Nanometer delivery system with sonodynamic and immune regulation dual effects and preparation method and application thereof

Abstract

The invention relates to the field of medicines, and discloses a nano delivery system with sonodynamic and immune regulation dual effects as well as a preparation method and application of the nano delivery system. The nano delivery system comprises a precious metal modified inorganic nano sound-sensitive agent, a linker structure containing disulfide bonds and / or sulfydryl and an immunomodulatory unit, the precious metal modified inorganic nano sound-sensitive agent has a Janus heterostructure, and the immunomodulatory unit is in conjugation connection with the precious metal through the disulfide bonds and / or sulfydryl in the linker structure. The nano delivery system disclosed by the invention can release immunomodulatory molecules at a lesion part according to the characteristics of a lesion microenvironment or characteristic enzyme response to play an immunoregulation role, and the inorganic nano sound-sensitive agent can generate a large amount of ROS (Reactive Oxygen Species) through ultrasonic treatment to achieve a treatment purpose, so that sonodynamic-immune integrated treatment is realized.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and particularly to a nano-delivery system with dual functions of sonodynamic and immunomodulatory effects, and a preparation method and use thereof. Background Art

[0002] Sonodynamic therapy (SDT) is a new non-invasive treatment method developed from photodynamic therapy (PDT), and has advantages such as non-invasiveness, strong penetrability, and high specificity. Compared with photodynamic therapy, sonodynamic therapy has deeper tissue penetration and weaker systemic toxic reactions, which broadens its clinical application value. Titanium dioxide nanoparticles (TiO 2 NPs), as representatives of inorganic nano-sensitizers, exhibit high physiochemical stability and biocompatibility, and are good potential carriers for disease treatment drugs. However, the electrons (e 2 ) and holes (h - ) structures generated by pure TiO + NPs will quickly recombine, resulting in a decrease in the quantum yield of reactive oxygen species (ROS), thereby limiting the application of traditional TiO 2 particles in SDT. For this reason, researchers have optimized the inorganic sensitizer TiO 2 , and formed composite nanoparticles by integrating TiO 2 with noble metals (such as Pt, Au, Ag), in order to use the surface plasmon resonance of noble metals to increase the absorption spectrum of the composite material, promote the interfacial electron transfer in the composite, thereby avoiding electron-hole recombination and increasing the quantum yield of ROS. However, in traditional composite nanoparticles induced by ultraviolet lamps, tiny metals are likely to aggregate and nucleate on the surface of TiO 2 , which cannot play the role of avoiding electron-hole recombination, and thus is not conducive to the improvement of the ROS quantum yield.

[0003] Immunotherapy, as a hot field in the research and development of biopharmaceuticals in recent years, has small side effects on the human body and has a long-term anti-disease immune effect, and is an ideal auxiliary treatment mode for diseases. Due to the special microenvironment formed by the special metabolic mode of diseases, different immune suppression or promotion phenomena of the body are formed. Therefore, an important way to treat diseases is to regulate the special immune abnormalities of diseases, that is, to use immunomodulators for treatment. However, free small molecule preparation drugs lack selective targeting of the lesion site, have large toxic and side effects, and are easily cleared by blood circulation, thereby reducing the treatment effect of diseases.

[0004] It can be seen that for a variety of diseases (including but not limited to tumors, inflammation, cardiovascular diseases, Alzheimer's disease), a single treatment mode is difficult to completely cure, and there are defects such as drug resistance, incomplete treatment, and immunosuppression. Therefore, overcoming the deficiencies of a single treatment mode, seeking the combined treatment of multiple therapies, and improving the treatment effect of diseases are the directions of efforts in the field of nanomedicine research at the present stage. Summary of the Invention

[0005] In view of this, the present invention provides a nanodelivery system with dual sonodynamic and immunomodulatory effects to solve the problems of non-targeted selectivity, large toxic and side effects, and low treatment efficiency of immunomodulators.

[0006] In a first aspect, the present invention provides a nanodelivery system with dual sonodynamic and immunomodulatory effects, comprising a noble metal-modified inorganic nanosensitizer, a linker structure containing disulfide bonds and / or thiol groups, and an immunomodulatory unit;

[0007] The noble metal-modified inorganic nanosensitizer has a Janus heterostructure;

[0008] The immunomodulatory unit is conjugated to the noble metal through the disulfide bonds and / or thiol groups in the linker structure.

[0009] In an alternative embodiment, the immunomodulator undergoes a condensation reaction with a compound containing disulfide bonds and / or thiol groups to obtain an immunomodulatory unit linked with a linker structure;

[0010] The chemical structure of the immunomodulator contains a carboxyl group, and the chemical structure of the compound containing disulfide bonds and / or thiol groups further contains an amino group or a hydroxyl group; or, the chemical structure of the immunomodulator contains an amino group or a hydroxyl group, and the chemical structure of the compound containing disulfide bonds and / or thiol groups further contains a carboxyl group.

[0011] In an alternative embodiment, the compound containing disulfide bonds and / or thiol groups is selected from at least one of lipoic acid and 4-mercaptobutyric acid.

[0012] In an alternative embodiment, the immunomodulator is at least one of 6-diazo-5-oxo-L-norleucine, atezolizumab, pembrolizumab, BMS-8, BMS-200, pidotimod, ubenimex, GS-4224, sulfamonomethoxine, sulfamethizole, INCB086550, and mannan peptide.

[0013] In an alternative embodiment, the noble metal-modified inorganic nano-sonosensitizer is prepared by the method of focusing the excitation light, and its process conditions include: using a continuous Ar+ laser, the beam power range at the laser head outlet gradually increases from PL = 0 to 300 mW, and irradiating the reaction solution with the laser in the light control mode.

[0014] In an alternative embodiment, the inorganic nano-sonosensitizer is TiO 2 、ZnO、Fe 2 O 3 、SnO 2 at least one of them.

[0015] In an alternative embodiment, the noble metal is at least one of Au, Ag, and Pt.

[0016] In an alternative embodiment, the particle size of the noble metal-modified inorganic nano-sonosensitizer is 50 nm - 300 nm.

[0017] In a second aspect, the present invention also provides a preparation method of a nano-delivery system with dual sonodynamic and immunomodulatory effects, including the following steps:

[0018] Prepare an immunomodulatory unit connected with a linker structure: dissolve the immunomodulator in a non-polar organic solvent, and add a compound containing a disulfide bond and / or a thiol group for condensation reaction; wherein, the chemical structure of the immunomodulator contains a carboxyl group, and the chemical structure of the compound containing a disulfide bond and / or a thiol group also contains an amino group or a hydroxyl group; or, the chemical structure of the immunomodulator contains an amino group or a hydroxyl group, and the chemical structure of the compound containing a disulfide bond and / or a thiol group also contains a carboxyl group;

[0019] Prepare a noble metal-modified inorganic nano-sonosensitizer: uniformly mix an aqueous solution containing the inorganic nano-sonosensitizer with a noble metal salt solution under an inert atmosphere, add a hole scavenger and a pH regulator, use a continuous Ar+ laser, and gradually increase the beam power range at the laser head outlet from PL = 0 to PL = 300 mW, and carry out the reaction by laser irradiation in the light control mode;

[0020] Disperse the prepared noble metal-modified inorganic nano-sonosensitizer and the immunomodulatory unit connected with a linker structure in ultrapure water, stir, and carry out a conjugation reaction to obtain a nano-delivery system with dual sonodynamic and immunomodulatory effects.

[0021] In an alternative embodiment, the molar ratio of the immunomodulator to the compound containing a disulfide bond and / or a thiol group is 1:1 - 3.

[0022] In an alternative embodiment, the molar ratio of the inorganic nano-sonosensitizer to the noble metal in the noble metal salt solution is 1:0.3 - 1.0.

[0023] In an alternative embodiment, the pH value of the reaction system of the aqueous solution containing the inorganic nano-sonosensitizer and the noble metal salt solution is 3.5.

[0024] In an alternative embodiment, the temperature of the conjugation reaction is 10°C - 50°C, and the reaction time is 12 h - 48 h.

[0025] In a third aspect, the nano-delivery system with dual sonodynamic and immunomodulatory effects according to the first aspect of the present invention or the nano-delivery system with dual sonodynamic and immunomodulatory effects prepared by the preparation method according to the second aspect of the present invention is used in the preparation of drugs for treating tumors, inflammation, cardiovascular diseases, and Alzheimer's disease.

[0026] In a fourth aspect, the nano-delivery system with dual sonodynamic and immunomodulatory effects according to the first aspect of the present invention or the nano-delivery system with dual sonodynamic and immunomodulatory effects prepared by the preparation method according to the second aspect of the present invention is used in the preparation of type I reactive oxygen species generators and / or type II reactive oxygen species generators.

[0027] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0028] The nano-delivery system with dual sonodynamic and immunomodulatory effects provided by the present invention, by using a noble metal-modified inorganic nano-sonosensitizer with a Janus heterostructure, can avoid the aggregation of noble metal particles on the surface of the sonosensitizer. Thus, the surface plasmon resonance of the noble metal can be utilized to increase the absorption spectrum of the sonosensitizer, promote the interfacial electron transfer of the sonosensitizer, prevent the rapid recombination of electrons and holes, and effectively improve the ROS quantum yield. At the same time, the present invention also introduces a linker structure containing disulfide bonds and / or thiol groups into the chemical structure of the immunomodulator, and uses this linker structure to conjugate and connect with the noble metal on the surface of the inorganic nano-sonosensitizer, thereby coupling the sonosensitizer and the immunomodulator together. Thus, the immunomodulator can be specifically enriched at the lesion site by virtue of the characteristics of the sonosensitizer nano-material, solving the problems of non-targeted selectivity, large toxic and side effects, and low treatment efficiency of the immunomodulator.

[0029] The nano-delivery system with dual sonodynamic and immunomodulatory effects provided by the present invention can release immunomodulatory molecules in response to the characteristics or characteristic enzymes of the lesion microenvironment at the lesion site, thereby exerting an immunoregulatory effect; and, the inorganic nano-sonosensitizer, as the source of sonodynamic therapy, can generate ROS through ultrasonic treatment to achieve the treatment purpose, realizing sonodynamic-immunological integrated therapy.

[0030] The preparation method of the nano-delivery system with dual functions of sonodynamic and immunomodulatory provided by the present invention has a short synthetic route, mild reaction conditions, simple operation, high efficiency, rapidity and good reproducibility, and is suitable for large-scale industrial production. Description of the Drawings

[0031] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0032] Figure 1 It is the mass spectrometry diagram of LA-DON prepared in Example 1 of the present invention;

[0033] Figure 2 It is the TEM image of TiO 2 -Au Janus heterostructure prepared in Example 1 of the present invention;

[0034] Figure 3 It is the schematic diagram of the synthetic route of TiO 2 -Au@DON prepared in Example 1 of the present invention;

[0035] Figure 4a It is the ROS ultraviolet absorption spectrum diagram of TiO 2 prepared in Example 1 of the present invention;

[0036] Figure 4b It is the ROS ultraviolet absorption spectrum diagram of TiO 2 -Au prepared in Example 1 of the present invention;

[0037] Figure 5 It is the superoxide anion detection diagram of the nano-delivery system prepared in Example 1 of the present invention;

[0038] Figure 6 It is the confocal laser scanning microscopy image of cell uptake of TiO 2 -Au@DON prepared in Example 1 of the present invention;

[0039] Figure 7 It is the flow cytometry image of cell uptake of TiO 2 -Au@DON prepared in Example 1 of the present invention;

[0040] Figure 8 It is the enrichment diagram of TiO 2 -Au@DON at the lesion site in mice prepared in Example 1 of the present invention;

[0041] Figure 9 Distribution map of TiO 2 -Au@DON in various organs of mice;

[0042] Figure 10 Tumor volume growth curve of the nano-delivery system prepared in Example 1 of the present invention in a tumor disease model;

[0043] Figure 11 Detection map of CD8+ and CD4+ T cells of the nano-delivery system prepared in Example 1 of the present invention in a tumor disease model;

[0044] Figure 12 Quantitative analysis map of BCL-6, CD62L and CD122 of the nano-delivery system prepared in Example 1 of the present invention in a tumor disease model. Detailed implementation manners

[0045] The following examples are provided to better further understand the present invention. They are not limited to the best implementation manners, and do not limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with those of other existing technologies falls within the protection scope of the present invention.

[0046] For those not specifying specific experimental steps or conditions in the examples, the operations or conditions of the conventional experimental steps described in the literature in this field can be followed. For the reagents or instruments not specifying the manufacturer, they are all conventional reagent products that can be obtained through commercial purchase.

[0047] To solve the problems existing in the above related technologies, according to the first aspect of the present invention, a nano-delivery system with dual functions of sonodynamic and immunomodulation is provided, including a noble metal-modified inorganic nano-sensitizer, a linker structure containing disulfide bonds and / or thiol groups, and an immunomodulation unit;

[0048] The noble metal-modified inorganic nano-sensitizer has a Janus heterostructure;

[0049] The immunomodulation unit is conjugated to the noble metal through the disulfide bonds and / or thiol groups in the linker structure.

[0050] The nano-delivery system with dual sonodynamic and immunomodulatory effects provided by the present invention uses an inorganic nano-sensitizer modified with noble metals having a Janus heterostructure, which can avoid the aggregation of noble metal particles on the surface of the sensitizer. Thus, the surface plasmon resonance of noble metals can be utilized to increase the absorption spectrum of the sensitizer, promote the interfacial electron transfer of the sensitizer, prevent the rapid recombination of electrons and holes, and effectively improve the ROS quantum yield. At the same time, the present invention also introduces a linker structure containing disulfide bonds and / or thiol groups into the chemical structure of the immunomodulator, and conjugates the linker structure with the noble metals on the surface of the inorganic nano-sensitizer, thereby coupling the sensitizer with the immunomodulator. Thus, the immunomodulator can be specifically enriched at the lesion site by virtue of the characteristics of the nano-sensitizer material, solving the problems of non-targeted selectivity, large toxic and side effects, and low treatment efficiency of the immunomodulator.

[0051] Therefore, the nano-delivery system with dual sonodynamic and immunomodulatory effects provided by the present invention can release immunomodulatory molecules in response to the characteristics or characteristic enzymes of the lesion microenvironment at the lesion site, thereby exerting an immunomodulatory effect; and, the inorganic nano-sensitizer, as the source of sonodynamic therapy, can generate ROS through ultrasonic treatment to achieve the therapeutic purpose, realizing sonodynamic-immunological integrated therapy.

[0052] In an alternative embodiment, the immunomodulator undergoes a condensation reaction with a compound containing disulfide bonds and / or thiol groups to obtain an immunomodulatory unit linked with a linker structure;

[0053] The chemical structure of the immunomodulator contains a carboxyl group, and the chemical structure of the compound containing disulfide bonds and / or thiol groups further contains an amino group or a hydroxyl group; or, the chemical structure of the immunomodulator contains an amino group or a hydroxyl group, and the chemical structure of the compound containing disulfide bonds and / or thiol groups further contains a carboxyl group.

[0054] It can be understood that the nano-delivery system of the present invention can be applied to any immunomodulator containing carboxyl groups, amino groups or hydroxyl groups that are easy to modify and link the linker, including but not limited to 6-diazo-5-oxo-L-norleucine (DON), atezolizumab, pembrolizumab, carboxyl-containing immunomodulators (such as BMS-8, BMS-200, pidotimod, ubenimex), carboxyl-containing immunomodulators (such as GS-4224, sulfamonomethoxine, sulfamethizole), hydroxyl-containing immunomodulators (INCB086550, mannan peptide), etc. These exemplified immunomodulators generally have significant curative effects.

[0055] Accordingly, the compound containing a disulfide bond and / or a thiol group must also contain a group capable of undergoing condensation with the carboxyl group, amino group, or hydroxyl group of the chemical structure of the immunomodulator. In an alternative embodiment, the compound containing a disulfide bond and / or a thiol group is selected from at least one of lipoic acid and 4-mercaptobutyric acid. The structures of these compounds are relatively simple and have little effect on the immunomodulator.

[0056] In an alternative embodiment, the noble metal-modified inorganic nanosensitizer is prepared by the method of focusing excitation light, and its process conditions include: using a continuous Ar + laser, the beam power range at the laser head outlet gradually increases from PL = 0 to PL = 300 mW, and the reaction solution is irradiated with laser under the light control mode.

[0057] Compared with the traditional ultraviolet lamp and xenon lamp induction methods, the method of focusing excitation light adopted in the present invention can prepare a single-metal-modified inorganic nanoparticle with a Janus heterostructure, thereby avoiding the aggregation of noble metal particles on the surface of the sensitizer. Therefore, the surface plasmon resonance of the noble metal can be used to increase the absorption spectrum of the sensitizer, promote the interfacial electron transfer of the sensitizer, prevent the rapid recombination of electrons and holes, effectively improve the quantum yield of ROS, and further improve the therapeutic effect.

[0058] In an alternative embodiment, the inorganic nanosensitizer is TiO 2 、ZnO、Fe 2 O 3 、SnO 2 or at least one of them. These sensitizers not only have good sonodynamic efficacy but also have good biocompatibility.

[0059] In an alternative embodiment, the noble metal is at least one of Au, Ag, and Pt. These noble metals have low toxicity, and the surface plasmon resonance of the noble metal can enhance the separation of electrons and holes of the sensitizer, thereby enhancing the sonodynamic effect.

[0060] In an alternative embodiment, the particle size of the noble metal-modified inorganic nanosensitizer is 50 nm - 300 nm. Within this range, it can reach the lesion site well. If the particle size of the sensitizer is too small or too large, it is easily metabolized.

[0061] According to the second aspect of the present invention, a preparation method of a nano-delivery system having dual functions of sonodynamic and immunomodulation is provided, including the following steps:

[0062] Prepare an immunomodulatory unit connected with a linker structure: dissolve the immunomodulator in a non-polar organic solvent, and add a compound containing a disulfide bond and / or a thiol group for condensation reaction;

[0063] Among them, the chemical structure of the immunomodulator contains a carboxyl group, and the chemical structure of the compound containing a disulfide bond and / or a thiol group further contains an amino group or a hydroxyl group; or, the chemical structure of the immunomodulator contains an amino group or a hydroxyl group, and the chemical structure of the compound containing a disulfide bond and / or a thiol group further contains a carboxyl group;

[0064] Preparation of noble metal-modified inorganic nano-sonosensitizers: Mix an aqueous solution containing inorganic nano-sonosensitizers with a noble metal salt solution evenly under an inert atmosphere, add a hole scavenger and a pH regulator, and use a continuous Ar + laser, and the beam power range at the laser head outlet gradually increases from PL = 0 to 300 mW, and the reaction is carried out by laser irradiation in the light control mode;

[0065] Disperse the prepared noble metal-modified inorganic nano-sonosensitizers and the immunomodulatory unit connected with a linker structure in ultrapure water, stir, and carry out a conjugation reaction to obtain a nano-delivery system with dual sonodynamic and immunomodulatory effects.

[0066] The synthesis route of the preparation method of the present invention is short, the reaction conditions are mild, the operation is simple, efficient, fast, and the reproducibility is good, and it is suitable for large-scale industrial production.

[0067] In an optional embodiment, the molar ratio of the immunomodulator to the compound containing a disulfide bond and / or a thiol group is 1:1-3, which is beneficial to the progress of the reaction and maximizes the yield.

[0068] In an optional embodiment, the molar ratio of the inorganic nano-sonosensitizer to the noble metal in the noble metal salt solution is 1:0.3-1.0, which is beneficial to maximizing the yield and generating single-metal-modified nanoparticles.

[0069] In an optional embodiment, the pH value of the reaction system of the aqueous solution containing inorganic nano-sonosensitizers and the noble metal salt solution is 3.5, which is beneficial to the progress of the reaction and maximizes the yield.

[0070] In an optional embodiment, the hole scavenger is at least one of methanol, isopropanol, trichloroethylene, and n-propyl bromide, and the dosage of the hole scavenger is 0.1-5 mL. Its function is to effectively scavenge holes, thereby prolonging the service life of the material and improving the performance of the material.

[0071] In an optional embodiment, the pH regulator is an acidic aqueous solution, and its dosage is 1-10 mL. Its function is to adjust the acidity and alkalinity of the reaction system solution to facilitate the progress of the reaction and maximize the yield.

[0072] In an alternative embodiment, the mass ratio of the inorganic nano-sonosensitizer to the immunomodulatory unit linked with a linker structure is 50 to 500, so as to ensure that the immunomodulator can fully bind to the sonosensitizer.

[0073] In an alternative embodiment, the temperature of the conjugation reaction is 10°C - 50°C, and the reaction time is 12 h - 48 h, which is conducive to a complete reaction and improves the binding degree of the immunomodulator to the sonosensitizer.

[0074] Exemplarily, the present invention provides a preparation method of a TiO 2 -M@DON Janus nanoparticle complex, which generally includes the following steps:

[0075] 1) Synthesis of an immunomodulator (LA-DON) containing a disulfide bond or a mercapto group

[0076] ① Take 0.01 mol - 0.086 mol of fluorenylmethoxycarbonyl-aspartic acid-β-tert-butyl ester (Fmoc-Asp(OtBu)-OH) in a reaction flask, add 100 ml - 250 ml of absolute ethanol for dissolution. After thorough mixing, add 0.68 ml - 5.5 ml of thionyl chloride and react at room temperature.

[0077] ② Take 0.001 mol - 0.006 mol of the product obtained in the first step in a reaction flask, add 10 ml - 30 ml of dichloromethane and stir for dissolution, then add 0.1 ml - 0.7 ml of trifluoroacetic acid and react.

[0078] ③ Take 0.001 - 0.01 mol of the product obtained in the second step in a reaction flask, add 10 ml - 50 ml of tetrahydrofuran for dissolution, and add 1 - 4 equivalents of triethylamine; then add 1.0 - 3.0 equivalents of isobutyl chloroformate. After reacting for a period of time, perform preliminary suction filtration and concentration, dissolve with acetonitrile, and add 3.0 - 6.0 equivalents of trimethylsilyldiazomethane and react overnight.

[0079] ④ Take 0.1 mmol - 0.5 mmol of the product obtained in the third step in a reaction flask, dissolve it with 2 ml - 10 ml of dichloromethane, add 30 μL - 200 μL of piperidine and stir for reaction to obtain the immunomodulator DON.

[0080] ⑤ Place the immunomodulator DON obtained in the fourth step in a reaction flask, add N,N-dimethylformamide for dissolution, and then add 1 - 3 equivalents of lipoic acid or 4-mercaptobutyric acid for reaction to obtain LA-DON.

[0081] 2) Preparation of TiO 2 -M Janus heterostructure

[0082] ① First, 0.01 mol - 0.2 mol of titanium isopropoxide (TIPO) and 0.02 mol - 0.4 mol of triethanolamine (TEOA) are mixed and stirred under an argon stream. After reacting for 12 - 36 hours, 70 mL - 300 mL of deionized water is added to obtain a stock solution of [Ti 4+ . Then, 5 mL - 20 mL of this stock solution is diluted with 5 mL - 20 mL of water and grown at 50 °C - 100 °C for 24 hours. The solution is then transferred to an autoclave and grown at 120 °C - 200 °C for 72 hours. Finally, the autoclave is naturally cooled to room temperature, and the product is collected by centrifugation for 45 minutes. The precipitated product is washed several times by sonication / centrifugation with sodium hydroxide, nitric acid aqueous solution, and deionized water to obtain TiO 2 nanoparticles.

[0083] ② The prepared TiO 2 nanoparticles are formulated into a TiO 2 aqueous solution (10 - 30 mM, 0.3 - 1.0 mL), which is mixed uniformly with a noble metal salt solution (5 - 15 mM, 0.3 - 1.0 mL) under an argon stream. 0.5 - 1 mL of a hole scavenger and an acid solution (pH 3.3, 2.352 - 7.84 mL) are added. A continuous Ar + laser is used, and the power range of the light is gradually changed within 0 - 300 mW. Noble metal M (including but not limited to Au, Ag, Pt) particles are loaded onto the mesoporous titanium dioxide particles by laser exposure deposition to form 100 nm - 300 nm of TiO 2 -M Janus heterostructure nanoparticles.

[0084] 3) Preparation of TiO 2 -M@DON Janus nanoparticle complex (nanodelivery system)

[0085] The LA-DON prepared in step 1) and the TiO 2 -M Janus heterostructure nanoparticles prepared in step 2) are dispersed in ultrapure water. The immunomodulator DON (0.1 - 1 mg) is conjugated to the noble metal M surface of the TiO 2 -M Janus nanoparticles (5 - 500 mg) through a modified disulfide bond or thiol group after stirring, and finally a TiO 2 -M@DON Janus nanoparticle complex is obtained.

[0086] It is understandable that the above preparation method provided by the present invention can be applicable to any immunomodulator containing carboxyl, amino or hydroxyl groups that are easy to modify and link the linker, including but not limited to 6-diazo-5-oxo-L-norleucine (DON), atezolizumab, pembrolizumab, carboxyl-containing immunomodulators (such as BMS-8, BMS-200, pidotimod, ubenimex), carboxyl-containing immunomodulators (such as GS-4224, sulfamonomethoxine, sulfamethizole), hydroxyl-containing immunomodulators (INCB086550, mannan peptide), etc.

[0087] It is understandable that the above preparation method provided by the present invention is not only applicable to TiO 2 photosensitizer, but also applicable to ZnO, Fe 2 O 3 , SnO 2 and other photosensitizers.

[0088] According to the third aspect of the present invention, there is provided the use of the nano-delivery system having dual functions of sonodynamic and immunomodulatory described in the first aspect of the present invention or the nano-delivery system having dual functions of sonodynamic and immunomodulatory prepared by the preparation method described in the second aspect of the present invention in the preparation of drugs for treating tumors, inflammation, cardiovascular diseases, and Alzheimer's disease.

[0089] According to the fourth aspect of the present invention, there is provided the use of the nano-delivery system having dual functions of sonodynamic and immunomodulatory described in the first aspect of the present invention or the nano-delivery system having dual functions of sonodynamic and immunomodulatory prepared by the preparation method described in the second aspect of the present invention in the preparation of type I reactive oxygen species generators and / or type II reactive oxygen species generators.

[0090] The nano-delivery system having dual functions of sonodynamic and immunomodulatory provided by the present invention can release immunomodulatory molecules in response to the characteristics or characteristic enzymes of the lesion microenvironment at the lesion site, thereby exerting an immunomodulatory effect; and, the inorganic nano-photosensitizer, as the source of sonodynamic therapy, can generate ROS through ultrasonic treatment to achieve the therapeutic purpose, realizing sonodynamic-immunological integrated therapy.

[0091] The following further describes the present invention in detail with specific examples, and these examples should not be construed as limiting the scope claimed by the present invention.

[0092] Example 1

[0093] (1) Preparation of immunomodulatory small molecule LA-DON:

[0094] ① Take 17.7 g of Fmoc-Asp(OtBu)-OH in a reaction flask, add 180 ml of absolute ethanol for dissolution. After thorough mixing, add 2.75 ml of thionyl chloride and react at room temperature.

[0095] ② Place the 2.69 g of product obtained in step ① in a reaction flask, add 20 ml of dichloromethane and stir for dissolution, then add 0.7 ml of trifluoroacetic acid for reaction.

[0096] ③ Place the 2 g of product obtained in step ② in a reaction flask, add 25 ml of tetrahydrofuran for dissolution, and add 1.05 equivalents of triethylamine. Subsequently, add 1.05 equivalents of isobutyl chloroformate. After reacting for a period of time, conduct preliminary suction filtration and concentration. Then dissolve with 25 ml of acetonitrile and add 3.5 equivalents of trimethylsilyldiazomethane to react overnight.

[0097] ④ Place the 100 mg of product obtained in step ③ in a reaction flask, dissolve with 4 ml of dichloromethane, add 60 μL of piperidine and stir for reaction to obtain 6-diazo-5-oxo-L-norleucine (DON).

[0098] ⑤ Place the 200 mg of immunomodulator DON obtained in ④ in a reaction flask, add 2 mL of N,N-dimethylformamide for dissolution, and then add 1 equivalent of lipoic acid for reaction to obtain LA-DON, and its mass spectrum is as Figure 1 shown.

[0099] (2) Preparation of TiO 2 -Au Janus heterostructure:

[0100] ① First, mix 0.1 mol of TIPO and 0.2 mol of TEOA under an argon stream and stir for reaction for 24 hours. Then add 144 mL of deionized water to obtain a stock solution of [Ti 4+ . Dilute 10 mL of this stock solution with 10 mL of water in a 35 mL glass bottle and grow at 100 °C for 24 hours. Then transfer the solution to an autoclave and grow at 140 °C for 72 hours. Finally, naturally cool the autoclave to room temperature and collect the product by centrifugation for 45 minutes. The precipitated product is washed several times by sonication / centrifugation with sodium hydroxide (pH = 12), 2M HNO 3 aqueous solution and deionized water to obtain TiO 2 nanoparticles.

[0101] ② In the ultraviolet range of λ 0 = 363.8 nm and within the anatase bandgap (Eg ~ 3.2 eV) of TiO 2 , using continuous Ar +The laser, the beam power range at the laser head outlet can gradually increase from PL = 0 to 160 mW. Mix 10 mL of the reaction solution in an argon environment: prepare TiO aqueous solution (19 mM, 580 μL) diluted with 3.92 mL of HNO solution with a pH of 3.3, and mix it evenly with freshly prepared KAuCl aqueous solution (10 mM, 500 μL) under an argon stream, then add 0.5 mL of anaerobic hole scavenger methanol (volume fraction 50%), and adjust the pH to 3.5 with nitric acid (0.1 M). Under the light control mode, irradiate the mixture with laser, and prepare TiO-Au Janus heterostructure nanoparticles by laser exposure deposition. 3 aqueous solution 2 aqueous solution (19 mM, 580 μL), and mix it evenly with freshly prepared KAuCl 4 aqueous solution (10 mM, 500 μL) under an argon stream, then add 0.5 mL of anaerobic hole scavenger methanol (volume fraction 50%), and adjust the pH to 3.5 with nitric acid (0.1 M). Under the light control mode, irradiate the mixture with laser, and prepare TiO 2 -Au Janus heterostructure nanoparticles.

[0102] The electron micrograph of the purified TiO 2 -Au Janus heterostructure nanoparticles can be seen in Figure 2 , and it can be seen from Figure 2 that the shape of TiO 2 polyhedra is close to a rotating ellipsoid, their length is 34 ± 8 nm, width is 21 ± 4 nm, and at most 1 Au particle is connected to 1 TiO 2 particle, and the size of the connected Au particle is about 16 nm.

[0103] (3) Preparation of nanoparticle complex (nanodelivery system) and verification of confocal uptake

[0104] Add 20 mg of the immunomodulatory molecule LA-DON to 1 mL of the prepared TiO 2 -Au (0.05 mM) ultrapure aqueous solution, and stir the reaction for 2 days. After the reaction is completed, dialyze and concentrate the reaction solution to obtain TiO 2 -Au@DON Janus nanoparticle complex, and the schematic synthesis route can be seen in Figure 3 .

[0105] Experimental example

[0106] To verify the ability of TiO 2 and TiO 2 -Au complex to generate reactive oxygen species (ROS) under ultrasonic conditions, the present invention uses 1,3-diphenylisobenzofuran (DPBF) to detect it. DPBF is an in vitro ROS probe, which can be oxidized under the action of ROS, resulting in a decrease in the ultraviolet absorption intensity at about 410 nm. Set blanks, TiO 2 and TiO 2- Au in three groups. At a working concentration of 10 mM of DPBF, the ultraviolet intensity absorption at 410 nm of these three groups was detected respectively after 10 min, 20 min, 30 min, and 40 min of ultrasound. The results are as Figures 4a - 4b shown. The results indicate that TiO 2 (see Figure 4a ) and TiO 2 - Au (see Figure 4b ) can both generate ROS that oxidize DPBF after ultrasonic treatment. Detection with a UV spectrophotometer shows a trend of decreasing absorption at 410 nm in the ultraviolet region, and TiO 2 - Au generates more and decreases faster than TiO 2 , indicating that the modified sonosensitizer nanoparticles have a stronger sonodynamic effect.

[0107] Before conducting in vitro cell studies, a superoxide anion content detection kit was used to detect the superoxide anions (O 2 , TiO 2 - Au, and TiO 2 - Au@DON nanomaterials generated under ultrasonic (US) irradiation. As 2- shown, TiO Figure 5 , TiO 2 , and TiO 2 - Au and TiO 2 - Au@DON nanomaterials can generate a large amount of superoxide anions under US induction. Compared with TiO 2 NPs, the O 2 - Au and TiO 2 - Au@DON nanomaterials have an O 2- generation activity approximately twice that of TiO 2 NPs, indicating that the present invention has better therapeutic potential.

[0108] To verify cell uptake and in vivo distribution, a dye Cy5.5 with a thiol group was added during the preparation of the final preparation (TiO 2 - Au@DON). 4T1 cells were revived and the cultured cells were evenly seeded in 8-well chambers. After overnight culture, the medium was replaced with fresh DMEM medium containing TiO 2 - Au@DON nanoparticles. After incubation for 1 h, 2 h, and 4 h respectively, they were washed twice with PBS, the cell nuclei were stained, and the 8-well plate was placed under a laser scanning confocal microscope for observation. The results are as Figure 6 . As can be seen from Figure 6 , as the incubation time increases, more TiO 2 - Au@DON nanoparticles are taken up by the cells, showing a time-dependent cell uptake pattern.

[0109] 4T1 cells in good condition were inoculated in a 6-well plate. After being treated in the same manner as described above, they were detected by flow cytometry. The results are as Figure 7 shown. Figure 7 It was further demonstrated that with the increase of incubation time, the detected fluorescence intensity was higher and the cellular uptake was greater.

[0110] To verify the in vivo distribution of the nanoparticle complex, 4T1 cells were resuscitated, cultured, and collected. The cells were inoculated on the lower right side of Balb / c mice. When the tumor volume reached 300 - 500 mm 3 , the mice were randomly divided into 2 groups, and the nanoparticle complex TiO 2 -Au@DON / Cy5.5 containing a fluorescent dye was injected via the tail vein. Using the NIR-II fluorescence in vivo small animal imaging system, NIR-II images were captured at 1 h, 3 h, 6 h, 9 h, 12 h, 24 h, and 48 h respectively, with an excitation wavelength of 675 nm. The results are as Figures 8 - 9 .

[0111] Figure 8 In 2 , it can be seen from the fluorescence intensity of the lesion sites monitored at different times that the nanoparticle complex TiO

[0112] prepared in the present invention had a good enrichment effect at the lesion sites after intravenous injection. Figure 9 It can be seen from 2 that 48 hours after the nanoparticle complex TiO

[0113] prepared in the present invention was injected into mice, it had a good enrichment effect at the lesion sites and relatively less accumulation in other organ tissues. This indicates that the present invention can achieve good enrichment at the lesion sites and has relatively low toxic and side effects. 2 To evaluate the therapeutic effect of the prepared nanoparticle complex TiO Figure 10 -Au@DON on diseases, in this invention, taking the tumor model as an example, a 4T1 tumor animal model was established and treated. The therapeutic effect was judged by observing the size of the tumor volume during the treatment process. The results are as Figure 10 shown. It can be seen from 2 that the experimental group TiO 2 -Au@DON + US had the best therapeutic effect and could significantly inhibit the growth of tumors. The inhibitory effect of TiO

[0114] -Au + US on tumors demonstrated that the prepared nanoparticles indeed had sonodynamic therapeutic effects.To clarify the potential mechanisms of the immune response after different treatments, the T cells in tumor tissues were first evaluated by flow cytometry on the third day after ultrasound treatment. CD8+ and CD4+ T cells were observed and immune markers such as BCL-6, CD62L, and CD122 were monitored. The results are as Figures 11 - 12 shown. As can be seen from Figures 11 - 12 , the immune effect of the experimental group TiO 2 -Au@DON+US was better than that of other groups, with more activated T cells and obvious significant differences. This indicates that the nanoparticle complex TiO 2 -Au@DON of the present invention has good immunotherapeutic effects.

[0115] Obviously, the above embodiments are merely examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or variations can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. And the obvious changes or variations derived therefrom are still within the protection scope of the present invention.

Claims

1. A nano-delivery system with dual functions of sonodynamic and immunomodulatory effects, characterized in that, it includes a noble metal-modified inorganic nano-sensitizer, a linker structure containing disulfide bond and / or mercapto group, and an immunomodulatory unit; the noble metal-modified inorganic nano-sensitizer has a Janus heterostructure; the immunomodulatory unit is conjugated to the noble metal through the disulfide bond and / or mercapto group in the linker structure.

2. The nano-delivery system with dual functions of sonodynamic and immunomodulatory effects according to claim 1, characterized in that, the immunomodulator reacts with a compound containing disulfide bond and / or mercapto group through a condensation reaction to obtain an immunomodulatory unit connected with a linker structure; the chemical structure of the immunomodulator contains a carboxyl group, and the chemical structure of the compound containing disulfide bond and / or mercapto group also contains an amino group or a hydroxyl group; or, the chemical structure of the immunomodulator contains an amino group or a hydroxyl group, and the chemical structure of the compound containing disulfide bond and / or mercapto group also contains a carboxyl group.

3. The nano-delivery system with dual functions of sonodynamic and immunomodulatory effects according to claim 2, characterized in that, the compound containing disulfide bond and / or mercapto group is selected from at least one of lipoic acid and 4-mercaptobutyric acid; and / or, the immunomodulator is at least one of 6-diazo-5-oxo-L-norleucine, atezolizumab, pembrolizumab, BMS-8, BMS-200, pidotimod, ubenimex, GS-4224, sulfamonomethoxine, sulfamethizole, INCB086550, and mannan peptide.

4. The nano-delivery system with dual functions of sonodynamic and immunomodulatory effects according to claim 1, characterized in that, the noble metal-modified inorganic nano-sensitizer is prepared by the method of focusing excitation light, and its process conditions include: Using a continuous Ar + laser, the beam power at the laser head outlet gradually increases from PL = 0 to PL = 300 mW, and the reaction solution is irradiated with laser in the optical control mode.

5. The nano-delivery system with dual functions of sonodynamic and immunomodulatory effects according to claim 1, characterized in that, The inorganic nano-sonosensitizer is TiO 2 , ZnO, Fe 2 O 3 , SnO 2 or at least one of them; and / or, the noble metal is at least one of Au, Ag, and Pt; and / or, the particle size of the noble metal-modified inorganic nano-sensitizer is 50nm - 300nm.

6. A preparation method of a nano-delivery system with dual functions of sonodynamic and immunomodulatory effects, characterized in that, it includes the following steps: Preparing an immunomodulatory unit connected with a linker structure: dissolving the immunomodulator in a non-polar organic solvent, and adding a compound containing disulfide bond and / or mercapto group for a condensation reaction; wherein, the chemical structure of the immunomodulator contains a carboxyl group, and the chemical structure of the compound containing disulfide bond and / or mercapto group also contains an amino group or a hydroxyl group; or, the chemical structure of the immunomodulator contains an amino group or a hydroxyl group, and the chemical structure of the compound containing disulfide bond and / or mercapto group also contains a carboxyl group; Preparation of noble metal-modified inorganic nanosonosensitizers: An aqueous solution containing inorganic nanosonosensitizers is mixed evenly with a noble metal salt solution under an inert atmosphere, a hole scavenger and a pH regulator are added, and a continuous Ar + laser is used. The beam power range at the laser head outlet gradually increases from PL = 0 to 300 mW, and the reaction is carried out by laser irradiation in a light control mode; Dispersing the prepared noble metal-modified inorganic nano-sensitizer and the immunomodulatory unit connected with a linker structure in ultrapure water, stirring, and carrying out a conjugation reaction to obtain a nano-delivery system with dual functions of sonodynamic and immunomodulatory effects.

7. The preparation method of the nano-delivery system with dual functions of sonodynamic and immunomodulatory according to claim 6, characterized in that, the temperature of the conjugation reaction is 10°C - 50°C, and the reaction time is 12h - 48h.

8. The preparation method of the nano-delivery system with dual functions of sonodynamic and immunomodulatory according to claim 6, characterized in that, the molar ratio of the inorganic nano-sensitizer to the noble metal in the noble metal salt solution is 1:0.3 - 1.0; and / or, the molar ratio of the immunomodulator to the compound containing disulfide bond and / or mercapto group is 1:1 - 3; and / or, the pH value of the reaction system of the aqueous solution containing the inorganic nano-sensitizer and the noble metal salt solution is 3.

5.

9. Use of the nano-delivery system with dual functions of sonodynamic and immunomodulatory according to any one of claims 1 - 5 or the nano-delivery system with dual functions of sonodynamic and immunomodulatory prepared by the preparation method according to any one of claims 6 - 8 in the preparation of drugs for treating tumors, inflammation, cardiovascular diseases, and Alzheimer's disease.

10. Use of the nano-delivery system with dual functions of sonodynamic and immunomodulatory according to any one of claims 1 - 5 or the nano-delivery system with dual functions of sonodynamic and immunomodulatory prepared by the preparation method according to any one of claims 6 - 8 in the preparation of type I reactive oxygen species generator and / or type II reactive oxygen species generator.

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