TW202317652A - A bio-inert material and its application for preventing biofouling from metal surface - Google Patents

Abstract

The invention discloses a copolymer and its application for preventing biofouling from metal surface. The copolymer structure comprises a first segment and a second segment. The first segment comprises phosphonic acid groups, the second segment comprises pyridine sulfobetaine groups or pyridine carboxybetaine groups and the first segment bond with the second segment by covalent bonding to form the copolymer. Particularly, the phosphonic acid groups form coordinate covalent bonds with metal surface for anchoring function and the pyridine sulfobetaine groups or pyridine carboxybetaine groups prevent biofouling from the metal surface.

Description

A kind of copolymer and its application in preventing metal surface from adhering biomolecules Methods

The invention discloses a copolymer and a method for its application in preventing metal surfaces from adhering biomolecules. In particular, the structure of the copolymer contains a plurality of phosphoric acid groups and a plurality of pyridinesulfobetaine or pyridinecarboxybetaine.

The stent medical materials widely used in the treatment of cardiovascular diseases in the market include Bare Metal Stent (BMS), which is a simple metal stent, and its function is to open the stenosis of the blood vessel, but it has the disadvantage of causing the restenosis of the blood vessel in the future. . The other is a drug-coated stent (Drug Eluting Stent, DES), which is coated with a layer of biodegradable polymers containing anti-cell proliferation drugs on the surface of the metal stent, and gradually releases drugs through slow biodegradation, making vascular endothelial cells The growth is inhibited. Although the restenosis of blood vessels can be avoided, the blood coagulation reaction continues to occur. Anticoagulant drugs must be taken for life, and the number of drugs on the stent can only be maintained for 5 to 10 years. This is a major disadvantage. The above All scaffold materials have different disadvantages that need to be overcome in order to reduce their side effects after being implanted in a living body.

According to the above, in order to overcome the lack of side effects of current scaffold materials and implanted organisms, research and development of a material with good structural stability and biological inertness is an urgent research and development topic for the current biomedical material industry.

According to the aforementioned background of the invention and industrial needs, the technical means and effects of the present invention disclose a copolymer with a novel structure, which contains a phosphoric acid group capable of anchoring/fixing the substrate, and a cracking-resistant Biologically inert pyridinesulfobetaine or pyridinecarboxybetaine functional groups. In particular, the present invention uses the above-mentioned copolymer structure to manufacture design-related biologically inert materials and apply them to prevent the adhesion of biomolecules on the surface of metal materials.

The first object of the present invention is to provide a novel copolymer. The structure of the copolymer comprises a first segment and a second segment, the first segment comprises a plurality of phosphoric acid groups, and the second segment comprises a plurality of pyridinesulfone Acid-based betaine or pyridinecarboxylic acid-based betaine, and the above-mentioned first segment and second segment are connected by covalent bonds to form the copolymer.

Specifically, the copolymer is ethylene produced by radical polymerization using vinylphosphonic acid (VPA) and 4-vinylpyridine sulfobetaine (4-vinylpyridine sulfobetaine, 4VPPS) without ester functional groups. Phosphate-4-vinylpyridinesulfonate betaine copolymer.

Specifically, the vinyl phosphate-4-vinylpyridine sulfobetaine copolymer can form a coordination covalent bond with the surface of the metal material, thereby being anchored on the surface of the metal material.

Specifically, the above-mentioned copolymer has the technical effects of biocompatibility, anti-biomolecular adhesion, easy anchoring on the surface of the substrate and stable molecular structure. The results can be widely used in the preparation of biologically inert materials.

The second object of the present invention is to provide a biologically inert material, which includes a film layer and a substrate, the film layer is composed of the copolymer described in the first object, and is covalently bonded by coordination or The chemical graft is anchored to the surface of the substrate, thereby forming the bioinert material.

In particular, the film layer also includes an interpenetrating polymer network structure.

Specifically, the structure of the copolymer includes a first segment and a second segment, the first segment includes a plurality of phosphoric acid groups, and the second segment includes a plurality of pyridine sulfobetaine or pyridinecarboxylate betaine The base, and the above-mentioned first chain segment and second chain segment are connected by covalent bonds to form the copolymer.

Specifically, the above-mentioned bioinert materials have the technical effects of biocompatibility, anti-biomolecular adhesion and stable material structure, and can be widely used in the field of biomedical materials.

The third object of the present invention is to provide a method for preventing the adhesion of biomolecules to the metal surface, which includes covalently bonding the copolymer described in the first object of the present invention on the metal surface or forming interpenetrating polymerization by chemical grafting The network structure of the object can prevent the metal surface from adhering to biomolecules.

Specifically, the structure of the copolymer includes a first segment and a second segment, the first segment includes a plurality of phosphoric acid groups, and the second segment includes a plurality of pyridine sulfobetaine or pyridinecarboxylate betaine The base, and the above-mentioned first chain segment and second chain segment are connected by covalent bonds to form the copolymer.

Specifically, the coordinate covalent bond is formed by the phosphoric acid group and the metal surface Formed by the chromium contained in the surface.

In summary, the technical features and effects of the present invention include: (1) provide a copolymer with phosphoric acid group and pyridine sulfonate betaine or pyridine carboxylate betaine structure, wherein the phosphoric acid group forms a complex with the surface of the material Covalent bonding or interpenetrating polymer network structure, thereby anchored on the material to form a stable copolymer film structure; The copolymer film layer achieves the biological inert effects such as anti-biomolecular adhesion; (2) provide a novel biological inert material, especially a biological inert material with metal as the base material, the biological inert material comprises a phosphate group and a pyridine The film layer composed of a copolymer of sulfobetaine or pyridinecarboxylate betaine structure has the technical effects of biocompatibility, anti-biomolecule adhesion and stable material structure; The method of sticking biomolecules on the surface, through coordination covalent bonding or chemical grafting to form an interpenetrating polymer network structure to make the above-mentioned ones with the structure of phosphate group and pyridine sulfonate betaine or pyridine carboxylate betaine The copolymer is anchored on the surface of the metal material, thereby achieving the purpose of preventing the surface of the metal material from adhering to biomolecules, and can be widely used in the field of metal medical materials.

[Figure 1] is the FTIR spectrum of the vinyl phosphate-4-vinylpyridinesulfonate betaine copolymer poly(VPA- r -4VPPS) of the present invention; the control group is Poly(VPA- r -SBMA) copolymer.

[Fig. 2] is a bar graph of the water contact angle of the vinylphosphoric acid-4-vinylpyridinesulfonate betaine copolymer poly(VPA- r -4VPPS) of the present invention.

[Fig. 3] is a bar graph of the hydration content of the vinylphosphoric acid-4-vinylpyridinesulfonate betaine copolymer poly(VPA- r -4VPPS) of the present invention.

〔Fig. 4〕is the micrograph of the vinyl phosphate-4-vinylpyridine sulfonate betaine copolymer poly(VPA- r -4VPPS) of the present invention and the bar graph of the relative attachment amount of human fibroblasts; the control group It is SBMA water glue.

[Fig. 5] is a bar chart of the coagulation time of the vinylphosphoric acid-4-vinylpyridinesulfonate betaine copolymer poly(VPA- r -4VPPS) of the present invention.

[Fig. 6] is a bar graph of the hemolysis test percentage of vinylphosphoric acid-4-vinylpyridinesulfonate betaine copolymer poly(VPA- r -4VPPS) of the present invention.

[Fig. 7] is the microscope image and grading of cytotoxicity analysis of vinylphosphoric acid-4-vinylpyridinesulfonate betaine copolymer poly(VPA- r -4VPPS) of the present invention.

[Fig. 8] is the thermogravimetric analysis (TGA) spectrum of vinyl phosphate-4-vinylpyridinesulfonic acid betaine copolymer poly(VPA- r -4VPPS) of the present invention; the control group is Poly(VPA- r- SBMA) copolymer.

[Fig. 9] Microscope images of human whole blood before and after sterilization of vinyl phosphate-4-vinylpyridinesulfonic acid betaine copolymer poly(VPA- r -4VPPS) of the present invention and the relative attachment amount of human whole blood Bar graph; control group is SBMA hydrogel.

[Fig. 10] is the bar chart of the relative amount of human fibroblasts attached to the film layer of vinylphosphoric acid-4-vinylpyridinesulfonic acid betaine copolymer PPA70 and its copolymer interpenetrating network (PPA70-PVA) of the present invention ; The control group was SBMA hydrogel.

〔Figure 11〕It is vinyl phosphoric acid-4-vinylpyridinesulfonic acid betaine copolymerization of the present invention The bar chart of the relative attachment amount of green fluorescent protein Escherichia coli on the film layer of PPA70 and its copolymer interpenetrating network (PPA70-PVA); the control group is SBMA water glue.

Although the present invention has been described above with specific examples, the scope of the present invention is not limited thereto. Those skilled in the art understand that various modifications or changes can be made without departing from the intent and scope of the present invention. In addition, the abstract and the title are only used to assist the search of patent documents, and are not used to limit the scope of rights of the present invention.

According to the aforementioned content of the invention, the first embodiment of the present invention is to provide a copolymer whose structure includes a first chain segment and a second chain segment, the first chain segment contains a plurality of phosphoric acid groups, and the second chain segment contains a plurality of pyridine The sulfobetaine or pyridinecarboxybetaine, and the above-mentioned first chain segment and second chain segment are connected by covalent bonds to form the copolymer.

In one embodiment, the copolymer has the following general structural formula, wherein m is an integer of 5-500, n is an integer of 5-500; R is a functional group with carboxylic acid or sulfonic acid base; k is a carbon chain with 1~3 repetitions.

Formula 1)

In a specific embodiment, the copolymer is vinylphosphoric acid-4-vinylpyridinesulfonic acid betaine copolymer, its chemical structure is shown in formula (2), wherein m is an integer of 20 to 400, and n is 30 Integer of ~300.

Formula (2)

In a specific embodiment, the weight average molecular weight of the vinyl phosphate-4-vinylpyridine sulfobetaine copolymer is less than 120 kDa, preferably, the range is 50-120 kDa.

In a specific embodiment, the vinyl phosphate-4-vinylpyridine sulfobetaine copolymer is prepared by free radical polymerization of vinyl phosphate and 4-vinylpyridine sulfobetaine, the vinyl The reaction molar ratio of phosphonyl phosphoric acid and 4-vinylpyridine sulfobetaine ranges from about 9:1 to 1:9. Preferably, it is 7:3 to 3:7.

The second embodiment of the present invention is to provide a biologically inert material, which includes a film layer and a substrate, the film layer includes the copolymer as provided in the first embodiment of the present invention, and the film layer is covalently Bonding or chemical grafting is anchored to the surface of the substrate, thereby forming the bioinert material.

In a specific embodiment, the copolymer is vinyl phosphate-4-vinylpyridine sulfobetaine copolymer, and its weight average molecular weight is about 50-120 kDa.

In a specific embodiment, the vinyl phosphate-4-vinylpyridine sulfobetaine copolymer is prepared by free radical polymerization of vinyl phosphate and 4-vinylpyridine sulfobetaine, the vinyl The reaction molar ratio of phosphonyl phosphoric acid and 4-vinylpyridine sulfobetaine ranges from about 7:3 to 3:7.

In a specific embodiment, the water contact angle of the film layer is about less than 60 degrees, preferably 30-50 degrees.

In another specific embodiment, the film layer also includes an interpenetrating polymer network structure formed by polyvinyl alcohol and vinyl phosphoric acid as shown in formula (3), wherein X is an integer of 5-500, preferably, is an integer of 20-400, Y is an integer of 5-7000, preferably, Y is an integer of 30-1500.

Formula (3)

In another specific embodiment, the substrate comprises stainless steel, titanium alloy, glass, silicon wafer, ceramic, polyethylene terephthalate, polypropylene, polydimethylsiloxane, polytetrafluoroethylene or poly Vinylidene fluoride.

In another specific embodiment, the biologically inert material is applied to the preparation of cardiac catheters, vascular stents, endoscopes or surgical instruments.

The third embodiment of the present invention is to provide a method for preventing the metal surface from adhering to biomolecules, including covalently bonding the copolymer described in the first embodiment of the present invention on the metal surface or forming interpenetration by chemical grafting The polymer network structure prevents the metal surface from adhering to biomolecules, and the coordination covalent bond is formed by phosphoric acid groups and chromium contained in the metal surface.

In particular, the technical features and functions mentioned in the above embodiments can solve the current problems of bare metal stents and drug-coated stents. Using the copolymer material of the present invention to create a layer of anti-biomolecule adhesion film layer on the metal stent or modify its surface to make it biocompatible and able to resist cell adhesion without inhibiting cell growth; On the one hand, the copolymer of the present invention and the film layer formed by it can also inhibit the attachment of blood cells, avoid reactions such as coagulation and hemolysis, and further reduce the taking of anticoagulant drugs. Accordingly, the copolymer, biologically inert material and application method of the present invention have unexpected effects in the biomedical industry, especially in the invasive medical material industry.

The following examples and experimental examples are experiments carried out according to the content described in the above-mentioned content of the invention and the embodiments, and are used as effect verification and description of the present invention accordingly.

Example 1: Vinyl Phosphate-4-Vinylpyridine Sulfonate Betaine Polymer (poly(VPA- r -4VPPS)) synthesis

Synthesize poly(VPA- r -4VPPS) by free radical polymerization (Free Radical Polymerization), and control the molar ratio of vinylphosphoric acid (VPA): 4-vinylpyridinesulfobetaine (4VPPS), as shown in Table 1 As shown, and the theoretical molecular weight is controlled at about 50kDa, the actual molecular weight is determined by gel permeation chromatography (Gel Permeation Chromatography, GPC). The reaction of copolymerization macromolecule synthesis is as shown in paragraph [0036], VPA and 4VPPS monomer are put into polypropylene (PP) reaction bottle, with 1M NaCl aqueous solution as solvent dissolution monomer, add ammonium persulfate (Ammonium persulphate, APS ) as the starting agent and dissolved, ventilate with argon for 15 minutes to remove oxygen and moisture in the solution, then seal the bottle mouth, and place it in an oil bath at 70° C. to stir for 48 hours. The finished product is soaked in an ice bath to terminate the reaction, and acetone is used as a non-solvent to precipitate the polymer, and the precipitated copolymer polymer is placed in a vacuum drying device to remove the residual solvent, and the synthesis of the copolymer polymer is completed. The control group of the present invention is Poly(VPA- r- SBMA) copolymer synthesized from vinyl phosphoric acid (VPA) and sulfobetaine methacrylate monomer (SBMA).

Table 1

Reaction equation

FT-IR Analysis of Vinyl Phosphate-4-Vinylpyridine Sulfonate Betaine Copolymer (poly(VPA- r -4VPPS))

FT-IR was used to detect the bonds and functional groups in the copolymer structure. In the Poly(VPA- r -4VPPS) copolymer, the characteristic functional groups could be detected. The analysis spectrum is shown in Figure 1. The wavenumber of Pyridine is 1571~1423cm ^-1 ; the wavenumber of Poly(VPA- r -SBMA) copolymer is 1725cm ^-1 C=O characteristic functional group; To the CN ⁺ bond with a wave number of 1641~1643cm ^-1 , the SO ₃ bond with 1201~1043cm ^-1 and the PO bond with 983~925cm ^-1 , it is proved that the example 1 uses a phosphoric acid group monomer and double ion The Poly(VPA- r -4VPPS) copolymer of the present invention is successfully prepared by the free radical polymerization method of the monomer.

Analysis of Water Contact Angle of Surface Modified by Vinyl Phosphate-4-Vinylpyridine Sulfonate Betaine Copolymer (poly(VPA- r -4VPPS))

In this experiment, stainless steel was used as the material to be surface modified to endow it with biological inertness. Water contact angle analysis was performed on the stainless steel surface before and after modification, thereby proving the technical efficacy of the copolymer of the present invention that can form a stable structure on the surface of the material. Specifically, using 1M NaCl as the solvent, the Poly(VPA- r -4VPPS) copolymerized polymer was configured into a polymer solution at a ratio of 40 mg/mL, and a stainless steel ingot was placed in each grid in a 24-well porous plate. And add 1mL of polymer solution, cover the porous plate with Teflon antidiarrheal tape, put it in a 60°C oven to react for 48 hours, when the reaction is completed, wash it repeatedly with 1M NaCl solution, and remove the ungrafted high Molecules are removed, and then cleaned with deionized water to wash away the residual NaCl, and then the water is blotted dry to complete the surface modification of stainless steel. The water contact angle detection method in this experiment is to drop 4 μL of deionized water onto the surface of the stainless steel sample at room temperature, wait for it to balance for 3 minutes, and then measure its water contact angle. The experimental results are shown in Figure 2 shown. Among them, the contact angle of the stainless steel modified by Poly(VPA- r -4VPPS) copolymer is significantly reduced by about 60° compared with the unmodified stainless steel, and the better condition is 36.8° of PPA70, which proves that the surface of the hydrophobic stainless steel has been modified. into a hydrophilic surface. However, PPA100 is a pure VPA homopolymer, and the two -OH functional groups will bond with the chromium oxide (Cr ₂ O ₃ ) on the stainless steel surface, leaving only one hydrophilic P=O functional group in the structure, so the water contact angle The downward trend is significantly smaller than for the copolymers of the present invention. P4VPPS is a homopolymer of 4VPPS. In the absence of phosphate-based monomer (VPA), it cannot form a stable bond with the stainless steel surface. After the modification, the cleaning and purification process is washed, resulting in an ineffective water contact angle. Therefore, the copolymer without phosphoric acid group cannot form a stable structure with the surface of the material, so as to achieve the purpose of having good hydrophilicity after the surface of the material is modified.

Analysis of Hydration Ability of the Surface of Modified Materials with Vinyl Phosphate-4-Vinylpyridine Sulfonate Betaine Copolymer (poly(VPA- r -4VPPS))

Hydration capacity is an indicator used to evaluate the biological inertia of the material surface. The higher the hydration capacity, the better the anti-biomolecule adhesion effect on the surface of the material, which can capture more water molecules in the biological environment and form hydration on its surface. The layer can effectively reduce the intermolecular force between biomolecules and the material surface, thereby increasing the anti-biomolecular adhesion properties of the material surface. The specific experimental results of the present invention are shown in Figure 3. Compared with the unmodified surface, the surface of stainless steel modified by poly(VPA- r -4VPPS) significantly improves the hydration capacity of the stainless steel surface. The optimal condition is Compared with unmodified stainless steel, PPA70 has a hydration capacity of about 200%. Specifically, the average hydration capacity of stainless steel modified by PPA70 is 0.097 mg/cm ² .

One of the Bioinert Efficacy Analysis of Vinyl Phosphate-4-Vinylpyridine Sulfonate Betaine Copolymer (poly(VPA- r -4VPPS))

The cells used in this experiment were human fibroblasts (HT1080). The 75T Flask cultured to 90% full cells was reacted with 1mL trypsin-EDTA with the extracellular matrix for 6 minutes to suspend the cells, and 9mL of cell culture medium was added to stop the trypsin reaction, and the cell suspension was dissolved in 15mL After the centrifuge tube was centrifuged at 290 rcf for 5 minutes, the supernatant was removed, and the cells were washed away with 10 mL of cell culture medium. Count the number of cells under a microscope with a cell counting disc, and dilute the number of cells to 50,000 cells/mL with culture medium. Place the sample in a 24well plate and inflate it with PBS for 12 hours, sterilize it with UV light for 30 minutes, put it in a biological safety cabinet, remove the PBS in the well plate, wash the sample twice with 1mL of PBS, and pour 1mL The diluted cell solution was placed in a constant humidity and constant temperature incubator at 37°C, 5% CO ₂ for 24 hours. The cultured samples were washed 3 times with PBS, and glutaraldehyde was added as a cell fixative. After reacting for 1 hour, they were washed once with PBS, and the amount of cells attached to the surface of the material was observed with a fluorescent microscope. Using computer software Do quantitative analysis. The experimental results are shown in Figure 4, which is the analysis chart of HT1080 attached to the surface of the material and the picture under the fluorescent microscope, showing that PPA70 is one of the better anti-cell attachment conditions, and the stainless steel modified by PPA70 On the surface, compared with the cell attachment amount of unmodified stainless steel, the average number of cells per square centimeter is greatly reduced by 15,000, forming an effective anti-cell adhesion surface.

Bio-inert performance analysis of vinyl phosphate-4-vinylpyridine sulfonate betaine copolymer (poly(VPA- r -4VPPS))

In this coagulation analysis experiment, stainless steel ingots were soaked in 1 mL of platelet poor plasma (PPP), and equilibrated in a water bath at 37°C for 1 hour, and the equilibrated PPP was drawn into the sample tube, and the coagulation time analyzer ( CA-600 series), set the detection items as prothrombin time (Prothrombin Time, PT) and activated partial thromboplastin time (Activated Partial Thromboplastin Time, APTT), the instrument will automatically inject the detection reagent mixed with PPP, and the wavelength 660nm for detection of light scattering intensity. The difference in the normal value reaction time between the PPP of the soaked sample and the normal value of PPP was compared. According to the biocompatibility specification of ISO 10993, coagulation analysis must be carried out for medical devices that come into contact with blood. In this study, the relationship between prothrombin time (PT) and activity of stainless steel was detected. The activated partial thromboplastin time (Activated Partial Thromb oplastin Time, APTT) is used to detect extrinsic coagulation and intrinsic coagulation respectively. The normal value of PT is 12-16 seconds, and the normal value of APTT is 24-36 seconds; Comparing the measured values of PPP immersed in stainless steel and pure PPP, as shown in Figure 5, the surface of stainless steel modified by copolymerized polymer does not cause blood coagulation reaction.

Bioinert performance analysis of vinyl phosphate-4-vinylpyridine sulfonate betaine copolymer (poly(VPA- r -4VPPS))

The whole blood was first diluted with PBS to a total volume of 800 μL of diluents of different concentrations, and 200 μL was added to 3.33 mL of PBS, and 200 μL was taken to detect the absorbance at a wavelength of 542 nm, and the concentration value of OD ₅₄₂ =0.7~0.8 was found. Use an appropriate concentration to prepare whole blood dilution, add 200 μL of the dilution to a 15mL centrifuge tube containing 3.33mL PBS and stainless steel samples, and prepare PBS and DI water as control conditions for coagulation. Place the centrifuge tube in a 37°C water bath to keep the temperature constant for 1 hour. After the constant temperature, the centrifuge tube is centrifuged at a speed of 180 rcf for 10 minutes. Take 200 μL of the centrifuged supernatant to detect the absorbance at a wavelength of 542 nm, which is detected by PBS and DI water The OD value is set as 0% and 100%. When the degree of hemolysis is lower than 5%, it is considered that the sample will not cause hemolysis. The following formula is used to calculate the degree of hemolysis under different conditions.

Hemolysis=(OD _SUS -OD _PBS )/(OD _DI -OD _PBS )×100%

OD _SUS : the OD value of the stainless steel in each condition measured by the hemolysis test; OD _PBS : the OD value measured by the PBS of the negative control group; OD _DI : the OD value measured by the DIWater of the positive control group.

In the above-mentioned hemolysis analysis, take PBS as the negative control group that will not hemolysis, and deionized water as the positive control group that will cause hemolysis, soak the copolymer material of the present invention in the blood, and use UV-VIS to observe whether the blood cells are affected by the hemolysis. The hemolysis caused by the copolymer material leads to the dissolution of hemoglobin into the PBS that dilutes the blood, and the degree of hemolysis caused by the stainless steel of each condition is calculated by the formula. As shown in Figure 7, the stainless steel modified with the copolymer of the present invention does not cause hemolysis.

Bioinert performance analysis of vinyl phosphate-4-vinylpyridine sulfonate betaine copolymer (poly(VPA- r -4VPPS))

The cytotoxicity of the copolymer provided by the present invention is detected according to the standard process stipulated in the regulations ISO 10993-5, and the cells used are mouse fibroblasts (L929) for the experiment, which is divided into 3 days and carried out in total. It is used for qualitative observation by microscope and quantitative detection by XTT reagent. The first day is divided into cell culture and sample toxicity extraction. Cell culture: Take out the L929 cells cultured in the cell culture incubator, wash them twice with PBS; add 1 mL of trypsin (Trypsin-EDTA) to react for 6 minutes to detach the cells on the Flask; use a microscope to observe that the cells have completely detached , add 9 mL of cell culture medium to terminate the trypsin reaction; transfer the desorbed cells to a 15 mL centrifuge tube, and centrifuge at 290 rcf for 5 minutes; remove the supernatant after centrifugation, and refill with 10 mL of cell culture Wash away the cells at the bottom of the centrifuge tube; use a cell counting disc to count the number of cells, and dilute the cells with cell culture medium; the cell concentration of the sample to be observed under a microscope is 150,000 cells/mL, add 1 mL of each to a 12-well multi-well plate Cell dilution solution was added to and cultured in a cell culture incubator for 24 hours; the concentration of cells to be detected by XTT reagent was 100,000 cells/mL, and 0.1 mL was added to a 96-well multi-well plate and cultured in a cell culture incubator for 24 hours. Sample toxicity extraction: The sample was sterilized by UV lamp for 30 minutes, and the L929 cell culture solution was extracted with stainless steel at a ratio of 6cm ² /mL, and at the same time, HDPE was extracted at a ratio of 0.2g/mL as the cytotoxicity The positive control group was extracted with ZDEC at a ratio of 0.1 g/mL, and as the negative control group of cytotoxicity, the extraction was performed at 37°C and 150 rpm. On the second day, replace the culture medium of the cultured cells on the first day with the extract of the extracted samples, add 1 mL to each 12-well plate, and add 0.1 mL to each 96-well plate, and continue to culture in a cell culture incubator for 24 hours. The third day is divided into qualitative and quantitative, qualitative: the cells in the 12-well plate were washed twice with cell culture medium, and the growth of the cells was observed under a microscope. Quantification: Mix XTT Reagent and XTT Activity Solution at a ratio of 50:1, add 51 μL of the mixed XTT Reagent to each grid of a 96-well plate, and put it in the cell culture phase at 37°C for 3 hours; The plate is read by UV-VIS for absorbance at 450 nm. The experimental results are shown in Figure 6. The qualitative analysis of the toxicity of stainless steel under various conditions and the toxicity grade converted by the XTT reagent, HDPE and ZDEC are the positive and negative control groups, and the control group Blank cultured in culture medium is defined as 100%, healthy cells are in the shape of a healthy spindle under the microscope, and are covered in the well plate, while ZDEC in the negative control group will cause cell death, and the cell shape will change from a spindle shape to a spherical shape, and suspended in solution. From the qualitative analysis of microscope and the quantitative analysis of XTT reagent, it can be known that each modified condition has a good cell shape under the microscope, and under the quantitative analysis of XTT reagent, the cell survival rate is greater than 80%, and the cytotoxicity is 0 grade, indicating that The stainless steel modified by polymer is not cytotoxic.

Thermal Stability Analysis of Vinyl Phosphate-4-Vinylpyridine Sulfonate Betaine Copolymer

This experimental example compares the pyrolysis temperature of copolymerized polymers containing 4VPPS and SBMA respectively. Weigh 5 mg of PPA30 and SBMA70, and use a thermogravimetric analysis (TGA) to measure the thermal stability of the material. The detection procedure is as follows: Maintain 120°C for 15 minutes to remove the moisture in the copolymerized polymer. After the water removal is completed, the temperature is raised to 800°C at a rate of 10°C/min. The weight of the water removal is taken as 100% by weight. The temperature at % is the thermal cracking temperature of the material. The thermal cracking temperature is measured by a thermogravimetric analyzer, and the temperature at the time of 10% weight loss is defined as the thermal cracking temperature of the copolymerized polymer. The experimental results are shown in Figure 8. The PPA30 of the present invention does not contain ester functional groups The thermal cracking temperature is 332°C, which is about 50°C higher than the 283°C of the control group SBMA70 containing ester functional groups, which proves that the copolymer structure design of the present invention can improve thermal stability. More importantly, when the pyrolysis temperature is higher than 300°C, it is more conducive to the processing of various materials, especially the surface modification of metal materials.

Analysis of Anti-Bioinert Efficacy Decay of Vinyl Phosphate-4-Vinylpyridine Sulfonate Betaine Copolymer

This experimental example uses a moist heat sterilization procedure to test and compare the copolymerization of the present invention The technical effect of anti-biologically inert performance decay that the substance has, the specific method and result are as follows.

Use PPA30, SBMA70 modified stainless steel and SBMA hydrogel for moist heat sterilization, and compare the copolymer with ester functional group and the copolymer without ester functional group by the amount of human whole blood (Human Whole Blood) attachment Differences in thermal stability properties between. Soak the modified stainless steel and water-gel control group in PBS, and perform moist heat sterilization at a temperature of 121°C for 20 minutes. Put the non-sterilized sample and the sterilized sample in a 24-well multi-well plate, and swell with PBS in an oven at 37°C for 12 hours; remove the swelled PBS, and wash once with PBS; each grid in the 24-well plate Add 1mL of whole blood, and attach the blood in a shaking oven at 37°C and 120rpm; wash the adhered samples with PBS for 3 times, move the samples to a new 24-well plate, and wash with PBS for 3 times. Fix the blood cells with glutaraldehyde in an oven at 37°C for 1 hour, and stain the blood with SYTO-9 dye; wash once with PBS, and use a Confocal Laser Scanning Microscope (Confocal Laser Scanning Microscope) , CLSM) to photograph blood cells and perform quantitative analysis with computer software. The results are shown in Figure 9. The unmodified stainless steel surface is defined as 100% of the attached amount. Obviously, the blood attached amount of the sterilized SBMA hydrogel increased by 1,633% compared with that before sterilization; the SBMA70 modified stainless steel The amount of blood attached to the surface after sterilization increased by 947% compared with the amount of blood attached before sterilization. Indicates that it loses its anti-adhesive effect after moist heat sterilization. However, the surface of the stainless steel modified by PPA30 of the present invention does not contain ester functional groups in the structure, and the amount of blood attached does not change significantly before and after sterilization. Chemicalized, indicating that its structure still retains its anti-sticking effect after sterilization. Accordingly, it is proved that the copolymer of the present invention and the formed film layer have the technical effect of resisting biological inert performance decay.

Analysis of Bioinert Performance of Interpenetrating Polymer Network Structure Formed by Vinyl Phosphate-4-Vinylpyridine Sulfonate Betaine Copolymer

The phosphoric acid group of the vinylphosphoric acid-4-vinylpyridinesulfonic acid betaine copolymer (PPA70) of the present invention and the hydroxyl group of polyvinyl alcohol (PVA) can form a covalent bond by reacting, making mutual Wear a polymer network structure, as shown in formula (3), wherein X is an integer of 20 to 400, and Y is an integer of 30 to 1500.

Formula (3)

The film or substrate with the interpenetrating polymer network structure film layer made of the above-mentioned copolymer and the copolymer and PVA is tested for biological inertness, and the experimental results are shown in Figures 10 and 11. Vinyl phosphoric acid of the present invention- The film layer coated with 4-vinylpyridinesulfonate betaine copolymer (PPA70) on metal and ceramic materials is less than 2% for Escherichia coli. And through the above interpenetrating polymer network shown in formula (2) The cell attachment amount of the polyethylene terephthalate, polypropylene, polytetrafluoroethylene or polyvinylidene fluoride film modified by the network structure is almost 0, which is lower than that of the film without modification. The plastic film modified by the interpenetrating polymer network structure represented by formula (2) has very good anti-cell adhesion properties. In particular, the polytetrafluoroethylene or polyvinylidene fluoride film modified with the interpenetrating polymer network structure shown in formula (2) showed a very good anti-adhesion effect on Escherichia coli, and its attachment amount was also close to 0. Accordingly, the film layer or material with the interpenetrating polymer network structure made of the copolymer of the present invention exhibits good anti-biological molecular adhesion effect, and is an innovative biologically inert material.

Although the present invention has been described above with specific examples, the scope of the present invention is not limited thereto. Those skilled in the art understand that various modifications or changes can be made without departing from the intent and scope of the present invention. In addition, the abstract and the title are only used to assist the search of patent documents, and are not used to limit the scope of rights of the present invention.

Claims (10)

A copolymer, the structure of the copolymer includes a first segment and a second segment, the first segment includes a plurality of phosphoric acid groups, and the second segment includes a plurality of pyridine sulfobetaine or pyridine carboxylic acid groups Betaine, and the above-mentioned first segment and second segment are connected by covalent bonds to form the copolymer. As the copolymer described in claim item 1, the chemical structure of the copolymer is as shown in formula (1):

Wherein, R is a functional group with carboxylic acid or sulfonic acid; k is a carbon chain repeated from 1 to 3; m is an integer of 5 to 500, and n is an integer of 5 to 500. The copolymer as claimed in item 2, which is vinylphosphoric acid-4-vinylpyridinesulfonate betaine copolymer, and its weight average molecular weight is less than 120kDa. The copolymer as described in claim 3, the vinyl phosphate-4-vinyl Pyridine sulfobetaine copolymer is prepared by free radical polymerization of vinyl phosphoric acid and 4-vinylpyridine sulfobetaine, the reaction of vinyl phosphoric acid and 4-vinylpyridine sulfobetaine The molar ratio ranges from about 9:1 to 1:9. A biologically inert material, comprising a film layer and a substrate, the film layer is composed of a copolymer as described in Claims 1 to 4, and is anchored by coordination covalent bonding or chemical grafting On the surface of the substrate, thereby forming said bioinert material. For the biologically inert material as claimed in item 5, the water contact angle of the film layer is about less than 60 degrees. As the biological inert material described in claim item 5, the film layer also includes an interpenetrating polymer network structure formed by polyvinyl alcohol and vinyl phosphoric acid as shown in formula (2):

Where X is an integer from 5 to 500, and Y is an integer from 5 to 7000. The biologically inert material as described in Claim 5, the base material includes stainless steel, titanium alloy, glass, silicon wafer, ceramics, polyethylene terephthalate, polypropylene, polydimethylsiloxane, polytetrafluoroethylene Vinyl fluoride or polyvinylidene fluoride. The biologically inert material as described in Claim 5 is applied to the preparation of cardiac catheters, vascular stents, endoscopes or surgical instruments. A method for preventing metal surfaces from adhering to biomolecules, the steps of which include covalently bonding the copolymer described in claims 1 to 4 on the metal surface or forming an interpenetrating polymer network structure by chemical grafting, whereby To prevent the metal surface from adhering to biomolecules, the coordination covalent bond is formed by the phosphoric acid group and the chromium contained in the metal surface.

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