Intelligently designed oral targeting ferroptosis biological agent

Abstract

The invention belongs to the technical field of biological medicines, and relates to an intelligently designed oral targeting ferroptosis biological preparation. The preparation comprises a core function unit, a carrier unit and a targeted delivery unit. The core function unit is a ferroptosis targeted intelligent peptide which is reasonably designed through an AlphaFold2 and Anand generation model AI and is introduced with non-natural amino acid; the vector unit is a recombinant lentiviral vector which is optimized by AI and carries a ferroptosis regulation gene; and the targeted delivery unit is a probiotic source exosome which is subjected to engineering modification by virtue of an AS1411 aptamer, Fe3O4 (at) PDA nanoparticles and an anti-EGFR monoclonal antibody fragment. The intelligent peptide and the recombinant lentiviral vector form a functional compound through click chemistry, and the functional compound is efficiently loaded in the modified exosome to prepare the oral capsule. According to the preparation, the bioavailability and the targeting property of the medicine are improved, and the preparation can be used for treating ferroptosis pathway disorder related diseases such as intestinal tumors and inflammatory bowel diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to an intelligently designed oral targeted ferroptosis biological agent. Background Technology

[0002] Ferroprelation is a programmed cell death mode driven by iron-dependent lipid peroxidation that has been discovered and widely studied in recent years. It differs from traditional cell death modes such as apoptosis and necrosis. By regulating intracellular iron metabolism, lipid peroxidation balance and antioxidant system function, it plays a key role in the occurrence and development of various diseases. In particular, in intestinal-related diseases such as intestinal tumors and inflammatory bowel disease, abnormal activation or inhibition of the ferroptosis pathway directly affects the survival and proliferation of diseased cells. Therefore, targeted regulation of the ferroptosis pathway has become a highly promising therapeutic direction in the biomedical field.

[0003] However, despite the well-validated theoretical value of ferroptosis-targeted therapy, its clinical translation has encountered three major technological hurdles, preventing existing therapies from meeting clinical needs. At the drug molecule design level, a core challenge lies in molecular-scale adaptation: ferroptosis targets are mostly located intracellularly or on the cell membrane surface. If the designed drug molecule is too small, it lacks the structural basis for specific binding to the target, making precise recognition impossible and resulting in extremely poor therapeutic specificity. If the molecule is too large, it struggles to overcome the phospholipid bilayer barrier of the cell membrane, preventing intracellular penetration and creating a design dilemma where specificity and penetration are mutually exclusive. Simultaneously, traditional drug design relies on experimental screening and experience accumulation, lacking precise structural prediction and sequence optimization methods. This not only results in lengthy development cycles, often requiring several years to obtain candidate molecules, but also makes it difficult to precisely control the binding affinity between the drug and the target, further exacerbating the difficulty of drug design.

[0004] In terms of therapeutic efficacy, existing ferroptosis-targeted therapies generally suffer from a single mechanism of action, regulating only a key node in the ferroptosis pathway. With long-term use, diseased cells easily develop resistance through the activation of compensatory pathways or gene mutations, leading to a gradual decline in therapeutic efficacy and failing to achieve long-term regulation of the ferroptosis pathway. Furthermore, due to the lack of a precise targeting system, existing drugs are widely distributed in the body, affecting not only diseased tissues but also healthy cells in normal tissues, causing off-target damage and a series of adverse reactions. This significantly reduces treatment safety and patient tolerability, limiting their widespread clinical application.

[0005] At the delivery system level, oral administration has become the preferred clinical method of drug delivery due to its convenience and high patient compliance. However, oral delivery of ferroptosis-targeting agents faces multiple obstacles. On the one hand, the strong acid and digestive enzymes in the gastrointestinal tract can degrade drug molecules, causing them to lose their biological activity before reaching the target site in the intestine. On the other hand, the intestinal mucosal barrier, as an important physiological barrier in the human body, prevents the arbitrary penetration of foreign substances, making it difficult for drugs to cross this barrier and enter the diseased tissue to exert their effects. More importantly, existing oral formulations lack effective targeting navigation systems and cannot accurately identify ferroptosis targets in intestinal lesions, resulting in non-specific drug distribution in the body, oral bioavailability of less than 5%, and most drugs failing to exert their therapeutic effects.

[0006] The triple technical bottlenecks of design, maintenance, and delivery mentioned above, combined with each other, have made the clinical translation of ferroptosis-targeted therapy extremely difficult. Currently, no mature oral targeted ferroptosis biologics have been approved for marketing. Therefore, developing an oral biologic that can overcome existing technological barriers and possesses precise target recognition, efficient and stable delivery, and long-term safe regulation characteristics has become a crucial issue that urgently needs to be addressed in the biopharmaceutical field, and is of great significance for promoting the clinical application of ferroptosis-targeted therapy. Summary of the Invention

[0007] In response to the above situation and to overcome the shortcomings of the prior art, the present invention provides an intelligently designed oral targeted ferroptosis biological agent. This agent is designed through artificial intelligence and multi-unit synergistic integration, aiming to simultaneously solve the three major technical bottlenecks of ferroptosis targeted therapy in drug design, long-term regulation and oral delivery.

[0008] To achieve the above objectives, the following technical solution is adopted: This invention provides an intelligently designed oral targeted ferroptosis biological agent, comprising a core functional unit, a carrier unit, and a targeted delivery unit. The core functional unit is an AI-rationally designed ferroptosis-targeting intelligent peptide, the carrier unit is an AI-optimized recombinant lentiviral vector, and the targeted delivery unit is an engineered probiotic-derived exosome. The recombinant lentiviral vector carries a ferroptosis regulatory gene, and the intelligent peptide and the recombinant lentiviral vector form a functional complex through a specific linkage. The functional complex is loaded into the engineered probiotic exosome, and the surface of the exosome is modified with a targeting ligand.

[0009] Furthermore, the design method of the AI-rational ferroptosis-targeting smart peptide includes: predicting the target protein backbone structure based on AlphaFold2, constructing a multi-task design model by combining the Anand generative model, fixing the target protein backbone, iteratively sampling the side chain conformations of natural amino acids, and obtaining an iterative smart peptide through 36 rounds of sequence optimization; 2-5 non-natural amino acids are introduced into the amino acid sequence of the smart peptide, and the non-natural amino acids are selected from ornithine, citrulline, and leucine, and are located between the target binding domain and the membrane penetration domain of the smart peptide.

[0010] Furthermore, the AI-optimized recombinant lentiviral vector consists of a vector plasmid, a packaging plasmid, and a regulatory plasmid; the vector plasmid carries a fusion fragment of a CAR gene and a ferroptosis regulatory gene, wherein the ferroptosis regulatory gene is selected from the GPX4 repressor gene and the SLC7A11 interference gene; the packaging plasmid carries a VSV-G gene variant; and the regulatory plasmid embeds a tandem element of a hypoxia-responsive promoter and a CMV promoter; the recombinant lentiviral vector is jointly optimized by full-dimensional dynamic convolution and a high-efficiency capsule network.

[0011] Furthermore, the preparation method of the engineered probiotic-derived exosomes includes: exosomes isolated from Lactobacillus plantarum CGMCC1.396, which are sequentially coupled with AS1411 aptamer, encapsulated with Fe3O4@PDA nanoparticles, and modified with anti-EGFR monoclonal antibody fragments. The exosomes have a particle size of 40-60 nm and a surface zeta potential of -25 to -18 mV.

[0012] Furthermore, the smart peptide has an amino acid sequence length of 15-30 amino acids, with an N-terminus containing a 7-amino acid signal peptide sequence, namely Met-Ala-Ser-Leu-Ile-Lys-Arg, and a C-terminus containing a 7-amino acid membrane permeation domain, namely Trp-Phe-Leu-Val-Arg-Pro-Gly. The central region is a ferroptosis target binding domain. The smart peptide forms a cyclic structure through two intramolecular disulfide bonds, namely Cys 6 -Cys 21 and Cys 10 -Cys 17 .

[0013] Furthermore, the oral formulation is a capsule, the capsule shell of which is made of hydroxypropyl methylcellulose, trehalose and polyethylene glycol 6000 in a mass ratio of 5:3:2. The capsule filling material includes an exosome-carrier-smart peptide complex, mannitol, magnesium stearate and an intestinal mucus penetration promoter. The intestinal mucus penetration promoter is selected from chitosan quaternary ammonium salt. The mass percentage of each component is 40-50% complex, 35-45% mannitol, 2-5% magnesium stearate and 3-8% intestinal mucus penetration promoter.

[0014] Furthermore, the regulatory plasmid also contains a tetracycline regulatory element, which is tandemly linked to the ferroptosis regulatory gene. The expression intensity of the ferroptosis gene can be regulated by oral administration of a tetracycline drug, wherein the tetracycline drug is selected from doxycycline.

[0015] Furthermore, the functional complex is loaded onto probiotic exosomes using a combined loading method of electroporation and ultrasound assistance. The electroporation voltage is 120-150V and the pulse time is 5-8ms. The ultrasound assistance frequency is 40kHz and the power is 100-150W, with a loading efficiency of ≥85%. The exosomes contain IL-10, TGF-β1 anti-inflammatory factor, and glutathione peroxidase inhibitor.

[0016] Furthermore, the specific connection between the smart peptide and the recombinant lentiviral vector is a click chemical connection, specifically, an azide group is introduced at the C-terminus of the smart peptide, an alkyne group is modified on the capsid protein of the recombinant lentiviral vector, and covalent connection is achieved through a Cu(I)-catalyzed azide-alkyne cycloaddition reaction.

[0017] Furthermore, the formulation is used to treat diseases related to ferroptosis pathway disorders, including intestinal tumors and inflammatory bowel disease, including ulcerative colitis and Crohn's disease.

[0018] The beneficial effects of this invention are:

[0019] This approach addresses the core contradiction in traditional ferroptosis-targeting drug design by significantly improving drug-target compatibility through AI-driven rational design technology. Based on AlphaFold2 target structure prediction and Anand generation model multi-task optimization, the resulting smart peptide, obtained through 36 rounds of sequence iteration, not only effectively balances the relationship between molecular size, target binding specificity, and cell membrane permeability, but also further enhances the in vivo stability of the smart peptide by introducing non-natural amino acids and intramolecular disulfide bonds to form a cyclic structure, preventing enzymatic degradation and laying the foundation for efficient ferroptosis regulation.

[0020] This study achieves long-term and precise regulation of the ferroptosis pathway, overcoming the shortcomings of existing therapies such as drug resistance and off-target damage. The AI-optimized recombinant lentiviral vector is jointly optimized through multi-dimensional dynamic convolution and high-efficiency capsule network. Combined with the fusion design of CAR genes and ferroptosis regulatory genes in the vector plasmid, the modification of the VSV-G gene variant in the packaging plasmid, and the introduction of tandem elements of hypoxia response promoters and CMV promoters and tetracycline regulatory elements in the regulatory plasmid, it not only significantly improves the efficiency of targeted ferroptosis regulation, but also achieves temporal controllability and long-term maintenance of gene expression, effectively reducing the risk of drug resistance. At the same time, the precise targeting design greatly reduces off-target damage to healthy intestinal epithelial cells, improving treatment safety.

[0021] This breakthrough overcomes the technical bottleneck of low oral delivery efficiency, significantly improving formulation bioavailability and targeted delivery precision: probiotic-derived exosomes conjugated with AS1411 aptamers, encapsulated in Fe3O4@PDA nanoparticles, and modified with anti-EGFR monoclonal antibody fragments possess extremely strong intestinal mucosal adsorption and penetration capabilities; combined with a combined loading method of electroporation and ultrasound-assisted loading, efficient loading of functional complexes is achieved, and the IL-10, TGF-β1 anti-inflammatory factors, and glutathione peroxidase inhibitor contained in the exosomes can synergistically enhance the therapeutic effect while reducing intestinal inflammatory response; this delivery system enables the formulation to accurately identify ferroptosis targets in intestinal lesions after oral administration, significantly breaking through the intestinal mucosal barrier, significantly improving oral bioavailability, and solving the problem of difficult delivery of traditional formulations. Attached Figure Description

[0022] Figure 1 This is a flowchart illustrating the preparation process of an intelligently designed oral targeted ferroptosis biological agent according to the present invention.

[0023] 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

[0024] 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.

[0025] 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.

[0026] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are commercially available. The preparation process of the oral targeted ferroptosis biological agent in the embodiments of the present invention is shown in the appendix. Figure 1 As shown.

[0027] Example

[0028] I. Material Preparation

[0029] (a) Reagents and bacterial strains:

[0030] Strains: Lactobacillus plantarum CGMCC1.396, purchased from China General Microbiological Culture Collection Center.

[0031] Plasmids and gene fragments: CAR gene fragment, GPX4 repressor gene fragment, SLC7A11 interference gene fragment, VSV-G gene variant fragment, hypoxia response promoter (HRE) fragment, CMV promoter fragment, and tetracycline regulatory element (TRE) fragment were all custom-synthesized by a gene synthesis company; vector plasmid pLVX-Puro, packaging plasmid psPAX2, and regulatory plasmid pMD2.G were purchased from Addgene.

[0032] Amino acid and peptide synthesis reagents: natural amino acids, non-natural amino acids (ornithine, citrulline, leucine), solid-phase synthesis resins, activators, condensing agents, etc., purchased from Sigma-Aldrich.

[0033] Exosome modification-related reagents: AS1411 aptamer, Fe3O4@PDA nanoparticles, anti-EGFR monoclonal antibody fragment, conjugation reagents (EDC, NHS), etc., were purchased from Shanghai Sangon Biotech Co., Ltd.

[0034] Other reagents include: hydroxypropyl methylcellulose, trehalose, polyethylene glycol 6000, mannitol, magnesium stearate, chitosan quaternary ammonium salt, electroporation buffer, ultrasonic disruption buffer, cell culture reagents (DMEM medium, fetal bovine serum, trypsin, etc.), ferroptosis detection kit, and inflammatory factor detection kit, all of which are commercially available analytical grade or biological grade reagents.

[0035] (II) Instruments and Equipment:

[0036] The system includes an AlphaFold2 structure prediction server, an Anand generative model running workstation, a full-dimensional dynamic convolution and high-efficiency capsule network analysis system, a solid-phase peptide synthesizer, a high-performance liquid chromatograph (HPLC), a mass spectrometer, an ultracentrifuge, a transmission electron microscope, a nanoparticle tracking analyzer, an electroporator, an ultrasonic cell disruptor, a real-time quantitative PCR instrument, an enzyme-linked immunosorbent assay (ELISA) reader, a cell culture incubator, an animal in vivo imaging system, and a high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS) system.

[0037] II. Core Functional Unit: Preparation of AI-Rationally Designed Iron Death-Targeting Intelligent Peptides

[0038] (I) Target protein structure prediction

[0039] Key target proteins of the intestinal ferroptosis pathway (GPX4 protein and SLC7A11 protein) were selected. The amino acid sequences of the target proteins were input through the AlphaFold2 server. The structure prediction parameters were set (prediction rounds of 5, template coverage threshold of 80%) to obtain the three-dimensional structure model of the target proteins. The model with the highest confidence was selected as the target backbone structure for subsequent design.

[0040] (II) Construction of Multi-Task Design Model and Intelligent Peptide Sequence Optimization

[0041] Based on the Anand generative model, a multi-task design model was constructed. The target protein backbone structure data, the natural amino acid side chain conformation database, and the ferroptosis-regulated active peptide sequence feature data were input. The model training parameters were set (learning rate 0.001, number of iterations 1000, batch size 32) to complete the model training.

[0042] The target protein backbone structure was fixed, and the natural amino acid side chain conformations were sampled through model iteration. Combining the structural requirements of the ferroptosis target binding domain and membrane permeation domain, 36 rounds of sequence optimization were performed. After each round of optimization, the sequence with the lowest target binding energy was screened through molecular docking simulation, ultimately obtaining the amino acid sequence of the iterative smart peptide.

[0043] (III) Introduction of non-natural amino acids and intelligent peptide synthesis

[0044] Three non-natural amino acids (ornithine, citrulline, and leucine) were introduced between the target binding domain and the membrane penetration domain of the smart peptide to adjust the sequence arrangement and ensure molecular conformation stability.

[0045] Intelligent peptides were synthesized using a solid-phase synthesis method: Fmoc-protected starting amino acids were linked to a solid-phase resin, and the protection was sequentially removed and subsequent amino acids were added for condensation reactions. Non-natural amino acids were activated by a special activator before being added to the reaction system. The condensation reaction temperature was 25°C and the reaction time was 2 hours.

[0046] After synthesis, the resin was cleaved with a cleavage reagent to obtain crude peptide, which was then purified by HPLC (C18 column, acetonitrile-water mobile phase, gradient elution). The target peak was collected, and after mass spectrometry verification that the molecular weight was correct, it was freeze-dried for later use.

[0047] (iv) Formation of intelligent peptide cyclic structure

[0048] The purified linear smart peptide was dissolved in buffer (PBS buffer, pH 7.4), oxidized glutathione was added, and the reaction was carried out at room temperature for 4 hours to promote Cys 6 With Cys 21 Cys 10 With Cys 17 Intramolecular disulfide bonds are formed, resulting in a cyclic structure. After the reaction is complete, the peptide is purified again by HPLC to obtain a high-purity cyclic smart peptide.

[0049] III. Vector Unit: Preparation of AI-Optimized Recombinant Lentiviral Vectors

[0050] (I) Construction of recombinant plasmids

[0051] Vector plasmid construction: The vector plasmid pLVX-Puro was double-digested with restriction endonucleases EcoRI and BamHRI, and the vector backbone was recovered. The CAR gene fragment and the ferroptosis regulatory gene fragment (GPX4 repressor gene or SLC7A11 interference gene) were spliced ​​into a fusion fragment by overlap extension PCR. After being digested with the same endonuclease, the fragment was ligated to the vector backbone, transformed into E. coli DH5α competent cells, positive clones were screened, and the recombinant vector plasmid was obtained by sequencing.

[0052] Packaging plasmid construction: The packaging plasmid psPAX2 was modified by enzyme digestion, and a VSV-G gene variant fragment (alanine at position 435 was mutated to glycine) was inserted. After ligation, it was transformed, screened, and sequenced for verification to obtain the recombinant packaging plasmid.

[0053] Construction of regulatory plasmid: Using the regulatory plasmid pMD2.G as the backbone, the hypoxia response promoter and the CMV promoter were inserted in tandem through enzyme digestion and ligation. At the same time, the tetracycline regulatory element (TRE) was inserted and tandem with the ferroptosis regulatory gene to construct the recombinant regulatory plasmid. After sequencing verification, it was put into use.

[0054] (II) AI Optimization of Recombinant Lentiviral Vectors

[0055] The sequence information of recombinant vector plasmids, packaging plasmids, and regulatory plasmids, target gene expression data, and vector packaging efficiency data are input into a multi-dimensional dynamic convolutional and high-efficiency capsule network joint analysis system. Optimization parameters are set (feature extraction dimension is 256, capsule network iteration is 500 times), and the best plasmid sequence combination and vector construction scheme are screened through model analysis to optimize the vector's targeting binding ability and gene expression efficiency.

[0056] (III) Packaging and purification of recombinant lentiviruses

[0057] 293T cells in the logarithmic growth phase were seeded in 10cm cell culture dishes and cultured until the cell confluence reached 70%-80%.

[0058] The plasmid mixture was transfected into 293T cells using a liposome transfection method at a mass ratio of recombinant vector plasmid: recombinant packaging plasmid: recombinant regulatory plasmid = 4:3:1.

[0059] Cell supernatant was collected at 48h and 72h after transfection, filtered through a 0.45μm filter membrane, and then purified by ultracentrifugation (100000g, 4℃, 2h). The lentiviral particles were resuspended in viral preservation solution to obtain recombinant lentiviral vector.

[0060] Viral titer was determined using real-time quantitative PCR to ensure a titer ≥ 1 × 10⁻⁶. 8 TU / mL.

[0061] IV. Targeted Delivery Unit: Preparation of Engineered Probiotic-Derived Exosomes

[0062] (a) Probiotic culture and exosome isolation

[0063] Lactobacillus plantarum CGMCC1.396 was inoculated into MRS medium and cultured at 37°C under anaerobic conditions for 24 h to obtain seed culture. The seed culture was then inoculated into expansion medium at a ratio of 1:100 and cultured for another 48 h before the culture was collected.

[0064] The culture was centrifuged at 8000g for 10 min to remove the bacterial precipitate and the supernatant was collected. The supernatant was filtered through a 0.22μm filter membrane and then centrifuged at 10000g for 30 min and 100000g for 2 h in sequence. The precipitate was collected and resuspended in PBS buffer to obtain natural probiotic exosomes.

[0065] (II) Identification of exosomes

[0066] Transmission electron microscopy observation: The exosome resuspension was dropped onto a copper grid, negatively stained, and then dried. The morphology of the exosomes was observed by transmission electron microscopy, confirming that they were typical cup-shaped structures.

[0067] Nanoparticle tracking analysis: The particle size distribution and zeta potential of exosomes were detected using a nanoparticle tracking analyzer to ensure that the particle size of natural exosomes is in the range of 40-60 nm and the surface zeta potential is -25 to -18 mV.

[0068] (III) Engineering modification of exosomes

[0069] AS1411 aptamer coupling: Exosomes and AS1411 aptamer were mixed at a mass ratio of 10:1, and EDC and NHS were added as coupling agents. The mixture was reacted at room temperature for 2 hours. Unbound aptamers were removed by ultrafiltration centrifugation to obtain aptamer-modified exosomes.

[0070] Fe3O4@PDA nanoparticle encapsulation: aptamer-modified exosomes were mixed with Fe3O4@PDA nanoparticles at a volume ratio of 5:1 and slowly added dropwise to the reaction system under stirring. The reaction was carried out at 37°C for 4 hours. The product was collected by centrifugation, and free nanoparticles were removed.

[0071] Anti-EGFR monoclonal antibody fragment modification: The above exosomes and anti-EGFR monoclonal antibody fragments were mixed at a mass ratio of 20:1, a coupling reagent was added, and the mixture was reacted overnight at 4°C. The mixture was then purified by gel filtration chromatography to obtain probiotic exosomes with triple engineering modification.

[0072] V. Preparation and Loading of Functional Complexes

[0073] (i) Click chemical linking of smart peptides and recombinant lentiviral vectors

[0074] Introducing an azide group at the C-terminus of the smart peptide: The purified cyclic smart peptide was dissolved in a reaction buffer, an azide modification reagent was added, and the reaction was carried out at room temperature for 1 hour. The azide-modified smart peptide was obtained by HPLC purification.

[0075] Alkyne modification on the capsid protein of recombinant lentiviral vector: The recombinant lentiviral vector and alkyne modification reagent were mixed at a volume ratio of 10:1, reacted at 4°C for 2 h, and the unbound modification reagent was removed by ultrafiltration and centrifugation to obtain the alkyne-modified recombinant lentiviral vector.

[0076] Click on the chemical link: The azide-modified smart peptide and the alkynyl-modified recombinant lentiviral vector were mixed at a molar ratio of 3:1, Cu(I) catalyst was added, and the reaction was carried out at room temperature for 3 h. The smart peptide-recombinant lentiviral vector functional complex was obtained by gel filtration chromatography and the linkage efficiency was verified by HPLC to be ≥90%.

[0077] (ii) Co-loading of functional complexes

[0078] A mixed system of functional complex and engineered modified exosomes was prepared, with a mass ratio of exosomes to functional complex of 10:1.

[0079] A combined loading method using electroporation and ultrasound assistance was adopted: the mixed system was added to an electroporation cup, and the electroporation parameters were set to voltage 130V and pulse time 6ms for electroporation treatment; then the system was transferred to an ultrasound reaction vessel, and the ultrasound parameters were set to frequency 40kHz and power 120W for ultrasound treatment for 5min.

[0080] After loading was completed, exosomes of the loaded functional complex were collected by ultracentrifugation (100,000 g, 4 °C, 1 h), resuspended in PBS buffer, and the loading efficiency was ≥85%. At the same time, the structural integrity of the exosomes was ≥95% as determined by transmission electron microscopy.

[0081] VI. Preparation of Oral Capsules

[0082] (I) Preparation of capsule shells

[0083] Weigh hydroxypropyl methylcellulose, trehalose, and polyethylene glycol 6000 in a mass ratio of 5:3:2, add an appropriate amount of purified water, and stir until completely dissolved to form a uniform capsule shell material solution. Pour the solution into a capsule shell mold, dry and shape it at 60°C, and demold it after cooling to obtain a composite capsule shell for later use.

[0084] (II) Preparation of filler materials

[0085] Weigh out the exosomes (45%), mannitol (40%), magnesium stearate (3%), and chitosan quaternary ammonium salt (5%) according to their mass percentages.

[0086] First, mannitol and chitosan quaternary ammonium salt are mixed evenly and then prepared into a mixed powder with a particle size of 100-200μm by a pulverizer. Then, exosomes of the loaded functional complex and magnesium stearate are added and mixed in a three-dimensional mixer for 15 minutes to obtain a uniform filling material.

[0087] (III) Capsule filling and packaging

[0088] A fully automated capsule filling machine is used to fill the composite capsule shell with the filler material, controlling the filling amount of each capsule to be 200mg; after filling, the capsule is sealed by a capsule sealing machine to remove unqualified capsules and obtain oral targeted ferrode biological agent capsules.

[0089] 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.

[0090] 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 smartly designed oral targeted ferroptosis biological agent, characterized in that: The invention comprises a core functional unit, a carrier unit, and a targeted delivery unit. The core functional unit is an AI-designed ferroptosis-targeting smart peptide. The carrier unit is an AI-optimized recombinant lentiviral vector. The targeted delivery unit is an engineered probiotic-derived exosome. The recombinant lentiviral vector carries a ferroptosis regulatory gene. The smart peptide and the recombinant lentiviral vector form a functional complex through a specific linkage. The functional complex is loaded into the engineered probiotic exosome, and the surface of the exosome is modified with a targeting ligand.

2. The intelligently designed oral targeted ferroptosis biological agent according to claim 1, characterized in that: The design method of the AI-rational ferroptosis-targeting smart peptide includes: predicting the target protein backbone structure based on AlphaFold2, constructing a multi-task design model by combining the Anand generative model, fixing the target protein backbone, iteratively sampling the side chain conformations of natural amino acids, and obtaining an iterative smart peptide through 36 rounds of sequence optimization; 2-5 non-natural amino acids are introduced into the amino acid sequence of the smart peptide, and the non-natural amino acids are selected from ornithine, citrulline, and leucine, and are located between the target binding domain and the membrane penetration domain of the smart peptide.

3. The intelligently designed oral targeted ferroptosis biological agent according to claim 2, characterized in that: The AI-optimized recombinant lentiviral vector consists of a vector plasmid, a packaging plasmid, and a regulatory plasmid. The vector plasmid carries a fusion fragment of a CAR gene and a ferroptosis regulatory gene, wherein the ferroptosis regulatory gene is selected from the GPX4 repressor gene and the SLC7A11 interference gene. The packaging plasmid carries a VSV-G gene variant. The regulatory plasmid contains a tandem element of a hypoxia-responsive promoter and a CMV promoter. The recombinant lentiviral vector is jointly optimized by full-dimensional dynamic convolution and a high-efficiency capsule network.

4. The intelligently designed oral targeted ferroptosis biological agent according to claim 3, characterized in that: The method for preparing the engineered probiotic-derived exosomes includes: exosomes isolated from Lactobacillus plantarum CGMCC1.396, which are sequentially coupled with AS1411 aptamer, encapsulated with Fe3O4@PDA nanoparticles, and modified with anti-EGFR monoclonal antibody fragments. The exosomes have a particle size of 40-60 nm and a surface zeta potential of -25 to -18 mV.

5. The intelligently designed oral targeted ferroptosis biological agent according to claim 4, characterized in that: The smart peptide has an amino acid sequence length of 15-30 amino acids, with a 7-amino acid signal peptide sequence at the N-terminus (Met-Ala-Ser-Leu-Ile-Lys-Arg) and a 7-amino acid membrane permeation domain at the C-terminus (Trp-Phe-Leu-Val-Arg-Pro-Gly). The central region is a ferroptosis target binding domain. The smart peptide forms a cyclic structure through two intramolecular disulfide bonds (Cys). 6 -Cys 21 and Cys 10 -Cys 17 .

6. The intelligently designed oral targeted ferroptosis biological agent according to claim 5, characterized in that: The oral formulation is a capsule. The capsule shell is made of hydroxypropyl methylcellulose, trehalose and polyethylene glycol 6000 in a mass ratio of 5:3:

2. The capsule filling material includes an exosome-carrier-smart peptide complex, mannitol, magnesium stearate and an intestinal mucus penetration promoter. The intestinal mucus penetration promoter is selected from chitosan quaternary ammonium salt. The mass percentage of each component is 40-50% complex, 35-45% mannitol, 2-5% magnesium stearate and 3-8% intestinal mucus penetration promoter.

7. The intelligently designed oral targeted ferroptosis biological agent according to claim 6, characterized in that: The regulatory plasmid also contains a tetracycline regulatory element, which is tandem with a ferroptosis regulatory gene. The expression intensity of the ferroptosis gene can be regulated by oral administration of a tetracycline drug, which is selected from doxycycline.

8. The intelligently designed oral targeted ferroptosis biological agent according to claim 7, characterized in that: The functional complex is loaded onto probiotic exosomes using a combined loading method of electroporation and ultrasound assistance. The electroporation voltage is 120-150V and the pulse time is 5-8ms. The ultrasound assistance frequency is 40kHz and the power is 100-150W, with a loading efficiency of ≥85%. The exosomes contain IL-10, TGF-β1 anti-inflammatory factor, and glutathione peroxidase inhibitor.

9. The intelligently designed oral targeted ferroptosis biological agent according to claim 8, characterized in that: The specific connection between the smart peptide and the recombinant lentiviral vector is a click chemical connection. Specifically, an azide group is introduced at the C-terminus of the smart peptide, an alkyne group is modified on the capsid protein of the recombinant lentiviral vector, and covalent connection is achieved through a Cu(I)-catalyzed azide-alkyne cycloaddition reaction.

10. The intelligently designed oral targeted ferroptosis biological agent according to claim 9, characterized in that: The formulation is used to treat diseases related to ferroptosis pathway disorders, including intestinal tumors and inflammatory bowel disease, including ulcerative colitis and Crohn's disease.

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