Manufacturing Method of Catechol-Modified Hyaluronic Acid-Based Hydrogel Encapsulating Cu@SiO2
Patent Information
- Authority / Receiving Office
- KR · KR
- Patent Type
- Patents
- Current Assignee / Owner
- DONAM NANO BIO LAB CO LTD
- Filing Date
- 2025-12-18
- Publication Date
- 2026-08-03
Smart Images
Figure 112025143781274-PAT00001_ABST
Abstract
Description
Technology Field
[0001] The present invention relates to a method for preparing a catechol-modified hyaluronic acid-based hydrogel encapsulated with Cu@SiO2, and more specifically, to a method for preparing a catechol-modified hyaluronic acid-based hydrogel encapsulated with Cu@SiO2 with enhanced antibacterial and bioadhesive properties by catechol-modifying sea urchin-shaped copper-coated silica nanoparticles (Cu@SiO2) and embedding them in a photopolymerizable hyaluronic acid hydrogel. Background Technology
[0002] Hydrogel-based patch systems are increasingly recognized for their wound healing efficacy due to their biodegradability, porous structure, ability to contain growth factors, and controlled release of therapeutic agents. These characteristics make them superior to conventional wound dressing materials such as cotton or gauze; hydrogels consist of hydrophilic three-dimensional polymer networks possessing high absorbency, controllable chemical and physical properties, and excellent biocompatibility. This three-dimensional mesh structure of hydrogels enables the encapsulation of various cells, nanomaterials, or proteins. Hydrogel-based patches adhere firmly to the wound site to effectively absorb wound exudate and help prevent secondary infections by creating a complete coverage and antimicrobial environment. Beyond wound dressings, hydrogel systems can introduce biological functions that accelerate wound healing by delivering essential growth factors or providing structural support for efficient tissue regeneration. Due to this versatility, hydrogel systems are attracting attention as potential candidates for various wound healing applications.
[0003] Hyaluronic acid (HA) is a major natural polymer found in the extracellular matrix, and its role in tissue engineering and regenerative medicine has been extensively studied. In addition to biocompatibility, HA offers excellent modulation and ease of functionalization, which can significantly expand its range of applications. For example, HA-based hydrogels can be photochemically responsive by attaching methacrylate groups to the backbone or pH-sensitive by introducing reactive peptides into the hydrogel polymer network. Furthermore, they can change their mechanical properties over time, making them suitable for use as injectable hydrogels. Nevertheless, a major drawback of using HA-based hydrogels for wound dressings is their poor adhesion to natural tissues. Further research is needed on HA-based hydrogels that address the issue of adhesion to natural tissues and possess enhanced antimicrobial properties. Prior art literature
[0004] Korean Registered Patent Publication No. 10-2739553 Korean Registered Patent Publication No. 10-2846107 Korean Registered Patent Publication No. 10-2829586 Korean Published Patent Publication No. 10-2025-0153788 The problem to be solved
[0005] The present invention aims to solve the above-mentioned problems by providing a method for manufacturing a catechol-modified hyaluronic acid-based hydrogel with enhanced antibacterial and bioadhesive properties, in which Cu@SiO2 encapsulated with Cu@SiO2 is modified by catechol and embedded in a photopolymerizable hyaluronic acid hydrogel. means of solving the problem
[0006] The method for preparing a catechol-modified hyaluronic acid-based hydrogel encapsulated with Cu@SiO2 according to the present invention to achieve the above-mentioned purpose comprises deionized water, NH 3·A first step in which SiO2 spherical particles are prepared using a mixture of H2O, ethanol, and TEOS (tetraethyl orthosilicate); a second step in which copper silicate salt is prepared by adding a mixed solution of Cu(CH3COO)2·H2O and NH4Cl to the SiO2 spherical particles; a third step in which copper silicate salt is loaded into a quartz tube and purged with an H2 / Ar gas mixture to prepare copper-coated silica nanoparticles (Cu@SiO2); and a fourth step in which MA-HA is prepared by adding methacrylic anhydride (MA) to a hyaluronic acid (HA) solution, and MA-HA-CA is synthesized by reacting MA-HA with EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide). A fifth step of preparing a Cu@SiO2 mixed polymer precursor solution by dissolving PEG4SH (4-arm poly(ethylene glycol) thiol) and the MA-HA-CA of the fourth step in PBS (Dulbecco's phosphate-buffered saline) and adding EY (Eosin Y) and TEOA (Triethanolamine); and a sixth step of preparing a HA-based hydrogel encapsulated with Cu@SiO2 by injecting the Cu@SiO2 mixed polymer precursor solution into a cylindrical well made of PDMS (Polydimethylsiloxane) and inducing photopolymerization.
[0007] The first step above is deionized water, NH 3·The method comprises a first step 1-1 in which H2O and ethanol are mixed and stirred at 200 to 300 rpm for 5 to 15 minutes, and then a mixture with added TEOS is stirred for 3 to 5 hours to produce a white product, and a second step 1-2 in which the white product is washed with ethanol and deionized water, centrifuged twice at 2,000 to 3,000 rpm for 10 to 20 minutes, and the resulting product is dried in an oven at 50 to 70°C for 7 to 9 hours to produce SiO2 spherical particles.
[0008] In the above 1-1 step, deionized water, NH 3· It is preferable that the mixing ratio of H2O, ethanol, and TEOS be 10:30:200:8.
[0009] In the above Step 1-1, deionized water, NH 3· An alkaline environment is created by mixing H2O and ethanol. Here, NH 3· H2O acts as a catalyst to promote the hydrolysis and condensation reactions of TEOS.
[0010] In the above 1-1 step, TEOS reacts with water to proceed with hydrolysis, followed by a condensation reaction in which Si-O-Si bonds are formed and a colloidal white SiO2 sol is produced, and spherical SiO2 begins to form.
[0011] In the above first and second steps, the white product is washed with ethanol and deionized water to remove reaction byproducts such as ethanol, ammonia, and unreacted TEOS. Additionally, impurities are removed by centrifugation, and the solvent is completely removed by drying in an oven to obtain pure spherical SiO2 particles.
[0012] The second step comprises: Step 2-1, preparing an SiO2-deionized water / ethanol mixture by dispersing the SiO2 spherical particles from Step 1-2 in a deionized water / ethanol mixture and ultrasonically treating for 10 to 30 minutes; Step 2-2, forming a transparent crystalline solution by adding Cu(CH3COO)2·H2O and NH4Cl to deionized water and stirring for 10 to 30 minutes; and adding NH4Cl to the crystalline solution of Step 2-2. 3· The method comprises a second step of adding H2O dropwise, then slowly adding the SiO2-deionized water / ethanol mixture of the second step while stirring, transferring it to a Teflon-coated stainless steel autoclave and maintaining it at 155 to 165°C for 12 to 18 hours, and a second step of the precipitate produced in the second step of
[0013] In the above 2-1 step, it is preferable that the volume of the deionized water / ethanol mixture be 1:1.
[0014] In the above Step 2-1, spherical SiO2 particles are dispersed in a deionized water / ethanol mixture to prevent particle aggregation. Subsequently, a uniformly dispersed SiO2 colloidal solution is formed by ultrasonic treatment.
[0015] In the above 2-2 step, it is preferable that the weight ratio of Cu(CH3COO)2·H2O and NH4Cl is 4:6.
[0016] In the above Step 2-2, Cu(CH3COO)2·H2O and NH4Cl are added to deionized water to form a transparent crystalline solution, where NH4Cl acts as a buffer and complex-forming agent to stabilize copper ions.
[0017] In the above 2-3 steps, NH 3·H2O is added dropwise to raise the pH of the solution, causing copper ions to begin precipitating in the form of copper hydroxide (Cu(OH)2). Additionally, as the SiO2 dispersion is slowly added while stirring, copper compounds grow and coat the surface of the SiO2. Subsequently, hydrothermal synthesis is carried out using an autoclave, forming a copper silicate layer on the surface of the SiO2 particles.
[0018] The third step comprises: a third step 3-1 in which the copper silicate from the second to fourth steps is placed in an alumina boat and loaded into a quartz tube, and the quartz tube is purged with a 3 to 7 vol% H2 / Ar gas mixture for 20 to 40 minutes; and a third step 3-2 in which the temperature of the quartz tube is gradually increased at a rate of 4 to 8°C / min to reach 700 to 900°C and maintained for 1 to 2 hours to convert the copper silicate into copper-coated silica nanoparticles (Cu@SiO2).
[0019] The above third step is a process of converting copper silicate into copper-coated silica nanoparticles (Cu@SiO2) by treating it under conditions of a reducing atmosphere (H2 / Ar mixed gas) and high temperature heat treatment (700~900℃).
[0020] The above 3-1 step creates a reducing environment by removing oxygen inside the quartz tube with H2 gas, and Cu2 + The ions are reduced to metallic copper (Cu 0 It is a preparatory stage for transitioning to ).
[0021] In the above 3-2 step, copper silicate (CuSiO3 or Cu2SiO4, etc.) is decomposed at high temperature, and Cu2 by H2 + → Cu 0 A reduction reaction with (metallic copper) occurs, and metallic copper particles are precipitated on the SiO2 matrix.
[0022] In the above 3-3 step, reduced copper metal is uniformly dispersed and coated on the SiO2 surface, and finally, a Cu@SiO2 structure is obtained.
[0023] The copper-coated silica nanoparticles (Cu@SiO2) generated in the above 3-2 step are characterized by having a porous, sea urchin-shaped structure. Cu@SiO2 is a nanostructure in which multiple high aspect ratio spikes or protrusions extend outward from a central core, resembling the shape of a sea urchin. This unique structure increases the surface area-to-volume ratio, enhances reactivity, and facilitates interaction with biological materials. The sea urchin-shaped protrusion structure provides mechanical anchor points to which cells can attach, and the porous surface facilitates the adsorption of extracellular matrix proteins, aiding cell proliferation and differentiation. Through this, the porous, sea urchin-shaped structure of Cu@SiO2 simultaneously enhances cell affinity, drug delivery, and antimicrobial functions. Furthermore, nanoparticles with high aspect ratio spikes 1) enable more controlled and sustained release of metal ions that disrupt bacterial metabolism, and 2) strengthen the interaction between highly positively charged surface charges and negatively charged bacterial membranes, thereby destroying the bacterial membrane upon contact, resulting in cell membrane rupture and bacterial death.
[0024] The above Cu@SiO2 is characterized by exhibiting distinct intensity peaks corresponding to the (111), (200), and (220) diffraction planes at 2θ values of 43.2°, 50.2°, and 73.9°, respectively. These diffraction peaks are representative diffraction planes corresponding to metallic copper with a cubic (FCC) structure, which is evidence that well-crystalline copper nanoparticles exist within Cu@SiO2. In other words, this means that SiO2 encapsulates copper particles to stably maintain the crystallinity of copper.
[0025] The above-mentioned fourth step comprises: Step 4-1, in which sodium hyaluronate (HA sodium) is dissolved in deionized water at a weight ratio of 10:1 to prepare a 1% (w / v) HA solution; Step 4-2, in which methacrylic anhydride (MA) is slowly added to the HA solution in a molar excess of 4 to 6 times, and the mixture is maintained in an ice batch for 10 to 14 hours to promote further reaction within a pH range of 8.0 to 11 to produce MA-HA; Step 4-3, in which cold ethanol equivalent to 9 to 11 times the volume of the MA-HA is added to precipitate the solution, and subsequently, the solution is centrifuged at 4,000 to 6,000 rpm at 3 to 5°C for 3 to 7 minutes; and Step 4-3, in which the HA-MA, obtained by redissolving the precipitate in deionized water after removing the supernatant, is dialyzed with deionized water for 2 to 4 days using a 3.5 kDa molecular weight barrier. Step 4-4, which involves freeze-drying for an additional 2 to 4 days; Step 4-5, in which the MA-HA from Step 4-4 is dissolved in deionized water and the pH is adjusted to 4.3 to 4.7 using 0.8 to 1.2 M HCl; Step 4-6, in which EDC and NHS are added to the MA-HA solution and the pH is adjusted to 4.8 to 5.2, and a dopamine hydrochloride solution is slowly added to the mixture to adjust the pH to 4.3 to 4.7; Step 4-7, in which the mixture from Step 4-6 is purged with nitrogen gas for 10 to 20 minutes and the EDC and NHS are reacted at 20 to 30°C for 10 to 14 hours; and Step 4-7, in which the deionized water acidified to a pH of 4 to 5 is dialyzed with 95 to 105 mM NaCl for 2 days, then dialyzed with the deionized water alone for 3 to 5 hours, followed by freeze-drying to synthesize MA-HA-CA. Includes steps 4-8.
[0026] The above fourth step is a process of synthesizing a functional polymeric biomaterial called MA-HA-CA by chemically bonding methacrylic acid (MA) and catecholamine (CA) to hyaluronic acid (HA). In the above 4-1 step, an HA solution to be used in the reaction is prepared, and in the above 4-2 step, an excess amount of methacrylic anhydride (MA) is added to introduce methacrylic groups to the hydroxyl / carboxyl groups of HA to produce MA-HA. In the above 4-3 step, impurities are removed with cold ethanol, and MA-HA is recovered by centrifugation. In the above 4-4 step, low molecular weight by-products are removed by dialysis with a 3.5 kDa barrier membrane, and pure MA-HA polymer is obtained by freeze-drying. In the above 4-5 step, the MA-HA solution is acidified with HCl to create conditions suitable for the subsequent EDC / NHS reaction. In step 4-6 above, EDC / NHS is added to activate the carboxyl group, and dopamine hydrochloride is added to combine the carboxyl group of MA-HA with the amine group of dopamine to produce catechol-functionalized hyaluronic acid. In step 4-7 above, oxygen is removed with nitrogen gas to prevent oxidation of dopamine, and a stable amide bond is formed by reacting at 20-30°C for 10-14 hours. In step 4-8 above, residual reaction products are removed by dialysis with acidic deionized water and NaCl, and the final MA-HA-CA polymer is obtained through additional dialysis and freeze-drying.
[0027] The EDC and NHS reactions in steps 4-7 above are characterized as being EDC / NHS coupling reactions. The EDC / NHS coupling reaction connects a carboxyl group (-COOH) and an amine group (-NH2) to form a stable amide bond (-CONH-). EDC activates the carboxyl group, and NHS stabilizes it, allowing for efficient binding to the surfaces of proteins, peptides, antibodies, nanoparticles, etc. EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) reacts with the carboxyl group to produce an O-acylisourea intermediate, which is unstable and can be rapidly hydrolyzed. Additionally, the addition of NHS (N-hydroxysuccinimide) forms an NHS ester, which is relatively stable and provides sufficient time to react with the amine. The amine group (-NH2) attacks the NHS ester to form an amide bond (-CONH-). While EDC alone is unstable, it has the advantage of being able to form a stable NHS ester by adding NHS.
[0028] The above 5th step comprises: Step 5-1, in which PEG4SH and the MA-HA-CA of Steps 4-8 are dissolved in 0.008 to 0.012 M DPBS to prepare a precursor solution containing 3% to 7% (w / v) of a polymer component with a thiol / acrylate composition ratio of 1:1 molar ratio; Step 5-2, in which 0.008 to 0.012% (w / v) EY and 0.08 to 0.12% TEOA (v / v) are added to the precursor solution, respectively, and the pH of the precursor solution is adjusted to 7.6 to 8 using 0.8 to 1.2 N NaOH; and Step 5-2, in which the precursor solution is sterilized by filtering it through a 0.18 to 0.22 μm filter, and then the precursor solution is dissolved in deionized water containing 0.18 to 0.22% (w / v) of the above 3-2 It includes step 5-3, in which a Cu@SiO2 mixed polymer precursor solution is prepared by mixing with Cu@SiO2.
[0029] In the above 5-1 step, PEG4SH and MA-HA-CA are dissolved in DPBS, a buffer solution, and the molar ratio of thiol (-SH) to acrylate (-C=C-) is adjusted to 1:1. Then, crosslinking is enabled through a thiol-ene click reaction, and a hydrogel network forming precursor is prepared by adjusting the final concentration to 3~7% (w / v) to create a concentration suitable for hydrogel formation.
[0030] In the above 5-2 step, a photoinitiator and a co-initiator are prepared and the pH is adjusted to create conditions optimized for the photoinitiation reaction and the thiol-ene reaction.
[0031] In the above 5-3 step, microorganisms are removed from the precursor solution by filter filtration to ensure a sterile state, and Cu@SiO2 nanoparticles are added to form a Cu@SiO2 mixed polymer precursor solution.
[0032] The above 6th step comprises 6-1, in which a cylindrical well is fabricated using PDMS, and the Cu@SiO2 mixed polymer precursor solution is injected into the cylindrical well, and 200 mW cm⁻¹ is applied to induce photopolymerization. -2 , includes step 6-2, in which a Cu@SiO2-hydrogel is prepared by exposing it to green LED light of λ=525 nm for 4 to 6 minutes.
[0033] The PDMS of step 6-1 above is used as a mold for hydrogels due to its biocompatibility, transparency, and ease of processing, and a cylindrical hydrogel can be obtained by injecting a Cu@SiO2 mixed polymer precursor solution.
[0034] The FT-IR of the Cu@SiO2-hydrogel of Step 6-2 above is 1,410 cm⁻¹ -1 , 1,520 cm -1 , 1,630 cm -1 , 1,720 cm -1 , 2,850 cm -1 It is characterized by exhibiting a peak at 1,410 cm⁻¹. -1The peak at is attributed to CO stretching vibrations within the HA backbone and indicates ester or alcoholic CO bonds within the HA backbone, which implies that the polysaccharide structure of HA is maintained. Next, at 1,520 cm⁻¹ -1 The peak at indicates the inclusion of an aromatic structure through catechol conjugation on the HA framework due to C=C ring stretching vibrations. Next, at 1,630 cm⁻¹ -1 The peak at corresponds to the stretching vibration of the methacrylate group on the HA, which implies that the photopolymerizable reactive group for hydrogel formation has been successfully introduced into the HA backbone. Next, at 1,720 cm⁻¹ -1 The peak at is attributed to C=O elongation vibrations within the HA framework, which reflects the inherent structural characteristics of HA and demonstrates that the basic framework is maintained even after deformation. Next, at 2,850 cm -1 The peak at [location] signals the presence of CH bonds of benzene rings on the HA backbone due to catechol conjugation, which indicates that the catechol group has been successfully bound to HA. The results of the FTIR analysis above prove that methacrylate and catechol groups were successfully introduced into HA, and that the hydrogel structure combined with Cu@SiO2 was formed as desired.
[0035] In the above 5th or 6th step, as the concentration of the precursor solution increases, the crosslinking density increases, which is characterized by an increase in the storage modulus and a decrease in the ESR value. The storage factor of the Cu@SiO2-hydrogel increases to 1,860 ± 40 Pa at a polymer content of 3% (w / v) and to 4,580 ± 120 Pa at a polymer concentration of 7% (w / v). As the polymer concentration increases from 3% (w / v) to 7% (w / v), the ESR decreases from 65.6 ± 9.6 to 30.7 ± 3.9.
[0036] In addition, the mechanical properties of the Cu@SiO2 hydrogel are characterized by being improved as the concentration of the precursor solution increases. The properties were improved by 20% when the polymer content was 3% (w / v), 30% when it was 5% (w / v), and 30% when it was 7% (w / v). In other words, the improvement in the mechanical properties of the hydrogel is due to the increased interaction between the Cu(II) ions released from Cu@SiO2 and the carboxyl groups of the polymer backbone.
[0037] The present invention comprises a Cu@SiO2-hydrogel patch prepared by a method for manufacturing a catechol-modified hyaluronic acid-based hydrogel encapsulated with Cu@SiO2. The Cu@SiO2-hydrogel patch can be used as a wound dressing due to its high adhesion to natural tissue and high antimicrobial activity. The Cu@SiO2-hydrogel patch adheres firmly to the wound site, effectively absorbs wound exudate, and helps prevent secondary infection by creating a complete coverage and antimicrobial environment. In addition, beyond wound dressing, the hydrogel patch accelerates wound healing by delivering essential growth factors or providing structural support for efficient tissue regeneration. Effects of the invention
[0038] As described above, the present invention has lower cytotoxicity compared to nickel-based nanoflowers and excellent biocompatibility by encapsulating it within a catechol-modified HA hydrogel through photopolymerization.
[0039] In addition, the present invention introduces catechol groups to exhibit high tissue adhesion.
[0040] In addition, the present invention exhibits an excellent bactericidal effect, showing about 100% bactericidal effect against three bacterial strains (K. pneumoniae, MRSA, and S. mutans). Brief explanation of the drawing
[0041] Figure 1 shows the manufacturing process of a photocrosslinkable and bioadhesive HA-based hydrogel. Figure 2 shows a schematic process of the Cu@SiO2 synthesis process. Figure 3 shows the X-ray diffraction patterns of Cu@SiO2 (gray) and a Cu standard (JCPDS 04-0836, orange). Figure 4 shows the TGA profile of Cu@SiO2. Figure 5 shows an SEM image of Cu@SiO2 including the elemental distribution map for copper (Cu, red), silicon (Si, green), and oxygen (O, blue). Figure 6 shows TEM images of Cu@SiO2. Scale bars: 100 nm (left) and 50 nm (right) Figure 7 shows the process of HA-MA synthesis through an ester exchange reaction between hyaluronic acid and methacrylic anhydride. Figure 8 shows the synthesized MA-HA and MA-HA-CA and HA. 1 It shows the H NMR spectrum. Figure 9 shows the UV-Vis spectrophotometers of HA, MA-HA, and MA-HA-CA, where the catechol conjugate peak appears at 280 nm. Figure 10 shows the absorption spectrum of eosin Y (EY). Figure 11 shows Cu@SiO2 dispersed in a polymer precursor solution after mixing PEG4SH and MA-HA-CA with EY in a 1:1 molar ratio. Figure 12 shows the transfer of a Cu@SiO2 mixed polymer solution onto a substrate. Figure 13 shows the adhesive properties of the Cu@SiO2-HA hydrogel. Figure 14 shows the PXRD patterns comparing the control hydrogel (gray), Cu@SiO2 (orange), and Cu@SiO2-hydrogel (red). Figure 15 shows the FT-IR spectra of the control hydrogel and the Cu@SiO2-hydrogel. Figure 16 shows the TGA profile comparing the control hydrogel (gray) and the Cu@SiO2-hydrogel (red). Figure 17 shows SEM images of the control hydrogel and Cu@SiO2-hydrogel and the corresponding elemental distribution maps. Figure 18 shows the EDS spectra of the control hydrogel and the Cu@SiO2-hydrogel, with carbon (C, red), oxygen (O, green), sulfur (S, yellow), copper (Cu, purple), and silicon (Si, blue) highlighted. Figure 19 shows a rheological analysis comparing a control hydrogel (gray) and a Cu@SiO2-hydrogel (red) at various polymer concentrations (3%, 5%, 7%) within the hydrogel. Figure 20 is a graph showing the average storage modulus of the control hydrogel (gray) and Cu@SiO2-hydrogel (red) at various polymer concentrations. Figure 21 is a graph showing the ESR (equilibrium expansion rate) of the control hydrogel (gray) and Cu@SiO2-hydrogel (red) at various polymer concentrations. Figure 22 is a photographic image showing a control hydrogel (top) and a Cu@SiO2-hydrogel (bottom). Figure 23 shows a schematic diagram of a lab-shear test performed to demonstrate the bioadhesive properties of the hydrogel. Figure 24 shows the average adhesion strength (shear stress) data of the control hydrogel (blue) and Cu@SiO2-hydrogel (red). Figure 25 shows the Cu(II) ion emission profiles of Cu@SiO2 (orange) and Cu@SiO2-hydrogel (red). Figure 26 shows photographs of bacteria grown after 24 hours of culture with various sample types (from left: blank, control hydrogel, Cu@SiO2, Cu@SiO2-hydrogel) (from top to bottom: K. pneumonia, MRSA, S. mutans). Figure 27 shows the bactericidal efficiency of the control hydrogel (gray), Cu@SiO2 (orange), and Cu@SiO2-hydrogel (red) against K. pneumonia, MRSA, and S. mutans (NS: not significant, *** P<0.001). Figure 28 shows representative images of MEFs stained with live and dead stains after 24 hours of exposure to normal culture medium, in the order of control (positive control), Cu@SiO2, hydrogel (control hydrogel), Cu@SiO2-hydrogel, and EtOH (negative control). Figure 29 shows the analysis of cellular metabolic activity of MEFs exposed to various conditions, in the order of normal medium (Blank; white), ethanol (EtOH; black), control hydrogel-derived culture medium (gray), Cu@SiO2 (orange), and Cu@SiO2-hydrogel (red). Specific details for implementing the invention
[0042] 1. Introduction
[0043] The marine mussel (*Mytilus edulis*) has developed a powerful adhesion mechanism capable of adhering to various surfaces in demanding aquatic environments. Mussels secrete proteins containing 3,4-dihydroxyphenyl-1-alanine (DOPA) from their foot glands. Catechol, a key component of DOPA, forms strong bonds with organic and inorganic groups on various surfaces. Several studies have been published demonstrating how catechol improves the bioadhesive properties of various polymers, such as chitosan, gelatin, poloxamers, and polyethylene glycol (PEG). Furthermore, it has been proven that functionalizing hyaluronic acid (HA) with catecholamines using carbodiimide coupling reactions can enhance adhesion properties for nerve regeneration. However, the strong adhesive power of catechol can attract microorganisms, potentially increasing the risk of biofilm formation and infection. Therefore, for applications in wound healing, the development of HA-based systems that combine strong adhesion with antimicrobial properties is essential.
[0044] Nanomaterials, particularly those made of metal oxides, are attracting significant attention as potential alternatives to antibiotics due to their antimicrobial properties. Metal oxide-based nanoparticles are preferred over conventional antibiotics because they 1) act as effective antibiotic carriers, 2) prevent biofilm formation, and (3) inhibit bacterial growth. The antimicrobial effect of nanomaterials depends on various factors, such as composition, size, shape, and surface characteristics. Extensive research has confirmed the potent antimicrobial effects of various metals, including silver, gold, iron, zinc, copper, and nickel. These metals or their oxides exhibit much stronger antimicrobial properties at the nanoscale compared to their bulk forms. Silicon-supported nickel oxide (NiO) nanoflowers were fabricated as antimicrobial agents and demonstrated to possess sufficient antimicrobial properties when encapsulated within a HA-based hydrogel. Sea urchin-shaped nanoparticles are nanostructures that resemble the shape of a sea urchin, consisting of multiple high aspect ratio spikes or protrusions extending outward from a central core. This unique structure has received significant attention due to its ability to increase the surface area-to-volume ratio, enhance reactivity, and facilitate interaction with biological materials, and is highly effective in applications such as antimicrobial treatment, catalysis, and drug delivery. In the present invention, a hydrogel encapsulated with sea urchin-shaped copper-coated silica nanoparticles was prepared.
[0045] Combining sea urchin-shaped nanoparticles with a hydrogel improves the biocompatibility of the system because encapsulation within the hydrogel mitigates the toxicity of the nanoparticles. Although further research is needed regarding the toxicity of nanomaterials, these substances can easily enter the human body through inhalation or skin contact and potentially accumulate in sensitive organs such as the lungs or liver. To mitigate this risk, encapsulating nanoparticles within a hydrogel enables the indirect application of antimicrobial nanomaterials and can reduce toxicity by controlling the release of nanomaterials from the hydrogel network.
[0046] Accordingly, the present invention aims to develop a photocrosslinkable adhesive HA hydrogel as a wound dressing patch by encapsulating antimicrobial nanomaterials. The research process involves synthesizing sea urchin-shaped copper-coated silica nanoparticles (Cu@SiO2) as an antimicrobial agent, and then encapsulating them within an adhesive HA-based hydrogel via visible light-induced photocrosslinking. Meanwhile, Eosin Y, which possesses excellent water solubility and cellular compatibility, was used as a photoinitiator, and 4-am PEG-thiol (PEG4SH), which possesses structural stability and biocompatibility, was used as a crosslinking agent. A comprehensive evaluation of the chemical composition, mechanical properties, biocompatibility, adhesion, and antimicrobial properties of the HA hydrogel encapsulated with Cu@SiO2 is performed to assess its potential as an effective material for wound healing applications.
[0048] 2. Materials and Methods
[0049] 2.1. Materials
[0050] The materials and reagents used in the present invention include tetraethyl orthosilicate (TEOS, 98%), copper acetate monohydrate (Cu(CH3COO)2·H2O, 99.99%), methacrylic anhydride (MA), deuterium water (D2O), ethanol (EtOH, 99.5%), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), ammonium chloride (NH4Cl, 99.0%), ammonia solution (NH3·H2O, 28%), dopamine hydrochloride (DA), hyaluronidase extracted from bovine testes (HAse), and eosin Y (EY) purchased from Sigma-Aldrich (St. Louis, Missouri, USA). In addition, sodium hyaluronate (40 kDa, HA) was purchased from Bloomage Freda Biopharm Co., Ltd. (Jinan, China), and 4-am poly(ethylene glycol) thiol (10 kDa, PEG4SH) was purchased from Laysan Inc. (Arab, Alabama, USA). Other materials included Dulbecco Modified Eagle Medium (DMEM), collagen type I, Dulbecco Phosphate Buffered Saline (DPBS, pH 7.4), deionized water (DIW), penicillin / streptomycin, mouse embryonic fibroblasts (MEFs), fetal bovine serum (FBS), and Thermo Fisher Scientific's (Waltham, Massachusetts, USA) LIVE / DEAD™ viability / cytotoxicity kit. High-purity argon gas (Ar, 99.999%) and hydrogen gas (H2, 99.999%) were supplied by Dong-A Specialty Gases (Seoul, Korea). In addition, the MTS analysis kit from Abcam (Waltham, Massachusetts, USA) was also used.
[0052] 2.2. Synthesis of Cu@SiO2
[0053] Sea urchin-shaped copper-coated silica nanoparticles (Cu@SiO2) were synthesized using a previously reported method. Briefly, silicon nanoparticles were encapsulated within porous sea urchin-shaped copper silicate via a hydrothermal reaction. This structure was then reduced to create a core-shell structure, and the core was coated with a porous copper composite sheet.
[0054] Step 1. Manufacture of SiO2 spheres
[0055] First, deionized water (10 mL), NH3·H2O (30 mL), and ethanol (200 mL) were mixed and stirred at 250 rpm for 10 minutes. Then, 8 mL of TEOS was added, and the mixture was stirred continuously for 4 hours. The resulting white product was washed with ethanol and deionized water and centrifuged twice at 2,500 rpm for 15 minutes. Finally, the product was dried in a 60°C oven for 8 hours.
[0056] Step 2. Preparation of copper silicate salt
[0057] Spherical SiO2 particles (0.4 g) were dispersed in a deionized water / ethanol mixture (40 mL, volume ratio 1:1) and sonicated for 20 minutes to prepare a SiO2-deionized water / ethanol mixture. Cu(CH3COO)2·H2O (1.84 g) and NH4Cl (2.73 g) were added to DIW (80 mL) and stirred for 20 minutes. After a clear crystalline solution was formed, NH3·H2O (10 mL) was added dropwise, followed by the gradual addition of the SiO2-deionized water / ethanol mixture while vigorously stirring. After 10 minutes, the solution was transferred to a 200 mL Teflon-coated stainless steel autoclave and maintained at 160°C for 15 hours. The resulting precipitate was filtered three times using ethanol and deionized water, and then dried overnight in a 60°C oven.
[0058] Step 3. Preparation of sea urchin-shaped copper-coated silica nanoparticles (Cu@SiO2)
[0059] Copper silicate powder (0.4 g) was placed in an alumina boat and loaded into a quartz tube. The quartz tube was purged with a 5 vol% H2 / Ar gas mixture for 30 minutes. After purging, the temperature of the quartz tube was gradually increased at a rate of 6°C / min until it reached 800°C, at which point it was maintained for 1.5 hours. Through this thermal reduction process, the copper silicate was converted to a Cu@SiO₂ structure. After the reduction step, the quartz tube was allowed to cool naturally.
[0061] 2.3. Preparation of HA Hydrogel Encapsulated with Cu@SiO2 (Cu@SiO2 Hydrogel)
[0062] Step 1. Synthesis of MA-HA and MA-HA-CA
[0063] HA was modified to a photoreactive methacrylate (MA) group according to the conventional method. First, a 1% (w / v) HA solution was prepared by dissolving 200 mg of HA sodium in 20 mL of DIW. Then, MA was slowly added to this solution in a 5-fold molar excess, and the mixture was kept on an ice batch for 12 hours to promote further reactions within the pH range of 8.0 to 11. The resulting product (MA-HA) was precipitated by adding cold ethanol equivalent to 10 times its volume. The solution was then centrifuged at 5,000 rpm at 4°C for 5 minutes. After removing the supernatant, the precipitate was redissolved in 20 mL of deionized water. HA-MA was dialyzed with deionized water for 3 days using a 3.5 kDa molecular weight barrier (Spectrum Laboratories, Rancho Dominguez, California, USA), followed by freeze-drying for an additional 3 days.
[0064] Subsequently, bioadhesive catechol groups were conjugated to the carboxylic acid groups of the MA-HA backbone via an EDC / NHS coupling reaction. Purified MA-HA (200 mg, 0.5 mmol) was dissolved in 18 mL of deionized water, and the pH was adjusted to 4.5 using 1 M HCl. Then, EDC (2.5 mmol) and NHS (0.75 mmol) were added to the MA-HA solution, and the pH was adjusted to 5.0. Afterward, dopamine hydrochloride solution (0.75 mmol dissolved in 2 mL of DIW) was slowly added to the mixture (pH 4.5). To minimize catechol oxidation, the mixture was purged with nitrogen gas for 15 minutes. The reaction was carried out overnight at 25°C. The solution was first dialyzed with acidified deionized water (pH 4.5) with 100 mM NaCl for 2 days, then dialyzed with deionized water alone for 4 hours, and finally lyophilized. The degree of MA and catechol modification is proton nuclear magnetic resonance ( 1 It was evaluated using ¹H NMR spectroscopy (DMX 360, Bruker, Massachusetts, USA). Catechol content was quantified using ultraviolet-visible (UV-Vis) spectroscopy, which measures absorbance at 280 nm using a dopamine standard solution.
[0065] Step 2. Preparation of Cu@SiO2 mixed polymer precursor solution
[0066] The precursor solutions required for the photocrosslinking gelation of the antimicrobial Cu@SiO2 hydrogel were prepared as follows. PEG4SH and MA-HA-CA were dissolved in 0.01 M DPBS to achieve a thiol / acrylate composition ratio of 1:1 molar ratio, thereby preparing solutions containing 3%, 5%, or 7% (w / v) of polymer components. Subsequently, 0.01% (w / v) EY (photoinitiator) and 0.1% (v / v) TEOA (co-initiator) were added to each precursor solution type. The pH of the final precursor solution was adjusted to 7.8 using 1 N NaOH. The precursor solution was then sterilized by filtering it through a 0.2 μm filter (Mannasas, Corning, New York, USA). After sterilization, the solution was mixed with 0.2% (w / v) Cu@SiO2 dissolved in deionized water and transferred to a plastic syringe equipped with a 25-gauge needle.
[0067] Step 3. Preparation of HA-based hydrogel encapsulated with Cu@SiO2 (Cu@SiO2-hydrogel):
[0068] Cylindrical wells with a diameter of 1.2 cm and a depth of 1 cm were fabricated using polydimethylsiloxane (PDMS) according to a conventional method. A polymer solution mixed with Cu@SiO2 was injected into these wells, and a green LED light (200 mW cm⁻¹) was applied to induce photopolymerization. -2 The material was exposed to λ=525 nm for 5 minutes. The resulting material was referred to as Cu@SiO2-hydrogel in the case of a hydrogel containing Cu@SiO2, and as a control hydrogel in the case of a hydrogel not containing Cu@SiO2.
[0070] 2.4. Characterization of Cu@SiO2
[0071] Powder X-ray Diffraction (PXRD) patterns were obtained using a diffractometer (SmartLab, Rigaku, Tokyo, Japan) set to 30 mA and 40 kV for 4° min -1Recordings were performed at a scan rate and 0.02° intervals. Thermogravimetric analysis (TGA) was performed using a NETZSCH TG 209 F3 Tarsus instrument (Burlington, Massachusetts, USA). Si nanoparticles and Cu@SiO2 were observed using scanning electron microscopy with energy-dispersive X-ray spectroscopy (SEM-EDS) and transmission electron microscopy (TEM). SEM-EDS was performed using a Hitachi SU8230 microscope (Hitachi High-Tec, Tokyo, Japan) at an acceleration voltage of 10 kV, and TEM imaging was performed using a Tecnai G2 F20 TWIN TMP (FEI, Hillsboro, Oregon, USA) at an acceleration voltage of 120 kV. The specific surface area of the samples was measured at -196°C using the Brunauer-Emmett-Teller (BET) method with a 3FLEX surface characterization and physicosorption analyzer from Micromeritics (Atlanta, Georgia, USA). The pore size distribution was analyzed in the adsorption branch using the Barrett-Joyner-Halenda (BJH) method.
[0073] 2.5. Characterization of Cu@SiO2 Hydrogel
[0074] Fourier transform infrared (FT-IR) spectra of Cu@SiO2, control hydrogel, and Cu@SiO2-hydrogel were acquired using a Tensor II spectrometer (Bruker, Billerica, Massachusetts, USA). Additionally, a thermogravimetric analyzer was used to compare the thermogravimetric properties of the control hydrogel and the Cu@SiO2-hydrogel. Freeze-dried hydrogel samples were observed using a scanning electron microscope (SEM). Prior to SEM imaging, the control hydrogel and Cu@SiO2-hydrogel were sputter-coated with 5 nm thick gold (EMS150R ES, Electron Microscopy Sciences, Hatfield, Pennsylvania, USA). The coated samples were analyzed using a benchtop SEM (TM4000Plus II; Hitachi, Tokyo, Japan) at an acceleration voltage of 10 kV. The elemental composition of all samples, including carbon (C), oxygen (O), sulfur (S), copper (Cu), and silicon (Si), was measured using Energy Dispersive Spectroscopy (EDS). After acquiring the EDS spectra, the elemental content was quantified using AZtec software (Oxford Instruments; Concord, Massachusetts, USA).
[0075] The rheological properties of the Cu@SiO2 hydrogel were determined using a rheometer (MCR302, Anton Paar; Graz, Austria) at a constant shear strain of 0.1% and 0.1–10 rad sec -1 Evaluations were performed within the frequency sweep range. Various hydrogel samples were prepared at polymer concentrations of 3%, 5%, and 7% (w / v), and tests were conducted for each concentration with and without the addition of Cu@SiO2. This allowed for the evaluation of the effects of polymer concentration and Cu@SiO2 addition on the rheological properties of the hydrogels. To ensure the accuracy of the final analysis, all samples were measured three times.
[0076] To evaluate the swelling behavior of the control group and Cu@SiO2-hydrogel, five fresh hydrogel samples of each were prepared and immersed in DPBS for 2 days to achieve maximum absorption and swelling. After this period, the hydrogels were removed from the DPBS, gently wiped to remove excess surface liquid, and then immediately weighed (W s ) was measured. Subsequently, the hydrogel was freeze-dried for 24 hours and re-measured in the dry state to obtain the dry weight (W d ) was calculated. The Equilibrium Expansion Ratio (ESR) was calculated using Equation 1:
[0077] (Equation 1) ESR = W s / W d
[0078] Adhesion testing was performed using a lab-shear test, in which the hydrogel was placed between two pieces of pig skin replacing human skin tissue. Cu@SiO2-hydrogel specimens (30 mm × 25 mm) were prepared and inserted between the pig skin pieces, with each skin piece attached to one end of a glass slide. This assembly was mounted on a universal testing machine (68SC-05, Instron; Norwood, Massachusetts, USA). The testing machine operated at a displacement rate of 5 mm min -1 It was set to operate, and the maximum force at the point of hydrogel rupture was recorded as the adhesive strength of the sample. The adhesive ability of the MA-HA-CA based hydrogel (photocrosslinkable and adhesive hydrogel) was evaluated by comparing its performance with that of the MA-HA hydrogel (photocrosslinkable but non-adhesive) used as a control.
[0079] 5% (w / v) samples were used to evaluate the degradation characteristics of the control group and Cu@SiO2 hydrogels. The hydrogels were first immersed in DPBS and incubated for 48 hours to induce swelling. Subsequently, the samples were gently wiped to remove residual surface liquid, and then weighed. After weighing, the samples were placed in 10–1,000 IU mL of DPBS dissolved in -1The hydrogel samples were immersed in an enzyme solution containing HAse. Subsequently, these hydrogel samples were incubated in a vibrating vessel at 37°C and a vibration speed of 80 rpm. The solution was periodically replaced to maintain enzyme activity. At specific time intervals, the hydrogel samples were gently wiped to remove surface residue, and their weight was measured. The weight loss rate was calculated using Equation 2.
[0080] (Equation 2) Weight reduction rate (%) = (W0--W t ) / W0× 100
[0081] Here, W0 represents the initial weights and W t is the weight at time t.
[0083] 2.6. Metal Ion Emission Test
[0084] To evaluate the release of metal ions from Cu@SiO2 and Cu@SiO2-hydrogels, 5 mg of each sample was added to 1 mL of DPBS and stirred at 25°C for 6 to 48 hours. The solution was then centrifuged at 5 m / 0 rpm, and the resulting supernatant was collected. The concentration of released Cu(II) ions was measured using inductively coupled plasma emission spectroscopy (ICP-OES) with an Agilent Marker 7700 instrument (Tokyo, Japan) operating at an RF generator output of 1,550 W.
[0086] 2.7. Antimicrobial Test
[0087] According to existing methods, the antimicrobial activity of three different materials—a control hydrogel, Cu@SiO2, and Cu@SiO2-hydrogel—was evaluated against the following three bacterial strains: Klebsiella pneumoniae (ATCC 4352), methicillin-resistant Staphylococcus aureus (MRSA) (ATCC 33,591), and Streptococcus mutans (ATCC 25,175). A Cu@SiO2-hydrogel sheet measuring 5 cm × 5 cm was prepared as a test sample. Cu@SiO2 and the control hydrogel were prepared using the same procedure, and a stomacher film was prepared as a blank control.
[0088] The surface of the test sample was wiped with ethanol 2 to 3 times and then dried. 1–4 × 10⁶ test bacteria 5 Inoculations were performed within the colony-forming unit (CFU) / milliliter (mL) concentration range. Subsequently, 200 μL of each sample was inoculated into individual test solutions. To ensure uniform distribution, a drop of bacteria was gently pressed onto the film. All samples and test strains were incubated at 37°C for 24 hours. After inoculation with the test strains, the other uncoated specimens were separated using sterile tweezers, and the test bacteria were washed with fresh medium. The washed medium was analyzed immediately to count the number of viable cells. The bactericidal activity was calculated using Equation 3.
[0089] (Equation 3) Sterilization activity rate (%) = (C--S) / C × 100
[0090] Here, C represents the CFU of bacteria cultured on a stomacher film, and S represents the CFU of bacteria cultured on a control hydrogel, Cu@SiO2, or Cu@SiO2-hydrogel.
[0092] 2.8. Biocompatibility of Cu@SiO2 Hydrogel
[0093] MEF monolayers were used to evaluate the biocompatibility of Cu@SiO2-hydrogels. First, MEFs were cultured on glass slides (1.25 cm × 1.25 cm) coated with Collagen I and placed in 24-well plates. This process followed the protocol presented in prior studies. After the monolayers were formed, the prepared Cu@SiO2-hydrogel and control hydrogels were carefully placed on top of the cell monolayers, and Cu@SiO2 (0.2% (w / v)) was carefully mixed with the cell monolayers. The cells were then cultured for 24 hours. In this invention, the positive control (blank) consisted of MEFs cultured as a monolayer under normal culture conditions, and the negative control consisted of the same MEF monolayers exposed to 10% ethanol.
[0094] Cell viability was evaluated using the live-dead staining method, and cells were stained with calcein-AM and ethidium bromide and observed under an inverse fluorescence microscope (IX83; Olympus, Center Valley, Pennsylvania, USA). Total cell viability was calculated using Equation 4.
[0095] (Equation 4) Cell viability (%) = Number of living cells / Total number of cells × 100
[0096] In addition to qualitative evaluation, cell viability was quantitatively confirmed through metabolic activity analysis. Conditioned media were prepared by incubating control samples, Cu@SiO2 and Cu@SiO2-hydrogel, in complete culture medium at 37°C for 24 hours. Subsequently, MEF monolayer cultures were established in 24-well plates and exposed to each type of conditioned media (500 μL) for 24 hours. After the exposure period, the conditioned media was replaced with fresh MTS solution diluted 1:10 with fresh media, and incubated at 37°C for 4 hours. Absorbance was measured at 490 nm using a spectrophotometer (Synergy H1; BioTek, Winooski, Vermont, USA). Cell viability via MTS analysis was calculated using Equation 5.
[0097] (Equation 5) Cell viability (%) = (OD sample --OD blank ) / (OD contro l--OD blank ) × 100
[0098] Here, OD sample represents the optical density (i.e., absorbance) of the sample solution, and OD control OD is the absorbance of fresh medium. blank represents the absorbance of the empty well.
[0100] 2.9. Statistical Analysis
[0101] Three repeated experiments were performed to calculate the mean and standard deviation. Statistical significance was determined using a t-test, and the significance levels were set to * p<0.05, ** p<0.01, *** p<0.001.
[0103] 3. Results and Discussion
[0104] Figure 1 illustrates the main objectives of the present invention. 1) Synthesis of antimicrobial nanoparticles: Urchin-shaped copper-based antimicrobial nanoparticles (Cu@SiO2) with multiple high aspect ratio spikes or protrusions extending outward from a central core were synthesized via a hydrothermal method. 2) Preparation of photocrosslinkable and bioadhesive hyaluronic acid: Photocrosslinkable and bioadhesive hyaluronic acid was synthesized separately. To enable photopolymerization, methacrylate groups (MA) were introduced to the hydroxyl groups of hyaluronic acid via an ester exchange reaction. Additionally, bioadhesive catechol groups (CA) were functionalized to the carboxyl groups of hyaluronic acid using an EDC / NHS chemical reaction. 3) Preparation of antimicrobial hydrogel: To prepare an antimicrobial hydrogel, a precursor solution for photocrosslinking was prepared as follows. First, methacrylated and catechol-modified hyaluronic acid (MA-HA-CA) and a crosslinking agent (PEG4SH, thiol / methacrylate 1:1 molar ratio) were sequentially dissolved in PBS. Next, Eosin Y was added as a photoinitiator, and Cu@SiO2 nanoparticles were added and mixed into the polymer solution. The Cu@SiO2 hydrogel was formed through thiol-ene photopolymerization between the thiol groups of the crosslinking agent and the methacrylate groups of MA-HA-CA, initiated by visible light. Meanwhile, the catechol groups on the HA backbone provide excellent surface adhesion, allowing the hydrogel to adhere easily to the skin while maintaining excellent antibacterial properties.
[0106] 3.1. Analysis of Cu / SiO2 Characteristics
[0107] Figure 2 shows that the synthesis of sea urchin-shaped Cu@SiO2 nanoparticles underwent several steps. In the initial hydrothermal reaction process, Si nanoparticles were coated with porous flower-shaped copper silicate. These structures were subsequently reduced to form a core-shell structure, which was then wrapped in a porous copper composite sheet. In the final step, sea urchin-shaped Cu@SiO2 nanoparticles were produced by sequential heating to 800°C in an H2 / Ar mixed gas atmosphere.
[0108] Figure 3 shows the X-ray diffraction (XRD) pattern of Cu@SiO2, exhibiting distinct intensity peaks corresponding to the (111), (200), and (220) diffraction planes at 2θ values of 43.2°, 50.2°, and 73.9°, respectively. These peaks are very similar to those observed in the Cu standard (JCPDS No. 04-0836). Figure 4 presents the TGA results, showing an increase in the weight of Cu@SiO2 with increasing temperature. This is likely due to the formation of CuO. Figure 5 shows the SEM image of Cu@SiO2 and the corresponding EDS results. Cu, Si, and O elements were uniformly distributed within the nanoparticles, which exhibited a distinct sea urchin-like structure. The TEM image (Figure 6) confirmed that the thickness of the Cu shell surrounding the Si core was uniform at approximately 10 ± 3 nm. This sea urchin-like structure remained intact even after the subsequent annealing process.
[0109] High-Angle Darkfield (HAADF) scanning transmission electron microscopy (STEM) and energy-dispersive X-ray spectroscopy (EDS) mapping demonstrate that the elemental composition of Cu, Si, and O within Cu@SiO2 was confirmed. These results strongly demonstrate that a Cu coating was successfully deposited on Si nanoparticles via a hydrothermal reaction.
[0111] 3.2. Analysis of Pore Structure Characteristics
[0112] The pore structure and surface area of SiO2 and Cu@SiO2 were compared using the N2 adsorption-desorption isotherm obtained at -196°C. Hollow pore volume (V meso ) and specific surface area (S BET ) was measured by the BET method, and the average pore size was evaluated in the adsorption branch using the BJH method. As shown in Table 1, after producing Cu@SiO2 by coating SiO2, V meso , S BET and the average pore size increased significantly. Specifically, the value of Cu@SiO2 (0.24 cm²) 3 / g, 58.41 m2 / g, 16.93 nm) was 11.4 times, 5.6 times, and 14.2 times larger than the values of SiO2, respectively.
[0113] Sample name V meso (cm 3 / g) S BET (m 2 / g) Average pore size (nm) Cu@SiO2 0.24 58.41 16.93 SiO2 0.023 10.35 1.19
[0115] 3.3. Synthesis and Characterization of MA-HA-CA
[0116] In the present invention, HA was selected as the main polymer backbone due to its well-established biocompatibility and biodegradability. The versatility of HA is attributed to its abundant hydroxyl and carboxylic acid groups. To introduce photopolymerizable groups into the backbone, primary hydroxyl groups were modified into methacrylate groups via a transesterification reaction. Additionally, the goal was to introduce bioadhesive groups, specifically catechol groups, into MA-HA. This was achieved by conjugating dopamine to carboxyl groups via carbodiimide coupling using EDC and NHS (Fig. 7).
[0117] The degree of modification of HA, MA-HA, and MA-HA-CA 1 The results were quantified using ¹H NMR spectroscopy. The degree of methacrylation and catechol substitution was determined by comparing the areas of the N-acetylmethyl peak of HA at 1.9 ppm, the methacrylate peaks of MA-HA and MA-HA-CA at 5.6 and 6.1 ppm, and the catechol peak of MA-HA-CA at 6.5–7.0 ppm. The degree of methacrylation and catechol substitution was confirmed to be 50% and 18%, respectively (Fig. 8). The catechol content was quantified by UV-Vis spectroscopy using a catechol concentration standard curve constructed with a dopamine standard solution at 280 nm. The absorbance of MA-HA-CA was significantly higher than that of HA and MA-HA, indicating that approximately 19% of HA was successfully conjugated with catechol (Fig. 9).
[0119] 3.4. Preparation of Cu@SiO2 Hydrogel
[0120] Figure 10 shows that EY absorbs light at 515 nm, which enables photocrosslinking under conditions more favorable to cells than other UV photoinitiators such as Irgacure 2959.
[0121] Cu@SiO2-hydrogels were synthesized via visible light-initiated photopolymerization. This process was carried out by reacting PEG4SH with HA-MA-CA in the addition of 0.01% EY as a photoinitiator and 0.1% TEOA as a co-initiator. Both EY and TEOA are known for their excellent biocompatibility and water solubility, while the PEG4SH crosslinker is known for its structural stability and biocompatibility (Figs. 11 and 12). Upon exposure to 524 nm visible light, EY is excited to extract hydrogen atoms from PEG4SH and generate thiyl radicals. The generated thiyl radicals can react with the methacrylic groups of MA-HA-CA to form carbon radicals. These carbon radicals can propagate through other methacrylic groups within MA-HA-CA or continue to extract hydrogen atoms from additional thiol groups of PEG4SH. These reactions proceeded collectively to form a photocrosslinked Cu@SiO2-hydrogel. The catechol groups present in the hydrogel allow for anchoring to the natural nucleophilic groups of the substrate, thereby providing stable bioadhesion. Figure 13 shows a representative image of a photocurable bioadhesive hydrogel anchored between the thumb and index finger for visualization.
[0123] 3.5. Analysis of Chemical Characteristics
[0124] The chemical composition of the hydrogels was characterized using PXRD and FT-IR techniques. Figure 14 shows that in the PXRD pattern, the control and the Cu@SiO2-hydrogel exhibit two distinct peaks at 2θ = 19.2° and 23.1°, respectively, which correspond to the backbone polymer. Additionally, the Cu@SiO2-hydrogel exhibited additional peaks at 2θ = 43.2°, 50.2°, and 73.9°, which are presumed to be due to the presence of coated Cu@SiO2 (Figure 3).
[0125] Figure 15 shows the FT-IR results. 1,630 cm⁻¹ -1 The absorption peak at corresponds to the stretching vibration of the methacrylate group on the HA phase, and at 1,720 cm⁻¹ -1 and 1,410 cm -1 The peaks at are attributed to the C=O and CO stretching vibrations within the HA framework, respectively. Additionally, at 2,850 cm⁻¹ -1 CH vibrations at and 1,520 cm -1 The C=C ring stretching vibration in represents catechol junctions on the HA framework.
[0126] The thermal stability and decomposition behavior of the control group and Cu@SiO2 hydrogels were evaluated via TGA. As shown in Figure 16, these hydrogels exhibited weight losses of approximately 11.2% and 4.2%, respectively, up to 200°C, which is primarily attributed to the evaporation of absorbed moisture. Subsequent additional weight loss occurring at approximately 350°C is attributed to the decomposition of side chains (particularly carboxylate and hydroxyl groups) within the polymer backbone. At this point, the control group and Cu@SiO2 hydrogels lost 50% of their initial weight at 304°C and 306°C, respectively. At 900°C, they lost 93.0% and 82.5% of their weight, respectively. The introduction of Cu@SiO2 significantly increased the thermal decomposition temperature.
[0127] Detailed information regarding the chemical composition and morphology of the hydrogels was obtained through SEM-EDS analysis. Figure 17 shows that both hydrogels exhibited a moderately porous structure with an irregular pattern, indicating that Cu@SiO2 particles were effectively encapsulated within the hydrogel network. Figure 18 shows that C, O, and S elements were distributed in both networks, and the Cu@SiO2-hydrogel also showed the presence of Cu and Si in the EDS spectrum. These observations confirm that Cu@SiO2 was successfully integrated into the hydrogel matrix, as evidenced by the SEM and EDS results (Figures 17 and 18).
[0129] 3.6. Analysis of Mechanical Properties
[0130] The mechanical properties of the control hydrogel and the Cu@SiO2-hydrogel were evaluated by measuring the storage modulus. These rheological properties are crucial for the functionality of the hydrogel and are directly related to the crosslinking density of the polymer forming the three-dimensional network within the hydrogel. Furthermore, increasing the concentration of the precursor solution generally leads to higher crosslinking density, which in turn increases the storage modulus and decreases the ESR value. According to our findings, the storage modulus of the Cu@SiO2-hydrogel increased from 1,860 ± 40 Pa to 4,580 ± 120 Pa (Figs. 19 and 20), and the ESR decreased from 65.6 ± 9.6 to 30.7 ± 3.9 as the polymer content increased from 3% (w / v) to 7% (w / v) (Fig. 21). A similar trend was observed in the control hydrogel as the polymer concentration increased. The storage modulus increased from 1,520 ± 160 Pa to 3,670 ± 70 Pa (Figs. 19 and 20), and the ESR decreased from 98.4 ± 12.5 to 46.6 ± 6.9 (Fig. 21). In addition, the effect of Cu@SiO2 encapsulation on mechanical properties was evaluated by comparing the two hydrogel types at similar polymer concentrations. The mechanical properties of the Cu@SiO2 hydrogel showed significant improvement compared to the control hydrogel, with improvements of 20% at a polymer content of 3% (w / v), 30% at 5% (w / v), and 30% at 7% (w / v) (Fig. 20). The improvement in the mechanical properties of the hydrogel appears to be due to increased interaction between Cu(II) ions released from Cu@SiO2 and the carboxyl groups of the polymer backbone.
[0131] The observed improvement in mechanical properties appears to be due to increased intermolecular interactions and enhanced stiffness of the hydrogel network. At the same time, ESR and pore size decreased. Figure 22 shows a photograph of the hydrogel fully expanded in an aqueous solution. The Cu@SiO2-hydrogel exhibited a uniform distribution of Cu@SiO2, whereas the control hydrogel appeared porous and transparent. Furthermore, the sea urchin-shaped nanoparticles remained intact within the Cu@SiO2-hydrogel even after continuous immersion in the aqueous solution for 48 hours. These observations confirm the stable encapsulation and preservation of Cu@SiO2 throughout the photopolymerization process.
[0133] 3.7. Tissue Adhesion Properties
[0134] To develop effective wound-healing hydrogel patches using HA, it is essential to improve adhesive properties due to the inherent lack of adhesion in HA. Research has focused on significantly enhancing tissue adhesion by introducing catechol groups into hydrogel-based hydrogels. To evaluate the adhesion of these hydrogels, lap-shear tests were performed.
[0135] Initially, the hydrogel was placed between layers of porcine skin replacing human skin tissue (Fig. 23). Despite the application of tensile forces in opposite directions, the tissue layers remained stably bonded thanks to the strong adhesive properties of the hydrogel. However, as the tensile force increased, the hydrogel eventually failed to maintain cohesion, resulting in a clear separation point. This separation point was measured as the maximum adhesive strength. To evaluate the adhesive properties of hydrogels containing methacrylate and catechol groups, non-adhesive MA-HA-based hydrogels lacking catechol groups were also prepared using the same photocuring method. Fig. 24 shows that the adhesive hydrogel exhibited an adhesive strength of approximately 101.5 ± 3.5 kPa, which is significantly higher than the 8.3 ± 2.4 kPa measured in the non-adhesive hydrogel. This substantial difference highlights the superior bioadhesion provided by catechol groups, which enhance adhesion by interacting with nucleophilic groups on the tissue surface.
[0137] 3.8. Decomposition Analysis
[0138] To enable the potential in vivo applications of HA-based hydrogels (e.g., tissue engineering scaffolds, wound healing patches, drug delivery systems), it is important to ensure that the implanted material is biodegradable, thereby eliminating the need for a second surgery for removal. Considering therapeutic properties and suitability for in vivo application, the biodegradation tendency of a control hydrogel and a Cu@SiO2-hydrogel in a HAse-based solution was evaluated.
[0139] Cu@SiO2-hydrogel and control hydrogel were mixed with 0.01 M DPBS or 10–1000 IU mL -1They were immersed in enzyme solutions with HAse concentrations within a specified range. Table 2 shows the weight loss measured at designated time intervals to evaluate the degradation characteristics. Degradation was clearly observed for both hydrogel types in the enzyme solution, and the degradation rate increased with higher HAse concentrations. However, the control hydrogel degraded much faster and more extensively than the Cu@SiO2-hydrogel. For example, 10 IU mL -1 In a solution containing HAse, the control hydrogel showed a weight loss rate of 2.31% per day, whereas the weight loss rate of the Cu@SiO2-hydrogel was significantly slower at 0.94%. 100 IU mL -1 When using HAse, the control hydrogel showed a weight loss of 4.54%, whereas the Cu@SiO2 hydrogel maintained a lower rate of 1.06%. In addition, at a higher concentration of 1,000 IU mL -1 In HAse, the control hydrogel experienced a significant daily weight loss of 6.24%, whereas the degradation rate of the Cu@SiO2-hydrogel remained relatively low at 1.58%. When immersed in DPBS, both hydrogels exhibited only negligible weight loss. The relatively slow degradation rate of the Cu@SiO2-hydrogel is presumed to be due to the presence of Cu@SiO2. This enhances the hydrogel's resistance to enzymatic degradation. The encapsulation of Cu@SiO2 within the hydrogel strengthens the intermolecular interactions between copper ions and HA through coordination reactions. This can lead to increased stiffness and lower sensitivity to enzymatic degradation.
[0140] Daily weight loss rate % 10 IU mL -1 HAse 100 IU mL -1 HAse 1000 IU mL -1 HAse Control group hydrogel 2.31 4.54 6.24 Cu@SiO2-hydrogel 0.94 1.06 1.58
[0142] 3.9. Ion Emission Test
[0143] The ion emission profiles of Cu@SiO2 and Cu@SiO2-hydrogel were compared. Figure 25 shows that Cu@SiO2 emitted an amount of Cu(II) ions that increased from 1.59 ppm at 6 hours to 3.79 ppm at 48 hours while maintaining structural integrity in DPBS. In contrast, the Cu@SiO2-hydrogel showed significantly lower Cu(II) ion emission, decreasing 9.8-fold to only 0.39 ppm after 48 hours. This reduction in ion emission may be due to Cu@SiO2 directly interacting with the polymer network to limit metal ion release. The encapsulation of Cu@SiO2 within the hydrogel network may have resulted in reduced ion emission by blocking direct contact with the aqueous solution. Additionally, the ion emission of the control hydrogel was negligible.
[0145] 3.10. Antimicrobial activity
[0146] The antimicrobial efficacy of the control hydrogel, Cu@SiO2, and Cu@SiO2-hydrogel was evaluated. The initial antimicrobial activity of the control hydrogel was tested against Klebsiella pneumoniae, methicillin-resistant Staphylococcus aureus (MRSA), and Streptococcus mutans by incubation at 37°C for 24 hours. The results showed that the control hydrogel exhibited varying levels of antimicrobial activity, with the efficacy rankings as follows: K. pneumoniae (63.8%), MRSA (91.1%), and S. mutans (99.9%) (Figs. 26 and 27). Both Cu@SiO2 and Cu@SiO2-hydrogel demonstrated an impressive bactericidal rate of 99.9% against all three strains. These results are consistent with the following findings from previous studies: the introduction of catechol groups into HA-based hydrogels significantly enhances bactericidal activity compared to pure HA hydrogels. Furthermore, nanoparticles with high aspect ratio spikes 1) enable more controlled and sustained release of metal ions that disrupt bacterial metabolism, and 2) enhance the interaction between highly positively charged surface charges and negatively charged bacterial membranes, destroying the membrane upon contact to lead to membrane rupture and bacterial death. Copper ions also exhibit stronger interactions and enhanced toxicity due to their higher affinity for bacterial membranes compared to other ions (e.g., silver and nickel). Consequently, positively charged Cu@SiO2 at 200 μg mL⁻¹ is superior to neutral-charged nanoparticles such as nano-sized Ag / Cu-graphene. -1 It exhibited a stronger bactericidal effect at the concentration. In addition, the Cu@SiO2-hydrogel showed lower ion release than Cu@SiO2 alone, which is mainly due to the encapsulation of sea urchin-shaped nanoparticles within the hydrogel matrix. Cu@SiO2-hydrogel at 2 μg mL -1 It maintained a sterilization rate of 99.9% even at low concentrations.
[0147] Therefore, we hypothesized that the antimicrobial effect of Cu@SiO2-hydrogels is primarily derived from direct interactions between the hydrogel and cells, rather than the release of Cu(II) ions from Cu@SiO2 into cells. Numerous studies have been conducted on functional hydrogels containing various antimicrobial agents to control bacterial infections. Research on the antimicrobial properties of hydrogel matrices and nanoparticles has expanded their potential application fields and is already widely used in the biomedical field as a multifunctional material.
[0149] 3.11. Cytotoxicity
[0150] The cytocompatibility of the Cu@SiO2-hydrogel was evaluated through direct exposure of the sample to cells or indirect interaction with the culture medium. MEFs were used as cell lines for in vitro studies as they play a crucial role in wound healing. Toxicity was evaluated by comparing the effects of cell viability among the control group, Cu@SiO2, and Cu@SiO2-hydrogel. Live-death staining was performed to assess viability under various conditions. Figure 28 shows that the viability of MEFs exposed to Cu@SiO2 was less than 60%. This is because high levels of Cu(II) ions (e.g., 3.79 ppm) induce apoptosis through the oxidation of proteins, nucleic acids, and lipids. In contrast, normal culture (blank), control, or Cu@SiO2-hydrogel-treated cells exhibited viability of over 90%. Additionally, as expected, most MEFs died upon exposure to 10% ethanol (EtOH). These results suggest that the encapsulated Cu@SiO2 delays the rapid release of metal ions, indicating that the Cu@SiO2-hydrogel is non-cytotoxic.
[0151] MTS analysis was used to evaluate the level of biocompatibility (Fig. 29). Culture mediums of Cu@SiO2, control hydrogel, and Cu@SiO2-hydrogel were each diluted with fresh cell culture medium at a 1:3 ratio and added to MEF monolayer cultures for 24 hours. Cells treated with the control hydrogel and Cu@SiO2-hydrogel showed excellent viability of over 90%. In contrast, cells exposed to the diluted culture medium derived from Cu@SiO2 showed a decrease in viability to 65%. Upon exposure to ethanol (EtOH), viability dropped sharply to less than 10%, and near-complete cell death was observed. These results are consistent with viability analysis results, reaffirming the excellent biocompatibility of Cu@SiO2 hydrogel and demonstrating its potential as a reliable biomaterial.
[0153] 4. Conclusion
[0154] In this invention, an antimicrobial and bioadhesive hydrogel system for wound dressings using sea urchin-shaped Cu@SiO2 nanoparticles was successfully developed, and this is expected to be a promising candidate material compared to nickel-based nanomaterials reported in previous studies. While approximately 20 ppm of nickel ions were released from Si@NiO2 nanoflowers over 50 hours, only 4 ppm of copper ions were released from Cu@SiO2 nanoparticles during the same period, and this figure was further reduced to approximately 0.5 ppm when encapsulated in a hydrogel system. This ensures that the cytotoxicity of sea urchin-shaped Cu@SiO2 nanoparticles is lower than that of nickel-based nanoflowers, and biocompatibility and usability were further enhanced by encapsulating them within a catechol-modified HA hydrogel via photopolymerization. As indicated by the increase in storage modulus in the rheological analysis results of the hydrogel platform, the introduction of Cu@SiO2 significantly improved the mechanical properties of the hydrogel. TGA and SEM-EDS analyses further confirmed that Cu@SiO2 was successfully incorporated into the hydrogel. The uniform and homogeneous dispersion of sea urchin-shaped nanoparticles was clearly observed even with the naked eye. Furthermore, the hydrogel demonstrated approximately 100% bactericidal efficacy against three bacterial strains (K. pneumoniae, MRSA, and S. mutans).
[0155] To verify the practicality of the hydrogel as a wound dressing patch, we fabricated a hydrogel in which the HA backbone was modified with catecholamine groups to impart bioadhesive properties. The bioadhesive properties were confirmed through lab-shear tests with porcine skin, exhibiting an adhesion strength more than 10 times higher than that of non-adhesive hydrogels. In addition to the antimicrobial properties imparted by encapsulated Cu@SiO2 nanoparticles, this Cu@SiO2-hydrogel possesses great potential as a wound dressing patch because its bioadhesiveness allows it to properly cover the wound and support the wound healing process without falling off for a critical period. Although further in vitro and in vivo studies are required to fully explore its therapeutic potential, the present invention demonstrates the potential for using the Cu@SiO2-hydrogel as an effective wound dressing patch.
Claims
Claim 1 Deionized water, NH 3· A first step in which SiO2 spherical particles are prepared using a mixture of H2O, ethanol, and TEOS (tetraethyl orthosilicate); a second step in which a copper silicate salt is prepared by adding a mixed solution of Cu(CH3COO)2·H2O and NH4Cl to the SiO2 spherical particles from the first step; a third step in which copper silicate salt from the second step is loaded into a quartz tube and purged with a mixture of H2 gas and Ar gas to prepare copper-coated silica nanoparticles (Cu@SiO2); and a method in which MA-HA is prepared by adding methacrylic anhydride (MA) to a hyaluronic acid (HA) solution, carboxyl groups are activated by adding EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide) to the MA-HA, and dopamine hydrochloride is added to combine the carboxyl groups of MA-HA with the amine groups of dopamine. A method for preparing a catechol-modified hyaluronic acid-based hydrogel encapsulated with Cu@SiO2, comprising: a fourth step of synthesizing MA-HA-CA (catecholamine); a fifth step of preparing a Cu@SiO2 mixed polymer precursor solution by dissolving PEG4SH (4-arm poly(ethylene glycol) thiol) and the MA-HA-CA of the fourth step in PBS (phosphate-buffered saline), and mixing EY (Eosin Y), TEOA (Triethanolamine), and the copper-coated silica nanoparticles (Cu@SiO2) of the third step; and a sixth step of preparing a HA-based hydrogel encapsulated with Cu@SiO2 by injecting the Cu@SiO2 mixed polymer precursor solution of the fifth step into a cylindrical well made of PDMS (Polydimethylsiloxane) and inducing photopolymerization. Claim 2 In claim 1, the first step comprises deionized water, NH 3· A method for preparing a catechol-modified hyaluronic acid-based hydrogel encapsulated with Cu@SiO2, comprising: Step 1-1, in which H2O and ethanol are mixed and stirred at 200 to 300 rpm for 5 to 15 minutes, and then a mixture with added TEOS is stirred for 3 to 5 hours to produce a white product; and Step 1-2, in which the white product is washed with ethanol and deionized water, centrifuged twice at 2,000 to 3,000 rpm for 10 to 20 minutes, and the resulting product is dried in an oven at 50 to 70°C for 7 to 9 hours to produce SiO2 spherical particles. Claim 3 In claim 2, the second step comprises: a second step 2-1 of preparing a SiO2-deionized water-ethanol mixture by dispersing the SiO2 spherical particles of the first step 1-2 in a deionized water-ethanol mixture and ultrasonically treating for 10 to 30 minutes; a second step 2-2 of forming a crystalline solution by adding Cu(CH3COO)2·H2O and NH4Cl to the deionized water and stirring for 10 to 30 minutes; and NH4Cl to the crystalline solution of the second step 2-2. 3· A method for preparing a catechol-modified hyaluronic acid-based hydrogel encapsulated with Cu@SiO2, comprising: a second step of adding H2O dropwise, then adding the SiO2-deionized water and ethanol mixture of the second step while stirring, transferring to a Teflon-coated stainless steel autoclave and maintaining at 155 to 165°C for 12 to 18 hours, and a second step of filtering the precipitate generated in the second step using the ethanol and deionized water mixture, and then drying in an oven at 50 to 70°C for 10 to 14 hours to produce copper silicate. Claim 4 A method for preparing a catechol-modified hyaluronic acid-based hydrogel encapsulated with Cu@SiO2 according to claim 3, wherein the third step comprises a third step of placing the copper silicate from the second-fourth steps into an alumina boat and loading it into a quartz tube, and then purging the quartz tube with a mixture of 3 to 7 vol% H2 and Ar gas for 20 to 40 minutes, and a third step of increasing the temperature of the quartz tube at a rate of 4 to 8°C / min to reach 700 to 900°C and maintaining it for 1 to 2 hours so that the copper silicate is converted into copper-coated silica nanoparticles (Cu@SiO2), wherein the copper-coated silica nanoparticles (Cu@SiO2) produced in the third step have a porous sea urchin-shaped structure. Claim 5 In claim 4, the fourth step comprises: a 4-1 step of preparing a 1% (w / v) HA solution by dissolving sodium hyaluronate (HA) in deionized water at a weight ratio of 10:1; a 4-2 step of adding methacrylic anhydride (MA) to the HA solution in a molar excess of 4 to 6 times and maintaining the mixture in an ice batch for 10 to 14 hours to promote further reaction within a pH range of 8.0 to 11 to produce MA-HA; a 4-3 step of adding cold ethanol equivalent to 9 to 11 times the volume of the MA-HA to precipitate it, and subsequently centrifuging the solution at 4,000 to 6,000 rpm at 3 to 5°C for 3 to 7 minutes; and a 4-3 step of removing the supernatant and redissolving the precipitate in deionized water, wherein the HA-MA is then hydrated in deionized water using a 3.5 kDa molecular weight barrier for 2 to 4 days. Step 4-4, which involves freeze-drying for an additional 2 to 4 days after dialysis; Step 4-5, which involves dissolving the MA-HA from Step 4-4 in deionized water and adjusting the pH to 4.3 to 4.7 using 0.8 to 1.2 M HCl; Step 4-6, which involves adding EDC and NHS to the MA-HA solution and adjusting the pH to 4.8 to 5.2, and adding a dopamine hydrochloride solution to the mixture to adjust the pH to 4.3 to 4.7; Step 4-7, which involves purging the mixture from Step 4-6 with nitrogen gas for 10 to 20 minutes and reacting the EDC and NHS at 20 to 30°C for 10 to 14 hours; and Step 4-7, which involves dialyzing the deionized water acidified to a pH of 4 to 5 with 95 to 105 mM NaCl for 2 days, then dialyzing the deionized water alone for 3 to 5 hours, followed by freeze-drying. A method for preparing a catechol-modified hyaluronic acid-based hydrogel encapsulated in Cu@SiO2, comprising steps 4-8 in which MA-HA-CA is synthesized. Claim 6 In claim 5, the fifth step comprises: a 5-1 step of preparing a precursor solution containing 3% to 7% (w / v) of a polymer component with a thiol / acrylate composition ratio of 1:0.5 to 1 molar ratio by dissolving PEG4SH and the MA-HA-CA of the 4-8 steps in PBS; a 5-2 step of adding 0.008 to 0.012% (w / v) EY and 0.08 to 0.12% TEOA (v / v) to the precursor solution of the 5-1 step, and adjusting the pH of the precursor solution to 7.6 to 8; and a 5-3 step of preparing a Cu@SiO2 mixed polymer precursor solution by filtering the precursor solution of the 5-2 step through a 0.18 to 0.22 μm filter to sterilize it, and then mixing it with the Cu@SiO2 of the 3-2 step. Method for manufacturing a hyaluronic acid-based hydrogel. Claim 7 In claim 6, the 6th step comprises a 6-1 step in which a cylindrical well is fabricated using PDMS, and the Cu@SiO2 mixed polymer precursor solution is injected into the cylindrical well, and 200 mW cm⁻¹ is applied to induce photopolymerization. -2 A method for preparing a catechol-modified hyaluronic acid-based hydrogel encapsulated with Cu@SiO2, comprising step 6-2, in which a Cu@SiO2-hydrogel is prepared by exposing it to green LED light of λ=525 nm for 4 to 6 minutes. Claim 8 delete