Method for preparing extracellular membrane vesicles by using high-voltage pulsed electric field

Abstract

The invention discloses a method for preparing extracellular membrane vesicles by using a high-voltage pulsed electric field. The method comprises the following steps: preparing a living tumor tissue specimen into a single-cell suspension; treating the single-cell suspension by using a unipolar high-voltage pulsed electric field to induce bubbling of an extracellular membrane; stripping extracellular membrane vesicles from the foamed cells by using a bipolar high-voltage pulse electric field; sorting the extracellular membrane vesicles by using specific magnetic beads; eluting the specific magnetic beads to obtain specific extracellular membrane vesicles; and carrying out gradient centrifugal concentration to obtain the extracellular membrane vesicles in a predetermined particle size range. The method has the advantages that the obtained extracellular membrane vesicles are formed by stripping an extracellular membrane under the action of a high-voltage pulse electric field; the extracellular membrane vesicles carrying predetermined antigen molecules or immune signal molecules can be sorted; the extracellular membrane vesicles with preset particle sizes can be enriched and separated. The invention can be used for developing a specific anti-tumor or immunomodulatory preparation so as to be used for precise treatment of predetermined diseases.

Description

Technical Field

[0001] The present application relates to the field of medical technology, and in particular to a method for preparing extracellular membrane vesicles using a high-voltage pulse electric field. Background Art

[0002] The cell outer membrane (plasmalemma, also known as the cytoplasmic membrane or cell membrane) wraps the protoplasm in the cell and maintains the relative stability of the intracellular environment. Some oligosaccharides or polysaccharides are covalently bound to membrane lipids or proteins on the non-cytoplasmic side of the cell outer membrane to form glycolipids and glycoproteins, forming a cell coat on the outer surface of the cell outer membrane. Under physiological conditions, the oligosaccharides or polysaccharides in the outer coat of human cells usually carry a negative charge, which can prevent the cells from being adhered to the outer coat of endothelial cells that also carry a negative charge in the circulatory system, thereby preventing them from being cleared by endothelial cells or adsorbed in large quantities on the surface of endothelial cells in the ducts to block the duct system.

[0003] The monosaccharide sequence of the specifically ordered oligosaccharides or polysaccharides on the cell outer membrane can become an antigenic determinant recognized by other cells or proteins, and can also combine with transmitters or hormones to achieve specific functions. The cluster of differentiation on the cell outer membrane surface is a glycoprotein or protein that appears on the cell membrane surface at different stages. It can not only be used as a surface marker for cell identification and separation, but also widely participates in cell growth, maturation, differentiation, development, migration, and activation. Antigenic determinants or differentiation clusters participate in the formation of antigen molecules or immune signaling molecules on the cell outer membrane.

[0004] The cell's biological membrane system includes the extracellular membrane and the endomembrane system. The endomembrane system includes the envelope membranes of organelles such as the endoplasmic reticulum, Golgi apparatus, lysosomes, endosomes, secretory vesicles and exosomes. There is no extracellular membrane on the surface of the endomembrane system.

[0005] In previous studies, cell clumps or tissue samples were often mechanically ground or cells were subjected to hypotonic, ultrasonic or repeated freeze-thaw treatment to obtain cell biomembrane fragments. Cell membrane-derived vesicles were prepared from the cell biomembrane fragments and used as carriers to deliver small molecules or nucleic acid sequences for the treatment of diseases such as tumors.

[0006] Currently, there is no report on the method of preparing cell membrane outer membrane vesicles. Summary of the invention

[0007] In order to overcome the above shortcomings, the present invention provides a method for preparing extracellular membrane vesicles using a high-voltage pulse electric field.

[0008] According to an embodiment of the present application, a method for preparing extracellular membrane vesicles using a high-voltage pulsed electric field is provided, comprising: Prepare live tumor tissue specimens into single-cell suspensions; Treating the single cell suspension with a unipolar high voltage pulse electric field to induce cell outer membrane blebbing; Apply bipolar high-voltage pulse electric field to the vesicled cells to remove the extracellular membrane vesicles; Use specific magnetic beads to sort out extracellular membrane vesicles labeled with specific proteins; Elute specific magnetic beads to obtain specific extracellular membrane vesicles; Extracellular membrane vesicles with a predetermined particle size range are obtained by gradient centrifugation.

[0009] Optionally, the living tumor tissue specimen is a surgical resection specimen, a biopsy specimen or a tissue fluid specimen.

[0010] Optionally, the type of the living tumor tissue specimen is lung cancer, liver cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, nervous system cancer, cervical cancer, nasopharyngeal cancer, lymphoma, thyroid cancer or prostate cancer.

[0011] Optionally, the unipolar high-voltage pulse electric field is a positive polarity pulse electric field, a negative polarity pulse electric field or a differential pulse electric field.

[0012] Optionally, the intensity of the unipolar high-voltage pulse electric field is 1-30 kV / cm, the pulse electric field width is 1 nanosecond to 500 microseconds, the pulse frequency is 1 Hz to 10 kHz, and the number of pulses is 1-2000 times.

[0013] Optionally, the pulse electric field of the unipolar high-voltage pulse electric field is a nanosecond pulse, a microsecond pulse, or a nanosecond-microsecond combined pulse.

[0014] Optionally, the positive and negative polarities of the bipolar high-voltage pulse electric field appear alternately, and the time interval between the positive and negative polarities is 1 microsecond to 1 millisecond.

[0015] Optionally, the peak-to-peak value of the pulse voltage of the bipolar high-voltage pulse electric field is 100 V to 10 kV, the pulse electric field width is 1 microsecond to 100 microseconds, the pulse frequency is 1 Hz to 10 kHz, and the number of pulses is 1-2000 times.

[0016] In the present application, there are two steps of high-voltage pulse treatment. The first pulse treatment is to use a unipolar high-voltage pulse electric field treatment to make the charged groups outside the cell membrane move in one direction, causing bubbling on the cell membrane surface; the second pulse electric field treatment is a bipolar pulse electric field treatment to make the charged groups outside the cell membrane move back and forth, and the vesicles formed in the first step are peeled off (pulled) from the cell membrane surface.

[0017] Optionally, the specific markers on the specific magnetic beads are anti-human phosphatidylinositol protein glypican 3 (Homo sapiens Glypican-3, hGPC-3), anti-human carcinoembryonic antigen (Homo sapiens carcinoembryonic antigen, hCEA), anti-human programmed death receptor-1 (Homo sapiens programmed death receptor-1, hPD-1), anti-programmed death ligand-1 (Homo sapiens programmed death ligand-1, hPD-L1), and anti-cytotoxic T lymphocyte associated protein (Homo sapiens cytotoxic T lymphocyte associated antigen-4, CTLA-4). These specific markers can specifically bind to the corresponding proteoglycans or proteins on the surface of the vesicles, and the vesicles with specific molecules are enriched and separated by magnetic bead sorting technology.

[0018] The technical solution provided by the embodiments of the present application may have the following beneficial effects: As can be seen from the above examples, the extracellular membrane vesicles prepared from living tumor tissue cells by this method are consistent with the personalized immune matching of the patient, and are used to develop specific anti-tumor or immunomodulatory preparations, which can avoid non-specific clearance by immune cells. The electric field force of the charge carried on the surface of the cell outer membrane is exerted by a unipolar high-voltage pulsed electric field, and the cell outer membrane is caused to bleb under the action of the electric field force; and the bipolar pulsed electric field is further used to generate a reciprocating force on the microbubbles formed on the cell outer membrane, and these microbubbles are peeled off to form extracellular membrane vesicles. Its advantages are: 1) By utilizing the characteristic of high-voltage pulsed electric field to form electric field force on the net charge of the cell coat, there is a repulsive force between the prepared extracellular membrane vesicles and the endothelial cell coat carrying the same charge, which can avoid non-specific adsorption of extracellular membrane vesicles to endothelial cells, and then be cleared and absorbed by endothelial cells or even cause blockage of the ducts; 2) Extracellular membrane vesicles carrying specific antigen molecules or immune signaling molecules can specifically bind to the above-mentioned molecular ligands or antibodies on target cells, thereby regulating the function of target cells; 3) Nanoscale extracellular membrane vesicles can enter the tissue through the gaps between endothelial cells, and the specific antigen molecules or immune signaling molecules they carry transmit the regulatory signals to the target cells in the target tissue, thereby developing new methods for precise treatment of specific diseases on this basis.

[0019] It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and, together with the description, serve to explain the principles of the present application.

[0021] Figure 1 The present invention is a flow chart showing the preparation of extracellular membrane vesicles using a high-voltage pulsed electric field according to an exemplary embodiment.

[0022] Figure 2 is a schematic diagram of a unipolar high-voltage pulse voltage waveform for inducing cell membrane blistering according to an exemplary embodiment.

[0023] Figure 3 is a schematic diagram of a bipolar high-voltage pulse voltage waveform for stripping vesicles according to an exemplary embodiment.

[0024] Figure 4 It is a schematic diagram showing the method of obtaining vesicles from a single cell suspension by treating it with a unipolar and bipolar high voltage pulse electric field according to an exemplary embodiment.

[0025] Figure 5 2 is a scanning electron microscope image of a hepatocellular carcinoma cell according to an exemplary embodiment, wherein (a) is a scanning electron microscope image of a hepatocellular carcinoma cell before being treated with a unipolar high-voltage pulse electric field; and (b) is a scanning electron microscope image of a hepatocellular carcinoma cell after being treated with a unipolar high-voltage pulse electric field.

[0026] Figure 6 The figures are 3D scanning reconstruction images of hepatocellular carcinoma cells using a laser confocal microscope according to an exemplary embodiment, wherein (a) is a 3D scanning reconstruction image of hepatocellular carcinoma cells using a laser confocal microscope; (b) is a 3D scanning reconstruction image of hepatocellular carcinoma cells in which vesicles are induced by a unipolar high-voltage pulse electric field; and (c) is a 3D scanning reconstruction image of hepatocellular carcinoma cells in which vesicles are stripped by a bipolar high-voltage pulse electric field.

[0027] Figure 7 is a graph showing the particle size distribution of extracellular membrane vesicles obtained after centrifugation at 100,000 g according to an exemplary embodiment.

[0028] Figure 8 FIG. 1 is a diagram showing extracellular membrane vesicles carrying signaling molecules separated by gradient centrifugation according to an exemplary embodiment. DETAILED DESCRIPTION

[0029] In order to describe the present invention more specifically, the technical solution of the present invention is described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0030] Figure 1 FIG. 1 is a flow chart showing a method for preparing extracellular membrane vesicles using a high voltage pulse electric field according to an exemplary embodiment. Figure 1 As shown, the method may include the following steps: S1: Prepare living tumor tissue specimens into single cell suspensions; Specifically, the specimen tissues of surgically removed lung cancer, liver cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, nervous system cancer, cervical cancer, nasopharyngeal cancer, lymphoma, thyroid cancer, and prostate cancer specimens are mechanically decomposed into small tissue blocks, and then digested with working fluids such as hyaluronidase, collagenase, protease, and DNase to obtain small tissue blocks, and then filtered and washed with a cell mesh to obtain a single cell suspension. The extracellular membrane vesicles prepared from living tumor tissue cells are consistent with the patient's personalized immune matching and are used to develop specific anti-tumor or immunomodulatory preparations to avoid nonspecific clearance by immune cells.

[0031] S2: treating the single cell suspension with a unipolar high voltage pulse electric field to induce cell outer membrane blebbing; Specifically, a device for treating a single cell suspension with a unipolar high voltage pulse electric field to induce cell outer membrane blebbing is as follows: Figure 4 As shown, the unipolar high voltage pulse voltage waveform is as follows Figure 2 Taking hepatocellular carcinoma cells as an example, the scanning electron microscope image of cells induced by unipolar high voltage pulse electric field treatment is shown in Figure 5 As shown, Figure 5 (a) shows two hepatocellular carcinoma cells; Figure 5 (b) shows a hepatocellular carcinoma cell, where the white arrow indicates a bleb on the cell outer membrane surface. The cell coat outside the outer membrane of living cells carries a large amount of net negative charge under physiological conditions, and the electric field force exerted on these negative charges by a unipolar high-voltage pulse electric field can cause blebs on the cell outer membrane.

[0032] S3: Apply bipolar high voltage pulse electric field to the vesicled cell membrane to remove the extracellular membrane vesicles; Specifically, the bipolar high voltage pulse voltage waveform is as follows Figure 3 As shown, bipolar high voltage pulse electric field strips the cell extracellular membrane vesicles. Figure 6 As shown, the three-dimensional reconstruction of the hepatocellular carcinoma cell image was obtained by scanning layer by layer using a laser confocal microscope. Figure 6 (a) shows a hepatocellular carcinoma cell that has not been treated with an electric field, as indicated by the arrow in the figure; Figure 6 (b) shows a hepatocellular carcinoma cell treated with a unipolar high-voltage pulse electric field, and each arrow indicates a bleb on the cell membrane caused by the unipolar high-voltage pulse electric field. Figure 6(c) in the figure shows a hepatocellular carcinoma cell that has been treated with a unipolar high-voltage pulse electric field and then stripped of the cell outer membrane using a bipolar high-voltage pulse electric field to form an extracellular membrane vesicle. Each arrow indicates one of the stripped extracellular membrane vesicles. After the microbubbles are formed on the cell outer membrane, the net negative charge carried on the cell outer membrane on its surface is subjected to the action of the bipolar high-voltage pulse electric field with alternating positive and negative polarities, and the microbubbles are stripped from the cell outer membrane by the reciprocating electric field force. After centrifugation at 2000g to remove live cells and cell debris, an extracellular membrane vesicle suspension is obtained. By utilizing the characteristics of the high-voltage pulse electric field that generates electric field force on the charge carried by the cell outer membrane, the prepared extracellular membrane vesicles have charges of the same charge repulsion with the surface of endothelial cells that also have a cell outer membrane, avoiding nonspecific adsorption of extracellular membrane vesicles and endothelial cells, which in turn causes the extracellular membrane vesicles to block the endothelial cell lumen or be cleared and absorbed by endothelial cells.

[0033] S4: Use specific magnetic beads to sort extracellular membrane vesicles labeled with specific proteins; Specifically, the extracellular membrane vesicle suspension prepared from the hepatocellular carcinoma cell suspension, taking anti-PD-L1 magnetic bead labeling as an example, adds PD-L1 magnetic beads to the suspension, so that it binds to the extracellular membrane vesicles carrying PD-L1, and then uses magnetic bead separation technology to obtain the suspension of extracellular membrane vesicles carrying PD-L1 labeled with magnetic beads. The stripped extracellular membrane vesicles are only a part of the cell membrane, containing only part or no specific molecules on the extracellular membrane, such as antigen determinant molecules or differentiation cluster molecules, such as programmed death ligand-1 (PD-L1) molecules. The extracellular membrane vesicle suspension obtained by separating S3 with magnetic beads coupled to anti-PD-L1 antibodies can separate the extracellular membrane vesicles carrying PD-L1 molecules from the extracellular membrane vesicles not carrying PD-L1 molecules.

[0034] S5: Elute the specific magnetic beads to obtain specific extracellular membrane vesicles.

[0035] Specifically, the extracellular membrane vesicles carrying PD-L1 labeled with the above magnetic beads are eluted with a magnetic bead dissociation agent, and a suspension of extracellular membrane vesicles carrying PD-L1 is obtained after removing the magnetic beads bound to the extracellular membrane vesicles. S4 obtains that the PD-L1 molecules on the extracellular membrane vesicles are bound to the magnetic beads coupled to anti-PD-L1, and the magnetic beads coupled to PD-L1 are removed with a magnetic bead elution solution to obtain a suspension of extracellular membrane vesicles carrying PD-L1 molecules with the labeled magnetic beads removed. Extracellular membrane vesicles carrying predetermined antigen molecules or immune signaling molecules can specifically bind to the above-mentioned molecular ligands or antibodies on the target cells, thereby regulating the function of the target cells.

[0036] S6: Extracellular membrane vesicles of a predetermined size range are obtained by gradient centrifugation and concentration.

[0037] Specifically, the particle size distribution of extracellular membrane vesicles obtained after gradient centrifugation at 100,000 g is as follows: Figure 7 As shown in Figure 1, centrifugation at 100,000 g yielded a suspension of extracellular membrane vesicles enriched and concentrated with an average particle size of about 100 nm. The extracellular membrane vesicles carrying signal molecules were immunogold labeled and observed under a transmission electron microscope. Figure 8 As shown, Figure 8 The white arrow in the middle indicates the PD-L1 molecule detected on the surface of one of the extracellular membrane vesicles. S5 obtained an extracellular membrane vesicle solution. After gradient centrifugation at different centrifugal forces such as 10,000g, 30,000g and 100,000g, the precipitate can be enriched with a suspension of extracellular membrane vesicles in a predetermined particle size range. Nanoscale (1-1000nm) extracellular membrane vesicles, especially 100nm extracellular membrane vesicles, can enter the tissue through the endothelial cell gap, thereby achieving precise treatment of predetermined diseases.

[0038] It can be seen from the above technical scheme that the present application utilizes living tumor tissue to obtain a large number of single-cell suspensions of individual-specific living cells; the cell coat outside the outer membrane of living cells carries a large amount of net negative charge under physiological conditions, and the electric field force exerted on these negative charges by a unipolar high-voltage pulse electric field can cause blistering of the cell outer membrane; after blistering on the surface of the cell outer membrane to form microbubbles, the net negative charge carried on the cell coat is peeled off from the cell outer membrane by the reciprocating electric field force under the action of a bipolar high-voltage pulse electric field with alternating positive and negative polarities; the peeled-off cell outer membrane vesicles are only a part of the cell membrane, carrying part (or no) of specific macromolecular substances on the cell outer membrane, including antigenic determinant molecules or differentiation cluster molecules, such as Homo sapiens Glypican-3 (hGPC-3), human carcinoembryonic antigen (hCEA), and human programmed death receptor-1 (Homo sapiens programmed death Receptor-1, hPD-1), programmed death ligand-1 (Homo sapiensprogrammed death ligand-1, hPD-L1), cytotoxic T lymphocyte associated antigen-4 (Homo sapienscytotoxic T lymphocyte associated antigen-4, CTLA-4), etc. Magnetic beads coupled to antibodies of specific macromolecules can be used to separate extracellular membrane vesicles carrying related molecules or not. Extracellular membrane vesicles carrying specific macromolecules can be obtained after elution and removal of magnetic beads to obtain molecularly specific extracellular membrane vesicles; gradient centrifugation can be used to obtain concentrated extracellular membrane vesicles of a predetermined particle size. This forms a new method for preparing extracellular vesicles with the potential for precise treatment of diseases.

[0039] The extracellular membrane vesicles prepared from living tumor tissue cells are consistent with the patient's personalized immune matching and are used to develop specific anti-tumor or immunomodulatory preparations, which can avoid non-specific clearance by immune cells. High-voltage pulsed electric fields are used to act on the cell coat to carry electric charges, generating electric field forces to prepare extracellular membrane vesicles, which are of the same charge as the extracellular coat of endothelial cells, thus avoiding non-specific electrostatic adsorption of extracellular membrane vesicles by the extracellular coat of endothelial cells, and then being cleared and absorbed by endothelial cells, and even causing blockage of endothelial ducts. Extracellular membrane vesicles carrying specific antigen molecules or immune signaling molecules can specifically bind to the above-mentioned molecular ligands or antibodies on target cells, thereby regulating the function of target cells. Nanoscale (1-1000nm) extracellular membrane vesicles, especially 100nm extracellular membrane vesicles, can enter the tissue through the gaps between endothelial cells, thereby binding to ligands or antibodies on specific cells in the target tissue, thereby achieving precise treatment of related diseases.

[0040] Those skilled in the art will readily appreciate other embodiments of the present application after considering the description and practicing the contents disclosed herein. The present application is intended to cover any modification, use or adaptation of the present application, which follows the general principles of the present application and includes common knowledge or customary techniques in the art that are not disclosed in the present application. The description and examples are intended to be exemplary only, and the true scope and spirit of the present application are indicated by the claims.

[0041] It should be understood that the present application is not limited to the precise structures that have been described above and shown in the drawings, and that various modifications and changes may be made without departing from the scope thereof. The scope of the present application is limited only by the appended claims.

Claims

1. A method for preparing extracellular membrane vesicles using a high voltage pulse electric field, comprising: Prepare live tumor tissue specimens into single-cell suspensions; Treating the single cell suspension with a unipolar high voltage pulse electric field to induce cell outer membrane blebbing; Apply bipolar high-voltage pulse electric field to the vesicled cells to remove the extracellular membrane vesicles; Use specific magnetic beads to sort out extracellular membrane vesicles labeled with specific proteins; Elute the magnetic beads to obtain specific extracellular membrane vesicles; Extracellular membrane vesicles with a predetermined particle size range are obtained by gradient centrifugation.

2. The method according to claim 1, characterized in that The living tumor tissue specimen is a surgical resection specimen, a biopsy specimen or a tissue fluid specimen.

3. The method according to claim 1, characterized in that The type of the living tumor tissue specimen is lung cancer, liver cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, nervous system cancer, cervical cancer, nasopharyngeal cancer, lymphoma, thyroid cancer or prostate cancer.

4. The method according to claim 1, characterized in that The unipolar high-voltage pulse electric field is a positive polarity pulse electric field, a negative polarity pulse electric field or a differential pulse electric field.

5. The method according to claim 1, characterized in that The intensity of the unipolar high-voltage pulse electric field is 1-30 kV / cm, the pulse electric field width is 1 nanosecond to 500 microseconds, the pulse frequency is 1 Hz to 10 kHz, and the pulse number is 1-2000 times.

6. The method according to claim 1, characterized in that The pulse electric field of the unipolar high-voltage pulse electric field is a nanosecond pulse, a microsecond pulse or a nanosecond-microsecond combined pulse.

7. The method according to claim 1, characterized in that The positive and negative polarities of the bipolar high-voltage pulse electric field appear alternately, and the time interval between the positive polarity and the negative polarity is 1 microsecond to 1 millisecond.

8. The method according to claim 1, characterized in that The peak-to-peak value of the pulse voltage of the bipolar high-voltage pulse electric field is 100 V to 10 kV, the pulse electric field width is 1 microsecond to 100 microseconds, the pulse frequency is 1 Hz to 10 kHz, and the number of pulses is 1-2000 times.

9. The method according to claim 1, characterized in that: The specific markers on the specific magnetic beads are anti-human phosphatidylinositol proteoglycan 3 (Homo sapiens Glypican-3, hGPC-3), anti-human carcinoembryonic antigen (Homo sapiens carcinoembryonic antigen, hCEA), anti-human programmed death receptor-1 (Homo sapiens programmed death receptor-1, hPD-1), anti-programmed death ligand-1 (Homo sapiens programmeddeath ligand-1, hPD-L1), and anti-cytotoxic T lymphocyte associated antigen-4 (Homo sapiens cytotoxic Tlymphocyte associated antigen-4, CTLA-4).

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