Preparation Method and Application of Acryloylphenylalanine-N-Vinylpyrrolidone Chiral Hydrogel Film

Abstract

The present invention belongs to the field of biocompatible material preparation, and specifically discloses a preparation method and application of an acryloylphenylalanine-N-vinylpyrrolidone chiral hydrogel film. Based on polyethylene glycol diacrylate as a crosslinking agent, the present invention obtains a chiral copolymer gel film based on acryloylphenylalanine and N-vinylpyrrolidone by ultraviolet light-induced copolymerization of monomer acryloylphenylalanine and monomer N-vinylpyrrolidone. The present invention introduces chirality into the gel film through a gel preparation method to obtain a chiral gel interface suitable for cell culture. The chiral gel interface has better advantages similar to the extracellular matrix (ECM), and the chiral gel surface containing a large amount of water can regulate cell adhesion and proliferation.

Description

Technical Field

[0001] The present invention belongs to the field of chiral hydrogels in biotechnology. Specifically, it relates to a method for preparing a chiral hydrogel film of (L / D)-acryloylphenylalanine and N-vinylpyrrolidone and its applications. Background Art

[0002] In the field of tissue engineering, the interaction between multicells and the matrix is essentially crucial for the development and growth of healthy cell clusters. These interactions are not only determined by extracellular matrix (ECM) proteins, but the physical and chemical properties of the substrates used are also involved in the regulation together. The ability of cells to interact better with substrate materials with certain characteristics has been particularly concerned to regulate cell fate. Molecular chirality, as an inherent property of biology and cell chemistry, often shows a preference for specific symmetries, which is reflected at the most basic molecular symmetry level in the building blocks of life (the D-isomer of nucleic acid and the L-isomer of amino acid). Therefore, interfacial chirality has an important impact on the behavior of various cell-related biomolecules. In addition, controlling the surface properties of functional materials to regulate the behavior of cells and other biomolecules is an important basis for developing new biomaterials and devices, and chiral interfaces provide this possibility.

[0003] Pyrrolidine is one of the most common five-membered non-aromatic nitrogen heterocycles and has important applications in the fields of medicine, food, tissue engineering, etc. The five-membered lactam ring structure in its basic skeleton is stable and plastic, and a rich variety of pyrrolidone derivatives with broad biological activities can be generated therefrom. Therefore, it is of great significance to fully develop and utilize it. In view of some problems of current hydrogel materials in biomedicine, such as complex synthesis routes, certain toxicity of crosslinking agents, and difficulty in regulating cell growth of hydrogels, the research group synthesized a class of N-vinylpyrrolidone chiral macromolecular gel materials. The chiral macromolecular gels form a three-dimensional network structure, have high water content, are similar to the cell microenvironment in vivo, have good biocompatibility, and the chiral effect of the chiral macromolecular gels is significantly amplified relative to molecular chirality, and it has been successfully used to regulate cell adhesion and proliferation. Summary of the Invention

[0004] In view of the above problems, the present invention provides a method for preparing an acryloylphenylalanine and N-vinylpyrrolidone chiral hydrogel film based on a polyethylene glycol diacrylate crosslinking agent and its applications.

[0005] The object of the present invention is achieved by the following technical solutions

[0006] The present invention provides a method for preparing a copolymer hydrogel of acryloylphenylalanine and N-vinylpyrrolidone, comprising the following steps:

[0007] 1) Dissolve acryloylphenylalanine (L-PHEOH or D-PHEOH) and monomer N-vinylpyrrolidone (NVP) uniformly in N-methylpyrrolidone solution. Subsequently, add photoinitiator hydroxycyclohexyl phenyl ketone (I184) and crosslinker polyethylene glycol diacrylate and stir to dissolve. Deoxygenate by passing nitrogen at room temperature to obtain a reaction solution. Drop a small amount of the reaction solution onto a silanized substrate (cover glass), cover it with a film (PET film), gently press to extrude air bubbles, and irradiate with 365 nm ultraviolet light for 1 h to initiate the reaction.

[0008] 2) After the ultraviolet irradiation is completed, place the substrate in an oven at 50 °C for 4 h to dry, tear off the film, and obtain an acryloylphenylalanine-N-vinylpyrrolidone copolymer hydrogel film, which is immersed and stored in PBS buffer solution.

[0009] Furthermore, in step (1), acryloylphenylalanine (L-PHEOH or D-PHEOH) is obtained by reacting L-phenylalanine or D-phenylalanine with acryloyl chloride at a molar ratio of 1:1.5 - 2.2.

[0010] Furthermore, in step (1), the molar ratio of acryloylphenylalanine (L-PHEOH or D-PHEOH) to N-vinylpyrrolidone is (1 - 4):(1 - 2).

[0011] Furthermore, in step (2), the photoinitiator is selected as hydroxycyclohexyl phenyl ketone, and the dosage of the initiator is 2% - 4% of the sum of the amounts of the two monomers in terms of amount of substance. The crosslinker is polyethylene glycol diacrylate, and the dosage of the crosslinker is 5% - 8% of the sum of the amounts of the two monomers in terms of amount of substance. The polymerization initiation temperature is room temperature.

[0012] Furthermore, the molecular weight of polyethylene glycol diacrylate is 400 - 1200; preferably, the molecular weight of polyethylene glycol diacrylate is 1000.

[0013] Furthermore, the ratio of the mass of the N-methylpyrrolidone solution in step (2) to the sum of the masses of the two monomers is (2.5 - 4):1, and the total solid content is 25% - 35%; the solid content is the proportion of the total mass of monomer acryloylphenylalanine and N-vinylpyrrolidone in the total mass. If the solid content is too low, it will be difficult to form a gel, and if it is too high, the crosslinking will be too tight and it will be difficult to swell.

[0014] Furthermore, the application of acryloyl-(L / D)-phenylalanine / N-vinylpyrrolidone chiral hydrogel as a biomaterial. In the present invention, acryloylphenylalanine is copolymerized with N-vinylpyrrolidone to transfer monomer chirality to the polymer, and through a gel preparation method, chirality is combined with the gel film to obtain a chiral gel interface suitable for cell culture. Compared with the polymer chiral interface, the chiral gel interface has better advantages similar to the extracellular matrix (ECM), and the matrix surface containing a large amount of water is also more advantageous for cell culture. Therefore, based on the preparation of L / D-phenylalanine-co-N-vinylpyrrolidone chiral gel film, the present invention explores the influence of polymer chiral gel on regulating cell behavior. Description of the Drawings

[0015] Figure 1 It is the reaction equation and structural formula of the chiral (L / D) APHEOH-co-NVP copolymer gel prepared in the present invention.

[0016] Figure 2 It is the nuclear magnetic resonance hydrogen spectrum of L / D acryloylphenylalanine (APHEOH) prepared in the present invention. Figure 3 It is the Fourier transform infrared spectroscopy (FTIR) of acryloylphenylalanine monomer and chiral (L / D) APHEOH-co-NVP copolymer gel prepared in the present invention.

[0017] Figure 4 It is the circular dichroism CD curve of the chiral (L / D) APHEOH-co-NVP copolymer gel prepared in the present invention.

[0018] Figure 5 It is the morphology observed by scanning electron microscopy (SEM) of the freeze-dried chiral (L / D) APHEOH-co-NVP copolymer gel prepared in the present invention. A, B, and C are the SEM images of the gel film surfaces prepared in Examples 1, 2, and 3 at 1000×, respectively, and D, E, and F are the SEM images of the cracks in the gel films prepared in Examples 1, 2, and 3 after freeze-drying at 1000×, respectively.

[0019] Figure 6 It is the rheological analysis of the chiral (L / D) APHEOH-co-NVP copolymer gel prepared in the present invention. A, B, and C are the amplitude tests of DAPHEOH-co-NVP gels in Examples 1, 2, and 3, respectively, and D, E, and F are the frequency tests of LAPHEOH-co-NVP gels in Examples 1, 2, and 3, respectively.

[0020] Figure 7 It is the cell viability of the chiral (L / D) APHEOH-co-NVP copolymer gel film prepared in the present invention for the culture of mouse fibroblasts (L929).

[0021] Figure 8 This is the live / dead cell staining of mouse fibroblasts (L929) cultured on the chiral (L / D) APHEOH-co-NVP copolymer gel film prepared by the present invention for 48 hours. Detailed implementation manners

[0022] The technical solution of the present invention will be further described below through specific examples. For the experimental methods without specific conditions noted in the examples, they are usually carried out according to the conventional conditions and the conditions described in the manuals, or according to the conditions recommended by the manufacturers; for the general equipment, materials, reagents, etc., if not otherwise specified, they can be obtained from commercial channels.

[0023] Preparation of acryloylphenylalanine

[0024] Dissolve 3.3 g of L-phenylalanine or D-phenylalanine in 40 mL of 1 M NaOH solution. Under stirring in an ice-water bath, slowly dropwise add 3.98 g of acryloyl chloride, and continuously add 2 M NaOH to keep the pH of the solution around 10. After the dropwise addition, keep the ice-water bath for 30 min and react at room temperature for 3 h. Acidify the solution to pH 2 - 3 with 2 M HCl, precipitate white precipitate and stir for 30 min to make it fully uniform. Filter the turbid liquid and wash it with deionized water. Then, extract the solid with ethyl acetate three times, separate the ethyl acetate solution and dry the ethyl acetate solution with anhydrous magnesium sulfate. Filter the anhydrous magnesium sulfate to obtain a clear solution, place it in a rotary evaporator, evaporate most of the solvent, and then dry it in a vacuum oven to obtain L-acryloylphenylalanine monomer or D-acryloylphenylalanine monomer L / D-APHEOH. Figure 2 This is the 1H NMR spectrum of the L / D acryloylphenylalanine (APHEOH) prepared above, indicating the successful synthesis of (L / D) acryloylphenylalanine (APHEOH).

[0025] Example 1

[0026] 0.108 g of monomer acryloylphenylalanine (L / D-APHEOH) and 0.027 g of monomer N-vinylpyrrolidone (NVP) with a molar ratio of 2:1 were both dissolved in 380 μL of N-methylpyrrolidone solution. Subsequently, 0.006 g of initiator I184 and 0.015 g of crosslinker polyethylene glycol diacrylate with a molecular weight of 400 were added and stirred until dissolved. Nitrogen was passed through to remove oxygen at room temperature to obtain a reaction solution. A small amount of the reaction solution was dropped onto a silanized cover glass, covered with a PET film, gently pressed to squeeze out air bubbles, and irradiated with 365 nm ultraviolet light for 1 h to initiate the reaction. After the ultraviolet irradiation ended, the cover glass was placed at 50 °C for 4 h, and the PET film was torn off with tweezers to obtain an acryloylphenylalanine-N-vinylpyrrolidone copolymer hydrogel film ((L / D)APHEOH-co-NVP), which was immersed in PBS buffer solution three times for storage.

[0027] Example 2

[0028] 0.593 g of monomer acryloylphenylalanine (L / D-APHEOH) and 0.148 g of monomer N-vinylpyrrolidone (NVP) with a molar ratio of 2:1 were uniformly dissolved in 2.03 mL of N-methylpyrrolidone solution. Subsequently, 0.032 g of initiator I184 and 0.205 g of crosslinker polyethylene glycol diacrylate with a molecular weight of 1000 were added and stirred until dissolved. Nitrogen was passed through to remove oxygen at room temperature to obtain a reaction solution. A small amount of the reaction solution was dropped onto a silanized cover glass, covered with a PET film, gently pressed to squeeze out air bubbles, and irradiated with 365 nm ultraviolet light for 1 h to initiate the reaction. After the ultraviolet irradiation ended, the cover glass was placed at 50 °C for 4 h, and the PET film was torn off with tweezers to obtain an acryloylphenylalanine-N-vinylpyrrolidone copolymer hydrogel film ((L / D)APHEOH-co-NVP), which was immersed in PBS buffer solution three times for storage.

[0029] Example 3

[0030] 3.0 g of monomer acryloylphenylalanine (L / D-APHEOH) and 0.75 g of monomer N-vinylpyrrolidone (NVP) with a molar ratio of 2:1 were uniformly dissolved in 10.27 mL of N-methylpyrrolidone solution. Subsequently, 0.167 g of initiator I184 and 2.08 g of crosslinker polyethylene glycol diacrylate with a molecular weight of 2000 were added and stirred until dissolved. Nitrogen was passed through to remove oxygen at room temperature to obtain a reaction solution. A small amount of the reaction solution was dropped onto a silanized cover glass, covered with a PET film, gently pressed to squeeze out air bubbles, and irradiated with 365 nm ultraviolet light for 1 h to initiate the reaction. After the ultraviolet irradiation ended, the cover glass was placed at 50 °C for 4 h, and the PET film was torn off with tweezers to obtain an acryloylphenylalanine-N-vinylpyrrolidone copolymer hydrogel film ((L / D)APHEOH-co-NVP), which was immersed in PBS buffer solution three times for storage.

[0031] (L / D) APHEOH-co-NVP Gel Infrared Spectroscopy Analysis

[0032] The FTIR spectra of acryloylphenylalanine (APHEOH) and acryloylphenylalanine-N-vinylpyrrolidone copolymer gel (APHEOH-NVP) are as Figure 3 shown. For acryloylphenylalanine, the peaks at 3343, 2920, 1712, 1650, 1596, 1536 cm -1 are due to N-H stretching vibration, O-H stretching vibration in carboxyl group, C=O stretching vibration, amide I band, COO － antisymmetric stretching vibration and amide II band respectively. For acryloylphenylalanine-N-vinylpyrrolidone copolymer gel, the peaks at 1720, 1655, 1601, 1382, 1086 are due to C=O stretching vibration on APHEOH and NVP, amide I band on APHE, COO － antisymmetric stretching vibration on APHEOH, C-C stretching vibration on NVP, and C-N stretching vibration on NVP respectively. In addition, since the C=C stretching vibration on APHEOH is suppressed by the amide I band and COO － antisymmetric stretching vibration and does not appear, and also does not peak on APHEOH-NVP after polymerization, it indicates the successful reaction of acryloylphenylalanine and N-vinylpyrrolidone.

[0033] (L / D) APHEOH-co-NVP Gel Membrane Circular Dichroism CD Curve Analysis

[0034] To study the chirality of the copolymer gel, the chirality of the synthesized (L / D)

[0035] APHEOH-co-NVP copolymer gel membrane was tested by a circular dichroism spectrometer (CD). The results are as Figure 4 shown. LAPHEOH-co-NVP and DAPHEOH-co-NVP show completely opposite chiral signals. The maximum positive Cotton effect peak is at 232 nm, and the maximum negative Cotton effect peak is also at 228 nm. The absolute values of the peaks are also basically the same, indicating the existence of chirality at the phenylalanine position after copolymerization of APHEOH-co-NVP and presenting a good chiral mirror image relationship at the characteristic peak of phenylalanine.

[0036] (L / D) APHEOH-co-NVP Copolymer Gel Scanning Electron Microscopy (SEM) Analysis

[0037] To observe the surface morphology of the gel, a field emission scanning electron microscope (SEM) from FEI Company, USA was used to characterize the (L / D) APHEOH-co-NVP copolymer gel. The results are as Figure 5As shown, A, B, and C are the SEM images of the surfaces of the gel films prepared in Examples 1, 2, and 3, respectively. It can be seen that the surfaces of the gel films under the three crosslinking agents all exhibit a continuous and uniform surface layer, which can serve as a substrate for cell adhesion. D, E, and F are the SEM images of the cracks in the freeze-dried gel films of Examples 1, 2, and 3, respectively. It can be seen that as the molecular weight of the crosslinking agent increases, the gel pores become smaller and smaller and the density becomes larger and larger, forming a pore structure with a pore diameter between 1 and 10 μm, indicating that the interior of the gel contains a large amount of water.

[0038] Rheological analysis of APHEOH-co-NVP copolymer gel.

[0039] To study the rheological properties of the gel, a rotational rheometer was used to perform rheological tests on the gel. A flat plate with a diameter of 25 mm was used. At room temperature, dynamic strain tests were carried out at a frequency of 10 rad / s within a stress range of 0.1% - 1000%, and dynamic frequency tests were carried out at a stress of 1% within a frequency range of 0.1 rad / s - 100 rad / s. The results are shown in Figure 6. A, B, and C are the amplitude tests of the DAPHEOH-co-NVP gels of Examples 1, 2, and 3, respectively. The elastic modulus of the gel film basically reaches more than 1000 Pa. Before the stress reaches 10%, the elastic modulus (G’) of the gel is always greater than the loss modulus (G”), and the gel exhibits a solid-like rheological property. As the stress increases, G’ gradually becomes less than G”, and the gel exhibits a liquid-like rheological property, indicating that the gel undergoes a gel-sol transition. D, E, and F are the frequency tests of the LAPHEOH-co-NVP gels of Examples 1, 2, and 3, respectively. The figure shows that the intensities of the elastic modulus and the loss modulus of the L / D gels are basically the same, indicating that the mechanical properties of the L / D gels are equivalent.

[0040] (L / D)APHEOH-co-NVP copolymer gel cytotoxicity analysis To study the effect of chirality on cell culture, (L929) mouse fibroblasts were selected to perform cell culture on the (L / D)APHEOH-co-NVP copolymer gel film, and the cytotoxicity of the cells at 24 h was tested. The results are as Figure 7As shown, at the same monomer molar ratio, NVP-400, NVP-1000, and NVP-2000 chiral gels prepared by copolymerization of cross-linking agents with different molecular weights correspond to Examples 1, 2, and 3, respectively, and the cell activity after cell culture on NVP-400, NVP-1000, and NVP-2000 L / D gel membranes. After culturing on the chiral gel films prepared in Examples 1 (NVP-400), 2 (NVP-1000), and 3 (NVP-2000) for 24 hours, it can be seen that the chiral gel film prepared by NVP-1000 in Example 2 has the best cell activity, the activity of D-APHEOH-co-NVP gel film reaches 151%, the activity of L-APHEOH-co-NVP gel film also reaches 94%, the difference in cell activity caused by chirality reaches 57%, and the activity difference of the chiral gel films of Examples 1 and 3 also reaches 51% and 54%. However, considering the overall activity of the cells, the activities of the L-APHEOH-co-NVP gel films of Examples 1 and 3 are only 71% and 66%, which are relatively low. Therefore, the chiral gel film of Example 2 is most suitable for cell research on chiral films.

[0041] The cell proliferation of the chiral gel membrane of Example 2 was subjected to live-dead staining (eg Figure 8 ), Figures A, B, and C are live cell staining images of the D-APHEOH-co-NVP, L-APHEOH-co-NVP gel membranes and coverslips of the same size selected from Example 2, and Figures D, E, and F are dead cell staining images of the D-APHEOH-co-NVP, L-APHEOH-co-NVP gel membranes and coverslips, respectively.

[0042] After 48 hours of culture, the cells on the D-type gel membrane (A) can be seen to be in the shape of rice grains under live cell staining, and the cell density and cell spreading area are greater than those of the blank cells, indicating that the D-type gel membrane has a promoting effect on the culture of mouse fibroblasts. The cells on the L-type gel membrane (B) are mostly spherical in shape, and the cell proliferation density is much lower than that of the blank group, indicating that the cell adhesion and proliferation are poor. Figures D, E, and F are the dead cell staining images of the D-type, L-type, and control groups, respectively. It can be seen that the number of dead cells on the L / D membrane is consistent with that of the control group, indicating that the cell mortality rate is low and the gel has almost no toxicity to cells. In addition to the chirality difference, the physical and chemical properties of the L / D gels are basically the same, so it can be explained that the difference in cell proliferation is caused by the chirality of the gel.

Claims

1. Preparation method of acryloylphenylalanine-N-vinylpyrrolidone chiral hydrogel film, characterized in that L-acryloylphenylalanine or D-acryloylphenylalanine and monomer N-vinylpyrrolidone are uniformly dissolved in N-methylpyrrolidone solution, then initiator and polyethylene glycol diacrylate are added and stirred to dissolve. Nitrogen is passed through to remove oxygen at room temperature to obtain a reaction solution. The reaction solution is dropped on a substrate and covered with a film, and bubbles are gently squeezed out. It is placed under ultraviolet light for irradiation to initiate a polymerization reaction; the molecular weight of polyethylene glycol diacrylate is 400 - 1200; After the ultraviolet irradiation ends, an L-acryloylphenylalanine-N-vinylpyrrolidone copolymer hydrogel film or a D-acryloylphenylalanine-N-vinylpyrrolidone copolymer hydrogel film, namely L-APHEOH-co-NVP or D-APHEOH-co-NVP, is obtained from the reacted substrate.

2. The preparation method of the acryloylphenylalanine-N-vinylpyrrolidone chiral hydrogel film according to claim 1, characterized in that: L-acryloylphenylalanine or D-acryloylphenylalanine is obtained by reacting L-phenylalanine or D-phenylalanine and acryloyl chloride as monomers in a molar ratio of 1:1.5 - 2.

2.

3. The preparation method of the acryloylphenylalanine-N-vinylpyrrolidone chiral hydrogel film according to claim 1, characterized in that, The dosage of the polyethylene glycol diacrylate is 5% - 8% of the total molar amount of the monomers; the temperature for initiating the polymerization reaction is room temperature.

4. The preparation method of the acryloylphenylalanine-N-vinylpyrrolidone chiral hydrogel film according to claim 1, characterized in that, The molar ratio of L-acryloylphenylalanine or D-acryloylphenylalanine to N-vinylpyrrolidone is 1 - 4:1 - 2.

5. The preparation method of the acryloylphenylalanine-N-vinylpyrrolidone chiral hydrogel film according to claim 1, characterized in that, The molecular weight of polyethylene glycol diacrylate is 1000.

6. Application of the acryloylphenylalanine-N-vinylpyrrolidone chiral hydrogel film prepared by the method according to any one of claims 1 - 5 as a chiral biomaterial interface.

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