Preparation method and application of hydrogel based on cellulose nanocrystals

Abstract

The invention provides a preparation method and application of hydrogel based on cellulose nanocrystals. The invention provides a preparation method of hydrogel, which comprises the following steps: (1) adding cerium ions into a cellulose nanocrystal CNC suspension, and controlling the pH value to be neutral to obtain a cerium oxide modified CNC suspension; (2) mixing the CNC suspension modified in the step (1) with the CNC suspension not modified in the step (1) to obtain a CNC mixed suspension; and (3) mixing the CNC mixed suspension in the step (2) with a prepolymer, and performing ultraviolet irradiation to obtain the hydrogel.

Description

Technical Field

[0001] The invention belongs to the technical field of hydrogel materials, and particularly relates to a preparation method and application of a hydrogel based on cellulose nanocrystals. Background Art

[0002] In recent years, various synthetic or natural hydrogel materials have great application potential in the treatment of corneal injuries due to their physical properties similar to those of soft tissues.

[0003] Poly(hydroxyethyl methacrylate) and poly(methyl methacrylate) are synthetic hydrogel materials that are often used in the eye and can be used as corneal contact lens materials. They have high transparency and stable physical and chemical properties, but their cell affinity is low.

[0004] Cellulose nanocrystals (CNCs) are rod-shaped crystalline residues obtained after acid hydrolysis of cellulose. Due to their low cost, biocompatibility, nanoscale size, abundant hydroxyl groups and unique rod-shaped morphology, they can be used as reinforcing agents and crosslinking agents in biomedical hydrogel materials. Above a certain critical concentration, CNCs can self-assemble into a cholesteric phase with a regular helical structure, which has a certain effect on cell adhesion, proliferation and differentiation. More importantly, this high similarity to the microscopic topological configuration of the corneal stroma makes it possible to continue to maintain the light transmittance of the material. However, when it is actually applied to the biomedical field, since CNCs themselves do not have any therapeutic functions, the functional properties and structural properties of the hydrogels formed by pure CNCs cannot meet the requirements. Summary of the invention

[0005] The purpose of the present invention is to provide a preparation method and application of a hydrogel based on cellulose nanocrystals.

[0006] A first aspect of the present invention provides a method for preparing a hydrogel, the method comprising:

[0007] (1) mixing a cellulose nanocrystal suspension with cerium ions and controlling the pH to be neutral to obtain a cerium oxide-modified cellulose nanocrystal suspension;

[0008] (2) mixing the cellulose nanocrystal suspension modified in step (1) with the cellulose nanocrystal suspension not modified in step (1) to obtain a cellulose nanocrystal mixed suspension;

[0009] (3) mixing the mixed suspension of cellulose nanocrystals in step (2) with a monomer, and obtaining a hydrogel after polymerization of the monomer; preferably, mixing the mixed suspension of cellulose nanocrystals in step (2) with a prepolymer containing a monomer and an initiator, and obtaining a hydrogel after initiation with the initiator.

[0010] In one or more embodiments, the cellulose nanocrystals are cellulose nanocrystals obtained from acid hydrolysis of plant materials.

[0011] In one or more embodiments, the acid comprises an inorganic acid or an organic acid. Preferably, the acid is a strong acid. More preferably, the acid is sulfuric acid or hydrochloric acid. More preferably, the concentration of the acid is 40-60%.

[0012] In one or more embodiments, the plant raw material includes: cotton, hemp or wood pulp.

[0013] In one or more embodiments, the ratio of the plant material to the acid is: 1 g of plant material: (1-100 ml) of acid solution, preferably 1 g of plant material: (1-20 ml) of acid solution.

[0014] In one or more embodiments, the concentration of the cellulose nanocrystal suspension is 1-10 wt %, preferably 2-6 wt %.

[0015] In one or more embodiments, in step (1), the cerium ion is Ce 3+ , preferably derived from Ce(NO3)3·6H2O.

[0016] In one or more embodiments, the neutral pH refers to a pH value of 6.8 to 8, preferably a pH value of 7 to 8.

[0017] In one or more embodiments, in step (2), the volume ratio of the modified cellulose nanocrystal suspension to the cellulose nanocrystal suspension not modified with cerium oxide is 3:1 to 1:3, preferably 3:1 to 1:1 or 2:1 to 1:1.

[0018] In one or more embodiments, in step (3), the prepolymer comprises: a monomer and an initiator.

[0019] In one or more embodiments, the monomer is substituted or unsubstituted hydroxyethyl acrylate and / or substituted or unsubstituted methyl acrylate, preferably, the monomer is alkyl-substituted hydroxyethyl acrylate and / or alkyl-substituted methyl acrylate, more preferably hydroxyethyl methacrylate and / or methyl methacrylate.

[0020] In one or more embodiments, the initiator is a UV light initiator, more preferably one or more of 2-hydroxy-2-methyl-1-phenylpropanone, 1-hydroxycyclohexylphenyl ketone, 2-methyl-2-(4-morpholinyl)-1-[4-(methylthio)phenyl]-1-propanone, and 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone.

[0021] In one or more embodiments, the molar ratio of the monomer to the initiator is 1:(0.01-0.02).

[0022] In one or more embodiments, in step (3), the volume ratio of the cellulose nanocrystal mixed suspension to the prepolymer is 5:5 to 9:1, preferably 6:4 to 9:1, 7:3 to 9:1 or 8:2 to 9:1.

[0023] In one or more embodiments, the step (3) further comprises: mixing the mixed suspension of cellulose nanocrystals in step (2) with a prepolymer, standing for at least 12 hours, and initiating polymerization with ultraviolet light to obtain a hydrogel; more preferably, the ultraviolet light is 365 nm ultraviolet light.

[0024] In one or more embodiments, after the cellulose nanocrystal mixed suspension is mixed with the prepolymer, the mixture is allowed to stand at a constant temperature and humidity under light-shielding conditions for at least 12 hours, such as 24 hours, 48 ​​hours or 72 hours.

[0025] In one or more embodiments, the polymerization is initiated by ultraviolet light under nitrogen protection.

[0026] The second aspect of the present invention provides a hydrogel comprising cellulose nanocrystals modified with cerium oxide and a matrix, wherein the matrix is ​​a polymer formed by polymerization of monomers. Preferably, the hydrogel also comprises cellulose nanocrystals that are not modified with cerium oxide.

[0027] In one or more embodiments, the polymer is a hydrophilic polymer.

[0028] In one or more embodiments, the monomer is substituted or unsubstituted hydroxyethyl acrylate and / or substituted or unsubstituted methyl acrylate, preferably, the monomer is alkyl-substituted hydroxyethyl acrylate and / or alkyl-substituted methyl acrylate, more preferably hydroxyethyl methacrylate and / or methyl methacrylate.

[0029] In one or more embodiments, in the hydrogel, the cerium oxide-modified cellulose nanocrystals are dispersed in the matrix.

[0030] In one or more embodiments, in the cerium oxide-modified cellulose nanocrystals, the mass ratio of cerium to cellulose nanocrystals is 1:30 to 1:50, preferably 1.3:40.

[0031] In one or more embodiments, the cerium oxide is CeOx, and x is 1.5-2.

[0032] In one or more embodiments, the hydrogel comprises 45% to 90% by volume of cerium oxide-modified cellulose nanocrystals.

[0033] In one or more embodiments, the hydrogel comprises 45% to 67.5% by volume of cerium oxide-modified cellulose nanocrystals, and the hydrogel comprises 22.5% to 45% by volume of cellulose nanocrystals that are not modified with cerium oxide.

[0034] In one or more embodiments, the cellulose nanocrystals that are not modified with cerium oxide are unmodified cellulose nanocrystals.

[0035] In one or more embodiments, the hydrogel has a thickness of 50 to 200 μm.

[0036] In one or more embodiments, the hydrogel is prepared by the preparation method described in any embodiment of the present invention.

[0037] In one or more embodiments, the hydrogel also has one or more of the following properties:

[0038] (1) Tensile strength at break 800KPa or above,

[0039] (2) The elongation is greater than 200%,

[0040] (3) Light transmittance is more than 80%,

[0041] (4) Moisture content is more than 50%,

[0042] (5) Anti-oxidation

[0043] (6) Anti-inflammatory

[0044] (7) It can resist angiogenesis,

[0045] (8) It has the biological activity of promoting the rapid repair of corneal epithelial damage, preferably promoting the adhesion and proliferation of corneal epithelial cells.

[0046] The third aspect of the present invention provides a composition, wherein the composition comprises the hydrogel described in any embodiment of the present invention, or comprises the hydrogel prepared by the preparation method described in any embodiment of the present invention.

[0047] In one or more embodiments, the composition further comprises an active ingredient or an inactive ingredient, preferably the active ingredient or the inactive ingredient is within and / or on the surface of the hydrogel.

[0048] A fourth aspect of the present invention provides a biomaterial, a transplant or a tissue engineering matrix for growing cells and tissues in vivo or in vitro, wherein:

[0049] The biomaterial comprises the hydrogel described in any embodiment of the present invention, or comprises the hydrogel prepared by the preparation method described in any embodiment of the present invention,

[0050] The transplant or tissue engineering matrix for growing cells and tissues in vivo or in vitro comprises the hydrogel described in any embodiment of the present invention, or comprises the hydrogel obtained by the preparation method described in any embodiment of the present invention, or is formed by the hydrogel described in any embodiment of the present invention, or is formed by the hydrogel obtained by the preparation method described in any embodiment of the present invention.

[0051] In one or more embodiments, the biomaterial, transplant or tissue engineering matrix for growing cells and tissues in vivo or in vitro further comprises cells or tissues, preferably corneal epithelial cells or corneal tissues, more preferably the corneal epithelial cells or corneal tissues are within and / or on the surface of the hydrogel.

[0052] A fifth aspect of the present invention provides an application selected from the following:

[0053] (1) Use of the hydrogel described in any embodiment of the present invention, or the hydrogel prepared by the preparation method described in any embodiment of the present invention, as a delivery carrier in the preparation of a drug;

[0054] (2) Use of the hydrogel described in any embodiment of the present invention, or the hydrogel prepared by the preparation method described in any embodiment of the present invention, in the preparation of biomaterials, transplants, or tissue engineering matrices for growing cells and tissues in vivo or in vitro;

[0055] (3) Use of the hydrogel described in any embodiment of the present invention, or the hydrogel prepared by the preparation method described in any embodiment of the present invention, in the preparation of ophthalmic drugs, preferably the ophthalmic drugs include eye implants, eye drops, injections, eye ointments, and tablets;

[0056] (4) Use of the hydrogel described in any embodiment of the present invention, or the hydrogel prepared by the preparation method described in any embodiment of the present invention, in the preparation of a drug for treating corneal damage.

[0057] Other aspects of the present invention will be apparent to those skilled in the art in view of the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 , Atomic force microscopy photograph of the surface of the hydrogel prepared by the method of Example 3. Scale bar = 400 nm.

[0059] Figure 2, the antioxidant (SOD activity, CAT activity), anti-inflammatory (IL-1β), and anti-angiogenic (TNF-α, angiopoietin) properties of the hydrogels prepared by the methods of Examples 1-6, respectively, and the supernatant of human corneal epithelial cells not co-cultured with the hydrogels was used as the control group.

[0060] Figure 3 , Staining photos of human corneal epithelial cells cultured on the hydrogels prepared by the methods of Examples 1-6, respectively. DAPI marks the cell nucleus, and phalloidin marks the cytoskeleton. PHEMA is used as the control group. Scale bar = 100 μm. DETAILED DESCRIPTION

[0061] After in-depth research, the inventors have provided a method for preparing a bioactive hydrogel material based on cellulose nanocrystals (CNC). The prepared hydrogel has antioxidant, anti-inflammatory and anti-angiogenic functionality, good mechanical properties and high transparency, and has the biological activity of promoting rapid repair of corneal epithelial damage, and can be used in the treatment of corneal damage.

[0062] Preparation method of hydrogel

[0063] The present invention provides a method for preparing a hydrogel, comprising:

[0064] (1) mixing a cellulose nanocrystal (CNC) suspension with cerium ions and controlling the pH to be neutral to obtain a cerium oxide-modified cellulose nanocrystal (CNC) suspension;

[0065] (2) mixing the cellulose nanocrystal (CNC) suspension modified in step (1) with the cellulose nanocrystal (CNC) suspension not modified in step (1) to obtain a cellulose nanocrystal (CNC) mixed suspension;

[0066] (3) mixing the cellulose nanocrystal (CNC) mixed suspension in step (2) with a monomer, and obtaining a hydrogel after polymerization of the monomer;

[0067] Preferably, the cellulose nanocrystal (CNC) mixed suspension in step (2) is mixed with a prepolymer containing a monomer and an initiator, and the hydrogel is obtained after initiation with the initiator.

[0068] In the present invention, the "cellulose nanocrystal" and "CNC" can be used interchangeably. It is a polymer material with at least one dimension of nanometer scale made by cellulose through chemical, mechanical, biological and other processing methods, usually in a crystalline form. In some embodiments, the CNC is prepared from plant raw materials by chemical methods, such as acid hydrolysis. Acid hydrolysis methods include inorganic acid hydrolysis, organic acid hydrolysis and the like. The acid in the acid hydrolysis is usually a strong acid, such as sulfuric acid or hydrochloric acid. Exemplarily, the plant raw material can be mixed with the acid and incubated to obtain a cellulose nanocrystal suspension. In some embodiments, the plant raw material is a natural plant raw material containing cellulose, such as but not limited to: cotton, hemp, wood pulp. In some embodiments, the ratio of plant raw material to acid is 1g plant raw material: (1-100ml) acid solution, preferably 1g plant raw material: (1-20ml) acid solution. In some embodiments, the concentration of the acid solution is 40-60%, for example 50%. In some specific embodiments, the cellulose nanocrystal suspension is prepared by the following method: the plant raw material is mixed with 40-60% strong acid solution according to the ratio of 1g plant raw material: (1-100ml) acid solution, incubated at 40-100°C (preferably 65-100°C, more preferably heated in a water bath) for at least 60min (preferably 60-120min), deionized water is added to terminate the reaction, dialyzed (preferably 14000Da dialysis), and ultrasonically dispersed to obtain a cellulose nanocrystal suspension with a concentration of 1-10wt% (preferably 2-6wt%).

[0069] In this article, "modification" refers to the process of improving, optimizing or giving new properties to the original properties of cellulose nanocrystals by physical, chemical or biological methods. In this article, "cerium oxide modified cellulose nanocrystals" refers to cellulose nanocrystals loaded with cerium oxide nanoparticles, which can make cellulose nanocrystals have antioxidant, anti-inflammatory and anti-angiogenic functions.

[0070] In the present invention, the cerium ion generally refers to Ce 3+ , for example, from Ce(NO3)3·6H2O.

[0071] In step (1) of the present invention, the neutral pH generally refers to a pH value of 6.8 to 8. In some embodiments, the neutral pH preferably has a pH value of 7 to 8. Under neutral pH conditions, cerium ions are added to the cellulose nanocrystal suspension, and cerium oxide nanoparticles can be grown in situ by controlled hydrolysis to obtain a cerium oxide-modified cellulose nanocrystal suspension.

[0072] In step (2) of the present invention, the volume ratio of the cerium oxide modified cellulose nanocrystal suspension to the unmodified cellulose nanocrystal suspension can be 3:1 to 1:3. In some embodiments, the volume ratio of the cerium oxide modified cellulose nanocrystal suspension to the unmodified cellulose nanocrystal suspension is 3:1 to 1:1, preferably 2:1 to 1:1.

[0073] In step (3) of the present invention, the prepolymer includes a monomer and an initiator. In some embodiments, the monomer is a high molecular weight monomer. In some embodiments, the monomer is substituted or unsubstituted hydroxyethyl acrylate and / or substituted or unsubstituted methyl acrylate, alkyl-substituted hydroxyethyl acrylate and / or alkyl-substituted methyl acrylate, for example, hydroxyethyl methacrylate and / or methyl methacrylate. In some embodiments, the initiator is an ultraviolet light initiator, for example, selected from 2-hydroxy-2-methyl-1-phenylpropanone, 1-hydroxycyclohexylphenyl ketone, 2-methyl-2-(4-morpholinyl)-1-[4-(methylthio)phenyl]-1-propanone, 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone. One or more. In some embodiments, the molar ratio of the high molecular weight monomer to the initiator is 1:(0.01-0.02). In some embodiments, the volume ratio of the cellulose nanocrystal mixed suspension to the prepolymer is 5:5 to 9:1, for example, 6:4 to 9:1, 7:3 to 9:1 or 8:2 to 9:1.

[0074] In addition to the photoinitiator, the initiators that can be used in this article also include: thermal initiators that can decompose under heating conditions to produce free radicals, thereby initiating polymerization reactions, such as azobisisobutyronitrile (AIBN) will initiate monomer polymerization in the temperature range of 45-80°C; dibenzoyl peroxide (BPO) initiates polymerization of vinyl monomers at 60-80°C. Redox initiators are composed of oxidants and reductants, and are systems that initiate polymerization reactions by generating free radicals through redox reactions, such as potassium persulfate-sodium bisulfite systems and hydrogen peroxide-ferrous salt systems. Radiation initiators can produce active species under the action of high-energy radiation (such as gamma rays, electron beams, etc.), thereby initiating polymerization reactions. For example, methyl methacrylate can spontaneously undergo polymerization reactions under gamma ray radiation, and benzophenone can initiate monomer polymerization under radiation conditions. Bioinitiators are substances or systems that use biologically active substances such as enzymes or microorganisms to initiate polymerization reactions. For example, horseradish peroxidase (HRP) can initiate the polymerization of monomers containing functional groups such as phenolic hydroxyl groups in the presence of hydrogen peroxide; certain microorganisms such as yeast can also produce some enzymes or metabolites under specific conditions to initiate polymerization reactions of specific monomers. Chemical initiators (non-oxidation-reduction type) initiate polymerization reactions by generating free radicals or other active species through their own chemical reactions. For example, in the triethylboron-water system, triethylboron reacts with water to generate active species such as ethyl free radicals, which initiate polymerization reactions of monomers such as ethylene; some metal organic compounds such as alkyl aluminum compounds can also initiate polymerization reactions of olefins in synergistic action with co-catalysts.

[0075] In some embodiments, the mixing can be carried out by stirring (e.g., magnetic stirring), ultrasound, etc. In some embodiments, the step (3) comprises: mixing the cellulose nanocrystal mixed suspension in step (2) with the prepolymer, standing for at least 12 hours (e.g., 72 hours), initiating polymerization with ultraviolet light (e.g., 365nm ultraviolet light), and obtaining a hydrogel. In some embodiments, the cellulose nanocrystal mixed suspension is mixed with the prepolymer and then standing for at least 12 hours under constant temperature and humidity shading conditions. In some embodiments, the polymerization is initiated by ultraviolet light under nitrogen protection. In some specific embodiments, the step (3) comprises: mixing the cellulose nanocrystal mixed suspension in step (2) with the prepolymer, standing for at least 12 hours (e.g., 72 hours) under constant temperature and humidity shading conditions, initiating polymerization with ultraviolet light (e.g., 365nm ultraviolet light) under nitrogen protection conditions, and obtaining a hydrogel.

[0076] Hydrogel

[0077] Herein, "hydrogel" is made based on monomer polymerization. The term "polymer" refers to a macromolecule comprising connected monomer molecules. The term "homopolymer" is used herein to refer to a polymer derived from a polymer monomer substance. The term "copolymer" refers to a polymer derived from two (or more) monomer polymer substances. The polymerization of monomers can be formed by a variety of conditions, such as by initiators. In some embodiments, the polymer is a hydrophilic polymer.

[0078] In the present invention, the hydrogel comprises cellulose nanocrystals modified with cerium oxide and a matrix, wherein the matrix is ​​a polymer formed by polymerization of monomers. Preferably, the hydrogel further comprises cellulose nanocrystals that are not modified with cerium oxide.

[0079] In the hydrogel, the monomer is usually a high molecular weight monomer. In some embodiments, the monomer is substituted or unsubstituted hydroxyethyl acrylate and / or substituted or unsubstituted methyl acrylate, alkyl-substituted hydroxyethyl acrylate and / or alkyl-substituted methyl acrylate, for example, hydroxyethyl methacrylate and / or methyl methacrylate.

[0080] In some embodiments, the hydrogel comprises one or more features selected from the group consisting of:

[0081] (A) the cerium oxide-modified cellulose nanocrystals are dispersed in the matrix,

[0082] (B) In the cerium oxide-modified cellulose nanocrystals, the mass ratio of cerium to cellulose nanocrystals is 1:30 to 1:50, preferably 1.3:40,

[0083] (C) the cerium oxide is CeOx, x is 1.5-2,

[0084] (D) The hydrogel contains 45 vol% to 90 vol% of cerium oxide-modified cellulose nanocrystals.

[0085] In some embodiments, the hydrogel comprises 45 vol% to 67.5 vol% of cerium oxide-modified cellulose nanocrystals, and the hydrogel comprises 22.5 vol% to 45 vol% of cellulose nanocrystals that are not modified with cerium oxide.

[0086] In some embodiments, the thickness of the hydrogel is 50-200 μm, for example, 100±10 μm.

[0087] The present invention also provides a hydrogel prepared by the preparation method described in any embodiment of the present invention. The hydrogel of the present invention has anti-oxidation, anti-inflammatory and anti-angiogenic functionality, good mechanical properties and high transparency, and has biological activity of promoting rapid repair of corneal epithelial damage.

[0088] In the present invention, the "antioxidant" refers to the ability of the hydrogel to inhibit the generation of reactive oxygen species (ROS) in the body or in cells. Exemplary indicators for evaluating the antioxidation of hydrogels include: the activity of superoxide dismutase (SOD), the activity of catalase (CAT), the activity of reactive oxygen species (ROS) and the activity of malondialdehyde (MDA). Taking SOD activity as an example, the stronger the SOD activity, the stronger the antioxidant capacity of the hydrogel.

[0089] In the present invention, the term "anti-inflammatory" refers to the ability of the hydrogel to inhibit inflammatory responses. The anti-inflammatory ability of the hydrogel can be measured by measuring the degree to which the hydrogel inhibits inflammation-related factors (such as IL-1β, etc.).

[0090] In the present invention, the term "anti-angiogenesis" refers to the ability of the hydrogel to inhibit angiogenesis. The anti-angiogenesis ability of the hydrogel can be measured by measuring the degree to which the hydrogel inhibits TNF-α or angiogenin.

[0091] In the present invention, the hydrogel also has excellent tensile strength (e.g., above 800 KPa), high elongation (e.g., greater than 200%), high light transmittance (e.g., above 80%), and high water content (e.g., above 50%). The hydrogel of the present invention also has biological activity that promotes rapid repair of corneal epithelial damage, for example, it can promote the adhesion and proliferation of corneal epithelial cells.

[0092] The hydrogels of the present invention may be present in a variety of forms, including beads, solid matrices, blocks, sheets, or any other shaped objects.

[0093] Applications of hydrogels

[0094] The hydrogel of the present invention can be used as a delivery carrier to provide a hydrogel containing one or more active ingredients or inactive ingredients to a subject. Therefore, the present invention also provides the use of the hydrogel prepared by the method of the present invention as a delivery carrier in the preparation of a drug. Based on the high biocompatibility of the hydrogel with the subject's tissue, when the hydrogel contacts the subject's tissue (such as skin, cornea, etc.), the active or inactive ingredients therein are provided to the subject. The present invention also provides a composition comprising the hydrogel prepared by the method of the present invention. The composition may also include one or more active ingredients or inactive ingredients, such as pharmaceutical active ingredients. The hydrogel of the present invention may also be used as a drug release platform for controllably and effectively delivering active or inactive ingredients (such as drugs, therapeutic agents or cosmetic agents) to the subject's tissue.

[0095] The hydrogel of the present invention can also be used to provide a suitable environment for the growth, survival and proliferation of living cells (including living cells in vivo and living cells in vitro). Such cells can come from a variety of tissues, such as cornea, lung, liver, kidney, thymus, thyroid, heart, brain, pancreas, etc., as well as cultured cell populations. Non-limiting examples of cells include corneal endothelial cells, fibroblasts, human umbilical vein endothelial cells, chondrocytes and osteoblasts.

[0096] The present invention also provides the use of the hydrogel prepared by the method of the present invention in the preparation of biomaterials. The present invention also provides a biomaterial, which is or contains the hydrogel described in the present invention. The biomaterial can be used as a matrix for cell and tissue generation. The biomaterial can also form a transplant for implantation into a subject (e.g., a mammal). The transplant described in the present invention can be any transplant containing the hydrogel described in the present invention. The transplant containing the hydrogel described in the present invention can be transplanted onto the damaged tissue (e.g., cornea) of the subject to promote cell adhesion and proliferation, thereby producing a therapeutic effect, such as promoting wound closure and promoting the repair of damaged tissue. In the present invention, the transplant can also contain cells, such as corneal epithelial cells. The cells can be contained in and / or on the surface of the hydrogel.

[0097] The present invention also provides the use of the hydrogel prepared by the method of the present invention in the preparation of ophthalmic drugs. The ophthalmic drugs include but are not limited to: ocular implants, eye drops, injections, eye ointments, tablets. In some embodiments, the present invention also provides the use of the hydrogel prepared by the method of the present invention in the preparation of drugs for treating corneal damage. The present invention also provides a method for treating corneal damage, the method comprising: delivering a composition or implant containing the hydrogel of the present invention to the site of corneal damage in a subject.

[0098] The beneficial effects of the present invention include:

[0099] 1. The surface of cellulose nanocrystals is negatively charged, which makes it easy to adsorb cerium ions and hydrolyze in situ to generate cerium oxide nanoparticles under neutral pH conditions. Cellulose nanocrystals modified with cerium oxide nanoparticles exhibit antioxidant, anti-inflammatory and anti-angiogenic functional properties, which are beneficial for inhibiting inflammation and fibrosis at the site of injury.

[0100] 2. Cellulose nanocrystals contain sulfonic acid groups and hydroxyl groups on their surfaces, which are highly dispersible in aqueous solutions. Mixing cellulose nanocrystals modified with cerium oxide nanoparticles with unmodified cellulose nanocrystals enhances the dispersibility of the nanoparticles in the hydrogel matrix, prevents the agglomeration of the nanoparticles, and ensures that the modified cellulose nanocrystals can self-assemble. At the same time, the tensile strength and elongation at break of the hydrogel can be improved by incorporating unmodified rigid rod-shaped cellulose nanocrystals.

[0101] 3. The surface of cellulose nanocrystals is rich in hydroxyl groups, which are easy to form hydrogen bonds with polymers, forming physical crosslinking points in the hydrogel network. Cellulose nanocrystals are mixed with monomers and initiators. Monomers such as hydroxyethyl methacrylate and / or methyl methacrylate provide hydrogels with good mechanical properties and high transparency.

[0102] 4. Cellulose nanocrystals induce self-assembly through evaporation, forming a chiral helical regular structure in the hydrogel, ensuring a high degree of similarity with the microscopic topological configuration of the corneal stroma. The hydrogel prepared by the method of the present invention has excellent light transmittance and has biological activity of promoting the adhesion and proliferation of corneal epithelial cells, and can be used in the treatment of corneal injuries.

[0103] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. The reagents not otherwise specified in the following examples are usually conventional reagents in the art.

[0104] Example 1

[0105] (1) Natural plant raw materials such as cotton, hemp or wood pulp were added to a concentrated sulfuric acid solution diluted 1:1 with deionized water. 20 ml of sulfuric acid solution was added per gram of raw material for acid hydrolysis, heated in a water bath at 65°C for 90 min, and 10 times the volume of deionized water was added to dilute the sulfuric acid solution to terminate the reaction. The mixture was allowed to stand overnight, centrifuged, dialyzed (14000 Da) for 3 days, concentrated, and ultrasonically dispersed to obtain a CNC suspension with a concentration of 4 wt%.

[0106] (2) Take a CNC suspension, heat it to 65°C, add Ce(NO3)3·6H2O (0.004 g / ml), adjust the pH with ammonia water, keep pH=7.5, and control the hydrolysis rate by pH to regulate the in situ growth of nanoparticles. Stir for 2 h in dark, add polyethylene glycol to terminate the reaction, dialyze (14000 Da) for three days to obtain nanoparticles CeOx (x is 1.5-2) modified CNC, and mix the modified CNC with the unmodified CNC suspension in a volume ratio of 3:1 to obtain a CNC mixture.

[0107] (3) uniformly mixing the polymer monomer hydroxyethyl methacrylate and the photoinitiator 2-hydroxy-2-methyl-1-phenylpropanone at a molar ratio of 1:0.01 to obtain a prepolymer;

[0108] (4) The CNC mixture in (2) and the prepolymer in (3) were uniformly mixed at a volume ratio of 9:1, magnetically stirred for 15 minutes, ultrasonically treated for 15 minutes, poured into a culture dish, and allowed to stand at 25°C for 72 hours. Self-assembly was induced by evaporation, and CNC self-assembled into a helical regular structure during this concentration process. Under nitrogen protection, the mixture was irradiated with 365nm ultraviolet light for 30 minutes to obtain a hydrogel with a helical regular microstructure.

[0109] Example 2

[0110] In step (2), the modified CNC and the unmodified CNC suspension are mixed in a volume ratio of 2:1, and the other steps are the same as in Example 1.

[0111] Example 3

[0112] In step (2), the modified CNC and the unmodified CNC suspension are mixed in a volume ratio of 1:1, and the other steps are the same as in Example 1.

[0113] Example 4

[0114] In step (2), Ce(NO3)3·6H2O is replaced by FeCl3·6H2O and FeSO4·7H2O (molar ratio 1:1, concentration of FeCl3·6H2O is 0.00135 g / ml, concentration of FeSO4·7H2O is 0.00139 g / ml), and other steps are the same as in Example 1.

[0115] Example 5

[0116] In step (4), the prepolymer is allowed to stand at 25° C. for 24 h, and the other steps are the same as those in Example 1, to obtain a hydrogel that is not assembled into an ordered structure.

[0117] Example 6

[0118] In step (2), pH=11, and other steps are the same as in Example 1.

[0119] Test Example 1: Tensile strength, light transmittance and moisture content test

[0120] The cellulose nanocrystal-based hydrogels prepared by the methods of Examples 1-5 were subjected to stretching, light transmittance and water content tests.

[0121] Tensile test: A hydrogel sample with a size of 30 mm×10 mm×2 mm was stretched to break at a speed of 2 mm / min using a tensile testing machine at room temperature (humidity of 60%).

[0122] Light transmittance test: The sample was cut into discs with a diameter of 30 mm and measured by UV-visible spectrophotometer.

[0123] Moisture Content Test: The equilibrium water content (EWC) was determined by immersing the freeze-dried samples in PBS at 35°C and calculated using the following formula:

[0124] EWC(%)=(We-Wd) / We×100%,

[0125] Where We is the weight of the hydrogel at equilibrium and Wd is the weight of the dry hydrogel.

[0126] The test results are shown in Table 1.

[0127] Table 1

[0128]

[0129] As can be seen from Table 1, the tensile strength and elongation at break of the hydrogel are related to the content of unmodified CNC. Compared with the hydrogels of Examples 1, 2, 3, 4 and 5, the relative content of CNC increases, the tensile strength of the hydrogel increases, the presence of functionalized modified nanoparticles has little effect on the tensile strength, and the short standing time leads to insufficient physical ordered crosslinking, which reduces the tensile strength. The results show that the addition of cellulose nanocrystals can significantly improve the tensile strength of the hydrogel, but the increase in the physical crosslinking density of the molecular chain makes the molecular chain slip more difficult, and its elongation decreases. At the same time, the water content is generally close to the water content of the human cornea, which meets the requirements of eye applications. The transmittance of the hydrogels of Examples 1, 2, and 3 at 555nm decreases slightly with the increase in the content of modified CNC, and all reach more than 80%, while the transmittance of the hydrogel formed by CNC modified with iron oxide nanoparticles in Example 4 is less than 10%, the transmittance of the hydrogel in Example 5 is less than 10% due to the short standing time, and the transmittance of the hydrogel that failed to successfully form an ordered assembly structure is less than 10%, and the particle size in Example 6 is large, and the transmittance of the hydrogel is less than 30%. The results show that the addition of CNC and fully ordered assembly ensure the light transmittance of the hydrogel, which meets the needs of ocular applications, and the in situ growth of CeOx in functional nanoparticles has little effect on light transmittance.

[0130] Test Example 2: Microscopic observation of hydrogel

[0131] Taking the hydrogel prepared by the method of Example 3 as an example, the hydrogel was freeze-dried and then fixed on a glass slide, and the morphology was photographed by atomic force microscopy and observed with the naked eye.

[0132] The results are as follows Figure 1 As shown, the results show that the hydrogel surface presents a nanofiber network structure.

[0133] Test Example 3: Antioxidant, anti-inflammatory and anti-angiogenic test

[0134] The hydrogels were prepared by the methods of Examples 1-6, respectively, and cut into discs with a diameter of about 15 mm. The hydrogels were then tested for their antioxidant, anti-inflammatory and anti-angiogenic effects.

[0135] Antioxidant test: The active oxygen scavenging performance of the hydrogel was determined by superoxide dismutase (SOD) activity assay kit and catalase (CAT) activity assay kit (Solarbio Technology).

[0136] Anti-inflammatory test: using a multiplex sandwich ELISA quantitative array platform array (RayBiotech) to detect the inflammatory cytokine IL-1β in the supernatant of corneal epithelial cells co-cultured with the materials.

[0137] Anti-angiogenesis test: using a multiplex sandwich ELISA quantitative array platform array (RayBiotech) to detect angiogenic cytokines TNF-α and angiopoietin in the supernatant of corneal epithelial cells co-cultured with the materials.

[0138] The control group was the supernatant of corneal epithelial cells not co-cultured with hydrogel.

[0139] The results are as follows Figure 2 As shown, compared with the control group, the hydrogels prepared by the methods of Examples 1-3 exhibited significant antioxidant, anti-inflammatory and anti-angiogenic properties.

[0140] Test Example 4: Corneal biocompatibility test

[0141] The polymer monomer hydroxyethyl methacrylate and the photoinitiator 2-hydroxy-2-methyl-1-phenylpropanone were uniformly mixed at a molar ratio of 1:0.01 to obtain a prepolymer, which was irradiated with 365nm ultraviolet light for 30 minutes under nitrogen protection to obtain PHEMA as a control group.

[0142] Human corneal epithelial cells (HCECs) (obtained from primary culture) were cultured on the hydrogels prepared by the methods of Examples 1-6, respectively, with PHEMA as a control. The hydrogels were placed in a 24-well plate, the cells were digested and centrifuged, and then the cells were centrifuged at 5*10 4 / cm 2 The cells were seeded at a density of 1.54 μg / mL on the surface of the hydrogel and cultured in a 37°C incubator overnight. The medium was changed after 24 hours. After 48 hours of culture, the cells were fluorescently stained with DAPI and phalloidin. DAPI marked the cell nucleus and phalloidin marked the cytoskeleton.

[0143] The results are as follows Figure 3As shown, the density of HCECs grown on pure PHEMA is low, and the cell morphology is wrinkled, indicating that the cells on the surface of the material failed to successfully adhere and proliferate, while the density of HCECs on the hydrogels prepared by the methods of Examples 1-3 and 6 is significantly higher, and most of the cells extend pseudopodia, showing migration and proliferation capabilities. The density of HCECs on the hydrogels prepared by the methods of Examples 4 and 5 is low, which is not conducive to the adhesion and proliferation of HCECs.

[0144] Comparing PHEMA with the hydrogel prepared by the method of Examples 1-3, human corneal epithelial cells cultured on the hydrogel showed excellent adhesion and proliferation ability, indicating that the introduction of helically assembled CNC significantly improved the biocompatibility of the material in the eye.

[0145] In summary, the hydrogel prepared by the methods of Examples 1-3 of the present invention has antioxidant, anti-inflammatory and anti-angiogenic functionalities, biological activity of promoting rapid repair of corneal epithelial damage, and has good mechanical properties and high transparency. The preparation process is safe, simple and low-cost, and can be used as a therapeutic corneal contact lens to promote rapid repair of corneal damage.

[0146] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims. At the same time, all the documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference separately.

Claims

1. A method for preparing a hydrogel, characterized in that: The method comprises: (1) mixing a cellulose nanocrystal suspension with cerium ions to obtain a cerium oxide-modified cellulose nanocrystal suspension under neutral pH conditions; (2) mixing the cellulose nanocrystal suspension modified in step (1) with the cellulose nanocrystal suspension not modified in step (1) to obtain a cellulose nanocrystal mixed suspension; (3) mixing the mixed suspension of cellulose nanocrystals in step (2) with a monomer, and obtaining a hydrogel after polymerization of the monomer; preferably, mixing the mixed suspension of cellulose nanocrystals in step (2) with a prepolymer containing a monomer and an initiator, and obtaining a hydrogel after initiation with the initiator.

2. The preparation method according to claim 1, characterized in that: The cellulose nanocrystals are obtained by acid hydrolysis of plant raw materials. Preferably, the acid comprises an inorganic acid or an organic acid, more preferably the acid is a strong acid, more preferably the acid is sulfuric acid or hydrochloric acid, more preferably the concentration of the acid is 40-60%; and / or, Preferably, the plant raw materials include: cotton, hemp or wood pulp; More preferably, the ratio of the plant material to the acid is: 1 g of plant material: (1-100 ml) of acid solution, preferably 1 g of plant material: (1-20 ml) of acid solution; More preferably, the concentration of the cellulose nanocrystal suspension is 1-10 wt%, preferably 2-6 wt%.

3. The preparation method according to claim 2, characterized in that: In step (1), the cerium ion refers to Ce 3+ , preferably derived from Ce(NO3)3·6H2O; and / or, The neutral pH refers to a pH value of 6.8 to 8, preferably a pH value of 7 to 8.

4. The preparation method according to claim 2 or 3, characterized in that: In step (2), the volume ratio of the modified cellulose nanocrystal suspension to the unmodified cellulose nanocrystal suspension is 3:1 to 1:3, preferably 3:1 to 1:1 or 2:1 to 1:

1.

5. The preparation method according to claim 2, characterized in that: In step (3), the prepolymer comprises: a monomer and an initiator, Preferably, the monomer is substituted or unsubstituted hydroxyethyl acrylate and / or substituted or unsubstituted methyl acrylate, more preferably, the monomer is alkyl-substituted hydroxyethyl acrylate and / or alkyl-substituted methyl acrylate, further preferably hydroxyethyl methacrylate and / or methyl methacrylate; and / or, Preferably, the initiator is an ultraviolet light initiator, more preferably one or more of 2-hydroxy-2-methyl-1-phenylpropanone, 1-hydroxycyclohexylphenyl ketone, 2-methyl-2-(4-morpholinyl)-1-[4-(methylthio)phenyl]-1-propanone, and 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone; More preferably, the molar ratio of the monomer to the initiator is 1:(0.01-0.02).

6. The preparation method according to claim 2, characterized in that: In step (3), the volume ratio of the cellulose nanocrystal mixed suspension to the prepolymer is 5:5 to 9:1, preferably 6:4 to 9:1, 7:3 to 9:1 or 8:2 to 9:1; and / or, The step (3) further comprises: mixing the cellulose nanocrystal mixed suspension in step (2) with the prepolymer, standing for at least 12 hours, and initiating polymerization with ultraviolet light to obtain a hydrogel; more preferably, the ultraviolet light is 365nm ultraviolet light.

7. A hydrogel comprising cerium oxide-modified cellulose nanocrystals and a matrix, wherein the matrix is ​​a polymer formed by polymerization of monomers, Preferably, the hydrogel further comprises cellulose nanocrystals which are not modified with cerium oxide.

8. The hydrogel according to claim 7, characterized in that The polymer is a hydrophilic polymer, Preferably, the monomer is substituted or unsubstituted hydroxyethyl acrylate and / or substituted or unsubstituted methyl acrylate, More preferably, the monomer is alkyl-substituted hydroxyethyl acrylate and / or alkyl-substituted methyl acrylate, more preferably hydroxyethyl methacrylate and / or methyl methacrylate.

9. The hydrogel according to claim 7 or 8, characterized in that The hydrogel comprises one or more characteristics selected from the following: (A) the cerium oxide-modified cellulose nanocrystals are dispersed in the matrix, (B) In the cerium oxide-modified cellulose nanocrystals, the mass ratio of cerium to cellulose nanocrystals is 1:30 to 1:50, preferably 1.3:40, (C) the cerium oxide is CeOx, x is 1.5-2, (D) the hydrogel comprises 45% to 90% by volume of cerium oxide-modified cellulose nanocrystals, Preferably, The hydrogel contains 45 volume % to 67.5 volume % of cerium oxide-modified cellulose nanocrystals, The hydrogel comprises 22.5 volume % to 45 volume % of cellulose nanocrystals that are not modified with cerium oxide, and / or The thickness of the hydrogel is 50 to 200 μm.

10. The hydrogel according to claim 7 or 8, characterized in that The hydrogel is prepared by the preparation method according to any one of claims 1 to 6; Preferably, the hydrogel also has one or more of the following properties: (1) Tensile strength at break 800KPa or above, (2) The elongation is greater than 200%, (3) Light transmittance is more than 80%, (4) Moisture content is more than 50%, (5) Anti-oxidation (6) Anti-inflammatory (7) It can resist angiogenesis, (8) It has the biological activity of promoting the rapid repair of corneal epithelial damage, preferably promoting the adhesion and proliferation of corneal epithelial cells.

11. A composition, characterized in that The composition comprises the hydrogel according to any one of claims 7 to 10, or comprises the hydrogel prepared by the preparation method according to any one of claims 1 to 6; Preferably, the composition further comprises an active ingredient or an inactive ingredient, more preferably the active ingredient or the inactive ingredient is within and / or on the surface of the hydrogel.

12. A biomaterial, a graft or a tissue engineering matrix for growing cells and tissues in vivo or in vitro, characterized in that: The biomaterial comprises the hydrogel according to any one of claims 7 to 10, or comprises the hydrogel prepared by the preparation method according to any one of claims 1 to 6, The transplant or tissue engineering matrix for growing cells and tissues in vivo or in vitro comprises the hydrogel according to any one of claims 7 to 10, or comprises the hydrogel obtained by the preparation method according to any one of claims 1 to 6, or is formed by the hydrogel according to any one of claims 7 to 10, or is formed by the hydrogel obtained by the preparation method according to any one of claims 1 to 6; Preferably, the biomaterial, transplant or tissue engineering matrix for growing cells and tissues in vivo or in vitro further comprises cells or tissues, more preferably corneal epithelial cells or corneal tissues, more preferably the corneal epithelial cells or corneal tissues are in and / or on the surface of the hydrogel.

13. Select from the following applications: (1) Use of the hydrogel according to any one of claims 7 to 10, or the hydrogel prepared by the preparation method according to any one of claims 1 to 6, as a delivery carrier in the preparation of a drug; (2) Use of the hydrogel according to any one of claims 7 to 10, or the hydrogel prepared by the preparation method according to any one of claims 1 to 6, in the preparation of biomaterials, transplants, or tissue engineering matrices for growing cells and tissues in vivo or in vitro; (3) Use of the hydrogel according to any one of claims 7 to 10, or the hydrogel prepared by the preparation method according to any one of claims 1 to 6, in the preparation of ophthalmic drugs, preferably the ophthalmic drugs include eye implants, eye drops, injections, eye ointments, and tablets; (4) Use of the hydrogel according to any one of claims 7 to 10, or the hydrogel prepared by the preparation method according to any one of claims 1 to 6, in the preparation of a drug for treating corneal damage.

Images