Photoisomerization chiral hydrogel material as well as preparation method and application thereof

Abstract

The invention relates to a photoisomerization chiral hydrogel material as well as a preparation method and application thereof, and belongs to the field of supramolecular materials. The photoisomerization chiral hydrogel material is synthesized by adopting a stilbene derivative to modify a symmetric phenylalanine derivative through a three-step liquid phase reaction (amidation, trifluoroacetic acid deprotection and amidation), and the structural formula of the gel material is shown as a formula (I) or a formula (II), # imgabs0 #; the prepared nanogel material with uniform chirality successfully simulates a chiral helical structure of an extracellular microenvironment, the preparation method is simple and convenient, and the obtained hydrogel has good biocompatibility, can realize non-destructive three-dimensional packaging and light-operated release of cells, and has good application prospects. The material has a good application prospect in the fields of drug controlled release carriers, tissue engineering scaffold materials and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of supramolecular materials, and in particular relates to a photoisomerized chiral hydrogel material and a preparation method and application thereof. Background Art

[0002] Hydrogel is a cross-linked system that is fully diluted with water. It has a three-dimensional network cross-linked polymer structure. It contains a large number of hydrophilic groups that can be filled with water, making the overall mechanical properties of the hydrogel similar to those of soft tissue. As a result, the hydrogel has the properties of both solids and liquids, and has the ability to change its shape in response to the external environment. When used as a tissue engineering material, hydrogel has the following advantages: (1) The hydrogel contains a large amount of water, which effectively maintains the balance of the tissue microenvironment; (2) It has good biocompatibility and low tissue irritation; (3) It can be gelled in situ or injected to fill cavities of any shape. The above characteristics make hydrogel have broad application prospects in biomedicine, such as biological scaffolds, cell culture, drug controlled release, biosensors and other fields.

[0003] Chirality is one of the most important chemical signals in nature. It not only has an important influence on the replication and transcription of genetic information, the formation of higher-order protein structures and protein functionalization, but also plays a decisive role in maintaining cell adhesion, growth, proliferation and normal function in the extracellular matrix (ECM). Helical chirality is an important form of chirality in living organisms. It participates in many important physiological processes in organisms and has an important influence on the biological effects of chiral molecules. The helical chiral structure is formed by biomacromolecules such as proteins, polysaccharides and nucleic acids through non-covalent bonding such as π-π stacking, hydrogen bonding, electrostatic interaction and van der Waals force. These non-covalent bonding is the main force for constructing supramolecular structures and is the key factor for the natural system to transfer and amplify the molecular chirality of the basic unit of the organism into supramolecular chiral structures. Supramolecular chiral hydrogel materials with helical chirality can provide a three-dimensional (3D) microenvironment similar to ECM in three-dimensional cell culture due to their unique chiral helical structure, good biocompatibility, controllable chemical composition, easy functionalization and dynamic regulation, so that cells can grow and differentiate in a state closer to the natural state in vivo. Therefore, constructing new supramolecular chiral hydrogel materials is one of the current research hotspots in the field of biomimetic materials.

[0004] Patent CN 114106305A discloses a method for preparing a chirally uniform and controllable supramolecular hydrogel factor, comprising the following steps: S1, dissolving p-phenylacyl chloride or isocyanate and phenylalanine methyl ester hydrochloride in an organic solvent to generate a diester intermediate product under an alkaline environment; S2, demethylating the intermediate product and adjusting the pH value with hydrochloric acid to generate a white precipitate, filtering, rinsing, and vacuum drying to obtain a diacid intermediate product; S3, condensing the intermediate product with diglycolamine or 3-amino-1,2-propanediol under the action of an activator and an amide condensation agent, and obtaining a supramolecular hydrogel factor after separation and purification. It is suitable for commercial cell three-dimensional culture scaffold materials, etc. In the S2 step of the method, strong base is used for demethylation, and improper control of the amount of strong base and reaction time will lead to racemization of the product. In addition, the subsequent treatment of the S3 step is to remove the solvent, directly add water to form a hydrogel, filter, and wash with water. The activator is easily entrained during the hydrogel formation process, and it is not easy to remove it by washing with water, which increases the difficulty of purification.

[0005] In addition, photoisomerization is a phenomenon in which the chemical structure of a molecule changes under light conditions, resulting in different changes in its macroscopic properties. Photoisomerization has shown great application potential in the fields of light-driven molecular motors, photochromic probes, optical information storage, and optical switches. Summary of the invention

[0006] The purpose of the present invention is to develop a photoresponsive distilbene-based gel material, provide a photoisomerized chiral hydrogel material and its preparation method and application. The present invention utilizes supramolecular self-assembly to amplify and transfer the chiral molecules of phenylalanine to the supramolecular structure, utilizes the photoisomerization of distilbene to change the molecular conformation of the supramolecular structure and the morphology of the supramolecular hydrogel, and realizes the three-dimensional adhesion and release of the hydrogel material to the cells.

[0007] The objective of the present invention is achieved through the following technical solutions:

[0008] A photoisomerizable chiral hydrogel material, wherein the structural formula of the photoisomerizable chiral hydrogel material is shown in formula (I) or formula (II):

[0009]

[0010] The method for preparing the photoisomerized chiral hydrogel material comprises the following steps:

[0011] (1) Boc-L-phenylalanine or Boc-D-phenylalanine, diglycolamine and N,N-diisopropylethylamine (DIPEA), 1-hydroxybenzotriazole (HOBT) are dissolved in dichloromethane (organic solvent I), first reacted in an ice bath, then added with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) to continue the reaction, and after impurity removal and post-treatment, an amide condensation product is obtained, wherein N,N-diisopropylethylamine (DIPEA) provides an alkaline environment, 1-hydroxybenzotriazole (HOBT) is an activator, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) is a condensation agent. The structural formula of the amide condensation product is shown in formula (III) Boc-LF or formula (IV) Boc-DF respectively:

[0012]

[0013] The reaction equation is:

[0014]

[0015] (2) The amide condensation product obtained in step (1) is added to a mixed solution of dichloromethane (organic solvent I) and trifluoroacetic acid, and reacted at room temperature for 3 hours to remove the protecting group (Boc-), and then the solution and the remaining trifluoroacetic acid are removed by rotary evaporation to obtain trifluoroacetate, the structural formula of which is shown in formula (V) LF-trifluoroacetate or formula (VI) DF-trifluoroacetate:

[0016]

[0017] (3) Under the protection of inert gas, 4,4'-stilbene dicarboxylic acid and dithionyl chloride are subjected to chlorination reaction, and after post-treatment and impurity removal, 4,4'-stilbene dicarboxylic acid chloride is obtained, the structure of which is shown in formula (VII):

[0018]

[0019] (4) dissolving the trifluoroacetate and 4,4'-stilbene dichloride in dichloromethane, reacting them in an ice bath, then adding an organic base triethylamine to react in the dark, and then filtering, washing, and drying to obtain a photoisomerizable chiral hydrogel material.

[0020] In the step (1), the molar ratio of Boc-L-phenylalanine or Boc-D-phenylalanine, diglycolamine, DIPEA, HOBT and EDCI is 1:(1-3):(1-4):(1-3):(1-4).

[0021] The ice bath reaction time in step (1) is 20-50 min, and the reaction is continued in the ice bath for 20-50 min, then heated to 20-40° C. and reacted for 12-24 h. The dichloromethane solvent (organic solvent I) in steps (1), (2) and (4) can also be replaced by N-methylformamide or tetrahydrofuran.

[0022] The impurity removal post-treatment in step (1) includes washing with a saturated ammonium chloride solution, a saturated sodium bicarbonate solution, and a saturated sodium chloride solution in sequence; drying by rotary evaporation; recrystallization by ethyl acetate and petroleum ether, and then pumping dry with an oil pump.

[0023] In the step (2), the molar ratio of the amide condensation product to trifluoroacetic acid is 1:(1-3); the volume ratio of trifluoroacetic acid to organic solvent I is 1:(1-5); and the reaction conditions are room temperature for 30 min-4 h.

[0024] A catalyst is added to the chlorination reaction in step (3), wherein the catalyst is dimethylformamide (DMF), and the conditions for the chlorination reaction are reflux at 60-100° C. for 3-10 h; preferably, reaction at 75-85° C. for 3-10 h; thionyl chloride is in excess relative to 4,4′-stilbene dicarboxylic acid, and the molar ratio of 4,4′-stilbene dicarboxylic acid, dithionyl chloride and catalyst DMF is 1:(3-7):(0.3-0.6), and the post-treatment impurity removal process is to remove thionyl chloride (dithionyl chloride) by reduced pressure evaporation.

[0025] In the step (4), the molar ratio of 4,4'-stilbene dichloride: trifluoroacetate: triethylamine is 1:(2.0-2.5):(10-15).

[0026] The concentration of the trifluoroacetate intermediate product in step (4) is 0.02-0.3 mmol / mL; the light-proof reaction time is 16-24 hours; and the washing method includes washing with deionized water and dilute hydrochloric acid (0.1-1 mol / L) in sequence.

[0027] The photoisomerizable chiral hydrogel material is used in three-dimensional cell culture and release or in drug controlled release carriers.

[0028] The present invention has the following beneficial effects:

[0029] (1) The present invention utilizes divinyl-phenylalanine derivatives to self-assemble in a trans conformation to form a light-responsive hydrogel system. The aromatic group is larger and has a stronger aromatic stacking effect, and the resulting hydrogel is more stable.

[0030] (2) The addition of diphenylethylene in the present invention makes the hydrogel photoresponsive. The prepared hydrogel can be used for 3D cell encapsulation. Under light (λ=365nm), diphenylethylene isomerizes from the trans conformation to the cis conformation, causing the hydrogel to collapse and triggering cell release. This work allows light to manipulate material properties or chemistry in real time, and the material has good biocompatibility, degradability, and intelligence. Light-induced changes in gel structure or gel geometry affect its gel properties, and the microenvironment of the encapsulated cells is controlled in space and time through precisely controlled "on-off".

[0031] (3) The photoisomerized chiral hydrogel material for three-dimensional cell culture and release of the present invention is a chiral nanofiber constructed from a single enantiomer, which has the advantages of easy chirality, simple synthesis, good biocompatibility, and degradability, and is expected to have unique applications in cell tissue culture and other medical fields.

[0032] (4) The photoisomerized chiral hydrogel material for three-dimensional cell culture and release in the present invention can achieve cell adhesion and release under physiological conditions, is easy to operate, and has practical application value.

[0033] (5) The photoisomerized chiral hydrogel material of the present invention avoids the reaction conditions of long time and strong alkaline environment during the preparation process, reducing the possibility of chiral racemization; realizes the optimization of reaction steps, avoids impurities such as activators that are difficult to remove during post-treatment, and is relatively easy to purify. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The hydrogen spectrum of the photoisomerized chiral hydrogel gel factor L-SDF obtained by three-dimensional cell culture and release in Example 1 of the present invention ( 1 HNMR);

[0035] Figure 2 The hydrogen spectrum of the photoisomerized chiral hydrogel gel factor D-SDF obtained by three-dimensional cell culture and release in Example 2 of the present invention ( 1 HNMR);

[0036] Figure 3 This is a gel photograph of the photoisomerized chiral hydrogel material for three-dimensional cell culture and release in Example 1 and Example 2 of the present invention;

[0037] Figure 4 The ultraviolet spectra of the photoisomerized chiral hydrogel materials for three-dimensional cell culture and release in Examples 1 and 2 of the present invention change with illumination time (the left picture is L-SDF, and the right picture is D-SDF);

[0038] Figure 5Circular dichroism (CD) spectra of the photoisomerized chiral hydrogel materials for three-dimensional cell culture and release according to Examples 1 and 2 of the present invention that change with illumination time;

[0039] Figure 6 Vibrational circular dichroism (VCD) spectra of the photoisomerized chiral hydrogel materials for three-dimensional cell culture and release in Examples 1 and 2 of the present invention (the upper figure is the D-SDF of Example 2, and the lower figure is the L-SDF of Example 1);

[0040] Figure 7 Scanning electron microscope images of the photoisomerized chiral hydrogel material L / D-SDF before and after illumination of the photoisomerized chiral hydrogel material cell three-dimensional culture and release in Example 1 and Example 2 of the present invention (a and c are respectively before illumination in Example 1 and Example 2, and b and d are after illumination in Example 1 and Example 2);

[0041] Figure 8 Cytotoxicity test of the photoisomerized chiral hydrogel materials of Examples 1 and 2 of the present invention;

[0042] Fig. 9 The bright field and live-dead cell staining photos of the cells adhered to the two-dimensional film of the photoisomerized chiral hydrogel material of Examples 1 and 2 of the present invention (D is the D-SDF of Example 2, and L is the L-SDF of Example 1);

[0043] Fig.10 The live and dead cell staining pictures of the cell three-dimensional encapsulation culture experiment of the photoisomerized chiral hydrogel material of Example 1 and Example 2 of the present invention are shown. It can be seen from the pictures that NIH3T3 cells have no deposition in the two gels and are in good growth state, indicating that the two gel materials have good cell three-dimensional culture capabilities;

[0044] Fig.11 After 3D encapsulation and culture of cells in the photoisomerized chiral hydrogel material of Example 1 and Example 2 of the present invention, the cells released by photoisomerization after ultraviolet irradiation for 30 minutes were re-seeded on the PS plate, and the bright field photos and corresponding fluorescent staining photos at different culture times were taken. DETAILED DESCRIPTION

[0045] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0046] The room temperature in the embodiments of the present invention is 20-40°C.

[0047] Example 1

[0048] This embodiment provides a method for preparing supramolecular hydrogel L-SDF, comprising the following steps:

[0049] (1) Boc-L-phenylalanine (10 mmol) and 1-hydroxybenzotriazole (13 mmol, 1.3 eq) were dissolved in dichloromethane, cooled in an ice bath, diglycolamine (12 mmol, 1.2 eq) and N,N-diisopropylethylamine (20 mmol, 2 eq) were added, and stirred in an ice bath for 30 minutes. EDCI (20 mM, 2 eq) was added, and continued to stir in an ice bath for 30 minutes; the reaction solution was slowly warmed to room temperature and reacted at room temperature for 12 h; it was washed with saturated ammonium chloride solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution in sequence, and then dried by spin drying. It was recrystallized with ethyl acetate and petroleum ether, and pumped dry with an oil pump to prepare an amide condensation product Boc-LF. The reaction formula is shown below:

[0050]

[0051] (2) adding the amide condensation product Boc-LF prepared in step (1) to a solution having a volume ratio (V / V) of dichloromethane / trifluoroacetic acid of 3 / 1, wherein the molar / volume ratio of Boc-LF to trifluoroacetic acid is 1:1; reacting at room temperature for 2 hours, and then rotary evaporating the solution to completely remove the solvent and excess trifluoroacetic acid to prepare LF-trifluoroacetate. The reaction process is shown in the following formula:

[0052]

[0053] (3) Under nitrogen protection, 4,4'-stilbene dicarboxylic acid (5 mmol) was added to 15 mmol of dichlorothionyl) and then catalyst DMF (0.2 mL) was added. The mixture was heated under reflux at 85°C for 5 h to generate 4,4'-stilbene dicarboxylic acid.

[0054] Acid chloride; remove excess thionyl chloride in vacuum, the reaction process is as follows:

[0055]

[0056] (4) Dissolve 4,4'-stilbene dichloride (3 mmol) in dichloromethane (33 mL), add the generated LF-trifluoroacetate (6.6 mmol) and TEA (39 mmol) under ice bath conditions, and the concentration of LF-trifluoroacetate in the solution is 0.2 mmol / mL; react for 16 hours in the dark. Filter the obtained colloid with a Buchner funnel, wash with deionized water and 1M HCl in turn, and dry the sample in an oven to obtain a white solid target product, namely, the photoisomerized chiral hydrogel material L-SDF:

[0057]

[0058] Example 2 Preparation of supramolecular hydrogel D-SDF

[0059] The preparation of supramolecular hydrogel D-SDF in this embodiment includes the following steps:

[0060] (1) Boc-D-phenylalanine (10 mmol) and 1-hydroxybenzotriazole (13 mmol, 1.3 eq) were dissolved in dichloromethane, cooled in an ice bath, diglycolamine (12 mmol, 1.2 eq) and N,N-diisopropylethylamine (20 mmol, 2 eq) were added, and stirred in an ice bath for 30 minutes. EDCI (20 mmol, 2 eq) was added, and continued to stir in an ice bath for 30 minutes; the reaction solution was slowly warmed to room temperature and reacted at room temperature for 12 h; it was washed with saturated ammonium chloride solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution in turn, and then spin-dried and recrystallized with ethyl acetate and petroleum ether to prepare an amide condensation product Boc-DF, the reaction formula of which is shown below:

[0061]

[0062] (2) adding the amide condensation product Boc-DF prepared in step (1) to a solution of dichloromethane / trifluoroacetic acid (V / V) of 3 / 1, wherein the molar ratio of Boc-DF to trifluoroacetic acid is 1:2; after reacting at room temperature for 2 hours, the solution is rotary evaporated to completely remove the solvent and excess trifluoroacetic acid, to prepare DF-trifluoroacetate, and the reaction process is shown in the following formula:

[0063]

[0064] (3) Under nitrogen protection, 4,4'-stilbene dicarboxylic acid (5 mmol) was added to an excess of thionyl chloride (15 mmol), and then a catalyst DMF (0.2 mL) was added. The reaction temperature was 85°C and refluxed for 5 hours to generate 4,4'-stilbene dicarboxylic acid chloride. The excess thionyl chloride was removed in vacuum. The reaction process is as follows:

[0065]

[0066] (4) 4,4'-stilbene dichloride (3 mmol) was suspended in dichloromethane (33 mL), and the generated LD-trifluoroacetate (6.6 mmol) and TEA (39 mmol) were added under ice bath conditions. The concentration of LF-trifluoroacetate in the solution was 0.2 mmol / mL. The reaction was carried out in the dark for 16 hours. The obtained colloid was filtered through a Buchner funnel, washed with deionized water and 1M HCl in sequence, and the sample was dried in an oven to obtain a white solid target product, namely, the photoisomerized chiral hydrogel material D-SDF: The reaction process equation is as follows:

[0067]

[0068] Example 3

[0069] This embodiment provides a method for preparing supramolecular hydrogel L-SDF, comprising the following steps:

[0070] (1) Boc-L-phenylalanine (10 mmol) and 1-hydroxybenzotriazole (10 mmol, 1.0 eq) were dissolved in dichloromethane, cooled in an ice bath, diglycolamine (30 mmol, 3 eq) and N,N-diisopropylethylamine (10 mmol, 1 eq) were added, and stirred in an ice bath for 30 minutes, and EDCI (40 mM, 4 eq) was added, and continued to stir in an ice bath for 50 minutes; the reaction solution was slowly heated to 20° C. and reacted at room temperature for 24 hours; it was washed with saturated ammonium chloride solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution in sequence, and then spin-dried, and recrystallized with ethyl acetate and petroleum ether, and pumped dry with an oil pump to prepare an amide condensation product Boc-LF;

[0071] (2) adding the amide condensation product Boc-LF prepared in step (1) to a solution of dichloromethane / trifluoroacetic acid (V / V) of 5 / 1, wherein the molar ratio of Boc-LF to trifluoroacetic acid is 1:3; after reacting at room temperature for 2 hours, the solution is rotary evaporated to completely remove the solvent and excess trifluoroacetic acid, to prepare LF-trifluoroacetate;

[0072] (3) Under nitrogen protection, 4,4'-stilbene dicarboxylic acid (5 mmol) was added to excess thionyl chloride (12.5 mmol), and then catalyst DMF (1.5 mmol) was added. The temperature was 60°C and refluxed for 10 hours to generate 4,4'-stilbene dicarboxylic acid chloride; excess thionyl chloride was removed in vacuo;

[0073] (4) 4,4'-stilbene dichloride (3 mmol) was suspended in dichloromethane (DCM), and the generated LF-trifluoroacetate (6.0 mmol) and TEA (30 mmol) were added under ice bath conditions. The concentration of LF-trifluoroacetate in the solution was 0.15 mol / L. The reaction was carried out in the dark for 24 hours. The obtained colloid was filtered through a Buchner funnel, washed with deionized water and 0.1 M HCl in sequence, and the sample was dried in an oven to obtain a white solid target product, namely, the photoisomerized chiral hydrogel material L-SDF.

[0074] Example 4

[0075] This embodiment provides a method for preparing supramolecular hydrogel L-SDF, comprising the following steps:

[0076] (1) Boc-L-phenylalanine (10 mmol) and 1-hydroxybenzotriazole (30 mmol, 1.0 eq) were dissolved in dichloromethane, cooled in an ice bath, diglycolamine (10 mmol, 1 eq) and N,N-diisopropylethylamine (40 mmol, 4 eq) were added, and stirred in an ice bath for 30 minutes, and EDCI (10 mM, 1 eq) was added, and continued to stir in an ice bath for 50 minutes; the reaction solution was slowly raised to 50° C. and reacted at room temperature for 12 h; washed with saturated ammonium chloride solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution in sequence, spin-dried, recrystallized with ethyl acetate and petroleum ether, and pumped dry with an oil pump to prepare an amide condensation product Boc-LF;

[0077] (2) adding the amide condensation product Boc-LF prepared in step (1) to a solution of dichloromethane / trifluoroacetic acid (V / V) of 5 / 1, wherein the molar ratio of Boc-LF to trifluoroacetic acid is 1:1; after reacting at room temperature for 2 hours, the solution is rotary evaporated to completely remove the solvent and excess trifluoroacetic acid, to prepare LF-trifluoroacetate, and the reaction process is shown in the following formula:

[0078] (3) Under nitrogen protection, 4,4'-stilbene dicarboxylic acid (5 mmol) was added to excess thionyl chloride (25 mmol), and then catalyst DMF (3 mmol) was added. The temperature was 100°C and refluxed for 3 hours to generate 4,4'-stilbene dicarboxylic acid chloride; excess thionyl chloride was removed in vacuo;

[0079] (4) 4,4'-stilbene dichloride (3 mmol) was suspended in dichloromethane (DCM), and the generated LF-trifluoroacetate (7.5 mmol) and TEA (45 mmol) were added under ice bath conditions. The concentration of LF-trifluoroacetate in the solution was 0.3 mol / L. The reaction was carried out in the dark for 16 hours. The obtained colloid was filtered through a Buchner funnel, washed with deionized water and 1M HCl in sequence, and the sample was dried in an oven to obtain a white solid target product, namely, the photoisomerized chiral hydrogel material L-SDF.

[0080] Example 5

[0081] This embodiment provides a method for preparing supramolecular hydrogel D-SDF, comprising the following steps:

[0082] (1) Boc-D-phenylalanine (10 mmol) and 1-hydroxybenzotriazole (10 mmol, 1.0 eq) were dissolved in dichloromethane, cooled in an ice bath, diglycolamine (30 mmol, 3 eq) and N,N-diisopropylethylamine (10 mmol, 1 eq) were added, and stirred in an ice bath for 30 minutes, and EDCI (40 mM, 4 eq) was added, and continued to stir in an ice bath for 50 minutes; the reaction solution was slowly heated to 20° C. and reacted at room temperature for 24 hours; washed with saturated ammonium chloride solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution in sequence, spin-dried, recrystallized with ethyl acetate and petroleum ether, and pumped dry with an oil pump to prepare an amide condensation product Boc-DF;

[0083] (2) adding the amide condensation product Boc-DF prepared in step (1) to a solution of dichloromethane / trifluoroacetic acid (V / V) of 4 / 1, wherein the molar ratio of Boc-DF to trifluoroacetic acid is 1:2; after reacting at room temperature for 30 minutes, the solution is rotary evaporated to completely remove the solvent and excess trifluoroacetic acid, to prepare DF-trifluoroacetate;

[0084] (3) Under nitrogen protection, 4,4'-stilbene dicarboxylic acid (5 mmol) was added to excess thionyl chloride (10 mmol), and then catalyst DMF (2 mmol) was added. The temperature was 60°C and refluxed for 10 hours to generate 4,4'-stilbene dicarboxylic acid chloride; excess thionyl chloride was removed in vacuo;

[0085] (4) 4,4'-stilbene dichloride (3 mmol) was suspended in dichloromethane (DCM), and the generated DF-trifluoroacetate (6.6 mmol) and TEA (40 mmol) were added under ice bath conditions. The concentration of DF-trifluoroacetate in the solution was 0.02 mol / L. The reaction was carried out in the dark for 24 hours. The obtained colloid was filtered through a Buchner funnel, washed with deionized water and 1M HCl in sequence, and the sample was dried in an oven to obtain a white solid target product, namely, the photoisomerized chiral hydrogel material D-SDF.

[0086] Example 6

[0087] This embodiment provides a method for preparing supramolecular hydrogel D-SDF, comprising the following steps:

[0088] (1) Boc-D-phenylalanine (10 mmol) and 1-hydroxybenzotriazole (30 mmol, 1.0 eq) were dissolved in dichloromethane, cooled in an ice bath, diglycolamine (10 mmol, 1 eq) and N,N-diisopropylethylamine (40 mmol, 4 eq) were added, and stirred in an ice bath for 30 minutes, and EDCI (10 mM, 1 eq) was added, and continued to stir in an ice bath for 50 minutes; the reaction solution was slowly heated to 50° C. and reacted at room temperature for 12 h; washed with saturated ammonium chloride solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution in sequence, spin-dried, recrystallized with ethyl acetate and petroleum ether, and pumped dry with an oil pump to prepare an amide condensation product Boc-DF;

[0089] (2) adding the amide condensation product Boc-DF prepared in step (1) to a solution of dichloromethane / trifluoroacetic acid (V / V) of 4 / 1, wherein the molar ratio of Boc-DF to trifluoroacetic acid is 1:2; after reacting at room temperature for 1.5 hours, the solution is rotary evaporated to completely remove the solvent and excess trifluoroacetic acid, to prepare LF-trifluoroacetate, and the reaction process is shown in the following formula:

[0090] (3) Under nitrogen protection, 4,4'-stilbene dicarboxylic acid (5 mmol) was added to excess thionyl chloride (20 mmol), and then catalyst DMF (1.5 mmol) was added. The temperature was 100°C and refluxed for 3 hours to generate 4,4'-stilbene dicarboxylic acid chloride; excess thionyl chloride was removed in vacuo;

[0091] (4) 4,4'-stilbene dichloride (3 mmol) was suspended in dichloromethane (DCM), and the generated DF-trifluoroacetate (7.0 mmol) and TEA (40 mmol) were added under ice bath conditions. The concentration of DF-trifluoroacetate in the solution was 0.15 mol / L. The reaction was carried out in the dark for 20 hours. The obtained colloid was filtered through a Buchner funnel, washed with deionized water and 1M HCl in sequence, and the sample was dried in an oven to obtain a white solid target product, namely, the photoisomerized chiral hydrogel material D-SDF.

[0092] according to Figure 1 The hydrogen nuclear magnetic resonance spectrum shows that the white solid prepared in Example 1 is the target product with the structural formula of L-SDF;

[0093] according to Figure 2 The hydrogen nuclear magnetic resonance spectrum shows that the white solid prepared in Example 1 is the target product with the structural formula D-SDF;

[0094] pass Figure 3 From the photos, it can be seen that the L-SDF of Example 1 and the D-SDF photoisomerized chiral hydrogel materials of Example 2 are very stable hydrogels, and the transparency of the two is slightly different;

[0095] Figure 4 The ultraviolet spectra of the photoisomerized chiral hydrogel materials for three-dimensional cell culture and release in Example 1 and Example 2 of the present invention as a function of illumination time (the left picture is L-SDF, and the right picture is D-SDF); it can be seen from the figure that the two gels achieve maximum photoisomerization in about 30 minutes;

[0096] Figure 5 The circular dichroism (CD) spectra of the photoisomerized chiral hydrogel materials of cell three-dimensional culture and release in Example 1 and Example 2 of the present invention changing with the illumination time; it can be seen from the figure that the two gels have strong Cotton effect peaks and are hydrogels with chiral optical signals;

[0097] Figure 6 The vibration circular dichroism (VCD) spectra of the photoisomerized chiral hydrogel materials for three-dimensional cell culture and release in Examples 1 and 2 of the present invention (the upper figure is the D-SDF of Example 2, and the lower figure is the L-SDF of Example 1). It can be seen from the figure that the D-SDF signal is positive first and then negative, which is a left-handed helical fiber network, and the L-SDF signal is negative first and then positive, which is a right-handed helical fiber network;

[0098] Figure 7The scanning electron microscope images of the photoisomerized chiral hydrogel material L / D-SDF before and after illumination for three-dimensional cell culture and release of photoisomerized chiral hydrogel materials in Examples 1 and 2 of the present invention (a and c are before illumination for Example 1 and Example 2, respectively, and b and d are after illumination for Example 1 and Example 2); it can be seen from the figure that both can assemble into spiral fibers before illumination, and after illumination, the gel disintegrates, and the fibers become nanoparticles after photoisomerization.

[0099] Application Examples

[0100] Application of photoisomerized chiral hydrogel materials in cell culture

[0101] Adhesion of NIH 3T3 cells on the surface of photoisomerized chiral hydrogel nanofiber film and three-dimensional adhesion and release of hydrogel.

[0102] The nanofiber film used in this application example and the chiral uniform distilbene derivative gel factor three-dimensional hydrogel prepared in the above-mentioned Example 1 and Example 2 are self-assembled.

[0103] Experimental Example 1: Cytotoxicity test of two-dimensional fiber film:

[0104] First, the two gel factors of application examples 1 and 2 were prepared into 0.3 mg / mL ultrapure water dilute solutions, cooled and self-assembled, and 100 L of 0.3 mg / mL hydrogel dilute solution was added to each well of the cell culture plate (96-well plate), dried at 37°C for 12 hours, and irradiated with ultraviolet light for 30 minutes. Well-grown NIH 3T3 cells were seeded on a 96-well plate covered with L-SDF and D-SDF dry gel films (about 5000 cells / well), placed in a 37°C, 5% CO2 cell culture incubator for 24 hours, and then 10 μL CCK-8 reagent was added to each well (three groups of parallels were performed for each result at the same time), incubated in a humidified incubator at 37°C, 5% CO2 for 1 hour, and the absorbance value was detected at 450nm using an enzyme reader, as shown in Figure 2. Figure 8 .Depend on Figure 8 It can be seen that the adhesion amount of NIH 3T3 cells on the L-SDF two-dimensional fiber film is equivalent to that of the control group, and the adhesion amount of the D-SDF two-dimensional fiber film is higher than that of the control group, indicating that the two chiral materials have good biocompatibility.

[0105] The specific operation of live-dead cell staining is as follows: first, the culture medium in the culture plate is aspirated and rinsed twice with PBS buffer. Subsequently, a PBS solution containing calcein-AM (final concentration of 2 μM) and propidium iodide (final concentration of 4 μM) is added to the cell culture plate, and incubated in a humidified incubator under standard culture conditions (37°C, 5% CO2) for 15 minutes, and then PBS buffer is added to wash away the staining solution, and the cells are observed under a fluorescence microscope. The green cells observed under the microscope represent live cells, and the red cells represent dead cells.

[0106] The live-dead cell double staining method was used to test the cytotoxicity of ultraviolet light irradiation for 30 minutes: the two gel factors were prepared into 0.3 mg / mL ultrapure water dilute solutions, 1 mL of 0.3 mg / mL hydrogel dilute solution was added to each well of the cell culture plate (24-well plate), dried at 37°C for 24 hours, irradiated with ultraviolet light for 30 minutes, rinsed twice with PBS buffer, and well-grown NIH 3T3 cells were seeded on 24-well plates covered with L-SDF and D-SDF dry gel films in the application example (about 10,000 cells / well), and incubated in a 37°C, 5% CO2 cell culture incubator for 24 hours.

[0107] Invert the image (such as Fig. 9 Then the 24-well plate with NIH 3T3 cells adhered was irradiated under UV light (365 nm) for 30 min (as shown in a) in D and a) in L). Fig. 9 As shown in D (b) and L (b), it was observed that the cell tentacles adhered to the dry gel membrane shrank, the spindle-shaped cells became round, and the cells were detached by gently blowing (as shown in Fig. 9 The detached cells were re-seeded on a 24-well cell culture plate and incubated in a 37°C, 5% CO2 cell culture incubator for 12 h. The cells were then stained for live-dead cells and imaged using a Leica DMI8 inverted fluorescence microscope (as shown in FIG. Fig. 9 As shown in d), e) in D and d), e) in L). Fig. 9 The results showed that 30 minutes of 365nm ultraviolet irradiation caused little damage to the cells, and the cells still maintained good vitality.

[0108] Experimental Example 2 Three-dimensional cell culture:

[0109] First, 50 μL of DMSO concentrate containing gel factor (300 mg / mL) and 950 μL of cell suspension (cell number about 1.0x10 6 100 μg / mL) were mixed evenly in a 24-well plate, placed in an incubator for 15 min to allow gel formation, and then 1 mL of DMEM cell culture medium was slowly added. The cells were incubated in an incubator at 37°C and 5% CO2. The culture medium on the top of the gel was replaced twice every 24 h.

[0110] Live-dead cell staining was performed using Ca-AM / PI, and imaging was performed using the Nikon / A1+N-SIM structured illumination ultra-high resolution microscopy system ( Fig.10 a and b correspond to the cell culture experiment diagrams of L-SDF and D-SDF gel materials in the application examples, respectively). Fig.10 The results showed that cells encapsulated in D-SDF and L-SDF hydrogels grew well, and cells encapsulated in D-SDF hydrogels proliferated faster than those encapsulated in L-SDF hydrogels, indicating that D-SDF hydrogels were more conducive to inducing cell adhesion and proliferation. D-SDF hydrogel-encapsulated cells were selected and irradiated under 365nm UV light. After 60 minutes of illumination, the gel collapsed and the cells were released. They were re-seeded in a culture dish and placed in an incubator at 37°C and 5% CO2 for different culture times (12h, 24h, 36h), and then live-dead cell staining and inverted fluorescence microscopy imaging were performed. ( Fig.11 a, d, g are the bright field images corresponding to 12h, 24h, and 36h, b, e, h are the live cell staining images corresponding to 12h, 24h, and 36h, c, f, i are the dead cell staining images corresponding to 12h, 24h, and 36h, respectively). Fig.11 The results showed that the number of cells continued to increase with time, indicating that the cells still maintained good activity and proliferation ability after being three-dimensionally encapsulated in hydrogel materials and released under 365nm ultraviolet light irradiation.

[0111] In summary, the chiral uniform nanogel material prepared in this embodiment successfully mimics the chiral helical structure of the extracellular microenvironment, and cells are cultured in an in vitro environment. The adhesion and release of cells are regulated by light. It has the advantages of simple synthesis, good biocompatibility, and degradability. In addition, it is a chiral nanofiber constructed from a single enantiomer, and the chirality is easy to control. It is expected to have unique applications in cell tissue culture and other medical fields.

[0112] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A photoisomerized chiral hydrogel material, characterized in that: The structural formula of the photoisomerizable chiral hydrogel material is shown in formula (I) or formula (II): 。 2. The method for preparing the photoisomerized chiral hydrogel material according to claim 1, characterized in that: The steps include: (1) Boc-L-phenylalanine or Boc-D-phenylalanine, diglycolamine, N,N-diisopropylethylamine and 1-hydroxybenzotriazole are dissolved in an organic solvent I, reacted under ice bath conditions, and then 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is added to continue the reaction. After impurity removal and post-treatment, an amide condensation product is obtained, the structural formula of which is shown in formula (III) Boc-LF or formula (IV) Boc-DF: (2) The amide condensation product obtained in step (1), trifluoroacetic acid and organic solvent I are mixed and reacted, and then impurities are removed by rotary evaporation to obtain trifluoroacetate. The structural formula of the trifluoroacetate is shown in formula (V) LF-trifluoroacetate or formula (VI) DF-trifluoroacetate: (3) Under the protection of inert gas, 4,4'-stilbene dicarboxylic acid and dithionyl chloride are subjected to chlorination reaction, and after post-treatment and impurity removal, 4,4'-stilbene dichloride is obtained, the structure of which is shown in formula (VII): (4) The trifluoroacetate and 4,4'-stilbene dichloride are dissolved in an organic solvent I, reacted in an ice bath, and then triethylamine is added to react overnight in the dark, filtered, washed, and dried to obtain a photoisomerizable chiral hydrogel material.

3. The method for preparing the photoisomerized chiral hydrogel material according to claim 2, characterized in that: In the step (1), the molar ratio of Boc-L-phenylalanine or Boc-D-phenylalanine, diglycolamine, N,N-diisopropylethylamine, 1-hydroxybenzotriazole, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:(1-3):(1-4):(1-3):(1-4).

4. The method for preparing the photoisomerized chiral hydrogel material according to claim 2, characterized in that: The ice bath reaction time in step (1) is 20-50 min, and the conditions for continuing the reaction are: reacting in the ice bath for 20-50 min, then heating to 20-40° C. and reacting for 12-24 h. The organic solvent I in steps (1), (2) and (4) is dichloromethane, N-methylformamide or tetrahydrofuran.

5. The method for preparing the photoisomerized chiral hydrogel material according to claim 2, characterized in that: The impurity removal post-treatment in step (1) includes washing with a saturated ammonium chloride solution, a saturated sodium bicarbonate solution, and a saturated sodium chloride solution in sequence; drying by rotary evaporation; recrystallization by ethyl acetate and petroleum ether, and then pumping dry with an oil pump.

6. The method for preparing the photoisomerized chiral hydrogel material according to claim 2, characterized in that: In the step (2), the molar ratio of the amide condensation product to trifluoroacetic acid is 1:(1-3); the volume ratio of trifluoroacetic acid to organic solvent I is 1:(1-5); and the reaction conditions are reaction at room temperature for 30 min-4 h.

7. The method for preparing the photoisomerized chiral hydrogel material according to claim 2, characterized in that: In the step (3), a catalyst is added to the chlorination reaction, the catalyst is dimethylformamide, and the conditions of the chlorination reaction are 75-85° C. for 3-10 hours; the molar ratio of 4,4′-stilbene dicarboxylic acid, dithionyl chloride and the catalyst is 1:(2.5-5):(0.3-0.6), and the post-treatment impurity removal process is to remove the dithionyl chloride by reduced pressure evaporation.

8. The method for preparing the photoisomerized chiral hydrogel material according to claim 2, characterized in that: In the step (4), the molar ratio of 4,4'-stilbene dichloride, trifluoroacetate and triethylamine is 1:(2.0-2.5):(10-15); the concentration of trifluoroacetate is 0.02-0.3 mmol / mL.

9. The method for preparing the photoisomerized chiral hydrogel material according to claim 2, characterized in that: The light-protected reaction time in step (4) is 12-24 hours; the washing method includes washing with deionized water and diluted hydrochloric acid with a concentration of 0.1-1 mol / L in sequence.

10. Use of the photoisomerizable chiral hydrogel material according to claim 1 in three-dimensional cell culture and release or in a drug controlled release carrier.

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