Medical composite material for regulating and controlling lipid content of macrophages as well as preparation method and application of medical composite material

Abstract

The invention relates to the technical field of medical materials, in particular to a medical composite material for regulating and controlling the lipid content of macrophages as well as a preparation method and application of the medical composite material. The medical composite material comprises an amino-rich surface material grafted with polyamidoamine and a liver X receptor stimulant, wherein the liver X receptor stimulant is loaded on the amino-rich surface material. The medical composite material can effectively inhibit proliferation of inflammatory macrophages and adjust the lipid content of the macrophages, can be further used for in-situ treatment of focuses of atherosclerosis patients, and has a good anticoagulant effect.

Description

Technical Field

[0001] The present invention relates to the technical field of medical materials, and in particular to a medical composite material for regulating the lipid content of macrophages, a preparation method and application thereof. Background Art

[0002] Implant materials and devices, especially minimally invasive interventional materials and devices, have become the most effective tools for treating cardiovascular diseases, such as vascular stents, drug-eluting balloons, artificial blood vessels, and injectable anti-heart failure hydrogels. Cardiovascular diseases such as atherosclerosis are mainly treated with drugs in the early stages, and interventional therapy is the main treatment method when they develop to the middle and late stages. The research on cardiovascular implant materials mainly focuses on promoting endothelial growth and anti-smooth muscle proliferation, while there are fewer studies on abnormal macrophage lipid metabolism. The imbalance between the uptake and excretion of lipids in macrophages is one of the main causes of cardiovascular disease. The accumulation of lipids in macrophages can lead to their dysfunction, which in turn affects the anti-inflammatory and lipid metabolism functions of macrophages. Therefore, there is an urgent need for a cardiovascular disease surface material based on lipid regulation in macrophages.

[0003] In view of this, the present invention is proposed. Summary of the invention

[0004] The purpose of the present invention is to provide a medical composite material for regulating the lipid content of macrophages, a preparation method and application thereof. The medical composite material provided by the embodiment of the present invention can effectively inhibit the proliferation of inflammatory macrophages and regulate the lipid content of macrophages, and can be used for in situ treatment of lesions in patients with atherosclerosis, and at the same time, it has a good anticoagulant effect.

[0005] The present invention is achieved in that:

[0006] In a first aspect, the present invention provides a medical composite material for regulating the lipid content of macrophages, comprising: an amine-rich surface material grafted with polyamidoamine and a liver X receptor agonist, wherein the liver X receptor agonist is loaded on the amine-rich surface material.

[0007] In an alternative embodiment, the liver X receptor agonist is selected from GW3965.

[0008] In a second aspect, the present invention provides a method for preparing a medical composite material for regulating the lipid content of macrophages according to the aforementioned embodiment, comprising: mixing an amine-rich surface material grafted with polyamidoamine and a liver X receptor agonist, so that the liver X receptor agonist is loaded on the amine-rich surface material.

[0009] In an optional embodiment, the method comprises: mixing the liver X receptor agonist with a hydroxyl activator, and then mixing the mixture with the amine-rich surface material grafted with polyamidoamine;

[0010] Preferably, the concentration of the liver X receptor agonist is 1-1000ug / ml;

[0011] Preferably, the conditions for mixing the liver X receptor agonist and the hydroxy activator include: a temperature of 35-37° C. and a time of 15-30 minutes;

[0012] Preferably, the conditions for mixing with the amine-rich surface material grafted with polyamidoamine include: a temperature of 4-40° C. and a time of 8-24 hours.

[0013] In a third aspect, the present invention provides a use of the medical composite material for regulating the lipid content of macrophages described in the aforementioned embodiment in the preparation of a regulator for regulating the lipid content of macrophages.

[0014] In a fourth aspect, the present invention provides a use of the medical composite material for regulating the lipid content of macrophages as described in the aforementioned embodiment in the preparation of a treatment for diseases caused by abnormal lipid content of macrophages.

[0015] In alternative embodiments, the disease comprises cardiovascular disease.

[0016] In alternative embodiments, the disease comprises atherosclerosis.

[0017] In a fifth aspect, the present invention provides a use of the medical composite material for regulating the lipid content of macrophages described in the aforementioned embodiment in the preparation of an anticoagulant.

[0018] The present invention has the following beneficial effects: In the embodiment of the present invention, the liver X receptor agonist is loaded on the amine-rich surface material, so that the medical composite material can effectively inhibit the proliferation of inflammatory macrophages and regulate the lipid content of macrophages, and can be used for in situ treatment of lesions in patients with atherosclerosis. At the same time, it has a good anticoagulant effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0020] Figure 1 A flow chart of the preparation of a medical composite material for regulating the lipid content of macrophages provided in Example 1 of the present invention;

[0021] Figure 2 The test result diagram provided for Experimental Example 1 of the present invention;

[0022] Figure 3 The test result diagram provided for Experimental Example 2 of the present invention;

[0023] Figure 4 The test result diagram provided for Experimental Example 3 of the present invention;

[0024] Figure 5 The test result diagram provided for Experimental Example 4 of the present invention;

[0025] Figure 6 The test result diagram provided for Experimental Example 5 of the present invention;

[0026] Figure 7 This is a test result diagram provided for Experimental Example 6 of the present invention. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0028] The embodiment of the present invention provides a medical composite material for regulating the lipid content of macrophages, comprising: an amine-rich surface material grafted with polyamidoamine and a liver X receptor agonist, wherein the liver X receptor agonist is loaded on the amine-rich surface material.

[0029] Wherein, the liver X receptor agonist is selected from GW3965. The selection of the liver X receptor agonist is only an example of the embodiment of the present invention, and other liver X receptor agonists may also be selected.

[0030] The amine-rich surface material is formed by reacting polyamidoamine with a surface material having active hydroxyl groups.

[0031] In a second aspect, the present invention provides a method for preparing the medical composite material for regulating the lipid content of macrophages as described in the aforementioned embodiment, comprising:

[0032] The preparation step of the surface material for forming active hydroxyl groups includes: mixing the load material with the alkaline solution for reaction; wherein the reaction conditions include: temperature of 70-90°C and time of 8-12h. For example, the temperature is any value between 70°C, 80°C and 90°C, and the time is any value between 8 hours, 10 hours and 12 hours.

[0033] The alkaline solution may be a hydroxide solution such as a sodium hydroxide solution and a potassium hydroxide solution.

[0034] Specifically, the loaded material is immersed in an alkaline solution with a concentration of 2.5-3.5 mol / L, and reacted at 70-90°C for 8-12 hours to obtain an alkali-activated material, i.e., a surface material with active hydroxyl groups. During the alkali activation process, hydroxyl groups are introduced onto the surface of the loaded material to make the surface of the material strongly negatively charged, which is convenient for the subsequent introduction of amine groups. The alkali-activated material is ultrasonically washed 2-4 times, kept at 70-90°C for 8-12 hours, ultrasonically cleaned again, and dried.

[0035] The preparation steps of the amine-rich surface material include: mixing and reacting polyamidoamine (also known as PAMAM) with a surface material having active hydroxyl groups; the reaction conditions include: temperature of 4-40°C, time of 6-24 hours, and molecular weight of polyamidoamine of 6000-10000. Specifically, the alkali-activated material obtained above is immersed in a PAMAM solution to obtain an amine-rich surface. When the pH of the solution is less than 10, the amine groups in PAMAM can be protonated to carry a positive charge, and thus can interact with the negatively charged surface through electrostatic bonding. The reaction temperature is 4-40°C, the time is 6-24h, the concentration of the PAMAM solution is 0.5-3mg / ml, and the molecular weight is 6000-10000.

[0036] The liver X receptor agonist is mixed with a hydroxyl activator, wherein the hydroxyl activator is selected from EDC and NHS; the molar ratio of EDC and NHS to the liver X receptor agonist is: 1.5:1.1:1-3:1.5:1; the concentration of the liver X receptor agonist is 1-1000ug / ml; the conditions for mixing the liver X receptor agonist with the hydroxyl activator include: temperature of 35-37°C and time of 15-30 minutes. Then, it is mixed with the amine-rich surface material grafted with polyamide amine. The conditions for mixing with the amine-rich surface material grafted with polyamide amine include: temperature of 4-40°C and time of 8-24 hours.

[0037] Specifically, a liver X receptor agonist (LXR), such as GW3965, is dissolved in DMSO to obtain a solution concentration of 1-1000ug / ml, and the obtained solution is mixed with a carboxyl activator EDC / NHS, wherein the molar ratio of EDC:NHS:GW3965 is 1.5:1.1:1-3:1.5:1, and the reaction is carried out at 35-37°C for 15-30min, and then the amine-rich surface material is immersed in the above solution and reacted at 4-40°C for 8-24h. The carboxyl group activated by EDC / NHS can be introduced to the surface through a covalent reaction with the amine group. In addition, GW3965 can also enter the hydrophobic cavity of PAMAM through hydrophobic interaction to increase its surface introduction amount.

[0038] The medical composite material prepared in the embodiment of the present invention can regulate the lipid content in macrophages, increase cholesterol efflux, and has a good anticoagulant effect.

[0039] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0040] Example 1

[0041] The present invention provides a method for preparing a medical composite material (denoted as PG20) for regulating the lipid content of macrophages. Figure 1 ,include:

[0042] After polishing and cleaning, the titanium sheet was immersed in a 3 mol / L sodium hydroxide solution, allowed to stand at 80°C for 12 hours, then ultrasonically cleaned with RO water 3 times, allowed to stand at 80°C for 12 hours, ultrasonically cleaned with RO water 3 times, and then dried at 50°C for use. The alkali-activated material was reacted in a 1 mol / L PAMAM solution at room temperature for 12 hours, washed with RO water 3 times, and dried with an ear-washing bulb for use. GW3965 was dissolved in DMSO at a concentration of 20ug / ml, and EDC and NHS solutions were prepared with a molar ratio of 1.5:1.1:1 for EDC:NHS:GW3965. EDC and NHS were added to the GW3965 solution successively, reacted at 37°C for 30 minutes, and the mixed solution was added to the surface of the PAMAM-modified titanium material, reacted at 37°C for 12 hours, washed with RO water 3 times, and dried with an ear-washing bulb for use.

[0043] Example 2

[0044] The present invention provides a method for preparing a medical composite material (denoted as PG100) for regulating the lipid content of macrophages, comprising:

[0045] After polishing and cleaning, the titanium sheet was immersed in a 3 mol / L sodium hydroxide solution, allowed to stand at 80°C for 12 hours, then ultrasonically cleaned with RO water 3 times, allowed to stand at 80°C for 12 hours, ultrasonically cleaned with RO water 3 times, and then dried at 50°C for use. The alkali-activated material was reacted in a 1 mol / L PAMAM solution at room temperature for 12 hours, washed with RO water 3 times, and blown dry with an ear-washing bulb for use. GW3965 was dissolved in DMSO at a concentration of 100ug / ml, and EDC and NHS solutions were prepared with a molar ratio of EDC: NHS: GW3965 of 1.5:1.1:1. EDC and NHS were added to the GW3965 solution successively, reacted at 37°C for 30 minutes, and the mixed solution was added to the surface of the PAMAM-modified titanium material, reacted at 37°C for 12 hours, washed with RO water 3 times, and blown dry with an ear-washing bulb for use.

[0046] Example 3

[0047] The present invention provides a method for preparing a medical composite material (denoted as PG300) for regulating the lipid content of macrophages, comprising:

[0048] After polishing and cleaning, the titanium sheet was immersed in a 3 mol / L sodium hydroxide solution, allowed to stand at 80°C for 12 hours, then ultrasonically cleaned with RO water 3 times, allowed to stand at 80°C for 12 hours, ultrasonically cleaned with RO water 3 times, and then dried at 50°C for use. The alkali-activated material was reacted in a 1 mol / L PAMAM solution at room temperature for 12 hours, washed with RO water 3 times, and blown dry with an ear-washing bulb for use. GW3965 was dissolved in DMSO at a concentration of 300ug / ml, and EDC and NHS solutions were prepared with a molar ratio of EDC: NHS: GW3965 of 1.5:1.1:1. EDC and NHS were added to the GW3965 solution successively, reacted at 37°C for 30 minutes, and the mixed solution was added to the surface of the PAMAM-modified titanium material, reacted at 37°C for 12 hours, washed with RO water 3 times, and blown dry with an ear-washing bulb for use.

[0049] Example 4

[0050] The present invention provides a method for preparing a medical composite material (denoted as PG500) for regulating the lipid content of macrophages, comprising:

[0051] After polishing and cleaning, the titanium sheet was immersed in a 3 mol / L sodium hydroxide solution, allowed to stand at 80°C for 12 hours, then ultrasonically cleaned with RO water 3 times, allowed to stand at 80°C for 12 hours, ultrasonically cleaned with RO water 3 times, and then dried at 50°C for use. The alkali-activated material was reacted in a 1 mol / L PAMAM solution at room temperature for 12 hours, washed with RO water 3 times, and dried with an ear-washing bulb for use. GW3965 was dissolved in DMSO at a concentration of 500ug / ml, and EDC and NHS solutions were prepared with a molar ratio of EDC: NHS: GW3965 of 1.5:1.1:1. EDC and NHS were added to the GW3965 solution successively, reacted at 37°C for 30 minutes, and the mixed solution was added to the surface of the PAMAM-modified titanium material, reacted at 37°C for 12 hours, washed with RO water 3 times, and dried with an ear-washing bulb for use.

[0052] Comparative Example 1

[0053] This comparative example provides a method for preparing a medical composite material (referred to as OHP), comprising:

[0054] After polishing and cleaning, the titanium sheet was immersed in a 3 mol / L sodium hydroxide solution, left at 80°C for 12 hours, then ultrasonically cleaned with RO water for 3 times, left at 80°C for 12 hours, ultrasonically cleaned with RO water for 3 times, and then dried at 50°C for use. The alkali-activated material was reacted in a 1 mol / L PAMAM solution at room temperature for 12 hours, washed with RO water 3 times, and dried with an ear-cleaning bulb for use.

[0055] Comparative Example 2

[0056] This comparative example provides a method for preparing a medical composite material (referred to as TiOH), comprising:

[0057] After polishing and cleaning, the titanium sheet was immersed in a 3 mol / L sodium hydroxide solution, allowed to stand at 80°C for 12 h, then ultrasonically cleaned with RO water for 3 times, allowed to stand at 80°C for 12 h, ultrasonically cleaned with RO water for 3 times, and then dried at 50°C for standby use.

[0058] Experimental Example 1

[0059] XPS was used to test the surface element content of the samples prepared in Example 4 and Comparative Examples 1 and 2, and a titanium sheet was used for comparison. The results are as follows: Figure 2 In the figure, Ti represents a titanium sheet without any treatment, TiOH represents comparative example 2, OHP represents comparative example 1, and PG represents example 4.

[0060] Depend on Figure 2 The XPS full spectrum results show that the surface of the sample loaded with GW3965 has an F1s peak at a binding energy of 685 eV, indicating that GW3965 has been successfully introduced into the sample surface. Figure 2 The XPS element quantitative results show that when PAMAM is introduced, the N element content increases from 2.46% to 12.14%. This is because PAMAM contains a large number of amine groups, and the introduction of PAMAM increases the N element content; when GW3965 is introduced, the F element content increases from 1.05% to 8.28%. This is because GW3965 also has F atoms, and the introduction of GW3965 increases the F element content. This result is consistent with the full spectrum results, both of which indicate that materials with adjustable lipid content have been successfully prepared.

[0061] Experimental Example 2

[0062] The grafting amount of GW3965 of the samples prepared in Examples 1-4 was tested by UV-Vis spectrophotometer. The results are as follows: Figure 3 In the figure, PG20 represents embodiment 1, PG100 represents embodiment 2, PG300 represents embodiment 3, and PG500 represents embodiment 4.

[0063] Depend on Figure 3The UV results show that the density of GW3965 on the sample surface is dose-dependent with the concentration of introduced GW3965, indicating that GW3965 was successfully introduced into the material surface and that the surface concentration was different.

[0064] Experimental Example 3

[0065] The effects of the samples prepared in Examples 1-4 and Comparative Examples 1 and 2 on the lipid content in macrophages were tested.

[0066] The test method is: first, the macrophages are treated and co-cultured with oxLDL to make the macrophages foam, and then the treated macrophages with the same concentration are planted on the surface of different samples, the intracellular lipids are stained with BODIPY fluorescent dye, and the fluorescence image of the intracellular lipid content on the surface of the material is tested using a fluorescence microscope. The fluorescence intensity is positively correlated with the lipid content, that is, the stronger the fluorescence intensity, the higher the intracellular lipid content, and the weaker the fluorescence intensity, the lower the intracellular lipid content.

[0067] The results are as follows Figure 4 As shown, Figure 4 Fluorescence images of lipid content in macrophages on the surface of different samples. Figure 4 It can be seen that with the introduction of GW3965, the fluorescence intensity gradually weakened, the intracellular lipid content decreased, and the amount of lipid reduction was dose-dependent with the concentration of GW3965.

[0068] Experimental Example 4

[0069] The effects of the sample surfaces prepared in Examples 1-4 and Comparative Examples 1 and 2 on the number of macrophage adhesion were tested.

[0070] The test method is: firstly, the macrophages are treated and co-cultured with oxLDL, then the treated macrophages with the same concentration are planted on the surface of different samples, and the fluorescence image of the number of macrophages on the surface of the material is tested using a fluorescence microscope.

[0071] The results are as follows Figure 5 As shown, Figure 5 The fluorescence images of the number of macrophages adhering to the surface of different samples are shown in Figure 2. Figure 5 It can be seen that after the introduction of the GW3965 molecule, the number of inflammatory macrophages decreased, indicating that GW3965 can inhibit the proliferation of inflammatory macrophages.

[0072] Experimental Example 5

[0073] The effects of the sample surfaces prepared in Examples 1-4 and Comparative Examples 1 and 2 on macrophage inflammatory factors were tested.

[0074] The test method is: firstly, the macrophages are treated and co-cultured with oxLDL, then the same concentration of treated macrophages are planted on the surface of different samples, and the content of TNF-α, a macrophage inflammatory factor, on the surface of the material is tested using an enzyme-linked immunosorbent assay kit.

[0075] The results are as follows Figure 6 As shown, Figure 6 The quantitative results of the content of TNF-α, an inflammatory factor in macrophages on the surface of different samples. Figure 6 It can be seen that after the introduction of the GW3965 molecule, the inflammatory factor TNF-α secreted by inflammatory macrophages was significantly reduced, indicating that GW3965 has an anti-inflammatory effect.

[0076] Experimental Example 6

[0077] The effects of the samples prepared in Examples 1-4 and Comparative Examples 1 and 2 on the number of platelet adhesion were tested.

[0078] The test method is: first collect blood with a blood collection tube, collect platelet-rich plasma by centrifugation, then add the same dose of platelet-rich plasma on the surface of different samples, let it stand at 37°C for 45 minutes, then wash and fix, dehydrate, spray gold, and take 6 pictures of different fields of view under a scanning electron microscope at 1,000 times, and count and statistics.

[0079] The results are as follows Figure 7 As shown, Figure 7 The following is a scanning diagram and a statistical diagram of platelet adhesion on the sample surface. Figure 7 It can be seen that compared with titanium and comparative examples 1 and 2, the number of platelets adhering to the surface of the samples in the embodiment group is reduced, and the activation of platelets is inhibited.

[0080] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A medical composite material for regulating the lipid content of macrophages, characterized in that: It includes: An amine-rich surface material grafted with polyamidoamine and a liver X receptor agonist, wherein the liver X receptor agonist is loaded on the amine-rich surface material.

2. The medical composite material for regulating the lipid content of macrophages according to claim 1, characterized in that: The liver X receptor agonist is selected from GW3965.

3. A method for preparing a medical composite material for regulating macrophage lipid content according to claim 1, characterized in that: include: The amine-rich surface material grafted with polyamidoamine and the liver X receptor agonist are mixed, so that the liver X receptor agonist is loaded on the amine-rich surface material.

4. The preparation method according to claim 3, characterized in that: include: The liver X receptor agonist is mixed with a hydroxyl activator, and then mixed with the amine-rich surface material grafted with polyamidoamine; Preferably, the concentration of the liver X receptor agonist is 1-1000ug / ml; Preferably, the conditions for mixing the liver X receptor agonist and the hydroxy activator include: a temperature of 35-37° C. and a time of 15-30 minutes; Preferably, the conditions for mixing with the amine-rich surface material grafted with polyamidoamine include: a temperature of 4-40° C. and a time of 8-24 hours.

5. Use of the medical composite material for regulating the lipid content of macrophages according to claim 1 in preparing a regulator for regulating the lipid content of macrophages.

6. Use of the medical composite material for regulating macrophage lipid content according to claim 1 in the preparation of a treatment for diseases caused by abnormal macrophage lipid content.

7. The use according to claim 6, characterized in that: The diseases include cardiovascular diseases.

8. The use according to claim 6, characterized in that: Such diseases include atherosclerosis.

9. Use of the medical composite material for regulating the lipid content of macrophages according to claim 1 in the preparation of an anticoagulant.

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