Glycol chitosan derivative capable of double crosslinking

Abstract

An aqueous solution of a glycol chitosan derivative of the present invention exhibited a thermoreversible sol-gel transition in the range of 21-54°C, and the gelation temperature could be controlled by means of the polymer concentration and the degree of substitution. By performing photocrosslinking using ultraviolet light on a glycol chitosan derivative hydrogel, a chemically crosslinked hydrogel with improved mechanical properties could be prepared. A thermogel containing the glycol chitosan derivative showed thermosensitive sol-gel transition characteristics in the concentration range of 2-6 wt%. In addition to the thermosensitive characteristics, the chemically crosslinked thermogel can have various property ranges depending on various photocrosslinking times, polymer concentrations, and substitution rates, and thus is expected to be useful for various biomedical applications.

Description

Glycol chitosan derivatives capable of double cross-linking

[0001] This invention claims the benefit of Korean Patent Application No. 10-2023-0158764 filed on November 16, 2023 with the Korean Intellectual Property Office, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to a glycol chitosan derivative capable of double crosslinking, and more specifically, to a glycol chitosan containing an alkenyl group capable of physical crosslinking and chemical crosslinking.

[0003] Hydrogels have attracted significant research interest for decades due to their promising properties, including biocompatibility, high water content, tunable physicochemical properties, and sensitivity to stimuli. Among hydrogels, thermogelling polymers, which undergo a heat-sensitive sol-gel transition in aqueous media, have attracted significant attention as injectable delivery systems. These hydrogels can readily encapsulate cells or drugs in a sol state, enable direct injection into target sites, and offer longer-lasting results through heat-induced gelation at body temperature. In clinical applications, injectable hydrogels are particularly suitable for treating irregularly shaped tissue areas, eliminating the need for customized scaffold design. Depending on the materials used, injectable hydrogels can be formed by UV irradiation, ionic cross-linking, pH, or temperature changes. Natural polymers such as alginate, hyaluronic acid, dextran, and chitosan, polysaccharides, have been proposed for biomedical applications due to their biodegradability and biocompatibility. In particular, chitosan is widely studied for drug delivery, cell encapsulation, and tissue engineering due to its antibacterial, biodegradable, biocompatible, and adhesive properties.

[0004] However, the low water solubility of chitosan itself under physiological conditions is a major drawback for its widespread use as a hydrogel material. Glycol chitosan, a water-soluble chitosan derivative, readily dissolves in water under physiological conditions due to its glycol functional group, while maintaining chitosan's advantageous non-toxic and biodegradable properties, making it suitable for pharmaceutical and biomedical applications. A glycol chitosan-based thermogel polymer synthesized by the simple N-hexanoyl group of glycol chitosan (HGC) has been reported. While conventional thermogel polymers are based on synthetic block copolymer structures, this thermogel polymer is the only polysaccharide-based polymer capable of exhibiting thermosensitivity around body temperature (~37°C) without the need for chemical crosslinkers, grafting, or blending systems. HGC exhibits thermoreversible sol-gel transition behavior in aqueous solutions at low polymer concentrations. However, conventional thermosetting polymers, such as glycol chitosan-based thermogels, have poor mechanical properties and rapid degradation under physiological conditions, limiting their practical clinical applications.

[0005] The technical challenge of the present invention is to provide a glycol chitosan derivative as a novel functional thermogel platform capable of exhibiting both thermosetting and chemical functionality. The alkenyl groups of the glycol chitosan derivative are hydrophobic, enabling physical crosslinking through hydrophobic interactions, and the double bonds enable UV crosslinking. The chemical and physical properties of the glycol chitosan derivative, which is capable of dual crosslinking, have been finely tuned for thermosetting and UV crosslinking. The physicochemical and biological properties were investigated for biomedical applications such as injectable delivery systems and tissue engineering.

[0006] However, the problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

[0007] One embodiment of the present invention provides a glycol chitosan derivative having a double bond reactive group capable of physical crosslinking and chemical crosslinking by heat, having a structure represented by the following chemical formula 1:

[0008] [Chemical Formula 1]

[0009] .

[0010] (In the above chemical formula 1, R1 includes one selected from the group consisting of an alkenyl group having 1 to 18 carbon atoms, a haloalkenyl group having 1 to 18 carbon atoms, a cycloalkenyl group having 3 to 8 carbon atoms, and an alkenylcarbonyl group having 1 to 8 carbon atoms, and the three monomers modified by x, y, and z are randomly arranged, and x, y, and z are average mole fractions of each monomer, and 0.1≤x≤0.7, 0.1≤y≤0.5, and 0.1≤z≤0.7, and x + y + z = 1)

[0011] In one embodiment of the present invention, R1 is , and R2 and R3 may each independently be H, an alkyl group having 1 to 5 carbon atoms, or OH.

[0012] In one embodiment of the present invention, R2 may be -CH3, and R3 may be H.

[0013] In one embodiment of the present invention, 0.46≤z≤0.53 may be satisfied.

[0014] In one embodiment of the present invention, the degree of crosslinking may be 30% to 75% after the photocrosslinking reaction.

[0015] In one embodiment of the present invention, the degree of crosslinking can be 37% to 72% after the photocrosslinking reaction.

[0016] In one embodiment of the present invention, the weight average molecular weight may be 200 to 5,000,000.

[0017] One embodiment of the present invention is represented by the following reaction formula 1, and provides a method for producing a glycol chitosan derivative having a double bond reactive group capable of physical crosslinking and chemical crosslinking by heat as in chemical formula 4 by reacting an N-acetylated glycol chitosan derivative of chemical formula 2 with an R4COX derivative of chemical formula 3:

[0018] [Reaction Formula 1]

[0019] .

[0020] (In the above reaction formula 1, the R4 includes one selected from the group consisting of an alkenyl group having 1 to 18 carbon atoms, a haloalkenyl group having 1 to 18 carbon atoms, a cycloalkenyl group having 3 to 8 carbon atoms, and an alkenylcarbonyl group having 1 to 8 carbon atoms, and the X is a leaving group, and the two monomers modified by m and n are randomly arranged, and m and n are average mole fractions of each monomer of the above chemical formula 2, and 0.1≤m≤0.7, 0.3≤n≤0.9, and m + n = 1, and the three monomers modified by o, p, and q are randomly arranged, and the o, p, and q are average mole fractions of each monomer of the above chemical formula 4, and 0.1≤o≤0.7, 0.1≤p≤0.5, and 0.1≤q≤0.7, and o + p + q = 1)

[0021] In one embodiment of the present invention, X may be a hydroxy group; a halogen substituent; an alkoxy group having 1 to 4 carbon atoms; or -OC(=O)-R4.

[0022] In one embodiment of the present invention, R4 is , and R5 and R6 may each independently be H, an alkyl group having 1 to 5 carbon atoms, or OH.

[0023] In one embodiment of the present invention, R5 may be -CH3 and R6 may be H.

[0024] One embodiment of the present invention provides a drug and cell delivery vehicle comprising the glycol chitosan derivative.

[0025] One embodiment of the present invention provides a hydrogel comprising the glycol chitosan derivative, wherein the hydrogel is capable of carrying a drug or cell.

[0026] In one embodiment of the present invention, the glycol chitosan derivative is photocrosslinked, and the elastic modulus (G') of the hydrogel may be 10 to 5000 Pa.

[0027] In one embodiment of the present invention, the concentration of the glycol chitosan derivative is 2 to 10 wt%, and the hydrogel can undergo a sol-gel transition in the range of 19 to 54°C.

[0028] The aqueous solution of the glycol chitosan derivative of the present invention exhibited a thermoreversible sol-gel transition in the range of 21-54°C, and the gelation temperature could be controlled by the polymer concentration and degree of substitution. Photocrosslinking of the glycol chitosan derivative hydrogel using ultraviolet light enabled the preparation of a chemically crosslinked hydrogel with improved mechanical properties. Thermogels containing the glycol chitosan derivative exhibited thermosensitive sol-gel transition characteristics in the concentration range of 2-6 wt%. Cytotoxicity studies using the MTT and Live&Dead assays revealed that the hydrogel containing the glycol chitosan derivative was not cytotoxic.

[0029] Figure 1 shows the double cross-linking system of MGC thermogel.

[0030] Figure 2 shows the synthesis process of methacryl glycol chitosan.

[0031] Figure 3a shows the GC and MGC 1 Shows the H-NMR spectrum.

[0032] Figure 3b shows the ATR-FTIR spectra of GC and MGC.

[0033] Figure 4a is a sol-gel transition phase diagram of an MGC solution obtained by the tube inversion method.

[0034] Figure 4b shows the degree of methacrylation (DM) according to the supply molar ratio of methacrylic anhydride.

[0035] Figure 5a shows the temperature-dependent rheological behavior of MGC39.

[0036] Figure 5b shows the temperature-dependent rheological behavior of MGC46.

[0037] Figure 5c shows the temperature-dependent rheological behavior of MGC50.

[0038] Figure 5d shows the temperature-dependent rheological behavior of MGC53.

[0039] Figure 5e shows the temperature-dependent rheological behavior of MGC59.

[0040] Figure 6a shows the viscoelastic properties according to the increase in temperature at an MGC50 concentration of 3 wt%.

[0041] Figure 6b shows the viscoelastic properties according to the increase in temperature at an MGC50 concentration of 4 wt%.

[0042] Figure 6c shows the viscoelastic properties according to the increase in temperature at a MGC50 concentration of 5 wt%.

[0043] Figure 6d shows the viscoelastic properties according to the increase in temperature at a MGC50 concentration of 6 wt%.

[0044] Figure 7 shows the changes in the ATR-FTIR spectrum for the photocrosslinked MGC hydrogel.

[0045] Figure 8 shows the deconvolution of the absorption band of the methacrylic group.

[0046] Figure 9 shows the change in elastic modulus of the photocrosslinked MGC hydrogel according to frequency change.

[0047] Figure 10a shows the results of a physical stability test of MGC hydrogel and photocrosslinked MGC50-30 and MGC50-60 hydrogels in PBS solution.

[0048] Figure 10b shows the results of in vitro biodegradability tests of MGC50-30 and MGC50-60 hydrogels.

[0049] Figure 11a shows the internal structure of a freeze-dried MGC50 hydrogel sample.

[0050] Figure 11b shows the internal structure of the freeze-dried photocrosslinked MGC50-30 hydrogel sample.

[0051] Figure 11c shows the internal structure of the freeze-dried photocrosslinked MGC50-90 hydrogel sample.

[0052] In this specification, when a part is said to "include" a certain component, this does not mean that it excludes other components, but rather that it may include other components, unless otherwise specifically stated.

[0053] In this specification, “A and / or B” means “A and B, or A or B.”

[0054] Hereinafter, the present invention will be described in more detail.

[0055] One embodiment of the present invention provides a glycol chitosan derivative having a double bond reactive group capable of physical crosslinking and chemical crosslinking by heat, having a structure represented by the following chemical formula 1:

[0056] [Chemical Formula 1]

[0057] .

[0058] (In the above chemical formula 1, R1 includes one selected from the group consisting of an alkenyl group having 1 to 18 carbon atoms, a haloalkenyl group having 1 to 18 carbon atoms, a cycloalkenyl group having 3 to 8 carbon atoms, and an alkenylcarbonyl group having 1 to 8 carbon atoms, and the three monomers modified by x, y, and z are randomly arranged, and x, y, and z are average mole fractions of each monomer, and 0.1≤x≤0.7, 0.1≤y≤0.5, and 0.1≤z≤0.7, and x + y + z = 1)

[0059] One embodiment provides a thermogel comprising the glycol chitosan derivative. The glycol chitosan derivative of the present invention is capable of dual crosslinking, and may be capable of physical crosslinking and chemical crosslinking. The physical crosslinking may be thermal crosslinking, and the chemical crosslinking may be photocrosslinking. Fig. 1 illustrates a dual crosslinking system of a thermogel comprising the glycol chitosan derivative of the present invention. Thermal crosslinking increases hydrophobic interactions, and photocrosslinking generates covalent bonds between alkenyl groups.

[0060] In one embodiment of the present invention, R1 is , and R2 and R3 may each independently be H, an alkyl group having 1 to 5 carbon atoms, or OH.

[0061] In one embodiment of the present invention, R2 may be -CH3, and R3 may be H.

[0062] In one embodiment of the present invention, z may be 0.46≤z≤0.53. In one embodiment of the present invention, z is 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49 or 0.50 or more, or 0.50, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, It may be 0.67, 0.68, 0.69, or 0.70 or less. In one embodiment of the present invention, the z may be 0.39, 0.46, 0.50, 0.53, or 0.59.

[0063] In one embodiment of the present invention, after the photocrosslinking reaction, it may have a crosslinking degree of 30% to 75%. In one embodiment of the present invention, after the photocrosslinking reaction, it may have a crosslinking degree of 37% to 72%. In one embodiment of the present invention, after the photocrosslinking reaction, it may have a crosslinking degree of 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, or 45% or more, or a crosslinking degree of 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, or 75% or less.

[0064] In one embodiment of the present invention, the weight average molecular weight may be 200 to 5,000,000.

[0065] One embodiment of the present invention is represented by the following reaction formula 1, and provides a method for producing a glycol chitosan derivative having a double bond reactive group capable of physical crosslinking and chemical crosslinking by heat as in chemical formula 4 by reacting an N-acetylated glycol chitosan derivative of chemical formula 2 with an R4COX derivative of chemical formula 3:

[0066] [Reaction Formula 1]

[0067] .

[0068] (In the above reaction formula 1, the R4 includes one selected from the group consisting of an alkenyl group having 1 to 18 carbon atoms, a haloalkenyl group having 1 to 18 carbon atoms, a cycloalkenyl group having 3 to 8 carbon atoms, and an alkenylcarbonyl group having 1 to 8 carbon atoms, and the X is a leaving group, and the three monomers modified by m and n are randomly arranged, and m and n are average mole fractions of each monomer of the above chemical formula 2, and 0.1≤m≤0.7, 0.3≤n≤0.9, and m + n = 1, and the three monomers modified by o, p, and q are randomly arranged, and the o, p, and q are average mole fractions of each monomer of the above chemical formula 4, and 0.1≤o≤0.7, 0.1≤p≤0.5, and 0.1≤q≤0.7, and o + p + q = 1)

[0069] The N-acetylated glycol chitosan derivative as a starting material has part of the amine group at the 5-position N-acetylated, and can be directly manufactured using a known method or purchased and used commercially. When directly manufactured, the N-acetylated glycol chitosan derivative is manufactured by reacting glycol chitosan with an acetylating agent. The acetylating agent can be selected from acetic anhydride and acetic chloride, and acetic anhydride is preferably used.

[0070] At this time, the reaction is carried out at -10 to 60°C, preferably 15 to 50°C, and the reaction time is 10 to 50 hours, preferably 18 to 30 hours.

[0071] In one embodiment of the present invention, X may be a hydroxy group; a halogen substituent; an alkoxy group having 1 to 4 carbon atoms; or -OC(=O)-R4.

[0072] In one embodiment of the present invention, R4 is , and the R5 and R6 may each independently be H, an alkyl group having 1 to 5 carbon atoms, or OH. In one embodiment of the present invention, the R5 may be -CH3, and the R6 may be H.

[0073] One embodiment of the present invention provides a drug and cell delivery vehicle comprising the glycol chitosan derivative. The drug delivery vehicle may encapsulate and then release a drug. The drug and cell delivery vehicle may be a hydrogel. The hydrogel comprises the glycol chitosan derivative, and the hydrogel can support a drug or cell.

[0074] In the hydrogel comprising the glycol chitosan derivative according to the present invention, the glycol chitosan derivative may be photocrosslinked, and the elastic modulus (G') of the hydrogel may be 10 to 5000 Pa. The elastic modulus is a storage modulus and may be a value measured according to a frequency sweep of 0 to 10 Hz at 37°C. Preferably, the elastic modulus of the hydrogel may be 10 to 4000 Pa.

[0075] In the hydrogel comprising the glycol chitosan derivative according to the present invention, the concentration of the glycol chitosan derivative is 2 to 10 wt%, and the hydrogel can undergo a sol-gel transition in the range of 19 to 54°C. Specifically, the concentration of the glycol chitosan derivative with respect to the total weight of the hydrogel may be 2 to 10 wt%, preferably 2 to 6 wt%. Within the above-described concentration range, the thermoreversible sol-gel phase transition of the hydrogel can be controlled by controlling the concentration. Specifically, the hydrogel can undergo a sol-gel transition in the range of 19 to 54°C, preferably 21 to 54°C. For example, the hydrogel can support a drug or cell for delivery to a treatment target, and then release the drug or cell at a temperature specifically occurring at the body temperature of the treatment target or a target site.

[0076] The glycol chitosan derivative according to the present invention can be applied in various fields such as medicine, biotechnology, and electronics due to its thermoresponsive and photoresponsive properties. For example, it can be applied in various fields such as drug delivery systems for encapsulating and delivering drugs, cell delivery systems for culturing, supporting, and delivering cells, tissue engineering supports, gas storage systems, gas filters, catalyst carriers for chemical reactions, and temperature-sensitive sensors.

[0077] Specifically, considering that most drugs are hydrophobic and poorly soluble, the glycol chitosan derivative of the present invention can easily encapsulate such hydrophobic and poorly soluble drugs due to the hydrophobic groups in the derivative. In particular, the hydrophobic block containing the hydrophobic groups forms self-assemblies to form micelles in an aqueous solution, enabling encapsulation with a high content of hydrophobic and poorly soluble drugs. In addition, since it has sol-gel transition properties, the drug can be released by converting to a gel state by temperature control after encapsulation and then to a sol state by temperature control again, thereby enabling use as an effective drug delivery vehicle.

[0078] At this time, the derivative of the present invention also includes a hydrophilic group (amine group, glycol group), so that hydrophilic drugs can also be easily incorporated.

[0079] The available hydrophilic, hydrophobic and poorly soluble drugs are not particularly limited in the present invention, and any drug known in the art may be used. Representative water-soluble drugs include betamethasone phosphate, dexamethasone phosphate, prednisolone phosphate, prednisolone succinate, hydrocortisone succinate, vancomycin, vincristine, vinblastine, chloramphenicol succinate, latamoxef, cefpirome, carumonam, clindamycin phosphate, and abacavir, while hydrophobic and poorly soluble drugs include testosterone enanthate, testosterone propionate, testosterone, estradiol, estradiol valerate, estradiol benzoate, dexamethasone acetate, betamethasone, betamethasone dipropionate, betamethasone valerate, prednisolone acetate, cyclosporine, tacrolimus, paclitaxel, irinotecan hydrochloride, cisplatin, methotrexate, carmofur, These include tegafur, doxorubicin, clarithromycin, aztreonam, cefnidil, nalidixic acid, ofloxacin, norfloxacin, ketoprofen, flurbiprofen, flurbiprofenaxetil, chlorpromazine, diazepam, nifedipine, nicardipine hydrochloride, amurodipine besilate, candesartan cilexetil, acyclovir, vidarabine, efavirenz, alprostadil, dinoprostone, ubidecarenone, vitamin A (retinol), vitamin D, vitamin E, and vitamin K.

[0080] The glycol chitosan derivative having a hydrophobic substituent according to the present invention can control reversible sol-gel transition depending on temperature as well as biocompatibility and amphiphilic properties, and thus can be applied to bio-fields such as cell delivery vehicles.

[0081] Due to the amphiphilicity of the derivative according to the present invention, cells having either hydrophilic or hydrophobic properties are supported. When the temperature is controlled beyond the sol-gel critical temperature, the supported cells can be detached due to the sol-gel transition of the derivative, allowing its use as a cell delivery vehicle. Furthermore, due to the hydrogel properties of the derivative, it can be used for the culture of various cells.

[0082] The cells that can be used are not particularly limited in the present invention, and any cells, growth factors, peptides, etc. known in this field can be used, and for example, epithelial cells, fibroblasts, osteoblasts, chondrocytes, hepatocytes, human-derived umbilical cord blood cells, and human bone marrow-derived mesenchymal stem cells can be used, and preferably, human bone marrow-derived mesenchymal stem cells can be used. Additionally, transforming growth factor-β (TGF), insulin-like growth factor (IGF), epidermal growth factor (EGF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), and bone morphogenetic protein (BMP) are possible growth factors.

[0083] Hereinafter, the present invention will be described in detail using examples. However, the examples according to the present invention may be modified in various ways, and the scope of the present invention is not limited to the examples described below. The examples in this specification are provided to more fully explain the present invention to those of ordinary skill in the art.

[0084]

[0085] Example

[0086] ingredient

[0087] Glycol chitosan (GC) (DP≥400) was purchased from Fujifilm (Japan). Methacrylic anhydride (97%), lysozyme (63,564 units / mg), dimethyl sulfoxide (DMSO), and 2-hydroxy-2-methylpropiophenone were purchased from Sigma-Aldrich (St. Louis, MO, USA). PBS, a solvent, was purchased from Gibco. TM Dulbecco's phosphate buffered saline was purchased from Thermo Fisher Scientific (USA). Methanol and acetone, the organic solvents used in the synthesis, were purchased from Samjeon Chemical Co., Ltd. (Korea). Triple-distilled water was used, and all other reagents were used without further purification. The dialysis membrane (molecular weight cutoff: 12–14 kDa) was from Spectrum Laboratories (USA).

[0088]

[0089] Example 1. Synthesis and characterization of methacryl glycol chitosan (MGC)

[0090] Methacrylic glycol chitosan (MGC) was synthesized by methacrylation of GC under mild conditions. Figure 2 illustrates the synthetic process of methacrylic glycol chitosan. 1 g of GC was dissolved in 100 mL of distilled water and diluted with 40 mL of methanol. A predetermined amount of methacrylic anhydride was added to the GC solution using magnetic stirring under a nitrogen atmosphere. After continuous stirring at 40°C for 24 h, the polymer was collected by precipitation in acetone. The polymer was then dialyzed against distilled water using a dialysis membrane (molecular weight cutoff: 12–14 kDa) for 2 days and then freeze-dried. The freeze-dried polymer was finally collected and stored in a refrigerator (below 4°C) until use. The chemical composition of MGC was analyzed by proton NMR spectroscopy at 600 MHz using an AVANCE III 600 spectrometer (Bruker, Germany). GC and MGC were dissolved in 1.0 wt% D2O in each tube. The D2O peak at 4.85 ppm was set as the reference peak. The chemical composition of MGC was also confirmed by ATR-FTIR characterization using Nicolet iS 5 (Thermo Scientific, USA). The ATR-FTIR spectra of GC and MGC were obtained in the range of 4000-750 cm -1 4 cm in the frequency range -1 Recorded using 32 scans of resolution.

[0091] MGC with various degrees of N-methacrylate were synthesized from glycol chitosan through a one-step reaction procedure under mild conditions. In one step of the reaction, methacrylic anhydride reacted with the amine group of glycol chitosan through amide bond formation to form MGC. The chemical structures of GC and MGC are 1 It was characterized through H-NMR and ATR-IR spectral analysis. Figure 3a shows the GC and MGC spectra confirming that the methacryl group was successfully introduced into the glycol chitosan backbone. 1H-NMR spectrum is shown. The D2O peak at 4.65 ppm was used as the reference peak. The overlapping peaks at 3-4 ppm are attributed to the protons of the glucopyranosyl ring at positions 2-8 (H-2~H-8). The peak at 2.6 ppm arises from the protons of the primary amine moiety. In the methacrylate derivative, additional peaks at 5.4 and 5.6 ppm appear, indicating the alkenyl double bond of the methacrylate moiety conjugated to glycol chitosan. In addition, the additional peak at 1.84 ppm in the spectrum of methacrylic glycol chitosan is due to the -CH3 of the methacrylic group. The ratio of the average intensity of the proton peak of the methacrylate group (δ=5.4-5.6 ppm) to the proton peak of the chitosan glucosamine (δ=3.0-3.8 ppm) provides the degree of methacrylation according to the following mathematical formula:

[0092] [Mathematical Formula 1]

[0093] Methacrylation (%) = [(integral of methacrylate protons at 5.5 & 5.6 ppm / 2) / (integral of H2 - H8 protons / 10)] × 100

[0094] As shown in Fig. 3b, the chemical structures of GC and MGC were also confirmed by ATR-FTIR spectroscopy. 3400 cm -1 The broadband was assigned to the stretching vibration of the hydroxyl group, which overlapped with the NH stretching vibration in the same region. 2890 cm -1 The absorption peak at 1596 cm is attributed to the CH2 group. -1 The absorption peak at 1596 cm is due to the amino bending vibration of GC. -1 The amino vibration band disappears at 1555 cm after methacrylation. -1 The appearance of amide II band at 1700 cm indicates that MGC was successfully synthesized. Also, -1The absorption peak in is due to the methacrylate carbon-to-carbon double bond after methacrylation. A series of MGCs were synthesized by controlling the degree of methacrylation from 39% to 59% by varying the feed molar ratio of methacrylic anhydride as shown in Table 1 below (yield: 72-76%).

[0095] Sample Methacrylic Anhydride / Glucosamine Residue DM (%) Sol-Gel Transition (℃) Yield (%) T gel a (℃)T gel b (℃)MGC390.739-(Sol)-(Sol)75MGC461.046544372MGC501.250333675MGC531.453212374MGC592.059-(Gel)-(Gel)76

[0096]

[0097] The degree of methacrylation (DM) of MGC increased to 39%, 46%, 50%, 53%, and 59% depending on the molar ratio of methacrylic anhydride supplied (Fig. 4b). T gel a was measured by the tube inversion method at 4 wt%, and T gel b The results were measured using rheological equipment. MGC39, the MGC with the lowest DM (39%), did not exhibit any noticeable heat-sensitive gelation behavior, which is thought to be due to its low content of hydrophobic methacrylic groups. However, MGC59, the MGC with the highest DM (59%), gelled below 4°C, likely due to the increased hydrophobicity caused by the introduction of excessive methacrylic groups. Therefore, in the next stage of experiments, further investigations were conducted on MGC46, MGC50, and MGC59.

[0098]

[0099] Example 2. Study of temperature-sensitive sol-gel transition behavior

[0100] The heat-sensitive sol-gel transition behavior of MGC solutions was investigated using the tube inversion method at a heating rate of 0.2°C / min. Polymer solutions were prepared by dissolving MGC in phosphate-buffered saline (PBS) solution (pH 7.4, 0.01 M) at 4°C, with concentrations of 3.6–5.0 wt%. The solution-gel transition temperature was measured under flowing or no-flow conditions with the tube inverted for 60 s, and each data value represents the average of three measurements with the standard deviation (SD). Sol-gel transition phase diagrams obtained using this method are known to have an accuracy of ±1°C. The sol-gel transition temperature of MGC was also investigated using a rheometer (Waters TA Instruments, Milford, MA, USA). The MGC aqueous solution was placed between parallel plates with a diameter of 20 mm and a gap of 1000 μm. The frequency was optimized to 1 Hz, as determined using frequency sweep experiments. A constant stress of 25 Pa was used for the measurements.

[0101] The heat-sensitive sol-gel transition characteristics of MGC solutions were investigated using the tube-inversion method, viscometry, and rheology. Figure 4a shows the sol-gel transition phase diagram of MGC solutions obtained by the tube-inversion method. Various MGC solutions were prepared by dissolving MGC with DM ranging from 39 to 59% in PBS solution (pH 7.4, 0.01 M) at concentrations ranging from 2 to 6 wt%. The sol-gel transition temperatures of the MGC solutions could be well controlled from 19 to 54°C by varying the DM and MGC concentrations. A decrease in the sol-gel transition temperature was observed with increasing DM or polymer concentration, which may be due to the higher physical cross-linking density formed by enhanced hydrophobic interactions between the methacrylic groups of MGC.

[0102] The viscoelastic properties, storage modulus (G′), and loss modulus (G″) of MGC39, MGC46, MGC50, MGC53, and MGC59 were studied through rheological experiments at temperatures ranging from 10 to 45°C. Figures 5a to 5e show the temperature-dependent rheological behaviors of 4 wt% aqueous solutions of a, b, c, d, and e, MGC59, respectively. This rheological analysis yielded more reproducible and quantitative results than the tube inversion method. As the temperature increased and gelation progressed, both the storage modulus (G′) and the loss modulus (G″) increased, and in particular, the rate of increase of G′ was much faster than that of G″. The difference in the rates of increase of G′ and G″ creates a crossover point (defined as the gelation point), which indicates the transition from a viscous liquid phase to an elastic solid phase (Journal of Rheology, 30(2), 367-382). The G′ values ​​of MGC39, MGC46, MGC50, and MGC53 were initially lower than G″, but increased rapidly with increasing temperature. The point where the G′ and G″ values ​​intersect is similar to the previous results of the tube inversion method. In the case of MGC59, since G″ was initially higher than G′, it is assumed that the sol-gel phase transition occurred below 10℃.

[0103] Figure 6 shows the viscoelastic properties according to the increase in temperature at MGC50 concentrations of a 3 wt%, b 4 wt%, c 5 wt%, and d 6 wt%. It can be seen that the crossover point decreases as the MGC50 concentration increases. It can be seen that the higher the MGC50 concentration, the more hydrophobic parts there are, so the hydrophobic interaction increases and the sol-gel phase transition temperature decreases. For this reason, MGC50 (6 wt%) has the lowest sol-gel phase transition temperature of 23°C.

[0104]

[0105] Example 3. Photocrosslinking of MGC

[0106] An aqueous solution of MGC in phosphate buffered saline (PBS, pH 7.4, 4.0 wt%) containing photoinitiator I2959 (0.05 wt%) was poured into a cylindrical silicone mold (diameter 10 mm × thickness 2 mm). Then, the solution was exposed to ultraviolet light (365 nm, 20 mW / cm 2 ) for 30–60 min to prepare chemically cross-linked MGC hydrogels. The photocross-linking reaction of MGC was tracked by ATR-FTIR measurements, observing the change in characteristic peaks corresponding to methacrylate groups as a function of UV irradiation time.

[0107] MGC aqueous solution can solidify to form a non-flowable transparent gel at temperatures below 37°C, similar to body temperature. The MGC thermogel was further modified through UV crosslinking to obtain a chemically crosslinked elastic hydrogel. The thermogel was exposed to UV light for 30 and 60 minutes, respectively, and the degree of crosslinking reaction was determined by identifying the methacrylate groups according to the UV irradiation time. The changes in the ATR-FTIR spectra of the photocrosslinked MGC hydrogel are shown in Figure 7. 1650 cm -1 The decrease in the vibrational band in is due to the conversion of the methacrylic group.

[0108] For the kinetic study of the crosslinking reaction, a deconvolution process was performed to obtain a single Gaussian curve corresponding to the carbon-carbon double bond band. Figure 8 shows the deconvolution for the absorption band of the methacrylic group at 1650 cm at different times. -1 The degree of crosslinking (DC) can be calculated using the integral value A of the absorption band according to the following mathematical formula 2: (Macromolecules, 35(16), 6377-6383). MGC 50 in Fig. 8 is not irradiated with UV light, and MGC 50-30, MGC 50-60, MGC 50-90, and MGC 50-120 represent absorption for 30 minutes, 60 minutes, 90 minutes, and 120 minutes, respectively.

[0109] [Equation 2]

[0110] degree of crosslinking (DC) = 1 - (A t / A0)

[0111] The study on MGC50 revealed that the double bond peak gradually decreased over time upon UV irradiation. MGC 50-30, MGC 50-60, MGC 50-90, and MGC 50-120 showed crosslinking degrees (DC) of 37%, 45%, 72%, and 69%, respectively. The critical time was identified as 90 minutes (crosslinking degree = 72%).

[0112]

[0113] Example 4. Rheological analysis

[0114] The rheological behavior of MGC hydrogels and photocrosslinked MGC hydrogels was investigated using a HR10 rheometer (Waters TA Instruments, Milford, USA) with 20 mm titanium parallel plates at 37°C to measure frequency sweeps. MGC solution (4 wt%, PBS) was applied to the rheometer, and the upper plate was lowered with a measurement step size of 500 μm. The storage modulus (G´) and loss modulus (G”) were measured as a function of frequency. In the same manner, the frequency sweep was studied for the photocrosslinked MGC hydrogel after UV irradiation. The frequency sweep for the hydrogels was performed from 0.01 to 10 Hz at a strain of 0.01%.

[0115] The mechanical properties of physically cross-linked MGC and chemically cross-linked (photocross-linked) MGC hydrogels were investigated and compared through oscillatory rheology experiments at 37°C. The storage modulus, which represents the formation of an elastic network, was measured for all hydrogel groups. The elastic modulus (G′ component) in Figure 9 was determined as a function of frequency sweep at 37°C. Referring to Figure 9, the hydrogels containing photocross-linked glycol chitosan derivatives can have an elastic modulus (G′) of 10 to 500 Pa. The photocross-linked MGC hydrogels exhibited significantly higher G′ values ​​than the physically cross-linked MGC thermogels across the entire frequency range due to the formation of a robust network structure through strong covalent bonds. In addition, the G′ of the MGC50-60 hydrogel was found to be slightly higher than that of the MGC50-30 hydrogel, indicating that the higher content of methacrylate groups leads to a denser cross-linked network, resulting in higher elasticity. In summary, the results show that chemically cross-linked hydrogels have significantly higher storage modulus than physically cross-linked hydrogels, and that prolonged UV irradiation can further increase the cross-linking density of the hydrogels.

[0116]

[0117] Example 5. In vitro gel stability and enzymatic biodegradation

[0118] The biodegradation of MGC50 hydrogel and photocrosslinked MGC50 hydrogel was investigated in vitro by measuring the weight change of MGC hydrogel during incubation in the presence or absence of lysozyme. MGC50 solution (PBS, 1 mL, 4 wt%) was placed in a 5 mL vial and incubated in a water bath (series BS-21, Lab Companion, Korea) at 37°C for 15 min to form a gel. MGC50 solution (PBS, 1 mL, 4 wt%) containing photoinitiator I2959 (0.05 wt%) was prepared and poured into a cylindrical silicone mold (10 mm in diameter × 2 mm in thickness). Then, the solution was exposed to UV light (365 nm, 20 mW / cm) for 30–60 min. 2 ) were exposed to chemically cross-linked MGC hydrogels. The hydrogels were incubated in 2 mL of PBS solution containing either no lysozyme or 5 mg / mL lysozyme. The hydrogels were weighed at predetermined time intervals and re-immersed in fresh PBS. All experiments were performed in triplicate. The percentage of residual hydrogel weight was calculated using the following equation:

[0119] [Equation 3]

[0120] Residual weight (%) = (W s - W i ) / W i × 100(%)

[0121] Here W i is the initial weight of the hydrogel, and W s is the weight of the hydrogel sample as a function of time.

[0122] To determine the physical stability of the MGC hydrogel and the photocrosslinked MGC50-30 and MGC50-60 hydrogels, stability tests were performed in PBS solution. As shown in Fig. 10a, MGC showed a gradual weight increase due to initial swelling for up to 2 days. However, after 3 days, the residual weight decreased rapidly due to mass loss of the MGC hydrogel. In contrast, the photocrosslinked MGC hydrogels did not show significant mass loss for both MGC50-30 and MGC50-60 over 50 days of culture, as shown in Fig. 10a. In conclusion, it was confirmed that the photocrosslinked MGC hydrogels exhibited excellent physical stability.

[0123] To investigate the in vitro biodegradability of MGC hydrogels and photocrosslinked MGC50-30 and MGC50-60 hydrogels, hydrogel samples were incubated in PBS solution containing lysozyme (5 mg / mL) at 37°C. Figure 10b shows the weight changes over time of MGC hydrogels and photocrosslinked MGC50-30 and MGC50-60 hydrogels for biodegradability investigation. MGC hydrogels exhibited very rapid dissolution behavior, losing 100% of their total mass within 1 day, whereas a continuous mass loss rate was observed for photocrosslinked MGC50-30 and MGC50-60 hydrogels. The MGC50-60 hydrogel with a higher crosslinking density was found to degrade more slowly than the MGC50-30 hydrogel. The sustained mass loss profiles of the MGC50-60 and MGC50-30 hydrogels compared to the MGC hydrogels indicate that the introduction of a photocrosslinking system can delay degradation due to the enhanced steric hindrance effect caused by the formation of a chemical bonding network. Therefore, the enzymatic degradation of the hydrogels can be controlled by adjusting the degree of crosslinking. These tunable properties of the MGC hydrogels may be useful in various biomedical applications, such as drug delivery systems and tissue engineering.

[0124]

[0125] Example 6. Morphological study

[0126] The morphological structure of the MGC hydrogels was visualized using a field-emission transmission electron microscope (FE-SEM; S-4800, Hitachi, Japan) operating at 10 kV. The hydrogel samples were frozen in liquid nitrogen, rapidly fractured to reveal the internal structure, and then freeze-dried for 4 days. All samples were coated with platinum for 60 s.

[0127] To investigate the morphological characteristics of the MGC50 hydrogel, the cross-sectional morphology of the freeze-dried hydrogel samples was observed using FE-SEM. Figure 11 shows the internal structure of the freeze-dried hydrogel samples. Figures a, b, and c in Figure 11 represent MGC50, photocross-linked MGC50-30, and photocross-linked MGC50-90, respectively. The MGC50 hydrogel was observed to have a microporous structure with irregular pore sizes. Compared to the thermogel before photocross-linking, the photocross-linked hydrogel exhibited a well-developed pore structure with relatively regular sizes and shapes. After photocross-linking, the MGC50-30 and MGC50-90 hydrogels exhibited a sponge-like microporous structure with pore sizes of 20-25 and 10-15 μm, respectively. The pores were interconnected to form an open channel structure in a three-dimensional network. As observed in the SEM images, the pore size of MGC50-90 was smaller than that of MGC50-30. This difference in pore structure may be due to the introduction of chemical crosslinking and the difference in crosslinking density. The relatively high crosslinking density of the photocrosslinked MGC-90 hydrogel may reduce the pore size.

Claims

1. A glycol chitosan derivative having a double bond reactive group capable of physical crosslinking and chemical crosslinking by heat, having a structure represented by the following chemical formula 1: [Chemical Formula 1] . (In the above chemical formula 1, Above R 1 It comprises one selected from the group consisting of an alkenyl group having 1 to 18 carbon atoms, a haloalkenyl group having 1 to 18 carbon atoms, a cycloalkenyl group having 3 to 8 carbon atoms, and an alkenylcarbonyl group having 1 to 8 carbon atoms, The three monomers, which are expressed as x, y, and z above, are arranged randomly, The above x, y, and z are the average mole fractions of each monomer, 0.1≤x≤0.7, 0.1≤y≤0.5, and 0.1≤z≤0.7, and x + y + z = 1.

2. In the first paragraph, the R 1 silver And, the above R 2 and R 3 A glycol chitosan derivative, wherein each independently represents H, an alkyl group having 1 to 5 carbon atoms, or OH.

3. In the second paragraph, the R 2 is -CH 3 And, the above R 3 A glycol chitosan derivative characterized by being H.

4. A glycol chitosan derivative characterized in that 0.46≤z≤0.53 in the first paragraph.

5. A glycol chitosan derivative characterized in that it has a crosslinking degree of 30% to 75% after the photocrosslinking reaction in the first paragraph.

6. A glycol chitosan derivative according to claim 1, characterized in that the weight average molecular weight is 200 to 5,000,000.

7. It is represented by the following reaction formula 1, N-acetylated glycol chitosan derivative of chemical formula 2 and R of chemical formula 3 4 A method for producing a glycol chitosan derivative having a double bond reactive group capable of physical crosslinking and chemical crosslinking by heat as shown in chemical formula 4 by reacting a COX derivative: [Reaction Formula 1] . (In the above reaction formula 1, Above R 4 It includes one selected from the group consisting of an alkenyl group having 1 to 18 carbon atoms, a haloalkenyl group having 1 to 18 carbon atoms, a cycloalkenyl group having 3 to 8 carbon atoms, and an alkenylcarbonyl group having 1 to 8 carbon atoms, The above X is a leaving group, The two monomers, which are expressed as m and n above, are arranged randomly, The above m and n are the average molar fractions of each monomer of the above chemical formula 2, 0.1≤m≤0.7, 0.3≤n≤0.9, and m + n = 1. The three monomers, formulated as o, p, and q above, are arranged randomly, The above o, p, and q are the average mole fractions of each monomer of the above chemical formula 4, 0.1≤o≤0.7, 0.1≤p≤0.5, and 0.1≤q≤0.7, and o + p + q = 1.

8. In the 7th paragraph, X is a hydroxyl group; a halogen substituent; an alkoxy group having 1 to 4 carbon atoms; or -OC(=O)-R 4 A method for producing a glycol chitosan derivative characterized by:

9. In paragraph 7, the R 4 Is And, the above R 5 and R 6 A method for producing a glycol chitosan derivative, wherein each independently represents H, an alkyl group having 1 to 5 carbon atoms, or OH.

10. In paragraph 9, the R 5 is -CH 3 And, the above R 6 A method for producing a glycol chitosan derivative, characterized in that it is H.

11. A hydrogel comprising a glycol chitosan derivative of clause 1, The above hydrogel is a hydrogel capable of carrying a drug or cell.

12. In the 11th paragraph, the glycol chitosan derivative is photocrosslinked, A hydrogel having an elastic modulus (G') of 10 to 5000 Pa.

13. In the 11th paragraph, the concentration of the glycol chitosan derivative is 2 to 10 wt%, The above hydrogel is a hydrogel capable of undergoing a sol-gel transition in the range of 19 to 54°C.

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