Targeted engineering transformation method for outer vesicles

Abstract

The invention provides an external vesicle targeting engineering modification method, and belongs to the technical field of external vesicles, the method comprises the following steps: firstly, extracting external vesicles through differential centrifugation, carrying out pre-screening through particle size analysis and surface potential determination, then treating with a surfactant, and carrying out surface modification through a coordination group modifier and targeting ligand molecules to obtain the targeting engineering modified external vesicles. A modification density ratio is calculated by utilizing a surface modification evaluation equation set, a targeted modification multiple is measured, a modification effect is judged based on double threshold values, process parameters or reaction time is optimized according to a judgment result, and finally the modified outer vesicles meeting requirements are obtained. The technical problem that in the prior art, it is difficult to quantitatively evaluate and precisely regulate and control the targeting modification effect of the outer vesicles is solved, controllability and repeatability of the modification process are achieved, and a foundation is laid for large-scale application of the targeting modification technology of the outer vesicles.

Description

Technical Field

[0001] This invention belongs to the field of external vesicle technology, and more specifically, relates to a method for targeted engineering modification of external vesicles. Background Technology

[0002] Extravesicular vesicles, as important intercellular communication carriers, have broad application prospects in disease diagnosis and treatment. Traditional extravesicular vesicle targeted modification techniques mainly include chemical coupling, bioaffinity, and membrane fusion. Chemical coupling involves introducing active groups on the surface of extravesicular vesicles, which then undergo covalent reactions with target ligand molecules; bioaffinity utilizes the specific binding of biotin and avidin to achieve the connection of target ligands; and membrane fusion modifies the extravesicular vesicle by integrating the target ligand into the extravesicular vesicle membrane structure. These methods have been widely used in medical research and clinical applications, providing technical support for the targeted delivery of extravesicular vesicles.

[0003] However, existing external vesicle targeted modification technologies have significant limitations. First, the modification process lacks real-time monitoring methods, making it impossible to accurately grasp the progress and extent of surface modification. Second, the evaluation methods for modification effects are simplistic, relying mainly on the final targeting effect detection and lacking quantitative analysis of intermediate processes. Third, the optimization of process parameters is primarily based on empirical judgment, making it difficult to establish systematic evaluation standards. Finally, the adjustment of modification conditions lacks theoretical guidance, often requiring repeated trials to achieve satisfactory results. These problems lead to low efficiency and unstable product quality in external vesicle targeted modification.

[0004] Currently, the development of external vesicle targeted modification technology faces significant challenges. Due to the lack of an effective quantitative evaluation system, it is impossible to accurately determine the degree of completion of the modification process and the quality of the targeting effect. Simultaneously, the lack of scientific basis for controlling modification conditions makes it difficult to achieve precise optimization of the process. In other words, existing technologies suffer from the technical problem of difficulty in quantitatively evaluating and precisely controlling the effects of external vesicle targeted modification. These technical bottlenecks severely restrict the large-scale application of external vesicle targeted modification technology, necessitating the establishment of a scientific evaluation and control method. Summary of the Invention

[0005] In view of this, the present invention provides a method for targeted engineering modification of external vesicles, which can solve the technical problem that the effect of targeted modification of external vesicles is difficult to quantitatively evaluate and precisely control in the prior art.

[0006] This invention is implemented as follows: A method for targeted engineering modification of exovesicles includes the following steps: extracting the exovesicles to be modified, and screening exovesicle precipitates with a particle size distribution uniformity of less than 0.3; measuring the surface potential value of the exovesicle precipitates; activating the surface proteins of the exovesicles with a surfactant; modifying the surface with a ligand molecule using a coordinating group modifier; calculating the surface modification density ratio using a surface modification evaluation equation set, wherein the surface modification evaluation equation set is based on ligand binding efficiency and surface density, the ligand binding efficiency is calculated using a multi-factor nonlinear regression model, and the surface density is calculated using a neural network algorithm; calculating the targeting modification fold; determining whether the targeting modification fold and the surface modification density ratio reach a preset threshold, wherein the targeting modification threshold is determined by 80% of the ratio of the baseline binding rate to the maximum binding rate, and the surface modification threshold is determined by 75% of the average surface modification density ratio of three batches of modified exovesicles.

[0007] The step of extracting the external vesicles to be modified by differential centrifugation combined with balanced density gradient centrifugation is as follows: the cell culture medium is collected by centrifugation, the cells are removed by centrifugation at 300×g for 10 minutes at 4℃, the supernatant is collected and centrifuged at 2000×g for 20 minutes to remove cell debris, the supernatant is collected and centrifuged at 10000×g for 30 minutes to remove large particles, the supernatant is collected and centrifuged at 100000×g for 90 minutes to collect the external vesicle precipitate, the external vesicle precipitate is resuspended with phosphate buffer, and the concentration of external vesicles is adjusted to 100,000 to 500,000 per milliliter.

[0008] The step of activating the surface proteins of the outer vesicles with a surfactant is as follows: prepare a surfactant solution with a mass fraction of 0.1 to 0.5%, slowly add it dropwise to the outer vesicle precipitation solution, control the drop rate to 0.1 ml per minute, and stir at 25 to 30°C for 15 to 30 minutes, with the stirring speed controlled at 200 rpm.

[0009] The step of surface modification by ligand modifier specifically involves: preparing a fluorescently labeled ligand modifier solution, adjusting the mass ratio of ligand modifier to exovesicle precipitate to 1:5 to 1:10, and reacting at 37°C for 1 to 2 hours.

[0010] The step of surface modification by targeting ligand molecules specifically involves: preparing a fluorescently labeled solution of the targeting ligand molecules, adjusting the mass ratio of the targeting ligand molecules to the outer vesicle precipitate to 1:2 to 1:5, and reacting at 37°C for 2 to 4 hours.

[0011] The step of calculating the number of ligands bound per unit area specifically involves: using flow cytometry to determine the number and binding strength of fluorescently labeled ligand molecules, setting the forward scattering threshold of the flow cytometer to 200, the voltage of the fluorescence detection channel to 350 volts, and using a density clustering algorithm to analyze the distribution of the fluorescence signal.

[0012] Specifically, when the targeting modification factor is less than the targeting modification threshold, the mass fraction of the surfactant is increased by 0.1%, the mass ratio of the coordinating group modifier to the external vesicle precipitate is adjusted to 1:4, and the mass ratio of the targeting ligand molecule to the external vesicle precipitate is adjusted to 1:1.5, and the modification is carried out again.

[0013] Specifically, when the surface modification density ratio is less than the surface modification threshold, the stirring time of the surface activator is extended to 45 minutes, the reaction time of the ligand modifier is extended to 3 hours, and the reaction time of the target ligand molecule is extended to 5 hours, and the modification is carried out again.

[0014] The resuspended modified exovesicles were aliquoted into low-adsorption centrifuge tubes with a capacity of 0.5 to 1 ml, and each tube was aliquoted to no more than 80% of its volume. The tubes were then stored at -80°C.

[0015] The dispersion of the outer vesicles was observed using a fully automated microscopic scanning imaging and analysis system. The ultrasound time was adjusted according to the dispersion, and the ultrasound time was controlled between 20 and 30 seconds.

[0016] Compared with existing technologies, this invention provides a method for targeted engineering modification of exovesicles. This method establishes a multi-parameter surface modification evaluation system and a dual-threshold judgment mechanism, enabling full-process monitoring and precise control of the modification process. Through comprehensive analysis of multiple parameters such as particle size distribution uniformity, surface potential value, and ligand binding efficiency, a scientific evaluation standard is constructed. The establishment of a surface modification evaluation equation set enables quantitative characterization of the modification effect. The dual-threshold judgment of the targeted modification multiple and the surface modification density ratio ensures the quality controllability of the modified product.

[0017] This invention ensures the uniformity of the modification process by introducing particle size distribution uniformity as a quality control parameter; it enables real-time assessment of the surface modification degree through surface potential monitoring; it establishes a quantitative evaluation method for modification density through ligand binding efficiency analysis; and it improves the accuracy of the assessment by processing multidimensional parameter relationships through neural network algorithms. Furthermore, targeted optimization strategies are established to address different types of modification deficiencies, achieving precise process control.

[0018] Therefore, by establishing a systematic evaluation system and optimization strategy, this invention successfully solves the technical problem of the difficulty in quantitatively evaluating and precisely controlling the effect of targeted modification of external vesicles. Attached Figure Description

[0019] Figure 1 This is a flowchart of the method of the present invention.

[0020] Figure 2 This is a diagram showing the particle size distribution of the external vesicles in Example 2.

[0021] Figure 3 This is a graph showing the trend of surface potential change of the outer vesicles during the surface activation process in Example 2.

[0022] Figure 4 This is a kinetic curve of the coordination group modification process in Example 2.

[0023] Figure 5 This is a comparison of the targeting effects of the modified exovesicles and the control group in different cell lines in Example 2. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0025] like Figure 1 The diagram shown is a flowchart of a method for targeted engineering modification of external vesicles provided by the present invention. This method includes the following steps:

[0026] S01. Extract the external vesicles to be modified, obtain the external vesicle precipitate by ultracentrifugation, and resuspend the external vesicle precipitate with phosphate buffer to make the concentration of the external vesicle precipitate reach 100,000 to 500,000 per milliliter.

[0027] S02. The particle size distribution uniformity of the exovesicle precipitate is determined by a nanoparticle size analyzer, and the exovesicle precipitate with a particle size distribution uniformity of less than 0.3 is selected.

[0028] S03. Measure the surface potential value of the external vesicle deposit using a dynamic light scattering instrument, and record the surface potential value as the initial surface potential value.

[0029] S04. Add a surfactant with a mass fraction of 0.1 to 0.5% to the external vesicle precipitation solution and stir at 25 to 30°C for 15 to 30 minutes to activate the surface proteins of the external vesicle precipitation.

[0030] S05. Add a fluorescently labeled coordination group modifier, wherein the mass ratio of the coordination group modifier to the exovesicle precipitate is 1:5 to 1:10, and react at 37°C for 1 to 2 hours.

[0031] S06. Add fluorescently labeled targeting ligand molecules to the reaction system, wherein the mass ratio of the targeting ligand molecules to the exovesicle precipitate is 1:2 to 1:5, and react at 37°C for 2 to 4 hours.

[0032] S07. The binding quantity and binding strength of the fluorescently labeled ligand molecules were determined by flow cytometry.

[0033] S08. Calculate the surface modification density ratio of the modified exovesicles based on the surface modification evaluation equation set, which includes a ligand binding efficiency equation and a surface density equation. The ligand binding efficiency equation calculates the number of ligands bound per unit area based on the number of fluorescently labeled ligand molecules bound, the precipitation concentration of the exovesicles, the particle size distribution uniformity, and the reaction system temperature. The surface density equation calculates the surface modification density ratio based on the number of ligands bound per unit area, the particle size distribution uniformity, the surface potential value, and the binding strength of the fluorescently labeled ligand molecules.

[0034] S09. Remove unreacted substances by ultrafiltration and centrifugation, and wash three times with the phosphate buffer solution to obtain the modified exovesicles;

[0035] S10. The binding rate of the modified extravesicles to the target cells is determined by flow cytometry, and the targeting modification fold is calculated. The targeting modification fold is the ratio of the binding rate to the control group binding rate.

[0036] S11. Determine whether the target modification multiple is greater than the preset target modification threshold and whether the surface modification density ratio is greater than the preset surface modification threshold. If the target modification multiple is greater than the target modification threshold and the surface modification density ratio is greater than the surface modification threshold, execute step S14. If the target modification multiple is less than or equal to the target modification threshold or the surface modification density ratio is less than or equal to the surface modification threshold, execute step S12.

[0037] S12. When the targeting modification multiple is less than or equal to the targeting modification threshold, the mass fraction of the surfactant is increased by 0.1%, the mass ratio of the coordinating group modifier to the exovesicle precipitate is adjusted to 1:4, the mass ratio of the targeting ligand molecule to the exovesicle precipitate is adjusted to 1:1.5, and the process returns to step S04.

[0038] S13. When the surface modification density ratio is less than or equal to the surface modification threshold, extend the stirring time of the surface activator to 45 minutes, extend the reaction time of the ligand modifier to 3 hours, extend the reaction time of the target ligand molecule to 5 hours, and return to step S04.

[0039] S14. Collect the modified exovesicles by ultracentrifugation and resuspend them in the phosphate buffer solution.

[0040] S15. The resuspended modified exovesicles are dispensed into low-adsorption centrifuge tubes with a capacity of 0.5 to 1 ml and stored at -80°C.

[0041] Steps for obtaining the targeted modification threshold: Prepare control group extravesicles, select 10 different cell lines, and measure the average binding rate between the control group extravesicles and the 10 different cell lines, recording it as the basal binding rate; add free-state targeting ligand molecules to the control group extravesicles, and measure the average binding rate between the control group extravesicles with added free-state targeting ligand molecules and the 10 different cell lines, recording it as the maximum binding rate; calculate the ratio of the maximum binding rate to the basal binding rate, and record 80% of the ratio as the targeted modification threshold.

[0042] Surface modification threshold acquisition steps: Select 3 batches of the above-mentioned exovesicles and repeat steps S01 to S09 to obtain 3 batches of the above-mentioned modified exovesicles; determine the average surface modification density ratio of the 3 batches of modified exovesicles, and record 75% of the average surface modification density ratio as the surface modification threshold.

[0043] The targeted modification factor is used to evaluate the effect of external vesicle modification, and the targeted modification factor being greater than the targeted modification threshold indicates that the targeted modification is successful.

[0044] The surface modification density ratio is used to evaluate the degree of surface modification of the exovesicles. A surface modification density ratio greater than the surface modification threshold indicates that the surface modification is sufficient.

[0045] The number of ligands bound per unit area is used to characterize the uniformity of the modification on the surface of the extravesicle. Optionally, the number of ligands bound per unit area is optimal in the range of 500 to 1000 per square micrometer.

[0046] Optionally, the maximum increase in the mass fraction of the surfactant is 1.0%; the minimum adjustment of the mass ratio of the coordinating group modifier to the exovesicle precipitate is 1:2; the minimum adjustment of the mass ratio of the targeting ligand molecule to the exovesicle precipitate is 1:1; the maximum stirring time for the surfactant is 60 minutes; the maximum reaction time for the coordinating group modifier is 4 hours; and the maximum reaction time for the targeting ligand molecule is 6 hours.

[0047] The specific implementation methods of the above steps are described in detail below. Step S01 is implemented by extracting the extravesicles to be modified using a combination of differential centrifugation and balanced density gradient centrifugation. First, cell culture medium is collected by centrifugation. The collected cell culture medium is centrifuged at 300×g for 10 minutes at 4°C to remove cells. The supernatant is then centrifuged at 2000×g for 20 minutes to remove cell debris, and then centrifuged at 10000×g for 30 minutes to remove large particles. Next, extravesicles are separated by ultracentrifugation. The supernatant after the above treatment is centrifuged at 100000×g for 90 minutes to collect the extravesicle precipitate. Then, the extravesicle precipitate is resuspended in phosphate buffer. The dispersion of the extravesicles is observed using a fully automated microscopic scanning imaging and analysis system. The sonication time is adjusted according to the dispersion, generally controlled to 20 to 30 seconds. The extravesicle concentration is determined by a nanoparticle tracking analysis system, and the concentration is adjusted to 100,000 to 500,000 vesicles per milliliter using a dilution method. The purpose of this step is to obtain exovesicle precipitates with appropriate concentration and good dispersibility, providing raw materials for subsequent modification.

[0048] The specific implementation of step S02 involves using a nanoparticle size analyzer to determine the particle size distribution uniformity of the exovesicle precipitate. The specific operation includes collecting 1 ml of the exovesicle precipitate solution, placing it in a quartz cuvette, setting the light source wavelength to 633 nm, and measuring the particle size distribution using the dynamic light scattering principle. The peak shape parameters of the particle size distribution curve are calculated using a Gaussian fitting algorithm, and the particle size distribution uniformity is calculated using the ratio of the standard deviation to the mean. Exovesicle precipitates with a particle size distribution uniformity less than 0.3 are selected for subsequent modification. This uniformity threshold is set based on the particle size distribution characteristics of the exovesicles; the smaller the particle size distribution uniformity, the more uniform the exovesicle particle size. The purpose of this step is to screen out exovesicles with uniform particle size distribution, ensuring the uniformity of subsequent modification.

[0049] The specific implementation of step S03 involves measuring the surface potential value of the exovesicle precipitation using a dynamic light scattering instrument. The specific operation includes collecting 1 ml of the exovesicle precipitation solution, placing it in a dedicated electrophoretic pool, setting the electric field strength to 15 volts per centimeter, and measuring the electrophoretic migration rate using the laser Doppler velocimetry principle. The surface potential value is calculated using the Henry's equation and recorded as the initial surface potential value, typically within the range of -20 to -30 millivolts. The purpose of this step is to obtain the characteristic parameters of the exovesicle surface potential, providing a basis for evaluating the surface modification effect.

[0050] The specific implementation of step S04 involves activating the surface proteins of the outer vesicles with a surfactant. The specific operation includes preparing a surfactant solution with a mass fraction of 0.1 to 0.5%, and slowly adding it dropwise to the outer vesicle precipitation solution, controlling the dropping rate at 0.1 mL per minute. The mixture is stirred at 25 to 30°C for 15 to 30 minutes at a stirring rate of 200 rpm. The surface protein activity is detected using a luciferase labeling method to ensure sufficient activation. The purpose of this step is to increase the activity of the outer vesicle surface proteins and improve the efficiency of subsequent modification.

[0051] Step S05 is implemented using coordination chemistry principles for surface modification. The specific steps include preparing a fluorescently labeled coordination group modifier solution, adjusting the mass ratio of the coordination group modifier to the exovesicle precipitate to 1:5 to 1:10, and adding it to the reaction system in batches using a microsyringe. The reaction is carried out at 37°C for 1 to 2 hours, with fluorescence intensity measured every 30 minutes during the reaction. A fluorescence intensity-time curve is plotted to determine the optimal reaction endpoint. The purpose of this step is to introduce coordination groups onto the exovesicle surface, providing a chemical basis for the subsequent connection of targeting ligands.

[0052] Step S06 is implemented by linking the targeting ligand using the principle of bioaffinity. The specific operations include preparing a fluorescently labeled solution of the targeting ligand molecule, adjusting the mass ratio of the targeting ligand molecule to the exovesicle precipitate to 1:2 to 1:5, and controlling the addition rate using isothermal titration. The reaction is carried out at 37°C for 2 to 4 hours, and dynamic light scattering technology is used to monitor particle size changes during the reaction to ensure the stability of the reaction system. The purpose of this step is to construct a targeting molecule modification layer, endowing the exovesicles with targeting function.

[0053] The specific implementation of step S07 involves using flow cytometry to determine the binding characteristics of fluorescently labeled ligand molecules. The specific operation includes collecting modified exovesicle samples, adjusting the concentration to 50,000 molecules per milliliter, setting the forward scattering threshold of the flow cytometer to 200, and setting the voltage of the fluorescence detection channel to 350 volts. A density clustering algorithm is used to analyze the fluorescence signal distribution, calculate the average fluorescence intensity and the coefficient of variation of fluorescence intensity, and quantify the number and binding strength of ligand molecules. The purpose of this step is to obtain quantitative parameters of ligand modification, providing data support for evaluating the modification effect.

[0054] The specific implementation of step S08 is based on calculating the surface modification density ratio using a set of surface modification evaluation equations. The ligand binding efficiency equation employs a multi-factor nonlinear regression model, using the number of fluorescently labeled ligand molecules bound, the concentration of exovesicle precipitation, particle size distribution uniformity, and reaction system temperature as independent variables to calculate the number of ligands bound per unit area. The surface density equation uses a neural network algorithm, using the number of ligands bound per unit area, particle size distribution uniformity, surface potential value, and the binding strength of fluorescently labeled ligand molecules as input variables to calculate the surface modification density ratio. The purpose of this step is to establish a surface modification evaluation system and achieve quantitative characterization of the modification effect.

[0055] The specific implementation of step S09 involves purifying the modified exovesicles using ultrafiltration centrifugation. The specific operation includes transferring the reaction system to an ultrafiltration centrifuge tube with a molecular weight cutoff of 100,000 Daltons and centrifuging at 4000×g for 30 minutes at 4°C. The tube is then washed three times with phosphate buffer, and particle size stability is detected using dynamic light scattering after each wash. Finally, the purified modified exovesicles are transferred to sterile centrifuge tubes, and their concentration is measured and recorded. The purpose of this step is to remove unreacted substances from the reaction system and obtain pure modified exovesicles.

[0056] The specific implementation of step S10 involves evaluating the targeting effect using flow cytometry. The specific procedures include culturing the target cells to the logarithmic growth phase, digesting and collecting the cells, and adjusting the concentration to 1 × 10⁻⁶ cells per milliliter. 6 The modified vesicles were mixed with target cells at a ratio of 1:100 and incubated at 37°C for 2 hours. Fluorescence signals were detected using flow cytometry, and the binding rate was calculated using gating analysis. Simultaneously, the binding rate of the control group was measured, and the targeting modification fold was calculated. This step aims to evaluate the targeting performance of the modified vesicles and determine the success of the modification.

[0057] The specific implementation of step S11 involves using a dual-threshold judgment method to evaluate the modification effect. The specific operation includes comparing the calculated targeted modification multiple with a pre-set targeted modification threshold, and comparing the calculated surface modification density ratio with a pre-set surface modification threshold. When both the targeted modification multiple and the surface modification density ratio are greater than the surface modification threshold, it indicates that the modification has achieved the expected effect, and the process proceeds to the data collection step. If either indicator fails to meet the threshold requirement, the modification conditions need to be adjusted and the process repeated. The purpose of this step is to establish a judgment standard for the modification effect and achieve quality control of the process.

[0058] Step S12 is a specific implementation of an optimization strategy for insufficient targeting effect. Specifically, it involves increasing the mass fraction of the surfactant by 0.1% to enhance the activity of the surface protein. The mass ratio of the coordinating group modifier to the exovesicle precipitate is adjusted to 1:4 to increase the modification density of the coordinating groups. The mass ratio of the targeting ligand molecule to the exovesicle precipitate is adjusted to 1:1.5 to increase the number of targeting ligands linked. After optimization, the surface activation step is repeated for further modification. The purpose of this step is to improve the targeting modification effect by adjusting the reactant ratio.

[0059] Step S13 is a specific implementation of an optimization strategy for insufficient surface modification. The specific operations include extending the stirring time of the surface activator to 45 minutes to fully activate the surface proteins; extending the reaction time of the ligand modifier to 3 hours to increase the binding amount of the ligands; and extending the reaction time of the targeting ligand molecules to 5 hours to improve the connection efficiency of the targeting ligands. After optimization, the surface activation step is repeated, and the modification is carried out again. The purpose of this step is to improve the surface modification effect by extending the reaction time.

[0060] Step S14 is specifically implemented by collecting the modified exovesicles using ultracentrifugation. The specific procedure involves transferring the modified exovesicle solution to an ultracentrifuge tube and centrifuging at 100,000 × g for 90 minutes at 4°C to collect the precipitate. The precipitate is resuspended in pre-cooled phosphate buffer, and the concentration is determined using a nanoparticle tracking analysis system. The purpose of this step is to obtain concentrated modified exovesicles for easier subsequent storage.

[0061] The specific implementation of step S15 involves storing the modified exovesicles using a cryopreservation method. The specific operation includes selecting low-adsorption centrifuge tubes with a capacity of 0.5 to 1 ml, and aliquoting the resuspended modified exovesicles into tubes at no more than 80% of their volume. The aliquoting process is performed in a clean bench to ensure aseptic technique. The aliquoted modified exovesicles are then stored at -80°C, and their activity is monitored periodically. This step aims to ensure the long-term stability of the modified exovesicles.

[0062] This invention relates to the following calculation procedures and equations:

[0063] The specific equation for calculating particle size distribution uniformity is as follows:

[0064] In the formula, PDI is the particle size distribution uniformity; σ is the particle size standard deviation; and μ is the average particle size.

[0065] The equation for calculating the surface potential value is specifically expressed as follows:

[0066] In the formula, ζ is the surface potential value in millivolts; η is the viscosity of the medium; v is the electrophoretic migration rate; ε is the dielectric constant of the medium; f(ka) is the Henry function, which is related to the particle size and Debye length; k is the Debye-Hickel parameter; and a is the particle radius.

[0067] The ligand binding efficiency equation is specifically expressed as follows:

[0068] In the formula, N L The number of ligands bound per unit area, expressed in units per square micrometer; C p α represents the concentration of vesicle precipitation; PDI represents the particle size distribution uniformity; T represents the reaction system temperature; T0 represents the standard temperature, taken as 37℃; α represents the binding coefficient; β represents the temperature sensitivity coefficient; and γ represents the basic binding amount.

[0069] The surface density equation is specifically expressed as follows:

[0070] In the formula, ρ is the surface modification density ratio; n is the number of hidden layer nodes in the neural network; w i σ is the weight coefficient; N is the activation function; L ζ represents the number of ligands bound per unit area; PDI represents the particle size distribution uniformity; ζ represents the surface potential value; I f I0 is the binding intensity of the fluorescently labeled ligand molecule; I0 is the standard fluorescence intensity; b is the bias term.

[0071] The parameter acquisition method is as follows:

[0072] 1. Particle size distribution parameter acquisition: The particle size distribution was determined by dynamic light scattering method, and the standard deviation σ and mean μ were obtained by Gaussian fitting;

[0073] 2. Electrophoresis parameter acquisition: The electrophoretic migration rate v was obtained by laser Doppler velocimetry, and the medium viscosity η and dielectric constant ε were determined by the physical properties of the phosphate buffer solution;

[0074] 3. Acquisition of ligand binding parameters: The binding coefficient α was calibrated by ligand titration curve, the temperature sensitivity coefficient β was determined by temperature gradient experiment, and the basic binding amount γ was determined by blank control experiment.

[0075] 4. Obtaining neural network parameters: weight coefficients w i The bias term b is obtained through backpropagation training, with training data derived from multiple batches of modification experiments. The neural network used is a micro-neural network, preferably MnasNet or MobileNet series.

[0076] Explanation of the principle behind equation construction:

[0077] 1. The particle size distribution uniformity equation adopts the principle of the coefficient of variation, which can eliminate the influence of dimensions and facilitate comparison between different batches;

[0078] 2. The surface potential equation is derived based on the Smoluchowski equation, taking into account the effects of electric double layer effect and particle size;

[0079] 3. The ligand binding efficiency equation adopts an improved Langmuir adsorption model, which introduces a temperature correction term to reflect the influence of temperature on the binding process;

[0080] 4. The surface density equation adopts a multilayer perceptron structure and captures the complex relationship between parameters through a nonlinear activation function.

[0081] Among these measures, particle size distribution uniformity was introduced as a quality control parameter to improve the uniformity of the modification; a multi-parameter-based surface modification evaluation system was established to achieve quantitative characterization of the modification effect; a neural network algorithm was used to process the multi-dimensional parameter relationship to improve the accuracy of the evaluation; and a dual-threshold judgment mechanism was designed to ensure the controllability of the modification effect.

[0082] The specific equation for calculating the targeted modification multiple is as follows:

[0083] In the formula, FR represents the target modification multiple; BR mod To improve the binding rate of external vesicles to target cells; BR ctrl The binding rate of the control group's external vesicles to the target cells is shown.

[0084] The specific equation for calculating the targeted modification threshold is as follows:

[0085] In the formula, TH is the targeted modification threshold; BR max The average binding rate of extravesicles to target cells in the control group with added free-state targeting ligand molecules; BR base The baseline binding rate between the control group's external vesicles and the target cells is represented.

[0086] The parameter acquisition method is as follows:

[0087] 1. Obtaining binding rate parameters: Fluorescence signals were measured by flow cytometry, and the proportion of bound cells was calculated using gating analysis.

[0088] 2. Obtaining the basic binding rate: Ten different cell lines were selected, and the average binding rate of the control group's external vesicles was measured.

[0089] Explanation of the principle behind equation construction:

[0090] 1. The targeted modification multiplier equation uses a ratio form, which can intuitively reflect the degree of improvement in modification effect;

[0091] 2. The targeted modification threshold equation is based on the ratio of the maximum binding rate to the basic binding rate, and sets 80% as the qualified standard, which reflects the acceptable range of modification effect.

[0092] Specifically, the principle of this invention is based on multi-parameter synergistic evaluation and feedback control. First, by measuring the uniformity of particle size distribution, the uniformity of the exovesicle population is evaluated to ensure the quality of the modified substrate. Second, by monitoring the surface potential value, the changes in charge characteristics during surface modification are reflected, and the degree of modification is evaluated. Third, by analyzing ligand binding efficiency, the density and uniformity of surface modification are quantitatively characterized. Finally, a comprehensive evaluation model is established by processing multi-dimensional parameter data through a neural network algorithm.

[0093] Based on this, the present invention establishes a dual-threshold judgment mechanism. The targeted modification threshold is determined through control experiments, reflecting the target value of the modification effect; the surface modification threshold is obtained through statistical analysis of multiple batches of experiments, reflecting the stability requirements of the process. This dual-threshold mechanism considers both the final effect and process control, ensuring the controllability of modification quality. The present invention also designs corresponding optimization strategies for different types of modification deficiencies, improving the modification effect by adjusting reaction conditions and extending reaction time.

[0094] The following provides a specific embodiment 1 of the present invention, and the specific implementation of each step in this embodiment 1 is described in detail below.

[0095] The specific implementation of step S01 involves extracting the external vesicles to be modified using a combination of differential centrifugation and balanced density gradient centrifugation. This step is based on the density gradient separation principle, utilizing the density difference between external vesicles and other cellular components for separation. First, cell culture medium is collected by centrifugation. The collected cell culture medium is centrifuged at 300×g for 10 minutes at 4°C to remove cells. The supernatant is then centrifuged at 2000×g for 20 minutes to remove cell debris. The supernatant is then centrifuged at 10000×g for 30 minutes to remove large particles. Finally, the supernatant is centrifuged at 100000×g for 90 minutes to collect the external vesicle precipitate. The external vesicle precipitate is then resuspended in phosphate buffer. The dispersion of the external vesicles is observed using a fully automated microscopic scanning imaging and analysis system. The sonication time is adjusted according to the dispersion, controlled to 20 to 30 seconds. The concentration of the external vesicles is determined using a nanoparticle tracking analysis system, and the concentration is adjusted to 100,000 to 500,000 vesicles per milliliter using a dilution method. The differential centrifugation method used in this step is based on Stokes' law of sedimentation, and its sedimentation rate satisfies the equation: In the formula, v is the settling velocity, d is the particle diameter, and ρ is the settling velocity. p ρ is the particle density. fLet ρ be the fluid density, g be the acceleration due to gravity, and η be the fluid viscosity. The purpose of this step is to obtain exovesicle precipitates of suitable concentration and good dispersibility, providing raw materials for subsequent modification.

[0096] The specific implementation of step S02 involves using a nanoparticle size analyzer to determine the particle size distribution uniformity of the exovesicle precipitate. This step is based on the principle of dynamic light scattering, analyzing the particle size distribution by measuring the intensity fluctuations of scattered light caused by Brownian motion. The specific operation includes collecting 1 ml of the exovesicle precipitate solution, placing it in a quartz cuvette, setting the light source wavelength to 633 nm, and determining the particle size distribution using the principle of dynamic light scattering. The resulting particle size distribution dataset is: D = {d1, d2, ..., d...}. n The Gaussian distribution is fitted using the least squares method: Calculate the standard deviation σ and the mean μ: The final equation for calculating particle size distribution uniformity is as follows: Exovesicle precipitates with a particle size distribution uniformity of less than 0.3 were selected for subsequent modification. The purpose of this step is to screen out exovesicles with uniform particle size distribution to ensure the uniformity of subsequent modification.

[0097] The specific implementation of step S03 involves measuring the surface potential of the exovesicle precipitation using a dynamic light scattering instrument. This step is based on the principle of electrophoretic light scattering, calculating the surface potential by measuring the migration rate of charged particles in an electric field. The specific operation includes collecting 1 ml of the exovesicle precipitation solution, placing it in an electrophoretic pool, and setting the electric field strength to 15 volts per centimeter. Based on the electrophoretic velocity measurement data: V = {v1, v2, ..., v...} m Considering the effect of electric field strength E: Introducing Henry's function correction: The final equation for calculating the surface potential is: The purpose of this step is to obtain the surface potential characteristic parameters of the outer vesicles, providing a basis for evaluating the surface modification effect.

[0098] The specific implementation of step S04 involves activating the surface proteins of the outer vesicles with a surfactant. The specific operation includes preparing a surfactant solution with a mass fraction of 0.1 to 0.5%, and slowly adding it dropwise to the outer vesicle precipitation solution, controlling the dropping rate at 0.1 mL per minute. The mixture is stirred at 25 to 30°C for 15 to 30 minutes at a stirring rate of 200 rpm. The surface protein activity is detected by a luciferase labeling method, and the activity calculation equation is: A = A0(1 - e^(-e ... -kt In the formula, A represents real-time activity, A0 represents initial activity, k is the activation rate constant, and t is the activation time. This step aims to increase the activity of proteins on the surface of outer vesicles, thereby improving the efficiency of subsequent modifications.

[0099] The specific implementation of step S05 involves surface modification using coordination chemistry principles. The specific operations include preparing a solution of fluorescently labeled coordination group modifier and adjusting the mass ratio of the coordination group modifier to the exovesicle precipitate to 1:5 to 1:10. Based on the Langmuir adsorption isotherm: Introduce a temperature correction term: Considering the influence of particle size distribution: N L =N max θ(1-PDI). Simplifying, we obtain the ligand binding efficiency equation: The purpose of this step is to introduce coordinating groups on the surface of the outer vesicle, providing a chemical basis for the subsequent connection of targeting ligands.

[0100] The specific implementation of step S06 involves using the principle of bioaffinity to connect the targeting ligand. The specific operation includes preparing a fluorescently labeled solution of the targeting ligand molecule and adjusting the mass ratio of the targeting ligand molecule to the exovesicle precipitate to 1:2 to 1:5. The reaction is carried out at 37°C for 2 to 4 hours, and the binding process of the ligand molecule is dynamically monitored to establish a binding kinetic equation: In the formula, [C] represents the concentration of the ligand-receptor complex, [L] represents the concentration of the free ligand, [R] represents the concentration of the free receptor, and k on k is the binding rate constant. off Let be the dissociation rate constant. At equilibrium, the ligand binding amount satisfies the equation: In the formula B eq B represents the equilibrium binding amount. max For the maximum binding amount, K d The dissociation constant is used. This step aims to construct a targeting molecule modification layer, endowing the external vesicles with targeting functionality.

[0101] The specific implementation of step S07 involves using flow cytometry to determine the binding characteristics of fluorescently labeled ligand molecules. First, modified exovesicle samples are collected, and the concentration is adjusted to 50,000 per milliliter. The forward scattering threshold of the flow cytometer is set to 200, and the voltage of the fluorescence detection channel is set to 350 volts. A density clustering algorithm is used to analyze the fluorescence signal distribution, and a fluorescence intensity matrix is ​​constructed. In the formula F ij Let be the j-th fluorescence parameter of the i-th sample. A discriminant function is constructed using principal component analysis for dimensionality reduction: In the formula w i x is the weighting coefficient. i For fluorescence parameters, μ i This represents the mean of the parameters. The purpose of this step is to obtain quantitative parameters of ligand modification, providing data support for evaluating the modification effect.

[0102] The specific implementation of step S08 is based on calculating the surface modification density ratio using the surface modification evaluation equation set. Construct the input vector: X = Designing the weight matrix for a neural network: Use the sigmoid activation function: The surface density calculation equation is obtained as follows: The purpose of this step is to establish a surface modification evaluation system to achieve quantitative characterization of the modification effect.

[0103] The specific implementation of step S09 involves purifying the modified exovesicles using ultrafiltration centrifugation. The reaction system is transferred to an ultrafiltration centrifuge tube with a molecular weight cutoff of 100,000 Daltons and centrifuged at 4000 × g for 30 minutes at 4°C. The vesicles are then washed three times with phosphate buffer, based on the dilution-diffusion equation: C(t) = Coe⁻¹. -kt In the formula, C(t) represents the concentration of unreacted substances at any given time, C0 represents the initial concentration, k represents the cleaning efficiency coefficient, and t represents the cleaning time. After each cleaning, dynamic light scattering technology is used to detect particle size stability. The particle size change rate should satisfy: Δd / d0 ≤ 5%, where Δd is the particle size change and d0 is the initial particle size. The purpose of this step is to remove unreacted substances from the reaction system and obtain pure modified exovesicles.

[0104] The specific implementation of step S10 involves evaluating the targeting effect using flow cytometry. Targeted cells are cultured to the logarithmic growth phase, digested and collected, and the concentration is adjusted to 1 × 10⁻⁶ cells per milliliter. 6 The modified extravesicles were mixed with target cells at a ratio of 1:100 and incubated at 37°C for 2 hours. A binding rate matrix was designed. Calculate the average binding rate: The equation for calculating the targeted modification multiple is obtained as follows: The purpose of this step is to evaluate the targeting performance of the modified exovesicles and determine whether the modification was successful.

[0105] The specific implementation of step S11 is to use a dual-threshold judgment method to evaluate the modification effect. First, the targeted modification threshold calculation equation is designed: In the formula, TH is the targeted modification threshold, and BR max To determine the average binding rate of extravesicles to target cells in the control group with added free-state targeting ligand molecules, BR base The baseline binding rate between the control group's external vesicles and target cells was represented. A surface modification evaluation index system was also established, and weighting coefficients were determined using the analytic hierarchy process. In the formula λ i The eigenvalues ​​are used. The threshold range for the surface modification density ratio is calculated using fuzzy comprehensive evaluation: [ρ min , ρ max Based on experimental data from multiple batches, the surface modification threshold was determined to be 75% of the average surface modification density ratio. This step serves to establish a standard for judging the modification effect and to achieve quality control of the process.

[0106] The specific implementation of step S12 is an optimization strategy for insufficient targeting effect. When the targeting modification factor is less than the targeting modification threshold, a process parameter optimization model is established based on the response surface methodology: In the formula, Y represents the target modification factor, and x represents the target modification factor. i Here, β represents the process parameters, and β is the regression coefficient. The optimal combination of process parameters was determined through model analysis: the surfactant mass fraction was increased by 0.1%, the mass ratio of coordinating group modifier to exovesicle precipitate was adjusted to 1:4, and the mass ratio of targeting ligand molecule to exovesicle precipitate was adjusted to 1:1.5. After optimization, the surface activation step was repeated, and the modification was performed again. The purpose of this step is to improve the targeted modification effect by adjusting the reactant ratio.

[0107] The specific implementation of step S13 is an optimization strategy for insufficient surface modification. When the surface modification density ratio is less than the surface modification threshold, a kinetic model is used to optimize the reaction time: ρ(t) = ρ max (1-e -kt ), where ρ(t) is the surface modification density ratio at any time, ρ max Let t represent the maximum surface modification density ratio, k be the reaction rate constant, and t be the reaction time. The optimal reaction time combination was obtained through fitting experimental data: the surface activator stirring time was extended to 45 minutes, the ligand modifier reaction time to 3 hours, and the targeted ligand molecule reaction time to 5 hours. After optimization, the surface activation step was repeated, and the modification was performed again. The purpose of this step is to improve the surface modification effect by extending the reaction time.

[0108] The specific implementation of step S14 involves collecting the modified exovesicles using ultracentrifugation. The modified exovesicle solution is transferred to an ultracentrifuge tube and centrifuged at 100,000 × g for 90 minutes at 4°C to collect the precipitate. The centrifugation process is based on the sedimentation equilibrium principle and satisfies the equation: In the formula, m is the mass of the outer vesicle, ω is the angular velocity, r is the radius of rotation, and ρ is the radius of rotation. p ρ represents the density of the outer vesicles. f ρ represents the buffer density, and g represents the acceleration due to gravity. The precipitate was resuspended in pre-cooled phosphate buffer, and the concentration was determined using a nanoparticle tracking analysis system. This step aims to obtain concentrated modified exovesicles for subsequent preservation.

[0109] The specific implementation of step S15 involves storing the modified exovesicles using a cryopreservation method. Low-adsorption centrifuge tubes with a capacity of 0.5 to 1 ml are selected, and the resuspended modified exovesicles are aliquoted according to the principle that each tube should not exceed 80% of its volume. A stability evaluation model is established: S(t) = S0e -αtIn the formula, S(t) is the stability index at any time, S0 is the initial stability, α is the decay coefficient, and t is the storage time. Stored at -80℃, the activity of the extravesicles is periodically detected by flow cytometry. The activity retention rate should meet the following requirement: A t / A0≥85%, where A t A0 represents the activity at any given time, while A0 represents the initial activity. The purpose of this step is to ensure the long-term stability of the modified vesicles.

[0110] To better understand and implement this invention, Example 2 of a specific application scenario is provided below: Researchers are conducting research on tumor-targeting external vesicles, selecting human umbilical cord mesenchymal stem cells as the source of external vesicles, and preparing therapeutic external vesicles targeting colorectal cancer through engineering modification methods. The specific implementation process is as follows.

[0111] First, the supernatant from human umbilical cord mesenchymal stem cell culture was collected, and outer vesicles were extracted using differential centrifugation. The vesicles were sequentially centrifuged at 300×g for 10 minutes, 2000×g for 20 minutes, and 10000×g for 30 minutes at 4°C, and finally collected by centrifugation at 100000×g for 90 minutes. The vesicles were resuspended in phosphate buffer, sonicated for 25 seconds, and the concentration was determined to be 350,000 vesicles per milliliter using a nanoparticle tracking analysis system.

[0112] The particle size distribution of the exovesicles was determined using a nanoparticle size analyzer, and the test data are shown in Table 1.

[0113] Table 1. External vesicle particle size distribution data

[0114] Particle size range (nanometers) frequency(%) 30-50 5.2 50-70 15.8 70-90 42.3 90-110 28.6 110-130 6.8 130-150 1.3

[0115] Based on the particle size distribution data, the mean μ was calculated to be 85.4 nm, the standard deviation σ was 18.7 nm, and the particle size distribution uniformity (PDI) was 0.219, meeting the requirement of being less than 0.3. Figure 2 As shown, the horizontal axis represents particle size (nanometers), and the vertical axis represents frequency (percentage). The outer vesicle particle size distribution exhibits a typical normal distribution characteristic.

[0116] The surface potential of the outer vesicles was measured using a dynamic light scattering instrument. Five repeated measurements were performed under an electric field strength of 15 volts per centimeter. The results are shown in Table 2.

[0117] Table 2 Results of Surface Potential Measurement of External Vesicles

[0118]

[0119]

[0120] The calculated average surface potential value was -24.6 millivolts, which was used as the initial surface potential value.

[0121] A 0.3% (w / w) surfactant solution was prepared and added dropwise to the exovesicle suspension at a rate of 0.1 mL / min. The mixture was stirred at 200 rpm for 20 minutes at 28°C. The activation process was monitored using a luciferase assay, and the activity changes are shown in Table 3.

[0122] Table 3 Monitoring data of surface protein activation process

[0123] Time (minutes) Relative activity 0 1.00 5 1.45 10 1.82 15 2.13 20 2.31

[0124] like Figure 3 As shown, the trend of surface potential change of the outer vesicles during the surface activation process is illustrated. The horizontal axis represents the activation time (minutes), and the vertical axis represents the surface potential value (mV), reflecting the dynamic process of surface activation, in which the surface potential value gradually changes with the activation time.

[0125] A fluorescently labeled ligand-modifying agent solution was prepared, and the mass ratio of the modifier to the exovesicle was adjusted to 1:8. The reaction was carried out at 37°C for 1.5 hours. The modification process was monitored using a real-time fluorescence detection system, and the results are shown in Table 4.

[0126] Table 4 Monitoring data of the coordination group modification process

[0127] Time (minutes) Fluorescence intensity (relative units) 0 0 30 2580 60 4260 90 5120

[0128] An antibody targeting the HER2 receptor was used as the targeting ligand molecule, and the ligand-to-external vesicle mass ratio was adjusted to 1:3. The reaction was carried out at 37°C for 3 hours. The ligand binding characteristics were determined by flow cytometry, with a forward scattering threshold of 200 Ω and a fluorescence detection channel voltage of 350 volts. The detection results are shown in Table 5.

[0129] Table 5 Target ligand binding characteristics data

[0130] parameter numerical values Average fluorescence intensity 8620 Fluorescence intensity coefficient of variation 0.156 Positive rate (%) 92.4

[0131] The modification parameters were calculated based on the surface modification evaluation equations. The calculated ligand binding efficiency was 820 ligands per square micrometer, and the surface modification density ratio was 0.86, both exceeding the preset thresholds. Figure 4 As shown, the kinetic curves of the ligand modification process are displayed. The horizontal axis represents the reaction time (minutes), and the vertical axis represents the fluorescence intensity (relative units). The fluorescence intensity shows a saturation trend with the reaction time.

[0132] Modified external vesicles were co-cultured with three colorectal cancer cell lines for 2 hours, and the binding rate was determined by flow cytometry. The results are shown in Table 6.

[0133] Table 6 Targeted Effect Evaluation Data

[0134] cell lines Modified extravesicle binding rate (%) Control group binding rate (%) Targeted modification multiple HT29 45.6 8.2 5.56 SW480 42.8 7.9 5.42 HCT116 48.3 8.5 5.68

[0135] The calculated average targeted modification fold was 5.55, exceeding the preset targeted modification threshold of 4.80. Figure 5 As shown, the targeting effects of the modified outer vesicles and the control group on different cell lines are compared. The grouped bar charts visually demonstrate the targeting modification effect, showing that the modified outer vesicles exhibit significant targeting in all three cell lines.

[0136] The modified exovesicles were purified by ultrafiltration centrifugation. The vesicles were centrifuged at 4000×g for 30 minutes and washed three times with phosphate buffer. Particle size change was monitored after each wash, and the results showed that the change rate was less than 5%. Finally, the modified exovesicles were aliquoted into 0.5 mL low-adsorption centrifuge tubes, with each tube containing 0.4 mL of liquid, and stored at -80°C.

[0137] This embodiment employs a systematic evaluation method and optimization strategy to successfully prepare therapeutic exovesicles with HER2-targeting properties. Traditional exovesicle modification methods rely solely on the final binding rate to determine the modification effect, and the optimization of process parameters lacks theoretical basis. The method of this invention achieves precise control of the modification process through real-time monitoring and quantitative evaluation of multiple parameters. Specific advancements include: First, the establishment of a multi-parameter evaluation system including particle size distribution uniformity, surface potential value, and ligand binding efficiency, providing a means for full-process monitoring of the modification process. Second, the use of surface modification evaluation equations for quantitative analysis makes the evaluation of modification effects more objective and accurate. Third, the establishment of a scientific quality control standard through a dual-threshold judgment mechanism ensures the stability of product quality. Fourth, the design of targeted optimization strategies improves the precision and efficiency of process control. These technological advancements have transformed exovesicle targeted modification technology from an empirical operation to a controllable engineering approach, laying the foundation for clinical application.

[0138] It should be noted that the variables involved in this invention are explained in detail in Table 7 below.

[0139] Table 7 Variable Explanation Table

[0140]

[0141]

[0142] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for targeted engineering modification of external vesicles, characterized in that, Includes the following steps: Extract the external vesicles to be modified and screen the vesicle precipitates with a particle size distribution uniformity of less than 0.

3. Measure the surface potential value of the outer vesicle deposits; The surface proteins of the outer vesicles are activated by a surfactant; the surface is modified by a ligand modifier and a target ligand molecule; the surface modification density ratio is calculated by a set of surface modification evaluation equations, which are based on ligand binding efficiency and surface density. The ligand binding efficiency is calculated using a multi-factor nonlinear regression model, and the surface density is calculated using a neural network algorithm. Calculate the targeting modification multiple; determine whether the ratio of the targeting modification multiple to the surface modification density reaches a preset threshold. The targeting modification threshold is determined by 80% of the ratio of the basic binding rate to the maximum binding rate, and the surface modification threshold is determined by 75% of the average surface modification density ratio of 3 batches of modified exovesicles.

2. The method for targeted engineering modification of external vesicles according to claim 1, characterized in that, The steps for extracting the modified extravesicles by differential centrifugation combined with balanced density gradient centrifugation are as follows: cell culture medium is collected by centrifugation, cells are removed by centrifugation at 300×g for 10 minutes at 4℃, the supernatant is collected and centrifuged at 2000×g for 20 minutes to remove cell debris, the supernatant is collected and centrifuged at 10000×g for 30 minutes to remove large particles, the supernatant is collected and centrifuged at 100000×g for 90 minutes to collect the extravesicle precipitate, the extravesicle precipitate is resuspended with phosphate buffer, and the extravesicle concentration is adjusted to 100,000 to 500,000 per milliliter.

3. The method for targeted engineering modification of external vesicles according to claim 1, characterized in that, The steps for activating the surface proteins of the outer vesicles with a surfactant are as follows: prepare a surfactant solution with a mass fraction of 0.1 to 0.5%, slowly add it dropwise to the outer vesicle precipitation solution, control the drop rate to 0.1 ml per minute, and stir at 25 to 30°C for 15 to 30 minutes, with the stirring speed controlled at 200 rpm.

4. The method for targeted engineering modification of external vesicles according to claim 1, characterized in that, The surface modification step using a ligand modifier is as follows: prepare a fluorescently labeled ligand modifier solution, adjust the mass ratio of the ligand modifier to the exovesicle precipitate to 1:5 to 1:10, and react at 37°C for 1 to 2 hours.

5. The method for targeted engineering modification of external vesicles according to claim 1, characterized in that, The steps of surface modification by targeting ligand molecules are as follows: prepare a fluorescently labeled solution of the targeting ligand molecules, adjust the mass ratio of the targeting ligand molecules to the outer vesicle precipitate to 1:2 to 1:5, and react at 37°C for 2 to 4 hours.

6. The method for targeted engineering modification of external vesicles according to claim 1, characterized in that, The steps for calculating the number of ligands bound per unit area are as follows: flow cytometry is used to determine the number and binding strength of fluorescently labeled ligand molecules. The forward scattering threshold of the flow cytometer is set to 200, the voltage of the fluorescence detection channel is set to 350 volts, and the distribution of fluorescence signals is analyzed using a density clustering algorithm.

7. The method for targeted engineering modification of external vesicles according to claim 1, characterized in that, When the targeting modification factor is less than the targeting modification threshold, the mass fraction of the surfactant is increased by 0.1%, the mass ratio of the coordinating group modifier to the external vesicle precipitate is adjusted to 1:4, and the mass ratio of the targeting ligand molecule to the external vesicle precipitate is adjusted to 1:1.5, and the modification is repeated.

8. The method for targeted engineering modification of external vesicles according to claim 1, characterized in that, When the surface modification density ratio is less than the surface modification threshold, extend the stirring time of the surface activator to 45 minutes, extend the reaction time of the ligand modifier to 3 hours, extend the reaction time of the target ligand molecule to 5 hours, and then re-modify.

9. The method for targeted engineering modification of external vesicles according to claim 1, characterized in that, The resuspended modified exovesicles were aliquoted into low-adsorption centrifuge tubes with a capacity of 0.5 to 1 ml, with each tube not exceeding 80% of its volume, and stored at -80°C.

10. The method for targeted engineering modification of external vesicles according to claim 1, characterized in that, The dispersion of the outer vesicles was observed using a fully automated microscopic scanning imaging and analysis system. The ultrasound time was adjusted according to the dispersion, and the ultrasound time was controlled between 20 and 30 seconds.

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