CN101361992B - A method for immobilizing heparin multilayer film on the surface of TiO2 - Google Patents

Abstract

The invention discloses a method for immobilizing a heparin multilayer film on the surface of _TiO2 . A self-assembled monolayer method is used to prepare an intermediate layer between the _TiO2 surface and biotin to form a chemical bond with the two, so as to improve the stability of the immobilized heparin multilayer film. Hexamethylenediamine is used as a linker to react with the carboxyl group of heparin and the carboxyl group of biotin respectively to form biotinylated heparin, thereby having the recognition site of avidin. The self-assembled monolayer modified TiO ₂ is coated with biotin azide, the biotin is firmly immobilized on the surface of the TiO ₂ by photochemical method, and the biotin-avidin recognition function is used to assemble and form heparin multilayer layer film. The method can firmly fix the heparin multilayer film on the surface of the TiO ₂ thin layer on the surface of the titanium-based metal biomaterial or the surface of other materials after depositing the TiO ₂ film, so that the surface can obtain excellent anticoagulant function. Moreover, the process is simple and the realization is easy.

Description

A method for immobilizing heparin multilayer film on the surface of TiO2

Technical field

The invention relates to a method for biochemically modifying the surface of an inorganic material, in particular to a method for fixing a heparin multilayer film on the surface of _TiO2 .

Background technique

Titanium-based metals have been widely used in biomaterials, and a layer of oxide layer TiO ₂ will naturally form on the surface of titanium-based metal biomaterials. This TiO ₂ layer has been proved to be the main reason for the good biocompatibility of titanium-based metal biomaterials, but The anticoagulant properties of thin _TiO2 layers on the surface of titanium-based metal biomaterials are still insufficient.

Heparin is an anticoagulant drug commonly used clinically. It achieves anticoagulant effect by terminating certain steps in blood coagulation. Immobilizing heparin on the surface of titanium-based metal materials can improve the anticoagulant performance of the materials. The prior art adopts a simple physical coating method to adsorb a layer of heparin on the surface of titanium-based metals. The binding force between the two is poor, and because heparin is water-soluble, it will be lost quickly in the blood flow environment, and it is difficult to achieve the expected resistance. Coagulation effect. The chemical method of silane coupling to immobilize heparin on the surface of TiO ₂ is also difficult to be applied clinically due to the instability of the silane coupling layer under physiological conditions.

Contents of the invention

The purpose of the present invention is to provide a method for firmly fixing heparin multilayer film on the surface of _TiO2 , which can deposit _TiO2 thin film on the _surface of titanium-based metal biomaterials or other materials to firmly fix heparin multilayer on the surface membrane, so that its surface can obtain excellent anticoagulant function. Moreover, the process is simple and the realization is easy.

The present invention realizes above invention object, and the adopted technical solution is, a kind of method for fixing heparin multilayer film on the surface of _TiO2 , and its steps are:

1. A method for fixing a heparin multilayer film on the surface of _TiO , the steps of which are:

A. Preparation of biotinylated heparin

Add heparin, NHS and EDC sequentially to the MES solution with a concentration of 50mM, so that the ratio of carboxyl groups in heparin, NHS and EDC is 1:0.6:1. The ratio of the amount of hexamethylenediamine is 1.2:1, add hexamethylenediamine, and dialyze to remove small molecules after reaction to obtain heparin-hexamethylenediamine solution;

In addition, biotin with an amount equal to that of hexamethylenediamine was added to the MES solution with a concentration of 50 mM, and then NHS and EDC were added to it in turn, so that the ratio of the three substances of biotin, NHS and EDC was 1: 0.6:1, the biotin activation solution is obtained;

Add biotin activation solution to heparin-hexamethylenediamine solution, dialyze to remove small molecules after reaction, and then freeze-dry to obtain biotinylated heparin;

B. Self-assembly of monomolecular layers of organic phosphonic acid Soak the clean _TiO2 material in an organic phosphonic acid or phosphoric acid monoester solution with a temperature of 50-120°C and a concentration of 1-50mmol/L to carry out monomolecular formation on the surface of _TiO2 Layer self-assembly, soaking time is 1 to 24 hours, remove TiO ₂ and then clean;

C. Immobilizing biotin Put the monomolecular layer self-assembled TiO ₂ obtained in step B into an ethanol solution with a concentration of 0.1-0.6 mg/ml azide biotin (photobiotin), and after the surface is completely wetted, take out , dry in the shade, and irradiate with ultraviolet light for 1 to 10 minutes to fix biotin on the surface of TiO ₂ ;

D. Assemble avidin Soak the biotin-immobilized _TiO2 obtained in step C in an avidin solution with a concentration of 0.1-1 mg/ml and assemble for 0.5-24 hours;

E. Fix the heparin membrane. Prepare the biotinylated heparin prepared in step A into a solution with a concentration of 1-10 mg/ml, and then put the TiO ₂ assembled with avidin obtained in step D into the solution for 0.5- 24 hours, obtain the heparin film;

F. Repeat steps D and E to obtain TiO ₂ with 1 to 50 layers of heparin film immobilized

The mechanism of each step of the present invention is:

Through the B-step self-assembled monolayer technology, an intermediate layer of organic phosphonic acid is constructed between the _TiO2 layer and biotin on the surface of titanium-based metal biomaterials. The intermediate layer first forms a firm chemical bond with _TiO2 , and then in C In the step, the intermediate layer forms a strong chemical bond with the biotin layer, so that the subsequent immobilized heparin multilayer film is firmly combined with the base material TiO ₂ . In step D, biotin and avidin are molecularly recognized, and their binding constant is 10 ¹⁵ M ^-1 , which is an irreversible binding with strong binding force. At the same time, since each avidin molecule contains four equivalent biotin binding sites, three biotin binding sites remain on the surface of avidin after assembly; while heparin molecules contain many carboxyl groups, through A Hexamethylenediamine is used as a linking agent to make it react with the carboxyl group of heparin and the carboxyl group of biotin to form biotinylated heparin, so that one molecule of biotinylated heparin is connected with more biotin, and these biotins are in turn Combined with avidin, thereby immobilizing a layer of biotinylated heparin film on the surface of _TiO2 . Since there is also residual biotin on the surface of the biotinylated heparin membrane, which can be combined with avidin, and avidin produces redundant biotin binding points, so the fixation of avidin and biotinylated heparin membrane can be repeated on the surface. Combined, thereby firmly immobilizing more than 6 layers of heparin multilayer films on the surface of the TiO ₂ .

Compared with prior art, the beneficial effect of the present invention is:

The biotin and _TiO2 surface are connected by chemical bonds through the intermediate layer of organic phosphonic acid, and then avidin is fixed on the _TiO2 surface by biotin/avidin biorecognition, and finally through avidin/biotinylated heparin Repeated assembly of heparin by bio-recognition to obtain a firm multilayer film, so that the heparin immobilized on the surface of TiO ₂ is firmly bound, not easy to fall off, and the number and density are also significantly increased, and the anticoagulant function is excellent. Experiments also prove that the _TiO2 surface of the heparin multilayer film fixed by the method of the present invention has longer partial thromboplastin time and less platelet adhesion quantity, and has good anticoagulant function.

All operations in the present invention are carried out under solution conditions, the process is simple, no special expensive equipment is required, and it is easy to realize. There is no restriction on the shape and structure of materials or implanted devices, and various biomedical applications with complex body structures can be realized in industry. The heparin multilayer film on the surface of the device is immobilized.

The aforementioned TiO ₂ is anatase crystal TiO ₂ or anatase crystal TiO ₂ mixed with rutile.

From the perspective of chemical thermodynamics, rutile is a more stable phase than anatase, and phosphonic acid or phosphoric acid monoester forms a monolayer self-assembly on the surface, that is, forms chemical adsorption on the surface, which is bound to be accompanied by the surface part _{of TiO2} . The dissociation of Ti-O bonds and the formation of new Ti-OPO(OH)R or Ti-OPO(OH)OR bonds. Compared with rutile, the dissociation of Ti-O bonds on the surface of anatase crystal film is relatively easy , can generate more reactive sites and, therefore, induce more surface chemisorption of phosphonic acid or phosphate monoester. Of course, the method of the present invention can also immobilize heparin multilayer films on the surface of amorphous TiO ₂ .

The operation in the above step C is carried out in a dark room. This is because azide biotin will be partially photolyzed under natural light conditions, and the operation in the dark room can reduce the photolysis of azide biotin, so that more biotin can be fixed on the surface of the material.

Water may also be added to the ethanol in which the azido-modified biotin is dissolved in the above step C, and the volume ratio of the added water to ethanol is 0.1-1:3. The presence of a small amount of water is beneficial to better maintain the biological activity of biotin.

The solution dissolved in the above two steps of D and E is a phosphate buffer solution. Phosphate buffer solution replaces water to dissolve avidin and biotinylated heparin, and its pH value is 7.4, which is consistent with the pH of normal human body, which is beneficial to maintain the biological activity of avidin and biotinylated heparin.

The method of the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments.

Description of drawings

Fig. 1 is a scanning electron microscope image of platelet adhesion on the surface of a TiO ₂ material with a heparin multilayer film fixed by the method of Example 2.

Fig. 2 is a scanning electron microscope image of platelet adhesion on the surface of TiO ₂ material without fixed heparin multilayer film.

Detailed ways

Example 1:

A method for immobilizing a heparin multilayer film on the surface of _TiO , the steps of which are:

A. Preparation of biotinylated heparin

Add heparin, NHS (N-hydroxysuccinimide) and EDC (1-(3-dimethylaminopropyl) base)-3-ethylcarbodiimide), so that the ratio of the amount of carboxyl groups in heparin, NHS and EDC is 1:0.6:1, and then the ratio of the amount of carboxyl groups in hexamethylenediamine to heparin The ratio is 1.2:1, add hexamethylenediamine, and dialyze to remove small molecules after reaction to obtain heparin-hexamethylenediamine solution;

In addition, biotin with an amount equal to that of hexamethylenediamine was added to the MES solution with a concentration of 50 mM, and then NHS and EDC were added to it in turn, so that the ratio of the three substances of biotin, NHS and EDC was 1: 0.6:1, the biotin activation solution is obtained;

Add biotin activation solution to heparin-hexamethylenediamine solution, dialyze to remove small molecules after reaction, and then freeze-dry to obtain biotinylated heparin;

B. Self-assembly of organic phosphonic acid monolayer Soak the clean anatase _TiO2 material in a 3-aminopropylphosphonic acid solution with a temperature of 80°C and a concentration of 10mmol/L for 12 hours to perform TiO ₂ The monolayer self-assembles on the surface, and the TiO ₂ is removed and cleaned.

C. Immobilizing biotin In a dark room, put the monolayer self-assembled TiO ₂ obtained in step B into ethanol/ In the aqueous solution, after the surface is completely wetted, take it out, dry it in the shade, irradiate with ultraviolet light for 5 minutes, and the radiation energy is 8mW/cm ² , so that biotin can be fixed on the surface of TiO ₂ .

D. Assembly of avidin The biotin-immobilized TiO ₂ obtained in step C was soaked in a phosphate buffer solution with a concentration of 1 mg/ml of avidin and assembled for 24 hours.

E, fix the heparin membrane The biotinylated heparin prepared in step A is configured into a solution with a concentration of 10 mg/ml, and then the _TiO assembled with avidin obtained in step D is placed in the solution for 24 hours to obtain Heparin film;

F. Repeat steps D and E 5 times to obtain TiO ₂ immobilized with 6 layers of heparin film.

The in vitro partial thromboplastin time (APTT) experiment proved that the TiO ₂ on the 6-layer heparin film fixed by the method in this example has excellent anticoagulant function. Its specific experimental operation is:

(1) Preparation of platelet-poor plasma: centrifuge the whole blood of three healthy persons at 3000 rpm for 15 minutes, take the light yellow liquid above, and mix to obtain platelet-poor plasma (PPP).

(2) Put the TiO ₂ and blank TiO ₂ samples (10×10mm) of the 6-layer heparin film to be tested into a 24-well plate and a blank 24-well plate respectively, add 500 μL of PPP, incubate at 37°C for 30 minutes, and draw 250 μL of incubated PPP was used to detect APTT on an ACL200 multi-channel coagulation instrument.

The results are shown in Table 1. Partial thromboplastin time (APTT) is a commonly used screening test for the intrinsic coagulation pathway and the preferred index for clinical monitoring of heparin therapy. The longer the APTT time, the better the anticoagulant performance of the material.

Table 1 APTT of heparin multilayer film with _TiO2

Table 1 shows that the partial thromboplastin time of the TiO ₂ immobilized with 6 layers of heparin film of the present invention is significantly longer than that of TiO ₂ without heparin film, indicating that its anticoagulant function is better.

Example 2:

A method for immobilizing a heparin multilayer film on the surface of _TiO , the steps of which are:

A. Preparation of biotinylated heparin This step is exactly the same as step A in Example 1.

B. Self-assembly of organic phosphonic acid monolayer Soak the clean anatase _TiO2 material in the isooctane solution of monododecyl phosphate at a temperature of 120 ° C and a concentration of 1 mmol/L 1 Hours, monolayer self-assembly on the _TiO2 surface was performed, and the _TiO2 was removed and cleaned.

C. Immobilize biotin in a dark room, put the monolayer self-assembled TiO ₂ obtained in step B into ethanol/ In the aqueous solution, after the surface is completely wetted, take it out, dry it in the shade, irradiate with ultraviolet light for 10 minutes, and the radiation energy is 8mW/cm ² , so that biotin can be fixed on the surface of TiO ₂ .

D. Assembly of avidin The biotin-immobilized TiO ₂ obtained in step C was soaked in a phosphate buffer solution with a concentration of 0.1 mg/ml of avidin and assembled for 12 hours.

E. Fix the heparin membrane. Prepare the biotinylated heparin prepared in step A into a phosphate buffer solution with a concentration of 1 mg/ml, and then place the _TiO2 assembled with avidin obtained in step D in the solution for 12 hours. , to obtain a heparin film;

F. Repeat steps D and E 19 times to obtain TiO ₂ immobilized with 20 layers of heparin film.

The platelet adhesion experiment proves that TiO ₂ fixed with 20 layers of heparin film has excellent anticoagulant function:

The aggregation and activation of platelets on the surface of materials is generally considered to be an important link in the coagulation process. The higher the degree of aggregation and activation of platelets on the surface of materials, the easier it is to cause blood coagulation. In the following experiment, TiO ₂ fixed with 20 layers of heparin film and blank TiO ₂ were used for in vitro platelet adhesion experiment in this example, and the specific operation was as follows:

Centrifuge fresh human whole blood at 1500rpm for 15 minutes, and the light yellow liquid in the upper part is platelet-rich plasma (PRP). The material was cut into 10×10mm size and put into 24-well culture plate, 500 μL of PRP was added respectively, and cultured at 37°C for 2 hours. After taking it out, wash it twice with phosphate buffer solution (PBS), then fix it with 0.2% glutaraldehyde for 1 hour and 0.5% glutaraldehyde for 6 hours, and then fix it with 50%, 50%, 75%, 90% and 100% ethanol (water is a solvent) solution was dehydrated for 15 minutes respectively, then dealcoholized with 50%, 50%, 75%, 90% and 100% isoamyl acetate (ethanol was a solvent) solution for 15 minutes respectively, and finally used _CO2 critical point drying of samples. After drying, the number and morphology of platelet adhesion were observed by scanning electron microscope (SEM).

Fig. 1 is a scanning electron micrograph of platelet adhesion on the surface of a TiO ₂ material with a heparin multilayer film fixed by the method of this example. Fig. 2 is a scanning electron micrograph of platelet adhesion on the surface of a blank TiO ₂ material without immobilized heparin multilayer film. Figures 1 and 2 show that the number of platelets adhered to the _TiO2 material of the heparin multilayer film fixed by the method of this example is significantly reduced compared with that of the blank _TiO2 material, and the platelets adhered to the form are complete without activation, indicating that its surface has Excellent anticoagulant function.

Example 3:

A method for immobilizing a heparin multilayer film on the surface of _TiO , the steps of which are:

A. Preparation of biotinylated heparin This step is exactly the same as step A in Example 1.

B. Self-assembly of organic phosphonic acid monolayer Soak the clean anatase _TiO2 material mixed with rutile in isooctyl monododecyl phosphate at a temperature of 50°C and a concentration of 50mmol/L In the alkane solution for 18 hours, the monolayer self-assembly on the surface of TiO ₂ was carried out, and the TiO ₂ was removed and cleaned.

C. Immobilizing biotin In a dark room, put the monolayer self-assembled TiO ₂ obtained in step B into ethanol/ In the aqueous solution, after the surface is completely wetted, take it out, dry it in the shade, irradiate with ultraviolet light for 1min, and the radiation energy is 8mW/cm ² , so that biotin can be fixed on the surface of TiO ₂ .

D. Assembly of avidin The biotin-immobilized TiO ₂ obtained in step C was soaked in a phosphate buffer solution with a concentration of 0.5 mg/ml of avidin for 0.5 hour assembly.

E. Fix the heparin membrane. Prepare the biotinylated heparin prepared in step A into a phosphate buffer solution with a concentration of 5 mg/ml, and then place the _TiO2 assembled with avidin obtained in step D in the solution for 0.5 hours. , to obtain a heparin film;

F. Repeat steps D and E 49 times to obtain TiO ₂ immobilized with 50 layers of heparin film.

Example 4:

A method for immobilizing a heparin multilayer film on the surface of _TiO , the steps of which are:

A. Preparation of biotinylated heparin This step is exactly the same as step A in Example 1.

B. Self-assembly of organic phosphonic acid monolayer Soak the clean amorphous _TiO2 material in a 4-aminobutylphosphonic acid solution with a temperature of 100°C and a concentration of 30mmol/L for 24 hours to carry out the _TiO2 surface The monolayer self-assembled, and the _TiO2 was removed and cleaned.

C. Immobilizing biotin Put the monomolecular layer self-assembled TiO ₂ obtained in step B into an ethanol solution with a concentration of 0.5 mg/ml azide biotin (photobiotin), and after the surface is completely wetted, take it out and dry it in the shade , and irradiate with ultraviolet light for 3 minutes, and the radiation energy is 12mW/cm ² , so that biotin is fixed on the surface of TiO ₂ .

D. Assembly of avidin The biotin-immobilized TiO ₂ obtained in step C was soaked in an aqueous solution of avidin with a concentration of 0.5 mg/ml for 24 hours.

E, fix the heparin membrane The biotinylated heparin prepared in step A is configured into an aqueous solution with a concentration of 5 mg/ml, and then the _TiO assembled with avidin obtained in step D is placed in the solution for 24 hours to obtain Single-layer heparin film;

In step A, when preparing the heparin-hexamethylenediamine solution, the ratio of the amount of carboxyl groups, NHS and EDC is 1:0.6:1, and the ratio of the amount of biotin, NHS and EDC when preparing the biotin activation solution It is 1:0.6:1; the ratio of the amount of these substances is the optimal ratio required by the theory, of course, the amount of some of them can be changed, but there is a surplus of substances exceeding this ratio, which will be removed by dialysis , and cause waste, therefore, changing the ratio of these substances belongs to the equivalent deterioration replacement of the present invention, and still belongs to the protection scope of the present invention.

The organic phosphonic acid or phosphoric acid monoester in the B step of the present invention can also select any existing organic phosphonic acid or any phosphoric acid monoester for use except the selected organic phosphonic acid or phosphoric acid monoester used in the above examples. Any ester can be used, because any organic phosphonic acid or any phosphoric acid monoester can provide hydroxyl groups to form a chemical bond with the _TiO2 surface.

Claims (5)

1. one kind at TiO ₂The method of surface fixing heparin multilayer film the steps include:

The preparation of A, biotinylation heparin

, concentration adds heparin, N-hydroxy-succinamide and 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide successively in being 2-(N-morphine quinoline) the ethyl sulfonic acid solution of 50mM; Making the ratio of carboxyl amount, N-hydroxy-succinamide and 1-in the heparin (3-dimethylaminopropyl)-three kinds of amount of substances of 3-ethyl carbodiimide is 1: 0.6: 1; Ratio by the amount of substance of carboxyl in hexamethylene diamine and the heparin is 1.2: 1 again; Add hexamethylene diamine; Micromolecule is removed in the dialysis of reaction back, obtains heparin-hexamethylene diamine solution;

The biotin that in addition amount of substance is equated with hexamethylene diamine; Join in the 2-that concentration is 50mM (N-morphine quinoline) the ethyl sulfonic acid solution; Again successively to wherein adding N-hydroxy-succinamide and 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide; Making the ratio of biotin, N-hydroxy-succinamide and 1-(3-dimethylaminopropyl)-three kinds of amount of substances of 3-ethyl carbodiimide is 1: 0.6: 1, promptly obtains the biotin activating solution;

The biotin activating solution is joined in heparin-hexamethylene diamine solution, and micromolecule is removed in the dialysis of reaction back, and lyophilization then obtains the biotinylation heparin;

The self assembly of B, organic phospho acid monolayer is with the TiO of cleaning ₂It is that 50～120 ℃, concentration are to carry out TiO in organic phospho acid or the phosphate monoester solution of 1～50mmol/L that material is immersed in temperature ₂The monolayer self assembly on surface, soak time is 1～24 hour, takes out TiO ₂The back is cleaned;

C, fixed biologically element go on foot B the TiO after the monolayer self assembly that obtains ₂, put into the alcoholic solution that concentration is 0.1～0.6mg/ml Azide biotin, after the surperficial complete wetting, take out, dry in the shade, irradiation under ultraviolet ray made biotin be fixed on TiO in 1～10 minute ₂The surface;

The TiO that has fixed biotin that D, assembling Avidin obtain the C step ₂Be immersed in concentration and be in the Avidin solution of 0.1～1mg/ml and assembled 0.5～24 hour;

E, fixing heparin film go on foot prepared biotinylation heparin with A and are configured to the solution that concentration is 1～10mg/ml, the assembling that again D step is obtained the TiO of Avidin ₂Place this solution to assemble 0.5～24 hour, obtain the heparin film;

F, repetition D, the operation in two steps of E, the TiO of 1～50 layer of heparin film has been fixed in acquisition ₂

2. according to claim 1 at TiO ₂The method of surface fixing heparin multilayer film is characterized in that said TiO ₂TiO for anatase crystal ₂Or be mixed with the anatase crystal TiO of rutile ₂

3. according to claim 1 at TiO ₂The method of surface fixing heparin multilayer film is characterized in that, can also add entry, the water of adding and alcoholic acid volume ratio 0.1～1: 3 in the ethanol of said C dissolving azido modified biological element in the step.

4. according to claim 1 at TiO ₂The method of surface fixing heparin multilayer film is characterized in that, said C operating in the darkroom in the step carried out.

5. according to claim 1 at TiO ₂The method of surface fixing heparin multilayer film is characterized in that, said D, E dissolve Avidin and biotinylation heparin in two steps solution is phosphate buffer solution.

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