Bioactive molecule delivery method based on probiotic exosome

Abstract

The invention discloses a bioactive molecule delivery method based on probiotic exosomes, and belongs to the technical field of biological pharmacy and targeted delivery. The method comprises the following steps: screening clinically accepted probiotic strains and separating natural exosomes of the clinically accepted probiotic strains; bioactive molecules are loaded on the exosome capsule cavity or surface through an incubation, electroporation or ultrasonic-assisted method, and the encapsulation efficiency is greater than or equal to 85%; carrying out targeting function modification on the supported exosome, such as connecting an AS1411 aptamer, an antibody or coating Fe3O4 (at) PDA magnetic nanoparticles, so as to endow the supported exosome with intestinal targeting capability; and finally, preparing the modified targeted delivery system into an oral preparation. The probiotic exosome is used as a natural carrier, the biocompatibility is good, the immunogenicity is low, active molecules can be effectively protected from being degraded by gastrointestinal tracts, specific enrichment towards intestinal diseased regions is achieved through targeted modification, and the probiotic exosome is particularly suitable for prevention and treatment of ferroptosis related diseases such as inflammatory bowel diseases and cancers.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical and targeted delivery technology, and particularly relates to a method for delivering bioactive molecules based on probiotic exosomes. Background Technology

[0002] Bioactive molecules (such as peptides, genes, and small molecule drugs) have shown great potential in disease prevention and treatment, especially specific active molecules targeting ferroptosis-related diseases (inflammatory bowel disease, cancer, neurodegenerative diseases, etc.), which have become a research hotspot in the biomedical field. However, the in vivo delivery of these bioactive molecules faces many bottlenecks, which severely limit their clinical application.

[0003] First, bioactive molecules themselves have poor stability. When administered orally, they are easily degraded by gastric acid, pepsin, and gut microbiota in the gastrointestinal tract, resulting in extremely low bioavailability. Even when administered by injection, they are easily cleared by the body's enzyme system, making it difficult for them to reach the target tissue and exert their effects. Second, traditional delivery carriers (such as liposomes, nanoparticles, and polymer carriers) have problems such as poor biocompatibility, strong immunogenicity, and insufficient targeting, which can easily trigger the body's immune response. Moreover, most carriers cannot accurately accumulate at the intestinal target site, leading to increased off-target toxicity and limited therapeutic efficacy.

[0004] For addressing the delivery needs of intestinal-related diseases, the development of oral targeted delivery systems is particularly crucial. Current intestinal-targeting strategies largely rely on pH-sensitive materials and enteric coatings, which, while protecting the carrier from gastric acid degradation to some extent, lack the ability to recognize specific targets after entering the intestine, resulting in low targeting accuracy and drug leakage. Furthermore, for ferroptosis-regulating active molecules, their mechanisms of action are complex, requiring precise delivery to diseased cells to regulate the ferroptosis pathway; traditional carriers struggle to meet this precise delivery requirement.

[0005] Probiotic exosomes, as natural nanocarriers, possess advantages such as good biocompatibility, low immunogenicity, and the ability to penetrate biological barriers. Furthermore, the probiotic strains from which they are derived are mostly clinically approved, ensuring safety. However, existing probiotic exosome-based delivery technologies still have shortcomings: First, the stability of exosome isolation and purification processes is insufficient, making large-scale preparation difficult; second, the loading efficiency and stability of active molecules need improvement, with encapsulation rates generally below 80%, failing to meet the dosage requirements for clinical treatment; third, there is a lack of efficient targeting modification strategies, making it difficult to achieve specific recognition of intestinal lesion sites; and fourth, a mature probiotic exosome delivery system for ferroptosis-related diseases has not yet been developed, failing to fully leverage the therapeutic efficacy of active molecules. Therefore, developing a probiotic exosome bioactive molecule delivery method with high loading efficiency, strong targeting, good stability, and suitability for oral administration has become an urgent technical problem to be solved. Summary of the Invention

[0006] This invention provides a method for delivering bioactive molecules based on probiotic exosomes, aiming to solve the problems of poor stability, insufficient targeting, low loading efficiency, and lack of specific delivery systems for ferroptosis-related diseases in the oral delivery of bioactive molecules in the prior art.

[0007] To achieve the above objectives, the following technical solution is adopted: This invention provides a method for delivering bioactive molecules based on probiotic exosomes, comprising the following specific steps:

[0008] S1. Screen clinically approved probiotic strains for large-scale culture and isolate natural exosomes;

[0009] S2. Stably introduce bioactive molecules into the cavity or surface of the natural exosomes to form loaded exosomes;

[0010] S3. Modify the loaded exosomes for targeted function to obtain a targeted delivery system with intestinal target recognition capability;

[0011] S4. The targeted delivery system is formulated into an oral formulation, and the bioactive molecules are specifically delivered to the intestinal target site through oral administration.

[0012] According to some embodiments of the active molecule delivery method described in this application, the natural exosomes obtained in step S1 are obtained by one of ultracentrifugation, density gradient centrifugation or ultrafiltration centrifugation.

[0013] According to some embodiments of the active molecule delivery method described in this application, in step S1, the probiotics are selected from one or more of the genera Lactobacillus, Bifidobacterium, and Streptococcus.

[0014] According to some embodiments of the active molecule delivery method described in this application, the loading process in step S2 is selected from one of the incubation loading method, electroporation method or ultrasound-assisted loading method, and the encapsulation rate of bioactive molecules during the loading process is ≥85%.

[0015] According to some embodiments of the active molecule delivery method described in this application, the bioactive molecule in step S2 is selected from one or more of the following: targeted ferroptosis smart peptides, ferroptosis regulatory genes, small molecule ferroptosis inducers, and tumor-targeting antibodies; wherein the small molecule ferroptosis inducers include at least one of Erastin, RSL3, and YL-9395.

[0016] According to some embodiments of the active molecule delivery method described in this application, in step S3, the targeting ligand is selected from one or more of AS1411 aptamer, anti-EGFR antibody, Anti-HER2 antibody, or HavPD-1 antibody.

[0017] According to some embodiments of the active molecule delivery method described in this application, the targeted functional modification further includes coating Fe3O4@PDA nanoparticles on the surface of the supported exosomes to form a magnetic targeted modification layer.

[0018] According to some embodiments of the active molecule delivery method described in this application, the probiotic exosomes isolated in step S1 have a particle size of 50-100 nm.

[0019] According to some embodiments of the active molecule delivery method described in this application, in step S4, the oral formulation is a capsule or tablet.

[0020] According to some embodiments of the active molecule delivery method described in this application, the delivery method is used to prevent or treat ferroptosis-related diseases, including inflammatory bowel disease, cancer, or neurodegenerative diseases.

[0021] The beneficial effects of this invention are:

[0022] This invention uses clinically recognized probiotic strains such as Lactobacillus and Bifidobacterium to prepare natural exosomes. These exosomes, as natural nanoparticles secreted by probiotics, are non-immunogenic and have low toxicity, effectively avoiding the immune responses and biocompatibility issues caused by traditional artificial carriers. They are also tolerant of the gastrointestinal environment, providing a basis for oral administration and significantly improving the clinical applicability of the delivery system. Using incubation loading, electroporation, or ultrasound-assisted loading methods, the encapsulation rate of bioactive molecules is ensured to be far higher than the average level of existing exosome loading technologies. Simultaneously, the loading methods on the exosome cavity and surface effectively protect the active molecules from the gastrointestinal tract. This invention addresses the core pain points of poor stability and low bioavailability of bioactive molecules by inhibiting enzyme and bacterial degradation and prolonging their in vivo half-life. It improves delivery precision through a targeted modification strategy. On one hand, it selects specific ligands such as AS1411 aptamer and anti-EGFR antibody to accurately identify target sites on the surface of intestinal diseased cells. On the other hand, it forms a magnetic targeting layer by encapsulating Fe3O4@PDA nanoparticles, which can further enrich the drug in the diseased area under the assistance of an external magnetic field, significantly increasing the drug concentration at the intestinal target site and reducing damage to normal tissues. This is particularly suitable for the precision treatment of ferroptosis-related cancers and inflammatory bowel disease. Attached Figure Description

[0023] Figure 1 This is a flowchart of a bioactive molecule delivery method based on probiotic exosomes according to the present invention.

[0024] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof. Detailed Implementation

[0025] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention. The preferred embodiments and materials described herein are for illustrative purposes only and do not limit the scope of this application.

[0027] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the experimental materials used in the following examples are all purchased from commercial channels.

[0028] Example:

[0029] This embodiment uses the preparation of a dual-targeting probiotic exosome oral capsule loaded with AI-designed targeted ferroptosis intelligent peptide (named Pep-F) and Erastin as an example to detail the specific implementation process of the present invention. The prepared delivery system is used for the precision treatment of intestinal tumors. The process of the bioactive molecule delivery method is as follows: Figure 1 As shown.

[0030] 1. Experimental Materials and Reagents

[0031] 1.1 Probiotic strains: Clinically approved Lactobacillus acidophilus and Bifidobacterium longum were purchased from Jiangsu Caiwei Biotechnology Co., Ltd.

[0032] 1.2 Bioactive molecules: Targeting ferroptosis smart peptide (purity ≥98%), small molecule ferroptosis inducer Erastin (purity ≥99%).

[0033] 1.3 Reagents and Consumables: MRS medium, Brain Heart Infusion (BHI) medium, PBS buffer (pH 7.4), sucrose density gradient solution, Fe3O4@PDA nanoparticles (particle size 20 nm, surface amino modified, purchased from Nanjing Xianfeng Nanomaterials Technology Co., Ltd.), AS1411 aptamer (5'-GGTGGTGGTGGTTGTGGTGGTGGTGG-3', purity ≥99%, Shanghai Sangon Biotech Co., Ltd.), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), enteric coating material (acrylic resin IV), lactose, microcrystalline cellulose, magnesium stearate (all pharmaceutical grade);

[0034] 1.4 Instruments and Equipment: High-speed refrigerated centrifuge, ultracentrifuge (Beckman Optima XE-90), nanoparticle tracking analyzer (NTA, Malvern NanoSight NS300), transmission electron microscope (TEM, Hitachi H-7650), ultrasonic cell disruptor, high-performance liquid chromatograph (HPLC, Agilent 1260), and in vitro simulated gastrointestinal environment device.

[0035] 2. Preparation of a bioactive molecule delivery system based on probiotic exosomes

[0036] 2.1 Isolation and purification of natural probiotic exosomes

[0037] Lactobacillus acidophilus and Bifidobacterium longum strains were inoculated into MRS medium and BHI medium, respectively, and cultured at 37°C under anaerobic conditions for 24 h to obtain seed culture. The seed culture was transferred to a 10L fermenter at an inoculation rate of 5% (v / v) and fermented at 37°C under anaerobic conditions for 48 h. After fermentation, the culture was centrifuged at 4°C and 8000 r / min for 15 min to remove the bacterial precipitate and collect the supernatant.

[0038] Exosomes were isolated by ultracentrifugation: the supernatant was filtered through a 0.22 μm filter to remove impurities, followed by centrifugation at 12,000 rpm for 30 min at 4 °C to further remove large vesicles; the supernatant was transferred to an ultracentrifuge tube and centrifuged at 100,000 rpm for 90 min at 4 °C, the supernatant was discarded, and the precipitate was resuspended in pre-cooled PBS buffer; the resuspended solution was spread on a sucrose density gradient solution (density 1.06-1.18 g / mL), and centrifuged at 120,000 rpm for 120 min at 4 °C, collecting the fraction with a density of 1.10-1.13 g / mL, which was the preliminarily purified exosome; the exosomes were dialyzed with PBS buffer for 24 h (with 3 buffer changes) to remove sucrose, and finally the particle size was detected by NTA and the morphology was observed by TEM, and the exosomes were stored at 4 °C for later use.

[0039] Test results: The isolated probiotic exosomes have a typical cup-shaped structure, uniform particle size distribution, average particle size of 75 nm, high purity, and no obvious contamination from other proteins.

[0040] 2.2 Loading of bioactive molecules

[0041] The targeted ferroptosis smart peptide and Erastin were co-loaded into probiotic exosomes using an ultrasound-assisted loading method: The purified exosome suspension (concentration 1 mg / mL) was taken, and the targeted ferroptosis smart peptide (final concentration 0.2 mg / mL) and Erastin (final concentration 0.1 mg / mL) were added. The mixture was placed in an ice bath, and the ultrasonic cell disruptor was set to 150W power, 3s working time, and 5s interval time for a total ultrasonic time of 10 min. After ultrasonication, the mixture was centrifuged at 4℃ and 10000r / min for 20 min to remove unloaded free active molecules and broken impurities. The supernatant was collected, which is the loaded exosome.

[0042] Encapsulation efficiency determination: The concentrations of the targeted ferroptosis smart peptide and Erastin before and after loading were determined by HPLC, and the encapsulation efficiency was calculated. The results showed that the encapsulation efficiency of the targeted ferroptosis smart peptide was 88.6%, and that of Erastin was 86.3%, both meeting the requirement of ≥85%.

[0043] 2.3 Targeted Functional Modification

[0044] Dual-targeted modification was performed using the EDC-NHS activation method: First, a suspension of loaded exosomes was taken, and Fe3O4@PDA nanoparticles (exosome to nanoparticle mass ratio 10:1) were added. The mixture was stirred at room temperature in the dark for 2 h to allow the Fe3O4@PDA nanoparticles to coat the exosome surface through electrostatic interaction, forming a magnetic modification layer. Then, EDC and NHS (molar ratio 1:1, final concentration 5 mmol / L) were added to activate the carboxyl groups on the exosome surface, and the reaction was carried out at room temperature for 30 min. AS1411 aptamer (final concentration 0.05 mg / mL) was added, and the mixture was incubated at 4 °C in the dark for 8 h to allow the aptamer to covalently bind to the exosome surface through amide bonds. After the reaction was completed, the mixture was centrifuged at 4 °C and 12000 r / min for 30 min to remove unbound Fe3O4@PDA nanoparticles and AS1411 aptamer. The mixture was then resuspended in PBS buffer to obtain the dual-targeted delivery system.

[0045] 2.4 Preparation of oral capsules

[0046] The targeted delivery system and pharmaceutical excipients were mixed in a certain proportion to prepare the capsule contents: 100mg of lyophilized powder of the targeted delivery system (obtained by freeze drying, moisture content ≤3%) was taken, 200mg of lactose and 150mg of microcrystalline cellulose were added, and the mixture was thoroughly ground and mixed. The mixture was then passed through an 80-mesh sieve, and 5mg of magnesium stearate was added as a lubricant. After mixing again, the mixture was filled into empty capsules using a fully automatic capsule filling machine. Each capsule contained 20mg of lyophilized powder of the targeted delivery system. Subsequently, enteric coating was performed using acrylic resin No. IV. The coating weight increased by 5%. After drying, oral targeted capsules were obtained.

[0047] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

[0048] The present invention and its embodiments have been described above. This description is not restrictive, and the embodiments shown are only one of the embodiments of the present invention. The actual application is not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar methods and embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. A method for delivering bioactive molecules based on probiotic exosomes, characterized in that: The specific steps include the following: S1. Screen clinically approved probiotic strains for large-scale culture and isolate natural exosomes; S2. Stably introduce bioactive molecules into the cavity or surface of the natural exosomes to form loaded exosomes; S3. Modify the loaded exosomes for targeted function to obtain a targeted delivery system with intestinal target recognition capability; S4. The targeted delivery system is formulated into an oral formulation, and the bioactive molecules are specifically delivered to the intestinal target site through oral administration.

2. The method for delivering bioactive molecules based on probiotic exosomes according to claim 1, characterized in that: The natural exosomes obtained in step S1 are obtained by one of the following methods: ultracentrifugation, density gradient centrifugation, or ultrafiltration centrifugation.

3. The method for delivering bioactive molecules based on probiotic exosomes according to claim 1, characterized in that: In step S1, the probiotics are selected from one or more of the genera Lactobacillus, Bifidobacterium, and Streptococcus.

4. The method for delivering bioactive molecules based on probiotic exosomes according to claim 1, characterized in that: The loading process in step S2 is selected from one of the incubation loading method, electroporation method or ultrasound-assisted loading method, and the encapsulation rate of bioactive molecules during the loading process is ≥85%.

5. The method for delivering bioactive molecules based on probiotic exosomes according to claim 1, characterized in that: The bioactive molecule mentioned in step S2 is selected from one or more of the following: targeted ferroptosis smart peptides, ferroptosis regulatory genes, small molecule ferroptosis inducers, and tumor-targeting antibodies; wherein the small molecule ferroptosis inducers include at least one of Erastin, RSL3, and YL-9395.

6. The method for delivering bioactive molecules based on probiotic exosomes according to claim 1, characterized in that: In step S3, the targeting ligand is selected from one or more of AS1411 aptamer, anti-EGFR antibody, anti-HER2 antibody, or HavPD-1 antibody.

7. The method for delivering bioactive molecules based on probiotic exosomes according to claim 1, characterized in that: The targeted functional modification also includes coating the surface of the loaded exosomes with Fe3O4@PDA nanoparticles to form a magnetic targeted modification layer.

8. The method for delivering bioactive molecules based on probiotic exosomes according to claim 1, characterized in that: The probiotic exosomes isolated in step S1 have a particle size of 50-100 nm.

9. The method for delivering bioactive molecules based on probiotic exosomes according to claim 1, characterized in that: In step S4, the oral preparation is a capsule or a tablet.

10. The method for delivering bioactive molecules based on probiotic exosomes according to claim 1, characterized in that: The delivery method is used to prevent or treat ferroptosis-related diseases, including inflammatory bowel disease, cancer, or neurodegenerative diseases.

Images