Development of paintable hydrogel system for therapeutic factor delivery

Abstract

The present invention relates to a paintable hydrogel system. The hydrogel system possesses adhesive properties and carries therapeutic factors. According to the present invention, the hydrogel system overcomes limitations such as tissue damage and fibrosis associated with methods for delivering a substance for injection or patch-type treatment, and can be applied without limitation to the shape and size of a target site. In addition, the system can be easily and stably applied in transplantation fields involving synthetic drugs, stem cells, healthcare electronic devices, etc.

Description

Development of a Paintable Hydrogel System for Therapeutic Factor Delivery

[0001] The present invention relates to a paintable hydrogel system, wherein the hydrogel system can have adhesive properties and can contain therapeutic factors. This application claims the benefit of priority to Korean Patent Application No. 10-2024-0023638, filed February 19, 2024, the entire disclosure of which is incorporated herein by reference.

[0002] Existing therapeutic substance delivery methods have primarily involved injecting the substance into organs through injection or applying it to organs in the form of hydrogel patches. Medical patches typically lack tissue adhesion, requiring suturing to secure them to the tissue.

[0003] However, these methods have limitations in that they require needles or suturing, which can cause tissue damage such as hematoma formation in the organ. Adhesive patches have been developed to overcome these problems, but these also have limitations such as unnecessary adhesion to surrounding organs and fibrous adhesion. Furthermore, existing patches require an additional process of manufacturing them in a size and shape that fits the damaged area before transplantation. Natural adhesives, which are being further studied, have the advantage of being human-friendly, but because their adhesiveness cannot be controlled, they have the problem of possibly inhibiting the tissue regeneration effect due to the possibility of unnecessary adhesion to other organs or fibrosis.

[0004] Therefore, a hydrogel system that overcomes limitations such as tissue damage and fibrosis of systems requiring injection needles and sutures, has controllable adhesiveness, and is easier to apply and more stable due to no restrictions on shape and size is required.

[0005] An object of the present invention is to provide a paintable hydrogel and system thereof for delivering therapeutic factors.

[0006] To solve the above problem, the present invention provides a paintable hydrogel composition for tissue regeneration, comprising a decellularized extracellular matrix (dECM), a polymer containing a phenol derivative, and tyrosinase derived from Streptomyces.

[0007] According to one embodiment, the polymer including the phenol derivative may be a compound obtained by introducing the phenol derivative into hyaluronic acid, alginate, chondroitin sulfate, chitosan, collagen, fibrin, glycol chitosan, or polyethylene glycol.

[0008] According to one embodiment, the polymer comprising the phenol derivative may be a compound obtained by introducing the phenol derivative into a polymer hyaluronic acid having a molecular weight of 1000 kDa to 1500 kDa.

[0009] According to one embodiment, the polymer comprising the phenol derivative may be present at a concentration of 3% to 6% (w / v).

[0010] According to one embodiment, the tyrosinase may be derived from Streptomyces avermitilis.

[0011] According to one embodiment, the phenol derivative may be tyrosine, tyramine, dopamine, pyrogallol, caffeic acid, 3,4-dihydroxybenzylamine, 4-hydroxyphenylacetic acid or 3-(4-hydroxyphenyl)propionic acid.

[0012] According to a specific embodiment, the phenol derivative may be tyrosine or tyramine.

[0013] According to one embodiment, the adhesiveness of the composition can be adjusted by washing with a solution of bovine serum albumin (BSA), penicillamine or cysteine.

[0014] According to another embodiment of the present invention, there is provided a method for preparing a composition comprising: mixing a polymer comprising a decellularized extracellular matrix (dECM) and a phenol derivative with a tyrosinase derived from Streptomyces to obtain a pre-gelling composition; and

[0015] A method for producing a paintable tissue regeneration hydrogel composition is provided, comprising a step of contacting the above-mentioned pre-gelling composition with oxygen for 10 to 20 minutes to obtain a gelled hydrogel composition.

[0016] According to one embodiment, the hydrogel composition can have a storage modulus (G') of 11 to 13 kPa in a dry state and 4 to 6 kPa in a swollen state, depending on the amplitude and frequency sweep measurement method.

[0017] According to one embodiment, the hydrogel composition may have a viscosity of 100 to 500 Pa.s.

[0018] According to one embodiment, the pre-gelling composition can exhibit an adhesion stress of 0.6 to 0.9 kPa within 1 hour after mixing, as measured by a viscoelasticity measurement method.

[0019] Specific details of other embodiments of the present invention are included in the detailed description below.

[0020] According to the present invention, limitations such as tissue damage and fibrosis caused by injection or patch-type therapeutic substance delivery methods can be overcome, enabling application without restrictions on the shape and size of the target area. Furthermore, it can be easily and reliably applied to transplantation fields such as synthetic pharmaceuticals, stem cells, and healthcare electronic devices.

[0021] Figure 1 is a drawing showing an example of applying a paintable hydrogel composition to a heart.

[0022] Figure 2 is a photograph showing a heart that has been decellularized according to decellularization conditions.

[0023] Figure 3 is a graph showing the change in weight of a heart subjected to decellularization treatment according to the decellularization conditions.

[0024] Figure 4 is a microscopic photograph showing the density of extracellular matrix after staining a decellularized heart under each decellularization condition.

[0025] Figure 5 is a graph showing the quantification of extracellular matrix (ECM) components for each decellularized heart under each decellularization condition.

[0026] Figure 6 is a 1HNMR analysis graph for tyramine-conjugated hyaluronic acid (HA_t).

[0027] Figure 7 is an FT-IR analysis graph for tyramine-conjugated hyaluronic acid (HA_t).

[0028] Figure 8 is a graph evaluating the specific activity of SA_Ty against monophenolic substances (L-tyrosine and tyramine) using the Beer-Lambert law and the Michaelis-Menten equation.

[0029] Figure 9 is a photograph of application according to the concentration of tyramine-conjugated hyaluronic acid (HA_t).

[0030] Figure 10 is a photograph of painting and molding a hydrogel composition.

[0031] Figure 11 is a graph confirming viscosity according to the presence or absence of decellularized extracellular matrix.

[0032] Figure 12 is a photograph showing a composition adhered to a pig heart.

[0033] Figure 13 shows the results of confirming the compressive stress of the composition before and after swelling.

[0034] Figure 14 is a graph showing the storage modulus (G') before and after swelling.

[0035] Figure 15 shows the results of confirming the shear stress of a composition using porcine epicardial tissue.

[0036] Figure 16 shows the electron spin resonance (ESR) analysis results for the composition.

[0037] Figure 17 is a graph showing the results of Raman spectrometry analysis for the composition.

[0038] Figure 18 shows the results of a standard tensile test performed by treating the composition on porcine epicardium.

[0039] Figure 19 is a graph showing the maximum adhesive stress obtained by treating the composition on the porcine epicardium.

[0040] Figure 20 is a weight measurement analysis graph for verifying the in vivo decomposition behavior of the composition.

[0041] Figure 21 is a photograph showing the composition applied to a myocardial infarction (MI) rat model.

[0042] Figure 22 is a photograph showing the composition applied to the heart of a beating rat.

[0043] Figure 23 shows the results of decomposition scoring of the composition applied to the rat heart.

[0044] Figure 24 is a photograph of the left ventricle (LV) of a rat heart.

[0045] Figure 25 shows the results of observing angiogenesis in the rat heart according to application of the composition.

[0046] Figure 26 shows the results confirming that the paintable hydrogel can be painted in multiple layers.

[0047] The present invention is susceptible to various modifications and embodiments. Specific embodiments are illustrated in the drawings and described in detail in the detailed description. However, this is not intended to limit the present invention to specific embodiments, but rather to encompass all modifications, equivalents, and alternatives falling within the spirit and technical scope of the present invention. In describing the present invention, detailed descriptions of related known technologies will be omitted if they are deemed to obscure the gist of the present invention.

[0048] Hereinafter, a paintable hydrogel system for delivering therapeutic factors according to an embodiment of the present invention will be described in more detail.

[0049] The present invention utilizes a painting method and a catechol functional group to achieve easier and more stable adhesion to the application site, and to utilize this as a delivery system for synthetic drugs, stem cells, healthcare electronic devices, etc. By painting an adhesive hydrogel onto a defect site, tissue damage during the transplantation process is minimized, and application to defect sites of any size or shape is possible without limitation. Furthermore, the adhesive hydrogel enables stable adhesion to deliver therapeutic substances while simultaneously allowing the adhesiveness of the hydrogel to be controlled, thereby preventing unnecessary adhesion to surrounding organs after the procedure.

[0050] Catechol functional groups are converted to quinone functional groups through an oxidation process, which not only forms a hydrogel through bonding between different quinone functional groups, but also forms bonds with amine or thiol functional groups on the tissue surface, enabling the hydrogel to stably adhere to a wet tissue surface. The catechol of the present invention is an adhesive material derived from mussels and exhibits stable adhesion even in a wet environment. Since catechol exhibits adhesive properties by chemically converting its structure into quinone through an oxidation process, it has beneficial effects such as antioxidant effects. Therefore, active research has been conducted recently on adhesive patches using catechol. However, adhesive patches using catechol also have problems in that they cannot control adhesiveness, resulting in unnecessary adhesion to other organs or fibrosis, which inhibits tissue regeneration or induces new tissue regeneration without inducing it. In the present invention, we attempted to control adhesiveness by utilizing the fact that catechol-based patches adhere to organs through the quinone functional group created through an oxidation process. First, by removing the radicals generated during the oxidation process, the reaction in which catechol is transformed into quinone can be prevented to some extent. Second, the formed quinone functional groups combine with amine or thiol functional groups on the tissue surface to form tissue adhesion. By treating the patch surface with a solution containing a large amount of amine or thiol functional groups to remove quinone functional groups that will react with the tissue in advance, the adhesiveness of the catechol patch can be controlled.

[0051] Representative chemical cross-linking agents include carbodiimide (EDC), genipin, glutaraldehyde, NaIO4, and HRP / H2O2, which are primarily used to cross-link various biomaterials such as hydrogels. However, these chemical cross-linking agents can remain directly in the product, causing cytotoxicity or inhibiting the physiological activity of the decellularized material. In contrast, enzymatic cross-linking agents do not directly participate in network formation between biomaterial components, but rather induce stable covalent bonds between chemical functional groups. This allows for securing desired properties without the use of hazardous chemicals. Due to their specific binding to the substrate, enzymatic cross-linking agents do not remain in the cross-linking inducer, or even if they do remain, they are biocompatible.

[0052] Specifically, the present invention provides a paintable hydrogel composition for tissue regeneration comprising a decellularized extracellular matrix (dECM), a polymer comprising a phenol derivative, and tyrosinase derived from Streptomyces. According to the present invention, additional tissue damage and undesirable tissue adhesion can be minimized.

[0053] According to one embodiment, the decellularized extracellular matrix can be derived from the heart, lung, skin, bladder, cartilage, liver, blood vessels, brain, kidney, nerve ducts, muscle, intestine, or bone. The tissue-specific decellularized extracellular matrix can promote regeneration by mimicking the biophysical and biochemical composition of the tissue, and can provide cells with signals that promote and regulate the growth, function, and repair of the tissue, and can promote tissue formation at the transplant site. For example, the cardiac tissue-specific decellularized extracellular matrix can be used to treat myocardial infarction because it can reduce cardiac hypertrophy and fibrosis by modulating inflammation, apoptosis, and cardiac metabolism. The tissue-specific decellularized extracellular matrix containing angiogenic factors can play an important role in tissue remodeling by promoting angiogenesis from the composite after implantation in vivo. Furthermore, the mechanical properties of the composition can be increased by including the decellularized extracellular matrix.

[0054] According to one embodiment, the polymer comprising a phenol derivative may be a compound obtained by introducing a phenol derivative into hyaluronic acid, alginate, chondroitin sulfate, chitosan, collagen, fibrin, glycol chitosan, or polyethylene glycol. For example, the phenol derivative may include tyramine, tyrosine, dopamine, pyrogallol, caffeic acid, 3,4-dihydroxybenzylamine, 4-hydroxyphenylacetic acid, or 3-(4-hydroxyphenyl)propionic acid, and specifically, for example, the phenol derivative may be tyrosine or tyramine. The phenol derivative is not limited as long as it includes phenol, catechol, or pyrogallol.

[0055] According to one embodiment, the polymer comprising a phenol derivative may be a compound obtained by introducing a phenol derivative into a polymeric hyaluronic acid having a molecular weight of 1000 to 1500 kDa, for example, 1000 to 1200 kDa.

[0056] According to one embodiment, the polymer comprising a phenol derivative may be present in the hydrogel composition at a concentration of 3 to 6% (w / v), for example 3 to 5% (w / v), or 4 to 6% (w / v). The present invention may not include a gelatin mixture.

[0057] In one embodiment, the tyrosinase may be Streptomyces avermitilis tyrosinase. Tyrosinase is a polyphenol oxidase that converts phenol to catechol by introducing a hydroxyl group to the aromatic ring in the presence of oxygen. It also generates quinones from catechol through an additional oxidation reaction. Highly reactive quinones rapidly combine with amine or thiol groups through a nucleophilic reaction, allowing them to form carbon-carbon, carbon-oxygen, carbon-nitrogen, and carbon-sulfur bonds. Since this reactivity of quinones occurs in the presence of oxygen without any other cofactors, the reaction can proceed under mild reaction conditions. Tyrosinase from Streptomyces avermitilis has its active site exposed on the surface of its protein structure compared to those from Bacillus megaterium and Burkholderia thailandensis, making it easy to access large, structurally complex polymer substrates. Tyrosinase (SA_ty) from Streptomyces avermitilis has a cross-linking time that is more than 40 times shorter than that from Bacillus megaterium and Burkholderia thailandensis.

[0058] According to one embodiment, in order to control the adhesiveness of a composition having the above-described structure, the surface of the composition can be washed with a bovine serum albumin (BSA), penicillamine, or cysteine ​​solution. Specifically, for example, when washing the composition of the present invention with bovine serum albumin, penicillamine, or cysteine, quinones can be blocked, and the material used for washing is not limited as long as it contains an amine or thiol functional group. For example, amino acids such as penicillamine or cysteine ​​can be used for washing the composition, and the use of proteins such as BSA is also not limited. When the quinone functional group that reacts with tissue is removed from the composition, the adhesive stress is reduced as a result, so that the adhesiveness can be controlled by utilizing this property. In addition, the composition of the present invention can prevent stenosis from the application target by easily controlling the adhesiveness through washing. For example, when the application target is the heart and its surrounding area, unnecessary adhesion with nearby organs such as the thorax and lungs can be prevented.

[0059] According to one aspect, a method for producing a paintable tissue regeneration hydrogel composition is provided, comprising: mixing a polymer including a decellularized extracellular matrix (dECM) and a phenol derivative with tyrosinase derived from Streptomyces to obtain a pre-gelling composition; and reacting the pre-gelling composition with oxygen for 10 to 20 minutes to obtain a gelled hydrogel composition.

[0060] As an example, Fig. 1 shows the application of a paintable hydrogel (a hydrogel containing cardiac tissue-specific decellularized extracellular matrix (hdECM)) to a myocardial infarction (MI) model heart according to one aspect. Stable wet tissue adhesion can be expected from the myocardial infarction model applied with the paintable hydrogel by the SA_Ty-mediated oxidation process.

[0061] According to one embodiment, the pre-gelling composition can exhibit a shear stress of 0.6 to 0.9 kPa and a tensile strength of 20 to 30 kPa within 1 hour after mixing.

[0062] The hydrogel composition prepared by the above method can have a storage modulus (G') of 11 to 13 kPa in a dry state and 4 to 6 kPa in a swollen state, depending on the amplitude and frequency sweep measurement method. In addition, the hydrogel composition of the present invention can have a viscosity of 100 to 500 Pa.s, for example, 100 to 300 Pa.s, or 150 to 300 Pa.s, 200 to 400 Pa.s. These viscosity characteristics are higher than those of existing injectable or sprayable bioadhesive compositions, and the present invention has a viscosity suitable for painting, and can also be used by laminating and molding.

[0063] According to one embodiment, the adhesive properties of the hydrogel composition can be developed within 10 to 20 minutes and completely gelled within 1 hour. The hydrogel composition can remain non-biodegradable while sufficiently performing the roles of delivering and adhesive properties of tissue-derived decellularized material.

[0064] The hydrogel composition according to the present invention is not limited in size and shape, and can prevent adhesion by controlling adhesiveness, thereby overcoming limitations such as tissue damage and fibrosis, thereby providing a more easily applicable and stable hydrogel system. Since the hydrogel composition of the present invention includes a decellularized extracellular matrix, it can deliver tissue-derived decellularized materials. Therefore, it can be easily applied to various diseases such as skin care and myocardial infarction, and can be applied to transplantation fields such as synthetic pharmaceuticals, stem cells, and healthcare electronic devices.

[0065]

[0066] Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily implement them. However, the present invention may be implemented in various different forms and is not limited to the embodiments described herein.

[0067]

[0068] Manufacturing Example 1: Manufacturing of cardiac tissue-specific decellularized extracellular matrix (hdECM)

[0069] Porcine hearts were minced into 3 mm thick pieces and rinsed in cold water for 24 h. The hearts were then treated with 0, 0.1, 1, and 3% sodium dodecyl sulfate (SDS) concentrations for 48 h to remove cells, and then treated with 1% Triton X-100 for 6 h to remove residual SDS. The decellularized hearts were sterilized with 0.1% peracetic acid in 4% ethanol for 4 h, rinsed with sterile distilled water for 48 h, and lyophilized. The lyophilized decellularized hearts were digested with pepsin in 0.5 M acetic acid (decellularized heart: pepsin = 10:1, weight ratio). The digested solution was neutralized to physiological pH (pH 7.0) and lyophilized. The lyophilized hdECM was ground in a blender to obtain hdECM powder.

[0070] The photographs of the hearts decellularized for 48 hours are shown in Fig. 2, and the weight changes are shown in Fig. 3. From Fig. 2, it was confirmed that the transparency of the hearts treated with 1% and 3% SDS increased. Comparing the hearts treated with 1% and 3% SDS, the overall size of the heart treated with 3% SDS decreased, and the extracellular matrix (ECM) decreased more than that of the heart treated with 1% SDS. In addition, from Fig. 3, it was confirmed that the hearts treated with 0, 0.1, and 1% SDS showed slight degradation, with weights of 88.4±3.3, 84.8±4.10, and 69.1±4.50%, respectively. In the case of the heart treated with 3% SDS, more than half of the initial weight was lost, indicating that the extracellular matrix was significantly reduced.

[0071] The presence of cells and the maintenance of the extracellular matrix (ECM) were confirmed by hematoxylin & eosin (H&E) staining and immunohistochemistry using DAPI and collagen type 1, and are shown in Figs. 4 and 5. In the 0 and 0.1% SDS groups, a large number of cells were confirmed in the extracellular matrix, whereas no cells were observed in the 1% and 3% SDS treatment groups. Compared to the 1% SDS treatment group, the 3% SDS treatment group showed a lower density of extracellular matrix (Fig. 4).

[0072] Hydroxyproline analysis, dimethyl methylene blue (DMMB) analysis, and PicoGreen analysis were performed to quantify extracellular matrix (ECM) components such as collagen, glycosaminoglycans (GAGs), and DNA, respectively, and the results are shown in Fig. 5. The 3% SDS-treated group showed a decrease in ECM content due to ECM degradation. The DNA content of the 1% and 3% SDS groups was reduced by more than 90% compared to the original heart tissue, resulting in a final concentration of less than 50 ng per mg of ECM dry weight.

[0073]

[0074] Manufacturing Example 2: Hydrogel Manufacturing

[0075] Hyaluronic acid with a molecular weight of 1,000 to 1,200 kDa was used. The hyaluronic acid (HA)-tyramine conjugate was prepared through a carbodiimide reaction involving Schiff base condensation and amide bond formation between the carboxyl group of HA and the amine of tyramine. DMTMM was reacted at 70°C for 1 hour (molar ratio: HA:DMTMM=1:2). Tyramine hydrochloride (molar ratio: HA:tyramine=1:4) was added and reacted for 24 hours. The HA_tyr solution thus formed was dialyzed for 48 hours and then lyophilized. The degree of tyramine substitution was measured by 1H NMR (AVANCE III HD 300 MHz). FT-IR spectra were acquired at a wavelength of 400–4000 cm-1 using an FT-IR spectrometer (Bruker TENSOR27, Germany).

[0076] To determine the degree of substitution of tyramine-linked hyaluronic acid (HA_t), 1H NMR was performed and is shown in Figure 6. Three proton peaks of the N-acetyl group of HA appeared at approximately 2.0 ppm. Four aromatic proton peaks at the ortho and meta positions of tyramine were detected at 6.8 and 7.2 ppm, respectively. The integrated areas of the aromatic proton peaks were 0.36 and 0.64, respectively, compared to those of the N-acetyl peak. Based on the integrated areas, the tyramine substitution rate was calculated to be 75.2%.

[0077] To confirm the presence of conjugated tyramine residues, additional FT-IR analysis was performed and is shown in Figure 7. The broad band at 3290 cm-1 represents the hydroxyl group of HA. The additional peaks at 1634 and 1045 cm-1 of HA_t correspond to the C=C and C-O bonds of the aromatic ring, respectively.

[0078] The specific activity of tyrosinase (SA_Ty) from Streptomyces avermitilis toward monophenolic substances (L-tyrosine and tyramine) was evaluated using the Beer-Lambert law and the Michaelis-Menten equation and is shown in Fig. 8. The Kcat and Km values ​​were calculated by measuring the reaction rate of tyrosinase in the presence of various concentrations of monophenolic substrates and are shown in Table 1.

[0079]

[0080] The affinity for the substrate and the reaction rate of the product were expressed as Km and Kcat, respectively. While SA_Ty showed a lower affinity for tyramine than for L-tyrosine, the enzyme reaction rate constants for the two substrates were identical due to the faster reaction rate of tyramine. SA_Ty, a highly reactive cross-linker, catalyzes the ortho-quinone conversion before the coupling process initiated by tyrosinase-mediated oxidation is completed. This process is shown in Chemical Formula 1.

[0081]

[0082]

[0083] Manufacturing Example 3: Manufacturing of tyrosinase (SA_ty) derived from Streptomyces avermitilis

[0084] Tyrosinase E. coli was inoculated into autoclaved LB medium with ampicillin and grown overnight in a 37°C shaking incubator. The cultured E. coli was transferred to fresh LB medium and cultured at 37°C for 3 h. When the OD600 value of the bacterial solution was between 0.6 and 0.8, protein expression was induced by adding 1 M isopropyl β-D-1-thiogalactopyranoside (IPTG) and 1 M CuSO4*5H2O, and the culture was stored in a shaking incubator at 18°C ​​for 20 h. The cell pellet was then collected by ultracentrifugation (4,000 rpm for 10 min) and washed twice with 50 mM Tris-HCl buffer (pH 8.0) (5 mL). Cells were lysed using an ultrasonicator (VC505, USA) at 4°C for 20 minutes and then centrifuged at 4°C for 30 minutes. The expressed enzyme was purified through His-tag purification with Ni-NTA agarose beads. The obtained tyrosinase (SA_Ty) was filtered using a 0.22 μm syringe filter, collected in a 10 kDa filter tube, and mixed with autoclaved 75% glycerol solution. The concentration of the purified enzyme was calculated using the Bradford assay. After adding pH 8.0 Tris buffer, 10 nM CuSO4, tyrosinase, and 2 mM L-tyrosine, SA_Ty was incubated at 37°C for 30 minutes, and the absorbance was measured at 475 nm. The specific activity of SA_ty was calculated using the Beer-Lambert law and the Michaelis-Menten equation. The activity of tyrosinase in response to various concentrations of L-tyrosine and tyramine at 37°C was measured using UV spectrophotometry (475 nm).

[0085]

[0086] Example 1: Preparation of paintable hydrogels

[0087] Paintable hydrogels for therapeutic factor delivery must have sufficient viscosity, like a flexible and sticky paste. A pre-gelling composition was prepared by mixing 1% (w / v) hdECM treated with 1% SDS and 1 to 6% (w / v) of a tyramine-conjugated hyaluronic acid (HA_t) solution, to which a 1% (v / v) Sa_Ty solution was added, followed by cross-linking by contact with oxygen for 10 to 20 minutes to prepare a paintable hydrogel (pdHA_t).

[0088] The results of hydrogel application according to each HA_t concentration are shown in Fig. 9. HA_t solutions prepared at low concentrations of 1 to 3% (w / v) had low viscosity and high shrinkage, making it difficult to apply the hydrogel with a commercial brush. Furthermore, high-concentration groups of 5 and 6% (w / v) HA_t failed to form a coating of uniform thickness, making it difficult to paint. On the other hand, the paintable hydrogel (pdHA_t) prepared with 4% HA_t, hdECM, and SA_Ty was easy to apply and formed a uniform, thin hydrogel layer on the substrate.

[0089]

[0090] Example 2: Molding of paintable hydrogels

[0091] A paintable hydrogel (pdHA_t) prepared with 4% (w / v) HA_t, 1% (w / v) hdECM, and 1% (v / v) SA_Ty can easily fill a polydimethylsiloxane (PDMS) mold using a commercial brush. As shown in Fig. 10, the hydrogel of the present invention is paintable and moldable. These results demonstrate that the composition of the present invention has no theoretical limitations in size or shape.

[0092]

[0093] Experimental Example 1: Bonding Time

[0094] A rheological analysis was performed to determine the viscosity of the paintable hydrogel (pdHA_t) compared to the composition used in Example 2 and the hydrogel (pHA_t) without cardiac tissue-specific decellularized extracellular matrix (hdECM). pHA_t is the same as the composition in Example 2 except that it does not contain hdECM. The viscosities of pHA_t and pdHA_t hydrogels were measured in a Demo lab (Anton-Paar Korea). After adding SA_Ty to the HA_t solutions (W and W / O hdECM), a shear rate of 1 1 / s was applied to the hydrogels for 1 hour to obtain viscosity measurements. As shown in Fig. 11, the viscosity of pdHA_t increased significantly compared to pHA_t, indicating that pdHA_t quickly reached a sticky paste form. These results indicate that the adhesion time of the composition of the present invention is within 1 hour.

[0095]

[0096] Experimental Example 2: Pig Heart Bonding

[0097] The paintable hydrogel (pdHA_t) was successfully applied to a pig heart with stable adhesion under various conditions, and the results are shown in Fig. 12.

[0098]

[0099] Experimental Example 3: Swelling Evaluation

[0100] The prepared hydrogels were required to exhibit minimal swelling to prevent cardiac tamponade, so the degree of swelling was evaluated. pHA_t and pdHA_t hydrogels were molded into holes (d: 8 mm and h: 2 mm) of a PDMS mold. The prepared hydrogel pellets were immersed in PBS (10 mL) and weighed for 24 hours. The swelling ratio was calculated using the equation: swelling ratio = (Ws-Wi) / Wi, where Wi and Ws are the initial weight and the weight of the swollen hydrogel, respectively. The composition used was the same as that in Experimental Example 1.

[0101] After swelling in phosphate-buffered saline (PBS) for 24 h, the size of pHA_t significantly increased compared to the initial size. Furthermore, the swelling ratio, defined as the ratio of the original hydrogel weight to the swollen hydrogel weight, increased to 4000±319.2% for pHA_t. In contrast, pdHA_t showed a minimal difference in size, exhibiting a swelling ratio of 452±19.3%.

[0102] Based on the swelling behavior of pHA_t and pdHA_t, the mechanical properties of the hydrogels before and after swelling were measured and further analyzed. The compressive strength of hydrogel pellets with a size of 8 mm X 2 mm (diameter X height) was measured and is shown in Fig. 13. Both pHA_t and pdHA_t showed a decrease in maximum compressive stress after swelling, but pdHA_t showed a greater compressive strength than pHA_t before and after swelling.

[0103] Dynamic rheological measurements were performed by performing amplitude and frequency sweep analyses before and after swelling the hydrogel pellets. The rheological analyses were evaluated in a demo lab (Anton-Paar Korea) using a rheometer. The amplitude sweep measured the storage modulus G' and the loss modulus G" as the shear strain (%) increased from 0.1 to 100 at a fixed frequency of 1 Hz. The frequency sweep measured the storage modulus G' and the loss modulus G' as the frequency increased from 0.1 to 10 Hz at a fixed strain of 1%. The results are shown in Fig. 14.

[0104] In both amplitude and frequency sweeps, pdHA_t exhibited higher storage moduli (G') of approximately 12.04±0.61 kPa and 12.36±0.75 kPa in the dry state and 5.78±0.28 kPa and 4.73±0.48 kPa in the swollen state, respectively. In contrast, pHA_t exhibited significantly lower rheological behavior in both the dry and swollen states over the entire strain and frequency ranges.

[0105]

[0106] Experimental Example 4: Adhesive Stress

[0107] Wet adhesion to cardiac tissue was evaluated using a standard lap shear test using porcine epicardial tissue, and the results are shown in Fig. 15. The shear stress was measured by applying the hydrogel between epicardial tissues of a certain size, bonding them, and then pulling them apart. The pellets were stretched at a rate of 5 mm min-1 until failure while recording the load and displacement using a universal tensile machine (UTM-Shimazu, EZ-SX STD, Japan). The pdHA_t and pHA_t hydrogels were applied to epicardial tissues measuring 2.5 cm in width and 2.5 cm in length for both tensile and lap shear tests. Both adhesion tests were performed using the UTM (Shimazu, EZ-SX STD, Japan) module according to the ASTM standard tensile (ASTM F2258) and lap shear (ASTM F2255) tests, respectively. To analyze the loss of adhesion during hydrogel washing, hydrogels applied to the epicardium were washed with DW and BSA solutions before testing. For tensile testing, hydrogels applied to the epicardium without washing served as a control.

[0108] pdHA_t showed maximum shear stresses of 0.33±0.10 kPa and 0.78±0.08 kPa before and after crosslinking, respectively. This indicates a significant difference in shear stress after crosslinking pdHA_t. pHA_t showed maximum shear stresses of 0.28±0.03 kPa and 0.48±0.13 kPa before and after crosslinking.

[0109] To prevent unnecessary adhesion to surrounding organs, a method was applied to remove free radicals forming quinone groups as in Chemical Formula 2 and to block quinone groups that interact with amine or thiol groups on the tissue surface to induce wet tissue adhesion.

[0110]

[0111] Radicals were measured using electron spin resonance (ESR) analysis. For residual radical measurements, pdHA_t hydrogel pellets (d: 4 mm and h: 1 mm) were fabricated using a PDMS mold. The residual radicals present in the hydrogel pellets were measured before and after washing. ESR measurements were performed at the National Center for Inter-university Research Facility (NCIRF) using an EMXplus-9.5 / 12 / P / L system (Bruker, Germany).

[0112] Electron spin resonance (ESR) demonstrated that washing the pdHA_t surface with DW reduced the amount of free radicals by half compared to the unwashed surface, and the results are shown in Fig. 16. The surface of pdHA_t was washed with DW, BSA solution, etc. to block the existing quinones. After washing the hydrogel pellets with DW and BSA solutions, the quinone amounts of the untreated hydrogel pellets (control) and the washed hydrogel pellets were measured using a Raman spectrometer (RAMAN spectrometer, DXR2xi, Thermo Fisher Scientific, USA), and the results of the Raman spectrometer analysis are shown in Fig. 17. When the pdHA_t surface was washed with a BSA solution, the quinone intensity within the band at 1630 cm-1 was significantly reduced.

[0113] To evaluate the bonding strength, a standard tensile test was performed to evaluate the separation strength of pdHA_t between porcine epicardium, which is shown in Fig. 18. In addition, the maximum bonding stress was determined and is shown in Fig. 19. Compared to the control and DW-treated pdHA_t, which had maximum bonding stresses of 23.35±1.75 kPa and 17.11±0.37 kPa, respectively, pdHA_t treated with BSA solution was confirmed to have a maximum bonding stress reduced by 4.68±0.51 kPa.

[0114]

[0115] Experimental Example 5: Decomposition Behavior

[0116] The pHA_t and pdHA_t samples, which reached swelling equilibrium within 24 hours, were weighed and then transplanted. Sprague Dawley (SD) rats (6 weeks old, male) were prepared. After making an incision in the skin, three samples from each group were transplanted subcutaneously at regular intervals and sutured with 5-0 silk (Ethicon; Somerville, NJ, USA). Samples were collected on days 7, 14, 21, and 28 after transplantation, and as much tissue as possible was removed. The weight of the hydrogel samples with the tissue removed was measured and compared with the weight before transplantation.

[0117] The weights of pHA_t and pdHA_t were measured on day 0 after transplantation, and the weights of samples collected on days 7, 14, 21, and 28 after subcutaneous transplantation into mice were measured and shown in Fig. 20. Compared to the weight of the hydrogel before transplantation, both groups showed a significant weight% decrease on day 7, but there was no significant difference between the groups (PHA_t: 31.26±14.02% vs. pdHA_t: 32.62±3.09%). Interestingly, after day 7, the weight of pHA_t (day 14: 48.24±4.37%, day 21: 44.56±8.68%, day 28: 20.07±10.05%) continuously decreased over time, whereas the specific gravity of pdHA_t (day 14: 79.88±1.30%, day 21: 89.80±0.46%, day 28: 93.07±0.94%) gradually increased. On day 28, the weight of pdHA_t was similar to that of pdHA_t observed before transplantation.

[0118]

[0119] Experimental Example 6: Hydrogel Painting and Angiogenesis Induction in a Myocardial Infarction (MI) Model

[0120] After ligation of the left anterior descending artery (LAD) from the heart of a rat to induce myocardial infarction (MI), the white area of ​​the left ventricle was clearly visible 28 days later in the control group due to the deprivation of nutrients and oxygen. To determine whether the hydrogel that can be applied in the MI model can actually exhibit a therapeutic effect, pHA_t or pdHA_t was applied to the left ventricle (LV) using a commercial brush. As shown in Fig. 21, pdHA_t adhered well to the beating heart. From Fig. 15, it was confirmed that there was a difference in the maximum shear stress between pHA_t and pdHA_t after crosslinking, and pdHA_t was easier to paint and adhered better to the wet LV area than pHA_t.

[0121] Paintable hydrogels were successfully applied to the MI area of ​​a beating heart. The applied hydrogel rapidly crosslinked and adhered only to the application site, with no adhesion observed in the surrounding areas. These results are depicted in Figure 22. This highlights the importance of using paintable hydrogels for MI treatment compared to conventional suture techniques. These results demonstrate that the paintable or paintable hydrogels prevented the lesion adhesions that commonly occur after surgery and did not restrict the movement of the beating heart. To compare the degree of adhesion of pHA_t and pdHA_t to the heart 28 days after MI induction and hydrogel application, degradation scoring (S1: complete degradation, S2: >50% degradation, S3: non-degraded) was performed and is depicted in Figure 23. Similar to the degradation behavior described above after subcutaneous implantation, pdHA_t (S2: 12.5% ​​and S3: 87.5%, n= 7 / 8) was hardly degraded and maintained at the cardiac application site, unlike pHA_t (S1: 40%, S2: 40%, S3: 20%).

[0122] To closely examine the lesions and hydrogel adhesion in the left ventricle, the heart was dissected. The observation images are shown in Figure 24. The control group exhibited a thin, white left ventricle (LV) wall at the lesion site. In contrast, the pdHA_t group showed fewer lesions, and adherent, paintable hydrogel was observed on the LV wall.

[0123] There are two main processes in new blood vessel formation: 1) angiogenesis, which consists primarily of capillaries dominated by endothelial cells (ECs), and 2) vascularization, which primarily forms blood vessels in which smooth muscle cells (SMCs) surround ECs, such as arteries and veins. The results of co-staining for CD31 and cTnT are shown in Fig. 25 . pdHA_t formed more CD31+ capillaries than Control and pHA_t in the MI heart region (area where cTnT was sparsely or densely expressed) (Fig. 25 C, E). CD31+ capillaries with lumens were also formed in the painted hydrogels and were distributed much more abundantly in pdHA_t than in pHA_t. Comparing vascularization by staining for α-smooth muscle actin (αSMA; a SMC-specific marker), pdHA_t formed significantly more αSMA+ vessels than Control and pHA_t in the MI heart (Fig. 25 D, F) and in the painted hydrogel area.

[0124] Furthermore, cTnT staining revealed a decrease in myocardium due to MI-induced necrosis in the control group, whereas pdHA_t prevented severe myocardial collapse. Notably, as shown in Figure 25, the paintable hydrogels (pHA_t and pdHA_t) exhibited migration and reorganization of angiogenic cells, but not cardiomyocytes (CM). Figure 26 shows the results demonstrating that the paintable hydrogels can be painted in multiple layers.

[0125] As described above, the paintable hydrogel composition can enhance physical properties such as adhesive stress and biodegradability by introducing adhesive and decellularized extracellular matrix into the hydrogel, and can prevent adverse effects by rapidly controlling the degree of adhesiveness. Furthermore, the paintable hydrogel composition induces blood vessel formation and tissue remodeling, promoting angiogenesis from the composite after implantation and aiding in tissue remodeling.

[0126] The above description is merely an illustrative illustration of the technical idea of ​​the present invention, and those skilled in the art can make various modifications and variations without departing from the essential characteristics of the present invention. In addition, the embodiments disclosed in the present invention are not intended to limit the technical idea of ​​the present invention, but rather to explain it, and the scope of the technical idea of ​​the present invention is not limited by these embodiments. The protection scope of the present invention should be interpreted by the following claims, and all technical ideas within a scope equivalent thereto should be interpreted as being included in the scope of the rights of the present invention.

Claims

1. A paintable hydrogel composition for tissue regeneration comprising a decellularized extracellular matrix (dECM), a polymer containing a phenol derivative, and tyrosinase derived from Streptomyces.

2. In paragraph 1, A hydrogel composition, wherein the polymer containing the above phenol derivative is a compound obtained by introducing a phenol derivative into hyaluronic acid, alginate, chondroitin sulfate, chitosan, collagen, fibrin, glycol chitosan or polyethylene glycol.

3. In paragraph 2, A hydrogel composition, wherein the polymer containing the above phenol derivative is a compound obtained by introducing a phenol derivative into a polymer hyaluronic acid having a molecular weight of 1000 kDa to 1500 kDa.

4. In paragraph 2, A hydrogel composition wherein the polymer containing the above phenol derivative is present at a concentration of 3% to 6% (w / v).

5. In paragraph 1, A hydrogel composition wherein the above tyrosinase is derived from Streptomyces avermitilis.

6. In paragraph 1, A hydrogel composition wherein the phenol derivative is tyrosine, tyramine, dopamine, pyrogallol, caffeic acid, 3,4-dihydroxybenzylamine, 4-hydroxyphenylacetic acid or 3-(4-hydroxyphenyl)propionic acid.

7. In paragraph 1, A hydrogel composition wherein the phenol derivative is tyrosine or tyramine.

8. In paragraph 1, A hydrogel composition, wherein the adhesiveness of the composition is controlled by washing with a bovine serum albumin (BSA), penicillamine or cysteine ​​solution.

9. A step of mixing a polymer containing decellularized extracellular matrix (dECM) and a phenol derivative with tyrosinase derived from Streptomyces to obtain a pre-gelling composition; and A method for producing a paintable tissue regeneration hydrogel composition, comprising a step of leaving the above-mentioned pre-gelling composition for 10 to 20 minutes to obtain a gelled hydrogel composition.

10. In paragraph 9, A method for producing a hydrogel composition, wherein the hydrogel composition has a storage modulus (G') of 11 to 13 kPa in a dry state and 4 to 6 kPa in a swollen state according to an amplitude and frequency sweep measurement method.

11. In paragraph 9, A method for producing a hydrogel composition, wherein the hydrogel composition has a viscosity of 100 to 300 Pa.s.

12. In paragraph 9, A method for producing a hydrogel composition, wherein the composition before gelation exhibits a shear stress of 0.6 to 0.9 kPa and a tensile strength of 20 to 30 kPa within 1 hour after mixing, as measured according to a viscoelasticity measurement method.

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