Application of lipidosome with high phase transition temperature

Abstract

The invention discloses an application of a high-phase-transition-temperature liposome, and particularly relates to an application of the high-phase-transition-temperature liposome in preparation of a medicine for treating diseases related to interburial effects of phagocytes. The lipidosome with different phase-change temperatures is prepared by selecting phospholipid materials with different phase-change temperatures through a film dispersion method, and the influence of the lipidosome with different phase-change temperatures on the interburial effect of phagocytes is further investigated. Results show that the DPPC and DPSC lipidosome with the high phase transition temperature can be used as a carrier material, the apoptotic cell phagocytosis capacity of phagocytes is remarkably enhanced, the cell burial effect is promoted, and the problem of plaque accumulation caused by cell burial damage in atherosclerosis can be relieved.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine technology, and in particular relates to the application of high phase transition temperature liposomes in the preparation of medicines for diseases related to phagocytic cell efferocytosis. Background Art

[0002] Atherosclerosis (AS) is the leading cause of cardiovascular disease with the highest number of deaths worldwide, and the continuous accumulation of apoptotic cells and necrotic cell fragments is an important cause of the formation of necrotic cores and vulnerable plaques in AS. The process of professional phagocytes (such as macrophages) or non-professional phagocytes (such as neighboring cells) of the body to engulf and remove apoptotic cells is called efferocytosis. Efferocytosis is a necessary process for maintaining tissue homeostasis in normal physiological state and restoring tissue homeostasis after disease state (Doran, AC et al. Nat Rev Immunol 2020, 20 (4), 254-267.). Removing apoptotic cells and necrotic cells is an important way to reduce the area of ​​AS plaques and maintain plaque stability. Therefore, restoring the damaged efferocytosis in AS plaques and removing apoptotic cells in time are of great clinical significance for the treatment of atherosclerosis.

[0003] At present, there are no drugs or treatments that can restore the damaged phagocytic function in clinical practice. Studies have shown that the process of macrophages ingesting nanoparticles affects the cell membrane morphology, physiological function and phagocytic function of macrophages. In addition, Wang et al. (Wang Y, et al. Sci Adv. 2024, Jun 7; 10 (23)) found that hardness can increase the phagocytic function of macrophages. Therefore, it is beneficial to find a carrier material with suitable hardness to promote the phagocytic function of macrophages, which is beneficial to the treatment of damaged phagocytic function in atherosclerosis. Summary of the invention

[0004] The purpose of the present invention is to provide the use of high phase transition temperature liposomes in the preparation of medicines for diseases related to phagocytic cell efferocytosis.

[0005] Furthermore, the drug promotes phagocytosis of phagocytes.

[0006] Furthermore, the disease associated with phagocyte efferocytosis is atherosclerosis.

[0007] Furthermore, the raw materials of the high phase transition temperature liposomes include phospholipids and DSPE-PEG 2000 Phospholipids and DSPE-PEG 2000 The mass ratio is 19:1.

[0008] Preferably, the phospholipid is dipalmitoylphosphatidylcholine (DPPC) or distearoylphosphatidylcholine (DSPC), more preferably DSPC.

[0009] Furthermore, the raw material of the high phase transition temperature liposome also includes cholesterol, and the mass of cholesterol does not exceed 40% of the total mass of the liposome.

[0010] The hardness of liposomes is related to the phase transition temperature (T m ), the higher the phase transition temperature of phospholipids, the greater the hardness of the liposomes. Therefore, the present invention is designed to construct liposomes with different phase transition temperatures to investigate the effect of promoting phagocytic cell efferocytosis, and further use it to treat impaired efferocytosis in atherosclerosis.

[0011] The present invention selects dioleoylphosphatidylcholine (DOPC, T m =-20°C), dimyristoylphosphatidylcholine (DMPC, T m =23°C), dipalmitoylphosphatidylcholine (DPPC, T m =41°C), distearoylphosphatidylcholine (DSPC, T m =53°C) were prepared by thin film dispersion method to obtain liposomes with different phase transition temperatures, and the effects of liposomes with different phase transition temperatures on phagocytic efferocytosis were further investigated. The experimental results show that DPPC and DPSC liposomes with high phase transition temperatures can be used as carrier materials, significantly enhancing the ability of phagocytes to engulf apoptotic cells, promoting efferocytosis, and helping to alleviate the problem of plaque accumulation caused by impaired efferocytosis in atherosclerosis. In addition, the high phase transition temperature liposomes constructed by the present invention are blank liposomes, which can also be further loaded with active drugs for the treatment of diseases related to phagocytic efferocytosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Particle size distribution and zeta potential of liposomes prepared from different phospholipids.

[0013] Figure 2 is the phase transition temperature of liposomes prepared from different phospholipids.

[0014] Figure 3 Effects of liposomes prepared with different phospholipids on the clearance of apoptotic cells by macrophage efferocytosis.

[0015] Figure 4 The effect of different amounts of DPPC liposomes on the clearance of apoptotic cells by macrophage efferocytosis.

[0016] Figure 5 The effect of different amounts of DSPC liposomes on the clearance of apoptotic cells by macrophage efferocytosis.

[0017] Figure 6 Particle size distribution and Zeta potential of DSPC liposomes with different cholesterol contents.

[0018] Figure 7 The effect of DSPC liposomes with different cholesterol contents on the clearance of apoptotic cells by macrophages. DETAILED DESCRIPTION

[0019] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0020] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0021] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0022] Examples 1-4

[0023] Liposome preparation with different phospholipids

[0024] 76 mg of DSPC / DPPC / DMPC / DOPC phospholipids and 4 mg of DSPE-PEG 2000 Dissolve in 3 mL of chloroform. Add the phospholipid solution to a 100 mL eggplant-shaped bottle, remove the organic solvent under reduced pressure at 37°C to form a lipid film, and then vacuum dry for 4 hours. Hydrate the resulting lipid film with 2 mL of PBS (1×, pH 7.4) at 60°C for 10 min. Extrude the resulting liposomes 13 times under a 400 nm film and then 13 times under a 200 nm film to obtain liposomes.

[0025] 1. Determine the particle size distribution and Zeta potential of liposomes prepared with different phospholipids. The test results are as follows: Figure 1 As shown, the particle size of liposomes prepared by different phospholipids is similar, about 200nm, which meets the conditions for in vivo administration. Liposomes prepared by different phospholipids are all negatively charged and are not easy to produce toxicity to cells.

[0026] 2. Differential scanning calorimetry (DSC) is used to measure the phase transition temperature (T m ). 10 μL of liposome preparation was placed on an aluminum pan, which was then sealed and heated at a rate of 1°C / min. The scanning results were recorded in the temperature range of -30°C to 60°C. Figure 2 As shown, the phase transition temperatures of liposomes prepared from different phospholipids are the same as those of the corresponding phospholipid materials.

[0027] 3. Evaluation of the effects of different phospholipid liposomes on macrophage efferocytosis and clearance of apoptotic cells at the cellular level

[0028] CellTracker Deep Red was used to label macrophages RAW264.7, and CellTracker Orange was used to label RAW264.7 apoptotic cells. Liposomes prepared with different types of phospholipids were added to 180,000 macrophages RAW264.7 and 900,000 apoptotic cells, so that the final concentration of liposome particles was 2.5×10 3 1 macrophage, mixed evenly in 500 μL of DMEM medium, and then placed in an incubator at 37°C and 5% CO2 for 4 hours. After the incubation, the proportion of RAW264.7 macrophages in the double positive area was detected by flow cytometry to determine the efferocytosis effect.

[0029] The experimental results are as follows Figure 3 As shown, liposomes prepared with DPPC and DSPC phospholipids have a significant ability to promote macrophage efferocytosis and clearance of apoptotic cells.

[0030] 4. Evaluation of the effects of different concentrations of DPPC liposomes and DSPC liposomes on the clearance of apoptotic cells by macrophage efferocytosis at the cellular level

[0031] Macrophages RAW264.7 were labeled with CellTracker Deep Red, and apoptotic cells were labeled with CellTrackerOrange. 180,000 macrophages RAW264.7 and 900,000 apoptotic cells were added with different numbers of DPPC liposomes or DSPC liposomes to make the final concentration of liposome particles 0 and 0.25×10 3 , 0.5×10 3 , 1.25×10 3 , 2.5×10 3 , 5×10 3 , 12.5×10 3 , 25×10 3 1 macrophage, mixed evenly in 500 μL of DMEM medium, and then placed in an incubator at 37°C and 5% CO2 for 4 hours. After the incubation, the proportion of RAW264.7 macrophages in the double positive area was detected by flow cytometry to determine the efferocytosis.

[0032] The experimental results are as follows Figure 4 and Figure 5 As shown, liposomes prepared from DPPC and DSPC phospholipids were 3 -2.5×10 3It has the ability to significantly promote macrophage efferocytosis and clear apoptotic cells in the concentration range of 1 macrophage / 1 macrophage.

[0033] Example 5

[0034] Preparation of DSPC liposomes with different cholesterol contents

[0035] According to the prescription in Table 1, DSPC, DSPE-PEG 2000 Dissolve with cholesterol in 3 mL of chloroform. Add the phospholipid solution to a 100 mL eggplant-shaped bottle, remove the organic solvent under reduced pressure at 37°C to form a lipid film, and then vacuum dry for 4 hours. Hydrate the obtained lipid film with 2 mL of PBS at 60°C for 10 minutes. Extrude the obtained liposomes 13 times under a 400 nm film and then 13 times under a 200 nm film to obtain liposomes.

[0036] The particle size distribution, Zeta potential and cholesterol content of liposomes with different cholesterol contents were measured. The test results are shown in Table 2 and Figure 6 As shown, the prepared liposomes with different cholesterol contents were detected by HPLC, and the actual cholesterol content in the liposomes was close to the theoretical value, proving that the DSPC liposomes with different cholesterol contents were successfully prepared. The particle size of the DSPC liposomes containing different cholesterol contents was similar, about 200nm, which met the conditions for in vivo administration. Liposomes prepared from different phospholipids were all negatively charged and not easy to produce toxicity to cells.

[0037] Table 1 DSPC liposome formulations with different cholesterol contents

[0038]

[0039] Table 2 Actual cholesterol concentration in DSPC liposomes with different cholesterol contents determined by HPLC

[0040]

[0041] Evaluation of the effect of DSPC liposomes with different cholesterol contents on the clearance of apoptotic cells by macrophage efferocytosis at the cellular level

[0042] Macrophages RAW264.7 were labeled with CellTracker Deep Red, and apoptotic cells were labeled with CellTrackerOrange. Liposomes prepared with different types of phospholipids were added to 180,000 macrophages RAW264.7 and 900,000 apoptotic cells, so that the final concentration of liposome particles was 2.5×10 31 macrophage, mixed evenly in 500 μL DMEM medium, and then placed in an incubator at 37°C and 5% CO2 for 4 hours. After the incubation, the proportion of RAW264.7 macrophages in the double positive area was detected by flow cytometry to determine the cell burial effect. The experimental results are shown in Figure 7 As shown, DSPC liposomes with different cholesterol contents have a significant ability to promote macrophage efferocytosis and clearance of apoptotic cells.

Claims

1. Application of high phase transition temperature liposomes in the preparation of drugs for diseases related to phagocyte efferocytosis.

2. The use according to claim 1, characterized in that: The drug promotes phagocytosis of phagocytes.

3. The use according to claim 2, characterized in that: The disease associated with phagocyte efferocytosis is atherosclerosis.

4. The use according to claim 1, characterized in that: The raw materials of the high phase transition temperature liposomes include phospholipids and DSPE-PEG 2000 , phospholipids and DSPE-PEG 2000 The mass ratio is 19:

1.

5. The use according to claim 4, characterized in that: The phospholipid is dipalmitoylphosphatidylcholine or distearoylphosphatidylcholine.

6. The use according to claim 4, characterized in that: The raw materials of the high phase transition temperature liposome also include cholesterol, and the mass of cholesterol does not exceed 40% of the total mass of the liposome.

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