Cell multi-band ultraviolet protection agent with sinapinic acid-derived copolymer structure and preparation method and application thereof

Abstract

The present invention belongs to the field of polymer cell UV protectants and discloses a cell wave-band UV protectant with a sinapinic acid derivative copolymer structure, as well as a preparation method and application. The preparation method comprises the following steps: (1) mixing a sinapinic acid derivative structural monomer and polyethylene glycol methacrylate in an anhydrous organic solvent to react to obtain component A; (2) mixing a free radical initiator with component A in an inert atmosphere to react to obtain component B, which is then separated from component B to obtain a sinapinic acid derivative copolymer having a structure as shown in Formula I. #imgabs0# In Formula I, R is an oxygen atom (O) or an imino group (NH); m is the degree of polymerization of the sinapinic acid derivative structural monomer, n is the degree of polymerization of PEGMA; x is the number of carbon atoms in the sinapinic acid derivative structural monomer, and y is the number of repeating units of ethylene glycol in the PEGMA monomer. The polymer of the present invention exhibits good cell safety and multi-band UV damage resistance in cell experiments.

Description

Technical Field

[0001] The invention belongs to the field of polymer cell ultraviolet protective agents, and particularly relates to a cell multi-band ultraviolet protective agent with a sinapinic acid-derived copolymer structure, a preparation method and an application thereof. Background Art

[0002] Sunburn and even skin cancer caused by excessive ultraviolet radiation have gradually become a threat to public health.

[0003] Traditional sunscreens primarily consist of inorganic nanoparticles and organic aromatic small molecules, which inherently suffer from limitations such as poor dispersibility, thermal stability, and easy skin penetration. Polymeric UV absorbers are gaining increasing attention as they can effectively reduce the toxicity of small molecules that penetrate the skin.

[0004] The current method mainly uses a variety of small molecule sunscreens such as dibenzophenones, triazines, and benzotriazoles as monomers and grafts them onto water-soluble polymer skeletons such as polyethylene glycol and polyethylene glycol monomethyl ether.

[0005] However, the polymer UV absorbers reported so far generally have shortcomings such as difficulty in synthesis and certain safety risks of active functional groups in their structures, making them difficult to put into practical application.

[0006] Therefore, it is necessary to develop a multi-band UV protection agent for cells with a sinapinic acid-derived copolymer structure to overcome the above technical problems. Summary of the Invention

[0007] The purpose of the present invention is to provide a multi-band ultraviolet protection agent for cells with a sinapinic acid-derived copolymer structure, as well as a preparation method and application. The preparation method of the present invention uses different sinapinic acid derivatives and polyethylene glycol methacrylate (PEGMA) to carry out free radical polymerization under the initiation of a free radical initiator to obtain a sinapinic acid-derived copolymer with good water solubility. The polymer exhibits good cell safety and multi-band anti-ultraviolet damage effect in cell experiments.

[0008] The technical solution adopted in the present invention is as follows:

[0009] In a first aspect, the present invention provides a sinapinic acid-derived copolymer, the structural formula of the sinapinic acid-derived copolymer is shown in Formula I:

[0010]

[0011] In the formula I, R is an oxygen atom (O) or an imino group (NH); n:m=1:1-5, m is the degree of polymerization of the sinapinic acid derivative structural monomer, 20≤m; n is the degree of polymerization of PEGMA, 20≤n; x is the number of carbon atoms in the sinapinic acid derivative structural monomer, x is an integer between 1 and 5; y is the number of repeating units of ethylene glycol in the PEGMA monomer, and the value of y is any integer from 4 to 5, 9 to 10, 19 to 20, or 31 to 33;

[0012] The PEGMA is polyethylene glycol methacrylate.

[0013] Preferably, the sinapinic acid-derived copolymer of the present invention is a compound as shown in Formula IV:

[0014]

[0015] Its number average molecular weight M n =5.84×10 4 g mol -1 , weight average molecular weight M w =11.33×10 4 g mol -1 , the molecular weight distribution coefficient is 1.94.

[0016] In a second aspect, the present invention provides a method for preparing the sinapinic acid-derived copolymer described in the first aspect, comprising the following steps:

[0017] (1) In an anhydrous organic solvent, a sinapinic acid derivative structural monomer, polyethylene glycol methacrylate and a free radical initiator are mixed and reacted to obtain component A; the molar ratio of the sinapinic acid derivative structural monomer to the polyethylene glycol methacrylate is 1:0.2 to 1; the molar ratio of the free radical initiator to the sinapinic acid derivative structural monomer can be 0.01 to 1:1

[0018] (2) removing air from the component A system, and then reacting at 50-100° C. to obtain component B; and separating the sinapic acid-derived copolymer from the component B;

[0019] The specific structure of the sinapinic acid derivative structural monomer is shown in Formula II:

[0020]

[0021] In the formula II, R is an oxygen atom (O) or an imino group (NH); x is the number of carbon atoms in the monomer structure of the sinapinic acid derivative, and x is an integer between 1 and 5;

[0022] The average molecular weight of the polyethylene glycol methacrylate is 300 to 2500.

[0023] Preferably, the reaction time in step (1) can be 10 to 20 minutes; and the reaction time in step (2) can be 6 to 20 hours.

[0024] Preferably, in step (2), air in component A is removed by bubbling with an inert gas or performing a liquid nitrogen freeze-vacuum-thaw cycle operation.

[0025] Preferably, in step (1), the anhydrous organic solvent is at least one of dioxane, tetrahydrofuran, N,N-dimethylformamide and acetonitrile.

[0026] Preferably, the free radical initiator includes an azo initiator and / or a peroxide initiator; the azo initiator is selected from azobisisobutyronitrile (AIBN) and azobisisoheptanenitrile (ABVN), and the peroxide initiator is selected from dibenzoyl peroxide (BPO).

[0027] Preferably, the preparation method of the sinapinic acid derivative structural monomer comprises the following steps:

[0028] In an anhydrous organic solvent, sinapinic acid is mixed with a methacrylic acid compound, a dehydrating agent, and a catalyst to react to obtain component C, and then a sinapinic acid derivative structural monomer is separated from the component C;

[0029] The structural formula of the methacrylic acid compound is shown in Formula III:

[0030]

[0031] In formula III, R' is a hydroxyl group (OH) or an amino group (NH2); x is the number of carbon atoms, and x is an integer between 1 and 5;

[0032] The molar ratio of the sinapinic acid to the methacrylic acid compound is 1:0.5-1;

[0033] The dehydrating agent includes any one of a carbodiimide dehydrating agent and a molecular sieve, or a mixture of two or more thereof; the carbodiimide dehydrating agent is selected from any one of dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC); the molar ratio of the dehydrating agent to the methacrylic acid compound is 1:1 to 2.

[0034] The catalyst is an acyl transfer catalyst, selected from any one of 4-dimethylaminopyridine (DMAP), dimethyltin dichloride, and aluminum chloride, or a mixture of two or more thereof; the molar ratio of the catalyst to the raw material 1 can be 1:0.1-1.

[0035] The reaction temperature for obtaining the component C is 25 to 60° C., and the reaction time is 12 to 48 hours.

[0036] Preferably, the operation of isolating the structural monomer of the sinapinic acid derivative is as follows: washing the component C with deionized water, saturated NaHCO3 solution and saturated brine, respectively, combining the organic layers, adding anhydrous MgSO4 to dry, filtering, and rotary evaporation to obtain a crude product, purifying it by column chromatography, and finally freeze-drying it.

[0037] Furthermore, the column chromatography purification uses 100-500 mesh silica gel powder; the eluent used is one or a mixture of dichloromethane, methanol, ethyl acetate and petroleum ether, with a volume ratio of 1:0.01-100.

[0038] Furthermore, the freeze-drying temperature is -20 to -50°C, and the vacuum degree is 0.1 to 30Pa.

[0039] In a third aspect, the present invention provides a use of the sinapinic acid-derived copolymer described in the first aspect as a cell ultraviolet protective agent.

[0040] The present invention has the following advantages:

[0041] The method of the present invention uses different sinapinic acid derivative structural monomers and polyethylene glycol methacrylate (PEGMA) to carry out free radical polymerization under the initiation of a free radical initiator to obtain a sinapinic acid-derived copolymer with good water solubility. The polymer shows good cell safety and multi-band anti-ultraviolet damage effect in cell experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 is a synthetic route of the sinapinic acid-derived copolymer of the present invention;

[0043] Figure 2 The H NMR spectrum of the synthetic sinapinic acid derivative structure monomer used in the examples ( 1 H-NMR);

[0044] Figure 3 The H NMR spectrum of the sinapic acid-derived copolymer synthesized in Example 1 of the present invention is ( 1 H-NMR);

[0045] Figure 4 Gel permeation chromatography (GPC) of the sinapinic acid-derived copolymer synthesized in Example 1 of the present invention;

[0046] Figure 5 This is a cell fluorescence staining photo of plate A1 in Example 2 of the present invention (without UV irradiation), where Figure 5 (a) is a photograph of living cells stained with calcein acetyl phosphate buffer. Figure 5(b) Photograph of dead cells stained with propidium iodide-phosphate buffer;

[0047] Figure 6 This is a fluorescence staining photo of cells on plate A2 in Example 2 of the present invention (after 365nm UV irradiation), where Figure 6 (a) is a photograph of living cells stained with calcein acetyl phosphate buffer. Figure 6 (b) Photograph of dead cells stained with propidium iodide-phosphate buffer;

[0048] Figure 7 This is a cell fluorescence staining photograph of plate A3 in Example 2 of the present invention (303nm ultraviolet irradiation), where Figure 7 (a) is a photograph of living cells stained with calcein acetyl phosphate buffer. Figure 7 (b) Photograph of dead cells stained with propidium iodide-phosphate buffer;

[0049] Figure 8 This is a fluorescence staining photograph of cells on an A4 plate in Example 2 of the present invention (after 254nm UV irradiation), where Figure 8 (a) is a photograph of living cells stained with calcein acetyl phosphate buffer. Figure 8 (b) Photograph of dead cells stained with propidium iodide-phosphate buffer;

[0050] Figure 9 This is a fluorescent staining photo of cells on plate B1 in Example 2 of the present invention (without UV irradiation), where Figure 9 (a) is a photograph of living cells stained with calcein acetyl phosphate buffer. Figure 9 (b) Photograph of dead cells stained with propidium iodide-phosphate buffer;

[0051] Figure 10 This is a fluorescence staining photograph of cells on plate B2 in Example 2 of the present invention (after 365nm UV irradiation), where Figure 10 (a) is a photograph of living cells stained with calcein acetyl phosphate buffer. Figure 10 (b) Photograph of dead cells stained with propidium iodide-phosphate buffer;

[0052] Figure 11 This is a fluorescence staining photograph of cells on plate B3 in Example 2 of the present invention (303 nm UV irradiation), where Figure 11 (a) is a photograph of living cells stained with calcein acetyl phosphate buffer. Figure 11 (b) Photograph of dead cells stained with propidium iodide-phosphate buffer;

[0053] Figure 12 This is a fluorescence staining photograph of cells on plate B4 in Example 2 of the present invention (after 254nm UV irradiation), where Figure 12(a) is a photograph of living cells stained with calcein acetyl phosphate buffer. Figure 12 (b) is a photograph of dead cells stained with propidium iodide-phosphate buffer. DETAILED DESCRIPTION

[0054] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0055] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0056] The present invention will be further described below with reference to the embodiments and accompanying drawings.

[0057] The nitrogen used in the following examples is all high-purity gas with a mass concentration of ≥99.999%; other chemical reagents used are obtained through conventional commercial channels unless otherwise specified.

[0058] like Figure 1 As shown, the preparation of the sinapic acid derivative structural monomer used in the following examples includes the following steps:

[0059] 1) Add 2.50 g of erucic acid, 1.14 g of aminoethyl methacrylate, 2.68 g of dicyclohexylcarbodiimide (DCC), 0.02 g of 4-dimethylaminopyridine (DMAP), and 40 mL of dichloromethane as solvent to a 100 mL reaction flask and place in a 30°C oil bath for reaction.

[0060] 2) The mixture in step 1) after 24 hours of reaction was washed with deionized water, saturated NaHCO3 solution and saturated brine respectively, the organic layers were combined, dried over anhydrous MgSO4, filtered and rotary evaporated to obtain a crude product, which was purified by column chromatography (eluent: methanol / dichloromethane = 1:20-30), collected, rotary evaporated, and freeze-dried at -50 ° C for 24 hours to obtain 2.87 g of a light yellow powdery sinapinic acid derivative structural monomer, which was respectively 1 H-NMR characterization showed that its structure is shown in Formula II, wherein R is imino (NH) and x is 1.

[0061] Depend on Figure 2 It can be seen that the obtained sinapinic acid derivative structural monomer corresponds one-to-one with its structural characteristic peak in the nuclear magnetic resonance spectrum, indicating that the product is very pure and contains no other impurities.

[0062] Example 1

[0063] Sinapic acid derivative copolymers were synthesized by copolymerization of sinapic acid derivative monomers with PEGMA:

[0064] In a 10 mL reaction flask, 0.34 g of the aforementioned sinapinic acid derivative structural monomer and 0.95 g of PEGMA (average molecular weight 950 g mol) were added.-1 ), 10.0 mg of azobisisoheptanenitrile (ABVN) and 2 mL of N, N-dimethylformamide (DMF) as solvents, add a magnetic bar and stir evenly. The reaction bottle is purged with nitrogen (flow rate of 20 mL / min), bubbled for 15 minutes, and the air in the system is removed. The reaction bottle is placed in a constant temperature oil bath at 70°C, and after reacting for 12 hours, it is added dropwise to ether with rapid stirring (speed of 800 r / min) to precipitate, filter, collect the filter residue, add ether to wash three times to obtain a polymer, and freeze-dry at -50°C for 24 hours to obtain 1.10 g of a copolymer (i.e., a multi-band ultraviolet protective agent for cells with a mustard acid-derived copolymer structure), the structural formula of which is shown in Formula IV. 1 H-NMR and GPC characterization.

[0065]

[0066] Depend on Figure 3 It can be seen that the hydrogen nuclear magnetic spectrum of the copolymer contains characteristic peaks of two monomers, namely, the sinapinic acid derivative structural monomer and the PEGMA monomer: the peaks at chemical shifts of 8.84, 6.86, and 3.80 ppm are respectively attributed to the phenolic hydroxyl group, benzene ring, and methoxy group connected to the benzene ring in the sinapinic acid derivative structural monomer; the peaks at chemical shifts of 3.45-3.80 and 3.30 ppm are respectively attributed to the ether bond and terminal methyl group in PEGMA, proving that copolymerization reaction has occurred.

[0067] Depend on Figure 4 It can be seen that the molecular weight distribution of the polymer obtained by synthesis is a typical normal distribution, and the GPC test results are: number average molecular weight M n =5.84×10 4 g mol -1 , weight average molecular weight M w =11.33×10 4 g mol -1 , the molecular weight distribution coefficient is 1.94.

[0068] Example 2

[0069] The sinapic acid-derived copolymer prepared in Example 1 was used as a multi-band UV-resistant test for cells:

[0070] 1) Human lens epithelial cells (hLECs) were obtained at a cell density of 5 × 10 per ml. 5Cells were plated into eight 96-well plates, divided into two groups, A and B, designated as plates A1, A2, A3, and A4, and plates B1, B2, B3, and B4. Standard commercially available DMEM medium was added to plates A1-A4, while a 5 mg / mL solution of the copolymer prepared in Example 1 of the present invention (prepared in commercially available DMEM medium) was added to plates B1-B4. All plates were placed in an incubator (37°C, 5% CO2) and incubated for 24 hours.

[0071] 2) After 24 hours, irradiate plates A2 and B2 under UV light (365 nm) for 30 minutes, plates A3 and B3 under UV light (303 nm) for 10 minutes, and plates A4 and B4 under UV light (265 nm) for 2 minutes. Plates A1 and B1 remain unirradiated. After irradiation, replace the culture medium with a new copolymer culture medium solution or standard culture medium of the same concentration. Continue culturing in the incubator. After 24 hours, remove the culture medium from the 96-well plate and wash three times with phosphate buffer.

[0072] 3) Add 5 μg / mL calcein acetyl methyl ester and 3 μg / mL propidium iodide in phosphate buffer to all wells, excite with 450-490 nm blue light and 515-560 nm green light, and observe the cell staining under a fluorescence microscope. The photos of plates A1-A4 and B1-B4 are shown below. Figures 5 to 12 As shown (green represents living cells, red represents dead cells).

[0073] From the above Figure 5 、 Figure 9 As shown in A1 and B1, without UV irradiation, cells survived well in the culture medium solution of sinapinic acid-derived copolymer, just like in ordinary culture medium, indicating that the copolymer had no obvious toxicity to cells. Figures 5 to 8 As shown in A1, A2, A3, and A4, after ultraviolet irradiation, cells cultured in ordinary culture medium underwent apoptosis in large quantities (red color increased significantly); Figures 10 to 12 It can be seen from B2, B3 and B4 in that the culture medium solution of the sinapinic acid-derived copolymer can play a good role in protecting cells from ultraviolet rays, and the cells still survive well after being irradiated with ultraviolet rays of multiple bands, which proves that the cell ultraviolet protective agent of the sinapinic acid-derived copolymer of the present invention does have a good multi-band anti-ultraviolet effect on cells and can be used as a multi-band ultraviolet protective agent for cells.

Claims

1. A sinapic acid-derived copolymer, characterized in that: The structural formula of the sinapic acid-derived copolymer is shown in Formula I: Formula I In Formula I, R is an oxygen atom (O) or an imino group (NH); n:m = 1:1-5, where m is the degree of polymerization of the sinapinic acid derivative structural monomer, and m ≥ 20; n is the degree of polymerization of PEGMA (polyethylene glycol methacrylate), and n ≥ 20; x is the number of carbon atoms in the sinapinic acid derivative structural monomer, and x is an integer between 1 and 5; and y is the number of repeating units of ethylene glycol in the PEGMA monomer, and the value of y is any integer between 4-5, 9-10, 19-20, and 31-33.

2. The sinapic acid-derived copolymer according to claim 1, characterized in that The sinapic acid-derived copolymer is a compound as shown in Formula IV, wherein m, n, and y are as defined in claim 1. Formula IV.

3. A method for preparing the sinapic acid-derived copolymer according to claim 1 or 2, characterized in that: The steps include: (1) In an anhydrous organic solvent, a sinapinic acid derivative structural monomer, polyethylene glycol methacrylate and a free radical initiator are mixed and reacted to obtain component A; the molar ratio of the sinapinic acid derivative structural monomer to the polyethylene glycol methacrylate is 1:0.2~1; the molar ratio of the free radical initiator to the sinapinic acid derivative structural monomer can be 0.01~1:1 (2) removing air from the component A system, and then reacting at 50-100°C to obtain component B; and separating the sinapic acid-derived copolymer from the component B; The specific structure of the sinapinic acid derivative structural monomer is shown in Formula II: Formula II In the formula II, R is an oxygen atom (O) or an imino group (NH); x is the number of carbon atoms in the monomer structure of the sinapinic acid derivative, and x is an integer between 1 and 5; The average molecular weight of the polyethylene glycol methacrylate is 300-2500.

4. The method for preparing the sinapic acid-derived copolymer according to claim 3, wherein: The reaction time in step (1) is 10 to 20 minutes; the reaction time in step (2) is 6 to 20 hours.

5. The method for preparing the sinapic acid-derived copolymer according to claim 3, wherein: In step (2), the air in component A is removed by bubbling with inert gas or performing a liquid nitrogen freeze-vacuum-thaw cycle operation.

6. The method for preparing the sinapic acid-derived copolymer according to claim 3, wherein: In the step (1), the anhydrous organic solvent is at least one of dioxane, tetrahydrofuran, N, N-dimethylformamide and acetonitrile.

7. The method for preparing the sinapic acid-derived copolymer according to claim 3, wherein: The free radical initiator includes an azo initiator and / or a peroxide initiator; the azo initiator includes any one of azobisisobutyronitrile (AIBN) and azobisisoheptanenitrile (ABVN); the peroxide initiator includes dibenzoyl peroxide (BPO).

8. The method for preparing the sinapic acid-derived copolymer according to claim 3, wherein: The preparation method of the sinapinic acid derivative structural monomer comprises the following steps: In an anhydrous organic solvent, sinapinic acid is mixed with a methacrylic acid compound, a dehydrating agent, and a catalyst to react to obtain component C, and then a sinapinic acid derivative structural monomer is separated from the component C; The structural formula of the methacrylic acid compound is shown in Formula III: Formula III In formula III, R' is a hydroxyl group (OH) or an amino group (NH2); x is the number of carbon atoms, and x is an integer between 1 and 5; The molar ratio of the sinapinic acid to the methacrylic acid compound is 1:0.5 to 1; The dehydrating agent includes any one of a carbodiimide dehydrating agent and a molecular sieve, or a mixture of two or more thereof; the carbodiimide dehydrating agent is selected from any one of dicyclohexylcarbodiimide (DCC), N, N'-diisopropylcarbodiimide (DIC), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC); the molar ratio of the dehydrating agent to the methacrylic acid compound is 1:1-2; The catalyst is an acyl transfer catalyst, selected from any one of 4-dimethylaminopyridine (DMAP), dimethylstannous dichloride, and aluminum chloride, or a mixture of two or more thereof; The reaction temperature for obtaining the component C is 25-60° C., and the reaction time is 12-48 h.

9. The method for preparing the sinapic acid-derived copolymer according to claim 8, characterized in that: The operation of isolating the structural monomer of the sinapinic acid derivative is as follows: washing the component C with deionized water, saturated NaHCO3 solution and saturated brine, respectively, combining the organic layers, adding anhydrous MgSO4 for drying, filtering, and rotary evaporating to obtain a crude product, purifying it by column chromatography, and finally freeze-drying it; the column chromatography purification uses 100-500 mesh silica gel powder; the eluent used is one or a mixture of two of dichloromethane, methanol, ethyl acetate and petroleum ether, with a volume ratio of 1:0.01-100; the freeze-drying temperature is -20 to -50°C, and the vacuum degree is 0.1 to 30 Pa.

10. Use of the sinapinic acid-derived copolymer according to claim 1 or 2 for preparing a cell ultraviolet protective agent.

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