Patent Application
20250222169
This application claims the benefit of Korean Patent Application No. 10-2024-0002005 filed on Jan. 5, 2024, in the Korean Intellectual Property Office, the entire disclosure of which is incorporated herein by reference for all purposes.
The present disclosure relates to a photoinitiator, a bioink including the same, and a method of manufacturing a hydrogel.
3D bioprinting is widely recognized as an innovative method for precisely replicating the structural complexity of native corneas. It enables the production patient-specific corneal structures. Compared to other tissues, the cornea is completely avascular requires minimal metabolism, and has a relatively uniform cell composition, making it particularly suitable for 3D bioprinting. Bioinks used in 3D bioprinting must exhibit appropriate biocompatibility and optical properties. Hydrogel-based bioinks demonstrate high cytocompatibility with living cells, excellent mechanical stability after printing, and outstanding printing resolution.
3D printing involves simple processes that ensure low cost, and it allows for the production of variety products in a small quantity without restrictions on manufacturing forms. However, in to the context of 3D bioprinting for medical applications, significant limitations remain in the selection of bioink materials.
Hyaluronic acid (HA) is a natural linear polysaccharide found in various connective tissues, including the skin, umbilical cord, and vitreous. HA-based hydrogels are highly suitable as bioprinting materials due to their high cellular compatibility, biodegradability, and the presence of numerous functional groups in HA. To utilize HA-based hydrogels for bioprinting, they must be rapidly manufactured through photocrosslinking.
A photoinitiator is a critical factor influencing the photocrosslinking rate and properties of hydrogels. For application in bioprinting, such as with HA-based hydrogels, the photoinitiator must exhibit high water solubility, perform photo-crosslinking under visible light, demonstrate fast photoreactivity, and ensure superior cell compatibility.
An object of the present disclosure is to provide a photoinitiator.
Another object of the present disclosure is to provide a bioink including the photoinitiator.
Still another object of the present disclosure is to provide a method of manufacturing a hydrogel.
To achieve above objects, an embodiment of the present disclosure provides a photoinitiator which has a chemical structure of the following Chemical Formula 1 and is water-soluble and activated by visible light to form free radicals:
In addition, an embodiment of the present disclosure provides a bioink composition including distilled water, a methacrylated hyaluronic acid (HAMA) compound dissolved in the distilled water and has a plurality of molecular weights, and a photoinitiator dissolved in the distilled water with the chemical structure of the following Chemical Formula 1:
wherein, in the Chemical Formula 1, R1 is H or F, and R2 is Li+ or Mg2+.
In addition, an embodiment of the present disclosure provides a method for manufacturing a hydrogel, including: dissolving a methacrylated hyaluronic acid (HAMA) compound having a plurality of molecular weights and a photoinitiator with a chemical structure of the following Chemical Formula 1 in distilled water to prepare a bioink composition; printing the bioink composition while irradiating visible light to crosslink the methacrylated hyaluronic acid (HAMA) compound:
wherein, in the Chemical Formula 1, R1 is H or F, and R2 is Li+ or Mg2+.
According to the present disclosure, a photoinitiator of the present disclosure may have water solubility, high molar extinction coefficient, and low cytotoxicity. In addition, a method of manufacturing a hydrogel of the present disclosure may have high cell viability by increasing 3D printing efficiency due to the fast photo-curing rate.
Hereinafter, embodiments of the present disclosure will be described in detail with reference to the accompanying drawings. The present disclosure may have various modifications and forms, and thus specific embodiments will be illustrated in the drawings and described in detail in the description. However, it is not intended to limit the present disclosure to a specific form of disclosure, but should be understood to include all modifications, equivalents, or substitutes included in the spirit and technical scope of the present disclosure. In describing each drawing, similar reference numerals are used to refer to similar components. In the accompanying drawings, the enlarged dimensions are given in the constructures to ensure clarity of the present disclosure.
Terms such as first and second may be used to describe various components, but such components should not be limited by such terms. The terms are used only for distinguishing one component from another. For example, without departing from the scope of the present disclosure, the first component may be named the second component, and similarly, the second component may also be named the first component.
The terms used in this application are used only to describe particular embodiments, but not intended to limit the present disclosure. Singular expressions include plural expressions, unless the context explicitly means otherwise. As used herein, it should be understood that terms such as “comprise”, “include” or “have” are intended to specify the presence of a feature, number, step, operation, component, part, or combination thereof described in the specification, but do not exclude in advance the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof.
On the other hand, unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by those with ordinary skill in the art to which the present disclosure pertains. Terms as defined in commonly used dictionaries should be interpreted as having a meaning consistent with those in the context of the relevant art but not be interpreted in an idealized or overly formal sense, unless expressly defined otherwise in this application.
A photoinitiator according to the present disclosure has a chemical structure of the following Chemical Formula 1 and is water-soluble and activated by visible light to form free radicals:
wherein, in the Chemical Formula 1, R1 is H or F, and R2 is Li+ or Mg2+.
In one embodiment, a solubility of the photoinitiator in water at 25° C. may be 160 to 200 mM, but is not limited thereto.
In one embodiment, a molar extinction coefficient of the photoinitiator may be 40 to 80 M−1 cm−1, but is not limited thereto.
A bioink composition according to the present disclosure may include distilled water, a methacrylated hyaluronic acid (HAMA) compound that is dissolved in the distilled water, and a photoinitiator which is dissolved in the distilled water and has a chemical structure of the following Chemical Formula 1:
wherein, in the Chemical Formula 1, R1 is H or F, and R2 is Li+ or Mg2+.
In one embodiment, the methacrylated hyaluronic acid compound may have a molecular weight of about 1 to 2000 kDa.
As one embodiment, the methacrylated hyaluronic acid compound may include a compound of a single molecular weight in a range of about 1 to 2000 kDa.
As another embodiment, the methacrylated hyaluronic acid compound may include a first methacrylated hyaluronic acid compound with a molecular weight of 1 to 20 kDa and a second methacrylated hyaluronic acid compound with a molecular weight of 50 to 2000 kDa. For example, the methacrylated hyaluronic acid compound may include the first methacrylated hyaluronic acid compound with a molecular weight of about 10 kDa and the second methacrylated hyaluronic acid compound with a molecular weight of about 100 kDa.
In one embodiment, the methacrylated hyaluronic acid compound may include the first methacrylated hyaluronic acid compound and the second methacrylated hyaluronic acid compound in a molar ratio of 2 to 7:8 to 3.
In one embodiment, the bioink composition may include about 5 to 15 mass % of the methacrylated hyaluronic acid compound and about 0.02 to 5 mass % of the photoinitiator.
In one embodiment, viscosity of the bioink composition may be about 30 to 70 mPa·s, but is not limited thereto.
In one embodiment, the bioink composition may further include a photoabsorber. As an embodiment, tartrazine may be used as the photoabsorber.
In one embodiment, the bioink composition may further include a cell. As one embodiment, a corneal stromal cell may be used as the cell.
In one embodiment, the bioink composition may further include collagen.
In one embodiment, the bioink composition may be a composition for corneal formation.
Referring to
wherein, in the Chemical Formula 1, R1 is H or F, and R2 is Li+ or Mg2+.
In one embodiment, the methacrylated hyaluronic acid (HAMA) compound may have a single or a plurality of molecular weights within a range of about 1 to 2000 kDa.
In one embodiment, a thickness of the hydrogel may be about 300 to 700 μm. As an embodiment, the thickness of the hydrogel may be about 500 μm.
In one embodiment, the hydrogel may have a transmittance of about 70 to 98% in light with a wavelength of about 450 to 600 nm.
In one embodiment, tensile strength of the hydrogel may be about 150 to 250 kPa, elongation may be about 20 to 55%, and toughness may be about 10 to 40 KJ/m3, but are not limited thereto.
Hereinafter, the present disclosure will be described in more detail through examples to help understanding of the present disclosure. However, examples below are merely intended to illustrate the content of the present disclosure, and the scope of the present disclosure is not limited to the following examples. Examples of the present disclosure are provided to more completely explain the present disclosure to those skilled in the art.
Hyaluronic acid (HA, about 0.5 g, molecular weight: about 100 kDa) was dissolved in distilled water (5 mL), and sodium hydroxide (about 1 N) was used at about 0 to 5° C. to adjust the pH to 8.0 to prepare a mixture. To the mixture, 4 equivalents of anhydrous methacrylate were added dropwise for about 30 minutes based on the disaccharide unit of HA. The mixture was precipitated in ethanol followed by filtration, and the obtained solid was washed with ethanol, frozen at about −30° C., and then lyophilized to be stored at −20° C. until use.
Except that the molecular weight of hyaluronic acid in Preparation Example 1 was about 10 kDa, the HAMA was prepared using the same method as in Preparation Example 1.
Dimethyl phenylphosphonite (about 187 mg, about 1.1 mmol) was added to benzoyl chloride (about 140 mg, about 1 mmol) and 2-butanone (about 10 ml) and stirred in the presence of nitrogen for about 24 hours at room temperature to prepare a first mixture. Lithium bromide (about 95 mg, about 1.1 mmol) was added to 2-butanone (about 10 mmol) solution to prepare a second mixture. The first mixture and the second mixture were mixed, heated at about 60° C. for about 20 minutes, and cooled to room temperature to produce a reactant. The reactant was washed with 2-butanone and diethyl ether in turn, and then dried under vacuum to obtain a photoinitiator for bioprinting, which was named LBP (about 237 mg, about 94% yield).
1H NMR (600 MHZ, D2O): 8.09 (d, 2H), 7.61-7.65 (m, 2H), 7.58 (t, 1H), 7.49 (t, 1H), 7.46-7.42 (m, 4H). 13C NMR (151 MHz, D2O): 212.39, 211.61, 135.72, 135.43, 134.53, 133.54, 132.66, 131.91, 131.89, 131.70, 131.64, 129.18, 129.16, 128.83, 128.46, 128.36. 31P NMR (243 MHz, D2O): 18.37. HRMS (ESI MS) m/z: calculated: 252.13. found: 253.06 ([M+H]+detected).
Except that 4-fluorobenzoyl chloride (about 158 mg, about 1 mmol) was used instead of the benzoyl chloride in Example 1, a photoinitiator for bioprinting was prepared using the same method as in Example 1 and named LFBP.
1H NMR (600 MHZ, D2O): 8.18-8.17 (m, 2H), 7.64-7.61 (m, 2H), 7.50 (t, 1H), 7.44-7.42 (m, 2H), 7.17 (t, 2H). 13C NMR (151 MHz, D2O): 210.53, 209.74, 167.03, 165.34, 133.41, 132.53, 132.27, 132.21, 131.99, 131.97, 131.95, 131.93, 131.71, 131.65, 128.46, 128.39. 31P NMR (243 MHz, D2O): 18.59. HRMS (ESI MS) m/z: calculated, 270.12. found: 271.05 ([M+H]+detected).
Except that magnesium bromide (about 203 mg, about 1.1 mmol) was used instead of the lithium bromide in Example 1, a photoinitiator for bioprinting was prepared using the same method as in Example 1 and named MBP.
1H NMR (600 MHZ, D2O): 8.14-8.13 (d, 2H), 7.70-7.65 (m, 2H), 7.65-7.60 (t, 1H), 7.44-7.42 (t, 2H), 7.51-744 (m, 4H).
Bioink was prepared by dissolving LBP (about 1.0 g), Preparation Example 1 (about 5.0 g), Preparation Example 2 (about 5.0 g), and tartrazine (about 3 mM) in about 100 ml of distilled water.
LBP (about 1.0 g), Preparation Example 1 (about 5.0 g), and Preparation Example 2 (about 5.0 g) were dissolved in about 100 ml of PBS.
Bioink was prepared by dissolving LBP (about 1.0 g), Preparation Example 1 (about 9.0 g), and Preparation Example 2 (about 1.0 g) in about 100 ml of distilled water. The bioink was used to prepare a hydrogel through light irradiation (about 405 nm, ˜6.8 mW/cm2, about 15 seconds) using a DLP printer (Perfactory Micro Plus HD, EnvisionTEC, Germany) in a dog-bone shape with a thickness of about 1 mm according to the ASTM D1708 test method.
Bioink was prepared by dissolving LBP (about 1.0 g), Preparation Example 1 (about 7.0 g), and Preparation Example 2 (about 3.0 g) in about 100 ml of distilled water. The bioink was used to prepare a hydrogel through light irradiation (about 405 nm, ˜6.8 mW/cm2, about 15 seconds) using the DLP printer (Perfactory Micro Plus HD, EnvisionTEC, Germany) in a dog-bone shape with a thickness of about 1 mm according to the ASTM D1708 test method.
Bioink was prepared by dissolving LBP (about 1.0 g), Preparation Example 1 (about 5.0 g), and Preparation Example 2 (about 5.0 g) in about 100 ml of distilled water. The bioink was used to prepare a hydrogel through light irradiation (about 405 nm, ˜6.8 mW/cm2, about 15 seconds) using the DLP printer (Perfactory Micro Plus HD, EnvisionTEC, Germany) in a dog-bone shape with a thickness of about 1 mm according to the ASTM D1708 test method.
Bioink was prepared by dissolving LBP (about 1.0 g), Preparation Example 1 (about 3.0 g), and Preparation Example 2 (about 7.0 g) in about 100 ml of distilled water. The bioink was used to prepare a hydrogel through light irradiation (about 405 nm, ˜6.8 mW/cm2, about 15 seconds) using the DLP printer (Perfactory Micro Plus HD, EnvisionTEC, Germany) in a dog-bone shape with a thickness of about 1 mm according to the ASTM D1708 test method.
Bioink was prepared by dissolving LBP (about 1.0 g), Preparation Example 1 (about 5.0 g), and Preparation Example 2 (about 5.0 g) in about 100 ml of distilled water. The bioink was used to prepare a hydrogel through light irradiation (about 405 nm, ˜6.8 mW/cm2, about 15 seconds) using the DLP printer (Perfactory Micro Plus HD, EnvisionTEC, Germany) with an average thickness of about 500 μm (hereinafter referred to as bHAMA).
Lithium phenyl-2,4,6-trimethylbenzoylphosphination (LAP), a commercial photoinitiator for bioprinting, was used (hereinafter referred to as LAP).
Bioink was prepared by dissolving LBP (about 1.0 g), Preparation Example 1 (about 10.0 g), and tartrazine (about 3 mM) in about 100 ml of distilled water.
Bioink was prepared by dissolving LBP (about 1.0 g), Preparation Example 2 (about 10.0 g), and tartrazine (about 3 mM) in about 100 ml of distilled water.
LBP (about 1.0 g) and gelatin methacryloyl (GelMA, about 10 g) were dissolved in about 100 ml of PBS.
LBP (about 1.0 g) and poly (ethylene glycol) diacrylate (PEGDA, about 10 g) were dissolved in about 100 ml of PBS.
Bioink was prepared by dissolving LBP (about 1.0 g) and Preparation Example 1 (about 10.0 g) in about 100 ml of distilled water. The bioink was used to prepare a hydrogel through light irradiation (about 405 nm, ˜6.8 mW/cm2, about 15 seconds) using the DLP printer (Perfactory Micro Plus HD, EnvisionTEC, Germany) in a dog-bone shape with a thickness of about 1 mm according to the ASTM D1708 test method.
Bioink was prepared by dissolving LBP (about 1.0 g) and Preparation Example 2 (about 10.0 g) in about 100 ml of distilled water. The bioink was used to prepare a hydrogel through light irradiation (about 405 nm, ˜6.8 mW/cm2, about 15 seconds) using the DLP printer (Perfactory Micro Plus HD, EnvisionTEC, Germany) in a dog-bone shape with a thickness of about 1 mm according to the ASTM D1708 test method.
Bioink was prepared by dissolving gelatin methacryloyl (GelMA, about 10 g) in about 100 ml of distilled water. The bioink was used to prepare a hydrogel through light irradiation (about 405 nm, ˜6.8 mW/cm2, about 15 seconds) using the DLP printer (Perfactory Micro Plus HD, EnvisionTEC, Germany) in an average thickness of about 500 μm (hereinafter referred to as GelMA).
Bioink was prepared by dissolving poly (ethylene glycol) diacrylate (PEGDA, about g) in about 100 ml of distilled water. The bioink was used to prepare a hydrogel through light irradiation (about 405 nm, ˜6.8 mW/cm2, about 15 seconds) using the DLP printer (Perfactory Micro Plus HD, EnvisionTEC, Germany) in an average thickness of about 500 μm (hereinafter referred to as PEGDA).
All animal testing procedures were approved by the Institutional animal care and use Committee of Pusan National University Hospital (Approval Number: PNUH-2020-162). Rabbit eyes (adult white New Zealand rabbits) were removed from euthanized animals to obtain rabbit corneal stromal cells. Rabbit corneal stromal cells were cultured in a cell culture dish with a size of about 100 mm along with DMEM supplemented with about 10% FBS and penicillin-streptomycin (about 100 UI/ml). The cultured cells were kept at about 37° C. in a sterile culture machine (Labogene, Seoul, Korea) containing about 5% CO2 until reaching the confluence.
Murine NIH-3T3 fibroblasts purchased from ATCC (Manassas, VA, USA) were cultured in a cell culture dish in a size of about 100 mm along with DMEM supplemented with about 10% FBS and penicillin-streptomycin (about 100 UI/ml). The cultured cells were kept at about 37° C. in a sterile culture machine (Labogene, Seoul, Korea) containing about 5% CO2 until reaching the confluence.
The cytotoxicity of LBP, MBP, and LAP was assessed by measuring cell viability after exposure to various concentrations. To conduct cell viability analysis, fibroblasts were seeded into 24-well-plates (2×104 cells/well) and cultured in a sterile culture machine at about 37° C. for about 12 hours, and then the media were removed. The LBP, MBP and LAP were diluted in media having various concentrations (0, 0.025, 0.05, 0.1, and 0.2% (w/v)), and then the fibroblasts were cultured for about 24 hours in a sterile incubator at about 37° C., followed by washing with PBS (1×).
Rabbit corneal stromal cells cultured in Experimental Example 2 were used. Rabbit corneal stromal cells were cultured in LBP (about 1% (w/v)) and washed with PBS (1×) at intervals of about 30, 60, 120, 240, and 360 minutes. To measure the cell viability, cells were supplemented with about 10 μl of WST-1 reagent (EZ-Cytox) and cultured for about 1 to 2 hours, and then cell viability was measured via a colorimetric assay at about 450 nm using a microplate reader (AMR-100, Allsheng Co., Ltd., China).
The mechanical properties of Example 6 to Example 9, Comparative Example 6, and Comparative Example 7 were measured using a tensile tester (34SC-1, Instron, USA) at a strain of about 1 mm/min in tensile mode, which is shown in
While preferred embodiments of the present disclosure have been described above, those skilled in the art will appreciate that various modifications and changes can be made in the present disclosure within a scope without departing from the spirit and scope of the present disclosure set forth in the appended claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2024-0002005 | Jan 2024 | KR | national |
