Milk exosome loaded erythromycin nano-preparation, and preparation method and application thereof
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- DEYONGSHAN MICRO (GUANGDONG) BIOTECHNOLOGY CO LTD
- Filing Date
- 2026-02-14
- Publication Date
- 2026-05-26
Smart Images
Figure CN122075680A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the interdisciplinary field of nanomedicine formulations and tumor immunotherapy, specifically relating to a milk exosome-loaded erythromycin nanoformulation, its preparation method, and its application. Background Technology
[0002] Malignant tumors remain one of the leading causes of death worldwide. Although immune checkpoint inhibitors, represented by PD-1 / PD-L1 antibodies, have made breakthrough progress in the treatment of various solid tumors in recent years, their clinical efficacy has significant limitations. First, PD-L1 monoclonal antibodies have a molecular weight as high as 150 kDa, resulting in poor penetration into tumor tissue and difficulty in reaching deep hypoxic areas of the tumor. Second, the Fc fragment of the antibody can mediate ADCC / CDC effects, inducing immune-related adverse reactions such as pneumonia and colitis. Third, the antibody half-life is as long as 20 days, making it difficult to quickly eliminate toxicity once it occurs. More importantly, the objective response rate of single immunotherapy in solid tumors is only 20-30%, with most patients failing treatment due to primary or acquired resistance. Therefore, developing novel PD-L1 blockers with smaller molecular weights, stronger penetration, and lower toxicity, and combining them with strategies that directly kill tumors, has become a key issue that urgently needs to be addressed in the field of tumor immunotherapy.
[0003] Erythromycin, a macrolide antibiotic used clinically for over 70 years, has recently been found to possess significant antitumor activity. Its mechanisms of action include: inhibiting protein synthesis by binding to the 50S subunit of tumor cell ribosomes; downregulating the expression of hypoxia-inducible factor HIF-1α and its downstream vascular endothelial growth factor VEGF, thereby blocking tumor angiogenesis; and activating the mitochondrial apoptosis pathway, inducing programmed cell death in tumor cells. However, erythromycin faces severe challenges in its drug development: its plasma half-life is only 1.5-2 hours, requiring frequent administration to maintain effective blood drug concentrations; after intravenous injection, the drug is mainly distributed in normal tissues such as the liver and kidneys, with less than 1% accumulation at tumor sites; and when the dose is increased to produce an antitumor effect, it easily induces serious side effects such as QT interval prolongation and elevated liver enzymes. These shortcomings severely limit the clinical development and application of erythromycin as an antitumor drug.
[0004] Nanoparticle drug delivery systems offer a novel approach to overcoming the pharmacokinetic deficiencies of erythromycin, with milk-derived exosomes (mExo) emerging as an ideal carrier due to their unique advantages. Milk exosomes are naturally occurring 30-200 nm nanovesicles found in cow's milk. Their phospholipid bilayer structure is highly similar to that of mammalian cell membranes, exhibiting excellent biocompatibility and extremely low immunogenicity. Compared to synthetic liposomes, milk exosomes are rich in natural ligands such as lactoside and integrins on their surface, enabling passive targeting of tumor tissues through receptor-ligand interactions. Their internal acidic microenvironment is conducive to the stable encapsulation and sustained release of the weakly basic drug erythromycin. More importantly, milk is widely available and inexpensive, allowing for kilogram-scale preparation through a mild process combining differential centrifugation and ultrafiltration, laying the foundation for industrial-scale production. However, relying solely on the natural targeting capabilities of milk exosomes limits their tumor tissue enrichment efficiency and lacks the function of regulating the immune microenvironment, making it difficult to overcome the tumor immunosuppressive barrier.
[0005] The emergence of nanobodies has provided a new tool for precision immunotherapy. VHH antibodies, derived from camels, have a molecular weight of only 12-15 kDa, one-tenth that of traditional antibodies, yet retain complete antigen-binding capacity. PD-L1 nanobodies can specifically bind to PD-L1 molecules on the surface of tumor cells, blocking the PD-1 / PD-L1 immune checkpoint pathway and restoring T-cell anti-tumor activity. Compared with monoclonal antibodies, nanobodies have several advantages: they lack an Fc fragment, avoiding ADCC / CDC-related immunotoxicity; they have strong tissue permeability, reaching deep into the tumor core; and they are highly stable, tolerating pH fluctuations and shear forces during milk exosome extraction. However, free PD-L1 nanobodies have a short in vivo half-life (<2 hours), requiring frequent high-dose administration, and lack a tumor-specific enrichment mechanism.
[0006] Currently, there is a significant technological divide in the field of cancer treatment: on the one hand, research on chemotherapy drugs loaded onto milk exosomes focuses solely on drug delivery efficiency, neglecting the regulation of the immune microenvironment; on the other hand, the application of PD-L1 nanobodies remains at the level of single immune intervention, without being organically combined with direct killing methods. This "spatiotemporal separation" treatment model is unlikely to produce synergistic effects, and it cannot solve the problems of tumor heterogeneity and immune escape. Therefore, this invention is derived from this. Summary of the Invention
[0007] To address at least one of the aforementioned technical problems, the present invention aims to provide a milk exosome nanoformulation, its preparation method, and its application. By integrating the dual functions of "PD-L1 nanobody active targeting + immune checkpoint blockade" and "erythromycin direct killing" on the same nanocarrier, it achieves synergistic treatment with spatiotemporal synchronization and complementary mechanisms, significantly improving anti-tumor efficacy and reducing systemic toxicity, and providing a safe, efficient, and scalable novel combination therapy strategy for clinical use.
[0008] The technical solution of this invention is:
[0009] One object of the present invention is to provide a milk exosome-loaded erythromycin nanoformulation, the formulation comprising milk exosomes, erythromycin and immune checkpoint inhibitor nanobodies, wherein the erythromycin is encapsulated in the cavity of the milk exosomes and the immune checkpoint inhibitor nanobodies are modified on the surface of the milk exosome membrane.
[0010] Preferably, the immune checkpoint inhibitor nanobody is a PD-L1 nanobody, a LAG-3 nanobody, a TIM-3 nanobody, or a CTLA-4 nanobody.
[0011] Preferably, the immune checkpoint inhibitor nanobody is a PD-L1 nanobody, which is a single-domain antibody or a fragment thereof with a molecular weight of 12-15 kDa. It is modified onto the surface of milk exosomes by an enzyme-mediated chemical coupling method, and the enzyme used is a CD36-modified ligase.
[0012] Preferably, the milk exosomes are derived from cow's milk, have a particle size of 30-200 nm, and express positive markers such as CD36, PIGR, and BT1A1 on their surface.
[0013] Preferably, the erythromycin is a macrolide antibiotic with antitumor activity, which is encapsulated in milk exosomes by a combination of electroporation and co-incubation.
[0014] The characteristics of the drug-loaded exosomes obtained by encapsulating erythromycin in milk exosomes are as follows: encapsulation efficiency >65%.
[0015] Another object of the present invention is to provide a method for preparing a nano-formulation, wherein the nano-formulation is any of the nano-formulations described above, and the preparation method includes the following steps:
[0016] 1) Extraction of exosomes from cow's milk;
[0017] 2) Erythromycin was encapsulated in milk exosomes via electroporation and co-incubation to obtain drug-loaded exosomes;
[0018] 3) Immunoimmune checkpoint inhibitor nanobodies were modified onto the surface of drug-loaded exosome membranes of milk exosomes using an enzyme-mediated chemical coupling method;
[0019] 4) Purify to obtain the nano-formulation.
[0020] Preferably, in step 3), the enzyme-mediated chemical coupling method includes:
[0021] Immune checkpoint inhibitor nanobodies were mixed with milk exosomes, and then linked to CD36, the most abundant protein on the milk exosome membrane, by CD36-modifying ligase.
[0022] Another object of this invention is to provide the application of the nano-formulations described in any one of the above claims or the nano-formulations prepared by any one of the above-described methods in the preparation of antitumor drugs. The nano-formulations are administered via intravenous injection, subcutaneous injection, or intratumoral injection. The immune checkpoint inhibitor nanobody is a PD-L1 nanobody, suitable for PD-L1-positive breast cancer, lung cancer, colorectal cancer, melanoma, and lymphoma, and can be used alone or in combination with radiotherapy or chemotherapy.
[0023] Preferably, the dosage of the nano-formulation is 0.1-10 mg / kg body weight for erythromycin and 0.05-5 mg / kg body weight for immune checkpoint inhibitor nanobodies.
[0024] Compared with the prior art, the advantages of the present invention are:
[0025] Synergistic effect: PD-L1 nanobody blocks immune checkpoints and restores T cell activity; erythromycin inhibits protein synthesis and induces apoptosis; the two are released spatiotemporally synchronously on the same carrier, producing a synergistic effect of "immune activation + direct killing", with a tumor inhibition rate of >85%.
[0026] High safety: Nanobodies do not have Fc fragments (crystallizable fragments, which are functional structural units produced by the hydrolysis of immunoglobulin G (IgG) molecules by proteases), avoiding the ADCC / CDC toxicity of traditional monoclonal antibodies; milk exosomes have excellent biocompatibility, reducing the dosage of erythromycin by 70% for the same therapeutic effect, and significantly reducing cardiotoxicity.
[0027] Precise targeting: PD-L1 nanobodies actively recognize tumors, increasing drug enrichment by 5–8 times and reducing exposure to normal tissues.
[0028] The process is mild: the CD36-modified ligase reaction conditions are mild (pH 7.4, 37℃), which does not damage the integrity of the exosome membrane and the activity of erythromycin, making it suitable for large-scale production.
[0029] Low cost: Milk is widely available; a kilogram of milk source can yield >10 kilograms of milk.14 Granules meet the needs of green pharmaceutical manufacturing and industrialization.
[0030] High versatility: The "CD36-modified ligase-mediated exosome-nanobody specific conjugation" strategy proposed in this invention can provide a paradigm for the combined application of other immune checkpoint inhibitors and natural active molecules, and promote the repositioning of antibiotics in tumor treatment. Attached Figure Description
[0031] The present invention will be further described below with reference to the accompanying drawings and embodiments:
[0032] Figure 1 This is a schematic diagram illustrating the principle and process of the present invention.
[0033] Figure 2 Extraction and characterization of milk exosomes; where A represents the particle size of milk exosomes extracted by nanoflow cytometry, B represents the detection of milk exosome protein markers, C represents the number of milk exosome particles obtained from 3 batches, D represents the electron micrograph of milk exosomes, and E represents the CD36 positivity rate on milk exosomes.
[0034] Figure 3 Erythromycin was loaded onto milk exosomes; where A is the encapsulation rate, B is the loading rate, and C is the drug leakage rate after storage at 4°C for 30 days.
[0035] Figure 4 PD-L1 nanobodies were modified on the surface of milk exosomes via CD36-modifying ligase; where A represents the PD-L1 modification positivity rate, B represents the particle size of the modified exosomes, and C represents the Western blot (WB) detection of the PD-L1 nanobodies and CD36 fusion protein.
[0036] Figure 5 The assays included in vitro targeting and T cell activation detection; A represented the exosome uptake rate of PD-L1-high and low expression cells, B represented the IFN-γ release from co-cultured T cells, and C represented the T cell proliferation index.
[0037] Figure 6 The in vitro antitumor activity is represented by A, where A is the tumor cell survival rate and B is the tumor cell apoptosis rate.
[0038] Figure 7 This represents in vivo targeting and biodistribution; where A is in vivo imaging and B is in vivo tumor fluorescence quantification.
[0039] Figure 8 This represents the in vivo antitumor activity; where A is the tumor inhibition rate analysis, B is the median survival time of mice after treatment, and C is the CD8 infiltrating tumor after treatment. + T cell abundance.
[0040] Figure 9To verify its universality with other immune checkpoint nanobodies; where A is LAG-3 nanobody and B is TIM-3 nanobody. Detailed Implementation
[0041] To make the technical means, objectives and effects of the present invention easier to understand, the present invention is further illustrated by the following examples, but the present invention is not limited to the examples.
[0042] The milk exosome-loaded erythromycin nanoformulation of this invention comprises: using milk exosomes as the core carrier, with erythromycin loaded in the lumen, and specifically linking PD-L1 nanobody (hereinafter referred to as Nb-PD-L1, where Nb is the abbreviation of the English word nanobody) to the CD36 protein of the milk exosome membrane via a CD36-modified ligase; the PD-L1 nanobody has dual functions of tumor tissue targeting recognition and immune checkpoint inhibition.
[0043] The preparation method of the milk exosome-loaded erythromycin nanoparticle formulation according to the present invention is described in the following embodiment: Figure 1 This includes the following steps:
[0044] (1) Extraction of milk exosomes: Exosomes were separated and purified from milk using a differential centrifugation combined with the Huixin EXODUS equipment.
[0045] (2) Erythromycin encapsulation: Erythromycin was loaded into the exosome cavity by a combination of electroporation and co-incubation, with an encapsulation rate of ≥65%;
[0046] (3) Surface modification: CD36 ligase was used to catalyze the specific covalent linking of PD-L1 nanobodies with CD36 protein under physiological conditions. The reaction conditions were pH 7.4, 37℃, and 30-60 minutes, with a linking efficiency ≥80%.
[0047] (4) Purification: Free components are removed using Huixin EXODUS equipment to obtain nano-formulations with uniform particle size and PDI < 0.25. PDI (Polymer dispersity index) is the polymer dispersibility index.
[0048] The present invention relates to the application of nano-formulations or nano-formulations prepared by the present invention in the preparation of antitumor drugs. The nano-formulations are administered via intravenous injection, subcutaneous injection, or intratumoral injection. The immune checkpoint inhibitor nanobody is a PD-L1 nanobody, suitable for PD-L1-positive breast cancer, lung cancer, colorectal cancer, melanoma, and lymphoma, and can be used alone or in combination with radiotherapy or chemotherapy. The dosage of the nano-formulation is 0.1-10 mg / kg body weight for erythromycin and 0.05-5 mg / kg body weight for the PD-L1 nanobody.
[0049] The following detailed description uses PD-L1 nanobody as an example of an immune checkpoint inhibitor nanobody and lung cancer as an example. The invention can be better understood through the following embodiments. However, those skilled in the art will readily understand that the specific material ratios, process conditions, and results described in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as described in the claims.
[0050] Example 1: Preparation and Identification of Milk Exosomes
[0051] 1.1 Milk pretreatment: Take fresh Holstein milk and centrifuge at 3000 g for 30 min at 4℃ to remove milk fat, then centrifuge at 10000 g for 45 min at 4℃ to remove cell debris. Then adjust the milk to pH 4.6 with hydrochloric acid to precipitate protein, and collect the supernatant by centrifugation at 10000 g for 60 min at 4℃.
[0052] 1.2 Ultrafiltration concentration: The solution was concentrated 20 times by passing it through a 0.22 µm microfiltration filter and a 100 kDa hollow fiber column.
[0053] 1.3 Extraction using Huixin EXODUS equipment: The above concentrate is added to the extraction chip, and milk exosomes are purified using the machine's preset program.
[0054] 1.4 Characterization: such as Figure 2 As shown, nanoflow cytometry determined the particle size of the purified milk exosomes (hereinafter referred to as mExo) to be 80.7 ± 20.1 nm. Figure 2 As shown in Figure A); Western blotting (WB, also known as immunoblotting) showed positive results for CD36, PIGR (polyimmunoglobulin receptor), and BT1A1 (gene name btn1a1), but negative for Calnexin (calc-linked protein). Figure 2 (As shown in B); approximately 5 × 10⁻⁶ ppm can be obtained per milliliter of raw milk. 13 Bovine exosome granules ( Figure 2 As shown in C), the CD36 positivity rate is >85% ( Figure 2 (As shown in E).
[0055] Example 2: Erythromycin Encapsulation
[0056] 2.1 Take mExo 1×10 12 The particles were co-suspended with erythromycin hydrochloride (Sigma) at a mass ratio of 1:0.2 in 400 µL of electroporation buffer (250 mM sucrose, 1 mM MgCl2, 10 mM KH2PO4, pH 7.4).
[0057] 2.2 2 mm shock cup, 400 V, 125 µF, ∞ Ω, single pulse; immediately place at 37℃ for 30 min for resuscitation.
[0058] 2.3 The Huixin EXODUS device removes the free drug, yielding drug-loaded exosomes mExo-EM.
[0059] 2.4 As Figure 3 As shown, HPLC analysis revealed that the mExo-EM encapsulation efficiency of the drug-loaded exosomes was 72%. Figure 3 As shown in Figure A), the drug loading was 9.1% ( Figure 3 As shown in Figure B); drug leakage <8% after 30 days of storage at 4℃ (as shown in Figure B). Figure 3 (As shown in C).
[0060] Example 3: CD36-specific coupling mediated by CD36-modified ligase
[0061] 3.1 Reaction system: mExo-EM 1×10 12 Particles, Nb-PD-L1 100 µg, CD36-modified ligase 5 µM, CaCl2 5 mM, total volume 500 µL, pH 7.4.
[0062] 3.2 Shake gently at 37℃ for 45 min, then add 20 mM EDTA to terminate the reaction.
[0063] 3.3 The Huixin EXODUS equipment removes free Nb and enzymes to obtain the final product mExo-EM-Nb-PD-L1.
[0064] 3.4 Characterization: such as Figure 4 As shown, the Nb positivity rate of the exosome surface of the final product mExo-EM-Nb-PD-L1 detected by nanoflow cytometry was 78%. Figure 4 As shown in Figure A), the particle size is 74.1 ± 16.6 nm. Figure 4 (As shown in B); Western blotting showed a band of Nb and CD36 fusion protein at 68 kDa (as shown in B). Figure 4 (As shown in C).
[0065] Example 4: Validation of in vitro targeted and immune checkpoint blockade
[0066] 4.1 Cells: Human lung adenocarcinoma cells A549 (PD-L1) + ) and normal human bronchial epithelial cells BEAS-2B (PD-L1) - ).
[0067] 4.2 Targeting: DiR (deep red fluorescence) labeled exosomes were co-incubated with cells for 2 h, such as... Figure 5 As shown in Figure A, flow cytometry revealed an A549 uptake rate of 87%, while BEAS-2B showed only 11%; confocal microscopy showed significant A549 enrichment in the film.
[0068] 4.3 Blockade: Exosomes (containing 2 µg / mL Nb) were added and co-cultured with activated human T cells (effective cells). ELISA was used to measure the supernatant IFN-γ: mExo-EM-Nb-PD-L1 level, which was 4.6 times higher in the mExo-EM-Nb-PD-L1 group than in the PBS group (blank group). Figure 5 As shown in Figure B), it is equivalent to Atezolizumab (10 µg / mL); the T cell proliferation CFSE assay showed a 3.1-fold increase in the proliferation index (…). Figure 5 (As shown in C).
[0069] Example 5: In vitro synergistic cytotoxicity experiment
[0070] 5.1 A549 cells were prepared at a rate of 5 × 10⁻⁶. 3 / wells were seeded into 96-well plates and, after adhesion, were divided into five groups: ①PBS; ② free EM; ③ mExo-EM; ④ mExo empty + Nb; ⑤ mExo-EM-Nb-PD-L1.
[0071] 5.2 Erythromycin final concentration 5 µg / mL, CCK-8 assay after 48 h: (e.g., ...) Figure 6 As shown, the survival rate of group ⑤ was 28%, significantly lower than that of group ② (62%) and group ③ (48%) (P<0.01). Figure 6 As shown in Figure A); Calcein-AM / PI staining showed that the apoptosis rate in group ⑤ was 65%, which was higher than that in group ② (31%). Figure 6 (As shown in B).
[0072] 5.3 The synergy index CI = 0.68 (CompuSyn) confirms the synergy effect.
[0073] Example 6: In vivo distribution study in mice
[0074] 6.1 Establishment of A549-Luc subcutaneous tumor BALB / c nude mice (n=3 / group, tumor volume ≈100 mm) 3 () Figure 7 (As shown in A).
[0075] 6.2 DiR-labeled exosomes were injected via tail vein at a dose equivalent to erythromycin 2 mg / kg; IVIS imaging showed that the mExo-EM-Nb-PD-L1 group exhibited fluorescence accumulation in the tumor area within 2 hours, and the signal intensity at 24 hours was 5.4 times that of free DiR-EM; the tumor / heart fluorescence ratio in ex vivo organs reached 9.1:1 (…). Figure 7 (As shown in B).
[0076] Example 7: In vivo efficacy evaluation
[0077] 7.1 Model: C57BL / 6-PD-L1 humanized mice, with 1×10⁶ MC38-PD-L1 overexpressing cells subcutaneously injected into the right axilla. 6 .
[0078] 7.2 Grouping (n=8): ① Normal saline; ② Free EM 5 mg / kg; ③ mExo-EM 5 mg / kg; ④ mExo-EM-Nb-PD-L1 (EM 5 mg / kg + Nb 2 mg / kg); ⑤ Atezolizumab 10 mg / kg. Administered every other day for a total of 7 doses. Atezolizumab is an immune checkpoint inhibitor targeting PD-L1.
[0079] 7.3 Results: As Figure 8 As shown, the tumor inhibition rate in group ④ was 87%, significantly higher than that in group ③ (54%) and group ⑤ (52%). Figure 8 (As shown in A); median survival time: group ④ > 60 days, group ① 21 days ( Figure 8 (As shown in Figure B); Flow cytometry shows tumor infiltration CD8. + The proportion of T cells increased from 8.1% in group ① to 31.4% in group ④. Figure 8 (As shown in group C); there were no differences in serum ALT, CK-MB and body weight compared with group ①, and no obvious immune-related toxicity was observed.
[0080] Example 8: Formulation stability and scale-up test
[0081] 8.1 When mExo-EM-Nb-PD-L1 was placed in PBS at 4 ℃ for 90 days, the particle size change was <10%, and Nb maintained >80% of its antigen-binding activity.
[0082] 8.2 The process of Examples 1 to 4 was repeated three times using a 10 L raw milk scale. The final product particle yield, drug loading, and Nb linkage efficiency RSD were all less than 8%, meeting the consistency requirements. After freeze-drying and reconstitution with a freeze-drying protectant (5% trehalose + 2% sucrose), the particle size and activity remained greater than 90%, meeting the needs of industrial production and long-distance transportation.
[0083] Example 9: Verification of universality with other immune checkpoint nanobodies
[0084] Replace the PD-L1 nanobody with LAG-3-VHH (i.e., LAG-3 nanobody) or TIM-3-VHH (i.e., TIM-3 nanobody), and couple them using the same CD36-modified ligase protocol to obtain mExo-EM-Nb-LAG-3 or mExo-EM-Nb-TIM-3. For example... Figure 9 As shown, in vitro T cell function experiments and CT26 tumor-bearing mice efficacy experiments showed that the tumor inhibition rate reached 79% ( Figure 9 (as shown in A) and 82% ( Figure 9 As shown in Figure B), the CD36-ligase platform can be extended to a variety of immune checkpoint inhibitors, demonstrating its broad applicability.
[0085] It should be understood that the specific embodiments described above are merely illustrative or explanatory of the principles of the invention and do not constitute a limitation thereof. Therefore, any modifications, equivalent substitutions, improvements, etc., made without departing from the spirit and scope of the invention should be included within the protection scope of the invention. Furthermore, the appended claims are intended to cover all variations and modifications falling within the scope and boundaries of the appended claims, or equivalent forms of such scope and boundaries.
Claims
1. A milk exosome-loaded erythromycin nanoparticle formulation, characterized in that, The formulation comprises milk exosomes, erythromycin, and immune checkpoint inhibitor nanobodies, wherein the erythromycin is encapsulated within the lumen of the milk exosomes, and the immune checkpoint inhibitor nanobodies are modified on the surface of the milk exosome membranes.
2. The nano-formulation according to claim 1, characterized in that, The immune checkpoint inhibitor nanobody is a PD-L1 nanobody, a LAG-3 nanobody, a TIM-3 nanobody, or a CTLA-4 nanobody.
3. The nano-formulation according to claim 2, characterized in that, The immune checkpoint inhibitor nanobody is a PD-L1 nanobody, which is a single-domain antibody or a fragment thereof with a molecular weight of 12-15 kDa. It is modified onto the surface of milk exosomes by an enzyme-mediated chemical coupling method, and the enzyme used is a CD36-modified ligase.
4. The nano-formulation according to any one of claims 1-3, characterized in that, The milk exosomes are derived from cow's milk, with a particle size of 30-200 nm, and their surface expresses positive markers such as CD36, PIGR, and BT1A1.
5. The nano-formulation according to claim 4, characterized in that, The erythromycin is a macrolide antibiotic with antitumor activity, which is encapsulated in milk exosomes by a combination of electroporation and co-incubation. The characteristics of the drug-loaded exosomes obtained by encapsulating erythromycin in milk exosomes are as follows: encapsulation efficiency >65%.
6. A method for preparing a nano-formulation, characterized in that, The nano-formulation is the nano-formulation according to any one of claims 1-5, and the preparation method includes the following steps: 1) Extraction of exosomes from cow's milk; 2) Erythromycin was encapsulated in milk exosomes via electroporation and co-incubation to obtain drug-loaded exosomes; 3) Immunoimmune checkpoint inhibitor nanobodies were modified onto the surface of drug-loaded exosome membranes of milk exosomes using an enzyme-mediated chemical coupling method; 4) Purify to obtain the nano-formulation.
7. The preparation method according to claim 6, characterized in that, In step 3), the enzyme-mediated chemical coupling method includes: Immune checkpoint inhibitor nanobodies were mixed with milk exosomes, and then linked to CD36, the most abundant protein on the milk exosome membrane, by CD36-modifying ligase.
8. The use of the nanoformulation according to any one of claims 1-5 or the nanoformulation prepared by the preparation method according to any one of claims 6-7 in the preparation of antitumor drugs.
9. The application according to claim 8, characterized in that, The tumors mentioned include breast cancer, lung cancer, colon cancer, melanoma, or lymphoma.
10. The application according to claim 8, characterized in that, The nanoformulation is administered via intravenous injection, subcutaneous injection, or intratumoral injection; and / or The dosage of the nano-formulation is 0.1-10 mg / kg body weight for erythromycin and 0.05-5 mg / kg body weight for immune checkpoint inhibitor nanobodies.