Preparation method and application of carboplatin-unsaturated fatty acid liposome

Abstract

The invention belongs to the field of anti-tumor compounds, and particularly relates to a preparation method of carboplatin-unsaturated fatty acid liposome, which comprises the following steps: S1, preparing lecithin into a lecithin solution; preparing unsaturated fatty acid into a fatty acid solution; preparing carboplatin into a carboplatin solution; s2, adding a lecithin solution and a fatty acid solution into an eggplant-shaped bottle, then adding chloroform or dichloromethane, uniformly mixing, and carrying out rotary evaporation to obtain a phospholipid membrane; s3, taking and preheating a carboplatin solution, adding the carboplatin solution into the phospholipid membrane after preheating, and performing ultrasonic hydration to obtain a liposome solution; s4, performing ultrasonic crushing on the liposome solution; and S5, filtering the ultrasonically crushed liposome solution to obtain the liposome. The invention also discloses an application of the carboplatin-unsaturated fatty acid liposome prepared by the preparation method in preparation of antitumor drugs. The problem that in the prior art, the curative effect and safety of a carboplatin drug are difficult to consider at the same time can be solved, and metastatic tumor cells can be effectively inhibited at low drug concentration.

Description

Technical Field

[0001] This invention relates to the field of antitumor compounds, and more particularly to a method for preparing carboplatin-unsaturated fatty acid liposomes and their applications. Background Technology

[0002] Ovarian cancer is one of the leading causes of death among malignant tumors of the female reproductive system. It is characterized by its insidious early symptoms, with more than half of cases diagnosed at an advanced stage, often showing metastatic lesions in the peritoneum, omentum, and other sites. Carboplatin, as a second-generation platinum-based antitumor drug, is widely used in chemotherapy for ovarian cancer due to its lower nephrotoxicity and gastrointestinal toxicity compared to cisplatin, as well as better clinical tolerability. It is considered a first-line treatment for ovarian cancer.

[0003] In current clinical applications, carboplatin is typically administered intravenously as a water-soluble small molecule formulation. It distributes to tumor tissue via the bloodstream and inhibits tumor cell proliferation and induces cell death by forming adducts with tumor cell DNA. Carboplatin's entry into tumor cells mainly relies on passive diffusion or limited cell membrane transporter-mediated pathways. However, after ovarian cancer metastasizes, especially in peritoneal and omental metastases, the membrane structure and lipid composition of tumor cells change. The expression of transporters associated with platinum drug uptake decreases, while the expression of transporters associated with drug efflux increases. Simultaneously, metastatic tumor cells are often in a low-proliferative state or undergo metabolic reprogramming. These factors combined result in conventional carboplatin formulations being difficult for metastatic ovarian cancer cells to efficiently take up, leading to insufficient intracellular effective drug concentrations.

[0004] In addition, metastatic ovarian cancer cells usually have enhanced DNA damage repair capabilities and strong antioxidant defense systems, which increases their tolerance to single DNA damage stimuli. As a result, under conventional drug dosages, it is difficult to significantly increase the level of reactive oxygen species in metastatic tumor cells and effectively trigger oxidative stress-related cell death pathways, thus limiting the therapeutic sensitivity of carboplatin to metastatic ovarian cancer.

[0005] Therefore, in order to improve the therapeutic effect of carboplatin, it is often necessary to increase the dosage or extend the treatment period in clinical practice. However, this can easily lead to adverse reactions such as systemic toxicity and bone marrow suppression, making it difficult to balance efficacy and safety. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing carboplatin-unsaturated fatty acid liposomes and their application, so as to solve the technical problems mentioned in the background art.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A method for preparing carboplatin-unsaturated fatty acid liposomes includes the following steps: S1: Prepare a lecithin solution with a concentration of 60 mg / mL using a lecithin solvent; prepare a fatty acid solution with a concentration of 6 mg / mL using a fatty acid solvent; prepare a carboplatin solution with a concentration of 0.5-2 mg / mL using a carboplatin solvent; S2: Add 0.9 mL of lecithin solution and 1 mL of fatty acid solution to a flask, then add 3 mL of chloroform or dichloromethane, place it in an ultrasonic cleaner and mix it evenly by ultrasonication. After ultrasonic mixing, perform rotary evaporation to obtain a phospholipid membrane. S3: Take 6 mL of carboplatin solution, preheat it in an ultrasonic cleaner, add it to the phospholipid membrane after preheating, and perform ultrasonic hydration to obtain a liposome solution; S4: Transfer the liposome solution to a centrifuge tube and place it in an ultrasonic grinder for ultrasonic grinding; S5: Filter the liposome solution after ultrasonic pulverization in S4 to obtain the carboplatin-unsaturated fatty acid liposomes.

[0008] Furthermore, in S1, the unsaturated fatty acid is one of oleic acid, linoleic acid, linolenic acid, and arachidonic acid.

[0009] Furthermore, in S1, the lecithin solvent is chloroform or dichloromethane; the fatty acid solvent is chloroform or dichloromethane; and the carboplatin solvent is PBS or physiological saline.

[0010] Furthermore, in S2, the ultrasonic cleaner has a power of 120W and an ultrasonic time of 2 minutes; the rotary evaporation temperature is 35℃ and the rotary evaporation time is 15-30 minutes.

[0011] Furthermore, in S3, the ultrasonic cleaner has a power of 120W, and the carboplatin solution is preheated to 55℃; the ultrasonic hydration power is 120W, and the time is 5-30min.

[0012] Furthermore, in S4, the power of the ultrasonic pulverizer is 300W, and the ultrasonic pulverization time is 6 minutes.

[0013] Another object of the present invention is to provide the application of carboplatin-unsaturated fatty acid liposomes prepared by the above preparation method in the preparation of antitumor drugs.

[0014] The advantages of this invention compared to the prior art are as follows: 1. This invention enhances the uptake capacity of carboplatin liposomes in ovarian cancer metastatic cells by adding unsaturated fatty acids to liposomes, thereby increasing the effective drug concentration within the cells. Simultaneously, it promotes the increase of intracellular reactive oxygen species levels by increasing intracellular unsaturation, rapidly inducing immunogenic death in ovarian cancer metastatic cells. This achieves effective killing of metastatic ovarian cancer at lower drug concentrations and improves the immunosuppressive microenvironment of metastatic ovarian cancer, thus enhancing the efficacy of carboplatin against metastatic ovarian cancer through multiple mechanisms.

[0015] 2. This invention utilizes the affinity of metastatic ovarian cancer cells for fatty acids to enhance the uptake of drugs in metastatic ovarian cancer cells, thereby increasing the effective drug concentration within the cells. Within 2 hours of administration, the level of intracellular reactive oxygen species can be rapidly increased, and within 6 hours, more than 50% of metastatic tumor cells are rapidly induced to undergo immunogenic death. This invention treats metastatic ovarian cancer by promoting cell killing and enhancing immunogenicity, achieving effective inhibition of metastatic tumor cells at relatively low drug concentrations. Attached Figure Description

[0016] Figure 1 This is a process diagram of the preparation of the present invention; Figure 2 These are images of liposome products prepared according to the present invention; wherein, A is an image of the liposome product prepared in Example 1; B is an image of the liposome product prepared in Example 2; C is an image of the liposome product prepared in Example 3; D is an image of the liposome product prepared in Example 4; and E is an image of the liposome product prepared in Comparative Example 1. Figure 3 These are cell micropore imaging observations of ID8 metastatic tumor cells at different time points after administration of liposomes in each group of the present invention. Blue: cell nucleus; red: DiI-labeled liposomes; scale bar: 300 μm. Figure 4 This is a line graph showing the quantitative analysis of ID8 metastatic tumor cell uptake at different time points after liposome administration in each group of the present invention, n=3, ****: p<0.0001; Figure 5 This is a bar chart showing the quantitative analysis of the uptake ratio of ID8 metastatic tumor cells at different time points after liposome administration in each group of the present invention, n=3, ****: p<0.0001; Figure 6 These are cell micropore imaging observations of ID8 metastatic tumor cells at different time points after liposome administration in each group of the present invention. Blue: cell nucleus; green: ROS; scale bar: 300 μm. Figure 7 This is a line graph showing the quantitative analysis of ROS content in ID8 metastatic tumor cells at different time points after liposome administration in each group of the present invention. n=3, ns: no statistical difference, ****: p<0.0001; Figure 8 This is a bar chart showing the cell viability of ID8 metastatic tumor cells after liposome administration in each group of the present invention; Figure 9 This is a bar chart showing the half-maximal inhibitory concentration (IC50) of each group of liposomes administered to ID8 metastatic tumor cells after the invention. n=6, ns: no statistical difference, ****: p<0.0001; Figure 10 These are flow cytometry chromatograms of calreticulin detection in ID8 metastatic tumor cells after liposome administration in each group of the present invention; wherein, A is a flow cytometry chromatogram of calreticulin detection in ID8 metastatic tumor cells without drug administration; B is a flow cytometry chromatogram of calreticulin detection in ID8 metastatic tumor cells after CBP administration; C is a flow cytometry chromatogram of calreticulin detection in ID8 metastatic tumor cells after CBP-Lip administration; and D is a flow cytometry chromatogram of calreticulin detection in ID8 metastatic tumor cells after CBP-LNA-Lip administration. Figure 11 This is a bar chart showing the proportion of calreticulin eversion in ID8 metastatic tumor cells after liposome administration in each group of the present invention. ****: p<0.0001; Figure 12 This is a diagram illustrating the modeling and drug administration regimen of the ovarian cancer peritoneal metastasis model of the present invention; Figure 13 These are images showing the tumor metastasis in the greater omentum, peritoneum, and diaphragm of mice after administration of the drug in each group according to the present invention. Figure 14 This is a bar chart showing the number of tumor metastases in mice after administration of the drug in each group according to the present invention; n=5, *: p<0.05, ***: p<0.001, ****: p<0.0001; Figure 15 These are images of ascites volume in mice after administration of the drug in each group according to the present invention; Figure 16 This is a bar chart showing the ascites volume of mice after administration in each group according to the present invention, n=5, *: p<0.05, ***: p<0.001, ****: p<0.0001; Figure 17 These are H&E staining images of various tissues and organs of mice after administration of the drug in each group according to the present invention. Figure 18 The graphs show the renal function indicators of mice after each group of drugs administered according to the present invention, n=5, ns: no statistical difference, where A is a bar chart of urea nitrogen content and B is a bar chart of creatinine content. Figure 19 The graphs show the liver function indicators of mice after administration of the drug in each group according to the present invention. n=5, ns: no statistical difference, *: P<0.05; **: P<0.01; ***: P<0.001; where A is a bar chart of alanine aminotransferase content and B is a bar chart of aspartate aminotransferase content. Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and preferred embodiments. However, it should be noted that many details listed in the specification are merely to provide the reader with a thorough understanding of one or more aspects of the invention, and these aspects of the invention can be achieved even without these specific details.

[0018] like Figure 1 As shown, a method for preparing carboplatin-unsaturated fatty acid liposomes includes the following steps: S1: Prepare a lecithin solution with a concentration of 60 mg / mL using chloroform or dichloromethane; prepare a fatty acid solution with a concentration of 6 mg / mL using chloroform or dichloromethane; the unsaturated fatty acids include oleic acid, linoleic acid, linolenic acid, and arachidonic acid; prepare a carboplatin solution with a concentration of 0.5-2 mg / mL using phosphate buffered saline (PBS) or physiological saline. S2: Add 0.9 mL of lecithin solution and 1 mL of fatty acid solution to a flask, then add 3 mL of chloroform or dichloromethane, place it in an ultrasonic cleaner, and sonicate at 120 W at room temperature for 2 min to mix evenly. After ultrasonic mixing, rotary evaporate at 35 °C for 15-30 min to obtain a phospholipid film. S3: Take 6 mL of carboplatin solution and place it in an ultrasonic cleaner. Preheat the carboplatin solution to 55°C at a power of 120W. After preheating, add it to the phospholipid membrane and perform ultrasonic hydration at a power of 120W and room temperature for 5-30 minutes to obtain a liposome solution. S4: Transfer the liposome solution to a centrifuge tube, place it in an ultrasonic grinder, and ultrasonically grind it for 6 minutes at a power of 300W and room temperature. S5: The liposome solution after ultrasonic pulverization in S4 is filtered through a 0.22μm filter to obtain the carboplatin-unsaturated fatty acid liposomes.

[0019] The principle of the preparation method of this invention is as follows: S1: The purpose of this step is to prepare a homogeneous solution of each component. Lecithin, as the liposome framework material, ultimately forms a bilayer vesicle structure. Unsaturated fatty acids, as functional regulators, enhance membrane fluidity, promote cellular uptake, and induce ROS production. Carboplatin, as the main drug and the antitumor active ingredient, is ultimately encapsulated in the inner aqueous phase.

[0020] S2: The purpose of this step is to induce the self-assembly of phospholipid molecules through the evaporation of organic solvents, forming a dry phospholipid film and establishing a structural basis for subsequent hydration steps. Ultrasonic mixing utilizes the cavitation effect and mechanical vibration of ultrasound to thoroughly and uniformly mix lecithin and unsaturated fatty acids in the organic solvent, forming a homogeneous dispersion system at the molecular level. Rotary evaporation, under reduced pressure, rapidly evaporates chloroform or dichloromethane. After losing solvent support, the phospholipid molecules spontaneously align themselves into an ordered bilayer film through hydrophobic interactions and van der Waals forces. The hydrophilic head groups of the phospholipids face the aqueous phase, the hydrophobic fatty acid chains interweave inwards, and the unsaturated fatty acids insert between the phospholipid molecules, forming a mixed film structure.

[0021] S3: The purpose of this step is to hydrate and expand the dried phospholipid film, causing it to spontaneously curl into closed vesicles, while simultaneously encapsulating the carboplatin solution within the aqueous phase of the vesicles. Preheating: The carboplatin solution is preheated to 55 degrees Celsius, above the phospholipid phase transition temperature, to reduce the viscosity of the aqueous phase, increase molecular mobility, and promote hydration efficiency. Ultrasonic hydration: Ultrasonic energy provides the hydration driving force, allowing water molecules to penetrate into the interlayer of the phospholipid film. The phospholipid bilayer expands, bends, and closes, forming multilayered vesicles. The carboplatin solution is physically encapsulated within the aqueous phase inside the vesicles and in the interlayer aqueous phase.

[0022] S4: The purpose of this step is to break down and homogenize large-sized multilayer vesicles to meet the requirements of nanomedicine delivery. High-power ultrasound generates a strong cavitation effect and shear force, which physically breaks down the layered structure of the multilayer vesicles, causing large vesicles to split into smaller vesicles. The microjets and shock waves generated when the cavitation bubbles collapse cause the liposome membrane to deform, break down, and reorganize, ultimately forming thermodynamically more stable small vesicles.

[0023] S5: The purpose of this step is to remove large particulate impurities, control the final particle size distribution, and perform aseptic filtration to obtain a formulation that meets the requirements.

[0024] The following description uses more specific examples.

[0025] Example 1 A method for preparing carboplatin-unsaturated fatty acid liposomes includes the following steps: S1: Lecithin was prepared into a lecithin solution with a concentration of 60 mg / mL using dichloromethane; oleic acid (OA) was prepared into a fatty acid solution with a concentration of 6 mg / mL using dichloromethane; carboplatin was prepared into a carboplatin solution with a concentration of 0.5 mg / mL using PBS; S2: Add 0.9 mL of lecithin solution and 1 mL of fatty acid solution to a flask, then add 3 mL of dichloromethane, place it in an ultrasonic cleaner, and ultrasonically mix for 2 min at 120 W and room temperature. After ultrasonic mixing, rotary evaporate at 35 °C for 15 min to obtain a phospholipid film. S3: Take 6 mL of carboplatin solution and place it in an ultrasonic cleaner. Preheat the carboplatin solution to 55°C at a power of 120W. After preheating, add it to the phospholipid membrane and perform ultrasonic hydration at a power of 120W and room temperature for 30 min to obtain a liposome solution. S4: Transfer the liposome solution to a centrifuge tube, place it in an ultrasonic grinder, and ultrasonically grind it for 6 minutes at a power of 300W and room temperature. S5: The liposome solution after ultrasonic pulverization in S4 is filtered through a needle filter with a pore size of 0.22 μm to obtain the following... Figure 2 The carboplatin-oleic acid liposome (CBP-OA-Lip) shown in Figure A contains one unsaturated double bond.

[0026] Example 2 A method for preparing carboplatin-unsaturated fatty acid liposomes includes the following steps: S1: Lecithin was prepared into a lecithin solution with a concentration of 60 mg / mL using chloroform; linoleic acid (LA) was prepared into a fatty acid solution with a concentration of 6 mg / mL using chloroform; carboplatin was prepared into a carboplatin solution with a concentration of 0.5 mg / mL using PBS; S2: Add 0.9 mL of lecithin solution and 1 mL of fatty acid solution to a flask, then add 3 mL of chloroform, place it in an ultrasonic cleaner, and ultrasonically mix for 2 min at 120 W at room temperature. After ultrasonic mixing, rotary evaporate at 35 °C for 20 min to obtain a phospholipid film. S3: Take 6 mL of carboplatin solution and place it in an ultrasonic cleaner. Preheat the carboplatin solution to 55°C at a power of 120W. After preheating, add it to the phospholipid membrane and perform ultrasonic hydration at a power of 120W and room temperature for 20 min to obtain a liposome solution. S4: Transfer the liposome solution to a centrifuge tube, place it in an ultrasonic grinder, and ultrasonically grind it for 6 minutes at a power of 300W and room temperature. S5: The liposome solution after ultrasonic pulverization in S4 is filtered through a needle filter with a pore size of 0.22 μm to obtain the following... Figure 2 The carboplatin-linoleic acid liposome (CBP-LA-Lip) shown in Figure B contains two unsaturated double bonds.

[0027] Example 3 A method for preparing carboplatin-unsaturated fatty acid liposomes includes the following steps: S1: Lecithin was prepared into a lecithin solution with a concentration of 60 mg / mL using chloroform; linolenic acid (LNA) was prepared into a fatty acid solution with a concentration of 6 mg / mL using chloroform; carboplatin was prepared into a carboplatin solution with a concentration of 0.5 mg / mL using PBS; S2: Add 0.9 mL of lecithin solution and 1 mL of fatty acid solution to a flask, then add 3 mL of chloroform, place it in an ultrasonic cleaner, and ultrasonically mix for 2 min at 120 W and room temperature. After ultrasonic mixing, rotary evaporate at 35 °C for 30 min to obtain a phospholipid film. S3: Take 6 mL of carboplatin solution and place it in an ultrasonic cleaner. Preheat the carboplatin solution to 55°C at a power of 120W. After preheating, add it to the phospholipid membrane and perform ultrasonic hydration at a power of 120W and room temperature for 5 min to obtain a liposome solution. S4: Transfer the liposome solution to a centrifuge tube, place it in an ultrasonic grinder, and ultrasonically grind it for 6 minutes at a power of 300W and room temperature. S5: The liposome solution after ultrasonic pulverization in S4 is filtered through a needle filter with a pore size of 0.22 μm to obtain the following... Figure 2 The carboplatin-linolenic acid liposome (CBP-LNA-Lip) shown in C contains three unsaturated double bonds.

[0028] Example 4 A method for preparing carboplatin-unsaturated fatty acid liposomes includes the following steps: S1: Lecithin was prepared into a lecithin solution with a concentration of 60 mg / mL using dichloromethane; arachidonic acid (AA) was prepared into a fatty acid solution with a concentration of 6 mg / mL using dichloromethane; carboplatin was prepared into a carboplatin solution with a concentration of 0.5 mg / mL using PBS; S2: Add 0.9 mL of lecithin solution and 1 mL of fatty acid solution to a flask, then add 3 mL of dichloromethane, place it in an ultrasonic cleaner, and ultrasonically mix for 2 min at 120 W and room temperature. After ultrasonic mixing, rotary evaporate at 35 °C for 20 min to obtain a phospholipid film. S3: Take 6 mL of carboplatin solution and place it in an ultrasonic cleaner. Preheat the carboplatin solution to 55°C at a power of 120W. After preheating, add it to the phospholipid membrane and perform ultrasonic hydration at a power of 120W and room temperature for 15 min to obtain a liposome solution. S4: Transfer the liposome solution to a centrifuge tube, place it in an ultrasonic grinder, and ultrasonically grind it for 6 minutes at a power of 300W and room temperature. S5: The liposome solution after ultrasonic pulverization in S4 is filtered through a needle filter with a pore size of 0.22 μm to obtain the following... Figure 2 The carboplatin-arachidonic acid liposome (CBP-AA-Lip) shown in D contains four unsaturated double bonds.

[0029] Example 5 A method for preparing carboplatin-unsaturated fatty acid liposomes includes the following steps: S1: Lecithin was prepared into a lecithin solution with a concentration of 60 mg / mL using dichloromethane; linolenic acid (LNA) was prepared into a fatty acid solution with a concentration of 6 mg / mL using dichloromethane; carboplatin was prepared into a carboplatin solution with a concentration of 2 mg / mL using physiological saline. S2: Add 0.9 mL of lecithin solution and 1 mL of fatty acid solution to a flask, then add 3 mL of dichloromethane, place it in an ultrasonic cleaner, and ultrasonically mix for 2 min at 120 W and room temperature. After ultrasonic mixing, rotary evaporate at 35 °C for 20 min to obtain a phospholipid film. S3: Take 6 mL of carboplatin solution and place it in an ultrasonic cleaner. Preheat the carboplatin solution to 55°C at a power of 120W. After preheating, add it to the phospholipid membrane and perform ultrasonic hydration at a power of 120W and room temperature for 30 min to obtain a liposome solution. S4: Transfer the liposome solution to a centrifuge tube, place it in an ultrasonic grinder, and ultrasonically grind it for 6 minutes at a power of 300W and room temperature. S5: The liposome solution after ultrasonic pulverization in S4 is filtered through a needle filter with a pore size of 0.22 μm to obtain carboplatin-linolenic acid liposomes (CBP-LNA-Lip) containing 3 unsaturated double bonds.

[0030] Example 6 A method for preparing carboplatin-unsaturated fatty acid liposomes includes the following steps: S1: Lecithin was prepared into a lecithin solution with a concentration of 60 mg / mL using dichloromethane; linolenic acid (LNA) was prepared into a fatty acid solution with a concentration of 6 mg / mL using dichloromethane; carboplatin was prepared into a carboplatin solution with a concentration of 2 mg / mL using PBS; S2: Add 0.9 mL of lecithin solution and 1 mL of fatty acid solution to a flask, then add 3 mL of dichloromethane, place it in an ultrasonic cleaner, and ultrasonically mix for 2 min at 120 W and room temperature. After ultrasonic mixing, rotary evaporate at 35 °C for 15 min to obtain a phospholipid film. S3: Take 6 mL of carboplatin solution and place it in an ultrasonic cleaner. Preheat the carboplatin solution to 55°C at a power of 120W. After preheating, add it to the phospholipid membrane and perform ultrasonic hydration at a power of 120W and room temperature for 15 min to obtain a liposome solution. S4: Transfer the liposome solution to a centrifuge tube, place it in an ultrasonic grinder, and ultrasonically grind it for 6 minutes at a power of 300W and room temperature. S5: The liposome solution after ultrasonic pulverization in S4 is filtered through a needle filter with a pore size of 0.22 μm to obtain carboplatin-linolenic acid liposomes (CBP-LNA-Lip) containing 3 unsaturated double bonds.

[0031] Comparative Example 1 The results are basically the same as in Example 1, except that in S1, unsaturated fatty acids were not added, and the prepared product was as follows. Figure 2 The carboplatin liposome (CBP-Lip) shown in E is free of unsaturated fatty acids.

[0032] 1. Characterization of carboplatin-unsaturated fatty acid liposomes The liposomes prepared in Examples 1-4 and Comparative Example 1 were characterized by measuring particle size, PDI and potential. The results are shown in Table 1.

[0033] Table 1. Characterization results of liposomes with different carboplatin-unsaturated fatty acids As shown in Table 1, the particle size of all liposomes was around 100 nm, which is within the ideal particle size range for liposomes; the PDI was less than 0.3, indicating uniform particle size distribution and good formulation quality; and the potential was around -50 mV, demonstrating excellent stability. Furthermore, the addition of different unsaturated fatty acids did not significantly affect the formation, particle size, or potential of the liposomes, indicating that the addition of unsaturated fatty acids does not alter the physical properties of the liposomes and that high-performance liposomes can be prepared.

[0034] 2. In vitro experiments of carboplatin-unsaturated fatty acid liposomes Examples 1-4 and Comparative Example 1 were divided into CBP-OA-Lip group, CBP-LA-Lip group, CBP-LNA-Lip group, CBP-AA-Lip group and CBP-Lip group for in vitro experiments.

[0035] 2.1 Uptake of carboplatin-unsaturated fatty acid liposomes An ID8 peritoneal metastatic tumor model was established in C57BL female mice. ID8 metastatic tumor cells were extracted from ascites fluid and cultured. After drug administration, liposomes in each group were stained with DiI dye. Cell micropore imaging and flow cytometry were used to quantify the uptake (calculated as the mean fluorescence intensity MFI of DiI) and the percentage of cellular uptake, examining the uptake of liposomes by metastatic tumor cells. The results are as follows: Figure 3-5 As shown.

[0036] like Figure 3As shown, the number and intensity of red fluorescence of all liposomes increased over time, and the number and intensity of red fluorescence of all liposomes containing unsaturated fatty acids were higher than those of the CBP-Lip group without fatty acids at all time points.

[0037] The results are as follows Figure 4-5 As shown, the intake and rate of liposomes containing unsaturated fatty acids were significantly higher than those of the CBP-Lip group without unsaturated fatty acids. Meanwhile, the percentage and amount of intake of the CBP-LA-Lip group, CBP-LNA-Lip group, and CBP-AA-Lip group containing multiple double bonds were higher than those of the CBP-OA-Lip group containing a single double bond.

[0038] This indicates that adding unsaturated fatty acids to liposomes can promote the uptake of liposomes by ID8 metastatic tumor cells, and that polyunsaturated fatty acids are superior to monounsaturated fatty acids. However, more double bonds in polyunsaturated fatty acids are not necessarily better. Among polyunsaturated fatty acids, the CBP-LNA-Lip group containing linolenic acid with three double bonds has the strongest uptake-promoting ability, and its uptake is stronger than all other liposomes throughout the entire time period.

[0039] 2.2 Investigation into the ability of unsaturated fatty acid-carboplatin liposomes to induce ROS production in ID8 metastatic tumor cells An ID8 peritoneal metastatic tumor model was established in C57BL female mice. ID8 metastatic tumor cells were extracted from ascites and cultured. Carboplatin raw material (CBP) was designated as the CBP group as a control. DCFH-DA was used as a ROS probe to detect the ROS level of ID8 metastatic tumor cells in each group after drug administration. The ROS fluorescence of metastatic tumor cells in each group was observed by micropore imaging 2-6 h after drug administration, and the ROS content was quantified by flow cytometry. The results are as follows: Figure 6-7 As shown.

[0040] from Figure 6 It can be seen that the number of green fluorescent cells and fluorescence intensity induced by the CBP-OA-Lip group, CBP-LA-Lip group, CBP-LNA-Lip group, and CBP-AA-Lip group containing unsaturated fatty acids were higher than those of the CBP-Lip group and CBP group without fatty acids throughout the entire time period. Furthermore, the number of green fluorescent cells and fluorescence intensity induced by the CBP-LA-Lip group, CBP-LNA-Lip group, and CBP-AA-Lip group containing multiple double bonds were higher than those of the CBP-OA-Lip group containing a single double bond throughout the entire time period.

[0041] from Figure 7As can be seen, the ROS content of liposomes containing unsaturated fatty acids was significantly higher than that of other groups. Among them, the ROS content of liposomes containing multiple double bonds was significantly higher than that of the CBP-OA-Lip group containing a single double bond. However, there was no statistically significant difference in ROS content among the CBP, CBP-Lip and CBP-OA-Lip groups. This indicates that unsaturated fatty acids containing multiple double bonds in liposomes are a key factor in promoting ROS production in ID8 metastatic tumor cells.

[0042] This indicates that adding unsaturated fatty acids to liposomes can enhance the induction of ROS production in ID8 metastatic tumor cells, and polyunsaturated fatty acids are superior to monounsaturated fatty acids. However, more double bonds in polyunsaturated fatty acids are not necessarily better. Among polyunsaturated fatty acids, the CBP-LNA-Lip group, which contains linolenic acid with three double bonds, has the strongest ROS induction effect. The ROS content induced at 2, 4, and 6 hours after administration is 13.47, 30.81, and 42.04 times that of CBP, respectively.

[0043] 2.3. Toxicity of unsaturated fatty acid-carboplatin liposomes against ID8 metastatic tumor cells 6×10⁶ mice were injected intraperitoneally into female C57BL / 6J mice. 6 ID8 ovarian cancer cells were collected, and primary ID8 metastatic tumor cells were extracted after the formation of substantial ascites. Carboplatin (CBP) was used as a control group. However, considering that arachidonic acid is an inflammatory mediator and its ability to induce ROS and its safety, its killing ability against metastatic ovarian cancer cells was not further investigated. Cell viability and half-maximal inhibitory concentration (IC50) of metastatic tumor cells in the CBP group and other groups 48 hours after liposome administration were determined using the CCK-8 assay. 50 The toxicity of ID8 metastatic tumor cells was examined, and the results were as follows: Figure 8-9 As shown.

[0044] from Figure 8 As can be seen, when the carboplatin dose is greater than 108 μM, all carboplatin liposomes containing unsaturated fatty acids have a stronger killing effect on ID8 ovarian cancer metastases than the CBP group and the CBP-Lip group, while the toxicity of CBP-LNA-Lip is higher than that of other groups at all administration concentrations.

[0045] Further analysis of IC50 in each treatment group 50 Perform calculations, from Figure 9 As can be seen, the IC50 of the CBP-Lip group, which does not contain unsaturated fatty acids, is... 50 The IC50 value was 411.52±26.26 μM, which was not significantly different from the 426.3±12.68 μM value of the CBP group containing the active pharmaceutical ingredient. However, the IC50 values ​​of the CBP-OA-Lip group, CBP-LA-Lip group, and CBP-LNA-Lip group containing unsaturated fatty acids were significantly higher. 50The concentrations were 277.2±8.90μM, 157.68±8.07μM, and 98.28±7.27μM, respectively, which were significantly lower than those of the active pharmaceutical ingredient.

[0046] The results showed that adding unsaturated fatty acids to liposomes could significantly improve the toxicity of carboplatin to metastatic ovarian cancer cells, with LNA being the best, increasing toxicity by 4.19 times.

[0047] 2.4. Effect of unsaturated fatty acid-carboplatin liposomes on inducing immunogenic cell death in ID8 metastatic tumor cells 6×10⁶ mice were injected intraperitoneally into female C57BL / 6J mice. 6 ID8 ovarian cancer cells were collected, and after the formation of a large amount of ascites, primary ID8 metastatic tumor cells were extracted. After treatment with 108 μM CBP, CBP-Lip group, and CBP-LNA-Lip group for 6 h, ID8 metastatic tumor cells were collected. At the same time, untreated ID8 metastatic tumor cells were collected as a control. After incubation with calreticulin (CRT) flow cytometry antibody for 30 min, the cells were detected by flow cytometry.

[0048] from Figure 10-11 It can be seen from this that Figure 10 The D-value in the study showed that the CBP-LNA-Lip group induced immunogenic death in ID8 cells, manifested as calreticulin eversion. Figure 11 The results showed that the proportion of CRT-positive cells in the CBP-LNA-Lip group increased to 55.36%, which was 325, 798, and 1114 times that of the ID8 cells, CBP, and CBP-Lip groups, respectively.

[0049] 3. In vivo experiments of carboplatin-unsaturated fatty acid liposomes As can be seen from the in vitro experiments, the CBP-LNA-Lip group of Example 3 showed the best killing effect on metastatic ovarian cancer in vitro. Therefore, this liposome was used for in vivo experiments. However, since the amount of carboplatin required for in vivo experiments was relatively high, the CBP-LNA-Lip group of Example 6 was used instead.

[0050] 6×10⁶ mice were injected intraperitoneally into female C57BL / 6J mice. 6 An ovarian cancer peritoneal metastasis model was created using ID8 ovarian cancer cells. Five animals were divided into groups: the CBP-LNA-Lip group (Example 6), the CBP-Lip group (Comparative Example 1) without unsaturated fatty acid liposomes, the carboplatin raw material group (CBP), and the PBS buffer group (PBS).

[0051] Mice developed ascites on day 21 after modeling. They were then administered the drug at a dose of 25 mg / mL, once every 3 days for a total of 5 administrations. Mice were sacrificed 38 days after modeling. The modeling and administration regimens are as follows: Figure 12 As shown.

[0052] 3.1 In vivo efficacy of unsaturated fatty acid-carboplatin liposomes in an ID8 metastatic tumor model After euthanizing the mice, the metastatic tumors in all organs of the abdominal cavity were counted, and the ascites fluid was collected and its volume was calculated. The results are as follows: Figure 13-16 As shown.

[0053] Experiments have shown that in mice, the most common sites of metastasis are the greater omentum, peritoneum, and diaphragm. Figure 13 As shown in the figure, the white arrows point to larger metastatic lesions. The greater omentum, peritoneum and diaphragm of the PBS group, CBP group and CBP-Lip group all have different degrees of metastasis, while the CBP-LNA-Lip group has no obvious large metastatic lesions in these three organs.

[0054] from Figure 14 As can be seen from the statistical results of the number of metastatic tumors, the number of metastases in the CBP-LNA-Lip group was significantly lower than that in other groups, approximately 11 times, 5.75 times, and 4.25 times lower than that in the PBS group, CBP group, and CBP-Lip group, respectively.

[0055] from Figure 15-16 As can be seen, the ascites volume in the CBP-LNA-Lip group was significantly lower than that in the other groups, indicating that the CBP-LNA-Lip group can significantly reduce the production of malignant ascites.

[0056] This indicates that the CBP-LNA-Lip group effectively inhibits the metastasis of ovarian cancer and the production of malignant ascites in vivo.

[0057] 3.2 In vivo safety study of unsaturated fatty acid-carboplatin liposomes in an ID8 metastatic tumor model.

[0058] H&E pathological staining was performed on the major organs of mice in each group: heart, liver, spleen, lungs, and kidneys. The results are as follows: Figure 17 As shown in the figure. Liver function was evaluated after drug administration using aspartate aminotransferase (AST) and alanine aminotransferase (ALT), while kidney function was evaluated using blood urea nitrogen (BUN) and creatinine (Crea). A healthy control group was added for comparison. The results are as follows. Figure 18-19 As shown.

[0059] from Figure 17 As can be seen, there was no difference in the H&E staining pathological sections of the major organs of mice in each group, and no pathological conditions such as disordered cell arrangement and cell vacuoles were observed. That is, the CBP-LNA-Lip group does not cause pathological damage to major organs such as heart, liver, spleen, lungs and kidneys, and has good safety.

[0060] from Figure 18-19 As can be seen, CBP and CBP-Lip groups cause an increase in aspartate aminotransferase, such as Figure 19 As shown in B, all indicators in the CBP-LNA-Lip group were not different from those in the healthy group after administration, indicating that it improved the adverse effects of CBP administration on liver and kidney function.

[0061] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing carboplatin-unsaturated fatty acid liposomes, characterized in that, Includes the following steps: S1: Prepare a lecithin solution with a concentration of 60 mg / mL using a lecithin solvent; prepare a fatty acid solution with a concentration of 6 mg / mL using a fatty acid solvent; prepare a carboplatin solution with a concentration of 0.5-2 mg / mL using a carboplatin solvent; S2: Add 0.9 mL of lecithin solution and 1 mL of fatty acid solution to a flask, then add 3 mL of chloroform or dichloromethane, place it in an ultrasonic cleaner and mix it evenly by ultrasonication. After ultrasonic mixing, perform rotary evaporation to obtain a phospholipid membrane. S3: Take 6 mL of carboplatin solution, preheat it in an ultrasonic cleaner, add it to the phospholipid membrane after preheating, and perform ultrasonic hydration to obtain a liposome solution; S4: Transfer the liposome solution to a centrifuge tube and place it in an ultrasonic grinder for ultrasonic grinding; S5: Filter the liposome solution after ultrasonic pulverization in S4 to obtain the carboplatin-unsaturated fatty acid liposomes.

2. The method for preparing carboplatin-unsaturated fatty acid liposomes according to claim 1, characterized in that: In S1, the unsaturated fatty acid is one of oleic acid, linoleic acid, linolenic acid, and arachidonic acid.

3. The method for preparing carboplatin-unsaturated fatty acid liposomes according to claim 1, characterized in that: In S1, the lecithin solvent is chloroform or dichloromethane; the fatty acid solvent is chloroform or dichloromethane; and the carboplatin solvent is PBS or physiological saline.

4. The method for preparing carboplatin-unsaturated fatty acid liposomes according to claim 1, characterized in that: In S2, the ultrasonic cleaner has a power of 120W and an ultrasonic time of 2 minutes; the rotary evaporation temperature is 35℃ and the rotary evaporation time is 15-30 minutes.

5. The method for preparing carboplatin-unsaturated fatty acid liposomes according to claim 1, characterized in that: In S3, the ultrasonic cleaner has a power of 120W, and the carboplatin solution is preheated to 55℃; the ultrasonic hydration power is 120W, and the time is 5-30min.

6. The method for preparing carboplatin-unsaturated fatty acid liposomes according to claim 1, characterized in that: In S4, the ultrasonic pulverizer has a power of 300W and an ultrasonic pulverization time of 6 minutes.

7. The use of carboplatin-unsaturated fatty acid liposomes prepared by the method according to any one of claims 1-6 in the preparation of antitumor drugs.

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