Disulfiram derivative CPD12C15 folic acid targeted liposome as well as preparation method and application thereof

Abstract

The invention relates to the technical field of drug nano delivery, and discloses a disulfiram derivative CPD12C15 folic acid targeted liposome as well as a preparation method and application thereof. The lipidosome is of a closed vesicle-shaped structure, and a lipid shell layer of the lipidosome is enclosed by a lipid bilayer which is jointly formed by lecithin, cholesterol and a DSPE lipophilic end of DSPE-PEG2000-Fa; a PEG-folic acid hydrophilic end of the DSPE-PEG2000-Fa extends and is exposed on the outer surface of a lipid shell layer, so that a folic acid receptor is endowed with a targeting function; the inner water phase core is a water-based cavity enclosed by a lipid shell layer, and the active component CPD12C15 is encapsulated in the inner water phase core. Compared with the prior art, the liposome disclosed by the invention has the advantages of high encapsulation efficiency, high drug loading capacity, pH-sensitive controlled release and targeted enrichment in hepatocellular carcinoma tissues, can remarkably inhibit tumor proliferation, migration and angiogenesis and induce apoptosis, and improves the curative effect and reduces the toxicity by regulating a PI3K / AKT pathway.

Description

Technical Field

[0001] The present application relates to the field of pharmaceutical preparations, and in particular to a disulfiram derivative CPD12C15 folic acid-targeted liposome and its preparation method and application. 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] Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. HCC poses a serious threat to human health due to its insidious onset, difficulty in early diagnosis, limited therapeutic efficacy, high metastasis rate, and high recurrence rate. Currently, HCC treatments primarily include surgery, chemotherapy, radiotherapy, interventional therapy, and targeted therapy. However, these treatment options all have limitations: surgery is ineffective for patients with advanced HCC, nonspecific adverse reactions to traditional chemotherapy are severe, and the response rate to immunotherapy is low.

[0004] Sorafenib is an FDA-approved first-line treatment for HCC, but it faces serious drug resistance and skin toxicity in clinical use. Therefore, the development of highly effective and low-toxic drugs for the treatment of hepatocellular carcinoma is of great clinical value.

[0005] Drug repositioning strategies have garnered significant attention in recent years. Disulfiram (DSF), an approved drug for the treatment of alcohol dependence, has been shown to exhibit antitumor activity. However, poor water solubility and a high risk of neurotoxicity limit its clinical application in the antitumor setting. Structural modification of DSF to enhance its antitumor activity and reduce its toxicity is a key area of ​​current research.

[0006] Liposomes, as a mature nano-drug delivery system, have been widely used to deliver a variety of drugs. Currently, key challenges in the field of nano-delivery systems remain to be addressed, including improving drug accumulation and release at the tumor site, reducing systemic drug toxicity, and overcoming tumor drug resistance. The development of highly efficient, low-toxic, and highly targeted drug delivery systems is crucial for improving the therapeutic efficacy of hepatocellular carcinoma. Summary of the Invention

[0007] The present invention aims to solve the problems of low specificity, large side effects and insufficient drug delivery efficiency of existing drugs for the treatment of hepatocellular carcinoma. Sorafenib, as an FDA-approved first-line drug for the treatment of hepatocellular carcinoma, has serious drug resistance and skin toxicity. About 40% of patients need to reduce the dose or even stop taking the drug due to hand-foot skin syndrome, which greatly reduces the therapeutic effect. Although the disulfiram derivative CPD12C15 has potential anti-tumor activity, its direct application in tumor treatment faces many challenges due to its poor water solubility, low bioavailability and lack of specific targeting ability. Although traditional liposomes can increase drug solubility, they are easily cleared by the reticuloendothelial system during circulation in the body, and lack the ability to actively target tumor tissues, resulting in insufficient enrichment of the drug at the tumor site and limited efficacy.

[0008] To overcome the above problems, the present invention provides a folic acid-targeted CPD12C15 liposome and a preparation method thereof. The liposome is composed of lecithin, cholesterol and DSPE-PEG2000-Fa to form a lipid bilayer, forming a closed vesicle-like structure, wherein the DSPE lipophilic end of DSPE-PEG2000-Fa is embedded in the lipid bilayer, and the PEG2000-Fa hydrophilic end extends out of the liposome surface to provide a targeting function; CPD12C15 is loaded in the aqueous phase within the liposome. The present invention establishes a stable and reproducible preparation process by systematically optimizing parameters such as the molar ratio of lecithin to cholesterol, calcium acetate concentration, drug-lipid molar ratio, phospholipid concentration, DSPE-PEG2000-Fa content, incubation temperature and time. This process combines drug forced loading technology with surface targeted modification strategy to achieve efficient encapsulation and targeted delivery of CPD12C15. In addition, the present invention also designs a medical kit so that folic acid-targeted CPD12C15 liposomes and copper salts can be administered simultaneously, separately or sequentially to meet different clinical needs.

[0009] Experimental results show that the folic acid-targeted CPD12C15 liposomes prepared by the present invention have the following technical effects: (1) significantly improving the solubility and stability of CPD12C15, achieving a sustained release effect of the drug, and effectively solving the problems of poor water solubility and low bioavailability; (2) through folate receptor-mediated endocytosis, active targeting of hepatocellular carcinoma cells expressing folate receptors is achieved, and in vivo fluorescence imaging shows that its enrichment at the tumor site is significantly increased compared with non-targeted liposomes; (3) exhibiting pH-responsive release characteristics under the weak acid conditions of the tumor microenvironment, achieving targeted release at the tumor site; (4) when used in combination with copper salts, it exhibits significant proliferation inhibition, migration inhibition and invasion inhibition activity against hepatocellular carcinoma cells, and the in vivo tumor inhibition rate reaches a level comparable to or better than that of the standard therapeutic drug sorafenib; (5) by reducing the level of inflammatory factors in serum, it effectively reduces the common skin inflammatory toxicity of sorafenib; (6) the hemolysis rate is low, and there are no obvious pathological changes in major organs, showing good biosafety.

[0010] In summary, the present invention has constructed a folate-targeted CPD12C15 liposome system that simultaneously exhibits high encapsulation efficiency, controlled release, active targeting, and good biocompatibility. This system effectively overcomes the shortcomings of CPD12C15, such as poor water solubility and lack of targeting, through folate receptor-mediated targeted delivery. This significantly enhances its anti-tumor efficacy while reducing inflammatory responses, providing a new approach for overcoming sorafenib resistance and skin inflammation-related toxicity. This invention not only provides an innovative strategy for the precision treatment of hepatocellular carcinoma but also provides an important reference for the development of targeted delivery systems for similar anti-tumor drugs.

[0011] Specifically, the present invention provides the following technical solutions.

[0012] In a first aspect, the present invention provides a folic acid-targeted CPD12C15 liposome, which is a vesicle-like structure and comprises:

[0013] The lipid shell is a lipid bilayer formed by phosphatidylcholine, cholesterol and the DSPE lipophilic end of DSPE-PEG2000-Fa, and encloses the vesicle;

[0014] A targeting functional layer comprising DSPE-PEG2000-Fa co-membraned with the lipid shell, wherein the lipophilic end of DSPE is embedded in the lipid bilayer, and the hydrophilic end of PEG2000-Fa extends and is exposed on the outer surface of the liposome, thereby imparting targeting properties;

[0015] The inner aqueous core is an aqueous cavity enclosed by the lipid shell, which contains the active ingredient CPD12C15;

[0016] Wherein, the structure of the CPD12C15 is:

[0017]

[0018] In some embodiments of the present invention, the active ingredient CPD12C15 can be partially embedded in the lipid shell to form a multi-site distribution state, which is beneficial to improving drug loading efficiency and stability.

[0019] In a preferred embodiment, the active ingredient, CPD12C15, is not only distributed in the inner aqueous core but also partially embedded in the lipid shell, forming a multi-site distribution. This amphiphilic nature enables CPD12C15 to coexist in both the inner aqueous phase and the lipid shell, enhancing drug loading efficiency and providing a biphasic release profile, significantly different from traditional strategies that only load drugs in a single region.

[0020] In some embodiments, the molar ratio of phosphatidylcholine to cholesterol in the liposomes of the present invention is 1:1 to 5:1, preferably 2:1. This ratio range can balance the fluidity and stability of the lipid bilayer. Too high a cholesterol content increases membrane rigidity, hindering drug penetration and release; too low a cholesterol content reduces the stability and drug retention capacity of the liposomes.

[0021] In some embodiments, in the liposomes of the present invention, the molar ratio of CPD12C15 to lipid is 1:1 to 1:8, preferably 1:2. This ratio range can fully utilize the drug loading capacity while maintaining the integrity of the liposome structure and avoiding liposome instability caused by excessive drug loading.

[0022] The molar ratio of CPD12C15 to lipid refers to the ratio of the molar amount of CPD12C15 to the total molar amount of all lipid components (including phosphatidylcholine, cholesterol, and DSPE-PEG2000-Fa) in the liposome, and the ratio ranges from 1:1 to 1:8, and is more preferably 1:2. This ratio affects the control of drug loading, maintaining liposome structural stability, and achieving ideal drug release characteristics. When the ratio is lower than 1:1, excessive drug loading will destroy the lipid bilayer structure; when the ratio is higher than 1:8, the drug loading efficiency is too low, which is not conducive to achieving an effective therapeutic concentration.

[0023] In another preferred embodiment, the DSPE-PEG2000-Fa content in the liposomes of the present invention is 0.5-2.0% by mole of total lipids, more preferably 1.25%. "Total lipids" herein refers to the total molar amount of all lipid components comprising the liposome membrane, namely, the sum of the molar amounts of phosphatidylcholine, cholesterol, and DSPE-PEG2000-Fa. This content range provides sufficient targeting while avoiding the congestion effect of excessive PEG chains that could affect liposome stability. A content below 0.5% results in insignificant targeting, while a content above 2.0% may interfere with the integrity of the lipid bilayer.

[0024] In some embodiments, the folate-targeted CPD12C15 liposomes described herein have the following physicochemical properties: a particle size of 80-130 nm, more preferably 100-120 nm; a polydispersity index (PDI) of less than 0.3, more preferably less than 0.2; and a surface potential of -1.0 to -2.5 mV, more preferably -1.8 ± 0.5 mV. This particle size range facilitates enhanced tumor accumulation through the EPR effect while preventing rapid clearance by the reticuloendothelial system. A low PDI value indicates a uniform particle size distribution, which facilitates consistent in vivo behavior. A slight negative charge helps maintain liposome dispersibility without overactivating the reticuloendothelial system.

[0025] In some embodiments, the folate-targeted CPD12C15 liposomes of the present invention have an encapsulation efficiency of 75-95%, more preferably 90-95%, and a drug loading of 10-20%, more preferably 12-17%. The high encapsulation efficiency and appropriate drug loading ensure sufficient drug loading efficiency and effective therapeutic concentration.

[0026] According to the folic acid-targeted CPD12C15 liposomes described in the present invention, the PEG chains on the surface of the liposomes form a three-dimensional barrier to reduce serum protein adsorption; the folic acid ligand specifically binds to the folate receptor overexpressed on the surface of tumor cells, promoting receptor-mediated endocytosis; after endocytosis, the liposomes release CPD12C15 in the acidic environment of the lysosome, and the latter can bind to intracellular copper ions, inhibit the ubiquitin proteasome system, and induce tumor cell apoptosis.

[0027] Compared with the existing technology, the folic acid-targeted CPD12C15 liposomes of the present invention achieve active targeting of hepatocellular carcinoma cells through a triple structure design (lipid shell-targeting functional layer-inner aqueous phase core); by optimizing the content and distribution of DSPE-PEG2000-Fa in the liposomes, the targeting efficiency and liposome stability are improved; and the multi-site distribution of drugs in the inner aqueous phase and lipid bilayer is achieved, which is different from the traditional strategy of loading drugs only in a single area.

[0028] In a second aspect of the present invention, a method for preparing folic acid-targeted CPD12C15 liposomes is provided, comprising the following steps:

[0029] Phosphatidylcholine, cholesterol and DSPE-PEG2000-Fa were mixed and dissolved in an organic solvent and rotary evaporated to form a uniform lipid film;

[0030] The lipid film was hydrated with calcium acetate solution, and after ultrasonic treatment, the blank calcium acetate liposomes containing DSPE-PEG2000-Fa were obtained by filtration;

[0031] The blank liposomes were dialyzed to establish a pH gradient;

[0032] The dialyzed blank liposomes were mixed with CPD12C15 and incubated, and then filtered to obtain folate-targeted CPD12C15 liposomes.

[0033] In some embodiments, the organic solvent is chloroform, the molar ratio of phosphatidylcholine to cholesterol is 1:1 to 5:1, preferably 2:1; and the content of DSPE-PEG2000-Fa is 0.5-2.0% of the total lipid molar number, preferably 1.25%. Precise control of these parameters is crucial for forming a structurally stable lipid film. The phosphatidylcholine to cholesterol ratio directly affects the fluidity and rigidity of the lipid bilayer, while the DSPE-PEG2000-Fa content determines the density of targeting ligands on the liposome surface.

[0034] In some embodiments, the concentration of the calcium acetate solution is 80-200 mmol / L, preferably 120 mmol / L; the phospholipid concentration is 10-40 mg / mL, preferably 20 mg / mL. The calcium acetate concentration is a key factor in establishing an effective pH gradient; too low a concentration may not generate sufficient transmembrane driving force, while too high a concentration may affect the stability of the liposomes. An appropriate phospholipid concentration ensures the formation of liposomes with uniform particle size while maximizing drug loading capacity.

[0035] In some embodiments, the ultrasonic treatment is performed intermittently in an ice bath, for example, using 50W power, sonication for 2 seconds on, 2 seconds off, for a total of 2 minutes. This intermittent ultrasonication scheme can effectively reduce local overheating and prevent lipid oxidative degradation, while ensuring adequate hydration and initial homogenization of liposome particle size.

[0036] In some embodiments, the dialysis is performed using a dialysis bag with a molecular weight cut-off of 3500 Da, and the dialysis medium is a sodium sulfate solution of equal concentration to the calcium acetate solution. This dialysis process aims to establish a transmembrane pH gradient—the acidic calcium acetate environment inside the liposomes is maintained, while the external medium is replaced by a neutral sodium sulfate solution. This pH gradient is the core driving force for the active loading of CPD12C15.

[0037] In some embodiments, the molar ratio of CPD12C15 to lipid is 1:1 to 1:8, preferably 1:2; the incubation temperature is 30-60°C, preferably 40°C; the incubation time is 40-70 minutes, preferably 60 minutes; and the filtration is performed using a 0.22 μm microporous filter membrane. These parameters are key control points during the drug loading process: an appropriate CPD12C15 to lipid ratio ensures efficient loading while maintaining liposome structural integrity; an appropriate incubation temperature provides sufficient membrane fluidity without disrupting the liposome structure; and an appropriate incubation time allows for sufficient drug permeation and redistribution.

[0038] In this study, a pH gradient-driven remote loading technique was employed, leveraging the physicochemical properties of CPD12C15, enabling it to penetrate the lipid membrane and accumulate in the inner aqueous phase. Furthermore, the internal calcium acetate environment may help improve drug retention. Compared to traditional passive loading methods, this active loading method helps improve encapsulation efficiency.

[0039] The preparation method of the present invention is controllable and reproducible. By precisely controlling parameters such as the lecithin to cholesterol ratio, DSPE-PEG2000-Fa content, calcium acetate concentration, incubation temperature, and time, batch-to-batch consistency in product quality is ensured. Optimization of the ultrasound and extrusion processes ensures uniform liposome particle size and distribution, which is crucial for in vivo distribution and targeting efficiency.

[0040] Compared with the existing technology, the uniqueness of the preparation method of the present invention lies in: for the first time, a specific pH gradient remote loading scheme was designed based on the physicochemical properties of CPD12C15; the calcium acetate concentration and lipid component ratio were optimized to achieve the coordinated distribution of CPD12C15 in the inner aqueous phase and the lipid bilayer; and low-temperature intermittent ultrasound and precisely controlled incubation conditions were used to maintain the activity of the folate targeting ligand while ensuring a high encapsulation efficiency.

[0041] The third aspect of the present invention provides a pharmaceutical composition comprising the folic acid-targeted CPD12C15 liposomes according to the first aspect and a pharmaceutically acceptable carrier or excipient, for treating hepatocellular carcinoma.

[0042] In a preferred embodiment, the pharmaceutical composition further comprises a copper salt selected from one or more of copper chloride, copper sulfate, copper acetate, and copper gluconate, more preferably copper gluconate. The addition of the copper salt is based on the synergistic anti-tumor effect exhibited by CPD12C15 and copper ions, and their combined use significantly enhances the killing ability against hepatocellular carcinoma cells.

[0043] In some embodiments, the molar ratio of the folic acid-targeted CPD12C15 liposomes to the copper salt is 1:0.5 to 1:2, more preferably 1:1. This ratio range is determined based on the results of in vitro cell experiments and in vivo animal experiments, while ensuring a synergistic effect while avoiding the toxic side effects that may be caused by excessive copper ions.

[0044] In some embodiments, the pharmaceutical composition can be prepared in a dosage form suitable for intravenous administration, such as an injection, lyophilized powder injection, or liposomal suspension. The choice of different dosage forms will be determined based on factors such as stability requirements, clinical convenience, and medical kit design. Injectable dosage forms ensure drug bioavailability and avoid the first-pass effect and digestive tract degradation issues that may arise with oral administration.

[0045] In some embodiments, the pharmaceutical composition contains CPD12C15 at a concentration of 0.5-5 mg / mL, more preferably 1-2 mg / mL, in the folate-targeted CPD12C15 liposomes; and the copper salt contains copper at a concentration of 0.1-1.0 mg / mL, more preferably 0.3-0.5 mg / mL. This concentration range ensures both a therapeutic dose and the physical stability of the formulation.

[0046] In some embodiments, the pharmaceutical composition may contain pharmaceutically acceptable excipients as needed, which can be selected from: isotonicity regulators, such as sodium chloride, mannitol, glucose, sucrose, etc.; pH regulators, such as citrate buffer, phosphate buffer, acetate buffer, etc.; excipients, such as trehalose, sucrose, mannitol, lactose, etc.; surfactants, such as polysorbate 80, polyethylene glycol 400, phospholipids, etc.; antioxidants, such as vitamin E, vitamin C, sodium thiosulfate, etc.; preservatives, such as benzyl alcohol, parahydroxybenzoic acid esters, etc.; and other conventional pharmaceutical excipients.

[0047] The selection and dosage of the above excipients can be adjusted according to the specific dosage form and clinical needs to optimize the physical and chemical properties of the pharmaceutical composition, improve ease of use, and ensure long-term storage stability. The preparation method adopts conventional aseptic preparation processes in the field, including but not limited to sterile filtration, sterile filling, lyophilization, etc. The lyophilized powder injection should be reconstituted with an appropriate solvent (such as water for injection, 0.9% sodium chloride injection, etc.) before use.

[0048] The pharmaceutical composition can be prepared into a formulation suitable for clinical use using standard pharmaceutical preparation techniques. For example, an injection can be obtained by mixing the folic acid-targeted CPD12C15 liposomes with suitable injection excipients, adjusting the isotonicity and pH value, and then sterile filtering and filling. A lyophilized powder injection can be obtained by adding a suitable lyoprotectant, pre-freezing, primary drying, and secondary drying. A liposome suspension can be prepared by controlling the liposome concentration and adding a suitable pH adjuster and isotonicity adjuster.

[0049] In some embodiments, the present invention provides a medical kit comprising:

[0050] (a) a first container containing folate-targeted CPD12C15 liposomes;

[0051] (b) a second container containing a copper salt; and

[0052] (c) instructions for different dosing regimens.

[0053] The design of this medical kit allows for flexible dosing of the folic acid-targeted CPD12C15 liposomes and copper salts according to clinical needs:

[0054] Mixed administration: For example, in one embodiment, the folate-targeted CPD12C15 liposomes in a first container and the copper salt solution in a second container are mixed in a proportional ratio (e.g., a 1:1 volume ratio). The mixture is gently inverted 5-10 times, allowed to stand for 5-10 minutes, and then administered via intravenous infusion. This regimen is suitable for situations where a direct synergistic effect between CPD12C15 and copper ions is desired.

[0055] Split dosing: On the same treatment day, administer the folic acid-targeted CPD12C15 liposomes in the first container first, followed by the copper salt solution in the second container 2-6 hours later. This approach may help minimize direct interactions between the two components and is suitable for situations where stability after mixing is a concern.

[0056] Sequential dosing: The two components are administered on different days of the treatment cycle, such as copper salt solution on days 1, 3, and 5, and folic acid-targeted CPD12C15 liposomes on days 2, 4, and 6. This regimen may help first establish copper ion levels in the body and then provide CPD12C15 that can work synergistically with them.

[0057] Physicians can choose the most appropriate dosing regimen based on the patient's specific condition, tumor status, and treatment response. This flexible design significantly improves the individualization of treatment and also provides the possibility for clinical research on the efficacy differences of different dosing regimens.

[0058] Another advantage of this medical kit is that packaging the two components separately can avoid possible interactions during long-term storage, prolong the stability of the product, and meet the usage needs of different medical institutions and clinicians.

[0059] In some embodiments, the composition of the present invention can be used by intravenous administration, and the specific dosage and administration regimen can be determined individually by a physician based on factors such as body weight, liver function status, and tumor load.

[0060] The pharmaceutical composition of the present invention can be used to treat hepatocellular carcinoma, particularly providing a new treatment option for patients who have not responded well to existing treatment strategies. As clinical research progresses, its therapeutic value and precise dosing regimens in different patient subpopulations are expected to be further clarified. The unique advantages of the pharmaceutical composition of the present invention are: precise targeting of hepatocellular carcinoma expressing folate receptors is achieved through a folate-targeted delivery system; the synergistic effect of CPD12C15 and copper ions significantly enhances anti-tumor activity; the liposome carrier system improves the water solubility and bioavailability of CPD12C15 and reduces its systemic toxicity; and the design of the medical kit increases flexibility and convenience of use.

[0061] In some embodiments, the pharmaceutical composition can also be used in combination with other anti-tumor drugs, wherein the anti-tumor drugs are selected from one or more of sorafenib, doxorubicin, capecitabine, and oxaliplatin. Combined use can exert a synergistic anti-tumor effect, reduce the development of single-drug resistance, and improve the overall therapeutic effect.

[0062] In summary, the folic acid-targeted CPD12C15 liposome pharmaceutical composition provided by the present invention provides a new option for the precision treatment of hepatocellular carcinoma through an innovative drug delivery system, optimized component ratio and flexible set design, and has important clinical value, especially for patient groups who are insensitive to existing treatments or have developed resistance.

[0063] In the fourth aspect of the present invention, there is provided the use of the folic acid-targeted CPD12C15 liposomes described in the first aspect or the pharmaceutical composition or medical kit described in the third aspect in the preparation of the following drugs: (1) a drug for treating hepatocellular carcinoma; (2) a drug for reversing sorafenib resistance or reducing sorafenib-related skin toxicity; (3) a drug for reducing the levels of IL-6 and / or TNF-α in chronic inflammation associated with hepatocellular carcinoma; (4) a drug for inhibiting angiogenesis in hepatocellular carcinoma; (5) a drug for inhibiting the migration or invasion of hepatocellular carcinoma cells; (6) a drug for inducing apoptosis of hepatocellular carcinoma cells; and (7) a drug for treating hepatocellular carcinoma by inhibiting the PI3K / AKT signaling pathway.

[0064] The present invention verifies the above application value of folic acid-targeted CPD12C15 liposomes through a series of experiments.

[0065] Drugs for treating hepatocellular carcinoma: The preparation of the present invention exhibits potent proliferation inhibitory activity against various human hepatocellular carcinoma cells (such as HepG2 and Huh7) in vitro. 50 The concentrations were generally lower than 1 μM, which was significantly better than non-targeted liposomes and free drugs. In the in vivo HepG2 tumor-bearing nude mouse model, the tumor inhibition rate of the preparation group was as high as 83.82%, which was significantly better than the sorafenib group, showing good overall anti-tumor potential.

[0066] Drugs for reducing IL-6 and / or TNF-α levels in chronic inflammation associated with hepatocellular carcinoma: ELISA analysis showed that the preparation of the present invention can effectively reduce the levels of IL-6 and TNF-α in tumor cell culture supernatant and the serum of tumor-bearing mice, regulate the inflammation-related microenvironment, help weaken tumor-promoting factors, and delay disease progression.

[0067] Drugs for inhibiting angiogenesis in hepatocellular carcinoma: In angiogenesis-related experiments, the preparation of the present invention significantly inhibited the tube-forming ability of HUVEC cells and simultaneously downregulated the expression of HIF-1α and VEGF in tumor tissues, suggesting that it can weaken the blood supply to the tumor through an anti-angiogenesis mechanism, thereby limiting its growth and spread.

[0068] Drugs for inhibiting the migration or invasion of hepatocellular carcinoma cells: Experiments have shown that the preparation of this invention can significantly reduce the migration and invasion capabilities of hepatocellular carcinoma cells. The molecular mechanism is associated with downregulating the expression of MMP-2 and MMP-9. This effect helps inhibit local tumor infiltration and distant metastasis, enhancing the overall therapeutic effect.

[0069] Drugs for inducing apoptosis in hepatocellular carcinoma cells: Flow cytometry and Western blot analysis revealed that the preparation of the present invention can induce significant tumor cell apoptosis, accompanied by upregulation of pro-apoptotic proteins (such as Bax, cleaved caspase-3, and cleaved PARP) and downregulation of anti-apoptotic protein Bcl-2, indicating that it can regulate programmed cell death through the mitochondrial pathway.

[0070] Drugs for treating hepatocellular carcinoma by inhibiting the PI3K / AKT signaling pathway: The preparation of the present invention can effectively inhibit the phosphorylation of signaling molecules such as PI3K, p-AKT and p-mTOR, reduce the expression levels of downstream Bcl-2, VEGF, and HIF-1α, and promote the upregulation of Bax, indicating that its anti-tumor effect is partially mediated by the PI3K / AKT pathway and is suitable for tumor types with active this signaling pathway.

[0071] In addition, the present invention also has the potential application value of reversing sorafenib resistance or reducing its related toxicity. As the current first-line drug for hepatocellular carcinoma, the efficacy of sorafenib is limited by the rapid development of drug resistance and adverse reactions such as skin toxicity, which seriously affects clinical compliance. The preparation of the present invention has an anti-tumor mechanism different from sorafenib, including targeting the ubiquitin-proteasome system and regulating the PI3K / AKT signaling pathway. It has shown a proliferation inhibition effect superior to sorafenib in multiple in vitro and in vivo experiments, suggesting that it can be used as an alternative treatment strategy after drug resistance.

[0072] Furthermore, the formulation of the present invention significantly reduced the expression levels of tumor-associated inflammatory factors IL-6 and TNF-α in animal experiments, while the sorafenib-treated group showed the opposite trend. This anti-inflammatory effect not only helps inhibit tumor immune escape but also may alleviate sorafenib-induced side effects such as inflammatory skin reactions. Therefore, the pharmaceutical composition of the present invention can be used not only as a standalone treatment but also in combination with sorafenib to enhance efficacy and reduce toxicity, providing a new treatment option for patients with drug resistance or intolerance.

[0073] In summary, the present invention has established an anti-hepatocellular carcinoma formulation system with multiple synergistic mechanisms and rich targets by designing folic acid-modified CPD12C15 liposomes and combining them with copper ions. This formulation not only has excellent targeting and anti-tumor activity, but can also effectively regulate the inflammatory microenvironment, inhibit tumor angiogenesis and cell migration, promote apoptosis, and intervene in key signaling pathways, showing its application prospects in the multidimensional treatment of hepatocellular carcinoma. This invention is particularly suitable for patient groups with poor first-line treatment effects or drug resistance problems, and has good application value and translational potential.

[0074] Compared with the prior art, the advantages of the present invention include:

[0075] Compared with existing treatments for hepatocellular carcinoma, the folic acid-targeted CPD12C15 liposomes and their pharmaceutical compositions provided by the present invention have significant advantages in terms of mechanism of action, formulation performance, targeted delivery efficiency, and clinical application suitability.

[0076] First, in terms of mechanism of action, the formulation of this invention, through targeted folic acid delivery and copper salt synergistic enhancement, can simultaneously inhibit tumor cell proliferation, inhibit angiogenesis, reduce inflammatory cytokine levels, inhibit migration and invasion, induce cell apoptosis, and regulate key signaling pathways such as PI3K / AKT in both in vitro and in vivo models, demonstrating a multi-target synergistic anti-liver cancer effect. Compared with the existing first-line drug sorafenib, the formulation of this invention has complementary and alternative mechanisms at multiple levels and has the potential to overcome drug resistance.

[0077] Secondly, the present invention utilizes a folic acid modification strategy in formulation design, significantly enhancing the targeting and uptake efficiency of liposomes for liver cancer cells. Related experiments have shown that in cells expressing folate receptors, the uptake of these targeted liposomes is 2–4 times that of non-targeted formulations. In vivo fluorescence imaging results show that the drug is continuously enriched in tumor tissue, with peak intensity 2–5 times that of the control group and a retention time exceeding 72 hours, demonstrating the synergistic advantages of "long circulation + active targeting."

[0078] In terms of controlled drug release, the liposomes of this invention exhibit excellent physicochemical stability and pH-responsive release properties. The liposome particle size is concentrated in the 80–130 nm range, with a polydispersity index (PDI) below 0.2, a moderate surface potential, and good blood circulation stability. The release rate is significantly accelerated in the weakly acidic tumor microenvironment, with a cumulative release rate of 60–75% over 72 hours, while under neutral conditions, it remains at 30–45%, achieving a balance between concentrated drug efficacy at the tumor site and reduced toxicity to normal tissues.

[0079] In terms of preparation technology, this invention utilizes an optimized calcium acetate gradient method to achieve efficient loading of CPD12C15. Liposome encapsulation efficiencies of 75–95% and drug loading of 10–20% are achieved, significantly outperforming traditional passive drug delivery methods, improving drug utilization while reducing production costs. This process requires no specialized equipment and is compatible with existing pharmaceutical production lines. Stable batch production can be achieved through conventional sterilization and packaging procedures, providing a promising foundation for industrialization.

[0080] Furthermore, to enhance clinical convenience and drug stability, the present invention also designed a complete medical kit solution. This solution supports the storage and flexible administration of liposomes and copper salts, and is compatible with dual-chamber syringes and dual-bottle packaging, allowing physicians to tailor the combination formulation to the patient's condition and treatment plan. This design effectively addresses the stability issues of the combination formulation during long-term storage, extending the drug's shelf life and enhancing the product's practical application value.

[0081] In terms of safety, the formulation of the present invention exhibits excellent biocompatibility. Animal studies revealed no significant toxicity, with a hemolysis rate below 5%. Long-term administration did not result in major organ dysfunction. Comparative studies with sorafenib demonstrated that the composition not only exhibited superior anti-tumor efficacy but also significantly reduced inflammatory cytokine levels, thereby alleviating sorafenib-related skin toxicity and supporting improved patient tolerance and compliance.

[0082] Taking into account the above technical features, the folate-targeted CPD12C15 liposomes and their pharmaceutical compositions are not only suitable for monotherapy of hepatocellular carcinoma, but can also be used as preoperative and postoperative adjuvant therapy, combination therapy, and advanced palliative treatment options. They are particularly suitable for patients who are intolerant to or have developed resistance to sorafenib. Furthermore, their preparation method and delivery strategy have the potential for application in other malignant tumors that express folate receptors, such as ovarian and breast cancer, and possess significant scientific significance and industrial value. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] 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:

[0084] Figure 1 : Encapsulation efficiency and drug loading capacity of CPD12C15 liposomes under different phosphatidylcholine-cholesterol ratios, calcium acetate concentrations, drug-to-lipid ratios, phospholipid concentrations, incubation temperatures, and incubation times.

[0085] Figure 2 : Transmission electron microscopy image, particle size distribution and potential map of CPD12C15-Falip.

[0086] Figure 3 : In vitro release curves of CPD12C15-Falip in PBS solution with pH 7.4 (left) and PBS solution with pH 6.8 (right) at 37°C (n=3).

[0087] Figure 4 : Preliminary stability study of CPD12C15-Falip.

[0088] Figure 5 : Results of CPD12C15-Falip in vitro hemolysis experiment. The left figure is a photo of hemolysis samples of CPD12C15-Falip at different concentrations. The right figure shows the hemolysis rate of CPD12C15-Falip at different concentrations.

[0089] Figure 6 : In vitro cytotoxicity results of CPD12C15+Cu, CPD12C15-Lip+Cu, CPD12C15-Falip+Cu (left) and Sorafenib (right) on HepG2 cells.

[0090] Figure 7 : In vitro cytotoxicity results of CPD12C15+Cu, CPD12C15-Lip+Cu, CPD12C15-Falip+Cu (left) and Sorafenib (right) on Huh7 cells.

[0091] Figure 8 : HepG2 cell uptake results, wherein, a: HepG2 cell uptake fluorescence microscopy (scale: 200 μm); b: HepG2 cell uptake flow cytometry quantitative determination; c: HepG2 cell uptake flow cytometry results fluorescence signal intensity histogram.

[0092] Figure 9 : Huh7 cell uptake results, wherein, a: Huh7 cell uptake fluorescence microscopy (scale: 200 μm); b: Huh7 cell uptake flow cytometry quantitative determination; c: Huh7 cell uptake flow cytometry results fluorescence signal intensity histogram.

[0093] Figure 10 :Scratch images of HepG2 cells after treatment with different drugs (scale: 500μm) and scratch rate. Compared with the Control group, * p<0.05, ** p<0.01, *** p<0.001.

[0094] Figure 11 :Huh7 cell scratch images (scale: 500μm) and scratch rates after treatment with different drugs. Compared with the Control group, * p<0.05,** p<0.01, *** p<0.001.

[0095] Figure 12 :Invasion images of HepG2 cells after treatment with different drugs (scale: 100μm) and relative invasion rates. Compared with the Control group, *** p<0.001.

[0096] Figure 13 :Huh7 cell invasion images (scale: 100μm) and relative invasion rates after treatment with different drugs. Compared with the Control group, *** p<0.001.

[0097] Figure 14 :Flow cytometry was used to detect the apoptosis of HepG2 cells after treatment with different drugs and to quantify the apoptosis rate. Compared with the Control group, *** p<0.001.

[0098] Figure 15 :Flow cytometry was used to detect the apoptosis of Huh7 cells after treatment with different drugs and the quantification of apoptosis rate. Compared with the Control group, * p<0.05, *** p<0.001.

[0099] Figure 16 :HUVEC cell tube formation diagram (scale: 100μm) and tubular branch number statistics after different drug treatment. Compared with the Control group, *** p<0.001.

[0100] Figure 17 :Changes in the expression of key proteins of the PI3K / AKT signaling pathway (p-PI3K, HIF-1α, Bcl-2, p-AKT, VEGF, Bax) in HepG2 cells after treatment with different drugs. Compared with the Control group, * p<0.05, ** p<0.01, *** p<0.001.

[0101] Figure 18 :Changes in the expression of key proteins of the PI3K / AKT signaling pathway (p-PI3K, HIF-1α, Bcl-2, p-AKT, VEGF, Bax) in Huh7 cells after treatment with different drugs. Compared with the Control group, * p<0.05, ** p<0.01, *** p<0.001.

[0102] Figure 19In vivo imaging and fluorescence intensity quantitative analysis of nude mice injected with DiR, DiR-Lip, and DiR-Falip at different time points

[0103] (The white circle is the tumor site).

[0104] Figure 20 :24 hours after nude mice were injected with DiR, DiR-Lip and DiR-Falip via tail vein, the tumor tissues and major organs were imaged and the fluorescence intensity was quantitatively analyzed. Compared with the DiR group, *** p<0.001; compared with DiR-Lip group, ### p<0.001.

[0105] Figure 21 : In vivo anti-tumor activity evaluation results; a: nude mouse tumor photo; b: nude mouse time-tumor volume change diagram; c: nude mouse tumor weight in different groups; d: nude mouse time-weight change diagram; e: nude mouse tumor volume in different groups. Compared with the Model group, ** p<0.01, *** p<0.001; compared with the CPD12C15-Lip group, ### p<0.001.

[0106] Figure 22 : H&E staining results of major organs.

[0107] Figure 23 : H&E staining results of tumor mass.

[0108] Figure 24 :TUNEL apoptosis test results include TUNEL apoptosis graph and relative TUNEL positive cell number bar graph in nude mouse tumor tissue. Compared with the Model group, *** p<0.001; compared with the CPD12C15-Lip group, ### p<0.001.

[0109] Figure 25 : Immunohistochemistry results, where a: immunohistochemistry images; b: p-PI3K immunohistochemistry quantitative results; c: p-AKT immunohistochemistry quantitative results; d: Bcl-2 immunohistochemistry quantitative results; e: Bax immunohistochemistry quantitative results; f: VEGF immunohistochemistry quantitative results. Compared with the Model group, * p<0.05, ** p<0.01, *** p<0.001.

[0110] Figure 26:The results of Elisa assay for changes in the levels of inflammatory factors IL-6 and TNF-α in the serum of nude mice. Compared with the Model group, * p<0.05, ** p<0.01, *** p<0.001. DETAILED DESCRIPTION

[0111] 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.

[0112] 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.

[0113] CPD12C15 was prepared according to the method disclosed in Chinese patent CN116444408A, with a purity >95%. The folic acid-modified DSPE-PEG2000 phospholipid (DSPE-PEG2000-FA) used in this invention has an average molecular weight of 2000 Daltons for the PEG segment. This material is commercially available from Shanghai Yuanye Biotechnology Co., Ltd. and can be directly used in the preparation of liposome formulations. It can also be prepared by condensing DSPE-PEG2000-NH2 with folic acid using conventional coupling methods.

[0114] Example 1 Preparation of CPD12C15 liposomes

[0115] 1. Preparation method and process optimization

[0116] 1.1 Preparation method

[0117] Thin Film Dispersion Method (TFV): Weigh a specific ratio of lecithin, cholesterol, and CPD12C15 into an eggplant-shaped flask. Add 15 mL of chloroform, seal with parafilm, and puncture five small holes in the film with a needle. Dissolve the mixture by sonication. Place the flask in a rotary evaporator and heat in a 40°C water bath under a negative pressure of 0.09 MPa until the organic solvent is completely evaporated, forming a uniform film on the flask wall. Weigh the flask every 5 minutes. If there is no change in mass, the chloroform is completely evaporated, and the evaporation is stopped. Add 10 mL of purified water for hydration and shake until the film falls off and is evenly dispersed in the water. Ultrasonicate the flask with a probe for 2 minutes (50 W, 2 seconds on, 2 seconds off). Finally, filter through a 0.22 μm aqueous microporous filter membrane, and collect the filtrate to obtain a CPD12C15 liposome (CPD12C15-Lip) solution with a blue opalescence.

[0118] Calcium Acetate Gradient (CAG) Method: Weigh a specific ratio of lecithin and cholesterol into an eggplant-shaped flask. Add 10 mL of chloroform, seal with parafilm, and puncture five small holes in the parafilm with a needle. Dissolve the flask by sonication. Place the flask in a rotary evaporator and heat in a 40°C water bath under a negative pressure of 0.09 MPa until the organic solvent is completely evaporated and a uniform film forms on the flask wall. Weigh the flask every 5 minutes. If there is no change in mass, the chloroform is completely evaporated, and the evaporation is stopped. Add a specific concentration of calcium acetate solution for hydration and shake until the film falls off and is evenly dispersed in the solution. Ultrasonicate the flask with a probe for 2 minutes (50W, 2 seconds on, 2 seconds off). Then, filter the flask through a 0.22 μm aqueous microporous filter to obtain blank calcium acetate liposomes. Place the blank calcium acetate liposomes into a dialysis bag and dialyze overnight at room temperature using a sodium sulfate solution of the same concentration as the calcium acetate solution. The dialyzed calcium acetate blank liposomes were mixed with CPD12C15 at a certain drug-lipid ratio (calculated based on the CPD12C15 concentration pre-determined by HPLC), and incubated at a certain temperature for a period of time to obtain a CPD12C15 liposome (CPD12C15-Lip) solution.

[0119] 1.2 Process Optimization: CPD12C15 liposomes were prepared according to the aforementioned preparation method, see Preparation Example 1-23, and the preparation parameters are shown in Table 1.

[0120] Table 1 Preparation Examples 1-32

[0121]

[0122]

[0123] 1.3 Process evaluation method

[0124] Determination of CPD12C15 content: The content of CPD12C15 was determined by high performance liquid chromatography.

[0125] Chromatographic conditions: A Phenomenex Luna C18 column (5 μm, 250 mm × 4.6 mm) was used; a mixed mobile phase consisting of 0.2% formic acid aqueous solution and acetonitrile was prepared in a volume ratio of 40:60; the detection wavelength of CPD12C15 was 284 nm; the flow rate of the mobile phase in the column was 1 mL / min; the column temperature was maintained at 30°C during the measurement, and the injection volume was 10 μL each time.

[0126] Preparation of stock solution: Accurately weigh 5 mg of CPD12C15 sample into a 10 mL volumetric flask. First, add acetonitrile to completely dissolve CPD12C15. Add acetonitrile to the marked scale of the volumetric flask and mix to obtain a 0.5 mg / mL CPD12C15 stock solution. Seal and store in a refrigerator at 4°C.

[0127] Establishment of the standard curve and linear relationship: Accurately measure volumes of 6 mL, 3 mL, 1.5 mL, 0.75 mL, 0.375 mL, 0.1875 mL, and 0.09375 mL of the stock solution into a 10 mL volumetric flask, add acetonitrile to the volume, and shake thoroughly to obtain CPD12C15 standard solutions with final concentrations of 300, 150, 75, 37.5, 18.75, 9.375, and 4.6875 μg / mL. Then filter each solution through a 0.22 μm microporous filter membrane, perform HPLC analysis, and record the peak area data of each concentration standard solution in detail. The horizontal axis of the linear regression analysis is the concentration of CPD12C15, and the vertical axis is the average of the peak areas measured at different concentrations. Plot the standard curve and calculate the regression equation.

[0128] The test method of encapsulation efficiency and drug loading was as follows: take an appropriate amount of liposome solution, add acetonitrile to dilute a certain multiple, ultrasonically break the emulsion for 20 minutes, filter through a 0.22 μm organic microporous filter membrane, and detect the peak area by HPLC and record it. Each sample was measured in parallel three times, and the concentration was calculated by the standard curve regression equation, which was recorded as C 总 .

[0129] Take an appropriate amount of liposome solution in an ultrafiltration tube and centrifuge at 5000 rpm for 15 minutes. Take the liquid at the bottom of the centrifuge tube and dilute it with acetonitrile to a certain multiple. After filtering through a 0.22 μm organic microporous filter membrane, use HPLC to detect the peak area and record it. Each sample is measured in parallel three times. The concentration is calculated by the standard curve regression equation and recorded as C 游 The formulas for calculating encapsulation efficiency and drug loading are as follows:

[0130]

[0131] 1.4 Process optimization results

[0132] (1) Preparation Example 1 and Preparation Example 2 were prepared using the thin film dispersion method (TFV) and the calcium acetate gradient method (CAG), respectively. The encapsulation efficiency and drug loading of the liposomes prepared by the two methods were measured by ultrafiltration centrifugation. The results are shown in Table 2.

[0133] Table 2 Encapsulation efficiency and drug loading of liposomes prepared by thin film dispersion method and calcium acetate gradient method

[0134]

[0135] Compared with the thin film dispersion method, the calcium acetate gradient method for preparing CPD12C15 liposomes has higher encapsulation efficiency and drug loading. Therefore, the calcium acetate gradient method is preferred for preparing CPD12C15 liposomes and CPD12C15 folic acid liposomes.

[0136] (2) Effects of different phosphatidylcholine-cholesterol molar ratios on the encapsulation efficiency and drug loading of CPD12C15 liposomes

[0137] Taking encapsulation efficiency and drug loading as evaluation indicators, the drug-lipid molar ratio was fixed at 1:4, the calcium acetate concentration was 120mmol / L, the phospholipid concentration was 10mg / mL, the incubation temperature was 40℃, and the incubation time was 60min. The effects of the phospholipid-cholesterol molar ratio on liposome encapsulation efficiency and drug loading were investigated at 1:1 (Preparation Example 3), 2:1 (Preparation Example 4), 3:1 (Preparation Example 2), 4:1 (Preparation Example 5), and 5:1 (Preparation Example 6). The results are shown in Figure 1 The encapsulation efficiency of CPD12C15 liposomes increases with decreasing phosphatidylcholine-cholesterol molar ratios. This is because cholesterol can regulate membrane fluidity, increasing liposome stability and improving drug encapsulation efficiency. However, excessive cholesterol (1:1) disrupts the formation of the liposomal phospholipid bilayer, increasing liposome membrane permeability and reducing drug loading. A comprehensive consideration of encapsulation efficiency and drug loading suggests a phosphatidylcholine-cholesterol molar ratio of 2:1 is optimal.

[0138] (3) Effects of different calcium acetate concentrations on the encapsulation efficiency and drug loading of CPD12C15 liposomes

[0139] Taking encapsulation efficiency and drug loading as evaluation indicators, the phospholipid-cholesterol molar ratio was fixed at 2:1, the drug-lipid molar ratio was 1:4, the phospholipid concentration was 10 mg / mL, the incubation temperature was 40°C, and the incubation time was 60 min. The effects of calcium acetate concentrations of 80 (Preparation Example 7), 120 (Preparation Example 4), 160 (Preparation Example 8), and 200 mmol / L (Preparation Example 9) on the liposome encapsulation efficiency and drug loading were investigated. The results are shown in Figure 1When the calcium acetate concentration is too low, an effective calcium acetate transmembrane gradient cannot be formed between the inner and outer aqueous phases. However, when the calcium acetate concentration is too high, liposome aggregation occurs. A calcium acetate concentration of 120 mmol / L is optimal, considering both encapsulation efficiency and drug loading.

[0140] (4) Effects of different drug-lipid ratios on the encapsulation efficiency and drug loading of CPD12C15 liposomes

[0141] The drug-lipid molar ratio (CPD12C15: lipid) refers to the molar ratio between CPD12C15 and the total lipid components (lecithin + cholesterol) in the liposome. Taking the encapsulation efficiency and drug loading as evaluation indicators, the lecithin-cholesterol molar ratio was fixed at 2:1, the calcium acetate concentration was 120mmol / L, the phospholipid concentration was 10mg / mL, the incubation temperature was 40℃, and the incubation time was 60min. The effects of the drug-lipid molar ratio on the liposome encapsulation efficiency and drug loading were investigated when they were 1:1 (Preparation Example 10), 1:2 (Preparation Example 11), 1:3 (Preparation Example 12), 1:4 (Preparation Example 4), 1:5 (Preparation Example 13), and 1:8 (Preparation Example 14). The results are shown in Figure 1 The encapsulation efficiency increases as the drug-to-lipid ratio decreases, but the corresponding drug loading decreases. Therefore, while maximizing encapsulation efficiency, it is also important to ensure that the drug loading meets the required drug concentration. A comprehensive consideration of encapsulation efficiency and drug loading found a drug-to-lipid molar ratio of 1:2 to be optimal.

[0142] (5) Effects of different phospholipid concentrations on the encapsulation efficiency and drug loading of CPD12C15 liposomes

[0143] Since the phospholipid concentration has a great influence on the particle size and PDI of the liposomes, the encapsulation efficiency, drug loading, particle size, and PDI were used as evaluation indicators. The phospholipid-cholesterol molar ratio was fixed at 2:1, the drug-lipid molar ratio was 1:2, the calcium acetate concentration was 120 mmol / L, the incubation temperature was 40°C, and the incubation time was 60 min. The effects of the phospholipid concentrations of 10 (Preparation Example 11), 20 (Preparation Example 15), 30 (Preparation Example 16), and 40 mg / mL (Preparation Example 17) on the liposome encapsulation efficiency, drug loading, particle size, and PDI were investigated. The results are shown in Figure 1 Increasing the phospholipid concentration from 10 mg / mL to 20 mg / mL significantly increased the encapsulation efficiency and drug loading; above 20 mg / mL, both the encapsulation efficiency and drug loading remained largely unchanged. Table 3 shows that the particle size and PDI of the liposomes increased with increasing phospholipid concentration, while the liposomes' stability decreased and aggregation tended to occur. Based on a comprehensive consideration of encapsulation efficiency, drug loading, particle size, and PDI, a phospholipid concentration of 20 mg / mL was selected as the optimal level.

[0144] Table 3 Effects of different phospholipid concentrations on the particle size and PDI of CPD12C15 liposomes

[0145]

[0146] (6) Effects of different incubation temperatures on the encapsulation efficiency and drug loading of CPD12C15 liposomes

[0147] Taking encapsulation efficiency and drug loading as evaluation indicators, the phospholipid-cholesterol molar ratio was fixed at 2:1, the drug-lipid molar ratio was 1:2, the phospholipid concentration was 20 mg / mL, and the incubation time was 60 min. The effects of incubation temperatures of 30°C (Preparation Example 18), 40°C (Preparation Example 15), 50°C (Preparation Example 19), and 60°C (Preparation Example 20) on the liposome encapsulation efficiency and drug loading were investigated. The results are shown in Figure 1 As the temperature rises, the lipid membrane permeability increases, making it easier for drugs to translocate into the membrane. The encapsulation efficiency increases with increasing incubation temperature, reaching the highest encapsulation efficiency and drug loading at 40°C. A comprehensive consideration of encapsulation efficiency and drug loading revealed that an incubation temperature of 40°C was optimal.

[0148] (7) Effects of different incubation times on the encapsulation efficiency and drug loading of CPD12C15 liposomes

[0149] Taking encapsulation efficiency and drug loading as evaluation indicators, the phospholipid-cholesterol molar ratio was fixed at 2:1, the drug-lipid molar ratio was 1:2, the phospholipid concentration was 20 mg / mL, the incubation temperature was 40°C, and the effects of incubation time on liposome encapsulation efficiency and drug loading were investigated at 40 min (Preparation Example 21), 50 min (Preparation Example 22), 60 min (Preparation Example 15), and 70 min (Preparation Example 23). The results are shown in Figure 1 If the incubation time is too short, complete drug encapsulation cannot be guaranteed. If the incubation time is too long, the drug will be more likely to leak out when the drug encapsulation reaches equilibrium due to the increased permeability of the membrane at the phase transition temperature. Considering the encapsulation efficiency and drug loading, an incubation time of 60 minutes is optimal.

[0150] Based on the above results, the optimal preparation method for CPD12C15 liposomes was determined to be the calcium acetate gradient method. The optimal process parameters were: phosphatidylcholine:cholesterol molar ratio = 2:1, calcium acetate concentration = 120 mmol / L, drug-lipid molar ratio = 1:2, phospholipid concentration = 20 mg / mL, incubation temperature = 40°C, and incubation time = 60 min. CPD12C15 liposomes prepared under these conditions exhibited excellent encapsulation efficiency (78.35 ± 1.05%) and drug loading (17.24 ± 0.28%).

[0151] Example 2 Preparation and characterization of CPD12C15 folic acid liposomes

[0152] Based on the optimal preparation conditions for CPD12C15 liposomes determined in Example 1, this example further investigated the effect of DSPE-PEG2000-Fa modification on liposome performance. Preliminary experiments demonstrated that the addition of an appropriate amount of DSPE-PEG2000-Fa did not significantly alter the key preparation parameters of the basic CPD12C15 liposomes. Therefore, this example used the optimal parameters determined in Example 1 to prepare CPD12C15 folic acid liposomes.

[0153] 1. Preparation of folic acid-targeted CPD12C15 liposomes:

[0154] Preparation of lipid film: Phosphatidylcholine and cholesterol (2:1 molar ratio, total lipid mass 20 mg / mL) and varying amounts of DSPE-PEG2000-Fa were weighed into an eggplant-shaped flask (the amount of DSPE-PEG2000-Fa added was 0.5%, 1.25%, and 2.0% of the total lipid content (phosphatidylcholine + cholesterol + DSPE-PEG2000-Fa), respectively). 10 mL of chloroform was added to fully dissolve the mixture. The flask was placed in a 40°C water bath and evaporated on a rotary evaporator at 150 rpm under a negative pressure of 0.09 MPa until a uniform film formed. The chloroform was weighed periodically to confirm complete evaporation. (During this process, the lipid portion of DSPE-PEG2000-Fa, DSPE, will form a uniform lipid film along the flask wall, along with the phosphatidylcholine and cholesterol.)

[0155] Preparation of blank liposomes: Add 10 mL of calcium acetate solution (120 mmol / L) to the film and shake at room temperature to completely detach the film and evenly disperse it. The suspension was probe-ultrasonicated in an ice bath (50 W, 2 seconds on, 2 seconds off, total time 2 minutes), and then filtered through a 0.22 μm microporous filter to obtain blank calcium acetate liposomes containing DSPE-PEG2000-Fa. (During this process, when the film is hydrated with calcium acetate solution, the DSPE portion is embedded in the formed lipid bilayer membrane, while the PEG chain and folic acid portion extend into the aqueous phase and are exposed on the liposome surface.)

[0156] Establish pH gradient: Blank liposomes were placed in a dialysis bag (molecular weight cut-off 3500 Da), 120 mmol / L sodium sulfate solution was used as the dialysis medium, and dialyzed for 12 h at room temperature with magnetic stirring (100 rpm) to form a transmembrane pH gradient.

[0157] Drug loading: After dialyzation, blank liposomes were removed and their lipid concentration was determined by HPLC. The liposomes were then mixed with CPD12C15 at a 1:2 molar ratio and incubated in a 40°C water bath for 60 minutes. After cooling to room temperature, the mixture was filtered through a 0.22 μm microporous membrane to obtain folate-targeted CPD12C15 liposomes (CPD12C15-Falip).

[0158] Table 4

[0159]

[0160] 2. Effect of different DSPE-PEG2000-Fa contents on the performance of CPD12C15-Falip

[0161] The present invention adopts a range exploration method to optimize the content of DSPE-PEG2000-Fa. Experiments show that when the addition amount of DSPE-PEG2000-Fa is in the range of 0.5%-2.0%, folic acid-targeted CPD12C15 liposomes with good encapsulation efficiency and appropriate particle size can be obtained. Too low a modification content (<1%) is insufficient to provide effective targeting ability, while too high a modification content (>2%) may lead to unstable lipid membrane structure and decreased drug loading capacity. In particular, after three batches of reproducible experiments, when the DSPE-PEG2000-Fa content was 1.25%, the prepared CPD12C15-Falip showed the best comprehensive performance. The results are shown in Table 5.

[0162] Table 5: Reproducible results of three batches of CPD12C15-Falip (DSPE-PEG2000-Fa content 1.25%)

[0163]

[0164]

[0165] 3. Characterization of CPD12C15 folic acid liposomes

[0166] 3.1 Morphological examination results: Transmission electron microscopy observation of the morphology of CPD12C15-Falip Figure 2 As shown, the morphology is round, the dispersion is good and there is no adhesion, and the particle size is between 100 and 120 nm.

[0167] 3.2 Particle size, PDI and potential measurement results: CPD12C15-Falip particle size distribution and potential are as follows Figure 2 As shown, the particle size is 105.33±0.38nm, and the liposomes are evenly distributed; the PDI is 0.17±0.03, which is less than 0.2; and the potential is -1.81±0.48mV, which is slightly negative.

[0168] 3.3 In vitro release experiment: The release of CPD12C15-Falip in different pH media at 37°C was investigated by dialysis. Different pH release media were used to simulate the normal body fluid environment (pH 7.4) and tumor microenvironment (pH 6.8). The in vitro release results are as follows: Figure 3As shown. Free CPD12C15 exhibited a burst release in release media of varying pH, with the cumulative release rate reaching 90% within 4 hours. However, compared to free CPD12C15, CPD12C15-Falip exhibited sustained and slow drug release over a 24-hour period. At 24 hours, the cumulative release rates of CPD12C15-Falip in release media at pH 7.4 and pH 6.8 were 71.18% and 84.16%, respectively, indicating that CPD12C15-Falip significantly reduced the drug release rate and prolonged the in vivo circulation time of the liposomes. A further 20% to 30% of the drug in CPD12C15-Falip remained within the liposomes, making it difficult to release. This is presumably because CPD12C15 encapsulated in the lipid bilayer slowly crosses the membrane. CPD12C15-Falip was released in a medium with a pH of 6.8 for 24 hours, and the release rate and release degree of CPD12C15 were higher than those in a medium with a pH of 7.4, indicating that a low pH value can accelerate the release of CPD12C15. It can be seen that CPD12C15-Falip is more conducive to drug release in tumor cells.

[0169] 3.4 Preliminary stability study: The stability of CPD12C15-Falip stored at 4°C was investigated. Figure 4 As shown in the figure, the particle size and PDI did not change much within 4 weeks, and no obvious aggregation or precipitation of CPD12C15-Falip was observed, indicating that CPD12C15-Falip had good storage stability at 4°C.

[0170] 3.5 In vitro hemolysis test: The preliminary safety of the preparation is investigated using the red blood cell hemolysis test. Figure 5 As shown in the data, within the concentration range of 2.5 to 40 μg / mL, CPD12C15-Falip had no obvious hemolysis phenomenon, and the hemolysis rate was less than 5%, indicating that CPD12C15-Falip had good safety.

[0171] Example 3 In vitro anti-hepatocellular carcinoma efficacy of CPD12C15 folic acid liposomes combined with copper

[0172] Experimental cell lines: HepG2 and Huh7 human hepatocellular carcinoma cells were provided by the School of Basic Medical Sciences, Shandong University; human umbilical vein endothelial cells (HUVECs) were provided by the School of Pharmacy, Shandong University. HepG2 and Huh7 human hepatocellular carcinoma cells were cultured in DMEM supplemented with 10% FBS, while HUVECs were cultured in complete HUVEC medium. All cells were cultured in a cell culture incubator at 37°C and 5% CO2.

[0173] Experimental solution preparation:

[0174] MTT solution: Precisely weigh 200.00 mg of MTT powder in a dark place and dissolve it in 40 mL of PBS. Filter through a 0.22 μm sterile filter membrane in a clean bench, sterilize, and aliquot. Wrap the solution in tin foil and store in a -20°C refrigerator until used.

[0175] CuCl2 ˙ 2H2O solution: Accurately weigh 1.368 mg CuCl2 ˙ 2H2O powder, dissolve in 4mL purified water to obtain 2mmol / L CuCl2 ˙ 2H2O solution. After filtering, sterilizing and aliquoting, store in a -20℃ refrigerator until use.

[0176] CPD12C15 solution: Accurately weigh 1.36 mg of CPD12C15 and add 2 mL of DMSO. Dissolve thoroughly by sonication to obtain a 2 mmol / L CPD12C15 solution. Filter, sterilize, and aliquot the solution. Store in a -20°C refrigerator until ready for use.

[0177] CPD12C15-Lip and CPD12C15-Falip solutions were prepared according to the optimal process and formulation of Examples 1 and 2. After filtration, sterilization, and sub-packaging, they were stored in a refrigerator at 4°C for future use.

[0178] Sorafenib solution: Accurately weigh sorafenib powder and dissolve in DMSO to prepare a 160 mmol / L stock solution. Filter, sterilize, and aliquot, then store in a -20°C freezer until ready for use.

[0179] DMEM complete medium: Aliquot 45 mL of DMEM complete medium into a 50 mL sterile EP tube and add 10% fetal bovine serum and 1% double-stranded antibody. Store in a refrigerator at 4°C until needed.

[0180] HUVECs culture medium: Take 45 mL of F12K culture medium and dispense it into a 50 mL sterile EP tube. Add 10% fetal bovine serum, 1% double-stranded antibody, and vascular endothelial growth supplement / heparin (ECGS / H) (v / v) and store in a refrigerator at 4°C until use.

[0181] Coumarin-6 solution: Dissolve 400 μg of Coumarin-6 powder in 1 mL of DMSO in the dark. Filter, sterilize, and aliquot. Dilute to 400 ng / mL in DMEM complete medium for storage.

[0182] Preparation of free folic acid: Weigh an appropriate amount of folic acid powder in the dark, add it to DMEM complete culture medium to prepare a 1 mmol / L free folic acid solution, and store it in a 4°C refrigerator until use.

[0183] 1. Experimental methods

[0184] 1.1 Cell culture

[0185] 1.1.1 Cell Thawing: Turn on the UV sterilizer in the cell clean bench and sterilize the pipette tips, EP tubes, and culture flasks used in the experiment for 30 minutes. Heat a constant temperature water bath to 37°C. Remove the cryovial from the liquid nitrogen and quickly transfer it to a constant temperature water bath at 37°C to thaw. Once the cells are completely thawed, transfer them to a centrifuge tube and centrifuge at 1000 rpm for 3 minutes. Discard the cryovial solution, add 1 mL of DMEM complete medium, mix thoroughly, transfer to a cell culture flask, and add 4 mL of DMEM complete medium. Gently shake the flask to evenly disperse the cells, and culture in a cell incubator.

[0186] 1.1.2 Cell passaging: Passage is performed when the cells in the culture flask have an appropriate growth density and are in good condition. Turn on the ultraviolet light of the cell clean bench for sterilization, and take out the DMEM complete medium and trypsin in advance and let them reach room temperature. Aspirate the medium in the culture flask and add PBS buffer to wash the cells. Add 1mL of trypsin and place it in the incubator for digestion, and observe the cell status at any time. When the cells become round and bright, add 2mL of complete medium to stop digestion, transfer the cells to a centrifuge tube, and centrifuge at 1000r / min for 3min. After aspirating the upper layer of liquid, add 2mL of DMEM complete medium to resuspend the cells, and gently blow them with a pipette to disperse them evenly. Then divide the cell suspension equally into two culture flasks, add 4mL of DMEM complete medium to each flask, and place them in a cell culture incubator for culture.

[0187] 1.1.3 Cell Cryopreservation: Select cells in good growth condition for cryopreservation. Follow the same steps as in "1.1.2 Cell Passaging" before centrifugation. After centrifugation, carefully aspirate the upper layer of culture medium and prepare a cell freezing solution with a ratio of FBS:DMSO = 9:1. Add 1 mL of cell freezing solution and gently pipette the cell pellet. Transfer the cell pellet to a cryovial. Place the cell pellet in a cryovial cooled to room temperature in a cryovial box and freeze at -80°C overnight. The next day, transfer the cryovial to liquid nitrogen for long-term storage.

[0188] 1.1.4 Cell counting: Take cells in the logarithmic growth phase, and the steps before centrifugation are the same as "1.1.2 Cell passaging". After centrifugation, carefully discard the liquid in the centrifuge tube, and add DMEM complete medium to suspend the cells evenly. Wipe the cover slip and blood cell counting plate with 75% alcohol in advance, add 10μL of cell suspension on the counting plate, and be careful not to produce bubbles to prevent interference with the counting. Then observe under an optical microscope, follow the counting principle of "record the top and not the bottom, count the left and not the right", and use a counter to count the total number of cells in the four squares. The number of cells contained in each mL of cell suspension = total number of cells / 4×10 4 .

[0189] 1.2 MTT assay: The in vitro cytotoxicity of CPD12C15, CPD12C15-Lip, CPD12C15-Falip, and Sorafenib was investigated using the MTT assay. The seeding densities of HepG2 and Huh7 cells in 96-well plates were 6 × 10 cells / well, respectively. 3 and 5×10 3 The volume of cell suspension in each well was 100 μL. After the cells adhered to the wall, 100 μL of drugs of different concentrations were added to each well and incubated for 48 hours. The experimental drug addition groups were as follows: CPD12C15+Cu group, CPD12C15-Lip+Cu group and CPD12C15-Falip+Cu group: CuCl2 with the same molar concentration as CPD12C15 was added to CPD12C15 solution, CPD12C15-Lip solution and CPD12C15-Falip solution respectively. ˙ 2H2O solutions were mixed to obtain CPD12C15+Cu, CPD12C15-Lip+Cu, and CPD12C15-Falip+Cu mixtures, respectively. These mixtures were diluted to the desired concentrations in complete culture medium. The final CPD12C15 concentrations in each well were 50, 100, 200, 400, 800, and 1600 nmol / L, respectively. For the sorafenib group, the prepared sorafenib stock solution was diluted with DMEM complete culture medium to a final sorafenib concentration of 2.5, 5, 10, 20, 40, 80, and 160 μmol / L, respectively. A control group and a blank group were set up, with three replicate wells in each group. The experiment was repeated three times. After incubation for 48 hours, 20 μL of MTT solution was added to each well in the dark, and the cells were incubated in a cell culture incubator for another 4 hours. After discarding the liquid in the 96-well plate, add 150 μL of DMSO and shake at low speed on a shaker for 15 minutes. Preheat the microplate reader in advance, measure the absorbance of each well at 490 nm, and calculate the survival rate of each group of cells according to the formula:

[0190] 1.3 Cellular uptake experiment: Coumarin 6 was selected as a tracer to evaluate the cell-targeting ability of liposomes. Coumarin 6 fluorescent liposomes C6-Lip and fluorescent targeting liposomes C6-Falip were prepared using the calcium acetate gradient method (the preparation of C6-Lip and C6-Falip was based on the preparation methods of CPD12C15 liposomes and CPD12C15-Falip, except that the drug CPD12C15 was replaced with the fluorescent tracer coumarin 6. The remaining preparation steps and process parameters remained unchanged). HepG2 and Huh7 cells were seeded in six-well plates at a density of 1×10 5 / well and incubate overnight. When the cells reached an appropriate density, the medium in each well was aspirated and discarded. Empty DMEM complete medium (Control group), free coumarin 6 (free C6 group), coumarin 6 fluorescent liposomes (C6-Lip group), coumarin 6 fluorescent targeted liposomes (C6-Falip group), and coumarin 6 fluorescent targeted liposomes containing free folic acid (Fa+C6-Falip group) were added to each well in sequence. The final concentration of C6 in each group was 50 ng / mL, and the concentration of free folic acid in the folic acid blocking group was 1 mmol / L. After incubation in a cell culture incubator for 4 hours, the medium was aspirated and washed twice with PBS buffer. The cells were fixed with 4% paraformaldehyde for 10 minutes in the dark and washed twice with PBS buffer. DAPI (4',6-diamidino-2-phenylindole) was added for staining for 10 minutes, and the cells were washed twice with PBS buffer. The uptake of the two hepatocellular carcinoma cells was recorded using an inverted fluorescence microscope. At the same time, flow cytometry was used to quantitatively evaluate cell uptake. After incubation in a cell culture incubator for 4 hours, the culture medium was aspirated and washed twice with PBS buffer. The cells were digested with EDTA-free trypsin, and DMEM complete medium was added to terminate the digestion. The cells were centrifuged at 1000 rpm / min for 3 minutes, and the cell pellet was resuspended in 500 μL PBS buffer. The uptake of the two cells was quantitatively analyzed by flow cytometry.

[0191] 1.4 Cell scratch test: The seeding density of HepG2 and Huh7 cells in six-well plates was 3×10 per well. 5 and 4×10 5 2 mL of cell suspension was added to each well. When the cell density in each well was appropriate, the culture medium was aspirated and vertical lines were made with a 200 μL sterile pipette tip. PBS buffer was added to wash away the detached cells, and the width of the scratches in each well was recorded with an inverted optical microscope at 0 h. After the photography was completed, the cells were divided into five groups for drug addition, including the blank control group (Control group): 2 mL of serum-free DMEM culture medium was added to each well; the CPD12C15+Cu group, CPD12C15-Lip+Cu group and CPD12C15-Falip+Cu group: CuCl2 with the same molar concentration as CPD12C15 was added respectively. ˙2H2O solution was mixed to obtain CPD12C15+Cu mixtures, CPD12C15-Lip+Cu mixtures, and CPD12C15-Falip+Cu mixtures. These mixtures were diluted with serum-free DMEM (complete DMEM supplemented with antibiotics but without serum) to a final concentration of 600 nmol / L for each HepG2 cell group and 400 nmol / L for each Huh7 cell well. For the sorafenib group, the prepared sorafenib stock solution was diluted with serum-free DMEM to a final concentration of 30 μmol / L for HepG2 cells and 10 μmol / L for Huh7 cells. After incubation for 24 hours in a cell culture incubator, the six-well plates were aspirated, and the cells were gently washed three times with PBS buffer. Changes in the scratch width were observed under a microscope after 24 hours. The experiment was repeated three times, and the cell migration rates of the two cell lines were calculated.

[0192] 1.5 Cell invasion assay: First, Matrigel was diluted with serum-free DMEM medium at a ratio of 8:1, and 100 μL was added to the upper compartment of the Transwell. The compartment was then placed in a 24-well plate and placed in an incubator to wait for the Matrigel to solidify. HepG2 and Huh7 cells in the logarithmic growth phase were resuspended in serum-free DMEM medium at a seeding density of 3×10 cells per well. 4 and 5×10 4 100 μL of cell suspension was added to each well and incubated in the upper chamber of the Transwell. Then 100 μL of drug solution of different groups was added. Among them, the blank control group (Control group): serum-free DMEM medium was added to the upper chamber; the CPD12C15+Cu group, CPD12C15-Lip+Cu group and CPD12C15-Falip+Cu group: CuCl2 with the same molar concentration as CPD12C15 was added ˙2H2O solutions were mixed to obtain CPD12C15+Cu, CPD12C15-Lip+Cu, and CPD12C15-Falip+Cu mixtures. These mixtures were diluted with serum-free DMEM to a final concentration of 600 nmol / L for the upper chamber of each HepG2 cell group and 400 nmol / L for each Huh7 cell group. For the sorafenib group, the prepared sorafenib stock solution was diluted with serum-free DMEM to a final concentration of 30 μmol / L for HepG2 cells and 10 μmol / L for Huh7 cells. 800 μL of complete DMEM medium was added to the lower chamber of the Transwell. The chamber was gently shaken to evenly disperse the cells and incubated in a cell culture incubator for 24 hours. The culture medium in the upper and lower chambers was aspirated and washed three times with PBS. The cells were fixed with 4% paraformaldehyde for 15 minutes and washed three times with PBS. Finally, 2% crystal violet was added for staining for 30 minutes, followed by washing three times with PBS. Unmigrated cells were removed with a cotton swab. After the chambers were clear of residual liquid, the invasion of HepG2 and Huh7 cells was observed microscopically. Three randomly selected areas were photographed, and the number of cells invading the lower chamber was counted for each group.

[0193] 1.6 Cell apoptosis experiment: HepG2 and Huh7 cells were evenly seeded in a six-well plate at an appropriate density and incubated, and 2 mL of cell suspension was added to each well. When the cell density in each well was higher than 70%, the culture medium in each well was discarded and washed three times with PBS buffer. The cells were divided into five groups for drug addition, including the blank control group (Control group): 2 mL of DMEM complete medium was added to each well; the CPD12C15+Cu group, CPD12C15-Lip+Cu group and CPD12C15-Falip+Cu group: CuCl2 with the same molar concentration as CPD12C15 was added respectively. ˙The 2H2O solutions were mixed uniformly to obtain CPD12C15+Cu, CPD12C15-Lip+Cu, and CPD12C15-Falip+Cu mixtures. These mixtures were then diluted with DMEM complete medium to a final concentration of 600 nmol / L for each HepG2 cell group and 400 nmol / L for each Huh7 cell well. For the sorafenib group, the prepared sorafenib stock solution was diluted with DMEM complete medium to a final concentration of 30 μmol / L for HepG2 cells and 10 μmol / L for Huh7 cells. The cells were cultured in a cell culture incubator for 48 hours. After incubation, the culture medium from each well was collected, and the cells were trypsinized with EDTA-free trypsin. DMEM complete medium was added to terminate the digestion, and the cells were centrifuged at 1000 rpm for 3 minutes. The supernatant was discarded, and the cells were washed twice with PBS buffer. Add 100 μL Binding Buffer to resuspend the cell pellet. Under light-protected conditions, add 5 μL of FITC-Annexin V and PI dye in sequence according to the kit, mix well by pipetting, and incubate. Finally, add 400 μL Binding Buffer and mix evenly. After that, the proportion of apoptotic cells in each group was detected by flow cytometry.

[0194] 1.7 HUVEC tube formation experiment: Pre-cool a sterilized 200 μL pipette tip and a 96-well plate in a 4°C refrigerator. Place Matrigel on ice and add the Matrigel to the 96-well plate using a pre-cooled 200 μL pipette tip, avoiding bubbles. Add 60 μL of Matrigel to each well. Set up three replicates for each group, and then place the 96-well plate in a cell culture incubator for activation for 1 hour. Take HUVEC cells that are in good growth condition and resuspend them in F12K complete medium to adjust the cell density to 2×10 5 Cells were plated at 400 μL / mL and 100 μL of cell suspension was added to each well. Next, 100 μL of HepG2-conditioned medium treated with CPD12C15+Cu, CPD12C15-Lip+Cu, or CPD12C15-Falip+Cu solutions was added, maintaining a consistent final CPD12C15 concentration in each group. A control group was set up and incubated in a cell culture incubator for 5 hours. Observation and photography were performed under a microscope, and data were analyzed and statistically analyzed using Image J.

[0195] 1.8 Western Blot detection of related protein expression levels

[0196] 1.8.1 Cell culture and treatment: HepG2 and Huh7 cells were evenly inoculated in six-well plates at appropriate concentrations and incubated, and 2 mL of cell suspension was added to each well. The culture medium in each well was discarded the next day and washed three times with PBS buffer. The cells were divided into five groups for drug addition, including the blank control group (Control group): 2 mL of DMEM complete medium was added to each well; the CPD12C15+Cu group, CPD12C15-Lip+Cu group and CPD12C15-Falip+Cu group: CuCl2 with the same molar concentration as CPD12C15 was added respectively. ˙ Mix the 2H2O solution to obtain CPD12C15+Cu mixed solution, CPD12C15-Lip+Cu mixed solution, and CPD12C15-Falip+Cu mixed solution. Dilute with DMEM complete medium to a final CPD12C15 concentration of 600 nmol / L in each HepG2 cell group and 400 nmol / L per well in Huh7 cells. Incubate in a cell culture incubator for 48 hours.

[0197] 1.8.2 Extraction of total cell protein: Prepare protein lysis buffer at a volume ratio of RIPA cell lysis buffer: phosphatase inhibitor: PMSF: protease inhibitor = 97:1:1:1 and place on ice until ready to use. Remove the 6-well plate and place on ice. Aspirate the culture medium and wash the cells three times with PBS buffer. Add 120 μL of protein lysis buffer to each well, scrape the cells, and lyse on ice for 25 minutes. Carefully transfer the liquid from each well to a 1.5 mL EP tube and centrifuge at 12,000 rpm for 25 minutes at 4°C. Aspirate the supernatant and quantify the protein using a BCA kit. Add SDS-PAGE loading buffer (5×) and an appropriate amount of protein supernatant, mix thoroughly, turn on the metal bath and heat to 98°C. Heat the protein sample for 10 minutes to denature it, cool to room temperature, and store at -20°C.

[0198] 1.8.3 Determination of protein concentration using a BCA kit: Determine the protein content in the protein extract according to the instructions of the BCA kit. First, prepare a BCA working solution (BCA:Cu = 50:1, v / v) by combining BCA reagent and Cu reagent based on the number of samples and standards. Dissolve the protein standard and dilute it with PBS to 0.5 mg / mL. Add 0, 2, 4, 6, 8, 12, 16, and 20 μL to a 96-well plate, and make up to 20 μL with PBS. Dilute the sample and add 20 μL per well to a 96-well plate. Add 200 μL of BCA working solution to each standard and sample well and incubate at 37°C for 20 min. Measure the absorbance of each well at 562 nm using a microplate reader. Plot a standard curve and calculate the protein concentration of the sample.

[0199] 1.8.4 SDS-PAGE gel electrophoresis: Rinse the glass plate and check for leaks. Select gel preparation kits of different concentrations according to the molecular weight of different proteins and prepare the gel according to the instructions. Insert the prepared gel plate into the electrophoresis tank, add electrophoresis buffer, and align the positive and negative poles. After removing the bubbles inside the electrophoresis tank, slowly pull out the comb, and load the Maker and protein in turn. Run electrophoresis at 80V constant voltage for 30 minutes. After the proteins have run smoothly, run electrophoresis at 120V constant voltage for 50 minutes to separate proteins of different molecular weights. Place the PVDF membrane cut to the appropriate size in methanol for activation for 10 minutes, and then place it in the transfer solution to keep it moist. After the electrophoresis is completed, cut the gel of the target protein according to the position of the Maker. Prepare a "sandwich" structure using the "black glue, white flour" principle. From cathode to anode, place a sponge, transfer filter paper, target gel strip, target PVDF membrane, transfer filter paper, and sponge in order. Gently press to remove any air bubbles between the gel strip and the PVDF membrane. Ensure the transfer solution fully submerges the sandwich. Transfer the membrane at a constant current of 200 mA for 90 minutes on an ice bath. After transfer, block the PVDF membrane with 5% skim milk powder on a shaker for 3 hours at room temperature. After blocking, wash with TBST three times for 10 minutes each. Place the membrane face-up in the primary antibody and incubate at 4°C for 16 hours. After incubation, remove the strips and wash with TBST three times for 10 minutes each. Add the corresponding secondary antibody based on the primary antibody used in the strips and incubate at room temperature for 50 minutes. After the timer expires, wash the strips with TBST three times for 10 minutes each. Prepare the developer by mixing equal parts of Solution A and Solution B from the ECL developer solution. Place the PVDF membrane on the developing plate of the developer and drip the developer solution until the strips are fully submerged. The gel imaging system was used to develop and record the developed bands, and the protein bands were quantitatively analyzed using Lane 1D analysis software.

[0200] 1.9 Statistical Analysis: GraphPad Prism software was used to process the experimental data and plot the results. The results are expressed as mean ± standard deviation, n = 3. Statistical analysis was performed using one-way analysis of variance and Tukey's Multiple Comparison Test. * p<0.05, ** p<0.01, *** p<0.001 indicates a significant difference compared with the Control group.

[0201] 2. Experimental results

[0202] 2.1 In vitro cytotoxicity test results: In vitro cytotoxicity evaluation was performed using the MTT test. Figure 6 、 Figure 7As shown. CPD12C15+Cu, CPD12C15-Lip+Cu, and CPD12C15-Falip+Cu can all inhibit the proliferation of hepatocellular carcinoma cells HepG2 and Huh7 in a concentration-dependent manner. Compared with the CPD12C15-Lip combined with copper group, CPD12C15-Falip combined with copper can further enhance the drug's killing effect on hepatocellular carcinoma cells, indicating that CPD12C15-Falip combined with copper has a stronger in vitro anti-tumor effect. Table 6 shows the IC values ​​of CPD12C15+Cu, CPD12C15-Lip+Cu, CPD12C15-Falip+Cu and sorafenib in HepG2 and Huh7 cells 50 Combined with the chart, it can be seen that CPD12C15+Cu, CPD12C15-Lip+Cu, and CPD12C15-Falip+Cu all exhibit excellent anti-hepatocellular carcinoma activity, especially the IC values ​​of CPD12C15-Falip+Cu in HepG2 and Huh7 cells. 50 The values ​​were 0.66±0.01μM and 0.39±0.01μM respectively, and their activity was significantly better than that of the positive drug Sorafenib.

[0203] Table 6 IC values ​​of CPD12C15+Cu, CPD12C15-Lip+Cu, CPD12C15-Falip+Cu and Sorafenib in HepG2 and Huh7 cells 50 value

[0204]

[0205] According to IC 50 The values ​​were used to determine the drug concentrations used in subsequent cell experiments. In HepG2 cells, the CPD12C15 concentration in the CPD12C15, CPD12C15-Lip, and CPD12C15-Falip combined with copper groups was 0.6 μmol / L, and the sorafenib concentration was 25 μmol / L. In Huh7 cells, the CPD12C15 concentration in the CPD12C15, CPD12C15-Lip, and CPD12C15-Falip combined with copper groups was 0.4 μmol / L, and the sorafenib concentration was 10 μmol / L.

[0206] 2.2 In vitro cell uptake experimental results: The fluorescence microscopy observation results and flow cytometry quantitative determination of fluorescence intensity of the two cells are shown in Figure 2. Figure 8 、 Figure 9As shown, the fluorescence intensity of the C6-Lip group was slightly enhanced compared to the free C6 group. The fluorescence intensity of C6-Falip after uptake by hepatocellular carcinoma cells HepG2 and Huh7 was significantly higher than that of the free C6 group, C6-Lip group, and Fa+C6-Falip group, and was time-dependent, indicating that folic acid modification can increase the targeting ability of liposomes to hepatocellular carcinoma cells. The addition of free folic acid to the Fa+C6-Falip group can competitively bind to folate receptors, blocking C6-Falip from entering tumor cells and weakening the fluorescence intensity of coumarin, indicating that liposomes are internalized into tumor cells by binding to folate receptors highly expressed on the surface of tumor cells.

[0207] 2.3 Cell migration experiment results: After scratching, HepG2 and Huh7 hepatocellular carcinoma cells were treated with different drug groups, and photos were taken at 0h and 48h. The scratch healing rate was calculated based on the scratch width. Figure 10 、 Figure 11 As shown in the results, compared with the control group, CPD12C15 combined with copper, CPD12C15-Lip combined with copper, CPD12C15-Falip combined with copper and sorafenib could significantly inhibit the migration of HepG2 and Huh7 cells. In HepG2 and Huh7 cells, the scratch healing rates of the control group were (34.23±0.87)% and (24.86±0.57)%, respectively. The CPD12C15-Falip combined with copper group had the strongest anti-migratory ability against the two hepatocellular carcinoma cells ( *** p<0.001), and the wound healing rates were (12.39±1.33)% and (10.54±1.32)%, respectively, which were significantly better than those of the positive drug sorafenib.

[0208] 2.4 Cell invasion assay results: Matrigel-Transwell assay was used to investigate the ability of CPD12C15 and its preparations combined with copper to inhibit the invasion of hepatocellular carcinoma cells. Transwell chambers containing Matrigel can simulate the external environment of cells. The results of three parallel experiments are shown in Figure 2. Figure 12 、 Figure 13 As shown in the results, the number of hepatocellular carcinoma cells passing through the Transwell chamber in the CPD12C15 combined with copper, CPD12C15-Lip combined with copper, and CPD12C15-Falip combined with copper groups was significantly reduced, indicating that the invasion ability of HepG2 and Huh7 hepatocellular carcinoma cells was inhibited, and the CPD12C15-Falip combined with copper group had the strongest anti-invasion ability against the two hepatocellular carcinoma cells ( *** p<0.001), the relative invasion rates of HepG2 and Huh7 hepatocellular carcinoma cells decreased to (12.34±0.94)% and (12.24±1.88)%, respectively, and the inhibitory effect was better than that of the positive drug sorafenib.

[0209] 2.5 Cell apoptosis experimental results: Flow cytometry was used to investigate the effects of CPD12C15 and its preparations combined with copper on the apoptosis of hepatocellular carcinoma cells. The first quadrant (UL) represents cell necrosis, the second quadrant (UR) corresponds to late apoptosis, the third quadrant (LR) indicates that cells are in the early apoptosis state, and the fourth quadrant (LL) represents living cells. The total apoptosis rate is the cumulative value of the early apoptosis rate and the late apoptosis rate. The results are shown in Figure 14 、 Figure 15 Compared with the Control group, CPD12C15+Cu, CPD12C15-Lip+Cu, and CPD12C15-Falip+Cu groups could induce apoptosis in HepG2 and Huh7 hepatocellular carcinoma cells. The CPD12C15-Falip combined with copper group had the strongest ability to promote apoptosis in both hepatocellular carcinoma cells ( *** The apoptosis rates in HepG2 and Huh7 cells were (36.28±1.25)% and (59.57±0.77)%, respectively. These results demonstrate that CPD12C15-Falip combined with copper has a strong ability to induce apoptosis in HepG2 and Huh7 cells.

[0210] 2.6 Results of HUVEC tubule formation experiment: Hepatocellular carcinoma is a highly vascularized tumor. Angiogenesis in tumor tissue generally begins with the release of growth factors by cancer cells, which send signals to vascular endothelial cells. These signals can promote the proliferation and migration of vascular endothelial cells, causing them to sprout and form blood vessels in the tumor matrix. Vascular endothelial cells (HUVEC) retain the characteristics of rapid proliferation, division, and migration of endothelial cells on Matrigel, and can respond to angiogenesis signals. Therefore, the HUVEC cell in vitro angiogenesis experiment can simply and reliably detect in vitro whether a drug has the effect of inhibiting angiogenesis. The results can be seen Figure 16 In the control group, HUVEC cells formed a complete capillary network of tubules. However, after adding HepG2 conditioned medium treated with CPD12C15+Cu, CPD12C15-Lip+Cu, or CPD12C15-Falip+Cu, the complete tubular structures of HUVEC cells were significantly reduced. Almost no obvious tubular structures were formed in the CPD12C15-Falip combined with copper group. By quantitatively counting the number of tubular branches, the number of tubular branches in the CPD12C15+Cu, CPD12C15-Lip+Cu, and CPD12C15-Falip+Cu groups was 96, 41, 17, and 12, respectively, indicating that CPD12C15-Falip combined with copper has the ability to significantly inhibit HUVEC cell angiogenesis.

[0211] 2.7 Western Blot results: Studies have shown that activation of the PI3K / AKT signaling pathway can regulate multiple biological processes in hepatocellular carcinoma cells, including cell invasion and metastasis, apoptosis, cell cycle, and angiogenesis. In order to further elucidate the potential molecular biological mechanism by which CPD12C15 and its preparations combined with copper inhibit the proliferation, metastasis, invasion, and angiogenesis of HepG2 and Huh7 cells, Western Blot experiments were performed to detect changes in the expression of key proteins in the PI3K / AKT signaling pathway of HepG2 and Huh7 cells. The results of Western Blot experiments are shown in Figure 2. Figure 17 、 Figure 18 As shown, the CPD12C15+Cu, CPD12C15-Lip+Cu, and CPD12C15-Falip+Cu groups inhibited the phosphorylation of p-PI3K and p-AKT in HepG2 and Huh7 cells, thereby reducing the expression of HIF-1α and VEGF proteins, inhibiting angiogenesis and proliferation. Furthermore, they increased the expression of the pro-apoptotic protein Bax and decreased the expression of the anti-apoptotic protein Bcl-2, promoting cell apoptosis. In summary, CPD12C15 folic acid liposomes combined with copper may inhibit the proliferation, invasion, and angiogenesis of hepatocellular carcinoma cells by inhibiting the expression of key proteins in the PI3K / AKT signaling pathway, thereby promoting apoptosis and exerting anti-hepatocellular carcinoma efficacy.

[0212] Example 4 In vivo efficacy of CPD12C15 folic acid liposomes combined with copper against hepatocellular carcinoma

[0213] Experimental cells and animals: Human hepatocellular carcinoma cells (HepG2) were provided by the School of Basic Medical Sciences, Shandong University. SPF-qualified Balb / c-nu nude mice, 6-7 weeks old and weighing 20-23 g, were purchased from the Beijing Huafukang Laboratory Animal Center. They were housed in an independent ventilation system at a temperature of 25 ± 2°C and a relative humidity of (50 ± 15%), with a 12 / 12 hour day and night cycle, and received normal chow and drinking water. Experiments were performed after one week of stable housing. All experimental procedures adhered to the guidelines of the Animal Experimentation Center of Shandong University.

[0214] Preparation of experimental reagents:

[0215] CPD12C15-Lip, CPD12C15-Falip, DiR-Lip, and DiR-Falip solutions: CPD12C15-Lip and CPD12C15-Falip were prepared with reference to the optimal process and formulation of Examples 1 and 2. DiR-Lip and DiR-Falip were prepared with reference to the preparation methods of CPD12C15-Lip and CPD12C15-Falip, except that the drug CPD12C15 was replaced with the near-infrared fluorescent dye DiR (i.e., 1,1'-dioctyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) during the lipid film formation stage. The remaining preparation steps and process parameters remained unchanged.

[0216] Copper gluconate solution: Accurately weigh 1.00 mg of copper gluconate powder, add 10 mL of normal saline, and sonicate to fully dissolve it to obtain a 100 μg / mL copper gluconate sulfate aqueous solution.

[0217] Sorafenib solution: Accurately weigh an appropriate amount of sorafenib powder and dissolve it in the prepared solvent (90% corn oil + 5% anhydrous ethanol + 5% DMSO) to prepare a 2 mg / mL stock solution.

[0218] 1. Experimental methods

[0219] 1.1 Cell culture: The cell culture method is the same as that in Example 3.

[0220] 1.2 Establishment of nude mouse subcutaneous tumor model: HepG2 cells in the logarithmic growth phase were digested and collected, and resuspended in sterile PBS to adjust the cell density to 1×10 7 The skin of the nude mice under the armpits was disinfected and 200 μL of cell suspension was inoculated subcutaneously in each nude mouse. The length (L) and width (W) of the tumor were recorded with a vernier caliper every day and the formula was used. The tumor volume was calculated for subsequent research.

[0221] 1.3 Investigation of the in vivo distribution behavior of the preparation: In order to verify the targeting of liposomes in vivo, the near-infrared fluorescent dye DiR was selected as a liposome marker, and the drug distribution in tumor-bearing nude mice was monitored in real time by in vivo imaging. DiR-Lip and DiR-Falip were prepared respectively. Nine nude mice bearing HepG2 subcutaneous tumors with successful modeling were selected and randomly divided into three groups: (1) DiR group, (2) DiR-Lip group, and (3) DiR-Falip group, with three mice in each group. When the tumor volume reached 300mm 3The above drugs were injected into the tail vein at a DiR concentration of 1 mg / kg. Images were taken using a small animal in vivo imaging device 2, 4, 8, 12, and 24 hours after injection, using 745 nm as the excitation wavelength and ICG (indocyanine green) as the emission channel. Fluorescence distribution was observed and photographed. After 24 hours of imaging, the nude mice were anesthetized and sacrificed, and the heart, liver, spleen, lung, kidney, and tumor were removed. The isolated organs and tumor tissues were cleaned with PBS, dried with filter paper, and placed in a small animal in vivo imaging device for scanning and photography. The distribution of DiR, DiR-Lip, and DiR-Falip in the isolated tissues and organs 24 hours after administration was recorded.

[0222] 1.4 In vivo antitumor activity evaluation: When the tumor volume of nude mice grows to 100 mm 3 Thirty nude mice bearing HepG2 subcutaneous tumors were randomly divided into five groups of six. The model group received a 2 mg / kg oral gluconate solution in the morning and a 100 μL / mouse tail vein injection of normal saline in the evening. The CPD12C15+Cu, CPD12C15-Lip+Cu, and CPD12C15-Falip+Cu groups received a 2 mg / kg oral gluconate solution in the morning and a 10 mg / kg tail vein injection in the evening, every two days. The positive control group received 10 mg / kg sorafenib daily. Body weight and tumor volume were measured every other day, and tumor volume and body weight curves were plotted. After 14 days of dosing, before sacrificing the nude mice, blood was collected from the eyeballs and heparin pretreated blood vessels were collected. The resulting whole blood was centrifuged (3000 rpm, 10 min, 4°C), and 100 μL of serum was collected in a centrifuge tube and stored in a -80°C refrigerator until use. The nude mice were euthanized, and the heart, liver, spleen, lung, kidney, and tumor mass were removed and fixed in 4% paraformaldehyde and stored in a 4°C refrigerator until use. The tumor weight of each group was weighed and the average was calculated. The tumor inhibition rate of each group was calculated according to the formula:

[0223] 1.5 H&E Staining: Nude mice's major organs and tumor masses were fixed with 4% paraformaldehyde for 24 hours. After fixation, the sections were removed and dehydrated with graded ethanol. After xylene was added to clear the tissue, the sections were embedded in liquid paraffin to create paraffin blocks. The blocks were sectioned at 4-5 μm thickness, flattened with hot water, stretched, and mounted on glass slides. The sections were deparaffinized and then rehydrated with graded ethanol. The sections were then rinsed with distilled water until transparent. Hematoxylin solution was added to the sections for 8-10 minutes. Excess stain was removed by slow water flow. Separation was performed with 1% hydrochloric acid and ammonia solution, followed by a water rinse. Dehydration was performed with 80% and then 90% ethanol, followed by further staining with eosin solution for 3-8 minutes. The slides were dehydrated with varying concentrations of ethanol (70%, 80%, and 95%) and then permeabilized again with xylene until transparent. Neutral gum was applied to the sections, sealed, mounted, and dried before microscopic examination.

[0224] 1.6 TUNEL apoptosis assay: Deparaffinize tumor sections and rinse with PBS buffer three times for 5 minutes each. Incubate sections in 3% H₂O₂ solution at room temperature for 20 minutes, then rinse thoroughly with PBS. Add proteinase K working solution to the sections, incubate at 37°C for 15-25 minutes, and rinse thoroughly with PBS. Add the prepared TUNEL reaction solution dropwise to the sections, incubate at 37°C in the dark for 60 minutes, and rinse three times with PBS for 5 minutes each. Add an appropriate amount of Conventer-POD reagent dropwise, incubate at 37°C in the dark for 60 minutes, and rinse three times with PBS for 5 minutes each. Add an appropriate amount of DAB staining solution to the sections and observe the staining under a microscope. When the staining is satisfactory, rinse thoroughly with PBS to stop staining. Add hematoxylin staining solution and stain for 5 minutes. Remove excess stain by running water at a low speed. Separate the sections with 1% hydrochloric acid and ammonia solution, then rinse with water. The sections were dehydrated with gradient ethanol, immersed in xylene to permeabilize until transparent, and sealed with neutral gum. After drying, they were placed under a microscope for photography, and the expression levels of positive cells were counted using Image J software.

[0225] 1.7 Immunohistochemistry: Deparaffinized tissue sections were placed in EDTA buffer (pH 9.0) and kept boiling for antigen retrieval. After 20–30 minutes, the sections were cooled to room temperature and rinsed three times with PBS buffer (5 minutes each). A 3% H₂O₂ solution was added to the sections and incubated at room temperature for 25 minutes. The sections were then rinsed three times with PBS buffer (5 minutes each). The sections were blocked in 3% BSA solution at room temperature for 1 hour. The blocking solution was discarded and the primary antibodies for p-PI3K, p-AKT, Bcl-2, Bax, and VEGF were diluted according to the IHC ratio specified in the manufacturer's instructions and applied dropwise to the sections. The sections were incubated overnight at 4°C. The sections were rinsed with PBS buffer and, after gently shaking to dry, the corresponding secondary antibodies were added. The sections were incubated at room temperature for 50 minutes and then rinsed again with PBS buffer. An appropriate amount of DAB staining solution was added to the sections and the staining was observed under a microscope. Once the staining was adequate, the sections were rinsed thoroughly with PBS to stop staining. The sections were counterstained with hematoxylin for 3 minutes, then washed with water to remove excess stain. The sections were then differentiated with 1% hydrochloric acid and alcohol and rinsed with water. The sections were then dehydrated with graded ethanol, permeabilized in xylene until transparent, and mounted with neutral gum. Once dry, the sections were placed under a microscope for photography.

[0226] 1.8 ELISA Assay: Prepare standard curves for IL-6 and TNF-α according to the ELISA kit instructions. Remove the prepared serum from the -80°C freezer, dilute it to the appropriate dilution, and process the samples according to the instructions. Measure absorbance and calculate the concentrations of IL-6 and TNF-α in each serum group.

[0227] 1.9 Statistical analysis: GraphPad Prism (8.0) software was used to analyze the data, and the results were expressed as mean ± SD. The data were analyzed using t-test and one-way analysis of variance to compare the differences between the groups. * p<0.05, ** p<0.01, *** p<0.001 indicates significant difference from the Model group; # p<0.05, ## p<0.01, ### p<0.001 indicates a significant difference compared with the CPD12C15-Lip group.

[0228] 2. Experimental results

[0229] 2.1 Results of in vivo targeting ability study of the preparation

[0230] The targeted distribution of fluorescently labeled liposomes in tumor-bearing nude mice was investigated through small animal in vivo imaging experiments. Figure 19The qualitative and quantitative results of in vivo imaging show that free DiR is widely distributed in the nude mice due to its lack of tumor targeting. There is no obvious signal at the tumor site, and the fluorescence at the liver and spleen is strong; as time goes on, the fluorescence signal in the nude mice weakens. The DiR-Lip group has obvious accumulation at the tumor site at 4 hours due to the EPR effect. As time goes on, the accumulation of DiR-Lip at the tumor site increases, and the fluorescence intensity at the tumor site reaches the strongest at 12 hours; as time goes on, the fluorescence signal gradually weakens after 12 hours. At 2 hours, the fluorescence of the DiR-Falip group was mainly concentrated in the liver, but compared with the first two groups, the DiR-Falip group had a small amount of accumulation at the tumor site. Similarly, as time goes on, the fluorescence intensity at the tumor site increases, and after 24 hours, there is still strong fluorescence at the tumor tissue. Figure 19 It can be seen that at all time points, the fluorescence intensity of DiR-Falip at the tumor site was stronger than that of the free DiR and DiR-Lip groups, and the retention time in the body was longer, indicating that folic acid-modified liposomes can actively target the tumor site and increase the accumulation of drugs at the tumor site.

[0231] The nude mice were anesthetized and sacrificed, and the heart, liver, spleen, lung, kidney and tumor were taken for imaging. The fluorescence distribution of different preparations in the isolated tissues and organs was analyzed 24 hours after administration. The experimental results are shown in the figure. Figure 20 As shown, compared with free DiR ( *** p<0.001) and DiR-Lip( ### Compared to the DiR-Lip group (p<0.001), folate liposomes exhibited the strongest fluorescence at the tumor site, with a fluorescence signal approximately 4.3 times that of the DiR-Lip group and 22 times that of the free DiR group. These results demonstrate the excellent in vivo targeting ability of folate liposomes. This excellent in vivo targeting is due to the fact that the folic acid on the surface of folate liposomes can specifically bind to folate receptors overexpressed in tumor cells, enabling the liposomes to precisely target tumor cells and enhance the drug's efficacy.

[0232] 2.2 In vivo antitumor activity evaluation results

[0233] After the administration, the tumors of the nude mice in each group were removed and photographed. The weight of the nude mice and tumors recorded during the experiment were analyzed. The results are as follows: Figure 21 As shown. Figure 21 In (a), it can be seen intuitively that the tumor size of nude mice in different groups is the smallest in the CPD12C15-Falip+Cu group, and the tumor size of the CPD12C15-Lip+Cu group is similar to that of the positive drug Sorafenib group. Figure 21The results of the tumor volume changes in each group (b) show that the tumor in the Model group grew rapidly, the CPD12C15 group had a certain inhibitory effect on tumor growth, and the tumor growth in the CPD12C15-Lip+Cu group, the Sorafenib group, and the CPD12C15-Falip+Cu group was relatively slow. The average tumor weight and average tumor volume of each group are shown in Figure 2. Figure 21 As shown in (c) and (e), the average tumor volume of the Model group was 916.23±81.61mm 3 The average tumor weight was 0.92±0.13 g; the average tumor volume in the CPD12C15+Cu group was 554.21±12.61 mm 3 The average tumor weight was 0.42±0.10 g; the average tumor volume in the CPD12C15-Lip+Cu group was 400.05±55.42 mm 3 The average tumor weight was 0.35±0.05g; the average tumor volume in the CPD12C15-Falip+Cu group was 179.61±43.57mm 3 The average tumor weight was 0.15±0.06 g; the average tumor volume in the Sorafenib group was 369.83±48.65 mm 3 , the average tumor weight was 0.37±0.05g. Calculation showed that compared with the Model group, the relative tumor inhibition rate of the CPD12C15+Cu group was 54.55%, the relative tumor inhibition rate of the CPD12C15-Lip+Cu group was 61.82%, the relative tumor inhibition rate of the CPD12C15-Falip+Cu group was 83.82%, and the relative tumor inhibition rate of the Sorafenib group was 60%. The above results show that CPD12C15+Cu, CPD12C15-Lip+Cu and CPD12C15-Falip+Cu can inhibit tumor growth. Compared with the CPD12C15-Lip+Cu group, the anti-tumor effect of CPD12C15-Falip+Cu is better ( ## In summary, folate targeting enhances the specific uptake of CPD12C15-Falip by tumor cells by specifically binding to folate receptors on the surface of tumor cells, thereby improving the anti-tumor effect of CPD12C15 in vivo.

[0234] During the administration process, the nude mice were weighed every two days. Figure 21 As shown in (d), there was no significant change in the body weight of nude mice in each group, indicating that the drug had no obvious systemic toxicity and had good safety.

[0235] 2.3 H&E staining results

[0236] After the administration, the main organs (heart, liver, spleen, lung, kidney) and tumor masses of each group of nude mice were removed, and the toxic effects of CPD12C15-Falip combined with copper on the tissues in nude mice and the anti-tumor effects of different groups of drugs were evaluated by H&E staining. Figure 22 As shown, compared with the Model group, there was no obvious pathological change or inflammatory infiltration in the heart, liver, spleen, lung and kidney of nude mice in the CPD12C15-Falip+Cu group, indicating that CPD12C15-Falip combined with copper has the best anti-tumor effect and also has good in vivo safety and biocompatibility.

[0237] The H&E staining results of the tumor masses in each group are as follows Figure 23 As shown, cells in the Model group grew densely and the tumor proliferated vigorously; cells in the CPD12C15+Cu group, CPD12C15-Lip+Cu group, and Sorafenib group showed gaps, indicating that it had an inhibitory effect on tumor growth; cells in the CPD12C15-Falip+Cu group showed a large amount of necrosis and vacuoles, and the cell nuclei shrank, indicating a strong killing effect on tumor cells.

[0238] 2.4 Tumor TUNEL staining results: TUNEL apoptosis assay was used to investigate the effects of different preparations on tumor cell apoptosis in vivo. Figure 24 The TUNEL apoptosis images and quantitative analysis results of different groups showed that almost all cells in the Model group were negative, indicating that the tumor cells grew vigorously; the apoptosis of tumor cells in nude mice in the CPD12C15+Cu group was not obvious, and there was no significant difference compared with the Model group; the number of apoptosis-positive cells in the CPD12C15-Lip+Cu group, CPD12C15-Falip+Cu group and Sorafenib group increased significantly ( *** p<0.001), the CPD12C15-Falip+Cu group had a more significant effect on promoting the apoptosis of hepatocellular carcinoma cells than the CPD12C15-Lip+Cu group ( ### p<0.001). In summary, CPD12C15-Falip combined with copper can promote apoptosis in hepatocellular carcinoma cells in vivo, and its pro-apoptotic ability is superior to that of the same dose of unmodified liposomal CPD12C15-Lip combined with copper and the positive drug sorafenib. Specific targeting of folic acid is conducive to precisely enhancing the anti-tumor effect of CPD12C15.

[0239] 2.5 Immunohistochemistry results: In order to confirm the effect of CPD12C15 and its preparations combined with copper on the PI3K / AKT signaling pathway in vivo, immunohistochemistry was used to detect the effects of CPD12C15+Cu group, CPD12C15-Lip+Cu group and CPD12C15-Falip+Cu group on the expression of key proteins in the PI3K / AKT signaling pathway, such as p-PI3K, p-AKT, VEGF, Bcl-2 and Bax. Figure 25 As shown in the results, compared with the Model group, the p-PI3K, p-AKT, VEGF, and Bcl-2 protein levels in the CPD12C15+Cu group, the CPD12C15-Lip+Cu group, and the CPD12C15-Falip+Cu group were significantly decreased, and the CPD12C15-Falip+Cu group had a more significant effect on increasing the expression of the pro-apoptotic protein Bax. Therefore, it was found that CPD12C15-Falip combined with copper inhibited the proliferation and angiogenesis of hepatocellular carcinoma cells and promoted the apoptosis of hepatocellular carcinoma cells by affecting the expression of key proteins in the PI3K / AKT signaling pathway, which is consistent with the results of in vitro experiments.

[0240] 2.6 ELISA test results: The levels of inflammatory factors TNF-α and IL-6 in the serum of nude mice were determined by ELISA kits. Figure 26 As shown, compared with the Model group, the positive drug sorafenib group increased the expression of TNF-α and IL-6, while the CPD12C15+Cu group, CPD12C15-Lip+Cu group, and CPD12C15-Falip+Cu group were able to significantly inhibit the expression of pro-inflammatory factors, and the CPD12C15-Falip combined with copper group had the lowest expression level of pro-inflammatory factors. Compared with the positive drug sorafenib, CPD12C15-Falip combined with copper has excellent anti-inflammatory effects in vivo.

[0241] 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 folic acid-targeted CPD12C15 liposome, characterized in that: It is a vesicle-like structure containing: The lipid shell is a lipid bilayer formed by phosphatidylcholine, cholesterol and the DSPE lipophilic end of DSPE-PEG2000-Fa, and encloses the vesicle; A targeting functional layer comprising DSPE-PEG2000-Fa co-membraned with the lipid shell, wherein the lipophilic end of DSPE is embedded in the lipid bilayer, and the hydrophilic end of PEG2000-Fa extends and is exposed on the outer surface of the lipid shell, thereby imparting targeting properties; The inner aqueous core is the aqueous cavity enclosed by the lipid shell, and the active ingredient CPD12C15 is encapsulated in the inner aqueous core; Wherein, the structure of the CPD12C15 is:

2. The folic acid-targeted CPD12C15 liposome according to claim 1, characterized in that wherein the active ingredient CPD12C15 is partially embedded in the lipid shell; Preferably, the molar ratio of lecithin to cholesterol is 1:1 to 5:1, preferably 2:1; Preferably, the molar ratio of CPD12C15 to lipid is 1:1 to 1:8, preferably 1:2; Preferably, the content of DSPE-PEG2000-Fa is 0.5-2.0% of the total lipid molar number, preferably 1.25%.

3. The folic acid-targeted CPD12C15 liposome according to claim 1 or 2, characterized in that: The particle size of the folic acid-targeted CPD12C15 liposome is 80-130 nm, preferably 100-120 nm; Preferably, the polydispersity index (PDI) of the folic acid-targeted CPD12C15 liposomes is less than 0.3, preferably less than 0.2; Preferably, the surface potential of the folic acid-targeted CPD12C15 liposomes is -1.0 to -2.5 mV, preferably -1.8±0.5 mV; Preferably, the encapsulation efficiency of the folic acid-targeted CPD12C15 liposomes is 75-95%, preferably 90-95%; Preferably, the drug loading of the folic acid-targeted CPD12C15 liposome is 10-20%, preferably 12-17%.

4. A method for preparing folic acid-targeted CPD12C15 liposomes, comprising the following steps: Phosphatidylcholine, cholesterol and DSPE-PEG2000-Fa were mixed and dissolved in an organic solvent and rotary evaporated to form a uniform lipid film; The lipid film was hydrated with calcium acetate solution, and after ultrasonic treatment, the blank calcium acetate liposomes containing DSPE-PEG2000-Fa were obtained by filtration; The blank liposomes were dialyzed to establish a pH gradient; The dialyzed blank liposomes were mixed with CPD12C15 and incubated, and then filtered to obtain folate-targeted CPD12C15 liposomes.

5. The preparation method according to claim 4, characterized in that The organic solvent is chloroform; Preferably, the molar ratio of lecithin to cholesterol is 1:1 to 5:1, preferably 2:1; Preferably, the molar ratio of CPD12C15 to lipid is 1:1 to 1:8, preferably 1:2; Preferably, the content of DSPE-PEG2000-Fa is 0.5-2.0% of the total lipid molar amount, preferably 1.25%; Preferably, the concentration of the calcium acetate solution is 80-200 mmol / L, preferably 120 mmol / L; Preferably, the lecithin concentration is 10-40 mg / mL, preferably 20 mg / mL; Preferably, the incubation temperature is 30-60°C, preferably 40°C; Preferably, the incubation time is 40-70 minutes, preferably 60 minutes. A pharmaceutical composition comprising the folic acid-targeted CPD12C15 liposome according to any one of claims 1 to 3.

7. The pharmaceutical composition according to claim 6, characterized in that include: a) Folate-targeted CPD12C15 liposomes according to any one of claims 1 to 3; 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.

8. A pharmaceutical preparation comprising the folate-targeted CPD12C15 liposome according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier.

9. A medical kit comprising: A first container containing the folic acid-targeted CPD12C15 liposomes according to any one of claims 1 to 3; a second container containing a pharmaceutically acceptable copper salt solution; The two containers are designed for simultaneous, separate or sequential administration.

10. Use of the folic acid-targeted CPD12C15 liposomes according to any one of claims 1 to 3, the pharmaceutical composition according to claim 6 or 7, the pharmaceutical preparation according to claim 8, or the medical kit according to claim 9 in the preparation of the following drugs: (1) Drugs used to treat hepatocellular carcinoma; (2) drugs used to reverse sorafenib resistance or reduce sorafenib-related skin toxicity; (3) drugs for reducing IL-6 and / or TNF-α levels in chronic inflammation associated with hepatocellular carcinoma; (4) drugs used to inhibit angiogenesis in hepatocellular carcinoma; (5) drugs used to inhibit the migration or invasion of hepatocellular carcinoma cells; (6) Drugs used to induce apoptosis in hepatocellular carcinoma cells; (7) Drugs for treating hepatocellular carcinoma by inhibiting the PI3K / AKT signaling pathway.

Images