WO2024214274A1 - Polymer material containing chemically modified polysaccharide - Google Patents

Abstract

[Problem] To provide a biological material using chitosan, especially a hydrogel material, having mechanical properties applicable to various uses while maintaining the antibacterial properties of chitosan. [Solution] It was discovered that, by using a polymer material containing a hydrophilic chitosan derivative and two kinds of neutral, hydrophilic polysaccharides capable of forming a hydrogen bond between the chitosan derivative and the molecule, a hydrogel having mechanical properties applicable to various uses while maintaining the antibacterial properties of chitosan can be obtained by a means such as photocrosslinking.

Description

Polymeric materials containing chemically modified polysaccharides

The present invention relates to polymeric materials comprising chemically modified polysaccharides that form antibacterial hydrogels by crosslinking with each other, bioprinting matrices comprising the polymeric materials, and other uses.

Bacterial infections have become one of the world's largest public healthcare challenges. Specifically, wound infections, one of the most commonly occurring infections, are a major cause of morbidity and mortality. Meanwhile, contamination in cell cultures by bacteria and other microorganisms remains a major concern for researchers, especially in 3D culture systems where detection by visual tracking is more complicated than in regular 2D cultures. One of the most common means to prevent bacterial contamination in vitro is the use of antibiotics. However, previous studies have raised concerns that antibiotics may induce changes in gene expression and regulation in cells.

Chitosan, a cationic polysaccharide derived from chitin, is a renewable polymer that is biodegradable, biocompatible, non-toxic, and antibacterial, and is expected to find applications in fields such as medicine, cosmetics, the food industry, and agriculture (Non-Patent Document 1, etc.). However, because most of the sugar units of chitosan contain primary amino groups, it is insoluble in water, organic solvents, and alkaline solutions, although it is soluble in dilute acids.

As a result, it has traditionally been difficult to create materials such as hydrogels that contain water as a solvent while maintaining the antibacterial properties of chitosan.

Dash et al., Progress in Polymer Science, Vol. 36, 981-1014, 2011

The present invention aims to provide a biomaterial, particularly a hydrogel material, that uses chitosan and has mechanical properties that can be used for a variety of purposes while maintaining the antibacterial properties of chitosan.

As a result of intensive research aimed at solving the above problems, the inventors discovered that by using a polymer material containing two types of polysaccharides, a hydrophilic chitosan derivative and a neutral hydrophilic polysaccharide capable of forming intermolecular hydrogen bonds with the hydrophilic chitosan derivative, it is possible to obtain a hydrogel that has mechanical properties applicable to a variety of uses by means of photocrosslinking and the like while maintaining the antibacterial properties of chitosan, and thus completed the present invention.

を少なくとも部分的に含むガラクトシル化キトサンである、上記＜１＞に記載のポリマー材料；
＜１４＞前記第２の多糖類が、中性の水溶性多糖類の修飾多糖類である、上記＜１＞に記載のポリマー材料；
＜１５＞前記中性の水溶性多糖類が、グルコース、ガラクトース、マンノース、及びそれらの組み合わせよりなる群から選択される繰り返しユニットを含む、上記＜１３＞に記載のポリマー材料；
＜１６＞前記第２の多糖類が、デキストラン、キトサン、ローカストビーンガム、カレギーナン、及びそれらの誘導体よりなる群から選択される、上記＜１＞に記載のポリマー材料；
＜１７＞前記第２の多糖類が、以下の繰り返しユニット：

を少なくとも部分的に含むメタクリル化デキストランである、上記＜１＞に記載のポリマー材料；
＜１８＞前記第２の多糖類が、以下の繰り返しユニット：

を少なくとも部分的に含むメタクリル化キトサンである、上記＜１＞に記載のポリマー材料；
＜１９＞前記第１の多糖類と前記第２の多糖類のモル比が、１：０．１～１：１０の範囲である、上記＜１＞に記載のポリマー材料；
＜２０＞前記第１の多糖類及び前記第２の多糖類が、同じポリマー骨格を有しており、場合により互いに異なる化学修飾を有する、上記＜１＞に記載のポリマー材料；
＜２１＞架橋剤をさらに含む、上記＜１＞に記載のポリマー材料；
＜２２＞前記架橋剤が、水溶性の光架橋剤である、上記＜２１＞に記載のポリマー材料；及び
＜２３＞バイオプリンティングにおけるマトリクス；タンパク質、粒子、若しくはエクソソームのカプセル化；又は、ドラッグデリバリーに用いるための、上記＜１＞に記載のポリマー材料
を提供するものである。 That is, in one aspect, the present invention provides
<1> A polymer material comprising: a) a first polysaccharide having a repeating unit including two types of sugar units, d-glucosamine and N-acetyl-d-glucosamine, the first polysaccharide having a structure in which at least a portion of the amino groups in the d-glucosamine are substituted with a site having a carboxyl group to form an amide bond; and b) a second polysaccharide having a structure in which at least a portion of the hydroxyl groups in the repeating unit are substituted with a functional group selected from the group consisting of an acryl group and a methacryl group, the first polysaccharide and the second polysaccharide both being water-soluble, and the first polysaccharide and the second polysaccharide being capable of forming a hydrogel by crosslinking with each other;
<2> The polymer material according to claim 1, wherein a hydrogen bond is formed between the first polysaccharide and the second polysaccharide;
<3> The polymer material according to <1> above, wherein the site having a carboxyl group in the first polysaccharide contains a sugar acid obtained by oxidizing a monosaccharide and/or a disaccharide;
<4> The polymer material according to the above <2>, wherein the sugar acid contains a galactosyl group; <5> The polymer material according to the above <2>, wherein the sugar acid is selected from the group consisting of lactobionic acid, threonic acid, xylonic acid, and gluconic acid derivatives having one or more sugar structures;
<6> The polymer material according to the above <1>, wherein an introduction rate of the carboxyl group-containing site in the first polysaccharide is 1 to 10%;
<7> The polymer material according to <1> above, wherein the functional group in the second polysaccharide is a methacryl group;
<8> The polymer material according to <1> above, wherein the first polysaccharide has a weight average molecular weight (Mw) in the range of 50 to 2000 kDa;
<9> The polymer material according to <1> above, wherein the second polysaccharide has a weight average molecular weight (Mw) in the range of 4 to 2000 kDa;
<10> The polymer material according to <1> above, wherein the first polysaccharide has antibacterial activity;
<11> The polymer material according to the above <1>, wherein the first polysaccharide is a modified polysaccharide of a cationic polysaccharide;
<12> The polymer material according to the above <10>, wherein the cationic polysaccharide is chitosan or a chitosan derivative;
<13> The first polysaccharide comprises the following repeating units:

The polymer material according to the above item <1>, which is a galactosylated chitosan at least partially comprising:
<14> The polymer material according to the above <1>, wherein the second polysaccharide is a modified polysaccharide of a neutral water-soluble polysaccharide;
<15> The polymer material according to the above <13>, wherein the neutral water-soluble polysaccharide contains a repeating unit selected from the group consisting of glucose, galactose, mannose, and combinations thereof;
<16> The polymer material according to the above <1>, wherein the second polysaccharide is selected from the group consisting of dextran, chitosan, locust bean gum, careginan, and derivatives thereof;
<17> The second polysaccharide has the following repeating unit:

The polymer material according to the above item <1>, which is a methacrylated dextran at least partially comprising:
<18> The second polysaccharide has the following repeating unit:

The polymer material according to the above item <1>, which is a methacrylated chitosan at least partially comprising:
<19> The polymer material according to <1> above, wherein the molar ratio of the first polysaccharide to the second polysaccharide is in the range of 1:0.1 to 1:10;
<20> The polymer material according to <1> above, wherein the first polysaccharide and the second polysaccharide have the same polymer backbone and, optionally, different chemical modifications from each other;
<21> The polymer material according to the above <1>, further comprising a crosslinking agent;
<22> The polymer material according to <21> above, wherein the crosslinking agent is a water-soluble photocrosslinking agent; and <23> the polymer material according to <1> above for use as a matrix in bioprinting; for encapsulation of proteins, particles, or exosomes; or for drug delivery.

In another aspect, the present invention relates to a bioprinting matrix comprising the above gel material and a use thereof,
<24> A matrix for bioprinting, comprising the polymer material according to any one of <1> to <23> above, wherein the first polysaccharide and the second polysaccharide are crosslinked with each other during bioprinting to form a hydrogel;
<25> The bioprinting matrix according to <24> above, which has antibacterial activity and low toxicity; and <26> use of the polymer material according to any one of <1> to <23> above or the matrix according to <24> or <25> above in bioprinting; encapsulation of proteins, particles, or exosomes; or drug delivery.

According to the present invention, it is possible to obtain an antibacterial composite hydrogel that has mechanical properties applicable to various applications by means of photocrosslinking or the like while maintaining the antibacterial properties of chitosan. The mechanical properties of such a hydrogel are improved by the formation of hydrogen bonds between the two types of polysaccharides contained in the polymer material.

In addition, the polymer material of the present invention is biocompatible, water-soluble, and antibacterial, and can be easily gelled by light irradiation, etc., making it useful as a matrix for 3D bioprinting. These properties make it applicable to a wide range of applications, including encapsulation of proteins and drug delivery, tissue engineering, cell culture, drug discovery and screening, in vitro research, tissue regeneration, and regenerative medicine.

FIG. 1 shows the FTIR spectra of chitosan (CH) and modified chitosan (LACH). Figure 2 is a graph showing the inhibition activity of E. coli growth in the presence of chitosan (CH) and modified chitosan (LACH). The final concentration of polysaccharide in the graph is in % wt/v. FIG. 3A is a graph showing the growth rate of bacteria in the presence of polysaccharides, and FIG. 3B is an image of CFUs on an agar plate after 24 hours of incubation. FIG. 4 is a graph showing the decomposition behavior of a hydrogel by an enzyme. FIG. 5 shows the printing properties of the LACH/DEXMA solution: FIG. 5A is an image of continuous filament formation (shear thinning action) as the precursor polymer solution is extruded; FIG. 5B is a screenshot of the print preview; FIG. 5C is an image of the bioprinting process; FIG. 5D is an image of the printed construct; and FIG. 5E is an image of the square area used to calculate the printing properties. FIG. 6 is a graph showing cell viability in gel-forming polysaccharides (gel precursor polymers). FIG. 7 is a graph showing cell viability in photocrosslinked gels. FIG. 8 is a flow diagram showing the procedure of the protein release assay in Example 7. FIG. 9 is a graph showing the kinetic profile of protein release from photocrosslinked gels.

Below, an embodiment of the present invention will be described. The scope of the present invention is not limited to these descriptions, and other than the following examples, the present invention can be modified and implemented as appropriate without departing from the spirit of the present invention.

1. Polymer Material of the Present Invention The polymer material of the present invention is characterized in that it contains a) a first polysaccharide and b) a second polysaccharide defined below, both of which are water-soluble and capable of forming a hydrogel by crosslinking with each other.

1-1. First polysaccharide The first polysaccharide of the present invention is a polysaccharide having repeating units containing two types of sugar units, d-glucosamine and N-acetyl-d-glucosamine. The first polysaccharide has a structure in which at least a part of the amino groups in the d-glucosamine is substituted with a site having a carboxyl group to form an amide bond. This chemical modification can increase the water solubility of the first polysaccharide.

In a preferred embodiment, the "site having a carboxyl group" includes a sugar acid formed by oxidizing a monosaccharide and/or a disaccharide. Such a sugar acid may preferably include a galactosyl group. For example, the sugar acid may be selected from the group consisting of lactobionic acid, threonic acid, xylonic acid, and gluconic acid derivatives having one or more sugar structures.

In a preferred embodiment, the first polysaccharide can be a modified cationic polysaccharide. Such cationic polysaccharides can typically include, but are not limited to, chitosan or chitosan derivatives. In terms of having antibacterial activity, chitosan or chitosan derivatives are preferred.

A non-limiting example of the first polysaccharide is a galactosylated chitosan that at least partially comprises the following repeating units:

The introduction rate of the carboxyl group-containing moiety in the first polysaccharide can be preferably 1 to 10%. This can increase the water solubility of the first polysaccharide while maintaining the inherent properties of the first polysaccharide, such as antibacterial activity. Here, "introduction rate" refers to the proportion of amino groups in the first polysaccharide that are substituted by the carboxyl group-containing moiety.

The first polysaccharide preferably has a weight average molecular weight (Mw) in the range of 50 to 2000 kDa.

1-2. Second polysaccharide The second polysaccharide in the present invention has a structure in which at least a part of the hydroxyl groups in the repeating unit is substituted with a functional group selected from the group consisting of an acrylic group and a methacrylic group. The functional group is a methacrylic group. By having such a functional group, the water solubility of the second polysaccharide can be increased.

The second polysaccharide is preferably a modified polysaccharide of a neutral water-soluble polysaccharide. That is, the first polysaccharide is preferably a cationic polysaccharide, whereas the second polysaccharide is preferably neutral and uncharged. This is preferable from the viewpoint of preventing electrostatic interactions with the first polysaccharide and avoiding pH dependency in solubility.

The neutral water-soluble polysaccharide may be, for example, a polysaccharide containing a repeating unit selected from the group consisting of glucose, galactose, mannose, and combinations thereof. More specifically, the second polysaccharide may be selected from the group consisting of dextran, chitosan, locust bean gum, carrageenan, and derivatives thereof, and may have the above-mentioned functional groups. Preferably, the second polysaccharide may be dextran, chitosan, or a derivative thereof.

（式中、ｎは２～１０００の自然数である。） A non-limiting example of the second polysaccharide may be a methacrylated dextran that includes, at least in part, the following repeating units:

(In the formula, n is a natural number from 2 to 1000.)

In another preferred embodiment, the second polysaccharide may include, but is not limited to, methacrylated chitosan, which at least in part comprises the following repeating units:

The second polysaccharide may preferably have a weight average molecular weight (Mw) in the range of 4 to 2000 kDa.

In the present invention, the first polysaccharide and the second polysaccharide are both water-soluble polysaccharides. Typically, the first polysaccharide is the above-mentioned galactosylated chitosan; and the second polysaccharide is methacrylated dextran or methacrylated chitosan.

For example, when the first polysaccharide is a chitosan and the second polysaccharide is a dextran, hydrogen bonds can be formed between the amino groups of the chitosan and the hydroxyl groups of the dextran. These hydrogen bonds are weaker than the electrostatic interactions of other polyanionic polymers. Here, the antibacterial properties of chitosan are derived from the electrostatic interactions between the amino groups of the sugar moiety and the negatively charged cell walls of bacterial cells, so the antibacterial properties of chitosan can be maintained by using a neutral dextran or the like as the first polysaccharide.

In another preferred embodiment, the first polysaccharide and the second polysaccharide have the same polymer backbone and may have different chemical modifications. Specifically, the first polysaccharide may be a galactosylated chitosan and the second polysaccharide may be a methacrylated chitosan.

In a preferred embodiment, the molar ratio of the first polysaccharide to the second polysaccharide in the polymer material of the present invention is preferably in the range of 1:0.1 to 1:10. However, the molar ratio can be changed appropriately depending on the type of polysaccharide, etc., from the viewpoint of ease of forming a hydrogel, which will be described later. For example, the first polysaccharide can have a concentration typically in the range of 10 to 100 mg/ml, preferably 25 to 50 mg/ml, and the second polysaccharide can have a concentration typically in the range of 30 to 200 mg/ml, preferably 50 to 100 mg/ml.

In a preferred embodiment, the first polysaccharide and the second polysaccharide have the same polymer backbone and, optionally, different chemical modifications.

1-3. Other Components The first polysaccharide and the second polysaccharide contained in the polymer material of the present invention can form a hydrogel by crosslinking with each other. Therefore, the polymer material of the present invention can contain an optional crosslinking agent in addition to the first and second polysaccharides. The crosslinking agent can be added at a concentration of preferably 1 to 10 mg/ml, more preferably 2 to 5 mg/ml.

As such a crosslinking agent, any agent known in the art can be used, for example, a photopolymerization initiator or a water-soluble azo polymerization initiator such as 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959) or 2,2'-azobis[2-methyl-N-(2-hydroxyethyl)propionamide (VA-086) can be used.

The crosslinking agent contained in the polymer material of the present invention is preferably a water-soluble photocrosslinking agent. The light irradiation time for photocrosslinking is not particularly limited, but typically can be in the range of 1 to 30 minutes.

In this specification, "gel" generally refers to a dispersion system of polymers that has high viscosity and has lost fluidity, and in which the storage modulus G' and loss modulus G" satisfy the relationship G' ≥ G". A gel that uses water as a solvent is called a "hydrogel".

1-4. Applications The polymeric material of the present invention can be applied to a wide range of applications, such as tissue engineering, cell culture, drug discovery and screening, in vitro research, tissue regeneration and regenerative medicine, etc. More specifically, such applications can include, for example, matrices in bioprinting; encapsulation of proteins, particles, or exosomes; or drug delivery.

In particular, the polymer material of the present invention is biocompatible, water-soluble, and antibacterial, and can be easily gelled by light irradiation or the like, making it useful as a matrix for 3D bioprinting. Therefore, the present invention also provides a matrix for bioprinting that includes the above-mentioned polymer material and forms a hydrogel by crosslinking the first polysaccharide and the second polysaccharide with each other during bioprinting. The matrix is characterized by having antibacterial activity and low toxicity.

In another aspect, the present invention provides the use of the polymeric material or matrix in bioprinting; encapsulation of proteins, particles or exosomes; or drug delivery.

The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these.

1. Preparation of Polymer Material The polymer material of the present invention was prepared and a hydrogel was formed from the polymer material according to the following procedure.

[Reaction 1: Synthesis of water-soluble chitosan]
A water-soluble chitosan material corresponding to the first polysaccharide was prepared by introducing a hydrophilic functional group into chitosan. Specifically, as shown in Scheme 1, chitosan was reacted with lactobionic acid (galactosylation reaction) to modify the amino groups in the chitosan molecule. This reaction yielded a soluble galactosylated chitosan that was soluble up to 60 mg/ml in a buffer in the physiological pH range.

The formation of galactosylated chitosan was confirmed by FTIR. The wavenumber region used for the measurements was in the range of 4000-1000 cm ^-1 . Figure 1 shows the FTIR spectra of chitosan before (left panel: CH) and after modification (right panel: LACH) with lactobionic acid (LA). The most representative changes that confirm the successful implementation of chemical modification are the shift of the absorption band from 1653 cm ^-1 to 1625 cm ^-1 , corresponding to the stretching of the C-O bond of the acetamide group of chitosan, and the shift of the absorption band from 1597 cm ^-1 to 1532 cm ^-1 , corresponding to the amino group of the polysaccharide. These changes are due to the formation of new amide bonds between the amino group of CH and the carboxyl group of LA.

Reaction 2: Modification of the second polysaccharide
The second polysaccharide of the present invention includes methacrylation by reaction of hydroxyl groups present in the starting polysaccharide with methacrylic anhydride (MA) or glycidyl methacrylate (GMA), which introduces double bond moieties into the second polysaccharide that can be converted to crosslinks by UV light activation.

Specifically, dextran (DEX) was reacted with glycidyl methacrylate (GMA) to obtain methacrylated dextran (DEXMA) as shown in Scheme 2. The reaction was confirmed by NMR.

The reaction was confirmed by ¹ H-NMR (solvent: DMSO-d ₆ ). As a result, the proton peak in the region of δ5-4.4 ppm represents the anomeric proton in the anhydroglucose unit of dextran. The peak of the acryl group (-CH=CH2), which does not exist in the dextran before modification, appeared in the range of δ5.6-6.3 ppm, indicating that methacrylated dextran was obtained.

[Reaction 3: Mixing of the first and second polysaccharides]
Next, the water-soluble chitosan (LACH: first polysaccharide) synthesized above was mixed with methacrylated dextran (DEXMA: second polysaccharide), which resulted in the formation of hydrogen bonds between the amino groups of LACH and the hydroxyl groups of DEXMA, as shown in Scheme 3.

[Reaction 4: Addition of photocrosslinker]
A water-soluble photocrosslinker was added to the mixture of the first and second polysaccharides, and the mixture was gelled by short-term irradiation with light. The photocrosslinker can be a compound that is activated by UV or visible light wavelengths.

2. Study of gelation conditions The first and second polysaccharides, LACH and DEXMA, synthesized above, were dissolved in PBS and photocrosslinked in the presence of Irgacure 2959 or VA-086 (2,2'-azobis[2-methyl-N-(2-hydroxyethyl)propionamide]) as a crosslinking agent. Photocrosslinking was performed by irradiating a 30 ul polymer droplet with 365 nm light at an intensity of 5 mW/ ^cm2 at a distance of 5 cm from the light source. Table 1 shows the results of photocrosslinking using multiple concentrations of LACH with various molecular weights and DEXMA with various concentrations. Note that Irgacure 2959 and VA-086 gave the same results at all concentrations.

As shown in Table 1, the molecular weight of chitosan, the raw material of LACH, was found not to affect gelation. Under the LACH conditions used in the experiment, the minimum DEXMA concentration at which gel formation was achieved was 50 mg/ml. When the photoinitiator concentration was low or the DEXMA concentration was low, the exposure time required for gelation tended to be longer. Under the highest photoinitiator concentration (5 mg/ml), gel formation was achieved within 1 minute at almost all LACH/DEXMA ratios. The optimal condition (number 7) in Table 1 was selected taking into consideration the potential toxicity of the photoinitiator, gelation time, and polymer ratio.

3. Antibacterial Properties Chitosan is known to have antibacterial properties. To confirm that the modified chitosan (LACH) synthesized above also maintained its antibacterial properties, its bactericidal activity against the gram-negative bacterium Escherichia coli (E. coli) was examined. The optical density (OD) measurement method of bacterial culture was used to estimate the density of cells in liquid culture in the presence or absence of polysaccharide samples at various time points. Specifically, bacteria were resuspended in LB medium at a concentration of ^1x108 CFU/ml, and 300 μl aliquots of bacterial broth were added to 2700 μl of medium containing CH, LACH or control. The cultures were incubated at 37°C with moderate agitation. Thereafter, 100 uL of culture medium was sampled at 1-hour intervals (first 6 hours including time zero) and the absorbance at 640 nm was measured by a spectrophotometer. If the medium was turbid, the sampled culture medium was diluted with PBS.

The measurement results are shown in Figure 2. As a result, it was confirmed that both chitosan (CH) and modified chitosan (LACH) have inhibitory activity against the growth of E. coli. Furthermore, the higher the concentration of these polysaccharides, the stronger the antibacterial effect.

Next, the bactericidal effects of CH, LACH, DEXMA, and the LACH/DEXMA composite (0.5% wt/v) against Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria were evaluated. The optical density was measured after 24 hours of incubation in the presence of polysaccharides (Figure 3A). The results were normalized to the optical density measured at time zero (100% cultured bacteria). 100 μl of each culture broth was diluted (1:100,000) and plated on nutrient agar, and images of bacterial colony forming units (CFU) were taken after 24 hours of incubation (Figure 3B).

As shown in Figure 3, CH, LACH, and LACH/DEXMA composites were found to exhibit antibacterial activity against both E. coli and S. aureus. In contrast, the control sample (containing no polysaccharides) and DEXMA showed high bacterial growth rates and no antibacterial activity. These results demonstrate that both LACH and the LACH/DEXMA composite have antibacterial activity against two of the most common bacteria found in clinical settings.

4. In Vitro Degradation Test Hydrogels used in vivo must be degraded in a controlled manner to allow the target tissue to regenerate to its natural structure, morphology, and function. Chitosan is easily degraded by enzymes, particularly lysozyme, which is naturally present in various parts of the human body, via cleavage of glycosidic bonds. In addition, dextran is known to be susceptible to enzymatic degradation by lysozyme. Therefore, the degradability of the LACH/DEXMA gel of the present invention was examined in the presence of lysozyme.

Hydrogels were formed by irradiating a gel precursor solution containing LACH/DEXMA with 5 mW/ ^cm2 UV light (365 nm) for 2 minutes at a distance of 5 cm from the light source. Low and medium molecular weight LACH were used. The composition of the gel precursor solution is as shown in Table 1 above. VA-086 (2 mg/ml) was added to the gel precursor solution (PBS solution) prior to UV crosslinking. As a comparative example, a hydrogel consisting of only DEXMA without LACH was prepared.

For degradation experiments, 100 ul drops were crosslinked in a 24-well plate. After gelation, 500 ul of PBS containing 4 mg/ml lysozyme was added to each well and incubated at 37°C with gentle agitation. At predetermined time intervals, the lysozyme solution was removed and excess surface liquid from the gel samples was dried with filter paper. After recording the weight of the gel samples, the gel samples were placed back into the wells with freshly prepared lysozyme solution.

The degree of decomposition of the gel (% DD) was calculated from the measured weight change of the gel sample according to the following formula.

In this formula, "%DD" is the degree of decomposition of the gel, "W0" is the initial weight of the gel, and "W(t)" is the weight of the gel at the time of measurement.

The results of the degradation behavior of hydrogels due to lysozyme activity are shown in Figure 4. It was found that the hydrogels containing LACH were more easily degraded over time than the gels containing DEXMA alone. In addition, after 48 hours, different degradation behaviors were observed between gels containing low molecular weight LACH and gels containing medium molecular weight LACH. That is, the hydrogels derived from low molecular weight chitosan showed accelerated degradation behavior compared to those containing medium molecular weight chitosan. However, after 144 hours of incubation, both of these gels were degraded by about 50%. These results confirmed that the hydrogels of the present invention have a degradation profile suitable for tissue engineering and that the degradation characteristics can be adjusted by the molecular weight of LACH.

5. Evaluation of Printing Characteristics Next, a 3D bioprinting test was carried out using the LACH/DEXMA composite material of the present invention.

　Generally, hydrogel-based inks with shear-thinning properties are used to prevent excessive shear stress during the bioprinting process, which may affect cell viability. In this regard, the LACH/DEXMA composite solution (gel precursor) of the present invention has a lower viscosity than the final hydrogel after gelation and exhibits shear-thinning behavior at an optimal composition, as shown in Figure 5A.

The bioprinting test was carried out as follows: Bioprinting was performed using a 3d Cultures Tissue Scribe bioprinter. A polymer solution (composition 7 in Table 1) containing modified chitosan (LACH), modified dextran (DEXMA) and VA-086 was loaded into a 3 ml syringe (BD Luer-Lok™) with a nozzle (inner diameter 0.8 mm). A square object of 30 x 30 mm with a height of 5 mm was designed and loaded into the Repeater Host program (Figure 5B). The height was adjusted by scaling the Z axis to 0.1. The nozzle was 0.8 mm, the layer thickness was 0.8 mm, the initial layer and the filling layer were 0.5 mm, the filling speed was 2 mm/s and the filling rate was 60%. A printing temperature of 25 °C was used. A representative photograph of the printing process is shown in Figure 5C.

After the printing process was completed, the resulting grid area was irradiated with 5 mW/ ^cm2 UV light (365 nm) for 2 min at a distance of 5 cm from the light source to form a gel by photocrosslinking (Figure 5D). The printed structures were photographed by a digital camera, and the printing properties were evaluated by the following equations:

In this formula, "A'" is the measured area of the square-like regions in the printed structure, and "A" is the theoretical area of the square as originally designed. When the bioink (LACH/DEXMA solution) is in an ideal gelation state, the extruded filaments will show a well-defined morphology with a three-dimensional smooth surface and consistent width, and the printed structure will form a regular grid and square holes. A structure printed using the LACH/DEXMA gel of the present invention is shown in Figure 9E. The printing characteristic in this case was Pr = 0.93 ± 0.05.

In an ideal printing process, the structures would exhibit a perfect square shape and the printing characteristic Pr would have a value of 1. A higher Pr value would result in a greater degree of gelation of the bioink, whereas a lower Pr value would result in a lesser degree of gelation of the bioink. In general, a printing characteristic Pr in the range of 0.9 to 1.1 indicates that the bioink is suitable for use in bioprinting applications. Thus, the polymer material of the present invention has been demonstrated to be suitable as a bioink for 3D bioprinting applications.

6. Evaluation of cytotoxicity in cell culture The polymeric material of the present invention was then evaluated for cytotoxicity. Specifically, modified chitosan (LACH) and modified dextran (DEXMA) polysaccharides were tested for cytotoxicity against human dermal fibroblasts (HDFa, ATCC) using either individually or in combination. Cell viability, proliferation and cytotoxicity were evaluated using the XTT assay, a standard colorimetric assay.

Cells were plated in 96-well plates at a final density of ^5x103 cells per well in 100 μL of DMEM containing 10% FBS and allowed to attach to the bottom of the well overnight. The medium was then replaced with DMEM containing 0.5% w/ ^v of the polymer sample (same concentration as in Example 3) under serum-free conditions. The cells were incubated for 48 hours at 37°C in 5% CO2. After the incubation period, XTT reagent (BIOTIUM, Cat: 10060y Lot: 17X0824) was added and the cells were incubated for another 2 hours. This allows only metabolically active cells to reduce the yellow tetrazolium salt (XTT: sodium 3'-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzenesulfonic acid hydrate) to an orange formazan dye. The resulting color change was measured at 460 nm (reference 650 nm) using a plate reader. Data was normalized to untreated wells (100% viability).

As shown in Figure 6, cells treated with the polymeric material of the present invention showed nearly 100% viability after the incubation period, with no significant differences between groups. This result indicates that the polymeric materials (gel precursor polymers) of the present invention, either individually or in combination, do not exhibit cytotoxicity.

Next, we verified whether the encapsulated cells could survive and grow in the hydrogel after photocrosslinking. A polymer material (precursor solution) containing LACH and DEXMA was prepared according to the composition in Table 1, and mesenchymal stem cells were encapsulated (1 x ¹⁰⁶ /ml). Then, 30 μl droplets were dispensed into wells of a 96-well plate. As a comparative example, the same cell density was resuspended in methacrylated gelatin (Gelma), the most common photocrosslinking polymer used as bioink. Finally, the plate was irradiated with 5 mW/ ^cm2 UV light (365 nm) for 2 minutes at a distance of 5 cm from the light source. The cell viability was evaluated using the XXT assay described above.

As shown in Figure 7, no significant differences were observed in cells resuspended in hydrogels crosslinked with the polymeric material of the present invention compared to the comparative methacrylated gelatin (Gelma). Both samples showed nearly 100% viability after the incubation period. This result indicates that the LACH/DEXMA hydrogel of the present invention is not cytotoxic and is a suitable material for bioprinting of live cells.

7. Evaluation of protein release capacity Protein release assay was performed using bovine serum albumin (BSA). The procedure is shown in Figure 8. After a given time, all collected samples were treated with known concentrations of BSA in PBS, and the protein concentration was determined using a standard curve.

The obtained dynamic release profile is shown in Figure 9. During the first 24 hours, a strong release of protein occurs, known as the burst effect. Thereafter, a sustained protein release is observed from 24 to 168 hours. In particular, the release of BSA becomes stable after 48 hours. The result of a burst effect value of approximately 30% suggests that the hydrogel of the present invention can provide both the targeted initial delivery of the drug at the required dose and the subsequent long-term maintenance of a constant therapeutic concentration in the internal environment.

8. Formation of photocrosslinked gels using other combinations of polysaccharides
To demonstrate the applicability of the system using other methacrylated derivatives, the second polysaccharide was changed to methacrylated chitosan (MACH) to form hydrogels and their gelling properties and cell viability were evaluated. MACH was synthesized by reacting medium molecular weight chitosan with methacrylic anhydride.

MACH was synthesized by reacting medium molecular weight chitosan with methacrylic anhydride. The formation of MACH was confirmed by analyzing the peaks in the ¹ H-NMR spectrum of the reaction product. The characteristic peaks of chitosan were observed at δ 2.0 ppm (-CH ₃ of the acetylated part of chitosan) and δ 3.1-4 ppm, which represent protons from the glucosamine ring. The peaks of acrylic groups (-CH=CH ₂ ), which are not present in unmodified chitosan, were observed at δ 5.6-6.3 ppm, confirming the formation of methacrylated chitosan MACH.

LACH (medium molecular weight) and MACH were dissolved in PBS and photocrosslinked in the presence of the crosslinker VA-086. Photocrosslinking was performed by irradiating a 30 μl drop of the polymer mixture with 365 nm light at an intensity of 5 mW/ ^cm2 and a distance of 5 cm to the light source. The results obtained using various polymer ratios are shown in Table 2.

As shown in Table 2, the use of MACH as the second polysaccharide in combination with LACH as the first polysaccharide also demonstrated the formation of hydrogels within minutes of UV light exposure over a wide range of concentration ratios. Furthermore, the resulting gels are transparent and may be suitable for several biomedical applications, such as vitreous substitutes.

Claims (26)

A polymeric material comprising:
a) a first polysaccharide having a repeating unit containing two types of sugar units, d-glucosamine and N-acetyl-d-glucosamine, the first polysaccharide having a structure in which at least a portion of the amino groups in the d-glucosamine are substituted with a site having a carboxyl group to form an amide bond; and b) a second polysaccharide having a structure in which at least a portion of the hydroxyl groups in the repeating unit are substituted with a functional group selected from the group consisting of an acryl group and a methacryl group,
The first polysaccharide and the second polysaccharide are both water-soluble, and the first polysaccharide and the second polysaccharide are capable of forming a hydrogel by crosslinking with each other.
The polymeric material. The polymer material of claim 1, wherein hydrogen bonds are formed between the first polysaccharide and the second polysaccharide. The polymer material according to claim 1, wherein the carboxyl group-containing portion of the first polysaccharide contains a sugar acid formed by oxidizing a monosaccharide and/or a disaccharide. The polymer material of claim 3, wherein the sugar acid comprises a galactosyl group. The polymer material of claim 3, wherein the sugar acid is selected from the group consisting of lactobionic acid, threonic acid, xylonic acid, and gluconic acid derivatives having one or more sugar structures. The polymer material according to claim 1, wherein the introduction rate of the carboxyl group-containing site in the first polysaccharide is 1 to 10%. The polymer material of claim 1, wherein the functional group in the second polysaccharide is a methacryl group. The polymer material of claim 1, wherein the first polysaccharide has a weight average molecular weight (Mw) in the range of 50 to 2000 kDa. The polymer material of claim 1, wherein the second polysaccharide has a weight average molecular weight (Mw) in the range of 4 to 2000 kDa. The polymeric material of claim 1, wherein the first polysaccharide has antibacterial activity. The polymer material of claim 1, wherein the first polysaccharide is a modified cationic polysaccharide. The polymer material according to claim 11, wherein the cationic polysaccharide is chitosan or a chitosan derivative. The first polysaccharide comprises the following repeating units:

The polymeric material of claim 1 , which is a galactosylated chitosan comprising at least in part: The polymer material of claim 1, wherein the second polysaccharide is a modified polysaccharide of a neutral water-soluble polysaccharide. The polymeric material of claim 14, wherein the neutral water-soluble polysaccharide comprises repeating units selected from the group consisting of glucose, galactose, mannose, and combinations thereof. The polymeric material of claim 1, wherein the second polysaccharide is selected from the group consisting of dextran, chitosan, locust bean gum, carrageenan, and derivatives thereof. The second polysaccharide comprises the following repeat unit:

2. The polymeric material of claim 1, which is a methacrylated dextran comprising at least in part: The second polysaccharide comprises the following repeat unit:

2. The polymeric material of claim 1, which is a methacrylated chitosan comprising at least in part: The polymer material according to claim 1, wherein the molar ratio of the first polysaccharide to the second polysaccharide is in the range of 1:0.1 to 1:10. The polymeric material of claim 1, wherein the first polysaccharide and the second polysaccharide have the same polymer backbone and optionally have different chemical modifications. The polymeric material of claim 1 further comprising a crosslinking agent. The polymer material of claim 21, wherein the crosslinking agent is a water-soluble photocrosslinking agent. The polymeric material of claim 1 for use as a matrix in bioprinting; for encapsulating proteins, particles or exosomes; or for drug delivery. A matrix for bioprinting comprising the polymer material according to any one of claims 1 to 23, in which the first polysaccharide and the second polysaccharide are crosslinked with each other during bioprinting to form a hydrogel. The bioprinting matrix of claim 24, which has antibacterial activity and low toxicity. Use of the polymeric material according to any one of claims 1 to 23 or the matrix according to claim 24 or 25 in bioprinting; encapsulation of proteins, particles or exosomes; or drug delivery.