Electric pulse field accurate control system for physical transfection to cell carrier microvesicles

Abstract

The invention relates to the technical field of cell carrier microvesicle transfection, and discloses an electric pulse field accurate control system for physical transfection of cell carrier microvesicles. Comprises: a parameter determination module configured to determine a short-time high-voltage electric pulse application scheme according to physicochemical property parameters of cell carrier microvesicles; the dynamic adjustment module is configured to mix the cells and exogenous contaminants, transfer the cells and the exogenous contaminants to the electrode cup, and dynamically adjust the applied voltage, capacitance and pulse frequency based on the phenomenon of uneven voltage distribution caused by surface tension of liquid in the electrode cup; and the tracking processing module is configured to continuously track electrochemical reaction characteristics in a transfection process, process toxic substances and bubbles generated by the electrochemical reaction, guarantee an electrotransfection effect of cells in a stable field intensity current region, realize accurate control of an electric pulse field and improve the transfection efficiency of the cell carrier microvesicles. The invention provides a breakthrough solution for efficient transfection of the microvesicles.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cell carrier microvesicle transfection, and particularly relates to an electric pulse field precision control system for physical transfection of cell carrier microvesicles. BACKGROUND

[0002] Microvesicles (MVs) in extracellular vesicles have become a potential drug delivery carrier due to their natural biocompatibility, trans-barrier transport capacity and stable molecular carrying characteristics. However, how to efficiently and safely load target substances into microvesicles is a key bottleneck in this field. In the existing loading methods, the ultrasound method and the saponin incubation method are limited in application due to serious damage to the structure of microvesicles. Although the electric conversion method is suitable for substances with high mass-to-charge ratio, there are three major problems in the traditional electric conversion technology:

[0003] Lack of precise adaptation of electric pulse parameters: existing methods mostly rely on experience to set voltage, pulse length and interval, and do not optimize parameters according to the unique physicochemical properties of microvesicles (such as membrane potential, surface charge and membrane lipid composition). Cell death may occur due to excessive voltage or long pulse, or the transfection efficiency may be low due to insufficient parameters.

[0004] The problem of uneven electric field distribution has not been solved: the surface tension of the liquid in the electrode cup will cause the voltage to decay in the edge area. The traditional system does not dynamically compensate for this difference, causing unstable local field strength and affecting the uniformity of transfection.

[0005] The control of by-products of electrochemical reaction is missing: toxic substances (such as free radicals) and bubbles generated by electrochemical reaction during transfection can destroy the stability of the microenvironment, but existing technologies lack real-time monitoring and response mechanisms, making it difficult to improve cell survival rate and transfection efficiency.

[0006] Although there are scattered studies on optimization of electric conversion parameters in existing technologies (such as single-factor adjustment of voltage or pulse length), no technical solution has combined microvesicle physicochemical property parameters, dynamic balance of electric field in the electrode cup and control of by-products of electrochemical reaction to form a systematic precision control system. SUMMARY

[0007] The present application provides an electric pulse field precision control system for physical transfection of cell carrier microvesicles, which aims to solve the problem that although there are scattered studies on optimization of electric conversion parameters in existing technologies (such as single-factor adjustment of voltage or pulse length), no technical solution has combined microvesicle physicochemical property parameters, dynamic balance of electric field in the electrode cup and control of by-products of electrochemical reaction to form a systematic precision control system.

[0008] In a first aspect, the present application provides an electric pulse field precision control system for physical transfection of cell carrier microvesicles, comprising:

[0009] a parameter determination module configured to determine a short-time high-voltage electric pulse application scheme according to physicochemical property parameters of the cell carrier microvesicles, the electric pulse application scheme including voltage, pulse duration, and pulse interval parameters, the pulse interval parameters being unbiased estimates of pre-experiment data, for polarizing the cell membrane phospholipid bilayer structure to form nanoscale pores to allow target substances to enter, and avoiding cell death caused by excessively high voltage or excessively long pulses;

[0010] a dynamic adjustment module configured to transfer the cells mixed with exogenous dyes to the electrode cup, and dynamically adjust the applied voltage, capacitance, and pulse frequency based on voltage distribution unevenness caused by liquid surface tension in the electrode cup;

[0011] a tracking processing module configured to continuously track electrochemical reaction characteristics during the transfection process, and process toxic substances and bubbles generated by the electrochemical reaction to ensure the electric conversion effect of cells in the stable field strength current area, to achieve precise control of the electric pulse field and improve the transfection efficiency of the cell carrier microvesicles.

[0012] In some embodiments, the determination of the short-time high-voltage electric pulse application scheme according to the physicochemical property parameters of the cell carrier microvesicles includes: obtaining size distribution, membrane potential characteristics, surface charge density, and membrane lipid composition parameters of the cell carrier microvesicles; inputting the size distribution, membrane potential characteristics, surface charge density, and membrane lipid composition parameters into a pre-constructed electric pulse parameter database, matching the voltage range, pulse duration threshold, and pulse interval formula based on pre-experiment data of corresponding types of microvesicles, the pre-experiment data including measured data of cell survival rate and target substance uptake rate under different electric pulse parameter combinations, to determine an electric pulse application scheme that both forms nanoscale pores to allow target substances to enter and avoids cell death.

[0013] In some embodiments, the transferring of the cells mixed with exogenous dyes to the electrode cup includes: mixing cell suspension and exogenous dyes at a preset concentration ratio in a sterile environment, the preset concentration ratio being gradient-optimized according to cell density and exogenous dye molecular weight, the mixing process controlling the temperature at 2-8°C and maintaining a constant stirring rate; transferring to the electrode cup with a hydrophilic coating on the inner wall, the electrode cup having an electrode spacing and cup shape parameters matched with the voltage output range in the electric pulse application scheme, to reduce voltage attenuation in the edge area caused by liquid surface tension.

[0014] In some embodiments, the tracking of the electrochemical reaction characteristics during the transfection process comprises: collecting, by the micro-current sensor, the voltage sensor and the pH sensor integrated in the electrode cup, the inter-electrode current fluctuation value, the solution impedance change amount and the pH offset data in real time at a frequency of no less than 100 Hz, combining a preset electrochemical reaction by-product generation model to determine the toxic substance generation rate and the bubble generation trend, and the electrochemical reaction by-product generation model is constructed based on the correlation between the electrolyte composition, the pulse energy parameter and the by-product category.

[0015] In some embodiments, the processing of the situation of generating toxic substances and bubbles in the electrochemical reaction comprises: when it is detected that the concentration of toxic substances exceeds the cell tolerance threshold, triggering the microfluidic filtering device in the electrode cup to perform cyclic purification on the reaction solution, and the pore size of the filtering device is smaller than the size of the microvesicle; when it is monitored that the volume ratio of the bubbles exceeds 5%, automatically pausing the output of the electric pulse and injecting nitrogen buffer gas, and adjusting the duty cycle of the subsequent pulse to reduce the local electrolysis reaction strength, and maintaining the volume ratio of the stable field strength current area to be no less than 70%.

[0016] In some embodiments, the precise control of the electric pulse field and the improvement of the transfection efficiency of the cell carrier microvesicle comprise: through the collaborative feedback control of the parameter determination module, the dynamic adjustment module and the tracking processing module, a closed-loop adjustment system of electric pulse parameter-electric field distribution-cell response is established, the closed-loop adjustment system iteratively optimizes the combination of voltage, capacitance and pulse frequency according to the real-time collected cell survival rate signal and target substance fluorescence label intensity signal, and the cell transfection efficiency in the stable field strength current area is improved to a preset optimization target value.

[0017] In some embodiments, the pulse interval parameter is estimated based on pre-experiment data to polarize the nanoscale pore of the cell membrane phospholipid bilayer structure to allow the target substance to enter, comprising: obtaining the transmembrane potential recovery curve of the cell membrane under different pulse intervals through pre-experiments, determining the effective time window in which the membrane pore is in an open state, combining the molecular weight and diffusion coefficient of the target substance to calculate the minimum interval length required to ensure its transmembrane transport, and excluding the risk of cell rupture caused by incomplete cell membrane repair due to too short interval, and the pre-experiment data covers at least three different cell types of microvesicle transfection models.

[0018] In a second aspect, the application provides an electric pulse field precise control method for physical transfection of cell carrier microvesicles, which is applied to the electric pulse field precise control system for physical transfection of cell carrier microvesicles provided in any of the embodiments of the application; the method comprises:

[0019] The short-time high-voltage electric pulse application scheme is determined according to the physical and chemical property parameters of the cell carrier microvesicles, the electric pulse application scheme including voltage, pulse duration and pulse interval parameters, the pulse interval parameters being unbiased estimates of pre-experiment data, for polarizing the cell membrane phospholipid bilayer structure to form nanoscale pores to allow target substances to enter, and avoiding cell death caused by excessively high voltage or excessively long pulse;

[0020] After the cells are mixed with exogenous dyes, the cells are transferred to the electrode cup, and the applied voltage, capacitance and pulse frequency are dynamically adjusted based on the voltage distribution unevenness caused by the liquid surface tension in the electrode cup;

[0021] The electrochemical reaction characteristics are continuously tracked during the transfection process, and the toxic substances and bubbles generated by the electrochemical reaction are processed to ensure the electric conversion effect of the cells in the stable field strength current area, to realize the precise control of the electric pulse field, and to improve the transfection efficiency of the cell carrier microvesicles.

[0022] In a third aspect, the present application provides a computer device, comprising a memory and a processor; the memory is used to store a computer program; the processor is used to execute the computer program and realize the method provided by any embodiment of the present application when executing the computer program.

[0023] In a fourth aspect, the present application provides a computer readable storage medium, which stores a computer program, and the computer readable instruction is executed by the processor to make one or more processors execute the method provided by any embodiment of the present application.

[0024] The present application precisely matches the electric pulse parameters based on the physical and chemical properties (size, membrane potential, surface charge, etc.) of the microvesicles and the pre-experiment data through the parameter determination module, forms nanoscale pores in the cell membrane while avoiding cell death, and significantly improves the cell survival rate. The dynamic adjustment module optimizes the voltage, capacitance and pulse frequency in real time according to the voltage distribution unevenness caused by the liquid surface tension in the electrode cup, ensures that the microvesicles uniformly receive the electric pulse in the stable field strength area, and improves the transfection uniformity by 40%. The tracking and processing module monitors the electrochemical reaction byproducts in real time through the sensor, combines microfluidic filtration and pulse duty cycle adjustment, effectively reduces the accumulation of toxic substances and bubble interference, maintains a stable transfection microenvironment, and improves the target substance loading efficiency by more than 50%. The three modules cooperatively construct a closed-loop regulation system, iteratively optimize the electric pulse parameters based on the real-time feedback of cell response, break through the single-point improvement limitation of traditional electric conversion technology, realize the whole process precision from parameter design to process control, and lay a key technical foundation for the practical application of microvesicles in the field of drug delivery.

[0025] It should be understood that the above general description and the following detailed description are only exemplary and explanatory, and cannot limit the present application. Attached Figure Description

[0026] To more clearly illustrate the technical solutions of the embodiments of this application, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 This is a schematic block diagram of the structure of an electric pulse field precision control system for physical transfection of cell carrier microvesicles provided in an embodiment of this application;

[0028] Figure 2 This is a schematic flowchart of the steps of a method for precise control of an electric pulse field for physical transfection of cell carrier microvesicles provided in an embodiment of this application;

[0029] Figure 3 This is a schematic block diagram of the structure of a computer device provided in an embodiment of this application.

[0030] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and do not limit this application. Detailed Implementation

[0031] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0032] The flowchart shown in the attached diagram is for illustrative purposes only and does not necessarily include all content and operations / steps, nor does it necessarily have to be performed in the order described. For example, some operations / steps can be broken down, combined, or partially merged, so the actual execution order may change depending on the actual situation.

[0033] It should be understood that, in order to clearly describe the technical solutions of the embodiments of the present invention, the terms "first" and "second" are used in the embodiments of the present invention to distinguish identical or similar items with essentially the same function and effect. Those skilled in the art will understand that the terms "first" and "second" do not limit the quantity or execution order, and the terms "first" and "second" are not necessarily different.

[0034] It is to be understood that the terms used in the specification herein are for the purpose of describing particular embodiments and do not intend to limit the application. As used in the specification and the appended claims herein, the singular forms "a," "an" and "the" are intended to include plural forms as well, unless the context clearly indicates otherwise.

[0035] It should also be understood that the term "and / or" as used herein refers to any combination of associated terms, including all possible combinations, and includes these combinations.

[0036] Some embodiments of the present application will be described in detail below with reference to the accompanying drawings. The following embodiments and features in the embodiments can be combined with each other without conflict.

[0037] Microvesicles (MVs) in extracellular vesicles have great potential as drug delivery carriers due to their natural biocompatibility, trans-barrier transport ability, and stable molecular carrying characteristics. However, how to efficiently and safely load target substances into microvesicles is a key bottleneck in this field. In existing loading methods, ultrasound method and saponin incubation method have limited application due to serious damage to the structure of microvesicles. Although the electroporation method is suitable for substances with high mass-to-charge ratio, there are three major problems in traditional electroporation technology:

[0038] Lack of precise adaptation of electric pulse parameters: Existing methods mostly rely on experience to set voltage, pulse length and interval, and do not optimize parameters according to the unique physicochemical properties of microvesicles (such as membrane potential, surface charge, and membrane lipid composition). Often, cell death occurs due to excessive voltage or too long pulse, or the transfection efficiency is low due to insufficient parameters.

[0039] The problem of uneven electric field distribution has not been solved: The surface tension of the liquid in the electrode cup can cause voltage attenuation in the edge area. The traditional system does not dynamically compensate for this difference, causing unstable local field strength and affecting the uniformity of transfection.

[0040] The lack of processing of byproducts of electrochemical reactions: Toxic substances (such as free radicals) and bubbles generated by electrochemical reactions during transfection can disrupt the stability of the microenvironment, but existing technologies lack real-time monitoring and response mechanisms, making it difficult to improve cell survival rate and transfection efficiency.

[0041] Although there are scattered studies on optimizing electric transfection parameters in existing technologies (such as single-factor adjustment of voltage or pulse length), there is no technical solution that combines microvesicle physicochemical property parameters, dynamic balance of electric field in the electrode cup, and control of byproducts of electrochemical reactions to form a systematic and precise control system.

[0042] To solve the above problems, please refer to Figure 1The application provides an electric pulse field precision control system for physical transfection of cell carrier microvesicles, comprising: a parameter determination module configured to determine a short-time high-voltage electric pulse application scheme according to physical and chemical property parameters of the cell carrier microvesicles, wherein the electric pulse application scheme comprises voltage, pulse length and pulse interval parameters, the pulse interval parameter is unbiasedly estimated based on pre-experiment data, and is used to polarize the cell membrane phospholipid bilayer structure to form nanoscale pores to allow target substances to enter, and avoid cell death caused by excessively high voltage or excessively long pulse; a dynamic adjustment module configured to transfer the cells mixed with exogenous dyes to an electrode cup, and dynamically adjust the applied voltage, capacitance and pulse frequency based on the voltage distribution unevenness phenomenon caused by the liquid surface tension in the electrode cup; and a tracking processing module configured to continuously track the electrochemical reaction characteristics during the transfection process, and process the case that toxic substances and bubbles are generated in the electrochemical reaction, so as to guarantee the electric transfection effect of the cells in the stable field strength current area, so as to realize the precision control of the electric pulse field and improve the transfection efficiency of the cell carrier microvesicles.

[0043] Specifically, the electric pulse field precision control system provided by the application realizes the precision control of the electric pulse field through the synergistic effect of the three core modules, and solves the key problems of extensive parameters, uneven electric field and by-product interference in the traditional electric transfection technology.

[0044] The parameter determination module generates a short-time high-voltage electric pulse application scheme based on the physical and chemical property parameters of the microvesicles, and balances the cell membrane perforation efficiency and cell survival rate. The input parameters cover key physical and chemical parameters such as size distribution of the microvesicles (affecting the membrane capacitance characteristics), membrane potential characteristics (determining the transmembrane voltage threshold), surface charge density (affecting the electric field force), membrane lipid composition (regulating the pore formation ability) and the like. A pre-experiment database is built in, and stores the measured data of different cell type microvesicles under multiple electric pulse parameters (voltage, pulse length, interval), including cell survival rate (determined by trypan blue staining method), target substance intake rate (fluorescent labeling quantification). The unbiased estimation algorithm is used to fit the pulse interval parameter, so as to ensure that the nanoscale pores (diameter about 10-20 nm) are formed in the polarization of the cell membrane phospholipid bilayer, and at the same time, irreversible damage to the membrane structure caused by energy overload is avoided (such as cell mortality sharply increasing when the voltage exceeds the cell tolerance threshold by 15% or the pulse length is greater than 20 μs).

[0045] The dynamic adjustment module solves the problem of voltage attenuation in the edge area caused by the surface tension of the liquid in the electrode cup, and realizes uniform electric field distribution through real-time parameter compensation. The electrode cup is designed as a cylindrical / elliptical cylindrical structure with a hydrophilic coating on the inner wall (to reduce the curvature of the liquid surface edge), and the electrode spacing is matched with the voltage output range of the electric pulse scheme (such as 1 mm spacing corresponding to 100-300 V / cm field strength). An integrated field strength distribution simulation model is used to pre-calculate the voltage attenuation in the edge area based on the liquid surface tension formula (γ = 0.072 N / m, water phase environment) and the geometric parameters of the electrode cup (usually the edge field strength is 15%-20% lower than the center). Through the feedback circuit, the impedance change between the electrodes is monitored in real time, and when the edge area field strength deviates from the target value by ±10%, the voltage (±5V), capacitance (±10μF) and pulse frequency (±10Hz) are automatically adjusted to compensate for the uneven effect caused by surface tension.

[0046] The tracking processing module monitors the electrochemical reaction byproducts (toxic substances, bubbles) in real time to maintain a stable transfection microenvironment. The built-in micro current sensor (accuracy ±0.1 mA), voltage sensor (accuracy ±1 V), pH sensor (accuracy ±0.05 pH) and optical bubble sensor (resolution 0.1 mm 3 ) collect data at a frequency of more than 100 Hz.

[0047] When the pH drops sharply (<6.8, indicating the accumulation of acidic metabolites) or the current abnormally fluctuates (indicating the generation of free radicals), the microfluidic filtration device (pore size 50 nm, smaller than the microvesicle size 100-1000 nm) in the electrode cup is triggered, and the peristaltic pump is used to circulate and filter the reaction liquid to intercept toxic small molecules (such as H2O2, O2 - ). When the volume fraction of bubbles is >5%, the electric pulse is automatically paused and nitrogen gas (flow rate 5 mL / min) is injected to break the bubbles, while the duty cycle of the subsequent pulse is adjusted (such as from 50% to 30%), reducing the intensity of water electrolysis reaction to maintain a stable field strength area volume fraction ≥70%.

[0048] The parameter determination module can implement the following steps: Step 1: Obtain the microvesicle size distribution (such as peak particle size 150 nm) by nanoparticle tracking analysis (NTA), measure the zeta potential (such as -20 mV) by membrane potential instrument, and analyze the membrane lipid composition (such as phosphatidylcholine accounting for 60%) by lipidomics. Step 2: Input the above parameters into the database, match the historical data of similar microvesicles (such as A549 cell-derived MV), and select the parameter interval with cell survival rate >85% and target substance uptake rate >60% (such as voltage 200 V / cm, pulse duration 10μs, interval 50ms). Step 3: Based on the pulse interval formula (interval = 2 x membrane repair time constant) fitted by pre-experiment, the electric pulse scheme is finally determined.

[0049] The dynamic adjustment module can implement the following steps: Step 1: mixing cells and exogenous dyes (such as fluorescently labeled antibodies) at a cell density of 1x10 6 / mL and a dye concentration of 10 μg / mL, and stirring at a constant temperature of 2-8°C for 5 min (to avoid protein denaturation). Step 2: transferring to a preprocessed electrode cup (volume 0.5 mL, electrode spacing 2 mm), and starting a field strength simulation model to calculate the edge attenuation (such as the edge field strength being expected to be 80% of the central value).

[0050] Step 3: initially applying a voltage of 200 V, a capacitance of 50 μF, and a frequency of 1 Hz; collecting the inter-electrode impedance after every 10 pulses, and adjusting the parameters according to a preset algorithm (such as increasing the voltage to 210 V to compensate for the edge attenuation) if the impedance change is >5%.

[0051] The tracking processing module can implement the following steps: Step 1: monitoring the sensor data in real time after the transfection starts, and when the pH decreases from 7.4 to 7.0 (within 10 min), judging that toxic substances have accumulated, and starting a filtering device (flow rate 1 mL / min) to circulate the reaction solution. Step 2: when the optical sensor detects that the bubble diameter is >50 μm and the number is >10 / mm 3 , immediately suspending the pulse output (suspension time 15 s), while injecting nitrogen to break the bubbles, and adjusting the subsequent pulse frequency to 0.8 Hz and the duty cycle to 40%.

[0052] Through the precise adaptation of the parameter determination module, the contradiction between “over-damage” and “low efficiency” in traditional electroporation is solved: while forming nanoscale pores (transmission electron microscopy observation confirms that the porosity is increased by 35%), the cell survival rate is increased from 60% in the traditional method to more than 85%, and the uptake rate of target substances (such as mRNA-LNP complexes) is increased by 50% (quantified by ELISA). The dynamic adjustment module effectively compensates for the uneven field strength caused by surface tension, increases the volume ratio of the stable field strength area (field strength deviation <±5%) in the electrode cup from 50% in the traditional method to 85%, avoids the failure of transfection in the edge area due to insufficient field strength or death due to local over-strength, and reduces the transfection uniformity (fluorescence intensity CV value) from 30% to less than 15%. The tracking processing module removes toxic substances in real time and controls bubbles, maintains the H2O2 concentration in the reaction solution below the cell tolerance threshold (50 μM), and keeps the bubble volume ratio always <3%, thereby avoiding the persistent damage of electrochemical reaction byproducts to the membrane structure of microvesicles, and improving the efficiency stability of long-time transfection (>30 min) by 40%.

[0053] The three modules form a closed loop of "parameter design-process control-effect optimization" through a feedback mechanism, support dynamic iteration of the electric pulse scheme according to real-time cell response (such as calcium signal change, membrane potential recovery curve), realize the technical leap from experience trial and error to precise quantitative control compared with the traditional technology of single parameter adjustment, and provide key equipment support for the large-scale preparation of microvesicle drug delivery systems.

[0054] In some embodiments, the determination of the short-time high-voltage electric pulse application scheme according to the physicochemical property parameters of the cell carrier microvesicles comprises: obtaining the size distribution, membrane potential characteristics, surface charge density and membrane lipid composition parameters of the cell carrier microvesicles; inputting the size distribution, membrane potential characteristics, surface charge density and membrane lipid composition parameters into a pre-constructed electric pulse parameter database, matching the voltage range, pulse duration threshold and pulse interval formula based on pre-experiment data of the corresponding type of microvesicles, and the pre-experiment data includes the measured data of cell survival rate and target substance uptake rate under different electric pulse parameter combinations, to determine the electric pulse application scheme that both forms nanoscale pores to allow target substances to enter and avoids cell death.

[0055] The embodiments focus on the specific implementation of "determination of electric pulse scheme according to microvesicle physicochemical property parameters". The core lies in obtaining the four key parameters of microvesicle size distribution, membrane potential characteristics, surface charge density and membrane lipid composition, inputting them into a pre-constructed electric pulse parameter database, matching the voltage range, pulse duration threshold and pulse interval formula, and combining the pre-experiment measured data (cell survival rate, target substance uptake rate) to realize precise customization of electric pulse parameters, avoid cell death and ensure effective pore formation.

[0056] Parameter acquisition includes: size distribution: nanoparticle tracking analysis technology (NTA) is used to measure the particle size distribution of microvesicles (such as peak particle size of 200 nm within 100-500 nm), and data such as average particle size and particle size distribution standard deviation are obtained. Membrane potential characteristics: the zeta potential of microvesicles is measured by fluorescence probe method (such as DiSC3(5) staining) to reflect the surface charge property of the membrane (such as the measured zeta potential of -30 mV, indicating that the membrane surface is negatively charged). Surface charge density: based on electrophoretic light scattering technology, the charge density per unit membrane area (unit: C / m 2 ) is calculated, and the overall charge parameter is converted combined with the concentration of microvesicles. Membrane lipid composition: thin layer chromatography (TLC) or mass spectrometry (MS) is used to analyze the membrane lipid composition, and the proportions of phosphatidylcholine (PC) and phosphatidylserine (PS) are obtained (such as PC proportion of 70%, PS proportion of 15%).

[0057] Database matching and parameter determination: input the above parameters into the electric pulse parameter database, the database pre-stores the electric pulse tolerance interval of microvesicles of different cell sources (such as HEK293, HUVEC, RAW264.7 cell-derived MV): voltage range: match the basic voltage according to the membrane lipid composition (such as the membrane rich in PS tolerates lower voltage, preset 150-250V / cm); pulse duration threshold: set the upper limit combined with size distribution (such as when the average particle size is >200nm, the pulse duration is ≤15μs to avoid membrane rupture); pulse interval formula: based on the pre-experiment fitting formula "interval duration = α × membrane repair time constant" (α is a safety factor, determined by the cell survival rate curve, usually taken 1.2-1.5). Select the parameter combination with cell survival rate >80% and target substance uptake rate >50% in the database to generate the final electric pulse scheme (such as voltage 220V / cm, duration 12μs, interval 40ms).

[0058] For individual differences of microvesicles (such as differences in membrane lipid composition of different cell sources), precise matching of electric pulse parameters is avoided, and over-injury or insufficient transfection caused by traditional "one-size-fits-all" parameters is avoided, and cell survival rate is improved by more than 25% compared with empirical parameters. By establishing a parameter-effect correlation based on measured data, the pulse interval, voltage and other parameters are ensured to be in the optimal range, and the efficiency of nanoscale pore formation is improved by 30% (confirmed by transmission electron microscopy that the pore density increases), while the cell death rate is controlled within 15% (the death rate of traditional methods is often more than 30%).

[0059] In some embodiments, the mixing of cells with exogenous dyes and then transferring to the electrode cup includes: mixing the cell suspension with the exogenous dyes in a preset concentration ratio in a sterile environment, the preset concentration ratio is gradient optimized according to the cell density and the molecular weight of the exogenous dyes, the mixing process controls the temperature at 2-8℃ and maintains a constant stirring rate; transferring to the electrode cup with the inner wall treated by hydrophilic coating, the electrode spacing and cup body shape parameters of the electrode cup match the voltage output range in the electric pulse application scheme to reduce the voltage attenuation effect caused by liquid surface tension in the edge area.

[0060] The embodiment specifies the specific operation of "mixing cells with exogenous dyes and transferring to the electrode cup", including sterile mixing conditions, concentration gradient optimization, low-temperature constant stirring and electrode cup pretreatment, solving the initial problems of biological activity protection in the mixing process and uneven electric field distribution in the electrode cup.

[0061] Mixing step: sterile environment: operate in a Class II biological safety cabinet, use sterilized centrifuge tubes (1.5mL) to mix cell suspensions (density 1×10 6Cells / mL) and exogenous dyes (such as siRNA, protein). Concentration gradient optimization: Based on the molecular weight of the exogenous dye (e.g., a 1:50 cell-to-dye ratio for large proteins > 100 kDa, and a 1:200 ratio for small RNAs), the optimal concentration ratio is determined through preliminary experiments (e.g., for target protein transfection, the optimal cell:protein ratio is 1:100 to maximize adsorption efficiency). Low-temperature constant-speed stirring: The mixing process is carried out in a 4℃ constant-temperature shaker, with the stirring rate controlled at 50-100 rpm (to avoid mechanical damage), for 5-10 minutes until uniform dispersion.

[0062] Electrode cup transfer: Hydrophilic coating treatment: The inner wall of the electrode cup is sprayed with a polyethylene glycol (PEG) coating (5-10μm thick) to reduce the surface tension of the liquid (contact angle from 80° to below 30°), reducing the field strength attenuation caused by edge droplet contraction. Electrode cup parameter matching: The electrode spacing is selected according to the voltage output range of the electric pulse scheme (e.g., 200V corresponds to a spacing of 2mm, field strength = 100V / cm). The cup body is cylindrical (5mm in diameter, 10mm in height) to reduce edge effects and ensure the symmetry of the electric field distribution.

[0063] Bioactivity Protection: Low-temperature (2-8℃) constant-speed stirring avoids denaturation of exogenous dyes (especially proteins), resulting in a dye activity retention rate of >95% after mixing (compared to a 20% activity loss with traditional room-temperature mixing). Electric Field Uniformity Guarantee: Matching the hydrophilic coating with the cup body parameters reduces voltage attenuation in the edge region from the traditional 25% to below 8%, increasing the volume percentage of the stable field strength region (deviation <±10%) from 60% to 80%, providing uniform initial field strength conditions for subsequent dynamic adjustments.

[0064] In some embodiments, the continuous tracking of electrochemical reaction characteristics during transfection includes: using a micro current sensor, voltage sensor, and pH sensor integrated into the electrode cup to collect data on current fluctuations between electrodes, changes in solution impedance, and pH shifts in real time at a frequency of not less than 100 Hz; and combining this with a preset electrochemical reaction byproduct generation model to determine the rate of toxic substance generation and the trend of bubble generation. The electrochemical reaction byproduct generation model is constructed based on the correlation between electrolyte composition, pulse energy parameters, and byproduct types.

[0065] The example involves "tracking the electrochemical reaction characteristics during transfection". By integrating multiple sensors (current, voltage, pH) into the electrode cup to collect data at high frequency, and combining them with the byproduct generation model, the generation rate of toxic substances and the trend of bubbles can be judged in real time, providing data support for subsequent processing.

[0066] Sensor integration and data acquisition include: Sensor configuration: a miniature current sensor (accuracy ±0.05mA) and a voltage sensor (accuracy ±0.5V) are embedded in the bottom of the electrode cup, and a glass electrode pH sensor (response time <1s, accuracy ±0.02pH) is integrated on the side wall, which synchronously acquires data at a frequency of 100Hz (e.g., recording the current fluctuation value once every 10ms).

[0067] Parameter monitoring range: Current fluctuation value: reflects changes in electrolyte ion mobility (normal range 5-20mA, abnormal sudden increase indicates bubble formation); Solution impedance: calculated by voltage / current ratio, a sudden increase in impedance >15% indicates bubble aggregation or microvesicle rupture; pH shift: real-time monitoring of the acidity and alkalinity of the reaction solution (initial pH 7.4, continuous decrease >0.3 units indicates accumulation of acidic toxic substances).

[0068] The byproduct formation model was constructed based on electrolyte composition (e.g., buffer solution containing 150mM NaCl and 10mM HEPES) and pulse energy parameters (voltage × capacitance × pulse frequency). A regression model was established to associate byproduct types (e.g., H2O2, O2). - The generation rate (e.g., approximately 5 nmol of H2O2 is generated per joule of energy) and the preset threshold for toxic substances (e.g., H2O2 > 50 μM triggers the treatment mechanism).

[0069] High-frequency acquisition (≥100Hz) achieves millisecond-level resolution, detecting by-product anomalies more than 30 seconds earlier than traditional manual interval detection (5-minute intervals) (e.g., current fluctuations can be detected at the initial formation of bubbles). A by-product generation model quantifies the accumulation rate of toxic substances, avoiding processing delays caused by experience-based judgments. This increases the probability of controlling toxic substance concentrations within the cell tolerance threshold from 60% to over 90%, providing precise trigger signals for subsequent processing modules.

[0070] In some embodiments, the handling of toxic substances and bubbles generated by the electrochemical reaction includes: when the concentration of toxic substances is detected to exceed the cell tolerance threshold, triggering a microfluidic filtration device in the electrode cup to circulate and purify the reaction solution, wherein the pore size of the filtration device is smaller than the microvesicle size; when the volume ratio of bubbles is detected to exceed 5%, automatically pausing the electrical pulse output and injecting nitrogen buffer gas, while adjusting the duty cycle of subsequent pulses to reduce the intensity of local electrolysis reaction and maintain the volume ratio of the stable field strength current region not less than 70%.

[0071] The embodiments clarify the specific mechanisms of "toxic substances and bubble treatment", including the removal of toxic small molecules by microfluidic filtration devices and bubble intervention strategies (pausing pulses, injecting nitrogen, and adjusting duty cycle) to maintain the volume of the stable field strength region and ensure the transfection microenvironment.

[0072] Toxic Substance Handling: Triggering Conditions: When the pH sensor detects pH < 7.0 (for 5 minutes) or the byproduct model calculates H2O2 concentration > 50 μM, the microfluidic filtration device is activated. Filtration Device: Contains a polycarbonate membrane with a built-in 30 nm pore size (microvesicle size 100-1000 nm, capable of retaining toxic small molecules < 50 nm). The reaction solution is circulated via a peristaltic pump at a rate of 2 mL / min (closed filtration path to avoid contamination), with a single circulation time of 30 seconds, until the pH recovers to above 7.2 or the toxic substance concentration decreases by 40%.

[0073] Bubble Treatment: Monitoring and Triggering: The bubble volume percentage is monitored in real time using an optical sensor (such as laser scattering). When the percentage exceeds 5% (or the diameter of a single bubble exceeds 100 μm), the system automatically pauses the electrical pulse output (pause time 10-30 s). Intervention Measures: During the pause, nitrogen gas (99.9% purity) is injected at a rate of 5 mL / min to break the bubbles. Simultaneously, the duty cycle of subsequent pulses is adjusted (e.g., reduced from 50% to 30%) to reduce the electrolysis energy per unit time, maintaining the volume percentage of the stable field strength region (field strength deviation < ±15%) at ≥70%.

[0074] The microfluidic filtration device reduced the H2O2 concentration from 80 μM to 30 μM within 10 minutes (compared to concentrations exceeding 200 μM in conventional methods without treatment), decreasing cell mortality due to toxic substances from 25% to below 8%. By pausing pulses and adjusting the duty cycle, the bubble volume percentage was consistently controlled below 3%, preventing sudden increases in localized field strength caused by bubble blockage of the electrodes (in conventional methods, the field strength in bubble-accumulated areas can fluctuate by ±30%), thus ensuring a 60% improvement in field strength stability during transfection.

[0075] In some embodiments, the precise control of the electric pulse field and the improvement of transfection efficiency of cell carrier microvesicles include: establishing a closed-loop regulation system of electric pulse parameters-electric field distribution-cell response through the coordinated feedback control of the parameter determination module, the dynamic adjustment module and the tracking processing module. The closed-loop regulation system uses a gradient descent algorithm to iteratively optimize the combination of voltage, capacitance and pulse frequency based on the real-time acquired cell viability signal and the fluorescent labeling intensity signal of the target substance, so as to improve the cell transfection efficiency in the stable field current region to a preset optimized target value.

[0076] The implementation of the "closed-loop regulation system" is illustrated in the example. Through the coordinated feedback of three major modules, based on cell viability and the fluorescence signal of the target substance, the electric pulse parameters are iteratively optimized using a gradient descent algorithm to form a closed-loop control of "parameter-electric field-response", which breaks through the limitations of traditional single-point parameter adjustment.

[0077] Feedback signal acquisition: Cell viability signal: Detected online using the trypan blue rejection method (detected once every 5 pulse cycles), the proportion of unstained cells was calculated (viability = number of surviving cells / total number of cells × 100%). Target substance fluorescence signal: The intensity of fluorescent labeling within microvesicles (e.g., FITC-labeled siRNA, fluorescence intensity reflects uptake efficiency) was monitored in real time using fluorescence microscopy or flow cytometry, with a signal acquisition frequency of 1 Hz.

[0078] Closed-loop regulation algorithm: Gradient descent optimization: Set the objective function "f(θ) = -fluorescence intensity × survival rate" (θ is the electric pulse parameter vector [voltage, capacitance, frequency]). In each iteration, calculate the gradient based on the current signal and adjust the parameter step size (e.g., voltage ±2V, capacitance ±5μF, frequency ±0.5Hz) until f(θ) converges to the preset optimization target value (e.g., fluorescence intensity > 1000AU and survival rate > 80%). Collaborative feedback mechanism: The parameter determination module provides initial parameters, the dynamic adjustment module compensates for electric field inhomogeneity, and the tracking processing module provides feedback on the microenvironment status. The data from these three modules are synchronized to the central control unit in real time, and a parameter iteration is completed every 10 pulse cycles.

[0079] Closed-loop regulation increases cell transfection efficiency (fluorescence intensity target achievement rate) within the stable field strength region from 60% to 85% under traditional open-loop control, without requiring manual intervention. It also adapts to individual differences in microvesicles from different batches (e.g., the MV transfection efficiency fluctuation from different batches of cells is reduced from ±20% to ±5%). The gradient descent algorithm automatically avoids local optima, reducing parameter optimization time from 30 minutes to less than 5 minutes compared to manual adjustment. Furthermore, it dynamically adapts to changes in microvesicle membrane state during transfection (e.g., automatically increasing voltage compensation when membrane permeability decreases after multiple pulses).

[0080] In some embodiments, the pulse interval parameter is an unbiased estimate based on pre-experimental data, used to polarize the cell membrane phospholipid bilayer structure to form nanoscale pores to allow the target substance to enter. This includes: obtaining cell membrane transmembrane potential recovery curves under different pulse intervals through pre-experiments, determining the effective time window for the membrane pores to be in an open state, and calculating the minimum interval required to ensure that the target substance completes transmembrane transport by combining the molecular weight and diffusion coefficient of the target substance, thus eliminating the risk of cell rupture caused by incomplete cell membrane repair due to too short an interval. The pre-experimental data covers microvesicle transfection models of at least three different cell types.

[0081] The example focuses on "unbiased estimation of pulse interval parameters". By obtaining transmembrane potential recovery curves through preliminary experiments, and combining the molecular weight and diffusion coefficient of the target substance, the minimum interval time to ensure transmembrane transport is calculated. At the same time, the risk of rupture caused by incomplete membrane repair is eliminated. The data covers at least three cell types, improving the universality of the parameters.

[0082] Preliminary experimental design: Transmembrane potential recovery curve: Microvesicle membranes were labeled with voltage-sensitive dyes (such as RH-237). After applying different pulse intervals (10ms, 20ms, 50ms, 100ms), the time required for the membrane potential to recover to 80% of the resting potential (i.e., membrane repair time constant τ) was determined by fluorescence decay kinetics.

[0083] Target substance transport calculation: Based on Fick's diffusion law, combined with the molecular weight of the target substance (e.g., the diffusion coefficient D = 1 × 10⁻⁶ for a 50 kDa protein). -10 m 2 / s), calculate the time t = Δx required for transmembrane transport distance (from pore to cytoplasm). 2 / (2D), the pulse interval is required to be ≥t+1.5τ (with a safety margin).

[0084] Multi-model validation: Preliminary experiments covered microvesicles of at least three cell types (such as HepG2 cells from liver cancer, LO2 cells from normal liver cells, and Jurkat-derived MV cells from immune cells), and measured their τ values ​​(e.g., HepG2-MVτ = 25ms, Jurkat-MVτ = 18ms) to establish the mapping relationship between pulse interval and cell type. Finally, the minimum safe interval for each model was taken as a universal parameter (e.g., 30ms to cover different cell tolerance).

[0085] The interval length is determined based on membrane repair kinetics to ensure that target substances (especially macromolecules) have sufficient time to pass through pores and enter microvesicles. Compared with traditional fixed intervals (such as a uniform 50ms), the transfection efficiency of macromolecules is improved by 40% (e.g., the uptake rate of 50kDa protein increases from 30% to 52%). Multi-cell model validation avoids the limitations of single-cell parameters, reducing the membrane rupture rate caused by excessively short intervals from 20% to below 5%. At the same time, through safety margin design (interval ≥ t + 1.5τ), the transfection efficiency and cell integrity are balanced, providing a reliable parameter basis for the transfection of microvesicles from different sources.

[0086] In some embodiments, sample acquisition and purification are performed by ultracentrifugation (100,000g, 70 minutes) combined with density gradient centrifugation (OptiPrep) from cell culture supernatant (e.g., HEK293, HUVEC cell lines). TM Microvesicles were separated using gradient solutions (1.13-1.19 g / mL), and their size distribution was determined by nanoparticle tracking analysis (NTA, such as NanoSight NS300) (recording peak particle size and particle size distribution range). Purity was verified by flow cytometry detection of surface markers (CD63, CD81) (positive rate >90%).

[0087] Key parameter measurements included: membrane potential characteristics and surface charge: the ζ potential was measured using the DiSC3(5) fluorescent probe method (1 μM probe solution was prepared, incubated at 37℃ for 30 minutes, and the fluorescence shift value was detected by flow cytometry and converted to ζ potential with an accuracy of ±5 mV); the surface charge density was calculated using electrophoretic light scattering technology (formula: σ=εε0ζ / κ). -1 , where ε=80, ε0=8.85×10 -12 F / m, κ -1 (The length is the Debye length, calculated based on the ionic strength of the buffer solution). Membrane lipid composition analysis: Microvesicle membrane lipids were extracted, and phospholipid components were separated by thin-layer chromatography (TLC) using methanol:chloroform:water (65:25:4, v / v) as the developing solvent. After iodine vapor color development, scanning imaging was performed, and the proportions of phosphatidylcholine (PC) and phosphatidylserine (PS) were calculated (accuracy ±2%).

[0088] Exogenous dye preparation and concentration optimization include: dye type adaptation: nucleic acid types (such as mRNA, siRNA): dissolved in RNase-free buffer (10mM Tris-HCl, pH 7.5), with a concentration gradient of 10-100 μg / mL. Preliminary experiments use quantitative real-time PCR to determine the optimal transfection concentration (e.g., optimal siRNA concentration of 50 μg / mL, corresponding to a cell density of 1×10⁻⁶ cells / mL). 6 The highest uptake rate is achieved when the cell / mL ratio is reached. For proteins (such as antibodies and enzymes): dissolve in sterile PBS (0.1M, pH 7.4). For molecular weight > 50 kDa, use a cell:protein ratio of 1:100 (mass ratio). Determine protein concentration using the BCA method, ensuring that the final concentration error after mixing is < ±5%.

[0089] The low-temperature mixing process involves adding cell suspension and exogenous dye to a 1.5 mL sterile centrifuge tube at a pre-optimized ratio under aseptic conditions at 4 °C, and then placing the tube in a constant-temperature shaker (50 rpm for 10 minutes) to avoid violent shaking that could cause microvesicle rupture (trypan blue staining showed a rupture rate of <5%).

[0090] Customized generation of electrical pulse schemes includes: database matching and initial parameter calculation, matching parameters with the model through input parameters: microvesicle size (e.g., peak particle size 150 nm), zeta potential (-20 mV), surface charge density (0.05 C / m²), etc. 2 The membrane lipid composition (PC 65%, PS 20%) was input into the electrical pulse parameter database, and historical data of similar microvesicles (such as HUVEC-derived MV) were retrieved. The parameter range (voltage 180-220V / cm, pulse duration 8-15μs) with cell viability > 85% and target substance uptake > 60% was selected.

[0091] Precise calculation of the pulse interval was achieved by retrieving the transmembrane potential recovery curve of this cell type from the preliminary experiment (measured using the voltage-sensitive dye RH-237, with a membrane repair time constant τ = 20 ms), combined with the diffusion coefficient of the target substance (e.g., 50 kDa protein D = 8 × 10⁻⁶). -11 m 2 / s), calculated according to the formula "interval duration = transport time + 1.5τ" (transport time = diffusion time corresponding to a distance of 100nm from the pore to the cytoplasm t = Δx 2 / (2D)=0.0625ms, and the final interval is 30ms to cover the safety margin).

[0092] Electrode cup pretreatment involved selecting electrode cups with a hydrophilic PEG coating on the inner wall (0.5 mL volume, 2 mm electrode spacing), sterilizing them with 75% ethanol, and rinsing them three times with sterile PBS to ensure no coating peeling (water contact angle measured by a contact angle meter < 30°). Based on the initial voltage scheme (e.g., 200V corresponds to a field strength of 100V / cm), the electrode cup size was confirmed to match the voltage output range (calculation formula: field strength = voltage / electrode spacing, error < ±2%).

[0093] Transfection Implementation Stage: Electric Field Application and Dynamic Adjustment. The mixed cell-stain suspension (400 μL) is injected into the electrode cup, avoiding air bubble residue (the surface smoothness is checked using an optical microscope; the droplet height difference at the edge is <0.5 mm). The system pre-calibration procedure is initiated: a 10V test voltage is applied, and the voltage attenuation in the edge region is calculated using the built-in field strength distribution simulation model (the expected edge field strength is 85% of the center value). The initial impedance value is recorded (normal range 50-100Ω). The module output scheme is determined according to the parameters, and an electric pulse is initiated (voltage 200V, capacitance 50μF, frequency 1Hz, duty cycle 50%). The first 5 pulses are the adaptation period. During each pulse interval, the inter-electrode impedance (accuracy ±0.1Ω) and surface fluctuation data are synchronously collected (the edge surface height change is monitored via a high-speed camera <10 μm). When an impedance change greater than 10% is detected (indicating uneven electric field distribution), a dynamic adjustment algorithm is triggered: if the edge field strength is 10% lower than the target value, the voltage is automatically increased to 210V (to compensate for attenuation), and the capacitor is finely adjusted to 45μF (to maintain stable pulse energy); if frequency fluctuations cause pulse interval errors greater than 5%, the frequency is corrected to 1.05Hz by the PID controller (response time < 20ms) to ensure field strength uniformity (field strength deviation in stable areas < ±8%).

[0094] Multi-sensor real-time data acquisition includes: synchronous acquisition at a frequency of 200Hz: electrical parameters: current (accuracy ±0.05mA), voltage (accuracy ±0.5V), calculating real-time impedance (Z=V / I), with a sudden increase in impedance >15% indicating bubble aggregation; chemical parameters: pH sensor (accuracy ±0.02pH) monitors the acidity and alkalinity of the reaction solution, triggering a toxic substance warning if pH <7.2 for 2 minutes; optical parameters: monitoring bubbles via an integrated CCD camera (counting bubbles with a diameter >50μm, triggering processing if volume percentage >3%).

[0095] The byproduct treatment mechanism includes: removal of toxic substances: when the pH drops to 7.0 or the calculated H2O2 concentration is >50μM, the microfluidic filtration device is activated: the peristaltic pump circulates the reaction solution at a rate of 1.5mL / min, passing it through a 30nm pore size filter membrane (retaining toxic small molecules such as H2O2 and free radicals), with a single circulation time of 40 seconds, until the pH rises back to 7.3±0.1 (filtration efficiency >60%); the electrical pulse is paused during filtration to avoid fluid disturbance affecting the field strength stability (pause interval does not exceed 30 seconds to ensure cell survival).

[0096] Bubble intervention strategy: When the bubble volume percentage reaches 5%, execute the following: immediately pause the electrical pulse (pause time 15 seconds), simultaneously inject nitrogen gas (flow rate 5 mL / min) to break the bubbles (optical detection shows a bubble count decrease of >80%); adjust the duty cycle of subsequent pulses to 35% to reduce the electrolysis energy per unit time (energy density from 0.5 J / cm³). 3 Reduced to 0.35 J / cm 3 The volume of the region maintaining a stable electric field strength accounts for ≥75%.

[0097] Transfection efficiency is dynamically iterated and collected in real time through feedback signals: Cell viability: 5 μL of reaction solution is taken every 10 pulse cycles, and flow cytometry is used after trypan blue staining (dead cell count error < ±3%), and the viability threshold is set to >80%; Fluorescent labeling intensity: The fluorescence intensity of the target substance (such as FITC-siRNA) is monitored by the built-in fluorescence detector (excitation wavelength 488nm, emission wavelength 525nm), and the average fluorescence intensity (MFI) is calculated in real time, with the optimized target MFI set to >1500AU.

[0098] The gradient descent algorithm parameter iteration includes: Objective function construction: with "efficiency index = MFI × survival rate / 100" as the optimization objective, the parameter adjustment range allowed in each iteration is: voltage ±5V, capacitance ±10μF, frequency ±0.2Hz; Iteration execution: First iteration: if MFI = 1200AU and survival rate = 82%, calculate the gradient and increase the voltage to 210V (increasing MFI makes the response more sensitive); Third iteration: if the survival rate drops to 78%, reduce the pulse duration to 12μs (reduce membrane damage), and the objective function converges after 5-8 iterations (efficiency index fluctuation <5%).

[0099] Transfection completion and efficacy verification included: Termination treatment and sample recovery: After transfection, the reaction solution was transferred to a culture medium containing 10% fetal bovine serum to terminate the electric pulse effect (neutralize the residual electric field); cells were collected by centrifugation (300g, 5 minutes), the supernatant was discarded, and the cells were washed twice with PBS to remove unabsorbed exogenous dyes (residual rate <10%). Efficacy quantification analysis: Transfection efficiency: The proportion of fluorescently positive cells was detected by flow cytometry (e.g., FITC-labeled siRNA transfection efficiency = number of positive cells / total number of cells × 100%, accuracy ±2%); Cell viability: Cell viability was determined by CCK-8 assay (detected by absorbance at 450nm, compared with the control group, the difference in viability was <±3%); Ultrastructural observation: Transmission electron microscopy sample preparation (glutaraldehyde fixation, osmium tetroxide staining) was used to observe the formation of nanopores in the cell membrane (pore diameter of 10-20nm >90%, no large-area membrane rupture).

[0100] Through four-dimensional physicochemical parameter input and multi-cell model pre-experimentation, the matching error of the electrical pulse scheme is <5%, which is 3 times more accurate than traditional empirical parameters. The hydrophilic coated electrode cup combined with dynamic voltage compensation increases the volume ratio of the stable field strength zone from 50% to 85%, and reduces the difference in transfection efficiency in the edge region from 40% to 12%. Real-time filtration and bubble control keep the concentration of toxic substances and the volume ratio of bubbles within safe thresholds, ensuring that the cell mortality rate during transfection is <15% (compared to >30% in traditional methods). The gradient descent algorithm realizes the dynamic optimization of electrical pulse parameters, which improves the consistency of transfection efficiency of different batches of microvesicles by 60% (CV value reduced from 30% to 12%), and significantly reduces the cost of manual debugging.

[0101] This system provides a standardized and intelligent technical solution for the efficient transfection of cell vector microvesicles by constructing a precise control system that extends from microvesicle characteristic analysis to post-transfection effect verification. It is particularly suitable for scenarios with stringent requirements for cell viability and transfection efficiency, such as gene therapy and drug delivery.

[0102] This invention utilizes a parameter determination module to precisely match electrical pulse parameters based on the physicochemical properties of microvesicles (size, membrane potential, surface charge, etc.) and preliminary experimental data. This prevents cell death while simultaneously creating nanoscale pores in the cell membrane, significantly improving cell viability. A dynamic adjustment module addresses uneven voltage distribution caused by the surface tension of the liquid within the electrode cup by optimizing voltage, capacitance, and pulse frequency in real time. This ensures that microvesicles receive electrical pulses uniformly within a stable field strength region, improving transfection uniformity by 40%. A tracking and processing module monitors electrochemical reaction byproducts in real time using sensors. Combined with microfluidic filtration and pulse duty cycle adjustment, this effectively reduces the accumulation of toxic substances and bubble interference, maintaining a stable transfection microenvironment and increasing target material loading efficiency by over 50%. These three modules collaboratively construct a closed-loop regulation system. Based on real-time feedback from cell response, the electrical pulse parameters are iteratively optimized, overcoming the limitations of single-point improvements in traditional electroporation technology. This achieves precision across the entire process, from parameter design to process control, laying a crucial technological foundation for the practical application of microvesicles in drug delivery.

[0103] like Figure 2 As shown, the provided method includes steps S101 to S103. The computer device can be a handheld terminal, a laptop computer, a wearable device, or a robot, etc. This is used to implement steps S101 to S103 and their corresponding embodiments.

[0104] Step S101. Determine the short-time high-voltage electric pulse application scheme based on the physicochemical properties of the cell carrier microvesicles. The electric pulse application scheme includes voltage, pulse duration and pulse interval parameters. The pulse interval parameter is an unbiased estimate based on pre-experimental data. It is used to polarize the cell membrane phospholipid bilayer structure to form nanoscale pores to allow the target substance to enter, and avoid cell death due to excessive voltage or pulse duration.

[0105] Step S102. After mixing the cells with the exogenous dye, transfer them to the electrode cup. Based on the uneven voltage distribution caused by the surface tension of the liquid in the electrode cup, dynamically adjust the applied voltage, capacitance and pulse frequency.

[0106] Step S103. During the transfection process, continuously track the characteristics of the electrochemical reaction and deal with the situation where toxic substances and bubbles are generated by the electrochemical reaction to ensure the electrotransfection effect of cells in the stable field current region, so as to achieve precise control of the electric pulse field and improve the transfection efficiency of cell carrier microvesicles.

[0107] In some embodiments, determining the short-term high-voltage electrical pulse application scheme based on the physicochemical properties of the cell carrier microvesicles includes: acquiring the size distribution, membrane potential characteristics, surface charge density, and membrane lipid composition parameters of the cell carrier microvesicles; inputting the size distribution, membrane potential characteristics, surface charge density, and membrane lipid composition parameters into a pre-constructed electrical pulse parameter database, matching the voltage range, pulse duration threshold, and pulse interval formula fitted based on pre-experimental data for the corresponding type of microvesicle, wherein the pre-experimental data includes measured data on cell viability and target substance uptake under different combinations of electrical pulse parameters, in order to determine an electrical pulse application scheme that both forms nanoscale pores to allow target substances to enter and avoids cell death.

[0108] In some embodiments, the step of mixing cells with exogenous dyes and then transferring them to an electrode cup includes: mixing cell suspensions and exogenous dyes at a preset concentration ratio under sterile conditions, wherein the preset concentration ratio is optimized according to cell density and the molecular weight of the exogenous dyes, and controlling the temperature at 2-8°C and maintaining a constant stirring rate during the mixing process; transferring the mixture to an electrode cup whose inner wall is treated with a hydrophilic coating, wherein the electrode spacing and cup shape parameters of the electrode cup are matched with the voltage output range in the electrical pulse application scheme to reduce the voltage attenuation effect in the edge region caused by liquid surface tension.

[0109] In some embodiments, the continuous tracking of electrochemical reaction characteristics during transfection includes: using a micro current sensor, voltage sensor, and pH sensor integrated into the electrode cup to collect data on current fluctuations between electrodes, changes in solution impedance, and pH shifts in real time at a frequency of not less than 100 Hz; and combining this with a preset electrochemical reaction byproduct generation model to determine the rate of toxic substance generation and the trend of bubble generation. The electrochemical reaction byproduct generation model is constructed based on the correlation between electrolyte composition, pulse energy parameters, and byproduct types.

[0110] In some embodiments, the handling of toxic substances and bubbles generated by the electrochemical reaction includes: when the concentration of toxic substances is detected to exceed the cell tolerance threshold, triggering a microfluidic filtration device in the electrode cup to circulate and purify the reaction solution, wherein the pore size of the filtration device is smaller than the microvesicle size; when the volume ratio of bubbles is detected to exceed 5%, automatically pausing the electrical pulse output and injecting nitrogen buffer gas, while adjusting the duty cycle of subsequent pulses to reduce the intensity of local electrolysis reaction and maintain the volume ratio of the stable field strength current region not less than 70%.

[0111] In some embodiments, the precise control of the electric pulse field and the improvement of transfection efficiency of cell carrier microvesicles include: establishing a closed-loop regulation system of electric pulse parameters-electric field distribution-cell response through the coordinated feedback control of the parameter determination module, the dynamic adjustment module and the tracking processing module. The closed-loop regulation system uses a gradient descent algorithm to iteratively optimize the combination of voltage, capacitance and pulse frequency based on the real-time acquired cell viability signal and the fluorescent labeling intensity signal of the target substance, so as to improve the cell transfection efficiency in the stable field current region to a preset optimized target value.

[0112] In some embodiments, the pulse interval parameter is an unbiased estimate based on pre-experimental data, used to polarize the cell membrane phospholipid bilayer structure to form nanoscale pores to allow the target substance to enter. This includes: obtaining cell membrane transmembrane potential recovery curves under different pulse intervals through pre-experiments, determining the effective time window for the membrane pores to be in an open state, and calculating the minimum interval required to ensure that the target substance completes transmembrane transport by combining the molecular weight and diffusion coefficient of the target substance, thus eliminating the risk of cell rupture caused by incomplete cell membrane repair due to too short an interval. The pre-experimental data covers microvesicle transfection models of at least three different cell types.

[0113] It should be noted that those skilled in the art will clearly understand that, for the sake of convenience and brevity, the specific working process of the above-described method for precise control of the electric pulse field for physical transfection of cell carrier microvesicles and each step can be referred to the corresponding process in the embodiments of the above-described precise control system for electric pulse field for physical transfection of cell carrier microvesicles, and will not be repeated here.

[0114] Please see Figure 3 , Figure 3 This is a schematic block diagram of the structure of a computer device provided in an embodiment of this application. The computer device includes a processor, a memory, and a network interface connected via a device bus, wherein the memory may include a storage medium and internal memory.

[0115] The storage medium may store operating devices and computer programs. The computer program includes program instructions that, when executed, cause the processor to perform any embodiment of a method for precisely controlling an electrical pulse field for the physical transfection of cell carrier microvesicles.

[0116] The processor provides computing and control capabilities, supporting the operation of the entire computer device.

[0117] Internal memory provides an environment for the execution of computer programs in non-volatile storage media. When executed by a processor, the computer program enables the processor to execute any method based on an electrical pulse field precision control system for the physical transfection of cell carrier microvesicles.

[0118] This network interface is used for network communication, such as sending assigned tasks. Those skilled in the art will understand that... Figure 3 The structure shown is merely a block diagram of a portion of the structure related to the present application and does not constitute a limitation on the terminal to which the present application is applied. Specific computer devices may include more or fewer components than those shown in the figure, or combine certain components, or have different component arrangements.

[0119] It should be understood that the processor can be a Central Processing Unit (CPU), but it can also be other general-purpose processors, digital signal processors (DSPs), application-specific integrated circuits (ASICs), field-programmable gate arrays (FPGAs), or other programmable logic devices, discrete gate or transistor logic devices, discrete hardware components, etc. Among these, a general-purpose processor can be a microprocessor or any conventional processor.

[0120] In one embodiment, the processor is configured to run a computer program stored in memory to perform the following steps:

[0121] Step S101. Determine the short-time high-voltage electric pulse application scheme based on the physicochemical properties of the cell carrier microvesicles. The electric pulse application scheme includes voltage, pulse duration and pulse interval parameters. The pulse interval parameter is an unbiased estimate based on pre-experimental data. It is used to polarize the cell membrane phospholipid bilayer structure to form nanoscale pores to allow the target substance to enter, and avoid cell death due to excessive voltage or pulse duration.

[0122] Step S102. After mixing the cells with the exogenous dye, transfer them to the electrode cup. Based on the uneven voltage distribution caused by the surface tension of the liquid in the electrode cup, dynamically adjust the applied voltage, capacitance and pulse frequency.

[0123] Step S103. During the transfection process, continuously track the characteristics of the electrochemical reaction and deal with the situation where toxic substances and bubbles are generated by the electrochemical reaction to ensure the electrotransfection effect of cells in the stable field current region, so as to achieve precise control of the electric pulse field and improve the transfection efficiency of cell carrier microvesicles.

[0124] In some embodiments, determining the short-term high-voltage electrical pulse application scheme based on the physicochemical properties of the cell carrier microvesicles includes: acquiring the size distribution, membrane potential characteristics, surface charge density, and membrane lipid composition parameters of the cell carrier microvesicles; inputting the size distribution, membrane potential characteristics, surface charge density, and membrane lipid composition parameters into a pre-constructed electrical pulse parameter database, matching the voltage range, pulse duration threshold, and pulse interval formula fitted based on pre-experimental data for the corresponding type of microvesicle, wherein the pre-experimental data includes measured data on cell viability and target substance uptake under different combinations of electrical pulse parameters, in order to determine an electrical pulse application scheme that both forms nanoscale pores to allow target substances to enter and avoids cell death.

[0125] In some embodiments, the step of mixing cells with exogenous dyes and then transferring them to an electrode cup includes: mixing cell suspensions and exogenous dyes at a preset concentration ratio under sterile conditions, wherein the preset concentration ratio is optimized according to cell density and the molecular weight of the exogenous dyes, and controlling the temperature at 2-8°C and maintaining a constant stirring rate during the mixing process; transferring the mixture to an electrode cup whose inner wall is treated with a hydrophilic coating, wherein the electrode spacing and cup shape parameters of the electrode cup are matched with the voltage output range in the electrical pulse application scheme to reduce the voltage attenuation effect in the edge region caused by liquid surface tension.

[0126] In some embodiments, the continuous tracking of electrochemical reaction characteristics during transfection includes: using a micro current sensor, voltage sensor, and pH sensor integrated into the electrode cup to collect data on current fluctuations between electrodes, changes in solution impedance, and pH shifts in real time at a frequency of not less than 100 Hz; and combining this with a preset electrochemical reaction byproduct generation model to determine the rate of toxic substance generation and the trend of bubble generation. The electrochemical reaction byproduct generation model is constructed based on the correlation between electrolyte composition, pulse energy parameters, and byproduct types.

[0127] In some embodiments, the handling of toxic substances and bubbles generated by the electrochemical reaction includes: when the concentration of toxic substances is detected to exceed the cell tolerance threshold, triggering a microfluidic filtration device in the electrode cup to circulate and purify the reaction solution, wherein the pore size of the filtration device is smaller than the microvesicle size; when the volume ratio of bubbles is detected to exceed 5%, automatically pausing the electrical pulse output and injecting nitrogen buffer gas, while adjusting the duty cycle of subsequent pulses to reduce the intensity of local electrolysis reaction and maintain the volume ratio of the stable field strength current region not less than 70%.

[0128] In some embodiments, the precise control of the electric pulse field and the improvement of transfection efficiency of cell carrier microvesicles include: establishing a closed-loop regulation system of electric pulse parameters-electric field distribution-cell response through the coordinated feedback control of the parameter determination module, the dynamic adjustment module and the tracking processing module. The closed-loop regulation system uses a gradient descent algorithm to iteratively optimize the combination of voltage, capacitance and pulse frequency based on the real-time acquired cell viability signal and the fluorescent labeling intensity signal of the target substance, so as to improve the cell transfection efficiency in the stable field current region to a preset optimized target value.

[0129] In some embodiments, the pulse interval parameter is an unbiased estimate based on pre-experimental data, used to polarize the cell membrane phospholipid bilayer structure to form nanoscale pores to allow the target substance to enter. This includes: obtaining cell membrane transmembrane potential recovery curves under different pulse intervals through pre-experiments, determining the effective time window for the membrane pores to be in an open state, and calculating the minimum interval required to ensure that the target substance completes transmembrane transport by combining the molecular weight and diffusion coefficient of the target substance, thus eliminating the risk of cell rupture caused by incomplete cell membrane repair due to too short an interval. The pre-experimental data covers microvesicle transfection models of at least three different cell types.

[0130] It should be noted that those skilled in the art will clearly understand that, for the sake of convenience and brevity, the specific working process of the processor described above can be referred to the corresponding process in the method embodiments of the above embodiments, and will not be repeated here.

[0131] The embodiments of this application also provide a computer-readable storage medium storing a computer program, the computer program including program instructions, and the processor executing the program instructions to implement the steps of the method for precise control of the electric pulse field for physical transfection of cell carrier microvesicles provided in the above embodiments of this application.

[0132] The computer-readable storage medium may be an internal storage unit of the computer device described in the foregoing embodiments, such as the hard disk or memory of the computer device. The computer-readable storage medium may also be an external storage device of the computer device, such as a plug-in hard disk, SmartMedia Card (SMC), Secure Digital (SD) card, or Flash Card equipped on the computer device.

[0133] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in this application, and these modifications or substitutions should all be covered within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A precise control system for an electric pulse field for physical transfection of cell carrier microvesicles, characterized in that, include: The parameter determination module is configured to determine a short-time high-voltage electric pulse application scheme based on the physicochemical properties of the cell carrier microvesicles. The electric pulse application scheme includes voltage, pulse duration, and pulse interval parameters. The pulse interval parameter is estimated unbiasedly based on pre-experimental data. It is used to polarize the cell membrane phospholipid bilayer structure to form nanoscale pores to allow the target substance to enter, and to avoid cell death due to excessive voltage or pulse duration. The dynamic adjustment module is configured to transfer cells to an electrode cup after mixing them with exogenous dyes. Based on the uneven voltage distribution caused by the surface tension of the liquid in the electrode cup, the applied voltage, capacitance, and pulse frequency are dynamically adjusted. The tracking and processing module is configured to continuously track the electrochemical reaction characteristics during transfection and to handle situations where toxic substances and bubbles are generated by the electrochemical reaction, so as to ensure the electrotransfection effect of cells in the stable field current region, thereby achieving precise control of the electric pulse field and improving the transfection efficiency of cell carrier microvesicles.

2. The system according to claim 1, characterized in that, The method for determining the short-time high-voltage pulse application scheme based on the physicochemical properties of the cell carrier microvesicles includes: To obtain the size distribution, membrane potential characteristics, surface charge density, and membrane lipid composition parameters of cell carrier microvesicles; The size distribution, membrane potential characteristics, surface charge density, and membrane lipid composition parameters are input into a pre-constructed electrical pulse parameter database. The voltage range, pulse duration threshold, and pulse interval formula fitted based on the pre-experimental data are matched for the corresponding type of microvesicle. The pre-experimental data includes measured data on cell survival rate and target substance uptake rate under different combinations of electrical pulse parameters, in order to determine an electrical pulse application scheme that can both form nanoscale pores to allow target substances to enter and avoid cell death.

3. The system according to claim 1, characterized in that, The process of mixing cells with exogenous dyes and then transferring them to an electrode cup includes: Under sterile conditions, cell suspension and exogenous dye are mixed at a preset concentration ratio. The preset concentration ratio is optimized according to cell density and molecular weight of exogenous dye. The mixing process is controlled at 2-8℃ and a constant stirring rate is maintained. The electrode is transferred to an electrode cup whose inner wall is treated with a hydrophilic coating. The electrode spacing and cup shape parameters of the electrode cup are matched with the voltage output range in the electric pulse application scheme to reduce the voltage attenuation effect in the edge region caused by liquid surface tension.

4. The system according to claim 1, characterized in that, The continuous monitoring of electrochemical reaction characteristics during transfection includes: By integrating a miniature current sensor, voltage sensor, and pH sensor into the electrode cup, the current fluctuation value between electrodes, the change in solution impedance, and the acid-base deviation data are collected in real time at a frequency of not less than 100Hz. Combined with a preset electrochemical reaction byproduct generation model, the generation rate of toxic substances and the trend of bubble generation are determined. The electrochemical reaction byproduct generation model is constructed based on the correlation between electrolyte composition, pulse energy parameters, and byproduct types.

5. The system according to claim 1, characterized in that, The treatment of situations where electrochemical reactions produce toxic substances and bubbles includes: When the concentration of toxic substances exceeds the cell tolerance threshold, a microfluidic filtration device in the electrode cup is triggered to circulate and purify the reaction solution. The pore size of the filtration device is smaller than the microvesicle size. When the volume percentage of bubbles exceeds 5%, the electrical pulse output is automatically paused and nitrogen buffer gas is injected. At the same time, the duty cycle of subsequent pulses is adjusted to reduce the intensity of local electrolysis reaction and maintain the volume percentage of the stable field strength current region at no less than 70%.

6. The system according to claim 1, characterized in that, The method for achieving precise control of the electrical pulse field and improving the transfection efficiency of cell carrier microvesicles includes: Through the collaborative feedback control of the parameter determination module, dynamic adjustment module, and tracking processing module, a closed-loop regulation system of electric pulse parameters, electric field distribution, and cell response is established. The closed-loop regulation system uses a gradient descent algorithm to iteratively optimize the combination of voltage, capacitance, and pulse frequency based on the real-time acquired cell viability signal and the fluorescence labeling intensity signal of the target substance, so as to improve the cell transfection efficiency in the stable field current region to the preset optimized target value.

7. The system according to claim 1, characterized in that, The pulse interval parameter, estimated unbiased based on pre-experimental data, is used to polarize the cell membrane phospholipid bilayer structure, creating nanoscale pores to allow target substances to enter, including: By obtaining cell membrane transmembrane potential recovery curves under different pulse intervals through preliminary experiments, the effective time window for membrane pores to be in an open state is determined. Combined with the molecular weight and diffusion coefficient of the target substance, the minimum interval required to ensure its completion of transmembrane transport is calculated to eliminate the risk of cell rupture caused by incomplete cell membrane repair due to too short an interval. The preliminary experimental data covers microvesicle transfection models of at least three different cell types.

8. A method for precise control of an electrical pulse field for physical transfection of cell carrier microvesicles, characterized in that, The method, applied to the precise control system for physical transfection of cell carrier microvesicles according to any one of claims 1-7, comprises: The short-time high-voltage electric pulse application scheme is determined based on the physicochemical properties of the cell carrier microvesicles. The electric pulse application scheme includes voltage, pulse duration and pulse interval parameters. The pulse interval parameter is estimated unbiasedly based on pre-experimental data. It is used to polarize the cell membrane phospholipid bilayer structure to form nanoscale pores to allow the target substance to enter, and avoid cell death due to excessive voltage or pulse duration. Cells were mixed with exogenous dyes and transferred to an electrode cup. Based on the uneven voltage distribution caused by the surface tension of the liquid in the electrode cup, the applied voltage, capacitance and pulse frequency were dynamically adjusted. During the transfection process, the characteristics of the electrochemical reaction are continuously monitored, and measures are taken to address the generation of toxic substances and bubbles in the electrochemical reaction. This ensures the electrotransfection effect of cells within the stable field current region, thereby achieving precise control of the electric pulse field and improving the transfection efficiency of cell carrier microvesicles.

9. A computer device, characterized in that, The computer device includes a memory and a processor; The memory is used to store computer programs; The processor is configured to execute the computer program and, in executing the computer program, implement the method as described in claim 8.

10. A computer-readable storage medium, characterized in that, The computer-readable storage medium stores a computer program that, when executed by a processor, causes the processor to implement the method as described in claim 8.

Images