PH and light dual-response nano diagnosis and treatment agent as well as preparation method and application thereof

Abstract

The invention provides a pH and light dual-response nano diagnosis and treatment agent as well as a preparation method and application thereof. The pH and light dual-response nano diagnosis and treatment agent comprises a carrier formed by self-assembly of an amphiphilic compound as shown in a formula I and near-infrared fluorescent dye entrapped in the carrier. The pH and light dual-response nano diagnosis and treatment agent provided by the invention can stably exist in a non-tumor environment, has a proper particle size, is convenient for drug delivery and treatment, has relatively high encapsulation efficiency and near-infrared fluorescent dye loading capacity, and gradually amplifies oxidative stress reaction in a tumor acid environment and under a near-infrared illumination condition to synergistically realize tumor treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and specifically relates to a pH and light dual-responsive nano diagnostic and therapeutic agent and a preparation method and application thereof. Background Art

[0002] Phototherapy such as photodynamic therapy (PDT) and photothermal therapy (PTT) has the characteristics of minimal invasiveness, high selectivity and safety, and has become an important means of cancer treatment. The PDT process will quickly consume oxygen and form an oxygen-deficient environment. The process will also consume NADPH and reduce the synthesis of GSH. In addition, compared with normal tissues, tumor tissues usually present a more acidic environment. pH-responsive nanomaterials have also received widespread attention in tumor treatment due to their unique properties and functions.

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

[0004] Based on the above, the development of a nano-diagnostic agent with dual responses to pH and light is of great significance for tumor treatment and fluorescence imaging. Summary of the invention

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

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a pH and light dual-responsive nano-diagnostic agent, wherein the pH and light dual-responsive nano-diagnostic agent comprises a carrier formed by self-assembly of an amphiphilic compound as shown in Formula I, and a near-infrared fluorescent dye encapsulated in the carrier;

[0008]

[0009] The pH and light dual-responsive nano-diagnostic agent provided by the present invention has a bilayer structure similar to that of liposomes, and the nitro group, Fmoc group, cinnamaldehyde group of the amphiphilic compound and the near-infrared fluorescent dye form a hydrophobic middle layer of the nanoparticles through π-π stacking. (1) In the acidic environment of the tumor, the acetal bond of the amphiphilic compound breaks, cinnamaldehyde is released, the H2O2 level increases, and the first ROS amplification occurs; (2) Under the irradiation of near-infrared light, the near-infrared fluorescent dye consumes oxygen, produces singlet oxygen, forms an oxygen-deficient environment, and the second ROS amplification occurs; at this time, the nitro group is reduced to an amino group, the hydrophilicity is enhanced, the nanoparticles are disassembled, and the near-infrared fluorescent dye is released; at the same time, a large amount of NADPH is consumed during the nitro reduction process, the synthesis of glutathione (GSH) is reduced, and the proliferation of tumor cells is inhibited; (3) After the near-infrared fluorescent dye is released, photodynamic therapy is fully exerted to produce singlet oxygen, and the third ROS amplification occurs; at the same time, the near-infrared fluorescent dye exerts photothermal therapy, converts light energy into heat energy, makes the local temperature too high, and kills tumor cells. The hypoxic environment formed by the first ROS amplification and the second ROS amplification synergistically promotes the reduction of nitro groups, promotes the disassembly of nanoparticles and the occurrence of the third ROS amplification reaction. The pH and light dual-responsive nanodiagnostic agent provided by the present invention can gradually amplify the oxidative stress response and synergistically improve the anti-tumor effect.

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

[0011] Preferably, the hydrated particle size of the pH and light dual-responsive nanodiagnostic and therapeutic agent is 100-500 nm, for example, it may 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 near-infrared fluorescent dye in the pH and light dual-responsive nanodiagnostic and therapeutic agent is 4%-7%, for example, it 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 ​​within the above numerical ranges can be selected and will not be described in detail here.

[0014] In a second aspect, the present invention provides a method for preparing a pH and light dual-responsive nano-diagnostic agent as described in the first aspect, the preparation method comprising:

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

[0016] The mixed solution is mixed with an alkaline solution and self-assembled to obtain the pH and light dual-responsive nano-diagnostic agent.

[0017] The preparation method of the present invention can efficiently load the near-infrared fluorescent dye inside the amphiphilic compound to form nanoparticles that are stable in a non-tumor environment, have a suitable particle size for drug delivery and treatment, and have a 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 and will not be described in detail here.

[0021] In the present invention, the pH and light dual-responsive nano-diagnostic and therapeutic agent prepared by using the above-mentioned concentration of amphiphilic compounds and near-infrared fluorescent dyes 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 of methanol, N,N-dimethylformamide, dimethyl sulfoxide, ethanol, acetone or n-butanol, or a combination of at least two thereof.

[0023] Particle size has an important impact on cellular uptake efficiency, blood circulation time, etc. A suitable particle size is conducive to improving drug absorption and targeting, but too small a particle size may cause the drug to be quickly cleared by the kidneys, and a larger particle size may cause the immune system to recognize and phagocytize it. In the present invention, the pH and light dual-responsive nanodiagnostic agent prepared in the above-mentioned organic solvent has a suitable particle size to facilitate drug delivery and transportation to the tumor site to achieve targeted therapy.

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

[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 ​​within the above numerical ranges can be selected and will not be described in detail here.

[0028] In the present invention, the pH and light dual-responsive nano-diagnostic and therapeutic agent prepared in an alkaline solution within the above-mentioned pH range has high stability in a non-tumor environment, will not cause premature release or leakage of the drug during the delivery process, and ensures 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 ​​within the above numerical ranges can be selected and will not be described in detail here.

[0031] In the present invention, the pH and light dual-responsive nano-diagnostic and therapeutic agent prepared by the volume ratio of the above-mentioned 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: adding the mixed solution dropwise 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°C, for example, it can be 20°C, 22°C, 24°C, 26°C, 28°C, 30°C, 32°C, 34°C, 36°C, 38°C, 40°C, etc.

[0035] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

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

[0037] Preferably, the purification method is dialysis.

[0038] Preferably, the purification time is 8-12 h, for example, it can be 8 h, 8.5 h, 9 h, 9.5 h, 10 h, 10.5 h, 11 h, 11.5 h, 12 h, etc.

[0039] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0040] In a third aspect, the present invention provides the use of the pH and light dual-responsive nano-diagnostic and therapeutic agent as described in the first aspect in tumor treatment and tumor fluorescence imaging.

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

[0042] (1) The pH and light dual-responsive nano-diagnostic and therapeutic agent provided by the present invention can gradually amplify the oxidative stress response under the conditions of tumor acidic environment and near-infrared light, and synergistically achieve tumor treatment;

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

[0044] Figure 1 The synthetic route of the amphiphilic compound FPLC in Preparation Example 1 is shown in FIG.

[0045] Figure 2 This is a transmission electron microscopy image of the pH and light dual-responsive nano-diagnostic agent FPLC@IR in Example 1;

[0046] Figure 3 This is the particle size distribution diagram of the pH and light dual-responsive nano-diagnostic agent FPLC@IR in Example 1;

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

[0048] Figure 5 This is a graph showing the apoptosis flow cytometry results of the pH and light dual-responsive nano-diagnostic agent FPLC@IR killing Cal27 cells in Example 1;

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

[0050] Figure 7 This is a graph showing the tumor volume growth curve after the pH and light dual-responsive nano-diagnostic agent FPLC@IR in Example 1 was injected into the CDX model. DETAILED DESCRIPTION

[0051] In order to further explain the technical means and effects adopted by the present invention, the technical solution of the present invention is further described below in conjunction with the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.

[0052] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0053] Preparation Example 1 Preparation of amphiphilic compounds FPLC

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

[0055] (1) 1-phenyl-N-(piperidin-4-ylmethyl)methaniline (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 at 120°C for 20 minutes, cooled to room temperature, and ethanol was removed by rotary evaporation. The mixture was mixed with 3 mL of 1.2 N aqueous hydrochloric acid at 40°C for 4 hours. The reaction mixture was extracted with dichloromethane (4×10 mL). The lower layer of liquid obtained in this step was an unwanted impurity. Then, the aqueous phase was adjusted to pH 11 with 500 μL of 40% aqueous NaOH solution, extracted with dichloromethane (4×10 mL), and the organic phase was dried over anhydrous magnesium sulfate. After filtration, the solvent was evaporated and the residue was dried under vacuum overnight to obtain compound PMND as a yellow oil.

[0056] (2) 198.5 mg of Nα-fluorenylmethoxycarbonyl-Nε-tert-butyloxycarbonyl-L-lysine (Fmoc-Lys(Boc)-OH, 0.424 mmol) was dissolved in 6 mL of dichloromethane, and 131.08 mg of dicyclohexylcarbodiimide (DCC, 0.635 mmol) and 48.7 mg of N-hydroxysuccinimide (NHS, 0.424 mmol) were added. The mixture was stirred at room temperature for 3 hours, and the reaction progress was monitored by thin layer chromatography (TLC). When the reaction endpoint was reached, the precipitate produced during the activation process was removed by filtration. The filtrate was then concentrated to about 2 mL using a rotary evaporator.

[0057] (3) 100.0 mg PMND (0.353 mmol) was dissolved in 2 mL dichloromethane and added to the solution of step (2). The mixture was stirred at room temperature for 3 hours and the reaction progress was monitored by TLC. After the reaction was completed, the solvent was removed by rotary evaporator, and then the product was purified by silica gel column chromatography (dichloromethane / methanol=5 / 1), and the solvent was removed. Finally, 188.5 mg of compound FL(B)-P was obtained.

[0058] (4) 20.0 mg of compound FL(B)-P (0.027 mmol) was dissolved in 1 mL of 25% trifluoroacetic acid solution (0.25 mL of trifluoroacetic acid and 0.75 mL of dichloromethane), stirred at room temperature for 0.5 hours, and the reaction progress was monitored by TLC. After the reaction was completed, the above solution was slowly added dropwise to ice ether, and after precipitation, the ether was removed by centrifugation, and the precipitate was retained and dried in a vacuum drying oven overnight. Finally, 15.1 mg of compound FLP was obtained, with a yield of 87.4%.

[0059] (5) 44.3 mg of carboxylated cinnamaldehyde derivative CA-D-COOH (0.133 mmol), 70.0 mg of compound FLP (0.110 mmol) and 55.3 mg of O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU, 0.146 mmol) were dissolved in 4 mL of ultra-dry N,N-dimethylformamide (DMF), and 57.7 μL of N,N-diisopropylethylamine was added. The mixture was stirred at room temperature for 2 hours, and the reaction progress was monitored by TLC plate. After the reaction was completed, the reaction solution was dropped into 12 mL of sodium hydroxide aqueous solution to precipitate the product, the supernatant was discarded by centrifugation, and the precipitate was freeze-dried to remove moisture. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 40 / 3), and after removing the solvent, 53.5 mg of the final product amphiphilic compound (abbreviated as FPLC) was obtained, with a yield of 51.1%.

[0060] Example 1

[0061] This embodiment provides a pH and light dual-responsive nano-diagnostic agent, and its preparation method is as follows:

[0062] (1) Weigh 20 mg of the amphiphilic compound FPLC and 2 mg of the near-infrared fluorescent dye IR780 and dissolve them in 1 mL of methanol to obtain a mixed solution;

[0063] (2) The mixed solution was added dropwise to 10 mL of sodium hydroxide aqueous solution (pH = 7.5), and the mixture was stirred at 2000 rpm at 25 °C for 2 h. After the reaction, the reaction product was dialyzed in sodium hydroxide aqueous solution for 12 h to remove impurities, thereby obtaining a pH and light dual-responsive nanodiagnostic agent (abbreviated as FPLC@IR).

[0064] Example 2

[0065] This embodiment provides a pH and light dual-responsive nano-diagnostic agent, and its preparation method is as follows:

[0066] (1) Weigh 10 mg of the amphiphilic compound FPLC and 1 mg of the near-infrared fluorescent dye IR780 and dissolve them in 1 mL of N,N-dimethylformamide to obtain a mixed solution;

[0067] (2) The mixed solution was added dropwise to 20 mL of potassium hydroxide aqueous solution (pH = 7), and the mixture was stirred at 1000 rpm at 20 °C for 1 h. After the reaction, the reaction product was dialyzed in potassium hydroxide aqueous solution for 8 h to remove impurities, thereby obtaining the pH and light dual-responsive nanodiagnostic agent FPLC@IR.

[0068] Example 3

[0069] This embodiment provides a pH and light dual-responsive nano-diagnostic agent, and its preparation method is as follows:

[0070] (1) Weigh 50 mg of the amphiphilic compound FPLC and 10 mg of the near-infrared fluorescent dye IR780 and dissolve them in 1 mL of dimethyl sulfoxide to obtain a mixed solution;

[0071] (2) The mixed solution was added dropwise to 5 mL of sodium carbonate aqueous solution (pH = 8), and the mixture was stirred at 3000 rpm at 40 °C for 6 h. After the reaction, the reaction product was dialyzed in sodium carbonate aqueous solution for 10 h to remove impurities, thereby obtaining the pH and light dual-responsive nanodiagnostic agent FPLC@IR.

[0072] Example 4

[0073] This embodiment provides a pH and light dual-responsive nano-diagnostic agent, and its preparation method is as follows:

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

[0075] (2) The mixed solution was added dropwise to 15 mL of sodium bicarbonate aqueous solution (pH = 10), and the mixture was stirred at 4000 rpm at 30 °C for 4 h. After the reaction, the reaction product was dialyzed in 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 embodiment provides a pH and light dual-responsive nano-diagnostic agent, and its preparation method is as follows:

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

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

[0080] Example 6

[0081] This embodiment provides a pH and light dual-responsive nano-diagnostic agent, and its preparation method is as follows:

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

[0083] (2) The mixed solution was added dropwise to 9 mL of sodium hydroxide aqueous solution (pH = 8.5), and the mixture was stirred at 1500 rpm at 23 °C for 5 h. After the reaction, the reaction product was dialyzed in 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 embodiment provides a pH and light dual-responsive nanodiagnostic and therapeutic agent, which differs from Example 1 only in that during the preparation process, "20 mg of amphiphilic compound FPLC and 2 mg of near-infrared fluorescent dye IR780" are replaced with "5 mg of amphiphilic compound FPLC and 0.5 mg of near-infrared fluorescent dye IR780", and other steps remain unchanged.

[0086] Example 8

[0087] This embodiment provides a pH and light dual-responsive nanodiagnostic and therapeutic agent, which differs from Example 1 only in that during the preparation process, "20 mg of amphiphilic compound FPLC and 2 mg of near-infrared fluorescent dye IR780" are replaced with "80 mg of amphiphilic compound FPLC and 8 mg of near-infrared fluorescent dye IR780", and other steps remain unchanged.

[0088] Example 9

[0089] This embodiment provides a pH and light dual-responsive nanodiagnostic and therapeutic agent, which differs from Example 1 only in that "10 mL of sodium hydroxide aqueous solution" is replaced by "3 mL of sodium hydroxide aqueous solution" during the preparation process, and other steps remain unchanged.

[0090] Example 10

[0091] This embodiment provides a pH and light dual-responsive nanodiagnostic and therapeutic agent, which differs from Example 1 only in that "10 mL of sodium hydroxide aqueous solution" is replaced by "25 mL of sodium hydroxide aqueous solution" during the preparation process, and other steps remain unchanged.

[0092] Embodiment 11

[0093] This embodiment provides a pH and light dual-responsive nanodiagnostic and therapeutic agent, which differs from Example 1 only in that "sodium hydroxide aqueous solution (pH=7.5)" is replaced by "sodium hydroxide aqueous solution (pH=5)" during the preparation process, and other steps remain unchanged.

[0094] Test Example 1: Detection of hydrated particle size, encapsulation efficiency and near-infrared fluorescent dye loading of FPLC@IR

[0095] The pH and light dual-responsive nano-diagnostic agent FPLC@IR prepared in Example 1-11 was used for the following tests: (1) The morphology of FPLC@IR was detected under a transmission electron microscope, and its particle size distribution was statistically analyzed; (2) Drug-loading efficiency (DL) of near-infrared fluorescent dye was determined: 5 mL of FPLC@IR was freeze-dried, and the mass of the freeze-dried solid was accurately weighed to obtain the total weight of FPLC@IR; then the mass of IR780 in 5 mL of FPLC@IR solution was calculated based on the demulsification results; DL = (mass of IR780 in FPLC@IR) / (total weight of FPLC@IR) × 100%; (3) Encapsulation efficiency (EE) was determined: EE = (mass of IR780 in FPLC@IR) / (mass of IR780 initially input) × 100%.

[0096] Test results: Figure 2This is the transmission electron microscopy image of the FPLC@IR prepared in Example 1, from which it can be seen that the FPLC@IR is spherical; Figure 3 The particle size distribution diagram of FPLC@IR prepared in Example 1 shows that the hydrated particle size of FPLC@IR prepared in the present invention 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 1 291.4nm 58.7％ 6.8％ Example 2 350.6nm 55.3％ 6.4％ Example 3 305.6nm 51.6％ 5.2％ Example 4 390.7nm 50.9％ 5.1％ Example 5 463.4nm 45.9％ 4.7％ Example 6 401.6nm 48.2％ 4.9％ Example 7 80nm 23.7％ 2.1％ Example 8 675.2nm 28.2％ 2.4％ Example 9 684.3nm 25.6％ 2.6％ Example 10 649.8nm 22.3％ 2.2％ Embodiment 11 No nanoparticles formed / /

[0099] It can be seen from the data in Table 1 that the FPLC@IR prepared in Examples 1-6 of the present invention has a suitable particle size, is convenient for drug delivery and treatment, and has a high encapsulation rate and near-infrared fluorescent dye loading. It can be seen from Examples 7-8 that when the carrier concentration is too low, the system is unstable, resulting in a smaller particle size, a lower encapsulation rate, and a reduced near-infrared fluorescent dye loading; when the carrier concentration is too high, the interaction between the systems is enhanced, and aggregation is prone to occur, resulting in an increase in particle size, a decrease in encapsulation rate, and a decrease in near-infrared fluorescent dye loading. It can be seen from Examples 9-10 that when the volume of the mixed solution is too much, aggregation is prone to occur, resulting in an increase in particle size, a decrease in encapsulation rate, and a decrease in near-infrared fluorescent dye loading; when the volume of the alkaline solution is too much, the concentration of the drug carrier is diluted, the system is unstable, the particle size becomes larger, the encapsulation rate decreases, and the near-infrared fluorescent dye loading decreases. It can be seen from Example 11 that when the pH value of the alkaline solution is too low, the acetal bond of the carrier breaks prematurely and nanoparticles cannot be formed.

[0100] Test Example 2: Detection of the Tumor Cell Fluorescence Imaging Capability of FPLC@IR

[0101] Human tongue squamous cell carcinoma Cal27 was cultured at 1×10 5 The cells were inoculated in a confocal dish and cultured overnight. When the cell density reached about 60%, the culture medium was aspirated and fresh culture medium containing free IR780 or FPLC@IR prepared in Example 1 was added (the concentration of IR780 was 1 μg / mL). After incubation for 4 hours, the drug-containing culture medium was removed and 1 mL of phenol red-free DMEM culture medium was added. One group of culture dishes containing FPLC@IR was irradiated with 808 nm laser (0.5 W / cm 2, 5 minutes). Remove the culture medium, dilute the mitochondrial green fluorescence (MTG) probe with PBS at a dilution ratio of 1:10000, take 1 mL and add it to the confocal dish, incubate with the cells in a 37°C incubator for 30 minutes to stain the mitochondria. 5 minutes before the end of the incubation, add the corresponding volume of Hoechst 33342 staining solution at a dilution ratio of 1:100 to stain the cell nucleus. Remove the PBS containing the MTG probe and Hoechst 33342 staining solution, and then wash twice with PBS, each time for 3 minutes. Use a laser confocal microscope to observe the fluorescence of IR780 in the cells, record and plot.

[0102] The results of FPLC@IR imaging of Cal27 cells are shown in Figure 4 As shown, cells treated with FPLC@IR exhibited obvious IR780 fluorescence, and laser irradiation could further promote the release of IR780 and further enhance the fluorescence signal, demonstrating the tumor cell light-responsive fluorescence imaging properties of FPLC@IR.

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

[0104] Human tongue squamous cell carcinoma Cal27 cells were plated at 3×10 5 The cells were seeded in a 6-well plate and cultured for 24 hours. Three replicate wells were set up for each group. The culture medium was removed, and fresh culture medium containing free IR780 (1 μg / mL) or FPLC@IR prepared in Example 1 was added. The culture medium without drugs was used as the control group. Incubated in a 37°C incubator for 4 hours. The drug-containing culture medium was removed, the cells were collected by trypsin digestion, the cell pellet was collected by centrifugation, 500 μL of phenol red-free DMEM was added to resuspend the cells, and the illumination group was irradiated with 808 nm laser (0.6 W / cm 2 ,5min).

[0105] Centrifuge at 1200rpm for 5 minutes, discard the supernatant, add 195μL Annexin V-FITC binding solution to resuspend the cells again, add 5μL Annexin V-FITC and 10μL propidium iodide staining solution, and mix well. After incubation at room temperature in the dark for 15 minutes, perform cell apoptosis flow cytometry. Calculate the cell apoptosis rate (early apoptosis + late apoptosis) between different groups, and perform statistical analysis of the differences between groups.

[0106] The tumor cell killing effect of FPLC@IR is as follows Figure 5 As shown, compared with the control group, the FPLC@IR prepared in Example 1 can significantly induce tumor cell apoptosis under laser irradiation and exhibit obvious cytotoxicity, which indicates that the FPLC@IR of the present invention has a good tumor killing effect under laser irradiation.

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

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

[0109] The results of the in vivo fluorescence imaging test of tumor cells are as follows: Figure 6 As shown, FPLC@IR exhibits time-dependent biodistribution, and the fluorescence signal intensity in the tumor area gradually increases with time. 24 hours after injection, the fluorescence intensity in the tumor site reaches a peak, indicating that FPLC@IR can achieve tumor fluorescence imaging at the in vivo level.

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

[0111] Human tongue squamous cell carcinoma Cal27 was inoculated subcutaneously into the back of nude mice to establish the CDX model. 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), ③ FPLC@IR laser irradiation group (abbreviated as FPLC@IR+L) (IR780: 1.5 mg / kg) prepared in Example 1. 24 hours after tail vein injection, 808 nm laser (0.3 W / cm 2 All treatment groups were observed for 14 days, during which the tumor volume of mice was monitored every other day.

[0112] The results of the in vivo tumor inhibition test are as follows Figure 7 As shown, at the end of 14 days of treatment, the FPLC@IR laser irradiation group could effectively inhibit the growth of tumors compared with the saline group, indicating that the FPLC@IR of the present invention has an excellent anti-tumor effect under laser irradiation.

[0113] The applicant declares that the present invention illustrates a pH and light dual-responsive nano-diagnostic agent and its preparation method and application through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0114] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0115] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A pH and light dual-responsive nano-diagnostic agent, characterized in that: The pH and light dual-responsive nano-diagnostic agent comprises a carrier formed by self-assembly of an amphiphilic compound as shown in Formula I, and a near-infrared fluorescent dye encapsulated in the carrier; 2. The pH and light dual-responsive nano-diagnostic agent according to claim 1, characterized in that: The near-infrared fluorescent dye is selected from any one of IR780, IR825, IR755 or IR813.

3. The pH and light dual-responsive nano-diagnostic agent according to claim 1 or 2, characterized in that: The hydrated particle size of the pH and light dual-responsive nano-diagnostic agent is 100-500 nm; Preferably, the loading amount of the near-infrared fluorescent dye in the pH and light dual-responsive nano-diagnostic and therapeutic agent is 4%-7%.

4. A method for preparing a pH and light dual-responsive nano-diagnostic agent according to any one of claims 1 to 3, 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; The mixed solution is mixed with an alkaline solution and self-assembled to obtain the pH and light dual-responsive nano-diagnostic agent.

5. The method for preparing the pH and light dual-responsive nano-diagnostic agent according to claim 4, characterized in that: The concentration of the amphiphilic compound in the mixed solution is 10-50 mg / mL; Preferably, the concentration of the near-infrared fluorescent dye in the mixed solution is 0.5-10 mg / mL.

6. The method for preparing the pH and light dual-responsive nano-diagnostic agent according to claim 4 or 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.

7. The method for preparing a pH and light dual-responsive nano-diagnostic agent according to any one of claims 4 to 6, characterized in that: The alkaline solution is selected from any one or a combination of at least two of an aqueous sodium hydroxide solution, an aqueous potassium hydroxide solution, an aqueous sodium carbonate solution, an aqueous sodium bicarbonate solution or aqueous ammonia; Preferably, the pH value of the alkaline solution is 6.8-10; Preferably, the pH value of the alkaline solution is 7-8.

8. The method for preparing a pH and light dual-responsive nano-diagnostic agent according to any one of claims 4 to 7, characterized in that: The volume ratio of the mixed solution to the alkaline solution is 1:5-1:20; Preferably, the mixing method is: adding the mixed solution dropwise into the alkaline solution under stirring; Preferably, the stirring speed is 500-4000 rpm.

9. The method for preparing a pH and light dual-responsive nano-diagnostic agent according to any one of claims 4 to 8, characterized in that: The mixing temperature is 20-40°C; Preferably, after the self-assembly is completed, the reaction product is purified; Preferably, the purification method is dialysis; Preferably, the purification time is 8-12 hours.

10. Use of the pH and light dual-responsive nano-diagnostic and therapeutic agent according to any one of claims 1 to 3 in tumor treatment and tumor fluorescence imaging.

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