CN113004306A - Near-infrared two-region fluorescent molecule containing benzodithiadiazole, preparation method thereof, fluorescent nanoparticles, and preparation method and application thereof - Google Patents

Near-infrared two-region fluorescent molecule containing benzodithiadiazole, preparation method thereof, fluorescent nanoparticles, and preparation method and application thereof Download PDF

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CN113004306A
CN113004306A CN202110256220.0A CN202110256220A CN113004306A CN 113004306 A CN113004306 A CN 113004306A CN 202110256220 A CN202110256220 A CN 202110256220A CN 113004306 A CN113004306 A CN 113004306A
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孙跃
梁晓翠
高千词
闵雪红
赵兴霖
彭凯
荣誉
陶明洁
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South Central Minzu University
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Abstract

The invention provides a near-infrared two-region fluorescent molecule containing benzobithiadiazole, a preparation method thereof, a fluorescent nanoparticle, a preparation method and application thereof, and relates to the technical field of fluorescent materials. The near-infrared two-region fluorescent molecule provided by the invention has the characteristics of charge transfer in distorted molecules and aggregation-induced luminescence, can improve the quantum yield, effectively avoids the problem of fluorescence quenching, and has good fluorescence stability. The invention also provides a fluorescent nanoparticle which comprises a natural protein nanocage and a near-infrared fluorescent molecule encapsulated in a cavity of the natural protein nanocage.

Description

Near-infrared two-region fluorescent molecule containing benzodithiadiazole, preparation method thereof, fluorescent nanoparticles, and preparation method and application thereof
Technical Field
The invention relates to the technical field of fluorescent materials, in particular to a near-infrared two-region fluorescent molecule containing benzodithiadiazole, a preparation method thereof, a fluorescent nanoparticle, a preparation method and an application thereof.
Background
Near-infrared imaging has become an important tool for in vivo fluorescence imaging and image-guided surgery because of its advantages of high sensitivity, high spatial resolution, high signal-to-noise ratio (S/B), noninvasive control, deep tissue penetration, and real-time visualization. Especially, near infrared two-region (NIR-II) organic fluorescent nanoparticles have the excellent characteristics of clinical transformation, small cytotoxicity and easy preparation, and are researched more and more widely.
At present, most of organic fluorescent nanoparticles adopt organic fluorescent molecules, and fluorescence quenching effect is caused by strong intermolecular interaction, so that the fluorescence stability is low, and most of the organic fluorescent nanoparticles adopt artificially synthesized polymers (such as amphoteric polymers and triblock copolymers) as an encapsulation matrix, so that the biocompatibility of the organic fluorescent nanoparticles is poor, and the popularization and application of the organic fluorescent nanoparticles are limited.
Disclosure of Invention
In view of the above, the present invention aims to provide a near-infrared two-region fluorescent molecule containing benzodithiadiazole, a preparation method thereof, a fluorescent nanoparticle, a preparation method thereof, and applications thereof. The near-infrared two-region fluorescent molecule provided by the invention can effectively avoid the problem of fluorescence quenching, has good fluorescence stability, and forms fluorescent nanoparticles with good biocompatibility by taking a protein nanocage as an encapsulation matrix. In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a near-infrared two-region fluorescent molecule containing benzobithiadiazole, which has a structure shown in a formula I or a formula II:
Figure BDA0002967351150000021
the invention provides a preparation method of a near-infrared two-region fluorescent molecule containing benzodithiadiazole in the technical scheme, which comprises the following steps:
(1) under the anhydrous and oxygen-free conditions, carrying out Vilsmeier-Haack reaction on 4-bromotriphenylamine and phosphorus oxychloride in N, N-dimethylformamide, and separating by a chromatographic column to respectively obtain a compound shown in a formula III and a compound shown in a formula IV;
Figure BDA0002967351150000022
(2) carrying out Witting reaction on a compound shown as a formula III or a compound shown as a formula IV and (triphenylphosphine alkene) ethyl acetate in toluene under anhydrous and oxygen-free conditions to obtain a compound shown as a formula V or a compound shown as a formula VI;
Figure BDA0002967351150000023
(3) under the anhydrous and oxygen-free conditions, carrying out SuZuki coupling reaction on a compound shown as a formula V or a compound shown as a formula VI and pinacol diboron in dioxane under the action of tetrakis (triphenylphosphine) palladium and potassium acetate to obtain a compound shown as a formula VII or a compound shown as a formula VIII;
Figure BDA0002967351150000031
(4) under the oxygen-free condition, carrying out SuZuki coupling reaction on a compound shown as a formula VII or a compound shown as a formula VIII and 4, 7-dibromobenzo [1,2-C:4,5-C ] bis ([1,2,5] thiadiazole) in a mixed solvent of water and tetrahydrofuran under the action of tetrakis (triphenylphosphine) palladium and potassium carbonate to obtain the near-infrared fluorescent molecule with the structure shown as a formula I or a formula II.
Preferably, the mole ratio of the 4-bromotriphenylamine to the phosphorus oxychloride in the step (1) is 1: 7-11; the temperature of the Vilsmeier-Haack reaction is 95-105 ℃, and the time is 12-16 h.
Preferably, the molar ratio of the compound shown in the formula III in the step (2) to the ethyl (triphenylphosphine ene) acetate is 1: 2-3, wherein the molar ratio of the compound shown in the formula IV to ethyl (triphenylphosphine alkene) acetate is 1: 2-3; the temperature of the Witting reaction is room temperature, and the time is 48-60 hours.
Preferably, the molar ratio of the compound shown in the formula V in the step (3) to the sodium pinacol biborate, the palladium tetrakis (triphenylphosphine) and the potassium acetate is 37:58: 1-2: 100-110;
the molar ratio of the compound shown in the formula VI to the pinacol diboron, the tetrakis (triphenylphosphine) palladium and the potassium acetate is 37:58: 2-3: 100-110; the temperature of the SuZuki coupling reaction is 95-105 ℃, and the time is 12-16 h.
Preferably, the molar ratio of the compound shown in the formula VII in the step (4) to 4, 7-dibromobenzo [1,2-C:4,5-C ] bis ([1,2,5] thiadiazole), tetrakis (triphenylphosphine) palladium and potassium carbonate is 20: 6-9: 1: 60;
the molar ratio of a compound shown as a formula VIII to 4, 7-dibromobenzo [1,2-C:4,5-C ] bis ([1,2,5] thiadiazole), tetrakis (triphenylphosphine) palladium and potassium carbonate is 20: 8-12: 1: 60; the volume ratio of water to tetrahydrofuran in the mixed solvent is preferably 1: 5; the temperature of the SuZuki coupling reaction is 60-68 ℃, and the time is 48-60 h.
The invention provides a fluorescent nanoparticle, which comprises a protein nanocage and a near-infrared fluorescent molecule encapsulated in a cavity of the protein nanocage; the near-infrared fluorescent molecule is the near-infrared two-region fluorescent molecule containing the benzodithiadiazole according to the technical scheme or the near-infrared two-region fluorescent molecule containing the benzodithiadiazole prepared by the preparation method according to the technical scheme.
Preferably, the protein nanocage is a monkey virus-like particle; the mass ratio of the protein nano cage to the near-infrared fluorescent molecules is 3.5-5.5: 1.
the invention provides a preparation method of the fluorescent nano-particles in the technical scheme, which comprises the following steps:
dissolving the protein nano cage by using a depolymerization buffer solution to obtain a pentameric protein solution;
and adding the near-infrared fluorescent molecules into the pentameric protein solution, and dialyzing the obtained mixture in an assembly buffer solution to obtain the fluorescent nanoparticles.
Preferably, the depolymerization buffer comprises Tris-HCl, NaCl, EDTA, beta-mercaptoethanol and glycerol, the concentration of the Tris-HCl, the NaCl, the EDTA, the beta-mercaptoethanol and the glycerol in the depolymerization buffer is respectively 9.5-10 mmol/L, 195-200 mmol/L, 1.8-2 mmol/L, 28-30 mmol/L and 5-7.5 wt%, and the pH value of the depolymerization buffer is 8.6-8.8;
the assembly buffer solution comprises Tris-HCl and CaCl2NaCl and glycerol, the Tris-HCl, CaCl2The concentration of NaCl and glycerol in the assembly buffer solution is respectively 9-10 mmol/L, 1-1.2 mmol/L, 250-260 mmol/L and 5-7.5 wt%; the pH value of the assembly buffer solution is 7.0-7.3.
The invention provides application of the fluorescent nanoparticles in the technical scheme or the fluorescent nanoparticles prepared by the preparation method in the technical scheme in preparation of a blood vessel imaging agent or a fluorescent tracer.
The invention provides a near-infrared two-region fluorescent molecule containing benzobithiadiazole, which has a structure shown in a formula I or a formula II. The near-infrared two-region fluorescent molecule containing the benzodithiadiazole has the characteristics of distorted intramolecular charge transfer (TICT) and Aggregation Induced Emission (AIE), can improve the quantum yield, effectively avoids the problem of fluorescence quenching, and has good fluorescence stability; and can be encapsulated in native protein nanocages.
The invention provides a fluorescent nanoparticle, which comprises a protein nanocage and a near-infrared fluorescent molecule encapsulated in a cavity of the protein nanocage; the near-infrared fluorescent molecule is the near-infrared two-region fluorescent molecule containing the benzodithiadiazole in the technical scheme. The fluorescent nano-particles obtained by encapsulating the near-infrared fluorescent molecules by the protein nano-cage have the advantages of bright fluorescence, good fluorescence stability, high biocompatibility and uniform size, show excellent performance in-vivo blood vessel imaging and image-guided surgery, and can be widely used for preparing blood vessel imaging agents or fluorescent tracer agents.
The invention also provides a preparation method of the fluorescent nano-particles, which has the advantages of simple process, easy operation and convenient large-scale production.
Drawings
FIG. 1 is a schematic diagram of the process of forming fluorescent nanoparticles according to the present invention;
FIG. 2 is a hydrogen spectrum of the reaction product obtained in example 2;
FIG. 3 is a carbon spectrum of the reaction product obtained in example 2;
FIG. 4 is a mass spectrum of the reaction product obtained in example 2;
FIG. 5 is a TEM image and a distribution image of the particle size of the fluorescent nanoparticles obtained in example 3, wherein (a) in FIG. 5 is a TEM image of the fluorescent nanoparticles, and (b) is a distribution image of the particle size of the fluorescent nanoparticles;
FIG. 6 is a graph showing the fluorescence intensity changes of the fluorescent nanoparticles obtained in example 3 at three time points of 2min, 4h and 19h after incubation in water, phosphate buffer and fetal bovine serum, respectively;
FIG. 7 is a graph showing the effect of fluorescence brightness of the fluorescent nanoparticles obtained in example 3 in pure DMSO, a mixed solution of DMSO and water at a volume ratio of 1:1, and a phosphate buffer;
FIG. 8 is a graph showing the effect of the fluorescent nanoparticles obtained in example 3 on the cell viability of HeLa cells, MCF-7 cells, SKBR3 cells, Vero cells and HepG2 cells;
FIG. 9 is a graph showing the effect of injecting different concentrations of the fluorescent nanoparticles of example 3 and PBS solution on the body weight of mice;
FIG. 10 is a graph showing the effect of injecting different concentrations of the fluorescent nanoparticles of example 3 and PBS solution on the histopathology of mice;
FIG. 11 is an image of the blood vessel of the hind leg of a mouse after tail vein injection of the fluorescent nanoparticles of example 3, wherein (a) in FIG. 11 is a near infrared fluorescence map of the blood vessel, and (b) is a cross-sectional fluorescence distribution and Gaussian fitting distribution map of the blood vessel;
FIG. 12 is a graph showing the effect of the fluorescence of example 3 on the time course at the tumor site when the fluorescent nanoparticles were injected into the tumor site of a mouse, and in FIG. 12, (a) is a graph showing the change of the fluorescence of the tumor site with time, (b) is the tumor resection site in a bright field, (c) is a graph showing the fluorescence of the tumor site after resection, (d) is the main organ in a bright field, and (e) is a graph showing the fluorescence of the main organ;
fig. 13 is a tem image and a distribution diagram of the particle size of the fluorescent nanoparticles obtained in example 4, and (a) in fig. 13 is a tem image of the fluorescent nanoparticles, and (b) is a distribution diagram of the particle size of the fluorescent nanoparticles.
Detailed Description
The invention provides a near-infrared two-region fluorescent molecule containing benzobithiadiazole, which has a structure shown in a formula I or a formula II:
Figure BDA0002967351150000061
the near-infrared two-region fluorescent molecule containing the benzodithiadiazole has the characteristics of distorted intramolecular charge transfer (TICT) and Aggregation Induced Emission (AIE), can improve the quantum yield, effectively avoids the problem of fluorescence quenching, and has good fluorescence stability; and can be encapsulated in native protein nanocages.
The invention provides a preparation method of a near-infrared two-region fluorescent molecule containing benzodithiadiazole, which comprises the following steps:
(1) under the anhydrous and oxygen-free conditions, carrying out Vilsmeier-Haack reaction on 4-bromotriphenylamine and phosphorus oxychloride in N, N-dimethylformamide, and separating by a chromatographic column to respectively obtain a compound shown in a formula III and a compound shown in a formula IV;
Figure BDA0002967351150000062
(2) carrying out Witting reaction on a compound shown as a formula III or a compound shown as a formula IV and (triphenylphosphine alkene) ethyl acetate in toluene under anhydrous and oxygen-free conditions to obtain a compound shown as a formula V or a compound shown as a formula VI;
Figure BDA0002967351150000071
(3) under the anhydrous and oxygen-free conditions, carrying out SuZuki coupling reaction on a compound shown as a formula V or a compound shown as a formula VI and pinacol diboron in dioxane under the action of tetrakis (triphenylphosphine) palladium and potassium acetate to obtain a compound shown as a formula VII or a compound shown as a formula VIII;
Figure BDA0002967351150000072
(4) under the oxygen-free condition, carrying out SuZuki coupling reaction on a compound shown as a formula VII or a compound shown as a formula VIII and 4, 7-dibromobenzo [1,2-C:4,5-C ] bis ([1,2,5] thiadiazole) in a mixed solvent of water and tetrahydrofuran under the action of tetrakis (triphenylphosphine) palladium and potassium carbonate to obtain the near-infrared fluorescent molecule with the structure shown as a formula I or a formula II.
The invention carries out Vilsmeier-Haack reaction on 4-bromotriphenylamine and phosphorus oxychloride in N, N-dimethylformamide under anhydrous and anaerobic conditions, and the compound shown in the formula III and the compound shown in the formula IV are respectively obtained by separation of a chromatographic column. In the present invention, the molar ratio of the 4-bromotriphenylamine to the phosphorus oxychloride is preferably 1: 7-11; the N, N-dimethylformamide is used as a solvent, and the method has no special requirement on the dosage of the N, N-dimethylformamide and can ensure that the reaction is smoothly carried out; in order to ensure anhydrous and anaerobic conditions, the N, N-dimethylformamide is preferably subjected to oxygen removal and drying treatment before being added. In the invention, the temperature of the Vilsmeier-Haack reaction is preferably 95-105 ℃, more preferably 100-105 ℃, and the time is preferably 12-16 h, more preferably 12-15 h; the Vilsmeier-Haack reaction is preferably carried out with stirring.
In the present invention, the particular operation of the Vilsmeier-Haack reaction is preferably: adding 4-bromotriphenylamine into a three-neck flask with magnetons, vacuumizing the three-neck flask filled with the 4-bromotriphenylamine, and then sequentially adding N, N-dimethylformamide and phosphorus oxychloride into the three-neck flask under the condition of ice bath stirring; stirring the obtained mixed material liquid for 1 hour at room temperature, and then heating to 95-105 ℃ to carry out Vilsmeier-Haack reaction; the phosphorus oxychloride is preferably added dropwise, and the dropping rate is preferably 1 drop/second. After the Vilsmeier-Haack reaction is finished, preferably, the obtained reaction liquid is cooled to room temperature, then a proper amount of ice water is added, the obtained solution is neutralized to be neutral by NaOH solution (2mol/L) and then is filtered, and a solid crude product is obtained; dissolving the solid in dichloromethane, adding anhydrous sodium sulfate to remove water, filtering the obtained solution, and spin-drying by using a rotary evaporator; and (4) carrying out chromatographic column separation on the cyclone-dried product to respectively obtain a compound shown as a formula III and a compound shown as a formula IV. In the invention, the eluent used for the chromatographic column separation is preferably a mixed solution of dichloromethane and petroleum ether, and the volume ratio of the dichloromethane to the petroleum ether is preferably 2: 1; in the process of the chromatographic column separation, the compound shown in the formula III is firstly separated, and then the compound shown in the formula IV is separated. In the present invention, the Vilsmeier-Haack reaction has a reaction formula shown in formula 1:
Figure BDA0002967351150000081
the compound shown in the formula III or the compound shown in the formula IV and (triphenylphosphine alkene) ethyl acetate are subjected to Witting reaction in toluene under the anhydrous and oxygen-free conditions to obtain the compound shown in the formula V or the compound shown in the formula VI. In the present invention, the molar ratio of the compound represented by the formula iii to ethyl (triphenylphosphine ene) acetate is preferably 1: 2-3, and the molar ratio of the compound shown in the formula IV to the ethyl (triphenylphosphine ene) acetate is preferably 1: 2-3. In the invention, the toluene is used as a solvent, and the invention has no special requirement on the addition amount of the toluene and can ensure that the reaction is smoothly carried out; in order to ensure anhydrous and anaerobic conditions, the invention preferably performs the drying and oxygen removal treatment on the toluene before adding. In the invention, the temperature of the Witting reaction is preferably room temperature, the time is preferably 48-60 h, and more preferably 48-55 h; the Witting reaction is preferably carried out with stirring.
In the present invention, the Witting reaction is preferably performed by: adding a compound shown as a formula III or a compound shown as a formula IV and (triphenylphosphine alkene) ethyl acetate into a three-neck flask with magnetons, vacuumizing the three-neck flask, adding toluene, and carrying out a Witting reaction on the obtained mixed feed liquid at room temperature (i.e. without additional heating or cooling). After the Witting reaction is finished, the obtained Witting reaction liquid is preferably cooled to room temperature, and is subjected to chromatographic column separation after being dried by a rotary evaporator to obtain the compound shown in the formula V or the compound shown in the formula VI. In the present invention, the Witting reaction is represented by formula 2 or formula 3:
Figure BDA0002967351150000091
after obtaining the compound shown as the formula V and the compound shown as the formula VI, the compound shown as the formula V or the compound shown as the formula VI and the pinacol ester diborate are subjected to SuZuki coupling reaction in dioxane under the action of palladium tetrakis (triphenylphosphine) and potassium acetate to respectively obtain the compound shown as the formula VII or the compound shown as the formula VIII. In the invention, the molar ratio of the compound shown in the formula V to the pinacol ester diborate, the tetrakis (triphenylphosphine) palladium and the potassium acetate is preferably 37:58: 1-2: 100-110, and the molar ratio of the compound shown in the formula VI to the pinacol ester diborate, the tetrakis (triphenylphosphine) palladium and the potassium acetate is preferably 37:58: 2-3: 100-110; the potassium carbonate is preferably anhydrous potassium carbonate. In the invention, the dioxane is a reaction solvent, and the addition amount of the dioxane has no special requirement, so that the reaction can be carried out smoothly; to ensure anhydrous and anaerobic conditions, the dioxane is preferably subjected to oxygen removal and drying before being added. In the invention, the temperature of the SuZuki coupling reaction is preferably 95-105 ℃, more preferably 100 ℃, and the time is preferably 12-16 h, more preferably 12-15 h; the SuZuki coupling reaction is preferably carried out under stirring conditions.
In the present invention, the specific operation of the SuZuki coupling reaction is preferably: adding a compound shown as a formula V or a compound shown as a formula VI, pinacol diboron diboride, tetrakis (triphenylphosphine) palladium and potassium acetate into a three-neck flask with magnetons, vacuumizing the three-neck flask, adding dioxane into the three-neck flask, and heating to 95-105 ℃ to perform SuZuki coupling reaction. After the SuZuki coupling reaction is completed, the SuZuki coupling reaction liquid is preferably dried by a rotary evaporator and then is subjected to chromatographic column separation to obtain the compound shown as the formula VII or the compound shown as the formula VIII. In the present invention, the reaction formula of the SuZuki coupling reaction is represented by formula 4 or formula 5:
Figure BDA0002967351150000101
Figure BDA0002967351150000111
after the compound shown in the formula VII and the compound shown in the formula VIII are obtained, under the oxygen-free condition, the compound shown in the formula VII or the compound shown in the formula VIII and 4, 7-dibromobenzo [1,2-C:4,5-C ] bis ([1,2,5] thiadiazole) are subjected to SuZuki coupling reaction in a mixed solvent of water and tetrahydrofuran under the action of tetrakis (triphenylphosphine) palladium and potassium carbonate to obtain the near-infrared fluorescent molecule shown in the formula I or the formula II. In the invention, the molar ratio of the compound shown in the formula VII to 4, 7-dibromobenzo [1,2-C:4,5-C ] bis ([1,2,5] thiadiazole), tetrakis (triphenylphosphine) palladium and potassium carbonate is preferably 20: 6-9: 1:60, and the molar ratio of the compound shown in the formula VIII to 4, 7-dibromobenzo [1,2-C:4,5-C ] bis ([1,2,5] thiadiazole), tetrakis (triphenylphosphine) palladium and potassium carbonate is preferably 20: 8-12: 1: 60. In the present invention, the volume ratio of water to tetrahydrofuran in the mixed solvent is preferably 1: 5; the invention has no special requirement on the adding amount of the mixed solvent, and can ensure that the reaction is carried out smoothly; in order to ensure the oxygen-free condition, the mixed solvent is preferably subjected to oxygen removal treatment before being added. In the invention, the temperature of the SuZuki coupling reaction is preferably 60-68 ℃, more preferably 65-68 ℃, and the time is preferably 48-60 h, more preferably 48-55 h; the SuZuki coupling reaction is preferably carried out with stirring.
In the present invention, the specific operation of the SuZuki coupling reaction is preferably: adding a compound shown as a formula VII or a compound shown as a formula VIII, 4, 7-dibromobenzo [1,2-C:4,5-C ] bis ([1,2,5] thiadiazole), tetrakis (triphenylphosphine) palladium and potassium carbonate into a three-neck flask with magnetons, vacuumizing the three-neck flask, adding the mixed solvent, and heating to 60-68 ℃ to perform SuZuki coupling reaction. After the SuZuki coupling reaction is completed, the obtained coupling reaction solution is preferably evaporated to dryness by using a rotary evaporator and then subjected to chromatographic column separation to obtain the near-infrared fluorescent molecule with the structure shown in the formula I or the formula II. In the present invention, the reaction formula of the SuZuki coupling reaction is represented by formula 6 or formula 7:
Figure BDA0002967351150000121
the invention provides a fluorescent nanoparticle, which comprises a protein nanocage and a near-infrared fluorescent molecule encapsulated in a cavity of the protein nanocage; the near-infrared fluorescent molecule is the near-infrared two-region fluorescent molecule containing the benzodithiadiazole according to the technical scheme or the near-infrared two-region fluorescent molecule containing the benzodithiadiazole prepared by the preparation method according to the technical scheme. In the present invention, the protein nanocage is preferably a monkey virus-like particle (i.e. SV40 VLPS), which is a highly symmetric protein nanocage consisting of 60 subunits, with an outer diameter of 24nm and an inner diameter of 10nm (i.e. with a hollow structure); the mass ratio of the protein nano cage to the near-infrared fluorescent molecule is preferably 3.5-5.5: 1.
according to the invention, the protein nanocages are used for encapsulating the near-infrared fluorescent molecules, so that the obtained fluorescent nanoparticles are bright in fluorescence, good in fluorescence stability, and uniform in size, and have the advantages of biodegradability and biocompatibility.
The invention provides a preparation method of the fluorescent nano-particles in the technical scheme, which comprises the following steps:
dissolving the protein nano cage by using a depolymerization buffer solution to obtain a pentameric protein solution;
and adding the near-infrared fluorescent molecules into the pentameric protein solution, and dialyzing the obtained mixture in an assembly buffer solution to obtain the fluorescent nanoparticles.
The invention dissolves natural protein nano cage with depolymerization buffer solution to obtain pentameric protein solution. In the invention, the depolymerization buffer solution preferably comprises Tris-HCl, NaCl, EDTA, beta-mercaptoethanol and glycerol, the concentration of the Tris-HCl, the NaCl, the EDTA, the beta-mercaptoethanol and the glycerol in the depolymerization buffer solution is preferably 9.5-10 mmol/L, 195-200 mmol/L, 1.8-2 mmol/L, 28-30 mmol/L and 5-7.5 wt%, and the pH value of the depolymerization buffer solution is 8.6-8.8; the amount of the depolymerization buffer added is not particularly required, and the natural protein nanocages can be sufficiently dissolved. In the invention, the dissolving temperature is preferably 4 ℃, and the dissolving time is preferably 12 hours; the dissolution is preferably carried out under stirring. After the dissolution is finished, the obtained feed liquid is preferably centrifuged, and the obtained supernatant is the pentameric protein solution; the rotating speed of the centrifugation is preferably 50000rpm, and the time of the centrifugation is preferably 1 h. The invention dissolves natural protein nano cages by depolymerization buffer solution, and depolymerizes the natural protein nano cages into pentameric protein by the depolymerization buffer solution.
After the solution of the pentameric protein is obtained, the invention adopts the pentameric proteinAnd adding the near-infrared fluorescent molecules into the polymer protein solution, and dialyzing the obtained mixture in an assembly buffer solution to obtain the fluorescent nanoparticles. In the present invention, the assembly buffer preferably comprises Tris-HCl, CaCl2NaCl and glycerol, the Tris-HCl, CaCl2The concentration of NaCl and glycerol in the assembly buffer solution is preferably 9-10 mmol/L, 1-1.2 mmol/L, 250-260 mmol/L and 5-7.5 wt% respectively; the pH value of the assembly buffer solution is preferably 7.0-7.3, and more preferably 7.2. The present invention does not require any particular method for carrying out the dialysis, and a dialysis method known to those skilled in the art may be used. According to the invention, through dialysis of the obtained mixture in an assembly buffer solution, the pentameric protein can be self-assembled into spherical virus-like particles with uniform size, and meanwhile, the near-infrared fluorescent molecules are encapsulated in cavities of the virus-like particles. After the dialysis is finished, the invention also preferably purifies the reserved solution obtained by dialysis in sequence to obtain the fluorescent nanoparticles; the purification method is preferably sucrose cushion centrifugation, and the mass ratio of the sucrose cushion from top to bottom in the sucrose cushion centrifugation is preferably 15%: 25%: 50 percent, the temperature of the centrifugation is preferably 4 ℃, the rotating speed is preferably 38000rpm, and the time is preferably 1 h. In the present invention, the formation process of the fluorescent nanoparticles is shown in fig. 1.
The preparation method of the fluorescent nano-particles provided by the invention is simple in process, easy to operate and convenient for large-scale production.
The invention also provides the application of the fluorescent nano-particles in the technical scheme or the fluorescent nano-particles prepared by the preparation method in the technical scheme in the preparation of blood vessel imaging agents and fluorescent tracers. In the invention, the fluorescent nanoparticles use 808nm laser as an excitation light source, and the method for applying the fluorescent nanoparticles is not particularly required, and can be applied by a method well known to those skilled in the art. The fluorescent nano-particles provided by the invention show excellent performance in-vivo blood vessel imaging and fluorescence-labeled tumor resection, and can be widely used for preparing blood vessel imaging agents and fluorescence tracers.
The present invention provides a near-infrared two-region fluorescent molecule containing benzodithiadiazole and its preparation method, and fluorescent nanoparticles and their preparation method and application, which are described in detail below with reference to the following examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Preparation of a near-infrared two-region fluorescent molecule containing benzodithiadiazole of formula I:
(1) 11.5g of 4-bromotriphenylamine was added to a three-necked flask containing magnetons. Vacuumizing for three times, adding 80mL of dry N, N-dimethylformamide by using a constant-pressure separating funnel under the stirring of an ice bath, then slowly dripping 30mL of phosphorus oxychloride, stirring for 1h at normal temperature, raising the temperature to 95 ℃, continuing to react for 12h, cooling the obtained reaction solution to room temperature after the reaction is finished, adding 330mL of ice water, and then neutralizing by using 2mol/L of NaOH solution to be neutral. And (2) collecting the precipitate after suction filtration, dissolving the precipitate with dichloromethane, adding anhydrous sodium sulfate to remove water, filtering the solution, spin-drying the solution by using a rotary evaporator, and separating the spin-dried product by using a chromatographic column (the eluent is a mixed solution of dichloromethane and petroleum ether (the volume ratio is 2: 1)) to obtain a compound of the formula III and a compound of the formula IV.
(2) 2.15g of the compound of the formula III obtained in the step (1), 3.72g of ethyl (triphenylphosphine ene) acetate were put in a 200mL three-necked flask equipped with magnetons. Vacuumizing for three times, adding 100mL of deoxygenated toluene by using a syringe, stirring at room temperature for reaction for 48 hours, cooling to room temperature, performing spin-drying by using a rotary evaporator, and separating by using a chromatographic column to obtain the compound shown in the formula V.
(3) And (3) adding 1.56g of the compound shown in the formula V obtained in the step (2), 1.52g of pinacol diboron, 0.19g of tetrakis (triphenylphosphine) palladium and 1.02g of potassium acetate into a 150mL three-neck flask with magnetons, vacuumizing for three times, adding 65mL deoxygenated dioxane into the flask by using a syringe, reacting at the temperature of 100 ℃, stirring, performing reflux reaction for 12 hours, performing spin-drying by using a rotary evaporator, and separating by using a chromatographic column to obtain the compound shown in the formula VII.
(4) And (3) adding 1.21g of the compound shown in the formula VII obtained in the step (3), 0.36g of 4, 7-dibromobenzo [1,2-C:4,5-C ] bis ([1,2,5]) thiadiazole), 0.15g of tetrakis (triphenylphosphine) palladium and 1.07g of anhydrous potassium carbonate into a 150mL three-neck flask filled with magnetons, vacuumizing for three times, adding a mixed solvent of deoxygenated water and tetrahydrofuran (volume ratio of 1:5) by using an injector, reacting at 60 ℃, stirring, performing reflux reaction for 48 hours, then spin-drying by using a rotary evaporator, and separating by using a chromatographic column to obtain the near-infrared fluorescent molecule containing the benzodithiadiazole in the formula I.
Example 2
Preparation of a near-infrared two-region fluorescent molecule containing benzobisthiadiazole as shown in formula II:
(1) taking 1.34g of the compound shown in the formula IV obtained in the step (1) in the example 1, and 2.79g of ethyl (triphenylphosphine ene) acetate, adding the mixture into a 200mL three-neck flask filled with magnetons, vacuumizing for three times, adding 70mL of deoxygenated toluene by using a syringe, stirring and reacting at room temperature for 48 hours, cooling to room temperature, drying by using a rotary evaporator, and separating by using a chromatographic column to obtain the compound shown in the formula VI.
(2) And (3) adding 1.93g of the compound shown in the formula VI obtained in the step (1), 1.48g of pinacol diboron, 0.28g of tetrakis (triphenylphosphine) palladium and 1.09g of potassium acetate into a 150mL three-neck flask with magnetons, vacuumizing for three times, adding 60mL of deoxygenated dioxane into the flask by using an injector, reacting at the temperature of 100 ℃, stirring, performing reflux reaction for 12 hours, performing spin-drying by using a rotary evaporator, and separating by using a chromatographic column to obtain the compound shown in the formula VIII.
(3) Taking 1.10g of the compound shown in the formula VIII obtained in the step (2), 0.352g of 4, 7-dibromobenzo [1,2-C:4,5-C ] bis ([1,2,5]) thiodiazole), 0.112g of tetrakis (triphenylphosphine) palladium and 0.802g of anhydrous potassium carbonate, adding the mixture into a 150mL three-neck flask filled with magnetons, vacuumizing the three times, adding a mixed solvent of deoxygenated water and tetrahydrofuran (in a volume ratio of 1:5) into the mixture by using an injector, stirring the mixture at the reaction temperature of 60 ℃, carrying out reflux reaction for 48 hours, carrying out spin-drying by using a rotary evaporator, and separating the mixture by using a chromatographic column to obtain a reaction product, namely the near-infrared fluorescent molecule with the structure shown in the formula II.
5mg of the reaction product obtained in example 2 was dissolved in 600. mu.L of deuterated chloroform, and the resulting solution was measured by a 400MHz NMR spectrometer to obtain a hydrogen spectrum of the reaction product, as shown in FIG. 2.
10mg of the reaction product obtained in example 2 was dissolved in 600. mu.L of deuterated chloroform and tested by a 400MHz NMR spectrometer to obtain a carbon spectrum of the reaction product, as shown in FIG. 3.
0.5mg of the reaction product of example 2 was dissolved in 100. mu.L of methanol, and the molecular weight was measured by a mass spectrometer to obtain a mass spectrum of the reaction product, as shown in FIG. 4.
As is clear from FIGS. 2 to 4, the reaction product obtained in example 2 has a structure corresponding to that shown in formula II.
Example 3
A fluorescent nanoparticle is prepared by the following steps:
dissolving 15mg of monkey virus-like particles in a disaggregation buffer (10mM Tris-HCl, 200mM NaCl, 2mM EDTA, 30mM beta-mercaptoethanol and 5 wt% glycerol, pH 8.8), slowly stirring overnight at 4 ℃, then centrifuging at 50000rpm at 4 ℃ for 1h, and taking the supernatant to obtain a pentamer protein solution; to a solution of the pentameric protein 1.62mg of the fluorescent molecule of formula I prepared in example 1 in dimethyl sulfoxide was added and the resulting mixture was placed in assembly buffer (10mM Tris-HCl, 1mM CaCl)2250mM NaCl and 5 wt% glycerol, pH 7.2), and passing the retentate obtained by dialysis through a sucrose cushion (from top to bottom, mass ratio 15%: 25%: 50 percent) and centrifuging at 38000rpm for 1h at the temperature of 4 ℃ to obtain fluorescent nano particles; the mass ratio of the protein nanocages to the near-infrared fluorescent molecules in the fluorescent nanoparticles is 5: 1, the quantum yield was found to be 13.03%.
Dropping the fluorescent nanoparticle solution obtained in example 3 on a copper mesh, incubating for 2min, then negatively staining for 2min with 20 μ L of 2.0% phosphotungstate, and observing the sample with a Hitachi H7000 transmission electron microscope; meanwhile, characterizing the particle size of the particles by using a Malvern particle sizer; the test results are shown in fig. 5, in which (a) is a projection electron microscope image of the fluorescent nanoparticles and (b) is a particle size distribution diagram of the fluorescent nanoparticles in fig. 5. As can be seen from fig. 5, the fluorescent nanoparticles prepared in example 3 were spherical and uniform in particle size, and the number of particles at 23.2nm was the largest.
The fluorescent nanoparticles obtained in example 3 were incubated in Water (Water), Phosphate Buffered Saline (PBS) and Fetal Bovine Serum (FBS), respectively, and their fluorescence intensities were measured at three time points of 2min, 4h and 19h, respectively, at an excitation wavelength of 808nm, and the results are shown in fig. 6. As can be seen from FIG. 6, the fluorescence intensity at different time points is not substantially changed, indicating that the fluorescent nanoparticles have good fluorescence stability.
The fluorescence brightness of the fluorescent nanoparticles obtained in example 3 in pure dimethylsulfoxide, a mixed solution of dimethylsulfoxide and water at a volume ratio of 1:1, and a phosphate buffer was measured at an excitation wavelength of 808nm, and the measurement results are shown in fig. 7. As can be seen from fig. 7, the fluorescent nanoparticles showed weak NIR-II fluorescence in pure dimethyl sulfoxide; in a mixed solution of dimethyl sulfoxide and water in a volume ratio of 1:1, although fluorescence is very strong, the fluorescence is not uniform; the fluorescent nanoparticles fluoresce strongly and uniformly in phosphate buffer.
Incubation of HeLa cells, MCF-7 cells, SKBR3 cells, Vero cells and HepG2 cells with the fluorescent nanoparticle solutions of example 3 at different concentrations (0. mu.M, 5. mu.M, 10. mu.M and 20. mu.M), respectively, and setting 4 sets of parallel experiments; after 24h, the viability of the cells was checked using the CCK-8 kit. The test results are shown in fig. 8. As can be seen from FIG. 8, the fluorescent nanoparticles have very low toxicity to HeLa cells, MCF-7 cells, SKBR3 cells, Vero cells and HepG2 cells, and even the concentration of the fluorescent nanoparticles reaches 20 μm, each cell still has vigorous cell activity.
Set up 3 groups of mouse experiments, injecting 45.6 μ M, 22.8 μ M of the fluorescent nanoparticle in PBS solution of example 3 and the same volume (100 μ L) of PBS solution as a control, and monitor the body weight of the mouse for 30 days; at 30 days, mice were dissected and histopathological analysis was performed on the mouse heart, liver, spleen, lung and kidney. The test results are shown in fig. 9 and 10, respectively, fig. 9 is a graph of the effect of injecting fluorescent nanoparticles with different concentrations and PBS solution on the body weight of mice, fig. 10 is a graph of the effect of injecting fluorescent nanoparticles with different concentrations and PBS solution on the histopathology of mice, in fig. 9-10, "Low" indicates injecting 22.8 μ M fluorescent nanoparticles, "High" indicates injecting 45.6 μ M fluorescent nanoparticles, and "CH 1-SV40 dots" indicates fluorescent nanoparticles. As can be seen from fig. 9, the body weight change tendency of the mice in the experimental group and the control group was substantially the same, and as can be seen from fig. 10, no pathology occurred in the heart, liver, spleen, lung, and kidney of the mice at 30 days after the injection of the fluorescent nanoparticles at different concentrations.
As can be seen from fig. 8 to 10, the fluorescent nanoparticles obtained in example 3 have good biocompatibility.
The fluorescent nanoparticles of example 3 (100 μ L, 450mM) were injected into mice via the tail vein, immediately followed by hind leg vascular imaging at an excitation wavelength of 808nm, and the fluorescence imaging signal-to-noise ratio was calculated by fluorescence measurement and gaussian simulation, and the results of the measurement are shown in fig. 11, in which (a) is a vascular near-infrared fluorescence map and (b) is a vascular cross-sectional fluorescence distribution and gaussian fit distribution map. As can be seen from FIG. 11, the fluorescent nanoparticles of example 3 were used to image the blood vessels of the hind legs of mice, and the signal-to-noise ratio obtained by fluorescence measurement and Gaussian simulation was 10.88, which is much higher than most of the current near-infrared nano-fluorescent materials.
The fluorescent nanoparticles of example 3 (500 μ M, 20 μ L) were injected into mouse tumor sites, followed by monitoring the change in fluorescence at the tumor sites over time, and the results of the test are shown in fig. 12, where all imaging was performed at an excitation wavelength of 808nm, and in fig. 12, (a) is a graph of the change in fluorescence at the tumor sites over time, (b) is a tumor resection site in a bright field, (c) is a graph of the fluorescence at the tumor sites after resection, (d) is a main organ in a bright field, and (e) is a graph of the fluorescence of the main organ. As can be seen from fig. 12, the fluorescence does not diffuse within 5 hours and is always present at the tumor site, while the fluorescence is not monitored in the surrounding normal tissues, and the tumor can be successfully resected under the tracing of the fluorescence, which indicates that the fluorescent nanoparticles obtained in example 3 exhibit excellent performance in the tumor resection with the tracing of the fluorescence.
Example 4
A fluorescent nanoparticle is prepared by the following steps:
dissolving 15mg of monkey virus-like particles in a disaggregation buffer (10mM Tris-HCl, 200mM NaCl, 2mM EDTA, 30mM beta-mercaptoethanol and 5 wt% glycerol, pH 8.8), slowly stirring overnight at 4 ℃, then centrifuging at 50000rpm at 4 ℃ for 1h, and taking the supernatant to obtain a pentamer protein solution; adding the solution of pentameric protein dissolved in dimethyl sulfoxide1.98mg of the fluorescent molecule of formula II prepared in example 2, the resulting mixture was placed in assembly buffer (10mM Tris-HCl, 1mM CaCl)2250mM NaCl and 5 wt% glycerol, pH 7.2), and passing the retentate obtained by dialysis through a sucrose cushion (from top to bottom, mass ratio 15%: 25%: 50 percent) and centrifuging at 38000rpm for 1h at the temperature of 4 ℃ to obtain fluorescent nano particles; the mass ratio of the protein nanocages to the near-infrared fluorescent molecules in the fluorescent nanoparticles is 4: 1, the quantum yield was found to be 4.2%.
Dropping the fluorescent nanoparticle solution obtained in example 4 on a copper mesh, incubating for 2 minutes, then carrying out negative staining for 2 minutes by using 20 mu L of 2.0% phosphotungstate, and observing a sample by using a Hitachi H7000 transmission electron microscope; meanwhile, the particle size of the particles is characterized by using a Malvern particle sizer. As can be seen from fig. 13, in fig. 13, (a) is a tem image of the fluorescent nanoparticles, and (b) is a distribution graph of the particle size of the fluorescent nanoparticles. As can be seen from fig. 13, the fluorescent nanoparticles prepared in example 4 were spherical and uniform in particle size, and the number of particles at 23.4nm was the largest.
The fluorescent nanoparticles obtained in example 4 were incubated in water, phosphate buffer and fetal bovine serum, respectively, and their fluorescence intensities were measured at three time points of 2min, 4h and 19h, respectively, at an excitation wavelength of 808nm, with the following results: the fluorescence intensity at different time points is basically unchanged, which shows that the fluorescent nanoparticles have good fluorescence stability.
The fluorescence brightness of the fluorescent nanoparticles obtained in example 4 in pure dimethyl sulfoxide, a mixed solution of dimethyl sulfoxide and water in a volume ratio of 1:1, and a phosphate buffer solution was tested at an excitation wavelength of 808nm, and the test results were as follows: the fluorescent nanoparticles show weak NIR-II fluorescence in pure dimethyl sulfoxide; in a mixed solution of dimethyl sulfoxide and water in a volume ratio of 1:1, although fluorescence is very strong, the fluorescence is not uniform; the fluorescent nanoparticles fluoresce strongly and uniformly in phosphate buffer.
Incubation of HeLa cells, MCF-7 cells, SKBR3 cells, Vero cells and HepG2 cells with the fluorescent nanoparticle solutions of example 4 at different concentrations (0. mu.M, 5. mu.M, 10. mu.M and 20. mu.M), respectively, and setting 4 sets of parallel experiments; after 24h, the viability of the cells was checked using the CCK-8 kit. And (3) testing results: the fluorescent nanoparticles have low toxicity to HeLa cells, MCF-7 cells, SKBR3 cells, Vero cells and HepG2 cells, and even the concentration of the fluorescent nanoparticles reaches 20 mu m, each cell still has vigorous cell activity.
Set up 3 groups of mouse experiments, injecting 45.6 μ M, 22.8 μ M of the fluorescent nanoparticle in PBS solution of example 4 and the same volume (100 μ L) of PBS solution as a control, and monitor the body weight of the mouse for 30 days; at 30 days, mice were dissected and histopathological analysis was performed on the mouse heart, liver, spleen, lung and kidney. And (3) testing results: by injecting fluorescent nanoparticles at different concentrations, no pathology appeared in the heart, liver, spleen, lung and kidney of the mice at 30 days.
The fluorescent nanoparticles of example 4 (100 μ L, 450mM) were injected into mice via the tail vein, immediately followed by hind leg vascular imaging at an excitation wavelength of 808nm, and the fluorescence imaging signal-to-noise ratio was calculated by fluorescence measurement and gaussian simulation and tested to give a signal-to-noise ratio of 4.8.
Fluorescent nanoparticles of example 4 (500 μ M, 20 μ L) were injected into mouse tumor sites, followed by monitoring the change in fluorescence at the tumor sites over time (imaging was performed at an excitation wavelength of 808 nm). And (3) testing results: fluorescence is not diffused within 5 hours and is always in the tumor part, and the surrounding normal tissues do not monitor fluorescence, so that the tumor can be successfully resected under the tracing of the fluorescence, which shows that the fluorescent nanoparticles obtained in example 4 have excellent performance in the tumor resection of the fluorescence tracing.
The embodiments show that the fluorescent nanoparticles obtained by encapsulating the near-infrared fluorescent molecules with the structure shown in formula I or formula II by using the natural protein nanocages as the template have the advantages of bright fluorescence, good fluorescence stability, high biocompatibility, uniform size and excellent performance in-vivo blood vessel imaging and image-guided surgery.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (10)

1. The near-infrared two-region fluorescent molecule containing benzo-bi-thiadiazole is characterized by having a structure shown in a formula I or a formula II:
Figure FDA0002967351140000011
2. the method for preparing the near-infrared two-region fluorescent molecule containing the benzodithiadiazole of claim 1, which is characterized by comprising the following steps:
(1) carrying out Vilsmeier-Haack reaction on 4-bromotriphenylamine and phosphorus oxychloride in N, N-dimethylformamide under anhydrous and oxygen-free conditions, and separating by a chromatographic column to obtain a compound shown in a formula III and a compound shown in a formula IV;
Figure FDA0002967351140000012
(2) carrying out Witting reaction on a compound shown as a formula III or a compound shown as a formula IV and (triphenylphosphine alkene) ethyl acetate in toluene under anhydrous and oxygen-free conditions to obtain a compound shown as a formula V or a compound shown as a formula VI;
Figure FDA0002967351140000013
(3) under the anhydrous and oxygen-free conditions, carrying out SuZuki coupling reaction on a compound shown as a formula V or a compound shown as a formula VI and pinacol diboron in dioxane under the action of tetrakis (triphenylphosphine) palladium and potassium acetate to obtain a compound shown as a formula VII or a compound shown as a formula VIII;
Figure FDA0002967351140000021
(4) under the oxygen-free condition, carrying out SuZuki coupling reaction on a compound shown as a formula VII or a compound shown as a formula VIII and 4, 7-dibromobenzo [1,2-C:4,5-C ] bis ([1,2,5] thiadiazole) in a mixed solvent of water and tetrahydrofuran under the action of tetrakis (triphenylphosphine) palladium and potassium carbonate to obtain the near-infrared fluorescent molecule with the structure shown as a formula I or a formula II.
3. The preparation method according to claim 2, wherein the molar ratio of 4-bromotriphenylamine to phosphorus oxychloride in the step (1) is 1: 7-11; the temperature of the Vilsmeier-Haack reaction is 95-105 ℃, and the time is 12-16 h;
the molar ratio of the compound shown in the formula III in the step (2) to the ethyl (triphenylphosphine ene) acetate is 1: 2-3, wherein the molar ratio of the compound shown in the formula IV to ethyl (triphenylphosphine alkene) acetate is 1: 2-3; the temperature of the Witting reaction is room temperature, and the time is 48-60 hours.
4. The preparation method according to claim 2, characterized in that the molar ratio of the compound shown in formula V in the step (3) to the pinacol ester diboron, tetrakis (triphenylphosphine) palladium and potassium acetate is 37:58: 1-2: 100-110;
the molar ratio of the compound shown in the formula VI to the pinacol diboron, the tetrakis (triphenylphosphine) palladium and the potassium acetate is 37:58: 2-3: 100-110; the temperature of the SuZuki coupling reaction is 95-105 ℃, and the time is 12-16 h.
5. The preparation method according to claim 2, wherein the molar ratio of the compound shown in the formula VII in the step (4) to 4, 7-dibromobenzo [1,2-C:4,5-C ] bis ([1,2,5] thiadiazole), tetrakis (triphenylphosphine) palladium and potassium carbonate is 20: 6-9: 1: 60;
the molar ratio of a compound shown as a formula VIII to 4, 7-dibromobenzo [1,2-C:4,5-C ] bis ([1,2,5] thiadiazole), tetrakis (triphenylphosphine) palladium and potassium carbonate is 20: 8-12: 1: 60; the volume ratio of water to tetrahydrofuran in the mixed solvent is preferably 1: 5; the temperature of the SuZuki coupling reaction is 60-68 ℃, and the time is 48-60 h.
6. A fluorescent nanoparticle comprising a protein nanocage and a near-infrared fluorescent molecule encapsulated in a cavity of the protein nanocage; the near-infrared fluorescent molecule is the near-infrared two-region fluorescent molecule containing the benzodithiadiazole according to claim 1 or the near-infrared two-region fluorescent molecule containing the benzodithiadiazole prepared by the preparation method according to any one of claims 2 to 5.
7. The fluorescent nanoparticle of claim 6, wherein the protein nanocage is a monkey virus-like particle; the mass ratio of the protein nano cage to the near-infrared fluorescent molecules is 3.5-5.5: 1.
8. the method for preparing fluorescent nanoparticles according to claim 6 or 7, comprising the steps of:
dissolving the protein nano cage by using a depolymerization buffer solution to obtain a pentameric protein solution;
and adding the near-infrared fluorescent molecules into the pentameric protein solution, and dialyzing the obtained mixture in an assembly buffer solution to obtain the fluorescent nanoparticles.
9. The method according to claim 8, wherein the depolymerization buffer comprises Tris-HCl, NaCl, EDTA, beta-mercaptoethanol and glycerol, the concentration of the Tris-HCl, NaCl, EDTA, beta-mercaptoethanol and glycerol in the depolymerization buffer is 9.5 to 10mmol/L, 195 to 200mmol/L, 1.8 to 2mmol/L, 28 to 30mmol/L and 5 to 7.5 wt%, respectively, and the pH value of the depolymerization buffer is 8.6 to 8.8;
the assembly buffer solution comprises Tris-HCl and CaCl2NaCl and glycerol, the Tris-HCl, CaCl2The concentration of NaCl and glycerol in the assembly buffer solution is respectively 9-10 mmol/L, 1-1.2 mmol/L, 250-260 mmol/L and 5-7.5 wt%; the pH value of the assembly buffer solution is 7.0 to7.3。
10. Use of the fluorescent nanoparticle according to any one of claims 6 to 7 or the fluorescent nanoparticle prepared by the preparation method according to any one of claims 8 to 9 in preparation of a blood vessel imaging agent or a fluorescent tracer.
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