Mannose-modified CPD12C15 lipid nanoparticles and application thereof in preparation of anti-gastric cancer drugs

Abstract

The invention relates to the field of pharmaceutical preparations, in particular to mannose-modified CPD12C15 lipid nanoparticles and application of the mannose-modified CPD12C15 lipid nanoparticles in preparation of anti-gastric cancer drugs. The mannose modified CPD12C15 lipid nanoparticle disclosed by the invention is prepared from CPD12C15, phospholipid, mannose modified DSPE-PEG2000 (Distearoyl Pyrrolidone-Polyethylene Glycol 2000) and a surface active agent, wherein the CPD12C15 is encapsulated in the lipid nanoparticle, and the mannose part of the mannose modified DSPE-PEG2000 is exposed on the surface of the lipid nanoparticle; the problems that CPD12C15 is poor in water solubility, low in-vivo stability, low in bioavailability and the like are solved, meanwhile, by means of specific binding of mannose and a mannose receptor over-expressed on the surface of gastric cancer cells, active targeting delivery of drugs to tumor cells is achieved, and the drug delivery efficiency is improved. And a new thought and an experimental basis are provided for clinical application of CPD12C15 in gastric cancer treatment.

Description

Technical Field

[0001] The present application relates to the field of pharmaceutical preparations, and specifically to mannose-modified CPD12C15 lipid nanoparticles and their application in the preparation of anti-gastric cancer drugs. Background Art

[0002] Any discussion of the prior art throughout the specification should not be considered as an admission that such prior art is widely known or forms part of the common general knowledge in the field.

[0003] Gastric cancer is one of the most common malignant tumors worldwide, and its treatment presents numerous challenges. Although surgery is the primary treatment for early-stage gastric cancer, due to the lack of obvious early symptoms, most patients are diagnosed in the advanced stages, missing the opportunity for surgery. Chemotherapy is the primary treatment option for patients with advanced gastric cancer, but traditional chemotherapy drugs generally suffer from serious drawbacks such as poor selectivity, high systemic toxicity, and the development of drug resistance. This results in a low five-year survival rate for patients, necessitating the development of new, highly effective, and low-toxic anti-gastric cancer drugs.

[0004] Cisplatin, as a first-line chemotherapy drug for gastric cancer treatment, has a good initial response rate, but faces two major challenges. The first is severe toxic side effects, including nephrotoxicity, neurotoxicity, ototoxicity, and severe gastrointestinal reactions such as nausea and vomiting. About 30-40% of patients cannot tolerate these side effects and need to reduce the dose or even interrupt treatment. The second is the problem of acquired drug resistance. Studies have shown that about 50-70% of gastric cancer patients develop resistance after 4-6 cycles of cisplatin treatment, leading to treatment failure. The mechanisms of cisplatin resistance mainly include reduced drug uptake, increased drug efflux, enhanced DNA repair capacity, and inhibition of apoptosis. How to overcome cisplatin resistance has become a key challenge in the treatment of gastric cancer.

[0005] The "repurposing" strategy for old drugs has garnered significant attention in recent years in the field of anti-cancer drug development. Disulfiram and its derivatives, as a class of compounds with promising anti-tumor potential, have demonstrated anticancer activity in various tumor models. However, these compounds suffer from significant drawbacks: poor water solubility, low stability in the bloodstream, susceptibility to metabolic inactivation, and limited bioavailability. In particular, the disulfiram derivative CPD12C15, while superior to disulfiram itself in antitumor activity, suffers from the same pharmacokinetic issues mentioned above, resulting in its in vivo anti-tumor efficacy far inferior to in vitro results, severely limiting its clinical application prospects.

[0006] Nano drug delivery systems are seen as a potential solution to the problem of poorly soluble drug delivery. By encapsulating drugs in nanocarriers, the solubility and stability of drugs can theoretically be improved. However, existing nano delivery systems still have many drawbacks: First, the preparation process of many nanoformulations is complex, requiring the use of large amounts of organic solvents, and making large-scale production difficult; second, the encapsulation efficiency of nanocarriers is generally low, resulting in low drug utilization; third, traditional nanoformulations have poor stability in the body's circulation and are easily cleared by the reticuloendothelial system, making it difficult to maintain effective blood drug concentrations; most importantly, most nanocarriers rely solely on the EPR effect for passive targeting, which is insufficient for targeting specific types of tumor cells, making it difficult to achieve precise drug delivery.

[0007] Existing treatment strategies are particularly inadequate for gastric cancer, a specific tumor type. Gastric cancer tissue and normal tissue differ in physiological and pathological characteristics, but existing delivery systems fail to effectively utilize these differences for targeted delivery. Furthermore, the gastric cancer microenvironment is complex, and existing delivery systems often fail to effectively respond to the characteristics of the tumor microenvironment, making it difficult to release drugs at specific locations. In particular, there is insufficient research on targeted delivery of specific receptors on the surface of gastric cancer cells, failing to fully utilize the advantages of active targeting, resulting in insufficient drug accumulation in gastric cancer tissue and limited therapeutic efficacy. Summary of the Invention

[0008] The present invention aims to solve the key technical problems faced by existing gastric cancer therapeutics, such as poor water solubility, low targeting, and high systemic toxicity. As a potential anti-tumor drug, the disulfiram derivative CPD12C15 has shown good anti-cancer activity in in vitro studies. However, due to its extremely low water solubility, poor bioavailability, and lack of specific targeting, its direct application in in vivo treatment faces major challenges. At the same time, traditional nano drug delivery systems mostly rely solely on the EPR effect for passive tumor targeting and lack an active targeting mechanism, resulting in limited drug enrichment efficiency in tumor tissue. In addition, many existing lipid nanoparticles have complex preparation processes and low drug encapsulation rates, making it difficult to meet the needs of efficient, scalable production and clinical application.

[0009] To overcome the above problems, the present invention provides a mannose-modified CPD12C15 lipid nanoparticle and its preparation method, composition, kit and application. The lipid nanoparticle uses lecithin as the main lipid and mannose-modified DSPE-PEG2000 as the targeting lipid. By presenting mannose ligands on the particle surface, it achieves active targeting of gastric cancer cells and tumor-associated macrophages that highly express mannose receptors (MR). The present invention precisely controls the molar ratio of CPD12C15 to lipid components (1:2 to 1:5, preferably 1:2) and the molar ratio of soy lecithin to mannose-modified DSPE-PEG2000 (20:1 to 35:1, preferably 30:1). Through this precise ratio design, the efficient encapsulation ability and stable structure of the drug delivery system are ensured. At the same time, the physicochemical parameters of the nanoparticles are precisely controlled, including particle size (within the range of 60-75nm), PDI (less than 0.2), Zeta potential (between -20 and -30mV), encapsulation efficiency (70-80%) and drug loading (12-15%). These parameter optimizations are based on a deep understanding of tumor penetration and drug release kinetics, laying the foundation for efficient targeted delivery.

[0010] The present invention adopts a low-temperature nanoprecipitation-ultrasound combined method, combined with a surface modification strategy, to establish a stable, efficient and reproducible preparation process. This process slowly adds the organic phase (containing CPD12C15) dropwise to the aqueous phase (containing lipids and surfactants), and ensures that the nanoparticles have an ideal particle size distribution and structural stability by precisely controlling the ultrasonic time (preferably 3 minutes), stirring conditions and organic solvent removal process. Compared with traditional thin film dispersion methods or emulsification-solvent evaporation methods, this process is simple to operate, has mild conditions, low energy consumption, good scalability, and significantly reduces the amount of organic solvent used, making it more environmentally friendly and economical. Three batches of reproducible experiments showed that the fluctuation of the key quality indicators of the prepared nanoparticles was less than 5%, confirming that the process is robust and reliable and suitable for large-scale production. In addition, the present invention also innovatively designed a medical kit containing lipid nanoparticles and copper salts, based on the mechanism of action of CPD12C15 and copper ions synergistically inducing endoplasmic reticulum stress and inhibiting the ubiquitin-proteasome system, supporting flexible combined drug delivery modes to maximize the synergistic anti-tumor effect.

[0011] Systematic experimental studies have shown that the mannose-modified CPD12C15 lipid nanoparticles of the present invention have achieved significant technical effects. First, the system significantly improved the water solubility and blood circulation stability of CPD12C15, achieved a pH-responsive sustained-release effect, and maintained stable physical and chemical properties for 14 days at 4°C, providing a solid foundation for clinical application. Secondly, through mannose receptor-mediated active targeting, the uptake efficiency of nanoparticles by gastric cancer cells was increased by about 2.5 times. The mannose receptor competitive inhibition experiment confirmed the specificity of the targeting mechanism, and in vivo imaging further showed that the enrichment of the drug in gastric cancer tissue was significantly increased compared with non-targeted nanoparticles. Third, in terms of anti-tumor activity, the IC of M-LNP-CPD12C15 combined with copper ions on gastric cancer cells was 0.0447 W / v. 50 The values (MKN-45: 0.304±0.007μM, SGC-7901: 0.335±0.025μM) were significantly lower than those of unencapsulated CPD12C15 and the traditional chemotherapy drug cisplatin. In a nude mouse gastric cancer xenograft model, the high-dose M-LNP-CPD12C15 combined with copper ions achieved a tumor inhibition rate of 73.81%, significantly superior to the cisplatin group (53.92%) and the unmodified CPD12C15 group, indicating that this system can also achieve good anti-tumor effects in vivo.

[0012] The pharmaceutical composition of the present invention also has excellent biocompatibility and safety. Hemolysis experiments showed that the hemolysis rate was less than 5% at different concentrations, confirming that intravenous administration is safe. Compared with cisplatin, there were no obvious pathological changes in the main organs of nude mice after high-dose administration, renal function indicators were normal, and there was no abnormal increase in the expression of NF-κB and inflammatory factors, completely avoiding the common nephrotoxicity problem of cisplatin. Mechanism of action studies have shown that M-LNP-CPD12C15 combined with copper ions inhibits the ubiquitin-proteasome system, causing the accumulation of ubiquitinated proteins, thereby inducing irreversible endoplasmic reticulum stress, activating the p-EIF2α / ATF4 / CHOP apoptosis pathway, and ultimately leading to apoptosis of gastric cancer cells through caspase-3 activation. This unique mechanism of action enables the drug system of the present invention to have a significant inhibitory effect on gastric cancer cells resistant to traditional chemotherapy, providing a new strategy for overcoming chemotherapy resistance.

[0013] In summary, the present invention has constructed a mannose-modified CPD12C15 lipid nanoparticle system that combines high encapsulation efficiency, excellent particle size distribution, active targeting, and excellent biocompatibility. Through mannose receptor-mediated active targeted delivery and a copper ion synergistic mechanism, this system successfully overcomes key technical challenges in traditional CPD12C15 applications, such as poor water solubility, insufficient targeting, and high systemic toxicity. This system provides an innovative strategy for the precision treatment of gastric cancer and establishes a technical platform for the development of nanomedicines for the targeted delivery of disulfiram derivatives. The design concepts and technical solutions of this invention can be expanded to the development of delivery systems for other poorly soluble anti-tumor drugs, demonstrating broad application prospects and industrial potential.

[0014] Specifically, the present invention provides the following technical solutions.

[0015] The first aspect of the present invention provides a mannose-modified CPD12C15 lipid nanoparticle comprising the following components:

[0016] Active ingredient CPD12C15;

[0017] phospholipids, preferably soy lecithin;

[0018] Mannose-modified DSPE-PEG2000;

[0019] The surfactant is preferably a nonionic surfactant, more preferably polysorbate 80 (also known as Tween 80).

[0020] The lipid nanoparticles have a spherical structure, CPD12C15 is encapsulated inside the lipid nanoparticles, and the mannose portion of the mannose-modified DSPE-PEG2000 is exposed on the surface of the nanoparticles, thereby giving the lipid nanoparticles the ability to actively target tumor cells and tumor-associated macrophages that highly express the mannose receptor (MR).

[0021] The structure of CPD12C15 is as follows:

[0022]

[0023] Furthermore, to optimize the stability and drug loading performance of lipid nanoparticles, in some embodiments, the molar ratio of CPD12C15 to the lipid component (i.e., the total amount of soy lecithin and mannose-modified DSPE-PEG2000) is 1:2 to 1:5, preferably 1:2. This ratio ensures the stability of the liposome structure while balancing efficient drug loading and particle size control, avoiding lipid bilayer destruction or abnormal particle size increase caused by excessive drug loading.

[0024] In some preferred embodiments, the molar ratio of soybean lecithin to mannose-modified DSPE-PEG2000 in the lipid nanoparticles is controlled between 20:1 and 35:1, preferably 30:1. This setting can ensure the stability of the lipid nanoparticles while giving them excellent targeting, taking into account both particle size control and blood circulation time, and further improving the efficiency of drug accumulation in tumor tissue.

[0025] Furthermore, in a preferred embodiment of the present invention, the lipid nanoparticles have the following physicochemical properties:

[0026] The average particle size is 60-75 nm, preferably 65-70 nm. This particle size range can take into account the EPR effect to promote tumor accumulation, while reducing the clearance of the reticuloendothelial system, increasing the circulation time in the blood and the accumulation in tumor tissue.

[0027] The zeta potential is -20 to -30 mV, preferably -20 to -25 mV. A moderately negative potential helps prevent the nanoparticles from self-aggregating, maintain good dispersion, and avoid serum protein adsorption and clearance caused by strong negative charge.

[0028] The encapsulation efficiency of CPD12C15 is 60-80%, preferably 70-80%. A high encapsulation efficiency can effectively improve drug utilization and reduce drug loss.

[0029] The drug loading of CPD12C15 is 10-15%, preferably 12-15%. A moderate drug loading can ensure the therapeutic dose while maintaining the structural integrity and physiological stability of the lipid nanoparticles.

[0030] The concentration of the surfactant polysorbate 80 is 0.5-2.0%, preferably 2.0%. A reasonable concentration of surfactant can further optimize particle size control and particle size distribution, improve formulation uniformity, and avoid the formation of overly large aggregates.

[0031] In some preferred embodiments, the mannose-modified DSPE-PEG2000 is prepared by reacting DSPE-PEG2000-NH2 with α-D-mannopyranosylphenylisothiocyanate (MPITC), and the specific reaction steps include:

[0032] MPITC was dissolved in methanol and triethylamine was added as a catalyst. DSPE-PEG2000-NH2 was dissolved in dichloromethane. The DSPE-PEG2000-NH2 solution was added dropwise to the MPITC solution and reacted at room temperature. After the reaction, the organic solvent was removed and the product was dissolved in DMSO and water. After dialysis to remove small molecule impurities, the product was freeze-dried to obtain mannose-modified DSPE-PEG2000.

[0033] In a second aspect of the present invention, a method for preparing mannose-modified CPD12C15 lipid nanoparticles is provided, which comprises: dissolving phospholipids and mannose-modified DSPE-PEG2000 in an aqueous solution containing a surfactant to form an aqueous phase; dissolving CPD12C15 in an organic solvent to form an organic phase; slowly adding the organic phase dropwise to the aqueous phase under stirring; ultrasonically treating the mixed solution; stirring to volatilize the organic solvent; and filtering to obtain mannose-modified CPD12C15 lipid nanoparticles.

[0034] The low-temperature nanoprecipitation-ultrasound combined method adopted by the present invention has significant advantages over traditional thin film dispersion methods or emulsification-solvent evaporation methods, such as simple operation, mild conditions, low energy consumption, and good scalability. In addition, the amount of organic solvent used is significantly reduced, making it more environmentally friendly and economical. During the preparation process, the preferred embodiment uses a non-ionic surfactant, more preferably polysorbate 80, whose concentration is controlled within the range of 0.5-2.0%, preferably 2.0%. Experimental results show that as the concentration of polysorbate 80 increases from 0.5% to 2.0%, the encapsulation efficiency increases from 57.28% to 71.31%, indicating that an appropriate concentration of surfactant can significantly improve the formation efficiency and drug encapsulation ability of nanoparticles.

[0035] In some embodiments of the present invention, the organic solvent is one or more of methanol, ethanol, acetone or acetonitrile, preferably methanol. These low-toxicity solvents can not only effectively dissolve CPD12C15, but also facilitate subsequent removal, reducing the impact of residual solvents on biosafety. The ultrasonic treatment step is crucial for controlling the particle size distribution of the nanoparticles, and is preferably controlled within the range of 1-7 minutes, more preferably 3 minutes. Experiments have shown that when the ultrasonic time is extended from 1 minute to 3 minutes, the encapsulation efficiency increases from 68.91% to 73.24%; but when it is further extended to 5 minutes and 7 minutes, the encapsulation efficiency decreases, indicating that 3 minutes is the optimal ultrasonic treatment time.

[0036] Complete removal of the organic solvent is also crucial for producing high-quality nanoparticles. Therefore, stirring to volatilize the organic solvent is preferably performed for 4-8 hours, more preferably 6 hours. This step utilizes magnetic stirring at an appropriate speed (typically 200-400 rpm) to ensure uniform solvent evaporation without disrupting the nanostructure. Finally, the mixture is filtered through a 0.22 μm membrane to remove any large particle aggregates and obtain a uniform final product size distribution.

[0037] The nanoparticles prepared by the above method have the characteristics of small particle size, narrow distribution, high encapsulation efficiency, and good targeting. The entire process is simple and easy to control, suitable for large-scale production. The results of three batches of reproducible experiments showed that the encapsulation efficiency of the prepared nanoparticles was 73.02±0.70%, the drug loading was 12.06±0.55%, the particle size was 67.33±2.53nm, the PDI was 0.20±0.04, and the Zeta potential was -23.27±3.94mV. The fluctuation of each indicator was less than 5%, confirming that the process is robust and reliable, with good reproducibility and industrialization potential. In addition, stability studies showed that the encapsulation efficiency and drug loading of the prepared nanoparticles did not change significantly within 14 days at 4°C, the particle size only increased slightly, and the appearance remained clear, indicating that the product has good storage stability.

[0038] In the third aspect of the present invention, a pharmaceutical composition is provided, comprising the mannose-modified CPD12C15 lipid nanoparticles described in the first aspect.

[0039] In some embodiments of the present invention, the pharmaceutical composition comprises: a) the mannose-modified CPD12C15 lipid nanoparticles of claim 1 or 2; and b) a pharmaceutically acceptable copper salt. This combination, based on the synergistic anti-tumor effects of CPD12C15 and copper ions, can significantly enhance therapeutic efficacy.

[0040] In some embodiments of the present invention, in the pharmaceutical composition, the molar ratio of the copper salt to CPD12C15 is 0.5-2:1, preferably 1:1. Experimental results show that when copper ions and CPD12C15 are combined at a molar ratio of 1:1, the proliferation inhibition effect on gastric cancer cells MKN-45 and SGC-7901 is the best, IC 50 The values were 0.304±0.007μM and 0.335±0.025μM, respectively, which were significantly lower than those of CPD12C15 alone or the traditional chemotherapy drug cisplatin.

[0041] Regarding the choice of copper salt, the present invention preferably uses copper gluconate, copper chloride, or a combination thereof. These copper salts have good water solubility and biocompatibility, making them suitable for clinical administration. In particular, copper gluconate, a commonly used copper supplement with extensively validated safety, is suitable for oral administration; while copper chloride is suitable for preparing injectable solutions, providing options for different routes of administration.

[0042] Considering that the long-term coexistence of copper ions and CPD12C15 may affect formulation stability, the present invention prefers to mix component a) and component b) immediately prior to use. This design not only addresses the issue of long-term storage stability but also provides clinicians with the flexibility to adjust the copper salt dosage based on individual patient needs, enhancing personalized treatment.

[0043] The results of in vitro and in vivo experiments fully confirmed the synergistic effect of copper salt and mannose-modified CPD12C15 lipid nanoparticles. In terms of gastric cancer cell proliferation inhibition, migration inhibition, invasion inhibition, and apoptosis induction, the M-LNP-CPD12C15+Cu group showed significantly better effects than the single drug group and cisplatin group. The mechanism of action study showed that this synergistic effect mainly increases ROS and Ca 2+ levels, inducing sustained endoplasmic reticulum stress and activating the p-EIF2α / ATF4 / CHOP apoptosis pathway, providing new strategies and ideas for the treatment of gastric cancer.

[0044] The fourth aspect of the present invention provides a pharmaceutical preparation comprising the aforementioned mannose-modified CPD12C15 lipid nanoparticles and a pharmaceutically acceptable carrier. The preparation can be designed into different dosage forms such as injections and lyophilized powder injections according to clinical needs, providing a variety of options for clinical applications. Pharmaceutically acceptable carriers may include, but are not limited to, physiological saline, glucose injection, water for injection, etc. These carriers have good biocompatibility and are suitable for intravenous administration. As a preferred route of administration, intravenous injection can avoid gastrointestinal degradation that may result from oral administration, ensure sufficient drug bioavailability, and utilize the targeting properties of mannose-modified nanoparticles to achieve efficient enrichment of the drug in gastric cancer tissue. In the fifth aspect of the present invention, a medical kit is provided, comprising: a first container containing the mannose-modified CPD12C15 lipid nanoparticles according to claim 1 or 2; and a second container containing a pharmaceutically acceptable copper salt solution; the two containers are designed for simultaneous, separate, or sequential administration.

[0045] In clinical applications, this set design provides physicians with flexible dosing options, allowing them to adjust dosing regimens based on individual patient conditions for personalized treatment. For example, a combination of intravenous CPD12C15 and oral copper gluconate can be used. Oral copper ion absorption is stable and safe, while intravenous nanoparticles can directly enter the bloodstream, avoiding the first-pass effect and maximizing drug bioavailability. In addition, the set comes with instructions for different dosing regimens, providing detailed dosage recommendations, dosing intervals, and precautions to ensure safe and effective use.

[0046] Preferably, the pharmaceutically acceptable copper salt is selected from copper gluconate, copper chloride, or a combination thereof. Copper gluconate, a commonly used copper supplement, has been extensively validated for safety and is suitable for oral administration; copper chloride, on the other hand, can be used to formulate injectable solutions, providing options for different routes of administration. Both copper salts can synergize with CPD12C15, promoting apoptosis in gastric cancer cells by inhibiting the ubiquitin-proteasome system.

[0047] In the sixth aspect of the present invention, the use of the mannose-modified CPD12C15 lipid nanoparticles described in the first aspect, the pharmaceutical composition described in the third aspect, the pharmaceutical preparation described in the fourth aspect, or the medical kit described in the fifth aspect in the preparation of the following drugs is provided: (1) a drug for treating tumors with high mannose receptor expression; (2) a drug for inhibiting the proliferation of gastric cancer cells; (3) a drug for inhibiting the migration or invasion of gastric cancer cells; (4) a drug for inducing apoptosis of gastric cancer cells; (5) a drug for reducing gastric cancer-related ROS levels and maintaining cellular GSH homeostasis; (6) a drug for reducing the expression levels of gastric cancer-related inflammatory factors IL-6 and / or IL-1β; (7) a drug for reversing cisplatin resistance or reducing cisplatin-related nephrotoxicity. In some embodiments of the present invention, the tumor with high mannose receptor expression is gastric cancer. Based on the results of Western blot experiments, it was confirmed that the expression of mannose receptor in MKN-45 and SGC-7901 gastric cancer cells was significantly higher than that in normal HUVEC cells, providing a biological basis for mannose-targeted design. In addition, the preferred route of administration is intravenous injection to ensure that the drug efficiently reaches the tumor tissue and exerts a therapeutic effect.

[0048] In an in vivo tumor model experiment, the relative tumor proliferation rate of the high-dose M-LNP-CPD12C15 combined with copper ions group was only 26.19%, equivalent to a tumor inhibition rate of 73.81%, significantly better than the cisplatin group (53.92%) and the unmodified CPD12C15 combined with copper ions group (64.44%). At the same time, safety evaluation confirmed that compared with the cisplatin group, the M-LNP-CPD12C15 combined with copper ions group did not cause nephrotoxicity, serum renal function indicators were normal, there were no obvious pathological changes in renal tissue, and there was no abnormal increase in the expression of NF-κB and inflammatory factors, providing safety guarantees for clinical application.

[0049] It is worth noting that while this invention is primarily designed for gastric cancer, since mannose receptors are highly expressed in a variety of tumors, such as liver cancer and non-small cell lung cancer, the technical solution of this invention is expected to be expanded to the treatment of various solid tumors and has broad applicability. Furthermore, the design concept and technical solution of this invention can also be used to develop delivery systems for other poorly soluble anti-tumor drugs, providing an innovative strategy and technical platform for overcoming the challenges of poorly soluble drug delivery.

[0050] Compared with the prior art, the advantages of the present invention include:

[0051] Preparation Process Advantages: This method utilizes a combined nanoprecipitation-ultrasound method to prepare M-LNP-CPD12C15. Compared to traditional thin-film dispersion or emulsification-solvent evaporation methods, this method offers ease of operation, milder conditions, lower energy consumption, and improved scalability. It also eliminates the need for large amounts of organic solvents, making it more environmentally friendly and economical. The optimized preparation process demonstrates excellent reproducibility, with fluctuations in encapsulation efficiency and drug loading across three batches of preparations less than 5%, demonstrating a robust and reliable process.

[0052] Physical and chemical performance advantages: The M-LNP-CPD12C15 prepared by the present invention has a small particle size (approximately 67 nm), which is significantly smaller than traditional liposomes (usually ≥100 nm), making it easier to penetrate the gaps between tumor vascular endothelial cells and deep tissues. At the same time, the surface Zeta potential is moderate (around -23 mV), which can maintain good dispersion stability while preventing blood component interactions due to excessive charge. In addition, the encapsulation efficiency (up to 73% and above) and drug loading (up to 12% and above) of the present invention are both higher than those of existing related nanoformulations (usually encapsulation efficiency <60% and drug loading <10%), thereby improving drug utilization efficiency.

[0053] Targeting Advantages: Compared to unmodified or other modified (e.g., PEG-modified) nanoparticles, the mannose-modified strategy of this invention specifically recognizes the mannose receptors highly expressed on the surface of gastric cancer cells, enabling active targeted delivery and significantly improving drug uptake efficiency in target cells (by approximately 2.5 times). While many studies have used targeting ligands such as folic acid or RGD peptides, these receptors are also expressed to some extent in normal tissues. The highly specific expression of mannose receptors on gastric cancer cells provides this invention with more precise targeting capabilities.

[0054] Advantages of drug release characteristics: The M-LNP-CPD12C15 of the present invention exhibits pH-responsive release characteristics in in vitro release experiments. The release rate at the pH of the tumor microenvironment (about 6.8) is higher than that at the physiological pH (about 7.4). This intelligent responsive release mode is superior to the nonspecific release of traditional nanocarriers. While ensuring the stability of the drug blood circulation, it can achieve selective release at the tumor site, further improving the therapeutic index.

[0055] Clinical Application Potential: By encapsulating CPD12C15 within mannose-modified lipid nanoparticles, this invention not only addresses the poor water solubility and low stability of the original drug but also improves its tumor targeting, potentially reducing systemic toxicity while enhancing efficacy. Furthermore, the materials used in this invention are all FDA-approved excipients for injections with excellent biocompatibility. Hemolysis experiments have confirmed their safety, demonstrating promising clinical translational potential.

[0056] Scope of application advantage: Although the present invention is mainly designed for gastric cancer cells, since mannose receptors are highly expressed in a variety of tumors, such as liver cancer and non-small cell lung cancer, the technical solution of the present invention is expected to be expanded to the treatment of a variety of solid tumors and has wide applicability.

[0057] In summary, the present invention not only overcomes the inherent defects of CPD12C15 in the delivery of anti-tumor drugs, but also achieves efficient targeted delivery to gastric cancer cells through a precisely designed mannose-targeted modification strategy, providing an innovative technical platform for the development of new, efficient, and low-toxic gastric cancer treatment options. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] The drawings constituting part of the present application are provided to provide a further understanding of the present application. The illustrative embodiments and their descriptions of the present application are provided to explain the present application and do not constitute an undue limitation on the present application. The following describes the implementation scheme of the present application in detail in conjunction with the drawings, wherein:

[0059] Figure 1 :Synthesis route of Man-DSPE-PEG2000, DSPE-PEG2000-NH2, MPITC and Man-DSPE-PEG2000 1 H NMR spectrum.

[0060] Figure 2 : Preparation Examples 1-13 encapsulation efficiency and drug loading results.

[0061] Figure 3 : Particle size distribution and potential diagram of M-LNP-CPD12C15.

[0062] Figure 4 : Visual image of M-LNP-CPD12C15 solution and TEM morphology observation.

[0063] Figure 5 : Release profiles of CPD12C15 drug substance and M-LNP-CPD12C15 at pH 7.4 and pH 6.8.

[0064] Figure 6 : Hemolysis of different concentrations of M-LNP-CPD12C15 (5, 10, 20, 30, 40 μg / mL).

[0065] Figure 7 : Expression of mannose receptor in gastric cancer cells and normal cells.

[0066] Figure 8 : Experimental results of C6-M-LNP uptake in MKN-45 cells.

[0067] Figure 9: Cytotoxicity of M-LNPs against MKN-45 and SGC-7901 cells.

[0068] Figure 10 : Cytotoxicity experimental results of CPD12C15+Cu (left) and DDP (right) on MKN-45 cells.

[0069] Figure 11 : Cytotoxicity experimental results of CPD12C15+Cu (left) and DDP (right) on SGC-7901 cells.

[0070] Figure 12 Effects of different drugs on MKN-45 cell migration. (a) Image of MKN-45 cell scratch healing after drug administration (scale: 500 μm); (b) Changes in MKN-45 cell wound healing rate in each group after drug administration. *p < 0.05, ***p < 0.001 compared with the control group.

[0071] Figure 13 Effects of different drugs on SGC-7901 cell migration. (a) SGC-7901 cell scratch healing images after drug administration (scale: 500 μm). (b) Changes in the wound healing rate of SGC-7901 cells in each group after drug administration. **p<0.01, ***p<0.001 compared with the control group.

[0072] Figure 14 Effects of different drugs on the invasion ability of MKN-45 cells. (a) MKN-45 cell invasion image after drug administration (scale: 100 μm); (b) Changes in the number of MKN-45 cells in each group after drug administration. ***p < 0.001 compared with the control group.

[0073] Figure 15 Effects of different drugs on the invasion ability of SGC-7901 cells. (a) SGC-7901 cell invasion image after drug administration (scale: 100 μm). (b) Changes in the number of invasive SGC-7901 cells in each group after drug administration. ***p < 0.001 compared with the control group.

[0074] Figure 16 Figure 3: Results of different drugs promoting MKN-45 cell apoptosis. (a) MKN-45 cell apoptosis after drug administration. (b) Changes in MKN-45 cell apoptosis rate in each group after drug administration. ***p<0.001 compared with the control group.

[0075] Figure 17 Figure 3: Results of different drugs promoting apoptosis in SGC-7901 cells. (a) SGC-7901 cell apoptosis after drug administration; (b) Changes in the apoptosis rate of SGC-7901 cells in each group after drug administration. ***p < 0.001 compared with the control group.

[0076] Figure 18 : Results of different drugs upregulating ROS in MKN-45 cells, compared with the control group, ***p<0.001.

[0077] Figure 19 : Results of different drugs upregulating ROS in SGC-7901 cells, compared with the control group, **p<0.01, ***p<0.001.

[0078] Figure 20 :Different drugs upregulate Ca2+ in MKN-45 cells 2+ ***p<0.001 compared with the control group.

[0079] Figure 21 :Different drugs upregulate Ca in SGC-7901 cells 2+ ***p<0.001 compared with the control group.

[0080] Figure 22 : Results of M-LNP-CPD12C15+Cu-induced changes in the endoplasmic reticulum of MKN-45 and SGC-7901 cells.

[0081] Figure 23 : Changes of ubiqutin in gastric cancer cells after drug administration. (a) Ubiqutin expression and quantitative analysis in MKN-45 cells after drug administration; (b) Ubiqutin expression and quantitative analysis in SGC-7901 cells after drug administration. **p<0.01, ***p<0.001 compared with the control group.

[0082] Figure 24 : Changes in p-EIF2α, ATF4, ATF6, CHOP, and cleaved caspase 3 in gastric cancer cells MKN-45 (a) and SGC-7901 (b) after drug administration, compared with the control group, *p<0.05, **p<0.01, ***p<0.001.

[0083] Figure 25 Figure 3: Changes in nude mouse tumors after drug administration in each group. (a) Photos of tumors in each group after drug administration; (b) Changes in tumor volume after drug administration; (c) Tumor weight after drug administration; (d) H&E staining of tumors (scale: 100 μm). **p<0.01, ***p<0.001 indicate significant differences compared with the Model group.

[0084] Figure 26: Tumor apoptosis in nude mice after drug administration in each group, including TUNEL apoptosis images of tumor tissue (scale 100μm) and TUNEL apoptosis quantitative analysis results. Compared with the Model group, **p<0.01, ***p<0.001.

[0085] Figure 27 Ubiqutin, CHOP, and cleaved caspase 3 expression levels in nude mouse tumors, including immunohistochemistry and quantitative analysis. *p<0.05, **p<0.01, ***p<0.001 compared to the model.

[0086] Figure 28 In vivo safety assessment results after administration of the drug to each group, including changes in nude mouse body weight and H&E staining of major organs in different groups after administration. **p<0.01 compared with the Model group.

[0087] Figure 29 : Results of renal injury index detection. Compared with the Model group, **p<0.01, ***p<0.001.

[0088] Figure 30 : Expression and quantitative analysis of NF-κB in renal tissue after drug administration, as well as detection of inflammatory factors IL-6 and IL-1β in renal tissue. Compared with the Model group, *p<0.05, **p<0.01. DETAILED DESCRIPTION

[0089] The present application will be further described in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present application only and are not intended to limit the scope thereof. Experimental methods for which specific conditions are not specified in the examples are generally performed under conventional conditions or conditions recommended by the manufacturer.

[0090] Unless otherwise defined, all technical terms and scientific terms used in this application should have the meaning familiar to those skilled in the art. Unless otherwise specified, the reagents or raw materials used in this application can be obtained by conventional means and used in accordance with conventional methods or product specifications in this area. In addition, any content similar to or equivalent to the methods or materials described can be applied to the methods of this application. The preferred embodiments and materials described in this application are for illustrative purposes only.

[0091] CPD12C15 was prepared according to the method disclosed in Chinese patent CN116444408A, with a purity of >95%.

[0092] Example 1 Synthesis and Characterization of Mannose-Modified DSPE-PEG2000 (Man-DSPE-PEG2000)

[0093] 5 mg of α-D-mannopyranosylphenyl isothiocyanate (MPITC) (Beijing Mairida Technology Co., Ltd.) was dissolved in 2 mL of methanol, and triethylamine was added as a catalyst. 15.5 mg of DSPE-PEG2000-NH2 (Beijing Mairida Technology Co., Ltd.) was dissolved in 0.5 mL of dichloromethane and added dropwise to the methanol solution containing MPITC under magnetic stirring, and reacted at room temperature for 24 hours. After the reaction, the organic solvent was removed by rotary evaporation, a small amount of DMSO and water were added to dissolve the product, and it was placed in a dialysis bag for 48 hours to remove unreacted MPITC, and the dialysate was collected and freeze-dried to obtain the final product. 1 H-NMR (deuterated DMSO) verifies the product structure. Figure 1 The results showed that the product contained characteristic peaks on DSPE-PEG2000 and hydrogen on the phenyl group of MPITC, and the chemical shift of hydrogen was different from that of the phenyl group on the raw material MPITC, indicating that Man-DSPE-PEG2000 was successfully synthesized.

[0094] Example 2 Preparation of CPD12C15 lipid nanoparticles

[0095] Preparation method:

[0096] Accurately weigh appropriate amounts of soybean lecithin and DSPE-PEG2000 and ultrasonically dissolve them in 1% Tween 80 aqueous solution. Accurately weigh appropriate amounts of CPD12C15 powder and dissolve it in methanol. Slowly add methanol dropwise to the above aqueous solution using a 1 mL syringe under magnetic stirring. Continue stirring for 10 minutes after the addition is completed. Ultrasonicate with a probe for 1 minute in an ice bath, then magnetically stir at room temperature for 6 hours to evaporate the organic solvent. Pass the solution through a 0.22 μm filter membrane to obtain a lipid nanoparticle solution with a light blue opalescence. Seal the prepared lipid nanoparticles and store them in a refrigerator at 4°C.

[0097] CPD12C15 lipid nanoparticles were prepared according to the aforementioned preparation method. The preparation example is shown in Table 1.

[0098] Table 1

[0099]

[0100]

[0101] Determination of CPD12C15 content: The content of CPD12C15 was determined by high performance liquid chromatography.

[0102] Chromatographic conditions: chromatographic column: Phenomenex Luna 5u C18 column (5 μm, 250×4.6 mm); mobile phase: 0.2% formic acid water-acetonitrile (40:60, v / v); flow rate: 1 mL / min; detection wavelength: 284 nm; column temperature: 30°C; injection volume: 10 μL.

[0103] Preparation of stock solution: Accurately weigh 50 mg of CPD12C15 drug into a 50 mL volumetric flask, add acetonitrile to dissolve, and dilute to the scale with acetonitrile. After thorough mixing, a 1 mg / mL CPD12C15 stock solution is obtained. Store at 4°C until use.

[0104] To establish a standard curve: Use a pipette to precisely measure 0.125, 0.25, 0.50, 1.00, 2.00, 3.00, and 4.00 mL of CPD12C15 stock solution into a 10 mL volumetric flask. Dose to volume with acetonitrile and shake well to prepare a series of standard solutions with concentrations of 12.5, 25, 50, 100, 200, 300, and 400 μg / mL, respectively. Inject samples according to the chromatographic conditions and record the peak areas. Perform regression analysis using drug concentration as the abscissa and peak area as the ordinate to establish a standard curve.

[0105] Ultracentrifugation is used to determine the encapsulation efficiency and drug loading. Take an appropriate amount of lipid nanoparticle solution and add it to the ultrafiltration tube, centrifuge at 5000r / min for 10 minutes, dilute the solution obtained by centrifugation 25 times, pass it through a 0.22μm filter membrane, and use HPLC to determine the content. The peak area obtained is averaged and then substituted into the standard curve to calculate the concentration of free drugs. The total drug concentration is determined by the demulsification method. The lipid nanoparticle solution is diluted 25 times with acetonitrile, ultrasonically demulsified for 30 minutes, and then passed through a 0.22μm filter membrane. It is analyzed by HPLC. The peak area obtained is averaged and then substituted into the standard curve to calculate the concentration to obtain the total drug concentration. The encapsulation efficiency and drug loading are calculated according to the following formula: Encapsulation efficiency % = W drug / W total drug ×100%; drug loading % = W drug / (W total drug +W lipids )×100%. Among them, W drug W: The weight of the drug successfully encapsulated by the lipid nanoparticles, that is, the amount of CPD12C15 encapsulated in the lipid nanoparticles. total drug W: The total weight of the drug added during the preparation process, i.e. the total amount of CPD12C15 added. lipidsThe total weight of lipid materials used in the preparation of lipid nanoparticles, including the weight of all lipid components such as soybean lecithin, Man-DSPE-PEG2000, etc. The encapsulation efficiency and drug loading of each preparation example were investigated to screen out the optimal preparation process.

[0106] Drug-lipid ratio: CPD12C15, soybean lecithin, and DSPE-PEG2000 were accurately weighed to prepare CPD12C15 lipid nanoparticle solutions with different drug-lipid ratios, namely 1:2 (Preparation Example 1), 1:3 (Preparation Example 2), 1:4 (Preparation Example 3), and 1:5 (Preparation Example 4). Other factors were set as Tween 80 concentration of 1%, soybean lecithin to DSPE-PEG2000 ratio of 25:1, and ultrasonic time of 1 min.

[0107] Tween 80 concentration: CPD12C15, soybean lecithin, and DSPE-PEG2000 were accurately weighed to prepare CPD12C15 lipid nanoparticle solutions with different Tween 80 concentrations. The Tween 80 concentrations were 0.5% (Preparation Example 5), 1.0% (Preparation Example 1), 1.5% (Preparation Example 6), and 2.0% (Preparation Example 7). Other factors were set to a drug-to-lipid ratio of 1:2, a ratio of soybean lecithin to DSPE-PEG2000 of 25:1, and an ultrasonic time of 1 min.

[0108] Molar ratio of soybean lecithin and DSPE-PEG2000: CPD12C15, soybean lecithin, and DSPE-PEG2000 were accurately weighed to prepare different CPD12C15 lipid nanoparticle solutions. The molar ratios of soybean lecithin and DSPE-PEG2000 were 20:1 (Preparation Example 8), 25:1 (Preparation Example 7), 30:1 (Preparation Example 9), and 35:1 (Preparation Example 10), respectively. Other factors were set to a drug-lipid ratio of 1:2, a Tween 80 concentration of 2.0%, and an ultrasonic time of 1 min.

[0109] Ultrasonic time: CPD12C15, soybean lecithin, and DSPE-PEG2000 were accurately weighed to prepare lipid nanoparticle solutions with different ultrasonic times, namely 1 min (Preparation Example 9), 3 min (Preparation Example 11), 5 min (Preparation Example 12), and 7 min (Preparation Example 13). Other factors were set to a drug-lipid ratio of 1:2, a Tween 80 concentration of 2.0%, and a ratio of soybean lecithin to DSPE-PEG2000 of 30:1.

[0110] The results of the investigation are as follows Figure 2As shown in the figure, the results showed that as the drug-to-lipid ratio increased, the encapsulation efficiency remained unchanged, while the drug loading significantly decreased. To meet the in vivo animal dosing requirements, a drug-to-lipid ratio of 1:2 was selected as the optimal factor. Increasing Tween 80 concentration increased both the encapsulation efficiency and drug loading. However, the typical dosage of Tween 80 in pharmaceuticals does not exceed 5%, so a Tween 80 concentration of 2% was selected as the optimal factor. As the DSPE-PEG2000 ratio decreased, the encapsulation efficiency initially increased and then decreased, so a soy lecithin:DSPE-PEG2000 ratio of 30:1 was selected as the optimal factor. As the ultrasonication time increased, the encapsulation efficiency and drug loading of lipid nanoparticles initially increased and then decreased, so a 3-minute ultrasonication time was selected as the optimal factor.

[0111] Example 3 Preparation and characterization of M-LNP-CPD12C15

[0112] Based on the optimal preparation process for CPD12C15 lipid nanoparticles determined in Example 2, this example further investigated the effect of mannose-modified DSPE-PEG2000 on liposome performance. Preliminary experiments showed that adding an appropriate amount of mannose-modified DSPE-PEG2000 did not significantly change the main preparation parameters of the basic CPD12C15 liposomes. Therefore, this example continued to use the optimal parameters determined in Example 2 to prepare M-LNP-CPD12C15.

[0113] 1. Preparation of M-LNP-CPD12C15

[0114] Accurately weigh the prescribed amount of soybean lecithin and Man-DSPE-PEG2000 (molar ratio 30:1) and ultrasonically dissolve them in 2% Tween aqueous solution. Accurately weigh the prescribed amount of CPD12C15 (drug-lipid molar ratio 1:2, that is, the molar ratio between CPD12C15 and total lipid components soybean lecithin + Man-DSPE-PEG2000) and dissolve it in methanol. Under magnetic stirring, methanol was slowly added dropwise to the above aqueous solution with a 1 mL syringe. After the addition was completed, stirring was continued for 10 minutes. The probe was ultrasonicated in an ice bath for 3 minutes, and then magnetically stirred at room temperature for 6 hours to volatilize the organic solvent. The M-LNP-CPD12C15 was filtered through a 0.22 μm membrane, and three batches of reproducibility experiments were carried out.

[0115] 2. Characterization of M-LNP-CPD12C15

[0116] 2.1 Determination of encapsulation efficiency and drug loading: The determination method is the same as Example 2. The results are shown in Table 2.

[0117] Table 2 Results of three batches of reproducibility experiments of M-LNP-CPD12C15

[0118] batch Encapsulation efficiency (%) Drug loading (%) 1 73.69 11.67 2 72.29 11.82 3 73.07 12.69 Mean ± SD 73.02±0.70 12.06±0.55

[0119] The results showed that the prepared M-LNP-CPD12C15 had good batch consistency, with an average encapsulation efficiency of 73.02±0.70% and a drug loading of 12.06±0.55%, indicating that this method has high encapsulation and drug loading capacity and good reproducibility. It can be used to encapsulate CPD12C15 and provide a stable and reliable formulation basis.

[0120] 2.2 Determination of particle size, PDI and Zeta potential: The prepared M-LNP-CPD12C15 was appropriately diluted and then the particle size, PDI and Zeta potential were measured. The measurement method was as follows: 1 mL of M-LNP-CPD12C15 sample moderately diluted with ultrapure water was added to the particle size sample pool, placed in the particle size potential meter, and the particle size and PDI of the sample were measured. Take an appropriate amount of diluted M-LNP-CPD12C15 sample solution, slowly inject it into the Zeta potential cell with a 1 mL syringe, place it in the instrument detection chamber, and detect the Zeta potential of the sample solution. The detection temperature was 25 ° C, and the data image was recorded and exported. The results are shown in Table 3 and Figure 3 The particle size of M-LNP-CPD12C15 was 67.33±2.53 nm, the PDI was 0.20±0.04, and the Zeta potential was -23.27±3.94 mV.

[0121] Table 3 Particle size, PDI, and Zeta potential measurement results

[0122]

[0123] 2.3 Morphological observation

[0124] The morphology of M-LNP-CPD12C15 was observed by TEM. The specific operation process is as follows: (1) Preparation: Clamp the edge of the copper mesh and place it on the filter paper. Use a dropper to absorb the M-LNP-CPD12C15 sample solution and drop it on the copper mesh. The filter paper absorbs the excess liquid; (2) Drying: Use infrared light to dry the copper mesh; (3) Observation: Use TEM to observe and record. The results are shown in Figure 2. Figure 4 As shown in the figure, the M-LNP-CPD12C15 solution is clear and transparent with a light blue opalescence. The M-LNP-CPD12C15 lipid nanoparticles are spherical and well dispersed.

[0125] 2.4 Stability test

[0126] Mannose-modified lipid nanoparticles were prepared according to the preparation method and optimal process of M-LNP-CPD12C15 and stored in a refrigerator at 4°C. A certain amount of the lipid nanoparticle solution was taken out and diluted at 1, 2, 4, 7, and 14 days, and the encapsulation efficiency and drug loading were measured and calculated according to the determination method in Example 2. The particle size of the lipid nanoparticles was detected according to the aforementioned method, and the stability of the lipid nanoparticles over time was observed and recorded. The results are shown in Table 4. As can be seen from the results, the encapsulation efficiency and drug loading of the lipid nanoparticles did not change significantly over 14 days, the particle size increased slightly, and the appearance remained clear with a light blue opalescence. The results indicate that the prepared lipid nanoparticles have good stability.

[0127] Table 4 Stability test results of M-LNP-CPD12C15

[0128]

[0129] 2.5 In vitro release experiment

[0130] Dialysis is a commonly used method for in vitro drug release studies. Its principle is to exploit the osmotic pressure difference between the inside and outside of a dialysis bag, causing small molecule drugs to diffuse from the high-concentration side to the low-concentration side. The drug concentration at different time points is measured by HPLC, and the cumulative release rate is calculated to determine the in vitro drug release rate. 3.00 mg of CPD12C15 powder and the prepared M-LNP-CPD12C15 (containing 3.00 mg of CPD12C15) were added to a pre-activated dialysis bag, followed by 1 mL of distilled water. The dialysis bag was tied with string and placed in an EP tube containing 30 mL of release medium. The EP tube was placed in a thermostatic oscillator at 37°C with an air bath and an oscillation frequency of 100 rpm. At 10, 20, 30, 45, 60, 90, 120, 180, 240, 360, 720, and 1440 min, 0.5 mL of solution was removed from the EP tube and replenished with 0.5 mL of release medium. The solution was filtered and analyzed by HPLC to calculate the content and cumulative release rate of CPD12C15 and draw the release curve. According to the above method, the release behavior of CPD12C15 API and its preparation in two release media with pH 7.4 (body fluid circulation pH) and 6.8 (tumor microenvironment pH) was tested respectively. The results are shown in Figure 2. Figure 5The results showed that due to the water solubility of CPD12C15, the API exhibited a burst release at both pH levels, with rapid release and complete release within approximately 1.5 hours. M-LNP-CPD12C15, on the other hand, exhibited a slower release compared to the CPD12C15 API, with complete release within approximately 6 hours and sustained release for 24 hours. These results demonstrate that M-LNP-CPD12C15 can be slowly released in both body fluids and the weakly acidic tumor microenvironment, demonstrating a sustained-release effect.

[0131] 2.6 Red blood cell hemolysis test

[0132] Take rat blood and place it in a centrifuge tube moistened with sodium heparin, add an appropriate amount of physiological saline and stir evenly, centrifuge at 3000r / min for 10min at 4°C, discard the supernatant, wash with physiological saline until the supernatant is blood-red, and obtain the red blood cell pellet. Add physiological saline to the red blood cell pellet to prepare a 2% red blood cell suspension. Prepare a series of M-LNP-CPD12C15 solutions, take 1mL of preparation samples of different concentrations in centrifuge tubes, add 1mL of 2% red blood cell suspension and mix evenly, and set up a negative control group and a positive control group at the same time. Place the sample in a 37°C air bath constant temperature oscillator and incubate for 3h, centrifuge at 3000r / min for 10min, take the supernatant, and use an enzyme marker to measure the OD value at 545nm, and calculate the hemolysis rate according to the formula: Hemolysis rate % = (OD 实验组 –OD 阴性对照组 ) / (OD 阳性对照组 –OD 阴性对照组 )×100%.

[0133] The results are as follows Figure 6 The results showed that after treatment with different concentrations of M-LNP-CPD12C15 solution, no obvious hemolysis occurred in the experimental groups, and the hemolysis rate in each group was less than 5%, demonstrating that tail vein injection of M-LNP-CPD12C15 was safe and did not cause hemolysis.

[0134] Cell experiment results of 2.7M-LNP-CPD12C15

[0135] 2.7.1 Cell culture methods

[0136] MKN-45 and SGC-7901 gastric cancer cells are adherent cells and cultured in 1640 medium (10% fetal bovine serum, 1% penicillin-streptomycin); HUVEC cells are cultured in DMEM medium (10% fetal bovine serum, 1% penicillin-streptomycin). They are cultured in a cell culture incubator at 37°C, 5% CO2, and passaged every 1-2 days.

[0137] (1) Cell recovery: Take out the cell cryopreservation tube from the liquid nitrogen tank, quickly transfer it to a preheated (37°C) water bath to melt, then disinfect it with alcohol, place it in a clean bench, transfer the cell suspension to a centrifuge tube, and centrifuge it at 1000r / min for 3min. The cells will settle at the bottom, discard the supernatant, add culture medium to resuspend the cells, pipet evenly, transfer to a culture dish with culture medium, shake it gently, and place it in the incubator for culture.

[0138] (2) Cell passaging: When the cell density reaches 90%, the cells can be passaged. Aspirate the culture medium, wash with sterile PBS, add 1 mL of trypsin, and then place in the incubator for digestion for 2 minutes. When the cells become round and bright, add 2 mL of complete culture medium to terminate the digestion. Transfer the cells to an EP tube by blowing them off and centrifuge at 1000 r / min for 3 minutes. Discard the supernatant, add 3 mL of culture medium to resuspend, aspirate 1 mL and add it back to the culture dish, add culture medium to 8 mL, shake gently and place in the incubator for culture.

[0139] (3) Cell cryopreservation: When the cells are in good growth condition and the density is greater than 80%, the cells can be cryopreserved. After digestion and centrifugation according to the cell passaging method, the supernatant is discarded. The cell pellet at the bottom of the EP tube is resuspended with 1 mL of cell freezing solution and then transferred to a cryopreservation tube. The cryopreservation tube is placed in a freezing box and frozen at -80°C overnight. The next day, it is transferred to a liquid nitrogen tank for long-term storage.

[0140] (4) Cell counting: Wipe the cover glass and hemocytometer clean with alcohol, take 10 μL of cell suspension and add it to the hemocytometer, and observe and count under a microscope. Calculate the cell density according to the following formula, and take a certain amount of cell suspension for subsequent experiments according to experimental requirements. Total number of cells / mL = (total number of cells in four squares / 4) × 10 4 pieces / mL.

[0141] 2.7.2 Cellular uptake experiments

[0142] Coumarin-6 (C6) is a hydrophobic fluorescent marker that plays an important role in biological research. It is particularly suitable for in vivo tracking analysis, as well as for exploring the mechanism of cellular uptake and the working principle of microparticle drug delivery systems. CPD12C15 is a lipophilic drug, so C6 can be used instead of CPD12C15 to prepare lipid nanoparticles and explore the differences in the ability of cells to uptake nanoparticles under different formulations and different incubation times. According to the preparation method and optimal formulation of lipid nanoparticles described in Examples 2 and 3, C6 was used instead of CPD12C15 to prepare unmodified lipid nanoparticles and mannose-modified lipid nanoparticles, respectively. MKN-45 cells in the logarithmic growth phase were passaged to obtain a cell suspension, which was then counted and inoculated into a 6-well plate at a cell density of 3×10 5Each well was gently shaken and then placed in an incubator for further culture. When the cell density reached approximately 60%, the culture medium was discarded and different preparations were added, with a C6 concentration of 30 ng / mL. The experiment was divided into four groups: free C6, unmodified lipid nanoparticles (LNP-C6), mannose-modified LNPs (M-LNP-C6), and Mannan + mannose-modified LNPs (Mannan is a mannose receptor inhibitor). In the inhibitor group, cells were pretreated with Mannan solution (1 mM) for 45 minutes, washed three times with PBS, and then incubated with mannose-modified LNPs. Two time points were set to examine the differences in cellular uptake of the different preparations at 1 hour and 4 hours. After incubation, cells were washed three times with PBS to remove excess C6 probe, fixed with paraformaldehyde for 20 minutes, labeled with DAPI for nuclei, stained for 20 minutes, washed three times with PBS, and photographed using a fluorescence microscope. After completion, cells were trypsinized, harvested, resuspended in PBS, and quantitatively analyzed by flow cytometry.

[0143] 2.7.3 Western blot detection of mannose receptor expression in various cell lines

[0144] (1) Cell culture: Gastric cancer cells MKN-45 and SGC-7901 in the logarithmic growth phase and human umbilical vein endothelial cells (HUVEC) were digested with trypsin to prepare cell suspensions. 3 × 10 cells were added to each well. 5 The cells were seeded in a 6-well plate at a density of 100 cells / well, shaken gently to evenly distribute, and placed in an incubator for further culture. When the cell density reached 90%, the total cell protein could be extracted.

[0145] (2) Extraction of total cell protein: 1) Add 1% PMSF, protease inhibitors and phosphatase inhibitors to RIPA cell lysis buffer to prepare 1 mL of cell lysis buffer, mix well and place on ice for use. 2) Remove the 6-well plate, discard the culture medium, wash twice with PBS, add 120 μL of cell lysis buffer to each well, scrape the cells with a 200 μL pipette tip, and lyse on ice for 25 minutes. 3) Precool the centrifuge to 4°C, transfer the cell lysate to a 1.5 mL centrifuge tube, and centrifuge at 12000 r / min for 25 minutes. 4) After centrifugation, take the supernatant to a new centrifuge tube, add 1 / 4 of the supernatant volume of loading buffer to each tube, and mix well. 5) Place the protein sample in a 98°C metal bath for heating denaturation for 10 minutes. After heating, take out the sample and store it in a -20°C refrigerator when it cools to room temperature.

[0146] (3) BCA protein quantification: 1) First, establish a standard curve according to the instructions. 2) Dilute the protein sample appropriately, mix well, and add 20 μL to a 96-well plate. 3) Prepare an appropriate amount of BCA working solution based on the number of protein samples. Add 200 μL of BCA working solution to each well and incubate at 37°C for 20 minutes. 4) Measure the absorbance at 562 nm using a microplate reader and calculate the protein concentration of the sample based on the standard curve.

[0147] (4) SDS-PAGE gel electrophoresis: 1) Leak detection: Clean the glass plate in advance, fix it and check whether it leaks. 2) Glue preparation: Select gel kits with different ratios according to the molecular weight of the protein. Prepare the separation gel according to the instructions, add it to the fixed glass plate, and press it flat with anhydrous ethanol when it is added to the appropriate position. Let it stand at room temperature for 30 minutes. After the separation gel solidifies, pour out the anhydrous ethanol, absorb the residual ethanol with filter paper, and then add the concentrated gel prepared according to the instructions, insert the comb, and let it stand at room temperature for 30 minutes. 3) Sample loading: Place the glass plate with the gel into the electrophoresis tank, fill it with the newly prepared electrophoresis solution, slowly pull out the comb, add the protein sample according to the calculated amount, and add 2.5μL protein pre-stained marker on both sides. 4) Electrophoresis: Set the voltage to 80V, run the electrophoresis for 30 minutes, and when the protein sample runs to the separation gel, change the voltage to 120V and continue the electrophoresis for 50 minutes. The electrophoresis ends when the protein sample runs to the bottom of the separation gel. 5) Transfer: Pre-cut a PVDF membrane of appropriate size based on the size of the gel and activate it in methanol for 3 minutes. After electrophoresis, remove the gel and arrange it on the blackboard of the transfer cassette in the order of sponge, filter paper, gel, PVDF membrane, filter paper, sponge, and transparent plate. Transfer the membrane to a transfer tank, fill it with transfer buffer, set the current to 200mA, and transfer the membrane for 90 minutes. 6) Blocking: After transfer, soak the PVDF membrane in 5% skim milk powder and block it at room temperature for 2 hours. 7) Primary Antibody Incubation: After blocking, wash the PVDF membrane three times with 1× TBST (10 minutes each). Incubate the strips in the corresponding primary antibody and incubate overnight at 4°C. 8) Secondary Antibody Incubation: Wash the PVDF membrane three times with 1× TBST (10 minutes each). Incubate the strips in the secondary antibody and incubate at room temperature for 1 hour. 9) Development: Wash the PVDF membrane three times with 1× TBST (10 minutes each). Prepare a developer solution in a 1:1 ratio of ECL luminescent solution A:solution B. Mix thoroughly and allow the solution to evenly soak the PVDF membrane. Develop the solution using a gel imaging system and take photos. 10) Analysis: Quantitatively analyze protein bands using Lane 1D software.

[0148] 2.7.4 Results

[0149] Western blot experiments were performed to detect the expression of MR in two human gastric cancer cells, MKN-45 and SGC-7901, and normal human umbilical vein endothelial cells (HUVECs). Figure 7As shown in Figure 2, the expression of mannose receptors in gastric cancer cells is much higher than that in normal cells. Therefore, mannose can be selected as the target head to modify lipid nanoparticles and actively target gastric cancer cells.

[0150] The uptake results were qualitatively observed using a fluorescence microscope. DAPI can mark the nuclei of MKN-45 cells and appear blue under a fluorescence microscope. C6 appears green under a fluorescence microscope after being taken up by cells. Figure 8 In (a), it can be seen that C6 can be more absorbed into the cells when loaded into lipid nanoparticles, and the green fluorescence of the mannose-modified lipid nanoparticle group is more obvious. As time goes on, the fluorescence intensity becomes more and more obvious, indicating that more C6 is absorbed into the cells. In order to further explore the mechanism by which mannose modification improves the uptake of lipid nanoparticles, MKN-45 cells were pre-treated with the mannose inhibitor mannoside and then incubated with M-LNP-C6. It was found that the fluorescence intensity was significantly reduced. The results showed that mannoside can competitively bind to the mannose receptors on the surface of MKN-45 cells, partially blocking the entry of M-LNP-C6 into the cells, resulting in a decrease in fluorescence intensity. The treated cells were quantitatively analyzed by flow cytometry to further investigate the cellular uptake efficiency of lipid nanoparticles, as shown in Figure 3. Figure 8 Consistent results were obtained in (bc), indicating that lipid nanoparticles can promote cellular uptake of drugs, and mannose-modified lipid nanoparticles can increase drug uptake efficiency through active targeting.

[0151] Example 4 In vitro anti-gastric cancer efficacy of M-LNP-CPD12C15 combined with copper

[0152] Experimental cell lines: Human gastric cancer MKN-45 and SGC-7901 cells were provided by the School of Basic Medical Sciences of Shandong University.

[0153] Experimental solution preparation

[0154] (1) CPD12C15 solution: Accurately weigh 1.36 mg of CPD12C15 powder and dissolve it in 2 mL of DMSO to prepare a 2 mM CPD12C15 solution. Sterilize the solution under UV light in a laminar flow hood for 30 min, aliquot into 1.5 mL centrifuge tubes, and store at -20°C.

[0155] (2) LNP-CPD12C15 and M-LNP-CPD12C15 Solutions: Unmodified LNP-CPD12C15 and mannose-modified M-LNP-CPD12C15 were prepared according to the lipid nanoparticle preparation methods and optimized processes described in Examples 2 and 3. The encapsulation efficiency and drug loading were determined by HPLC. 1 mL of a solution containing 2 mM CPD12C15 was prepared. The solution was sterilized by UV in a laminar flow hood for 30 min before use and stored at 4°C.

[0156] (3) CuCl₂·2H₂O solution: Accurately weigh 1.36 mg of CuCl₂·2H₂O powder and add 4 mL of ultrapure water to prepare a 2 mM CuCl₂·2H₂O solution. Sterilize the solution by filtration through a 0.22 μm microporous filter in a clean bench. Transfer the filtered solution to a 1.5 mL centrifuge tube and store at -20°C.

[0157] (4) Cisplatin (DDP) solution: Accurately weigh 1.19 mg of DDP powder and dissolve it in 2 mL of sterile saline to prepare a 2 mM DDP solution. Filter the solution through a 0.22 μm microporous filter in a clean bench to sterilize. Transfer the filtered solution to a 1.5 mL centrifuge tube and store at -20°C.

[0158] (5) MTT solution: Accurately weigh an appropriate amount of MTT powder and dissolve it in sterile PBS by ultrasonication to a final concentration of 5 mg / mL. Sterilize the solution by filtration using a 0.22 μm microporous filter in a clean bench under dark conditions. Aliquot the solution into 10 mL EP tubes, wrap in tin foil to protect from light, and store at -20°C.

[0159] 1. Experimental methods

[0160] Cell culture: The operation method is the same as Example 3.

[0161] 1.1 MTT assay to detect cell viability: MKN-45 and SGC-7901 cells were taken out and passaged to obtain cell suspension. Cell count was performed, and 5×10 cells were contained in 100 μL cell suspension per well. 3Cells were seeded at a density of 100 μM in 96-well plates and cultured in an incubator. After cells adhered, different concentrations of drugs were added for 48 hours. First, the cytotoxicity of the blank formulation against both cell lines was investigated. The drug-treated groups were divided into four groups: CPD12C15+Cu, LNP-CPD12C15+Cu, M-LNP-CPD12C15+Cu, and DDP. CPD12C15 and its formulation were mixed with a CuCl2.2H2O solution of the same concentration in a 1:1 ratio, then diluted with culture medium to different concentrations (0.05, 0.1, 0.2, 0.4, 0.8, and 1.6 μM), and 100 μL of drug was added to each well. For the DDP group, the prepared DDP solution was diluted with culture medium to different concentrations (0.5, 1, 2, 4, 8, and 16 μM), and 100 μL of drug was added to each well. Three replicates were set for each concentration. A blank and control group were also established. 48 hours after drug addition, 20 μL of MTT solution was added to each well and incubated at 37°C in the dark for 4 hours. After 4 hours, the culture medium was slowly discarded and 150 μL of DMSO was added to each well. The cells were shaken on a shaker for 15 minutes to dissolve the precipitate. The absorbance at 490 nm was then measured using a microplate reader, and the cell viability was calculated according to the formula: Cell viability % = (OD 实验组 -OD 空白组 ) / (OD 对照组 -OD 空白组 )×100%.

[0162] 1.2 Cell scratch test: MKN-45 and SGC-7901 cells were passaged and counted, and then seeded into 6-well plates at a cell density of 5×10 5 Each well was gently shaken and placed in an incubator for further culture. When the cell density reached approximately 90%, the culture medium was discarded and a 200 μL sterilized pipette tip was used to scratch the bottom of each well. Cell debris was washed with PBS. Six empty plates were photographed under a microscope, and the scratch width at 0 h was recorded. Subsequently, the cells were treated with drugs and divided into the following five groups: Control group, CPD12C15+Cu group, LNP-CPD12C15+Cu group, M-LNP-CPD12C15+Cu group, and DDP group. In MKN-45 cells, the CPD12C15+Cu concentration in different preparations was 0.35 μM, and the DDP concentration was 2 μM. In SGC-7901 cells, the CPD12C15+Cu concentration in different preparations was 0.4 μM, and the DDP concentration was 4.5 μM. Each well was filled with 2 mL of the system. The 6-well plate was returned to the incubator and cultured for 24 hours. After 24 hours, the culture medium was discarded, and the wounds were washed three times with PBS. The scratch width was then photographed under a microscope and recorded over 24 hours. Data were analyzed using Image J software, and cell migration rate was calculated according to the formula: Cell migration rate (%) = (initial scratch area - final scratch area) / initial scratch area × 100%.

[0163] 1.3 Cell invasion assay: Dilute Matrigel 10-fold with serum-free 1640 medium and slowly add 100 μL to the bottom of each chamber. Place the chamber in a 24-well plate and transfer to an incubator to allow the Matrigel to solidify. MKN-45 and SGC-7901 cells were starved for 12 h in serum-free medium. After counting the cells, dilute with serum-free medium and seed the chambers at a cell density of 7 × 10 for MKN-45 cells. 4 cells / well; SGC-7901 cells: 5×10 4 Each well was filled with 100 μL of drug diluted to a specific concentration. The experiment was divided into five groups: control, CPD12C15+Cu, LNP-CPD12C15+Cu, M-LNP-CPD12C15+Cu, and DDP. In MKN-45 cells, the CPD12C15+Cu concentration for all formulations was 0.35 μM, and the DDP concentration was 2 μM. In SGC-7901 cells, the CPD12C15+Cu concentration for all formulations was 0.4 μM, and the DDP concentration was 4.5 μM. 800 μL of complete culture medium was added to the 24-well plate, and the chamber was slowly replaced to avoid creating bubbles. The 24-well plate was transferred to an incubator and cultured for a further 48 h. The culture medium in the upper chamber was discarded, the chamber was washed twice with PBS, 4% paraformaldehyde was added inside and outside the chamber to fix for 20 min, PBS was repeatedly washed, and the cells were stained with crystal violet solution for 20 min. After cleaning, the cells in the upper chamber were gently wiped off with a cotton swab, and photographed with an inverted microscope. The number of cells invading the lower layer of the chamber was counted using Image J software.

[0164] 1.4 Cell apoptosis experiment: MKN-45 and SGC-7901 cells in good growth condition were cultured at a rate of 5×10 5Cells were seeded at a density of 100 μg / well in a 6-well plate, gently shaken, and placed in an incubator for further culture. When the cell density reached approximately 60%, the culture medium was discarded and drugs were added according to experimental grouping. The experiment was divided into five groups: control, CPD12C15+Cu, LNP-CPD12C15+Cu, M-LNP-CPD12C15+Cu, and DDP. For MKN-45 cells, the CPD12C15+Cu concentration for all formulations was 0.35 μM, and the DDP concentration was 2 μM; for SGC-7901 cells, the CPD12C15+Cu concentration for all formulations was 0.4 μM, and the DDP concentration was 4.5 μM. A 2 mL volume was added to each well of each group. The 6-well plate was returned to the incubator and cultured for an additional 24 hours. After the incubation period, the culture medium was collected, and the cells were digested with EDTA-free trypsin. Digestion was terminated by adding the corresponding culture medium. The cells were then transferred to a centrifuge tube and centrifuged at 1000 rpm for 5 minutes to collect the cells. Wash the cells twice with pre-chilled PBS. Add 100 μL of 1× Binding Buffer to the cell pellet and resuspend the cells. Add 5 μL of Annexin V-FITC and PI dye to each tube, mix gently, and incubate at room temperature in the dark for 15 minutes. Simultaneously, set up a blank group without dye, Annexin V-FITC single-stained groups, and PI single-stained groups for gating and compensation. After staining, add 400 μL of 1× Binding Buffer to each tube, mix gently, and transfer to a flow cytometer. Analyze on a flow cytometer within 1 hour.

[0165] 1.5 Determination of Intracellular Reactive Oxygen Species: MKN-45 and SGC-7901 cells in the logarithmic growth phase were passaged and seeded at appropriate density in 6-well plates and cultured overnight in an incubator. When the cell density reached approximately 60%, the culture medium was discarded, and drugs were added according to experimental grouping. Four groups were divided into the experiment: control, CPD12C15+Cu, LNP-CPD12C15+Cu, and M-LNP-CPD12C15+Cu. The CPD12C15+Cu concentration for all formulations was 0.35 μM for MKN-45 cells, and 0.4 μM for all formulations for SGC-7901 cells. 2 mL of solution was added to each well of each group. The 6-well plates were returned to the incubator and cultured for an additional 24 hours. After incubation, wash twice with PBS. Dilute DCFH-DA 1000-fold with serum-free medium to a final concentration of 10 μM. Add 1 mL of diluted DCFH-DA to each well and incubate at 37°C in the dark for 20 min. After incubation, discard the medium, wash three times with serum-free medium, collect the cells, wash once with PBS, resuspend in 500 μL of PBS, transfer to a flow cytometer, and analyze using a flow cytometer.

[0166] 1.6 Intracellular Ca 2+ Assay: MKN-45 and SGC-7901 cells in the logarithmic growth phase were passaged and seeded at appropriate density in 6-well plates and cultured overnight in an incubator. When the cell density reached approximately 60%, the culture medium was discarded and the drug was added according to the experimental group. Four groups were divided into the experiment: control, CPD12C15+Cu, LNP-CPD12C15+Cu, and M-LNP-CPD12C15+Cu. The CPD12C15+Cu concentration for all formulations was 0.35 μM for MKN-45 cells, and 0.4 μM for all formulations for SGC-7901 cells. 2 mL of solution was added to each well of each group. The 6-well plates were returned to the incubator and cultured for an additional 24 hours. Discard the culture medium, wash twice with PBS, and dilute the Fluo-3 AM calcium ion fluorescent probe 2500-fold in serum-free culture medium to a final concentration of 2 μM. Add 600 μL of the diluted probe to each well and incubate at 37°C in the dark for 45 minutes. Remove the Fluo-3 AM working solution, wash twice with PBS, and add 1 mL of serum-free culture medium to each well. Incubate at 37°C in the dark for 15 minutes. After incubation, wash the cells with PBS, digest them, collect them, wash once with PBS, and resuspend them in 500 μL of PBS. Transfer the cells to a flow cytometer and analyze them using a flow cytometer.

[0167] 1.7 Live cell imaging with fluorescent tracers

[0168] MKN-45 and SGC-7901 cells in the logarithmic growth phase were seeded in confocal microplates at a cell density of 5 × 10 3 Each confocal microplate was cultured overnight. After cells adhered, the medium was discarded and drugs were added according to experimental grouping. The experiment was divided into a control group and an M-LNP-CPD12C15+Cu group. MKN-45 cells were treated with 0.35 μM, while SGC-7901 cells were treated with 0.4 μM. Each confocal microplate was filled with 1 mL of solution. After drug addition, the cells were returned to the incubator and cultured for 24 hours. After 24 hours, the medium was discarded, and ER-Tracker and Hoechst 33342 were diluted 1:2000 with ER-Tracker diluent. After mixing, 800 μL of the diluted working solution was added to each microplate. The cells were incubated at 37°C in the dark for 30 minutes, gently shaking the culture plate every 10 minutes to ensure sufficient contact between the probe and the cells. After the incubation period, the medium was discarded, the cells were washed three times with PBS, and then observed and imaged using a confocal microscope.

[0169] 1.8 Western Blot detection of related protein expression levels

[0170] (1) Cell culture and drug treatment

[0171] MKN-45 and SGC-7901 cells in the logarithmic growth phase were passaged and seeded at appropriate density in 6-well plates and cultured overnight in an incubator. When the cell density reached approximately 60%, the culture medium was discarded, and drugs were added according to experimental grouping. The experiment was divided into four groups: control, CPD12C15+Cu, LNP-CPD12C15+Cu, and M-LNP-CPD12C15+Cu. The CPD12C15+Cu concentration for all formulations was 0.35 μM for MKN-45 cells, and 0.4 μM for all formulations for SGC-7901 cells. 2 mL of solution was added to each well of each group. The 6-well plates were returned to the incubator and cultured for an additional 48 hours, after which total cell protein was extracted.

[0172] (2) Extraction of total cell protein and SDS-PAGE gel electrophoresis: The specific operation is the same as that in Example 3.

[0173] 1.9 Statistical Analysis: Experimental data were processed and plotted using GraphPad Prism 8 software. Results are presented as mean ± standard deviation (SD). Data were analyzed by one-way analysis of variance to assess inter-group differences. P < 0.05 indicated a significant difference compared with the control group; *p < 0.05, **p < 0.01, and ***p < 0.001 were used.

[0174] 2. Experimental results

[0175] 2.1 MTT assay to detect cell viability

[0176] First, the cytotoxicity of blank formulation M-LNP was investigated in MKN-45 and SGC-7901 cells. Figure 9 As shown in the results, M-LNP had no toxicity to both cells in the range of 0.05-1.6 μM. Then, the effects of CPD12C15 and its preparations combined with copper on the proliferation of two human gastric cancer cells were investigated, and cisplatin was used as a positive control. The results are shown in the figure below. Figure 10 、 Figure 11 As shown, CPD12C15+Cu, LNP-CPD12C15+Cu, M-LNP-CPD12C15+Cu and DDP can inhibit the proliferation of MKN-45 and SGC-7901 cells in a concentration-dependent manner. Table 5 shows the IC values of various drugs in the two cell lines. 50It can be seen that CPD12C15 and its preparations combined with copper showed a strong inhibitory effect on the proliferation of gastric cancer cells, and the inhibitory ability was better than DDP. In addition, the killing ability of the CPD12C15+Cu group, LNP-CPD12C15+Cu group and M-LNP-CPD12C15+Cu group on two gastric cancer cells was compared. Under the same concentration and treatment time, the cell viability of the two cells in the M-LNP-CPD12C15+Cu group was the lowest. The IC of M-LNP-CPD12C15+Cu on MKN-45 and SGC-7901 cells was 50 The concentrations of mannose-modified lipid nanoparticles were 0.304±0.007μM and 0.335±0.025μM, respectively. The efficacy was significantly better than that of the raw material drug, indicating that mannose-modified lipid nanoparticles were more absorbed into cells and exhibited a stronger killing effect.

[0177] Table 5 IC values of various drugs on MKN-45 and SGC-7901 cells 50

[0178]

[0179] Based on IC of different groups of drugs 50 To determine the drug administration concentration for cell experiments, the concentration of CPD12C15+Cu was selected as 0.35μM and the concentration of DDP was 2μM in MKN-45 cells; the concentration of CPD12C15+Cu was selected as 0.4μM and the concentration of DDP was 4.5μM in SGC-7901 cells.

[0180] 2.2 Discussion of cell migration and invasion results

[0181] The cell wound healing experiment was conducted to investigate the effects of CPD12C15 and its preparations combined with copper on the migration ability of two gastric cancer cells. After the scratch was made, CPD12C15 with different preparations combined with copper and DDP were respectively exposed to MKN-45 and SGC-7901 cells for 24 hours, and the phenomenon of wound width reduction was inhibited. Figure 12 and 13As shown, compared with the control group, CPD12C15 and its preparation combined with copper inhibited the migration of both gastric cancer cells (*p<0.05), and the LNP-CPD12C15+Cu and M-LNP-CPD12C15+Cu groups showed stronger inhibitory abilities (***p<0.001). In MKN-45 and SGC-7901 cells, the wound healing rates in the control group were 31.43±1.09% and 32.40±1.41%, respectively; in the M-LNP-CPD12C15+Cu group, the migration rates of the two cell types were 13.05±2.60% and 12.99±3.39%, respectively; and in the DDP group, the wound healing rates were 19.39±4.58% and 23.28±1.83%, respectively. The experimental results show that M-LNP-CPD12C15+Cu has a stronger ability to inhibit gastric cancer cell migration and is more effective than DDP.

[0182] Matrigel experiments were conducted to investigate the effects of CPD12C15 and its preparations combined with copper on the invasion ability of two gastric cancer cells. Figure 14 and 15 As shown, compared with the control group, different formulations of CPD12C15 combined with copper and DDP can inhibit the invasion of both gastric cancer cells, and the number of cells invading into the lower chamber after administration is significantly reduced (***p < 0.001). In MKN-45 and SGC-7901 cells, the number of cells invading into the lower chamber in the control group is 313.33 ± 27.47 and 142 ± 5.29, respectively. In the M-LNP-CPD12C15 + Cu group, the number of cells invading into the lower chamber is 76 ± 7.21 and 43.67 ± 2.08, respectively. In the DDP group, the number of cells invading into the lower chamber is 151.33 ± 18.04 and 57.33 ± 5.69, respectively. The experimental results show that M-LNP-CPD12C15 + Cu has a stronger ability to inhibit gastric cancer cell invasion and is more effective than DDP.

[0183] 2.3 Discussion of cell apoptosis results

[0184] In order to explore the effects of CPD12C15 and its preparations combined with copper on the apoptosis of two gastric cancer cells, flow cytometry was used to detect the differences in cell apoptosis rates in each group after administration. Figure 16 and 17As shown, compared with the control group, the sum of early and late apoptosis of both cell types was upregulated after administration (***p<0.001). In MKN-45 and SGC-7901 cells, the apoptosis rates of the control group were 5.56±0.19% and 9.14±0.64%, respectively, while those of the M-LNP-CPD12C15+Cu group were 40.70±0.33% and 36.19±1.35%, respectively, and those of the DDP group were 32.50±1.11% and 16.20±1.06%, respectively. The experimental results show that CPD12C15 and its preparations combined with copper can induce apoptosis in tumor cells. Among them, M-LNP-CPD12C15+Cu has a stronger ability to promote apoptosis of gastric cancer cells, and its effect is better than that of the positive drug DDP.

[0185] 2.4 Changes in ROS content in gastric cancer cells

[0186] ROS is an important factor in inducing endoplasmic reticulum stress, which can induce tumor cell apoptosis and is closely related to the occurrence and development of tumors. In order to explore whether CPD12C15 and its preparations combined with copper can induce tumor cells to produce reactive oxygen species, a reactive oxygen species detection kit was used to detect the changes in ROS in cells after administration. Figure 18 、 Figure 19 Compared to the control group, the drug treatment increased ROS levels in both cell types. In MKN-45 cells, ROS levels were 3-5 times higher than in the control group; in SGC-7901 cells, ROS levels were 1-1.5 times higher than in the control group. The M-LNP-CPD12C15+Cu group showed the strongest ability to induce ROS production in tumor cells. These results demonstrate that CPD12C15 and its preparations, combined with copper, can upregulate ROS levels in gastric cancer cells, affecting redox balance, disrupting cellular structure, and promoting apoptosis.

[0187] 2.5 Ca in gastric cancer cells 2+ Content changes

[0188] The endoplasmic reticulum is an important Ca 2+ Reservoir, Ca 2+ The dynamic balance of Ca is the basis for cells to maintain normal structure and function. 2+ The steady-state balance is broken, resulting in a large amount of Ca 2+ Ca flows into cells and mitochondria, affecting the activity of Bcl-2 family proteins, further triggering the activation of Caspase cascade reactions and inducing cell apoptosis. 2+ The release of Ca is a key feature of endoplasmic reticulum stress, so flow cytometry was used to detect the Ca release in cells after drug administration. 2+ The content changes, the results are as follows Figure 20 、 Figure 21 As shown, in MKN-45 cells, compared with the Control group, the Ca 2+ The content of Ca2+ in SGC-7901 cells was increased by two times (***p<0.001); in SGC-7901 cells, the CPD12C15+Cu group had no significant change compared with the Control group (p>0.05), while the Ca2+ content in LNP-CPD12C15+Cu and M-LNP-CPD12C15+Cu cells was significantly increased (***p<0.001). 2+ The concentration increased significantly (***p<0.001), which may be due to the stress of endoplasmic reticulum and the increase of Ca 2+ Homeostasis is disrupted, and a large amount of Ca 2+ Enter the cell.

[0189] 2.6 Changes in endoplasmic reticulum morphology

[0190] Literature reports that endoplasmic reticulum stress can cause cell vacuolization and induce cell apoptosis. Studies have shown that disulfiram combined with copper can induce endoplasmic reticulum stress in malignant tumors such as pancreatic cancer, liver cancer, and breast cancer, thereby inducing tumor cell autophagy and apoptosis, and inhibiting tumor growth. To determine the effect of M-LNP-CPD12C15+Cu on the endoplasmic reticulum, ER-Tracker (red) was used to label the endoplasmic reticulum, and Hoechst 33342 was used to label the cell nucleus (blue). Live cell staining was performed on the two gastric cancer cells after administration, and the morphological changes of the endoplasmic reticulum of the two gastric cancer cells were observed by confocal microscopy. The results are as follows. Figure 22 As shown in the data, the endoplasmic reticulum of the control group cells remained intact and continuous, while the endoplasmic reticulum of the M-LNP-CPD12C15+Cu group cells showed vacuolation and discontinuity, and the endoplasmic reticulum structure was severely damaged, indicating that M-LNP-CPD12C15+Cu can induce endoplasmic reticulum stress in gastric cancer cells. Therefore, it is speculated that the mechanism of inducing tumor cell apoptosis may be through inducing the endoplasmic reticulum stress pathway.

[0191] 2.7 Discussion of Western blot results

[0192] The endoplasmic reticulum (ER) is the primary organelle responsible for protein synthesis, modification, folding, and secretion. Under conditions of cellular stress, the risk of disrupting protease homeostasis and protein misfolding may increase, activating the ERS and triggering the UPR, leading to restoration of cellular homeostasis or cell death. ER stress and the unfolded protein response (UPR) have been shown to play key roles in the pathogenesis and progression of various cancers. Gastric cancer is one of the most aggressive types of cancer, and inducing ER stress and activating pro-apoptotic signaling pathways can inhibit its development. This section will explore the effects of CPD12C15+Cu on the unfolded protein response and ER stress signaling pathways in two gastric cancer cells to elucidate its anti-tumor mechanism of action.

[0193] Ubiqutin is a ubiquitinated protein. Inhibition of the proteasome system impairs protein synthesis and degradation in cells, preventing waste proteins from being cleared promptly and becoming ubiquitinated. This leads to the accumulation of large amounts of waste proteins, triggering the UPR and causing irreversible ER stress. CPD12C15, a derivative of DSF, inhibits the ubiquitination proteasome system, affecting cellular protein homeostasis. Therefore, changes in ubiqutin expression in two gastric cancer cell lines after administration were measured to assess whether waste proteins accumulate in cells. The typical UPR consists of three ER transmembrane stress sensors: IRE1, PERK, and ATF6. Under resting conditions, these three sensors bind to the ER chaperone, glucose-regulated protein GRP78 / BiP, remaining inactive. Under stress, GRP78 dissociates from these UPR sensors, allowing their oligomerization and autophosphorylation, as well as activation of ATF6. This leads to activation of the UPR and downstream signaling pathways, initiating the caspase cascade and inducing apoptosis in gastric cancer cells.

[0194] Western Blot was used to detect the changes in ubiquitinated proteins and several key proteins in the ERS signaling pathway after administration. The results are as follows Figure 23 As shown in the figure, after a period of administration, the expression of Ubiqutin in the two gastric cancer cells increased, and the increase in the M-LNP-CPD12C15+Cu group was the most obvious. Figure 24 It can be seen that the expression of related proteins p-EIF2α, ATF4, and ATF6 in the endoplasmic reticulum stress pathway signaling pathway increased after administration, indicating that CPD12C15+Cu inhibits UPS, leading to the accumulation of ubiquitinated proteins in cells, thereby inducing ERS. Under endoplasmic reticulum stress conditions, CHOP is one of the key regulatory factors of apoptosis signal transduction, and its expression level is closely related to cell fate. When the stress response inside the cell reaches the irreversible stage, the three branches of UPR can promote the expression of CHOP, thereby activating caspase 3 and inducing apoptosis of gastric cancer cells. From Figure 24 It can be seen that the expressions of CHOP and activated caspase 3 increased after administration, demonstrating that CPD12C15 and its preparations combined with copper may induce apoptosis of gastric cancer cells through the endoplasmic reticulum stress pathway.

[0195] Example 5 In vivo anti-gastric cancer efficacy of M-LNP-CPD12C15 combined with copper

[0196] Experimental cell line: Human gastric cancer MKN-45 cells were provided by the School of Basic Medical Sciences of Shandong University.

[0197] Experimental animals: 25 female Balb / C nude mice, 6-7 weeks old and weighing approximately 18-20 g each, were purchased from Jinan Xingkang Biological Co., Ltd. Five mice were housed in individually ventilated cages at a temperature of 25°C and a humidity of 50-60%. They were fed and watered daily at normal doses. Experiments were conducted after one week of maintenance. All experiments were conducted in accordance with the guidelines of the Animal Experimentation Center of Shandong University.

[0198] Experimental solution preparation:

[0199] (1) CPD12C15 solution: Accurately weigh 20.00 mg of CPD12C15 powder and dissolve it in 10 mL of normal saline. Ultrasonicate and dissolve to obtain a 2 mg / mL CPD12C15 solution.

[0200] (2) M-LNP-CPD12C15 solution: M-LNP-CPD12C15 was prepared according to the preparation method and the optimal process of Example 3. The encapsulation efficiency and drug loading were determined by HPLC, and M-LNP-CPD12C15 solutions of different concentrations were prepared.

[0201] (3) DDP solution: Dissolve lyophilized cisplatin powder for injection (containing 20 mg of cisplatin) in 33.33 mL of normal saline and dissolve by ultrasonication to obtain a 0.6 mg / mL DDP solution.

[0202] (4) Copper gluconate solution: Accurately weigh 8.00 mg of copper gluconate powder and dissolve it in 10 mL of normal saline. Ultrasonicate dissolution is performed to obtain a 0.8 mg / mL copper gluconate solution.

[0203] 1. Experimental methods

[0204] Cell culture: Same as Example 3.

[0205] Establishment of nude mouse subcutaneous tumor model: MKN-45 cells with good growth status were obtained, digested with trypsin to obtain single cell suspension, washed twice with PBS, and counted to adjust the cell density to 2×10 7 The treated cell suspension was placed on ice for later use. The skin of the nude mouse armpit was disinfected, and then 100 μL of the cell suspension was taken with a 1 mL sterile syringe and injected into the subcutaneous area of the right armpit of the nude mouse. The growth of the tumor was observed and recorded with a vernier caliper. When the tumor volume grew to 80-100 mm 3 At 3:00 pm, group dosing began.

[0206] Animal grouping and dosing

[0207] Twenty-five nude mice pre-inoculated with MKN-45 cells were randomly divided into five groups: the Model group received saline via tail vein injection; the CPD12C15 group received a 2 mg / kg copper gluconate solution orally in the morning and a 10 mg / kg CPD12C15 solution orally in the evening; the M-LNP-CPD12C15 low-dose group (L) received a 2 mg / kg copper gluconate solution orally in the morning and a 10 mg / kg M-LNP-CPD12C15 solution orally in the evening; and the M-LNP-CPD12C15 high-dose group (H) received a 2 mg / kg copper gluconate solution orally in the morning and a 20 mg / kg M-LNP-CPD12C15 solution orally in the evening. These drugs were administered every other day for a total of seven doses over two weeks. The positive drug DDP group received a 3 mg / kg DDP solution orally in the evening and a 5 dose every other day over two weeks.

[0208] Calculation of relative tumor inhibition rate: All nude mice were grouped and weighed every 2 days after drug administration, and the size of the tumor in each group was measured with a vernier caliper and recorded until the end of drug administration. On the 16th day, all experimental animals were bled after being killed, and the tumors and major organs of each group were dissected, weighed and recorded. The relative tumor growth rate of each group was calculated according to the formula: Relative tumor growth rate (%) = tumor weight of drug administration group / tumor weight of model group × 100%.

[0209] 1.1 Hematoxylin and Erythrocyte Embedding (H&E) Staining: Tumor masses from each group were cleaned with saline and wiped clean. They were then fixed in 4% paraformaldehyde and dehydrated in 85%, 95%, and 100% ethanol solutions, then immersed in xylene. When the tissue became transparent, the sections were embedded in paraffin blocks. The paraffin blocks were cut into 4-5 μm thin sections, attached to glass slides, and baked in a 90°C oven for 30 min. Dewaxing was performed in xylene, followed by hydration with decreasing ethanol concentrations and then rinsed with distilled water. The sections were stained in hematoxylin for 5-10 min, rinsed with distilled water, differentiated with 1% hydrochloric acid-ethanol, rinsed with distilled water, then blued with 1% ammonia, dehydrated with 80% ethanol, and stained with eosin for 3-5 min. The stained slides were then dehydrated in graded ethanol solutions, permeabilized with xylene, and mounted with a neutral resin. Histopathological changes were observed under a microscope.

[0210] 1.2 TUNEL apoptosis assay: Dewax the embedded paraffin sections and wash them three times with distilled water. Add proteinase K working solution to the sections, incubate at 37°C for 30 minutes, and wash three times with PBS. Then, immerse the sections in blocking solution, block at room temperature for 20 minutes, and wash three times with PBS. TUNEL staining is then performed. Add 100 μL of Streptavidin-HRP working solution to each section and react at 37°C for 60 minutes. Rinse three times with PBS. Finally, add DAB colorimetric solution and incubate for an appropriate time depending on the staining. Wash three times with PBS. Dehydrate using a gradient of ethanol, then place in xylene to make it transparent. Finally, mount the sections with neutral gum and observe under a microscope.

[0211] 1.3 Immunohistochemistry: The expressions of ubiquitin, CHOP, and cleaved caspase 3 in tumor tissues of each group were detected by immunohistochemistry to explore the mechanism of CPD12C15 and its preparations combined with copper in the treatment of gastric cancer.

[0212] After dewaxing the embedded paraffin blocks, sections were washed three times with distilled water for 5 minutes each. Sections were incubated in 3% H₂O₂ for 10 minutes and washed three times with PBS. Antigen retrieval was then performed by boiling the sections in citrate buffer for 3 minutes. After cooling to room temperature, the sections were removed and washed three times with PBS. Sections were then incubated in 5% BSA solution for 60 minutes at room temperature. The blocking solution was removed and a solution of primary antibodies, Ubiquitin, CHOP, and cleaved caspase 3, diluted to the specified ratio was added dropwise to the sections and incubated overnight at 4°C. Sections were washed three times with PBS and then the secondary antibody was added. The sections were incubated at room temperature for 50 minutes and washed three times with PBS. DAB working solution was added and the staining was observed under a microscope. After staining, the sections were immediately destained with distilled water. Counterstaining was performed with hematoxylin solution for 2 minutes. The stain was then washed off with distilled water and the sections were differentiated in 1% hydrochloric acid-alcohol solution for several seconds and washed again with water. Then dehydrate in gradient ethanol solution, place in xylene to permeabilize the slices, and finally seal with neutral gum before observing under a microscope.

[0213] 1.4 In vivo toxicity studies: While many anti-tumor drugs exert their anti-tumor effects, they generally present significant toxicity issues, such as hepatotoxicity, neurotoxicity, and bone marrow suppression. DDP, one of the most widely used and successful chemotherapy drugs, also carries risks during clinical use. The most serious toxic reaction of DDP is nephrotoxicity, which is caused by oxidative stress, inflammation, renal tubular damage, and apoptosis. The in vivo safety of the M-LNP-CPD12C15+Cu high-dose group and the DDP group was investigated by tissue H&E staining and serum creatinine and urea nitrogen levels.

[0214] 1.5 Tissue H&E Staining: H&E staining was performed on the major organs (heart, liver, spleen, lung, and kidney) of nude mice to observe pathological changes in each group and evaluate the in vivo safety of CPD12C15 and M-LNP-CPD12C15 combined with copper and DDP. The H&E staining procedure was the same as in Example 1.1.

[0215] 1.6 Renal function evaluation index detection: serum samples stored at -80°C were removed and returned to room temperature. The levels of serum creatinine and urea nitrogen were determined according to the kit instructions.

[0216] 1.7 Immunohistochemical detection of NF-κB expression in renal tissue: The operation method is the same as that in 1.3 of this example.

[0217] 1.8 Determination of IL-6 and IL-1β Content in Renal Tissue

[0218] Sample Preparation: Cool the tissue grinder in advance. Weigh a certain amount of kidney tissue and add 9 volumes of normal saline as the homogenizer at a weight (mg): volume (mL) ratio of 1:9. Place the tissue in an EP tube, add two magnetic beads to each tube, and place it in a pre-cooled grinding tank. Set the grinding power to 60 Hz and grind for 10 seconds each time for a total of five times. After grinding, centrifuge at 12,000 rpm for 15 minutes at 4°C. The supernatant should be stored at -80°C.

[0219] Determination of inflammatory factor content: First, standard curves were established according to the instructions of the IL-6 and IL-1β detection kits, and then the contents of IL-6 and IL-1β in the renal tissue homogenates of different experimental groups were detected.

[0220] 1.9 Statistical Analysis: Experimental data were processed and plotted using GraphPad Prism 8 software. Results are presented as mean ± standard deviation (SD). Data were analyzed by one-way analysis of variance to assess inter-group differences. p < 0.05 indicated a significant difference from the Model group. *p < 0.05, **p < 0.01, ***p < 0.001 were used.

[0221] 2. Experimental results

[0222] 2.1M-LNP-CPD12C15 combined with copper inhibits tumor growth in nude mice

[0223] The volume of nude mouse tumors was recorded during the administration period. After the administration, the tumors were removed and divided into groups for photographing and weighing. Figure 25 As shown, from Figure 25In (a), it can be found that the tumor volume of the drug-treated group was significantly smaller than that of the model group, indicating that CPD12C15 combined with copper can inhibit tumor growth. Among them, the inhibitory effect of M-LNP-CPD12C15+Cu on tumors was better than that of CPD12C15+Cu at the same dose. The tumor in the M-LNP-CPD12C15+Cu(H) group was the smallest, indicating that M-LNP-CPD12C15 can better exert its anti-tumor efficacy in a dose-dependent manner. Figure 25 (b) and (c) show the volume change of the tumor during the experiment and the final tumor mass weight, respectively. It can be seen that the tumor grew slowly after administration. The final volume of the tumor mass in the Model group was 1048.46±182.36mm 3 The tumor mass weight was 0.96±0.12 g, and the final volume of the tumor mass in the CPD12C15+Cu group was 590.25±151.39 mm 3 , the tumor mass weight was 0.61±0.14 g, and the final volume of the tumor mass in the M-LNP-CPD12C15+Cu(L) group was 393.44±167.99 mm 3 The tumor mass was 0.40±0.18 g, and the final volume of the tumor mass in the M-LNP-CPD12C15+Cu(H) group was 225.40±42.01 mm 3 The tumor weight was 0.25±0.08 g, and the final volume of the tumor in the DDP group was 426.36±183.38 mm 3 The tumor mass weighed 0.44±0.18g. Calculations showed that the relative tumor growth rates were 64.44% for the CPD12C15+Cu group, 41.48% for the M-LNP-CPD12C15+Cu(L) group, 26.19% for the M-LNP-CPD12C15+Cu(H) group, and 46.08% for the DDP group. This indicates that the inhibitory effect of M-LNP-CPD12C15 combined with copper on tumors was superior to that of the positive drug DDP and the same dose of CPD12C15+Cu, and was clearly dose-dependent. Figure 25 (d) shows the H&E staining results of the tumor mass. The Model group tumor cells are dense and growing vigorously. After drug administration, tumor cell density decreases. The M-LNP-CPD12C15+Cu(H) group has the lowest cell density, shrunken nuclei, and numerous vacuoles in the intercellular spaces, demonstrating the strongest inhibitory effect on tumor tissue. These results indicate that M-LNP-CPD12C15 combined with copper has a strong killing effect on tumor cells and can effectively inhibit tumor growth.

[0224] 2.2M-LNP-CPD12C15 combined with copper promotes apoptosis of gastric cancer cells

[0225] The apoptosis of gastric cancer cells in subcutaneous tumors was detected by TUNEL. Figure 26 The results of TUNEL staining showed that all cells in the Model group were blue, indicating a negative reaction. After drug treatment, the number of positive apoptotic cells increased. Among them, the number of apoptotic cells in the CPD12C15+Cu and DDP groups did not increase significantly (p>0.05). The efficacy of M-LNP-CPD12C15+Cu in promoting the apoptosis of gastric cancer cells was more significant (**p<0.01) and showed concentration dependence.

[0226] Exploration of the mechanism of apoptosis of gastric cancer cells induced by 2.3M-LNP-CPD12C15 combined with copper

[0227] Endoplasmic reticulum stress plays an important role in the occurrence and development of gastric cancer. It can regulate the apoptosis and autophagy of gastric cancer cells, initiate the caspase cascade reaction, and mediate cell apoptosis. The results of in vitro cell experiments showed that CPD12C15 and its preparations combined with copper can induce irreversible ERS in gastric cancer cells and promote apoptosis. In order to further determine the effect of M-LNP-CPD12C15+Cu on this pathway, the expression of Ubiquitin, CHOP and cleaved caspase 3 in tumor tissues after administration was detected by immunohistochemistry. Figure 27 The results show that after administration, the expression of ubiquitin, endoplasmic reticulum stress marker protein CHOP, and pro-apoptotic protein cleaved caspase 3 increased, indicating that CPD12C15+Cu can inhibit the ubiquitination proteasome system, so that waste proteins in tumor cells cannot be processed, resulting in an increase in ubiquitinated proteins, thereby inducing ERS in tumor cells, promoting apoptosis of gastric cancer cells, and inhibiting their proliferation.

[0228] 2.4 In vivo safety analysis

[0229] During the experiment, the nude mice were weighed and recorded every 3 days to observe drug toxicity during administration. After administration, H&E staining of important tissues and organs of the nude mice was performed for histopathological analysis to investigate the in vivo safety of M-LNP-CPD12C15. Figure 28 shown. Figure 28 The figure shows the changes in the body weight of nude mice during the administration process. It can be seen that CPD12C15 and its preparations combined with copper have no significant effect on the body weight of nude mice, and the body weight fluctuation range is small and relatively stable; while the body weight of nude mice in the DDP group decreased significantly (**p<0.01), and the nude mice gradually lost weight during the administration process. Figure 28The results of H&E staining of the heart, liver, spleen, lung, and kidney of nude mice in each group are shown. It can be seen that the tissue sections of the M-LNP-CPD12C15+Cu(H) group did not produce obvious pathological changes compared with the Model group, while the renal cortex of the renal tissue in the DDP group showed mild atrophy of multiple renal tubules, reduced volume of renal tubular epithelial cells, decreased cytoplasmic eosinophilia (black arrows), and mild connective tissue hyperplasia (yellow arrows) in the interstitium. The experimental results show that M-LNP-CPD12C15+Cu has good in vivo safety while exerting anti-tumor effects, while DDP has certain nephrotoxicity and can induce renal tissue lesions during use. It can be seen that M-LNP-CPD12C15+Cu has more obvious advantages over the traditional chemotherapy drug DDP.

[0230] 2.5 In vivo nephrotoxicity analysis

[0231] DDP is one of the most widely used chemotherapeutic drugs in clinical practice, used to treat a variety of cancers. Its antitumor activity primarily involves blocking DNA transcription and replication. However, its clinical application is limited by its severe side effects, the most serious of which is nephrotoxicity, manifested as acute kidney injury (AKI), sodium or magnesium loss, and decreased urine concentrating ability. Clinical studies have shown that 8-10% of patients experience elevated serum creatinine and urinary albumin, as well as decreased glomerular filtration, within 10 days of DDP treatment. Recent studies have shown that DDP toxicity is caused by inflammation. It has been found to induce proinflammatory signaling and elevated inflammatory markers in nephrotoxicity, neurotoxicity, and ototoxicity. NF-κB is a key signaling pathway in inflammatory responses, upregulating inflammatory cytokines and causing renal damage.

[0232] 2.5.1 Renal function index test results

[0233] Creatinine and urea nitrogen in serum are the main indicators for evaluating renal function. Serum creatinine (CRE) is a product of muscle metabolism and is excreted through the kidneys. An increase in serum creatinine concentration can reflect a decrease in glomerular filtration function. Blood urea nitrogen (BUN) is a metabolite generated by protein metabolism and is excreted through the kidneys. An increase in blood urea nitrogen concentration can indicate a decrease in renal excretion function. The experimental results are as follows: Figure 29 As shown in the data, CRE and BUN in the DDP group were significantly higher than those in the Model group (***p<0.001, **p<0.01), while there was no change in the CPD12C15+Cu and M-LNP-CPD12C15+Cu(H) groups, indicating that DDP can cause renal dysfunction and has certain nephrotoxicity, while M-LNP-CPD12C15+Cu has good safety.

[0234] 2.5.2 Detection results of NF-κB and inflammatory factors in renal tissue

[0235] The expression of NF-κB in the kidney tissue of nude mice was detected by immunohistochemistry, and the contents of IL-6 and IL-1β in the kidney tissue homogenate were then detected. Figure 30 As shown in the data, there were no significant differences in the expression of NF-κB and the contents of IL-6 and IL-1β in the CPD12C15+Cu and M-LNP-CPD12C15+Cu(H) groups compared with the Model group, while the expression of NF-κB in the DDP group was significantly enhanced, and the contents of IL-6 and IL-1β were also increased (**p<0.01, *p<0.05), indicating that DDP promoted the production of inflammatory factors by upregulating the expression of NF-κB, causing renal damage, while M-LNP-CPD12C15+Cu had no effect on the expression of NF-κB in renal tissue and had better in vivo safety.

[0236] The above description is merely a preferred embodiment of the present application and is not intended to limit the scope of the present application. Although the present application has been described in detail with reference to the above embodiments, those skilled in the art may, after reading this description, make various modifications to the technical solution or replace some of the technical features with equivalents. Any modification, equivalent replacement, or improvement made within the spirit and principles of the present application shall be deemed to fall within the scope of protection of the present application.

Claims

1. A mannose-modified CPD12C15 lipid nanoparticle, characterized in that: The lipid nanoparticles comprise CPD12C15, phospholipids, mannose-modified DSPE-PEG2000, and a surfactant; wherein CPD12C15 is encapsulated inside the lipid nanoparticles, and the mannose portion of the mannose-modified DSPE-PEG2000 is exposed on the surface of the lipid nanoparticles; wherein the structure of CPD12C15 is as follows:

2. The lipid nanoparticle according to claim 1, wherein The phospholipid is soybean lecithin; the surfactant is a nonionic surfactant, preferably a polysorbate surfactant, more preferably polysorbate 80 (also known as Tween 80); Preferably, the molar ratio of the CPD12C15 to the lipid component is 1:2 to 1:5, preferably 1:2, wherein the lipid component refers to the total amount of the phospholipid and the mannose-modified DSPE-PEG2000; Preferably, the molar ratio of soybean lecithin to mannose-modified DSPE-PEG2000 is 20:1 to 35:1, preferably 30:1; Preferably, the average particle size of the lipid nanoparticles is 60-75 nm, preferably 65-70 nm; Preferably, the zeta potential of the nanoparticles is -20 to -30 mV, preferably -20 to -25 mV; Preferably, the encapsulation efficiency of CPD12C15 is 60-80%, preferably 70-80%; Preferably, the drug loading of the CPD12C15 is 10-15%, preferably 12-15%; Preferably, the concentration of polysorbate 80 is 0.5-2.0%, preferably 2.0%; Preferably, the mannose-modified DSPE-PEG2000 is synthesized by reacting DSPE-PEG2000-NH2 with α-D-mannopyranosylphenylisothiocyanate (MPITC); Preferably, the reaction of DSPE-PEG2000-NH2 and MPITC comprises: dissolving MPITC in methanol, adding triethylamine as a catalyst, dissolving DSPE-PEG2000-NH2 in dichloromethane, then adding the DSPE-PEG2000-NH2 solution dropwise to the MPITC solution, reacting at room temperature, removing the organic solvent after the reaction, adding DMSO and water to dissolve the product, dialyzing, and freeze-drying to obtain mannose-modified DSPE-PEG2000.

3. A method for preparing mannose-modified CPD12C15 lipid nanoparticles, characterized in that: include: Phospholipids and mannose-modified DSPE-PEG2000 are dissolved in an aqueous solution containing a surfactant to form an aqueous phase; CPD12C15 is dissolved in an organic solvent to form an organic phase; the organic phase is slowly added dropwise to the aqueous phase under stirring; the mixed solution is ultrasonically treated; the organic solvent is evaporated by stirring; and filtration is performed to obtain mannose-modified CPD12C15 lipid nanoparticles.

4. The method according to claim 3, characterized in that The surfactant is a nonionic surfactant, preferably polysorbate 80, and the concentration of polysorbate 80 is 0.5-2.0%, preferably 2.0%; Preferably, the organic solvent is one or more of methanol, ethanol, acetone or acetonitrile, preferably methanol; Preferably, the ultrasonic treatment time is 1-7 minutes, preferably 3 minutes; Preferably, the stirring time for volatilizing the organic solvent is 4-8 hours, preferably 6 hours; Preferably, the stirring is magnetic stirring, and the stirring time is 4-8 hours, preferably 6 hours; Preferably, the filtration uses a 0.22 μm filter membrane. 5 . A pharmaceutical composition comprising the mannose-modified CPD12C15 lipid nanoparticles according to claim 1 or 2 .

6. The pharmaceutical composition according to claim 5, characterized in that include: a) the mannose-modified CPD12C15 lipid nanoparticles according to claim 1 or 2; b) pharmaceutically acceptable copper salts; Preferably, the molar ratio of the copper salt to CPD12C15 is 0.5-2:1, preferably 1:1; Preferably, the copper salt is selected from copper gluconate, copper chloride or a combination thereof; Preferably, component a) is mixed with component b) immediately before use.

7. A pharmaceutical preparation comprising the mannose-modified CPD12C15 lipid nanoparticles according to claim 1 or 2 and a pharmaceutically acceptable carrier.

8. A medical kit comprising: A first container containing the mannose-modified CPD12C15 lipid nanoparticles according to claim 1 or 2; a second container containing a pharmaceutically acceptable copper salt solution; The two containers are designed for simultaneous, separate or sequential administration.

9. The medical kit according to claim 8, characterized in that: The pharmaceutically acceptable copper salt is selected from copper gluconate, copper chloride or a combination thereof.

10. Use of the mannose-modified CPD12C15 lipid nanoparticles according to claim 1 or 2, or the pharmaceutical composition according to claim 5 or 6, or the pharmaceutical preparation according to claim 7, or the medical kit according to claim 8 in the preparation of a medicament for treating tumors with high mannose receptor expression; Preferably, the tumor with high mannose receptor expression is gastric cancer; Preferably, the drug is administered by intravenous injection.

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