TWI663190B - A zwitterionic hydrogel and application thereof - Google Patents

Abstract

The present invention provides a double ion type water gel which is composed of a monomer formed by reacting vinylpyridine and a derivative thereof and an alkyl sultone and a crosslinking agent, and the above single The concentration of the body in the polymerization is 10 to 50 wt.%. Secondly, the present invention also discloses the use of the dual-ionic water gel to prevent the decay of the bio-molecular adhesion effect of the material.

Description

Double ion type water glue and application thereof

The invention relates to a double ion type water glue, which is a double ion type water glue with high heat stability. Secondly, the present invention also discloses the use of the dual-ionic water gel to prevent the decay of the bio-molecular adhesion effect of the material.

For a long time, ideal biomedical materials require good compatibility between materials and complex biological systems. In addition to biocompatibility, it is still necessary to improve mechanical properties, thermal stability, etc., in order to provide more highly functional biomedical materials.

In general, diionic polymers have the property of resisting the adhesion of non-specific biomolecules and provide good blood compatibility. When the surface of the material is surface-modified with a double-ion polymer, the general modification reaction temperature is lower than 100 ° C. The modified interface does have excellent antifouling properties. However, many medical materials need to be formed at high temperatures. Made in the environment, such as thermoplastic and thermosetting process temperatures are usually higher than 100 ° C, even some scalpels, biochips, metal stents, etc. need to be implanted in the human body High temperature saturated steam sterilization at about 121 ° C was performed. In recent years, it has been found that general diionic polymer materials cannot retain good antifouling properties after high temperature treatment.

Document ACS Appl. Mater. Interfaces, 2015.7(19): p.10096-10107 indicates that the sulfobetaine functional group (SBMA) diionic polymer is annealed at 200 ° C and then chemically confirmed by surface chemistry. The acid group (-SO ₃ ^- ) is cleaved under high temperature conditions. Therefore, the sulfobetaine Methacrylate (SBMA) type double ion material does not have good thermal stability in a high temperature process, and loses the anti-biomolecule adhesion effect after high temperature treatment.

In order to meet the demand for high temperature treatment of materials, it is an urgent bottleneck for the development of an anti-biomolecular adhesive material with high thermal stability and excellent biocompatibility.

In summary, it is a cutting-edge technology to use diionic molecules as the main anti-biomolecule-adhesive component and to design this dual-ion structure with high thermal stability. In the future, it can be used as a heat-stable antifouling coating, a high-temperature injection material required for 3D printing technology, a metal surface modification coating, high-temperature thermosetting or thermoplastic processing, or even when the medical material is reused. The required sterilization requirements, etc., are a potential development material and an industrial subject to be studied.

In view of the above background of the invention, in order to meet the industrial needs, a first object of the present invention is to provide a double ion type water gel which is reacted with vinylpyridine and its derivatives and alkyl sultone. A polymer composed of a monomer and a crosslinking agent formed by polymerization.

In a preferred embodiment, the concentration of the above monomer in the polymerization reaction is 10 to 50 wt.%.

Specifically, the diionic water gel of the present invention is a 4-vinylpyridine propyl sulfobetaine and a crosslinker N formed by the reaction of 4-vinylpyridine and propyl sultone. , N'-Methylene bis acrylamide (NMBA) is a polymer composed of a polymerization reaction, and the monomer 4-vinylpyridinium propylamine (4-vinylpyridine) The concentration of propyl sulfobetaine in the polymerization is 10 to 50 wt.%. The thermal decomposition temperature of the double ion type water gel of the invention is up to 340 ° C or more, and the double ion type water gel can maintain excellent anti-biomolecular adhesion effect after heat treatment at 120 ° C.

In a representative example, the above-mentioned monomer 4-vinylpyridine propyl sulfobetaine (4VPPS) has the following structural formula.

A second object of the present invention is to provide a method for preventing decay of an anti-biomolecule adhesion effect of a material, which comprises providing a double-ionic water gel which is reacted with vinylpyridine and alkyl sultone. The formed monomer and a crosslinking agent are polymerized to form a polymer; and the diionic water-based glue is fixed on the surface of the material to prevent the bio-molecular adhesion effect of the material from decaying.

Specifically, the diionic hydrocolloid is a 4-vinylpyridine propyl sulfobetaine and a crosslinking agent N, N formed by the reaction of 4-vinylpyridine and propyl sultone. '-N-N'-methylene-bis-acrylamide (NMBA) is a polymer composed of a polymerization reaction, and the above-mentioned double-ionic water gel is fixed by chemical grafting. The material is designed to prevent the material from decaying due to heat treatment.

In summary, the present invention discloses a dual ion type water gel which is a monomer formed by reacting vinylpyridine and its derivatives with alkyl sultone and a crosslinking agent for polymerization. Preferably, the concentration of the above monomer in the polymerization is 10 to 50 wt.%. Place The thermal decomposition temperature of the double-ionic water gel is as high as 340 ° C or higher, and the double-ionic water gel can maintain excellent anti-biomolecular adhesion after heat treatment at 120 ° C. Secondly, the present invention also provides a method for preventing the decay of the bio-molecular adhesion effect of a material by immobilizing the dual-ionic water gel on the surface of a material to prevent the bio-molecular adhesion of the material. decay.

The above and other technical contents, features and advantages of the present invention will be apparent from the following detailed description of the preferred embodiments. In order to thoroughly understand the present invention, detailed steps and compositions thereof will be set forth in the following description. Obviously, the practice of the invention is not limited to the specific details that are apparent to those skilled in the art. On the other hand, well-known components or steps are not described in detail to avoid unnecessarily limiting the invention. The preferred embodiments of the present invention are described in detail below, but the present invention may be widely practiced in other embodiments, and the scope of the present invention is not limited by the scope of the following patents. .

According to a first embodiment of the present invention, the present invention provides a double ion type water gel which is a monomer formed by reacting vinylpyridine and a derivative thereof with an alkyl sultone and a crosslinking agent. Polymer formed by polymerization

In one embodiment, the concentration of the above monomer in the polymerization reaction is 10 to 50 wt.%.

In one embodiment, the diionic hydrocolloid is a 4-vinylpyridine propyl sulfobetaine formed by the reaction of 4-vinylpyridine and propyl sultone. a polymer formed by a polymerization reaction of a N,N'-methylene-bis-acrylamide (NMBA), in one embodiment, the monomer 4-ethylene The concentration of 4-vinylpyridine propyl sulfobetaine in the polymerization is 10 to 50 wt.%.

In one embodiment, the monomer has the following structural formula.

In one embodiment, the diionic aqueous gel is attached to the surface of a substrate by chemical grafting or coating.

In one embodiment, the substrate comprises titanium metal, tantalum, plastic, ceramic, and stainless steel.

In a specific embodiment, the dual ion type water gel system is used for preparing a thermostatic anti-molecular bio-adhesive material, and the heat-stable anti-molecular bio-adhesive material has an operable temperature range of less than 340 ° C. . Specifically, the thermostable bio-molecular adhesive material is prepared by using the double-ionic water gel, which is formed by reacting vinyl pyridine and alkyl sultone. a polymer composed of a monomer and a crosslinking agent, which is thermally stable The operational temperature range of the anti-molecular bioadhesive material is less than 340 °C.

According to a second embodiment of the present invention, the present invention provides a method for preventing decay of an anti-biomolecule adhesion effect of a material, which comprises providing a double-ionic water gel which is composed of vinylpyridine and alkylsulfonate. a polymer formed by a polymerization reaction between a monomer formed by a lactone reaction and a crosslinking agent; and a surface of the above-mentioned double-ionic water-based glue is fixed on the surface of the material to prevent the bio-molecular adhesion effect of the material from decaying.

In one embodiment, the diionic hydrocolloid is a 4-vinylpyridine propyl sulfobetaine and a crosslinker formed by the reaction of 4-vinylpyridine and propyl sultone. N,N'-methylenebisbisacrylamide (NMBA) is a polymer composed of a polymerization reaction.

In one embodiment, the concentration of the above monomer in the polymerization reaction is 10 to 50 wt.%.

In one embodiment, the monomer 4-vinylpyridine propyl sulfobetaine has the following structural formula.

In one embodiment, the method of immobilizing the above-described dual ion type water gel on the surface of a material includes chemical grafting.

In one embodiment, the material is titanium, tantalum, plastic, ceramic or stainless steel.

In a specific embodiment, the dual ion type water gel system is used for preparing a thermostatic anti-molecular bio-adhesive material, and the heat-stable anti-molecular bio-adhesive material has an operable temperature range of less than 340 ° C. .

In one embodiment, the thermally stable anti-molecular bioadhesive material is one of a coating composition of a part or all of a surface of a medical device, which is a sterilization and sterilization device, a surgical instrument, an endoscope, and a living body. Wafers, wound dressings or dental implants.

The invention is illustrated by the following detailed examples, but the experimental examples disclosed are merely preferred examples of the invention and are not intended to limit the scope of the invention.

Experimental Example 1: Synthesis of 4VPPS monomer

The synthetic reaction formula is shown in Figure 1.

4 vinylpyridine (4VP) and propyl sultone (PPS) were dissolved in DMF in a ratio of 1:2, and placed in an oil bath at 60 ° C, and the reaction was stirred for 24 hours. After 24 hours, the monomer was precipitated with acetone, purified with methanol, and the excess unreacted 4VP and PPS were removed, and then the solvent was removed to obtain a powder 4VPPS monomer.

Structure Identification of 4VPPS Monomer Hydrogen Nuclear Magnetic Resonance Spectroscopy

The use of an external RF field to provide applied energy causes a jump in the direction of the magnetic flux of the nucleus, which may also be referred to as a resonance phenomenon. A certain applied magnetic field energy is generated for the sample to be tested, and the spin precession frequency of the nucleus is detected, and the functional groups in the molecular structure are analyzed by this characteristic.

In this experiment, PPS was used to carry out ring-opening reaction to generate free radicals, and 4VPPS was synthesized by reaction with 4VP. The chemical element composition was analyzed by NMR spectrometer. According to the ¹ H NMR spectrum, the peak position of 4VP was shown in Fig. 2(a). The signal at 8.7 and 7.5 ppm is a characteristic peak of the pyridine structure. According to Fig. 2(b), the peak positions of the PPS, the signals at 4.2 and 3.1 and 2.4 ppm are the characteristic peaks of A, B and C on the PPS structure, respectively. According to Fig. 2(c), the peak position of the 4VPPS is 8.0 and 9,0ppm, and the signal of the peak of the peak is 3.75ppm, and the signal of the peak of the 3.75ppm is the tail of the 4VPPS carbon chain. Location. After 4VP and PS were synthesized into 4VPPS, the area under the peak line was calculated. From the integration result, the structure of the front-end pyridine and the structure of the terminal sulfur trioxide were consistent, which proved that the 4VPPS monomer was successfully synthesized.

Experimental Example 2: Preparation of 4VPPS water gel

In this experimental example, water gelatin of four different monomer reaction concentrations were prepared, and the monomer 4VPPS and NaCl (1M aqueous solution) were separately mixed in different proportions, wherein the weight percentage concentration of the monomer 4VPPS in the polymerization reaction was 10 wt.%, 15 wt. .%, 20wt.% and 30wt.%, after stirring for 10 minutes, add cross-linking agent (NMBA) for cross-linking reaction, and evenly stir for ten minutes, then add the initiator (APS) and stir for ten minutes. Add catalyst (TEMED), draw the mixture with a suitable syringe, inject the liquid into the water gel mold, store it in an oven at 60 ° C, perform free radical polymerization, wait 12 hours, then remove the water gel The mold was taken out, immersed in deionized water, and the water gel was cut into a circular shape with a circular cutter and stored in deionized water at 4 °C. The four water gels obtained in this experimental example are represented by the codes of 4VPPS10, 4VPPS15, 4VPPS20, and 4VPPS30, respectively.

Experimental Example 3: Preparation of HEMA, 4VP water gel

The monomer HEMA and 4VP were mixed with methanol in different proportions, and uniformly stirred for ten minutes. Then, a crosslinking agent (NMBA) was added for crosslinking reaction, and the mixture was uniformly stirred for ten minutes, and then the initiator (APS) was added and stirred. After a minute, immediately add the catalyst (TEMED), draw the mixture with a suitable syringe, inject the liquid into the water gel mold, store it at room temperature, perform free radical polymerization, wait for 1 hour, then remove the water gel from The mold was taken out, immersed in deionized water, and the water gel was cut into a circular shape with a circular cutter and stored in deionized water at 4 °C.

Experimental Example 4: Preparation of PEGMA, SBMA water gel

The monomer PEGMA and SBMA were mixed in different proportions and mixed with deionized water. After uniformly stirring for ten minutes, a crosslinking agent (NMBA) was added for crosslinking reaction, and evenly stirred for ten minutes, then the initiator (APS) was added. After stirring for ten minutes, immediately add the catalyst (TEMED), draw the mixture with a suitable syringe, inject the liquid into the water gel mold, store it at room temperature, perform free radical polymerization, wait for 1 hour, then put the water The glue was taken out of the mold, immersed in deionized water, and the water gel was cut into circular shapes with a circular cutter and stored in deionized water at 4 °C.

Thermal analysis of water gel

The DSC is used to measure the relationship between the temperature and the heating rate at the same temperature between the sample to be tested and the reference sample. During the heating process, the sample will undergo a phase change, so that the correct sample glass transition temperature, melting temperature, etc. can be obtained. Temperature parameter.

Using TGA and DSC to carry out the thermal properties analysis of the water gel prepared in the above experimental examples, it can be seen from Table 1 that except for Poly (HEMA), the Tg points of the other water gels are higher than 100 ° C, and it can be seen from Table 1 The higher the molecular weight of Poly(4VPPS), the higher the Tg and Tm points, and the temperature parameters of Poly(SBMA), which is also a diionic polymer, are quite close. When the temperature continues to rise and reaches the Td point, it can be found that the molecular weight of Poly (4VPPS) does not affect the Td parameter value, and the thermal cracking condition is observed by Td. The thermal stability of Poly (4VPPS) is also better than that of Poly (SBMA). High; Poly (4VP) also has good thermal stability. It is inferred that Poly (4VPPS) having a pyridine structure increases the steric hindrance of the molecule, and therefore has a characteristic that it is difficult to be cleaved compared to the linear structure of Poly (SBMA), and thus has high thermal stability.

In Table 1, Mw* represents the theoretical molecular weight; Tg represents the glass transition temperature; Tm represents the melting temperature; and Td represents the thermal decomposition temperature.

Water gel surface contact angle measurement

Place the water gel in a clean and transparent small glass container filled with deionized water, place it on the surface of the surface contact angle measuring instrument, use diiodomethane as the test liquid, and draw 4 μl onto the surface of the water gel. Observing again, the hydrophobicity of the surface of the water gel was obtained. The greater the contact angle, the more hydrophilic the surface of the material; the more hydrophobic it is.

In order to clearly compare the hydrophilic and hydrophobic physical properties of the 4VPPS water-based rubber material, the monomer was made into a network-like polymer monohydrate, and the surface contact angle measuring instrument was used to analyze the water gel and other monomer components of different 4VPPS monomer ratios. The difference in hydrophilicity between water gels and whether there is a difference in physical properties of water gel after heat treatment at 121 °C. However, the environment in which the water gel measures the contact angle is different from the measurement method of the contact angle of the general air phase. The common water gel is a hydrophilic material. Therefore, if the contact angle is measured by using the water droplet, the water absorption is too fast and the measurement cannot be accurately performed. Contact angle. Therefore, this experiment uses diiodomethine titration to conduct contact angle test in the aqueous phase. When the contact angle is larger, it means that the surface of the water gel is more hydrophilic; otherwise, the more hydrophobic. When the water phase contact angle is greater than 100 degrees, it indicates that the surface of the material has good hydrophilicity.

It can be seen from Fig. 3 that the higher the 4VPPS content of the water gel before heat treatment, the higher the hydrophilic property. The monomer 4VP material which is not converted into a double ion is relatively hydrophobic, so the measurement result is also relatively hydrophobic. In the other control groups, the double-ion SBMA water gel was the most hydrophilic, but in fact the contact angles of the different generations of antifouling materials were very close. However, after heat treatment, the hydrophilic properties of all materials decreased, especially PEGMA water gel. After heat treatment, the decrease was the highest. The reason can be explained by the literature. When the water gel is heated at high temperature, the original cross-linking will be improved. Therefore, the physical structure of the water gel is changed, thereby affecting the hydrophilic property.

Water gel swelling test

The human body needs water to be a physiological function. Many human organs need to have a water-rich environment. However, water glue has become a good medical material, and the biggest feature is high water content. The application of water gel is as an example: water gel can be made into contact lenses, and the surface of human eye contains a certain amount of water layer. Water-filled water is used as a contact lens, except that it can bring enough moisture to the eyeball. The water layer on the surface of the eyeball is absorbed, and sufficient water can also provide oxygen dissolved in the water, so that the oxygen content of the eyeball is increased, and the contact lens is worn for a long time without causing dry eye discomfort. Therefore, the water content test of water gel is quite important. Using the difference in weight between the dry and wet state of the water gel, a test for the water swelling of the water gel can be obtained.

Water gel swelling test method

Soak the water gel in deionized water for one day, take out the water gel, use the filter paper to dry the excess moisture on the surface of the glue, then weigh it (Weight _W ), then use the vacuum oven to dry the glue for 1 day. It can be weighed (Weight _D ), and the calculation formula of water gel swelling degree is as follows.

As shown in FIG. 4, the test results of the swelling degree of the water gel prepared in the above Experimental Examples 2 to 4 have a higher degree of swelling of the virgin gel before the heat treatment, and the sterilized gel after the heat treatment. Usually, the crosslinking reaction is promoted to reduce the degree of swelling; in addition, it is known from Fig. 4 that when the 4VPPS content is increased to 30%, the swelling degree is maintained after heat treatment, but the conventional PEGMA water gel is promoted by heat treatment. The traditional water gel is crosslinked, and its swelling degree is drastically reduced to 200%. Accordingly, it was confirmed that the 4VPPS water gel of the present invention has an unpredictable effect compared to the conventional application of PEGMA water gel for eye care and maintenance.

Scanning electron microscope surface analysis

The SEM is used to measure the image of the sample to provide a high-resolution image of the surface of the sample, which can identify the fine structure and the surface type of the object, and its resolution can reach the nanometer scale. Thereby, the size of the hole, the distribution of the holes, and the like on the surface of the water gel can be detected.

In this analysis, the physical structure of the surface of the material was observed by scanning electron microscopy (SEM) at an accelerating voltage of 15 kV and a magnification of 3 K. The pore size and distribution of the surface of the water gel prepared in each of the above experimental examples can be clearly observed. The degree can reach the nanometer scale, and the image of the image is shown in Fig. 5(A)~(H). The surface morphology of the water gel before the high temperature heat treatment at 121 °C can be seen, as shown in Fig. 5(E)~(H). When the 4VPPS content is higher, the cross-linking degree of the hydrogel decreases, so many pores are formed on the surface. The larger the pore size, the more the number of pores increases, while the other control groups have a dense surface before reheating. Therefore, the values of the contact angles are quite close.

Next, as shown in Fig. 6 (a) to (h), it is a scanning electron microscope image obtained by subjecting the water gel prepared in each of the experimental examples to a high temperature of 121 ° C. As shown in Fig. 6(e)~(h), the 4VPPS water gel originally filled with holes also began to become dense, thus verifying that high temperature heating helps to increase the degree of crosslinking and form a dense surface, causing the water gel to swell. Degree drops. As shown in Fig. 6(c), the surface structure of the PEGMA water gel is cracked, so that after heating at a high temperature, the hydrophilicity and swelling degree of the PEGMA water gel are lowered. The surface morphology of 4VP which has not been converted into double ions is almost unchanged before and after heating; however, the surface morphology of HEMA water gel and traditional double ion PSBMA water gel is high. The situation in which cracks are broken after the occurrence is as shown in Fig. 6(b) and Fig. 6(d). It is thus proved that the 4PGPS water gel phase of the present invention has unpredictable efficacy compared with the conventional double ion PSBMA water gel in an operating environment requiring high temperature treatment, so the water gel of the 4VPPS of the present invention is a novel heat stable. Sexual dual-ionic water gel.

Application experiment of water gel antibiotic bioadhesive

Plasma separation

The whole blood was placed in a centrifuge tube, placed in a centrifuge, the rotation speed was adjusted to 3000 rpm, the acceleration was set to 5, the deceleration was set to 2, and the centrifugation time was set to 10 minutes. After the end of the centrifugation, there was significant stratification in the centrifuge tube. The upper clarified liquid was platelet-poor plasma (Platelet Poor Plasma, PPP), and the concentration was diluted with PBS.

Red blood cells and platelets are separated

The whole blood was placed in a centrifuge tube, placed in a centrifuge, the rotation speed was adjusted to 1200 rpm, the acceleration was set to 5, the deceleration was set to 2, and the centrifugation time was set to 10 minutes. After the end of the centrifugation, there is obvious stratification in the centrifuge tube. The upper layer of liquid is platelet-rich plasma (Platelet Rich Plasma, PRP), and the lower layer of red liquid is red blood cell thick solution.

White blood cell separation

After uniformly mixing 10 ml of whole blood with 10 ml of DPBS buffer, slowly add a 10 ml mononuclear cell gradient separation solution (Ficoll-Paque) to the centrifuge tube and avoid mixing the blood with Ficoll-Paque. Otherwise, it is not easy to effectively separate white blood cells. After that, it was placed in a centrifuge, the rotation speed was adjusted to 400 rpm, the acceleration was set to 2, the deceleration was set to 0, and the centrifugation time was set to 30 minutes. After that, you can get obvious blood stratification. From top to bottom, it is plasma, mononuclear leukocyte, Ficoll-Paque, red blood cell thick liquid.

It can be seen from Fig. 7 that the best anti-erythrocyte adhesion effect is that the 4VPPS content is 30% and the SBMA water gel is not heat-treated, so the stable double ion structure is beneficial to the anti-erythrocyte adhesion, and the 4VPPS water gel is heat treated. The surface structure is denser and therefore exhibits better resistance to sticking. However, after heat treatment, the charge of SBMA is unstable and it is impossible to maintain good electrical neutrality. Therefore, a negatively charged red blood cell produces a large amount of attached SBMA water gel after heat treatment. However, it can be proved from the past literature that although HEMA and PEGMA are also good anti-adhesive water-adhesive materials, HEMA water gel is easy to cause adhesion problems with blood cells, while PEGMA water gel is easy to be oxidized, especially after heat treatment. The surface structure has been destroyed, so the amount of red blood cells attached is also high. The 4VP water gel does not have anti-sticking ability before and after heat treatment.

Blood cell attachment test

The blood cell type to be tested was placed in a 37 ° C oven for 30 minutes, and the sample to be tested was washed three times with PBS buffer. After the constant temperature was over, 800 μl of blood cells were added to each sample and placed in a 37 ° C oven. After standing for 1 hour, the blood in the sample was taken out and washed several times with PBS buffer, and then 1 ml of 2.5 wt% Gludaric dialdehyde was added to each sample, and observation and photographing were performed after 2 hours.

Bacterial attachment test

The culture liquid of the cultured bacteria is added to the sample to be tested, placed in a culture oven at a constant temperature of 37 ° C, and the rotation speed is 100 rpm, and attached according to the set attachment time (this experiment was attached for 3 hours, 24 times respectively). Hours), after the end of the attachment time, the bacterial solution was removed and washed three times with deionized water to remove unattached bacteria.

Cell attachment test

The cultured cells (HT1080) cell solution (20000 cells/ml) were added to the sample to be tested, placed in a culture oven at 37 ° C, and attached according to the set attachment time (this experiment was attached for 1 day). , 3 days), after the end of the attachment time, the cell liquid was removed and washed three times with PBS buffer to remove unattached cells.

Cytotoxicity test

The procedure is as follows: (a) The cultured cells (L929) cell solution (15000 cells/ml) are placed on a clean 12-well plate (Tissue Culture Well Plate), placed in a culture oven at 37 ° C, and cultured for one day. (b) Preparation of sample extraction liquids, according to ISO 10993-5 according to different sample properties have different ways of extracting liquid configuration. Preparation of standard extract liquid, with HDPE as negative control (0.2 g/ml); ZDEC powder (0.1 g/ml) was used as a positive control group. After all the extraction liquids are configured, place them in a shaker, set the temperature to 37 ° C, set the rotation speed to 150 rpm, and mix the sample with the culture solution for one day; (c) After the next day, place the cells on the 12-well plate. The liquid is removed, and the extracted sample and the standard extract are added, and then the cells are cultured at a constant temperature of 37 ° C in a culture oven; (d) after the third day, the extract on the 12-well plate is removed. The cells were washed once with PBS buffer, and then a new culture solution was added, after which the cells were qualitatively observed; and (e) the cytotoxicity was judged based on the difference in cell type and number between the sample and the control group. .

Blood cell attachment test results

The test results are shown in Fig. 7. When the content of 4VPPS reaches 30%, it can effectively resist the adhesion of blood cells, and the high temperature treated 4VPPS water gel can improve the antifouling effect. However, HEMA, PEGMA and SBMA water gels are lost after heat treatment. Anti-adhesive ability, attached to a variety of blood cells. Therefore, using the whole blood attachment experiment, it is possible to clearly understand the various blood cells attached to the medical material in the real human blood environment.

As shown in Figure 8, PEGMA and SBMA water gels exhibit anti-platelet adhesion properties before heat treatment. However, when 4VPPS water gel is not heat treated, large pores are formed on the surface. Therefore, platelet adhesion test, platelets Since the diffusion effect easily enters the deep layer of the water gel and is not easy to discharge, a large amount of adhesion is formed. However, after the heat treatment of the 4VPPS water gel, the surface structure is more dense, which can effectively block the platelet from entering the interior of the water gel, thus exhibiting good resistance. Adhesion ability. However, after heat treatment, the charge of SBMA is unstable, it is unable to maintain good electrical neutrality, and it is easy to stimulate platelet activation. A high stickiness occurs. However, it can be proved from the past literature that HEMA and PEGMA are also good anti-adhesive water-adhesive materials, but HEMA water gel is easy to stick with blood cells, and PEGMA is easy to oxidize. Especially after heat treatment, the surface structure of water gel has been Damaged, rough surface makes the amount of attachment high. The 4VP water gel does not have anti-sticking ability before and after heat treatment.

The white blood cell attachment experiment uses a mononuclear ball separation solution (Ficoll) to separate white blood cells, and the whole blood is taken out of a white blood cell thick liquid of lymphocytes and mononuclear cells. In this experiment, a conjugated-focus laser scanning electron microscope was used to detect the type of white blood cells attached to the surface of the water gel.

As shown in Fig. 8, when the 4VPPS content is more, the anti-white blood cell adhesion effect is better. When the 4VPPS content reaches 30%, the ultra-low adhesion rate can be achieved, and the high-temperature treated 4VPPS water gel strengthens the anti-sticking effect. Stickiness, however, 4VP, which has not been converted to double ions, does not have anti-adhesion ability, so the number of white blood cells attached is high. The past literature also records that HEMA does not exhibit good anti-adhesion effect on blood cells, so Before and after heat treatment, HEMA water gel has a high number of white blood cell attachments. PEGMA water gel is heated at high temperature, easily oxidatively decomposed, and reduces the ability to resist white blood cell adhesion. The white blood cells have a negative charge, so the SBMA hydrogel charge after high temperature treatment is unstable, causing a large number of white blood cells to attach.

Bacterial attachment test results

E. coli

Escherichia coli, scientific name: Escherichia coli, is a Gram-negative Brevibacterium, which has a negative electric charge. It is a common bacteria in the intestines of humans and animals. Its shape is obtuse at both ends, and its diameter is about 1-2μm. Feces are released and spread to the environment and can survive in the human body.

As shown in Fig. 9, it can be observed that E-Coli is attached for 3 hours, and the higher the content of 4VPPS without heat treatment, the less the amount of E-Coli attached, except for 4VP water gel. Other commonly used anti-adhesive materials show superior anti-staining ability. However, after high-temperature heat treatment at 121 °C, the number of bacteria attached to 4VPPS water gel is significantly reduced. On the contrary, other control groups lose their original anti-adhesion due to heat treatment. characteristic. In particular, after the heat treatment of SBMA water gel, the double ionic structure begins to be unstable, so the amount of bacterial adhesion is greatly improved. However, after attaching for 3 hours, only the condition of E. coli adhering to the surface of the material can be observed. If the experiment is extended to 24 hours, the bacteria grow or aggregate after the Escherichia coli is attached, and finally the biofilm is formed. On the surface of the material. If the surface of the material has a biofilm or a large amount of bacteria, the material cannot continue to exhibit its functionality, and even cause health damage to the human body. Therefore, it can be seen from Fig. 9 that the 4VPPS water gel can exhibit excellent antibacterial adhesion properties for the bacterial test after 24 hours of heat treatment before and after heat treatment; the opposite heat resistance of HEMA water gel after heat treatment The adhesion characteristics are the worst. From the SEM image of Fig. 6-(b), it can be observed that the surface of the water gel has been destroyed. There are many cracks on the original dense surface. The colony accumulation can be clearly observed from Fig. 9; while SBMA is heated by high temperature. This leads to the destruction of the isoelectric structure of the molecule, which causes the negatively charged E. coli to produce a large amount of stickiness.

Stenotrophomonas maltophilia, scientific name: Stenotrophomonas maltophilia; S.M

The strain of Streptomyces maltophilia is a Gram-negative bacterium with a negatively charged charge and flagella, which is less than 1 μm in diameter and is persistent in human infectious diseases and difficult to treat, and is found in waters, soils and plants. It is ubiquitous and has a profound impact on human health.

It can be seen from Fig. 10 that S.M is attached for 3 hours. Before the heat treatment, the higher the content of 4VPPS, the less the amount of S.M attached, except for the 4VP water gel which is not double ionized. The materials all show superior anti-fouling ability, similar to the results of attaching E. coli, but the traditional SBMA water gel after heat treatment, the double ionic charge is unstable, resulting in a large amount of bacterial attachment on the surface of SBMA water gel.

In order to observe the amount of bacterial attachment over a long period of time, the attachment time was extended to 24 hours to form colonies or biofilms on the surface of the material. Therefore, it can be seen from Fig. 10 that the 4VPPS water gel has a bacterial adhesion after the bacteria are attached for 24 hours, but if the 4VPPS content is increased and the heat treatment is performed, the amount of the bacteria attached can be lowered. On the contrary, SBMA water gel almost completely lost its anti-sticking effect after heat treatment, and many bacterial attachments were observed on the surface. Therefore, the 4VPPS content is increased and 4VPPS water gel after heat treatment has an unpredictable effect on preventing bacterial adhesion.

Cell attachment test results

In this experiment, the attached cell-human fibrosarcoma cell (HT1080) was used. This type of cell can spontaneously fluoresce, and the fluorescence microscopy can be used to observe the attachment of cells to water gel, which is widely used in biomedical research. Under normal conditions, the cells will begin to attach to the surface of the material after 6 hours and will be attached after 12 hours. From Fig. 11, it can be observed that the cells are attached to the water gel for one day, and it is found that the more the 4VPPS content in the 4VPPS water gel, the more the cell attachment is reduced, and the heat-treated water gel can also reduce the number of attachments, and 4VP The surface of the water gel is more hydrophobic, resulting in a large amount of cell attachment. HEMA, PEGMA, and SBMA water gels also exhibited excellent anti-cell adhesion characteristics before heat treatment. However, after heat treatment, there was obvious cell attachment. When the cell attachment time is extended to the fifth day, the cell proliferates after cell differentiation, so the number of cells increases under normal conditions until the cell growth environment is unfavorable to cell growth or reaches the apoptotic phase. A lot of death. It can be observed from Fig. 11 that most of the cell attachment amount is increased, except that the 4VPPS content is 30% of the water gel and is not heated. The treated SBMA water gel is in a state of almost zero attachment, and the rest has a significant increase in the amount of adhesion, especially HEMA, PEGMA, SBMA water gel which has lost the anti-sticking ability after heat treatment. However, when the negative charge of the material gradually increases, the cell attachment pattern changes from round to shuttle length or polygon, and the number of cell attachments increases as the charge increases, so the SBMA water gel is heated and the molecule is negative. Charge generation, causing the amount of cell attachment to rise

ISO 10993-5 cytotoxicity test

All biomedical materials must be tested for toxicity before being used in humans, and their safety can be determined to be open to humans. Therefore, the final test of medical materials is usually one of the most important tests. This toxicity test is to use cytotoxicity to detect the compatibility between materials and cells. When cells are exposed to toxic substances released by materials, unnatural cell death or inhibition of cell growth occurs. In addition to the specific morphology of healthy cells, the number of growth must be considered. Therefore, in addition to using the microscope to observe the cell type, the number of cells needs to be calculated. In order to optimize the accuracy of this experiment, the cell viability test is also used. The method (XTT assay), which performs cell quantitative analysis, can accurately calculate the cell survival rate. When the cell survival rate is lower, it means that the material toxicity is strong.

When the water gel is in contact with the cell culture solution for one day, if the extract contains any toxicity, it will cause cell death or inhibit cell growth. Figure 12 shows HEMA, PEGMA, SBMA water gel, whether or not heat treated, cell type The state of the cells is intact, and the cell survival rate in the XTT test is also very high. Therefore, it is judged that HEMA, PEGMA, and SBMA water gel have no toxicity. The cytotoxicity of the unheated 4VPPS water gel has a cell coverage of about 85%. The test results in the XTT test, as shown in Figure 13, have a cell viability of 83%. . although The coverage and survival rate of the heat-treated 4VPPS water gel increased, but it was still in the range of primary toxicity. Since 4VPPS water gel contains 4VPPS monomer which is not completely reacted, a small amount of 4VPPS monomer is dissolved in the cytotoxic extract, resulting in primary toxicity. The first-grade toxicity is the acceptable range of toxicity in biomedical materials. The results are shown in Table 2.

Medical component application

The double-ionic water gel with pyridine structure of the invention can be widely used in the process of thermoplastic type, thermosetting type medical material and medical material due to good blood compatibility, bio-molecular adhesion and high thermal stability. Use high temperature sterilization to remove all biomolecules from the medical material.

In order to further apply the 4VPPS novel dual ion water gel of the present invention to the biomedical industry, the 4VPPS water gel material of the present invention can be directly grafted on a metal material, such as stainless steel metal commonly used in surgical instruments, and grafted on the surface. 4VPPS molecules, which make the metal surface have good biocompatibility, and can use high temperature sterilization to purify surgical equipment without restriction and reuse. In addition to surgical equipment, it can also be applied to bio-wafers. Generally, wafers are hot-pressed, and feature components such as polymer materials are placed in the machine. The bio-wafer is finally implanted into the living body, so it is also necessary to consider Biocompatibility, while hot pressing is usually higher than 100 ° C, such a process must not affect its biocompatibility, so the grafting of 4 VPPS water gel to the silicon wafer can maintain biocompatible properties. In the field of medical treatment, another cutting-edge technology, such as tumor ablation technology, also known as electrocautery treatment, accurately inserts a very fine electrode needle into the tumor area. When the treatment starts, the needle emits radio frequency waves, which are generated by ion agitation. The heat energy will increase the temperature by about 60 to 100 ° C in the treatment area. Once the temperature reaches a high temperature, the tumor cells will be necrotic and the tumor removal effect will be achieved. Such a technology is bound to pay attention to biocompatibility and thermal stability. The titanium alloy commonly used in electric burning equipment, whether it is an electric burning needle or an electric burning knife, can be surface-modified with 4VPPS water glue. Improve the biocompatible stability of this medical device. Accordingly, the present invention grafts the water gel of Experimental Example 2 onto different substrates, and performs various attachment experiments on various biomolecules. The branching method comprises a physical method and a chemical method, and the attaching test step of each biomolecule is as in the respective attaching test steps of the water gel according to the present invention.

The test results are shown in Fig. 14. When 4VPPS water gel is grafted with various metals and tantalum wafers, it is attached to various biomolecules and compared with the same antifouling double ion material SBMA water gel. Differences before and after heat treatment. It can be seen that the 4VPPS water gel is grafted on the metal and tantalum wafers, and it exhibits excellent biocompatibility regardless of heat treatment. However, the metal and tantalum wafers modified by SBMA water gel cannot withstand heat treatment. Consistent with previous water gel and biomolecular attachment experiments, 4VPPS water gel can maintain a perfect anti-sticking effect.

In summary, the heat-stable double-ion water gel provided by the present invention has the following characteristics and effects: (1). Since 4VPPS water gel has a stable pyridine structure, poly(4VPPS) exhibits good thermal stability and cracking. The temperature can reach 340 °C. (2). The sulfonate structure of the hydrophilic group in the VPPS water gel makes it have good hydrophilic properties, and it is tested in water hydration and moisturizing, which proves that it has strong water absorption and water lock, which is beneficial to the application of water in water absorption. (3). The 30% content of 4VPPS water gel can exhibit the best anti-adhesive properties, especially after heat treatment, the anti-fouling ability of 4VPPS water gel is greatly improved, while HEMA, PEGMA, SBMA water gel is heat treated. Loss of anti-stick properties. (4). 4VPPS water gel grafted on different metals and tantalum wafers, even after receiving high temperature and heat, it can maintain biocompatibility, and can be applied to scalpels, biochips, electric appliances, and even new technologies in the future. Develop a 3D printing, using high The molecular thermoforming type and the thermosetting type are made into medical materials, so that the medical materials can be personalized and have good biocompatibility after repeated use.

The present invention has been described with reference to the particular embodiments of the invention, and the scope of the invention is not limited thereto, and it is understood that various modifications and changes can be made without departing from the spirit and scope of the invention. In addition, the abstract sections and headings are only used to assist in the search of patent documents and are not intended to limit the scope of the invention.

1 is a reaction synthesis formula of Experimental Example 1 of the present invention; FIG. 2 is a hydrogen-NMR spectrum of 4VPPS prepared in Experimental Example 1 of the present invention, wherein FIG. 2(a) ¹ H NMR spectrum of 4VP in D-MeOH; 2 (b) is a PPS D-MeOH in ¹ H NMR spectrum, and FIG. 2 (c) is in 4VPPS ¹ H NMR spectrum in D ₂ O; Figure 3 is an embodiment of the present invention, the water gel test prepared by two to four A comparison chart of the effects of high temperature treatment on the contact angle of the surface; FIG. 4 is a comparison diagram of the strips affected by the high temperature treatment of the water gel prepared in Experimental Examples 2 to 4 of the present invention; FIG. 5 is an experimental example 2 of the present invention. Electron microscopy (SEM) image of four prepared water gels, wherein Figure 5 (A) is 4VP water gel; Figure 5 (B) is HEMA water gel; Figure 5 (C) is PEGMA water gel; Figure 5 (D) ) is SBMA water gel; Figure 5 (E) is 4VPPS10 water gel; Figure 5 (F) is 4VPPS15 water gel; Figure 5 (G) is 4VPPS20 water gel and Figure 5 (H) is 4VPPS30 water gel; SBA-15 Fourier infrared spectrum of size porous coffin (black line), SBA-15_NH(CH ₂ ) ₂ NH ₂ _Ag composite (dashed line) and SBA-15_NH ₂ _Ag (dotted line) composite; FIG. 6 is an experiment of the present invention The water gel prepared in Examples 2 to 4 was subjected to a high temperature of 121 ° C. Scanning electron microscope image, wherein Fig. 6(a) is 4VP water gel; Fig. 6(b) is HEMA water gel; Fig. 6(c) is PEGMA water gel; Fig. 6(d) is SBMA water gel; Figure 6 (e) is 4VPPS10 water gel; Figure 6 (f) is 4VPPS15 water gel; Figure 6 (g) is 4VPPS20 water gel and Figure 6 (h) is 4VPPS30 water gel; Figure 7 is the experimental example 2 of the present invention A conjugated-focus laser scanning micrograph attached to the red blood cells of the water gel and the whole blood; FIG. 8 is a conjugated-focus laser scanning microscope image of the water gel platelets and white blood cells attached to the experimental examples 2 to 4 of the present invention. Figure 9 is a conjugated-focus laser scanning micrograph of E. coli attached to water gel of Experimental Examples 2 to 4 of the present invention for 3 hours and 24 hours; Figure 10 is a water gel of Experimental Examples 2 to 4 of the present invention. A 3-hour and 24-hour conjugated-focus laser scanning micrograph is attached to the Stenotrophomonas maltophilia; FIG. 11 is a conjugate of a cell paste of the water gel of Experimental Examples 2 to 4 of the present invention for one day and five days. a laser scanning scanning micrograph; FIG. 12 is a cell distribution and a microscopic image of the cytotoxicity of the water gel of Experimental Examples 2 to 4 of the present invention; FIG. 13 is an experimental example 2 to the present invention. A comparison chart of the cell viability of the water gel cytotoxicity XTT test of the water gel of the fourth embodiment; and FIG. 14 is a biomolecule attachment test of the metal and tantalum wafer material after the surface modification of the water gel of the experimental example of the present invention. Yoke-focus laser scanning microscope.

Claims (9)

A thermostable bio-molecular adhesive material is prepared by using a double-ionic water gel which is a monomer formed by reacting vinyl pyridine and alkyl sultone and The polymer formed by the polymerization of the crosslinking agent has an operable temperature range of less than 340 ° C for the thermally stable anti-molecular bio-adhesive material. The invention relates to a method for preventing decay of anti-biomolecule adhesion effect of a material, which comprises providing a double-ionic water-based glue which is a monomer formed by reacting vinylpyridine and alkyl sultone and a cross-linking The polymer formed by the polymerization of the crosslinking agent; and the surface of the above-mentioned double-ionic water-based glue is fixed on the surface of the material to prevent the bio-molecular adhesion effect of the material from decaying. The method for preventing the bio-molecular adhesion effect of the material from decaying according to the scope of claim 2, wherein the double-ionic water-based glue is a monomer 4-vinylpyridine formed by the reaction of 4-vinylpyridine and propyl-sultone. A polymer composed of a polymerization reaction of 4-vinylpyridine propyl sulfobetaine and a crosslinking agent N, N'methylene-bis-acrylamide (NMBA). The method of preventing the decay of the anti-biomolecular adhesion effect of the material according to the scope of claim 2, wherein the monomer has the following structural formula: The method for preventing the biomolecule adhesion effect of the material from decaying as described in Patent Application No. 2, wherein the concentration of the monomer in the polymerization reaction is 10 to 50 wt.%. The method for preventing the anti-biomolecular adhesion effect of the material from decaying as described in Patent Application No. 2, wherein the method of fixing the above-mentioned diionic water-based glue to a surface of a material comprises chemical grafting. A method for preventing the bio-molecular adhesion of a material from decaying as described in Patent Application No. 6, which is titanium, tantalum, plastic, ceramic or stainless steel. The method for preventing the bio-molecular adhesion of a material from decaying according to the scope of claim 2, wherein the dual-ionic water-based adhesive is used for preparing a thermally stable anti-molecular bio-adhesive material, which is thermally stable. The operational temperature range of the anti-molecular bioadhesive material is less than 340 °C. The method of preventing the bio-molecular adhesion of a material from decaying as described in Patent Application No. 8, wherein the heat-resistant molecular anti-molecular bio-adhesive material is one of coating compositions of a part or all of a surface of a medical device. The medical device is a sterilization and sterilization device, a surgical instrument, an endoscope, a biochip, a wound dressing or a dental implant device.