Bionic hybrid nano regulator for enhancing cell copper death as well as preparation method and application of bionic hybrid nano regulator

Abstract

The invention discloses a bionic hybrid nano regulator for enhancing cell copper death and a preparation method and application thereof, and belongs to the technical field of biomedical materials. The invention designs a bionic nano-platform which simultaneously loads a lactic acid regulator cyclostilbene Su 3118 and a copper ion carrier disulfiram DSF, and the bionic nano-platform is combined with hybrid macrophages and tumor cell membranes to construct a nano-regulator SCTDM. The nano regulator can exert multiple functions, including tumor targeting, in-situ photothermal therapy, lactic acid regulation and enhanced copper death. The nano regulator provided by the invention is simple and convenient to manufacture, is combined with in-situ photothermal therapy and lactic acid regulation and is used for enhancing cell death immunotherapy of copper death so as to eliminate primary tumors and delay relapse, and a technical support is provided for regulating immunity by enhancing copper-induced cell death through metabolic reprogramming.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials, and in particular to a biomimetic hybrid nanoregulator that enhances copper cell death, its preparation method, and its application. Background Technology

[0002] Colorectal cancer is a common malignant tumor of the gastrointestinal tract, and its traditional treatments include surgery, chemotherapy, and radiotherapy. However, among patients diagnosed with colorectal cancer, 20% have metastatic disease; 40% experience recurrence after previous local disease treatment, resulting in a poor prognosis and a 5-year survival rate of less than 20%. Therefore, it is of great significance to develop a precision medicine platform that addresses the pathological characteristics and clinical needs of colorectal cancer, achieving highly efficient killing of cancer cells, precise treatment, and avoiding the sequelae such as systemic functional impairment caused by traditional treatments.

[0003] Copper death, a newly discovered mode of cell death, unravels the mystery of the relationship between copper ions and cellular energy metabolism through the mitochondrial pathway. Excessive accumulation of copper ions can directly bind to lipoylated proteins, thereby interfering with the tricarboxylic acid (TCA) cycle and ultimately inducing programmed immunogenic cell death (ICD). Copper death in tumor cells releases damage-associated molecular patterns (DAMPs), thereby activating anti-tumor immune responses. However, obstacles to copper death-induced immune responses lie in the effective accumulation of intracellular copper ions, cellular sensitivity to copper death, and the enhancement of a robust immune response. Therefore, there is a need to develop a precise and effective delivery platform to achieve effective copper death-immunotherapy.

[0004] Copper death primarily occurs in tumor cell types that rely on mitochondrial respiration. However, cancer cells reprogram their energy metabolism, shifting to aerobic glycolysis and bypassing the mitochondrial tricarboxylic acid (TCA) cycle, leading to a loss of sensitivity to copper death. Therefore, inhibiting glycolysis may be a potential strategy to redirect tumor energy metabolism back to the TCA cycle, restoring its sensitivity to copper death. Lactate is the most important metabolite in glycolysis, so restricting lactate transport would be an effective therapeutic approach to interfere with glycolysis. Inhibiting lactate efflux by suppressing the activity of monocarboxylic acid transporters 1 and 4 (MCT1 / 4) can increase intracellular lactate concentration, subsequently forcing lactate to be converted to pyruvate, entering the TCA cycle and restoring mitochondrial respiration. Furthermore, inhibiting lactate efflux can reduce tumor microenvironment acidification and immunosuppression, activating anti-tumor immune responses. Therefore, combining lactate regulation with copper death would be a synergistic therapeutic strategy, enhancing efficacy by increasing copper death sensitivity and amplifying the immune response. Summary of the Invention

[0005] The purpose of this invention is to provide a biomimetic hybrid nanoregulator that enhances copper death in cells, its preparation method, and its applications, thereby addressing the problems existing in the prior art. The biomimetic hybrid nanoregulator of this invention achieves effective tumor accumulation, tumor cell recognition, and intracellular delivery through a surface-coated hybrid macrophage membrane and tumor cell membrane. By regulating and enhancing the copper death sensitivity of tumor cells through in-situ photothermal activity and lactate modulators, it achieves tumor immunogenic cell death, activates anti-tumor immune responses, reverses the tumor immunosuppressive microenvironment, and inhibits tumor proliferation and postoperative tumor recurrence.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] In a first aspect, the present invention provides a method for preparing a biomimetic hybrid nanoregulator that enhances copper cell death, comprising the following steps:

[0008] Take ciprosilidine Su 3118, tannic acid TA, NaOH, CuCl2·2H2O, and disulfiram DSF, mix them, stir to obtain SCTD nanoparticles, centrifuge, resuspend, and centrifuge again to obtain SCTD nano-regulators.

[0009] Macrophages and tumor cells were collected and swollen in water. The cells were lysed by quick-freezing and re-thawing. After centrifugation, the supernatant was collected and centrifuged again. The precipitate was resuspended in water to obtain the macrophage membrane and the tumor cell membrane. The macrophage membrane and the tumor cell membrane were mixed to obtain the hybrid cell membrane.

[0010] The SCTD nanoregulator was mixed with a hybrid cell membrane at a mass ratio of 2:1 to 15:1, and then subjected to sonication and centrifugation to obtain the biomimetic hybrid nanoregulator SCTDM.

[0011] Preferably, the following steps are performed: 100-300 μL of Su 3118 (1-5 mg / mL) and 50-100 μL of TA (10-100 mg / mL) are mixed; 25-65 μL of NaOH (1-5 mg / mL) is added and mixed; 20-60 μL of CuCl2·2H2O (5-15 mg / mL) is added and mixed; finally, 100-300 μL of DSF (0.05-0.15 mg / mL) is added and mixed. The mixture is stirred at 800-1200 rpm for 10-20 min at room temperature to obtain SCTD nanoparticles. Unreacted material is removed by centrifugation at 10000-15000 rpm for 5-15 min, the mixture is resuspended in water, and then centrifuged at 10000-15000 rpm for 5-15 min to obtain the SCTD nanoregulator.

[0012] Preferably, the following steps are performed: 200 μL of Su 3118 (2 mg / mL) and 80 μL of TA (50 mg / mL) are mixed, 45 μL of NaOH (4 mg / mL) is added, 40 μL of CuCl2·2H2O (10 mg / mL) is added, and finally 200 μL of DSF (0.1 mg / mL) is added. The mixture is stirred at 1000 rpm for 15 min at room temperature to obtain SCTD nanoparticles. Unreacted material is removed by centrifugation at 13000 rpm for 10 min, the mixture is resuspended in water, and then centrifuged at 13000 rpm for 10 min to obtain the SCTD nanoregulator.

[0013] Preferably, the macrophages and tumor cells are collected and swollen in water for 12-36 hours, then lysed by quick-freezing and thawing, repeated three times. The resulting mixture is centrifuged at 1500-2500×g for 5-15 minutes, and the supernatant is centrifuged again at 15000-25000×g for 10-50 minutes. The precipitate is then resuspended in water to obtain the macrophage membrane and tumor cell membrane.

[0014] Preferably, the macrophages and tumor cells are collected and swollen in water for 24 hours, then lysed by quick freezing and thawing, repeated three times. The resulting mixture is centrifuged at 2000×g for 10 minutes, and the supernatant is centrifuged again at 20000×g for 30 minutes. The precipitate is then resuspended in water to obtain the macrophage membrane and tumor cell membrane; the mass ratio of tumor cell membrane to macrophage membrane in the hybrid cell membrane is 1:1.

[0015] Preferably, the SCTD nanoregulator is mixed with the hybrid cell membrane at a mass ratio of 10:1.

[0016] Secondly, the present invention also provides a biomimetic hybrid nanoregulator SCTDM prepared according to the preparation method described above.

[0017] Thirdly, the present invention also provides the application of the aforementioned biomimetic hybrid nanomodulator SCTDM in the preparation of drugs for treating cancer and / or inhibiting cancer recurrence, wherein the cancer types include, but are not limited to, colon cancer.

[0018] Fourthly, the present invention also provides a drug that enhances copper death in tumor cells, the drug comprising the biomimetic hybrid nanoregulator SCTDM.

[0019] Preferably, the product also contains a pharmaceutically acceptable carrier or excipient.

[0020] The present invention discloses the following technical effects:

[0021] To achieve synergistic lactate regulation and copper death, precise delivery of therapeutic agents and copper ions to tumor sites is required. Cell membrane camouflage, as a promising strategy for constructing biomimetic platforms, has significant therapeutic potential. By fusing cell membranes from two different sources to construct hybrid membrane biomimetic nanosystems, the characteristics of both cell types can be amplified. Therefore, biomimetic nanoplatforms coated with macrophage and tumor cell membranes exhibit excellent biocompatibility and homologous targeting effects due to the tumor tropism of macrophages and their specific affinity for parental cancer cells. Meanwhile, tannic acid-metal complexes have attracted considerable attention due to their high copper ion loading capacity, ease of drug loading, precise drug delivery, and simple preparation process. Tannic acid can react with multivalent metal ions (such as Cu)... 2+ It chelates to form pH-sensitive hydrophobic tannic acid-metal complexes, achieving sustained dissolution under acidic conditions and meeting the requirements for controlled release. Furthermore, it can interact with proteins on cell membranes, facilitating the creation of biomimetic nanoplatforms with membrane camouflage. With these desirable capabilities, it is feasible to fabricate biomimetic nanomodulators that cleverly combine tumor targeting, lactate regulation, copper death, and photothermal therapy to enhance therapeutic efficacy.

[0022] Based on the above considerations, this invention designs a biomimetic nanoplatform loaded with the lactate regulator ciloshepped (Su 3118) and the copper ion carrier disulfiram (DSF). This platform is then combined with hybrid macrophage membranes and tumor cell membranes to prepare a biomimetic hybrid nanoregulator, SCTDM, which can perform multiple functions, including tumor targeting, in situ photothermal therapy, lactate regulation, and enhanced copper death. The coated macrophage membrane achieves efficient tumor accumulation, while the surface tumor cell membrane actively recognizes CT26 cells for targeting of homologous tumor cells, releasing the loaded Su 3118 and copper ions in a pH-responsive manner. Subsequently, copper ions are translocated to mitochondria via DSF to induce copper death. Under the activation of high intracellular H2S, the generated copper sulfide complex undergoes photothermal therapy via 808nm laser irradiation, increasing reactive oxygen species levels to enhance apoptosis. Furthermore, the released Su 3118 restricts lactate efflux by inhibiting the activity of lactate transporters MCT1 / 4 and increases intracellular lactate levels to reverse glycolysis and generate pyruvate. Excessive pyruvate enters the TCA cycle to restore mitochondrial respiration and enhance copper death sensitivity. Synergistic PTT (photothermal therapy) and lactate modulation enhance cellular copper death, leading to immunogenic cell death and the release of DMAPs, which in turn activates the immune response, including dendritic cell maturation, T cell infiltration, and regulatory T cell suppression, to alleviate the tumor immunosuppressive microenvironment and thus promote immunotherapy. This invention provides a biomimetic hybrid nanomodulator SCTDM with a simple fabrication process. Combining in situ photothermal therapy and lactate modulation, this invention provides a method for enhancing copper death-induced cell death immunotherapy to eliminate primary tumors and delay recurrence, providing technical support for modulating immunity through metabolic reprogramming to enhance copper death. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments 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 creative effort.

[0024] Figure 1 The hydration particle size of different nano-regulators;

[0025] Figure 2 The zeta potential results are for different nano-modulators;

[0026] Figure 3 TEM images and EDS elemental spectra of SCTD and SCTDM;

[0027] Figure 4Photothermal heating curves of SCTDM mixed with different concentrations of sodium sulfide under laser irradiation;

[0028] Figure 5 The UV absorbance spectrum of SCTDM glutathione consumption (a), Cu + Fluorescence intensity at 610 nm (b) and UV absorption spectrum of methylene blue consumption (c); where the concentrations in the figures are labeled as the concentrations of SCTDM added;

[0029] Figure 6 The photothermal effect of SCTDM on copper death is shown; (a) represents the glutathione (GSH) consumption of SCTDM under normal conditions and under laser irradiation after reaction with sodium sulfide, and (b) represents the Cu content of SCTDM under normal conditions and under laser irradiation after reaction with sodium sulfide. + The amount produced, (c) is the amount of methylene blue consumed under normal conditions and laser irradiation after the reaction of SCTDM with sodium sulfide;

[0030] Figure 7 The uptake of SCTD and SCTDM by CT26 colon cancer cells;

[0031] Figure 8 Lactate levels inside and outside colon cancer cells CT26 were treated with different nanomodulators; where (a) represents intracellular lactate content and (b) represents extracellular lactate content.

[0032] Figure 9 Treatment of intracellular pyruvate levels in CT26 colon cancer cells with different nanomodulators;

[0033] Figure 10 Western blot analysis was performed on the expression levels of copper death-related proteins in CT26 cells treated with different nanomodulators.

[0034] Figure 11 Survival rates of CT26 cells after treatment with different nanomodulators;

[0035] Figure 12 To evaluate the safety of the nanomodulator SCTDM in an in vitro hemolysis assay;

[0036] Figure 13 In vivo imaging of fluorescently labeled SCTD and SCTDM in mice;

[0037] Figure 14 The results of in vivo treatment with different nanomodulators are shown; (a) is the tumor volume growth curve during treatment, (b) is the final tumor weight, and (c) is the mouse body weight change curve during treatment.

[0038] Figure 15The results show the inhibition of tumor recurrence by the nano-modulator; (a) is an optical image of the recurrent tumor, and (b) is the tumor weight. Detailed Implementation

[0039] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0040] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0041] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0042] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0043] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0044] Example 1: Fabrication of a biomimetic hybrid nanocontroller

[0045] 1. Fabrication of CT nanomodulators

[0046] Tannic acid (TA) (80 μL, 50 mg / mL), CuCl2·2H2O (80 μL, 10 mg / mL), and NaOH (45 μL, 4 mg / mL) were sequentially added to a vial containing 1.795 mL of pure water. Stable CT nanoparticles were obtained by stirring at 1000 rpm for 1 h at room temperature. Unreacted material was removed by high-speed centrifugation (13000 rpm, 10 min). The nanoparticles were then resuspended in pure water and centrifuged again (under the same conditions as before, 13000 rpm, 10 min) to achieve a single water wash, thus obtaining the CT nano-regulator. The obtained nano-regulator was dispersed in pure water for subsequent use.

[0047] The particle size of nanoscale regulators prepared under different conditions will vary, but they are all uniform and stable.

[0048] 2. Fabrication of CTD nanocontrollers (single copper death nanocontrollers)

[0049] TA (80 μL, 50 mg / mL), CuCl2·2H2O (80 μL, 10 mg / mL), and NaOH (45 μL, 4 mg / mL) were added to a vial containing 1.595 mL of pure water. The mixture was stirred at 1000 rpm for 1 h at room temperature. Then, DSF (200 μL, 0.1 mg / mL) was added, and the mixture was stirred at 1000 rpm for 15 min at room temperature to obtain stable CTD nanoparticles. Unreacted material was removed by high-speed centrifugation (13000 rpm, 10 min). The nanoparticles were then resuspended in pure water and centrifuged again (under the same conditions as before, 13000 rpm, 10 min) to achieve a single water wash, thus obtaining the CTD nanoregulator. The obtained nanoregulator was dispersed in pure water for subsequent use.

[0050] The particle size of nanoscale regulators prepared under different conditions will vary, but they are all uniform and stable.

[0051] 3. Preparation of SCT nanoregulators (single lactate-regulated nanoregulators)

[0052] Su3118 (200 μL, 2 mg / mL) and TA (80 μL, 50 mg / mL) were added to a bottle containing 1.635 mL of pure water. The mixture was stirred at 1000 rpm for 10 min at room temperature. NaOH (45 μL, 4 mg / mL) was added to adjust the pH of the system to approximately 7, and the mixture was stirred at 1000 rpm for 10 min at room temperature. CuCl2·2H2O (40 μL, 10 mg / mL) was added, and the mixture was stirred at 1000 rpm for 15 min at room temperature to prepare stable SCT nanoparticles. Unreacted material was removed by high-speed centrifugation (13000 rpm, 10 min). The nanoparticles were then resuspended in pure water and centrifuged again (under the same conditions as before, 13000 rpm, 10 min) to achieve a single water wash, thus obtaining the SCT nano-regulator. The obtained nano-regulator was dispersed in pure water for subsequent use.

[0053] The particle size of nanoscale regulators prepared under different conditions will vary, but they are all uniform and stable.

[0054] 4. Preparation of SCTD nanoregulators (nanoregulators for synergistic lactate regulation and copper death)

[0055] Su3118 (200 μL, 2 mg / mL) and TA (80 μL, 50 mg / mL) were added to a bottle containing 1.435 mL of pure water. The mixture was stirred at 1000 rpm for 10 min at room temperature. NaOH (45 μL, 4 mg / mL) was added to adjust the pH of the system to approximately 7, and the mixture was stirred at 1000 rpm for 10 min at room temperature. CuCl2·2H2O (40 μL, 10 mg / mL) was added, and the mixture was stirred at 1000 rpm for 15 min at room temperature. DSF (200 μL, 0.1 mg / mL) was added, and the mixture was stirred again at 1000 rpm for 15 min at room temperature to obtain stable SCTD nanoparticles. Unreacted material was removed by high-speed centrifugation (13000 rpm, 10 min). The nanoparticles were then resuspended in pure water and centrifuged again (under the same conditions as before, 13000 rpm, 10 min) to achieve a single water wash, thus obtaining the SCTD nano-regulator. The obtained nano-regulator was dispersed in pure water for subsequent use.

[0056] Preparation of IR-820-labeled SCTD nanocontrollers: IR-820 (100 μL, 2 mg / mL), Su3118 (200 μL, 2 mg / mL), and TA (80 μL, 50 mg / mL) were added to a bottle containing 1.335 mL of pure water. The other preparation procedures were the same as above. After preparation, the IR-820-labeled SCTD nanocontrollers were dispersed in pure water, and the content of IR-820 in the system was measured using a UV-Vis spectrophotometer for subsequent use.

[0057] The particle size of nanoscale regulators prepared under different conditions will vary, but they are all uniform and stable.

[0058] 5. Preparation of SCTDM nanoregulators (biomimetic nanoregulators for synergistic lactate regulation and copper death)

[0059] 5.1 Extraction of cell membrane Membrane

[0060] CT26 tumor cells and RAW 264.7 cells were collected and swollen in pure water (containing PMSF) for 24 h. Cells were lysed by flash freezing in liquid nitrogen and rehydration in a 37°C water bath, repeated three times. The resulting mixture was centrifuged at 2000×g for 10 min, and the supernatant was centrifuged at 20000×g for 30 min. The precipitate was resuspended in pure water to obtain the cell membrane solution. Protein concentration of the cell membrane solution was determined using a BCA assay kit, and the solutions were aliquoted and stored at -80°C for later use. Hybrid cell membranes were obtained by mixing CT26 tumor cell membranes and RAW 264.7 cell membranes at a 1:1 mass ratio.

[0061] 5.2 Fabrication of SCTDM Nanocontrollers

[0062] The SCTD nanoregulators prepared above were mixed with hybrid cell membranes at a mass ratio of 2:1 to 15:1, with a preferred ratio of 10:1. The mass ratio of CT26 tumor cell membrane to RAW 264.7 cell membrane in the hybrid cell membrane was 1:1. The mixture was then sonicated for 10 min and centrifuged to obtain the cell membrane-modified nanoregulator SCTDM. The obtained nanoregulators were dispersed in pure water for subsequent use.

[0063] Preparation of IR-820-labeled SCTDM nanocontrollers: The SCTD was replaced with an IR-820-labeled SCTD nanocontroller, and the other preparation process was the same as above.

[0064] The particle size and potential distribution of the prepared CT, CTD, SCT, SCTD, and SCTDM nanoscale modulators were measured using a Malvern particle size analyzer. The morphology of SCTD and SCTDM was observed by TEM, and characteristic elemental analysis was performed. The results are shown below. Figure 1 , Figure 2 and Figure 3 .from Figure 1 Particle size and Figure 2 The potential distribution results show that the SCTD nanometers have a particle size of approximately 245.8 ± 20 nm and a potential of -51 ± 4.2 mV. After being coated with a cell membrane, the SCTDM nanometers have a particle size of approximately 256.3 ± 8 nm and a potential of -50 ± 3 mV. From the TEM results ( Figure 3As can be seen from the data, the SCTD nanoregulator has a core-shell structure. The elemental distribution map shows the presence of elements such as Cu, S, and N, indicating the successful loading of copper ions, disulfiram, and cilosépine into the system framework. A distinct cell membrane layer is visible on the surface of the SCTDM nanoregulator, which preliminarily proves that the cell membrane is coated on it. The presence of P in the elemental spectrum analysis also confirms this result, proving that the cell membrane has been successfully modified.

[0065] Example 2: Performance Measurement of the Nano Regulator

[0066] 1. Evaluation of in vitro photothermal conversion performance

[0067] Dilute SCTDM with pure water to Cu 2+ The concentration is 0.5-2 mg / mL, and the concentration of Na2S solution is 10 mg / mL. This is done according to the reaction of Na2S and Cu... 2+ The reaction solutions were mixed in molar ratios of 4:1, 2:1, 1:1, and 1:2. After reacting for 10 minutes, the solutions were analyzed using an 808nm laser at a speed of 1.0W / cm². 2 Irradiate at a certain power for 200 seconds, and take a picture every 20 seconds to record the temperature.

[0068] The addition of Na2S enables the nano-modulator to produce a copper sulfide composite with photothermal properties. From Figure 4 The photothermal heating curve shows that: as Cu... 2+ As the ratio increases, the system heats up faster and the photothermal effect is better. This indicates that more copper sulfide with photothermal properties is generated within the system, leading to an increased photothermal conversion heating rate. Furthermore, starting from a 1:1 ratio, the photothermal temperature rises to over 40°C, meeting the conditions for mild photothermal therapy, demonstrating the system's good photothermal therapeutic potential.

[0069] 2. In vitro evaluation of the effect of photothermal promotion of copper death by the nano-regulator SCTDM

[0070] SCTDM was diluted with pure water to a concentration of 0-200 μg / mL to prepare 0.5 mM GSH. 1000 μL of each 0.5 mM GSH solution was reacted with 1000 μL of SCTDM at different concentrations at room temperature for 12 h, followed by centrifugation at 13000 rpm for 10 min. 800 μL of the supernatant was collected, and 25 μL of LTNB solution (2.5 mg / mL) was added. The UV absorption was measured at 412 nm to assess the in vitro GSH consumption. Another 800 μL of the supernatant was collected, and the fluorescence intensity at 610 nm was measured using a fluorescence spectrophotometer to assess the formation of monovalent copper ions in the system. The precipitate was resuspended, and H2O2 and MB (methylene blue) solution were added. The reaction continued at room temperature for 12 h, followed by centrifugation at 13000 rpm for 10 min. The UV absorption at 660 nm was measured using a UV spectrophotometer to assess the formation of hydroxyl radicals in the Fenton-like reaction.

[0071] The experimental results are shown in Figure 5 The experimental results show that the higher the concentration of the nano-regulator SCTDM, the lower the UV absorption intensity at 412 nm, the less residual GSH content in the system, and the greater the GSH consumption. Simultaneously, the stronger the fluorescence absorption intensity at 610 nm, indicating an increase in monovalent copper formation. The absorption intensity of MB at 660 nm decreases with increasing SCTDM concentration, indicating that SCTDM has good Fenton-like reactivity, catalyzing the decomposition of H₂O₂ to generate hydroxyl radicals, leading to MB degradation.

[0072] Both are used to detect the effects of photothermal activity on material consumption of GSH and formation of Cu. + To promote the generation of ·OH, SCTDM was first reacted with Na2S (molar ratio 1:1) for 10 min, and then the system was irradiated with a laser (808 nm, 1.0 W / cm²). 2 (10 min), and then continue the experiment according to the above method to detect the effect of photothermal activity on the material's GSH consumption and Cu formation. + And the degradation that produces MB.

[0073] Depend on Figure 6 It can be seen that after irradiation with an 808nm laser, the consumption of GSH by the nano-modulator SCTDM increases. Figure 6 In (a), the production of monovalent copper increased significantly. Figure 6 (b) and the degradation of methylene blue increased significantly. Figure 6 c) indicates that photothermal irradiation promotes the reactivity of SCTDM, which is beneficial to enhancing the copper death effect in its cells.

[0074] 3. Uptake of nanomodulators by CT26 colon cancer cells

[0075] To track intracellular nanoregulators, the hydrophobic drug Nile Red was encapsulated within the nanoregulators SCTD and SCTDM, enabling fluorescence tracking. CT26 cells were seeded at a rate of 100,000 per dish on confocal microscopes and cultured for 24 hours. Afterward, fluorescently labeled nanoregulators were applied. Six hours later, cells were collected, and the nuclei were labeled using Hoechst staining. The fluorescence intensity of Nile Red within the cells was observed under a confocal microscope to record the content of the intracellular nanoregulators.

[0076] from Figure 7The results showed that obvious red fluorescence was visible in both cells, indicating that CT26 cells could effectively take up SCTD and SCTDM. However, SCTDM showed stronger intracellular fluorescence, indicating that it had a higher content of intracellular nanoregulators. This can be attributed to the fact that the hybrid macrophage-tumor cell membrane coated on the surface of the nanoregulators enhanced the cells' recognition and uptake of the biomimetic nanoregulator SCTDM through homologous membrane targeting performance.

[0077] 4. Evaluation of the performance of nanomodulators in regulating lactate metabolism in colon cancer cells CT26

[0078] CT26 cells were seeded at 300,000 per well in six-well plates and cultured for 24 hours. Different nanoregulators prepared in Example 1 were then added, with an equal volume of PBS used as a control group. The drug concentration (diluted with culture medium) was determined according to Cu... 2+ The drug was administered at a concentration of 2.5 μg / mL. Cells were collected 12 h after drug administration, and the intracellular and extracellular lactate content was detected using an L-LA lactate assay kit.

[0079] The results are as follows Figure 8 As shown in the figure: Compared with group G1 (control group), the intracellular lactate content of cells treated in group G4 was significantly increased. Figure 8 In the middle (a), the extracellular lactate content was significantly reduced ( Figure 8 Figure b) shows that Su3118, an inhibitor of the activity of the lactate transporter MCT1 / 4 loaded in the nanoregulator, can be released intracellularly, thereby inhibiting intracellular lactate efflux and regulating lactate metabolism. Compared with group G4, group G5 showed a further decrease in extracellular lactate content and an increase in intracellular content, indicating that copper death can synergize with the effect of Su3118, enhancing the regulatory effect on lactate metabolism. Group G6 exhibited the strongest lactate metabolism regulatory capacity, which can be attributed to the membrane coating enhancing the content of the intracellular nanoregulator, increasing the corresponding drug concentration, and improving the therapeutic effect.

[0080] 5. Nanomodulators affect intracellular pyruvate levels in CT26 colon cancer cells.

[0081] CT26 cells were seeded at 300,000 per well in six-well plates and cultured for 24 hours. Different nanoregulators prepared in Example 1 were then added, with an equal volume of PBS used as a control group. The drug concentration (diluted with culture medium) was determined according to Cu... 2+ The drug was administered at a concentration of 2.5 μg / mL. Cells were collected 12 h after drug administration, and the intracellular pyruvate content was detected using a pyruvate content assay kit.

[0082] The results are as follows Figure 9As shown in the figure, compared with the control group G1, the intracellular pyruvate content in groups G3, G4, G5, and G6 was significantly increased, accompanied by a significant increase in intracellular lactate concentration. These results indicate that inhibition of cellular lactate transport can increase lactate content. As the final product of glycolysis, the increase in lactate concentration can reverse the pyruvate-lactic acid conversion process during glycolysis, block the lactate generation step in glycolysis, enhance pyruvate production, force excess pyruvate into the mitochondrial tricarboxylic acid cycle (TCA), enhance mitochondrial respiration, thereby increasing its copper death sensitivity and improving the therapeutic effect.

[0083] 6. Western blot analysis (WB) of expression of copper death-related proteins in colon cancer cells CT26.

[0084] CT26 cells were seeded at 300,000 per well in six-well plates and cultured for 24 hours before drug treatment. An equal volume of PBS was used as a control group. Different nanometers prepared in Example 1 were added to the control group. Simultaneously, a laser-treated SCTDM sample group was selected. The drug concentration (diluted with culture medium) was determined according to Cu... 2+ The drug was administered at a concentration of 5 μg / mL. Six hours after administration, the SCTDM sample group was irradiated with laser for 5 minutes (SCTDM+L group, 1.0 W / cm²). 2 Cells were collected at 808 nm. Proteins were extracted by lysing the cells using RIPA lysis buffer. Protein concentration was detected using a BCA protein assay kit. 20 μg of protein sample was loaded each time, and the required volume was calculated. The total loading volume was 10 μL. 5× Loading buffer was added at a total volume ratio of 4:1 (sample:buffer volume ratio of 4:1). Any remaining insufficient volume was made up with lysis buffer. The mixture was boiled at 100°C for 10 min. SDS-PAGE was performed at 90V for 90 min. After electrophoresis, wet transfer was performed at 300 mA for 120 min. After transfer, the membrane was blocked with 5% BSA on a shaker at room temperature for 1 h. After blocking, the membrane was washed three times with TBST. Add the corresponding primary antibody, incubate overnight at 4°C, wash 3 times with TBST, then add the corresponding species' secondary antibody, incubate for 2 hours at room temperature, wash 3 times with TBST, and finally use a high-sensitivity ECL chemiluminescence kit to mix the colorimetric solution A:B at a volume ratio of 1:1 and drop it evenly onto the membrane, then expose and image using a chemiluminescence imager.

[0085] The results are as follows Figure 10As shown, the G3 group of colorectal cancer CT26 cells showed decreased intracellular FDX1 and LIAS expression and increased DLAT expression, while the G5, G6, and G7 groups of colorectal cancer CT26 cells further significantly decreased FDX1 and LIAS expression and increased DLAT expression. This result indicates that nanoregulators can effectively induce copper death in cells, and lactate regulation and photothermal therapy can synergistically enhance the effect of copper death.

[0086] 7. In vitro pharmacodynamic evaluation

[0087] The in vitro tumor cell therapy efficacy of the nanoregulator was determined using the CCK8 assay. CT26 cells were seeded at 5000 cells / well in 96-well plates and cultured for 24 h. Afterward, the culture medium was removed, and the cells were incubated for another 24 h with culture medium containing different concentrations (diluted with culture medium) of the nanoregulator prepared in Example 1. Simultaneously, 12 h after drug administration, the SCTDM sample group was treated with 808 nm laser irradiation for 5 min as the laser therapy group (SCTDM+L group, 808 nm, 1.0 W / cm²). 2 After irradiation, the cells were incubated for 12 hours. After incubation, CCK8 reagent was added and the cells were incubated at 37°C for 1 hour. The absorbance of each sample was measured at 450 nm, and the cell viability was plotted.

[0088] The results are as follows Figure 11 As shown, the cell viability of CT26 cells decreased with increasing nanoregulator concentration. At concentrations of 1.25 μg / mL and above, the cell viability of SCTD and SCTDM in the treatment group was lower than that of CTD and SCT in the treatment group, indicating that lactate regulation enhanced the effect of copper cell death. The laser treatment group SCTDM+L showed a significant ability to reduce cell viability, indicating that after the drug concentration is increased to a certain level, sufficient photosensitizer copper sulfide complex is generated intracellularly, initiating effective photothermal therapy and further enhancing the therapeutic effect on cells. Colorectal cancer contains high concentrations of hydrogen sulfide, which can react in situ with copper ion delivery systems to generate photothermally responsive copper sulfide complexes. External laser radiation enables in situ photothermal therapy of the tumor, improving treatment precision.

[0089] 8. Hemolytic properties

[0090] Healthy mice (BALB / c mice, female, SPF grade, 18-20g, Beijing Huafukang Biotechnology Co., Ltd.) underwent enucleation to collect blood. Heparin sodium injection was added pre-treated to the ep ... 2+ A 0.8 mg / mL solution (diluted with pure water) was prepared. 0.3 mL of the material solution was added to every 0.7 mL of 2% RBCs solution. Pure water and physiological saline were used as positive and negative controls, respectively. TA and Cu were used as skeleton material controls. The mixture was incubated at room temperature for 2 h, centrifuged at 2000 rpm for 10 min, photographed, and the absorbance at 541 nm was measured from the supernatant. The degree of hemolysis was calculated.

[0091] Depend on Figure 12 It is known that SCTDM has good biocompatibility, does not cause hemolysis, and can be used for intravenous injection.

[0092] 9. In vivo targeted therapy

[0093] CT26 cell suspension (2 × 10⁶ cells per mouse) was subcutaneously injected into the right back of mice (BALB / c mice, female, SPF grade, 18-20g, Beijing Huafukang Biotechnology Co., Ltd.) 6 A CT-26 tumor-bearing model was established using individual cells. When the tumor volume reached 150 mm², the tumor was further investigated. 3 In the above scenario, CT26 tumor-bearing mice were randomly divided into two groups: SCTD and SCTDM, with four mice in each group. SCTD and SCTDM nanomodulators were labeled with an IR-820 fluorescent probe. The preparation methods of the IR-820-labeled SCTD and SCTDM nanomodulators are described in Example 1. 0.1 mL of the fluorescently labeled nanomodulator was injected via the tail vein at a dose of IR-820 2 mg / kg. Mice were anesthetized 12 and 24 hours after injection, and CRI imaging was used to capture images to detect the fluorescence intensity of IR-820 at the tumor site, followed by statistical analysis.

[0094] The results are as follows Figure 13 As shown in the figure, the membrane-coated nanomodulator SCTDM exhibits strong IR-820 fluorescence at the tumor site, indicating its enhanced tumor targeting, which can be attributed to the tumor tropism of the coated macrophage membrane.

[0095] 10. Internal treatment

[0096] The CT26 tumor-bearing mouse model was constructed as described in "9. In vivo targeting," when the tumor volume reached 80 mm. 3 At that time, tumor-bearing mice were randomly divided into 5 groups of 5 mice each, and administered the drug via tail vein. The groups were as follows: g1 was the PBS group, g2 was the CTD group, g3 was the SCTD group, g4 was the SCTDM group, and g5 was the SCTDM+Laser group. The dosage was as follows: Cu 2+ The drug was administered at a concentration of 4 mg / kg. It was given once every 4 days for three treatment cycles. Group g5 received SCTDM first, followed by laser irradiation of the tumor site for 5 minutes (808 nm, 1.0 W / cm²) on days 1 and 2 after administration. 2 ).

[0097] The length and width of the tumor were measured daily, and the weight of the tumor-bearing mice was recorded. The tumor volume was calculated using the formula: length × width × width / 2. On day 12, the mice were sacrificed, the tumor tissue was isolated, and weighed.

[0098] The results are as follows Figure 14 As shown in Figure 1, compared to the control group, tumor growth was slower and tumor weight was reduced in group G2. Group G3 further inhibited tumor proliferation, indicating that lactate regulation can increase the therapeutic effect of copper death. Group G4 enhanced the inhibition of tumor proliferation, attributed to membrane-mediated tumor targeting, which increased tumor accumulation on the treatment platform. Laser treatment in group G5 almost completely inhibited tumor proliferation, indicating that photothermal therapy, as an adjunct, can further enhance the therapeutic effect of copper death in tumors. The mouse weight curve did not show a significant decrease in body weight, indicating the biocompatibility of the nanoregulator.

[0099] 11. Suppression of tumor recurrence after surgery

[0100] The CT26 tumor-bearing mouse model was constructed as described in "9. In vivo targeting," when the tumor volume reached 80 mm. 3 At that time, tumor-bearing mice were randomly divided into three groups: PBS group (control group), SCTDM group, and SCTDM+Laser group, with five mice in each group. The drugs were administered via tail vein injection at the dosage specified by Cu. 2+ The concentration was 4 mg / kg. The administration and treatment method were the same as in "10. In vivo treatment". On day 12, the mouse tumor tissue was surgically removed. The mice were fed until day 29. The mice were then sacrificed, the tumor tissue was separated, photographed, weighed, and the recurrence rate after surgery was assessed.

[0101] The results are as follows Figure 15As shown in the figure, compared with the control group, the mice treated with SCTDM had significantly fewer recurrent tumors, indicating that SCTDM can delay postoperative tumor recurrence. In the SCTDM+Laser group, no recurrent tumors were observed in 3 mice, and the weight of the recurrent tumors was further reduced. These results indicate that the SCTDM+Laser group effectively achieves photothermal-lactic acid regulation, induces copper death, enhances the body's anti-tumor immune response and immune memory function, thereby effectively inhibiting postoperative tumor recurrence.

[0102] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing a biomimetic hybrid nanoregulator that enhances copper cell death, characterized in that, Includes the following steps: Take ciprosilidine Su 3118, tannic acid TA, NaOH, CuCl2·2H2O, and disulfiram DSF, mix them, stir to obtain SCTD nanoparticles, centrifuge, resuspend, and centrifuge again to obtain SCTD nano-regulators. Macrophages and tumor cells were collected and swollen in water. The cells were lysed by quick-freezing and re-thawing. After centrifugation, the supernatant was collected and centrifuged again. The precipitate was resuspended in water to obtain the macrophage membrane and the tumor cell membrane. The macrophage membrane and the tumor cell membrane were mixed to obtain the hybrid cell membrane. The SCTD nanoregulator was mixed with a hybrid cell membrane at a mass ratio of 2:1 to 15:1, and then subjected to sonication and centrifugation to obtain the biomimetic hybrid nanoregulator SCTDM.

2. The preparation method according to claim 1, characterized in that, The following steps are performed: 100-300 μL of Su 3118 (1-5 mg / mL) and 50-100 μL of TA (10-100 mg / mL) are mixed, followed by 25-65 μL of NaOH (1-5 mg / mL), 20-60 μL of CuCl2·2H2O (5-15 mg / mL), and finally 100-300 μL of DSF (0.05-0.15 mg / mL). The mixture is stirred at 800-1200 rpm for 10-20 min at room temperature to obtain SCTD nanoparticles. Unreacted material is removed by centrifugation at 10000-15000 rpm for 5-15 min, the mixture is resuspended in water, and then centrifuged at 10000-15000 rpm for 5-15 min to obtain the SCTD nanoregulator.

3. The preparation method according to claim 2, characterized in that, The following steps were performed: 200 μL of Su 3118 (2 mg / mL) and 80 μL of TA (50 mg / mL) were mixed, followed by 45 μL of NaOH (4 mg / mL) and 40 μL of CuCl2·2H2O (10 mg / mL). Finally, 200 μL of DSF (0.1 mg / mL) was added and mixed. The mixture was stirred at 1000 rpm for 15 min at room temperature to obtain SCTD nanoparticles. Unreacted material was removed by centrifugation at 13000 rpm for 10 min, the mixture was resuspended in water, and then centrifuged at 13000 rpm for 10 min to obtain the SCTD nanoregulator.

4. The preparation method according to claim 1, characterized in that, The macrophages and tumor cells were collected and swollen in water for 12-36 hours. The cells were then lysed by quick-freezing and thawing, and this process was repeated three times. The resulting mixture was centrifuged at 1500-2500×g for 5-15 minutes. The supernatant was then centrifuged again at 15000-25000×g for 10-50 minutes. The precipitate was resuspended in water to obtain the macrophage membrane and tumor cell membrane.

5. The preparation method according to claim 4, characterized in that, The macrophages and tumor cells were collected and swollen in water for 24 hours. The cells were then lysed by quick freezing and thawing, and this process was repeated three times. The resulting mixture was centrifuged at 2000×g for 10 minutes, and the supernatant was centrifuged again at 20000×g for 30 minutes. The precipitate was then resuspended in water to obtain the macrophage membrane and the tumor cell membrane. The mass ratio of the tumor cell membrane to the macrophage membrane in the hybrid cell membrane was 1:

1.

6. The preparation method according to claim 1, characterized in that, The SCTD nanoregulator is mixed with the hybrid cell membrane at a mass ratio of 10:

1.

7. A biomimetic hybrid nanocontroller SCTDM prepared by the preparation method according to any one of claims 1-6.

8. The use of the biomimetic hybrid nanomodulator SCTDM according to claim 7 in the preparation of drugs for treating cancer and / or inhibiting cancer recurrence, characterized in that, The types of cancer mentioned include, but are not limited to, colon cancer.

9. A drug that enhances copper death in tumor cells, characterized in that, The drug contains the biomimetic hybrid nanoregulator SCTDM as described in claim 7.

10. The product according to claim 9, characterized in that, The product also contains pharmaceutically acceptable carriers or excipients.

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