Multifunctional double-drug delivery system based on metal collaborative treatment and application of multifunctional double-drug delivery system

Abstract

The invention discloses a multifunctional double-drug delivery system based on metal collaborative treatment and application of the multifunctional double-drug delivery system, and belongs to the technical field of biological medicine. The double-drug delivery system is double-drug delivery lipidosome and comprises metal ions, a lipidosome membrane and active ingredients, the metal ions are adsorbed on the surface of the lipidosome membrane, and the active ingredients are wrapped in the lipidosome membrane; the metal ions are divalent metal ions; the active component consists of an STING agonist and a traditional Chinese medicine active monomer component. According to the double-drug delivery system, the STING agonist and the traditional Chinese medicine anti-inflammatory components are co-loaded through lipidosome, and multi-dimensional remodeling of a tumor microenvironment is achieved through the double effects of exciting tumor immunity and inhibiting inflammatory reaction; and meanwhile, by introducing metal ions, the lysosome escape efficiency of a liposome drug delivery system is effectively enhanced, so that the intracellular delivery efficiency of the drug is improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a multifunctional dual-drug delivery system based on metal synergistic therapy and application thereof. BACKGROUND

[0002] Osteosarcoma is a malignant tumor originating from bone tissue, which often occurs in adolescents and young adults. The current clinical standard treatment regimen includes preoperative chemotherapy, surgical resection, and postoperative adjuvant chemotherapy. Although surgical treatment can effectively remove tumor tissue, it is difficult to achieve complete resection, and the resection margin is prone to residual tumor cells, while postoperative resection is often accompanied by complications such as traumatic inflammation, hypoxia, and infection. Traditional postoperative treatment methods have obvious limitations in simultaneously addressing anti-tumor therapy and postoperative complications.

[0003] In the prior art, drug delivery systems have become a research hotspot for postoperative treatment of osteosarcoma. Patent CN120381455A discloses a drug composition combining neomycin and STING agonists, which verifies that neomycin and STING agonists (one or more of diABZI, cGAMP, CMA) have synergistic therapeutic effects through various experimental methods, and achieves the delivery of STING agonists such as cGAMP; by inhibiting the interference of lipopolysaccharide (LPS) on the STING signaling pathway, the anti-tumor immune response is enhanced. However, this delivery system lacks lysosome escape ability, resulting in a large amount of degradation of STING agonists in lysosomes and low bioavailability.

[0004] On the other hand, traditional Chinese medicine active ingredients exhibit unique advantages in bone inflammatory diseases. According to the Nano Letters (2022, 20, 7728-7736), emodin has great application value in bone-related inflammatory diseases. The report achieves the targeted accumulation and release of drug active ingredients at the inflammatory site of mice by delivering emodin with nanomicelles. The study provides a treatment strategy for rheumatoid arthritis and verifies the good anti-inflammatory application value of emodin. However, this conventional drug delivery system also lacks lysosome escape function, resulting in low intracellular drug delivery efficiency.

[0005] The prior art shows that single-drug therapy is difficult to achieve multiple goals of immune activation and tumor microenvironment regulation. There are significant differences in physicochemical properties between STING agonists (hydrophilic) and traditional Chinese medicine active ingredients (mostly hydrophobic), which pose great challenges to co-encapsulation and synergistic release. In addition, traditional liposomes lack specific targeting ability to tumor cells and tumor-associated macrophages, as well as the ability to escape endosome-lysosome clearance, which further limits the therapeutic effect of existing lipid delivery systems.

[0006] At present, there is no liposome system reported that can realize lysosome escape, double-drug delivery and specific targeting at the same time. Especially in the field of postoperative treatment of osteosarcoma, how to design an intelligent liposome system that can overcome the lysosome barrier and realize the synergistic delivery of STING agonists and active ingredients of traditional Chinese medicine is still a technical problem to be solved in the field. SUMMARY

[0007] One of the purposes of the present application is to provide a double-drug delivery liposome, comprising a metal ion, a liposome membrane and an active ingredient, wherein the metal ion is adsorbed on the surface of the liposome membrane, and the active ingredient is loaded in the liposome membrane. The metal ion is a divalent metal ion. The active ingredient is composed of a STING agonist and a traditional Chinese medicine active monomer component.

[0008] Further, the divalent metal ion is selected from Zn 2+ , Mn 2+ , Mg 2+ , Fe 2+ , and Ca 2+ .

[0009] Further, the STING agonist is a cyclic dinucleotide or a cyclic dinucleotide analogue, selected from ADU-S100, cGAMP, c-di-GMP, c-di-AMP, 2',3'-cGAMP; and the traditional Chinese medicine active monomer component is celastrol, curcumin, baicalein, cryptotanshinone, honokiol, oleuropein, lycepersicin, and diosgenin.

[0010] Further, the liposome membrane is made of lecithin and cholesterol.

[0011] Further, the mass ratio of the cholesterol and lecithin is 1:5-1:20; the mass ratio of the traditional Chinese medicine active monomer component and lecithin is 1:10-1:20; the molar ratio of the STING agonist and traditional Chinese medicine active monomer component is 1:2; and the molar ratio of the metal ion and lecithin is 1:1-4:1.

[0012] In a specific embodiment of the present application, the mass ratio of the cholesterol and lecithin is 1:10, the mass ratio of celastrol and soy lecithin is 1:15, the molar ratio of ADU-S100 and celastrol (Celastrol) is 1:2, and the molar ratio of the metal ion compound and lecithin is 2:1.

[0013] The second purpose of the present application is to provide a preparation method of the above-mentioned double-drug delivery liposome, comprising the following steps: Step 1, dissolving lecithin, cholesterol and traditional Chinese medicine active monomer component in an organic solvent, and rotary evaporation to obtain a film; Step 2, dissolving the film in an aqueous solution to obtain a double-drug delivery liposome. Step 2, adding an aqueous solution of STING agonist to the film obtained in step 1, hydrating and ultrasonicating to obtain a liposome solution; Step 3, adding an aqueous solution containing metal ions to the liposome solution of step 2, hydrating and adsorbing to obtain the dual-drug delivery liposome.

[0014] A third object of the present application is to provide the use of the above-mentioned dual-drug delivery liposome in the preparation of a tumor treatment drug. Preferably, the tumor is osteosarcoma.

[0015] The present application constructs a synergistic treatment system of "immune activation-inflammation regulation", and realizes the multi-dimensional remodeling of tumor microenvironment by co-loading STING agonist and traditional Chinese medicine anti-inflammatory ingredients in liposomes. This composite delivery system forms a delicate treatment balance at the molecular level: STING agonist activates type I interferon response through the cGAS-STING pathway, promotes CD8 + T cell infiltration; at the same time, traditional Chinese medicine ingredients selectively inhibit the pro-inflammatory pathway of NF-κB, and polarize tumor-associated macrophages from M2 type to M1 type.

[0016] The delivery system of the present application co-loads STING agonist and traditional Chinese medicine anti-inflammatory ingredients in liposomes, and realizes the multi-dimensional remodeling of tumor microenvironment through the dual effects of stimulating tumor immunity and inhibiting inflammatory response. In addition, the system effectively enhances the lysosome escape efficiency of the liposome drug delivery system by introducing metal ions, thereby improving the intracellular delivery efficiency of the drug. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 The particle size of L@CA prepared for different ratios of cholesterol to bile acid (1:5, 1:10, 1:15 and 1:20).

[0018] Figure 2 Encapsulation efficiency (EE%) and (B) drug loading (LD%) of Celastrol (A-B) and ADU-S100 (C-D) in L@CA prepared for different ratios of cholesterol to bile acid.

[0019] Figure 3 Encapsulation efficiency (EE%) and (B) drug loading (LD%) of Celastrol (A-B) and ADU-S100 (C-D) in L@CA prepared for different ratios of cholesterol to bile acid.

[0020] Figure 4 Surface charge changes of different metal ions coordinated with L@CA at different ratios.

[0021] Figure 5 Particle size changes of different metal ions coordinated with L@CA at a ratio of 2:1.

[0022] Figure 6 The effect of different metal ions adsorption on the drug loading and encapsulation efficiency of L@CA nano-preparation.

[0023] Figure 7 The results of lysosome co-localization of double-drug lipid delivery system after adsorption of different metal ions.

[0024] Figure 8 The schematic diagram of flow cytometry results of cell uptake of double-drug lipid delivery system after adsorption of different metal ions.

[0025] Figure 9 The schematic diagram of confocal results of cell uptake of double-drug lipid delivery system after adsorption of different metal ions.

[0026] Figure 10 The schematic diagram of apoptosis induction results of double-drug lipid delivery system after adsorption of different metal ions.

[0027] Figure 11 The schematic diagram of apoptosis proportion statistics results of double-drug lipid delivery system after adsorption of different metal ions.

[0028] Figure 12 The observation results of the effect of double-drug lipid delivery system after adsorption of different metal ions on cell viability.

[0029] Figure 13 The results of in vivo drug efficacy of double-drug lipid delivery system after adsorption of different metal ions.

[0030] Figure 14 The tumor growth curve of in vivo drug efficacy of double-drug lipid delivery system after adsorption of different metal ions.

[0031] Figure 15 The statistics results of in vivo drug efficacy of double-drug lipid delivery system after adsorption of different metal ions.

[0032] Figure 16 The statistics results of in vivo drug efficacy of double-drug lipid delivery system after adsorption of different metal ions. DETAILED DESCRIPTION

[0033] Liposomes have unique advantages in the field of drug delivery, not only because of their excellent biocompatibility, but also because of their ability to achieve tumor tissue-specific delivery through PEG modification or coupling with targeting ligands. This characteristic has enabled liposomes to be widely used in drug delivery, gene therapy, and transdermal drug delivery. Liposome drug delivery systems not only improve the therapeutic effect of drugs, but also significantly reduce systemic toxicity. However, the endosome-lysosome entrapment problem faced by traditional liposomes severely restricts their drug delivery efficiency. After entering cells, liposomes are often trapped in the endosome-lysosome pathway, where they are degraded in the acidic environment and lysosomal enzymes, leading to premature release and inactivation of the drug.

[0034] To address this key challenge, metal ion modification strategies have shown unique value. By integrating metal ions such as Ca²⁺ and Mn²⁺ or metal compounds onto the surface of liposomes, their lysosome escape ability can be significantly enhanced. This process involves multiple synergistic mechanisms: in the acidic environment of the lysosome, metal ions act as efficient proton buffers, greatly consuming H + , causing a large influx of Cl - and water, leading to an increase in osmotic pressure and rupture of the lysosome; at the same time, the specific binding of divalent metal ions to phospholipid phosphate groups can change the membrane fluidity, promoting the fusion or local destruction of the liposome and lysosome membranes; in addition, the dissolution of certain metal compounds (such as ZnO) can locally buffer the acidic environment and inhibit lysosomal enzyme activity, providing protection for the integrity of the drug. These mechanisms work together not only to solve the lysosomal degradation problem of traditional liposomes, but also to endow the system with multiple response characteristics such as pH / ROS / magnetic field, enabling more precise drug control release.

[0035] STING agonists are emerging as new target drugs for tumor immunotherapy, but their clinical application faces three key challenges: poor drug stability, insufficient targeting, and significant systemic toxicity. Enamin is an anti-inflammatory active ingredient with broad application prospects, but it has low bioavailability, significant side effects, and insufficient targeting.

[0036] To address these bottlenecks, the present invention innovatively designs a multifunctional liposome delivery system that achieves efficient delivery of STING agonists and traditional Chinese medicine active ingredients through nanotechnology, providing a new strategy for the postoperative treatment of osteosarcoma. This delivery system co-delivers STING agonists and traditional Chinese medicine anti-inflammatory ingredients through liposomes, achieving multi-dimensional remodeling of the tumor microenvironment through the dual effects of stimulating tumor immunity and suppressing inflammatory response. In addition, the system effectively enhances the lysosome escape efficiency of the liposome drug delivery system by introducing metal ions, thereby improving the intracellular delivery efficiency of the drug.

[0037] The delivery system has three core advantages: first, its phospholipid bilayer structure can effectively protect the STING agonist from nuclease degradation, thereby significantly prolonging the drug half-life; second, selective accumulation at the tumor site is achieved through the EPR effect and active targeting modification, which significantly improves the drug concentration at the targeted site; more importantly, the specially designed cationic liposome can promote endosome escape by "proton sponge effect", significantly improve the intracellular delivery efficiency of the drug, and improve the activation efficiency of the STING pathway in dendritic cells, while reducing the systemic inflammatory response.

[0038] The preferred embodiments of the present application will be described in detail below with reference to the examples. It should be understood that the following examples are given only for the purpose of illustration and are not intended to limit the scope of the present application. Those skilled in the art can make various modifications and substitutions to the present application without departing from the spirit and principles of the present application.

[0039] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0040] The materials, reagents, etc. used in the following examples can be obtained commercially unless otherwise specified.

[0041] The lecithin used in the following examples is all soybean lecithin. Example 1

[0042] 1. Investigation of the cholesterol-phospholipid ratio during preparation The cholesterol-phospholipid ratio (mass ratio) was used as a single-factor analysis variable, and the drug-phospholipid ratio (mass ratio) = 1:15 and the triptolide / ADU-S100 (molar ratio) = 2:1 were used as single-factor analysis invariants. The double-drug-loaded lipid delivery system was prepared according to the cholesterol-phospholipid mass ratio of 1:5, 1:10, 1:15 and 1:20.

[0043] Under the action of ultrasonic waves, cholesterol (36 mg, 18 mg, 12 mg and 9 mg), lecithin (180 mg) and triptolide (12 mg) were completely dissolved in 18 mL chloroform in a round-bottom flask. Then the solution was subjected to rotary evaporation at 40°C (rotation speed 60 / vacuum degree controlled between 0.06~0.07). A uniform film was formed. Subsequently, it was placed in a vacuum drying box overnight, 24 mL of deionized water containing 9.36 mg of ADU-S100 was added to the round-bottom flask, and the hydration was stirred at 50°C for 2 hours. After hydration, the solution was treated with an ultrasonic probe at 100W for 3min, and then filtered with 0.80μm, 0.45μm and 0.22μm microporous membranes in turn, to obtain the nano-preparation L@CA with different cholesterol-phospholipid ratios.

[0044] The sample L@CA solution to be tested with a concentration of 1.5 mg / mL was placed in a 1 cm particle size cuvette and a Malvern potential cell, respectively. The particle size, PDI and Zeta potential were determined by using BeNano 90 nanoparticle size analyzer (Better BT), and the determination was carried out in triplicate.

[0045] Table 1 L@CA particle size and potential statistics table prepared by different cholesterin ratios (x ± SD, n = 3)

[0046] As shown in Table 1 and Figure 1 It can be seen that when the cholesterin mass ratio is taken as a single factor analysis variable, the particle size is 77.14 ± 1.01 nm, the Zeta potential value is -26.04 ± 1.60 Mv, and the PDI value of L@CA is obviously large (0.397 ± 0.014), indicating that the content of cholesterin is relatively high, which affects the dispersity and stability of L@CA, resulting in a certain degree of aggregation (as shown in Figure 1 When the cholesterin mass ratio is 1:10, the particle size of the prepared L@CA is 93.97 ± 2.10 nm, the Zeta potential value is -33.87 ± 0.10 Mv, and the PDI value of L@CA is 0.283 ± 0.008, indicating that the L@CA nano-preparation prepared under this condition is uniform in size and more uniform in dispersion, and has good dispersity and stability (as shown in Figure 1 When the cholesterin mass ratio is 1:15 and 1:20, the particle size of L@CA increases significantly, which is 173.93 ± 2.01 nm and 191.51 ± 3.57 nm, respectively, and the PDI value of L@CA is 0.323 ± 0.021 and 0.336 ± 0.018, respectively. The particle size of the nano-preparation prepared under this condition is relatively large. Therefore, the cholesterin mass ratio of 1:10 is selected as the best ratio for preparing L@CA nano-preparation for subsequent investigation.

[0047] 2. Investigation of the drug loading (DL) and encapsulation efficiency (EE) of celastrol in L@CA Standard stock solution: 20 mg of celastrol was accurately weighed into a 20 mL volumetric flask, and an appropriate amount of methanol was added to ultrasonically dissolve it completely. The volume was adjusted to the mark with methanol, and the mixture was shaken to obtain a control stock solution with a concentration of 1000 μg / mL.

[0048] Standard sample solution: Respectively, take the linear stock solution into a suitable volumetric flask to get 1, 10, 100, 200, 300, 500 μg / mL of linear sample solution, pass through 0.22 μm organic microporous filter membrane, inject 10 μL, perform HPLC analysis according to the above chromatographic conditions, and record the peak area. Linear regression is performed on the peak area A of celastrol and the concentration C (μg / mL) to draw a standard curve. The standard curve is drawn with the concentration (μg / mL) as the horizontal coordinate and the absorbance value (F) as the vertical coordinate, and linear regression is performed.

[0049] Test sample solution: In order to calculate the drug loading (DL) and encapsulation efficiency (EE) of L@CA, free celastrol is separated by ultrafiltration. Briefly, the solution of L@CA is centrifuged in an ultrafiltration tube (1-kDa cutoff) at 12000 rpm for 30 minutes. Pass through 0.22 μm organic microporous filter membrane, inject 10 μL, perform HPLC analysis according to the above chromatographic conditions, and record the peak area A. According to the standard curve formula, the content of free celastrol in the filtrate is calculated. And according to the following formula, the drug loading (LD) and encapsulation efficiency (EE) are calculated.

[0050]

[0051]

[0052] wherein W loaded Cel , W total Cel and W nanoparticles represent the mass of loaded Cel, total Cel and nano-preparation, respectively.

[0053] In order to prepare L@CA nano-preparation with high drug loading and encapsulation efficiency, the effects of different proportions of cholesterol ratio (w / w) and drug-lipid ratio (w / w) on the drug loading and encapsulation efficiency of celastrol were studied by single factor analysis.

[0054] According to the experimental results Figure 2As shown in Table A and B, when the cholesterol-phospholipid ratio (w / w) was taken as a single factor analysis variable, the drug loading and encapsulation efficiency of L@CA were 75.67% ± 0.93 and 4.03% ± 0.93, respectively, when the cholesterol-phospholipid ratio was 1:5. As the proportion of cholesterol decreased, the drug loading and encapsulation efficiency of L@CA were 94.67% ± 0.78 and 4.97% ± 0.02, respectively, when the cholesterol-phospholipid ratio was 1:10, indicating that the drug loading and encapsulation efficiency were higher at this ratio. However, when the cholesterol-phospholipid ratio was reduced to 1:15 and 1:20, the drug loading and encapsulation efficiency of L@CA decreased significantly, which may be due to the decrease in the content of cholesterol, leading to drug leakage and reduced stability of phospholipid oxidation in L@CA liposomes. Therefore, the drug loading and encapsulation efficiency results of L@CA further demonstrated that the cholesterol-phospholipid ratio of 1:10 can be used as the optimal ratio for preparing L@CA nanofomulations, and can be used for subsequent investigations.

[0055] According to the experimental results Figure 3 As shown in Table A and B, when the cholesterol-phospholipid ratio (w / w) was taken as a single factor analysis variable, the drug loading and encapsulation efficiency of L@CA were 75.67% ± 0.93 and 4.03% ± 0.93, respectively, when the cholesterol-phospholipid ratio was 1:5. As the proportion of cholesterol decreased, the drug loading and encapsulation efficiency of L@CA were 94.67% ± 0.78 and 4.97% ± 0.02, respectively, when the cholesterol-phospholipid ratio was 1:10, indicating that the drug loading and encapsulation efficiency were higher at this ratio. However, when the cholesterol-phospholipid ratio was reduced to 1:15 and 1:20, the drug loading and encapsulation efficiency of L@CA decreased significantly, which may be due to the decrease in the content of cholesterol, leading to drug leakage and reduced stability of phospholipid oxidation in L@CA liposomes. Therefore, the drug loading and encapsulation efficiency results of L@CA further demonstrated that the cholesterol-phospholipid ratio of 1:10 can be used as the optimal ratio for preparing L@CA nanofomulations, and can be used for subsequent investigations.

[0056] 3. Investigation of ADU-S100 drug loading (DL) and encapsulation efficiency (EE) in L@CA Standard stock solution: 10 mg of ADU-S100 was accurately weighed into a 10 mL volumetric flask, and an appropriate amount of DMSO was added to completely dissolve it. The volume was adjusted to the mark with DMSO, and the mixture was shaken to obtain a control stock solution with a concentration of 1000 μg / mL.

[0057] Standard sample solution: The linear stock solution was diluted to 1, 10, 50, 100, 200, 500 μg / mL in a suitable volumetric flask, and the absorbance was recorded using a MμLtiskan FC microplate reader (Thermo Scientific) with an excitation wavelength of 345 nm and an emission wavelength of 419 nm. The standard curve was plotted by linear regression of the absorbance (A) of ADU-S100 versus the concentration (μg / mL).

[0058] Test sample solution: To calculate the drug loading (DL) and encapsulation efficiency (EE) of ADU-S100, free ADU-S100 was separated by ultrafiltration. Briefly, the solution of L@CA was directly centrifuged in an ultrafiltration tube (1-kDa cutoff) at 12000 rpm for 30 min. At a specific time point, 1 mL of solution was collected, and then 1 mL of PBS was added to the solution. The absorbance was recorded using a MμLtiskan FC microplate reader (Thermo Scientific) with an excitation wavelength of 345 nm and an emission wavelength of 419 nm. The content of free ADU-S100 in the filtrate was calculated according to the standard curve formula. The drug loading (LD) and encapsulation efficiency (EE) were calculated according to the following formula.

[0059]

[0060]

[0061] where W loaded ADU-S100 , W total ADU and W nanoparticles represent the mass of ADU-S100 loaded, ADU-S100 input, and nanoformulation, respectively.

[0062] To prepare L@CA nanoformulation with high drug loading and encapsulation efficiency, the effect of different ratios of cholesterol to lipid (w / w) and drug to lipid (w / w) on the drug loading and encapsulation efficiency of ADU-S100 was studied by single-factor analysis.

[0063] According to the experimental results Figure 2From Tables C and D, when the cholesterol-phospholipid ratio (w / w) was used as a single factor analysis variable, the entrapment efficiency and drug loading of ADU-S100 in L@CA were 63.70 ± 3.96 and 2.94 ± 0.18, respectively, when the cholesterol-phospholipid ratio was 1:5. As the proportion of cholesterol decreased, the drug loading and entrapment efficiency of L@CA were 84.04 ± 3.23 and 3.85 ± 0.14, respectively, when the cholesterol-phospholipid ratio was 1:10, indicating that the drug loading and entrapment efficiency were higher at this ratio. However, when the cholesterol-phospholipid ratio was reduced to 1:15 and 1:20, the drug loading and entrapment efficiency of ADU-S100 in L@CA decreased significantly, which may be due to the decrease in cholesterol content, leading to drug leakage and decreased stability of phospholipid oxidation in L@CA liposomes. This result is consistent with the entrapment efficiency and drug loading of celastrol. When the cholesterol-phospholipid ratio was 1:10, the entrapment efficiency and drug loading of celastrol and ADU-S100 were the highest. Therefore, the drug loading and entrapment efficiency of ADU-S100 further demonstrate that the cholesterol-phospholipid ratio of 1:10 is the optimal ratio for preparing L@CA nanofomulations, and can be used for subsequent investigations.

[0064] According to the experimental results Figure 3 From Tables C and D, when the cholesterol-phospholipid ratio (w / w) was used as a single factor analysis variable, the entrapment efficiency and drug loading of ADU-S100 in L@CA were 63.70 ± 3.96 and 2.94 ± 0.18, respectively, when the cholesterol-phospholipid ratio was 1:5. As the proportion of cholesterol decreased, the drug loading and entrapment efficiency of L@CA were 84.04 ± 3.23 and 3.85 ± 0.14, respectively, when the cholesterol-phospholipid ratio was 1:10, indicating that the drug loading and entrapment efficiency were higher at this ratio. However, when the cholesterol-phospholipid ratio was reduced to 1:15 and 1:20, the drug loading and entrapment efficiency of ADU-S100 in L@CA decreased significantly, which may be due to the decrease in cholesterol content, leading to drug leakage and decreased stability of phospholipid oxidation in L@CA liposomes. This result is consistent with the entrapment efficiency and drug loading of celastrol. When the cholesterol-phospholipid ratio was 1:10, the entrapment efficiency and drug loading of celastrol and ADU-S100 were the highest. Therefore, the drug loading and entrapment efficiency of ADU-S100 further demonstrate that the cholesterol-phospholipid ratio of 1:10 is the optimal ratio for preparing L@CA nanofomulations, and can be used for subsequent investigations. Examples 2-6

[0065] 1、Zn 2+ Preparation of adsorbed lipid delivery systems Soy lecithin (180 mg), cholesterol (18 mg) and tripterine (12 mg) were completely dissolved in 18 mL of chloroform under the action of ultrasonic waves. Then the solution was subjected to rotary evaporation under reduced pressure at 40°C until a uniform film was formed. Subsequently, the film obtained by rotary evaporation under reduced pressure was placed in a vacuum drying oven and dried overnight. After drying, 24 mL of deionized water containing 9.36 mg of ADU-S100 was added to a round-bottom flask, and the hydration was stirred at 50°C for 1 hour. After hydration, the ultrasonic probe was used to cycle the ultrasonic waves at a power of 100 W for 3 min.

[0066] The corresponding ZnCl2·6H2O and L@CA nano-preparation were complexed and self-assembled according to the conditions of metal compound / phospholipid molar ratio 0:1, 0.1:1, 0.2:1, 0.5:1, 1:1, 2:1, 4:1. The test process is as follows: respectively, ZnCl2·6H2O was accurately weighed into 10 mL of deionized water and dissolved to obtain a ZnCl2·6H2O solution, the metal ion solution and the above obtained L@CA solution were sequentially added to a vial containing PB (sodium phosphate buffer (Na2HPO4) (2 mL, 10 mM)), and incubated at room temperature for 2 hours by magnetic stirrer. Subsequently, Zn-L@CA was obtained by membrane filtration.

[0067] The sample Zn-L@CA solution to be tested with a concentration of 1.5 mg / mL was respectively placed in a 1 cm particle size cuvette and a Malvern potential cuvette. The particle size, PDI and Zeta potential were measured by using BeNano 90 nanoparticle size analyzer (Better BT), and the determination was carried out in parallel for 3 times.

[0068] 2、Mn 2+ Preparation of adsorbed lipid delivery system Soy lecithin (180 mg), cholesterol (18 mg) and tripterine (12 mg) were completely dissolved in 18 mL of chloroform under the action of ultrasonic waves. Then the solution was subjected to rotary evaporation under reduced pressure at 40°C until a uniform film was formed. Subsequently, the film obtained by rotary evaporation under reduced pressure was placed in a vacuum drying oven and dried overnight. After drying, 24 mL of deionized water containing 9.36 mg of ADU-S100 was added to a round-bottom flask, and the hydration was stirred at 50°C for 1 hour. After hydration, the ultrasonic probe was used to cycle the ultrasonic waves at a power of 100 W for 3 min.

[0069] MnCl2·4H2O and L@CA nano-preparation were complexed and self-assembled according to the metal ion / phospholipid molar ratio of 0:1, 0.1:1, 0.2:1, 0.5:1, 1:1, 2:1, and 4:1. The test process was as follows: MnCl2·4H2O was precisely weighed into 10 mL deionized water and dissolved to obtain a MnCl2·4H2O solution. The metal ion solution and the obtained L@CA solution were sequentially added to a vial containing PB (sodium phosphate buffer (Na2HPO4) (2 mL, 10 mM)) and incubated at room temperature for 2 hours by a magnetic stirrer. Subsequently, the membrane was obtained to obtain Mn-L@CA.

[0070] The sample solution to be tested, Mn-L@CA, with a concentration of 1.5 mg / mL, was placed in a 1 cm particle size cuvette and a Malvern potential cuvette. The particle size, PDI, and Zeta potential were measured by BeNano 90 nanoparticle size analyzer (Better BT), and three parallel measurements were performed.

[0071] 3, Mg 2+ Preparation of adsorbed lipid delivery system Under the action of ultrasonic waves, soybean lecithin (180 mg), cholesterol (18 mg), and tripterine (12 mg) were completely dissolved in 18 mL of chloroform. Then the solution was subjected to rotary evaporation under reduced pressure at 40°C until a uniform film was formed. Subsequently, the film obtained by rotary evaporation under reduced pressure was placed in a vacuum drying box and dried overnight. After drying, 24 mL of deionized water containing 9.36 mg of ADU-S100 was added to a round-bottom flask, and the hydration was performed at 50°C for 1 hour. After hydration, the ultrasonic probe was used to cycle ultrasonic waves at a power of 100 W for 3 min.

[0072] MgCl2·6H2O and L@CA nano-preparation were complexed and self-assembled according to the metal ion / phospholipid molar ratio of 0:1, 0.1:1, 0.2:1, 0.5:1, 1:1, 2:1, and 4:1. The test process was as follows: MgCl2·6H2O was precisely weighed into 10 mL deionized water and dissolved to obtain a MgCl2·6H2O solution. The metal ion solution and the obtained L@CA solution were sequentially added to a vial containing PB (sodium phosphate buffer (Na2HPO4) (2 mL, 10 mM)) and incubated at room temperature for 2 hours by a magnetic stirrer. Subsequently, the membrane was obtained to obtain Mg-L@CA.

[0073] The sample solution to be tested, Mg-L@CA, with a concentration of 1.5 mg / mL, was placed in a 1 cm particle size cuvette and a Malvern potential cuvette. The particle size, PDI, and Zeta potential were measured by BeNano 90 nanoparticle size analyzer (Better BT), and three parallel measurements were performed.

[0074] 4、Fe 2+ Preparation of adsorptive lipid delivery system Under the action of ultrasonic waves, soybean lecithin (180 mg), cholesterol (18 mg), and tripterine (12 mg) were completely dissolved in 18 mL of chloroform. Then, the solution was subjected to rotary evaporation under reduced pressure at 40°C until a uniform film was formed. Subsequently, the film obtained by rotary evaporation under reduced pressure was placed in a vacuum drying box and dried overnight. After drying was completed, 24 mL of deionized water containing 9.36 mg of ADU-S100 was added to a round-bottom flask, and the hydration was stirred at 50°C for 1 hour. After hydration, the ultrasonic probe was used to cyclically ultrasonic at a power of 100 W for 3 min.

[0075] FeCl2·4H2O and L@CA nano-preparation were complexed and self-assembled according to the metal ion / phospholipid molar ratio of 0:1, 0.1:1, 0.2:1, 0.5:1, 1:1, 2:1, and 4:1. The test process was as follows: FeCl2·4H2O was precisely weighed, added to 10 mL of deionized water, and dissolved to obtain a FeCl2·4H2O solution. The metal ion solution and the L@CA solution obtained above were sequentially added to a vial containing PB (sodium phosphate buffer (Na2HPO4)) (2 mL, 10 mM), and incubated at room temperature for 2 hours by a magnetic stirrer. Subsequently, Fe-L@CA was obtained by membrane filtration.

[0076] The sample Fe-L@CA solution to be tested with a concentration of 1.5 mg / mL was respectively placed in a 1 cm particle size cuvette and a Malvern potential cuvette. The BeNano 90 nanoparticle size analyzer (Better BT) was used to determine the particle size, PDI, and Zeta potential, and the determination was performed in parallel for 3 times.

[0077] 5、Ca 2+ Preparation of adsorptive lipid delivery system Under the action of ultrasonic waves, soybean lecithin (180 mg), cholesterol (18 mg), and tripterine (12 mg) were completely dissolved in 18 mL of chloroform. Then, the solution was subjected to rotary evaporation under reduced pressure at 40°C until a uniform film was formed. Subsequently, the film obtained by rotary evaporation under reduced pressure was placed in a vacuum drying box and dried overnight. After drying was completed, 24 mL of deionized water containing 9.36 mg of ADU-S100 was added to a round-bottom flask, and the hydration was stirred at 50°C for 1 hour. After hydration, the ultrasonic probe was used to cyclically ultrasonic at a power of 100 W for 3 min.

[0078] The corresponding CaCl2 and L@CA nano-preparation coordination self-assembly was prepared according to the metal ion / phospholipid molar ratio of 0:1, 0.1:1, 0.2:1, 0.5:1, 1:1, 2:1, and 4:1. The test process is as follows: precisely weigh CaCl2, add it to 10 mL deionized water and dissolve to obtain a CaCl2 solution, then sequentially add the metal ion solution and the obtained L@CA solution to a vial containing PB (sodium phosphate buffer (Na2HPO4) (2 mL, 10 mM)) and incubate at room temperature for 2 hours by magnetic stirrer. Then filter to obtain Ca-L@CA.

[0079] The sample Ca-L@CA solution to be tested with a concentration of 1.5 mg / mL was placed in a 1 cm particle size cuvette and a Malvern potential cuvette. The particle size, PDI and Zeta potential were measured by BeNano 90 nanoparticle size analyzer (Better BT), and the determination was carried out in triplicate.

[0080] Table 2 Zeta potential value statistics table of different metal ions and L@CA at different ratios (x ± SD, n = 3)

[0081] As shown in Table 2 and Figure 4 It can be seen that when the metal ion-phospholipid ratio is 0:1 (i.e. without adding any metal ion), the Zeta potential value of L@CA is -34.85 ± 1.61, which indicates that the surface of L@CA itself carries a negative charge and can coordinate with metal ions through electrostatic adsorption. Compared with the group of metal ion-phospholipid ratio = 0:1, as the metal ion-phospholipid ratio gradually increases, the surface charge of L@CA self-assembled with different metal ions changes to different degrees, and even shows a positively charged surface, which indicates that several metal ions have different degrees of coordination reaction with L@CA.

[0082] Among them, the surface charge of Mn-L@CA group changes most significantly, when the Mn-phospholipid ratio is 2:1, the Zeta potential value changes from -34.85 ± 1.61 to 34.43 ± 2.36, indicating that the introduction of Mn 2+ increases the surface charge of L@CA (as shown in Tab.4-1). But when the Mn-phospholipid ratio is 4:1, the surface charge of Mn-L@CA does not change significantly (the Zeta potential value is 34.05 ± 0.73), which may be that the phospholipid coordination phosphoric acid group on the surface of L@CA is saturated by Mn 2+Completely occupied; at the same time, the surface charge of Fe-L@CA group and Zn-L@CA group changed significantly, when the metal-phospholipid ratio was 4:1, the Zeta potential value of Fe-L@CA group was 22.53±1.85, and the Zeta potential value of Zn-L@CA group was 16.48±1.87; in addition, compared with Mn-L@CA group, the surface charge change effect of Mg-L@CA group and Ca-L@CA group was not obvious, when the metal-phospholipid ratio was 4:1, the Zeta potential value of Mg-L@CA group and Ca-L@CA group was only 0.44±0.32 and 3.12±0.32. This result shows that MnCl2 has excellent self-assembly coordination with L@CA, and makes the surface charge of L@CA change to positive charge. When the Mn-phospholipid ratio is 2:1, the Zeta potential value is 34.43±2.36, and Mn-L@CA group shows higher positive charge and better stability compared with other groups.

[0083] Zeta potential analysis results also show that, compared with the metal-phospholipid ratio of 2:1, when the metal-phospholipid ratio is increased to 4:1, the surface charge of M-L@CA does not change significantly, so the metal-phospholipid ratio of 2:1 is selected as the best ratio for preparing M-L@CA nano-preparation for subsequent investigation.

[0084] Table 3 Particle size statistics of different metal ions coordinated with L@CA at a ratio of 2:1 (x ± SD, n = 3)

[0085] As shown in Figure 5 , when the metal ion-phospholipid ratio is 2:1 (i.e. without adding any metal ion), the particle size of L@CA is 93.97±2.10, and the PDI value is 0.283±0.01, indicating that the particle size of L@CA nano-preparation before metal coordination is uniform and has good dispersibility and stability; after self-assembly of different metal ions with L@CA, the particle sizes of Zn 2+ , Mn 2+ , Mg 2 + , Fe 2+ and Ca 2+ adsorbed L@CA nano-preparation are 118.46±1.21, 121.83±2.29, 109.83±1.75, 125.24±5.83 and 122.67±2.16, respectively (as shown in Table 3).

[0086] The above results show that L@CA nano-preparations after coordination with different metals all increase to different degrees, further proving that metal ions are anchored on the surface of L@CA nano-preparation through self-assembly coordination. Example 7

[0087] Drug loading determination of metal ion adsorbed lipid delivery system In order to investigate the influence of different metal ions on the drug loading and encapsulation efficiency of L@CA nanofomulation, the drug loading and encapsulation efficiency of L@CA prepared by different metal ions were determined by the above analysis method. The mass ratio of cholesterol to lipid was fixed at 1:10, the mass ratio of drug to lipid was fixed at 1:15, and the molar ratio of metal compound to lipid was fixed at 2:1. The influence of metal ion adsorption on the ion was analyzed by single factor analysis. The specific method is as follows.

[0088] Under the action of ultrasonic wave, soybean lecithin (180 mg), cholesterol (18 mg) and triptolide (12 mg) were completely dissolved in 18 mL chloroform. Then the solution was subjected to rotary evaporation under reduced pressure at 40°C until a uniform film was formed. Subsequently, the film obtained by rotary evaporation under reduced pressure was placed in a vacuum drying box for drying overnight. After drying, 24 mL of deionized water containing 9.36 mg of ADU-S100 was added to a round-bottom flask, and the hydration was stirred at 50°C for 1 hour. After hydration, the ultrasonic probe was used to cycle ultrasonic wave at a power of 100 W for 30 min.

[0089] According to the molar ratio of metal compound to phospholipid of 2:1, the corresponding ZnCl2·6H2O, MnCl2·4H2O, MgCl2·6H2O, FeCl2·4H2O and CaCl2 were weighed and dissolved in 10 mL deionized water. The metal ion solution and the L@CA solution obtained above were sequentially added to a vial containing PB (sodium phosphate buffer (Na2HPO4) (2 mL, 10 mM)), and the mixture was incubated at room temperature for 2 hours by a magnetic stirrer. Subsequently, the corresponding metal ion adsorbed liposomes were obtained by membrane filtration. Drug loading determination was performed.

[0090] As shown in Figure 6 When the metal ion-phospholipid ratio was 0:1 (i.e. no metal ion was added), the drug loading and encapsulation efficiency of L@CA were 95.63%±0.83 and 5.04%±0.04, respectively, indicating that L@CA before metal coordination had a high drug loading (LE) and encapsulation efficiency (EE). After different metals were coordinated with L@CA to self-assemble, the drug loading and encapsulation efficiency of triptolide in Zn-L@CA group, Mn-L@CA group, Mg-L@CA group and Ca-L@CA group were 93.28%±1.44 and 4.93%±0.07, 92.93%±1.46 and 4.91%±0.06, 92.87%±1.40 and 4.91%±0.07, and 92.83%±1.74 and 4.90%±0.09, respectively (as shown in Figure 6No significant changes were found compared with the L@CA group, which indicated that the metal ions at this ratio had little effect on the drug loading and encapsulation efficiency of L@CA after anchoring on the surface of L@CA nano-preparation. However, the drug loading and encapsulation efficiency of the Fe-L@CA group were slightly reduced (drug loading and encapsulation efficiency were 84.27% ± 1.82 and 4.47% ± 0.09), which might be due to the aggregation of Fe 2+ Irreversible destruction of the phospholipid molecular structure, leading to drug leakage in the lipid nano-preparation. Example 8

[0091] Observation experiment of lysosome escape of dual-drug combination delivery system based on metal ion synergistic therapy The logarithmic growth phase K7M2 cells were used for preparation and lysosome co-localization observation experiment. After placing a sterile cover glass in a six-well plate, the logarithmic growth phase K7M2 cells were configured into a 2.5 × 10 5 / mL cell suspension, 2 mL of complete DMEM medium cell suspension was added to each well, and the cells were cultured in a cell incubator for 24 h after labeling. After the culture was completed, the old culture medium was discarded.

[0092] L@CA and M-L@CA (Zn-L@CA, Mn-L@CA, Mg-L@CA, Fe-L@CA, Ca-L@CA) labeled with coumarin 6 (C6) dye were used for cell drug intervention at a dose of 100 μg / mL for 24 h.

[0093] The specific prescription of coumarin 6 labeled L@CA liposomes is as follows: under the action of ultrasonic waves, soybean lecithin (180 mg), cholesterol (18 mg), tripterine (12 mg) and coumarin 6 (0.5 mg) were completely dissolved in 18 mL chloroform. Then the solution was rotary evaporated under reduced pressure at 40°C until a uniform film was formed. Subsequently, the film obtained by rotary evaporation under reduced pressure was placed in a vacuum drying box and dried overnight. After drying, 24 mL of deionized water containing 9.36 mg of ADU-S100 was added to a round-bottom flask, and the hydration was stirred at 50°C for 1 hour. After hydration, the ultrasonic probe was used to cycle ultrasonic waves at a power of 100 W for 3 min. Then it was filtered with 0.80 μm, 0.45 μm and 0.22 μm microporous membranes in turn, and the coumarin 6 labeled L@CA was obtained.

[0094] The specific prescription of coumarin 6 labeled M-L@CA liposomes is as follows: under the action of ultrasonic waves, soybean lecithin (180 mg), cholesterol (18 mg), gynura root red (12 mg) and coumarin 6 (0.5 mg) are completely dissolved in 18 mL chloroform. Then the solution is subjected to rotary evaporation under reduced pressure at 40°C until a uniform film is formed. Subsequently, the film obtained by rotary evaporation under reduced pressure is placed in a vacuum drying box and dried overnight. After drying, 24 mL of deionized water containing 9.36 mg of ADU-S100 is added to a round-bottom flask, and the hydration is carried out at 50°C for 1 hour. After hydration, the ultrasonic probe is used to cycle ultrasonic waves at a power of 100 W for 3 min. Then, the corresponding ZnCl2·6H2O, MnCl2·4H2O, MgCl2·6H2O, FeCl2·4H2O and CaCl2 are weighed and dissolved in 10 mL of deionized water according to the molar ratio of metal compound to phospholipid of 2:1. The metal ion solution and the L@CA solution obtained above are sequentially added to a vial containing PB (sodium phosphate buffer (Na2HPO4) (2 mL, 10 mM)), and the mixture is incubated at room temperature for 2 hours by a magnetic stirrer. Subsequently, the corresponding metal ion adsorbed liposomes are obtained by membrane filtration.

[0095] After the administration culture is completed, the old culture medium is discarded, and the cells are washed 2-3 times with pre-cooled PBS solution and incubated with LysoTracker Red (100 nM) for 45 min. The cells are washed 2-3 times with pre-cooled PBS solution, and then 1 mL of 4% paraformaldehyde is added to each well to fix the cells for 10 min. The paraformaldehyde is discarded, and the cells are washed 2-3 times with pre-cooled PBS. After washing, 1 mL of DAPI working solution (5 μg / mL) is added to each well to stain in the dark for 15 min. After staining, each well is washed 2-3 times with pre-cooled PBS.

[0096] After staining, the cover glass is mounted. After the clean glass slide is wiped clean, an appropriate amount of anti-fluorescence quencher is added, and the cover glass is removed from the six-well plate, with the side where the cells are adherently growing facing the direction of the anti-fluorescence quencher. Then the cover glass is mounted.

[0097] After mounting, the co-localized fluorescence signals are detected by a confocal laser scanning microscope at a specific wavelength, and the degree of co-localization of the fluorescence intensity of C6 labeled M-L@CA nanofactories and LysoTracker Red labeled lysosomes is analyzed.

[0098] As Figure 7As shown, after co-incubation of L@CA with K7M2 cells for 24 h, C6-labeled M-L@CA green fluorescence signal was observed to be co-localized with lysosome red fluorescence, characterized by the emission of punctate yellow fluorescence and strong positive correlation peaks at the co-localization site. However, compared with the L@CA treatment group, after co-incubation with L@CA nano-preparations coordinated with different metals, different degrees of separation between LysoTracker red (lysosome) and green (C6) fluorescence signals were observed, indicating that the metal-coordinated L@CA nano-preparations can be transported through the lysosome pathway and overcome lysosome escape. Notably, compared with other metals, a significant fluorescence separation was observed in the Mn-coordinated L@CA nano-preparation group (Mn-L@CA), with the most significant decrease in the intensity of overlapping yellow fluorescence as the intensity of green fluorescence in the cells increased, indicating that Mn-L@CA effectively destroyed the stability of the lysosome membrane, thereby facilitating the entry of C6-labeled Mn-L@CA into the cytoplasm. Example 9

[0099] Cell uptake observation experiment (flow cytometry) of the dual-drug delivery system based on metal ion synergistic therapy Logarithmic growth phase K7M2 cells were configured into a 2.5 x 10 5 / mL cell suspension, 2 mL of complete DMEM medium cell suspension was added to each well, and the cells were cultured in the cell incubator for 24 h after labeling. After the culture was completed, the old culture medium was discarded.

[0100] L@CA and M-L@CA (Zn-L@CA, Mn-L@CA, Mg-L@CA, Fe-L@CA, Ca-L@CA, see Example 8 for specific prescriptions) labeled with coumarin 6 (C6) dye were administered to the cells at a dose of 100 μg / mL for 24 h.

[0101] After the administration was completed, the supernatant culture medium was discarded, and ice-cold PBS was washed twice, making sure that the PBS was discarded cleanly to avoid affecting the 4% paraformaldehyde fixation of the cells; 2 mL of 4% paraformaldehyde was added to each well for fixation at room temperature for 10 min; the 4% paraformaldehyde was discarded, and ice-cold PBS was washed twice; 1 mL of PBS was added to each well, and the cells were gently blown down; the blown-down cell suspension was centrifuged at 1200 RPM for 3 min; the supernatant was discarded, and 400 μL of polyethylene glycol was added to each well to resuspend the cells; flow cytometry was used for detection.

[0102] According to Figure 8As shown, the cell uptake efficiency was different between different groups. Compared with the blank PBS group, the cell uptake efficiency of the liposome without metal ion coordination was the lowest, the uptake efficiency of Zn-L@CA and Mn-L@CA was the highest, the uptake efficiency of Mg-L@CA and Fe-L@CA was the second, and the uptake efficiency of Ca-L@CA was crossed in the metal coordination liposome. It is speculated that the reason is that these ions can help the liposome escape the clearance of lysosomes, thereby more effectively delivering the liposome into the cell interior. Example 10

[0103] Cell uptake observation experiment (confocal) of the dual-drug delivery system based on metal ion synergistic therapy After placing a sterile cover glass in a six-well plate, K7M2 cells in the logarithmic growth phase were configured into a 2.5 x 10 5 / mL cell suspension, 2 mL of complete DMEM medium cell suspension was added to each well, and after labeling, it was placed in a cell incubator for 24 h. After the culture was completed, the old culture medium was discarded.

[0104] L@CA and M-L@CA (Zn-L@CA, Mn-L@CA, Mg-L@CA, Fe-L@CA, Ca-L@CA) labeled with rhodamine B dye were administered to the cells at a dose of 100 μg / mL for 24 h.

[0105] The specific prescription of rhodamine B labeled L@CA liposome is as follows: under the action of ultrasonic waves, soybean lecithin (180 mg), cholesterol (18 mg), triptolide (12 mg) and rhodamine B (0.6 mg) were completely dissolved in 18 mL chloroform. Then the solution was rotary evaporated under reduced pressure at 40°C until a uniform film was formed. Subsequently, the film obtained by rotary evaporation under reduced pressure was placed in a vacuum drying box and dried overnight. After drying, 24 mL of deionized water containing 9.36 mg of ADU-S100 was added to the round-bottom flask, and the hydration was stirred at 50°C for 1 hour. After hydration, the ultrasonic probe was used to cycle ultrasonic waves at a power of 100 W for 3 min. Then it was filtered with 0.80 μm, 0.45 μm and 0.22 μm microporous membranes in turn, and rhodamine B labeled L@CA was obtained.

[0106] The specific formulation of Rhodamine B-labeled ML@CA liposomes is as follows: Soy lecithin (180 mg), cholesterol (18 mg), triptolide (12 mg), and Rhodamine B (0.6 mg) were completely dissolved in 18 mL of chloroform under ultrasonic treatment. The solution was then subjected to vacuum rotary evaporation at 40 °C until a uniform film was formed. Subsequently, the film obtained by vacuum rotary evaporation was dried overnight in a vacuum drying oven. After drying, 24 mL of deionized water containing 9.36 mg ADU-S100 was added to a round-bottom flask, and the mixture was stirred and hydrated at 50 °C for 1 hour. After hydration, the mixture was circulated and sonicated for 3 min using an ultrasonic probe with a power of 100 W. Then, ZnCl2·6H2O, MnCl2·4H2O, MgCl2·6H2O, FeCl2·4H2O, and CaCl2 were accurately weighed according to a 2:1 molar ratio of metal compounds to phospholipids and dissolved in 10 mL of deionized water. The metal ion solution and the L@CA solution obtained above were sequentially added to a vial containing PB (sodium phosphate buffer (Na2HPO4) (2 mL, 10 mM)), and the mixture was incubated at room temperature for 2 hours using a magnetic stirrer. The resulting solution was then filtered through a membrane to obtain the corresponding rhodamine-labeled ML@CA.

[0107] After drug administration, discard the old culture medium, wash three times with pre-chilled PBS solution, and add 1 mL of 4% paraformaldehyde to each well to fix cells for 10 min. Remove the paraformaldehyde, wash three times with pre-chilled PBS, stain with DAPI working solution (5 μg / mL) in the dark for 15 min, and wash three times with pre-chilled PBS. After washing, remove the cleaned slides, add a drop of 50% glycerol to the slides, and remove the coverslips from the six-well plate. Carefully identify the side with cells, and invert the coverslip with the cell-containing side facing the glycerol. Tilt the coverslip so that one side is soaked in 50% glycerol, gradually reducing the angle of invasion. Slowly attach the coverslip to the well, and fix it with nail polish at the four corners. After fixation, use filter paper to absorb any excess 50% glycerol. After preparation, perform confocal microscopy for analysis.

[0108] according to Figure 9 As shown, the cellular uptake efficiency varied among different groups. Compared with the blank PBS group, liposomes without metal ion coordination had the lowest cellular uptake efficiency, while Zn-L@CA and Mn-L@CA had the highest uptake efficiency, followed by Mg-L@CA and Fe-L@CA. Ca-L@CA uptake efficiency was mixed among metal-coordinated liposomes. We hypothesize that this is because these ions can help liposomes escape lysosomal clearance, thus delivering liposomes more effectively into the cell. Example 11

[0109] Apoptosis induction experiment of the dual-drug delivery system based on metal ion synergistic therapy The K7M2 cells in the logarithmic growth phase were prepared for lysosome co-localization observation experiment. The K7M2 cells in the logarithmic growth phase were configured into a 2.5 x 10 5 / mL cell suspension, 2 mL of complete DMEM medium cell suspension was added to each well, and it was labeled and placed in a cell incubator for 24 h. After the culture was completed, the old culture medium was discarded. The cells were dosed with L@CA and M-L@CA (Zn-L@CA, Mn-L@CA, Mg-L@CA, Fe-L@CA, Ca-L@CA) at a dosing dose of 100 μg / mL for 24 h.

[0110] The specific prescription of L@CA liposome is as follows: under the action of ultrasonic wave, soybean lecithin (180 mg), cholesterol (18 mg), and tripterine (12 mg) are completely dissolved in 18 mL of chloroform. Then, the solution is subjected to rotary evaporation under reduced pressure at 40°C until a uniform film is formed. Subsequently, the film obtained by rotary evaporation under reduced pressure is placed in a vacuum drying box for drying overnight. After drying is completed, 24 mL of deionized water containing 9.36 mg of ADU-S100 is added to a round-bottom flask, and the water is stirred at 50°C for 1 hour. After hydration, the ultrasonic probe is used to cycle ultrasonic wave at a power of 100 W for 3 min. Then, the obtained L@CA is filtered with 0.80 μm, 0.45 μm, and 0.22 μm microporous membranes in sequence.

[0111] The specific prescription of M-L@CA liposome is as follows: under the action of ultrasonic wave, soybean lecithin (180 mg), cholesterol (18 mg), and tripterine (12 mg) are completely dissolved in 18 mL of chloroform. Then, the solution is subjected to rotary evaporation under reduced pressure at 40°C until a uniform film is formed. Subsequently, the film obtained by rotary evaporation under reduced pressure is placed in a vacuum drying box for drying overnight. After drying is completed, 24 mL of deionized water containing 9.36 mg of ADU-S100 is added to a round-bottom flask, and the water is stirred at 50°C for 1 hour. After hydration, the ultrasonic probe is used to cycle ultrasonic wave at a power of 100 W for 3 min. Then, the corresponding ZnCl2·6H2O, MnCl2·4H2O, MgCl2·6H2O, FeCl2·4H2O, and CaCl2 are weighed and dissolved in 10 mL of deionized water under the condition of a molar ratio of 2:1 between the metal compound and phospholipid. The metal ion solution and the obtained L@CA solution are sequentially added to a vial containing PB (sodium phosphate buffer (Na2HPO4)) (2 mL, 10 mM), and the mixture is incubated at room temperature for 2 hours by a magnetic stirrer. Subsequently, the corresponding metal ion adsorbed liposome is obtained by membrane filtration.

[0112] After the administration intervention is completed, the supernatant culture medium is discarded, and ice-cold PBS is washed twice, and the PBS is carefully discarded to avoid affecting the subsequent experimental results; 100 μL of trypsin is added to each well, and after room temperature digestion for 1 min, three times the volume of complete medium is added to terminate digestion, and the wells are blown up, down, left and right three times, and after the blowing is completed, the cell suspension is collected, centrifuged at 1,800 rpm (300 x g) at 4°C for 5 min, and the supernatant is discarded; the cells are washed twice with pre-cooled PBS, each time at 1,800 rpm (300 x g) at 4°C for 5 min; after centrifugation is completed, 100 μL of 1 x Binding Buffer is added to each sample, and then 5 μL of Annexin V-FITC is added to each sample for 15 min, and then 5 μL of PI Staining Solution is added and gently mixed; incubate at room temperature for 10 min in the dark; add 400 μL of 1 x Binding Buffer and mix gently. The stained sample is detected by flow cytometry within 1 h.

[0113] According to Figure 10 and Figure 11 As shown by the experimental results, the effects of liposomes adsorbed with different metal ions on K7M2 cells showed obvious differences. Among them, the induction ability of Zn²⁺, Mn²⁺ and Fe²⁺ components was the strongest, and we speculated that the reason was that these ions could help liposomes escape lysosomal clearance, thereby more effectively exerting tumor killing effect. It is worth noting that the killing power of Ca²⁺ and Mg²⁺ components is relatively weak, and the blank liposome without metal ion adsorption has the worst effect. The above gradient phenomenon shows that the lysosomal clearance mechanism is likely to be the key to the difference in killing effect between groups. Example 12

[0114] Cell viability observation experiment of double-drug combination delivery system based on metal ion synergistic therapy Logarithmic growth period K7M2 cells were taken for preparation and lysosome co-localization observation experiment. The logarithmic growth period K7M2 cells were configured into 2.5 x 10 5 / mL cell suspension, 2 mL of complete DMEM medium cell suspension was added to each well, and after labeling, it was placed in a cell incubator for 24 h. After the culture was completed, the old culture medium was discarded. L@CA and M-L@CA (Zn-L@CA, Mn-L@CA, Mg-L@CA, Fe-L@CA, Ca-L@CA) were used for administration intervention on cells at a dose of 100 μg / mL for 24 h.

[0115] The specific prescription of L@CA liposome is as follows: under the action of ultrasonic wave, soybean lecithin (180 mg), cholesterol (18 mg) and tripterine (12 mg) are completely dissolved in 18 mL chloroform. Then the solution is subjected to rotary evaporation under reduced pressure at 40°C until a uniform film is formed. Subsequently, the film obtained by rotary evaporation under reduced pressure is placed in a vacuum drying box for drying overnight. After drying is completed, 24 mL of deionized water containing 9.36 mg of ADU-S100 is added to a round-bottom flask, and the hydration is stirred at 50°C for 1 hour. After hydration, the ultrasonic probe is used to cycle ultrasonic wave at a power of 100 W for 3 min. Then, the obtained L@CA solution is filtered with 0.80 μm, 0.45 μm and 0.22 μm microporous membranes in sequence to obtain the L@CA.

[0116] The specific prescription of M-L@CA liposome is as follows: under the action of ultrasonic wave, soybean lecithin (180 mg), cholesterol (18 mg) and tripterine (12 mg) are completely dissolved in 18 mL chloroform. Then the solution is subjected to rotary evaporation under reduced pressure at 40°C until a uniform film is formed. Subsequently, the film obtained by rotary evaporation under reduced pressure is placed in a vacuum drying box for drying overnight. After drying is completed, 24 mL of deionized water containing 9.36 mg of ADU-S100 is added to a round-bottom flask, and the hydration is stirred at 50°C for 1 hour. After hydration, the ultrasonic probe is used to cycle ultrasonic wave at a power of 100 W for 3 min. Then, the corresponding ZnCl2·6H2O, MnCl2·4H2O, MgCl2·6H2O, FeCl2·4H2O and CaCl2 are weighed and dissolved in 10 mL of deionized water according to the molar ratio of metal compound to phospholipid of 2:1. The metal ion solution and the obtained L@CA solution are sequentially added to a vial containing PB (sodium phosphate buffer (Na2HPO4)) (2 mL, 10 mM), and the mixture is incubated at room temperature for 2 hours by a magnetic stirrer. Subsequently, the corresponding metal ion adsorbed liposome is obtained by membrane filtration.

[0117] The staining working solution is prepared in advance before staining, and the specific staining process of the staining working solution is as follows. The staining working solution needs to be freshly prepared before use, and the whole process is operated under light shielding condition. First, the Calcein AM and PI stock solution are taken out from the frozen environment, and are balanced at room temperature for thirty minutes, and are subjected to short centrifugation to ensure that the liquid is collected at the bottom of the tube.

[0118] Next, take 5 μL of PI stock solution and add it to 10 mL of phosphate buffer or serum-free culture medium. Mix thoroughly using a vortex mixer to prepare an 8 μM PI working solution. Then, take another 5 μL of Calcein AM stock solution and add it to the above 10 mL PI working solution. Vortex again to obtain staining working solutions containing 2 μM and 8 μM PI.

[0119] After drug administration, discard the used culture medium, wash once with preheated PBS, and discard the used PBS. After washing, add sufficient staining working solution to each well and incubate at room temperature in the dark for 45 min. After incubation, the solution can be directly used for observation under a fluorescence microscope.

[0120] according to Figure 12 As shown, liposomes adsorbed with different metal ions exhibited significantly different cytotoxic effects on K7M2 cells. The Zn²⁺, Mn²⁺, and Fe²⁺ components showed the strongest cytotoxic activity, presumably because these ions help the liposomes escape lysosomal clearance, thus more effectively exerting their tumor-killing effect. Notably, the Ca²⁺ and Mg²⁺ components showed relatively weaker cytotoxic activity, while the blank liposomes without metal ion adsorption showed the worst effect. This gradient phenomenon suggests that the lysosomal clearance mechanism is likely the key factor leading to the differences in cytotoxic effects among the groups. Example 13

[0121] Pharmacodynamic evaluation of dual-drug delivery systems based on metal ion synergistic therapy Fifteen healthy 6-8 week old female BALB / c mice were selected and subcutaneously injected with K7M2 wt cells (2,000,000 cells / mouse) into the right leg while the mice were crawling naturally. When the tumor volume reached 150 mm... 3 At that time, 70% of the tumor volume was removed to simulate a mouse osteosarcoma postoperative tumor model.

[0122] In this experiment, each group consisted of 3 mice: the control group (PBS), the L@CA group (see Example 1 for the specific preparation process), and the ML@CA group (see Examples 2-4 for the specific preparation process).

[0123] The dosage is calculated according to the liposome-encapsulated ADU-S100, and the target is to inject each mouse with 20 μg ADU-S100-containing liposomes intratumorally. The dosage conversion process is as follows: the L@CA liposome concentration is 10 mg / mL, the L@CA liposome encapsulation rate is 5.04%, and the liposome administration weight is 400 μg of liposomes, which is converted to a volume of 40 μL of liposomes with a concentration of 10 mg / mL. The liposome concentration of the remaining groups is 10 mg / mL, and the specific administration volume can be converted according to the conversion process and the data given in Example 7.

[0124] The postoperative administration is performed every two days, and the tumor volume at the operation site is measured every 1 day, and the tumor growth curve and the weight change of the mice are recorded.

[0125] Figures 13-16 It is shown that the Mn-LNPs formulation group observes a better tumor recurrence inhibition effect. Specifically, compared with the blank control group, the tumor volume of the animals in this group grows the least, and the tumor weight is the lightest. These evaluation indexes in terms of tumor load and immune response consistently prove the synergistic anti-tumor effect of the combined preparation.

Claims

1. A dual-drug delivery liposome, characterized in that, It includes metal ions, liposome membranes, and active ingredients, wherein the metal ions are adsorbed on the surface of the liposome membranes, and the active ingredients are encapsulated within the liposome membranes; The metal ion is a divalent metal ion; The active ingredient consists of a STING agonist and active monomeric components from traditional Chinese medicine.

2. The dual-drug delivery liposome according to claim 1, characterized in that, The divalent metal ions are selected from Zn. 2+ Mn 2+ Mg 2+ Fe 2+ Ca 2+ .

3. The dual-drug delivery liposome according to claim 1, characterized in that, The STING agonist is a cyclic dinucleotide or a cyclic dinucleotide analogue, selected from ADU-S100, cGAMP, c-di-GMP, c-di-AMP, 2',3'-cGAMP; the active monomeric components of the traditional Chinese medicine are tripterygium wilfordii, curcumin, baicalin, cryptotanshinone, magnolol, oleandrin, saural flavin, and diosgenin.

4. The dual-drug delivery liposome according to claim 1, characterized in that, The liposome membrane is made of lecithin and cholesterol.

5. The dual-drug delivery liposome according to claim 4, characterized in that, The mass ratio of cholesterol to lecithin is 1:5-1:20; the mass ratio of the active monomeric component of traditional Chinese medicine to lecithin is 1:10-1:20; the molar ratio of the STING agonist to the active monomeric component of traditional Chinese medicine is 1:2; and the molar ratio of the metal ion to lecithin is 1:1-4:

1.

6. The dual-drug delivery liposome according to claim 4, characterized in that, The active monomeric component of the traditional Chinese medicine is triptolide, and the STING agonist is ADU-S100; the mass ratio of cholesterol to lecithin is 1:10, the mass ratio of triptolide to soybean lecithin is 1:15, the molar ratio of ADU-S100 to triptolide is 1:2, and the molar ratio of metal ion compound to lecithin is 2:

1.

7. The method for preparing dual-drug delivery liposomes according to any one of claims 1-6, characterized in that, Includes the following steps: Step 1: Dissolve lecithin, cholesterol, and active monomers of traditional Chinese medicine in an organic solvent, and then rotary evaporate to obtain a thin film; Step 2: Add an aqueous solution of STING agonist to the film obtained in Step 1, hydrate and sonicate to obtain a liposome solution; Step 3: Add an aqueous solution containing metal ions to the liposome solution from Step 2 for hydration and adsorption to obtain the dual-drug delivery liposomes.

8. The preparation method according to claim 7, characterized in that, The organic solvent is chloroform.

9. The preparation method according to claim 7, characterized in that, In step 3, hydration adsorption is carried out in a phosphate buffer system with a pH of 6.2-7.

8.

10. Use of the dual-drug delivery liposome according to any one of claims 1-6 in the preparation of tumor therapeutic drugs.

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