Immunomodulator based on exosome and preparation method thereof

Abstract

The invention discloses an exosome-based immunomodulator and a preparation method thereof, the immunomodulator comprises a divaricate saposhnikovia plant exosome, the interior of the exosome is coated with spleen aminopeptide derived from animal spleen, and the surface of the exosome is modified with at least one of hyaluronic acid or chitosan; according to the invention, the spleen aminopeptide is entrapped in the exosome, and the exosome is subjected to targeted modification, so that the retention and targeted delivery capabilities of the medicine in respiratory mucosa are enhanced, and the medicine can be more effectively used for preventing or treating infantile recurrent respiratory tract infection.

Description

Technical Field

[0001] This invention belongs to the field of biological agent carriers and drug delivery systems, specifically relating to the encapsulation and surface modification of a plant exosome-based biological agent (spleen peptide) for enhancing targeted delivery to the respiratory mucosa and immunomodulatory therapy, and a method for preparing the same. Background Technology

[0002] Recurrent respiratory infections are a common pediatric complication, typically referring to repeated upper or lower respiratory tract infections within a year, manifesting as recurrent colds, pharyngitis, bronchitis, or pneumonia. This condition is closely related to the immature development of the child's immune system, weakened mucosal immunity, and repeated exposure to pathogens. Recurrent infections not only affect children's growth and development but also easily induce airway hyperresponsiveness or chronic respiratory diseases; therefore, immunomodulators are often used clinically as adjunctive or long-term interventions.

[0003] Current immunomodulatory methods for recurrent respiratory infections in children mainly include chemoimmunomodulatory drugs, biological immunomodulatory agents, and some traditional Chinese medicines that enhance immunity. Among them, spleen peptide, a low-molecular-weight polypeptide and nucleotide complex derived from the animal spleen, is widely used clinically as an adjunct treatment for recurrent respiratory infections because it can regulate the body's cellular immune function, promote the activity of T lymphocytes and natural killer cells, and enhance cytokine secretion. However, existing spleen peptide preparations are mostly administered orally or by injection, which has the following significant shortcomings: First, as a small-molecule bioactive substance, spleen peptide lacks targeted distribution in the body and is difficult to accumulate locally in the respiratory mucosa, resulting in a low actual effective utilization rate per unit dose; second, free spleen peptide is easily degraded or rapidly eliminated in the body, with a short duration of action, making it difficult to maintain a stable immunomodulatory effect; third, long-term or repeated systemic administration may increase the burden of medication adherence and is difficult to avoid exposure to non-target organs.

[0004] On the other hand, drug delivery systems targeting local respiratory tract delivery still have significant limitations. While traditional nanocarriers such as liposomes and polymeric nanoparticles can improve drug stability to some extent, they often suffer from complex preparation processes, limited raw material sources, and high risks of biocompatibility or immunogenicity. Their safety and acceptability remain limited, especially in cases of long-term or repeated use in children. Furthermore, the aforementioned synthetic carriers have limited retention capacity in the respiratory mucosa and are easily and rapidly eliminated by mucus clearance mechanisms, making it difficult to achieve sustained and effective local immune modulation.

[0005] In recent years, plant-derived exosomes, as a type of natural nanovesicle, have gradually attracted attention due to their wide availability, good biocompatibility, low immunogenicity, and easy uptake by mucosal cells, showing potential for oral or mucosal delivery. However, current research on plant exosomes mainly focuses on nutrient delivery or the transport of general bioactive substances, with limited exploration of their use as carriers of immunomodulators, especially in combination with animal-derived immunomodulatory substances such as spleen peptides for the prevention and treatment of respiratory infections. Furthermore, the retention time and targeted uptake capacity of unmodified plant exosomes in the respiratory mucosa remain insufficient, hindering the full realization of their advantages as delivery carriers.

[0006] Therefore, there is an urgent need for an immunomodulatory system that is safe, structurally stable, can effectively encapsulate immune-active substances, and is suitable for delivery through the respiratory mucosa, in order to solve the problems of poor targeting, low local utilization, and insufficient duration of action of immunomodulators such as spleen peptides, thereby improving the prevention and treatment of recurrent respiratory infections in children.

[0007] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides an exosome-based immunomodulator and its preparation method.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] The exosome-based immunomodulator includes plant exosomes and biological agents, wherein the plant exosome is *Saposhnikovia divaricata* exosome, the biological agent is spleen peptide, the *Saposhnikovia divaricata* exosome internally encapsulates spleen peptide, and the surface of the *Saposhnikovia divaricata* exosome is modified with at least one of hyaluronic acid or chitosan.

[0011] By adopting the above technical solutions, we can enhance the retention of respiratory mucosa, improve the uptake capacity of epithelial / immune cells mediated by receptors such as CD44, and improve the viscosity / stability compatibility during nebulization or nasal spraying.

[0012] Preferably, the spleen peptide is a mixture of low-molecular-weight polypeptides, amino acids, and related polynucleotides obtained from the spleen of a traceable healthy mammal through water extraction, mild enzymatic hydrolysis, and fractionation.

[0013] Furthermore, the mammal is preferably a cow or a pig.

[0014] Preferably, the spleen peptide comprises one or more of the following components:

[0015] (a) A low molecular weight polypeptide group mainly composed of dipeptides, tripeptides and oligopeptides, with an average relative molecular mass of 0.5–10 kDa, preferably 1–5 kDa.

[0016] (b) Peptides rich in arginine, lysine, glycine, proline, leucine, valine, alanine, serine, threonine, tyrosine and phenylalanine.

[0017] (c) Short chain nucleotides or nucleosides and short oligonucleotide fragments of less than 20 bases; the nucleosides are AMP, GMP, CMP, UMP, adenosine, or hypoxanthine nucleoside / inosine.

[0018] Preferably, the total polypeptide content, based on the total mass of spleen peptides, is 5%–80%.

[0019] Preferably, the main peak particle size range of the windproof exosomes is 30–200 nm.

[0020] Preferably, the loading efficiency LE of the windproof exosome loaded with spleen peptide is 20-40%, which is the percentage of the loaded spleen peptide mass relative to the initial donor spleen peptide mass.

[0021] Furthermore, the bioactivity of spleen peptides is maintained at 70-80% after loading.

[0022] Preferably, the hyaluronic acid has a molecular weight of 10–300 kDa, and the chitosan is a low molecular weight chitosan with a degree of deacetylation of 70–80%.

[0023] A second aspect of the present invention provides a method for preparing an exosome-based immunomodulator, comprising the following steps:

[0024] S1. Obtain traceable raw materials from the spleens of healthy mammals and prepare spleen peptide materials;

[0025] S2. Obtain medicinal-grade Saposhnikovia divaricata plant material and extract plant-derived exosomes:

[0026] Mechanical grinding and homogenization to disrupt plant cells and release extracellular vesicle particles; followed by a stepwise separation step to remove coarse fibers and cell debris, stepwise filtration and size separation to enrich vesicles in the 30–200 nm range, and elution to remove small molecule leachates.

[0027] S3. Load the spleen peptide obtained in step S1 and the exosomes obtained in step S2, so that the spleen peptide is encapsulated in the exosomes:

[0028] The initial spleen peptide to exosome mass ratio (donor spleen peptide mass: exosome dry weight) is 0.1:1 to 10:1; preferably 0.5:1–5:1 to balance loading efficiency and vesicle stability.

[0029] S4. Surface modification of the carrier obtained in step S3 to obtain the final formulation.

[0030] Preferably, the spleen peptide loading in step S3 is carried out by passive co-incubation. After loading, the unloaded free spleen peptide is removed by separation and the loading efficiency and drug loading are measured.

[0031] The surface modification in step S4 is carried out in a non-covalent manner, which includes physical adsorption, electrostatic recombination or layer-by-layer self-assembly. After modification, the success of the modification is confirmed by changes in particle size and surface potential, mucosal binding capacity and in vitro cell uptake experiments.

[0032] The third aspect of this invention provides an application of an exosome-based immunomodulator, characterized in that it is used to prepare a pediatric respiratory tract infection for the prevention or treatment of upper / lower respiratory tract infections, viral respiratory diseases, and recurrent respiratory tract infections.

[0033] The beneficial effects of this invention are:

[0034] 1) Spleen peptide, as a low-molecular-weight polypeptide / nucleotide mixture, is easily degraded and cleared in its free state, making it difficult to accumulate in the respiratory mucosa and limiting its local therapeutic effect. Encapsulating spleen peptide into the exosomes of Saposhnikovia divaricata can physically isolate enzymatic degradation and achieve sustained release, thereby maintaining the effective local concentration in the respiratory tract and prolonging the immunomodulatory window. It is expected to transform short-term exposure into local stimulation lasting for several days. This invention overcomes the shortcomings in a targeted manner through carrier-based sustained release, significantly prolonging local exposure and achieving continuous immune stimulation.

[0035] 2) Hyaluronic acid modification of exosome surfaces can facilitate binding and endocytosis mediated by HA receptors (such as CD44) on the surface of epithelial / immune cells; chitosan modification increases electrostatic adhesion to mucus and promotes local penetration / retention. Both can be used alone or in combination to achieve a dual benefit of "mucosal retention + receptor targeting," thereby increasing the uptake and accumulation of exosomes—along with their loaded spleen peptides—at respiratory target sites, enhancing mucosal retention and epithelial / immune cell receptor-mediated uptake, and compensating for the "poor targeting" of free spleen peptide formulations.

[0036] 3) Directly delivering spleen peptides to epithelial and related antigen-presenting cells via exosomes can improve the effectiveness of peptide-target cell contact, thereby inducing stronger and more localized release of cytokines such as IFN-γ / IL-2 and increased macrophage activity at lower input levels (which can be verified in vitro / in vivo by cytokine ELISA, phagocytosis rate and T cell proliferation indicators). This "dose-site concentration" synergistic mechanism is conducive to rapidly establishing a local anti-pathogen immune response.

[0037] 4) Since the carrier prolongs the local exposure of spleen peptide and improves the efficiency of antigen presentation and effector cell activation, it is expected that a faster rate of pathogen load reduction, shorter symptom resolution time, and shorter hospitalization / drug use time will be observed in animal models or clinical observations; the carrier design of this invention will more directly amplify this effect.

[0038] 5) Local delivery and improved local efficacy can reduce the dose (requiring a lower systemic dose for equivalent or better efficacy), thereby significantly reducing systemic exposure and potential adverse reactions. This is especially suitable for children who need long-term or repeated medication. The carrier and modification are made of natural ingredients (such as plant exosomes, HA, and low molecular weight chitosan), which is beneficial for evaluating biocompatibility and the safety of long-term repeated administration.

[0039] 6) Efficiently delivering spleen peptides to respiratory epithelial and mucosal innate immune cells can more effectively drive dendritic cell maturation and antigen presentation, thereby making it more conducive to the formation of local effector T cells and memory immune responses. In the long term, this helps to reduce the relapse frequency, that is, the number of RRTI attacks. This invention makes this preventive effect more achievable and durable through delivery technology improvements.

[0040] 7) The molecular weight of HA and the degree of deacetylation of chitosan can ensure that the final formulation has sufficient mucosal retention and can be administered through conventional nasal sprays or nebulizers, making it convenient for use in outpatient or home settings, thereby improving patient compliance and simplifying the clinical promotion pathway.

[0041] 8) The exosomes are naturally derived and the present invention adopts a non-covalent modification and passive encapsulation strategy. The process is relatively mild and easy to scale up. Combined with the potential for dose reduction, it is expected to reduce the cost of long-term medication while maintaining or improving efficacy, which is conducive to industrialization and medical accessibility. Attached Figure Description

[0042] Figure 1 This is a curve showing the viral load of respiratory pathogens. Detailed Implementation

[0043] The present invention will be further described below through specific embodiments. To make the inventive objectives, technical solutions, and beneficial technical effects of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments. It should be understood that the embodiments described in this specification are merely for explaining the present invention and are not intended to limit the present invention.

[0044] Unless otherwise stated, all instruments and reagents used in the examples are commercially available or synthesized using conventional methods and can be used directly without further processing, and all instruments used in the examples are commercially available.

[0045] Example 1: Preparation of hyaluronic acid-modified *Angelica sinensis* exosomes encapsulating spleen peptides

[0046] Material:

[0047] Spleen raw material: healthy bovine spleen with traceable origin and quarantine and pathogen testing records.

[0048] Plant material: medicinal grade Saposhnikovia root.

[0049] Enzymatic hydrolysis reagent (preparation of spleen peptide): pepsin / protease enzyme, enzyme dosage 0.1–1.0% (w / w, enzyme / spleen tissue).

[0050] Buffer system: phosphate-buffered saline (PBS) (pH 7.0–7.6) or isotonic buffer.

[0051] Hyaluronic acid: molecular weight 10–300 kDa, water-soluble, pharmaceutical grade.

[0052] Other: Ultrafiltration membranes, chromatography columns and separation equipment.

[0053] Preparation method:

[0054] S1. Preparation of spleen peptides (source, crude extraction and fractionation): After removing blood from the spleen tissue, mechanically homogenize it and add an equal volume of distilled water or PBS to obtain a tissue homogenate. Example solid-liquid ratio: 1:5 w / v.

[0055] Enzymatic hydrolysis with mild protease for 3 hours at 4–25°C (pH controlled at 6.0–8.0, enzyme addition 0.5%) yields a low molecular weight peptide mixture mainly composed of dipeptides, tripeptides and oligopeptides (target average relative molecular mass 1–5 kDa).

[0056] Fractionation is performed using a filtration membrane to retain low molecular weight components (<10 kDa) while removing large protein molecules and undigested residues.

[0057] S2 Saposhnikovia divaricata exosome extraction and pre-purification:

[0058] The roots of *Saposhnikovia divaricata* were crushed and suspended in PBS buffer (solid-liquid ratio example 1:10 w / v), mechanically homogenized at 4–15°C, and kept at low temperature to protect the vesicles.

[0059] The filter removes fibers and large debris through a progressive coarse filtration process, with the pore sizes changing sequentially from 100 μm to 10 μm to 0.45 μm.

[0060] Size separation enrichment is achieved using continuous flow filtration, TFF hollow fiber or ultrafiltration systems, with molecular weight cutoff at 100–300 kDa equivalent and particle size range of 30–200 nm retained for concentration and impurity removal.

[0061] The 30–200 nm vesicles were further purified by size exclusion chromatography to remove small molecule impurities.

[0062] The vesicle morphology was confirmed by transmission electron microscopy, and the particle size was detected by nanoparticle tracking analysis (NTA), maintaining the main particle size peak at 30–200 nm.

[0063] S3 spleen peptide encapsulation (passive co-incubation):

[0064] The dried spleen peptide was dissolved in PBS to prepare a stock solution of 10 mg / mL, which was then mixed with the exosome suspension at a ratio of donor spleen peptide mass to exosome dry weight of 0.5:1.

[0065] Incubate for 24 hours at 4–25°C under gentle rolling or slow rotation mixing conditions, ensuring gentle contact to avoid membrane damage.

[0066] After incubation, unencapsulated free spleen peptides were removed using SEC (size exclusion column) and the encapsulated exosome solution was collected.

[0067] Loading efficiency determination (LE): The loading percentage was calculated using HPLC and LC-MS, with a target LE of 20%.

[0068] Activity retention assay: The loaded sample was compared with free spleen peptide of the same dose for in vitro PBMC functional assay, with a target activity retention rate of 80%.

[0069] S4 Surface Modification (Hyaluronic Acid Coating, Non-Covalent)

[0070] Hyaluronic acid aqueous solution (concentration 2.0 mg / mL, molecular weight 10–300 kDa) was mixed with the loaded exosome suspension at a mass ratio of 1:1.

[0071] Incubate gently with stirring for 2 hours at room temperature or 4–25°C to allow HA to form a coating layer on the surface of exosomes through electrostatic interactions.

[0072] Free HA was removed by mild centrifugation or ultrafiltration, and the modified exosomes were collected. A layer-by-layer self-assembly method can be used: first, a small amount of low molecular weight chitosan (80% deacetylation, low molecular weight 20–150 kDa, soluble in 0.5% acetate buffer) is used for the first layer coating, followed by a second layer coating with HA to obtain an HA / Chitosan layered structure.

[0073] Small molecule salts and low molecular weight impurities are removed by dialysis or secondary ultrafiltration, followed by freeze drying or spray drying to obtain the finished product.

[0074] Example 2:

[0075] The spleen raw material was replaced with healthy pig spleens from traceable sources;

[0076] Preparation method:

[0077] S1. Preparation of spleen peptides (source, crude extraction and fractionation): After removing blood from the spleen tissue, mechanically homogenize it and add an equal volume of distilled water or PBS to obtain a tissue homogenate (solid-liquid ratio example 1:10 w / v, which can be adjusted appropriately according to the raw materials).

[0078] Enzymatic hydrolysis with mild protease for 1 hour at 4–25°C (pH controlled at 6.0–8.0, enzyme addition 0.1%) yields a low molecular weight peptide mixture mainly composed of dipeptides, tripeptides and oligopeptides (target average relative molecular mass 5–10 kDa).

[0079] Fractionation is performed using an ultrafiltration membrane with a 3–10 kDa cutoff, retaining low molecular weight components <10 kDa while removing large protein molecules and undigested residues.

[0080] S2 Saposhnikovia divaricata exosome extraction and pre-purification:

[0081] The roots of *Saposhnikovia divaricata* were crushed and suspended in PBS buffer (solid-liquid ratio example 1:5 w / v), mechanically homogenized at 4–15°C, and kept at low temperature to protect the vesicles.

[0082] The filter removes fibers and large debris through a progressive coarse filtration process, with the pore sizes changing sequentially from 100 μm to 10 μm to 0.45 μm.

[0083] Continuous flow cytometry was used for size separation enrichment of TFF hollow fibers with a molecular weight cutoff of 100–300 kDa equivalent and a particle size range of 30–200 nm for concentration and impurity removal.

[0084] The 30–200 nm vesicles were further purified by size exclusion chromatography to remove small molecule impurities.

[0085] The vesicle morphology was confirmed by transmission electron microscopy, and the particle size was detected by nanoparticle tracking analysis (NTA), maintaining the main particle size peak at 30–200 nm.

[0086] S3 spleen peptide encapsulation (passive co-incubation):

[0087] The dried spleen peptide was dissolved in PBS to prepare a stock solution of 25 mg / mL, which was then mixed with the exosome suspension at a ratio of donor spleen peptide mass to exosome dry weight of 3:1.

[0088] Incubate for 12 hours at 4–25°C under gentle rolling or slow rotation mixing conditions, ensuring gentle contact to avoid membrane damage.

[0089] After incubation, unencapsulated free spleen peptides were removed by ultrafiltration (MWCO 100–300 kDa), and the encapsulated exosome solution was collected.

[0090] Loading efficiency determination (LE): The loading percentage was calculated using HPLC and LC-MS, with a target LE of 20%.

[0091] Activity retention assay: The loaded sample was compared with free spleen peptide of the same dose for in vitro PBMC functional assay, with a target activity retention rate of 80%.

[0092] S4 Surface Modification (Hyaluronic Acid Coating, Non-Covalent)

[0093] Hyaluronic acid aqueous solution (concentration 0.1 mg / mL, molecular weight 10–300 kDa) was mixed with the loaded exosome suspension at a mass ratio of 0.1:1.

[0094] Gently stir and incubate for 4 hours at room temperature or 4–25°C to allow HA to form a coating layer on the surface of exosomes through electrostatic interactions.

[0095] Free HA was removed by mild centrifugation or ultrafiltration, and the modified exosomes were collected. A layer-by-layer self-assembly method can be used: first, a small amount of low molecular weight chitosan (80% deacetylation, low molecular weight 20–150 kDa, soluble in 0.2% acetate buffer) is used for the first layer coating, followed by a second layer coating with HA to obtain an HA / Chitosan layered structure.

[0096] Small molecule salts and low molecular weight impurities are removed by dialysis or secondary ultrafiltration, followed by freeze drying or spray drying to obtain the finished product.

[0097] Example 3:

[0098] S1. Preparation of spleen peptides (source, crude extraction and fractionation): After removing blood from the spleen tissue, mechanically homogenize it and add an equal volume of distilled water or PBS to obtain a tissue homogenate (solid-liquid ratio example 1:3 w / v, which can be adjusted appropriately according to the raw materials).

[0099] Enzymatic hydrolysis with mild protease for 6 hours at 4–25°C (pH controlled at 6.0–8.0, enzyme addition 1.0%) yields a low molecular weight peptide mixture mainly composed of dipeptides, tripeptides and oligopeptides (target average relative molecular mass 0.5–10 kDa).

[0100] Fractionation is performed using a filtration membrane to retain low molecular weight components (<10 kDa) while removing large protein molecules and undigested residues.

[0101] S2 Saposhnikovia divaricata exosome extraction and pre-purification:

[0102] The roots of *Saposhnikovia divaricata* were crushed and suspended in PBS buffer (solid-liquid ratio example 1:20 w / v), mechanically homogenized at 4–15°C, and kept at low temperature to protect the vesicles.

[0103] The filter removes fibers and large debris through a progressive coarse filtration process, with the pore sizes changing sequentially from 100 μm to 10 μm to 0.45 μm.

[0104] Size separation enrichment is achieved using continuous flow filtration, TFF hollow fiber or ultrafiltration systems, with molecular weight cutoff at 100–300 kDa equivalent and particle size range of 30–200 nm retained for concentration and impurity removal.

[0105] The 30–200 nm vesicles were further purified by size exclusion chromatography to remove small molecule impurities.

[0106] The vesicle morphology was confirmed by transmission electron microscopy, and the particle size was detected by nanoparticle tracking analysis (NTA), maintaining the main particle size peak at 30–200 nm.

[0107] S3 spleen peptide encapsulation (passive co-incubation):

[0108] The dried spleen peptide was dissolved in PBS to prepare a stock solution of 50 mg / mL, which was then mixed with the exosome suspension at a ratio of donor spleen peptide mass to exosome dry weight of 5:1.

[0109] Incubate for 4 hours at 4–25°C under gentle rolling or slow rotation mixing conditions, ensuring gentle contact to avoid membrane damage.

[0110] After incubation, unencapsulated free spleen peptides were removed using SEC (size exclusion column) and the encapsulated exosome solution was collected.

[0111] Loading efficiency determination (LE): The loading percentage was calculated using the protein / peptide quantification method, with a target LE of 30%.

[0112] Activity retention assay: The loaded sample was compared with free spleen peptide of the same dose for in vitro PBMC functional assay, with a target activity retention rate of 70%.

[0113] S4 Surface Modification (Hyaluronic Acid Coating, Non-Covalent)

[0114] Hyaluronic acid aqueous solution (concentration 1.0 mg / mL, molecular weight 10–300 kDa) was mixed with the loaded exosome suspension at a mass ratio of 0.5:1.

[0115] Incubate with gentle stirring for 0.5 hours at room temperature or 4–25°C to allow HA to form a coating layer on the surface of exosomes through electrostatic interactions.

[0116] Free HA was removed by mild centrifugation or ultrafiltration, and the modified exosomes were collected. A layer-by-layer self-assembly method can be used: first, a small amount of low molecular weight chitosan (75% deacetylation, low molecular weight 20–150 kDa, soluble in 0.1% acetate buffer) is used for the first layer coating, followed by a second layer coating with HA to obtain an HA / Chitosan layered structure.

[0117] Small molecule salts and low molecular weight impurities are removed by dialysis or secondary ultrafiltration, followed by freeze drying or spray drying to obtain the finished product.

[0118] Comparative Example 1: Unmodified exosomes carrying spleen peptide

[0119] Exosomes were prepared according to steps S1–S3 of Example 1, but step S4 (modification) was omitted. These were used to compare mucosal retention, cellular uptake, and in vitro immunological effects, serving as comparative data in the specification to demonstrate the enhancement effect of the modification.

[0120] Examples 1-3 were compared with Comparative Example 1 in the following experiments. The experimental methods included:

[0121] Experiment 1: Successful modification of exosomes and verification of their physicochemical properties, verifying that the modification and encapsulation process of hyaluronic acid did not damage the exosome structure and that the modification was successful;

[0122] Experimental groups: Samples from Examples 1, 2, and 3 and Comparative Example 1 (unmodified exosomes carrying spleen peptides)

[0123] Experimental procedure: Samples from each group were taken, and the particle size distribution and concentration were determined using a nanoparticle tracking analyzer; the surface potential was determined using a Zeta potential analyzer; vesicle morphology was observed using transmission electron microscopy, and the results are as follows:

[0124] sample Main peak particle size (nm) Zeta potential (mV) Vesicle morphology Example 1 118 ± 12 −18.6 ± 2.1 Circular, complete Example 2 132 ± 15 −16.3 ± 1.9 Circular, complete Example 3 145 ± 18 −14.8 ± 2.4 Circular, complete

[0125] The results showed that the exosome size increased slightly and the surface potential shifted towards neutral after hyaluronic acid modification, proving that the modification was successful, and no vesicle rupture or aggregation was observed.

[0126] II. Experiment 2: Mucosal retention and mucin binding experiment, demonstrating that HA modification significantly enhances the retention capacity of exosomes in the respiratory mucosal environment.

[0127] Experimental methods: A mucin binding assay was used. Samples from each group were added to a buffer system containing mucin; unbound portions were washed after incubation; and the proportion of exosomes bound to mucin was quantitatively determined.

[0128] sample Mucin binding rate (%) Example 1 68.4 ± 5.2 Example 2 62.1 ± 4.8 Example 3 71.3 ± 6.0

[0129] This indicates that the mucosal binding capacity of the HA-modified group was significantly higher than that of the unmodified group (2.1–2.4 times higher), demonstrating that the modification significantly enhances mucosal retention.

[0130] III. Experiment 3: Cellular uptake and CD44-mediated verification experiment, demonstrating that HA modification enhances epithelial / immune cell uptake through CD44 receptor.

[0131] Experimental model: respiratory epithelial cells (BEAS-2B) and macrophage line (RAW264.7)

[0132] Experimental procedure: Exosomes were fluorescently labeled; incubated with cells for 4 hours; uptake rate was measured by flow cytometry; CD44 antibody was added for blocking. The results are shown in the table below:

[0133] sample Intake rate (%) Example 1 64.7 ± 6.1 Example 2 58.3 ± 5.4 Example 3 69.2 ± 6.8 Comparative Example 1 31.5 ± 4.2

[0134] This indicates that HA modification significantly enhances the ability of exosomes to be taken up by respiratory-associated cells, supporting the CD44-mediated targeted delivery mechanism.

[0135] IV. Experiment 4: Experiment on the preservation of the bioactivity of spleen peptide, which proved that the encapsulation and modification process did not significantly damage the immune activity of spleen peptide.

[0136] Experimental methods: In vitro stimulation of human PBMCs and measurement of IFN-γ and IL-2; experimental results are as follows:

[0137] sample IFN-γ relative activity (%) Free spleen peptides 100 Example 1 81.6 ± 6.3 Example 2 79.2 ± 5.9 Example 3 72.8 ± 6.7

[0138] This indicates that the activity of spleen peptide remained at 70–80% after encapsulation and modification.

[0139] V. Experiment on Synergistic Immune Enhancement Effect

[0140] The experimental groups were: blank control, free spleen peptide.

[0141] Unmodified exosomes encapsulating spleen peptide (Comparative Example 1)

[0142] HA-modified exosomes encapsulating spleen peptides (Examples 1–3) yielded the following results:

[0143] Group IFN-γ (pg / mL) IL-2 (pg / mL) blank 45 ± 8 38 ± 6 Free spleen peptides 92 ± 11 85 ± 9 Comparative Example 1 118 ± 14 102 ± 12 Example 1 186 ± 19 164 ± 17 Example 2 172 ± 18 151 ± 16 Example 3 193 ± 21 169 ± 18

[0144] The results show that HA-modified exosomes encapsulating spleen peptides induce significantly higher levels of immune factors than free spleen peptides and unmodified encapsulated systems at the same spleen peptide dose, indicating that the technical solution of the present invention has a significant synergistic effect in immune regulation.

[0145] In summary, the above experiments demonstrate that by encapsulating spleen peptides in the exosomes of Saposhnikovia divaricata and modifying the surface with hyaluronic acid, this invention significantly enhances the retention capacity and cellular uptake efficiency of spleen peptides in the respiratory mucosa while maintaining their biological activity. As a result, it exhibits significantly better immune enhancement effects than free spleen peptides and unmodified encapsulated systems in in vitro immune models.

[0146] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An exosome-based immunomodulator, including plant exosomes and biological agents, characterized in that, The plant exosome is *Saposhnikovia divaricata* exosome, the biological agent is spleen peptide, the *Saposhnikovia divaricata* exosome internally encapsulates spleen peptide, and the surface of the *Saposhnikovia divaricata* exosome is modified with at least one of hyaluronic acid or chitosan.

2. The exosome-based immunomodulator according to claim 1, characterized in that, The spleen peptide is a mixture of low-molecular-weight polypeptides, amino acids, and related polynucleotides obtained from the spleen of a traceable healthy mammal through water extraction, mild enzymatic hydrolysis, and fractionation.

3. The exosome-based immunomodulator according to claim 1, characterized in that, The spleen peptide contains one or more of at least the following components: (a) A low molecular weight polypeptide group mainly composed of dipeptides, tripeptides and oligopeptides, with an average relative molecular mass of 0.5–10 kDa. (b) Peptides rich in arginine, lysine, glycine, proline, leucine, valine, alanine, serine, threonine, tyrosine and phenylalanine. (c) Short chain nucleotides or nucleosides and short oligonucleotide fragments of less than 20 bases.

4. The exosome-based immunomodulator according to claim 3, characterized in that, The total polypeptide content, based on the total mass of spleen peptides, is 5%–80%.

5. The exosome-based immunomodulator according to claim 1, characterized in that, The main peak particle size range of the exosomes is 30–200 nm.

6. The exosome-based immunomodulator according to claim 5, characterized in that, The loading efficiency (LE) of the windproof exosome loaded with spleen peptide is 20-40%, which is the percentage of the loaded spleen peptide mass relative to the initial donor spleen peptide mass. Furthermore, the bioactivity of spleen peptides is maintained at 70-80% after loading.

7. The exosome-based immunomodulator according to claim 1, characterized in that, The hyaluronic acid has a molecular weight of 10–300 kDa, and the chitosan is a low molecular weight chitosan with a degree of deacetylation of 70–80%.

8. A method for preparing an exosome-based immunomodulator as described in any one of claims 1-7, characterized in that, Includes the following steps: S1. Obtain traceable raw materials from the spleens of healthy mammals and prepare spleen peptide materials; S2. Obtain medicinal-grade Saposhnikovia divaricata plant material and extract plant-derived exosomes; S3. Load the spleen peptide obtained in step S1 and the exosomes obtained in step S2, so that the spleen peptide is encapsulated in the exosomes. S4. Surface modification of the carrier obtained in step S3 to obtain the final formulation.

9. The method for preparing an exosome-based immunomodulator according to claim 8, characterized in that, The spleen peptide loading in step S3 is carried out by passive co-incubation. After loading, the unloaded free spleen peptide is removed by separation and the loading efficiency and drug loading are measured. The surface modification in step S4 is performed in a non-covalent manner, which includes one of physical adsorption, electrostatic recombination, or layer-by-layer self-assembly.

10. The application of an exosome-based immunomodulator, characterized in that, Used to prepare pediatric respiratory tract infections for the prevention or treatment of upper / lower respiratory tract infections and viral respiratory diseases.

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