Construction and application of supramolecular nanoamplifiers regulated by multiple interactions

Abstract

This invention discloses the construction and application of a multi-interaction-regulated supramolecular nanoamplifier, belonging to the field of biopharmaceutical technology. For the first time, this invention utilizes 5,10,15,20-tetrakis(4-aminophenyl)porphyrin photosensitizer (TAPP) and manganese dioxide nanozyme (MnO2) to assemble with poly-α-lipoic acid (PαLA) through electrostatic adsorption and metal coordination, respectively, to develop a multi-interaction-regulated GSH-stimulated supramolecular nanoamplifier, PαLA@TAPP-MnO2. This achieves specific delivery and release of the photosensitizer, disrupting redox homeostasis within tumors, thereby enhancing the PDT-mediated anti-tumor efficacy. This has been verified both in vitro and in vivo experiments. The supramolecular nanoamplifier developed in this study provides new insights into the design of anticancer nanomedicines by regulating redox homeostasis within tumors.

Description

Technical Field

[0001] The present invention belongs to the field of biopharmaceutical technology, and in particular relates to the construction and application of a supramolecular nanoamplifier regulated by multiple interactions. Background Art

[0002] Colorectal cancer (CRC) has become the third most common malignant tumor in the world, with an incidence rate second only to lung cancer and female breast cancer. In 2020, it has become the second leading cause of cancer-related deaths, and its incidence rate in my country has also been increasing year by year.

[0003] Currently, the primary clinical treatment for colorectal cancer is complete tumor resection. However, patients with advanced colorectal cancer are prone to recurrence and metastasis after surgery, resulting in a 5-year survival rate of less than 10%. Therefore, traditional radiotherapy and chemotherapy remain the main treatments for advanced colorectal cancer. Even with current targeted therapies and immunotherapies, due to their anti-tumor mechanisms, these treatments can still cause intolerable side effects for patients. Photodynamic therapy (PDT) has attracted widespread attention due to its advantages such as minimal invasiveness, controllability, high efficacy, and low adverse reactions. PDT utilizes toxic reactive oxygen species (ROS) such as singlet oxygen and free radicals generated by photosensitizers to induce cytotoxicity, leading to tumor cell damage and even cell death. Despite its many advantages, PDT has not yet been widely used in clinical healthcare. The main reason is that the intrinsic properties of photosensitizer molecules, such as poor targeting, low solubility, and high hydrophobicity, have resulted in a severe shortage of photosensitizers that are effective in tumor treatment.

[0004] Chemodynamic therapy, sonodynamic therapy, radiotherapy, and PDT are effective anticancer strategies that promote cellular oxidative stress by promoting the production of toxic ROS within cancer cells. Unfortunately, the levels of ROS that trigger oxidative stress are counteracted by upregulated antioxidant systems. To prevent damage caused by excessive oxidative stress, tumor cells appropriately regulate various antioxidants to control ROS production, thereby limiting ROS accumulation and developing resistance to ROS therapy. The delicate balance between ROS and antioxidant levels is crucial for maintaining redox homeostasis within tumor cells. Intracellular antioxidants are categorized as enzymatic and nonenzymatic. Enzymatic antioxidants include catalase, superoxide dismutase, and members of the peroxidase family, while nonenzymatic antioxidants primarily include reduced glutathione (GSH), tocopherol, and ascorbic acid. Reduced GSH is considered the most abundant antioxidant in cells and is essential for scavenging ROS. Therefore, glutathione depletion can prevent ROS scavenging and lead to increased ROS accumulation. The resulting alteration of redox homeostasis within tumors makes tumor growth more susceptible to oxidative stress and enhances the efficacy of ROS-based tumor therapy. Therefore, it is necessary to develop an effective strategy to deliver photosensitizer molecules to specific sites or targeted areas of tumors to achieve glutathione depletion. Summary of the Invention

[0005] In light of the shortcomings of existing technologies, the present invention provides a supramolecular nanoamplifier with multiple interactions to disrupt redox homeostasis within tumors, thereby enhancing the anti-tumor efficacy of PDT-mediated therapy. TAPP, combined with MnO2, which has the ability to phagocytose GSH, synergistically increases ROS production in tumor cells, disrupting intracellular redox homeostasis and making tumor growth more susceptible to oxidative stress, thereby improving the therapeutic efficacy of colon cancer cells.

[0006] In order to achieve the above-mentioned purpose of the invention, the technical solution adopted by the present invention is as follows.

[0007] The present invention provides a supramolecular nanoamplifier regulated by multiple interactions, characterized in that the supramolecular nanoamplifier is PαLA@TAPP-MnO2.

[0008] Further, it is prepared by the following method:

[0009] 5,10,15,20-tetrakis(4-aminophenyl)porphyrin photosensitizer (TAPP) and manganese dioxide nanozyme (MnO2) were encapsulated in disulfide-bonded poly-α-lipoic acid (PαLA) to construct the supramolecular nanoamplifier PαLA@TAPP-MnO2.

[0010] Furthermore, the method specifically includes the following steps:

[0011] S1. Preparation of poly-α-lipoic acid (PαLA): αLA was added to a dry flask and stirred at 80°C for 2 h under a nitrogen flow; the obtained PαLA was then dissolved in N,N-dimethylformamide (DMF) to obtain a PαLA solution;

[0012] S2. Preparation of MnO2 nanozyme: Potassium permanganate was added to water and stirred for 30 minutes to obtain a solution; oleic acid was then added to the solution to form a stable emulsion; the emulsion was further stirred at room temperature until brown-black blocks appeared, indicating the synthesis of the MnO2 nanozyme; the obtained MnO2 nanozyme was washed three times by centrifugation with alcohol to remove the remaining reactants, and then dried MnO2 nanozyme was obtained by vacuum freeze-drying;

[0013] S3. Preparation of TAPP solution: TAPP was added to DMF and dissolved by ultrasonication to prepare TAPP solution;

[0014] S4. Preparation of supramolecular nanoamplifier:

[0015] (1) Weigh the MnO2 nanozyme prepared in S2 and fully disperse the MnO2 in DMF using ultrasonication to prepare a MnO2 suspension;

[0016] (2) The PαLA solution prepared in S1 and the TAPP solution prepared in S3 were mixed and added to a dry penicillin bottle, and magnetically stirred at room temperature for 2 h in a dark environment to obtain suspension A; then the MnO2 suspension prepared in (1) was added to suspension A, and the mixture was stirred at room temperature for 2 h to obtain suspension B; DSPE-PEG 2000 (20 wt %) was added to suspension B and the mixture was stirred for 5 min to obtain suspension C;

[0017] (3) Take the mixed solution C prepared in (2) to another dry vial and slowly drip it into deionized water under magnetic stirring, and stir at room temperature for 5 minutes; remove DMF by ultracentrifugation to prepare the supramolecular nanoamplifier (PαLA@TAPP-MnO2).

[0018] Furthermore, any of the above-mentioned supramolecular nanoamplifiers regulated by multiple interactions is used in the preparation of a tumor-targeted drug delivery system.

[0019] Furthermore, any one of the above-mentioned supramolecular nanoamplifiers regulated by multiple interactions is used in the preparation of anti-tumor nanomedicines.

[0020] Furthermore, any of the above-mentioned supramolecular nanoamplifiers regulated by multiple interactions is used in the preparation of drug carriers that respond to the high GSH environment in tumor cells.

[0021] The present invention also provides a photodynamic therapy anti-tumor nanomedicine, characterized in that the drug is the PαLA@TAPP-MnO2 described in claim 1.

[0022] Furthermore, the laser wavelength for photodynamic therapy of the tumor is 660 nm.

[0023] Furthermore, the tumor is intestinal cancer.

[0024] Compared with the prior art, the present invention has the following advantages and beneficial effects.

[0025] (1) The present invention adopts a simple preparation process and a "one-step nanoprecipitation method" to allow TAPP with positively charged amino groups to electrostatically modify the negatively charged carboxyl groups on the polymer side chains. At the same time, manganese dioxide nanozyme (MnO2) is used to coordinate the carboxyl groups to prepare the supramolecular nanoamplifier PαLA@TAPP-MnO2, which is simple and easy to implement.

[0026] (2) PαLA@TAPP-MnO2 has good stability under simulated physiological conditions (10% FBS + pH 7.4 PBS buffer) and exhibits good responsiveness to high GSH in a simulated tumor high GSH environment (10% FBS + pH 7.4 + 10 mM DTT PBS buffer), and is effectively cleaved under reducing conditions.

[0027] (3) The effective uptake of PαLA@TAPP-MnO2 by cells is a prerequisite for its anti-tumor effect. TAPP and MnO2 co-assembled into nanoamplifiers have stronger in vitro synergistic anti-tumor ability, which is mainly reflected in: PαLA@TAPP-MnO2 induces cells to produce more ROS through Fenton-like reaction and photodynamic therapy; has stronger cytotoxicity; induces more cell death; and can significantly induce cell apoptosis. The PαLA carrier has no toxic effect on cells and has good biocompatibility.

[0028] (4) Compared with the DiR solution, the accumulation and retention of PαLA@TAPP-MnO2 in the tumor site were significantly enhanced. The highest accumulation in the tumor was 12 hours after administration. These results ensure the therapeutic effect of the prepared nanoamplifier in vivo.

[0029] (5) The nanoamplifier PαLA@TAPP-MnO2 has the most efficient synergistic anti-tumor effect in subcutaneous tumors. PαLA@TAPP-MnO2 does not cause systemic toxic side effects and meets the criteria of an ideal anti-tumor drug, namely, high efficiency and low toxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1Preparation and characterization of supramolecular nanoamplifiers. Figure A shows the synthesis process of PαLA@TAPP-MnO2; Figure B shows the particle size and potential of MnO2, PαLA@MnO2, PαLA@TAPP, and PαLA@TAPP-MnO2; Figure C shows the TEM observation of the morphology of PαLA@TAPP-MnO2 and MnO2; Figure D shows the synthesis of PαLA and its molecular docking studies with TAPP and MnO2; Figure E shows UV absorption spectroscopy.

[0031] Figure 2 Colloidal stability, GSH responsiveness, and in vitro release of PαLA@TAPP-MnO2. Figure A shows the colloidal stability of PαLA@TAPP-MnO2 in PBS containing 10% FBS, pH 7.4; Figure B shows the particle size changes of PαLA@TAPP-MnO2 after treatment with PBS containing or without 10 mM DTT for 12 hours; Figure C shows the TEM image of PαLA@TAPP-MnO2 after treatment with PBS containing 10 mM DTT for 12 hours; Figure D shows the in vitro accumulation and release of PαLA@TAPP-MnO2 after treatment with different concentrations of DTT for 24 hours.

[0032] Figure 3 Cellular uptake of the nanoamplifier PαLA@TAPP-MnO2. A shows the uptake in CT26 cells observed by laser scanning confocal microscopy; B shows the quantitative analysis of intracellular fluorescence intensity; and C shows the quantitative analysis of cellular uptake by flow cytometry.

[0033] Figure 4 Cell viability assay results. A shows the relative cell viability of CT26 cells incubated with different concentrations of PaLA for 24 hours using the MTT assay; B shows the relative cell viability of CT26 cells treated with different treatments using the MTT assay; C shows CLSM fluorescence images of CT26 cells treated with different treatments after live-dead staining; D shows flow cytometric analysis of PaLA@TAPP-MnO2 and PaLA@TAPP cell apoptosis; E shows a schematic diagram of the supramolecular nanoamplifier's GSH-responsive drug release, TAPP-mediated PDT, Mn2-mediated Fenton-like reactions, and synergistic disruption of intratumoral redox homeostasis.

[0034] Figure 5 Detection of intracellular reactive oxygen species generation. A is a CLSM fluorescence image of ROS generation in CT26 cells; B is a fluorescence intensity histogram of ROS in CT26 cells analyzed by flow cytometry; C is a fluorescence intensity histogram of ROS in CT26 cells analyzed by flow cytometry.

[0035] Figure 6Tissue distribution of nanoamplifiers. A shows in vivo DiR fluorescence images of CT26 tumor-bearing mice at 6, 12, and 24 hours after intravenous injection of DiR solution and DiR-labeled NPs. B shows in vitro fluorescence images of major organs and tumor tissues 24 hours after injection of DiR solution and DiR-labeled NPs.

[0036] Figure 7 Pharmacodynamic study of the nanoamplifier in a subcutaneous tumor model. A is a schematic diagram of the treatment of a CT26 tumor-bearing mouse model; B shows the tumor volume growth curve of CT26 tumor-bearing mice after intravenous injection of different formulations (n=6); C shows the weight of the ex vivo tumor on day 9 after different dosing methods; D shows an image of the ex vivo tumor tissue after the last treatment; E shows a TUNEL analysis image of the tumor section after the last treatment.

[0037] Figure 8 In vivo safety assessment of PaLA@TAPP-MnO2. A shows the weight changes of tumor-bearing mice from day 0 to 9 after administration of different formulations; B shows the blood biochemical parameters of CT26 tumor-bearing mice after treatment with different formulations; and C shows H&E staining of the main organ tissues of CT26 tumor-bearing mice after treatment. DETAILED DESCRIPTION

[0038] The present invention will be further described in detail below by means of specific examples and accompanying drawings. The following examples are only provided to further illustrate the present invention and should not be construed as limiting the present invention.

[0039] 1. Materials and methods

[0040] 1. Cell.

[0041] Mouse colon cancer cell line CT26, (Nanjing Kebai Biotechnology Co., Ltd.); mouse colon cancer cell-luciferase marker (CT26-Luc) (Beijing Yita Biotechnology Co., Ltd.).

[0042] 2. Animals.

[0043] SPF BALB / c mice and C57BL / 6 mice (approved by the Experimental Animal Ethics Committee of Jinzhou Medical University), female, 18-22 g.

[0044] 2. Experimental methods

[0045] (a) Preparation and characterization of supramolecular nanoamplifiers.

[0046] 1. Preparation of supramolecular nanoamplifier.

[0047] Preparation of poly-α-lipoic acid (PαLA): αLA can be self-polymerized by heating above its melting point without any initiator. Briefly, 2.06 g of αLA was added to a dry flask and stirred at 80°C for 2 hours under a nitrogen stream. The resulting PαLA was then dissolved in 10 mL of N,N-dimethylformamide (DMF) to obtain a PαLA solution.

[0048] Preparation of MnO2: To synthesize MnO2 nanoflowers, 250 mg of potassium permanganate was added to 125 mL of water and stirred for 30 minutes to obtain a solution. Then, 2.5 mL of oleic acid was added to form a stable emulsion. The emulsion was further stirred at room temperature for 24 hours, until brown-black clumps appeared, indicating the synthesis of the MnO2 nanozyme. The solution was then centrifuged at 3000 g for 10 minutes, washed three times with alcohol to remove any remaining reactants, and finally freeze-dried in a vacuum to obtain the dried MnO2 nanozyme.

[0049] Preparation of supramolecular nanoamplifiers: The nanoamplifiers were prepared using a one-step nanoprecipitation method. An appropriate amount of MnO2 was weighed and thoroughly dispersed in DMF using ultrasonication. 2 mg of TAPP was weighed, added to 1 mL of DMF, and sonicated to dissolve. 1 mL of the previously prepared PαLA solution and 1 mL of the TAPP solution were mixed and added to a dry vial. The mixture was magnetically stirred at room temperature in the dark for 2 h. 1 mL of the MnO2 suspension was then added, and stirring continued at room temperature for 2 h. DSPE-PEG 2000 (20 wt %) was added to the stirred solution, and stirring continued for 5 min. Finally, 200 μL of the mixed solution was transferred to another dry vial and slowly added dropwise to 2 mL of deionized water under magnetic stirring. The mixture was stirred at room temperature for 5 min. The DMF was removed by ultracentrifugation to yield the supramolecular nanoamplifier (PαLA@TAPP-MnO2). To prepare single-component nanoparticles PαLA@TAPP and PαLA@MnO2, 1 mL of TAPP solution and MnO2 suspension were diluted half with DMF and added to 1 mL of PαLA solution respectively, and magnetically stirred at room temperature for 2 h. The remaining steps referred to the above operation process.

[0050] 2. Particle size, potential and appearance morphology of supramolecular nanoamplifier.

[0051] The prepared PEG-modified nanoparticles (PαLA@TAPP, PαLA@MnO2, and PαLA@TAPP-MnO2) and MnO2 were diluted with deionized water and placed in a particle size cup and a potentiometer. The particle size distribution, zeta potential, and polydispersity index (PDI) were analyzed using a Malvern Zetasizer. The morphology of PαLA@TAPP-MnO2 was observed using transmission electron microscopy (TEM). TEM sample preparation was as follows: the PαLA@TAPP-MnO2 solution and the MnO2 suspension were diluted to a certain amount and then dropwise added to a carbon-coated copper TEM grid. After the solution dried at room temperature, the morphology of the nanoamplifiers was observed and photographed using a transmission electron microscope.

[0052] 3. Study on the assembly mechanism of nanoamplifiers.

[0053] Molecular docking simulations of the nanoamplifier were investigated. Molecular simulations, a widely used method for predicting molecular interactions, were used to investigate the details of the co-assembly of PαLA, TAPP, and MnO2. Sybyl software was used to construct the three-dimensional structures and intermolecular forces of PαLA, TAPP, and MnO2. The obtained results were analyzed using Discovery Studio 2017 Visualizer software.

[0054] 4. Ultraviolet absorption spectrum determination.

[0055] The UV-visible absorption spectra of DMF solutions of PαLA, TAPP, MnO2, and PαLA@TAPP-MnO2 (6 μg / mL TAPP and 0.6 mg / mL PαLA) were obtained by scanning in the range of 280–650 nm using a Varioskan LUX multimode microplate reader (Thermo Fisher Scientific, USA).

[0056] 5. Investigation of colloidal stability and glutathione responsiveness.

[0057] To monitor the stability of the nanoamplifier, we investigated the particle size changes of PαLA@TAPP-MnO2 in PBS buffer (pH 7.4) containing 10% FBS. The following steps were performed: PαLA@TAPP-MnO2 was placed in the aforementioned PBS buffer and incubated in a shaker (37°C) in the dark with constant shaking (100 rpm) for 12 hours. Samples were taken at predetermined times (0, 2, 4, 6, 8, 10, and 12 hours) and their particle size changes were monitored using a Zetasizer. PαLA@TAPP-MnO2 was then mixed with PBS buffer (pH 7.4) containing 10% FBS and 10 mM dithiothreitol (DTT) and incubated for 12 hours with constant shaking. The particle size was then measured. Transmission electron microscopy was also used to analyze the degradation of PαLA@TAPP-MnO2 after DTT treatment to assess the GSH responsiveness of the nanoamplifier.

[0058] 6. In vitro release of drug-loaded nanoamplifiers.

[0059] The in vitro drug release behavior of PαLA@TAPP-MnO2 was determined using a dialysis method. To prevent TAPP precipitation, all release media contained 20% (v / v) DMF. Briefly, 1 mL of PαLA@TAPP-MnO2 (equivalent to 60 μg TAPP) was transferred to a dialysis bag and then immersed in 30 mL of different release media. The media were incubated at 37°C with shaking at 100 rpm. The release media included pH 7.4 PBS, pH 7.4 PBS containing 1 mM DTT, and pH 7.4 PBS containing 10 mM DTT. At various predetermined time points, 1 mL of release media was collected and replaced with an equal volume of fresh media. The cumulative release of TAPP from PαLA@TAPP-MnO2 was measured by fluorescence spectroscopy (ex / em = 434 / 682 nm). The release rate of TAPP was determined based on the fluorescence intensity.

[0060] (2) Cellular level research on supramolecular nanoamplifiers.

[0061] 1. Preparation of test solution.

[0062] RPMI 1640 culture medium: Dissolve one 1L bag of RPMI 1640 medium powder in 1L of triple-distilled water. Add 2.0g of sodium bicarbonate, penicillin (100 units / mL), and streptomycin (100μg / mL). Stir magnetically for 2 hours until the powder is fully dissolved. Then, add an appropriate amount of concentrated hydrochloric acid to adjust the pH to approximately 7.2. Sterilize the solution through a 0.22μm filter in a clean room. Aliquot and seal the solution, then store in a refrigerator at 4°C until needed. Before use, add 10% (v / v) fetal bovine serum and sterilize the solution through a 0.22μm filter.

[0063] Preparation of pancreatic enzyme: Add 2.5g pancreatic enzyme and 0.2g EDTA to 1L PBS buffer and stir to dissolve completely. After sterile filtration through a 0.22μm filter membrane in a clean bench, seal the aliquots and store at 4℃ until use.

[0064] Preparation of MTT solution: Keep sterile and away from light throughout the process. Dissolve an appropriate amount of MTT powder in PBS solution to prepare a 5 mg / mL MTT solution. Sterilize the solution by filtering through a 0.22 μm filter membrane and store at -20°C away from light. Dilute to 1 mg / mL with serum-free culture medium before use.

[0065] 2. Cell culture.

[0066] CT26 cells were cultured in RPMI 1640 medium supplemented with 10% (v / v) fetal bovine serum. The specific steps were as follows: cells frozen at -80°C in liquid nitrogen were removed and rapidly thawed in a 37°C water bath. After thawing, the cell suspension was gently aspirated to create a single-cell suspension. This suspension was then aspirated and transferred to a centrifuge tube containing 10 mL of culture medium. The suspension was centrifuged at 1000 rpm / min for 3 minutes. The supernatant was discarded, and the cells were resuspended in 1 mL of culture medium. The suspension was then transferred to a culture dish containing 9 mL of culture medium. Gently agitated in a clean hood to ensure even distribution of the cells, the cells were then incubated in a CO2 incubator (at 37°C, 5% CO2, and 90% relative humidity). When the cells reached 80% confluence in the culture dish, they were trypsinized and passaged as described above.

[0067] 3. Cellular uptake.

[0068] CT26 cells that had grown to about 80% were digested into single-cell suspensions and inoculated into 12-well plates at a cell seeding density of 1×10 5Cells were plated at 400 μg / well and grown in a CO2 incubator for 12 hours until fully adhered. The old culture medium was then removed and fresh culture medium containing either TAPP solution or PαLA@TAPP-MnO2 solution (TAPP equivalent concentration: 10 μg / mL) was added. The cells were then incubated in the incubator for 1, 2, and 4 hours. After incubation, the old drug-containing culture medium was aspirated and the cells were washed three times with ice-cold PBS to remove residual drug from the cell surface and terminate cellular uptake. 500 μL of 4% paraformaldehyde was then added to each well, and the cells were fixed at 37°C for 10 minutes. The cells were then rinsed three times with cold PBS, and 300 μL of DAPI solution was added, followed by incubation for 15 minutes in the dark. After nuclear staining, the solution was discarded, the cells were washed with cold PBS, and intracellular fluorescence was observed using a laser scanning confocal microscope. To quantify cellular uptake, PαLA@TAPP-MnO2 uptake was assessed by flow cytometry. Plating and cell treatment were performed as described above. After incubation, the cells were washed three times with cold PBS and then digested with trypsin. After digestion was terminated with fresh culture medium, the cells were collected in a 2 mL EP tube, the supernatant was removed by centrifugation, and the cells were resuspended in PBS. The fluorescence intensity in the cells was measured by flow cytometry.

[0069] 4. Cell viability detection.

[0070] (1) MTT method was used to investigate the cytotoxic effect of PαLA@TAPP-MnO2 on cells. The MTT method was used to investigate the in vitro cytotoxicity of different formulations on CT26 cells. First, the cytotoxicity of different concentrations of PαLA was evaluated. 5×10 3 CT26 cells were seeded into 96-well plates at a density of 10 cells / well and incubated in a carbon dioxide incubator for 24 hours. Fresh culture medium was used to prepare PαLA-containing culture medium with different concentrations. After the incubation, the old culture medium was replaced with it and incubated for another 24 hours. To investigate the cytotoxicity of TAPP, MnO2, PαLA@TAPP, PαLA@MnO2 and PαLA@TAPP-MnO2 (TAPP equivalent concentration 10μg / mL), fresh culture medium was used to prepare drug-containing culture medium containing various formulations to replace the old cell culture medium. 150μL of drug-containing culture medium was added to each well, and 3 parallel wells were set up for each group. Fresh RPMI 1640 culture medium was still added to the blank control group. After incubation with drugs for 4 hours, the cells in the 96-well plate were exposed to laser irradiation (wavelength 660nm, power 20mW / cm 2, 5 minutes), then returned to the CO2 incubator for another 20 hours. After incubation, the 96-well plate was removed, the drug-containing culture medium discarded, and 150 μL of 1 mg / mL MTT solution was added to each well and incubated in the incubator for 4 hours. After 4 hours, the MTT solution was discarded, and 100 μL of DMSO solution was added to each well. The cells were shaken on a shaker for 10 minutes to dissolve the resulting blue-purple crystals. The absorbance of each well was measured at 490 nm using a multi-function microplate reader.

[0071] (2) Live and dead cell staining method to observe the survival status of cells. Calcein AM itself has no fluorescence. After passing through the living cell membrane and reacting with the esterase in the living cell, the AM group is removed and the generated Calcein emits a strong green fluorescence signal. Propidium iodide (PI) can only enter the dead cell when the living cell membrane is damaged and chimera with nucleic acid to produce a conspicuous red fluorescence, so dead cells can be detected with red fluorescence. Calcein AM and PI emit different fluorescence under different laser excitations, so they are used to double-stain living and dead cells. The operation steps are as follows: CT26 cells are plated at 1×10 5 Cells were seeded into 12-well plates at a density of 10 μg / well. After 12 hours of culture, fresh culture medium containing TAPP, MnO2, PαLA@TAPP, PαLA@MnO2, and PαLA@TAPP-MnO2 (TAPP equivalent concentration 10 μg / mL) was added and incubated for another 4 hours. Fresh RPMI 1640 culture medium was also added to the blank control group. After 4 hours, the cells were directly irradiated with laser for 5 minutes (wavelength 660 nm, power 20 mW / cm 2 ) Continue incubating at 37°C for 20 hours. After incubation, discard the drug-containing culture medium and gently wash the cells three times with PBS to remove active esterases. Add enough staining solution prepared according to the instructions to cover the cell monolayer and incubate at 37°C for 30 minutes. Finally, remove the staining solution and add an appropriate amount of PBS to submerge the cells to prevent cell lysis. Observe the cell fluorescence using a laser scanning confocal microscope.

[0072] (3) Annexin V-FITC / PI apoptosis kit was used to detect cell apoptosis. Annexin V-FITC / PI staining kit was used to investigate the apoptosis of CT26 cells induced by PαLA@TAPP-MnO2. The specific steps were as follows: CT26 cells were seeded into 12-well plates (2×10 5The cells were placed in a 37°C incubator and incubated for 12 hours. After the cells were fully attached, the old cell culture medium was replaced with fresh culture medium without drugs, culture medium containing PαLA@TAPP or PαLA@TAPP-MnO2 (TAPP equivalent concentration 10μg / mL), and the blank control group was still added with fresh RPMI 1640 culture medium. After incubation for 4 hours, the cells in the 12-well plate were exposed to a wavelength of 660nm and a power of 20mW / cm 2 After 5 minutes of laser irradiation, the cells were placed in a CO2 incubator and cultured for another 20 hours. The cells were then trypsinized and harvested by centrifugation at 1000 rpm for 5 minutes. The cells were washed three times with ice-cold PBS. Each sample was resuspended in 500 μL of Binding Buffer and transferred to a flow cytometer. The cells were stained with Annexin V-FITC / Propidium Iodide (PI) for 15 minutes according to the manufacturer's instructions and analyzed by flow cytometry.

[0073] 5. Detection of intracellular reactive oxygen species generation.

[0074] DCFH-DA was used as a reactive oxygen species (ROS) probe to detect ROS generated by PαLA@TAPP-MnO2 in CT26 cells. DCFH-DA itself does not produce fluorescence. After being hydrolyzed by esterase in cells, it exists as a non-fluorescent form, DCFH. This can be specifically oxidized by ROS to produce the fluorescent substance DCF, and the fluorescence intensity depends on the level of ROS in the cells. The operation process is as follows: CT26 cells are plated at 1×10 5 Cells were seeded into 12-well plates at a density of 1 mL / well. After 12 hours of culture, the cells were replaced with 1 mL of fresh culture medium containing TAPP, MnO2, PαLA@TAPP, PαLA@MnO2, and PαLA@TAPP-MnO2 (TAPP equivalent concentration 10 μg / mL) and incubated for another 4 hours. The blank control group was also incubated with fresh RPMI 1640 culture medium. After 4 hours of incubation, the drug-containing culture medium was removed, the cells were washed with cold PBS, and the DCFH-DA probe diluted in culture medium (10 μg / mL) was added and incubated for another 30 minutes. Afterwards, the drug-containing culture medium was discarded, blank PBS was added, and the cells were placed at a wavelength of 660 nm and a power of 20 mW / cm 2 The cells were irradiated with laser for 3 minutes or without illumination. Finally, the cells were washed three times with ice-cold PBS, fixed with 4% paraformaldehyde for 10 minutes, stained with DAPI for 15 minutes in the dark, and the fluorescence intensity of DCF in the cells was observed using a laser scanning confocal microscope.

[0075] The steps for determining intracellular ROS levels by flow cytometry are as follows: plating and cell treatment are the same as above. After incubation, the cells are washed three times with cold PBS and digested with trypsin. After terminating the digestion with fresh culture medium, the cells are collected in a 2 mL EP tube. After centrifugation to remove the supernatant, the cells are resuspended in PBS and transferred to a flow tube. The fluorescence intensity of intracellular DCF is measured by flow cytometry.

[0076] (3) Tissue distribution of nanoamplifiers.

[0077] 1. Preparation of DiR-labeled nanoamplifiers.

[0078] TAPP solution, PαLA solution, and MnO2 suspension were prepared according to the previously described method. 1 mL of PαLA solution and 1 mL of TAPP solution were mixed and added to a dry vial. The mixture was magnetically stirred at room temperature in the dark for 2 hours. Subsequently, 1 mL of MnO2 suspension was added, and stirring continued at room temperature for 2 hours. DSPE-PEG 2000 (20 wt%) and 4 mg of DiR were weighed and mixed with the above-mentioned solution, stirring continued for 5 minutes. Finally, 200 μL of the mixed solution was transferred to another dry vial and slowly added dropwise to 2 mL of deionized water under magnetic stirring. The mixture was stirred at room temperature for 5 minutes. This yielded DiR-labeled nanoamplifiers (DiR-labeled PαLA@TAPP-MnO2), abbreviated as DiR-labeled NPs. The organic solvent, DMF, was removed by ultracentrifugation.

[0079] 2. Establish a CT26 tumor-bearing mouse model.

[0080] Select CT26 cells in good condition, digest them from the culture dish with trypsin, transfer them to a 15 mL centrifuge tube, add RPMI 1640 cell culture medium (containing 10% v / v fetal bovine serum) to terminate cell digestion, centrifuge (1000 rpm / min, 3 min), discard the cell supernatant containing trypsin, re-add cell culture medium containing fetal bovine serum, count and collect the cell pellet by centrifugation, resuspend the cells in an appropriate amount of PBS, and blow until the cells are evenly dispersed and have a density of 2 × 10 7 cells / mL of single cell suspension. Use an insulin syringe to inject 100 μL of single cell suspension (containing 2×10 6 The cells were inoculated subcutaneously on the right posterior flank or lumbar abdomen of 6-week-old female BALB / c mice to establish a mouse CT26 colon cancer xenograft model.

[0081] 3. Distribution of DiR-labeled PαLA@TAPP-MnO2 in vivo.

[0082] When the subcutaneous tumor volume of tumor-bearing mice approaches 300 mm 3Mice were randomly divided into two groups and injected via the tail vein with either free DiR solvent or DiR-labeled NPs at a dose of 2 mg / kg. Six, 12, and 24 hours after administration, the mice were anesthetized and fluorescence images were observed using an in vivo imaging system (IVIS Spectrum Small Animal In vivo Imaging System). Twenty-four hours after administration, the mice were sacrificed, and the major organs (heart, liver, spleen, lung, and kidney) and tumors of the tumor-bearing mice were dissected and collected. Fluorescence images of the ex vivo tissues were captured using a small animal in vivo imaging system.

[0083] (IV) Pharmacological study of nanoamplifiers in subcutaneous tumor models.

[0084] The synergistic therapeutic effect of PαLA@TAPP-MnO2 on tumors in vivo was studied in the CT26 tumor-bearing female BALB / c mouse model established above. 3 Around 24 hours after treatment, the mice were marked and randomly divided into 6 groups, with 6 mice in each group, namely PBS group, MnO2 suspension group, TAPP solution + Laser group, PαLA@MnO2 group, PαLA@TAPP+Laser group and PαLA@TAPP-MnO2+Laser group. The corresponding drug preparations were injected into each group of mice through the tail vein. The drug was administered once every two days, for a total of 4 times. For the treatment groups requiring laser irradiation (TAPP solution + Laser group, PαLA@TAPP+Laser group and PαLA@TAPP-MnO2+Laser group), 12 hours after each administration, a laser with a wavelength of 660nm and a power of 100mW / cm 2 The light source was used to irradiate the tumor site for 5 minutes. During the entire treatment cycle, the weight of the mice and the short diameter (a) and long diameter (b) of the tumor were recorded at the same time every day to calculate the average tumor volume. On the last day of the in vivo efficacy experiment, the mice were killed by cervical vertebrae dissection, the tumor tissue was peeled off and weighed, and finally the tumor tissues of each group after treatment were photographed. Combined with the tumor growth curve (that is, the growth curve of tumor volume over time, tumor volume V=a×a×b / 2), the effects of different preparations on tumor growth were analyzed and compared. The apoptosis of tumor tissue was observed by TUNEL immunofluorescence staining.

[0085] Safety evaluation included plotting the time course of mouse body weight changes, liver and kidney function parameters, and H&E staining.

[0086] Plot a mouse weight-time curve. That is, throughout the treatment process, weigh and record the weight changes of tumor-bearing mice daily until the last day. The systemic toxicity of the drug can be assessed based on the changing trend.

[0087] To assess liver and kidney function, on the last day of the efficacy study, blood was collected from tumor-bearing mice using a coagulant tube and centrifuged at 4000 rpm for 15 minutes. Serum samples were then collected and tested. Key liver and kidney function indicators tested included aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine (CREA), and blood urea nitrogen (BUN). Liver and kidney function was assessed based on the test results.

[0088] For H&E staining, tumor-bearing mice were sacrificed, and major organs (heart, liver, spleen, lungs, and kidneys) were isolated and immersed in 4% paraformaldehyde solution for dehydration and fixation. The tissues were then sent to Wuhan Sewell Biotechnology Co., Ltd. for paraffin embedding and paraffin sectioning. Finally, the tissue sections were stained with H&E. The sections were observed and photographed using an upright microscope to assess the pathological status of each tissue.

[0089] 3. Experimental results.

[0090] (a) Preparation and characterization of supramolecular nanoamplifiers.

[0091] 1. Preparation of supramolecular nanoamplifier.

[0092] We used the "one-step nanoprecipitation method" to prepare a supramolecular nanoamplifier with polymer, TAPP and MnO2 as the main components to achieve a synergistic anti-tumor effect. Figure 1 As shown in A.

[0093] 2. Particle size, potential and appearance morphology of supramolecular nanoamplifier.

[0094] Dynamic light scattering was used to further characterize the particle size, potential and PDI of PαLA@TAPP-MnO2. Figure 1 As shown in B: The particle sizes of PαLA@TAPP and PαLA@MnO2 are 138±1.5nm and 197±5.7nm, respectively. In comparison, the particle size of PαLA@TAPP-MnO2 increased slightly to 226±2.4nm, and the PDI was 0.107±0.084. The negative Zeta potential of PαLA@TAPP-MnO2 was detected to be -13.5±0.82mV, which is also higher than that of PαLA@TAPP and PαLA@MnO2. TEM was then used to observe the morphology of PαLA@TAPP-MnO2 and MnO2. The image showed that PαLA@TAPP-MnO2 had a nearly spherical structure with a uniform size distribution and a particle size of about 230nm, which is consistent with the DLS results ( Figure 1 C). It was also observed that the dispersed MnO2 showed a flower-like morphology in the TEM image, as shown in Figure 1 C.

[0095] 3. Study on the assembly mechanism of nanoamplifiers.

[0096] The molecular docking experiment preliminarily analyzed the mechanism of PαLA, TAPP and MnO2 co-assembly to form PαLA@TAPP-MnO2 and investigated the intermolecular forces. Figure 1 As shown in Figure D, there is an electrostatic adsorption force between the amino group of the TAPP molecule and the carboxyl group of the PαLA molecule, while there is a metal coordination force between the MnO2 molecule and the carboxyl group of the PαLA molecule.

[0097] 4. Ultraviolet absorption spectrum determination.

[0098] Poly-α-lipoic acid (PαLA) was synthesized by ring-opening polymerization of αLA monomer at 80°C for 2h. According to the literature, the UV-visible absorption spectrum of disulfane in αLA monomer shows a characteristic peak at 330nm. However, the UV-visible spectrum of PαLA obtained in this study did not show a clear characteristic peak. Figure 1 As shown in Figure E, this means that all monomers are ring-opening polymerized to form PαLA. In addition, compared with the TAPP solution, the characteristic peak of TAPP was observed in the UV absorption spectrum of PαLA@TAPP-MnO2, and an obvious red shift occurred.

[0099] 5. Investigation of colloidal stability and glutathione responsiveness.

[0100] In order to evaluate the stability of PαLA@TAPP-MnO2 under simulated physiological conditions and its reactivity in the tumor-mimicking intracellular environment, we used DLS to observe the changes in particle size under different conditions. Figure 2 As shown in Figure A, after incubation for 12 hours in PBS buffer (pH 7.4) containing 10% FBS, the particle size of PαLA@TAPP-MnO2 did not change significantly within 12 hours and remained relatively stable, indicating that it has good colloidal stability. We then tested the particle size of PαLA@TAPP-MnO2 after incubation for 12 hours in PBS buffer (pH 7.4) containing 10mM DTT (a commonly used GSH mimetic agent) and 10% FBS. It was found that in the presence of 10mM DTT, the particle size of PαLA@TAPP-MnO2 changed significantly after 12 hours ( Figure 2 B). At the same time, TEM images also show that the PαLA@TAPP-MnO2 structure is destroyed and the intact nanoparticles are cracked ( Figure 2 C).

[0101] 6. Release in vitro.

[0102] The in vitro reductive release behavior of PαLA@TAPP-MnO2 was further evaluated using dialysis, anticipating that the decomposition of the nanoamplifier in the presence of high levels of GSH would trigger drug release in the tumor-specific microenvironment. A PBS buffer solution containing 20% ​​(v / v) DMF was selected as the release medium, and the in vitro release of PαLA@TAPP-MnO2 was investigated using TAPP as a standard. Figure 2 D shows the results of in vitro reductive drug release in the presence and absence of DTT. In the absence of DTT and at a pH of 7.4, only approximately 5% of TAPP was released from PαLA@TAPP-MnO2 within 24 hours. Conversely, the cumulative release of TAPP increased with increasing DTT concentration. A significant increase in TAPP release was observed at a DTT concentration of 10 mM, reaching 75% of the cumulative release within 24 hours. This result demonstrates that PαLA@TAPP-MnO2 exhibits excellent GSH reduction-sensitive drug release properties.

[0103] (2) Cellular level research on supramolecular nanoamplifiers.

[0104] 1. Cellular uptake.

[0105] We used mouse colon cancer CT26 cells as an in vitro tumor cell model and used flow cytometry and laser confocal microscopy to detect the TAPP fluorescence intensity in CT26 cells at different time points to evaluate the cell uptake of PαLA@TAPP-MnO2 and investigate the difference between its uptake and that of free TAPP solution. Figure 3 As shown in A, blue fluorescence represents DAPI, i.e., the cell nucleus, and red fluorescence refers to TAPP. The results showed that the fluorescence intensity of TAPP in both groups of cells increased over time, indicating that the uptake of free TAPP and PαLA@TAPP-MnO2 by cells was time-dependent. However, PαLA@TAPP-MnO2 showed stronger intracellular red fluorescence intensity than free TAPP at different time points (1h, 2h, and 4h), indicating that cells had a stronger uptake efficiency of PαLA@TAPP-MnO2 ( Figure 3 B). In addition, it can be observed that as time goes on, the red fluorescence and blue fluorescence in cells treated with PαLA@TAPP-MnO2 tend to overlap, which means that after the cells take up PαLA@TAPP-MnO2, TAPP released in response to the higher concentration of GSH in tumor cells can enter the cell nucleus. These results indicate that PαLA@TAPP-MnO2 has good cellular internalization ability and effective intracellular drug molecule release ability. Flow cytometry results also show that PαLA@TAPP-MnO2 has time-dependent cellular uptake ( Figure 3 C).

[0106] 2. Cell viability detection.

[0107] (1) MTT method was used to investigate the cytotoxic effect of PαLA@TAPP-MnO2 on cells. We used the MTT method to detect the biocompatibility of PαLA in CT26 cells. Figure 4 As shown in A, after treating cells with different concentrations of PαLA for 24 hours, no significant inhibition of cell viability was observed even at high concentrations of PαLA, indicating good biocompatibility. We used CT26 cells as a tumor cell model to evaluate the effects of TAPP solution, MnO2 suspension, PαLA@TAPP, PαLA@MnO2 and PαLA@TAPP-MnO2 on the growth of CT26 cells and to study the synergistic therapeutic effect of PαLA@TAPP-MnO2 on tumor cells in vitro. Figure 4 As shown in Figure B, PαLA@TAPP-MnO2 and other components showed different degrees of cytotoxicity to CT26 cells. Among them, the cytotoxicity caused by MnO2 suspension and PαLA@MnO2 may be due to the redox reaction of MnO2 with high concentrations of GSH in CT26 cells, which generates GSSG and Mn 2+ , then Mn 2+ It mediates a Fenton-like reaction, catalyzing the overexpressed hydrogen peroxide (H2O2) in cells to generate highly reactive hydroxyl radicals (•OH). •OH is a type of ROS, thereby inhibiting the activity of tumor cells. In TAPP-mediated PDT, the degree of tumor cell killing depends on 1 We observed that the TAPP solution + Laser group showed cytotoxicity similar to that of the PαLA@TAPP + Laser group, which may be due to the unique therapeutic properties of TAPP solution under laser irradiation - that is, TAPP mediates PDT in extracellular and intracellular 1 O2 and PαLA@TAPP-MnO2 both contribute to oxidative damage in tumor cells. Because cells are more resistant to PαLA@TAPP-MnO2 than TAPP, this may lead to greater cytotoxicity. Therefore, the PαLA@TAPP-MnO2+Laser group exhibited the strongest cytotoxicity.

[0108] (2) Live and dead cell staining method to observe the survival status of cells. Use the live and dead cell staining kit to stain the cells after different sample treatments to more intuitively observe the distribution of live and dead cells. Its fluorescence image is as follows Figure 4As shown in Figure C (G1, blank control group; G2, MnO2 suspension group; G3, TAPP solution + laser group; G4, PaLA@MnO2 group; G5, PaLA@TAPP + laser group; G6, PaLA@TAPP-MnO2 + laser group, Scale Bar = 50 μm), green fluorescence represents live cells and red fluorescence represents dead cells, respectively. As can be seen in the figure, cell growth in the untreated control group was unaffected. Cells treated with TAPP solution, MnO2 suspension, PαLA@TAPP, PαLA@MnO2, and PαLA@TAPP-MnO2 all showed cell death. PαLA@TAPP-MnO2 caused the most significant cell death, consistent with the cytotoxicity results.

[0109] (3) Annexin V-FITC / PI apoptosis kit was used to detect cell apoptosis. Next, we compared the anti-proliferative properties of PαLA@TAPP-MnO2 and PαLA@TAPP in CT26 cells. After drug treatment, CT26 cells were labeled with Annexin V-FITC / PI, and flow cytometry was used to study their apoptosis induced by laser irradiation. Figure 4 D is the flow cytometry analysis result of cell apoptosis. Under the action of laser, PαLA@TAPP-MnO2 can more efficiently induce apoptosis of CT26 cells. The positive cell rate is 90.3%, which is higher than the apoptosis rate of PαLA@TAPP (70.1%). This finding strongly supports the 2+ The synergistic effect of auxiliary enhanced TAPP-mediated photodynamic therapy-induced oxidative stress amplification maximized the anti-tumor therapeutic effect. Figure 4 E illustrates the potential therapeutic mechanism of nanoamplifiers to synergistically amplify oxidative stress and disrupt redox dynamic balance.

[0110] 3. Detection of intracellular reactive oxygen species generation.

[0111] In order to study the redox state in cells, DCFH-DA was used as a typical ROS probe to detect the content of ROS generated by PαLA@TAPP-MnO2 in CT26 cells. DCFH-DA can be oxidized by ROS in cells to DCF, which appears as a green fluorescence signal in the CLSM fluorescence image, as shown in Figure 2. Figure 5As shown in A (G1, Blank control group; G2, MnO2 suspension group; G3, TAPP solution + Laser group; G4, PaLA@MnO2 group; G5, PaLA@TAPP + Laser group; G6, PaLA@TAPP-MnO2 + Laser group). Fluorescence signals can be detected in CT26 cells treated with MnO2 suspension and PαLA@MnO2, indicating that MnO2 can induce the consumption of GSH and the conversion of GSH to Mn. 2+ The conversion of H2O2 to •OH was catalyzed by a Fenton-like reaction. Compared with cells treated with TAPP solution, the PαLA@TAPP group showed brighter green fluorescence, indicating that the nanostructures formed by TAPP can be taken up more by cells and produce higher levels of ROS under laser irradiation. Among these treatment groups, we observed the strongest green fluorescence in cells treated with PαLA@TAPP-MnO2, indicating that PαLA@TAPP-MnO2 produced the largest ROS content in cancer cells. This finding provides strong evidence that PαLA@TAPP-MnO2 has superior properties for synergistically generating ROS. Flow cytometry analysis also yielded the same results ( Figure 5 B, 5C).

[0112] (3) Tissue distribution of nanoamplifiers.

[0113] We used the CT26 tumor-bearing mouse model to investigate the tissue distribution of DiR-labeled PαLA@TAPP-MnO2 in the tumor-bearing mice at set time points of 6h, 12h, and 24h. In vivo fluorescence images showed ( Figure 6 A) DiR-labeled PαLA@TAPP-MnO2 (DiR-labeled NP) and DiR solution showed the maximum accumulation of DiR in the tumor at 12 hours after injection, while the fluorescence intensity in the tumor gradually decreased from 12 hours to 24 hours. This shows that PαLA@TAPP-MnO2 reaches the maximum accumulation in the tumor 12 hours after intravenous injection. Secondly, at different detection time points, the fluorescence intensity of DiR-labeled NPs in tumor tissue is higher than that of intravenously injected DiR solution. 24 hours after administration, the mouse organs were collected for in vitro observation, as shown in Figure 2. Figure 6 Figure B (1, tumor; 2, heart; 4, spleen; 5, lung; 6, kidney). The fluorescence signal of the DiR solution was concentrated in the liver, lungs, spleen, and kidneys, while the fluorescence intensity of the DiR-labeled NPs group at the tumor site remained significantly stronger than that of the DiR solution group even 24 hours after administration. These results indicate that PαLA@TAPP-MnO2 favors tumor accumulation and prolonged retention.

[0114] (IV) Pharmacological study of nanoamplifiers in subcutaneous tumor models.

[0115] Based on the cytotoxicity of the nanoamplifier in the CT26 tumor model and the good drug accumulation behavior at the tumor site, we are encouraged to further evaluate the in vivo synergistic anti-tumor effect of PαLA@TAPP-MnO2 in the CT26 tumor implantation model. Using CT26 tumor-bearing mice as the animal model, PBS, MnO2 suspension, TAPP solution, PαLA@MnO2, PαLA@TAPP and PαLA@TAPP-MnO2 were intravenously injected once every two days for a total of 4 times. The specific dosing regimen is as follows. Figure 7 A. The growth curve of mouse tumor volume over time is shown in Figure 7 As shown in B (G1, Blank control group; G2, MnO2 suspension group; G3, TAPP solution + Laser group; G4, PaLA@MnO2 group; G5, PaLA@TAPP + Laser group; G6, PaLA@TAPP-MnO2 + Laser group). The tumors in the PBS group grew rapidly and increased in size. On the 9th day after administration, the tumor volume of the mice was >1000mm. 3 . The TAPP solution and MnO2 suspension treatment groups showed certain anti-tumor ability, but the inhibition rate of tumor growth was limited, suggesting that the free drug was less enriched in the tumor after intravenous injection and the degree of inhibition of tumor growth was limited. In contrast, PαLA-based nanoparticles (PαLA@TAPP, PαLA@MnO2) showed significantly enhanced anti-tumor activity, which was attributed to the favorable characteristics of the nanostructure: longer in vivo circulation time and EPR effect. As expected, compared with the other groups, the PαLA@TAPP-MnO2 group significantly delayed the growth progression of tumor volume, and the tumor volume hardly increased, showing the strongest tumor inhibitory effect. In addition, compared with the PBS group, the average weight of the isolated tumor reached 1.26g, the tumor weight of the PαLA@TAPP-MnO2 group was significantly reduced ( Figure 7 C), Photograph of ex vivo tumor ( Figure 7 D) also proved this point. In addition, TUNEL immunofluorescence staining was used to detect the level of apoptosis of mouse tumor cells induced by each treatment method. The test results are shown in Figure 4. Figure 7 As shown in E (Scale bar = 100 μm), the green fluorescence signal in the tumor tissue sections of mice treated with PαLA@TAPP-MnO2 was the strongest, indicating that PαLA@TAPP-MnO2 has the most significant ability to induce cell apoptosis, which is consistent with the therapeutic results of in vivo anti-tumor experiments.

[0116] Next, we conducted a further systematic investigation on the safety of PαLA@TAPP-MnO2 in vivo. We mainly investigated the changes in body weight, liver and kidney function parameters, and H&E staining of tumor-bearing mice after drug treatment to evaluate the in vivo safety of PαLA@TAPP-MnO2. Figure 8 As shown in A, no significant weight loss was found in any group of mice after drug treatment compared with the PBS group. Figure 8 As shown in Figure B, there was no statistically significant difference in aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine (CREA), and blood urea nitrogen (BUN) among the groups, and there was a lack of obvious toxicity indicators, indicating that the hepatotoxicity of PαLA@TAPP-MnO2 to mice was negligible. In addition, H&E staining of various organs showed that no substantial histological pathological damage or inflammatory response was observed in the main organs of mice in each experimental group ( Figure 8 C), indicating that PαLA@TAPP-MnO2 has good in vivo biosafety (G1, Blank control group; G2, MnO2 suspension group; G3, TAPP solution + Laser group; G4, PaLA@MnO2 group; G5, PaLA@TAPP + Laser group; G6, PaLA@TAPP-MnO2 + Laser group).

[0117] In summary, the prepared PαLA@TAPP-MnO2 was characterized using transmission electron microscopy and particle size analysis. The results demonstrated successful preparation of PαLA@TAPP-MnO2, with a particle size of 226±2.4 nm, a nearly spherical structure, and a uniform size distribution. Molecular docking experiments revealed that electrostatic adsorption and metal coordination drove the co-assembly of TAPP, MnO2, and PαLA. Furthermore, in a simulated physiological environment, the nanoparticle size fluctuated little within 12 hours but cleaved in a high GSH environment. In vitro release results also demonstrated that PαLA@TAPP-MnO2 exhibited a higher cumulative release in a high GSH environment, reaching 75% of its release after 24 hours. Cellular uptake assays demonstrated that PαLA@TAPP-MnO2 exhibited superior cellular uptake efficiency and demonstrated the strongest cytotoxicity against CT26 cells. Live-dead staining further confirmed that PαLA@TAPP-MnO2 induced the most significant cell death. Cell apoptosis experiments revealed that the apoptosis rate in the PαLA@TAPP-MnO2 group was 90.3%, significantly higher than the 70.1% apoptosis rate in the PαLA@TAPP group. Confocal microscopy and flow cytometry demonstrated that PαLA@TAPP-MnO2 induced increased ROS production in CT26 cells. Fluorescence imaging revealed that PαLA@TAPP-MnO2 efficiently accumulated in tumor tissue. The antitumor effects of MnO2 suspension, TAPP solution, PαLA@MnO2, PαLA@TAPP, and PαLA@TAPP-MnO2 were evaluated in a subcutaneous ectopic tumor-bearing mouse model. PαLA@TAPP-MnO2 demonstrated the best tumor growth inhibition efficacy and demonstrated good in vivo biosafety.

Claims

1. A supramolecular nanoamplifier regulated by multiple interactions, characterized in that: The supramolecular nanoamplifier is PαLA@TAPP-MnO2; The supramolecular nanoamplifier is prepared by the following method: The 5,10,15,20-tetrakis(4-aminophenyl)porphyrin photosensitizer TAPP and manganese dioxide nanozyme MnO2 were encapsulated in disulfide-bonded poly-α-lipoic acid PαLA to construct the supramolecular nanoamplifier PαLA@TAPP-MnO2.

2. The multi-interaction-regulated supramolecular nanoamplifier according to claim 1, characterized in that: The specific steps include: S1. Preparation of poly-α-lipoic acid (PαLA): αLA was added to a dry flask and stirred at 80° C. for 2 h under a nitrogen flow; the obtained PαLA was then dissolved in N,N-dimethylformamide (DMF) to obtain a PαLA solution; S2. Preparation of MnO2 nanozyme: Potassium permanganate was added to water and stirred for 30 minutes to obtain a solution; oleic acid was then added to the solution to form a stable emulsion; the emulsion was further stirred at room temperature until brown-black blocks appeared, indicating the synthesis of the MnO2 nanozyme; the obtained MnO2 nanozyme was washed three times by centrifugation with alcohol to remove the remaining reactants, and then dried MnO2 nanozyme was obtained by vacuum freeze-drying; S3. Preparation of TAPP solution: TAPP was added to DMF and dissolved by ultrasonication to prepare TAPP solution; S4. Preparation of supramolecular nanoamplifier: (1) Weigh the MnO2 nanozyme prepared in S2 and fully disperse the MnO2 in DMF using ultrasonication to prepare a MnO2 suspension; (2) The PαLA solution prepared in S1 and the TAPP solution prepared in S3 were mixed and added to a dry penicillin bottle, and magnetically stirred at room temperature for 2 h in a dark environment to obtain suspension A; then the MnO2 suspension prepared in (1) was added to suspension A, and the mixture was stirred at room temperature for 2 h to obtain suspension B; 20 wt% DSPE-PEG 2000 was added to suspension B and stirred for 5 min to obtain suspension C; (3) Take the mixed solution C prepared in (2) to another dry vial and slowly drip it into deionized water under magnetic stirring, and stir at room temperature for 5 minutes; use ultracentrifugation to remove DMF to obtain the supramolecular nanoamplifier PαLA@TAPP-MnO2.

3. Use of the multi-interaction-regulated supramolecular nanoamplifier according to any one of claims 1 to 2 in the preparation of anti-tumor nanomedicines.

4. Use of the multi-interaction-regulated supramolecular nanoamplifier according to any one of claims 1 to 2 in the preparation of a drug carrier responsive to the high GSH environment in tumor cells.

5. A photodynamic therapy anti-tumor nanomedicine, characterized in that: The drug is the supramolecular nanoamplifier according to claim 1; the laser wavelength for photodynamic therapy of the tumor is 660 nm.

6. The drug according to claim 5, characterized in that The tumor is intestinal cancer.

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