Copper-containing polymer, preparation thereof and application of copper-containing polymer in intracellular delivery of functional protein

Abstract

The invention discloses a copper-containing polymer, namely an application of a copper-coordinated 1-Boc-4-(4-formylphenyl) piperazine modified dendrimer (CMD) in intracellular delivery of a functional protein. The invention also discloses a method for inducing copper death of tumor cells through protein delivery of the copper-containing polymer, and copper death of the tumor cells is efficiently induced by delivering Cu < 2 + > into the cells. The CMD protein delivery system disclosed by the invention not only is obviously superior to the existing commercial delivery system in protein delivery efficiency, but also can maintain an efficient delivery effect under the conditions of low dosage and high serum interference. The CMD and LOX protein transfection compound CMD / L provided by the invention can obviously enhance the occurrence of tumor cell copper death. The CMD protein delivery system and the protein transfection compound CMD / L thereof provided by the invention provide a new and effective strategy for cancer treatment. The invention has a good clinical application prospect.

Description

Technical Field

[0001] The present invention relates to the fields of biotechnology, polymer chemistry, cell biology, and in particular to copper-containing polymers, their preparation, and their application in the intracellular delivery of functional proteins. Background Art

[0002] In the late 20th century and early 21st century, cancer treatments have been continuously developed, including chemotherapy, radiotherapy and targeted therapy. However, these treatments are often accompanied by serious side effects, and cancer cells often develop drug resistance. Therefore, scientists have been looking for new treatment approaches. In recent years, copper death has attracted widespread attention as a new type of regulated cell death pathway. Copper death depends on copper ions and high levels of mitochondrial respiration. It directly binds copper ions to key enzymes of the tricarboxylic acid cycle (TCA), especially dihydrolipoyl succinyltransferase (DLAT), leading to protein aggregation and dysfunction, thereby inducing cell death. There are already some copper coordination carriers in the prior art for increasing intracellular copper ion levels and solving the above problems. However, these methods still have several limitations. First, many copper coordination carriers fail to effectively enhance mitochondrial respiration while increasing copper ion levels, making it impossible to fully induce copper death. Secondly, cancer cells usually rely on glycolysis (Warburg effect) rather than oxidative phosphorylation (OXPHOS) to obtain energy, which makes them less sensitive to copper death. In addition, high levels of glutathione (GSH) in tumors can chelate copper ions, further inhibiting the occurrence of copper death.

[0003] In summary, to the best of the applicant's knowledge, the present invention is the first to demonstrate a biotherapy that simultaneously targets glycolysis inhibition and enhances mitochondrial respiration as the basis for inducing copper death. The present invention not only addresses the high glycolysis levels commonly seen in cancer cells, but also enhances mitochondrial respiration, which is essential for inducing copper-dependent cell death. The method of the present invention overcomes the problems of existing copper coordination carriers by inhibiting glycolysis and enhancing mitochondrial respiration, effectively improving the application prospects of copper death in cancer treatment. Summary of the invention

[0004] In order to solve these problems, the present invention proposes a novel protein delivery system, which uses a copper-containing polymer, namely a copper-coordinated 1-Boc-4-(4-formylphenyl)piperazine modified dendrimer (CMD) to deliver functional proteins. The copper-coordinated 1-Boc-4-(4-formylphenyl)piperazine modified dendrimer can effectively bind to proteins through electrostatic, hydrogen bonding and hydrophobic interactions, and form a stable nanoscale complex. The 4-formylphenylpiperazine also coordinates with copper ions through the N atom at position 1 close to the benzene ring, which not only achieves efficient intracellular delivery of functional proteins of copper ions, but also enhances protein binding and stability through the coordination of copper ions with proteins. The present invention proposes for the first time that the protein transfection complex CMD / L formed by the coordination of CMD and LOX can convert lactate into pyruvate and H2O2, which not only reprograms energy metabolism, but also increases the availability of copper ions by consuming GSH in cells, thereby enhancing copper death. In addition, H2O2 generated by LOX can also induce an iron-independent Fenton-like reaction, further enhancing cytotoxicity and inducing ferroptosis.

[0005] Through material library screening and in vitro and in vivo experiments, the novel protein delivery system of the present invention has shown significant advantages in enhancing mitochondrial function, inducing copper death and iron death, and has shown good therapeutic potential in tumor cells. The delivery system of the present invention has not only achieved success at the cellular level, but also shown significant anti-tumor effects in animal models, and has good clinical application prospects.

[0006] In order to solve the problems of low efficiency and poor specificity of existing protein delivery systems in cancer treatment, the present invention innovatively provides a method for delivering functional proteins through CMD to enhance the copper death effect of tumor cells. The CMD is composed of 1-Boc-4-(4-formylphenyl)piperazine covalently linked to the PAMAM-G5 dendrimer (MD), and the N atom at position 1 near the benzene ring is connected to the Cu 2+ The delivery system achieves efficient protein delivery at low protein doses and material usage, and exhibits significant therapeutic effects in a variety of cancer cell models.

[0007] The CMD is complexed with LOX (CMD / L), and promotes the binding and delivery of LOX through coordination, thereby remodeling tumor cell mitochondrial respiration and enhancing copper death.

[0008] During the conception and development of the present invention, the main technical difficulties overcome include the following aspects:

[0009] 1. Realization of efficient copper ion delivery: Existing copper ion delivery systems often have low efficiency in cells, especially when copper ions need to be highly concentrated in specific cell regions to induce copper death. In the present invention, the design of 4-formylphenylpiperazine modified dendrimer (CMD) must ensure that it can effectively pass through the cell membrane, efficiently deliver copper ions into the cell, and ensure the stable release of copper ions in the cell, which is crucial to achieve copper death.

[0010] 2. Stability of protein-copper complex: In the internal environment of the cell, maintaining the stability of the protein-copper complex is a challenge, because the complex biochemical environment inside the cell can easily lead to the destruction of the protein structure or the dissociation of copper ions. The design of CMD needs to consider how to effectively coordinate the copper ions with the N atom at position 1 close to the benzene ring to ensure the structural stability and functionality of the protein in the cell.

[0011] 3. Intracellular copper ion toxicity: Although copper ions can induce cancer cell death, they may also be toxic to normal cells. Therefore, how to accurately control the delivery and release of copper ions to maximize the killing effect on cancer cells while minimizing damage to normal cells is a major technical challenge.

[0012] 4. Reshape the energy metabolism of tumor cells: Cancer cells usually rely on aerobic glycolysis to obtain energy. The present invention not only achieves protein delivery through the complex of CMD and LOX (CMD / L), but also reprograms the energy metabolism of tumor cells from glycolysis to OXPHOS. This shift is critical for enhancing copper death. To achieve this, precise control of LOX delivery and activity maintenance is required.

[0013] The present invention provides a copper-containing polymer, which is a copper-coordinated 1-Boc-4-(4-formylphenyl)piperazine modified dendrimer (CMD). The copper-containing polymer comprises a polyamide-amine dendrimer (PAMAM-G5) and a 1-Boc-4-(4-formylphenyl)piperazine functional group, and the 1-Boc-4-(4-formylphenyl)piperazine functional group is connected to the polyamide-amine dendrimer through a covalent bond; the structure of the copper-containing polymer is shown in formula (1):

[0014]

[0015] Wherein, R is a polyamide-amine dendrimer; x is the number of connections of 1-Boc-4-(4-formylphenyl)piperazine functional groups, which is an integer between 5 and 128.

[0016] Specifically, the polyamide-amine dendrimer is as shown in formula (2):

[0017]

[0018] Wherein, M is the core of the dendrimer, ethylenediamine; and n is an integer between 1-5.

[0019] Specifically, when n=1, x is an integer between 5 and 8; when n=2, x is an integer between 7 and 16; when n=3, x is an integer between 16 and 32; when n=4, x is an integer between 17 and 64; when n=5, x is an integer between 60 and 128.

[0020] The present invention also provides application of the copper-containing polymer in intracellular delivery of functional proteins.

[0021] Wherein, the functional protein is one or more of monoclonal antibody drugs, cytokine drugs, oxidoreductase drugs, etc.; preferably, it is LOX.

[0022] The present invention also provides the use of the copper-containing polymer in preparing medicines for treating and / or preventing tumors.

[0023] The present invention also provides a method for preparing the copper-containing polymer as described above, the preparation method comprising the following steps:

[0024] In the first step, a polyamidoamine dendrimer is reacted with 1-methyl-4-phenylpiperazine via a Schiff base to form a 1-Boc-4-(4-formylphenyl)piperazine modified dendrimer;

[0025] In the second step, the 1-Boc-4-(4-formylphenyl)piperazine modified dendrimer obtained in the first step is reduced by sodium cyanoborohydride, and then coordinated with CuCl2 in anhydrous methanol to prepare copper-coordinated 1-Boc-4-(4-formylphenyl)piperazine modified dendrimer CMD.

[0026] In a specific embodiment, the CMD is prepared as follows:

[0027] In the first step, PAMAM-G5 dendrimer and 1-Boc-4-(4-formylphenyl)piperazine were reacted at room temperature for 12 hours in anhydrous methanol at a molar ratio of 1:90. Then sodium cyanoborohydride was added at a molar ratio of 1.5:1 to 1-Boc-4-(4-formylphenyl)piperazine, and the reaction was continued at room temperature for 6 hours. The product was dialyzed with deionized water for more than 10 times, and the product obtained after vacuum freeze drying was a white powder and was named MD. The product was characterized by nuclear magnetic resonance hydrogen spectroscopy (H NMR, JEOL, 500MHz, Japan) to determine the average number of benzylpiperazine grafted per dendrimer.

[0028] In the second step, the dendrimer modified with CuCl2 and benzylpiperazine was heated in anhydrous methanol with different Cu 2+ The mixture was added at a molar ratio of 1:1 to 1:1 and reacted at room temperature for 12 h. The methanol was then removed by a rotary evaporator at 30 °C. The sample was redissolved in deionized water and vacuum freeze-dried to obtain a blue product named CMD, which was stored at -20 °C for later use.

[0029] The present invention also provides a delivery system for inducing copper death of tumor cells. The delivery system comprises the copper-containing polymer as described above, which effectively induces copper death of tumor cells.

[0030] The present invention provides a transfection complex, which comprises the co-assembled complex of the copper-containing polymer and the functional protein as described above.

[0031] In a specific embodiment, the functional protein is LOX.

[0032] The present invention also provides a delivery system for enhancing copper death of tumor cells, wherein the delivery system comprises the transfection complex as described above, wherein the transfection complex delivers LOX into the cell, enhances mitochondrial respiration by generating pyruvate, activates pyruvate dehydrogenase activity, inhibits pyruvate kinase function, and consumes glutathione by generating H2O2, thereby increasing the sensitivity of tumor cells to copper death.

[0033] In the present invention, the transfection complex also generates hydroxyl radicals ·OH through an iron-independent Fenton-like reaction, thereby inducing ferroptosis in tumor cells.

[0034] In the present invention, the transfection complex induces ferroptosis, induces the release of damage-associated molecular patterns, promotes immunogenic cell death, and triggers the activation of dendritic cells.

[0035] The present invention also provides a pharmaceutical composition, which comprises the copper-containing polymer as described above.

[0036] In the present invention, the pharmaceutical composition can be used alone, and / or in combination with monoclonal antibody drugs, cytokine drugs, oxidoreductase drugs, etc.

[0037] The present invention also provides a drug carrier, which comprises the copper-containing polymer as described above.

[0038] The present invention also provides the use of the delivery system for inducing copper death of tumor cells as described above, or the transfection complex as described above, or the delivery system for enhancing copper death of tumor cells as described above, or the pharmaceutical composition as described above, or the preparation method as described above in the preparation of drugs for treating and / or preventing tumors, and intracellular delivery of functional proteins.

[0039] Preferably, the drug for treating and / or preventing tumors includes drugs that induce copper death in tumor cells, increase the sensitivity of tumor cells to copper death, and induce ferroptosis in tumor cells.

[0040] In the present invention, the delivery effect and mitochondrial function regulation of the CMD and CMD / L in 143B cells are as follows:

[0041] 1. Availability of copper ions:

[0042] After entering the cell, the CMD complex promotes the accumulation of intracellular copper ions. At the same time, it consumes intracellular GSH by generating H2O2, thereby increasing the availability of intracellular copper ions, further inducing DLAT aggregation, and ultimately leading to cell death through the copper death pathway.

[0043] 2. Enhance mitochondrial respiration:

[0044] The CMD / L transfection complex significantly enhanced the mitochondrial respiration of cancer cells by generating pyruvate, activating the activity of pyruvate dehydrogenase (PDH) and inhibiting the function of pyruvate kinase (PK). This effect effectively improved the energy metabolism of mitochondria.

[0045] 3. Inducing ferroptosis:

[0046] In addition to copper death, the CMD / L of the present invention also induces ferroptosis through a non-iron-dependent Fenton-like reaction. This method enhances the cell death effect in many aspects and improves the therapeutic effect.

[0047] 4. Immunogenic cell death:

[0048] The CMD / L promotes immunogenic cell death by triggering the activation of dendritic cells (DCs), thereby enhancing the anti-tumor immune response.

[0049] The beneficial effect of the present invention is that the delivery system CMD of the present invention not only shows efficient protein delivery effect in in vitro experiments, but also shows significant anti-tumor effect in in vivo tumor models, and has good clinical application prospects. In particular, in the osteosarcoma model, CMD / L showed excellent anti-tumor activity, providing a new and effective strategy for cancer treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without paying any creative work.

[0051] Figure 1 This is the synthetic route for preparing CMD in Example 1 of the present invention.

[0052] Figure 2 is the CMD in Example 1 of the present invention 1 H NMR spectrum.

[0053] Figure 3 is the ultraviolet-visible spectrophotometer (UV-Vis) absorption spectrum of CMD in Example 1 of the present invention.

[0054] Figure 4 The delivery efficiency of the complex formed by the co-assembly of CMD with different copper coordination ratios and green fluorescent protein (eGFP) in Example 2 of the present invention in NIH3T3 cells was observed by laser confocal microscopy and compared with the delivery efficiency of commercial protein delivery vectors.

[0055] Figure 5 These are the cytotoxicity evaluation results of the CMD / RNaseA and CMD / saporin complexes in 143B cells in Example 3 of the present invention.

[0056] Figure 6 The figure shows the change of oxygen consumption rate (OCR) of the CMD / L complex in 143B cells in Example 4 of the present invention.

[0057] Figure 7 This is the effect of the CMD / L complex on GSH consumption in 143B cells in Example 4 of the present invention.

[0058] Figure 8 This is the effect of the CMD / L complex on cell death in 143B cells in Example 4 of the present invention.

[0059] Fig. 9 This is the effect of the CMD / L complex in Example 4 of the present invention on immunogenic cell death in 143B cells.

[0060] Fig.10 The figure shows the change in tumor volume of mice after injection of the CMD / L complex in Example 5 of the present invention. DETAILED DESCRIPTION

[0061] The present invention is further described in detail in conjunction with the following specific examples and drawings, and the protection content of the present invention is not limited to the following examples. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention, and the attached claims are the scope of protection. The process, conditions, reagents, experimental methods, etc. for implementing the present invention, except for the contents specifically mentioned below, are all common knowledge and common common sense in the art, and the present invention does not specifically limit the content.

[0062] The following description of at least one exemplary embodiment is actually only illustrative and is by no means intended to limit the present invention and its application or use. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0063] In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limiting. Therefore, other examples of the exemplary embodiments may have different values.

[0064] The present invention provides a method for inducing copper death of tumor cells by copper-coordinated 1-Boc-4-(4-formylphenyl)piperazine modified dendrimer (CMD) protein delivery system. By delivering Cu2+ into the cell, copper ions are accumulated in the tumor cell, thereby efficiently inducing copper death of the tumor cell. CMD binds to functional proteins through coordination to form a protein transfection complex, and efficiently delivers functional proteins into the cell. CMD combines with LOX to form a LOX transfection complex (CMD / L), which can generate pyruvic acid and H2O2, significantly enhance mitochondrial respiration, and consume glutathione (GSH) in the cell, thereby increasing the availability of copper ions and inducing DLAT aggregation, and finally causing cell death through copper death and iron death pathways. The CMD / L delivery system of the present invention realizes efficient protein intracellular delivery under low-dose conditions, and significantly enhances the copper death effect of tumor cells. In addition, CMD / L also induces iron death through a non-iron-dependent Fenton-like reaction, promotes immunogenic cell death (ICD), and triggers the activation of DCs. In cell experiments, the CMD / L complex can effectively enter cells through multiple pathways and exhibit excellent endosome escape ability. In animal experiments, the CMD / L delivery system showed significant tumor suppression effects, especially excellent anti-tumor activity in tumor models, and has good clinical application prospects. The copper coordination polymer of the present invention is not only significantly superior to existing commercial delivery systems in protein delivery efficiency, but also can maintain efficient delivery effects under low-dose and high serum interference conditions. The transfection complex CMD / L of CMD and LOX protein can significantly enhance the occurrence of copper death in tumor cells. Taking into account its high efficiency, safety and wide application potential, the CMD protein delivery system and its protein transfection complex CMD / L provided by the present invention provide a new and effective strategy for cancer treatment, especially the treatment of tumors.

[0065] Unless otherwise specified, the test materials used in the examples are all conventional biochemical reagents.

[0066] Example 1: Preparation and characterization of CMD

[0067] In this embodiment, PAMAM-G5 dendrimer and 1-Boc-4-(4-formylphenyl)piperazine were used to prepare MD by Schiff base reaction, and then the MD was reacted with CuCl2 to obtain CMD (see attached Figure 1 ).

[0068] Synthesis method:

[0069] 1. Preparation of MD: a. Dissolve 500 mg of PAMAM-G5 in 50 mL of anhydrous methanol and stir at room temperature until completely dissolved; b. Add 1.5 g of 1-methyl-4-phenylpiperazine to the solution and continue stirring at room temperature for 24 hours to perform a Schiff base reaction to generate MD; c. Reduce the generated C=N bond with 0.5 g of sodium cyanoborohydride and continue stirring for 4 hours; d. After the reaction is completed, dialyze the solution in deionized water for 48 hours using a dialysis bag (molecular weight cutoff: 3500 Da, Spectra / Por) to remove unreacted reagents and by-products; e. Freeze-dry the dialyzed solution to obtain a light yellow MD.

[0070] 2. Preparation of CMD: a. Dissolve 100 mg of MD in 10 mL of anhydrous methanol and stir at room temperature until completely dissolved; b. Add 0.25 molar ratio, 0.5 molar ratio and 0.75 molar ratio of CuCl2 to the solution and react for 12 hours respectively to obtain polymer carriers with different copper coordination ratios, including MD-0.25Cu2+, MD-0.5Cu2+, MD-0.75Cu2+ and CMD; c. After the reaction is completed, dialyze the solution in deionized water for 48 hours using a dialysis bag to remove unreacted CuCl2 and by-products; d. Freeze-dry the dialyzed solution to obtain light green CMD.

[0071] Characterization methods: 1. Nuclear magnetic resonance spectroscopy ( 1 H NMR): MD was characterized using a Bruker AVANCE III 400MHz NMR instrument. 20 mg of MD was dissolved in 0.5 mL of D2O, and the spectrum was recorded to determine the number of 1-Boc-4-(4-formylphenyl)piperazine groups. 2. UV-Vis: CMD was characterized using a Thermo Scientific Evolution 300. 10 mg of CMD was dissolved in 10 mL of deionized water, and the absorption spectrum in the range of 200-800 nm was recorded to confirm the presence of Cu 2+ 3. X-ray Photoelectron Spectroscopy (XPS): CMD was characterized using a Kratos Axis Ultra DLD X-ray Photoelectron Spectrometer. The CMD samples were prepared into thin films, and the XPS spectra of C1s, N 1s and Cu 2p were recorded to analyze the copper coordination mechanism.

[0072] Experimental results: See the attached Figure 2 To the attached Figure 3 1. 1 H NMR results (see Appendix Figure 2 ): 1The H NMR spectrum shows that there are characteristic peaks of benzene rings in the range of δ = 7.2-7.5 ppm, proving that 1-Boc-4-(4-formylphenyl)piperazine is successfully modified on PAMAM-G5. By integral calculation, on average, about 82 1-Boc-4-(4-formylphenyl)piperazine groups are grafted on each PAMAM-G5 molecule. 2. UV-Vis results (see Appendix Figure 3 ): UV-Vis spectrum shows that CMD has a significant absorption peak at 260nm, indicating that Cu 2+ Successfully coordinated with MD.

[0073] Through Example 1, CMD was successfully prepared and 1 H NMR, UV-Vis, and XPS characterizations confirmed its structure and composition, laying the foundation for subsequent protein delivery experiments.

[0074] Example 2: Study on intracellular delivery of proteins using copper-coordinated polymer carriers

[0075] In this example, eGFP was used as a model protein to evaluate the protein delivery efficiency of CMD.

[0076] Materials and instruments: eGFP; dynamic light scattering (DLS): Malvern Zetasizer Nano ZS; sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) (Bio-Rad Mini-PROTEAN system); flow cytometer (BD FACSCalibur); laser confocal microscope (Leica TCS SP8); fetal bovine serum (FBS) (16140071, Gibco)

[0077] Methods: 1. Cell culture: NIH3T3 cells were seeded in 24-well plates, 1 mL of DMEM medium containing 10% FBS was added to each well, and the cells were cultured in an incubator at 37°C and 5% CO2 for 24 hours until the cell density reached 80-90%. Preparation of CMD / eGFP complex: a. eGFP was dissolved in PBS buffer at a concentration of 1 mg / mL; b. 20 μg CMD was dissolved in 1 mL of sterile water and stirred at room temperature until completely dissolved; c. At room temperature, equal volumes of CMD solution and eGFP solution were mixed, gently mixed, and incubated for 30 minutes to form CMD / eGFP complex. 2. Transfection process: a. Place 0.2μg eGFP (2μL, 1mg / mL) in a 1.5mL EP tube; b. Dilute CMD with serum-free medium to a total liquid volume of 50μL, pipette and mix the mixed solution, and incubate at room temperature for 15 minutes; c. Add 200μL serum-free medium to a total volume of 250μL; d. Remove the original medium in the cell well and add the above 250μL transfection solution; e. After 6 hours of transfection, add 500μL of medium containing 10% FBS; f. After 24 hours of transfection, remove the medium, add 250μL PBS solution, and observe the cells with an inverted fluorescence microscope. 3. Cell observation and fluorescence intensity measurement: a. Take photos of the cells using an inverted fluorescence microscope; b. After digesting the cells with trypsin, collect the cells and quantify the average fluorescence intensity of the cells by flow cytometry.

[0078] Experimental results: See the attached Figure 4 The experimental results showed that the best delivery efficiency of CMD to eGFP occurred when the molar ratio of copper ions to methylpiperazine was 1:1. The efficiency of CMD to eGFP was significantly higher than that of MD and the commercial transfection reagent PULSin (see Appendix Figure 4 ), which further validates the effectiveness of copper coordination in enhancing protein delivery performance.

[0079] Through the above examples, the high efficiency and superiority of CMD in protein delivery were verified, laying a foundation for further research on functional protein delivery.

[0080] Example 3: Study on intracellular delivery of functional proteins RNAse and saporin using copper-coordinated polymer carriers

[0081] In this example, RNase A and saporin were used as model proteins to evaluate the protein delivery ability and biological effects of CMD.

[0082] Materials and reagents: RNase A (R6513, Sigma-Aldrich); Saporin (PR-01, Sigma-Aldrich); CCK8 cell activity detection kit (MA0218, Meilunbio).

[0083] Methods: 1. Cell culture is the same as in Example 2 of the present invention; 2. Preparation of CMD / functional protein complex: a. RNaseA and saporin are dissolved in PBS buffer, respectively, with a concentration of 1 mg / mL; b. 20 μg CMD is dissolved in 1 mL sterile water and stirred at room temperature until completely dissolved; c. At room temperature, equal volumes of CMD solution are mixed with RNaseA or saporin solution, gently mixed, and incubated for 30 minutes to form CMD / RNase A and CMD / saporin complexes. 3. The transfection process is the same as in Example 2. 4. Cytotoxicity test: a. CCK8 is used to detect cell viability. After 24 hours of transfection, 10 μL of CCK8 reagent is added to each well, incubated at 37°C for 2 hours, and the absorbance is measured at 450 nm; b. Compare the cytotoxicity of CMD / RNaseA, CMD / saporin, CMD alone, RNaseA and saporin.

[0084] Experimental results: See the attached Figure 5 Cytotoxicity evaluation: CCK8 assay results showed that the cytotoxicity of CMD / RNase A and CMD / saporin complexes to 143B cells was significantly higher than that of CMD, RNaseA and saporin alone; under the same conditions, the cell viability of CMD / RNaseA and CMD / saporin complexes was reduced to 50% and 45%, respectively, while RNaseA and saporin alone had less effect on cells.

[0085] Through the above examples, the high efficiency and superiority of CMD in functional protein delivery were verified, laying a foundation for further research on LOX delivery and biological effects.

[0086] Example 4: Study on the delivery and biological effects of LOX by copper-coordinated polymer carriers

[0087] In this example, the delivery effect of CMD on LOX and its biological effect in tumor cells were studied.

[0088] Materials and reagents: LOX (S25769, Yuanye Bio-Technology); transmission electron microscope (HT7700, Hitachi); OCR analyzer (Seahorse XF24, Agilent); GSH detection kit (S0053, Beyotime Inst. Biotech).

[0089] Methods: 1. Cell culture was the same as in Example 2. 2. Preparation of CMD / L complex was the same as in Example 2. 3. Transfection process was the same as in Example 2. 4. OCR analysis: a. OCR of 143B cells was measured using Seahorse XF24 oxygen consumption rate analyzer. Transfected cells were inoculated in Seahorse XF24 cell culture plates, 10^4 cells per well, and incubated for 24 hours; b. Basal OCR, maximum OCR and spare respiratory capacity of cells were measured according to the instrument operation manual. 5. GSH consumption determination: a. GSH content in cells was detected using a GSH detection kit. 24 hours after transfection, cells were collected and GSH concentration was determined according to the kit operation manual. 6. DLAT aggregation determination: DLAT aggregation was detected using Western blot. 24 hours after transfection, cell lysates were collected, SDS-PAGE electrophoresis and Western blot analysis were performed, and DLAT antibody (13426-1-AP, Proteintech) was used for detection. 7. Evaluation of cell death and immunogenicity Cell death effect: a. Detect cell survival rate using CCK8 kit. 24 hours after transfection, add 10 μL CCK8 reagent to each well, incubate at 37°C for 2 hours, and measure absorbance at 450 nm; b. Detect Annexin V and PI staining using flow cytometry to evaluate cell apoptosis and necrosis; c. Detect cell surface CRT protein expression using immunocytofluorescence technology, detect extracellular ATP level using ATP detection kit, and evaluate ICD effect; d. Detect BMDC cell activation level using flow cytometry to evaluate the activation effect of ICD effect on dendritic cells.

[0090] Experimental results: See the attached Figures 6 to 9 1. DLS and TEM characterization: a. The average particle size of CMD / L complex in aqueous solution is about 141nm, and the surface charge is positive; b. TEM images show that CMD / L complex is uniform spherical particles. 2. OCR analysis (see attached Figure 6 ): a. After transfection with CMD / L complex, the basal OCR and maximum OCR of 143B cells increased significantly within 6 hours, and the spare respiratory capacity was enhanced, indicating that the mitochondrial respiratory activity of cells was significantly enhanced in the early stage of treatment; 3. GSH consumption (see Appendix Figure 7): After transfection with CMD / L complex, the GSH concentration in 143B cells decreased significantly, indicating that the complex can effectively consume GSH in cells and increase the availability of copper ions. 4. DLAT oligomerization: Western blot results showed that the aggregation of DLAT in cells increased significantly, indicating that CMD / L complex can effectively induce DLAT aggregation and cause cell death through the copper death pathway. 5. Cell death and ICD effect: a. CCK8 detection results showed that the cytotoxicity of CMD / L complex to 143B cells was significantly higher than that of CMD and LOX alone, and the cell survival rate was reduced to about 20% (attached Figure 8 ); b. Flow cytometry results showed that CMD / L complex could significantly induce cell apoptosis and necrosis; c. ATP level test results showed that after transfection of CMD / L complex, ATP content in cell culture medium increased significantly; d. Immunocytofluorescence test results showed that CRT expression on cell surface increased significantly; e. Flow cytometry results showed that BMDC cell activation increased significantly (Appendix Fig. 9 ). The above results indicate that the complex can induce ICD and activate dendritic cells.

[0091] The above examples verify the high efficiency and superiority of CMD in LOX delivery and biological effects, providing strong support for the application of CMD in anti-tumor therapy.

[0092] Example 5: In vivo study of membrane-encapsulated copper-coordinated polymer carriers delivering LOX, investigating the ability of the complex to deliver LOX and kill tumors in mice

[0093] In this example, the effect of delivering LOX (MCMD / L) in mice by using a membrane-encapsulated copper-coordinated polymer carrier and its killing effect on tumors were studied.

[0094] Materials and reagents: BALB / c mice were purchased from Shanghai Sigerui Experimental Animal Co., Ltd.

[0095] Methods: 1. Establishment of animal model: a. Suspend 1×10^6 143B cells in 100 μL PBS and inoculate into the tibial bone marrow cavity of 6-week-old female BALB / c mice to establish an orthotopic xenograft tumor model; b. Wait for the tumor to grow to about 100 mm 3 2. Monitoring of tumor volume and survival rate: The tumor size was measured with a vernier caliper every other day, and the tumor volume was calculated according to the following formula:

[0096]

[0097] Where L is the longitudinal length of the tumor and AP is the anterior-posterior diameter of the fluid.

[0098] Experimental results: Tumor volume: The tumor volume of mice in the MCMD / L complex group was significantly smaller than that in the other groups (see Appendix Fig.10 ).

[0099] The above examples verify the high efficiency and superiority of CMD in delivering LOX in vivo. The CMD / L complex can induce tumor cell death in mice, thereby achieving significant anti-tumor effects, providing strong support for its clinical application.

[0100] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0101] As used in the present invention, the terms "include" and "comprising" are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

[0102] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0103] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the attached claims.

Claims

1. A copper-containing polymer, characterized in that: The copper-containing polymer is a copper-coordinated 1-Boc-4-(4-formylphenyl)piperazine modified dendrimer CMD, comprising a polyamide-amine dendrimer PAMAM-G5 and a 1-Boc-4-(4-formylphenyl)piperazine functional group, wherein the 1-Boc-4-(4-formylphenyl)piperazine functional group is covalently bonded to the polyamide-amine dendrimer; the structure of the copper-containing polymer is shown in formula (1): Wherein, R is PAMAM-G5; x is the number of connections of 1-Boc-4-(4-formylphenyl)piperazine functional groups, which is an integer between 5 and 128.

2. The copper-containing polymer according to claim 1, characterized in that The PAMAM-G5 is shown in formula (2): Wherein, M is the core of the polyamide-amine dendrimer, which is ethylenediamine; and n is an integer between 1-5.

3. The copper-containing polymer according to claim 2, characterized in that When n=1, x is an integer between 5 and 8; or, when n=2, x is an integer between 7 and 16; or, when n=3, x is an integer between 16 and 32; or, when n=4, x is an integer between 17 and 64; or, when n=5, x is an integer between 60 and 128.

4. A delivery system for inducing copper death of tumor cells, characterized in that: The delivery system comprises the copper-containing polymer as described in any one of claims 1 to 3, which effectively induces copper death of tumor cells.

5. A transfection complex, characterized in that The transfection complex comprises the co-assembled complex of the copper-containing polymer and the functional protein as described in any one of claims 1 to 3.

6. A delivery system for enhancing copper death of tumor cells, characterized in that: The delivery system comprises the transfection complex as described in claim 5, which delivers lactate oxidase (LOX) into the cell, enhances mitochondrial respiration by generating pyruvate, activates pyruvate dehydrogenase activity, inhibits pyruvate kinase function, and consumes glutathione by generating H2O2, thereby increasing the sensitivity of tumor cells to copper death.

7. The delivery system according to claim 6, wherein The transfection complex also generates hydroxyl radicals ·OH through a non-iron-dependent Fenton-like reaction, thereby inducing ferroptosis in tumor cells; and / or, the transfection complex induces the release of damage-associated molecular patterns by inducing ferroptosis, thereby promoting immunogenic cell death and triggering activation of dendritic cells.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the copper-containing polymer as described in any one of claims 1 to 3.

9. A method for preparing a copper-containing polymer as claimed in claim 1, characterized in that: The preparation method comprises the following steps: In the first step, a polyamidoamine dendrimer is reacted with 1-methyl-4-phenylpiperazine via a Schiff base to form a 1-Boc-4-(4-formylphenyl)piperazine modified dendrimer; In the second step, the 1-Boc-4-(4-formylphenyl)piperazine modified dendrimer obtained in the first step is reduced by sodium cyanoborohydride, and then coordinated with CuCl2 in anhydrous methanol to prepare copper-coordinated 1-Boc-4-(4-formylphenyl)piperazine modified dendrimer CMD.

10. Use of the copper-containing polymer according to any one of claims 1 to 3, the delivery system according to claim 4, the transfection complex according to claim 5, the delivery system according to claim 6 or 7, the pharmaceutical composition according to claim 8, or the preparation method according to claim 9 in the preparation of drugs for treating and / or preventing tumors, and intracellular delivery of functional proteins; preferably, the drugs for treating and / or preventing tumors include drugs that induce copper death in tumor cells, increase the sensitivity of tumor cells to copper death, and induce ferroptosis in tumor cells.

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