Shellac modified object

Abstract

To provide a shellac modified object which can be changed from a state where cell adhesiveness to a mammalian cell is high, to a state where the cell adhesiveness is low.SOLUTION: A shellac modified object is expressed by a formula (1). In the formula, Z is a part excluding a carboxy group modified by a functional group Y in the structure of the shellac, X1 to X4 are a dialkylamino group, alkylamino group, hydroxy group, halogen group, H or the like.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present disclosure relates to shellac modified products.

Background Art

[0002] Conventionally, shellac has been used in a wide range of fields. Patent Document 1 discloses a shellac modified product. In the shellac modified product, at least one of the hydroxyl group and the carboxyl group in the structure of shellac is chemically modified. The shellac modified product has cell adhesiveness to mammalian cells. The shellac modified product can be used, for example, as a material for products in the medical field.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] It is preferable that the shellac modified product can change from a state with high cell adhesiveness to mammalian cells to a state with low cell adhesiveness. In this case, the applications of the shellac modified product are expanded. In one aspect of the present disclosure, it is preferable to provide a shellac modified product that can change from a state with high cell adhesiveness to mammalian cells to a state with low cell adhesiveness.

Means for Solving the Problems

[0005] One aspect of the present disclosure is a shellac modified product represented by formula (1).

[0006]

Chemical Formula

[0007] In formula (1), Z is the portion of the structure of the sella turcica excluding the carboxy group modified by the functional group Y. X1, X2, X3, and X4 in formula (1) are each independently, a dialkylamino group, an alkylamino group, a hydroxy group, a halogen group, an alkoxy group, an amino group, an acyl group, an alkyl group, an alkanate group, or hydrogen.

[0008] One aspect of the present disclosure, the sella turcica modified product, can change from a state with high cell adhesiveness to mammalian cells to a state with low cell adhesiveness. Another aspect of the present disclosure is a sella turcica modified product represented by formula (2).

[0009]

Chemical formula

[0010] In formula (2), Z is the portion of the structure of the sella turcica excluding the carboxy group modified by the functional group Y. X1, X2, X3, and X4 in formula (2) are each independently a dialkylamino group, an alkylamino group, a hydroxy group, a halogen group, an alkoxy group, an amino group, an acyl group, an alkyl group, an alkanate group, or hydrogen.

[0011] Another aspect of the present disclosure, the sella turcica modified product, can change from a state with high cell adhesiveness to mammalian cells to a state with low cell adhesiveness. Another aspect of the present disclosure is a sella turcica modified product represented by formula (3).

[0012]

Chemical formula

[0013] In formula (3), Z is the part of the shellac structure excluding the carboxy group modified by the functional group Y. In formula (3), X5 is a dialkylamino group, an alkylamino group, a hydroxy group, a halogen group, an alkoxy group, an amino group, an acyl group, an alkyl group, an alkanate group, or hydrogen.

[0014] Another aspect of the present disclosure, the shellac-modified product, can change from a state with high cell adhesiveness to mammalian cells to a state with low cell adhesiveness. Another aspect of the present disclosure is a shellac-modified product represented by formula (4).

[0015] [Chemical formula]

[0016] In formula (4), Z is the part of the shellac structure excluding the carboxy group modified by the functional group Y. Group. Another aspect of the present disclosure, the shellac-modified product, can change from a state with high cell adhesiveness to mammalian cells to a state with low cell adhesiveness. [Brief Description of the Drawings]

[0017]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Embodiments for Carrying Out the Invention

[0018] Exemplary embodiments of the present disclosure will be described with reference to the drawings. 1. Composition of the shellac modified product The shellac modified product of the present disclosure is one in which the carboxy groups in the structure of shellac are chemically modified. Shellac is a natural product purified from a resinous substance secreted by lac insects, which are about 0.6 mm in body length, on the branches of specific trees such as leguminous plants and mulberry trees. The main structure of shellac is, for example, one in which about 1 to 5 structural units are oligomerized by esterification. The structural unit is, for example, one in which a long-chain fatty acid such as aleuritic acid and a resin acid such as jalapinic acid or laccijalaric acid are ester-bonded.

[0019] The shellac modified product of the present disclosure is represented, for example, by formula (1).

[0020]

Chemical formula

[0021] In formula (1), Z is the part of the shellac structure excluding the carboxy group modified by the functional group Y. X1, X2, X3, and X4 in formula (1) are each independently a dialkylamino group, an alkylamino group, a hydroxy group, a halogen group, an alkoxy group, an amino group, an acyl group, an alkyl group, an alkanate group, or hydrogen.

[0022] The shellac modified product of the present disclosure is represented, for example, by formula (2).

[0023]

Chemical formula

[0024] In formula (2), Z is the portion of the shellac structure excluding the carboxy group modified by the functional group Y. X1, X2, X3, and X4 in formula (2) are each independently a dialkylamino group, an alkylamino group, a hydroxy group, a halogen group, an alkoxy group, an amino group, an acyl group, an alkyl group, an alkanate group, or hydrogen.

[0025] The shellac modified product of the present disclosure is represented, for example, by formula (3).

[0026]

Chemical formula

[0027] In formula (3), Z is the portion of the shellac structure excluding the carboxy group modified by the functional group Y. X5 in formula (3) is a dialkylamino group, an alkylamino group, a hydroxy group, a halogen group, an alkoxy group, an amino group, an acyl group, an alkyl group, an alkanate group, or hydrogen.

[0028] The shellac modified product of the present disclosure is represented, for example, by formula (4).

[0029]

Chemical formula

[0030] In formula (4), Z is the portion of the shellac structure excluding the carboxy group modified by the functional group Y. Z in formulas (1) to (4) has, for example, the structure shown in FIG. 1. R1 in FIG. 1 is CHO or COOH. R2 in FIG. 1 is CH3 or CH2OH. n in FIG. 1 is a natural number of 0 or more and 15 or less. When producing the shellac modified product, the carboxy group on the right side of Z in FIG. 1 is modified by the functional group Y.

[0031] Examples of the dialkylamino group in X1 to X5 include a diethylamino group and the like. Examples of the alkylamino group in X1 to X5 include an ethylamino group and the like. Examples of the halogen group in X1 to X5 include a chloro group and the like. Examples of the alkoxy group in X1 to X5 include a methoxy group and the like. Examples of the acyl group in X1 to X5 include an acetyl group and the like. Examples of the alkyl group in X1 to X5 include a methyl group and the like. Examples of the alkanate group in X1 to X5 include a methanoate group and the like.

[0032] The shellac-modified product of the present disclosure has cell adhesiveness to mammalian cells. However, when the shellac-modified product is irradiated with light, the cell adhesiveness to mammalian cells decreases compared to before the light irradiation. Therefore, the shellac-modified product of the present disclosure can change from a state of high cell adhesiveness to mammalian cells to a state of low cell adhesiveness. Note that the state of low cell adhesiveness to mammalian cells is not necessarily limited to a state where there is no cell adhesiveness at all. For example, if it is lower than the cell adhesiveness when not irradiated with light, it corresponds to the state of low cell adhesiveness.

[0033] When reducing the cell adhesiveness of the shellac-modified product, the wavelength of the light irradiated on the shellac-modified product is, for example, 230 to 600 nm. The irradiation time of the light is, for example, 1 to 720 minutes. When the shellac-modified product of the present disclosure is irradiated with light, the functional group Y detaches from the shellac-modified product. After the functional group Y detaches, the shellac has reduced cell adhesiveness to mammalian cells.

[0034] The shellac-modified product of the present disclosure can be used, for example, as a material for products in the medical field. Examples of products in the medical field include DDS carriers and the like. The shellac-modified product of the present disclosure is excellent, for example, in terms of thermosetting properties, film-forming properties, electrical insulation properties, high miscibility with added components, biodegradability, biocompatibility, and the property of sustained release of pharmaceuticals.

[0035] The cerac modified product of the present disclosure can be used, for example, as follows. A film of the cerac modified product is formed. Next, mammalian cells are seeded on the surface of the film. Next, the mammalian cells are cultured on the film. Note that since the cerac modified product has cell adhesiveness to mammalian cells in a state where it is not irradiated with light, it is possible to culture mammalian cells on the film of the cerac modified product.

[0036] Next, the film is irradiated with light. At this time, the functional group Y is detached from the cerac modified product constituting the film. After the light irradiation, the cell adhesiveness of the film to mammalian cells decreases compared to before the light irradiation. As a result, the mammalian cells cultured on the film are peeled off from the film while alive. For example, a plurality of mammalian cells peeled off from the film are alive and form a mass. Next, the mammalian cells peeled off from the film and alive are recovered.

[0037] 2. Examples (2-1) Synthesis of DEAC-modified cerac 5.00 g of raw material cerac (purified cerac PEARL-N811; manufactured by Gifu Ceramics Co., Ltd.) was dissolved in 40 mL of dehydrated dimethylformamide (FUJIFILM Wako Pure Chemical Industries, Ltd.) to prepare a cerac solution. Next, 2.29 g of 4-(bromomethyl)-7-(diethylamino) coumarin (4-(Bromomethyl)-7-(diethylamino) coumarin, Tokyo Chemical Industry Co., Ltd.) and 1.66 g of potassium carbonate (FUJIFILM Wako Pure Chemical Industries, Ltd.) were added to the cerac solution. The addition amount of 4-(bromomethyl)-7-(diethylamino) coumarin was 1.1 equivalents to the acid value of cerac. The addition amount of potassium carbonate was 2 equivalents to the acid value of cerac.

[0038] Next, heating and stirring were carried out at 60 °C for 3 hours under light shielding. Next, after allowing to cool to room temperature, the reaction solution was filtered. At this time, most of the potassium carbonate was filtered off. Next, the solvent of the filtrate was distilled off under reduced pressure. Next, the residue obtained by distillation under reduced pressure was dropped into hexane to insolubilize and precipitate diethylaminocoumarin-modified shellac (hereinafter referred to as DEAC-modified shellac). The state of the precipitated DEAC-modified shellac was a viscous oily substance.

[0039] Next, the precipitate composed of DEAC-modified shellac was separated from the supernatant component. Next, the precipitate was dissolved in ethanol and filtered to remove insoluble matter. Next, the filtrate was dropped into hexane again, the supernatant was removed, and the precipitate was dried in vacuo to obtain 4.93 g of DEAC-modified shellac. DEAC-modified shellac is a shellac-modified product represented by formula (4). The functional group Y is a diethylaminocoumarin group. In the obtained DEAC-modified shellac, Z is represented in Figure 1. R1 in Z is a formyl group or a carboxylic acid group, R2 is a hydroxymethyl group or a methyl group, and n is about 0 to 10.

[0040] When DEAC-modified shellac is formed, the bromomethyl group contained in 4-(bromomethyl)-7-(diethylamino) coumarin reacts with the carboxy group contained in the structure of shellac. When reacting, the hydrogen of the carboxy group and the bromine of the bromomethyl group are eliminated. The carboxy group contained in the structure of shellac is modified by the diethylaminocoumarin group.

[0041] (2-2) Calculation of functional group introduction rate The functional group introduction rate of DEAC-modified shellac is the ratio of the molar amount of the modified carboxyl groups in DEAC-modified shellac to the molar amount of the carboxyl groups in the raw material shellac. The functional group introduction rate was calculated by the following method. In DEAC-modified shellac, the molar amount of the carboxyl groups remaining unmodified (hereinafter referred to as the residual molar amount) was determined by titration using a pH indicator and TFA. Based on the determined residual molar amount, the functional group introduction rate was calculated. The functional group introduction rate was 94%.

[0042] (2-3)Confirmation of the synthesis of DEAC-modified shellac Using electrospray ionization mass spectrometry (ESI-MS), it was confirmed that DEAC-modified shellac was successfully synthesized. Specifically, the following operations were performed.

[0043] An ethanol solution of the raw material shellac was prepared. The concentration of the raw material shellac in the ethanol solution was 4 mg / mL. The ethanol solution of the raw material shellac was subjected to ESI-MS (Synapt G2 HDMS; Waters) for mass spectrometry.

[0044] An ethanol solution of DEAC-modified shellac was also prepared. The concentration of DEAC-modified shellac in the ethanol solution was 4 mg / mL. Next, the ethanol solution of DEAC-modified shellac was subjected to ESI-MS for mass spectrometry.

[0045] The results of the mass spectrometry of the raw material shellac and the mass spectrometry of DEAC-modified shellac are shown in Figure 2. Shellac is a mixture of oligoesters with different degrees of polymerization. In the results of the mass spectrometry of DEAC-modified shellac, compared with the results of the mass spectrometry of the raw material shellac, [M-H] + , [M-Na] + , [M-K] + and other peaks corresponding to the diethylaminocoumarin group An increase in the mass to be produced (i.e., the mass of ~129) was observed. The diethylaminocoumarin group corresponds to the functional group Y. From this, it was confirmed that DEAC-modified serac could be synthesized.

[0046] (2-4) Confirmation that the functional group Y is eliminated from the DEAC-modified serac when irradiated with light An ethanol solution of DEAC-modified serac was prepared. The concentration of DEAC-modified serac in the ethanol solution was 4 mg / mL. Next, without irradiating with light, the ethanol solution of DEAC-modified serac was subjected to ESI-MS for mass spectrometry.

[0047] In addition, the ethanol solution of DEAC-modified serac prepared as described above was irradiated with light having a wavelength of 380 nm at room temperature for 2 hours using an LED light source (manufactured by Asahi Spectra). Next, the ethanol solution of DEAC-modified serac after irradiation with light was subjected to ESI-MS for mass spectrometry.

[0048] The results of mass spectrometry without irradiation with light and mass spectrometry with irradiation with light are shown in Fig. 3. In the result of mass spectrometry with irradiation with light, compared with the result of mass spectrometry without irradiation with light, [M-H] derived from each oligoester + , [M-Na] + , [M-K] + At peaks such as , a decrease in the mass corresponding to the diethylaminocoumarin group (i.e., the mass of ~129) was observed. From this, it was confirmed that by irradiating with light having a wavelength of 380 nm, the diethylaminocoumarin group is eliminated from the DEAC-modified serac and the structure of the raw material serac is restored.

[0049] (2-5) Cell proliferation evaluation (Part 1) (a1) In the case of the raw material serac An ethanol solution of the raw material serac was prepared. The concentration of the raw material serac in the ethanol solution was 50 (w / v)%. This solution and a spin coater (MS-B100, Mikasa Co., Ltd.) Using a spin coater (manufactured by [manufacturer name]), a spin-coated film was fabricated on a round cover glass with a diameter of 15 mm.

[0050] After the spin-coated film was dried, the round cover glass was set on the bottom of a 24-well plate. Next, NIH3T3 cells were seeded on the spin-coated film at a density of 1×10 4 cells / well. NIH3T3 cells correspond to mammalian cells.

[0051] Next, the NIH3T3 cells were cultured in a CO2 incubator under conditions of a CO2 concentration of 5% and a temperature of 37°C for a predetermined period of time. The culture medium was changed 3 and 5 days after the start of the culture. The number of cells 1, 3, and 5 days after the start of the culture was measured using a Cell Counting Kit-8 (manufactured by Dojindo Laboratories ).

[0052] (b1) In the case of DEAC-modified chitosan without light irradiation Basically, cell proliferation was evaluated in the same manner as in the case of (a1) above. However, DEAC-modified chitosan was used instead of the raw chitosan. Note that, unlike (c1) described later, light with a wavelength of 380 nm was not irradiated onto the spin-coated film.

[0053] (c1) In the case of DEAC-modified chitosan with light irradiation Basically, cell proliferation was evaluated in the same manner as in the case of (b1) above. However, after the round cover glass was set on the bottom of the 24-well plate, the spin-coated film was irradiated with light having a wavelength of 380 nm using an LED light source (manufactured by Asahi Spectra Co., Ltd.) at room temperature for 30 minutes. After the light irradiation, NIH3T3 cells were seeded on the spin-coated film.

[0054] (d1) In the case of the control A control slide glass was set on the bottom of a 24-well plate. Next, NIH3T3 cells were seeded on the surface of the slide glass at a density of 1×10 4 cells / well. Next, in a CO2 incubator, the CO2 concentration was 5% and the temperature was 37°C NIH3T3 cells were cultured for the specified time under the above conditions. The medium was replaced 3 and 5 days after the start of culture. The cell count was counted 1, 3, and 5 days after the start of culture using Cell Counting Kit-8 (Dojindo Chemical Industries, Ltd.). The measurements were performed using a ferroelectric liquid crystal display (manufactured by the Institute of Electrical and Electronics Engineering).

[0055] The measurement results of (a1) to (d1) are shown in FIG. 4. In the case of DEAC-modified shellac that was not irradiated with light, the number of cells increased. This confirmed that NIH3T3 cells could grow at a sufficient growth rate on DEAC-modified shellac that was not irradiated with light. The fact that NIH3T3 cells grew indicates that DEAC-modified shellac that was not irradiated with light has high cell adhesiveness to NIH3T3 cells. On the other hand, in the case of raw shellac and DEAC-modified shellac that was irradiated with light, the number of cells was difficult to increase. The fact that the number of cells was difficult to increase indicates that raw shellac and DEAC-modified shellac that was irradiated with light have low cell adhesiveness to NIH3T3 cells.

[0056] (2-6) Cell proliferation evaluation (part 2) (a2) In the case of raw shellac Cell proliferation was evaluated in the same manner as in (a1) above.

[0057] (b2) DEAC-modified shellac without light irradiation Cell proliferation was evaluated in the same manner as in (b1) above. (c2) DEAC-modified shellac exposed to light The cell proliferation evaluation was carried out basically in the same manner as in (c1) above. However, the method of irradiating the spin-coated film with light was different from that in (c1) above. Specifically, the round cover glass was immersed in phenol red-free DEM medium, and the spin-coated film was irradiated with light of 380 nm wavelength using an LED light source (manufactured by Asahi Spectroscopy) at room temperature for 30 minutes. The volume of the DEM medium was 500 μL. After the light irradiation, NIH3T3 cells were seeded on the spin-coated film.

[0058] (d2) Control In the same manner as (d1), cell growth was evaluated. The measurement results of (a2) to (d2) are shown in Fig. 5. In the case of DEAC-modified serac that was not irradiated with light, the number of cells increased. From this, it was confirmed that NIH3T3 cells can proliferate at a sufficient growth rate on DEAC-modified serac without light irradiation. The fact that NIH3T3 cells proliferated indicates that DEAC-modified serac without light irradiation has high cell adhesiveness to NIH3T3 cells. On the other hand, in the case of the raw material serac and the DEAC-modified serac irradiated with light, it was difficult for the number of cells to increase. The difficulty in increasing the number of cells indicates that the raw material serac and the DEAC-modified serac irradiated with light have low cell adhesiveness to NIH3T3 cells. (2-7) Implementation of cell growth and detachment An ethanol solution of DEAC-modified serac was prepared. The concentration of DEAC-modified serac in the ethanol solution was 50 (w / v)%. Using this solution and a spin coater (MS-B100, manufactured by Mikasa), a spin-coated film was prepared on a round cover glass with a diameter of 15 mm. After drying the spin-coated film, the round cover glass was set on the bottom of a 24-well plate.

[0059] Next, NIH3T3 cells were seeded on the spin-coated film at 1×10 4 cells / well. Next, NIH3T3 cells were cultured for 7 days in a CO2 incubator under the condition that the CO2 concentration was 5%. Next, the medium was replaced with DEM medium without phenol red. Next, the spin-coated film was irradiated with light at 380 nm for 30 minutes at room temperature using an LED light source.

[0060] Next, the cells detached from the spin-coated film were suspended by pipetting and collected. A cell double staining kit (Dojindo Laboratories) was added to the collected cells, and the ratio of live cells to dead cells was evaluated by confocal microscopy observation.

[0061] In living cells, Calcein-AM is introduced and shows green fluorescence. In dead cells, PI binds to DNA and shows red fluorescence. Most of the detached cell clumps showed green fluorescence. From this, it was confirmed that by irradiating light on the membrane of DEAC-modified cellulose, cells can be detached from the membrane of DEAC-modified cellulose while remaining alive.

[0062] Also, the detached cells were transferred to another culture plate and observed with a differential interference microscope. Figure 6 shows the image of the differential interference microscope and the image diagram of the detached cells. Fibroblasts such as NIH3T3 cells secrete extracellular matrix outside the cell. Therefore, as shown in the part of the image diagram in Figure 6 and Figure 7, the cells 3 on the culture plate 1 are adhered to each other through the extracellular matrix 5. The cells 3 detached from the membrane of DEAC-modified cellulose were in the form of a mass of multiple cells 3. This result indicated that the cells 3 were detached from the culture plate 1 because the affinity (i.e., cell adhesiveness) between the cells 3 and the culture plate 1 changed significantly due to light irradiation.

[0063] 3. Other Embodiments As described above, the embodiments of the present disclosure have been described. However, the present disclosure is not limited to the above-described embodiments and can be implemented with various modifications.

[0064] (1) A cellulose-modified product represented by any of formulas (1) to (4). Cellulose-modified products other than DEAC-modified cellulose can basically be produced in the same manner as in the examples. In this case, for example, instead of 4-(bromomethyl)-7-(diethylamino)coumarin, a raw material containing the target functional group Y can be used. The raw material containing the target functional group Y contains, for example, a bromomethyl group. The bromomethyl group reacts with the carboxyl group provided in the structure of cellulose. As a result of this reaction, the hydrogen of the carboxyl group and the bromine of the bromomethyl group are eliminated. The carboxyl group provided in the structure of cellulose is modified by the functional group Y.

[0065] (2) The functions of one component in each of the above embodiments may be shared among a plurality of components, or the functions of a plurality of components may be exerted by one component. Also, a part of the configuration of each of the above embodiments may be omitted. Further, at least a part of the configuration of each of the above embodiments may be added to, replaced with, etc., the configuration of other of the above embodiments.

[0066] (3) In addition to the above-described shellac-modified product, the present disclosure can also be realized in various forms such as a product having the shellac-modified product as a component, a method for producing the shellac-modified product, and the like.

Explanation of Reference Numerals

[0067] 1... culture plate, 3... cell, 5... extracellular matrix, Y... functional group, Z... part of the shellac structure excluding the carboxy group modified by the functional group Y

Claims

1. A shellac-modified compound represented by formula (1). 【Chemical 1】 In formula (1), Z is the part of the structure of the shellac excluding the carboxy group modified by the functional group Y. X in formula (1) １ , X ２ , X ３ , X ４ is each independently a dialkylamino group, an alkylamino group, a hydroxy group, a halogen group, an alkoxy group, an amino group, an acyl group, an alkyl group, an alkanate group, or hydrogen.

2. A shellac-modified compound represented by formula (2). [Chemical Formula 2] In formula (2), Z is a carboxyl group in the structure of shellac that is modified by the functional group Y. It is the part excluding the Si group. X in formula (2) １ , X ２ , X ３ , X ４ are each independently a dialkylamino group, an alkylamino group, a hydroxy group, a halogen group, an alkoxy group, an amino group, an acyl group, an alkyl group, an alkanate group, or hydrogen.

3. A shellac-modified compound represented by formula (3). 【Chemical 3】 In formula (3), Z is the part of the structure of the cerac that excludes the carboxy group modified by the functional group Y. X in formula (3) ５ is a dialkylamino group, an alkylamino group, a hydroxy group, a halogen group, an alkoxy group, an amino group, an acyl group, an alkyl group, an alkanate group, or hydrogen.

4. A shellac-modified compound represented by formula (4). 【Chemical 4】 In formula (4), Z is the part of the structure of shellac excluding the carboxyl group modified by the functional group Y.

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