Hydrogel material with elasticity, electrical conductivity and photothermal properties and method for manufacturing the same

Abstract

One embodiment of the present invention provides a bio-ink comprising hyaluronic acid methacrylate and PDRN. The bio-ink according to the embodiment of the present invention is characterized by having good physical properties with improved elasticity compared to conventional hydrogel materials using HAMA, and provides a material of excellent performance that can be used in various fields by possessing excellent biocompatibility and bio-healing ability.

Description

Technology Field

[0001] The present invention relates to bio-ink, and more specifically, to a hyaluronic acid methacrylate-based bio-ink with improved elasticity and a method for manufacturing the same. Background Technology

[0002] As 3D printing is being fully integrated into the medical field, it is driving a major transformation in medical technology, and accordingly, bio-ink technology is attracting attention.

[0003] Bioink is produced by combining biomaterials or biomolecules and is used in bioprinting technology that fabricates three-dimensional structures by fusing various types of cells, biomaterials, and biomolecules.

[0004] Polymer hydrogels are widely used as biocompatible materials utilizing bioinks, but bioactivity may vary depending on the composition of the hydrogel, and it is important to have physical and chemical properties similar to body tissues to ensure bioactivity.

[0005] Regarding 3D printing bioink materials for creating such in vivo tissues, our research team focused on the previously proposed HAMA hydrogel. HAMA (Hyaluronic acid methacrylate) is a widely used 3D printable hydrogel material, but due to its poor elasticity, it was not suitable for use in human body parts such as the knee or elbow. Prior art literature

[0006] Republic of Korea Registered Patent No. 10-1848997 The problem to be solved

[0007] The present invention provides a hydrogel patch with improved elasticity by introducing PDRN (Poly Deoxy Ribo Nucleotide) into a conventional HAMA hydrogel as a method to solve the problems of the aforementioned prior art, and with improved functionality by additionally introducing MXene.

[0008] The technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned technical problems will be clearly understood by those skilled in the art to which the present invention belongs from the description below. means of solving the problem

[0009] To achieve the above technical problem, one embodiment of the present invention provides a bio-ink.

[0010] A bio-ink according to one embodiment of the present invention is characterized by comprising hyaluronic acid methacrylate; PDRN (Poly Deoxy Ribo Nucleotide); and a photoinitiator.

[0011] In an embodiment of the present invention, the bio-ink may be characterized by further including MXene.

[0012] In an embodiment of the present invention, the bio-ink may be characterized by containing 1 to 8 wt% of the hyaluronic acid methacrylate and 0.5 to 5 wt% of the PDRN based on the total weight.

[0013] In an embodiment of the present invention, the MXene may be a bioink characterized by being selected from Ti2C, Ti2N, Ti3C2, Ti3N2, Ti3CN, Ti4N3, Nb4C3, Nb2C, Nb2N, V2C, V2N, Ta4C3, Mo2C, Mo2TiC2, Mo2Ti2C3, Cr2TiC2, (Ti0.5, Nb0.5)2C, Zr3C2, Hf3C2, Mo2TiC2, Cr2TiC2, or a combination thereof.

[0014] In an embodiment of the present invention, the bioink may be characterized by containing 0.1 to 3 mg / ml of MXene with respect to the bioink.

[0015] In an embodiment of the present invention, the photoinitiator may be a bioink characterized by being at least one selected from the group consisting of lithium phenyl-2,4,6-trimethylbenzoyl phosphinate (LAP), 4-(2-hydroxyethoxy)phenyl-(2-hydroxy-2-propyl)ketone, Eoxin-Y, rose bengal, and riboflavin (vitamin B2).

[0016] To achieve the above technical problem, another embodiment of the present invention provides a method for manufacturing bio-ink.

[0017] A method for manufacturing a bio-ink according to one embodiment of the present invention comprises: a step of preparing hyaluronic acid methacrylate, PDRN, and a photoinitiator; a step of adding the PDRN and the photoinitiator to distilled water and mixing them; and a step of adding the hyaluronic acid methacrylate to the distilled water and mixing them to manufacture a bio-ink.

[0018] In an embodiment of the present invention, the method for manufacturing a bio-ink may be characterized by adding MXene in the step of adding and mixing the PDRN and photoinitiator in distilled water.

[0019] In an embodiment of the present invention, the step of preparing the hyaluronic acid methacrylate and PDRN may be a method for manufacturing a bio-ink, wherein the hyaluronic acid methacrylate is prepared by synthesizing it by adding methacrylic anhydride (MA) to an aqueous solution of sodium hyaluronic acid salt (HA).

[0020] In an embodiment of the present invention, the step of preparing the hyaluronic acid methacrylate and PDRN may be a method for manufacturing a bio-ink, wherein the hyaluronic acid methacrylate is prepared by synthesizing it by adding methacrylic anhydride (MA) to an aqueous solution of sodium hyaluronic acid salt (HA).

[0021] In an embodiment of the present invention, the hydrogel may be prepared using the bio-ink. Effects of the invention

[0022] According to an embodiment of the present invention, through a bio-ink comprising hyaluronic acid methacrylate, PDRN, and a photoinitiator, it is possible to provide an excellent bio-3D printing material having high physical properties with improved elasticity compared to conventional hyaluronic acid methacrylate-based hydrogel materials, as well as excellent biocompatibility and bio-healing ability.

[0023] In addition, according to another embodiment of the present invention, by further adding MXene to the bio-ink, there is an advantage of being able to provide a 3D printing material with various functionalities that possesses both conductive and photothermal properties.

[0024] The effects of the present invention are not limited to the effects described above, and should be understood to include all effects that can be inferred from the configuration of the invention described in the detailed description of the invention or the claims. Brief explanation of the drawing

[0025] FIG. 1 is a flowchart illustrating a method for manufacturing bio-ink according to one embodiment of the present invention. Figure 2 shows the results of Fourier transform infrared spectral analysis of HA and HAMA prepared in an experimental example according to one embodiment of the present invention. Figure 3 shows the results of nuclear magnetic resonance spectroscopy analysis of HA and HAMA prepared in an experimental example of one embodiment of the present invention. Figure 4 is a photograph showing bio-ink and hydrogel produced by a bio-ink manufacturing method according to one embodiment of the present invention. Figure 5 is a graph showing the results of a tensile test according to the PDRN content of a hydrogel according to one embodiment of the present invention. Figure 6 shows the results of a tensile test according to the MXene concentration of a hydrogel according to one embodiment of the present invention. Figure 7 shows the photothermal properties of a hydrogel according to one embodiment of the present invention, showing the temperature change and the maximum temperature reached according to the MXene concentration. Figure 8 shows the change in resistance value according to the MXene concentration of a hydrogel according to one embodiment of the present invention. FIG. 9 is a diagram showing the electrical conductivity of HAMA, HX, HD, and a hydrogel according to one embodiment of the present invention. Figure 10 is an image of a wound healing assay according to DNA concentration of a hydrogel according to one embodiment of the present invention. FIG. 11 is a diagram showing cell viability data of a hydrogel according to one embodiment of the present invention. Specific details for implementing the invention

[0026] The present invention will be described below with reference to the attached drawings. However, the present invention may be implemented in various different forms and is therefore not limited to the embodiments described herein. Furthermore, in order to clearly explain the present invention in the drawings, parts unrelated to the explanation have been omitted, and similar parts throughout the specification have been given similar reference numerals.

[0027] Throughout the specification, when it is stated that a part is "connected (connected, in contact, combined)" with another part, this includes not only cases where they are "directly connected," but also cases where they are "indirectly connected" with other members interposed between them. Furthermore, when it is stated that a part "includes" a certain component, this means that, unless specifically stated otherwise, it does not exclude other components but rather allows for the inclusion of additional components.

[0028] The terms used herein are merely for describing specific embodiments and are not intended to limit the invention. Singular expressions include plural expressions unless the context clearly indicates otherwise. In this specification, terms such as “comprising” or “having” are intended to indicate the presence of the features, numbers, steps, actions, components, parts, or combinations thereof described in the specification, and should be understood as not precluding the existence or addition of one or more other features, numbers, steps, actions, components, parts, or combinations thereof.

[0029] Embodiments of the present invention will be described in detail below with reference to the attached drawings.

[0031] A bio-ink according to one embodiment of the present invention is described.

[0032] A bio-ink according to one embodiment of the present invention is characterized by comprising hyaluronic acid methacrylate, PDRN, and a photoinitiator.

[0033] In the above bio-ink, hyaluronic acid methacrylate is a substance in which hyaluronic acid and methacrylate are combined, and it is known as a substance capable of chemically modifying methacrylate while maintaining the properties of hyaluronic acid.

[0034] At this time, the aforementioned hyaluronic acid is a natural polymer found in the skin and joints, and is known to have excellent moisture absorption and retention capabilities, as well as functions such as enhancing skin elasticity and protecting joints. On the other hand, methacrylate is chemically stable and has the characteristic of being able to combine with various substances to create new materials.

[0035] The above hyaluronic acid methacrylate combines the advantages of these two substances and is a material that possesses both the moisture absorption and retention capabilities of hyaluronic acid and the chemical stability of methacrylate. Due to these characteristics, hyaluronic acid methacrylate is utilized in various fields such as cosmetics, pharmaceuticals, and medical devices, and has been primarily used in bio-inks for biohydrogel materials. In the following specification, “HAMA” refers to hyaluronic acid methacrylate, and “HA” refers to hyaluronic acid.

[0036] The present invention is characterized by combining PDRN, a DNA material, with hyaluronic acid methacrylate, which has been used as a bio-ink for biomedicine as described above.

[0037] At this time, the above PDRN is a substance called Poly Deoxy Ribo Nucleotide, which corresponds to a regenerative material obtained by extracting DNA fragments from germ cells such as the sperm or testes of salmon or trout. It is known as a tissue regeneration material similar to human DNA and is a substance that exists in human cells and acts to physiologically stimulate regeneration and metabolic activity. In the present invention, wound healing and regeneration effects can be enhanced by including the above PDRN in the bio-ink.

[0038] In addition, surprisingly, when PDRN is combined with the hyaluronic acid methacrylate, the elasticity, which was a problem of the existing hyaluronic acid methacrylate material, is improved, and a property of being able to stretch well is obtained. Specifically, as the content of PDRN in the material increases, the degree of deformation and fracture strength of the hydrogel material according to the present invention increase in proportion.

[0039] At this time, the weight ratio of the hyaluronic acid methacrylate and PDRN may preferably be 0.5:1 to 3:1, and with respect to the total weight of the bio-ink, the hyaluronic acid methacrylate may be included in an amount of 1 to 8 wt% and the PDRN in an amount of 0.5 to 5 wt%, and other substances or distilled water may be further included.

[0040] In the above bio-ink, the photoinitiator may be a photoinitiator known in the art, and may be at least one selected from the group consisting of, for example, lithium phenyl-2,4,6-trimethylbenzoyl phosphinate (LAP), 4-(2-hydroxyethoxy)phenyl-(2-hydroxy-2-propyl)ketone, Eoxin-Y, rose bengal, and riboflavin (vitamin B2), but is not limited thereto.

[0041] In addition, in an embodiment of the present invention, the bio-ink may be characterized by further including MXene.

[0042] The above MXene is a transition metal carbide, transition metal nitride, or transition metal carbonitride having two-dimensional crystallinity, and can satisfy the following chemical formula 1.

[0044] (Chemical Formula 1)

[0045] M n+1 X n

[0047] In the above chemical formula 1, M is a transition metal selected from one or more of groups IIIB, IVB, VB, VIB, and VIIB, X is carbon (C), nitrogen (N), or a combination thereof, and n is 1, 2, or 3.

[0048] In one embodiment, the transition metal M may be titanium (Ti), zirconium (Zr), hafnium (Hf), vanadium (V), chromium (Cr), manganese (Mn), scandium (Sc), molybdenum (Mo), niobium (Nb), tantalum (Ta), yttrium (Y), tungsten (W), or a combination thereof, but is not limited thereto. X may be carbon, nitrogen, or carbon and nitrogen, and in one embodiment may be carbon. n may be 1, 2, or 3.

[0049] Representative examples of such MXenes include Ti2C, Ti2N, Ti3C2, Ti3N2, Ti3CN, Ti4N3, Nb4C3, Nb2C, Nb2N, V2C, V2N, Ta4C3, Mo2C, Mo2TiC2, Mo2Ti2C3, Cr2TiC2, (Ti 0.5 , Nb 0.5 Examples include )2C, Zr3C2, Hf3C2, Mo2TiC2, Cr2TiC2, or combinations thereof. In this case, in the bio-ink of the present invention, the MXene may be any one of the examples described above or a combination thereof, and a specific example may be Ti3C2, but is not limited thereto.

[0050] The aforementioned MXene has excellent biocompatibility, electrical conductivity, and photothermal effects, and thus possesses additional functionality; therefore, when used as a bioink, it can be used in various fields such as phototherapy and treatment using electrical stimulation.

[0051] At this time, it may be preferable to include 0.1 to 3 mg / ml of MXene relative to the total bio-ink.

[0053] To achieve the above technical problem, another embodiment of the present invention provides a method for manufacturing bio-ink.

[0054] FIG. 1 is a flowchart illustrating a method for manufacturing bio-ink according to one embodiment of the present invention.

[0055] A method for manufacturing bio-ink according to one embodiment of the present invention is characterized by comprising the steps of preparing hyaluronic acid methacrylate, PDRN, and a photoinitiator (S100), as illustrated in FIG. 1, adding the PDRN and the photoinitiator to distilled water and mixing them (S200), and adding the hyaluronic acid methacrylate to distilled water and mixing them to manufacture bio-ink (S300).

[0056] The step (S100) of preparing the hyaluronic acid methacrylate and PDRN may utilize a known method for preparing hyaluronic acid methacrylate. For example, hyaluronic acid methacrylate can be prepared by adding hyaluronic acid and methacrylic anhydride. Specifically, a method may be selected in which methacrylic anhydride is added to an aqueous solution of sodium hyaluronic acid salt (HA), synthesized under constant temperature and pH conditions, and then freeze-dried to obtain the methacrylic anhydride.

[0057] In the step (S200) of adding the above PDRN and the photoinitiator to distilled water and mixing, the photoinitiator may be a photoinitiator known in the art as described above, and may be, for example, at least one selected from the group consisting of lithium phenyl-2,4,6-trimethylbenzoyl phosphinate (LAP), 4-(2-hydroxyethoxy)phenyl-(2-hydroxy-2-propyl)ketone, Eoxin-Y, rose bengal, and riboflavin (vitamin B2), but is not limited thereto.

[0059] At this stage, MXene may be further added to impart functionality. This is as described above in the bio-ink according to the present invention.

[0060] The step (S300) of preparing a solution by adding the hyaluronic acid methacrylate to the distilled water and mixing it is a step of preparing a bio-ink. At this time, as the overall composition of the bio-ink, PDRN may be included in an amount of 0.5 to 5 wt%, hyaluronic acid methacrylate in an amount of 1 to 8 wt%, and a photoinitiator in an amount of 0.01 to 0.5 wt%, and if MXene is added, it may be preferable for the MXene to be included in an amount of 0.1 to 2 wt%. In this case, the remainder may consist of distilled water or other substances and distilled water.

[0062] The present invention will be explained in more detail below through manufacturing examples, comparative examples, and experimental examples. However, the present invention is not limited to the following manufacturing examples and experimental examples.

[0064] Preparation Example 1: HAMA Synthesis

[0065] To synthesize HA into HAMA, methacrylic anhydride (MA) was added while maintaining the temperature at 4°C, and the pH during synthesis was maintained using NaOH. The synthesis was carried out for 24 hours. Afterward, the mixture was precipitated with anhydrous ethanol, followed by dialysis through a dialysis tube for 3 days, and freeze-dried to obtain HAMA powder.

[0066] To confirm whether the above HAMA was synthesized, HA and HAMA were compared using Fourier transform infrared spectroscopy and nuclear magnetic resonance spectroscopy.

[0067] Figure 2 shows the results of Fourier transform infrared spectral analysis of HA and HAMA prepared in an experimental example according to one embodiment of the present invention.

[0068] Figure 3 shows the results of nuclear magnetic resonance spectroscopy analysis of HA and HAMA prepared in an experimental example of one embodiment of the present invention.

[0069] As shown in Figures 2 and 3, the HAMA synthesized using the HAMA synthesis method specified above was proven to be HAMA through Fourier transform infrared spectroscopy and nuclear magnetic resonance spectroscopy.

[0071] Preparation Example 2: Preparation of bio-ink and 3D-printed hydrogel material

[0072] PDRN, a photocuring agent, and MXene were added to distilled water and mixed, after which HAMA was added. At this time, the composition contained 3 wt% HAMA, 2 wt% PDRN, 1 wt% MXene, and 0.1 wt% LAP (Lithium phenyl-2,4,6-trimethylbenzoylphosphinate), respectively. A scaffold structure measuring 10 mm x 10 mm x 1 mm was printed using the prepared bio-ink via a pneumatic extrusion printer and cured by irradiating with 405 nm ultraviolet light for 60 seconds.

[0073] Figure 4 is a photograph showing bio-ink and hydrogel produced by a bio-ink manufacturing method according to one embodiment of the present invention.

[0074] Hereinafter, the hydrogel prepared according to one embodiment of the present invention is referred to as HDX(HAMA-DNA-MXene).

[0075] In addition, as comparative examples, HAMA hydrogel, HAMA-MXene hydrogel, and HAMA-DNA(PDRN) hydrogel were prepared, and these are denoted as HAMA, HX, and HD, respectively.

[0077] Experimental Example 1: Evaluation of Tensile Strength According to PDRN and MXene Content

[0078] To verify the physical properties of the hydrogel according to the present invention, dumbbell-shaped specimens were prepared to evaluate the tensile strength and compressive strength using the prepared hydrogel, and these were measured using a universal testing machine.

[0079] Figure 5 is a graph showing the results of a tensile test according to the PDRN content of a hydrogel according to one embodiment of the present invention.

[0080] Figure 6 shows the results of a tensile test according to the MXene concentration of a hydrogel according to one embodiment of the present invention.

[0081] As shown in Figure 5 above, it was confirmed that the degree of deformation increased as the PDRN content increased. In addition, by observing the increase in fracture strength, it was confirmed that PDRN can withstand stronger forces.

[0082] In addition, as shown in Figure 6 above, as the MXene content increased, the fracture strength decreased, but the degree of deformation did not change significantly, and it was confirmed that the degree of deformation was not significantly changed by MXene.

[0083] As such, by adding PDRN to HAMA, a remarkable effect of increased elasticity was obtained in existing HAMA-based hydrogel materials, and it is predicted that the bio-ink and hydrogel according to the present invention can be used in various fields.

[0085] Experimental Example 2: Photothermal Properties Experiment

[0086] To verify photothermal properties, an 808nm laser was irradiated for 60 seconds and captured and analyzed using a thermal imaging camera.

[0087] Figure 7 shows the photothermal properties of a hydrogel according to one embodiment of the present invention, showing the temperature change and the maximum temperature reached according to the MXene concentration.

[0088] As shown in Figure 7 above, it can be confirmed that the maximum temperature increased as the concentration of MXene increased, and it can be confirmed that the thermal sensitivity increased starting from a concentration of MXene of 1.0 mg / ml.

[0090] Experimental Example 3: Electrical Conductivity Experiment

[0091] To verify the effect of MXene on electrical conductivity, the resistance value was measured using an LCR meter.

[0092] Figure 8 shows the change in resistance value according to the MXene concentration of a hydrogel according to one embodiment of the present invention.

[0093] FIG. 9 is a diagram showing the electrical conductivity of HAMA, HX, HD, and a hydrogel according to one embodiment of the present invention.

[0094] As shown in Figures 8 and 9, it was confirmed that the group containing DNA and MXene together (HDX) had lower resistance values ​​than HAMA, HX, and HD, indicating increased electrical conductivity.

[0096] Experimental Example 4: Cell-based Wound Healing Test and Cytotoxicity Evaluation

[0097] To evaluate the intracellular wound healing ability of PDRN, fluorescence imaging was performed after a wound healing assay.

[0098] FIG. 10 is an image of a wound healing assay according to DNA concentration of a hydrogel according to one embodiment of the present invention.

[0099] In the above Figure 10, the NT group, which is the untreated group, showed the least cell migration and proliferation, and it can be confirmed that the cell migration and proliferation rates increased as the PDRN concentration increased.

[0100] FIG. 11 is a diagram showing cell viability data of a hydrogel according to one embodiment of the present invention.

[0101] As shown in Figure 11 above, it can be confirmed that the cell viability of the hydrogel groups containing DNA (PDRN) is higher.

[0103] According to the embodiments of the present invention described above, a bio 3D printing material having excellent biocompatibility and bio-healing ability can be provided through a hydrogel material comprising hyaluronic acid methacrylate and PDRN, which has high physical properties with improved elasticity compared to conventional hyaluronic acid methacrylate-based hydrogel materials.

[0104] Additionally, according to another embodiment of the present invention, by further adding MXene to the hydrogel material, there is an advantage of being able to provide a 3D printing material with various functionalities that possesses both conductive and photothermal properties.

[0105] In addition, according to an embodiment of the present invention, it includes an effect that can aid in the regeneration of biological tissue by including a component that accelerates wound healing.

[0107] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. For example, each component described as a single unit may be implemented in a distributed manner, and components described as distributed may likewise be implemented in a combined form.

[0108] The scope of the present invention is defined by the claims set forth below, and all modifications or variations derived from the meaning and scope of the claims and equivalent concepts thereof should be interpreted as being included within the scope of the present invention.

Claims

Claim 1 A bio-ink characterized by comprising hyaluronic acid methacrylate; PDRN (Poly Deoxy Ribo Nucleotide); and a photoinitiator, wherein the hyaluronic acid methacrylate is present in an amount of 1 to 8 wt% and the PDRN is present in an amount of 0.5 to 5 wt% based on the total weight. Claim 2 A bio-ink characterized by further including MXene in claim 1. Claim 3 delete Claim 4 In paragraph 2, the above MXene is Ti2C, Ti2N, Ti3C2, Ti3N2, Ti3CN, Ti4N3, Nb4C3, Nb2C, Nb2N, V2C, V2N, Ta4C3, Mo2C, Mo2TiC2, Mo2Ti2C3, Cr2TiC2, (Ti 0.5 , Nb 0.5 A bio-ink characterized by being selected from )2C, Zr3C2, Hf3C2, Mo2TiC2, Cr2TiC2, or a combination thereof. Claim 5 A bioink according to claim 2, characterized in that the MXene comprises 0.1 to 3 mg / ml with respect to the bioink. Claim 6 A bioink according to claim 1, characterized in that the photoinitiator is at least one selected from the group consisting of lithium phenyl-2,4,6-trimethylbenzoyl phosphinate (LAP), 4-(2-hydroxyethoxy)phenyl-(2-hydroxy-2-propyl)ketone, Eoxin-Y, rose bengal, and riboflavin (vitamin B2). Claim 7 A method for manufacturing a bio-ink, comprising the steps of: preparing hyaluronic acid methacrylate, PDRN, and a photoinitiator; adding the PDRN and the photoinitiator to distilled water and mixing them; and adding the hyaluronic acid methacrylate to distilled water and mixing them to manufacture the bio-ink; wherein the hyaluronic acid methacrylate comprises 1 to 8 wt% and the PDRN comprises 0.5 to 5 wt% based on the total weight. Claim 8 A method for manufacturing bio-ink according to claim 7, characterized by adding MXene in the step of adding and mixing the PDRN and photoinitiator in distilled water. Claim 9 In claim 8, the above MXene is Ti2C, Ti2N, Ti3C2, Ti3N2, Ti3CN, Ti4N3, Nb4C3, Nb2C, Nb2N, V2C, V2N, Ta4C3, Mo2C, Mo2TiC2, Mo2Ti2C3, Cr2TiC2, (Ti 0.5 , Nb 0.5 A method for manufacturing bio-ink characterized by being selected from )2C, Zr3C2, Hf3C2, Mo2TiC2, Cr2TiC2, or a combination thereof. Claim 10 A method for manufacturing bio-ink according to claim 7, wherein the step of preparing the hyaluronic acid methacrylate and PDRN is to synthesize and prepare hyaluronic acid methacrylate by adding methacrylic anhydride (MA) to an aqueous solution of hyaluronic acid sodium salt (HA). Claim 11 Hydrogel manufactured with bio-ink according to paragraph 1.

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