A composition, its preparation method and applications

Abstract

The present invention provides a composition, a preparation method and an application thereof, belonging to the technical field of biomaterials. The composition comprises a first component and a second component. The first component is polyethylene glycol NHS-PEG-NHS with N-hydroxysuccinimide ester groups at both ends, and the second component is a random copolymer polypeptide formed by random copolymerization of serine and lysine. The hydrogel composition composed of NHS-PEG-NHS and the random copolymer polypeptide can effectively resist protein adsorption and cell attachment due to the strong double hydrogen bond hydration formed by amide bonds on the main chain and hydroxyl groups on the side chain, and has antifouling properties, thereby resisting foreign body reactions and the ability to resist foreign body reactions in vivo. When cells are added for encapsulation, NHS-PEG-NHS is used to covalently bind the amino groups on the cell surface to the amino groups on the copolymer side chain to construct a high-strength hydrogel network, and the strength of the hydrogel composition is regulated by the density of the encapsulated cells.

Description

Technical Field

[0001] The present invention relates to the field of biomaterials, and particularly to a composition, a preparation method thereof, and an application thereof. Background Art

[0002] Natural extracellular matrix (ECM) consists of a network of substances around cells in tissues, which is the supporting structure and microenvironment for tissue cells. Its physical properties are heterogeneous and its biochemical components are very complex. Therefore, in related technologies, efforts have been made to find materials with simple structures that can replace ECM for culturing cells.

[0003] Hydrogels can provide an aqueous environment similar to natural ECM (usually with a water content of >70%), and can effectively support the exchange of nutrients, metabolic wastes, and signaling molecules, and can also prevent the entry of immune-related molecules such as immune cells or antibodies, thereby maintaining cell survival and physiological functions. The properties of hydrogels can be simply customized through synthetic chemical strategies to simulate specific tissue environments. However, such implantable hydrogels are usually hindered by the foreign body response (FBR), which causes the host to recognize and subsequently form a dense fibrous capsule, which blocks the diffusion of oxygen and nutrients, resulting in implant failure. Non-specific protein adsorption on the surface of the implant is considered to be the first and key step in triggering FBR. Therefore, it is necessary to develop an implantable hydrogel that can reduce FBR. Summary of the Invention

[0004] In view of this, in order to at least partially solve the above-mentioned technical problems, the present invention provides a composition, comprising a first component and a second component. The first component is polyethylene glycol NHS-PEG-NHS having N-hydroxysuccinimide ester groups at both ends, and the second component is a random copolymer polypeptide formed by random copolymerization of serine and lysine.

[0005] According to another embodiment of the present invention, a kit comprising the above composition is provided.

[0006] According to another embodiment of the present invention, a preparation method of the above composition is provided, comprising: dissolving the first component and the second component in a solution to obtain a hydrogel composition.

[0007] According to another embodiment of the present invention, an application of the above composition or kit is provided, and the application includes at least one of the following: (1) preparing a cell therapy preparation; (2) preparing a tissue or organ repair preparation; (3) preparing a drug delivery carrier; (4) preparing a biosensor; (5) encapsulating cells.

[0008] According to an embodiment of the present invention, a hydrogel composition composed of a poly(ethylene glycol) with N-hydroxysuccinimide ester groups at both ends and a random copolymer polypeptide formed by the random copolymerization of serine and lysine can effectively resist protein adsorption and cell attachment due to the strong "double hydrogen bond hydration" formed by the amide bonds on the main chain and the hydroxyl groups on the side chains, and has anti-fouling properties, thus resisting FBR and the ability to resist foreign body reactions in vivo; further, when cells are added for encapsulation, through a cell crosslinking strategy, NHS-PEG-NHS is used to covalently bind the amino groups on the cell surface to the amino groups on the side chains of the copolymer, constructing a high-strength hydrogel network. The strength of the cell hydrogel composition can be directly regulated by the density of the encapsulated cells. After encapsulation, it can be directly injected into the body through a medical syringe to avoid secondary injuries caused by surgery or ultraviolet irradiation, etc., and the operation is simple and non-toxic. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 Schematic diagram of the synthesis process of the random copolymer polypeptide in the embodiment of the present invention;

[0010] Figure 2 1H NMR spectrum of L-lysine N-carboxyanhydride in the embodiment of the present invention;

[0011] Figure 3 1H NMR spectrum of L-serine N-carboxyanhydride in the embodiment of the present invention;

[0012] Figure 4 1H NMR spectrum of D-serine N-carboxyanhydride in the embodiment of the present invention;

[0013] Figure 5 1H NMR spectrum of the random copolymer polypeptide in the side chain protection stage in the embodiment of the present invention;

[0014] Figure 6 Molecular weight diagram of the random copolymer polypeptide in the side chain protection stage measured by gel permeation chromatography in the embodiment of the present invention;

[0015] Figure 7 1H NMR spectrum of the deprotected random copolymer polypeptide in the embodiment of the present invention;

[0016] Figure 8 1H NMR spectrum of poly(ethylene glycol) with N-hydroxysuccinimide ester groups at both ends in the embodiment of the present invention;

[0017] Figure 9 Schematic diagram of the preparation process of the cell-containing hydrogel composition in the embodiment of the present invention;

[0018] Figure 10 Fourier transform infrared spectroscopy characterization comparison diagram in the embodiment of the present invention;

[0019] Figure 11It is a comparison chart of the swelling degree in 24 hours for the embodiment of the present invention;

[0020] Figure 12 It is a comparison chart of the swelling degree in 7 days for the embodiment of the present invention;

[0021] Figure 13 It is a combined chart of hydrogels for the determination of compression modulus in the embodiment of the present invention;

[0022] Figure 14 It is a comparison chart of stress-strain curves in the compression test of the hydrogel composition in the embodiment of the present invention;

[0023] Figure 15 It is a comparison chart of the compression modulus of the hydrogel composition in the embodiment of the present invention;

[0024] Figure 16 It is a comparison chart of the change in storage modulus of the hydrogel composition in the embodiment of the present invention;

[0025] Figure 17 It is a comparison chart of the average value of the storage modulus of the hydrogel composition in the embodiment of the present invention;

[0026] Figure 18 It is a fluorescence image of live and dead cells of the hydrogel composition containing cells in the embodiment of the present invention;

[0027] Figure 19 It is a result chart of lactate dehydrogenase cytotoxicity detection of the hydrogel composition containing cells in the embodiment of the present invention;

[0028] Figure 20 It is a comparison chart of in vitro protein adsorption between the hydrogel composition without cells and polyethylene glycol hydrogel in the embodiment of the present invention;

[0029] Figure 21 It is a comparison chart of in vitro cell adhesion between the hydrogel composition and polyethylene glycol hydrogel in the embodiment of the present invention;

[0030] Figure 22 It is an inflammation reaction chart two weeks after in vivo implantation of the hydrogel composition in the embodiment of the present invention. Detailed implementation manners

[0031] Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. However, it should be understood that these descriptions are merely exemplary and are not intended to limit the scope of the present invention. In the following detailed description, for the sake of explanation, many specific details are set forth to provide a comprehensive understanding of the embodiments of the present invention. However, obviously, one or more embodiments can be implemented without these specific details. In addition, in the following description, descriptions of well-known structures and technologies are omitted to avoid unnecessarily confusing the concepts of the present invention.

[0032] The terms used herein are merely for describing specific embodiments and are not intended to limit the present invention. The term "comprising" used herein indicates the presence of features, steps, operations, but does not exclude the presence or addition of one or more other features.

[0033] The term "treatment" means that after a subject has contracted a disease, the subject is exposed (such as administered) to a drug, composition, etc. based on the present invention, so that the symptoms of the disease are alleviated compared with the situation without exposure, and it does not necessarily mean that the symptoms of the disease are completely suppressed. Contracting a disease means that the body shows symptoms of the disease.

[0034] The term "prevention" means that before a subject contracts a disease, by exposing (such as administering) the subject to a drug, composition, etc. based on the present invention, the symptoms after contracting the disease are alleviated compared with the situation without exposure, and it does not necessarily mean that getting sick is completely suppressed.

[0035] In the process of implementing the inventive concept, it is found that artificial cell culture systems have a wide range of applications in the field of biomedicine. For example, encapsulated islet cells are implanted into the body to treat type 1 diabetes. However, the hydrogel of the artificial cell culture system formed above needs to be crosslinked by ultraviolet light, which will inevitably cause damage to cells. In addition, implanting the hydrogel into an organism usually requires secondary or multiple surgical operations. During the treatment process, repeated open surgeries increase the medical risk and the pain of patients.

[0036] Furthermore, since hydrogels usually achieve the "sol-gel" transition through the crosslinking of precursor solutions, they can be designed as injectable biomaterials without damaging their original functions, reducing the damage to surrounding tissues. However, injectable hydrogels usually have poor mechanical properties due to weak non-covalent interactions, and may cause the hydrogel to rupture during or after injection, resulting in cell escape. In particular, the escape of stem cells has the risk of forming teratomas, thus limiting their application in the field of cell encapsulation.

[0037] Taking the above factors into consideration, preparing an injectable hydrogel with in vivo anti-foreign body reaction and high mechanical strength can be used as a cell encapsulation material to achieve subsequent treatment and other corresponding functions.

[0038] Specifically, according to an embodiment of one aspect of the present invention, a composition is provided, which comprises a first component and a second component. The first component is polyethylene glycol NHS-PEG-NHS with N-hydroxysuccinimide ester groups at both ends, and the second component is a random copolymer polypeptide formed by the random copolymerization of serine and lysine.

[0039] According to an embodiment of the present invention, a hydrogel composition composed of a random copolymer polypeptide formed by the random copolymerization of polyethylene glycol with N-hydroxysuccinimide ester groups at both ends, serine, and lysine can effectively resist protein adsorption and cell attachment due to the strong "double hydrogen bond hydration" formed by amide bonds on the main chain and hydroxyl groups on the side chain, and has anti-fouling properties, thereby resisting FBR and the ability to resist foreign body reactions in vivo; further, when cells are added for encapsulation, through a cell crosslinking strategy, NHS-PEG-NHS is used to covalently bind the amino groups on the cell surface to the amino groups on the copolymer side chain to construct a high-strength hydrogel network. The strength of the cell hydrogel composition can be directly regulated by the encapsulated cell density. After encapsulation, it can be directly injected into the body through a medical syringe to avoid secondary injuries caused by surgery or ultraviolet irradiation, etc., and the operation is simple and non-toxic.

[0040] According to an embodiment of the present invention, the random copolymer polypeptide includes L-serine, D-serine, and L-lysine.

[0041] According to an embodiment of the present invention, serine in the random copolymer polypeptide includes L-type and D-type. Since the binding between poly-L-serine segments is strong and can form β-sheet aggregates, this rigid structure results in very low water solubility (<0.1 mg / mL). Therefore, by introducing D-type serine, the tendency to form β-sheets is broken, and the entire polymer forms a random coil structure, which can improve its water solubility and make the prepared hydrogel more suitable for the in vivo environment.

[0042] According to an embodiment of the present invention, the molar ratio of L-serine, D-serine, and L-lysine is (0.5 - 1.5):(0.5 - 1.5):(1.5 - 2.5).

[0043] According to an embodiment of the present invention, the molar ratio of L-serine, D-serine, and L-lysine can be 0.5:1:1, 0.5:1.5:1.5, 0.5:1:1.5, 0.5:0.5:1, 1:0.5:2, 1:1:2, 1:1.5:1.5, 1.5:1.5:2, 1:1:2.5, but is not limited to the listed values, and other unlisted values within this numerical range are equally applicable.

[0044] According to an embodiment of the present invention, the adjustment of the amino acid ratio can affect the supramolecular assembly behavior of the random copolymer polypeptide; the synergistic effect of L-serine and D-serine can promote the formation of a uniform fiber network to form double hydrogen bond hydration, while the addition of L-lysine can enhance the connectivity between fibers to play a crosslinking role and form a more stable network structure. By adjusting the molar ratio, the performance of the polypeptide can be customized to meet different biomedical application requirements, such as drug delivery, tissue engineering, etc.

[0045] According to an embodiment of the present invention, the above-mentioned composition is a hydrogel composition, and the hydrogel composition comprises a solution of a first component and a second component, and the solution comprises a cell or tissue preservation solution of the first component and the second component.

[0046] According to an embodiment of the present invention, (1) in the hydrogel composition, the mass concentration of the first component is 5-15 wt%, and the mass concentration of the second component is 25-35 wt%, and / or (2) the volume ratio of the solution of the first component and the second component is (0.8-1.2):(0.8-1.2).

[0047] According to an embodiment of the present invention, the mass concentration of the first component can be 5 wt%, 10 wt%, 15 wt%, and the mass concentration of the second component can be 25 wt%, 30 wt%, 35 wt%, but is not limited to the listed values, and other unlisted values within this numerical range are equally applicable.

[0048] According to an embodiment of the present invention, the volume ratio of the solution of the first component and the second component can be 0.8:0.8, 0.8:1, 0.8:1.2, 1:0.8, 1:1, 1:1.2, 1.2:0.8, 1.2:1, but is not limited to the listed values, and other unlisted values within this numerical range are equally applicable.

[0049] In some specific embodiments of the present invention, the hydrogel composition is an injectable hydrogel composition, and the first component and the second component are dissolved in a cell or tissue preservation solution formed in Hank's Balanced Salt Solution (HBSS buffer), Phosphate Buffered Saline (PBS), Dulbecco's Phosphate Buffered Saline (DPBS), Earle's Balanced Salt Solution (EBSS buffer), etc.

[0050] Specifically, the solution of the first component and the second component is an alkaline solution, which provides an alkaline environment for the Michael addition of the amino group and the NHS ester. The ε-amino group (pKa≈10.5) of the lysine residue is partially deprotonated (in the form of -NH2) at pH 8.0, enhancing its nucleophilicity, and thus more easily undergoes covalent cross-linking with the active ester group (NHS ester) at the end of NHS-PEG-NHS. The NHS ester has higher stability under neutral to weak alkaline conditions and slower hydrolysis at pH 7-8, but too high pH (>8.5) will cause its rapid hydrolysis and failure. pH 8.0 is a balance point, which can both activate the amino group and avoid premature degradation of the NHS ester.

[0051] According to an embodiment of the present invention, the hydrogel composition has good biocompatibility and is suitable for the in vivo environment, such as for a cell or tissue preservation solution.

[0052] According to an embodiment of the present invention, the composition further comprises cells, and the density of the cells is (0.1 to 10) × 10 7 cells / mL.

[0053] According to an embodiment of the present invention, the NHS-PEG-NHS crosslinker can simultaneously react with a large number of amino groups on the cell surface and amino groups on the side chains of polyamino acids to obtain a hydrogel with cells as crosslinking points. Such large-volume crosslinking points can enhance the mechanical properties of the hydrogel. The strength of the cell hydrogel composition can be directly regulated by the encapsulated cell density, which is suitable for the requirements of different human tissues. For example, in the application of treating intervertebral discs, the mechanical properties of the required hydrogel are stronger, so a larger cell density can be used to crosslink to obtain a hydrogel with greater strength; while in the treatment of brain diseases, a lower cell density is required, thus obtaining a hydrogel with more suitable mechanical properties, ranging from the level of brain tissue in pascals (Pa) to the level of bone tissue in megapascals (MPa).

[0054] According to an embodiment of the present invention, the density of the cells can be 0.1 × 10 7 、1 × 10 7 、2 × 10 7 、4 × 10 7 、5 × 10 7 、6 × 10 7 、7 × 10 7 、8 × 10 7 、10 × 10 7 cells / mL, but is not limited to the listed values, and other unlisted values within this numerical range are equally applicable.

[0055] According to an embodiment of one aspect of the present invention, there is also provided a kit, comprising a first component and a second component. The first component is polyethylene glycol NHS-PEG-NHS having N-hydroxysuccinimide ester groups at both ends, and the second component is a random copolymer polypeptide formed by random copolymerization of serine and lysine, and may further comprise cells.

[0056] According to an embodiment of the present invention, the composition in the kit exists in a form that allows the biological activity of the active ingredients contained therein to be effective, and may further include other auxiliary components or mediators for forming the kit, such as liquid or solid fillers, diluents, excipients, solvents or encapsulating materials.

[0057] According to an embodiment of one aspect of the present invention, there is also provided a method for preparing a composition, comprising: dissolving the first component and the second component in a solution to obtain a hydrogel composition.

[0058] According to an embodiment of the present invention, the dissolved solution includes a cell or tissue preservation solution of a first component and a second component, for example, a cell or tissue preservation solution formed by dissolving the first component and the second component in Hank's Balanced Salt Solution (HBSS buffer), phosphate buffer solution (PBS), Dulbecco's phosphate buffer solution (DPBS), Earle's Balanced Salt Solution (EBSS buffer), etc.

[0059] Specifically, the preparation method includes cross-linking a random copolymer polypeptide of polyethylene glycol, serine, and lysine having N-hydroxysuccinimide ester groups at both ends, and obtaining a hydrogel composition after standing.

[0060] According to an embodiment of the present invention, it further includes dissolving the first component, the second component, and cells in a solution to obtain a cell-containing hydrogel composition.

[0061] Specifically, the preparation method includes mixing a random copolymer polypeptide of polyethylene glycol, serine, and lysine having N-hydroxysuccinimide ester groups at both ends with target encapsulated cells for cross-linking reaction, and obtaining a cell-containing hydrogel composition after standing.

[0062] Furthermore, in the above cross-linking reaction, the reaction environment is an alkaline environment, and the alkaline environment can be a pH value of 7 or above, 7.2 or above, 7.4 or above, 7.6 or above, 7.8 or above, 8 or above, etc.

[0063] According to an embodiment of the present invention, the preparation method of the hydrogel of the present invention has mild reaction conditions and a moderate reaction rate, does not require the addition of small molecule cross-linking agents with biological / cellular toxicity, and has low biological toxicity.

[0064] According to an embodiment of one aspect of the present invention, there is also provided an application of a hydrogel composition and a hydrogel composition kit, including at least one of the following: (1) preparing a cell therapy preparation; (2) preparing a tissue or organ repair preparation; (3) preparing a drug delivery carrier; (4) preparing a biosensor; (5) for encapsulating cells.

[0065] According to an embodiment of the present invention, the hydrogel composition of the present invention can replace the extracellular matrix and can be used in one or more of, for example, biological dressings, human biomimetic materials, plastic and cosmetic materials, organoid culture materials, cardiovascular stent materials, coating materials, tissue injection filling materials, ophthalmic materials, obstetrics and gynecology biological materials, nerve repair and regeneration materials, liver tissue materials and blood vessel repair and regeneration materials, 3D printing artificial organ biological materials, cosmetic raw materials, pharmaceutical excipients, and food additives.

[0066] The following will further explain the solution of the present invention in combination with specific embodiments. In the following embodiments, if not otherwise stated, all are conventional commercially available reagents.

[0067] Example 1 Synthesis of Nε-tert-butoxycarbonyl-L-lysine N-carboxyanhydride (Boc-L-Lys NCA)

[0068] Figure 1 This is a schematic diagram of the synthesis process of the random copolymer polypeptide in the embodiment of the present invention.

[0069] The reaction process is as Figure 1 shown. In a thick-walled pressure vessel, Nε-Boc-L-lysine (Boc-L-lysine) (4.0 g, 16.4 mmol), 80 mL of tetrahydrofuran, 11.2 mL of methyloxirane, 162.4 mmol, and 2.48 g of triphosgene, 8.4 mmol were added in sequence, and the container was immediately sealed. The reaction was stirred at room temperature for 3 h.

[0070] After the reaction was completed, 40 mL of cold water at about 4 °C was added and stirred for 3 min to quench the excess triphosgene. The mixture was extracted with ethyl acetate (80 mL×2) at room temperature. The combined organic phases were washed with saturated brine and dried over anhydrous magnesium sulfate overnight. The solvent was removed by rotary evaporation under vacuum at 45 °C from the dried solution to obtain the crude product.

[0071] The crude product was recrystallized and purified in tetrahydrofuran / n-hexane to obtain a white solid. The 1H NMR spectrum was characterized using deuterated dimethyl sulfoxide as the solvent, as Figure 1 shown.

[0072] Figure 2 This is the 1H NMR spectrum of L-lysine N-carboxyanhydride in the embodiment of the present invention.

[0073] According to Figure 2 it can be seen that L-lysine N-carboxyanhydride was successfully synthesized.

[0074] Example 2 Synthesis of O-tert-butyl-D,L-serine N-carboxyanhydride (tBu-D,L-Ser NCA) monomer

[0075] The reaction process is as Figure 1As shown, under a N2 atmosphere and in an ice-water bath, add tert-butyl-D / L-serine (tBu-D / L-Sering) (4.03 g, 25 mmol), α-pinene (10.5 mL, 66 mmol), and anhydrous tetrahydrofuran (75 mL) to a dry three-necked flask. Separately, dissolve triphosgene (3.26 g, 11 mmol) in anhydrous tetrahydrofuran (15 mL) and add it to the mixed solution. Stir the reaction at 50 °C for 2 h under a nitrogen atmosphere until the solution becomes clear, and then rotary evaporate under reduced pressure to remove tetrahydrofuran at 45 °C.

[0076] Dissolve the reaction mixture in ethyl acetate (50 mL), wash it three times with ice water (50 mL), and once with ice-saturated brine (50 mL). Dry the collected organic solution over anhydrous magnesium sulfate overnight. After drying, rotary evaporate the solution under vacuum at 45 °C to remove the solvent to obtain the crude product. Recrystallize and purify the crude product three times with anhydrous ethyl acetate and anhydrous n-hexane in a glove box to obtain white needle-like crystals. Perform 1H NMR characterization using deuterated dimethyl sulfoxide as the solvent, as Figure 3 , 4 shown.

[0077] Figure 3 This is the 1H NMR spectrum of the L-serine N-carboxyanhydride in the example of the present invention; Figure 4 This is the 1H NMR spectrum of the D-serine N-carboxyanhydride in the example of the present invention.

[0078] According to Figure 3 and Figure 4 , it can be seen that the L-serine N-carboxyanhydride and D-serine N-carboxyanhydride were successfully synthesized.

[0079] Example 3 Synthesis of random copolymer polypeptide (poly(D,L-serine)-random-poly(L-lysine), Poly(D,L-Serine)-r-poly(L-Lysine))

[0080] The synthesis process is as Figure 1 shown. Weigh the L-serine N-carboxyanhydride and D-serine N-carboxyanhydride (each 0.94 g, 5 mmol) prepared in Example 2 and the L-lysine N-carboxyanhydride (2.72 g, 10 mmol) prepared in Example 1, and dissolve them in tetrahydrofuran (90 mL). Immediately add a solution of lithium hexamethyldisilazide (0.1 mmol / mL, 10 mL) in anhydrous tetrahydrofuran to the solution. After stirring the reaction at room temperature for 10 min, add the reaction mixture dropwise to anhydrous cold diethyl ether (1 L) to obtain a white flocculent precipitate, collect it by centrifugation (5000 rpm, 4 °C), and preliminarily dry it under a nitrogen stream.

[0081] The collected solid was dissolved in tetrahydrofuran (100 mL), reprecipitated, and the dissolution-precipitation process was repeated three times, followed by drying under vacuum to obtain the white solid Poly(tBu-D,L-Serine)-r-poly(Boc-L-Lysine) of the side chain protecting group.

[0082] 1H NMR characterization was carried out using deuterated dimethyl sulfoxide as the solvent, as Figure 5 shown.

[0083] Figure 5 This is the 1H NMR spectrum of the random copolymer polypeptide in the side chain protection stage of the embodiment of the present invention.

[0084] 5 mg of the dried poly(O-tert-butyl-D,L-serine)-random-poly(Nε-tert-butoxycarbonyl-L-lysine) (Poly(tBu-D,L-Serine)-r-poly(Boc-L-Lysine)) was dissolved in N,N-dimethylformamide (DMF) to a concentration of 1 mg / mL. Molecular weight determination was carried out using a gel permeation chromatography (GPC) instrument with DMF supplemented with 0.01 M LiBr as the mobile phase and a flow rate of 1 mL / min, as Figure 6 shown.

[0085] Figure 6 This is the molecular weight diagram of the random copolymer polypeptide in the side chain protection stage measured by gel permeation chromatography in the embodiment of the present invention.

[0086] The dried Poly(tBu-D,L-Serine)-r-poly(Boc-L-Lysine) was dissolved in 80 mL of trifluoroacetic acid. After stirring and reacting for 2 h, trifluoroacetic acid was removed by rotary evaporation under vacuum to obtain a yellow oil. It was dissolved in methanol (40 mL) and dropped into methyl tert-butyl ether (400 mL) to precipitate a white flocculent solid. The solid was collected by centrifugation (5000 rpm, 4 °C) and preliminarily dried under a nitrogen stream. The collected solid was dissolved in methanol (40 mL), reprecipitated, and after three dissolution / precipitation cycles, the deprotected random copolymer polypeptide Poly(D,L-Serine)-r-poly(L-Lysine) was obtained. 1H NMR characterization was carried out using deuterated water as the solvent, as Figure 7 shown.

[0087] Figure 7 This is the 1H NMR spectrum of the deprotected random copolymer polypeptide in the embodiment of the present invention.

[0088] According to Figures 5 - 7It can be seen that the random copolymer polypeptide (Poly(D,L-Serine)-r-poly(L-Lysine)) was successfully synthesized. It can be seen that the ratio of serine units to lysine units in the random copolymer polypeptide is approximately 1:1, that is Figure 1 in the chemical formula, x:y≈1:1. According to the measured molecular weight of 61.2 kDa, it can be obtained that x≈y≈165.

[0089] Example 4 Synthesis of Polyethylene Glycol with N-Hydroxysuccinimide Ester Groups at Both Ends (NHS-PEG-NHS)

[0090] Polyethylene glycol (PEG) (Mn = 4000 Da, 20 g, 5 mmol) was dissolved in anhydrous dichloromethane (250 mL) and stirred for 10 min under a nitrogen atmosphere. Separately, N,N'-disuccinimidyl carbonate (6.4 g, 25 mmol) was dissolved in anhydrous acetonitrile (100 mL). The two solutions were mixed, and finally pyridine (1 mL, 12.5 mmol) was added. After stirring at room temperature for 24 h under a nitrogen atmosphere, the mixture was filtered and dried under vacuum. After drying, the sample was dissolved in chloroform (50 mL) and dropped into anhydrous cold ether (200 mL) to precipitate a white flocculent solid. The solid was collected by centrifugation (5000 rpm, 4 °C) and preliminarily dried under a nitrogen stream. The collected solid was dissolved in chloroform (50 mL) and precipitated again. After three dissolution / precipitation cycles, NHS-PEG-NHS was obtained. The proton nuclear magnetic resonance spectrum was characterized using deuterated chloroform as the solvent, as Figure 8 shown.

[0091] Figure 8 This is the proton nuclear magnetic resonance spectrum of polyethylene glycol with N-hydroxysuccinimide ester groups at both ends in the examples of the present invention.

[0092] According to Figure 8 it can be seen that NHS-PEG-NHS was successfully synthesized.

[0093] Example 5 Preparation and Characterization of Hydrogel Compositions Containing Cells

[0094] The synthesis process is as Figure 9 shown. The cell mass of the mouse pancreatic islet β cell line (Min6) (consisting of 1×10 6The precipitate (cell clusters formed by cells) was washed three times with HBSS buffer (pH 7.2) and then evenly dispersed in 50 μL of the HBSS buffer (pH 8.0) of NHS-PEG-NHS prepared in Example 4 (30 wt%), and then 50 μL of the HBSS buffer (pH 8.0) of the copolymer polypeptide prepared in Example 3 (10 wt%) was added. The cells and the solution were gently mixed evenly, and the precursor solution was added to a glass bottle and incubated at 37 °C to obtain a cell-containing hydrogel composition.

[0095] Figure 9 It is a schematic diagram of the preparation process of the cell-containing hydrogel composition in the embodiment of the present invention.

[0096] Fourier transform infrared spectroscopy (FTIR) was used to analyze the copolymer polypeptide (Peptide), NHS-PEG-NHS and the hydrogel composition (cell-free, denoted as NCCH) in the range of 4000 cm -1 to 400 cm -1 , as well as the cell-containing hydrogel compositions with cell densities of 1×10 7 and 2×10 7 cells / mL, denoted as CCH-1 and CCH-2 respectively, and the spectral resolution was 4 cm -1 . For this purpose, the freeze-dried samples were thoroughly ground and mixed with potassium bromide, and then pressed into tablets for further characterization, and the results are as Figure 10 shown.

[0097] Figure 10 It is a comparative diagram of Fourier transform infrared spectroscopy characterization in the embodiment of the present invention.

[0098] According to Figure 10 , it can be seen that NHS-PEG-NHS has stretching vibration peaks of -CH2 on the PEG main chain and stretching vibration peaks of C-O-C at 2888 cm -1 and 1109 cm -1 respectively, and the stretching vibration peak of C=O of the NHS group is at 1740 cm -1 . The random copolymer polypeptide has stretching vibration peaks of C=O on the main chain and stretching vibration peaks of C-N at 1650 cm -1 and 1540 cm -1 respectively. After the hydrogel is formed and undergoes swelling and freeze-drying, it can be found that the peak shapes of NCCH, CCH-1 and CCH-2 are basically the same, and peaks are observed at 2888 cm -1 , 1109 cm -1 , 1650 cm -1 and 1540 cm -1 , while a peak is observed at 1740 cm -1No peak was observed at this position, indicating the formation of the CO-NH bond and that the NHS group was washed away after complete reaction. These results indicate that the hydrogel was crosslinked from NHS-PEG-NHS and random copolymer polypeptides.

[0099] Example 6 Characterization of the Swelling Performance of the Hydrogel Composition

[0100] For the cell-free hydrogel composition NCCH and the cell-containing hydrogel compositions CCH-1 and CCH-2, the liquid was blotted dry with filter paper, and their initial masses (M0) were recorded. Then, the samples were incubated in PBS buffer at 37 °C for a specified time, and the excess liquid of the swollen hydrogel was blotted dry with filter paper, and the samples were weighed again (Mt). Fresh buffer was replaced, and the swelling ratio was calculated: ((Mt - M0)) / M0, and the equilibrium swelling ratio was recorded when the mass of the hydrogel no longer increased. The swelling ratios were calculated respectively, and the results are as Figure 11 , 12 shown.

[0101] Figure 11 This is the comparison chart of the swelling degree of Example 24 hours of the present invention; Figure 12 This is the comparison chart of the swelling degree of Example 7 days of the present invention. In Figure 11 , **** indicates that the P value < 0.0001, and two-way ANOVA with Tukey multiple comparison test was used.

[0102] According to Figure 11 and Figure 12 it can be seen that the cell-free hydrogel composition NCCH reached swelling equilibrium within 5 h, with a swelling degree of about 150%. After 7 days, no obvious swelling was found compared with the swelling at 24 h; the cell-containing hydrogel compositions CCH-1 and CCH-2 had swelling degrees of only about 80% and 50% respectively, greatly reducing the swelling of the hydrogel. From the above data, it can be seen that compared with NCCH, CCH has a greater degree of crosslinking and a denser crosslinking network, which is attributed to the large number of amino groups on the cell surface.

[0103] Example 7 Determination of the Compression Modulus of the Hydrogel Composition

[0104] After washing the cell pellet and dispersing it evenly in 100 μL of HBSS solution (pH 8.0) of 10 wt% copolymer polypeptide, 100 μL of HBSS solution (pH 8.0) of 30 wt% NHS-PEG-NHS was added. The cells and the solution were gently mixed evenly, and the precursor solution was dropped into a hollow cylindrical polytetrafluoroethylene mold with an inner diameter of 8 mm and incubated at 37 °C for five minutes, as Figure 13 shown.

[0105] Disk-shaped gels (about 8 mm in diameter and about 4 mm in height, n = 5) were subjected to compression tests in a universal mechanical testing machine (Instron) equipped with a 50 N load cell. After reaching a preloading force of 0.001 N, mechanical compression was performed at a crosshead speed of 1 mm / min, and the test results are as Figure 14 and Figure 15 shown.

[0106] Figure 13 Composite graph of hydrogels for the determination of compression modulus in the embodiments of the present invention; Figure 14 Comparison graph of stress-strain curves in the compression test of the hydrogel composition in the embodiments of the present invention; Figure 15 Comparison graph of compression moduli of the hydrogel composition in the embodiments of the present invention. In Figure 15 , *** indicates P value = 0.0002 (CCH-1 vs. NCCH), *** indicates P value = 0.0003 (CCH-2 vs. CCH-1), **** indicates P value < 0.0001 (CCH-2 vs. NCCH), and one-way ANOVA with Tukey's multiple comparison test was used.

[0107] According to Figures 13 - 15 it can be seen that the hydrogel compositions CCH-1, CCH-2 and the cell-free hydrogel composition NCCH all have high compression strains (86%, 75% and 88% respectively), but the compression modulus increases with the increase of cell density. The compression modulus of CCH-2 is 4.1 times that of NCCH, and this result directly shows the enhancement of the compression performance of the hydrogel by the cell cross-linking sites.

[0108] Example 8 Determination of Compression Modulus of Hydrogel Composition

[0109] Amplitude sweep rheological studies were carried out on a rheometer to characterize the storage modulus. The Figure 13 disk-shaped hydrogels shown (about 8 mm in diameter and about 2 mm in height, n = 5) were placed on the rheometer plate, incubated at 37 °C for 3 min, and the shear strain range of 0.1 - 100% was measured at a frequency of 0.01 Hz. The upper parallel geometry plate (8 mm in diameter, smooth surface), and sampling was performed every 20 seconds. The storage modulus (G') is the average of the first five data points, and the results are as Figure 16 and Figure 17 shown.

[0110] Figure 16 Comparison graph of the change of storage modulus of the hydrogel composition in the embodiments of the present invention; Figure 17 Comparison graph of the average value of the storage modulus of the hydrogel composition in the embodiments of the present invention. In Figure 17Among them, * indicates P value = 0.0436 (CCH-1 vs. NCCH), * indicates P value = 0.0129 (CCH-2 vs. CCH-1), *** indicates P value = 0.0001 (CCH-2 vs. NCCH), and one-way ANOVA with Tukey's multiple comparison test was used.

[0111] According to Figure 16 and Figure 17 it can be seen that the cell crosslinking strategy also enhanced the storage modulus. The storage modulus of CCH-2 was 1.8 times that of NCCH, further indicating that compared with short chemical bonds, cells as crosslinking points, this large structure can enhance the mechanical properties of the hydrogel network.

[0112] Example 9 Biosecurity Test of Hydrogel Composition

[0113] Cell viability test: Add the hydrogel composition containing cells (diameter 8 mm, thickness 2 mm) to a 24-well plate, and culture it in DMEM high-glucose medium containing 10% fetal bovine serum (FBS), 1% penicillin-streptomycin (P / S), and 1% MEM non-essential amino acid solution (NEAA) at 37 °C and 5% CO2 for 7 days. Wash the hydrogel twice with PBS at specific time points, and stain it with a cell viability staining solution for 30 min, and image it under a fluorescence microscope. The results are as Figure 18 shown.

[0114] Figure 18 This is the cell viability fluorescence image of the hydrogel composition containing cells in the embodiment of the present invention.

[0115] According to Figure 18 it can be seen that basically all cell clusters showed green fluorescence within 7 days, indicating high cell viability and the hydrogel composition of the present invention has biosecurity.

[0116] Lactate dehydrogenase (LDH) cytotoxicity detection: Add the hydrogel composition containing cells (diameter 8 mm, thickness 2 mm) to a 24-well plate, and culture it in 2 mL of DMEM high-glucose medium containing 10% FBS, 1% P / S, and 1% NEAA at 37 °C and 5% CO2 for 48 hours. To measure the maximum LDH release, 200 μL of LDH release reagent was added to the control wells containing the same number of cell clusters 1 h before adding the LDH detection reagent. To measure the amount of released LDH, 120 μL of the supernatant was taken after centrifugation of each well and transferred to a new 96-well plate, 60 μL of LDH detection reagent was added, and then incubated at room temperature for 30 min. Finally, the absorbance was measured at 490 nm using a microplate reader, and a dual-wavelength measurement was performed using a 600 nm wavelength as the reference wavelength. The results are as Figure 19as shown

[0117] Figure 19 This is the result diagram of the lactate dehydrogenase cytotoxicity detection of the hydrogel composition containing cells in the embodiment of the present invention. In Figure 19 , ** indicates that the P value = 0.0031, and one-way ANOVA with Tukey's multiple comparison test was used.

[0118] According to Figure 19 it can be seen that the LDH released by the cells in CCH-1 is only 2.5% relative to the maximum group, indicating that the hydrogel composition is basically non-toxic to cells. The percentage of LDH released by the cells in CCH-2 is 8.3%, probably because the number of cell clusters in it is relatively large, and the amount of LDH released will also increase correspondingly. The above data indicate that the hydrogel composition containing cells has good cytocompatibility.

[0119] Example 10 Fibrinogen Adsorption Assay of Hydrogel Composition

[0120] A hydrogel composition without cells (about 8 mm in diameter and about 1 mm in height) was prepared. The commonly used biocompatible material PEG was selected as the control. The tetra-arm PEG-NH2 (molecular weight 5000 Da) and NHS-PEG-NHS were dissolved in HBSS buffer (pH 8.0) at 50 wt% respectively. 25 μL of the tetra-arm PEG-NH2 solution and 25 μL of the NHS-PEG-NHS solution were mixed evenly, and the precursor solution was dropped into a hollow cylindrical polytetrafluoroethylene mold with an inner diameter of 8 mm and incubated at 37 °C for five minutes to obtain a PEG hydrogel as the control group.

[0121] The protein adsorption on the hydrogel was detected by enzyme-linked immunosorbent assay (ELISA). Fibrinogen (Fg) was selected as the test protein to examine the anti-protein adhesion ability of the hydrogel sample. The samples were initially placed in a 24-well plate and underwent a 5-minute equilibration period in PBS buffer. Next, the samples were immersed in 1 mL of PBS buffer containing 1 mg / mL Fg for 1 h, and then washed thoroughly three times with PBS buffer. Then the samples were transferred to fresh wells and immersed in 1 mL of PBS solution containing 1 μg / mL horseradish peroxidase (HRP)-conjugated anti-fibrinogen antibody for 0.5 h. After washing five more times with PBS buffer, all the samples were transferred to new wells. Subsequently, 1 mL of o-phenylenediamine (OPD) chromogenic solution was added. After incubating for 15 min, an equal volume of 1N hydrochloric acid (HCl) was added to stop the enzymatic reaction. Then the absorbance readings at 490 nm were recorded using a microplate reader, and the results are as Figure 20 as shown

[0122] Figure 20This is a comparison graph of in vitro protein adsorption between the cell-free hydrogel composition and polyethylene glycol hydrogel of the embodiments of the present invention. In Figure 20 **** indicates that the P value < 0.0001, and one-way ANOVA with Tukey's multiple comparison test was used.

[0123] According to Figure 20 It can be seen that compared with the PEG hydrogel, the polypeptide hydrogel composition of the present invention exhibits excellent anti-fouling ability, significantly reducing fibrinogen adsorption, and the adsorption amount is only 30% of that of the PEG hydrogel.

[0124] Example 11 Cell Adhesion Assay of Hydrogel Composition

[0125] A cell-free hydrogel composition (about 8 mm in diameter and about 1 mm in height) was prepared. The PEG hydrogel was used as a control group. Under the conditions of 37 °C and 5% CO2, a mouse embryonic fibroblast cell line (NIH 3T3 fibroblasts) was cultured using DMEM containing 10% FBS, 100 U / mL penicillin, 100 mg / mL streptomycin, and 2 mM L-glutamine. Cells at about 80% confluence in the culture dish were separated using 0.05% trypsin and 0.02% EDTA, centrifuged, and resuspended in the medium to a final concentration of 1.0×10 5 cells / mL. The hydrogel was placed at the bottom of a 12-well plate, and 1 mL of the NIH 3T3 cell suspension at a concentration of 1.0×10 5 cells / mL was added to each well, and incubated at 37 °C for 3 days. After incubation, the hydrogel was transferred to a new 12-well plate, and each well contained sterile PBS. The hydrogel was stained with a cell viability staining solution for 30 min and imaged under a wide-field fluorescence microscope, and the results are as Figure 21 shown.

[0126] Figure 21 This is a comparison graph of in vitro cell adhesion between the hydrogel composition and polyethylene glycol hydrogel of the embodiments of the present invention.

[0127] According to Figure 21 It can be seen that there was slight cell adhesion on the surface of the PEG hydrogel, while almost no cells were observed on the surface of the hydrogel composition, indicating that the hydrogel composition has excellent anti-cell adhesion ability.

[0128] Example 12 In Vivo Anti-FBR Test of Hydrogel Composition

[0129] At 6 weeks of age, 100 μL of cell-free cross-linked polypeptide hydrogel and 100 μL of PEG hydrogel were subcutaneously injected into the back of male C57BL / 6 mice. Each hydrogel was repeatedly implanted into three mice, one on each side of the back subcutaneously. After 2 weeks of implantation and feeding, the mice were sacrificed, and the hydrogel samples and surrounding tissues were excised and collected. The transplanted samples were fixed in 4% paraformaldehyde overnight and embedded in paraffin.

[0130] Each sample was cut into sections 3 - 5 µm thick and mounted on glass slides for histological staining. The inflammatory response was examined by staining the tissue sections with hematoxylin and eosin (H&E), which stains cell nuclei blue and cytoplasm pink. The formation and distribution of collagen were examined by staining the tissue sections with Masson's trichrome (M&T), which stains collagen blue, cytoplasm red, and cell nuclei black. The adhesion of macrophages was examined by staining the tissue sections with F4 / 80. Hematoxylin stains cell nuclei blue, and rabbit anti-mouse F4 / 80 monoclonal antibody and enzyme-labeled goat anti-mouse / rabbit IgG polymer stain macrophages brown. The results are as Figure 22 shown.

[0131] Figure 22 This is the inflammatory response diagram two weeks after in vivo implantation of the hydrogel composite of the embodiment of the present invention.

[0132] According to Figure 22 it can be seen that H&E staining shows an obvious inflammatory response around the PEG hydrogel, while the inflammatory response around the random copolymer polypeptide hydrogel composite is very low. The purple part pointed by the arrow indicates the cell nucleus; M&T staining shows that the collagen deposition of the random copolymer polypeptide hydrogel composite is significantly lower than that of the PEG hydrogel. The blue part pointed by the arrow indicates collagen; F4 / 80 staining shows that a large number of macrophages adhere to the surface of the PEG hydrogel. The brown part pointed by the arrow indicates macrophages, while the random copolymer polypeptide hydrogel composite significantly inhibits cell overgrowth. The above data indicate that the random copolymer polypeptide hydrogel composite is an anti-FBR material.

[0133] The specific embodiments described above have further elaborated on the purpose, technical solutions, and beneficial effects of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not used to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A composition, characterized in that, The composition is a hydrogel composition, comprising cells, a first component and a second component. The first component is polyethylene glycol NHS-PEG-NHS with N-hydroxysuccinimide ester groups at both ends, and the second component is a random copolymer polypeptide formed by the random copolymerization of serine and lysine; wherein, the random copolymer polypeptide comprises L-serine, D-serine and L-lysine.

2. The composition according to claim 1, wherein the molar ratio of L-serine, D-serine and L-lysine is (0.5~1.5):(0.5~1.5):(1.5~2.5).

3. The composition according to claim 1, wherein The hydrogel composition comprises a solution of the first component and the second component, and the solution comprises a cell or tissue preservation solution of the first component and the second component.

4. The composition according to claim 3, wherein (1) In the hydrogel composition, the mass concentration of the first component is 5~15 wt%, and the mass concentration of the second component is 25~35 wt%, and / or (2) The volume ratio of the solution of the first component to the solution of the second component is (0.8~1.2):(0.8~1.2).

5. The composition according to claim 3, wherein The density of the cells is (0.1~10)×10 7 cells / mL.

6. A kit comprising the composition according to any one of claims 1~5.

7. A method for preparing the composition according to any one of claims 1 to 5, characterized in that, It includes: Dissolve the first component, the second component and the cells in a solution to obtain a cell-containing hydrogel composition.

8. Use of the composition according to any one of claims 1 to 5 or the kit according to claim 6, characterized in that, The applications include at least one of the following: (1) Preparing a cell therapy preparation; (2) Preparing a tissue or organ repair preparation; (3) Preparing a drug delivery carrier; (4) Preparing a biosensor.

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