CN1322115C - Ectobody loaded with exogenous ligand molecule and its preparation method and application - Google Patents

Abstract

The present invention discloses an exosome loaded with an exogenous ligand molecule, and the preparation method and the application of the exosome. The exosome loded with an exogenous ligand molecule is composed of an exogenous ligand molecule and an exosome, wherein the exogenous ligand molecule joints at the surface of the membrane of the exosome or is wrapped in the cavity of the exosome, or exists at both places. The exogenous ligand molecule is albumen or a molecule which are synthesized by a nontarget cell, or the exogenous ligand molecule can directly act on a target cell or can be absorbed by the target cell via endocytosis. The exosome loaded with an exogenous ligand molecule of the present invention can transfer the exogenous ligand molecule with biological activity (exogenous protein, exogenous toxin or exogenous medicine molecules) between isogenous cells or allogenous cells in a specificity mode; an active molecule is led to the target cell. The exosome loaded with an exogenous ligand molecule of the present invention can be used for regulating the activity of the isogenous target cells or the allogenous target cells, and can be used for preparing medicines (such as a medicine for treating tumors).

Description

Extracellular body loaded with exogenous ligand molecule and its preparation method and application

technical field

The invention relates to an extracellular body loaded with exogenous ligand molecules, a preparation method and application thereof.

Background technique

Many animal cells, including a variety of tumor cells, can secrete a small membrane vesicle of endocytic origin called "exosome". It is composed of phospholipid bimolecular membrane and its wrapped substances, with a diameter of 30-100nm and a buoyant density of about 1.1-1.2g/ml, carrying some characteristic molecules, such as transferrinreceptor, flotillin, tsg101, rab5, etc. (all or part of it). The extracellular body is produced by the fusion of the multivesicular body (MVB) in the cell and the plasma membrane of the cell. The specific formation process is shown in Figure 1: 1. Cells absorb extracellular substances through endocytosis to form endocytic vesicles (endocytic vesicle, EV). 2. Endocytic vesicles further fuse into early endosomes (early endosomes, EE). 3. During the maturation process of early endosomes, the restrictive membranes of the endosomes are inwardly depressed and bud out to form small membrane vesicles in the lumen; the organelles with multilayer membrane structures formed at this time are called multivesicular bodies (MVB). 4. The mature MVB fuses with the plasma membrane (plasma membrane, PM) and releases the membrane vesicles in the lumen into the extracellular environment to form exosomes.

Exosomes carry specific membrane-integrated or membrane-bound proteins from the plasma membrane or endosomes, as well as some soluble proteins from the cytoplasm. Exosomes secreted into the cell matrix can be passed between adjacent cells and selectively bind to surrounding homologous or heterologous cells, thereby mediating the exchange of membrane components and cytoplasmic components between cells, or activating Specific signaling responses (Stoorvogel, W., Kleijmeer, M.J., Geuze, H.J., and Raposo, G. (2002). The biogenesis and functions of exosomes. Traffic 3, 321-330; Thery, C., Zitvogel, L., and Amigorena, S. (2002). Exosomes: composition, biogenesis and function. Nat Rev Immunol 2, 569-579).

Trichosanthin (TCS) is a toxin protein isolated from the root tuber of Trichosanthus chinensis, a traditional Chinese medicine plant. It has RNA N-glycosidase activity and belongs to type I ribosome-inactivating proteins (RIPs). After binding to target cells, TCS can invade the cytoplasm, inactivate large ribosomal subunits, block protein biosynthesis, and lead to the death of target cells. TCS mainly acts on syncytiotrophoblast cells, some tumor cells - such as human uterine choriocarcinoma epithelial cells (JAR cells), leukemia cells (K562 cells), melanoma cells, etc., and immune-related cells infected by viruses - such as HIV invasion. stained macrophages and lymphocytes. Therefore, TCS and its derived immunotoxins are widely used in midterm labor induction, anti-tumor and anti-HIV treatments.

Contents of the invention

One object of the present invention is to provide an extracellular body loaded with exogenous ligand molecules.

The exosome (cargo-loaded exosome, CLE) provided by the present invention is composed of exogenous ligand molecule and extracellular body; The surface of the membrane, or wrapped in the cavity of the extracellular body, or exists in both positions; the exogenous ligand molecule is a protein or molecule synthesized by the non-target cell itself, which can directly act on the target cell or be passed by the target cell through the internal Absorption by swallowing.

The exogenous ligand molecules can specifically be exogenous proteins, toxins or drug molecules, such as trichosanthin or trichosanthin derivatives; the target cells can be syncytiotrophoblast cells, tumor cells or virus-infected immune-related cells .

The tumor cells can be human uterine choriocarcinoma epithelial cells, leukemia cells or melanoma cells, etc.

The immune-related cells include lymphocytes, hematopoietic stem cells, monocytes and phagocytes.

Another object of the present invention is to provide two methods for preparing extracellular bodies loaded with exogenous ligand molecules, wherein, one is to prepare natural extracellular bodies loaded with exogenous ligand molecules, and the other is to prepare Recombinant extracellular bodies loaded with exogenous ligand molecules.

The method for preparing natural extracellular bodies loaded with exogenous ligand molecules provided by the present invention is to culture target cells in a medium containing exogenous ligand molecules for 4-12 hours, collect the culture mixture, and centrifuge to remove the cells and cell debris, the obtained supernatant is filtered with a filter diameter of 0.2 μM, and the obtained filtrate is centrifuged at 60000g-100000g for 1-2 hours, and the obtained precipitate is the extracellular body loaded with exogenous ligand molecules.

In this method, the precipitate obtained by centrifugation at 60000g-100000g for 1-2 hours can be washed with PBS buffer to obtain purified extracellular bodies loaded with exogenous ligand molecules.

The exogenous ligand molecule can be TCS; the target cells can be K562 cells, JAR cells; the ratio of TCS to target cells can be 0.5-1× ¹⁰⁷ cells add 0.2-1μM TCS/8ml culture medium .

The method for preparing recombinant extracellular bodies carrying exogenous ligand molecules provided by the present invention is to first extract the extracellular bodies secreted by the target cells, and incubate the extracellular bodies in a medium with a pH of 4.5-5.5 for 30- After 50 minutes, add exogenous ligand molecules to the medium, incubate for another 2-6 hours, and centrifuge at 60,000g-100,000g for 1-2 hours, and the obtained precipitate is the extracellular body loaded with exogenous ligand molecules.

The extracellular body was incubated for 30-50 minutes in a medium consisting of the following components: pH5.0 RPMI 1640 medium containing 10mM Hepes, 50mM NaAc, and 10% (mass percentage) bovine serum.

In this method, the precipitate obtained by centrifugation at 60000g-100000g for 1-2 hours can be washed with PBS buffer to obtain purified extracellular bodies loaded with exogenous ligand molecules.

The exogenous ligand molecule can be TCS; the target cell can be K562 cell; the concentration of added trichosanthin can be 0.5-2.0 μM.

The results of the research on the mechanism of action of TCS in JAR cells and K562 cells showed that: 1. TCS molecules endocytosed by JAR cells or K562 cells can be enriched in MVB in large quantities, and the toxin-bound extracellular body (TCS-loaded exosome, TLE) form is secreted extracellularly. 2. The secreted TLE can be transmitted between adjacent cells. This transmission is selective. Both the TLE produced by JAR cells and K562 cells can be transmitted between homologous cells; and heterologous cells can also be transmitted between JAR cells and K562 cells. TLE exchange, in addition, TLE produced by K562 cells can also combine with Hela cells, but not with 293T cells and SP2/0 cells. 3. TLE can transport the carried TCS molecules to the target cells, and the latter exerts a toxic effect on the target cells and kills them. 4. TLE secreted by K562 cells can be isolated and purified from the culture supernatant of tumor cells treated with TCS. 5. The isolated and purified TLE maintains the selective binding ability to homologous K562 cells or heterologous JAR cells. 6. Purified TLE can effectively introduce the carried TCS molecules into the cytoplasm of target cells, resulting in cell death. The direct toxicity of TLE to target cells is 3-4 times that of the same amount of free TCS molecules. 7. To simulate the environment in intracellular MVB, incubate TCS with purified extracellular bodies derived from normal K562 cells in a cell-free system to obtain recombinant TLE. Recombinant TLE has similar properties to TLE secreted by cells.

The above research results show that the extracellular body secreted by cells can be used as a "natural" carrier to specifically deliver biologically active soluble ligand molecules between homologous or heterologous cells, and effectively introduce them into target cells In order to exert the corresponding cell regulation effect. Compared with the free diffusion of ligand molecules between cells, or the delivery method using artificial liposomes as carriers, the use of extracellular bodies to deliver soluble active molecules between cells has many advantages:

1. Exosomes can protect active molecules from being decomposed or destroyed by enzymes in the environment during delivery.

2. Many exogenous active molecules have strong immunogenicity and can cause allergic reactions. Carried by extracellular bodies, active molecules will not be directly dissociated in the extracellular environment, which can avoid contact with the immune system.

3. Compared with artificial liposomes, extracellular bodies are "organelles" secreted by cells themselves, which will not cause rejection by the body.

4. Compared with liposomes, as a kind of organelle, extracellular bodies have better self-stability and fluidity between cells, and are not easily damaged.

5. Exosomes have natural specificity in the recognition of target cells, and can deliver active molecules to specific cells in a directional manner without harming other cells.

6. Using extracellular bodies produced by different types of cells, different "targeted" transporters can be prepared.

7. As a membrane-wrapped organelle, the extracellular body can efficiently pass through the cell membrane barrier and transport the active molecules it carries to the target site that plays a biological role.

8. The exosomes loaded with active molecules can be directly extracted from cell culture or recombined in vitro, which is simple and easy.

The exogenous ligand molecule-loaded extracellular body of the present invention can specifically transfer exogenous, biologically active ligand molecules (exogenous proteins, toxins or drug molecules) between homologous or heterologous cells, and will Active molecules are introduced into target cells. The extracellular body loaded with exogenous ligand molecules of the present invention can be used to regulate the activity of homologous or heterologous target cells and prepare medicines (such as medicines for treating tumors). The extracellular body loaded with exogenous ligand molecules of the present invention has very wide application prospects and development value in the fields of scientific research, drug preparation, disease treatment and the like.

Description of drawings

Figure 1 is a schematic diagram of the production process of extracellular bodies

Figure 2 is the dynamic production process of TLE in JAR cells

Figure 3 is the dynamic production process of TLE in K562 cells

Figure 4A is an immunoelectron micrograph of polyvesicles containing endocytosed TCS molecules in JAR cells

Figure 4B is the immunoelectron micrograph of JAR cells secreting TLE

Figure 5A is an immunoelectron micrograph of polyvesicles containing endocytosed TCS molecules in K562 cells

Figure 5B is an immunoelectron micrograph of K562 cells secreting TLE

Figure 6 is the fluorescence microscope observation of TLE secreted by purified K562 cells

Figure 7 is the immunoelectron microscope observation of TLE secreted by purified K562 cells

Figure 8 is the continuous density gradient centrifugation analysis of TLE secreted by K562 cells

Figure 9 is an analysis of the protein composition of TLE secreted by K562 cells

Figure 10 is the distribution of TCS molecules in TLE secreted by K562 cells

Figure 11 is the trypsin digestion analysis of TLE secreted by K562 cells

Figure 12 is the Western blotting analysis of recombinant TLE

Figure 13 is a fluorescence microscope photo of recombinant TLE

Figure 14A and Figure 14B are the distribution of TCS in recombinant TLE

Figure 15A shows that TLE mediates the delivery of TCS in JAR cells

Figure 15B is TLE-mediated transmission of TCS in K562 cells

Figure 16 shows that TTLE mediates the transfer of TCS between heterologous cells

Figure 17 shows the selectivity of TLE-mediated intercellular molecular delivery

Figure 18 is the selective combination of recombinant TLE and target cells

Figure 19 shows the introduction of TCS molecules carried by TLE into target cells

Figure 20 is the electron microscope observation of TLE endocytosis and fusion in target cells

Figure 21 is the direct toxicity of purified TLE to target cells

Detailed ways

The experimental methods in the following examples are conventional methods unless otherwise specified.

The percentages in the following examples are all mass percentages unless otherwise specified.

Example 1, TCS-treated JAR cells and K562 cells secrete natural TCS-loaded exosomes (TCS-loaded exosome, TLE)

1. Preparation of FITC-TCS

Dissolve TCS in 100-500 μl pH9.5, 0.1M Na ₂ CO ₃ -NaHCO ₃ buffer solution with a final concentration of 2-10 mg/ml; add FITC (sigma company) to make the molar ratio of TCS to FITC 1:5- 1:10, mix well, incubate at 37°C for 4-6 hours or 4°C for 12-16 hours; dialyze with 2-4L PBS buffer to remove free FITC to obtain FITC-TCS.

2. Fluorescence microscopy to observe the production process of TLE

Add 0.2 μM FITC-TCS to JAR cells or K562 cells grown in RPMI-1640 medium, and incubate at 37°C for 10 min or 30 min, so that FITC-TCS molecules are endocytosed by the cells. Then remove the medium containing TCS, add fresh medium, and continue to incubate the cells at 37°C for 0-120min. The results are shown in Figures 2 and 3. After binding to target cells, TCS can be rapidly absorbed into primary endocytic vesicles at the cell edge (Figure 2A and Figure 3A), and then transported to early endosomes (Figure 2B and Figure 3B). After 60 to 90 min of endocytosis, a large number of TCS molecules accumulated into the pomegranate-like MVB. There, TCS molecules were fully bound to the luminal membrane structures of MVB (Fig. 2C and Fig. 3C). With the prolongation of incubation time, TCS molecules further aggregated to the luminal membrane vesicles of MVB; at the same time, mature MVB moved to the cell surface (Fig. 2D and Fig. 3D). At about 120-150min, some MVBs fuse with the plasma membrane of the cell, and release the luminal vesicles bound with TCS molecules into the extracellular environment, forming TLE (TCS-loaded exosome, TLE, TCS-loaded extracellular body) (Fig. 2E and 3E). In Fig. 2 and Fig. 3, 10' and 30' represent cells incubated with FITC-TCS for 10 min or 30 min respectively; 30'+30', 30'+60', 30'+90', 30'+120' represent respectively After the cells were co-incubated with FITC-TCS for 30 minutes, add fresh medium and continue to incubate for 0 minutes, 30 minutes, 60 minutes, 90 minutes, and 120 minutes; the picture on the left is a fluorescence photo, and the picture on the right is a photo of fluorescence and differential interference contrast Superimposed photos.

3. Immunoelectron microscopy to observe the generation of TLE

JAR and K562 cells were briefly incubated with 0.2 μMTCS for 30 min as before, and the basic medium used was RpMI 1640. The medium containing TCS was removed, fresh medium was added, and the cells were further incubated at 37° C. for 60 ( FIG. 4A and FIG. 5A ) or 120 min ( FIG. 4B and FIG. 5B ). Cells were collected, rinsed with PBS, and then routinely embedded in samples for electron microscopy. The brief steps are as follows: fix the cells with 4% paraformaldehyde, 2.5% glutaraldehyde, and 1% tetraoxide; then fix them with 30%, 40%, 50%, 70%, 80%, 90%, and % acetone for dehydration, and finally embedded with epoxy resin Epon812. Embed the cell sample for ultrathin sectioning, and obtain slices with a thickness of about 70 nm, and pick them up on a nickel grid coated with formvar film (polyvinyl formal film). After the section was etched with hydrogen peroxide, rabbit anti-TCS polyclonal antibody (prepared according to conventional methods, that is, rabbits were immunized with purified TCS as an antigen to obtain antiserum, and the polyclonal antibody of TCS was obtained by protein-G affinity purification) at 37 Incubate at ℃ for 4-6 hours for labeling; after the sample is fully rinsed, it is incubated with a gold-labeled goat anti-rabbit secondary antibody (5nm colloidal gold, purchased from Jackson ImmunoResearch). After the marked samples were fully rinsed, they were stained with uranyl acetate and lead citrate, and observed after drying. For TCS and flotillin double-labeled samples, the slices were first labeled with goat anti-flotillin polyclonal antibody (purchased from Santa Cruz Biotechnology Company) at 37°C for 4-6 hours; after rinsing, gold-labeled protein A (10nm colloidal gold, purchased from in sigma company) mark; then carry on the mark of TCS as above.

Under the electron microscope, the detailed process of TLE production can be observed. After TCS endocytosis for 60-90min, a considerable number of TCS molecules can be seen bound to the 30-100nm small membrane vesicles in the lumen of MVB. Also contained on these vesicles was flotillin, a common extracellular body protein. When MVB fuses with the plasma membrane, TCS and flotillin molecules are released to the extracellular space along with the luminal vesicles of MVB (Fig. 4A, Fig. 4B, Fig. 5A and Fig. 5B). Figure 4A and Figure 5A show multivesicles containing endocytosed TCS molecules, TCS molecules (immunolabeled with 5nm gold particles) and flotillin molecules (immunolabeled with 10nm gold particles) bound to the luminal membrane vesicles of MVB; Figure 4B and Figure 5B shows the secretion process of TLE, the MVB containing TCS fuses with the plasma membrane of the cell, and releases the luminal membrane vesicles bound with TCS molecules to the extracellular space. The scale bar in Figure 4A, Figure 4B, Figure 5A and Figure 5B is 100 nm.

Example 2, Isolation, purification and identification of TLE secreted by K562 cells

1. Separation and purification of TLE

1. Place 2×10 ⁸ K562 cells in fresh 100ml RPMI-1640 medium, add 0.2μM FITC-TCS (for fluorescence microscope detection) or TCS (for other detection), and incubate at 37°C for 1h. Then, the medium containing TCS was removed, fresh RPMI 1640 medium was added, and the cells were further incubated at 37° C. for 2 h.

2. Centrifuge the incubated culture mixture at 300 g for 10 min to remove cells, and take 100 ml of supernatant.

3. The supernatant was centrifuged for 3 times in sequence, the first two times were centrifuged at 800g for 15min, and the third time was centrifuged at 10000g for 30min to remove cell debris.

4. After centrifugation, the supernatant was filtered through a 0.2 μM filter membrane and concentrated to 20 ml by ultrafiltration.

5. After concentration, the supernatant was centrifuged at a speed of 60000g-100000g for 2 hours to obtain a membrane precipitate.

6. Rinse the membrane pellet with PBS once, and resuspend with an appropriate amount of PBS (0.5-1ml), which is TLE.

2. Identification of TLE

The TLE purified from the FITC-TCS-treated cell sample was directly observed under a fluorescence microscope, and it was found that the TLE was a small vesicle loaded with a large number of FITC-TCS molecules (Figure 6). The picture on the left is a fluorescence picture, and the picture on the right is a picture of differential interference contrast, and the scale bar is 1 μM.

Drop the resuspended TLE onto a nickel grid for electron microscopy, and carry out immunogold labeling. The method is the same as in Example 1, that is, the sample is labeled with TCS, flotillin, or tsg101 primary antibody, and then labeled with gold-labeled secondary antibody or protein A. The primary antibody of TCS was prepared according to the method in Example 1, and the antibodies of flotillin and tsg101 were purchased from Santa Cruz Biotechnology Company. After labeling, the sample was negatively stained with uranyl acetate and observed with electron microscope. The results showed that the TLEs obtained by centrifugation were small membrane vesicles with a diameter of 30-100 nm, loaded with a considerable amount of TCS molecules, and contained both flotillin and tsg101 - two common extracellular body proteins (Fig. 7). In Figure 7, A is 10nm gold particles immunolabeled flotillin (Flo); B is 10nm gold particles immunolabeled tsg101 (Tsg); C is 5nm gold particles and 10nm gold particles immunolabeled TCS and flotillin; D is 5nm gold particles and 10 nm gold particles immuno-double-labeled TCS and tsg101; scale bar is 100 nm.

The isolated TLE was subjected to continuous sucrose density gradient centrifugation, and the results showed that the secreted TLE was mainly distributed in the low buoyancy density equilibrium area, and the buoyancy density was in the range of 1.1-1.2g/ml. As a control, free TCS molecules had a higher The buoyant density is mainly distributed at the bottom of the gradient, and the corresponding density value is about 1.3g/ml (Figure 8).

The isolated TLE and K562 whole cell lysates (Cell) were subjected to 12.5% SDS-PAGE and Western blotting. Among them, the primary antibodies used in Western blotting were: TCS antibody, prepared according to the method in Example 1; TfR, flotillin, tsg101, rab5, calnexin and rab6 antibodies were purchased from Santa Cruz Biotechnology Company. The secondary antibody was horseradish peroxidase-labeled antibody, which was purchased from Beijing Zhongshan Biotechnology Co., Ltd. The results are shown in Figure 9, indicating that the isolated TLE and K562 cell lysate (Cell) have different protein composition profiles; in addition to carrying a large number of TCS molecules, TLE also contains a variety of extracellular characteristic molecules, as described above flotillin and tsg101 as well as transferrin receptor (TfR) and rab5, but the isolated TLE did not contain rab6 and calnexin - specific marker molecules present in the Golgi complex and ER, respectively, indicating that cells were better avoided during the purification process Debris and other contamination of membrane structures (Figure 9).

The isolated TLE was incubated with 0.1% Triton X-100 for 30 min at room temperature. Centrifuge at 60000-80000g for 1-2h to separate the permeabilized supernatant (S) and membrane fraction (P). The supernatant (S) and membrane fraction (P) were subjected to 12.5% SDS-PAGE and Western blotting, wherein the primary antibodies used in Western blotting were: TCS antibody, prepared according to the method of Example 1; TfR, flotillin The antibodies were purchased from Santa Cruz Biotechnology, and the secondary antibodies were horseradish peroxidase-labeled antibodies, purchased from Beijing Zhongshan Biotechnology Co., Ltd. The results are shown in Figure 10. The membrane-integrated proteins transferrin receptor (TfR) and flotillin are distributed in the membrane precipitate after permeabilization, most of the TCS is also bound to the membrane of the extracellular body, and a small amount of TCS molecules appear after permeabilization In the supernatant, it was shown that part of the TCS was located in the lumen of the extracellular body. Non, membrane pellet centrifuged from the culture supernatant of TCS-treated K562 cells without permeabilization.

The isolated TLE was digested with 1 mg/ml trypsin at 37°C, the buffer was PBS buffer or PBS buffer containing 0.1% Triton X-100, and Western blotting was used to detect the amount of residual TCS after digestion for different times. The primary antibody used in Western blotting was TCS antibody, which was prepared according to the method in Example 1; the secondary antibody was horseradish peroxidase-labeled antibody, which was purchased from Beijing Zhongshan Biotechnology Co., Ltd. As a control (Control), the same amount of free TCS was digested with 1 mg/ml trypsin at 37°C, and the buffer was PBS buffer or PBS buffer containing 0.1% Triton X-100. The results are shown in Figure 11, indicating that free TCS can be completely degraded within 15 min regardless of whether Triton X-100 is added or not; and most of the TCS molecules carried by TLE can also be degraded within 15 min, indicating that they are exposed on the surface of TLE. However, a small amount of TCS molecules (accounting for about 1/10 of the total amount) still remained after 60 minutes of digestion, and only after 0.1% Triton X-100 permeabilized the extracellular bodies, this part of TCS molecules could be digested and degraded, indicating that its Located in the lumen of the TLE. Non, samples that have not been digested.

Permeabilization experiments with 0.1% Triton X-100 and trypsin digestion experiments showed that most of the TCS molecules were bound to the membrane surface of the extracellular body, while a small amount of TCS molecules were wrapped in the cavity of the extracellular body, which was in the middle of the two distribution modes. The number ratio of TCS molecules is about 9:1.

Example 3, Preparation and Identification of Recombinant TLE in Non-Cell System

By simulating the environment in intracellular MVB, recombinant TLE can be produced in a cell-free system. The specific operation is as follows:

1. The extracellular bodies secreted by normal K562 cells were extracted from the 12h cell culture supernatant, and the specific steps were as follows:

(1) Culture 2×10 ⁸ K562 cells in RPMI-1640 medium at 37°C for 12-24 hours, centrifuge the culture mixture at 300 g for 10 min, remove the cells, and take about 100 ml of supernatant.

(2) The supernatant was centrifuged for 3 times in sequence, the first two times were centrifuged at 800g for 15min, and the third time was centrifuged at 10000g for 30min to remove cell debris.

(3) After centrifugation, the supernatant was filtered through a 0.2 μM filter membrane, concentrated by ultrafiltration to 20 ml, and the concentrated supernatant was centrifuged at an ultra-speed 60000g-100000g for 2 hours to obtain a membrane precipitate. The membrane pellet was rinsed once with PBS to obtain extracellular bodies.

2. Take the purified K562 extracellular body, place it in pH 5.0 RPMI-1640 medium (10mM Hepes, 50mM NaAc, 10% bovine serum), and incubate at 37°C for 40min.

3. Add different concentrations (0.1, 0.2, 0.5 or 1 μM) of TCS (or FITC-TCS, for fluorescence microscope detection) to the above extracellular bodies, and incubate at 37° C. for 4 hours.

4. Centrifuge at 100000 g for 2 hours to obtain extracellular body pellet (P) and supernatant (S).

5. Rinse the precipitated extracellular bodies once with PBS.

The supernatant (S) obtained in step 4 and the extracellular precipitate (P) obtained in step 5 were subjected to 12.5% SDS-PAGE and Western blotting, wherein the primary antibody used in Western blotting was the antibody of TCS, according to Example 1 prepared by the method; the secondary antibody was a horseradish peroxidase-labeled antibody, which was purchased from Beijing Zhongshan Biotechnology Co., Ltd. The results are shown in Figure 12. At low concentrations, the binding of TCS to extracellular bodies was very weak. When increased to 0.5μM or above, TCS molecules can fully bind to extracellular bodies.

The exosomes treated with FITC-TCS were observed under a fluorescence microscope, and the results are shown in Figure 13, which showed that a considerable number of FITC-TCS molecules were bound to most of the exosomes. In Figure 13, the scale bar is 1 μM; the picture on the left is a differential interference contrast (DIC) photo, the middle picture is a fluorescence photo, and the right picture is a superimposed photo of a differential interference contrast photo and a fluorescence photo.

The results of Western blotting and fluorescence microscopy showed that when the concentration of TCS in the bulk phase was higher than 0.5 μM, TCS molecules could fully and spontaneously bind to the extracellular body of normal K562 cells.

According to the method of Example 2, 0.1% Triton X-100 permeation test and trypsin digestion test and detection were carried out on the recombined TLE, which showed that in the recombined TLE, TCS molecules were also distributed in two different positions: most of the molecules were bound in The membrane surface of the extracellular body, while a small number of molecules are wrapped in the cavity of the extracellular body, and the ratio of the number of TCS in these two distributions is also about 9:1, which is very similar to the natural TLE secreted by cells (Fig. 14A and Fig. 14B). Figure 14A, the recombinant TLE was permeabilized with 0.1% Triton X-100 as before, after permeabilization, the supernatant (S1) and membrane pellet (P1) were separated by centrifugation. Non is recombinant TLE that has not been permeabilized. Figure 14B, recombinant TLE was digested with 1mg/ml trypsin in PBS (buffer) or PBS containing 0.1% Triton X-100 for 60min at 37°C, the results showed that most of the TCS molecules were distributed on the membrane surface of recombinant TLE , while a small amount of molecules are wrapped in the cavity of TLE; Non is the recombinant TLE that has not been digested.

Example 4, TLE mediates the transfer of TCS between cells

1. Transmission between homologous cells

1. Get A, B two groups of JAR cells or K562 cells, add 0.2 μ M FITC-TCS (preparation method is the same as Example 1) in A group, add 0.2 μ M TRITC-TCS (preparation method is the same as FITC-TCS in the B group, mark When replacing FITC with TRITC), incubate at 37°C for 30min, the medium used is RPMI 1640.

2. Remove the medium containing TCS, add fresh medium, and continue to cultivate for 120 minutes.

3. After fully rinsing the cells with pre-cooled PBS, take an equal amount of cells from groups A and B, mix them, and co-culture them in fresh RPMI 1640 medium for 30-60 minutes.

The results are shown in Figure 15A and Figure 15B, indicating that secreted TLE can be passed between adjacent cells, resulting in the transport of FITC-TCS-containing TLE and TRITC-TCS-containing TLE from cells in group A and group B, respectively, to the same cell. cells (enlarged inset). In Fig. 15A and Fig. 15B, the long arrow indicates TLE containing FITC-TCS, the short arrow indicates TLE containing TRITC-TCS, the scale bar is 3 μM, the picture on the left is a fluorescence picture, and the picture on the right is a picture of differential interference contrast.

2. Transfer between heterologous cells

According to the operation of step 1, the JAR cells treated with FITC-TCS and the K562 cells treated with TRITC-TCS were mixed and cultured. The results are shown in Figure 16, indicating that the TLE carrying FITC-TCS molecules secreted by JAR cells can be delivered to K562 cells (lower row); at the same time, the TLE carrying TRITC-TCS molecules secreted by K562 cells can be delivered to JAR cells (upper row) Row) on. In Figure 16, the long arrow indicates TLE containing FITC-TCS (secreted by JAR), the short arrow indicates TLE containing TRITC-TCS (secreted by K562 cells), the scale bar is 3 μm, the inset is enlarged, and the picture on the left is a fluorescent photo, The picture on the right is a differential interference contrast photo.

3. The selectivity of TLE transmission between cells

According to the operation of step 1, the same amount of FITC-TCS-treated K562 cells were co-incubated with JAR cells, Hela cells, 293T cells and sp2/0 cells respectively. The results are shown in Figure 17, indicating that TLE secreted by K562 cells can be effectively delivered to JAR cells and partially delivered to Hela cells, but there is no obvious interaction with 293T cells and sp2/0 cells. In Fig. 17, the scale bar is 3 μm, the upper row of pictures are fluorescence photos, and the lower row of pictures are differential interference contrast photos.

Example 5. The isolated and purified natural TLE and the TLE recombined in vitro maintain the ability to selectively bind to target cells

According to the operation in Example 2, natural TLE containing FITC-TCS was isolated and purified from the culture supernatant of K562 cells treated with FITC-TCS. Equal amounts of purified TLE were added to K562 cells, JAR cells, 293T cells or sp2/0 cells, and incubated in RPMI-1640 medium at 37°C for 10-30min. The results are shown in Figure 19, indicating that TLE can selectively bind to the surface of syngeneic K562 cells or heterologous JAR cells (Figure 19A and D), but has no significant interaction with 293T and sp2/0 cells.

Similarly, according to the operation in Example 3, the recombinant TLE containing FITC-TCS was prepared in vitro and incubated with the above cells, and similar results were obtained. Recombinant TLE selectively bound to the surface of K562 cells and JAR cells, but not 293T and sp2/0 cells (Figure 18). In Fig. 18, the first row of pictures are fluorescence photos, the second row of pictures are differential interference contrast photos, and the scale bar is 3 μm.

Example 6, TLE effectively introduces the carried TCS molecules into target cells

According to the operation in Example 2, natural TLE containing FITC-TCS was isolated and purified from the culture supernatant of K562 cells treated with FITC-TCS. Add the isolated and purified TLE to K562 cells or JAR cells, and incubate at 37°C for 10 minutes to allow TLE to bind to the target cells. The medium used is RPMI-1640. Remove the TLE not combined with the cells, add fresh medium, and continue to culture the cells at 37°C for 60-120min. Observe the cell sample with a fluorescence microscope (Figure 19) or prepare an ultrathin section and carry out immunogold standard (as in Example 1) and observe with an electron microscope (Figure 20)

The results indicated that TLE could rapidly bind to the cell surface and be captured in endocytic pits (Fig. 19A and D, Fig. 20A and D). After 60 min of incubation, a large amount of bound TLE was taken up by the target cells and appeared in endocytic organelles close to the nucleus (Fig. 19B and E, Fig. 20B and E). Endocytosed TLEs can fuse with the limiting membrane of the organelle, releasing the carried TCS molecules into the cytoplasm of target cells (Fig. 20C and E, arrows). After approximately 120 min of incubation, almost all the endocytosed TLEs were fused with the target cells, and only a small amount of residual TLEs could be observed in the cells at this time (Fig. 19C and F). TCS molecules released from TLE can diffuse to various parts of the cytoplasm, especially near the nucleus, where the rough endoplasmic reticulum is distributed, and a large number of ribosomes are bound to the target sites of TCS attack.

In Fig. 19, 10', 10'+60', and 10'+120' respectively represent the photos of cells incubated with TLE for 10 minutes, and then adding fresh medium and continuing to incubate for 0 minutes, 60 minutes, and 120 minutes; the first row and The third row of pictures is the fluorescence photos, the second and fourth row of pictures are superimposed photos of fluorescence photos and differential interference contrast photos, pictures A-C are JAR cells, pictures D-F are K562 cells, the scale bar is 3 μm, and Nu is the nucleus.

In Fig. 20, A-C are JAR cells, D-E are K562 cells, the scale bar, 200nm, Nu is the nucleus, PM is the plasma membrane. After binding to target cells, TLEs are taken up into endocytic pits (A and D), and subsequently, TLEs are endocytosed into organelles adjacent to the nucleus (B and E), and endocytosed TLEs can fuse with the limiting membrane of organelles, The carried TCS molecules are released into the cytoplasm of target cells (C and E, arrows).

The efficiency with which TLE carries TCS molecules into the cytoplasm of target cells can be measured by its direct lethality to target cells, because when TCS molecules enter the cytoplasm, they can attack ribosomes and cause the death of target cells. According to the method in Example 2, TLE was isolated and purified from the culture supernatant of TCS-treated K562 cells. The co-incubation of purified TLE loaded with 85ng TCS with JAR cells and K562 cells for 48h resulted in the death of about 19% of JAR cells and about 16% of K562 cells. Its cytotoxicity is equivalent to 675ng of free TCS molecule (48h action can cause about 22% JAR cell death, 18% K562 cell death). In contrast, the cytotoxicity of 85ng of free TCS was only about 1/3-1/4 of that of TLE ( FIG. 21 ). This result also fully proves that TLE can effectively transport the TCS molecules it carries into the cytoplasm of target cells.

Claims (10)

1, is loaded with born of the same parents' ectosome of exogenous ligand molecule, forms by exogenous ligand molecule and born of the same parents' ectosome; Described exogenous ligand molecule is combined in the film surface of born of the same parents' ectosome, perhaps is wrapped in the chamber of born of the same parents' ectosome, perhaps all exists two positions;

Described exogenous ligand molecule is non-target cell self synthetic albumen or molecule, can directly act on target cell or is absorbed by endocytosis by target cell.

2, the born of the same parents' ectosome that is loaded with exogenous ligand molecule according to claim 1 is characterized in that: described exogenous ligand molecule is albumen, toxin or the drug molecule of external source; Described target cell is the fit immunity-associated cell of growing germinal layer cell, tumour cell or virus infection.

3, the born of the same parents' ectosome that is loaded with exogenous ligand molecule according to claim 1 and 2, it is characterized in that: described exogenous ligand molecule is a Trichosanthin.

4, the born of the same parents' ectosome that is loaded with exogenous ligand molecule according to claim 2 is characterized in that: described tumour cell behaviour uterus villioma epithelial cell, leukemia cell or melanoma cells.

5, the born of the same parents' ectosome that is loaded with exogenous ligand molecule according to claim 2, it is characterized in that: described immunity-associated cell is lymphocyte, hemopoietic stem cell, monocyte or phagocytic cell.

6, a kind of described method that is loaded with born of the same parents' ectosome of exogenous ligand molecule of claim 1 for preparing, be that target cell was cultivated 4-12 hour in containing the substratum of exogenous ligand molecule, collect culturing mixt, centrifugal cell and the cell debris removed, it is the filtration of 0.2 μ M that the supernatant liquor that obtains is filtered the footpath, at the centrifugal 1-2 of 60000g-100000g hour, the precipitation that obtains was the born of the same parents' ectosome that is loaded with exogenous ligand molecule with the filtrate that obtains.

7, method according to claim 6 is characterized in that: described exogenous ligand molecule is a Trichosanthin; Described target cell is K562 cell or jar cell; The ratio of described Trichosanthin and target cell is 0.5-1 * 10 ⁷Individual cell 0.2-1 μ M Trichosanthin/8ml substratum.

8, a kind of described method that is loaded with born of the same parents' ectosome of exogenous ligand molecule of claim 1 for preparing, be to extract target cell excretory born of the same parents ectosome earlier, born of the same parents' ectosome was hatched in the substratum of pH4.5-5.5 30-50 minute, in substratum, add exogenous ligand molecule again, hatched again 2-6 hour, the centrifugal 1-2 of 60000g-100000g hour, the precipitation that obtains was the born of the same parents' ectosome that is loaded with exogenous ligand molecule.

9, method according to claim 8 is characterized in that: described exogenous ligand molecule is a Trichosanthin; Described target cell is K562 cell or jar cell; The concentration of the Trichosanthin that is added is 0.5-2.0 μ M.

10, the described born of the same parents' ectosome that is loaded with exogenous ligand molecule of claim 1 is being regulated homology or allos target cell activity and the application in the preparation medicine.

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