Method for producing temperature-responsive hydrogels and their particles

Abstract

This invention provides a temperature-responsive material that can provide a sufficient response even in environments where water is present. [Solution] The temperature-responsive hydrogel has a structure in which a temperature-responsive polymer is bonded via an amide bond derived from the carboxyl group of at least one alginic acid selected from alginic acid, alginic acid salts, and their derivatives.

Description

[Technical Field]

[0001] This invention relates to a temperature-responsive hydrogel and a method for producing its particles. [Background technology]

[0002] Temperature-responsive polymers are being explored for application in the development of intelligent materials in the fields of biomaterials and drug delivery systems. Temperature responsiveness refers to the property of changing shape and / or properties in response to temperature (thermal) changes, both before and after the stimulus.

[0003] For example, Reference 1 discloses a temperature-responsive material consisting of a temperature-responsive polymer, fibers having a specific fiber diameter, and water, in which an attempt is made to achieve both ease of application and high mechanical properties. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] International Publication No. 2013 / 118605 [Overview of the project] [Problems that the invention aims to solve]

[0005] However, when temperature-responsive materials are used in environments where water is present, such as in living organisms, or in environments where organic solvents are present, depending on the material's composition, components may leach out, resulting in a failure to obtain the expected performance. [Means for solving the problem]

[0006] One embodiment of the temperature-responsive hydrogel according to the present invention is: The structure has a temperature-responsive polymer bonded via an amide bond derived from the carboxyl group of at least one alginic acid selected from alginic acid, alginic acid salts, and their derivatives.

[0007] One embodiment of the method for producing temperature-responsive hydrogel particles according to the present invention is: At least one alginic acid selected from alginic acid, alginic acid salts, and their derivatives, A temperature-responsive polymer in which an amino group is formed, Condensing agent and, Condensation accelerator and, The process of reacting and A step of removing the unreacted temperature-responsive polymer, It holds. [Brief explanation of the drawing]

[0008] [Figure 1] A graph showing the volume change of an unmodified hydrogel (Alg50-pNIPAM0). [Figure 2] A graph showing the volume change of Alg50-pNIPAM0.5. [Figure 3] A graph showing the volume change of Alg50-pNIPAM0.75. [Figure 4] A graph showing the volume change of Alg50-pNIPAM1. [Figure 5] A graph showing the volume change of Alg50-pNIPAM2.5. [Figure 6] A graph showing the volume change of Alg50-pNIPAM5. [Figure 7] A graph showing the volume change of Alg + Alg50-pNIPAM5 in a 1:1 ratio. [Figure 8] A graph comparing the volume changes of Alg50-pNIPAM0, Alg50-pNIPAM0.5, Alg50-pNIPAM2.5, and Alg50-pNIPAM5. [Modes for carrying out the invention]

[0009] Embodiments of the present invention will be described below. The embodiments described below illustrate examples of the present invention. The present invention is not limited to the following embodiments, and also includes various modified forms implemented within the scope of not changing the gist of the present invention. Note that not all of the configurations described below are essential configurations of the present invention.

[0010] 1. Temperature-responsive hydrogel The temperature-responsive hydrogel according to the present embodiment has a structure in which a temperature-responsive polymer is bonded via an amide bond derived from a carboxy group of at least one alginic acid selected from alginic acid, salts of alginic acid, and derivatives thereof.

[0011] 1.1. Alginic acid, salts of alginic acid, and derivatives thereof Alginic acid is a β-1,4-linked heteropolymer composed of D-mannuronic acid (M) and L-guluronic acid (G). Alginic acid can be extracted from algae. Alginic acid is one of the acidic polysaccharides present in the extracellular matrix of brown algae.

[0012] Examples of salts of alginic acid include ammonium salts, potassium salts, calcium salts, and sodium salts, and it is not necessary for all carboxy groups of alginic acid to be in the form of salts. The calcium salt of alginic acid forms a cross-linked structure between two carboxy groups of alginic acid by divalent calcium ions and gels, but as long as there are carboxy groups not involved in the cross-linking, it can form an amide bond with the temperature-responsive polymer described below. However, when selecting a salt of alginic acid, it is preferable to choose a non-calcium salt because it is less likely to form a cross-linked structure and can be advantageous for the reaction described below. The salt of alginic acid may be a mixture of ammonium ions, potassium ions, calcium ions, and sodium ions.

[0013] Alginic acid or a salt of alginic acid may be derivatized. Examples of derivatization include esterification, and some carboxy groups of alginic acid or a salt of alginic acid may be ester groups. Examples of the compound that forms an ester bond with them include propylene glycol, ethylene glycol, diethylene glycol, and the like.

[0014] 1.2. Temperature-responsive polymer In the temperature-responsive hydrogel of the present embodiment, a temperature-responsive polymer is chemically bonded via an amide bond derived from the carboxy group of the above-mentioned alginic acids. The temperature-responsive polymer can be selected from an LCST-type temperature-responsive polymer and a UCST-type temperature-responsive polymer.

[0015] As the temperature-responsive polymer, it is preferable to use an LCST-type temperature-responsive polymer, and it is more preferable that it is at least one selected from poly(N-alkylacrylamide), poly(N-vinylalkylamide), polyvinyl alkyl ether, polyethylene glycol / polypropylene glycol block copolymer, poly(2-alkyl-2-oxazoline), and mixtures thereof.

[0016] Furthermore, in the temperature-responsive hydrogel of the present embodiment, it is more preferable to use poly(N-alkylacrylamide) as the temperature-responsive polymer, and it is particularly preferable to use poly(N-isopropylacrylamide). Using such a temperature-responsive polymer makes it easy to obtain a hydrogel having a phase transition temperature closer to the human body temperature, and also makes it possible to make the hysteresis of physical properties such as biocompatibility and volume change smaller, which is preferable. In particular, poly(N-isopropylacrylamide) is also preferable in terms of easy availability.

[0017] Temperature-responsive polymers preferably have amino groups so that they can form amide bonds with the carboxyl groups of alginic acid. These amino groups react with the carboxyl groups of alginic acid to form amide bonds. If the temperature-responsive polymer does not have amino groups, amino groups can be introduced using appropriate reagents such as 2-aminoethanethiol. Alternatively, commercially available temperature-responsive polymers with introduced amino groups may be obtained and used. The position of the amino groups in the temperature-responsive polymer is not particularly limited, but it is more preferable for them to be introduced at the ends of the polymer.

[0018] Examples of commercially available temperature-responsive polymers with incorporated amino groups include poly(N-isopropylacrylamide), amine-terminated (manufactured by Sigma-Aldrich) (pNIPAM-NH2).

[0019] A temperature-responsive hydrogel can be obtained by reacting the amino groups of a temperature-responsive polymer with the carboxyl groups of alginic acid, alginic acid salts, or derivatives thereof, in which the two are chemically bonded via amide bonds.

[0020] The proportion of alginates and temperature-responsive polymers in a temperature-responsive hydrogel can be adjusted by changing the ratio of the molar equivalents of carbonyl groups in the alginates to the molar equivalents of amino groups in the temperature-responsive polymers.

[0021] The ratio of alginates to temperature-responsive polymers is preferably 100:1 to 10:1, more preferably 50:1 to 15:1, and more preferably 30:1 to 20:1, where (A) is the molar equivalent of the carbonyl group of the alginate and (B) is the molar equivalent of the amino group of the temperature-responsive polymer. When the ratio A:B is below the upper limit, the shape stability of the temperature-responsive hydrogel is better, and when it is above the lower limit, the temperature responsiveness is more pronounced.

[0022] 1.3. Effects and Effects In this embodiment, the temperature-responsive hydrogel has a temperature-responsive polymer chemically bonded via amide bonds derived from the carboxyl groups of alginic acid. Therefore, the elution of the temperature-responsive polymer into the environment (water, organic solvents, biological materials) is suppressed. This reduces the degradation of temperature responsiveness in such environments, extending the material's lifespan.

[0023] 2. Method for producing temperature-responsive hydrogel particles The temperature-responsive hydrogel particles described above can be manufactured, for example, as follows:

[0024] The method for producing temperature-responsive hydrogel particles according to this embodiment comprises the steps of reacting at least one alginic acid selected from alginic acid, alginic acid salts, and their derivatives with a temperature-responsive polymer on which an amino group has been formed, a condensing agent, and a condensation accelerator, and removing the unreacted temperature-responsive polymer.

[0025] Alginic acid derivatives, temperature-responsive polymers, and methods for introducing amino groups into temperature-responsive polymers have already been described.

[0026] An example of a condensing agent is, but is not limited to, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (WSC). Similarly, an example of a condensation accelerator is, but is not limited to, H-hydroxysuccinimide (NHS).

[0027] The reaction can be carried out, for example, in pure water. This is done by stirring a solution of alginates and a temperature-responsive polymer to obtain a homogeneous solution, and then adding WSC and NHS and stirring. The amounts of WSC and NHS to be added can be set as appropriate, but it is preferable that the amount of WSC be 2 to 10 times, preferably 4 to 6 times, the molar equivalent of the molar equivalent of the amino groups of the temperature-responsive polymer (B), and that the amount of NHS be 5 to 50 times, preferably 10 to 30 times, the molar equivalent of the molar equivalent of the amino groups of the temperature-responsive polymer (B).

[0028] The step of removing unreacted temperature-responsive polymers can be carried out, for example, by dropping the hydrogel obtained above into an aqueous solution of calcium bromide (e.g., a 2% by mass aqueous solution) or an aqueous solution of calcium chloride (e.g., a 2% by mass aqueous solution) and stirring. Such a washing operation can be performed multiple times as needed.

[0029] In this way, the temperature-responsive hydrogel particles of this embodiment can be easily manufactured.

[0030] 3. Examples, etc. The present invention will be described in more detail below with reference to experimental examples, but the present invention is not limited to these examples. Unless otherwise specified, "%" below refers to mass.

[0031] 3.1.Materials Sodium alginate (Alg) (80-120 cp, Wako, Osaka, Japan) was used as the alginic acid, and poly(N-isopropylacrylamide),amine terminated (pNIPAM-NH2) (Mn; 5,500, Sigma-Aldrich, USA) was used as the temperature-responsive polymer. 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide,hydrochloride (WSC) (TCI, Tokyo, Japan) was used as the condensing agent, and N-hydroxysuccinimide (NHS) (Wako, Osaka, Japan) was used as the auxiliary agent.

[0032] 3.2. Preparation of temperature-responsive hydrogels Alg was added to Milli-Q water to a concentration of 50 mmol / L (moles of carbonyl groups) and pNIPAM-NH2 to a concentration of 5 mmol / L (moles of amino groups), and the mixture was reacted at room temperature for 24 hours with stirring. Subsequently, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was used as a coupling agent. Add 25 mmol / L of (EDC) and 100 mmol / L of N-hydroxysuccinimide (NHS) as a condensation aid, and react at room temperature for 24 hours with stirring to form a hydrogel (Sample name: Alg 50 -pNIPAM5) was obtained. The obtained reaction product was added dropwise to a 2 wt% CaCl2 aqueous solution, and Alg 50 -pNIPAM5 hydrogel beads were prepared. Subsequently, the hydrogel beads were washed three times by replacing the 2wt% CaCl2 aqueous solution and stirring at room temperature for 24 hours.

[0033] The amount of Alg charged was fixed at 50 mmol / L (number of moles of carbonyl groups), the amount of pNIPAM-NH2 charged was changed to 0 mmol / L (unmodified), 0.5 mmol / L, 0.75 mmol / L, 1 mmol / L, and 2.5 mmol / L, and the amounts of EDC and NHS added were changed as shown in Table 1.

[0034] [Table 1]

[0035] Otherwise, proceed as described above, Alg 50 -pNIPAM 0.5 Hydrogel beads, Alg 50 -pNIPAM 0.75 Hydrogel beads, Alg 50 -pNIPAM 2.5 Hydrogel beads were each created.

[0036] 3.3. Evaluation of volume change and strength Each of the washed hydrogel beads prepared above was placed in an assembled cell, and the gap was filled with an aqueous CaCl2 solution. The cell was then set on a thermoplate and kept at 50 °C. Subsequently, the temperature was repeatedly increased to 50 °C or decreased to 25 °C multiple times, and the change in the projected area was observed under a microscope. The degree of volume change was quantified by measuring the projected area in an image. The projected area at 50 °C was normalized with a value of 1. Thus, the volume change of each hydrogel in an aqueous solution was evaluated. Since the lower critical solution temperature (LCST) of pNIPAM is around 32 °C, 50 °C and 25 °C were selected as the temperatures for the temperature cycle.

[0037] Note that, as a control, a hydrogel of unmodified Alg alone (Alg 50 -pNIPAM0), and a gel obtained by mixing unmodified Alg (Alg 50 -pNIPAM0) and Alg 50 -pNIPAM5 at a ratio of 1:1 were similarly subjected to evaluation.

[0038] Each hydrogel bead was sandwiched between cover glasses, and a simple strength comparison was visually performed. Also, in each evaluation, the number of samples (n number) was set to 3.

[0039] 3.4. Evaluation Results Figs. 1 to 8 show the volume change of the hydrogel beads obtained above. In the experiment, the temperature cycle was photographed in the order of 25 °C initial state → 50 °C for 1 hour → 25 °C for 1 hour ···. The initial state was denoted as (0), 1 hour after heating as (1), and 1 hour after cooling as (2) in sequence. In each figure, the horizontal axis was denoted as "50(1)" for 50 °C for 1 hour or "25(2)" for 25 °C for 1 hour. Also, the vertical axis of each figure was normalized with the volume at the time of "50(1)".

[0040] Looking at Fig. 1, it can be seen that the hydrogel of alginic acid into which pNIPAM has not been introduced hardly undergoes a volume change due to a temperature change. The particle shape of unmodified alginic acid was maintained, and there was no problem with mechanical strength.

[0041] Figure 2 shows the temperature dependence of the volume of a hydrogel where the molar equivalent of the carbonyl groups of alginates is (A) and the molar equivalent of the amino groups of pNIPAM is (B), with a ratio of A:B of 50:0.5. A volume change of approximately 1-2% was observed with temperature changes. The shape of the hydrogel particles was maintained.

[0042] Figure 3 shows the temperature dependence of the volume of a hydrogel with a ratio of A:B of 50:0.75. A volume change of approximately 3-5% was observed with temperature changes. The shape of the hydrogel particles was maintained.

[0043] Figure 4 shows the temperature dependence of the volume of a hydrogel with a ratio of A:B of 50:1. A volume change of approximately 3-5% was observed with temperature changes. The shape of the hydrogel particles was maintained.

[0044] Figure 5 shows the temperature dependence of the volume of a hydrogel with a ratio of A:B of 50:2.5. A volume change of approximately 100% was observed with temperature changes. The hydrogel particles were somewhat difficult to mold and had slightly insufficient mechanical strength.

[0045] Figure 6 shows the temperature dependence of the volume of a hydrogel with a ratio of A:B of 50:5. A volume change of approximately 100% was observed with temperature changes. The hydrogel particles were somewhat difficult to mold and had slightly insufficient mechanical strength.

[0046] Figure 7 shows the temperature dependence of the volume of a hydrogel obtained by mixing unmodified alginate with a hydrogel having an A:B ratio of 50:5 in a 1:1 weight ratio. A volume change of approximately 50-60% was observed with temperature changes. The shape of the hydrogel particles was maintained.

[0047] Figure 8 compares the temperature dependence of the volume of hydrogels with A:B ratios of 50:0, 50:0.5, 50:2.5, and 50:5. The graphs in Figure 8 are displayed with the vertical axis scales aligned. It was found that a clear temperature response is obtained when the amount of pNIPAM introduced is 50:2.5 or higher.

[0048] The embodiments and variations described above are merely examples and are not limited thereto. For example, each embodiment and each variation can be combined as appropriate.

[0049] The present invention includes configurations substantially identical to those described in the embodiments, for example, configurations with the same function, method, and results, or configurations with the same purpose and effect. Furthermore, the present invention includes configurations in which non-essential parts of the configurations described in the embodiments are replaced. Furthermore, the present invention includes configurations that produce the same effects or achieve the same purpose as those described in the embodiments. Finally, the present invention includes configurations that add known technology to the configurations described in the embodiments.

Claims

1. A temperature-responsive hydrogel having a structure in which a temperature-responsive polymer is bonded via an amide bond derived from the carboxyl group of at least one alginic acid selected from alginic acid, alginic acid salts, and derivatives thereof.

2. In claim 1, The amide bond is derived from an amino group formed on the temperature-responsive polymer, resulting in a temperature-responsive hydrogel.

3. In claim 1 or claim 2, The temperature-responsive polymer is at least one selected from poly(N-alkylacrylamide), poly(N-vinylalkylamide), polyvinyl alkyl ether, polyethylene glycol / polypropylene glycol block copolymer, poly(2-alkyl-2-oxazoline), and mixtures thereof, in a temperature-responsive hydrogel.

4. In claim 1 or claim 2, The temperature-responsive polymer is poly(N-isopropylacrylamide), and the hydrogel is temperature-responsive.

5. At least one alginic acid selected from alginic acid, alginic acid salts, and their derivatives, A temperature-responsive polymer in which an amino group is formed, Condensing agent and, Condensation accelerator and, The process of reacting and A step of removing the unreacted temperature-responsive polymer, A method for producing temperature-responsive hydrogel particles having [a certain characteristic].

6. In claim 5, A method for producing temperature-responsive hydrogel particles, further comprising the step of forming amino groups in the temperature-responsive polymer.

7. In claim 5 or claim 6, A method for producing temperature-responsive hydrogel particles, wherein the temperature-responsive polymer is at least one selected from poly(N-alkylacrylamide), poly(N-vinylalkylamide), polyvinyl alkyl ether, polyethylene glycol / polypropylene glycol block copolymer, poly(2-alkyl-2-oxazoline), and mixtures thereof.

8. In claim 5 or claim 6, A method for producing temperature-responsive hydrogel particles, wherein the temperature-responsive polymer is poly(N-isopropylacrylamide).

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