A double modified colchicine liposome targeting M1 type macrophages and a preparation method thereof

Abstract

The application discloses a double-modified colchicine liposome targeting M1 macrophages and a preparation method thereof, which comprises the following components in parts by weight: colchicine 0.1-2 parts, hydrogenated soybean phosphatidylcholine 10-40 parts, cholesterol 1-20 parts, phosphatidyl ethanolamine 1-5 parts, phospholipid polyethylene glycol folic acid 0.4-2 parts, phospholipid polyethylene glycol arginine 0.4-2 parts, and buffer 10-100 parts. The double-modified colchicine liposome targeting M1 macrophages prepared by the application 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.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of liposome preparation, and particularly relates to colchicine liposomes targeting M1 macrophages and a preparation method thereof. BACKGROUND

[0002] Colchicine is a tricyclic alkaloid, which is extracted from Colchicum autumnale L. of Liliaceae, and has various pharmacological effects. For example, colchicine can reduce the levels of cytokines such as IL-1β, IL-6 and TNF-α, reduce inflammatory cell infiltration, and inhibit uric acid sodium-induced arthritis, and thus can be used as an effective drug for treating gouty arthritis, relieving pain and subsiding inflammation. In addition, colchicine can also be used for treating pancreatitis, hepatitis and pneumonia. Colchicine is currently approved by FDA for acute gout and familial Mediterranean fever. Colchicine is closely related to macrophages. Colchicine can inhibit the polymerization of microtubules, thereby reducing the generation of ROS and the release of NO and IL-1β of macrophages, inhibiting the chemotaxis, migration and adhesion of macrophages, inhibiting the activation of NLRP3 inflammasome of macrophages, and inhibiting the transformation of macrophages to M1 type and promoting the polarization of macrophages to M2 type and reducing pyroptosis of macrophages, thereby playing an anti-inflammatory role. However, the application of colchicine also has some limitations, such as a narrow therapeutic window, and many side effects, such as gastrointestinal reactions such as nausea, vomiting and diarrhea, bone marrow suppression, and liver and kidney function damage. In addition, the effective plasma steady-state concentration of colchicine is close to the toxic concentration, and 7 mg of colchicine taken per day can cause human death. Therefore, the dose used in clinical practice is difficult to reach the maximum therapeutic dose, and it is difficult to exert the maximum anti-inflammatory effect.

[0003] Liposomes are a kind of bilayer structure assembled by hydrophilic groups and hydrophobic groups, have high targeting property, can directly deliver drugs to inflammatory tissues, reduce damage to normal tissues, can effectively protect the encapsulated drugs and controllably release the drugs, prolong the action time of the drugs in the body, are easy to degrade in the body, significantly improve the therapeutic effect of the drugs, and at the same time, the particle size of the liposomes can be controlled to improve the in vivo distribution behavior of the drugs and reduce the toxic side effects.

[0004] Wang Linzhen encapsulated colchicine in liposomes by active drug loading method, and the encapsulation rate was 58.9%. The optimal preparation process method was as follows: colchicine 0.5 mg / mL, phospholipid 50 mg / mL, cholesterol 8 mg / mL, VB 1 mg / mL, and the pH value of the external aqueous phase was 6.4, the incubation time was 10 min, and the drug loading order was to first add the blank liposomes into the drug solution, and then add NaHCO3 to adjust the pH value (Wang Linzhen, Preparation of colchicine liposomes. Strait Pharmaceutical Journal, 2011, 23 (7): 27-29.). However, the encapsulation rate is low, and the liposomes have no targeting property.

[0005] M1 type macrophages are activated by IFN-gamma and TNF, mainly mediate cellular immunity and anti-infection activity, have the functions of killing microorganisms, participating in inflammation and anti-tumor effect. M1 type macrophages highly up-regulate folate receptor beta (FR-beta). The differential expression of FR-beta provides a way for selective delivery of 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) mediating L-arginine uptake is highly expressed on the surface of M1 type macrophages, therefore, L-arginine modified liposomes have the potential to achieve targeted drug delivery to M1 type macrophages.

[0006] Since colchicine belongs to a fat-soluble drug, it is soluble in water and easily soluble in ethanol, and 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 type macrophages has become a problem to be solved in the field of colchicine disease treatment. SUMMARY

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

[0008] In order to achieve the above-mentioned purpose, the present application provides a double-modified colchicine liposome targeting M1 type macrophages, which comprises the following raw materials by weight: colchicine 0.1-2 parts, phosphatidylcholine 10-40 parts, cholesterol 1-20 parts, phosphatidyl ethanolamine 1-5 parts, phospholipid polyethylene glycol folate 0.4-2 parts, phospholipid polyethylene glycol arginine 0.4-2 parts, and buffer 10-100 parts.

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

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

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

[0012] Preferably, the molar ratio of the phosphatidyl ethanolamine to the phospholipid polyethylene glycol arginine and the phospholipid polyethylene glycol folate 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 application also provides a preparation method of the double modified colchicine liposome targeting M1 type macrophages, comprising the following steps:

[0015] (1) Preparation of organic phase: phosphatidylcholine, cholesterol, phosphatidyl ethanolamine, phosphatidyl polyethylene glycol arginine, phosphatidyl polyethylene glycol folic acid are mixed and dissolved in anhydrous ethanol to obtain an organic phase;

[0016] (2) Preparation of water phase: preparation of buffer solution as water phase;

[0017] (3) Dissolution: colchicine is dissolved in anhydrous ethanol to obtain a colchicine solution, and the organic phase is added;

[0018] (4) Preparation of drug-loaded liposome: the organic phase and the water phase are mixed, and a drug-loaded liposome is prepared by microfluidic method;

[0019] (5) Ultrafiltration: the drug-loaded liposome is purified by ultrafiltration to remove anhydrous ethanol and free colchicine, and a double modified colchicine liposome is obtained.

[0020] Preferably, the concentration of the organic phase in step (1) is 30-45 mg / mL; the concentration of the water phase in step (2) is 0.01-1 M.

[0021] 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.

[0022] Further preferably, the lipid is composed of hydrogenated soybean phosphatidylcholine, cholesterol, phosphatidyl ethanolamine, phosphatidyl polyethylene glycol folic acid and phosphatidyl polyethylene glycol arginine.

[0023] Preferably, the flow rate ratio of the organic phase to the water phase in the microfluidic method in step (4) is 1-6:6-18; and the ultrafiltration condition in step (5) is that the molecular cut-off rate is 100 kD and the ultrafiltration rotation speed is 5000 g / min.

[0024] The beneficial effects of the present application are that by coordinating the several components of colchicine, hydrogenated soy phosphatidylcholine, cholesterol, phosphatidylethanolamine, phospholipid polyethylene glycol folic acid, phospholipid polyethylene glycol arginine and adjusting the flow rate of microfluidics, the encapsulation rate of the double-modified colchicine liposome targeting M1 macrophages is further improved. The double-modified colchicine liposome targeting M1 macrophages prepared has good targeting, PDI and target particle size, and has a longer half-life, reduces the toxicity of colchicine, and enhances the anti-inflammatory efficacy of colchicine. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 The particle size distribution graph of the double-modified colchicine liposome prepared in Example 1.

[0026] Figure 2 The graph of the relationship between the drug-lipid ratio and the encapsulation rate of the double-modified colchicine liposome in Examples 1, 6, 7, 8 and Comparative Examples 1 and 2.

[0027] Figure 3 The drug release curve graph of the colchicine liposome obtained in Examples 1, Comparative Examples 6, 7 and 8 in PBS buffer at 37℃ and pH=7.4.

[0028] Figure 4 The fluorescence graph of the unmodified and modified colchicine liposomes targeting M1 macrophages in Example 13.

[0029] Figure 5 The fluorescence intensity column graph of the unmodified and modified colchicine liposomes targeting M1 macrophages in Example 13.

[0030] Figure 6 The cytotoxicity graph of the unmodified colchicine liposome at different concentrations to M1 macrophages in Example 14.

[0031] Figure 7 The cytotoxicity graph of the unmodified and modified colchicine liposomes and colchicine raw drug at the same concentration to M1 macrophages in Example 14.

[0032] Figure 8 The IL-1β release column graph of the unmodified and modified colchicine liposomes and colchicine raw drug at the same concentration to M1 macrophages in Example 15. DETAILED DESCRIPTION

[0033] The technical solutions of the present application are further explained in combination with the drawings and specific embodiments. It is worth noting that the following embodiments are only preferred embodiments of the present application, and should not be understood as limiting the present application. The protection scope of the present application should be based on the content recorded in the claims. The modifications and replacements of the technical solutions of the present application made by those skilled in the art without creative labor fall within the protection scope of the present application.

[0034] In the following examples, phospholipid polyethylene glycol folic acid is purchased from Shanghai Pingsuo Biotechnology Co., Ltd., and phospholipid polyethylene glycol arginine is purchased from Chongqing Yusi Pharmaceutical Technology Co., Ltd.

[0035] Example 1

[0036] A double-modified colchicine liposome targeting M1 type macrophages, the formula includes colchicine 0.5 parts, hydrogenated soybean phosphatidylcholine 24 parts, cholesterol 11 parts, phosphatidyl ethanolamine 3 parts, phospholipid polyethylene glycol arginine 1 part, phospholipid polyethylene glycol folic acid 1 part, and buffer 30 parts by weight;

[0037] Preparation method:

[0038] (1) Preparation of organic phase: according to the formula, hydrogenated soybean phosphatidylcholine, cholesterol, phosphatidyl ethanolamine, phosphatidyl ethanolamine-L-arginine, and phospholipid polyethylene glycol folic acid are placed in anhydrous ethanol and dissolved under ultrasonic mixing to obtain an organic phase with a concentration of 40 mg / mL;

[0039] (2) Preparation of aqueous phase: PBS buffer is prepared, and the concentration of the PBS buffer is 0.01M;

[0040] (3) Dissolution: colchicine is dissolved in anhydrous ethanol to obtain a colchicine solution (10 mg / mL), 50 μL of the colchicine solution is added to 1 mL of the organic phase to obtain an organic phase solution containing colchicine; the drug-lipid ratio is 1:80;

[0041] (4) Preparation of drug-loaded liposomes: at 60°C, the organic phase solution and the aqueous phase are mixed at a volume ratio of 1:3 and a flow rate ratio of 3:9 to obtain drug-loaded liposomes by microfluidization;

[0042] (5) Ultrafiltration: the drug-loaded liposome solution prepared in step (4) is loaded into a 100kD ultrafiltration tube, diluted 15 times with PBS buffer, and centrifuged at 5000g / min to remove free drugs, anhydrous ethanol and other substances outside the liposomes, to obtain a double-modified colchicine liposome solution.

[0043] Example 2

[0044] A double modified colchicine liposome targeting M1 type macrophages, the formula includes colchicine 0.5 parts, hydrogenated soybean phosphatidylcholine 24 parts, cholesterol 11 parts, phosphatidyl ethanolamine 4 parts, phospholipid polyethylene glycol arginine 0.4 parts, phospholipid polyethylene glycol folic acid 0.4 parts, buffer 30 parts by weight, the preparation method is the same as example 1.

[0045] Example 3

[0046] A double modified colchicine liposome targeting M1 type macrophages, the formula includes colchicine 0.5 parts, hydrogenated soybean phosphatidylcholine 24 parts, cholesterol 11 parts, phosphatidyl ethanolamine 1 part, phospholipid polyethylene glycol arginine 2 parts, phospholipid polyethylene glycol folic acid 2 parts, buffer 30 parts by weight, the preparation method is the same as example 1.

[0047] Example 4

[0048] A double modified colchicine liposome targeting M1 type macrophages, the formula and preparation method are the same as example 1, the flow rate ratio of organic phase and aqueous phase in step (4) is 2:6.

[0049] Example 5

[0050] A double modified colchicine liposome targeting M1 type macrophages, the formula and preparation method are the same as example 1, the flow rate ratio of organic phase and aqueous phase in step (4) is 6:18.

[0051] Example 6

[0052] A double modified colchicine liposome targeting M1 type macrophages, the formula includes colchicine 0.5 parts, hydrogenated soybean phosphatidylcholine 27 parts, cholesterol 12 parts, phosphatidyl ethanolamine 3.4 parts, phospholipid polyethylene glycol arginine 1.3 parts, phospholipid polyethylene glycol folic acid 1.3 parts, buffer 30 parts by weight;

[0053] The preparation method is the same as example 1, wherein the organic phase concentration in step (1) is 50 mg / mL, and the concentration of colchicine solution in step (3) is 0.5 mg / mL, that is, the drug-lipid ratio is 1:90.

[0054] Example 7

[0055] A double modified colchicine liposome targeting M1 type macrophages, the formula includes colchicine 0.5 parts, hydrogenated soybean phosphatidylcholine 21 parts, cholesterol 9.6 parts, phosphatidyl ethanolamine 2.6 parts, phospholipid polyethylene glycol arginine 0.9 parts, phospholipid polyethylene glycol folic acid 0.9 parts, buffer 30 parts by weight;

[0056] The preparation method is the same as that in Example 1, wherein the concentration of the organic phase in step (1) is 35 mg / mL, and the concentration of the aqueous colchicine solution in step (3) is 1 mg / mL, so that the drug-lipid ratio is 1:70.

[0057] Example 8

[0058] A double-modified colchicine liposome targeting M1 macrophages, which comprises, by weight fraction, colchicine 0.5 part, hydrogenated soybean phosphatidylcholine 12 parts, cholesterol 5.5 parts, cultured phosphatidyl ethanolamine 1.5 parts, phospholipid polyethylene glycol arginine 0.5 part, phospholipid polyethylene glycol folic acid 0.5 part;

[0059] The preparation method is the same as that in Example 1, wherein the concentration of the organic phase in step (1) is 20 mg / mL, and the concentration of the aqueous colchicine solution in step (3) is 0.5 mg / mL, so that the drug-lipid ratio is 1:60.

[0060] Comparative Example 1

[0061] A double-modified colchicine liposome targeting M1 macrophages, which comprises, by weight fraction, colchicine 0.5 part, hydrogenated soybean phosphatidylcholine 15 parts, cholesterol 6.9 parts, cultured phosphatidyl ethanolamine 1.9 parts, phospholipid polyethylene glycol arginine 0.6 part, phospholipid polyethylene glycol folic acid 0.6 part, buffer 30 parts;

[0062] The preparation method is the same as that in Example 1, wherein the concentration of the organic phase in step (1) is 10 mg / mL, and the concentration of the aqueous colchicine solution in step (3) is 0.5 mg / mL, so that the drug-lipid ratio is 1:50.

[0063] Comparative Example 2

[0064] A double-modified colchicine liposome targeting M1 macrophages, which comprises, by weight fraction, colchicine 0.5 part, hydrogenated soybean phosphatidylcholine 30 parts, cholesterol 13.8 parts, cultured phosphatidyl ethanolamine 3.8 parts, phospholipid polyethylene glycol arginine 1.2 part, phospholipid polyethylene glycol folic acid 1.2 part, buffer 30 parts;

[0065] The preparation method is the same as that in Example 1, wherein the concentration of the organic phase in step (1) is 50 mg / mL, and the concentration of the aqueous colchicine solution in step (3) is 1 mg / mL, so that the drug-lipid ratio is 1:100.

[0066] Comparative Example 3

[0067] A double modified colchicine liposome targeting M1 type macrophages, the formula includes colchicine 0.5 parts, hydrogenated soybean phosphatidylcholine 24 parts, cholesterol 11 parts, phosphatidyl ethanolamine 3 parts, phospholipid polyethylene glycol arginine 0.2 parts, phospholipid polyethylene glycol folic acid 0.2 parts, buffer 30 parts by weight; the preparation method is the same as example 1.

[0068] Comparative example 4

[0069] A double modified colchicine liposome targeting M1 type macrophages, the formula includes colchicine 0.5 parts, hydrogenated soybean phosphatidylcholine 24 parts, cholesterol 11 parts, phosphatidyl ethanolamine 1 part, phospholipid polyethylene glycol arginine 2.5 parts, phospholipid polyethylene glycol folic acid 2.5 parts, buffer 30 parts by weight; the preparation method is the same as example 1.

[0070] Result detection: take the colchicine liposome prepared in the above examples and comparative examples, detect its performance, and the results are shown in table 1:

[0071] Table 1 Performance of colchicine liposome

[0072]

[0073] From table 1 and Figure 2 It can be seen that: with the increase of drug-lipid ratio, the encapsulation efficiency of 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 concentration of lipid, so the optimal drug-lipid ratio is 1:80. When the microfluidic flow rate ratio is 3:9, the particle size and PDI are better, so the optimal flow rate ratio is 3:9.

[0074] Example 9

[0075] A double modified colchicine liposome, the formula and preparation method are the same as example, only the molar ratio of hydrogenated soybean phosphatidylcholine to cholesterol in step (1) is changed to 2:1.

[0076] Example 10

[0077] A double modified colchicine liposome, the formula and preparation method are the same as example, only the molar ratio of hydrogenated soybean phosphatidylcholine to cholesterol in step (1) is changed to 3:1.

[0078] Example 11

[0079] A double modified colchicine liposome, the formula and preparation method are the same as example, only the molar ratio of hydrogenated soybean phosphatidylcholine to cholesterol in step (1) is changed to 4:1.

[0080] Comparative example 5

[0081] A double modified colchicine liposome, the formula and preparation method are the same as the embodiment, only the molar ratio of hydrogenated soybean phosphatidylcholine to cholesterol in step (1) is changed to 1:2.

[0082] Result detection: Take the colchicine liposomes prepared in the above examples and comparative examples to detect their performance, and the results are shown in Table 2:

[0083] Table 2 Influence of phospholipid to cholesterol ratio on colchicine liposome performance

[0084]

[0085] As shown in Table 2, when the phospholipid to cholesterol ratio increases, the liposome particle size increases, the PDI becomes larger, and the liposome becomes unstable, therefore, the most suitable phospholipid to cholesterol ratio is 1:1.

[0086] Comparative example 6

[0087] A single modified colchicine liposome targeting M1 type macrophages, the formula includes colchicine 0.5 parts, hydrogenated soybean phosphatidylcholine 24 parts, cholesterol 11 parts, cultured phosphatidyl ethanolamine 4 parts, phospholipid polyethylene glycol arginine 1 part, buffer 30 parts by weight, and the colchicine liposome is prepared.

[0088] Comparative example 7

[0089] A single modified colchicine liposome targeting M1 type macrophages, the formula includes colchicine 0.5 parts, hydrogenated soybean phosphatidylcholine 24 parts, cholesterol 11 parts, cultured phosphatidyl ethanolamine 4 parts, phospholipid polyethylene glycol folic acid 1 part, buffer 30 parts by weight, and the colchicine liposome is prepared.

[0090] Comparative example 8

[0091] An unmodified colchicine liposome, the formula includes colchicine 0.5 parts, hydrogenated soybean phosphatidylcholine 24 parts, cholesterol 11 parts, cultured phosphatidyl ethanolamine 5 parts, buffer 30 parts by weight, and the colchicine liposome is prepared.

[0092] Result detection: Take the colchicine liposomes prepared in comparative examples 6-8 to detect their performance, and the results are shown in Table 3:

[0093] Table 3 Influence of modification on liposome performance

[0094]

[0095] The results show that the colchicine liposome particle size becomes larger after modification, the encapsulation efficiency slightly decreases, and the influence of folic acid on particle size is greater than that of arginine. However, different modifications have no significant influence on the drug loading capacity of the liposome, therefore, the liposome can be subjected to multiple modifications to improve the performance of the liposome.

[0096] Example 12

[0097] The colchicine liposomes prepared in Example 1, Comparative Example 6, 7 and 8 were placed in PBS buffer at pH = 7.4 and incubated at 37°C, with the volume ratio of colchicine liposomes to PBS buffer being 1:20; samples were taken at 0.25h, 1h, 4h, 8h, 16h and 24h, and the cumulative release percentage of colchicine was detected by ultrafiltration; the results are shown in Figure 3 As compared with the unmodified colchicine liposomes, the release curve of the modified colchicine liposomes was gentler, indicating a slower release rate, but both eventually tended to the same level; and the release curves of the colchicine liposomes modified in different ways were close to each other, with the release rate of the FA-modified colchicine liposomes being the lowest, the release rate of the ARG-modified colchicine liposomes being the highest, and the release level of the ARG-FA double-modified colchicine liposomes being between the two single modifications, with no significant difference.

[0098] Example 13

[0099] (1) Peripheral blood mononuclear cells were obtained from normal human peripheral blood by density gradient centrifugation, and the mononuclear cell concentration was adjusted to 2x10 6 / mL in a 6-well plate, and macrophage colony-stimulating factor M-CSF was given, and after 7 days, primary human macrophages were polarized and generated;

[0100] (2) LPS+IFN-γ was given to the mature primary human macrophages to polarize the proinflammatory macrophages M1, and after 24 hours, the proinflammatory macrophages were polarized and matured;

[0101] (3) Unmodified, ARG-modified, FA-modified and FA+ARG-modified siRNA liposomes loaded with FAM fluorescent dye were prepared, and the preparation scheme was the same as that in Example 1, Comparative Examples 6-8, wherein the PBS buffer in step (2) was replaced by a PBS buffer containing 1.3 μg / ml of FAM fluorescent dye-labeled siRNA, and step (3) was omitted, and each group of liposomes was added to the 6-well plate.

[0102] (3) After 1h of incubation, the excess fluorescence was removed by washing with PBS buffer, and the green fluorescence in the cells was observed under an inverted fluorescence microscope, and the entire experiment was carried out in a light-protected environment, and each experiment was repeated 3 times.

[0103] As can be seen from Figure 4 the fluorescence intensity, 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.

[0104] Fluorescence intensity analysis was performed using ImageJ software, and a histogram of the average fluorescence intensity of M1 macrophages was plotted. The results are as follows: Figure 5 As shown, the average fluorescence intensity of M1 macrophages treated with dual-modified liposomes was significantly better than that of single-modified liposomes. The folate receptor on the surface of M1 macrophages is different from the L-arginine receptor. Dual modification can increase the targeting synergistic effect by targeting different receptors of M1 macrophages.

[0105] Example 14

[0106] Cytotoxicity assay:

[0107] 1. Prepare unmodified colchicine liposomes at different concentrations and investigate their toxic effects on M1 macrophages:

[0108] (1) The concentration of peripheral blood mononuclear cells was set at 1×10⁻⁶. 5 / wells were seeded in 96-well plates and polarized into pro-inflammatory macrophages M1;

[0109] (2) Prepare stock solutions of unmodified colchicine liposomes with different high concentrations, and add them to cells at a ratio of liposomes:complete culture medium = 1:9, with concentrations of 0.1, 0.5, 0.75, 1, and 5 μM, 100 μL per well;

[0110] (3) After 24 h of treatment, add 10 μL of CCK8 to each well, incubate in the dark for 1 h, and measure its OD value at 450 nm.

[0111] Depend on Figure 6 It can be seen that the survival rate of M1 macrophages is >80% at a concentration of 0.5 μM, therefore 0.5 μM was selected for subsequent cell experiments.

[0112] 2. Prepare 0.5 μM concentrations of unmodified and modified colchicine liposomes and colchicine technical, and investigate their toxic effects on M1 macrophages:

[0113] (1) The concentration of peripheral blood mononuclear cells was set at 1×10⁻⁶. 5 / wells were seeded in 96-well plates and polarized into pro-inflammatory macrophages M1;

[0114] (2) Prepare high concentrations of unmodified and modified colchicine liposomes and colchicine technical (COL). Add 0.5 μM of liposomes and colchicine technical to complete culture medium at a ratio of 1:9 to cells, 100 μL per well.

[0115] (3) After 24 h of treatment, add 10 μL of CCK8 to each well, incubate in the dark for 1 h, and measure its OD value at 450 nm.

[0116] Depend on Figure 7It can be seen that, in the same concentration, the toxicity of unmodified and modified colchicine liposomes to M1 macrophages is less than that of the original drug colchicine, indicating that unmodified and modified colchicine liposomes have attenuated effect compared with the original drug colchicine, and the double modification and single modification have similar cytotoxicity to M1 macrophages, with no statistical difference, which is lower than that of colchicine, indicating that the double modified liposome can significantly improve the targeting while maintaining the cell viability at a high level, and reduce the impact on normal life activities.

[0117] Example 15

[0118] Cellular inflammatory factor detection:

[0119] The supernatant of the cell culture after 24h of Example 15-2 was centrifuged at 1000xg for 20 minutes, and the IL-1β level was detected according to the requirements of the human interleukin 1β enzyme-linked immunosorbent assay kit.

[0120] From Figure 8 It can be seen that, in the same concentration, the IL-1β level of unmodified and modified colchicine liposomes to M1 macrophages is less than that of the original drug colchicine, indicating that unmodified and modified colchicine liposomes have stronger anti-inflammatory effect compared with the original drug colchicine, and the double modification has stronger anti-inflammatory effect compared with the single modification, with statistical significance, and the double modification has stronger targeting to M1 macrophages, producing the strongest anti-inflammatory effect.

Claims

1. A colchicine liposome with dual modification targeting M1 macrophages, characterized in that: The raw materials include the following parts by weight: colchicine 0.1-2 parts, phosphatidylcholine 10-40 parts, cholesterol 1-20 parts, phosphatidylethanolamine 1-5 parts, phosphatidyl-polyethylene glycol folic acid 0.4-2 parts, phosphatidyl-polyethylene glycol arginine 0.4-2 parts, and buffer solution 10-100 parts; the molar ratio of phosphatidylethanolamine to phosphatidyl-polyethylene glycol arginine and phosphatidyl-polyethylene glycol folic acid is 0.6-1:0.1-0.4:0.1-0.

4.

2. The colchicine liposome with dual modification targeting M1 macrophages according to claim 1, characterized in that: The phosphatidylcholine is any one of hydrogenated soybean phosphatidylcholine, distearyl phosphatidylcholine, glycerol phosphatidylcholine, dimyristoyl phosphatidylcholine, and dilauryl phosphatidylcholine.

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

4. The colchicine liposome with dual modification 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 colchicine liposome with dual modification targeting M1 macrophages according to claim 1, characterized in that: The buffer solution is any one of PBS buffer solution, citrate-sodium citrate solution, sodium carbonate-sodium bicarbonate buffer solution, acetic acid-sodium acetate solution, lactate-sodium lactate solution, and glycine-sodium glycine solution.

6. A method for preparing colchicine liposomes targeting M1 macrophages with dual modification as described in any one of claims 1-5, characterized in that: Includes the following steps: (1) Preparation of organic phase: Phosphatidylcholine, cholesterol, phosphatidylethanolamine, phosphatidylpolyethylene glycol arginine, phosphatidylpolyethylene glycol folic acid are mixed and dissolved with anhydrous ethanol to obtain organic phase; (2) Preparation of the aqueous phase: Prepare a buffer solution as the aqueous phase; (3) Dissolution: Dissolve colchicine in anhydrous ethanol to obtain a colchicine solution, and add an organic phase; (4) Preparation of drug-loaded liposomes: The organic phase and the aqueous phase were mixed and drug-loaded liposomes were prepared by microfluidic method; (5) Ultrafiltration: The drug-loaded liposomes were purified by ultrafiltration to remove anhydrous ethanol and free colchicine, resulting in double-modified colchicine liposomes.

7. The preparation method according to claim 6, 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.

8. The preparation method according to claim 6, 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-lipid ratio of colchicine to lipid is 1:60-90.

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

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