Preparation of an albumin nano-delivery system with hypoxic-active oxygen dual response and application thereof in preparation of a colorectal cancer drug

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to preparation of an anaerobic-active oxygen dual-response albumin nano delivery system and application thereof in preparation of colorectal cancer drugs. The system is used for mediating high-efficiency uptake of tumor cells by MC4A modified BSA, and BSA-C4A is used for loading prodrugs by virtue of host-guest action of MC4A, is stable in normal oxygen environment, and is enriched in a tumor anaerobic microenvironment, wherein an azo bond in MC4A is cleaved by reductase, the prodrugs are released, and are targeted to mitochondria. The prodrugs are coupled with a photosensitizer Nile Blue derivative and a chemotherapeutic drug camptothecin (NB-TK-CPT) by taking a thio-ketone as an active oxygen sensitive linker. Under laser irradiation of a specific wavelength, by virtue of the type I photodynamic effect of NB, killing active oxygen is generated in an anaerobic area through a non-oxygen-dependent electron transfer pathway, and the TK linker is broken to realize directional release of CPT, so that the synergistic antitumor effect of photodynamic therapy and chemotherapy is amplified.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the preparation of a hypoxia-reactive oxygen species dual-response albumin nanodelivery system and its application in the preparation of colorectal cancer drugs. Background Technology

[0002] Colorectal cancer is a highly prevalent malignant tumor, with approximately 1.93 million new cases worldwide each year, and its incidence in my country has been steadily increasing in recent years. The tumor microenvironment of colorectal cancer generally exhibits significant hypoxia, a key factor contributing to treatment failure. Hypoxic environments not only induce resistance to chemotherapy drugs in tumor cells but also severely inhibit the efficacy of oxygen-dependent photodynamic therapy (PDT), making it difficult for single treatment modalities to effectively eliminate tumors.

[0003] Photodynamic therapy (PDT), a non-invasive tumor treatment, works by using photosensitizers to generate reactive oxygen species (ROS) under specific wavelengths of light. ROS can directly kill tumor cells and trigger apoptosis or necrosis by inducing mitochondrial dysfunction and endoplasmic reticulum stress, representing the core and most direct anti-tumor mechanism of PDT. Mitochondria, as the core organelle for cellular energy metabolism and apoptosis regulation, have been shown in studies to amplify the local effects of ROS by precisely delivering photosensitizers, thus significantly enhancing cytotoxicity. However, traditional PDT is highly dependent on the oxygen concentration within tumor tissue, generating singlet oxygen through a type II mechanism (energy transfer). In hypoxic regions, ROS generation efficiency significantly decreases, severely limiting its ability to kill tumors in hypoxic areas within solid tumors. Although type I PDT can generate free radicals through oxygen-independent electron transfer pathways, potentially overcoming hypoxia limitations to some extent, effectively integrating it with chemotherapy drugs and precisely delivering it to hypoxic regions and targeting mitochondria remains a critical technical challenge.

[0004] Chemotherapy is an important treatment for colorectal cancer, and chemotherapeutic drugs such as camptothecin (CPT) exert their anti-tumor effects by regulating the tumor microenvironment. However, single chemotherapeutic drugs are insufficient to overcome tumor drug resistance and lack precise release strategies targeting hypoxic microenvironments, making it difficult to achieve efficient killing of tumors in hypoxic regions. Therefore, developing novel treatment strategies that can overcome hypoxia limitations and achieve synergistic effects between photodynamic therapy and chemotherapy is of great significance for improving the treatment outcomes of colorectal cancer.

[0005] In recent years, nanodelivery systems have been widely used for targeted delivery of antitumor drugs. Among them, albumin-based nanocarriers have attracted much attention due to their good biocompatibility, long in vivo circulation time, and tumor targeting. However, existing albumin-based nanodelivery systems still have room for improvement in addressing the hypoxic tumor microenvironment: on the one hand, most existing carriers have relatively limited specific response capabilities to the hypoxic microenvironment, and the precise release of drugs in hypoxic regions and the realization of subsequent cascade reactions still face certain challenges; on the other hand, existing photodynamic therapies mostly rely on type II mechanisms, and the efficiency of reactive oxygen species generation is limited under hypoxic conditions, while cascade-response platforms based on type I photodynamic mechanisms and hypoxia-responsive drug release functions integrated into albumin nanoparticles have relatively limited research reports. Therefore, there is an urgent need for a novel albumin supramolecular nanodelivery system that can stably accumulate and specifically respond to release in the hypoxic tumor microenvironment, and achieve synergistic effects between type I photodynamic therapy and chemotherapy, in order to overcome treatment resistance caused by hypoxia in colorectal cancer. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides a hypoxia-reactive oxygen species dual-response albumin nanodelivery system and its application in the preparation of colorectal cancer drugs. The nanodelivery system enables precise co-delivery of photosensitizers and chemotherapeutic drugs in a hypoxic tumor microenvironment. Furthermore, it amplifies the synergistic antitumor effect of photodynamic therapy and chemotherapy through a hypoxia-responsive release and reactive oxygen species-triggered cascade mechanism, thereby enhancing the therapeutic effect on hypoxic tumors. This system has significant scientific and practical value.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of this invention provides a method for preparing a hypoxia-reactive oxygen species dual-response prodrug nanodelivery system based on maleimide calixarene supramolecular (MC4A) modified bovine serum albumin (BSA), comprising the following steps: Step S1: Dissolve monocarboxylic acid trisulfonate azocalixarene in N,N-dimethylformamide as solvent, then add buffer and condensing agent, and continuously stir and activate at room temperature. Then add 1-(2-(2-aminoethoxy)ethyl)-1H-pyrrole-2,5-dione-2,2,2-trifluoroacetate, and continue stirring and react at room temperature under nitrogen protection. Then remove the solvent by rotary evaporation under reduced pressure, and finally recrystallize in a mixed solution of water and methanol to obtain product MC4A. Step S2: Dissolve BSA and reducing agent in ultrapure water and stir continuously at room temperature to expose the thiol groups inside BSA. Obtain thiol-exposed BSA chains by dialysis and freeze-drying. Step S3: Redissolve the thiol-exposed BSA chain, add MC4A, stir continuously at room temperature, dialyze in ultrapure water using a dialysis bag, and freeze-dry to obtain product BSA-C4A; Step S4: Dissolve 1-naphthylamine and methyl 3-bromopropionate in anhydrous ethanol, heat to reflux, and after the reaction is complete, evaporate the solvent and separate and purify by column chromatography to obtain compound 1; Step S5: Prepare an aqueous solution containing aluminum sulfate and add N,N-diethyl-p-phenylenediamine to it. While stirring, add sodium thiosulfate and zinc chloride to the reaction system in sequence. Then transfer it to an ice bath to cool. After the temperature stabilizes, add an aqueous solution containing oxidant dropwise at a uniform rate. Continue the reaction at low temperature until a viscous substance is observed to form. Filter the solution and wash the filter cake with detergent to obtain a crude product. Finally, place the crude product in methanol solvent and heat under reflux. After filtering again, obtain a gray powdery solid, namely compound 2. Step S6: Dissolve compound 1 and compound 2 in methanol, add silver carbonate, heat to reflux, after the reaction is complete, remove the solvent by rotary evaporation, remove impurities by redissolved extraction, then acidify and dry, and finally separate and purify by column chromatography to obtain compound 3. Step S7: Compound 3 was dissolved in 1,4-dioxane solution and hydrolyzed with sodium hydroxide solution. After the reaction was completed, the organic matter was extracted, the solvent was removed by rotary evaporation, acidified, dried and purified by column chromatography to obtain compound 4. Step S8: Dissolve thioglycolic acid in acetone and then pass hydrogen chloride gas through it. Stir the reaction at room temperature. After the reaction is complete, place the mixture in an ice bath, filter and wash it, and vacuum dry it overnight. The resulting white powdery solid is compound 5. Step S9: Under low temperature conditions, compound 5 was dissolved in tetrahydrofuran, lithium aluminum hydride was added and stirred at room temperature, then ultrapure water was added dropwise, filtered, washed, and the solvent was removed by rotary evaporation, and compound 6 was further separated and purified by silica gel column chromatography. Step S10: CPT and bis(trichloromethyl) carbonate are dissolved in dichloromethane, and then a dichloromethane solution containing 4-dimethylaminopyridine is added dropwise. The reaction is continued to be stirred at room temperature. Then, an anhydrous tetrahydrofuran solution containing compound 6 is added dropwise. The reaction is continued to be stirred at room temperature. After the reaction is completed, the mixture is washed, the solvent is removed by rotary evaporation, and compound 7 is further purified by column chromatography. Step S11: Dissolve compounds 4 and 7 in N,N-dimethylformamide, then add condensing agent and catalyst, and continue stirring the reaction. After the reaction is completed, extract the crude product and purify it by column chromatography to obtain compound 8, namely the responsive prodrug NB-TK-CPT. Step S12: Redissolve BSA-C4A and add it to NB-TK-CPT solution in a molar ratio. Shake to obtain TK NPs nanoparticles loaded with ROS-responsive prodrug NB-TK-CPT, which is the hypoxia-reactive oxygen dual-response prodrug nanodelivery system based on MC4A-modified BSA.

[0008] The nanodelivery system utilizes MC4A-modified BSA to mediate efficient uptake by tumor cells. The prodrug is encapsulated via the host-guest interaction of MC4A, remaining stable in normoxic environments. When enriched in the hypoxic tumor microenvironment, the azo bonds within MC4A are specifically cleaved by reductases, causing the carrier to disintegrate and precisely release the prodrug, which is then targeted to the mitochondria. The prodrug uses thioketal (TK) as a reactive oxygen species (ROS)-sensitive linker to conjugate a photosensitizer, Nile Blue (NB), with the chemotherapeutic drug CPT (NB-TK-CPT). Under specific wavelength laser irradiation, the system leverages the unique type I photodynamic effect of NB to effectively generate a large amount of cytotoxic ROS in the hypoxic region through an oxygen-independent electron transfer pathway. Simultaneously, it induces the TK linker to break, achieving targeted release of CPT, thereby amplifying the synergistic antitumor effect of photodynamic therapy and chemotherapy.

[0009] Further, in step S1, the buffer is N,N-diisopropylethylamine, the condensing agent is 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylhexafluorophosphate, the activation time under continuous stirring at room temperature is 20-40 min, the molar ratio of the monocarboxylic acid trisulfonate azocalixarene, N,N-diisopropylethylamine, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylhexafluorophosphate and 1-(2-(2-aminoethoxy)ethyl)-1H-pyrrole-2,5-dione-2,2,2-trifluoroacetate is 3-4:1-2:1:2-3, and the reaction time under nitrogen protection at room temperature with continuous stirring is 10-14 h.

[0010] Further, in step S2, the reducing agent is tris(2-carboxyethyl)amine hydrochloride, the reaction time with continuous stirring at room temperature is 20-40 min, and the molar ratio of BSA to tris(2-carboxyethyl)amine hydrochloride is 1:110-120; in step S3, the solvent for resolubilizing the thiol-exposed BSA chain is PBS, the reaction time with continuous stirring at room temperature is 12-16 h, the dialysis time is 36-72 h, and the molar ratio of the thiol-exposed BSA chain to MC4A is 1:30-35.

[0011] Further, in step S4, the reflux temperature is 95~105 °C, the reflux duration is 12~20 h, and the eluent for column chromatography is dichloromethane and petroleum ether in a volume ratio of 5~1:1; in step S5, the oxidant is potassium dichromate, the low temperature is 0 °C, the reaction duration at the low temperature is 1~4 h, the reflux temperature is 65~100 °C, the duration is 1~5 h, the washing agent is acetone, and the molar ratio of aluminum sulfate, N,N-diethyl-p-phenylenediamine, sodium thiosulfate, potassium dichromate, and zinc chloride is 2~5:2~5:3~6:1:2~5.

[0012] Further, in step S6, the molar ratio of compound 1, compound 2, and silver carbonate is 1.5~3∶1∶1.5~3, the reflux temperature is 70~100 ℃, and the duration is 1~3 h. The extractant for the redissolution extraction is dichloromethane and saturated sodium carbonate, the acidifying agent for the acidification is concentrated hydrochloric acid, and the eluent for the column chromatography is dichloromethane and methanol in a volume ratio of 100~20:1. In step S7, the acidifying agent for the acidification is hydrochloric acid, the molar ratio of compound 3, sodium hydroxide, and hydrochloric acid is 1∶2~5∶2~5, the hydrolysis duration is 2~6 h, the extraction solvent for the extraction is dichloromethane, and the eluent for the column chromatography is dichloromethane and methanol in a volume ratio of 20~8:1.

[0013] Further, in step S8, the stirring reaction at room temperature lasts for 4-8 hours, and the washing agent for filtration is ultrapure water and n-hexane; in step S9, the low temperature is 0-5 °C, the molar ratio of compound 5 to aluminum hydroxide is 1:2-6, the washing agent is tetrahydrofuran, and the eluent for column chromatography is ethyl acetate and dichloromethane in a volume ratio of 1:4-1.

[0014] Further, in step S10, the molar ratio of CPT, bis(trichloromethyl) carbonate, 4-dimethylaminopyridine and compound 6 is 2~4∶1∶7~10∶15~20, the reaction times for the two reactions are 20~60 min and 8~14 h, respectively, the washing agent is hydrochloric acid and saturated brine, and the eluent for column chromatography is ethyl acetate.

[0015] Further, in step S11, the condensing agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, the catalyst is 4-dimethylaminopyridine, and the molar ratio of compound 4, compound 7, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine is 3~6∶2~4∶2~4∶1; in step S12, the solvent for the BSA-C4A resolution and the NB-TK-CPT solution is PBS solution, and the molar ratio of MC4A to NB-TK-CPT is 1∶1~5.

[0016] A second aspect of the present invention provides a hypoxia-reactive oxygen species dual-response prodrug nanodelivery system based on MC4A-modified BSA prepared by the above-described preparation method.

[0017] A third aspect of the present invention provides the application of the above-mentioned hypoxia-reactive oxygen species dual-response prodrug nanodelivery system based on MC4A-modified BSA in the preparation of colorectal cancer drugs.

[0018] Compared with the prior art, the beneficial effects of the present invention are: This invention constructs a hypoxia-reactive oxygen species dual-effect prodrug delivery system using MC4A-modified BSA as a carrier, overcoming the limitations of the hypoxic tumor microenvironment and achieving synergistic effects of photodynamic therapy and chemotherapy. This provides a promising translational solution for overcoming treatment resistance caused by the hypoxic microenvironment of colorectal cancer. Specifically, this invention has the following beneficial effects: (1) This invention innovatively constructs a hypoxia-reactive oxygen species dual-response drug release system, effectively overcoming the technical bottleneck of precise drug delivery in the hypoxic tumor microenvironment. The hypoxia / reactive oxygen species dual-response cascade drug release system utilizes MC4A-modified BSA as a carrier, which remains highly stable in a normoxic environment and releases the prodrug NB-TK-CPT only in the hypoxic tumor microenvironment through azo bond cleavage. The prodrug NB-TK-CPT uses TK as a reactive oxygen species sensitive linker to couple NB and CPT. Under hypoxic conditions, free NB-TK-CPT undergoes ROS-specific cleavage of the TK linker by a photosensitizer, directionally releasing CPT, thereby achieving precise delivery of photosensitizers and chemotherapeutic drugs to the hypoxic region inside the solid tumor.

[0019] (2) This invention achieves synergistic treatment of photodynamic therapy and chemotherapy. After release in response to hypoxia, the NB-TK-CPT can target mitochondria. Under irradiation with a specific wavelength of laser, the ROS generated by NB not only destroys mitochondria and significantly enhances the cytotoxicity of ROS, but also triggers the breakage of TK linkers, releasing the chemotherapeutic drug CPT to further enhance cytotoxicity. This forms a cascade amplification effect of "hypoxia-responsive drug release—targeting mitochondria—type I photodynamic therapy to generate ROS—ROS-triggered release of chemotherapeutic drugs", which significantly improves the precision and synergistic efficiency of combined photodynamic therapy and chemotherapy.

[0020] (3) This invention reduces non-specific drug exposure through carrier encapsulation and a dual-response mechanism. NB-TK-CPT remains relatively stable in the carrier and releases CPT when hypoxia-related reducing environment and light conditions are met sequentially, thereby reducing premature drug release from non-target sites. Under the dosage and administration conditions shown in the examples, the drug delivery system exhibits good inhibitory effects and biocompatibility on colorectal tumors, providing a new technical solution and translational application prospect for multimodal synergistic treatment of hypoxic solid tumors. Attached Figure Description

[0021] Figure 1 This is a schematic diagram illustrating the preparation of the TK NPs prodrug nanodelivery system, which has a dual response to hypoxia and reactive oxygen species.

[0022] Figure 2 This is the proton NMR spectrum of MC4A.

[0023] Figure 3 This is the carbon NMR spectrum of MC4A.

[0024] Figure 4 This is the mass spectrum of MC4A.

[0025] Figure 5 In the diagram, a is the UV absorption curve of MC4A; b is the UV standard curve of MC4A; and c is the UV absorption curve of BSA-C4A and BSA.

[0026] Figure 6 Synthesis roadmap for MC4A, NB-TK-CPT, and NB-C6-CPT.

[0027] Figure 7 The image shows the proton NMR spectrum of compound 1.

[0028] Figure 8 The image shows the proton NMR spectrum of compound 3.

[0029] Figure 9 The image shows the proton NMR spectrum of compound 4.

[0030] Figure 10This is the mass spectrum of compound 4.

[0031] Figure 11 The image shows the proton NMR spectrum of compound 6.

[0032] Figure 12 The image shows the proton NMR spectrum of compound 7.

[0033] Figure 13 The image shows the proton NMR spectrum of compound 8.

[0034] Figure 14 This is the mass spectrum of compound 8.

[0035] Figure 15 The image shows the proton NMR spectrum of compound 9.

[0036] Figure 16 The image shows the proton NMR spectrum of compound 10.

[0037] Figure 17 This is the mass spectrum of compound 10.

[0038] Figure 18 In the figure, a is the UV-Vis absorption spectrum of BSA-C4A, NB-TK-CPT and TK NPs; b is the fluorescence emission spectrum of NB-TK-CPT, BSA-C4A and TK NPs under 670 nm excitation; c is the TEM image of TK NPs; d is the Zeta potential and particle size distribution of TK NPs; e and f are the particle size change curve and PDI change curve of TK NPs after 7 days of storage in PBS (n = 3).

[0039] Figure 19 In the figure, ac represents the uptake and quantitative results of B, BT, and BC by CT26 cells at different time points (scale bar: 25 μm); d represents the uptake and penetration of B, BT, and BC by CT26 three-dimensional tumor spheres (scale bar: 200 μm); e represents the fluorescence imaging of mice in vivo and major organs (heart, liver, spleen, lung, and kidney).

[0040] Figure 20 In the figure, a represents the cumulative drug release rate of TK NPs within 40 min under different oxygen conditions (n ​​= 3), b represents the quantitative analysis results of intracellular ROS detected by flow cytometry after the same drug treatment, and c and d represent the confocal fluorescence images (scale bar: 20 μm) and flow cytometry results of intracellular ROS generation after different drug treatments under normoxic and hypoxic conditions, respectively.

[0041] Figure 21In the figures, ac represents confocal images and quantitative results of CT26 cells uptake after co-incubation with TK NPs for different times (n = 3) (scale bar: 40 μm); d represents cell viability under normoxic and hypoxic conditions with different concentrations of NB-TK-CPT, NB-C6-CPT, TK NPs, and C6 NPs under 660 nm laser irradiation; e represents cell viability after drug treatment in the absence of light under normoxic and hypoxic conditions; f represents the apoptosis level of CT26 cells treated with different drugs under normoxic and hypoxic conditions; g represents the apoptosis rate (n = 3); h represents confocal images of live / dead cells stained with Calcein AM / PI after different drug treatments (scale bar: 100 μm).

[0042] Figure 22 In the middle, a) on the left is the CLSM image and analysis of mitochondrial colocalization in CT26 cells after treatment with PBS and TK NPs, and on the right is the corresponding Pearson correlation coefficient (Pearson's R) scatter plot and fluorescence intensity distribution curve across the cell region (scale bar: 20 μm); b) is the JC-1 staining confocal image after treatment with different drugs under normoxic and hypoxic conditions (scale bar: 20 μm) (n = 3).

[0043] Figure 23 In the figure, a is the flowchart of the in vivo anti-tumor experiment; b is the tumor volume growth curve of each group of mice during the treatment cycle; c is the ex vivo tumor photograph of each group at the end of treatment; d is the tumor inhibition rate of each group (n = 5); e is the H&E (scale bar: 200 μm), Ki67 (scale bar: 100 μm) and TUNEL (scale bar: 100 μm) stained sections of tumor tissues from different groups.

[0044] Figure 24 In the table, a represents the relative weight change curves of mice in each group during the treatment period; b represents the H&E stained histopathological images of the major organs (heart, liver, spleen, lung, and kidney) of mice in each group at the end of treatment (scale bar: 200 μm); c and d represent the serum biochemical and routine blood index test results of mice in each group (n = 3). Detailed Implementation

[0045] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0046] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0047] Example 1: Preparation and characterization of a hypoxia-reactive oxygen species dual-response prodrug nanodelivery platform based on maleimide calixarene supramolecular modification of bovine serum albumin. 1. Synthesis of MC4A Monocarboxylic acid trisulfonate azocalixarene (809 mg, 0.68 mmol) was dissolved in (for specific synthesis methods, refer to the literature "Wu, Xueyao, et al. "Antibody–Calixarene Drug Conjugate: A General Drug Delivery Platform for Tumor-Targeted Therapy." ACS Applied Materials & Interfaces 16.32 (2024): 41939-41948."), N,N-dimethylformamide (DMF, 25 mL), followed by N,N-diisopropylethylamine (DIPEA, 44 mg, 0.34 mmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylhexafluorophosphate (HATU, 78 mg, 0.21 mmol), and stirred at room temperature for 30 min. Then, 1-(2-(2-aminoethoxy)ethyl)-1H-pyrrole-2,5-dione-2,2,2-trifluoroacetate (170 mg, 0.57 mmol) was added to the reaction system, and the mixture was stirred at room temperature for 12 h under nitrogen protection. After the reaction was completed, the solvent was removed by vacuum drying, and finally recrystallized in water and methanol to obtain a brick-red powder product with a yield of 75%. The 1H NMR spectrum of the product is shown below. Figure 2 As shown: 1 ¹H NMR (400 MHz, DMSO-d⁶) δ: 7.92 (d, J = 6.3 Hz, 2H), 7.85–7.76 (m, 10H), 7.74–7.66 (m, 12H), 6.97 (s, 2H), 4.31 (s, 4H), 3.96 (s, 4H), 3.53 (dq, J = 16.0, 5.6, 4.9 Hz, 6H), 3.43–3.25 (m, 2H) ppm; its carbon NMR spectrum is as follows: Figure 3 As shown: 13C10 NMR (100 MHz, DMSO-d6) δ: 170.96, 165.66, 158.14, 158.05, 153.47, 151.87, 149.24, 144.92, 135.16, 134.56, 130.19, 128.37, 126.56, 124.31, 124.06, 121.67, 121.52, 68.35, 66.85, 36.80, 31.53 ppm; its high-resolution mass spectra are as follows: Figure 4 As shown: HRMS (ES) - : m / z calculated value [C 61 H 49 N 10 O 17 S3] - The value is: 1289.24 [MH] - The actual value is 1289.24003. MC4A was prepared into solutions of different concentrations (3, 6, 7.5, 15, 21, and 30 μM), and the UV absorption spectra of each solution were measured. The results are as follows: Figure 5 As shown in Figure a, MC4A exhibits characteristic absorption at 356 nm, and a concentration-dependent UV standard curve for MC4A is established. Figure 5 (as shown in b).

[0048] 2. Preparation of BSA-C4A Couplings BSA (40 mg, 6.02 × 10⁻⁶) -4 mmol) and Tris(2-carboxyethyl) phosphine hydrochloride (TCEP) (20 mg, 6.98 × 10 mmol) -2 7.722 mL of the above solution was added to 20 mL of ultrapure water and stirred at room temperature for 30 min to fully reduce the disulfide bonds in BSA, resulting in a solution exposing the internal thiol-containing BSA chains. Subsequently, this solution (7.722 mL) was mixed with ultrapure water (13.878 mL) and 10×PBS (4.4 mL), followed by the addition of 300 μM ultrapure water of MC4A, and the mixture was stirred overnight at room temperature. After the reaction, the solution was dialyzed in ultrapure water for 48 h using a dialysis bag (3.5 kDa) to thoroughly remove unreacted small molecule impurities. During dialysis, the dialysate was changed every 8 h to ensure effective removal of impurities. Finally, the reaction product was lyophilized using a freeze dryer and stored at -80 °C to ensure its stability during long-term storage. The UV absorption spectrum of BSA-C4A was measured, and the results are shown below. Figure 5As shown in Figure c, BSA-C4A exhibits the characteristic UV absorption of MC4A, proving its successful modification. Based on the UV absorbance-concentration standard curve (… Figure 5 As shown in b), the modification rate of MC4A in BSA-C4A was determined to be approximately 90%.

[0049] 3. Preparation of ROS-responsive prodrug NB-TK-CPT and non-responsive prodrug NB-C6-CPT (1) Synthesis of Compound 1: 1-Naphthylamine (4 g, 28.00 mmol) and methyl 3-bromopropionate (4.7 g, 28.00 mmol) were added to a 250 mL round-bottom flask, followed by 50 mL of anhydrous ethanol. The mixture was heated to reflux at 100 °C for 15 h. After the reaction was complete, the solvent was evaporated, and the mixture was purified by column chromatography using a dichloromethane:petroleum ether system (v / v, 1:1) to give a brown oily liquid with a yield of 36.4%. The proton NMR spectrum is shown below. Figure 7 As shown: 1 H NMR (500 MHz, DMSO-d6) δ 8.13-8.10 (m, 1H), 7.76 (dd, J = 7.9, 1.6 Hz, 1H), 7.45-7.39 (m, 2H), 7.30 (t, J = 7.9Hz, 1H), 7.13 (d, J = 8.1 Hz, 1H), 6.54 (dd, J = 7.6, 1.0 Hz, 1H), 6.17 (s, 1H), 3.64 (s, 3H), 3.49 (t, J = 7.0 Hz, 2H), 2.76 (t, J = 6.9 Hz, 2H).

[0050] (2) Synthesis of Compound 2: First, a 10 mL aqueous solution containing aluminum sulfate (2.075 g, 3.29 mmol) was prepared, and N,N-diethyl-p-phenylenediamine (2 g, 12.18 mmol) was added to it. Sodium thiosulfate (2.4 g, 15 mmol) and zinc chloride (1.77 g, 13 mmol) were added sequentially to the reaction system under continuous stirring. The apparatus was then transferred to an ice bath and cooled to 0 °C. An aqueous solution containing potassium dichromate (1 g, 3.40 mmol) was added dropwise at a uniform rate over 20 min. The reaction was continued at low temperature for 2 h. The precipitate was retained by vacuum filtration, and the filter cake was thoroughly washed with acetone to initially obtain the crude product. Finally, the crude product was heated under reflux in methanol for 3 h, and after filtration again, a gray powdery solid was obtained with a yield of 57.2%.

[0051] (3) Synthesis of Compound 3: Compound 1 (0.78 g, 3.40 mmol) and Compound 2 (0.44 g, 1.48 mmol) were dissolved in 50 mL of methanol. After heating to reflux temperature, silver carbonate (0.94 g, 3.40 mmol) was slowly added, and the mixture was refluxed at 85 °C for 2 h. After the reflux was completed, the solvent was removed by rotary evaporation. The crude product was redissolved in 50 mL of dichloromethane, and saturated sodium carbonate aqueous solution was introduced to wash away impurities in the system. The organic phase was separated, dried with anhydrous sodium sulfate, filtered, and then acidified with 0.9 mL of concentrated hydrochloric acid solution. The mixture was dried in a fume hood for 12 h, and then purified by column chromatography using a dichloromethane:methanol system (v / v, 100:1-20:1) for gradient elution. A dark blue powdery solid was collected, with a yield of 73.8%. The proton NMR spectrum is shown below. Figure 8 As shown: 1 H NMR (500 MHz, DMSO-d6) δ 9.88 (9.73 (m, 1H), 9.03 (dd, J = 8.4, 2.5 Hz, 1H), 8.58 (d, J = 8.3 Hz, 1H), 8.05 (dd, J = 9.4, 4.0 Hz, 1H), 7.97 (t, J = 7.6 Hz, 1H), 7.89 (t, J = 7.6 Hz, 1H), 7.63 (d, J = 2.5 Hz, 1H), 7.46 (td, J = 5.8, 2.9 Hz, 1H), 3.94 (dq, J = 18.3, 6.6 Hz, 2H), 3.83-3.53 (m, 7H), 2.89 (dt, J = 42.7, 6.9 Hz, 2H), 1.26 (d, J = 6.7Hz, 6H).

[0052] (4) Synthesis of compound 4: Compound 3 (0.12 g, 0.29 mmol) and 10 mL of 1,4-dioxane solution were added to a 100 mL round-bottom flask. While stirring, 1 M sodium hydroxide solution was added for hydrolysis. After 4 h of reaction, the organic matter was extracted with dichloromethane, the solvent was removed by rotary evaporation, and 0.2 mL of 1 M hydrochloric acid solution was added to the crude product for acidification. The solution was dried in a fume hood for 12 h, and then separated by column chromatography using a gradient elution system of dichloromethane:methanol (v / v, 20:1-8:1) to obtain compound 4 with a yield of 76.9%. Its 1H NMR spectrum is shown below. Figure 9 As shown: 1¹H NMR (500 MHz, DMSO-d⁶) δ 8.62 (d, J = 7.9 Hz, 1H), 8.35 (d, J = 7.9 Hz, 1H), 7.64 (dq, J = 26.3, 10.0, 8.7 Hz, 3H), 7.19 (s, 1H), 6.90–6.86 (m, 1H), 6.83 (s, 1H), 3.83 (t, J = 6.8 Hz, 2H), 3.49 (d, J = 7.2 Hz, 4H), 2.69 (t, J = 6.7 Hz, 2H), 1.25 (d, J = 5.3 Hz, 6H); its high-resolution mass spectra are as follows: Figure 10 As shown: HRMS (ES) + : m / z calculated value [C 23 H 23 N3O2S] + 406.15 [M+H] + ; Measured value: 406.1582.

[0053] (5) Synthesis of compound 5: Thioglycolic acid (27.60 g, 0.30 mol) was dissolved in 45 mL of acetone, and then hydrogen chloride gas was introduced. The reaction was stirred at room temperature for 6 h. After the reaction was completed, the mixture was placed in an ice bath, filtered, and the filter cake was washed with frozen ultrapure water and n-hexane. After vacuum drying overnight, a white powdery solid was collected with a yield of 32.9%.

[0054] (6) Synthesis of Compound 6: Compound 5 (1.50 g, 6.70 mmol) was fully dissolved in 150 mL of anhydrous tetrahydrofuran under ice bath conditions. Lithium aluminum hydride (40 mL, 26.70 mmol) was slowly added over 30 min, and the mixture was stirred at room temperature for 2 h. Ultrapure water was added dropwise to the reaction mixture until no more bubbles were generated. The mixture was filtered and washed with anhydrous tetrahydrofuran. The solvent was removed by rotary evaporation, and the mixture was then dissolved in anhydrous dichloromethane (10 mL). Anhydrous sodium sulfate was added and the mixture was left overnight to remove residual water. The crude product obtained by vacuum drying of the solvent was further purified by silica gel column chromatography using an ethyl acetate:dichloromethane system (v / v, 1:1) gradient elution. A pale yellow oily liquid was collected, with a yield of 34.9%. Its 1H NMR spectrum is shown below. Figure 11 As shown: 1 H NMR (400 MHz, Chloroform-d) δ 3.82 (t, J = 6.4 Hz, 4H), 2.91 (td, J = 6.3, 2.3 Hz, 4H), 1.66 (s, 6H).

[0055] (7) Synthesis of Compound 7: Weigh CPT (300 mg, 0.86 mmol) and bis(trichloromethyl) carbonate (86 mg, 0.29 mmol), add them together to a 50 mL round-bottom flask, and add 6 mL of anhydrous dichloromethane for dissolution and dispersion. Then, add 2 mL of anhydrous dichloromethane solution containing 4-dimethylaminopyridine (DMAP, 315 mg, 2.58 mmol) dropwise to the above reaction solution at a uniform rate, and continue stirring at room temperature for 45 min. Dissolve Compound 6 (1.00 g, 5.16 mmol) in 5 mL of anhydrous tetrahydrofuran beforehand, and then slowly add the solution dropwise to the above reaction system, and continue stirring at room temperature for 12 h. After the reaction is complete, wash the reaction system three times with 50 mL of 0.05 M hydrochloric acid and 50 mL of saturated saline solution. After the organic layer was treated with an appropriate amount of anhydrous sodium sulfate to remove residual moisture, it was rotary evaporated to dryness under reduced pressure. The crude product was further purified by column chromatography, eluted with ethyl acetate, to obtain a white powdery solid with a yield of 68.6%. Its 1H NMR spectrum is shown below. Figure 12 As shown: 1 H NMR (400 MHz, Chloroform-d) δ 8.43- 8.39 (m, 1H), 8.27-8.22 (m, 1H), 7.95 (ddd, J = 8.0, 6.4, 1.4 Hz, 1H), 7.84 (dtd, J = 8.3, 6.8, 1.5 Hz, 1H), 7.69 (d, J = 8.2 Hz, 1H), 5.73 (dd, J = 24.4, 16.8 Hz, 1H), 5.42 -5.29 (m, 3H), 4.30 (t, J = 6.7 Hz, 1H), 3.80 (s, 4H), 2.89 (s, 4H), 1.91 (ddt, J = 21.6, 14.2, 7.2 Hz, 2H), 1.54 (d, J = 1.0 Hz, 6H), 1.03 (dt, J = 22.6, 7.5 Hz, 3H).

[0056] (8) Synthesis of compound 8, i.e., NB-TK-CPT: Compound 4 (50 mg, 0.12 mmol) and compound 7 (43.5 mg, 0.08 mmol) were thoroughly dissolved and dispersed in 20 mL of N,N-dimethylformamide. Then, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (17.30 mg, 0.09 mmol) was added sequentially to the system as a condensing agent, and a small amount of 4-dimethylaminopyridine (3 mg, 0.025 mmol) was added as a catalytic medium. The reaction was stirred continuously at room temperature for 24 h. After the reaction was complete, the crude product was transferred to a separatory funnel, washed with saturated sodium chloride aqueous solution, and extracted with ethyl acetate. The combined organic phases were subjected to anhydrous sodium sulfate to remove residual water, and the solvent was evaporated under reduced pressure. Finally, the crude product was purified by column chromatography using a gradient elution of dichloromethane:methanol (v / v, 100–12:1) to obtain a dark blue powdery solid in 29.2% yield. Its 1H NMR spectrum is shown below. Figure 13 As shown: 1 H NMR (500 MHz, DMSO-d6) δ 8.58 (s, 1H), 8.42 (d, J = 8.4 Hz, 1H), 8.07 (d, J = 8.4 Hz, 1H), 8.03 (d, J = 8.3 Hz, 1H), 7.91 (s, 1H), 7.79 (t, J = 7.7 Hz, 1H), 7.64 (t, J = 7.5 Hz, 1H), 7.50 (s, 1H), 7.41 (d, J = 8.7 Hz, 1H), 7.36 (d, J = 15.1 Hz, 1H), 7.26 (dd, J = 8.7, 2.5Hz, 1H), 7.19 (d, J = 3.8 Hz, 1H), 7.05 (s, 1H), 6.90 (s, 1H), 5.50 (d, J = 7.4 Hz, 1H), 5.21 (s, 1H), 4.22 (d, J = 6.4 Hz, 1H), 4.17 (t, J = 6.6 Hz, 1H), 3.92 (s, 2H), 3.67 (d, J = 10.1Hz, 4H), 3.10 (d, J = 8.0 Hz, 2H), 2.88 (d, J = 5.8 Hz, 2H), 2.78 (d, J = 15.2 Hz, 4H), 2.18 (d, J = 7.3 Hz, 2H), 1.35 (d, J = 8.0 Hz, 6H), 0.93 (t, J = 7.3 Hz, 3H), 0.86 (t, J =6.7 Hz, 6H). Its high-resolution mass spectrometry ( Figure 14 ): HRMS (ES) + : m / z calculated value [C51 H 51 N5O8S3] + 958.29 [M+H] + ; Measured value: 958.2959.

[0057] (9) Synthesis of compound 9: CPT (100 mg, 0.29 mmol) and DMAP (105.2 mg, 0.86 mmol) were dissolved in 25 mL of anhydrous dichloromethane to a final volume of 50 mL. Then, bis(trichloromethyl) carbonate (28.1 mg, 0.10 mmol) was added to the reaction mixture, and the mixture was stirred at room temperature for 90 min to activate it. Next, a solution of hexanediol (170 mg, 1.44 mmol) pre-dissolved in 5 mL of anhydrous tetrahydrofuran was slowly added dropwise, and the mixture was stirred at room temperature in the dark for 24 h. After the reaction was complete, the solvent was evaporated to dryness under reduced pressure, and the dichloromethane was redissolved. The system was washed 2-3 times with ultrapure water, 0.5 M HCl solution, and saturated saline solution, respectively, to separate and retain the organic layer. Residual water was removed by anhydrous sodium sulfate, and the solvent was evaporated. The crude product was then purified by column chromatography (ethyl acetate: petroleum ether = 2:1, v / v) to obtain a white solid powder with a yield of 86.2%. Its 1H NMR spectrum is shown below. Figure 15 As shown: 1 H NMR (500 MHz, Chloroform-d) δ 8.41 (s, 1H), 8.23 ​​(d, J = 8.6 Hz, 1H), 7.95 (d, J = 8.2 Hz, 1H), 7.85 (t, J = 7.7 Hz, 1H), 7.68 (t, J = 7.6 Hz, 1H), 7.36 (s, 1H), 5.70 (d, J = 17.2 Hz, 1H), 5.40 (d, J = 17.2 Hz, 1H), 5.31-5.30 (m, 1H), 4.13 (ddt, J = 10.8, 6.9, 4.3 Hz, 2H), 3.60 (t, J = 6.5 Hz, 2H), 1.64 (s, 4H), 1.53 (t, J = 6.8 Hz, 2H), 1.39-1.37 (m, 2H), 1.27 (d, J = 14.7 Hz, 4H), 1.00 (t, J = 7.5 Hz, 3H).

[0058] (10) Synthesis of compound 10, i.e., NB-C6-CPT: Compound 4 (50 mg, 0.12 mmol) and compound 9 (39.5 mg, 0.08 mmol) were fully dissolved in 20 mL of N,N-dimethylformamide. Then, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (17.30 mg, 0.09 mmol) was added sequentially to the system as a condensing agent, and a small amount of 4-dimethylaminopyridine (3 mg, 0.025 mmol) was added as a catalytic medium. The mixture was stirred continuously at room temperature for 24 h. After the reaction was completed, the crude product was washed sequentially with saturated sodium chloride solution and purified and extracted with ethyl acetate multiple times. The combined organic phases were purified by removing residual water with anhydrous sodium sulfate. The solvent was then evaporated under reduced pressure. The crude product was purified by column chromatography using a dichloromethane:methanol system (v / v, 100:1-12:1) with gradient elution to obtain a dark blue solid powder with a yield of 26.5%. Its proton nuclear magnetic resonance spectrum is as follows Figure 16 As shown: 1 H NMR (500 MHz, Methanol-d4) δ 9.12 (d, J = 8.1 Hz, 1H), 8.31 (d, J = 8.2 Hz, 1H), 8.09 (d, J = 9.5 Hz, 1H), 7.92 (t, J = 7.7 Hz, 1H), 7.83 (t, J = 7.7 Hz, 1H), 7.50 -7.38 (m, 4H), 7.31 (d, J = 2.8 Hz, 1H), 7.22 (dd, J = 8.6, 2.5 Hz, 1H), 5.83-5.77 (m, 1H), 5.47 -5.16 (m, 2H), 3.99 (t, J = 6.7 Hz, 2H), 3.78-3.66 (m, 6H), 2.93 (t, J = 6.7 2.26 (t, J = 7.6 Hz, 2H), 2.00 (dt, J = 29.2, 7.1 Hz, 6H), 0.89 (t, J = 6.8 Hz, 9H). Its high-resolution mass spectra... Figure 17 As shown: HRMS (ES) + : m / z calculated value [C 51 H 51 N5O8S3] + : 880.33 [M+H] + ; Measured value: 880.3369.

[0059] 4. Synthesis of TK NPs and C6 NPs 2 μM NB-TK-CPT and MC4A solutions were prepared using PBS. Then, the PBS-C4A and NB-TK-CPT solutions were mixed at a molar ratio of MC4A:NB-TK-CPT = 1:1. The mixture was shaken thoroughly for 2 h to obtain the TK NP composite system. The ROS-non-responsive C6 NP composite system was prepared using the same method. The construction of the TK NPs was characterized by UV-Vis absorption spectroscopy. Figure 18 As shown in Figure a, the blank carrier BSA-C4A exhibits characteristic absorption near 370 nm, while the free prodrug NB-TK-CPT displays two significant absorption features in the visible light region, located at 540 nm and 670 nm, with maximum UV absorption at 670 nm. After forming the nanodrug delivery system TK NPs, its absorption spectrum completely retains the carrier's signal at 370 nm, while the characteristic absorption of the prodrug molecule is significantly reduced, confirming that NB-TK-CPT has been successfully loaded into the BSA-C4A carrier and maintains structural integrity. Notably, the encapsulated drug exhibits a significant absorption peak at 540 nm, and its relative intensity exceeds the absorption peak at 670 nm, which is dominant in the free state. This change may be attributed to the forced tight aggregation of the prodrug molecules within the confined space inside the BSA-C4A carrier, resulting in strong π-π stacking between their conjugated backbones, thus forming typical H-aggregates. This dense arrangement of molecules causes the splitting of excited-state energy levels, leading to a shift of the main absorption band towards shorter wavelengths. This characteristic spectral change effectively demonstrates the high-density encapsulation of the prodrug molecule within the carrier, directly proving the successful preparation of the TK NPs nanoparticle drug delivery system. Fluorescence spectroscopy can effectively evaluate the loading performance of BSA-C4A on the drug NB-TK-CPT. Figure 18As shown in b, at an excitation wavelength of 670 nm, the free NB-TK-CPT solution exhibits a strong characteristic fluorescence emission peak at 707 nm. However, when the drug is encapsulated by BSA-C4A to form the TK NPs nanoparticle drug delivery system, under the same detection conditions, the fluorescence signal of the free drug undergoes significant quenching, with a fluorescence quenching efficiency as high as 99.87%. This dramatic change in fluorescence signal strongly demonstrates that NB-TK-CPT molecules have been successfully and efficiently loaded into the nanostructure formed by BSA-C4A. The fluorescence quenching mechanism is mainly attributed to the significant fluorescence quenching triggered by the formation of a host-guest complex between the calixarene and the dye after the drug is loaded with calixarene. Therefore, the disappearance of the characteristic fluorescence of the drug in TK NPs strongly proves that the drug is not a simple physical mixture, but is efficiently loaded into the carrier, qualitatively confirming the extremely high encapsulation efficiency of the system. Further characterization of its morphology, particle size, and surface charge was performed using transmission electron microscopy (TEM) and dynamic light scattering (DLS). Figure 18 As shown in Figure d, DLS analysis revealed that the average hydrated particle size of the nanoparticles was 155.4 nm, and their polydispersity index (PDI) was less than 0.3, which is conducive to their passive targeted aggregation within tumor tissues in vivo based on the EPR effect. The zeta potential of the nanosystem was measured to be -19.64 ± 2.04 mV. The moderate negative charge endows the nanoparticle surface with a strong electrostatic repulsion force, thereby inhibiting particle aggregation to a certain extent and improving the dispersion stability of the system. At the same time, this negatively charged surface is conducive to reducing non-specific adsorption with plasma proteins, which helps to prolong the circulation time of the nano-drug delivery system in the blood. Further stability studies showed that the particle size of the nanoparticles did not change significantly within 7 days of storage in a PBS buffer system. Figure 18 As shown in Figure e), this indicates that no significant aggregation or degradation occurred in the system. Meanwhile, the PDI value remained stable and consistently below 0.3 (as shown in Figure e). Figure 18 As shown in f), this indicates that the particle distribution of the system remains highly uniform and has good dispersion stability.

[0060] Test Example 1: Evaluation of cellular uptake, permeation, and in vivo biodistribution of the albumin-calixarene drug-loaded material prepared in Example 1. To evaluate the performance of MC4A-modified BSA in terms of cellular uptake, permeation, and in vivo biodistribution in this invention, empty nanoparticles B, BT, and BC were prepared using Cy5-labeled raw BSA, BSA chains reduced by TCEP to untie disulfide bonds and expose thiol groups, and BSA-C4A conjugates, respectively. CT26 cells were seeded in 6-well plates or confocal culture dishes (2×10⁻⁶). 5CT26 cells were incubated in 96-well ultra-low adsorption plates (5 × 10⁶ cells / well) for 12 h, then replaced with a dedicated medium containing B, BT, and BC. After incubation under hypoxic (1% O₂) conditions for the set time, cell uptake was analyzed using flow cytometry and confocal microscopy. 3 3D tumor spheroids were formed by centrifugation at 500×g for 10 min for 5 days. The culture medium was then replaced with a special medium containing B, BT, and BC, and the spheroids were incubated under hypoxic conditions for 24 h. The 3D tumor spheroids were then imaged using a confocal microscope. Twelve female BALB / c mice were randomly divided into four groups (3 mice per group), and subsequently injected with PBS, B, BT, and BC via the tail vein, respectively. Imaging was performed at different time points after injection under isoflurane anesthesia using an in vivo imaging system. Forty-eight hours later, all mice were sacrificed, and tumors and major organs (heart, liver, spleen, lung, and kidney) were harvested for fluorescence imaging. Fluorescence intensity was analyzed using Living Image® 4.4 software.

[0061] Confocal image display ( Figure 19 (a) Compared to groups B and BT, groups BC showed significant drug uptake by cells 4 h after drug administration, with uptake gradually increasing over 12 h. Flow cytometry quantification and analysis ( Figure 19 (b) and (c) also showed that cellular uptake of BC was significantly higher than that of B and BT. 3D tumor spheroid imaging ( Figure 19 (d) Results showed that the tumor penetration depth in group BC was as high as 120 nm, and the overall fluorescence intensity was the strongest. In vivo imaging results showed ( Figure 19 In group BC, the drug rapidly accumulated at the tumor site 2 hours after administration, exhibiting a higher degree of drug enrichment at the tumor site compared to groups B and BT. This indicates that the carrier used in the hypoxia-ROS dual-response albumin nanodelivery platform of this invention demonstrates significant advantages in cellular uptake efficiency, tumor penetration depth, and in vivo tumor enrichment capacity, thereby achieving more efficient drug delivery performance and providing favorable technical support for anti-tumor therapy.

[0062] Test Example 2: Testing the drug release performance and ROS generation capacity of the hypoxia-ROS dual-response albumin nanodelivery platform prepared in Example 1. To simulate the difference in oxygen partial pressure between tumor hypoxia and normal tissue, the drug release behavior of TK NPs under hypoxic conditions was evaluated using a rat liver microsome-NADPH enzymatic system. Liver microsomes provided the enzymatic system primarily composed of cytochrome P450 reductase, while NADPH, as an electron donor, was more conducive to reduction reactions under hypoxic conditions, thus triggering the cleavage of hypoxia-responsive groups in the nanoparticles. Under normoxic conditions, oxidation reactions predominated, and the system remained stable overall. At 37°C, TK NPs nanoparticle solution was uniformly mixed with rat liver microsomes (40 μg / mL) and NADPH (40 μM), and incubated in normoxic (21% O2) or hypoxic (1% O2) environments, respectively. At set time points (5, 10, 15, 20, 25, 30, and 40 min), the reaction solution was transferred to a 3 kDa ultrafiltration tube and centrifuged for 5 min to effectively retain unreleased nanoparticles. Finally, the lower ultrafiltrate was collected to determine the actual release amount of NB-TK-CPT. For example... Figure 20 As shown in Figure a, under hypoxic conditions, the drug in the TK NPs is rapidly released, with a release rate exceeding 80% within 15 minutes and a cumulative release rate reaching 95% at 40 minutes. In contrast, under normoxic conditions, the drug is barely released, with a cumulative release rate of less than 5%. These results indicate that the TK NPs of this invention possess good drug loading stability and significant hypoxia-responsive controlled-release capability, which helps reduce non-specific drug release and improve therapeutic selectivity.

[0063] To verify the ability of the nanodelivery system to generate ROS in the hypoxic tumor microenvironment, this test case used the DCFH-DA probe to assess the ROS levels in each group of cells. CT26 cells were seeded in confocal dishes (5 × 10⁻⁶ cells / year). 5 Cells / well), after overnight incubation, the original medium was replaced with medium containing 0.5 μM of different drugs, and incubated for 8 h under normoxic (21% O2) or hypoxic (1% O2) conditions. Subsequently, the fluorescent indicator DCFH-DA was introduced into the cell system and co-incubated for 1 h. For the light-illuminated experimental group, a 660 nm laser (20 mW / cm²) was used. 2 Irradiate for 3 min. Fix with 4% paraformaldehyde and stain with DAPI solution in the dark for 10 min. Observe and acquire images using a laser confocal scanning microscope. Digest and collect cells, and perform flow cytometry analysis (FITC: 488 nm / 525 nm) within a 1 h window. Figure 20As shown in Figure 2, under hypoxic and normoxic conditions, almost no obvious green fluorescence signal was observed in the PBS and TK NPs groups, indicating that the basal ROS of the cells was at a low level and the carrier itself did not induce oxidative stress. The NB-TK-CPT+Laser group showed strong green fluorescence, indicating that the drug could efficiently induce ROS generation under light irradiation. Furthermore, the ROS generation capacity of the nanoparticle group showed a significant hypoxia response. In normoxic environments, both groups of nanoparticles showed almost no green fluorescence and low quantitative fluorescence intensity, fully demonstrating their high structural stability and low off-target damage risk under normal physiological conditions. However, once in a hypoxic environment, bright green fluorescence erupted in the cells of this group, and the fluorescence intensity of the TK NPs+Laser group was stronger than that of the C6 NPs+Laser group, indicating that TK NPs have superior photodynamic conversion efficiency in hypoxic microenvironments. In summary, the hypoxia-ROS dual-response prodrug nanosystem constructed in this invention can efficiently induce ROS generation under hypoxic conditions, thereby effectively overcoming the oxygen dependence of traditional photodynamic therapy.

[0064] Test Example 3: Evaluation of cellular uptake, toxicity, and apoptosis of the hypoxia-ROS dual-response albumin nanodelivery platform prepared in Example 1. To investigate the cellular uptake behavior of TK NPs under hypoxic conditions, CT26 cells were seeded in 6-well plates or confocal culture dishes (2 × 10⁶ cells / wells). 5 Incubate in medium (cells / well) for 12 h, then replace with medium containing TK NPs. After incubation under hypoxic (1% O2) conditions for the set time, observe intracellular distribution at different time points using laser confocal scanning microscopy (CLSM), and perform quantitative analysis using flow cytometry. CLSM results showed ( Figure 21 (a) With prolonged incubation, the intensity of intracellular red fluorescence gradually increased, with significant nanoparticle uptake observed from 2 h onwards, and a significant enhancement of the fluorescence signal at 6-10 h. This indicates that TK NPs exhibit a clear time-dependent uptake characteristic. Flow cytometry results showed ( Figure 21 The mean fluorescence intensity (MFI) of samples b and c increased over time and stabilized after about 8 h, indicating that TK NPs can be efficiently taken up by cells in a hypoxic microenvironment.

[0065] Next, the cytotoxicity of TK NPs was evaluated. CT26 cells were seeded in 96-well plates (5 × 10⁻⁶ cells / well). 3 Cells were transferred to wells and incubated overnight at 37 °C. After cell attachment, the culture medium was replaced with fresh medium containing appropriate concentration gradients of drugs, and incubated for 8 h under normoxic (21% O2) or hypoxic (1% O2) conditions. The light-illuminated group was irradiated with a 660 nm laser (20 mW / cm²). 2After incubation for 3 min, the culture medium was continued for 24 h. After incubation, the old medium was discarded and replaced with pre-prepared medium containing CCK-8 detection solution, and incubated at 37 ℃ for 60 min. Finally, the absorbance value (OD450) of each well was obtained at 450 nm wavelength using a microplate reader. Cell viability was calculated using the formula: Viability (%) = (OD treated group - OD blank background) / (OD untreated group - OD blank background) × 100%. The experimental results showed that when laser irradiation was introduced ( Figure 21 As shown in d), the cytotoxic effects of the small molecule prodrugs NB-TK-CPT and NB-C6-CPT after light exposure were not significantly affected by oxygen concentration. Compared to normoxic environments, the cytotoxic effects of TK NPs and C6 NPs were significantly enhanced under hypoxic conditions. Under light-free conditions ( Figure 21 As shown in Figure e), neither the TK NPs nor the C6 NPs nanoparticles exhibited significant cytotoxicity (cell viability > 80%) under normoxic or hypoxic conditions, indicating that the system possesses good biocompatibility and structural stability under light-free conditions. In contrast, free NB-TK-CPT showed some dark cytotoxicity at a drug concentration of 2 μM, indicating that the albumin-calixarene nanocarrier can reduce the dark cytotoxicity of NB-TK-CPT. Under hypoxic conditions, the cell-killing effect of TK NPs after light exposure was significantly better than that of the non-ROS-responsive C6 NPs control group, with the half-maximal inhibitory concentration (IC50) of TK NPs being significantly higher. 50 ICs with C6 NPs as low as 405 nM 50 The value is 721 nM. This is because the ROS-responsive TK linker introduced in the NB-TK-CPT system can break under the action of reactive oxygen species induced by light, thereby promoting the effective release of the chemotherapy drug CPT and achieving a synergistic enhancement effect between photodynamic therapy and chemotherapy; while the C6 linker does not have ROS-responsive characteristics and cannot release CPT, thus weakening its anti-tumor effect.

[0066] Further apoptosis was detected using Annexin V-FITC / PI double staining flow cytometry. CT26 cells were seeded in 6-well plates (5 × 10⁻⁶ cells / well). 5 (Number of NPs / well), incubated overnight at 37 ℃. The original medium was replaced with medium containing 0.4 μMTK NPs, and incubated for 8 h under normal aerobic (21% O2) or hypoxic (1% O2) conditions. The light group was irradiated with a 660 nm laser (20 mW / cm²). 2Cells were cultured for 24 h after 3 min of incubation. Cells were then gently digested with EDTA-free trypsin and collected. After washing with PBS, the cells were resuspended in Annexin V Binding Buffer, and Annexin V-FITC Reagent and PI Reagent were added respectively. After gentle mixing, the cells were stained at room temperature in the dark for 20 min. Flow cytometry analysis was performed within a 1 h window, and four-quadrant statistical analysis was performed using FlowJo software. Flow cytometry results. Figure 21 As shown in f and g, under normoxic and hypoxic conditions, the total apoptosis rate of both the PBS control group and the TK NPs group without light remained below 10%, indicating that hypoxia itself does not significantly induce cell apoptosis, and TK NPs do not exhibit significant dark toxicity under light-free conditions. After the introduction of light, the nanoparticle groups exhibited varying degrees of cell-killing activity and showed obvious hypoxia-responsive characteristics. The apoptosis rate of the TK NPs+Laser group was 19.61±2.61% under normoxic conditions, while it increased to 54.06±1.72% under hypoxia; the apoptosis rate of the C6 NPs+Laser group also increased from 14.05±1.28% under normoxic conditions to 34.14±4.49% under hypoxia, while the apoptosis rate of the free drug NB-TK-CPT+Laser group did not show obvious oxygen-dependent characteristics. These results fully demonstrate that in a normal physiological environment with sufficient oxygen, TK NPs can maintain high structural stability and effectively prevent the premature release of internal photosensitizers and chemotherapeutic drugs. Only under the triggering of the tumor-specific hypoxic microenvironment can TK NPs dissociate and precisely release internal drugs, effectively inducing apoptosis and exerting anti-tumor effects.

[0067] To further evaluate the cell-killing effect of the nanomedicine delivery system in the simulated complex microenvironment of solid tumors, CT26 cells in good logarithmic growth phase were selected and subjected to treatment at a rate of 5 × 10⁻⁶ cells / year. 3 Cells were initially seeded at a density of cells / well in low-adsorption 96-well plates. After sealing the plates, the cells were centrifuged at 500-600×g for 5 min to induce rapid sedimentation and aggregation at the bottom of the wells. The plates were then transferred to a 37 ℃ incubator for incubation, with half-volume medium replacement every two days. After 5-7 days of continuous culture, 3D tumor spheres with a diameter of approximately 400 μm, regular morphology, and smooth edges were formed. The old culture medium for the tumor spheres was replaced with medium containing 0.8 μM of different drugs, and incubated for 8 h under normoxic (21% O2) or hypoxic (1% O2) conditions. The light-illuminated group was irradiated with a 660 nm laser (20 mW / cm²). 2After 3 min, the culture was continued for 24 h. Then, 500 μL of Calcein AM / PI double staining reagent was added to each well, and the wells were incubated at 37 ℃ in the dark for 30 min. Finally, images were acquired using a laser confocal scanning microscope (Calcein AM: 490 nm / 515 nm; PI: 535 nm / 617 nm). Figure 21 As shown in h, the tumor spheres in the PBS control group and the unilluminated TK NPs group mainly exhibited strong and uniform green fluorescence signals, with only a small amount of red fluorescence, indicating that the overall cell viability was good. In contrast, under light irradiation, all treatment groups showed varying degrees of tumor-killing effects. Among them, the free prodrug group NB-TK-CPT+Laser was limited by its ability to penetrate the dense barrier, and some cells in the tumor core area still survived. The control nanogroup C6 NPs+Laser, due to the lack of CPT release, could not exert a synergistic chemotherapy effect, and its killing effect was relatively limited, with some residual green fluorescence still observed. In contrast, the TK NPs+Laser group was almost completely covered by red fluorescence, and the green fluorescence was basically gone, indicating that it could induce large-scale cell death in the 3D tumor model, exhibiting the most significant cytotoxicity, suggesting its potential advantage in overcoming the tumor microenvironment barrier and achieving deep cell killing.

[0068] Test Example 4: Functional evaluation of the photosensitizer in the hypoxia-ROS dual-response albumin nanodelivery platform prepared in Example 1 on mitochondrial targeting and mitochondrial membrane potential damage. To further investigate the targeting ability of TK NPs to mitochondria, fluorescence colocalization analysis was performed using laser confocal scanning microscopy (CLSM). CT26 cells were seeded in confocal dishes (2 × 10⁻⁶ cells / year). 5 Cells were incubated overnight (cells / plate). The original medium was replaced with medium containing 0.25 μM TK NPs, and incubated for 8 h under hypoxic (1% O2) conditions. Cells were washed 2-3 times with PBS buffer. Subsequently, cells were stained with Mito-Tracker Green staining working solution for 30 min in the dark. After staining, cells were fixed with 4% paraformaldehyde for 10 min, followed by DAPI staining for another 10 min in the dark. Cells were observed and photographed under a laser confocal microscope, and fluorescence images were extracted using ImageJ software. Pearson's correlation coefficient (R value) was calculated to assess the degree of colocalization. (Mito-Tracker: 488 nm / 550 nm; NB-TK-CPT: 640 nm / 720 nm). Results are shown below. Figure 22As shown in Figure a, in the PBS control group, only normal nuclear and mitochondrial fluorescence was observed. In the TK NPs treatment group, the cytoplasmic region of tumor cells exhibited strong red fluorescence, indicating that the nanoparticles were efficiently taken up by the cells. Furthermore, the red fluorescence of NB-TK-CPT highly overlapped with the green fluorescence of the mitochondrial probe, presenting a large area of ​​bright yellow fluorescence signal. Simultaneously, the Pearson correlation coefficient (Pearson's R) of the red-green fluorescence channels in the TK NPs group was as high as 0.81, and the fluorescence intensity curves across the cell region also showed that the fluorescence peaks of the two highly overlapped spatially, further verifying the enrichment of NB-TK-CPT in the mitochondrial region. These data demonstrate that TK NPs possess excellent mitochondrial targeting capabilities. This precise mitochondrial targeting characteristic ensures that the nanoparticles can directly destroy mitochondria and induce oxidative stress under light irradiation, thereby achieving highly efficient killing of tumor cells.

[0069] To investigate the disruptive effect of ROS generated by the nanodelivery system on mitochondrial function in tumor cells, the JC-1 fluorescent probe was used to detect the effect of each treatment group on mitochondrial membrane potential. CT26 cells were seeded in confocal dishes (5 × 10⁻⁶). 5 (each sample per dish), incubated overnight. The original medium was replaced with a medium containing 0.25 μM of different drugs, and incubated for 8 h under normal aerobic (21% O2) or hypoxic (1% O2) conditions. The light group was irradiated with a 660 nm laser (20 mW / cm²). 2 After washing with PBS, cells were incubated with JC-1 staining working solution at 37 °C for 30 min, then washed with pre-cooled JC-1 buffer and PBS. Fluorescence images of the cells were then captured using a laser confocal scanning microscope. During the detection process, dual-channel imaging was performed on both JC-1 monomers (set parameters 490 nm / 530 nm) and JC-1 aggregates (set parameters 525 nm / 590 nm) to analyze the effect of each nanoparticle on the mitochondrial membrane potential. Results Figure 22As shown in b, under normoxic conditions, the PBS group and each nanoparticle group mainly exhibited red fluorescence, indicating a high mitochondrial membrane potential and structural stability. Notably, under normoxic conditions, the nanoparticle-light-exposed group only showed a slight decrease in red fluorescence and no obvious green fluorescence signal, indicating that it did not induce a significant decrease in mitochondrial membrane potential. This suggests that the nanosystem maintains high stability in a normoxic environment and is difficult to activate effectively. Under hypoxic conditions, the control group and the unexposed TK NPs group maintained strong red fluorescence, while the nano-drug delivery system showed a significant increase in green fluorescence under light exposure, especially the TKNPs+L group, which showed explosive green fluorescence, indicating a drastic decrease in mitochondrial membrane potential and mitochondrial damage. In summary, this nano-drug delivery system has limited impact on mitochondrial function under normoxic conditions, but can significantly induce a decrease in mitochondrial membrane potential after light excitation in a hypoxic environment, exhibiting a clear environmental response characteristic. This result indicates that the system can be effectively activated in the hypoxic tumor microenvironment, thereby enhancing the destruction of mitochondrial function and promoting apoptosis.

[0070] Test Example 5: Evaluation of the in vivo antitumor effect and in vivo biosafety of the hypoxia-ROS dual-response albumin nanodelivery platform prepared in the examples. To further evaluate the antitumor effect of the nanoplatform in vivo, a CT26 tumor-bearing mouse model was established and randomly divided into four groups (n = 5): PBS group, TK NPs group, C6 NPs+Laser group, and TK NPs+Laser group. The administration regimen was as follows: [Details omitted]. Figure 23 As shown in Figure a, after treatment, all mice were euthanized. During the experiment, mouse weight and tumor volume were recorded every two days. Whole blood samples were collected from the treated mice for routine blood tests and blood biochemistry analysis, and tumor samples were also collected. Simultaneously, the heart, liver, spleen, lungs, and kidneys were collected for H&E staining to assess the histomorphological changes of major organs in different treatment groups. Figure 23 As shown in Figure 2, compared to the PBS group, the TK NPs group without laser irradiation showed only a very limited tumor-suppressing trend. In contrast, all groups receiving phototherapy showed varying degrees of tumor growth inhibition. Among them, the TK NPs+Laser group had the smallest tumor volume, with more than half of the mice experiencing complete tumor regression, and the average tumor volume was successfully limited to 32 mm at the experimental endpoint. 3Around 97% of the tumors were inhibited; while the C6 NPs+Laser group showed some tumor-suppressing effect, some tumors still relapsed and grew in the later stages of treatment. This significant difference in efficacy indicates that the stimulus-responsive prodrug strategy combined with PDT exhibits the best anti-tumor effect. External light not only stimulates photosensitizers to generate a large amount of ROS that directly kills tumors, but more importantly, the in-situ generated ROS induces the breakage of TK linkers, enabling precise and controllable release of chemotherapy drugs at the tumor site, thus amplifying the therapeutic effect. H&E staining ( Figure 23 Results e) showed that tumor cells in the PBS group and the unexposed TK NPs group were tightly packed, morphologically intact, and showed no obvious necrosis areas. However, the C6 NPs+Laser group exhibited some degree of tissue structure destruction and local necrosis, indicating that photodynamic therapy had a certain damaging effect on tumor tissue. The TK NPs+Laser treatment group showed the most significant tissue damage, with extensive cytoplasmic loss and tissue structure destruction, indicating its most significant tumor-killing effect. In Ki67 staining to assess cell proliferation activity, sections of the control group and the unexposed TK NPs group were filled with dense brown positive granules, indicating that tumor cells were in a highly proliferative state. In the TK NPs+Laser group, the Ki67 positive signal was significantly downregulated, and the brown area was greatly reduced, confirming that it could effectively inhibit tumor cell proliferation and promote tumor cell death. Furthermore, TUNEL fluorescence detection results showed that the TKNPs+Laser group exhibited the highest intensity of yellow fluorescence signal (representing apoptotic cells), and its distribution range was significantly wider than that of other groups. This indicates that TK NPs can achieve highly efficient killing of tumor tissues by inhibiting cell proliferation and inducing large-scale apoptosis under photoexcitation.

[0071] like Figure 24 As shown in Figure a, the body weight of mice in each treatment group did not change significantly throughout the treatment period, indicating that the nano-drug delivery system and its combined treatment regimen did not cause significant systemic toxicity, and the overall physiological state of the animals was good. Further histological evaluation of the major organs (heart, liver, spleen, lung, and kidney) was performed using H&E staining, as shown in Figure a. Figure 24 As shown in b, the tissue morphology of major organs such as the heart, liver, spleen, lungs, and kidneys in each treatment group was intact and the texture was clear. No pathological cell necrosis, inflammatory infiltration, or tissue damage was observed. A comprehensive analysis of peripheral blood biochemistry and routine blood parameters was performed on the mice in each group after treatment. The results... Figure 24As shown in Figures c and d, compared with healthy mice and control mice, the liver function-related indicators (ALT, AST) and kidney function-related indicators (UREA, CREA) of mice treated with nanoparticles were all within the normal physiological thresholds, indicating that the treatment process did not cause significant abnormalities in liver and kidney function. No significant differences were observed in the levels of RBC, WBC, HGB, Lymph, PLT, HCT, MCV, MCH, MCHC, MPV, PDW, and Gran% among the groups. Erythrocyte-related indicators (RBC, HGB, HCT, MCV, MCH, and MCHC) were normal, indicating that the nanoparticles did not significantly affect the body's oxygen-carrying capacity or erythrocyte morphology; white blood cell-related indicators (WBC, Lymph, and Gran%) were normal, indicating that the nanoparticles did not cause significant acute inflammatory reactions or immunotoxicity in vivo during the treatment; platelet-related indicators (PLT, MPV, and PDW) showed minimal changes, indicating that coagulation function was not significantly interfered with. The above results demonstrate that the nanodelivery system has good biocompatibility and in vivo safety, achieving highly effective anti-tumor treatment without causing serious toxic side effects on the body, and has good potential for clinical translation.

[0072] In summary, this invention can effectively overcome the limitations of the hypoxic microenvironment of tumors and enhance the synergistic effect of photodynamic therapy combined with chemotherapy. It has good biocompatibility and provides a promising application strategy for overcoming treatment resistance caused by the hypoxic microenvironment inside colorectal cancer.

[0073] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. A method for preparing a hypoxia-reactive oxygen species dual-response prodrug nanodelivery system based on MC4A-modified BSA, characterized in that, Includes the following steps: Step S1: Dissolve monocarboxylic acid trisulfonate azocalixarene in N,N-dimethylformamide as solvent, then add buffer and condensing agent, and continuously stir and activate at room temperature. Then add 1-(2-(2-aminoethoxy)ethyl)-1H-pyrrole-2,5-dione-2,2,2-trifluoroacetate, and continue stirring and react at room temperature under nitrogen protection. Then remove the solvent by rotary evaporation under reduced pressure, and finally recrystallize in a mixed solution of water and methanol to obtain product MC4A. Step S2: Dissolve BSA and reducing agent in ultrapure water and stir continuously at room temperature to expose the thiol groups inside BSA. Obtain thiol-exposed BSA chains by dialysis and freeze-drying. Step S3: Redissolve the thiol-exposed BSA chain, add MC4A, stir continuously at room temperature, dialyze in ultrapure water using a dialysis bag, and freeze-dry to obtain product BSA-C4A; Step S4: Dissolve 1-naphthylamine and methyl 3-bromopropionate in anhydrous ethanol, heat to reflux, and after the reaction is complete, evaporate the solvent and separate and purify by column chromatography to obtain compound 1; Step S5: Prepare an aqueous solution containing aluminum sulfate and add N,N-diethyl-p-phenylenediamine to it. While stirring, add sodium thiosulfate and zinc chloride to the reaction system in sequence. Then transfer it to an ice bath to cool. After the temperature stabilizes, add an aqueous solution containing oxidant dropwise at a uniform rate. Continue the reaction at low temperature until a viscous substance is observed to form. Filter the solution and wash the filter cake with detergent to obtain a crude product. Finally, place the crude product in methanol solvent and heat under reflux. After filtering again, obtain a gray powdery solid, namely compound 2. Step S6: Dissolve compound 1 and compound 2 in methanol, add silver carbonate, heat to reflux, after the reaction is complete, remove the solvent by rotary evaporation, remove impurities by redissolved extraction, then acidify and dry, and finally separate and purify by column chromatography to obtain compound 3. Step S7: Compound 3 was dissolved in 1,4-dioxane solution and hydrolyzed with sodium hydroxide solution. After the reaction was completed, the organic matter was extracted, the solvent was removed by rotary evaporation, acidified, dried and purified by column chromatography to obtain compound 4. Step S8: Dissolve thioglycolic acid in acetone and then pass hydrogen chloride gas through it. Stir the reaction at room temperature. After the reaction is complete, place the mixture in an ice bath, filter and wash it, and vacuum dry it overnight. The resulting white powdery solid is compound 5. Step S9: Under low temperature conditions, compound 5 was dissolved in tetrahydrofuran, lithium aluminum hydride was added and stirred at room temperature, then ultrapure water was added dropwise, filtered, washed, and the solvent was removed by rotary evaporation, and compound 6 was further separated and purified by silica gel column chromatography. Step S10: CPT and bis(trichloromethyl) carbonate are dissolved in dichloromethane, and then a dichloromethane solution containing 4-dimethylaminopyridine is added dropwise. The reaction is continued to be stirred at room temperature. Then, an anhydrous tetrahydrofuran solution containing compound 6 is added dropwise. The reaction is continued to be stirred at room temperature. After the reaction is completed, the mixture is washed, the solvent is removed by rotary evaporation, and compound 7 is further purified by column chromatography. Step S11: Dissolve compounds 4 and 7 in N,N-dimethylformamide, then add condensing agent and catalyst, and continue stirring the reaction. After the reaction is completed, extract the crude product and purify it by column chromatography to obtain compound 8, namely the responsive prodrug NB-TK-CPT. Step S12: Redissolve BSA-C4A and add it to NB-TK-CPT solution in a molar ratio. Shake to obtain TK NPs nanoparticles loaded with ROS-responsive prodrug NB-TK-CPT, which is the hypoxia-reactive oxygen dual-response prodrug nanodelivery system based on MC4A-modified BSA.

2. The method for preparing a hypoxia-reactive oxygen species dual-response prodrug nanodelivery system based on MC4A-modified BSA according to claim 1, characterized in that, In step S1, the buffer is N,N-diisopropylethylamine, the condensing agent is 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylhexafluorophosphate, the activation time under continuous stirring at room temperature is 20-40 min, the molar ratio of monocarboxylic acid trisulfonate azocalixarene, N,N-diisopropylethylamine, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylhexafluorophosphate and 1-(2-(2-aminoethoxy)ethyl)-1H-pyrrole-2,5-dione-2,2,2-trifluoroacetate is 3-4:1-2:1:2-3, and the reaction time under nitrogen protection at room temperature with continuous stirring is 10-14 h.

3. The method for preparing a hypoxia-reactive oxygen species dual-response prodrug nanodelivery system based on MC4A-modified BSA according to claim 1, characterized in that, In step S2, the reducing agent is tris(2-carboxyethyl)amine hydrochloride, the reaction time with continuous stirring at room temperature is 20-40 min, and the molar ratio of BSA to tris(2-carboxyethyl)amine hydrochloride is 1:110-120; in step S3, the solvent for resolubilizing the thiol-exposed BSA chain is PBS, the reaction time with continuous stirring at room temperature is 12-16 h, the dialysis time is 36-72 h, and the molar ratio of the thiol-exposed BSA chain to MC4A is 1:30-35.

4. The method for preparing a hypoxia-reactive oxygen species dual-response prodrug nanodelivery system based on MC4A-modified BSA according to claim 1, characterized in that, In step S4, the reflux temperature is 95~105 °C, the reflux duration is 12~20 h, and the eluent for column chromatography is dichloromethane and petroleum ether in a volume ratio of 5~1:

1. In step S5, the oxidant is potassium dichromate, the low temperature is 0 °C, the reaction duration at the low temperature is 1~4 h, the reflux temperature is 65~100 °C, the duration is 1~5 h, the washing agent is acetone, and the molar ratio of aluminum sulfate, N,N-diethyl-p-phenylenediamine, sodium thiosulfate, potassium dichromate, and zinc chloride is 2~5:2~5:3~6:1:2~5.

5. The method for preparing a hypoxia-reactive oxygen species dual-response prodrug nanodelivery system based on MC4A-modified BSA according to claim 1, characterized in that, In step S6, the molar ratio of compound 1, compound 2, and silver carbonate is 1.5~3∶1∶1.5~3; the reflux temperature is 70~100 °C; the duration is 1~3 h; the extractant for the redissolution extraction is dichloromethane and saturated sodium carbonate; the acidifying agent for the acidification is concentrated hydrochloric acid; and the eluent for the column chromatography is dichloromethane and methanol in a volume ratio of 100~20:

1. In step S7, the acidifying agent for the acidification is hydrochloric acid; the molar ratio of compound 3, sodium hydroxide, and hydrochloric acid is 1∶2~5∶2~5; the hydrolysis duration is 2~6 h; the extraction solvent for the extraction is dichloromethane; and the eluent for the column chromatography is dichloromethane and methanol in a volume ratio of 20~8:

1.

6. The method for preparing a hypoxia-reactive oxygen species dual-response prodrug nanodelivery system based on MC4A-modified BSA according to claim 1, characterized in that, In step S8, the stirring reaction at room temperature lasts for 4-8 hours, and the washing agent for filtration is ultrapure water and n-hexane; in step S9, the low temperature is 0-5 °C, the molar ratio of compound 5 to aluminum hydroxide is 1:2-6, the washing agent is tetrahydrofuran, and the eluent for column chromatography is ethyl acetate and dichloromethane in a volume ratio of 1:4-1.

7. The method for preparing a hypoxia-reactive oxygen species dual-response prodrug nanodelivery system based on MC4A-modified BSA according to claim 1, characterized in that, In step S10, the molar ratio of CPT, bis(trichloromethyl) carbonate, 4-dimethylaminopyridine and compound 6 is 2~4∶1∶7~10∶15~20, the reaction times for the first and second reactions are 20~60 min and 8~14 h, respectively, the washing agent is hydrochloric acid and saturated brine, and the eluent for column chromatography is ethyl acetate.

8. The method for preparing a hypoxia-reactive oxygen species dual-response prodrug nanodelivery system based on MC4A-modified BSA according to claim 1, characterized in that, In step S11, the condensing agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, the catalyst is 4-dimethylaminopyridine, and the molar ratio of compound 4, compound 7, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine is 3~6∶2~4∶2~4∶1; in step S12, the solvent for the BSA-C4A reconstitution and the NB-TK-CPT solution is PBS solution, and the molar ratio of MC4A to NB-TK-CPT is 1∶1~5.

9. A hypoxia-reactive oxygen species dual-response prodrug nanodelivery system based on MC4A-modified BSA prepared by the preparation method according to any one of claims 1 to 8.

10. The application of the hypoxia-reactive oxygen species dual-response prodrug nanodelivery system based on MC4A-modified BSA as described in claim 9 in the preparation of colorectal cancer drugs.

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