A nano-fluorescent temperature indicator, its preparation method and application

Abstract

The present invention provides a nano-fluorescent temperature indicator and its preparation method and application, belonging to the technical fields of polymer materials and biomedical engineering. The nano-fluorescent temperature indicator of the present invention is composed of a diblock dual-temperature-responsive fluorescent polymer. The nano-fluorescent temperature indicator includes a temperature-responsive fluorescent polymer A with a polar-responsive fluorescent molecule modified at the end and a lower critical solution temperature of 40 °C, and a temperature-responsive fluorescent polymer B with a non-polar-responsive fluorescent molecule that can be excited by the temperature-responsive fluorescent polymer A modified at the end and a lower critical solution temperature of 46 °C. The nano-fluorescent temperature indicator has no fluorescence when the ambient temperature is lower than 40 °C, shows green fluorescence when the temperature rises to 40 °C - 46 °C, and shows red fluorescence when the temperature is higher than 46 °C. The nano-fluorescent temperature indicator has good applications in temperature detection of cells or sub-cells and real-time temperature measurement and temperature control of local hyperthermia.

Description

Technical Field

[0001] The present invention belongs to the technical fields of polymer materials and biomedical engineering, and particularly relates to a nano-fluorescent temperature indicator, a preparation method thereof, and an application thereof. Background Art

[0002] At present, non-destructive detection and monitoring of the temperature and temperature field distribution of organisms are the difficulties in clinical hyperthermia research. Traditional temperature measurement methods mainly include thermocouples, thermistors, fiber optic temperature sensors, etc. Their advantages are that the technology is mature and the temperature measurement is accurate. However, the above temperature measurement methods belong to destructive temperature measurement. Not only is surface tissue required during temperature measurement, but they can only reflect the temperature at the temperature measurement point and cannot comprehensively feedback the temperature change and temperature field distribution of the hyperthermia target area. Based on the demand for non-destructive temperature measurement in clinical hyperthermia, the most commonly used non-destructive temperature measurement method in clinical practice is infrared imaging temperature measurement. Infrared imaging temperature measurement can relatively comprehensively feedback the temperature change and temperature field distribution of the hyperthermia target area, but it is easily affected by the temperature of the measured object itself and the ambient temperature, so the accuracy of temperature measurement is poor.

[0003] To solve the above problems, existing research has developed a variety of nano-thermometers that can use fluorescence changes for non-destructive temperature measurement, including quantum dots, rare earth metal nanoparticles, temperature-sensitive fluorescent nanogels, etc. These nano-thermometers reflect the height and distribution of temperature by constructing the correlation between the fluorescence signal and temperature, such as the change in the intensity of the fluorescence emission wavelength related to temperature change or the shift (red shift or blue shift) of the fluorescence emission wavelength. Through the recognition of the fluorescence signal by a high-precision detection device, this kind of nano-thermometer can achieve highly sensitive non-destructive measurement of temperature. However, the above nano-thermometers are not suitable for the temperature measurement and monitoring of tumor mild hyperthermia.

[0004] Since the temperature monitoring of clinical tumor hyperthermia has certain particularities, that is, the hyperthermia temperature within a certain range (between 40°C and 46°C) can be regarded as effective, lower than this temperature is regarded as ineffective, and higher than this temperature range is considered overheating, which may cause other serious side effects. Therefore, the monitoring of the temperature level and temperature field distribution of clinical tumor hyperthermia is not only a single temperature measurement, but more importantly, it is necessary to effectively distinguish ineffective hyperthermia (<40°C), effective hyperthermia (40°C - 46°C), and excessive hyperthermia (>46°C), and the existing temperature measurement technologies cannot meet the above requirements. Therefore, it is necessary to construct a temperature indicator that can meet the temperature requirements of clinical hyperthermia and realize real-time monitoring of the temperature change and temperature field distribution during the hyperthermia process. Summary of the Invention

[0005] In view of some deficiencies existing in the prior art, the present invention provides a nano-fluorescent temperature indicator, a preparation method thereof, and an application thereof. The present invention is based on a two-block dual thermosensitive fluorescent polymer to form a nano-fluorescent temperature indicator. The nano-fluorescent temperature indicator includes a thermosensitive fluorescent polymer A with a polar-responsive fluorescent molecule modified at the end and a lower critical solution temperature of 40 °C, and a thermosensitive fluorescent polymer B with a non-polar-responsive fluorescent molecule that can be excited by the thermosensitive fluorescent polymer A modified at the end and a lower critical solution temperature of 46 °C. The nano-fluorescent temperature indicator has no fluorescence when the ambient temperature is lower than 40 °C, shows green fluorescence when the temperature rises to 40 °C - 46 °C, and shows red fluorescence when the temperature is higher than 46 °C. The nano-fluorescent temperature indicator has good applications in temperature detection of cells or sub-cells and real-time temperature measurement and temperature control of local hyperthermia.

[0006] In order to achieve the above technical objectives, the present invention adopts the following technical means:

[0007] The present invention first provides a nano-fluorescent temperature indicator, which is a two-block dual thermosensitive fluorescent polymer obtained by coupling a thermosensitive fluorescent polymer A and a thermosensitive fluorescent polymer B modified with maleimide.

[0008] The thermosensitive fluorescent polymer A is composed of a thermosensitive polymer with a carboxyl group at the end and a lower critical solution temperature of 40 °C and a hydroxyl group-modified polar-responsive fluorescent molecule.

[0009] The thermosensitive fluorescent polymer B is composed of a thermosensitive polymer with a carboxyl group at the end and a lower critical solution temperature of 46 °C and a maleimide-modified non-polar-responsive fluorescent molecule that can be excited by the thermosensitive fluorescent polymer A.

[0010] Preferably, under an excitation wavelength of 400 nm - 450 nm, when the ambient temperature is lower than 40 °C, the nano-fluorescent temperature indicator presents purple of the excitation wavelength.

[0011] When the ambient temperature is between 40 °C and 46 °C, the nano-fluorescent temperature indicator presents green emission light.

[0012] When the ambient temperature is higher than 46 °C, the nano-fluorescent temperature indicator presents red emission light.

[0013] Preferably, the hydroxyl group-modified polar-responsive fluorescent molecule includes nitrobenzofuran hydroxyl derivative (NBD-OH), N,N-dimethylaminosulfonylbenzofuran hydroxyl derivative (DBD-OH), 1-anilino-8-naphthalenesulfonic acid hydroxyl derivative (ANS-OH), aminosulfonylbenzofuran hydroxyl derivative (ABD-OH), or 4-N,N-dimethylamino-1,8-naphthalimide hydroxyl derivative (4-DMN-OH).

[0014] Preferably, the non-polar responsive fluorescent molecules that can be excited by the temperature-sensitive fluorescent polymer A modified with maleimide include sulforhodamine B maleimide derivative (Sulfo-RhB-Mal), Texas Red maleimide derivative, sulfonated cyanine 3 maleimide derivative of anthocyanin (Sulfo-Cy3-Mal), or sulfonated cyanine 3.5 maleimide derivative of anthocyanin (Sulfo-Cy3.5-Mal).

[0015] The present invention also provides a preparation method of the above-mentioned nano-fluorescent temperature indicator, which specifically includes the following steps:

[0016] (1) Preparation of the temperature-sensitive fluorescent polymer A:

[0017] A chain transfer agent containing a carboxyl group is used to prepare a temperature-sensitive polymer A with a lower critical solution temperature of 40 °C through reversible addition-fragmentation chain transfer (RAFT) polymerization reaction; through an esterification reaction, a polar responsive fluorescent molecule modified with a hydroxyl group is coupled to the carboxyl end of the temperature-sensitive polymer A to obtain the temperature-sensitive fluorescent polymer A.

[0018] The specific preparation steps are as follows: A chain transfer agent containing a carboxyl group, a temperature-sensitive monomer, and a regulating monomer are added to a reaction solvent, stirred and dissolved at room temperature, and then the air is replaced with argon to ensure that the temperature is raised to 60 °C - 80 °C under an anaerobic condition for a reversible addition-fragmentation chain transfer (RAFT) polymerization reaction. After the reaction, a temperature-sensitive polymer A with a lower critical solution temperature of 40 °C is obtained;

[0019] The temperature-sensitive polymer A and the polar responsive fluorescent molecule modified with a hydroxyl group are dissolved in an anhydrous solvent for an esterification reaction. After the reaction, the temperature-sensitive fluorescent polymer A is obtained.

[0020] (2) Preparation of the temperature-sensitive fluorescent polymer B:

[0021] A chain transfer agent containing a carboxyl group is selected, and then a temperature-sensitive polymer B with a lower critical solution temperature of 46 °C is prepared by using a reversible addition-fragmentation chain transfer (RAFT) polymerization reaction; both ends of the temperature-sensitive polymer B are a carboxyl group and a thioester respectively;

[0022] Through click chemistry, the thioester at the end of the temperature-sensitive polymer B is coupled with a non-polar responsive fluorescent molecule modified with maleimide that can be excited by the temperature-sensitive fluorescent polymer A to obtain the temperature-sensitive fluorescent polymer B.

[0023] The specific preparation steps are as follows: The preparation method of the thermosensitive fluorescent polymer B includes: adding a chain transfer agent containing a carboxyl group, a thermosensitive monomer, and a regulating monomer into a solvent, stirring and dissolving at room temperature, deoxygenating according to the deoxygenation method of the thermosensitive polymer A, and then heating to 60°C to 80°C for reversible addition-fragmentation chain transfer (RAFT) polymerization reaction. After the reaction ends, a thermosensitive polymer B with a lower critical solution temperature of 46°C and carboxyl and thioester groups at both ends is obtained.

[0024] Dissolve the thermosensitive polymer B and a non-polar responsive fluorescent molecule modified with maleimide and excited by the thermosensitive fluorescent polymer A in a solvent, deoxygenate by passing argon gas, and then carry out a click chemical reaction. After the reaction ends, the thermosensitive fluorescent polymer B is obtained.

[0025] (3) Preparation of maleimide-modified thermosensitive fluorescent polymer B:

[0026] Using amidation reaction, couple maleimide containing an amino group to the carboxyl end of the thermosensitive fluorescent polymer B to obtain maleimide-modified thermosensitive fluorescent polymer B.

[0027] The specific steps are as follows: Add maleimide containing an amino group and the thermosensitive fluorescent polymer B into a reaction solvent, and carry out an amidation reaction at room temperature to obtain maleimide-modified thermosensitive fluorescent polymer B.

[0028] (4) Preparation of the nano-fluorescent temperature indicator:

[0029] Using click chemical reaction, react the thermosensitive fluorescent polymer A with the maleimide-modified thermosensitive fluorescent polymer B to obtain a two-block double thermosensitive fluorescent polymer, that is, the nano-fluorescent temperature indicator.

[0030] The specific steps of step (4) are as follows: Add the thermosensitive fluorescent polymer A and the maleimide-modified thermosensitive fluorescent polymer B into a reaction medium, fully deoxygenate, and then carry out a click chemical reaction at room temperature, stir and react for 4 to 168 hours to obtain a two-block double thermosensitive fluorescent polymer, that is, the nano-fluorescent temperature indicator.

[0031] Preferably, in steps (1) and (2), the chain transfer agent containing a carboxyl group includes 2-(dodecylthiosulfonylthio)-2-methylpropanoic acid, 3-benzylmercaptothiocarbonylpropanoic acid, 2-(ethylthiosulfonylthio)-2-methylpropanoic acid, 4-cyano-4-(dodecylsulfanylthiosulfonyl)pentanoic acid, S-(thiobenzoyl)thioacetic acid, or 4-cyano-4-(thiobenzoylthio)pentanoic acid;

[0032] The conditions of the reversible addition-fragmentation chain transfer polymerization reaction are all under anaerobic conditions, stirring and reacting at 60°C to 80°C for 12 to 48 hours.

[0033] Preferably, in step (1), the temperature-sensitive monomer includes N-n-propylacrylamide (NNPAM, 21 °C), N-isopropylacrylamide (NIPAM, 32 °C), N-vinylcaprolactam (VCL, 30 °C - 32 °C);

[0034] The regulating monomer includes N,N-dimethylacrylamide (DMAM);

[0035] The molecular weight of the temperature-sensitive polymer A is 20,000 - 50,000, and the molecular weight distribution is 1.0 - 1.2.

[0036] Preferably, in step (1), the molar ratio of the carboxyl-containing chain transfer agent, the temperature-sensitive monomer, and the regulating monomer is 1:300:60 - 1:1000:500;

[0037] The molar ratio of the temperature-sensitive polymer A to the hydroxyl-modified polar responsive fluorescent molecule is 1:1 - 1:100;

[0038] The conditions for the esterification reaction are stirring and reacting for 2 - 72 hours at 10 °C - 50 °C under anhydrous conditions.

[0039] Preferably, in step (2), the temperature-sensitive monomer includes N-isopropylacrylamide (NIPAM, 32 °C), N-vinylcaprolactam (VCL, 30 °C - 32 °C), methyl vinyl ether (MVE, 37 °C), or N-isopropylmethacrylamide (NIPMAM, 46 °C);

[0040] The regulating monomer includes N,N-dimethylacrylamide (DMAM);

[0041] The molecular weight of the temperature-sensitive polymer B is 60,000 - 100,000, and the molecular weight distribution is 1.0 - 1.2.

[0042] Preferably, in step (2), the molar ratio of the carboxyl-containing chain transfer agent, the temperature-sensitive monomer, and the regulating monomer is 1:1200:0 - 1:2500:1000;

[0043] The molar ratio of the temperature-sensitive polymer B to the maleimide-modified non-polar responsive fluorescent molecule that can be excited by the temperature-sensitive fluorescent polymer A is 1:1 - 1:100;

[0044] The conditions for the click reaction are stirring and reacting for 1 - 72 hours at room temperature under anaerobic conditions.

[0045] Preferably, in step (3), the maleimide containing an amino group includes N-(2-aminopropyl) maleimide, N-(2-aminoethyl) maleimide, or N-(4-aminophenyl) maleimide;

[0046] The mass ratio of the amino-containing maleimide to the thermosensitive fluorescent polymer B is 1:1 to 50:1;

[0047] The conditions for the amidation reaction are to react at room temperature for 1 to 72 hours.

[0048] Preferably, in step (4), the dosage ratio of the thermosensitive fluorescent polymer A to the maleimide-modified thermosensitive fluorescent polymer B is 1:2 to 1:200;

[0049] The reaction conditions are an anaerobic condition. Stir and react at room temperature for 12 to 168 hours, and then dialyze using a dialysis bag with a molecular weight cut-off of 500,000 to 1,000,000 at 42 to 44 °C to remove the excessive maleimide-modified thermosensitive fluorescent polymer B.

[0050] The present invention also provides the application of the above-mentioned nano-fluorescent temperature indicator in the temperature detection of cells or sub-cells.

[0051] The present invention also provides the application of the above-mentioned nano-fluorescent temperature indicator in the real-time temperature measurement and temperature control of local hyperthermia. Compared with the prior art, the beneficial effects of the present invention are as follows:

[0052] Compared with using a single fluorescent dye, the present invention uses two different organic fluorescent dyes, which can make full use of the properties of the polarity-responsive fluorescent dye and can also utilize the FRET effect between the two dyes, thereby realizing various fluorescence changes. Compared with the existing thermosensitive polymers, the present invention uses a two-block thermosensitive polymer with two different phase transition points, so that a single polymer chain can characterize the changes in two temperature segments. The nano-fluorescent temperature indicator of the present invention combines different thermosensitive segments with fluorescent dyes with different fluorescence activation mechanisms, so that a single two-block thermosensitive polymer can respectively connect different phase transition temperatures with the fluorescence response behaviors of polar fluorescence response and polar fluorescence tandem FRET, thereby realizing the changes in the fluorescence emission wavelength and fluorescence emission intensity in different temperature segments, that is, the change in fluorescence color in visual observation, so as to achieve the purpose of monitoring different temperature ranges.

[0053] The excitation wavelength of the nano-fluorescent temperature indicator described in the present invention is in the range of 400 nm to 450 nm. When the ambient temperature of the nano-fluorescent temperature indicator is lower than 40 °C, excited by the excitation wave of 400 nm to 450 nm, the nano-fluorescent temperature indicator presents the purple color of the excitation wave. When the ambient temperature of the nano-fluorescent temperature indicator is in the range of 40 °C to 46 °C, excited by the excitation wave of 400 nm to 450 nm, the nano-fluorescent temperature indicator presents green emission light. When the ambient temperature of the nano-fluorescent temperature indicator is higher than 46 °C, excited by the excitation wave of 400 nm to 450 nm, the nano-fluorescent temperature indicator presents red emission light. Therefore, it can be used as a nano-fluorescent thermometer to extend the detection of cell or subcellular temperature to the real-time temperature measurement and temperature control of local hyperthermia, while improving the curative effect of local tumor hyperthermia, avoiding the adverse reactions and harm to the human body caused by high fever.

[0054] The preparation method of the nano-fluorescent temperature indicator described in the present invention is simple, does not require special equipment, operates under normal temperature and pressure, has strong controllability and good repeatability. Description of the Drawings

[0055] Figure 1 Schematic diagram of the structure and function of the nano-fluorescent temperature indicator prepared in Example 1.

[0056] Figure 2 Excitation spectra and emission spectra of the temperature-sensitive fluorescent polymer A and the temperature-sensitive fluorescent polymer B prepared in Example 2.

[0057] Figure 3 Graph of the transmittance of the temperature-sensitive fluorescent polymer A, the temperature-sensitive fluorescent polymer B, and the diblock double temperature-sensitive fluorescent polymer prepared in Example 3 varying with temperature.

[0058] Figure 4 Photos of the nano-fluorescent temperature indicator prepared in Example 4 under different temperature conditions under ultraviolet light irradiation. Detailed Embodiments

[0059] The present invention will be further described below in conjunction with the drawings and specific embodiments, but the protection scope of the present invention is not limited thereto.

[0060] Example 1: Preparation of Nano-Fluorescent Temperature Indicator

[0061] (1) Preparation of temperature-sensitive fluorescent polymer A:

[0062] In this reaction, 3-benzylmercapto thiocarbonyl propionic acid is used as a chain transfer agent, N-isopropylacrylamide (NIPAM) is used as a temperature-sensitive monomer, and N,N-dimethylacrylamide (DMAM) is used as a regulating monomer to prepare a temperature-sensitive polymer P(NIPAM-co-DMAM) with a lower critical solution temperature of 40 °C. The specific steps are as follows:

[0063] Using dimethyl sulfoxide as the reaction medium, 3-benzylmercapto thiocarbonyl propionic acid (1 mmol), N-isopropylacrylamide (NIPAM, 300 mmol), and N,N-dimethylacrylamide (DMAM, 60 mmol) were added in a molar ratio of 1:300:60. After evacuating and replacing with argon three times, the reaction was carried out at 60 °C for 12 hours under argon protection to obtain a thermosensitive polymer P(NIPAM-co-DMAM) with a lower critical solution temperature of 40 °C, a molecular weight of 20,800, and a molecular weight distribution of 1.02.

[0064] The nitrobenzofuran hydroxyl derivative (NBD-OH) and P(NIPAM-co-DMAM) were added to anhydrous dimethyl sulfoxide in a molar ratio of 100:1, and stirred at 50 °C under anhydrous conditions for 2 hours to obtain a thermosensitive fluorescent polymer A, denoted as NBD-P(NIPAM-co-DMAM).

[0065] (2) Preparation of thermosensitive fluorescent polymer B:

[0066] In this reaction, 2-(ethylmercapto thiocarbonylthio)-2-methylpropionic acid was used as a chain transfer agent, and N-isopropylmethacrylamide (NIPMAM) was used as a thermosensitive monomer to prepare a thermosensitive polymer PNIPMAM with a lower critical solution temperature of 46 °C. The specific steps are as follows:

[0067] Using dioxane as the reaction solvent, 2-(ethylmercapto thiocarbonylthio)-2-methylpropionic acid (0.5 mmol) and N-isopropylmethacrylamide (NIPMAM, 600 mmol) were added in a molar ratio of 1:1200. After purging with high-purity argon for 30 minutes, the temperature was raised to 80 °C under argon protection, and the reaction was carried out for 12 hours to obtain a thermosensitive polymer PNIPMAM with a lower critical solution temperature of 46 °C, a molecular weight of 61,000, and a molecular weight distribution of 1.04.

[0068] The sulfonated rhodamine B maleimide derivative (Sulfo-RhB-Mal) and PNIPMAM were added to N,N-dimethylformamide in a molar ratio of 1:1. After evacuating and replacing with argon three times, the reaction was stirred under argon protection for 72 hours to obtain a thermosensitive fluorescent polymer B, denoted as PNIPMAM-Sulfo-RhB.

[0069] (3) Preparation of maleimide-modified thermosensitive fluorescent polymer B:

[0070] Using dimethyl sulfoxide as the reaction medium, N-(2-aminopropyl) maleimide and thermosensitive fluorescent polymer B were reacted at a molar ratio of 1:1. The reaction conditions were room temperature and a reaction time of 70 hours to obtain thermosensitive fluorescent polymer B modified with maleimide, denoted as Mal-PNIPMAM-Sulfo-RhB.

[0071] (4) Preparation of nano-fluorescent temperature indicator:

[0072] NBD-P(NIPAM-co-DMAM) and Mal-PNIPMAM-Sulfo-RhB were added to dioxane at a molar ratio of 1:2, and high-purity argon was introduced to remove oxygen for 30 minutes. Then, under argon protection, the reaction was carried out at room temperature for 160 hours. Subsequently, dialysis was performed using a dialysis bag with a molecular weight cut-off of 500,000 at 42 °C to obtain a diblock dual-thermosensitive fluorescent polymer, namely the nano-fluorescent temperature indicator, denoted as NBD-P(NIPAM-co-DMAM)-b-PNIPMAM-Sulfo-RhB.

[0073] Figure 1 The structure and functional schematic diagram of the nano-fluorescent temperature indicator prepared in this example are shown. It can be seen from the figure that when the temperature is below 40 °C, the nano-fluorescent temperature indicator is randomly dispersed in water, and at this time, it shows the purple color of the excitation wave when excited with an excitation wavelength of 400 nm - 450 nm; when the temperature rises to 40 °C, the nano-fluorescent temperature indicator becomes amphiphilic, and the polar-responsive fluorescent molecules are located in the hydrophobic core, and at this time, it shows green when excited with an excitation wavelength of 400 nm - 450 nm; when the temperature rises above 46 °C, the nano-fluorescent temperature indicator completely forms micelles, and at this time, it shows red when excited with an excitation wavelength of 400 nm - 450 nm.

[0074] In this example, the segmented indication of the above-prepared nano-fluorescent temperature indicator for the hyperthermia temperature range was also investigated. The specific investigation steps are as follows:

[0075] The above nano-fluorescent temperature indicator was dispersed in water to a concentration of 5 mg / mL, and then the temperature was raised starting from 36 °C. The color of the nano-fluorescent temperature indicator aqueous solution was observed at an excitation wavelength of 410 nm every 1 °C. When the ambient temperature is below 40 °C, the nano-fluorescent temperature indicator aqueous solution shows the purple color of the excitation wave; when the ambient temperature is between 40 °C and 45 °C, the nano-fluorescent temperature indicator aqueous solution shows green; when the ambient temperature reaches or is higher than 46 °C, the nano-fluorescent temperature indicator aqueous solution shows red. This indicates that the prepared nano-fluorescent temperature indicator can achieve segmented indication of the hyperthermia temperature range.

[0076] Example 2: Preparation of nano-fluorescent temperature indicator

[0077] (1) Preparation of thermosensitive fluorescent polymer A:

[0078] In this reaction, 2-(dodecylthiosulfonylthio) - 2-methylpropanoic acid was used as a chain transfer agent, N-n-propylacrylamide (NNPAM) as a thermosensitive monomer, and N,N-dimethylacrylamide (DMAM) as a regulating monomer to prepare a thermosensitive polymer P(NNPAM-co-DMAM) with a lower critical solution temperature of 40 °C. The specific steps are as follows:

[0079] Using dimethyl sulfoxide as the reaction medium, 2-(dodecylthiosulfonylthio) - 2-methylpropanoic acid (1 mmol), N-n-propylacrylamide (NNPAM, 1000 mmol), and N,N-dimethylacrylamide (DMAM, 500 mmol) were added according to a molar ratio of 1:1000:500. After evacuation / argon replacement three times, under argon protection, the reaction was carried out at 70 °C for 24 hours to obtain a thermosensitive polymer P(NNPAM-co-DMAM) with a lower critical solution temperature of 40 °C, a molecular weight of 49500, and a molecular weight distribution of 1.19.

[0080] N,N-dimethylaminosulfonylbenzofuran hydroxyl derivative (DBD-OH) and P(NNPAM-co-DMAM) were added to anhydrous dimethyl sulfoxide according to a molar ratio of 70:1 and vigorously stirred at 20 °C for 20 hours to obtain a thermosensitive fluorescent polymer A, denoted as DBD-P(NNPAM-co-DMAM).

[0081] (2) Preparation of thermosensitive fluorescent polymer B:

[0082] In this reaction, 4-cyano-4-(dodecylsulfanylthio) - 4-(thiosulfonylthio) pentanoic acid was used as a chain transfer agent, N-isopropylacrylamide (NIPAM) as a thermosensitive monomer, and N,N-dimethylacrylamide (DMAM) as a regulating monomer to prepare a thermosensitive polymer P(NIPAM-co-DMAM) with a lower critical solution temperature of 46 °C. The specific steps are as follows:

[0083] Using dioxane as the reaction solvent, 4-cyano-4-(dodecylsulfanylthio) - 4-(thiosulfonylthio) pentanoic acid (0.5 mmol), N-isopropylacrylamide (NIPAM, 1250 mmol), and N,N-dimethylacrylamide (DMAM, 500 mmol) were added according to a molar ratio of 1:2500:1000. After purging with high-purity argon for 40 minutes, under argon protection, the temperature was raised to 70 °C and the reaction was carried out for 28 hours to obtain a thermosensitive polymer P(NIPAM-co-DMAM) with a lower critical solution temperature of 46 °C, a molecular weight of 99800, and a molecular weight distribution of 1.2.

[0084] Then, Texas Red maleimide and P(NIPAM-co-DMAM) were added to N,N-dimethylformamide at a molar ratio of 1:50. After evacuating and replacing with argon three times, the mixture was stirred under argon protection for 34 hours to obtain a temperature-sensitive fluorescent polymer B, denoted as P(NIPAM-co-DMAM)-TR.

[0085] (3) Preparation of maleimide-modified temperature-sensitive fluorescent polymer B:

[0086] Using dimethyl sulfoxide as the reaction medium, N-(2-aminoethyl) maleimide and temperature-sensitive fluorescent polymer B were reacted at a molar ratio of 20:1. The reaction conditions were room temperature and a reaction time of 50 hours to obtain maleimide-modified temperature-sensitive fluorescent polymer B, denoted as Mal-P(NIPAM-co-DMAM)-TR.

[0087] (4) Preparation of nano-fluorescent temperature indicator:

[0088] DBD-P(NNPAM-co-DMAM) and Mal-P(NIPAM-co-DMAM)-TR were added to dioxane at a molar ratio of 1:50. High-purity argon was introduced to remove oxygen for 35 minutes, and then the mixture was reacted at room temperature for 100 hours under argon protection. Then, dialysis was carried out using a dialysis bag with a molecular weight cut-off of 1,000,000 at 42 °C to obtain a diblock dual-temperature-sensitive fluorescent polymer, namely the nano-fluorescent temperature indicator, denoted as DBD-P(NNPAM-co-DMAM)-b-P(NIPAM-co-DMAM)-TR.

[0089] Figure 2 The excitation and emission spectra of the temperature-sensitive fluorescent polymer A and the temperature-sensitive fluorescent polymer B in this example are shown. It can be seen from the figure that an excitation wavelength of 400 nm to 450 nm can excite the temperature-sensitive fluorescent polymer A but not the temperature-sensitive fluorescent polymer B; there is a large overlap between the emission spectrum of the temperature-sensitive fluorescent polymer A and the excitation spectrum of the temperature-sensitive fluorescent polymer B, and fluorescence resonance energy transfer can occur between them. In this example, the segmented indication of the above-prepared nano-fluorescent temperature indicator for the hyperthermia temperature range was also investigated. The specific investigation steps are as follows:

[0090] Disperse the above-mentioned nano-fluorescent temperature indicator in water to a concentration of 5 mg / mL, and then start heating from 36 °C. Observe the color of the nano-fluorescent temperature indicator aqueous solution at an excitation wavelength of 410 nm every 1 °C. When the ambient temperature is lower than 40 °C, the nano-fluorescent temperature indicator aqueous solution shows the purple color of the excitation wave. When the ambient temperature is between 40 °C and 45 °C, the nano-fluorescent temperature indicator aqueous solution shows green. When the ambient temperature reaches or is higher than 46 °C, the nano-fluorescent temperature indicator aqueous solution shows red. This shows that the prepared nano-fluorescent temperature indicator can achieve segmented indication of the hyperthermia temperature range.

[0091] Example 3: Preparation of nano-fluorescent temperature indicator

[0092] (1) Preparation of thermosensitive fluorescent polymer A:

[0093] In this reaction, S-(thiobenzoylthio) acetic acid is used as a chain transfer agent, N-vinylcaprolactam (VCL) is used as a thermosensitive monomer, and N,N-dimethylacrylamide (DMAM) is used as a regulating monomer to prepare a thermosensitive polymer P(VCL-co-DMAM) with a lower critical solution temperature of 40 °C. The specific steps are as follows:

[0094] Using N,N-dimethylformamide as the reaction medium, add S-(thiobenzoylthio) acetic acid (1 mmol), N-vinylcaprolactam (VCL, 700 mmol), and N,N-dimethylacrylamide (DMAM, 160 mmol) according to a molar ratio of 1:700:160. After evacuating / argon displacement 3 times, under argon protection, react at 70 °C for 35 hours to obtain a thermosensitive polymer P(VCL-co-DMAM) with a lower critical solution temperature of 40 °C, a molecular weight of 41900, and a molecular weight distribution of 1.16.

[0095] Add the sulfamoylbenzofuran hydroxyl derivative (ABD-OH) and P(VCL-co-DMAM) to anhydrous dimethyl sulfoxide according to a molar ratio of 70:1, and stir at 30 °C under anhydrous conditions for 36 hours to obtain a thermosensitive fluorescent polymer A, denoted as ABD-P(VCL-co-DMAM).

[0096] (2) Preparation of thermosensitive fluorescent polymer B:

[0097] In this reaction, 4-cyano-4-(thiobenzoylthio) pentanoic acid is used as a chain transfer agent, N-vinylcaprolactam (VCL) is used as a thermosensitive monomer, and N,N-dimethylacrylamide (DMAM) is used as a regulating monomer to prepare a thermosensitive polymer P(VCL-co-DMAM) with a lower critical solution temperature of 46 °C. The specific steps are as follows:

[0098] Using dioxane as the reaction solvent, 4-cyano-4-(thiobenzoylthio) pentanoic acid (0.5 mmol), N-vinylcaprolactam (VCL, 750 mmol), and N,N-dimethylacrylamide (DMAM, 250 mmol) were added in a molar ratio of 1:1500:500. After purging with high-purity argon for 45 minutes, the temperature was raised to 80 °C under argon protection and reacted for 33 hours to obtain a thermosensitive polymer P(VCL-co-DMAM) with a lower critical solution temperature of 46 °C, a molecular weight of 70500, and a molecular weight distribution of 1.12.

[0099] The anthocyanin sulfonated Cy3 maleimide derivative (Sulfo-Cy3-Mal) and P(VCL-co-DMAM) were added to N,N-dimethylformamide in a molar ratio of 30:1. After evacuating / argon displacement three times, the mixture was stirred and reacted for 24 hours under argon protection to obtain a thermosensitive fluorescent polymer B, denoted as P(VCL-co-DMAM)-Sulfo-Cy3.

[0100] (3) Preparation of maleimide-modified thermosensitive fluorescent polymer B:

[0101] Using dimethyl sulfoxide as the reaction medium, N-(4-aminophenyl) maleimide and thermosensitive fluorescent polymer B were reacted in a molar ratio of 35:1. The reaction conditions were room temperature and a reaction time of 45 hours to obtain maleimide-modified thermosensitive fluorescent polymer B, denoted as Mal-P(VCL-co-DMAM)-Sulfo-Cy3.

[0102] (4) Preparation of nano-fluorescent temperature indicator:

[0103] ABD-P(VCL-co-DMAM) and Mal-P(VCL-co-DMAM)-Sulfo-Cy3 were added to dioxane in a molar ratio of 1:120. After purging with high-purity argon to remove oxygen for 50 minutes, the mixture was reacted at room temperature for 24 hours under argon protection, and then dialyzed using a dialysis bag with a molecular weight cut-off of 1000000 at 42 °C to obtain a diblock dual-thermosensitive fluorescent polymer, namely the nano-fluorescent temperature indicator, denoted as ABD-P(VCL-co-DMAM)-b-P(VCL-co-DMAM)-Sulfo-Cy3.

[0104] Figure 3The transmittance-temperature change graphs of the temperature-sensitive fluorescent polymer A, temperature-sensitive fluorescent polymer B, and diblock dual-temperature-sensitive fluorescent polymer prepared in this example are shown. It can be seen from the figure that the transmittances of the temperature-sensitive fluorescent polymer A, temperature-sensitive fluorescent polymer B, and diblock dual-temperature-sensitive fluorescent polymer all decrease with the increase of temperature. According to the change curves, the lower critical solution temperatures of the temperature-sensitive fluorescent polymer A and temperature-sensitive fluorescent polymer B are 40 °C and 46 °C respectively. At the same time, there are two inflection points in the diblock dual-temperature-sensitive fluorescent polymer, indicating that the diblock dual-temperature-sensitive fluorescent polymer has two lower critical solution temperatures of 40 °C and 46 °C.

[0105] In this example, the segmented indication of the above-prepared nano-fluorescent temperature indicator for the hyperthermia temperature range was also investigated. The specific investigation steps are as follows:

[0106] The above nano-fluorescent temperature indicator was dispersed in water to a concentration of 5 mg / mL, and then the temperature was raised starting from 36 °C. The color of the nano-fluorescent temperature indicator aqueous solution was observed every 1 °C at an excitation wavelength of 410 nm. When the ambient temperature is below 40 °C, the nano-fluorescent temperature indicator aqueous solution shows the purple color of the excitation wave. When the ambient temperature is between 40 °C and 45 °C, the nano-fluorescent temperature indicator aqueous solution shows green. When the ambient temperature reaches or is higher than 46 °C, the nano-fluorescent temperature indicator aqueous solution shows red. This shows that the prepared nano-fluorescent temperature indicator can achieve segmented indication of the hyperthermia temperature range.

[0107] Example 4: Preparation of nano-fluorescent temperature indicator

[0108] (1) Preparation of temperature-sensitive fluorescent polymer A:

[0109] In this reaction, 2-(dodecylthiosulfonylthio) - 2-methylpropionic acid was used as a chain transfer agent, N-isopropylacrylamide (NIPAM) as a temperature-sensitive monomer, and N,N-dimethylacrylamide (DMAM) as a regulating monomer to prepare a temperature-sensitive polymer P(NIPAM-co-DMAM) with a lower critical solution temperature of 40 °C. The specific steps are as follows:

[0110] Using dimethyl sulfoxide as a reaction medium, 2-(dodecylthiosulfonylthio) - 2-methylpropionic acid (1 mmol), N-isopropylacrylamide (NIPAM, 500 mmol), and N,N-dimethylacrylamide (DMAM, 100 mmol) were added according to a molar ratio of 1:500:100. After evacuating / argon replacement 3 times, under argon protection, the reaction was carried out at 80 °C for 24 hours to obtain a temperature-sensitive polymer P(NIPAM-co-DMAM) with a lower critical solution temperature of 40 °C, a molecular weight of 30300, and a molecular weight distribution of 1.08.

[0111] 1-Anilino-8-naphthalenesulfonic acid hydroxy derivative (ANS-OH) and P(NIPAM-co-DMAM) were added to anhydrous dimethyl sulfoxide in a molar ratio of 1:1, and stirred at 50 °C under anhydrous conditions for 72 hours to obtain a thermosensitive fluorescent polymer A, denoted as ANS-P(NIPAM-co-DMAM).

[0112] (2) Preparation of thermosensitive fluorescent polymer B:

[0113] In this reaction, 2-(dodecylthiosulfonylthio)propionic acid was used as a chain transfer agent, methyl vinyl ether (MVE) was used as a thermosensitive monomer, and N,N-dimethylacrylamide (DMAM) was used as a regulating monomer to prepare a thermosensitive polymer P(MVE-co-DMAM) with a lower critical solution temperature of 46 °C. The specific steps were as follows:

[0114] Dioxane was used as the reaction solvent. According to the molar ratio of 1:1200:400, 2-(dodecylthiosulfonylthio)propionic acid (0.5 mmol), methyl vinyl ether (MVE, 600 mmol), and N,N-dimethylacrylamide (DMAM, 200 mmol) were added. After purging with high-purity argon for 60 minutes, the temperature was raised to 75 °C under argon protection and reacted for 48 hours to obtain a thermosensitive polymer P(MVE-co-DMAM) with a lower critical solution temperature of 46 °C, a molecular weight of 82100, and a molecular weight distribution of 1.18.

[0115] The mass ratio of the thermosensitive polymer B to the non-polar responsive fluorescent molecule modified with maleimide and excited by the thermosensitive fluorescent polymer A was 1:1 to 1:100; the conditions for the click reaction were stirring reaction for 1 to 72 hours at room temperature under anaerobic conditions.

[0116] Anthocyanin sulfonated Cy3.5 maleimide derivative (Sulfo-Cy3.5-Mal) and P(MVE-co-DMAM) were added to N,N-dimethylformamide in a molar ratio of 100:1. After evacuation / argon replacement for 3 times, the reaction was stirred for 1 hour under argon protection to obtain a thermosensitive fluorescent polymer B, denoted as P(MVE-co-DMAM)-Sulfo-Cy3.5.

[0117] (3) Preparation of maleimide-modified thermosensitive fluorescent polymer B:

[0118] The molar ratio of maleimide containing an amino group to the thermosensitive fluorescent polymer B was 1:1 to 50:1;

[0119] The conditions for the amidation reaction were reaction at room temperature for 1 to 72 hours.

[0120] Using dimethyl sulfoxide as the reaction medium, N-(2-aminopropyl) maleimide and thermosensitive fluorescent polymer B were reacted at a molar ratio of 50:1. The reaction conditions were room temperature and a reaction time of 1 hour to obtain maleimide-modified thermosensitive fluorescent polymer B, denoted as Mal-P(MVE-co-DMAM)-Sulfo-Cy3.5.

[0121] (4) Preparation of the nano-fluorescent temperature indicator:

[0122] ANS-P(NIPAM-co-DMAM) and Mal-P(MVE-co-DMAM)-Sulfo-Cy3.5 were added to dioxane at a molar ratio of 1:200, and high-purity argon was introduced to remove oxygen for 60 minutes. Then, under argon protection, the reaction was carried out at room temperature for 12 hours. Subsequently, dialysis was performed using a dialysis bag with a molecular weight cut-off of 1,000,000 at 42 °C to obtain a diblock dual-thermosensitive fluorescent polymer, i.e., the nano-fluorescent temperature indicator, denoted as ANS-P(NIPAM-co-DMAM)-b-P(MVE-co-DMAM)-Sulfo-Cy3.5.

[0123] Figure 4 This is a photo of the nano-fluorescent temperature indicator prepared in this example under ultraviolet light irradiation at different temperatures. It can be seen from the figure that the excitation wavelength of the nano-fluorescent temperature indicator is in the range of 400 nm - 450 nm. When the environmental temperature of the nano-fluorescent temperature indicator is lower than 40 °C and excited by an excitation wavelength of 400 nm - 450 nm, the nano-fluorescent temperature indicator shows the purple color of the excitation wave. When the environmental temperature of the nano-fluorescent temperature indicator is in the range of 40 °C - 46 °C and excited by an excitation wavelength of 400 nm - 450 nm, the nano-fluorescent temperature indicator shows green emission light. When the environmental temperature of the nano-fluorescent temperature indicator is higher than 46 °C and excited by an excitation wavelength of 400 nm - 450 nm, the nano-fluorescent temperature indicator shows red emission light. Therefore, it can be used as a nano-fluorescent thermometer to extend the temperature detection of cells or sub-cells to real-time temperature measurement and temperature control in local hyperthermia, while improving the efficacy of tumor local hyperthermia and avoiding adverse reactions and harm to the human body caused by high fever.

[0124] In this example, the segmented indication of the nano-fluorescent temperature indicator prepared above for the hyperthermia temperature range was also investigated. The specific investigation steps are as follows:

[0125] Disperse the above-mentioned nano-fluorescent temperature indicator in water to make the concentration 5 mg / mL, and then start heating from 36 °C. Observe the color of the nano-fluorescent temperature indicator aqueous solution at an excitation wavelength of 410 nm every 1 °C. When the ambient temperature is lower than 40 °C, the nano-fluorescent temperature indicator aqueous solution presents the purple of the excitation wave. When the ambient temperature is between 40 °C and 45 °C, the nano-fluorescent temperature indicator aqueous solution presents green. When the ambient temperature reaches or is higher than 46 °C, the nano-fluorescent temperature indicator aqueous solution presents red. This shows that the prepared nano-fluorescent temperature indicator can achieve segmented indication of the hyperthermia temperature range.

[0126] In summary, the present invention is based on a two-block dual-thermosensitive fluorescent polymer to form a nano-fluorescent temperature indicator. The nano-fluorescent temperature indicator includes a thermosensitive fluorescent polymer A with a polar-responsive fluorescent molecule modified at the end and a lower critical solution temperature of 40 °C, and a thermosensitive fluorescent polymer B with a non-polar-responsive fluorescent molecule that can be excited by the thermosensitive fluorescent polymer A modified at the end and a lower critical solution temperature of 46 °C; the nano-fluorescent temperature indicator has no fluorescence when the ambient temperature is lower than 40 °C, presents green fluorescence when the temperature rises to 40 °C - 45 °C, and presents red fluorescence when the temperature is higher than 46 °C; the nano-fluorescent temperature indicator has good applications in temperature detection of cells or sub-cells and real-time temperature measurement and temperature control of local hyperthermia.

[0127] The above embodiments are the preferred embodiments of the present invention, but the present invention is not limited to the above embodiments. Without departing from the substantial content of the present invention, any obvious improvements, substitutions or variations that those skilled in the art can make all belong to the protection scope of the present invention.

Claims

1. A nano-fluorescent temperature indicator, characterized in that, The nano-fluorescent temperature indicator is a two-block dual thermosensitive fluorescent polymer obtained by coupling a thermosensitive fluorescent polymer A and a thermosensitive fluorescent polymer B modified with maleimide; The thermosensitive fluorescent polymer A is obtained by coupling a thermosensitive polymer a with a carboxyl-terminated and a lower critical solution temperature of 40 °C and a polar-responsive fluorescent molecule modified with a hydroxyl group; The polar-responsive fluorescent molecule includes any one of nitrobenzofuran hydroxyl derivatives, 1-aniline-8-naphthalene sulfonic acid hydroxyl derivatives, aminosulfonylbenzofuran hydroxyl derivatives, or 4-N,N-dimethylamino-1,8-naphthalimide hydroxyl derivatives; The thermosensitive fluorescent polymer B is obtained by coupling a thermosensitive polymer b with a carboxyl-terminated and a lower critical solution temperature of 46 °C and a non-polar-responsive fluorescent molecule modified with maleimide and excitable by the thermosensitive fluorescent polymer A; The non-polar-responsive fluorescent molecule excitable by the thermosensitive fluorescent polymer A includes any one of sulfonated rhodamine B maleimide derivatives, Texas Red maleimide derivatives, anthocyanin sulfonated Cy3 maleimide derivatives, or anthocyanin sulfonated Cy3.5 maleimide derivatives; Under an excitation wavelength of 400 nm to 450 nm, when the ambient temperature is lower than 40 °C, the nano-fluorescent temperature indicator exhibits a purple excitation wavelength; When the ambient temperature is between 40 °C and 46 °C, the nano-fluorescent temperature indicator exhibits green emission light; When the ambient temperature is higher than 46 °C, the nano-fluorescent temperature indicator exhibits red emission light.

2. The preparation method of the nano-fluorescent temperature indicator according to claim 1, characterized in that, Including: (1) Preparation of the thermosensitive fluorescent polymer A: A chain transfer agent containing a carboxyl group is used to prepare a thermosensitive polymer a with a lower critical solution temperature of 40 °C through reversible addition-fragmentation chain transfer polymerization; Through an esterification reaction, a polar-responsive fluorescent molecule modified with a hydroxyl group is coupled to the carboxyl end of the thermosensitive polymer a to obtain the thermosensitive fluorescent polymer A; (2) Preparation of the thermosensitive fluorescent polymer B: A chain transfer agent containing a carboxyl group is selected, and then a thermosensitive polymer b with a lower critical solution temperature of 46 °C is prepared by using reversible addition-fragmentation chain transfer polymerization; The two ends of the thermosensitive polymer b are a carboxyl group and a thioester, respectively; Through click chemistry, the thioester at the end of the thermosensitive polymer b is coupled with a non-polar-responsive fluorescent molecule modified with maleimide and excitable by the thermosensitive fluorescent polymer A to obtain the thermosensitive fluorescent polymer B; (3) Preparation of the maleimide-modified thermosensitive fluorescent polymer B: Using an amidation reaction, a maleimide containing an amino group is coupled to the carboxyl end of the thermosensitive fluorescent polymer B to obtain the maleimide-modified thermosensitive fluorescent polymer B; (4) Preparation of the nano-fluorescent temperature indicator: Using click chemistry reaction, the thermosensitive fluorescent polymer A and the maleimide-modified thermosensitive fluorescent polymer B are reacted to obtain a two-block dual thermosensitive fluorescent polymer, that is, the nano-fluorescent temperature indicator.

3. The preparation method of the nano-fluorescent temperature indicator according to claim 2, wherein, In steps (1) and (2), the carboxyl-containing chain transfer agent includes any one of 2-(dodecylsulfanylthiocarbonylthio)-2-methylpropanoic acid, 3-benzylsulfanylthiocarbonylpropanoic acid, 2-(ethylsulfanylthiocarbonylthio)-2-methylpropanoic acid, 4-cyano-4-(dodecylsulfanylthiocarbonyl)sulfanyl pentanoic acid, S-(thiobenzoyl)thioacetic acid or 4-cyano-4-(thiobenzoylthio)pentanoic acid; The conditions for the reversible addition-fragmentation chain transfer polymerization are all under anaerobic conditions, with stirring reaction at 60 °C to 80 °C for 12 to 48 hours.

4. The preparation method of the nano-fluorescent temperature indicator according to claim 2, characterized in that, In step (1), the molecular weight of the thermosensitive polymer a is 20,000 to 50,000, and the molecular weight distribution is 1.0 to 1.2; the molar ratio of the thermosensitive polymer a to the hydroxyl-modified polar responsive fluorescent molecule is 1:1 to 1:100; the conditions for the esterification reaction are stirring reaction at 10 °C to 50 °C and anhydrous conditions for 2 to 72 hours; In step (2), the molecular weight of the thermosensitive polymer b is 60,000 to 100,000, and the molecular weight distribution is 1.0 to 1.2; the molar ratio of the thermosensitive polymer b to the non-polar responsive fluorescent molecule that can be excited by the thermosensitive fluorescent polymer A modified with maleimide is 1:1 to 1:100; the conditions for the click reaction are stirring reaction at room temperature under anaerobic conditions for 1 to 72 hours.

5. The preparation method of the nano-fluorescent temperature indicator according to claim 2, wherein, In step (3), the maleimide containing an amino group includes N-(2-aminopropyl)maleimide, N-(2-aminoethyl)maleimide or N-(4-aminophenyl)maleimide; The molar ratio of the maleimide containing an amino group to the thermosensitive fluorescent polymer B is 1:1 to 50:1; The conditions for the amidation reaction are reaction at room temperature for 1 to 72 hours.

6. The preparation method of the nano-fluorescent temperature indicator according to claim 2, characterized in that In step (4), the dosage ratio of the thermosensitive fluorescent polymer A to the maleimide-modified thermosensitive fluorescent polymer B is 1:2 to 1:200; The reaction conditions are anaerobic conditions, stirring reaction at room temperature for 12 to 168 hours, and then dialysis using a dialysis bag with a molecular weight cut-off of 500,000 to 1,000,000 at 42 to 44 °C to remove the excessive maleimide-modified thermosensitive fluorescent polymer B.

7. The application of the nano-fluorescent temperature indicator described in claim 1 and the nano-fluorescent temperature indicator prepared by the method described in any one of claims 2 to 6 for non-therapeutic and diagnostic purposes in temperature detection of cells or sub-cells.

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