Temperature-sensitive hydrogel using chitosan-based triblock copolymer and method for preparing the same

Abstract

The temperature-sensitive hydrogel of the present invention includes a temperature-sensitive polymer at a specific concentration. Thus, since the hydrogel may be useful for phase transition control at room temperature, allow gelation temperature and time control depending on a hydrophilicity ratio and a concentration change, and also, grow cells without toxicity, it may be applicable to various biomedical fields such as a tissue engineering scaffold.

Description

TEMPERATURE-SENSITIVE HYDROGEL USING CHITOSAN-BASED TRIBLOCK COPOLYMER AND METHOD FOR PREPARING THE SAME

[0001] The present invention relates to a temperature-sensitive hydrogel using a chitosan-based triblock copolymer and a method for preparing the same.

[0002]

[0003] Tissue engineering is a technology to create and restore a scaffold which may replace a defective part in the human body caused by accident, illness, or the like. According to recent studies, a scaffold which may effectively grow cells of the defective part in the human body are being actively studied.

[0004] The scaffold should have conditions of excellent biocompatibility and physical properties appropriate for the intended use. Also, the scaffold should have porosity, low cell toxicity, and biodegradability so that cells required for regeneration grow well. A representative material used for the scaffold is a natural polymer and a synthetic polymer which have biocompatibility and non-toxic properties. A natural polymer is chitosan, collagen, and the like, which have excellent biocompatibility. A synthetic polymer has a merit of easily adjustable physical properties, and poly(lactic-co-glycolic), acid(PLGA), polyglycolic acid(PGA), polylactic acid(PLA), polycaprolactone(PCL), and copolymers thereof are being mainly used.

[0005] As a scaffold production technology, a method for forming pores in a scaffold by gas foaming and salt removal methods after molding a biocompatible polymer is mainly used, and when the method is applied in the human body, there are problems such as surgical operation and reproducibility of shape. In order to supplement the problem, a scaffold using a hydrogel which shows a sol-to-gel transition phenomenon by an external stimulus such as temperature and pH is being currently studied.

[0006] A hydrogel is used in anti-adhesion of tissues, regeneration of organs / tissues, a release system of a bioactive material from a medical filler, and the like in the medical field, and since it may transfer a therapeutic gene only to a specific disease site which provides stimulation in the genetic field, it may decrease side effects and increase treatment efficiency. In addition, due to the characteristics of being biocompatible, injected without surgical operation, and fixed within a target tissue to deliver a certain drug to surrounding tissues, the hydrogel is also applied in the drug delivery field.

[0007] In order to inject the hydrogel into the human body, a material which is in a solution state in vitro and may form a gel when injected into the body is required. Therefore, a stimuli-responsive polymer involving a phase change depending on temperature and pH is being mainly used as a hydrogel material.

[0008] A temperature-sensitive polymer is a polymer of which the properties change at a specific temperature, and may be, representatively, an amphiphilic polymer, and among the amphiphilic polymers, a polymer material applicable to the scaffold includes Poly(N-isopropylacrylamide) (PNIPAAm), Pluronic F127, poly(lactic-co-glycolic acid) (PLGA), and the like. Though PNIPAAm may respond more rapidly to external temperature changes by introducing various materials, such as grafting of oligo-NIPAAm or PEG, it is not biodegradable. In addition, PLGA has low strength and stability in using it as a scaffold and its decomposition time is short. However, F127 is FDA approved and biocompatible, and is biodegradable and applied in production of food additives, pharmaceutical raw materials, and agricultural products. In addition, Pluronic F127 is composed of a hydrophilic block and a hydrophobic block, and shows sol-to-gel properties by adjusting transition temperature depending on hydrophilic and hydrophobic block lengths and compositions of the copolymer, but is unfavorable due to the weak mechanical strength, high penetrability, non-degradability, and the like.

[0009] Meanwhile, chitosan is a natural polysaccharide polymer obtained by deacetylating chitin formed of two units of 2-amino-2-deoxy-(1,4)-β-D-glucopyranose and N-acetyl-D-glucosamine, and has various physiological activities such as antibacterial properties, immune activity, anti-cancer metastasis, and wound healing effects. In addition, since it has biocompatibility, biodegradability, low toxicity, and one amine group per unit, it is widely used as drug and gene transporter materials and in the biomedical field such as tissue engineering.

[0010] Therefore, it is intended to develop a temperature-sensitive hydrogel which is dissolved in an aqueous solution well, is biocompatible, has biodegradability, and also, may be easily used at room temperature and also promote cell growth, by showing a sol-to-gel transition behavior at a higher temperature than the sol-to-gel transition temperature of the temperature-sensitive hydrogel in the conventional art.

[0011]

[0012] An object of the present invention is to provide a temperature-sensitive polymer which shows sol-to-gel transition behavior at a higher temperature than the conventional art and a temperature-sensitive hydrogel including the polymer.

[0013] Another object of the present invention is to provide a temperature-sensitive hydrogel which may be used in tissue regeneration engineering and a cell scaffold using the same.

[0014]

[0015] In order to achieve the above objects, the present inventors studied continuously, and as a result, found that by including a temperature-sensitive polymer satisfying a specific structure, a hydrogel is formed at a higher temperature than a conventional technology, so that it is useful in phase transition control and, simultaneously, may grow cells without toxicity, and thus, the it may be used as a tissue engineering scaffold, thereby completing the present invention.

[0016] In one general aspect, a temperature-sensitive hydrogel includes a polymer represented by the following Chemical Formula 1:

[0017] [Chemical Formula 1]

[0018]

[0019] wherein

[0020] R1and R4are independently of each other a substituted or unsubstituted amino group,

[0021] R2, R3, R5, and R6are independently of one another hydrogen or a hydroxyl group, but are not all hydrogen,

[0022] R7is alkyl,

[0023] a, b, and c are independently of one another an integer of 1 or more, and

[0024] n and m are independently of each other an integer of 1 or more.

[0025] According to an exemplary embodiment, R1and R4may be independently of each other an amino group, an alkylamino group, or an alkylcarbonylamino group.

[0026] According to an exemplary embodiment, R2, R3, R5, and R6may be a hydroxyl group.

[0027] According to an exemplary embodiment, a, b, and c may be independently of one another 10 to 300.

[0028] According to an exemplary embodiment, a and c may be independently of each other 40 to 100.

[0029] According to an exemplary embodiment, b may be 80 to 150.

[0030] According to an exemplary embodiment, n and m may be independently of each other 1 to 100.

[0031] According to an exemplary embodiment, R7may be methyl or ethyl.

[0032] According to an exemplary embodiment, the polymer may have a weight average molecular weight of 2 to 50 kDa.

[0033] According to an exemplary embodiment, the polymer may be included at 10 to 25 wt%.

[0034] According to an exemplary embodiment, the hydrogel may have a viscosity measured at 25℃ of 100 Pa·s to 2000 Pa·s.

[0035] According to an exemplary embodiment, a temperature (Tgel) of the hydrogen at which a sol-to-gel transition phenomenon by a vial invert method occurs may be 22℃ or higher.

[0036] According to an exemplary embodiment, a temperature (Ttanδ=1) at which an elastic modulus (G') and a viscous modulus (G") intersect in evaluation of rheological properties of the hydrogel may be 21℃ or higher.

[0037] According to an exemplary embodiment, a time (tgel) when the elastic modulus (G') is at its highest point in evaluation of rheological properties of the hydrogel may be within 1 minute.

[0038] According to an exemplary embodiment, the hydrogel may have a low critical solution temperature of 19 to 23℃.

[0039] In another general aspect, a cell scaffold includes the temperature-sensitive hydrogel described above.

[0040] According to an exemplary embodiment, the hydrogel may have pores of 1 to 50 μm.

[0041] Since the temperature-sensitive hydrogel of the present invention may be useful for phase transition control at room temperature, allow gelation temperature and time control depending on a hydrophilicity ratio and a concentration change, and also, grow cells without toxicity, it may be applicable to various biomedical fields such as a tissue engineering scaffold.

[0042]

[0043] The above and other objects, features and advantages of the present invention will become apparent from the following description of preferred embodiments given in conjunction with the accompanying drawings, in which:

[0044] FIG. 1 shows a reaction formula for a method for preparing a hydrogel according to an exemplary embodiment.

[0045] FIG. 2 is FT-IR spectra of F127 and Preparation Examples 1, 2, 4, and 5.

[0046] FIG. 3 is 1H-NMR spectra of F127 and Preparation Examples 1, 2, 4, and 5.

[0047] FIG. 4 shows results of analyzing rheological properties of a hydrogel prepared according to an exemplary embodiment using a rheometer and is a graph showing changes in an elastic modulus (G') and a viscous modulus (G") depending on temperature.

[0048] FIG. 5 is an FE-SEM image in which the morphology of the hydrogel prepared according to an exemplary embodiment was analyzed.

[0049] FIG. 6 shows results of a cell live / dead assay test for using the hydrogel prepared according to an exemplary embodiment as a cell scaffold.

[0050]

[0051] Hereinafter, the present invention will be described in more detail with reference to specific examples and exemplary embodiments including the accompanying drawings. However, the following specific examples or exemplary embodiments are only a reference for describing the present invention in detail, and the present invention is not limited thereto, and may be implemented in various forms.

[0052] In addition, unless otherwise defined, all technical terms and scientific terms have the same meanings as those commonly understood by one of those skilled in the art to which the present invention pertains. The terms used herein are only for effectively describing a certain specific example and are not intended to limit the present invention.

[0053] In addition, the singular form used in the specification and claims appended thereto may be intended to include a plural form also, unless otherwise indicated in the context.

[0054] In addition, units used in the present specification without particular mention are based on weights, and as an example, a unit of % or ratio refers to a wt% or a weight ratio and wt% refers to wt% of any one component in a total composition, unless otherwise defined.

[0055] In addition, in the present specification, unless explicitly described to the contrary, "comprising" any elements will be understood to imply further inclusion of other elements rather than exclusion of any other elements.

[0056] In addition, the numerical range used in the present specification may include all values within the range including the lower limit and the upper limit, increments logically derived in a form and span in a defined range, all double limited values, and all possible combinations of the upper limit and the lower limit in the numerical range defined in different forms. Unless otherwise particularly defined in the present specification, values which may be outside a numerical range due to experimental error or rounding off of a value are also included in the defined numerical range.

[0057] The term "polymer" in the present specification includes a polymer and a copolymer.

[0058] The term "copolymer" in the present specification generally refers to any polymer derived from more than one monomer species, in which the polymer includes a repeating unit corresponding to more than one species. The copolymer is a reaction product of two or more monomers, and thus, may include corresponding two or more repeating unit species. The copolymer may be present as a block copolymer, a random copolymer, and / or an alternating copolymer.

[0059] In addition, "substituted" in the present specification means that a hydrogen atom in the substituted part is replaced with a substituent. The substituent may be any known substituent without limitation. An example of the substituent may be one or more selected from hydroxy, halogen, nitro, cyano, amino, carboxyl, carboxylate, C1-20alkyl, C2-20alkenyl, C2-20alkynyl, C1-20haloalkyl, C1-20alkoxy, C1-20alkoxycarbonyl, C3-30cycloalkyl, (C6-30)ar(C1-20)alkyl, C6-30aryl, C3-30heteroaryl, and the like, but is not limited thereto.

[0060] The term "residue" in the present specification refers to a remaining part excluding a specific functional group from a polymer, and the type of polymer is not particularly limited.

[0061] The term "alkyl" in the present specification includes both straight chain or branched chain forms, and may have 1 to 30, specifically 1 to 20 carbon atoms.

[0062] The term "amino" in the present specification refers to -NH2, and "alkylnylamino" refers to -NHalkyl radical, in which "alkyl" is as defined above. An example of the alkylamino includes methylamino, ethylamino, isopropyl amino, propyl amino, butyl amino, isobutyl amino, t-butyl amino, and the like, but is not limited thereto.

[0063] The term "alkylcarbonylamino" in the present specification refers to a -NHC(=O)alkyl radical, in which "alkyl" is as defined above. An example of the alkylcarbonylamino radical includes methylcarbonylamino, ethylcarbonylamino, isopropylcarbonyl amino, propylcarbonylamino, butylcarbonylamino, isobutylcarbonylamino, t-butylcarbonylamino, and the like, but is not limited thereto.

[0064] The term "temperature-sensitive hydrogel" in the present specification refers to a polymer having a characteristic capable of sol-to-gel phase transition of being in a solution state at room temperature and being in a viscous gel state near body temperature.

[0065] Hereinafter, the temperature-sensitive hydrogel using a chitosan-based triblock copolymer according to an exemplary embodiment of the present invention and a method for preparing the same will be described in more detail.

[0066] The present invention provides a temperature-sensitive hydrogel including a polymer represented by the following Chemical Formula 1:

[0067] [Chemical Formula 1]

[0068]

[0069] wherein R1and R4are independently of each other a substituted or unsubstituted amino group, R2, R3, R5, and R6are independently of one another hydrogen or a hydroxyl group, but are not all hydrogen, R7is alkyl, a, b, and c are independently of one another an integer of 1 or more, and n and m are independently of each other an integer of 1 or more.

[0070] According to an exemplary embodiment of the present invention, in Chemical Formula 1, R1and R4may be independently of each other an amino group, an alkylamino group, or an alkylcarbonylamino group, specifically an amino group, a C1-5 alkylamino group, or a C1-5 alkylcarbonylamino group. More specifically, R1and R4may be independently of each other an amino group (-NH2), a methylamino group (-NHCH3), or a methylcarbonylamino group (-NHC(=O)CH3). Preferably, R1and R4may be an amino group (-NH2).

[0071] According to an exemplary embodiment of the present invention, in Chemical Formula 1, R2, R3, R5, and R6may be independently of one another hydrogen or a hydroxyl group, or may be all a hydroxyl group. Otherwise, at least two or more of R2, R3, R5, and R6may be a hydroxyl group.

[0072] According to an exemplary embodiment of the present invention, in Chemical Formula 1, a, b, and c may be independently of one another 5 to 500, 10 to 300, or 20 to 200.

[0073] According to an exemplary embodiment of the present invention, in Chemical Formula 1, a and c may be independently of each other 5 to 200, 20 to 150, 40 to 100, or 50 to 80.

[0074] According to an exemplary embodiment of the present invention, in Chemical Formula 1, b may be 20 to 500, 50 to 200, 80 to 150, or 90 to 120.

[0075] According to an exemplary embodiment of the present invention, in Chemical Formula 1, n and m may be independently of each other 1 to 100, 5 to 50, 10 to 30, or 10 to 20.

[0076] According to an exemplary embodiment of the present invention, in Chemical Formula 1, R7may be alkyl, specifically C1-20 alkyl, and more specifically C1-7 alkyl, and R7may be methyl or ethyl.

[0077] According to an exemplary embodiment of the present invention, the polymer may have a form in which poly(ethylene oxide-C2 or higher alkylene oxide-ethylene oxide) in the center and a chitosan or chitin is bonded to the end. The ethylene oxide, chitosan, or chitin is a hydrophilic block, and the C2 or higher alkylene oxide acts as a hydrophobic block and may form a hydrogel at a certain temperature or higher in an aqueous solution. The hydrophilic block and the hydrophobic block may be adjusted to adjust the certain temperature, and also, may easily adjust the time to form a hydrogel. Thus, the polymer may be applied to various biomedical fields such as tissue engineering scaffold.

[0078] In addition, the triblock copolymer positioned at the center part of the polymer is an amphipathic copolymer, and since the principle of forming a hydrogel is well known in the art, it will be omitted.

[0079] According to an exemplary embodiment of the present invention, the polymer may have a weight average molecular weight of 2 to 50 kDa, 5 to 35 kDa, or 10 to 25 kDa.

[0080] In addition, the repeating unit (n or m) of the end part of the polymer is as described above, and the repeating unit may be a repeating unit derived from a polymer having a weight average molecular weight of 0.1 to 10 kDa, 0.5 to 6 kDa, or 0.8 to 5 kDa.

[0081] According to an exemplary embodiment of the present invention, the hydrogel may include the polymer and the solvent described above, and the polymer may be included at 5 to 50 wt%, 10 to 25 wt%, or 13 to 20 wt%. The solvent is preferably distilled water (water), and may further include a buffer commonly used for pH adjustment, but is not limited thereto.

[0082] According to an exemplary embodiment of the present invention, the hydrogel may have a viscosity measured at 25℃ of 50 Pa·s to 5000 Pa·s, 100 Pa·s to 2000 Pa·s, or 200 Pa·s to 1000 Pa·s (Pa).

[0083] According to an exemplary embodiment of the present invention, a temperature (Tgel) of the hydrogel at which a sol-to-gel transition phenomenon occurs by a vial invert method according to the method described later may be 22℃ or higher, 22 to 30℃, 23 to 30℃, or 24 to 28℃.

[0084] According to an exemplary embodiment of the present invention, a temperature (Ttanδ=1) at which an elastic modulus (G') and a viscous modulus (G") intersect in evaluation of rheological properties of the hydrogel according to the method described later may be 20℃ or higher, 21℃ or higher, 21 to 30℃, 22 to 28℃, or 22.5 to 28℃.

[0085] According to an exemplary embodiment of the present invention, a time (tgel) when the elastic modulus (G') is at its highest point in evaluation of rheological properties of the hydrogel according to the method described later may be within 1 minute, 1 second to 60 seconds, 10 seconds to 55 seconds, 10 seconds to 45 seconds, or 10 seconds to 35 seconds.

[0086] According to an exemplary embodiment of the present the hydrogel may have a low critical solution temperature in accordance with the measurement method described later of 19 to 23℃, 19.5 to 22℃, or 20 to 22℃.

[0087] Since the temperature-sensitive hydrogel satisfies the conditions described above, the sol-to-gel transition behavior is shown at a higher temperature than the conventional art, and thus, there are quite a lot of benefits in that the hydrogel may be freely handled in a wider range of temperature and has biocompatibility, biodegradability, and low toxicity.

[0088] The present invention provides a cell scaffold including the temperature-sensitive hydrogel described above. As a method for producing a cell scaffold, a method for producing a porous cell scaffold from a commonly used or known hydrogel may be used without limitation. The scaffold produced from the hydrogel is non-toxic, has a cell growth promoting effect as compared with the conventional art, and may be produced into various forms without limitation of its form, and the scaffold produced from the hydrogel may have pores of 1 to 50 μm, 5 to 30 μm, or 5 to 20 μm. The detailed description of the cell scaffold will be replaced with that equivalent to common technology.

[0089] Hereinafter, the present invention will be described in more detail with reference to the examples and the comparative examples. However, the following examples and comparative examples are only an example for describing the present invention in more detail, and do not limit the present invention in any way.

[0090] [Method for evaluating physical properties]

[0091] 1) Structural analysis

[0092] Structural analysis was performed using a nuclear magnetic resonance device (1H NMR, 400 mHz, Bruker, Germany) and an infrared spectrometer (FTIR 8700, Shimadzu, Japan). The analyzed FT-IR spectrum is shown in FIG. 2, and the analyzed1H-NMR spectrum is shown in FIG. 3.

[0093] FT-IR analysis was performed in a wavelength range of 400 to 4000 cm-1to analyze a specific peak, after mixing separated CFC and KBr at a ratio of about 1 / 100 (wt. / wt.) well to prepare a pellet.

[0094] 1H-NMR analysis was performed through chemical shift in a range of 1 to 10 ppm after dissolving 4 mg of CFC2K or CFC4K in 700 μl of a D2O solvent.

[0095] 2) Rheological property analysis

[0096] - Vial invert method

[0097] The prepared hydrogel solution was added to a water bath at 15℃, the vial was inverted while heating at 1℃ intervals, and a state of no flow was measured as a gelation temperature (Tgel).

[0098] - Method of using rheometer

[0099] 0.8 mL of a CFC hydrogel was put and spread on a rheometer plate, and an elastic modulus (G') and a viscous modulus (G") were measured in a range from 15℃ to 40℃ while heating at 1℃ intervals (frequency = 2 Hz, plate diameter = 25 mm, gap = 1 mm, shear deformation = 0.4%). At this time, a cross-point of G' and G" shown by an increase in temperature was measured as a gelation temperature (Ttanδ=1). A graph showing the measurement results is shown in FIG. 4 (A).

[0100] In addition, the viscosity was measured identically to the conditions described above at frequency = 2 Hz and room temperature (25℃) using the rheometer.

[0101] 3) Morphology analysis

[0102] Morphology properties were observed using a scanning electron microscope (filed emission scanning electron microscopy, FE-SEM, HITACHI S-4800, Japan). A CFC hydrogel was lyophilized for 2 days to completely remove moisture. A cross-section of the dried sample was taken and coated with platinum for 30 seconds for FE-SEM analysis. Thereafter, cross-sectional microstructure images obtained by observing shapes of cross sections at an acceleration voltage of 3 kV at various magnifications are shown in FIG. 5.

[0103] 4) Cell live / dead assay evaluation

[0104] The prepared temperature-sensitive hydrogel was used as a cell scaffold, and evaluation of performance such as biocompatibility thereon was performed. The prepared polymer was dissolved in a DMEM medium to prepare a hydrogel solution, 200 μl of the solution was taken, dispensed into a 24-well, and allowed to stand in an incubator at 36℃ for gel formation, and 5Х103cell / well of cultured AGS cells were dispensed, respectively. After each culture for 72 hours, the cells were washed with PBS and treated with 4 μM calcein-AM, and after 1 hour, the survival of the cells was confirmed by a fluorescence microscope. The cell AGS used in the live / dead assay was received from American Type Culture Collection (ATCC, KOREA) and cultured using Dulbecco's modified Eagle's medium (DMEM, SIGMA-ALDRICH, USA) containing a 10% fetal bovine serum (FBS, gibco, USA). The calcein-AM used in the live / dead assay was purchased from SIGMA-ALDRICH (USA). In addition, the measurement results are shown in FIG. 6.

[0105] [Preparation Example 1]

[0106] 1 g (0.00008 mol) of Pluronic F127 ((poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) [PEO-b-PPO-b-PEO], Mw: 12.6 kDa, BASF) was added to a reactor combined with a condenser, 30 mL of dichloromethane (MC) was added and completely dissolved, 65 μl (0.000646 mol) of triethylamine (TEA) was added, stirring was performed for 1 hour, 29 μl (0.00032 mol) of acryloyl chloride (AC, SIGMA-ALDRICH) was added, and the reaction was performed in a cooling reflux device at 30℃ for 4 hours in a N2environment. After the reaction was completed, unreacted materials were removed by deposition in cold ether and washing 3 times, the product was dried at room temperature using centrifugation, and then a F127 macromere was recovered.

[0107] The dried F127 macromere was dialyzed in 3rd distilled water for 48 hours using a MWCO 10 kDa cellulose membrane for completely removing low-molecular weight reactants, solvents, and the like, and then lyophilization was performed to obtain a F127 macromere.

[0108] As a result of analyzing the structure of the F127 macromere, as seen in (A) of FIG. 2, acryloyl chloride was introduced to a F127 -OH group in the FT-IR spectrum and the -C=C- characteristic peak of acryloyl chloride was observed at about 1800 cm-1, and also, as seen in (A) of FIG. 3, the CH2=CH hydrogen characteristic peak of acryloyl chloride was shown at 6.0-6.5 ppm in the 1H-NMR spectrum and it was confirmed that the F127 macromere was successfully synthesized.

[0109] The process of Preparation Example 1 is shown in (A) of FIG. 1.

[0110] [Preparation Example 2]

[0111] 600 mg of COS2K (chitosan lactate (chitosan oligosaccharide, COS, Mw: 2 kDa, KITTOLIFE)) was dissolved in 16 mL of distilled water and 120 mg of potassium persulfate (KPS) was dissolved in 4 mL of distilled water completely, respectively, the KPS aqueous solution was slowly added to the COS2K aqueous solution, and the reaction was performed at 60℃ for 1 hour in a N2environment to prepare a chitosan solution in which the COS2K radical species was activated.

[0112] The process of Preparation Example 2 is shown in (B) of FIG. 1.

[0113] [Preparation Example 3]

[0114] The process was performed in the same manner as in Preparation Example 2, except that COS4K (chitosan lactate (chitosan oligosaccharide, COS, Mw: 4 kDa, KITTOLIFE)) was used instead of COS2K.

[0115] [Preparation Example 4]

[0116] 3780 mg of the F127 macromere obtained in Preparation Example 1 was dissolved in 50 mL of 3rd distilled water, the solution was slowly added dropwise to a chitosan solution prepared in Preparation Example 2, and then synthesis was performed at room temperature in a N2environment overnight. After the reaction was completed, dialysis was performed for 48 hours in 3rd distilled water using the MWCO 10 kDa cellulose membrane for removing unreacted COS2K, F127 macromere, and the like, and lyophilization was performed to obtain final CFC2K.

[0117] As a result of structural analysis of CFC2K, as seen in (B) of FIG. 2, in the FT-IR spectrum, it was confirmed that a C=C bond present in acryloyl chloride was changed to a C-C single bond by a radical reaction and a C=C characteristic peak disappeared in acryloyl chloride at 1800 cm-1, and simultaneously, a characteristic peak by CH3at 2900-3000 cm-1overlapped a characteristic peak shown by chitosan, and as seen in (B) of FIG. 3, in1H-NMR spectrum, the CH2=CH hydrogen characteristic peak of acryloyl chloride disappeared at 6.0-6.5 ppm, the characteristic peak of hydrogen at position 2 of chitosan at 3.1 ppm which was not shown at F127 was newly shown in CFC, and the hydrogen characteristic peak of F127 macromere was observed near 1.2 ppm which was not present in chitosan, whereby it was confirmed that the CFC2K polymer was successfully synthesized.

[0118] The process of Preparation Example 4 is shown in (C) of FIG. 1.

[0119] [Preparation Example 5]

[0120] A final CFC4K polymer was obtained in the same manner as in Preparation Example 4, except that the chitosan solution prepared in Preparation Example 3 was used instead of the chitosan solution prepared in Preparation Example 2. In addition, the structural analysis results of CFC4K were identical or similar to those of CFC2K, and it was confirmed that CFC4K was successfully synthesized.

[0121] [Examples 1 to 6]

[0122] As shown in Table 1, CFC2K or CFC4K prepared in Preparation Examples 4 or 5 was dissolved in a phosphate-buffered saline (PBS) 7.4 buffer solution so that the concentrations were 15, 18, and 20%, thereby preparing hydrogel solutions.

[0123] [Comparative Example 1]

[0124] A hydrogel solution was prepared by using F127 of Preparation Example 1 instead of CFC2K or CFC4K in Example 1 and a concentration of 20%.

[0125] [Comparative Example 2]

[0126] A hydrogel solution was prepared by using F127 of Preparation Example 1 instead of CFC2K or CFC4K in Example 1 and a concentration of 15%.

[0127] [Evaluation Example 1] Evaluation of sol-to-gel transition behavior

[0128] As a method for determining sol-to-gel transition behavior, rheological properties may be analyzed, and specifically, there is a method of observing changes in an elastic modulus (G') and a viscous modulus (G") using a vial invert method and a rheometer ((Rotational) rheometer, Kinexus lab+, Malvern, UK). A temperature (Tgel) at which a sol-to-gel transition phenomenon by the vial invert method occurred and a temperature (Ttanδ=1) at which the elastic modulus (G') and the viscous modulus (G") intersected using a rheometer ((Rotational) rheometer, Kinexus lab+, Malvern, UK) for the polymers of each example and comparative example were recorded in the following Table 1.

[0129] In addition, the time when the elastic modulus was the highest was considered to be perfect gel formation, measured as a gelation time (tgel), and recorded in the following Table 1.

[0130] In addition, changes in the elastic modulus depending on an increase in temperature of each hydrogel were graphed using a rheometer, and an intersection obtained by plotting at the lowest temperature was defined as a low critical solution temperature (LCST) and recorded in the following Table 1.

[0131] Polymerconcentration [%]Tg [℃]Ttanδ=1[℃]tgel[sec]LCST [℃]Example 1CFC2K152423.993020.83Example 2CFC2K182221.954019.65Example 3CFC2K202220.876019.20Example 4CFC4K152525.475021.57Example 5CFC4K182422.885020.49Example 6CFC4K202221.847020.01Comparative Example 1F1272022---Comparative Example 2F12715----

[0132] As seen in Table 1, it was found that in the same material, as the concentration was higher, the sol-to-gel transition phenomenon was shown at a lower temperature. In addition, upon comparison depending on the amount of the hydrophilic group in the molecular structure, it was found that at the same concentration, as the molecular weight of the hydrophilic group was higher, the sol-to-gel transition temperature was increased. It was expected that the hydrophilic group was increased by introducing chitosan into a molecule and a gelation temperature was increased.In particular, upon comparison of Comparative Example 2 with Examples 1 and 4, it was confirmed that in Comparative Example 2, gelation did not proceed, but in Examples 1 and 4, phase transition occurred and gelation proceeded at a lower concentration and a higher temperature than F127, and thus, it was found that the temperature-sensitive hydrogel according to an exemplary embodiment was more favorable for application to a human body as compared with the conventional art.

[0133] In addition, in the same material, it was confirmed that the gelation time was long when the concentration was high. It was expected that at a high concentration, more time was needed for hydrophilic / hydrophobic balance and hydrophobic interaction, and it was confirmed that CFC4K having more hydrophilic groups had longer gelation time, and this was expected due to the decreased hydrophobic interaction by the hydrophilic group.

[0134] In particular, upon comparison of temperature when the elastic modulus was the highest, the gelation temperature of CFC2K was lowered from 28℃ to 20℃ as the concentration was increased. In addition, the temperature of CFC4K was lowered from 30℃ to 22℃. In addition, it was confirmed that Tgelof Comparative Example 1 is lower than Examples 1 and 4, and so gelation in Comparative Example 1 proceeded at a lower temperature than Examples 1 and 4, and, in particular, in Comparative Example 2, gelation did not proceed due to the low concentration. Thus, it was confirmed that when the hydrogel of an exemplary embodiment was used as a biomedical use, phase transition control was more useful than the comparative example.

[0135] Furthermore, both CFC2K and CFC4K showed LCST near about 20℃. In the case of the same material, LCST was lowered as the concentration was increased, and at the same concentration, LCST was increased as the hydrophilic group was increased. It may be expected from the results that pure F127 to which no hydrophilic group was introduced showed LCST at a lower temperature. It may be expected from the results that synthesized CFC is useful for phase transition control at room temperature and may be widely used as a tissue engineering scaffold.

[0136] (B) of FIG. 4 shows a graph of the results showing the sol-to-gel transition phenomenon depending on the hydrophilic / hydrophobic ratio and the concentration. As seen from the results, in the same materials, a phase transition temperature was shown at a lower temperature as the concentration was increased. Further, it was found that at the same concentration, the CFC4K having more hydrophilic groups had an increased phase transition temperature. It was expected that as chitosan was introduced into the molecule, the hydrophilic group was increased and gelation temperature was increased.

[0137] [Evaluation Example 2] Morphology analysis of hydrogel

[0138] The morphology of the polymer of each of the examples and the comparative examples was analyzed and the FE-SEM images are shown in FIG. 5. As seen in FIG. 5, in the hydrogel of the same material, a pore size was decreased as the concentration was increased, and at the same concentration, it was observed that CFC4K having more hydrophilic groups had larger pores. The results are due to the fact that hydrophobic interaction was more strongly shown as the concentration was increased, and thus, the microstructure was denser. In addition, when there were more hydrophilic groups, hydrophobic interaction was relatively weak and the microstructure became bulky.

[0139] [Evaluation 3] Cell live / dead assay test

[0140] The survival of cells was observed using calcein-AM in order to confirm the availability of the prepared CFC2K and CFC4K hydrogels as a cell transfer scaffold. As seen in FIG. 6, it was confirmed that fluorescence was expressed similarly to the control (a state in which the sample was not treated and there were only cells) in both CFC2K and CFC4K, and fluorescence was expressed by calcein-AM embedded in cells and calcein-AM hydrolyzed by an esterase enzyme in cells, so that cell was survived. It was confirmed from the results that the prepared CFC hydrogel is a material which may promote cell growth without toxicity to cells.

[0141] Since cell survival in a CFC hydrogel was observed by a fluorescence microscope through a live / dead assay, cells may grow without toxicity, and thus, it was proved that the hydrogel may be used as a tissue engineering scaffold. It is expected from the results that the temperature-sensitive hydrogel according to an exemplary embodiment of the present invention is useful for phase transition control at room temperature and may control gelation temperature and time depending on the hydrophilic ratio and concentration change and may be applied to various biomedical fields.

[0142] Hereinabove, although the present invention has been described by specified matters and specific exemplary embodiments, they have been provided only for assisting in the entire understanding of the present invention. Therefore, the present invention is not by the specific matters limited to the exemplary embodiments. Various modifications and changes may be made by those skilled in the art to which the present invention pertains from this description.

[0143] Therefore, the spirit of the present invention should not be limited to the above-described exemplary embodiments, and the following claims as well as all modifications equal or equivalent to the claims are intended to fall within the scope and spirit of the invention.

Claims

1.A temperature-sensitive hydrogel comprising a polymer represented by the following Chemical Formula 1:[Chemical Formula 1]whereinR1and R4are independently of each other a substituted or unsubstituted amino group,R2, R3, R5, and R6are independently of one another hydrogen or a hydroxyl group, but are not all hydrogen,R7is alkyl,a, b, and c are independently of one another an integer of 1 or more, andn and m are independently of each other an integer of 1 or more.2.The temperature-sensitive hydrogel of claim 1, wherein R1to R4are independently of one another an amino group, an alkylamino group, or an alkylcarbonylamino group.3.The temperature-sensitive hydrogel of claim 1, wherein R2, R3, R5, and R6are a hydroxyl group.4.The temperature-sensitive hydrogel of claim 1, wherein a, b, and c are independently of one another 10 to 300.5.The temperature-sensitive hydrogel of claim 1, wherein a and c are independently of each other 40 to 100.6.The temperature-sensitive hydrogel of claim 1, wherein b is 80 to 150.7.The temperature-sensitive hydrogel of claim 1, wherein n and m are independently of each other 1 to 100.8.The temperature-sensitive hydrogel of claim 1, wherein R7is methyl or ethyl.9.The temperature-sensitive hydrogel of claim 1, wherein the polymer has a weight average molecular weight of 2 to 50 kDa.10.The temperature-sensitive hydrogel of claim 1, wherein the polymer is included at 10 to 25 wt%.11.The temperature-sensitive hydrogel of claim 1, wherein the temperature-sensitive hydrogel has a viscosity measured at 25℃ of 100 Pa·s to 2000 Pa·s.12.The temperature-sensitive hydrogel of claim 1, wherein a temperature (Tgel) at which a sol-to-gel transition phenomenon by a vial invert method occurs is 22℃ or higher.13.The temperature-sensitive hydrogel of claim 1, wherein a temperature (Ttanδ=1) at which an elastic modulus (G') and a viscous modulus (G") intersect in evaluation of rheological properties is 21℃ or higher.14.The temperature-sensitive hydrogel of claim 1, wherein a time (tgel) when an elastic modulus (G') is at its highest point in evaluation of rheological properties is within 1 minute.15.The temperature-sensitive hydrogel of claim 1, wherein the temperature-sensitive hydrogel has a low critical solution temperature of 19 to 23℃.16.A cell scaffold comprising the temperature-sensitive hydrogel of any one of claims 1 to 15.17.The cell scaffold of claim 16, wherein the hydrogel has pores of 1 to 50 μm.

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