CN116236614A - TiO for catalyzing and releasing CO 2 Nanotube material, preparation method and application thereof - Google Patents

Abstract

The invention relates to the field of biomedical nano materials and implantation instruments, and discloses TiO for catalyzing and releasing CO ₂ Preparation method and application of nanotube material, preparing regular titanium dioxide nanotube array on pure titanium surface, covalent grafting sodium alginate/carboxymethyl chitosan composite polymer (SA/CS coating) on nanotube surface on self-assembled aminosilane, grafting CO release molecule (CORM-401) on material surface, thereby obtaining TiO for catalyzing and releasing CO ₂ Nanotube material. TiO for catalyzing and releasing CO prepared by the invention ₂ The nanotube material not only has excellent hydrophilic performance, but also can enhance the anticoagulation performance of the material under the condition of catalyzing and releasing CO, promote the adhesion, growth and functional expression of vascular endothelial cells, and can be applied to the surface modification of implantable biological materials or medical instruments such as blood contact and the like.

Description

A kind of TiO2 nanotube material that catalyzes releasing CO, its preparation method and application

technical field

The invention belongs to the field of biomedical nanomaterials and implantation devices, and in particular relates to a _TiO2 nanotube material that catalyzes CO release, its preparation method and application.

Background technique

Titanium and its alloys have excellent physical and chemical properties such as low elastic modulus, good physiological corrosion resistance and high specific strength, and have a wide range of applications in blood contact medical devices (such as vascular stents, heart valves, thrombus filters, etc. application. Titanium can still maintain its chemical stability when placed in the human body for a long time, and it will basically not cause immune rejection in the human body. However, when it is left in the human body for a long time or permanently, its poor blood compatibility can easily induce thrombosis and cause Implantation failed. In addition, for intravascular implant materials or devices, the delayed healing of endothelialization often leads to the difficulty of repairing damaged blood vessels, which leads to a series of clinical side effects. Although oral anticoagulants can prevent blood clots in the human body caused by poor hemocompatibility of materials, this method usually requires patients to take anticoagulants for life and increases the risk of bleeding in patients, and cannot effectively promote the rapid recovery of damaged endothelium. heal. After the material is implanted into the human body, human blood and endothelial cells (ECs) first contact the surface of the material. Therefore, surface modification can improve the anticoagulant performance of the material and promote the repair and regeneration of damaged vascular endothelium. Titanium-based blood contact materials clinical application is of great significance.

Studies have shown that the biocompatibility of materials has a lot to do with their surface topology, surface wettability, and surface bioactive molecules. Generally speaking, a material surface with specific void geometry and better hydrophilicity can induce cell adhesion and proliferation, and also exhibit better blood compatibility. Anodizing is a surface modification technology with simple process and low cost. Titanium dioxide nanotube arrays (TNTs) with unique micro-nano structures can be obtained in situ by anodizing titanium. TNT presents a porous, rough, and highly hydrophilic morphology, which is conducive to the adhesion and proliferation of cells and the improvement of blood compatibility. In addition, TNT's unique nanotube structure and size-controllable porosity provide a good platform for loading bioactive molecules or drugs to further enhance its surface bioactivity. For example, by loading gentamicin on the surface of TNT through co-deposition of dopamine, the release time of the drug can be as long as more than 30 days, and it can play an effective antibacterial role and improve biocompatibility. In order to prevent inflammation and possible complications caused by inflammation, chelating copper, zinc, silver and other metal ions on the surface of TNT is one of the effective methods. In addition, the covalent grafting of sodium alginate (SA), carboxymethyl chitosan (CS) and other substances with good biological activity on the surface of TNT can effectively improve the biocompatibility of the TNT surface.

Studies in recent years have found that CO has shown great potential in the treatment of cardiovascular diseases. As an endogenous gas signal molecule, CO can increase cyclic guanosine monophosphate (cGMP) by activating soluble guanylate cyclase (sGC). Level to inhibit thrombosis and intimal hyperplasia, thereby contributing to vascular endothelial regeneration. In addition, CO can also inhibit the endothelial injury and monocyte aggregation caused by HO-1 (heme oxygenase-1) deficiency, as well as the proliferation and migration of vascular smooth muscle cells (SMCs). Endothelial repair and regeneration, but also show good anticoagulant properties. However, direct inhalation of CO gas may trigger potential toxic reactions. Studies have found that CO can form CO-releasing molecules (CORMs) with some transition metal elements (such as Mn, Ru) through special coordination bonds. However, due to the lack of sufficient reducing agents, the release of CORMs in the blood environment is very low. For the cardiovascular microenvironment, studies have shown that cysteine containing sulfhydryl groups can act as a reducing agent to catalyze CORMs to release CO, while the human body Cells contain a large number of sulfhydryl compounds. These compounds cannot catalyze CORMs to release CO due to their low concentration in human blood. Therefore, when CORMs are applied to cardiovascular materials, they have sufficient stability in the blood and will not cause toxic reactions, but In the physiological environment of cells, it can catalyze the release of CO, thereby promoting the endothelial repair of the material surface. It can be seen that the introduction of CORMs to the surface of titanium alloy can catalyze the release of CO by utilizing the physiological microenvironment of the growth of vascular ECs in vivo and the cell physiological microenvironment of the tissue around the implant, improving the anticoagulant performance of the material and promoting the intima of the blood vessel. Repair regeneration.

Contents of the invention

Purpose of the invention: for the problems existing in the prior art, the present invention provides a kind of _TiO2nanotube material that catalyzes CO release, its preparation method and application, on the basis of the _TiO2nanotube (TNT) that anodic oxidation makes, First, introduce amino groups on the surface of nanotubes by self-assembled aminosilane, further immobilize the sodium alginate/chitosan complex in situ, and finally immobilize CO releasing molecules (CORM401) on the surface of the material, thereby constructing a catalytic release CO in TiO ₂ nanotube materials. The _TiO2 nanotube material that catalyzes the release of CO prepared by the present invention not only exhibits excellent hydrophilic properties, but also in the case of catalytic release of CO, the released CO gas molecules can not only enhance the anticoagulant performance of the material, but also promote blood vessel Adhesion, growth, and functional expression of endothelial cells have applications in surface modification of implantable biomaterials such as blood contact or medical devices.

Technical solution: The present invention provides a method for preparing a TiO _nanotube material that catalyzes CO release, comprising the following steps:

Step 1, preparing TiO _nanotubes in situ on the titanium surface by anodic oxidation;

Step 2, carrying out silanization treatment to the material obtained in step 1, to obtain silane- _modified TiO nanotubes;

Step 3, covalently grafting sodium alginate/carboxymethyl chitosan composite macromolecule on the surface of the silane-modified TiO _nanotube ;

Step 4: Covalently graft CO releasing molecules on the surface of the nanotubes obtained in Step 3 to obtain a TiO ₂ nanotube material that catalyzes releasing CO.

Further, the fourth step is to immerse the nanotubes obtained in the third step in the CORM-401 solution, add carbodiimide and N-hydroxysuccinimide, wash and dry after reacting for 2 to 5 hours, and obtain the nanotube that catalyzes the release of CO TiO ₂ nanotube material.

Preferably, the concentration of the CORM-401 solution is 0.05-1 mg/mL;

And/or, the mass ratio of the carbodiimide to the N-hydroxysuccinimide is 3-6:1.

Further, step 1 is to anodize the ultrasonically cleaned and dried titanium sheet in an electrolyte solution containing 0.2-1%wt NH ₄ F, 2-10mL deionized water and 200-300mL ethylene glycol solution for 0.5-2 Hours, the anodized product was ultrasonically cleaned with absolute ethanol and deionized water, dried, and then annealed at 450° C. in air for 2 to 4 hours at a high temperature to obtain the TiO ₂ nanotubes.

Further, the second step is to immerse the material obtained in the first step into a solution of 3-aminopropyltrimethoxysilane or 3-aminopropyltriethoxysilane, shake and react for 10-24 hours, and then cool it at 60-100°C Heating for 8 to 12 hours, washing and drying the heat-treated gain with absolute ethanol to obtain the silane-modified TiO _nanotubes ;

Further, the concentration of the 3-aminopropyltrimethoxysilane or 3-aminopropyltriethoxysilane solution is 5-15mM.

Further, the third step is to immerse the silane- _modified TiO nanotube into the mixed solution of sodium alginate and carboxymethyl chitosan, then add carbodiimide and N-hydroxysuccinate in the mixed solution imide, stirred and reacted for 10 to 14 hours, and the resulting nanotubes were washed with deionized water and dried.

Preferably, the sodium alginate solution concentration is 1 ~ 10mg/mL;

And/or, the carboxymethyl chitosan solution concentration is 1 ~ 10mg/mL;

And/or, the volume ratio of the sodium alginate solution to the carboxymethyl chitosan solution is 1:1;

And/or, the mass ratio of the carbodiimide to the N-hydroxysuccinimide is 3-6:1.

The present invention also provides an application of the TiO ₂ nanotube material that catalyzes the release of CO prepared by the method described in any one of the above in the preparation of the artificial vascular graft material that catalyzes the release of CO.

The present invention also provides an application of the _TiO2 nanotube material that catalyzes CO release prepared by the method described in any one of the above in the preparation of catalysis-induced CO release vascular stent coating.

The theoretical basis of the _TiO2 nanotube material that catalyzes CO release in the present invention can be represented by the following process:

In the present invention, the great potential of CO in the treatment of cardiovascular diseases has been confirmed by theory and experiment. CO not only has the effect of promoting vascular endothelial repair and regeneration, but also exhibits good anticoagulant properties, but direct inhalation CO is potentially toxic due to its strong binding to hemoglobin. Studies have found that CO can form CO-releasing molecules (CORMs) through special coordination bonds with some transition metal elements (such as Mn, Ru), and cysteine containing sulfhydryl groups can be used as a reducing agent to catalyze CORMs to release CO. Human cells contain a large number of thiol compounds, which exist in large quantities in human cells, but the concentration in human blood is very low, which can ensure that when CORMs are applied to cardiovascular materials, CORMs can be absorbed by thiols in the physiological environment of cells. The compound catalyzes the release of CO, thereby promoting endothelial repair on the surface of the material, but in the blood, due to the lack of sufficient mercapto compounds as reducing agents, it shows sufficient stability, and will not be catalyzed to release CO, causing potential toxic reactions. It can be seen that the introduction of CORMs to the surface of titanium alloy can catalyze the release of CO by using the physiological microenvironment of the growth of vascular ECs in vivo and the cell physiological microenvironment of the tissue around the implant, improving the anticoagulant performance of the material and promoting the intima of the blood vessel. Repair regeneration.

Beneficial effect: compared with the prior art, the present invention has the following significant advantages:

(1) The _TiO2 nanotubes used have strong hydrophilicity, the 3-aminopropyltrimethoxysilane used is a good cross-linking coupling agent, and the sodium alginate and carboxymethyl chitosan used have Good anticoagulant performance and antibacterial effect, the CORM-401 used can catalyze the release of CO after adding cysteine, reduce the adhesion and activation of platelets, improve the hemolysis rate of materials, promote the proliferation and adhesion of endothelial cells and The expression of VEGF and NO showed a better effect.

(2) The technical route of the present invention has mild reaction conditions, easy-to-obtain material sources, and the reaction does not require special equipment. The surface-immobilized bioactive molecules and grafted CORMs can be carried out at room temperature. Therefore, the process cost is low and the controllability is strong , the effect is remarkable. Moreover, the substances used in the present invention have good biocompatibility, so as nanomaterials or nanomedicines, they will not cause cytotoxicity, and have good biosafety and biocompatibility.

(3) The _TiO2 nanotube material that catalyzes the release of CO according to the present invention is widely used, has excellent anticoagulant properties and the function of releasing CO, and can be used not only in the preparation of artificial vascular graft materials, but also in blood vessels Surface coating preparation of implanted devices (such as vascular stents, cardiac pacemakers) is used to improve the anticoagulation performance and endothelial repair performance of the device.

Description of drawings

Fig. 1 is the surface feature of TNT under electron microscope scanning in the present invention;

Fig. 2 is the surface feature of TNTA under electron microscope scanning in the present invention;

Fig. 3 is the surface feature of TNTA-A under electron microscope scanning in the present invention;

Fig. 4 is the surface feature of TNTA-SC under electron microscope scanning in the present invention;

Fig. 5 is the surface feature of TNTA-CO under electron microscope scanning in the present invention;

Fig. 6 is the XPS high-resolution spectrogram of main element of TNTA surface among the present invention;

Fig. 7 is the XPS high-resolution spectrogram of main element of TNTA-A surface among the present invention;

Fig. 8 is the XPS high-resolution spectrogram of main element of TNTA-SC surface among the present invention;

Fig. 9 is the XPS high-resolution spectrogram of the main elements on the surface of TNTA-CO in the present invention;

Fig. 10 is the CO release content figure of TNTA-CO in the present invention;

Figure 11 is a platelet adhesion drawing of Ti under electron microscope scanning in the present invention;

Fig. 12 is a platelet adhesion drawing of TNT under electron microscope scanning in the present invention;

Fig. 13 is a platelet adhesion drawing of TNTA under electron microscope scanning in the present invention;

Fig. 14 is the platelet adhesion drawing of TNTA-A under scanning electron microscope in the present invention;

Figure 15 is a platelet adhesion drawing of TNTA-SC under electron microscope scanning in the present invention;

Figure 16 is a platelet adhesion drawing of TNTA-CO under electron microscope scanning in the present invention;

Figure 17 is a platelet adhesion drawing of TNTA-C under electron microscope scanning in the present invention;

Figure 18 is the cell adhesion diagram of Ti on the first day (left) and third day (right) under the fluorescence microscope in the present invention;

Figure 19 is the cell adhesion diagram of the first day (left) and the third day (right) of TNT under the fluorescence microscope in the present invention;

Figure 20 is the cell adhesion pictures of the first day (left) and the third day (right) of TNTA under the fluorescence microscope in the present invention;

Fig. 21 is a picture of cell adhesion of TNTA-A on the first day (left) and third day (right) under a fluorescence microscope in the present invention;

Figure 22 is a picture of cell adhesion of TNTA-SC on the first day (left) and third day (right) under a fluorescence microscope in the present invention;

Figure 23 is the cell adhesion diagram of TNTA-CO on the first day (left) and third day (right) under the fluorescence microscope in the present invention;

Figure 24 is the cell adhesion diagram of the first day (left) and the third day (right) of TNTA-C under the fluorescence microscope in the present invention;

Fig. 25 is a schematic diagram of the preparation method of the TiO ₂ nanotube material that catalyzes CO release in Embodiment 1 of the present invention.

Detailed ways

The present invention will be described in detail below in conjunction with the accompanying drawings.

Implementation mode 1:

(1) Pure titanium foil (TA2, 0.2 mm thick, 99.6% pure) was first cut into 70 mm × 50 mm titanium sheets, then ultrasonically cleaned with acetone and absolute ethanol for 10 minutes, rinsed with deionized water and air Blow dry in medium temperature, and record as Ti. The cleaned titanium sheet was immersed in 250 mL electrolyte (containing 0.5%wt NH ₄ F, 5 mL deionized water and 245 mL ethylene glycol solution), and was oxidized at 45 V with the titanium sheet as the anode for 1 h, and the sample was dehydrated with absolute ethanol It was fully washed with deionized water, and named TNT after drying. In order to transform the anodized _TiO2 nanotubes into anatase structure, the TNTs were treated in air at 450 °C for 3 hours, which was denoted as TNTA.

(2) Firstly, immerse the cleaned TNTA in 50mL of 10mM 3-aminopropyltrimethoxysilane (APTMS) ethanol solution, shake and react for 12 hours, then take out the sample and put it in a 60℃ oven for heat treatment After 12 hours, it was washed with absolute ethanol and dried to obtain 3-aminopropyltrimethoxysilane-modified _TiO2 nanotubes, denoted as TNTA-A.

(3) Submerge TNTA-A into a mixed solution of sodium alginate (SA) (25mL, 10mg/mL) and chitosan (CS) (25mL, 10mg/mL), and add N-(3-dimethyl Aminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) 0.5751g and N-hydroxysuccinimide (NHS) 0.1150g, stirred for 14 hours, rinsed with deionized water and kept at room temperature Blow-dried under the hood to obtain TiO ₂ nanotubes covalently grafted with sodium alginate/carboxymethyl chitosan composite polymer, named TNTA-SC.

(4) In order to graft CORMs (CORM-401), TNTA-SC was immersed in 20mL of CORM-401 solution with a concentration of 0.1mg/mL, EDC0.2300g and NHS0.0460g were added, and after 4 hours of reaction at room temperature, Washed with deionized water and blown dry at room temperature to obtain the _TiO2 nanotube material that catalyzes the release of CO, denoted as TNTA-CO.

The preparation method of this embodiment is shown in FIG. 25 .

Implementation mode 2:

(1) Pure titanium foil (TA2, 0.2 mm thick, 99.6% pure) was first cut into 70 mm × 50 mm titanium sheets, then ultrasonically cleaned with acetone and absolute ethanol for 10 minutes, rinsed with deionized water and air Blow dry in medium temperature, and record as Ti. The cleaned titanium sheet was immersed in 250 mL electrolyte (containing 0.5%wt NH ₄ F, 5 mL deionized water and 245 mL ethylene glycol solution), and was oxidized at 45 V with the titanium sheet as the anode for 1 h, and the sample was dehydrated with absolute ethanol It was fully washed with deionized water, and named TNT after drying. In order to transform the anodized _TiO2 nanotubes into anatase structure, the TNTs were treated in air at 450 °C for 3 hours, which was denoted as TNTA.

(2) Firstly, immerse the cleaned TNTA in 50mL of 10mM 3-aminopropyltrimethoxysilane (APTMS) ethanol solution, shake and react for 12 hours, then take out the sample and put it in a 60℃ oven for heat treatment After 12 hours, it was washed with absolute ethanol and dried to obtain 3-aminopropyltrimethoxysilane-modified _TiO2 nanotubes, denoted as TNTA-A.

(3) Submerge TNTA-A into a mixed solution of sodium alginate (SA) (25mL, 10mg/mL) and chitosan (CS) (25mL, 10mg/mL), and add N-(3-dimethyl Aminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) 0.5751g and N-hydroxysuccinimide (NHS) 0.1150g, stirred for 14 hours, rinsed with deionized water and kept at room temperature Blow-dried under the hood to obtain 3-aminopropyltrimethoxysilane-modified TiO ₂ nanotubes covalently grafted with sodium alginate/carboxymethyl chitosan composite polymer, named TNTA-SC.

(4) In order to graft CORMs (CORM-401), TNTA-SC was immersed in 20mL of CORM-401 solution with a concentration of 0.1mg/mL, EDC0.2300g and NHS0.0460g were added, and after 4 hours of reaction at room temperature, Washed with deionized water and blown dry at room temperature to obtain the _TiO2 nanotube material that catalyzes the release of CO, denoted as TNTA-CO.

Implementation mode 3:

(1) Pure titanium foil (TA2, 0.2 mm thick, 99.6% pure) was first cut into 70 mm × 50 mm titanium sheets, then ultrasonically cleaned with acetone and absolute ethanol for 10 minutes, rinsed with deionized water and air Blow dry in medium temperature, and record as Ti. The cleaned titanium sheet was immersed in 250 mL electrolyte (containing 0.5%wt NH ₄ F, 5 mL deionized water and 245 mL ethylene glycol solution), and was oxidized at 45 V with the titanium sheet as the anode for 1 h, and the sample was dehydrated with absolute ethanol It was fully washed with deionized water, and named TNT after drying. In order to transform the anodized _TiO2 nanotubes into anatase structure, the TNTs were treated in air at 450 °C for 3 hours, which was denoted as TNTA.

(2) Firstly, immerse the cleaned TNTA in 50mL of 10mM 3-aminopropyltrimethoxysilane (APTMS) ethanol solution, shake and react for 12 hours, then take out the sample and put it in a 60℃ oven for heat treatment After 12 hours, it was washed with absolute ethanol and dried to obtain 3-aminopropyltrimethoxysilane-modified _TiO2 nanotubes, denoted as TNTA-A.

(3) Submerge TNTA-A into a mixed solution of sodium alginate (SA) (25mL, 10mg/mL) and chitosan (CS) (25mL, 10mg/mL), and add N-(3-dimethyl Aminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) 0.5751g and N-hydroxysuccinimide (NHS) 0.1150g, stirred for 14 hours, rinsed with deionized water and kept at room temperature Blow-dried under the hood to obtain 3-aminopropyltrimethoxysilane-modified TiO ₂ nanotubes covalently grafted with sodium alginate/carboxymethyl chitosan composite polymer, named TNTA-SC.

(4) In order to graft CORMs (CORM-401), TNTA-SC was immersed in 20mL of CORM-401 solution with a concentration of 1mg/mL, EDC0.2300g and NHS0.0460g were added, and reacted at room temperature for 4 hours, then used After washing with deionized water and blowing dry at room temperature, a _TiO2 nanotube material that catalyzes the release of CO was obtained, denoted as TNTA-CO.

Observe the surface morphology of the samples of TNT, TNTA, TNTA-A, TNTA-SC, and TNTA-CO obtained in Embodiment 1 through a scanning electron microscope (SEM, FEI Quanta 250):

Figures 1-5 show SEM images of titania nanotube arrays with different surface modifications. It can be seen that anodic oxidation has obtained a regular nanotube array structure on the titanium surface. After different surface modification treatments, the surface nanotube structure still maintains its integrity. The inner diameter of the nanotube array formed after anodic oxidation is between 60-80nm and the outer diameter is about 105nm, as shown in Figure 1. After annealing at 450°C, the diameter of the nanotubes expands outward by about 5nm, accompanied by thickening of the tube wall, such as figure 2. This is because the annealing treatment at 450 °C transforms the amorphous nanotubes into an anatase structure after anodic oxidation, and the size of anatase grains increases with the increase of temperature during the formation stage, which causes the size of nanotubes to change. expand. Compared with TNTA, immobilizing APTMS made the nanotube array surface rougher and the inner diameter reduced by 3-5nm, as shown in Figure 3, indicating that the aminosilane self-assembly partially filled the inner wall of the nanotubes and reduced the inner diameter of the nanotubes. However, after covalently grafting SA/CS on the surface, the wall thickness continued to increase, the inner diameter continued to decrease by about 5nm, and some nanotubes were sealed, as shown in Figure 4, which indicated that the SA/CS composite was in the presence of carbodiimide coupling agent. successfully immobilized in the silane-modified nanotube arrays. Finally, after CORM-401 was grafted on the surface, although the inner diameter of the nanotubes did not change much, the surface roughness further increased, as shown in Figure 5. The immobilized CORM-401 can catalyze the release of CO gas signal molecules in the physiological environment of cells, thereby enhancing the anticoagulant performance of the material and promoting the adhesion and proliferation of endothelial cells.

The TNT, TNTA, TNTA-A, TNTA-SC, and TNTA-CO obtained in Embodiment 1 are measured by X-ray photoelectron spectroscopy (XPS, VG Science, UK) to measure the binding state of surface atoms of different modified samples, and the results are shown in the figure 6-9 shows:

Figure 6 is the XPS high-resolution spectrum of the main elements on the surface of TNTA. The surface of TNTA is mainly composed of Ti and O elements, and the atomic content ratio of O and Ti is about 2:1, indicating that TiO ₂ is mainly formed after anodic oxidation. The two doublets in the Ti2p spectrum are mainly due to the presence of Ti-O bonds and a small amount of Ti=O bonds. Similarly, two peaks with binding energies of 529.92 eV and 531.29 eV in the O1s spectrum are mainly attributed to the oxide TiO ₂ . Figure 7 is the XPS high-resolution spectrum of the main elements on the surface of TNTA-A. For TNTA-A samples, the C1s spectrum can be fitted into three peaks: binding energy 284.80eV (CH/CC/Si-C), CO-Si 286.36eV (CO) and 288.14eV (CN) in . The high-resolution N1s XPS spectrum showed strong peaks at binding energies 397.09 eV, 399.88 eV, and 401.58 eV, which were attributed to NC bonds and NH bonds, respectively. Figure 8 is the XPS high-resolution spectrum of the main elements on the surface of TNTA-SC. After covalently grafting SA/CS, the C1s peak (286.45eV) in TNTA-SC is enhanced, and the peak area increases from 2542.95 to 4699.01, which is similar to that of SA , CS contains a large number of CO bonds. In addition, the N1s peak (397.40 eV) was also enhanced due to the presence of a large amount of -NH2 in the polymer CS, and the peak area increased from 156.70 to 556.35. Figure 9 is the XPS high-resolution spectrum of the main elements on the surface of TNTA-CO. After grafting CORM-401, the N1s peak (397.29eV) was weakened (the peak area was reduced from 556.35 to 274.83) because the original surface was covered. These results demonstrate the successful synthesis of the target polymer material.

Determination of CO release behavior using the myoglobin assay:

First, 15 mL of myoglobin (Mb) solution with a concentration of 2 mg/mL was prepared in PBS, degassed by bubbling with N ₂ for 30 minutes, and then 20 mg of sodium dithionite (Na ₂ S ₂ O ₃ ) was added to convert it into deoxygenated myoglobin (Mb) For red protein (DeoMb), take 1mL DeoMb solution and add excess CORM-401 and 0.5mg cysteine to convert all DeoMb into carboxyhemoglobin (Mb-CO), and record the absorbance of Mb-CO at 500-600nm. Then add the sample TNTA-CO obtained in Embodiment 1 to the DeoMb solution, and measure the change of solution absorbance (500-600nm) under the conditions of adding and not adding cysteine, respectively, to characterize the release behavior of CO, adding Cysteine samples were designated as TNTA-C. Calculated as follows:

Where OD ₅₄₀ is the absorbance of the Mb-CO solution at 540nm, ΔOD ₅₄₀ is equal to the change in absorbance of the solution after adding sample TNTA-CO and cysteine (or no cysteine) at 540nm, ΔOD _iso510 is the isoabsorbance at 510nm The change of point absorbance, ε=15.4 mM ^-1 cm ^-1 is the extinction coefficient of Mb-CO.

The results are shown in Figure 10, which is the CO release content map of TNTA-CO. There is still CO released in TNTA-CO without adding cysteine as _a CO catalyst, _this is because the residual _Na2S2O3 can also catalyze the release of CO, but after adding cysteine, the amount of CO released is obvious It is proved that cysteine plays a role in catalyzing CORM-401 to release CO. TNTA-C will release CO quickly within 12h, and after 12h, the CO release rate will gradually decrease and tend to be stable, and then there will be a stable stage of CO release that lasts up to 7 days, which indicates that the release of CO in TNTA-C can exceed 7 days.

The TNTA-CO samples obtained in Embodiment 1 were respectively treated with Ti, TNT, TNTA, TNTA-A, TNTA- SC is the control material, and the in vitro platelet adhesion experiment is carried out:

Whole fresh healthy human blood (provided by Huaian Second People's Hospital) was centrifuged at 1500 r/min for 10 minutes to obtain platelet-rich plasma (PRP). 200 μL of PRP was completely covered on the surface of each sample, and incubated in a 37°C incubator for 3 hours, and then the samples were washed 2 times with normal saline. Adherent platelets were fixed with 2.5% glutaraldehyde solution (prepared in normal saline) at 4°C for 12 hours, and then the samples were washed 1-2 times with normal saline. The samples were sequentially dehydrated with 50%, 70%, 90% and 100% ethanol solutions for 15 min each, and dried in air. After spraying gold on the surface, the morphology of platelets was observed by SEM (FEI Quanta 250), and the results are shown in Figure 11-17.

Figures 11-17 are the platelet adhesion drawings of Ti, TNT, TNTA, TNTA-A, TNTA-SC, and TNTA-CO. It can be seen from Figure 11 that a large number of platelets adhere to the surface of Ti, and the adhered platelets are in a spread state, pseudo The extension of the foot indicates that the platelets may have been activated, which also indicates that the unmodified blank titanium has poor anticoagulant performance. It can be seen from Figure 12 that after anodizing, the number of platelets adhered to the surface of TNT decreased. The surface nanotube morphology obtained by anodic oxidation has excellent hydrophilicity and unique micro-nano structure, which can reduce the adhesion and aggregation of platelets. It can be seen from Figure 13 that after annealing, the hydrophilicity of the surface of TNTA is further improved, and at the same time, it shows stronger selective adsorption to albumin, so the amount of platelet adhesion on the surface of TNTA is further reduced, and the morphology of platelets is also more complete, indicating blood compatibility. be further improved. It can be seen from Figure 14 that the amount of platelet adhesion on the surface of TNTA-A is slightly increased by fixing APTMS, which is because the surface of TNTA-A promotes the adsorption of albumin and fibrinogen at the same time, and has a stronger adsorption to fibrinogen , promote the adhesion and aggregation of platelets to a certain extent. CS is a good biocompatible material. The carboxylate group in SA can chelate calcium ions in the blood to interrupt the coagulation cascade reaction and play an anticoagulant effect. Therefore, after grafting SA/CS , the amount of platelet adhesion on the surface of TNTA-SC was significantly reduced, and the platelets also showed a more regular spherical state, as shown in Figure 15. It can be seen from Figure 16 that after further grafting of CO-releasing molecules, the number of platelets adhered to the surface of TNTA-CO decreased significantly, probably because reducing groups such as thiol in blood catalyzed CORM-401 to release CO, but the reducing The group content is small and cannot fully catalyze CORM-401, so after adding CO catalyst (cysteine), the number of platelets adhered to the surface of TNTA-C was further reduced, and no aggregation and activation of platelets was found, as shown in Figure 17. It shows that the TiO ₂ nanotube material that catalyzes the release of CO prepared by the present invention releases CO after being catalyzed by cysteine, and CO also has a certain anticoagulant effect.

The TNTA-CO samples obtained in Embodiment 1 were respectively treated with Ti, TNT, TNTA, TNTA-A, TNTA- SC is the control material, and the endothelial cell adhesion experiment is carried out:

Place the samples in a 24-well culture plate, sterilize the samples with ultraviolet light on the ultra-clean bench, and then add 0.5 mL of endothelial cells (5× ¹⁰⁴ cells/mL, ECV304, purchased from Chongqing Biosain Biotechnology Development Co., Ltd.) suspension and 1.5mL cell culture medium. After cultured at 37°C and 5% CO ₂ for 1 and 3 days, the samples were washed 3 times with saline. Add 200 μL of rhodamine in PBS solution (10 μg/mL) to the surface of each sample to stain for 20 min, and then wash the sample 3 times with normal saline. Finally, 200 μL of 4',6-diamidino-2-phenylindole (DAPI) (500 ng/mL) was added to the surface of the sample, stained for 10 min, and the sample was washed 3 times with normal saline. The number and shape of the cells were observed with a fluorescent microscope (Zeiss, invertedA2) in the stained samples, and the results are shown in Figures 18-24.

Figures 18-24 are the cell adhesion diagrams of Ti, TNT, TNTA, TNTA-A, TNTA-SC, and TNTA-CO. The number of endothelial cell adhesion on the surface of each sample generally increased from the first day to the third day. In addition, it can be seen that the cells on the Ti sheet have less adhesion and are basically round, as shown in Figure 18, indicating that endothelial cells have not adhered well to the surface, and the spreading performance of the adhered cells is poor, indicating that the cells on the Ti sheet Insufficient ability to migrate and proliferate. It can be seen from Figure 19 that the nanotube structure and the improvement of hydrophilicity after anodizing are beneficial to the adhesion of cells. However, the amount of adhesion on the surface of TNT is slightly reduced, which is related to the residual fluorine and other impurity elements on the surface of nanotubes. It can be seen from Figure 20 that after annealing, the porosity of nanotube arrays further increases, impurities are effectively removed, and the surface is rougher. The porous and rough surface structure can increase the effective contact area between TNTA and endothelial cells, thereby improving the stability of endothelial cells in TNTA. surface adhesion. It can be seen from FIG. 21 that after self-assembly of 3-aminopropyltrimethoxysilane, endothelial cells exhibit better adhesion on the surface. It can be seen from Figure 22 that after grafting SA/CS coating, the number of endothelial cells adhered to the surface of TNTA-SC was more, and the spreading performance of cells was improved. It can be seen from Figure 23 that after CORM-401 was further grafted, the adhesion of endothelial cells on the surface was weakened, and most of the cells were round. However, after adding cysteine to catalyze the release of CO, the adhesion effect of endothelial cells on the surface of TNTA-C is the best, as shown in Figure 24, indicating that the CO released by surface catalysis can significantly promote the adhesion and proliferation of endothelial cells.

In summary, on the basis of the _TiO2 nanotubes obtained by anodic oxidation, the present invention first introduces amino groups on the surface of the nanotubes through self-assembled aminosilane, and further immobilizes the compound of sodium alginate/chitosan in situ simultaneously, Finally, the CO-releasing molecule (CORM401) was immobilized on the surface of the material to construct a TiO ₂ nanotube material TNTA-CO that catalyzes the release of CO, and the biocompatibility of the material was improved through the sustained catalytic release of CO in vivo. Compared with Ti, TNT, TNTA, TNTA-A and TNTA-SC, TNTA-CO has stronger anticoagulant performance in the case of catalyzing the release of CO, and can further promote the adhesion, growth and functional expression of vascular endothelial cells. It can be used not only for the preparation of artificial vascular graft materials, but also for the surface coating preparation of intravascular implanted devices (such as vascular stents, cardiac pacemakers), to improve the anticoagulant performance of the device and promote endothelial repair performance.

The above-mentioned embodiments are only for illustrating the technical concept and characteristics of the present invention, and its purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly, and not to limit the scope of protection of the present invention. All equivalent changes or modifications made according to the spirit of the present invention shall fall within the protection scope of the present invention.

Claims (10)

1. a kind of TiO that catalyzes release CO The preparation method of _nanotube material is characterized in that, comprises the steps: Step 1, preparing TiO _nanotubes in situ on the titanium surface by anodic oxidation; Step 2, carrying out silanization treatment to the material obtained in step 1 to obtain silane- _modified TiO nanotubes; Step 3, covalently grafting sodium alginate/carboxymethyl chitosan composite macromolecule on the surface of the silane-modified TiO _nanotube ; Step 4: Covalently graft CO releasing molecules on the surface of the nanotubes obtained in Step 3 to obtain a TiO ₂ nanotube material that catalyzes releasing CO. 2. the TiO of catalytic release CO according to claim ₁ The preparation method of nanotube material is characterized in that, step 4 is that the nanotube of step 3 gained is immersed in CORM-401 solution, adds carbodiimide and N-hydroxysuccinimide was reacted for 2 to 5 hours, washed and dried to obtain a TiO ₂ nanotube material that catalyzes the release of CO. 3. Catalyzing and releasing CO according to claim ₂ TiO The preparation method of the nanotube material is characterized in that, the CORM-401 solution concentration is 0.05-1mg/mL; And/or, the mass ratio of the carbodiimide to the N-hydroxysuccinimide is 3-6:1. 4. according to claim 1, the preparation method of the _TiO2nanotube material that catalyzes CO release is characterized in that, step 1 is to put the titanium sheet after ultrasonic cleaning and drying into containing 0.2～1%wt _NH4F , Anodize in the electrolyte of 2~10mL deionized water and 200~300mL ethylene glycol solution for 0.5~2 hours, clean the anodized product with absolute ethanol and deionized water ultrasonically, dry it and put it in the air at 450 °C high-temperature annealing for 2-4 hours to obtain the TiO ₂ nanotubes. 5. the TiO of catalytic release CO according to claim ₁ The preparation method of nanotube material is characterized in that, step 2 is that described step 1 gained material is immersed in 3-aminopropyltrimethoxysilane or 3- Aminopropyltriethoxysilane solution, shaking reaction for 10-24 hours, heating at 60-100°C for 8-12 hours, washing the heat-treated product with absolute ethanol, drying to obtain the modified silane properties of TiO ₂ nanotubes. 6. the TiO of catalytic release CO according to claim 5 The preparation method of _nanotube material is characterized in that, described 3-aminopropyltrimethoxysilane or 3-aminopropyltriethoxysilane solution concentration 5~15mM. 7. the preparation method of the TiO _nanotube material that catalyzes release CO according to claim 1, is characterized in that, step 3 is that described silane modified _TiO nanotube is immersed in sodium alginate and carboxymethyl chitosan Carbodiimide and N-hydroxysuccinimide are added to the mixed solution of sugar, and after stirring and reacting for 10-14 hours, the obtained nanotubes are washed with deionized water and dried. 8. the TiO of catalytic release CO according to claim 7 The preparation method of _nanotube material, it is characterized in that, described sodium alginate solution concentration is 1～10mg/mL; And/or, the carboxymethyl chitosan solution concentration is 1 ~ 10mg/mL; And/or, the volume ratio of the sodium alginate solution to the carboxymethyl chitosan solution is 1:1; And/or, the mass ratio of the carbodiimide to the N-hydroxysuccinimide is 3-6:1. 9. An application of the _TiO2 nanotube material that catalyzes CO release prepared by the method according to any one of claims 1 to 8 in the preparation of catalysis-induced CO release artificial vascular graft material. 10. A _TiO nanotube material that catalyzes CO release prepared by the method according to any one of claims 1 to 8 is used in the preparation of catalysis-induced CO release vascular stent coating.

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