Application of ultrasound microfluidics in drug encapsulation in exosomes

Abstract

The present invention belongs to the field of biopharmaceuticals, and is particularly related to the application of ultrasonic microfluidics in exosome (exo) drug encapsulation. The drug loading efficiency of exo-siRNA prepared by ultrasonic microfluidics is about 1-2 times that of electroporation, common ultrasonic method and incubation method; Ultrasonic microfluidics uses cavitation to quickly mix the entire mixed system uniformly, so that siRNA quickly enters exosomes, with high drug loading efficiency, and compared with common ultrasonic methods, ultrasonic microfluidics can complete the encapsulation of siRNA in a shorter time through a single low-power ultrasound, and has less effect on the membrane structure of exosomes and the structure of siRNA; 293F-exo prepared by ultrasonic microfluidics 293F-exo-siRNA can be quickly taken up by cells, therefore, ultrasonic microfluidics provides a new and effective encapsulation method for exosomes as drug carriers.

Description

Technical Field

[0001] The present invention belongs to the field of biopharmaceuticals, and specifically relates to the application of ultrasonic microfluidics in exosome drug encapsulation. Background Art

[0002] Exosomes can be used as a communication tool between cells. Almost all types of cells in the human body can secrete exosomes. Exosomes are widely present and distributed in various body fluids (such as blood, saliva, urine, cerebrospinal fluid and milk). They carry a variety of proteins, mRNA, miRNA and lipid substances. Small interfering RNA (siRNA), as one of the classic tools of RNAi therapy, can "silence" the expression of specific genes with complementary sequences. siRNA binds to the target gene messenger RNA (mRNA) in a complementary base pairing manner and exerts a gene silencing effect, thereby being used for the treatment of specific diseases. As of 2022, three siRNA drugs have been approved for marketing by the US Food and Drug Administration (FDA), and more than a dozen small interfering RNA drugs are in the clinical research stage. siRNA has the technical advantage of quickly targeting any gene. As a good delivery carrier, exosomes have been successfully used for the delivery of siRNA in preclinical and clinical trial stages to treat various diseases, including neurological diseases, tumors, eye diseases and kidney diseases. Currently, relevant literature reports that the drug loading efficiency of exosome-loaded siRNA is distributed between 15% and 25%. We should explore more effective drug loading methods to solve the problem of low drug loading efficiency and maximize the potential of exosomes in treating diseases.

[0003] At present, there are two main ways for siRNA to enter exosomes: electroporation and ultrasound. In addition to optimizing the voltage setting and time to achieve load consistency, electroporation will cause certain damage to exosomes and siRNA. Sample aggregation and precipitation can be clearly seen under a microscope, which will affect subsequent experimental observations. The ultrasound method puts the exosomes into water bath ultrasound multiple times, which will cause the reorganization / deformation of the exosomes and destroy the integrity of the exosomes. The simple incubation method has low drug loading efficiency and is not favored by the majority of scientific researchers. Today, many researchers are developing exosome treatment or diagnostic methods, but if exosomes are to be ultimately used in disease treatment, in-depth research is needed on how to effectively load drugs into exosomes. Therefore, exploring new loading methods can give full play to the greater application value of exosomes.

[0004] The ultrasonic microfluidic reactor consists of an automatic sampling pump, an ultrasonic generator and an ultrasonic microfluidic channel. The microfluidic channel structure is designed to match the ultrasonic field. Ultrasonic waves produce cavitation in the fluid. Cavitation bubbles move, oscillate and collapse rapidly under the action of ultrasonic waves, and produce acoustic streaming, jets and other phenomena, quickly mixing the fluid like a miniature stirrer. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides an application method of ultrasonic microfluidics in exosome drug encapsulation.

[0006] Including: Application of ultrasonic microfluidics in exosome drug encapsulation.

[0007] Furthermore, the exosomes are 293F cell exosomes (293F-exo), and the drug is siRNA.

[0008] Furthermore, the exosomes are milk exosomes or mesenchymal stem cell exosomes.

[0009] Furthermore, the drug is mRNA, protein or small molecule drug.

[0010] Furthermore, the conditions of the ultrasonic microfluidics are a power of 30 W and a mass ratio of exosomes to siRNA of 1:1.

[0011] Furthermore, the ultrasonic microfluidic process is achieved by an ultrasonic microfluidic reactor.

[0012] Furthermore, the ultrasonic microfluidic reactor model is Mozi No. 1, purchased from Moge Microfluidics Technology (Shantou) Co., Ltd.

[0013] The beneficial effects of the present invention are: 1) The present invention provides an application of ultrasonic microfluidics in drug encapsulation in exosomes; 2) The drug loading efficiency of exo-siRNA prepared by ultrasonic microfluidics is about 1.2 times that of electroporation and ultrasonic methods, and 1.44 times that of incubation method; 3) The ultrasonic microfluidics method uses cavitation to quickly mix the entire mixed system uniformly, so that siRNA quickly enters the exosomes, and has a higher drug loading efficiency. Compared with the ordinary ultrasonic method, the ultrasonic microfluidics method can complete the encapsulation of siRNA in a shorter time through a single low-power ultrasound, and has less damage to the membrane structure of the exosome and the structure of the siRNA; 4) The 293F-exo-siRNA prepared by the ultrasonic microfluidics method can be quickly taken up by cells. Therefore, the ultrasonic microfluidics method provides an effective encapsulation method for exosomes as drug carriers to deliver siRNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Electron micrographs of exosomes extracted from 293F cells;

[0015] Figure 2 Western blot of marker proteins of 293F-exo;

[0016] Figure 3 Particle size and quantity distribution of exosomes extracted from 293F cells;

[0017] Figure 4 Drug loading efficiency of various methods;

[0018] Figure 5 This is the electron microscopy image of 293F-exo after drug loading by ultrasonic microfluidics;

[0019] Figure 6 The potential measured by the nanoparticle size potential analyzer before and after exosome drug loading;

[0020] Figure 7 NTA measures the particle size and quantity distribution of exosomes after drug loading;

[0021] Figure 8 Co-localization of 293F-exo and cells;

[0022] Fig. 9 The co-localization image of siRNA cells successfully delivered by 293F-exo-FAMsiRNA obtained by ultrasound microfluidics DETAILED DESCRIPTION

[0023] The ultrasonic microfluidic reactor used in the present invention is Mozi No. 1, which was purchased from Moge Microfluidics Technology (Shantou) Co., Ltd. and FreeStyle TM 293-F cell line was purchased from Thermo fisher scientific, 293F cell exosomes were obtained by ultracentrifugation, exosome-related marker protein antibodies were purchased from Abcom (Shanghai, China), and the siRNA used was luciferase siRNA, 1.5 mg / ml.

[0024] Example 1: Method for extracting and characterizing exosomes

[0025] 1.293F exosome extraction and separation

[0026] (1) Differential centrifugation

[0027] The cell suspension was evenly divided into centrifuge tubes in a clean bench; the speed was set to 300g and centrifuged for 10 minutes; after the centrifugation was completed, the speed was set to 1200g and centrifuged for 10 minutes; after the centrifugation was completed, the speed was set to 10000g and centrifuged for 20 minutes; after the centrifugation was completed, the operation was carried out in a clean bench and the filter was filtered with a sterile filter;

[0028] (2) Ultracentrifugation extraction

[0029] Set the temperature to "4°C", the centrifugal force to "100000g", the time to "70min", and click "vacuum" to cool down; divide the clarified cell supernatant into ultracentrifuge tubes, balance them, place the ultracentrifuge tubes in the ultracentrifuge tube sleeves, and perform ultracentrifugation;

[0030] (3) PBS washing

[0031] After centrifugation, take out the ultracentrifuge tube with tweezers, pour 10 mL of PBS into the ultracentrifuge tube, blow the bottom of the ultracentrifuge tube several times with a pipette, balance it with PBS, and repeat the ultracentrifugation step;

[0032] (4) Resuspension

[0033] After centrifugation, use tweezers to take out the ultracentrifuge tube, pour the supernatant into the waste tank, take 300 μl PBS and add it to the ultracentrifuge tube, use a pipette to blow the bottom of the ultracentrifuge tube several times, transfer it to a centrifuge tube, and place it in a -80°C refrigerator.

[0034] 2. Western blot

[0035] (1) Glue preparation: Prepare 10% polyacrylamide gel separation gel, pour it into the middle of the gel plate, add concentrated gel, and insert the comb;

[0036] (2) Electrophoresis: After the concentrated gel solidifies, add 8 μg of exosome sample to the loading well, run the concentrated gel at 80 V for 30 min, and run to the bottom at 120 V;

[0037] (3) Transfer: After electrophoresis, remove the gel, activate the PVDF membrane in methanol, and place it on the transfer clip in the order of blackboard-sponge-filter paper-gel-PVDF membrane-filter paper-sponge-whiteboard. Insert the transfer clip into the transfer tank and transfer the membrane at a constant current of 300 mA in ice for 90 min.

[0038] (5) Blocking: After transfer, place the PVDF membrane in blocking solution for 20 min;

[0039] (6) Primary antibody incubation: Use TBST to rinse the blocked PVDF membrane, cut out the approximate band where the protein is located according to the location of the marker, and incubate it in the diluted primary antibody at 4°C overnight;

[0040] (7) Rinse the PVDF membrane with TBST three times, 10 minutes each time;

[0041] (8) Secondary antibody incubation: Place the PVDF membrane in the diluted secondary antibody and incubate for four hours;

[0042] (9) Rinse the PVDF membrane again with TBST for three times, 15 minutes each time;

[0043] (10) Add an appropriate amount of developer onto the membrane and expose it for imaging on a chemiluminescence imager.

[0044] 3. Electron microscopy (TEM)

[0045] Use tweezers to pick up a copper mesh and place it on a glass slide covered with a sealing film; perform hydrophilic treatment on the mesh, and after completion, use self-locking tweezers to pick up the copper mesh and place it on a culture dish so that the copper mesh is suspended in the air, and 4 μl of sample is added dropwise onto the copper mesh, and allowed to stand for 1 min. Place filter paper close to the mesh to absorb excess sample; 4 μl of phosphotungstic acid is added dropwise onto the copper mesh, and allowed to stand for about 45 s. The filter paper absorbs the dye solution, and the mesh is dried and observed.

[0046] 4. Particle size distribution

[0047] The nanoparticle tracking analyzer (NTA Nanosight 3000, NS300) uses a laser light source to illuminate the exosome suspension, and the Brownian motion of particles with scattered light can be clearly observed. Combined with the scattering intensity of the particles, the NTA software can draw a distribution diagram of the particle size and the corresponding number.

[0048] 5. Zeta potential

[0049] The zeta potential of exosomes was detected using a nanoparticle size potential analyzer (DLS). About 1.5 ml of exosomes resuspended in PBS were added to the sample measurement cell for Zeta potential detection.

[0050] Figure 1 This is an electron micrograph of exosomes extracted from 293F cells, showing that the extracted exosomes are in a regular bowl shape; Figure 2 This is a Western blot image of the marker protein of 293F-exo, indicating that exosomes were successfully separated from cells; Figure 3 This is the particle size and quantity distribution diagram of exosomes extracted from 293F cells, which shows that the particle size and quantity distribution diagram of the extracted exosomes has a single peak with a peak value of 86nm.

[0051] Example 2 Cell experiment

[0052] 1. Cell Culture

[0053] Take out a tube of frozen cells and revive them in a T25 culture flask. Observe the adhesion of the cells the next day. When the cells expand to 90%, wash them twice with PBS, add trypsin digestion solution, and pass them at a ratio of 1:3. When the cells are in good condition, they can be plated for the next experiment.

[0054] 2. Cellular Uptake

[0055] (1) Cells were plated in 24-well plates with approximately 6 × 10 4cells, add 1 ml of culture medium and culture overnight;

[0056] (2) DiR-labeled exosomes: Take 20 μl of exosomes. Similarly, if you want to see the uptake of 293F-exo-FAM-siRNA by cells, take the drug-loaded exosomes, add 2.5 μl of DiR dye working solution, mix by ultrasound for 1 min, incubate at 37°C for 30 min, terminate the reaction at 4°C for 30 min, add PBS to replenish the liquid, and remove the unbound dye through an ultrafiltration tube;

[0057] (3) Sample addition: add 5 μl of serum-free culture medium containing exosomes to each well;

[0058] (4) Fix the cell samples for a certain period of time, aspirate the liquid in the wells, wash twice with PBS, fix with 500 μl 4% paraformaldehyde, and let stand for 15 min;

[0059] (5) Nuclear staining: Add 100 μL of DAPI dye to each well, let it stand for 30-40 min, then remove excess liquid, wash three times with PBS, and observe.

[0060] Example 3 Loading siRNA into exosomes

[0061] Method 1: Ultrasonic microfluidics

[0062] The exosome and siRNA solution were filtered through a 0.22 μm microporous filter membrane; the exosomes and siRNA were mixed in a certain proportion and sucked into the syringe; the syringe and the introduction tube were connected and installed on the automatic injection pump; the specification of the injection pump was set to 1 ml, the transmission liquid volume was 1.000 ml, the injection flow rate was 200 μl / min, the power was set, and the operation was clicked. When the total flow rate of the outflow tube was observed to be greater than the dead volume of 1 residence time (about 200 μl), the sample was started, and the outflowing liquid was collected with a collection tube. The collection was stopped 1-2 s before the end of the injection (about 100 μl), and the total dead volume was considered to be 300 μl; the mixture was incubated at room temperature for 1 hour to measure the encapsulation efficiency.

[0063] Firstly, the encapsulation efficiency of ultrasonic microfluidics under different conditions was investigated, with the power set at 15 W, 30 W, and 45 W, and different exosome and siRNA mixing ratios (w / w) set at 1:1 and 1:3, respectively.

[0064] After measurement,

[0065] When the power was 15 W and the mass ratio of exosomes to siRNA was 1:1, the encapsulation efficiency was 30.6%;

[0066] When the power was 15 W and the mass ratio of exosomes to siRNA was 1:3, the encapsulation efficiency was 22.7%;

[0067] When the power was 30 W and the mass ratio of exosomes to siRNA was 1:1, the encapsulation efficiency was 31.1%;

[0068] When the power was 30 W and the mass ratio of exosomes to siRNA was 1:3, the encapsulation efficiency was 21.0%;

[0069] When the power was 45 W and the mass ratio of exosomes to siRNA was 1:1, the encapsulation efficiency was 24.5%;

[0070] When the power was 45 W and the mass ratio of exosomes to siRNA was 1:3, the encapsulation efficiency was 18.7%;

[0071] Therefore, when the conditions of the ultrasonic microfluidics method were set to a power of 30 W, a mass ratio of exosomes to siRNA of 1:1, and an injection flow rate of the automatic injection pump of 200 μl / min, the optimal encapsulation efficiency was 31.1%.

[0072] When the power was 30W and the mass ratio of exosomes to siRNA was 1:1, the average encapsulation efficiency of siRNA loaded with milk exosomes was 32.3%.

[0073] When the power was 30 W and the mass ratio of exosomes to siRNA was 1:1, the average encapsulation efficiency of siRNA loaded by mesenchymal stem cell exosomes was 29.94%.

[0074] Method 2: Simple incubation method

[0075] Thaw exo and siRNA on ice; pipette exo and siRNA and mix them in PBS according to a certain ratio; incubate at room temperature for 3.5 hours and vortex every 30 minutes.

[0076] Method 3: Electroporation

[0077] Thaw exo and siRNA on ice; pipette exo and siRNA into PBS in a certain proportion; pre-cool the electroporation cup on ice; transfer the mixture to the electroporation cup and use the electroporation system (Gene PulserXcell TM , BioRad, US), and electroporation was performed under the conditions of voltage 160 V, pulse time 10.0 ms, pulse number 3 times, and time interval 2 s. The mixture was incubated at room temperature for 1 hour to restore the membrane structure, and then the encapsulation efficiency was measured.

[0078] Method 4: Ultrasound

[0079] The exo and siRNA mixed in a certain ratio were placed in a water bath ultrasonic box (Shanghai Bilang Instrument Manufacturing Co., Ltd., BILON10-300B, China) with a power of 100 W and an ultrasonic time of 30 s, and then placed on ice for 2 min, for 4 cycles; the mixture was incubated at room temperature for 1 hour to restore the membrane structure, and then the encapsulation efficiency was measured.

[0080] Encapsulation efficiency determination method: Take two portions of the mixture after encapsulation, add 2% TritonX100 (Biyuntian ST795) solution to one portion for "demulsification", and use RNA fluorescence quantitative kit (Quant-iT RiboGreen RNA kit) to detect the total siRNA content after free and demulsification. Use PBS containing only exosomes as a control to detect the concentration of siRNA therein.

[0081]

[0082] Ultrasonic microfluidics, electroporation, incubation, and ultrasound were used to load siRNA. The encapsulation rates of various methods were measured. The results are as follows: Figure 4 As shown, when the power is 30 W and the mass ratio of exosomes to siRNA is 1:1, the encapsulation efficiency of the ultrasonic microfluidics method is 31.1%, the electrotransfer method is 25.96%, the incubation method is 21.57%, and the ultrasonic method is 25.79%. The drug loading efficiency of the ultrasonic microfluidics method is about 1.2 times that of the electrotransfer and ultrasonic methods, and 1.44 times that of the simple incubation method.

[0083] like Figure 5 As shown, the transmission electron microscopy image of exosomes after drug loading is shown. The morphology and particle size of 293F-exo did not change after drug loading by ultrasonic microfluidics, and its original physical characteristics were maintained.

[0084] like Figure 6 As shown, the potential changes before and after exosome loading are represented, indicating that the potential change of 293F-exo-siRNA prepared by electroporation is the most obvious.

[0085] like Figure 7 As shown, the particle size and number distribution diagram of exosomes after drug loading showed that the number of exosomes after drug loading decreased and the particle size generally increased, especially for the exosomes obtained by electroporation.

[0086] Ultrasonic microfluidics is more efficient than other drug delivery methods.

[0087] The ultrasonic microfluidics method uses cavitation to quickly mix the entire mixed system uniformly, allowing siRNA to quickly enter the exosomes, with a higher drug loading efficiency. Compared with the ordinary ultrasonic method, the ultrasonic microfluidics method can complete the encapsulation of siRNA in a shorter time through a single low-power ultrasound, with less damage to the membrane structure of the exosomes and the structure of siRNA.

[0088] Example 4 Analysis of the results of cell experiments

[0089] 293F-exo and 293F-exo-FAMsiRNA prepared by ultrasound microfluidics can be taken up by cells in large quantities

[0090] 293F-exo refers to exosomes that are extracted from cells without any processing. 293F-exo-FAMsiRNA refers to siRNA labeled with FAM fluorescence in order to observe the entry of the drug into the exosomes of cells. FAMsiRNA refers to siRNA labeled with fluorescent FAM.

[0091] 293F-exo was taken up by A549, Beas-2B, L929, and HFL-1 cells after 6 hours. Figure 8 The co-localization of 293F-exo and cells indicates that exo can enter cells in a short time, showing the advantages of exo in drug delivery.

[0092] After the drug loading by ultrasonic microfluidics, the unloaded siRNA was removed by ultrafiltration, and the uptake of 293F-exo-FAMsiRNA by cells at different time points was measured. Fig. 9 As shown, 293F-exo-FAM siRNA was taken up in large quantities, and the fluorescence intensity at 24 h was increased compared with that at 12 h, indicating that 293F-exo-siRNA entered the cells in a time-dependent manner.

[0093] DAPI labels the cell nucleus and displays blue fluorescence, DIR-exo indicates that 293F-exo labeled with DIR displays red light, FAMsiRNA indicates that FAM-labeled siRNA displays green light, and Merge indicates that the two fluorescent signals at the same position in the cell overlap.

[0094] The drug loading efficiency of exo-siRNA prepared by ultrasound microfluidics is about 1.2 times that of electroporation and incubation method, and 1.44 times that of simple incubation method. In addition, ultrasound microfluidics has the advantages of less damage to exosomes and siRNA, and is fast and simple compared with the other three methods. 293F-exo-siRNA prepared by ultrasound microfluidics can be quickly taken up by cells. Therefore, ultrasound microfluidics provides a new reference for exosomes as drug carriers to deliver siRNA.

Claims

1. The application of ultrasonic microfluidics in exosome drug encapsulation is characterized by: The exosomes are 293F cell exosomes, the drug is siRNA, the ultrasonic microfluidics condition is a power of 30W, the mass ratio of exosomes to siRNA is 1:1, the ultrasonic microfluidics process is achieved through an ultrasonic microfluidics reactor, and the ultrasonic process is a single ultrasonic process.

2. A method for applying ultrasonic microfluidics in exosome drug encapsulation, characterized in that: The 293F cell exosomes and siRNA solution were filtered through a micron microporous filter membrane; the 293F cell exosomes and siRNA were mixed at a mass ratio of 1:1, and the mixture was sucked into a syringe; the syringe and the introduction tube were connected and installed on an automatic injection pump; the specifications of the injection pump were set to 1ml, the transmission liquid volume was 1.000ml, the injection flow rate was 200μl / min, the ultrasonic power was set to 30W, and the run was clicked. When the total flow rate of the outflow tube was observed to be greater than the dead volume of 1 residence time, the sample was started, and the outflowing liquid was collected with a collection tube. The collection was stopped 1-2 seconds before the end of the injection, and the dead volume was totaled; the mixture was incubated at room temperature, and the encapsulation efficiency was measured.

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