CN-122005493-A - Bioactive molecule delivery method based on probiotics exosomes

Abstract

The invention discloses a bioactive molecule delivery method based on a probiotic exosome, and belongs to the technical field of biopharmaceuticals and targeted delivery. The method comprises the steps of screening clinically approved probiotic strains and separating natural exosomes, loading bioactive molecules on the exosome capsule cavity or surface through an incubation, electroporation or ultrasound assisted method, wherein the encapsulation rate is more than or equal to 85%, carrying out targeted functional modification on the loaded exosomes, such AS connection of AS1411 aptamer, antibody or coated Fe 3 O 4 @PDA magnetic nano particles, so AS to endow intestinal targeting capability, and finally preparing the modified targeted delivery system into an oral preparation. The invention uses the probiotics exosome as natural carrier, has good biocompatibility and low immunogenicity, can effectively protect active molecules from being degraded by gastrointestinal tract, realizes specific enrichment to intestinal lesions through targeted modification, and is especially suitable for preventing and treating iron death related diseases such as inflammatory bowel disease, cancer and the like.

Inventors

• GAO CHENZHE
• WANG HAITAO
• ZHANG LIN
• LAN Shuting
• Lv Zuokai
• ZHANG XINGCAN
• XU WENJING
• LIU JIAHUI
• PANG YUE
• ZHANG HAIYAN

Assignees

• 东北林业大学

Dates

Publication Date

20260512

Application Date

20260225

Claims (10)

1. 1. A method for delivering bioactive molecules based on probiotics exosomes is characterized by comprising the following specific steps: s1, screening clinically approved probiotic strains for amplification culture, and separating to obtain natural exosomes; s2, stably introducing bioactive molecules into the capsule cavity or the surface of the natural exosome to form a load-type exosome; s3, carrying out targeted function modification on the load exosomes to obtain a targeted delivery system with intestinal target recognition capability; s4, preparing the targeted delivery system into an oral preparation, and realizing the specific delivery of the bioactive molecules to intestinal target sites through an oral administration mode.
2. 2. A method of delivering a bioactive molecule based on a probiotic exosome according to claim 1, wherein: the natural exosomes obtained in step S1 are obtained by one of ultracentrifugation, density gradient centrifugation or ultrafiltration centrifugation.
3. 3. A method of delivering a bioactive molecule based on a probiotic exosome according to claim 1, wherein: in step S1, the probiotic is selected from one or more of lactobacillus, bifidobacterium, streptococcus.
4. 4. The method for delivering bioactive molecules based on the exosome of probiotics as set forth in claim 1, wherein the loading process in the step S2 is one of incubation loading method, electroporation method or ultrasound-assisted loading method, and the encapsulation rate of bioactive molecules in the loading process is not less than 85%.
5. 5. The method for delivering bioactive molecules based on the exosomes of probiotics as claimed in claim 1, wherein the bioactive molecules in the step S2 are selected from one or more of targeted iron death intelligent peptides, iron death regulatory genes, small molecule iron death inducers and tumor targeting antibodies, wherein the small molecule iron death inducers comprise at least one of Erastin, RSL3 and YL-9395.
6. 6. The method of claim 1, wherein in step S3, the targeting ligand is selected from one or more of an AS1411 aptamer, an Anti-EGFR antibody, an Anti-HER2 antibody, or a HavPD-1 antibody.
7. 7. The method for delivering bioactive molecules based on the exosome of probiotics as set forth in claim 1, wherein the targeting function modification further comprises coating Fe 3 O 4 @PDA nanoparticles on the surface of the loaded exosome to form a magnetic targeting modification layer.
8. 8. The method for delivering bioactive molecules based on the exosomes of probiotics as claimed in claim 1, wherein the particle size of the exosomes of probiotics isolated in step S1 is 50-100nm.
9. 9. The method of claim 1, wherein in step S4, the oral preparation is a capsule or a tablet.
10. 10. A method of delivering a probiotic exosome-based bioactive molecule according to claim 1, wherein said method of delivering is for preventing or treating iron death-related diseases, including inflammatory bowel disease, cancer or neurodegenerative diseases.

Description

Bioactive molecule delivery method based on probiotics exosomes Technical Field The invention belongs to the technical field of biopharmaceuticals and targeted delivery, and particularly relates to a bioactive molecule delivery method based on a probiotic exosome. Background Bioactive molecules (such as polypeptides, genes, small molecule drugs, etc.) have shown great potential in disease prevention and treatment, especially specific active molecules against iron death-related diseases (inflammatory bowel disease, cancer, neurodegenerative diseases, etc.), have become research hotspots in the biomedical field. However, such bioactive molecules face a number of bottlenecks in the in vivo delivery process, severely limiting their clinical application. Firstly, the bioactive molecules have poor self stability, are easily degraded by gastric acid, pepsin and intestinal flora in the gastrointestinal tract when orally taken, so that the bioavailability is extremely low, and even if the bioactive molecules are taken by injection, the bioactive molecules are easily and rapidly cleared by an in-vivo enzyme system, and are difficult to reach target tissues to play a role. Secondly, the traditional delivery carrier (such as liposome, nanoparticle, polymer carrier and the like) has the problems of poor biocompatibility, strong immunogenicity, insufficient targeting and the like, is easy to trigger immune response of an organism, and most of the carriers cannot be accurately enriched in intestinal target sites, so that the drug off-target toxicity is increased and the treatment effect is limited. The development of oral targeted delivery systems is particularly critical to the delivery needs of gut-related diseases. The existing intestinal targeting strategy mostly depends on pH sensitive materials, enteric coating and other technologies, and can protect a carrier from being damaged by gastric acid to a certain extent, but the existing intestinal targeting strategy lacks the recognition capability for specific targets after entering the intestinal tract, and still has the problems of low targeting precision, medicine leakage and the like. Meanwhile, for the iron death control active molecules, the action mechanism is complex, and the iron death control active molecules need to be accurately delivered to pathological cells to control an iron death pathway, so that the traditional carrier is difficult to meet the accurate delivery requirement. As a natural nano-carrier, the probiotics exosome has the advantages of good biocompatibility, low immunogenicity, penetrability of biological barriers and the like, and the safety of the probiotics strains from which the probiotics exosome is clinically approved is guaranteed. However, the existing delivery technology based on the probiotics exosomes still has the defects that firstly, the stability of the separation and purification technology of the exosomes is insufficient, the large-scale preparation is difficult to realize, secondly, the loading efficiency and stability of the active molecules are required to be improved, the encapsulation rate is generally lower than 80%, the dosage requirement of clinical treatment cannot be met, thirdly, the efficient targeting modification strategy is lacking, the specific identification of intestinal lesions is difficult to realize, and fourthly, a mature scheme is not formed in the probiotics exosomes delivery system aiming at iron death related diseases, and the treatment efficacy of the active molecules cannot be fully exerted. Therefore, developing a probiotic exosome bioactive molecule delivery method which has high loading efficiency, strong targeting property and good stability and is suitable for oral administration becomes a technical problem to be solved urgently. Disclosure of Invention The invention provides a bioactive molecule delivery method based on a probiotic exosome, and aims to solve the problems of poor oral delivery stability, insufficient targeting, low loading efficiency, lack of a specific delivery system for iron death related diseases and the like of bioactive molecules in the prior art. In order to achieve the aim, the invention provides a bioactive molecule delivery method based on a probiotic exosome, which comprises the following specific steps: s1, screening clinically approved probiotic strains for amplification culture, and separating to obtain natural exosomes; s2, stably introducing bioactive molecules into the capsule cavity or the surface of the natural exosome to form a load-type exosome; s3, carrying out targeted function modification on the load exosomes to obtain a targeted delivery system with intestinal target recognition capability; s4, preparing the targeted delivery system into an oral preparation, and realizing the specific delivery of the bioactive molecules to intestinal target sites through an oral administration mode. According to some embodiments of the method of active molecule deliver