An adhesive hydrogel, its preparation method and application

Abstract

The present application relates to the technical field of biomedical materials, and particularly relates to an adhesive hydrogel and a preparation method and application thereof. The adhesive hydrogel comprises a hydrogel and exosomes loaded in the hydrogel, the hydrogel is formed by konjac glucomannan and alpha-lipoic acid, and the exosomes internally load small interfering RNA targeting ACTC1 genes. The adhesive hydrogel of the present application adopts specific konjac glucomannan and alpha-lipoic acid to form a hydrogel, has good biocompatibility, no toxic side effects, good adhesion, can effectively reduce tissue tension, and can gradually degrade under physiological conditions to achieve slow release of the exosomes loaded internally; meanwhile, the slow-released exosomes internally load small interfering RNA targeting ACTC1 genes, which can inhibit the proliferation and migration of keloid fibroblasts and can inhibit the formation of keloids in vivo, so that seamless healing after keloid surgery can be achieved, and the adhesive hydrogel can be well used in the treatment of keloids.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to an adhesive hydrogel, its preparation method, and its applications. Background Technology

[0002] Keloids are a common benign skin tumor, clinically manifesting as large, hard, raised scars that occur after skin injury, accompanied by itching and pain. They can easily lead to cosmetic deformities, impaired daily functioning, reduced quality of life, and psychological burdens such as anxiety and depression. Keloids commonly occur in areas of high tension, such as the chest, shoulders, back, and earlobes.

[0003] Traditional treatments for keloids, such as intralesional injection of corticosteroids, 5-fluorouracil, or bleomycin, combined with surgical excision and radiation therapy, are often ineffective, time-consuming, expensive, and have significant side effects, including erythema, edema, keloid progression, and even malignant transformation. Surgical excision is the preferred treatment; however, increased suture tension and suture irritation after surgery can lead to lesion recurrence. Current combined surgical methods, such as tension-reducing sutures and tension-reducing dressings, also have unsatisfactory results. Therefore, there is an urgent need to develop effective tension-reducing closure techniques for keloid surgery, providing a new strategy for keloid treatment. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the purpose of this invention is to provide an adhesive hydrogel, its preparation method and application, in order to solve the problem of unsatisfactory efficacy of existing postoperative tension-reducing treatments for keloids.

[0005] The technical solution of the present invention is as follows:

[0006] In a first aspect, the present invention provides an adhesive hydrogel comprising a hydrogel and exosomes loaded within the hydrogel, the hydrogel being formed of konjac glucomannan and α-lipoic acid, the exosomes being internally loaded with small interfering RNA targeting the ACTC1 gene.

[0007] Optionally, the nucleotide sequence of the sense strand of the small interfering RNA targeting the ACTC1 gene is SEQ ID NO.1, and the nucleotide sequence of the antisense strand of the small interfering RNA targeting the ACTC1 gene is SEQ ID NO.2.

[0008] Optionally, the nucleotide sequence in the ACTC1 gene targeted by the small interfering RNA is SEQ ID NO.3.

[0009] Optionally, the exosomes are mesenchymal stem cell exosomes.

[0010] A second aspect of the present invention provides a method for preparing the adhesive hydrogel described herein, comprising:

[0011] Step S1: Mix exosomes, small interfering RNA targeting the ACTC1 gene, exosome transfection reagent and buffer for the first incubation, then add exosome extraction reagent for the second incubation, and then centrifuge and resuspend to obtain exosomes loaded with small interfering RNA targeting the ACTC1 gene.

[0012] Step S2: Dissolve konjac glucomannan in water to prepare a solution, then add α-lipoic acid, catalyst and condensing agent, mix and stir to react, then dialyze and freeze dry to obtain konjac glucomannan-α-lipoic acid;

[0013] Step S3: Dissolve the konjac glucomannan-α-lipoic acid obtained in step S2 in water to prepare a solution, then add the exosomes obtained in step S1 that are internally loaded with small interfering RNA targeting the ACTC1 gene, mix and stir, and then irradiate with light to obtain the adhesive hydrogel.

[0014] Optionally, in step S1, the conditions for the first incubation include: an incubation temperature of 37°C and an incubation time of 10 min;

[0015] The conditions for the second incubation include: an incubation temperature of 0°C and an incubation time of 30 minutes.

[0016] Optionally, in step S2, the catalyst is 4-dimethylaminopyridine, the condensing agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and the mass ratio of konjac glucomannan, α-lipoic acid, 4-dimethylaminopyridine and 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 25:46.25:1:43.

[0017] Optionally, in step S2, the conditions for the mixing and stirring reaction include: a reaction temperature of room temperature and a reaction time of 24 hours.

[0018] Optionally, in step S3, the conditions for the mixing and stirring reaction include: the reaction temperature is room temperature and the reaction time is 5 min;

[0019] The conditions for light irradiation include: a light wavelength of 365nm and a light power density of 10mW / cm². 2 The irradiation time is 5 minutes.

[0020] A third aspect of the present invention provides the use of the adhesive hydrogel described herein in the preparation of a medicament for treating keloids.

[0021] Beneficial Effects: This invention provides an adhesive hydrogel formed using specific konjac glucomannan and α-lipoic acid. It exhibits good biocompatibility, no toxic side effects, excellent adhesion, and effectively reduces tissue tension. Its three-dimensional structure provides ample space for loaded exosomes, allowing for gradual degradation under physiological conditions to achieve sustained release of the internally loaded exosomes. Simultaneously, the sustained-released exosomes are loaded with small interfering RNA targeting the ACTC1 gene, which inhibits the proliferation and migration of keloid fibroblasts and suppresses keloid formation in vivo. This enables seamless healing after keloid surgery, making it well-suited for keloid treatment. Furthermore, the preparation method of the provided adhesive hydrogel is simple, requires minimal equipment, and is easily mass-produced. Attached Figure Description

[0022] Figure 1 This is a scanning electron microscope image of the siACTC1-Exo composite adhesive hydrogel in Example 1.

[0023] Figure 2 The image shows a scanning electron microscope (SEM) image of siACTC1-Exo obtained in step 2 of Example 1.

[0024] Figure 3 The Fourier transform infrared spectra of the konjac glucomannan powder in Example 1 and the konjac glucomannan-α-lipoic acid powder obtained in step 3 are shown.

[0025] Figure 4 The image shows the immunofluorescence of the siACTC1-Exo composite adhesive hydrogel labeled with PKH26 in Example 2.

[0026] Figure 5 The figures show the cytotoxicity of the PBS control group, the exosome Exo treatment group, and the adhesive hydrogel treatment group in Example 3.

[0027] Figure 6 This is a diagram showing the migration of fibroblasts in keloids in the PBS control group and the siACTC1-Exo composite adhesive hydrogel treatment group in Example 4.

[0028] Figure 7 This image shows apoptosis of fibroblasts in keloids from the PBS control group and the siACTC1-Exo composite adhesive hydrogel treatment group in Example 5. Detailed Implementation

[0029] This invention provides an adhesive hydrogel, its preparation method, and its applications. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0030] For postoperative treatment of keloids, reducing postoperative tension during closure is crucial to preventing recurrence. However, current methods such as tension-reducing sutures and tension-reducing dressings have limited efficacy. Recent biomechanical studies have revealed that tension promotes keloid development by influencing the cytoskeleton. Research shows that mechanical tension stimulates fibroblast cytoskeleton stretching and reorganization, thereby activating cell proliferation and extracellular matrix synthesis, ultimately promoting keloid formation. Actin Alpha Cardiac Muscle 1 (ACTC1) is a crucial cytoskeleton protein that regulates cell proliferation and migration. Furthermore, small interfering RNA (siRNA) is a type of double-stranded RNA typically 20-25 nucleotides in length. Once in vivo, it interferes with mRNA translation, silencing the protein synthesis of the gene. siRNA carries a negative surface charge, has poor cell membrane permeability, and is easily degraded by endonucleases and cleared by the immune system. Therefore, specific carriers are needed to enhance cellular uptake and prolong its release. Compared to other carriers, mesenchymal stem cell exosomes (Exo) possess natural cell targeting capabilities and are free of toxic side effects and immunogenicity, enabling them to carry and target siRNA to cells. Based on this, this invention designs and develops an adhesive hydrogel loaded with siACTC1-Exo and konjac glucomannan-α-lipoic acid, which inhibits the proliferation and migration of keloid fibroblasts, achieving seamless suture healing.

[0031] In this article, the small interfering RNA targeting the ACTC1 gene is referred to as siACTC1, and the exosomes containing the small interfering RNA targeting the ACTC1 gene are referred to as siACTC1-Exo.

[0032] This invention provides an adhesive hydrogel comprising a hydrogel and exosomes loaded within the hydrogel. The hydrogel is formed from konjac glucomannan and α-lipoic acid, and the exosomes are loaded with small interfering RNA targeting the ACTC1 gene.

[0033] In this embodiment, a hydrogel is formed by using specific konjac glucomannan and α-lipoic acid. This hydrogel exhibits good biocompatibility, no toxic side effects, and good adhesion, effectively reducing tissue tension. Its three-dimensional structure provides ample space for loaded exosomes, allowing for gradual degradation under physiological conditions to achieve sustained release of the internally loaded exosomes. Simultaneously, the sustained-released exosomes are loaded with small interfering RNA targeting the ACTC1 gene, which inhibits the expression of cardiac actin α1, thereby inhibiting the proliferation and migration of keloid fibroblasts and suppressing keloid formation in vivo. This enables seamless healing after keloid surgery, making it highly effective in keloid treatment and possessing significant social and clinical application value.

[0034] The adhesive hydrogel provided in this invention has at least the following advantages:

[0035] (1) Good biocompatibility and no toxic side effects: Unlike traditional medical tissue adhesives, the adhesive hydrogel of the present invention is composed of konjac glucomannan and α-lipoic acid. Konjac glucomannan, as a natural material, has good biocompatibility with organisms and degrades in the body; α-lipoic acid is a fatty acid that is naturally present in every cell of the human body, so it has the advantages of good biocompatibility and no toxic side effects.

[0036] (2) Good adhesion: Compared with the mechanical tension and foreign body stimulation of sutures, the adhesive hydrogel of the present invention can quickly close wounds, effectively reduce tissue mechanical tension, and has no rejection reaction. The adhesive strength of the gel is mainly determined by its biological adhesive properties and cohesive force. The aldehyde group of konjac glucomannan in the adhesive hydrogel of the present invention can form Schiff base with the amino group of the tissue to produce tissue adhesion, and the hydroxyl group on the main chain of konjac glucomannan has a complexation effect with calcium ions to enhance cohesive force, thus exhibiting excellent adhesion.

[0037] (3) Sustained release: The hydrogel has a three-dimensional structure, which can provide sufficient space for the loaded exosomes. Under physiological conditions, it can be gradually degraded to achieve sustained release of exosomes.

[0038] (4) Inhibits keloid proliferation: The siACTC1 loaded inside the exosome can inhibit the expression of myocardial actin α1, thereby inhibiting the proliferation and migration of keloid fibroblasts, and can also inhibit the formation of keloids in vivo.

[0039] In some embodiments, the nucleotide sequence of the sense strand of the small interfering RNA targeting the ACTC1 gene is SEQ ID NO.1, and the nucleotide sequence of the antisense strand of the small interfering RNA targeting the ACTC1 gene is SEQ ID NO.2.

[0040] The small interfering RNA targeting the ACTC1 gene used in this invention was designed and synthesized by Guangzhou Ruibo Biotechnology Co., Ltd., and its nucleotide sequences of the sense and antisense strands are as follows:

[0041] Sense strand: 5'-GAAGGACUCCUACGUAGGU-dTdT-3' (SEQ ID NO.1);

[0042] Antisense strand: 5'-ACCUACGUAGGAGUCCUUC-dTdT-3' (SEQ ID NO.2).

[0043] In some embodiments, the nucleotide sequence in the ACTC1 gene targeted by the small interfering RNA is SEQ ID NO. 3.

[0044] The nucleotide sequence of SEQ ID NO.3 is as follows:

[0045] GAAGGACTCCTACGTAGGT.

[0046] In some embodiments, the exosomes are mesenchymal stem cell exosomes. These mesenchymal stem cell exosomes can be mesenchymal stem cell exosomes from different sources (such as bone marrow, adipose tissue, or umbilical cord blood) or extracellular vesicles such as liposomes.

[0047] This invention provides a method for preparing the adhesive hydrogel described in any of the foregoing embodiments, comprising:

[0048] Step S1: Mix exosomes, small interfering RNA targeting the ACTC1 gene, exosome transfection reagent and buffer for the first incubation, then add exosome extraction reagent for the second incubation, and then centrifuge and resuspend to obtain exosomes loaded with small interfering RNA targeting the ACTC1 gene.

[0049] Step S2: Dissolve konjac glucomannan in water to prepare a solution, then add α-lipoic acid, catalyst and condensing agent, mix and stir to react, then dialyze and freeze dry to obtain konjac glucomannan-α-lipoic acid;

[0050] Step S3: Dissolve the konjac glucomannan-α-lipoic acid obtained in step S2 in water to prepare a solution, then add the exosomes obtained in step S1 that are internally loaded with small interfering RNA targeting the ACTC1 gene, mix and stir, and then irradiate with light to obtain the adhesive hydrogel.

[0051] In step S1, in some embodiments, the exosomes are bone marrow-derived mesenchymal stem cell exosomes, and the buffer solution is PBS buffer.

[0052] In some embodiments, the method for preparing the exosomes includes the steps of:

[0053] The mesenchymal stem cell culture medium was centrifuged at 2000g for 20 minutes, and then... 4 Centrifuge for 30 min at g, then centrifuge the supernatant at 1×10 g. 6 Centrifuge at g for 90 min, discard the supernatant after centrifugation, and resuspend the precipitate with PBS to obtain exosomes.

[0054] In some embodiments, the conditions for the first incubation include: an incubation temperature of 37°C and an incubation time of 10 minutes.

[0055] In some embodiments, the conditions for the second incubation include: an incubation temperature of 0°C and an incubation time of 30 minutes.

[0056] In step S2, in some embodiments, the catalyst is 4-dimethylaminopyridine (DMAP) and the condensing agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC).

[0057] In some embodiments, the mass ratio of the konjac glucomannan, α-lipoic acid, 4-dimethylaminopyridine and 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 25:46.25:1:43.

[0058] In some embodiments, the conditions for the mixing and stirring reaction include: a reaction temperature of room temperature and a reaction time of 24 hours.

[0059] In some embodiments, the dialysis and freeze-drying are conventional techniques in the art and will not be described in detail here. Preferably, the dialysis conditions can be dialysis in water (preferably deionized water) for three days, and the freeze-drying conditions can be freeze-drying for three days.

[0060] In step S3, in some embodiments, the conditions for the mixing and stirring reaction include: a reaction temperature of room temperature and a reaction time of 5 minutes.

[0061] In some embodiments, the conditions for light irradiation include: a light wavelength of 365 nm and a light power density of 10 mW / cm². 2 The irradiation time is 5 minutes.

[0062] This invention provides an application of the adhesive hydrogel described in any of the foregoing embodiments in the preparation of a medicament for treating keloids.

[0063] This invention provides an application of the adhesive hydrogel described in any of the foregoing embodiments in the preparation of a drug for postoperative tension reduction in the treatment of keloids.

[0064] The adhesive hydrogel provided in this invention has excellent biocompatibility and adhesion, and can effectively reduce tissue tension and inhibit keloid proliferation. Therefore, it can be well used in the treatment of keloids, especially in the treatment of postoperative tension reduction of keloids.

[0065] The present invention will be further described below through specific embodiments.

[0066] Example 1

[0067] This embodiment provides a method for preparing an adhesive hydrogel (siACTC1-Exo composite adhesive hydrogel), as detailed below:

[0068] Step 1, Preparation of exosomes (Exo): First, centrifuge 500 mL of bone marrow mesenchymal stem cell culture medium at 2000 g for 20 min, then... 4 Centrifuge for 30 min at g, then centrifuge the supernatant at 1×10 g. 6 After centrifugation for 90 min under the given conditions, the separated exosomes Exo were resuspended in 100 μL of PBS buffer.

[0069] Step 2, Preparation of siACTC1-Exo: Using Exo-Fect TM The Exosome Transfection Kit (purchased from System Biosciences, catalog number EXO-FECT-1) was used to mix 150 μL of the transfection reaction system: 10 μL of exosome transfection reagent + 20 μL of siACTC1 (purchased from Guangzhou Ruibo Biotechnology Co., Ltd.) + 70 μL of PBS buffer + 50 μL of Exo obtained in step 1; then incubate at 37°C for 10 min; then add 30 μL of ExoQuick-TC (exosome extraction reagent) and incubate at 0°C on ice for 30 min; then centrifuge at 14000 rpm for 3 min, discard the supernatant, and resuspend in 300 μL of PBS buffer to obtain siACTC1-Exo.

[0070] Step 3: Preparation of konjac glucomannan-α-lipoic acid: First, 1g of konjac glucomannan was dissolved in 140mL of deionized water at 130℃. Then, 0.04g of 4-dimethylaminopyridine (DMAP) was added and reacted for 1h. Next, 1.85g of α-lipoic acid and 1.72g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) were added. After stirring the reaction at room temperature for 24h, it was dialyzed in deionized water for three days (MWCO: 3.5kDa). Finally, the reaction mixture was freeze-dried for three days to obtain konjac glucomannan-α-lipoic acid.

[0071] Step 4: Preparation of siACTC1-Exo composite adhesive hydrogel: Dissolve 0.1 g of konjac glucomannan-α-lipoic acid powder obtained in Step 3 in 4 mL of deionized water to obtain a 2.5% (w / v) konjac glucomannan-α-lipoic acid solution; then add 300 μL of siACTC1-Exo obtained in Step 2, stir at room temperature for 5 min, and let stand at 4 °C. Finally, examine the hydrogel using 365 nm light (light power density of 10 mW / cm²). 2 Irradiate the solution for 5 minutes to obtain siACTC1-Exo composite adhesive hydrogel.

[0072] Figure 1 This is a scanning electron microscope image of the siACTC1-Exo composite adhesive hydrogel. According to the image, both the konjac glucomannan-α-lipoic acid and siACTC1-Exo composite adhesive hydrogel exhibit a porous network structure, and the addition of siACTC1-Exo does not change the network structure of the hydrogel.

[0073] Figure 2 The image shows a scanning electron microscope (SEM) image of siACTC1-Exo obtained in step 2. According to the image, the morphological structure of siACTC1-Exo is complete.

[0074] Figure 3 The Fourier transform infrared spectra of konjac glucomannan powder and konjac glucomannan-α-lipoic acid powder obtained in step 3 are shown. According to the figure, the signals of methyl and methylene groups of cycloalkyl groups can be observed at 1.2-1.6 ppm, and the signal of methylene protons adjacent to disulfide bonds can be seen at 2.0-3.0 ppm. This indicates that α-lipoic acid has successfully bound to konjac glucomannan.

[0075] Example 2

[0076] This embodiment investigates the distribution of siACTC1-Exo in the siACTC1-Exo composite adhesive hydrogel prepared in Example 1, as detailed below:

[0077] 300 μL of siACTC1-Exo obtained in step 2 of Example 1 was mixed with the working solution of PKH26 dye at a ratio of 1 μL of PKH26 dye per 100 μL of exosome suspension. After gentle mixing, the mixture was incubated at room temperature for 5 min. Subsequently, to terminate the labeling reaction, an equal volume of 1% BSA (bovine serum albumin) solution or serum-free culture medium was added to the mixture, and incubation at room temperature was continued for 2 min. Finally, unbound PKH26 dye was removed by centrifugation at 10000g and 2-4℃ for 2 min to obtain fluorescently labeled siACTC1-Exo. Then, the fluorescently labeled siACTC1-Exo was added to a konjac glucomannan-α-lipoic acid solution and stirred at room temperature for 5 min, followed by exposure to 365 nm light (10 mW / cm²). 2 The solution was irradiated for 5 minutes to form a fluorescently labeled siACTC1-Exo composite adhesive hydrogel. The distribution of siACTC1-Exo in the hydrogel was then observed using a confocal microscope.

[0078] Figure 4 This is an immunofluorescence image of the PKH26-labeled siACTC1-Exo composite adhesive hydrogel. According to the image, the PKH26-labeled siACTC1-Exo is uniformly distributed in the hydrogel.

[0079] Example 3

[0080] This embodiment investigates the cytotoxicity of exosomes Exo and konjac glucomannan-α-lipoic acid adhesive hydrogels loaded with exosomes Exo, as detailed below:

[0081] Keloid fibroblasts were treated at a rate of 2×10 5 Up to 3×10 5 Cells were seeded at a density per well in 12-well plates. The PBS control group received no additional treatment. The exosome Exo treatment group received 10 μg / mL of exosome Exo (exosome Exo obtained in step 1 of Example 1), and the adhesive hydrogel treatment group received 10 μg / mL of konjac glucomannan-α-lipoic acid adhesive hydrogel loaded with exosome Exo (exosome Exo did not contain siACTC1). Cells were cultured for three days, followed by the addition of 10 μL of CCK-8 solution to each well and incubation for another 2 hours. The absorbance of each well at 450 nm was measured using a microplate reader to assess cell proliferation.

[0082] Figure 5The figure shows the cytotoxicity of the PBS control group, the exosome Exo treatment group, and the adhesive hydrogel treatment group. According to the figure, both the exosome Exo and the konjac glucomannan-α-lipoic acid adhesive hydrogel loaded with exosome Exo showed low cytotoxicity, indicating that they have good biocompatibility.

[0083] Example 4

[0084] This example investigates the effect of the siACTC1-Exo composite adhesive hydrogel prepared in Example 1 on inhibiting the migration of fibroblasts in keloids, as detailed below:

[0085] Transwell assay for cell migration: Transwell chambers were used for transwell assays in 24-well plates. The PBS control group received 700 μL of complete culture medium containing 10% exosome-free serum in the lower chamber. The siACTC1-Exo composite adhesive hydrogel treatment group received 700 μL of complete culture medium containing 10% exosome-free serum and 10 μg / mL of siACTC1-Exo composite adhesive hydrogel in the lower chamber. Then, keloid fibroblasts (8 × 10⁶ cells / well) were... 4 Cells (per well) were seeded into the upper chamber containing 200 μL of serum-free medium. After culturing for 24 hours, the chambers were washed with PBS and fixed with 4% PFA at room temperature for 15 minutes. After washing 2–3 times with PBS, the cells were stained with 0.1% crystal violet for 15 minutes. After washing 2–3 times with PBS, any remaining cells in the upper chamber were wiped away with a cotton swab. Images were observed and captured using an inverted fluorescence microscope.

[0086] Figure 6 The images show the migration of fibroblasts in keloids in the PBS control group and the siACTC1-Exo composite adhesive hydrogel treatment group. The left image is the PBS control group, and the right image is the siACTC1-Exo composite adhesive hydrogel treatment group. According to the images, the siACTC1-Exo composite adhesive hydrogel can effectively inhibit the migration of fibroblasts in keloids.

[0087] Example 5

[0088] This example investigates the effect of the siACTC1-Exo composite adhesive hydrogel prepared in Example 1 on promoting apoptosis of keloid fibroblasts, as detailed below:

[0089] First, the fibroblasts of the keloid were treated with 2×10 5 Up to 3×10 5Cells were seeded at a density of 10 μg / mL in 12-well plates. The PBS control group received no additional treatment, while the siACTC1-Exo composite adhesive hydrogel treatment group received 10 μg / mL of siACTC1-Exo composite adhesive hydrogel and cultured for three days. Cells were then collected by transferring the culture medium to centrifuge tubes, washing the cells once with PBS, digesting them with trypsin, and collecting the digested cells in centrifuge tubes. The collected culture medium and cells were then mixed and centrifuged at 4°C, 300g for 5 min, and the supernatant was discarded. The cells were then washed twice with pre-cooled PBS, centrifuged at 4°C, 300g for 5 min each time. 500 μL of fixative was added. 1 × 10⁻⁶ cells were then used. 5 Resuspend the cells in a flow cytometry tube, add 1 μL of annexin labeled with a fluorescent dye and 1 μL of propidium iodide, and mix gently. Incubate at room temperature in the dark for 15-20 min. Resuspend the cells in 300 μL of PBS and analyze them by flow cytometry within 1 hour.

[0090] Figure 7 The images show apoptosis of fibroblasts in keloids in the PBS control group and the siACTC1-Exo composite adhesive hydrogel treatment group. The left image is the PBS control group, and the right image is the siACTC1-Exo composite adhesive hydrogel treatment group. According to the images, the siACTC1-Exo composite adhesive hydrogel can effectively promote apoptosis of fibroblasts in keloids.

[0091] In summary, the adhesive hydrogel provided by this invention, formed using specific konjac glucomannan and α-lipoic acid, exhibits good biocompatibility, no toxic side effects, excellent adhesion, and effectively reduces tissue tension. Its three-dimensional structure provides ample space for loaded exosomes, allowing for gradual degradation under physiological conditions to achieve sustained release of the internally loaded exosomes. Simultaneously, the sustained-released exosomes are loaded with small interfering RNA targeting the ACTC1 gene, which inhibits the proliferation and migration of keloid fibroblasts and suppresses keloid formation in vivo, thereby achieving seamless healing after keloid surgery. Therefore, it is well-suited for the treatment of keloids. Furthermore, the preparation method of the provided adhesive hydrogel is simple, requires minimal equipment, and is easily mass-produced.

[0092] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. An adhesive hydrogel, characterized in that, The hydrogel and the exosome loaded inside the hydrogel, the hydrogel is formed by konjac glucomannan and alpha-lipoic acid, the inside of the exosome is loaded with small interfering RNA targeting ACTC1 gene; The nucleotide sequence of the sense strand of the small interfering RNA targeting ACTC1 gene is SEQ ID NO. 1, and the nucleotide sequence of the antisense strand of the small interfering RNA targeting ACTC1 gene is SEQ ID NO.

2.

2. The adhesive hydrogel of claim 1, wherein, The nucleotide sequence of the ACTC1 gene targeted by the small interfering RNA is SEQ ID NO.

3.

3. The adhesive hydrogel of claim 1, wherein, The exosome is a mesenchymal stem cell exosome.

4. A method of preparing the adhesive hydrogel of claim 1, wherein, It comprises: Step S1, mixing exosome, small interfering RNA targeting ACTC1 gene, exosome transfection reagent and buffer for the first incubation, then adding exosome extraction reagent for the second incubation, then centrifuging and resuspending to obtain exosome loaded with small interfering RNA targeting ACTC1 gene inside; Step S2, dissolving konjac glucomannan in water to prepare a solution, then adding alpha-lipoic acid, catalyst and condensing agent for mixing and stirring reaction, then dialysis and freeze-drying to obtain konjac glucomannan-alpha-lipoic acid; Step S3, dissolving konjac glucomannan-alpha-lipoic acid obtained in step S2 in water to prepare a solution, then adding exosome loaded with small interfering RNA targeting ACTC1 gene inside obtained in step S1 for mixing and stirring reaction, then light irradiation to obtain the adhesive hydrogel.

5. The method of preparing an adhesive hydrogel according to claim 4, wherein In the step S1, the conditions of the first incubation include: the incubation temperature is 37℃, and the incubation time is 10 min; The conditions of the second incubation include: the incubation temperature is 0℃, and the incubation time is 30 min.

6. The method of preparing an adhesive hydrogel according to claim 4, wherein In the step S2, the catalyst is 4-dimethylaminopyridine, the condensing agent is 1-ethyl-(3-dimethylaminopropyl) carbonyldiimide, and the mass ratio of konjac glucomannan, alpha-lipoic acid, 4-dimethylaminopyridine and 1-ethyl-(3-dimethylaminopropyl) carbonyldiimide is 25:46.25:1:

43.

7. The method of preparing an adhesive hydrogel according to claim 4, wherein In the step S2, the conditions of the mixing and stirring reaction include: the reaction temperature is room temperature, and the reaction time is 24 h.

8. The method of preparing an adhesive hydrogel according to claim 4, wherein In the step S3, the conditions of the mixing and stirring reaction include: the reaction temperature is room temperature, and the reaction time is 5 min; The conditions of the light irradiation include: light wavelength of 365 nm, light power density of 10 mW / cm 2 , and irradiation time of 5 min.

9. Use of the adhesive hydrogel of claim 1 in the preparation of a medicament for treating keloid.

Images