A method for preparing filler particles for targeted adsorption of low-density lipoprotein from patients with atherosclerosis and its application.

Abstract

This invention discloses a method for preparing filler particles for targeted adsorption of low-density lipoprotein (LDL) in patients with atherosclerosis. The steps include: 1) washing D101 macroporous resin with ultrapure water and physiological saline; 2) adding fibrin and PBS solution for soaking; adding thrombin factor II; adding zinc gluconate during the reaction; 3) repeating step 2) at least three times and drying the D101 macroporous resin; 4) adding physiological saline and D101 macroporous resin to lyophilized protein ApoB100 and mixing thoroughly; 5) lyophilizing the resin obtained in step 4) while controlling the humidity at 65±5% to obtain the finished product. This application utilizes this adhesion effect to target the adsorption of foam cells in atherosclerosis, adsorbing foam cells through hemoperfusion, removing redundant blood lipid components in the body, reducing macrophage infiltration of organs and blood vessels, and reducing pathological changes in organs and blood vessels.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a method for preparing filler particles for targeted adsorption of low-density lipoprotein from patients with atherosclerosis and its application. Background Technology

[0002] Atherosclerosis is a disease in which lipid plaques (atherosclerotic plaques) form on the walls of medium-sized or large arteries, leading to reduced blood flow or obstruction of blood flow. Atherosclerosis can affect medium-sized and large arteries in the brain, heart, kidneys, other vital organs, and legs. It is the most important and common type of arteriosclerosis. Atherosclerosis is a general term for several diseases characterized by thickened arterial walls and reduced elasticity.

[0003] The causes of atherosclerosis: The arterial wall is composed of several layers; the intima, or inner lining (endothelium), is usually smooth and intact. Atherosclerosis begins with a damaged or diseased intima. Subsequently, some white blood cells called monocytes and T cells (called low-density lipoprotein (LDL), a blood lipid closely related to cardiovascular disease) are activated and leave the bloodstream, passing through the arterial intima into the arterial wall. Inside the intima, LDL transforms into foam cells, which are cells that collect fatty substances, primarily cholesterol.

[0004] Current technologies primarily address the elimination of foam cells, but they are not very effective in controlling the growth of foam cells. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide a method for preparing filler particles for targeted adsorption of low-density lipoprotein from patients with atherosclerosis. Another objective of this invention is to provide applications for these filler particles.

[0006] To address the above problems, the adopted technical solution is: a method for preparing filler particles for targeted adsorption of low-density lipoprotein from patients with atherosclerosis, the steps of which include:

[0007] 1) Clean the D101 macroporous resin with ultrapure water at least 3 times, and then clean the D101 macroporous resin with physiological saline at least 2 times.

[0008] 2) Add fibrin and PBS solution to the D101 macroporous resin after cleaning in step 1), soak for 24±1h, and control the temperature at 18~27℃; then add thrombin factor II and control the temperature at 18~27℃; add zinc salt of gluconate during the reaction.

[0009] 3) Repeat step 2) at least three times to dry the D101 macroporous resin after drying at a temperature of 40±5℃ and a humidity of 55±5%.

[0010] 4) Add physiological saline and the D101 macroporous resin treated in step 3) to the freeze-dried protein ApoB100, and mix well.

[0011] 5) Freeze-dry the resin obtained in step 4) for 8±0.5h at a humidity of 65±5% to obtain filler particles that directionally adsorb low-density lipoprotein from patients with atherosclerosis.

[0012] The fibrin accounts for 0.5±0.1‰ of the mass of the D101 macroporous resin; the thrombin factor II accounts for 0.03±0.005‰ of the mass of the D101 macroporous resin; and the zinc salt of gluconate accounts for 0.002±0.0005‰ of the mass of the D101 macroporous resin.

[0013] The amount of PBS solution used is 20 mL ± 1 mL for every 10 g of D101 macroporous resin.

[0014] In step 4), 1 mg of the lyophilized protein ApoB100 is added to 1 mL ± 0.1 mL of physiological saline, and 4 g ± 1 g of the resin treated in step 3).

[0015] The pH of the PBS solution is 7.5 ± 0.5.

[0016] The filler particles for targeted adsorption of low-density lipoprotein from patients with atherosclerosis are stored at 2-8°C.

[0017] In step 1), the purity of the ultrapure water reaches 18.2 MΩ·cm.

[0018] The second technical problem to be solved by this application is the application of the filler particles prepared by the method for preparing filler particles for directional adsorption of low-density lipoprotein in patients with atherosclerosis as fillers in a hemoperfusion device.

[0019] Beneficial effects: Fibrin glue is an absorbable blood-derived product and a biological tissue adhesive that mimics the final stage of the coagulation cascade.

[0020] The role of fibrin glue is to more effectively fix the enzyme protein onto the resin. The combination of these two substances can uniformly cross-link ApoB100 onto the resin, forming a stable structure. ApoB100 is an immune protein that has a directional binding effect on low-density lipoprotein (LDL), and is used to target and clear LDL.

[0021] Principle: The coagulation pathway involves thrombin converting fibrinogen into fibrin glue, which can adhere to resin. Zinc ions can promote the conversion of fibrinogen into fibrin glue by thrombin, which can effectively adhere to biological factors.

[0022] This application utilizes this adhesion effect to target and adsorb foam cells in atherosclerosis. By using blood perfusion to adsorb foam cells, redundant blood lipid components in the body are removed, macrophage infiltration into organs and blood vessels is reduced, and pathological changes in organs and blood vessels are reduced. Detailed Implementation

[0023] The present application is further illustrated below by way of examples, but these examples do not limit the present invention.

[0024] Example 1

[0025] A method for preparing filler particles for targeted adsorption of low-density lipoprotein from patients with atherosclerosis, comprising the following steps:

[0026] 1) Take 1000g of D101 type macroporous resin (CAS No.: 2393-92-2), wash it 3 times with ultrapure water; then wash it 2 times with physiological saline.

[0027] 2) Add 500mg of fibrin and 200mL of PBS solution (phosphate buffer solution) (pH=7.0) to the cleaned D101 macroporous resin in step 1), soak for 24h, add 30mg of thrombin factor II, and control the temperature at 25℃ (add 2mg of zinc gluconate during the reaction, zinc ions promote the formation of fibrin biogel).

[0028] 3) Repeat step 2) three times to dry the resin at a temperature of 40℃ and a humidity of 55%;

[0029] 4) Take 50 mg of lyophilized protein ApoB100, add 50 mL of physiological saline, and 200 g of the resin treated in step 3). Stir with a magnetic stirrer for 20 min.

[0030] 5) The resin obtained in step 4) is freeze-dried for 8 hours at a humidity of 65%. The filler particles prepared for directional adsorption of low-density lipoprotein in patients with atherosclerosis are stored at 2-8℃ for later use.

[0031] Example 2

[0032] A method for preparing filler particles for targeted adsorption of low-density lipoprotein from patients with atherosclerosis, comprising the following steps:

[0033] 1) Take 1000g of D101 type macroporous resin (CAS No.: 2393-92-2), wash it 3 times with ultrapure water; then wash it 2 times with physiological saline.

[0034] 2) After cleaning in step 1), add 600 mg of fibrin and 1500 mL of PBS solution (pH = 7.5) to the D101 macroporous resin, control the temperature at 22℃, and soak for 24 ± 1 h; keep the temperature at 22℃ and add 35 mg of thrombin factor II; add 2.5 mg of zinc gluconate during the reaction.

[0035] 3) Repeat step 2) three times to dry the resin at a temperature of 40℃ and a humidity of 55%;

[0036] 4) Take 50 mg of lyophilized protein ApoB100, add 55 mL of physiological saline, and 200 g of the resin treated in step 3), and stir with a magnetic stirrer for 20 min.

[0037] 5) The resin obtained in step 4) is freeze-dried for 8 hours at a humidity of 65%. The filler particles prepared for directional adsorption of low-density lipoprotein in patients with atherosclerosis are stored at 2-8℃ for later use.

[0038] Example 3

[0039] A method for preparing filler particles for targeted adsorption of low-density lipoprotein from patients with atherosclerosis, comprising the following steps:

[0040] 1) Take 1000g of D101 type macroporous resin (CAS No.: 2393-92-2), wash it 3 times with ultrapure water; then wash it 2 times with physiological saline.

[0041] 2) After cleaning in step 1), add 400 mg of fibrin and 1800 mL of PBS solution (pH = 7.5) to the D101 macroporous resin, control the temperature at 22℃, and soak for 24 ± 1 h; keep the temperature at 22℃ and add 25 mg of thrombin factor II; add 2.5 mg of zinc gluconate during the reaction.

[0042] 3) Repeat step 2) three times to dry the resin at a temperature of 40℃ and a humidity of 55%;

[0043] 4) Take 50 mg of lyophilized protein ApoB100, add 45 mL of physiological saline, 180 g of the resin treated in step 3), and stir with a magnetic stirrer for 20 min.

[0044] 5) The resin obtained in step 4) is freeze-dried for 8 hours at a humidity of 65%. The filler particles prepared for directional adsorption of low-density lipoprotein in patients with atherosclerosis are stored at 2-8℃ for later use.

[0045] The adsorption effect of D101 type macroporous resin filler on foam cells:

[0046] filler 150g 200g Adsorption effect 4％ 5％

[0047] Results: The D101 type macroporous resin filler itself has a very low adsorption efficiency for foam cells.

[0048] Application of the filler particles obtained in Examples 1-3 as fillers in blood perfusion devices:

[0049] The filler particles prepared in the example for targeted adsorption of low-density lipoprotein from patients with atherosclerosis were placed in a hemoperfusion device. Foam cells were adsorbed by hemoperfusion to remove redundant blood lipid components in the body. The present invention has an effective removal rate of 70% for foam cells formed by low-density lipoprotein with 200 mg of resin.

[0050] Filler content (g) Before clearance (mg / dl) After clearance (mg / dl) 120 183 150 200 218 90 250 223 81 300 208 63 350 229 50

[0051] Filtration efficiency

[0052]

[0053] The higher the adhesion rate of ApoB100, the higher the clearance rate of foam cells.

Claims

1. A method for preparing filler particles for targeted adsorption of low-density lipoprotein from patients with atherosclerosis, comprising the following steps: 1) Clean the D101 macroporous resin with ultrapure water at least 3 times, and then clean the D101 macroporous resin with physiological saline at least 2 times. 2) Add fibrin and PBS solution to the D101 macroporous resin after cleaning in step 1), control the temperature at 18~27℃, and soak for 24±1h; keep the temperature at 18~27℃ and add thrombin factor II; add zinc salt of gluconate during the reaction. 3) Repeat step 2) at least three times to dry the D101 macroporous resin after drying, controlling the drying temperature at 40±5℃ until the humidity is 55±5%; 4) Add physiological saline and the D101 macroporous resin treated in step 3) to the freeze-dried protein ApoB100, and mix well; 5) Freeze-dry the resin obtained in step 4) for 8±0.5h, controlling the humidity at 65±5%, to obtain filler particles that directionally adsorb low-density lipoprotein from patients with atherosclerosis.

2. The method for preparing filler particles for targeted adsorption of low-density lipoprotein from patients with atherosclerosis according to claim 1, characterized in that: The fibrin accounts for 0.5±0.1‰ of the mass of the D101 macroporous resin; the thrombin factor II accounts for 0.03±0.005‰ of the mass of the D101 macroporous resin; and the zinc salt of gluconate accounts for 0.002±0.0005‰ of the mass of the D101 macroporous resin.

3. The method for preparing filler particles for targeted adsorption of low-density lipoprotein from patients with atherosclerosis according to claim 1 or 2, characterized in that: The amount of PBS solution used is 2 mL ± 1 mL for every 10 g of D101 macroporous resin.

4. The method for preparing filler particles for targeted adsorption of low-density lipoprotein from patients with atherosclerosis according to claim 1 or 2, characterized in that: In step 4), 1 mg of the lyophilized protein ApoB100 is added to 1 mL ± 0.1 mL of physiological saline, and 4 g ± 1 g of the resin treated in step 3).

5. The method for preparing filler particles for targeted adsorption of low-density lipoprotein from patients with atherosclerosis according to claim 1 or 2, characterized in that: The pH of the PBS solution is 7.5 ± 0.

5.

6. The method for preparing filler particles for targeted adsorption of low-density lipoprotein from patients with atherosclerosis according to claim 1 or 2, characterized in that: The filler particles for targeted adsorption of low-density lipoprotein from patients with atherosclerosis are stored at 2-8°C.

7. A method for preparing filler particles for targeted adsorption of low-density lipoprotein from patients with atherosclerosis according to claim 1 or 2, characterized in that: In step 1), the purity of the ultrapure water reaches 18.2 MΩ·cm.

8. The filler particles prepared by the method for preparing filler particles for targeted adsorption of low-density lipoprotein in patients with atherosclerosis according to any one of claims 1 to 7 are used as fillers in hemoperfusion devices.

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