A nano-diagnosis and treatment agent with pH and light dual response and a preparation method and application thereof

Abstract

The application provides a pH and light dual response nanodiagnosis and treatment agent, a preparation method and application thereof, and the pH and light dual response nanodiagnosis and treatment agent comprises a carrier self-assembled by an amphiphilic compound as shown in formula I, and a near-infrared fluorescent dye loaded in the carrier. The pH and light dual response nanodiagnosis and treatment agent provided by the application can exist stably in a non-tumor environment, has a suitable particle size, is convenient for drug delivery and treatment, has a high encapsulation efficiency and a high near-infrared fluorescent dye loading, can gradually amplify oxidative stress under a tumor acid environment and near-infrared light irradiation, and realizes tumor treatment in cooperation.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medical materials, and particularly relates to a pH and light dual-responsive nanodiagnosis and treatment agent as well as a preparation method and application thereof. BACKGROUND

[0002] Photodynamic therapy (PDT) and photothermal therapy (PTT) and other phototherapies have the characteristics of minimally invasive, high selectivity and safety, and have become an important means for cancer treatment. The PDT process rapidly consumes oxygen to form an oxygen-deficient environment, and also consumes NADPH to reduce the synthesis of GSH. In addition, compared with normal tissues, tumor tissues usually present a more acidic environment. pH-responsive nanomaterials have also been widely concerned in tumor treatment due to their unique characteristics and functions.

[0003] Cinnamaldehyde (CA) is a safe and non-toxic plant extract approved by the US Food and Drug Administration. In recent years, it has been found that it can stimulate endogenous H2O2 production and inhibit the proliferation of tumor cells as a ROS regulator. Near-infrared fluorescent dyes have various uses, can be used for photodynamic therapy, photothermal therapy and real-time imaging of tumors, and due to their hydrophobicity, can be easily loaded into nanomicelles.

[0004] Based on the above, it is of great significance to develop a nanodiagnosis and treatment agent with pH and light dual response for tumor treatment and fluorescence imaging. SUMMARY

[0005] In view of the deficiencies of the prior art, the purpose of the present application is to provide a pH and light dual-responsive nanodiagnosis and treatment agent and a preparation method and application thereof. The pH and light dual-responsive nanodiagnosis and treatment agent provided by the present application can exist stably in a non-tumor environment, has a suitable particle size for drug delivery and treatment, has a high encapsulation efficiency and near-infrared fluorescent dye loading capacity, and can amplify oxidative stress under tumor acidic environment and near-infrared light conditions, thereby achieving synergistic tumor treatment.

[0006] To achieve the purpose of the present application, the following technical solutions are adopted:

[0007] In a first aspect, the present application provides a pH and light dual-responsive nanodiagnosis and treatment agent, which comprises a carrier formed by self-assembly of an amphiphilic compound as shown in formula I, and a near-infrared fluorescent dye loaded in the carrier.

[0008]

[0009] The pH and light dual-responsive nanodiagnosis and treatment agent provided by the application has a similar bilayer structure to a liposome, and the nitro group, Fmoc group and cinnamaldehyde group of the amphiphilic compound and the near-infrared fluorescent dye form a hydrophobic intermediate layer of the nanoparticle through π-π stacking action. (1) In the acidic environment of a tumor, the acetal bond of the amphiphilic compound is broken, cinnamaldehyde is released, the level of H2O2 is increased, and the first ROS amplification occurs; (2) Under the irradiation of near-infrared light, the near-infrared fluorescent dye consumes oxygen to generate singlet oxygen, forming a hypoxic environment, and the second ROS amplification occurs; at this time, the nitro group is reduced to an amino group, the hydrophilicity is enhanced, the nanoparticle is disassembled, and the near-infrared fluorescent dye is released; at the same time, a large amount of NADPH is consumed in the process of reduction of the nitro group, the synthesis of glutathione (GSH) is reduced, and the proliferation of tumor cells is inhibited; (3) After the near-infrared fluorescent dye is released, the photodynamic therapy is fully exerted to generate singlet oxygen, and the third ROS amplification occurs; at the same time, the near-infrared fluorescent dye exerts the photothermal therapy to convert light energy into heat energy, so that the local temperature is too high to kill tumor cells. The reduction of the nitro group is promoted by the hypoxic environment formed by the first ROS amplification and the second ROS amplification, and the disassembly of the nanoparticle and the occurrence of the third ROS amplification reaction are promoted. The pH and light dual-responsive nanodiagnosis and treatment agent provided by the application can amplify the oxidative stress reaction step by step, and synergistically improve the antitumor effect.

[0010] Preferably, the near-infrared fluorescent dye is selected from any one of IR780, IR825, IR755 or IR813.

[0011] Preferably, the hydration particle size of the pH and light dual-responsive nanodiagnosis and treatment agent is 100-500 nm, for example, can be 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, etc.

[0012] Preferably, the loading amount of the near-infrared fluorescent dye in the pH and light dual-responsive nanodiagnosis and treatment agent is 4%-7%, for example, can be 4%, 4.2%, 4.5%, 4.8%, 5%, 5.2%, 5.5%, 5.8%, 6%, 6.2%, 6.5%, 6.8%, 7%, etc.

[0013] Other specific point values in the above numerical range can be selected, which will not be described here.

[0014] In the second aspect, the application provides a preparation method of the pH and light dual-responsive nanodiagnosis and treatment agent according to the first aspect, and the preparation method comprises:

[0015] The amphiphilic compound and the near-infrared fluorescent dye are dissolved in an organic solvent to obtain a mixed solution;

[0016] Mixing the mixed solution with the alkaline solution to perform self-assembly to obtain the pH and light dual-responsive nanodiagnosis and treatment agent.

[0017] The preparation method of the application can efficiently load near-infrared fluorescent dyes into the amphiphilic compound to form nanoparticles that are stable in non-tumor environment, have suitable particle size for drug delivery and treatment, and have high encapsulation rate and near-infrared fluorescent dye loading.

[0018] Preferably, the concentration of the amphiphilic compound in the mixed solution is 10-50 mg / mL, for example, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, etc.

[0019] Preferably, the concentration of the near-infrared fluorescent dye in the mixed solution is 0.5-10 mg / mL, for example, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, etc.

[0020] Other specific point values within the above numerical ranges can be selected, which will not be described here.

[0021] In the application, the pH and light dual-responsive nanodiagnosis and treatment agent prepared by using the above concentrations of amphiphilic compound and near-infrared fluorescent dye has a suitable particle size for drug delivery and treatment, and has a high encapsulation rate and near-infrared fluorescent dye loading.

[0022] Preferably, the organic solvent is selected from any one or a combination of at least two of methanol, N,N-dimethylformamide, dimethyl sulfoxide, ethanol, acetone, or n-butanol.

[0023] Particle size has an important influence on cell uptake efficiency, blood circulation time, etc., and a suitable particle size is beneficial to improve drug absorption and targeting, but too small particle size may lead to rapid drug clearance by the kidney, and larger particle size may lead to recognition and phagocytosis by the immune system. In the application, the pH and light dual-responsive nanodiagnosis and treatment agent prepared in the above organic solvent has a suitable particle size for drug delivery, which is beneficial to transportation to the tumor site and realization of targeted therapy.

[0024] Preferably, the alkaline solution is selected from any one or a combination of at least two of sodium hydroxide aqueous solution, potassium hydroxide aqueous solution, sodium carbonate aqueous solution, sodium bicarbonate aqueous solution, or ammonia water.

[0025] Preferably, the pH value of the alkaline solution is 6.8-10, for example, it can be 6.8, 7, 7.2, 7.5, 7.8, 8, 8.2, 8.5, 8.8, 9, 9.2, 9.5, 9.8, 10, etc.

[0026] Preferably, the pH value of the alkaline solution is 7-8, for example, it can be 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, etc.

[0027] Other specific point values in the above numerical ranges can be selected, which will not be repeated here.

[0028] In the present application, the pH and light dual-responsive nanodiagnosis and treatment agent prepared in the above-mentioned pH value range of the alkaline solution has high stability in a non-tumor environment and will not cause premature release or leakage of the drug during delivery, ensuring stable delivery to the tumor site.

[0029] Preferably, the volume ratio of the mixed solution to the alkaline solution is 1:5-1:20, for example, it can be 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, etc.

[0030] Other specific point values in the above numerical ranges can be selected, which will not be repeated here.

[0031] In the present application, the pH and light dual-responsive nanodiagnosis and treatment agent prepared in the above-mentioned volume ratio of the mixed solution to the alkaline solution has a suitable particle size for drug delivery and treatment, and has a high encapsulation rate and near-infrared fluorescent dye loading.

[0032] Preferably, the mixing method is to drop the mixed solution into the alkaline solution under stirring.

[0033] Preferably, the stirring speed is 500-4000 rpm, for example, it can be 500 rpm, 800 rpm, 1000 rpm, 1500 rpm, 2000 rpm, 2500 rpm, 3000 rpm, 3500 rpm, 4000 rpm, etc.

[0034] Preferably, the mixing temperature is 20-40℃, for example, it can be 20℃, 22℃, 24℃, 26℃, 28℃, 30℃, 32℃, 34℃, 36℃, 38℃, 40℃, etc.

[0035] Other specific point values in the above numerical ranges can be selected, which will not be repeated here.

[0036] Preferably, the reaction product is further purified after the self-assembly is completed.

[0037] Preferably, the purification is performed by dialysis.

[0038] Preferably, the purification is performed for 8-12 hours, for example, 8 hours, 8.5 hours, 9 hours, 9.5 hours, 10 hours, 10.5 hours, 11 hours, 11.5 hours, 12 hours, etc.

[0039] Other specific point values within the above-mentioned numerical ranges can also be selected, which will not be repeated here.

[0040] In a third aspect, the present application provides a use of the pH and light dual-responsive nanodiagnostic and nanotherapeutic agent in the first aspect in tumor treatment and tumor fluorescence imaging.

[0041] Compared with the prior art, the present application has the following beneficial effects:

[0042] (1) The pH and light dual-responsive nanodiagnostic and nanotherapeutic agent provided by the present application can amplify oxidative stress reaction step by step under the conditions of tumor acidic environment and near-infrared light irradiation, and cooperatively achieve tumor treatment.

[0043] (2) The preparation method of the present application can efficiently load near-infrared fluorescent dye inside the amphiphilic compound, and the formed nanoparticles are stable in non-tumor environment, have appropriate particle size for drug delivery and treatment, have an encapsulation efficiency of 40%-60%, and have a high near-infrared fluorescent dye loading capacity. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 The synthesis route chart of the amphiphilic compound FPLC in Preparation Example 1 is shown in the figure;

[0045] Figure 2 The transmission electron microscope image of the pH and light dual-responsive nanodiagnostic and nanotherapeutic agent FPLC@IR in Example 1 is shown in the figure;

[0046] Figure 3 The particle size distribution chart of the pH and light dual-responsive nanodiagnostic and nanotherapeutic agent FPLC@IR in Example 1 is shown in the figure;

[0047] Figure 4 The fluorescence imaging detection result chart of the pH and light dual-responsive nanodiagnostic and nanotherapeutic agent FPLC@IR in Cal27 cells in Example 1 is shown in the figure;

[0048] Figure 5 The apoptosis flow cytometry detection result chart of the pH and light dual-responsive nanodiagnostic and nanotherapeutic agent FPLC@IR on Cal27 cell killing in Example 1 is shown in the figure;

[0049] Figure 6This is a graph showing the in vivo fluorescence imaging results of the pH and light-responsive nanotherapeutic agent FPLC@IR in the CDX model in Example 1.

[0050] Figure 7 This is a tumor volume growth curve after the pH and light-responsive nanotherapeutic agent FPLC@IR was injected into the CDX model in Example 1. Detailed Implementation

[0051] To further illustrate the technical means and effects of the present invention, the following describes the technical solution of the present invention in conjunction with preferred embodiments of the present invention. However, the present invention is not limited to the scope of the embodiments.

[0052] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0053] Preparation Example 1: Preparation of Amphiphilic Compounds in FPLC

[0054] The synthetic route of the amphiphilic compound FPLC is as follows: Figure 1 As shown:

[0055] (1) 1-Phenylon-N-(piperidin-4-ylmethyl)methylimine (357 mg, 1.765 mmol) and 1-(2,3-epoxypropyl)-2-nitroimidazole (250 mg, 1.478 mmol) were mixed in ultra-dry ethanol (10 mL), heated in a microwave at 120 °C for 20 minutes, cooled to room temperature, and the ethanol was removed by rotary evaporation. The mixture was then mixed with 3 mL of 1.2 N hydrochloric acid aqueous solution at 40 °C for 4 hours. The reaction mixture was extracted with dichloromethane (4 × 10 mL), and the lower layer obtained from this extraction step was an unwanted impurity. Then, the aqueous phase was adjusted to pH 11 with 500 μL of 40% NaOH aqueous solution, extracted with dichloromethane (4 × 10 mL), and the organic phase was dried over anhydrous magnesium sulfate. After filtration, the solvent was selectively evaporated, and the residue was dried under vacuum overnight to obtain compound PMND, which was a yellow oil.

[0056] (2) Take 198.5 mg of Nα-fluorenylmethoxycarbonyl-Nε-tert-butyloxycarbonyl-L-lysine (Fmoc-Lys(Boc)-OH, 0.424 mmol) and dissolve in 6 mL of dichloromethane, add 131.08 mg of dicyclohexyl carbodiimide (DCC, 0.635 mmol) and 48.7 mg of N-hydroxysuccinimide (NHS, 0.424 mmol), and stir the reaction at room temperature for 3 hours, and monitor the progress of the reaction by a thin layer chromatography (TLC) plate. When the reaction reaches the end point, remove the precipitate produced in the activation process by filtration. Then, concentrate the filtrate to about 2 mL using a rotary evaporator.

[0057] (3) Take 100.0 mg of PMND (0.353 mmol) and dissolve in 2 mL of dichloromethane, and add the solution of step (2), and stir the reaction at room temperature for 3 hours, and monitor the progress of the reaction by a TLC plate. After the reaction is completed, remove the solvent using a rotary evaporator, and then purify the product using a silica gel column chromatography (dichloromethane / methanol = 5 / 1), and remove the solvent. Finally, obtain 188.5 mg of compound F-L(B)-P.

[0058] (4) Take 20.0 mg of compound F-L(B)-P (0.027 mmol) and dissolve in 1 mL of a 25% trifluoroacetic acid solution (0.25 mL of trifluoroacetic acid and 0.75 mL of dichloromethane), and stir the reaction at room temperature for 0.5 hours, and monitor the progress of the reaction by a TLC plate. After the reaction is completed, slowly drop the above solution into diethyl ether, and remove the diethyl ether by centrifugation after the precipitate is separated, and collect the precipitate, and dry in a vacuum drying oven overnight. Finally, obtain 15.1 mg of compound F-L-P at a yield of 87.4%.

[0059] (5) Take 44.3 mg of a carboxylated cinnamaldehyde derivative CA-D-COOH (0.133 mmol), 70.0 mg of compound F-L-P (0.110 mmol), and 55.3 mg of O-benzotriazol- tetramethyluronium hexafluorophosphate (HBTU, 0.146 mmol), and dissolve in 4 mL of super dry N,N-dimethylformamide (DMF), and then add 57.7 μL of N,N-diisopropylethylamine, and stir the reaction at room temperature for 2 hours, and monitor the progress of the reaction by a TLC plate. After the reaction is completed, drop the above reaction solution into 12 mL of an aqueous sodium hydroxide solution to separate the product, and remove the supernatant by centrifugation, and freeze-dry the precipitate to remove water. Purify the crude product by a silica gel column chromatography (dichloromethane / methanol = 40 / 3), and remove the solvent to obtain the final product, an amphiphilic compound (abbreviated as FPLC) 53.5 mg at a yield of 51.1%.

[0060] Example 1

[0061] The embodiment provides a pH and light dual-response nano-diagnosis and treatment agent, and a preparation method thereof is as follows:

[0062] (1) 20 mg of an amphiphilic compound FPLC and 2 mg of a near-infrared fluorescent dye IR780 are weighed and dissolved in 1 mL of methanol to obtain a mixed solution;

[0063] (2) The mixed solution is added dropwise into 10 mL of a sodium hydroxide aqueous solution (pH=7.5), and stirring is performed at 2000 rpm at 25 DEG C for 2 h; after the reaction is completed, the reaction product is dialyzed in the sodium hydroxide aqueous solution for 12 h to remove impurities, and a pH and light dual-response nano-diagnosis and treatment agent (abbreviated as FPLC@IR) is obtained.

[0064] Embodiment 2

[0065] The embodiment provides a pH and light dual-response nano-diagnosis and treatment agent, and a preparation method thereof is as follows:

[0066] (1) 10 mg of an amphiphilic compound FPLC and 1 mg of a near-infrared fluorescent dye IR780 are weighed and dissolved in 1 mL of N,N-dimethylformamide to obtain a mixed solution;

[0067] (2) The mixed solution is added dropwise into 20 mL of a potassium hydroxide aqueous solution (pH=7), and stirring is performed at 1000 rpm at 20 DEG C for 1 h; after the reaction is completed, the reaction product is dialyzed in the potassium hydroxide aqueous solution for 8 h to remove impurities, and a pH and light dual-response nano-diagnosis and treatment agent FPLC@IR is obtained.

[0068] Embodiment 3

[0069] The embodiment provides a pH and light dual-response nano-diagnosis and treatment agent, and a preparation method thereof is as follows:

[0070] (1) 50 mg of an amphiphilic compound FPLC and 10 mg of a near-infrared fluorescent dye IR780 are weighed and dissolved in 1 mL of dimethyl sulfoxide to obtain a mixed solution;

[0071] (2) The mixed solution is added dropwise into 5 mL of a sodium carbonate aqueous solution (pH=8), and stirring is performed at 3000 rpm at 40 DEG C for 6 h; after the reaction is completed, the reaction product is dialyzed in the sodium carbonate aqueous solution for 10 h to remove impurities, and a pH and light dual-response nano-diagnosis and treatment agent FPLC@IR is obtained.

[0072] Embodiment 4

[0073] The embodiment provides a pH and light dual-response nano-diagnosis and treatment agent, and a preparation method thereof is as follows:

[0074] (1) 40 mg of the amphiphilic compound FPLC and 5 mg of the near-infrared fluorescent dye IR825 were weighed and dissolved in 1 mL of acetone to obtain a mixed solution;

[0075] (2) The mixed solution was added dropwise to 15 mL of a sodium bicarbonate aqueous solution (pH = 10), and the reaction was stirred at 4000 rpm at 30°C for 4 h. After the reaction was completed, the reaction product was dialyzed in the sodium bicarbonate aqueous solution for 11 h to remove impurities, thereby obtaining the pH and light dual-responsive nanodiagnostic agent FPLC@IR.

[0076] Example 5

[0077] This example provides a pH and light dual-responsive nanodiagnostic agent, and the preparation method is as follows:

[0078] (1) 15 mg of the amphiphilic compound FPLC and 3.5 mg of the near-infrared fluorescent dye IR755 were weighed and dissolved in 1 mL of ethanol to obtain a mixed solution;

[0079] (2) The mixed solution was added dropwise to 18 mL of ammonia (pH = 6.8), and the reaction was stirred at 500 rpm at 35°C for 3 h. After the reaction was completed, the reaction product was dialyzed in the ammonia for 9 h to remove impurities, thereby obtaining the pH and light dual-responsive nanodiagnostic agent FPLC@IR.

[0080] Example 6

[0081] This example provides a pH and light dual-responsive nanodiagnostic agent, and the preparation method is as follows:

[0082] (1) 35 mg of the amphiphilic compound FPLC and 8 mg of the near-infrared fluorescent dye IR813 were weighed and dissolved in 1 mL of n-butanol to obtain a mixed solution;

[0083] (2) The mixed solution was added dropwise to 9 mL of a sodium hydroxide aqueous solution (pH = 8.5), and the reaction was stirred at 1500 rpm at 23°C for 5 h. After the reaction was completed, the reaction product was dialyzed in the sodium hydroxide aqueous solution for 10 h to remove impurities, thereby obtaining the pH and light dual-responsive nanodiagnostic agent FPLC@IR.

[0084] Example 7

[0085] This example provides a pH and light dual-responsive nanodiagnostic agent, which is different from Example 1 only in that "20 mg of the amphiphilic compound FPLC and 2 mg of the near-infrared fluorescent dye IR780" are replaced by "5 mg of the amphiphilic compound FPLC and 0.5 mg of the near-infrared fluorescent dye IR780" in the preparation process, and the other steps remain unchanged.

[0086] Example 8

[0087] The embodiment provides a pH and light dual-response nanodiagnosis and treatment agent, which is different from the embodiment 1 only in that "20 mg of amphiphilic compound FPLC and 2 mg of near-infrared fluorescent dye IR780" is replaced by "80 mg of amphiphilic compound FPLC and 8 mg of near-infrared fluorescent dye IR780" in a preparation process, and other steps remain unchanged.

[0088] Embodiment 9

[0089] The embodiment provides a pH and light dual-response nanodiagnosis and treatment agent, which is different from the embodiment 1 only in that "10 mL of sodium hydroxide aqueous solution" is replaced by "3 mL of sodium hydroxide aqueous solution" in a preparation process, and other steps remain unchanged.

[0090] Embodiment 10

[0091] The embodiment provides a pH and light dual-response nanodiagnosis and treatment agent, which is different from the embodiment 1 only in that "10 mL of sodium hydroxide aqueous solution" is replaced by "25 mL of sodium hydroxide aqueous solution" in a preparation process, and other steps remain unchanged.

[0092] Embodiment 11

[0093] The embodiment provides a pH and light dual-response nanodiagnosis and treatment agent, which is different from the embodiment 1 only in that "sodium hydroxide aqueous solution (pH=7.5)" is replaced by "sodium hydroxide aqueous solution (pH=5)" in a preparation process, and other steps remain unchanged.

[0094] Test example 1 detects the hydration particle size, encapsulation rate and near-infrared fluorescent dye loading of FPLC@IR

[0095] The pH and light dual-response nanodiagnosis and treatment agent FPLC@IR prepared in the embodiments 1-11 is used for the following detection: (1) the morphology of FPLC@IR is detected under a transmission electron microscope, and the particle size distribution is counted; (2) near-infrared fluorescent dye loading (Drug-loading efficiency, DL) determination: 5 mL of FPLC@IR is freeze-dried, the mass of the solid after freeze-drying is accurately weighed to obtain the total weight of FPLC@IR; then the mass of IR780 in 5 mL of FPLC@IR solution is calculated according to the demulsification result; DL=(the mass of IR780 in FPLC@IR) / (the total weight of FPLC@IR) x 100%; (3) encapsulation rate (Encapsulation efficiency, EE) determination: EE=(the mass of IR780 in FPLC@IR) / (the mass of IR780 initially input) x 100%.

[0096] Detection results: Figure 2The transmission electron microscope image of FPLC@IR prepared in Example 1 shows that FPLC@IR is spherical; Figure 3 The particle size distribution graph of FPLC@IR prepared in Example 1 shows that the hydrated particle size of FPLC@IR prepared in the application is 100-500 nm; the hydrated particle size, encapsulation efficiency and near-infrared fluorescent dye loading of FPLC@IR prepared in Examples 1-11 are shown in Table 1.

[0097] Table 1

[0098] FPLC@IR Hydrated particle size Encapsulation efficiency Near infrared fluorescent dye loading Example 1 291.4 nm 58.7％ 6.8％ Example 2 350.6 nm 55.3％ 6.4％ Example 3 305.6 nm 51.6％ 5.2％ Example 4 390.7 nm 50.9％ 5.1％ Example 5 463.4 nm 45.9％ 4.7％ Example 6 401.6 nm 48.2％ 4.9％ Example 7 80 nm 23.7％ 2.1％ Example 8 675.2 nm 28.2％ 2.4％ Example 9 684.3 nm 25.6％ 2.6％ Example 10 649.8 nm 22.3％ 2.2％ Example 11 No nanoparticles formed / /

[0099] From the data in Table 1, it can be seen that the FPLC@IR prepared in Examples 1-6 of the application has a suitable particle size, is convenient for drug delivery and treatment, and has a high encapsulation efficiency and near-infrared fluorescent dye loading. From Examples 7-8, it can be seen that when the carrier concentration is too low, the system is unstable, resulting in a decrease in particle size, encapsulation efficiency and near-infrared fluorescent dye loading; when the carrier concentration is too high, the interaction between the systems is enhanced, aggregation is easy to occur, resulting in an increase in particle size, a decrease in encapsulation efficiency and a decrease in near-infrared fluorescent dye loading. From Examples 9-10, it can be seen that when the volume of the mixed solution is too large, aggregation is easy to occur, resulting in an increase in particle size, a decrease in encapsulation efficiency and a decrease in near-infrared fluorescent dye loading; when the volume of the alkaline solution is too large, the drug carrier concentration is diluted, the system is unstable, the particle size increases, the encapsulation efficiency decreases and the near-infrared fluorescent dye loading decreases. From Example 11, it can be seen that when the pH value of the alkaline solution is too low, the acetal bond of the carrier is broken prematurely and cannot form nanoparticles.

[0100] Test Example 2 detects the tumor cell fluorescence imaging ability of FPLC@IR

[0101] Human tongue squamous cell carcinoma cells Cal27 were inoculated in a confocal dish at 1×10 5 cells per dish, and cultured overnight. When the cell density reached about 60%, the culture medium was removed, fresh culture medium containing free IR780 or FPLC@IR prepared in Example 1 (the concentration of IR780 was 1 μg / mL) was added, and after 4 hours of incubation, the drug-containing culture medium was removed, 1 mL of phenol-free DMEM culture medium was added, and one group of culture dishes containing FPLC@IR received 808 nm laser irradiation (0.5 W / cm 2, 5 minutes). The medium was removed and the Mito-Tracker Green (MTG) probe was diluted with PBS at a dilution ratio of 1:10000, 1 mL of which was added to the confocal dish and incubated with the cells in a 37°C incubator for 30 minutes to stain the mitochondria. 5 minutes before the end of the incubation, the corresponding volume of Hoechst 33342 staining solution was added at a dilution ratio of 1:100 to stain the cell nucleus. The PBS containing the MTG probe and the Hoechst 33342 staining solution was removed, and then the cells were washed twice with PBS, each time for 3 minutes. The fluorescence of IR780 in the cells was observed by laser confocal microscopy, and the results were recorded and plotted.

[0102] The results of imaging Cal27 cells by FPLC@IR are shown in Figure 4 The cells treated by FPLC@IR showed obvious IR780 fluorescence, and laser irradiation could further promote the release of IR780, making the fluorescence signal further enhanced, which proved the light-responsive fluorescence imaging property of FPLC@IR for tumor cells.

[0103] Test Example 3: Detection of the tumor cell killing ability of FPLC@IR

[0104] Human tongue squamous carcinoma cells Cal27 were inoculated in a 6-well plate at a density of 3x10 5 cells per well, and cultured for 24 hours. Three replicate wells were set up for each group. The medium was removed, and fresh medium containing free IR780 (1 μg / mL) or FPLC@IR prepared in Example 1 was added, and the medium without drugs was used as a control group. The cells were incubated in a 37°C incubator for 4 hours. The drug-containing medium was removed, the cells were collected by trypsinization, and the cell pellets were collected by centrifugation. 500 μL of phenol-free DMEM was added to resuspend the cells, and the light exposure group received 808 nm laser irradiation (0.6 W / cm 2 , 5 min).

[0105] The cells were centrifuged at 1200 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended by adding 195 μL of Annexin V-FITC binding solution. 5 μL of Annexin V-FITC and 10 μL of propidium iodide staining solution were added, and the mixture was mixed well. After incubation at room temperature for 15 minutes in the dark, the cells were subjected to flow cytometry for apoptosis detection. The apoptosis rate (early apoptosis + late apoptosis) was calculated for different groups, and statistical analysis of the differences between groups was performed.

[0106] The tumor cell killing effect of FPLC@IR is shown in Figure 5 Compared with the control group, the FPLC@IR prepared in Example 1 could obviously induce tumor cell apoptosis under laser irradiation, showing obvious cytotoxicity, which indicated that the FPLC@IR of the present application had good tumor killing effect under laser irradiation.

[0107] Test Example 4 detects the fluorescence imaging ability of FPLC@IR on tumor cells at the in vivo level

[0108] Human tongue squamous carcinoma cells Cal27 were subcutaneously inoculated on the back of nude mice to establish a CDX model. When the tumor grew to a volume of 120mm 3 , 100 μL of FPLC@IR (IR780: 1.5 mg / kg) prepared in Example 1 was injected through the tail vein. Fluorescence imaging was performed at different time points (1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 48 h) using a small animal optical 3D in vivo imaging system, and the excitation wavelength of the in vivo imager was set to 745 nm and the emission wavelength was set to 820 nm. Under isoflurane anesthesia, the mice were placed in the imager, and fluorescence images were collected for observation.

[0109] The fluorescence imaging ability test results of FPLC@IR on tumor cells at the in vivo level are shown in Figure 6 , FPLC@IR showed a time-dependent biodistribution, and the fluorescence signal intensity in the tumor area gradually increased over time. After 24 hours of injection, the fluorescence intensity at the tumor site reached a peak, indicating that FPLC@IR can achieve tumor fluorescence imaging at the in vivo level.

[0110] Test Example 5 detects the tumor inhibition effect of FPLC@IR at the in vivo level

[0111] Human tongue squamous carcinoma cells Cal27 were subcutaneously inoculated on the back of nude mice to establish a CDX model. When the tumor grew to a volume of 120mm 3 , the CDX model mice were randomly divided into: ① control group (abbreviated as CTR, normal saline), ② FPLC@IR group prepared in Example 1 (IR780: 1.5 mg / kg), and ③ FPLC@IR laser irradiation group (abbreviated as FPLC@IR+L) (IR780: 1.5 mg / kg). Twenty-four hours after tail vein injection, the tumor area of the ③ group was irradiated with an 808 nm laser (0.3 W / cm 2 , 15 minutes). All treatment groups were observed for 14 days, and the tumor volume of the mice was monitored every other day during this period.

[0112] The tumor inhibition effect test results of FPLC@IR at the in vivo level are shown in Figure 7 , at the end of the 14-day treatment, compared with the normal saline group, the FPLC@IR laser irradiation group effectively inhibited the growth of the tumor, indicating that the FPLC@IR of the present application has excellent anti-tumor effect under laser irradiation.

[0113] The applicant declares that the present application is illustrated by the above-mentioned embodiments of a pH and light dual-responsive nanodiagnostic and therapeutic agent, a preparation method and application thereof, but the present application is not limited to the above-mentioned embodiments, i.e. it does not mean that the present application must rely on the above-mentioned embodiments to be implemented. It should be understood by those skilled in the art that any improvement of the present application, equivalent replacement of each raw material of the product of the present application, addition of auxiliary ingredients, selection of specific modes, etc. all fall within the protection scope and disclosure scope of the present application.

[0114] The preferred embodiments of the present application are described in detail above, but the present application is not limited to the specific details in the above-mentioned embodiments, and within the technical concept scope of the present application, various simple modifications can be made to the technical solutions of the present application, and these simple modifications all belong to the protection scope of the present application.

[0115] In addition, it should be noted that each specific technical feature described in the above-mentioned specific embodiments can be combined in any suitable manner without contradiction, and in order to avoid unnecessary repetition, the present application will not further describe various possible combination manners.

Claims

1. A pH and light dual-responsive nanodiagnostic agent, characterized in that, The pH and light dual-responsive nanodiagnostic and nanotherapeutic agent comprises a carrier self-assembled from an amphiphilic compound as shown in Formula I, and a near-infrared fluorescent dye loaded in the carrier. 2.The pH and light dual-responsive nanodiagnostic and nanotherapeutic agent of claim 1, wherein, The near-infrared fluorescent dye is selected from any one of IR780, IR825, IR755 or IR813. 3.The pH and light dual-responsive nanodiagnostic and nanotherapeutic agent of claim 1, wherein, The pH and light dual-responsive nanodiagnostic and nanotherapeutic agent has a hydrated particle size of 100-500 nm. 4.The pH and light dual-responsive nanodiagnostic and nanotherapeutic agent of claim 1, wherein, The pH and light dual-responsive nanodiagnostic and nanotherapeutic agent has a loading amount of the near-infrared fluorescent dye of 4%-7%.

5. A method for preparing the pH and light dual-responsive nanodiagnostic and nanotherapeutic agent according to any one of claims 1 to 4, characterized in that, The preparation method comprises: dissolving the amphiphilic compound and the near-infrared fluorescent dye in an organic solvent to obtain a mixed solution; mixing the mixed solution with an alkaline solution to perform self-assembly to obtain the pH and light dual-responsive nanodiagnostic and nanotherapeutic agent. 6.The method for preparing the pH and light dual-responsive nanodiagnostic and nanotherapeutic agent according to claim 5, characterized in that, The concentration of the amphiphilic compound in the mixed solution is 10-50 mg / mL. 7.The method for preparing the pH and light dual-responsive nanodiagnostic and nanotherapeutic agent according to claim 5, characterized in that, The concentration of the near-infrared fluorescent dye in the mixed solution is 0.5-10 mg / mL. 8.The method for preparing the pH and light dual-responsive nanodiagnostic and nanotherapeutic agent according to claim 5, characterized in that, The organic solvent is selected from any one of methanol, N,N-dimethylformamide, dimethyl sulfoxide, ethanol, acetone or n-butanol or a combination of at least two thereof. 9.The method for preparing the pH and light dual-responsive nanodiagnostic and nanotherapeutic agent according to claim 5, characterized in that, The alkaline solution is selected from any one of a sodium hydroxide aqueous solution, a potassium hydroxide aqueous solution, a sodium carbonate aqueous solution, a sodium bicarbonate aqueous solution or ammonia water or a combination of at least two thereof. 10.The method for preparing the pH and light dual-responsive nanodiagnostic and nanotherapeutic agent according to claim 5, characterized in that, The pH value of the alkaline solution is 6.8-10. 11.The method for preparing the pH and light dual-responsive nanodiagnostic and nanotherapeutic agent according to claim 5, characterized in that, The pH value of the alkaline solution is 7-8.

12. The method for preparing the pH and light-responsive nanotherapeutic agent according to claim 5, characterized in that, The volume ratio of the mixed solution to the alkaline solution is 1:5-1:

20.

13. The method for preparing the pH and light-responsive nanotherapeutic agent according to claim 5, characterized in that, The mixing is performed by dropwise adding the mixed solution into the alkaline solution under stirring.

14. The method for preparing the pH and light-responsive nanotherapeutic agent according to claim 13, characterized in that, The stirring speed is 500-4000 rpm.

15. The method for preparing the pH and light-responsive nanotherapeutic agent according to any one of claims 5, characterized in that, The mixing temperature is 20-40℃.

16. The method of preparing the pH and light dual-responsive nanodiagnostic and nanotherapeutic agent according to any one of claim 5, wherein, The reaction product is further purified after the self-assembly.

17. The method of preparing the pH and light dual-responsive nanodiagnostic and nanotherapeutic agent according to any one of claim 16, wherein, The purification is performed by dialysis.

18. The method for preparing the pH and light-responsive nanotherapeutic agent according to any one of claims 16, characterized in that, The purification time is 8-12 h.

19. Use of the pH and light dual-responsive nanodiagnostic and nanotherapeutic agent of any one of claims 1-4 in the preparation of a drug for tumor treatment and tumor fluorescence imaging.

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