A dual-ion-based nanoparticle for treating p53-mutant tumors, its preparation method and application

Abstract

This invention discloses a dual-ion-based nanoparticle for treating p53-mutant tumors, its preparation method, and its application, relating to the field of tumor drug technology. The dual-ion-based nanoparticle comprises liposomes and Zn... 2+ Co 2+ The inner and outer tumor cell membranes; liposomes have a bilayer; Zn 2+ Encased within the liposome, the inner layer is the tumor cell membrane and Co 2+ The complex is formed and coated on the outer layer of the liposome, with the outer layer of tumor cell membrane covering the outside of the complex. This application connects mutant p53 and the cGAS-STING pathway, promoting the cGAS-STING innate immune pathway by reducing the expression of mutant p53, restoring the function of the p53 tumor suppressor gene, achieving better tumor treatment effects, and has good application value in tumor treatment.

Description

Technical Field

[0001] This invention relates to the field of tumor drug technology, and more specifically, to a dual-ion-based nanoparticle for treating p53-mutant tumors, its preparation method, and its application. Background Technology

[0002] Malignant tumors, as one of the most difficult-to-cure diseases, seriously threaten human life and health. p53 is the tumor suppressor gene most strongly associated with human tumorigenesis discovered to date, because the protein encoded by this gene is a transcription factor that controls the initiation of the cell cycle. The p53 gene is mutated in more than 50% of human cancers, and in some intractable malignant tumors, the mutation rate is as high as 80%. Mutated p53 genes not only lose their tumor suppressor function but also develop new functions that promote cancer development and progression, such as promoting tumor growth, invasion, and metastasis; blocking innate immune signaling pathways to promote tumor immune escape; and causing chemotherapy resistance and poor prognosis. Therefore, the highly prevalent and complex p53 mutations in tumors pose a significant challenge to the effective treatment of cancer.

[0003] Current treatment strategies for p53-mutant tumors include chemotherapy to directly induce the degradation of the mutant p53 protein or help it restore the function of the wild-type. However, due to the easy uptake of these drugs by normal tissues, leading to unavoidable toxic side effects and the high incidence of multidrug resistance, their clinical application for treating mutant p53 tumors remains difficult. Gene therapy strategies aim to correct or compensate for the mutated p53 gene; however, the risks of nuclear localization and insertional mutations in DNA, as well as the immunogenicity and instability of RNA, remain unresolved issues. Existing immunotherapy methods for p53-mutant tumors generally suffer from unsatisfactory treatment effects, limited applicability to multiple p53 mutants, and poor targeted delivery of antibodies and immune factors.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide a dual-ion-based nanoparticle for treating p53-mutant tumors, its preparation method, and its application.

[0006] This invention is implemented as follows:

[0007] In a first aspect, the present invention provides a dual-ion-based nanoparticle for treating p53-mutant tumors, comprising liposomes and Zn. 2+ Co 2+ The liposome has an inner tumor cell membrane and an outer tumor cell membrane, and the Zn... 2+Enclosed within the liposome, the inner tumor cell membrane and the Co 2+ The complex is formed and coated on the outer layer of the liposome, with the outer tumor cell membrane covering the outside of the complex.

[0008] In an optional embodiment, the particle size of the dual-ion-based nanoparticles is 100-150 nm.

[0009] In an optional embodiment, the ion release pH of the dual-ion nanoparticles is less than or equal to 5.5.

[0010] Secondly, the present invention provides a method for preparing dual-ion-based nanoparticles for treating p53-mutant tumors, comprising:

[0011] (1) Containing Zn 2+ The salt solution was hydrated with the liposome membrane, and the precipitate was separated and collected to prepare LIZn nanoparticles.

[0012] (2) Containing Co 2+ The salt solution was mixed with the inner tumor cell membrane, the precipitate was separated and collected, and the MCo complex was obtained.

[0013] (3) The LIZn nanoparticles were mixed with the MCo complex, the precipitate was separated and collected to obtain LIZn@MCo nanoparticles;

[0014] (4) The LIZn@MCo nanoparticles are mixed with the outer tumor cell membrane, the precipitate is separated and collected to obtain LIZn@MCo@M nanoparticles.

[0015] In an optional implementation, the Zn-containing 2+ Zn in salt solution 2+ The concentration is 8-12 mg / mL, and the Zn content is... 2+ The volume-to-mass ratio of the salt solution compound to the DPPC in the raw material of the liposome membrane is 2 ml: 5-10 mg;

[0016] Preferably, the one containing Co 2+ Co in salt solution 2+ The concentration is 8-15 mg / mL, and the substance containing Co 2+ The volume ratio of the compound to the inner tumor cell membrane is 400-600 μL: 1 ml;

[0017] Preferably, the volume ratio of the LIZn nanoparticles to the MCo composite is 1:1-1.5;

[0018] Preferably, the volume ratio of the LIZn@MCo nanoparticles to the outer tumor cell membrane is 1.5-2.5:1.

[0019] In an optional embodiment, the preparation method of the inner tumor cell membrane and the outer tumor cell membrane includes: centrifuging the tumor cells for 3-8 min, washing them 2-4 times in buffer solution, adding sterile water to rupture the cells, sonicating them with a cell disruptor until the centrifuge tube wall is slightly heated, centrifuging at 5000-9000 rpm for 3-8 min, and taking the supernatant to obtain the membrane.

[0020] Preferably, the buffer solution is PBS with pH = 7-8.

[0021] In an optional implementation, the tumor cells are pancreatic cancer cells.

[0022] In an optional implementation, the mixing in steps (1) to (4) is carried out under eddy current and ultrasonic conditions;

[0023] Preferably, the separation in steps (1) to (4) all includes centrifugation at 10,000-20,000 rpm for 20-40 min;

[0024] Preferably, step (3) further includes filtering with 0.7-0.9μM, 0.4-0.5μM, and 0.1-0.2μM filter membranes 8-12 times in sequence before separation;

[0025] Preferably, step (4) includes filtering with 0.7-0.9μM, 0.4-0.5μM and 0.1-0.2μM filter membranes 4-6 times in sequence before separation.

[0026] In an optional embodiment, the method for preparing the liposomes includes: dissolving DPPC and cholesterol in an organic solvent to form a reaction solution, and evaporating the reaction solution under reduced pressure to obtain a liposome film, wherein the mass-volume ratio of DPPC, cholesterol and organic solvent is 5-10 mg: 1-5 mg: 10 ml.

[0027] Thirdly, the present invention provides the use of dual-ion-based nanoparticles for treating p53-mutant tumors prepared by any of the foregoing embodiments, or dual-ion-based nanoparticles for treating p53-mutant tumors prepared by any of the foregoing embodiments, in the preparation of medicaments for treating or improving tumors with high expression of mutant p53 protein.

[0028] The present invention has the following beneficial effects:

[0029] The dual-ion-based nanoparticles (LIZn@MCo@M nanoparticles) provided in this application for treating p53-mutant tumors have an outermost tumor cell membrane that can target the tumor site, resulting in higher drug efficacy, while also protecting the inner Co layer.2+ Reduce Co 2+ Loss during the targeting process. The outermost tumor cell membrane has homologous targeting capabilities, allowing LIZn@MCo@M to enter the cell via endocytosis. In the acidic tumor cell environment, some Zn is released. 2+ and Co 2+ This application utilizes a liposome-based biomimetic delivery system. Liposomes, as artificial cell membrane vesicles, possess high biosafety, good biocompatibility, and biodegradability, and can be used to target ions at tumor sites, thereby improving ion utilization and therapeutic efficacy. Furthermore, liposomes are relatively stable, a common organic drug delivery carrier, and possess nanoscale, biomembrane-like structures and excellent biocompatibility. Liposomes enter lysosomes, releasing Zn... 2+ and Co 2+ Both ions promote the production of ROS in cells and ubiquitinate and degrade the protein content of mutant p53, thereby relieving the inhibition of the cGAS-STING signaling pathway by mutant p53. Furthermore, Zn... 2+ and Co 2+ It can synergistically promote the cGAS-STING signaling pathway, activate the body's innate immunity, and ultimately achieve an anti-tumor effect. Therefore, this application connects mutant p53 and the cGAS-STING pathway, promotes the cGAS-STING innate immune pathway by reducing the expression of mutant p53, restores the function of the p53 tumor suppressor gene, achieves better tumor treatment effects, and has good application value in tumor treatment. Furthermore, this LIZn@MCo@M nanoparticle can be widely used in various p53 mutant tumors. Attached Figure Description

[0030] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 This is a schematic diagram of the structure of LIZn@MCo@M nanoparticles provided in Embodiment 1 of this application;

[0032] Figure 2 This is a transmission electron microscope image of the LIZn@MCo@M nanoparticles provided in Example 1 of this application;

[0033] Figure 3 This is a schematic diagram of the particle size distribution of LIZn@MCo@M nanoparticles provided in Example 1 of this application;

[0034] Figure 4 Potential diagrams of different nanoparticles involved in the preparation process of the LIZn@MCo@M nanoparticles provided in Example 1 of this application;

[0035] Figure 5 The graphs show the release curves of LIZn@MCo@M nanoparticles under different pH conditions in Experiment Example 1 of this application. In the graphs, a is the release curve of zinc ions over time, and b is the release curve of cobalt ions over time.

[0036] Figure 6 This is a schematic diagram of KPC cell survival rate in Experiment Example 2 of this application, where a is a schematic diagram of the effect of LIZn@MCo@M nanoparticle concentration on KPC cell survival rate, and b is a schematic diagram of the effect of different treatment groups on KPC cell survival rate.

[0037] Figure 7 This is a schematic diagram of protein expression in Experimental Example 3 of this application. In this diagram, a is a schematic diagram of the expression of p53 and Mutp53 in KPC cells treated with different nanoparticles detected by immunoblotting, and b is a schematic diagram of the expression of TBK1, p-TBK1, STING and p-STING in KPC cells treated with different nanoparticles detected by immunoblotting.

[0038] Figure 8 This is a schematic diagram of the results of the anti-cancer experiment in Experiment Example 4 of this application. In this diagram, a is a curve showing the change in tumor volume of KPC model mice treated with different nanoparticles, b is a curve showing the change in tumor weight of KPC model mice treated with different nanoparticles, and c is a schematic diagram showing the changes in tumor of KPC model mice treated with different nanoparticles.

[0039] Figure 9 This is a schematic diagram of the results of the flow cytometry experiment of immune cells in Experiment Example 5 of this application. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0041] Experimental reagents used in this invention:

[0042] Cholesterol was purchased from Sigma-Aldrich (St. Louis, MO, USA). 1,2-Dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC) was purchased from AVT Pharmaceutical Tech CO., Ltd. (Shanghai, China). Zinc chloride (ZnCl2) was purchased from Beijing Solai Biotechnology Co., Ltd. (Beijing, China). Cobalt chloride (CoCl2) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Tumor cells were purchased from Fubai Ao (Suzhou) Biomedical Technology Co., Ltd. All chemicals were used as is without further processing.

[0043] This invention provides a dual-ion-based nanoparticle for treating p53-mutant tumors, comprising liposomes and Zn. 2+ Co 2+ The inner and outer tumor cell membranes; liposomes have a bilayer; Zn 2+ Encased within the liposome, the inner layer is the tumor cell membrane and Co 2+ The complex is formed and coated on the outer layer of the liposome, with the outer tumor cell membrane covering the outside of the complex.

[0044] The particle size of the dual-ion nanoparticles is 100-150 nm. The ion release pH of the dual-ion nanoparticles is less than or equal to 5.5.

[0045] The present invention also provides a method for preparing the above-mentioned dual-ion-based nanoparticles for treating p53-mutant tumors, which includes the following steps:

[0046] (1) Preparation of LIZn nanoparticles.

[0047] DPPC and cholesterol were dissolved in an organic solvent to form a reaction solution, which was then evaporated under reduced pressure to obtain a liposome film (at this stage, it is only a bilayer film). The mass-to-volume ratio of DPPC, cholesterol, and organic solvent was 5-10 mg: 1-5 mg: 10 ml. A Zn-containing... 2+ Liposome membranes were hydrated with a salt solution under eddy current and ultrasonic conditions. After centrifugation at 10,000-20,000 rpm for 20-40 min, the precipitate was separated and collected. The hydrated liposome membranes formed spherical liposomes, thus preparing LIZn nanoparticles. 2+ Zn in salt solution 2+ The concentration is 8-12 mg / mL, containing Zn 2+ The volume-to-mass ratio of the salt solution compound to DPPC is 2 ml: 5-10 mg.

[0048] (2) Preparation of tumor cell membranes.

[0049] Centrifuge tumor cells for 3-8 minutes, wash 2-4 times in buffer solution, add sterile water to rupture the cells, sonicate using a cell disruptor until the centrifuge tube wall is slightly warm, centrifuge at 5000-9000 rpm for 3-8 minutes, and collect the supernatant; the buffer solution is PBS with pH=7.4. The tumor cells are pancreatic cancer cells.

[0050] (3) Preparation of MCo complex.

[0051] Will contain Co 2+ The salt solution was mixed with the inner tumor cell membrane under vortex and sonication conditions, centrifuged at 10,000-20,000 rpm for 20-40 min, and the precipitate was separated and collected to obtain the MCo complex; containing Co 2+ Co in salt solution 2+ The concentration is 8-15 mg / mL, containing Co 2+ The volume ratio of the compound to the inner tumor cell membrane is 400-600 μL: 1 ml.

[0052] (4) Preparation of LIZn@MCo nanoparticles.

[0053] LIZn nanoparticles and MCo composites were mixed at a volume ratio of 1:1-1.5 under eddy current and ultrasonic conditions. The mixture was then filtered 8-12 times through 0.7-0.9 μM, 0.4-0.5 μM, and 0.1-0.2 μM filter membranes. The mixture was centrifuged at 10,000-20,000 rpm for 20-40 min to separate and collect the precipitate, thus obtaining LIZn@MCo nanoparticles.

[0054] (5) Preparation of LIZn@MCo@M nanoparticles.

[0055] LIZn@MCo nanoparticles were mixed with the outer tumor cell membrane (volume ratio 1.5-2.5:1) under eddy current and sonication conditions, and then filtered 4-6 times sequentially through 0.7-0.9 μM, 0.4-0.5 μM, and 0.1-0.2 μM filter membranes. The mixture was then centrifuged at 10,000-20,000 rpm for 20-40 min to separate and collect the precipitate, thus obtaining LIZn@MCo@M nanoparticles.

[0056] This application utilizes the hydrophilicity and hydrophobicity of the liposomal phospholipid bilayer, as well as the electrostatic adsorption of the cell membrane, to achieve the transfer of Zn 2+ Encapsulated within the liposomes, while the inner layer consists of tumor cell membranes and Co. 2+By electrostatic adsorption, the complex is formed and coated on the outer layer of the liposome. The outer layer of the tumor cell membrane is coated on the outside of the complex, forming the dual-ion nanoparticles (LIZn@MCo@M nanoparticles) of this application. The LIZn@MCo@M nanoparticles can be widely used in the preparation of drugs for the treatment or improvement of tumors with high expression of mutant p53 protein.

[0057] The mechanism of action of LIZn@MCo@M nanoparticles in this application is as follows:

[0058] After injection of LIZn@MCo@M nanoparticles, the outermost tumor cell membrane can target the tumor site, internalize into the cell, and release Zn in the acidic tumor cell environment. 2+ and Co 2+ Zn 2+ and Co 2+ It promotes the production of ROS in cells, ubiquitinates and degrades mutant p53 protein, thereby relieving the inhibition of the cGAS-STING signaling pathway by mutant p53. Furthermore, Zn... 2+ and Co 2+ It can synergistically promote the cGAS-STING signaling pathway, activate the body's own innate immunity, and ultimately achieve an anti-tumor effect.

[0059] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0060] Example 1

[0061] This embodiment provides a dual-ion-based nanoparticle for treating p53-mutant tumors, the preparation method of which includes the following steps:

[0062] (1) Preparation of LIZn nanoparticles.

[0063] 7 mg DPPC and 3 mg cholesterol were dissolved in 10 mL chloroform to form a reaction solution, which was then evaporated to dryness at 56 °C, yielding a liposome film on the flask wall. Zinc chloride solution (2.726 mg dissolved in 2 mL deionized water) was added to fully hydrate the liposome film. After hydration, the solution was mixed under vortex and sonication conditions until a clear liquid was obtained. The mixture was centrifuged at 15000 rpm for 20 min, the precipitate was collected, and then reconstituted in 2 mL of water by sonication to obtain LIZn nanoparticles.

[0064] (2) Preparation of tumor cell membranes.

[0065] Two 50ml culture flasks of pancreatic cancer cells (KPC cells) were centrifuged for 5 minutes, washed three times in PBS at pH 7.4, and 1mL of sterile water was added to rupture the cells. The cells were then sonicated until the centrifuge tube wall was slightly heated using a cell disruptor, centrifuged at 7000rpm for 5 minutes, and the supernatant was collected to obtain the tumor cell membrane.

[0066] (3) Preparation of MCo complex.

[0067] 500 μL of cobalt chloride solution (10 mg / mL) was added dropwise to 1 mL of tumor cell membrane solution, followed by repeated vortexing and sonication. The mixture was then centrifuged at 15,000 rpm for 30 min, the precipitate was collected, and dispersed in 1 mL of water to obtain the MCo complex.

[0068] (4) Preparation of LIZn@MCo nanoparticles.

[0069] LIZn nanoparticles were added dropwise to the MCo composite (volume ratio 1:1), mixed under eddy current and ultrasonic conditions, filtered 10 times sequentially through 0.8 μM, 0.45 μM and 0.2 μM filter membranes, centrifuged at 15000 rpm for 30 min, collected the precipitate, and washed repeatedly to obtain LIZn@MCo nanoparticles.

[0070] (5) Preparation of LIZn@MCo@M nanoparticles.

[0071] 2 mL of LIZn@MCo nanoparticles were mixed with 1 mL of tumor cell membrane under eddy current and sonication conditions. The mixture was then filtered five times through 0.8 μM, 0.45 μM, and 0.2 μM filters. After centrifugation at 15000 rpm for 30 min, the precipitate was collected and washed three times to obtain LIZn@MCo@M nanoparticles with an average particle size of 149 nm.

[0072] The structure of the obtained LIZn@MCo@M nanoparticles is as follows: Figure 1 As shown, liposomes have a bilayer, Zn 2+ Encased within the liposome, the inner layer is the tumor cell membrane and Co 2+ The complex is formed and coated on the outer layer of the liposome, with the outer tumor cell membrane covering the outside of the complex.

[0073] The obtained LIZn@MCo@M nanoparticles were examined by transmission electron microscopy and their particle size was analyzed. Figure 2 As can be seen, the bilayer cell membrane was successfully coated onto the outer layer of the lipid body, and the LIZn@MCo@M nanoparticles were successfully prepared. Figure 3 It can be seen that the particle size distribution of LIZn@MCo@M nanoparticles is uniform and relatively concentrated.

[0074] Furthermore, by performing potential analysis on multiple intermediates formed during the preparation of LIZn@MCo@M nanoparticles in Example 1, from... Figure 4 It can be seen that the tumor cell membrane (CM) alone is significantly negatively charged, while the liposomes (LIVN) are slightly charged with no obvious potential, and are coated with Zn. 2+ Liposomes (LIZn) contain positively charged Zn. 2+ Since LIZn is encapsulated within liposomes, it retains a slight charge, with a potential not significantly different from LIVN. However, coating LIZn with tumor cell membranes (LIZn@M) reveals that LIZn@M is distinctly negatively charged. Conversely, coating LIZn with Co... 2+ The complex (LIZn@MCo) that binds to the tumor cell membrane contains positively charged Co. 2+ When combined with the negatively charged tumor cell membrane, the potential decreases significantly. Then, by coating the surface of LIZn@MCo with a layer of tumor cell membrane, the potential increases significantly, exhibiting a clearly negatively charged potential. Potential analysis fully demonstrates that Example 1 effectively synthesized LIZn@MCo@M nanoparticles.

[0075] Example 2

[0076] This embodiment provides a dual-ion-based nanoparticle for treating p53-mutant tumors, the preparation method of which includes the following steps:

[0077] (1) Preparation of LIZn nanoparticles.

[0078] 5 mg DPPC and 1 mg cholesterol were dissolved in 10 mL chloroform to form a reaction solution. The solution was then evaporated to dryness at 56 °C, resulting in a liposome film on the flask wall. Zinc chloride solution (2.726 mg dissolved in 2 mL deionized water) was added to fully hydrate the liposome film. After hydration, the solution was mixed under vortex and sonication conditions until a clear liquid was obtained. The mixture was centrifuged at 10,000 rpm for 30 min, the precipitate was collected, and then reconstituted in 2 mL of water by sonication to obtain LIZn nanoparticles.

[0079] (2) Preparation of tumor cell membranes.

[0080] Two 50ml culture flasks of pancreatic cancer cells (KPC cells) were centrifuged for 5 minutes, washed twice in PBS at pH 7.4, and 1mL of sterile water was added to rupture the cells. The cells were then sonicated until the centrifuge tube wall was slightly heated using a cell disruptor, and centrifuged at 5000rpm for 8 minutes. The supernatant was collected to obtain the tumor cell membrane.

[0081] (3) Preparation of MCo complex.

[0082] 500 μL of cobalt chloride solution (8 mg / mL) was added dropwise to 1 mL of tumor cell membrane solution, followed by repeated vortexing and sonication. The mixture was then centrifuged at 10,000 rpm for 40 min, the precipitate was collected, and dispersed in 1 mL of water to obtain the MCo complex.

[0083] (4) Preparation of LIZn@MCo nanoparticles.

[0084] LIZn nanoparticles were added dropwise to the MCo composite (volume ratio 1:1.2), mixed under eddy current and ultrasonic conditions, filtered eight times sequentially through 0.7 μM, 0.4 μM, and 0.1 μM filter membranes, centrifuged at 10,000 rpm for 40 min, collected the precipitate, and washed repeatedly to obtain LIZn@MCo nanoparticles.

[0085] (5) Preparation of LIZn@MCo@M nanoparticles.

[0086] 1.5 mL of LIZn@MCo nanoparticles were mixed with 1 mL of tumor cell membrane under eddy current and sonication conditions. The mixture was then filtered four times sequentially through 0.7 μM, 0.4 μM, and 0.1 μM filters. After centrifugation at 10,000 rpm for 40 min, the precipitate was collected and washed three times to obtain LIZn@MCo@M nanoparticles with an average particle size of 149 nm.

[0087] Example 3

[0088] This embodiment provides a dual-ion-based nanoparticle for treating p53-mutant tumors, the preparation method of which includes the following steps:

[0089] (1) Preparation of LIZn nanoparticles.

[0090] 10 mg DPPC and 5 mg cholesterol were dissolved in 10 mL chloroform to form a reaction solution. The solution was then evaporated to dryness at 56 °C, yielding a liposome film on the flask wall. Zinc chloride solution (2.726 mg dissolved in 2 mL deionized water) was added to fully hydrate the liposome film. After hydration, the solution was mixed under vortex and sonication conditions until a clear liquid was obtained. The mixture was centrifuged at 20,000 rpm for 20 min, the precipitate was collected, and then reconstituted in 2 mL of water by sonication to obtain LIZn nanoparticles.

[0091] (2) Preparation of tumor cell membranes.

[0092] Two 50ml culture flasks of pancreatic cancer cells (KPC cells) were centrifuged for 5 minutes, washed four times in PBS at pH 7.4, and 1mL of sterile water was added to rupture the cells. The cells were then sonicated until the centrifuge tube wall was slightly heated using a cell disruptor, centrifuged at 9000rpm for 3 minutes, and the supernatant was collected to obtain the tumor cell membrane.

[0093] (3) Preparation of MCo complex.

[0094] 500 μL of cobalt chloride solution (15 mg / mL) was added dropwise to 1 mL of tumor cell membrane solution, followed by repeated vortexing and sonication. The mixture was then centrifuged at 20,000 rpm for 20 min, the precipitate was collected, and dispersed in 1 mL of water to obtain the MCo complex.

[0095] (4) Preparation of LIZn@MCo nanoparticles.

[0096] LIZn nanoparticles were added dropwise to the MCo composite (volume ratio 1:1.5), mixed under eddy current and ultrasonic conditions, filtered 12 times sequentially through 0.9 μM, 0.5 μM and 0.2 μM filter membranes, centrifuged at 20,000 rpm for 20 min, collected the precipitate, and washed repeatedly to obtain LIZn@MCo nanoparticles.

[0097] (5) Preparation of LIZn@MCo@M nanoparticles.

[0098] 2.5 mL of LIZn@MCo nanoparticles were mixed with 1 mL of tumor cell membrane under eddy current and sonication conditions. The mixture was then filtered six times through 0.7 μM, 0.4 μM, and 0.1 μM filters. After centrifugation at 20,000 rpm for 20 min, the precipitate was collected and washed three times to obtain LIZn@MCo@M nanoparticles with an average particle size of 149 nm.

[0099] Comparative Example 1

[0100] This comparative example provides a zinc chloride solution (3.667 mg dissolved in 2 mL of deionized water).

[0101] Comparative Example 2

[0102] This comparative example provides a cobalt chloride solution (10 mg / mL).

[0103] Comparative Example 3

[0104] This comparative example provides a mixture of zinc chloride solution (3.667 mg dissolved in 2 mL of deionized water) and zinc chloride solution (10 mg / mL) in a volume ratio of 1:1.

[0105] Comparative Example 4

[0106] This comparative example provides a liposome, the preparation method of which can be found in step (1) of Example 1, specifically including: dissolving 7 mg DPPC and 3 mg cholesterol in 10 mL chloroform to form a reaction solution, rotary evaporating at 56 °C until dry, obtaining a liposome film on the bottle wall, adding water for hydration to obtain the liposome, denoted as LIVN.

[0107] Comparative Example 5

[0108] This comparative example provides a LIZn nanoparticle, the preparation method of which can be found in step (1) of Example 1.

[0109] Comparative Example 6

[0110] This comparative example provides a method for preparing LIVN@MCo nano-ions, including:

[0111] 7 mg DPPC and 3 mg cholesterol were dissolved in 10 mL chloroform to form a reaction solution. The solution was then evaporated at 56 °C until dry. A liposome film was obtained on the flask wall. After hydration with water, liposomes were obtained, denoted as LIVN.

[0112] LIVN nanoparticles were added dropwise to the MCo composite prepared according to steps (2) and (3) in Example 1 (volume ratio 1:1), mixed under eddy current and ultrasonic conditions, filtered 10 times sequentially through 0.8 μM, 0.45 μM and 0.2 μM filter membranes, centrifuged at 15000 rpm for 30 min, collected the precipitate, and washed repeatedly to obtain LIVN@MCo nanoparticles.

[0113] Experimental Example 1: Ion Release Effect

[0114] The experimental methods included: detecting the ion release effect of the LIZn@MCo@M nanoparticles obtained in Example 1 under different pH conditions.

[0115] Please refer to the experimental results. Figure 5 .

[0116] The results showed that LIZn@MCo@M nanoparticles at pH 7.4 exhibited good Zn content. 2+ and Co 2+ The release rate is low, approximately 35% after 24 hours, while under acidic conditions at pH 5.5, Zn... 2+ and Co 2+ The release rate is high, reaching over 85% within 24 hours. This indicates that LIZn@MCo@M nanoparticles can be released more effectively in the acidic environment of tumors.

[0117] Experiment Example 2: Lethality Experiment

[0118] Nanoparticle killing experiments were conducted on pancreatic cancer KPC cells with high expression of mutant p53 protein in different treatment groups.

[0119] Different treatment groups included: blank control group (CON), comparative example 1 (Zn) 2+ Comparative Example 2 (Co) 2+ Comparative Example 3 (Zn) 2+ +Co 2+Comparative Examples 4 (LIVN), 5 (LIZn), 6 (LIVN@MCo), and 1 (LIZn@MCo@M) were included, with the blank control group consisting of culture medium containing 1% serum.

[0120] The experimental methods included: evaluating the cytotoxicity of LIZn@MCo@M using mouse pancreatic cancer cells (KPC) via the MTT assay kit. All cells (8,000 cells / well) were seeded in 96-well plates and incubated for 24 hours (37°C, 5% CO2). KPC cells were incubated with LIZn@MCo@M at different ion concentrations (0, 3, 5, 10, 15, 20 μg / mL) for 24 hours. After removing the LIZn@MCo@M-containing medium, 100 μL of complete medium containing 10 μL of MTT was added to each well, and the cells were incubated for 4 hours. The absorbance at 490 nm was measured.

[0121] The products provided in Comparative Examples 1-6 were diluted to ion concentrations of 20 μg / mL for ZnCl2, CoCl2, ZnCl2+CoCl2, LIVN, LIZn, LIVN@MCo, and LIZn@MCo@M, and then treated with KPC cells again using the same method described above.

[0122] Please refer to the experimental results. Figure 6 .

[0123] from Figure 6 As can be seen from Figure a, the survival rate of KPC cells gradually decreases with increasing concentration of LIZn@MCo@M nanoparticles. Therefore, the optimal concentration of LIZn@MCo@M nanoparticles is 3-20 μg / mL. Figure 6 As can be seen from b, Zn alone 2+ and Co 2+ It has some killing ability against KPC cells, but the killing ability is relatively small. Zn... 2+ and Co 2+ The combination can significantly enhance the killing ability against KPC cells. Liposomes and cell membranes, which have no killing ability against KPC cells, were used to target Zn... 2+ and Co 2+ Encapsulation can also significantly enhance the killing ability against KPC cells. The LIZn@MCo@M nanoparticles in Example 1, with Zn encapsulated via liposomes... 2+ And Co 2+ Loaded onto the cell membrane, and utilizing Co-loaded 2+ The liposomes are coated with cell membranes, and then coated with an outermost layer of blank cell membrane, thus achieving double membrane coating. This results in better killing ability against KPC cells, with a killing ability of 70%, which is more than twice that of other treatment groups, demonstrating significant effects.

[0124] Experiment Example 3: Protein Expression Experiment.

[0125] The expression of p53(DO-1), TBK1, p-TBK1, STING, and p-STING was examined in pancreatic cancer KPC cells with high expression of mutant p53 protein under different treatment groups.

[0126] Different treatment groups included: blank control group (Control), comparative example 4 (LIVN), comparative example 5 (LIZn), comparative example 6 (LIVN@MCo), and example 1 (LIZn@MCo@M).

[0127] The experimental protocol included: Western blot assay: Cell lysates were loaded onto an SDS-PAGE gel and transferred to a PVDF membrane (Millipore). The designated primary antibody was incubated overnight, and HRP-linked goat anti-rabbit IgG (BaoKe) was used as the secondary antibody. The blot signal was visualized via ECL using an Amersham Imager 600 system (GE Healthcare Life Sciences, Pittsburgh, USA).

[0128] Please refer to the experimental results. Figure 7 .

[0129] from Figure 7 As can be seen from a and b, LIZn@MCo@M nanoparticles can downregulate the expression of mutant p53 and upregulate the expression of p-TBK1 and p-STING.

[0130] Experiment Example 4: Anti-cancer experiment.

[0131] Different treatment groups were used in drug-loaded nanoparticle anticancer experiments in pancreatic cancer KPC model mice.

[0132] Different treatment groups included: blank control group (Control), comparative example 4 (LIVN), comparative example 5 (LIZn), comparative example 6 (LIVN@MCo), and example 1 (LIZn@MCo@M).

[0133] The experimental protocol included: using C57BL / 6 mice (6 weeks old, male) as an animal model. KPC cells were subcutaneously injected into the mice. When the tumor volume was 75-100 nm... 3 At that time, tumor-bearing mice were randomly divided into five groups: (1) control group injected with saline, (2) intravenously injected with LIVN, (3) intravenously injected with LIZn, (4) intravenously injected with LIVN@MCo, and (5) intravenously injected with LIZn@MCo@M. Injections were given every three days. The Zn content in each injection of all formulations was... 2+ and Co2+ The dosage is equal. Tumor volume is calculated as: width 2 × length / 2. After a 14-day treatment period, mice were sacrificed and tumors were obtained.

[0134] Please refer to the experimental results. Figure 8 .

[0135] from Figure 8 As can be seen from Figure a, the tumor volume in KPC model mice treated with LIZn@MCo@M nanoparticles was significantly lower than that in other treatment groups. Figure 8 As can be seen from Figure b, the tumor weight in KPC model mice treated with LIZn@MCo@M nanoparticles was also significantly lower than that in other treatment groups. Figure 8 As can be seen from the results, the changes in the tumor were more significant, with the treatment effect being nearly 8 times better than that of the blank control group.

[0136] Experimental Example 5: Flow Cytometry Experiment of Immune Cells.

[0137] Flow cytometry experiments were performed on immune cells in different treatment groups in pancreatic cancer KPC model mice.

[0138] Different treatment groups included: blank control group (Control), comparative example 4 (LIVN), comparative example 5 (LIZn), comparative example 6 (LIVN@MCo), and example 1 (LIZn@MCo@M).

[0139] The experimental protocol included: using C57BL / 6 mice (6 weeks old, female) as an animal model. KPC cells were subcutaneously injected into the mice. When the tumor volume was 75-100 nm... 3 At that time, tumor-bearing mice were randomly divided into five groups: (1) control group injected with saline, (2) intravenously injected with LIVN, (3) intravenously injected with LIZn, (4) intravenously injected with LIVN@MCo, and (5) intravenously injected with LIZn@MCo@M. Injections were given every three days. The Zn content in each injection of all formulations was... 2+ and Co 2+ The dosage is equal. Tumor volume is calculated as: width 2 × length / 2. After a 14-day treatment period, mice were sacrificed and tumors were obtained for flow cytometry immunocellular analysis. CD4+ T cells and CD8+ T cells in tumor tissues were analyzed by immunofluorescence staining using flow cytometry.

[0140] Please refer to the experimental results. Figure 9 .

[0141] from Figure 9It can be seen that the immune cells of LIZn@MCo@M nanoparticles are significantly activated, and the LIZn@MCo@M nanoparticle group has the highest number of activated immune cells.

[0142] In summary, the dual-ion-based nanoparticles (LIZn@MCo@M nanoparticles) provided in this application for treating p53-mutant tumors can target the tumor site through their outermost tumor cell membrane, resulting in higher efficacy, while also protecting the inner Co layer. 2+ Reduce Co 2+ Loss during the targeting process. The outermost tumor cell membrane can homologously target tumor cells, enter the cell via endocytosis, and release Zn in the acidic tumor cell environment. 2+ and Co 2+ This application utilizes a liposome-based biomimetic delivery system. Liposomes, as artificial cell membrane vesicles, possess high biosafety, good biocompatibility, and biodegradability, and can be used to target ions at tumor sites, thereby improving ion utilization and therapeutic efficacy. Furthermore, liposomes are relatively stable, a common organic drug delivery carrier, and possess nanoscale, biomembrane-like structures and excellent biocompatibility. Liposomes enter lysosomes, releasing Zn... 2+ and Co 2+ Zn 2+ and Co 2+ It promotes the production of ROS in cells, ubiquitinates and degrades mutant p53 protein, thereby relieving the inhibition of the cGAS-STING signaling pathway by mutant p53. Furthermore, Zn... 2+ and Co 2+ It can synergistically promote the cGAS-STING signaling pathway, activate the body's innate immunity, and ultimately achieve an anti-tumor effect. Therefore, this application connects mutant p53 and the cGAS-STING pathway, promotes the cGAS-STING innate immune pathway by reducing the expression of mutant p53, restores the function of the p53 tumor suppressor gene, achieves better tumor treatment effects, and has good application value in tumor treatment. Furthermore, the LIZn@MCo@M nanoparticles can be widely used in various p53 mutant tumors, without limitation on the type of tumor.

[0143] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A dual-ion-based nanoparticle for treating p53-mutant tumors, characterized in that, It includes liposomes, Zn 2+ Co 2+ The liposome has an inner tumor cell membrane and an outer tumor cell membrane, and the Zn... 2+ Enclosed within the liposome, the inner tumor cell membrane and the Co 2+ The complex is formed and coated on the outer layer of the liposome. The outer tumor cell membrane is coated on the outside of the complex. Both the inner and outer tumor cell membranes are pancreatic cancer cell membranes.

2. The dual-ion-based nanoparticles for treating p53-mutant tumors according to claim 1, characterized in that, The particle size of the dual-ion-based nanoparticles is 100-150 nm.

3. The dual-ion-based nanoparticles for treating p53-mutant tumors according to claim 1, characterized in that, The ion release pH of the dual-ion nanoparticles is less than or equal to 5.

5.

4. A method for preparing dual-ion-based nanoparticles for treating p53-mutant tumors as described in any one of claims 1-3, characterized in that, It includes: (1) Containing Zn 2+ The salt solution was hydrated with the liposome membrane, and the precipitate was separated and collected to prepare LIZn nanoparticles. (2) Containing Co 2+ The salt solution was mixed with the inner tumor cell membrane, the precipitate was separated and collected, and the MCo complex was obtained. (3) The LIZn nanoparticles are mixed with the MCo composite, the precipitate is separated and collected to obtain LIZn@MCo nanoparticles; (4) The LIZn@MCo nanoparticles are mixed with the outer tumor cell membrane, the precipitate is separated and collected to obtain LIZn@MCo@M nanoparticles.

5. The method for preparing dual-ion-based nanoparticles for treating p53-mutant tumors according to claim 4, characterized in that, The Zn-containing 2+ Zn in salt solution 2+ The concentration is 8-12 mg / mL, and the Zn content is... 2+ The volume-to-mass ratio of the salt solution to the DPPC in the raw material of the liposome membrane is 2 ml: 5-10 mg.

6. The method for preparing dual-ion-based nanoparticles for treating p53-mutant tumors according to claim 4, characterized in that, The containing Co 2+ Co in salt solution 2+ The concentration is 8-15 mg / mL, and the substance containing Co... 2+ The volume ratio of the salt solution to the inner tumor cell membrane is 400-600 μL: 1 mL.

7. The method for preparing dual-ion-based nanoparticles for treating p53-mutant tumors according to claim 4, characterized in that, The volume ratio of the LIZn nanoparticles to the MCo composite is 1:1-1.

5.

8. The method for preparing dual-ion-based nanoparticles for treating p53-mutant tumors according to claim 4, characterized in that, The volume ratio of the LIZn@MCo nanoparticles to the outer tumor cell membrane is 1.5-2.5:

1.

9. The method for preparing dual-ion-based nanoparticles for treating p53-mutant tumors according to claim 4, characterized in that, The preparation methods of the inner tumor cell membrane and the outer tumor cell membrane include: centrifuging the tumor cells for 3-8 minutes, washing them 2-4 times in buffer solution, adding sterile water to rupture the cells, sonicating them with a cell disruptor until the centrifuge tube wall is slightly heated, centrifuging at 5000-9000 rpm for 3-8 minutes, and collecting the supernatant.

10. The method for preparing dual-ion-based nanoparticles for treating p53-mutant tumors according to claim 9, characterized in that, The buffer solution is PBS with a pH of 7-8.

11. The method for preparing dual-ion-based nanoparticles for treating p53-mutant tumors according to claim 4, characterized in that, The mixing in steps (1) to (4) is carried out under eddy current and ultrasonic conditions.

12. The method for preparing dual-ion-based nanoparticles for treating p53-mutant tumors according to claim 4, characterized in that, The separation in steps (1) to (4) all involve centrifugation at 10,000-20,000 rpm for 20-40 minutes.

13. The method for preparing dual-ion-based nanoparticles for treating p53-mutant tumors according to claim 4, characterized in that, Step (3) before separation includes filtration 8-12 times sequentially using 0.7-0.9μM, 0.4-0.5μM, and 0.1-0.2μM filter membranes.

14. The method for preparing dual-ion-based nanoparticles for treating p53-mutant tumors according to claim 4, characterized in that, Step (4) before separation includes filtration 4-6 times using 0.7-0.9μM, 0.4-0.5μM, and 0.1-0.2μM filter membranes in sequence.

15. The method for preparing dual-ion-based nanoparticles for treating p53-mutant tumors according to claim 4, characterized in that, The method for preparing the liposome film includes: dissolving DPPC and cholesterol in an organic solvent to form a reaction solution, and evaporating the reaction solution under reduced pressure to obtain a liposome film, wherein the mass-volume ratio of DPPC, cholesterol and organic solvent is 5-10 mg: 1-5 mg: 10 ml.

16. The use of the dual-ion-based nanoparticles for treating p53-mutant tumors prepared by the method of preparing dual-ion-based nanoparticles for treating p53-mutant tumors as described in any one of claims 1-3, or the dual-ion-based nanoparticles for treating p53-mutant tumors as described in any one of claims 4-15, in the preparation of a medicament for treating p53-mutant tumors, wherein the p53-mutant tumor is pancreatic cancer.

Images