Preparation method and application of functionalized konjac glucomannan / silsesquioxane composite sponge dressing

Abstract

The invention discloses a preparation method and application of a functionalized konjac glucomannan / silsesquioxane composite sponge dressing, and belongs to the field of biomedical materials. The preparation method comprises the following steps: oxidizing konjac glucomannan by sodium periodate to prepare formylation modified konjac glucomannan; the preparation method comprises the following steps of: mixing an aqueous solution of the compound with an aqueous solution of polyamino silsesquioxane (POSS) and salts thereof, controlling the molar ratio of aldehyde groups to amino groups, carrying out chemical crosslinking by utilizing a Schiff base reaction, and homogenizing and freeze-drying to obtain the composite sponge dressing. The konjac glucomannan skeleton in the dressing has good biocompatibility, liquid absorption and healing promotion potential, and the covalent cross-linked POSS nano unit can efficiently adsorb endotoxin (LPS) in wound exudate by virtue of dense positive charge clusters on the surface of the covalent cross-linked POSS nano unit, so that the inflammation microenvironment is cooperatively regulated and controlled. The method can be used for preparing wound repair products.

Description

Preparation method and application of functionalized konjac glucomannan / silsesquioxane composite sponge dressing Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a composite functional sponge dressing for chronic wound repair. In particular, it relates to a porous sponge material with aldehyde-modified konjac glucomannan as its backbone, formed by dynamic covalent bonds with polyamino cage-type silsesquioxanes. This material integrates multiple repair functions, including endotoxin (LPS) adsorption, reactive oxygen species (ROS) scavenging, and immune regulation. Background Technology

[0002] Chronic wounds (such as diabetic foot ulcers) remain in a non-healing state for a long time due to persistent inflammation caused by endotoxins (LPS), high levels of reactive oxygen species (ROS), and microenvironmental imbalance dominated by macrophage M1 polarization. Although ordinary foam dressings and hydrocolloid dressings are widely used in clinical practice, they mostly focus on maintaining physical barriers or a moist environment, lacking active regulation of the wound's biochemical microenvironment. While some advanced active dressings attempt to load functional components through physical embedding or coating, they still have significant limitations in practical applications.

[0003] First, their functional synergy is insufficient, making it difficult to cope with complex microenvironments. Existing technologies mostly target single pathological factors, lacking multi-target synergistic mechanisms. For example, Chinese patent document CN204364499U discloses a silver ion antibacterial functional dressing, which achieves bacterial killing by introducing silver ions into the dressing layer and supplemented with an oil layer to prevent adhesion; another example is Chinese patent document CN102078636A, which discloses a hydrogel dressing containing recombinant human epidermal growth factor (rh-EGF), the core of which lies in using growth factors to promote epidermal cell proliferation. However, these existing technologies only focus on pathogen clearance or simple cell proliferation, respectively. When facing complex chronic wounds such as diabetic foot, such single mechanisms cannot simultaneously achieve multiple synergistic interventions of LPS adsorption, ROS clearance, and immune regulation at the molecular level, resulting in limited overall repair effects on chronic, difficult-to-heal wounds.

[0004] Secondly, the structural stability is poor, posing a challenge to balancing safety and effectiveness. Existing functional dressings mostly employ physical methods to load active ingredients, resulting in weak binding forces. For example, Chinese patent document CN201389128Y discloses a nano-silver antibacterial dressing, which uses coating or infiltration to attach nano-silver to a substrate; another example is Chinese patent document CN103623453A, which relates to a preparation method of directly mixing a silver compound solution into a polymer matrix. These techniques, using physical mixing or surface coating, lack a stable chemical bond between the functional components and the matrix. Under the continuous flushing of large amounts of wound exudate, the active substances are prone to burst release or loss. This not only shortens the effective action time of the dressing but also poses a significant risk of cytotoxicity from locally high concentrations of active substances (such as excessively released metal ions), making it difficult to balance long-term effectiveness and biocompatibility.

[0005] Third, the lack of bio-inducing function in matrix materials limits endogenous repair. Currently used dressing matrices are mostly bio-inert materials, lacking the ability to actively regulate cell behavior. For example, Chinese patent document CN101730515B discloses a medical polyurethane foam dressing and its preparation method, focusing on optimizing physical properties such as foaming process, air permeability, and liquid absorption ratio; Chinese patent document CN106916333B discloses a polyvinyl alcohol (PVA) sponge for negative pressure drainage, emphasizing the material's pore structure and mechanical strength. However, whether polyurethane (PU) or polyvinyl alcohol (PVA), these materials are essentially bio-inert matrices, only providing physical support or exudate absorption. They cannot actively mediate the transformation of macrophages into repair-type (M2 type) cells, nor can they effectively promote the directional migration of fibroblasts, thus limiting the material's endogenous role in promoting wound healing.

[0006] Therefore, developing a novel dressing that can adaptively regulate the pathological microenvironment, synergistically achieve efficient LPS clearance, oxidative stress relief, and immune repair functions, while possessing high structural stability and bioactivity, is a key technical problem urgently needing to be solved in this field. It is also the core challenge that this invention aims to address. Summary of the Invention

[0007] The purpose of this invention is to address the technical deficiencies of existing wound dressings, such as limited biochemical microenvironment regulation capabilities (unable to simultaneously address LPS adsorption and immune regulation), poor stability of functional components (easily lost due to burst release from physical load), and lack of bioactivity in matrix materials. This invention provides a functionalized konjac glucomannan / sesquioxane composite sponge dressing and its preparation method.

[0008] Design concept and theoretical analysis of the present invention

[0009] Konjac glucomannan (KGM) is a natural polysaccharide with unique biological activities. It is not only an excellent substrate for constructing sponge dressings, but also possesses the ability to scavenge reactive oxygen species (ROS), promote the polarization of wound macrophages from M1 to M2 types, and recruit and promote fibroblast migration. This makes it an ideal matrix with inherent healing potential. Through controlled aldehyde modification of KGM, its biological activity can be preserved while endowing it with the key characteristic of an active carrier—namely, anchoring and binding other functional components through stable Schiff base covalent bonds.

[0010] Cage-type silsesquioxanes are organic-inorganic hybrid nanoparticles that can be precisely designed at the molecular scale. By introducing multi-amino functional groups into the vertices of the POSS cages and then protonating them using acid treatment (such as hydrochloric acid) or under specific pH physiological conditions, dense clusters of positively charged ammonium salts (-NH3) are formed on their surfaces. + The structure utilizes electrostatic attraction to strongly adsorb negatively charged LPS molecules, making it an ideal endotoxin scavenger.

[0011] This invention creatively combines the above-mentioned materials to propose a novel functionalized konjac glucomannan sponge dressing. Its core concept lies in utilizing the active aldehyde groups of aldehyde-modified konjac glucomannan to undergo a Schiff base reaction with the amino groups of polyamino cage-type silsesquioxanes, thereby covalently anchoring POSS nano-LPS adsorbents in situ within the KGM three-dimensional network, followed by freeze-drying to form a porous sponge.

[0012] Technical solution of the present invention

[0013] A method for preparing a functionalized konjac glucomannan / sesquioxane composite sponge dressing includes the following steps:

[0014] Step 1: Dissolve konjac glucomannan powder in deionized water, add sodium periodate solution, and react for 4-12 hours under light-protected conditions. By controlling the mass ratio of sodium periodate to konjac glucomannan, an aldehyde-modified konjac glucomannan (OKGM) solution is obtained.

[0015] Step 2: The solution obtained in Step 1 is placed into a dialysis bag for dialysis purification, and then freeze-dried to obtain OKGM solid;

[0016] Step 3: Dissolve the solid obtained in Step 2 in water, add an aqueous solution of polyamino cage-type silsesquioxane or its salt, adjust the molar ratio of aldehyde group to amino group to carry out Schiff base crosslinking reaction, homogenize and inject into mold, freeze dry to obtain composite sponge dressing.

[0017] Furthermore, in step one, the mass ratio of sodium periodate to konjac glucomannan is 1:75-1:2; the oxidation degree of the vicinal diol unit in the obtained OKGM is controlled at 1%-60%.

[0018] Furthermore, in step one, the reaction is carried out under magnetic stirring conditions.

[0019] Furthermore, in step two, the dialysis time is 3-5 days, and the dialysis water is changed every 4-8 hours.

[0020] Furthermore, in step two, the aldehyde content is determined by hydroxylamine hydrochloride-potential titration.

[0021] Furthermore, in step three, the concentration of the reconstituted OKGM solution is 5-50 mg / mL; the molar ratio of aldehyde to amino groups is between 1:8 and 10:1. By adjusting the concentration of OKGM, the porosity and microstructure of the sponge dressing can be directionally controlled.

[0022] Furthermore, in step three, after injection into the mold, the material is pre-frozen at -80°C to -20°C, and then subjected to vacuum freeze-drying.

[0023] This invention also provides the application of the functionalized konjac glucomannan / sesquioxane composite sponge dressing prepared by the above method in the preparation of chronic wound repair products.

[0024] Advantages and beneficial effects of the present invention:

[0025] This invention utilizes a specific technical solution to prepare a multifunctional composite sponge dressing through dynamic covalent crosslinking of aldehyde-based konjac glucomannan (OKGM) with polyamino cage-type silsesquioxanes and their salts (POSS). Compared to existing technologies, the advantages and beneficial effects of this invention are specifically reflected in the following aspects:

[0026] 1. Achieving Precise Control and High Adaptability of Dressing Physical Structure: This invention achieves continuous gradient control of sponge porosity by adjusting the oxidation degree of aldehyde-modified konjac glucomannan (1%-60%) and the initial solution concentration (5 mg / mL to 50 mg / mL). Example results show that as the polymer concentration increases, the sponge structure transforms from loose and porous to relatively dense. This controllability allows the dressing to provide customized physical support and liquid adsorption environment for wounds with different exudate levels, meeting the individualized needs of complex wound repair.

[0027] 2. Employing a mild dynamic covalent cross-linking mechanism ensures structural stability and safety. This invention utilizes a Schiff base reaction to construct a stable covalent cross-linked network between aldehyde-modified KGM and POSS, eliminating the need for additional toxic chemical cross-linking agents. FT-IR analysis confirmed the formation of imine bonds (-CH=N-). This cross-linking method not only ensures the stable grafting of POSS nanounits onto the framework, preventing the physical loss of functional components, but also preserves the biocompatibility of the matrix, significantly reducing the potential cytotoxicity of the material.

[0028] 3. Possessing highly efficient endotoxin adsorption capacity, regulating the inflammatory microenvironment at its source. This invention creatively introduces POSS nanounits, utilizing their densely distributed positively charged clusters on their surface to efficiently adsorb endotoxins (LPS) from wound exudate through electrostatic interactions. Fluorescent labeling experiments confirmed that the composite sponge group had a significantly better adsorption capacity for endotoxins than the control group. By eliminating the inflammatory factor LPS at its source, the cascade amplification reaction of wound inflammation is effectively blocked, creating a favorable microenvironment for subsequent tissue repair.

[0029] 4. Synergistic Antioxidant and Immunomodulatory Functions, Actively Inducing Wound Healing from Inflammation to Proliferation: This invention fully leverages the free radical scavenging ability of KGM and the bioactivity of its complex structure. DPPH and cellular ROS scavenging experiments both confirmed its significant antioxidant properties; simultaneously, immunofluorescence staining showed that this dressing downregulated the M1 macrophage marker (CD86) and upregulated the M2 macrophage marker (CD206). This synergistic effect effectively alleviates oxidative stress in wounds, induces macrophages to polarize towards anti-inflammatory and repair mechanisms, and shortens the inflammatory phase of chronic wounds.

[0030] 5. Significantly Promotes Cell Migration and High-Quality Tissue Regeneration: In vitro experiments (Transwell and cell scratch assays) confirmed that the composite sponge prepared in this invention can significantly promote the directional migration of fibroblasts. In a diabetic rat wound model, this dressing exhibited excellent healing-promoting effects. Histopathological analysis (H&E and Masson staining) showed that the treatment group was significantly superior to the control group in terms of granulation tissue growth, re-epithelialization rate, and the orderliness of collagen deposition.

[0031] In summary, the composite sponge dressing prepared by this invention integrates multiple functions such as physical liquid absorption, structural support, endotoxin adsorption, inflammation regulation, and promotion of tissue regeneration. The preparation process is simple and controllable, and it has broad application prospects in the field of chronic and difficult-to-heal wound care (such as diabetic foot ulcers). Attached Figure Description

[0032] Figure 1 is a schematic diagram of the chemical reaction principle in which aldehyde-modified konjac glucomannan (OKGM) forms Schiff base bonds with polyamino cage-type silsesquioxanes and their salts (POSS) in this invention.

[0033] Figure 2 shows the Fourier transform infrared spectra of konjac glucomannan, aldehyde-modified konjac glucomannan, and konjac glucomannan / sesquioxane composite sponge dressing in the embodiments of the invention.

[0034] Figure 3 shows the morphology of the konjac glucomannan / sesquioxane composite sponge dressing under a scanning electron microscope in the embodiment of the invention. In the figure, A represents an OKGM concentration of 10 mg / mL, B represents an OKGM concentration of 20 mg / mL, C represents an OKGM concentration of 25 mg / mL, and D represents an OKGM concentration of 30 mg / mL.

[0035] Figure 4 shows the test results of the endotoxin adsorption performance of the composite sponge dressing in the embodiment of the invention, where A is the blank control group and B is the composite sponge dressing group.

[0036] Figure 5 shows the in vitro antioxidant performance test results of the composite sponge dressing in the embodiment of the invention, where A is the blank control group and B is the composite sponge dressing group.

[0037] Figure 6 shows the immunofluorescence staining results of the effect of composite sponge dressing on macrophage polarization regulation in the embodiment of the invention, where A is the blank control group and B is the composite sponge dressing group.

[0038] Figure 7 shows the experimental results of the composite sponge dressing promoting fibroblast migration in the embodiments of the invention, where A is the blank control group and B is the composite sponge dressing group.

[0039] Figure 8 is a schematic diagram of the wound healing process in diabetic rats in the embodiment of the invention, where A is the blank control group and B is the composite sponge dressing group.

[0040] Figure 9 shows the H&E staining and Masson staining results of wound tissue in the embodiment of the invention, where A is the blank control group and B is the composite sponge dressing group. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the scope of protection of the invention.

[0042] Example 1:

[0043] In this embodiment, the present invention provides a method for preparing a konjac glucomannan / silsesquioxane composite sponge dressing, wherein the molecular structure of konjac glucomannan is shown in formula (I). The molecular structure of aldehyde-modified konjac glucomannan is shown in formula (II). The molecular structures of polyamino cage-like silsesquioxanes and their salts are shown in formula (III). The molecular structures of aldehyde-modified konjac glucomannan crosslinked with polyamino cage-like silsesquioxanes and their salts are shown in formula (IV).

[0044] Formula (Ⅰ).

[0045] Formula (II).

[0046] Formula (Ⅲ).

[0047] Formula (Ⅳ).

[0048] In one embodiment, the present invention provides a method for preparing a konjac glucomannan / sesquioxane composite sponge dressing, comprising the following steps:

[0049] Step 1: Preparation and characterization of aldehyde-modified konjac glucomannan.

[0050] Weigh 1.0 g of konjac glucomannan (KGM) powder and dissolve it completely in 100 mL of deionized water, stirring until completely transparent. To achieve a theoretical oxidation degree of 5%, weigh 0.066 g of sodium periodate, dissolve it in deionized water, and slowly add it to the KGM solution under light-protected and magnetically stirred conditions. React at room temperature for 6 hours.

[0051] After the reaction, the mixture was placed in a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed with deionized water for 3 days (changing the water every 6 hours) to remove byproducts and impurities. The dialysate was freeze-dried to obtain a white flocculent solid product (OKGM). The aldehyde content of the product was determined by hydroxylamine hydrochloride-potentiometric titration, and its actual oxidation degree was found to be 4.83%. Fourier transform infrared spectroscopy (FT-IR) characterization was performed, as shown in Figure 2. The product was precipitated at 1721 cm⁻¹. -1 The presence of a distinct carbonyl (C=O) stretching vibration absorption peak at the aldehyde group indicates that the aldehyde group was successfully introduced.

[0052] Step 2: Crosslinking preparation and structural control of composite sponge dressings.

[0053] The OKGM solid prepared in step one was redissolved in deionized water at a mass concentration of 25 mg / mL. Based on the actual measured degree of oxidation (4.83%), the corresponding mass of polyamino cage-type silsesquioxane and its salt (POSS) was calculated and weighed and dispersed in water according to the molar ratio of aldehyde:amino = 2:3. After mixing the two, the mixture was thoroughly homogenized using a homogenizer, injected into a mold (e.g., 1 mL per well of a 24-well plate), pre-frozen at -80℃ for 12 hours, and finally freeze-dried to obtain the composite sponge dressing.

[0054] By maintaining the aldehyde to amino molar ratio (2:3) constant and changing the initial concentration of OKGM in step two (from 5 mg / mL to 50 mg / mL), a composite sponge dressing with a continuous gradient porosity can be obtained. As shown in the scanning electron microscope (SEM) image in Figure 3, the sponge structure changes from the loose and porous structure of group A to the relatively dense structure of group D as the concentration increases.

[0055] Step 3: Verification of the cross-linking structure of the sponge dressing.

[0056] The obtained composite sponge was subjected to FT-IR analysis (as shown in Figure 2). Compared with OKGM, the composite sponge showed better performance at 1721 cm⁻¹. -1 The characteristic peak intensity at 1638 cm⁻¹ is significantly reduced, while at 1638 cm⁻¹... -1 A distinct imine bond (-CH=N-) stretching vibration absorption peak was observed. This result confirms that a Schiff base reaction successfully occurred between the aldehyde group of OKGM and the amino group of POSS, forming a stable dynamic covalent crosslinking network (Formula IV).

[0057] Step 4: Characterization of endotoxin adsorption performance

[0058] The performance of the sponge was evaluated using a fluorescently labeled endotoxin adsorption experiment. As shown in Figure 4, group A was the FITC-LPS solution control group, and group B was the composite sponge dressing group. Fluorescence detection showed that the residual fluorescence intensity of group B was significantly lower than that of group A, proving that the dressing has the ability to efficiently adsorb endotoxins.

[0059] Step 5: Characterization of in vitro antioxidant properties

[0060] The DPPH free radical scavenging experiment results in Figure 5 confirm that the konjac glucomannan / sesquioxane composite sponge dressing has significant antioxidant capacity. Group A was the saline control group, and Group B was the sponge dressing treatment group. The results showed that the free radical scavenging rate of Group B (69.9±2.7)% was significantly higher than that of Group A (0.5±1.2)%. Figure 5 further validates the antioxidant effect of the sponge dressing through the intracellular reactive oxygen species (ROS) scavenging experiment. Group A was the hydrogen peroxide-only stimulation group, and Group B was the sponge dressing and hydrogen peroxide co-treatment group. Quantitative fluorescence analysis showed that the ROS level in Group B was significantly lower than that in Group A.

[0061] Step Six: Characterization of Macrophage Polarization Regulation Performance

[0062] Figure 6 illustrates that the immunofluorescence staining experiment confirmed that the sponge dressing can effectively regulate macrophage polarization. Group A was the LPS-stimulated group alone, and Group B was the sponge dressing and LPS co-treatment group. The results showed that the expression of the M1 marker CD86 was decreased and the expression of the M2 marker CD206 was increased in Group B.

[0063] Step 7: Characterization of fibroblast migration-promoting properties

[0064] Figure 7 illustrates that the Transwell and cell scratch assays confirmed the significant ability of the sponge dressing to promote fibroblast migration. Group A was the blank control group (using ordinary cell culture medium), and Group B was the sponge dressing treatment group. The Transwell assay results showed that the number of cells that passed through the microporous membrane in Group B (367.3±11.2) was significantly higher than that in Group A (57.0±10.5). The cell scratch assay results showed that the scratch healing rate in Group B (24.0±0.6)% was significantly higher than that in Group A (2.4±0.4)%. Both experiments collectively confirmed that the sponge dressing effectively promotes the directional migration of fibroblasts.

[0065] Step 8: Verification of wound repair effect in animals

[0066] Figure 8 illustrates the healing-promoting effect of the sponge dressing in the full-thickness skin defect model of diabetic rats. Part A is the blank control group, and part B is the sponge dressing treatment group. The results observed on the 8th day after surgery showed that the wound healing of part B was significantly better than that of part A.

[0067] Step Nine: Histopathological Analysis and Verification

[0068] Figure 9 illustrates the tissue repair effect of the composite sponge dressing, as further verified by histopathological staining analysis. Part A represents the wound tissue of the blank control group, while Part B represents the wound tissue of the composite sponge dressing treatment group. H&E staining and Masson staining results show that Part B is significantly better than Part A in terms of granulation tissue growth, re-epithelialization, and collagen deposition.

[0069] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a functionalized konjac glucomannan / sesquioxane composite sponge dressing, characterized in that, Includes the following steps: Step 1: Dissolve konjac glucomannan powder in water, add sodium periodate solution, and react under light-protected conditions for 4-12 hours. By controlling the mass ratio of sodium periodate to konjac glucomannan, obtain an aldehyde-modified konjac glucomannan solution. Step 2: Purify the solution obtained in Step 1 by dialysis and freeze-dry to obtain aldehyde-modified konjac glucomannan solid. Step 3: Redissolve the solid obtained in Step 2 in water, add an aqueous solution of polyamino cage-type silsesquioxane or its salt, adjust the molar ratio of aldehyde to amino groups to 1:8-10:1, carry out Schiff base crosslinking reaction, homogenize, inject into a mold, and freeze-dry to obtain composite sponge dressing.

2. The preparation method according to claim 1, characterized in that: In step one, the mass ratio of sodium periodate to konjac glucomannan is 1:75-1:

2.

3. The preparation method according to claim 1, characterized in that: In the aldehyde-modified konjac glucomannan, the oxidation degree of the vicinal diol unit of konjac glucomannan is 1%-60%.

4. The preparation method according to claim 1, characterized in that: In step two, the dialysis time is 3-5 days, and the dialysis water is changed every 4-8 hours.

5. The preparation method according to claim 1, characterized in that, In step three, the concentration of the reconstituted aldehyde-modified konjac glucomannan solution is 5-50 mg / mL.

6. A functionalized konjac glucomannan / sesquioxane composite sponge dressing, characterized in that: The dressing is prepared by the method described in any one of claims 1 to 5; or, the dressing is composed of a three-dimensional porous network structure formed by covalent cross-linking of aldehyde-based konjac glucomannan and polyamino cage-type silsesquioxanes and their salts through Schiff base bonds.

7. The application of the functionalized konjac glucomannan / sesquioxane composite sponge dressing according to claim 6 in the preparation of chronic wound repair products.

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