Carboxymethyl pachyman hydrogel, and preparation method and application thereof

Abstract

The application belongs to the technical field of biomedical materials, and particularly discloses a carboxymethyl pachyman polysaccharide hydrogel as well as a preparation method and application thereof. The preparation method comprises the following steps: dissolving pachyman polysaccharide polymers with a carboxymethyl substitution degree of 0.65-0.70 and sodium citrate in water to obtain a polysaccharide solution; adding calcium chloride into the polysaccharide solution, adjusting the pH, and then performing heating and stirring reaction; collecting first supernatant after the reaction is completed, adding an alcohol solution to perform low-temperature precipitation, separating the precipitate and redissolving the precipitate; then performing dialysis purification and freeze-drying to obtain the carboxymethyl pachyman polysaccharide hydrogel. The hydrogel has a porous network structure, good liquid absorption performance, biocompatibility and tissue adhesion performance, and has a rapid self-healing ability; after the hydrogel is damaged by shearing, the time required for self-healing to restore more than 75% of the original structure is not more than 25 s. The hydrogel can be used for preparing hemostatic medical dressings, and is suitable for the fields of wound hemostasis, postoperative hemostasis and tissue repair.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to a carboxymethyl poria cocos polysaccharide hydrogel, its preparation method, and its application. Background Technology

[0002] Natural polysaccharides (such as various plant polysaccharides, animal polysaccharides, and microbial polysaccharides) exhibit great application potential in the development of wound dressings, tissue adhesives, and hemostatic materials due to their excellent biocompatibility, biodegradability, low immunogenicity, and unique tissue adhesion and biochemical hemostatic activities. Preparing polysaccharides into three-dimensional network structures as polysaccharide hydrogels can not only maintain a moist wound environment but also mimic the extracellular matrix, promoting wound healing.

[0003] However, existing polysaccharide hydrogels, when used to promote wound hemostasis and healing, generally face the following pressing technical bottlenecks: Natural polysaccharide macromolecular chains rely primarily on weak hydrogen bonds in water, resulting in physical gels with insufficient mechanical strength. To improve stability and mechanical properties, current technologies typically require the introduction of chemical cross-linking agents such as glutaraldehyde and epichlorohydrin. These cross-linking agents often exhibit significant cytotoxicity and irritation, and residual cross-linking agents can easily cause secondary biochemical damage to the wound surface. Many active polysaccharides require treatment with strong alkalis (such as deacetylation, depolymerization, or alkaline swelling) during conventional extraction or modification. Traditional alkali treatment often leads to disordered and excessive degradation of polysaccharide molecular chains, a sharp decrease in molecular weight, and severe disruption of their spatial conformation, resulting in the final product losing its in-situ gelling ability or forming only fragmented solids lacking mechanical elasticity. Furthermore, while some existing polysaccharide hydrogels formed through physical self-assembly possess certain injectability properties (shear thinning), their internal broken polysaccharide spatial networks require a considerable amount of time to re-entangle and recover after being subjected to external shear forces such as syringe compression or wound spraying.

[0004] Therefore, how to develop a polysaccharide gel with a mild preparation process, no need for external toxic crosslinking agents, and ideal mechanical strength and self-healing properties is a common technical problem that urgently needs to be solved in the field of biomedical materials. Summary of the Invention

[0005] The purpose of this invention is to address the aforementioned shortcomings of the prior art by providing a carboxymethyl Poria cocos polysaccharide hydrogel, its preparation method, and its applications. 2+ The successful coordination of ions with the carboxyl groups in CMP not only enhances the thermal stability of the CMP-Ca complex and alters its surface morphology, but also endows it with gel-forming ability. Simultaneously, the metal coordination bonds ( - COO-Ca 2+ -OOC - The dynamic reversibility of ) endows carboxymethyl poria cocos polysaccharide hydrogel with self-healing and injectability.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of this invention is to provide a method for preparing carboxymethyl Poria cocos polysaccharide hydrogel, comprising the following steps: A polysaccharide polymer with a degree of substitution of 0.65 to 0.70 and sodium citrate were dissolved in water to obtain a polysaccharide solution. Calcium chloride was added to the polysaccharide solution, the pH was adjusted with an alkaline solution, and the reaction was stirred under heating conditions. After the reaction was completed, the first supernatant was collected. The mass ratio of the Poria cocos polysaccharide polymer to the calcium chloride was (111~444):1000. An alcohol solution was added to the first supernatant for low-temperature precipitation. The mixed solution after alcohol precipitation was subjected to solid-liquid separation to collect the precipitate. The precipitate was then dissolved again to obtain a reconstituted product. The reconstituted product was purified by dialysis to remove free ions, and then the resulting dialysate was freeze-dried to obtain the CMP-Ca complex, namely the carboxymethyl poria cocos polysaccharide hydrogel.

[0007] Furthermore, the mass ratio of the Poria cocos polysaccharide polymer to the sodium citrate is 1:(0.6~0.8).

[0008] Furthermore, the alkaline solution is a NaOH solution with a concentration of 0.1~0.12 mol / L, and the pH is adjusted to 9.5~10.5.

[0009] Furthermore, the heating conditions are 65~75℃, and the stirring reaction time is 2~4h.

[0010] Further, the alcohol solution is an ethanol solution with a volume fraction of 90%~98%; the volume ratio of the added ethanol solution to the first supernatant is (9~10):1; the temperature of the low-temperature precipitation is 2~8℃, and the time is 18~30h.

[0011] Furthermore, the solid-liquid separation is centrifugal separation, with a rotation speed of 3500~4500 rpm / min and a centrifugation time of 10~20min.

[0012] Furthermore, the dialysis purification uses a dialysis bag with a molecular weight cutoff of 3000~4000 Da and a dialysis time of 48~96 h.

[0013] A second aspect of the present invention is to provide a carboxymethyl poria cocos polysaccharide hydrogel prepared by the above-described preparation method.

[0014] Furthermore, after being sheared and damaged, the carboxymethyl poria cocos polysaccharide hydrogel requires no more than 25 seconds to self-heal and recover to more than 75% of its original structure.

[0015] A third aspect of the present invention is to provide the application of the aforementioned carboxymethyl poria cocos polysaccharide hydrogel in the preparation of hemostatic medical dressings.

[0016] Compared with the prior art, the beneficial effects of the present invention are: (1) This invention regulates Ca 2+ The concentration of ions, the intensity of synergistic alkali treatment, and heat treatment were used to fine-tune the conformation of the CMP molecular chain, avoiding the disordered and excessive degradation of the molecular chain caused by traditional strong alkali treatment. This resulted in the preparation of the CMP-Ca complex via Ca... 2+ Ion replacement of Na + Ions enter CMP and form metal coordination bonds with carboxyl groups ( - COO-Ca 2+ -OOC - With increasing CaCl2 addition, the calcium chelating ability of the CMP-Ca complex gradually increased, reaching a maximum chelating capacity of 69.80 mg / g. 2+ Successful coordination of ions with carboxyl groups in CMP not only enhances the thermal stability of the CMP-Ca complex and alters its surface morphology, but also endows it with gel-forming ability.

[0017] (2) The metal coordination bonds in the CMP-Ca complex provided by this invention form intramolecular calcium bridges in the aqueous system, which promotes the formation of a stable three-dimensional network structure and thus drives the gelation of the CMP-Ca complex. At the same time, hydrogen bonds still exist in the gel, and these interactions together enhance the stability of the hydrogel network structure.

[0018] (3) The hydrogel formed by the CMP-Ca composite provided by this invention has excellent self-healing and injectability. After being subjected to external shear damage, its internal broken physical cross-linked network can spontaneously reconstruct within a very short time of 25 seconds, restoring more than 75% of the original structure. This is all attributed to Ca 2+ The dynamic reversibility of metal coordination interactions between ions and carboxyl groups. Specifically, the fracture of hydrogels leads to the partial loss of Ca2+. 2+ The ion dissociates from its original coordination bond with the carboxyl group. Subsequently, the reconnection of the broken interface allows the free Ca to dissociate. 2+ The ions re-establish coordination bonds with the exposed carboxyl groups, thereby restoring the structural integrity of the hydrogel.

[0019] (4) The hydrogel formed by the CMP-Ca complex provided by the present invention has dynamic healing ability, which can maintain the integrity of the dressing in the state of wound movement and avoid the wound being re-exposed due to the dressing breaking or displacement.

[0020] (5) The CMP-Ca complex provided by the present invention has good biocompatibility and rapid self-healing ability. It can accelerate the aggregation of blood components by utilizing its specific spatial conformation, thereby effectively shortening the complete coagulation time of blood and serving as a safe and effective hemostatic medical dressing. Attached Figure Description

[0021] Figure 1 The figure shows the preparation of the CMP-Ca complex. A represents the synthesis process of the CMP-Ca complex, B represents the effect of different CaCl2 concentrations on the calcium chelating ability of the CMP-Ca complex, and C represents the energy spectrum of CMP and the CMP-Ca complex.

[0022] Figure 2 Scanning electron microscope image of CMP and CMP-Ca complex (×500).

[0023] Figure 3 The ¹H NMR spectrum (A) and ¹³C NMR spectrum (B) of CMP and its calcium complex are shown.

[0024] Figure 4 The HSQC spectrum of the CMP-Ca complex (A) and the potential metal coordination bonds in the CMP-Ca complex (B).

[0025] Figure 5 The figure shows the stability analysis results of CMP and CMP-Ca chelate. In the figure, AB represents the solution stability of CMP and CMP-Ca chelate, and C represents the zeta potential.

[0026] Figure 6 Thermal stability (A), XRD (B), and FT-IR (C) of CMP and CMP-Ca complex.

[0027] Figure 7 For the rheological analysis of the CMP-Ca complex, in the figure, A represents the difference in hydrogel-forming ability and calcium chelation ability under different polysaccharide concentrations, and B represents free Ca. 2+ The effect on the fluidity of CMP solution, where C represents the apparent viscosity of CMP and CMP-Ca complex solutions at different concentrations.

[0028] Figure 8 The oscillation amplitude scan curve of CMP solution and CMP-Ca composite gel is shown in Figure (A); the oscillation frequency scan curve of CMP solution and CMP-Ca composite gel is shown in Figure (BE).

[0029] Figure 9The tests included thixotropic (A), creep and rebound (B), three-stage structure rebound (C), self-healing ability demonstration (D), injectability demonstration (E), and gel strength test (F) of the CMP-Ca hydrogel.

[0030] Figure 10 Scanning electron microscope (A) and energy dispersive spectroscopy (EDS) images of CMP-Ca hydrogels with different calcium chelation contents (B).

[0031] Figure 11 This is a schematic diagram of the gelation and self-healing mechanism of the CMP-Ca complex of the present invention.

[0032] Figure 12 The graph shows the in vitro coagulation performance results of the CMP-Ca complex of the present invention.

[0033] Figure 13 This is a graph showing the in vitro hemolysis test results of the CMP-Ca complex of the present invention.

[0034] Figure 14 Images of the in vitro hemolysis test of the CMP-Ca complex of this invention. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of the present invention clearer, embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0036] This invention provides a method for preparing carboxymethyl Poria cocos polysaccharide hydrogel, which involves reacting carboxymethylated Poria cocos polysaccharide (CMP) with Ca... 2+ Ions are cross-linked to construct a CMP-Ca complex with a three-dimensional network structure. This hydrogel exhibits good biocompatibility, self-healing ability, and hemostatic properties, and can be used in wound hemostasis, tissue repair, and biomedical dressings. Specifically, the preparation method includes the following steps: (1) Preparation of polysaccharide solution First, weigh out a polysaccharide polymer of Poria cocos with a carboxymethyl substitution degree of 0.65~0.70 and dissolve sodium citrate in deionized water. The amount of sodium citrate added can be 60%~80% of the mass of the polysaccharide polymer of Poria cocos. Stir magnetically at room temperature for 2~6 h. (2) Crosslinking reaction A calcium chloride solution is slowly added to the above polysaccharide solution, and an ionic cross-linking reaction is carried out under stirring. The mass ratio of the Poria cocos polysaccharide polymer to the calcium chloride is (111~444):1000, preferably 55:1000.

[0037] After adding calcium chloride, the pH of the system is adjusted using an alkaline solution. The alkaline solution can be any one of sodium hydroxide solution, sodium carbonate solution, or ammonia water, preferably a 0.1~0.2 mol / L sodium hydroxide solution, more preferably a 0.1 mol / L sodium hydroxide solution. The pH of the system is preferably adjusted to 9.5~10.5, more preferably 10.

[0038] The reaction is then carried out under heating conditions with stirring. The heating temperature can be 65~75℃, preferably 70℃; the stirring time can be 2~4h, preferably 3h.

[0039] In this process, Ca 2+ Sodium citrate coordinates with the carboxyl groups on the carboxyl group of the Poria cocos polysaccharide molecular chain, gradually forming an ionic cross-linked network structure. 2+ It has a certain complexing effect and can slow down the Ca2+ metabolism. 2+ The instantaneous crosslinking rate avoids rapid local gelation and improves the uniformity of the system.

[0040] After the reaction is complete, the reaction system is allowed to stand or centrifuged, and the first supernatant is collected.

[0041] (3) Low-temperature alcohol precipitation and redissolution An alcohol solution is added to the first supernatant for low-temperature precipitation to promote the precipitation of the CMP-Ca complex. The alcohol solution may be one or more of methanol, ethanol, and isopropanol, preferably an ethanol solution with a volume fraction of 90% to 98%, more preferably a 95% ethanol solution; the volume ratio of the added ethanol solution to the first supernatant is (9~10):1, preferably 9.5:1.

[0042] In some embodiments, the alcohol precipitation process is carried out at a low temperature, which can be 2-8°C, preferably 4°C; the precipitation time can be 18-30 hours, preferably 24 hours. Low-temperature alcohol precipitation helps to improve the precipitation efficiency of the complex and reduce the degradation of polysaccharide chains.

[0043] The mixture after alcohol precipitation is then subjected to solid-liquid separation to collect the precipitate. The solid-liquid separation method can be centrifugation, filtration, or vacuum filtration, preferably centrifugation at a speed of 3500-4500 rpm / min for 10-20 min. The obtained precipitate is then redissolved in deionized water to obtain the reconstituted product.

[0044] (4) Dialysis purification and freeze drying The reconstituted product was placed in a dialysis bag for dialysis purification to remove free Ca from the system. 2+ Cl -1Citrate ions and unreacted small molecule impurities. The molecular weight cutoff of the dialysis bag can be 3000~4000 Da, preferably 3500 Da.

[0045] The dialysis process can be carried out in deionized water for 48 to 96 hours, preferably 72 hours, and the dialysis fluid should be changed regularly, preferably every 4 to 12 hours.

[0046] After dialysis, the dialysate is obtained and then freeze-dried. The freezing temperature can be -40℃ to -80℃, the freeze-drying vacuum degree can be 1 to 100 Pa, and the freeze-drying time can be 12 to 72 h.

[0047] The CMP-Ca complex, namely the carboxymethyl poria cocos polysaccharide hydrogel, was finally obtained.

[0048] The obtained carboxymethyl Poria cocos polysaccharide hydrogel exhibited a uniform porous structure, and the pore size decreased with increasing calcium chelation content. This phenomenon is attributed to Ca... 2+ Specific coordination interactions between ions and carboxyl groups on polysaccharide chains. These interactions promote interchain crosslinking, thereby increasing the density and mechanical strength of the gel network. In some embodiments, the resulting carboxymethyl Poria cocos polysaccharide hydrogel possesses self-healing capabilities. When the hydrogel is mechanically damaged, its internal dynamic ionic coordination bonds can reform under minimal external stimulation or static conditions, thereby restoring the continuity of the overall structure. This self-healing mechanism is attributed to Ca... 2+ The dynamic reversibility of the metal coordination interaction between ions and carboxyl groups allows the fractured interface to rebuild a cross-linked network.

[0049] Specifically, under room temperature conditions, after the hydrogel is subjected to high-speed shearing or cutting and then left to stand, the time required for it to self-heal and recover to more than 75% of its original structure is no more than 25 seconds.

[0050] In some embodiments, "recovering to more than 75% of the original structure" means that after the hydrogel is damaged, one or more of its storage modulus (G′), compressive strength, viscoelastic properties, structural continuity, or macroscopic morphology recovers to more than 75% of its undamaged state.

[0051] In some embodiments, the self-healing performance can be characterized by rheological testing. Specifically, a high-low strain cyclic scanning mode can be used, where the hydrogel network structure is disrupted under high strain conditions, followed by restoration to low strain conditions, and the recovery of the storage modulus (G′) is recorded. When the recovery G′ reaches more than 75% of the initial G′ and the required time does not exceed 25 s, it indicates that the hydrogel has rapid self-healing capability.

[0052] In some embodiments, the resulting carboxymethyl poria cocos polysaccharide hydrogel can undergo structural dissociation under shear force and quickly recover to a gel state after shearing stops, thus possessing certain injectability and plasticity, making it easy to apply to irregular wounds or deep hemostatic sites.

[0053] This invention also provides the application of the carboxymethyl pachymansia polysaccharide hydrogel in the preparation of hemostatic medical dressings. The carboxymethyl pachymansia polysaccharide hydrogel can be used alone as an effective hemostatic material, or it can be further compounded with other medical excipients to form composite hemostatic dressings. The excipients include, but are not limited to, one or more of the following: collagen, hyaluronic acid, chitosan, alginate, gelatin, polyvinyl alcohol, polylactic acid fiber, medical nonwoven fabric, or antibacterial components.

[0054] In some embodiments, the carboxymethyl pachymansia hydrogel can be prepared into medical dressings of different forms through methods such as freeze-drying, molding, electrospinning, or 3D printing. Among them, the porous hemostatic sponge formed after freeze-drying has a high specific surface area and rapid blood absorption capacity, and can be used in acute massive hemorrhage scenarios.

[0055] In some embodiments, the carboxymethyl pachymansia hydrogel medical dressing can be stored in aseptic packaging and can be terminally sterilized by gamma ray sterilization, ethylene oxide sterilization or low-temperature plasma sterilization to meet the requirements for use as medical materials.

[0056] The invention has now been generally described, and will be more readily understood by referring to the following embodiments, which are provided by way of example and not by way of limitation.

[0057] The experimental methods used in the embodiments of this invention are as follows: Determination of calcium ion chelation content: 100 mg of the CMP-Ca complex was accurately weighed and placed in an Erlenmeyer flask. 10 mL of nitric acid and 0.5 mL of perchloric acid were added. The Erlenmeyer flask was placed on a hot plate (DB-1AB, Tianjin Gongxing, China) for digestion. The digestion conditions were set as follows: digestion at 120℃ for 1 hour, followed by increasing the temperature to 180℃ for 4 hours, and finally heating to 200℃ until white fumes were produced and the digestion solution was colorless and transparent. The Erlenmeyer flask was removed and diluted with ultrapure water to 25 mL. To eliminate potential systematic errors in the experimental setup, a reagent without the CMP-Ca complex was used as a blank control. The chelated calcium content of the CMP-Ca complex was determined by atomic absorption spectrometry (AAS, AA-6300C, Shimadzu, Japan). During the determination, an appropriate amount of lanthanum oxide solution (20 g / L) was added to the test solution to adjust the final lanthanum oxide concentration to 1 g / L. A standard curve was plotted using calcium carbonate (CaCO3) as the standard solution, with the following equation: Y = 0.0358X + 0.0373, R² = 0.9988, where Y is the absorbance and X is the calcium concentration (mg / L). All experiments were performed in triplicate, with each replicate containing three technical replicates. Calcium chelating ability of the CMP-Ca complex (1).

[0058] X= (1) Where X represents calcium chelating capacity, defined as the mass of calcium bound per unit mass of CMP-Ca complex (mg Ca / g complex, i.e., mg / g); C represents the calcium concentration of the sample solution (mg / L); C0 represents the calcium concentration of the blank control (mg / L); f is the dilution factor of the digested sample solution; V represents the total volume of the sample solution after constant volume adjustment (L); and m represents the mass of the CMP-Ca complex (mg).

[0059] Solution stability analysis: The solution stability of CMP and CMP-Ca complex was determined using a Turbiscan stability analyzer (Formulaction, France). The sample concentration was 10 mg / mL, and the transmitted light and backscattered light detectors were scanned every 15 min to monitor the changes in transmittance of the sample over 24 h. The Turbiscan stability index (TSI) was calculated using Turbisoft software (Equation (2)).

[0060] TSI = (2) X i The average intensity of scattered light recorded at each scanning time point. x T All x i The overall average, n: Total number of scans.

[0061] Zeta potential analysis: CMP or CMP-Ca complex was weighed and dissolved in deionized water to prepare an aqueous solution with a concentration of 1.0 mg / mL. The zeta potential of CMP and CMP-Ca complex was determined using a Zeta potential analyzer (Zetasizer Nano ZS, Malvern Ltd., UK).

[0062] Fourier transform infrared spectroscopy (FT-IR) analysis: 5 mg of CMP or CMP-Ca was weighed and ground together with 500 mg of dry potassium bromide powder, and further prepared into transparent thin films. FT-IR spectroscopy (ThermoNicolet, USA) was performed in the range of 4000 to 500 cm⁻¹. - ¹Sampling within the wavenumber range, at a depth of 4 cm - ¹64 cumulative scans at resolution.

[0063] X-ray diffraction (XRD) analysis: CMP and CMP-Ca complex were analyzed using an X-ray diffractometer (D8 ADVANCE, Bruker) equipped with a Cu Kα radiation source (wavelength λ = 0.1542 nm). The 2θ scan range was set from 5° to 80°, and the scan rate was 5° / min. The XRD principle is based on the diffraction phenomenon of X-rays by crystalline materials, and its diffraction law is described by Bragg's law (Equation (3)): nλ=2d sinθ(3) Where θ is the Bragg angle, d is the interplanar spacing, λ is the X-ray wavelength, and n is the diffraction order.

[0064] Scanning electron microscopy (SEM) and energy-dispersive spectroscopy (EDS) analysis: CMP and CMP-Ca composites were placed on a sample platform covered with conductive tape and subjected to a 5-minute sputtering gold plating treatment. Surface morphology was observed using a scanning electron microscope (SEM, SU3800, Hitachi, Japan). The elemental composition and content of the samples were characterized using a field emission scanning electron microscope (FE-SEM, SU8010, Tianmei, China) combined with an energy-dispersive spectroscopy system (EDS, EDAX, USA) in point analysis mode. All test data were processed and analyzed using TEAM (Texture and Elemental Analysis Microscopy) software.

[0065] Nuclear magnetic resonance (NMR) analysis: 1D NMR of CMP and CMP-Ca complex ( 1 H and 13 C) and 2D nuclear magnetic resonance (HSQC) spectra were acquired using a Bruker AV-600 nuclear magnetic resonance spectrometer at 25°C. 25 mg of each sample was dissolved in 0.55 mL of deuterated water (D₂O, 99.9% deuterium content). All chemical shifts are expressed in ppm (δ). Hydrogel preparation and gel-forming ability analysis: Different masses (600 mg, 800 mg, 1000 mg, 1200 mg, and 1600 mg) of CMP and CMP-Ca complex were weighed. Each sample was dispersed in 20 mL of deionized water under magnetic stirring to ensure complete dissolution, resulting in solutions with concentrations of 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, and 80 mg / mL, respectively. These solutions were then incubated at 25 °C for 24 hours to observe the hydrogel formation process.

[0066] Rheological properties were determined: CMP and the CMP-Ca complex were dissolved in deionized water to prepare solutions with concentrations of 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, and 80 mg / mL. All rheological experiments were performed using a HAAKE MARS 40 rotational rheometer (Thermo Fisher Scientific, USA), equipped with a parallel plate geometry (35 mm in diameter, 1 mm gap). Data analysis was performed using HAAKE RheoWin software.

[0067] Apparent viscosity test: Viscosity refers to the property of a fluid to resist flow under shear force. Steady-state shear tests were performed on CMP and CMP-Ca complexes with concentrations ranging from 30 to 80 mg / mL. The shear rate ( The range is 0.1 to 500 s. - ¹. Viscosity (η) and applied shear rate ( The curve showing the relationship between ) is called the flow curve.

[0068] Oscillation Amplitude Scan: Amplitude scan testing is recognized as an effective method for determining the linear viscoelastic region (LVR) of a material. LVR tests were performed on CMP solutions and CMP-Ca hydrogels (concentrations 50–80 mg / mL) at a frequency of 1 Hz: the shear stress range for CMP solutions was 0.001–50 Pa, and for CMP-Ca hydrogels, it was 0.001–100 Pa.

[0069] Oscillating frequency scanning: The storage modulus (G′) and loss modulus (G″) of CMP solution and CMP-Ca hydrogel (50-80 mg / mL) were analyzed by frequency scanning in oscillating mode. Frequency scanning measurements were performed at 20 °C (in LVR) under constant shear stress of 0.5 Pa (CMP solution) and 10 Pa (CMP-Ca hydrogel), with angular frequencies (ω) ranging from 1 to 100 rad / s.

[0070] Thixotropic rings: Thixotropic ring experiments were conducted on CMP-Ca hydrogels with different calcium chelation contents at 20℃. The shear rate was initially set at 0.0001 s⁻¹. - ¹Increase to 100 s - ¹, then at 100 s - ¹ Maintain a constant position for 20 seconds, then start from 100 seconds. - ¹Decrease back to 0.0001 s - ¹. The closed curve of shear stress versus shear rate obtained in this process is defined as a thixotropic ring, and its enclosing area (A) is used as a quantitative index for evaluating the thixotropic strength of the gel (Q. Meng et al., 2023). The specific expression is shown in Equation (4): A = τ﹒ (Pa·s)(4) Where τ is the shear stress (Pa) and the shear rate (s). - ¹).

[0071] Creep and Recovery: Creep is defined as the increase in strain (γ) over time in a viscoelastic material under sustained stress (τ0). After the stress is removed, some time-dependent deformation can recover over time, typically characterized by two consecutive experimental phases: the creep phase and the recovery phase. Creep strain (γ) C ) by instantaneous strain (γ) in ), delayed elastic strain (γ) d ) and viscous strain (γ) v The material is composed of various components. Mathematically, the time-varying creep strain of a material can be expressed as equation (5): (5) During the recovery phase, the instantaneous elastic strain (γ) e ) can recover immediately, while delayed elastic strain (γ) d ) undergoes a gradual recovery process, viscous flow strain (γ) v The elastic strain (γ) is irreversible. For linear viscoelastic materials, the strain that can be recovered after the removal of a constant stress is called elastic strain (γ). e ), where γ e =γ in Therefore, the recovery strain (γ) varies with time. R It can be expressed as equation (6): (6) Creep and recovery experiments were conducted on CMP-Ca hydrogels with different calcium chelating agent contents. For 5 mM CMP-Ca hydrogels, a creep stage was applied under a constant stress (τ0) of 5 Pa; for 10 mM and 20 mM CMP-Ca hydrogels, a creep stage was applied under a constant stress of 10 Pa, with a holding time of 100 seconds, followed by a 300-second recovery period under 0 Pa conditions.

[0072] Three-stage structure recovery: Structure recovery experiments were performed on CMP-Ca hydrogel (80 mg / mL). The test procedure was carried out at 20°C according to the following protocol: initial shear rate of 1 s⁻¹. - ¹It lasts for 30 seconds, then increases to 100 seconds. - ¹Lasts for 30 seconds, then returns to 1 second. - ¹Lasts for 30 seconds.

[0073] Texture analysis (TPA) of CMP-Ca hydrogel: CMP-Ca hydrogel (80 mg / mL) was transferred to the testing platform and analyzed using a texture analyzer (TAXT plus, Stable Co., UK) with a P / 0.5 probe. The test program was set to gel mode, strain rate 45%, pre-test speed 1.5 mm / s, and test and subsequent test speeds 1 mm / s.

[0074] Scanning electron microscopy and energy dispersive spectroscopy analysis of CMP-Ca hydrogel: CMP-Ca hydrogel (80 mg / mL) was freeze-dried. Its surface morphology was observed by scanning electron microscopy (SEM), and the elemental distribution was analyzed by energy dispersive spectroscopy (EDS) in mapping mode.

[0075] Statistical analysis: Data are expressed as mean ± standard deviation (SD, n=3). Statistical significance was assessed using one-way ANOVA with SPSS Statistics 25.0 software. Values ​​labeled with different letters indicate statistically significant differences (P<0.05). Structural models were constructed using ChemDraw 21.0.

[0076] The Poria cocos polysaccharide polymer (CMP polymer) used in this invention was purchased from Wuhan Runge Biotechnology Co., Ltd., with a purity of 85% and a carboxymethyl substitution degree of 0.69. Calcium carbonate standard solution was purchased from the China Nonferrous Metals and Electronic Materials Analysis and Testing Center. Deuterated water (D2O) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. All other reagents used were of analytical grade. Example 1 Obtain the CMP-Ca complex.

[0077] Weigh 1 g of CMP and 0.7 g of sodium citrate (C6H5Na3O7) and dissolve them in 200 mL of deionized water, then heat to 70 °C. Add different masses of calcium chloride sequentially to the prepared polysaccharide solution: 111 mg (5 mM), 222 mg (10 mM), 333 mg (15 mM), 444 mg (20 mM), and 555 mg (25 mM). Adjust the pH of the solution to 10 with 0.1 mol / L NaOH, then stir in a 70 °C water bath for 3 h. Collect the supernatant, add 950 mL of 95% ethanol, and precipitate at 4 °C for 24 h. Filter the reaction product and collect the supernatant. Centrifuge at 4000 rpm / min for 15 min to collect the precipitate, then redissolve it in deionized water. The solution was dialyzed with distilled water (3500 Da) for 72 h to remove free ions. Finally, the dialysate was freeze-dried to obtain CMP-Ca complexes, which were named 5 mM CMP-Ca, 10 mM CMP-Ca, 15 mM CMP-Ca, 20 mM CMP-Ca, and 25 mM CMP-Ca, respectively.

[0078] The synthesis process of CMP-Ca complex is as follows: Figure 1 As shown in A, CMP itself does not contain calcium ions. With increasing CaCl2 concentration, the calcium chelating ability of the CMP-Ca complex gradually increases, reaching its maximum at a CaCl2 concentration of 20 mM (e.g., ...). Figure 1 (As shown in B in the figure). Specifically, the calcium chelating abilities of the complexes prepared using 5 mM, 10 mM, 15 mM, and 20 mM CaCl2 were 37.53 mg / g, 58.57 mg / g, 66.77 mg / g, and 69.80 mg / g, respectively (P < 0.05). However, when the CaCl2 concentration was further increased to 25 mM, the calcium chelating ability of the CMP-Ca complex decreased. This phenomenon can be attributed to the fact that excessively high CaCl2 concentrations hinder the binding of calcium ions to polysaccharide chains, thereby reducing the calcium chelating efficiency of the complex.

[0079] like Figure 1 Figure C shows the energy dispersive spectroscopy (EDS) spectrum of CMP and the CMP-Ca complex. A significant amount of sodium (Na₂O₃) is observed in the EDS spectrum of CMP. + Characteristic peaks. With increasing CaCl2 concentration, the Ca in the complex... 2+ The relative chelation content of ions gradually increases, while Na + The content of ions decreases accordingly. When the CaCl2 concentration reaches 20 mM, the calcium ion content reaches its peak, while the sodium ion (Na) content decreases. + The characteristic peaks of Ca were almost undetectable. Overall, the EDS results confirm that Ca...2+ Ions and Na bound to CMP + There is competition among ions. Specifically, Ca... 2+ Ions through Na + The ions were displaced from their original binding sites and successfully integrated into the CMP, forming a new CMP-Ca complex. This differs from the preparation of the chondroitin sulfate-calcium complex, which first removes Na+ through cation exchange. + The ions were then chelated with CaCl2. This study achieved Na+ chelation during the chelation process. + Ions and Ca 2+ Direct substitution of ions.

[0080] like Figure 2 As shown, significant differences in size and morphology were observed between CMP and the CMP-Ca complex samples. CMP exhibited a cylindrical or spherical shape, while the CMP-Ca complex showed an outward-extending antenna-like structure at its edges. Notably, the outward-extending antenna-like morphology of the CMP-Ca complex gradually expanded with increasing chelated calcium content. Simultaneously, the CMP surface was rough and had a fine texture, while the surface of the CMP-Ca complex appeared smooth and dense. These results confirm that CMP successfully chelated Ca. 2+ The calcium ions then combine to form the CMP-Ca chelate. The morphological differences observed between CMP and the CMP-Ca complex can be attributed to the bridging effect of calcium ions between the polysaccharide chains, which enhances intermolecular interactions.

[0081] The binding sites of polysaccharides and metal ions can be determined based on the differences in their NMR spectra with the corresponding metal complexes. The structural characteristics of CMP and the CMP-Ca complex were elucidated in detail using ¹H NMR, ¹³C NMR, and HSQC spectra, and their chemical shifts were comprehensively identified. Based on the distribution of anomeric proton chemical shifts, the signal at δ 4.9–5.5 corresponds to the α-configuration, while the chemical shift range at δ 4.3–4.9 corresponds to the β-configuration. As shown in the ¹H NMR spectrum (Figure 3A), the anomeric proton signals of CMP and the CMP-Ca complex exhibit the β-configuration, consistent with the results of the FT-IR spectrum. In the ¹³C NMR spectrum (Figure 3B), CMP shows a clear single peak within the anomeric carbon chemical shift range (95–110 ppm). The six strong signal peaks at δ 102.31, 83.87, 75.52, 73.27, 68.03, and 60.66 are attributed to C-1, C-3, C-5, C-2, C-4, and C-6 on the (1→3)-β-D-glucan ring, respectively. Combined with the HSQC spectrum of CMP, the H-6 / C-6 signals at δ 3.87 / 60.66 and δ 3.70 / 60.66 shift to δ 4.30 / 71.03 and δ 4.08 / 71.03, respectively, indicating the presence of a β-(1→6) side chain structure. Therefore, it is confirmed that CMP possesses a β-(1→3)-D-glucan backbone and β-(1→6) side chains. Three main linkage modes were identified and named units A, B, and C, respectively. The signal peak at δ 177.99 was attributed to the C=O group in the carboxymethyl group. The signal at δ 3.93 / 70.22 was attributed to the -CH2 group in the carboxymethyl fragment. Furthermore, the shift of the H-5 / C-5 signal from δ 3.46 / 75.52 to δ 3.58 / 74.35 indicates that carboxymethyl substitution mainly occurs at the hydroxyl group attached to the C-6 (R6) site of the CMP. The relative proportions of various linkage modes were determined by integrating the 1H and 13C cross-peaks. Calculations showed that the ratio of β-D-(1→3) to β-D-(1→6) glycosidic bonds in the CMP was approximately 4:1.

[0082] In the ¹H NMR (shown in Figure 3A) and ¹³C NMR (shown in Figure 3B) spectra of CMP-Ca complexes (5mM CMP-Ca, 10mM CMP-Ca, and 20mM CMP-Ca), four new proton signals were detected at δ values ​​of 2.70, 2.67, 2.52, and 2.49, respectively. A new carbon signal was also observed at δ 44.80, compared to CMP. Combined with HSQC spectral data (e.g., ... Figure 4As shown in Figure A), the relevant signals of CMP-Ca complexes with different calcium chelation contents are summarized as follows: For 5 mM CMP-Ca, the signal peaks are at δ 4.79 / 102.57 (H-1 / C-1), 3.79 / 84.13 (H-3 / C-3), 3.50 / 75.56 (H-5 / C-5), 3.54 / 73.45 (H-2 / C-2), 3.50 / 68.11 (H-4 / C-4), 3.93 / 60.83, 3.73 / 60.83 (H-6 / C-6), 4.32 / 71.02, and 4.08 / 71.02; For the 10 mM CMP-Ca system, the detected signal peaks are at δ 4.74 / 102.87 (H-1 / C-1), 3.73 / 84.13, and 4.08 / 71.02. (H-3 / C-3), 3.46 / 75.56 (H-5 / C-5), 3.51 / 73.45, 3.54 / 68.11 (H-4 / C-4), 3.86 / 60.83 and 3.68 / 60.83 (H-6 / C-6), 4.30 / 71.03 and 4.08 / 71.03; for 20 mM CMP-Ca, the signal was recorded at δ 4.71 / 102.39 (H-1 / C-1), 3.70 / 84.02 (H-3 / C-3), 3.43 / 75.57 (H-5 / C-5), 3.48 / 73.71, 3.44 / 68.07 (H-4 / C-4), 3.84 / 60.66 and 3.65 / 60.66. (H-6 / C-6), 4.30 / 70.86, and 4.06 / 70.86. These results indicate that the CMP-Ca complexes with different calcium chelation contents all have β-(1→3)-D-glucan as the backbone and contain β-(1→6) side chains. After calcium ions bind to CMP, the H-6 / C-6 signal is enhanced and shifts from δ 3.87 / 60.83 and 3.70 / 60.83 (CMP) to δ 3.93 / 60.83 and 3.73 / 60.83 (5 mM CMP-Ca), δ 3.86 / 60.83 and 3.68 / 60.83 (10 mM CMP-Ca), and δ 3.84 / 60.66 and 3.65 / 60.66 (20 mM CMP-Ca).Furthermore, newly observed signals were observed at δ 2.70 / 44.82, 2.68 / 44.82, 2.53 / 44.66, and 2.44 / 44.66 (5 mM CMP-Ca), δ 2.68 / 44.82, 2.65 / 44.82, 2.49 / 44.66, and 2.46 / 44.66 (10 mM CMP-Ca), and δ 2.65 / 44.82, 2.62 / 44.82, 2.46 / 44.66, and 2.43 / 44.66 (20 mM CMP-Ca). Therefore, based on the above analysis, calcium ions successfully chelated with CMP, mainly through the carboxyl group (…). - COO-Ca 2+ -OOC - They form metallic coordination bonds, as shown in B in Figure 4.

[0083] Example 2 The stability of the CMP-Ca complex prepared in Example 1 was examined.

[0084] (1) Solution stability Solution stability The stability of CMP and CMP-Ca chelates in aqueous solution for 24 hours was evaluated using a Tabiscan stability analyzer. The horizontal axis represents the height of the sample cell, with the left end corresponding to the bottom and the right end to the top. Figure 5 As shown in Figure A, the CMP solution exhibited significant precipitation at the bottom, while the top became correspondingly clear. In contrast, CMP-Ca chelate solutions with varying calcium chelation contents maintained excellent stability throughout the test. The Tabiscan Stability Index (TSI) is a quantitative indicator reflecting the dispersion uniformity and stability of a solution system. A lower TSI value indicates better dispersibility and stability. Consistent with visual observation, the CMP-Ca chelate demonstrated significantly better dispersion uniformity than CMP, which is evidenced by its lower TSI value (e.g., ...). Figure 5 (As shown in B in the figure). This result confirms that Ca 2+ The ions successfully chelated with CMP, thereby modifying the intermolecular interactions of CMP molecules and enhancing the stability of the solution.

[0085] (2) Zeta potential like Figure 5 As shown in C, the zeta potentials of CMP and CMP-Ca complexes (containing different calcium chelation contents) are all negative. Notably, 5 mM CMP-Ca exhibits the highest absolute zeta potential. With increasing Ca chelation... 2+ As the concentration of ions increases, the absolute value of the zeta potential gradually decreases, indicating that lower levels of Ca... 2+Ion chelation helps improve the solution stability of CMP-Ca.

[0086] (3) Thermal stability like Figure 6 As shown in Figure A, the thermal degradation process of all samples underwent two main stages. The initial mass loss occurred below 100 °C, which may be attributed to the evaporation of adsorbed free water in the CMP and CMP-Ca complexes. The second significant mass loss occurred at 252.18 °C, 277.65 °C, 282.10 °C, and 323.67 °C, corresponding to CMP, 5 mM CMP-Ca, 10 mM CMP-Ca, and 20 mM CMP-Ca, respectively. This may be related to the random breaking of glycosidic bonds and the thermal decomposition of the polysaccharide backbone. Notably, the CMP-Ca complex exhibited rapid mass loss at a higher temperature than CMP, indicating that the complex has better thermal stability than CMP. The three CMP-Ca complexes showed subtle differences in thermal stability, attributed to their varying calcium chelating abilities. The enhanced thermal stability of the CMP-Ca complex may be due to the presence of Ca. 2+ The coordination of ions with CMP, in which Ca 2+ It replaced the Na group in the carboxyl group of the polysaccharide chain. + Or hydrogen atoms.

[0087] To verify the crystalline or amorphous properties of CMP and the CMP-Ca complex, and to investigate the effect of calcium chelating ability on the crystallinity of the CMP-Ca complex, XRD measurements were performed under conditions ranging from 5° to 80°. Figure 6 (As shown in B in the figure). The XRD patterns of CMP and CMP-Ca complex did not show obvious diffraction peaks, indicating that both CMP and CMP-Ca complex are amorphous substances. Broad diffraction peaks of CMP, 5 mM CMP-Ca, 10 mM CMP-Ca and 20 mM CMP-Ca were observed at 2θ values ​​of approximately 19.63°, 21.72°, 22.49° and 23.23°. Compared with CMP, the θ value of CMP-Ca complex increased, which indicates that the d value decreased according to Bragg's law (equation (3)). In addition, the θ value increased with increasing calcium chelate content, which may be attributed to the coordination reaction between calcium ions and CMP. CMP remained amorphous after coordination with calcium ions, which may be related to the complex chemical structure of CMP. Previous studies have shown that CaCl2 is a crystalline substance and exhibits characteristic diffraction peaks at approximately 2θ = 33°. However, no comparable peaks were detected in the XRD pattern of the CMP-Ca complex. These results further confirm that calcium ions and CMP are not merely physically mixed, but rather that Ca... 2+Ions were successfully integrated into CMP, forming a novel polysaccharide-metal complex.

[0088] To identify the Ca involved in CMP 2+ The chelated functional groups were characterized by Fourier transform infrared spectroscopy (FTIR) for CMP and its CMP-Ca complexes (e.g., Figure 6 (As shown in C). 3435.35 cm⁻¹ in the CMP spectrum. -1 The broad peak at that point corresponds to the OH stretching vibration. Ca 2+ After binding with CMP, it was observed at 3392.75 cm⁻¹ (5 mM CMP-Ca) and 3404.42 cm⁻¹. -1 (10 mM CMP-Ca) and 3400.94 cm -1 A broad and intense peak was observed at (20 mM CMP-Ca). Compared to CMP, the OH stretching vibration peak of the CMP-Ca complex shifted to a lower wavenumber and became sharper and more intense. For CMP, the peak was at 1327.77 cm⁻¹. -1 The peak at [value missing] is attributed to the bending vibration of OH. The corresponding peaks for 5 mM CMP-Ca, 10 mM CMP-Ca, and 20 mM CMP-Ca appear at 1328.95 cm⁻¹, respectively. -1 1331.68 cm -1 and 1331.81 cm -1 Location. Similarly, 2922.00 cm. -1 The peak at 2914.45 cm⁻¹ is attributed to the CH stretching vibration in the CMP. -1 2913.99 cm -1 and 2914.29 cm -1 In addition, 1605.04 cm in CMP. - The peak at ¹ is attributed to the C=O stretching vibration of the carboxyl group, which shifts to 1601.44 cm⁻¹ in 5 mM CMP-Ca, 10 mM CMP-Ca, and 20 mM CMP-Ca, respectively. -1 The ions were transferred to 1601.44 cm⁻¹ in 5 mM CMP-Ca, 10 mM CMP-Ca, and 20 mM CMP-Ca, respectively. -1 1597.93 cm -1 and 1597.56 cm -1 This provides direct evidence for the involvement of carboxyl groups in the chelation process. CMP and CMP-Ca complexes are located at 1424.73–1427.80 cm⁻¹. -1The peak at [value] is attributed to the CH stretching vibration of the -COOCH2- group. Furthermore, the peak values ​​of CMP and CMP-Ca complexes are in the range of 1069.5–1077.51 cm⁻¹. -1 The absorption peak at 889.51-891.92 cm⁻¹ indicates the presence of a pyranose ring. The CMP and CMP-Ca complex show absorption peaks in the range of 889.51-891.92 cm⁻¹. -1 The absorption peak observed is a typical characteristic of β-D-glucosidic bonds. Previous studies have shown that in polysaccharide-Fe(III) complexes, Fe(III) is mainly coordinated with the carboxyl group. 2+ The ions form coordination bonds with the carboxyl groups in CMP, thereby generating O-Ca-O bonds that connect the CMP chains and promoting the formation of the CMP-Ca complex.

[0089] Example 3 The rheological properties of the CMP-Ca complex prepared in Example 1 were examined.

[0090] (1) Hydrogel forming ability like Figure 7 As shown in Figure A, CMP failed to form a gel across the entire tested concentration range (30-80 mg / mL). In contrast, the CMP-Ca complex exhibited gel-forming ability at different concentrations: 80 mg / mL for 5 mM CMP-Ca, 60 mg / mL for 10 mM CMP-Ca, and 50 mg / mL for 20 mM CMP-Ca. Notably, no gel formation was observed when 20 mM CaCl2 was directly mixed with an 80 mg / mL CMP solution (e.g., ...). Figure 7 (As shown in B in the diagram). Therefore, the unique gel properties of the CMP-Ca complex originate from Ca. 2+ The ions successfully chelated with CMP. This conclusion is consistent with that of Ca. 2+ Different ions have different effects on sodium alginate. Adding CaCl2 directly to a sodium alginate solution will lead to rapid gel formation.

[0091] (2) Analysis of flow behavior like Figure 7Figure C shows the apparent viscosity curves of CMP and the CMP-Ca complex at concentrations of 30, 40, 50, 60, and 80 mg / mL. Both CMP and the CMP-Ca complex exhibit shear-thinning non-Newtonian fluid characteristics. The apparent viscosity of both CMP and the CMP-Ca complex gradually increases with increasing solution concentration. This phenomenon can be attributed to the arrangement of the polysaccharide chains in solutions of different concentrations. In low-concentration systems, the chains are randomly dispersed with minimal inter-chain contact, resulting in intramolecular hydrodynamic interactions only within individual polymer chains. Conversely, in high-concentration systems, the polysaccharide chains extensively overlap and entangle, promoting the formation of numerous intermolecular interactions. At a concentration of 30 mg / mL, the apparent viscosity of CMP is higher than that of the CMP-Ca complex. However, with increasing concentration, the apparent viscosity of the CMP-Ca complex gradually surpasses that of CMP. Furthermore, with increasing calcium chelate content, the apparent viscosity at the same concentration changes in the following order: 5 mM CMP-Ca < 10 mM CMP-Ca < 20 mM CMP-Ca. Appropriate concentration and calcium chelate content can increase the apparent viscosity of CMP-Ca hydrogels.

[0092] (3) LVR analysis Oscillatory amplitude scans were performed on CMP solution and CMP-Ca hydrogel to determine the boundary between the linear and nonlinear viscoelastic regions (e.g., Figure 8 (As shown in A in the figure). The linear viscoelastic region (LVR) was determined by plotting G′ against applied stress, within which the storage modulus (G′) is independent of the magnitude of the applied stress. It was observed that the LVR range of the CMP-Ca hydrogel expands with increasing calcium chelating ability. This phenomenon can be attributed to the fact that calcium ions involved in the chelating interaction enhance the G′ value and viscosity of the polysaccharide hydrogel, thereby enabling it to withstand greater deformation.

[0093] (4) Oscillation frequency scan G′ reflects the elastic strain energy temporarily retained during rheological testing, which can be recovered after the applied stress is removed. In contrast, the loss modulus (G″) represents the irreversible energy loss consumed to initiate material flow, which is mainly converted into heat generated by shear. When G′ is greater than G″, the sample exhibits predominantly elastic, solid-like rheological behavior. Conversely, when G′ is less than G″, the sample exhibits predominantly viscous, liquid-like rheological properties. As shown in Figure 8B, in the concentration range of 50-80 mg / mL, the G″ of CMP is consistently higher than G′ across the entire frequency range, indicating its liquid-like rheological behavior. For 5 mM, 10 mM, and 20 mM CMP-Ca solutions, the G′ values ​​exceed the G″ values ​​at concentrations of 80 mg / mL, 60 mg / mL, and 50 mg / mL, respectively, which is a recognized indicator of gelation (as shown in Figure 8CE). This result is consistent with the macroscopic appearance of the corresponding solutions (Figure 7A), further confirming the interaction between CMP and Ca. 2+ Effective ion chelation results in significant gel-forming properties. Notably, the critical gelation concentration of the CMP-Ca complex is closely related to its calcium chelating ability: the stronger the chelating ability, the lower the concentration threshold for gel formation. However, CMP lacks the ability to form a gel network.

[0094] Example 4 The self-healing properties of the CMP-Ca complex prepared in Example 1 were examined.

[0095] (1) Thixotropy like Figure 9 As shown in Figure A, the CMP-Ca hydrogel exhibits thixotropic behavior within the tested concentration range. With increasing calcium ion content chelated with CMP, the area of ​​the thixotropic ring increases at the same concentration. These phenomena can be attributed to Ca... 2+ The coordination between ions and carboxyl groups promotes the formation of a stable three-dimensional network structure. When shear stress decreases, the CMP-Ca hydrogel requires a longer time to recover its three-dimensional network. Therefore, the higher the calcium chelate content in CMP, the more significant the thixotropic behavior of the CMP-Ca hydrogel.

[0096] (2) Creep and Recovery like Figure 9 As shown in Figure B, the effect of different calcium chelating agent contents on the structural properties of CMP-Ca hydrogels was investigated. Creep reflects the strength of the internal structure of the gel, while recovery indicates its ability to resist sliding deformation. For CMP-Ca hydrogels, with increasing chelating agent concentration, γ eThe value shows a decreasing trend. This phenomenon indicates that higher chelating agent concentrations enhance the structural stability of the hydrogel, which may be attributed to the tight interweaving between polysaccharide chains under high concentration conditions. At a fixed concentration, with the increase of calcium chelation content in CMP-Ca hydrogel, γ e The value gradually decreased. This indicates that the increased calcium chelate content improved the gel's elasticity and enhanced the stability of the internal three-dimensional network structure. Notably, during the recovery phase, when the calcium chelate content in the CMP-Ca complex reached 58.57 mg / g, the γ-ray concentration decreased. R Exceeding γ e This result shows that the hydrogel can recover to its initial state after the applied stress (τ=0) is removed, further confirming the stability of the gel network structure. According to equation (3), it can be known that γ exists in the hydrogel during the recovery stage. d Therefore, it can be inferred that the polysaccharide chains have rearranged, giving the CMP-Ca hydrogel its self-healing ability.

[0097] (3) Three-stage structural restoration The self-healing ability of CMP-Ca hydrogel was quantitatively evaluated using a three-stage structural recovery experiment. Specifically, this method aims to investigate the self-healing ability of CMP-Ca hydrogel at low shear rates (1 s⁻¹). -1 (First stage), high shear rate (100 s) -1 (Second stage) and again at a low shear rate (1 s) -1 (Third stage) time-dependent viscosity changes (e.g.) Figure 9 (As shown in C in the diagram). In the first stage, the CMP-Ca hydrogel exhibits high viscosity, indicating that the primary network structure has been fully formed. In the second stage, a sudden application of a high shear rate leads to a sharp decrease in viscosity, reflecting the disruption of the gel network structure and the occurrence of shear thinning. Subsequently, in the third stage, the shear rate rapidly recovers to its initial low value, and the viscosity gradually recovers, corresponding to the reconstruction of the gel network. Specifically, the times required for 5 mM CMP-Ca hydrogel, 10 mM CMP-Ca hydrogel, and 20 mM CMP-Ca hydrogel to recover 75% of their original structure are 20.89 s, 19.83 s, and 24.04 s, respectively. These results demonstrate that CMP-Ca hydrogel exhibits rapid structural recovery capability.

[0098] This ability can be attributed to the metal-coordinate bonds formed when calcium ions chelate with carboxyl groups on the polysaccharide chains. Increased shear stress induces the breakage of these metal-coordinate bonds, which rapidly reform once the shear stress is removed. This dynamic “break-reconstruction” exchange process occurs over a specific timescale, demonstrating the self-healing capability of the CMP-Ca hydrogel network. Furthermore, Figure 9Figure D demonstrates the self-healing properties of CMP-Ca hydrogel. Specifically, when a CMP-Ca hydrogel is cut in half and the fresh cut is in contact at room temperature, the two halves of the gel rapidly recombine into a single unit. In summary, these results confirm that CMP-Ca hydrogel possesses a significant self-healing capability.

[0099] like Figure 9 E in the figure demonstrates the injectability of the CMP-Ca hydrogel. Therefore, Ca 2+ Ions formed metal coordination bonds with the carboxyl groups on the CMP chain through a chelation reaction, successfully preparing a hydrogel with both self-healing and injectable capabilities.

[0100] like Figure 9 As shown in F, the study indicates that with the chelation of Ca in CMP... 2+ With increasing ion content, the gel strength of CMP-Ca hydrogel significantly improved (P<0.05). This result confirms that Ca... 2+ The chelation of ions enhanced the structural stability of the gel network. These findings are consistent with rheological measurements, further confirming the role of Ca... 2+ Successful ion chelation plays a crucial role in regulating the mechanical properties of CMP-Ca hydrogels.

[0101] Furthermore, the pore structure morphology of the CMP-Ca hydrogel was visualized using scanning electron microscopy (SEM), and the spatial distribution of its elemental components was analyzed using energy dispersive spectroscopy (EDS). Figure 10 As shown in Figure A, the CMP-Ca hydrogel exhibits a uniform porous structure, and the pore size decreases with increasing calcium chelation content. This phenomenon is attributed to the presence of Ca. 2+ Specific coordination interactions between ions and carboxyl groups on polysaccharide chains. These interactions promote interchain crosslinking, thereby increasing the density and mechanical strength of the gel network. EDS results further reveal that Ca... 2+ Ions are mainly distributed in the internal cavities of the gel network. Figure 10 (As shown in B in the diagram).

[0102] In this invention, Ca 2+ Ions replace Na + Ions enter CMP and form metal coordination bonds with carboxyl groups ( - COO-Ca 2+ -OOC -The metal coordination bonds within the CMP-Ca complex form intramolecular calcium bridges in the aqueous system. These bridging structures, anchored to the classic "eggbox" framework, further promote the formation of a stable three-dimensional network structure, thereby driving the gelation of the CMP-Ca complex. Notably, not all carboxyl groups participate in the chelation reaction with calcium ions. Therefore, hydrogen bonds still exist in the gel, and these interactions collectively enhance the stability of the hydrogel network structure. The self-healing ability of the hydrogel formed by the CMP-Ca complex can be attributed to Ca... 2+ The dynamic reversibility of metal coordination interactions between ions and carboxyl groups. Specifically, the fracture of hydrogels leads to the partial loss of Ca2+. 2+ The ion dissociates from its original coordination bond with the carboxyl group. Subsequently, the reconnection of the broken interface allows the free Ca to dissociate. 2+ The ions re-establish coordination bonds with the exposed carboxyl groups, thereby restoring the structural integrity of the hydrogel. Figure 11 shows a schematic diagram of the gelation and self-healing mechanism of CMP-Ca hydrogel.

[0103] Example 5 The hemostatic properties of the CMP-Ca complex prepared in Example 1 were examined.

[0104] (1) In vitro coagulation performance test Accurately pipette 100 μL of anticoagulated rat blood into a 1 mL EP tube, and add the same volume of 0.1 mol / L [solvent name missing]. -1 After mixing the CaCl2 solution thoroughly, 50 μL of the prepared mixture was added dropwise to the surface of 5 mM CMP-Ca, 10 mM CMP-Ca, 15 mM CMP-Ca and 20 mM CMP-Ca chelate hydrogels. The mixture was preheated at 37°C for 5 min and the timing was started. The experimental phenomena were observed every few seconds until the blood coagulated and showed no signs of flow. This was the whole blood clotting time (BCT).

[0105] (2) Hemolysis test The CMP-Ca complex (1 mg / mL) was mixed with 2% erythrocyte suspension (20 μL) to prepare the test sample, which was then incubated at 37°C for 1 h. The sample was then centrifuged at 3000 r / min for 5 min, and the supernatant was collected. The absorbance at 540 nm was measured using a UV spectrophotometer. Distilled water was used as a positive control, and PBS solution was used as a negative control. The hemolysis rate of the hydrogel was calculated using the following formula: Hemolysis rate (%) = ×100%.

[0106] like Figure 12As shown, the whole blood coagulation times of the chelate hydrogels of 5 mM CMP-Ca, 10 mM CMP-Ca and 20 mM CMP-Ca were 232 s, 157 s and 46 s, respectively, which were significantly lower than those of the negative control group (591 s), indicating that the prepared hydrogels have a certain procoagulant effect.

[0107] Hemolysis refers to the process by which red blood cells (RBCs) are damaged and release hemoglobin due to the interaction between RBCs and biomaterials or extracts of biomaterials in physiological saline. Hemolysis testing is also a commonly used method to determine the blood compatibility of biomedical materials. Generally, a hemolysis rate of less than 5% is considered acceptable for application. Figures 13-14 It can be seen that the hemolysis rate in the positive control test (distilled aqueous solution) was 100%, and the hemolysis rate of different CMP-Ca chelate hydrogel groups was close to 0%. This indicates that the prepared hydrogel meets the safety requirements for biomaterial applications.

[0108] For any points not covered above, existing technologies shall apply.

[0109] Although specific embodiments of the present invention have been described in detail by way of examples, those skilled in the art should understand that the above examples are for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to replace them, without departing from the direction of the invention or exceeding the scope defined by the appended claims. Those skilled in the art should understand that any modifications, equivalent substitutions, improvements, etc., made to the above embodiments based on the technical essence of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing carboxymethyl Poria cocos polysaccharide hydrogel, characterized in that, Includes the following steps: A polysaccharide polymer of Poria cocos with a degree of carboxymethyl substitution of 0.65~0.70 and sodium citrate were dissolved in water to obtain a polysaccharide solution; Calcium chloride was added to the polysaccharide solution, the pH was adjusted with an alkaline solution, and the reaction was stirred under heating conditions. After the reaction was completed, the first supernatant was collected. The mass ratio of the Poria cocos polysaccharide polymer to the calcium chloride was (111~444):1000. An alcohol solution was added to the first supernatant for low-temperature precipitation. The mixed solution after alcohol precipitation was subjected to solid-liquid separation to collect the precipitate. The precipitate was then dissolved again to obtain a reconstituted product. The reconstituted product was purified by dialysis to remove free ions, and then the resulting dialysate was freeze-dried to obtain the CMP-Ca complex, namely the carboxymethyl poria cocos polysaccharide hydrogel.

2. The preparation method according to claim 1, characterized in that, The mass ratio of the Poria cocos polysaccharide polymer to the sodium citrate is 1:(0.6~0.8).

3. The preparation method according to claim 1, characterized in that, The alkaline solution is a NaOH solution with a concentration of 0.1~0.2 mol / L, and the pH is adjusted to 9.5~10.

5.

4. The preparation method according to claim 1, characterized in that, The heating conditions are 65~75℃, and the stirring reaction time is 2~4h.

5. The preparation method according to claim 1, characterized in that, The alcohol solution is an ethanol solution with a volume fraction of 90%~98%; the volume ratio of the added ethanol solution to the first supernatant is (9~10):1; the low-temperature precipitation is carried out at a temperature of 2~8℃ for 18~30h.

6. The preparation method according to claim 5, characterized in that, The solid-liquid separation is performed by centrifugation, with a rotation speed of 3500~4500 rpm / min and a centrifugation time of 10~20 min.

7. The preparation method according to claim 1, characterized in that, The dialysis purification process uses dialysis bags with a molecular weight cutoff of 3000~4000 Da and a dialysis time of 48~96 h.

8. A carboxymethyl poria cocos polysaccharide hydrogel prepared by the preparation method according to any one of claims 1-7.

9. The carboxymethyl Poria cocos polysaccharide hydrogel according to claim 8, characterized in that, The carboxymethyl poria cocos polysaccharide hydrogel, after being sheared and damaged, requires no more than 25 seconds to self-heal and recover to more than 75% of its original structure.

10. The use of the carboxymethyl poria cocos polysaccharide hydrogel according to claim 8 or 9 in the preparation of hemostatic medical dressings.

Images