JP7664614B2 - Structure with hydroxyapatite crystal layer and method for producing same - Google Patents

Description

This application relates to a structure that has a hydroxyapatite crystal layer on the surface of the fiber and can adsorb and separate biopolymers such as proteins, peptides, and viruses.

The development of materials that adsorb biopolymers such as proteins is important for applications in masks, air filters, underwater filtration filters, artificial bones, cell culture carriers, and virus adsorbents. Furthermore, the ability to desorb adsorbed biopolymers under mild conditions is important for applications in adsorption/separation materials such as carriers for liquid chromatography, separation materials for columns, and protein concentration materials for in vitro diagnostic reagents. Hydroxyapatite, a type of calcium phosphate ceramic, has attracted attention as a material that adsorbs biopolymers such as proteins.

In order to impart flexibility to this hydroxyapatite, a composite of fibrous polymers and hydroxyapatite has attracted attention. In order to use a composite of fibrous polymers and hydroxyapatite as an adsorption/separation material, it is important that the surface of the fibrous polymer is covered with a thin and uniform layer of hydroxyapatite so that the area of contact between the hydroxyapatite and the biopolymer is increased. In addition, it is desirable for this composite to be cotton-like so that it can be easily manipulated by hand or with tweezers. Furthermore, when a biopolymer is adsorbed in water, it is desirable for the composite not to disperse in water so that it can be recovered without using special equipment such as a centrifuge or filter.

Patent Document 1 describes a composite in which a coating liquid containing hydroxyapatite microparticles, an aqueous urethane resin, and a self-emulsifying isocyanate compound is applied to nonwoven fibers. However, the composite in Patent Document 1 has a structure in which the hydroxyapatite particles are attached to the nonwoven fibers. In other words, the hydroxyapatite particles only cover a portion of the nonwoven fibers, and this composite has poor performance as an adsorbent.

Patent Document 2 describes a composite in which a fibrous polymer substrate is coated with a thin, uniform ceramic layer. This composite can impart chemical resistance, chemical resistance, oxidation resistance, wear resistance, corrosion resistance, and the like to the substrate, and can also provide the substrate with high fracture strength, high toughness, high toughness, high hardness, high elasticity, and functional gradient properties, and can achieve both lightness and strength. However, the composite of Patent Document 2 disperses when used in water because there is no bonding between the fibrous polymer substrate or the composites themselves.

To be used in water, it is necessary to render the fibrous polymer substrate or composite water-insoluble so that it does not disperse in water. To make fibrous polymer substrates water-insoluble, chemical methods such as crosslinking are often used. However, when a crosslinking agent is used for the fibrous polymer substrate, unreacted crosslinking agent or by-products resulting from the crosslinking agent may cause biotoxicity, so there is a demand for a method of manufacturing a composite without using a crosslinking agent.

JP 2013-204209 A International Publication No. 2018/143120

The objective of this application is to provide a structure that has a hydroxyapatite crystal layer on the surface of the fiber, can adsorb and separate biopolymers such as proteins, peptides, and viruses, and is less likely to disperse in water.

The structure of the present application has a plurality of fibrous composites each including a fiber and a hydroxyapatite crystal layer provided on the surface of the fiber, and the hydroxyapatite crystal layers of the plurality of fibrous composites are partially bonded to each other to form a cotton-like structure. The adsorbent of the present application has the structure of the present application as a member for adsorbing biopolymers.

The method for producing a structure according to the present application is a method for producing a structure having a fibrous composite comprising fibers and a hydroxyapatite crystal layer provided on the surface of the fibers, and includes a mixing step of mixing and stirring a dispersion liquid in which the fibers are dispersed with a first solution containing calcium ions to obtain a mixed liquid, a drying step of drying the mixed liquid to obtain a dried body comprising the fibers and calcium, and a structure forming step of immersing the dried body in a liquid and adding a second solution containing phosphate ions to obtain a structure.

In the structure of the present application, the hydroxyapatite crystal layers of the multiple fibrous composites are partially bonded to each other. Therefore, the structure of the present application hardly separates in liquid. The adsorbent of the present application has the structure of the present application as an adsorption member. Therefore, the adsorbent of the present application can adsorb biopolymers and easily separate the biopolymers as necessary. According to the manufacturing method of the structure of the present application, a structure that has fibers and fibrous composites with hydroxyapatite crystal layers provided on the surfaces of the fibers and hardly separates in liquid can be obtained without using a crosslinking agent.

1 is a transmission electron microscope image of the composite of Example 1. Graph showing the relationship between the equilibrium protein concentration and the amount of protein adsorption in Example 4.

The structure, adsorbent, and method for manufacturing the structure of the present application will be described below based on embodiments and examples. Note that duplicated explanations will be omitted as appropriate. In addition, when a numerical range is expressed by writing "to" between two numerical values, these two numerical values are also included in the numerical range. The structure of the embodiment of the present application comprises a plurality of fibrous composites. The fibrous composites comprise fibers and a hydroxyapatite crystal layer provided on the surface of the fibers. The fibers are thin and long materials, for example, whose length is 100 times or more larger than their thickness.

The fiber may be a fibrous polymer. In an embodiment, the fiber is a fibrous polymer, and the fibrous polymer has an anionic functional group. Examples of the anionic functional group include, but are not limited to, a carboxyl group, a sulfo group, and a phosphate group. The fibrous polymer is preferably a polysaccharide. Examples of the polysaccharide include pectin, hyaluronic acid, cellulose, cellulose derivatives, and chitin chitosan derivatives such as carboxymethyl chitosan. Examples of the cellulose derivative include carboxymethyl cellulose, hydroxyethyl cellulose, TEMPO oxidized cellulose nanofiber, and hydroxypropyl methyl cellulose.

At least a portion of the calcium or phosphate contained in the hydroxyapatite in the hydroxyapatite crystal layer is chemically bonded, such as by ionic bonding, to the functional groups on the surface of the fiber. In the embodiment, the calcium or phosphate present in the inner portion of the hydroxyapatite crystal layer is chemically bonded to the functional groups derived from the anionic groups present on the surface of the fiber. For this reason, the hydroxyapatite crystal layer of the embodiment is not easily peeled off from the fiber and is not easily broken.

The thickness of the hydroxyapatite crystal layer is preferably 0.3 nm to 50 nm, more preferably 0.3 nm to 20 nm, and even more preferably 0.3 nm to 10 nm. In this way, because the hydroxyapatite crystal layer is thin, the structure of the embodiment can impart the adsorption and separation ability of hydroxyapatite to the biopolymers without impairing the lightness and flexibility of the fiber. In other words, the adsorbent of the embodiment of the present application has the structure of the embodiment as a member that adsorbs biopolymers, that is, as an active ingredient that adsorbs biopolymers. Examples of biopolymers include lysozyme or spike proteins such as those of viruses.

In the structure of the embodiment, the hydroxyapatite crystal layers of the multiple fibrous composites are partially bonded together. That is, the fibers or the fibrous composites are bonded together by the hydroxyapatite or the calcium contained in the hydroxyapatite. Therefore, the structure of the embodiment does not substantially disperse in water. "Does not substantially disperse in water" means that even if the entire structure is placed in water and ultrasonically cleaned for 10 minutes with an ultrasonic cleaner (manufactured by AS ONE, ASU-3M, oscillation frequency 42 kHz, output 80 W) that is widely used as a scientific instrument, the structure can be picked up with a mass of 95% or more of the mass before ultrasonic cleaning. In addition, the structure of the embodiment is cotton-like in that multiple fibrous composites are entangled with each other. Therefore, the structure of the embodiment is easy to operate and can be held by hand.

The structure of the embodiment is obtained, for example, by chemically bonding the contact points between multiple fibers with calcium to form a cotton-like material, and then laminating a hydroxyapatite crystal layer on the surface of this cotton-like material. It is preferable that the laminated structure of this hydroxyapatite crystal layer is such that the hydroxyapatite is laminated in the c-axis direction toward the outside of the fiber, that is, the crystal plane on the surface of the hydroxyapatite crystal layer is the c-plane.

This is because the hydroxyapatite layers are stacked in the c-axis direction toward the outside of the fiber, making the hydroxyapatite layer stronger and enabling the fiber to be stably held in place. It is preferable that this stacked structure exists over the entire surface of the hydroxyapatite layer, for example, that the outermost shell of the ceramic crystal is composed of hydroxyapatite with a regular crystal structure.

The method for manufacturing a structure according to an embodiment of the present application is a method for manufacturing a structure having a fibrous composite including fibers and a hydroxyapatite crystal layer provided on the surface of the fibers, and includes a mixing step, a drying step, and a structure forming step. In the mixing step, a dispersion in which the fibers are dispersed is mixed with a first solution containing calcium ions and stirred to obtain a mixed solution. The fiber dispersion can be prepared by mixing the fibers with a dispersion medium and stirring, as necessary, using a propeller-type stirrer or a magnetic stirrer.

The dispersion is preferably an aqueous dispersion because it is easy to handle. For the same reason, the first solution is preferably an aqueous solution. Examples of the first solution that is an aqueous solution include, but are not limited to, an aqueous calcium nitrate tetrahydrate solution, an aqueous calcium chloride solution, an aqueous calcium chloride monohydrate solution, an aqueous calcium chlorate dihydrate solution, an aqueous calcium perchlorate solution, an aqueous calcium bromide solution, and an aqueous calcium acetate solution.

The calcium ion concentration of the first solution cannot be generally set because it must be changed depending on the concentration and amount of the fibers. However, the calcium ion concentration of the mixture obtained after mixing the fiber dispersion with the first solution is preferably 1×10 ⁻⁷ mol/L to 2×10 ⁻¹ mol/L, and more preferably 1×10 ⁻⁶ mol/L to 2×10 ⁻² mol/L. If the concentration is too high, the precipitation of calcium phosphate proceeds excessively in the structure formation step, making it difficult to form a thin, uniform hydroxyapatite crystal layer on the surface of the fibers.

In the drying process, the mixture is dried, for example, by pouring the mixture into a mold and drying it, to obtain a dried body that is a precursor of the structure. This dried body comprises fibers and calcium. Drying causes the functional groups of the fibers to bond with the calcium, and the fibers can be bonded at their contact points. In other words, in this dried body, at least a portion of the contact points between the fibers are chemically bonded by calcium to form a cotton-like structure. Note that this calcium is not an elemental metal, but a calcium component such as calcium ions. Drying methods include heat drying using a dryer and freeze drying.

In the structure formation process, the dried body is immersed in a liquid, and a second solution containing phosphate ions is added to obtain a structure. At least one of the solvents of the liquid and the second solution is preferably water, since it is easy to handle. Examples of the second solution that is an aqueous solution include an aqueous solution of diammonium hydrogen phosphate, an aqueous solution of ammonium dihydrogen phosphate, an aqueous solution of disodium hydrogen phosphate, an aqueous solution of sodium dihydrogen phosphate monohydrate, an aqueous solution of sodium dihydrogen phosphate dihydrate, an aqueous solution of potassium phosphate, an aqueous solution of dipotassium hydrogen phosphate, an aqueous solution of potassium dihydrogen phosphate, and an aqueous solution of phosphoric acid, but there is no particular limitation.

The ratio of the amount of substance of calcium ions in the first solution to the amount of substance of phosphate ions in the second solution to be added, that is, the molar ratio, is preferably 0.1 to 20. This is because if the molar ratio of the hydroxyapatite crystal layer to be precipitated is 1.7 and the molar ratio of the materials is also approximately the same, the hydroxyapatite crystal layer can be precipitated efficiently. Note that a dried body that is dried without adding the first solution to the fiber dispersion disintegrates when immersed in water. In contrast, a dried body that is dried after mixing the first solution with the fiber dispersion as in the embodiment does not disintegrate even when immersed in water for 24 hours.

In the structure formation process, it is preferable to leave the mixture to stand after adding the second solution. The standing time is, for example, 10 minutes to 120 hours, and is preferably 1 hour to 72 hours to promote the precipitation of calcium phosphate on the surface of the fiber. The obtained structure is preferably desalted by washing with water and then dried. The method for manufacturing the structure of the embodiment can be carried out at room temperature and normal pressure, which can suppress denaturation of the fiber and has a low environmental impact. Heating can be carried out in some or more steps as necessary.

The structure-forming step chemically bonds calcium ions and phosphate ions present on the surface of the precursor of the structure to form a hydroxyapatite crystal layer. After the structure-forming step, the first solution containing calcium ions and the second solution containing phosphate ions may be added to the structure repeatedly. Since the structure that has undergone the structure-forming step is substantially indispersible in water, a drying step between the addition of the first solution and the addition of the second solution is not necessary. In this way, a structure that does not contain unreacted crosslinking agent and by-products of the crosslinking agent is obtained. The fact that the structure does not contain crosslinking agent and by-products of the crosslinking agent can be determined by component analysis of the structure, spectroscopic analysis, color analysis using a reagent, or the like.

Example 1
4.0 g of cellulose fiber (manufactured by Daiichi Kogyo Seiyaku, Leocrysta I-2SX, 2 wt%) was dispersed in 16 mL of distilled water. 0.4 mL of 0.20 mol/L calcium acetate aqueous solution was added to this dispersion and stirred. 2 mL of the liquid after stirring was placed in a cylindrical container with an inner diameter of about 15 mm, and the solid content was freeze-dried to obtain a cylindrical cotton-like dried body with a diameter of about 15 mm and a thickness of about 10 mm. This dried body was immersed in 4 mL of distilled water, 0.08 mL of 0.12 mol/L diammonium hydrogen phosphate aqueous solution was added, and the mixture was left to stand at room temperature for 72 hours. After washing with distilled water to desalt, the mixture was freeze-dried to obtain a cotton-like structure with a diameter of about 15 mm and a thickness of about 10 mm.

This structure had a cotton candy or cylindrical cotton-like shape. A transmission electron microscope image of this structure is shown in Figure 1. It can be seen from Figure 1 that a uniform hydroxyapatite layer with a thickness of about 3 nm covers the surface of the fibrous polymer. In addition, the distance between the crystal faces of this hydroxyapatite crystal layer in the thickness direction was 0.34 nm, which indicated that the crystal face on the surface of the hydroxyapatite crystal layer was a c-plane. From the shape of the structure and the transmission electron microscope image, it is believed that in this structure, multiple fibrous composites comprising cellulose fibers and hydroxyapatite crystal layers provided on the surfaces of the cellulose fibers are bonded to each other at parts of the hydroxyapatite crystal layers, and are entangled and cotton-like.

Example 2
The stability of the structure of Example 1 in water was evaluated. A cylindrical cotton-like structure with a diameter of about 15 mm and a thickness of about 10 mm was placed in a sample bottle, immersed in 3 mL of distilled water, and then dispersed for 10 minutes using an ultrasonic cleaner (ASU-3M, manufactured by AS ONE, oscillation frequency 42 kHz, output power 80 W (hereinafter the same)). Even after this ultrasonic dispersion, the structure did not collapse and maintained a cylindrical cotton-like structure with a diameter of about 15 mm and a thickness of about 10 mm. The structure could be pinched with tweezers and removed from the sample bottle while maintaining this structure. The structure removed with the tweezers was freeze-dried, and the mass was measured to be 7.7 mg.

Because the mass of the structure of Example 1 before ultrasonic dispersion in water was 7.8 mg, it was found that 99% of the mass of the structure was retained even after ultrasonic dispersion in Example 2. In other words, it was found that the structure of Example 1 is insoluble in water, in other words, it has the property of not dissolving in water and not dispersing in water. From this result, it is considered that in the structure of Example 1, parts of the hydroxyapatite crystal layers of the multiple fibrous complexes are bonded together, that is, parts of the hydroxyapatite crystal layers of the multiple fibrous complexes are integrated together, and the multiple fibrous complexes are entangled together.

Comparative Example 1
An aggregate of a composite having cellulose fibers and a hydroxyapatite crystal layer was obtained by the same procedure as in Example 1, except that after the calcium acetate aqueous solution was added, the diammonium hydrogen phosphate aqueous solution was added without freeze-drying. The aggregate was placed in a sample bottle, immersed in 3 mL of distilled water, and then dispersed for 10 minutes in an ultrasonic cleaner. The aggregate was uniformly dispersed in the water, and could not be removed from the water.

Example 3
Protein adsorption evaluation of the structure of Example 1 was performed. 1 mL of a solution containing 0.3 mg of lysozyme was added to 3 mg of the structure and stirred for 24 hours in an incubator at an internal temperature of 4°C. Then, the mixture was centrifuged at 12,000 rpm for 5 minutes to precipitate the lysozyme adsorbed to the structure. The lysozyme concentration in the supernatant after centrifugation was quantified by the Bradford method, and the amount of lysozyme adsorbed to the structure was quantified from the value. The amount of lysozyme adsorbed per 1 mg of the structure was 93 μg, and 93% of the amount of lysozyme was adsorbed.

Comparative Example 2
For comparison, a commercially available hydroxyapatite crystal (Taihei Chemical Industry Co., Ltd., HAP-100, particle size 200 nm (hereinafter the same)) was evaluated for protein adsorption in the same manner as in Example 3. The amount of lysozyme adsorbed per mg of this hydroxyapatite crystal was 29 μg, which was 29% of the amount of lysozyme adsorbed.

Example 4
The maximum adsorption capacity of the protein to the structure of Example 1 was evaluated. 1 mL of each solution containing 0.5, 1.0, 2.0, 3.0, 4.0, or 5.0 mg of lysozyme was added to 1.5 mg of the structure. The lysozyme concentration was then quantified in the same manner as in Example 3. From these quantified values, an adsorption isotherm of lysozyme to the structure was created. FIG. 2 is an adsorption isotherm showing the relationship between the equilibrium concentration of the protein and the amount of protein adsorption. The adsorption mode of lysozyme to the structure was well fitted to the Langmuir type indicating monolayer adsorption (coefficient of determination R ² =0.97), and the maximum adsorption amount calculated from the Langmuir formula was 1667 μg per 1 mg of the structure.

Comparative Example 3
For comparison, the maximum adsorption amount of lysozyme to commercially available hydroxyapatite crystals was calculated in the same manner as in Example 4. The maximum adsorption amount of lysozyme per mg of the hydroxyapatite crystals was 57 μg.

Example 5
Protein desorption evaluation of the structure of Example 1 was carried out. The amount of lysozyme adsorbed to the structure was quantified in the same manner as in Example 3. Thereafter, the structure and the lysozyme adsorbed to the structure were taken out, immersed in phosphate buffer (pH 7.4) with a concentration of 0.01, 0.20, or 0.30 mol/L, and stirred for 24 hours in an incubator at an internal temperature of 4°C. Further, the structure was centrifuged at 12,000 rpm for 5 minutes, and the lysozyme concentration contained in the supernatant after centrifugation was quantified by the Bradford method, and the proportion of lysozyme desorbed from the structure was calculated.

When immersed in 0.10 mol/L phosphate buffer, approximately 12% of lysozyme was released. In contrast, when immersed in 0.20 mol/L and 0.30 mol/L phosphate buffer, approximately 93% and 99% of lysozyme were released, respectively. Since the protein was released in a near-neutral buffer at pH 7.4, it was found that the protein could be released from the structure without denaturing it.

Example 6
The structure of Example 1 was evaluated for viral spike protein adsorption. 0.1 mL of a solution containing 5 μg of coronavirus (SARS-CoV-2) spike protein (SARS-CoV-2 Spike Protein (S-RBD) (aa319-541) mFc Tag Recombinant Protein, RP-8770, manufactured by Invitrogen) was added to 2 mg of the structure and allowed to stand at room temperature for 1 hour. Thereafter, the concentration of the spike protein in the solution was quantified by polyacrylamide electrophoresis (SDS-PAGE), and the mass of the spike protein adsorbed to the structure was quantified from the value. As a result, the mass of the spike protein adsorbed to 2 mg of the structure was 3.6 μg, and it was found that 72% of the mass of the spike protein was adsorbed.

Claims (8)

A plurality of fibrous composites each comprising a fiber and a hydroxyapatite crystal layer having a thickness of 0.3 nm to 50 nm provided directly on the surface of the fiber;
The hydroxyapatite crystal layers of the plurality of fibrous composites are partially bonded to each other to form a cotton-like structure. In claim 1,
The structure is free of unreacted crosslinker and crosslinker by-products. In claim 1 or 2,
A structure that is virtually non-dispersive in water. In any one of claims 1 to 3,
A structure in which the crystal plane on the surface of the hydroxyapatite crystal layer is a c-plane. In any one of claims 1 to 4,
The structure wherein the fibers are cellulose fibers. 6. An adsorbent having the structure according to claim 1 as a member for adsorbing a biopolymer. In claim 6,
The adsorbent, wherein the biopolymer is lysozyme or a viral spike protein. A method for producing a structure having a fibrous composite including a fiber and a hydroxyapatite crystal layer provided on a surface of the fiber, comprising:
a mixing step of mixing and stirring the dispersion liquid in which the fibers are dispersed with a first solution containing calcium ions to obtain a mixed liquid;
a drying step of drying the mixture to obtain a dried body including fibers and calcium;
a structure-forming step of immersing the dried body in a liquid and adding a second solution containing phosphate ions to obtain the structure;
A method for manufacturing a structure having the above structure.

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