DNA hydrogel dressing with skin-like structure and function as well as preparation method and application of DNA hydrogel dressing

Abstract

The invention belongs to the technical field of medical dressings, and relates to a DNA hydrogel dressing with a skin-like structure and function as well as a preparation method and application of the DNA hydrogel dressing. The dressing is structurally characterized in that a skin-like three-layer structure, namely an epidermal layer, a corium layer and a subcutaneous tissue layer, is constructed by regulating and controlling the cross-linking degree of chain segments and combining a directional freezing technology, and an extracellular matrix microenvironment is provided for physiological activities of cells. In function, an aptamer sequence with a specific function is introduced, and stem cells are induced to directionally differentiate into cells required by tissue repair; the subcutaneous tissue-like layer is loaded with resveratrol, and fascia is activated to induce regeneration of skin appendages; in view of the fact that chronic ulcerative wounds are easily attacked by bacteria, the antibacterial peptide and a functional DNA sequence of targeted bacteria form an antibacterial peptide-DNA composite chain segment through a click reaction, and the dressing is endowed with targeted antibacterial performance. The prepared dressing has a skin-imitating three-dimensional structure and performance, can guide skin appendages such as regeneration of hair follicles and sweat glands, and can be widely applied to repair treatment of chronic wounds.

Description

Technical Field

[0001] This invention belongs to the field of medical dressing technology, specifically relating to DNA hydrogel dressings with skin-like structures and functions, their preparation methods, and applications. Background Technology

[0002] Globally, the cost of caring for non-healing skin wounds (commonly known as ulcers) is as high as US$13-15 billion per year, with the cost of caring for burn ulcers ranging from US$10.58 to US$125,597.86. Wound healing is regulated by the body's genes, and its healing process follows its own inherent rhythms. The body can tolerate a certain degree of adverse systemic or local factors. However, once systemic diseases or local pathological changes exceed the body's regulatory capacity, wound healing may be delayed or non-healing, resulting in an "out-of-control" situation in wound repair. Among all the factors leading to "out-of-control" and difficult-to-heal wounds, high-metabolic burns are one of the most concerning issues. The essence of difficult-to-heal burn wounds is the severe damage to the skin and appendages caused by deep burns, accompanied by complex pathophysiological processes such as inflammation and infection, microcirculatory disturbances, immune disorders, and wound contraction scar formation. These "harmful factors" intertwine, disrupting the normal tissue repair microenvironment, hindering the orderly healing of the wound, and ultimately leading to delayed or non-healing.

[0003] From a skin developmental perspective, wound healing is a result of the synergistic action of multiple cells. Cell therapy is a treatment method that utilizes cells. It involves transplanting normal or bioengineered cells into the patient's body, primarily aiming to replace damaged cells and enhance immune function to guide the body's adaptive regulation, thereby restoring the vitality or physiological function of damaged tissues. It is also one of the most effective methods for replacing non-homologous skin. Stem cells, extracted from the human body, possess regenerative, multi-directional differentiation, paracrine, and homing potential, and are considered an ideal seed source for tissue repair, reconstruction, and regeneration. Stem cells can reconstruct the tissue microenvironment at the site of injury, enhance the body's adaptive regulatory capacity, and promote the transmission of necessary healing factors to achieve tissue repair and regeneration. However, stem cells lack a clearly defined directional differentiation microenvironment, leading to low differentiation efficiency and uncontrolled structural reconstruction in wounds, making it difficult to form a functional layered structure of epidermis, dermis, and subcutaneous tissue. Worse still, the stemness of stem cells gradually diminishes during in vitro culture.

[0004] Furthermore, existing clinical tissue engineering scaffolds have limited functionality and typically cannot simulate the complex three-dimensional structure of the skin and the arrangement of cells requiring repair, making it difficult to guide tissue reconstruction. Meanwhile, commonly used clinical growth factor release systems suffer from difficulties in dosage control and high levels of side effects, making it difficult to precisely regulate the repair process and potentially inducing side effects such as fibrosis.

[0005] Therefore, based on the tissue repair mechanism, the required functional dressing should be able to ensure that pluripotent stem cells are directed to differentiate into the phenotype cells required for healing, so as to guide the body to adaptively regulate the microenvironment. This important principle will help to adopt reasonable regenerative medicine technologies and achieve truly perfect repair and regeneration. Summary of the Invention

[0006] To address the shortcomings of existing clinical medical dressings in terms of structure, functional specificity, and bioregulation, this invention provides a DNA hydrogel dressing with skin-like structure and function, its preparation method, and its application. The DNA hydrogel dressing provided by this invention aims to mimic the multi-layered structure of natural skin, regulate the directed differentiation of stem cells, activate fascia to promote the formation of skin appendages, and optimize the microenvironment of chronic wounds, thereby accelerating high-quality healing and tissue regeneration of refractory wounds.

[0007] This invention is achieved through the following technical solution: A method for preparing a DNA hydrogel dressing with skin-like structure and function, the method comprising: (1) Preparation of Y monomer: Mix the three DNA strands Y1, Y2 and Y3, place them in a PCR instrument and anneal them to obtain a Y monomer solution with a Y-type three-arm structure; (2) Preparation of L monomer: based on L-type linker chain L4 0 L-shaped connecting sub-chain L5 and derived segments L4, L4', L4'', L4''', and L5' were used to prepare L4. 0 / L5 dual-mixed monomer solution, L4 / L4' / L5 / L5' multi-mixed monomer solution, L4' / L4'' / L4''' / L5 / L5' multi-mixed monomer solution and L4' / L4'' / L5 / L5' multi-mixed monomer solution; (3) Preparation of skin-like epidermal hydrogel: Mix the Y monomer solution prepared in step (1) with the adipose-dermal mesenchymal stem cell solution to obtain solution A1; use the L4 prepared in step (2) 0 Solution B1 was prepared by mixing L5 dual-mixed monomer solution and L4 / L4' / L5 / L5' multi-mixed monomer solution; solution A1 and solution B1 were mixed to form a skin-like epidermal hydrogel; wherein, to ensure differentiation effect, stem cells of generation 6 (G6) or earlier are preferred.

[0008] (4) Preparation of skin-like dermal hydrogel: Mix the Y monomer solution prepared in step (1) with the adipose-dermal stem cell solution to obtain solution A2; use the L4 prepared in step (2) 0 Solution B2 was prepared by mixing L5 dual-mixed monomer solution and L4' / L4'' / L4''' / L5 / L5' multi-mixed monomer solution; solution A2 and solution B2 were mixed to form a skin-like dermal hydrogel; (5) Preparation of skin-like subcutaneous gel: Mix the Y monomer solution prepared in step (1) with the adipose-dermal mesenchymal stem cell solution to obtain solution A3; use the L4 prepared in step (2) 0 / L5 dual-mixed monomer solution and L4' / L4'' / L5 / L5' multi-mixed monomer solution are added to saturated resveratrol solution to obtain solution B3; solution A3 and solution B3 are mixed to form a skin-like subcutaneous gel. (6) Add the skin-like subcutaneous gel, skin-like dermal hydrogel, and skin-like epidermal hydrogel into the orientation mold in the order from bottom to top and solidify them; place the orientation mold horizontally and freeze it in an orientation to induce the lateral growth of ice crystals, forming a transversely arranged skin-like porous ordered structure, and obtain a DNA hydrogel dressing with skin-like structure and function.

[0009] Further, step (1) specifically involves mixing the three DNA strands Y1, Y2, and Y3 in equal molar ratios, placing them in a PCR instrument, heating at 90-98℃ for 1-5 min, and then slowly annealing to 4-30℃ for 60-100 min to form a Y monomer solution with a Y-type three-arm structure. The nucleotide sequence of Y1 is shown in SEQ ID: NO.1, the nucleotide sequence of Y2 is shown in SEQ ID: NO.2, and the nucleotide sequence of Y3 is shown in SEQ ID: NO.3.

[0010] Further, in step (2), the L4 0 The preparation method of the L5 dual-mixed monomer solution is as follows: L4 0 DNA segments and L5 DNA segments were mixed in equimolar ratio and placed in a PCR instrument for annealing to form L4. 0 / L5 dual-mixed monomer solution; The L4 / L4' / L5 / L5' mixed monomer solution is prepared by mixing L4 DNA segments, L4' DNA segments, L5 DNA segments and L5' DNA segments in an equimolar ratio and placing them in a PCR instrument for annealing to form the L4 / L4' / L5 / L5' mixed monomer solution. The L4' / L4'' / L4''' / L5 / L5' mixed monomer solution is prepared by mixing L4' DNA segments, L4'' DNA segments, L4''' DNA segments, L5 DNA segments, and L5' DNA segments in an equimolar ratio and placing them in a PCR instrument for annealing to form the L4' / L4'' / L4''' / L5 / L5' mixed monomer solution. The L4' / L4'' / L5 / L5' mixed monomer solution is prepared by mixing L4' DNA segments, L4'' DNA segments, L5 DNA segments and L5' DNA segments in an equimolar ratio and placing them in a PCR instrument for annealing to form the L4' / L4'' / L5 / L5' mixed monomer solution. Among them, L4 0 The nucleotide sequences of the L4 DNA segment are shown in SEQ ID: NO.4, the L4' DNA segment in SEQ ID: NO.5, the L4' DNA segment in SEQ ID: NO.6, the L4'' DNA segment in SEQ ID: NO.7, the L4''' DNA segment in SEQ ID: NO.8, and the L5 DNA segment in SEQ ID: NO.9; the L5' derived segment is an antimicrobial peptide-L5 complex segment, and the amino acid sequence of the antimicrobial peptide is shown in SEQ ID: NO.10.

[0011] Furthermore, the preparation method of the L5' derivative segment is as follows: the alkyne-modified antimicrobial peptide and the 3'-terminal azide-modified L5 segment are mixed at a molar ratio of 1:(1.0~1.8), and a click catalyst copper(I) ions (Cu⁺, concentration of 30-55 µM) are added to form the L5' derivative segment.

[0012] Further, step (3) specifically involves: taking 1-5 μL of L1 monomer solution and adding 1-4 μL of adipose-derived mesenchymal stem cell solution to obtain solution A1; taking 1-5 μL of L4 0 The L5 dual-mixed monomer solution and 3-15 μL of the L4 / L4' / L5 / L5' multi-mixed monomer solution are mixed to obtain solution B1; solution A1 and solution B1 are mixed to form a skin-like epidermal hydrogel.

[0013] Furthermore, step (4) specifically involves: Take 2-10 μL of L4 monomer solution and add 1-4 μL of adipose-derived mesenchymal stem cell solution to obtain solution A2; take 2-10 μL of L4... 0 Solution B2 is obtained by mixing L5 dual-mixed monomer solution and 6-30 μL of L4' / L4'' / L4''' / L5 / L5' multi-mixed monomer solution; solution A2 and solution B2 are mixed to form a skin-like dermal hydrogel.

[0014] Further, step (5) specifically involves: taking 3-15 μL of L1 monomer solution and adding 1-4 μL of adipose-derived mesenchymal stem cell solution to obtain solution A3; taking 4-20 μL of L4 0Solution B3 is obtained by mixing L5 dual-mixed monomer solution, 6-30 μL of L4' / L4'' / L5 / L5' multi-mixed monomer solution and 2-10 μL of resveratrol solution with a concentration of 100-200 μmol / L; solution A3 and solution B3 are mixed to form a skin-like subcutaneous gel.

[0015] Furthermore, step (6) specifically involves: The skin-like subcutaneous gel, skin-like dermal hydrogel, and skin-like epidermal hydrogel are added to the orientation mold in a bottom-up order and stacked slowly in a vertical direction, with a solidification interval of 30 seconds to 1 minute. The orientation mold is then placed horizontally and directionally frozen at -40°C to -20°C for 12-24 hours to induce the lateral growth of ice crystals, forming a transversely arranged porous and ordered skin-like structure, thus obtaining a DNA hydrogel dressing with skin-like structure and function.

[0016] A DNA hydrogel dressing with skin-like structure and function, the DNA hydrogel dressing having a skin-like three-layer structure, namely, an epidermal layer, a dermal layer and a subcutaneous tissue layer; in the preparation process of the epidermal layer, dermal layer and subcutaneous tissue layer, specific functional DNA aptamer sequences are introduced respectively to induce stem cells to differentiate into cells required for tissue repair; In this invention, monomer solutions containing epidermal growth factor aptamer (EGF) and transforming growth factor β1 aptamer (TGF-β1) are added during the preparation of the epidermal layer; monomer solutions containing transforming growth factor β1 aptamer (TGF-β1), vascular endothelial growth factor aptamer (VEGF), and basic fibroblast growth factor aptamer (bFGF) are added during the preparation of the dermal layer; and monomer solutions containing transforming growth factor β1 aptamer (TGF-β1) and vascular endothelial growth factor aptamer (VEGF) are added during the preparation of the subcutaneous tissue layer. This invention achieves the directed differentiation capability of stem cells by modifying four functional aptamers into L4-type connectives, namely L4, L4', L4'', and L4''', respectively, and by designing different functional aptamers in the L4 connectives and adding them to different structural layers.

[0017] The total concentration of DNA segments in the DNA hydrogel dressing is 2-15 mM. At this concentration, the elastic modulus of the DNA hydrogel dressing is similar to that of human skin (4.5-8 kPa) and the physiological microenvironment of chronic wounds (0.1-10 kPa).

[0018] The application of DNA hydrogel dressings involves using the DNA hydrogel dressings for the development of skin appendages in acute or chronic wounds, including for the regeneration of hair follicles, sweat glands, and sebaceous glands, or for the repair and treatment of diabetic ulcers, burns, pressure sores, or chronic refractory wounds.

[0019] Beneficial technical effects of the present invention: (1) This invention constructs a DNA hydrogel system with a skin-like three-layer biomimetic ordered structure; by precisely controlling the length, sequence and cross-linking mode of DNA segments, combined with directional cryo-molding technology, a three-layer biomimetic structure of epidermis, dermis and subcutaneous layer is prepared, which achieves a high degree of matching with natural skin in terms of three-dimensional tissue structure, elastic modulus and viscoelasticity and other physical and mechanical properties, providing effective structural support and microenvironment regulation basis for wound repair process.

[0020] (2) The DNA hydrogel dressing provided by the present invention introduces functional aptamers into DNA segments to induce stem cells to differentiate into different skin cell phenotypes, effectively mimics the tissue development mechanism, reconstructs the regenerative microenvironment of the wound area, and enhances the tissue's self-repair ability.

[0021] (3) The DNA hydrogel dressing provided by the present invention achieves stable loading in the subcutaneous tissue layer based on the π-π stacking effect between DNA segments and resveratrol, and controls the slow release of resveratrol in the subcutaneous layer, thereby activating the fascia during tissue regeneration, promoting the regeneration of skin appendages such as hair follicles, sebaceous glands and sweat glands, and accelerating the high-quality and intact healing of wounds.

[0022] (4) This invention significantly enhances the protective ability of hydrogels in infectious chronic wound environments by integrating antimicrobial peptides into DNA hydrogel systems, thereby reducing the risk of drug-resistant strains growing.

[0023] (5) Based on the excellent sequence designability and high-precision molecular recognition capability of DNA molecules, the DNA hydrogel constructed by this invention has unique characteristics. At the same time, the DNA hydrogel dressing provided by this invention has high compositional purity, good biocompatibility, and low immunogenicity, and has good clinical application prospects. It is suitable for the repair and treatment of diabetic ulcers, burns, pressure sores, and chronic non-healing wounds. Attached Figure Description

[0024] Figure 1 This describes the principle of DNA hydrogel synthesis mechanism in the embodiments of the present invention.

[0025] Figure 2 This is a morphological characterization of the three-layer ordered structure of the DNA hydrogel in this embodiment of the invention. (a) and (b) show the layered microstructure of the DNA hydrogel, (c) is an enlarged view of the skin-like structural layer, and (d) shows that stem cells can deliver factors within this three-dimensional network structure.

[0026] Figure 3 In this embodiment of the invention, cell immunofluorescence is used to assess stem cell stemness, wherein DAPI labels the cell nucleus, CD29 identifies stem cells, and Nanog detects stemness markers.

[0027] Figure 4This is a performance evaluation of the directed differentiation of JB6 (Anti-Keratin marker protein), L929 (Anti-Vinculin marker protein), and HUVEC (CD31 marker protein) cells by co-culturing DNA hydrogel for 144 h in this embodiment of the invention.

[0028] Figure 5 This invention provides a performance evaluation of the directed differentiation of JB6 (Anti-Keratin marker protein), L929 (Anti-Vinculin marker protein), and HUVEC (CD31 marker protein) cells by co-culturing DNA hydrogel for 216 h.

[0029] Figure 6 In this embodiment of the invention, H&E staining was performed on mouse skin on days 5 and 28 to assess the development of hair follicles and sebaceous glands. On day 28, the functional DNA hydrogel / resveratrol / adipose mesenchymal stem cell (ADSC) group was compared with the control group (untreated group), and it was found that it promoted the regeneration of hair follicles and sebaceous glands.

[0030] Figure 7 This is a diagram showing the antibacterial effect of the DNA hydrogel against common pathogenic bacteria (Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginose) in the embodiments of the present invention (n=3). Detailed Implementation

[0031] The technical solution of the present invention will be further described below with reference to specific embodiments and accompanying drawings.

[0032] Based on the excellent biocompatibility, programmable structure, modifiability, designability, and molecular recognition capabilities of DNA molecules, this invention prepares a DNA hydrogel with a three-layer biomimetic structure consisting of an epidermal layer, a dermal layer, and a subcutaneous layer. This hydrogel possesses skin-like mechanical properties (hardness and viscoelasticity), a three-dimensional ordered network structure, and multiple biological functions. It can provide structural support and microenvironment regulation for chronic wounds, promoting the orderly occurrence of cell migration, differentiation, and tissue remodeling.

[0033] Example 1: A method for preparing a DNA hydrogel dressing with skin-like structure and function, such as Figure 1 As shown, the method includes: (1) Preparation of Y monomer: Mix the three DNA strands Y1, Y2 and Y3, place them in a PCR instrument and anneal them to obtain a Y monomer solution with a Y-type three-arm structure; (2) Preparation of L monomer: based on L-type linker chain L4 0 L-shaped connecting sub-chain L5 and derived segments L4, L4', L4'', L4''', and L5' were used to prepare L4.0 / L5 dual-mixed monomer solution, L4 / L4' / L5 / L5' multi-mixed monomer solution, L4' / L4'' / L4''' / L5 / L5' multi-mixed monomer solution and L4' / L4'' / L5 / L5' multi-mixed monomer solution; (3) Preparation of skin-like epidermal hydrogel: Mix the Y monomer solution prepared in step (1) with the adipose-dermal mesenchymal stem cell solution to obtain solution A1; use the L4 prepared in step (2) 0 Solution B1 was prepared by mixing L5 dual-mixed monomer solution and L4 / L4' / L5 / L5' multi-mixed monomer solution; solution A1 and solution B1 were mixed to form a skin-like epidermal hydrogel; (4) Preparation of skin-like dermal hydrogel: Mix the Y monomer solution prepared in step (1) with the adipose-dermal stem cell solution to obtain solution A2; use the L4 prepared in step (2) 0 Solution B2 was prepared by mixing L5 dual-mixed monomer solution and L4' / L4'' / L4''' / L5 / L5' multi-mixed monomer solution; solution A2 and solution B2 were mixed to form a skin-like dermal hydrogel; (5) Preparation of skin-like subcutaneous gel: Mix the Y monomer solution prepared in step (1) with the adipose-dermal mesenchymal stem cell solution to obtain solution A3; use the L4 prepared in step (2) 0 / L5 dual-mixed monomer solution and L4' / L4'' / L5 / L5' multi-mixed monomer solution are added to saturated resveratrol solution to obtain solution B3; solution A3 and solution B3 are mixed to form a skin-like subcutaneous gel. (6) Add the skin-like subcutaneous gel, skin-like dermal hydrogel, and skin-like epidermal hydrogel into the orientation mold in the order from bottom to top and solidify them; place the orientation mold horizontally and freeze it in an orientation to induce the lateral growth of ice crystals, forming a transversely arranged skin-like porous ordered structure, and obtain a DNA hydrogel dressing with skin-like structure and function.

[0034] In this embodiment, step (1) specifically involves mixing the three DNA strands Y1, Y2, and Y3 in an equimolar ratio (preferably 5-15 μL each), placing them in a PCR instrument, heating at 90-98°C for 1-5 min, and then slowly annealing to 4-30°C for 60-100 min to form a Y monomer solution with a Y-type three-arm structure. The nucleotide sequence of Y1 is shown in SEQ ID: NO.1, the nucleotide sequence of Y2 is shown in SEQ ID: NO.2, and the nucleotide sequence of Y3 is shown in SEQ ID: NO.3.

[0035] In this embodiment, in step (2), the L4 0 The preparation method of the L5 dual-mixed monomer solution is as follows: L40 DNA segments and L5 DNA segments were mixed in equimolar ratio and placed in a PCR instrument for annealing (95-20℃, 60-120 min) to form L4. 0 / L5 dual-mixed monomer solution; specifically, 12-60 μL of each DNA strand; The L4 / L4' / L5 / L5' mixed monomer solution is prepared as follows: L4 DNA segment, L4' DNA segment, L5 DNA segment and L5' DNA segment are mixed in equimolar ratio and placed in a PCR instrument for annealing (95-20℃, 60-120 min) to form L4 / L4' / L5 / L5' mixed monomer solution; specifically, 12-60 μL of each DNA segment is used. The L4' / L4'' / L4''' / L5 / L5' mixed monomer solution is prepared as follows: L4' DNA segments, L4'' DNA segments, L4''' DNA segments, L5 DNA segments, and L5' DNA segments are mixed in an equimolar ratio and placed in a PCR instrument for annealing (95-20℃, 60-120 min) to form the L4' / L4'' / L4''' / L5 / L5' mixed monomer solution; specifically, 12-60 μL of each DNA segment is used. The L4' / L4'' / L5 / L5' mixed monomer solution is prepared as follows: L4' DNA fragments, L4'' DNA fragments, L5 DNA fragments, and L5' DNA fragments are mixed in an equimolar ratio and placed in a PCR instrument for annealing (95-20℃, 60-120 min) to form the L4' / L4'' / L5 / L5' mixed monomer solution; specifically, 12-60 μL of each DNA fragment is used. Among them, L4 0 The nucleotide sequences of the L4 DNA segment are shown in SEQ ID: NO.4, the L4' DNA segment in SEQ ID: NO.5, the L4' DNA segment in SEQ ID: NO.6, the L4'' DNA segment in SEQ ID: NO.7, the L4''' DNA segment in SEQ ID: NO.8, and the L5 DNA segment in SEQ ID: NO.9; the L5' derived segment is an antimicrobial peptide-L5 complex segment, and the amino acid sequence of the antimicrobial peptide is shown in SEQ ID: NO.10.

[0036] Furthermore, the L4 DNA segment contains the epidermal growth factor aptamer (EGF); the nucleotide sequence of the EGF aptamer is shown in SEQ ID: NO.11; the L4' DNA segment contains the transforming growth factor β1 aptamer (TGF-β1); the nucleotide sequence of the TGF-β1 aptamer is shown in SEQ ID: NO.12; the L4'' DNA segment contains the vascular endothelial growth factor aptamer (VEGF); the nucleotide sequence of the VEGF aptamer is shown in SEQ ID: NO.13; and the L4''' DNA segment contains the basic fibroblast growth factor aptamer (bFGF); the nucleotide sequence of the bFGF aptamer is shown in SEQ ID: NO.14. The nucleotide sequences of the above DNA segments and related aptamers are shown in Table 1.

[0037] Table 1. Sequence List of DNA Segments, Related Aptamers, and Antimicrobial Peptides In this embodiment, the L5' derivative segment preparation method is as follows: the alkyne-modified antimicrobial peptide (alkynyl-LHISIKFP) and the 3'-terminal azide-modified L5 segment are mixed at a molar ratio of 1:(1.0~1.8), and a click catalyst copper(I) ions (Cu⁺, 30-55 µM) are added to form the L5' derivative segment; the L5' derivative segment is used to construct a skin-like structure to achieve broad-spectrum antimicrobial activity of the three-layer structure.

[0038] In this embodiment, step (3) specifically includes: Take 1-5 μL of L1 monomer solution and add 1-4 μL of adipose-derived mesenchymal stem cell solution to obtain solution A1; take 1-5 μL of L4 0 Solution B1 is obtained by mixing L5 dual-mixed monomer solution and 3-15 μL of L4 / L4' / L5 / L5' multi-mixed monomer solution; solution A1 and solution B1 are then mixed to form a skin-like epidermal hydrogel. The cell density of the adipose-dermal mesenchymal stem cell solution is approximately 1×10⁻⁶ cells / mL. 5 -1×10 6 cells / mL.

[0039] In this embodiment, step (4) specifically includes: Take 2-10 μL of L4 monomer solution and add 1-4 μL of adipose-derived mesenchymal stem cell solution to obtain solution A2; take 2-10 μL of L4... 0 Solution B2 is obtained by mixing L5 dual-mixed monomer solution and 6-30 μL of L4' / L4'' / L4''' / L5 / L5' multi-mixed monomer solution; solutions A2 and B2 are then mixed to form a skin-like dermal hydrogel; the cell density of the adipose-dermal stem cell solution is approximately 1×10⁻⁶. 5 -1×10 6 cells / mL.

[0040] In this embodiment, step (5) specifically involves: taking 3-15 μL of L1 monomer solution and adding 1-4 μL of adipose-derived mesenchymal stem cell solution to obtain solution A3; taking 4-20 μL of L4 0 Solution B3 is prepared by mixing L5 dual-mixed monomer solution, 6-30 μL of L4' / L4'' / L5 / L5' multi-mixed monomer solution, and 2-10 μL of resveratrol solution with a concentration of 100-200 μmol / L. Solution A3 and solution B3 are then mixed to form a skin-like subcutaneous gel. The cell density of the adipose-dermal mesenchymal stem cell solution is approximately 1 × 10⁻⁶ cells / mL. 5 -1×10 6 (cells / mL) In this embodiment, step (6) specifically includes: The skin-like subcutaneous gel, skin-like dermal hydrogel, and skin-like epidermal hydrogel are added to the orientation mold in a bottom-up order and stacked slowly in a vertical direction, with a solidification interval of 30 seconds to 1 minute. The orientation mold is then placed horizontally and directionally frozen at -40°C to -20°C for 12-24 hours to induce the lateral growth of ice crystals, forming a transversely arranged porous and ordered skin-like structure, thus obtaining a DNA hydrogel dressing with skin-like structure and function.

[0041] The DNA hydrogel dressing prepared in this embodiment was characterized by scanning electron microscopy (SEM) of the lyophilized structure: a cross section of the prepared lyophilized DNA hydrogel sample was taken, and after metal sputtering treatment, its surface and cross-sectional structure were observed using field emission scanning electron microscopy. Figure 2 The results showed that after freeze-drying, the hydrogel formed a multi-layered porous structure arranged along the surface of the skin-like structure. The pore size and pore network were similar to the dermis and epidermis of natural skin, and the pores were well connected, which was conducive to cell migration and nutrient exchange, fully verifying the formation and maintenance of the three-dimensional ordered multi-layered structure.

[0042] The DNA hydrogel dressing prepared in this embodiment was characterized for stem cell stemness: stem cells regulate immunity and angiogenesis through paracrine function, their homing ability enables them to migrate to the injury site, and their differentiation potential facilitates their transformation into the desired cell type to participate in tissue repair. Maintaining stem cell stemness is fundamental for them to perform these functions; loss of stemness can lead to premature cell differentiation or functional abnormalities, affecting healing outcomes. To evaluate the impact of different materials on stem cell stemness, adipose-derived mesenchymal stem cells were co-incubated with functional DNA hydrogel, and the expression of stemness markers was subsequently detected using immunofluorescence staining. Figure 3 The results showed that ADSC exhibited high-intensity positive expression of the stem cell marker (Nanog), indicating that the material can effectively maintain the stem cell nature and provide a key guarantee for the stem cells to continue to function in the chronic wound microenvironment.

[0043] The DNA hydrogel dressing prepared in this embodiment was characterized for stem cell-directed differentiation performance: one of the key characteristics of stem cell differentiation is the dynamic changes in cell morphology, cytoskeleton structure, and biomarker expression. ADSCs were co-cultured with functional DNA hydrogels (including hydrogels for keratinocyte differentiation, fibroblast differentiation, and vascular endothelial differentiation). Cell immunofluorescence staining was used to detect relevant phenotypic biomarkers before and after ADSC differentiation, and changes in cell morphology were observed. Figure 4 and Figure 5 The results showed that after 144 h of culture, the expression level of CD29, a surface stem cell marker, in ADSCs was significantly reduced. Simultaneously, significant changes occurred in cell morphology: keratinocytes gradually became spindle-shaped, fibroblasts changed from a flattened, multi-protruding shape to an interwoven star-shaped or polygonal shape, and vascular endothelial cells changed from a spindle-shaped to a rhomboid shape. By 216 h, the CD29 positive signal almost disappeared, and the morphological characteristics of the three types of differentiated cells became clearer and more stable; vascular endothelial cells even formed typical vascular lumen structures. These results indicate that DNA hydrogels with different functional layers have a good ability to induce stem cells to differentiate into keratinocytes, fibroblasts, and vascular endothelial cells, respectively, providing structural support and a biological signaling environment for tissue regeneration, dermal reconstruction, and angiogenesis, demonstrating promising application prospects.

[0044] The DNA hydrogel dressing prepared in this embodiment was used to verify its skin appendage regeneration function: To evaluate the effect of resveratrol loaded in the hydrogel on appendage regeneration, the functional DNA dressing prepared in this invention was applied to a mouse model of grade III burn skin wounds. Samples were taken on days 5 and 20 post-injury for H&E staining to observe tissue reconstructive activity. Figure 6 The results showed that by day 28 post-injury, skin appendages such as hair follicles and sebaceous glands had appeared in the experimental group, significantly better than in the control group. These results indicate that the dressing optimized the wound microenvironment, activated fascia tissue, and promoted the regeneration of skin appendages.

[0045] The antibacterial properties of the DNA hydrogel dressing prepared in this embodiment were tested: the antibacterial effect of the functional DNA hydrogel against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa was tested using the plate diffusion method. The DNA hydrogel containing the antimicrobial peptide complex was co-cultured with bacterial culture. Figure 7The results showed that the antimicrobial peptide-DNA hydrogel sample exhibited the best antibacterial effect against Pseudomonas aeruginosa, and also had significant inhibitory effects against Staphylococcus aureus and Escherichia coli, with significantly better antibacterial effects than the control sample without antimicrobial peptides. This indicates that the multifunctional DNA hydrogel can effectively kill common pathogens in wounds and reduce the risk of infection.

[0046] Example 2: A DNA hydrogel dressing with skin-like structure and function, prepared using the method described in Example 1. The DNA hydrogel dressing has a skin-like three-layer structure, namely, an epidermal layer, a dermal layer, and a subcutaneous tissue layer. During the preparation of the epidermal layer, dermal layer, and subcutaneous tissue layer, specific functional DNA aptamer sequences are introduced to induce stem cells to differentiate into cells required for tissue repair. In this invention, monomer solutions containing epidermal growth factor aptamer (EGF) and transforming growth factor β1 aptamer (TGF-β1) are added during the preparation of the epidermal layer; monomer solutions containing transforming growth factor β1 aptamer (TGF-β1), vascular endothelial growth factor aptamer (VEGF), and basic fibroblast growth factor aptamer (bFGF) are added during the preparation of the dermal layer; and monomer solutions containing transforming growth factor β1 aptamer (TGF-β1) and vascular endothelial growth factor aptamer (VEGF) are added during the preparation of the subcutaneous tissue layer. This invention achieves the directed differentiation capability of stem cells by modifying four functional aptamers into L4-type connectives, namely L4, L4', L4'', and L4''', respectively, and by designing different functional aptamers in the L4 connectives and adding them to different structural layers.

[0047] In this embodiment, the total concentration of DNA segments in the DNA hydrogel dressing is 2-15 mM. At this time, the elastic modulus of the DNA hydrogel dressing is similar to that of human skin (4.5-8 kPa) and the physiological microenvironment of chronic wounds (0.1-10 kPa).

[0048] Example 3: Application of the DNA hydrogel dressing prepared according to the method described in Example 1, the DNA hydrogel dressing is used for the regeneration of skin appendages in acute or chronic wounds, including for the regeneration of hair follicles, sweat glands and sebaceous glands, or for the repair and treatment of diabetic ulcers, burns, pressure sores or chronic refractory wounds.

[0049] The DNA hydrogel dressing provided by this invention, structurally, constructs a skin-like three-layer structure—epidermis, dermis, and subcutaneous tissue—by regulating the degree of cross-linking of DNA segments and combining it with directional cryotherapy, providing an extracellular matrix-like microenvironment for cellular physiological activities. Functionally, by introducing DNA aptamer sequences with specific functions, it induces stem cells to differentiate into cells required for tissue repair; resveratrol is loaded into the subcutaneous tissue-like layer of the DNA hydrogel to activate the fascia and induce the regeneration of skin appendages; given that chronic ulcerative wounds are susceptible to bacterial invasion, antimicrobial peptides and functional DNA sequences targeting bacteria are combined through a click reaction to form antimicrobial peptide-DNA complex segments, endowing the dressing with targeted antibacterial properties. The prepared DNA hydrogel dressing possesses both a skin-like three-dimensional structure and properties, and guides the regeneration of skin appendages, such as hair follicles and sweat glands. It has multiple functions including stem cell induction, antibacterial activity, mitochondrial activation, and appendage regeneration, and can be widely used in the repair and treatment of diabetic ulcers, burns, and other chronic, difficult-to-heal wounds.

[0050] The functionalized DNA hydrogel biomimetic dressing prepared by this invention not only simulates the multi-layered tissue structure of natural skin in terms of structure, but also integrates multiple functions such as stem cell regulation, antibacterial protection and skin appendage regeneration, showing excellent comprehensive performance and broad clinical translation prospects in the field of tissue repair and regenerative medicine.

[0051] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, modifications or variations can still be made to the technical solutions described above, and these modifications and variations all fall within the protection scope of the present invention.

Claims

1. A method for preparing a DNA hydrogel dressing with skin-like structure and function, characterized in that, The method includes: (1) Preparation of Y monomer: Mix the three DNA strands Y1, Y2 and Y3, place them in a PCR instrument and anneal them to obtain a Y monomer solution with a Y-type three-arm structure; (2) Preparation of L monomer: based on L-type linker chain L4 0 L-shaped connecting sub-chain L5 and derived segments L4, L4', L4'', L4''', and L5' were used to prepare L4. 0 / L5 dual-mixed monomer solution, L4 / L4' / L5 / L5' multi-mixed monomer solution, L4' / L4'' / L4''' / L5 / L5' multi-mixed monomer solution and L4' / L4'' / L5 / L5' multi-mixed monomer solution; (3) Preparation of skin-like epidermal hydrogel: Mix the Y monomer solution prepared in step (1) with the adipose-dermal mesenchymal stem cell solution to obtain solution A1; use the L4 prepared in step (2) 0 Solution B1 was prepared by mixing L5 dual-mixed monomer solution and L4 / L4' / L5 / L5' multi-mixed monomer solution; solution A1 and solution B1 were mixed to form a skin-like epidermal hydrogel; (4) Preparation of skin-like dermal hydrogel: Mix the Y monomer solution prepared in step (1) with the adipose-dermal stem cell solution to obtain solution A2; use the L4 prepared in step (2) 0 Solution B2 was prepared by mixing L5 dual-mixed monomer solution and L4' / L4'' / L4''' / L5 / L5' multi-mixed monomer solution; solution A2 and solution B2 were mixed to form a skin-like dermal hydrogel; (5) Preparation of skin-like subcutaneous gel: Mix the Y monomer solution prepared in step (1) with the adipose-dermal mesenchymal stem cell solution to obtain solution A3; use the L4 prepared in step (2) 0 / L5 dual-mixed monomer solution and L4' / L4'' / L5 / L5' multi-mixed monomer solution are added to saturated resveratrol solution to obtain solution B3; solution A3 and solution B3 are mixed to form a skin-like subcutaneous gel. (6) Add the skin-like subcutaneous gel, skin-like dermal hydrogel, and skin-like epidermal hydrogel into the orientation mold in the order from bottom to top and solidify them; place the orientation mold horizontally and freeze it in an orientation to induce the lateral growth of ice crystals, forming a transversely arranged skin-like porous ordered structure, and obtain a DNA hydrogel dressing with skin-like structure and function.

2. The method for preparing the DNA hydrogel dressing with skin-like structure and function according to claim 1, characterized in that, Step (1) is as follows: Mix the three DNA strands Y1, Y2 and Y3 in equal molar ratio, place them in a PCR instrument, heat at 90-98℃ for 1-5 min, and then slowly anneal to 4-30℃ for 60-100 min to form a Y monomer solution with a Y-type three-arm structure. The nucleotide sequence of Y1 is shown in SEQ ID: NO.1, the nucleotide sequence of Y2 is shown in SEQ ID: NO.2, and the nucleotide sequence of Y3 is shown in SEQ ID: NO.

3.

3. The method for preparing the DNA hydrogel dressing with skin-like structure and function according to claim 1, characterized in that, In step (2), the L4 0 The preparation method of the L5 dual-mixed monomer solution is as follows: L4 0 DNA segments and L5 DNA segments were mixed in equimolar ratio and placed in a PCR instrument for annealing to form L4. 0 / L5 dual-mixed monomer solution; The L4 / L4' / L5 / L5' mixed monomer solution is prepared by mixing L4 DNA segments, L4' DNA segments, L5 DNA segments and L5' DNA segments in an equimolar ratio and placing them in a PCR instrument for annealing to form the L4 / L4' / L5 / L5' mixed monomer solution. The L4' / L4'' / L4''' / L5 / L5' mixed monomer solution is prepared by mixing L4' DNA segments, L4'' DNA segments, L4''' DNA segments, L5 DNA segments, and L5' DNA segments in an equimolar ratio and placing them in a PCR instrument for annealing to form the L4' / L4'' / L4''' / L5 / L5' mixed monomer solution. The L4' / L4'' / L5 / L5' mixed monomer solution is prepared by mixing L4' DNA segments, L4'' DNA segments, L5 DNA segments and L5' DNA segments in an equimolar ratio and placing them in a PCR instrument for annealing to form the L4' / L4'' / L5 / L5' mixed monomer solution. Among them, L4 0 The nucleotide sequences of the L4 DNA segment are shown in SEQ ID: NO.4, the L4' DNA segment in SEQ ID: NO.5, the L4' DNA segment in SEQ ID: NO.6, the L4'' DNA segment in SEQ ID: NO.7, the L4''' DNA segment in SEQ ID: NO.8, and the L5 DNA segment in SEQ ID: NO.9; the L5' derived segment is an antimicrobial peptide-L5 complex segment, and the amino acid sequence of the antimicrobial peptide is shown in SEQ ID: NO.

10.

4. The method for preparing the DNA hydrogel dressing with skin-like structure and function according to claim 3, characterized in that, The preparation method of L5' derivative segments is as follows: the alkyne-modified antimicrobial peptide and the 3'-terminal azide-modified L5 segment are mixed at a molar ratio of 1:(1.0~1.8), and the click catalyst copper(I) ions are added to form L5' derivative segments.

5. The method for preparing the DNA hydrogel dressing with skin-like structure and function according to claim 3, characterized in that, Step (3) specifically involves: taking 1-5 μL of L1 monomer solution and adding 1-4 μL of adipose-derived mesenchymal stem cell solution to obtain solution A1; taking 1-5 μL of L4 0 The L5 dual-mixed monomer solution and 3-15 μL of the L4 / L4' / L5 / L5' multi-mixed monomer solution are mixed to obtain solution B1; solution A1 and solution B1 are mixed to form a skin-like epidermal hydrogel.

6. The method for preparing the DNA hydrogel dressing with skin-like structure and function according to claim 1, characterized in that, Step (4) is as follows: Take 2-10 μL of L4 monomer solution and add 1-4 μL of adipose-derived mesenchymal stem cell solution to obtain solution A2; take 2-10 μL of L4... 0 The L5 dual-mixed monomer solution and 6-30 μL of L4' / L4'' / L4''' / L5 / L5' multi-mixed monomer solution are mixed to obtain solution B2; Mixing solutions A2 and B2 forms a skin-like dermal hydrogel.

7. The method for preparing the DNA hydrogel dressing with skin-like structure and function according to claim 1, characterized in that, Step (5) specifically involves: taking 3-15 μL of L1 monomer solution and adding 1-4 μL of adipose-derived mesenchymal stem cell solution to obtain solution A3; taking 4-20 μL of L4 0 Solution B3 is obtained by mixing L5 dual-mixed monomer solution, 6-30 μL of L4' / L4'' / L5 / L5' multi-mixed monomer solution and 2-10 μL of resveratrol solution with a concentration of 100-200 μmol / L. Mixing solutions A3 and B3 forms a skin-like subcutaneous gel.

8. The method for preparing a DNA hydrogel dressing with skin-like structure and function according to claim 1, characterized in that, Step (6) specifically involves: The skin-like subcutaneous gel, skin-like dermal hydrogel, and skin-like epidermal hydrogel are added to the orientation mold in a bottom-up order and stacked slowly in a vertical direction, with a solidification interval of 30 seconds to 1 minute. The orientation mold is then placed horizontally and directionally frozen at -40°C to -20°C for 12-24 hours to induce the lateral growth of ice crystals, forming a transversely arranged porous and ordered skin-like structure, thus obtaining a DNA hydrogel dressing with skin-like structure and function.

9. A DNA hydrogel dressing with skin-like structure and function, prepared by the method according to any one of claims 1-8, characterized in that, The DNA hydrogel dressing has a skin-like three-layer structure, namely the epidermis, dermis, and subcutaneous tissue layer; specific functional DNA aptamer sequences are introduced into the preparation process of the epidermis, dermis, and subcutaneous tissue layer to induce stem cells to differentiate into cells required for tissue repair. The total concentration of DNA segments in the DNA hydrogel dressing is 2-15 mM.

10. The application of the DNA hydrogel dressing prepared according to any one of claims 1-8, characterized in that, The DNA hydrogel dressing is used for the development of skin appendages in acute or chronic wounds, including for the regeneration of hair follicles, sweat glands, and sebaceous glands, or for the repair and treatment of diabetic ulcers, burns, pressure sores, or chronic refractory wounds.

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