Colchicine lipidosome for dual-modified targeted M1 type macrophages and preparation method of colchicine lipidosome

Abstract

The invention discloses colchicine lipidosome for dual-modified targeted M1 type macrophages and a preparation method of the colchicine lipidosome. The colchicine lipidosome is prepared from the following components in parts by weight: 0.1 to 2 parts of colchicine, 10 to 40 parts of hydrogenated bean phosphatidylcholine, 1 to 20 parts of cholesterol, 1 to 5 parts of phosphatidyl ethanolamine, 0.4 to 2 parts of phospholipid polyethylene glycol folic acid, 0.4 to 2 parts of phospholipid polyethylene glycol arginine and 10 to 100 parts of buffer solution. The colchicine lipidosome of the dual-modified targeted M1 type macrophage prepared by the invention has good targeting property, PDI and target particle size, and has a longer half-life period, the toxicity of colchicine is reduced, and the anti-inflammatory curative effect of colchicine is enhanced.

Description

Technical Field

[0001] The invention belongs to the technical field of liposome preparation, and particularly relates to a double-modified colchicine liposome targeting M1 macrophages and a preparation method thereof. Background Art

[0002] Colchicine is a tricyclic alkaloid, originally extracted from the lily family plant Colchicum, and has multiple pharmacological effects, such as reducing the levels of cytokines such as IL-1β, IL-6, and TNF-α, reducing inflammatory cell infiltration, and inhibiting sodium urate-induced arthritis. Therefore, it can be used as an effective drug for the treatment of gouty arthritis, relieve pain, and subside inflammation. It can also fight pancreatitis, hepatitis, and pneumonia. Colchicine is currently approved by the FDA for acute gout and familial Mediterranean fever. Colchicine is closely related to macrophages. Colchicine inhibits the polymerization of microtubules, thereby reducing the generation of macrophage ROS and the release of NO and IL-1β, inhibiting macrophage chemotaxis, migration, and adhesion, inhibiting the activation of macrophage NLRP3 inflammasomes, and inhibiting the transformation of macrophages to M1 type, promoting macrophage polarization to M2 type and reducing macrophage pyroptosis, etc. to exert anti-inflammatory effects. However, the application of colchicine also has some limitations, such as a narrow therapeutic window and more side effects, such as gastrointestinal reactions such as nausea, vomiting, and diarrhea, as well as bone marrow suppression, liver and kidney function damage, etc.; the effective plasma steady-state concentration of colchicine is close to the toxic concentration, and taking 7 mg per day can cause death. Therefore, the dose used clinically is difficult to reach its maximum therapeutic dose and it is difficult to exert its maximum anti-inflammatory effect.

[0003] Liposomes are a bilayer structure composed of hydrophilic groups and hydrophobic groups. They are highly targeted and can deliver drugs directly to inflammatory tissues, reducing damage to normal tissues. They can effectively protect the encapsulated drugs and controllably release the drugs, prolonging the drug's action time in the body. They are easily degraded in the body and significantly improve the drug's therapeutic effect. At the same time, by controlling the particle size of liposomes, the drug's distribution behavior in the body can be improved, reducing toxic side effects.

[0004] Wang Linzhen encapsulated colchicine in liposomes using active drug loading method, and the encapsulation rate was measured to be 58.9%. The optimal preparation process was colchicine 0.5 mg / mL, phospholipid 50 mg / mL, cholesterol 8 mg / mL, VB 1 mg / mL, external water phase pH 6.4, incubation time 10 min, and the order of drug addition during drug loading was to first add blank liposomes to the drug solution, and then add NaHCO3 to adjust the pH value (Wang Linzhen, Preparation of colchicine liposomes. Strait Pharmacy, 2011, 23 (7): 27-29.). However, its encapsulation rate was low and it had no targeting.

[0005] M1 macrophages are activated by IFN-γ and TNF, mainly mediating cellular immunity and anti-infection activities, and have the effects of killing microorganisms, participating in inflammation and anti-tumor. M1 macrophages highly upregulate folate receptor β (FR-β). This differential expression of FR-β provides a way to selectively deliver therapeutic drugs using folate as a targeting molecule. In addition, L-arginine plays a key role in regulating the immune function of macrophages, and the receptor (CAT-2) that mediates L-arginine uptake is highly expressed on the surface of M1 macrophages. Therefore, L-arginine-modified liposomes have the potential to achieve targeted drug delivery to M1 macrophages.

[0006] Since colchicine is a fat-soluble drug that is soluble in water and easily soluble in ethanol, its physical and chemical properties result in a low encapsulation rate in liposomes. Therefore, how to further improve the encapsulation rate of colchicine in liposomes so that it can better target M1 macrophages has become an urgent problem to be solved in the field of disease treatment of colchicine. Summary of the invention

[0007] In view of the above technical problems, the present invention provides a double-modified colchicine liposome targeting M1 macrophages and a preparation method thereof.

[0008] In order to achieve the above object, the present invention provides a double-modified colchicine liposome targeting M1 macrophages, comprising the following raw materials in parts by weight: 0.1-2 parts of colchicine, 10-40 parts of phosphatidylcholine, 1-20 parts of cholesterol, 1-5 parts of cultured phosphatidylethanolamine, 0.4-2 parts of phospholipid polyethylene glycol folic acid, 0.4-2 parts of phospholipid polyethylene glycol arginine, and 10-100 parts of buffer.

[0009] Preferably, the phosphatidylcholine is any one of hydrogenated soybean phosphatidylcholine, distearoyl phosphatidylcholine, glyceryl phosphatidylcholine, dimyristoyl phosphatidylcholine and dilauroyl phosphatidylcholine.

[0010] Preferably, the molecular weight of the phospholipid polyethylene glycol folic acid is any one of 1000, 2000, 3400, 5000, and 10000.

[0011] Preferably, the molecular weight of the phospholipid phospholipid polyethylene glycol arginine is any one of 1000, 2000, 3400, 5000, and 10000.

[0012] Preferably, the molar ratio of the cultured phosphatidylethanolamine to the phospholipid polyethylene glycol arginine and the phospholipid polyethylene glycol folic acid is 0.6-1:0.1-0.4:0.1-0.4.

[0013] Preferably, the buffer is any one of PBS buffer solution, citric acid-sodium citrate solution, sodium carbonate-sodium bicarbonate buffer, acetic acid-sodium acetate solution, lactic acid-sodium lactate solution and aminoacetic acid-sodium aminoacetate solution.

[0014] The present invention also provides a method for preparing a double-modified colchicine liposome targeting M1 macrophages, comprising the following steps: (1) Preparing an organic phase: mixing and dissolving phosphatidylcholine, cholesterol, cultured phosphatidylethanolamine, phospholipid polyethylene glycol arginine, phospholipid polyethylene glycol folic acid and anhydrous ethanol to obtain an organic phase; (2) Preparing the aqueous phase: preparing a buffer solution as the aqueous phase; (3) Dissolving: dissolving colchicine in anhydrous ethanol to obtain a colchicine solution, and adding the solution to the organic phase; (4) Preparation of drug-loaded liposomes: mixing the organic phase and the aqueous phase, and preparing drug-loaded liposomes by microfluidics; (5) Ultrafiltration: The drug-loaded liposomes are purified by ultrafiltration to remove anhydrous ethanol and free colchicine to obtain double-modified colchicine liposomes.

[0015] Preferably, the concentration of the organic phase in step (1) is 30-45 mg / mL; the concentration of the aqueous phase in step (2) is 0.01-1 M.

[0016] Preferably, the concentration of colchicine in step (3) is 0.1-2 mg / mL, the pH value is 5.9, and the drug-lipid ratio of colchicine to lipid is 1:60-90.

[0017] More preferably, the lipids are composed of hydrogenated soybean phosphatidylcholine, cholesterol, hydrogenated phosphatidylethanolamine, phospholipid polyethylene glycol folic acid, and phospholipid polyethylene glycol arginine.

[0018] Preferably, the flow rate ratio of the organic phase to the aqueous phase in the microfluidic system in step (4) is 1-6:6-18; and the ultrafiltration conditions in step (5) are a molecular retention rate of 100 kD and an ultrafiltration speed of 5000 g / min.

[0019] The beneficial effect of the present invention is that the encapsulation rate of the double-modified colchicine liposome targeting M1 macrophages is further improved by coordinating the components of colchicine, hydrogenated soybean phosphatidylcholine, cholesterol, cultured phosphatidylethanolamine, phospholipid polyethylene glycol folic acid, and phospholipid phospholipid polyethylene glycol arginine and adjusting the flow rate of microfluidics. The prepared double-modified colchicine liposome targeting M1 macrophages has good targeting, PDI and target particle size, and has a longer half-life, reduces the toxicity of colchicine, and enhances the anti-inflammatory effect of colchicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the particle size distribution diagram of the double-modified colchicine liposomes prepared in Example 1.

[0021] Figure 2 This is a relationship diagram of the effect of the drug-lipid ratio of the double-modified colchicine liposomes on the encapsulation efficiency in Examples 1, 6, 7, 8 and Comparative Examples 1 and 2.

[0022] Figure 3 The drug release curves of colchicine liposomes obtained in Example 1 and Comparative Examples 6, 7 and 8 in PBS buffer at 37° C. and pH=7.4 are shown.

[0023] Figure 4 These are fluorescence images of unmodified and modified colchicine liposomes targeting M1 macrophages in Example 13.

[0024] Figure 5 This is a fluorescence intensity bar graph of unmodified and modified colchicine liposomes targeting M1 macrophages in Example 13.

[0025] Figure 6 This is a graph showing the cytotoxicity of different concentrations of unmodified colchicine liposomes on M1 macrophages in Example 14.

[0026] Figure 7 This is a graph showing the cytotoxicity of unmodified and modified colchicine liposomes and colchicine original drug to M1 macrophages at the same concentration in Example 14.

[0027] Figure 8 This is a bar graph showing the release of IL-1β from M1 macrophages by unmodified and modified colchicine liposomes and colchicine original drug at the same concentration in Example 15. DETAILED DESCRIPTION

[0028] The technical solution of the present invention is further explained below in conjunction with the accompanying drawings and specific embodiments. It is worth noting that the following embodiments are only preferred embodiments of the present invention and should not be understood as limiting the present invention. The protection scope of the present invention shall be based on the contents recorded in the claims. Any modification or replacement made by those skilled in the art to the technical solution of the present invention without creative work shall fall within the protection scope of the present invention.

[0029] In the following examples, phospholipid polyethylene glycol folic acid was purchased from Shanghai Pengshuo Biotechnology Co., Ltd., and phospholipid polyethylene glycol arginine was purchased from Chongqing Yusi Pharmaceutical Technology Co., Ltd.

[0030] Example 1 A double-modified colchicine liposome targeting M1 macrophages, the formula comprising, by weight, 0.5 parts of colchicine, 24 parts of hydrogenated soybean phosphatidylcholine, 11 parts of cholesterol, 3 parts of cultured phosphatidylethanolamine, 1 part of phospholipid polyethylene glycol arginine, 1 part of phospholipid polyethylene glycol folic acid, and 30 parts of buffer; Preparation method: (1) Preparing an organic phase: according to the formula, hydrogenated soybean phosphatidylcholine, cholesterol, cultured phosphatidylethanolamine, cultured phosphatidylethanolamine-L-arginine, and phospholipid polyethylene glycol folic acid are placed in anhydrous ethanol, mixed and dissolved under ultrasound to obtain an organic phase with a concentration of 40 mg / mL; (2) Preparing the aqueous phase: preparing PBS buffer, wherein the concentration of PBS buffer is 0.01 M; (3) Dissolution: Dissolve colchicine in anhydrous ethanol to obtain a colchicine solution (10 mg / mL), take 50 μL of the colchicine solution and add it to 1 mL of the organic phase to obtain an organic phase solution containing colchicine; that is, the drug-lipid ratio is 1:80; (4) Preparation of drug-loaded liposomes: At 60°C, the volume ratio of organic phase solution to aqueous phase was 1:3 and the flow rate ratio was 3:9 to obtain drug-loaded liposomes by microfluidics; (5) Ultrafiltration: The drug-loaded liposome solution prepared in step (4) was loaded into a 100 kD ultrafiltration tube, diluted 15 times with PBS buffer, and centrifuged at 5000 g / min to remove free drugs and anhydrous ethanol and other substances outside the liposomes to obtain a double-modified colchicine liposome solution.

[0031] Example 2 A doubly modified colchicine liposome targeting M1 macrophages, comprising, by weight, 0.5 parts of colchicine, 24 parts of hydrogenated soybean phosphatidylcholine, 11 parts of cholesterol, 4 parts of cultured phosphatidylethanolamine, 0.4 parts of phospholipid polyethylene glycol arginine, 0.4 parts of phospholipid polyethylene glycol folic acid, and 30 parts of buffer, and the preparation method is the same as that of Example 1.

[0032] Example 3 A double-modified colchicine liposome targeting M1 macrophages, the formula of which includes, by weight, 0.5 parts of colchicine, 24 parts of hydrogenated soybean phosphatidylcholine, 11 parts of cholesterol, 1 part of cultured phosphatidylethanolamine, 2 parts of phospholipid polyethylene glycol arginine, 2 parts of phospholipid polyethylene glycol folic acid, and 30 parts of buffer, and the preparation method is the same as that of Example 1.

[0033] Example 4 A double-modified colchicine liposome targeting M1 macrophages, the formulation and preparation method are the same as those in Example 1, and the flow rate ratio of the organic phase to the aqueous phase in step (4) is 2:6.

[0034] Example 5 A double-modified colchicine liposome targeting M1 macrophages, the formulation and preparation method are the same as those in Example 1, and the flow rate ratio of the organic phase to the aqueous phase in step (4) is 6:18.

[0035] Example 6 A double-modified colchicine liposome targeting M1 macrophages, the formula comprising, by weight, 0.5 parts of colchicine, 27 parts of hydrogenated soybean phosphatidylcholine, 12 parts of cholesterol, 3.4 parts of cultured phosphatidylethanol, 1.3 parts of phospholipid polyethylene glycol arginine, 1.3 parts of phospholipid polyethylene glycol folic acid, and 30 parts of buffer; The preparation method is the same as that of Example 1, wherein the concentration of the organic phase in step (1) is 50 mg / mL, and the concentration of the colchicine solution in step (3) is 0.5 mg / mL, that is, the drug-to-lipid ratio is 1:90.

[0036] Example 7 A double-modified colchicine liposome targeting M1 macrophages, the formula comprising, by weight, 0.5 parts of colchicine, 21 parts of hydrogenated soybean phosphatidylcholine, 9.6 parts of cholesterol, 2.6 parts of cultured phosphatidylethanolamine, 0.9 parts of phospholipid polyethylene glycol arginine, 0.9 parts of phospholipid polyethylene glycol folic acid, and 30 parts of buffer; The preparation method is the same as that of Example 1, wherein the concentration of the organic phase in step (1) is 35 mg / mL, and the concentration of the colchicine aqueous solution in step (3) is 1 mg / mL, so that the drug-lipid ratio is 1:70.

[0037] Example 8 A double-modified colchicine liposome targeting M1 macrophages, the formula comprising, by weight, 0.5 parts of colchicine, 12 parts of hydrogenated soybean phosphatidylcholine, 5.5 parts of cholesterol, 1.5 parts of cultured phosphatidylethanolamine, 0.5 parts of phospholipid polyethylene glycol arginine, and 0.5 parts of phospholipid polyethylene glycol folic acid; The preparation method is the same as that of Example 1, wherein the concentration of the organic phase in step (1) is 20 mg / mL, the concentration of the colchicine aqueous solution in step (3) is 0.5 mg / mL, and the drug-lipid ratio is 1:60.

[0038] Comparative Example 1 A double-modified colchicine liposome targeting M1 macrophages, the formula comprising, by weight, 0.5 parts of colchicine, 15 parts of hydrogenated soybean phosphatidylcholine, 6.9 parts of cholesterol, 1.9 parts of cultured phosphatidylethanolamine, 0.6 parts of phospholipid polyethylene glycol arginine, 0.6 parts of phospholipid polyethylene glycol folic acid, and 30 parts of buffer; The preparation method is the same as that of Example 1, wherein the concentration of the organic phase in step (1) is 10 mg / mL, and the concentration of the colchicine solution in step (3) is 0.5 mg / mL, that is, the drug-to-lipid ratio is 1:50.

[0039] Comparative Example 2 A double-modified colchicine liposome targeting M1 macrophages, the formula comprising, by weight, 0.5 parts of colchicine, 30 parts of hydrogenated soybean phosphatidylcholine, 13.8 parts of cholesterol, 3.8 parts of cultured phosphatidylethanolamine, 1.2 parts of phospholipid polyethylene glycol arginine, 1.2 parts of phospholipid polyethylene glycol folic acid, and 30 parts of buffer; The preparation method is the same as that of Example 1, wherein the concentration of the organic phase in step (1) is 50 mg / mL, and the concentration of the colchicine solution in step (3) is 1 mg / mL, that is, the drug-to-lipid ratio is 1:100.

[0040] Comparative Example 3 A doubly modified colchicine liposome targeting M1 macrophages, comprising, by weight, 0.5 parts of colchicine, 24 parts of hydrogenated soybean phosphatidylcholine, 11 parts of cholesterol, 3 parts of cultured phosphatidylethanolamine, 0.2 parts of phospholipid polyethylene glycol arginine, 0.2 parts of phospholipid polyethylene glycol folic acid, and 30 parts of buffer; the preparation method is the same as that of Example 1.

[0041] Comparative Example 4 A double-modified colchicine liposome targeting M1 macrophages, the formula of which includes, by weight, 0.5 parts of colchicine, 24 parts of hydrogenated soybean phosphatidylcholine, 11 parts of cholesterol, 1 part of cultured phosphatidylethanolamine, 2.5 parts of phospholipid polyethylene glycol arginine, 2.5 parts of phospholipid polyethylene glycol folic acid, and 30 parts of buffer; the preparation method is the same as that of Example 1.

[0042] Results: The colchicine liposomes prepared in the above examples and comparative examples were tested for their properties. The results are shown in Table 1: Table 1 Properties of colchicine liposomes

[0043] From Table 1 and Figure 2 It can be seen that: with the increase of drug-lipid ratio, the encapsulation rate of the double-modified colchicine liposome gradually increases. When the drug-lipid ratio reaches 1:100, the lipid will precipitate from the organic phase due to the high lipid concentration, so the optimal drug-lipid ratio is 1:80. When the microfluidic flow rate ratio is 3:9, it has a better particle size and PDI, so the optimal flow rate ratio is 3:9.

[0044] Example 9 A double-modified colchicine liposome, the formula and preparation method are the same as those in the embodiment, except that the molar ratio of hydrogenated soybean phosphatidylcholine to cholesterol in step (1) is changed to 2:1.

[0045] Example 10 A double-modified colchicine liposome, the formula and preparation method are the same as those in the embodiment, except that the molar ratio of hydrogenated soybean phosphatidylcholine to cholesterol in step (1) is changed to 3:1.

[0046] Embodiment 11 A double-modified colchicine liposome, the formula and preparation method are the same as those in the embodiment, except that the molar ratio of hydrogenated soybean phosphatidylcholine to cholesterol in step (1) is changed to 4:1.

[0047] Comparative Example 5 A double-modified colchicine liposome, the formula and preparation method are the same as those in the embodiment, only the molar ratio of hydrogenated soybean phosphatidylcholine to cholesterol in step (1) is changed to 1:2.

[0048] Results: The colchicine liposomes prepared in the above examples and comparative examples were tested for their properties. The results are shown in Table 2: Table 2 Effect of phosphorus-bile ratio on the performance of colchicine liposomes

[0049] It can be seen from Table 2 that when the phosphorus-bile ratio increases, the liposome particle size increases, the PDI becomes larger, and the liposome becomes unstable. Therefore, the most suitable phosphorus-bile ratio is 1:1.

[0050] Comparative Example 6 A single-modified colchicine liposome targeting M1 macrophages comprises, by weight, 0.5 parts of colchicine, 24 parts of hydrogenated soybean phosphatidylcholine, 11 parts of cholesterol, 4 parts of cultured phosphatidylethanolamine, 1 part of phospholipid polyethylene glycol arginine, and 30 parts of buffer, to prepare the colchicine liposome.

[0051] Comparative Example 7 A single-modified colchicine liposome targeting M1 macrophages comprises, by weight, 0.5 parts of colchicine, 24 parts of hydrogenated bean phosphatidylcholine, 11 parts of cholesterol, 4 parts of cultured phosphatidylethanolamine, 1 part of phospholipid polyethylene glycol folic acid, and 30 parts of buffer, to prepare colchicine liposomes.

[0052] Comparative Example 8 The invention discloses an unmodified colchicine liposome, which comprises 0.5 parts of colchicine, 24 parts of hydrogenated bean phosphatidylcholine, 11 parts of cholesterol, 5 parts of phosphatidylethanolamine and 30 parts of buffer solution by weight, and the colchicine liposome is prepared.

[0053] Results: The colchicine liposomes prepared in Comparative Examples 6-8 were tested for their performance. The results are shown in Table 3: Table 3 Effects of modification on liposome properties

[0054] The results showed that the particle size of the modified colchicine liposomes became larger, the encapsulation efficiency slightly decreased, and the effect of folic acid on the particle size was greater than that of arginine. However, different modifications had no significant effect on the drug loading capacity of the liposomes, so the liposomes can be modified multiple times to improve the performance of the liposomes.

[0055] Example 12 The colchicine liposomes prepared in Example 1, Comparative Examples 6, 7, and 8 were placed in a PBS buffer solution with a pH of 7.4 and incubated at 37° C., wherein the volume ratio of colchicine liposomes to PBS buffer solution was 1:20; samples were taken at 0.25 h, 1 h, 4 h, 8 h, 16 h, and 24 h, respectively, and the relationship between the cumulative release percentage of colchicine and time was detected by ultrafiltration; the results are as follows Figure 3 As shown in the figure, compared with the unmodified colchicine liposomes, the release curve of the modified colchicine liposomes is relatively flat, indicating that the release rate is slower, but they all tend to the same level in the end; the release curves of the colchicine liposomes after different modifications are similar, among which the release rate of FA modification is the lowest, the release rate of ARG modification is the highest, and the release level of ARG-FA dual-modified colchicine liposomes is between the two single-modified ones, and the difference is not significant.

[0056] Example 13 (1) Peripheral blood mononuclear cells were obtained by density gradient centrifugation from normal human peripheral blood and the mononuclear cell concentration was adjusted to 2×10 6 / mL in a 6-well plate, and macrophage colony-stimulating factor M-CSF was given, and primary human macrophages were polarized and generated after 7 days; (2) LPS + IFN-γ was given to mature primary human macrophages to polarize them into pro-inflammatory macrophage M1 cells. After 24 hours, the pro-inflammatory macrophages were polarized and mature. (3) Unmodified, ARG-modified, FA-modified, and FA+ARG-modified liposomes encapsulating siRNA labeled with FAM fluorescent dye were prepared respectively. The preparation scheme was the same as that of Example 1 and Comparative Examples 6-8, wherein the PBS buffer in step (2) was replaced with a PBS buffer containing siRNA labeled with FAM fluorescent dye at a concentration of 1.3 μg / ml, and step (3) was omitted. Each group of liposomes was added to a 6-well plate respectively; (3) After incubation for 1 h, the cells were washed with PBS buffer to remove excess fluorescence and the green fluorescence in the cells was observed under an inverted fluorescence microscope. The entire experiment was carried out in a light-proof environment and each experiment was repeated 3 times.

[0057] Depend on Figure 4It can be seen that the fluorescence intensity of the double-modified liposomes>the single-modified liposomes>the unmodified liposomes, indicating that the targeting of the double-modified liposomes is better than that of the single-modified liposomes.

[0058] ImageJ software was used to analyze the fluorescence intensity of the fluorescence image and a histogram of the mean fluorescence intensity of M1 macrophages was drawn. The results are shown in Figure 5 As shown, the average fluorescence intensity of M1 macrophages treated with double-modified liposomes was significantly better than that of single-modified liposomes. The folate receptor and L-arginine receptor on the surface of M1 macrophages are different. Double modification can increase the targeting synergistic effect by targeting different receptors of M1 macrophages.

[0059] Embodiment 14 Cytotoxicity assay: 1. Prepare different concentrations of unmodified colchicine liposomes to investigate their toxic effects on M1 macrophages: (1) The concentration of peripheral blood mononuclear cells was 1×10 5 / well were plated in 96-well plates and polarized into pro-inflammatory macrophages M1; (2) Prepare different concentrations of unmodified colchicine liposome stock solutions at a ratio of liposome: complete medium = 1:9, and add 0.1, 0.5, 0.75, 1, and 5 μM into the cells, 100 μL per well; (3) After 24 h of action, add 10 μL of CCK8 to each well and incubate in the dark for 1 h. Measure the OD value at 450 nm.

[0060] Depend on Figure 6 It can be seen that at a concentration of 0.5μM, the survival rate of M1 macrophages is >80%, so 0.5μM was selected for subsequent cell experiments.

[0061] 2. Prepare unmodified and modified colchicine liposomes and colchicine stock drug at a concentration of 0.5 μM and investigate their toxic effects on M1 macrophages: (1) The concentration of peripheral blood mononuclear cells was 1×10 5 / well were plated in 96-well plates and polarized into pro-inflammatory macrophages M1; (2) Prepare high concentrations of unmodified and modified colchicine liposomes and colchicine stock drug (COL), and add 0.5 μM to the cells at a ratio of liposomes, colchicine stock drug, and complete culture medium = 1:9, with 100 μL per well; (3) After 24 h of action, add 10 μL of CCK8 to each well and incubate in the dark for 1 h. Measure the OD value at 450 nm.

[0062] Depend on Figure 7It can be seen that under the same concentration, the toxicity of unmodified and modified colchicine liposomes to M1 macrophages is less than that of the original colchicine, indicating that the unmodified and modified colchicine liposomes have a reduced toxicity effect compared with the original colchicine. The double modification and single modification have similar cytotoxicity to M1 macrophages, with no statistical difference, and both are lower than the original colchicine, indicating that the double modified liposomes can significantly improve the targeting while maintaining cell viability at a high level, reducing the impact on the normal life activities of cells.

[0063] Embodiment 15 Cellular inflammatory factor detection: The cell culture supernatant of Example 15-2 after 24 hours of action was taken and centrifuged at 1000×g for 20 minutes, and the IL-1β level was detected according to the requirements of the human interleukin 1β enzyme-linked immunosorbent assay kit.

[0064] Depend on Figure 8 It can be seen that under the same concentration, the level of IL-1β released by unmodified and modified colchicine liposomes on M1 macrophages is lower than that of the original colchicine, indicating that the unmodified and modified colchicine liposomes have stronger anti-inflammatory effects than the original colchicine, and the double modification has a stronger anti-inflammatory effect than the single modification. The statistical analysis is significant, and the double modification has a stronger targeting effect on M1 macrophages and produces the strongest anti-inflammatory effect.

Claims

1. A dual-modified colchicine liposome targeting M1 macrophages, characterized in that: The invention comprises the following raw materials in parts by weight: 0.1-2 parts of colchicine, 10-40 parts of phosphatidylcholine, 1-20 parts of cholesterol, 1-5 parts of cultured phosphatidylethanolamine, 0.4-2 parts of phospholipid polyethylene glycol folic acid, 0.4-2 parts of phospholipid polyethylene glycol arginine and 10-100 parts of buffer solution.

2. The dual-modified colchicine liposome targeting M1 macrophages according to claim 1, characterized in that: The phosphatidylcholine is any one of hydrogenated bean phosphatidylcholine, distearoyl phosphatidylcholine, glyceryl phosphatidylcholine, dimyristoyl phosphatidylcholine and dilauroyl phosphatidylcholine.

3. The dual-modified colchicine liposome targeting M1 macrophages according to claim 1, characterized in that: The molecular weight of the phospholipid polyethylene glycol folic acid is any one of 1000, 2000, 3400, 5000 and 10000.

4. The dual-modified colchicine liposome targeting M1 macrophages according to claim 1, characterized in that: The molecular weight of the phospholipid polyethylene glycol arginine is any one of 1000, 2000, 3400, 5000 and 10000.

5. The dual-modified colchicine liposome targeting M1 macrophages according to claim 1, characterized in that: The molar ratio of the cultured phosphatidylethanolamine to the phospholipid polyethylene glycol arginine and the phospholipid polyethylene glycol folic acid is 0.6-1:0.1-0.4:0.1-0.

4.

6. The dual-modified colchicine liposome targeting M1 macrophages according to claim 1, characterized in that: The buffer is any one of PBS buffer solution, citric acid-sodium citrate solution, sodium carbonate-sodium bicarbonate buffer solution, acetic acid-sodium acetate solution, lactic acid-sodium lactate solution and aminoacetic acid-sodium aminoacetate solution.

7. A method for preparing the double-modified colchicine liposome targeting M1 macrophages according to any one of claims 1 to 6, characterized in that: The steps include: (1) Preparing an organic phase: mixing and dissolving phosphatidylcholine, cholesterol, cultured phosphatidylethanolamine, phospholipid polyethylene glycol arginine, phospholipid polyethylene glycol folic acid and anhydrous ethanol to obtain an organic phase; (2) Preparing the aqueous phase: preparing a buffer solution as the aqueous phase; (3) Dissolving: dissolving colchicine in anhydrous ethanol to obtain a colchicine solution, and adding the solution to the organic phase; (4) Preparation of drug-loaded liposomes: mixing the organic phase and the aqueous phase, and preparing drug-loaded liposomes by microfluidics; (5) Ultrafiltration: The drug-loaded liposomes are purified by ultrafiltration to remove anhydrous ethanol and free colchicine to obtain double-modified colchicine liposomes.

8. The preparation method according to claim 7, characterized in that: The concentration of the organic phase in step (1) is 30-45 mg / mL; the concentration of the aqueous phase in step (2) is 0.01-1 M.

9. The preparation method according to claim 7, characterized in that: The concentration of colchicine in step (3) is 0.1-2 mg / mL, the pH value is 5.9, and the drug-to-lipid ratio of colchicine to lipid is 1:60-90.

10. The preparation method according to claim 7, characterized in that: The flow rate ratio of the organic phase to the aqueous phase in the microfluidic system described in step (4) is 1-6:6-18; the ultrafiltration conditions described in step (5) are a molecular retention rate of 100 kD and an ultrafiltration speed of 5000 g / min.

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