A hydrogel based on radix trichosanthis crude polysaccharide and a preparation method and application thereof

Abstract

The present application relates to a kind of water gel based on crude polysaccharide of Radix Trichosanthis and its preparation method and application.The water gel based on crude polysaccharide of Radix Trichosanthis is prepared by the following method:1) preparing crude polysaccharide of Radix Trichosanthis;2) making aldehyde group of crude polysaccharide of Radix Trichosanthis to obtain aldehyde group of crude polysaccharide of Radix Trichosanthis;And 3) aldehyde group of crude polysaccharide of Radix Trichosanthis and carboxymethyl cellulose are covalently combined to form the water gel by Schiff base reaction.The multifunctional water gel of the present application is expected to become the candidate dressing of clinical protection and treatment of cachexia wound and other immunosuppressive chronic wound.

Description

Technical Field

[0001] This application belongs to the field of polysaccharide drugs, specifically relating to a hydrogel based on crude polysaccharide from Trichosanthes kirilowii pollen, its preparation method, and its uses. Background Technology

[0002] Cachexia, also known as cachexia syndrome, is a worsening state of chronic diseases such as cancer, AIDS, and inflammatory diseases. Its characteristics include weight loss, decreased albumin synthesis, anemia, immunosuppression, and poor wound healing. Wounds caused by surgery or bedsores in cachexia patients are difficult to heal due to inhibited collagen synthesis, weakened immunity, and recurrent local inflammation, severely impacting their quality of life and posing a major challenge for clinical healthcare professionals. [1] Developing safe and effective wound dressings for cachexia is key to solving the problem.

[0003] Wound healing is divided into four stages: the coagulation phase, the inflammation phase, the proliferative phase, and the scar remodeling phase. The coagulation phase occurs immediately after the wound appears, when platelets aggregate at the wound site to promote hemostasis. The inflammation phase primarily involves clearing pathogenic microorganisms and necrotic tissue. In cachexia, due to insufficient immunity, the inflammation phase can be prolonged, manifested as persistent infiltration of inflammatory factors such as TNF-α and IL-6 at the wound site. [2] During the proliferative phase, shortly after the onset of inflammation, new cells, fibroblasts, endothelial cells, and other cells appear at the wound site, along with newly formed capillaries. These elements collectively constitute proliferative granulation tissue, filling and covering the wound, thus forming a scar. In cachexia, protein synthesis is inhibited, and collagen production is hindered, leading to delayed wound healing. [3] During the scar remodeling phase, the wound has begun to heal, and the newly formed scar tissue gradually adjusts to physiological function over time and with changes in lifestyle, ultimately improving the appearance and function of the injured area. This stage is the longest; reportedly, scar remodeling and hyperplasia can take as long as 5–10 years. [4] Hypertrophic scar tissue not only affects appearance, but its likelihood of re-damage is also far greater than that of normal tissue, and it can also impair joint mobility in joints. Therefore, cytokines such as TNF-α, IL-6, and TGF-β are closely related to the healing of cachexia wounds and the process of scar hyperplasia. Studies have shown that TGF-β induces increased expression of the scar hyperplasia-related gene Engrailed-1 (En-1) in skin fibroblasts, exacerbating scar hyperplasia, while inhibiting En-1 expression can reduce scar hyperplasia.

[0004] Currently, an increasing number of plant polysaccharides are being used to prepare hydrogels because their superior pharmacological activity and good biocompatibility enable drug delivery strategies that combine carriers and drugs. [5]Hydrogels possess many superior properties, such as adjustable hardness (soft or hard), good biocompatibility, and slow drug release. [6] Suitable for use as a wound dressing, it can isolate infection and promote cell proliferation and tissue repair. [7] Polysaccharide hydrogels combine the properties of both polysaccharides and hydrogels, and can improve immunosuppression in cachectic wounds and promote tissue repair.

[0005] Trichosanthes root is the dried root of *Trichosanthes kirilowii* Maxim or *Trichosanthes rosthornii* Harms, belonging to the Cucurbitaceae family. It possesses properties of clearing heat and detoxifying, reducing swelling and draining pus, and tonifying deficiency and calming the mind. Based on this, it is necessary to study the effective polysaccharide components of Trichosanthes root and develop its hydrogel medicinal form, thereby creating products with potential applications in the treatment of cachectic wounds.

[0006] [1]NG MF Y.Cachexia-an intrinsic factor in wound healing[J].IntWound J, 2010,7(2):107-13.

[0007] [2] Qiu Genquan, Zhao Xusheng, Sun Ye, et al. Experimental study on the effect of Atractylodes macrocephala volatile oil on cancer cachexia [J]. Journal of Xi'an Jiaotong University (Medical Edition), 2006, (05): 477-9.

[0008] [3]ABE Y,IMAMURAK,OGAWAY,et al.Tumor-derived components wereresponsible for suppression ofornithine decarboxylase activity in the ratwounded skin[J].J Surg Oncol,1994,55(3):135-42.

[0009] [4]OGAWAR.Keloid and Hypertrophic Scars Are the Result of ChronicInflammation in the ReticularDermis[J].Int J Mol Sci, 2017,18(3).

[0010] [5]EL HOSARY R,EL-MANCY SMS,EL DEEB KS,et al.Efficient wound healing composite hydrogelusing Egyptian Avena sativa L.polysaccharidecontainingβ-glucan[J].International Journal of BiologicalMacromolecules,2020,149:1331-8.

[0011] [6]YANG Z,HUANG R,ZHENG B,et al.Highly Stretchable,Adhesive,Biocompatible,and AntibacterialHydrogel Dressings for Wound Healing[J].Advanced Science(Weinheim,Baden-Wurttemberg,

[0012] Germany), 2021, 8(8): 2003627.

[0013] [7]WU J,PAN Z,ZHAO ZY,et al.Anti-Swelling,Robust,and AdhesiveExtracellular Matrix-MimicckingHydrogel Used as Intraoral Dressing[J].AdvMater,2022,34(20):e2200115. Summary of the Invention

[0014] Technical Purpose

[0015] The technical objective of this invention is to provide a method for preparing pollen polysaccharide-chitosan (CMOT) hydrogel.

[0016] Another technical objective of this invention is to provide a pollen polysaccharide-chitosan hydrogel prepared by the above method.

[0017] Another technical objective of the present invention is to provide the use of the above-mentioned CMOT hydrogel in the preparation of a medicament for promoting the healing of cancer cachexia wounds.

[0018] Technical solution

[0019] On one hand, the present invention provides a method for preparing a pollen polysaccharide-chitosan (CMOT) hydrogel, the method comprising the following steps:

[0020] 1) Preparation of crude polysaccharide from Trichosanthes kirilowii pollen:

[0021] 1-1) Extract the powder of Trichosanthes kirilowii using ultrasonic water, concentrate it, and centrifuge to remove insoluble starch;

[0022] 1-2) Add anhydrous ethanol to the extract from which insoluble starch has been removed, let stand, and centrifuge to remove the supernatant;

[0023] 1-3) Dissolve the precipitate after removing the supernatant by adding water, and then add trifluoroacetic acid to precipitate the protein;

[0024] 1-4) The supernatant after protein precipitation was placed in a 7kD dialysis bag for dialysis to remove trifluoroacetic acid, concentrated and lyophilized to obtain crude polysaccharide from Trichosanthes kirilowii.

[0025] 2) Aldehydeation of crude polysaccharides from Trichosanthes kirilowii:

[0026] Sodium periodate, TEMPO (2,2,6,6-tetramethylpiperidine-1-oxy radical), or manganese dioxide (MnO2) were added to the crude polysaccharide of Trichosanthes kirilowii prepared in step 1) and the reaction was carried out in the dark to obtain aldehyde-modified crude polysaccharide of Trichosanthes kirilowii.

[0027] 3) Preparation of CMOT hydrogel:

[0028] The aldehyde-modified trichosanthes pollen crude polysaccharide obtained in step 2) is covalently bonded to carboxymethyl chitosan via a Schiff base reaction to form a CMOT hydrogel.

[0029] In a specific implementation, in step 1-1), the powder of Trichosanthes kirilowii is soaked in water for 0.5-2 hours and then ultrasonically extracted at 35-50°C for 1-3 hours.

[0030] In a specific implementation, in step 1-1), the powder of Trichosanthes kirilowii is extracted in water by ultrasonic extraction, then the supernatant is collected by centrifugation, and the precipitate residue is extracted again by ultrasonic extraction in a water bath. The extracts from the two extractions are combined.

[0031] In a specific embodiment, in step 1-2), anhydrous ethanol is added to the extract after removing insoluble starch so that the volume fraction of ethanol reaches more than 85%.

[0032] In a specific embodiment, in step 2), sodium periodate is added to the crude polysaccharide of Trichosanthes kirilowii prepared in step 1), and a light-protected reaction is carried out at 20-35°C to generate aldehyde-modified crude polysaccharide of Trichosanthes kirilowii. The molar ratio of crude polysaccharide of Trichosanthes kirilowii to sodium periodate is 1:10 to 1:1000.

[0033] In a specific embodiment, in step 2), the oxidation degree of the aldehyde-modified crude polysaccharide of Trichosanthes kirilowii is 1 to 5 mmol / g TPS, preferably 5 mmol / g TPS, wherein the oxidation degree (DO) refers to the number of moles of aldehyde groups oxidized per gram of polysaccharide.

[0034] In a specific embodiment, in step 3), the degree of deacetylation of carboxymethyl chitosan is ≥90%.

[0035] In a specific embodiment, in step 3), an aqueous solution of carboxymethyl chitosan is added to the aqueous solution of aldehyde-modified pollen polysaccharide. The concentration of the aqueous solution of aldehyde-modified pollen polysaccharide is 1%–3%, and the concentration of the aqueous solution of carboxymethyl chitosan is 1%–3% (g / mL), with a volume ratio of 1:1. The ratio of carboxymethyl chitosan to aldehyde-modified pollen polysaccharide affects the performance and function of the hydrogel. As the proportion of aldehyde-modified pollen polysaccharide increases, the hardness of the formed hydrogel increases, and the swelling performance of the hydrogel decreases.

[0036] In a specific embodiment, in step 3), the amino groups in carboxymethyl chitosan react with the dialdehyde groups of aldehyde-modified pollen polysaccharide to form C=N bonds and achieve cross-linking.

[0037] In a specific implementation, in step 3), the crosslinking time is 0.5 to 3 minutes.

[0038] On the other hand, the present invention provides a pollen polysaccharide-chitosan hydrogel prepared by the above method.

[0039] On the other hand, the present invention provides a pharmaceutical composition comprising at least the above-mentioned pollen polysaccharide-chitosan hydrogel and pharmaceutically acceptable excipients.

[0040] In a specific embodiment, the pharmaceutical composition is in the form of a hydrogel dressing, a hydrogel patch, or a hydrogel spray.

[0041] In another aspect, the present invention provides the use of the above-mentioned pollen polysaccharide-chitosan hydrogel or the above-mentioned pharmaceutical composition in the preparation of a medicament for treating cachectic wounds and other immunosuppressive chronic trauma.

[0042] In specific embodiments, the above-mentioned pollen polysaccharide-chitosan hydrogel or the above-mentioned pharmaceutical composition can promote the regeneration of blood vessels, collagen and epithelial tissues and reduce inflammation.

[0043] In specific embodiments, the above-mentioned pollen polysaccharide-chitosan hydrogel or the above-mentioned pharmaceutical composition can regulate the local immune microenvironment of cachexia wounds.

[0044] In specific embodiments, cachexia includes cachexia caused by cancer, AIDS, or inflammatory diseases.

[0045] In a specific embodiment, the other immunosuppressive chronic trauma includes diabetic wounds.

[0046] Beneficial effects

[0047] This invention proposes the preparation of a safe, non-toxic, and biodegradable hydrogel as a wound dressing using Trichosanthes kirilowii pollen polysaccharide and chitosan as raw materials. This hydrogel treats cachexia-related, difficult-to-heal wounds by modulating local wound immunity and promoting epithelial migration and granulation tissue growth. First, the polysaccharide TPS was extracted from Trichosanthes kirilowii pollen, and its monosaccharide composition, molecular weight distribution, and zeta potential were analyzed. TPS was oxidized with sodium periodate to form aldehyde-modified polysaccharide oTPS, which was then covalently bonded to carboxymethyl chitosan via a Schiff base reaction to form CMOT hydrogel. In a mouse model of full-thickness skin wounds associated with cancer cachexia, the CMOT hydrogel exhibited a good promoting effect on wound healing by modulating the local immune microenvironment of the cachexia wound.

[0048] This application further demonstrates that *Trichosanthes kirilowii* polysaccharides possess immunomodulatory effects. The prepared *Trichosanthes kirilowii* polysaccharide hydrogel exhibits good biocompatibility and excellent hydrogel properties, including good healing performance, stable mechanical parameters, and water retention capacity. In a mouse model of full-thickness skin injury, the CMOT2 hydrogel with high oxidation degree showed superior effect to the CMOT1 hydrogel in accelerating wound healing. Furthermore, the CMOT2 hydrogel can effectively promote the healing of cancer cachexia wounds by modulating the inflammatory microenvironment at the wound site, promoting the regeneration of blood vessels, collagen, and epithelial tissue. Therefore, the multifunctional hydrogel dressing CMOT shows promise as a potential candidate dressing for the clinical protection and treatment of cachexia wounds and other immunosuppressive chronic wounds.

[0049] In a mouse model of full-thickness skin injury, CMOT2 hydrogel, with its high oxidation state, demonstrated superior efficacy compared to CMOT1 hydrogel in accelerating wound healing. Furthermore, CMOT2 hydrogel effectively promotes the healing of cancer cachexia wounds by modulating the inflammatory microenvironment at the wound site, thereby facilitating the regeneration of blood vessels, collagen, and epithelial tissue. Therefore, the multifunctional hydrogel dressing CMOT shows promise as a potential candidate dressing for the clinical protection and treatment of cachexia wounds and other immunosuppressive chronic trauma. Attached Figure Description

[0050] Figure 1 Extraction of crude polysaccharide (TPS) from Trichosanthes kirilowii pollen and determination of total sugar content. (A) Extraction process of TPS; (B) Standard curve established using glucose as standard; (C) Number of extractions, and corresponding yield and sugar content results.

[0051] Figure 2 Characterization of TPS molecular weight. (A) Molecular weight distribution of TPS detected by HPGPC, (B) Molecular weight distribution of TPS by MALDI-TOF-MS, (C) Potential of TPS.

[0052] Figure 3 Preparation and characterization of aldehyde-modified Trichosanthes kirilowii polysaccharides (oTPS) with different oxidation degrees. (A) Principle of oTPS preparation; (B) Infrared spectra of oTPS and TPS; (C) Standard curve established using glutaraldehyde as a standard; (D) Aldehyde content and sodium periodate dosage ratio in different oTPS; (E) Schiff reagent test results of oTPS and TPS; (F) Reaction principle of residual sodium periodate in sodium iodide detection system; (G) Detection results of residual sodium periodate.

[0053] Figure 4 Preparation, healing properties, and porous structure characterization of Trichosanthes kirilowii polysaccharide-chitosan (CMOT) hydrogel. (A) Schematic diagram of CMOT hydrogel preparation principle, (B) Solid state and healing property characterization results of CMOT hydrogel, (C) SEM image of CMOT hydrogel, scale bar = 300 μm.

[0054] Figure 5 Characterization of CMOT hydrogels in terms of swelling (A), degradation (B), water retention (C), and mechanical properties (D, E) (n=3).

[0055] Figure 6 Infrared spectral characterization of CMOT hydrogel.

[0056] Figure 7 Results of antioxidant studies on CMOT hydrogel. (A) Photograph of the results of superoxide anion radical scavenging, (B) Statistical analysis of superoxide anion radical scavenging activity, (C) Statistical analysis of DPPH radical scavenging activity (n=3).

[0057] Figure 8 Biocompatibility results of CMOT. (A) Procedure for administration of CMOT extract to RAW 264.7 cells, (B) Calcein / PI staining images of RAW 264.7 cells after administration of CMOT extract (scale bar = 100 μm), and (C, D) flow cytometry plots, (E) CCK8 assay for toxicity of CMOT extract to C2C12 cells and BMDM cells, (F) hemolytic activity of CMOT hydrogel, (G) HE section of subcutaneous tissue after subcutaneous implantation of CMOT hydrogel (scale bar = 300 μm), n = 3.

[0058] Figure 9Inhibitory activity of CMOT against Staphylococcus aureus (A, C) and Escherichia coli (B, D) (n=3).

[0059] Figure 10 Results of CMOT's immunomodulatory activity. Effects of CMOT on (A) M1 macrophages and (B) M2 macrophages and (C) their relative ratio, and on (D) CD3... + (E)CD49b + (F)CD8 + The effect of lymphocyte Ki67 proliferation factor expression (n=3).

[0060] Figure 11 Comparison of the effects of CMOT hydrogels with different oxidation levels on promoting skin healing in mice. (A) Animal experimental protocol for skin healing, (B) Photographs of mouse skin healing, (C) Statistics on skin healing rate, (D) HE-stained and Masson-stained sections of skin, scale bar = 250 μm, (E) Statistical analysis of collagen volume fraction in skin based on Masson staining of skin sections, (n = 3).

[0061] Figure 12 (A) Schematic diagram of the experimental protocol for treating non-healing wounds in cachectic mice with CMOT hydrogel, (B) Changes in food intake between normal mice and C26 tumor mice, (C) Changes in tumor volume in C26 tumor mice, (D) Changes in body weight without tumors and (E) Changes in body weight, (F) Images of wound healing in cachectic mice and (G) Statistics on wound healing rate, (n=3).

[0062] Figure 13 Results of CMOT hydrogel promoting skin healing in cachectic mice. (A) HE-stained images of skin wound sections from cachectic mice at different application times, (B) Masson staining images, (C, D) immunohistochemical images of IL-6 and TNF-α, and (E, F) statistical analysis of immunohistochemical images, scale bar = 100 μm, (n = 3). Detailed Implementation

[0063] The technical content of this application is described in detail below through specific embodiments to enable those skilled in the art to better understand this application. However, the provision of these embodiments is not intended to constitute any limitation on the scope of protection of this application.

[0064] Example 1: Extraction and Characterization of Trichosanthes kirilowii Polysaccharides

[0065] 1. Materials and Instruments

[0066] Material

[0067] Trichosanthes kirilowii root slices (provided free of charge by Bozhou Yonggang Medicinal Herbs Factory Co., Ltd.)

[0068] Anhydrous ethanol, trichloroacetic acid (TCA), phenol, concentrated sulfuric acid (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China)

[0069] Dextran dextran with different molecular weights (molecular weights of 6100, 9600, 21100, 47100, 107000, 194000, 337000, and 642000 Da, respectively) (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China)

[0070] Acetonitrile, trifluoroacetic acid, sinapic acid (SA), Congo red (Shanghai Saiyi Biotechnology Co., Ltd., Shanghai, China)

[0071] instrument

[0072] DK-S26 Thermostatic Water Bath (Shanghai Hualian Medical Instrument Co., Ltd., Shanghai, China)

[0073] SBL water bath ultrasonic instrument (Ningbo Xinzhi Biotechnology Co., Ltd., China)

[0074] MALDI-TOF-MS AB Sciex 5800 mass spectrometer (Framingham, Massachusetts, USA)

[0075] High-performance liquid chromatograph (HPLC) Agilent 1260, KS804-KS802 column (Agilent, California, USA)

[0076] Multiskan full-wavelength microplate reader (Thermo Fisher Scientific, Massachusetts, USA)

[0077] MS105DU electronic balance with a strength of 0.0000 (Mettler-Toledo Instruments GmbH, Zurich, Switzerland)

[0078] BDS HYPESIL C18 column (250 × 4.60 mm, 6 μm) (Thermo Fisher Scientific, Massachusetts, USA)

[0079] Malvern Zetasizer Nano-90ZS laser particle size analyzer (Malvern, UK)

[0080] Gary 630 infrared spectrometer (Agilent, California, USA)

[0081] 2 Experimental Methods

[0082] 2.1 Extraction of Trichosanthes polysaccharides (TPS)

[0083] The *Trichosanthes kirilowii* herb was pulverized into powder using a grinder and stored in a sealed container at room temperature. 150g of the powder was weighed into a 1L Erlenmeyer flask, and ultrapure water was added at a ratio of 50g / 250mL. The mixture was soaked for 30 minutes and then ultrasonically extracted in a 45℃ water bath for 2 hours, with continuous stirring during the ultrasonic process to improve extraction efficiency. After extraction, the supernatant was collected by centrifugation at 15000×g. The precipitate was washed with 100mL of water and ultrasonicated for 10 minutes. The supernatant was collected again by centrifugation, and this process was repeated once more. The extract and washings were combined and concentrated in a 60℃ water bath to a concentration of 50g of herb per 50mL volume. Insoluble starch was removed by centrifugation. Anhydrous ethanol was added to bring the ethanol volume fraction to 85%, and the mixture was shaken thoroughly and allowed to stand overnight. Centrifuge and discard the supernatant. Dissolve the precipitate in 10 mL of water and add trichloroacetic acid (TCA) to achieve a mass-volume fraction of 6%. Incubate overnight at 4°C to precipitate the protein. Centrifuge to remove the protein precipitate. Add 1 g of TCA again to precipitate for 3 hours. No protein precipitate is found. Place the supernatant in a 7 kDa dialysis bag and dialyze for 3 days to remove TCA. Concentrate in a water bath to 120 mL and freeze-dry to obtain total polysaccharide TPS, which is also referred to as "Crude Polysaccharide of Trichosanthes kirilowii" in this application.

[0084] 2.2 Determination of Total Sugar Content in TPS by Phenol-Sulfuric Acid Method

[0085] Detection principle: Polysaccharides are first hydrolyzed into monosaccharides under the action of sulfuric acid, and then rapidly dehydrated to generate uronic acid derivatives. These derivatives then react with phenol to form an orange-yellow compound, which has maximum absorption at 490 nm.

[0086] 2.2.1 Preparation of glucose standard solution and 5% phenol solution

[0087] Preparation of D-glucose standard solution: Weigh 10 mg of glucose standard dried to constant weight and dilute to 100 mL in a volumetric flask to obtain glucose standard solution (0.1 mg / mL).

[0088] Preparation of 5% phenol solution: Weigh 5.0g of phenol, dissolve it in heated distilled water, and dilute to 100mL in a brown volumetric flask to obtain a 5% phenol solution.

[0089] 2.2.2 Construction of the glucose standard curve

[0090] Accurately pipette 0.1 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL of glucose standard solution into 10 mL test tubes, respectively. Add distilled water to a final volume of 1 mL, then add 1 mL of 5% phenol solution and 5 mL of concentrated sulfuric acid to each tube. Shake for 2 min, incubate in a 100°C water bath for 20 min, remove and cool to room temperature. Use 1.0 mL of distilled water as a blank control. Measure the absorbance at 490 nm and plot a standard curve.

[0091] 2.2.3 Polysaccharide content determination

[0092] Accurately weigh 0.01 g of dried pollen polysaccharide to constant weight, and dilute to a volumetric flask of 100 mL. Accurately transfer 1.0 mL of the solution to a test tube, add 1 mL of 5% phenol solution, and then quickly add 5 mL of concentrated sulfuric acid. Shake thoroughly and incubate at 100 °C for 20 min in a water bath. After cooling to room temperature, use 1.0 mL of distilled water as a blank control and measure the absorbance at 490 nm. Substitute the absorbance into the standard curve to calculate the polysaccharide content.

[0093] Polysaccharide yield (%) = M crude polysaccharide / M medicinal material × 100

[0094] Polysaccharide content (%) = M0 / M crude polysaccharide × 100

[0095] (M0 represents the mass of the polysaccharide obtained by substituting it into the standard curve)

[0096] 2.3 Detection of TPS molecular weight by HPLC

[0097] Weigh 20 mg of each standard molecular weight polysaccharide, dissolve in 10 mL of 0.2 M NaCl mobile phase, centrifuge (10000×g, 10 min), and collect the supernatant for later use. Weigh 20 mg of TPS sample, dissolve in 10 mL of 0.2 M NaCl, centrifuge (10000×g, 10 min), and collect the supernatant for later use. Inject the standard and sample solutions sequentially, determine the corresponding liquid chromatograms, and calculate the molecular weight distribution of the samples using GPC software.

[0098] 2.4 Detection of TPS molecular weight by MALDI-TOF-MS

[0099] Weigh 10 mg of TPS, dissolve in 1 mL of ultrapure water, centrifuge at 10000 × g for 5 min, and collect the supernatant. Weigh 10 mg of CHCA, dissolve in 1 mL of acetonitrile (containing 0.1% trifluoroacetic acid), centrifuge, and collect the supernatant. Mix 10 μL of TPS solution with an equal volume of SA solution, and spot 1 μL into the well of the MALDI-TOF-MS sample plate. Allow to evaporate naturally before loading onto the instrument and detect in "Linear mid-mass positive" mode.

[0100] 2.5 TPS zeta potential detection

[0101] Add 1 mL of a 2 mg / mL TPS solution (prepared with ultrapure water) to a potentiometer. Measure the potential of the TPS solution using the potential detection mode of a Malvern particle size analyzer. Repeat the detection three times.

[0102] 3 Results and Analysis

[0103] 3.1 Extraction of TPS and determination of sugar content

[0104] Polysaccharides were extracted using a water bath ultrasonic method, with the temperature controlled at approximately 40℃ to avoid damaging the polysaccharide structure. The addition of ethanol to the polysaccharide extract produced a large amount of precipitate with high viscosity, indicating successful extraction. However, the precipitate still contained pigments and water-soluble proteins that needed to be removed. The precipitate was redissolved in water, and the addition of trichloroacetic acid at low temperature produced a large amount of protein precipitate until no new precipitate formed, indicating that the protein had been largely removed. Experimental results showed that the standard curve R of the phenol-sulfuric acid method... 2 A value of 0.999 indicates a good linear relationship. The extraction method was repeated three times, yielding an average TPS yield of 13.7% (RSD 4.4%) and an average sugar content of 90.6% (RSD 5.6%). See [link to extraction details]. Figure 1 This indicates that the extraction and detection method is relatively stable and can be used to evaluate polysaccharide yield and content.

[0105] 3.2 Characterization of TPS

[0106] The molecular weight of the polysaccharide was characterized using high-performance gel permeation chromatography (HPGPC), and the results are as follows: Figure 2 As shown in Figure A, the TPS polysaccharide has two molecular weight distribution peaks. The first peak appearing in the liquid chromatography spectrum (far right) is the peak of the mobile phase NaCl. According to the molecular weight calculation using HPGPC software, the weight-average molecular weight of the first peak in the liquid chromatography spectrum is 5.7 kDa (middle), and the average molecular weight of the second peak is 72.1 kDa (far left), indicating that TPS contains two polysaccharides with different molecular weights. Using the sum of the two peak areas as the denominator and the area of ​​each peak as the numerator, Image J calculations show that the proportion of the first peak is 86.8%, and the proportion of the second peak is 13.2%.

[0107] Molecular weight characterization of TPS using MALDI-TOF-MS revealed that TPS eluted at approximately 53 kDa. Figure 2 B) represents the absolute molecular weight of the polysaccharide, but its response value is low, exhibiting a peak resembling a steaming top. Various matrix solutions were tried, including sinapic acid (SA), 5-hydroxysalicylic acid (DHB), and α-cyanocinonic acid (CHCA). SA showed the best effect, suggesting that the low ionic strength due to the limited number of active functional groups in the polysaccharide may be the cause of the steaming top peak.

[0108] The potential detection results show that TPS is a negative potential. Figure 2 C) The average potential is -7.98±0.23mV. This is due to the electrostatic repulsion between charges on the polysaccharide chain, or it may be due to the presence of ionizable acidic groups on the polysaccharide, such as carbonyl groups.

[0109] Example 2: Preparation and characterization of Trichosanthes kirilowii pollen polysaccharide-chitosan hydrogel (CMOT)

[0110] 1 Materials and Instruments

[0111] Material

[0112] Carboxymethyl chitosan (CMCS) (viscosity: 0.01–0.08 Pa·s, degree of carboxylation ≥80%, 150–800 kDa) (Shanghai Yuanye Biotechnology Co., Ltd., Shanghai, China)

[0113] Schiff's reagent, glutaraldehyde, sodium iodide, starch (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China)

[0114] Methylene blue, sodium periodate (NaIO4) (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China)

[0115] instrument

[0116] Gary 630 infrared spectrometer (Agilent, California, USA)

[0117] Scanning electron microscope Quanta 250FEG, FEI (Thermo Fisher Scientific, Massachusetts, USA)

[0118] Semi-micro dual-range balance ES225SM-DR(E) (Presica, Zurich, Switzerland)

[0119] Rheology tester MCR301 (Anton Paar, Graz, Austria)

[0120] ZQTY-70V Thermostatic Shaker (Shanghai Zhichu Instrument Co., Ltd., Shanghai, China)

[0121] Multiskan full-wavelength microplate reader (Thermo Fisher Scientific, Massachusetts, USA)

[0122] Inverted fluorescence microscope BX53 (Olympus, Tokyo, Japan)

[0123] 2. Experimental Methods

[0124] 2.1 Preparation of aldehyde-modified TPS

[0125] TPS was oxidized to aldehyde-modified TPS (oTPS) using a sodium periodate oxidation method. 1 g of TPS was weighed into separate EP tubes, dissolved in 20 mL of ultrapure water. Then, 50 mg of sodium periodate was weighed and dissolved in 1 mL of ultrapure water, and added to one TPS tube. The mixture was stirred at room temperature in the dark for 4 hours to obtain oTPS1 (TPS with a lower degree of oxidation). For the other TPS, 500 mg of sodium periodate was weighed and dissolved in 1 mL of water, and the mixture was added to the tube and reacted in the dark for 4 hours to obtain oTPS2. After the reaction was complete, 1 mL of ethylene glycol was added and stirring was continued for 1 hour to terminate the reaction. The product was dialyzed in a 14 kDa dialysis bag for 48 hours and then freeze-dried to obtain oTPS1 and oTPS2.

[0126] The Schiff reagent was used to detect the dialdehyde content in oTPS, with glutaraldehyde as the standard reference. The principle is that the condensate formed by the condensation of sulfite groups and aldehyde groups in the Schiff reagent reacts with a fuchsin solution, exhibiting maximum absorption at 550 nm. Glutaraldehyde standard solutions of different molar concentrations were prepared: 0, 1, 2, 4, 8, and 16 mM. 2 mg / mL oTPS1 and oTPS2 solutions were also prepared. 0.5 mL of the analyte was placed in a 10 mL EP tube, and 2.5 mL of Schiff reagent was added and mixed thoroughly. The absorbance was immediately measured at 550 nm. A standard curve was constructed with the glutaraldehyde concentration on the x-axis and the absorbance values ​​on the y-axis. The aldehyde content was then calculated by substituting the absorbance values ​​of the sample into the curve.

[0127] To detect whether there is residual sodium periodate in oTPS, the reaction of sodium periodate with sodium iodide to generate elemental iodine is used. The elemental iodine then undergoes a color reaction with a 1% starch solution for qualitative identification.

[0128] 2.2 Preparation of Trichosanthes kirilowii pollen polysaccharide-chitosan (CMOT) hydrogel

[0129] Weigh 3g of carboxymethyl cellulose (CMCS) and 3g of oTPS1 / oTPS2, add 100mL of ultrapure water and dissolve completely to obtain a 30mg / mL polysaccharide solution. Mix equal volumes of the CMCS and oTPS solutions thoroughly, and allow to stand to obtain CMOT hydrogel. CMOT1 hydrogel was prepared using oTPS1 and CMCS, while CMOT2 hydrogel was prepared by reacting oTPS2 with CMCS. The difference between the two hydrogels lies in the degree of oxidation of the pollen polysaccharides.

[0130] 2.3 Performance Characterization of CMOT Hydrogel

[0131] 2.3.1 Gelation Time

[0132] The gelation time of CMOT hydrogels was determined using the inverted vial method. The hydrogel was considered gelled if it stopped flowing within 30 seconds of inverting the vial. The time from preparation to gelation was recorded as the gelation time.

[0133] 2.3.2 FTIR Characterization

[0134] TPS (2 mg), oxidized TPS (2 mg), CMCS (2 mg), CMCS+TPS (physical mixture), and CMOT hydrogel were each mixed with KBr powder (100 mg), then pressed into tablets and analyzed by FTIR at 4000–400 cm⁻¹. -1 Analysis within the wavelength range.

[0135] 2.3.3 Characterization of self-healing properties

[0136] Different colored CMOT hydrogels were prepared: one using DMEM medium and the other using methylene blue solution, to investigate the self-healing properties of the CMOT hydrogels. Both gels were cut, and the resulting surfaces were brought into contact with each other without force for 20 minutes. Healing was then examined. Additionally, the CMOT hydrogels were preloaded into syringes and extruded using a 25G needle to check injectability.

[0137] 2.3.4 Scanning Electron Microscopy Characterization

[0138] After freeze-drying, the CMOT hydrogel was sectioned, a thin layer of gold was coated on the cross section, and observed under a scanning electron microscope (SEM).

[0139] 2.3.5 Swelling and Degradation

[0140] Swelling test: The freshly prepared CMOT hydrogel was weighed (recorded as W0) and immersed in 10 mL of PBS (pH 7.2) for swelling at 37°C. At regular intervals, the CMOT hydrogel was removed, residual water was blotted off with filter paper, and then the hydrogel was weighed (recorded as Wt). The swelling ratio (SR) was calculated using the following equation:

[0141]

[0142] Degradation test: Freshly prepared CMOT hydrogels were dried in an oven until the weight difference between two weighings did not exceed 0.3 mg (1 g sample), which was considered constant weight. The hydrogels were then weighed (recorded as W0) and immersed in 10 mL of PBS (pH 7.2) at 37°C and 220 rpm for degradation. The hydrogels were removed at regular intervals, dried to constant weight, and weighed (recorded as Wt). The degradation rate (DR) of the hydrogels was calculated as follows:

[0143]

[0144] 2.3.6 Water holding capacity

[0145] First, the freshly prepared CMOT hydrogel was weighed (recorded as W0). Then, the hydrogel was exposed to air at 25°C and weighed at predetermined intervals (recorded as Wd). The water holding capacity (WR) of the CMOT hydrogel was calculated using the following equation:

[0146]

[0147] 2.3.7 Rheological properties

[0148] At 25°C, the hydrogel was added into parallel plates (PP25, d = 1 mm), and the rheological properties of the CMOT hydrogel were measured using a stress-controlled rheometer (MCR301, Anton Paar, Austria) with frequency scanning (0.1 Hz–10 Hz) and strain scanning (0.01%–10%). Storage modulus is denoted by G', and loss modulus by G”.

[0149] 3. Results and Analysis

[0150] 3.1 Preparation and Characterization of oTPS

[0151] The principle of preparing aldehyde-modified TPS (oTPS) is as follows: Figure 3 As shown in Figure A, different amounts of sodium periodate were added to obtain aldehyde polysaccharides oTPS1 and oTPS2 with different degrees of oxidation.

[0152] The infrared spectroscopy results show that oTPS exhibits a characteristic absorption at 1630 cm⁻¹ of the aldehyde group. -1 Large absorption was observed nearby, while TPS absorption was relatively small. Figure 3 B indicates that the polysaccharide was successfully aldehyde-substituted.

[0153] The results of Schiff's reagent for the quantitative detection of aldehyde groups showed that the standard curve R established using glutaraldehyde as a standard was effective. 2 It is 0.98 ( Figure 3 C), the linear correlation between glutaraldehyde concentration and absorbance at 550 nm was good. The aldehyde content in the polysaccharide was directly proportional to the sodium periodate dosage ratio; the aldehyde content of oTPS2 was approximately twice that of oTPS1. Figure 3 D) The higher the degree of oxidation of the polysaccharide, the deeper the purplish-red color. (See...) Figure 3 E.

[0154] Sodium periodate reacts with sodium iodide in a redox reaction to produce elemental iodine (brown). Elemental iodine reacts with starch to produce a blue color. This reaction can be used to determine whether a system contains sodium periodate. See [link to relevant documentation]. Figure 3F. The results showed that no residual sodium periodate was found in the oxidized polysaccharide. Figure 3 G), thereby eliminating its influence on subsequent experiments.

[0155] 3.2 Preparation and Characterization of CMOT Hydrogel

[0156] Chitosan is rich in amino groups, and oTPS contains a large number of dialdehyde groups. The amino groups can undergo Schiff base cross-linking with the aldehyde groups. Figure 4 A) When the cross-linked units are large enough, a three-dimensional network structure, i.e., a hydrogel, is formed. Furthermore, carboxymethylated chitosan is soluble in water, oTPS is readily soluble in water, the preparation process is safe and non-toxic, and the prepared hydrogel has good biocompatibility. Two hydrogels, CMOT1 and CMOT2, with different mechanical properties were prepared by cross-linking carboxymethyl chitosan (CMCS) with two different oxidation states of pollen polysaccharides, oTPS1 and oTPS2. The results of the inverted test tube experiment showed that the cross-linking time of CMOT1 was approximately 60 s, while that of CMOT2 was approximately 30 s, indicating that increasing the aldehyde content of the polysaccharides can shorten the cross-linking time of the hydrogel and allow for faster hydrogel formation.

[0157] When two different colored hydrogels, CMOT1 and CMOT2, were cut and then reapplied, it was found that both hydrogels possessed self-healing capabilities, and the time required for self-healing was not significantly different. Figure 4 B).

[0158] Next, the microstructure of the two hydrogels was observed using scanning electron microscopy. It was found that both hydrogels possessed the porous network microstructure characteristic of hydrogels. The pore size of CMOT1 (150 μm) was larger than that of CMOT2 (89 μm), approximately twice that of CMOT2. Figure 4 C.

[0159] Water absorption swelling ratio is an important parameter for characterizing hydrogels. It can predict the initial volume of the hydrogel after injection or implantation in the body, and for drug-loaded hydrogels, it can also help predict drug release. Swelling results showed that CMOT1 and CMOT2 hydrogels were swelled and stable after 1 hour, with a swelling ratio of 60% of their own mass. The swelling ratio of CMOT1 was slightly higher than that of CMOT2. Figure 5 A, this indirectly proves that CMOT1 has a large aperture.

[0160] In vitro degradation results showed that the degradation outcomes of the two hydrogels were essentially the same, with complete degradation occurring in approximately two weeks, but the degradation behaviors differed. CMOT1 began rapid degradation on day 5, while the degradation of CMOT2 was relatively gradual, with rapid degradation only occurring on day 9. Figure 5 A).

[0161] Water is essential for maintaining healthy skin; therefore, water-holding capacity is a necessary performance indicator for hydrogels used on skin wounds. In vitro water loss rate results showed that the two CMOT hydrogels had similar and relatively slow water loss rates, maintaining a water holding capacity of over 50% for 2–3 days under overall exposure conditions. Figure 5 C).

[0162] G' is the elastic modulus; a higher value indicates better elasticity of the tested object. G” is the viscous modulus, which is directly proportional to the viscosity of the tested object. Mechanical test results show that within the frequency range of 0.1–10 Hz, the elastic modulus G' of CMOT2 is higher than that of CMOT1, indicating that the CMOT2 hydrogel has greater elasticity. The square root of the sum of the squares of G' and G” can be used to represent the hardness of the hydrogel. Within a certain range, the closer the two values ​​are, the larger the square root value, and vice versa. The viscous modulus G” of CMOT2 is similar to that of G', indicating that CMOT2 has high hardness. Conversely, the greater the difference between the values ​​of G' and G” of CMOT1, the lower the hardness of CMOT1. Figure 5 D. Similar results were obtained in tests with strain ranging from 0.1% to 10%, see... Figure 5 E indicates the relative stability of the two hydrogels under stress.

[0163] The chemical crosslinking of CMOT hydrogel was verified using infrared spectroscopy. Figure 6 The results showed that CMOT at 1647 cm -1 and 1628cm -1 Absorption bands corresponding to C=O and C=N tensile vibrations were observed at the 3200-3600 cm⁻¹, confirming successful cross-linking of the polysaccharide chains via C=N bonds. Furthermore, the CMOT hydrogel exhibited absorption bands at 3200-3600 cm⁻¹. -1 The signal intensity attributable to the OH / NH tensile vibration is lower than that of the CMCS+TPS physical mixture group, indicating that Schiff base C=N bonds were formed in the prepared hydrogel.

[0164] Test Example 1: Evaluation of the bioactivity and safety of CMOT

[0165] 1 Materials and Instruments

[0166] Material

[0167] DMEM culture medium (Shanghai Saiyi Biotechnology Co., Ltd., Shanghai, China)

[0168] CFDA-SE staining solution, Calcein / PI staining kit (Yisheng Biotechnology Co., Ltd., Shanghai, China)

[0169] CCK8 solution (Yisheng Biotechnology Co., Ltd., Shanghai, China)

[0170] Escherichia coli, Staphylococcus aureus (Beijing BioBio Biotechnology Co., Ltd., Beijing, China)

[0171] 1,1-diphenyl-2-picrylhydrazyl (DPPH) (MCE, New Jersey, USA)

[0172] Assay Kit for Inhibition and Generation of Superoxide Anion Radicals (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China)

[0173] instrument

[0174] Inverted fluorescence microscope BX53 (Olympus, Tokyo, Japan)

[0175] ACEA NovoCyte 3000 flow cytometer (Agilent, California, USA)

[0176] Cells and animals

[0177] C2C12 cells (number ZKCC-X2072) and RAW264.7 cells (number ZK0833(XR)) were provided by the Shanghai Cell Bank of the Chinese Academy of Sciences.

[0178] BALB / c mice (4–6 weeks old, female, SPF grade, 20–22 g) were provided by Jiangsu Huachuang Xinno Pharmaceutical Technology Co., Ltd., Animal Qualification Certificate No.: SCXK(Su)2020-0009. They were housed in a specific pathogen-free (SPF) care facility under a 12-hour light / dark cycle, fed with sterile feed and distilled water, facility license number: SYXK(Hu)2020-0042. All animal experimental procedures were approved by the Institutional Animal Care and Use Committee of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences, IACUC approval number 2023-08-HYZ-149. Animals underwent one week of acclimatization before the experiments.

[0179] 2. Evaluation Content and Methods

[0180] 2.1 Effects of CMOT on cell viability

[0181] Cytotoxicity of CMOT extract: 30 mg of freshly prepared CMOT hydrogel was immersed in 10 mL of DMEM complete medium and incubated at 37 °C for 24 h to obtain CMOT extract solution.

[0182] BMDM and C2C12 (mouse myoblasts) cells were seeded in 96-well plates at 5000 cells / well and cultured for 24 h in a cell culture incubator before drug administration. CMOT was administered at concentrations of 3 mg / mL, 1.5 mg / mL, 0.75 mg / mL, 0.375 mg / mL, and 0 mg / mL. Cell viability was assessed 24 h after drug administration by adding CCK8 reagent. After 24 h of incubation, CCK8 was added and the cells were incubated at 37°C for 1 h. The OD value was measured at 450 nm using a microplate reader. Cell viability (CV) was calculated using the following equation.

[0183]

[0184] RAW264.7 cells were seeded in 24-well plates and injected with 1 mg / mL CMOT hydrogel extraction solution. After incubation for 24 h, the cytotoxicity of CMOT hydrogel to RAW264.7 cells was measured using a live / dead cell assay kit. Cells were stained with Calcein / PI at 37 °C for 30 min. Cells were observed under a fluorescence microscope and collected for flow cytometry analysis.

[0185] Toxicity of CMOT in direct contact with cells: Freshly prepared CMOT hydrogel was first layered into 24-well plates and sterilized by UV irradiation for 4 hours. RAW264.7 cells were then added to the plates and cultured. CFDA-SE staining solution was added to monitor cell proliferation activity. Cell viability was observed by fluorescence microscopy at different time points (1 hour, 24 hours, 48 ​​hours, and 72 hours). CFDA-SE exhibited green fluorescence under FITC channel irradiation; its fluorescence decreased due to cell division during cell proliferation.

[0186] 2.2 Effects of CMOT on subcutaneous tissue

[0187] Freshly prepared CMOT hydrogel was subcutaneously injected into normal BALB / c mice for 3 days. Skin tissue and CMOT hydrogel were collected for HE staining to assess the in vivo biocompatibility of the CMOT hydrogel.

[0188] 2.3 Hemolytic Properties of CMOT

[0189] Add 1 mL of freshly prepared red blood cell (RBC) suspension to a 1.5 mL EP tube containing 1 mg of CMOT hydrogel and incubate at 37°C for 4 h. Remove the CMOT hydrogel and centrifuge the treated RBC suspension at 2000 rpm for 5 min for imaging and OD540 detection. PBS and 1% Triton X-100 were used as negative and positive controls, respectively. Calculate the hemolysis rate using the following equation:

[0190]

[0191] 2.4 Antibacterial activity of CMOT

[0192] Frozen *Escherichia coli* or *Staphylococcus aureus* cultures were removed from -20°C and thawed at room temperature. 1 mL of the bacterial culture was transferred to a 10 mL EP tube in a clean bench, and 5 mL of LB medium was added. The culture was incubated at 220 rpm and 37°C for 1 hour to activate the culture. 200 mg of freshly prepared CMOT1 / CMOT2 hydrogel was weighed into a 4 mL EP tube, and bacterial culture diluted 1000 times with LB medium was added. A blank control group and a positive control group (100 μg / mL ampicillin sodium solution) were also set up. The culture was incubated at 220 rpm and 37°C for 4 hours, with three replicates per group. The cultured bacterial culture was diluted 100 times with LB medium, and 50 μL of the cultured culture was spread onto agarose gel plates, one plate per culture tube. The plates were incubated upside down at 37°C for 12 hours. Photos were taken, colony counts were recorded, and the inhibition rate was calculated.

[0193]

[0194] 2.5 CMOT's regulatory activity on BMDM and lymphocytes

[0195] DMEM complete medium was used as the extraction medium, and 1 mg / mL CMOT hydrogel was co-incubated at 37°C for 2 days to prepare the hydrogel extraction solution. A 1 mg / mL CMCS and TPS polysaccharide solution group was also prepared to detect the immunomodulatory activity of individual and combined polysaccharide hydrogels. BMDM cells were co-incubated with the extraction solutions of both hydrogels for 24 h, followed by flow cytometry staining with F4 / 80-BV510, CD86-PerCP-Cy5.5, and CD206-APC antibodies. Cells were analyzed by flow cytometry after 45 min.

[0196] Similarly, the polysaccharide solution and hydrogel extract were administered to mouse spleen-derived lymphocytes and co-cultured for 48 hours before being incubated with flow cytometry antibodies containing CD3-PerCP Cy5.5, CD8α-PE, Ki67-BV421, and CD49b-FITC. The antibodies were then detected by flow cytometry.

[0197] 2.6 CMOT activity in scavenging DPPH free radicals

[0198] Prepare 100 μM DPPH (20 mg / 500 mL ethanol), 0.5 mg / mL vitamin C solution (positive control), and a certain amount of CMOT hydrogel. Each group has three replicates, and samples are added according to the requirements of each group. Sample group: 100 μL sample solution + 100 μL DPPH solution; Blank group: 100 μL sample solution + 100 μL anhydrous ethanol; Control group: 100 μL water + 100 μL DPPH solution. Stir the mixture and incubate at room temperature in the dark for 30 min. Next, scan the DPPH at 518 nm using a UV-Vis spectrophotometer. DPPH degradation is calculated using the following formula:

[0199]

[0200] Where AB and AH are the absorptions of the blank group and the sample group, respectively.

[0201] 2.7CMOT superoxide anion radical (O2) .- Sweep experiment

[0202] The antioxidant capacity of CMOT hydrogel was determined according to the instructions for the superoxide anion radical detection kit (catalog number A052-1-1). The calculation formula used is:

[0203]

[0204] C standard: concentration of standard (0.15 mg / mL); N: dilution factor of sample before testing.

[0205] 3. Results and Analysis

[0206] 3.1 Antioxidant experiments showed that both types of CMOT hydrogels possessed antioxidant capabilities, able to scavenge DPPH free radicals and superoxide anion free radicals (…). Figure 7 B, C), among which CMOT2 hydrogel has a stronger antioxidant effect, and its ability to scavenge superoxide anions is similar to that of the positive control vitamin C, see [reference needed]. Figure 7 A.

[0207] 3.2 Biocompatibility Evaluation of CMOT Hydrogel

[0208] After co-culturing RAW264.7 cells with the extract of CMOT hydrogel, cell viability staining and fluorescence microscopy results showed that neither CMOT hydrogel extract was toxic to RAW264.7 cells. Figure 8 Flow cytometry results also showed that the proportion of viable cells in the CMOT extract was not different from that in the blank PBS group, with virtually no dead cells. Figure 8C, D). CCK8 assays were performed on mouse C2C12 myoblasts and bone marrow-derived macrophages (BMDM) administered with CMOT hydrogel. The results showed that 3 mg / mL of CMOT hydrogel extract was essentially non-toxic to both cell types, with cell viability approaching 100%. Figure 8 E).

[0209] Direct contact culture of RAW264.7 cells with CMOT hydrogel revealed that RAW264.7 cells could survive and proliferate well on the hydrogel. At 48 h, the cells grew into the hydrogel and embedded themselves in it. The cells were still surviving well at 72 h, indicating that the hydrogel had good softness and biocompatibility.

[0210] Co-culturing mouse erythrocytes with hydrogels revealed that in the positive control group, erythrocytes completely ruptured after incubation with 1% Triton solution, resulting in a red solution with almost no erythrocyte sedimentation in the lower layer after centrifugation. In contrast, the CMOT hydrogel group and the negative control group showed clear separation of erythrocytes and solution in the lower layer after centrifugation with PBS, with almost no erythrocyte rupture. Figure 8 F) indicates that the hydrogel has no hemolytic activity.

[0211] To assess the biocompatibility of CMOT hydrogel in vivo, CMOT was injected subcutaneously into mouse tissue. Three days later, skin tissue was harvested for pathological sectioning and HE staining. Figure 8 G) Pathological images showed that the cells in the hydrogel-filled subcutaneous tissue had good morphology, indicating that the hydrogel did not damage the skin tissue. A small number of intact immune cells were also observed to infiltrate, suggesting that the hydrogel may have a role in recruiting immune cells. Furthermore, a large amount of hydrogel was still present in the skin tissue taken on the third day, indicating that the CMOT hydrogel degrades slowly in vivo.

[0212] 3.3 Antibacterial activity of CMOT hydrogel

[0213] After co-culturing CMOT hydrogel with Staphylococcus aureus, the culture medium was spread, and it was found that CMOT2 hydrogel could inhibit the growth of Staphylococcus aureus by about 50%, showing significant antibacterial activity (P = 0.001), and its antibacterial potency was approximately equivalent to 50 μg / mL ampicillin sodium. Figure 9 A, C); while CMOT1 hydrogel showed virtually no inhibitory effect on the growth of Staphylococcus aureus. For Gram-negative Escherichia coli, CMOT2 exhibited good inhibitory effects, equivalent to 100 μg / mL ampicillin sodium (A, C); Figure 9 B, D); CMOT1 also showed an inhibitory effect on the growth of Escherichia coli. In summary, the overall antibacterial effect of CMOT2 hydrogel is better than that of CMOT1 hydrogel, and it can inhibit the growth of both Gram-positive and Gram-negative bacteria.

[0214] 3.4 Immunomodulatory Activity of CMOT Hydrogel

[0215] The relative balance between immunosuppression and immune activation in vivo is crucial for immune stability. Flow cytometry results showed that CMOT hydrogel has an immunomodulatory effect on BMDM and spleen-derived lymphocytes. CMOT hydrogel significantly increased the expression of CD206 and CD86 in BMDM-M0 (P<0.0001), and the difference was statistically significant. Figure 10 A, B), among which CD86 showed a higher increase ( Figure 10 C) indicates that CMOT hydrogel promotes the development of macrophages towards the M1 phenotype. Simultaneously, after CMOT hydrogel treatment, lymphocytes containing CD3... + CD49b + and CD8 + Lymphocytes begin to overexpress the proliferation index Ki67 ( Figure 10 (D, E, F) indicates that CMOT hydrogel promotes the proliferation of T lymphocytes and NK cells by modulating and reshaping the immune microenvironment, and has the potential to become a T cell immune enhancer.

[0216] Test Example 2: In vivo evaluation of the effects of CMOT on promoting wound healing in cachexia

[0217] 1. Materials and Instruments

[0218] Material

[0219] Surgical scissors, surgical forceps, ruler, marker (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China)

[0220] Electric shaver, depilatory cream, cotton balls, 75% ethanol (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China)

[0221] Isoflurane (Shenzhen Ruiward Life Technology Co., Ltd., Shenzhen, China)

[0222] DAPI staining solution, Triton X-100 (Yisheng Biotechnology Co., Ltd., Shanghai, China)

[0223] Alexa Fluor 488 - Goat Anti-Rabbit Secondary Antibody (Yisheng Biotechnology Co., Ltd., Shanghai, China)

[0224] Alexa Fluor 594 - Goat Anti-Mouse Secondary Antibody (Yisheng Biotechnology Co., Ltd., Shanghai, China)

[0225] Recombinant Anti-EN1 / Engrailed-1 rabbit primary antibody (Abcam, Cambridge, UK)

[0226] Anti-Fibronectin murine primary antibody (Abcam, Cambridge, UK)

[0227] Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mouse primary antibody (Abcam, Cambridge, UK)

[0228] TRIeasy TM Total RNA extraction reagent (Yisheng Biotechnology Co., Ltd., Shanghai, China)

[0229] Reverse transcription reagent III. 1st Strand cDNA Synthesis SuperMix for qPCR (gDNAdigester plus) (Yisheng Biotechnology Co., Ltd., Shanghai, China)

[0230] qPCR SYBR Green Master Mix (No Rox) (Yisheng Biotechnology Co., Ltd., Shanghai, China)

[0231] Hemmei Scar Silicone Gel (Anshili (China) Medical Co., Ltd., Guangzhou)

[0232] instrument

[0233] NanoZoomer pathology slide scanner (Hamamatsu Photonics Co., Ltd., Hamamatsu, Japan)

[0234] PCR instrument EPPENDORH 5331 (EPPENDORH, Hamburg, Germany)

[0235] BIO-RAD CFX384™ Real-Time PCR System (BIO-RAD, California, USA)

[0236] Super-resolution rotating confocal microscope SPIN SR10 (Olympus, Tokyo, Japan)

[0237] Cells and Animals

[0238] Mouse myoblasts C2C12 (number ZKCC-X2072) were provided by the Shanghai Cell Bank of the Chinese Academy of Sciences.

[0239] BALB / c mice (4–6 weeks old, female, SPF grade, 20–22 g) were provided by Jiangsu Huachuang Xinno Pharmaceutical Technology Co., Ltd., Animal Qualification Certificate No.: SCXK(Su)2020-0009. They were housed in a specific pathogen-free (SPF) care facility under a 12-hour light / dark cycle, fed with sterile feed and distilled water, facility license number: SYXK(Hu)2020-0042. All animal experimental procedures were approved by the Institutional Animal Care and Use Committee of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences, IACUC approval number 2023-08-HYZ-149. Animals underwent one week of acclimatization before the experiments.

[0240] 2 Experimental Methods

[0241] 2.1 The therapeutic effect of CMOT hydrogel on full-thickness damaged skin

[0242] Mice were anesthetized with the respiratory anesthetic isoflurane, and their backs were shaved to expose the skin. A 7mm diameter circle was drawn on the mouse's back using a marker and ruler. Surgical scissors and forceps were then used to cut the skin along the dotted line, creating a circular full-thickness skin defect on the mouse's back. CMOT hydrogel was applied. Photographs of the wound were taken on days 0, 3, 7, and 11, and skin samples from the wound site were harvested for HE and Masson staining analysis. The collagen volume fraction of the Masson-stained skin tissue sections was calculated using ImageJ.

[0243] 2.2 Therapeutic effect of CMOT hydrogel on wound healing in mice with cachexia

[0244] C26 cells were resuscitated in liquid nitrogen and expanded. The culture conditions for C26 cells were DMEM medium + 10% FBS + 1% penicillin-streptomycin solution. Digestion was performed by rinsing with trypsin digestion solution. After rinsing, the trypsin digestion solution was discarded, and the cells were incubated at room temperature for 1 min. Then, DMEM medium was added to resuspend the cells, and the cells were pipetted to form a single-cell suspension for cell counting. After centrifugation and discarding the medium, the cells were washed with PBS and resuspended in PBS to form a 10n saturated suspension. 6 Subcutaneous tumor transplantation was performed using 0.1 mL of single-cell suspension. First, the backs of BALB / c mice were shaved, and the depilatory cream was wiped off. Then, 0.1 mL of C26 single-cell suspension was subcutaneously injected into the backs of the mice using an insulin syringe. Before injection, the insulin syringe needle was wiped with 75% alcohol. Daily body weight, food intake, and tumor volume were recorded. A significant decrease in mouse body weight and food intake was used as an indicator of cachexia.

[0245] When mice exhibited significant weight loss and a marked decrease in food intake, they were considered to have reached a cachectic state, and a cachectic wound model was established, following the full-thickness skin injury model described above. Groups were set up as follows: a normal control group (Control, i.e., normal mice with full-thickness skin injury), a C26 group (cachectic mice with full-thickness skin injury), and a CMOT group (cachectic mice with CMOT hydrogel applied to the injured skin). The hydrogel was ensured to completely cover the wound surface, and then fixed with a breathable dressing. Wounds were photographed on days 0, 3, 7, and 14 after skin injury, and the wound healing rate was statistically analyzed using ImageJ. On days 3, 7, and 14, mice were anesthetized, and skin samples from the wound site were harvested for pathological sectioning, HE staining, and immunohistochemical staining to observe the microscopic recovery of the wound.

[0246] 3 Results and Analysis

[0247] 3.1 CMOT hydrogel promotes skin healing in mice

[0248] A full-thickness skin defect model was used to evaluate the effect of hydrogels on promoting wound healing. Figure 11 A) The results showed that, compared with the control group (no treatment), the wound area of ​​the CMOT1 and CMOT2 hydrogel groups was significantly reduced on day 3, and the CMOT2 gel group had a higher and more significant wound closure rate (P = 0.030), which was statistically significant. Figure 11 B, C). On day 7, the wound closure rates of both CMOT hydrogels were significantly higher than those of the blank control group (P<0.0001), and the differences were statistically significant, indicating that the hydrogels had a good effect on promoting wound healing. On day 11, all three groups of wounds were completely healed, with the CMOT2 hydrogel group showing the best healing and the least scar hyperplasia. Hematoxylin and eosin stained sections (HE staining) were used to assess the wound healing of skin tissue on day 11. The results showed that the hydrogel group exhibited thicker granulation tissue and higher epithelial regularity compared to the control group, and generated more blood vessels and hair follicles. Figure 11 D). Masson staining results showed that the CMOT hydrogel group formed more collagen fibers (blue) and had a higher collagen volume fraction (CVF). Figure 11 (D, E) indicate better healing. All results show that CMOT2 is superior to CMOT1 hydrogel in promoting skin healing, which may be due to the higher aldehyde content and stronger antibacterial activity of CMOT2, as well as its better viscoelasticity, which contributes to wound healing.

[0249] 3.2 Establishment of a cachectic, non-healing wound model

[0250] A mouse model of cachexia was established to study the therapeutic effect of CMOT hydrogel on poorly healing wounds in cachexia. Figure 12 A). The results showed that 18 days after subcutaneous injection of C26 cells, the tumor volume was approximately 1000 mm. 3 ( Figure 12 B), and observed a decrease in cumulative food intake and body weight in the mice ( Figure 12 B, E). After day 20, the tumor-free body weight and cumulative food intake of the mice decreased significantly (B, E). Figure 12 (D, B) indicates the successful establishment of the cachexia mouse model. Full-thickness skin lesions were created on day 23 and CMOT hydrogel was applied. Results showed that, compared to the normal group, the untreated C26 (cachexia) group had a significantly delayed wound healing rate on postoperative day 3 (P = 0.002), further demonstrating the successful establishment of the cachexia-related non-healing wound model.

[0251] 3.3CMOT promotes wound healing in cachexia

[0252] Previous experiments have optimized CMOT2 hydrogel as the best choice for improving wound healing in cachectic mice; therefore, the CMOT hydrogel used here is CMOT2 hydrogel. Macroscopic morphology of wound healing in cachectic mice (photographs) demonstrates the effect of CMOT hydrogel in promoting wound healing in cachectic mice. Figure 12 F); Statistical analysis revealed that on day 3, the wound healing rate of the CMOT hydrogel group was significantly higher than that of the C26 group (P<0.0001), and even higher than that of the control group (F). Figure 12 G); On days 5 and 7, the wound healing rate of the CMOT hydrogel group was still significantly higher than that of the C26 group (P<0.05), which may be attributed to the antioxidant and immunomodulatory effects of the multifunctional CMOT hydrogel.

[0253] Based on pathological changes, the CMOT hydrogel group also showed better healing effects than the C26 group. On day 3, all groups exhibited a mild acute inflammatory response, but compared to the control group and the C26 cachexia group, the CMOT hydrogel group showed more collagen fibers and inflammatory cells around the wound. Figure 13 (A, B) This is attributed to the healing-promoting and immunomodulatory effects of CMOT hydrogel. By day 7, all groups had formed a layer of epithelium, with the CMOT hydrogel group exhibiting thicker recovery tissue and higher epithelial regularity than the C26 and normal groups. By day 14, all groups had formed the basic structure of epithelium and dermis, while the C26 group had the fewest blood vessels and hair follicles. In conclusion, the thickened epithelial and dermal tissue, mature blood vessels, hair follicles, and well-proliferating fibroblasts all indicate that CMOT hydrogel has a wound-healing effect on cachectic mice.

[0254] Upregulation of IL-6 and TNF-α is a major driver of slow wound healing. [2] Following administration of CMOT hydrogel, IL-6 in the local wound initially increased compared to the C26 group (day 3, P < 0.0001), contributing to the inhibition of bacterial infection; it then decreased to normal levels (days 7 and 14, P < 0.01), while untreated cachectic mice still had high levels of IL-6 on days 7 and 14. Figure 13 C, E). Meanwhile, in the C26 group mice, TNF-α levels at the wound site remained elevated on day 7 (P < 0.0001 compared to the normal group) and day 14 (P = 0.006 compared to the normal group), while in the CMOT hydrogel treatment group, TNF-α levels were significantly downregulated on day 7 (P < 0.0001) and day 14 (P = 0.004), with statistically significant differences, approaching normal levels. Figure 13 D, F).

[0255] In summary, this application successfully extracted the pollen polysaccharide TPS and investigated its molecular weight and activity. TPS is a negatively charged polysaccharide. TPS can promote macrophage differentiation into M1 and M2 types, with a slightly higher proportion of M1 type, and possesses immunomodulatory functions. Simultaneously, TPS promotes the proliferation of peripheral blood lymphocytes and splenic lymphocytes, and increases CD4+. + CD8 + The ratio of NK cells to other cells has an immune-enhancing effect.

[0256] Two oxidized Trichosanthes kirilowii polysaccharides, oTPS1 and oTPS2, were obtained by oxidizing TPS with sodium periodate. The aldehyde content of oTPS2 was twice that of oTPS1. The aldehyde groups in the oxidized polysaccharides could react with the amino groups in carboxymethyl chitosan to prepare multifunctional Schiff base cross-linked hydrogels. Chitosan exhibits anti-inflammatory and antibacterial effects, while the Trichosanthes kirilowii polysaccharides have an immune-enhancing effect that promotes lymphocyte proliferation. The combination of these two polysaccharides has the effects of regulating inflammation at the wound site, anti-oxidation, antibacterial activity, and immunomodulation.

[0257] The prepared CMOT hydrogel exhibits good biocompatibility both in vitro and in vivo, and possesses the desirable properties of hydrogels, such as good healing performance, stable mechanical parameters, and water retention capacity. In a mouse model of full-thickness wound injury, the CMOT2 hydrogel with high oxidation levels was more effective than the CMOT1 hydrogel in accelerating wound healing, thus becoming the optimal hydrogel for subsequent experiments. Histological analysis showed that the CMOT2 hydrogel demonstrated immunomodulatory advantages in promoting the healing of cancer cachexia wounds by promoting the regeneration of blood vessels, collagen, and epithelial tissues and reducing inflammation. Therefore, the multifunctional hydrogel dressing CMOT is a promising candidate for clinical protection and treatment of cachexia wounds and immunosuppressive chronic trauma.

Claims

1. A method for preparing a pollen polysaccharide-chitosan hydrogel, the method comprising the following steps: 1) Preparation of crude polysaccharide from Trichosanthes kirilowii pollen: 1-1) Extract the powder of Trichosanthes kirilowii using ultrasonic water, concentrate it, and centrifuge to remove insoluble starch; 1-2) Add anhydrous ethanol to the extract from which insoluble starch has been removed, let stand, and centrifuge to remove the supernatant; 1-3) Dissolve the precipitate after removing the supernatant by adding water, and then add trifluoroacetic acid to precipitate the protein; 1-4) The supernatant after protein precipitation was placed in a 7 kD dialysis bag for dialysis to remove trifluoroacetic acid, concentrated and lyophilized to obtain crude polysaccharide from Trichosanthes kirilowii. 2) Aldehydeation of crude polysaccharides from Trichosanthes kirilowii: Sodium periodate, TEMPO (2,2,6,6-tetramethylpiperidine-1-oxy radical), or manganese dioxide (MnO2) are added to the crude polysaccharide of Trichosanthes kirilowii prepared in step 1) and reacted in the dark to obtain aldehyde-modified crude polysaccharide of Trichosanthes kirilowii with an oxidation degree of 1~5 mmol / g, wherein the oxidation degree refers to the number of moles of aldehyde groups oxidized per gram of polysaccharide; 3) Preparation of CMOT hydrogel: The aldehyde-modified trichosanthes pollen crude polysaccharide obtained in step 2) is covalently bonded to carboxymethyl chitosan via a Schiff base reaction to form a CMOT hydrogel.

2. The method according to claim 1, wherein, In step 1-1), the powdered Trichosanthes kirilowii root is soaked in water for 0.5-2 hours and then ultrasonically extracted at 35-50℃ for 1-3 hours; and / or In step 1-1), the powdered Trichosanthes kirilowii root is extracted in water by ultrasonication, followed by centrifugation to collect the supernatant. The precipitate residue is then extracted again by ultrasonication in a water bath, and the two extracts are combined; and / or In step 1-2), anhydrous ethanol is added to the extract after removing insoluble starch so that the volume fraction of ethanol reaches more than 85%.

3. The method according to claim 1, wherein, In step 2), Sodium periodate is added to the crude polysaccharide of *Trichosanthes kirilowii* prepared in step 1), and a light-protected reaction is carried out at 20-35°C to generate aldehyde-modified crude polysaccharide of *Trichosanthes kirilowii*, wherein the molar ratio of crude polysaccharide of *Trichosanthes kirilowii* to sodium periodate is 1:10 to 1:1000; and / or The oxidation degree of the aldehyde-modified crude trichosanthes pollen polysaccharide obtained was 5 mmol / g TPS.

4. The method according to claim 1, wherein, In step 3), The degree of deacetylation of carboxymethyl chitosan is ≥90%; and / or An aqueous solution of carboxymethyl chitosan was added to an aqueous solution of aldehyde-modified crude pollen polysaccharide, wherein the concentration of the aqueous solution of aldehyde-modified crude pollen polysaccharide was 1%–3%, and the concentration of the aqueous solution of carboxymethyl chitosan was 1%–3% (in g / mL), and the volume ratio of the two solutions was 1:1; and / or The amino groups in carboxymethyl chitosan react with the dialdehyde groups of aldehyde-modified pollen crude polysaccharide through a Schiff base reaction to form C=N bonds and achieve cross-linking, with a cross-linking time of 0.5~3 min.

5. A pollen polysaccharide-chitosan hydrogel prepared by the method according to any one of claims 1-4.

6. A pharmaceutical composition comprising at least the pollen polysaccharide-chitosan hydrogel as described in claim 5, and pharmaceutically acceptable excipients.

7. The pharmaceutical composition according to claim 6, wherein, The pharmaceutical composition is available in the form of a hydrogel dressing, hydrogel patch, or hydrogel spray.

8. Use of the pollen polysaccharide-chitosan hydrogel of claim 5 or the pharmaceutical composition of claim 6 or 7 in the preparation of a medicament for treating cachectic wounds.

9. The use according to claim 8, wherein, The pollen polysaccharide-chitosan hydrogel or the pharmaceutical composition can promote the regeneration of blood vessels, collagen and epithelial tissues and reduce inflammation, and regulate the local immune microenvironment of cachectic wounds.

10. The use according to claim 8, wherein, The cachexia mentioned includes cachexia caused by cancer, AIDS, and inflammatory diseases.

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