Electroporation regulation and control method of exosome loaded miRNA
By optimizing the electroporation method for loading miRNA onto exosomes, the aggregation problem after electroporation was solved, the optimal electroporation conditions were determined, the loading efficiency and molecular stability were improved, and efficient miRNA loading and exosome recovery were achieved.
Patent Information
- Application Number
- CN202511650733.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-12
- Publication Date
- 2026-02-06
AI Technical Summary
Existing electroporation methods for loading miRNA onto exosomes suffer from aggregation problems and lack standardized optimal electroporation conditions, resulting in insufficient loading efficiency and molecular stability.
By obtaining the relationship between the concentration of the analyte, pulse parameters, and aggregation parameters, electroporation parameters with an aggregation rate less than a first preset value and an aggregation rate greater than a second preset value are selected. Combined with fluorescence labeling and image analysis, the electroporation process is dynamically controlled to optimize electroporation conditions, thereby reducing aggregation and improving loading efficiency and molecular stability.
This approach achieves low aggregation rate and high migration rate of miRNAs in exosomes, improves electroporation efficiency and exosome recovery rate, reduces damage to exosomes during electroporation time, and ensures the integrity of exosome membrane structure.
Smart Images
Figure CN121472334A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of electroporation, in particular, to an electroporation method for loading miRNA into exosomes. BACKGROUND
[0002] Exosomes (EXOS) are extracellular lipid bilayer vesicles secreted by many cell types in prokaryotes and eukaryotes under physiological and pathological conditions. These vesicles are about 40 to 160 nm in diameter, with an average diameter of about 100 nm. EXOS can be released into the extracellular environment by osmotic action and play a key role in intercellular communication by transporting proteins, lipids and nucleic acids, including long non-coding RNA (lncRNA), microRNA (miRNA) and messenger RNA (mRNA). This transport is essential for maintaining the balance of proteins and lipids. Among the nucleic acids in exosomes, miRNA is the most important RNA species. miRNA is a small RNA molecule that does not encode proteins. The function of miRNA is to act as a guide molecule in RNA silencing and to regulate gene expression by promoting the degradation of information RNA (mRNA) or inhibiting mRNA translation. Exosome-transported miRNA can regulate gene expression because the encapsulation of exosomes protects miRNA from ribonuclease activity in the blood circulation. Many research results show that exosome-transported miRNA has therapeutic potential.
[0003] However, developing a safe and effective method to load miRNA into exosomes is still a key challenge for therapeutic applications. Many methods have been explored to optimize the loading of miRNA, and the method of electroporation is widely used for the loading of miRNA, although it is simple to operate, but due to the aggregation of nucleic acids after electroporation, it often leads to the aggregation of exosomes. The currently reported electroporation instrument is Bio-Rad's gene pulser II electric transfer system, however, there is no uniform standard for the electroporation conditions of exosomes (such as electroporation buffer, voltage and capacitance) of the same type of instrument, and the different electroporation conditions reported in different documents lead to the fact that researchers cannot choose an electroporation scheme with the highest loading efficiency in time when conducting similar researches, such as in the research on acute lung injury in sepsis and the research on the inhibition of BGC-823 cell proliferation by exosome-delivered miRNA-21 , the electroporation conditions used by the researchers are 150 V, 100 μF; in the research on myocardial ischemia and reperfusion injury , the electroporation conditions are 350 V, 150 μF; in a study of endometrium , the electroporation conditions are set to pulse 20 ms and 500v for electroporation. The type of electroporation buffer also plays a crucial role in drug loading of exosomes. Currently, the electroporation buffer used is mostly the instrument matching electroporation buffer, but it is proposed that using 50mM trehalose solution as the electroporation buffer can improve cell survival rate and transfection efficiency, and has the potential to maintain the stability of exosomes and reduce aggregation .
[0004] If the aggregation problem after electroporation can be solved and the optimal electroporation conditions are determined, the loading efficiency and molecular stability of this method can be improved, and the research efficiency can be improved. SUMMARY
[0005] The purpose of the present application is to provide an exosome loaded miRNA electroporation regulation method, which solves the aggregation problem after electroporation and determines the optimal electroporation conditions to improve the loading efficiency and molecular stability.
[0006] The embodiment of the present application is realized by the following technical solutions:
[0007] An exosome loaded miRNA electroporation regulation method, comprising:
[0008] S100, obtaining the relationship between the concentration of the test substance, the pulse parameters and the aggregation parameters of the test substance; the aggregation parameters include: aggregation speed and aggregation rate; the test substance includes: exosomes and miRNA;
[0009] S200, selecting the test substance concentration and pulse parameters corresponding to the aggregation rate less than the first preset value and the aggregation speed greater than the second preset value as the actual use of the electroporation parameters.
[0010] Preferably, the method for obtaining the aggregation parameters comprises:
[0011] The test substance is labeled with fluorescence, and the fluorescence distribution image is continuously obtained during the electroporation process to obtain an image group, and the aggregation parameters are obtained through the image group.
[0012] Preferably, the exosome loaded miRNA electroporation regulation method comprises: dividing the fluorescence distribution image into a plurality of blocks, and obtaining the position information and gray value corresponding to each block;
[0013] Obtain the gray value change of the blocks corresponding to the same position information in different fluorescence distribution images; and obtain the aggregation end time according to the gray value change.
[0014] Preferably, the exosome loaded miRNA electroporation regulation method comprises: obtaining the gray value deviation of the blocks and their surrounding blocks, and setting two blocks as the same aggregate when the gray value deviation of the two blocks is less than a preset deviation value.
[0015] Obtain the number of tiles corresponding to different clusters, and calculate the cluster area based on the number of tiles.
[0016] Preferably, the electroporation regulation method for exosome-loaded miRNA includes: acquiring stable patches, wherein the stable patches refer to patches whose grayscale value change rate is less than a preset change rate;
[0017] Set continuous stable tiles as stable clusters and obtain the growth status of stable clusters;
[0018] Adjust the electrotransfer parameters according to the growth of stable aggregates.
[0019] Preferably, the electroporation regulation method for exosomes loaded with miRNA includes: obtaining the relationship between the integrity of the exosome membrane structure and the electroporation parameters, and obtaining the maximum value of the pulse parameters while ensuring the integrity of the exosome membrane structure;
[0020] With the pulse parameter set to the maximum value, the growth of stable aggregates after their aggregation area reaches a preset area threshold is obtained.
[0021] If the aggregated area begins to decrease, the pulse parameter remains unchanged; if the aggregated area continues to increase, the time when the aggregated area reaches the preset area threshold is obtained, and the relationship between the decrease in the pulse parameter and the aggregated area of the stable aggregate is obtained; the time is the pulse parameter adjustment time.
[0022] Obtain the pulse parameter reduction value when the clustered area remains stable at or below the preset area threshold.
[0023] Preferably, the electroporation regulation method for exosome-loaded miRNA includes: acquiring blank control images continuously during electroporation when no analyte is added to the system; acquiring difference images between fluorescence distribution images and blank control images at the same time; and forming an image group from several difference images.
[0024] Preferably, the electroporation regulation method for exosome-loaded miRNA includes: obtaining the loading rate of the analyte by measuring the change in the distribution area of the analyte; and obtaining a preset area threshold based on the loading rate of the analyte.
[0025] The present invention has at least the following beneficial effects:
[0026] This invention continuously adjusts the analyte concentration and pulse parameters by detecting the aggregation parameters of electroporation results to obtain optimal electroporation parameters. This ensures that miRNAs exhibit a certain migration rate and a low aggregation rate during electroporation, while also reducing exosome aggregation and improving exosome recovery. A higher migration rate improves electroporation efficiency and reduces exosome damage caused by excessive electroporation time. A low aggregation rate promotes miRNA entry into exosomes, facilitating higher loading rates. To better track the migration and aggregation of analytes during electroporation, this invention uses existing technology for fluorescent labeling of the analytes. Fluorescence distribution images are continuously acquired during electroporation, and the migration and aggregation of analytes can be determined by analyzing the fluorescence distribution and intensity in these images. This allows for the acquisition of optimal electroporation parameters, improving electroporation efficiency and exosome recovery rates while ensuring adequate nucleic acid loading. Attached Figure Description
[0027] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.
[0028] Figure 1 Fluorescence distribution diagram of electroporation parameters when trehalose is added to the electroporation system;
[0029] Figure 2 The fluorescence distribution diagram shows the effect of adjusting the electrotransfer parameters when there is no trehalose in the electrotransfer system. Detailed Implementation
[0030] To make the objectives, methods, and advantages of the embodiments of the present invention clearer, the methods in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments.
[0031] Example 1: An electroporation regulation method for exosomes loaded with miRNA, comprising:
[0032] S100: Obtain the relationship between analyte concentration, pulse parameters, and analyte aggregation parameters; the aggregation parameters include aggregation rate and aggregation efficiency; the analytes include exosomes and miRNAs.
[0033] S200. Select the analyte concentration and pulse parameters corresponding to the aggregation rate being less than the first preset value and the aggregation rate being greater than the second preset value as the actual electroporation parameters used.
[0034] In practice, electroporation creates transient pores in the exosome membrane by applying an electric field, allowing nucleic acid drugs such as miRNA to rapidly enter the exosome. The aggregation of nucleic acids is due to at least the following reasons:
[0035] 1. During the application of an electric pulse, negatively charged nucleic acid molecules migrate electrophoretically towards the anode under the influence of an electric field. When they reach the vicinity of the exosome membrane, due to the negative charge of the transient hydrophilic pores formed on the membrane surface during electroporation, nucleic acids are adsorbed and enriched in large quantities around the membrane pores through electrostatic interactions, forming local high-concentration areas, thus resulting in visible aggregates.
[0036] 2. The nanoscale pores formed by electroporation typically have a diameter of 2-5 nm, which physically restricts the free diffusion of larger nucleic acid molecules. These macromolecules become congested and entangled when attempting to pass through the narrow pores, especially at high nucleic acid concentrations or long pulse durations, where this steric hindrance exacerbates aggregation.
[0037] 3. The composition of electroporation buffer has a significant impact on aggregation. Buffers with low ionic strength or containing specific sugars, such as trehalose, may reduce cell damage, but they may also alter the solvation state of nucleic acids, thus promoting their proximity and aggregation. While the ionic components in standard electroporation buffer can reduce electrostatic repulsion by shielding charges, aggregation can still occur under improper conditions, such as excessively high pulse parameters.
[0038] 4. Excessively long pulse duration or excessively high electric field strength can prolong the membrane pore opening time, causing more nucleic acids to continuously migrate and remain near the pores, increasing the risk of aggregation.
[0039] Following electroporation, nucleic acid aggregation forms large complexes that are difficult to penetrate the nanoscale pores created by electroporation, significantly reducing the success rate of exogenous gene delivery. High concentrations of nucleic acid aggregates also exert additional physical and chemical stress on the cell membrane, exacerbating cell damage and leading to decreased cell viability and increased mortality. Disruption of the exosome membrane structure increases surface hydrophobicity, promoting exosome adhesion and aggregation, ultimately resulting in decreased exosome recovery or reduced analyte loading efficiency. Therefore, simultaneously controlling the aggregation of analytes and exosomes through electroporation parameters can significantly improve electroporation efficiency.
[0040] Many factors can lead to analyte aggregation, such as analyte concentration, buffer composition, and pulse parameters. This can easily cause batch-to-batch fluctuations, making experimental data unstable and difficult to optimize and standardize electroporation conditions. Therefore, the applicant hopes to reduce analyte aggregation and improve exosome recovery and miRNA loading efficiency by controlling the electroporation process.
[0041] The applicant used the existing electroporation buffer Bio-Rad Gene Pulser Electroporation Buffer, mixing exosomes (2 μg / μL) with the buffer at a 1:1 volume ratio and incubating at room temperature for 5 minutes. Then, miRNA mimics were added to the mixture, thoroughly mixed, and transferred to a 0.2 cm cold electroporation cuvette for electroporation. The concentration of the analyte and the pulse parameters were continuously adjusted by monitoring the electroporation results (aggregation parameters) to obtain optimal electroporation parameters. This ensured that the miRNA exhibited a certain migration rate and a low aggregation rate during electroporation, and that the exosomes also had a low aggregation rate. A high migration rate improves electroporation efficiency and reduces exosome damage caused by excessive electroporation time. A low aggregation rate promotes miRNA entry into exosomes while maintaining the structural integrity of the exosomes, which is beneficial for improving loading and exosome recovery rates.
[0042] As an example, the pulse parameters can be any one of voltage, pulse duration, and pulse count.
[0043] Example 2: The method for obtaining the aggregation parameters includes:
[0044] Different analytes are labeled with different colors of fluorescence, and fluorescence distribution images are continuously acquired during the electroporation process. Aggregation parameters are obtained through the fluorescence distribution images.
[0045] In the specific implementation process, to better track the migration and aggregation of analytes during electroporation, this embodiment employs existing technologies for fluorescent labeling of the analytes. For example, the analytes are labeled with the fluorescent dye Cy5. The amino-modified analytes are reacted with NHS ester fluorescent dyes (such as Cy3-NHS) in a pH 8.5 buffer solution. After purification, the labeled product is obtained. Unbound fluorescent dyes are removed using methods such as dye removal columns or gel filtration to ensure a labeling efficiency >95%. The labeling ratio is quantified using a fluorescence spectrophotometer. During electroporation, fluorescence distribution images are continuously acquired, and the migration and aggregation of the analytes can be determined by analyzing the fluorescence distribution and intensity in the images.
[0046] When analyzing the aggregation of exosomes or miRNAs, different fluorescence can be excited to create a fluorescence distribution image that shows the aggregation of miRNAs or exosomes.
[0047] Example 3: In this example, the fluorescence distribution image is divided into several patches, and the location information and gray value of each patch are obtained; the gray value changes of the patches corresponding to the same location information in different fluorescence distribution images are obtained; and the aggregation end time is obtained based on the gray value changes.
[0048] In practice, after sorting different fluorescence distribution images by time series, the change in grayscale value of the same location patch over time can be easily obtained. The aggregation status can be judged based on the rate of change of patch grayscale value. For example, if the number of patches whose grayscale value changes still exceed a preset grayscale change threshold exceeds a preset percentage, it indicates that the migration of the object under test is still continuing. Conversely, it indicates that the migration has basically ended, and the aggregation end time can be characterized by the time of the fluorescence distribution image corresponding to the end of the migration.
[0049] One purpose of setting a grayscale change threshold is to eliminate systematic errors as much as possible. For example, the grayscale values obtained by the system may deviate under the same fluorescence intensity. Another purpose of setting a grayscale change threshold is that when the fluorescence intensity change is small, it may be due to fluorescence loss rather than the migration of the analyte. For example, the luminescence efficiency of the fluorophore decreases with the extension of irradiation time. Therefore, a grayscale change less than the threshold may indicate that the migration of the analyte may have ended.
[0050] The preset percentage can be set according to the actual situation, such as 90%.
[0051] Example 4: Obtain the grayscale value deviation between a tile and its surrounding tiles. When the grayscale value deviation between the two tiles is less than a preset deviation value, set the two tiles as the same aggregate.
[0052] Obtain the number of tiles corresponding to different clusters, and calculate the cluster area based on the number of tiles.
[0053] In practice, when the grayscale value deviation between two adjacent patches is less than a preset deviation value, the objects corresponding to the two patches are either fluorescently labeled analytes or system solutions. If the grayscale value determines that the patch is a fluorescently labeled analyte, adjacent patches with similar grayscale values can be considered as an aggregate, and the size of the aggregate, i.e., the aggregate area, can be represented by the number of patches.
[0054] As an example, the electrotransfer parameters corresponding to the maximum aggregation rate when the aggregate size is less than the threshold are selected as the parameters to be used in practice.
[0055] Example 5: The electroporation regulation method for exosome-loaded miRNA further includes: acquiring stable patches, wherein the stable patches refer to patches whose grayscale value change rate is less than a preset change rate;
[0056] Set continuous stable tiles as stable clusters and obtain the growth status of stable clusters;
[0057] Adjust the electrotransfer parameters according to the growth of stable aggregates.
[0058] In practice, the size of aggregates may vary at different times. This involves the migration of nucleic acid molecules toward exosomes and their entry into exosomes, as well as the aggregation process of exosomes. The difference between the aggregation rate of miRNAs and their entry into exosomes can also affect whether the area of the aggregates increases or decreases. Therefore, during electroporation, the degree of change in the size of some aggregates is difficult to directly characterize the aggregation rate.
[0059] Changes in aggregation rate can regulate the rate at which nucleic acid molecules enter exosomes. Therefore, the applicant aims to regulate the aggregation rate by dynamically varying electroporation parameters, thereby promoting nucleic acid entry into exosomes. Increasing the rate of nucleic acid entry into exosomes can reduce the impact of nucleic acid aggregates on the exosome membrane structure, thus reducing exosome aggregation.
[0060] In this embodiment, a continuous stable patch refers to any stable patch being adjacent to at least one stable patch within a stable aggregate. When the number of stable patches in a stable aggregate increases, it can be considered that new nucleic acids are accumulating in the stable aggregate, and the aggregation rate is greater than the rate at which nucleic acids from the stable aggregate enter the exosome. When the number of stable patches in a stable aggregate decreases, it can be simply considered that the stable aggregate is effectively entering the exosome. Since excessively large stable aggregates can hinder nucleic acid entry into the exosome, a maximum number of patches corresponding to a stable aggregate can be initially set. While ensuring the aggregation rate, the growth of the stable aggregate is monitored during electroporation. If the aggregation area begins to shrink before reaching the aforementioned maximum number, the pulse parameters, such as voltage, can be increased to a certain extent to increase the aggregation rate. When the aggregation area of the stable aggregate reaches the aforementioned maximum number and continues to increase, the pulse parameters can be decreased to a certain extent to reduce the aggregation rate. The maximum number of patches corresponding to a stable aggregate can be obtained through the exosome pore size or simple experimental methods. The experiment can obtain the relationship between the maximum number of patches corresponding to stable aggregates and the nucleic acid load in exosomes, and then select a value that will not affect the final load.
[0061] Example 6: The electroporation regulation method for exosomes loaded with miRNA includes: obtaining the relationship between exosome membrane structure integrity and electroporation parameters, and obtaining the maximum value of the pulse parameters while ensuring the exosome membrane structure integrity;
[0062] With the pulse parameter set to the maximum value, the growth of stable aggregates after their aggregation area reaches a preset area threshold is obtained.
[0063] If the aggregated area begins to decrease, the pulse parameter remains unchanged; if the aggregated area continues to increase, the time when the aggregated area reaches the preset area threshold is obtained, and the relationship between the decrease in the pulse parameter and the aggregated area of the stable aggregate is obtained; the time is the pulse parameter adjustment time.
[0064] Obtain the pulse parameter reduction value when the clustered area remains stable at or below the preset area threshold.
[0065] In practice, during the migration and aggregation of nucleic acids towards and around exosomes, nucleic acids can usually enter the exosomes effectively. Therefore, to improve process efficiency and the nucleic acid loading in the aforementioned process, the maximum voltage that does not affect the structural integrity of the exosome membrane can be selected. At higher voltages, the aggregation rate of nucleic acids towards exosomes is usually greater than the rate of nucleic acid entry into the exosomes, resulting in a gradual increase in the aggregation area of the aggregates. However, due to the limited pore size of the channels on the exosomes, an excessively large aggregation area can hinder nucleic acid entry into the exosomes, thus requiring a certain reduction in voltage. Furthermore, the large-scale aggregation of miRNAs outside the exosome membrane can also affect the structural integrity of the exosome membrane, further increasing the tendency for exosome aggregation. Reducing the voltage can weaken the aggregation of miRNAs and exosomes, further improving the exosome recovery rate and miRNA loading efficiency.
[0066] As an example, when the pulse parameters are reduced, the aggregation area can be kept below the preset area threshold, at which point the rate of nucleic acid entry into exosomes and the recovery rate of exosomes are ensured.
[0067] As an example, when the pulse parameter is reduced and the aggregation area can be maintained at a preset area threshold, the aggregation rate is comparable to the rate at which nucleic acids enter the exosomes. The value of the pulse parameter affects the aggregation rate; generally, the larger the pulse parameter, the greater the aggregation rate. Therefore, when the aggregation area can be maintained at the preset area threshold, the aggregation rate of exosomes is higher, and the corresponding rate of nucleic acid entering the exosomes is also faster. This can improve the efficiency of nucleic acid loading on exosomes, while reducing the aggregation of nucleic acids outside the exosomes can reduce the damage to the exosome membrane structure and improve the exosome recovery rate.
[0068] The methods for detecting the integrity of exosome membrane structures are existing technologies. For example, transmission electron microscopy can be used to directly observe the bilayer membrane structure of exosomes (which are saucer-shaped or hemispherical), and at a resolution of 0.1–0.2 nm, it is possible to visually determine whether the membrane is ruptured or deformed. Another method is to scan the membrane surface morphology and mechanical properties using probes. Yet another method is to treat exosomes with proteases; if the membrane is intact, the internal proteins (such as TSG101) are not degraded; if the membrane is damaged, the proteins are hydrolyzed.
[0069] Example 7: In the case of Examples 2-6, the electroporation regulation method for exosome-loaded miRNA in this example includes: acquiring blank control images continuously during electroporation when no analyte is added to the system; acquiring the difference images between the fluorescence distribution image and the blank control image at the same time; and forming an image group from several difference images.
[0070] In this specific implementation, to simplify the image processing, a blank control image was acquired. When acquiring the blank control image, no analyte, such as miRNA or exosome, was added to the electroporation system; all other conditions were the same as when acquiring the fluorescence distribution image. By subtracting the fluorescence distribution image from the blank control image at the same time point, the distribution of the analyte in the system can be obtained. This facilitates the analysis of the relationship between the analyte distribution and the electroporation parameters to obtain the optimal electroporation parameters.
[0071] As an example, when performing subtraction, the image features of exosomes in the blank control image and the image features of the system solution can be obtained. Subtracting the portions of the fluorescence distribution image that share the same image features as described above yields a differential image characterizing the distribution of miRNAs. Similarly, differential images characterizing the distribution of exosomes can also be obtained.
[0072] Example 8: The electroporation regulation method for exosome-loaded miRNA in this example includes: obtaining the nucleic acid loading rate by the change in the distribution area of miRNA; and obtaining a preset area threshold based on the nucleic acid loading rate.
[0073] In practice, the miRNA distribution area refers to the area of miRNA distribution outside the exosome. By monitoring the rate of decrease in the miRNA distribution area, the rate at which miRNA enters the exosome can be determined. This allows us to obtain the relationship between the aggregation area of stable aggregates and the rate of decrease in the miRNA distribution area. A minimum rate of miRNA entry into the exosome can be preset, and the aggregation area of stable aggregates corresponding to this minimum rate can be obtained as a preset area threshold. When the rate of nucleic acid entry into the exosome is ensured, exosome aggregation can be reduced to some extent.
[0074] Since miRNA aggregation and exosome aggregation have a certain mutual influence, in a miRNA-free system, by obtaining the optimal pulse parameters while ensuring the integrity of the exosome membrane structure, the influence of pulse parameters on exosome integrity can be avoided or reduced, thereby avoiding or reducing the influence of pulse parameters on exosome aggregation. Furthermore, by adjusting the pulse parameters according to the aggregation of exosomes and miRNAs, the miRNA loading efficiency can be ensured while improving the exosome recovery rate.
[0075] Figure 1 and Figure 2 All images show the distribution of the analytes during the regulation of electroporation parameters. Green fluorescence represents the fluorescence distribution of exosomes, red fluorescence represents the fluorescence distribution of miRNAs, and yellow fluorescence represents the fluorescence synthesis of both. The images demonstrate that regulating the pulse parameters significantly affects the aggregation of miRNAs and exosomes.
[0076] The above are merely preferred embodiments of the present invention and are not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.
Claims
1. A method for electroporation regulation of exosomes loaded with miRNA, characterized in that, include: S100: Obtain the relationship between the concentration of the analyte, pulse parameters, and the aggregation parameters of the analyte; The aggregation parameters include aggregation rate and aggregation speed; the analytes include exosomes and miRNAs. S200. Select the analyte concentration and pulse parameters corresponding to the aggregation rate being less than the first preset value and the aggregation rate being greater than the second preset value as the actual electroporation parameters used.
2. The electroporation regulation method for exosome-loaded miRNA according to claim 1, characterized in that, The method for obtaining the aggregation parameters includes: Different analytes are labeled with different colors of fluorescence, and fluorescence distribution images are continuously acquired during electroporation to obtain image sets. Aggregation parameters are obtained through these image sets.
3. The electroporation regulation method for exosome-loaded miRNA according to claim 2, wherein the fluorescence distribution image is segmented into several patches, and the position information and gray value corresponding to each patch are obtained; Obtain the grayscale value changes of the corresponding image patches at the same location in images with different fluorescence distributions; obtain the aggregation end time based on the grayscale value changes.
4. The electroporation regulation method for exosome-loaded miRNA according to claim 3, characterized in that, Obtain the grayscale value deviation between the tile and its surrounding tiles. When the grayscale value deviation between the two tiles is less than the preset deviation value, set the two tiles as the same aggregate. Obtain the number of tiles corresponding to different clusters, and calculate the cluster area based on the number of tiles.
5. The electroporation regulation method for exosome-loaded miRNA according to claim 4, characterized in that, include: Obtain stable patches, where a stable patch is a patch whose grayscale value change rate is less than a preset change rate; Set continuous stable tiles as stable clusters and obtain the growth status of stable clusters; Adjust the electrotransfer parameters according to the growth of stable aggregates.
6. The electroporation regulation method for exosome-loaded miRNA according to claim 5, characterized in that, The relationship between exosome membrane structure integrity and electrotransmission parameters was obtained, and the maximum value of the pulse parameters was obtained while ensuring the exosome membrane structure integrity. With the pulse parameter set to the maximum value, the growth of stable aggregates after their aggregation area reaches a preset area threshold is obtained. If the aggregation area begins to decrease, keep the pulse parameters unchanged; If the aggregated area continues to increase, the time when the aggregated area reaches the preset area threshold is obtained, and the relationship between the decrease value of the pulse parameter and the aggregated area of the stable aggregate is obtained; the time is the pulse parameter adjustment time. Obtain the pulse parameter reduction value when the clustered area remains stable at or below the preset area threshold.
7. The electroporation regulation method for exosome-loaded miRNA according to any one of claims 2-6, characterized in that, When no analyte is added to the system, blank control images are continuously acquired during the electroporation process; at the same time, the difference images between the fluorescence distribution image and the blank control image are acquired; several difference images are combined into an image group.
8. The electroporation regulation method for exosome-loaded miRNA according to claim 7, characterized in that, The loading speed of the test object is obtained by measuring the change in the distribution area of the test object; a preset area threshold is obtained based on the loading speed of the test object.