Hydrogel based on radix trichosanthis crude polysaccharide as well as preparation method and application of hydrogel

Abstract

The invention relates to hydrogel based on radix trichosanthis crude polysaccharide as well as a preparation method and application of the hydrogel. The hydrogel based on the radix trichosanthis crude polysaccharide is prepared by the following method: 1) preparing the radix trichosanthis crude polysaccharide; (2) carrying out hydroformylation on the radix trichosanthis crude polysaccharide to obtain hydroformylated radix trichosanthis crude polysaccharide; and 3) covalently binding the aldehyde crude trichosanthes root polysaccharide and carboxymethyl cellulose through a Schiff base reaction to form the hydrogel. The multifunctional hydrogel disclosed by the invention is expected to become a candidate dressing for clinically protecting and treating cachexia wounds and other immunosuppressive chronic wounds.

Description

Technical Field

[0001] The present application belongs to the field of polysaccharide drugs, and specifically relates to a hydrogel based on crude trichosanthes polysaccharide, and a preparation method and use thereof. Background Art

[0002] Cachexia, also known as cachexia syndrome, is a deterioration of chronic diseases such as cancer, AIDS, and inflammatory diseases, characterized by weight loss, reduced albumin synthesis, anemia, immunosuppression, and poor wound healing. Wounds caused by surgery or bedsores in cachexia patients are difficult to heal due to blocked collagen synthesis, low immunity, and repeated local inflammation, which seriously endangers the quality of life of patients and is a major problem that plagues medical staff in clinical practice. [1] Developing safe and effective cachexia wound dressings is the key to solving the problem.

[0003] Wound healing is divided into four stages: coagulation, inflammation, proliferation, and scar remodeling. The coagulation stage is when the wound first appears, and platelets will gather at the wound to promote hemostasis. The inflammation stage is mainly to remove pathogenic microorganisms and necrotic tissues. In cachexia, the inflammation stage will be prolonged due to insufficient immunity, which is manifested by the continuous infiltration of inflammatory factors such as TNF-α and IL-6 in the local wound. [2] . In the proliferative phase, soon after the onset of inflammation, new cells, fibroblasts, endothelial cells, etc. appear at the wound site, and new capillaries are formed at the same time, which together form hyperplastic granulation tissue, fill and cover the wound, thus forming a scar. In the cachectic state, protein synthesis is inhibited and collagen production is blocked, which leads to slow wound healing. [3] During the scar remodeling phase, the wound has initially healed, and the newly formed scar tissue will gradually adjust to physiological functions over time and with lifestyle habits, ultimately improving the appearance and function of the injured area. This phase takes the longest time, and it is reported that scar remodeling and hyperplasia may take as long as 5–10 years. [4] . Hyperplastic scar tissue not only affects the appearance, but also has a much greater chance of being damaged again than normal tissue. It also affects joint movement at the joints. Therefore, in the process of cachexia wound healing and scar hyperplasia, there is a close relationship with cytokines such as TNF-α, IL-6, and TGF-β. Studies have shown that TGF-β can induce increased expression of Engrailed-1 (En-1), a gene related to scar hyperplasia, in skin fibroblasts, aggravating scar hyperplasia, while inhibiting the expression of En-1 can reduce scar hyperplasia.

[0004] At present, more and more plant polysaccharides are used to prepare hydrogels, because the superior pharmacological activity and good biocompatibility of polysaccharides can realize the drug delivery strategy of combining carriers and drugs. [5]Hydrogels have many superior properties, such as adjustable hardness (soft or hard), good biocompatibility, slow drug release, etc. [6] , suitable for use as wound dressings, can isolate infection, promote cell proliferation and tissue repair [7] Polysaccharide hydrogel has the properties of both polysaccharides and hydrogels, which can improve the immunosuppression of cachectic wounds and promote tissue repair.

[0005] Radix Trichosanthes is the dried root of Trichosanthes kirilowii Maxim or Trichosanthes rosthornii Harms, a plant of the Cucurbitaceae family, which has the effects of clearing away heat and detoxifying, reducing swelling and draining pus, and replenishing deficiency and calming the stomach. Based on this, it is necessary to study the effective polysaccharide components of Radix Trichosanthes and develop its hydrogel medicinal form, so as to develop products with potential application prospects in the treatment of cachexia 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 treatment of cancer cachexia with volatile oil of Atractylodes macrocephala[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 purpose of the present invention is to provide a method for preparing trichosanthes polysaccharide-chitosan (CMOT) hydrogel.

[0016] Another technical purpose of the present invention is to provide a trichosanthes polysaccharide-chitosan hydrogel prepared by the above method.

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

[0018] Technical Solution

[0019] In one aspect, the present invention provides a method for preparing a CMOT hydrogel, the method comprising the following steps:

[0020] 1) Preparation of crude trichosanthes polysaccharide:

[0021] 1-1) Extracting the powder of Radix Trichosanthis with water ultrasonic, concentrating and centrifuging to remove insoluble starch;

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

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

[0024] 1-4) The supernatant after protein precipitation is placed in a 7 kD dialysis bag to remove trifluoroacetic acid, concentrated and freeze-dried to obtain crude Trichosanthes polysaccharide;

[0025] 2) Aldehydation of crude polysaccharide from Radix Trichosanthis:

[0026] Adding sodium periodate, TEMPO (2,2,6,6-tetramethylpiperidin-1-oxyl free radical) or manganese dioxide (MnO2) to the crude Trichosanthes polysaccharide prepared in step 1) to carry out a light-protected reaction to obtain aldehyde-modified crude Trichosanthes polysaccharide;

[0027] 3) Preparation of CMOT hydrogel:

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

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

[0030] In a specific embodiment, in step 1-1), powder of Radix Trichosanthis is extracted by ultrasonic in water, and then the supernatant is collected by centrifugation, and the precipitated residue is extracted again by ultrasonic in a water bath, and the two extracts are combined.

[0031] In a specific embodiment, in step 1-2), anhydrous ethanol is added to the extract from which insoluble starch has been removed 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 Trichosanthes kirilowii polysaccharide prepared in step 1), and a light-protected reaction is carried out at 20-35° C. to generate aldehyde-modified crude Trichosanthes kirilowii polysaccharide, wherein the molar ratio of crude Trichosanthes kirilowii polysaccharide to sodium periodate is 1:10-1:1000.

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

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

[0035] In a specific embodiment, in step 3), an aqueous solution of carboxymethyl chitosan is added to an aqueous solution of aldehyde-modified crude trichosan polysaccharide, wherein the aqueous solution of aldehyde-modified crude trichosan polysaccharide has a concentration of 1% to 3%, and the aqueous solution of carboxymethyl chitosan has a concentration of 1% to 3%, in g / mL, and the volume ratio of the two solutions is 1:1. The ratio of carboxymethyl chitosan to aldehyde-modified trichosan polysaccharide will affect the performance and effect of the formed hydrogel. As the ratio of aldehyde-modified trichosan polysaccharide increases, the hardness of the formed hydrogel will increase, and the swelling performance of the hydrogel will decrease.

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

[0037] In a specific embodiment, in step 3), the cross-linking time is 0.5 to 3 minutes.

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

[0039] On the other hand, the present invention provides a pharmaceutical composition, which at least comprises the above-mentioned Radix Trichosanthis 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 use of the above-mentioned trichosanthes polysaccharide-chitosan hydrogel or the above-mentioned pharmaceutical composition in the preparation of a drug for treating cachexia wounds and other immunosuppressive chronic wounds.

[0042] In a specific embodiment, the above-mentioned trichosan 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 a specific embodiment, the above-mentioned Trichosanthes polysaccharide-chitosan hydrogel or the above-mentioned pharmaceutical composition can regulate the local immune microenvironment of cachectic wounds.

[0044] In a specific embodiment, the cachexia includes cancer, AIDS, inflammatory disease cachexia.

[0045] In a specific embodiment, said otherwise immunosuppressive chronic wound comprises a diabetic wound.

[0046] Beneficial Effects

[0047] The present invention proposes to use trichosanthes polysaccharide and chitosan as raw materials to prepare a safe, non-toxic, biodegradable hydrogel as a wound dressing, and treat cachectic and difficult-to-heal wounds by regulating local wound immunity, promoting epithelial migration and granulation growth. First, the polysaccharide TPS was extracted from trichosanthes, and the monosaccharide composition, molecular weight distribution and zeta potential of TPS were analyzed. TPS was oxidized with sodium periodate to form an aldehyde-modified polysaccharide oTPS, which was then covalently bound to carboxymethyl chitosan through a Schiff base reaction to form a CMOT hydrogel. In a mouse full-thickness skin wound model associated with cancer cachexia, the CMOT hydrogel showed a good healing-promoting effect by regulating the local immune microenvironment of cachectic wounds.

[0048] The present application further demonstrates that Trichosanthes polysaccharide has an immunomodulatory effect. The prepared Trichosanthes polysaccharide hydrogel has good biocompatibility and excellent hydrogel properties, including good healing properties, stable mechanical parameters and water storage capacity. In a full-thickness skin injury mouse model, CMOT2 hydrogel with a high degree of oxidation showed superior effects than CMOT1 hydrogel in accelerating wound healing. In addition, CMOT2 hydrogel can also promote the regeneration of blood vessels, collagen and epithelial tissue by regulating the inflammatory microenvironment at the wound site, thereby effectively promoting the healing of cancer cachexia wounds. Therefore, the multifunctional hydrogel dressing CMOT is expected to become a potential candidate dressing for the clinical protection and treatment of cachexia wounds and other immunosuppressive chronic wounds.

[0049] In a full-thickness skin injury mouse model, CMOT2 hydrogel with a high degree of oxidation showed superior effects to CMOT1 hydrogel in accelerating wound healing. In addition, CMOT2 hydrogel can also effectively promote the healing of cancer cachexia wounds by regulating the inflammatory microenvironment at the wound site and promoting the regeneration of blood vessels, collagen, and epithelial tissue. Therefore, the multifunctional hydrogel dressing CMOT is expected to become a potential candidate dressing for the clinical protection and treatment of cachexia wounds and other immunosuppressive chronic wounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 :Extraction of Radix Trichosanthis Polysaccharides (TPS) and determination of total sugar content. (A) TPS extraction process; (B) Standard curve established with glucose as standard; (C) Number of extractions, and corresponding yield and sugar content results.

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

[0052] Figure 3 :Preparation and characterization of aldehyde-modified Trichosanthes polysaccharide (oTPS) with different oxidation degrees. (A) Principle of preparation of oTPS; (B) IR spectra of oTPS and TPS; (C) Standard curve established with glutaraldehyde as standard; (D) Aldehyde content in different oTPS and sodium periodate dosage ratio; (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 polysaccharide-chitosan (CMOT) hydrogel. (A) Schematic diagram of the preparation of CMOT hydrogel, (B) Solid-state and healing properties characterization results of CMOT hydrogel, (C) SEM image of CMOT hydrogel, scale bar = 300 μm.

[0054] Figure 5 : Swelling (A), degradation (B), water holding capacity (C), and mechanical properties (D, E) characterization of CMOT hydrogels (n=3).

[0055] Figure 6 : Infrared spectroscopic characterization of CMOT hydrogel.

[0056] Figure 7 :The results of the antioxidant study of CMOT hydrogel. (A) Photograph of the results of anti-superoxide anion free radical, (B) Statistics of anti-superoxide anion free radical activity, (C) Statistics of anti-DPPH free radical activity, (n=3).

[0057] Figure 8 : Biosafety results of CMOT. (A) Process of administering CMOT extract to RAW 264.7, (B) Calcein / PI staining images of RAW 264.7 cells after administration of CMOT extract (scale bar = 100 μm) and (C, D) flow cytometry, (E) CCK8 detection of toxicity of CMOT extract to C2C12 cells and BMDM, (F) Hemolytic activity of CMOT hydrogel, (G) HE sections of subcutaneous tissue after subcutaneous implantation of CMOT hydrogel (scale bar = 300 μm), n = 3.

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

[0059] Fig.10 :The results of immunomodulatory activity of CMOT. The effects of CMOT on (A) M1 macrophages and (B) M2 macrophages and their (C) relative ratio, and on (D) CD3 + , (E)CD49b + , (F)CD8 + Effect of the expression of Ki67 proliferation factor on lymphocytes (n=3).

[0060] Fig.11 :Comparison of the effects of CMOT hydrogels with different oxidation degrees on promoting mouse skin healing. (A) Animal experiment scheme for skin healing, (B) Photos of mouse skin healing, (C) Statistics of skin healing rate, (D) HE staining and Masson staining sections of skin, scale bar = 250 μm, (E) Statistics of skin collagen volume fraction based on Masson staining of skin sections, (n = 3).

[0061] Fig.12 :(A) Schematic diagram of the experimental scheme for treating difficult-to-heal wounds in cachectic mice with CMOT hydrogel, (B) changes in food intake of normal mice and mice with C26 tumor, (C) changes in tumor volume of mice with C26 tumor, (D) changes in tumor-free body weight and (E) body weight, (F) pictures of wound healing in cachectic mice and (G) statistics of wound healing rate, (n=3).

[0062] Fig.13 : Results of CMOT hydrogel promoting skin healing in cachectic mice. (A) HE staining images of cachectic mouse skin wound sections at different times of CMOT hydrogel application, (B) Masson staining images, (C, D) immunohistochemical images of IL-6 and TNF-α and (E, F) statistics of immunohistochemical images, scale bar = 100 μm, (n = 3). DETAILED DESCRIPTION

[0063] Hereinafter, the technical content of the present application is described in detail through specific implementation methods so that those skilled in the art can better understand the present application. However, the provision of these embodiments is not intended to constitute any limitation on the protection scope of the present application.

[0064] Example 1: Extraction and characterization of Trichosanthes polysaccharide

[0065] 1. Materials and Instruments

[0066] Material

[0067] Radix Trichosanthis decoction pieces (provided free of charge by Bozhou Yonggang Decoction Pieces Factory Co., Ltd.)

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

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

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

[0071] instrument

[0072] Constant temperature water bath DK-S26 (Shanghai Hualian Medical Instrument Co., Ltd., Shanghai, China)

[0073] Water bath sonicator SBL (Ningbo Xinzhi Biotechnology Co., Ltd., China)

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

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

[0076] Full wavelength microplate reader Multiskan (Thermo Fisher scientific, Massachusetts, USA)

[0077] 1 / 100,000 electronic balance MS105DU (Mettler-Toledo Instruments, Zurich, Switzerland)

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

[0079] Zetasizer Nano-90ZS, Malvern, UK

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

[0081] 2 Experimental methods

[0082] 2.1 Extraction of Trichosanthes polysaccharides (TPS)

[0083] The Radix Trichosanthis was crushed into powder using a grinder and sealed at room temperature. Weigh 150g of Radix Trichosanthis powder into a 1L conical flask, add ultrapure water at a ratio of 50g / 250mL, soak for 30min, and ultrasonically extract in a 45℃ water bath for 2h. Stir continuously during the ultrasonic process to improve the extraction efficiency. After the extraction is completed, centrifuge at 15000×g to collect the supernatant, add 100mL of water to the precipitated residue and ultrasonically wash it for 10min, continue to centrifuge to collect the supernatant, repeat this process once, combine the extract and the washing liquid, concentrate in a 60℃ water bath to a volume of 50g of medicinal material / 50mL, and centrifuge to remove insoluble starch. Add anhydrous ethanol to make the ethanol volume fraction reach 85%, shake it fully, and let it stand overnight. The supernatant was discarded after centrifugation, 10 mL of water was added to dissolve the precipitate, trichloroacetic acid (TCA) was added to make the mass volume fraction reach 6%, and the protein was precipitated at 4°C overnight. The protein precipitate was removed by centrifugation, and 1 g of TCA was added again to precipitate for 3 hours. If there was no protein precipitate, the supernatant was placed in a 7 kD dialysis bag and dialyzed for 3 days to remove TCA. The product was concentrated to 120 mL in a water bath and lyophilized to obtain total polysaccharide TPS, which is also referred to as "Trichosanthes crude polysaccharide" in this application.

[0084] 2.2 Detection 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 form aldehyde derivatives, which then react with phenol to form orange-yellow compounds with maximum absorption at 490nm.

[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 that has been dried to constant weight and add to a 100 mL volumetric flask to obtain glucose standard solution (0.1 mg / mL).

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

[0089] 2.2.2 Drawing of glucose standard curve

[0090] Accurately pipette 0.1mL, 0.2mL, 0.4mL, 0.6mL, 0.8mL, and 1.0mL of glucose standard solution into 10mL test tubes, add distilled water to 1mL, then add 1mL of 5% phenol solution and 5mL of concentrated sulfuric acid, shake for 2min, keep warm in a 100℃ water bath for 20min, take out and cool to room temperature, use 1.0mL of distilled water as a blank control, measure the absorbance at 490nm, and draw a standard curve.

[0091] 2.2.3 Determination of polysaccharide content

[0092] Accurately weigh 0.01 g of Radix Trichosanthis polysaccharide dried to constant weight, make up to volume in a 100 mL volumetric flask, accurately transfer 1.0 mL from it to a test tube, add 1 mL of 5% phenol solution, and then quickly add 5 mL of concentrated sulfuric acid, shake thoroughly, keep warm in a water bath at 100 ° C for 20 min, take out and cool to room temperature, use 1.0 mL of distilled water as a blank control, measure the absorbance at 490 nm, and substitute it 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 is the polysaccharide mass obtained by substituting into the standard curve)

[0096] 2.3HPGPC detection of molecular weight of TPS

[0097] Weigh 20 mg of each standard molecular weight polysaccharide, dissolve it with 10 mL of 0.2 M NaCl mobile phase, centrifuge (10000 × g, 10 min), and take the supernatant for use. Weigh 20 mg of TPS sample, add 10 mL of 0.2 M NaCl to dissolve, centrifuge (10000 × g, 10 min), and take the supernatant for use. Take the standard and sample solutions and inject them in sequence, determine the corresponding liquid chromatogram, and calculate the sample molecular weight distribution using GPC software.

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

[0099] Weigh 10 mg of TPS, add 1 mL of ultrapure water to dissolve, centrifuge at 10,000 × g for 5 min, and take the supernatant for later use. Weigh 10 mg of CHCA, add 1 mL of acetonitrile (containing 0.1% trifluoroacetic acid) to dissolve, centrifuge and take the supernatant for later use. Pipette 10 μL of TPS solution and an equal volume of SA solution and mix them evenly, then prick 1 μL and spot it in the hole of the MALDI-TOF-MS special sample plate. After natural evaporation, load it onto the machine and perform detection in the "Linear mid mass positive" mode.

[0100] 2.5 Zeta potential detection of TPS

[0101] Take 1 mL of TPS solution (prepared with ultrapure water) with a concentration of 2 mg / mL and add it to the potentiometric cup. Use the potentiometric detection mode of the Malvern particle size analyzer to perform the potentiometric detection on the TPS solution. Repeat the detection 3 times.

[0102] 3 Results and analysis

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

[0104] Polysaccharides were extracted by water bath ultrasound. To avoid destroying the polysaccharide structure, the temperature was controlled at about 40°C. After adding ethanol to the polysaccharide water extract, a large amount of precipitate was produced. The precipitate was very sticky, indicating that the polysaccharide extraction was successful, but it also contained pigments and water-soluble proteins that needed to be removed. The precipitate was redissolved in water, and a large amount of protein precipitate was produced after adding trichloroacetic acid at low temperature until no new precipitate was produced, indicating that the protein had been basically removed. The experimental results show that the standard curve R of the phenol-sulfuric acid method 2 The value was 0.999, indicating a good linear relationship. The TPS was extracted three times using this extraction method, and the average TPS yield was 13.7% (RSD was 4.4%) and the average sugar content was 90.6% (RSD was 5.6%). Figure 1 , indicating that the extraction and detection method is relatively stable and can be used to evaluate the yield and content of polysaccharides.

[0105] 3.2 Characterization of TPS

[0106] The molecular weight of the polysaccharide was characterized by high performance gel permeation chromatography (HPGPC). Figure 2 As shown in A, TPS polysaccharide has two molecular weight distribution peaks. The first peak (rightmost) in the liquid phase spectrum is the peak of the mobile phase NaCl. According to the HPGPC software to calculate the molecular weight, the weight average molecular weight of the first peak in the liquid phase spectrum is 5.7kDa (middle), and the average molecular weight of the second peak is 72.1kDa (leftmost), indicating that TPS contains two polysaccharides with different molecular weights. Using the sum of the two peak areas as the denominator and the single peak area as the numerator, Image J calculated that the first peak accounted for 86.8% and the second peak accounted for 13.2%.

[0107] The molecular weight of TPS was characterized by MALDI-TOF-MS, and it was found that TPS peaked at about 53 kDa ( Figure 2 B) is the absolute molecular weight of the polysaccharide, but the response value is low, showing a steamed-bun peak. A variety of matrix solutions were tried, including sinapinic acid (SA), 5-hydroxysalicylic acid (DHB), and α-cyanocinnamic acid (CHCA), among which SA had the best effect. Therefore, it is speculated that the steamed-bun peak may appear due to the low ionic strength caused by the small number of active functional groups in the polysaccharide.

[0108] The potential detection results show that TPS is negative potential ( Figure 2 C), the average potential is -7.98±0.23 mV, which is caused by the electrostatic repulsion between the charges on the polysaccharide chains, and may also be caused by the ionizable acidic groups on the polysaccharide, such as carbonyl groups.

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

[0110] 1 Materials and Instruments

[0111] Material

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

[0113] Schiff 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] Infrared spectrometer Gary 630 (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] Rheometer MCR301 (Anton Paar, Graz, Austria)

[0120] Constant temperature shaker ZQTY-70V (Shanghai Zhichu Instrument Co., Ltd., Shanghai, China)

[0121] Full wavelength microplate reader Multiskan (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 TPS (Oxidized TPS, oTPS) by sodium periodate oxidation method. Weigh 1g of TPS into EP tubes, add 20mL ultrapure water to each portion to dissolve, then weigh 50mg of sodium periodate, add 1mL ultrapure water to dissolve, add to one portion of TPS, stir at room temperature in the dark for 4h to obtain oTPS1 (TPS with lower oxidation degree). For another portion of TPS, weigh 500mg of sodium periodate, add 1mL of water to dissolve, then add in the dark for 4h to react, to obtain oTPS2. After the reaction is completed, add 1mL of ethylene glycol and continue stirring for 1h to terminate the reaction. The product is dialyzed in a 14kDa dialysis bag for 48h and freeze-dried to obtain oTPS1 and oTPS2.

[0126] The Schiff reagent was used to detect the dialdehyde content in oTPS, and glutaraldehyde was used as the standard reference for calculation. The principle is that the condensate formed by the condensation of the sulfite group and the aldehyde group in the Schiff reagent is colored with the fuchsin solution, and has a maximum absorption at 550nm. Prepare standard solutions of glutaraldehyde with different molar concentrations: 0, 1, 2, 4, 8, and 16mM. Then prepare 2mg / mL oTPS1 and oTPS2 solutions. Take 0.5mL of the substance to be tested in a 10mL EP tube, then add 2.5mL of Schiff reagent and mix evenly, and immediately measure the absorbance at 550nm. Use the concentration of glutaraldehyde as the horizontal axis and the absorbance value as the vertical axis to establish a standard curve, and then substitute the absorbance value of the sample to solve the aldehyde content.

[0127] In order to detect whether there is residual sodium periodate in oTPS, sodium periodate is reacted with sodium iodide to generate iodine, and iodine reacts with 1% starch solution to produce a color reaction for qualitative judgment.

[0128] 2.2 Preparation of Radix Trichosanthis Polysaccharide-Chitosan (CMOT) Hydrogel

[0129] Weigh 3g of carboxymethyl cellulose (CMCS) and 3g of oTPS1 / oTPS2, add 100mL of ultrapure water to dissolve completely to obtain a 30mg / mL polysaccharide solution. Take equal volumes of CMCS solution and oTPS solution and mix them evenly, and then stand to obtain CMOT hydrogel. CMOT1 hydrogel is prepared by using oTPS1 and CMCS, and CMOT2 hydrogel is prepared by reacting oTPS2 and CMCS. The difference between the two hydrogels is that the oxidation degree of Trichosanthes polysaccharide is different.

[0130] 2.3 Characterization of CMOT hydrogel properties

[0131] 2.3.1 Gel time

[0132] The gel time of CMOT hydrogel was measured by the vial inversion method. When the hydrogel stopped flowing within 30 seconds after the vial was inverted, it was considered to be gelled, and the time from the preparation to gelation was recorded as the gel time.

[0133] 2.3.2 FTIR characterization

[0134] TPS (2 mg), oxidized TPS (2 mg), CMCS (2 mg), CMCS+TPS (physical mixing), and CMOT hydrogel were mixed with KBr powder (100 mg), respectively, and then pressed into tablets. The results were analyzed in an FTIR analyzer at 4000–400 cm -1 Analysis over a wide wavelength range.

[0135] 2.3.3 Characterization of self-healing performance

[0136] CMOT hydrogels of different colors were prepared: one was prepared with DMEM medium and the other was prepared with methylene blue solution to study the self-healing properties of CMOT hydrogels. The two gels were cut, and the planes obtained by cutting were in contact with each other for 20 minutes without force. The healing was then examined. In addition, the CMOT hydrogel was preloaded into a syringe and extruded with a 25G needle to examine injectability.

[0137] 2.3.4 Scanning electron microscopy characterization

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

[0139] 2.3.5 Swelling and degradation

[0140] Swelling test: Weigh the freshly prepared CMOT hydrogel (recorded as W0), immerse it in 10 mL PBS (pH 7.2), and swell it at 37°C. Take out the CMOT hydrogel at regular intervals, absorb the residual water on the surface with filter paper, and then weigh the hydrogel (recorded as Wt). Calculate the swelling rate (SR) using the following equation:

[0141]

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

[0143]

[0144] 2.3.6 Water holding capacity

[0145] First, the freshly prepared CMOT hydrogel was weighed (recorded as W0), and then the hydrogel was exposed to air at 25°C and weighed at predetermined day intervals (recorded as Wd). The water holding rate (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 to a parallel plate (PP25, d = 1 mm), and the hydrogel was subjected to frequency sweep (0.1 Hz–10 Hz) and strain sweep (0.01%–10%) using a stress-controlled rheometer (MCR301, Anton Paar, Austria) to measure the rheological properties of the CMOT hydrogel. G' represents the storage modulus and G" represents the loss modulus.

[0149] 3. Results and Analysis

[0150] 3.1 Preparation and characterization of oTPS

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

[0152] The infrared spectrum results show that oTPS has a characteristic absorption of 1630 cm -1 There is a large absorption near the TPS, while the absorption is small. Figure 3 B, indicating that the polysaccharide was successfully aldehyde-formylated.

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

[0154] Sodium periodate reacts with sodium iodide to produce iodine (brown). Iodine reacts with starch to produce blue. This reaction can determine whether the system contains sodium periodate. Figure 3F. The results showed that there was no residual sodium periodate 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 crosslinking with aldehyde groups ( Figure 4 A), when the cross-linked unit is large enough, a three-dimensional network structure, i.e., a hydrogel, will be formed. Moreover, carboxymethylated chitosan is soluble in water, oTPS is easily soluble in water, the preparation process is safe and non-toxic, and the prepared hydrogel has good biocompatibility. Two types of hydrogels CMOT1 and CMOT2 with different mechanical properties were prepared by cross-linking two types of Trichosanthes polysaccharides oTPS1 and oTPS2 with different oxidation degrees with carboxymethyl chitosan CMCS. The results of the test tube inversion experiment showed that the cross-linking time of CMOT1 was about 60s, while the cross-linking time of CMOT2 was about 30s, indicating that increasing the aldehyde content of the polysaccharide can shorten the cross-linking time of the hydrogel and form the hydrogel faster.

[0157] After cutting the two different colored hydrogels, CMOT1 and CMOT2, and then putting them back together, it was found that both hydrogels had the ability to self-heal, and the time required was not much different ( Figure 4 B).

[0158] Then, the microstructures of the two hydrogels were observed using a scanning electron microscope, and it was found that both hydrogels have a porous network microstructure unique to hydrogels. The pore size of CMOT1 (150 μm) is larger than that of CMOT2 (89 μm), which is about twice that of CMOT2. Figure 4 C.

[0159] The water absorption swelling rate is an important parameter for characterizing hydrogels. It can predict the initial volume of hydrogels when injected or buried in the body. For drug-loaded hydrogels, it can also help predict the release of drugs. The swelling results show that CMOT1 and CMOT2 hydrogels have swollen stably after 1 hour, and the swelling rate is 60% of their own mass. The swelling rate of CMOT1 is slightly higher than that of CMOT2. Figure 5 A, This indirectly proves that the pore size of CMOT1 is larger.

[0160] The in vitro degradation results showed that the degradation results of the two hydrogels were basically the same, and they were completely degraded in about two weeks, but their degradation behaviors were different. CMOT1 began to degrade rapidly on the 5th day, while CMOT2 degraded relatively slowly, and only began to degrade rapidly on the 9th day ( Figure 5 A).

[0161] Water is essential for maintaining skin health, so water retention is a necessary test indicator for hydrogels used on skin wounds. The results of in vitro water loss rate showed that the water loss rates of the two CMOT hydrogels were similar and slow, and the water retention rate could be maintained at more than 50% for 2 to 3 days under overall exposure conditions. Figure 5 C).

[0162] G' is the elastic modulus, and the larger its value is, the better the elasticity of the tested object is; G" is the viscous modulus, and its value is proportional to the viscosity of the tested object. The mechanical test results show that within the frequency range of 0.1 to 10 Hz, the elastic modulus G' of CMOT2 is higher than G' of CMOT1, indicating that the elasticity of CMOT2 hydrogel is greater. 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 is, and vice versa. The viscous modulus G" of CMOT2 is close to G', indicating that CMOT2 has a large hardness. On the contrary, the distance between the values ​​of G' and G" of CMOT1 is far, indicating that CMOT1 has a small hardness, see Figure 5 D. Similar results were obtained in the test with strain in the range of 0.1 to 10%, see Figure 5 E, shows the relative stability of the two hydrogels under stress.

[0163] The chemical cross-linking of CMOT hydrogel was verified by infrared spectroscopy. Figure 6 The results show that CMOT is at 1647cm -1 and 1628cm -1 The absorption bands corresponding to C=O and C=N stretching vibrations were shown at 3200-3600 cm -1 The signal intensity attributed to the OH / NH stretching vibration is lower than that of the physical mixture of CMCS and TPS (CMCS+TPS), indicating that Schiff base C=N bonds are formed in the prepared hydrogel.

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

[0165] 1 Materials and instruments

[0166] Material

[0167] DMEM 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 Biotech Co., Ltd., Beijing, China)

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

[0172] Inhibition and generation of superoxide anion free radical assay kit (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China)

[0173] instrument

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

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

[0176] Cells and animals

[0177] C2C12 cells (No. ZKCC-X2072) and RAW264.7 cells (No. ZK0833(XR)) were provided by the Shanghai Cell Bank, Chinese Academy of Sciences.

[0178] BALB / c mice (4–6 weeks old, female, SPF grade, 20–22 g) were provided by Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd., animal qualification certificate number: SCXK (Su) 2020-0009. They were fed with sterile feed and distilled water in a specific pathogen-free (SPF) care facility under a 12-h light / dark cycle, and the experimental facility license number is SYXK (Shanghai) 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, with IACUC approval number 2023-08-HYZ-149. The animals were fed adaptively for one week before the experiment.

[0179] 2. Evaluation content and methods

[0180] 2.1 Effect of CMOT on cell activity

[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 cells (mouse myoblasts) were plated in 96-well plates, 5000 cells / well, and administration began after 24 hours of culture in a cell culture incubator. The concentrations of CMOT were 3 mg / mL, 1.5 mg / mL, 0.75 mg / mL, 0.375 mg / mL, and 0 mg / mL. CCK8 reagent was added 24 hours after administration for cell viability detection. After 24 hours of incubation, CCK8 was added and incubated at 37°C for 1 hour, and 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 a 24-well plate and given 1 mg / mL of CMOT hydrogel extract. After 24 hours of incubation in an incubator, the cytotoxicity of CMOT hydrogel on RAW264.7 cells was measured using a live / dead assay kit. The cells were stained with Calcein / PI at 37°C for 30 minutes. The cells were observed by fluorescence microscopy and collected for flow cytometry analysis.

[0185] Toxicity of CMOT in direct contact with cells: First, freshly prepared CMOT hydrogels were spread into 24-well plates and sterilized by ultraviolet irradiation for 4 hours, and then RAW264.7 cells were added to the plates and cultured. CFDA-SE staining solution was added to monitor cell proliferation activity, and cells were photographed under a fluorescence microscope at different time periods of 1h, 24h, 48h, and 72h to observe cell activity. CFDA-SE exhibited green fluorescence under FITC channel irradiation, and its fluorescence would weaken due to cell division when cells proliferated.

[0186] 2.2 Effects of CMOT on subcutaneous tissue

[0187] Freshly prepared CMOT hydrogels were subcutaneously injected into normal BALB / c mice for 3 days. Skin tissues and CMOT hydrogels were collected for HE staining to evaluate the in vivo biosafety of CMOT hydrogels.

[0188] 2.3 Hemolytic properties of CMOT

[0189] 1 mL of freshly prepared red blood cell (RBC) suspension was added to CMOT hydrogel (1 mg) in a 1.5 mL EP tube and incubated at 37 °C for 4 h. The CMOT hydrogel was removed and the treated RBC suspension was centrifuged at 2000 rpm for 5 min for photography and OD540 detection. PBS and 1% Triton X-100 were used as negative and positive controls, respectively. The hemolysis rate was calculated using the following equation:

[0190]

[0191] 2.4 Antibacterial activity of CMOT

[0192] Take out the frozen Escherichia coli or Staphylococcus aureus from -20℃, thaw at room temperature, draw 1mL of bacterial solution into a 10mL EP tube in a clean bench, add 5mL of LB medium, and culture at 220rpm, 37℃ for 1h. Weigh 200mg of freshly prepared CMOT1 / CMOT2 hydrogel into a 4mL EP tube, add bacterial solution diluted 1000 times with LB medium, set up a blank control group and a positive control group (100μg / mL ampicillin sodium solution) at 220rpm, 37℃ for 4h, and set up 3 replicates for each group. Dilute the cultured bacterial solution 100 times with LB, then draw 50μL of the cultured bacterial solution and apply it to agarose solid plates. Each culture tube corresponds to a plate, and culture it upside down at 37℃ for 12h, take pictures, count the number of colonies, and calculate the antibacterial rate:

[0193]

[0194] 2.5 Regulatory activity of CMOT on BMDM and lymphocytes

[0195] DMEM complete medium was used as the extraction medium and co-incubated with 1 mg / mL CMOT hydrogel at 37°C for 2 days as the hydrogel extract, and 1 mg / mL CMCS and TPS polysaccharide solution groups were set up to detect the immune activity of single polysaccharide and composite polysaccharide hydrogels. BMDM cells were co-incubated with the extracts of the two hydrogels for 24 hours, and then flow cytometry staining was performed with flow cytometry antibodies F4 / 80-BV510, CD86-PerCP-Cy5.5, and CD206-APC, and the test was performed on the machine after 45 minutes.

[0196] Similarly, the above-mentioned polysaccharide solution and hydrogel extract were administered to mouse spleen-derived lymphocytes, and after 48 hours of co-culture, flow cytometry antibody incubation was performed, and the flow cytometry antibodies contained CD3-PerCP Cy5.5, CD8α-PE, Ki67-BV421, and CD49b-FITC, followed by flow cytometry detection.

[0197] 2.6 DPPH radical scavenging activity of CMOT

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

[0199]

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

[0201] 2.7CMOT superoxide anion radical (O2 .- ) Clear Experiment

[0202] The antioxidant capacity of CMOT hydrogel was determined according to the instructions of the superoxide anion free radical detection kit (Cat. No. A052-1-1). The calculation formula used is:

[0203]

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

[0205] 3. Results and Analysis

[0206] 3.1 Antioxidant experiments showed that both CMOT hydrogels have antioxidant capacity and can 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. Figure 7 A.

[0207] 3.2 Biosafety evaluation of CMOT hydrogel

[0208] The extracts of CMOT hydrogel were co-cultured with RAW264.7 cells and then cell death and viability staining was performed. The results of fluorescence microscopy showed that the extracts of the two CMOT hydrogels were non-toxic to RAW264.7 cells ( Figure 8 A, B), flow cytometry results also showed that the proportion of surviving cells in the CMOT extract was no different from that in the blank group PBS, and there were basically no dead cells ( Figure 8C, D). CCK8 test was performed on mouse myoblasts C2C12 and bone marrow macrophages BMDM administered with CMOT hydrogel. The results showed that 3 mg / mL CMOT hydrogel extract was basically non-toxic to both cells, and the cell survival rate was close to 100% ( Figure 8 E).

[0209] Direct contact culture of CMOT hydrogel and RAW264.7 cells revealed that RAW264.7 cells could survive and proliferate well on the hydrogel. At 48 h, the cells grew into the hydrogel and embedded in the hydrogel. At 72 h, the cells still survived well, indicating that the hydrogel had good softness and biosafety.

[0210] The hydrogel was co-cultured with mouse red blood cells and found that in the positive control group, the red blood cells were completely ruptured after incubation with 1% Triton, and the solution was red after centrifugation, with almost no red blood cell precipitation in the lower layer. In the CMOT hydrogel group and the negative control group, after PBS centrifugation, the red blood cells in the lower layer were clearly separated from the solution, and almost no red blood cells were ruptured ( Figure 8 F), indicating that the hydrogel has no hemolytic activity.

[0211] To test the biosafety of CMOT hydrogel in vivo, CMOT was injected into the subcutaneous tissue of mice, and the skin tissue was removed 3 days later for pathological sectioning and HE staining ( Figure 8 G), pathological images show that the cell morphology in the subcutaneous tissue filled with hydrogel is good, indicating that the hydrogel does not damage the skin tissue. At the same time, a small amount of complete immune cell infiltration was observed, indicating that the hydrogel may have the function of recruiting immune cells. In addition, a large amount of hydrogel still exists in the skin tissue removed on the third day, indicating that the degradation of CMOT hydrogel in the body is slow.

[0212] 3.3 Antibacterial activity of CMOT hydrogel

[0213] After CMOT hydrogel was co-cultured with Staphylococcus aureus, the culture fluid was applied and it was found that CMOT2 hydrogel could inhibit the growth of Staphylococcus aureus by about 50%, with significant antibacterial activity (P=0.001). Its antibacterial potency was approximately equivalent to 50 μg / mL of ampicillin sodium ( Fig. 9 A, C); CMOT1 hydrogel was basically unable to inhibit the growth of Staphylococcus aureus. For Gram-negative bacteria Escherichia coli, CMOT2 had a good inhibitory effect, equivalent to 100 μg / mL of ampicillin sodium ( Fig. 9 B, D); CMOT1 also showed the effect of inhibiting the growth of Escherichia coli. In short, the overall antibacterial effect of CMOT2 hydrogel is better than that of CMOT1 hydrogel, and it can inhibit the growth of both Gram-positive bacteria 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 to the stability of immunity in vivo. Flow cytometry results showed that CMOT hydrogel had 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 ( Fig.10 A, B), among which CD86 increased more ( Fig.10 C), indicating that CMOT hydrogel has the effect of promoting the development of macrophages to M1 phenotype. At the same time, after CMOT hydrogel treatment, lymphocytes including CD3 + 、CD49b + and CD8 + Lymphocytes begin to overexpress the proliferation index Ki67 ( Fig.10 D, E, F), indicating that CMOT hydrogel promotes the proliferation of T lymphocytes and NK cells by regulating 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 effect of CMOT in promoting cachexia wound healing

[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, 75% ethanol (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China)

[0221] Isoflurane (Shenzhen Reward Life Science 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 mouse 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 reagents Ⅲ1st 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] Scar Silicone Gel (Anshili (China) Medical Co., Ltd., Guangzhou)

[0232] instrument

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

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

[0235] Fluorescence quantitative PCR instrument BIO-RAD CFX384TM (BIO-RAD, California, USA)

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

[0237] Cells and Animals

[0238] Mouse myoblast C2C12 cells (No. ZKCC-X2072) were provided by the Shanghai Cell Bank, Chinese Academy of Sciences.

[0239] BALB / c mice (4–6 weeks old, female, SPF grade, 20–22 g) were provided by Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd., animal qualification certificate number: SCXK (Su) 2020-0009. They were fed with sterile feed and distilled water in a specific pathogen-free (SPF) care facility under a 12-h light / dark cycle, and the experimental facility license number is SYXK (Shanghai) 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, with IACUC approval number 2023-08-HYZ-149. The animals were fed adaptively for one week before the experiment.

[0240] 2 Experimental methods

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

[0242] After the mice were anesthetized with the respiratory anesthetic isoflurane, the backs of the mice were depilated to expose the back skin. A circle with a diameter of 7 mm was drawn on the back of the mouse using a marker pen and a ruler, and then the mouse skin was cut along the outer periphery of the dotted line using surgical scissors and forceps to create a circular full-thickness skin defect on the back of the mouse, and CMOT hydrogel was applied. Photos of the wounds were taken on days 0, 3, 7, and 11, and the skin at the wound site was cut for HE staining and Masson staining analysis. Image J was used to calculate the collagen volume fraction of the Masson-stained skin tissue sections.

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

[0244] C26 cells were revived from liquid nitrogen and expanded. The culture conditions of C26 cells were DMEM medium + 10% FBS + 1% penicillin-streptomycin solution, and the digestion conditions were rinsed with trypsin digestion solution. After rinsing, the trypsin digestion solution was discarded, and the cells were allowed to stand at room temperature for 1 minute. Then, DMEM medium was added to resuspend the cells, and single cell suspension was formed by pipetting and counting. The culture medium was discarded by centrifugation, and the cells were washed with PBS and then resuspended in PBS to form a 10 6 / 0.1mL of single cell suspension was used for subcutaneous tumor transplantation. First, the back of BALB / c mice was depilated, the depilatory cream was wiped clean, and 0.1mL of C26 single cell suspension was injected subcutaneously into the back of the mice using an insulin syringe. The needle of the insulin syringe was wiped with 75% alcohol cotton before injection. The daily weight, food intake and tumor volume of the mice were recorded, and a significant decrease in the weight and food intake of the mice was used as an indicator of cachexia.

[0245] When the mice showed significant weight loss and a significant decrease in food intake, it was judged that the mice reached a cachectic state, and cachectic wound modeling was started. The modeling method was referred to the full-thickness skin injury model above. The groups were set as the normal control group Control (i.e., normal mice with full-thickness skin injury), the C26 group (i.e., cachectic mice with full-thickness skin injury), and the CMOT group (CMOT hydrogel was applied to cachectic mice with damaged skin), ensuring that the hydrogel was fully covered on the wound surface, and then fixed with a breathable dressing. The wounds were photographed on the 0th, 3rd, 7th, and 14th days of skin injury, and the wound healing rate was calculated using Image J. On the 3rd, 7th, and 14th days, the mice were anesthetized and the skin at the wound site was cut for pathological sections, HE staining, and immunohistochemical staining to observe the microscopic recovery of the wound.

[0246] 3 Results and analysis

[0247] 3.1CMOT hydrogel promotes mouse skin healing

[0248] A full-thickness skin defect model was used to evaluate the effect of the hydrogel on wound healing. Fig.11 A), the results showed that compared with the control group (without any treatment), the wound area of ​​CMOT1 and CMOT2 hydrogel groups was significantly reduced on the third day, and the CMOT2 hydrogel group had a higher and more significant wound closure rate (P = 0.030), and the difference was statistically significant ( Fig.11 B, C). On the 7th day, the wound closure rates of the two CMOT hydrogels were significantly higher than those of the blank control group (P<0.0001), and the difference was statistically significant, indicating that the hydrogel has a good effect in promoting wound healing. On the 11th day, the wounds of the three groups were completely healed, among which the CMOT2 hydrogel group healed the best and had the least scar hyperplasia. The wound healing of the skin tissue on the 11th day was evaluated using hematoxylin and eosin stained sections (HE staining). The results showed that the hydrogel group showed thicker granulation tissue and higher epithelial regularity than the control group, and generated more blood vessels and hair follicles ( Fig.11 D). Masson staining results showed that the CMOT hydrogel group formed more collagen fibers (blue) and had a higher collagen volume fraction (CVF) ( Fig.11 D, E), indicating better healing. All the results showed that CMOT2 was better than CMOT1 hydrogel in promoting skin healing, which may be due to the higher aldehyde content in CMOT2, stronger antibacterial activity, and better viscoelasticity of CMOT2, which is conducive to wound healing.

[0249] 3.2 Establishment of cachexia-resistant wound model

[0250] A cachexia mouse model was established to study the therapeutic effect of CMOT hydrogel on cachexia-resistant wounds. Fig.12 A). The results showed that the tumor volume was approximately 1000 mm 18 days after subcutaneous injection of C26 cells. 3 ( Fig.12 B), and observed a decrease in the cumulative food intake and body weight of mice ( Fig.12 B, E). After day 20, the tumor-free body weight and cumulative food intake of mice decreased significantly ( Fig.12 D, B), indicating that the cachexia mouse model was successfully established. Full-thickness skin injuries were made and CMOT hydrogel was applied on the 23rd day. The results showed that compared with the normal group, the wound healing rate of the C26 (cachexia) group without any treatment was significantly delayed on the 3rd day after surgery (P=0.002), which also indicated the successful establishment of the cachexia difficult-to-heal wound model.

[0251] 3.3CMOT promotes the healing of cachexia wounds

[0252] Previous experiments have optimized CMOT2 hydrogel as the best choice for improving wound healing in cachectic mice, so the CMOT hydrogel used here is CMOT2 hydrogel. The macroscopic morphology of wound healing in cachectic mice (photo) shows that CMOT hydrogel has the effect of promoting cachectic wound healing ( Fig.12 F); Statistics show that on the third day, 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 ( Fig.12 G); On the 5th and 7th days, 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 showed mild acute inflammatory responses, while the CMOT hydrogel group showed more collagen fibers and inflammatory cells around the wound compared with the control group and the C26 cachexia group ( Fig.13 A, B), which is attributed to the healing effect and good immunomodulatory effect of CMOT hydrogel. On the 7th day, all groups formed a layer of epithelium, and the CMOT hydrogel group had thicker recovery tissue and higher epithelial regularity than the C26 group and the normal group. By the 14th day, all groups had formed the basic structure of epithelium and dermis, while the C26 group had the least blood vessels and hair follicles. In short, the thickened epithelial and dermal tissues, mature blood vessels, hair follicles and well-proliferated fibroblasts all indicate that CMOT hydrogel has the effect of promoting wound healing in cachectic mice.

[0254] Upregulation of IL-6 and TNF-α is a major driver of slow wound healing [2] After CMOT hydrogel administration, IL-6 in the local wound initially increased compared with the C26 group (on day 3, P < 0.0001), which helped to inhibit bacterial infection; it then decreased to normal levels (on days 7 and 14, P < 0.01), while cachectic mice without any treatment still had high levels of IL-6 on days 7 and 14 ( Fig.13 C, E). Meanwhile, the TNF-α level at the wound site of mice in the C26 group remained high on the 7th day (compared with the normal group, P<0.0001) and the 14th day (compared with the normal group, P=0.006), while the TNF-α level in the CMOT hydrogel-treated group was significantly downregulated on the 7th day (P<0.0001) and the 14th day (P=0.004), and the difference was statistically significant, close to the normal level ( Fig.13 D, F).

[0255] In summary, this application successfully extracted Trichosanthes polysaccharide TPS and explored its molecular weight and activity. TPS is a negatively charged polysaccharide. TPS can promote the differentiation of macrophages into M1 and M2 types, among which the proportion of M1 type is slightly higher, and has immunomodulatory function. At the same time, TPS promotes the proliferation of peripheral blood lymphocytes and spleen lymphocytes, increases CD4 + 、CD8 + The ratio of NK cells has the effect of enhancing immunity.

[0256] TPS was oxidized with sodium periodate to obtain two oxidized Trichosanthes polysaccharides oTPS1 and oTPS2, of which the aldehyde content of oTPS2 was twice that of oTPS1. The aldehyde groups in the oxidized polysaccharide can react with the amino groups in carboxymethyl chitosan to prepare a multifunctional Schiff base cross-linked hydrogel. Chitosan has anti-inflammatory and antibacterial effects, and Trichosanthes polysaccharide has an immune enhancement effect that promotes lymphocyte proliferation. The combination of the two has the effects of regulating inflammation at the wound site, anti-oxidation, antibacterial and immunomodulatory.

[0257] The prepared CMOT hydrogels have good biocompatibility both in vitro and in vivo and possess qualified properties of hydrogels, such as good healing properties, stable mechanical parameters, and water storage capacity. In a mouse model of full-thickness wound injury, CMOT2 hydrogels with high oxidation were more effective than CMOT1 hydrogels in accelerating wound healing and thus became the best hydrogel for subsequent experiments. Histological analysis showed that CMOT2 hydrogels exhibited advantages in immunomodulatory function 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 the clinical protection and treatment of cachexia wounds and immunosuppressive chronic wounds.

Claims

1. A method for preparing a CMOT hydrogel, the method comprising the following steps: 1) Preparation of crude trichosanthes polysaccharide: 1-1) Extracting the powder of Radix Trichosanthis with water ultrasonic, concentrating and centrifuging to remove insoluble starch; 1-2) adding anhydrous ethanol to the extract from which insoluble starch has been removed, allowing the extract to stand, and centrifuging to remove the supernatant; 1-3) Add water to dissolve the precipitate after removing the supernatant, and then add trifluoroacetic acid to precipitate the protein; 1-4) The supernatant after protein precipitation is placed in a 7 kD dialysis bag to remove trifluoroacetic acid, concentrated and freeze-dried to obtain crude Trichosanthes polysaccharide; 2) Aldehydation of crude polysaccharide from Radix Trichosanthis: Adding sodium periodate, TEMPO (2,2,6,6-tetramethylpiperidin-1-oxyl free radical) or manganese dioxide (MnO2) to the crude Trichosanthes polysaccharide prepared in step 1) to carry out a light-protected reaction to obtain aldehyde-modified crude Trichosanthes polysaccharide; 3) Preparation of CMOT hydrogel: The aldehyde-modified Trichosanthes kirilowii crude polysaccharide obtained in step 2) is covalently bonded with carboxymethyl chitosan through a Schiff base reaction to form a CMOT hydrogel.

2. The method according to claim 1, wherein: In step 1-1), the powder of Radix Trichosanthis is soaked in water for 0.5-2h, and ultrasonically extracted at 35-50°C for 1-3; and / or In step 1-1), extracting Radix Trichosanthis powder in water with ultrasonic wave, collecting the supernatant by centrifugation, extracting the precipitate residue again in water bath with ultrasonic wave, and combining the two extracts; and / or In step 1-2), anhydrous ethanol is added to the extract from which insoluble starch has been removed so that the volume fraction of ethanol reaches more than 85%.

3. The method according to claim 1, wherein: In step 2), Adding sodium periodate to the crude Trichosanthes kirilowii polysaccharide prepared in step 1), and performing a reaction at 20 to 35° C. in the dark to generate aldehyded crude Trichosanthes kirilowii polysaccharide, wherein the molar ratio of the crude Trichosanthes kirilowii polysaccharide to sodium periodate is 1:10 to 1:1000; and / or The oxidation degree of the obtained aldehyde-modified Radix Trichosanthis crude polysaccharide is 1-5 mmol / g Radix Trichosanthis crude polysaccharide (TPS), preferably 5 mmol / g TPS, wherein the oxidation degree refers to the molar number of aldehyde groups oxidized per gram of polysaccharide.

4. The method according to claim 1, wherein: In step 3), The degree of deacetylation of carboxymethyl chitosan is ≥ 90%; and / or Adding an aqueous solution of carboxymethyl chitosan to an aqueous solution of aldehyde-modified crude polysaccharide of Radix Trichosanthis, wherein the concentration of the aqueous solution of aldehyde-modified crude polysaccharide of Radix Trichosanthis is 1% to 3%, the concentration of the aqueous solution of carboxymethyl chitosan is 1% to 3%, the unit is g / mL, and the volume ratio of the two solutions is 1:1; and / or The amino groups in the carboxymethyl chitosan react with the dialdehyde groups in the aldehyde-modified crude polysaccharide of Radix Trichosanthis to form C=N bonds to achieve cross-linking, and the cross-linking time is 0.5 to 3 minutes.

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

6. A pharmaceutical composition, comprising at least the trichosan polysaccharide-chitosan hydrogel according to claim 5, and pharmaceutically acceptable excipients.

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

8. Use of the trichosanthes polysaccharide-chitosan hydrogel according to claim 5 or the pharmaceutical composition according to claim 6 or 7 in the preparation of a medicament for treating cachectic wounds and other immunosuppressive chronic wounds.

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

10. The use according to claim 8, wherein The cachexia includes cancer, AIDS, inflammatory disease cachexia, and / or Such other immunosuppressive chronic wounds include diabetic wounds.

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