CN115590969A

CN115590969A - Method for enhancing cell uptake efficiency of exosome and application

Info

Publication number: CN115590969A
Application number: CN202211222679.XA
Authority: CN
Inventors: 原续波; 战琦; 康春生; 赵瑾; 侯信
Original assignee: Tianjin University
Current assignee: Tianjin University
Priority date: 2022-10-08
Filing date: 2022-10-08
Publication date: 2023-01-13

Abstract

The invention provides a method for enhancing the cell uptake efficiency of exosomes and application thereof. The tumor cell uptake efficiency of exosomes can be improved by alteration of exosome membrane lipid composition. Compared with the natural exosome, the lysophospholipid modified exosome not only has enhanced cellular uptake efficiency, but also has enhanced intracellular delivery efficiency after being loaded with RNA molecules or small molecule drugs. The invention provides a method for enhancing the cellular uptake efficiency of exosomes, which has the advantages of simple process and mild reaction conditions, improves the delivery efficiency of exosomes as drug delivery carriers, and has very important significance for research and application of exosomes as nano carriers.

Description

Method for enhancing cell uptake efficiency of exosome and application

Technical Field

The invention belongs to the technical field of biomedical materials, and particularly relates to a method for enhancing the cell uptake efficiency of exosomes and application thereof.

Background

The most efficient interaction with target cells and drug delivery is a goal pursued by various types of drug carriers. Exosomes have been extensively studied to deliver therapeutic molecules such as chemotherapeutic drugs, nucleic acids, proteins, and immunomodulators as cell-derived nanoscale vesicles, some of which have achieved positive therapeutic effects in early clinical trials. As separation and purification technologies mature, exosomes are advancing from laboratories to commercialization, and Evox Therapeutics and wutian pharmaceutical limited (Takeda) reached $ 8.82 million transactions to develop their exosome drug delivery technologies, which have been considered as a new generation of therapeutic nano-platforms for drug delivery and immunotherapy. In view of clinical application safety and quantification scale, exosomes derived from non-tumor cells such as mesenchymal stem cells and macrophages are currently reported to be used more exosomes, however, the interaction between the exosomes and target cells does not achieve high-efficiency delivery efficiency, and improving tumor targeting of the exosomes and delivery efficiency in the target cells are core problems of developing novel exosome medicaments. Modifying targeting factors (such as RGD tumor targeting peptide, magnetic particles and the like) on the surface of the exosome to optimize the tumor targeting is one of the common strategies, but the strategy has complicated operation process and harsh synthesis conditions in the synthesis process, and can damage the membrane structure of the exosome, thereby limiting the application of the exosome in industrialization and clinic. Therefore, a new entry point is needed and a method which is simple to operate and mild in reaction conditions is designed for engineering exosomes to improve the target cell uptake efficiency, which is of great significance for the uptake and utilization efficiency of exosomes in target cells and promoting clinical transformation of exosomes.

Disclosure of Invention

The invention aims to provide a method for enhancing the cellular uptake efficiency of exosomes and application thereof.

The invention provides an exosome modified by lysophospholipid molecules, wherein the lysophospholipid molecules are introduced into a membrane structure of the exosome, and the membrane lipid composition of the exosome is modified. The exosome comprises an exosome of natural source and also comprises an artificially modified exosome. All exosomes may be modified by a lysophospholipid molecule by the method provided by the invention to obtain lysophospholipid molecule modified exosomes as described in the invention. Wherein the exosomes of natural origin comprise exosomes extracted and purified from natural biological samples, such as exosomes obtained by separating and purifying body fluids (urine, blood, tissue fluids, milk and the like) and cell culture fluids. The artificially modified exosome comprises an exosome subjected to post-modification on a natural exosome, for example, an artificially modified exosome in which the membrane structure of the exosome is modified or the content of the exosome is changed, and the like.

The lysophospholipid molecules are added into the exosome solution, so that the lysophospholipid molecules are automatically integrated into the exosomes, and finally, new exosomes are formed. In the process of the lysophospholipid molecules automatically integrating into exosomes, the lysophospholipid molecules do not form liposomes or other structures but directly interact with exosomes, and the lysophospholipid molecules are inserted into exosome membranes through the interaction between the fatty acid chains of the lysophospholipid molecules and the hydrophobic domains of the exosome phospholipid bilayers.

Preferably, the lysophospholipid molecule is a single-chain phospholipid capable of entering the membrane structure of an exosome.

Any one of the above is preferably that the lysophospholipid molecule is at least one of Lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylserine (LPS), lysophosphatidylglycerol (LPG), lysophosphatidylinositol (LPI).

The invention also provides a preparation method of the exosome, which comprises the following steps: step (1): obtaining exosomes; step (2): preparing lysophospholipid molecule modified exosome; and (3): separating and purifying to obtain lysophospholipid modified exosome.

Preferably, the step (1) of obtaining the exosomes comprises the steps of separating, purifying and obtaining the exosomes; or an exosome obtained by modifying the exosome obtained by separation and purification;

preferably, in any of the above steps (2), the lysophospholipid molecule is a single-chain phospholipid capable of enhancing fluidity of cell membranes.

Any of the above is preferred, in the step (2), the exosome is homogeneously mixed with lysophospholipid to obtain lysophospholipid molecule-modified exosome.

Preferably, any of the above exosomes is milk-derived, bone marrow mesenchymal stem cells, macrophage-derived, or the like.

Preferably, in any one of the above, the lysophospholipid molecule is at least one of lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylserine, lysophosphatidylglycerol and lysophosphatidylinositol.

Preferably, in the step (2), the step of preparing the lysophospholipid-modified exosome comprises: step A: preparing an exosome solution; and B: preparing a lysophospholipid solution; and C: and D, adding the lysophospholipid solution obtained in the step B into the exosome solution obtained in the step A, maintaining the reaction temperature, and stirring.

Any of the above is preferred, wherein the exosomes are resuspended in PBS solution and mixed well in step a.

Any of the above is preferably prepared by dissolving lysophospholipid in sterile double distilled water and mixing well.

Any one of the above is preferred, the protein concentration of the exosome solution obtained in step a is 0.5-2mg/mL; further preferred protein concentrations are 0.5, 0.7, 0.9, 1.1, 1.3, 1.6, 1.8, 2.0mg/mL; it is further preferred that the protein concentration of the exosome PBS solution obtained in step a is 0.5, 0.7, 0.9, 1.1, 1.3, 1.6, 1.8, 2.0mg/mL.

Any one of the above preferred lysophospholipids obtained in step B have a concentration of 1-10mM; more preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10mM, and still more preferably the concentration of the aqueous lysophospholipid solution obtained in step B is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10mM.

Preferably, in any one of the above, the ratio of exosomes to lysophospholipids is (50-200 μ g): (10-100. Mu. Mol);

exosomes: the lysophospholipid is more preferably 50 μ g: 10. Mu. Mol, 50. Mu.g: 100. Mu. Mol, 50. Mu.g: 20. Mu. Mol, 50. Mu.g: 30. Mu. Mol, 50. Mu.g: 40. Mu. Mol, 50. Mu.g: 50 μmol,50 μ g: 60. Mu. Mol, 50. Mu.g: 70. Mu. Mol, 50. Mu.g: 80. Mu. Mol, 50. Mu.g: 90 mu mol; more preferably 100. Mu.g: 10. Mu. Mol, 100. Mu.g: 100. Mu. Mol, 100. Mu.g: 20. Mu. Mol, 100. Mu.g: 30. Mu. Mol, 100. Mu.g: 40. Mu. Mol, 100. Mu.g: 50. Mu. Mol, 100. Mu.g: 60. Mu. Mol, 100. Mu.g: 70. Mu. Mol, 100. Mu.g: 80. Mu. Mol, 100. Mu.g: 90 mu mol;

more preferably 150. Mu.g: 10. Mu. Mol, 150. Mu.g: 100. Mu. Mol, 150. Mu.g: 20. Mu. Mol, 150. Mu.g: 30. Mu. Mol, 150. Mu.g: 40. Mu. Mol, 150. Mu.g: 50 μmol,150 μ g: 60. Mu. Mol, 150. Mu.g: 70 μmol,150 μ g:80 μmol,150 μ g: 90. Mu. Mol.

Preferably, in any of the above steps, in step C, the reaction temperature is-4 to 40 ℃; more preferably, -4, -1, 0, 5, 10, 15, 20, 25, 30, 35, 40 ℃.

In any of the above-mentioned steps, the stirring time in step C is preferably 0.1 to 24 hours, more preferably 0.1, 1, 5, 12, 18, or 24 hours.

Any one of the above methods is preferably that in the step (1), the method for separating, purifying and obtaining exosome is at least one of ultracentrifugation, density gradient centrifugation, ultrafiltration centrifugation, immunomagnetic bead method, PEG precipitation method and molecular exclusion method;

any one of the above is preferably that the solution for obtaining exosomes is at least one of biological fluid, cell culture supernatant, tissue supernatant; further preferably, the solution for obtaining exosomes is preferably a biological body fluid, preferably blood, urine, emulsion, etc.; the solution for obtaining the exosome is preferably cell culture supernatant, and the cell culture supernatant is preferably mesenchymal stem cells, macrophages, immune cells and the like; the solution from which the exosomes are obtained is preferably a tissue supernatant, and more preferably a brain tissue.

Any one of the above methods is preferably that the method for separating and purifying lysophospholipid-modified exosomes in step (3) is at least one of ultracentrifugation, density gradient centrifugation, ultrafiltration centrifugation, immunomagnetic bead method, PEG precipitation method and molecular exclusion method;

any one of the above preferred methods, the lysophospholipid-modified exosomes obtained in step (3) are stored in the following manner: resuspended in PBS solution and placed at 4 ℃.

Preferably, any of the above is stored under refrigeration for a period of 0.5 to 15 days.

The invention also provides a method for enhancing the cellular uptake efficiency of exosomes, which inserts lysophospholipid molecules into membrane lipid bilayers of exosomes and improves the cellular uptake efficiency of the exosomes by modifying the membrane lipid composition of the exosomes.

Preferably, the preparation method comprises the preparation method of the exosome.

The invention also provides an exosome according to any one of the above or a method for enhancing the cellular uptake efficiency of an exosome or a method for applying a method for preparing an exosome, comprising at least one of the following steps:

step w: lysophospholipid modified exosomes are directly co-incubated with target cells,

step x: lysophospholipid modified exosomes are added to animal bodies,

step y: the lysophospholipid modified exosome after being loaded with the drug is incubated with a target cell,

step z: the lysophospholipid modified exosome after being loaded with the medicine is added into an animal body. Preferably, in step w, the target cell is at least one of tumor cell, stem cell, vascular endothelial cell and immune cell;

any one of the above is preferred, in step w, the lysophospholipid is at least one of Lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylserine (LPS), lysophosphatidylglycerol (LPG) and Lysophosphatidylinositol (LPI).

Preferably in any of the above, the medium concentration of the lysophospholipid-modified exosomes in step w when co-incubated with the target cells is 10-1000 μ g/mL. More preferably, the concentration is 10, 30, 60, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000. Mu.g/mL.

Preferably, in any of the above cases, the lysophospholipid-modified exosome in step w is LPC-Exos, and the target cell is a tumor cell, and more preferably a U87 cell.

Preferably in any one of the above, the lysophospholipid-modified exosomes in step w are LPE-modified exosomes and the target cell is a macrophage.

Preferably, in any of the above, the lysophospholipid-modified exosomes in step w are LPS-modified exosomes (LPS-Exos) and the target cells are tumor cells, preferably PC9 lung cancer cells.

Preferably, in step x, the lysophospholipid-modified exosomes are added in an amount of 0.1-100mg of exosome protein per kg of body weight. Further preferably, the amount of the exosome protein per kg of body weight is 0.1, 0.3, 0.7, 1.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0, 55.0, 60.0, 65.0, 70.0, 75.0, 80.0, 85.0, 90.0, 95.0, 100.0mg.

Any one of the above is preferred, in step x, the lysophospholipid is at least one of Lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylserine (LPS), lysophosphatidylglycerol (LPG), and Lysophosphatidylinositol (LPI).

Preferably, in step y, the target cell is at least one of tumor cell, stem cell, vascular endothelial cell and immune cell;

such tumor cells include, but are not limited to, breast cancer, lung cancer, glioma cells, and the like.

Preferably, in step y, the drug is at least one of a small molecule drug, a miRNA drug, and a siRNA drug;

any one of the above is preferable, in the step y, the lysophospholipid in the step w is at least one of Lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylserine (LPS), lysophosphatidylglycerol (LPG) and Lysophosphatidylinositol (LPI).

Any of the above preferred, after loading the drug, lysophospholipid-modified exosomes are incubated with cells to a final concentration of 10-1000 μ g/mL (mass not containing the loaded drug). More preferably, the concentration is 10, 30, 60, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000. Mu.g/mL.

Preferably, the drug carried in step z is at least one of a small molecule drug, a miRNA drug, and a siRNA drug.

Any one of the above is preferably that the lysophospholipid is at least one of Lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylserine (LPS), lysophosphatidylglycerol (LPG), lysophosphatidylinositol (LPI).

Preferably, any of the above, the loaded lysophospholipid-modified exosomes are administered to an animal at a dose of 0.1-100mg exosome protein amount per kg body weight (excluding the mass of drug loaded). Further preferably, the amount of the exosome protein per kg body weight is 0.1, 0.3, 0.7, 1.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0, 55.0, 60.0, 65.0, 70.0, 75.0, 80.0, 85.0, 90.0, 95.0, 100.0mg.

The micromolecule is a medicine with the molecular weight less than 1000D, and particularly is a chemically synthesized targeted medicine which comprises chemotherapeutic medicines (such as adriamycin, paclitaxel, gefitinib and the like), anti-inflammatory medicines (such as curcumin), metformin and the like.

Preferably, in step y or step z, the ratio of the drug to the lysophospholipid-modified exosomes added during drug loading is 20-80 μ g:100-400 mug.

In any of the above cases, the amount of the drug added in the drug loading process is preferably 20 to 80 μ g, and more preferably 20, 30, 40, 50, 60, 70, or 80 μ g.

Any of the above is preferably added in an amount of 100-400 μ g, more preferably 100, 150, 200, 250, 300, 350, 400 μ g, of the lysophospholipid-modified exosomes during drug loading.

Preferably, the weight ratio of the drug added in the drug loading process to the lysophospholipid-modified exosome is 1-8: 10, more preferably 1: 5. 1: 10. 4: 5. 2: 5. 3: 10. 1: 2. 3:5.

preferably in any of the above, the drug is doxorubicin.

In any of the above cases, the amount of doxorubicin added during drug loading is preferably 20 to 80 μ g, more preferably 20, 30, 40, 50, 60, 70, 80 μ g.

In any of the above, the amount of the lysophospholipid-modified exosomes added during the drug loading is preferably 100 to 400 μ g, and more preferably 100, 150, 200, 250, 300, 350, or 400 μ g.

Preferably, the weight ratio of the adriamycin added in the drug loading process to the lysophospholipid-modified exosome is 1-8: 10, more preferably 1: 5. 1: 10. 4: 5. 2: 5. 3: 10. 1: 2. 3:5.

any of the above preferred methods preferably have an effect concentration of doxorubicin of 0.2-4.0 μ g/mL, preferably 0.5-2.0 μ g/mL, preferably 0.2, 0.5, 0.8, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 μ g/mL during co-incubation of the drug-loaded lysophospholipid-modified exosomes with target cells.

Any of the above preferred, drug-loaded lysophospholipid-modified exosomes are added to animal subjects at a concentration of 0.1-10mg of Dox per kg body weight. Further preferably, the Dox per kg body weight is 0.1, 0.3, 0.5, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10mg.

The preferred technical scheme for preparing the lysophospholipid molecule modified exosome comprises the following steps:

a. separation and extraction of exosomes

Obtaining a certain amount of solution containing exosomes, such as cell culture supernatant, body fluid, etc., removing cells, cell debris, apoptotic bodies, microvesicles, etc., from the solution to purify the exosomes.

According to the scheme, the method for separating, purifying and obtaining the exosome can be as follows: ultracentrifugation, density gradient centrifugation, ultrafiltration centrifugation, immunomagnetic bead method, PEG precipitation, and size exclusion;

according to the above scheme, the solution for obtaining exosomes may be: biological fluid (such as blood, urine, lotion, etc.), cell culture supernatant (such as mesenchymal stem cells, macrophages, immune cells, etc.), tissue supernatant (such as brain tissue);

b. preparation of lysophospholipid-modified exosomes

1. Dissolving a certain amount of lysophospholipid molecules in double distilled water, and ultrasonically heating for 5-10min; the heating temperature is preferably 25 to 37 ℃ and more preferably 37 ℃.

2. Adding lysophospholipid molecular solution into exosome solution, and uniformly stirring the mixed solution, wherein the preferable stirring mode is that the mixed solution is placed in a rotary reactor for reaction;

3. separating and purifying the mixed solution to obtain lysophospholipid modified exosome, and refrigerating and storing.

According to the above scheme, the lysophospholipid molecule in step (b) may be: at least one of Lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylserine (LPS), lysophosphatidylglycerol (LPG) or Lysophosphatidylinositol (LPI), which may be a single compound or a mixture;

according to the above scheme, the concentration of the lysophospholipid molecule solution in the step (b) is 1-10mM;

according to the above scheme, the concentration of the exosome solution in step (b) is 0.5-2mg/mL;

according to the above scheme, the exosomes in step (b): the proportion of lysophospholipids is (50-200. Mu.g): (10-100. Mu. Mol);

according to the scheme, the reaction time in the step (b) is 0.1-24h, and the reaction temperature is-4-40 ℃;

according to the above scheme, the method for separating and extracting lysophospholipid-modified exosomes in step (b) may be: ultracentrifugation, density gradient centrifugation, ultrafiltration centrifugation, immunomagnetic bead method, PEG precipitation, and size exclusion;

c. cellular uptake and transfection of lysophospholipid-modified exosomes

1. Carrying out fluorescence labeling or cargo loading on the lysophospholipid modified exosome obtained by purification;

2. co-incubating the labeled or loaded exosomes with target cells;

3. the cellular uptake efficiency or transfection efficiency was measured.

According to the scheme, the lysophospholipid modified exosome in the step (c) can be loaded with water-soluble or fat-soluble small-fraction drugs, miRNA drugs, siRNA drugs and the like;

according to the above scheme, the target cells in step (c) can be tumor cells (such as breast cancer, lung cancer, glioma cells, etc.), stem cells, vascular endothelial cells, immune cells, etc.

The beneficial effect of the invention is that the cell uptake efficiency of the exosome is improved by modifying the membrane lipid composition of the exosome. The lysophospholipid molecule is used for modifying the membrane lipid composition of the exosome, the method has simple process and mild reaction condition, improves the uptake and delivery efficiency of the exosome in target cells, and has important significance for the research and application of directly using the exosome as a therapeutic agent or as a delivery carrier of the therapeutic agent.

Taking example 1 as an example, lysophosphatidylcholine (LPC) modified exosomes were prepared. As can be seen from fig. 1 and fig. 2, the particle size of LPC-modified exosomes was not significantly changed compared to unmodified exosomes, which indicates that the nano-vesicle structure of exosomes was not significantly changed by LPC modification of lysophospholipid molecules. As can be seen from fig. 3-5, the tumor cell intracellular fluorescence intensity of LPC-modified exosomes can be increased nearly three-fold compared to unmodified exosomes, indicating that LPC modification can significantly increase tumor cell uptake efficiency of exosomes. As can be seen from fig. 6 and 7, after the exosomes are loaded with the chemotherapeutic drug doxorubicin, the intracellular doxorubicin fluorescence intensity of the LPC-modified exosome group is enhanced and the drug delivery efficiency is significantly improved compared with the unmodified exosome group, and as can be seen from fig. 8, the LPC modification enhances the tumor cell proliferation inhibition effect of the drug-loaded exosome. By combining the above results, it can be shown that the lysophospholipid molecule modified exosome does not change the membrane structure and physicochemical properties of the exosome, completely retains the characteristics of the exosome, and can improve the target cell uptake efficiency of the exosome, thereby enhancing the delivery efficiency of the exosome and the action effect in the target cell after loading the therapeutic agent molecule.

Drawings

FIG. 1 is a particle size distribution diagram of exosomes (Exos) of preferred embodiment 1 of the present invention.

FIG. 2 is a particle size distribution diagram of LPC modified exosomes (LPC-Exos) according to a preferred embodiment of the present invention.

FIG. 3 is a cytofluorescence map of tumor cell uptake of exosomes (Exos) and LPC-modified exosomes (LPC-Exos) in preferred embodiment 1 of the present invention.

FIG. 4 is a statistic of intracellular fluorescence intensity taken up by tumor cells of exosomes (Exos) and LPC-modified exosomes (LPC-Exos) according to a preferred embodiment of the present invention 1.

FIG. 5 is a comparison of the tumor cell uptake fluorescence intensity of the exosomes (Exos) and LPC-modified exosomes (LPC-Exos) of the preferred embodiment 1 of the present invention.

FIG. 6 is a cell fluorescence diagram of tumor cell uptake of doxorubicin-loaded exosomes (Exos-Dox) and LPC-modified exosomes (LPC-Exos-Dox) in preferred embodiment 1 of the present invention.

FIG. 7 comparison of fluorescence intensity of tumor cell uptake of doxorubicin-loaded exosomes (Exos-Dox) and LPC-modified exosomes (LPC-Exos-Dox) in preferred embodiment 1 of the present invention.

FIG. 8 is a diagram showing the inhibition of tumor cell proliferation by the doxorubicin-loaded exosomes (Exos-Dox) and LPC-modified exosomes (LPC-Exos-Dox) according to the preferred embodiment 1 of the present invention.

Reference numbers in the drawings:

1 is the nucleus of tumor cells co-incubated with exosomes

2 fluorescence of exosomes taken up in the cytoplasm of exosome co-incubated tumor cells

Superposition of pictures (merge) 3 with 1 and 2

4 is the nucleus of tumor cells co-incubated with LPC modified exosomes

5 fluorescence of LPC-modified exosomes taken up in the cytoplasm of exosome-co-incubated tumor cells

6 superposition of pictures 4 and 5 (merge)

Detailed Description

The present invention will be more clearly and completely described in the following embodiments, which are however only a part of the embodiments of the present invention and are not all the embodiments described. The examples are provided to aid understanding of the present invention and should not be construed to limit the scope of the present invention.

Example 1

a. Separation and extraction of exosomes

Diluting 50mL of serum of a healthy animal in 200mLPBS, centrifuging the diluted serum solution for 10min by 300g, centrifuging for 20min by 2000g and centrifuging for 40min by 10000g, taking the supernatant, then placing the supernatant in an ultracentrifuge, centrifuging for 70min by 100000g, then discarding the supernatant, carrying out heavy suspension precipitation by PBS, centrifuging the resuspended solution for 70min by 100000g again, discarding the supernatant, wherein the precipitate is exosome from the serum source, re-suspending the exosome by 1mLPBS, and storing the solution at-80 ℃. The exosomes prepared in example 1 were characterized by dynamic light scattering instrument with particle size of 100 ± 24nm.

b. Preparation of lysophospholipid-modified exosomes

The protein amount and the particle amount of the exosome are quantified by using a BCA kit pair and a nanoparticle tracer technology (NTA). Lysophosphatidylcholine (LPC) powder was dissolved in sterile double distilled water and subjected to water bath sonication at 37 ℃ for 30min to prepare a 10mM LPC solution. mu.L of the exosome solution (1 mg/mL) and 5. Mu.L of the LPC solution (10 mM) were mixed in 100. Mu.L of PBS, and the mixed solution was reacted at 37 ℃ for 30min. Diluting the reacted mixed solution in 40mLPBS, placing the solution in an ultracentrifuge, centrifuging 100000g for 70min, then discarding supernatant, resuspending and precipitating with PBS, wherein the precipitate is LPC modified exosome, resuspending the exosome with 100 mu LPBS, and storing the solution at 4 ℃. The LPC modified exosomes prepared in example 1 were characterized by dynamic light scattering measurement and the particle size was 106 + -33 nm.

The results of the particle size distribution of exosomes (Exos) and LPC-modified exosomes (LPC-Exos) are shown in fig. 1 and fig. 2.

Tumor cell uptake of LPC modified exosomes

Exosomes (Exos) and LPC-modified exosomes (LPC-Exos) were fluorescently labeled with FITC to study their cellular uptake behavior. The U87 glioma cells were treated with 10 ⁵ Density per well was seeded on glass coverslips of 12-well plates and incubated for 24 hours. Next, cells were incubated with FITC-labeled Exos or LPC-Exos for 4h. Exos or LPC-Exo in co-incubationThe action concentration of s is 100. Mu.g/mL. Cells were then washed with PBS, fixed with 4% paraformaldehyde, DAPI stained, and mounted. Cell internalization of Exos and LPC-Exos in U87 cells was observed using confocal laser scanning microscopy. For quantitative analysis of cellular uptake, U87 cells were seeded into 12-well plates. After overnight incubation, the medium was replaced with fresh medium containing FITC-labeled Exos or LPC-Exos. After 4h incubation, cells were washed 3 times with PBS, detached with trypsin solution, and then dispersed in PBS for flow cytometry measurement. The U87 cell uptake efficiency of the LPC-modified exosomes prepared in example 1 was determined to be 2.9 times that of exosomes using flow cytometry.

The results of tumor cell uptake of exosomes (Exos) and LPC-modified exosomes (LPC-Exos) are shown in fig. 3-5.

d. Adriamycin loaded LPC modified exosomes for tumor cell uptake

Doxorubicin was loaded on the exosomes and detected using the same method as c.

FIGS. 6 and 7 show the results of tumor cell uptake of doxorubicin-loaded exosomes (Exos-Dox) and LPC-modified exosomes (LPC-Exos-Dox). The ability of tumor cells to take up doxorubicin through LPC-modified exosomes was significantly enhanced. Tumor cell uptake of doxorubicin was 3.5-fold greater than unmodified after doxorubicin delivery via LPC-modified exosomes.

FIG. 8 shows the results of tumor cell proliferation inhibition by doxorubicin-loaded exosomes (Exos-Dox) and LPC-modified exosomes (LPC-Exos-Dox).

Wherein, the addition amount of the exosome (or LPC modified exosome) in the drug loading process is 200 mug, and the addition amount of the adriamycin is 80 mug. In the process of co-incubation of the LPC modified exosome carrying the adriamycin and the tumor cell, the action concentration of the adriamycin is shown in figure 8, the inhibition effect of 0.5-2.0 mu g/mL on the proliferation of the tumor cell is obviously better than that of the unmodified exosome, and the effect is best when the action concentration of the adriamycin is 1.0 mu g/mL.

e. In vivo tumor treatment effect of doxorubicin-loaded LPC-modified exosomes

30 female BALB/c nude mice of 4-6 weeks were injected subcutaneously on their dorsal side with U87 cells (2X 10) ⁵ Cell/cell-Only) to construct tumor-bearing mice. The tumor volume is about 100mm ³ At the time, the mice were randomly divided into 3 groups of 10 mice each, and the groups were: PBS, exos-Dox, LPC-Exos-Dox. Each sample was injected intravenously every three days for 18 days at a dose of 5mg Dox per kg body weight. Changes in mouse tumor volume and mouse body weight were recorded. At the end of the experiment, mice were euthanized to obtain tumors, and the final mass of the tumors was determined. The results show that the volume and weight of the LPC-Exos-Dox group and the tumor are obviously reduced compared with the PBS and the Exos-Dox group. The Exos-Dox group had a therapeutic effect compared to the PBS group, and the therapeutic effect was significantly higher in the LPC-Exos-Dox group than in the Exos-Dox group.

The methods for loading adriamycin by exosomes, detecting uptake of tumor cells and detecting proliferation inhibition of tumor cells are the prior art in the field, and the methods for loading drugs by exosomes, detecting uptake of tumor cells and detecting inhibition of tumor cells in the prior art are all suitable for the invention.

Example 2

Separation and extraction of exosomes

Collecting 200mL of human mesenchymal stem cell supernatant, centrifuging for 10min at 300g, centrifuging for 20min at 2000g and centrifuging for 20min at 10000g in sequence for 40min, taking the supernatant, then placing the supernatant in an ultracentrifuge, centrifuging for 70min at 100000g, then discarding the supernatant, carrying out heavy suspension precipitation by PBS, centrifuging the resuspended solution for 70min at 100000g again, discarding the supernatant, obtaining the precipitate as the exosome from the mesenchymal stem cell source, carrying out heavy suspension by 1mL PBS, and preserving the solution at-80 ℃. The exosomes prepared in example 2 were characterized by dynamic light scattering and particle size was 93 ± 17nm.

Preparation of lysophospholipid-modified exosomes

The protein amount and the particle amount of the exosome were quantified by BCA kit pair and nanoparticle tracer technique (NTA). Lysophosphatidylethanolamine (LPE) powder was dissolved in sterile double distilled water, and subjected to water bath sonication at 37 ℃ for 30min to prepare a 10mM LPE solution. mu.L of the exosome solution (1 mg/mL) and 4. Mu.L of the LPE solution (10 mM) were mixed in 100. Mu.L of PBS, and the mixed solution was reacted at 37 ℃ for 30min. Diluting the reacted mixed solution in 40mLPBS, placing in an ultracentrifuge, centrifuging at 100000g for 70min, discarding the supernatant, resuspending the precipitate with PBS to obtain LPE modified exosome, resuspending the exosome with 100 mu LPBS, and storing the solution at 4 ℃. The LPE modified exosomes prepared in example 2 were characterized by dynamic light scattering instrument with particle size 95 ± 23nm.

Macrophage uptake of LPE-modified exosomes

Exosomes (Exos) and LPE-modified exosomes (LPE-Exos) were fluorescently labeled with FITC to study their cellular uptake behavior. RAW246.7 macrophage cell line 10 ⁵ Density per well was seeded on glass coverslips of 12-well plates and incubated for 24 hours. Next, cells were incubated with FITC-labeled Exos or LPE-Exos for 4h. The effect concentration of Exos or LPC-Exos in the co-incubation was 100. Mu.g/mL. Cells were then washed with PBS, fixed with 4% paraformaldehyde, DAPI stained, and mounted. Cellular internalization of Exos and LPE-Exos in RAW246.7 cells was observed using confocal laser scanning microscopy. For quantitative analysis of cellular uptake, RAW246.7 cells were seeded into 12-well plates. After overnight incubation, the medium was replaced with fresh medium containing FITC-labeled Exos or LPE-Exos. After 4h incubation, cells were washed 3 times with PBS, detached with trypsin solution, and then dispersed in PBS for flow cytometry. The RAW246.7 cell uptake efficiency of the LPE-modified exosome prepared in example 2 was determined to be 2.5 times that of the exosome using flow cytometry.

Macrophage inflammatory phenotype modulation of LPE-modified exosomes

RAW264.7 cells in 6-well plates at 10 per well ⁵ Individual cells were seeded at density and RAW264.7 cells were pretreated with lipopolysaccharide at a concentration of 100ng/mL for 24h for an inflammatory environment. RAW264.7 cells were treated with PBS, exosomes (Exos), LPE-modified exosomes (LPE-Exos) or iL-4 for 24h, with iL-4 at a concentration of 20ng/mL as a positive control, and detected by flow cytometry macrophage inflammatory phenotype. The cells were treated with 10 ⁶ The density of each tube was suspended in 100. Mu.L of PBS buffer and incubated with PE fluorescently labeled CCR7 antibody (marker for M1-type macrophages) and APC fluorescently labeled CD206 antibody (marker for M2-type macrophages), respectively, at 37 ℃ for 1h in the absence of light. Then, in the thin1mL of PBS was added to the cell suspension, and the cell suspension was centrifuged at 3000r/min for 10min, and then the supernatant was discarded and resuspended in 300. Mu.L of PBS. Samples were analyzed by flow cytometry and the results were processed using FlowJo software. The CCR7 positive rate under the effect of LPE-modified exosomes was reduced compared to the lipopolysaccharide-stimulated control group and was significantly lower than that of the unmodified exosome group; the CD206 positive rate was increased by the LPE modified exosomes and was significantly higher than the unmodified exosome group, indicating that the effect of macrophage conversion to M2 phenotype was significantly higher by the LPE modified exosomes than the unmodified exosome group. In order to detect the secretion of the macrophage phenotype-associated cytokine, enzyme-linked immunosorbent assay (ELISA) kit is used for detection. The cell culture step was performed as described above, and after 24h of stimulation, the supernatant was collected and centrifuged at 3000r/min to remove cell debris. The levels of TNF-. Alpha.and iL-10 cytokines in the supernatants were measured on a microplate reader using an ELISA kit according to the instructions. The results show that under the action of LPE modified exosomes, the expression of TNF-alpha (cytokine associated with M1 phenotype) is reduced, and is obviously lower than that of an unmodified exosome group; meanwhile, under the action of LPE modified exosomes, the expression of iL-10 (an M2 phenotype-associated cytokine) is increased and is obviously higher than that of an unmodified exosome group.

Macrophage phenotypic regulation of LPE-modified exosomes in vivo under bone microenvironment

Using a rat tibial defect model: 24 SD rats were evenly divided into a random group, a control group, an exosome (Exos) group and an LPE-modified exosome (LPE-Exos) group. SD rats were anesthetized and the surgical area was shaved and disinfected. An incision was then made to expose the tibia and a round cortical and medullary defect (3 mm diameter) was formed using a dental drill with constant irrigation with sterile saline. After suturing, 1mg of Exos or LPE-Exos was injected into the defect, and the untreated group served as a control group. Euthanasia was performed on days 1 and 3 after surgery. The tibia is obtained and fixed in 4% paraformaldehyde, a tissue section is prepared, the section is subjected to membrane rupture and closure in sequence, and then is incubated with iNOS (marker of M1 type macrophage) and CD206 antibody overnight and then is incubated with secondary antibody for 1h. After washing the mounting, the sections were imaged by confocal laser scanning microscopy to analyze macrophage phenotype. The result shows that under the action of LPE modified exosome, iNOS expression is reduced and is obviously lower than that of an unmodified exosome group; under the action of LPE modified exosomes, CD206 expression is remarkably increased and is remarkably higher than that of an unmodified exosome group. Under the action of LPE modified exosome, macrophage is transformed to M2 phenotype, and the technical effects of anti-inflammation and tissue repair are more obvious.

Example 3

a. Separation and extraction of exosomes

Collecting 200mL of macrophage supernatant, centrifuging for 10min at 300g, centrifuging for 20min at 2000g and centrifuging for 20min at 10000g in sequence for 40min at 10000g, taking the supernatant, then placing the supernatant in an ultracentrifuge, centrifuging for 70min at 100000g, then discarding the supernatant, carrying out heavy suspension precipitation by PBS, centrifuging the resuspended solution for 70min at 100000g again, discarding the supernatant, wherein the precipitate is the exosome derived from the macrophages, carrying out heavy suspension by 1mL PBS, and storing the solution at-80 ℃. The exosomes prepared in example 1 were characterized by dynamic light scattering and particle size was 97 ± 23nm.

b. Preparation of lysophospholipid-modified exosomes

The protein amount and the particle amount of the exosome were quantified by BCA kit pair and nanoparticle tracer technique (NTA). Lysophosphatidylserine (LPS) powder was dissolved in sterile double distilled water, and subjected to ultrasonic treatment in a water bath at 37 ℃ for 30min to prepare a 10mM LPS solution. mu.L of the exosome solution (1 mg/mL) and 7. Mu.L of the LPS solution (10 mM) were mixed in 100. Mu.L of PBS, and the mixed solution was reacted at 37 ℃ for 30min. Diluting the reacted mixed solution in 40mLPBS, placing the solution in an ultracentrifuge, centrifuging 100000g for 70min, then discarding the supernatant, carrying out resuspension precipitation by PBS (phosphate buffer solution), namely obtaining the LPS modified exosome, carrying out resuspension on the exosome by 100 mu LPBS, and storing the solution at 4 ℃. Characterization was performed by measuring the LPS-modified exosomes prepared in example 1 using a dynamic light scattering instrument, and the particle size was 96. + -. 33nm.

Tumor cell uptake of LPS-modified exosomes

Exosomes (Exos) and LPS-modified exosomes (LPS-Exos) were fluorescently labeled with FITC to study their cellular uptake behavior. Mixing PC9 lung cancer cell with 10 ⁵ Density per well was seeded on glass coverslips of 12-well plates and incubated for 24 hours. Next, the cells were incubated with FITC-labeled Exos or LPS-Exos for 4h. The effect concentration of Exos or LPC-Exos in the co-incubation was 100. Mu.g/mL. Cells were then washed with PBS, fixed with 4% paraformaldehyde, DAPI stained, and mounted. Cell internalization of Exos and LPS-Exos in PC9 cells was observed using confocal laser scanning microscopy. For quantitative analysis of cellular uptake, PC9 cells were seeded into 12-well plates. After overnight incubation, the medium was replaced with fresh medium containing FITC-labeled Exos or LPS-Exos. After 4h incubation, cells were washed 3 times with PBS, detached with trypsin solution, and then dispersed in PBS for flow cytometry. The efficiency of PC9 cell uptake of the LPS-modified exosomes prepared in example 3 was determined to be 2.5 times higher than that of exosomes using flow cytometry.

Similar to example 1, after the drug (e.g., doxorubicin) is delivered by LPS-modified exosomes, the uptake capacity of the drug by the cells is significantly increased compared to unmodified exosomes.

Claims

1. An exosome modified by a lysophospholipid molecule, wherein the lysophospholipid molecule is introduced into the membrane structure of the exosome.

2. Exosome according to claim 1, characterized in that the lysophospholipid molecule is a single chain phospholipid.

3. Exosome according to claim 2, characterized in that the lysophospholipid molecule is at least one of lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylserine, lysophosphatidylglycerol, lysophosphatidylinositol.

4. A method for producing an exosome according to any one of claims 1 to 3, comprising the steps of: step (1): obtaining exosomes; step (2): preparing lysophospholipid molecule modified exosome; and (3): separating and purifying to obtain lysophospholipid modified exosome.

5. The method of claim 4, wherein in the step (2), the step of preparing lysophospholipid-modified exosomes comprises: step A: suspending the exosome obtained in the step (1) to obtain an exosome solution; and B: preparing a lysophospholipid solution; and C: and D, adding the lysophospholipid solution obtained in the step B into the exosome solution obtained in the step A, maintaining the reaction temperature, and stirring.

6. The method according to claim 5, wherein the protein concentration of the exosome solution obtained in step a is 0.5-2mg/mL; the concentration of the lysophospholipid obtained in the step B is 1-10mM; exosomes: the proportion of lysophospholipids is (50-200. Mu.g): (10-100. Mu. Mol); the reaction temperature is-4-40 ℃.

7. The method according to claim 4, wherein in the step (1), the exosome is obtained by separation and purification, and the method for separating and purifying the exosome is at least one of ultracentrifugation, density gradient centrifugation, ultrafiltration centrifugation, immunomagnetic bead method, PEG precipitation method and molecular exclusion method; the solution for obtaining the exosome is at least one of biological body fluid, cell culture supernatant and tissue supernatant; the method for separating and purifying to obtain the lysophospholipid modified exosome in the step (3) is at least one of an ultracentrifugation method, a density gradient centrifugation method, an ultrafiltration centrifugation method, an immunomagnetic bead method, a PEG precipitation method and a molecular exclusion method; the storage mode of the lysophospholipid modified exosome obtained in the step (3) is as follows: resuspended in PBS solution and stored at 4 ℃.

8. A method of enhancing the cellular uptake efficiency of exosomes, comprising the method of any one of claims 4 to 7, wherein lysophospholipid molecules are introduced into the membrane structure of exosomes, and their cellular uptake efficiency is enhanced by engineering the membrane lipid composition of exosomes.

9. The method of using the method of enhancing cellular uptake efficiency of exosomes of claim 8, comprising at least one step of:

step x: lysophospholipid modified exosomes are added to animal bodies,

step y: the lysophospholipid modified exosome after carrying the medicine is incubated with a target cell,

step z: the lysophospholipid modified exosome after being loaded with the medicine is added into an animal body.

10. The method of claim 9, wherein in step w, the target cell is at least one of a tumor cell, a stem cell, a vascular endothelial cell, and an immune cell; in the step y, the target cell is at least one of tumor cell, stem cell, vascular endothelial cell and immune cell; in the step y, the medicine is at least one of micromolecular medicine, miRNA medicine and siRNA medicine; and the drug loaded in the step z is at least one of micromolecular drug, miRNA drug and siRNA drug.