KR102334955B1 - Poly(2-Methacryloyloxyethyl Phosphorylcholine)-Functionalized Hydrogel and Lens Using the Same - Google Patents

Abstract

In the present invention, the surface of a hydrogel using poly(hydroxyethylmethacrylate, p(HEMA)) as a main material is poly(2-methacryloyloxyethyl phosphorylcholine)(p(MPC)). It relates to a modified hydrogel and a lens composed of the hydrogel, and by modifying the surface of the hydrogel to p (MPC), it is possible to reduce the adsorption of proteins and bacteria to improve antifouling properties and to produce a hydrogel lens with excellent antifouling properties. .

Description

Poly(2-Methacryloyloxyethyl Phosphorylcholine)-Functionalized Hydrogel and Lens Using the Same}

The present invention relates to a hydrogel modified with poly(2-methacryloyloxyethyl phosphorylcholine) (p(MPC)) and a hydrogel lens composed of the hydrogel, more particularly, p(MPC) It relates to a hydrogel capable of reducing the adsorption of proteins and bacteria by giving functionality to the surface of the hydrogel using the hydrogel, and to a hydrogel lens with improved antifouling properties manufactured using the hydrogel.

Recently, functional biomaterials having various types of biocompatibility and antibiofouling properties have been developed. In particular, when biomaterials are prepared as hydrogels to be applied to biomedicine or industry, the hydrophobic polymer network having a three-dimensional network structure is useful because it is possible to capture a large amount of water and biological fluid. In particular, hydrogels made of poly(2-hydroxyethyl methacrylate) (p(HEMA)) are biomedical materials such as urinary catheters, neural tissue devices, dental adhesives, implantable drug delivery systems, contact lenses, and intraocular lenses. is being used as

This conventional hydrogel has a problem in that it is difficult to use as a biomaterial for insertion by attaching a protein and other foulants. This is because the adsorption of proteins and biomolecules causes various unwanted biological reactions such as inflammation, blood coagulation, biofilm formation, cell adhesion, bacterial adhesion, and bacterial infection. Therefore, there is a demand for the development of a new type of biomaterial capable of preventing non-specific protein adsorption, bacterial adhesion, and colony formation.

According to this demand, studies on surface modification using polyethylene glycol (PEG), polysaccharides, biomimetic zwitterionic polymers, etc. as secondary hydrophobic polymers are being conducted. Among these polymers, poly(2-methacryloyloxyethyl phosphorylcholine) (p(MPC)) is expected to have an excellent effect as an antifouling biomaterial. Since the cell membrane outer surface is rich in phospholipids, in particular, phosphatidylcholine, a polymer material containing MPC is expected to be useful as an antifouling agent.

The present invention has been devised in view of the prior art as described above, and an object of the present invention is to provide a hydrogel surface-modified with p(MPC).

In particular, an object of the present invention is to provide a hydrogel with improved antifouling properties by reducing the adsorption of proteins and bacteria.

Another object of the present invention is to provide a hydrogel contact lens obtained by molding the hydrogel.

The hydrogel modified with poly(2-methacryloyloxyethyl phosphorylcholine) of the present invention for solving the above problems is poly(hydroxyethylmethacrylate, p(HEMA)) as the main material It is characterized in that the surface of the hydrogel to be modified with poly (2-methacryloyloxyethyl phosphorylcholine) (p (MPC)).

At this time, the p (HEMA) hydrogel is prepared by dissolving ethylene glycol dimethacrylate (EGDMA) and azobisisobutyronitrile (AIBN) in hydroxyethyl methacrylate (HEMA) monomer to prepare a mixed solution, the It may be prepared by injecting the mixture into a mold and heating to prepare a polymer, and washing the polymer to prepare a hydrogel.

In addition, the hydrogel contact lens according to the present invention is characterized in that it is a hydrogel contact lens manufactured by molding the hydrogel modified with the poly(2-methacryloyloxyethyl phosphorylcholine).

The hydrogel surface-modified with p(MPC) according to the present invention exhibits the effect of improving antifouling properties by reducing the adsorption of proteins and bacteria by modifying the surface of the hydrogel with p(MPC).

In addition, by manufacturing a hydrogel lens using the hydrogel, it exhibits the effect of providing a lens excellent in antifouling properties.

1 is a schematic diagram showing a process for preparing a surface-modified hydrogel according to the present invention.
2 is a result of measuring the permeability of the hydrogel sample.
Figure 3 is a contact angle test results for the hydrogel sample p (HEMA) (a), Example 1 (b), Example 2 (c), Example 3 (d) is the measurement result.
4 is a result showing the protein adsorption per lens (a) and the relative protein adsorption rate (b) as the protein adsorption test results for the hydrogel sample.
5 is a result of a bacterial adsorption test for the sample of Example 3.

Hereinafter, preferred embodiments according to the present invention will be described in detail. However, the following examples are provided so that those of ordinary skill in the art can fully understand the present invention, and can be modified in various other forms, and the scope of the present invention is limited to the examples described below it is not going to be

The hydrogel modified with poly(2-methacryloyloxyethyl phosphorylcholine) according to the present invention is the surface of the hydrogel using poly(hydroxyethylmethacrylate, p(HEMA)) as a main material. It is characterized in that it is modified with poly(2-methacryloyloxyethyl phosphorylcholine) (p(MPC)).

The hydrogel containing p (HEMA) constituting the hydrogel as a main material is obtained by dissolving ethylene glycol dimethacrylate (EGDMA) and azobisisobutyronitrile (AIBN) in a hydroxyethyl methacrylate (HEMA) monomer. It may be prepared including preparing a mixture, injecting the mixture into a mold and heating to prepare a polymer, and washing the polymer to prepare a hydrogel.

To explain this in detail, the HEMA monomer is first used after vacuum distillation. 0.04 parts by weight of EGDMA and 0.04 parts by weight of AIBN were dissolved in 9.92 parts by weight of HEMA and mixed for 30 minutes. A polymer was obtained by pouring the resulting mixture into a square mold, sealing it, and placing it at 90° C. for 5 hours to react. After cooling to room temperature, the polymer was taken out from the mold, and placed in 400 ml of distilled water to completely remove unreacted monomers and reactants, and dialysis was performed. Dialysis was performed while changing distilled water 3 times a day over 2 days. Through this, a hexagonal hydrogel (10 × 10 × 2.4 mm) was obtained, which was placed in boiling water for 15 minutes and dried overnight at 40° C. to obtain a hydrogel containing p (HEMA) as a main material.

By modifying the surface of the hydrogel prepared by the above method, it is possible to obtain a hydrogel modified with the desired p(MPC) in the present invention.

To this end, p(HEMA) hydrogel containing p(HEMA) as a main material was impregnated with benzophenone solution and dried to prepare p(HEMA) hydrogel to which benzophenone was adsorbed, and then p(HEMA) to which benzophenone was adsorbed. ) The hydrogel is impregnated with 2-methacryloyloxyethyl phosphorylcholine (MPC) solution to prepare an impregnation solution. By irradiating ultraviolet light to the impregnation solution, a surface-modified hydrogel can be obtained, and then, by washing the surface-modified hydrogel, a hydrogel modified with p (MPC) as desired in the present invention can be obtained.

Specifically, the p(HEMA) hydrogel was impregnated in a solution of 10 mg/ml of benzophenone in acetone for 1 minute and vacuum dried for 1 hour to adsorb the benzophenone p(HEMA) hydrogel. was prepared. Then, the benzophenone adsorbed p (HEMA) hydrogel was impregnated in the MPC aqueous solution. The concentration of the MPC aqueous solution may be 0.1 to 0.5M. Thereafter, the impregnated hydrogel was irradiated with UV-B for 15 minutes, and the obtained product was washed with acetone and water to remove unreacted monomers, excess benzophenone, and by-product benzopinacol. In addition, the surface-modified hydrogel was treated by immersing it in distilled water overnight in order to additionally remove the polymer that did not form a covalent bond and excess acetone.

The physical properties of the surface-modified hydrogel were measured as follows.

In order to measure the transmittance of the surface-modified hydrogel, a sample having an average thickness of the hydrogel of 2.4 mm was prepared and measured using a UV spectrometer (UV-1650PC spectrophotometer, Shimadzu). The measurement was performed in a wavelength range of 300 to 700 nm at room temperature, and the average value was obtained by repeating measurement 4 times for each sample.

In addition, the equilibrium swelling ratio (ESR) of the surface-modified hydrogel was determined gravimetrically at room temperature. The surface-modified hydrogel sample was completely dried and then measured. The completely dried hydrogel was allowed to stand for 24 hours using PBS buffer (pH 7.4) to equilibrate. Thereafter, the PBS buffer was removed, the remaining moisture was removed, and the weight of the expanded hydrogel was measured. ESR was calculated according to the equation below.

ESR (%) = [(Ws - Wd)/Wd] × 100

Wherein Ws and Wd are the hydrogel weight in the equilibrium state and the hydrogel weight in the dry state.

In addition, in order to measure the contact angle of the surface-modified hydrogel, the sample was measured using a contact angle meter (DSA100, Kruss GmbH). After 4.5 μl of water was dropped on the surface of the hydrogel sample, the static contact angle was measured. The contact angle was obtained as an average value of 10 measurements.

In addition, in order to evaluate the protein desorption performance of the surface-modified hydrogel, the surface-modified hydrogel was put into a mold to prepare a hydrogel lens and used as a sample. The sample was immersed in 10 ml of PBS (pH 7.4). Thereafter, the cells were incubated at room temperature at a speed of 150 rpm for 24 hours on a constant-temperature shaker. An artificial tear solution containing 3.88 g/L bovine serum albumin (BSA) contained in PBS and 1.2 g/L egg white lysozyme were prepared, 10 ml of artificial tears were added to the sample, and then at 37° C. at 150 rpm for 12 hours. cultured. After washing the sample with PBS, unbound proteins were removed, immersed in an extraction solvent containing 0.1% trifluoroacetic acid in acetonitrile/water 1:1 (v/v), and incubated at room temperature in the dark for 24 hours. The extraction solution was diluted 1/10 using the mobile phase and then analyzed. The protein content desorbed from the lens was quantified by HPLC (Azura) measurement. About 20 liters of the mixed solution was injected into a C18 column (LUNA-C18, 4.6 × 250 mm, 5 μm; Phenomenex, Torrance) and separated using an isocratic mobile phase, 50 containing 0.1% trifluoroacetic acid and 50% water. % acetonitrile mixture was obtained. The runtime was 20 minutes, the flow rate was 1.0 ml/min, and a 220 nm UV detector (DAD 2.1L, Azura) was used.

In addition, in order to evaluate the bacterial desorption performance of the surface-modified hydrogel, the hydrogel lens was analyzed as a sample. All samples were placed in a 70% ethanol solution and placed in distilled water for 1 day, followed by a bacterial desorption test. Mueller-Hinton medium solution was obtained by culturing E. coli, about 1 ml of E. coli stock solution was sterilized with 50 ml of 21 g/L Mueller-Hinton medium solution, and cultured at 37° C. for 6 hours.

Then, about 2 ml of the cultured E. coli solution was inoculated into 400 ml of sterile Mueller-Hinton medium solution, and incubated at 37° C. for 12 hours on a thermostat. After incubation, the E. coli solution was transferred to a 1 L beaker and the sample was placed in a container and impregnated with the bacterial solution. The samples were placed on a rotary shaker at room temperature and bacteria were allowed to contact the surface of the samples. After incubation for 6 hours, the sample was washed with distilled water, placed in 20 ml of sterilized Mueller-Hinton medium solution, and treated at 37° C. at 150 rpm using a rotary shaker. Then, about 1 ml of a medium sample was taken and absorbance was measured at a wavelength of 595 nm using a UV spectrometer (TU-1800, Korea).

In order to measure the physical properties, a surface-modified hydrogel sample was prepared, which was prepared through a three-step process as shown in FIG. 1 . HEMA monomers were polymerized as described above using EGDMA and AIBN as crosslinkers and free radical sources. The next two steps are adsorption processes using benzophenone as a photosensitizer, and the MPC monomer was adsorbed by UV-induced free radical polymerization on the hydrogel surface. For surface modification, samples were prepared by varying the concentration of the MPC monomer in an aqueous environment to 0.10M (Example 1), 0.25M (Example 2), and 0.50M (Example 3).

Table 1 shows the properties of the surface-modified hydrogel.

Content of conjugated p(MPC)
(μmol/cm2) Equilibrium expansion ratio (%) Contact angle (°) p(HEMA) - 60.1±1.1 78.6±9.6 Example 1 18.0±1.2 61.0±2.9 74.7±9.7 Example 2 21.8±0.5 61.6±1.0 65.0±4.7 Example 3 24.1±1.8 68.3±1.3 59.2±7.0

Referring to Table 1, it can be seen that as the amount of MPC increases, the amount of p(MPC) adsorbed to the hydrogel surface increases. In addition, the amount of p(MPC) on the hydrogel surface was estimated to be 24.0, 21.8, and 18.0 μmol/cm 2 for Examples 1 to 3, respectively.

The effect of surface modification was evaluated by measuring the equilibrium swelling ratio (ESR) for PBS medium at room temperature. High equilibrium water content is a major characteristic of hydrogels and is a factor influencing hydrogel swelling. As shown in the results of Table 1, ESR showed a tendency to increase with the increase in the amount of MPC. The ESR of the p(HEMA) hydrogel was 60.1%, and when the surface was modified with p(MPC), it was found to increase from 61.0 to 68.3%.

In addition, the optical transmittance of the lens prepared from the surface-modified hydrogel was evaluated, and as a result, it exhibited a high transmittance of 94% or more for all hydrogel samples as in FIG. 2 . This is the result of meeting the transmittance (92%) required for contact lenses. In addition, the high permeability is also a result suggesting that the p(HEMA) hydrogel surface-modified with p(MPC) is not opaque or phase-separated. This is also due to the high miscibility of MPC with monomer solutions containing HEMA and EGDMA.

In addition, the surface wettability was evaluated by measuring the contact angle, and the results are shown in Table 1 and FIG. 3 . The surface-modified hydrogel has zwitterionic and hydrophilic MPC components, and the contact angle decreases with an increase in the amount of MPC, and the surface wettability is improved. A relatively high contact angle of 78.6° was observed in the p(HEMA) hydrogel sample, and it was confirmed that the contact angle decreased to 74.7, 65.0, and 59.2° as the amount of MPC increased. This improvement in surface wettability is due to ion hydration of phosphorylcholine groups in MPC.

In addition, protein and bacterial desorption tests were performed to evaluate the surface antifouling properties.

Protein desorption is due to various factors, and is affected by topology, protein structure, size, charge, and the like. Therefore, the adsorption of two protein models, BSA and lysozyme, was considered and tested.

The results are as shown in FIG. 4, and it was found that the amount of BSA adsorbed per lens increased to 3.13, 2.54, and 2.51 g as the MPC content increased. Compared with the unmodified hydrogel lens at 7.55 g, these results suggest 59, 66, and 67% reduction in BSA adsorption. In addition, it was shown that lysozyme adsorption was reduced by the surface modification. In the case of the surface-modified hydrogel lens, the adsorption amount of the lysozyme for the hydrogel lens that is not surface-modified was 3.87 g compared to the adsorption amount of 1.02 to 1.05 g.

However, in Examples 2 and 3, the adsorption amount of BSA and lysozyme was almost the same, and from this, for optimal surface modification, it is suitable to surface-modify the surface with 0.25 M MPC monomer, and through this, it is possible to obtain optimal surface antifouling properties. appear. The resistance to protein adsorption is due to the interaction between the phosphorylcholine moiety and the surrounding water, and the density of the hydration layer around the phosphorylcholine group is determined according to the content of free water on the substrate, and through this, a kind of barrier that inhibits protein adsorption. is thought to form

In addition, an experiment was conducted using Gram-negative E. coli for the bacterial adsorption test. The hydrogel sample was impregnated in the bacterial suspension and incubated at 37°C, which was then taken out and re-impregnated in the sterile medium. Thereafter, the content of bacteria was measured for 1 to 24 hours at preset time intervals. The measurement was performed by obtaining the optical density using a UV spectrometer.

The test results for the sample of Example 3 are the same as in FIG. 5, and the suspension including the surface-modified hydrogel exhibited low bacterial adsorption compared to the control. That is, the result was obtained that the amount of bacterial adsorption decreased by 10 to 73% compared to the control.

Similar to the protein desorption result, a decrease in the adsorption rate was also obtained for bacteria, which was thought to be due to the increased density of the hydration layer on the hydrogel surface.

From these results, it was confirmed that the hydrogel surface-modified with poly(2-methacryloyloxyethyl phosphorylcholine) according to the present invention exhibits high resistance to adsorption of proteins and bacteria, thereby exhibiting excellent antifouling properties. . In addition, when a contact lens is manufactured by molding such a surface-modified hydrogel, antifouling and antibacterial properties are improved due to the characteristics of the hydrogel, and it prevents eye infection and thereby reduces eye diseases that may occur when wearing contact lenses. It is expected to be preventable.

As described above, preferred embodiments of the present invention have been described in detail, but the present invention is not limited to the above embodiments, and various methods can be made by those of ordinary skill in the art within the scope of the technical spirit of the present invention. Transformation is possible.

Claims (3)

Characterized in modifying the surface of a hydrogel containing poly(hydroxyethylmethacrylate, p(HEMA)) with poly(2-methacryloyloxyethyl phosphorylcholine)(p(MPC)) As a hydrogel modified with poly (2-methacryloyloxyethyl phosphorylcholine),
The p (HEMA) hydrogel is,
Dissolving ethylene glycol dimethacrylate (EGDMA) and azobisisobutyronitrile (AIBN) in hydroxyethyl methacrylate (HEMA) monomer to prepare a mixed solution;
preparing a polymer by injecting the mixture into a mold and heating;
washing the polymer to prepare a hydrogel;
Manufactured including
The modification is
impregnating the p(HEMA) hydrogel in a benzophenone solution and drying to prepare a p(HEMA) hydrogel to which benzophenone is adsorbed;
preparing an impregnation solution by impregnating the benzophenone-adsorbed p (HEMA) hydrogel in an MPC solution;
irradiating ultraviolet rays to the impregnating liquid;
A hydrogel modified with poly (2-methacryloyloxyethyl phosphorylcholine), characterized in that it is prepared including a.
delete A hydrogel contact lens, characterized in that it is formed of a hydrogel modified with the poly(2-methacryloyloxyethyl phosphorylcholine) of claim 1.

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