Carrier synthesis preparation system and carrier synthesis preparation method

Abstract

The invention discloses a carrier synthesis preparation system and a carrier synthesis preparation method, and relates to the technical field of biomedicines.The carrier synthesis preparation system comprises a chip adaptation device, a liquid storage device and a liquid path system, a micro-fluidic chip device used for engineering modification of a platform using nano vesicles as a drug carrier is installed on a chip adaptation device and is communicated with a liquid storage device and a liquid path device pipeline, and a liquid path system adopts a constant flow pump set to pump sample solutions of the nano vesicles and molecular drugs in a first solution bottle into the micro-fluidic chip device to complete loading. And finally, collecting through a collecting bottle. The technical scheme provided by the invention is based on a microfluidic technology, and can be used as a universal technical platform for engineering transformation of nano vesicle carriers, and efficient, safe and large-scale synthesis and preparation can be performed on different engineering formulas (drug delivery carriers) of specific carriers; the problems that a traditional carrier engineering transformation method is complex and lengthy, lacks standardization and is difficult to produce on a large scale are solved.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a carrier synthesis preparation system and a carrier synthesis preparation method. Background Art

[0002] In recent years, with the deepening of research in precision medicine, vectors capable of effectively loading, transporting, and delivering a variety of therapeutic agents (nucleic acid drugs, gene editing tools, protein drugs, and chemical drugs) have become a key focus in drug development and clinical practice. However, while current mainstream drug delivery vectors, such as adeno-associated virus (AAV) and lipid nanoparticles (LNPs), have met drug delivery needs to some extent, they still have many shortcomings. Traditional vector engineering methods, such as bio-fusion expression, chemical synthesis, and gene editing, can improve vector properties to some extent, but the processes are complex and lengthy, lack standardized methods, and require customized technical routes for different vector types and application areas. Each vector technology has its own unique characteristics, and no single vector technology can meet most requirements in terms of safety, loading capacity, and targeting ability, making it impossible to serve as a universal technology platform. This increases the difficulty and cost of technology development and large-scale production. Microfluidics holds the potential to become a universal vector engineering platform. The core of microfluidics technology lies in the precise control of material transport, synthesis, and interaction at the micro- and nanoscale. It can achieve microscopic effects on micro- and nanoscale carriers that cannot be provided by macroscale platforms. It has been applied to a certain extent in the preparation of drug carriers such as liposomes and microspheres.

[0003] However, the application of microfluidic technology in related technologies to achieve nanovesicle loading of molecular drugs is time-consuming and labor-intensive, has low preparation efficiency, and is only suitable for small-scale preparation. Summary of the Invention

[0004] The main purpose of the present invention is to propose a carrier synthesis preparation system and carrier synthesis preparation method. The carrier synthesis preparation system is based on microfluidic technology and can serve as a universal technical platform for engineering the modification of nanovesicle-type carriers. It can efficiently, safely and scalably synthesize and prepare different engineered formulations of specific carriers (drug delivery carriers); it can solve the problems of traditional carrier engineering modification methods being complicated and lengthy, lacking standardization and difficult to scale up.

[0005] To achieve the above-mentioned purpose, the carrier synthesis preparation system proposed by the present invention comprises: A chip adapter device, comprising a chip stage and an adapter seat for mounting a microfluidic chip device, wherein the chip stage is movable relative to the adapter seat so that the microfluidic chip device can communicate with the adapter seat to form a flow path; a liquid storage device, which includes a first solution bottle and a collecting bottle; and The liquid circuit system includes a first constant flow pump group. The first solution bottle, the first constant flow pump group, the chip adapter device and the collection bottle are connected by a pipeline. The first constant flow pump group is used to allow the sample solution in the first solution bottle to enter the collection bottle after being loaded through the microfluidic chip device.

[0006] The technical solution of the present invention pumps a sample solution from a first solution bottle into a microfluidic chip device via a liquid circuit system, enabling nanovesicle loading of molecular drugs. Compared to solutions that manually draw a certain amount of sample solution through a syringe and then use microdevices such as micropumps and microvalves to deliver the drug to the target area, this solution supports large-scale production. Specifically, the micro-nanostructures within the microfluidic chip enable physical collision and high-speed fluid impact on the vesicles, thereby achieving morphological transformation and material reprogramming of the vesicles. The microfluidic chip can precisely control the ratio of reactants, mixing method, and reaction time, facilitating the synthesis of high-quality carriers. The microfluidic chip device is mounted on a chip carrier and docked with an adapter seat to form a flow path, ensuring that the microfluidic chip device can be stably and accurately connected to the liquid circuit system, ensuring smooth liquid flow. The liquid circuit system, through the first constant-current pump group and pipelines, can precisely control the flow rate and pressure of the sample solution. In summary, the existing production efficiency of nanovesicle carriers is low. Through modular design, this solution can easily replace different microfluidic chip devices to meet different engineered formulas (drug delivery carriers) and realize a variety of different synthetic preparation needs; the design of the chip adapter device liquid path system makes the system more stable and reliable, reducing the difficulty and complexity of operation; through the control of the first constant flow pump group and the liquid path system, the precise metering and mixing of the reactants are achieved, and the efficiency and accuracy of the reaction are improved. It can be used as a universal technical platform for efficient, safe and scalable synthetic preparation of different engineered formulas (drug delivery carriers) of specific carriers; it solves the problems of traditional carrier engineering modification methods being complicated and lengthy, lacking standardization and difficult to scale production.

[0007] In one embodiment, the carrier synthesis preparation system also includes a pressure sensing module and a flow sensing module, both of which are arranged on the pipeline. The pressure sensing module, the flow sensing module, and the first constant flow pump group are electrically connected, so that the liquid circuit system has multiple selectable working modes, and the multiple working modes include constant flow mode, constant pressure mode, variable flow mode and variable pressure mode.

[0008] In one embodiment, the carrier synthesis and preparation system also includes a microfluidic chip device, which includes a chip box body having a liquid inlet and a liquid outlet and a microfluidic chip sealed and installed in the chip box body, and the liquid inlet and the liquid outlet are used to dock and connect with the adapter seat; wherein, in the multiple working modes, the microfluidic chip has different loading effects on the sample solution in the first solution bottle.

[0009] In one embodiment, the first constant flow pump group includes at least two first plunger pumps connected in series or in parallel; and the multiple first plunger pumps in the first constant flow pump group can operate independently.

[0010] In one embodiment, the carrier synthesis preparation system also includes a first switching valve and a second switching valve, both of which are arranged on the pipeline and have multiple branch valve ports; the liquid storage device is also provided with a first circulation bottle and a second circulation bottle; the first switching valve pipeline connects the chip adapter device, the collection bottle, the first circulation bottle and the second circulation bottle; the second switching valve pipeline connects the first circulation bottle, the second circulation bottle, the first solution bottle and the first constant flow pump group; the chip adapter device can selectively connect the first solution bottle, the first circulation bottle and the second circulation bottle through the first switching valve and the second switching valve, so that the sample solution in the first solution bottle can be repeatedly loaded through the microfluidic chip device.

[0011] In one embodiment, the carrier synthesis preparation system also includes a third switching valve with multiple branch valve ports and a sample quantitative loop connected to a micro-injector, and the sample quantitative loop is connected to the third switching valve; the first constant flow pump group is connected to the chip adapter device pipeline through the third switching valve, and the third switching valve can optionally connect the sample quantitative loop and the first constant flow pump group.

[0012] In one embodiment, the liquid circuit system further includes a degassing device and a mixing device; the liquid circuit system further includes a second constant flow pump group; the liquid storage device further includes a second solution bottle; the second solution bottle, the degassing device, the second constant flow pump group and the mixing device pipeline are connected to form a second liquid circuit, and the second constant flow pump group is used to pump the solution in the second solution bottle; the degassing device is used to degas the solution pumped out of the first solution bottle and the second solution bottle; the first solution bottle, the degassing device, the first constant flow pump group and the mixing device pipeline are connected to form a first liquid circuit; the mixing device, the chip adapter and the collection bottle pipeline are connected to form a third liquid circuit; The solution pumped from the first liquid path by the first constant flow pump group and the solution pumped from the second liquid path by the second constant flow pump group are mixed through the mixing device, and then loaded from the third liquid path through the microfluidic chip and enter the collecting bottle.

[0013] The present invention also provides a carrier synthesis preparation system, comprising: A microfluidic chip device, comprising a chip box having a liquid inlet and a liquid outlet, and a microfluidic chip sealed and mounted in the chip box; A chip adapter device, comprising a chip stage and an adapter seat for mounting the chip cartridge, wherein the chip stage is movable relative to the adapter seat so that the liquid inlet and the liquid outlet are docked with the adapter seat to form a flow path; A liquid storage device, comprising a first solution bottle, a second solution bottle, a first circulation bottle, a second circulation bottle and a collection bottle, wherein the microfluidic chip is used for loading and processing the sample solution in the first solution bottle; and The hydraulic system includes: A first pipeline connects the second switching valve, the first solution bottle, the first constant flow pump group and the third switching valve to the liquid inlet, wherein the third switching valve is connected in parallel to the sample quantitative loop for switching the first pipeline straight-through or sample quantitative loop injection; A second pipeline is connected to the second solution bottle and the second constant flow pump assembly to the liquid inlet, and merges with the first pipeline in front of the liquid inlet; a third pipeline, connecting the liquid outlet to the first switching valve, the first switching valve being connected to the collecting bottle and the circulation pipeline group respectively; a circulation pipeline group, comprising a first branch connected to the first circulation bottle and a second branch connected to the second circulation bottle, wherein the circulation pipeline group forms a closed loop with the first pipeline via a second switching valve; The linkage control of the first switching valve, the second switching valve and the third switching valve realizes a non-circulating injection mode, a circulating injection mode or a micro-injection mode; The flow ratio control of the second constant flow pump group and the first constant flow pump group realizes the sample dilution function, and the dilution function can be operated in conjunction with the non-circulation injection mode and the circulation injection mode.

[0014] In one embodiment, the fluid system further includes a particle size measuring device and a main control system, wherein: The particle size measuring device is used to detect the particle size distribution of the carrier synthesis product in real time; The main control system is electrically connected to the particle size measuring device, the first constant flow pump group, and the second constant flow pump group, and is configured as follows: receiving real-time detection data of the particle size measuring device; Compare the detected data with the preset size range; The flow rate parameters of the first constant flow pump group and / or the second constant flow pump group are dynamically adjusted according to the comparison results so that the carrier particle size converges to a preset size range.

[0015] In one embodiment, the fluid system further comprises a component concentration analysis device for real-time monitoring of the concentration of a specific component of the carrier synthesis product, wherein the specific component is at least one selected from nucleic acids, polypeptides, proteins, enzymes, probe molecules, cytokines, and small molecule compounds; The main control system is electrically connected to the component concentration analysis device, the first constant flow pump group, and the second constant flow pump group, and is configured as follows: receiving a transmission signal from the component concentration analysis device; Calculate and obtain real-time concentration data based on the transmitted signal; Compare the real-time concentration data with the preset target concentration range; The flow rate of the first constant flow pump group and / or the second constant flow pump group is dynamically adjusted according to the comparison result so that the concentration of the specific molecule is within a preset concentration range.

[0016] In one embodiment, the carrier synthesis preparation system further includes a fourth switching valve having multiple branch valve ports, and the fourth switching valve connects the degassing module and the second solution bottle.

[0017] In one embodiment, the carrier synthesis preparation system also includes a temperature control device, which is used at least to regulate the ambient temperature in the liquid storage device; wherein the temperature control device includes a refrigeration module, an insulation module and a temperature sensing module, the refrigeration module is used to create a refrigerated environment; the insulation module is used to maintain the temperature and heat in the liquid storage device and reduce heat convection; the temperature sensing module is used to monitor the ambient temperature in the liquid storage device in real time and dynamically adjust the working state of the refrigeration module so that the ambient temperature in the liquid storage device is within a preset temperature threshold range.

[0018] The present invention also provides a method for synthesizing and preparing a carrier based on the above-mentioned carrier synthesis preparation system, comprising the following steps: Select a microfluidic chip device and dock the microfluidic chip device with the adapter seat; Adding a sample solution formed by mixing the nanovesicles and the molecular drug into the first solution bottle; Pumping the sample solution into the microfluidic chip device through a liquid system device for processing; Collect the engineered drug carrier solution after processing through the microfluidic chip. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying any creative work.

[0020] Figure 1 This is a schematic structural diagram of an embodiment of a carrier synthesis and preparation system provided by the present invention; Figure 2 A schematic diagram of the product structure of an embodiment of a carrier synthesis and preparation system provided by the present invention; Figure 3 For display Figure 2 A schematic structural diagram of an embodiment of an internal storage device and a liquid circuit system of a medium carrier synthesis and preparation system; Figure 4 for Figure 3 A schematic structural diagram of an embodiment of a chip adapter device; Figure 5 for Figure 4 A schematic structural diagram of the chip adapter from another perspective; Figure 6 for Figure 4 A schematic structural diagram of an embodiment of a chip stage; Figure 7 This is a schematic structural diagram of an embodiment of a carrier synthesis and preparation system; Figure 8 Schematic diagram of the process for synthesizing and preparing the carrier; Figure 9a This is a data graph of sample concentration and particle size obtained by nano-resistance pulse sensing technology before the experimental group passed through the preparation system; Figure 9b This is a data graph of sample concentration and particle size obtained by nano-resistance pulse sensing technology after the experimental group passed through the preparation system; Figure 10 The electron micrograph of prepared 293T exosomes (containing miR-146a) was obtained using transmission electron microscopy; Figure 11 This is a comparison chart of the background nucleic acid expression level of exosomes (Evs) obtained by polymerase chain reaction (PCR) and the nucleic acid expression level after chip processing and loading (EvsNP_@miR146a).

[0021] Description of Figure Numbers: 100, microfluidic chip device; 110, chip box; 111, liquid inlet; 112, liquid outlet; 130, communication module; 140, liquid leakage detection element; 141, liquid leakage control circuit board; 142, liquid leakage sensor; 200, chip adapter; 210, chip stage; 211, adapter slot; 220, adapter seat; 230, displacement module; 240, adapter seat; 250, adapter circuit board; 260, card box circuit board; 300, storage device; 310, temperature control device; 320, liquid storage device; 321, first solution bottle; 322, second solution bottle; 323, collection bottle; 324, first circulation bottle; 325, second circulation bottle; 326, cleaning solution bottle; 327, waste liquid bottle; 400, liquid circuit system; 410, first constant flow pump group; 420, second constant flow pump group; 430, degassing device; 440, mixing device; 450, pressure relief module; 510, pressure sensing module; 520, flow sensing module; 530, first liquid level sensor; 540, second liquid level sensor; 550, third liquid level sensor; 560, fourth liquid level sensor; 610, first switching valve; 620, second switching valve; 630, third switching valve; 640, fourth switching valve; 710, microinjector; 720, sample quantitative loop.

[0022] The purpose, features and advantages of the present invention will be further described with reference to the accompanying drawings and in conjunction with the embodiments. DETAILED DESCRIPTION

[0023] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0024] It should be noted that if the embodiments of the present invention involve directional indications (such as up, down, left, right, front, back, etc.), the directional indications are only used to explain the relative position relationship, movement status, etc. between the various components under a certain specific posture. If the specific posture changes, the directional indications will also change accordingly.

[0025] In addition, if there are descriptions involving "first", "second", etc. in the embodiments of the present invention, the descriptions of "first", "second", etc. are only for descriptive purposes and cannot be understood as indicating or suggesting their relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features limited to "first" and "second" may explicitly or implicitly include at least one of such features. In addition, if "and / or" or "and / or" appears in the full text, its meaning includes three parallel solutions. Taking "A and / or B" as an example, it includes solution A, solution B, or solutions that satisfy both A and B. In addition, the technical solutions between the various embodiments can be combined with each other, but it must be based on the ability of ordinary technicians in this field to implement. When the combination of technical solutions is mutually contradictory or cannot be implemented, it should be deemed that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0026] Microfluidics integrates the basic operational units of biological, chemical, and medical analysis processes, such as sample preparation, reaction, separation, and detection, onto a micron-scale chip, automating the entire analytical process. Depending on the design of the microfluidic chip, drug loading methods based on microfluidics generally include: electroporation (a technique that uses a brief high-voltage electric field to form tiny pores in the cell membrane, allowing drug molecules or other biomacromolecules to enter the cell), mechanical extrusion (physical pressure causes the cell membrane to deform, forming pores or cracks, allowing drug molecules to enter the cell), and microfluidic shear stress induction (applying shear stress to cells through microchannels in the microfluidic chip to induce cell membrane rupture or form tiny pores, thereby enabling drug loading).

[0027] At present, it has been applied to a certain extent in the preparation of drug carriers such as liposomes and microspheres, but it mainly uses micro devices such as micropumps and microvalves to deliver drugs to the target area, which is time-consuming and labor-intensive, has low preparation efficiency, and is only suitable for small-scale preparation.

[0028] The present invention proposes a carrier synthesis and preparation system. This carrier synthesis and preparation system is based on microfluidic technology and can serve as a universal technical platform for engineering the modification of nanovesicle-type carriers. It can efficiently, safely and scalably synthesize and prepare different engineered formulations of specific carriers (drug delivery carriers). It solves the problems of traditional carrier engineering modification methods that are complicated and lengthy, lack standardization and are difficult to scale up.

[0029] See also Figure 1In one embodiment of the present invention, the carrier synthesis and preparation system includes a microfluidic chip device 100, a chip adapter device 200, a liquid storage device 320 and a liquid circuit system 400. The microfluidic chip device 100 has a liquid inlet 111 and a liquid outlet 112; the chip adapter device 200 includes a chip stage 210 and an adapter seat 220, and the chip stage 210 is used to install the microfluidic chip device 100; the liquid inlet 111 and the liquid outlet 112 are respectively connected to the adapter seat 220; a first solution bottle 321 and a collection bottle 323 are provided in the liquid storage device 320; a flow path is formed in the adapter seat 220, and the adapter seat 220 has a pipeline interface corresponding to the liquid circuit system 400 and a pipeline interface connected to the microfluidic chip device 100. The chip stage 210 can move relative to the adapter seat 220, and the movement method is not limited, such as moving by a motor guide rail. The adapter seat 220, the first constant current pump group 410, the first solution bottle 321 and the collecting bottle 323 are connected by pipelines. Depending on the design of the microfluidic chip, different models of microfluidic chip devices 100 can be installed on the chip stage 210 and connected to the adapter seat 220 to form a flow path. In this way, under the action of the first constant current pump group 410, the sample solution in the first solution bottle 321 is loaded through the microfluidic chip device 100 and then enters the collecting bottle 323.

[0030] The sample solution refers to a mixed solution of nanovesicles and molecular drugs, and the ratio of nanovesicles to molecular drugs varies according to different preparation requirements.

[0031] Constant flow pumps typically provide precise flow control and stable pressure output, ensuring accurate liquid delivery. By controlling flow and flow rate through a constant flow pump assembly, which serves as the primary driving force in fluidic system 400, the constant flow pump assembly can meet the requirements for consistent system fluid parameters during high-flow, high-throughput production processes under high-pressure operating conditions. This ensures that nanovesicles experience sufficient extrusion and fluid shear forces within the microfluidic chip, while also meeting the precise and stable control of microfluidics in microfluidic reaction environments.

[0032] The technical solution of the present invention pumps the sample solution from the first solution bottle 321 into the microfluidic chip device 100 via the liquid circuit system 400, achieving the loading of nanovesicles with molecular drugs. Compared to solutions that manually draw a certain amount of sample solution through a syringe and use microdevices such as micropumps and microvalves to deliver the drug to the target area, this solution supports large-scale production. Specifically, the micro-nanostructures within the microfluidic chip can physically collide and impact the vesicles with high-speed fluid, thereby achieving morphological transformation and material reprogramming of the vesicles. The microfluidic chip can precisely control the ratio of reactants, mixing method, and reaction time, facilitating the synthesis of high-quality carriers. The microfluidic chip device 100 is mounted on the chip stage 210 and docked with the adapter seat 220 to form a flow path, ensuring that the microfluidic chip device 100 can be stably and accurately connected to the liquid circuit system 400, ensuring smooth liquid flow. The liquid circuit system 400, through the first constant flow pump unit 410 and pipelines, can precisely control the flow rate and pressure of the sample solution. In summary, the existing production efficiency of nanovesicle carriers is low. Through modular design, this solution can easily replace different microfluidic chip devices 100 to meet different engineered formulas (drug delivery carriers) and realize a variety of different synthetic preparation needs; the design of the chip adapter device 200 and the liquid path system 400 makes the system more stable and reliable, reducing the difficulty and complexity of operation; through the control of the first constant flow pump group 410 and the liquid path system 400, the precise metering and mixing of the reactants are achieved, and the efficiency and accuracy of the reaction are improved. It can be used as a universal technical platform for efficient, safe and scalable synthetic preparation of different engineered formulas (drug delivery carriers) of specific carriers; it solves the problems of traditional carrier engineering modification methods being complicated and lengthy, lacking standardization and difficult to scale production.

[0033] First, the structure and assembly relationship of the chip adapter 200 and the microfluidic chip are introduced.

[0034] Reference Figures 2 to 6 The chip adapter 200 ensures the accurate docking of the microfluidic chip device 100 and the liquid circuit system 400. The microfluidic chip device 100 is reliably connected to the liquid circuit system 400 through the chip adapter 200, completing the high-throughput screening requirements under high-pressure working scenarios. Specifically, the microfluidic chip device 100 includes a chip box 110 and a microfluidic chip, wherein the chip box 110 is provided with an adapter groove 211, the adapter groove 211 accommodates the microfluidic chip, and the chip box 110 is reserved with a liquid inlet 111 and a liquid outlet 112 of the microfluidic chip. The chip box 110 wraps the microfluidic chip therein by ultrasonic welding, thermal compression bonding, etc.

[0035] Reference Figure 6Furthermore, to automatically achieve reliable connection between the microfluidic chip device 100 and the chip adapter device 200, a liquid leakage detection module 140 is also provided on the chip stage 210. The liquid leakage detection module 140 includes a liquid leakage control circuit board 141 and a liquid leakage sensor 142 provided on the chip stage 210. The liquid leakage control circuit board 141 is electrically connected to the liquid leakage sensor 142. The liquid leakage control circuit board 141 is used to receive the electrical signal sent by the liquid leakage sensor 142 and transmit it to the host system, so that the main control circuit board of the experimental equipment controls the chip stage 210 to move toward the adapter seat 220.

[0036] The chip box body 110 is provided with a liquid inlet 111 and a liquid outlet 112; the adapter seat 220 is provided with at least one connecting pipe and two connecting protrusions corresponding to the liquid inlet 111 and the liquid outlet 112. When the connecting protrusions are connected with the liquid inlet 111 and the liquid outlet 112, the adapter seat 220 is connected with the chip box body 110.

[0037] The chip stage 210 is moved relative to the adapter base 220 via a displacement module 230. In one embodiment, the displacement module 230 comprises a base, a screw, a motor, and a slider. The adapter base 220 is fixed to the base; the screw is rotatably connected to the base; a motor is mounted on the base, and its output shaft is connected to one end of the screw; the slider is slidably connected to the screw; and the chip stage 210 is connected to the slider. Through the precise design and coordination of the base, screw, motor, slider, and adapter base 220, the displacement module 230 achieves high-precision and high-stability positioning and movement control of the chip stage 210.

[0038] The adapter circuit board 250 is mounted on the chip stage 210, which is equipped with an adapter seat 240. When the adapter seat 240 and the adapter circuit board 250 come into contact and conduction, the leakage control circuit board 141 is electrically connected to the main control circuit board of the experimental equipment. A built-in control algorithm also quickly instructs the chip stage 210 to stop operating. This prevents excessive compression between the chip stage 210 and the adapter seat 220, thereby reducing the risk of damage to the microfluidic chip.

[0039] The adapter seat 220 also includes a card box circuit board 260 installed at the installation position; a number of conductive pins are provided on the side of the chip box body 110 facing the card box circuit board 260, and the card box circuit board 260 is used to transmit the information stored in the microfluidic chip to the main control circuit board of the experimental equipment when it contacts and conducts with the number of conductive pins.

[0040] The adapter seat 220 encloses a mounting position with a specific size and shape. The mounting position is specially designed for the card box circuit board 260 to ensure that it can be stably installed and achieve efficient electrical connection.

[0041] When the conductive pin contacts the cartridge circuit board 260, the main control circuit board of the experimental equipment controls the liquid inlet device according to preset instructions to inject liquid into the adapter seat 220. This design makes the liquid inlet process of the adapter seat 220 controllable, and the liquid inlet status can be adjusted according to actual needs, improving the flexibility and safety of the system.

[0042] The microfluidic chip device 100 is also provided with a communication module 130, which is used to communicate with the host. The communication content includes reading the model, specification parameters and other information of the microfluidic chip in the chip box 110; specifically, the communication module 130 is connected to the adapter seat 220 through a Pin pin to increase communication stability. When the adapter seat 220 is connected to the microfluidic chip device 100 for communication, the leakage detection module 140 is started. In one embodiment, the chip stage 210 includes a placement seat and a moving stage, and the placement seat is located on one side of the adapter seat 220; the placement seat is provided with a mounting groove, and the mounting groove is used to place the microfluidic chip device 100, and the leakage sensor 142 is installed at the bottom of the mounting groove and is located below the microfluidic chip device 100; the main working process is that after the above-mentioned microfluidic chip is installed on the chip stage 210, the chip adapter device 20 The chip stage 210 is driven to move toward the adapter seat 220 through the displacement module 230. When the liquid inlet and outlet ports 112 of the microfluidic chip device 100 placed on the chip stage 210 and the pipeline interface on the adapter seat 220 contact each other, the displacement module 230 stops moving and completes chip identification (pin connection). The leakage detection module 140 can identify whether the microfluidic chip device 100 is sealed and connected to the pipeline interface on the adapter seat 220. Specifically, when the seal between the microfluidic chip device 100 and the adapter seat 220 is poor, the sample solution will leak from the interface between the two and drip onto the leakage detection module 140 below. The leakage detection module 140 sends a signal to the host system to detect the leakage of the liquid circuit, thereby automatically stopping the sampling process or issuing an alarm signal to remind the operator to deal with the abnormal state in time.

[0043] The microfluidic chip device 100 is key to achieving high-throughput screening and precise control. Different microchannel structures can be designed to accommodate different types of carriers, depending on experimental requirements. This allows the microfluidic chip device 100 to serve as a universal technology platform. The microfluidic chip serves as the system's core reaction module, determining the specific carrier construction and synthesis process for nanovesicles. Previous studies have shown that mechanical shear forces have varying degrees of effect on membrane-bound cells and smaller vesicle systems.

[0044] Excessive shear force can easily damage or even destroy the morphological structure of the vesicles; appropriate shear force can disturb the membrane structure of the vesicles, using the fluidity of the phospholipid membrane to produce instantaneous pores; or fully mix the dispersed phospholipid molecules and reassemble them into functional complete nanovesicles.

[0045] The microfluidic chip device 100 can be used in different operating modes within the carrier synthesis and preparation system to achieve varying effects on the morphology and structure of the nanovesicles loaded through the microfluidic chip device 100, thereby affecting the loading efficiency of molecular drugs into the nanovesicles. These various operating modes include constant flow mode, constant pressure mode, variable flow mode, and variable pressure mode. A detailed description of these various operating modes is provided below. Regarding the loading efficiency of molecular drugs into nanovesicles, by adjusting the flow rate and pressure of the fluidic system 400 without replacing the microfluidic chip device 100, different mechanical shear force ranges corresponding to different strategies can be achieved. This allows for highly controllable engineering processes such as deformation, pore opening, fragmentation, fusion, and restoration of natural / artificial vesicles and their phospholipid components, ultimately forming effective drug carriers. (The term "microfluidic chip" here does not specify a specific design, strategy, or even structure; it refers to a general-purpose chip. The chip structure and strategy can be optimized based on the specific back-end application.)

[0046] The chip adapter device 200 and the microfluidic chip device 100 are introduced above, and the fluid path system 400 is introduced below.

[0047] Reference Figure 1 and Figure 7 In a further embodiment, the carrier synthesis preparation system further includes a main control system for monitoring the screening results and key parameters in the production process; thereby realizing online regulation of the fluid parameters in the liquid circuit system 400, and using an automated control system to precisely control the time and conditions of each step, such as the solution delivery speed, mixing ratio, temperature, etc.

[0048] Reference Figure 7 Specifically, the main control system includes a pressure sensing module 510 and a flow sensing module 520. These modules are used for online control of fluid parameters in the fluid path system 400. Specifically, the pressure sensing module 510 monitors system pressure and determines the operating status of the microfluidic chip. For example, based on the pressure increase during drug loading of nanovesicles within the microfluidic chip, it can determine whether the chip is clogged or has exceeded its processing capacity. This can improve the lifespan of the microfluidic chip and precisely control the reaction process. The flow sensing module 520 monitors system flow and provides feedback on fluid parameters during the drug loading process through pressure and flow sensing.

[0049] Furthermore, the first constant flow pump assembly 410, in conjunction with the flow sensor module 520 and the pressure sensor module 510, can also enable the fluid system 400 to have different operating modes under the control of the host system: constant flow mode (constant operating flow rate), constant pressure mode (constant operating pressure), variable flow mode (specified operating flow rate curve), and variable pressure mode (specified operating pressure curve). It should be noted that any of the above operating modes can be selected as the operating mode, and the operating modes can be switched.

[0050] By monitoring pressure signals, the pressure sensing module 510 can determine the status of the microfluidic chip, such as whether there is clogging or throughput limitations. The flow sensing module 520 uses technologies such as differential pressure sensors, magnetic sensors, and ultrasonic sensors to measure the flow rate of liquid through the pump body and convert the flow rate value into an electrical signal for output. By monitoring the flow signal, it can provide feedback on fluid parameters during the drug loading process, providing real-time data to the control system. In the fluid circuit system 400, the constant flow pump group can accurately deliver according to the set flow rate value, ensuring the stability and repeatability of the experiment.

[0051] Specifically, different working modes are implemented as follows: Implementation of Constant Flow Mode: In constant flow mode, the control system sets a constant flow rate. Flow sensor module 520 monitors the flow rate in fluid system 400 in real time and compares the actual flow rate with the set value. If the actual flow rate deviates from the set value, the control system adjusts the current output of the constant flow pump assembly to bring the actual flow rate back to near the set value. This ensures that the flow rate in fluid system 400 remains constant, facilitating precise control and repeatability of experiments.

[0052] Implementation of the constant pressure mode: In the constant pressure mode, the control system sets a constant pressure value. The pressure sensing module 510 monitors the pressure in the liquid circuit system 400 in real time and compares the actual pressure value with the set value. If the actual pressure value deviates from the set value, the control system will adjust the current output of the constant flow pump group (or adjust the system pressure by other means) to return the actual pressure value to near the set value. Due to the complex relationship between pressure and flow (affected by various factors such as pipeline resistance and liquid properties), it may be necessary to combine the feedback of the flow sensing module 520 for more precise control. It has the advantages of maintaining a constant pressure in the liquid circuit system 400, helping to control the fluid dynamics behavior in the microchannel and improving the stability of the experiment.

[0053] Implementation of Variable Flow Mode: In variable flow mode, the control system sets a flow rate profile (e.g., a gradually increasing or decreasing flow rate). The flow sensor module 520 monitors the flow rate in the fluid system 400 in real time and compares the actual flow rate with the preset flow rate profile. Based on the comparison result, the control system adjusts the current output of the constant flow pump assembly to ensure that the actual flow rate follows the preset flow rate profile. Advantages: The ability to dynamically adjust the flow rate as needed helps optimize experimental conditions and improve experimental efficiency.

[0054] Implementation of variable pressure mode: In variable pressure mode, the control system sets a pressure profile (e.g., gradually increasing or decreasing pressure). The pressure sensing module 510 monitors the pressure in the fluid system 400 in real time and compares the actual pressure value with the preset pressure profile. Based on the comparison result, the control system adjusts the current output of the constant flow pump group (or adjusts the system pressure through other means) to ensure that the actual pressure value follows the preset pressure profile. Similarly, due to the complex relationship between pressure and flow, feedback from the flow sensing module 520 can be combined for more precise control. This has the advantage of being able to dynamically adjust the system pressure as needed, facilitating the exploration of experimental effects under different pressure conditions.

[0055] By combining the constant flow pump assembly, pressure sensing module 510, and flow sensing module 520, the fluidic system 400 can flexibly switch between different operating modes. This not only improves experimental accuracy and controllability but also broadens the possibilities for experimental design, thereby accelerating the drug development process and improving the performance of drug delivery systems. (As mentioned above, the same microfluidic chip device 100 can achieve different effects on the membrane structure of nanovesicles, such as deformation, pore opening, and fragmentation.) Specifically, in one embodiment, the first constant flow pump group 410 includes at least two first plunger pumps connected in series. In a specific embodiment, the number of first plunger pumps is two. In other embodiments, the number of first plunger pumps can be 3, 4, 5, etc. When multiple constant flow plunger pumps are used in series, they can jointly bear a larger pressure difference, thereby ensuring that the flow rate can remain stable under high pressure conditions. In addition, the series structure helps to reduce the flow fluctuations that may occur when each pump works alone, further improving the flow stability of the entire pump group.

[0056] When multiple constant flow plunger pumps are used in series, they can share a greater pressure differential, ensuring stable flow even under high pressure conditions. Furthermore, the series structure helps reduce the flow fluctuations that may occur when each pump operates independently, further improving the flow stability of the entire pump group.

[0057] Furthermore, using multiple constant-flow plunger pumps to form a pump system allows for redundancy. If one pump fails, the others continue to operate, ensuring continuous system operation. This improves system reliability and stability. Furthermore, during implementation, a single or multiple pumps can be used to drive a single (single-phase) or multiple (multi-phase) liquids, meeting the loading requirements of single or multiple medications. This modular design allows each pump to be independently disassembled and replaced, providing independent functionality and interfaces. For driving a single (single-phase) liquid, a single pump module can be selected. This constant-flow plunger pump typically offers precise flow control and stable pressure output, ensuring accurate liquid delivery. When handling multiple (multi-phase) liquids, this can be achieved by combining multiple pump modules. These pump modules can be used in series or parallel to provide varying flow and pressure characteristics, meeting the needs of complex liquid systems.

[0058] Reference Figure 3 and Figure 7 In a further scheme, the carrier synthesis preparation system can also realize the circulation of the sample solution, that is, the sample solution (a mixed solution of nanovesicles and molecular drugs) repeatedly passes through the microfluidic chip to carry out the nanovesicle drug loading process.

[0059] Specifically, the carrier synthesis preparation system also includes a first switching valve 610 and a second switching valve 620, both of which are arranged on the pipeline and have multiple branch valve ports; in this embodiment, the first switching valve 610 and the second switching valve 620 are high-pressure six-way switching valves; in other embodiments, they can be set to three-way switching valves, four-way switching valves, five-way switching valves, etc. according to design requirements.

[0060] A first circulation bottle 324 and a second circulation bottle 325 are also provided in the liquid storage device 320; the first switching valve 610 pipeline connects the chip adapter device 200, the collection bottle 323, the first circulation bottle 324 and the second circulation bottle 325; the second switching valve 620 pipeline connects the first circulation bottle 324, the second circulation bottle 325, the first solution bottle 321 and the first constant flow pump group 410; the chip adapter device 200 can selectively connect the first solution bottle 321, the first circulation bottle 324 and the second circulation bottle 325 through the first switching valve 610 and the second switching valve 620, so that the sample solution in the first solution bottle 321 can be repeatedly loaded through the microfluidic chip device 100.

[0061] Reference Figure 3 and Figure 7 , the carrier synthesis preparation system has a non-circulating injection function: When there is no circulation, the carrier synthesis preparation system realizes the process of loading molecular drugs into nanovesicles as follows: the first switching valve 610 is connected to the collection bottle 323, the second switching valve 620 is connected to the first solution bottle 321 (the first solution bottle 321 is pre-filled with the sample solution), the first constant flow pump group 410 is started (the flow rate and flow rate are set according to experimental requirements), the sample solution is pumped into the microfluidic chip device 100 for drug loading, and then into the collection bottle 323.

[0062] Reference Figure 3 and Figure 7 , the carrier synthesis preparation system has a cyclic injection function: When the sample solution needs to be processed multiple times in a cycle to improve the yield, the carrier synthesis preparation system can pre-set the number of cycles (one loading through the microfluidic chip device 100 is considered one cycle) during the process of nanovesicle loading of molecular drugs. For example, three cycles are performed (i.e., the sample needs to pass through the microfluidic chip device 100 three times): The first switching valve 610 is first connected to the first circulation bottle 324 (i.e., the microfluidic chip device 100 is only connected to the first circulation bottle 324 through the first switching valve 610); the second switching valve 620 is connected to the first solution bottle 321. At this time, the first solution bottle 321, the first constant current pump group 410, the microfluidic chip device 100, and the first circulation bottle 324 form a loop. The first constant current pump group 410 is started (the flow rate and flow rate are set according to experimental requirements) to pump the sample solution into the microfluidic chip device 100 for drug loading, and then into the first circulation bottle 324, completing the first cycle.

[0063] The first switching valve 610 is switched to connect to the second circulation bottle 325 (disconnecting the first circulation bottle 324), and the second switching valve 620 is switched to connect to the first circulation bottle 324 (disconnecting the first solution bottle 321). The first circulation bottle 324, the first constant flow pump group 410, the microfluidic chip device 100, and the second circulation bottle 325 form a loop. The first constant flow pump group 410 is started (the flow rate and flow rate are set according to experimental requirements), and the nanovesicle drug mixture loaded once in the first circulation bottle 324 is pumped into the microfluidic chip device 100 for drug loading for the second time, and then enters the second circulation bottle 325, completing the second cycle.

[0064] The first switching valve 610 is switched to connect to the first circulation bottle 324, and the second switching valve 620 is switched to connect to the second circulation bottle 325. At this time, the second circulation bottle 325, the first constant flow pump group 410, the microfluidic chip device 100, and the first circulation bottle 324 form a loop. The first constant flow pump group 410 is started to load the nanovesicle drug mixture twice in the second circulation bottle 325. After the mixture is pumped into the microfluidic chip device 100 for drug loading for the third time, it enters the first circulation bottle 324, completing the third cycle.

[0065] More cycles are set as above, and when the system reaches the preset number of cycles and completes loading, the system stops running. It should be noted that simply replacing the first circulation bottle 324 and the second circulation bottle 325 with the collection bottle 323 does not deviate from the design concept of the present invention.

[0066] Reference Figure 7 , the carrier synthesis preparation system also has the function of micro sample injection: Specifically, the carrier synthesis preparation system also includes a third switching valve 630 with multiple branch valve ports and a sample quantitative ring 720 connected to a micro-injector 710, and the sample quantitative ring 720 is connected to the third switching valve 630; the first constant flow pump group 410 is connected to the chip adapter device 200 pipeline through the third switching valve 630, and the third switching valve 630 can optionally connect the sample quantitative ring 720 and the first constant flow pump group 410.

[0067] Considering the contradiction between the input of micro-volume samples and the larger cavity volume caused by the longer pipelines in the fluidic system 400, a third switching valve 630 is added between the second switching valve 620 and the chip adapter 200. The sample loop 720 is connected to the micro-injector 710 and installed on the third switching valve 630. During use, the micro-sample is first pushed into the sample loop 720 through the micro-injector 710. After switching to the first beam pump group, the first constant flow pump group 410 pushes the previously input micro-sample into the microfluidic chip for loading.

[0068] Specifically, under normal circumstances, the third switching valve 630 is in a closed state or connected to other non-main liquid circuits to prevent the solution in the microinjector 710 from flowing directly into the main liquid circuit, that is, flowing into the circuit formed by the first solution bottle 321, the first constant flow pump group 410, the microfluidic chip device 100, and the collection bottle 323.

[0069] The microinjector 710 is used to remove a trace sample from a sample bottle and push it into the sample quantitative loop 720. The quantitative loop is a loop with a fixed volume, which is used to temporarily store the sample solution. When the microinjector 710 pushes the sample into the quantitative loop, the sample volume in the quantitative loop is fixed and will not change due to subsequent operations. The microinjector 710, also known as a microsyringe, is an indispensable sampling and injection tool for conducting microanalysis experiments. It usually has a variety of specifications, such as 5ul, 10ul, 25ul, 50ul, 100ul, etc., to meet the needs of different experiments. The sample quantitative loop 720 is a fixed-volume pipeline commonly used in laboratories, such as metal tubes, plastic tubes, etc., which is mainly used to accurately measure a certain volume of liquid in analytical chemistry experiments.

[0070] Specifically, the injection control method using the micro-injector 710 is as follows: Add buffer solution to the first solution bottle 321 and connect the first constant flow pump group 410 to the first solution bottle 321; Connect the second switching valve 620 to the first solution bottle 321 and the first switching valve 610 to the collection bottle 323; Use the micro-injector 710 to inject a small amount of sample into the system flow path through the sample loop 720, so that the sample liquid flows out of the loop of the third switching valve 630; Switch the third switching valve 630 to a passage mode, so that the first constant flow pump group 410 is connected to the sample quantitative loop 720; The flow rate of the first constant flow pump group 410 is set, and the buffer solution is started to be pumped. The buffer solution is pushed forward and the sample liquid in the injection needle flows forward. The sample liquid flows into the collection bottle 323 after passing through the microfluidic chip, and the injection is completed (the volume of buffer solution required for the micro sample to completely pass through the microfluidic chip is calculated based on the length of the liquid path. If the specified volume of buffer solution has been pumped, the injection is considered to be completed and the first constant flow group is stopped).

[0071] The above describes the non-circulation, circulation, and micro-injection functions of the carrier synthesis preparation system. In order to adjust the concentration of the sample solution without frequently replacing the sample solution in the first solution bottle 321 to meet different experimental requirements, based on the above scheme, in a further scheme: Combine Figure 3 and Figure 7 The liquid circuit system 400 further includes a degassing device 430 and a mixing device 440; the liquid circuit system 400 further includes a second constant flow pump group 420; the liquid storage device 320 further includes a second solution bottle 322; the second solution bottle 322, the degassing device 430, the second constant flow pump group 420 and the mixing device 440 are connected by pipelines to form a second liquid circuit, and the second constant flow pump group 420 is used to pump the solution in the second solution bottle 322; the degassing device 430 is used to pump the first solution bottle 321 and the second solution bottle 322 2. The first solution bottle 321, the degassing device 430, the first constant flow pump group 410 and the mixing device 440 are connected through pipelines to form a first liquid circuit; the mixing device 440, the chip adapter 200 and the collecting bottle 323 are connected through pipelines to form a third liquid circuit; wherein, the first constant flow pump group 410 pumps the solution of the first liquid circuit and the second constant flow pump group 420 pumps the solution of the second liquid circuit, which are mixed through the mixing device 440, and then enter the collecting bottle 323 after being loaded through the microfluidic chip from the third liquid circuit.

[0072] In one embodiment, the second solution bottle 322 is filled with a dilution solution, while the first solution bottle 321 is filled with a sample solution (a mixture of nanovesicles and a molecular drug). The dilution solution is used to dilute the molecular drug according to preparation requirements, thereby synthesizing and preparing different engineered formulations (drug delivery vehicles). In other embodiments, the first solution bottle 321 and the second solution bottle 322 can hold a nanovesicle solution and a molecular drug solution, respectively (or vice versa). The specific types of nanovesicles and molecular drugs are not limited; for example, human embryonic kidney 293T cells secreted as natural nanovesicles can be used. Of course, in some embodiments, the first solution bottle 321 or the second solution bottle 322 can be left empty.

[0073] The first and second liquid paths are used to transport the first and second solutions (such as vesicles and molecular drugs), respectively. The flow rate and flow rate are controlled by a constant flow pump. As the primary driving force for the reaction solution in the system, the constant flow pump must maintain a high flow rate to ensure that the nanovesicles are subjected to sufficient extrusion and fluid shear forces within the microfluidic chip. At the same time, it must meet the precise and stable control of microfluidics required in the microfluidic reaction environment. To ensure consistent system fluid parameters during high-throughput production, a constant flow pump is employed. A degasser 430 removes bubbles from the solution to prevent damage to the microfluidic chip device 100. The first and second liquid paths are used to transport the first and second solutions (such as nanovesicles and molecular drugs), respectively. The pressure and flow rate are controlled by the constant flow pump. A mixing device 440 is used to mix the two solutions and deliver them to the third liquid path through the microfluidic chip device 100 for collection in a collection bottle 323. Alternatively, the solutions can be collected in the first and second circulation bottles 324 and 325 through multiple circulations.

[0074] Reference Figure 7 Furthermore, in order to accurately measure the amount of reaction solution and monitor the remaining amount of each solution inside the liquid storage device 320 in real time, a liquid level sensor is provided to continuously monitor the liquid level changes in the solution bottle to ensure that the experimenter knows the remaining amount of the solution at any time. The setting of the liquid level sensor will be introduced later.

[0075] In a further embodiment, the carrier synthesis preparation system is further provided with a temperature control device 310, referring to Figure 2 and Figure 6 The temperature control device 310 in the storage device 300 can ensure the stability of the solution during storage and transportation; the design of the liquid storage device 320 should facilitate the use and replacement of the solution to meet the needs of large-scale production.

[0076] In one embodiment, the liquid storage device 320 contains a first solution bottle 321 , a second solution bottle 322 , a first circulation bottle 324 , a second circulation bottle 325 and a collection bottle 323 , and the microfluidic chip is used to load and process the sample solution in the first solution bottle 321 .

[0077] The fluid system 400 includes: a first pipeline (see Figure 7 In the embodiment, the pipeline between the first solution bottle 321 and the microfluidic chip device 100), the second pipeline (see Figure 7 , the pipeline between the second solution bottle 322 and the microfluidic chip device 100), the third pipeline (see Figure 7 The collection bottle 323 to the microfluidic chip device 100 and the circulation pipeline group (see Figure 7 , a pipeline between the first switching valve 610 and the second switching valve 620); A first pipeline connects the second switching valve 620, the first solution bottle 321, the first constant flow pump group 410 and the third switching valve 630 to the liquid inlet 111, wherein the third switching valve 630 is connected in parallel to the sample quantitative loop 720 for switching between direct injection through the first pipeline and injection through the sample quantitative loop 720; A second pipeline connects the second solution bottle 322 and the second constant flow pump assembly 420 to the liquid inlet 111 and merges with the first pipeline in front of the liquid inlet 111; A third pipeline connects the liquid outlet 112 to the first switching valve 610, and the first switching valve 610 is connected to the collection bottle 323 and the circulation pipeline group respectively; A circulation pipeline group, comprising a first branch connected to the first circulation bottle 324 and a second branch connected to the second circulation bottle 325, wherein the circulation pipeline group forms a closed loop with the first pipeline through the second switching valve 620; Among them: the linkage control of the first switching valve 610, the second switching valve 620 and the third switching valve 630 realizes the non-circulation injection mode, the circulation injection mode or the micro-injection mode; the flow ratio control of the second constant flow pump group 420 and the first constant flow pump group 410 realizes the sample dilution function, and the dilution function can be coordinated with the non-circulation or circulation injection mode.

[0078] Explanation of switching valve linkage: The third switching valve 630 (connected to the first pipeline) and the first switching valve 610 (connected to the third pipeline) are connected to the chip adapter device 200 between the first pipeline and the third pipeline, and are connected to the microfluidic chip in the microfluidic chip device 100 through the chip adapter device 200. The third switching valve 630 and the first switching valve 610 are synchronously controlled by mechanical linkage or electrical signals to form the following working state combination: No-loop injection mode: The third switching valve 630 is in the first working position (directly connected to the channel of the first solution bottle 321), the first switching valve 610 is in the first working position (connected to the channel of the collection bottle 323), and the sample solution flows unidirectionally through the microfluidic chip to the collection bottle 323.

[0079] Cyclic injection mode: The third switching valve 630 maintains the first working position (maintaining the passage to the first solution bottle 321), and the first switching valve 610 switches to the second working position (connecting to the circulation pipeline group). The sample solution flows back from the liquid outlet 112 through the circulation pipeline group to the second switching valve 620. The solution is stored back and forth between the first circulation bottle 324 and the second circulation bottle 325 through the alternating on and off of the second switching valve 620, forming a closed-loop circulation path.

[0080] Regarding micro-sampling: When the third switching valve 630 is switched to the second working position (connected to the sample quantitative loop 720 channel), the first switching valve 610 is in the first working position (connected to the collection bottle 323), and the second switching valve 620 prohibits the activation of the circulation pipeline group. The micro-injector 710 is connected to the sample quantitative loop 720 to inject the micro-sample into the first pipeline to ensure the complete processing of the micro-sample.

[0081] Explanation of the coordinated control of the dilution function: Basis for the dilution function: The second constant flow pump group 420 and the first constant flow pump group 410 independently control the flow rates of the second solution bottle 322 (diluent) and the first solution bottle 321 (sample solution), respectively, to achieve concentration adjustment in the following manner: Control the flow ratio of the two pump groups (Q1:Q2) to determine the ratio of sample to diluent in the mixed solution; The first pipeline and the second pipeline merge in the mixing device 440 before the liquid inlet 111; It should be noted that the dilution function can be run in conjunction with the injection function and the circulation function across different modes: The associated control of the dilution function and injection mode is as follows: No-circulation dilution mode: The first switching valve 610 remains connected to the collecting bottle 323, and the diluted mixed liquid is directly collected after a single treatment.

[0082] Cyclic dilution mode: The first switching valve 610 is switched to the circulation pipeline group, and the diluted mixed liquid repeatedly flows through the microfluidic chip in the circulation system to achieve dynamic continuous dilution.

[0083] When the dilution function is enabled, the second constant flow pump group 420 is started, and the system maintains the third switching valve 630 and the sample quantitative loop 720 in a disconnected state to ensure stable mixing of the diluent and the sample solution.

[0084] The above solution has the following technical effects: 1. Through the linkage control of the switching valve, the system can seamlessly switch between micro / conventional injection and single / cycle processing modes; 2. The independent flow ratio of the constant flow pump group and the decoupling and disconnection design of the valve body status make the dilution function a basic capability that can be superimposed, significantly improving the functional scalability of the system; 3. The pump / valve linkage mechanism avoids mode conflicts caused by manual operation and ensures operational reliability.

[0085] In a further embodiment, the carrier synthesis preparation system also includes a main control system for monitoring the screening results and key parameters in the production process; thereby realizing online regulation of the fluid parameters in the liquid path system 400, and using an automated control system to precisely control the time and conditions of each step, such as the solution delivery speed, mixing ratio, temperature, etc.

[0086] Specifically, the liquid circuit system 400 also includes a particle size measuring device and a main control system, wherein: The particle size measuring device is used to detect the particle size distribution of the carrier synthesis product in real time; The main control system is electrically connected to the particle size measuring device, the first constant flow pump group 410 and the second constant flow pump group 420, and is configured as follows: receiving real-time detection data of the particle size measuring device; Compare the detected data with the preset size range; The flow rate parameters of the first constant flow pump group 410 and / or the second constant flow pump group 420 are dynamically adjusted according to the comparison results, so that the carrier particle size converges to a preset size range.

[0087] The particle size measuring device is located in the liquid flow path for detecting the processed liquid and is set downstream of the liquid outlet 112, such as being set on the third pipeline, to ensure that the detection object is the final synthetic product and avoid interference in the intermediate process.

[0088] The linked control of the particle size measurement device and the fluid pump group forms a closed-loop feedback system. Through "detection-comparison-adjustment", the particle size measurement results are directly converted into pump group operation instructions (such as increasing the diluent flow rate to reduce particle size, or reducing the sample flow rate to reduce agglomeration).

[0089] Dynamic regulation: the regulation objects include the first constant flow pump group 410 (sample flow) and the second constant flow pump group 420 (dilution flow). The regulation methods include but are not limited to: proportional regulation (such as maintaining Q1 / Q2 constant, or adjusting Q1, changing Q2, etc.).

[0090] It should be noted that the closed-loop feedback system can operate in a non-loop / loop mode: No circulation mode: output to collection bottle 323 after single detection and adjustment; Circulation mode: Repeated testing and adjustment until the particle size meets the standard and then switch to the collection bottle 323.

[0091] Specifically, the particle size measuring device preferably adopts a dynamic light scattering instrument (DLS), whose detection unit is embedded in the transparent detection window of the third pipeline, and the detection frequency is 1-10 times per second; the control system has a built-in PID algorithm. When the D90 particle size value (refer to the diagram of particle size value and concentration ratio in Figure 9) is detected to exceed the preset threshold, the flow rate of the second constant flow pump group 420 is automatically increased and the flow rate of the first constant flow pump group 410 is synchronously reduced, and the adjustment amplitude is positively correlated with the deviation value.

[0092] Optionally, the particle size measuring device may also use laser diffraction measurement.

[0093] For reference, the main control system includes: Signal input unit: Communicates directly with a particle size measurement device (e.g., dynamic light scattering) to obtain real-time particle size distribution data of carrier particles (including characteristic values such as D50 and D90 (see the diagram of particle size and concentration ratio in Figure 9)). Data processing unit: Built-in database of preset size ranges (e.g. target particle size range 80-120nm (see the diagram of particle size value and concentration ratio in Figure 9)) and comparison algorithms (e.g. standard deviation calculation module) Instruction output unit: establishes a bidirectional control link with the first constant flow pump group 410 (controls the sample flow Q1) and the second constant flow pump group 420 (controls the diluent flow Q2); For reference, the control method steps are as follows: Step a, data reception, includes the following steps: The particle size measuring device sends a real-time detection data packet to the control system at a predetermined frequency (e.g., 5-10 Hz). The data packet contains: Current particle size distribution histogram and / or statistical parameters (such as average particle size, concentrated particle size, median particle size D50, maximum particle size, minimum particle size, particle size standard deviation (D90, D10, D90-D10), span, dispersion index (PDI), etc., see Figure 9); Signals transmit data (e.g. via digital signals (such as RS485 protocol) or analog signals, etc.).

[0094] Step b, data comparison, includes the following steps: Extract relevant parameters from the test data to calculate the current particle size value, such as the D90 particle size value.

[0095] Compare the D90 value to a preset upper threshold (e.g., 120 nm), and the average particle size to a target median value (e.g., 100 nm). Generate the deviation coefficient K (for reference, K = (measured value - target value) / target value × 100%).

[0096] Of course, it is also possible to receive real-time detection data from the particle size measuring device and compare the detection data with a preset size range; (e.g., the measured D90 data is compared with a preset D90 value, and if it is within the preset range, no adjustment is made.) Step c, dynamic adjustment, includes the following steps: According to the deviation coefficient K, the corresponding adjustment strategy is triggered; For reference, the adjustment strategy is: In fine-tuning mode (e.g., |K| ≤ 5%), only the second constant flow pump group 420 (diluent flow Q2) is adjusted; In the coordinated regulation mode (e.g., 5%<|K|≤20%), Q1 and Q2 are changed synchronously to keep the total flow Q1+Q2 constant. Intervention mode (e.g., |K|>20%): suspends the current process, initiates system self-test (e.g., microfluidic chip blockage detection), and issues an alarm.

[0097] Among them, the non-circulation mode: output to the collection bottle 323 after a single adjustment, suitable for rapid synthesis with small particle size deviation; Circulation mode: Multiple feedback adjustments (such as 3-5 cycles) are performed in the circulation pipeline until the particle size stabilizes within the preset range.

[0098] In a further embodiment, the fluid system 400 further includes a component concentration analysis device for real-time monitoring of the concentration of a specific component of the carrier synthesis product, wherein the specific component is at least one selected from nucleic acids, polypeptides, proteins, enzymes, probe molecules, cytokines, and small molecule compounds; The main control system is electrically connected to the component concentration analysis device, the first constant flow pump group 410 and the second constant flow pump group 420, and is configured as follows: receiving a transmission signal from the component concentration analysis device; Calculate and obtain real-time concentration data based on the transmitted signal; Compare the real-time concentration data with the preset target concentration range; The flow rate of the first constant flow pump group 410 and / or the second constant flow pump group 420 is dynamically adjusted according to the comparison result, so that the concentration of the specific molecule is within a preset concentration range.

[0099] The component concentration analyzer is used to detect the liquid flow path after treatment. Like the particle size measuring device, it is set downstream of the liquid outlet 112, such as on the third pipeline, to ensure that the detection object is the final synthetic product and avoid interference in the intermediate process.

[0100] Optionally, the component concentration analysis device includes a spectral analysis module, which performs quantitative analysis through the relationship between spectral characteristics (such as absorption, scattering, fluorescence, etc.) and component concentration, and combines the algorithm to feed back data to the main control system in real time to dynamically adjust the fluid pump parameters.

[0101] The transmission signal of the component concentration analysis device is an optical signal.

[0102] Optionally, the component concentration analysis device may also be an electrochemical sensor or a biochip detection unit, the transmitted signal being a current signal or a bioaffinity signal, and the real-time concentration data is obtained through conversion using a calibration curve or an algorithm model.

[0103] As a supplement, a third constant flow pump group can be added to increase the flow rate of the supplementary liquid (when a third solution bottle is present).

[0104] Specifically, receiving the real-time detection data of the particle size measuring device includes the following steps: Convert the original transmission signal (such as absorbance, current value, mass spectrum peak area) into a concentration-related electrical signal; As an example, a method for calculating and obtaining real-time concentration data based on the transmission signal; The absorbance value is obtained from the spectral signal, and the concentration is calculated using the Beer-Lambert law; alternatively, a current-concentration calibration curve is obtained from the electrochemical signal, and the molar concentration is output.

[0105] Dynamically adjusting the flow rate of the first constant flow pump group 410 and / or the second constant flow pump group 420 according to the comparison result so that the concentration of the specific molecule is within a preset concentration range, including: Set the target concentration range (the value of the particle amount of the detected component, refer to the values in Figure 9); The calculated real-time concentration is used to calculate the deviation coefficient from the target concentration (for reference, such as δ = (measured value - target value) / target value × 100%); According to the obtained concentration deviation coefficient δ, the corresponding adjustment strategy is triggered; For reference, the adjustment strategy is: If |δ|≤5% (the deviation between the real-time concentration and the target concentration is within 5%), the system does not make any adjustments, the concentration meets the requirements, and the current parameters are maintained. The system determines that the concentration is insufficient only when the following conditions are met at the same time: 3 or more consecutive tests are below the threshold, and Q2 is reduced or Q1 is increased to increase the concentration (that is, a comprehensive judgment is made based on the deviation size (|δ|) and duration (number of consecutive limit violations) to prevent misjudgment).

[0106] If 5% < |δ| ≤ 15%, only adjust Q2 (gradually correct the concentration deviation by fine-tuning the diluent flow rate (Q2).) If |δ|>15%, coordinately adjust the sample flow rate Q1 and the diluent flow rate Q2, and suspend the process and issue an alarm if necessary.

[0107] In summary, the linkage control of the component concentration analysis device, the particle size measurement device and the fluid pump group forms a multi-parameter linkage intelligent control system.

[0108] The technical solution of the present invention uses a liquid circuit system 400 to draw a mixed solution into a microfluidic chip and automatically complete the steps of mixing, processing, and collection. It also achieves high-throughput screening of nanovesicle drug carriers and supports large-scale production. Specifically, the micro-nanostructures within the microfluidic chip can be used to physically collide and impact the vesicles with high-speed fluid, thereby achieving morphological transformation and material reprogramming of the vesicles. The microfluidic chip can accurately control the ratio of reactants, mixing method, and reaction time, which is conducive to the synthesis of high-quality carriers. The chip stage 210 is used to support the microfluidic chip device 100. The adapter seat 220 is docked with the microfluidic chip device 100 to ensure that the microfluidic chip device 100 can be stably and accurately connected to the liquid circuit system 400 and ensure smooth flow of liquid. The storage device 300 can store and control the temperature of the reactants to ensure that the reaction is carried out at the optimal temperature, thereby improving the reaction efficiency and product quality, and facilitating the management and use of the reactants. The liquid circuit system 400 includes a first constant flow pump group 410, a second constant flow pump group 420, a degasser 430, and a mixing device 440. Mixing device 440 and degassing device 430 enable reactant degassing, mixing, and transport, ensuring the smooth progress of the reaction, improving reaction precision and controllability, and reducing the impact of bubbles on the reaction. Fluid system 400, through a constant-flow pump assembly and piping, precisely controls the flow rate and pressure of the mixed solution. In summary, existing methods for manufacturing nanovesicle-based carriers have low production efficiency, incompatible production principles, and a lack of a universal technological platform. Through modular design, this solution can easily replace different microfluidic chip devices 100 and reactants to meet a variety of different synthesis and preparation needs; the design of the chip adapter device 200 and the liquid system 400 makes the system more stable and reliable, reducing the difficulty and complexity of operation; through precise control of reaction conditions (such as temperature, reactant ratio, etc. (achieved by the temperature control device 310, degassing device 430, mixing device 440, etc.)), high-quality carriers can be synthesized; through microfluidic technology and precise control of the liquid system 400, precise metering and mixing of reactants are achieved, improving the efficiency and accuracy of the reaction, and can be used as a universal technology platform for efficient, safe, and scalable synthesis and preparation of different engineered formulations of specific carriers (drug delivery carriers); solving the problems of traditional carrier engineering modification methods being complicated and lengthy, lacking standardization, and difficult to scale production.

[0109] Specifically, in one embodiment, the second constant flow pump group 420 includes at least two second plunger pumps connected in series. In a specific embodiment, the number of the second plunger pumps is two. In other embodiments, the number of the plunger pumps can be 3, 4, 5, etc.

[0110] When multiple constant flow plunger pumps are used in series, they can share a greater pressure differential, ensuring stable flow even under high pressure conditions. Furthermore, the series structure helps reduce the flow fluctuations that may occur when each pump operates independently, further improving the flow stability of the entire pump group.

[0111] In another embodiment, the second constant-flow pump assembly 420 includes at least two parallel-connected constant-flow plunger pumps, while the first constant-flow pump assembly 410 includes at least two parallel-connected first plunger pumps. The parallel configuration is primarily used to increase flow rate. By connecting multiple constant-flow plunger pumps in parallel, flow rates can be easily superimposed, thereby meeting higher flow requirements. The parallel configuration also helps distribute the operating pressure of each pump, extending its service life. Furthermore, using multiple constant-flow plunger pumps to form a pump assembly allows for redundancy. If one pump fails, the remaining pumps can continue to operate, ensuring continuous system operation. This improves system reliability and stability. Furthermore, during implementation, a single or multiple pumps can be used to drive a single (single-phase) or multiple (multi-phase) liquids, enabling the loading of single or multiple medications. This modular design allows each pump to be independently disassembled and replaced, ensuring that each pump has independent functions and interfaces. For driving a single (single-phase) liquid, a single pump module can be used. This type of constant-flow plunger pump typically features precise flow control and stable pressure output, ensuring accurate liquid delivery. When handling multiple (multiphase) liquids, this can be achieved by combining multiple pump modules. These pump modules can be connected in series or parallel to provide varying flow and pressure characteristics, meeting the needs of complex liquid systems.

[0112] Reference Figure 3 and Figure 7 Furthermore, the carrier synthesis and preparation system also includes a fourth switching valve 640 with multiple branch valve ports. This fourth switching valve 640 connects the degassing module and the second solution bottle 322. The branch valve ports of the fourth switching valve 640 can be used to connect multiple functional bottles for various liquids, such as dilution solutions and cleaning solutions. Similarly, the first switching valve 610 and the second switching valve 620 can also be connected to different functional bottles, such as those for collecting waste liquids. The solution in the cleaning liquid bottle 326 can be used to clean the entire pipeline system 400, and the waste liquid is discharged to the waste liquid bottle 327 for collection.

[0113] In order to accurately measure the amount of reaction solution and monitor the remaining amount of each solution inside the liquid storage device 320 in real time, a first liquid level sensor 530 and a second liquid level sensor 540 are also provided. The first liquid level sensor 530 is arranged on the first liquid path to alarm when the solution in the first solution bottle 321 exceeds a preset threshold value; the second liquid level sensor 540 is arranged on the second liquid path to alarm when the solution in the second solution bottle 322 exceeds a preset threshold value.

[0114] First and second liquid level sensors 530 and 540 are respectively disposed in the first and second liquid paths, and are typically installed near or within first and second solution bottles 321 and 322. The sensors continuously monitor the liquid levels in the solution bottles, ensuring that the experimenter is aware of the remaining solution levels at all times.

[0115] When the first liquid level sensor 530 or the second liquid level sensor 540 detects that the liquid level in the solution bottle in its fluid circuit has reached a preset threshold, the sensor will sound an alarm. "Reaching the preset threshold" here can be understood as indicating that the liquid level is too high. If the preset threshold is set to the maximum safe capacity of the solution bottle, then when the liquid level exceeds this threshold, the sensor will sound an alarm, notifying the experimenter that the solution is about to overflow or has already overflowed.

[0116] Low liquid level: If you set a low liquid level threshold to monitor whether the solution is about to run out, the sensor can also sound an alarm when the liquid level falls below this threshold, reminding the experimenter to replenish the solution in time.

[0117] Specifically, the first circulation bottle 324 is further provided with a third liquid level sensor 560 , and the second circulation bottle 325 is provided with a fourth liquid level sensor 560 .

[0118] The first liquid path, the second liquid path, and the third liquid path can be combined to realize loading and injection. The specific control method includes the following steps: The fourth switching valve 640 connects the second solution bottle 322 and the second constant flow pump group 420 to communicate with the second liquid path; The second switching valve 620 connects the first solution bottle 321 and the first constant flow pump group 410 to communicate with the first liquid path; The first switching valve 610 connects the collecting bottle 323 and the chip adapter 200 to communicate with the third liquid path; The first solution bottle 321 contains the sample solution, and the second solution bottle 322 contains the dilution solution. Calculate the flow rates of the first constant flow pump group 410 and the second constant flow pump group 420 according to the dilution ratio and the set total flow rate; The first constant flow pump group 410 and the second constant flow pump group 420 are operated at their respective calculated flow rates. The first constant flow pump group 410 and the second constant flow pump group 420 respectively pump the solution from the first liquid path and the second liquid path into the mixer, where the solution is combined and mixed and flows into the third liquid path to the microfluidic chip device 100 for loading. Read the readings of the pressure sensor, flow sensor, etc. to determine whether the injection is proceeding normally and update the injection status; determine whether the injection process is completed, if completed, stop the constant flow pump, if not completed, continue to work until the constant flow pump is stopped; after completion, the engineered drug carrier solution is collected in the storage device 300, and in the case of no circulation and micro injection, it is collected in the collection bottle 323; in the circulation mode, it is collected in the first circulation bottle 324 or the second circulation bottle 325.

[0119] Furthermore, the carrier synthesis preparation system also includes a pressure relief module 450 for achieving pressure relief of the liquid circuit system 400. Specifically, when the pressure reaches the preset upper limit pressure of the system, the pressure relief module 450 will quickly relieve the pressure to ensure the safety of the components. In addition, for example, when there is no liquid infiltration in the first constant flow pump (that is, air is sucked into the pipeline) and liquid cannot be pumped, the syringe can be manually connected to the pressure relief module 450 to suck in liquid to infiltrate the constant flow pump (extract the air in the pipeline) to restore the constant flow pump to normal operation. The specific structure and form of the pressure relief module 450 are not limited. The pressure relief module 450 can be connected to different positions of the liquid circuit system 400 according to specific needs, such as the first liquid circuit, the second liquid circuit or the third liquid circuit; multiple ones can also be selectively connected.

[0120] Reference Figure 3 Specifically, the storage device 300 is equipped with a temperature control device 310, which includes a refrigeration module, a heat preservation module, and a temperature sensing module. The refrigeration module is used to create a refrigerated environment for the reagents. The heat preservation module is used to maintain the internal temperature and heat of the storage device 300 and reduce heat convection. The temperature sensing module is used to monitor the ambient temperature within the storage device 300 in real time and dynamically adjust the operating status of the refrigeration module to ensure that the ambient temperature within the storage device 300 remains within a certain range. Temperature control by the temperature control device 310 ensures that the reaction proceeds at the optimal temperature, which can improve reaction efficiency and product quality, while also facilitating the management and use of reactants. Precise temperature control helps ensure that samples are stored under optimal temperature conditions, reducing system instability and failure rates caused by temperature fluctuations, thereby improving the accuracy and reliability of experiments. In addition, different experiments have different requirements for sample storage temperatures. By providing refrigeration and heat preservation functions, as well as precise temperature control, the temperature control device enables the system to adapt to various experimental needs, improving the system's flexibility and versatility.

[0121] In one embodiment, in order to achieve rapid cooling of the system, a semiconductor refrigeration sheet may be used as a cooling source.

[0122] In one embodiment, to achieve good heat conduction, metal may be used as a heat conducting medium to connect the semiconductor cooling fins.

[0123] In one embodiment, the heat-conducting medium can also serve as a container for placing liquid storage reagent tubes, thereby achieving direct and effective heat conduction.

[0124] In one embodiment, the cooling module may further include a heat sink assembly and a fan to remove the heat generated by the hot end.

[0125] In some embodiments, a disinfection device may be provided in the temperature control device 310 of the storage device 300 to disinfect the interior. The disinfection device may be, but is not limited to, an ultraviolet lamp, an ozone generator, and the like.

[0126] Reference Figure 8 Specifically, the carrier synthesis preparation method includes the following steps: The sample solution in the first solution bottle 321 is drawn into the microfluidic chip device 100 through the liquid path system 400; The micro-nanostructures in the microfluidic chip device 100 are used to perform physical collision and high-speed fluid impact on the vesicles, thereby achieving morphological transformation and material reprogramming of the vesicles; Collect the engineered drug carriers after processing on the microfluidic chip.

[0127] In order to avoid the influence of bubbles on the microfluidic chip, in some embodiments, the sample solution needs to be degassed through a degassing device 430 before entering the microfluidic chip; in addition, in some embodiments, it is necessary to adjust the different concentration ranges of molecular drugs, so it is necessary to extract a dilution solution from the first solution bottle 321 for dilution and mix it through a mixing device 440; in order to ensure that the nanovesicles and molecular drugs are in a suitable environment, a temperature control device 310 is provided in the storage device 300 to regulate the temperature.

[0128] The solution of diluting the sample solution further includes the following steps before the sample solution is drawn into the microfluidic chip device 100 through the liquid path system 400: Select the microfluidic chip device 100 and dock the microfluidic chip device 100 with the adapter seat 220; mixing the nanovesicles and the molecular drug to form a sample solution; (The above two steps are in no particular order) Adding a sample solution formed by mixing the nanovesicles and the molecular drug into the first solution bottle 321; Under the control of the temperature control device 310 of the storage device 300, the diluted solution is added to the second solution bottle 322; (Understandably, the order of adding the dilution solution and the sample solution does not matter) The dilution solution and the sample solution are degassed by the degassing device 430 through the liquid system 400, and the two degassed solutions are combined in the mixing device 440 to form a mixed solution; Pumping the sample solution into the microfluidic chip device 100 through the liquid path system 400; The nanovesicles are loaded with molecular drugs using the micro-nanostructures within the microfluidic chip device 100; Collect the engineered drug carrier solution after processing through the microfluidic chip.

[0129] MiR-146 is one of the first miRNAs discovered to regulate the immune system, playing a crucial role in biological processes such as innate immunity, inflammatory responses, cell differentiation, and cancer development. By loading the exogenous substance, miR-146, into natural exosomes, the exosome-nucleic acid drug carrier is constructed. The principle is that when the exosomes are subjected to fluid shear forces within the micro-nanochannels of a microfluidic chip, the fluid membrane on the surface is disturbed, forming pores or gaps, allowing the exogenous nucleic acid to effectively enter the exosomes and form a composite carrier. Once the exosomes exit the micro-nanochannels, the fluid shear forces decrease, and the membrane returns to its intact structure, thus forming a stable drug carrier.

[0130] In one embodiment, the exosomes secreted by human embryonic kidney cells 293T are used as natural nanovesicle drug carriers to load miR-146a. The specific operation is as follows: 1. Prepare 293T exosomes extracted by ultracentrifugation (Ultracentrifugation parameters: Transfer 400 mL of cell culture supernatant to a fresh centrifuge tube and centrifuge at 300 g, 4°C for 10 min; transfer the supernatant to a fresh centrifuge tube and centrifuge at 2000 g, 4°C for 15 min; transfer the supernatant to a fresh centrifuge tube and centrifuge at 10,000 g, 4°C for 30 min; transfer the supernatant to a fresh ultracentrifuge tube and centrifuge at 100,000 g, 4°C for 90 min. Resuspension concentration: 2.15 × 109 particles / mL, volume: 5 mL).

[0131] 2. Add 50 μL of 10 μM miR-146a to 4.95 mL of 293T exosome solution.

[0132] 3. Mix 293T exosomes with miR-146a and divide them equally into two groups of samples, one as the incubation control group and the other as the experimental group.

[0133] It should be noted that the above preparation scheme is not exclusive, as long as 293T exosomes and miR146a can be obtained.

[0134] 4. Place the experimental group 293T exosome / miR-146a sample into the above-mentioned carrier synthesis preparation system, set the experimental parameters: (flow rate: 450 μL / min; cycle mode: 1 time), and start the synthesis preparation process.

[0135] 5. After the experimental group is prepared, perform ultrafiltration along with the control group, which has been incubated for the same length of time. After collecting the sample, add it to the corresponding ultrafiltration tube and centrifuge at 14,000 × g at 4°C for 10 minutes. Place the ultrafiltration column upside down in a new collection tube and centrifuge at 1,000 × g at 4°C for 1 minute. Collect the sample and transfer it to a new 1.5 mL low-absorption centrifuge tube. Add PBS to restore the initial volume.

[0136] 6. Add 1 μL RNase If enzyme + 9 μL Buffer to every 1 mL of sample and perform enzymatic hydrolysis in a 37°C water bath for 30 min. After the enzymatic hydrolysis is completed, place the exosome suspension on a metal heating device for 5 min to inactivate the RNase enzyme.

[0137] 7. Add 1 mL of Trizol reagent to each 1 mL sample tube, invert and mix thoroughly, and let it stand for 5 minutes; add 200 μL of chloroform to extract miRNA, invert and mix thoroughly, let it stand for 5 minutes, and until obvious stratification is visible; centrifuge at 12,000 rpm at 4°C for 15 minutes; prepare a new 1.5 mL centrifuge tube, add 500 μL of isopropanol to extract exosome RNA (be sure to aspirate it completely), invert and mix thoroughly, let it stand at room temperature for 3 minutes, and then place it in a -20°C refrigerator for 15 minutes; after standing, centrifuge at 12,000 rpm at 4°C for 15 minutes; discard the supernatant, invert the centrifuge tube for 3 minutes to pour out as much isopropanol or chloroform as possible; add pre-chilled anhydrous ethanol, centrifuge at 12,000 rpm at 4°C for 15 minutes, repeat the previous step to wash the RNA again, inverting to remove as much ethanol as possible; add 10 μL of RNase-free water, and store in a -80°C refrigerator; 8. Determine the concentration of the extracted RNA using a microspectrophotometer. Reverse transcribe 10 ng of RNA using the Takara RR037A kit, using U6 as the reference gene. The reaction system is as follows (reaction conditions: 37°C for 15 min, 85°C for 5 s, 4°C).

[0138] A two-step polymerase chain reaction (PCR) procedure was used: Step 1: 95°C, 30 seconds Step 2: 95°C, 5 seconds Step 3: 60°C, 30 s, set loop to 39.

[0139] qPCR detection was performed using the RR820A kit, and the reaction system was as follows:

[0140] 9.Reference Figure 9a , Figure 9a The graph below shows the data of sample concentration and particle size before the preparation system of the experimental group obtained using nano-resistance pulse sensing technology; the table below shows the particle size-related and concentration-related values before preparation.

[0141]

[0142] Reference Figure 9b , Figure 9b The graph below shows the data of sample concentration and particle size after the preparation system of the experimental group obtained using nano-resistance pulse sensing technology; the table below shows the particle size-related values and concentration-related values after preparation.

[0143]

[0144]

[0145] The experimental group was prepared before the system ( Figure 9a ) / back( Figure 9b ) of the sample concentration and particle size, by comparing Figure 7 The particle size analysis of the samples before and after preparation showed that the experimental group had Figure 9a ) / back( Figure 9b ), there was no significant change in sample concentration and particle size distribution. The lack of significant change in sample concentration indicated that there was no significant loss or change in the exosomes during the preparation process. The lack of significant change in particle size distribution indicated that the exosomes did not break or aggregate (otherwise the particle size would become larger) or decompose (the particle size would become smaller) due to microfluidic processing (shear force or other factors). The integrity of the exosomes was maintained after preparation, that is, the exosome structure was stable and the processing process did not destroy their membrane structure.

[0146] 10.Reference Figure 10 , Figure 10This is an electron micrograph of 293T exosomes (containing miR-146a) after preparation. Transmission electron microscopy (TEM) observation revealed that the exosomes treated with the above-mentioned vector preparation system did not undergo morphological changes and maintained an intact membrane structure, further indicating that the physical properties of the exosomes remained intact during the preparation process.

[0147] 11.Reference Figure 11 , Figure 11 This figure compares the background nucleic acid expression level (Evs) in exosomes obtained by polymerase chain reaction (PCR) with the nucleic acid expression level (EvsNP_@miR146a) after microarray processing and loading. Comparing the background nucleic acid expression level in exosomes with the nucleic acid expression level after microarray processing and loading by PCR can evaluate the efficiency and effectiveness of microarray loading technology. The experimental results obtained by PCR technology show that the nucleic acid expression level after microarray processing and loading is significantly higher than the background nucleic acid expression level in exosomes. This demonstrates that the vector preparation system can efficiently load nucleic acids into exosomes, thereby increasing their expression levels.

[0148] The above description is merely an exemplary embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural transformation made by utilizing the contents of the present invention's description and drawings under the technical concept of the present invention, or directly / indirectly applied in other related technical fields, is included in the patent protection scope of the present invention.

Claims

1. A carrier synthesis preparation system, characterized in that: include: A chip adapter device, comprising a chip stage and an adapter seat for mounting a microfluidic chip device, wherein the chip stage is movable relative to the adapter seat so that the microfluidic chip device can communicate with the adapter seat to form a flow path; a liquid storage device, which includes a first solution bottle and a collecting bottle; and The liquid circuit system includes a first constant flow pump group. The first solution bottle, the first constant flow pump group, the chip adapter device and the collection bottle are connected by a pipeline. The first constant flow pump group is used to allow the sample solution in the first solution bottle to enter the collection bottle after being loaded through the microfluidic chip device.

2. The carrier synthesis and preparation system according to claim 1, characterized in that: The carrier synthesis preparation system also includes a pressure sensing module and a flow sensing module, both of which are arranged on the pipeline. The pressure sensing module, the flow sensing module, and the first constant flow pump group are electrically connected, so that the liquid circuit system has multiple selectable working modes, and the multiple working modes include constant flow mode, constant pressure mode, variable flow mode and variable pressure mode.

3. The carrier synthesis and preparation system according to claim 2, characterized in that: The carrier synthesis and preparation system also includes a microfluidic chip device, which includes a chip box body with a liquid inlet and a liquid outlet and a microfluidic chip sealed and installed in the chip box body, and the liquid inlet and the liquid outlet are used to dock and connect with the adapter seat; wherein, in the multiple working modes, the microfluidic chip has different loading effects on the sample solution in the first solution bottle.

4. The carrier synthesis and preparation system according to claim 1, characterized in that: The first constant flow pump group includes at least two first plunger pumps connected in series or in parallel; Furthermore, the multiple first plunger pumps in the first constant flow pump group can all operate independently.

5. The carrier synthesis and preparation system according to claim 1, characterized in that: The carrier synthesis preparation system further includes a first switching valve and a second switching valve, both of which are arranged on the pipeline and have multiple branch valve ports; The liquid storage device is further provided with a first circulation bottle and a second circulation bottle; The first switching valve pipeline is connected to the chip adapter, the collecting bottle, the first circulation bottle and the second circulation bottle; the second switching valve pipeline is connected to the first circulation bottle, the second circulation bottle, the first solution bottle and the first constant flow pump group; The chip adapter device can selectively connect the first solution bottle, the first circulation bottle and the second circulation bottle through the first switching valve and the second switching valve, so that the sample solution in the first solution bottle can be repeatedly loaded through the microfluidic chip device.

6. The carrier synthesis and preparation system according to claim 5, characterized in that: The carrier synthesis preparation system also includes a third switching valve with multiple branch valve ports and a sample quantitative loop connected to a micro-injector, and the sample quantitative loop is connected to the third switching valve; the first constant flow pump group is connected to the chip adapter device pipeline through the third switching valve, and the third switching valve can optionally connect the sample quantitative loop and the first constant flow pump group.

7. The carrier synthesis and preparation system according to claim 1, characterized in that: The liquid circuit system further includes a degassing device and a mixing device; the liquid circuit system further includes a second constant flow pump group; the liquid storage device further includes a second solution bottle; the second solution bottle, the degassing device, the second constant flow pump group and the mixing device are connected through pipelines to form a second liquid circuit, the second constant flow pump group is used to pump the solution in the second solution bottle; the degassing device is used to degas the solution pumped out of the first solution bottle and the second solution bottle; The first solution bottle, the degassing device, the first constant flow pump group and the mixing device pipeline are connected to form a first liquid circuit; the mixing device, the chip adapter and the collection bottle pipeline are connected to form a third liquid circuit; The solution pumped from the first liquid path by the first constant flow pump group and the solution pumped from the second liquid path by the second constant flow pump group are mixed through the mixing device, and then loaded from the third liquid path through the microfluidic chip and enter the collecting bottle.

8. The carrier synthesis and preparation system according to claim 7, characterized in that: The carrier synthesis preparation system further includes a fourth switching valve with multiple branch valve ports, and the fourth switching valve is connected to the degassing module and the second solution bottle.

9. The carrier synthesis and preparation system according to claim 1, characterized in that: The carrier synthesis and preparation system also includes a temperature control device, which is used at least to regulate the ambient temperature in the liquid storage device; wherein, the temperature control device includes a refrigeration module, a heat preservation module and a temperature sensing module, the refrigeration module is used to create a refrigerated environment; the heat preservation module is used to maintain the temperature and heat in the liquid storage device and reduce heat convection; the temperature sensing module is used to monitor the ambient temperature in the liquid storage device in real time and dynamically adjust the working state of the refrigeration module so that the ambient temperature in the liquid storage device is within a preset temperature threshold range.

10. A carrier synthesis preparation system, characterized in that: include: A microfluidic chip device, comprising a chip box having a liquid inlet and a liquid outlet, and a microfluidic chip sealed and mounted in the chip box; A chip adapter device, comprising a chip stage and an adapter seat for mounting the chip cartridge, wherein the chip stage is movable relative to the adapter seat so that the liquid inlet and the liquid outlet are docked with the adapter seat to form a flow path; A liquid storage device is provided with a first solution bottle, a second solution bottle, a first circulation bottle, a second circulation bottle and a collection bottle, and the microfluidic chip is used to load and process the sample solution in the first solution bottle; and The hydraulic system includes: A first pipeline connects the second switching valve, the first solution bottle, the first constant flow pump group and the third switching valve to the liquid inlet, wherein the third switching valve is connected in parallel to the sample quantitative loop for switching the first pipeline straight-through or sample quantitative loop injection; A second pipeline is connected to the second solution bottle and the second constant flow pump assembly to the liquid inlet, and merges with the first pipeline in front of the liquid inlet; a third pipeline, connecting the liquid outlet to the first switching valve, the first switching valve being connected to the collecting bottle and the circulation pipeline group respectively; a circulation pipeline group, comprising a first branch connected to the first circulation bottle and a second branch connected to the second circulation bottle, wherein the circulation pipeline group forms a closed loop with the first pipeline via a second switching valve; The linkage control of the first switching valve, the second switching valve and the third switching valve realizes a non-circulating injection mode, a circulating injection mode or a micro-injection mode; The flow ratio control of the second constant flow pump group and the first constant flow pump group realizes the sample dilution function, and the dilution function can be operated in conjunction with the non-circulation injection mode and the circulation injection mode.

11. The carrier synthesis and preparation system according to claim 10, characterized in that: The liquid circuit system also includes a particle size measuring device and a main control system, wherein: The particle size measuring device is used to detect the particle size distribution of the carrier synthesis product in real time; The main control system is electrically connected to the particle size measuring device, the first constant flow pump group, and the second constant flow pump group, and is configured as follows: receiving real-time detection data of the particle size measuring device; Compare the detected data with the preset size range; The flow rate parameters of the first constant flow pump group and / or the second constant flow pump group are dynamically adjusted according to the comparison results so that the carrier particle size converges to a preset size range.

12. The carrier synthesis and preparation system according to claim 11, characterized in that: The liquid circuit system also includes a component concentration analysis device for real-time monitoring of the concentration of a specific component of the carrier synthesis product, wherein the specific component is at least one selected from nucleic acids, polypeptides, proteins, enzymes, probe molecules, cytokines and small molecule compounds; The main control system is electrically connected to the component concentration analysis device, the first constant flow pump group, and the second constant flow pump group, and is configured as follows: receiving a transmission signal from the component concentration analysis device; Calculate and obtain real-time concentration data based on the transmitted signal; Compare the real-time concentration data with the preset target concentration range; The flow rate of the first constant flow pump group and / or the second constant flow pump group is dynamically adjusted according to the comparison result so that the concentration of the specific molecule is within a preset concentration range.

13. A method for synthesizing a carrier, characterized in that: Using the carrier synthesis preparation system according to any one of claims 1 to 9, the carrier synthesis preparation method comprises the following steps: Select a microfluidic chip device and dock the microfluidic chip device with the adapter seat; Adding a sample solution formed by mixing the nanovesicles and the molecular drug into the first solution bottle; Pumping the sample solution into the microfluidic chip device through a liquid system device for processing; Collect the engineered drug carrier solution after processing through the microfluidic chip.

Images