A photoisomerizable chiral hydrogel material and its preparation method and application

Abstract

The present invention relates to a photoisomerizable chiral hydrogel material, its preparation method, and application, belonging to the field of supramolecular materials. The photoisomerizable chiral hydrogel material is synthesized by modifying a symmetrical phenylalanine derivative with a stilbene derivative through a three-step liquid-phase reaction (amidation, trifluoroacetic acid deprotection, and amidation). The structural formula of the hydrogel material is shown as (I) or (II). The chirally uniform nanogel material prepared by the present invention successfully mimics the chiral helical structure of the extracellular microenvironment. The preparation method is simple, and the resulting hydrogel has excellent biocompatibility and can achieve damage-free three-dimensional encapsulation and photocontrolled release of cells. It has promising application prospects in fields such as controlled-release drug carriers and tissue engineering scaffold materials.

Description

Technical Field

[0001] The present invention belongs to the technical field of supramolecular materials, and in particular relates to a photoisomerizable 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 fill water inside, making the overall mechanical properties of the hydrogel similar to those of soft tissue. As a result, the hydrogel has the properties of both solid and liquid, and has the ability to change its shape in response to the external environment. Hydrogel has the following advantages when used as a tissue engineering material: (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 nature's most important chemical signals. It not only plays a crucial role in the replication and transcription of genetic information, the formation of higher-order protein structures, and protein functionalization, but also plays a crucial role in maintaining cell adhesion, growth, proliferation, and normal function in the extracellular matrix (ECM). Helical chirality is a key form of chirality in living organisms, participating in many important physiological processes within the body and significantly influencing the biological effects of chiral molecules. Helical chiral structures are formed in biomacromolecules such as proteins, polysaccharides, and nucleic acids through non-covalent bonding interactions such as π-π stacking, hydrogen bonding, electrostatic interactions, and van der Waals forces. These non-covalent bonds are the primary forces in the construction of supramolecular structures and are key factors in the natural system's ability to transfer and amplify the chirality of the basic molecular units of organisms into supramolecular chiral structures. Supramolecular chiral hydrogels with helical chirality, due to their unique chiral helical structure, good biocompatibility, controllable chemical composition, ease of functionalization, and dynamic regulation, can provide a three-dimensional (3D) microenvironment similar to the ECM in 3D cell culture, enabling cell growth and differentiation in a state closer to that found in the body. Therefore, the construction of new supramolecular chiral hydrogels is currently a hot topic in the field of biomimetic materials.

[0004] Patent CN 114106305A discloses a method for preparing a supramolecular hydrogelator with uniform and controllable chirality. The method comprises the following steps: S1. Dissolving p-phenylyl chloride or isocyanate with phenylalanine methyl ester hydrochloride in an organic solvent to form a diester intermediate under alkaline conditions; S2. Demethylating the intermediate product and adjusting the pH with hydrochloric acid to produce a white precipitate, which is then filtered, rinsed, and vacuum-dried to obtain a diacid intermediate; S3. Condensing the intermediate product with diglycolamine or 3-amino-1,2-propanediol in the presence of an activator and an amide condensing agent. After separation and purification, the supramolecular hydrogelator is obtained. The method is suitable for commercial applications such as three-dimensional cell culture scaffolds. In step S2, a strong base is used for demethylation. Improper control of the base dosage and reaction time can lead to racemization of the product. Furthermore, the subsequent steps in step S3 involve removing the solvent, directly adding water to form the hydrogel, filtering, and washing. This process can easily entrain the activator and make it difficult to remove by washing, complicating purification.

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

[0006] The present invention aims to develop a photoresponsive stilbene-based gel material, providing a photoisomerizable chiral hydrogel material, its preparation method, and its application. This invention utilizes supramolecular self-assembly to amplify and transfer the chiral phenylalanine molecule into a supramolecular structure. The photoisomerization of stilbene modifies the molecular conformation of the supramolecular structure and the morphology of the supramolecular hydrogel, enabling three-dimensional cell adhesion and release.

[0007] The purpose 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 preparation method of the photoisomerizable chiral hydrogel material comprises the following steps:

[0011] (1) Boc-L-phenylalanine or Boc-D-phenylalanine, diglycolamine, N,N-diisopropylethylamine (DIPEA), and 1-hydroxybenzotriazole (HOBT) are dissolved in dichloromethane (organic solvent I), first reacted in an ice bath, and then added with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) to continue the reaction. 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 condensing 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-). The solution and the remaining trifluoroacetic acid are then removed by rotary evaporation to obtain a trifluoroacetate having the structural formula 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 thionyl 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 minutes, and the conditions for continuing the reaction are to react in the ice bath for 20-50 minutes, and then raise the temperature to 20-40° C. and react for 12-24 hours; 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). The conditions for the chlorination reaction are reflux at 60-100° C. for 3-10 hours; preferably, reaction at 75-85° C. for 3-10 hours; thionyl chloride is in excess relative to 4,4′-stilbene dicarboxylic acid, and the molar ratio of 4,4′-stilbene dicarboxylic acid, thionyl chloride and catalyst DMF is 1:(3-7):(0.3-0.6). The post-treatment impurity removal process is to remove thionyl chloride (thionyl 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 reaction time in the dark 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 groups are larger and have 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. The material has good biocompatibility, degradability, and intelligence. Light-induced changes in gel structure or gel geometry affect its gel properties, and through precisely controlled "on-off", the microenvironment of the encapsulated cells is controlled in space and time.

[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. The chirality is easy to control and has the advantages of simple synthesis, good biocompatibility, and degradability. It 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 the post-processing process, and is relatively easy to purify. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The hydrogen spectrum of the photoisomerizable 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 photoisomerizable 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 photoisomerizable chiral hydrogel material for three-dimensional cell culture and release in Examples 1 and 2 of the present invention;

[0037] Figure 4 The UV spectra of the photoisomerizable 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 figure is L-SDF, and the right figure is D-SDF);

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

[0039] Figure 6 Vibrational circular dichroism (VCD) spectra of the photoisomerizable 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 photoisomerizable chiral hydrogel material L / D-SDF before and after illumination of cells three-dimensionally cultured and released in Examples 1 and 2 of the present invention (a and c are before illumination in Examples 1 and 2, respectively; b and d are after illumination in Examples 1 and 2);

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

[0042] Figure 9 Bright field and live-dead cell staining photographs of cells adhered to the two-dimensional film of the photoisomerizable chiral hydrogel materials of Examples 1 and 2 of the present invention (D is D-SDF of Example 2, and L is L-SDF of Example 1);

[0043] Figure 10 Images of live and dead cell staining from the three-dimensional cell encapsulation culture experiments using photoisomerizable chiral hydrogel materials in Examples 1 and 2 of the present invention show that NIH3T3 cells showed no deposition and good growth in both gels, demonstrating the excellent three-dimensional cell culture capabilities of both gel materials.

[0044] Figure 11 After 3D encapsulation and culture of cells in the photoisomerized chiral hydrogel materials of Examples 1 and 2 of the present invention, the cells released by photoisomerization after 30 minutes of UV irradiation were re-seeded on the PS plate. Bright field photos and corresponding fluorescent staining photos at different culture times. DETAILED DESCRIPTION

[0045] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention 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 and 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 stirred in an ice bath for another 30 minutes. The reaction solution was slowly warmed to room temperature and reacted at room temperature for 12 hours. The solution was washed with saturated ammonium chloride solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution in sequence, and the mixture was spin-dried. The mixture was recrystallized with ethyl acetate and petroleum ether and pumped dry with an oil pump to obtain the 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 h, 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 thionyl chloride (15 mmol), 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] Acyl chloride; remove excess thionyl chloride in vacuo. 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) in an ice bath. The concentration of LF-trifluoroacetate in the solution is 0.2 mmol / mL. The reaction is carried out in the dark for 16 hours. The obtained colloidal solution is filtered through a Buchner funnel, washed with deionized water and 1M HCl in sequence, and dried in an oven to obtain the target product as a white solid, i.e., the photoisomerizable 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 and 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 stirred in an ice bath for another 30 minutes. The reaction solution was slowly warmed to room temperature and reacted at room temperature for 12 hours. The solution was washed with saturated ammonium chloride solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution in sequence, dried by spin drying, and recrystallized with ethyl acetate and petroleum ether to prepare the amide condensation product Boc-DF. The reaction formula 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; reacting at room temperature for 2 hours, and then rotary evaporating the solution to completely remove the solvent and excess trifluoroacetic acid to prepare DF-trifluoroacetate. 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 the 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 vacuo. The reaction process is as follows:

[0065]

[0066] (4) 4,4'-Stilbene dichloride (3 mmol) was suspended in dichloromethane (33 mL). 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 colloidal product was filtered through a Buchner funnel, washed with deionized water and 1M HCl in sequence, and dried in an oven to obtain the target product as a white solid, i.e., the photoisomerizable 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 and 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. EDCI (40 mM, 4 eq) was added and stirred in an ice bath for 50 minutes. The reaction solution was slowly heated to 20°C and reacted at room temperature for 24 hours. The product was washed with saturated ammonium chloride solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution in sequence, and then dried by spin drying. The product was recrystallized with ethyl acetate and petroleum ether and pumped dry by an oil pump to obtain the 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 an excess of thionyl chloride (12.5 mmol), and then a catalyst, DMF (1.5 mmol), was added. The temperature was set at 60°C and refluxed for 10 hours to generate 4,4'-stilbene dicarboxylic acid chloride; the excess thionyl chloride was removed in vacuo;

[0073] (4) 4,4'-Stilbene dichloride (3 mmol) was suspended in dichloromethane (DCM). LF-trifluoroacetate (6.0 mmol) and TEA (30 mmol) were added under ice-cooling 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 resulting colloid was filtered through a Buchner funnel and washed with deionized water and 0.1 M HCl in sequence. The sample was dried in an oven to obtain the target product as a white solid, i.e., the photoisomerizable 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 and 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. EDCI (10 mM, 1 eq) was added and stirred in an ice bath for 50 minutes. The reaction solution was slowly heated to 50°C and reacted at room temperature for 12 hours. The solution was washed with saturated ammonium chloride solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution in sequence, and the mixture was spin-dried. The mixture was recrystallized with ethyl acetate and petroleum ether and dried with an oil pump to obtain the 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; reacting at room temperature for 2 h, 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:

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

[0079] (4) 4,4'-Stilbene dichloride (3 mmol) was suspended in dichloromethane (DCM). The resulting LF-trifluoroacetate (7.5 mmol) and TEA (45 mmol) were added under ice-cooling 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 resulting colloid was filtered through a Buchner funnel, washed with deionized water and 1 M HCl in sequence, and dried in an oven to obtain the target product as a white solid, i.e., the photoisomerizable 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 and 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. EDCI (40 mM, 4 eq) was added and stirred in an ice bath for 50 minutes. The reaction solution was slowly heated to 20°C and reacted at room temperature for 24 hours. The solution was washed with saturated ammonium chloride solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution in sequence, and the mixture was spin-dried. The mixture was recrystallized with ethyl acetate and petroleum ether and dried with an oil pump to obtain 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; reacting at room temperature for 30 minutes, and then rotary evaporating the solution 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 an excess of thionyl chloride (10 mmol), and then a catalyst, DMF (2 mmol), was added. The temperature was set at 60°C and refluxed for 10 hours to generate 4,4'-stilbene dicarboxylic acid chloride; the excess thionyl chloride was removed in vacuo;

[0085] (4) 4,4'-Stilbene dichloride (3 mmol) was suspended in dichloromethane (DCM). DF-trifluoroacetate (6.6 mmol) and TEA (40 mmol) were added under ice-cooling 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 resulting colloidal product was filtered through a Buchner funnel, washed with deionized water and 1 M HCl in sequence, and dried in an oven to obtain the target product as a white solid, i.e., the photoisomerizable 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 and 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. EDCI (10 mM, 1 eq) was added and stirred in an ice bath for 50 minutes. The reaction solution was slowly heated to 50°C and reacted at room temperature for 12 hours. The solution was washed with saturated ammonium chloride solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution in sequence, and the mixture was spin-dried. The mixture was recrystallized with ethyl acetate and petroleum ether and dried with an oil pump to obtain an amide condensation product, Boc-DF.

[0089] (2) The amide condensation product Boc-DF prepared in step (1) is added 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. The reaction process is shown in the following formula:

[0090] (3) Under nitrogen protection, 4,4'-stilbene dicarboxylic acid (5 mmol) was added to an excess of thionyl chloride (20 mmol), and then a 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; the excess thionyl chloride was removed in vacuo;

[0091] (4) 4,4'-Stilbene dichloride (3 mmol) was suspended in dichloromethane (DCM). DF-trifluoroacetate (7.0 mmol) and TEA (40 mmol) were added under ice-cooling 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 resulting colloidal product was filtered through a Buchner funnel, washed with deionized water and 1 M HCl in sequence, and dried in an oven to obtain the target product as a white solid, i.e., the photoisomerizable chiral hydrogel material D-SDF.

[0092] according to Figure 1 The H NMR spectrum showed that the white solid prepared in Example 1 was the target product with the structural formula L-SDF;

[0093] according to Figure 2 The H NMR spectrum showed that the white solid prepared in Example 1 was the target product with the structural formula D-SDF;

[0094] pass Figure 3 From the photos, we can see 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 UV spectra of the photoisomerizable chiral hydrogel materials for three-dimensional cell culture and release in Examples 1 and 2 of the present invention (L-SDF on the left and D-SDF on the right) change with illumination time. It can be seen from the figures that both gels achieve maximum photoisomerization in about 30 minutes.

[0096] Figure 5 Circular dichroism (CD) spectra of the photoisomerizable chiral hydrogel materials for three-dimensional cell culture and release in Examples 1 and 2 of the present invention as a function of illumination time; the figures show that both gels have strong Cotton effect peaks, indicating that they are hydrogels with chiral optical signals;

[0097] Figure 6 Vibrational circular dichroism (VCD) spectra of the photoisomerizable 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 first positive and then negative, indicating a left-handed helical fiber network, and the L-SDF signal is first negative and then positive, indicating a right-handed helical fiber network;

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

[0099] Application Examples

[0100] Application of photoisomerizable 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 films:

[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 the 0.3 mg / mL hydrogel dilute solution was added to each well of a cell culture plate (96-well plate), dried at 37°C for 12 hours, and irradiated with UV light for 30 minutes. Well-grown NIH 3T3 cells were seeded on a 96-well plate covered with L-SDF and D-SDF xerogel films (about 5000 cells / well), and incubated in a 37°C, 5% CO2 cell culture incubator for 24 hours. After that, 10 μL of CCK-8 reagent was added to each well (three groups of parallels were performed for each result at the same time), and incubated in a 37°C, 5% CO2 humidified incubator for 1 hour. The absorbance value was detected at 450 nm using a microplate reader, as shown in FIG. 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 comparable to that of the control group, and the adhesion amount on 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] Live-dead cell staining involves aspirating the culture medium from the culture plate and rinsing 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. After incubation for 15 minutes in a humidified incubator under standard culture conditions (37°C, 5% CO2), PBS buffer is added to wash away the staining solution, and the cells are observed under a fluorescence microscope. Green cells observed under the microscope represent live cells, while red cells represent dead cells.

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

[0107] Inverted imaging (such as Figure 9 Then, the 24-well plate with NIH 3T3 cells adhered was irradiated under UV light (365 nm) for 30 min (as shown in FIG. Figure 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 Figure 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. Figure 9 As shown in d), e) in D and d), e) in L). Figure 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 (300 mg / mL) containing gel factor and 950 μL of cell suspension (cell number about 1.0 x 10 6 Mix well in a 24-well plate (1000 cells / mL) and place in an incubator for 15 minutes to allow gel formation. Then, slowly add 1 mL of DMEM cell culture medium. Incubate in a 37°C, 5% CO2 incubator. Change the culture medium above the gel twice every 24 hours.

[0110] Live-dead cell staining was performed using Ca-AM / PI, and imaging was performed using a Nikon / A1+N-SIM structured illumination super-resolution microscopy system ( Figure 10 a and b correspond to the cell culture experiment diagrams of L-SDF and D-SDF gel materials in the application examples, respectively). Figure 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 are more conducive to inducing cell adhesion and proliferation. Cells encapsulated in D-SDF hydrogels were selected and irradiated under 365nm UV light. After 60 minutes of illumination, the gel collapsed and the cells were released. They were then re-seeded in culture dishes and placed in an incubator at 37°C and 5% CO2 for different culture times (12h, 24h, 36h). Live-dead cell staining and inverted fluorescence microscopy imaging were then performed. Figure 11 a, d, g are bright field images at 12h, 24h, and 36h, b, e, h are live cell staining images at 12h, 24h, and 36h, and c, f, i are dead cell staining images at 12h, 24h, and 36h, respectively. Figure 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, allows cell culture in an in vitro environment, and regulates cell adhesion and release by light. It has the advantages of simple synthesis, good biocompatibility, and degradability. Moreover, it is a chiral nanofiber constructed from a single enantiomer, and its 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 principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A photoisomerizable chiral hydrogel material, characterized by: The structural formula of the photoisomerizable chiral hydrogel material is shown in Formula (I) or Formula (II): 。 2. The method for preparing the photoisomerizable 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 and reacted under ice bath conditions. 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 dicarboxylic acid chloride 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 photoisomerizable chiral hydrogel material according to claim 2, wherein: 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 photoisomerizable chiral hydrogel material according to claim 2, wherein: 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 photoisomerizable chiral hydrogel material according to claim 2, wherein: 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 photoisomerizable chiral hydrogel material according to claim 2, wherein: In 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.

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

8. The method for preparing the photoisomerizable chiral hydrogel material according to claim 2, wherein: 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 photoisomerizable chiral hydrogel material according to claim 2, wherein: The light-proof 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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