Temperature-sensitive hydrogel as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicine, and provides temperature-sensitive hydrogel as well as a preparation method and application thereof. The temperature-sensitive hydrogel comprises a macromolecular matrix formed by acrylic monomers, polysaccharide macromolecules, collagen and water. The hydrogel disclosed by the invention is sensitive to temperature change, can keep a solution state in a low-temperature state, can quickly form a solid state at a relatively high temperature (30-50 DEG C), and has relatively high mechanical strength. In addition, the hydrogel has excellent biocompatibility, no toxicity and biodegradability, can be used for in-vitro cell culture, and can also be used as a drug carrier. In addition, the hydrogel has high moisture retention and light transmittance, and can be used as a biomedical material.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular, to a thermosensitive hydrogel and a preparation method and application thereof, and more particularly, to a thermosensitive collagen-hydroxypropylcellulose-polyacrylamide hydrogel and a preparation method and application thereof. Background Art

[0002] Thermosensitive hydrogel materials are a type of intelligent materials that are sensitive to temperature changes. They can undergo phase transitions with changes in the use conditions or the temperature of the use site, and form non-chemically cross-linked hydrogels from aqueous solutions. Their formation is often caused by changes in hydrogen bonds or hydrophobic interactions after temperature changes, which ultimately leads to changes in the physical state of the aqueous solution, that is, when the ambient temperature is lower than the lower critical solution temperature (LCST), it is in a liquid state, and when it is higher than the LCST, it is in a semi-solid hydrogel state. After in situ injection into the human body, the material can fill the tissue gap, rapidly undergo phase transitions in the body, and form a semi-solid hydrogel at the injection site. When the material is loaded with drugs (or active molecules), local administration can be achieved and the retention time of the drug at the injection site can be prolonged, achieving the purpose of controlled release to improve the efficacy of the drug.

[0003] In the prior art, thermosensitive hydrogel materials have problems such as poor controllability of gelation temperature, low mechanical strength, rapid dissolution of the formed hydrogel, short hydrogel state stability time, and non-biodegradability in the body, which have limited the application of thermosensitive hydrogel materials to a certain extent. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art to at least a certain extent.

[0005] In a first aspect of the present invention, the present invention provides a thermosensitive hydrogel. According to an embodiment of the present invention, the thermosensitive hydrogel comprises: a polymer matrix formed by acrylic monomers, a polysaccharide polymer, collagen and water.

[0006] The inventor unexpectedly discovered during the experiment that the hydrogel of the present invention is sensitive to temperature changes, maintains a solution state below 6°C, and can quickly form a semi-solid hydrogel state at a higher temperature (30°C-50°C), with strong mechanical strength. In addition, the hydrogel has excellent biocompatibility, non-toxicity and biodegradability, and can not only be used for in vitro cell culture, but also can be used as a drug carrier to encapsulate the drug therein, and by controlling the rate and duration of drug release, the sustained release and targeted drug delivery of the drug are achieved. In addition, the hydrogel has a high light transmittance and moisture retention. When used as a dressing, it not only helps to reduce the inflammatory response and infection risk during wound healing, but also can intuitively observe the degree of wound healing, meeting clinical application needs.

[0007] According to an embodiment of the present invention, the temperature-sensitive hydrogel may further include at least one of the following additional technical features:

[0008] According to an embodiment of the present invention, the acrylic monomer forms a polymer matrix under the action of a crosslinking agent and an initiator.

[0009] According to an embodiment of the present invention, the acrylic monomer includes an acrylamide monomer.

[0010] According to an embodiment of the present invention, the cross-linking agent includes N,N'-methylenebisacrylamide.

[0011] According to an embodiment of the present invention, the initiator includes at least one of potassium persulfate, ammonium persulfate, diethylenetriamine and trimethylolpropane.

[0012] According to an embodiment of the present invention, the polysaccharide polymer includes at least one of cellulose, alginic acid (ALG), hyaluronic acid (HA), xanthan gum and chitosan (CS).

[0013] According to an embodiment of the present invention, the collagen includes at least one of fish collagen, porcine collagen, bovine collagen and plant-derived collagen.

[0014] In the second aspect of the present invention, the present invention provides a method for preparing the thermosensitive hydrogel described in the first aspect. According to an embodiment of the present invention, the method comprises: mixing an acrylic monomer, a crosslinking agent, an initiator, a polysaccharide polymer, collagen and water to obtain a mixed solution; and reacting the mixed solution under a protective atmosphere to obtain the thermosensitive hydrogel.

[0015] Therefore, different functional group components can be introduced through the method of the present invention, so that the components in the composite hydrogel form cross-links, rapid temperature-sensitive gelation can be achieved, and it has high moisture retention and light transmittance, providing a new material for subsequent dressings; in addition, the hydrogel prepared by the method is non-toxic and can be used for cell proliferation and 3D cell culture; in addition, the hydrogel also has excellent biocompatibility and biodegradability, can be used as a drug carrier, injected into the body, form a semi-solid gel state in the body, and encapsulate the drug therein, and by controlling the rate and duration of drug release, the sustained release and targeted drug delivery of the drug can be achieved, the therapeutic effect can be improved, and the frequency of drug delivery can be reduced.

[0016] According to an embodiment of the present invention, the method for preparing a thermosensitive hydrogel may further include at least one of the following additional technical features:

[0017] According to an embodiment of the present invention, the mixing treatment includes: performing a first mixing treatment on the polysaccharide polymer and water to obtain a polysaccharide polymer solution; performing a second mixing treatment on the initiator and water to obtain an initiator solution; performing a third mixing treatment on the acrylic monomer and water to obtain an acrylic solution; performing a fourth mixing treatment on the acrylic solution, the polysaccharide polymer solution, the collagen, the cross-linking agent and the initiator solution to obtain the mixed solution.

[0018] According to an embodiment of the present invention, the concentration of the polysaccharide polymer in the mixed solution is 1 mg / ml-10 mg / ml.

[0019] According to an embodiment of the present invention, the concentration of the initiator solution in the mixed solution is 10 mg / ml-20 mg / ml.

[0020] According to an embodiment of the present invention, the concentration of the acrylic acid solution in the mixed solution is 10 mg / ml-50 mg / ml.

[0021] According to an embodiment of the present invention, the concentration of the collagen in the mixed solution is 1 mg / ml-10 mg / ml.

[0022] According to an embodiment of the present invention, the concentration of the cross-linking agent in the mixed solution is 5 mg / ml-20 mg / ml.

[0023] According to an embodiment of the present invention, the reaction treatment further comprises: subjecting the reaction treatment product to gelation treatment to obtain the thermosensitive hydrogel.

[0024] According to an embodiment of the present invention, the reaction treatment time is 1h-3h.

[0025] According to an embodiment of the present invention, the protective atmosphere is nitrogen.

[0026] According to an embodiment of the present invention, the temperature of the gelling treatment is 30°C-50°C.

[0027] According to an embodiment of the present invention, the gelling treatment time is 1 min-10 min.

[0028] In the third aspect of the present invention, the present invention proposes the use of the thermosensitive hydrogel described in the first aspect or the thermosensitive hydrogel prepared by the method described in the second aspect as a biomedical material, a drug carrier and in vitro cell culture. As mentioned above, the thermosensitive hydrogel of the present invention has non-toxicity, injectability, high moisture retention and high light transmittance, as well as good biocompatibility and biodegradability. Therefore, it can be used as a new material for dressings, for biomedical materials, as a drug carrier and for cell culture.

[0029] In the fourth aspect of the present invention, the present invention proposes a method for in vitro three-dimensional cell culture. According to an embodiment of the present invention, the method comprises: incubating the cells with the thermosensitive hydrogel described in the first aspect or the thermosensitive hydrogel prepared by the method described in the second aspect. As mentioned above, the thermosensitive hydrogel of the present invention has non-toxicity, injectability, high moisture retention and high light transmittance, as well as good biocompatibility and biodegradability. Therefore, the hydrogel of the present invention can be used to culture 3D cells.

[0030] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0032] Figure 1 A schematic diagram of the synthesis route for preparing a thermosensitive collagen-hydroxypropylcellulose-polyacrylamide hydrogel according to an embodiment of the present invention;

[0033] Figure 2 This is a SEM image of the thermosensitive collagen-hydroxypropylcellulose-polyacrylamide hydrogel according to an embodiment of the present invention;

[0034] Figure 3 This is a schematic diagram of the gelling results of the thermosensitive collagen-hydroxypropylcellulose-polyacrylamide hydrogel according to an embodiment of the present invention;

[0035] Figure 4 This is a schematic diagram of the temperature sensitivity and injectability of the thermosensitive collagen-hydroxypropylcellulose-polyacrylamide hydrogel according to an embodiment of the present invention;

[0036] Figure 5 This is a schematic diagram of the degradation test results of the thermosensitive collagen-hydroxypropylcellulose-polyacrylamide hydrogel according to an embodiment of the present invention;

[0037] Figure 6 This is a schematic diagram of the test results of the moisture retention of the thermosensitive collagen-hydroxypropylcellulose-polyacrylamide hydrogel according to an embodiment of the present invention;

[0038] Figure 7 This is a schematic diagram of the light transmittance test results of the thermosensitive collagen-hydroxypropylcellulose-polyacrylamide hydrogel according to an embodiment of the present invention;

[0039] Figure 8 This is a schematic diagram of the cytotoxicity results of the thermosensitive collagen-hydroxypropylcellulose-polyacrylamide hydrogel according to an embodiment of the present invention;

[0040] Fig. 9 This is a schematic diagram of the cell proliferation results of the thermosensitive collagen-hydroxypropylcellulose-polyacrylamide hydrogel according to an embodiment of the present invention. DETAILED DESCRIPTION

[0041] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present invention. The appearance of the phrase in various places in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0042] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0043] For simplicity, only some numerical ranges are specifically disclosed herein. However, any lower limit can be combined with any upper limit to form an unspecified range; and any lower limit can be combined with other lower limits to form an unspecified range, and any upper limit can be combined with any other upper limit to form an unspecified range. In addition, each separately disclosed point or single value can itself be combined as a lower limit or upper limit with any other point or single value or with other lower limits or upper limits to form an unspecified range.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by technicians in the technical field to which the present invention belongs; the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention; the terms "including" and "having" in the specification and claims of the present invention and the above-mentioned figure descriptions and any variations thereof are intended to cover non-exclusive inclusions.

[0045] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

[0046] If not otherwise specified, all embodiments and optional embodiments of the present invention can be combined with each other to form a new technical solution.

[0047] Bio-based hydrogels are a type of biomaterial that has attracted much attention in recent years. Compared with traditional synthetic hydrogels, bio-based hydrogels have many advantages, the most prominent of which is their environmental sustainability. Since their main raw materials are natural or renewable biomass, a large amount of waste and harmful substances will not be generated during the production process, thereby reducing pollution to the environment. In addition, bio-based hydrogels have good biocompatibility and can be used in organisms or in environments that contact organisms, such as for drug controlled release, tissue engineering, etc. Bio-based hydrogels also have the characteristics of good controllability and high efficiency. By adjusting the composition structure of the hydrogel, its mechanical properties, microstructure and swelling properties can be controlled, making it suitable for different applications. However, there are still some problems with bio-based smart hydrogels. First, the preparation method is relatively limited. The preparation of some biomass materials is difficult, which limits their use in hydrogel synthesis. Secondly, the performance stability of bio-based smart hydrogels needs to be further improved, especially their stability in different environments and applications. Furthermore, the preparation cost of bio-based smart hydrogels is relatively high, which is also one of the main reasons limiting their large-scale synthesis and use.

[0048] Thus, the present invention prepares a thermosensitive collagen-hydroxypropylcellulose-polyacrylamide hydrogel, which is in a solution state at low temperatures and can be transformed into a semi-solid gel state as the temperature rises. The hydrogel has good stability, high moisture retention and light transmittance, as well as excellent biocompatibility and biodegradability, and can be used in the field of biomedicine. The hydrogel, its preparation method and application will be introduced in detail below.

[0049] Thermosensitive hydrogel

[0050] In a first aspect of the present invention, the present invention provides a thermosensitive hydrogel. According to an embodiment of the present invention, the thermosensitive hydrogel comprises: a polymer matrix formed by acrylic monomers, a polysaccharide polymer, collagen and water.

[0051] The inventor unexpectedly discovered during the experiment that the hydrogel of the present invention is sensitive to temperature changes, maintains a solution state below 6°C, and can quickly form a semi-solid hydrogel state at a higher temperature (30°C-50°C), with strong mechanical strength. In addition, the hydrogel has excellent biocompatibility, non-toxicity and biodegradability, and can not only be used for in vitro cell culture, but also can be used as a drug carrier to encapsulate the drug therein, and by controlling the rate and duration of drug release, the sustained release and targeted drug delivery of the drug are achieved. In addition, the hydrogel has a high light transmittance and moisture retention. When used as a dressing, it not only helps to reduce the inflammatory response and infection risk during wound healing, but also can intuitively observe the degree of wound healing, meeting clinical application needs.

[0052] In some embodiments of the present invention, acrylic monomers form a polymer matrix under the action of a crosslinking agent and an initiator. The initiator can initiate a polymerization reaction of acrylic monomers; the crosslinking agent contains two or more reactive double bonds, which can react with growing polymer chains to connect different polymer chains to form a three-dimensional network structure of a hydrogel.

[0053] In some embodiments of the present invention, acrylic monomers refer to monomers having an acryloyl group (H2C=CH-C(=O)-) or a methacryloyl group (H2C=C(CH3)-C(=O)-) that can form a polymer (polymer) by polymerization. Acrylic monomers are monofunctional monomers having one polymerizable carbon-carbon double bond in the molecule and are non-crosslinking monomers.

[0054] The acrylic monomer is not particularly limited, but water-soluble monomers such as acrylamide monomers and acrylic esters are preferred, and acrylamide monomers are more preferred.

[0055] In some embodiments of the present invention, acrylamide monomers include, but are not limited to: N,N-dialkyl acrylamides such as acrylamide, N,N-dimethyl acrylamide, and N,N-diethyl acrylamide; N-alkyl acrylamides such as N-isopropyl acrylamide, N-methyl acrylamide, N-ethyl acrylamide, and N-propyl acrylamide; N-hydroxyalkyl acrylamides such as N-hydroxyethyl acrylamide and N-hydroxymethyl acrylamide; N-ethoxymethyl acrylamide, N-propoxymethyl acrylamide, n-butoxymethyl acrylamide, N-isobutoxymethyl acrylamide, N-pentyloxymethyl acrylamide, N-hexyloxymethyl acrylamide, N-heptyloxymethyl acrylamide, N-octyloxymethyl acrylamide, N-ethoxyethyl acrylamide, N-propoxyethyl acrylamide, and n-butoxyethyl acrylamide; cationic acrylamide compounds containing amino groups such as dimethylaminopropyl acrylamide; anionic acrylic monomers or their salts containing sulfonic acid groups such as 4-acryloylmorpholine and tert-butyl acrylamide sulfonic acid; and their derivatives, etc. Among them, at least one selected from acrylamide, N,N-dimethylacrylamide, N,N-diethylacrylamide, N-isopropylacrylamide, N-methylacrylamide, N-ethylacrylamide, N-propylacrylamide, N-hydroxyethylacrylamide, N-hydroxymethylacrylamide, dimethylaminopropylacrylamide, 4-acryloylmorpholine, tert-butylacrylamidesulfonic acid and salts thereof is preferred.

[0056] In some embodiments of the present invention, acrylates include, but are not limited to: alkyl acrylates having an alkyl carbon number of 1 to 18, such as methyl acrylate, ethyl acrylate, n-propyl acrylate, isopropyl acrylate, n-butyl acrylate, isobutyl acrylate, tert-butyl acrylate, n-hexyl acrylate, n-octyl acrylate, isooctyl acrylate, 2-ethylhexyl acrylate, n-nonyl acrylate, isononyl acrylate, n-pentyl acrylate, n-decyl acrylate, isodecyl acrylate, n-lauryl acrylate, tridecyl acrylate, n-stearyl acrylate, and the like; alkyl acrylates such as cyclohexyl acrylate, isobornyl acrylate, 1-adamantyl acrylate, and the like. Alicyclic acrylates; alkoxy-containing acrylates such as methoxypolyethylene glycol acrylate, methoxyethyl acrylate, methoxyethoxyethyl acrylate, methoxytriethylene glycol acrylate, etc.; hydroxyalkyl acrylates such as 2-hydroxyethyl acrylate, 2-hydroxypropyl acrylate, 2-hydroxy-3-phenoxypropyl acrylate, 2-hydroxybutyl acrylate, etc. (the hydroxyalkyl group is optionally bonded to an aromatic group via an ether bond); glyceryl monoacrylate; monoacrylate polyalkylene glycols such as monoacrylate polyethylene glycol and polyethylene glycol-polypropylene glycol copolymers; acrylates having an aromatic ring such as benzyl acrylate; and acrylates having a heterocyclic ring such as tetrahydrofurfuryl acrylate.

[0057] In some embodiments of the present invention, the acrylic monomer is acrylamide.

[0058] In some embodiments of the present invention, the crosslinking agent preferably uses a monomer having more than two polymerizable carbon-carbon double bonds in the molecule. Specifically, N, N'-methylenebisacrylamide, methylenebisacrylamide, ethylenebisacrylamide, (poly)ethylene glycol diacrylate, (poly)propylene glycol diacrylate, glycerol diacrylate, glycerol triacrylate and other multifunctional acrylamide or multifunctional acrylate, tetraallyloxyethane, diallyl ammonium chloride and the like can be cited. It should be noted that as the above-mentioned crosslinking agent having more than two polymerizable double bonds in the molecule. According to an embodiment of the present invention, the crosslinking agent is N, N'-methylenebisacrylamide. Thus, the above-mentioned types of crosslinking agents can accurately connect different polymer chains. This crosslinking method can effectively control the crosslinking density, and then control the physical properties of the generated hydrogel such as elasticity, hardness, and swelling performance.

[0059] The initiator used in the present invention is a conventional auxiliary agent conventionally used in the art for cross-linking acrylamide to obtain polyacrylamide hydrogel. In some embodiments of the present invention, the initiator includes but is not limited to at least one of potassium persulfate, ammonium persulfate, diethylenetriamine, and trimethylolpropane. Thus, the above-mentioned types of initiators can initiate polymerization of acrylic monomers.

[0060] In some embodiments of the present invention, the polysaccharide polymer includes but is not limited to at least one of cellulose, alginic acid (ALG), hyaluronic acid (HA), xanthan gum and chitosan (CS). Thus, the above-mentioned polysaccharide polymers can improve the overall properties of the hydrogel, such as biocompatibility, stability, mechanical properties, etc.

[0061] In some embodiments of the present invention, the cellulose includes but is not limited to hydroxypropyl cellulose (HPMC), hydroxymethyl cellulose (CMC), hydroxyethyl cellulose (HEC), and the like.

[0062] In some embodiments of the present invention, the polysaccharide polymer is hydroxypropyl cellulose, and the molecular weight of the hydroxypropyl cellulose is 100,000-400,000. For example, the molecular weight can be 100,000, 150,000, 200,000, 250,000, 300,000, 350,000, 300,000, etc., or can be a range consisting of any of the above values.

[0063] In some embodiments of the present invention, collagen includes but is not limited to at least one of fish collagen, porcine collagen, bovine collagen, and plant-derived collagen. Thus, the above-mentioned types of collagen can improve the biocompatibility of the hydrogel, reduce immune response and inflammation, and promote wound healing and the formation of new tissues.

[0064] Method for preparing thermosensitive hydrogel

[0065] In the second aspect of the present invention, the present invention provides a method for preparing the thermosensitive hydrogel described in the first aspect. According to an embodiment of the present invention, the method comprises: mixing an acrylic monomer, a crosslinking agent, an initiator, a polysaccharide polymer, collagen and water to obtain a mixed solution; and reacting the mixed solution under a protective atmosphere to obtain the thermosensitive hydrogel.

[0066] Therefore, the method of the present invention is simple to operate, low in cost, convenient for mass preparation, and can be used for industrial production. Through this method, different functional group components can be introduced to form cross-links in the components of the composite hydrogel, which can achieve rapid temperature-sensitive gelation, and has high moisture retention and light transmittance, providing a new material for subsequent dressings; in addition, the hydrogel prepared by this method is non-toxic and can be used for cell proliferation and 3D cell culture; in addition, the hydrogel also has excellent biocompatibility and biodegradability, can be used as a drug carrier, injected into the body, form a gel state in the body, and encapsulate the drug therein, and by controlling the rate and duration of drug release, the sustained release and targeted drug delivery of the drug can be achieved, the therapeutic effect can be improved, and the frequency of drug administration can be reduced.

[0067] In some embodiments of the present invention, the mixing process includes: performing a first mixing process on a polysaccharide polymer and water to obtain a polysaccharide polymer solution; performing a second mixing process on an initiator and water to obtain an initiator solution; performing a third mixing process on an acrylic monomer and water to obtain an acrylic solution; and performing a fourth mixing process on the acrylic solution, the polysaccharide polymer solution, collagen, a crosslinking agent, and the initiator solution to obtain the mixed solution. By separately configuring a polysaccharide polymer solution, an initiator solution, and an acrylic solution, and then mixing them with other components, it is possible to ensure that each component is fully dissolved in water to form a uniform solution, ensure uniform distribution of each component in the final hydrogel, avoid premature polymerization or decomposition, and ensure the quality and performance of the hydrogel.

[0068] In some embodiments of the present invention, the concentration of the polysaccharide polymer in the mixed solution is 1 mg / ml-10 mg / ml. For example, it can be 1 mg / ml, 2 mg / ml, 5 mg / ml, 8 mg / ml, 10 mg / ml, etc., or it can be a range composed of any of the above values. In this way, the prepared hydrogel can have temperature sensitivity, high moisture retention and light transmittance, as well as good biocompatibility and biodegradability.

[0069] In some embodiments of the present invention, the concentration of the initiator solution in the mixed solution is 10 mg / ml-20 mg / ml. For example, it can be 10 mg / ml, 12 mg / ml, 15 mg / ml, 18 mg / ml, 20 mg / ml, etc., or it can be a range composed of any of the above values. In this way, the prepared hydrogel can have temperature sensitivity, high moisture retention and light transmittance, as well as good biocompatibility and biodegradability.

[0070] In some embodiments of the present invention, the concentration of the acrylic acid solution in the mixed solution is 10 mg / ml-50 mg / ml. For example, it can be 10 mg / ml, 20 mg / ml, 30 mg / ml, 40 mg / ml, 50 mg / ml, etc., or can be a range of any of the above values. Thus, the prepared hydrogel can have temperature sensitivity, high moisture retention and light transmittance, as well as good biocompatibility and biodegradability.

[0071] In some embodiments of the present invention, the concentration of the collagen in the mixed solution is 1 mg / ml-10 mg / ml. For example, it can be 1 mg / ml, 3 mg / ml, 5 mg / ml, 8 mg / ml, 10 mg / ml, etc., or it can be a range composed of any of the above values. In this way, the prepared hydrogel can have temperature sensitivity, high moisture retention and light transmittance, as well as good biocompatibility and biodegradability.

[0072] In some embodiments of the present invention, the concentration of the cross-linking agent in the mixed solution is 5 mg / ml-20 mg / ml. For example, it can be 5 mg / ml, 10 mg / ml, 15 mg / ml, 20 mg / ml, etc., or it can be a range composed of any of the above values. In this way, the prepared hydrogel can have temperature sensitivity, high moisture retention and light transmittance, as well as good biocompatibility and biodegradability.

[0073] In some embodiments of the present invention, the reaction treatment time is 1-3 hours, for example, 1 hour, 2 hours, 3 hours, etc., or any range of the above values. In this way, the acrylic monomer and the crosslinking agent can fully form a polymer matrix under the action of the initiator.

[0074] In some embodiments of the present invention, the protective atmosphere is nitrogen, thereby providing an oxygen-free environment to prevent unnecessary side reactions between oxygen and reactants or intermediates, thereby protecting the reactants from oxidation.

[0075] In some embodiments of the present invention, the first mixing treatment is carried out under the condition of stirring in the dark. According to an embodiment of the present invention, the time of the first mixing treatment is 6h-10h, for example, it can be 6h, 7h, 8h, 9h, 10h, etc., or it can be a range composed of any of the above values. Thus, the polysaccharide polymer can be fully dissolved into a transparent viscous solution.

[0076] In some embodiments of the present invention, the second mixing treatment is carried out under the action of ultrasound. According to an embodiment of the present invention, the temperature of ultrasound is 30°C-50°C, for example, it can be 30°C, 32°C, 35°C, 38°C, 40°C, 42°C, 45°C, 48°C, 50°C, etc., or it can be a range composed of any of the above numerical values. According to an embodiment of the present invention, the time of ultrasound is 10min-30min, for example, it can be 10min, 12min, 15min, 18min, 20min, 22min, 25min, 28min, 30min, etc., or it can be a range composed of any of the above numerical values. Thus, a uniform initiator solution can be prepared.

[0077] In some embodiments of the present invention, the acrylic monomer includes an acrylamide monomer. According to an embodiment of the present invention, the acrylamide monomers include but are not limited to: acrylamide; N,N-dimethylacrylamide, N,N-diethylacrylamide and other N,N-dialkylacrylamide; N-isopropylacrylamide, N-methylacrylamide, N-ethylacrylamide, N-propylacrylamide and other N-alkylacrylamide; N-hydroxyethylacrylamide, N-hydroxymethylacrylamide and other N-hydroxyalkylacrylamide; N-ethoxymethylacrylamide, N-propoxymethylacrylamide, n-butoxymethylacrylamide, N-isobutoxymethylacrylamide, N-pentyloxymethylacrylamide, N-hexylmethylacrylamide, N-heptyloxymethylacrylamide, N-octyloxymethylacrylamide, N-ethoxyethylacrylamide, N-propoxyethylacrylamide, n-butoxyethylacrylamide and other N-alkoxyalkylacrylamide; cationic acrylamide compounds containing amino groups such as dimethylaminopropylacrylamide; anionic acrylic monomers or their salts containing sulfonic acid groups such as 4-acryloylmorpholine and tert-butylacrylamidesulfonic acid; and their derivatives, etc. Among them, at least one selected from acrylamide, N,N-dimethylacrylamide, N,N-diethylacrylamide, N-isopropylacrylamide, N-methylacrylamide, N-ethylacrylamide, N-propylacrylamide, N-hydroxyethylacrylamide, N-hydroxymethylacrylamide, dimethylaminopropylacrylamide, 4-acryloylmorpholine, tert-butylacrylamidesulfonic acid and salts thereof is preferred.

[0078] In some embodiments of the present invention, the crosslinking agent includes at least one of N,N'-methylenebisacrylamide, methylenebisacrylamide, ethylenebisacrylamide, (poly)ethylene glycol diacrylate, (poly)propylene glycol diacrylate, glycerol diacrylate, glycerol triacrylate and other multifunctional acrylamides or multifunctional acrylates, tetraallyloxyethane, diallyl ammonium chloride and the like.

[0079] In some embodiments of the present invention, the initiator includes but is not limited to at least one of potassium persulfate, ammonium persulfate, diethylenetriamine, and trimethylolpropane.

[0080] In some embodiments of the present invention, the polysaccharide polymer includes but is not limited to at least one of cellulose, alginic acid (ALG), hyaluronic acid (HA), xanthan gum and chitosan (CS).

[0081] In some embodiments of the present invention, the cellulose includes but is not limited to hydroxypropyl cellulose (HPMC), hydroxymethyl cellulose (CMC), hydroxyethyl cellulose (HEC), and the like.

[0082] In some embodiments of the present invention, the collagen includes but is not limited to at least one of fish collagen, porcine collagen, bovine collagen, and plant-derived collagen.

[0083] In some embodiments of the present invention, the reaction treatment further includes: gelling the reaction product to obtain the thermosensitive hydrogel. According to an embodiment of the present invention, the temperature of the gelling treatment is 30°C-50°C, for example, it can be 30°C, 32°C, 35°C, 37°C, 40°C, 42°C, 45°C, 48°C, 50°C, etc., or it can be a range composed of any of the above numerical values. According to an embodiment of the present invention, the time of the gelling treatment is 1min-10min, for example, it can be 1min, 2min, 4min, 6min, 8min, 10min, etc., or it can be a range composed of any of the above numerical values. Thereby, the gel solution can be converted into a semi-solid hydrogel state.

[0084] application

[0085] In the third aspect of the present invention, the present invention proposes the use of the thermosensitive hydrogel described in the first aspect or the thermosensitive hydrogel prepared by the method described in the second aspect as a biomedical material, a drug carrier and in vitro cell culture. As mentioned above, the thermosensitive hydrogel of the present invention has non-toxicity, injectability, high moisture retention and high light transmittance, as well as good biocompatibility and biodegradability. Therefore, it can be used as a new material for dressings, for biomedical materials, as a drug carrier and for cell culture.

[0086] The hydrogel of the present invention is an injectable hydrogel, and its formation mechanism is that the polymer network responds to temperature, so that chemical crosslinking or physical crosslinking occurs between the polymer networks to gel. Compared with preformed scaffolds in vitro, it can be formed in situ in vivo, can fill defects of any shape, can be conveniently mixed with therapeutic drugs, and as a drug carrier, it does not require surgical implantation, effectively reduces the pain of patients, and can be widely used in the fields of tissue engineering, drug controlled release, regenerative medicine, etc.

[0087] In the fourth aspect of the present invention, the present invention proposes a method for in vitro three-dimensional cell culture. According to an embodiment of the present invention, the method comprises: incubating the cells with the thermosensitive hydrogel described in the first aspect or the thermosensitive hydrogel prepared by the method described in the second aspect. As mentioned above, the thermosensitive hydrogel of the present invention has non-toxicity, injectability, high moisture retention and high light transmittance, as well as good biocompatibility and biodegradability. Therefore, the hydrogel of the present invention can be used to culture 3D cells or conduct scientific research (for example, studying the toxicity of the hydrogel, etc.).

[0088] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. If no specific techniques or conditions are specified in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used that do not specify the manufacturer are all conventional products that can be obtained commercially.

[0089] Example 1: Preparation of thermosensitive hydrogel

[0090] 1. Preparation of Hydroxypropyl Cellulose Solution

[0091] Weigh 0.5 g of hydroxypropyl cellulose powder, add 5 mL of water and stir for 6 h in the dark to fully dissolve the hydroxypropyl cellulose to obtain a hydroxypropyl cellulose solution.

[0092] 2. Preparation of potassium persulfate solution

[0093] 0.25 g of potassium persulfate powder was added to 4 mL of water and dissolved by ultrasonication at 40 °C for 30 min.

[0094] 3. Preparation of thermosensitive hydrogel

[0095] 4. Synthesis of thermosensitive collagen-hydroxypropylcellulose-polyacrylamide hydrogel by "one-pot method", e.g. Figure 1As shown, 0.5 g acrylamide was first dissolved in 10 mL of aqueous solution and stirred to dissolve, and then 1 mL of hydroxypropyl cellulose solution was added after dissolution, followed by 100 mg of fish collagen and 0.025 g of N,N'-methylenebisacrylamide, and finally 4 mL of potassium persulfate solution was added and stirred to mix. Finally, the mixture solution was fully reacted for 5 minutes under nitrogen protection, and then gelled at 37°C for 5 minutes to obtain a thermosensitive collagen-hydroxypropyl cellulose-polyacrylamide hydrogel.

[0096] Example 2: Characterization of the properties of thermosensitive hydrogels

[0097] 1. Scanning electron microscopy (SEM) observation of the surface morphology of thermosensitive hydrogel

[0098] In order to observe the surface morphology of the thermosensitive hydrogel after low-temperature drying in detail, this study used the following steps to prepare the sample for SEM examination. The dried hydrogel sample was freeze-dried, and then placed on a double-sided tape coated with an electrocarbon film, and gold-sprayed for 30 seconds using an ion sputtering instrument to improve the conductivity under the electron microscope. Finally, a scanning electron microscope was used to observe and record the surface structural details of the hydrogel in detail.

[0099] The microstructure of the hydrogel system was observed by scanning electron microscopy. Figure 2 As shown, the hydrogel scaffold has a dense structure and a rich reticular structure, and can load more drugs or protein small molecules.

[0100] 2. Gelation test

[0101] In Example 1, before the solution gelled, a 1 mL syringe was used to suck the gel solution into a vial, which was placed in a 37° C. oven and allowed to stand until the solution gelled and the time and state were recorded.

[0102] The experimental results are as follows Figure 3 As shown, the hydrogel can quickly form a hydrogel within 1-2 minutes at 37°C and present a colorless and transparent state.

[0103] 3. Temperature-sensitive and injectability testing

[0104] In Example 1, before the solution gels, a certain amount of the solution before gelling is sucked out using a 10 mL syringe, and then two vials are placed in water at 37° C. and refrigerated in a refrigerator at 4° C., respectively, and the solution in the syringe is slowly added to the two vials to observe the gelling time and state.

[0105] The experimental results are as follows Figure 4 As shown in Figure 1, Figure A shows that the hydrogel has turned into a gel after 2 minutes at 37°C, and Figure B shows that it is still liquid after 2 minutes at 6°C, indicating that the hydrogel has good temperature sensitivity and injectability.

[0106] 4. Hydrogel degradation performance testing

[0107] The hydrogels prepared in Example 1 were placed in culture dishes, a certain amount of water was added, and cultured in a 37° C. oven for 0, 1, 2, 3, 4, and 5 days, then taken out for freeze-drying, weighed, and the degradation rate was calculated.

[0108] The experimental results are as follows Figure 5 As shown, the hydrogel degraded by more than 35% after 5 days, showing good biodegradability.

[0109] 5. Water retention properties of hydrogel

[0110] The water retention performance of the prepared thermosensitive hydrogel was analyzed by detecting the moisturizing ability of the thermosensitive hydrogel at 37°C. 2 mL of thermosensitive collagen-hydroxypropylcellulose-polyacrylamide hydrogel was prepared and placed in a sterile and dry 6-well plate, and then slowly shaken at a constant speed in a 37°C constant temperature shaker. The weight of the hydrogel was weighed after 0, 2, 4, 6, 8, 10, 12, and 24 hours. The original weight of the hydrogel was recorded as Mc (g), and the weight at different times was recorded as Mt (g). Three parallel control experiments were set up at the same time, and the average value was taken. The water holding ratio of the hydrogel was calculated according to the following formula.

[0111] Water holding ratio (%)=Mc / Mt*100%

[0112] The experimental results are as follows Figure 6 As shown in the figure, the moisture retention rate of the hydrogel gradually decreases with the increase of time. At 2 hours, the moisture retention rate is 98%; at 4 hours, the moisture retention rate is 96%; at 6 hours, the moisture retention rate is 94%; at 8 hours, the moisture retention rate is 93%; at 10 hours, the moisture retention rate is 92%; at 12 hours, the moisture retention rate is 90%; at 24 hours, the moisture retention rate is 80%, which shows that the moisture retention performance is good.

[0113] 6. Transmittance test of hydrogel

[0114] The transmittance of the hydrogel prepared in Example 1 was measured by UV spectrometer to obtain the transmittance percentage. Figure 7 As shown, the light transmittance of the hydrogel is about 80%, which basically does not affect its high-magnification observation under a microscope.

[0115] Example 3: Biocompatibility testing of thermosensitive hydrogels

[0116] 1. Cytotoxicity test of hydrogel

[0117] The hydrogel prepared in Example 1 was made into a 2 mm thick sample, cut into a suitable size and placed in a cell culture dish, and sterilized. Then, 4T1 cells were cultured at 37°C and 5% CO2 for 24 h, and stained with calcein AM / propidium iodide PI, where live cells were marked in green and dead cells were marked in red.

[0118] The experimental results are as follows Figure 8 As shown, the 4T1 cells cultured on the hydrogel for 24 hours were alive, indicating that the prepared hydrogel was non-toxic and had good biosafety.

[0119] 2. Hydrogel cell proliferation test

[0120] The hydrogel prepared in Example 1 was made into a 2 mm thick sample, cut into a suitable size and placed in a cell culture dish, and sterilized. The sterilized hydrogel was placed in a culture dish, a certain amount of cell culture medium was added, and the CT26 cells were placed in an incubator for culture, and the cell state and culture medium were observed during the period and added at any time.

[0121] The experimental results are as follows Fig. 9 As shown, the hydrogel is covered with a large number of spherical cells, indicating that the hydrogel has a good effect on 3D cell culture and has good biocompatibility and non-toxicity.

[0122] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0123] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A thermosensitive hydrogel, characterized in that: include: A polymer matrix formed by acrylic monomers, polysaccharide polymers, collagen and water.

2. The thermosensitive hydrogel according to claim 1, characterized in that The acrylic monomer forms a polymer matrix under the action of a crosslinking agent and an initiator.

3. The thermosensitive hydrogel according to claim 2, characterized in that The acrylic monomers include acrylamide monomers; and / or, the cross-linking agent comprises N,N'-methylenebisacrylamide; and / or, the initiator comprises at least one of potassium persulfate, ammonium persulfate, diethylenetriamine and trimethylolpropane; And / or, the polysaccharide polymer includes at least one of cellulose, alginate, hyaluronic acid, xanthan gum and chitosan; And / or, the collagen includes at least one of fish collagen, porcine collagen, bovine collagen and plant-derived collagen.

4. A method for preparing the thermosensitive hydrogel according to any one of claims 1 to 3, characterized in that: include: Mixing acrylic monomers, a cross-linking agent, an initiator, a polysaccharide polymer, collagen and water to obtain a mixed solution; The mixed solution is subjected to reaction treatment under a protective atmosphere to obtain the thermosensitive hydrogel.

5. The method according to claim 4, characterized in that The mixing process includes: Performing a first mixing treatment on the polysaccharide polymer and water to obtain a polysaccharide polymer solution; Performing a second mixing process on the initiator and water to obtain an initiator solution; performing a third mixing process on the acrylic monomer and water to obtain an acrylic solution; The acrylic solution, the polysaccharide polymer solution, the collagen, the cross-linking agent and the initiator solution are subjected to a fourth mixing treatment to obtain the mixed solution.

6. The method according to claim 5, characterized in that The concentration of the polysaccharide polymer in the mixed solution is 1 mg / ml-10 mg / ml; and / or, the concentration of the acrylic acid solution in the mixed solution is 10 mg / ml-50 mg / ml; and / or, the concentration of the collagen in the mixed solution is 1 mg / ml-10 mg / ml; and / or, the concentration of the cross-linking agent in the mixed solution is 5 mg / ml-20 mg / ml; And / or, the concentration of the initiator solution in the mixed solution is 10 mg / ml-20 mg / ml.

7. The method according to claim 6, characterized in that The method further comprises the following steps after the reaction treatment: subjecting the reaction product to gelation treatment to obtain the thermosensitive hydrogel.

8. The method according to claim 7, characterized in that The reaction time is 1h-3h; And / or, the protective atmosphere is nitrogen; And / or, the temperature of the gelling treatment is 30°C-50°C; And / or, the gelling treatment time is 1 min-10 min.

9. Use of the thermosensitive hydrogel according to any one of claims 1 to 3 or the thermosensitive hydrogel prepared by the method according to any one of claims 4 to 8 as a biopharmaceutical material, a drug carrier and in vitro cell culture.

10. A method for in vitro three-dimensional cell culture, characterized in that: include: The cells are incubated with the thermosensitive hydrogel described in any one of claims 1 to 3 or the thermosensitive hydrogel prepared by the method described in any one of claims 4 to 8.

Images