Chitosan surface modified extracellular vesicle as well as preparation method and application thereof

Abstract

The invention discloses a preparation method of chitosan surface modified extracellular vesicles, which specifically comprises the following steps: S1, dissolving chitosan in water, adjusting the pH value to be acidic, and completely dissolving the chitosan to obtain a chitosan solution with the concentration of 10mg / mL; s2, adding N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride into the solution obtained in the step S1, and uniformly stirring and fully mixing the N-hydroxysuccinimide and the 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride; s3, the distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 is dissolved in formamide, and a distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 solution with the concentration being 1 mg / mL is prepared; s4, uniformly mixing the solution obtained in the step S2 and the solution obtained in the step S3, and stirring at room temperature for 12 hours; s5, transferring the mixed solution obtained in the step S4 into a dialysis bag with the specification of 3.5 KDa, and dialyzing in pure water for 24 hours; and S6, co-incubating the dialyzed solution obtained in the step S5 with an extracellular vesicle solution, and self-assembling to form the extracellular vesicle with the chitosan surface modified. The chitosan surface modified extracellular vesicles prepared by the method disclosed by the invention have relatively high encapsulation efficiency on hydrophobic drugs such as fucoxanthin and the like and relatively high stability in various environments. The method is suitable for embedding and delivering various bioactive substances, and has an important application prospect in the aspect of active substance delivery.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a chitosan-modified extracellular vesicle, its preparation method, and its application. Background Technology

[0002] Extracellular vesicles (EVs) are nanoscale membrane structures actively secreted by cells and are widely found in bacteria, plants, and mammals. Their natural phospholipid bilayer structure endows them with excellent biocompatibility, low immunogenicity, and the ability to cross biological barriers, thus showing great potential in drug delivery, disease diagnosis, and regenerative medicine. However, natural EVs still face significant challenges as delivery carriers: on the one hand, their limited surface functional groups make targeted delivery or efficient drug loading difficult; on the other hand, EVs exhibit low encapsulation efficiency for hydrophobic active substances (such as fucoxanthin and some drugs) and are easily degraded by enzymes or cleared by the immune system in complex physiological environments, leading to insufficient stability. These problems severely limit the practical application of EVs in the biomedical field.

[0003] Currently, functional modification of EVs mainly relies on techniques such as chemical cross-linking, genetic engineering, or physical extrusion. For example, chemical cross-linking involves introducing cross-linking agents such as maleimide or carbodiimide to covalently link functional molecules (e.g., antibodies, ligands) to the EV surface. However, such methods often involve toxic reagents, which may disrupt the integrity of the EV membrane structure and generate uncontrollable byproducts, affecting their biosafety. Genetic engineering involves modifying progenitor cells to secrete EVs carrying specific membrane proteins, but this method is complex, time-consuming, and costly, and may interfere with the natural function of EVs due to overexpression of exogenous proteins. Physical extrusion fuses EVs with modifying molecules using mechanical force, which can retain some of the EV's activity, but suffers from low modification efficiency and large batch-to-batch variability. Therefore, there is an urgent need to develop a mild, efficient, and reproducible EV surface modification strategy that can enhance the drug loading capacity and stability of EVs while avoiding damage to their structure and function.

[0004] In recent years, self-assembly technology based on amphiphilic molecules has provided new ideas for the modification of EVs. Phospholipid derivatives (such as DSPE-PEG) are widely used for EV surface functionalization due to their ability to spontaneously insert into the phospholipid bilayer of EVs through their hydrophobic tails. The introduction of polyethylene glycol (PEG) chains can form a hydrophilic protective layer, reducing immune recognition and prolonging the in vivo circulation time of EVs. However, single PEGylation modification can only improve stability and cannot significantly improve the drug loading efficiency or targeting ability of EVs. Therefore, researchers have begun to explore combining PEG with other functional molecules (such as polysaccharides and targeting peptides) to construct composite modification systems. Chitosan (CS), as a natural cationic polysaccharide, has excellent biocompatibility, degradability, and mucosal adhesion. Its positive surface charge can enhance the interaction with negatively charged biological membranes (such as intestinal epithelium and tumor cells), while improving the encapsulation ability of lipid-soluble drugs through hydrophobic interactions and hydrogen bonds. However, CS has a large molecular weight and strong hydrophilicity. Direct binding with EVs may lead to membrane structure damage or aggregation and precipitation. How to achieve stable anchoring of CS on the surface of EVs has become a technical challenge. Summary of the Invention

[0005] To address the aforementioned issues, this invention proposes an innovative solution: by combining CS with amphiphilic phospholipid-polyethylene glycol (DSPE-PEG), a DSPE-PEG-CS modification system is constructed. The hydrophobic end of DSPE spontaneously inserts into the EV membrane structure, achieving directional and mild modification of CS. This strategy cleverly combines the self-assembly characteristics of DSPE-PEG with the functional advantages of CS, avoiding the toxicity risks of traditional chemical crosslinking while overcoming the compatibility issues of CS modification alone. Specifically, DSPE-PEG acts as a "molecular bridge," with one end embedded in the EV membrane through hydrophobic interactions, and the other end providing functional groups through covalently coupled CS, thereby forming a stable CS-modified layer on the EV surface.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] The first objective of this invention is to provide a method for preparing chitosan-modified extracellular vesicles, comprising the following steps:

[0008] S1. Dissolve chitosan (hereinafter referred to as CS) in water, adjust the pH to acidic, and completely dissolve the chitosan to obtain a chitosan solution with a concentration of 10 mg / mL;

[0009] S2. Add N-hydroxysuccinimide (hereinafter referred to as NHS) with a concentration of 60 mmol / L and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (hereinafter referred to as EDC) with a concentration of 80 mmol / L to the solution obtained in step S1, and stir until fully mixed;

[0010] S3. Distearate phosphatidylethanolamine-polyethylene glycol 2000 (hereinafter referred to as DSPE-PEG2000) is dissolved in formamide to prepare a 1 mg / mL solution of distearate phosphatidylethanolamine-polyethylene glycol 2000.

[0011] Steps S2 and S3 have no specific order.

[0012] S4. Mix the solutions obtained in steps S2 and S3 thoroughly and stir at room temperature;

[0013] S5. Transfer the mixture obtained in step S4 to a 3.5 kDa dialysis bag and dialyze with pure water;

[0014] S6. The dialysis solution obtained in step S5 (i.e., DSPE-PEG-CS) is co-incubated with the extracellular vesicle (hereinafter referred to as EVs) solution to form chitosan-modified extracellular vesicles (hereinafter referred to as CS-EVs) through self-assembly.

[0015] Preferably, the extracellular vesicles include one or more of Lactobacillus plantarum and mammalian cells.

[0016] Preferably, step S6 specifically involves: taking the same volume of extracellular vesicle solution and mixing it evenly with the dialysis solution obtained in step S5, and shaking it at 37°C to obtain chitosan-modified extracellular vesicles.

[0017] A second objective of this invention is to provide extracellular vesicles with chitosan surface modification prepared by the above method.

[0018] A third objective of this invention is to provide the application of the above-mentioned chitosan-modified extracellular vesicles in the preparation of extracellular vesicle drug delivery carriers.

[0019] The beneficial effects of this invention are as follows:

[0020] 1. High efficiency and mildness: DSPE-PEG-CS inserts into the phospholipid structure of EVs through self-assembly, without the need for chemical cross-linking agents or high-pressure treatment. The reaction conditions are mild, with no harmful byproducts, while maintaining the structural integrity and bioactivity of EVs.

[0021] 2. Functional enhancement: The introduction of CS enhances the positive charge on the surface of EVs, improves the adhesion to negatively charged biomembranes (such as intestinal epithelium), and increases the encapsulation efficiency of hydrophobic drugs (such as FX).

[0022] 3. Stability: The CS-EV prepared by this invention has the advantages of high encapsulation rate and long-term stability under different external environmental factors such as temperature and ultraviolet light.

[0023] 4. Versatility: Applicable to EVs and active substances from a variety of sources. Attached Figure Description

[0024] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0025] Figure 1 This is a particle size distribution diagram of EV and CS-EV in Example 1 of the present invention.

[0026] Figure 2 The image shows the zeta potential diagrams of the EV and CS-EV in Embodiment 1 of the present invention.

[0027] Figure 3 The infrared spectra of the EV and CS-EV in Embodiment 1 of the present invention are shown.

[0028] Figure 4 The embedding rate of FX by EV and CS-EV in Embodiment 2 of the present invention.

[0029] Figure 5 This is to assess the stability of EV-FX and CS-EV-FX in Embodiment 2 of the present invention. Detailed Implementation

[0030] To better understand the present invention, the present invention will be further described in detail below with reference to the embodiments and accompanying drawings. However, those skilled in the art will understand that the following embodiments are not intended to limit the scope of protection of the present invention, and any changes and variations made on the basis of the present invention are within the scope of protection of the present invention.

[0031] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0032] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0033] Example 1: Preparation of CS-EV

[0034] (1) Preparation of EVs: bacterial suspension was centrifuged to collect bacterial cells, and the cells were treated with lysozyme for 24 h. Protoplasts were then centrifuged to precipitate the cells. The protoplasts were washed twice with PBS buffer. The resulting suspension was sonicated for 30 min. The supernatant was collected by centrifugation. The supernatant was centrifuged at 20000×g for 30 min. The resulting supernatant was centrifuged at 150000×g for 1 h to obtain membrane fragments. These fragments were resuspended in PBS and sonicated on ice for 30 min to form membrane vesicles (EVs).

[0035] (2) Synthesis of DSPE-PEG-CS complex:

[0036] S1. Dissolve chitosan (CS) in distilled water, adjust the pH to acidic, and ensure that the chitosan is completely dissolved to obtain a chitosan solution with a concentration of 10 mg / mL;

[0037] S2. Add N-hydroxysuccinimide (NHS) (60 mmol / L) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) (80 mmol / L) to the solution obtained in step S1, and stir until fully mixed;

[0038] S3. Distearate phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) in formamide to prepare a 1 mg / mL solution of distearate phosphatidylethanolamine-polyethylene glycol 2000.

[0039] S4. Mix the solutions obtained in step S2 and step S3 thoroughly and stir at room temperature for 12 hours;

[0040] S5. Transfer the mixture obtained in step S4 to a dialysis bag with a specification of 3.5 kDa and dialyze in pure water for 24 hours;

[0041] S6. The DSPE-PEG-CS obtained in step S5 was co-incubated with natural EVs at 37°C for 24 hours. The hydrophobic end of DSPE was spontaneously inserted into the phospholipid bilayer of EVs to achieve stable anchoring of CS on the surface of EVs, resulting in chitosan-modified extracellular vesicles (hereinafter referred to as CS-EVs).

[0042] Example 2: Encapsulation of FX by CS-EV

[0043] (1) Prepare a 1 mg / mL fucoxanthin (FX) ethanol solution;

[0044] (2) Mix the FX solution with the vesicles (EV / CS-EV) at a volume ratio of 1:10 until homogeneous;

[0045] (3) Sonicate the above solution in an ice bath (200W, 2min, turn on for 10s and turn off for 15s), and incubate in ice for 2min after sonication. Repeat the above steps 3 times.

[0046] (4) The product obtained in step (3) was placed in ice and incubated for 1 hour to restore the stability of the vesicles;

[0047] (5) A small amount of ethanol was removed by rotary evaporation at 37℃ to obtain the FX-embedded sample groups EV-FX and CS-EV-FX.

[0048] Identification of Experimental Example 1EV and CS-EV

[0049] The particle size distribution, zeta potential, and Fourier transform infrared spectra of the EV and CS-EV prepared in Example 1 were detected using a nanoparticle size analyzer and a Fourier transform infrared spectrometer.

[0050] The particle size potential detection procedure is as follows: 800 μL of EV and CS-EV are placed in a nanoparticle size analyzer for detection. Figure 1 and Figure 2 The results showed that the average particle size of EV was 112.1 nm and the zeta potential was -15.56 mV; the average particle size of CS-EV was 386.8 nm and the zeta potential was 2.14 mV.

[0051] The Fourier transform infrared spectroscopy detection procedure is as follows: Take a small amount of sample and dry potassium bromide, mix and grind it into a film, place it in the instrument for measurement, and the results are as follows. Figure 3 As shown. Figure 3 This indicates that the infrared spectra of EV and CS-EV are significantly different.

[0052] Experimental Example 2: Encapsulation efficiency and stability testing of FX by EV and CS-EV

[0053] A mixed solution of n-hexane and ethanol (2:1, V:V) was prepared. The prepared EV-FX and CS-EV-FX were sonicated for 30 min to release free FX. The above organic solution was then vortexed with the sample for 10 min to collect the n-hexane layer. The UV absorbance was measured at 450 nm. The results are as follows: Figure 4 As shown.

[0054]

[0055] According to the above formula, as Figure 4 As shown, the encapsulation rate of fucoxanthin in chitosan-modified extracellular vesicles (80%) was significantly higher than that in the natural vesicle group (53.96%).

[0056] EV-FX and CS-EV-FX were placed in an 80℃ water bath, and 1 mL was collected every 30 min; EV-FX and CS-EV-FX were placed under UV irradiation, and 1 mL was collected every 15 min; EV-FX and CS-EV-FX were placed in an acidic solution at pH 2, and 1 mL was collected every 10 min; EV-FX and CS-EV-FX were placed in an oxidant solution, and 1 mL was collected every 10 min; EV-FX and CS-EV-FX were placed in simulated gastric fluid (containing 3.2 mg / mL pepsin, 0.7% HCl, and 2 mg / mL bile salts), and 1 mL was collected every 30 min; EV-FX and CS-EV-FX were placed in simulated intestinal fluid (containing 150 mM NaCl, 10 mM CaCl2, 1.6 mg / mL trypsin, and 5 mg / mL bile salts), and 1 mL was collected every 1 h; the retention rate of FX was measured using the above experimental steps. The initial encapsulation rate was taken as 100%, and subsequent data were defined as the initial percentage. The results are as follows: Figure 5 As shown.

[0057] from Figure 5 The study found that the retention rate of free FX decreased the fastest, while the FX encapsulated in CS-modified EVs showed the highest stability, with the FX retention rate decreasing slowly and ultimately higher than that of natural EVs. This indicates that CS-EVs exhibit increased stability against temperature, UV, acid, oxidants, simulated gastric juice, and simulated intestinal juice after CS modification.

[0058] In summary, traditional EV modification techniques (such as chemical crosslinking and genetic engineering) rely on toxic reagents or complex operations, easily leading to structural damage or functional loss of EVs. This invention utilizes the amphiphilic properties of DSPE-PEG to directionally anchor CS onto the EV membrane surface. The reaction conditions are mild, requiring no chemical crosslinking agents or high-pressure treatment, and no byproducts are generated, maximizing the preservation of the natural bioactivity and structural integrity of EVs. Experiments show that the particle size potential of the modified CS-EV changes significantly, increasing the encapsulation efficiency of fucoxanthin (FX) to 80%, significantly better than unmodified EVs. Furthermore, the CS-EV of this invention exhibits higher stability in FX encapsulation under external environmental factors such as temperature and UV exposure. With its high encapsulation efficiency, long-term stability, and functional customizability, CS-EV shows broad application prospects: In the future, with further analysis of the EV drug delivery mechanism and iterative optimization of the modification system, this invention is expected to spawn various applications in precision medicine, functional foods, and smart materials, demonstrating significant value.

[0059] While specific embodiments of the present invention have been described above, those skilled in the art should understand that the specific embodiments described are merely illustrative and not intended to limit the scope of the present invention. Equivalent modifications and variations made by those skilled in the art in accordance with the spirit of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing chitosan-modified extracellular vesicles, characterized in that, Includes the following steps: S1. Dissolve chitosan in water, adjust the pH to acidic, and ensure that the chitosan is completely dissolved to obtain a chitosan solution with a concentration of 10 mg / mL; S2. Add 60 mmol / L N-hydroxysuccinimide and 80 mmol / L 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to the solution obtained in step S1, and stir until fully mixed. S3. Distearate distearate phosphatidylethanolamine-polyethylene glycol 2000 in formamide to prepare a distearate phosphatidylethanolamine-polyethylene glycol 2000 solution with a concentration of 1 mg / mL; S4. Mix the solutions obtained in steps S2 and S3 thoroughly and stir at room temperature; S5. Transfer the mixture obtained in step S4 to a 3.5 kDa dialysis bag and dialyze with pure water; S6. The dialysis solution obtained in step S5 is co-incubated with the extracellular vesicle solution to form chitosan-modified extracellular vesicles through self-assembly.

2. The chitosan-modified extracellular vesicles according to claim 1, characterized in that, The extracellular vesicles are one or more of Lactobacillus plantarum and mammalian cells.

3. The chitosan-modified extracellular vesicles according to claim 1, characterized in that, Step S6 specifically involves mixing the same volume of extracellular vesicle solution with the dialysis solution obtained in step S5, and shaking the mixture at 37°C to obtain chitosan-modified extracellular vesicles.

4. Chitosan-modified extracellular vesicles prepared by the method according to any one of claims 1-3.

5. The application of chitosan-modified extracellular vesicles according to claim 4 in the preparation of extracellular vesicle drug delivery carriers.

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