Thermogel composition comprising polyphenol

Abstract

This polyphenol-containing acyl glycol chitosan thermogel has lower cytotoxicity than a single component acyl glycol chitosan thermogel, and can alleviate conventional weak mechanical properties through physical-chemical crosslinking of polyphenols and acyl glycol chitosan thermogel. Self-healing was observed using pyrogallol groups of polyphenols, physical stability and biodegradability were easily controlled, and antioxidant and antibacterial properties, which had been difficult to achieve with a single component, could also be observed. As a result, by using polyphenols as a functional additive, limitations of single thermogel properties were compensated for, and the performance of a conventional thermogel was improved by imparting the properties of polyphenols.

Description

Thermogel composition containing polyphenol

[0001] This invention claims the benefit of patent application No. 10-2023-0146524 filed with the Korean Intellectual Property Office on October 30, 2023, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to a thermogel used as a drug delivery vehicle and a biomaterial for tissue engineering, and more particularly, to a thermogel composition comprising acyl glycol chitosan and polyphenol.

[0003] Thermogels, classified as temperature-sensitive hydrogels, possess a unique sol-gel transition capability, capable of changing their physical state in response to temperature changes. Typically composed of polymer chains intertwined to form a three-dimensional network, these thermogels exhibit a sol-gel transition at concentrations consistent with their lower critical concentration temperature (LCST). This transition dynamics significantly influences the material's physical properties and stability. Thermogels have diverse potential applications in fields such as drug delivery, tissue engineering, and wound healing. Despite this wide range of applications, conventional thermogels have been plagued by issues such as reduced gel stability, poor degradation resistance, and potential adverse effects in vivo.

[0004] The technical task of the present invention is to provide a thermogel composition having high mechanical strength, high gel stability, and high degradation resistance. Specifically, a thermogel composition comprising acyl glycol chitosan and polyphenol can be provided.

[0005] 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.

[0006] One embodiment of the present invention provides a thermogel composition comprising acyl glycol chitosan and polyphenol.

[0007] In one embodiment of the present invention, the polyphenol may be present in an amount of 0.1 to 10 parts by weight based on 100 parts by weight of the acyl glycol chitosan.

[0008] In one embodiment of the present invention, the polyphenol may be present in an amount of 5.0 to 10 parts by weight based on 100 parts by weight of the acyl glycol chitosan.

[0009] In one embodiment of the present invention, the acyl glycol chitosan may be hexanoyl glycol chitosan (HGC).

[0010] In one embodiment of the present invention, the acyl glycol chitosan may be octanoyl glycol chitosan (OGC).

[0011] In one embodiment of the present invention, the polyphenol may be tannic acid (TA).

[0012] In one embodiment of the present invention, the polyphenol may be gallic acid (GA).

[0013] In one embodiment of the present invention, it may have a compressive strength of 40 kPa or more.

[0014] In one embodiment of the present invention, it may have a radical scavenging ability of 80% or more in the DPPH analysis method.

[0015] In one embodiment of the present invention, it may have a radical scavenging ability of 95% or more in the ABTS analysis method.

[0016] In one embodiment of the present invention, it may have a colony forming unit (CFU) of 10% or less in a colony forming unit (CFU) experiment.

[0017] One embodiment of the present invention provides a drug delivery vehicle comprising the thermogel composition.

[0018] One embodiment of the present invention provides a biomaterial for tissue engineering comprising the thermogel composition.

[0019] Polyphenol-containing acyl glycol chitosan thermogels have lower cytotoxicity than single-component acyl glycol chitosan thermogels, and can improve the previously weak mechanical properties through the physical-chemical cross-linking of polyphenols and acyl glycol chitosan thermogels. Self-healing was observed due to the pyrogallol group of polyphenols, and physical stability and biodegradability were easily controlled. In addition, antioxidant and antibacterial properties, which were difficult to achieve with single components, were observed. Consequently, by using polyphenols as functional additives, the limitations of single thermogel properties were complemented, and the performance of existing thermogels was improved by imparting the properties of polyphenols.

[0020] Figure 1a shows 1H-NMR of glycol chitosan (GC) and HGC.

[0021] Figure 1b shows the characterization of the ATR-FTIR spectra of glycol chitosan (GC) and HGC.

[0022] Figure 2a shows 1H-NMR of GC and OGC.

[0023] Figure 2b shows the characterization of the ATR-FTIR spectra of GC and OGC.

[0024] Figure 3 shows the signal change of the hydroxyl characteristic peak after mixing HGC and TA.

[0025] Figure 4 shows the signal change of the hydroxyl characteristic peak after mixing OGC and GA.

[0026] Figure 5 is a schematic diagram showing physical and chemical bonding in HGC / TA thermogel.

[0027] Figure 6 shows the appearance of HGC and HGC / TA thermogels over time.

[0028] Figure 7 shows the appearance of OGC and OGC / GA thermogels over time.

[0029] Figure 8a shows the storage modulus of HGC and HGC / TA thermogels over time at 25°C.

[0030] Figure 8b shows the storage modulus of HGC and HGC / TA thermogels over time at 37°C.

[0031] Figure 9 shows the storage modulus of OGC and OGC / GA thermogels over time at 37°C.

[0032] Figure 10 shows a macroscopic self-healing test to verify whether the HGC / TA thermogel has self-healing ability.

[0033] Figure 11 shows the rheological recovery analysis results of HGC / TA.

[0034] Figure 12 shows the compressive strength of HGC / TA and HGC thermogels.

[0035] Figure 13 shows the in vitro gel stability test of HGC / TA and HGC thermogel in PBS at 37°C.

[0036] Figure 14 shows the enzymatic degradation profiles of HGC and HGC / TA thermogels in PBS containing 10 mg / mL lysozyme at 37°C.

[0037] Figure 15 is an FE-SEM image showing the cross-linked form of HGC / TA.

[0038] Figure 16a shows the results of DPPH analysis of a mixture of HGC and TA, showing the UV-vis spectrum of the DPPH solution after a contact time of 30 minutes with the thermogel.

[0039] Figure 16b shows the DPPH scavenging activity of HGC and HGC / TA thermogels.

[0040] Figure 16c is an image confirming the colors of DPPH and HGC / TA thermogels.

[0041] Figure 17a shows the results of DPPH analysis of a mixture of OGC and GA, and shows the UV-vis spectrum of the DPPH solution after contact with the thermogel.

[0042] Figure 17b shows the DPPH scavenging activity of OGC and OGC / GA thermogels.

[0043] Figure 18a shows the results of ABTS analysis of a mixture of HGC and TA, showing the UV-vis spectrum of the ABTS solution after a contact time of 30 minutes with the thermogel.

[0044] Figure 18b shows the ABTS scavenging activity of HGC and HGC / TA thermogels.

[0045] Figure 18c is an image confirming the colors of ABTS and HGC / TA thermogels.

[0046] Figure 19a shows the in vitro antibacterial activity of HGC / TA, showing the inhibition zone (left) and colonies (right) of E. coli and S. aureus for various samples.

[0047] Figure 19b shows the quantitative analysis of the inhibition zone, optical density and relative colony forming units (%).

[0048] Figure 20a shows the in vitro cytotoxicity of HGC / TA, showing survival / death images of NIH3T3 cells cultured with hydrogel dissolution for 24 and 48 hours.

[0049] Figure 20b shows the cell viability of HGC / TA in the survival / death image.

[0050] Figure 20c shows the cell viability of HGC / TA in the WST-8 assay.

[0051] 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.

[0052] In this specification, “A and / or B” means “A and B, or A or B.”

[0053] Hereinafter, the present invention will be described in more detail.

[0054] One embodiment of the present invention provides a thermogel composition comprising acyl glycol chitosan and polyphenol.

[0055] In one embodiment of the present invention, the polyphenol may be present in an amount of 0.1 to 10 parts by weight based on 100 parts by weight of acyl glycol chitosan. The polyphenol may be present in an amount of 5.0 to 10 parts by weight based on 100 parts by weight of acyl glycol chitosan. The ratio of acyl glycol chitosan to polyphenol may be 0.1 to 20, 0.5 to 15, 1.0 to 10, or 5.0 to 10. The ratio of acyl glycol chitosan to polyphenol may be 0.1 or more, 0.5 or more, 1.0 or more, or 5.0 or more. The ratio of acyl glycol chitosan to polyphenol may be 20.0 or less, 15.0 or less, or 10 or less. The ratio of acyl glycol chitosan to polyphenol may be 0.1, 0.5, 1.0, 5.0, or 10.

[0056] In one embodiment of the present invention, the acyl glycol chitosan may be hexanoyl glycol chitosan. In one embodiment of the present invention, the acyl glycol chitosan may be octanoyl glycol chitosan. The acyl glycol chitosan may be glycol chitosan substituted with various acyl groups such as acetyl, propionyl, butyryl, valeryl, hexanoyl, heptanoyl, octanoyl, and oleoyl groups.

[0057] In one embodiment of the present invention, the polyphenol may be tannic acid. In one embodiment of the present invention, the polyphenol may be gallic acid.

[0058] In one embodiment of the present invention, it may have a compressive strength of 40 kPa or more. In one embodiment of the present invention, the compressive strength may be 10 kPa or more, 20 kPa or more, 30 kPa or more, 40 kPa or more, or 50 kPa or more.

[0059] In one embodiment of the present invention, it may have a radical scavenging ability of 80% or more in the DPPH analysis method. The radical scavenging ability in the DPPH analysis method may be 70% or more, 80% or more, or 90% or more.

[0060] In one embodiment of the present invention, it may have a radical scavenging ability of 95% or more in the ABTS analysis method. The radical scavenging ability in the ABTS analysis method may be 70% or more, 80% or more, or 90% or more.

[0061] In one embodiment of the present invention, the colony forming unit (CFU) may be 10% or less in a colony forming unit (CFU) experiment. The colony forming unit may be 20% or less, 15% or less, 10% or less, or 5% or less.

[0062] One embodiment of the present invention provides a drug delivery vehicle comprising the thermogel composition.

[0063] One embodiment of the present invention provides a biomaterial for tissue engineering comprising the thermogel composition.

[0064]

[0065] 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.

[0066] Manufacturing example

[0067] ingredient

[0068] GC (DP ≥ 400) was purchased from Wako Pure Chemical Industries (Osaka, Japan). Tannic acid was purchased from Alfa Aesar Co. Ltd. (Tianjin, China). Hexanoic anhydride (97%) extracted from chicken egg white and lysozyme were procured from Sigma-Aldrich (St. Louis, MO, USA). DPPH (free radical, purity 95%), ABTS, and phosphate-buffered saline powder were provided by Gibco™ (Thermo Fisher Scientific, USA). Dialysis membrane (12–14 kDa) was purchased from Spectrum Laboratories (USA). Acetone and methanol were purchased from Samcheon Chemical (Korea). All distilled water was triple-distilled.

[0069] Manufacturing Example 1. HGC Synthesis

[0070] GC (3 g) was dissolved in 375 mL of distilled water and then diluted with 375 mL of methanol. A predetermined amount (1.06 mL) of hexanoic anhydride was added to the GC solution with stirring. After continuous stirring at room temperature for 24 h, the HGC product precipitated from acetone, collected, and then dialyzed against distilled water for 2 days using a dialysis membrane (MWCO = 12-14 kDa). Finally, HGC was obtained in powder form after lyophilization for 5 days.

[0071] Manufacturing Example 2. Manufacturing of HGC / TA thermogel

[0072] HGC and TA were prepared as shown in Table 1: First, HGC was dissolved in distilled water at a concentration of 4.5 wt%. Next, TA was dissolved in 0.1 ml of distilled water at HGC weight ratios of 0.1%, 0.5%, 1%, 5%, and 10%, according to the composition. A 0.1 ml TA solution was added to a 1 ml HGC standard solution through a mixing tube. Afterwards, the solution was oxidized in a water bath at 37°C for more than 4 days.

[0073] Sample HGC: TA (weight ratio) HGC (mg) TA (mg) Total HGC concentration (wt%) Total TA concentration (wt%) HGC 36 40 4 HGC / TA 0.1100 / 0.145 0.045 40.04 HGC / TA 0.5100 / 0.545 0.225 40.25 HGC / TA 1.0100 / 145 0.45 40.4 HGC / TA 5.0100 / 545 2.25 42.5 HGC / TA 10100 / 1045 4.5 4.0

[0074]

[0075] Manufacturing Example 3. OGC Synthesis

[0076] GC (1.5 g) was dissolved in 170 mL of distilled water and then diluted with 170 mL of methanol. A predetermined amount (0.401 mL) of octanoic anhydride was added to the GC solution with stirring. After stirring at room temperature for 24 h, the resulting OGC product was collected by precipitation from acetone and dialyzed against distilled water for 2 days using a dialysis membrane. Finally, OGC was obtained in powder form after lyophilization for 5 days.

[0077] Manufacturing Example 4. Manufacturing of OGC / GA thermogel

[0078] OGC was dissolved in distilled water at a concentration of 6 wt%. GA was then dissolved at concentrations of 0.1%, 1%, and 10% based on the weight of OGC. The GA solution was added to the OGC standard solution through a mixing tube. The solution was then stored in a water bath at 37°C.

[0079]

[0080] Example 1. Characterization of HGC and OGC

[0081] The chemical composition of HGC was characterized using 1H-NMR (AVANCE III 600, Bruker, Germany) operating at 600 MHz. HGC thermogel was dissolved in 0.5 wt% D2O. The D2O peak at 4.85 ppm was used as a reference peak. The degree of hexanoylation of HGC was calculated to be 35.8% by 1H-NMR spectroscopy.

[0082] HGC thermogel was synthesized through N-acylation of GC. The synthetic results of the synthesized HGC were confirmed by 1H-NMR and ATR-FTIR analyses. Figure 1a shows a comparison of the 1H-NMR analysis results of GC and HGC to confirm the introduction of a hexanoyl group. In the case of GC, characteristic peaks corresponding to H2-H8 of the glycopranosyl ring were observed in the range of δ = 3.9 to 3.5 ppm, whereas in the case of the HGC spectrum, characteristic peaks of hydrogen corresponding to the methyl and methylene groups of the introduced hexanoyl group were observed. These are δ = 0.9 ppm, δ = 1.3 ppm, δ = 1.6 ppm, and δ = 2.3 ppm, respectively. The degree of hexanoylation of the hexanoyl group was calculated to be 35.8% by comparing the hydrogen integral of the hexanoyl group in the 1H-NMR spectrum with the hydrogen integral of the glucopranosyl ring in GC. Similar results were obtained for conventional synthetic HGC.

[0083] Figure 1b shows the results of ATR-FTIR analysis confirming the synthesis of HGC. In both spectra of GC and HGC, characteristic peaks due to the stretching vibration of hydroxyl groups and NH groups are observed at 3300 cm -1 While it was widely observed in the vicinity, in the case of HGC, the hexanoyl peak was observed at 2870 cm -1 was observed in the vicinity. It was observed that a characteristic peak appeared due to the CH stretching vibration of the methyl group (-CH3) and methylene (-CH2-) present in the group. In addition, the bending vibration peak of the primary amine (1588 cm) was observed compared to GC after the acylation reaction for the introduction of the hexanoyl group. -1 ) decreased, while the bending vibration peak of the amide group (1558 cm -1 ) and the elongation vibration of the carbonyl group (1652 cm -1 ) could be observed to be clearly evident.

[0084] OGC thermogel was also synthesized through N-acylation of GC. The synthesis results of the synthesized OGC were confirmed through 1H-NMR and ATR-FTIR analyses. Figure 2a shows a comparison of the 1H-NMR analysis results of GC and OGC to confirm the introduction of an octanoyl group. In the case of the OGC spectrum, characteristic peaks of hydrogen corresponding to the methyl and methylene groups of the introduced octanoyl group were observed. The degree of octanoylation of the octanoyl group was calculated to be 16.8% by comparing the hydrogen integral of the octanoyl group in the 1H-NMR spectrum with the hydrogen integral of the glucopranosyl ring of GC.

[0085] Figure 2b shows the results of ATR-FTIR analysis confirming the synthesis of OGC. In the case of OGC, the octanoyl peak is 2890 cm -1 It was observed in the vicinity. It was observed that a characteristic peak appeared due to the CH stretching vibration of the methyl group (-CH3) and methylene (-CH2-) present in the group.

[0086]

[0087] Example 2. Characterization of HGC / TA and OGC / GA

[0088] Attenuated total reflectance ATR-FTIR spectra of HGC and HGC / TA thermogels were recorded using a Nicolet iS 5 spectrometer (Thermo Scientific, USA) in the range of 4000–750 cm -1 4cm in the frequency range -1 HGC and HGC / TA thermogels were identified by performing ATR-FTIR analysis with 32 scans at a resolution of .

[0089] HGC / TA thermogels were prepared by adding TA to HGC. This was confirmed through ATR-FTIR analysis of each thermogel. Figure 3 shows that the signal of the characteristic peak of hydroxyl group (3351-3369 ppm) was enhanced after the addition of TA. This was because TA contained a large amount of phenolic hydroxyl group (-OH). Figure 3 shows the characteristic peaks for a single HGC, b for HGC / TA0.1, c for HGC / TA0.5, d for HGC / TA1.0, e for HGC / TA5.0, f for HGC / TA10, and g for tannic acid. The signal intensity of the characteristic peak was 2920 cm in single HGC. -1 2929 cm at -1 The increase and shift of TA indicate that TA is successfully introduced into the CS structure. The bending vibration peak of NH bond (1555 ~ 1567 cm -1 ) exhibits a similar behavior to HGC and is attributed to the chemical bond between NH and tannic acid. These results indicate that HGC and tannic acid are cross-linked by chemical bonds.

[0090] OGC / GA thermogels were prepared by adding GA to OGC. This was confirmed through ATR-FTIR analysis of each thermogel. Figure 4 shows that the signal of the characteristic peak of hydroxyl group (3351-3369 ppm) was enhanced after the addition of GA. This was because there were many phenolic hydroxyl groups (-OH) in GA. Figure 4 shows the characteristic peaks for OGC alone, OGC / GA0.1, OGC / GA1.0, OGC / GA10, and GA alone. As the GA content increased, the signal at 1650 cm, which appears as the stretching vibration of carbonyl, increased. -1 Since the peak decreased, it can be seen that chemical crosslinking was successfully formed by the Schiff base reaction.

[0091]

[0092] Example 3. Rheological behavior of thermogels over time

[0093] To observe the time-dependent storage modulus due to oxidation, time-dependent rheological analysis of HGC and HGC / TA thermogels was performed using a rheometer (HR-10 rheometer, TA Instruments, New Castle, DE, USA). HGC (4 wt%, distilled water) and HGC / TA were placed between two parallel plates with a diameter of 20 mm and a gap of 1 mm under a constant stress of 10 Pa and a frequency of 1 Hz at 25°C and 37°C. The time-dependent storage modulus was analyzed using the time sweep method at a sweep time of 60 s. The rheological behaviors of each sample were measured after 0 h, 6 h, 12 h, 24 h, 48 h, 96 h, and 192 h.

[0094] HGC significantly increased in TA-treated hydrogels with increasing TA concentration due to the additional cross-linking effect provided by TA. This reaction begins with the oxidation of the phenolic groups of tannic acid to quinones. Cross-linking occurs through physical bonds, such as hydrogen bonds and hydrophobic bonds between the quinone and HGC, and chemical bonds, such as Michael additions and Schiff base reactions with the amine groups of HGC. Figure 5 is a schematic diagram showing the physical and chemical bonds in HGC / TA thermogels. Figure 6 shows the appearance of HGC and HGC / TA thermogels over time. It was confirmed that each sample turned dark brown and became opaque over time as the tannic acid content increased. Since this mechanism is affected by temperature according to the Arrhenius equation, it was observed by comparing room temperature (25°C) and body temperature (37°C). Figure 7 shows the appearance of OGC and OGC / GA thermogels over time. As the GA content increased, each sample was observed to turn dark brown, become opaque over time, and have reduced fluidity.

[0095] Figure 8 shows the storage modulus over time of HGC and HGC / TA thermogels at a 25℃ and b 37℃. As a result, the storage elastic modulus at 37℃ was twice as high as that at 25℃. As the tannic acid content increased, the storage modulus increased, and finally, through the 37℃ graph, it was confirmed that the storage modulus after 96 days was improved by up to about 100 times compared to the existing HGC. Figure 9 shows the storage modulus over time of OGC and OGC / GA thermogels at 37℃. In the case of OHC / GA10, the storage modulus increased significantly. It was confirmed that the weak mechanical properties of the existing thermogel were improved by increasing the storage modulus value. This is expected to be utilized as a biomaterial for wound healing and tissue engineering.

[0096]

[0097] Example 4. Self-healing properties

[0098] 4.1 Macroscopic recovery analysis of thermogels

[0099] To evaluate the self-healing ability of HGC / TA thermogels, a macroscopic self-healing test was performed. The HGC / TA thermogels were cut into two pieces and incubated together at 25°C. After 1 hour, the HGC / TA thermogels were lifted with tweezers to determine whether they could support their own weight.

[0100] HGC / TA thermogels possess unique self-healing properties, allowing them to autonomously repair and restore their structural integrity upon damage. The self-healing ability of HGC / TA thermogels is attributed to the reversible nature of the interactions between tannic acid molecules. This self-healing ability arises from intermolecular and intramolecular interactions between tannic acids bearing pyrrole-gallol groups or from numerous hydrogen bonds between these hydroxyl groups and the HGC backbone. To verify the self-healing ability of HGC / TA thermogels, a macroscopic self-healing test was first performed (Figure 10). Without external intervention, the HGC / TA thermogels were cut into two pieces, and the different hydrogel pieces were bonded together to form a whole body at 25°C. Complete healing occurred within 1 hour.

[0101] 4.2 Rheological recovery analysis of thermogels

[0102] A quantitative analysis of the self-healing ability of HGC / TA thermogels was performed using a rheometer. HGC / TA thermogels were loaded between parallel plates with a diameter of 20 mm and a gap of 1.0 mm. To obtain the critical strain point, strain amplitude sweep tests (strain [γ] = 0.1–5000%, fixed frequency of 1 Hz at 25°C) were performed. Alternating step strain sweep tests were then performed. The strain was switched from a small strain (γ = 0.1%) to a large strain (γ = 1000%) at a fixed frequency of 1 Hz at 25°C for 60 s. The strain range over which the thermogels maintained their integrity varied among samples, and the experiments were conducted based on the large strain value of 1000%.

[0103] The self-healing properties of HGC / TA thermogels were further evaluated using rheological recovery analysis based on the strain sweep results. The strain amplitude graph shows the pour point of the material, where a transition from an elastic gel state (G′>G″) to a viscous liquid-like state (G″>G′) is observed. In these experimental results, the G′ and G″ curves of the HGC / TA thermogels intersected at a strain of 1000%, indicating that the thermogel structure was completely destroyed when the strain was greater than 1000%. The rheological recovery behavior of the thermogels was then evaluated using continuous strain cycles from 0.1% to 1000%. Figure 11 shows the rheological recovery analysis results of HGC / TA. The strain cycles were continuously varied from 0.1% to 1000% at a constant frequency of 1 Hz with an interval of 60 s between each step. When exposed to a higher strain (1000%), the thermogel structure collapsed, but at a lower strain (0.1%), the hydrogel was able to recover its shape. As a result, the self-healing ability of each sample was confirmed.

[0104]

[0105] Example 5. Compressive strength test

[0106] The compressive strength tests of HGC and HGC / TA thermogels were performed using a universal testing machine (Texture analyzer (EZ-SX, 500 N, Shimadzu, Japan) equipped with a 500 kN load cell). For the HGC and HGC / TA thermogels to be tested, cylindrical thermogels measuring 10 mm (diameter) × 10 mm (height) were prepared. The samples were placed in the testing machine and compressed at a crosshead speed of 5 mm / min. Following the previous experiment, each compressive strength test was repeated three times, and the error bars show the variation of these data.

[0107] In addition to the hydrophobic bonds of the existing HGC, HGC thermogels containing tannic acid are cross-linked through interactions between tannic acids, physical bonds between tannic acids and the HGC backbone, and chemical bonds. As a result, the compressive strength also showed a higher strength as the storage modulus increased, which was inferred to be a thermogel with excellent mechanical properties. Figure 12 shows the compressive strength of HGC / TA and HGC thermogel. While the compressive strength of HGC was 1 kPa, HGC containing tannic acid showed a higher compressive strength than HGC. As the tannic acid content increased, the compressive strength increased up to a maximum of 47 kPa for HGC / TA5.0. However, in the case of HGC / TA10, the interaction between tannic acids increased, making the thermogel brittle. As a result, it was confirmed that the mechanical properties of HGC thermogels containing TA were improved through mutual interactions.

[0108]

[0109] Example 6. Physical stability test

[0110] HGC / TA thermogels were prepared by oxidizing at 37°C for more than 4 days. This experiment was conducted with reference to a previous experiment. 0.5 mL of HGC and HGC / TA thermogels were prepared in 5 mL vials and placed in an incubator maintained at 37°C to induce gelation. After 30 minutes, 2 mL of PBS at the same temperature was added to each vial and placed back in the incubator. The PBS was replaced at intervals of 6, 12, and 24 hours, and then replaced until the gel was completely dissolved. The residual weight of the thermogels was measured immediately before replacing the PBS. The ratio of the measured residual weight to the initial weight was converted to a percentage, and the following formula was used to calculate the residual amount (%) over time.

[0111] The gel stability of HGC / TA thermogels over time was evaluated at 37℃. Figure 13 shows the in vitro gel stability test of HGC / TA and HGC thermogels in PBS at 37℃. As shown in Figure 13, a single HGC was degraded in about 20 days, and HGC / TA0.1, which had the lowest tannic acid content, showed almost similar behavior. HGC / TA0.5 showed physical stability that was more than twice as high as that of the existing HGC, and HGC / TA1.0 showed swelling behavior that was more than four times as high, confirming that the physical stability was maintained even after 2 months. HGC / TA5.0 and HGC / TA10 maintained a similar state to the initial state even after 2 months. In this way, it was observed that the stability of the gel was greatly increased by the chemical bonding of HGC and TA, and the thermogel was maintained for more than 2 months. It was confirmed that the physical stability could be greatly improved compared to the existing synthetic thermogel by adding TA.

[0112]

[0113] Example 7. In vitro biodegradation test

[0114] The biodegradability of HGC / TA mixed thermogels was evaluated using PBS containing lysozyme (10 mg / mL) extracted from chicken egg white. Each sample was prepared in a 5-mL vial (0.5 mL). Each sample was stored in an incubator at 37°C and weighed after gelation. After weighing, 2 mL of a PBS solution containing lysozyme was added to each thermogel sample. The thermogels were weighed after 3, 6, 12, 24, 36, 48, and 60 h, and then an equal volume of fresh PBS was added. The residual amounts (%) of the thermogels over time were calculated and compared.

[0115] HGC manufactured with GC is biodegraded by lysozyme, similar to GC, which is known to have a variable biodegradation rate due to various chemical modifications. The degradability of thermogels containing various amounts of tannic acid was observed over time in the presence of a degrading enzyme and compared with that of a single thermogel. Figure 14 shows the enzymatic degradation profiles of HGC and HGC / TA thermogels in PBS containing 10 mg / mL lysozyme at 37°C. As shown in Figure 14, the HGC thermogel was completely degraded within 1 day. On the other hand, for HGC thermogels containing tannic acid, 0.1-1% was observed to degrade within 36 hours, 5% within 48 hours, and 10% within 60 hours. These results are presumed to be due to the slower degradation rate caused by the steric hindrance due to the addition of tannic acid. While the degradability of a single thermogel is determined by its own properties, the degradability of a tannic acid-containing thermogel can be controlled by the tannic acid content.

[0116]

[0117] Example 8. FE-SEM image of HGC / TA

[0118] Figure 15 is an FE-SEM image showing the cross-linked morphology of HGC / TA. Conventional HGC thermogels exhibited a porous, interconnected structure. We inferred that adding TA to HGC would alter the chemical and physical cross-linking morphology. Measurements confirmed that the pore size decreased after adding TA to the HGC thermogel. This diversification in morphology is due to the increased degree of cross-linking as the TA content increased.

[0119]

[0120] Example 9. Antioxidant activity

[0121] DPPH and ABTS assays were performed to evaluate the radical scavenging activity of HGC and HGC / TA thermogels. Briefly, a DPPH aqueous solution (0.2 mM) was prepared and stored in a dark room. 0.5 mL of thermogel was added to 5 mL of DPPH solution and left in the dark for 30 min. The thermogel was removed at regular intervals and the absorbance of the DPPH solution was recorded. Pure DPPH solution was used as a control. The absorbance of the DPPH solution was recorded at 517 nm using a UV-VIS spectrophotometer (SINCO, Korea), and the radical scavenging activity was measured from the difference in absorbance between the solutions before and after thermogel immersion (n = 3).

[0122] ABTS analysis was performed by mixing ABTS (7 mM) and potassium persulfate (2.4 mM) solutions in a 1:0.5 ratio (v / v) to prepare an ABTS solution, storing it in a dark room for 12-16 hours, and then diluting it with distilled water to obtain an absorbance of 0.7 (±0.1) at 734 nm.

[0123] Similar to the DPPH assay, 0.5 mL of thermogel was placed in 5 mL of ABTS solution and left in the dark for 30 minutes. The film sample was then removed and the absorbance of the ABTS solution recorded. Pure ABTS solution was used as a control, and radical scanning activity was analyzed based on the difference in absorbance between the solutions at 734 nm (n = 3). The antioxidant activity of thermogel was calculated using the following mathematical equation:

[0124] [Mathematical Formula 1]

[0125] Antioxidant activity (%) = [(Ai-Af) / Ai] × 100

[0126] Here, Ai is the absorbance of pure DPPH or ABTS solution and Af is the absorbance of the same solution after immersing the thermogel.

[0127] The DPPH radical (purple) is a stable free radical that can accept a hydrogen atom from an antioxidant compound to form a stable DPPH-H molecule (colorless). The DPPH assay is widely used to evaluate antioxidant activity, and Figure 16 shows data showing the results of a DPPH analysis at 517 nm. Figure 16a shows the UV-vis spectrum of the DPPH solution after 30 minutes of contact with the thermogel, and Figure 16b shows the DPPH scavenging activity of HGC and HGC / TA thermogels. While little antioxidant activity was observed for HGC alone, antioxidant activity was observed for TA. In the case of HGC / TA 5.0 or higher, a radical scavenging ability of over 80% was confirmed, confirming the possibility of antioxidant capacity. Figure 16c is an image confirming the color difference between DPPH and HGC / TA thermogel. Figure 17 shows the DPPH analysis results of OGC and GA mixture. Figure 17a shows the UV-vis spectrum of the DPPH solution after contact with thermogel, and Figure 17b shows the DPPH scavenging activity of OGC and OGC / GA thermogel.

[0128] In the ABTS assay, the ABTS+ radical (cyan) can accept electrons to become stable ABTS (colorless). ABTS analysis is an antioxidant capacity evaluation test used together with DPPH. Figure 18 shows the results of the ABTS analysis at 734 nm. Figure 18a shows the UV-vis spectrum of the ABTS solution after 30 minutes of contact with the thermogel, and figure 18b shows the ABTS scavenging activity of HGC and HGC / TA thermogels. An antioxidant capacity of nearly 100% was confirmed for HGC / TA 0.5 or higher. As expected, HGC without TA showed no activity or relatively low activity, confirming that the antioxidant capacity is recognized by the TA content. Figure 18c is an image confirming the color of ABTS and HGC / TA thermogels.

[0129]

[0130] Example 10. Antibacterial Experiment

[0131] The antibacterial activity of HGC and HGC / TA was tested using Gram-negative Escherichia coli (E. coli) DH5α and Gram-positive Staphylococcus aureus (S. aureus). Bacterial suspension (1.0 × 10 8 cells / mL forE. coli and 6.2×10 8 cells / mL for S. aureus) was diluted to 1 / 10 and used in antibacterial experiments. 100 μL of the diluted bacterial solution was dispensed onto LB / KAN solid medium (E. coli) and LB solid medium (S. aureus). After that, HGC and HGC / TA hydrogels were placed at the center of the solid medium and cultured at 37°C for 24 hours. After 24 hours, the inhibition zone was measured using a ruler. The hydrogels placed on the solid medium were removed and placed into 3 mL of LB / KAN medium solution (E. coli) and LB medium solution (S. aureus), and cultured at 37°C for 3 hours. The cultured solution was measured for absorbance at 600 nm using a UV spectrophotometer (Optizen pop) to confirm the degree of bacterial growth. Colony forming unit (CFU) experiments were performed using the cultured solution. The cultured solution was 10 5 After diluting to 100 μL, 100 μL was dispensed onto LB / KAN solid medium (E. coli) and LB solid medium (S. aureus) and plated. After culturing at 37°C for 21 hours, colony formation was quantified using the Image J program.

[0132] The antibacterial ability of HGC / TA was evaluated through inhibition zone and colony forming unit (CFU) experiments. Figure 19 shows the in vitro antibacterial activity of HGC / TA. Figure 19a shows the inhibition zone (left) and colonies (right) of E. coli and S. aureus for various samples. Figure 19b shows the quantitative analysis of inhibition zone, optical density, and relative colony forming unit (%). Statistical analysis was performed by one-way ANOVA (*p < 0.05; **p < 0.01, ***p < 0.001, NS; not significant). The inhibition zone tended to increase with tannic acid content against Gram-negative E. coli and Gram-positive S. aureus. When the hydrogel placed in the center was removed and cultured in the medium for 3 hours during the inhibition zone experiment, the optical density tended to decrease as the tannic acid content increased, confirming that HGC / TA inhibits bacterial growth. To confirm clear inhibition of bacterial growth, a colony formation unit experiment was conducted. Looking at the colony formation pattern, bacterial growth was not inhibited for the control group, HGC, but for E. coli, it was confirmed that bacterial growth was clearly inhibited from HGC / TA0.5 and for S. aureus from HGC / TA1.0.

[0133]

[0134] Example 11. Cytotoxicity evaluation

[0135] Biocompatibility tests of HGC and HGC / TA were performed using NIH3T3 cells derived from mouse fibroblasts. For quantitative evaluation and cell image observation, the WST-8 cell viability assay kit (Biomax) and LIVE / DEAD TMViability / cytotoxicity assay kit (Invitrogen) was used. HGC and HGC / TA were sterilized under UV irradiation for 3 h before the experiment and then eluted by placing them in a 24-well plate with 2 mL of Dulbecco's modified Eagle's medium (DMEM, Gibco) containing 10% (v / v) FBS and 1% (w / v) penicillin / streptomycin. In the WST-8 assay, NIH3T3 cells were plated in a 96-well culture plate (1X10 4 (cell / well) and cultured in a 37℃ incubator for 24 hours. After 24 hours of culture, the previously eluted solution was diluted 1 / 10 with DMEM solution and added to the cells, followed by culture at 37℃ for 24 hours. 10 μL of WST-8 solution was added to each well and cultured for an additional 2 hours at 37℃. The absorbance was measured at a wavelength of 450 nm using a microplate reader (VersaMax, Molecular Devices, Sunnyvale, CA, USA). Cell viability was quantified using the difference in absorbance between the sample and the control group at 450 nm according to the following mathematical equation 2.

[0136] [Equation 2]

[0137] Cell viability (%) = ( Abs sample / Abs control ) × 100 (%)

[0138] Cell viability / death assay was performed on NIH3T3 cells in 24-well culture plates (2X10 4After dispensing 10 cells / well, the cells were cultured for 24 hours. The diluted eluate was added to each well and cultured for 24 and 48 hours. After that, the cells were treated with a staining solution containing 2 μM Calcein-AM and 4 μM Ethidium homodimer-1 and maintained at 37°C for 15 minutes, washed with PBS, and the cell viability was calculated and analyzed using confocal laser scanning microscopy (LSM 880 with Airyscan, Zeiss) as shown in the following mathematical formula 3.

[0139] [Equation 3]

[0140] Cell viability (%) = {(viable cells) / (non-viable cells + viable cells)} × 100 (%)

[0141] The WST-8 assay results showed that all sample groups showed a cell viability rate of over 85% when compared to the untreated control group of HGC / TA samples, confirming the absence of toxicity. To confirm that the cells grew well without damage caused by the HGC / TA substance, the cell morphology was examined using live / dead staining. As a result, it was confirmed that the cells grew well by extending and adhering to each other. The cell morphology and quantitative analysis results confirmed that the HGC / TA substance was not toxic. Figure 20 shows the in vitro cytotoxicity of HGC / TA. Figure 20(a) shows the live / dead images of NIH3T3 cells cultured with hydrogel dissolution for 24 and 48 hours, and the scale bar represents 50 μm. Figure 20(b) shows the cell viability of HGC / TA in the live / dead images, and Figure 20(c) shows the cell viability of HGC / TA in the WST-8 assay. For WST-8 analysis, NIH3T3 cells were treated with HGC / TA elution for 24 h.

Claims

1. A thermogel composition comprising acyl glycol chitosan and polyphenol.

2. A thermogel composition characterized in that the polyphenol is present in an amount of 0.1 to 10 parts by weight based on 100 parts by weight of the acyl glycol chitosan in the first paragraph.

3. A thermogel composition characterized in that the polyphenol has 5.0 to 10 parts by weight based on 100 parts by weight of the acyl glycol chitosan in the second paragraph.

4. A thermogel composition according to claim 1, characterized in that the acyl glycol chitosan is hexanoyl glycol chitosan (HGC).

5. A thermogel composition according to claim 1, characterized in that the acyl glycol chitosan is octanoyl glycol chitosan (OGC).

6. A thermogel composition according to claim 1, characterized in that the polyphenol is tannic acid (TA).

7. A thermogel composition according to claim 1, characterized in that the polyphenol is gallic acid (GA).

8. A thermogel composition characterized in that it has a compressive strength of 40 kPa or more according to claim 1.

9. A thermogel composition characterized in that it has a radical scavenging ability of 80% or more in the DPPH analysis method according to claim 1.

10. A thermogel composition characterized in that it has a radical scavenging ability of 95% or more in the ABTS analysis method according to claim 1.

11. A thermogel composition characterized in that it has a colony forming unit (CFU) of 10% or less in a colony forming unit (CFU) experiment according to claim 1.

12. A drug delivery system comprising the thermogel composition of paragraph 1.

13. A biomaterial for tissue engineering comprising the thermogel composition of paragraph 1.

Images