CN118085168A - Preparation method and application of acryloylphenylalanine-N-vinyl pyrrolidone chiral hydrogel film - Google Patents
Preparation method and application of acryloylphenylalanine-N-vinyl pyrrolidone chiral hydrogel film Download PDFInfo
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Abstract
The invention belongs to the field of biological material preparation, and particularly discloses a preparation method and application of an acryloylphenyl alanine/N-vinyl pyrrolidone chiral hydrogel film. According to the invention, the chiral gel interface suitable for cell culture is obtained by introducing chirality into the gel membrane through a gel preparation method, and has the advantage of better resembling extracellular matrix (ECM), and the surface of the chiral gel containing a large amount of water can regulate cell adhesion and proliferation.
Description
Technical Field
The invention belongs to the technical field of biology, in particular to a preparation method and application of a chiral hydrogel film with (L/D) acryloylphenylalanine as a substrate and N-vinyl pyrrolidone.
Background
In the field of tissue engineering, interactions between multicellular and stroma are essentially critical to the development and growth of healthy cell clusters. These interactions are not solely determined by the extracellular matrix (ECM) proteins, but the physical and chemical properties of the substrates used are also involved in regulation. The ability of cells to exhibit better interactions with substrate materials having certain characteristics is of particular interest to regulate cell fate. Molecular chirality, an inherent property of biology and cytochemistry, often exhibits a preference for specific symmetry, reflected in the life building block at the most basic level of molecular symmetry (D-isomer of nucleic acid and L-isomer of amino acid), and thus interfacial chirality has a significant impact on the behavior of various cell-associated biomolecules. In addition, controlling the surface properties of functional materials to regulate the behavior of cells and other biomolecules is an important basis for the development of new biomaterials and devices, while chiral interfaces offer this possibility.
Pyrrolidine is one of the most common five-membered non-aromatic nitrogen heterocycles, has important application in the fields of medicine, food, tissue engineering and the like, has stable five-membered lactam ring structure in a basic framework and strong plasticity, can generate various pyrrolidone derivatives with wide biological activity, and has important significance in fully developing and utilizing the pyrrolidone derivatives. In view of the problems of complex synthetic route, certain toxicity of cross-linking agent, difficult regulation and control of cell growth of hydrogel and the like of the existing hydrogel materials in biomedicine, the problems are combined into the N-vinyl pyrrolidone chiral macromolecular gel material. The chiral macromolecular gel forms a three-dimensional network structure, has high water content, is similar to the microenvironment of cells in organisms, has better biocompatibility, obviously amplifies the chiral effect of the chiral macromolecular gel relative to the molecular chirality, and is successfully used for regulating the adhesion and proliferation of the cells.
Disclosure of Invention
In order to solve the problems, the invention provides a method for preparing an acryloylphenylalanine and N-vinyl pyrrolidone chiral hydrogel film based on a polyethylene glycol diacrylate cross-linking agent and application thereof.
The aim of the invention is achieved by the following technical proposal
The invention provides a preparation method of a copolymer hydrogel of acryloylphenylalanine and N-vinyl pyrrolidone, which comprises the following steps:
1) Uniformly dissolving acryloylphenylalanine (L-PHEOH or D-PHEOH) and monomer N-vinyl pyrrolidone (NVP) in N-methyl pyrrolidone solution, then adding a photoinitiator hydroxycyclohexanedione (I184) and a cross-linking agent polyethylene glycol diacrylate, stirring and dissolving, introducing nitrogen at normal temperature to remove oxygen to obtain a reaction solution, dripping a small amount of the reaction solution on a silanized substrate (cover glass), covering with a film (PET film), slightly extruding an air bubble, and irradiating for 1h under 365nm ultraviolet light to initiate reaction.
2) And after the ultraviolet irradiation is finished, the base material is dried for 4 hours at 50 ℃, the film is torn off, and the acrylamide-phenylalanine-N-vinyl pyrrolidone copolymerized hydrogel film is obtained and is placed in PBS buffer solution for soaking and preservation.
In the step (1), the acryloylphenylalanine (L-PHEOH or D-PHEOH) is obtained by reacting L-phenylalanine or D-phenylalanine with acryloyl chloride according to a molar ratio of 1:1.5-2.2.
Further, the molar ratio of the acryloylphenylalanine (L-PHEOH or D-PHEOH) to the N-vinylpyrrolidone in the step (1) is (1-4): 1-2.
Further, the photoinitiator in the step (2) is hydroxycyclohexane benzophenone, and the dosage of the initiator is 2% -4% of the sum of the two monomer substances. The cross-linking agent is polyethylene glycol diacrylate, and the dosage of the cross-linking agent is 5% -8% of the sum of the two monomer substances. The polymerization temperature was room temperature.
Further, the molecular weight of the polyethylene glycol diacrylate is 400-1200; preferably, the polyethylene glycol diacrylate has a molecular weight of 1000.
Further, the ratio of the mass of the N-methyl pyrrolidone solution in the step (2) to the sum of the mass of the two monomers is (2.5-4) 1, and the total solid content is 25% -35%; the solid content is the ratio of the total mass of the monomers of the acryloylphenylalanine and the N-vinyl pyrrolidone to the total mass, and the gel is difficult to form due to the fact that the solid content is too low, and the gel is too tight to swell due to too high crosslinking.
Further, the application of the chiral hydrogel of the acryloyl (L/D) phenylalanine/N-vinyl pyrrolidone as a biological material. According to the invention, the chiral gel interface suitable for cell culture is obtained by copolymerizing the acryloylphenylalanine and the N-vinyl pyrrolidone, transferring the chirality of the monomer into the polymer and combining the chirality with the gel membrane through a gel preparation method, and compared with the chiral interface of the polymer, the chiral gel interface has the advantage of better resembling an extracellular matrix (ECM), and the matrix surface containing a large amount of water has the advantage of cell culture. Therefore, the invention explores the influence of the chiral gel of the polymer on the regulation of the cell behavior based on the preparation of the chiral gel membrane of L/D phenylalanine-co-N-vinyl pyrrolidone.
Drawings
FIG. 1 shows the reaction equation and the structural formula of the chiral (L/D) APHEOH-co-NVP copolymer gel prepared by the invention.
FIG. 2 is a nuclear magnetic resonance hydrogen spectrum of L/D Acryloylphenylalanine (APHEOH) prepared by the present invention. FIG. 3 is a Fourier Transform Infrared (FTIR) chart of an acryloylphenylalanine monomer and chiral (L/D) APHEOH-co-NVP copolymer gel prepared according to the present invention.
FIG. 4 is a circular dichromatic CD curve of chiral (L/D) APHEOH-co-NVP copolymer gel prepared according to the present invention.
FIG. 5 shows the morphology of the chiral (L/D) APHEOH-co-NVP copolymer gel prepared by the invention observed by Scanning Electron Microscopy (SEM). A. B, C are SEM images of the surfaces of the gel films prepared in examples 1, 2 and 3 at 1000Xand D, E, F are SEM images of the cracks at 1000Xafter lyophilization of the gel films of examples 1, 2 and 3, respectively.
FIG. 6 is a rheological analysis of chiral (L/D) APHEOH-co-NVP copolymer gel prepared according to the present invention. A. B, C are amplitude tests of DAPHEOH-co-NVP gels of examples 1, 2, 3, respectively, and D, E, F are frequency tests of LAPHEOH-co-NVP gels of examples 1, 2, 3, respectively.
FIG. 7 shows the cell activity of the chiral (L/D) APHEOH-co-NVP copolymer gel film prepared by the present invention on mouse fibroblast (L929) culture.
FIG. 8 shows the staining of live and dead cells after culturing mouse fibroblasts (L929) on a chiral (L/D) APHEOH-co-NVP copolymer gel film prepared by the present invention for 48 hours.
Detailed Description
The technical scheme of the invention is further described by specific examples. The experimental methods for which specific conditions are not specified in the examples are generally as described in conventional conditions and handbooks, or as suggested by the manufacturer; the general equipment, materials, reagents, etc. used, unless otherwise indicated, are all commercially available.
Preparation of acryloylphenylalanine
3.3G of L-phenylalanine or D-phenylalanine was dissolved in 40ml of 1M NaOH solution, 3.98g of acryloyl chloride was slowly added dropwise with stirring in an ice water bath, and 2M NaOH was continuously added to maintain the pH of the solution at about 10. After the dripping is finished, the ice water bath is kept for 30min, the reaction is carried out for 3h at normal temperature, the pH of the solution is acidified to 2-3 by using 2M HCl, white precipitate is separated out, and the solution is stirred for 30min to make the solution fully uniform. Filtering the turbid liquid, washing the turbid liquid with deionized water, extracting the solid through ethyl acetate for 3 times, separating ethyl acetate solution, then drying the ethyl acetate solution through anhydrous magnesium sulfate, filtering the anhydrous magnesium sulfate to obtain a clear solution, placing the clear solution in a rotary evaporator, evaporating most of the solvent, and then placing the clear solution in a vacuum oven for drying to obtain L-acryloylphenyl alanine monomer or D-acryloylphenyl alanine monomer L/D-APHEOH. FIG. 2 is a nuclear magnetic resonance hydrogen spectrum of the above-prepared L/D Acryloylphenylalanine (APHEOH), showing successful synthesis of (L/D) Acryloylphenylalanine (APHEOH).
Example 1
0.108G of monomer acryloylphenylalanine (L/D-APHEOH) and 0.027g of monomer N-vinyl pyrrolidone (NVP) are dissolved in 380 mu L N-methyl pyrrolidone solution according to a molar ratio of 2:1, then 0.006g of initiator I184 and 0.015g of polyethylene glycol diacrylate with molecular weight of cross-linking agent 400 are added and stirred for dissolution, nitrogen is introduced at normal temperature for deoxidization, a reaction solution is obtained, a small amount of reaction solution is dripped on a silanized cover glass, the reaction solution is covered by a PET film, bubbles are extruded under light pressure, and the reaction is initiated by irradiation for 1h under 365nm ultraviolet light. After the ultraviolet irradiation is finished, the cover glass is placed at 50 ℃ for 4 hours, the PET film is torn off by forceps, and the acrylamide phenylalanine-N-vinyl pyrrolidone copolymerized hydrogel film ((L/D) APHEOH-co-NVP) is obtained and is placed in PBS buffer solution for three times of soaking and preservation.
Example 2
0.593G of monomer acryloylphenylalanine (L/D-APHEOH) and 0.148g of monomer N-vinyl pyrrolidone (NVP) are uniformly dissolved in 2.03mL of N-methyl pyrrolidone solution according to a molar ratio of 2:1, then 0.032g of initiator I184 and 0.205g of polyethylene glycol diacrylate with the molecular weight of the crosslinking agent of 1000 are added, stirred and dissolved, nitrogen is introduced at normal temperature for deoxidization, a reaction solution is obtained, a small amount of reaction solution is dripped on a silylated cover glass, the reaction solution is covered by a PET film, bubbles are extruded under light pressure, and the reaction is initiated by irradiation for 1h under 365nm ultraviolet light. After the ultraviolet irradiation is finished, the cover glass is placed at 50 ℃ for 4 hours, the PET film is torn off by forceps, and the acrylamide phenylalanine-N-vinyl pyrrolidone copolymerized hydrogel film ((L/D) APHEOH-co-NVP) is obtained and is placed in PBS buffer solution for three times of soaking and preservation.
Example 3
3.0G of monomer acryloylphenylalanine (L/D-APHEOH) and 0.75g of monomer N-vinyl pyrrolidone (NVP) are uniformly dissolved in a molar ratio of 2:1, then 0.167g of initiator I184 and 2.08g of polyethylene glycol diacrylate with a molecular weight of a cross-linking agent 2000 are added into the solution to be stirred and dissolved, nitrogen is introduced at normal temperature for deoxidization, a reaction solution is obtained, a small amount of reaction solution is dripped on a silanized cover glass, the reaction solution is covered by a PET film, bubbles are extruded under light pressure, and the solution is irradiated for 1h under 365nm ultraviolet light to initiate reaction. After the ultraviolet irradiation is finished, the cover glass is placed at 50 ℃ for 4 hours, the PET film is torn off by forceps, and the acrylamide phenylalanine-N-vinyl pyrrolidone copolymerized hydrogel film ((L/D) APHEOH-co-NVP) is obtained and is placed in PBS buffer solution for three times of soaking and preservation.
Infrared spectroscopic analysis of (L/D) APHEOH-co-NVP gel
FTIR spectra of Acryloylphenylalanine (APHEOH), acryloylphenylalanine-N-vinylpyrrolidone copolymer gel (APHEOH-NVP) are shown in fig. 3, with peaks at 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 for acryloylphenylalanine, 3343, 2920, 1712, 1650, 1596, 1536cm -1, respectively. For the acryloylphenylalanine-N-vinylpyrrolidone copolymer gel, the peaks at 1720, 1655, 1601, 1382, 1086 are caused by 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, C-N stretching vibration on NVP, respectively. In addition, since the C=C stretching vibration on APHEOH was not developed by the anti-symmetrical stretching vibration compaction of the amide I band and COO －, and the peak was not seen on APHEOH-NVP after polymerization, indicating successful reaction of acryloylphenylalanine and N-vinylpyrrolidone.
Circular dichromatic CD Curve analysis of (L/D) APHEOH-co-NVP gel films
To study the chirality of the copolymer gel, the synthesized (L/D) was tested by a round two Chromatograph (CD)
The result of the chiral of APHEOH-co-NVP copolymer gel film is shown in FIG. 4, LAPHEOH-co-NVP and DAPHEOH-co-NVP show completely opposite chiral signals, the maximum positive Ketone effect peak is at 232nm, the maximum negative Ketone effect peak is also at 228nm, the absolute values of the peaks are basically the same, the existence of the polymerized chirality of APHEOH-co-NVP after copolymerization at phenylalanine and good chiral mirror image relationship at the characteristic peaks of phenylalanine are shown.
Scanning Electron Microscope (SEM) analysis of (L/D) APHEOH-co-NVP copolymer gel
To observe the surface morphology of the gels, (L/D) APHEOH-co-NVP copolymer gels were characterized using field emission Scanning Electron Microscopy (SEM) from FEI company, USA. As shown in FIG. 5, A, B, C is an SEM image of the surface of the gel film prepared in examples 1, 2 and 3, respectively, it can be seen that the gel film surface under three crosslinking agents presents a continuous and uniform surface layer, which can be used as a substrate for cell adhesion, D, E, F is an SEM image of the break of the gel film prepared in examples 1, 2 and 3 after lyophilization, respectively, it can be seen that as the molecular weight of the crosslinking agent becomes larger and smaller and the density becomes larger and larger, a pore structure with a pore diameter of 1-10 μm is formed, indicating the environment in which the gel contains a large amount of water.
Rheological analysis of APHEOH-co-NVP copolymer gel.
To study the rheological properties of the gels, the gels were rheologically tested using a rotarheometer using plates 25mm in diameter, and were dynamically strain tested at room temperature at a frequency of 10rad/s over a stress range of 0.1% -1000% and dynamically frequency tested at a stress of 1% over a frequency range of 0.1rad/s-100 rad/s. As shown in FIG. 6, A, B, C is an amplitude test of DAPHEOH-co-NVP gels of examples 1, 2, and 3, respectively, the elastic modulus of the gel film reached substantially 1000Pa or more, the elastic modulus (G ') of the gel was always greater than the loss modulus (G') until the stress reached 10%, the gel exhibited solid-like rheological properties, and as the stress increased, the G 'was progressively less than the G', the gel exhibited liquid-like rheological properties, indicating that gel sol transformation occurred. D. E, F are frequency tests of LAPHEOH-co-NVP gels of examples 1, 2, 3, respectively. The graph shows that the elastic modulus of the L/D gel is basically consistent with the strength of the loss modulus, and the mechanical properties of the L/D gel are identical.
Cytotoxicity analysis of (L/D) APHEOH-co-NVP copolygel to investigate the effect of chirality on cell culture (L929) mouse fibroblasts were selected for cell culture on (L/D) APHEOH-co-NVP copolygel film, respectively, and cell cytotoxicity at 24h was tested. As shown in FIG. 7, the chiral gels of NVP-400, NVP-1000 and NVP-2000 prepared by copolymerizing the crosslinking agents with different molecular weights at the same monomer molar ratio correspond to the cell activities of the cells cultured on the NVP-400, NVP-1000 and NVP-2000L/D gel films of examples 1, 2 and 3, respectively. After 24 hours of incubation on the chiral gel films prepared in examples 1 (NVP-400), 2 (NVP-1000) and 3 (NVP-2000), it can be seen that the chiral gel film prepared in example 2 has the best cell activity, the activity of the D-APHEOH-co-NVP gel film reaches 151%, the activity of the L-APHEOH-co-NVP gel film reaches 94%, the difference in cell activity due to chirality reaches 57%, and the difference in chiral gel film activities in examples 1 and 3 reaches 51% and 54%, but the L-APHEOH-co-NVP gel films in examples 1 and 3 have only 71% and 66% in terms of the overall cell activity, and the activity is low, so that the chiral gel film in example 2 is most suitable for cell investigation of chiral films.
Cell proliferation of chiral gel films of example 2 were subjected to live-dead staining (see FIG. 8), FIG. A, B, C is a live-cell staining chart of D-APHEOH-co-NVP, L-APHEOH-co-NVP gel films and equally sized coverslips of example 2, respectively, and FIG. D, E, F is a dead-cell staining chart of D-APHEOH-co-NVP, L-APHEOH-co-NVP gel films and coverslips, respectively.
After 48h of culture, the cells (A) on the D-type gel film can be seen to be in the shape of rice grains under the staining of living cells, the cell density and the cell spreading area are larger than those of blank cells, which indicates that the D-type gel film has a promoting effect on the culture of mouse fibroblasts, the cells (B) on the L-type gel film mostly show a sphere shape, and the proliferation density of the cells is far lower than that of the blank cells, which indicates that the adhesion proliferation condition of the cells is poor. FIG. D, E, F is a graph of the staining of dead cells in the D-form, L-form and control groups, respectively, showing that the number of dead cells on the L/D membrane is consistent with that of the control group, indicating that the mortality of cells is low and that the toxicity of the gel to cells is almost absent. In addition to the chiral difference, the physical and chemical properties of the L/D gels were substantially identical, thus indicating that the difference in proliferation of cells was due to the chirality of the gels.
Claims (6)
1. A preparation method of an acryloylphenylalanine-N-vinyl pyrrolidone chiral hydrogel film is characterized in that,
Uniformly dissolving L-acryloylphenyl alanine or D-acryloylphenyl alanine and monomer N-vinyl pyrrolidone in N-methyl pyrrolidone solution, then adding an initiator and polyethylene glycol diacrylate, stirring and dissolving, introducing nitrogen at normal temperature to remove oxygen to obtain a reaction solution, dripping the reaction solution on a substrate, covering the substrate with a film, extruding an air bubble under light pressure, and irradiating under ultraviolet light to initiate polymerization reaction; the molecular weight of the polyethylene glycol diacrylate is 400-1200;
And obtaining the L-acryloylphenyl alanine-N-vinyl pyrrolidone copolymerized hydrogel film or the D-acryloylphenyl alanine-N-vinyl pyrrolidone copolymerized hydrogel film from the reacted substrate after ultraviolet irradiation is finished, wherein the L-APHEOH-co-NVP or the D-APHEOH-co-NVP is calculated.
2. The method for preparing the chiral hydrogel film of acryloylphenylalanine-N-vinylpyrrolidone according to claim 1, wherein the method comprises the following steps: the L-acryloylphenylalanine or D-acryloylphenylalanine is obtained by reacting L-phenylalanine or D-phenylalanine with acryloyl chloride as monomers according to the mol ratio of 1:1.5-2.2.
3. The method for preparing an acryloylphenylalanine-N-vinylpyrrolidone chiral hydrogel film according to claim 1, wherein the amount of polyethylene glycol diacrylate is 5% to 8% of the sum of the amounts of total monomer materials; the polymerization reaction temperature was room temperature.
4. The method for preparing the chiral hydrogel film of acryloylphenylalanine-N-vinylpyrrolidone according to claim 1, wherein the molar ratio of L-acryloylphenylalanine or D-acryloylphenylalanine to N-vinylpyrrolidone is 1-4:1-2.
5. The method for preparing an acryloylphenylalanine-N-vinylpyrrolidone chiral hydrogel film according to claim 1, wherein the molecular weight of the polyethylene glycol diacrylate is 1000.
6. Use of the method for preparing an acryloylphenylalanine-N-vinylpyrrolidone chiral hydrogel film prepared by the method according to any one of claims 1 to 5 as a chiral biomaterial interface.
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