvent is 190-210 mg/20 mL. The solvent (including but not limited to) is dry dichloromethane.

Preferably, in S81 to S82, the ratio of triphenylphosphine, N-bromosuccinimide and β-cyclodextrin is 9-10g:6-7g:2-3g. The concentration of triphenylphosphine in the solvent is 9-10g/30mL, and the concentration of β-cyclodextrin in the solvent is 2-3g/30mL. The solvent (including but not limited to) is anhydrous N,N-dimethylformamide (DMF).

Preferably, in S9, the usage ratio of the compound 8, thiourea and sodium hydroxide is 4-6 g: 2-3 g: 2-3 g. The concentration of the compound 8 in the solvent is 4-6 g/50 mL. The solvent (including but not limited to) is anhydrous N,N-dimethylformamide (DMF).

Preferably, in S10, the ratio of the compound 9, dimethylallylamine and azobisisobutyronitrile is 1-2 g: 3-4 g: 1-2 g. The concentration of the compound 9 in the solvent is 1-2 g/30 mL. The solvent (including but not limited to) is anhydrous N,N-dimethylformamide (DMF).

Preferably, in S11, the usage ratio of the 3,3,3-trifluoropropan-1-ol, anhydrous triethylamine and cyclic chloroethylene phosphate is 3-4 g: 3-4 mL: 4-5 g. The concentration of the 3,3,3-trifluoropropan-1-ol in the solvent is 3-4 g/30 mL. The solvent (including but not limited to) is dry dichloromethane.

Preferably, in S12, the usage ratio of the compound 10 to the compound 11 is 2-3 g:4-6 g. The solvent (including but not limited to) is anhydrous dimethyl sulfoxide (DMSO).

Preferably, in S13, the dosage ratio of compound 12 to compound 7 is 3-5 mg:1-2 mg. The concentration of compound 12 in the solvent is 3-5 mg/5 mL, and the concentration of compound 7 in the solvent is 1-2 mg/200 μL. The solvent (including but not limited to) is anhydrous dimethyl sulfoxide (DMSO).

The second aspect of the present invention also provides supramolecular self-assembled nanomicelles based on fluorinated cyclodextrin and porphyrin prepared by the preparation method described in the first aspect.

The supramolecular self-assembly nano-diagnostic agent based on fluorinated cyclodextrin and porphyrin prepared by the method of the present invention uses the fluorinated cyclodextrin of phosphorylcholine zwitterion as ¹⁹ F MRI function, and porphyrin is coupled to adamantane through a disulfide bond for specific response to glutathione and photodynamic therapy. In aqueous solution, adamantane can be complexed with cyclodextrin based on host-guest interaction to form an amphiphilic molecular structure with porphyrin as the hydrophobic end and fluorinated cyclodextrin as the hydrophilic end, followed by secondary assembly to form a nanostructure. After the nanostructure responds to glutathione, the system disintegrates and releases the fluorinated cyclodextrin small molecules, which enhances the responsiveness of the ¹⁹ F signal. Therefore, the present invention constructs a photodynamic therapy system that can effectively and specifically respond to ¹⁹ F MRI, which can be used not only in tumor diagnosis and treatment, but also in detection and catalysis, and has a high application value.

The third aspect of the present invention further provides the use of the supramolecular self-assembled nanomicelles based on fluorinated cyclodextrin and porphyrin described in the second aspect in the preparation of ¹⁹ F MRI contrast agents.

The third aspect of the present invention further provides the use of the supramolecular self-assembled nanomicelles based on fluorinated cyclodextrin and porphyrin described in the second aspect in the preparation of anti-tumor drugs.

Preferably, the tumor includes (but is not limited to) breast cancer.

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

The present invention discloses a method for preparing a supramolecular self-assembly nano-diagnostic agent based on fluorinated cyclodextrin and porphyrin, with zwitterionic fluorinated β-cyclodextrin and tetraphenylporphyrin as the basic structure, firstly modifying tetraphenylporphyrin into zinc porphyrin coupled adamantane, then utilizing the host-guest interaction between adamantane and β-cyclodextrin to construct an amphiphilic structure of porphyrin and hydrophilic fluorinated cyclodextrin, and then making the amphiphilic structure undergo secondary assembly to form a stable nanostructure. Among them, the disulfide bond structure in the obtained nanosystem can intelligently respond to glutathione, and the fluorinated cyclodextrin small molecules released after the disintegration of the nanosystem can realize the specific response amplification of the 19F signal, and the singlet oxygen generated by the porphyrin structure after illumination can be effectively used for photodynamic therapy. Therefore, the supramolecular self-assembled nano-diagnostic and therapeutic agent based on fluorinated cyclodextrin and porphyrin prepared in the present invention can be used for responsive 19F magnetic resonance imaging-guided photodynamic therapy, and is expected to be applied to the fields of tumor diagnosis, detection and catalysis, and has broad application prospects.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a schematic diagram of the structure of a supramolecular self-assembled nano-diagnostic agent based on fluorinated cyclodextrin and porphyrin;

FIG2 is a nuclear magnetic resonance hydrogen spectrum ( ¹ H NMR) of a porphyrin-coupled adamantane compound;

FIG3 is a comparison of nuclear magnetic resonance hydrogen spectra ( ¹ H NMR) of porphyrin coupled with adamantane compound before and after complexing zinc ions;

FIG4 is a hydrogen nuclear magnetic resonance spectrum ( ¹ H NMR) of fluorinated cyclodextrin;

FIG5 is a nuclear magnetic resonance fluorine spectrum ( ¹⁹ F NMR) of fluorinated cyclodextrin;

FIG6 is a schematic diagram of an assembly scheme of a supramolecular self-assembly type nano-diagnostic and therapeutic agent based on fluorinated cyclodextrin and porphyrin to form nano-micelles;

FIG7 is a graph showing the particle size distribution of the supramolecular self-assembled nano-diagnostic agent based on fluorinated cyclodextrin and porphyrin, and the changes in particle size and zeta potential during constant temperature incubation for a certain period of time;

FIG8 is a TEM image of a supramolecular self-assembled nano-theranostic agent based on fluorinated cyclodextrin and porphyrin;

FIG9 is a fluorescence emission spectrum of a supramolecular self-assembled nano-theranostic agent based on fluorinated cyclodextrin and porphyrin;

FIG10 is a ¹⁹ F NMR signal spectrum of a supramolecular self-assembled nano-diagnostic agent based on fluorinated cyclodextrin and porphyrin and co-incubated with GSH for a certain period of time;

FIG. 11 is a graph showing the change in fluorescence intensity when SOSG (singlet oxygen green fluorescence probe) detects singlet oxygen.

Figure 12 shows the dark toxicity and phototoxicity evaluation results of the supramolecular self-assembled nano-diagnostic and therapeutic agent based on fluorinated cyclodextrin and porphyrin on different cell lines (human umbilical vein endothelial cells HUVEC, mouse breast cancer cell line 4T1).

Detailed ways

The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in each embodiment of the present invention described below can be combined with each other as long as they do not conflict with each other.

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.

Example: A method for preparing a supramolecular self-assembled nano-diagnostic agent based on fluorinated cyclodextrin and porphyrin

As shown in Figure 1, the preparation method comprises the following steps:

1. Preparation of zinc porphyrin coupled to adamantane

(1) Preparation of compound 1 (1-hydroxytetraphenylporphyrin):

According to the above reaction formula, after the double-necked flask was dehydrated and deoxygenated, 5 mL of benzaldehyde and 2.2 g of p-hydroxybenzaldehyde were added under argon protection, and then 180 mL of propionic acid was added as a solvent, and then stirred at 135° C. in an oil bath for 4 hours.

Pyrrole needs to be distilled to remove water before use. Collect 5 mL of pyrrole, stir for 4 hours, add dry pyrrole (5 mL) dropwise to the propionic acid solution, and then continue to stir and react for 4 hours in an oil bath at 135°C. After the reaction is completed, most of the propionic acid solvent is removed by vacuum distillation, and then 100 mL of methanol is added, sealed and placed in a -20°C refrigerator to stand overnight, and then the precipitate is collected by suction filtration. The obtained precipitate is dissolved in dichloromethane, separated by silica gel column chromatography, vacuum distilled and vacuum dried to obtain a purple-black solid powder product (yield 4.2%).

(2) Preparation of compound 2 (dithiopyridinol):

According to the above reaction formula, after dehydrating and deoxygenating the double-necked flask, 6.27 g of disulfide dipyridine was added under argon protection, and then 20 mL of dry dichloromethane was added as a reaction solvent, and 0.8 g of mercaptoethanol was added under stirring conditions, and the reaction was stirred at room temperature for 2 hours. After the reaction was completed, it was washed once with a 10% NaOH aqueous solution, and then washed once with a NaCl aqueous solution, and then anhydrous magnesium sulfate was added to dry, and the excess solvent was removed by reduced pressure distillation, and finally separated by silica gel column chromatography (wherein a mixture of ethyl acetate: petroleum ether of 1:6 was used as a separation developing solvent) to obtain a light yellow oily liquid product (yield 87%).

(3) Preparation of compound 3 (p-nitrophenyl dithiopyridinium ester):

According to the above reaction formula, after dehydrating and deoxygenating the double-necked flask, 2.0 g of compound 2 (dithiopyridinol) was added to 50 mL of dry dichloromethane under argon protection, and then 268.4 mg of 4-dimethylaminopyridine (DMAP) was added, and then the system was cooled to 0° C. in an ice bath, and 3.42 g of p-nitrophenyl chloroformate was added under stirring, and the system was stirred at room temperature under argon protection for 24 hours. After the reaction was completed, the excess solvent was distilled off under reduced pressure, and then separated and dried by silica gel column chromatography to obtain a yellow viscous oily liquid product (yield: 63%).

(4) Preparation of compound 4 (dithiopyridyl porphyrin):

According to the above reaction formula, after the three-necked flask was dehydrated and deoxygenated, 628 mg of compound 1 (1-hydroxytetraphenylporphyrin) was added under argon protection, and 100 mL of dry dichloromethane was added as a solvent, and 423 mg of compound 3 (p-nitrophenyl dithiopyridinium ester) containing an active ester and 146.4 mg of 4-dimethylaminopyridine (DMAP) were added under stirring, and then condensed and refluxed in a 50° C. oil bath for 24 hours, during which the closed system prevented dichloromethane from volatilizing and escaping. After the reaction was completed, it was cooled to room temperature, and the excess solvent was removed by vacuum rotary evaporation, and then separated and dried by silica gel column chromatography to obtain a purple-black solid product (yield 82%).

(5) Preparation of compound 5 (dithiol porphyrin):

According to the above reaction formula, 680 mg of compound 4 (dithiopyridyl porphyrin) was added to a round-bottom flask, and 10 mL of dry dichloromethane was added as a reaction solvent, and then 50 mg of mercaptoethanol was added dropwise under stirring, and the resulting mixture was stirred and reacted for 2 hours at room temperature and in the dark. After the reaction was completed, a purple-black solid product (yield 47%) was obtained by silica gel column chromatography separation and drying.

(6) Preparation of compound 6 (porphyrin-coupled adamantane):

According to the above reaction formula, after the double-necked flask was dehydrated, 400 mg of compound 5 (dithiol porphyrin), 12 mg of 4-dimethylaminopyridine and 202 mg of dicyclohexylcarbodiimide were added under argon protection, and 10 mL of dry tetrahydrofuran was added as a reaction solvent, and then 166 mg of 1-adamantanecarboxylic acid was added, and the resulting reaction mixture was placed in a dark environment and stirred at room temperature for 24 hours. After the reaction was completed, the solvent was removed by rotary evaporation under reduced pressure, and then separated by silica gel column chromatography to obtain a purple-black solid product (yield: 68%).

(7) Preparation of compound 7 (zinc porphyrin coupled to adamantane):

According to the above reaction formula, after the double-necked flask was dehydrated and deoxygenated, 200 mg of compound 6 (porphyrin-coupled adamantane) was added to 50 mL of dry dichloromethane under argon protection, and 43 mg of anhydrous zinc acetate was added, and then stirred at room temperature for 5 hours. After the reaction was completed, the excess solvent was removed by vacuum distillation, and then separated and dried by silica gel column chromatography to obtain a purple-red solid product (yield 91%).

2. Preparation of Fluorinated Cyclodextrin

(1) Preparation of Compound 8 (Brominated Cyclodextrin):

According to the above reaction formula, under argon protection, 9.18 g of triphenylphosphine was dissolved in 30 mL of anhydrous N,N-dimethylformamide (DMF) and kept in an ice bath, and then 6.23 g of N-bromosuccinimide (NBS) was dissolved in 10 mL of anhydrous DMF using another dry reaction bottle, and then the NBS solution was added dropwise to the triphenylphosphine solution using a dropping funnel, and the resulting mixed solution was stirred at room temperature for 30 minutes.

β-cyclodextrin (2.8 g) was vacuum dried at 80°C overnight in advance and then dissolved in 30 mL of anhydrous DMF. The obtained triphenylphosphine and NBS mixed solution was added dropwise to the β-cyclodextrin solution using a dropping funnel, and the temperature was raised to 80°C after the addition, and the reaction was stirred overnight under argon protection. After the reaction, the heating was removed and cooled to room temperature, and then 10 mL of methanol was added to continue stirring for 30 minutes. After stirring, sodium methoxide was added to the solution under an ice bath to adjust the pH to about 9. After continuing to stir for 1 hour, the reaction solution was added to 1 liter of ice-water mixture to dilute a fine precipitate. Finally, the supernatant was removed, and the precipitate was washed three times with methanol, and a beige solid product (yield was 86%) was obtained after drying.

(2) Preparation of compound 9 (thiolated cyclodextrin):

According to the above reaction formula, 5g of compound 8 (brominated cyclodextrin) and 2.5g of thiourea were dissolved in 50mL of anhydrous DMF, and heated to 70°C under argon protection for 24 hours. After the reaction, DMF was removed by distillation under reduced pressure, and the obtained brown oil was dissolved in 200mL of water, and 2.22g of sodium hydroxide was added, and then the reaction solution was placed under argon protection and heated to 50°C, and refluxed at this temperature. After 1 hour, the obtained suspension was acidified with potassium hydrogen sulfate aqueous solution to form a white precipitate, which was filtered and thoroughly washed with water. Finally, a white powder product (yield of 71%) was obtained by drying.

(3) Preparation of Compound 10 (tertiary aminated cyclodextrin):

According to the above reaction formula, the thiol group and the carbon-carbon double bond undergo an addition reaction through a "click chemistry" reaction. Specifically, 1.5 g of compound 9 (thiolated cyclodextrin) and 3.4 g of dimethylallylamine are added to 30 mL of DMF under argon protection, and then 1.3 g of azobisisobutyronitrile is added, and then the reaction system is placed under argon protection at 70°C for 24 hours. After the reaction is completed, it is cooled to room temperature and precipitated in cold ether. The precipitate is collected and dried to obtain a white powder product (yield 68%).

(4) Preparation of compound 11 (ethylene trifluorophosphate):

According to the above reaction formula, 3.6g of 3,3,3-trifluoropropane-1-ol was added to a three-necked flask, and then 30mL of dry dichloromethane was added as a reaction solvent, and 3.75mL of anhydrous triethylamine was added. In addition, 4.28g of cyclochloroethylene phosphate was dissolved in 15mL of dry dichloromethane, and then the cyclochloroethylene phosphate solution was added to a dropping funnel and added dropwise to the solution in the above flask. After the addition was completed, ice bathed for 30 minutes, and then the ice bath was removed and stirred at room temperature for 4 hours. After the reaction was completed, it was washed with a saturated saline solution, and then anhydrous magnesium sulfate was added for drying to obtain a light yellow liquid, and then the excess solvent was removed by reduced pressure distillation to obtain a yellow oily liquid product (yield 64%).

(5) Preparation of Compound 12 (fluorinated cyclodextrin):

According to the above reaction formula, 2 g of dry compound 10 (tertiary aminated cyclodextrin) and 4.86 g of compound 11 (ethylene trifluorophosphate) were dissolved in 50 mL of anhydrous dimethyl sulfoxide (DMSO), heated to 90°C and stirred for reaction for 3 days. After the reaction was completed, it was cooled to room temperature and precipitated in cold ether, and the precipitate was collected to obtain a brown oily product (yield 57%).

The fluorinated cyclodextrin and zinc porphyrin-coupled adamantane (or porphyrin-coupled adamantane) prepared above were dissolved in deuterated solvents respectively, and their concentrations were maintained at 10-20 mg/mL, and then analyzed using a BRUKER AscendTM400 nuclear magnetic resonance analyzer to obtain hydrogen spectra ( ¹ H NMR) or fluorine spectra ( ¹⁹ F NMR), and then the MestReNova software was used to analyze the attribution and integration of characteristic peaks.

Figure 2 is the nuclear magnetic resonance hydrogen spectrum of the porphyrin-coupled adamantane compound. It can be seen that the h position is the characteristic peak of the porphyrin ring. Due to the rigid planar conjugated structure of the porphyrin ring, the hydrogen electron cloud density in the ring is very high, which has a strong shielding effect, resulting in a relative chemical shift of the characteristic peak of -2.86ppm. At the same time, f, e, and g are characteristic peaks of the adamantane structure, indicating that the chemical structure of the compound is correct. After the zinc ion is complexed, the characteristic peak of the porphyrin ring at the a position disappears, and the other characteristic peaks remain unchanged, indicating that the zinc ion is successfully complexed (as shown in Figure 3).

FIG4 is a hydrogen nuclear magnetic resonance spectrum of a fluorinated cyclodextrin compound, wherein the characteristic peak integration and attribution are shown in the figure. Meanwhile, its fluorine spectrum is shown in FIG5 , and it can be seen that there is a strong fluorine signal peak at -61.97 ppm, indicating that it has the potential to be used as an imaging probe.

3. Assembly of Nanodiagnostic Agents

Fluorinated cyclodextrin and zinc porphyrin coupled adamantane compounds are assembled into nanomicelles (TTF) (i.e., supramolecular self-assembled nano-theranostic agent based on fluorinated cyclodextrin and porphyrin) according to the scheme shown in FIG6 . The TTF mainly comprises four structures of zwitterionized trifluoromethyl, β-cyclodextrin, disulfide bond and tetraphenylporphyrin. The specific operation is as follows:

3.5 mg of fluorinated cyclodextrin was dissolved in 5 mL of water, and 1 mg of zinc porphyrin-coupled adamantane was dissolved in 200 μL of DMSO. Then, the zinc porphyrin-coupled adamantane solution was slowly added to the fluorinated cyclodextrin aqueous solution using a 1 mL syringe under rapid stirring at 600 rpm, and the stirring was continued for 2 hours after the addition. After that, the solvent DMSO was removed by dialysis in water (changing deionized water for more than three times), and finally a nanoassembly, also known as nanomicelle (TTF), was obtained, with a concentration of about 0.9 mg/mL.

The particle size of the obtained nanomicelles was tested by the Malvern Zetasizer Nano ZS90 laser particle size analyzer, as shown in Figure 7A. The dynamic light scattering particle size was 220nm and the polydispersity index (PDI) was 0.16. Then, the particles were incubated in a constant temperature shaker at 37°C for a certain period of time, and the changes in particle size and Zeta potential were tested by the Malvern Zetasizer Nano ZS90 laser particle size analyzer. The changes in particle size and Zeta potential with incubation time are shown in Figure 7B. It can be seen from the figure that after the TTF nanoparticles were incubated at a constant temperature for 48 hours, there was no significant change in particle size and Zeta potential. The particle size remained at about 230nm and the Zeta potential remained at about 21mV, indicating that the supramolecular self-assembled nanodiagnostic agent based on fluorinated cyclodextrin and porphyrin has good stability.

At the same time, a small amount of TTF solution (about 50 μg/mL) was taken as a test sample for transmission electron microscopy (TEM), and the obtained nanoparticles were scanned using a 200KV transmission electron microscope to obtain a TEM image. The transmission electron microscopy result of TTF is shown in Figure 8. From the electron microscope image, it can be seen that the morphology of the nanoparticles is self-assembled into a relatively regular sphere with a particle size of about 100 nm.

In addition, 1 mL of TTF solution (about 50 μg/mL) was taken as a test sample, and the fluorescence emission spectra of the nanomicelles and porphyrin molecules were detected using a FluoroMax-4 fluorescence spectrometer (excitation wavelength was 520 nm). The test results are shown in Figure 9, which have obvious fluorescence emission peaks at 660 nm and 725 nm, indicating that it has the potential for application as a fluorescence imaging probe.

Experimental example: Performance characterization of supramolecular self-assembled nano-theranostics based on fluorinated cyclodextrin and porphyrin

(1) Responsiveness to glutathione (GSH)

600 μL of TTF liquid (about 50 μg/mL) was taken as the test sample, and the fluorine spectrum of the nanoparticles was analyzed by BRUKER AscendTM400 nuclear magnetic resonance analyzer, and then the ¹⁹ F NMR fluorine spectrum was analyzed by MestReNova software. The ¹⁹ FNMR spectrum analysis results of TTF nanoparticles are shown in Figure 10. From the spectrum, it can be seen that there is an obvious signal peak at -64.41 ppm. At the same time, 10-20 mM glutathione (GSH) was added to the TTF liquid, and after incubation in a constant temperature incubator at 37°C for 6 hours, the supernatant was taken as the test sample, and the fluorine spectrum was obtained by BRUKER AscendTM400 nuclear magnetic resonance analyzer, and then the fluorine spectrum was analyzed by MestReNova software to obtain the ¹⁹ F NMR spectrum of the micelle fluorine signal change. The results are shown in Figure 10. After adding glutathione (GSH), the fluorine signal was significantly enhanced. The enhancement of fluorine signal is because the micelles disintegrate after responding to GSH, and the released fluorinated cyclodextrin has a more suitable fluorine density and hydration environment after dissociation, thus showing a stronger fluorine signal. This shows that the diagnostic agent has good GSH responsiveness and has the function of enhancing fluorine signal response, and can be effectively applied to ¹⁹ F magnetic resonance imaging.

(2) Singlet oxygen production capacity

The obtained TTF solution was diluted to a concentration of about 100 μg/mL, and 2 mL of the solution was taken for standby use. Another 2 μL of the storage solution of the singlet oxygen green fluorescent probe (SOSG) purchased from Shanghai Biyuntian Biotechnology Co., Ltd. was dispersed in 2 mL of the aqueous solution. Then, the mixed solution of 100 μg/mL TTF+SOSG was used as the detection sample. Under light-proof conditions, the fixed laser (660 nm) irradiation time was set to 30 s, and the irradiation power was set to 0 mw, 200 mw, 400 mw, 600 mw, 800 mw, and 1000 mw in sequence. The HORIBA Jobin Yvon FluoroMax-4 fluorescence spectrometer was used to detect the fluorescence intensity of the samples and the single SOSG sample under different irradiation powers, and the ORIGIN software was used to plot the change of fluorescence intensity with laser irradiation power.

The SOSG probe can specifically detect singlet oxygen. When singlet oxygen is present, the fluorescence intensity of SOSG at 525nm will increase, and the increase in fluorescence intensity is linearly related to the singlet oxygen content. As can be seen from Figure 11, with the increase of laser irradiation power, the fluorescence intensity of the TTF solution has a significant growth trend, indicating that the diagnostic and therapeutic agent TTF has a good ability to generate singlet oxygen and can be effectively used in photodynamic therapy.

(3) Evaluation of cytotoxicity and therapeutic performance of diagnostic and therapeutic agents

The MTT method was used to investigate the effect of the therapeutic agent TTF on the survival rate of Huvec cells and 4T1 cells. Huvec cells and 4T1 cells were inoculated in a 96-well cell culture plate (5×10 ³ cells/well) and cultured overnight. The original culture medium (DMEM) was then replaced with a culture medium containing the therapeutic agent TTF (TTF: 0-1 mg/mL), and the control group was added with fresh culture medium and incubated for 24 hours. After washing with PBS, it was replaced with a fresh culture medium containing MTT (5 mg/mL) and cultured for 4 hours. The culture medium was then removed and 150 μL DMSO was added, and the mixture was shaken in the dark for 10 minutes. The absorbance of each well was scanned in an ELISA instrument (wavelength of 490 nm), and the cell activity of the control group was set to 100%. The cell survival rate at each concentration was calculated based on this, and the results are shown in Figure 12A. It can be seen from the figure that the therapeutic agent TTF has low toxicity to normal endothelial cells.

At the same time, the dark toxicity of 4T1 cells with added TTF drugs can detect the cytotoxicity of the diagnostic and therapeutic agent TTF itself (after replacing the culture medium containing TTF, cover the well plate with tin foil and avoid light). As can be seen from Figure 12B, its cytotoxicity is weak when there is no light response, and the survival rate of 4T1 cells can be maintained at more than 80%. In addition, after laser irradiation (660nm, 1000mw, 5min) of the cells with added TTF drugs, the singlet oxygen produced by TTF is lethal to cells. It can be seen from the phototoxicity data in the figure that the survival rate of 4T1 cells decreases with the increase of TTF concentration, and the cell survival rate is less than 20% when the drug concentration is 50μg/mL. The dark toxicity and phototoxicity experiments of the above-mentioned diagnostic and therapeutic agent TTF on cells show that the diagnostic and therapeutic agent TTF has good cell compatibility and effective photodynamic therapy effect.

In summary, it can be seen that the supramolecular self-assembly nano-diagnostic agent based on fluorinated cyclodextrin and porphyrin prepared by the method of the present invention can undergo secondary self-assembly to form a nanostructure, which not only has good GSH responsiveness, but also has the function of enhancing fluorine signal response, and can be effectively applied to ¹⁹ F magnetic resonance imaging. At the same time, it also has a good ability to produce singlet oxygen and can be effectively applied to photodynamic therapy. Therefore, the supramolecular self-assembly nano-diagnostic agent based on fluorinated cyclodextrin and porphyrin prepared by the present invention integrates ¹⁹ F MRI and photodynamic therapy, and is expected to be applied to the fields of tumor diagnosis, detection and catalysis, and has broad application prospects.

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

Claims (10)

1. A method for preparing supramolecular self-assembled nanomicelles based on fluorinated cyclodextrin and porphyrin, characterized in that it comprises the following steps: S1. Preparation of compound 1: benzaldehyde and p-hydroxybenzaldehyde are added to a solvent under an inert gas atmosphere, and the mixture is stirred and reacted in an oil bath at 130-140°C for 3-5 hours, and then pyrrole is added dropwise, and then the mixture is stirred and reacted in an oil bath at 130-140°C for 3-5 hours. After the reaction is completed and the solvent is removed, methanol is added, and the mixture is allowed to stand at (-10)-(-30)°C overnight, and the precipitate is collected. The precipitate is separated by silica gel column chromatography and dried to obtain 1-hydroxytetraphenylporphyrin; S2, preparation of compound 2: under an inert gas atmosphere, dithiodipyridine is added to a solvent, and mercaptoethanol is added under stirring, and the mixture is stirred at room temperature for 1-3 hours, and then washed, dried, desolventized, and separated by silica gel column chromatography to obtain dithiopyridinol; S3, preparation of compound 3: under an inert gas atmosphere, compound 2 and 4-dimethylaminopyridine are added to a solvent, the temperature is lowered to 0°C, and then p-nitrophenyl chloroformate is added under stirring, the reaction is stirred at room temperature for 20-30 hours, the solvent is removed, and then separated and dried by silica gel column chromatography to obtain p-nitrophenyl dithiopyridine ester; S4, preparation of compound 4: Compound 1 is added to a solvent under an inert gas atmosphere, and compound 3 and 4-dimethylaminopyridine (DMAP) are added under stirring, followed by condensation reflux reaction at 45-55° C. in an oil bath for 20-30 hours, and after the reaction, the solvent is removed, and then separated and dried by silica gel column chromatography to obtain dithiopyridyl porphyrin; S5. Preparation of compound 5: Compound 4 is added to a solvent, and mercaptoethanol is added dropwise under stirring. After stirring and reacting for 1-3 hours at room temperature and in the dark, dithiol porphyrin is obtained by separation and drying through silica gel column chromatography; S6, preparation of compound 6: under an inert gas atmosphere, compound 5, 4-dimethylaminopyridine, dicyclohexylcarbodiimide and 1-adamantanecarboxylic acid are added to a solvent, stirred at room temperature for 20-30 hours under light-proof conditions, and then the solvent is removed, followed by separation by silica gel column chromatography to obtain porphyrin-coupled adamantane; S7, preparation of compound 7: compound 6 and anhydrous zinc acetate are added to a solvent under an inert gas atmosphere, stirred and reacted at room temperature for 4-6 hours, the solvent is removed, and then separated by silica gel column chromatography and dried to obtain zinc porphyrin-coupled adamantane; S8. Preparation of compound 8: S81, under an inert gas atmosphere, dissolve triphenyl phosphine in a solvent and keep it in an ice bath, separately dissolve N-bromosuccinimide (NBS) in a solvent, then dropwise add the N-bromosuccinimide solution to the triphenyl phosphine solution, and stir the reaction at room temperature for 20-40 minutes; S82, dissolving β-cyclodextrin in a solvent, and then dropping the mixed solution obtained in S81, heating to 75-85°C after dropping, stirring and reacting for 10-15 hours under an inert gas atmosphere, cooling to room temperature after the reaction, adding methanol and continuing to stir for 20-40 minutes, and after stirring, adjusting the pH of the solution to 8.5-9.5 in an ice bath, continuing to stir for 1-2 hours, diluting the reaction solution with ice water to produce a fine precipitate, and finally removing the supernatant, washing and drying to obtain brominated cyclodextrin; S9, preparation of compound 9: Compound 8 and thiourea are dissolved in a solvent under an inert gas atmosphere, and heated to 65-75° C. for reaction for 20-30 hours. After the reaction is completed and the solvent is removed, sodium hydroxide is added, and refluxed for 1 hour, acidified to form a white precipitate, which is then filtered, washed and dried to obtain thiolated cyclodextrin; S10, preparation of compound 10: compound 9, dimethylallylamine and azobisisobutyronitrile are dissolved in a solvent under an inert gas atmosphere, reacted at 65-75° C. for 20-30 hours, cooled to room temperature, and then precipitated and dried to obtain tertiary aminated cyclodextrin; S11, preparation of compound 11: dissolving 3,3,3-trifluoropropane-1-ol and anhydrous triethylamine in a solvent to prepare solution 1, dissolving cyclic vinyl chlorophosphate in a solvent to prepare solution 2, then adding solution 2 dropwise to solution 1, placing in an ice bath for 20-40 minutes after adding the sample, removing the ice bath and stirring at room temperature to react for 3-5 hours, and after the reaction, washing, drying and removing the solvent to obtain vinyl trifluorophosphate; S12, preparation of compound 12: dissolving compound 10 and compound 11 in a solvent, heating to 85-95° C., stirring and reacting for 2-4 days, cooling to room temperature, and then precipitating to obtain fluorinated cyclodextrin; S13. Assembly of nano-diagnostic and therapeutic agents: Compound 12 of S12 and compound 7 of S7 are dissolved in solvents respectively, and then the zinc porphyrin-coupled adamantane solution is slowly added dropwise to the fluorinated cyclodextrin solution under rapid stirring. After the addition, stirring is continued for 1-3 hours, and then the solvent is removed to obtain supramolecular self-assembled nano-micelles based on fluorinated cyclodextrin and porphyrin. 2. The method for preparing supramolecular self-assembled nanomicelles based on fluorinated cyclodextrin and porphyrin according to claim 1, characterized in that in S13, the dosage ratio of compound 12 to compound 7 is 3-5 mg:1-2 mg. 3. The method for preparing supramolecular self-assembled nanomicelles based on fluorinated cyclodextrin and porphyrin according to claim 1, characterized in that in S12, the amount ratio of compound 10 to compound 11 is 2-3g:4-6g. 4. The method for preparing a supramolecular self-assembled nanomicelle based on fluorinated cyclodextrin and porphyrin according to claim 1, characterized in that, in S11, the amount ratio of the 3,3,3-trifluoropropan-1-ol, anhydrous triethylamine and cyclic chloroethylene phosphate is 3-4g:3-4mL:4-5g. 5. The method for preparing a supramolecular self-assembled nanomicelle based on fluorinated cyclodextrin and porphyrin according to claim 1, characterized in that, in S10, the amount ratio of compound 9, dimethylallylamine and azobisisobutyronitrile is 1-2g:3-4g:1-2g. 6. The method for preparing supramolecular self-assembled nanomicelles based on fluorinated cyclodextrin and porphyrin according to claim 1, characterized in that in S9, the amount ratio of the compound 8, thiourea and sodium hydroxide is 4-6g:2-3g:2-3g. 7. The method for preparing supramolecular self-assembled nanomicelles based on fluorinated cyclodextrin and porphyrin according to claim 1, characterized in that, in S81 to S82, the amount ratio of triphenylphosphine, N-bromosuccinimide and β-cyclodextrin is 9-10g:6-7g:2-3g. 8. Supramolecular self-assembled nanomicelles based on fluorinated cyclodextrin and porphyrin prepared by the preparation method according to any one of claims 1 to 7. 9. Use of the supramolecular self-assembled nanomicelle based on fluorinated cyclodextrin and porphyrin according to claim 8 in the preparation of ¹⁹ F MRI contrast agent. 10. Use of the supramolecular self-assembled nanomicelle based on fluorinated cyclodextrin and porphyrin according to claim 8 in the preparation of anti-tumor drugs.