Preparation method and application of acid-responsive extracellular vesicles

Abstract

The invention provides a preparation method and application of an acid-responsive extracellular vesicle. The method comprises the following steps: separating the extracellular vesicle from a biological source material through a differential centrifugation method and an ultracentrifugation method; and carrying out acid responsive modification of polyethylene glycol on the extracellular vesicles by using a Schiff base bond. The preparation method is mild in reaction conditions, does not need strong acid or strong alkali conditions, and can maintain the stability of EVs. Meanwhile, polyethylene glycol modification can prolong the in-vivo circulation time of the extracellular vesicles, and the extracellular vesicles have acid response capability and can generate response bond breaking reaction and release in a weak acid microenvironment of tumors to achieve the aim of tumor targeting.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a preparation method and application of acid-responsive extracellular vesicles. Background Art

[0002] Cancer is one of the most lethal diseases worldwide, characterized by high recurrence rates, strong drug resistance, and robust metastatic potential, posing a serious threat to human health. Currently, the main treatments for cancer include chemotherapy, radiotherapy, and surgical resection, with chemotherapy being the most commonly used. However, traditional chemotherapy drugs suffer from low drug utilization, strong tissue resistance, and insufficient drug targeting, resulting in limited therapeutic efficacy and significant side effects.

[0003] Extracellular vesicles (EVs) are small, membrane-bound vesicles secreted by cells with excellent biocompatibility and low immunogenicity. Studies have shown that EVs can serve as drug carriers, loading a variety of drugs for delivery to tumors, thereby reducing chemoresistance and enhancing drug delivery efficiency and anti-tumor efficacy. EVs are widely available, with milk-derived EVs (mEVs) being abundant and inexpensive to produce. They can be loaded with a variety of functional molecules and possess important functions such as immunomodulation, anti-inflammatory, and antimicrobial properties, making them widely used in the intervention and treatment of various diseases. mEVs possess natural cell-targeting abilities, but this also makes them more susceptible to capture by non-target organs such as macrophages, the liver, and the lungs during circulation, reducing their tumor-targeting ability. Modifying mEVs with ligands is an important approach to further enhance targeting selectivity, but the introduction of exogenous ligands can also increase the immunogenicity of mEVs and reduce their circulation time. Therefore, how to maintain the long circulation characteristics of mEVs in the body while improving their target cell binding ability is the key to achieving tumor-targeted delivery. Summary of the Invention

[0004] The present invention aims to address, at least to some extent, one of the technical problems in the above-mentioned technologies, namely, to provide a method for preparing acid-responsive extracellular vesicles and its application. The extracellular vesicles prepared by this method have good stability, acid responsiveness, and tumor targeting ability, which can effectively improve drug delivery efficiency. The preparation method has mild reaction conditions, does not require strong acid or strong base conditions, can maintain the stability of EVs, and has acid-base reversible reactions, which can respond to bond cleavage reactions and release in the weakly acidic tumor microenvironment, achieving the purpose of tumor targeting.

[0005] To this end, in a first aspect of the present invention, the present invention provides a method for preparing acid-responsive extracellular vesicles, comprising the following steps: Extracellular vesicles were isolated from biological materials by differential centrifugation and ultracentrifugation; The extracellular vesicles are acid-responsively modified by using Schiff base bonds to obtain acid-responsive extracellular vesicles.

[0006] The tumor microenvironment (TME) refers to the complex ecosystem composed of tumor cells, their surrounding non-cancerous cells (such as immune cells, fibroblasts, endothelial cells, etc.), and the extracellular matrix (ECM). The pH of the TME is typically 6.5-7.0, with a low of 6.0, indicating a weak acidity; this property provides a practical basis for the acid-responsive modification of EVs. According to the present invention, a method for preparing acid-responsive extracellular vesicles utilizes Schiff-base bonds, which have mild reaction conditions and do not require strong acid or base conditions, to modify the EVs acid-responsively. This maintains the stability of EVs while exhibiting reversible acid-base reactions, allowing them to undergo bond cleavage reactions and release in the weakly acidic tumor microenvironment, achieving tumor targeting.

[0007] Optionally, the acid-responsive modification of the extracellular vesicles using Schiff base bonds is performed by mixing benzaldehyde-polyethylene glycol complexes with the extracellular vesicles and incubating them for acid-responsive modification, and then removing excess benzaldehyde-polyethylene glycol complexes by dialysis.

[0008] Optionally, the biological source material is cow's milk.

[0009] Furthermore, the molecular weight of polyethylene glycol of the benzaldehyde-polyethylene glycol complex is 2 k-10 k, and the concentration of the benzaldehyde-polyethylene glycol complex is 1 mM-100 mM.

[0010] Furthermore, the dialysis time is 9 h-18 h, and the cutoff volume of the dialysis bag is 8 k-13 k.

[0011] Optionally, the conditions of the differential centrifugation method are as follows: centrifugation at 4°C, 2000 g for 10 min, taking the supernatant; centrifugation at 4°C, 11000 g for 30 min, taking the supernatant; centrifugation at 4°C, 10000 g for 15 min, taking the supernatant; centrifugation at 4°C, 10000 g for 10 min, taking the supernatant; centrifugation at 4°C, 10000 g for 10 min, taking the supernatant.

[0012] Optionally, the ultracentrifugation method is performed at 4° C. and 150,000 g for 1 h, and the supernatant is discarded.

[0013] In a second aspect of the present invention, provided is the use of the acid-responsive extracellular vesicles prepared by the above preparation method in tumor-targeted delivery.

[0014] Optionally, the application is the preparation of anti-tumor targeted drugs.

[0015] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a flow chart for preparing acid-responsive extracellular vesicles according to an embodiment of the present invention; Figure 2 is a BSA standard curve of acid-responsive extracellular vesicles according to an embodiment of the present invention; Figure 3 This is a SEM image of acid-responsive extracellular vesicles according to an embodiment of the present invention; Figure 4 This is a WB picture of characteristic proteins of acid-responsive extracellular vesicles according to an embodiment of the present invention; Figure 5 After the acid-responsive extracellular vesicles were modified with benzaldehyde-polyethylene glycol at different concentrations according to the embodiments of the present invention, the PEG steric hindrance effect led to a decrease in GFP coupling efficiency. The GFP coupling efficiency effect was characterized by nanoflow cytometry (nFCM) to verify the degree of modification of the acid-responsive extracellular vesicles by benzaldehyde-polyethylene glycol; Figure 6 In order to verify the pH responsiveness of the responsive extracellular vesicles by adjusting the pH value of the solution according to an embodiment of the present invention, PEG falls after the extracellular vesicles respond to pH, the steric hindrance effect of PEG disappears, and the GFP coupling efficiency is improved. The GFP coupling efficiency effect is characterized by nano-flow cytometry (nFCM) to verify the pH responsiveness of the acid-responsive extracellular vesicles; Figure 7 The modification effect of membrane dye DiD at different concentrations on acid-responsive extracellular vesicles according to an embodiment of the present invention; Figure 8 FIG1 shows the uptake of acid-responsive extracellular vesicles by tumor cells according to an embodiment of the present invention. DETAILED DESCRIPTION

[0017] The technical solution of the present invention is described below through specific examples. It should be understood that the one or more method steps mentioned in the present invention do not exclude the presence of other method steps before and after the combination step or the insertion of other method steps between these explicitly mentioned steps; it should also be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. Moreover, unless otherwise specified, the numbering of each method step is only a convenient tool for identifying each method step, and is not intended to limit the order of arrangement of each method step or to define the scope of the present invention. Changes or adjustments in their relative relationships, without substantially changing the technical content, should also be regarded as the scope of the present invention.

[0018] In order to better understand the above technical solutions, exemplary embodiments of the present invention are described in more detail below. Although exemplary embodiments of the present invention are shown, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

[0019] The test materials used in the present invention are all common commercial products and can be purchased on the market. Among them, the benzaldehyde-polyethylene glycol compound was purchased from Xi'an Ruixi Biotechnology Co., Ltd.

[0020] The present invention is described below with reference to specific examples. It should be noted that these examples are merely illustrative and do not limit the present invention in any way.

[0021] Example 1 (I) Isolation of extracellular vesicles: Step 1: Take an appropriate amount of fresh milk and centrifuge it at 4°C and 2000 g for 10 min in a high-speed refrigerated centrifuge. Take the supernatant and remove the upper fat.

[0022] Step 2: Take the supernatant from step 1 and centrifuge it at 11,000 g for 30 min at 4°C in a high-speed refrigerated centrifuge. Take the supernatant and remove milk cells and cell debris.

[0023] Step 3: Take the supernatant from step 2, add glacial acetic acid, stir, let stand for 20 minutes, then centrifuge in a high-speed refrigerated centrifuge at 4°C, 10,000 g for 15 minutes, take the supernatant, and repeat twice to remove casein.

[0024] Step 4: Take the supernatant from step 3 and filter it through a 0.45 μm filter to remove impurities.

[0025] Step 5: Take the supernatant from step 4, centrifuge it at 150,000 g for 1 h at 4°C in an ultrahigh-speed refrigerated centrifuge, and discard the supernatant; resuspend the precipitate with a phosphate buffer solution at a pH of 7.8-8 to obtain extracellular vesicles.

[0026] (II) Acid-responsive modification of extracellular vesicles: Step 1: Take benzaldehyde-polyethylene glycol complex (polyethylene glycol molecular weight of 2 k-10 k, concentration of 1 mM-100 mM) and incubate it with the extracellular vesicles obtained above at a ratio of extracellular vesicles: benzaldehyde-polyethylene glycol = 5:1, pH 7.8, room temperature, and incubation time for 12 h.

[0027] Step 2: The solution after the reaction in step 1 is dialyzed for 12 h with a molecular weight cut-off of 8 k-13 k to obtain acid-responsive modified extracellular vesicles.

[0028] (III) Acid responsiveness verification: Step 1: Modify the acid-responsive extracellular vesicles with 2 mM tris(2-carboxyethyl)phosphine (TCEP) for 1 h at room temperature, and then centrifuge at 5000 g and 4°C for 20 min using a 10K molecular weight cutoff ultrafiltration tube to remove excess TCEP.

[0029] Step 2: Modify 50 μM green fluorescent protein (GFP) with 250 μM Sulfo-SMCC, react at room temperature for 30 min, and centrifuge at 5000 g and 4°C for 20 min using a 10K molecular weight cutoff ultrafiltration tube to remove excess Sulfo-SMCC.

[0030] Step 3: Mix the modified green fluorescent protein with acid-responsive extracellular vesicles in a 1:1 ratio and incubate at room temperature for 12 h to allow the surface of the extracellular vesicles to carry fluorescence.

[0031] Step 4: Detect extracellular vesicle concentration using the BCA assay: First, mix Solution A and Solution B from the BCA protein quantification kit at a ratio of 50:1 to prepare the BCA working solution. Next, prepare bovine serum albumin (BSA) standard solutions at varying concentrations from 2 mg / mL to 0.05 mg / mL. Add 50 mL of each BSA standard solution and the test sample to 200 mL of glacial acetone and incubate at 20°C for 30 minutes. After incubation, centrifuge in a high-speed refrigerated centrifuge. After evaporation of the acetone, add 50 mL of ddH2O to the precipitate. Add 100 mL of BCA working solution to each BSA standard and test sample. Incubate at 65°C for 20 minutes. Afterwards, measure the absorbance at 562 nm using a microplate reader. Plot a standard curve comparing BSA concentration and absorbance, and calculate the sample concentration to be 12 mg / mL.

[0032] In step 5, the fluorescent acid-responsive extracellular vesicles were treated with phosphate buffers at different pH values ​​(4.8-7). The particle size, concentration, and conjugation status were measured using nanofluidics (nFCM). The control was the acid-responsive modified extracellular vesicles modified at a pH of 7.2, yielding a reaction product of benzaldehyde-polyethylene glycol and the extracellular vesicles.

[0033] Step 6, nano-flow cytometry (nFCM): Turn on the Flow Nanoanalyzer to preheat, turn on the laser after the indicator light comes on, then perform high-pressure flushing and remove bubbles, use fluorescent silicon balls to adjust the instrument light path, dilute the sample to a concentration of approximately 108 particles / mL, and then load it for detection.

[0034] The results are as follows Figure 2 、 Figure 3 、 Figure 4 As shown, the acid-responsive extracellular vesicles prepared in this example have the characteristics of uniform particle size, high particle concentration and stable structure.

[0035] The results are as follows Figure 5 As shown in Figure 2, with the increase of Benzaldehyde-PEG concentration, the acid response modification effect is enhanced; the results are shown in Figure 2. Figure 6 As shown, the acid-responsive extracellular vesicles prepared in this example have excellent acid-responsiveness.

[0036] Example 2 Application of acid-responsive extracellular vesicles in tumor cell models A tumor cell model based on hepatocellular carcinoma cells (HepG2) was constructed and cultured in complete medium (DMEM90%+10%NCBS+1% penicillin-streptomycin) at 37°C in a 5% CO2 culture environment.

[0037] The acid-responsive extracellular vesicles prepared in Example 1 were modified with membrane dye DiD, and the final concentration was 10-50 μM DiD and 1×10 12 Particles / mL acid-responsive vesicles were mixed and incubated at 37°C for 1 h. The fluorescence signal of the PC5 channel was detected using a nano flow cytometer (nFCM).

[0038] Acid-responsive vesicles stained with 50 μM DiD were mixed with HepG2 cells in a 1:1 ratio and incubated for 2 h. The fluorescence intensity of acid-responsive extracellular vesicles in HepG2 cells was detected using a 633 nm helium-neon (He-Ne) laser confocal microscope.

[0039] The results are as follows Figure 7 As shown in , the optimal membrane dye modification concentration of the acid-responsive extracellular vesicles prepared in this application is 50 μM; Figure 8 The results show that the acid-responsive extracellular vesicles prepared in this application are responsive to the acidic environment of tumor cells in the liver cancer cell model, can be effectively taken up, and release drugs inside the cells. Compared with the unmodified extracellular vesicles, the intracellular fluorescence signal is increased from 1×10 4 Increased to 5.4×10 4 , demonstrating that the tumor targeting ability of acid-responsive extracellular vesicles was significantly improved.

[0040] In summary, according to the embodiments of the present invention, the present application separates extracellular vesicles from bovine milk by differential centrifugation and ultracentrifugation; uses the Schiff base reaction to modify the benzaldehyde-polyethylene glycol complex onto the extracellular vesicles to form acid-responsive extracellular vesicles; after verification, the acid-responsive extracellular vesicles improve the tumor targeting ability of the extracellular vesicles, enhance the drug delivery efficiency, and can utilize the acidic properties of the tumor microenvironment to achieve targeted release of drugs. Compared with unmodified extracellular vesicles, the tumor targeting ability of the acid-responsive extracellular vesicles is significantly improved.

[0041] In the description of this specification, reference to the terms "one embodiment," "some embodiments," "example," "specific example," or "some examples" means that the specific features, structures, materials, or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms should not be understood as necessarily referring to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in any suitable manner in any one or more embodiments or examples. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification.

[0042] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A method for preparing acid-responsive extracellular vesicles, characterized in that: The following steps are involved: Extracellular vesicles were isolated from biological materials by differential centrifugation and ultracentrifugation; The extracellular vesicles are acid-responsively modified by using Schiff base bonds to obtain acid-responsive extracellular vesicles.

2. The preparation method according to claim 1, wherein The acid-responsive modification of the extracellular vesicles using Schiff base bonds comprises the following steps: mixing benzaldehyde-polyethylene glycol complexes with the extracellular vesicles for acid-responsive modification, and then removing excess benzaldehyde-polyethylene glycol complexes by dialysis.

3. The preparation method according to claim 1, wherein The biological source material is cow's milk.

4. The preparation method according to claim 2, wherein The molecular weight of polyethylene glycol of the benzaldehyde-polyethylene glycol complex is 2 k-10 k, and the concentration of the benzaldehyde-polyethylene glycol complex is 1 mM-100 mM.

5. The preparation method according to claim 2, wherein The dialysis time is 9 h-18 h, and the cutoff volume of the dialysis bag is 8 k-13 k.

6. The preparation method according to claim 1, wherein The conditions of the differential centrifugation method are as follows: centrifugation at 4°C, 2000 g for 10 min, taking the supernatant; centrifugation at 4°C, 11000 g for 30 min, taking the supernatant; centrifugation at 4°C, 10000 g for 15 min, taking the supernatant; centrifugation at 4°C, 10000 g for 10 min, taking the supernatant; centrifugation at 4°C, 10000 g for 10 min, taking the supernatant.

7. The preparation method according to claim 1, wherein The ultracentrifugation conditions are as follows: 4°C, 150,000 g centrifugation for 1 h, and discarding the supernatant.

8. Use of the acid-responsive extracellular vesicles prepared by the preparation method according to claims 1-7 in tumor-targeted delivery.

9. The use according to claim 8, characterized in that The application is to prepare anti-tumor targeted drugs.

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