Targeting exosome preparation mixing device and method based on specific antibody coupling

Abstract

The invention discloses a targeted exosome preparation mixing device and method based on specific antibody coupling, and relates to the field of biomedicines.The targeted exosome preparation mixing device comprises an antibody coupling reaction module, an exosome purification and enrichment module, a mixed incubation and condition control module, a multi-channel fluid connection module, an exosome raw material pretreatment module and a control system module; the multi-channel fluid connection module is respectively communicated with the antibody coupling reaction module, the exosome purification and enrichment module, the mixed incubation and condition control module and the exosome raw material pretreatment module. Seamless series connection of links of exosome raw material pretreatment, antibody coupling reaction, purification and enrichment and functional mixing is realized by virtue of the multi-channel fluid connection module, meanwhile, a solid-phase carrier of the antibody coupling reaction module can realize directional efficient coupling of the exosome and a specific antibody, and the purification and enrichment module can effectively separate a free antibody; the preparation efficiency and the product purity of the targeting exosome are obviously improved.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a targeted exosome preparation mixing device and method based on specific antibody conjugation. Background Technology

[0002] Exosomes, as nanoscale vesicles secreted by cells, can carry a variety of bioactive substances such as nucleic acids, proteins, and lipids, showing great application potential in the fields of intercellular information transmission, disease diagnosis, and targeted therapy.

[0003] Currently, most methods for preparing targeted exosomes based on specific antibody conjugation employ a manual, step-by-step approach: first, pretreatment of cell secretions through centrifugation and filtration to obtain crude exosomes; then, conjugation of exosomes with specific antibodies in centrifuge tubes; followed by separation and purification of the conjugated products using density gradient centrifugation and magnetic bead sorting; and finally, adding functionalizing reagents for further incubation as needed. However, this approach suffers from several technical drawbacks: firstly, the manual step-by-step transfer of exosome suspensions easily leads to exosome loss and contamination, resulting in low product yield and poor purity; secondly, the reaction conditions for key steps such as antibody conjugation, purification and enrichment, and functionalization mixing rely on manual control, resulting in significant parameter fluctuations and inconsistent conjugation efficiency and targeting activity among different batches of prepared targeted exosomes, making it difficult to meet standardized production requirements; and thirdly, the lack of integrated equipment support for each operational step makes the preparation process cumbersome and time-consuming, unsuitable for large-scale targeted exosome preparation scenarios. Summary of the Invention

[0004] (a) Technical problems to be solved

[0005] To address the problems existing in the prior art, the present invention provides a targeted exosome preparation mixing device and method based on specific antibody conjugation.

[0006] (II) Technical Solution

[0007] To achieve the above objectives, the present invention provides the following technical solution: a targeted exosome preparation mixing device and method based on specific antibody conjugation, comprising an antibody conjugation reaction module, an exosome purification and enrichment module, a mixing incubation and condition control module, a multi-pathway fluid connection module, an exosome raw material pretreatment module, and a control system module; the multi-pathway fluid connection module is connected to the antibody conjugation reaction module, the exosome purification and enrichment module, the mixing incubation and condition control module, and the exosome raw material pretreatment module, respectively; the control system module is electrically connected to the antibody conjugation reaction module, the exosome purification and enrichment module, and the mixing incubation and condition control module via signal lines.

[0008] As a preferred embodiment of the targeted exosome preparation mixing device and method based on specific antibody conjugation described in this invention, the antibody conjugation reaction module includes an exosome immobilization unit, the surface of which is coated with a solid-phase carrier containing specific antibodies; the output end of the exosome raw material pretreatment module is connected to the input end of the antibody conjugation reaction module through a multi-channel fluid connection module.

[0009] As a preferred embodiment of the targeted exosome preparation mixing device and method based on specific antibody conjugation described in this invention, the input end of the exosome purification and enrichment module is connected to the output end of the antibody conjugation reaction module through a multi-channel fluid connection module, which is used to separate the conjugated targeted exosomes from the free antibody.

[0010] As a preferred embodiment of the targeted exosome preparation mixing device and method based on specific antibody conjugation described in this invention, the mixing incubation and condition control module includes a temperature adjustment unit and a rotation speed adjustment unit; the output end of the exosome purification and enrichment module is connected to the input end of the mixing incubation and condition control module through a multi-channel fluid connection module for subsequent functionalization mixing of targeted exosomes.

[0011] As a preferred embodiment of the targeted exosome preparation mixing device and method based on specific antibody conjugation described in this invention, the control system module is used to regulate the reaction time of the antibody conjugation reaction module, the separation parameters of the exosome purification and enrichment module, and the temperature and rotation speed of the mixing incubation and condition control module.

[0012] A method for preparing targeted exosomes based on specific antibody conjugation, comprising the following steps:

[0013] S1: After the exosome raw material is pretreated by the exosome raw material pretreatment module, it is input into the antibody conjugation reaction module through the multi-channel fluid connection module and incubated and conjugated with the specific antibody on the solid-phase carrier.

[0014] S2: The conjugated product is fed into the exosome purification and enrichment module through the multi-channel fluid connection module to separate and obtain targeted exosomes;

[0015] S3: Targeted exosomes are input into the mixing incubation and condition control module through the multi-channel fluid connection module, and functional mixing is completed under the control of the control system module.

[0016] As a preferred embodiment of the targeted exosome preparation method based on specific antibody conjugation according to the present invention, the incubation conjugation temperature in S1 is 4-25℃ and the time is 1-4h; the mixing incubation temperature in S3 is 20-37℃ and the rotation speed is 50-200rpm.

[0017] As a preferred embodiment of the targeted exosome preparation method based on specific antibody conjugation described in this invention, the specific antibody is at least one of anti-CD3 single-chain antibody and anti-PD-L1 nanobody; the exosome raw material is derived from the secretions of HEK293T cells or dendritic cells.

[0018] (III) Beneficial Effects

[0019] This invention provides a device and method for preparing and mixing targeted exosomes based on specific antibody conjugation. It has the following beneficial effects:

[0020] 1. By utilizing a multi-channel fluid connection module, the pretreatment of exosome raw materials, antibody conjugation reaction, purification and enrichment, and functionalization mixing are seamlessly connected, replacing the traditional manual stepwise transfer operation and reducing the risk of exosome loss and contamination. At the same time, the solid-phase carrier of the antibody conjugation reaction module can realize the directional and efficient conjugation of exosomes and specific antibodies, while the purification and enrichment module can effectively separate free antibodies, significantly improving the preparation efficiency and product purity of targeted exosomes.

[0021] 2. By relying on the control system module, the antibody conjugation reaction time, purification, enrichment and separation parameters, and mixing incubation temperature and rotation speed are precisely controlled in an integrated manner, avoiding parameter fluctuations caused by manual operation. This ensures that different batches of targeted exosomes maintain a high degree of uniformity in conjugation efficiency, activity and other indicators. At the same time, the adjustable parameters of each functional module also broaden the applicability of the device. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 This is a schematic diagram of the overall structure of the present invention;

[0024] Figure 2 This is a flowchart of the targeted exosome preparation method of the present invention.

[0025] In the diagram, 1 is the antibody-coupled reaction module; 2 is the exosome purification and enrichment module; 3 is the mixed incubation and condition control module; 4 is the multi-pathway fluid connection module; 5 is the exosome raw material pretreatment module; and 6 is the control system module. Detailed Implementation

[0026] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.

[0027] Reference Figures 1 to 2 As shown, this invention provides a technical solution: a targeted exosome preparation and mixing device based on specific antibody conjugation, comprising an antibody conjugation reaction module 1, an exosome purification and enrichment module 2, a mixing incubation and condition control module 3, a multi-pathway fluid connection module 4, an exosome raw material pretreatment module 5, and a control system module 6; the multi-pathway fluid connection module 4 is connected to the antibody conjugation reaction module 1, the exosome purification and enrichment module 2, the mixing incubation and condition control module 3, and the exosome raw material pretreatment module 5 respectively; the control system module 6 is electrically connected to the antibody conjugation reaction module 1, the exosome purification and enrichment module 2, and the mixing incubation and condition control module 3 via signal lines, and by setting the multi-pathway fluid connection module 4 as the fluid transport hub between the various functional modules, the device achieves... The seamless integration of the exosome raw material pretreatment module 5, antibody conjugation reaction module 1, exosome purification and enrichment module 2, and mixed incubation and condition control module 3 replaces the traditional manual step-by-step transfer operation, effectively reducing the loss and contamination risk of exosomes during the transfer process, and significantly improving the continuity and automation of the preparation process. At the same time, relying on the electrical connection between the control system module 6 and the antibody conjugation reaction module 1, exosome purification and enrichment module 2, and mixed incubation and condition control module 3, key process conditions such as antibody conjugation reaction time, exosome separation parameters, and mixed incubation temperature and speed can be precisely controlled in an integrated manner, avoiding parameter fluctuations caused by manual operation and ensuring the uniformity of different batches of targeted exosome products in terms of conjugation efficiency, purity, and activity.

[0028] Reference Figure 1As shown in this embodiment: the antibody conjugation reaction module 1 includes an exosome immobilization unit, and the surface of the immobilization unit is coated with a solid-phase carrier containing specific antibodies; the output end of the exosome raw material pretreatment module 5 is connected to the input end of the antibody conjugation reaction module 1 through a multi-channel fluid connection module 4, and the input end of the exosome purification and enrichment module 2 is connected to the output end of the antibody conjugation reaction module 1 through the multi-channel fluid connection module 4, which is used to separate the targeted exosomes and free antibodies after conjugation. By setting a solid-phase carrier coated with specific antibodies as an exosome immobilization unit in the antibody conjugation reaction module 1, the directional adsorption and efficient conjugation of exosomes and specific antibodies can be achieved, greatly improving the efficiency of antibody conjugation. This method enhances the binding efficiency and targeting specificity of antibodies to exosomes. Simultaneously, an ordered fluid pathway is constructed between the exosome raw material pretreatment module 5 and the antibody conjugation reaction module 1, and between the antibody conjugation reaction module 1 and the exosome purification and enrichment module 2, using a multi-pathway fluid connection module 4. This allows the pretreated exosome raw material to be directionally delivered to the antibody conjugation reaction module 1 to complete the conjugation reaction, while the conjugation product can be directly transported to the exosome purification and enrichment module 2. This achieves rapid and effective separation of targeted exosomes from free antibodies, reducing the risk of exosome loss and contamination from manual transfer operations, ensuring the high purity of the targeted exosomes, and laying a solid material foundation for subsequent functional mixing.

[0029] Reference Figure 2 As shown, specifically, the mixing incubation and condition control module 3 includes a temperature regulation unit and a rotation speed regulation unit; the output end of the exosome purification and enrichment module 2 is connected to the input end of the mixing incubation and condition control module 3 through a multi-pathway fluid connection module 4, for subsequent functional mixing of targeted exosomes. The control system module 6 is used to regulate the reaction time of the antibody conjugation reaction module 1, the separation parameters of the exosome purification and enrichment module 2, and the temperature and rotation speed of the mixing incubation and condition control module 3. By configuring the temperature regulation unit and the rotation speed regulation unit in the mixing incubation and condition control module 3, and constructing a fluid pathway between it and the exosome purification and enrichment module 2 based on the multi-pathway fluid connection module 4, the purified targeted exosomes can be mixed. Exosomes are directed to this module for subsequent functionalization mixing operations. The coordinated regulation of temperature and rotation speed provides a suitable reaction environment for the efficient binding of functionalized reagents and targeted exosomes. At the same time, the control system module 6 integrates and precisely regulates the reaction time of antibody conjugation module 1, the separation parameters of exosome purification and enrichment module 2, and the temperature and rotation speed parameters of mixing incubation and condition control module 3. This effectively avoids parameter errors caused by manual regulation and ensures the stability and consistency of process conditions for targeted exosomes throughout the entire process of conjugation, purification, and functionalization mixing. This significantly improves the uniformity and targeting activity of the final product and broadens the applicability of the device in different functionalized targeted exosome preparation scenarios.

[0030] A method for preparing targeted exosomes based on specific antibody conjugation, comprising the following steps:

[0031] S1: After the exosome raw material is pretreated by the exosome raw material pretreatment module 5, it is input into the antibody conjugation module 1 through the multi-channel fluid connection module 4 and incubated and conjugated with the specific antibody on the solid support.

[0032] S2: The coupling product is fed into the exosome purification and enrichment module 2 through the multi-channel fluid connection module 4 to separate and obtain targeted exosomes;

[0033] S3: Targeted exosomes are input to the mixing incubation and condition control module 3 through the multi-channel fluid connection module 4, and functional mixing is completed under the control of the control system module 6.

[0034] In S1, the incubation and coupling temperature is 4-25℃ and the time is 1-4h; in S3, the mixed incubation temperature is 20-37℃ and the rotation speed is 50-200rpm.

[0035] The specific antibody is at least one of anti-CD3 single-chain antibody and anti-PD-L1 nanobody; the exosome raw material is derived from the secretions of HEK293T cells or dendritic cells.

[0036] It should be noted that in this document, relational terms such as first and second are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations.

Claims

1. A targeted exosome preparation and mixing device based on specific antibody conjugation, characterized in that, include: The system comprises an antibody-coupled reaction module (1), an exosome purification and enrichment module (2), a mixed incubation and condition control module (3), a multi-pathway fluid connection module (4), an exosome raw material pretreatment module (5), and a control system module (6). The multi-pathway fluid connection module (4) is connected to the antibody-coupled reaction module (1), the exosome purification and enrichment module (2), the mixed incubation and condition control module (3), and the exosome raw material pretreatment module (5), respectively. The control system module (6) is electrically connected to the antibody-coupled reaction module (1), the exosome purification and enrichment module (2), and the mixed incubation and condition control module (3) through signal lines.

2. The apparatus according to claim 1, characterized in that, The antibody-coupled reaction module (1) includes an exosome immobilization unit, the surface of which is coated with a solid-phase carrier containing specific antibodies; the output of the exosome raw material pretreatment module (5) is connected to the input of the antibody-coupled reaction module (1) through a multi-channel fluid connection module (4).

3. The apparatus according to claim 1, characterized in that, The input end of the exosome purification and enrichment module (2) is connected to the output end of the antibody conjugation module (1) through the multi-pathway fluid connection module (4) for separating the conjugated targeted exosomes from the free antibody.

4. The apparatus according to claim 1, characterized in that, The mixing incubation and condition control module (3) includes a temperature regulation unit and a speed regulation unit; the output end of the exosome purification and enrichment module (2) is connected to the input end of the mixing incubation and condition control module (3) through a multi-channel fluid connection module (4) for subsequent functional mixing of targeted exosomes.

5. The apparatus according to claim 1, characterized in that, The control system module (6) is used to regulate the reaction time of the antibody-coupled reaction module (1), the separation parameters of the exosome purification and enrichment module (2), and the temperature and rotation speed of the mixed incubation and condition control module (3).

6. A method for preparing targeted exosomes based on the device of any one of claims 1-5, characterized in that, Includes the following steps: S1: After the exosome raw material is pretreated by the exosome raw material pretreatment module (5), it is input into the antibody conjugation module (1) through the multi-channel fluid connection module (4) and conjugated with the specific antibody on the solid-phase carrier. S2: The coupling product is fed into the exosome purification and enrichment module (2) through the multi-channel fluid connection module (4) to separate and obtain targeted exosomes; S3: Targeted exosomes are input to the mixing incubation and condition control module (3) through the multi-channel fluid connection module (4) and functional mixing is completed under the control of the control system module (6).

7. The method according to claim 6, characterized in that, The incubation and coupling temperature in S1 is 4-25℃ and the time is 1-4h; the mixed incubation temperature in S3 is 20-37℃ and the rotation speed is 50-200rpm.

8. The method according to claim 6, characterized in that, The specific antibody is at least one of anti-CD3 single-chain antibody and anti-PD-L1 nanobody; the exosome raw material is derived from the secretions of HEK293T cells or dendritic cells.

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