Exosome / liposome hybrid nano-vesicle biomotor and synthesis method thereof

Abstract

The invention provides an exosome / liposome hybrid nano-vesicle biomotor and a synthetic method thereof, and the synthetic method comprises the following steps: obtaining a functional exosome and a drug-loaded liposome of which the surface is modified with a focus targeting protein, mixing the two, incubating, and carrying out freeze-thaw cycle to obtain the exosome / liposome hybrid nano-vesicle biomotor. Adding the obtained first mixed nano-particles into a culture dish of which the bottom is fixed with an exosome biomarker antibody CD9 for incubation, washing and eluting, adding the obtained second mixed nano-particles into a culture dish of which the bottom is fixed with focus targeting protein for incubation, and washing to obtain the TP-DLL / Exo hybrid nano-vesicles; and modifying a urease motor on the TP-DLL / Exo hybrid nano-vesicle to obtain the exosome / liposome hybrid nano-vesicle biological motor. The antigen-antibody specific binding'similar ELISA (enzyme-linked immunosorbent assay) method 'is utilized, so that the separation degree of the exosome and drug-loaded liposome mixed hybridized nano-vesicle is higher and more accurate, meanwhile, the modification step of the biological motor is simplified, and the success rate and the accuracy of synthesizing the unilateral modified biological motor are higher.

Description

Technical Field

[0001] The present invention relates to the technical field of biomotors, and in particular to an exosome / liposome hybrid nanovesicle biomotors and a synthesis method thereof. Background Art

[0002] At present, the synthesis methods of exosome / liposome hybrid nanovesicle biomotors include magnetic bead sorting method and gradient centrifugation method. The magnetic bead sorting method uses antigen-labeled exosomes and uses immunomagnetic beads to sort out nanovesicles containing exosome marker proteins. However, the existing magnetic bead sorting method only uses exosome marker antibodies as a single screening method. Immunomagnetic beads can only separate nanovesicles containing exosomes, but cannot separate unhybridized and self-hybridized exosomes. It may cause a large amount of residues of single exosomes and self-hybridized exosomes that are not involved in hybridization with liposomes, which in turn leads to the low content and impurity of the separated correct exosome / liposome hybrid nanovesicles. On the other hand, the immunomagnetic beads used in this method are relatively expensive, complicated to operate, and cannot be used for the modification and synthesis of later biomotors.

[0003] The gradient centrifugation method uses the Nycodenz density gradient separation method, and its separation principle is to separate different samples by using the shape, size, density and centrifugal force of the separation sample, the density and viscosity coefficient of the gradient liquid. Due to the structural complexity of the exosomes produced by biological cells, the shape, size and density of the same exosome nanoparticles separated from the same batch are roughly similar, but there are slight differences. The hybridized nanovesicles also have this problem. It is difficult to correctly separate the hybridized nanovesicles by the Nycodenz density gradient separation method. The separation rate is low and the accuracy is poor. In addition, the reagents used in this method (Nycodenz separation solution) are relatively expensive, the operation is complicated, and the requirements for the level of experimental equipment are high, resulting in a high cost for obtaining the correct hybridized nanovesicles, and it cannot be used for the modification of biological motors. Summary of the invention

[0004] The purpose of the present invention is to provide an exosome / liposome hybrid nanovesicle biomotor and a preparation method thereof, which adopts an "ELISA-like" method of two antigen-antibody specific binding to synthesize an exosome / liposome hybrid nanovesicle biomotor (UM-(TP-DLL / Exo)). The separation degree of the mixed hybrid nanovesicles of exosomes and drug-loaded liposomes is higher, more accurate, and lower in cost. At the same time, the modification steps of the biomotor are simplified, so that the success rate and accuracy of synthesizing a unilaterally modified biomotor are higher.

[0005] The present invention solves the technical problem by adopting the following technical solutions.

[0006] The present invention provides a method for synthesizing an exosome / liposome hybrid nanovesicle biomotor, comprising the following steps: S1, obtaining functional exosomes (Exo); S2, obtaining a drug-loaded liposome (DLL), and modifying a lesion-targeting protein (TP) on the surface of the drug-loaded liposome to obtain TP-DLL; S3, mixing the functional exosomes and the TP-DLL, incubating the mixture, and subjecting the mixture to freeze-thaw cycles to obtain first hybrid nanoparticles; S4, adding the first hybrid nanoparticles to a culture dish with an exosome biomarker antibody CD9 fixed on the bottom for incubation, and washing to obtain second hybrid nanoparticles; S5, after the second hybrid nanoparticles are eluted, they are added to a culture dish with a lesion-targeting protein fixed on the bottom for incubation, and after washing, TP-DLL / Exo hybrid nanovesicles (TP-DLL / Exo) are obtained; S6. Modify the urease motor on the TP-DLL / Exo hybrid nanovesicle to obtain the exosome / liposome hybrid nanovesicle biomotor (UM-(TP-DLL / Exo)). The present invention uses an "ELISA-like method" of antigen-antibody specific binding to synthesize the exosome / liposome hybrid nanovesicle biomotor UM-(TP-DLL / Exo). First, functional exosomes (Exo) are extracted from biological cells. Then, drug-loaded liposomes (DLL) are synthesized, and lesion-targeting proteins (TP) are modified on the surface of the liposomes to obtain lesion-targeted exosome / liposome hybrid nanovesicles TP-DLL / Exo. Finally, the urease motor is modified on the TP-DLL / Exo hybrid nanovesicle to obtain the exosome / liposome hybrid nanovesicle biomotor. This method has higher separation, more accuracy and lower cost for mixed hybrid nanovesicles of exosomes and drug-loaded liposomes, and also simplifies the modification steps of the biomotor, so that the success rate and accuracy of synthesizing unilaterally modified biomotors are higher.

[0007] Further, in a preferred embodiment of the present invention, the steps of obtaining drug-loaded liposomes and modifying the lesion-targeting protein on the surface of the drug-loaded liposomes are: dissolving DSPE-PEG-2000-NHS in chloroform, evaporating the solvent to obtain a lipid film, adding an siRNA solution to the lipid film for hydration reaction, filtering three times, and obtaining drug-loaded liposomes loaded with siRNA drugs; The lesion targeting protein and the PBS solution of the drug-loaded liposome loaded with the siRNA drug are mixed in equal proportions and stirred, and the TP-DLL is obtained after dialysis reaction and freeze-drying. The lesion targeting protein is an anti-CD68 protein.

[0008] Furthermore, in a preferred embodiment of the present invention, the usage ratio of the siRNA solution to the lipid film is 1:15 (w / w), the temperature of the hydration reaction is 37° C., and the hydration reaction time is 2 h.

[0009] Furthermore, in a preferred embodiment of the present invention, the freeze-thaw cycle steps are: the functional exosomes and the TP-DLL are mixed in equal proportions and incubated at 37°C for 30 minutes, then frozen with liquid nitrogen for 5 minutes and thawed at room temperature for 15 minutes, and repeated three times to obtain the first mixed nanoparticles.

[0010] Furthermore, in a preferred embodiment of the present invention, the exosome biomarker antibody CD9 is fixed on the bottom of the culture dish by 0.01% L-polylysine.

[0011] Furthermore, in a preferred embodiment of the present invention, the elution uses an antibody elution solution.

[0012] Furthermore, in a preferred embodiment of the present invention, the lesion targeting protein is fixed on the bottom of the culture dish by 0.01% L-polylysine.

[0013] Furthermore, in a preferred embodiment of the present invention, the step of modifying the urease motor on the TP-DLL / Exo hybrid nanovesicle is: synthesizing urease-biotin, and then adding biotin, streptavidin and the urease-biotin in sequence to the CD68 antibody culture dish adhered to the TP-DLL / Exo nanovesicle for incubation, and after elution, obtaining the exosome / liposome hybrid nanovesicle biomotor.

[0014] Furthermore, in a preferred embodiment of the present invention, the step of synthesizing urease-biotin is: mixing sulfo-NHS-biotin with a urease solution, stirring at low temperature for 1 hour, and dialyzing in PBS with a 1 kDa dialysis bag for 24 hours to obtain the urease-biotin.

[0015] The present invention provides an exosome / liposome hybrid nanovesicle biomotor, which is synthesized according to the above-mentioned synthesis method.

[0016] The beneficial effects of the exosome / liposome hybrid nanovesicle biomotor and the synthesis method thereof according to the embodiment of the present invention are: The "similar ELISA method" of the present invention adopts two antigen-antibody combinations for exosomes and drug-loaded liposomes, that is, using exosome marker antibodies and drug-loaded liposome surface protein-specific antibodies respectively. First, unhybridized exosomes, self-hybridized exosomes and correctly hybridized exosome / liposome hybrid nanovesicles are separated from the mixed nanovesicles by exosome marker antibodies, and then the correctly hybridized exosome / liposome hybrid nanovesicles are separated from the remaining three mixed nanovesicles by drug-loaded liposome surface protein-specific antibodies. The "similar ELISA method" method of the present invention has a higher degree of separation, more accuracy and lower cost for mixed hybrid nanovesicles of exosomes and drug-loaded liposomes. The method also simplifies the modification steps of the biomotor, so that the success rate of synthesizing unilaterally modified biomotors is higher and the accuracy is higher. The "similar ELISA method" method has a wide range of applications, can be used to synthesize and separate different exosomes and different liposome hybrid nanovesicles, and is easier to modify biomotors to prepare micron- or nano-scale biorobots. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0018] Figure 1 Flow chart for the preparation of exosome / liposome hybrid nanovesicle biomotors; Figure 2 This is a transmission electron microscopy image of the UM-(TP-DLL / Exo) nanovesicles of Example 3 of the present invention; Figure 3 This is a confocal fluorescence microscopy image of the UM-(TP-DLL / Exo) nanovesicles of Example 3 of the present invention; Figure 4 The hybridization efficiency of Exo / FITC and anti-CD68-DLL / Cy5 was analyzed using a flow nanoanalyzer; Figure 5 Figure 2 is the detection result of miRNA in Exo, Exo-(anti-CD68-DLL) and UM-Exo-(anti-CD68-DLL); Figure 6 This is a comparison of the gel imaging of urease, Exo, TP-DLL / Exo, UM-(TP-DLL / Exo) and Marker. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0020] The exosome / liposome hybrid nanovesicle biomotor and its synthesis method according to the embodiment of the present invention are described in detail below.

[0021] Example 1 This example extracts and purifies functional exosomes from biological cells (bone marrow-derived macrophages (BMDM)), and the specific steps are as follows: C57BL / 6J mice were euthanized by cervical dislocation, and alcohol was sprayed all over the body. The limbs were separated, muscle tissue was removed, and the bones were soaked in PBS containing 1% penicillin-streptomycin. The bone marrow was blown several times with a syringe until the bone marrow cavity turned white. The bone marrow was added to 1640 medium, the medium was blown continuously, and then filtered with a 70 µm filter membrane, and the filtrate was centrifuged at 2000 rpm for 5 minutes. Take a 15 mL centrifuge tube, slowly add 3 mL of tissue water-1119 and tissue water-1077 separation solution along the wall of the tube, and then add an equal amount of cell suspension. Use a parallel centrifuge, 800 g, ACC: 1, Dec: 0, room temperature, centrifuge for 40 minutes. After centrifugation, carefully remove and aspirate the mononuclear cell layer. The mononuclear cell layer was mixed with 30% FBS RPMI1640 medium, placed in a 6-well plate, and incubated in an incubator for 12 hours. The culture flask was shaken to suspend the non-adherent cells, the medium was aspirated, and the cells were washed 2-3 times with D-Hanks buffer. The culture medium containing 40 ng / mL M-CSF (supplemented with 20 ng / mL IL-4) was used to continue the culture for 6 days. The medium was changed on the third day. On the sixth day, BMDM was obtained. The above-extracted BMDM was cultured in FBS-free medium (20 ng / mL IL-4) for 48 hours, and the supernatant was collected for exosome extraction. Exosomes were extracted by density gradient centrifugation, that is, 3 ml of 30% sucrose deuterium oxide solution was added to the bottom of a high-speed centrifuge tube. The cell culture supernatant was added and centrifuged at 120,000 g for 70 minutes at 4°C. Then, the sucrose layer solution was collected and centrifuged at 120,000 g for 70 minutes at 4°C. Finally, the supernatant was removed to obtain the exosome-rich precipitate, which was stored in PBS for later use.

[0022] Example 2 In this example, drug-loaded liposomes (DLL) were synthesized, and lesion-targeting proteins (TP) were modified on the surface of the liposomes. The specific steps were as follows: 20 mg DSPE-PEG-2000-NHS was dissolved in chloroform. The solvent was evaporated by a rotary evaporator at 35-45°C under vacuum conditions to form a lipid film. Then 4 mL siRNA solution (1 mg / mL, PBS) was added to the lipid film at 37°C, where siRNA solution: liposome = 1:15w / w, and the hydration reaction was carried out for 2 hours. The siRNA drug-loaded liposomes (DLL) were obtained by filtering three times through a polycarbonate membrane with a pore size of 200 nm and stored in PBS at 4°C.

[0023] 1 µg of anti-CD68 protein (TP) and 10 mg of DLL in PBS were mixed in equal proportions and stirred at 4°C for 12 h to covalently link the amino groups in the protein with the NHS on the surface of the DLL. Then, the reaction solution was dialyzed with water and a 2 kD dialysis bag for 24 h, the solution was changed three times, and lyophilized to obtain TP-DLL.

[0024] Example 3 Reference Figure 1 As shown, this example uses the functional exosomes of Example 1 and the TP-DLL of Example 2 to prepare the exosome / liposome hybrid nanovesicle biomotor UM-(TP-DLL / Exo), and the specific steps are as follows: (1) TP-DLL and Exo (6×109 nanoparticles / mL) were quickly mixed in a 1:1 ratio. The mixture was incubated at 37°C for 30 minutes, then frozen with liquid nitrogen for 5 minutes and thawed at room temperature for 15 minutes. After three freeze-thaw cycles, the lipid membranes between liposomes and exosomes fused to form five types of nanovesicles (Exo, Exo*Exo, EVLipo, TP-DLL / Exo, and TP-DLL*TP-DLL). In order to remove the wrong hybrid nanovesicles, the antigen-antibody binding principle was used. The mixed five nanovesicles were added to a culture dish with 0.01% L-polylysine fixed anti-CD9 on the bottom for 1 hour to retain the nanovesicles with high surface expression of CD9, a protein specifically expressed on the surface of exosomes.

[0025] (2) The nanovesicles that did not bind to the antibody CD9 (TP-DLL and TP-DLL*TP-DLL) were washed away with antibody elution buffer to obtain three types of nanovesicles (Exo, Exo*Exo, and TP-DLL / Exo).

[0026] (3) These three types of nanovesicles were added to a culture dish with 0.01% L-poly-lysine fixed to anti-CD68 at the bottom and incubated for 1 hour. The nanovesicles (Exo and Exo*Exo) that did not bind to the antibody CD68 were washed away with antibody elution solution. Finally, only TP-DLL / Exo nanovesicles adhered to the bottom of the culture dish modified with CD68 antibody.

[0027] (4) Surface modification of the urease-driven motor on the antibody-modified hybrid nanovesicle TP-DLL / Exo to obtain the urease-driven hybrid nanovesicle UM-(TP-DLL / Exo). First, synthesize urease linked to biotin (urease-biotin). Then, biotin, streptavidin, and urease-biotin are sequentially added to the CD68 antibody culture dish adhered with the TP-DLL / Exo nanovesicle to obtain the exosome / liposome hybrid nanovesicle biomotor UM-(TP-DLL / Exo). Finally, the UM-(TP-DLL / Exo) nanovesicle is separated using an antibody eluent. The specific steps are: Urease and sulfo-NHS-biotin were reacted to synthesize urease-biotin: sulfo-NHS-biotin (1 mL; PBS, 200 μM;) was mixed with urease solution (1 mL, 1 mg / mL) and stirred at low temperature for 1 h. After the reaction, the solution was dialyzed in PBS with a 1 kDa dialysis bag for 24 h. The obtained urease-biotin was dispersed in 2 mL PBS and stored at low temperature.

[0028] Urease-biotin modified hybrid nanovesicles: 400 µL TP-DLL / Exo hybrid nanovesicle solution (10 9 Nanoparticles (200 μM, 500 μL) were added to a 12-well plate and centrifuged at 900 RCF for 5 min to spin the nanovesicles onto a 0.01% L-polylysine (PLL) surface. The supernatant was removed and the plate was washed. The nanovesicles were then incubated with sulfo-NHS-biotin (200 μM, 500 μL), streptavidin (200 μM, 500 μL), and synthetic urease-biotin (Urease-Biotin, 500 μL) at 37°C for 1 h. The plate was washed three times with PBS and the UM-(TP-DLL / Exo) nanovesicles were separated using antibody elution buffer. Finally, the exosome / liposome hybrid nanovesicle biomotor UM-(TP-DLL / Exo) was obtained. To remove free drugs, the obtained nanovesicles were added to a 3500 kDa dialysis bag and dialyzed in PBS for 24 h. They were stored in PBS and kept in a refrigerator at 4°C.

[0029] Test Example 1 In this test example, transmission electron microscopy and confocal fluorescence microscopy were used to analyze the morphology of the UM-(TP-DLL / Exo) nanovesicles of Example 3. Scale bar: 100 nm.

[0030] like Figure 2 Shown is a transmission electron microscopy image of the UM-(TP-DLL / Exo) nanovesicles of Example 3. Figure 3 This is a confocal fluorescence microscopy image of the UM-(TP-DLL / Exo) nanovesicles of Example 3. Figure 2 and Figure 3 It can be seen that the four-step synthesis method similar to the ELISA method can obtain urease motor hybrid nanovesicles with better morphology. It also shows that this method is a reasonable and reliable method for preparing exosome / liposome hybrid nanovesicle biomotors.

[0031] Test Example 2 The "ELISA-like" method of the present invention only requires 4 steps to purify the hybridized nanovesicles and modify the biomotor on one side of the nanovesicles. In order to verify the rationality of this "ELISA-like" method, this test example uses a flow nanoanalyzer to analyze the hybridization rate of Exo and TP-DLL nanovesicles. First, FITC-loaded macrophage exosome Exo nanoparticles Exo / FITC, and Cy5-loaded and anti-CD68-DLL / Cy5 drug-loaded liposome nanoparticles modified with anti-CD68 were prepared. Then, according to the "ELISA-like" method, (Exo / FITC)-(anti-CD68-DLL / Cy5) hybrid nanovesicles were prepared, and the specific steps were as follows: (1) Preparation of FITC-loaded macrophage exosome nanoparticles Exo / FITC: Mix 1 mg FITC 50% DMSO aqueous solution with 10 mg exosome solution, ultrasonicate on ice for 15 min, stir at 4°C for 12 h, and dialyze in PBS for 24 h using a 1 kDa dialysis bag to obtain Exo / FITC.

[0032] (2) Preparation of anti-CD68-DLL / Cy5 drug-loaded liposome nanoparticles loaded with Cy5 and modified with anti-CD68: 20 mg DSPE-PEG-2000-NHS was dissolved in chloroform. The solvent was evaporated by a rotary evaporator at 35-45 °C under vacuum to form a lipid film. Then 1 mL Cy5 solution (1 mg / mL, chloroform) was added to the lipid film at 37 °C, drug: liposome = 1:20 w / w, and the hydration reaction was carried out for 2 hours. Cy5-loaded drug-loaded liposomes (DLL / Cy5) were obtained by filtering three times through a polycarbonate membrane with a pore size of 200 nm and stored in PBS at 4 °C.

[0033] 1 µg of anti-CD68 protein (anti-CD68) and 10 mg of DLL / Cy5 in PBS were mixed in equal proportions and stirred at 4°C for 12 h to covalently link the amino groups in the protein with the NHS on the surface of the DLL. The reaction solution was dialyzed with water and a 2 kD dialysis bag for 24 h, and the solution was changed three times and freeze-dried to obtain anti-CD68-DLL / Cy5.

[0034] (3) Preparation of (Exo / FITC)-(anti-CD68-DLL / Cy5) hybrid nanovesicles according to the “ELISA-like method”: (3.1) After Exo / FITC and anti-CD68-DLL / Cy5 were mixed in equal proportions (6×109 nanoparticles / mL), five mixed nanoparticles (Exo / FITC, Exo / FITC *2, (Exo / FITC)-(anti-CD68-DLL / Cy5), anti-CD68-DLL / Cy5, and anti-CD68-DLL / Cy5*2) were obtained through three repeated freeze-thaw cycles. The five mixed nanoparticle solutions were added to the culture dish, and the exosome biomarker antibody CD9 was fixed to the bottom of the culture dish with 0.01% L-polylysine for 1 hour.

[0035] (3.2) Wash the culture dish with antibody elution solution to remove any nanoparticles that are not adhered to the bottom of the culture dish, and obtain three types of nanoparticles (Exo / FITC, Exo / FITC*2, (Exo / FITC)-(anti-CD68-DLL / Cy5)).

[0036] (3.3) Wash off the three nanoparticles adhering to the bottom of the culture dish with antibody elution solution, and add them to the culture dish with 0.01% L-polylysine fixed TP on the bottom and incubate for 1 hour. Wash the culture dish with antibody elution solution to remove Exo / FITC and Exo / FITC *2 nanoparticles to obtain highly pure (Exo / FITC)-(anti-CD68-DLL / Cy5) hybrid nanovesicles.

[0037] like Figure 4The figure shows the hybridization efficiency of Exo / FITC and anti-CD68-DLL / Cy5 analyzed by flow nanoanalyzer. Figure 4 It can be seen that through the four-step synthesis method similar to the “ELISA method”, hybrid nanovesicles (Exo / FITC)-(anti-CD68-DLL / Cy5) with a purity of up to 99.8% were successfully obtained.

[0038] Test Example 3 This test example uses the Qsep100 nucleic acid protein analysis system to detect miRNA in Exo, Exo-(CD68-DLL) and UM-Exo-(CD68-DLL). The specific steps are as follows: First, RNA extraction: add 100 ul RNA lysis buffer to 100 ul RNA-free aqueous solution of Exo, TP-DLL / Exo and UM-(TP-DLL / Exo) at the same mass ratio, and store at -80°C. Thaw the RNA lysis buffer and place it at room temperature for 5 minutes. Add 140 μL chloroform and vortex to mix for 15 seconds. Incubate at room temperature for 3 minutes, centrifuge at 4°C and 12000 g for 15 minutes (pre-cool the centrifuge in advance, and return to room temperature immediately after this step). Transfer the upper aqueous phase to a new EP tube (avoid aspirating the middle layer), add 1.5 times the volume of anhydrous ethanol (usually 525 μL), and mix by pipetting. Pipette 700 μL of the mixture (including all precipitates), transfer to the RNeasy adsorption column, centrifuge at 8000 g for 15 seconds at room temperature, discard the filtrate (reuse the collection tube, the same below), and repeat this step with the remaining mixture. Add 700 μL Buffer RWT to wash the adsorption column, centrifuge at 8000 g for 15 s at room temperature, and discard the filtrate. Add 500 μL Buffer RPE to wash the adsorption column, centrifuge at 8000 g for 15 s at room temperature, and discard the filtrate. Add 500 μL Buffer RPE to wash the adsorption column, centrifuge at 8000 g for 2 min at room temperature, and discard the filtrate and collection tube (carefully remove the column to avoid touching the filtrate and residual alcohol). Transfer the adsorption column to a new 2 mL centrifuge tube (self-prepared), centrifuge at 12000 g for 1 min to dry, and discard the filtrate and collection tube. Transfer the adsorption column to a new 1.5 mL centrifuge tube, add 30 μL RNase-free water to the middle of the adsorption membrane, centrifuge at 8000 g for 1 min, and elute the RNA. Immediately transfer to a -80℃ refrigerator for freezing.

[0039] Then, RNA concentration was measured: 1 μL of the extracted RNA was taken out for staining, and the RNA concentration was measured on QuantusFluorometer. According to the RNA concentration, an appropriate amount of RNA stock solution was diluted with the NR1 cartridge supporting diluent, and after dilution, the Qsep100 nucleic acid protein analysis system was used to detect miRNA in Exo, TP-DLL / Exo and UM-(TP-DLL / Exo).

[0040] like Figure 5 The figure shows the detection results of miRNA in Exo, Exo-(anti-CD68-DLL) and UM-Exo-(anti-CD68-DLL). Figure 5 It can be seen that the nucleic acids contained in the three are very similar, indicating that the nucleic acid components in exosomes are successfully encapsulated in the hybrid nanovesicle biomotor.

[0041] Test Example 4 In this test example, protein silver staining analysis was performed on urease, Exo, TP-DLL / Exo, UM-(TP-DLL / Exo) and Marker, respectively. The specific steps are as follows: commercial 10% protein precast gel was used, and different sample urease, Exo, TP-DLL / Exo, UM-(TP-DLL / Exo) and other mass ratios were mixed with marker, and the sample loading volume was 20 ul. The gel plate was placed in the electrophoresis tank for protein electrophoresis. After the electrophoresis, the obtained gel was placed in a plastic container, 50 mL of formaldehyde fixative was added, and it was shaken slowly on a rotary shaker for 10 min. The fixative was poured off, and the gel was washed twice with water, each for 5 min, and shaken slowly. The water was poured off, and the gel was soaked in 0.2 g / L sodium thiosulfate solution for 1 min, and shaken slowly. The sodium thiosulfate solution was poured off, and the gel was washed twice with water, each for 20 s. The water was poured off, and the gel was soaked in 50 mL of silver nitrate for 10 min. Shake slowly. Pour off the silver nitrate solution, wash the gel with water, and then rinse with a small volume of thiosulfate developer. Soak the gel in 50 mL of freshly prepared thiosulfate developer and shake slowly until sufficient band color intensity is obtained. Add 5 ml of 2.3 mol / L citric acid per 100 mL of thiosulfate developer and shake slowly for 10 min. Pour off the liquid, wash the gel with water, and shake slowly for 10 min. Pour off the water and soak the gel in 50 mL of dry gel solution for 10 min. Place the gel between two wet dialysis membranes, clamp it in a glass plate, clamp the edges of the plate with clips, and dry it at room temperature overnight. Then use a gel imaging system for exposure.

[0042] like Figure 6Shown is a comparison of urease, Exo, TP-DLL / Exo, UM-(TP-DLL / Exo) and Marker gel imaging. Figure 6 It can be seen that this experiment also proved that the urease motor was successfully modified on the hybrid nanovesicles.

[0043] In summary, the results of Experimental Examples 1 to 4 all demonstrated the reliability and accuracy of synthesizing exosome / liposome hybrid nanovesicle biomotors using the “ELISA-like” method.

[0044] The embodiments described above are part of the embodiments of the present invention, rather than all of the embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Claims

1. A method for synthesizing an exosome / liposome hybrid nanovesicle biomotor, characterized in that: The following steps are involved: S1. Obtaining functional exosomes; S2, obtaining drug-loaded liposomes, and modifying the lesion-targeting protein on the surface of the drug-loaded liposomes to obtain TP-DLL; S3, mixing the functional exosomes and the TP-DLL, incubating the mixture, and subjecting the mixture to freeze-thaw cycles to obtain first hybrid nanoparticles; S4, adding the first hybrid nanoparticles to a culture dish with an exosome biomarker antibody CD9 fixed on the bottom for incubation, and washing to obtain second hybrid nanoparticles; S5, after the second hybrid nanoparticles are eluted, they are added to a culture dish with a lesion-targeting protein fixed on the bottom for incubation, and after washing, TP-DLL / Exo hybrid nanovesicles are obtained; S6. Modifying the urease motor on the TP-DLL / Exo hybrid nanovesicle to obtain the exosome / liposome hybrid nanovesicle biomotor.

2. The synthesis method according to claim 1, characterized in that In step S2, the steps of obtaining drug-loaded liposomes and modifying the lesion-targeting protein on the surface of the drug-loaded liposomes are as follows: dissolving DSPE-PEG-2000-NHS in chloroform, evaporating the solvent to obtain a lipid film, adding an siRNA solution to the lipid film for hydration reaction, and filtering three times to obtain drug-loaded liposomes loaded with siRNA drugs; The lesion targeting protein and the PBS solution of the drug-loaded liposome loaded with the siRNA drug are mixed in equal proportions and stirred, and the TP-DLL is obtained after dialysis reaction and freeze-drying. The lesion targeting protein is an anti-CD68 protein.

3. The synthesis method according to claim 2, characterized in that In step S2, the dosage ratio of the siRNA solution to the lipid film is 1:15 (w / w), the temperature of the hydration reaction is 37° C., and the hydration reaction time is 2 h.

4. The synthesis method according to claim 1, characterized in that In step S3, the freeze-thaw cycle is as follows: the functional exosomes and the TP-DLL are mixed in equal proportions and incubated at 37° C. for 30 min, then frozen with liquid nitrogen for 5 min and thawed at room temperature for 15 min, and the mixture is repeated three times to obtain the first mixed nanoparticles.

5. The synthesis method according to claim 1, characterized in that In step S4, the exosome biomarker antibody CD9 is fixed on the bottom of the culture dish by 0.01% L-polylysine.

6. The synthesis method according to claim 1, characterized in that In step S5, the elution uses an antibody elution solution.

7. The synthesis method according to claim 1, characterized in that In step S5, the lesion targeting protein is fixed to the bottom of the culture dish via 0.01% L-polylysine.

8. The synthesis method according to claim 1, characterized in that In step S6, the step of modifying the urease motor on the TP-DLL / Exo hybrid nanovesicle is: synthesizing urease-biotin, and then adding biotin, streptavidin and the urease-biotin in sequence to the CD68 antibody culture dish adhered to the TP-DLL / Exo nanovesicle for incubation, and after elution, obtaining the exosome / liposome hybrid nanovesicle biomotor.

9. The synthesis method according to claim 8, characterized in that The steps of synthesizing urease-biotin are as follows: mixing sulfo-NHS-biotin with urease solution, stirring at low temperature for 1 hour, and dialyzing in PBS for 24 hours using a 1 kDa dialysis bag to obtain the urease-biotin.

10. An exosome / liposome hybrid nanovesicle biomotor, characterized in that: The method according to any one of claims 1 to 9 is used for synthesis.

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