TPGS-modified CPD12C15 liposome as well as preparation method and application thereof

Abstract

The invention relates to the field of pharmaceutical preparations, and provides a TPGS modified CPD12C15 liposome as well as a preparation method and application thereof. The TPGS-modified CPD12C15 liposome is of a vesicle-shaped structure and comprises a lipid bilayer membrane which is of a closed membrane structure jointly formed by DPPC, SPC and cholesterol and forms a basic skeleton of the vesicle, and a TPGS-modified CPD12C15 liposome which is of a vesicle-shaped structure. The surface modification layer is formed by TPGS on the surface of the lipidosome, the hydrophobic end of the TPGS is inserted into the outer layer of the lipid bilayer membrane, and the hydrophilic end of the TPGS extends and is exposed on the outer surface of the lipidosome; and an internal aqueous phase, an aqueous chamber surrounded by a lipid bilayer, in which the active ingredient CPD12C15 is encapsulated; the structure of the CPD12C15 is # imgabs0 #. The liposome is prepared through a calcium acetate gradient method, the encapsulation efficiency reaches up to 79.84%, the drug loading capacity reaches 17.24%, the particle size is about 91 nm, and the PDI is lower than 0.2. The combination of the liposome and copper ions shows an excellent inhibition effect on non-small cell lung cancer cells, the IC50 value is 0.1331-0.199 [mu] mol / L and is far lower than that of docetaxel, and the multi-drug resistance of tumors can be effectively overcome.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical preparations, and in particular to TPGS-modified CPD12C15 liposomes, a preparation method thereof, and applications thereof. 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] Lung cancer is one of the most common malignant tumors worldwide, with high morbidity and mortality. Non-small cell lung cancer (NSCLC) accounts for over 80% of all lung cancer cases. While the development of molecular targeted therapy and immunotherapy has made some progress in the treatment of NSCLC, traditional chemotherapy remains the mainstay of treatment for some patients.

[0004] Among chemotherapy drugs, taxanes such as docetaxel (DTX) are widely used in the treatment of NSCLC. However, these drugs are often associated with severe side effects, including myelosuppression and neurotoxicity, and are prone to developing drug resistance, limiting their clinical effectiveness. To overcome these issues, current research has explored various strategies to improve the targeting, safety, and resistance-reversing capabilities of drug therapies.

[0005] Furthermore, research on drug delivery systems has also garnered significant attention. Liposomes, as nanoscale drug carriers, possess excellent biocompatibility and biodegradability, enhancing drug stability, prolonging circulation time, and, to a certain extent, improving drug tissue distribution. In recent years, liposomes have been increasingly employed in the development of delivery systems for a variety of anti-tumor drugs, aiming to optimize therapeutic efficacy and reduce systemic toxicity.

[0006] Although research has achieved some success in improving drug delivery efficiency and inhibiting tumor drug resistance, there are still urgent challenges to be addressed in terms of how to further enhance therapeutic efficacy, reduce adverse reactions, and improve tumor cell sensitivity to chemotherapeutic drugs. Therefore, there is still a need to provide a new drug delivery composition or treatment method to meet the clinical needs of NSCLC. Summary of the Invention

[0007] The present invention aims to provide a TPGS-modified CPD12C15 liposome and a preparation method thereof. The liposome has the characteristics of high encapsulation efficiency, suitable drug loading, good stability, strong targeting, etc., can effectively inhibit the proliferation, migration and invasion of non-small cell lung cancer cells, induce cell apoptosis, and reverse the multidrug resistance of tumor cells by inhibiting the Warburg effect and P-glycoprotein (P-gp) function.

[0008] Specifically, the present invention provides the following technical solutions:

[0009] In the first aspect of the present invention, a TPGS-modified CPD12C15 liposome is provided, which is a vesicle-like structure and comprises:

[0010] The lipid bilayer membrane is a closed membrane structure composed of dipalmitoylphosphatidylcholine (DPPC), soybean phosphatidylcholine (SPC) and cholesterol, forming the basic skeleton of the vesicle;

[0011] A surface modification layer is formed on the surface of the liposome by vitamin E polyethylene glycol succinate (TPGS), wherein the hydrophobic end of the vitamin E polyethylene glycol succinate is inserted into the outer layer of the lipid bilayer membrane, and the hydrophilic end extends and is exposed to the outer surface of the liposome;

[0012] The inner aqueous phase is an aqueous compartment surrounded by a lipid bilayer membrane, in which the active ingredient CPD12C15 is encapsulated;

[0013] Wherein, the structure of the CPD12C15 is:

[0014]

[0015] The composition and function of lipid bilayer membranes

[0016] In the present invention, the lipid bilayer membrane is composed of dipalmitoylphosphatidylcholine (DPPC), soy lecithin (SPC), and cholesterol, forming a closed membrane structure that forms the basic skeleton of the vesicle. Through systematic research, the inventors found that this multi-component membrane system has unique structural advantages and functional synergistic effects:

[0017] The synergistic combination of DPPC and SPC forms a complex phospholipid matrix with complementary physicochemical properties. DPPC, a saturated synthetic phospholipid, possesses a regular molecular arrangement and a high phase transition temperature, providing the membrane rigidity and structural stability required for liposomes. SPC, a naturally derived phospholipid mixture containing a variety of unsaturated phospholipid molecules, exhibits excellent membrane fluidity and film-forming properties, facilitating liposome formation and drug encapsulation. In the present invention, this complex phospholipid design achieves higher drug encapsulation efficiency and improved size uniformity compared to single phospholipid liposomes.

[0018] Cholesterol, as a membrane modulator, plays a key role in this complex system. Cholesterol molecules inserted into the phospholipid bilayer enhance the membrane's mechanical strength, reduce lipid mobility, and improve the liposome's stability in the body. Furthermore, cholesterol modulates membrane fluidity and permeability, optimizing drug loading and release characteristics. The inventors discovered that an appropriate ratio of phospholipids to cholesterol achieves a balance between membrane rigidity and fluidity, creating an ideal drug delivery vehicle.

[0019] This three-component lipid bilayer membrane not only provides a protective microenvironment for CPD12C15, but also achieves pH-responsive drug release through its specific phase change properties and membrane fluidity. It exhibits accelerated release characteristics in the acidic microenvironment of the tumor, while maintaining a lower release rate in the normal physiological environment, thereby improving the drug's targeting and utilization rate.

[0020] Characteristics and action mechanism of surface modification layer

[0021] In this invention, the surface modification layer is formed on the liposome surface by vitamin E polyethylene glycol succinate (TPGS), where the hydrophobic end of TPGS (the vitamin E portion) is inserted into the outer layer of the lipid bilayer membrane, while the hydrophilic end (the PEG segment) extends and is exposed on the outer surface of the liposome. This specific surface modification design has multiple functions and significant advantages:

[0022] TPGS, an amphiphilic nonionic surfactant, possesses a unique molecular structure and biological functions. Its hydrophobic portion, vitamin E, has a high affinity for lipid bilayer membranes, enabling stable insertion into the membrane structure. The hydrophilic portion, PEG, stretches in the aqueous phase to form a "brush" conformation, providing a steric barrier for liposomes. The inventors have discovered that this surface modification significantly improves the stability of liposomes in vitro and in vivo, reduces plasma protein adsorption, and reduces recognition and clearance by the reticuloendothelial system, thereby prolonging the liposome's circulation time in the body.

[0023] Compared to traditional PEG modifiers (such as PEG-DSPE), the TPGS-modified layer exhibits multifunctional synergistic properties. In the present invention, TPGS not only possesses the "stealth" effect of PEG but also inhibits the drug efflux function of P-glycoprotein (P-gp), effectively reversing multidrug resistance in tumor cells. Furthermore, in the present invention, TPGS has the ability to target mitochondria, promoting the mitochondrial localization of liposomes in tumor cells and enhancing the drug's intervention effect on the energy metabolism center of tumor cells.

[0024] TPGS surface modification also significantly improved the cellular uptake efficiency of liposomes. The inventors observed that TPGS-modified liposomes interacted more effectively with cell membranes, significantly increasing their efficiency in cellular entry via endocytosis. This enhanced cellular uptake is associated with the TPGS molecule's unique interfacial activity and its regulation of cell membrane fluidity, providing a highly efficient pathway for intracellular delivery of CPD12C15.

[0025] In summary, the composite phospholipid bilayer membrane constructed by DPPC, SPC, and cholesterol and the TPGS surface modification layer in the present invention form a drug delivery system with optimized structure and synergistic functions, which not only improves the stability and uniformity of the preparation, but also enhances the cellular uptake, tumor targeting, and anti-resistance effects of the drug through multiple mechanisms, providing an ideal platform for the efficient delivery of CPD12C15.

[0026] In some embodiments of the present invention, a combination of phospholipids (DPPC + SPC) and cholesterol is used to construct the lipid bilayer membrane. Through systematic research, the inventors discovered that the synergistic combination of DPPC and SPC offers significant advantages in liposome preparation: DPPC, as a synthetic phospholipid, has a high phase transition temperature and membrane structural rigidity, providing a stable membrane skeleton; while SPC, as a natural phospholipid, has a low phase transition temperature and good membrane fluidity, facilitating liposome formation and drug loading. Studies have observed that liposomes constructed with a single phospholipid have significant limitations: while liposomes constructed with DPPC alone are structurally stable, they exhibit low encapsulation efficiency; while liposomes constructed with SPC alone, while easy to form, lack stability, exhibit significant particle size growth during storage, and are prone to drug leakage. Attempts to substitute other phospholipids, such as DSPC or DOPE, often result in reduced encapsulation efficiency or poor compatibility with the calcium acetate gradient method due to differences in phase transition temperature or molecular configuration. Experiments have confirmed that a composite phospholipid formulation combining DPPC and SPC in an appropriate ratio can simultaneously optimize both encapsulation efficiency and drug loading, significantly improving formulation stability and pH-responsive release properties. Furthermore, the inventors discovered that DPPC, a natural component of lung surfactant, offers additional tissue affinity advantages for the treatment of non-small cell lung cancer. This systematically optimized composite phospholipid design provides key technical support for the efficient delivery of CPD12C15.

[0027] In some embodiments of the present invention, the molar ratio of dipalmitoylphosphatidylcholine (DPPC) to soy lecithin (SPC) is 1:1 to 1:7, preferably 1:5; this ratio range can ensure both fluidity and stability of the liposome membrane, which is conducive to the effective loading and controlled release of CPD12C15.

[0028] In some embodiments of the present invention, the molar ratio of phospholipids (DPPC + SPC) to cholesterol in the lipid bilayer membrane is 1:1 to 5:1, preferably 2:1. An appropriate cholesterol content can adjust the fluidity and rigidity of the membrane, ensuring liposome stability while not excessively affecting drug penetration and release.

[0029] In some embodiments of the present invention, the vitamin E polyethylene glycol succinate (TPGS) content is 3-15%, preferably 10%, of the total lipid molar amount, where the total lipid molar amount refers to the sum of the molar amounts of DPPC, SPC, cholesterol, and TPGS. The appropriate amount of TPGS modification can not only prolong the blood circulation time of liposomes, but also reverse tumor cell multidrug resistance by inhibiting P-gp function and promote liposome uptake by tumor cells.

[0030] Preferably, the total molar ratio of CPD12C15 to lipid components (DPPC, SPC, cholesterol and TPGS) is 1:1 to 1:5, preferably 1:2. This ratio range can ensure sufficient drug loading while maintaining the stability of the liposome structure.

[0031] The liposomes of the present invention have at least one of the following physicochemical properties:

[0032] Average particle size: 80-150 nm, preferably 90-110 nm; polydispersity index (PDI): 0.1-0.3, preferably less than 0.2; Zeta potential: -15 to 0 mV, preferably -10 to -5 mV; encapsulation efficiency: 70-90%, preferably 78-82%; drug loading: 10-25%, preferably 15-20%.

[0033] In particular, the liposomes of the present invention have a significantly faster release rate under tumor microenvironment-simulating conditions (pH 6.5-7.0) than under physiological conditions (pH 7.2-7.6), exhibiting a good pH-responsive release profile, which facilitates selective drug release at the tumor site. For example, in one embodiment, the liposomes have a cumulative release rate of 60-95%, preferably 80-90%, over 6 hours in a phosphate buffer at pH 6.5-7.0; and a cumulative release rate of 30-70%, preferably 40-50%, over 6 hours in a phosphate buffer at pH 7.2-7.6.

[0034] In a second aspect of the present invention, a method for preparing the TPGS-modified CPD12C15 liposomes described in the first aspect is provided, comprising:

[0035] (1) DPPC, SPC, cholesterol and TPGS were mixed and dissolved in an organic solvent, and rotary evaporated to form a uniform lipid film; (2) the lipid film was hydrated with calcium acetate solution, and after ultrasonic treatment, it was filtered to obtain blank liposomes; (3) the blank liposomes were dialyzed to establish a pH gradient; (4) the dialyzed blank liposomes were mixed with CPD12C15 and incubated, and filtered to obtain TPGS-modified CPD12C15 liposomes.

[0036] The present invention adopts the calcium acetate gradient method to prepare liposomes. Compared with the traditional thin film dispersion method, this method significantly improves the encapsulation efficiency and drug loading capacity of CPD12C15.

[0037] In some embodiments of the present invention, the organic solvent is a mixture of one or more of chloroform, methanol, and ethanol, preferably chloroform.

[0038] In some embodiments of the present invention, the rotary evaporation is performed at 30-50°C, preferably 40°C.

[0039] In some embodiments of the present invention, the concentration of the calcium acetate solution is 80-200 mmol / L, preferably 120 mmol / L.

[0040] In some embodiments of the present invention, the ultrasonic treatment is carried out in an ice bath, the ultrasonic power is 30-70 W, preferably 50 W, and the ultrasonic treatment is carried out in an intermittent ultrasonic manner, such as ultrasonicating for 2 seconds on and stopping for 2 seconds, with a total time of 1-5 minutes, preferably 2 minutes.

[0041] In some embodiments of the present invention, the dialysis uses a dialysis bag with a molecular weight cut-off of 3000-5000 Da, the dialysis medium is a sodium sulfate solution with an equal concentration to the calcium acetate solution, and the dialysis time is 8-16 hours, preferably 12 hours.

[0042] Preferably, the incubation temperature is 20-50° C., preferably 30° C.; the incubation time is 30-90 minutes, preferably 60 minutes.

[0043] In the third aspect of the present invention, a pharmaceutical composition is provided, comprising the TPGS-modified CPD12C15 liposomes described in the first aspect and a pharmaceutically acceptable carrier or excipient.

[0044] In some embodiments of the present invention, the pharmaceutical composition further comprises a copper salt, and the copper salt is selected from one or more of copper chloride, copper sulfate, copper acetate and copper gluconate, preferably copper chloride or copper gluconate.

[0045] In some embodiments of the present invention, the molar ratio of CPD12C15 to copper ions is 1:0.3 to 1:3, preferably 1: 1. Based on the synergistic mechanism of the two, their combined use can significantly enhance the killing effect on non-small cell lung cancer cells.

[0046] In some embodiments of the present invention, the pharmaceutical composition is in the form of an injection, a lyophilized powder injection, or a liposome suspension; wherein the content of CPD12C15 in the pharmaceutical composition is 0.05-20 mg / mL, preferably 0.5-5 mg / mL.

[0047] In a fourth aspect, the present invention provides a medical kit comprising: a first container containing the TPGS-modified CPD12C15 liposomes described in the first aspect; a second container containing a copper salt solution; and instructions for administration. The design of this medical kit allows for flexible administration of the CPD12C15 liposomes and copper salt, whether mixed, separate, or sequentially, depending on clinical needs.

[0048] In some embodiments of the present invention, the concentration of the copper salt solution is 50-500 μg / mL, preferably 100 μg / mL.

[0049] The present invention also includes a technical solution of using the above-mentioned TPGS-modified CPD12C15 liposomes in combination with a copper salt, wherein the copper salt can be an inorganic copper salt or an organic copper salt, wherein the inorganic copper salt is preferably copper chloride and the organic copper salt is preferably copper gluconate. The inventors have found that although the chemical forms of different copper salts are different, they are all effective in providing functional Cu 2+ The ions showed substantially the same effect in forming active complexes with CPD12C15, and the optimal molar ratio of CPD12C15 to copper ions was close to 1:1. In the specific implementation process of the present invention, copper chloride was preferably used in in vitro cell experiments, which has good solubility and stability in cell culture systems and can efficiently provide Cu 2+ ions interact with CPD12C15; copper gluconate is preferred in in vivo animal experiments because it has better biocompatibility and pharmacokinetic properties as an organic copper salt, and can maintain ideal anti-tumor effects while reducing systemic toxicity. Both copper salts significantly enhanced the anti-tumor activity of CPD12C15 liposomes in their respective application scenarios, verifying that Cu 2+ The key mechanism of the synergistic effect of ions and the liposome system of the present invention is that they show significant inhibitory effects on key enzymes responsible for metabolic reprogramming in tumor cells, such as PDK1 and P-glycoprotein that effluxes chemotherapy drugs, thereby achieving dual intervention on tumor growth and chemotherapy resistance.

[0050] The fifth aspect of the present invention provides the use of the TPGS-modified CPD12C15 liposomes described in the first aspect, the pharmaceutical composition described in the third aspect, or the medical kit described in the fourth aspect in the preparation of a drug for treating non-small cell lung cancer.

[0051] Particularly, the non-small cell lung cancer is drug-resistant non-small cell lung cancer, preferably non-small cell lung cancer resistant to docetaxel.

[0052] In particular, the drug is at least one of the following: (1) a drug for inhibiting the proliferation of non-small cell lung cancer cells; (2) a drug for inhibiting the migration of non-small cell lung cancer cells; (3) a drug for inhibiting the invasion of non-small cell lung cancer cells; (4) a drug for inducing apoptosis of non-small cell lung cancer cells; (5) a drug for inhibiting the Warburg effect in non-small cell lung cancer.

[0053] Compared with the prior art, the present invention has the following significant advantages:

[0054] The present invention has achieved an important breakthrough in the preparation process. The calcium acetate gradient method is used to prepare TPGS-modified CPD12C15 liposomes. Compared with the thin film dispersion method commonly used in the prior art, a high encapsulation efficiency of 79.84% and a drug loading capacity of 10.94% are achieved, far exceeding the efficiency of traditional methods (usually <60%). In addition, the process is carried out under mild conditions (30°C), avoiding the risk of thermal degradation of the drug. At the same time, through the "internal base and external acid" transmembrane gradient formation mechanism, efficient capture of the weakly acidic drug CPD12C15 is achieved. The physicochemical properties of the liposomes of the present invention are also significantly better than those of the prior art. The uniform nanostructure with an average particle size of 91.23±0.84nm and a PDI of 0.173±0.004 is more conducive to passive targeted enrichment at the tumor site through the EPR effect. Long-term storage stability tests show that the particle size increases by only 10.1% after storage at 4°C for 14 days, which is far better than existing single phospholipid liposomes (usually >30%), providing reliable guarantees for clinical application.

[0055] The present invention shows unique advantages in drug delivery and exhibits significant pH-responsive release characteristics. The cumulative release rate in a simulated tumor microenvironment (pH 6.8) reached 97.60% in 6 hours, while the release rate in 12 hours at physiological pH (7.4) was 86.58%. This significant pH response difference (nearly 40%) is far superior to the existing technology (usually <20%). The biphasic release behavior (a combination of an initial moderate burst release and subsequent sustained release) is superior to the single release mode of the existing technology and better meets the clinical efficacy requirements. In terms of cellular uptake and targeting ability, experiments have shown that the TPGS modified liposomes of the present invention have a cellular uptake rate approximately 3 times higher than that of free drugs, and have clear mitochondrial targeting ability (Pearson correlation coefficient>0.5, Mander overlap coefficient>0.6). This dual mechanism of EPR passive targeting and TPGS active mitochondrial targeting is significantly superior to the single targeting strategy of existing delivery systems and can more effectively deliver drugs to the energy metabolism center of tumor cells.

[0056] In terms of anti-tumor mechanism, the liposomes of the present invention exhibit multi-dimensional synergistic effects. Experiments have shown that the system significantly reduces the expression of PDK1, LDHA and HIF-1α in tumor cells, effectively inhibits the Warburg effect, and blocks the energy metabolism of tumor cells; at the same time, TPGS, as a P-gp inhibitor, forms a synergistic effect with CPD12C15, significantly enhancing the inhibition of drug-resistant related protein P-gp, while docetaxel further promotes P-gp expression and exacerbates drug resistance. Flow cytometry and TUNEL apoptosis experiments showed that the pro-apoptotic ability of the liposomes of the present invention (50.48% and 338.15%) was significantly higher than that of free CPD12C15 (24.87%) and docetaxel (48.23% and 184.59%). In the NCI-H460 subcutaneous tumor model, the tumor volume (271.13mm) after treatment with the liposomes of the present invention was significantly higher than that of free CPD12C15 (24.87%) and docetaxel (48.23% and 184.59%). 3 ) and weight (311.23 mg) were significantly lower in the docetaxel group (502.94 mm 3 , 562.03mg), the tumor inhibition effect increased by approximately 45%. This comprehensive mechanism of action surpasses existing single-mode anti-tumor drugs, acting simultaneously on tumor cells themselves and the tumor microenvironment, blocking multiple tumor growth and drug resistance mechanisms.

[0057] The present invention has excellent performance in terms of safety and embodies the design concept of reducing toxicity and increasing efficacy. During the administration period, the weight of animals in the liposome group of the present invention increased steadily, while the weight of the docetaxel group decreased; H&E staining proved that this system did not cause pathological changes in major organs such as the heart, liver, spleen, lungs, and kidneys. The most significant effect is the neuroprotective effect. Compared with docetaxel, the liposome group of the present invention can significantly reduce the MDA level in brain tissue (8.63 vs 17.51 nmol / mg prot), increase SOD activity (about 2 times that of the DTX group), downregulate GFAP expression, and upregulate TH expression. The TUNEL apoptosis experiment in brain tissue showed that the liposome of the present invention did not cause neuronal apoptosis (relative apoptosis rate of 77.86%), while the docetaxel group was as high as 622.16%, eliminating the neurotoxicity of traditional chemotherapy drugs. In vitro hemolysis experiments showed that the hemolysis rate at a concentration of 120 μg / mL was only 3.68%, far below the safety standard (5%). In terms of industrialization and clinical translational potential, the process employed in this invention is suitable for industrial scale-up production. The lipid materials used are all commercially available standards, avoiding complex synthesis steps, reducing production costs and quality control difficulties. Compared to docetaxel, which requires special solvents (such as polyoxyethylene castor oil), this system does not require allergenic excipients, significantly reducing the risk of allergic reactions during clinical use.

[0058] In summary, the TPGS-modified CPD12C15 liposomes of the present invention exhibit significant advantages over the prior art in terms of preparation process, physicochemical properties, drug delivery efficiency, cellular uptake and targeting ability, anti-tumor mechanism, in vivo effect, safety, and industrial transformation potential. Through the synergistic effect of multiple mechanisms and significant safety improvements, they provide an innovative treatment option for patients with non-small cell lung cancer, especially chemotherapy-resistant lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] The accompanying drawings, which constitute part of the present invention, are provided to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are provided to explain the present invention and do not constitute undue limitations thereon. The embodiments of the present invention will be described in detail below with reference to the accompanying drawings, wherein:

[0060] Figure 1 : Screening of CPD12C15-TLP preparation methods (n=3).

[0061] Figure 2 : CPD12C15-TLP optimization investigation results (n=3).

[0062] Figure 3 :CPD12C15-TLP morphology, including solution visualization and TEM morphology observation (scale is 0.6μm).

[0063] Figure 4 : Particle size distribution and potential diagram of CPD12C15-TLP.

[0064] Figure 5 : In vitro release curves of free CPD12C15 and CPD12C15-TLP in PBS solutions with pH of 6.8 (a) and 7.4 (b) and comparison (c) (n=3).

[0065] Figure 6 : Stability test results (n=3), including (a) encapsulation efficiency change, (b) drug loading change, (c) particle size change, and (d) PDI change.

[0066] Figure 7 : Cell uptake experimental results (n=3), including: (a) fluorescence analysis of NCI-H460 cell uptake (scale bar is 200 μm), (b) fluorescence analysis of NCI-H1299 cell uptake (scale bar is 200 μm), (c) quantitative analysis of NCI-H460 cell uptake by flow cytometry, and (d) quantitative analysis of NCI-H1299 cell uptake by flow cytometry.

[0067] Figure 8: Results of mitochondrial targeting experiments (n=3); including: (a) mitochondrial targeting in NCI-H460 cells (scale bar is 20 μm), (b) mitochondrial targeting in NCI-H1299 cells (scale bar is 20 μm), (c) colocalization of mitochondrial targeting in NCI-H460 cells, and (d) colocalization of mitochondrial targeting in NCI-H1299 cells.

[0068] Figure 9 : In vitro hemolysis experiment of CPD12C15-TLP (n=3).

[0069] Figure 10 :Effects of different concentrations of CPD12C15+Cu, CPD12C15-LP+Cu, CPD12C15-TLP+Cu and DTX on the proliferation of NCI-H460 and NCI-H1299 cells (n=3). Compared with the Control, ** p<0.01, *** p<0.001. (a) Effects of CPD12C15 and its preparations on the viability of NCI-H460 cells; (b) Effects of DTX on the viability of NCI-H460 cells; (c) Effects of CPD12C15 and its preparations on the viability of NCI-H1299 cells; (d) Effects of DTX on the viability of NCI-H1299 cells.

[0070] Figure 11 : Results of inhibition of migration of NCI-H460 and NCI-H1299 cells (n=3). * p<0.05, ** p<0.01, *** p<0.001, ### p<0.001. (a) Images of NCI-H460 cell migration (bar, 500 μm) and changes in the wound healing rate of NCI-H460 cells after treatment with CPD12C15, its preparations, and DTX. (b) Images of NCI-H1299 cell migration (bar, 500 μm) and changes in the wound healing rate of NCI-H1299 cells after treatment with CPD12C15, its preparations, and DTX.

[0071] Figure 12 : Results of inhibiting the invasion of NCI-H460 and NCI-H1299 cells (n=3). ** p<0.001, # p<0.05, ###p<0.001. (a) Invasion images of NCI-H460 cells (scale bar: 100 μm) and changes in the relative invasion rate of NCI-H460 cells after treatment with CPD12C15, its preparations, and DTX; (b) Invasion images of NCI-H1299 cells (scale bar: 100 μm) and changes in the relative invasion rate of NCI-H1299 cells after treatment with CPD12C15, its preparations, and DTX.

[0072] Figure 13 : Results of inducing apoptosis in NCI-H460 and NCI-H1299 cells (n=3). ** p<0.001, ### p<0.001. (a) Apoptosis images and apoptosis rate changes of NCI-H460 cells after treatment with CPD12C15, its preparations, and DTX; (b) Apoptosis images and apoptosis rate changes of NCI-H1299 cells after treatment with CPD12C15, its preparations, and DTX.

[0073] Figure 14 : Effects of CPD12C15 and its preparations combined with copper on glucose (a) and lactate levels (c) in NCI-H460 cells and glucose (b) and lactate (d) levels in NCI-H1299 cells (n=3). * p<0.05, ** p<0.01, *** p<0.001.

[0074] Figure 15 : qRT-PCR experiments were used to detect the effects of drug administration on the mRNA expression of related factors PDK1, HIF-1α, and caspase-3 in NCI-H460 cells and NCI-H1299 cells (n=3). * p<0.05, ** p<0.01, *** p<0.001.

[0075] Figure 16 :Western Blot assay was used to detect the effects of drug administration on the protein expressions of PDK1, LDHA, HIF-1α, and caspase-3 in NCI-H460 cells (n=3). * p<0.05, ** p<0.01, *** p<0.001.

[0076] Figure 17:Western Blot assay was used to detect the effects of drug administration on the protein expressions of PDK1, LDHA, HIF-1α, and caspase-3 in NCI-H1299 cells (n=3). * p<0.05, ** p<0.01, *** p<0.001.

[0077] Figure 18 : qRT-PCR assay was used to detect the expression of P-gp mRNA in NCI-H460 (left) and NCI-H1299 (right) cells after administration (relative gene expression) (n=3). * p<0.05, ** p<0.01, *** p<0.001.

[0078] Figure 19 :Western Blot assay was used to detect the protein expression of P-gp in NCI-H460 (a) and NCI-H1299 (b) cells after administration (n=3). * p<0.01, *** p<0.001.

[0079] Figure 20 : Uptake experiment of RH123 cells in NCI-H460 (a) and NCI-H1299 (b) after drug administration, including qualitative and flow cytometry analysis (n=3). ** p<0.001.

[0080] Figure 21 : Effects of CPD12C15+Cu, CPD12C15-LP+Cu, CPD12C15-TLP+Cu and DTX on tumors in nude mice, including: (a) images of tumors in nude mice after administration of different drugs; (b) changes in tumor volume; (c) tumor volume in nude mice; (d) tumor weight in nude mice; (e) H&E staining of tumors after administration of different drugs (scale bar is 100 μm).

[0081] Figure 22 : In vivo toxicity study results, including body weight changes of nude mice after administration of different drugs and H&E staining of major tissues after administration of different drugs (scale bar is 100 μm).

[0082] Figure 23 : TUNEL apoptosis results of tumors (n=3), including TUNEL apoptosis images of tumor masses (scale bar is 100 μm) and TUNEL apoptosis quantitative results. * p<0.05, *** p<0.001,# p<0.05, ### p<0.001.

[0083] Figure 24 : Immunohistochemical images of PDK1, HIF-1α and caspase-3 in tumors ((bar = 100 μm) and quantitative results (n = 3). * p<0.05, *** p<0.001.

[0084] Figure 25 : Immunohistochemical images of P-gp in tumors (scale bar = 100 μm) and quantitative results (n = 3). * p<0.05, *** p<0.001.

[0085] Figure 26 : Effects of drug administration on brain weight in nude mice.

[0086] Figure 27 : Pathological images of brain tissue (scale bar: 100 μm).

[0087] Figure 28 : Nissau staining results (scale bar: 100 μm).

[0088] Figure 29 : Effects of CPD12C15+Cu, CPD12C15-LP+Cu, CPD12C15-TLP+Cu and DTX on the levels of MDA (left) and SOD (right) in brain tissue (n=3). * p<0.05, *** p<0.001, ### p<0.001.

[0089] Figure 30 : Effects of CPD12C15+Cu, CPD12C15-LP+Cu, CPD12C15-TLP+Cu and DTX on the expression and quantification of GFAP in the hippocampus of brain tissue (a), and on the expression and quantification of TH in the hippocampus of brain tissue (n=3). # p<0.05, ### p<0.001.

[0090] Figure 31 : TUNEL apoptosis images of brain tissue (scale bar is 100 μm) and quantitative results (n=3). ### p<0.001. DETAILED DESCRIPTION

[0091] The present invention will be further described with reference to specific examples. It should be understood that these examples are intended to illustrate the present invention 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.

[0092] Unless otherwise defined, all technical and scientific terms used herein shall have the meanings familiar to those skilled in the art. Unless otherwise specified, the reagents or raw materials used in the present invention can be obtained through conventional routes and used in accordance with conventional methods in the art or product specifications. In addition, any content similar or equivalent to the methods or materials described herein can be applied to the methods of the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0093] CPD12C15 was prepared according to the method disclosed in Chinese patent CN116444408A, with a purity of >95%.

[0094] Example 1 Preparation of TPGS-modified CPD12C15 liposomes (CPD12C15-TLP)

[0095] 1. Preparation method and process optimization

[0096] This example provides a series of preparation examples, systematically examining the key process parameters affecting the preparation of CPD12C15-TLP, including the preparation method, DPPC to SPC molar ratio, phospholipid cholesterol molar ratio, TPGS content, calcium acetate concentration, drug-lipid molar ratio, phospholipid concentration, incubation temperature and incubation time, etc., to screen out the optimal preparation process.

[0097] Thin Film Dispersion Method (TFV): Precisely weigh a specific molar ratio of SPC (soy lecithin, Shanghai MacLean Biochemical Technology), DPPC (dipalmitoylphosphatidylcholine, Anhui Zesheng Technology), Chol (cholesterol, Shanghai MacLean Biochemical Technology), TPGS (vitamin E polyethylene glycol succinate, Shanghai Aladdin Biochemical Technology), and CPD12C15 into a 50 mL round-bottom flask. Add chloroform and sonicate to ensure dissolution. Connect the flask to a rotary evaporator via a splash guard, maintain the water bath at 40°C, and perform rotary evaporation under reduced pressure. Once there is no liquid in the flask and a uniform, transparent film has formed on the inner wall, immediately add ultrapure water for hydration. Once the phospholipid film has dissolved, a transparent solution with a light blue opalescence is formed. The solution is sonicated in an ice bath using an ultrasonic disruptor. After filtration through a water-based filter membrane, the solution is sealed in a glass bottle and stored at 4°C.

[0098] Calcium acetate gradient method (CAG): Precisely weighed molar ratios of SPC (soy lecithin, Shanghai MacLean Biochemical Technology), DPPC (dipalmitoylphosphatidylcholine, Anhui Zesheng Technology), Chol (cholesterol, Shanghai MacLean Biochemical Technology), and TPGS (vitamin E polyethylene glycol succinate, Shanghai Aladdin Biochemical Technology) were placed in a 50 mL round-bottom flask. Chloroform was added and sonicated to ensure dissolution. The water bath temperature was set to 40°C and rotary evaporation was performed under reduced pressure. Once no liquid remained in the flask and a uniform, transparent film formed on the inner wall, calcium acetate solution (120 mmol / L) was immediately added to hydrate the solution, forming a transparent solution with a light blue opalescence. Ultrasonication was then performed in an ice bath (50 W, 2 seconds on, 2 seconds off, for a total of 2 minutes). After filtration through a water-based filter membrane, the solution was transferred to a dialysis bag using sodium sulfate solution (120 mmol / L) as the dialysis medium and dialyzed on a magnetic stirrer for 12 h. After dialysis, the solution in the dialysis bag was transferred to a glass vial, a certain amount of CPD12C15 powder was weighed into it, and incubated in a water bath for 1 hour to obtain TPGS-modified CPD12C15 liposome (CPD12C15-TLP) solution.

[0099] CPD12C15-TLP was prepared according to the aforementioned preparation method, see Preparation Examples 1-24, and the preparation parameters are shown in Table 1.

[0100] Table 1 Preparation Examples 1-32

[0101]

[0102] 2. Test methods

[0103] The CPD12C15 content was determined by high performance liquid chromatography under the following conditions: chromatographic column: Phenomenex Luna 5u C18 column (5 μm, 250×4.6 mm); mobile phase: acetonitrile-0.2% formic acid water (60:40, V / V); flow rate: 1 mL / min; column temperature: 30°C; injection volume: 10 μL; detection wavelength: 284 nm.

[0104] Preparation of stock solution: Accurately weigh 50 mg of CPD12C15 drug into a 50 mL volumetric flask, add acetonitrile to dissolve, and dilute to the scale with acetonitrile. After thorough mixing, a 1 mg / mL CPD12C15 stock solution is obtained. Store at 4°C until use.

[0105] Standard curve and linear relationship: Pipette 3.2mL of CPD12C15 mother solution into a 10mL volumetric flask, add acetonitrile to make up the volume, shake thoroughly, pipette 5mL and dilute with the same volume of acetonitrile, mix well, and perform serial dilution to obtain CPD12C15 working solutions with final concentrations of 320, 160, 80, 40, 20, and 10μg / mL. All working solutions were filtered through a 0.22μm organic filter membrane and then tested by HPLC. The established methodology was used for analysis and the peak areas were recorded. With the CPD12C15 concentration as the horizontal axis and the corresponding average value of the chromatographic peak area as the vertical axis, a CPD12C15 standard curve was drawn and its linear relationship was analyzed. The experiment was repeated three times. With the CPD12C15 concentration as the horizontal axis and the average value of the peak area as the vertical axis, the linear equation was y=68.90x-410.2, R 2 =0.9991.

[0106] Determination of encapsulation efficiency and drug loading: Separate liposome solution and free drug by ultrafiltration centrifugation. Take an appropriate amount of liposome solution, dilute it 20 times with acetonitrile, perform ultrasonic demulsification for 15 minutes, filter it through a filter membrane, and inject it into HPLC analysis. Measure and record the peak area, substitute it into the standard curve to convert it into concentration, and multiply it by the dilution factor N (20) to obtain c total . Take an appropriate amount of liposome solution in an ultrafiltration tube, use ultrafiltration tube centrifugation method, centrifuge at 5000r / min for 15min, the free drug passes through the inner tube into the outer tube of the collection tube, while the liposomes remain in the inner tube due to the particle size. Carefully aspirate the free drug liquid in the outer tube, dilute it 20 times with acetonitrile, filter it through the membrane, inject it into HPLC analysis, measure and record the peak area, substitute it into the standard curve to convert the CPD12C15 content, and multiply it by the dilution factor N (20) to get c free . The encapsulation efficiency (EE) and drug loading (DL) were calculated according to the formula.

[0107]

[0108] 3. Experimental results

[0109] 3.1 Screening of CPD12C15-TLP Preparation Methods (See Preparation Examples 1-2)

[0110] Figure 1 The results of two preparation methods for CPD12C15-TLP are shown. The results show that the calcium acetate gradient method produces liposomes with higher encapsulation efficiency and drug loading than the thin film dispersion method, indicating that the calcium acetate gradient method is more suitable for the preparation of CPD12C15-TLP. Therefore, the calcium acetate gradient method was selected for subsequent experiments.

[0111] 3.2 CPD12C15-TLP preparation process optimization results

[0112] The DPPC to SPC molar ratio (Preparation Examples 3-6), phospholipid cholesterol molar ratio (Preparation Examples 3, 7-10), TPGS content (Preparation Examples 11-14), phospholipid concentration (Preparation Examples 8, 15-17), drug-lipid molar ratio (Preparation Examples 15, 18-21), and incubation temperature (Preparation Example 18) were screened by single-factor experiments. The results are as follows: Figure 2 shown. Figure 2 (a) shows that as the molar ratio of DPPC to SPC increases, the encapsulation efficiency and drug loading first decrease and then increase, reaching the maximum when the ratio is 1:5. Figure 2 Middle (b) shows that as the molar ratio of phospholipid cholesterol increases, the encapsulation first increases and then decreases, and the general trend of drug loading is the same, reaching the maximum when the molar ratio is 2:1. Figure 2 (c) shows that as the TPGS content increases, the encapsulation efficiency increases, the drug loading amount changes little, and the particle size decreases. Considering the particle size of the liposome, the TPGS content is selected as 10 mol%. Figure 2 Middle (d) shows that the encapsulation efficiency increases with the increase of phospholipid concentration, and the drug loading capacity first increases and then decreases. Considering that the solution will become viscous if the phospholipid concentration is too high, which is not conducive to in vivo administration, 20 mg / mL was selected. Figure 2 Middle (e) shows that as the drug-lipid molar ratio decreases, the encapsulation efficiency increases and the drug loading capacity decreases. Considering the dosage in animal experiments, the drug-lipid molar ratio is selected as 1:2. Figure 2 Middle (f) shows that as the incubation temperature increases, the encapsulation efficiency and drug loading capacity decrease slightly, and the effect is better at 30℃.

[0113] 3.3 Process verification experiment

[0114] According to the results of the single-factor experiment, a DPPC to SPC molar ratio of 1:5, a phospholipid cholesterol molar ratio of 2:1, a TPGS content of 10 mol%, a phospholipid concentration of 20 mg / mL, a drug-lipid molar ratio of 1:2, and an incubation temperature of 30°C were selected to prepare TPGS-modified CPD12C15 liposomes (CPD12C15-TLP). Three batches were repeated. As shown in Table 2, the average encapsulation efficiency and drug loading of CPD12C15-TLP prepared by this process were 79.84% and 10.94%, respectively.

[0115] Table 2 Verification of the optimal preparation process (n=3)

[0116]

[0117] Example 2 Characterization of CPD12C15-TLP

[0118] Encapsulation efficiency and drug loading are the two most basic characterization indicators of liposomes. Other characterizations can be used to evaluate the quality and stability of liposomes, thereby further expanding their application range. The characterization experiments in this example include: (1) TEM: to observe the morphology of liposomes and determine whether the liposome preparation is successful; (2) particle size and PDI measurement: related to the absorption and utilization of liposomes; (3) Zeta potential measurement: related to the stability of liposomes.

[0119] 1. Experimental Methods

[0120] 1. Morphological Observation of CPD12C15-TLP: TEM is commonly used to visualize liposome geometry, size, and other aspects. This experiment used TEM to observe the morphology of CPD12C15-TLP, using the following steps: 1) Preparation: Place a copper mesh on filter paper and drop a predetermined amount of CPD12C15-TLP solution onto the mesh; 2) Drying: Dry the sample solution using an infrared lamp; 3) Observation: Select an appropriate size and observe and record the sample using TEM.

[0121] 2. Determination of particle size, PDI, and Zeta potential of CPD12C15-TLP: PDI is used to characterize the uniformity of particle distribution. The smaller the PDI, the more uniform the particle distribution and the better the dispersibility of the solution. Generally speaking, a liposome PDI less than 0.20 is considered excellent. Appropriately dilute the CPD12C15-TLP sample with ultrapure water and transfer it to the particle size sample cell. Place it in the detection chamber of the nanoparticle size potentiometer. Measure and record the particle size and PDI, and export the relevant images. Zeta potential is used to characterize the surface charge and stability of particles. The higher the absolute value of the zeta potential, the more stable the particle solution. Appropriately dilute the CPD12C15-TLP solution with ultrapure water, draw it up with a syringe, and slowly inject it into the zeta potential cell. Place it in the detection chamber, measure and record the zeta potential, and export the relevant images.

[0122] 3. In vitro release experiment

[0123] Accurately measure the CPD12C15 stock solution and dilute it with PBS solutions at pH 6.8 and 7.4 to a series of solutions with concentrations of 160, 80, 40, 20, 10, and 5 μg / mL, respectively. Filter and analyze by HPLC. Analyze using established methodologies and record peak areas. Plot a CPD12C15 standard curve using CPD12C15 concentration as the abscissa and the corresponding average chromatographic peak area as the ordinate, and analyze its linearity.

[0124] In vitro release behavior was determined using the dialysis bag diffusion method. 4.0 mg of CPD12C15 powder and a certain amount of CPD12C15-TLP (equivalent to 4.0 mg of CPD12C15) were precisely weighed and placed in a dialysis bag (molecular weight cutoff, 3500 Da). A certain amount of PBS solution was added to equalize the two volumes. The dialysis bag was secured tightly and placed in 30 mL of PBS solution (pH 7.4). The bag was then shaken in a constant-temperature oscillator at 37°C and 100 rpm in an air bath. At each time point, 500 μL of solution was removed from the dialysis medium and the corresponding volume of release medium was replenished. The solutions were filtered and analyzed by HPLC. The peak areas were recorded and the CPD12C15 content was calculated. The cumulative release rate (CDR) at different time points was calculated according to Equation (2-4). Cumulative release curves of CPD12C15 and its formulation were plotted. Release experiments were conducted using the same method in PBS solution (pH 6.8). According to the above method, the release behavior in PBS solutions with pH of 6.8 and 7.4 was tested respectively.

[0125]

[0126] Where V0 is the total volume of release medium (mL), V i is the sampling volume (mL), c i and c n is the CPD12C15 concentration (μg / mL) measured when sampling at the i-th and n-th time points, and m is the mass of CPD12C15 in the input sample.

[0127] 4. Stability Test: The prepared CPD12C15-TLP is a composite phospholipid liposome (CPL) whose phospholipid components are SPC and DPPC. Therefore, a stability test was conducted to compare the stability of single phospholipid liposomes (SPL) with that of CPL. TPGS-modified CPD12C15 liposomes (TPGS-CPL) and single phospholipid liposomes (TPGS-SPL) with the same proportion but without DPPC were prepared according to the optimal formulation obtained in Example 1 and stored at 4°C. A certain amount of liposome solution was diluted and observed at 1, 2, 4, 7, and 14 days. The encapsulation efficiency and drug loading were measured and calculated according to the method in Example 1. The particle size and PDI of the two liposomes were also measured using the methods described above in this example to observe the stability of the two liposomes over time.

[0128] 5. Cell uptake experiment: Since CPD12C15 has no fluorescent properties, the hydrophobic green fluorescent dye Coumarin-6 (C6) was selected instead of CPD12C15 as a fluorescent probe to investigate the uptake of different preparations by NCI-H460 and NCI-H1299 cells.

[0129] 5.1 Cell culture

[0130] Cell Thawing: Remove frozen NCI-H460 and NCI-H1299 cells from a -80°C freezer and quickly transfer to a 37°C water bath for warming. Once thawed, transfer the cells to an EP tube and add 2 mL of RPMI-1640 complete medium (containing 10% fetal bovine serum). Centrifuge at 1000 rpm for 3 minutes. Discard the supernatant and resuspend the cells in 1 mL of complete medium. Transfer the cells to a culture dish, add a sufficient amount of complete medium, shake well, and incubate in an incubator (5% CO2, 37°C).

[0131] Cell passaging: Observe cell morphology under an inverted microscope. Cells can be passaged when the cell density reaches approximately 90%. Discard the culture medium in a clean bench, wash twice with sterile PBS solution, and then add 1 mL of trypsin for digestion. When the cells become brighter and rounder, add 2 mL of complete culture medium to terminate digestion. Discard the cells and transfer them to an EP tube. Centrifuge at 1000 rpm for 3 minutes. Discard the supernatant and resuspend in complete culture medium. Based on the passage density, transfer a certain volume of cell suspension to a culture dish. Add a certain amount of complete culture medium, shake well, and return to the incubator for continued culture.

[0132] Cell cryopreservation: Cryopreserve cells when they are in the logarithmic growth phase and in good condition. Discard the culture medium, wash the cells twice with sterile PBS, digest, centrifuge, and discard the supernatant. Resuspend the cells in 1 mL of freshly prepared freezing buffer (RPMI-1640 medium: FBS: DMSO = 5:4:1) and transfer to cryovials. Place the cryovials in a cryovial box and freeze overnight at -80°C. Transfer to liquid nitrogen for long-term storage.

[0133] Cell counting: Digest the cells, centrifuge to remove the supernatant, and resuspend in 3 mL of complete culture medium. After wiping the coverslip and hemocytometer clean with 75% alcohol, pipette 10 μL of cell suspension onto the counting plate and observe under a microscope. Following the principle of "count the top, not the bottom; count the left, not the right," record the number of cells N in the four large squares. Use the following formula to calculate the number of cells per milliliter of cell suspension. Calculate the required volume of cell suspension according to the experimental requirements and conduct subsequent experiments. The formula is:

[0134] 5.2 Preparation of a C6 fluorescence standard curve: Weigh a certain amount of C6 powder and dissolve it in dimethyl sulfoxide (DMSO) to prepare a 1 mg / mL stock solution. Then, take 16 μL of the stock solution into an EP tube and add 984 μL of acetonitrile to dilute to 16 μg / mL. By serial dilution, prepare a series of C6 solutions at 16, 8, 4, 2, 1, and 0.5 μg / mL. Set the excitation wavelength to 450.00 nm and the slit to 2.5 nm. Scan within the emission wavelength range of 469.00 nm to 600.00 nm, recording the fluorescence intensity at the highest point to create a standard curve.

[0135] 6. Preparation: Prepare blank liposomes according to the preparation method under Item 4 of this Example. Weigh a certain amount of C6 powder into the liposome solution. Heat and stir at 30°C for 1 hour to obtain TPGS-C6-LP and C6-LP. Store in the dark. Dissolve a certain amount of C6 in DMSO to prepare a C6-free solution. Dilute all three solutions 20-fold and measure the fluorescence intensity under the conditions under Item 5.2 of this Example to obtain the precise C6 concentration. Then, dilute to 50 ng / mL using complete culture medium. Store in the dark.

[0136] 7. Qualitative cell uptake experiment: NCI-H460 and NCI-H1299 cells were cultured at 1×10 5 Cells were seeded at a density of 100 μL / well in a 24-well plate. After 24 hours of culture, the culture medium was discarded. Three solutions (C6, C6-LP, and TPGS-C6-LP) were added at a concentration of 50 ng / mL, respectively, incubated in the dark for 0.5 and 4 hours. The culture medium was then discarded, the cells were washed three times with PBS, fixed with 4% paraformaldehyde for 10 minutes, washed with PBS, and stained with 200 μL of DAPI blue fluorescent dye for 10 minutes. The cells were washed three times with PBS, air-dried, and observed for qualitative uptake under an inverted fluorescence microscope. The exposure time was kept constant.

[0137] 8. Cell uptake quantitative experiment: Quantitative analysis was performed using flow cytometry. NCI-H460 and NCI-H1299 cells were grown at 1×10 6 The cells were seeded at a density of 1000 cells / well in a 6-well plate and cultured for 24 hours. The culture medium was discarded, and three solutions (C6, C6-LP, TPGS-C6-LP) with a concentration of 30 ng / mL were added respectively under light-proof conditions. After incubation in the incubator for 0.5 hours and 4 hours, the culture medium was aspirated, the cells were washed with PBS three times, digested with trypsin, centrifuged, resuspended in PBS, and transferred to a flow tube. The quantitative cell uptake was detected by flow cytometry.

[0138] 9. Mitochondrial targeting experiments

[0139] 9.1 Preparation: Prepare C6, C6-LP, and TPGS-C6-LP solutions according to the method in Section 6 of this Example. Dilute to 30 ng / mL in complete culture medium. Protect from light. Dilute the Mito-Tracker Red CMXRos probe to 100 nmol / L in culture medium according to the instructions and set aside.

[0140] 9.2 Confocal Observation of Mitochondrial Targeting: NCI-H460 and NCI-H1299 cells were respectively 5 The cells were seeded at a density of 100 μL / dish in confocal microplates and cultured for 24 h. The culture medium was discarded, and three solutions were added separately under light-proof conditions. After incubation for 4 h, the solution was discarded, and the cells were washed three times with PBS. 100 nmol / L probe was added to stain mitochondria for 15 min, the solution was discarded, and the cells were washed three times with PBS. The cells were fixed with 4% paraformaldehyde for 10 min, washed with PBS, and 200 μL DAPI dye was added to stain the cell nucleus for 10 min. After washing three times with PBS, the mitochondrial targeting of the preparation was observed under confocal high-resolution live cell imaging.

[0141] 10. In vitro hemolysis test

[0142] 10.1 Preparation of 2% Red Blood Cell Suspension: Collect 1-2 mL of whole blood from a SD rat and add it to a 1.5 mL centrifuge tube soaked in sodium heparin. Centrifuge in a refrigerated ultracentrifuge at 3000 rpm for 5 minutes at 4°C. Retain the red blood cells in the lower layer and transfer them to a 15 mL centrifuge tube. Add physiological saline and mix thoroughly. Centrifuge at 3000 rpm for 5 minutes. Repeat this process three times until the upper layer of blood is colorless and transparent. Then, dilute the washed red blood cells 50-fold with physiological saline to prepare a 2% red blood cell suspension.

[0143] 10.2 Preparation of Test Drug Solutions: Prepare CPD12C15-TLP solutions according to the optimized formulation in Example 1. Dilute with physiological saline to achieve CPD12C15 concentrations of 120, 60, 30, 15, 7.5, and 3.25 μg / mL, respectively. Pipette 500 μL of the erythrocyte suspension into a 5 mL centrifuge tube. Separately, pipette 500 μL of liposome solutions of varying concentrations and mix with an equal volume of the erythrocyte suspension for reaction. Seven groups of solutions were prepared using physiological saline as a negative control and deionized water as a positive control.

[0144] 10.3 Hemolysis Reaction and Calculation of Hemolysis Rate: The mixed solution was placed in a constant-temperature oscillator at 37°C and 100 rpm for 2 h. After the reaction, centrifuge at 3000 rpm for 5 min. 200 μL of the supernatant was transferred to a 96-well culture plate. The absorbance (optical density, OD) was measured at 545 nm using a microplate reader. The hemolysis rate was calculated according to the formula. Repeat three times for each sample.

[0145] 2. Experimental Results

[0146] 1. Morphological observation of CPD12C15-TLP: Figure 3 As shown, the CPD12C15-TLP solution is clear and transparent with light blue opalescence.

[0147] 2. Particle size, PDI and Zeta potential of CPD12C15-TLP: The particle size, PDI and Zeta potential of the prepared CPD12C15-TLP were measured. The results are shown in Table 3. Figure 4 As shown, the particle size of CPD12C15-TLP is 91.23±0.84 nm, the PDI is 0.173±0.004, and the Zeta potential is -6.17±1.00 mV.

[0148] Table 3 Optimal preparation process verification (n=3)

[0149]

[0150] 3. In vitro release experiment: The results of in vitro release experiment are as follows Figure 5 As shown. The release medium with a pH of 7.4 simulated the normal tissue environment, while the release medium with a pH of 6.8 simulated the tumor microenvironment. The experimental results showed that in the medium with a pH of 6.8, the CPD12C15 group released completely in 4 hours, with a cumulative release percentage of (87.19±2.34)%, while the CPD12C15-TLP group released completely in 6 hours, with a release rate of (97.60±1.99)%; in the medium with a pH of 7.4, the CPD12C15 group released completely in 6 hours, with a cumulative release percentage of (79.54±0.86)%, while the CPD12C15-TLP group released completely in 12 hours, with a release rate of (86.58±1.60)%. Figure 5 (a) and (b) show that in both media, the release rate of free CPD12C15 is much faster than that of CPD12C15-TLP, and the release degree of CPD12C15-TLP is more complete, indicating that liposomes can achieve a certain sustained release effect. Figure 5 (c) It can be seen that the release rate and extent of CPD12C15-TLP in a medium with a pH of 6.8 are greater than those in a medium with a pH of 7.4. The reason may be that the acidic environment is conducive to the hydrolysis of phospholipids in the liposomes, resulting in faster and more drug release.

[0151] 4. Stability test: The stability results are shown in Table 4 Figure 6 TPGS-SPL and TPGS-CPL were placed at 4℃ for 14 days. Figure 6(a) and (b) show that the encapsulation efficiency and drug loading of both TPGS-SPL and TPGS-CPL are slightly reduced, but overall the encapsulation efficiency and drug loading of TPGS-CPL are higher than those of TPGS-SPL. Figure 6 As shown in Figures (c) and (d), the particle size of TPGS-CPL remained relatively stable over 14 days, while that of TPGS-SPL gradually increased. Significant precipitation was observed at 14 days, and the PDI of TPGS-SPL later exceeded 0.2, indicating relatively poor particle uniformity. This indicates that compared with single phospholipid liposomes, composite phospholipid liposomes improve encapsulation efficiency and drug loading, and possess greater stability.

[0152] Table 4 Sexual experiment results

[0153]

[0154] 5. Cell uptake experiment: Use hydrophobic fluorescent dye C6 instead of CPD12C15 to prepare liposomes to study the uptake of the preparation by NCI-H460 and NCI-H1299 cells. Figure 7 As shown in (a), the fluorescence intensity of the drugs entering NCI-H460 cells increases from slowest to fastest, and becomes stronger with time, indicating that more and more drugs enter the cells. Flow cytometry was used for quantitative analysis. Figure 7 Option (b) can also be verified. Figure 7 (c) and (d) show similar results in NCI-H1299 cells, indicating that liposome preparation promoted drug uptake by cells, possibly because liposomes facilitated drug entry into cells, and the TPGS modified on the liposome surface facilitated liposome entry into cells.

[0155] 6. Mitochondrial targeting experiment: the results are as follows Figure 8 As shown. After 4 hours of incubation, liposomes containing C6 entered lung cancer cells. The green fluorescent liposomes overlapped with the red fluorescent mitochondria, appearing yellow, indicating that the liposomes were aggregated around the mitochondria. Figure 8 As shown in (a), a portion of TPGS-C6-LP accumulated in the mitochondria of NCI-H460 cells, while C6-LP and C6-Free did not enter the mitochondria. Figure 8(b) shows the same conclusion in NCI-H1299 cells. Image J software was used to analyze the correlation and overlap between channels using the Pearson correlation coefficient (PCC) and Mander's overlap coefficient (MOC) to characterize colocalization. Generally, a PCC > 0.5 and a MOC > 0.6 indicate colocalization. Figure 8 (c) and (d) show that C6-Free and C6-LP did not enter the mitochondria, while TPGS-C6-LP co-localized with mitochondria, indicating that TPGS promoted the aggregation of liposomes in the mitochondria of NCI-H460 and NCI-H1299 cells.

[0156] 7. In vitro hemolysis test: Figure 9 As can be seen, after centrifugation, the solution in the positive control group appeared red, indicating complete hemolysis. However, the solutions in the negative control group and each test drug group remained colorless and transparent after centrifugation, with red blood cells largely sedimenting at the bottom of the centrifuge tube. Hemolysis rate data showed that when the CPD12C15 concentration in the CPD12C15-TLP solution reached 120 μg / mL, the hemolysis rate was 3.68%, less than 5%. This result is consistent with visual observation, indicating that CPD12C15-TLP has no hemolytic effect within the range of 3.25 to 120 μg / mL and has a reasonable in vitro safety profile.

[0157] Example 3 In vitro anti-lung cancer efficacy of CPD12C15-TLP combined with copper

[0158] 1. Experimental Methods

[0159] Cell lines: Human non-small cell lung cancer NCI-H460 and NCI-H1299 cell lines were provided by the School of Pharmacy of Shandong University.

[0160] Experimental solution preparation:

[0161] CPD12C15 solution

[0162] Prepare 2 mmol / L CPD12C15 stock solution: accurately weigh 1.36 mg of CPD12C15 raw material and dissolve it in 2 mL of DMSO. Sonicate to dissolve it and store at -20°C.

[0163] CPD12C15-LP and CPD12C15-TLP liposome solutions

[0164] Liposomes were prepared according to the liposome preparation method and optimal formulation and process described in Example 1. 50.03 mg of SPC, 15.31 mg of Chol, 9.69 mg of DPPC, and / or 17.99 mg of TPGS were accurately weighed into a 50 mL round-bottom flask. 5 mL of chloroform was added and thoroughly dissolved by ultrasonication. The mixture was rotary evaporated in a 40°C water bath. When no liquid flowed in the flask and a uniform film formed on the flask wall, 3 mL of 120 mmol / L calcium acetate solution was added for hydration. After the film was completely hydrated, ultrasonic fragmentation was performed for 3 minutes. The mixture was filtered through a 0.22 μm aqueous filter and transferred to a dialysis bag for 12 hours of dialysis against a 120 mmol / L sodium sulfate solution. The dialyzed solution was transferred to a glass vial, 13.46 mg of CPD12C15 powder was added, and the mixture was stirred in a 30°C water bath for 1 hour to obtain CPD12C15-TLP. Without TPGS, a CPD12C15-LP solution was obtained. A certain amount of ultrapure water was added to dilute the CPD12C15 to a concentration of 2 mmol / L. The cells were sterilized by filtering with a 0.22 μm filter membrane in a clean bench and stored at 4°C until use.

[0165] Complete RPMI-1640 medium: Add 45 mL of RPMI-1640 medium and 5 mL of FBS to a 50 mL sterile EP tube, invert to mix, and store at 4°C. Preheat before use.

[0166] Cell freezing solution: Prepare according to the ratio of RPMI-1640:FBS:DMSO = 5:4:1, and prepare it according to the dosage each time.

[0167] CuCl2 solution: Prepare 2 mmol / L CuCl2 solution: Accurately weigh 2.04 mg of CuCl2·2H2O powder and dissolve it in 6 mL of ultrapure water. Filter the solution through a 0.22 μm filter membrane in a laminar flow hood and sterilize. Aliquot the solution into 1.5 mL EP tubes and store at -20°C.

[0168] DTX solution: Prepare 50 mmol / L DTX stock solution: accurately weigh 80.79 mg of DTX powder and dissolve it in 2 mL of DMSO. Sonicate to dissolve it and store at -20°C.

[0169] MTT solution: Prepare 5 mg / mL MTT solution: accurately weigh 200 mg of MTT powder and dissolve it in 40 mL of sterile PBS. Filter the solution through a 0.22 μm filter membrane in a clean bench in the dark and sterilize it. Then, divide the solution into 10 mL EP tubes and wrap them in tin foil. Store at -20°C for long-term storage or at 4°C for short-term storage. Protect from light before use.

[0170] RH123 mother solution: Prepare 5 mmol / L RH123 mother solution: accurately weigh 3.81 mg of RH123 powder and dissolve it in 2 mL of DMSO. Dissolve it by ultrasonication, wrap it in tin foil, and store it at -20°C in the dark.

[0171] 1. Experimental methods

[0172] Cell culture: The operation method is the same as Example 2.

[0173] 1.1 MTT assay to detect cell viability

[0174] (1) Prepare a cell suspension of NCI-H460 or NCI-H1299 cells in the logarithmic growth phase and count the cells to obtain a cell suspension of 5×10 3 Cells were plated at a density of 100 μL / well in a 96-well plate. Each well contained 100 μL of cells. A blank control containing only complete culture medium and no cells was plated. The outermost circle of the 96-well plate was excluded from the experiment to avoid edge effects. Cultured in an incubator.

[0175] (2) After the cells adhered to the wall, different concentrations of drug solution were added and acted for 48 hours, with a final volume of 200 μL. The control group was set up with cells but no drug. Three replicates were set up for each concentration. Drugs were added according to the following experimental groups: ① CPD12C15+Cu group: CPD12C15 solution was mixed with CuCl2 solution of equal molar ratio, and diluted with serum-free medium to make the final concentration of CPD12C15 0.05, 0.1, 0.2, 0.4, 0.8, 1.6 μmol / L; ② CPD12C15-LP+Cu group: CPD12C15-LP solution was mixed with CuCl2 solution of equal molar ratio, and diluted with serum-free medium to make the final concentration of CPD12C15 0.05, 0.1, 0.2, 0.4, 0.8, 1.6 μmol / L; ③ CPD1 2C15-TLP+Cu group: CPD12C15-TLP solution was mixed with CuCl2 solution in an equimolar ratio and diluted with serum-free medium to make the final CPD12C15 concentrations of 0.05, 0.1, 0.2, 0.4, 0.8, and 1.6 μmol / L; ④DTX group: DTX was diluted with serum-free medium to make the final DTX concentrations of 3.125, 6.25, 12.5, 25, 50, and 100 μmol / L in NCI-H460 cells and 12.5, 25, 50, 100, 200, and 400 μmol / L in NCI-H1299 cells.

[0176] (3) After incubation for 48 h, add 20 μL of MTT solution to each well in the dark. After 4 h, pour out the liquid in the 96-well plate, add 150 μL of DMSO solution to each well, and place the culture plate on a shaker for 15 min to promote the dissolution of the purple crystals.

[0177] (4) The OD value at 490 nm was measured using a microplate reader, and the cell viability of each group was calculated according to the formula. The half inhibitory concentration (IC) of the drug was calculated using GraphPad Prism 8.0.2. 50 The experiment was repeated three times.

[0178]

[0179] 1.2 Cell migration assay

[0180] The migration ability of lung cancer cells was investigated by cell scratch test. NCI-H460 and NCI-H1299 cells were respectively 6 Cells were evenly plated at a density of 100 μg / well in a 6-well plate and cultured until the density reached 90%. A sterile 200 μL pipette tip was then used to draw a straight line vertically across the bottom of each well, creating a scratch. The culture medium was then discarded, and residual cell debris was washed with PBS. The scratch width at 0 h was recorded under a light microscope. After the images were taken, drugs were added in a clean hood according to the following groups: ① Control group; ② CPD12C15 + Cu group (0.20 μmol / L); ③ CPD12C15-LP + Cu group (0.20 μmol / L); ④ CPD12C15-TLP + Cu group (0.20 μmol / L); and ⑤ DTX group (NCI-H460 cells: 5.0 μmol / L, NCI-H1299 cells: 15.0 μmol / L). All drug solutions were diluted with serum-free culture medium, and the final volume for each group was 2 mL. After 48 hours of drug treatment, the culture medium was discarded and the cells were washed twice with PBS. Microscopic images were taken at the location taken at 0 hours, and the width of the wound at 48 hours was recorded. The scratch area (S) was analyzed using Image J software, and the wound healing rate was calculated using the formula. The experiment was repeated three times.

[0181]

[0182] 1.3 Cell invasion assay

[0183] The invasive ability of lung cancer cells was examined using a Matrigel-Transwell assay. The Matrigel used in the experiment simulates the extracellular matrix. Matrix metalloproteins secreted by invasive cells can degrade the extracellular matrix, allowing them to penetrate the basement membrane and reach the subcellular compartment. Therefore, the number of cells in the subcellular compartment can represent the strength of the cell's invasive ability.

[0184] First, dilute Matrigel 10-fold with serum-free medium, take 100 μL and add it to the bottom of each chamber. Then place the chamber into a 24-well plate and transfer it to the incubator to wait for the Matrigel to solidify. Starve the cells with serum-free medium for 12 hours in advance. After making a cell suspension, resuspend NCI-H460 and NCI-H1299 cells in serum-free medium and dilute them to 3×10 5 100 μL of a cell suspension of 100 μg / mL was inoculated into the chamber. The specific experimental groups were: Control group, CPD12C15+Cu group (0.20 μmol / L), CPD12C15-LP+Cu group (0.20 μmol / L), CPD12C15-TLP+Cu group (0.20 μmol / L), and DTX group (NCI-H460 cells: 5.0 μmol / L, NCI-H1299 cells: 15.0 μmol / L). The above drug solutions were all diluted with serum-free culture medium, and 100 μL of drug solution was added to the chamber to make the final concentration the specified concentration. At the same time, 800 μL of culture medium containing 10% serum was added to the 24-well plate under the chamber and cultured for another 48 hours. The culture medium was discarded, and the chamber was washed three times with PBS. The cells were fixed with 4% paraformaldehyde inside and outside the chamber for 30 minutes. After washing with PBS, crystal violet was added for staining for 30 minutes. The chamber was washed three times with PBS. The cells on the upper layer of the chamber were wiped off with a cotton swab. After absorbing the water on the surface of the chamber, the chamber was observed under a microscope and photographed. The number of cells N that invaded the lower layer of the chamber was recorded using Image J software, and the relative invasion rate was calculated according to the formula.

[0185] 1.4 Cell apoptosis assay

[0186] Lung cancer cell apoptosis was detected by Annexin V / PI double staining. NCI-H460 and NCI-H1299 cells were plated at 1×10 6Cells were seeded evenly at a density of 100 cells / well in a 6-well plate and cultured until the density reached 70%. Drugs were then added according to the experimental groups: control group, CPD12C15+Cu group (0.20 μmol / L), CPD12C15-LP+Cu group (0.20 μmol / L), CPD12C15-TLP+Cu group (0.20 μmol / L), and DTX group (NCI-H460 cells: 5.0 μmol / L, NCI-H1299 cells: 15.0 μmol / L). After 48 h of incubation, the culture medium in the wells was collected and placed in a 5 mL EP tube. The cells were digested with EDTA-free trypsin, collected, transferred to the EP tube, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. The cells were washed twice with PBS and resuspended in 100 μL of 1× Binding Buffer (diluted 10-fold with DEPC water). Before staining, remove a certain amount of cell suspension from each EP tube and divide it equally into three tubes: a blank tube (no dye added), single-stain tube 1 (with FITC dye alone), and single-stain tube 2 (with PI dye alone). These tubes will be used for gating and compensation. In the dark, add 5 μL of FITC dye to each tube and incubate for 10 minutes. Then, add 5 μL of PI dye and incubate for 5 minutes. Finally, add 400 μL of 1× Binding Buffer to each tube, mix thoroughly, and transfer to a flow cytometer. Cell apoptosis rates will be calculated using a flow cytometer.

[0187] 1.5qRT-PCR experiments

[0188] Cell culture and treatment: NCI-H460 and NCI-H1299 cells were cultured at 1 × 10 6 Cells were seeded evenly in 6-well plates at a density of 100 cells / well. Cultures were cultured until the density reached 70%. Drugs were then added according to the experimental groups: Control, CPD12C15+Cu (0.20 μmol / L), CPD12C15-LP+Cu (0.20 μmol / L), CPD12C15-TLP+Cu (0.20 μmol / L), and / or DTX (5.0 μmol / L for NCI-H460 cells and 15.0 μmol / L for NCI-H1299 cells). A total of 2 mL of drug solution was added to each well. The cells were then cultured in an incubator for 48 hours.

[0189] RNA extraction: Discard the culture medium in the wells, wash the cells twice with sterile PBS, add 1 mL of Trizol to each well, pipette the cells down and transfer them to a 1.5 mL EP tube. Let them stand on ice for 5 minutes, then add 200 μL of chloroform, shake up and down to mix, let them stand for 5 minutes, then transfer them to an ultraspeed tabletop refrigerated centrifuge and centrifuge at 4°C and 12,000 rcf for 15 minutes. The sample can be observed to separate into three layers (the upper colorless liquid is RNA, the middle white film is DNA, and the lower red liquid is protein). Carefully pipette 400 μL of the supernatant into a new 1.5 mL EP tube, add an equal amount of isopropanol (to precipitate RNA), mix by inversion and let it stand for 10 minutes, then transfer them to a centrifuge and centrifuge at 4°C and 12,000 rcf for 10 minutes. Discard the supernatant and scrape the isopropanol off the inner wall of the EP tube with a 200μL pipette tip. Add 1mL of 75% ethanol (prepared with DEPC water for washing the RNA pellet) to each tube. After vortexing, centrifuge at 7500 rcf for 5 minutes at 4°C. Carefully discard the supernatant and air-dry the RNA pellet at room temperature for 5-10 minutes. Then, resuspend in 20μL of DEPC water to obtain the total RNA solution.

[0190] Measure RNA concentration: Place the resuspended RNA solution on ice and take 1 μL of it on a nucleic acid quantitative analyzer to detect and record the RNA concentration, A 260 With A 280 The ratio and A 260 With A 230 The ratio of 1:1 to 2:1 (used to assess the protein contamination and residual organic solvent content of the sample, respectively) was used to calculate the volume of RNA solution required for each sample. All consumables used throughout the process, including EP tubes and pipette tips, were sterile and RNase-free.

[0191] Reverse transcription: Prepare the reaction system in a 500 μL EP tube according to the reverse transcription kit instructions, as shown in Table 5. Set up the reaction system according to the program in the table and store the resulting cDNA at -20°C until use.

[0192] Table 5 Reverse transcription system

[0193]

[0194] Real-time fluorescence quantification: Keep the cDNA, DEPC water, SYBR dye, and primers required for the reaction on ice, away from light. Prepare the reaction system in eight strips, with a total system of 10 μL, as shown in Table 6. Mix thoroughly and centrifuge to concentrate the liquid on the inner wall at the bottom to reduce errors. Transfer to a PCR instrument and set the following settings: 95°C pre-denaturation, 3 minutes, 1 cycle; 95°C denaturation for 30 seconds, 60°C annealing for 30 seconds, 72°C extension for 30 seconds, and these three processes are cycled 39 times. Obtain C t After the data, use 2-ΔΔCT The target genes were quantitatively analyzed by relative quantification. The primers used were β-actin, PDK1, HIF-1α, P-gp, and caspase 3. The primer sequences are shown in Table 7.

[0195] Table 6 Real-time quantitative PCR system

[0196]

[0197] 1.6 Western Blot Experiment

[0198] Cell culture and treatment: The operation is the same as the cell culture and treatment under 1.5 of this embodiment.

[0199] To extract total cell protein: Prepare protein lysis buffer with a ratio of RIPA cell lysis buffer to protease inhibitors in a 99:1 ratio. Mix and place on ice until ready to use. Remove the culture plate after 48 hours of incubation and place on ice. Discard the culture medium and wash three times with PBS. Then, add 150 μL of the prepared lysis buffer to each well. Use a 200 μL pipette tip to scrape the cells and transfer all the cells to a 1.5 mL EP tube. Lyse on ice for 30 minutes. After lysis, transfer the cells to a centrifuge and centrifuge at 12,000 rpm at 4°C for 25 minutes. Transfer 120 μL of the supernatant to a new EP tube, add 30 μL of loading buffer, and mix thoroughly. Heat the protein sample in a metal bath at 98°C for 10 minutes to denature it. Cool to room temperature and store at -20°C.

[0200] SDS-PAGE gel electrophoresis: 1) Select and prepare the gel according to the molecular weight of the target protein: ① Leak detection: Clean the glass plate, secure it, and inspect it with ultrapure water for leaks. Let it stand for 10 minutes and observe whether the liquid level drops. If it does not, pour out the ultrapure water and remove any remaining liquid with filter paper. ② Gel preparation: Mix the lower gel (separating gel) solution and the modified coagulant according to the number of gel plates required. Immediately pour the solution into the glass plate to a certain height. Add anhydrous ethanol to press the gel. After the lower gel solidifies (a clear boundary is observed), pour out the anhydrous ethanol and remove any remaining liquid with filter paper. Mix the upper gel (concentrating gel) and the coagulant and pour it into the glass plate until it covers the short edge of the plate. Slowly insert a tooth comb vertically and wait 30 minutes for the upper gel to solidify. The prepared gel plates can be used directly or stored in a humidified environment at 4°C. 2) Loading protein samples: Place the gel plate into the electrophoresis tank, add electrophoresis buffer inside and outside the tank, remove bubbles, slowly pull out the comb, add a certain amount of protein sample (about 10μL) to the gel well, and add 4μL of protein pre-stained marker at each end of the protein sample to mark the protein molecular weight. 3) Electrophoresis: Set the voltage to 30V under constant voltage conditions, run the electrophoresis for 5 minutes, first align the protein samples; then 80V, electrophoresis for 30 minutes, at this time all proteins of different molecular weights reach the separation gel; then 120V, electrophoresis for 50 minutes, stop when the sample runs to the bottom of the glass plate. 4) Transfer: Cut the PVDF membrane and mark it in the lower right corner, and activate it in methanol for 5 minutes. After the electrophoresis is completed, remove the gel plate, soak it in pre-cooled transfer buffer, and cut the gel according to the marker position corresponding to the molecular weight of the target protein. Following the "black gel, white membrane" principle, place the sponge, filter paper, gel, PVDF membrane (front facing the gel), filter paper, and sponge on the black side of the transfer cassette in that order. After checking that there are no bubbles between the gel and PVDF membrane, clamp the transfer cassette to form a "sandwich" structure. Transfer the strips to a transfer chamber, fill with transfer buffer, and place on ice. Set the current to 200A under constant current conditions and transfer for 90 minutes. 5) Blocking: After transfer, place the strips in 5% skim milk (prepared with TBST) and block for 2 hours at room temperature on a shaker. Then, wash with TBST for 10 minutes, for a total of three washes. 6) Incubation with primary antibody: Place the strips in the prepared primary antibody and incubate at 4°C on a shaker at low speed for 16 hours. After incubation, wash with TBST for 10 minutes, for a total of three washes. 7) Incubation with secondary antibody: Place the strips in the secondary antibody and incubate for 60 minutes on a shaker at room temperature. After incubation, wash with TBST for three 10-minute washes. 8) Development: Prepare the developer solution by mixing equal volumes of Solution A and Solution B in the ECL luminescent solution. Evenly drip the developer solution onto the strips, covering them thoroughly. Then, develop the strips using a chemiluminescence imaging system. 9) Expression Analysis: Analyze the target protein bands using Lane 1D software.

[0201] 1.8 Determination of glucose and lactic acid content

[0202] Cell culture and treatment: The operation is the same as the cell culture and treatment method under 1.5 of this embodiment.

[0203] After culturing for 48 h, the cells were digested and centrifuged, and resuspended in a certain amount of PBS solution to obtain a cell suspension, which was then disrupted in an ultrasonic disruptor under ice water bath for 5 times, each time for 3 s, and then subjected to subsequent tests.

[0204] Determination of glucose content: prepare the reagent according to the kit instructions, react at 37°C for 10 min, measure the OD value at 505 nm using a microplate reader, and calculate the value.

[0205] Determination of lactic acid content: prepare enzyme working solution and color developer according to the kit instructions, mix all solutions, react in a 37°C water bath for 10 minutes, add stop solution, mix well, measure the OD value at 530 nm using a microplate reader, and calculate the value.

[0206] 1.9RH123 uptake experiment

[0207] RH123 uptake qualitative experiment: NCI-H460 and NCI-H1299 cells were cultured at a rate of 2×10 5 Cells were uniformly seeded at a density of 100 cells / dish in confocal microplates and cultured until the density reached 70%. Drugs were then added according to the experimental groups: Control group, CPD12C15+Cu group (0.20 μmol / L), CPD12C15-LP+Cu group (0.20 μmol / L), CPD12C15-TLP+Cu group (0.20 μmol / L), and DTX group (NCI-H460 cells: 5.0 μmol / L, NCI-H1299 cells: 15.0 μmol / L). Cells were placed in an incubator and cultured for 48 h.

[0208] After 48 hours, the culture medium was discarded, and 1 mL of 5 μg / mL RH123 solution was added and incubated in an incubator for 4 hours. After incubation, the solution was discarded, and the cells were washed three times with PBS. The cells were fixed with 4% paraformaldehyde for 10 minutes, washed with PBS, and photographed under a confocal microscope.

[0209] RH123 uptake quantitative experiment: The cells were cultured and treated in the same manner as in 1.5 of this Example.

[0210] After 48 hours of culture, the medium was discarded, and 1 mL of a 5 μg / mL RH123 solution was added and incubated in an incubator for 4 hours. The RH123 solution was discarded, and the cells were washed three times with PBS. The cells were trypsinized and transferred to a centrifuge tube. Centrifuged at 1000 rpm for 5 minutes, washed twice with PBS, and resuspended in 500 μL of PBS solution after centrifugation. The cells were transferred to a flow cytometer and the fluorescence intensity of intracellular RH123 was measured by flow cytometry.

[0211] 2.10 Statistical Analysis: Data were analyzed using GraphPad Prism 8.0.2 software. Results are presented as mean ± standard deviation (mean ± SD), with n = 3. Statistical analysis was performed using t-test and one-way ANOVA. * p<0.05, ** p<0.01, *** p<0.001 indicates that there is a significant difference between the CPD12C15 and its preparation groups and the Control group; # p<0.05, ## p<0.01, ### p<0.001 indicates that there are significant differences among the positive control drug DTX group, the Control group, and the CPD12C15 preparation group.

[0212] 2. Experimental Results

[0213] 1. CPD12C15-TLP combined with copper inhibits lung cancer cell proliferation

[0214] The MTT experiment was used to investigate the effects of CPD12C15 and its preparations combined with copper and DTX on the survival rates of NCI-H460 and NCI-H1299 cells. Figure 10 As shown in Table 8, the drug administration groups showed the effect of inhibiting the proliferation of NCI-H460 and NCI-H1299 cells in a concentration-dependent manner. Table 8 shows the IC 50 The value represents the ability to inhibit cell proliferation. The smaller the value, the stronger the inhibitory effect. Compared with DTX, the IC 50 The results showed that the CPD12C15-TLP+Cu group had a better anti-lung cancer efficacy. 50 The lowest, the best anti-lung cancer effect. This shows that CPD12C15 and its preparations combined with copper effectively inhibit the proliferation of lung cancer cells and exert good anti-lung cancer activity.

[0215] IC-based 50 The values were used to determine the subsequent cell experimental grouping and drug concentration.

[0216] In NCI-H460 cells: Control group, CPD12C15+Cu group (0.20 μmol / L), CPD12C15-LP+Cu group (0.20 μmol / L), CPD12C15-TLP+Cu group (0.20 μmol / L), and / or DTX group (5.0 μmol / L);

[0217] In NCI-H1299 cells: Control group, CPD12C15+Cu group (0.20 μmol / L), CPD12C15-LP+Cu group (0.20 μmol / L), CPD12C15-TLP+Cu group (0.20 μmol / L) and / or DTX group (15.0 μmol / L).

[0218] Table 8: IC values of drugs in NCI-H460 and NCI-H1299 cells 50 Value (n=3)

[0219]

[0220] 2. CPD12C15-TLP combined with copper inhibits the migration and invasion of lung cancer cells and induces apoptosis

[0221] The effects of CPD12C15 and its preparations combined with copper on the migration ability of NCI-H460 and NCI-H1299 cells were investigated by cell scratch test. Figure 11 As shown. Compared with the Control group, the drug-treated groups all showed the ability to inhibit cell migration (p < 0.001). In NCI-H460 and NCI-H1299 cells, the wound healing rates of the Control group were (21.46 ± 3.39)% and (51.02 ± 4.14)%, respectively; the wound healing rates of the CPD12C15 + Cu group were (7.63 ± 0.85)% and (22.77 ± 2.20)%, respectively; the wound healing rates of the CPD12C15-LP + Cu group were (7.08 ± 0.33)% and (18.18 ± 1.47)%, respectively; the wound healing rates of the CPD12C15-TLP + Cu group were (6.13 ± 1.33)% and (12.34 ± 1.12)%, respectively; and the wound healing rates of the DTX group were (8.67 ± 1.95)% and (17.27 ± 1.11)%. The comprehensive results showed that CPD12C15-TLP combined with copper inhibited the migration of lung cancer cells, and the inhibitory effect was better than DTX.

[0222] The effects of CPD12C15 and its preparations on the invasion ability of NCI-H460 and NCI-H1299 cells were investigated by Matrigel-Transwell assay. Figure 12As shown. Compared with the control group, the number of cells invading to the bottom of the chamber in the drug-treated group was significantly reduced, and both groups showed the ability to inhibit cell invasion (p < 0.001). In NCI-H460 and NCI-H1299 cells, the relative invasion rates of the CPD12C15+Cu group decreased to (68.54±6.14)% and (58.16±3.60)%, respectively; the relative invasion rates of the CPD12C15-LP+Cu group decreased to (41.07±6.39)% and (44.09±2.28)%, respectively; the relative invasion rates of the CPD12C15-TLP+Cu group decreased to (14.56±1.77)% and (31.09±0.54)%, respectively; and the relative invasion rates of the DTX group decreased to (18.64±2.59)% and (35.70±3.79)%, respectively. The comprehensive results showed that CPD12C15-TLP+Cu inhibited the invasion of lung cancer cells, and the inhibitory effect was better than DTX.

[0223] Flow cytometry was used to detect the number of apoptotic cells in NCI-H460 and NCI-H1299 cells after administration, and the effects of CPD12C15 and its preparations combined with copper on apoptosis of lung cancer cells were investigated. Figure 13 As shown. CPD12C15 and its preparations combined with copper and DTX increased the apoptosis rate of both cell lines (p < 0.001). In NCI-H460 and NCI-H1299 cells, the apoptosis rates of the control group were (9.21 ± 0.67)% and (15.79 ± 0.48)%, respectively; the apoptosis rates of the CPD12C15 + Cu group were (24.87 ± 0.66)% and (33.58 ± 0.87)%, respectively; the apoptosis rates of the CPD12C15-LP + Cu group were (29.81 ± 0.88)% and (40.63 ± 0.15)%, respectively; the apoptosis rates of the CPD12C15-TLP + Cu group were (50.48 ± 0.05)% and (42.51 ± 0.05)%, respectively; and the apoptosis rates of the DTX group were (48.23 ± 1.55)% and (31.90 ± 0.66)%, respectively. The comprehensive results showed that CPD12C15-TLP combined with copper promoted the apoptosis of lung cancer cells, and the effect was better than DTX.

[0224] 3. CPD12C15-TLP combined with copper inhibits the Warburg effect in lung cancer cells

[0225] The Warburg effect, also known as aerobic glycolysis, is the primary energy source for tumor cells. PDK1 and LDHA are key enzymes in the glycolytic pathway. Overexpression of PDK1 and abnormal upregulation of LDHA have been observed in lung cancer cells. Increased HIF-1α is closely associated with enhanced glycolysis, promoting the occurrence of the Warburg effect in tumor cells. Inhibiting the Warburg effect can inhibit cancer cell proliferation, migration, and invasion, and induce apoptosis. Therefore, we examined changes in lactate and glucose levels in NCI-H460 and NCI-H1299 cells. qRT-PCR and Western blot were used to determine the expression of PDK1, LDHA, and HIF-1α in these cells to investigate the effects of CPD12C15-TLP combined with copper on the Warburg effect.

[0226] The results of glucose and lactic acid test kits are as follows Figure 14 In NCI-H460 and NCI-H1299 cells, compared with the Control group, CPD12C15 and its preparations combined with copper downregulated the levels of glucose and lactate in lung cancer cells. Figure 15 As shown in Figure 2, in NCI-H460 and NCI-H1299 cells, compared with the Control group, CPD12C15 and its preparations combined with copper inhibited the expression of PDK1 and HIF-1α, promoted the expression of caspase-3, and the CPD12C15-TLP+Cu group had the best effect. Figure 16 、 Figure 17 As shown, in NCI-H460 and NCI-H1299 cells, compared with the control group, CPD12C15 and its preparations combined with copper downregulated the expression of PDK1, LDHA, and HIF-1α, and upregulated the expression of caspase-3, with the CPD12C15-TLP+Cu group showing the best effect. These results are similar to those of qRT-PCR experiments. These experimental results indicate that CPD12C15-TLP combined with copper inhibits the growth and proliferation of lung cancer cells and induces apoptosis by suppressing the Warburg effect.

[0227] 4. CPD12C15-TLP combined with copper inhibits the expression of drug resistance-related protein P-gp in lung cancer cells

[0228] Drug resistance may develop due to increased drug efflux, which reduces intracellular drug concentrations. P-gp, a broad-spectrum multidrug efflux pump, releases DTX from cells, so increased expression of P-gp can contribute to drug resistance. Studies have shown that the Warburg effect is associated with the development of tumor drug resistance. This may be due to the Warburg effect positively feedback looping on HIF-1α expression, which directly regulates P-gp expression, leading to drug resistance. qRT-PCR and Western blot were used to investigate P-gp expression in NCI-H460 and NCI-H1299 cells. Confocal microscopy and flow cytometry were used to qualitatively and quantitatively investigate RH123 uptake in NCI-H460 and NCI-H1299 cells.

[0229] The results of qRT-PCR are as follows Figure 18 As shown in the results, compared with the Control group, CPD12C15 and its preparations combined with copper inhibited the mRNA expression of P-gp in NCI-H460 and NCI-H1299 cells, and the CPD12C15-TLP+Cu group had the best inhibitory effect.

[0230] Western Blot results are as follows Figure 19 As shown in the results, compared with the Control group, CPD12C15 and its preparations combined with copper downregulated the protein expression of P-gp in NCI-H460 and NCI-H1299 cells, and the CPD12C15-TLP+Cu group had the best inhibitory effect, which was similar to the results of qRT-PCR experiments.

[0231] RH123 is a substrate of P-gp and can be used as a marker of P-gp. The stronger the P-gp inhibition, the more RH123 is taken up. Figure 20 shown. Figure 20 (a), (b) Confocal microscopy showed that the fluorescence intensity of CPD12C15 and its preparations combined with copper was enhanced, while the fluorescence intensity of DTX was weakened, and CPD12C15-TLP+Cu had the best effect. Fluorescence intensity was detected by flow cytometry. In NCI-H460 cells, the fluorescence intensity of the control group was (3.78±0.14)%, the fluorescence intensity of the CPD12C15+Cu group was (6.58±0.62)%, the fluorescence intensity of the CPD12C15-LP+Cu group was (8.18±0.82)%, and the fluorescence intensity of the CPD12C15-TLP+Cu group was (13.77±0.41)%. ***p<0.001, the fluorescence intensity of the DTX group was (3.22±0.21)%. In NCI-H1299 cells, the fluorescence intensity of the Control group was (2.29±0.26)%, the fluorescence intensity of the CPD12C15+Cu group was (9.87±0.38)%, the fluorescence intensity of the CPD12C15-LP+Cu group was (11.47±2.02)%, and the fluorescence intensity of the CPD12C15-TLP+Cu group was (16.49±0.55)%. *** The fluorescence intensity of the DTX group was (1.62±0.19)%. Similar to the results of the confocal microscopy qualitative experiment, the fluorescence intensity of CPD12C15 and its preparations combined with copper increased, while the fluorescence intensity of DTX decreased, and CPD12C15-TLP+Cu was the most effective. These experimental results indicate that CPD12C15-TLP combined with copper inhibits the expression of P-gp and produces resistance.

[0232] Example 4 In vivo anti-lung cancer efficacy of CPD12C15-TLP combined with copper

[0233] 1. Experimental Methods

[0234] Cell line: Human non-small cell lung cancer NCI-H460 cell line was provided by the School of Pharmacy of Shandong University.

[0235] Experimental animals: Female Balb / C nude mice, 4–5 weeks old, were purchased from Jinan Xingkang Biological Products, batch number SYXK(Lu)20200022. They were housed in individually ventilated cages at an ambient temperature of 23–27°C and a relative humidity of 40%–70% and received a normal daily diet and water. All experiments adhered to the guidelines of the Animal Experimentation Center Ethics Committee of Shandong University.

[0236] Experimental solution preparation

[0237] The preparation of CPD12C15 and its preparation, and DTX solution were the same as in Example 3.

[0238] Copper gluconate solution: Accurately weigh 8.00 mg of copper gluconate powder and dissolve it in 10 mL of normal saline. Ultrasonicate the solution to obtain a copper gluconate solution with a concentration of 0.8 mg / mL.

[0239] Cell culture: The operation method is the same as Example 2.

[0240] 1. Establishment of nude mouse subcutaneous tumor model: NCI-H460 cells in the logarithmic growth phase were prepared into a single cell suspension, and the cell density was adjusted to 2×10 7 100 μL of cell suspension was injected subcutaneously into the axilla of the nude mouse using a syringe.3 Tumor-bearing nude mice were randomly divided into five groups of six: Model, CPD12C15+Cu, CPD12C15-LP+Cu, CPD12C15-TLP+Cu, and DTX. The CPD12C15 and its preparation groups received 50 μL of copper (2 mg / kg) in the morning and 100 μL of CPD12C15 and its preparation (10 mg / kg) via tail vein injection in the evening, every two days for seven doses. The DTX group received 100 μL of the drug (10 mg / kg) via tail vein injection every three days for five doses. The dosing cycle lasted 14 days, and nude mouse body weight and tumor volume were recorded daily. After 14 days of dosing, tumors, major organs, and brain tissue were removed, and tumor volume and weight, as well as brain tissue weight, were recorded. Hematoxylin and eosin (H&E) staining, TUNEL apoptosis assay, and immunohistochemistry were performed.

[0241] 2. H&E staining experiment: The heart, liver, spleen, lung, kidney, tumor mass and brain tissues were cleaned with physiological saline, wiped dry, fixed with 4% paraformaldehyde, and then stained with H&E to detect the pathological changes and investigate the safety, efficacy and toxicity. The specific experimental procedures were as follows: 1) Embedding: The fixed tissues were removed and dehydrated with gradient ethanol (70%, 80%, 100%), then placed in xylene. After the tissues became transparent, they were immersed in liquid paraffin and embedded to form wax blocks; 2) Sectioning: The wax blocks were cut into uniform slices with a thickness of about 3 to 5 μm, stretched and smeared with hot water. Place on a glass slide and dry at 45°C; 3) Dewaxing: Dewax with xylene, hydrate with gradient ethanol (100%, 85%, 75%), and rinse with distilled water; 4) Staining: First stain the sections with hematoxylin for 5-10 minutes, separate with 1% hydrochloric acid and ethanol, then return to blue with 1% ammonia water, then add eosin and stain for 3-5 minutes, and rinse with ultrapure water; 5) Dehydration and mounting: Dehydrate with gradient ethanol, place in xylene to make it transparent, then add neutral gum to seal it, seal with a coverslip, and observe under a microscope after drying.

[0242] 3. TUNEL method to detect cell apoptosis: TUNEL staining was used to detect cell apoptosis in tumor and brain tissue to investigate drug efficacy and toxicity. The specific operations are as follows: 1) Embedding, sectioning, and dewaxing are the same as the experimental operations in Item 2 of this Example; 2) Permeabilization: Add 20 μg / mL proteinase K working solution to the slice sample, incubate at 37°C for 20 minutes, and wash 3 times with PBS; 3) Inactivate endogenous enzymes: Place the slice in 3% H2O2 solution and incubate at room temperature for 20 minutes, and wash with PBS; 4) Room temperature equilibration: Add 50 μL equilibration buffer, incubate at room temperature for 10 minutes, and wash 3 times with PBS; 5) Labeling: Prepare the labeling solution (TdT enzyme: dUTP marker: equilibration buffer = 1:5:50) according to the kit instructions, and add 56 μL Add TdT incubation buffer to the slice sample, incubate at 37°C in the dark for 60 minutes, and wash three times with PBS; 6) SA-HRP incubation: Add 100 μL of HRP reaction solution, incubate at 37°C for 30 minutes, and wash three times with PBS; 7) DAB color development: Add 50 μL of DAB staining solution to the slice tissue sample, observe the staining, and rinse with ultrapure water after positive staining; 8) Hematoxylin nuclear staining: First, counterstain with hematoxylin for 5 minutes, separate with 1% hydrochloric acid and ethanol, then return to blue with 1% ammonia water, and then rinse with ultrapure water; 9) Dehydration and mounting: Dehydrate with gradient ethanol, then place in xylene to make it transparent, then add neutral gum to seal it, use a coverslip to seal it, and observe it under a microscope after drying.

[0243] 4. Nissl staining: Nissl staining is a method of staining neural tissue with basic dyes to observe neuronal damage. The specific procedures are as follows: 1) Embedding, sectioning, and dewaxing are the same as the experimental procedures in Item 2 of this Example; 2) Staining: The sections are immersed in Nissl stain solution and incubated at 37°C for 30-60 minutes, and then washed with PBS; 3) Dehydration and mounting: Dehydrate with graded ethanol, then place in xylene to make it transparent, then add neutral gum to seal it, and use a cover glass to seal it. After drying, it can be observed under a microscope.

[0244] 5. Immunohistochemistry: Immunohistochemistry was used to detect the effects of CPD12C15 and its preparations combined with copper on the expression of PDK1, HIF-1α, caspase-3, and P-gp proteins in subcutaneous lung cancer cells, and to detect glial fibrillary acidic protein (GFAP) in the hippocampus and tyrosine hydroxylase (TH) in the substantia nigra in brain tissue to explore its mechanism of action and neurotoxicity. The specific steps are as follows: 1) Embedding, sectioning, and dewaxing are the same as the experimental procedures in item 2 of this example; 2) Antigen retrieval: Use pH Immerse the sections in 6.0% citric acid buffer, boil on high heat for 3 minutes, then heat on medium heat for 15 minutes, and wash three times with PBS after cooling to room temperature; 3) Inactivate endogenous enzymes: Place the sections in a 3% H2O2 solution and incubate at room temperature for 20 minutes, then wash with PBS; 4) Serum blocking: Place the sections in a 5% BSA solution and incubate at room temperature for 1 hour; 5) Incubate with primary antibodies: Discard the blocking solution, dilute the primary antibodies PDK1, HIF-1α, caspase-3, P-gp, GFAP, and TH solutions to a specific ratio, and add them dropwise to the section samples, incubate at 4°C overnight, and wash three times with PBS; 6) Incubate with secondary antibodies: Add secondary antibodies dropwise to the section samples, incubate at room temperature for 50 minutes, and wash three times with PBS; 7) DAB color development: Add an appropriate amount of DAB staining solution, observe the staining, and wash with ultrapure water after positive staining; 8) Hematoxylin staining, dehydration, and sealing are the same as the experimental procedures in item 3 of this example.

[0245] 6. Determination of oxidative stress indicators in brain tissue

[0246] 6.1 Brain Tissue Sample Preparation: Wash the brain tissue with physiological saline, mince it, add a certain amount of physiological saline, and grind it in a tissue grinder at 4°C at 60 Hz for 10 seconds each time, for a total of five times. Centrifuge it in a refrigerated ultracentrifuge at 12,000 rcf and 4°C for 10 minutes. Collect the supernatant for subsequent analysis.

[0247] 6.2 Brain Tissue MDA Detection: Prepare TBA solution and MDA working solution according to the kit instructions. Add the solutions as instructed, mix thoroughly, heat in a 100°C water bath for 15 min, cool to room temperature, and centrifuge at 1000 rcf for 10 min at room temperature. Add 200 μL of the supernatant to a 96-well plate and measure the OD value at 532 nm using a microplate reader. The MDA content can be calculated.

[0248] 6.3 Brain Tissue SOD Detection: Prepare WST-8 / enzyme working solution and reaction starting solution according to the kit instructions. Add the sample to be tested and other solutions to a 96-well plate in sequence according to the instructions. Mix thoroughly and incubate at 37°C for 30 min. Measure the OD value at 450 nm using a microplate reader. The SOD content can be calculated.

[0249] 7. Statistical analysis: Data were analyzed using GraphPad Prism 8.0.2 software. Results are expressed as mean ± SD, with n = 3. Statistical analysis was performed using t-test and one-way ANOVA. * p<0.05, ** p<0.01, *** p<0.001 indicated significant differences between the groups; # p<0.05, ## p<0.01, ### p<0.001 indicates that there are significant differences between the control positive drug DTX and the Model and CPD12C15 preparations.

[0250] 2. Experimental Results

[0251] 1. CPD12C15-TLP combined with copper inhibits tumor growth: The weight and tumor volume of nude mice were recorded during the administration period. After 14 days of administration, the tumors of nude mice were removed and photographed. The experimental results are as follows: Figure 21 As shown. Figure 21 As shown in (a), compared with the Model group, the tumors in the drug-treated groups were all smaller, among which the tumor in the CPD12C15-TLP+Cu group was significantly smaller than that in the other groups, showing the best effect. Figure 21 Middle (b) shows the changes in tumor volume after 14 days. The tumor in the CPD12C15-TLP+Cu group grew slowly, while the tumor in the other groups grew faster. Figure 21 (c) shows the final volume of the tumor mass. The tumor mass volume of the Model group was 1033.89±104.19mm 3 The tumor volume of the CPD12C15+Cu group was 638.94±78.39mm 3 The tumor volume of the CPD12C15-LP+Cu group was 512.01±50.44mm 3 The tumor volume of the CPD12C15-TLP+Cu group was 271.13±50.38mm 3 The tumor volume of the DTX group was 502.94±101.67mm 3 , the tumor volume in the CPD12C15-TLP+Cu group was the smallest; Figure 21(d) is the weight of the tumor. The tumor weight of the Model group was 1118.89±106.46 mg, the tumor weight of the CPD12C15+Cu group was 745.30±77.97 mg, the tumor weight of the CPD12C15-LP+Cu group was 562.95±91.06 mg, the tumor weight of the CPD12C15-TLP+Cu group was 311.23±40.04 mg, and the tumor weight of the DTX group was 562.03±128.60 mg. The tumor weight of the CPD12C15-TLP+Cu group was the smallest. Figure 21 (e) shows the H&E staining results of the tumor mass. The Model group showed dense cells and vigorous tumor cell growth. The drug group showed gaps between cells, indicating a certain inhibitory effect on tumor cells. In the CPD12C15-TLP+Cu group, tumor cells showed a large amount of necrosis and vacuoles, indicating a strong killing effect on tumor cells. These experimental results all demonstrate that CPD12C15 and its preparations combined with copper can inhibit tumor growth in vivo, and CPD12C15-TLP+Cu is more effective than DTX.

[0252] 2. In vivo safety evaluation of CPD12C15-TLP combined with copper: During the administration period, the nude mice were weighed once a day to assist in judging drug toxicity. After the administration, the main tissues of the nude mice were taken for H&E staining to determine the pathological changes of the tissues. The experimental results are as follows Figure 22 shown. Figure 22 The figure shows the changes in the body weight of nude mice during the experiment. During the entire administration process, the body weight of the Model group and the CPD12C15 and its preparation combined with copper group increased, which to a certain extent shows that the toxicity of CPD12C15 and its preparation combined with copper is relatively small; while the body weight of the DTX group decreased, indicating that DTX has certain toxicity. Figure 22 H&E staining of the heart, liver, spleen, lung, and kidney in each group is also shown. Compared with the model group, neither the CPD12C15-TLP+Cu group nor the DTX group showed significant pathological changes. These experimental results demonstrate that CPD12C15-TLP combined with copper has a certain safety profile while inhibiting lung cancer cell proliferation, while DTX is relatively toxic.

[0253] 3. CPD12C15-TLP combined with copper induced tumor cell apoptosis: TUNEL apoptosis was used to detect the apoptosis of subcutaneous lung cancer cells. The experimental results are as follows Figure 23As shown, the color of the Model group was basically blue (negative). After drug treatment, the positive expression increased. By Image J quantitative analysis, compared with the Model group, the relative positive expression of the CPD12C15+Cu group was (195.03±11.11)%, the relative positive expression of the CPD12C15-LP+Cu group was (233.63±10.54)%, and the relative positive expression of the CPD12C15-TLP+Cu group was (338.15±61.57)% ( *** p<0.001, the relative positive expression in the DTX group was (184.59±21.75)% ( # The CPD12C15-TLP+Cu group had the strongest apoptosis-inducing effect. These experimental results indicate that CPD12C15 and its preparations combined with copper induce apoptosis in subcutaneous lung cancer tumors, and the effect of CPD12C15-TLP combined with copper is superior to the positive control drug DTX.

[0254] 4. CPD12C15-TLP combined with copper inhibits the Warburg effect: The Warburg effect is the main source of energy for lung cancer cells, providing a large amount of energy for the growth and proliferation of lung cancer cells. To further confirm that CPD12C15 and its preparations combined with copper can inhibit the Warburg effect in vivo and induce tumor cell apoptosis, immunohistochemistry was used to detect the expression of PDK1, HIF-1α, and caspase-3 in tumor masses. The experimental results are as follows: Figure 24As shown. Compared with the Model group, the positive expressions of PDK1 and HIF-1α (brown) in the CPD12C15 and its preparation combined with copper group were less and less, and the positive expression of caspase-3 was increased. By Image J quantitative analysis, compared with the Model group, the relative positive expressions of PDK1, HIF-1α, and caspase-3 in the CPD12C15+Cu group were (78.50±7.75)%, (82.32±5.65)%, and (144.37±13.24)%, respectively; the relative positive expressions of PDK1, HIF-1α, and caspase-3 in the CPD12C15-LP+Cu group were (73.49±8.07)%, (48.17±5.68)%, and (209. The relative positive expression of PDK1, HIF-1α, and caspase-3 in the CPD12C15-TLP+Cu group was (49.36±8.72)%, (27.88±3.44)%, and (321.62±13.64)%, respectively. This indicates that after drug treatment, PDK1 and HIF-1α proteins in the tumor mass were inhibited, while caspase-3 protein expression was increased. The CPD12C15-TLP+Cu group showed the best effect, consistent with the results of in vitro mechanistic studies. These experimental results demonstrate that CPD12C15 and its preparations combined with copper can inhibit the Warburg effect in vivo and promote tumor cell apoptosis, thereby exerting anti-lung cancer efficacy, with CPD12C15-TLP+Cu showing the best effect.

[0255] 5. CPD12C15-TLP combined with copper to inhibit drug resistance: In order to explore whether CPD12C15 and its preparations combined with copper can produce anti-drug resistance in vivo, tumor masses were taken for immunohistochemistry experiments on drug resistance-related protein P-gp. The experimental results are as follows Figure 25 Compared with the Model group, the positive expression of P-gp in the CPD12C15 and its preparations combined with copper group was reduced, indicating that its protein expression was inhibited, which was consistent with the results of in vitro mechanism studies. By Image J quantitative analysis, compared with the Model group, the relative positive expression of P-gp in the CPD12C15+Cu group was (84.79±4.93)%, the relative positive expression of P-gp in the CPD12C15-LP+Cu group was (54.99±1.98)%, and the relative positive expression of P-gp in the CPD12C15-TLP+Cu group was (39.72±

[0256] The relative positive expression of P-gp in the DTX group was (5.54)%, and the relative positive expression of P-gp in the DTX group was (103.66±2.80)%. The above experimental results show that CPD12C15 and its preparations combined with copper can inhibit the expression of drug resistance-related protein P-gp in vivo, thereby exerting anti-drug resistance, and CPD12C15-TLP combined with copper has the best effect, while DTX can induce certain drug resistance and affect the efficacy of the drug.

[0257] 6. CPD12C15-TLP combined with copper shows no neurotoxicity: DTX is an effective and practical chemical anticancer agent that can be used as a single or combined therapy to inhibit NSCLC. However, it has significant side effects, including myelosuppression, peripheral neurotoxicity, hypersensitivity reactions, and the development of drug resistance. DTX has been reported to induce central nervous system toxicity, while CPD12C15 reduces neurotoxicity compared to DSF. Therefore, the neurotoxicity of CPD12C15 and its formulations combined with copper and DTX was evaluated using brain tissue weight, H&E staining, Nissl staining, immunohistochemistry, and the detection of oxidative stress markers such as MDA and SOD.

[0258] 6.1 Brain tissue weight: Generally speaking, brain weight is a relatively constant baseline weight of animals. After 14 days of administration, the brain tissues of each group were removed and weighed. The brain weight of the Model group was 542.55±39.47 mg, the brain weight of the CPD12C15+Cu group was 511.08±24.02 mg, the brain weight of the CPD12C15-LP+Cu group was 519.93±23.36 mg, the brain weight of the CPD12C15-TLP+Cu group was 533.75±31.39 mg, and the brain weight of the DTX group was 471.38±17.05 mg. The experimental results are as follows: Figure 26 Compared with the Model group, the brain weight of the CPD12C15 and preparation groups did not change much, while the brain weight of the positive drug DTX group was relatively light, which may have caused certain neurological damage.

[0259] 6.2 H&E staining of brain tissue: Figure 27 The hippocampus in the Model group and the CPD12C15 and its preparation combined with copper group was structurally intact, with larger cells and lighter staining, showing no signs of degeneration or necrosis. In the DTX group, however, neurons in the hippocampus of nude mice shrank, their cell bodies became smaller, and the staining became darker, indicating that the brain tissue of the nude mice in the DTX group had some pathological changes.

[0260] 6.3 Nissau staining of brain tissue: Nissau bodies are a type of basophilic substance in the cytoplasm that is widely distributed in various neurons and can be used to observe neuronal damage. Under normal circumstances, Nissau bodies are large and numerous. When nerve cells are damaged, the number of Nissau bodies will decrease. The experimental results are as follows: Figure 28In the Model group, CPD12C15 and its preparation combined with copper group, the number of nissoloids was large and arranged relatively neatly, while in the DTX group, the number of nissoloids was significantly reduced and arranged disorderly, indicating that neurons were damaged to a certain extent. This indicates that CPD12C15 and its preparation combined with copper have no obvious neurotoxicity, while DTX has certain neurotoxicity.

[0261] 6.4 Brain tissue MDA and SOD content

[0262] Neuronal cells have weak antioxidant capacity, and oxidative stress can easily cause damage to the central nervous system. Therefore, detecting oxidative stress-related indicators in brain tissue can be used to indicate whether a drug is neurotoxic. Malondialdehyde (MDA) is one of the hallmark products of lipid peroxidation, and its content represents the severity of oxidative stress damage. Figure 29 As shown in Figure 2, the MDA content in the brain tissue of the Model group was 10.94±0.47 nmol / mg prot; the MDA content in the brain tissue of the CPD12C15+Cu group was 9.68±0.24 nmol / mg prot, which was lower than that of the Model group ( * p<0.05); the MDA content in the brain tissue of the CPD12C15-LP+Cu group was 9.45±0.20nmol / mg prot, which was lower than that of the Model group; the MDA content in the brain tissue of the CPD12C15-TLP+Cu group decreased to 8.63±0.52nmol / mg prot, which was significantly lower than that of the Model group ( *** p<0.001); while the MDA content in the brain tissue of the DTX group increased to 17.51±0.82nmol / mg prot, which was significantly higher than that of the Model group ( ### p<0.001). The above experimental results indicate that CPD12C15 and its preparations combined with copper can reduce lipid peroxidation in brain tissue and have a certain neuroprotective effect; while DTX increases lipid peroxidation in brain tissue and has a certain neurotoxicity.

[0263] Superoxide dismutase (SOD) is an important antioxidant factor in the body, and its content can be used to study the antioxidant activity of drugs. Figure 29 As shown in Figure 2, the SOD content in the brain tissue of the Model group was 172.47±5.30U / mg prot; the SOD content in the brain tissue of the CPD12C15+Cu group was 186.23±3.40U / mg prot, which was slightly higher than that of the Model group ( *p<0.05); the SOD content in the brain tissue of the CPD12C15-LP+Cu group was 204.43±10.15U / mg prot, which was higher than that of the Model group; the SOD content in the brain tissue of the CPD12C15-TLP+Cu group increased to 8.63±0.52U / mg prot, which was significantly higher than that of the Model group ( *** p<0.001); while the SOD content in the brain tissue of the DTX group decreased to 116.10±10.38U / mg prot, which was significantly lower than that of the CPD12C15-TLP+Cu group ( ### p<0.001). The above experimental results indicate that CPD12C15 and its preparations combined with copper can increase the production of antioxidants in brain tissue and have a certain neuroprotective effect; while DTX reduces the production of antioxidants in brain tissue and has a certain neurotoxicity.

[0264] 6.5 Immunohistochemistry of brain tissue: Astrocytes are important regulators of brain function and play an important role in functional maintenance, neuronal metabolism and restoration of homeostasis. Glial fibrillary acidic protein (GFAP) is the main skeletal protein in astrocytes and plays an important role in neurological diseases. Its increased level indicates that the central nervous system may be damaged. Tyrosine hydroxylase (TH) is a marker of dopaminergic and noradrenergic neurons and can be used as an effective marker of neuronal maturation and differentiation. The experimental results are as follows: Figure 30 shown. Figure 30 (a) Observe the staining of GFAP in brain tissue. The staining of Model group, CPD12C15 and its preparation combined with copper group was relatively light, and the positive expression was sparse; while the positive expression of DTX group increased to a certain extent, and the distribution was relatively dense. The results of quantitative analysis by Image J are shown in Figure 5. Figure 30 In (a), compared with the Model group, the relative positive expression of GFAP in the CPD12C15+Cu group was (90.46±9.88)%, the relative positive expression in the CPD12C15-LP+Cu group was (86.16±5.30)%, the relative positive expression in the CPD12C15-TLP+Cu group was (85.52±1.68)%, and the relative positive expression in the DTX group was (160.07±6.25)%. ### p<0.001), indicating that CPD12C15 and its preparations combined with copper reduced the expression of GFAP in the hippocampus, while DTX increased the expression of GFAP. Figure 30(b) Observe the staining of TH in brain tissue. The Model group, CPD12C15 and its preparation combined with copper group were all stained darker, while the DTX group was the lightest, with sparse positive expression. The results were quantitatively analyzed by Image J. Figure 30 In (b), compared with the Model group, the relative positive expression of TH in the CPD12C15+Cu group was (108.20±10.25)%, that in the CPD12C15-LP+Cu group was (109.61±9.67)%, that in the CPD12C15-TLP+Cu group was (113.32±1.26)%, and that in the DTX group was (77.34±4.57)%. # p<0.05), indicating that CPD12C15 and its preparations combined with copper increased the expression of TH in the substantia nigra, while DTX decreased the expression of TH. Taken together, these results suggest that CPD12C15 and its preparations combined with copper can reduce DTX-related neurotoxicity.

[0265] 6.6 Brain TUNEL apoptosis: TUNEL apoptosis was used to detect the apoptosis of neurons in the brain tissue of nude mice. Figure 31 As shown, the color of the Model group is basically blue (negative). After DTX treatment, the number of positive apoptotic cells increased significantly. By quantitative analysis, compared with the Model group, the relative positive expression of the CPD12C15+Cu group was (124.35±40.86)%, the relative positive expression of the CPD12C15-LP+Cu group was (107.37±41.73)%, the relative positive expression of the CPD12C15-TLP+Cu group was (77.86±32.43)%, and the relative positive expression of the DTX group was (622.16±54.06)% ( ### (p<0.001). The results showed no significant difference in the effects of CPD12C15 and its preparations combined with copper, while the DTX group showed the strongest effect in inducing neuronal apoptosis and exhibiting some neurotoxicity. These experimental results suggest that CPD12C15 and its preparations combined with copper have a certain degree of neurotoxicity, while DTX has a certain degree of neurotoxicity.

[0266] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art, after reading this description, may make various modifications to the technical solution or replace some of the technical features with equivalents. Any modifications, equivalent replacements, or improvements made within the spirit and principles of the present invention shall be deemed to fall within the scope of protection of the present invention.

Claims

1. A TPGS-modified CPD12C15 liposome, characterized in that: It is a vesicle-like structure containing: The lipid bilayer membrane is a closed membrane structure composed of dipalmitoylphosphatidylcholine (DPPC), soybean phosphatidylcholine (SPC) and cholesterol, forming the basic skeleton of the vesicle; A surface modification layer is formed on the surface of the liposome by vitamin E polyethylene glycol succinate (TPGS), wherein the hydrophobic end of the vitamin E polyethylene glycol succinate is inserted into the outer layer of the lipid bilayer membrane, and the hydrophilic end extends and is exposed to the outer surface of the liposome; The inner aqueous phase is an aqueous compartment surrounded by a lipid bilayer membrane, in which the active ingredient CPD12C15 is encapsulated; Wherein, the structure of the CPD12C15 is:

2. The TPGS-modified CPD12C15 liposome according to claim 1, characterized in that The molar ratio of dipalmitoylphosphatidylcholine (DPPC) to soy lecithin (SPC) is 1:1 to 1:7, preferably 1:5; Preferably, in the lipid bilayer membrane, the molar ratio of phospholipids (DPPC+SPC) to cholesterol is 1:1 to 5:1, preferably 2:1; Preferably, the content of vitamin E polyethylene glycol succinate (TPGS) is 3-15% of the total lipid molar number, preferably 10%, wherein the total lipid molar number refers to the sum of the molar numbers of DPPC, SPC, cholesterol and TPGS; Preferably, the total molar ratio of CPD12C15 to lipid components (DPPC, SPC, cholesterol and TPGS) is 1:1 to 1:5, preferably 1:

2.

3. The TPGS-modified CPD12C15 liposome according to claim 1 or 2, characterized in that The liposome has at least one of the following physicochemical properties: Average particle size: 80-150 nm, preferably 90-110 nm; Polydispersity index (PDI): 0.1-0.3, preferably less than 0.2; Zeta potential: -15 to 0 mV, preferably -10 to -5 mV; Encapsulation efficiency: 70-90%, preferably 78-82%; Drug loading: 10-25%, preferably 15-20%.

4. A method for preparing the TPGS-modified CPD12C15 liposomes according to any one of claims 1 to 3, characterized in that: include: (1) DPPC, SPC, cholesterol, and TPGS were mixed and dissolved in an organic solvent and rotary evaporated to form a uniform lipid film; (2) hydrating the lipid film with calcium acetate solution, ultrasonically treating the film, and filtering to obtain blank liposomes; (3) dialyzing the blank liposomes to establish a pH gradient; (4) The dialyzed blank liposomes were mixed with CPD12C15 and incubated, and then filtered to obtain TPGS-modified CPD12C15 liposomes.

5. The method according to claim 4, characterized in that The organic solvent is a mixture of one or more of chloroform, methanol, and ethanol, preferably chloroform; Preferably, the rotary evaporation is carried out at 30-50°C, preferably 40°C; Preferably, the concentration of the calcium acetate solution is 80-200 mmol / L, preferably 120 mmol / L; Preferably, the ultrasonic treatment is carried out in an ice bath, with an ultrasonic power of 30-70 W, preferably 50 W, and is carried out in a manner of 2 seconds on and 2 seconds off, for a total time of 1-5 minutes, preferably 2 minutes; Preferably, the dialysis uses a dialysis bag with a molecular weight cut-off of 3000-5000Da, the dialysis medium is a sodium sulfate solution with an equal concentration to the calcium acetate solution, and the dialysis time is 8-16 hours, preferably 12 hours; Preferably, the incubation temperature is 20-50° C., preferably 30° C.; the incubation time is 30-90 minutes, preferably 60 minutes.

6. A pharmaceutical composition, characterized in that The invention comprises the TPGS-modified CPD12C15 liposome according to any one of claims 1 to 5 and a pharmaceutically acceptable carrier or excipient.

7. The pharmaceutical composition according to claim 6, characterized in that Also includes a copper salt, the copper salt is selected from one or more of copper chloride, copper sulfate, copper acetate and copper gluconate, preferably copper chloride or copper gluconate; Preferably, the molar ratio of CPD12C15 to copper ions is 1:0.3 to 1:3, preferably 1:1; Preferably, the pharmaceutical composition is in the form of an injection, a lyophilized powder injection or a liposomal suspension; Preferably, the content of CPD12C15 in the pharmaceutical composition is 0.05-20 mg / mL, preferably 0.5-5 mg / mL.

8. A medical kit, characterized in that: include: A first container containing the TPGS-modified CPD12C15 liposomes according to any one of claims 1 to 3; a second container containing a copper salt solution having a concentration of 50-500 μg / mL, preferably 100 μg / mL; and, Instructions for administration.

9. Use of the TPGS-modified CPD12C15 liposome according to any one of claims 1 to 3, the pharmaceutical composition according to claim 6 or 7, or the medical kit according to claim 8 in the preparation of a drug for treating non-small cell lung cancer.

10. The use according to claim 9, characterized in that The non-small cell lung cancer is drug-resistant non-small cell lung cancer, preferably non-small cell lung cancer that is resistant to docetaxel; Preferably, the drug is at least one of the following: (1) Drugs used to inhibit the proliferation of non-small cell lung cancer cells; (2) Drugs used to inhibit the migration of non-small cell lung cancer cells; (3) drugs used to inhibit the invasion of non-small cell lung cancer cells; (4) drugs used to induce apoptosis in non-small cell lung cancer cells; (5) Drugs used to inhibit the Warburg effect in non-small cell lung cancer.

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