Double-crosslinking ethylene glycol chitosan derivative

Abstract

The ethylene glycol chitosan derivative aqueous solution shows thermal reversible sol-gel transition within the range of 21 DEG C to 54 DEG C, and the gelling temperature can be controlled through polymer concentration and substitution degree. According to the present invention, the ethylene glycol chitosan derivative hydrogel is subjected to photo-crosslinking by using ultraviolet light so as to prepare the chemically-crosslinked hydrogel with improved mechanical characteristics; the temperature-sensitive gel comprising the ethylene glycol chitosan derivative exhibits a temperature-sensitive sol-gel transition characteristic in a concentration range of 2 wt% to 6 wt%. In addition to the temperature response characteristic, the chemically crosslinked temperature-sensitive gel can also have various physical performance ranges according to different photo-crosslinking times, polymer concentrations and substitution rates, and can be expected to be used for various biomedical applications.

Description

Technical Field

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

[0002] This invention relates to dual-crosslinkable glycolchitosan derivatives, specifically glycolchitosans containing alkenyl groups capable of both physical and chemical crosslinking. Background Technology

[0003] Hydrogels have garnered significant attention across various research fields over the past decades due to their promising properties, including biocompatibility, high water content, tunable physicochemical characteristics, and irritation sensitivity. Among hydrogels, thermosensitive gel polymers undergoing a heat-sensitive sol-gel transition in aqueous media have attracted considerable interest as injectable delivery systems. These hydrogels can readily encapsulate cells or drugs in a sol state, enabling direct delivery to the target site via injection, and can persist for extended periods through heat-induced gelation induced by body temperature. In clinical applications, injectable hydrogels are particularly suitable for treating irregularly shaped tissue sites, thus eliminating the need for custom scaffold designs. Injectable hydrogels can be formed through various materials, including UV irradiation, ionic cross-linking, pH changes, or temperature variations. Natural high-molecular-weight polysaccharides such as alginate, hyaluronic acid, dextran, and chitosan have been proposed for biomedical purposes due to their biodegradability and biocompatibility. In particular, chitosan has been widely studied for its antibacterial, biodegradable, biocompatible, and mucosal adhesive properties, and is used in drug delivery, cell encapsulation, and tissue engineering.

[0004] However, the low water solubility of chitosan itself under physiological conditions is one of the main drawbacks limiting its widespread application as a hydrogel material. Glycol chitosan, as a water-soluble chitosan derivative, readily dissolves in water under physiological conditions due to its glycol functional groups, while retaining the advantageous non-toxic and biodegradable properties of chitosan, making it suitable for pharmaceutical and biomedical applications. Thermosensitive gel polymers based on glycol chitosan (HGC) have been reported to be synthesized via simple N-hexanoylation. In contrast to existing thermosensitive gel polymers based on synthetic block copolymer structures, this thermosensitive gel polymer is the only polysaccharide-based polymer that exhibits temperature sensitivity around body temperature (~37°C) without the need for chemical crosslinking agents or grafting or blending systems. HGC exhibits thermally reversible sol-gel transition behavior in aqueous solutions with low polymer concentrations. However, existing thermosensitive gel polymers, such as glycol chitosan-based thermosensitive gels, have limitations in practical clinical applications due to their weak mechanical properties and rapid degradation in physiological environments. Summary of the Invention

[0005] Technical issues

[0006] The objective of this invention is to provide a novel functional thermosensitive gel platform, comprised of ethylene glycol chitosan derivatives, that exhibits not only thermosensitive gelling properties but also chemical functionality. The alkenyl groups of the ethylene glycol chitosan derivatives are hydrophobic, enabling physical cross-linking through hydrophobic interactions, and also allowing for UV cross-linking due to the double bonds. The chemical and physical properties of the doubly cross-linkable ethylene glycol chitosan derivatives have been fine-tuned for thermosensitive gelling and UV cross-linking. The physicochemical and biological properties have been studied for biomedical applications such as injectable delivery systems and tissue engineering.

[0007] However, the purpose of this invention is not limited to the above-mentioned purposes, and other purposes not mentioned can be clearly understood by those skilled in the art from the following description.

[0008] Solution to the problem

[0009] One embodiment of the present invention provides an ethylene glycol chitosan derivative having the structure of the following chemical formula 1 and containing double-bonded reactive groups capable of thermal physical crosslinking and chemical crosslinking:

[0010] [Chemical Formula 1]

[0011]

[0012] (In the above chemical formula 1, the above R1 includes one of the following groups: alkenyl with 1 to 18 carbon atoms, haloalkenyl with 1 to 18 carbon atoms, cycloalkenyl with 3 to 8 carbon atoms, and alkenylcarbonyl with 1 to 8 carbon atoms. The three monomers modified by the above x, y, and z are randomly arranged, where 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).

[0013] In one embodiment of the present invention, R1 is... R2 and R3 can each be H, an alkyl group having 1 to 5 carbon atoms, or OH.

[0014] In one embodiment of the present invention, R2 may be -CH3 and R3 may be H.

[0015] In one embodiment of the present invention, 0.46≤z≤0.53 can be satisfied.

[0016] In one embodiment of the present invention, the degree of crosslinking can be 30% to 75% after the photocrosslinking reaction.

[0017] In one embodiment of the present invention, the degree of crosslinking can be 37% to 72% after the photocrosslinking reaction.

[0018] In one embodiment of the invention, the weight-average molecular weight may be from 200 to 5,000,000.

[0019] One embodiment of the present invention provides a method for preparing an ethylene glycol chitosan derivative, which is represented by the following reaction formula 1, and by reacting an N-acetylated ethylene glycol chitosan derivative of chemical formula 2 with an R4COX derivative of chemical formula 3 to prepare an ethylene glycol chitosan derivative of chemical formula 4 having double bond reactive groups capable of thermally induced physical crosslinking and chemical crosslinking:

[0020] [Reaction Formula 1]

[0021]

[0022] (In the above reaction formula 1, the above R4 comprises one of the following groups: alkenyl with 1 to 18 carbon atoms, haloalkenyl with 1 to 18 carbon atoms, cycloalkenyl with 3 to 8 carbon atoms, and alkenylcarbonyl with 1 to 8 carbon atoms. The above X is a leaving group. The two monomers modified by the above m and n are randomly arranged, where m and n are the average mole fractions of each monomer in the above chemical formula 2, 0.1≤m≤0.7, 0.3≤n≤0.9, and m+n=1. The three monomers modified by the above o, p, and q are randomly arranged, where o, p, and q are the average mole fractions of each monomer in 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.)

[0023] In one embodiment of the present invention, X may be a hydroxyl group; a halogen substituent; an alkoxy group having 1 to 4 carbon atoms; or -OC (=O)-R4.

[0024] In one embodiment of the present invention, R4 may be... R5 and R6 can each be H, an alkyl group having 1 to 5 carbon atoms, or OH.

[0025] In one embodiment of the present invention, R5 may be -CH3 and R6 may be H.

[0026] One embodiment of the present invention provides a drug and a cell carrier comprising the above-mentioned ethylene glycol chitosan derivative.

[0027] One embodiment of the present invention provides a hydrogel comprising the above-mentioned ethylene glycol chitosan derivative, wherein the hydrogel is a hydrogel capable of loading drugs or cells.

[0028] In one embodiment of the present invention, the above-mentioned ethylene glycol chitosan derivative is photocrosslinked, and the elastic modulus (G') of the above-mentioned hydrogel can be from 10 Pa to 5000 Pa.

[0029] In one embodiment of the present invention, the concentration of the above-mentioned ethylene glycol chitosan derivative is 2% to 10% by weight, and the above-mentioned hydrogel is capable of undergoing sol-gel transition in the range of 19°C to 54°C.

[0030] The effects of the invention

[0031] The aqueous solution of the ethylene glycol chitosan derivative of this invention exhibits a thermally reversible sol-gel transition in the range of 21°C to 54°C, and the gelation temperature can be controlled by the polymer concentration and degree of substitution. By photocrosslinking the ethylene glycol chitosan derivative hydrogel with ultraviolet light, chemically crosslinked hydrogels with improved mechanical properties can be prepared. Thermosensitive gels containing ethylene glycol chitosan derivatives exhibit temperature-sensitive sol-gel transition characteristics in the concentration range of 2% to 6% by weight. In cytotoxicity studies using MTT (thiazolyl blue) and Live & Dead (live and dead cell staining) assays, the hydrogels containing ethylene glycol chitosan derivatives showed no cytotoxicity. Attached Figure Description

[0032] Figure 1 This illustrates a dual crosslinking system for MGC (Methacrylated Glycol Chitosan) thermosensitive gel.

[0033] Figure 2 The synthesis process of methacrylamide glycol chitosan is shown.

[0034] Figure 3a Showing GC and MGC 1 H-NMR spectrum.

[0035] Figure 3b The ATR-FTIR spectra of GC and MGC are shown.

[0036] Figure 4a This is the sol-gel phase transition diagram of the MGC solution obtained by tube reversal method.

[0037] Figure 4b The degree of methacrylation (DM) is shown based on the feed molar ratio of methacrylic anhydride.

[0038] Figure 5a The temperature-dependent rheological behavior of MGC39 is shown.

[0039] Figure 5b The temperature-dependent rheological behavior of MGC46 is shown.

[0040] Figure 5c The temperature-dependent rheological behavior of MGC50 is shown.

[0041] Figure 5d The temperature-dependent rheological behavior of MGC53 is shown.

[0042] Figure 5e The temperature-dependent rheological behavior of MGC59 is shown.

[0043] Figure 6aThe viscoelastic properties of MGC50 at a concentration of 3% by weight as temperature increases are shown.

[0044] Figure 6b The viscoelastic properties of MGC50 at a concentration of 4 wt% as temperature increases are shown.

[0045] Figure 6c The viscoelastic properties of MGC50 at a concentration of 5% by weight as temperature increases are shown.

[0046] Figure 6d The viscoelastic properties of MGC50 at a concentration of 6 wt% as temperature increases are shown.

[0047] Figure 7 The changes in the ATR-FTIR spectrum of the photocrosslinked MGC hydrogel are shown.

[0048] Figure 8 The deconvolution of the absorption band for methacryloyl is shown.

[0049] Figure 9 The change in elastic modulus of the photocrosslinked MGC hydrogel with frequency is shown.

[0050] Figure 10a The results show the physical stability of MGC hydrogel and photocrosslinked MGC50-30 and MGC50-60 hydrogels in PBS solution.

[0051] Figure 10b The results are for the in vitro biodegradability tests of MGC50-30 and MGC50-60 hydrogels.

[0052] Figure 11a The internal structure of the freeze-dried MGC50 hydrogel sample is shown.

[0053] Figure 11b The internal structure of the freeze-dried photocrosslinked MGC50-30 hydrogel sample is shown.

[0054] Figure 11c The internal structure of the freeze-dried photocrosslinked MGC50-90 hydrogel sample is shown. Detailed Implementation

[0055] In this specification, when a part is referred to as "including (contains)" a constituent element, unless otherwise stated, this does not mean that other constituent elements are excluded, but rather that other constituent elements may be further included.

[0056] In this specification, "A and / or B" means "A and B, or A or B".

[0057] The present invention will now be described in more detail.

[0058] One embodiment of the present invention provides an ethylene glycol chitosan derivative having the structure of the following chemical formula 1 and containing double-bonded reactive groups capable of thermal physical crosslinking and chemical crosslinking:

[0059] [Chemical Formula 1]

[0060]

[0061] (In the above chemical formula 1, the above R1 includes one of the following groups: alkenyl with 1 to 18 carbon atoms, haloalkenyl with 1 to 18 carbon atoms, cycloalkenyl with 3 to 8 carbon atoms, and alkenylcarbonyl with 1 to 8 carbon atoms. The three monomers modified by the above x, y, and z are randomly arranged, where 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).

[0062] One embodiment provides a thermosensitive gel comprising the above-described ethylene glycol chitosan derivative. The ethylene glycol chitosan derivative of the present invention is capable of dual crosslinking and can be a derivative capable of both physical and chemical crosslinking. The physical crosslinking can be thermal crosslinking, and the chemical crosslinking can be photocrosslinking. Figure 1 This illustrates a dual crosslinking system for a thermosensitive gel comprising the ethylene glycol chitosan derivative of the present invention. Hydrophobic interactions are increased through thermal crosslinking, and covalent bonds are formed between alkenyl groups through photocrosslinking.

[0063] In one embodiment of the present invention, R1 is... R2 and R3 can each be H, an alkyl group having 1 to 5 carbon atoms, or OH.

[0064] In one embodiment of the present invention, R2 may be -CH3 and R3 may be H.

[0065] In one embodiment of the present invention, z may satisfy 0.46 ≤ z ≤ 0.53. In another embodiment of the present invention, z may be 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 may be 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, 0.67, 0.68, 0.69 or less. In one embodiment of the present invention, z may be 0.39, 0.46, 0.50, 0.53 or 0.59.

[0066] In one embodiment of the present invention, the degree of crosslinking after the photocrosslinking reaction can be 30% to 75%. In another embodiment of the present invention, the degree of crosslinking after the photocrosslinking reaction can be 37% to 72%. In yet another embodiment of the present invention, the degree of crosslinking after the photocrosslinking reaction can be 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, or 45% or more, or less than 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, or 75%.

[0067] In one embodiment of the invention, the weight-average molecular weight may be from 200 to 5,000,000.

[0068] One embodiment of the present invention provides a method for preparing an ethylene glycol chitosan derivative, which is represented by the following reaction formula 1, and by reacting an N-acetylated ethylene glycol chitosan derivative of chemical formula 2 with an R4COX derivative of chemical formula 3 to prepare an ethylene glycol chitosan derivative of chemical formula 4 having double bond reactive groups capable of thermally induced physical crosslinking and chemical crosslinking:

[0069] [Reaction Formula 1]

[0070]

[0071] (In the above reaction formula 1, the above R4 comprises one of the following groups: alkenyl with 1 to 18 carbon atoms, haloalkenyl with 1 to 18 carbon atoms, cycloalkenyl with 3 to 8 carbon atoms, and alkenylcarbonyl with 1 to 8 carbon atoms. The above X is a leaving group. The three monomers modified by the above m and n are randomly arranged, where m and n are the average mole fractions of each monomer in the above chemical formula 2, 0.1≤m≤0.7, 0.3≤n≤0.9, and m+n=1. The three monomers modified by the above o, p, and q are randomly arranged, where o, p, and q are the average mole fractions of each monomer in 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.)

[0072] The N-acetylated ethylene glycol chitosan derivative, used as a starting material, is a derivative in which part of the amino group at position 5 is N-acetylated. It can be prepared directly using known methods or by purchasing commercially available products. In the case of direct preparation, the N-acetylated ethylene glycol chitosan derivative is prepared by reacting ethylene glycol chitosan with an acetylation agent. Acetic anhydride and acetyl chloride can be selected as the acetylation agent, with acetic anhydride being preferred.

[0073] At this time, the reaction temperature is -10°C to 60°C, preferably 15°C to 50°C, and the reaction time is 10 hours to 50 hours, preferably 18 hours to 30 hours.

[0074] In one embodiment of the present invention, X may be a hydroxyl group; a halogen substituent; an alkoxy group having 1 to 4 carbon atoms; or -OC (=O)-R4.

[0075] In one embodiment of the present invention, R4 may be... R5 and R6 can each be independently H, an alkyl group having 1 to 5 carbon atoms, or OH. In one embodiment of the present invention, R5 can be -CH3, and R6 can be H.

[0076] One embodiment of the present invention provides a drug and cell delivery carrier comprising the above-mentioned ethylene glycol chitosan derivative. The drug delivery carrier may be a carrier that encapsulates a drug for release. The drug and cell delivery carrier may be a hydrogel. The hydrogel comprises the above-mentioned ethylene glycol chitosan derivative and is capable of loading drugs or cells.

[0077] In the hydrogel containing the ethylene glycol chitosan derivative according to the present invention, the ethylene glycol chitosan derivative may be photocrosslinked, and the elastic modulus (G') of the hydrogel may be from 10 Pa to 5000 Pa. The above elastic modulus is a storage modulus, which may be a value measured at 37°C according to a frequency scan from 0 Hz to 10 Hz. Preferably, the elastic modulus of the hydrogel may be from 10 Pa to 4000 Pa.

[0078] In a hydrogel containing the ethylene glycol chitosan derivative according to the present invention, the concentration of the ethylene glycol chitosan derivative is from 2% to 10% by weight, and the hydrogel is capable of undergoing a sol-gel transition in the range of 19°C to 54°C. Specifically, based on the total weight of the hydrogel, the concentration of the ethylene glycol chitosan derivative can be from 2% to 10% by weight, preferably from 2% to 6% by weight. Within the above concentration range, the thermally reversible sol-gel phase transition of the hydrogel can be controlled by controlling the concentration. Specifically, the hydrogel is capable of undergoing a sol-gel transition in the range of 19°C to 54°C, preferably from 21°C to 54°C. For example, the hydrogel can be loaded with drugs or cells for delivery to a therapeutic subject and release the drugs or cells at the body temperature of the therapeutic subject or at a temperature specifically present at the target site.

[0079] The ethylene glycol chitosan derivatives of the present invention, due to their thermal and photoresponsive properties, can be widely used in the fields of medicine, biology, and electronics. For example, they can be used in various fields such as drug delivery carriers for encapsulating and delivering drugs, cell delivery carriers for culturing, supporting, and delivering cells, scaffolds for tissue engineering, gas reservoirs, gas filters, catalyst carriers for chemical reactions, and temperature-sensitive sensors.

[0080] Specifically, considering that most drugs are hydrophobic and poorly soluble, the ethylene glycol chitosan derivative of the present invention can readily encapsulate such hydrophobic and poorly soluble drugs due to the hydrophobic groups within the derivative. In particular, by forming self-assemblies through hydrophobic blocks containing hydrophobic groups, micelles can be formed in aqueous solutions, enabling the encapsulation of hydrophobic and poorly soluble drugs at high concentrations. Furthermore, due to its sol-gel transition properties, after encapsulation, the drug can be released by temperature-controlled conversion to a gel state and then back to a sol state, thus making it an effective drug delivery carrier.

[0081] At this point, the derivatives of the present invention also contain hydrophilic groups (amine groups, glycol groups), and therefore can easily encapsulate hydrophilic drugs.

[0082] The use of hydrophilic, hydrophobic, and poorly soluble drugs is not particularly limited in this invention; any drug known in the art may be used. Representative examples of water-soluble drugs include betamethasone sodium phosphate, dexamethasone sodium phosphate, prednisolone sodium phosphate, prednisolone sodium succinate, hydrocortisone sodium succinate, vancomycin, vincristine, vinblastine, chloramphenicol sodium succinate, latamoxef, cefpirome, carrumonam, clindamycin phosphate, and abacavir, etc. Examples of hydrophobic and poorly soluble drugs include testosterone enanthate, testosterone propionate, testosterone, estradiol, estradiol valerate, estradiol benzoate, dexamethasone acetate, betamethasone, betamethasone dipropionate, betamethasone valerate, etc. Prednisolone acetate, cyclosporine, tacrolimus, paclitaxel, irinotecan hydrochloride, cisplatin, methotrexate, carmoflu, tegafur, doxorubicin, clarithromycin, aztreonam, cefnicotinic acid, nalidixic acid, ofloxacin, norfloxacin, ketoprofen, flurbiprofen, flurbiprofen ester, chlorpromazine, diazepam, nifedipine, nicardipine hydrochloride, amlodipine besylate, candesartan medoxomil, acyclovir, vidarabine, efavirenz, alprostadil, dinoprostadone, ubiquitin, vitamin A (retinol), vitamin D, vitamin E, and vitamin K, etc.

[0083] The ethylene glycol chitosan derivatives with hydrophobic substituents according to the present invention not only have biocompatibility and amphiphilic properties, but also can undergo reversible sol-gel transitions according to temperature, and therefore can also be applied to biological fields such as cell delivery carriers.

[0084] Due to the amphiphilic nature of the derivatives according to the present invention, hydrophilic or hydrophobic cells are supported. When the temperature is adjusted away from the sol-gel critical temperature, the supported cells can detach due to the sol-gel transition of the derivatives, thus enabling their use as cell delivery carriers. Furthermore, due to the hydrogel properties of the derivatives, they can also be used for the culture of various cell types.

[0085] The cells that can be used are not particularly limited in this invention, and any cells, growth factors, peptides, etc., known in the art can be used, such as epithelial cells, fibroblasts, osteoblasts, chondrocytes, hepatocytes, human umbilical cord blood cells, and human bone marrow-derived mesenchymal stem cells, etc., preferably human bone marrow-derived mesenchymal stem cells. In addition, as growth factors, 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) can be used.

[0086] The present invention will be described in detail below through embodiments. However, embodiments of the present invention can be modified into many different forms, and the scope of the present invention should not be construed as limited to the following embodiments. The embodiments in this specification are provided to illustrate the present invention more completely to those skilled in the art.

[0087] Example

[0088] Material

[0089] Ethylene 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, purchased from Gibco, was used as the solvent. TMDulbecco's phosphate buffered saline (Thermo Fisher Scientific, USA) was used. Methanol and acetone, the organic solvents used in the synthesis process, were purchased from Samchun Chemicals Co., Ltd. (Korea). Triple-distilled water was used, and other reagents were used directly without additional purification. Dialysis membranes (molecular weight cutoff: 12-14 kDa) were used from Spectrum Laboratories (USA).

[0090] Example 1. Synthesis and Characterization of Methacrylamide Glycol Chitosan (MGC)

[0091] Methacrylamide glycol chitosan (MGC) was synthesized under mild conditions via GC methacrylation. Figure 2 The synthesis process of methacrylamide glycol chitosan (MGC) is illustrated. 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 under a nitrogen atmosphere using magnetic stirring. After continuous stirring at 40 °C for 24 hours, the polymer was collected by precipitation in acetone. The polymer was then dialyzed in distilled water for 2 days using a dialysis membrane (molecular weight cutoff: 12-14 kDa) and then lyophilized. The lyophilized polymer was finally collected and stored in a refrigerator (below 4 °C) until use. The chemical composition of MGC was analyzed using an AVANCE III 600 spectrophotometer (Bruck, Germany) at 600 MHz by proton NMR spectroscopy. GC and MGC were dissolved in D₂O at a concentration of 1.0 wt% in their respective tubes. 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 a Nicolet iS 5 (Thermo Scientific, USA). ATR-FTIR spectra of GC and MGC were used in the 4000-750 cm⁻¹ range. -1 Within the frequency range of 4cm -1 Recorded from 32 scans at the specified resolution.

[0092] MGCs with varying degrees of N-methacrylate were synthesized from ethylene glycol chitosan via a one-step reaction procedure under mild conditions. In one step of the reaction, methacrylic anhydride reacts with the amino groups of ethylene glycol chitosan via amide bond formation, thereby forming MGCs. The chemical structures of GCs and MGCs are described below. 1 Characterized by H-NMR and ATR-IR spectral analysis. Figure 3a Showing GC and MGC1 The H-NMR spectrum confirmed the successful introduction of the methacryloyl group into the ethylene glycol chitosan backbone. The D2O peak at 4.65 ppm was used as a reference peak. The overlapping peaks at 3-4 ppm are attributed to the protons of the glucopyranosyl ring located at positions 2-8 (H-2~H-8). The peak at 2.6 ppm originates from the protons of the primary amine residues. In the methacrylate derivatives, additional peaks at 5.4 ppm and 5.6 ppm indicate the alkenyl double bonds of the methacrylate groups coupled to ethylene glycol chitosan. Furthermore, the additional peak at 1.84 ppm in the spectrum of methacryloylated ethylene glycol chitosan is due to the -CH3 group of the methacryloyl group. The ratio of the average intensity of the chitosan glucamine protons (δ=3.0ppm-3.8ppm) to the proton peaks of the methacrylate groups (δ=5.4ppm-5.6ppm) provides the degree of methacryloylation according to the following mathematical formula 1:

[0093] [Mathematical Expression 1]

[0094] Methacrylation (%) = [(Integrated area of ​​methacrylate proton peaks at 5.5 ppm and 5.6 ppm / 2) / (Integrated area of ​​H2–H8 proton peaks / 10)] × 100%

[0095] like Figure 3b As shown, the chemical structures of GC and MGC were also confirmed by ATR-FTIR spectroscopy. (3400 cm⁻¹) -1 The broadband band at this location belongs to the stretching vibration of the hydroxyl group, which overlaps with the stretching vibration of NH in the same region. 2890 cm⁻¹ -1 The absorption peak at 1596 cm⁻¹ is attributed to the CH₂ group. -1 The absorption peak at 1596 cm⁻¹ is attributed to the amino bending vibration of GC. -1 The disappearance of the amino vibration band and the 1555 cm after methacrylation -1 The appearance of the amide II band indicates that MGC has been successfully synthesized. Furthermore, at 1700 cm⁻¹... -1 The absorption peak at the point is attributed to the carbon-carbon double bond of the methacrylate after methacrylation. As shown in Table 1 below, a series of MGCs (yields: 72%-76%) were synthesized by adjusting the degree of methacrylation from 39% to 59% by changing the feed molar ratio of methacrylic anhydride.

[0096]

[0097] The degree of methacrylation (DM) of MGC increased with the increase of the feed molar ratio of methacrylic anhydride to 39%, 46%, 50%, 53%, and 59%. Figure 4b T gela T represents the value determined by the tube reversal method at 4% by weight. gel ᵇ represents the values ​​measured using rheological equipment. MGC with the lowest DM (39%), namely MGC39, did not exhibit significant heat-sensitive gelation behavior, which is attributed to the low content of hydrophobic methacryloyl groups. However, MGC with the highest DM (59%), namely MGC59, gelled below 4°C due to the introduction of excess methacryloyl groups, as hydrophobicity increased. Therefore, further investigation was conducted on MGC46, MGC50, and MGC59 in the next phase of experiments.

[0098] Example 2. Study on temperature-sensitive sol-gel transition behavior

[0099] The thermosensitive 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 PBS solution (pH 7.4, 0.01 M) at 4 °C at concentrations ranging from 3.6 wt% to 5.0 wt%. Solution-gel transition temperatures were determined based on flow or non-flow conditions with the tube inverted for 60 seconds. Data values ​​represent the average of three measurements, including the standard deviation (SD). The sol-gel transition phase diagram obtained using this method is known to have an accuracy of ±1 °C. The sol-gel transition temperatures of MGC were also investigated using a rheometer (WatersTA Instruments, Milford, MA, USA). The MGC aqueous solution was arranged between parallel plates with a diameter of 20 mm and a spacing of 1000 mm. The frequency was optimized to 1 Hz based on results determined using frequency scanning experiments. A constant stress of 25 Pa was used in the measurements.

[0100] The heat-sensitive sol-gel transition characteristics of MGC solutions were studied using the tube inversion method, viscosity measurement, and rheology. Figure 4a The sol-gel transition phase diagram of MGC solutions obtained by tube reversal is shown. Various MGC solutions were prepared at concentrations ranging from 2 wt% to 6 wt% by dissolving MGC with DM in PBS solution (pH 7.4, 0.01 M) at concentrations ranging from 39% to 59%. The sol-gel transition temperature of the MGC solutions could be well controlled between 19 °C and 54 °C by varying the concentrations of DM and MGC. It was observed that the sol-gel transition temperature decreased with increasing DM or polymer concentration, which may be due to the higher physical crosslinking density formed by the enhanced hydrophobic interactions between the methacryloyl groups of MGC.

[0101] The viscoelastic properties, storage modulus (G'), and loss modulus (G'') of MGC39, MGC46, MGC50, MGC53, and MGC59 were investigated through rheological experiments conducted at temperature variations ranging from 10℃ to 45℃. Figures 5a to 5e The following figures show the temperature-dependent rheological behavior of MGC39, MGC46, MGC50, MGC53, and MGC59 in a 4% (wt%) aqueous solution. This rheological analysis yielded more reproducible and quantitative results compared to the tube reversal method. As the temperature increased, gelation proceeded, and both the storage modulus (G') and the loss modulus (G'') increased, especially with G' increasing at a much faster rate than G''. The difference in the rate of increase of G' and G'' resulted in a crossover point (defined as the gelation point), which indicates a transition from a predominantly viscous liquid phase to a predominantly 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 sharply with increasing temperature. The crossover point of G' and G'' values ​​was similar to previous results obtained using the tube reversal method. For MGC59, since the initial G'' is higher than G', it is speculated that a sol-gel phase transition occurred below 10 °C.

[0102] Figure 6 shows the concentration of MGC50 at 3 wt% ( Figure 6a ), 4% by weight ( Figure 6b ), 5% by weight ( Figure 6c ), 6% by weight ( Figure 6d The viscoelastic properties of MGC50 as temperature increases are shown. It can be seen that the higher the concentration of MGC50, the lower the crossover point. Since a higher concentration of MGC50 results in more hydrophobic components, hydrophobic interactions increase, leading to a lower sol-gel phase transition temperature. Therefore, the sol-gel phase transition temperature of MGC50 (6 wt%) is the lowest at 23 °C.

[0103] Example 3. Photocrosslinking of MGC

[0104] An aqueous solution of MGC containing photoinitiator I2959 (0.05 wt%) in phosphate-buffered saline (PBS, pH 7.4, 4.0 wt%) was poured into a cylindrical silicone mold (10 mm in diameter × 2 mm in thickness). The solution was then exposed to ultraviolet light (365 nm, 20 mW / cm²) for 30 to 60 minutes to prepare a chemically cross-linked MGC hydrogel. The photocrosslinking reaction of MGC was tracked by ATR-FTIR measurements, and the changes in the characteristic peaks corresponding to the methacrylate groups as a function of UV irradiation time were observed.

[0105] MGC aqueous solution can solidify at temperatures below 37°C, close to body temperature, forming a non-flowing, transparent gel. The MGC thermosensitive gel is further modified by UV crosslinking to obtain a chemically crosslinked elastic hydrogel. The degree of crosslinking reactivity with UV irradiation time was confirmed by identifying the methacrylate groups after exposing the thermosensitive gel to UV light for 30 minutes and 60 minutes, respectively. Changes in the ATR-FTIR spectrum of the photocrosslinked MGC hydrogel are shown below. Figure 7 1650cm -1 The reduction in the vibrational band is attributed to the transformation of the methacryloyl group.

[0106] To study the kinetics 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 The deconvolution of the absorption band for methacryloyl is shown. Using 1650 cm⁻¹ at different times... -1 The integral value A of the absorption band can be used to calculate the degree of crosslinking (DC) according to the following mathematical formula 2: (Macromolecules, 35(16), 6377-6383). Figure 8 MGC 50 represents the sample without UV irradiation. MGC 50-30, MGC 50-60, MGC 50-90, and MGC 50-120 represent the absorption at 30 minutes, 60 minutes, 90 minutes, and 120 minutes, respectively.

[0107] [Mathematical Expression 2]

[0108] Degree of Crosslinking (DC) = 1 – (A) t / A0)

[0109] The results of the study on MGC50 showed that the double bond peak gradually decreased under ultraviolet irradiation over time. MGC50-30, MGC50-60, MGC50-90, and MGC50-120 exhibited crosslinking degrees (DC) of 37%, 45%, 72%, and 69%, respectively. The critical time was determined to be 90 minutes (crosslinking degree = 72%).

[0110] Example 4. Rheological Analysis

[0111] The rheological behavior of MGC hydrogels and photocrosslinked MGC hydrogels was investigated using an HR10 rheometer (Waters TA Instruments, Milford, USA) equipped with a 20 mm titanium parallel plate at 37 °C. An MGC solution (4 wt%, PBS) was coated onto the rheometer, and the upper plate was lowered to a measurement gap of 500 mm. The storage modulus (G') and loss modulus (G'') were measured as a function of frequency. The photocrosslinked MGC hydrogels after UV irradiation were studied in the same manner. Frequency scans of the hydrogels were performed from 0.01 Hz to 10 Hz at 0.01% strain.

[0112] The mechanical properties of physically cross-linked MGC and chemically cross-linked (photocross-linked) MGC hydrogels were studied and compared using oscillatory rheological experiments at 37 °C. The storage modulus of elasticity, representing the formation of the elastic network, was measured for all hydrogel groups. Figure 9 The elastic modulus (G' component) was determined as a function of frequency scanning at 37°C. (Refer to...) Figure 9 The elastic modulus (G') of hydrogels containing photocrosslinked ethylene glycol chitosan derivatives can range from 10 Pa to 500 Pa. Photocrosslinked MGC hydrogels, due to their robust network structure formed by strong covalent bonds, exhibit significantly higher G' values ​​across the entire frequency range than physically crosslinked MGC thermosensitive gels. Furthermore, the G' of MGC50-60 hydrogels was confirmed to be slightly higher than that of MGC50-30 hydrogels, indicating a denser crosslinked network and higher elasticity due to the higher content of methacrylate groups. In summary, chemically crosslinked hydrogels exhibit significantly higher storage modulus compared to physically crosslinked hydrogels, and prolonged exposure to ultraviolet light can further increase the crosslinking density of the hydrogels.

[0113] Example 5. In vitro gel stability and enzymatic biodegradation

[0114] The biodegradation of MGC50 hydrogels and photocrosslinked MGC50 hydrogels was investigated by measuring the weight change of MGC hydrogels in vitro with and without lysozyme. MGC50 solution (PBS, 1 mL, 4 wt%) was placed in 5 mL vials and incubated at 37°C in a water bath (Series BS-21, Lab Companion, Korea) for 15 minutes to form a gel. MGC50 solution (PBS, 1 mL, 4 wt%) containing photoinitiator I2959 (0.05% relative to weight) was prepared and poured into a cylindrical silicone mold (10 mm diameter × 2 mm thickness). The solution was then exposed to ultraviolet light (365 nm, 20 mW / cm²) for 30 to 60 minutes to prepare chemically crosslinked MGC hydrogels. These hydrogels were cultured in 2 mL PBS solutions without lysozyme or containing 5 mg / mL lysozyme. The weight of the hydrogels was measured at predetermined time intervals, and then the gels were immersed again in fresh PBS. All experiments were performed in triplicate. The percentage of the weight of the residual hydrogel is calculated using the following mathematical formula 3:

[0115] [Mathematical Expression 3]

[0116] Residual weight (%) = (W s - W i ) / W i × 100 (%)

[0117] Among them, W i W is the initial weight of the hydrogel. s The weight of the hydrogel sample is a function of time.

[0118] To confirm the physical stability of the MGC hydrogel and the photocrosslinked MGC50-30 and MGC50-60 hydrogels, stability tests were performed in PBS solution. Figure 10a As shown, MGC exhibits a gradual weight increase due to initial swelling over a maximum of 2 days. However, after 3 days, the residual weight decreases sharply due to mass loss of the MGC hydrogel. On the other hand, photocrosslinked MGC hydrogels, such as... Figure 10a As shown, neither MGC50-30 nor MGC50-60 exhibited significant mass loss during the 50-day culture period. This confirms that the photocrosslinked MGC hydrogels possess excellent physical stability.

[0119] To investigate the in vitro biodegradability of MGC hydrogels and photocrosslinked MGC50-30 and MGC50-60 hydrogels, the hydrogel samples were cultured in PBS solution containing lysozyme (5 mg / mL) at 37 °C. Figure 10bThe image shows the weight changes over time of MGC hydrogels and photocrosslinked MGC50-30 and MGC50-60 hydrogels used in biodegradability studies. The MGC hydrogel exhibited very rapid dissolution behavior, losing 100% of its total mass within one day, while a sustained mass loss rate was observed for the photocrosslinked MGC50-30 and MGC50-60 hydrogels. The MGC50-60 hydrogel, with its higher crosslinking density, exhibited slower degradation than the MGC50-30 hydrogel. The sustained mass loss curves of the MGC50-60 and MGC50-30 hydrogels compared to the MGC hydrogel indicate that the introduction of the photocrosslinking system, due to the increased steric hindrance effect resulting from the formation of the chemically bonded network, can delay degradation. Therefore, the enzymatic degradation of the hydrogel can be regulated by adjusting the degree of crosslinking. This tunable property of the MGC hydrogel can play a role in various biomedical applications, such as drug delivery systems and tissue engineering.

[0120] Example 6. Morphological Study

[0121] The morphology and structure of the MGC hydrogels were visualized using a field emission scanning electron microscope (FE-SEM; S-4800, Hitachi, Japan) operating at 10 kV. Hydrogel samples were rapidly fractured after freezing in liquid nitrogen to expose the internal structure, and then freeze-dried for 4 days. All samples were coated with platinum for 60 seconds.

[0122] To investigate the morphological properties of MGC50 hydrogels, the cross-sectional morphology of the lyophilized hydrogel samples was observed using FE-SEM. Figure 11 shows the internal structure of the lyophilized hydrogel samples. Figure 11a , Figure 11b , Figure 11c MGC50, photocrosslinked MGC50-30, and photocrosslinked MGC50-90 are shown respectively. A microporous structure with irregular pore sizes was observed in the MGC50 hydrogel. Compared to the thermosensitive gel before photocrosslinking, a more developed pore structure with more regular size and shape was observed in the photocrosslinked hydrogel. The photocrosslinked MGC50-30 and MGC50-90 hydrogels exhibited sponge-like microporous structures with pore sizes of 20-25 mm and 10-15 mm, respectively. The pores are interconnected, forming an open channel structure in a three-dimensional network. As observed in the SEM images, the pore size of MGC50-90 is 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 enables a reduction in pore size.

Claims

1. A glycol-chitosan derivative, characterized in that, It possesses double-bonded reactive groups capable of undergoing thermo-induced physical and chemical cross-linking, and has the structure of the following chemical formula 1: [Chemical Formula 1] In the above chemical formula 1, The aforementioned R1 comprises one group 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 alkenyl carbonyl group having 1 to 8 carbon atoms. The three monomers modified by x, y, and z are randomly arranged. 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. The ethylene glycol chitosan derivative according to claim 1, characterized in that, The above R1 is R2 and R3 are each independently H, an alkyl group having 1 to 5 carbon atoms, or OH.

3. The ethylene glycol chitosan derivative according to claim 2, characterized in that, R2 above is -CH3, and R3 above is H.

4. The ethylene glycol chitosan derivative according to claim 1, characterized in that, 0.46≤z≤0.53。 5. The ethylene glycol chitosan derivative according to claim 1, characterized in that, It has a crosslinking degree of 30% to 75% after photocrosslinking reaction.

6. The ethylene glycol chitosan derivative according to claim 1, characterized in that, The weight-average molecular weight ranges from 200 to 5,000,000.

7. A method for preparing an ethylene glycol chitosan derivative, characterized in that, The following reaction formula 1 represents the preparation of an ethylene glycol chitosan derivative, represented by formula 4, having double-bonded reactive groups capable of undergoing thermo-induced physical and chemical crosslinking, by reacting an N-acetylated ethylene glycol chitosan derivative of formula 2 with an R4COX derivative of formula 3: [Reaction Formula 1] In the above reaction formula 1, The aforementioned R4 comprises one group 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 alkenyl carbonyl group having 1 to 8 carbon atoms. X above is a leaving group. The two monomers modified by m and n are randomly arranged. The above m and n are the average mole fractions of each monomer in chemical formula 2, where 0.1 ≤ m ≤ 0.7 and 0.3 ≤ n ≤ 0.9, and m + n = 1. The three monomers modified by o, p, and q are randomly arranged. 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. The method for preparing the ethylene glycol chitosan derivative according to claim 7, characterized in that, X above represents a hydroxyl group; a halogen substituent; an alkoxy group with 1 to 4 carbon atoms; or -OC (=O)-R4.

9. The method for preparing the ethylene glycol chitosan derivative according to claim 7, characterized in that, The above R4 is R5 and R6 are each independently H, an alkyl group having 1 to 5 carbon atoms, or OH.

10. The method for preparing the ethylene glycol chitosan derivative according to claim 9, characterized in that, R5 above is -CH3, and R6 above is H.

11. A hydrogel, characterized in that, Including the ethylene glycol chitosan derivative of claim 1, wherein the hydrogel is capable of loading drugs or cells.

12. The hydrogel according to claim 11, characterized in that, The above-mentioned ethylene glycol chitosan derivative is photocrosslinked, and the elastic modulus (G') of the above-mentioned hydrogel is 10 Pa to 5000 Pa.

13. The hydrogel according to claim 11, characterized in that, The concentration of the above-mentioned ethylene glycol chitosan derivative is 2% to 10% by weight, and the above-mentioned hydrogel can undergo sol-gel transition in the range of 19°C to 54°C.

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