Photo-thermal and hypoxia dual-sensitive nano carrier material as well as preparation method and application thereof

Abstract

The invention discloses a photo-thermal and hypoxia dual-sensitive nano carrier material and a preparation method and application thereof, and relates to the field of nano materials, the preparation method comprises the following steps: S1, preparing a carboxymethyl chitosan grafted azobenzene product CMCS-Azo; s2, respectively preparing an L-glutamic acid solution and a carboxymethyl chitosan grafted azobenzene nanoparticle solution, then stirring the L-glutamic acid solution and the carboxymethyl chitosan grafted azobenzene nanoparticle solution for reaction, and performing post-treatment on an obtained reaction solution b to obtain L-Gr-CMCS-Azo; s3, adding the L-Gr-CMCS-Azo into the DMF solution in which the metal compound is dissolved, carrying out ultrasonic treatment and stirring, carrying out post-treatment on the obtained reaction solution c, and finally, carrying out vacuum drying to obtain a solid L-Gr-CMCS-Azo (at) M; and S4, dissolving the L-Grub-CMCS-Azo (at) M material in a DMSO solution, carrying out ultrasonic assisted dissolution, slowly dropwise adding into deionized water, stirring at a low speed, filtering with a microfiltration membrane, and freeze-drying to obtain the nano-carrier material. Therefore, the photo-thermal and hypoxia dual-sensitive nano-carrier material as well as the preparation method and the application thereof are of great significance to the development of anti-cancer therapy.

Description

Technical Field

[0001] The present invention relates to the field of nanomaterials, and in particular to a nanocarrier material that is dual-sensitive to light, heat and hypoxia, and a preparation method and application thereof. Background Art

[0002] The development of cancer chemotherapy resistance remains a major challenge in cancer treatment. Ferroptosis is a unique type of cell death that differs from other forms of cell death in terms of mechanism and morphology. Ferroptosis plays a key role in inhibiting tumor growth and has provided new opportunities for the treatment of chemotherapy-insensitive tumors in recent years (Wang Y., Wu X., Ren Z. et al. Overcoming cancer chemotherapy resistance by the inducti on offerroptosis. Drug Resist. Updat. 2023; 66: 100916.). Hypoxia is a typical feature of most tumors. Tumor hypoxia stems from the fact that tumor cells consume a large amount of oxygen for rapid proliferation, while tumor blood vessels are malformed and abnormal, limiting the adequate supply of oxygen. Its unique microenvironment and physiological characteristics make it a specific target for cancer therapy. Many enzymes involved in reduction reactions and electron transfer are upregulated in hypoxic cancer cells (Kang N., Son S., Min S. et al. Stimuli-responsive ferroptosis for cancer therapy. Chem. Soc. Rev. 2023; 52: 3955-3972.). At the same time, photothermal therapy (PTT) is an emerging non-invasive cancer treatment method. In PTT, near-infrared laser light is absorbed by chromophores, converting light energy into heat energy to destroy tumor tissue (Malekzadeh R., Mortezazadeh T., Abdulsahib W.K. et al. Nanoarchitecture-based photothermalablation of cancer: A systematic review. Environ. Res. 2023; 236: 116526.). In addition, under high temperature conditions, the generation of reactive oxygen species (ROS) can also induce apoptosis of cancer cells. Tissue homeostasis relies on apoptosis to eliminate harmful cells. Other cell changes caused by high temperature (protein folding, cytoskeleton destruction, Golgi apparatus fragmentation, mitochondria, lysosomes and even nuclear damage) contribute to cell death (deMelo-Diogo D., Pais-Silva C., Dias D. R. et al. Strategies to improve cancer photothermal therapy mediated by nanomaterials. Adv. Healthcare Mater. 2017; 24: 1700073.). The present invention mainly designs and synthesizes a type of nanocarrier material that is dually sensitive to light, heat and hypoxia.Under hypoxic conditions, such materials can be cracked to release metals, undergo a Fenton-like reaction, and promote the death of tumor cells in vivo. On the other hand, under near-infrared (NIR) light irradiation, the present invention can generate enough heat to ablate tumors, which is of great significance for treating tumors. Summary of the invention

[0003] In order to solve the above problems, the present invention provides a nano-carrier material that is dual-sensitive to light, heat and hypoxia, and a preparation method and application thereof.

[0004] The present invention provides a nano-carrier material that is dual-sensitive to light, heat and hypoxia, and its structural formula is as follows:

[0005]

[0006] Wherein, M is one or more of Fe, Cu, Mn, and Co, and n is an integer.

[0007] The present invention provides a method for preparing a nano-carrier material that is dual-sensitive to light, heat and hypoxia, comprising the following preparation steps:

[0008] S1 Preparation of carboxymethyl chitosan grafted azobenzene product CMCS-Azo;

[0009] S2 prepares L-glutamic acid solution and carboxymethyl chitosan grafted azobenzene nanoparticle solution respectively, slowly drops the L-glutamic acid solution into the carboxymethyl chitosan grafted azobenzene nanoparticle CMCS-Azo NPs solution, stirs for reaction, and then post-treats the obtained reaction solution b to obtain the compound L-Gru-CMCS-Azo;

[0010] S3: Stirring the compound containing the metal element M and the DMF solution of N-dimethylformamide until it is clear, then adding L-Gru-CMCS-Azo to the DMF solution containing the above compound, ultrasonicating, stirring, and post-treating the obtained reaction solution c, and finally vacuum drying to obtain the solid L-Gru-CMCS-Azo@M;

[0011] S4: The L-Gru-CMCS-Azo@M material was dissolved in a dimethyl sulfoxide (DMSO) solution, dropped into deionized water, stirred, filtered with a microporous filter membrane, and freeze-dried to obtain the nanocarrier material L-Gru-CMCS-Azo@M NPs.

[0012] Preferably, the preparation step of step S1 comprises: stirring azobenzene-4,4-dicarboxylic acid Azo and dimethyl sulfoxide solution DMSO until clear, then dissolving carboxymethyl chitosan CMCS in a mixed solution of azobenzene-4,4-dicarboxylic acid Azo and dimethyl sulfoxide solution DMSO, condensing and refluxing, stirring and dissolving, and then dripping into anhydrous ethanol, stirring and swelling, adding EDC and NHS thereto, refluxing, stirring and reacting, and post-treating the obtained reaction solution a to obtain a carboxymethyl chitosan grafted azobenzene product CMCS-Azo; step S2 The preparation method of L-glutamic acid solution is as follows: dissolving L-glutamic acid in deionized water and stirring until it is clear, and then adjusting the pH of the solution to 7.3-7.5 with KOH or NaOH to obtain the L-glutamic acid solution; the preparation method of carboxymethyl chitosan grafted azobenzene nanoparticle solution is as follows: ultrasonically treating the carboxymethyl chitosan grafted azobenzene product CMCS-Azo in deionized water and dimethyl sulfoxide solution, adjusting the solution pH to 6.0 with KOH or NaOH, and stirring to obtain the carboxymethyl chitosan grafted azobenzene nanoparticle CMCS-Azo NPs solution.

[0013] Preferably, in step S1, the post-treatment method of the reaction solution a is: the reaction solution a is rotary evaporated and cooled to room temperature, then dialyzed, and freeze-dried to obtain the carboxymethyl chitosan grafted azobenzene product CMCS-Azo.

[0014] Preferably, in step S2, the post-treatment method of the reaction solution b is: the reaction solution b is centrifuged to collect the precipitate, and then the precipitate is ultrasonically dispersed in deionized water, and then the precipitate is centrifuged to collect the precipitate, and the process is repeated three times to remove the unattached L-glutamic acid, and the compound L-Gru-CMCS-Azo is obtained by vacuum drying.

[0015] Preferably, in step S1, the volume amount of the solvent in the dimethyl sulfoxide solution is 90-185 mL / g based on the mass of azobenzene-4,4-dicarboxylic acid Azo; the volume amount of the deionized water is 970-4878 mL / g based on the mass of carboxymethyl chitosan CMCS; and the volume amount of the anhydrous ethanol solvent is 4800-5000 mL / g based on the mass of carboxymethyl chitosan CMCS.

[0016] Preferably, in step S2, during the process of dissolving L-glutamic acid in deionized water and stirring until it is clear, the volume of deionized water used is 302-453 mL / g based on the mass of carboxymethyl chitosan grafted azobenzene CMCS-Azo; during the process of ultrasonicating the carboxymethyl chitosan grafted azobenzene product CMCS-Azo in deionized water and dimethyl sulfoxide solution DMSO, the volume of deionized water used is 84-170 mL / g based on the mass of L-glutamic acid.

[0017] Preferably, in step S3, the post-treatment process of the obtained reaction liquid c is: the reaction liquid c is centrifuged to collect the precipitate, the precipitate is ultrasonically dispersed in anhydrous ethanol, and then the precipitate is centrifuged to collect the precipitate, and the process is repeated three times, and the solid L-Gru-CMCS-Azo@M is obtained by vacuum drying.

[0018] Preferably, the volume amount of DMF solvent is 125-252 mL / g based on the mass of the compound L-Gru-CMCS-Azo.

[0019] The present invention provides an application of the above-mentioned photothermal and hypoxia dual-sensitive nanocarrier material in anti-tumor. Under near-infrared irradiation, the near-infrared laser is absorbed by the metal group, converting the light energy into heat energy to destroy tumor tissue. During the photothermal therapy, the metal nanoparticles increase the generation of reactive oxygen and damage DNA and mitochondria.

[0020] The present invention adopts the above-mentioned photothermal and hypoxia dual-sensitive nanocarrier material and its preparation method and application, which has the following beneficial effects:

[0021] (1) Through the azo-modified structure, the compound can effectively release drugs through the reductive cleavage of the nitrogen-nitrogen double bond under hypoxic conditions, and can release active drugs in the tumor area in a targeted manner, reducing toxicity and side effects to normal cells.

[0022] (2) Under the irradiation of near-infrared light (808nm), it can convert light energy into heat energy, effectively destroying tumor tissue. This photothermal effect can not only directly kill tumor cells, but also further damage the DNA and mitochondria of tumor cells by increasing the generation of reactive oxygen species, inducing cell apoptosis.

[0023] (3) As iron-loaded nanoparticles, they can release iron ions inside tumor cells to induce ferroptosis, which is a very effective anti-cancer mechanism. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Synthesis and characterization of the nanocarrier materials of this application;

[0025] Figure 2 This is the cumulative N=N cleavage diagram of L-Gru-CMCS-Azo@Fe of the present application in 0mM GSH, 10mM GSH and 25mM GSH environments;

[0026] Figure 3 The photothermal heating curve of L-Gru-CMCS-Azo@M under NIR irradiation of different powers (808nm). DETAILED DESCRIPTION

[0027] The present invention provides a nano-carrier material that is dual-sensitive to light, heat and hypoxia, and its structural formula is as follows:

[0028]

[0029] Wherein, M is one or more of Fe, Cu, Mn, and Co, and n is an integer.

[0030] The present invention provides a method for preparing a nano-carrier material that is dual-sensitive to light, heat and hypoxia, comprising the following preparation steps:

[0031] S1 Preparation of carboxymethyl chitosan grafted azobenzene product (CMCS-Azo): Azobenzene-4,4-dicarboxylic acid (Azo) of formula (3) and dimethyl sulfoxide solution (DMSO) are stirred until clear, then carboxymethyl chitosan CMCS is dissolved in a mixed solution of azobenzene-4,4-dicarboxylic acid Azo and dimethyl sulfoxide solution DMSO, condensed and refluxed, stirred and dissolved, and then dripped into anhydrous ethanol, stirred and swollen, EDC and NHS are added thereto, refluxed and stirred for reaction, the obtained reaction solution a is rotary evaporated and cooled to room temperature, and then placed in a dialysis bag for dialysis, and the water is changed every 4 hours. Finally, the liquid in the dialysis bag is freeze-dried to obtain a carboxymethyl chitosan grafted azobenzene product (CMCS-Azo). CS-Azo); in step S1, the volume amount of the solvent in the dimethyl sulfoxide solution is usually 90-185 mL / g based on the mass of azobenzene-4,4-dicarboxylic acid (Azo); the volume amount of the deionized water is usually 970-4878 mL / g based on the mass of carboxymethyl chitosan (CMCS); the volume amount of the anhydrous ethanol solvent is usually 4800-5000 mL / g based on the mass of carboxymethyl chitosan (CMCS);

[0032] S2: dissolving L-glutamic acid of formula (5) in deionized water and stirring until it is clear, the volume of deionized water used is usually 302-453 mL / g based on the mass of carboxymethyl chitosan grafted azobenzene (CMCS-Azo), and then adjusting the pH of the solution to 7.3-7.5 with KOH or NaOH to obtain an L-glutamic acid solution; ultrasonically treating the carboxymethyl chitosan grafted azobenzene product (CMCS-Azo) in deionized water and dimethyl sulfoxide solution, and then adjusting the pH of the solution to 6.0 with KOH or NaOH (0.1 M), the volume of deionized water used is usually 84-170 mL / g based on the mass of L-glutamic acid, and stirring after ultrasonication to obtain a solution of carboxymethyl chitosan grafted azobenzene nanoparticles (CMCS-Azo NPs) as shown in formula (4), and then slowly dripping the L-glutamic acid solution onto the carboxymethyl chitosan grafted azobenzene nanoparticles (CMCS-Azo NPs); NPs) solution, stirred for reaction, centrifuged to collect the precipitate obtained by the reaction solution b, and then ultrasonically dispersed the precipitate in deionized water, and then centrifuged to collect the precipitate, repeated three times, removed the unattached L-glutamic acid, and vacuum dried to obtain the compound (L-Gru-CMCS-Azo) as shown in formula (6);

[0033] S3: Stirring the compound containing the metal element M and the N-dimethylformamide (DMF) solution until it is clear, then adding L-Gru-CMCS-Azo to the DMF solution containing the above compound, ultrasonicating, stirring, collecting the precipitate by centrifugation of the obtained reaction solution c, and then ultrasonically dispersing the precipitate in anhydrous ethanol, and then centrifugally collecting the precipitate, repeating three times, and vacuum drying to obtain a solid (L-Gru-CMCS-Azo@M); the volume amount of the DMF solvent is usually 125-252mL / g based on the mass of the compound (L-Gru-CMCS-Azo);

[0034] S4: The L-Gru-CMCS-Azo@M material is dissolved in a dimethyl sulfoxide solution (DMSO) with ultrasonic dissolution aid, and then the solution is aspirated with a syringe and dripped into deionized water and stirred at a low speed for 5-6 days. The DMSO is taken away by volatile water. After 5-6 days, the solution is filtered with a microporous filter membrane and freeze-dried to obtain a nanocarrier material (L-Gru-CMCS-Azo@M NPs) as shown in formula (1).

[0035] The present invention provides a method for preparing a nanocarrier material that is dually sensitive to photothermal and hypoxia. The prepared nanocarrier material is used in anti-tumor applications. Under near-infrared irradiation, the near-infrared laser is absorbed by the metal group, converting light energy into heat energy to destroy tumor tissue. During photothermal therapy, metal nanoparticles increase the generation of reactive oxygen species, damaging DNA and mitochondria. The reaction formula of the present application is as follows:

[0036]

[0037] Example 1

[0038] In a 500ml round-bottom flask with a reflux condenser and an electric stirrer, add 54.048mg (0.2mmol) of azobenzene-4,4-dicarboxylic acid (Azo) and 5ml of dimethyl sulfoxide solution (DMSO), stir until clear, then use a 10ml syringe to draw this solution and drop it into 40ml of deionized water, one drop for 2s, 650rpm. Then weigh 41mg of carboxymethyl chitosan (CMCS) and dissolve it in the above solution. The mixture is condensed and refluxed at 60℃, and stirred and dissolved for 6h. After 6h, use a 10ml syringe to draw this solution and slowly drop it into 200ml of anhydrous ethanol, one drop for 2s, 600rpm. After the reaction is stirred (swelled) for 12h, 153.36mg (0.8mmol) of EDC and 115.09 (1mmol) of NHS are added thereto, and the reaction solution is refluxed and stirred at 60℃ for 12h. After the reaction was completed, the reaction solution was rotary evaporated at 60°C for 30 minutes, and after the reaction solution was cooled to room temperature, it was placed in a 2000Da dialysis bag for 4 days, and the water was changed every 4 hours. Finally, the liquid in the dialysis bag was freeze-dried to obtain the carboxymethyl chitosan grafted azobenzene product (CMCS-Azo).

[0039] 58.852 mg (0.4 mmol) of L-glutamic acid and 5 ml of deionized water were added to a 25 ml round-bottom flask, stirred at 50 ° C until clear, and then the solution pH was adjusted to 7.4 with KOH (0.1 M), stirred for 12 h, and L-glutamic acid solution was obtained. In another 50 ml round-bottom flask, 66.224 mg (0.1 mmol) of carboxymethyl chitosan grafted azobenzene product CMCS-Azo was added, and ultrasonicated in 20 ml of deionized water and 2 ml of ultra-dry dimethyl sulfoxide solution (DMSO) for 20 min, and then the solution pH was adjusted to 6.0 with NaOH (0.1 M), stirred at 50 ° C for 12 h, and a carboxymethyl chitosan grafted azobenzene nanoparticle (CMCS-Azo NPs) solution as shown in formula (4) was obtained. After 12 hours, the L-glutamic acid solution was slowly dripped into the solution of carboxymethyl chitosan grafted azobenzene nanoparticles (CMCS-Azo NPs) using a constant pressure funnel, one drop per second, and the reaction solution was stirred at 50°C for 12 hours. After the reaction was completed, the precipitate was collected by centrifugation at 10,000 rpm for 15 minutes, and then the precipitate was ultrasonically dispersed in deionized water, followed by centrifugation at 10,000 rpm for 15 minutes to collect the precipitate, and repeated three times to remove the ungrafted L-glutamic acid. Finally, the compound (L-Gru-CMCS-Azo) shown in formula (6) was obtained by vacuum drying.

[0040] Add 42.808 mg (0.2 mmol) of ferrocene carboxaldehyde and 10 ml of N, N-dimethylformamide (DMF) solution to a 25 ml round-bottom flask and stir until clear. Then add 79.137 mg (0.1 mmol) of L-Gru-CMCS-Azo to the DMF solution containing ferrocene carboxaldehyde and ultrasonicate for 20 minutes. After the ultrasonication, the reaction solution was stirred at a low speed at 30 ° C for 12 hours. After 12 hours, the precipitate was collected by centrifugation at 10000 rpm for 10 minutes, and then the precipitate was ultrasonically dispersed in anhydrous ethanol, followed by centrifugation at 10000 rpm for 15 minutes to collect the precipitate, and repeated three times. Finally, the black solid obtained by vacuum drying is the desired product, namely L-Gru-CMCS-Azo@Fe.

[0041] Weigh 10 mg of L-Gru-CMCS-Azo@Fe material and dissolve it in 10 ml of ultra-dry dimethyl sulfoxide solution (DMSO), and dissolve it by ultrasonication for 20 min. Then use a 10 ml syringe to draw this solution and drop it into 200 ml of deionized water (600 rpm), one drop for 2 seconds, and stir it at a low speed for 5-6 days. The volatile water takes away the DMSO. After 5-6 days, filter it with an 800 nm microporous filter membrane and freeze-dry it. Finally, freeze-dry it to obtain the nanocarrier material (L-Gru-CMCS-Azo@FeNPs) shown in formula (1).

[0042] Example 2

[0043] In a 500ml round-bottom flask with a reflux condenser and an electric stirrer, add 54.048mg (0.2mmol) of azobenzene-4,4-dicarboxylic acid (Azo) and 5ml of dimethyl sulfoxide solution (DMSO), stir until clear, then use a 10ml syringe to draw this solution and drop it into 40ml of deionized water, one drop for 2s, 650rpm. Then weigh 41mg of carboxymethyl chitosan (CMCS) and dissolve it in the above solution. The mixture is condensed and refluxed at 50℃, and stirred and dissolved for 6h. After 6h, use a 10ml syringe to draw this solution and slowly drop it into 200ml of anhydrous ethanol, one drop for 2s, 600rpm. After the reaction is stirred (swelled) for 12h, 153.36mg (0.8mmol) of EDC and 115.09 (1mmol) of NHS are added thereto, and the reaction solution is refluxed and stirred at 50℃ for 12h. After the reaction was completed, the reaction solution was rotary evaporated at 60°C for 30 minutes, and after the reaction solution was cooled to room temperature, it was placed in a 2000Da dialysis bag for 4 days, and the water was changed every 4 hours. Finally, the liquid in the dialysis bag was freeze-dried to obtain the carboxymethyl chitosan grafted azobenzene product (CMCS-Azo).

[0044] 58.852 mg (0.4 mmol) of L-glutamic acid and 5 ml of deionized water were added to a 25 ml round-bottom flask, and the mixture was stirred at 35 °C until clear. The pH of the solution was then adjusted to 7.4 with KOH (0.1 M) and stirred for 12 h to obtain an L-glutamic acid solution. 66.224 mg (0.1 mmol) of carboxymethyl chitosan grafted azobenzene product CMCS-Azo was added to another 50 ml round-bottom flask, and the mixture was ultrasonically treated in 20 ml of deionized water and 2 ml of dimethyl sulfoxide solution (DMSO) for 20 min. The pH of the solution was then adjusted to 6.0 with KOH (0.1 M) and stirred at 30 °C for 12 h to obtain a solution of carboxymethyl chitosan grafted azobenzene nanoparticles (CMCS-Azo NPs) as shown in formula (4). After 12 hours, the L-glutamic acid solution was slowly dripped into the solution of carboxymethyl chitosan grafted azobenzene nanoparticles (CMCS-Azo NPs) using a constant pressure funnel, one drop per second, and the reaction solution was stirred at 30°C for 12 hours. After the reaction was completed, the precipitate was collected by centrifugation at 10,000 rpm for 15 minutes, and then the precipitate was ultrasonically dispersed in deionized water, and then the precipitate was collected by centrifugation at 10,000 rpm for 15 minutes, and repeated three times to remove the ungrafted L-glutamic acid. Finally, the compound (L-Gru-CMCS-Azo) shown in formula (6) was obtained by vacuum drying.

[0045] Add 42.808 mg (0.2 mmol) of cyclopentadienyl iron aldehyde and 10 ml of N, N-dimethylformamide (DMF) solution to a 25 ml round-bottom flask and stir until clear. Then add 79.137 mg (0.1 mmol) of L-Gru-CMCS-Azo to the DMF solution containing cyclopentadienyl iron aldehyde and ultrasonicate for 20 minutes. After the ultrasonication, the reaction solution was stirred at a low speed at 50 ° C for 12 hours. After 12 hours, the precipitate was collected by centrifugation at 10000 rpm for 10 minutes, and then the precipitate was ultrasonically dispersed in anhydrous ethanol, followed by centrifugation at 10000 rpm for 15 minutes to collect the precipitate, and repeated three times. Finally, the solid obtained by vacuum drying is the desired product, namely L-Gru-CMCS-Azo@Fe.

[0046] Weigh 10 mg of L-Gru-CMCS-Azo@Fe material and dissolve it in 10 ml of dimethyl sulfoxide solution (DMSO), and dissolve it by ultrasonication for 20 min. Then use a 10 ml syringe to draw this solution and drop it into 200 ml of deionized water (600 rpm), one drop for 2 seconds, and stir it at a low speed for 5-6 days. The volatile water takes away the DMSO. After 5-6 days, filter it with an 800 nm microporous filter membrane and freeze-dry it. Finally, freeze-dry it to obtain the nanocarrier material (L-Gru-CMCS-Azo@FeNPs) shown in formula (1).

[0047] Example 3

[0048] In a 500ml round-bottom flask with a reflux condenser and an electric stirrer, add 54.048mg (0.2mmol) azobenzene-4,4-dicarboxylic acid (Azo) and 5ml dimethyl sulfoxide solution (DMSO), stir until clear, then use a 10ml syringe to draw this solution and drop it into 40ml deionized water, one drop for 2s, 650rpm. Then weigh 41mg carboxymethyl chitosan (CMCS) and dissolve it in the above solution. The mixture is condensed and refluxed at 50℃, and stirred and dissolved for 6h. After 6h, use a 10ml syringe to draw this solution and slowly drop it into 200ml anhydrous ethanol, one drop for 2s, 600rpm. After the reaction is stirred (swelled) for 12h, 153.36mg (0.8mmol) EDC and 115.09 (1mmol) NHS are added thereto, and the reaction solution is refluxed and stirred at 90℃ for 12h. After the reaction was completed, the reaction solution was rotary evaporated at 60°C for 30 minutes, and after the reaction solution was cooled to room temperature, it was placed in a 2000Da dialysis bag for 4 days, and the water was changed every 4 hours. Finally, the liquid in the dialysis bag was freeze-dried to obtain the carboxymethyl chitosan grafted azobenzene product (CMCS-Azo).

[0049] 58.852 mg (0.4 mmol) of L-glutamic acid and 10 ml of deionized water were added to a 25 ml round-bottom flask, and the mixture was stirred at 35 °C until clear. The pH of the solution was then adjusted to 7.4 with KOH (0.1 M) and stirred for 12 h to obtain an L-glutamic acid solution. 66.224 mg (0.1 mmol) of carboxymethyl chitosan grafted azobenzene product (CMCS-Azo) was added to another 50 ml round-bottom flask, and the mixture was ultrasonically treated in 30 ml of deionized water and 2 ml of dimethyl sulfoxide solution (DMSO) for 20 min. The pH of the solution was then adjusted to 6.0 with KOH (0.1 M) and stirred at 30 °C for 12 h to obtain a solution of carboxymethyl chitosan grafted azobenzene nanoparticles (CMCS-Azo NPs) as shown in formula (4). After 12 hours, the L-glutamic acid solution was slowly dripped into the solution of carboxymethyl chitosan grafted azobenzene nanoparticles (CM CS-Azo NPs) using a constant pressure funnel, one drop per second, and the reaction solution was stirred at 60°C for 12 hours. After the reaction was completed, the precipitate was collected by centrifugation at 10,000 rpm for 15 minutes, and then the precipitate was ultrasonically dispersed in deionized water, and then the precipitate was collected by centrifugation at 10,000 rpm for 15 minutes, and the precipitate was repeated three times to remove the ungrafted L-glutamic acid. Finally, the compound (L-Gru-CMCS-Azo) shown in formula (6) was obtained by vacuum drying.

[0050] Add 40.812 mg (0.2 mmol) of cyclopentadienyl manganese formaldehyde and 10 ml of N, N-dimethylformamide (DMF) solution to a 25 ml round-bottom flask and stir until clear. Then add 79.137 mg (0.1 mmol) of L-Gru-CMCS-Azo to the DMF solution containing cyclopentadienyl manganese formaldehyde and ultrasonicate for 20 minutes. After the ultrasonication, the reaction solution was stirred at a low speed at 60 ° C for 12 hours. After 12 hours, the precipitate was collected by centrifugation at 10000 rpm for 10 minutes, and then the precipitate was ultrasonically dispersed in anhydrous ethanol, followed by centrifugation at 10000 rpm for 15 minutes to collect the precipitate, and repeated three times. Finally, the solid obtained by vacuum drying is the desired product, namely L-Gru-CMCS-Azo@Mn.

[0051] Weigh 10 mg of L-Gru-CMCS-Azo@Mn material and dissolve it in 10 ml of dimethyl sulfoxide solution (DMSO), and dissolve it by ultrasonication for 20 min. Then use a 10 ml syringe to draw this solution and drop it into 200 ml of deionized water (600 rpm), one drop every 2 seconds, and stir it at a low speed for 5-6 days, and evaporate the water to take away the DMSO. After 5-6 days, filter it with an 800 nm microporous filter membrane and freeze-dry it. Finally, freeze-dry it to obtain the nanocarrier material (L-Gru-CMCS-Azo@MnNPs) shown in formula (1).

[0052] Example 4

[0053] In a 500ml round-bottom flask with a reflux condenser and an electric stirrer, add 54.048mg (0.2mmol) of azobenzene-4,4-dicarboxylic acid (Azo) and 10ml of dimethyl sulfoxide solution (DMSO), stir until clear, then use a 10ml syringe to draw this solution and drop it into 40ml of deionized water, one drop every 2s, 650rpm. Then weigh 41mg of carboxymethyl chitosan (CMCS) and dissolve it in the above solution. The mixture is condensed and refluxed at 50℃, and stirred and dissolved for 6h. After 6h, use a 10ml syringe to draw this solution and slowly drop it into 200ml of anhydrous ethanol, one drop every 2s, 600rpm. After the reaction is stirred (swelled) for 12h, 153.36mg (0.8mmol) of EDC and 115.09 (1mmol) of NHS are added thereto, and the reaction solution is refluxed and stirred at 50℃ for 12h. After the reaction was completed, the reaction solution was rotary evaporated at 60°C for 30 minutes, and after the reaction solution was cooled to room temperature, it was placed in a 2000Da dialysis bag for 4 days, and the water was changed every 4 hours. Finally, the liquid in the dialysis bag was freeze-dried to obtain the carboxymethyl chitosan grafted azobenzene product (CMCS-Azo).

[0054] 58.852 mg (0.4 mmol) of L-glutamic acid and 10 ml of deionized water were added to a 25 ml round-bottom flask, and the mixture was stirred at 35 °C until clear. The pH of the solution was then adjusted to 7.4 with KOH (0.1 M) and stirred for 12 h to obtain an L-glutamic acid solution. 66.224 mg (0.1 mmol) of carboxymethyl chitosan grafted azobenzene product (CMCS-Azo) was added to another 50 ml round-bottom flask, and the mixture was ultrasonically treated in 30 ml of deionized water and 2 ml of dimethyl sulfoxide solution (DMSO) for 20 min. The pH of the solution was then adjusted to 6.0 with KOH (0.1 M) and stirred at 30 °C for 12 h to obtain a solution of carboxymethyl chitosan grafted azobenzene nanoparticles (CMCS-Azo NPs) as shown in formula (4). After 12 hours, the L-glutamic acid solution was slowly dripped into the solution of carboxymethyl chitosan grafted azobenzene nanoparticles (CM CS-Azo NPs) using a constant pressure funnel, one drop per second, and the reaction solution was stirred at 60°C for 12 hours. After the reaction was completed, the precipitate was collected by centrifugation at 10,000 rpm for 15 minutes, and then the precipitate was ultrasonically dispersed in deionized water, and then the precipitate was collected by centrifugation at 10,000 rpm for 15 minutes, and the precipitate was repeated three times to remove the ungrafted L-glutamic acid. Finally, the compound (L-Gru-CMCS-Azo) shown in formula (6) was obtained by vacuum drying.

[0055] Add 42.808 mg (0.2 mmol) of ferrocene carboxaldehyde and 20 ml of N, N-dimethylformamide (DMF) solution to a 25 ml round-bottom flask and stir until clear. Then add 79.137 mg (0.1 mmol) of L-Gru-CMCS-Azo to the DMF solution containing ferrocene carboxaldehyde and ultrasonicate for 20 minutes. After the ultrasonication, the reaction solution was stirred at a low speed at 50 ° C for 12 hours. After 12 hours, the precipitate was collected by centrifugation at 10000 rpm for 10 minutes, and then the precipitate was ultrasonically dispersed in anhydrous ethanol, followed by centrifugation at 10000 rpm for 15 minutes to collect the precipitate, and repeated three times. Finally, the black solid obtained by vacuum drying is the desired product, namely L-Gru-CMCS-Azo@Fe.

[0056] Weigh 10 mg of L-Gru-CMCS-Azo@Fe material and dissolve it in 10 ml of dimethyl sulfoxide solution (DMSO), and dissolve it by ultrasonication for 20 min. Then use a 10 ml syringe to draw this solution and drop it into 200 ml of deionized water (600 rpm), one drop for 2 seconds, and stir it at a low speed for 5-6 days. The volatile water takes away the DMSO. After 5-6 days, filter it with an 800 nm microporous filter membrane and freeze-dry it. Finally, freeze-dry it to obtain the nanocarrier material (L-Gru-CMCS-Azo@FeNPs) shown in formula (1).

[0057] Example 5

[0058] In a 500ml round-bottom flask with a reflux condenser and an electric stirrer, add 54.048mg (0.2mmol) of azobenzene-4,4-dicarboxylic acid (Azo) and 5ml of dimethyl sulfoxide solution (DMSO), stir until clear, then use a 10ml syringe to draw this solution and drop it into 200ml of deionized water, one drop every 2s, 650rpm. Then weigh 41mg of carboxymethyl chitosan (CMCS) and dissolve it in the above solution. The mixture is condensed and refluxed at 50℃, and stirred and dissolved for 6h. After 6h, use a 10ml syringe to draw this solution and slowly drop it into 200ml of anhydrous ethanol, one drop every 2s, 600rpm. After the reaction is stirred (swelled) for 12h, 153.36mg (0.8mmol) of EDC and 115.09 (1mmol) of NHS are added thereto, and the reaction solution is refluxed and stirred at 50℃ for 12h. After the reaction was completed, the reaction solution was rotary evaporated at 60°C for 30 minutes, and after the reaction solution was cooled to room temperature, it was placed in a 2000Da dialysis bag for 4 days, and the water was changed every 4 hours. Finally, the liquid in the dialysis bag was freeze-dried to obtain the carboxymethyl chitosan grafted azobenzene product (CMCS-Azo).

[0059] 58.852 mg (0.4 mmol) of L-glutamic acid and 10 ml of deionized water were added to a 25 ml round-bottom flask, and the mixture was stirred at 35 °C until clear. The pH of the solution was then adjusted to 7.4 with KOH (0.1 M) and stirred for 12 h to obtain an L-glutamic acid solution. 66.224 mg (0.1 mmol) of carboxymethyl chitosan grafted azobenzene product (CMCS-Azo) was added to another 50 ml round-bottom flask, and the mixture was ultrasonically treated in 30 ml of deionized water and 2 ml of dimethyl sulfoxide solution (DMSO) for 20 min. The pH of the solution was then adjusted to 6.0 with KOH (0.1 M) and stirred at 30 °C for 12 h to obtain a solution of carboxymethyl chitosan grafted azobenzene nanoparticles (CMCS-Azo NPs) as shown in formula (4). After 12 hours, the L-glutamic acid solution was slowly dripped into the solution of carboxymethyl chitosan grafted azobenzene nanoparticles (CM CS-Azo NPs) using a constant pressure funnel, one drop per second, and the reaction solution was stirred at 60°C for 12 hours. After the reaction was completed, the precipitate was collected by centrifugation at 10,000 rpm for 15 minutes, and then the precipitate was ultrasonically dispersed in deionized water, and then the precipitate was collected by centrifugation at 10,000 rpm for 15 minutes, and the precipitate was repeated three times to remove the ungrafted L-glutamic acid. Finally, the compound (L-Gru-CMCS-Azo) shown in formula (6) was obtained by vacuum drying.

[0060] Add 42.808 mg (0.2 mmol) of ferrocene carboxaldehyde and 20 ml of N, N-dimethylformamide (DMF) solution to a 25 ml round-bottom flask and stir until clear. Then add 79.137 mg (0.1 mmol) of L-Gru-CMCS-Azo to the DMF solution containing ferrocene carboxaldehyde and ultrasonicate for 20 min. After the ultrasonication, the reaction solution was stirred at a low speed at 60 ° C for 12 h. After 12 h, centrifuge at 10000 rpm for 10 min to collect the precipitate, then ultrasonically disperse the precipitate in anhydrous ethanol, and then centrifuge at 10000 rpm for 15 min to collect the precipitate, and repeat three times. Finally, the black solid obtained by vacuum drying is the desired product, namely L-Gru-CMCS-Azo@Fe.

[0061] Weigh 10 mg of L-Gru-CMCS-Azo@Fe material and dissolve it in 10 ml of dimethyl sulfoxide solution (DMSO), and dissolve it by ultrasonication for 20 min. Then use a 10 ml syringe to draw this solution and drop it into 200 ml of deionized water (600 rpm), one drop for 2 seconds, and stir it at a low speed for 5-6 days. The volatile water takes away the DMSO. After 5-6 days, filter it with an 800 nm microporous filter membrane and freeze-dry it. Finally, freeze-dry it to obtain the nanocarrier material (L-Gru-CMCS-Azo@FeNPs) shown in formula (1).

[0062] Figure 1 The synthesis and characterization of the nanocarrier material of the present application; the nanocarrier material of the present invention, i.e., the compound as shown in formula (1) of the present invention, has the advantage of hypoxia sensitivity. Due to the rapid proliferation of cancer cells, the tumor microenvironment is hypoxic, and the GSH content is higher than that of normal cells. The nano drug carrier particles (1) carry azo-modified substances, and under hypoxic conditions, the nitrogen-nitrogen double bond undergoes reduction cleavage to release the drug. The degree of hypoxia in tumor cells affects the degree of cleavage of the nitrogen-nitrogen double bond. Figure 2 shown.

[0063] The nanocarrier material as shown in formula (1) of the present invention can also be used as a nanomedicine for tumor photothermal combined with iron therapy to induce ferroptosis in tumor cells. The application method is as follows: weigh 1 mg of the nanocarrier material as shown in formula (1), place it in a culture dish, and irradiate it with 808nm near-infrared radiation of 0.8W, 0.9W, 1W, 1.1W, and 1.2W respectively. The environment is closed, and the position and height of the lamp are fixed. Use an infrared thermal imager to take pictures every 5 seconds, record the temperature of each shot, and keep the distance between the lens and the culture dish consistent during each shot. The result is shown in the figure below. Figure 3 shown.

[0064] Therefore, the present invention provides a nanocarrier material that is dual-sensitive to light, heat and hypoxia, and a preparation method and application thereof, and provides a nanocarrier material that is highly efficient, highly targeted, and has few toxic side effects, which is of great significance to the development of anti-cancer therapy.

[0065] In the description of this specification, the description with reference to the terms "an experimental example", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the experimental example or example are included in at least one experimental example or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same experimental example or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more experimental examples or examples in a suitable manner.

[0066] Finally, it should be noted that the above experimental examples are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred experimental examples, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A nanocarrier material that is dual-sensitive to light, heat and hypoxia, characterized in that: Its structural formula is as follows: Wherein, M is one or more of Fe, Cu, Mn, and Co, and n is an integer.

2. The method for preparing a photothermal and hypoxia-sensitive nanocarrier material according to claim 1, characterized in that: The method comprises the following preparation steps: S1 Preparation of carboxymethyl chitosan grafted azobenzene product CMCS-Azo; S2 prepares L-glutamic acid solution and carboxymethyl chitosan grafted azobenzene nanoparticle solution respectively, slowly drops the L-glutamic acid solution into the carboxymethyl chitosan grafted azobenzene nanoparticle CMCS-Azo NPs solution, stirs for reaction, and then post-treats the obtained reaction solution b to obtain the compound L-Gru-CMCS-Azo; S3: Stirring the compound containing the metal element M and the DMF solution of N-dimethylformamide until it is clear, then adding L-Gru-CMCS-Azo to the DMF solution containing the above compound, ultrasonicating, stirring, and post-treating the obtained reaction solution c, and finally vacuum drying to obtain the solid L-Gru-CMCS-Azo@M; S4: The L-Gru-CMCS-Azo@M material was dissolved in a dimethyl sulfoxide (DMSO) solution, dropped into deionized water, stirred, filtered with a microporous filter membrane, and freeze-dried to obtain the nanocarrier material L-Gru-CMCS-Azo@M NPs.

3. The method for preparing a nanocarrier material that is dual-sensitive to light, heat and hypoxia according to claim 2, characterized in that: The preparation step of step S1 comprises: stirring azobenzene-4,4-dicarboxylic acid Azo and dimethyl sulfoxide solution DMSO until clear, then dissolving carboxymethyl chitosan CMCS in a mixed solution of azobenzene-4,4-dicarboxylic acid Azo and dimethyl sulfoxide solution DMSO, condensing and refluxing, stirring and dissolving, and then dripping into anhydrous ethanol, stirring and swelling, adding EDC and NHS thereto, refluxing, stirring and reacting, and post-treating the obtained reaction solution a to obtain a carboxymethyl chitosan grafted azobenzene product CMCS-Azo; in step S2, L -The preparation method of glutamic acid solution is as follows: dissolving L-glutamic acid in deionized water and stirring until it is clear, and then adjusting the pH of the solution to 7.3-7.5 with KOH or NaOH to obtain L-glutamic acid solution; The preparation method of carboxymethyl chitosan grafted azobenzene nanoparticle solution is as follows: after ultrasonicating the carboxymethyl chitosan grafted azobenzene product CMCS-Azo in deionized water and dimethyl sulfoxide solution, adjusting the solution pH to 6.0 with KOH or NaOH, and stirring to obtain carboxymethyl chitosan grafted azobenzene nanoparticle CMCS-Azo NPs solution.

4. The method for preparing a nanocarrier material that is dual-sensitive to light, heat and hypoxia according to claim 2, characterized in that: In step S1, the post-treatment method of the reaction solution a is: the reaction solution a is rotary evaporated and cooled to room temperature, then dialyzed, and freeze-dried to obtain a carboxymethyl chitosan grafted azobenzene product CMCS-Azo.

5. The method for preparing a nanocarrier material that is dual-sensitive to light, heat and hypoxia according to claim 2, characterized in that: In step S2, the post-treatment method of the reaction solution b is: the reaction solution b is centrifuged to collect the precipitate, and then the precipitate is ultrasonically dispersed in deionized water, and then the precipitate is centrifuged to collect the precipitate, and the process is repeated three times to remove the unattached L-glutamic acid, and the compound L-Gru-CMCS-Azo is obtained by vacuum drying.

6. The method for preparing a photothermal and hypoxia-sensitive nanocarrier material according to claim 3, characterized in that: In the step S1, the volume amount of the solvent in the dimethyl sulfoxide solution is 90-185 mL / g based on the mass of azobenzene-4,4-dicarboxylic acid Azo; the volume amount of the deionized water is 970-4878 mL / g based on the mass of carboxymethyl chitosan CMCS; and the volume amount of the anhydrous ethanol solvent is 4800-5000 mL / g based on the mass of carboxymethyl chitosan CMCS.

7. The method for preparing a photothermal and hypoxia-dual-sensitive nanocarrier material according to claim 2, characterized in that: In step S2, during the process of dissolving L-glutamic acid in deionized water and stirring until it is clear, the volume of deionized water used is 302-453 mL / g based on the mass of carboxymethyl chitosan grafted azobenzene CMCS-Azo; during the process of ultrasonicating the carboxymethyl chitosan grafted azobenzene product CMCS-Azo in deionized water and dimethyl sulfoxide solution DMSO, the volume of deionized water used is 84-170 mL / g based on the mass of L-glutamic acid.

8. The method for preparing a nano-carrier material that is dual-sensitive to light, heat and hypoxia according to claim 2, characterized in that: In step S3, the post-treatment process of the obtained reaction liquid c is as follows: the reaction liquid c is centrifuged to collect the precipitate, the precipitate is ultrasonically dispersed in anhydrous ethanol, and then the precipitate is centrifuged to collect the precipitate, and this is repeated three times, and the solid L-Gru-CMCS-Azo@M is obtained by vacuum drying.

9. The method for preparing a nano-carrier material that is dual-sensitive to light, heat and hypoxia according to claim 2, characterized in that: The volume amount of DMF solvent used is 125-252 mL / g based on the mass of the compound L-Gru-CMCS-Azo.

10. The use of a photothermal and hypoxia dual-sensitive nanocarrier material in anti-tumor according to claim 1, characterized in that: Under near-infrared irradiation, near-infrared laser is absorbed by metal groups, It can be converted into heat energy to destroy tumor tissue. During photothermal therapy, metal nanoparticles increase the generation of reactive oxygen species. Damage to DNA and mitochondria.

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