CN109432513B

CN109432513B - A kind of biological material with anti-thrombotic re-formation function and preparation method thereof

Info

Publication number: CN109432513B
Application number: CN201811516603.1A
Authority: CN
Inventors: 马勇
Original assignee: China Medical University
Current assignee: China Medical University
Priority date: 2018-12-12
Filing date: 2018-12-12
Publication date: 2021-03-23
Anticipated expiration: 2038-12-12
Also published as: CN109432513A

Abstract

The invention belongs to the field of medical materials, and specifically relates to a biological material for simulating endothelial cell anti-thrombotic re-formation and a preparation method thereof. The present invention provides a biological functional material with anti-thrombotic formation, which can fix a complete and stable liposome on the surface of a solid material by a chemical method, and a stable and ordered liposome covering layer on the surface of a specific material , using the selective specificity of "click chemistry" to further modify the surface of the liposome with thrombomodulin to realize the functionalization of the liposome, and to achieve the antithrombotic function of simulating endothelial cells. This surface-modified material with bioactive molecules will greatly reduce the incompatibility and immune system stress caused by the material of the interventional device and the surface after contacting the blood in the interventional therapy (such as coronary heart disease intervention, stent). resulting in re-thrombosis.

Description

Biological material with anti-thrombosis re-forming function and preparation method thereof

Technical Field

The invention belongs to the field of medical materials, and particularly relates to a biological material for simulating endothelial cell antithrombotic reformation and a preparation method thereof.

Background

The biological functional material with the nano structure shows attractive application prospect in a plurality of fields, so that the biological functional material becomes one of the most active research fields of the chemical and material disciplines in the 21 st century, particularly in the biomedical field. At present, for treating thrombotic vascular diseases, restoration of normal blood flow through interventional techniques such as stents, catheters and the like is the main treatment means. However, surface-induced incompatibility of the materials of interventional devices upon contact with blood and narrowing or occlusion of blood vessels due to thrombus reformation caused by immune system stress often limit the long-term use of such interventional devices. In the diseases of blood vessel circulation dysfunction caused by thrombosis, 80% of the thrombosis caused by equipment intervention accounts, and the treatment cost is more than 10 hundred million dollars per year. The failure of therapy due to such side effects is directly related to incompatibility of blood-biomaterial and tissue-biomaterial surfaces and biological stress response.

The blood meets with exogenous substances, and the coagulation system in the body is activated immediately. However, in the blood vessels covered by endothelial cells, blood does not clot, mainly due to the presence of some bioactive molecules present on the surface of endothelial cells (mainly thrombomodulin, thrombomodulin and heparin) that inhibit the stress response of thrombosis. With the further understanding of the mechanism of anti-thrombus of endothelial cells and the mechanism of thrombus formation of biomaterials and the development of medical technology, nanotechnology and surface chemistry, several models of non-thrombogenic surface materials have been proposed, such as direct phospholipid modification of surface materials for increasing blood compatibility, and particularly, methods of modifying the surface of biomaterials using phospholipid-containing high molecular polymers or phospholipid bilayer membranes based on biomembrane simulation have been proposed and widely studied. Surfaces composed of Phosphatidylcholine (PC), the major phospholipid component of cell membranes, exhibit excellent biocompatibility because they not only inhibit nonspecific adsorption of proteins, but also improve the stability of biomolecules present on the surface of the material. Such phospholipids and their high molecular polymers have been used for modification of various medical biomaterials such as dacron (Dacrons), artificial blood vessels, polyethylene artificial joints, medical stainless steel needles and coronary stents, and show good hemocompatibility and biocompatibility in vivo. However, a series of problems such as the orderly and close arrangement of phospholipids on the surface of the material and the stability of simulated biological membranes are still not solved.

Disclosure of Invention

In view of the defects existing in the prior art, the invention aims to provide a biological functional material with antithrombotic function, which is characterized in that complete and stable liposomes are fixed on the surface of a solid material by a chemical method, the surface of the specific material is provided with a stable and orderly arranged liposome covering layer, and the surface of the liposome is further modified by thrombomodulin by utilizing the selection specificity of 'click chemistry' so as to realize the functionalization of the liposome and realize the antithrombotic function of simulating endothelial cells.

In order to achieve the above object, the present invention adopts the following technical solutions.

A biomaterial having an antithrombotic property, which is characterized in that thrombomodulin and thrombin have a high affinity and can be produced by forming a mixture of 1: 1, activating protein C to complete the conversion from procoagulant blood coagulation to anticoagulative blood in a biological system, firstly modifying liposome to the surface of a solid material, changing the surface composition of the solid material, then modifying recombinant thrombomodulin to the surface of the liposome by using click chemistry, and finally forming the biological material with the function of antithrombotic reformation.

A biomaterial with an antithrombotic re-formation function, wherein the liposome comprises DPPC: cholesterol: DSPE-PEG2000-DBCO =2:1: 0.005.

A preparation method of a biological material with an antithrombotic re-formation function specifically comprises the following steps.

1) Taking azide PEG active ester N₃-PEG₆dissolving-NHS in tetrahydrofuran to form a tetrahydrofuran solution of active lipid, wherein the concentration of the active lipid is 5-10 mg/mL.

2) Adding N, N-diisopropylethylamine into a tetrahydrofuran solution of active lipid, then adding water to dilute so that the concentration of the active lipid is 3-5 mg/mL, the concentration of the N, N-diisopropylethylamine is 0.04-0.08 mL/mL, putting a solid material with an amino surface into the solution, slightly shaking at room temperature, taking out the solid material after the reaction is carried out for 8-12 hours, leaching, and drying by nitrogen.

3) Dissolving distearoylphosphatidylethanolamine-PEG 2000 in chloroform under argon protection, and adding DBCO-PEG₄dissolving-NHS in chloroform, mixing the two liquids under the protection of argon gas to obtain solution DSPE-PEG2000-NH₂The concentration of the DBCO-PEG is 20-30 mg/mL₄And (2) the concentration of-NHS is 3.0-8.0 mg/mL, the mixture is stirred at room temperature for reaction for 10-20 minutes, triethylamine is added to enable the concentration of the triethylamine to be 0.010-0.015 mL/mL, the reaction is completed after 45-60 hours, and the obtained mixture is purified to obtain DSPE-PEG 2000-DBCO.

4) The self-assembly of the liposome is completed by an extrusion mode, the size of the liposome is controlled by the pore diameter of the finally passed polycarbonate membrane, and the invention adopts a proton body with the diameter of 100nm and specifically comprises the following operations: DPPC, cholesterol and DSPE-PEG2000-DBCO were dissolved in chloroform, DPPC: cholesterol: DSPE-PEG2000-DBCO is 1: 1: 0.01-4: 1: 0.1, distilling at 40-45 deg.C under reduced pressure to obtain phospholipid membrane on the wall of round bottom flask, vacuum overnight to completely remove solvent, dissolving the obtained phospholipid membrane in phosphate buffer solution with pH7.4, repeatedly freezing and dissolving in liquid nitrogen and 65 deg.C water bath for 10 times, and sequentially extruding the obtained suspension through polycarbonate membrane to obtain liposome.

5) Liposome immobilization to solid material surface: will contain DPPC: CHL: diluting the DSPE-PEG2000-DBCO liposome to 2.0 mg/ml by PBS buffer solution with the pH value of 7.4, reacting the diluted DSPE-PEG2000-DBCO liposome with an azidated solid material at room temperature for 2-4 hours, fixing the liposome on the surface of the solid material by 'click chemistry' to form a surface with a 3D structure, removing the solution and unreacted liposome after the reaction is finished, and washing the solid material for 3 times by the PBS buffer solution.

6) And (3) recombination of thrombomodulin: 349-492 amino acid sequence containing thrombomodulin EGF-4, EGF-5 and EGF-6, wherein methionine of 388 sequence is replaced by leucine, nitrogen end and carbon end contain BamH I endonuclease, and the carbon end is connected with glycine GlyGlyMet as a variation orientation site is first embedded into a vector plasmid pET-39B (+), the vector plasmid pET-39B (+) containing thrombomodulin is transferred into Escherichia coli methionine auxotrophic sensory cell B834(DE3), 20-40 ml of transformed cells cultured overnight for about 8-12 hours are added into 500-1000 ml of M9 basic culture solution, when turbidity OD600 of the culture solution reaches 0.8, isopropyl thiogalactoside IPTG is added to make its concentration 0.5-1.0 mM, after 5 minutes of centrifugation, the intermediate product is exchanged to remove methionine, the cells are precipitated for 15-30 minutes under 3000-6000 g condition, discarding the supernatant, washing the cells with M9 culture solution, dissolving the cells in 500-1000 ml M9 culture solution to form suspension again, wherein the M9 culture solution does not contain methionine and is substituted by 50-150 mg of azidoalanine, and culturing the solution at 37 ℃ for 5 hours to obtain protein TM1, wherein the nitrogen end of the protein TM1 is connected with disulfide reductase Dsba and S-tags, and the protein can be analyzed by 4-20% SDS-PAGE gradient gel electrophoresis; and rapidly analyzing and quantifying the yield of the expressed protein by using S-tag, purifying the obtained protein TM1 by using metal affinity chromatography through TALON resin, and finally, cleaving and removing the combined tag by using enterokinase to obtain the target protein TM 2.

7) Preparing TM2 into a 1.0 nM solution with PBS buffer solution at pH7.4, culturing the solution with the solid material modified by the plasmid containing DSPE-PEG2000-DBCO at room temperature for 2-6 hours, and washing the solid material with PBS buffer solution to obtain the target material after the reaction is finished.

Compared with the prior art, the invention has the following beneficial effects.

The invention uses click chemistry to fix liposome on the surface of solid material completely and stably, while the biological active molecule is modified on the surface of material through the specific reaction with the specific functional group on the liposome surface, and the biological activity of the biological molecule is used to improve the compatibility of the biological material with blood and tissue, thus achieving the effects of reducing the stress reaction of immune system and inhibiting thrombosis. This goal is achieved by two parallel operations, "liposome intact, stable immobilization" and "functionalization of biofilm simulation". Firstly, the material surface covered by liposome simulates the characteristic of non-specific adsorption of phosphorus on the surface of a biological membrane, so that the compatibility of the material and blood is improved; the introduced biomolecules thrombomodulin and heparin secondly mimic the property of endothelial cells to inhibit thrombosis. The material with the surface modified with the bioactive molecules can greatly reduce the reformation of thrombus caused by surface incompatibility and immune system stress reaction after the material of an interventional device is contacted with blood in an interventional therapy (such as the intervention of coronary heart disease and stent) method.

Drawings

FIG. 1 shows the synthesis route of DSPE-PEG (2000) -DBCO.

FIG. 2 shows DSPE-PEG (2000) -DBCO¹³C NMR。

FIG. 3 shows the IR spectrum of DSPE-PEG (2000) -DBCO.

Fig. 4 shows the structure of TM 1.

Fig. 5 shows the structure of TM 2.

FIG. 6 is a standard curve of absorbance of thrombomodulin.

FIG. 7 is a standard curve of absorbance for liposome-modified thrombomodulin.

FIG. 8 is a standard curve of absorbance of solid material surface modified recombinant thrombomodulin liposome.

Detailed Description

The present invention will be described in detail below with reference to the accompanying drawings and examples, but the following examples do not limit the scope of the present invention.

Examples are given.

Mixing 30mg of N₃-PEG₆-NHS (azidopeg active ester) was dissolved in 4ml of THF (tetrahydrofuran), 0.5 ml of DIEA (N, N-diisopropyl-amine, N-diisopropylethylamine) was added and diluted to 8ml with water, the solid material having an amino surface was put into the solution, gently shaken at room temperature, the reaction was carried out for 8 hours and then taken out, rinsed with THF (3 times for 5 minutes) and water (3 times for 5 minutes), respectively, and dried with nitrogen to obtain a surface azidated solid material.

Adding distearoylphosphatidylethanolamine-PEG 2000 (DSPE-PEG 2000-NH)₂94.4 mg, 0.034 mmol) was dissolved in 3 mL of chloroform under argon protection, and 20 mg (0.029 mmol) of DBCO-PEG was added₄-NHS was dissolved in 1mL chloroform, the two liquids were mixed under argon protection, stirred at room temperature for 15 minutes and then triethylamine ((0.05 mL, 0.36 mmoles) was added thereto by syringe, and after 48 hours the reaction was completed, the resulting mixture was first distilled under reduced pressure and then purified by gel chromatography using methanol: chloroform = 1: 10 as eluent to give 0.077g DSPE-PEG (2000) -DBCO with a yield of 79%, and the synthetic route thereof is shown in fig. 1.¹³C NMR (CDCl3, 75 MHz), δ: 173.4, 173.0, 136.1, 132.1, 129.1, 128.6, 127.8, 125.6, 108.0, 93.2 (alkyne), 70.6 (PEG), 67.2, 55.5, 45.7, 39.1, 36.8, 34.1, 32.0, 29.71, 24.9, 22.7; FTIR (as shown in FIG. 3) (cm-1): 3490, 2915, 2870, 2850, 2091, 1714, 1651, 1542, 1466, 1349, 1249, 1092, 948, 843, 720.

DPPC (15mg, 20.44. mu. mol), cholesterol 4mg (10.2. mu. mol), DSPE-PEG (2000) -DBCO 0.5mg were dissolved in 3.0ml chloroform, distilled under reduced pressure at 40-45 ℃ to obtain a phospholipid membrane on the wall of a round-bottomed flask, and the solvent was completely removed overnight in vacuo. The obtained phospholipid membrane was dissolved in 3.0ml of a phosphate buffer solution (pH 7.4). Repeatedly freezing and dissolving in liquid nitrogen and 65 deg.C water bath for 10 times, and sequentially extruding the obtained suspension through 1000nm, 600nm, 400 nm, 200nm and 100nm polycarbonate membranes to obtain liposome (110 + -5 nm).

Will contain DPPC: CHL: the liposome of DSPE-PEG2000-DBCO is diluted to 2.0 mg/ml by PBS buffer solution, and reacts with the solid material of azide at room temperature for 4 hours, and the liposome is fixed on the surface of the solid material by 'click chemistry' to form the surface of a 3D structure. After the reaction was completed, the solution and the unreacted liposomes were removed, and the solid material was washed 3 times with the buffer (5 minutes/time, with gentle shaking) to obtain a solid material with liposome-coated surface, as shown in FIG. 1.

Comprises portions of thrombomodulin EGF-4, EGF-5 and EGF-6 (amino acid sequence 349-492 in which the methionine of the 388 sequence is substituted by leucine), the nitrogen and carbon termini of which contain an endonuclease of BamH I, and the carbon terminus of which is linked to glycine (GlyGlyMet) as a targeting site for variation is first inserted into the vector plasmid pET-39b (+), the vector plasmid pET-39b (+) containing thrombomodulin is transferred into Escherichia coli (E.coli)E. coli) Methionine auxotrophic sensory cell B834(DE 3).

To 1000ml of M9 basic culture solution (to which MgSO was additionally added)₄1mM, glucose 0.4%, ammonium chloride sulfate 1 mg, CaCl₂0.1mM, kanamycin 30mg, total protein amino acids 40 mg/L) was added to 40ml of the transformed cells which had been cultured overnight (about 10 hours). When the turbidity OD600 of the culture reached 0.8, isopropyl thiogalactoside (IPTG) was added to a concentration of 0.5 mM. After 5 minutes, the intermediate was exchanged to remove methionine, the cells were pelleted by centrifugation (4000 g for 20 minutes), the supernatant was discarded, and after washing the cells 2 times with M9 medium (400 ml each), the cells were dissolved in 1000ml M9 medium again to form a suspension, the M9 medium having the same composition as above but containing no methionine, instead 100 mg of azidoalanine. The solution was incubated at 37 ℃ for 5 hours to give protein TM1, whose nitrogen terminus was linked to disulfide reductase Dsba and S-tags as shown in FIG. 4. 4-20% ofProteins can be analyzed by SDS-PAGE gradient gel electrophoresis. And the yield of expressed protein (-15 mg/ml) was quantified by S-tag rapid analysis. The obtained protein TM1 was purified by metal affinity chromatography on talen resin, and eluted with imidazole in a gradient under natural conditions. The pool was centrifuged at 10000g for 30 min at 4 ℃ and then transferred to 75 mL of lysis buffer pH 8 (NaCl 300 mM, NaH)₂

PO

₄50 mM, glycerol 10%, lysozyme 1 mg/mL and protease inhibitor PMSF 10. mu.g/mL). After the solution was allowed to stand on ice for 30 minutes, the lysate was precipitated by centrifugation at 10000g for 20 minutes, and then the supernatant was purified by using a column packed with a metal affinity resin TALON. The specific operation is that a chromatographic column filled with TALON resin (30 ml) is balanced by a lysis buffer, and the protein with weaker affinity is eluted firstly (the eluent is 135ml and contains 300 mM of NaCl and NaH)₂

PO

₄50 mM, glycerol 10%, and imidazole 20 mM, pH = 8). Then 60 ml of eluent (containing 300 mM NaCl, NaH) is added₂

PO

₄50 mM, glycerol 10%, and imidazole 250 mM, pH = 8), and the chromatographic fractions can likewise be analyzed for protein by 4-20% SDS-PAGE gradient gel electrophoresis. Finally, bound tag was removed by enterokinase cleavage to obtain the target protein TM2, as shown in FIG. 5.

TM2 was dissolved in PBS buffer at pH7.4 to form a 1.0 nM solution, which was incubated with the solid material modified with DSPE-PEG (2000) -DBCO plastid for 4 hours at room temperature, after the reaction, the solid material was washed 3 times with PBS buffer to remove the unreacted solution TM2 to obtain the target material.

An apparent curve of active C protein was established.

Buffer tris-HCl (pH 7.5, 150 mM NaCl, BSA 0.1%, Ca)²⁺5 mM) to 500. mu.l (0.1. mu.g/μ l) of active protein C (4.2 mg/ml, 50. mu.g, 66.7. mu.M), transferring 0.4, 0.8, 1.6, 3.2, 6.4. mu.l of the solution to 5 cuvettes, adding a substrate CS-01 (38) 22.1. mu.l and diluting the solution to 1ml with the tris-HCl buffer solution to form a solution with a substrate concentration of 0.65, 1.29, 2.58, 5.16, 10.32 nM, and carrying out the reaction at 37 ℃ for 15 minutes, and then carrying out the reaction at 37 ℃ for 15 minutesThe absorbance at 405 nm was measured to obtain a standard working curve, see FIG. 6.

And (3) measuring the surface TM activity of the solid material.

The solid material modified with liposome and thrombomodulin on the surface was placed in tris-HCl (pH 7.5 containing 150 mM NaCl, BSA 0.1%, Ca)²⁺5 mM), thrombin (10 nM) and protein C (100 nM), after incubation at 37 ℃ for one hour, antithrombin III (300. mu.g/mL) is added to terminate the reaction, after incubation at 37 ℃ for 5 minutes, CS-01 (38) (0.2 mM) is added, the reaction is carried out at 37 ℃ for 15 minutes, and then the absorbance is measured at 405 nM, see FIGS. 7 and 8, to obtain the enzyme kinetic parametersKm andKcat, see table 1, shows that after thrombomodulin is introduced onto the surface of the material to be solid, its biological activity is not affected compared to that of the material in the form of free bodies and that of the material in the form of modified liposomes.

Claims

1. A method for preparing a biological material with anti-thrombotic re-formation function, characterized in that thrombomodulin and thrombin have higher affinity, and are accomplished by activating protein C by forming a stoichiometric 1:1 complex The biological system is transformed from pro-coagulation to anti-coagulation. First, the liposome is modified on the surface of the solid material to change the surface composition of the solid material, and then the recombinant thrombomodulin is modified on the surface of the liposome by "click chemistry". Finally, biomaterials with anti-rethrombotic function are formed;

The preparation method of the biological material with anti-thrombotic re-formation function specifically includes the following steps:

1) Dissolve azide PEG active lipid N ₃ -PEG ₆ -NHS in tetrahydrofuran to form a tetrahydrofuran solution of active lipid, and the active lipid concentration is 5-10 mg/mL;

2) Add N,N-diisopropylethylamine to the tetrahydrofuran solution of active lipid, and then dilute with water to make the active lipid concentration 3~5mg/mL, and the N,N-diisopropylethylamine concentration to be 0.04~ 0.08ml/mL, put the solid material with amino surface into the solution, shake gently at room temperature, take out the solid material after the reaction is carried out for 8-12 hours, rinse, and dry with nitrogen;

3) Distearoylphosphatidylethanolamine-PEG2000 was dissolved in chloroform under argon protection, then DBCO-PEG ₄ -NHS was dissolved in chloroform, and the two liquids were mixed under argon protection, and the resulting solution DSPE-PEG2000-NH The concentration of ₂ is 20~30mg/mL, the concentration of DBCO-PEG ₄ -NHS is 3.0~8.0 mg/mL, and the reaction is stirred at room temperature for 10~20 minutes, then triethylamine is added to make the concentration 0.010~0.015 ml/mL, 45 The reaction was completed after ~60 hours, and the resulting mixture was purified to obtain DSPE-PEG2000-DBCO;

4) The self-assembly of liposomes is accomplished by extrusion, and the size of liposomes is controlled by the pore size of the polycarbonate membrane that passes through at the end. Liposomes with a diameter of 100 nm are used. The specific operations are as follows: DPPC, cholesterol and DSPE- PEG2000-DBCO was dissolved in chloroform, DPPC: cholesterol: DSPE-PEG2000-DBCO was 1: 1: 0.01~4: 1: 0.1, after vacuum distillation at 40-45 °C, a phospholipid film was obtained on the wall of the round-bottomed flask, vacuumed Overnight to completely remove the solvent, the obtained phospholipid membranes were dissolved in phosphate buffer at pH 7.4, frozen-dissolved 10 times in liquid nitrogen and 65°C water bath repeatedly, and the obtained suspensions were sequentially extruded through the polymerization. Carbonate film, resulting in liposomes;

5) Immobilization of liposomes to the surface of solid materials: Dilute the liposomes containing DPPC:CHL:DSPE-PEG2000-DBCO with PBS buffer pH 7.4 to 2.0 mg/ml, and azide the solid materials at room temperature Under the reaction for 2 to 4 hours, the liposomes were immobilized on the surface of the solid material by "click chemistry" to form a surface of a 3D structure. After the reaction, the solution and unreacted liposomes were removed, and the solid material was washed 3 times with PBS buffer;

6) Recombination of thrombomodulin: It contains partial amino acid sequence 349-492 of thrombomodulin EGF-4, EGF-5 and EGF-6, of which the methionine of sequence 388 is replaced by leucine, and the nitrogen and carbon ends contain BamH I. Dicer, and glycine GlyGlyMet is connected to the carbon end as a directional site for variation. It was first embedded into the vector plasmid pET-39b(+), and the vector plasmid pET-39b(+) containing thrombomodulin was transferred into Escherichia coli In the methionine auxotrophic sensitive cells B834(DE3), add 20~40ml of transformed cells cultured overnight for 8~12 hours to 500~1000ml of M9 basic culture medium. When the turbidity OD600 of the culture medium reaches 0.8, add isopropyl The concentration of thiogalactoside IPTG was 0.5~1.0 mM, after 5 minutes, the intermediate product was exchanged to remove methionine, and the cell pellet was centrifuged at 3000~6000g for 15~30 minutes, and the supernatant was discarded. After washing with M9 medium, dissolve it in 500~1000ml M9 medium to form a suspension again. This M9 medium does not contain methionine, but instead contains 50~150 mg azidoalanine. The solution is incubated at 37ºC for 5 hours. The protein TM1 is obtained, and its nitrogen end is connected with disulfide bond reductase Dsba and S-tags. The protein can be analyzed by 4-20% SDS-PAGE gradient gel electrophoresis; and the yield of the expressed protein can be quantified by S-tag rapid analysis. , the obtained protein TM1 was purified by metal affinity chromatography through TALON resin, and finally the bound tag was removed by enterokinase cleavage to obtain the target protein TM2;

7) TM2 was prepared into a 1.0 nM solution with PBS buffer at pH 7.4, and incubated with the solid material modified with DSPE-PEG2000-DBCO liposomes at room temperature for 2-6 hours. After the reaction was over, the solid material was treated with PBS. The target material was obtained by buffer washing.