Intelligent response type in-situ copper ion adsorption nanometer delivery system as well as preparation method and application thereof

Abstract

The invention discloses an intelligent response type in-situ copper ion adsorption nanometer delivery system and a preparation method and application thereof.The intelligent response type in-situ copper ion adsorption nanometer delivery system comprises a nanometer liposome serving as a core, and a nitric oxide prodrug is loaded in the nanometer liposome; the surface is co-modified with drug molecules combined with copper ions and active targeting molecules. The nano-system prepared by the invention can utilize excellent carrier performance and biological safety of the liposome to enhance the dosage in cells, and through surface modification of targeting molecules, the in-vivo behavior and treatment effect of the drug delivery system are better regulated and controlled, and active targeting of the nano-liposome to a tumor site is promoted. More importantly, in the targeted delivery process, after entering a tumor microenvironment and / or cells, the nano-material shows specific response to adsorb autologous copper ions, so that the nano-material has no toxic or side effect on normal tissues, and precise treatment for inducing tumor cell copper death is realized.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomedical polymer materials, and particularly relates to an intelligent response type in-situ copper ion adsorption nano delivery system and preparation and application thereof. BACKGROUND

[0002] The treatment of malignant tumors mainly relies on surgical resection, radiotherapy and chemotherapy. At present, the clinical treatment method for tumors is to evaluate the advantages and disadvantages of different treatment methods in combination with the physiological and psychological state of patients according to different stages of tumor development. Although the treatment technology for malignant tumors is constantly improving, however, different types of therapy have some inherent limitations, which leads to the failure to achieve the ideal therapeutic effect. For example, surgical resection is one of the most commonly used treatment methods in clinical practice. However, it has inherent deficiencies such as great preoperative psychological pressure, incomplete resection during operation, long postoperative recovery period and unsuitability for metastatic cancer treatment. Compared with surgical treatment, chemotherapy and radiotherapy have a wider application range and are suitable for treating tumors at all stages of development. However, most of the chemotherapy and radiotherapy lack specificity, cannot accurately target lesions, and can cause serious systemic toxicity and adverse reactions. Therefore, it is urgent to develop new treatment methods that can specifically regulate the programmed death of tumor cells in view of the characteristics of the tumor cell microenvironment, which will effectively reduce the defects and shortcomings of traditional treatment, improve the quality of life of patients and prolong the survival rate of patients.

[0003] Metal elements play an important role in the human body. Among various ions, copper ions (Cu 2+ ) are essential trace elements for the human body, are redox cofactors for many enzymes to regulate cell metabolism, mediate a series of important physiological functions, including mitochondrial respiration, antioxidant defense, and biosynthesis of hormones, neurotransmitters and pigments. In order to meet the abnormal active capacity requirement of tumor cells, clinical studies have proved that the endogenous copper ion concentration in the tumor microenvironment is significantly higher than that in normal tissues. Therefore, it is an attractive new type of tumor treatment to try to cause cell damage by breaking the balance of metal ions in cells. Once the metabolism of free copper ions in cells is abnormal, i.e. copper balance is disrupted, serious cytotoxicity will occur. Copper overload is a typical manifestation of copper imbalance, which has been proved to be a key factor for inducing copper death. Unlike the mechanisms of other cell death processes (apoptosis, necrosis, ferroptosis, etc.), the mechanism of copper death involves the binding of excess copper ions to the lipidated tricarboxylic acid (TCA) cycle components in mitochondria, leading to lipidated protein aggregation and iron-sulfur cluster protein degradation, inducing protein toxic stress and inducing cell death. Therefore, disrupting copper homeostasis in tumor cells to achieve copper death is crucial for the treatment of cancer.

[0004] In order to destroy the tumor cell copper homeostasis, a series of carefully designed copper-based nanodelivery systems for tumor treatment that can induce or amplify copper death are emerging, aiming to release exogenous copper ions, induce cell copper overload, and then trigger copper death by utilizing the inherent stimulation of tumor-specific microenvironment or externally applied specific stimulation. However, it is a challenge for existing copper-based nanodelivery systems to achieve high concentration of intracellular copper ions to effectively activate copper death: (1) first, tumor cells pump out excess copper ions carried by copper-based nanodelivery systems to the outside of the cells through the membrane surface copper ion efflux pump ATP7B; (2) second, the high expression of reducing substances (such as glutathione GSH) in the tumor microenvironment weakens the activity of copper ions by chelating copper ions, thereby inhibiting the occurrence of copper death. These two cell self-protection pathways work together to prevent excessive accumulation of intracellular copper ions and achieve cell self-protection; more importantly, (3) as an exogenous copper ion supplier, excessive accumulation of copper-based nanodelivery systems may cause toxic reactions, leading to damage to normal tissues and organs. SUMMARY

[0005] The primary purpose of the present application is to provide an intelligent responsive in situ copper ion adsorption nanodelivery system, which can utilize the excellent carrier performance and biological safety of liposomes to enhance the drug delivery amount in cells, better regulate the in vivo behavior and therapeutic effect of the drug delivery system through surface modification of targeting molecules, and promote active targeting of nanoliposomes to tumor sites. More importantly, during its targeted delivery, it exhibits specific response to adsorb autologous copper ions after entering the tumor microenvironment and / or cells, thereby achieving precise treatment of inducing copper death of tumor cells without toxic side effects on normal tissues. The present application develops a nanodelivery system that does not rely on exogenous copper ions to induce copper imbalance and also has the function of inhibiting copper efflux, thereby achieving the induction of copper death of tumor cells.

[0006] The present application also provides a preparation method and application of the intelligent responsive in situ copper ion adsorption nanodelivery system.

[0007] Technical scheme: In order to achieve the above-mentioned purpose, the intelligent responsive in situ copper ion adsorption nanodelivery system comprises a nanoliposome as a core, the nanoliposome internally loaded with a nitric oxide prodrug, and the surface co-modified with a copper ion binding drug molecule and an active targeting molecule.

[0008] The nanoliposome is composed of phospholipids, and the phospholipids are any one or more of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, or artificially synthesized phospholipids, preferably the phospholipids are DPPC, DSPC, and DSPE-PEG2000 .

[0009] The nitric oxide (NO) prodrug is any one of JS-K, nitroglycerin, isosorbide dinitrate (ISDN), isosorbide mononitrate (ISMN), molar nitrate, sodium nitroprusside, or L-arginine.

[0010] The drug molecule that binds copper ions is either disulfiram (DSF) or Elesclomol; the active targeting molecule is either FTY720 or S1P.

[0011] The preparation method of the in-situ intelligent responsive copper ion adsorption nanodelivery system of the present invention includes the following steps:

[0012] (1) Preparation of nanoliposomes internally loaded with nitric oxide prodrug;

[0013] (2) The shell of the nanoliposome loaded with nitric oxide prodrug is modified with active targeting molecules and drug molecules that bind copper ions, forming a smart responsive in-situ copper ion adsorption nanodelivery system.

[0014] Furthermore, the prepared intelligent responsive in-situ copper ion adsorption nanodelivery system intelligently responds to tumor microenvironment stimulation after being injected into the body. Through in vivo adsorption, a nanoliposome drug delivery system with copper ions bound to its surface is obtained for the efficient treatment of tumors.

[0015] The preparation method of the nanoliposomes internally loaded with gaseous prodrugs in step (1) includes the following steps:

[0016] (1) Take phospholipid material and add it to an organic solvent to obtain a mixed phospholipid solution; take nitric oxide prodrug and add it to an organic solvent to obtain a gaseous precursor drug solution; mix the mixed phospholipid solution and the gaseous precursor drug solution thoroughly;

[0017] (2) The mixed solution was subjected to rotary vacuum distillation to obtain a mixed film of phospholipid and gaseous precursor drug; the mixed film was dried under vacuum overnight.

[0018] (3) Take out the mixed film after vacuum overnight drying, and perform rotational hydration to fully disperse the film and obtain a nanoliposome suspension loaded with gas prodrug.

[0019] (4) The nanoliposome suspension was repeatedly squeezed through a filter membrane with uniform pore size by extrusion membrane method, and then dialyzed to obtain nanoliposomes loaded with gas prodrugs.

[0020] In step (2), the solvent in the mixed solution is gradually removed by vacuum distillation under water bath conditions to obtain a mixed film of phospholipid and gaseous prodrug. Then, the mixed film is transferred to a vacuum drying oven and vacuum-treated overnight. In step (3), a neutral buffer solution is added to the rotary evaporator under water bath conditions, and after rotary hydration, a nanoliposome suspension loaded with gaseous prodrug is obtained. In step (4), the repeated squeezing through a filter membrane with uniform pore size requires that the nanoliposome suspension be passed through polycarbonate filter membranes with pore sizes of 800 nm, 400 nm and 220 nm in sequence, for no less than 20 times.

[0021] The method for modifying the shell of the nanoliposomes with active targeting molecules and copper-binding drug molecules in step (2) includes the following steps:

[0022] (1) Dissolve the active targeting molecule and the drug molecule that binds copper ions in organic solvents respectively;

[0023] (2) Mix the nanoliposomes containing gaseous prodrugs, the active targeting molecule solution, and the drug molecule solution containing copper ions evenly and stir to react;

[0024] (3) After dialysis, nanoliposomes containing gaseous prodrugs loaded inside a shell modified with active targeting molecules and drug molecules that bind copper ions were obtained.

[0025] In step (1), the active targeting molecule and the copper-binding drug molecule are dissolved in DMSO respectively; in step (2), the nanoliposomes loaded with gaseous prodrug, the active targeting molecule solution, and the copper-binding drug molecule solution are added together to a neutral buffer and mixed evenly, and stirred to react; the neutral buffer is a PBS buffer or NaCl buffer with a pH of 6.5 to 7.5.

[0026] The application of the intelligent responsive in-situ copper ion adsorption nanodelivery system described in this invention in the preparation of tumor therapeutic drugs.

[0027] This invention discloses a smart responsive in-situ copper ion adsorption nanodelivery system. This system uses liposomes as its core, internally loaded with a gaseous prodrug, and externally modified with copper-binding drug molecules and active targeting molecules. Upon in vivo injection, it intelligently responds to microenvironment stimulation, adsorbing copper ions to form the smart responsive in-situ copper ion adsorption nanodelivery system. This invention's smart responsive in-situ copper ion adsorption nanodelivery system, by loading a gaseous prodrug within the membrane shell and modifying the liposome surface with active targeting molecules and copper ion complexing molecules, can effectively promote the adsorption of copper ions in the tumor microenvironment, cellular uptake, and accumulation of intracellular copper ion concentration through the synergistic effect of the tumor microenvironment and high intracellular GSH concentration, ultimately improving therapeutic efficacy.

[0028] Specifically, the copper-chelating molecules modified on the surface of these nanoliposomes undergo structural transformation in response to GSH stimulation in the microenvironment, in situ chelating high concentrations of copper ions in the microenvironment. Subsequently, enhanced cellular uptake is achieved through active targeting molecule-mediated enhancement, thus enabling the in-situ reuse of copper ions. Simultaneously, the gaseous prodrug delivered concurrently by the liposomes can generate NO by consuming intracellular GSH, thereby inhibiting the activity of the cellular copper ion efflux pump. In summary, the in-situ adsorption and utilization of copper ions in the microenvironment, combined with the simultaneous inhibition of efflux, achieves effective accumulation of intracellular copper ion concentration, thus avoiding the toxic side effects of exogenous copper ion therapy and realizing green and precise tumor treatment.

[0029] The nanoliposomes described in this invention refer to nanoscale vesicle structures formed by the self-assembly of phospholipid molecules, with a particle size of approximately 10–200 nm. Specific phospholipid molecules can be phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, synthetic phospholipids, etc., or their inorganic or organic salts. Specific examples include DPPC, DSPC, DPPG, DOTAP, or DSPE-PEG. 2000 However, it is not limited to this. Preferably, in some specific embodiments of the present invention, the phospholipid molecules used include DPPC, DSPC, and DSPE-PEG. 2000 .

[0030] The gaseous prodrug loaded within the liposomes described in this invention refers to a drug structure capable of producing NO through an enzymatic reaction in tumor cells. Specific examples include JS-K, nitroglycerin, isosorbide dinitrate (ISDN), isosorbide mononitrate (ISMN), molar nitrate, sodium nitroprusside, or L-arginine. Preferably, in a specific embodiment of this invention, the gaseous prodrug used isosorbide dinitrate (ISDN). Compared to other types of gaseous prodrugs, ISDN, while possessing the function of releasing NO intracellularly, has ISMN as its primary metabolic product in vivo, which is also an effective NO donor. This results in ISDN having a longer-lasting NO production and release effect.

[0031] The externally modified copper-binding drug molecule described in this invention refers to a drug molecule that can complex copper ions in response to an increase in the concentration of GSH in the microenvironment. Specific examples that can be mentioned include DSF and Elesclomol.

[0032] Furthermore, the preparation method of the intelligent responsive in-situ copper ion adsorption nanodelivery system of the present invention mainly includes the following steps:

[0033] S1. Preparation of nanoliposomes internally loaded with gaseous prodrugs:

[0034] The nanoliposomes internally loaded with gaseous prodrugs can be prepared or obtained using methods known in the art. Here, a thin-film dispersion method is used as an example: First, phospholipid material is added to an organic solvent to obtain a mixed phospholipid solution, and a gaseous prodrug is added to the organic solvent to obtain a gaseous prodrug solution. Then, the mixed phospholipid solution and the gaseous prodrug solution are thoroughly mixed and subjected to rotary vacuum distillation under constant temperature water bath conditions to obtain a mixed thin film of phospholipids and gaseous prodrugs. Next, the mixed thin film is vacuum dried overnight, and under constant temperature water bath conditions, a neutral buffer solution is added for rotary hydration to fully disperse the film, resulting in a nanoliposome suspension loaded with gaseous prodrugs. Finally, the nanoliposome suspension is repeatedly extruded through a filter membrane with uniform pore size using an extrusion membrane method. After dialysis, nanoliposomes internally loaded with gaseous prodrugs are obtained.

[0035] The phospholipid materials can be phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, synthetic phospholipids, etc., with DPPC, DSPC, and DSPE-PEG being preferred. 2000 The preferred molar ratio of the substances is 8:1:1;

[0036] The gaseous precursor drug can be JS-K, nitroglycerin, isosorbide dinitrate (ISDN), isosorbide mononitrate (ISMN), molar nitrate, sodium nitroprusside, or L-arginine, with ISDN being the preferred candidate. Furthermore, DPPC, DSPC, and DSPE-PEG are also acceptable. 2000 The preferred molar ratio of the substance to ISDN is 8:1:1:1.

[0037] In the step of obtaining a mixed film of phospholipid and gaseous precursor drug, the temperature of the constant temperature water bath is 20-80℃, the rotary vacuum distillation speed is 10-200 rpm, and the vacuum distillation time can be adjusted as needed; the preferred temperature is 65℃, and the rotary vacuum distillation speed is 120 rpm.

[0038] In the step of fully dispersing the film, the temperature of the constant temperature water bath is 20-80℃, the rotational hydration speed is 10-200 rpm, and the duration can be adjusted as needed; the preferred temperature is 65℃, the added neutral buffer is PBS buffer, the rotational hydration speed is 100 rpm, and the duration can be adjusted as needed.

[0039] Among them, the filter membrane with uniform pore size that is repeatedly squeezed through requires the nanoliposome suspension to be passed through polycarbonate filter membranes with pore sizes of 800nm, 400nm and 220nm in sequence, no less than 20 times.

[0040] During dialysis, the drug is transferred to a dialysis bag with an MWCO of 30 kDa and dialyzed with deionized water for 4-48 hours to remove free drugs. Preferably, the dialysis time is 48 hours.

[0041] S2. The outer shell of the nanoliposomes containing the gaseous prodrug is modified with an active targeting molecule and a drug molecule that binds copper ions; the specific steps are as follows:

[0042] S21. Dissolve the active targeting molecule and the drug molecule that binds copper ions in DMSO or chloroform, respectively.

[0043] The active targeting molecule functions to endow the nanoliposomes with the structural ability to specifically adsorb onto the surface of tumor cells. Specific examples include FTY720 and S1P, with S1P being preferred. The copper-binding drug molecule enables the nanoliposomes to adsorb copper ions in the specific tumor microenvironment. Specific examples include DSF and Elesclomol, with DSF being preferred.

[0044] S22. Add the dialyzed nanoliposomes containing gaseous prodrugs, the chloroform solution of the active targeting molecules, and the chloroform solution of the drug molecules bound to copper ions to a neutral buffer solution, mix thoroughly, and stir to react.

[0045] Specifically, the pH of the reaction system is adjusted to be between 6.5 and 7.5 using a neutral buffer solution. Examples of suitable buffer solutions include phosphate buffer, physiological saline buffer, and PIPES (ethanesulfonic acid buffer), with phosphate buffer preferred. Subsequently, the mass ratio of the added active targeting molecule: drug molecule: nanoliposomes loaded with gaseous prodrug is 1–10:1–2:1–2, preferably 5:1:1. Further, in this step, the reaction is maintained at 40°C–70°C, preferably 50°C.

[0046] S23. Dialysis removes free active targeting molecules and drug molecules from the product, resulting in nanoliposomes with an outer shell modified with active targeting molecules and drug molecules bound to copper ions, and an internally loaded gas prodrug.

[0047] Specifically, the method of dialysis to remove free active targeting molecules and drug molecules involves dialysis of the reaction system generated in S22 with deionized water with a molecular weight cutoff of 30 kDa for 48 hours to remove excess unencapsulated active targeting molecules and drug molecules, thus obtaining an intelligent responsive in-situ copper ion adsorption nanodelivery system.

[0048] This invention proposes an in-situ intelligent responsive copper ion adsorption nanodelivery system and its anti-tumor application. The system uses a nanoliposome as the carrier core, preferably loaded with the nitric oxide (NO) prodrug isosorbide dinitrate (ISDN), and co-modified on the surface with disulfiram (DSF) and sphingosine 1-phosphate (S1P). S1P endows the carrier with the ability to cross the blood-brain barrier and target glioma cells.

[0049] The system of the present invention achieves precise treatment through a dual response mechanism of in-situ adsorption of copper ions and intracellular copper overload specifically triggered by the tumor microenvironment: (1) At the tumor site, high concentration of glutathione (GSH) promotes the conversion of DSF to diethyldithiocarbamate (DTC), which forms a stable complex with the copper ions overexpressed in the tumor microenvironment, thereby achieving in-situ enrichment of copper ions on the carrier surface; (2) After entering the tumor cells, ISDN maintains the stability of the copper complex by consuming intracellular GSH, while releasing nitric oxide (NO) to inhibit the activity of the copper ion efflux pump, thereby triggering continuous copper overload and activating the copper death pathway.

[0050] This invention constructs a multi-level response system that overcomes the challenges of targeted delivery and intracellular regulation in the treatment of gliomas through a microenvironment-triggered copper ion adsorption-retention mechanism, providing a new strategy for the treatment of malignant tumors.

[0051] This invention comprises a modular combination of tumor-targeting sphingosine-1-phosphate (S1P), the GSH-responsive nitric oxide (NO) technical drug isosorbide dinitrate (ISDN), and the copper chelator disulfiram (DSF), forming a DSF / S1P / ISDN-Lipos system. This system features a multi-stage therapeutic cascade: First, it utilizes the sphingosine-targeting molecule S1P to penetrate BBTB, achieving glioma-specific accumulation; second, instead of using exogenous copper ions, it leverages the glioma microenvironment rich in endogenous copper ions to trigger DSF / DSF on the surface of S1P / ISDN-Lipos is reduced to DTC, which chelates endogenous copper ions and forms a stable Cu(DTC)2 complex on the liposome surface, namely Cu(DTC)2 / S1P / ISDN-Lipos. Subsequently, after the liposomes are taken up by the cells, the released Cu(DTC)2 achieves an overload of intracellular copper ion concentration. The NO generated by the simultaneously released ISDN consumes GSH and inhibits the ATP7B efflux pump, preventing copper ions from being pumped out of the cell again, thus successfully inducing copper death in the cells.

[0052] This invention (1) abandons the use of exogenous copper ions to construct a copper-based nanodelivery system, and instead uses endogenous copper ions to construct a nanodelivery system to kill tumor cells, making the scheme safer; (2) while delivering copper ions into cells, it uses NO to simultaneously inhibit two cell self-protection pathways: glutathione (GSH) chelation of copper ions and ATP7B efflux of copper ions, thereby promoting the intracellular accumulation of copper ions and thus efficiently inducing copper death.

[0053] The system constructed in this invention eliminates the dependence on exogenous copper ions, realizes the reuse of excess copper ions in the tumor microenvironment in vivo, and can also prepare a nanoliposome drug delivery system with copper ions bound on its surface in vitro by mimicking the conditions of the tumor microenvironment in vivo.

[0054] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0055] 1. The intelligent responsive in-situ copper ion adsorption nanodelivery system prepared in this invention uses nanoliposomes as the core, loads gaseous prodrugs inside the liposomes, and modifies the outside with drug molecules that bind copper ions and active targeting molecules. After being injected into the body, the system intelligently responds to the stimulation of the microenvironment to adsorb copper ions, forming an intelligent responsive in-situ copper ion adsorption nanodelivery system. This solves the problems of non-specific toxicity and dosage control caused by the dependence of existing nanomedicine carriers on exogenous copper ions, as well as the difficulty in enriching and taking up in tumor sites, and the failure of inducing copper overload due to the re-excretion of copper ions by the cells after entering the tumor cells. Thus, it can induce copper death in tumor cells more safely, precisely and controllably.

[0056] 2. The intelligent responsive in-situ copper ion adsorption nanodelivery system prepared in this invention utilizes nanoliposomes. These nanoliposomes possess excellent biocompatibility, superior stability in physiological environments, modifiable external surfaces, large specific surface area, and high drug loading rates, making them suitable as carriers to improve drug encapsulation and loading rates. Furthermore, to achieve controllable adsorption and release of endogenous copper ions in the tumor microenvironment, this invention constructs GSH concentration-responsive copper ion adsorption nanoliposomes modified with DSF or Elesclomol. When the liposomes enter the tumor microenvironment and are stimulated by a high concentration of endogenous GSH, the DSF or Elesclomol modified on the liposome surface undergoes a complexation reaction with the high concentration of endogenous copper ions in the microenvironment to form a copper complex, thereby adsorbing a stable copper complex layer on the surface of the nanoliposomes. Simultaneously with the formation of the in-situ copper ion adsorption nanoliposomes, the targeting molecule S1P modified on the surface of the nanoliposomes mediates the active uptake of this nanosystem by tumor cells, ultimately inducing an overload of intracellular copper ion concentration, leading to copper death in the tumor cells. Furthermore, the hydrophobic shell of the nanoliposomes simultaneously loads a NO gas precursor drug, enabling the nanoliposomes to responsively produce and release NO in response to high intracellular GSH stimulation after cellular uptake. NO production depletes protective GSH, thereby inhibiting the tumor cells' self-protective ability against copper death. The large amount of NO produced also downregulates the expression of the tumor cell copper ion efflux pump ATP7B, weakening the tumor's copper efflux capacity. This effectively promotes the efficiency of the copper ion delivery nanosystem in inducing cellular copper overload and successfully induces cellular copper death. Attached Figure Description

[0057] Figure 1 This is a schematic diagram of the structure of the intelligent responsive in-situ copper ion adsorption nanodelivery system in this invention and its structural changes in different microenvironments;

[0058] Figure 2 This is a graph showing the changes in the absorption spectra of DSF / S1P / ISDN-Lipos under different concentrations of GSH stimulation, measured using an ultraviolet spectrophotometer in Example 2 of the present invention.

[0059] Figure 3 a is a transmission electron microscope (TEM) characterization image of the two nanodelivery systems in Example 1 of the present invention, DSF / S1P / ISDN-Lipos and Example 3, after incubation in different types of microenvironments.

[0060] Figure 3 b is an elemental distribution characterization diagram of the two nanodelivery systems after incubation in different types of microenvironments in Example 3 of the present invention;

[0061] Figure 3c is a hydrodynamic size distribution and zeta potential characterization diagram of two nanodelivery systems after incubation in different types of microenvironments in Example 3 of the present invention.

[0062] Figure 4 Changes in cell viability of GL261 cells and bEnd.3 cells after treatment with different types of nanodelivery systems;

[0063] Figure 5 Fluorescence imaging was used to characterize the intracellular NO levels in GL261 cells after they were treated with different nanodelivery systems.

[0064] Figure 6 Western blot characterization of ATP7B, an intracellular copper ion efflux protein, in GL261 cells after treatment with different nanodelivery systems.

[0065] Figure 7 Western Blot characterization of intracellular copper death-related proteins in GL261 cells after treatment with different nanodelivery systems;

[0066] Figure 8 Immunofluorescence staining characterizes copper death-related proteins in tumor tissue of mice with orthotopic gliomas after treatment with different nanodelivery systems. Detailed Implementation

[0067] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Experimental methods not specifically described in the examples are generally performed under standard conditions or as recommended by the manufacturer.

[0068] Among them, DPPC (dispalmitoylphosphatidylcholine, Sigmaaldrich, 850355C), DSPC (distearylphosphatidylcholine, Sigmaaldrich, 850365C), and DSPE-PEG 2000 (Disteazygosylphosphatidylethanolamine-polyethylene glycol, Suzhou Southeast Pharmaceutical Co., Ltd.); ISDN (Shanghai Aladdin Biochemical Technology Co., Ltd., S304813); DSF (Sigma-Aldrich, 1224008); S1P (Sphingosine-1-phosphate, Sigma, S9666); 800nm, 400nm and 220nm polycarbonate filter membranes (Whatman, Antos Nanotechnology (Suzhou) Co., Ltd.); NO-specific fluorescent probe DAF-FM DA (S0019S, Shanghai Beyotime Biotechnology Co., Ltd.); DiO (membrane green fluorescent probe, Shanghai Beyotime, C1038); DiR (membrane near-infrared fluorescent probe, Beijing Solarbio, IC6110); BALB / c mice (6-8 weeks old, Qinglongshan Animal Breeding Farm, Jiangning District, Nanjing).

[0069] Example 1

[0070] Preparation of a smart responsive in-situ copper ion adsorption nanodelivery system

[0071] This embodiment provides a smart responsive in-situ copper ion adsorption nanodelivery system and its preparation method. The system uses nanoliposomes as its core, internally loaded with a gaseous prodrug that generates active gas in response to the intracellular microenvironment. Its surface is modified with active targeting molecules and drug molecules that bind copper ions. The structure of this smart responsive nanodelivery system and its structural changes in different microenvironments are described below. Figure 1 As shown.

[0072] The specific fabrication steps of this intelligent responsive nanodelivery system are as follows:

[0073] S1. Preparation of ISDN-Lipos nanoliposomes internally loaded with gaseous prodrugs

[0074] Combine DPPC (12.5mg), DSPC (1.68mg), DSPE-PEG 2000 5.90 mg of phospholipids and 0.50 mg of ISDN were mixed in a molar ratio of 8:1:1:1 and placed in a 25 mL flask containing 3 mL of chloroform. The flask was then subjected to rotary evaporation at 120 rpm for 2 hours in a constant-temperature water bath at 65°C to obtain a dried phospholipid film. The film was then transferred to a vacuum drying oven and dried overnight. After overnight drying, 3 mL of PBS buffer (pH 7.4) was added to the flask, and the mixture was hydrated at 100 rpm for 1 hour in a constant-temperature water bath at 65°C to obtain a nanoliposome suspension. The liposome suspension was then repeatedly extruded through polycarbonate membranes with pore sizes of 800 nm, 400 nm, and 220 nm, with each pore size being extruded 50 times. Subsequently, the nanoliposome suspension was placed into a dialysis bag with a molecular weight cutoff of 30 kDa and dialyzed for 48 hours to remove excess unencapsulated gaseous prodrug, thus obtaining nanoliposomes ISDN-Lipos internally loaded with the gaseous prodrug ISDN.

[0075] S2. Preparation of nanoliposomes (DSF / S1P / ISDN-Lipos) with shells modified with active targeting molecules and drug molecules binding copper ions.

[0076] DSF and S1P were dissolved separately in chloroform solution and sonicated in a water bath at room temperature to promote complete dissolution before use. The ISDN-Lipos prepared in step S1 was dispersed in neutral PBS buffer (pH 7.4). Subsequently, at 50°C, DSF and S1P were added to the ISDN-Lipos solution according to a mass ratio of 5:1:1 for the active targeting molecule: drug molecule: internally loaded gaseous prodrug nanoliposomes, and the mixture was reacted at 50°C for 2 hours. After dialysis (MWCO = 30 kDa) for 48 hours, nanoliposomes with the outer shell modified with the active targeting molecule S1P and the copper-binding drug molecule DSF were obtained in the dialysis bag, i.e., DSF / S1P / ISDN-Lipos.

[0077] Example 2

[0078] Evaluation of the response of DSF / S1P / ISDN-Lipos to GSH concentration

[0079] Take 1 mL of the DSF / S1P / ISDN-Lipos prepared in Example 1 and add it to eight 10 mL flasks. Add GSH solution to each flask to achieve final GSH concentrations of 0, 1, 1.5, 2, 2.5, 3, 5, and 10 mM, respectively. After incubation at 25°C for 24 h, take 0.1 mL of the sample from each flask into an Eppendorf tube, add 0.9 mL of methanol, and perform vortex demulsification (1000 rpm, 5 min, 25°C). After demulsification, analyze the absorption spectrum of the solution using a UV spectrophotometer.

[0080] Figure 2 The results show that with increasing GSH concentration, the DSF modified on the surface of the nanodelivery system is gradually reduced and cleaved into the copper chelator DTC, with a significant increase in absorbance at its characteristic 210 nm peak. These results demonstrate that DSF / S1P / ISDN-Lipos possesses GSH concentration-dependent copper ion chelating ability: at physiological levels (~1 mM GSH), the structure of DSF / S1P / ISDN-Lipos is stable and cannot adsorb copper ions in response to structural changes in the microenvironment. However, in the high GSH microenvironment (~5 mM) of glioma, DSF / S1P / ISDN-Lipos is effectively activated, thereby achieving tumor microenvironment-responsive structural changes and acquiring the ability to adsorb copper ions from the environment.

[0081] Example 3

[0082] DSF / S1P / ISDN-Lipos adsorbs Cu under different microenvironments 2+ Performance evaluation

[0083] Two 10 mL flasks were used. 5 mL of PBS (pH 7.4) containing 1 mM GSH was added to each flask to simulate the blood microenvironment, and 5 mL of PBS (pH 6.5) containing 5 mM GSH was added to simulate the tumor microenvironment. Then, 1 mL of the DSF / S1P / ISDN-Lipos prepared in Example 1 was added to each flask, and the mixture was incubated at 25 °C for 24 h. After incubation, copper acetate solution was added to each flask to achieve a final copper ion concentration of 22.7 μg / mL, and incubation continued at 25 °C for 4 h. After the reaction, the samples in each flask were centrifuged at 10000 g for 20 min at 4 °C. The precipitates were collected and resuspended in 0.5 mL of PBS (pH 7.4) to obtain two different types of nanodelivery systems. The morphology of the DSF / S1P / ISDN-Lipos prepared in Example 1 and the two different types of nanodelivery systems obtained in this example were characterized using transmission electron microscopy. Figure 3 The data shows that DSF / S1P / ISDN-Lipos exhibits a uniform spherical structure with clear edges and interfaces. In the simulated blood microenvironment, its morphology is not significantly different from that of DSF / S1P / ISDN-Lipos, indicating that its surface cannot effectively adsorb copper ions in the simulated blood environment. However, in the simulated tumor microenvironment, while maintaining structural integrity, a translucent coating layer can be observed on the surface of DSF / S1P / ISDN-Lipos. This characteristic morphological difference confirms that a surface complex is formed after copper ion adsorption.

[0084] Furthermore, TEM-EDS was used to analyze two different types of nanodelivery systems co-incubated with copper ions in two different microenvironments. The results showed that in a simulated blood environment, no significant enrichment of copper was detected on the liposome surface, and the co-localization effect of copper and sulfur was weak; conversely, in a simulated tumor microenvironment, the DSF / S1P / ISDN-Lipos surface exhibited a significant cyclic copper distribution, and the co-localization effect of copper and sulfur was obvious. Figure 3 b). These results confirm the responsive in-situ copper ion adsorption capacity of DSF / S1P / ISDN-Lipos.

[0085] Dynamic light scattering method measurement shows ( Figure 3c) In a simulated blood environment, the size of the DSF / S1P / ISDN-Lipos was 174.32±4.28 nm. After co-incubation with copper ions in a simulated tumor microenvironment, the particle size of the nanodelivery system significantly increased to 185.58±2.59 nm, a size change consistent with TEM observations. Further zeta potential analysis revealed that the zeta potential of the DSF / S1P / ISDN-Lipos was -(22.15±2.94) mV, which decreased to -(17.85±1.59) mV after copper ion adsorption. This change in electrochemical property is directly related to the formation of the metal complex layer.

[0086] In summary, these results demonstrate that DSF / S1P / ISDN-Lipos can effectively adsorb copper ions in a simulated tumor microenvironment, forming Cu(DTC)2 / S1P / ISDN-Lipos, which significantly enhances the killing effect of this nanodelivery system on tumor cells, fully proving the rationality and superiority of the DSF / S1P / ISDN-Lipos design.

[0087] Example 4

[0088] Evaluation of the in vitro killing effect of DSF / S1P / ISDN-Lipos on glioma cells

[0089] Take a 10 mL round-bottom flask, add 5 mL of PBS (pH 6.5) containing 5 mM GSH, then add 1 mL of DSF / S1P / ISDN-Lipos prepared in Example 1 to the flask, and incubate at 25 °C for 24 h. After incubation, add copper acetate solution to the flask to achieve a final copper ion concentration of 22.7 μg / mL, and continue incubation at 25 °C for 4 h. After the reaction is complete, centrifuge the samples in each round-bottom flask at 10000 g for 20 min at 4 °C, collect the precipitate, and resuspend it in 0.5 mL of PBS (pH 7.4) to obtain DSF / S1P / ISDN-Lipos with copper ions adsorbed on the surface, i.e., Cu(DTC)2 / S1P / ISDN-Lipos.

[0090] GL261 glioma cells (5000 cells / well) were seeded into 96-well cell culture plates and cultured for 24 hours to allow cell attachment. After attachment, the original culture medium was aspirated and replaced with complete culture medium containing DSF / S1P / ISDN-Lipos or Cu(DTC)2 / S1P / ISDN-Lipos (containing 10% fetal bovine serum, phospholipid concentration of 1 mg / mL, and DSF concentration of 2 μg / mL), and cultured at 25°C for 0, 2, 4, 8, 10, 12, and 24 hours. Subsequently, the culture medium was aspirated, and the cells were washed three times with PBS. 100 μl of CCK-8 assay solution was added to each well, and the absorbance at 450 nm was measured using a microplate reader after 1 hour. Simultaneously, bEnd.3 mouse brain microvascular endothelial cells were used as a control group to verify the safety of the nanocarrier.

[0091] Experimental results are as follows Figure 4 The results showed that when bEnd.3 cells were treated with DSF / S1P / ISDN-Lipos and Cu(DTC)2 / S1P / ISDN-Lipos, the cell survival rate was greater than 85% within 24 hours, indicating that both prepared nanoliposomes had good biocompatibility. When DSF / S1P / ISDN-Lipos were co-incubated with GL261 cells, they exhibited time-dependent cytotoxicity. However, Cu(DTC)2 / S1P / ISDN-Lipos showed significantly better cell-killing effect than DSF / S1P / ISDN-Lipos at the same incubation time. This indicates that the nanocarrier with copper ions adsorbed on its surface can achieve a better tumor cell killing effect, and the copper complex released intracellularly after being taken up by cells can achieve a more efficient tumor cell killing purpose.

[0092] Example 5

[0093] Evaluation of the effects of DSF / S1P / ISDN-Lipos on NO production and NO inhibition of ATP7B expression in glioma cells.

[0094] Following the specific steps of Example 1, Lipos, DSF / Lipos, DSF / ISDN-Lipos, and DSF / S1P / ISDN-Lipos were prepared respectively.

[0095] GL261 cells in good growth condition were subjected to a 2×10⁻⁶ 3 Cells were seeded at a density of 1 cell per dish on a glass substrate with a bottom area of ​​9.6 cm². 2Cells were cultured in confocal microscope dishes at 37°C and 5% CO2 for 24 hours to allow them to adhere and grow. After cell adhesion, the culture medium was removed, and 1 mL of a 5 μM NO-specific fluorescent probe DAF-FM DA solution was added to the culture dish. The cells were pre-incubated at 37°C in the dark. After 6 hours, the cells were washed three times with PBS to remove any DAF-FM DA probes that had not entered the cells. Subsequently, 200 μL of Lipos, DSF / Lipos, DSF / ISDN-Lipos, and DSF / S1P / ISDN-Lipos solutions were mixed with 1.8 mL of culture medium and added to different confocal culture dishes (phospholipid final concentration 1 mg / mL, DSF final concentration 2 μg / mL, Cu...). 2+ The final concentration was 22.7 μg / mL, and the generation of green NO fluorescence in cells was monitored in real time using a fluorescence microscope.

[0096] Experimental results are as follows Figure 5 The results showed that the intracellular NO production level in GL261 cells was detected using the DAF-FM DA fluorescent probe method. The weak green fluorescence signal intensity in GL261 cells co-incubated with Lipos and DSF / Lipos indicated low NO levels. The DSF / ISDN-Lipos treatment group showed a significantly enhanced green fluorescence signal in the cytoplasm of GL261 cells. Furthermore, after further introduction of S1P targeting modification, the intracellular fluorescence intensity of GL261 cells in the DSF / S1P / ISDN-Lipos group was further increased compared to the non-targeted group, indicating that DSF / S1P / ISDN-Lipos can effectively generate a large amount of NO in glioma cells.

[0097] GL261 cells in good growth condition were subjected to a 2×10⁻⁶ 3 Cells were seeded at a density of 1 cell per dish on a glass substrate with a bottom area of ​​9.6 cm². 2 In a confocal microscope culture dish, cells were cultured at 37℃ and 5% CO2 for 24 h to allow them to adhere and grow. After cell adhesion, the culture medium was removed, and 200 μL of Lipos, DSF / Lipos, DSF / ISDN-Lipos, and DSF / S1P / ISDN-Lipos solutions (final phospholipid concentration 1 mg / mL; final DSF concentration 2 μg / mL, Cu) were added respectively. 2+ The final concentration (22.7 μg / mL) was mixed with 1.8 mL of culture medium and then added to different confocal culture dishes. After co-incubation for 24 h, the cells were washed and immunofluorescence staining of GL261 cells with ATP7B antibody was performed using conventional immunofluorescence staining methods.

[0098] Experimental results are as follows Figure 6The results showed that GL261 cells treated with Lipos- and DSF / Lipos exhibited significant ATP7B-specific green fluorescence, indicating a high ATP7B expression level in GL261 cells. In contrast, DSF / ISDNLipos treatment significantly reduced the ATP7B-specific fluorescence intensity, while DSF / S1P / ISDN-Lipos treatment further weakened the ATP7B fluorescence intensity. This result and Figure 5 The results collectively demonstrate that the S1P targeting molecule can significantly enhance the targeting effect of DSF / S1P / ISDN-Lipos on GL261 glioma cells. The NO generated by DSF / S1P / ISDN-Lipos in GL261 cells significantly inhibited the expression level of ATP7B protein, further proving the unique design of DSF / S1P / ISDN-Lipos.

[0099] Example 6

[0100] Evaluation of the effect of DSF / S1P / ISDN-Lipos on inducing copper death in glioma cells

[0101] Following the specific steps of Example 1, Lipos, DSF / Lipos, DSF / ISDN-Lipos, S1P / ISDN-Lipos and DSF / S1P / ISDN-Lipos were prepared respectively.

[0102] GL261 glioma cells (50,000 cells / well) were seeded into 6-well cell culture plates and cultured for 24 hours in a cell culture incubator until cell attachment. After cell attachment, the original culture medium was aspirated and replaced with complete culture medium containing Lipos, DSF / Lipos, DSF / ISDN-Lipos, S1P / ISDN-Lipos, or DSF / S1P / ISDN-Lipos (containing 10% fetal bovine serum, with a final phospholipid concentration of 1 mg / mL; and a final DSF concentration of 2 μg / mL, and containing Cu). 2+ The final concentration was 22.7 μg / mL, and the cells were cultured for another 24 h. Subsequently, the culture medium was aspirated and the cells were washed three times with PBS. The expression levels of FDX-1, LIAS, DLAT, and GAPDH proteins in GL261 cells after different treatments were detected by Western blotting.

[0103] Experimental results are as follows Figure 7The results showed that in GL261 cells treated with DSF / ISDN-Lipos or DSF / S1P / ISDN-Lipos, the expression of iron-sulfur cluster proteins (LIAS, FDX1) was significantly reduced compared to the Lipos and DSF / Lipos treatment groups, and the level of unesterified DLAT protein decreased. This indicates that the nitric oxide-regulated copper ion delivery system promotes the esterification and oligomerization of DLAT protein while inhibiting the biosynthesis of iron-sulfur clusters, ultimately inducing copper death. This confirms that the novel strategy of "in situ capture and transport of endogenous copper while blocking its efflux" can enhance the sensitivity of tumor cells to copper death. This example further demonstrates the importance of adding the NO prodrug ISDN to the DSF / S1P / ISDN-Lipos carrier. NO can more effectively enhance the copper death effect in GL261 cells by inhibiting the efflux of Cu ions by ATP7B. Compared to the DSF / Lipos group, the DSF / ISDN-Lipos group significantly improved the inhibitory effect on copper death-related proteins.

[0104] Example 7

[0105] Evaluation of the in vivo tumor copper death induction effect of DSF / S1P / ISDN-Lipos in glioma-bearing mice

[0106] Male BALB / c nude mice aged 5-6 weeks were weighed and anesthetized intraperitoneally with 10% chloral hydrate at a concentration of 0.1 mL per 10 g of body weight. After complete anesthesia, the mice were transferred to a stereotaxic instrument. The upper anterior teeth were fixed in the notches on the dental arch, a nasal protector was placed above the mouse's nose, and an ear rod was gently placed in the ear canal. The mouse's head was kept level using the scale lines on the stereotaxic instrument. Subsequently, the mouse's head was cleaned and sterilized by wiping it with a 70% alcohol pad.

[0107] An incision was made at the top of the skull behind the right eye of the nude mouse, diagonally back to the left posterolateral side of the skull. The incision was approximately 10-15 mm long. Using a cotton swab, the incision was gently separated to expose the skull. The membrane between the skull and skin was wiped away to expose the anterior fontanelle. Using a 1 mm diameter animal skull drill, the skull was carefully drilled 2 mm to the right and 1 mm anterior to the anterior fontanelle. A 5 μL / mouse suspension of GL261 in PBS was slowly injected into the brain via a microinfusion pump (inserting the needle 3.5 mm into the bone window, withdrawing it slightly by about 0.5 mm before injection, injecting for 10 minutes, stopping for 2 minutes after injection, and then slowly withdrawing the needle). The bone window was sealed with bone wax, the scalp was sutured, and the area was disinfected with iodine. The orthotopic glioma nude mouse model was obtained 14 days after injection.

[0108] Following the specific steps in Example 1, DSF / Lipos, DSF / ISDN-Lipos, and DSF / S1P / ISDN-Lipos were prepared respectively. BALB / c mice with successfully induced orthotopic gliomas were randomly divided into four groups. On days 7, 9, 11, 13, and 15 after modeling, 200 μL of PBS, DSF / Lipos, DSF / ISDN-Lipos, and DSF / S1P / ISDN-Lipos (final DSF concentration 2 μg / mL) were administered via tail vein. After treatment, the mice were euthanized by cervical dislocation, and the brain tumors were removed and fixed overnight at 4°C in 10 times their volume of 4% paraformaldehyde fixative. Immunofluorescence staining for H&E, TUNEL, FDX-1, DLAT, and ATP7B, and erythramine staining for copper ion distribution were then performed.

[0109] Experimental results are as follows Figure 8 As shown, H&E staining results indicated that GL261 glioma tissue treated with DSF / S1P / ISDN-Lipos exhibited extensive apoptotic / necrotic areas, characterized by severe membrane lysis and chromosomal fusion. TUNEL staining results showed that the level of apoptosis in brain tumor cells was increased in the DSF / S1P / ISDN-Lipos treatment group compared with the PBS, DSF / Lipos, and DSF / ISDN-Lipos treatment groups. To verify that DSF / S1P / ISDN-Lipos induced copper death in orthotopic GL261 gliomas, immunofluorescence staining was used to assess the expression of copper death-related proteins (DLAT, FDX1, and ATP7B) in tumor sections. Compared with the PBS, DSF / Lipos, and DSF / ISDN-Lipos treatment groups, the DSF / S1P / ISDN-Lipos group showed enhanced liponylated DLAT protein in tumor tissue, while the expression levels of copper chaperone protein FDX-1 and copper transporter ATP7B were significantly decreased. These molecular characteristics meet the biomarker criteria for copper death. Subsequent histochemical analysis based on a copper-specific chromogenic assay revealed strong reddish-brown copper deposits in the brain tumor tissue of the DSF / S1P / ISDN-Lipos group. Notably, the S1P-targeted modified DSF / S1P / ISDN-Lipos treatment group exhibited a stronger copper death-inducing effect. This synergistic effect was attributed to the combined effect of S1P-mediated enhanced brain-targeted delivery and ISDN-triggered tumor microenvironment acidification, effectively promoting the specific accumulation of copper ions within tumor cells, thereby constructing a potent copper death-inducing microenvironment. This demonstrates that the DSF / S1P / ISDN-Lipos vector, utilizing S1P, achieved effective accumulation of the vector within gliomas. Subsequently, the combination of S1P, ISDN, and DSF successfully induced copper death in an in vivo model.

Claims

1. A smart responsive in-situ copper ion adsorption nanodelivery system, characterized in that, The intelligent responsive in-situ copper ion adsorption nanodelivery system comprises a nanoliposome as its core, wherein the nanoliposome is loaded with a nitric oxide prodrug and its surface is co-modified with a drug molecule that binds to copper ions and an active targeting molecule.

2. The intelligent responsive in-situ copper ion adsorption nanodelivery system according to claim 1, characterized in that, The nanoliposomes are composed of phospholipids, which are any one or more of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, or synthetic phospholipids. Preferably, the phospholipids are DPPC, DSPC, and DSPE-PEG. 2000 .

3. The in-situ intelligent responsive copper ion adsorption nanodelivery system according to claim 1, characterized in that, The nitric oxide (NO) prodrug is any one of JS-K, nitroglycerin, isosorbide dinitrate (ISDN), isosorbide mononitrate (ISMN), molar nitrate, sodium nitroprusside, or L-arginine.

4. The in-situ intelligent responsive copper ion adsorption nanodelivery system according to claim 1, characterized in that, The drug molecule that binds copper ions is either disulfiram (DSF) or Elesclomol; the active targeting molecule is either FTY720 or S1P.

5. A method for preparing an in-situ intelligent responsive copper ion adsorption nanodelivery system according to any one of claims 1-4, characterized in that, Includes the following steps: (1) Preparation of nanoliposomes internally loaded with nitric oxide prodrug; (2) The shell of the nanoliposome loaded with nitric oxide prodrug is modified with active targeting molecules and drug molecules that bind copper ions, which is a smart responsive in-situ copper ion adsorption nanodelivery system.

6. The preparation method according to claim 5, characterized in that, The preparation method of the nanoliposomes internally loaded with gaseous prodrugs in step (1) includes the following steps: (1) Take phospholipid material and add it to an organic solvent to obtain a mixed phospholipid solution; take nitric oxide prodrug and add it to an organic solvent to obtain a gaseous precursor drug solution; mix the mixed phospholipid solution and the gaseous precursor drug solution thoroughly; (2) The mixed solution was subjected to rotary vacuum distillation to obtain a mixed film of phospholipid and gaseous precursor drug; the mixed film was dried under vacuum overnight. (3) Take out the mixed film after vacuum overnight drying, and perform rotational hydration to fully disperse the film and obtain a nanoliposome suspension loaded with gas prodrug. (4) The nanoliposome suspension was repeatedly squeezed through a filter membrane with uniform pore size by extrusion membrane method, and then dialyzed to obtain nanoliposomes loaded with gas prodrugs.

7. The preparation method according to claim 6, characterized in that, In step (2), the solvent in the mixed solution is gradually removed by vacuum distillation under water bath conditions to obtain a mixed film of phospholipid and gaseous prodrug. Then, the mixed film is transferred to a vacuum drying oven and vacuum-treated overnight. In step (3), a neutral buffer solution is added to the rotary evaporator under water bath conditions, and after rotary hydration, a nanoliposome suspension loaded with gaseous prodrug is obtained. In step (4), the repeated squeezing through a filter membrane with uniform pore size requires that the nanoliposome suspension be passed through polycarbonate filter membranes with pore sizes of 800 nm, 400 nm and 220 nm in sequence, for no less than 20 times.

8. The preparation method according to claim 5, characterized in that, Step (2) involves modifying the shell of the nanoliposomes with active targeting molecules and drug molecules that bind copper ions, and includes the following steps: (1) Dissolve the active targeting molecule and the drug molecule that binds copper ions in organic solvents respectively; (2) Mix the nanoliposomes containing gaseous prodrugs, the active targeting molecule solution, and the drug molecule solution containing copper ions evenly and stir to react; (3) After dialysis, nanoliposomes containing gaseous prodrugs loaded inside a shell modified with active targeting molecules and drug molecules that bind copper ions were obtained.

9. The preparation method according to claim 8, characterized in that, In step (1), the active targeting molecule and the copper-binding drug molecule are dissolved in DMSO or chloroform, respectively. In step (2), the nanoliposomes loaded with gaseous prodrug, the active targeting molecule solution, and the copper-binding drug molecule solution are added to a neutral buffer solution and mixed evenly, and stirred to react. The neutral buffer solution is PBS buffer or NaCl buffer with a pH of 6.5 to 7.

5.

10. The application of the intelligent responsive in-situ copper ion adsorption nanodelivery system according to claim 1 in the preparation of tumor therapeutic drugs.

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