Composition as well as preparation method and application thereof

Abstract

The invention provides a composition as well as a preparation method and application thereof, and belongs to the technical field of biological materials. 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 two ends, and the second component is random copolymerization polypeptide formed by random copolymerization of serine and lysine. The hydrogel composition formed by the NHS-PEG-NHS and the random copolymerized polypeptide can effectively resist protein adsorption and cell attachment due to the strong hydrogen bond hydration effect formed by amido bonds on a main chain and hydroxyl groups on side chains, and has the antifouling characteristic, so that foreign matter reaction resistance and in-vivo foreign matter reaction resistance are achieved. When the hydrogel composition is added into cells for encapsulation, amino groups on the surfaces of the cells and amino groups on side chains of the copolymer are covalently bound by utilizing NHS-PEG-NHS to construct a high-strength hydrogel network, and the strength of the hydrogel composition is regulated and controlled by the density of the encapsulated cells.

Description

Technical Field

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

[0002] The natural extracellular matrix (ECM) is composed of a network of materials surrounding cells in tissues. It is the support structure and microenvironment for the survival of tissue cells. Its physical properties are heterogeneous and its biochemical composition is very complex. Therefore, related technologies are committed to finding materials with simple structures that can replace ECM for cell culture.

[0003] Hydrogels can provide an aqueous environment similar to natural ECM (usually >70% water content), 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 function. 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 foreign body response (FBR), which leads to host recognition and subsequent formation of dense fibrous capsules, which block the diffusion of oxygen and nutrients and lead to implant failure. Nonspecific protein adsorption on the implant surface is considered to be the first and key step in triggering FBR. Therefore, it is necessary to develop an implantable hydrogel that can alleviate 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 yet another embodiment of the present invention, a kit comprising the above composition is provided.

[0006] According to another embodiment of the present invention, there is provided a method for preparing the above composition, 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, there is provided an application of the above-mentioned composition or kit, which 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) for encapsulating cells.

[0008] According to the embodiment of the present invention, the hydrogel composition composed of random copolymerized polypeptides formed by random copolymerization of polyethylene glycol and serine lysine having N-hydroxysuccinimide ester groups at both ends can effectively resist protein adsorption and cell attachment due to the strong "double hydrogen bond hydration" effect formed by the amide bonds on the main chain and the side chain hydroxyl groups, and has anti-fouling properties to resist FBR and the ability to resist foreign body reactions in the body; further, when cells are added for encapsulation, the amino groups on the cell surface are covalently bonded to the amino groups on the side chains of the copolymer using NHS-PEG-NHS through a cell cross-linking strategy to construct a high-strength hydrogel network, and 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 damage caused by surgery or ultraviolet irradiation, and the operation is simple and non-toxic. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 This is a schematic diagram of the random copolymerization polypeptide synthesis process of an embodiment of the present invention;

[0010] Figure 2 is the hydrogen nuclear magnetic resonance spectrum of L-lysine N-carboxy anhydride of the embodiment of the present invention;

[0011] Figure 3 is the hydrogen nuclear magnetic resonance spectrum of L-serine N-carboxylic anhydride of the embodiment of the present invention;

[0012] Figure 4 This is the hydrogen nuclear magnetic resonance spectrum of the D-serine N-carboxy anhydride of the embodiment of the present invention;

[0013] Figure 5 This is a hydrogen nuclear magnetic resonance spectrum of the random copolymerized polypeptide in the side chain protection stage of an embodiment of the present invention;

[0014] Figure 6 This is a graph showing the molecular weight of random copolymerized polypeptides at the side chain protection stage measured by gel permeation chromatography in an embodiment of the present invention;

[0015] Figure 7 This is a hydrogen nuclear magnetic resonance spectrum of the random copolymer polypeptide after deprotection in an embodiment of the present invention;

[0016] Figure 8 This is a hydrogen nuclear magnetic resonance spectrum of polyethylene glycol having N-hydroxysuccinimide ester groups at both ends in an embodiment of the present invention;

[0017] Fig. 9 Schematic diagram of the preparation process of the cell-containing hydrogel composition according to an embodiment of the present invention;

[0018] Fig.10 This is a comparison diagram of Fourier infrared spectroscopy characterization of an embodiment of the present invention;

[0019] Fig.11This is a comparison chart of the swelling degree of the embodiments of the present invention within 24 hours;

[0020] Fig.12 This is a comparison chart of the swelling degree of the embodiments of the present invention after 7 days;

[0021] Fig.13 This is a diagram of a hydrogel assembly used for measuring compression modulus in an embodiment of the present invention;

[0022] Fig.14 A comparison diagram of stress-strain curves in a compression test of a hydrogel composition according to an embodiment of the present invention;

[0023] Fig.15 This is a comparison chart of the compression modulus of the hydrogel composition of the embodiment of the present invention;

[0024] Fig.16 This is a comparison diagram of the storage modulus changes of the hydrogel composition according to the embodiment of the present invention;

[0025] Fig.17 This is a comparison chart of the average storage modulus of the hydrogel composition of the embodiment of the present invention;

[0026] Fig.18 This is a fluorescence image of cell life and death of a hydrogel composition containing cells according to an embodiment of the present invention;

[0027] Fig.19 This is a diagram showing the results of lactate dehydrogenase cytotoxicity test of a hydrogel composition containing cells according to an embodiment of the present invention;

[0028] Fig. 20 This is a comparative diagram of in vitro protein adsorption of a cell-free hydrogel composition and a polyethylene glycol hydrogel according to an embodiment of the present invention;

[0029] Fig.21 This is a comparison chart of in vitro cell adhesion between the hydrogel composition of the present invention and the polyethylene glycol hydrogel;

[0030] Fig. 22 This is a diagram showing the inflammatory response of the hydrogel composition of an example of the present invention two weeks after implantation in vivo. DETAILED DESCRIPTION

[0031] Below, embodiments of the present invention will be described with reference to the accompanying drawings. However, it should be understood that these descriptions are exemplary only and are not intended to limit the scope of the present invention. In the following detailed description, for ease of explanation, many specific details are set forth to provide a comprehensive understanding of embodiments of the present invention. However, it is apparent that one or more embodiments may also be implemented without these specific details. In addition, in the following description, descriptions of known structures and technologies are omitted to avoid unnecessary confusion of concepts of the present invention.

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

[0033] The term "treatment" means that after a subject has a disease, the subject is exposed to (e.g., administered) a drug, composition, etc. according to the present invention, thereby alleviating the symptoms of the disease compared to when the subject has not been exposed to the drug, composition, etc., and does not necessarily mean that the symptoms of the disease are completely suppressed. Suffering from a disease means that the body has symptoms of the disease.

[0034] The term "prevention" means that before a subject develops a disease, the symptoms after developing the disease are alleviated compared to when the subject does not develop the disease by contacting (eg administering) the drug, composition, etc. according to the present invention, but does not necessarily mean that the disease must be completely suppressed.

[0035] In the process of realizing the concept of the present invention, it is found that the artificial cell culture system has a wide range of applications in the biomedical field, such as encapsulating pancreatic islet cells and implanting them in the body to treat type 1 diabetes. However, the hydrogel of the artificial cell culture system formed above needs to be cross-linked by ultraviolet light, which will inevitably damage the cells. In addition, implanting the hydrogel into the body usually requires two or more open surgeries. During the treatment process, repeated open surgeries increase medical risks and patient suffering.

[0036] Furthermore, since hydrogels usually achieve a "sol-gel" transition through cross-linking of precursor solutions, they can be designed as injectable biomaterials without compromising their original functions, reducing damage to surrounding tissues. However, injectable hydrogels usually have poor mechanical properties due to weak non-covalent interactions, which may cause hydrogel rupture during or after injection, leading to cell escape, especially the risk of teratoma formation when stem cells escape, thus limiting their application in the field of cell encapsulation.

[0037] Taking all the above factors into consideration, an injectable hydrogel with anti-foreign body reaction in vivo and high mechanical strength is prepared, which 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, comprising a first component and a second component, the first component being polyethylene glycol NHS-PEG-NHS having N-hydroxysuccinimide ester groups at both ends, and the second component being a random copolymer polypeptide formed by random copolymerization of serine and lysine.

[0039] According to the embodiment of the present invention, the hydrogel composition composed of random copolymerized polypeptides formed by random copolymerization of polyethylene glycol, serine and lysine having N-hydroxysuccinimide ester groups at both ends can effectively resist protein adsorption and cell attachment due to the strong "double hydrogen bond hydration" effect formed by the amide bonds on the main chain and the side chain hydroxyl groups, and has anti-fouling properties to resist FBR and the ability to resist foreign body reactions in the body; further, when cells are added for encapsulation, the amino groups on the cell surface are covalently bonded to the amino groups on the side chains of the copolymer using NHS-PEG-NHS through a cell cross-linking strategy to construct a high-strength hydrogel network, and 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 damage caused by surgery or ultraviolet irradiation, and the operation is simple and non-toxic.

[0040] According to an embodiment of the present invention, the random copolypeptide comprises 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 poly-L-serine segments are firmly bound to each other, they can form β-folded aggregates. This rigid structure makes its water solubility very low (<0.1 mg / mL). Therefore, by introducing D-type serine, the tendency to form β-folded aggregates is broken, so that 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 the numerical range are equally applicable.

[0044] According to the embodiments 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, and the addition of L-lysine can enhance the connectivity between fibers and play a cross-linking role to 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 composition is a hydrogel composition, which 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 %, the mass concentration of the second component is 25-35 wt %, and / or (2) the solution volume ratio of the first component to 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 it is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0048] According to an embodiment of the present invention, the solution volume ratio 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 the numerical range are also 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 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. to form a cell or tissue preservation solution.

[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 (to -NH 2 NHS-PEG-NHS is a kind of PEG-NHS ester, which is a kind of PEG-NHS ester. It is a kind of PEG-NHS ester with a strong affinity to form a PEG-NHS ester. It is easy to covalently crosslink with the active ester group (NHS ester) at the end of NHS-PEG-NHS. NHS ester is more stable under neutral to weak alkaline conditions and hydrolyzes slowly at pH 7-8, but too high a pH (>8.5) will cause it to hydrolyze and become ineffective. pH 8.0 is a balance point that can activate the amino group while avoiding premature degradation of NHS ester.

[0051] According to an embodiment of the present invention, the hydrogel composition has good biocompatibility and is suitable for use in an in vivo environment, such as 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 (0.1~10)×10 7 cells / mL.

[0053] According to the embodiment of the present invention, the NHS-PEG-NHS crosslinker can react with a large number of amino groups on the cell surface and amino groups on the side chains of polyamino acids at the same time 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 density of the encapsulated cells, which is suitable for the needs of different tissues of the human body. For example, in the application of treating intervertebral discs, the mechanical properties of the required hydrogel are relatively strong, so a larger cell density can be used to crosslink to obtain a stronger hydrogel; while in the treatment of brain diseases, the required cell density is lower, thereby obtaining a hydrogel with more suitable mechanical properties, ranging from the Pa level of brain tissue to the MPa level of bone tissue.

[0054] According to an embodiment of the present invention, the density of 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 values ​​listed, and other values ​​not listed within the numerical range are also applicable.

[0055] According to an embodiment of one aspect of the present invention, a kit is also provided, comprising a first component and a second component, wherein 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 is in a form that allows the biological activity of the active ingredient contained therein to be effective, and may also include other auxiliary ingredients or vehicles used to constitute 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 a first component and a 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, such as 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 buffered saline (PBS), Dulbecco's phosphate buffered saline (DPBS), Earle's balanced salt solution (EBSS buffer), etc.

[0059] Specifically, the preparation method comprises cross-linking a random copolymerized 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, the method further comprises dissolving the first component, the second component and the 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 may have a pH value of 7 or more, 7.2 or more, 7.4 or more, 7.6 or more, 7.8 or more, 8 or more, etc.

[0063] According to the embodiment of the present invention, the method for preparing 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 / cytotoxicity, 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 for one or more purposes such as biological dressings, human bionic materials, plastic surgery materials, organoid culture materials, cardiovascular stent materials, coating materials, tissue injection filling materials, ophthalmic materials, obstetrics and gynecology biomaterials, nerve repair and regeneration materials, liver tissue materials and vascular repair and regeneration materials, 3D printing artificial organ biomaterials, cosmetic raw materials, pharmaceutical excipients and food additives.

[0066] The scheme of the present invention will be further explained below with reference to specific examples. Unless otherwise stated in the following examples, conventional commercial reagents are used.

[0067] Example 1 Synthesis of Nε-tert-butyloxycarbonyl-L-lysine N-carboxylic anhydride (Boc-L-Lys NCA)

[0068] Figure 1 Schematic diagram of the random copolymerization polypeptide synthesis process of an embodiment of the present invention.

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

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

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

[0072] Figure 2 The figure is the hydrogen nuclear magnetic resonance spectrum of L-lysine N-carboxy anhydride of the embodiment of the present invention.

[0073] according to Figure 2 It can be seen that L-lysine N-carboxy anhydride was successfully synthesized.

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

[0075] The reaction process is as follows Figure 1 As shown, in N 2Under a nitrogen atmosphere and 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 remove the tetrahydrofuran by vacuum rotary evaporation at 45 °C.

[0076] The reaction mixture was dissolved in ethyl acetate (50 mL), washed three times with ice water (50 mL), and washed once with ice saturated brine (50 mL). The collected organic solution was dried over anhydrous magnesium sulfate overnight. The dried solution was vacuum-rotated at 45 °C to remove the solvent to obtain a crude product. The crude product was recrystallized and purified three times in anhydrous ethyl acetate and anhydrous n-hexane in a glove box to obtain white needle-like crystals. Deuterated dimethyl sulfoxide was used as the solvent for H NMR characterization, such as Figure 3 , 4 shown.

[0077] Figure 3 is the hydrogen nuclear magnetic resonance spectrum of L-serine N-carboxylic anhydride of the embodiment of the present invention; Figure 4 The figure is the hydrogen nuclear magnetic resonance spectrum of the D-serine N-carboxy anhydride of the embodiment of the present invention.

[0078] according to Figure 3 and Figure 4 It can be seen that L-serine N-carboxy anhydride and D-serine N-carboxy anhydride were successfully synthesized.

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

[0080] The synthesis process is as follows Figure 1 As shown, the L-serine N-carboxy anhydride and D-serine N-carboxy anhydride prepared in Example 2 (0.94 g, 5 mmol each) and the L-lysine N-carboxy anhydride prepared in Example 1 (2.72 g, 10 mmol) were weighed and dissolved in tetrahydrofuran (90 mL). An anhydrous tetrahydrofuran solution of lithium hexamethyldisilazide (0.1 mmol / mL, 10 mL) was immediately added to the solution. After stirring at room temperature for 10 min, the reaction mixture was added dropwise to anhydrous cold ether (1 L) to obtain a white flocculent precipitate, which was collected by centrifugation (5000 rpm, 4 ° C) and preliminarily dried under a nitrogen stream.

[0081] The collected solid was dissolved in tetrahydrofuran (100 mL), precipitated again, and the dissolution-precipitation process was repeated three times, and dried under vacuum to obtain a white solid Poly(tBu- D,L -Serine)-r-poly(Boc-L-Lysine) with side chain protecting groups.

[0082] Deuterated dimethyl sulfoxide was used as solvent for H NMR characterization. Figure 5 shown.

[0083] Figure 5 This is a hydrogen nuclear magnetic resonance spectrum of the random copolymerized polypeptide in the side chain protection stage of an embodiment of the present invention.

[0084] Take 5 mg of dried poly(O-tert-butyl-D,L-serine)-random-poly(Nε-tert-butyloxycarbonyl-L-lysine) (Poly(tBu- D,L -Serine)-r-poly(Boc-L-Lysine)) and dissolve it in N,N-dimethylformamide (DMF) to a concentration of 1 mg / mL. Use a gel permeation chromatography (GPC) instrument, using DMF supplemented with 0.01 M LiBr as the mobile phase at a flow rate of 1 mL / min, to determine the molecular weight, as shown in Figure 2. Figure 6 shown.

[0085] Figure 6 This is a graph showing the molecular weight of random copolymerized polypeptides at the side chain protection stage measured by gel permeation chromatography according to an 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 for 2 h, the trifluoroacetic acid was removed by vacuum rotary evaporation to obtain a yellow oil. It was dissolved in methanol (40 mL) and added dropwise to 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 flow. The collected solid was dissolved in methanol (40 mL) and precipitated again. After three cycles of dissolution / precipitation, the deprotected random copolymer Poly(D,L-Serine)-r-poly(L-Lysine) was obtained. H-NMR characterization was performed using deuterated water as the solvent, such as Figure 7 shown.

[0087] Figure 7 This is the hydrogen nuclear magnetic resonance spectrum of the random copolymer polypeptide after deprotection in the embodiment of the present invention.

[0088] according to Figure 5~Figure 7It can be seen that the random copolymer polypeptide (poly(D,L-serine)-random-poly(L-lysine), 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 about 1:1, that is, Figure 1 In the chemical formula, x:y≈1:1. Based on the measured molecular weight of 61.2 kDa, we can obtain 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 nitrogen atmosphere. N,N'-disuccinimidyl carbonate (6.4 g, 25 mmol) was dissolved in anhydrous acetonitrile (100 mL). The two solutions were mixed and pyridine (1 mL, 12.5 mmol) was added at last. After stirring at room temperature for 24 h under nitrogen atmosphere, the mixture was filtered and dried under vacuum. After drying, the sample was dissolved in chloroform (50 mL) and added dropwise to 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 nitrogen flow. The collected solid was dissolved in chloroform (50 mL) and precipitated again. After three cycles of dissolution / precipitation, NHS-PEG-NHS was obtained. Deuterated chloroform was used as solvent for H NMR characterization. Figure 8 shown.

[0091] Figure 8 This is a hydrogen nuclear magnetic resonance spectrum of polyethylene glycol having N-hydroxysuccinimide ester groups at both ends according to an embodiment 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 cell-containing hydrogel compositions

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

[0095] Fig. 9 Schematic diagram of the preparation process of the cell-containing hydrogel composition according to an embodiment of the present invention.

[0096] Fourier transform infrared spectroscopy (FTIR) at 4000 cm -1 Up to 400 cm -1 Peptide, NHS-PEG-NHS and hydrogel compositions (cell-free, denoted as NCCH) were analyzed in the range of 1×10 7 and 2×10 7 The cell-containing hydrogel compositions were denoted as CCH-1 and CCH-2, respectively, with a spectral resolution of 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. Fig.10 shown.

[0097] Fig.10 This is a comparison chart of Fourier infrared spectroscopy characterization of the embodiments of the present invention.

[0098] according to Fig.10 It can be seen that NHS-PEG-NHS has a peak at 2888 cm -1 and 1109cm -1 The -CH 2 The stretching vibration peaks of COC and 1740 cm -1 The peak at 1650cm is the stretching vibration peak of C=O of NHS group. -1 and 1540 cm -1 The peaks at 2888 cm-1 are the stretching vibration peaks of C=O and CN on the main chain. After the hydrogel is formed and swelled and freeze-dried, it can be found that the peaks of NCCH, CCH-1 and CCH-2 are basically the same. -1 、1109 cm -1 、1650 cm -1 and 1540 cm -1 A peak was observed at 1740 cm-1 No peak was observed at the position, indicating the formation of CO-NH bonds and the complete reaction of NHS groups before washing out with water. These results indicate that the hydrogel is cross-linked by NHS-PEG-NHS and random copolymerized polypeptides.

[0099] Example 6 Characterization of Swelling Properties of Hydrogel Compositions

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

[0101] Fig.11 This is a comparison chart of the swelling degree of the embodiments of the present invention within 24 hours; Fig.12 This is a comparison chart of the swelling degree of the embodiments of the present invention after 7 days. Fig.11 In the table, **** indicates P value < 0.0001, and two-way ANOVA with Tukey's multiple comparison test was used.

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

[0103] Example 7 Determination of compression modulus of hydrogel composition

[0104] After washing the cell pellet, evenly disperse it in 100 μL of 10 wt% copolypeptide HBSS solution (pH 8.0), and then add 100 μL of 30 wt% NHS-PEG-NHS HBSS solution (pH 8.0). Gently mix the cells and solution, and drop the precursor solution into a hollow cylindrical polytetrafluoroethylene mold with an inner diameter of 8 mm, and incubate at 37 °C for five minutes. Fig.13 shown.

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

[0106] Fig.13 This is a diagram of a hydrogel assembly used for measuring compression modulus in an embodiment of the present invention; Fig.14 A comparison diagram of stress-strain curves in a compression test of a hydrogel composition according to an embodiment of the present invention; Fig.15 This is a comparison chart of the compression modulus of the hydrogel composition of the present invention. Fig.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), one-way ANOVA with Tukey's multiple comparison test was used.

[0107] according to Figure 13~Figure 15 It can be seen that the hydrogel compositions CCH-1, CCH-2 and the cell-free hydrogel composition NCCH all have high compressive 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. This result directly indicates that the cell cross-linking sites enhance the compression properties of the hydrogel.

[0108] Example 8 Determination of compression modulus of hydrogel composition

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

[0110] Fig.16 This is a comparison diagram of the storage modulus changes of the hydrogel composition according to the embodiment of the present invention; Fig.17 This is a comparison chart of the average storage modulus of the hydrogel composition of the present invention. Fig.17In the table, * 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), one-way ANOVA with Tukey's multiple comparison test was used.

[0111] according to Fig.16 and Fig.17 It can be seen that the cell cross-linking strategy also enhances the storage modulus. The storage modulus of CCH-2 is 1.8 times that of NCCH, which further indicates that compared with short chemical bonds, cells as cross-linking points and this large structure can enhance the mechanical properties of the hydrogel network.

[0112] Example 9 Biosafety Test of Hydrogel Composition

[0113] Cell life and death test: The hydrogel composition containing cells (8 mm in diameter and 2 mm in thickness) was added to a 24-well plate and incubated at 37 °C and 5% CO 2 Under the same conditions, the hydrogels were cultured 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) for 7 days. At specific time points, the hydrogels were washed twice with PBS and stained with cell death and viability staining solution (the hydrogels were stained for 30 min and imaged under a fluorescence microscope. The results are shown in Figure 2). Fig.18 shown.

[0114] Fig.18 This is a fluorescence image of cell life and death of a hydrogel composition containing cells according to an embodiment of the present invention.

[0115] according to Fig.18 It can be seen that within 7 days, substantially all cell clusters showed green fluorescence, indicating high cell viability and that the hydrogel composition of the present invention has biological safety.

[0116] Lactate dehydrogenase (LDH) cytotoxicity assay: The hydrogel composition containing cells (8 mm in diameter, 2 mm in thickness) was added to a 24-well plate and incubated at 37 °C and 5% CO. 2Under the same conditions, 2 mL of DMEM high-glucose medium containing 10% FBS, 1% P / S and 1% NEAA was used to culture 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 LDH released, 120 μL of the supernatant was transferred to a new 96-well plate after centrifugation of each well, 60 μL of LDH detection reagent was added, and then incubated at room temperature for 30 minutes. Finally, the absorbance was measured at 490 nm using a microplate reader, and a dual-wavelength measurement was performed using a wavelength of 600 nm as the reference wavelength. The results are shown in Figure 2. Fig.19 shown.

[0117] Fig.19 This is a graph showing the results of lactate dehydrogenase cytotoxicity test of a hydrogel composition containing cells according to an embodiment of the present invention. Fig.19 ** indicates P value = 0.0031, one-way ANOVA with Tukey's multiple comparison test.

[0118] according to Fig.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 the cells. The percentage of LDH released by the cells in CCH-2 is 8.3%, which may be because there are more cell clusters and the amount of LDH released will increase accordingly. The above data show that the cell-containing hydrogel composition has good cell compatibility.

[0119] Example 10 Determination of fibrinogen adsorption of hydrogel composition

[0120] A cell-free hydrogel composition (about 8 mm in diameter and 1 mm in height) was prepared. The commonly used biologically inert material PEG was used as a control. 2 (molecular weight 5000 Da) and NHS-PEG-NHS were dissolved in HBSS buffer (pH 8.0) at 50 wt%, and 25 μL of four-arm PEG-NH 2 The solution and 25 μL of 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 PEG hydrogel as a control group.

[0121] Protein adsorption on the hydrogel was detected by enzyme-linked immunosorbent assay (ELISA), and fibrinogen (Fg) was selected as the test protein to examine the anti-protein adhesion ability of the hydrogel samples. The samples were initially placed in a 24-well plate and subjected to a 5-min equilibration period in PBS buffer. Next, the samples were immersed in 1 mL, 1 mg / mL Fg in PBS buffer for 1 h and then thoroughly washed three times with PBS buffer. The samples were then transferred to fresh wells and immersed in 1 mL, 1 μg / mL horseradish peroxidase (HRP)-conjugated anti-fibrinogen antibody in PBS for 0.5 h. After washing five more times with PBS buffer, all samples were transferred to new wells. Subsequently, 1 mL of o-phenylenediamine (OPD) chromogen solution was added. After incubation for 15 minutes, an equal amount of 1N hydrochloric acid (HCl) was added to stop the enzymatic reaction. The absorbance readings at 490 nm were then recorded using a microplate reader, and the results are shown in Figure 2. Fig. 20 shown.

[0122] Fig. 20 The figure is a comparison of in vitro protein adsorption of the cell-free hydrogel composition and the polyethylene glycol hydrogel in the embodiment of the present invention. Fig. 20 In the table, **** indicates P value < 0.0001, and one-way ANOVA with Tukey's multiple comparison test was used.

[0123] according to Fig. 20 It can be seen that compared with PEG hydrogel, the polypeptide hydrogel composition of the present invention exhibits excellent antifouling ability and significantly reduces the adsorption of fibrinogen, with the adsorption amount being only 30% of that of PEG hydrogel.

[0124] Example 11 Hydrogel composition cell adhesion assay

[0125] A hydrogel composition without cells (about 8 mm in diameter and 1 mm in height) was prepared. PEG hydrogel was used as a control group. The cells were incubated at 37 ºC and 5% CO 2 Mouse embryonic fibroblast cell line (NIH 3T3 fibroblasts) was cultured in DMEM containing 10% FBS, 100 U / mL penicillin, 100 mg / mL streptomycin, and 2 mM L-glutamine under the same conditions. Cells at approximately 80% confluence in the culture dish were detached with 0.05% trypsin and 0.02% EDTA, centrifuged, and resuspended in culture medium to a final concentration of 1.0 × 10 5 The hydrogel was placed at the bottom of a 12-well plate and 1.0×10 51 mL of NIH 3T3 cell suspension at 100 cells / mL was added and incubated at 37 °C for 3 days. After incubation, the hydrogels were transferred to a new 12-well plate, each well of which contained sterile PBS. The hydrogels were stained with cell death and viability staining solution for 30 min and imaged under a wide-field fluorescence microscope. The results are shown in Figure 2. Fig.21 shown.

[0126] Fig.21 The figure is a comparison of in vitro cell adhesion between the hydrogel composition of the embodiment of the present invention and the polyethylene glycol hydrogel.

[0127] according to Fig.21 It can be seen that cells have a slight adhesion phenomenon on the surface of the PEG hydrogel, while almost no cells are observed on the surface of the hydrogel composition, which indicates that the hydrogel composition has excellent anti-cell adhesion ability.

[0128] Example 12 In vivo anti-FBR test of hydrogel composition

[0129] 100 μL of cell-free cross-linked peptide hydrogel and 100 μL of PEG hydrogel were injected subcutaneously in the back of 6-week-old C57BL / 6 male mice. Each hydrogel was implanted into three mice, one subcutaneously in each of the left and right backs. The mice were killed after 2 weeks of implantation, and the hydrogel samples and surrounding tissues were removed and collected. The transplanted samples were fixed in 4% paraformaldehyde overnight and embedded in paraffin.

[0130] Each sample was cut into 3-5 µm thick sections and mounted on slides for histological staining. The inflammatory response was examined by staining tissue sections with hematoxylin and eosin (H&E), which stains the nuclei of cells blue and the cytoplasm pink. The formation and distribution of collagen were examined by staining tissue sections with Masson's trichrome (M&T), which stains collagen blue, the cytoplasm red, and the nuclei of cells black. The adhesion of macrophages was examined by staining tissue sections with F4 / 80, which stains the nuclei of cells blue and the macrophages brown with rabbit anti-mouse F4 / 80 monoclonal antibody and enzyme-labeled goat anti-mouse / rabbit IgG polymer. The results are shown in Table 1. Fig. 22 shown.

[0131] Fig. 22 This is a diagram showing the inflammatory response of the hydrogel composition of an example of the present invention two weeks after implantation in vivo.

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

[0133] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific 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 protection scope of the present invention.

Claims

1. A composition, characterized in that The invention comprises a first component and a second component, wherein 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.

2. The composition according to claim 1, characterized in that The random copolypeptide comprises L-serine, D-serine and L-lysine.

3. The composition according to claim 2, characterized in that The molar ratio of L-serine, D-serine and L-lysine is (0.5~1.5):(0.5~1.5):(1.5~2.5).

4. The composition according to claim 1, characterized in that The composition is a hydrogel composition, which 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.

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

6. The composition according to claim 4, characterized in that The composition further comprises cells, the density of the cells is (0.1-10)×10 7 cells / mL.

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

8. A method for preparing the composition according to any one of claims 4 to 6, characterized in that: include: The first component and the second component are dissolved in a solution to obtain a hydrogel composition.

9. The preparation method according to claim 8, characterized in that: The first component, the second component and cells are dissolved in a solution to obtain a cell-containing hydrogel composition.

10. Use of the composition according to any one of claims 1 to 6 or the kit according to claim 7, characterized in that: The application includes at least one of the following: (1) Preparation of cell therapy preparations; (2) Preparation of tissue or organ repair preparations; (3) preparing drug delivery vehicles; (4) Preparation of biosensors; (5) Used for encapsulating cells.

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