Preparation and application of salvianic acid A sodium chitosan hydrogel and derivative thereof

Abstract

The invention relates to the technical field of biomedical materials, and discloses a salvianic acid A sodium chitosan hydrogel and preparation and application of derivatives of the salvianic acid A sodium chitosan hydrogel. Chitosan / salvianic acid A sodium (SDSS / CS) basic hydrogel serves as a core, and stable loading and controllable slow release of SDSS are achieved through hydrogen bond-static synergetic crosslinking. On the basis, chitin whiskers (CW) are introduced to prepare SDSS / CS / CW liquid crystal state double-layer composite hydrogel, and stage repair is realized by means of sequential degradation and structural guidance; furthermore, the M2 type macrophage exosome (M2-Exos) and the human umbilical cord mesenchymal stem cell exosome (hUCMSC-Exos) are loaded in a layered manner, so that a synergistic effect of physical guidance and biological regulation is formed. The hydrogel and the derivative thereof prepared by the invention have excellent micro-pores, anisotropy, antibacterial activity, immunoregulation and biocompatibility, and experiments show that the hydrogel and the derivative thereof can effectively promote healing and tissue regeneration of full-thickness skin defect infected wounds, and have wide application prospects in the fields of tissue engineering scaffolds and medical dressings.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials and tissue engineering technology, specifically to the preparation and application of a sodium tanshinone chitosan hydrogel and its derivatives. Background Technology

[0002] Skin wound repair, especially the repair of refractory wounds such as full-thickness skin defects, is a significant challenge in the fields of clinical biomedicine and tissue engineering. These wounds undergo complex and lengthy healing processes and are prone to complications such as infection, uncontrolled inflammation, and insufficient angiogenesis, ultimately leading to poor repair outcomes or disordered scar formation. Ideal wound repair materials must simultaneously meet the following requirements: safety and non-toxicity, ease of preparation, stable loading of active drugs, ability to guide orderly tissue regeneration, and suitability for the entire wound repair cycle from the inflammatory phase to the remodeling phase. However, existing repair materials struggle to achieve all these characteristics, necessitating the development of novel, multifunctional repair systems.

[0003] Chitosan (CS) is a natural cationic polysaccharide primarily derived from the exoskeletons of crustaceans such as shrimp and crab. As a renewable resource, CS possesses excellent biocompatibility, natural antibacterial properties, biodegradability, and low cost, thus finding wide application in sub-fields of biomedicine and tissue engineering, such as drug delivery, wound dressings, and tissue engineering scaffolds. However, existing chitosan-based hydrogels still face significant technical bottlenecks: on the one hand, CS itself is insoluble in water and has extremely poor solubility in neutral and alkaline environments. The preparation of existing CS hydrogels often relies on chemical modification or complex multi-step reactions with other materials, which not only significantly increases preparation costs and industrialization difficulty but may also introduce harmful substances due to chemical reagent residues, limiting its clinical translation; on the other hand, existing CS hydrogels are mostly isotropic and homogeneous structures, lacking topological guidance cues similar to the natural extracellular matrix (ECM), making it difficult to guide cell migration and proliferation. In the repair of full-thickness skin defects, this can easily lead to the formation of disordered scar tissue, severely affecting the repair effect. Therefore, there is an urgent need in the field of tissue engineering to develop a chitosan-based hydrogel system that is easy to prepare, safe and non-toxic, and can provide structural guidance.

[0004] Sodium Danshensu (SDSS) is the core water-soluble active ingredient of the traditional Chinese medicine Danshen (Salvia miltiorrhiza). It is the sodium salt of tanshinone and possesses well-defined pharmacological activities and a basis for clinical application. Its molecular structure contains an ortho-dihydroxy (1,2-cis-diol) benzene ring and a hydrophilic side chain, a specific structure that endows it with excellent anti-inflammatory, antioxidant, and chemical reactivity. SDSS can effectively scavenge reactive oxygen species (ROS) in wounds and inhibit excessive inflammatory responses. It can also improve the wound immune microenvironment by regulating macrophage polarization towards the M2 regenerative phenotype. Simultaneously, SDSS can promote vascular endothelial cell proliferation and angiogenesis, accelerating granulation tissue formation and providing crucial support for wound healing. Furthermore, its unique chemical structure endows it with strong chemical reactivity, providing a structural basis for its participation in the construction of hydrogel networks and demonstrating great application potential in the development of functionalized repair materials.

[0005] For example, Chinese invention patent CN116785270A discloses the application of SDSS in the preparation of drugs for treating chronic pain. This patent verifies that SDSS alleviates neuroinflammation by reducing TNF-α and IL-6 expression, exhibiting good analgesic effects on neuropathic pain. However, this invention mainly involves the use of SDSS as a drug alone (injection or oral administration). The drug is rapidly metabolized in the body, has a short half-life, and is difficult to maintain a long-lasting therapeutic concentration at the lesion site.

[0006] For example, Chinese invention patent CN117243928A (published on October 24, 2023) discloses a sprayable, thermosensitive hydrogel-coupled SDSS formulation. This technology utilizes the thermosensitive properties of poloxamer (PF-127) to prepare a thermosensitive spray for alleviating postoperative complications by physically blending SDSS and PF-127 solutions at low temperatures. However, this system is essentially a physical blend; SDSS is dispersed in the gel network only through weak intermolecular forces, lacking stable chemical bonding and anchoring. This leads to a rapid release of the drug under physiological conditions, making it difficult to maintain a long-term therapeutic concentration. Furthermore, PF-127, as a synthetically inert polymer, lacks the excellent bioadhesion and inherent antibacterial activity characteristic of natural polysaccharides (such as chitosan), limiting its application in complex wound repair.

[0007] For example, Chinese invention patent CN118750478A discloses the application of SDSS in the treatment of diabetic ulcers. This study used a solvent containing DMSO and PEG to dissolve SDSS and treated mouse ulcers via wet dressing. However, this liquid dressing lacks a three-dimensional porous structure, cannot provide a physical scaffold for cell growth, and exhibits poor moisture retention and antioxidant stability in the wound environment, limiting its application in the repair of deep or complex wounds.

[0008] Although both CS and SDSS have significant advantages in wound repair, existing technologies still face considerable bottlenecks and unresolved technical challenges. On the one hand, CS itself has inherent defects; it is insoluble in water and has extremely poor solubility in neutral and alkaline environments. The preparation of existing CS hydrogels often relies on chemical modification or the addition of toxic chemical cross-linking agents such as glutaraldehyde, which not only significantly increases preparation costs and industrialization difficulties but may also pose safety hazards due to chemical reagent residues, limiting its clinical translation and application. On the other hand, existing CS hydrogels are mostly isotropic homogeneous structures, lacking topological guidance structures similar to the natural extracellular matrix, making it difficult to guide the directional migration and orderly proliferation of wound repair-related cells, ultimately leading to disordered growth of scar tissue and severely affecting the repair effect.

[0009] On the other hand, although SDSS has shown great potential in anti-inflammation, immunomodulation, and tissue repair, its application as an active ingredient for wound repair still faces significant challenges. First, the o-dihydroxyl structure of SDSS itself is extremely unstable, easily affected by environmental factors (such as temperature, light, and pH) and undergoes oxidative degradation. Second, drug release is uncontrollable, with a tendency for burst release leading to low bioavailability. Furthermore, the administration methods of SDSS are still mostly simple physical blending or liquid wet dressings. This loading method not only fails to solve the aforementioned stability and controllable release problems of SDSS itself, but its loading carrier also has two major defects: first, weak mechanical support and insufficient adhesion to the wound surface, making it difficult to conform to irregular wounds and easily damaged and displaced by daily traction and friction. It cannot form a continuous wound protection barrier, nor can it provide a stable growth scaffold for the proliferation and remodeling of new tissue, directly limiting the repair effect; second, it lacks the ability to regulate skin repair in stages, failing to match the dynamic repair needs of early anti-inflammation, mid-term proliferation promotion, and late-stage scar reduction, ultimately severely restricting the full realization of SDSS's repair efficacy.

[0010] Currently, no technical solution has been found to deeply integrate CS and SDSS, leveraging the chemical activity of SDSS to construct a safe and stable physical cross-linking network, thus addressing the pain point of traditional CS hydrogel preparation relying on chemical modification or toxic cross-linking agents. Simultaneously, the structural advantages of the CS carrier enable stable loading and long-term controlled drug release of SDSS, while also endowing the material with topological guiding functions to guide orderly tissue regeneration, ultimately achieving the goal of precise wound repair throughout the entire lifecycle. Therefore, developing a novel multifunctional hydrogel system based on the deep integration of CS and SDSS has become a key direction for overcoming existing technological limitations and promoting the upgrading of wound repair materials. Summary of the Invention

[0011] The purpose of this invention is to provide an SDSS / CS hydrogel and its preparation method. Specifically, addressing the specific pain points of easy oxidation and inactivation of the o-diphenol hydroxyl groups of SDSS and uncontrollable drug release, the invention utilizes the multiple interactions between the amino and hydroxyl groups of chitosan and the o-diphenol hydroxyl and carboxyl groups of SDSS to achieve stable loading and controllable sustained release of SDSS. At the same time, relying on the excellent gelling properties, adhesion, repair-promoting and synergistic anti-inflammatory activities of CS, a composite hydrogel with functions of "drug stability - long-term drug release - mechanical support - synergistic repair" is constructed.

[0012] A further objective is to provide a derivative of the aforementioned hydrogel—a sodium tanshinone / chitosan / chitosan whisker (SDSS / CS / CW) liquid crystal hydrogel—that can achieve targeted proliferation regulation during the repair period through time-sequential degradation and structure guidance.

[0013] A further objective is to provide an SDSS / CS / CW combined exosome hydrogel (Exos@SDSS / CS / CW) that enhances the precise synergy between anti-inflammatory and repair-promoting effects by layering and loading M2 macrophage exosomes (M2-Exos) and human umbilical cord mesenchymal stem cell exosomes (hUCMSC-Exos).

[0014] To achieve the above objectives, the present invention adopts the following technical solution:

[0015] A method for preparing SDSS / CS hydrogel comprises the following steps: First, deionized water is added to SDSS and CS, and the mixture is stirred thoroughly to ensure complete dissolution of SDSS and complete dispersion of CS. Then, glacial acetic acid is gradually added, and the mixture is stirred continuously while adjusting the pH. Under room temperature or incubation conditions, the SDSS / CS hydrogel is formed through cross-linking via multiple intermolecular forces, including hydrogen bonding and electrostatic interactions. During incubation, the amino / hydroxyl groups on the CS molecular chain form an extensive hydrogen bond network with the carboxyl / phenolic hydroxyl groups on the SDSS molecules. Simultaneously, a strong electrostatic attraction occurs between the protonated amino groups of CS and the carboxyl groups of SDSS. These multiple intermolecular forces synergistically cross-link, allowing for the construction of a structurally stable SDSS / CS hydrogel network without the need for additional chemical cross-linking agents.

[0016] The concentration of CS in this invention is 0.01 g / mL to 0.1 g / mL; preferably 0.01 g / mL to 0.05 g / mL; more preferably 0.03 g / mL.

[0017] The concentration of SDSS described in this invention is 0.001 g / mL to 0.01 g / mL; preferably 0.002 g / mL to 0.006 g / mL; more preferably 0.004 g / mL.

[0018] The present invention uses glacial acetic acid to adjust the pH of the solution to 3-10; preferably 5-8; more preferably 7. The solution is a mixture of SDSS and CS.

[0019] The incubation temperature described in this invention is 4~50℃; preferably 20~50℃; more preferably 25~37℃.

[0020] The incubation time (i.e., the time for continuous stirring / standing until gelation after adjusting the pH value) of the present invention is 1~8h; preferably 2~4h; more preferably 4h.

[0021] Furthermore, although the aforementioned SDSS / CS hydrogel has addressed core issues such as the easy oxidation of SDSS, drug burst release, and insufficient mechanical support of the carrier, the drug release and degradation of the monolayer structure are difficult to precisely match the repair sequence, making it impossible to achieve phased regulation of "anti-inflammatory during the inflammatory phase and targeted induction during the proliferation phase." To further overcome this bottleneck, this invention introduces chitin whiskers (CW) into the SDSS / CS hydrogel to construct a tanshinone sodium / chitosan / chitin whisker (SDSS / CS / CW) liquid crystal bilayer hydrogel. Its core functionality is achieved through the difference in degradation rates between the two layers to realize timed functional release: the first SDSS / CS base layer degrades rapidly, releasing anti-inflammatory and antibacterial drugs in the early stage; after its degradation, the second liquid crystal layer loaded with CW is exposed, directionally inducing cell proliferation and orderly collagen deposition during the proliferation phase, reducing scarring, and achieving precise regulation of the entire repair cycle.

[0022] The preparation method of the derived sodium tanshinone, chitosan, and chitin whiskers (SDSS / CS / CW) liquid crystal bilayer composite hydrogel is as follows: 1) Preparation of the bottom layer hydrogel precursor solution (SDSS / CS precursor solution, the bottom layer serves as the wound contact layer): Deionized water is added to SDSS and CS, and the mixture is stirred thoroughly to ensure that SDSS is fully dissolved and CS is completely dispersed; then glacial acetic acid is gradually added, and the mixture is stirred and the pH value is adjusted to obtain the SDSS / CS bottom layer hydrogel precursor solution. 2) Preparation of the upper layer liquid crystal precursor solution (SDSS / CS / CW liquid crystal layer): Deionized water is added to SDSS and CS, and the mixture is stirred thoroughly to ensure that SDSS is fully dissolved and CS is completely dispersed; a small amount of glacial acetic acid is gradually added to dissolve CS and initiate preliminary cross-linking with SDSS; CW is added and ultrasonically dispersed; then glacial acetic acid is added again, the mixture is stirred and the pH value of the solution is adjusted to obtain the upper layer liquid crystal precursor solution (SDSS / CS / CW liquid crystal layer precursor solution). 3) Bilayer molding (SDSS / CS / CW liquid crystal bilayer hydrogel construction): The bottom layer hydrogel (SDSS / CS) precursor liquid is poured. When the SDSS / CS precursor liquid is in a semi-gel state (i.e., it loses its fluidity but the surface remains wet and viscous), the upper layer SDSS / CS / CW liquid crystal layer precursor liquid is poured. The unreacted active groups (amino and carboxyl groups) at the interface undergo cross-layer diffusion and secondary cross-linking to achieve overall solidification of the bilayer structure and strong bonding of the interface, ultimately forming a liquid crystal bilayer composite hydrogel with a "soft-hard" gradient.

[0023] In the SDSS / CS / CW liquid crystal layer precursor solution of the present invention, the concentration of CS is 0.01 g / mL to 0.1 g / mL; preferably 0.01 g / mL to 0.05 g / mL; more preferably 0.02 g / mL.

[0024] In the SDSS / CS / CW liquid crystal layer precursor solution of the present invention, the CW concentration is 2~40wt%, preferably 2~30wt%, and more preferably 5~20wt%.

[0025] The present invention describes the gradual addition of a small amount of glacial acetic acid. Taking a 10 mL reaction system as an example, the preferred amount added is 10~30 μL, and more preferably 25 μL.

[0026] The present invention further describes adding glacial acetic acid, stirring, and adjusting the pH of the solution to preferably pH=7.

[0027] The CW ultrasonic dispersion time described in this invention is 1 to 6 hours, preferably 3 hours.

[0028] The CW ultrasonic dispersion power described in this invention is 100~600W, preferably 300~500W.

[0029] The preferred pouring sequence of the present invention is as follows: first, pour the bottom layer SDSS / CS precursor liquid, and wait for the SDSS / CS precursor liquid to be incubated at 37°C for 10 to 60 minutes, more preferably 30 minutes, until it reaches a semi-gelled state (i.e., it loses its fluidity but the surface remains moist and viscous), and then pour the upper layer SDSS / CS / CW liquid crystal layer precursor liquid.

[0030] The preferred casting method of this invention is as follows: slowly add the upper SDSS / CS / CW liquid crystal layer precursor liquid along the mold wall, and then continue to incubate at 25~37℃ for 2~6 hours, more preferably 4 hours.

[0031] Furthermore, to enhance the functional specificity of each repair stage and improve the repair effect, this invention innovatively loads different types of exosomes in a layered liquid crystal bilayer hydrogel structure. This invention employs an "in-situ embedding" strategy, utilizing the gentle gelation process of the hydrogel precursor solution to stably anchor the exosomes within a three-dimensional network. Specifically, loading M2 macrophage exosomes (M2-Exos) in the rapidly degrading SDSS / CS layer synergistically enhances early anti-inflammatory efficiency; loading human umbilical cord mesenchymal stem cell exosomes (hUCMSC-Exos) in the liquid crystal layer, combined with the physical guidance of the ordered structure of the liquid crystal layer, forms a dual synergistic effect of "physical guidance + biological regulation," precisely promoting directed cell proliferation and tissue regeneration, achieving highly efficient regulation throughout the entire repair cycle.

[0032] The preparation method of the derived Exos@SDSS / CS / CW composite hydrogel is as follows:

[0033] 1) Exosome Extraction and Identification: Macrophages (RAW264.7) M2 polarization was stimulated using cytokines IL-4 or IL-13. M2-type macrophage exosomes (M2-Exos) and human umbilical cord mesenchymal stem cell exosomes (hUCMSC-Exos) were extracted using ultracentrifugation, analyzed, identified, and stored at -80℃ for later use. 2) Preparation of the M2-Exos@SDSS / CS Precursor Solution: Deionized water was added to SDSS and CS, and glacial acetic acid was added dropwise to dissolve and adjust the pH to 7. Then, a certain amount of the above M2-Exos suspension was added and stirred for 30 minutes to obtain a homogeneous M2-Exos@SDSS / CS precursor solution. 3) Preparation of the upper hUCMSC-Exos@SDSS / CS / CW precursor solution: Deionized water was added to SDSS and CS, and the mixture was stirred thoroughly to ensure that SDSS was fully dissolved and CS was completely dispersed. A small amount of glacial acetic acid was gradually added to dissolve CS and initiate its initial cross-linking with SDSS. CW was added and ultrasonically dispersed. Glacial acetic acid was then added, the mixture was stirred, and the pH was adjusted. A certain amount of the above hUCMSC-Exos suspension was added and stirred for 30 minutes to obtain a homogeneous upper hUCMSC-Exos@SDSS / CS / CW precursor solution. 4) Exos@SDSS / CS / CW hydrogel molding: The bottom layer M2-Exos@SDSS / CS precursor solution was poured into the bottom of a sterile mold and pre-gelled at 37°C for 30 minutes to form a semi-gelled M2-Exos@SDSS / CS layer. The upper hUCMSC-Exos@SDSS / CS / CW precursor solution was slowly poured to cover the surface of the M2-Exos@SDSS / CS layer and cross-linked at 37°C to obtain the Exos@SDSS / CS / CW composite hydrogel.

[0034] The final concentration of M2-Exos described in this invention is 1×10⁻⁶. 8 ~5×10 12 particles / mL, preferably 1×10⁻⁶ 10 ~5×10 12 particles / mL, more preferably 3×10⁻⁶ 10 particles / mL.

[0035] The final concentration of hUCMSC-Exos described in this invention is 1×10⁻⁶. 8 ~5×10 12 particles / mL, preferably 1×10⁻⁶ 10 ~5×10 12 particles / mL, more preferably 5 × 10⁻⁶ 10 particles / mL.

[0036] This invention utilizes chitosan (CS) as the core substrate material to construct a hydrogel material system covering the entire wound repair cycle. Starting from the core SDSS / CS physically cross-linked hydrogel, SDSS and CS are innovatively combined through hydrogen bonds and electrostatic attraction, achieving both stable loading and controllable release of SDSS. Simultaneously, leveraging the excellent gelling properties, adhesion, repair-promoting, and synergistic anti-inflammatory activity of CS, a composite hydrogel with functions of "drug stability – long-acting drug release – mechanical support – synergistic repair" is constructed. Furthermore, chitosan (CW) is introduced to prepare an SDSS / CS / CW liquid crystal hydrogel. Through the synergistic effect of the SDSS / CS network and CW, the material is further endowed with dual characteristics of temporal degradation and ordered structure guidance. Finally, M2-Exos and hUCMSC-Exos are layered and loaded within the SDSS / CS / CW bilayer structure, achieving precise synergy between anti-inflammatory and repair-promoting effects. The design logic of this material system perfectly aligns with the dynamic physiological process of wound healing. Each layer of material uses chitosan as the core carrier, with complementary functions and temporal continuity, laying a solid foundation for achieving excellent repair effects.

[0037] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0038] This invention addresses four core shortcomings of existing SDSS drug delivery systems: easy oxidation and inactivation of SDSS, uncontrollable drug release, poor carrier mechanical properties, and lack of dynamic regulation capabilities. The development of a tanshinone sodium chitosan (SDSS / CS) composite hydrogel overcomes these technical bottlenecks. This composite system achieves targeted solutions to multiple problems through precise design: First, by leveraging the strong interactions such as hydrogen bonds and electrostatic attraction between SDSS and CS molecules, stable loading and structural protection of SDSS are achieved, effectively inhibiting its oxidative inactivation. Second, relying on the three-dimensional network structure constructed by CS, the release rate of SDSS is regulated, avoiding burst release and significantly improving drug bioavailability. Third, utilizing the excellent properties of CS after gelation, the hydrogel possesses good mechanical strength and wound adhesion, allowing it to closely adhere to irregular wounds, resist traction and friction, and provide a stable growth microenvironment for new tissue. Fourth, through the synergistic anti-inflammatory, immunomodulatory, and repair-promoting activities of CS and SDSS, it covers the basic needs of the entire skin repair cycle.

[0039] All raw materials used in this invention are of natural origin: CS and CW are derived from marine organisms (shrimp and crab shells), and SDSS is derived from traditional Chinese medicine. These materials possess natural biocompatibility, biodegradability, and low immunogenicity, thus avoiding the immune rejection and toxicity risks of synthetic materials from the source. This lays a safe foundation for subsequent clinical applications and aligns with the industrial orientation of green chemistry and sustainable development.

[0040] This invention avoids the use of toxic chemical cross-linking agents such as glutaraldehyde, and innovatively endows SDSS with the dual roles of functional drug and cross-linking agent. By forming extensive hydrogen bonds and strong electrostatic attraction between the amino and hydroxyl groups on the CS chain and the carboxyl and phenolic hydroxyl groups of SDSS, a dual network is constructed, which not only significantly improves the antioxidant stability of SDSS and prevents its degradation and inactivation, but also achieves long-term and controllable sustained release of the drug, completely overcoming the technical defects of drug burst release in traditional physical mixing systems.

[0041] This invention is not a simple physical mixture, but rather achieves functional synergy between CS and SDSS through intermolecular interactions: the inherent antibacterial and tissue adhesive properties of CS complement the strong anti-inflammatory and antioxidant activities of SDSS. CS can adhere tightly to the wound surface and isolate infection, while SDSS can quickly remove reactive oxygen species (ROS) from the wound surface and inhibit inflammatory responses. At the same time, this synergistic effect can promote local cell proliferation in the wound, accelerate granulation tissue formation, and significantly improve basic repair efficiency.

[0042] This invention innovatively introduces CW to construct an anisotropic liquid crystal structure based on the SDSS / CS basic hydrogel network, endowing the hydrogel with multiple advanced functions: First, relying on the high aspect ratio and crystallinity of CW, the mechanical strength and anti-swelling properties of the hydrogel are significantly improved, enabling it to withstand the tension of skin wounds and adapt to actual clinical application scenarios; Second, the ordered structure of the liquid crystal state provides microscopic topological guidance for cells, promoting the growth of new tissue along an ordered direction and greatly reducing scar formation; Third, the dense network formed by CW can prolong the drug diffusion path and synergistically enhance the long-term sustained-release effect of SDSS with the basic cross-linked network.

[0043] This invention, based on the bilayer structure of a liquid crystal hydrogel, further layers two types of functionally specific exosomes to construct a "time-sequential precision repair system": M2-Exos are loaded onto the rapidly degrading SDSS / CS bottom layer, synergistically enhancing early anti-inflammatory efficiency with SDSS; hUCMSC-Exos are loaded onto the slowly degrading SDSS / CS / CW liquid crystal top layer, synergistically promoting targeted proliferation of cells and tissue regeneration during the proliferative phase in conjunction with the ordered structure of the liquid crystal. This dual synergy of "time-sequential material degradation + exosome functional targeting" precisely adapts to the dynamic physiological process of wound healing, significantly improving repair quality and efficiency.

[0044] In summary, this invention uses SDSS / CS hydrogel as its core technology foundation, constructing a progressively layered material system: First, through the innovative design of SDSS as both a "drug-crosslinking agent," a stable, safe, and effective basic hydrogel is formed, fundamentally addressing the core pain points of traditional repair materials. Building upon this, a liquid crystal structure is constructed by introducing CW, upgrading the material's mechanical properties and tissue-guiding function. Furthermore, by layering and loading functional exosomes, time-sequential, precise, and targeted repair is further enhanced, forming a progressive innovation of "basic hydrogel → liquid crystal-enhanced hydrogel → exosome-composite hydrogel." In this system, all advanced materials are derived from the structure and function of the core hydrogel, ensuring both the independence and creativity of the core technology while achieving functional diversification and precision through gradual optimization. The various technical solutions are logically coherent, highly reproducible, and perfectly meet the actual needs of clinical wound repair. Attached Figure Description

[0045] Figure 1 The diagram shows the preparation process and physical sample of the SDSS / CS hydrogel in this invention.

[0046] Figure 2 The image shown is a microstructure characterization diagram of the SDSS / CS hydrogel in this invention.

[0047] Figure 3 The image shown is a polarized light microscope image of the bilayer composite hydrogel in this invention.

[0048] Figure 4 The figure shown is a graph illustrating the in vitro antibacterial properties of the SDSS / CS hydrogel in this invention.

[0049] Figure 5 The diagram shown is a cell biocompatibility test image of the SDSS / CS hydrogel in this invention.

[0050] Figure 6 The figure shown is a graph illustrating the ability of the SDSS / CS hydrogel in this invention to test endothelial cell migration.

[0051] Figure 7 The figure shown is an evaluation diagram of the in vivo repair effect of the SDSS / CS hydrogel in the rat full-thickness skin defect (pressure ulcer) model.

[0052] Figure 8 The figure shown is a comparative verification experiment of SDSS / CS hydrogel gelation in this invention.

[0053] Figure 9The image shown is a transmission electron microscope (TEM) characterization of the bilayer composite hydrogel loaded with functional exosomes in this invention; where (a) is M2 macrophage exosomes (M2-Exos); and (b) is human umbilical cord mesenchymal stem cell exosomes (hUCMSC-Exos). Detailed Implementation

[0054] The specific embodiments of the present invention will be further described below to make the technical solution and its beneficial effects clearer and more explicit. The embodiments described below are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0055] Additional aspects and advantages of the invention will become apparent in the following description or may be learned by practice of the invention.

[0056] The basic concept of this invention is as follows: SDSS and CS are co-dissolved in an aqueous system with the assistance of acetic acid, and cross-linked under mild conditions through multiple intermolecular forces to form an SDSS / CS hydrogel. During this process, glacial acetic acid induces protonation of the amino groups in CS, causing a strong electrostatic attraction between CS and the carboxyl groups of SDSS; simultaneously, the amino / hydroxyl groups on the CS chain form an extensive hydrogen bond network with the carboxyl / phenolic hydroxyl groups of SDSS. These multiple non-covalent interactions work synergistically to ultimately construct a stable SDSS / CS composite hydrogel network. To make this invention clearer and easier to understand, detailed examples are provided below. Example 1

[0057] 300 mg of CS and 20 mg of SDSS were added to 10 mL of deionized water and stirred thoroughly to disperse them evenly. Glacial acetic acid was then gradually added dropwise to completely dissolve the chitosan. The addition was continued or fine-tuned to control the final pH to 7. The mixture was incubated at 25°C with continuous stirring for 4 hours until a homogeneous and stable hydrogel was formed, named SDSS / CS-2. A physical sample of SDSS / CS-2 can be found [link to sample description]. Figure 1 As shown. Example 2

[0058] 300 mg of CS and 40 mg of SDSS were added to 10 mL of deionized water and stirred thoroughly to disperse them evenly. Glacial acetic acid was then gradually added dropwise to completely dissolve the chitosan. The addition was continued or fine-tuned to control the final pH to 7. The mixture was incubated at 25°C with continuous stirring for 4 hours to obtain a physically cross-linked adhesive hydrogel, named SDSS / CS-4. A physical sample of SDSS / CS-4 can be found [link to sample description]. Figure 1 As shown. Example 3

[0059] 300 mg of CS and 60 mg of SDSS were added to 10 mL of deionized water and stirred thoroughly to disperse them evenly. Glacial acetic acid was then gradually added dropwise to completely dissolve the chitosan. The addition was continued or fine-tuned to control the final pH to 7.0. The mixture was incubated at 25°C with continuous stirring for 4 hours to obtain a physically cross-linked adhesive hydrogel, named SDSS / CS-6. A physical sample of SDSS / CS-6 can be found here. Figure 1 As shown. Example 4

[0060] 300 mg of CS and 80 mg of SDSS were added to 10 mL of deionized water and stirred thoroughly to disperse them evenly. Glacial acetic acid was then gradually added dropwise to completely dissolve the chitosan. The addition was continued or fine-tuned to control the final pH to 7.0. The mixture was incubated at 25°C with continuous stirring for 4 hours to obtain a physically cross-linked adhesive hydrogel, named SDSS / CS-8. A physical sample of SDSS / CS-8 can be found here. Figure 1 As shown. Example 5

[0061] First, 300 mg of CS and 40 mg of SDSS were added to 10 mL of deionized water and stirred thoroughly to disperse them evenly. Then, glacial acetic acid was added dropwise to completely dissolve the chitosan. The addition was continued or fine-tuned to control the final pH value to 6.8 (near neutral) to prepare the SDSS / CS-4 hydrogel precursor solution.

[0062] Next, 200 mg CS and 40 mg SDSS were added to 10 mL of deionized water, and glacial acetic acid (25 μL) was gradually added dropwise to dissolve CS and initiate its initial cross-linking with SDSS. Then, 1 g of chitin whiskers (CW) were added, and the whiskers were uniformly dispersed by ultrasonic dispersion (power: 500 W, time: 3 hours). Glacial acetic acid was added again, and the mixture was stirred and the pH was adjusted to 7 to obtain the SDSS / CS / CW liquid crystal layer precursor solution.

[0063] Finally, the SDSS / CS-4 hydrogel precursor solution was injected into the bottom of the mold and pre-gelled at 37°C for 30 minutes to form a semi-gelled SDSS / CS layer. Subsequently, the prepared SDSS / CS / CW liquid crystal layer precursor solution was poured onto the surface of the SDSS / CS layer gel and incubated at 25°C for 4 hours. Through molecular chain diffusion at the interface and overall solidification, the SDSS / CS / CW liquid crystal state bilayer composite hydrogel with a "soft-hard" gradient was finally obtained. Example 6

[0064] First, functional exosomes were prepared: M2 macrophages (RAW264.7) were stimulated with IL-4 / IL-13 to polarize. M2 macrophage exosomes (M2-Exos) and human umbilical cord mesenchymal stem cell exosomes (hUCMSC-Exos) were extracted using ultracentrifugation, analyzed, identified, and stored at -80℃ for later use. Transmission electron microscopy (TEM) images of the prepared functional exosomes are shown below. Figure 9 As shown, M2-Exos and hUCMSC-Exos have uniform morphology and exhibit a distinctive saucer-like membrane vesicle structure.

[0065] Next, 300 mg of CS and 40 mg of SDSS were added to 10 mL of deionized water and stirred thoroughly to disperse them evenly. Then, glacial acetic acid was gradually added dropwise to completely dissolve the CS. Glacial acetic acid was continued to be added dropwise to control the pH of the solution to 7. Finally, a certain amount of the above-mentioned M2-Exos (final content 3 × 10⁻⁶) was added. 10 (particles / mL), stir for 30 min to obtain a homogeneous bottom layer M2-Exos@SDSS / CS precursor solution.

[0066] Next, 200 mg CS and 40 mg SDSS were added to 10 mL of deionized water. Glacial acetic acid (25 μL) was gradually added dropwise to dissolve CS and initiate initial cross-linking with SDSS. Then, 1 g of chitin whiskers (CW) were added, and the mixture was ultrasonically dispersed (power: 500 W, time: 3 hours) to ensure uniform dispersion of the whiskers. Glacial acetic acid was then added, the mixture was stirred, and the pH was adjusted to 7. Finally, a certain amount of the above-mentioned hUCMSC-Exos (final content 5 × 10⁻⁶) was added. 10 (particles / mL), to prepare the upper layer hUCMSC-Exos@SDSS / CS / CW precursor solution.

[0067] Finally, the bottom layer M2-Exos@SDSS / CS precursor solution was injected into the bottom of the mold and pre-gelled at 37°C for 30 min to form a semi-gelled M2-Exos@SDSS / CS layer; then the prepared top layer hUCMSC-Exos@SDSS / CS / CW precursor solution was poured onto the surface of the bottom gel and incubated at 37°C for another 4 hours. Through molecular chain diffusion at the interface and overall solidification, the Exos@SDSS / CS / CW composite hydrogel was finally obtained.

[0068] Comparative experiments and verification of the gelation mechanism.

[0069] To further elucidate the gelation mechanism of the hydrogel of this invention and verify the irreplaceable roles of glacial acetic acid and sodium tanshinone, the following five sets of comparative experiments were designed, and the results are as follows: Figure 8 (As shown).

[0070] Comparative Experiment 1: A simple aqueous solution of sodium tanshinone (SDSS) cannot form a gel; it is a transparent liquid, such as... Figure 8 As shown in Figure a, this illustrates that sodium tanshinone alone dissolves only in water to form a dilute solution and has no self-assembly capability.

[0071] Comparative Experiment 2: A mixture of chitosan and deionized water, forming a white suspension, such as... Figure 8 As shown in Figure b.

[0072] Comparative Experiment 3: The step of adding sodium tanshinone was omitted; everything else was the same as in Example 1. The resulting product was a viscous liquid that did not gel. Figure 8 As shown in Figure c, the addition of glacial acetic acid successfully dissolved chitosan into a transparent, viscous liquid. However, due to the lack of a cross-linking agent, it exhibited significant fluidity when tilted and could not form a self-supporting gel network.

[0073] Comparative Experiment 4: The step of adding glacial acetic acid was omitted; everything else was the same as in Example 1. The resulting suspension was heterogeneous and could not crosslink. Figure 8 As shown in Figure d, without the addition of glacial acetic acid, chitosan cannot dissolve and protonate, and therefore cannot undergo molecular-level contact and reaction with sodium tanshinone. The system appears as a grayish-white turbidity, showing no signs of gelation. The addition of glacial acetic acid is a prerequisite for gelation, as it is responsible for transforming chitosan from a "suspended state" to a "dissolved state (positively charged)," thereby initiating subsequent reactions.

[0074] Comparative Experiment 5: Gallic acid was used instead of sodium tanshinone in Example 1, while other processes remained the same as in Example 1. The resulting product was a free-flowing brown liquid that could not crosslink into a gel, such as... Figure 8 Figure f in the diagram illustrates the irreplaceable role of sodium tanshinone (SDSS) in this system. Its unique molecular structure (specific distribution of carboxyl / phenolic hydroxyl groups) enables it to form extremely strong supramolecular interactions with chitosan. This highly efficient gelling ability is not possessed by common plant polyphenols (such as gallic acid), demonstrating the non-obviousness and inventiveness of the material selection in this invention.

[0075] Figure 8 Figure e in the figure shows the hydrogel prepared in this invention, for comparison.

[0076] Performance testing and effect verification.

[0077] To verify the performance of the SDSS / CS hydrogel and its liquid crystal bilayer composite scaffold SDSS / CS / CW prepared in this invention, comprehensive characterization and testing were carried out, including microstructure, molecular structure, whisker orientation, antibacterial properties, cell biocompatibility, and repair effect in a rat full-thickness skin defect model.

[0078] like Figure 2As shown in the figure, scanning electron microscopy (SEM) images reveal a highly interconnected three-dimensional porous network structure within the freeze-dried SDSS / CS hydrogel. This porous structure not only facilitates the transport of water and nutrients but also provides physical space for cell migration and colonization. Furthermore, Fourier transform infrared spectroscopy (FT-IR) analysis showed that the characteristic peaks shifted and broadened after the CS and SDSS were mixed, confirming the extensive hydrogen bonding and electrostatic interactions between the two components, forming a stable gel network.

[0079] like Figure 3 As shown, polarized light microscopy (POM) images of the SDSS / CS / CW hydrogel reveal bright birefringent fringes. This indicates that the CWs are not randomly arranged within the gel matrix, but rather retain their natural anisotropic characteristics. This microscopic orientation structure can provide contact guidance for attached cells, inducing fibroblasts to grow in an orderly manner along the whisker alignment direction, thus helping to reduce the formation of disordered scar tissue.

[0080] The bactericidal efficacy of the hydrogel against *Escherichia coli* and *Staphylococcus aureus* was quantitatively evaluated using the plating method. Figure 4 As shown, the bacterial suspension co-cultured with the hydrogel was diluted and spread onto agar plates. After 24 hours of incubation, the control group plates were covered with colonies, while the experimental group plates showed a significant reduction in colony count, indicating a very high inhibition rate. This confirms that the synergistic effect of SDSS and CS endows the material with excellent antibacterial activity and can effectively control bacterial growth in wounds.

[0081] like Figure 5 As shown, mouse fibroblasts (L929), endothelial cells (HUVECs), and macrophages (RAW264.7) were seeded on the surface of SDSS / CS hydrogels or in the extraction medium and cultured. CCK-8 assay results showed that the cells in each group exhibited good proliferation activity and no significant cytotoxicity; live / dead cell fluorescence images showed that the cells spread well on the gel surface and displayed normal morphological characteristics (green fluorescence indicates live cells). This confirms that the hydrogel has excellent cell biocompatibility and is suitable as a tissue engineering scaffold material.

[0082] like Figure 6 As shown, the effect of different ratios of SDSS / CS hydrogel on the migration behavior of human umbilical vein endothelial cells (HUVECs) was evaluated using a scratch assay. The results showed that the hydrogel could more effectively activate the motility of endothelial cells, indicating that the material has excellent pro-angiogenic potential in vivo and can accelerate the re-establishment of blood supply to granulation tissue, thereby shortening the healing cycle.

[0083] A full-thickness skin defect was established on the back of rats and Staphylococcus aureus was inoculated to construct an infected wound model. The SDSS / CS hydrogel of this invention was used for treatment, and the observation period was 21 days. Figure 7 As shown, in an infected environment, the control group exhibited slow wound healing and significant inflammation; while the treatment group showed a significant wound contraction and healing trend at day 21. By day 21, the wounds in the treatment group were almost completely closed, with smooth new tissue and significant hair follicle regeneration. Histological results indicate that this hydrogel not only effectively controlled infection but also promoted rapid growth of granulation tissue and re-epithelialization, achieving high-quality repair of infected wounds.

[0084] It should be noted that the technical features of the above embodiments can be combined arbitrarily. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0085] The above embodiments merely illustrate several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Parts not described in the specific embodiments are all prior art or common knowledge.

[0086] It should also be noted that the detailed description of the preferred embodiments and included examples in the description of this invention makes the content of this invention easier to understand. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In case of any conflict, the definitions in this specification shall prevail.

[0087] In this invention, the term "prepared from" is synonymous with "comprising". The terms "comprising", "including", "having", "containing", or any other variations thereof, as used in this invention, are intended to cover non-exclusive inclusion. For example, a composition, step, method, article, or apparatus comprising the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such a composition, step, method, article, or apparatus.

[0088] In this invention, when a quantity, concentration, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pair of any upper or preferred value with any lower or preferred value, regardless of whether the range is disclosed individually. For example, when the range “1 to 5” is disclosed, the described range should be interpreted as including ranges “1 to 4”, “1 to 3”, “1 to 2”, “1 to 2 and 4 to 5”, “1 to 3 and 5”, etc. When numerical ranges are described in this invention, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within that range.

[0089] Furthermore, the indefinite articles “a” and “an” preceding the elements or components of this invention do not impose any limitation on the quantity requirement (i.e., the number of times) of the elements or components. Therefore, “an” or “a” should be interpreted as including one or at least one, and the singular form of an element or component also includes the plural form, unless the quantity clearly refers to the singular form.

Claims

1. A sodium tanshinone chitosan hydrogel, characterized in that, It is prepared by the following method: (1) Mixing and dispersing: Add deionized water to the mixture of sodium tanshinone and chitosan, and stir thoroughly to completely dissolve sodium tanshinone and disperse chitosan evenly; (2) pH adjustment and cross-linking: Gradually add glacial acetic acid, stir and adjust the pH of the mixed solution to 3~10, and incubate at 4~50℃ for 1~8h. Hydrogen bond network is formed between the amino and hydroxyl groups on the chitosan molecular chain and the carboxyl and phenolic hydroxyl groups on the sodium tanshinone molecule. The protonated amino groups of chitosan and the carboxyl groups of sodium tanshinone generate electrostatic attraction, and synergistic cross-linking forms sodium tanshinone chitosan-based hydrogel. The concentration of chitosan is 0.01g / mL~0.1g / mL, and the concentration of sodium tanshinone is 0.001g / mL~0.01g / mL.

2. The sodium tanshinone chitosan hydrogel according to claim 1, characterized in that, The pH value of the mixed solution is 5-8, the incubation temperature is 20-50℃, and the incubation time is 2-4h; in the mixed solution, the concentration of chitosan is 0.01g / mL-0.05g / mL, and the concentration of sodium tanshinone is 0.002g / mL-0.006g / mL.

3. A liquid crystal bilayer composite hydrogel of sodium tanshinone, chitosan, and chitin whiskers, characterized in that, It is obtained by the following preparation method: (1) Preparation of the bottom layer hydrogel precursor solution: Deionized water was added to the mixture of sodium tanshinone and chitosan, and the mixture was stirred thoroughly to ensure that the sodium tanshinone was fully dissolved and the chitosan was completely dispersed; then glacial acetic acid was added gradually, and the mixture was stirred and the pH value was adjusted to 5-8 to obtain the bottom layer hydrogel precursor solution; in the bottom layer hydrogel precursor solution, the concentration of chitosan was 0.01 g / mL to 0.1 g / mL, and the concentration of sodium tanshinone was 0.001 g / mL to 0.01 g / mL; (2) Preparation of upper liquid crystal precursor solution: Deionized water was added to the mixture of sodium tanshinone and chitosan, and the mixture was stirred thoroughly to completely dissolve the sodium tanshinone and disperse the chitosan evenly. Glacial acetic acid was gradually added to initiate the initial cross-linking of sodium tanshinone and chitosan. Chitin whiskers were added and ultrasonically dispersed at a power of 100~600W for 1~6h. Then, the mixture was allowed to stand to allow the chitin whiskers to self-assemble into an anisotropic liquid crystal system. Glacial acetic acid was added to adjust the pH of the solution to 5~8 to obtain the upper liquid crystal precursor solution. In the upper liquid crystal precursor solution, the concentration of chitin whiskers was 2~40wt%, the concentration of chitosan was 0.01g / mL~0.1g / mL, and the concentration of sodium tanshinone was 0.001g / mL~0.01g / mL. (3) Double-layer molding: The bottom layer hydrogel precursor liquid obtained in step (1) is injected into the bottom of the mold and incubated until it is semi-gelled; then the upper layer liquid crystal precursor liquid obtained in step (2) is poured along the mold wall and incubated at 25~37℃ for 2~6h. Through the incomplete reaction of amino and carboxyl groups at the interface, cross-layer diffusion and secondary cross-linking occur to form an overall solidified double-layer composite hydrogel with a "soft-hard" gradient and an upper layer of liquid crystal structure.

4. The sodium tanshinone, chitosan, and chitin whisker liquid crystal bilayer composite hydrogel according to claim 3, characterized in that, In the bottom hydrogel precursor solution obtained in step (1), the concentration of chitosan is 0.01 g / mL to 0.05 g / mL, and the concentration of sodium tanshinone is 0.002 g / mL to 0.006 g / mL. In the upper liquid crystal precursor solution obtained in step (2), the concentration of chitin whiskers is 5~20wt%; the ultrasonic dispersion power is 300~500W, the dispersion time is 3h; and the concentration of chitosan is 0.01g / mL~0.05g / mL. In step (3), the incubation temperature is 25-37℃ and the incubation time is 10-600 min.

5. A liquid crystal bilayer composite hydrogel loaded with exosomes, characterized in that, It is obtained by the following preparation method: (1) Preparation of exosomes: RAW264.7 macrophages were stimulated to M2 polarize by cytokines IL-4 or IL-13. M2-Exos exosomes of M2 macrophages and hUCMSC-Exos exosomes of human umbilical cord mesenchymal stem cells were extracted by ultracentrifugation and stored at -80℃ for later use after identification. (2) Preparation of the bottom layer M2-Exos@SDSS / CS precursor solution: Deionized water was added to the mixture of sodium tanshinone and chitosan, and the mixture was stirred thoroughly to ensure that the sodium tanshinone was fully dissolved and the chitosan was completely dispersed; then glacial acetic acid was added gradually, and the mixture was stirred and the pH was adjusted to 5-8. The M2-Exos suspension of M2 macrophage exosomes was added and stirred to obtain the bottom layer M2-Exos@SDSS / CS precursor solution. The final concentration of M2-Exos in the bottom layer M2-Exos@SDSS / CS precursor solution was 1×10⁻⁶. 8 ~5×10¹² particles / mL; (3) Preparation of upper hUCMSC-Exos@SDSS / CS / CW precursor solution: Deionized water was added to the mixture of sodium tanshinone and chitosan, and the mixture was stirred thoroughly to completely dissolve the sodium tanshinone and disperse the chitosan evenly. Glacial acetic acid was gradually added to initiate the initial cross-linking of sodium tanshinone and chitosan. Chitosan whiskers were added and ultrasonically dispersed at 100~600W for 1~6h. The mixture was then allowed to stand to allow the chitosan whiskers to self-assemble into an anisotropic liquid crystal system. Glacial acetic acid was added to adjust the pH of the solution to 5~8. The human umbilical cord mesenchymal stem cell exosome hUCMSC-Exos suspension was added, stirred, and allowed to stand to form a liquid crystal system, thus obtaining the upper hUCMSC-Exos@SDSS / CS / CW precursor solution. The final concentration of human umbilical cord mesenchymal stem cell exosome hUCMSC-Exos in the upper hUCMSC-Exos@SDSS / CS / CW precursor solution was 1×10⁻⁶. 8 ~5×10¹² particles / mL; (4) Double-layer molding: The bottom layer M2-Exos@SDSS / CS precursor solution prepared in step (2) is injected into the bottom of the sterile mold and pregeled to a semi-gel state. The upper layer hUCMSC-Exos@SDSS / CS / CW precursor solution prepared in step (3) is poured along the mold wall and cross-linked and cured to obtain Exos@SDSS / CS / CW composite hydrogel.

6. The liquid crystal bilayer composite hydrogel loaded with exosomes according to claim 5, characterized in that, In step (2), the final concentration of M2 macrophage exosomes M2-Exos is 1×10⁻⁶. 10 ~5×10¹² particles / mL; In step (3), the final concentration of human umbilical cord mesenchymal stem cell exosomes hUCMSC-Exos is 1×10⁻⁶. 10 ~5×10¹² particles / mL.

7. The application of the chitosan-tanshinone sodium hydrogel as described in claim 1 or 2 in the preparation of wound repair dressings.

8. The application of the chitosan / sodium tanshinone / chitosan whisker liquid crystal bilayer composite hydrogel as described in claim 3 or 4 in the preparation of full-thickness skin defect repair materials or scar inhibition materials.

9. The application of the liquid crystal bilayer composite hydrogel loaded with exosomes as described in claim 5 or 6 in the preparation of a material for repairing refractory wounds, wherein the refractory wounds include diabetic ulcers and deep burn wounds.

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