A cross-linked nano-therapeutic agent, its preparation method and application

Abstract

The present invention discloses a crosslinked nano-therapeutic agent, a preparation method thereof and an application thereof. The crosslinked nano-therapeutic agent is crosslinked from raw materials including a reactive oxygen species (ROS)-responsive crosslinking agent and a functional protein. During the transportation process of the crosslinked nano-therapeutic agent provided by the present invention, the activity of the functional protein is inhibited due to crosslinking with the ROS-responsive crosslinking agent. After targeting to the tumor site, due to the ROS responsiveness of the ROS-responsive crosslinking agent, hydrogen peroxide in the tumor microenvironment can cleave the ROS-responsive crosslinking agent, causing the ROS-responsive crosslinking agent to break the bond, thereby dissolving the assembled functional protein nanoparticles and restoring the activity of the functional protein, realizing the selective control of the activity of the crosslinked nano-therapeutic agent. The crosslinked nano-therapeutic agent provided by the present invention significantly improves the drug loading capacity and bioavailability of the functional protein, can selectively control the activity of the functional protein, thereby realizing tumor targeting and selectivity, increasing the accumulation amount of the drug at the tumor site, and improving the curative effect.

Description

Technical Field

[0001] The present invention relates to the field of medical nanomaterials, and particularly to a cross-linked nano-therapeutic agent, a preparation method thereof, and an application thereof. Background Art

[0002] A nano-platform is a system integrating various functional components, including a nano-carrier and a loaded functional molecule. According to the composition of the nano-carrier, the nano-platform can be divided into various types, which may include inorganic carriers and organic carriers. Among them, the inorganic carriers include noble metal nanoparticles such as gold and silver, mesoporous silica, magnetic nanoparticles, and carbon; the organic carriers include metal-organic frameworks, polymers, and liposomes, etc. However, the inorganic carriers have disadvantages such as being difficult to degrade, having a large metabolic burden, and poor biosafety, which limit their application in clinics. The nano-platforms based on organic materials have a broader clinical application prospect due to their good biosafety and biodegradability. Many drug delivery systems have been approved for clinical treatment, such as doxorubicin hydrochloride liposome (Doxil), and more nano-drug delivery systems are in the clinical trial stage. However, the drug loading capacity of these nano-carriers is limited, and the bioavailability is still low, which restricts the further application of the nano-platform in clinics.

[0003] As an important part of the human body, proteins are composed of 20 amino acids in different proportions and have different characteristics and functions. The protein-based nano-platforms are assembled from natural or artificially modified proteins, and these proteins can self-assemble through the same protein subunits or through the combination and assembly of different proteins. Since proteins have good biocompatibility and biodegradability, the protein-based nano-carriers can reduce the systemic toxicity caused by the carriers themselves. Currently, many proteins have been successfully applied to the delivery of nano-drugs, mainly including ferritin, albumin, small heat shock protein, and elastin, etc. In addition, great progress has been made in biotherapeutic technologies for functional proteins in the past decade. However, the selective control of the targeting and activity of functional proteins has always been an important problem in protein therapy. The technology for selectively controlling the activity of functional proteins, so that the activity of functional proteins is inhibited during transportation and restored after being stimulated by exogenous stimuli or endogenous factors, is very lacking. Therefore, it is urgent to design and prepare a more effective controllable delivery strategy for functional proteins.

[0004] Therefore, the prior art still needs to be improved and developed. Summary of the Invention

[0005] In view of the above deficiencies of the prior art, the object of the present invention is to provide a cross-linked nano-therapeutic agent, a preparation method thereof, and an application thereof, aiming to solve the problems of low selectivity of the activity of existing functional proteins and poor curative effect.

[0006] The technical solution of the present invention is as follows:

[0007] In the first aspect of the present invention, a cross-linked nano-therapeutic agent is provided, wherein the cross-linked nano-therapeutic agent is cross-linked from raw materials including a reactive oxygen species (ROS)-responsive cross-linker and a functional protein.

[0008] Optionally, the cross-linked nano-therapeutic agent is cross-linked from a ROS-responsive cross-linker and a functional protein.

[0009] Optionally, the mass ratio of the ROS-responsive cross-linker to the functional protein is 51:1 to 70:1.

[0010] Optionally, the structural formula of the ROS-responsive cross-linker is:

[0011]

[0012] Optionally, the functional protein is selected from one or more of glucose oxidase (GOx), lactate oxidase (LOx), catalase (CAT), urate oxidase (UO), oxalate oxidase (OXO), protein kinase (PK), hyaluronidase (HAase), galactosidase (GAL), albumin (ALB), ferritin (SF), whey protein isolate (WPI), collagen (COL), silk fibroin (SF), lipoprotein (Lp), thrombin, and recombinant protein.

[0013] Optionally, the cross-linked nano-therapeutic agent is a nanoparticle, and the particle size of the nanoparticle is 50 to 100 nm.

[0014] In the second aspect of the present invention, a preparation method of the cross-linked nano-therapeutic agent as described above in the present invention is provided, which includes the steps of:

[0015] Providing a ROS-responsive cross-linker and a functional protein;

[0016] Mixing and stirring the ROS-responsive cross-linker and the functional protein to obtain the cross-linked nano-therapeutic agent.

[0017] Optionally, the step of mixing and stirring the ROS-responsive cross-linker and the functional protein specifically includes:

[0018] Adding the ROS-responsive cross-linker to dimethyl sulfoxide to obtain solution A;

[0019] Adding the functional protein to PBS buffer to obtain solution B;

[0020] Adding solution B to solution A for mixing and stirring.

[0021] Optionally, the preparation method of the ROS-responsive crosslinking agent includes the steps of:

[0022] Mix and stir mercaptoacetic acid, acetone, and p-toluenesulfonic acid, and obtain a first compound after the reaction;

[0023] Add the first compound and sodium borohydride to tetrahydrofuran, then add iodine, after the reaction, add sodium hydroxide and stir to obtain a second compound;

[0024] Add the second compound and N,N'-carbonyldiimidazole to dichloromethane and react to obtain the ROS-responsive crosslinking agent.

[0025] In the third aspect of the present invention, there is provided an application of the crosslinked nano-therapeutic agent as described above in the present invention in the preparation of a tumor therapeutic agent, and / or, there is provided an application of the crosslinked nano-therapeutic agent prepared by the preparation method as described above in the present invention in the preparation of a tumor therapeutic agent.

[0026] Beneficial effects: During the transportation process of the crosslinked nano-therapeutic agent provided by the present invention, due to the crosslinking with the ROS-responsive crosslinking agent, the activity of the functional protein is inhibited. After targeting to the tumor site, due to the ROS responsiveness of the ROS-responsive crosslinking agent, hydrogen peroxide in the tumor microenvironment can cleave the ROS-responsive crosslinking agent, causing the ROS-responsive crosslinking agent to break the bond, thereby dissolving the assembled functional protein nanoparticles and restoring the activity of the functional protein, realizing the selective control of the activity of the crosslinked nano-therapeutic agent. The crosslinked nano-therapeutic agent provided by the present invention significantly improves the drug loading amount and bioavailability of the functional protein, can selectively control the activity of the functional protein, thereby realizing tumor targeting and selectivity, increasing the drug accumulation amount at the tumor site, improving the curative effect, and reducing the toxic and side effects. Description of the Drawings

[0027] Figure 1 It is the synthesis route of the ROS-responsive crosslinking agent in Example 1 of the present invention.

[0028] Figure 2 It is the high-resolution mass spectrum of the ROS-responsive crosslinking agent prepared in Example 1 of the present invention.

[0029] Figure 3 It is the nuclear magnetic resonance hydrogen spectrum of the ROS-responsive crosslinking agent prepared in Example 1 of the present invention.

[0030] Figure 4 It is the TEM image of GOx NP prepared in Example 2 of the present invention.

[0031] Figure 5 It is the result diagram of the degradation effect of hydrogen peroxide on GOx NP in Example 4 of the present invention.

[0032] Figure 6 This is the evaluation result diagram of the enzyme secondary structure of GOx NP in Example 5 of the present invention.

[0033] Figure 7 This is the comparison diagram of the hydrogen peroxide generation ability of GOx and GOx NP in Example 6 of the present invention.

[0034] Figure 8 This is the cell survival rate diagram of different drug treatment groups in Example 7 of the present invention, where (a) is incubated with a sugar-free culture medium and (b) is incubated with a sugar-containing culture medium.

[0035] Figure 9 This is the fluorescence confocal diagram of cells taking up GOx and GOx NP respectively in Example 8 of the present invention. Detailed implementation manners

[0036] The present invention provides a cross-linked nano-therapeutic agent and its preparation method and application. To make the objectives, technical solutions and effects of the present invention clearer and more definite, the present invention is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0037] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the description of the present invention in this specification are only for the purpose of describing specific implementation manners and are not intended to limit the present invention.

[0038] An embodiment of the present invention provides a cross-linked nano-therapeutic agent, wherein the cross-linked nano-therapeutic agent is cross-linked from raw materials including a ROS-responsive cross-linking agent and a functional protein.

[0039] In this embodiment, during the transportation of the cross-linked nano-therapeutic agent, the activity of the functional protein is inhibited due to cross-linking with the ROS-responsive cross-linking agent. After targeting to the tumor site, due to the ROS responsiveness of the ROS-responsive cross-linking agent, hydrogen peroxide in the tumor microenvironment can cleave the ROS-responsive cross-linking agent, causing the ROS-responsive cross-linking agent to break the bond, thereby dissolving the assembled functional protein nanoparticles and restoring the activity of the functional protein, realizing the selective control of the activity of the cross-linked nano-therapeutic agent. The cross-linked nano-therapeutic agent provided in this embodiment significantly improves the drug loading capacity and bioavailability of the functional protein, can selectively control the activity of the functional protein, thereby realizing tumor targeting and selectivity, increasing the drug accumulation amount at the tumor site, improving the curative effect and reducing the toxic and side effects. The cross-linked nano-therapeutic agent truly realizes high drug loading capacity, tumor targeting and tumor microenvironment-activated protein activity, and will have good application prospects in the field of tumor treatment.

[0040] In one embodiment, the cross-linked nano-therapeutic agent is formed by cross-linking a ROS-responsive cross-linker and a functional protein.

[0041] In one embodiment, the mass ratio of the ROS-responsive cross-linker to the functional protein is 51:1 to 70:1. This ratio can better achieve the selective control of the activity of the cross-linked nano-therapeutic agent, so that the activity of the functional protein is inhibited during transportation. After targeting to the tumor site, the functional protein is released and the activity of the functional protein is restored.

[0042] In one embodiment, the structural formula of the ROS-responsive cross-linker is:

[0043]

[0044] In this embodiment, the cross-linked nano-therapeutic agent is formed by cross-linking the ROS-responsive cross-linker of the above structure and a functional protein. The amino groups of the functional protein react with the carbonyl imidazoles at both ends of the ROS-responsive cross-linker of the above structure, and any functional protein is connected at both ends by forming an ester bond to form cross-linked nanoparticles. The ROS-responsive cross-linker of the above structure contains a ROS-responsive thioacetal (TK) bond. When the cross-linked nano-therapeutic agent targets the tumor, hydrogen peroxide (as a kind of ROS) in the tumor microenvironment can break the TK bond into a mercapto group and acetone. Then, the mercapto group undergoes intramolecular cyclization to form a sulfur-containing ring and detach from the ester bond, thereby releasing the functional protein and restoring the activity of the functional protein, achieving the selective control of the activity of the cross-linked nano-therapeutic agent.

[0045] In one embodiment, the functional protein is selected from one or more of GOx, LOx, CAT, UO, OXO, PK, HAase, GAL, ALB, SF, WPI, COL, SF, Lp, thrombin, recombinant protein, etc., but not limited thereto.

[0046] Each functional protein has a different function. Taking GOx as an example, GOx has glucose-specific catalytic properties and can produce gluconic acid and hydrogen peroxide by oxidizing glucose, thereby consuming the nutrients of tumors and achieving the purpose of starving tumors. Therefore, it can be used for the starvation therapy of cancer. When the cross-linked nano-therapeutic agent is cross-linked by a ROS-responsive cross-linker and GOx, the cross-linked nano-therapeutic agent is a drug for cancer starvation therapy. Among them, after the cross-linked nano-therapeutic agent with inhibited GOx activity is targeted to the tumor site, hydrogen peroxide in the tumor microenvironment can break the bond of the ROS-responsive cross-linker (for example, the TK bond), so that the GOx nanoparticles are dispersed and the activity of GOx is restored. GOx consumes the nutrients in the tumor environment by oxidizing glucose at the tumor site, achieving the effect of starving tumors, and can realize the starvation therapy of cancer.

[0047] In one embodiment, the cross-linked nano-therapeutic agent is a nanoparticle, and the particle size of the nanoparticle is 50-100 nm. Within this particle size range, the cross-linked nano-therapeutic agent can better achieve tumor-targeted accumulation and tumor starvation therapy. Further, the cross-linked nano-therapeutic agent is a spherical nanoparticle.

[0048] The embodiment of the present invention also provides a preparation method of the cross-linked nano-therapeutic agent as described above in the embodiment of the present invention, which includes the steps:

[0049] S1. Provide a ROS-responsive cross-linker and a functional protein;

[0050] S2. Mix and stir the ROS-responsive cross-linker and the functional protein to obtain the cross-linked nano-therapeutic agent.

[0051] The preparation process provided by the embodiment of the present invention is simple, easy to operate, does not require complex and expensive equipment, and is easy to realize industrial production. The prepared cross-linked nano-therapeutic agent significantly improves the drug loading and bioavailability of the functional protein, can selectively control the activity of the functional protein, thereby realizing tumor targeting and selectivity, increasing the drug accumulation amount at the tumor site, improving the curative effect, and reducing the toxic and side effects. The cross-linked nano-therapeutic agent truly realizes high drug loading, tumor targeting, and protein activity activation in the tumor microenvironment, and will have good application prospects in the field of tumor treatment.

[0052] In step S1, in one embodiment, as Figure 1 shown, the preparation method of the ROS-responsive cross-linker includes the steps:

[0053] S11. Mix and stir mercaptoacetic acid, acetone, and p-toluenesulfonic acid (TsOH), and obtain a first compound after the reaction;

[0054] S12. Add the first compound and sodium borohydride to tetrahydrofuran, then add iodine. After the reaction, add sodium hydroxide and stir to obtain the second compound;

[0055] S13. React the second compound and N,N'-carbonyldiimidazole in dichloromethane to obtain the ROS-responsive crosslinker.

[0056] In step S12, in one embodiment, add the first compound and sodium borohydride to tetrahydrofuran, stir in an ice bath and then add iodine, then carry out a reflux reaction under nitrogen protection, then cool to room temperature, add methanol and continue to stir. After rotary evaporation, add sodium hydroxide and stir. Extract with ethyl acetate and dry with anhydrous sodium sulfate to obtain the second compound.

[0057] In step S13, in one embodiment, add the second compound and N,N'-carbonyldiimidazole to dichloromethane. After the reaction, carry out rotary evaporation and column chromatography purification to obtain the ROS-responsive crosslinker.

[0058] In step S2, in one embodiment, the step of mixing and stirring the ROS-responsive crosslinker and the functional protein specifically includes:

[0059] S21. Add the ROS-responsive crosslinker to dimethyl sulfoxide to obtain solution A;

[0060] S22. Add the functional protein to PBS buffer to obtain solution B;

[0061] S23. Add solution B to solution A for mixing and stirring.

[0062] The embodiments of the present invention also provide an application of the crosslinked nano-therapeutic agent as described above in the preparation of a tumor therapeutic agent. The embodiments of the present invention also provide an application of the crosslinked nano-therapeutic agent prepared by the preparation method as described above in the preparation of a tumor therapeutic agent. Specifically, the treatment can be starvation therapy. The crosslinked nano-therapeutic agent in this embodiment can achieve high drug loading, tumor targeting and tumor microenvironment-responsive protein activity, so as to achieve the effect of starvation therapy, and will have good application prospects in the field of tumor treatment.

[0063] The following is a detailed description through specific examples.

[0064] Example 1

[0065] Preparation of the ROS-responsive crosslinker (the synthesis route is as Figure 1 shown):

[0066] 5.52 g of thioglycolic acid was added to a 50 mL round-bottom flask, dissolved in 8.8 mL of anhydrous acetone, then 10 mg of TsOH was added, and the reaction was carried out with stirring at room temperature for 6 h. After the reaction was completed, stirring was continued under an ice bath, the precipitated white crystals were filtered by suction, and the crystals were washed with n-hexane cooled by an ice bath to obtain the first compound.

[0067] In a three-necked flask, 50 mL of tetrahydrofuran, 2.24 g of the first compound and 2.27 g of sodium borohydride were added, and the mixture was stirred and dissolved under an ice bath to obtain a mixed solution. 10.15 g of iodine was dissolved in 50 mL of tetrahydrofuran and added dropwise to the above mixed solution. After the addition was completed, the reaction was refluxed for 24 h under nitrogen protection. After cooling to room temperature, 25 mL of methanol was added until the solution became clear, and then stirring was continued for 45 min. The above reaction solution was rotary evaporated, 100 mL of an aqueous sodium hydroxide solution with a mass content of 25% was added, and stirring was continued for 5 h. Then, extraction was carried out 5 times with ethyl acetate, the organic phase was collected, dried with anhydrous sodium sulfate and rotary evaporated, and finally purified by column chromatography to obtain the second compound.

[0068] 0.196 g of the second compound and 0.389 mg of N,N'-carbonyldiimidazole were dissolved in 15 mL of dichloromethane, stirred at room temperature for 8 h, then rotary evaporated, and purified by column chromatography to obtain the ROS-responsive crosslinker.

[0069] The high-resolution mass spectrometry results of the ROS-responsive crosslinker are as Figure 2 shown, and the results show that the molecular weight of the ROS-responsive crosslinker is 381.11. The 1H NMR results of the ROS-responsive crosslinker are as Figure 3 shown, and the results confirm the molecular structure of the ROS-responsive crosslinker.

[0070] Example 2

[0071] Preparation of crosslinked nano-therapeutics:

[0072] 768 mg of the ROS-responsive crosslinker prepared in Example 1 was dissolved in 1 mL of dimethyl sulfoxide to obtain solution A;

[0073] 15 mg of GOx was dissolved in 100 mL of PBS (pH 7.4) to obtain solution B;

[0074] 10 μL of solution A was added to 1 mL of solution B, stirred at room temperature for 10 min, and finally the obtained nanoparticles were concentrated and washed through an ultrafiltration tube to obtain the crosslinked nano-therapeutics, denoted as GOx NP.

[0075] The TEM image of GOx NP is as Figure 4 shown. From Figure 4It can be seen that GOx and the ROS-responsive crosslinker can be assembled into spherical nanoparticles with a particle size of 50-100 nm.

[0076] Example 3

[0077] Preparation of crosslinked nano-therapeutics:

[0078] Dissolve 768 mg of the ROS-responsive crosslinker prepared in Example 1 in 1 mL of dimethyl sulfoxide to obtain Solution A;

[0079] Dissolve 15 mg of GOx in 100 mL of PBS (pH 7.4) to obtain Solution B;

[0080] Take 13.4 μL of Solution A and add it to 1 mL of Solution B, stir at room temperature for 10 min, and finally enrich and wash the obtained nanoparticles through an ultrafiltration tube to obtain the crosslinked nano-therapeutics, denoted as GOx NP.

[0081] Example 4

[0082] Evaluation of the degradation effect of hydrogen peroxide on GOx NP

[0083] Use TEM to evaluate the effect of hydrogen peroxide on the structure of GOx NP. Mix and co-incubate GOx NP in Example 2 with hydrogen peroxide, and perform TEM imaging on the mixture at different times. The results are as Figure 5 shown. After GOx NP is mixed and incubated with hydrogen peroxide for 2 h, its spherical structure is basically completely disassembled, indicating that the crosslinked nano-therapeutics have the property of hydrogen peroxide-responsive degradation.

[0084] The results after mixing and co-incubating GOx NP in Example 3 with hydrogen peroxide are similar to those after mixing and co-incubating GOx NP in Example 2 with hydrogen peroxide, and will not be elaborated here.

[0085] Example 5

[0086] Evaluation of the enzyme secondary structure of GOx and GOx NP

[0087] Use a circular dichroism spectrometer to evaluate the secondary structure of GOx in GOx NP. Measure the protein secondary structures of GOx and GOx NP in Example 2 respectively. The results are as Figure 6 shown. Compared with GOx, the corresponding secondary structure of GOx in GOx NP is hardly detectable, indicating that the secondary structure of GOx in GOx NP has changed and the activity of GOx is inhibited. After GOx NP is mixed and incubated with hydrogen peroxide, the protein secondary structure of GOx can be detected and the activity of GOx can be restored.

[0088] The evaluation results of the enzyme secondary structure of GOx NP in Example 3 are consistent with those of GOx NP in Example 2, and will not be elaborated here.

[0089] Example 6

[0090] Evaluation of the ability of GOx and GOx NP to produce hydrogen peroxide

[0091] The GOx NP in Example 2 was co-incubated with 10 mM hydrogen peroxide, and the pure nanoparticles were obtained by ultrafiltration washing at different times. After GOx, the GOx NP in Example 2, and the nanoparticles collected at the above different time points reacted with glucose at the same concentration for the same time, the hydrogen peroxide content in the mixture was detected by a hydrogen peroxide kit respectively to evaluate the enzyme activities of GOx, GOx NP, and the nanoparticles collected at the above different time points.

[0092] As Figure 7 shown, compared with the GOx group, the hydrogen peroxide produced after the incubation of GOx NP with glucose was less than 10 μM, and as the incubation time of GOx NP with hydrogen peroxide was prolonged in advance, the hydrogen peroxide produced by it became higher and higher, indicating that as the incubation time of GOx NP with hydrogen peroxide was prolonged, GOx NP gradually disassembled and its enzyme activity was gradually restored.

[0093] The ability of GOx NP in Example 3 to produce hydrogen peroxide after incubation with glucose is equivalent to that of GOx NP in Example 2 after incubation with glucose, and will not be elaborated here.

[0094] Example 7

[0095] Evaluation of the toxicity of different drug treatments to cells

[0096] The standard MTT method was used to evaluate the killing effect of starvation therapy on 4T1 cells.

[0097] Under the conditions of 37 °C and 5% CO2, 4T1 cells were seeded into a 96-well plate at a density of 5×10 3 and continued to be cultured for 24 h. Then, the old medium in the 96-well plate was aspirated, and sugar-free DMEM medium containing 0, 10, 20, 40, 60, and 80 ng / mL GOx, and glucose-containing DMEM medium containing 0, 2.5, 5, 10, 20, and 40 ng / mL GOx with a glucose concentration of 2 mM were added respectively; after continuing to culture for 24 hours, the old medium in the 96-well plate was aspirated, 100 μL of medium solution containing 5 mg / mL MTT was added to each well, and the culture was continued for 4 hours. Then, 150 μL of dimethyl sulfoxide was used to replace the old medium, and the OD value of each well was detected using a Bio-Tel EL microplate reader (the detection wavelength was 490 nm), denoted as the GOx group.

[0098] Under the conditions of 37 °C and 5% CO2, 4T1 cells were seeded into a 96-well plate at a density of 5×10 3 and further cultured for 24 h. Then, the old medium in the 96-well plate was aspirated, and sugar-free DMEM medium containing 0, 10, 20, 40, 60, and 80 ng / mL of GOx NP in Example 2, and glucose-containing DMEM medium containing 0, 2.5, 5, 10, 20, and 40 ng / mL of GOx NP in Example 2, with a glucose concentration of 2 mM, were added respectively; after continuous culture for 24 h, the old medium in the 96-well plate was aspirated, 100 μL of medium solution containing 5 mg / mL MTT was added to each well, and the culture was continued for 4 h. Then, the old medium was replaced with 150 μL of dimethyl sulfoxide, and the OD value of each well was measured using a Bio-Tel EL microplate reader (the detection wavelength was 490 nm), denoted as the GOxNP group.

[0099] Under the conditions of 37 °C and 5% CO2, 4T1 cells were seeded into a 96-well plate at a density of 5×10 3 and further cultured for 24 h. Then, the old medium in the 96-well plate was aspirated, and sugar-free DMEM medium containing 0, 10, 20, 40, 60, and 80 ng / mL of GOx NP in Example 2, and glucose-containing DMEM medium containing 0, 2.5, 5, 10, 20, and 40 ng / mL of GOx NP in Example 2, with a glucose concentration of 2 mM, and all media containing 10 μM hydrogen peroxide, were added respectively; after continuous culture for 24 h, the old medium in the 96-well plate was aspirated, 100 μL of medium solution containing 5 mg / mL MTT was added to each well, and the culture was continued for 4 h. Then, the old medium was replaced with 150 μL of dimethyl sulfoxide, and the OD value of each well was measured using a Bio-Tel EL microplate reader (the detection wavelength was 490 nm), denoted as the GOx NP + hydrogen peroxide group.

[0100] The cell viability of the above three groups was calculated using the following formula. Cell viability (%) = (OD490 value of the sample / OD490 value of the blank) × 100%, and the experimental results are shown in Figure 8 .

[0101] As Figure 8 shown in (a), the cell viability of different treatment groups incubated with sugar-free medium was significantly higher than that of the corresponding glucose-containing medium group in Figure (b). In Figure (b), the killing effect of GOx NP on cells was lower than that of GOx and the hydrogen peroxide-added group, and the addition of hydrogen peroxide had the strongest tumor cell inhibitory effect.

[0102] Cytotoxicity evaluation was performed using the GOx NPs in Example 3, and the results were similar to those of the GOx NPs in Example 2, which will not be elaborated here.

[0103] Example 8

[0104] Evaluation of the uptake ability of cells for GOx NPs

[0105] Fluorescent dye IR680 was covalently linked and modified to GOx and the GOx NPs in Example 2, and then the unreacted excess dye was removed by dialysis. Under the culture conditions of 37 °C and 5% CO2, 4T1 cells were seeded into a 96-well plate at a density of 1×105 cells per well. After 24 h, the old medium in the 96-well plate was aspirated, and IR680-labeled GOx and GOx NPs with the same concentration were added. After culturing for 4 h, the fluorescence changes were detected by using a fluorescence confocal microscope.

[0106] The results were as Figure 9 , and the fluorescence intensity of the GOx NP group was significantly higher than that of the GOx group. This indicates that cells have a stronger uptake effect on GOx NPs compared to GOx. It is proved that cells have a stronger uptake effect on the cross-linked nano-therapeutics provided by the present invention compared to pure proteins.

[0107] The uptake ability of cells for the GOx NPs in Example 3 was basically the same as that for the GOx NPs in Example 2, which will not be elaborated here.

[0108] In summary, the present invention provides a cross-linked nano-therapeutic agent, its preparation method and application. During the transportation process of the cross-linked nano-therapeutic agent, due to the cross-linking of the functional protein with the ROS-responsive cross-linker, the activity of the functional protein is inhibited. After targeting to the tumor site, due to the ROS responsiveness of the ROS-responsive cross-linker, hydrogen peroxide in the tumor microenvironment can cleave the ROS-responsive cross-linker, causing the ROS-responsive cross-linker to break the bond, so that the assembled functional protein nanoparticles are disassembled and the activity of the functional protein is restored, realizing the selective control of the activity of the cross-linked nano-therapeutic agent. The cross-linked nano-therapeutic agent provided by the present invention significantly improves the drug loading capacity and bioavailability of the functional protein, can selectively control the activity of the functional protein, thereby realizing tumor targeting and selectivity, increasing the drug accumulation amount at the tumor site, improving the curative effect and reducing the toxic and side effects.

[0109] It should be understood that the application of the present invention is not limited to the above examples. For those of ordinary skill in the art, improvements or transformations can be made according to the above description, and all such improvements and transformations should fall within the protection scope of the appended claims of the present invention.

Claims

1. A cross-linked nano-therapeutic agent, characterized in that, The cross-linked nano-therapeutic agent is cross-linked from raw materials including a reactive oxygen species-responsive cross-linker and a functional protein; The structural formula of the reactive oxygen species-responsive cross-linker is: The functional protein is selected from one of glucose oxidase, lactate oxidase, uricase, and oxalate oxidase.

2. The cross-linked nano-therapeutic agent according to claim 1, characterized in that, The cross-linked nano-therapeutic agent is cross-linked from a reactive oxygen species-responsive cross-linker and a functional protein.

3. The cross-linked nano-therapeutic agent according to claim 1, characterized in that, The mass ratio of the reactive oxygen species-responsive cross-linker to the functional protein is 51:1 to 70:

1.

4. The cross-linked nano-therapeutic agent according to claim 1, characterized in that, The cross-linked nano-therapeutic agent is a nanoparticle, and the particle size of the nanoparticle is 50 to 100 nm.

5. A method for preparing a cross-linked nano-therapeutic agent according to any one of claims 1-4, characterized in that, Including the steps: Providing a reactive oxygen species-responsive cross-linker and a functional protein; Mixing and stirring the reactive oxygen species-responsive cross-linker and the functional protein to obtain the cross-linked nano-therapeutic agent.

6. The preparation method according to claim 5, characterized in that, The step of mixing and stirring the reactive oxygen species-responsive cross-linker and the functional protein specifically includes: Adding the reactive oxygen species-responsive cross-linker into dimethyl sulfoxide to obtain solution A; Adding the functional protein into PBS buffer to obtain solution B; Adding solution B into solution A for mixing and stirring.

7. The preparation method according to claim 6, characterized in that, The preparation method of the reactive oxygen species-responsive cross-linker includes the steps: Mixing and stirring mercaptoacetic acid, acetone, and p-toluenesulfonic acid, and obtaining a first compound after reaction; Adding the first compound and sodium borohydride into tetrahydrofuran, then adding iodine, and after reaction, adding sodium hydroxide for stirring to obtain a second compound; Adding the second compound and N,N'-carbonyldiimidazole into dichloromethane for reaction to obtain the reactive oxygen species-responsive cross-linker.

8. Use of a cross-linked nano-therapeutic agent according to any one of claims 1-4 in the preparation of a tumor therapeutic preparation, and / or use of a cross-linked nano-therapeutic agent prepared by the preparation method according to any one of claims 5-7 in the preparation of a tumor therapeutic preparation.

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