Composite peptide fragment modified exosome as well as preparation method and application thereof

Abstract

The invention provides a composite peptide fragment modified exosome as well as a preparation method and application thereof, and belongs to the technical field of biomedicine. According to the exosome, a CP05 peptide fragment is selected as a basic peptide fragment modified by the exosome and is coupled with a CGSPGWVRC peptide fragment of a targeted lung, and the coupled composite peptide fragment is combined with a specific target spot on the surface of the exosome, so that the purpose of modifying the composite peptide fragment on the surface of the exosome is achieved, the purpose of regulating and controlling the guidance of the exosome is further achieved, the exosome reaches the lung in a targeted manner, and the lung targeting effect is achieved. Distribution in other tissues is reduced, and potential side effects are reduced. In addition, the method for coupling the two peptide fragments and modifying the two peptide fragments to the surface of the exosome has high feasibility and operability, and is suitable for large-scale preparation and application.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine and relates to an exosome modified with a composite peptide segment, a preparation method and an application thereof. Background Art

[0002] Exosomes are nanoscale vesicles secreted by cells and have the function of transmitting biological information between cells. Exosomes are involved in functions such as the body's immune response, antigen presentation, cell migration, cell differentiation, and tumor invasion, and thus have received extensive attention in the fields of drug delivery, disease diagnosis and treatment.

[0003] The lungs are an important respiratory organ of the human body and are also the sites where many diseases occur and develop. For example, diseases such as lung infections, pulmonary fibrosis, and lung cancer pose a serious threat to human health. If exosomes can be directed to accumulate in the lungs, not only can the efficiency of drug delivery be improved, but also the specificity of disease diagnosis and treatment can be enhanced, and the adverse effects on other tissues can be reduced. However, the distribution of exosomes in the body often lacks specificity, which limits their application effects in specific tissues or organs. Although there have been studies attempting to achieve their targeting by modifying the surface of exosomes, the exosome modification methods for lung enrichment still have deficiencies. Summary of the Invention

[0004] Based on the above problems to be solved, the purpose of the present invention is to provide an exosome modified with a composite peptide segment, a preparation method and an application thereof.

[0005] To achieve the above purpose, the present invention adopts the following technical solutions: The present application provides an exosome modified with a composite peptide segment. This exosome selects the CP05 peptide segment as the basic peptide segment for exosome modification, couples it with the CGSPGWVRC peptide segment targeting the lungs, and binds the coupled composite peptide segment to specific targets on the surface of the exosome, so as to achieve the purpose of modifying the composite peptide segment on the surface of the exosome, and further achieve the purpose of regulating the directivity of the exosome. In the present application, the specific targets on the surface of the exosome include membrane proteins, lipids, etc. The sequence of the CP05 peptide segment is CRHSQMTVTSRL, and the sequence of the CGSPGWVRC peptide segment is Cys-Gly-Ser-Pro-Gly-Trp-Val-Arg-Cys.

[0006] The CGSPGWVRC peptide segment consists of 9 amino acids and has high selectivity and high targeting ability, and can recognize specific molecular structures on the surface of lung endothelial cells. When it is modified on the surface of exosomes, it can enable the exosomes to target and accumulate in the lungs, improve the efficiency of drug delivery, and reduce the impact on non-target tissues.

[0007] The present application also provides a preparation method for an exosome modified with a composite peptide segment. This method includes: S01: After mixing the CP05 peptide segment with the CGSPGWVRC peptide segment, a coupling agent was added, the pH value of the reaction system was adjusted to 7.4, and the reaction was carried out at room temperature for 2 h to obtain the CP05-CGSPGWVRC peptide segment.

[0008] The CP05 peptide segment and the CGSPGWVRC peptide segment were mixed in an equimolar ratio, and N-hydroxysuccinimide ester (abbreviation: NHS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (abbreviation: EDC) were added as coupling agents. The pH value of the reaction system was adjusted to 7.4, and the reaction was carried out at room temperature for 2 h to obtain the CP05-CGSPGWVRC peptide segment. Among them, based on the total volume of the CP05 peptide segment and the CGSPGWVRC peptide segment, the amounts of NHS and EDC added per 1 mL volume were 5-10 mmol and 2.5-10 mmol respectively. Preferably, the amounts of NHS and EDC added per 1 mL volume were 5 mmol and 2.5 mmol respectively.

[0009] In the coupling reaction, the coupling agents NHS and EDC can connect the terminal amino group of the CP05 peptide segment and the terminal carboxyl group of the CGSPGWVRC peptide segment to form a stable amide bond, realizing the coupling of the two.

[0010] After the coupling reaction was completed, the CP05-CGSPGWVRC peptide segment was purified by high performance liquid chromatography, and the target peak product was collected to obtain the purified CP05-CGSPGWVRC peptide segment. Among them, the conditions of high performance liquid chromatography were: (1) Selection of chromatographic column: The chromatographic column was selected as a reverse chromatographic column such as a C18 column to facilitate the effective separation of the CP05-CGSPGWVRC peptide segment; A wide pore column with a particle size of 15-20 μm was selected, which could provide better separation effect and higher sample loading capacity; The appropriate chromatographic column size was selected according to the purification scale. The column selected in this experiment was 50 mm×300 mm.

[0011] (2) Selection of mobile phase: Phase A: The aqueous phase, usually adding a small amount of acid to improve the separation effect and peak shape. Preferably, 0.1% trifluoroacetic acid (TFA) was used. TFA can protonate the carboxyl group of the peptide segment, reduce secondary interactions, and at the same time form ion pairs with the positively charged functional groups in the peptide sequence, increasing the hydrophobic interaction between the peptide and the non-polar reverse phase chromatographic packing material.

[0012] Phase B: Acetonitrile, which can be used in combination with TFA and can effectively adjust the elution behavior of the peptide segment in reverse phase chromatography.

[0013] (3) Elution gradient Initial conditions: Usually start from a relatively low organic phase ratio, such as 0% - 5% phase B, to retain the peptide segments.

[0014] Gradient change: 0 - 5 minutes: 100% A 5 - 20 minutes: A linearly decreases from 100% to 50%, and B (acetonitrile) linearly increases from 0% to 50% 20 - 30 minutes: A linearly decreases from 50% to 0%, and B linearly increases from 50% to 100% 30 - 35 minutes: 100% B, for cleaning the chromatographic column (4) Flow rate: 0.5 - 1.0 mL / min; UV detection wavelength: 214 nm or 220 nm; Temperature: room temperature.

[0015] In this application, the CP05 peptide segment and the CGSPGWVRC peptide segment are respectively synthesized by the solid-phase peptide synthesis method. The specific method is as follows: The synthesis method of the CP05 peptide segment is: Soak the Wang resin in dichloromethane for treatment to make it fully expand. According to the amino acid sequence CRHSQMTVTSRL of the CP05 peptide segment, use 9-fluorenylmethoxycarbonyl protecting group (abbreviation: Fmoc) as the amino protecting group and attach it to the first amino acid C. Using HBTU and N,N-diisopropylethylamine (abbreviation: DIEA) as the coupling agent, couple the amino acid C with the attached amino protecting group to the fully expanded Wang resin. After the coupling is completed, wash the resin with N,N-dimethylformamide (abbreviation: DMF). Use 20% piperidine / DMF to remove Fmoc.

[0016] Sequentially add the subsequent amino acids with protecting groups and couple them to the Wang resin according to the peptide segment sequence, while removing Fmoc. After each coupling, wash the resin with DMF. Use trifluoroacetic acid (abbreviation: TFA) to cleave the synthesized peptide segment from the Wang resin, and use high-performance liquid chromatography to purify the peptide segment to obtain the CP05 peptide segment.

[0017] The synthesis method of the CGSPGWVRC peptide segment is the same as that of the CP05 peptide segment, only the amino acid sequence is different, and it will not be elaborated here.

[0018] S02: Combine the CP05-CGSPGWVRC peptide segment with a specific target on the surface of exosomes by chemical coupling method or physical adsorption method to obtain exosomes modified with composite peptide segments.

[0019] The purified CP05-CGSPGWVRC peptide is combined with specific targets on the surface of exosomes by chemical coupling or physical adsorption methods to immobilize the CP05-CGSPGWVRC peptide on the surface of exosomes, achieving directional modification of exosomes and obtaining exosomes modified with composite peptides. Among them, the molar ratio of the CP05-CGSPGWVRC peptide to exosomes is 2-6:1-2.

[0020] In this application, exosomes can be commercially available exosomes or extracted exosomes. When using extracted exosomes, existing methods can be used to extract exosomes.

[0021] In this application, the chemical coupling method is to use the biotin-streptavidin system to bind the CP05-CGSPGWVRC peptide to specific targets on the surface of exosomes. Specifically, (1) Collect the sample containing exosomes from the cell culture supernatant, centrifuge at 1000 xg for 30 min to remove cell debris and impurities, and collect the supernatant. Centrifuge the supernatant at 100000 xg for 70 min to precipitate exosomes. Resuspend the precipitated exosomes with phosphate buffer at pH 7.4 and then centrifuge at 100000 xg for 70 min again to wash the exosomes and remove residual impurities.

[0022] (2) Use biotin-N-hydroxysuccinimide ester (abbreviation: NHS-biotin) as a coupling agent to couple biotin to the membrane proteins or lipids on the surface of exosomes in phosphate buffer at pH 7.4. Centrifuge at 100000 xg for 70 min to remove free biotin and obtain pure biotinylated exosomes.

[0023] (3) Dissolve streptavidin (abbreviation: SA) in phosphate buffer at pH 7.4, incubate the biotinylated exosomes and streptavidin at pH 7.4 and room temperature to obtain SA-biotinylated exosomes.

[0024] (4) Covalently bind NHS-biotin to the CP05-CGSPGWVRC peptide to label the CP05-CGSPGWVRC peptide with biotin. Mix the biotin-labeled CP05-CGSPGWVRC peptide with SA-biotinylated exosomes and incubate at pH 7.4 and room temperature to obtain exosomes modified with composite peptides.

[0025] In this application, the process of binding the CP05-CGSPGWVRC peptide to specific targets on the surface of exosomes by physical adsorption method is as follows: (1)Collect the exosome-containing sample from the cell culture supernatant and centrifuge it at 1000 x g for 30 min to remove cell debris and impurities, and collect the supernatant. Centrifuge the supernatant at 100000 x g for 70 min to precipitate the exosomes. After resuspending the precipitated exosomes in phosphate buffer at pH 7.4, centrifuge them again at 100000 x g for 70 min to wash the exosomes and remove residual impurities, obtaining an exosome resuspension.

[0026] (2)Dissolve the CP05-CGSPGWVRC peptide in phosphate buffer at pH 7.4 to form a peptide solution.

[0027] (3)Mix the CP05-CGSPGWVRC peptide solution and the exosome resuspension at a volume ratio of 1:10 - 1:100, and place them in a constant temperature shaker at 37 °C for incubation for 1 - 2 h. During the incubation, through slow shaking or stirring, make the peptide fully contact with the exosomes to promote physical adsorption.

[0028] (4)After the incubation, ultracentrifuge at 100,000 g for 30 - 60 min, discard the supernatant, and retain the exosome precipitate. Wash the exosome precipitate 2 - 3 times with phosphate buffer at pH 7.4 to remove unbound peptides and other impurities. After each wash, perform ultracentrifugation again and discard the supernatant. Resuspend the washed exosome precipitate in phosphate buffer at pH 7.4 to obtain exosomes modified with composite peptides.

[0029] The above-prepared exosomes modified with composite peptides are used to target and enrich exosomes in the lungs, improve the drug delivery efficiency, and reduce the impact on non-target tissues.

[0030] In this application, it is inferred that the mechanism by which the CP05-CGSPGWVRC peptide targets and enriches exosomes in the lungs is as follows: (1)Specific binding to lung-related receptors Receptor-mediated targeting: Specific receptors or molecules may exist on the surface of lung cells. The CP05-CGSPGWVRC peptide may specifically bind to these receptors through its unique amino acid sequence and thus be taken up by lung cells.

[0031] Interaction with lung disease-related proteins: If there is a certain disease-related protein in the lungs and this protein has a certain affinity for the CP05-CGSPGWVRC composite peptide, then the peptide can bind to these proteins and be enriched in the diseased area of the lungs.

[0032] (2)Influence of the physiological characteristics of the lungs High permeability of the lungs: The capillary vessels and alveolar structures in the lungs have relatively high permeability, which allows some molecules to more easily enter the lung tissue through the blood circulation. The CP05-CGSPGWVRC peptide segment may utilize this property to be more easily enriched in the lungs.

[0033] Inflammation or pathological microenvironment in the lungs: In lung diseases such as lung cancer and pulmonary fibrosis, the local inflammation or pathological microenvironment can lead to changes in the expression of cell surface molecules or the generation of some new targets. The CP05-CGSPGWVRC peptide segment may have a certain responsiveness to these altered microenvironments, thus achieving targeting of the diseased lungs.

[0034] (3) Molecular structure and targeting Specificity of the amino acid sequence: The amino acid sequence of the peptide segment determines its spatial structure and chemical properties, and thus affects its interaction with various molecules in the body. The specific sequence of the CP05-CGSPGWVRC peptide segment may endow it with higher stability and affinity in the lung microenvironment.

[0035] Molecular weight and targeting: The molecular weight of the peptide segment may affect its distribution and targeting in the body. A smaller molecular weight may make it easier to enter the lung tissue through the capillary wall of the lungs.

[0036] The present invention has the following beneficial effects: (1) Enhancing the targeting of exosomes: After the CP05 peptide segment is conjugated with the CGSPGWVRC peptide segment targeting the lungs and binds to specific targets on the surface of exosomes, it can precisely regulate the orientation of exosomes, enabling them to target the lungs, reducing their distribution in other tissues, and lowering potential side effects.

[0037] (2) Improving the lung enrichment ability of exosomes: Exosomes modified with the CP05-CGSPGWVRC peptide segment can significantly improve the enrichment efficiency in the lungs, enhancing the application potential of exosomes in the treatment of lung diseases.

[0038] (3) Simple operation and easy implementation: The method of conjugation between the two peptide segments and modification onto the surface of exosomes has high feasibility and operability, and is suitable for large-scale preparation and application.

[0039] (4) Broad clinical application prospects: The present invention provides a new technical means for drug delivery of exosomes in the lungs, and is expected to promote the development of exosome-related research and clinical applications. Description of the drawings

[0040] Figure 1 It is a TEM detection diagram of exosomes purified in Example 1 and exosomes modified with the composite peptide segment. Among them, Figure A is the exosomes purified, and Figure B is the exosomes modified with the composite peptide segment; Figure 2 Comparison chart of the inhibitory effects of exosomes purified in Example 1 and exosomes modified with composite peptide segments on lung cancer A549 cells; Figure 3 Chart of changes in inflammatory factors before and after injecting exosomes purified in Example 1 and exosomes modified with composite peptide segments into an inflammatory mouse model; Figure 4 In vivo fluorescence imaging chart of the distribution of exosomes purified in Example 1 and exosomes modified with composite peptide segments in an inflammatory mouse model. Detailed implementation manners

[0041] The technical solutions of the present invention will be further explained and illustrated below through specific examples.

[0042] Example 1 The embodiments of the present application provide an exosome modified with a composite peptide segment. The preparation method of the composite peptide segment includes: S101: The CP05 peptide segment and the CGSPGWVRC peptide segment are respectively synthesized by solid-phase peptide synthesis. After mixing the CP05 peptide segment and the CGSPGWVRC peptide segment in an equimolar ratio, 5 mmol of NHS and 2.5 mmol of EDC are added to each 1 mL of the mixed solution as coupling agents, the pH value of the reaction system is adjusted to 7.4, and the reaction is carried out at room temperature for 2 h to obtain the CP05-CGSPGWVRC peptide segment. The CP05-CGSPGWVRC peptide segment is purified by high-performance liquid chromatography, the target peak product is collected, and the purified CP05-CGSPGWVRC peptide segment is obtained. Among them, the conditions of high-performance liquid chromatography are: (1) Selection of chromatographic column: The C18 column or other reverse-phase chromatographic columns are selected as the chromatographic column to facilitate the effective separation of the CP05-CGSPGWVRC peptide segment; A wide-pore column with a particle size of 15 μm is selected, which can provide better separation effect and higher sample loading capacity; The appropriate chromatographic column size is selected according to the purification scale. The column selected in this experiment is 50 mm×300 mm.

[0043] (2) Selection of mobile phase: Phase A: Deionized water + 0.1% trifluoroacetic acid (TFA). TFA can protonate the carboxyl group of the peptide segment, reduce secondary interactions, and at the same time form ion pairs with the positively charged functional groups in the peptide segment sequence, increasing the hydrophobic interaction between the peptide and the non-polar reverse-phase chromatographic packing material.

[0044] Phase B: Acetonitrile, which can be used in combination with TFA and can effectively adjust the elution behavior of the peptide segment in reverse-phase chromatography.

[0045] (3) Elution gradient 0 - 5 minutes: 100% A 5 - 20 minutes: A linearly decreases from 100% to 50%, and B (acetonitrile) linearly increases from 0% to 50% 20 - 30 minutes: A linearly decreases from 50% to 0%, and B linearly increases from 50% to 100% 30 - 35 minutes: 100% B, for cleaning the chromatographic column (4)Flow rate: 0.8 mL / min; UV detection wavelength: 214 nm; Temperature: room temperature.

[0046] S102: Extract exosomes from the culture supernatant of human alveolar epithelial cells and purify the exosomes by ultracentrifugation. Mix the purified CP05 - CGSPGWVRC peptide segment with the purified exosomes at a molar ratio of 6:2, and bind the CP05 - CGSPGWVRC peptide segment to the membrane protein on the surface of the exosomes through the biotin - streptavidin system to obtain exosomes modified with the composite peptide segment.

[0047] Example 2 The embodiment of the present application provides an exosome modified with a composite peptide segment, and the preparation method of the composite peptide segment includes: S201: Synthesize the CP05 peptide segment and the CGSPGWVRC peptide segment respectively by solid - phase peptide synthesis method. After mixing the CP05 peptide segment and the CGSPGWVRC peptide segment in an equimolar ratio, add 8 mmol NHS and 5 mmol EDC to each 1 mL of the mixed solution as coupling agents, adjust the pH value of the reaction system to 7.4, and react at room temperature for 2 h to obtain the CP05 - CGSPGWVRC peptide segment. Purify the CP05 - CGSPGWVRC peptide segment by high - performance liquid chromatography, collect the target peak product, and obtain the purified CP05 - CGSPGWVRC peptide segment. Among them, the conditions of high - performance liquid chromatography are: (1)Chromatographic column selection: Select a reverse - phase chromatographic column such as a C18 column for effective separation of the CP05 - CGSPGWVRC peptide segment; Select a wide - pore column with a particle size of 18 μm, which can provide better separation effect and higher sample loading capacity; Select a suitable chromatographic column size according to the purification scale. The column selected in this experiment is 50 mm×300 mm.

[0048] (2)Mobile phase selection: Phase A: Deionized water + 0.1% trifluoroacetic acid (TFA). TFA can protonate the carboxyl group of the peptide segment, reduce secondary interactions, and at the same time form ion pairs with the positively charged functional groups in the peptide segment sequence, increasing the hydrophobic interaction between the peptide and the non - polar reverse - phase chromatographic packing material.

[0049] Phase B: Acetonitrile, which can be used in combination with TFA and can effectively regulate the elution behavior of peptide segments in reverse-phase chromatography.

[0050] (3) Elution gradient 0 - 5 minutes: 100% A 5 - 20 minutes: A linearly decreases from 100% to 50%, and B (acetonitrile) linearly increases from 0% to 50% 20 - 30 minutes: A linearly decreases from 50% to 0%, and B linearly increases from 50% to 100% 30 - 35 minutes: 100% B, for cleaning the chromatographic column (4) Flow rate: 0.5 mL / min; UV detection wavelength: 214 nm; Temperature: room temperature.

[0051] S202: Extract exosomes from the culture supernatant of human alveolar epithelial cells and purify the exosomes by ultracentrifugation. Mix the purified CP05 - CGSPGWVRC peptide segments with the purified exosomes at a molar ratio of 2:1, and bind the CP05 - CGSPGWVRC peptide segments to the membrane proteins on the surface of exosomes through the biotin - streptavidin system to obtain exosomes modified with composite peptide segments.

[0052] Example 3 The embodiment of the present application provides an exosome modified with composite peptide segments, and the preparation method of the composite peptide segments includes: S301: Synthesize CP05 peptide segments and CGSPGWVRC peptide segments respectively by solid-phase peptide synthesis method. After mixing the CP05 peptide segments and CGSPGWVRC peptide segments in an equimolar ratio, add 10 mmol NHS and 10 mmol EDC to every 1 mL of the mixed solution as coupling agents, adjust the pH value of the reaction system to 7.4, and react at room temperature for 2 h to obtain CP05 - CGSPGWVRC peptide segments. Purify the CP05 - CGSPGWVRC peptide segments by high-performance liquid chromatography, collect the target peak products, and obtain the purified CP05 - CGSPGWVRC peptide segments. Among them, the conditions of high-performance liquid chromatography are: (1) Selection of chromatographic column: Select a reverse-phase chromatographic column such as a C18 column for the chromatographic column to facilitate the effective separation of CP05 - CGSPGWVRC peptide segments; Select a wide-pore column with a particle size of 20 μm, which can provide better separation effect and higher sample loading capacity; Select a suitable chromatographic column size according to the purification scale. The column selected in this experiment is 50 mm × 300 mm.

[0053] (2) Selection of mobile phase: Phase A: Deionized water + 0.1% trifluoroacetic acid (TFA). TFA can protonate the carboxyl groups of peptides, reducing secondary interactions. Meanwhile, it forms ion pairs with the positively charged functional groups in the peptide sequence, increasing the hydrophobic interaction between the peptide and the non-polar reversed-phase chromatography packing material.

[0054] Phase B: Acetonitrile, which can be used in combination with TFA and effectively regulate the elution behavior of peptides in reversed-phase chromatography.

[0055] (3) Elution gradient 0 - 5 minutes: 100% A 5 - 20 minutes: A linearly decreases from 100% to 50%, and B (acetonitrile) linearly increases from 0% to 50% 20 - 30 minutes: A linearly decreases from 50% to 0%, and B linearly increases from 50% to 100% 30 - 35 minutes: 100% B, for cleaning the chromatographic column (4) Flow rate: 1.0 mL / min; UV detection wavelength: 220 nm; Temperature: room temperature.

[0056] S302: Extract exosomes from the culture supernatant of human alveolar epithelial cells and purify the exosomes by ultracentrifugation. Mix the purified CP05 - CGSPGWVRC peptide with the purified exosomes at a molar ratio of 6:1, and bind the CP05 - CGSPGWVRC peptide to the membrane proteins on the surface of exosomes through the biotin - streptavidin system to obtain exosomes modified with the composite peptide.

[0057] Perform transmission electron microscopy (abbreviated as TEM) detection on the exosomes purified in Example 1 and the exosomes modified with the composite peptide respectively to observe the size and morphology of the exosomes, and obtain Figure 1 . From Figure 1 It can be seen that the morphology of the exosomes modified with the CP05 - CGSPGWVRC peptide has not changed significantly, indicating that the modification with the composite peptide does not affect the morphology of the exosomes.

[0058] To verify that the exosomes modified with the composite peptide prepared in the embodiments of the present application can target and enrich in the lungs and can deliver drugs to treat lung diseases, in vitro experiments, in vivo experiments, and in vivo fluorescence imaging detection are carried out with the purified exosomes in Example 1 and the exosomes modified with the composite peptide prepared.

[0059] 1. In vitro experiments Using lung cancer A549 cells without exosomes as the control group, purified exosomes and exosomes modified with the composite peptide segment prepared in Example 1 were respectively mixed with lung cancer A549 cells according to an effector-target ratio of 10:1 and incubated at room temperature for 48 h. At 4 h, 24 h, and 48 h of incubation, the tumor inhibition of lung cancer A549 cells was detected by the CCK8 method to evaluate the effectiveness of exosomes on lung diseases, and the following was obtained Figure 2 .

[0060] As can be seen from Figure 2 the attachment, both purified exosomes and exosomes modified with the composite peptide segment showed inhibitory effects on lung cancer A549 cells, and the anti-tumor ability of exosomes modified with the composite peptide segment was significantly enhanced. This experiment was repeated three times. From the different tumor inhibitory effects shown by exosomes, it can be known that the CP05-CGSPGWVRC composite peptide segment has been successfully loaded on the exosomes in the experimental group, and the following experiment can be continued.

[0061] 2. In vivo experiment After anesthetizing the mice, a bleomycin solution with a common dose of 3 U / kg was instilled into the trachea, and the inflammatory indexes were detected on the 7th, 14th, and 21st days after instillation. Using the mice without instilling the bleomycin solution as the control group, when the inflammatory indexes of the mice instilled with the bleomycin solution were higher than those of the control group, it indicated that the modeling was successful, and an inflammatory mouse model was obtained.

[0062] The inflammatory mouse model was divided into 3 groups. The control group was injected with normal saline into the inflammatory mouse model through the tail vein. The exosome group was injected with purified exosomes into the inflammatory mouse model through the tail vein. The modified exosome group was injected with exosomes modified with the composite peptide segment into the inflammatory mouse model through the tail vein. The contents of inflammatory indexes IL-6 and TNF-α in the 3 groups of inflammatory mouse models were detected respectively, and the following was obtained Figure 3 .

[0063] As can be seen from Figure 3 the attachment, before treatment, the inflammatory level of the control group mice was significantly increased, indicating that the mouse lung inflammation model was successfully established. After treatment, the inflammatory levels of the modified exosome group and the control group were basically the same, while the inflammatory level of the exosome group was still at a relatively high value. The main inflammation of this experiment was in the lungs. After treatment with the modified exosomes, the inflammatory level was greatly improved, which indicated that exosomes modified with the composite peptide segment could target and enrich in the lungs of mice, while the improvement of the exosome group was not significant, indicating that the modified exosome group had lung targeting ability.

[0064] 3. In vivo fluorescence imaging detection 3.1 Fluorescent labeling The fluorescent dye DiR was mixed with the purified exosomes in Example 1 and the exosomes modified with the prepared composite peptide segments respectively, and labeled according to the usage instructions of the fluorescent dye, and incubated at room temperature for 30 - 60 min respectively. After the incubation, the unbound dye was removed by centrifugation to obtain the stained exosomes and the exosomes modified with the composite peptide segments after staining.

[0065] 3.2 In vivo fluorescence imaging of mice Select the successfully constructed inflammatory mouse model in the in vivo experiment and fast it for 10 h to reduce the interference with fluorescence. The fasted mice were divided into two groups with equal numbers. One group injected the stained exosomes into the inflammatory mouse model through the caudal vein injection, and the other group injected the exosomes modified with the composite peptide segments after staining into the inflammatory mouse model through the caudal vein injection, with 150 μL injected into each mouse.

[0066] After 20 min of injection, when the fluorescence signal reached the stable plateau phase, the mice were placed in the dark room of the in vivo imaging instrument, and the fluorescence was excited with a suitable excitation light wavelength, and the fluorescence signal was collected by a CCD camera. During the imaging process, the best imaging effect was obtained by adjusting parameters such as the lens focal length and exposure time.

[0067] 3.3 Data processing The collected fluorescence images were processed using imaging software to remove the background fluorescence and enhance the signal contrast to obtain the attached Figure 4 .

[0068] It can be seen from the attached Figure 4 that a large amount of the purified exosomes and the exosomes modified with the composite peptide segments began to aggregate after 24 h. Although imaging was shown in a large range in the mice in the pictures, the strongest and brightest points of the exosomes modified with the composite peptide segments aggregated in the thoracic cavity. The only organ in the thoracic cavity is the lung, and with the extension of time, the aggregation degree of the exosomes modified with the composite peptide segments in this part is stronger. This indicates that the exosomes modified with the composite peptide segments can significantly enhance the aggregation degree of exosomes in the lungs of mice and achieve the purpose of targeted enrichment.

[0069] The above are only the preferred embodiments of the present application and are not used to limit the present application. For those skilled in the art, various changes and modifications can be made to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included within the protection scope of the present application.

Claims

1. A composite peptide-modified exosome, characterized in that, The exosome is formed by coupling the CP05 peptide segment with the CGSPGWVRC peptide segment and then binding to specific targets on the surface of the exosome; wherein, the sequence of the CP05 peptide segment is CRHSQMTVTSRL, and the sequence of the CGSPGWVRC peptide segment is Cys-Gly-Ser-Pro-Gly-Trp-Val-Arg-Cys.

2. The exosome modified with a composite peptide segment according to claim 1, characterized in that The specific targets include membrane proteins or lipids.

3. A method for preparing exosomes modified with a composite peptide segment, characterized in that, Including: After mixing the CP05 peptide segment with the CGSPGWVRC peptide segment, a coupling agent is added, the pH value of the reaction system is adjusted to 7.4, and the reaction is carried out at room temperature for 2 h to obtain the CP05-CGSPGWVRC peptide segment; The CP05-CGSPGWVRC peptide segment is combined with specific targets on the surface of the exosome by chemical coupling or physical adsorption to obtain exosomes modified with the composite peptide segment.

4. The preparation method of the exosomes modified with the composite peptide segment according to claim 3, wherein, The coupling agent includes N-hydroxysuccinimide ester and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

5. The preparation method of the exosome modified by the composite peptide segment according to claim 4, wherein, The dosages of the N-hydroxysuccinimide ester and the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide are 5-10 mmol and 2.5-10 mmol respectively.

6. The method for preparing the composite peptide segment-modified exosomes according to claim 3, wherein, The chemical coupling method uses the biotin-streptavidin system.

7. The method for preparing the composite peptide segment-modified exosomes according to claim 3, characterized in that, The CP05 peptide segment and the CGSPGWVRC peptide segment are mixed in an equimolar ratio.

8. The method for preparing the composite peptide segment-modified exosomes according to claim 3, wherein The molar ratio of the CP05-CGSPGWVRC peptide segment to the exosome is 2-6:1-2.

9. The exosome modified with the composite peptide segment according to claim 1 or 2 is used for targeting and enriching exosomes to the lungs.

Images