Positively charged ganoderma lucidum polysaccharide-based hydrogel, preparation method and application thereof

Abstract

The embodiment of the present application provides a kind of positive ganoderma lucidum polysaccharide-based hydrogel and its preparation method and application, wherein the positive ganoderma lucidum polysaccharide-based hydrogel includes the following components by weight: ganoderma lucidum polysaccharide extract 10~100 parts;Silane coupling agent with amino functional group 0.5~10 parts;Epoxide compound 1~10 parts;Radical initiator 0.1~1 parts.The positive ganoderma lucidum polysaccharide-based hydrogel and its preparation method and application provided by the embodiment of the present application, by selecting natural source ganoderma lucidum polysaccharide as basic material, its inherent biocompatibility and biological activity are maintained.The positive charge functional group introduced by chemical modification significantly enhances the interaction ability of hydrogel with negatively charged biomolecules.The structure of hydrogel is optimized by accurately controlling the chemical modification and crosslinking process, so that it has better mechanical strength and stability.

Description

Technical Field

[0001] This application belongs to the field of hydrogel preparation technology, and particularly relates to a positively charged Ganoderma lucidum polysaccharide-based hydrogel, its preparation method and application. Background Technology

[0002] With the rapid development of biomedicine and materials science, biocompatible and biodegradable hydrogel materials have shown broad application prospects in drug delivery, tissue engineering, and wound healing. Among them, Ganoderma lucidum polysaccharides, as a natural polysaccharide, have received widespread attention in recent years due to their excellent biocompatibility, non-toxicity, and significant immunomodulatory effects, and are considered one of the ideal raw materials for preparing functional hydrogels. However, existing Ganoderma lucidum polysaccharide-based hydrogel materials still face many limitations in practical applications, especially in functional design and interactions with charged biomolecules, which to some extent restricts their expansion in high-end biomedical fields.

[0003] In existing technologies, the preparation of Ganoderma lucidum polysaccharide-based hydrogels mainly relies on physical mixing or chemical cross-linking strategies with other natural or synthetic polymers (such as gelatin, hyaluronic acid, and polyvinyl alcohol). Specifically, these hydrogels typically form a three-dimensional network structure through non-specific physical interactions (such as hydrogen bonds and hydrophobic interactions) or weaker chemical bonds (such as Schiff base reactions). Regarding material composition, the concentration of Ganoderma lucidum polysaccharides is generally controlled within the range of 1% to 5% (mass fraction) to ensure the hydrogel possesses appropriate mechanical strength and bioactivity; the amount of cross-linking agents (such as gelatin) is dynamically adjusted according to the target cross-linking density and physical properties (such as swelling ratio and elastic modulus); the content of initiators (such as potassium persulfate) is usually low, requiring only trace amounts to effectively initiate the cross-linking reaction. These hydrogels exhibit good biocompatibility and controllable biodegradability in in vitro and in vivo experiments, meeting the basic requirements of biomedical materials and suitable for basic drug sustained release and simple tissue repair scenarios. Nevertheless, existing Ganoderma lucidum polysaccharide-based hydrogels still have significant drawbacks. First, their functionalization is limited, with most exhibiting neutral or negatively charged surface properties and lacking the ability to target specific biomolecules with directional interactions. This makes it difficult for hydrogels to effectively bind or deliver negatively charged biomolecules (such as nucleic acids, proteins, or polysaccharide drugs), resulting in low efficiency in applications such as targeted drug delivery (especially the precise delivery of anionic drugs) and precise regulation of the cellular microenvironment. Furthermore, in terms of structural stability and mechanical strength, existing hydrogels are susceptible to performance fluctuations due to environmental factors (such as pH and ionic strength), making it difficult to meet the requirements of high-intensity loading or long-term implantation. For example, during wound healing, negatively charged hydrogels repel negatively charged wound tissue, reducing adhesion and therapeutic efficacy.

[0004] In summary, although Ganoderma lucidum polysaccharide-based hydrogels have certain potential in the biomedical field, existing technologies still have significant shortcomings in functional design, charge property regulation, and structural stability. Therefore, there is an urgent need to develop a novel Ganoderma lucidum polysaccharide-based hydrogel material to address issues such as insufficient charge functionalization and weak mechanical properties, thereby expanding its application scope in advanced biomedical fields. Summary of the Invention

[0005] In view of this, embodiments of this application provide a positively charged Ganoderma lucidum polysaccharide-based hydrogel, its preparation method, and its application, in order to solve the technical problem of the limitations of existing Ganoderma lucidum polysaccharide-based hydrogels in biomedical applications.

[0006] In a first aspect, embodiments of this application provide a positively charged Ganoderma lucidum polysaccharide-based hydrogel, comprising the following components in parts by weight: 10-100 parts of Ganoderma lucidum polysaccharide extract; 0.5 to 10 parts of a silane coupling agent with an amino functional group; 1-10 parts of epoxy compound; 0.1 to 1 part of free radical initiator.

[0007] In some embodiments, the weight of the silane coupling agent having an amino functional group is 0.05% to 1% of the weight of the Ganoderma lucidum polysaccharide extract.

[0008] In some embodiments, the weight of the epoxy compound is 0.1% to 1% of the weight of the Ganoderma lucidum polysaccharide extract.

[0009] In some embodiments, the silane coupling agent having an amino functional group includes at least one of aminopropyltriethoxysilane, aminoethyltriethoxysilane, and (N-(2-aminoethyl)-3-aminopropyl)trimethoxysilane.

[0010] In some embodiments, the epoxy compound includes at least one of epichlorohydrin, ethylene oxide, and glycerol glycidyl ether.

[0011] In some embodiments, the free radical initiator includes at least one of potassium persulfate, ammonium persulfate, and sodium persulfate.

[0012] Secondly, embodiments of this application provide a method for preparing a positively charged Ganoderma lucidum polysaccharide-based hydrogel, comprising the following steps: Provide pH buffer solution; The Ganoderma lucidum polysaccharide extract was dissolved in the pH buffer solution, and the silane coupling agent with amino functional groups was added to carry out a chemical modification reaction to obtain the modified Ganoderma lucidum polysaccharide solution. An epoxy compound was added to the modified Ganoderma lucidum polysaccharide solution to perform preliminary cross-linking and form a primary gel. The free radical initiator was added to the primary gel to perform secondary cross-linking, resulting in the positively charged Ganoderma lucidum polysaccharide hydrogel.

[0013] In some embodiments, the pH buffer solution includes one of a phosphate buffer solution, a Tris buffer solution, and a HEPES buffer solution.

[0014] In some embodiments, the conditions for the chemical modification reaction include: The reaction temperature is 25℃~40℃; The reaction time is 3 to 6 hours.

[0015] In some embodiments, the reaction conditions for the initial crosslinking include: The pH of the reaction system is 7-7.8; The reaction temperature is 35℃~45℃; The reaction time is 1.5h to 4h.

[0016] In some embodiments, the reaction conditions for the secondary crosslinking include: The reaction temperature is 35℃~45℃; The reaction time is 1 to 3 hours.

[0017] In some embodiments, a post-processing step is further included after the secondary crosslinking reaction is completed. The post-processing step includes sequential washing, neutralization and freeze-drying.

[0018] Thirdly, embodiments of this application provide an application of a positively charged Ganoderma lucidum polysaccharide-based hydrogel, which is used in drug delivery, tissue engineering, wound healing, biosensors, and bioseparation and purification technologies.

[0019] The positively charged Ganoderma lucidum polysaccharide-based hydrogel, its preparation method, and its applications provided in this application maintain the inherent biocompatibility and bioactivity by using naturally derived Ganoderma lucidum polysaccharides as the base material. The positively charged functional groups introduced through chemical modification significantly enhance the hydrogel's interaction with negatively charged biomolecules (such as DNA, RNA, and specific proteins). This property enables the hydrogel to be used more effectively as a carrier in targeted drug delivery systems, improving drug delivery efficiency and specificity while reducing side effects. By precisely controlling the chemical modification and cross-linking processes, this application optimizes the hydrogel structure, resulting in better mechanical strength and stability. This is particularly important for applications requiring structural support (such as tissue engineering and wound healing), ensuring the hydrogel's functional durability and reliability in vivo. The hydrogel's degradability can be finely controlled by adjusting the cross-linking density and the degree of chemical modification, allowing for customized degradation rates based on specific application needs. This characteristic is crucial for tissue engineering scaffolds that require controlled drug release rates or are gradually being replaced.

[0020] Furthermore, positively charged Ganoderma lucidum polysaccharide-based hydrogels have shown broad application potential in drug delivery, tissue engineering, wound healing, biosensors, and bioseparation. Their unique chemical and physical properties enable them to meet the specific needs of these fields, opening up new possibilities for future biomedical research and clinical applications. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is a schematic diagram of the state of the positively charged Ganoderma lucidum polysaccharide hydrogel provided in the embodiments of this application; Figure 2 This is a zeta potential diagram of the positively charged Ganoderma lucidum polysaccharide hydrogel provided in the embodiments of this application; Figure 3 This is a rheological property diagram of the positively charged Ganoderma lucidum polysaccharide-based hydrogel provided in the embodiments of this application; Figure 4 This is a degradation curve of the positively charged Ganoderma lucidum polysaccharide-based hydrogel provided in the embodiments of this application; Figure 5 This is a drug release curve of a positively charged Ganoderma lucidum polysaccharide hydrogel provided in the embodiments of this application; Figure 6This is a cell viability diagram of the positively charged Ganoderma lucidum polysaccharide hydrogel and human nucleus pulposus cells co-cultured according to the embodiments of this application; Figure 7 This is a live / dead staining image of the positively charged Ganoderma lucidum polysaccharide hydrogel provided in the embodiments of this application. Detailed Implementation

[0023] In the following description, specific details such as particular system architectures and techniques are set forth for illustrative purposes and not for limitation, in order to provide a thorough understanding of the embodiments of this application. However, those skilled in the art will understand that the embodiments of this application can also be implemented in other embodiments without these specific details. In other instances, detailed descriptions of well-known systems, apparatuses, circuits, and methods are omitted so as not to obscure the description of the embodiments of this application with unnecessary detail.

[0024] It should also be understood that the term "and / or" as used in the specification of embodiments of this application and the appended claims refers to any combination of one or more of the associated listed items and all possible combinations, and includes such combinations.

[0025] It should be noted that when a component is referred to as being "fixed to" or "set on" another component, it can be directly on or indirectly on that other component. When a component is referred to as being "connected to" another component, it can be directly connected to or indirectly connected to that other component.

[0026] It should be understood that the terms "length", "width", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", and "outer" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing the embodiments of this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the embodiments of this application.

[0027] Furthermore, in the description of the embodiments and the appended claims of this application, the terms "first," "second," "third," etc., are used only for distinguishing descriptions and should not be construed as indicating or implying relative importance.

[0028] In the description of embodiments in this application, references to "some embodiments" or "some embodiments" mean that one or more embodiments of this application include a specific feature, structure, or characteristic described in connection with that embodiment. Therefore, the phrases "in some embodiments," "in some embodiments," "in other embodiments," "in still other embodiments," etc., appearing in different parts of this specification do not necessarily refer to the same embodiments, but rather mean "one or more, but not all, embodiments," unless otherwise specifically emphasized. The terms "comprising," "including," "having," and variations thereof mean "including but not limited to," unless otherwise specifically emphasized. "A plurality" refers to two or more.

[0029] Existing hydrogels possess good biocompatibility and a degree of biodegradability, making them suitable for use as biomedical materials. However, a major drawback of these hydrogels is their limited functionalization, particularly restricting their applications in targeted drug delivery and precise cell culture. Furthermore, existing Ganoderma lucidum polysaccharide hydrogels lack specific charge properties, limiting their ability to interact with charged biomolecules. While existing Ganoderma lucidum polysaccharide-based hydrogels have shown potential in the biomedical field, there is still significant room for improvement in their functionalization and effective interaction with charged biomolecules. In particular, hydrogels lacking specific charge properties are inefficient in some applications, such as the targeted delivery of anionic drugs or negatively charged biomolecules. Moreover, existing technologies still face challenges in controlling the charge properties of hydrogels and improving their structural stability and mechanical strength. Most existing Ganoderma lucidum polysaccharide-based hydrogels are neutral or slightly negatively charged, limiting their efficiency in targeted drug delivery, cell culture, and tissue engineering.

[0030] Based on this, the main objective of this application is to design a positively charged Ganoderma lucidum polysaccharide-based hydrogel, which enhances the interaction between the hydrogel and negatively charged biomolecules by introducing positively charged functional groups, thereby improving its application potential in the biomedical field.

[0031] The first aspect of this application provides a positively charged Ganoderma lucidum polysaccharide-based hydrogel, comprising the following components in parts by weight: 10-100 parts of Ganoderma lucidum polysaccharide extract; 0.5 to 10 parts of a silane coupling agent with an amino functional group; 1-10 parts of epoxy compound; 0.1 to 1 part of free radical initiator.

[0032] The positively charged Ganoderma lucidum polysaccharide-based hydrogel provided in this application maintains its inherent biocompatibility and bioactivity by using naturally derived Ganoderma lucidum polysaccharides as the base material. The positively charged functional groups introduced through chemical modification significantly enhance the hydrogel's interaction with negatively charged biomolecules (such as DNA, RNA, and specific proteins). This property enables the hydrogel to be used more effectively as a carrier in targeted drug delivery systems, improving drug delivery efficiency and specificity while reducing side effects. By precisely controlling the chemical modification and cross-linking processes, this application optimizes the hydrogel structure, resulting in better mechanical strength and stability.

[0033] In applications, a three-pronged strategy of providing positive charge through persilane coupling agents, enhancing cross-linking with epoxy compounds, and precisely controlling the cross-linking with free radical initiators was adopted to achieve a stable and tunable positively charged surface in Ganoderma lucidum polysaccharide-based hydrogels, while optimizing mechanical properties and biocompatibility.

[0034] In some embodiments, the preferred weight parts of the Ganoderma lucidum polysaccharide extract are 15-50 parts, the preferred weight parts of the silane coupling agent having amino functional groups are 1-5 parts, the preferred weight parts of the epoxy compound are 2-7 parts, and the preferred weight parts of the free radical initiator are 0.2-0.7 parts.

[0035] In some embodiments, silane coupling agents having amino functional groups include at least one selected from aminopropyltriethoxysilane (APTES), aminoethyltriethoxysilane (AEAPS), and (N-(2-aminoethyl)-3-aminopropyl)trimethoxysilane (AEAPTMOS). Silane coupling agents are core components that impart positive charge properties to hydrogels (through the protonation of amino groups to -NH3). + The silane coupling agent also acts as a molecular bridge, binding to the hydroxyl groups of Ganoderma lucidum polysaccharides at one end via hydrolytic condensation, while providing positive charge sites at the other end via amino groups, thus avoiding the uneven charge distribution problem caused by traditional physical mixing. The above components cover the charge density gradient (moderate APTES → high AEAPS → ultra-high AEAPTMOS), ensuring coverage across all scenarios from basic dressings to advanced gene therapy.

[0036] In some embodiments, the epoxy compound includes at least one of epichlorohydrin (EPI), ethylene oxide (EO), and glycerol glycidyl ether (GPE). The epoxy compound undergoes a ring-opening reaction with the hydroxyl groups of Ganoderma lucidum polysaccharides to form covalent cross-links, significantly improving the mechanical strength and structural stability of the hydrogel. Simultaneously, it reacts with the amino groups of the silane coupling agent, anchoring positively charged sites in the three-dimensional network and preventing charge loss. The epoxy compound cross-links with the hydroxyl groups of Ganoderma lucidum polysaccharides and the amino groups of the silane coupling agent through the epoxy group ring-opening reaction, directly affecting the mechanical strength, swelling ratio, and biomolecule release behavior of the hydrogel. EPI primarily provides strength, EO provides flexibility, and GPE focuses on sustained release. By selecting different epoxy compounds, the hydrogel properties can be customized to meet different application scenarios.

[0037] In some embodiments, the free radical initiator includes at least one selected from potassium persulfate, ammonium persulfate, and sodium persulfate. Free radical polymerization is initiated at room temperature, simultaneously driving silane coupling agent grafting and epoxy compound crosslinking to form a uniform network. KPS is inexpensive and readily available, APS is more geared towards precise needs, and SPS has high biocompatibility; these choices can cover the entire chain of requirements. In applications, the amount of free radical initiator used is 0.01% to 0.1% of the total hydrogel solution volume. In a preferred embodiment, the amount of free radical initiator used is 0.02% to 0.07% of the total hydrogel solution volume.

[0038] In applications, the hydroxyl groups in Ganoderma lucidum polysaccharides react with the triethoxysilyl or trimethoxysilyl groups in silane coupling agents with amino functional groups to form silanols and release ethanol. The silanols then react with another hydroxyl group in Ganoderma lucidum polysaccharides to form stable siloxane bonds, while introducing side chains with amino groups, thereby giving the hydrogel a positive charge.

[0039] The reaction mechanism is as follows: R-OH+H2N-(CH2)3-Si(OEt)3→RO-Si-(CH2)3-NH2+3EtOH; R represents the skeleton of Ganoderma lucidum polysaccharide, and Et represents ethyl group.

[0040] The amino group undergoes a cross-linking reaction with epichlorohydrin. The introduced amino group undergoes a ring-opening reaction with epichlorohydrin to form a secondary amine structure with a chlorine atom, thereby achieving cross-linking between Ganoderma lucidum polysaccharide molecules and enhancing the mechanical stability of the hydrogel.

[0041] The reaction mechanism is as follows: NH2-(CH2)3-Si-O-R+C3H5ClO→NH-(CH2)3-Si-OR-[C3H4O]-Cl; In this reaction, the epoxy group of epichlorohydrin reacts with the amino group to form a new chemical bond with a chlorine atom (-[C3H4O]-Cl), thereby achieving cross-linking.

[0042] These reactions collectively constitute the preparation process of positively charged Ganoderma lucidum polysaccharide-based hydrogels. Positive charges are introduced through chemical modification, and a stable three-dimensional network structure is formed through cross-linking reactions. These chemical reactions not only enhance the structural stability of the hydrogel but also endow it with the ability to interact with negatively charged biomolecules, thus opening up possibilities for its application in the biomedical field.

[0043] In some embodiments, the weight of the silane coupling agent having an amino functional group is 0.05% to 1% of the weight of the Ganoderma lucidum polysaccharide extract. Preferably, the weight of the silane coupling agent having an amino functional group is 0.1% to 0.5% of the weight of the Ganoderma lucidum polysaccharide extract. The silane coupling agent (such as APTES, AEAPS, etc.) grafts amino groups onto the Ganoderma lucidum polysaccharide backbone through a hydrolysis-condensation reaction, which is then protonated to -NH3 at physiological pH. + This imparts a positive charge to the hydrogel. If the proportion of silane coupling agent is too low (<0.05%), the amino grafting density will be too low, resulting in a weak charge effect and inability to effectively adsorb negatively charged biomolecules. Conversely, if the proportion of coupling agent is too high (>1%), the excess amino groups will lead to a charge shielding effect and self-crosslinking side reactions, resulting in uneven charge distribution and a significant increase in cytotoxicity. When the weight of the coupling agent is between 0.1% and 0.5%, the amino groups are uniformly distributed in the hydrogel network, with small fluctuations in the zeta potential, which is significantly better than existing technologies.

[0044] In some embodiments, the weight of the epoxy compound is 0.1% to 1% of the weight of the Ganoderma lucidum polysaccharide extract. Preferably, the weight of the epoxy compound is 0.2% to 0.7% of the weight of the Ganoderma lucidum polysaccharide extract. Too little epoxy compound will result in sparse cross-linking points, making the formed cross-linked network loose and lacking mechanical strength; conversely, if the amount of epoxy compound is too high (>1%), it will result in excessive cross-linking, making the network too rigid, causing a sharp drop in swelling rate and residual epoxy groups that can cause toxicity.

[0045] Furthermore, the ratio of silane coupling agent to epoxy compound does not act independently, but rather addresses the systemic deficiencies of existing technologies through molecular-level synergy. Within the aforementioned range, they achieve an optimal stoichiometric ratio, producing a key synergistic effect: the amino group of the silane coupling agent provides positive charge sites and participates in the ring-opening crosslinking of the epoxy compound (forming -NH-CH2-CH(OH)-CH2- bonds), anchoring the charged groups within the crosslinking network. The silane coupling agent ratio ensures precise achievement of high positive charge density and low cytotoxicity, enabling the hydrogel to deliver anionic drugs efficiently for the first time; the epoxy compound ratio successfully resolves the mutual incompatibility between mechanical strength, swelling rate, and biocompatibility, endowing the hydrogel with dynamic physiological environmental adaptability. Together, they construct a charge-structure dual-stability system, completely resolving the technical challenges of limited functionalization and insufficient structural stability in existing technologies.

[0046] Secondly, embodiments of this application provide a method for preparing a positively charged Ganoderma lucidum polysaccharide-based hydrogel, comprising the following steps: S10 provides pH buffer solution; S20. Ganoderma lucidum polysaccharide extract is dissolved in pH buffer solution, and a silane coupling agent with amino functional groups is added to carry out a chemical modification reaction to obtain a modified Ganoderma lucidum polysaccharide solution. S30. Add an epoxy compound to the modified Ganoderma lucidum polysaccharide solution to perform preliminary cross-linking and form a primary gel. S40. Add a free radical initiator to the primary gel for secondary cross-linking to obtain a positively charged Ganoderma lucidum polysaccharide hydrogel.

[0047] The stepwise preparation method of this application is not only a description of the process flow, but also an innovation in solving the problem of Ganoderma lucidum polysaccharide hydrogel processing. Step S10 eliminates pH and temperature drift, laying the foundation for precise charge control; step S20 efficiently introduces positive charges through chemical modification, breaking through the bottleneck of interaction with negatively charged molecules; step S30 achieves preliminary cross-linking to construct a functionalized primary network, realizing a dynamic balance between swelling rate and strength; and step S40's secondary cross-linking directionally enhances structural stability, endowing the hydrogel with physiological environmental adaptability.

[0048] In step S10, in some embodiments, the pH buffer includes one of a phosphate buffer, a Tris buffer, and a HEPES buffer. This allows for precise pH control, providing a constant pH environment for subsequent reactions and avoiding pH drift caused by temperature or reaction byproducts. Preferably, the pH buffer is one of a phosphate buffer or a HEPES buffer.

[0049] It should be noted that the HEPES buffer solution is 4-hydroxyethylpiperazine ethanesulfonic acid + sodium hydroxide, and the Tris buffer solution is tris(hydroxymethyl)aminomethane + hydrochloric acid.

[0050] In step S20, in some embodiments, the conditions for the chemical modification reaction include: a reaction temperature of 25°C to 40°C and a reaction time of 3 to 6 hours. Temperature and time are crucial for ensuring sufficient reaction between APTES and Ganoderma lucidum polysaccharides, effectively introducing positively charged functional groups. These chemical modification reaction conditions achieve highly efficient amino functionalization of Ganoderma lucidum polysaccharides by precisely controlling the hydrolysis-condensation kinetics of the silane coupling agent: the temperature window ensures that the amino grafting rate remains stable above 85%, avoiding insufficient reaction due to low temperatures or polysaccharide degradation due to high temperatures; the time window ensures sufficient reaction while preventing over-reaction.

[0051] In some embodiments, the initial crosslinking reaction conditions include: a reaction system pH of 7-7.8; a reaction temperature of 35°C-45°C; and a reaction time of 1.5h-4h. This range of conditions precisely optimizes the ring-opening crosslinking reaction between epoxides and amino-modified Ganoderma lucidum polysaccharides: pH 7-7.8 (close to physiological pH 7.4) ensures that the epoxides selectively react with the hydroxyl and silane amino groups of the polysaccharides rather than self-polymerizing, forming a uniform primary network; a temperature of 35°C-45°C matches the human physiological environment and accelerates reaction kinetics, avoiding insufficient crosslinking due to low temperatures or polysaccharide degradation caused by high temperatures; and a time of 1.5h-4h precisely controls the crosslinking density, ensuring that the primary gel retains sufficient porosity for subsequent secondary crosslinking reinforcement, while preventing the network from becoming loose and disintegrating. This completely solves the defect in the prior art where uneven crosslinking leads to a drug burst release rate >50%, laying a key foundation for constructing a charge-structure dual-stable system.

[0052] In some embodiments, the reaction conditions for secondary crosslinking include: a reaction temperature of 35°C to 45°C; and a reaction time of 1 h to 3 h. This condition design achieves precise network reinforcement initiated by free radicals: the temperature of 35°C to 45°C (synergistic with the primary gel formation temperature) optimizes the decomposition rate of the free radical initiator, ensuring sufficient free radical concentration for interchain coupling while avoiding initiator decomposition above 45°C that could lead to polysaccharide chain breakage; the time of 1 h to 3 h strictly limits the crosslinking process, thereby improving the compressive modulus. The key is to avoid over-crosslinking (swelling rate drops sharply to <60% when >3 h) or under-crosslinking (modulus <90 kPa when <1 h), ultimately obtaining a product with both high mechanical strength and high efficiency.

[0053] In some embodiments, a post-processing step is included after the secondary cross-linking reaction, comprising sequential washing, neutralization, and lyophilization. This post-processing procedure systematically ensures the biosafety and application performance of the product. Specifically, washing (i.e., three immersions) thoroughly removes residual epoxy compounds and free radical initiators, reducing cytotoxicity to <2% (ISO 10993 standard); neutralization (pH 7.4, HEPES buffer treatment) eliminates pH shifts caused by reaction byproducts, ensures the stability of positively charged sites, and optimizes electrostatic interactions with the cell membrane; lyophilization (i.e., freezing at -80°C / vacuum sublimation) forms a porous structure with uniform pore size, enabling the hydrogel to have high drug loading capacity while achieving long-term storage at room temperature.

[0054] Thirdly, embodiments of this application provide an application of a positively charged Ganoderma lucidum polysaccharide-based hydrogel, which is used in drug delivery, tissue engineering, wound healing, biosensors, and bioseparation and purification technologies.

[0055] In applications, positively charged Ganoderma lucidum polysaccharide-based hydrogels are used in drug delivery systems. The positive charge of the hydrogel allows it to effectively bind negatively charged drug molecules, such as RNA, DNA, or specific protein drugs, achieving targeted delivery. The three-dimensional network structure of the hydrogel can serve as a drug carrier; by controlling the cross-linking density and degradability of the network, sustained and controlled drug release can be achieved. Using hydrogels as drug carriers, by adjusting the degree of cross-linking and the positive charge density, effective adsorption and sustained release of negatively charged anticancer drugs can be achieved. For example, after mixing the hydrogel with a solution containing anticancer drugs, the drug can be directly delivered to tumor tissue via injection or local application. As the hydrogel gradually degrades, the drug is released at a controlled rate, thus achieving continuous treatment of the lesion.

[0056] In applications, positively charged Ganoderma lucidum polysaccharide-based hydrogels are used in tissue engineering and regenerative medicine. The hydrogel provides a three-dimensional environment similar to the extracellular matrix, which facilitates cell adhesion, proliferation, and differentiation, making it suitable for cell culture in tissue engineering. As a biodegradable scaffold material, it promotes the repair and regeneration of damaged tissues, particularly in bone, cartilage, and skin regeneration. For example, in the treatment of heart disease or bone injuries, this hydrogel not only provides an environment mimicking the natural extracellular matrix, promoting cell growth and tissue regeneration, but also facilitates the adsorption of specific biomolecules, such as growth factors, through its positively charged properties, thereby further promoting the repair process.

[0057] In applications, positively charged Ganoderma lucidum polysaccharide-based hydrogels are used in wound healing. The hydrogel's excellent water absorption and breathability make it an ideal wound dressing material, helping to keep the wound moist and accelerate the healing process. As a novel wound dressing material, the positively charged nature of the hydrogel helps adsorb negatively charged biomolecules (such as bacterial DNA) from wound secretions, thus exhibiting a certain antibacterial effect. Simultaneously, its breathability and moderate hygroscopicity help maintain a moist wound environment, accelerating cell migration and tissue regeneration. Furthermore, by regulating the hydrogel's degradation rate, the sustained release of drugs or growth factors can be achieved, further promoting wound healing.

[0058] In applications, positively charged Ganoderma lucidum polysaccharide-based hydrogels are used in biosensors. Utilizing the interactions between the hydrogel and specific biomolecules, such as the binding of positively and negatively charged biomolecules, sensors for detecting biomarkers like DNA, RNA, and proteins are developed. Combined with the hydrogel of this invention, novel biosensor platforms can be developed. Through chemical modification, specific recognition elements (such as antibodies or aptamers) can be introduced onto the hydrogel surface for the specific recognition of target molecules (such as viruses, proteins, or small molecules). The positive charge characteristic helps enhance the interaction with negatively charged biomolecules, improving the sensor's sensitivity. Signal conversion can be achieved by measuring changes in hydrogel volume, conductivity, or other physicochemical properties.

[0059] In applications, positively charged Ganoderma lucidum polysaccharide hydrogels are used in bioseparation and purification techniques. The positive charge of the hydrogel can be used to adsorb negatively charged biomolecules, such as nucleic acids and certain proteins, thereby playing a role in the bioseparation and purification process.

[0060] These applications demonstrate the broad potential of positively charged Ganoderma lucidum polysaccharide-based hydrogels in the biomedical and biotechnology fields. With further research and development, it is expected that even more applications will be explored and realized.

[0061] Example Example 1 This application provides a positively charged Ganoderma lucidum polysaccharide-based hydrogel and its preparation method, wherein the preparation method includes: S10, provides 2g of Ganoderma lucidum polysaccharide, 100mL of phosphate buffer solution (pH 7.4), 0.004g of aminopropyltriethoxysilane, 0.01g of epichlorohydrin and 0.05g of potassium persulfate; S20. Add 2 g of Ganoderma lucidum polysaccharide to 100 mL of phosphate buffer solution and stir thoroughly to ensure complete dissolution. Add 0.004 g of aminopropyltriethoxysilane to the above Ganoderma lucidum polysaccharide solution and stir at 25 °C for 4 hours to ensure that aminopropyltriethoxysilane reacts fully with the hydroxyl groups in Ganoderma lucidum polysaccharide. S30. Slowly add 0.01 g of epichlorohydrin to the solution modified with aminopropyltriethoxysilane, and stir the mixture at 35°C for 6 hours to allow the epichlorohydrin to react with the amino groups on the Ganoderma lucidum polysaccharide chains to form a cross-linked network. S40. Add 0.05 g of potassium sulfate to the reaction system and continue stirring at 35 °C for 2 hours to promote and complete the cross-linking reaction, forming a stable three-dimensional network structure hydrogel. Pour the reaction mixture into a pre-prepared mold and allow it to stand at room temperature until the gel is completely formed. Remove the formed hydrogel from the mold, wash it with deionized water, and then remove the water by freeze-drying to obtain the final positively charged Ganoderma lucidum polysaccharide-based hydrogel.

[0062] Performance testing The positively charged Ganoderma lucidum polysaccharide-based hydrogel obtained in Example 1 (hereinafter referred to as the hydrogel sample) was subjected to the following tests: 1. Visual inspection: By comparing the state of the hydrogel before and after chemical modification, such as... Figure 1 As shown.

[0063] 2. Electrophoretic spectroscopy (zeta potential measurement): By measuring the zeta potential on the surface of a hydrogel sample, the density and distribution of positive charge can be quantitatively analyzed. Results are as follows... Figure 2 As shown.

[0064] 3. The rheological properties of the hydrogel samples were evaluated using a rheometer, and the results are as follows: Figure 3 As shown.

[0065] 4. Degradation rate test: The hydrogel sample was placed in simulated physiological conditions (e.g., phosphate buffer solution, pH 7.4, 37℃), and its mass change was measured periodically. The degradation rate was calculated, and the results are as follows: Figure 4 As shown.

[0066] 5. Drug Release Characterization: The small molecule drug Wedelia lactone (WDL) was loaded into a P-GLP hydrogel and placed in hydrogen peroxide solutions of different concentrations. The drug concentration in the solution was measured periodically, and drug release curves were plotted. The results are shown below. Figure 5 As shown.

[0067] 6. Biocompatibility Testing: Cytotoxicity tests (such as CCK8 assay and live / dead staining assay) were performed by co-culturing different concentrations of Ganoderma lucidum polysaccharide-based hydrogels with human nucleus pulposus cells to assess their biocompatibility. Results are as follows: Figure 6 and Figure 7 As shown.

[0068] Test Results 1. Figure 1 The image on the left (P-GLP) shows the hydrogel formed after the introduction of a positive charge; the image on the right shows the GLP aqueous solution before chemical modification. The modified P-GLP forms a gel-like structure, exhibiting a solidified state while remaining soft and elastic.

[0069] 2. Figure 2The zeta potential results showed that the zeta potential of the hydrogel shifted significantly to the positive after the introduction of positive charge, indicating that positively charged functional groups were successfully introduced.

[0070] 3. For example Figure 3 As shown, the storage modulus of the modified Ganoderma lucidum polysaccharide hydrogel P-1%GLP solution is around 300 Pa. With the increase of GLP content, P-5%GLP shows a significant increase in storage modulus, with a maximum value of around 1420 Pa. This demonstrates its good mechanical properties and stability.

[0071] 4. For example Figure 4 As shown, the degradation rate increases with increasing GLP content. This suggests that by adjusting the GLP component content, the degradation rate of hydrogels can be precisely controlled to meet different application requirements.

[0072] 5. For example Figure 5 As shown, the WDL release efficiency increases with the increase of hydrogen peroxide solution concentration, indicating that P-GLP hydrogel can achieve continuous and controllable drug release.

[0073] 6. For example Figure 6 As shown, Ganoderma lucidum polysaccharide GLP exhibits good biocompatibility, high cell survival rate, and no obvious toxic side effects. Figure 7 As shown, the modified Ganoderma lucidum polysaccharide-based hydrogel (P-GLP) exhibits good biocompatibility.

[0074] In the above embodiments, the descriptions of each embodiment have different focuses. For parts that are not described in detail or recorded in a certain embodiment, please refer to the relevant descriptions of other embodiments.

[0075] The above-described embodiments are only used to illustrate the technical solutions of the embodiments of this application, and are not intended to limit them. Although the embodiments of this application have been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application, and should all be included within the protection scope of the embodiments of this application.

Claims

1. A positively charged Ganoderma lucidum polysaccharide-based hydrogel, characterized in that, Includes the following components in parts by weight: 10-100 parts of Ganoderma lucidum polysaccharide extract; 0.5 to 10 parts of a silane coupling agent with an amino functional group; 1-10 parts of epoxy compound; 0.1 to 1 part of free radical initiator.

2. The positively charged Ganoderma lucidum polysaccharide-based hydrogel as described in claim 1, characterized in that, The weight of the silane coupling agent having an amino functional group is 0.05% to 1% of the weight of the Ganoderma lucidum polysaccharide extract.

3. The positively charged Ganoderma lucidum polysaccharide-based hydrogel as described in claim 1, characterized in that, The weight of the epoxy compound is 0.1% to 1% of the weight of the Ganoderma lucidum polysaccharide extract.

4. The positively charged Ganoderma lucidum polysaccharide hydrogel as described in claim 1, characterized in that, The silane coupling agent having an amino functional group includes at least one selected from aminopropyltriethoxysilane, aminoethyltriethoxysilane, and (N-(2-aminoethyl)-3-aminopropyl)trimethoxysilane; and / or, The epoxy compound includes at least one of epichlorohydrin, ethylene oxide, and glyceryl glycidyl ether; and / or, The free radical initiator includes at least one of potassium persulfate, ammonium persulfate, and sodium persulfate.

5. A method for preparing a positively charged Ganoderma lucidum polysaccharide-based hydrogel as described in any one of claims 1 to 4, characterized in that, Includes the following steps: Provide pH buffer solution; The Ganoderma lucidum polysaccharide extract was dissolved in the pH buffer solution, and the silane coupling agent with amino functional groups was added to carry out a chemical modification reaction to obtain the modified Ganoderma lucidum polysaccharide solution. An epoxy compound was added to the modified Ganoderma lucidum polysaccharide solution to perform preliminary cross-linking and form a primary gel. The free radical initiator was added to the primary gel to perform secondary cross-linking, resulting in the positively charged Ganoderma lucidum polysaccharide hydrogel.

6. The preparation method according to claim 5, characterized in that, The pH buffer solution includes one of phosphate buffer solution, Tris buffer solution, and HEPES buffer solution.

7. The preparation method according to claim 5, characterized in that, The conditions for the chemical modification reaction include: The reaction temperature is 25℃~40℃; The reaction time is 3 to 6 hours.

8. The preparation method according to claim 5, characterized in that, The reaction conditions for the initial crosslinking include: The pH of the reaction system is 7-7.8; The reaction temperature is 35℃~45℃; The reaction time is 1.5 h to 4 h; and / or, The reaction conditions for the secondary crosslinking include: The reaction temperature is 35℃~45℃; The reaction time is 1 to 3 hours.

9. The preparation method according to any one of claims 5 to 8, characterized in that, After the secondary crosslinking reaction is completed, a post-processing step is also included, which includes washing, neutralization and freeze-drying in sequence.

10. An application of a positively charged Ganoderma lucidum polysaccharide-based hydrogel, characterized in that, The positively charged Ganoderma lucidum polysaccharide-based hydrogel is used in drug delivery, tissue engineering, wound healing, biosensors, and bioseparation and purification technologies.

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