JP5273675B2 - Care product for ophthalmic lens and method for hydrophilizing resin base material - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for hydrophilizing the surface of a hydrophobic resin base material, in particular, an eye lens, which utilizes a reaction with enzyme, and to provide a processing liquid for the hydrophilization, and a hydrophilized eye-lens. <P>SOLUTION: The present invention provides an eye-lens care supply containing a composite of hydroxycarboxylic acid and coenzyme, and polyhydroxy alkanoic acid (PHA) polymerase. The present invention also provides the method for hydrophilizing a resin base material. The method includes polymerizing a hydroxycarboxylic acid on the surface of the resin base material under the presence of coenzyme and polyhydroxy alkanoic acid (PHA) polymerase. The invention also provide the eye lens hydrophilized by this method. <P>COPYRIGHT: (C)2011,JPO&amp;INPIT

Description

  The present invention relates to a method for hydrophilizing a surface of a hydrophobic resin substrate, a treatment liquid for the hydrophilization (particularly, a care product for an ophthalmic lens), and an ophthalmic lens that has been hydrophilized.

  A 3-hydroxybutyric acid polymer or the like is known as a bioplastic that can be produced by microorganisms (Patent Documents 1 to 4). This bioplastic has been found to be hydrophilic.

Japanese Unexamined Patent Publication No. 6-14511 Japanese Patent Laid-Open No. 5-276934 JP-A-5-64592 International Publication WO2006 / 025375

  The main problems with oxygen-permeable hard contact lenses and silicone hydrogel lenses are discomfort during wearing and poor wettability. Although chemical and physical surface modification such as plasma treatment has been performed, there was a problem with durability during lens use. In addition, as a coating technology on the surface of contact lenses, artificially synthesized products and natural products (cellulose, PVA, etc.) are fixed with eye drops, applied, and tear solution, and adsorbed to the primary surface to impart hydrophilicity to the surface. However, when a lipid component, which is a tear fluid component, has already adhered to the lens surface, there is a problem that the interaction with the lens material is weakened and the effect is reduced.

  On the other hand, if an enzyme derived from a microorganism is used, it is expected that a monomer compound such as 3-hydroxybutyric acid can be polymerized on the surface of the resin substrate. Since this enzyme reaction can be carried out under mild conditions, it is expected to be suitable for the hydrophilic treatment of the resin substrate surface provided with functionality such as an ophthalmic lens.

  Accordingly, an object of the present invention is to provide a method for hydrophilizing a surface of a hydrophobic resin substrate using an enzyme reaction, particularly an ophthalmic lens, a treatment liquid for the hydrophilization, and an ophthalmic lens subjected to a hydrophilization treatment. That is.

That is, the present invention provides the following.
[1]
A care product for an ophthalmic lens comprising a complex of a hydroxycarboxylic acid and a coenzyme and a polyhydroxyalkanoic acid (PHA) polymerizing enzyme.

[2]
[1] The ophthalmic lens care product, wherein the complex of hydroxycarboxylic acid and coenzyme is a complex of hydroxyalkanoic acid and coenzyme A.

[3]
A method for hydrophilizing a resin base material, comprising polymerizing hydroxycarboxylic acid on the surface of the resin base material in the presence of a coenzyme and a polyhydroxyalkanoic acid (PHA) polymerizing enzyme.

[4]
The method according to [3], wherein the coenzyme and the hydroxycarboxylic acid form a complex.
[5]
Furthermore, the method according to [3] or [4], wherein the polymerization is performed in the presence of a polyhydric alcohol.

[6]
The method according to any one of [3] to [5], wherein the resin substrate is an ophthalmic lens.
[7]
An ophthalmic lens made hydrophilic by the method of [6].

  According to the present invention, the resin substrate surface can be hydrophilized under mild reaction conditions using an enzyme. This hydrophilization is achieved by growing a hydroxycarboxylic acid polymer on the surface of the resin substrate.

  In the case of using the ophthalmic lens care product of the present invention, the hydrophilization treatment is achieved, for example, in the lens cleaning and storage care process (for all-day wear). Carboxylic acid) is polymerized on the lens surface by the action of polyhydroxyalkanoic acid (PHA) polymerizing enzyme and coenzyme (for example, coenzyme A (coenzyme A)), and a part of the components of the care product works as a stop reaction. As a result, a moisturizing component (polyhydroxycarboxylic acid) is adsorbed on the lens surface. Moreover, this hydrophilic treatment can be repeated.

  More specifically, hydroxyalkanoic acid or the like added to a contact lens (CL) care product is primarily adsorbed on the lens, and a resin is produced by the action of polyhydroxyalkanoic acid (PHA) polymerizing enzyme and coenzyme (eg, coenzyme A). It is possible to form a hydrophilic polymer film or film by polymerizing on the surface of the substrate, particularly the plastic lens. The polymerized polyester grows with a hydrophobic surface on the lens side and a hydrophilic surface on the aqueous solution side, so that the entire surface exhibits hydrophilicity on the lens surface and also functions as a coating that prevents the lipid contained in tears from adsorbing. Furthermore, polymer thickeners such as polyethylene glycol (PEG) chains of the components included in CL care products and poloxamer components act as a polymerization termination reaction, and as a result, grafting of polymers such as poloxamers on the polyester chain ends is promoted. , Increase hydrophilicity. According to the present invention, a phenomenon occurring in a living body / cell can be performed using a CL care product, and CL can be hydrophilized.

  According to the ophthalmic lens care product of the present invention, for example, the ophthalmic lens is removed, the lens is washed with the care product, stored in a case and immersed in the care product, and then worn for the next day. In a special care system, the lipid attached to the lens is easily removed by washing, especially by rubbing, and the lens is hydrophilized by the adhesion of the PHA polymer while immersed in a storage solution overnight, and then washed the next morning. And a hydrophilized lens can be worn. In addition, the polymer attached to the lens also functions as a coating that prevents the lipid contained in the tear fluid from adsorbing. That is, according to the present invention, a lens having a clean and good wearing feeling can be used every day in a cycle of lens wearing → lens removal and cleaning (lipid removal) → storage (coating by PHA polymerization at this point) → lens wearing. .

It is a figure which shows the result of Nile Red observation. A: Dipped in 50 mM Tris-HCl (pH 7.5) (Comparative Example 1), B: PHA polymerization in 50 mM Tris-HCl (pH 7.5) (Example 1). It is a figure which shows the height image in an atomic force microscope (AFM) observation result. In the figure, immersion in 50 mM Tris-HCl (Comparative Example 1) and PHA polymerization (Example 1) are compared. It is a figure which shows the phase image and error signal in the AFM observation result. In the figure, immersion in 50 mM Tris-HCl (Comparative Example 1) and PHA polymerization (Example 1) are compared. It is a figure for demonstrating the preparation method of the PHA polymerase used in the Example. It is a figure which shows the amino acid sequence (sequence number 1) of PHA polymerase used in the Example.

  According to one aspect of the present invention, there is provided a method for hydrophilizing a resin base material, comprising polymerizing hydroxycarboxylic acid on the surface of the resin base material in the presence of a coenzyme and a polyhydroxyalkanoic acid (PHA) polymerizing enzyme. Provided.

[Hydroxycarboxylic acid]
Hydroxycarboxylic acid is a compound having at least one hydroxyl group and at least one carboxyl group in the molecule, and is not particularly limited as long as it can be polymerized by a polyhydroxyalkanoic acid (PHA) polymerizing enzyme. An intermolecular ester forming reaction occurs between the hydroxyl group and the carboxyl group to form a polyester polymer.

  Examples of hydroxycarboxylic acid include hydroxyalkanoic acid, hydroxyalkanoic acid substituted with halogen such as fluorine, chlorine, bromine, iodine, hydroxyl group- and carboxyl group-containing aromatic compound, hydroxyl group-containing unsaturated carboxylic acid, etc. It is done. The number of carbon atoms of the hydroxycarboxylic acid is preferably 3-18, more preferably 3-14, and in order to more effectively express the hydrophilicity-imparting effect of the present invention, the shorter chain carbon number. It is preferable that it is 3-8.

The following 58 types of hydroxycarboxylic acids are mentioned as the lipid component for forming a polyester whose reaction has been confirmed under an enzyme.
1 3-hydroxypropionic acid
2 3-hydroxybutanoic acid
3 3-Hydroxypentanoic acid
4 3-Hydroxyhexanoic acid
5 3-hydroxyheptanoic acid
6 3-hydroxyoctanoic acid
7 3-hydroxynonanoic acid
8 3-hydroxydecanoic acid
9 3-hydroxy-4-cyclohexylbutanoic acid
10 3-Hydroxydodecanoic acid
11 3-hydroxytetradodecanoic acid
12 3-hydroxy-2-butanoic acid
13 3-hydroxy-4-pentanoic acid
14 3-hydroxy-4-hexanoic acid
15 3-hydroxy-5-hexanoic acid
16 3-hydroxy-6-octanoic acid
17 3-hydroxy-7-octanoic acid
18 3-hydroxy-8-nonanoic acid
19 3-Hydroxy-9-decanoic acid
20 3-hydroxy-5-dodecanoic acid
21 3-Hydroxy-5-tetradecanoic acid
22 3-hydroxy-5,8-tetradecanedioic acid
23 3-hydroxy-2-methylbutanoic acid
24 3-hydroxy-2-methylpentanoic acid
25 3-hydroxy-2,6-dimethyl-5-heptanoic acid
26 3-Hydroxy-4-methylhexanoic acid
27 3-Hydroxy-5-methylhexanoic acid
28 3-Hydroxy-4-methyloctanoic acid
29 3-Hydroxy-5-methyloctanoic acid
30 3-hydroxy-6-methyloctanoic acid
31 3-hydroxy-7-methyloctanoic acid
32 3-hydroxy-6-methylnonanoic acid
33 3-hydroxy-7-methylnonanoic acid
34 3-Hydroxy-8-methylnonanoic acid
35 3-hydroxy-7-methyldecanoic acid
36 3-hydroxy-9-methyldecanoic acid
37 3-Hydroxy-7-fluoroheptanoic acid
38 3-hydroxy-9-fluorononanoic acid
39 3-Hydroxy-6-chlorohexanoic acid
40 3-hydroxy-8-chlorooctanoic acid
41 3-hydroxy-6-bromohexanoic acid
42 3-hydroxy-8-octanoic acid bromide
43 3-Hydroxy-11-brominated undecanoic acid
44 7-Cyano-3-hydroxyheptanoic acid
45 9-Cyano-3-hydroxynonanoic acid
46 3-Hydroxysuccinic acid methyl ester
47 3-Hydroxyadipic acid methyl ester
48 3-Hydroxysuccinic acid methyl ester
49 3-Hydroxysuccinic acid ethyl ester
50 3-Hydroxypimelic acid propyl ester
51 3-Hydroxysebacic acid benzyl ester
52 4-Hydroxybutanoic acid
53 4-Hydroxypentanoic acid
54 4-Hydroxyhexanoic acid
55 5-hydroxypentanoic acid
56 3,12-Dihydroxydodecanoic acid
57 Malic acid
58 3-Hydroxy-5-phenylpentanoic acid

[Coenzyme and polyhydroxyalkanoic acid (PHA) polymerization enzyme]
In the present invention, PHA polymerizing enzyme is used as a polymerization catalyst for hydroxycarboxylic acid. As the PHA polymerizing enzyme, those derived from bacteria such as hydrogen bacteria (Ralstonia eutropha) can be used. For example, poly-3-hydroxybutanoic acid polymerase is mentioned. Specific examples of the PHA polymerizing enzyme include those having the amino acid sequence of FIG. As can be easily understood by those skilled in the art, the PHA synthase that can be used in the present invention is not limited to the one having the amino acid sequence of FIG. 5, and includes one or several amino acids in the amino acid sequence of FIG. In which the amino acid sequence is deleted, substituted or added and has PHA polymerase activity.

In the present invention, it is necessary to use a coenzyme together with a PHA polymerizing enzyme. Examples of the coenzyme include coenzyme A (coenzyme A).
The coenzyme may form a complex with hydroxycarboxylic acid, and an example of this complex is 3-hydroxybutyryl coenzyme A.

[Polymerization reaction]
The outline of the polymerization reaction will be explained by the following chemical reaction formula using poly-3-hydroxybutanoic acid as an example.

  In the above chemical reaction formula, R is a methyl group, n is the degree of polymerization of poly-3-hydroxybutanoic acid, and (R) -3HA-CoA is a complex of 3-hydroxybutanoic acid and coenzyme A ( 3-hydroxybutyryl coenzyme A), PhaC is poly-3-hydroxybutanoic acid polymerase, and CoA-SH is coenzyme A. The polymerization reaction of PHA increases the degree of polymerization of poly-3-hydroxybutanoic acid by using coenzyme A as an acyl carrier as shown above.

[Resin substrate]
The resin base material to be hydrophilized in the present invention is not particularly limited as long as the hydroxycarboxylic acid polymer is physically attached or chemically bonded to the surface to increase the hydrophilicity of the surface. The resin base material may be a synthetic resin or a natural resin, and may be a thermoplastic resin or a thermosetting resin.

  Specific examples of the resin substrate include a resin substrate for medical devices, particularly an ophthalmic lens. Among the ophthalmic lenses, the present invention is particularly useful for oxygen permeable hard contact lenses, silicone hydrogel lenses, and the like, which are problematic in wearing discomfort and poor water wettability.

[Other ingredients]
Since the hydrophilic treatment of the present invention needs to be performed under mild reaction conditions, it is preferably performed in the presence of a buffer. Examples of the buffer include Tris-HCl buffer (Tris-HCl), TE buffer, Tris-buffered saline, and the like.

  The reaction system of the present invention includes components contained in ophthalmic lens care products, particularly polyhydric alcohol components (nonionic surfactants containing hydroxyl groups such as poloxamer, thickeners such as PEG, propylene glycol (PG), etc. And an isotonic agent, etc.) may be present. Due to the presence of such components, a higher hydrophilic effect can be obtained after the polymerization reaction.

  Poloxamer is a polyoxyethylene (POE) n-polyoxypropylene (POP) m block copolymer nonionic surfactant, and there are various compounds whose molecular weight and the like are changed by n and m. Among the poloxamers, those having both OH groups at the both ends become polyhydric alcohols, which contribute to the hydrophilization of the resin substrate of the present invention.

[Care products for ophthalmic lenses]
In the present invention, the care product for ophthalmic lenses means various care products defined in Article 2 of the Voluntary Safety Standards concerning contact lens cleaning agents, preservatives, cleaning preservatives and the like defined by the Japan Contact Lens Association, that is, contact lenses This refers to cleaning agents, preservatives, cleaning preservatives, and dissolved water, but may be in the form of granules, powders, tablets, gels and the like for use adjustment as shown in the above voluntary standards.

  The ophthalmic lens care product of the present invention comprises a complex of a hydroxycarboxylic acid and a coenzyme, and a polyhydroxyalkanoic acid (PHA) polymerizing enzyme. When the ophthalmic lens care product is a liquid care product such as a CL cleaning solution or a CL preservation solution, the amount of the complex of hydroxycarboxylic acid and coenzyme is 10 nM to 10 mM in the liquid care product, preferably Is about 100 μM, and the compounding amount of polyhydroxyalkanoic acid (PHA) polymerizing enzyme is 1 pM to 10 mM, preferably about 100 nM, in the concentration in the liquid care product. When the ophthalmic lens care product is a solid care product such as a powder, tablet or film, the compounding amount of the hydroxycarboxylic acid and coenzyme complex and polyhydroxyalkanoic acid (PHA) polymerizing enzyme in the solid care product is The amount is such that when dissolved in a liquid, the concentration is the same as the liquid care product.

Suitable ophthalmic lens care products of the present invention are exemplified below.
Care products for ophthalmic lenses containing coenzymes and PHA polymerizing enzymes.
Care product for ophthalmic lenses containing buffer, coenzyme and PHA polymerizing enzyme.

Care product for ophthalmic lenses containing thickener, coenzyme and PHA polymerizing enzyme.
Care products for ophthalmic lenses containing thickeners, buffers, coenzymes and PHA polymerizing enzymes.

Hydrophilization of contact lens surface (test sample)
55 parts by weight of tris (trimethylsiloxy) silylstyrene, 35 parts by weight of 2,2,2-trifluoroethyl methacrylate, and 10 parts by weight of ethylene glycol dimethacrylate were mixed, and 2,2′-azobis (2 , 4-dimethylvaleronitrile) was added in an amount of 0.3 parts by weight, and the mixture was well stirred. The mixture is poured into a plastic mold (diameter 12 mm, depth 5 mm), preliminarily polymerized at 50 ° C for 2 hours in a circulation drier in a nitrogen atmosphere, and then heated to 90 ° C. Then, the mixture was kept heated for 30 minutes to complete the polymerization.

(Preprocessing)
The test sample was immersed in a contact lens cleaning / preserving agent (O2 Care Neo (Menicon)) overnight to clean the lens surface. The next day, it was carefully washed with pure water so that no cleaning solution remained, and after wiping off the water with a Kimwipe, it was used for the experiment.

(Test method and results)
In the following description, all concentrations of the reaction solution are final concentrations. Moreover, unless otherwise indicated,% display means weight%. The results are shown in Tables 1 and 2.

Control reaction (Comparative Examples 1-4):
The test samples after the pretreatment were immersed in the solutions shown in Tables 1 and 2 below and left overnight at room temperature. The surface was washed with pure water, and then moisture was absorbed with Kimwipe. H ₂ O was dropped on the lens surface and photographed with a CCD camera, and the static contact angle was measured. The measurement was performed three times for each lens surface, and the average value was taken as the measurement result.

PHA polymerization reaction:
(1) System in which polymerization enzymes PhaC _Re and (R) -3HB-CoA are added simultaneously (Example 1)
Immerse the lens in 50 mM Tris-HCl or O2 care neo and add 100 μM (R) -3-hydroxybutyryl CoA [(R) -3HB-CoA (monomer)] and 100 nM PhaC _Re to room temperature. The PHA enzyme polymerization reaction was performed by incubating overnight (about 16 hours). The static contact angle was measured as in the comparative example.

<Preparation method of PhaC _Re >
Expression and purification of PHA synthase (PhaC _Re ) from Ralstonia eutropha
A strain in which pET15b :: phaC _Re was introduced into E. coli BL21 (DE3) (see FIG. 4) was cultured in an LB test tube medium containing 100 μg / mL ampicillin at 37 ° C. for 15 hours with shaking. 1 mL of the preculture was added to LB medium (100 ml) containing 100 μg / mL ampicillin to initiate main culture. After shaking culture at 37 ° C. for 2 hours, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1 mM, and further, shaking culture was performed at 30 ° C. for 4 hours. After cultivation, the cells were collected by centrifugation and stored at -80 ° C.

  The frozen cells were thawed on ice. The thawed bacterial body is 20 mM sodium phosphate buffer (pH 7.4) containing 20 mM imidazole, 500 mM sodium chloride, 5% glycerol, 0.05% 6-O- (N-Heptylcarbamoyl) methyl α-D-glucopyranoside (HECAMEG). ). After suspension, the cells were disrupted with a French press (3 times). The crude enzyme solution after crushing was centrifuged at 15000 g for 30 minutes. The supernatant was filtered through a 0.45 μm cellulose acetate filter (Advantech). The filtrate was injected into Ni Sepharose High Performance packed HiTrap ™ column (5 mL) manufactured by Amersham Bioscience. Using an AKTA explorer 10S system (Amersham Bioscience), elution was performed with a 20-500 mM linear concentration gradient (100 ml) of imidazole. The flow rate was 1 ml / min. The purification was performed while maintaining 4 ° C. The purified sample was dispensed into an eppen, frozen with liquid nitrogen and stored at -80 ° C.

The amino acid sequence (SEQ ID NO: 1) of the obtained enzyme is shown in FIG.

(2) System in which PhaC _Re and (R) -3HB-CoA are added separately (Examples 5 to 7)
First, 100 nM PhaC _Re was added to 50 mM Tris-HCl (pH 7.5), incubated at room temperature for 10 to 30 minutes, and then 100 μM (R) -3HB-CoA was added to polymerize PHA. In addition, (R) -3HB-CoA was added first and incubated at room temperature for 10 to 30 minutes, and then PhaC _Re was added and incubated overnight at room temperature to perform PHA enzyme polymerization reaction. The static contact angle was measured as in the comparative example.

(3) Influence of PEG200 and Px-PG (Examples 2 to 4, 8 to 10)
A system in which (R) -3HB-CoA, PhaC _Re and PEG200 are added simultaneously, a system in which (R) -3HB-CoA and PEG200 are added after adding PhaC _Re first, and a system in which PEG200 is added after PHA polymerization Went. In a system in which PEG200 was added after PHA polymerization, 100 nM PhaC _Re and 100 μM (R) -3HB-CoA were simultaneously added to a lens immersed in 50 mM Tris-HCl, and incubated overnight at room temperature to polymerize PHA. Later, PEG200 was added. A Px-PG solution containing polypropylene glycol was also examined in the same manner as PEG200. In all cases, the PHA enzyme polymerization reaction was performed by incubating overnight at room temperature. The static contact angle was measured as in the comparative example. When PEG200 and Px-PG were added after the PHA enzyme polymerization reaction, the contact angle was measured 4 hours after the addition of PEG200 and Px-PG.

  From the experimental results shown in Table 1 and Table 2, the contact lens surface subjected to the hydrophilization method of the present invention has a lower water contact angle and higher hydrophilicity than the contact lens surface not subjected to the method. You can see that

(Reference example)
Nile Red (a fluorescent dye that adsorbs to lipids and PHA) was added to the test sample after PHA polymerization, and the mixture was incubated at room temperature for 30 minutes and then observed under ultraviolet irradiation (FIG. 1). As a result, the sample just immersed in Tis-HCl did not emit fluorescence (FIG. 1A), whereas the surface of the sample subjected to PHA polymerization emitted fluorescence (FIG. 1B). It was found that a PHA coating was formed.

Further, the lens after PHA polymerization was observed with an atomic force microscope (AFM) (FIGS. 2 and 3). When the surface of the test sample before and after PHA polymerization was observed with AFM, a clear difference was observed. Assuming that the thickness of the test sample itself is around 10 nm, PhaC _Re or polymerized PHA is considered to be adsorbed in the portion that appears higher than that.

Claims (7)

  A care product for an ophthalmic lens comprising a complex of a hydroxycarboxylic acid and a coenzyme, and a polyhydroxyalkanoic acid (PHA) polymerizing enzyme.   The ophthalmic lens care product according to claim 1, wherein the complex of hydroxycarboxylic acid and coenzyme is a complex of hydroxyalkanoic acid and coenzyme A.   A method for hydrophilizing a resin base material, comprising polymerizing hydroxycarboxylic acid on the surface of the resin base material in the presence of a coenzyme and a polyhydroxyalkanoic acid (PHA) polymerizing enzyme.   The method of claim 3, wherein the coenzyme and the hydroxycarboxylic acid form a complex.   The method according to claim 3 or 4, wherein the polymerization is carried out in the presence of a polyhydric alcohol.   The method according to claim 3, wherein the resin substrate is an ophthalmic lens.   An ophthalmic lens hydrophilized by the method of claim 6.