CN-121987559-A - Preparation method of probiotics hydrogel material and application of probiotics hydrogel material in treatment of acute radiation intestinal injury

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a preparation method of a probiotic hydrogel material and application of the probiotic hydrogel material in treatment of acute radiation intestinal injury. Aiming at the clinical key problem of radioactive intestinal injury, the invention constructs a novel biological material TG-CCS@DY802 of thiolation synchronous modification chitosan hydrogel TG-CCS loaded 'localized high response' lactobacillus salivarius DY802. The material has excellent biological safety and tumor inhibition experiment performance when the synthetic process of the material increases efficiency and reduces, and can comprehensively repair four barriers of intestinal tracts, namely, the material can remove ROS, protect tight junction protein to repair physical barriers, form a hydrogel protective layer to strengthen chemical barriers, promote flora diversity, play a prebiotic effect to repair biological barriers, regulate and control sphingomyelin metabolism and ceramide abundance, inhibit abnormal infiltration of NK cells and remodel immune barriers. The probiotics hydrogel material has clear treatment mechanism and obvious clinical transformation potential and application value.

Inventors

• Ding Die
• GU XINYI
• YU LU
• CHEN ZHENHUI
• LIN ZHIYING

Assignees

• 南方医科大学南方医院

Dates

Publication Date

20260508

Application Date

20260302

Claims (10)

1. 1. The preparation method of the probiotic hydrogel material TG-CCS@DYS802 is characterized by comprising the following steps: S1, dispersing chitosan through an alkaline isopropanol solution to form a uniform flocculent dispersion without obvious caking; S2, adding a mixed solution consisting of chloroacetic acid and thioglycollic acid into the reaction system in the step S1, heating to 60-80 ℃, performing carboxymethylation reaction in an alkaline environment, cooling to 20-40 ℃, and performing thiolation reaction in a closed environment to finish carboxymethylation thiolation improvement synchronous modification of chitosan; s3, regulating the pH of the reaction product obtained in the step S2 to 6.5-7.0, adding ethanol, standing after uniformity to enable the carboxymethylated thiolated chitosan to be fully precipitated, filtering and collecting the precipitate, washing the precipitate with the ethanol until filtrate is colorless and transparent, dissolving the washing product in water, and dialyzing and drying to obtain purified carboxymethylated thiolated chitosan; S4, culturing lactobacillus salivarius DY802 by using an MRS culture medium, adjusting the bacterial suspension to OD 600 =0.8-1.0, and removing the culture medium to obtain purified DY802 viable bacteria; S5, dissolving the carboxymethylation thiolated chitosan purified by the S3 in PBS buffer solution, adding DY802 viable bacteria of the S4, adjusting the pH of the system to an acidic environment, and incubating to fully mix thalli and hydrogel to form stable electrostatic adsorption, thereby finally obtaining the probiotic hydrogel material TG-CCS@DY802 with bioactivity.
2. 2. The method for preparing the probiotic hydrogel material TG-CCS@DY802 according to claim 1, wherein the chitosan is a chitosan powder preparation with a deacetylation degree of more than 90%.
3. 3. The method for preparing the probiotic hydrogel material TG-CCS@DY802 according to claim 1, wherein the molar ratio of chloroacetic acid to chitosan is 1:4-6, and the molar ratio of thioglycollic acid to chitosan is 1:8-12.
4. 4. The method for preparing the probiotic hydrogel material TG-CCS@DY802 according to claim 1, wherein the carboxymethylation reaction is carried out under stirring for 2-4 h ℃ and the heating rate is 2-5 ℃ per min.
5. 5. The preparation method of the probiotic hydrogel material TG-CCS@DY802 according to claim 1, wherein the thiolation reaction is carried out under stirring at a stirring rate of 100-180 rpm, and the cooling rate of cooling to 20-40 ℃ after the carboxymethylation reaction is 0.5-1 ℃ per min.
6. 6. The method for preparing the probiotic hydrogel material TG-ccs@dy802 according to claim 1, wherein the dialysis is performed in a dialysis bag with a molecular weight cut-off of 9-11 kDa, and water is used as medium for dialysis until the dialysis external fluid is detected by AgNO 3 to be free of white precipitate, ensuring complete removal of Cl - residues.
7. 7. The method for preparing the probiotic hydrogel material TG-CCS@DY802 according to claim 1, wherein the concentration of the carboxymethylated thiolated chitosan in PBS buffer solution is 8-15 mg/mL, and the final concentration of DY802 viable bacteria is 10 8 CFU/mL.
8. 8. The method for preparing the probiotic hydrogel material TG-ccs@dy802 according to claim 1, wherein the pH of the acidic environment is 6.0-6.8.
9. 9. The probiotic hydrogel material TG-ccs@dy802 prepared by the preparation method of any one of claims 1 to 8.
10. 10. The use of the probiotic hydrogel material TG-ccs@dy802 of claim 9 in the preparation of a medicament for treating acute radiation intestinal injury.

Description

Preparation method of probiotics hydrogel material and application of probiotics hydrogel material in treatment of acute radiation intestinal injury Technical Field The invention belongs to the technical field of biological medicines, and particularly relates to a preparation method of a probiotic hydrogel material and application of the probiotic hydrogel material in treatment of acute radiation intestinal injury. Background Acute radiation-induced acute bowel injury, RARI) is an acute condition of the intestinal tract caused by radiation physical injury after a malignant tumor patient of the abdominal basin receives radiation treatment. In patients receiving radiotherapy, RARI can appear to various degrees in about 70% -80%, clinical symptoms are mainly abdominal pain, abnormal defecation and hematochezia, and even intestinal perforation rupture can occur when serious. Currently, the positive results of clinical researches such as PolishII, RAPIDO, STELLAR and the like are pushing the application of short-range radiotherapy (25 Gy/5F) to be increasingly popular, but the incidence and severity of acute radioactive intestinal injury are also obviously increased. In addition, the patients need comprehensive diagnosis and treatment means such as combined operation, chemotherapy, immunotherapy and the like, so that the treatment difficulty and risk of the disease are further improved, and the overall treatment progress and prognosis of tumors can be influenced by serious patients. At present, no practical and effective standardized and personalized treatment scheme exists in clinic aiming at RARI, the problem of insufficient sample size exists in the ongoing clinical research, and the related curative effect evidence is still deficient. Thus, alleviating and treating acute radiation intestinal injury is an important scientific problem to be solved in radiotherapy clinical practice. RARI occurs in that intestinal epithelial cells are highly sensitive to radiation, which can lead to impaired intestinal barrier, inflammatory cytokine release, immune cell recruitment, and thus secondary intestinal microbiota micro-ecological disorders. Based on this, the "inflammation relief" and "flora modulation" have become key principles for RARI treatments. According to the national institute of medical science and treatment (2023 edition) of Chinese radioactive intestinal injury, acute radioactive intestinal injury is usually treated by adopting a conservative method, such as montmorillonite powder, antibiotic combination or natural antioxidants (vitamin E/C, glutathione, anthocyanin and the like), and the traditional Chinese medicine prescription is orally taken or used for enema therapy. In the above treatments, limitations and side effects of each therapy are objectively present. The montmorillonite powder is easy to cause difficult defecation when being taken excessively, the traditional Chinese medicine preparation has complex components, various targets, obvious systemic effect and unstable curative effect. The combination of antibiotics is very easy to cause intestinal flora disturbance, secondary infectious enteritis or other systemic inflammatory diseases. Although natural antioxidants have certain efficacy, they are limited to scavenging active oxygen produced by radiation and have no efficacy in secondary intestinal function and microecological disorders. More and more clinical and basic research data in recent years show that intestinal flora plays an important role in delaying the disease progression of acute radiation intestinal injury, and supplementing relevant probiotics can play a remarkable role in clinical and pathological relief of the disease, so that a new strategy is provided for treatment RARI. Among them, fecal transplantation (Fecal microbiota transplantation, FMT) has been used more and more widely in recent 5 years as an emerging probiotic-related therapy. The method is to transplant functional flora in the feces of healthy people into the intestinal tract of RARI patients to reconstruct intestinal flora microecology. However, such therapies have problems associated with strain selection difficulties (poor engraftment, poor tolerance, difficult culture, unstable efficacy, etc.) and biosafety risks (bacteremia, translocation colonization, systemic adverse reactions, etc.), thus limiting their full clinical use. Existing studies have demonstrated that probiotic formulations can effectively alleviate RARI clinical symptoms. However, the probiotics have obvious differences of localization and individuation, so that the probiotic preparation customized for different people can exert the treatment effect to the maximum extent. Aiming at the patients with high response of colorectal radiotherapy and chemotherapy in the forefront stage, the subject group carries out the screening and separation work of fecal bacteria to successfully obtain the indigenous strain lactobacillus salivarius (Ligilactobacillus