Preparation method of tumor enhanced targeting and therapeutic function extracellular vesicles and chip device

Abstract

The invention belongs to the technical field of cell biology, and particularly relates to a preparation method of tumor enhanced targeting and therapeutic function extracellular vesicles and a chip device. The invention firstly provides an integrated chip device which is provided with a cell screening module and a functional preparation module and comprises a plurality of functional layers, wherein one functional layer is provided with a cell evolution screening area with gradient for cell screening, and the other functional layer is provided with a cell electric remodeling area for cell functionalization transformation. The invention innovatively proposes that tumor cells with strong metastasis ability are screened based on culture gradient, the tumor cells with stronger invasion ability are distinguished by utilizing spatial segmentation, then the screened cell subpopulation is transferred to a cell electric remodeling area in combination with overturning operation of the chip, and the cell subpopulation is transferred to the cell electric remodeling area in a manner of focusing an electric field through nanopores. And meanwhile, therapeutic molecules are efficiently delivered to realize cell transformation, so that the transformed cells can enhance the output of extracellular vesicles with a therapeutic function.

Description

Technical Field

[0001] The present invention belongs to the field of cell biotechnology, and particularly relates to a method for preparing tumor-enhanced targeted and therapeutic functional extracellular vesicles and a chip device. Background Art

[0002] Tumor metastasis is the main cause of death in cancer patients. How to effectively prevent and treat tumor metastasis is the key to prolonging the survival period of cancer patients. Due to the obvious heterogeneity of tumor cells, the tumor cells that form metastatic foci are mainly the subpopulation with strong invasion and migration ability in the tumor (Aggressive cells, AG cells). Sorting out these specific subpopulations for in-depth research may lead to breakthroughs in combating tumor metastasis.

[0003] For the methods of screening out viable cell subpopulations, the existing technologies mainly label the molecules specifically expressed by cells through fluorescence and other means, and combine sorting methods such as flow cytometry and optical tweezers to separate cell subpopulations. Although such technologies meet the requirements of cell separation, in the case where the molecules causing invasive differences cannot be determined, specific screening cannot be achieved, which greatly challenges the feasibility of such technologies and the workload is very large. In addition, without knowing the cell type, drug treatment methods are mainly used clinically for tumor metastasis, including chemotherapy drugs and immunotherapy drugs, etc. Although the drug treatment method has certain curative effects, due to the non-targeted delivery of drugs relying on the whole body blood circulation, the dose randomly reaching the metastatic tumor site is very limited or even unable to reach, so it is very difficult to effectively kill tumor metastatic cells and even brings greater side effects. How to select cell subpopulations with strong invasion and migration ability and design highly targeted drugs based on this has become the key to inhibiting tumor metastasis.

[0004] At present, there is no report that can screen out cell subpopulations with strong migration ability without damage under the natural growth state of cells and further carry out functional modification on them. To sum up, it is necessary to propose new method strategies to supplement the deficiencies of the existing technologies. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for preparing tumor-enhanced targeted and therapeutic functional extracellular vesicles and a chip device, which partially solve or alleviate the above deficiencies in the existing technologies. The present invention specifically adopts the following technical solutions.

[0006] The first aspect of the present invention is to provide an integrated chip device.

[0007] An integrated chip device for screening and functional modification of highly invasive cells, the integrated chip device is provided with two modules of cell screening and functional modification, and includes multiple functional layers; A plurality of micropores are provided on the first functional layer 100 for sample addition or sample output, including a first micropore 110, a second micropore 120, a third micropore 130, and a fourth micropore 140; A microchannel 210 is provided on the second functional layer 200 for liquid flow; A cell electro-remodeling region 310 and a cell evolution and screening region 320 with a hollow circular structure are provided on the third functional layer 300; wherein, the edge of the hollow circle of the cell evolution and screening region 320 has a slope to form two inner and outer circles, and the slope range is set to 20° - 80°; the diameter of the inner circle in the hollow circular structure of the cell evolution and screening region 320 is smaller than the diameter of the outer circle; a nano-film 330 is also provided on the third functional layer 300 and attached to the bottom of the cell electro-remodeling region 310; A reagent storage region 410 and a cell culture region 420 with a solid circular structure are provided on the fourth functional layer 400; wherein the reagent storage region 410 is provided below the cell electro-remodeling region 310; the cell culture region 420 is provided below the cell evolution and screening region 320; and the diameter of the cell culture region 420 is equal to the diameter of the inner circle of the cell evolution and screening region 320; The fifth functional layer 500 is set as an electrode; at least one pair of micropores are also provided on the fifth functional layer for sample addition or sample output.

[0008] Furthermore, the thickness of the third functional layer 300 is greater than that of other functional layers.

[0009] As a preference, the diameter of the hollow circle of the cell evolution and screening region 320 is smaller than the diameter of the hollow circle of the cell electro-remodeling region 310.

[0010] As a preference, the size of the nano-film 330 is not less than that of the cell electro-remodeling region 310.

[0011] As a preference, the slope of the cell evolution and screening region 320 is set to 30° - 50°.

[0012] Furthermore, the cell electro-remodeling region 310 is configured to deliver target substances to target cells by using the nano-pore focused electric field technology; the target substances include immunotherapy molecules and / or drugs.

[0013] In some specific embodiments, the immunotherapy molecules include IFNγ, PDL1 siRNA, or a combination thereof.

[0014] In addition, the immunotherapy molecules may further include tumor-specific antigens, immune adjuvants, cytokines, etc., and can be directly introduced into antigen-presenting cells such as dendritic cells to promote their maturation and efficiently present antigens to T cells, thereby triggering a strong anti-tumor immune response.

[0015] In addition to immune molecules, various other types of drugs can also be delivered, such as 1. Macromolecular drugs: polypeptides, proteins, and antibodies. These molecules are usually difficult to pass through cell membranes due to their large molecular weights, strong polarities, or specific three-dimensional structures. By using a nanopore-focused electric field to form transient and reversible nanopores in the cell membrane, these macromolecular drugs can more easily enter the interior of the cell; 2. Nucleic acid drugs: including DNA, RNA (such as mRNA and siRNA), etc. These nucleic acid molecules have important applications in gene therapy, vaccine development, and disease diagnosis. By means of a nanopore-focused electric field, genes encoding therapeutic proteins or RNA molecules with specific functions can be introduced into cells, thereby achieving gene expression regulation or disease treatment; 3. Nanodrugs; 4. Drugs encapsulated in liposomes or nanocarriers.

[0016] Further, the target cells include tumor cells, immune cells, and / or stem cells.

[0017] Delivering target substances to tumor cells can achieve the modification of extracellular vesicles. Delivering target substances to bacterial cells can be used for recombinant protein production, cloning, and other biotechnological applications.

[0018] Further, the material of the integrated chip device is a transparent material; the transparent material includes polydimethylsiloxane (PDMS) and / or polymethyl methacrylate (PMMA).

[0019] Further, the first functional layer 100 can seal the microchannels 210 provided on the second functional layer 200; alternatively, a plurality of micropores provided on the second functional layer 200 and the third functional layer 300 are connected and communicated with the micropores on the first functional layer 100.

[0020] Further, the fifth functional layer 500 is also provided with a fifth micropore 510 and a sixth micropore 520 for sample addition or sample extraction; alternatively, a plurality of drainage holes are also provided on the third functional layer 300 and the fourth functional layer 400 for draining liquid into the microchannels 210.

[0021] Further, the drainage holes 430 and 440 provided on the fourth functional layer 400 are respectively corresponding in position to 510 and 520.

[0022] In some preferred embodiments, the drainage holes provided on each functional layer are a group of two, and the distance between the two drainage holes is equal to the length of the microchannels 210.

[0023] On the other hand, the present invention provides a method for cell screening and functional modification / preparation.

[0024] A method for preparing tumor-enhanced targeted and therapeutic functional extracellular vesicles, which is implemented based on the above integrated chip device, and the specific steps are as follows: S01: Prepare a tumor cell suspension, inject the tumor cell suspension through the micropores on the first functional layer 100, and the tumor cells sequentially enter the cell culture area 420 through the through micropores provided on the second functional layer 200, the third functional layer 300, and the fourth functional layer 400; Observe that the tumor cells complete adherent growth, and supplement the cell culture medium to the cell screening area 320; S02: When it is observed that the tumor cells adhere and grow on the slope of the cell screening area 320, suck away the non-adherent tumor cells and the excess cell culture medium through the micropores on the first functional layer 100; S03: Flip the integrated chip device, inject a small amount of trypsin from the sixth micropore 520, so that the trypsin liquid level is only in the area of the cell screening area 320, and briefly incubate with the tumor cells at room temperature to digest the tumor cells, and continue to inject fresh cell culture medium from the sixth micropore 520 to terminate the digestion; S04: The tumor cells in the cell screening area 320 enter the microchannel 210 through the drainage holes on the fourth functional layer 400 and the third functional layer 300, and flow into the cell electro-remodeling area 310 through the microchannel 210; S05: Flip the integrated chip device so that the tumor cells entering the cell electro-remodeling area 310 adhere to the nanofilm 330; S06: Inject a reagent containing the target substance to be delivered by electroporation through the micropores on the first functional layer 100 into the reagent storage area 410, connect the electrodes of the electroporator on the first functional layer 100 and the fifth functional layer 500 respectively, and apply a pulsed electric field to the tumor cells in the cell electro-remodeling area 310 for electroporation; S07: After applying electroporation, continue to culture the tumor cells in situ, and suck out the supernatant of the tumor cell culture from the fifth micropore 510 for extracting functionalized extracellular vesicles.

[0025] Furthermore, the target substance includes immunotherapy molecules and / or drugs.

[0026] As a preference, the immunotherapy molecules include IFNγ and / or PD-L1 siRNA.

[0027] In some preferred embodiments, the immunotherapy molecule includes an IFNγ plasmid, and the sequence is as shown in SEQ ID NO.1; the immunotherapy molecule also includes siRNA targeting PDL1, and the sequences are as shown in SEQ ID NO.2-3.

[0028] Beneficial technical effects: The present invention first provides an integrated chip device, which is provided with two modules for cell screening and functional preparation, and is realized by multiple functional layers. One of the modules is provided with a cell evolution screening area with a slope for cell screening, and the other module is provided with a cell electro-remodeling area for cell functional transformation. The present invention innovatively proposes to screen tumor cells with strong metastasis ability based on the culture slope, use the spatial segmentation to distinguish tumor cells with stronger invasion ability, and then combine the flipping operation of the chip to transfer the screened cell subset to the cell electro-remodeling area. By means of the nano-pore focused electric field, the cell membrane is safely opened while efficiently delivering therapeutic molecules to achieve cell transformation, so that the transformed cells enhance the production of extracellular vesicles with therapeutic functions. This chip design takes into account the screening of cell invasion differences and avoids the disadvantages of the screening method based on molecular markers.

[0029] In addition, the present invention also proposes to efficiently load molecules (IFNγ and PD-L1 siRNA) for immunotherapy into the screened tumor cells by means of the nano-pore focused electric field, and at the same time significantly improve the EVs production. In addition, the EVs secreted by the transformed AG cells (EVs from Aggressive cells with Immunotherapy property, AI-EVs) can specifically recognize and efficiently bind to their parental cells (AG cells), and successfully deliver the immunotherapy molecules into the cells, so that the tumor cells increase IFNγ secretion and reduce PD-L1 expression at the same time. Verified by the mouse CDX and PDX tumor metastasis models, the AI-EVs produced by the method of the present invention successfully induced the killing effect of macrophages and immune T cells on tumor cells, and effectively prevented and treated tumor metastasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts do not necessarily draw according to the actual scale. Obviously, the following-described drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained according to these drawings without creative efforts.

[0031] Figure 1 It is an exploded view of the integrated chip device in one embodiment of the present invention; Figure 2 It is a layered schematic diagram and dimensions of the integrated chip device in one embodiment of the present invention; Figure 3Schematic diagram of the operation using the integrated chip device in one embodiment of the present invention; Figure 4 Determination of the screening slope and experimental results of slope screening in one embodiment of the present invention; Figure 5 Results of parental cells taking up EVs from cell sources screened at different slopes in one embodiment of the present invention; Figure 6 Homing ability of EVs from A549 and H1975 cell lines to parental cells in one embodiment of the present invention; Figure 7 Verification of the homing ability of EVs from evolutionarily screened A549 and H1975 cell lines at the animal level in one embodiment of the present invention; Figure 8 Comparison of different delivery methods in terms of cell viability, delivery efficiency, and EV yield in one embodiment of the present invention; Figure 9 Verification of EVs produced by modified cells in one embodiment of the present invention; Figure 10 Verification of the effect of EVs produced by modification on parental tumor cells in one embodiment of the present invention; Figure 11 Inhibitory effect of modified EVs on tumor metastasis in the CDX lung metastasis prevention model in one embodiment of the present invention; Figure 12 Verification of the treatment of tumor metastasis by modified EVs in the CDX lung metastasis treatment model in one embodiment of the present invention; Figure 13 Inhibitory effect of modified EVs on tumor metastasis in the PDX lung metastasis prevention model in one embodiment of the present invention; Figure 14 Verification of the treatment of tumor metastasis by modified EVs in the PDX lung metastasis treatment model in one embodiment of the present invention. Detailed implementation manners

[0032] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0033] As used herein, "and / or" includes any and all combinations of one or more of the listed related items.

[0034] In this text, "a plurality of" means two or more, that is, it includes two, three, four, five, etc.

[0035] As used in this specification, the term "about" typically represents + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0036] In this specification, certain embodiments may be disclosed in a format that is within a certain range. It should be understood that this description of "within a certain range" is merely for convenience and brevity and should not be construed as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and the individual numerical values within that range. For example, the description of the range 1 - 6 should be regarded as having specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as the individual numbers within that range, such as 1, 2, 3, 4, 5, and 6. The above rules apply regardless of the breadth of the range.

[0037] Partial detailed description of the drawings: Figure 4 a shows the analysis and verification of the evolutionary screening gradient of A549 cells and H1975 cells. Figure 4 b shows the verification of the migration ability of evolutionarily screened A549 cells and H1975 cells through scratch experiments; AG represents highly invasive cells; AM represents non-invasive cells; Control represents the cell control for planar growth. Figure 4 c shows the verification of the migration ability of evolutionarily screened A549 cells and H1975 cells through 3D invasion experiments.

[0038] Figure 5 a - b are respectively the fluorescence images and quantitative analysis of A549 cells taking up EVs from evolutionarily screened cells with different gradients. Figure 5 c - d are respectively the fluorescence images and quantitative analysis of H1975 cells taking up EVs from evolutionarily screened cells with different gradients.

[0039] Figure 6 a - b are respectively the fluorescence images and quantitative analysis of different cells taking up EVs secreted by A549. Figure 6 c - d are respectively the fluorescence images and quantitative analysis of different cells taking up EVs secreted by H1975.

[0040] Figure 7a-b show the in vivo distribution and quantitative analysis of EVs in BALB / c nude mice bearing A549 and H1975 tumors at different time points after tail vein injection of EVs from different evolutionarily screened cell sources. Figure 7 c-d show the in vitro fluorescence images and quantitative analysis of EVs distribution in major organs of BALB / c nude mice bearing A549 and H1975 tumors. Figure 7 e-f are frozen sections of the liver and tumor: cell nuclei and EVs are represented by blue and red fluorescence respectively (marked by green circles).

[0041] Figure 8 a-b show the comparison of cell viability and delivery efficiency of different delivery methods in A549 and H1975 cell lines. Figure 8 c-d show the comparison of EVs production of different delivery methods in A549 and H1975 cell lines.

[0042] Figure 9 a-b show the expression levels of IFNγ mRNA in EVs derived from modified A549 and H1975 respectively. Figure 9 c-d show the contents of PDL1 siRNA in EVs derived from modified A549 and H1975 respectively.

[0043] Figure 10 a shows the expression levels of IFNγ mRNA after co-incubation of EVs derived from A549 (left) and H1975 (right) with parental cells respectively. Figure 10 b shows the expression levels of PDL1 mRNA after co-incubation of EVs derived from A549 (left) and H1975 (right) with parental cells respectively. Figure 10 c-d show the expression levels of IFNγ and PDL1 proteins after co-incubation of EVs derived from A549 with parental cells. Figure 10 e-f show the expression levels of IFNγ and PDL1 proteins after co-incubation of EVs derived from H1975 with parental cells.

[0044] Figure 11 a shows the survival curves of different treatment groups in the CDX lung metastasis prevention mouse model. Figure 11 b shows the tumor volume statistics of different treatment groups. Figure 11 c shows the lung tissue photos after different treatments. Purple circles indicate the positions of nodules. Figure 11 d shows the statistics of the number of lung metastasis nodules detected in each group.

[0045] Figure 12 a shows the images of tumor size at the end of treatment in the lung metastasis treatment model. Figure 12 b shows the tumor volume statistics of different treatment groups.

[0046] Figure 13 a is the statistical result of tumor volume of different treatment groups in the PDX lung metastasis prevention model. Figure 13 b is the statistical result of the number of lung metastasis nodules detected in each group. Figure 13 c is a photograph of lung tissue after different treatments: the purple circles indicate the positions of the nodules.

[0047] Figure 14 a is an image of the tumor size at the end of treatment in the PDX lung metastasis treatment model. Figure 14 b is the statistical result of tumor volume of different treatment groups.

[0048] Summary of reference numerals: The first functional layer 100, the first micropore 110, the second micropore 120, the third micropore 130, the fourth micropore 140, the second functional layer 200, the microchannel 210, the third functional layer 300, the cell electro-remodeling region 310, the cell evolution and screening region 320, the nano-membrane 330, the first drainage hole 340, the fourth functional region 400, the reagent storage region 410, the cell culture region 420, the second drainage hole 430, the third drainage hole 440, the fifth functional layer 500, the fifth micropore 510, the sixth micropore 520.

[0049] Example 1 This example provides a schematic diagram of an integrated chip device.

[0050] An integrated chip device for the screening and functional modification of highly invasive cells, the integrated chip device is provided with two modules of cell screening and functional preparation, and is divided into the first functional layer 100, the second functional layer 200, the third functional layer 300, the fourth functional layer 400 and the fifth functional layer 500 from top to bottom. The integrated chip device is made of a transparent material for easy observation.

[0051] Among them, the first functional layer 100 can be set to have the function of a sealing layer, and a plurality of micropores are provided thereon for sample addition or sample extraction, including the first micropore 110, the second micropore 120, the third micropore 130 and the fourth micropore 140. Among them, the first micropore 110 is optionally set as the first reagent outlet, the second micropore 120 is optionally set as the second reagent inlet, the third micropore 130 is optionally set as the cell inlet, and the fourth micropore 140 is optionally set as the cell outlet.

[0052] A microchannel 210 is provided in the middle of the second functional layer 200, and the microchannel 210 is used for liquid circulation; a plurality of micropores are also provided on the second functional layer 200 and are in communication with the micropores on the first functional layer 100 to facilitate sample addition or sample extraction.

[0053] On the third functional layer 300, a cell electro-remodeling region (ES) 310 and a cell evolution screening region 320 are provided. Among them, both the cell electro-remodeling region 310 and the cell evolution screening region 320 are hollow circle structures, and the edge of the hollow circle of the cell evolution screening region 320 is set as a slope, and the slope is set to 20°-80°. In some preferred embodiments, the slope is set to 30°-50°. In some most preferred embodiments, the slope is set to 50°. The cell evolution screening region 320 is used to screen the cultured cells; the cell electro-remodeling region 310 is used for cell electrotransfection.

[0054] On the third functional layer 300, a plurality of micropores are also provided and are in communication with the micropores on the first functional layer 100 and the second functional layer 200, facilitating sample addition or sample extraction. On the third functional layer 300, at least one drainage hole (such as the first drainage hole 340) is also provided, which is used to drain the liquid into the microchannel 210. Optionally, the drainage hole can be set to one, located on one side of the microchannel 210, or can be set as a group, located on both sides of the microchannel 210. On the third functional layer 300, a nanomembrane 330 is also provided, and the nanomembrane 330 is attached to the bottom of the cell electro-remodeling region 310.

[0055] On the fourth functional layer 400, a reagent storage area 410 and a cell culture area 420 are provided, and the reagent storage area 410 and the cell culture area 420 are hollow circle structures. Among them, the reagent storage area 410 is arranged below the cell electro-remodeling region 310; the cell culture area 420 is arranged below the cell evolution screening region 320. On the fourth functional layer 400, a second drainage hole 430 and a third drainage hole 440 are also provided for introducing the liquid into the appropriate cavity. On the third functional layer 300, corresponding through-drainage holes are provided corresponding to those on the fourth functional layer 400, which are used to introduce or suck out the liquid from any side of the microchannel 210.

[0056] The fifth functional layer 500 is an electrode. In some alternative embodiments, the fifth functional layer 500 is also provided with a fifth micropore 510 and a sixth micropore 520 for sample addition or sample extraction.

[0057] It can be understood that the cell evolution screening region 320 and the cell culture area 420 constitute the cell screening module of the chip; the cell electro-remodeling region 310, the nanomembrane 330, the reagent storage area 410 and the fifth functional layer 500 constitute the functionalization module of the chip.

[0058] Among them, the first reagent contains a target delivery substance for cell electroporation. The second reagent is a buffer solution.

[0059] In some preferred embodiments, the delivery substance for cell electroporation is a plasmid containing INFγ and / or PDL1 siRNA, which is stored in a phosphate buffer solution. The second reagent is a phosphate buffer solution.

[0060] In some preferred embodiments, the culture medium for cell culture is a medium of 10% FBS F12K or 1640.

[0061] The sequences involved are shown in Table 1.

[0062] Table 1 Among them, the 3' ends of sequences NO.2 and NO.3 are modified with TsdT to enhance stability.

[0063] This embodiment also provides an example of the preparation method of the above integrated chip device.

[0064] The overall exploded view of the chip is as Figure 1 shown, and the size and structure schematic diagram are as Figure 2 shown. Specifically, the first functional layer 100 is made of polydimethylsiloxane (PDMS), and its preparation method is as follows: Mix PDMS with a curing agent and elastomer (Dow Corning, SylgardTM 184, MI, USA) at a ratio of 1:10, and degas it under vacuum for 30 minutes. After curing at 80°C for 30 minutes, peel the PDMS layer from the mold and punch holes to form inlets and outlets. The second functional layer 200, the third functional layer 300, and the fourth functional layer 400 are made of acrylic plates and manufactured using laser cutting. The fifth functional layer 500 is indium tin oxide (ITO) glass (with a resistance of 6 ohms) (NOZO, China). The first functional layer 100 and the second functional layer 200 are treated with oxygen plasma and then bonded at 80°C for 2 hours. The second functional layer 200, the third functional layer 300, the fourth functional layer 400, the fifth functional layer 500, and the nanofilm 330 are connected together with acrylic adhesive. After the chip is prepared, it is rinsed with 75% ethanol and sterile water, and then irradiated with ultraviolet light for 1 hour for disinfection.

[0065] This embodiment also provides a specific size example of the above integrated chip device.

[0066] The size of the first functional layer 100 is 100×50×1 mm (length × width × thickness). The diameter of the micropores provided on the first functional layer 100 is 2 mm, and the distance between its second micropore 120 and the third micropore 130 is 42 mm; the distance between the third micropore 130 and the fourth micropore 140 is 38 mm.

[0067] The size and micropore design of the second functional layer 200 are the same as those of the first functional layer 100. Among them, the length of the microchannel 210 is 82 mm, the width is 2 mm, and the depth is less than 1 mm.

[0068] The size of the third functional layer 300 is 100×50×4 mm (length×width×thickness). The microporous design on the third functional layer 300 is the same as that of the first functional layer 100. Among them, in the hollow circle structure of the cell evolution and screening area 320, the diameter of the inner circle is 20 mm, and the diameter of the outer circle is 26.71 mm. The diameter of the hollow circle in the cell electro-remodeling area 310 is 30 mm.

[0069] The size and microporous design of the fourth functional layer 400 are the same as those of the first functional layer 100. The diameter of the cell culture area 420 is 20 mm; and microchannels with a width of 2 mm extend from both the upper and lower ends of the cell culture area 420 to connect with the micropores. The diameter of the reagent storage area 410 is 30 mm, and microchannels with a width of 2 mm extend from both the upper and lower ends of the reagent storage area 410 to connect with the micropores.

[0070] The size of the fifth functional layer 500 is the same as that of the first functional layer 100. A group of micropores with a diameter of 2 mm are arranged on the fifth functional layer, and the distance between two micropores is the same as the length of the microchannel 210.

[0071] The content described above is only an example. According to the actual situation, when the overall size of the chip is enlarged or reduced, the sizes of its various structures are enlarged or reduced proportionally.

[0072] This embodiment also provides a method for preparing tumor-enhanced targeted and therapeutic functional extracellular vesicles based on the above integrated chip device. The operation schematic diagram is shown in Figure 3 , and the specific steps are as follows.

[0073] S01: Prepare a cell suspension, inject 2×10 6 tumor cells through the third micropore 130 on the first functional layer 100. The tumor cells sequentially enter the cell culture area 420 through the through-hole micropores on the second functional layer 200, the third functional layer 300, and the fourth functional layer 400; after culturing for about one week, it can be observed that the tumor cells complete adherent growth; S02: When it is observed that the cells on the slope of the cell screening area 320 have adhered, suck away the non-adherent tumor cells and excess culture medium from the fourth micropore 140; S03: Flip the integrated chip device, inject a small amount of 0.05% trypsin (G4011, Servicebio, China; pay attention to observe to ensure that the trypsin does not completely submerge the adherent tumor cells) from the sixth micropore 520, so that the trypsin solution is only in the area of the cell screening area 320. After incubating at room temperature for 5 minutes, continue to inject fresh culture medium from the sixth micropore 520 to terminate digestion; S04: The tumor cells in the cell screening area 320 enter the microchannel 210 through the drainage holes on the fourth functional layer 400 and the third functional layer 300, and flow into the cell electro-remodeling area 310 through the microchannel 210; S05: Flip the integrated chip device so that the tumor cells entering the cell electro-remodeling area 310 adhere to the nanofilm 330; S06: Inject a reagent containing the target substances (such as 5 μg IFNγ plasmid and 20 nM PD-L1 siRNA) to be delivered by electrotransfection into the reagent storage area 410 from the second micropore 120. After about 6 hours, insert a gold needle through the first functional layer 100 as the top electrode and connect it to the positive pole of an electroporator (ECM830, BTX, USA). At the same time, connect the fifth functional layer 500 to the negative pole of the electroporator, apply a pulsed electric field for electrotransfection, and then remove the top electrode; S07: After applying electrotransfection, continue to culture the tumor cells in situ for 2 days, and then aspirate the supernatant of the cell culture from the fifth micropore 510 for extracting functionalized extracellular vesicles.

[0074] It can be understood that the excess liquid before electrotransfection can be aspirated from the first micropore 110.

[0075] Example 2 Verify the integrated microfluidic chip system of Example 1.

[0076] In the cell screening area 320, the slope is an important factor affecting the cell screening effect. To determine the optimal screening slope, the present invention selects lung cancer, which has the largest number of patients and a very high probability of metastasis, as a research example, and cultures lung cancer (A549 and H1975) cells at slopes of 20° - 80°. The results show that within the same time, the migration distance of cells on the 30° slope is significantly decreased compared with that on the 20° slope; while when the slope is greater than 50° (50°, 60°, 70°, 80°), there is no significant difference in the migration distance of cells ( Figure 4 a).

[0077] Furthermore, the migration ability of the cells on the 50° slope is compared with that of the cells below the slope. According to the cell scratch assay ( Figure 4 b) and the 3D cell invasion assay ( Figure 4After the results of (c), after a long-term culture (72 h), the cells on the 50° slope showed stronger migration and invasion abilities compared to the cells below the slope (AM) and the cells cultured on a flat surface (Control). Therefore, 50° was selected as the critical condition for screening AG cells. At the same time, this experiment showed that after cell screening, the behavioral differences could be maintained for a long time after the environmental change, indicating that the differences between cells existed inherently. The screening module provided the environment for cell screening, rather than the stress changes of cells due to the environment. Therefore, this module was also called the evolutionary screening module.

[0078] Furthermore, the ability of EVs secreted by tumor cells after evolutionary screening to target parental tumor cells was verified. The results showed that the ability of EVs secreted by evolutionarily screened cells to enter the interior of parental cells was significantly improved, and this ability was significantly enhanced with the increase in slope ( Figure 5 ). Further, EVs were co-cultured with other non-parental cells respectively. In contrast, more EVs entered parental cells, indicating an obvious tendency of EVs to bind to parental cells ( Figure 6 ).

[0079] Example 3 Mouse tumor models of different tumor cells were established respectively, and EVs produced by tumor cells after slope evolutionary screening were injected into the tail veins of mice. It was found that the amounts of EVs-30° and EVs-50° aggregated at the tumor site were significantly higher than those of EVs-0°, and EVs-50° showed faster and higher tumor site aggregation ability, while most of EVs-0° stayed in the liver ( Figure 7 ).

[0080] In order to enable cells to secrete EVs with therapeutic functions, this example compared the performances of three cell modification methods, namely liposome (Lipo), bulk electroporation (BEP), and nanopore focused electric field, which are commonly used at present, in terms of cell activity, delivery efficiency, and EVs yield. Compared with the other two methods, the nanopore focused electric field could maintain higher cell activity (>90%) and delivery efficiency (>72%) ( Figure 8 a, b). This was because the nanopores of this method could generate a focused and directional electric field at low voltage, providing support for the transient reversible opening of the cell membrane and the directional and rapid movement of molecules to be delivered. In addition, the nanopore focused electric field could effectively promote the production of EVs ( Figure 8 c, d). Therefore, the 3E platform of the present invention selected the method of nanopore focused electric field to electro-remodel cells to obtain EVs with therapeutic functions.

[0081] Example 4 In Example 1, the EVs secreted by the screened tumor cells were also modified.

[0082] The method of Example 1 was used to electro-reshape the target cells, and the EVs secreted by the electro-reshaped cells were collected. After verification, the expression level of IFNγ mRNA and the content of PD-L1 siRNA in the EVs secreted by the cells after electro-delivery of IFNγ plasmid and PD-L1 siRNA were significantly increased ( Figure 9 ). This result indicates that the EVs were successfully loaded with IFNγ mRNA and PD-L1 siRNA.

[0083] Furthermore, the above-modified EVs were co-incubated with the parental cells, and then the expression of IFNγ and PD-L1 in the parental cells was identified. The results showed that at the RNA level, the IFNγ mRNA in the parental cells was significantly increased, and at the same time, the expression of PD-L1 mRNA was significantly decreased ( Figure 10 a, b). In terms of protein expression, the expression of IFNγ protein in the parental cells was significantly increased, and the expression of PD-L1 protein was significantly decreased ( Figure 10 d-f). The above results indicate that the modified EVs successfully achieved the effect on tumor cells. Among them, Control represents the parental cells after co-incubation with EVs derived from untreated cells; IFNγ represents the parental cells after co-incubation with EVs derived from cells carrying IFNγ plasmid; PD-L1 represents the parental cells after co-incubation with EVs derived from cells delivered with PD-L1 siRNA; IFNγ + PD-L1 represents the parental cells after co-incubation with EVs derived from cells delivered with IFNγ plasmid and PD-L1 siRNA simultaneously.

[0084] Example 5 To further verify the effect of the modified EVs in inhibiting tumor metastasis, two mouse CDX models were constructed according to two clinical situations of drugs used for tumor metastasis prevention and treatment.

[0085] For the reconstruction of the tumor metastasis prevention model, in this example, a BALB / c mouse subcutaneous xenograft model was first constructed. When the tumor volume reached 40 mm 3 , the macroscopically distinguishable tumor tissue was removed by surgical operation. Then, tumor cells were injected into the mouse tail vein to simulate the scenario of in vivo tumor metastasis. Subsequently, the mouse models were divided into 5 groups and different groups of treatments were carried out. The results of the survival rate of the treated mice showed that the EV@IFNγ + PD-L1 group significantly improved the survival rate of the mice compared with the Control group. The EV@IFNγ and EV@PD-L1 groups were lower than the EV@IFNγ + PD-L1 group in terms of improving the survival rate. The results of the EVs group were similar to those of the Control group and had no effect on improving the survival rate (Figure 11 a). Next, the recurrence of incompletely resected in situ tumors visually distinguishable was further evaluated. Among them, the inhibitory effect of the EV@IFNγ+PD-L1 group on tumor growth (inhibiting recurrence) was significantly better than that of the EV@IFNγ group and the EV@PD-L1 group, while the tumor volume growth of the Control group and the EV group was similar and significantly larger than that of the other three groups ( Figure 11 b). Further, the situation of tumor metastasis to the lungs was evaluated. According to the analysis results of lung nodules, compared with other groups, the EV@IFNγ+PD-L1 group had the fewest lung nodules ( Figure 11 c, d). Among them, Control represents EVs derived from untreated cells; IFNγ represents EVs derived from cells carrying the IFNγ plasmid; PD-L1 represents EVs derived from cells delivered with PD-L1 siRNA; IFNγ+PD-L1 represents EVs derived from cells delivered with the IFNγ plasmid and PD-L1 siRNA.

[0086] Subsequently, for the reconstruction of tumor metastasis treatment, first, the evolution screening module in the 3E platform of Example 1 was used to screen out more invasive cells from the mouse lung cancer cell line Lewis, and then a tumor xenograft model was constructed using these cells to mimic tumor metastasis lesions. When the tumor volume reached 80 - 100 mm 3 ³, these mice were randomly divided into 5 groups and received different treatment regimens. The grouping of treatment regimens was the same as that used in the reconstruction of the first type of treatment regimen. After treatment, compared with the tumor volumes of mice in the Control group and the EVs group, the tumor volumes of mice in the EVs group modified with immunotherapeutic molecules were smaller. Among them, the tumor volume of mice in the EV@IFNγ+PD-L1 group was significantly smaller than that of other groups ( Figure 12 ).

[0087] To be closer to the real tumor environment, in this example, lung cancer tumor tissues resected surgically from clinical lung cancer patients were further collected, and after in vitro primary cell culture and proliferation in NTG mice with autoimmune deficiency in vivo, a PDX model composed of the 3rd generation human lung cancer cells was constructed. Subsequently, the PDX model was used to deeply explore the preventive and therapeutic effects of EVs on tumor metastasis respectively. The method was to treat the mouse PDX model with the same 5 treatment methods as those used in the CDX model. The results of in situ tumors showed that compared with Control, there was no significant difference in the tumor volume growth of the EVs group, the EV@IFNγ and EV@PD-L1 groups had a certain inhibitory effect on tumor volume growth, while the EV@IFNγ+PD-L1 group had the most obvious inhibitory effect on tumor growth ( Figure 13a). The lung images of the mice and the statistical results of the number of nodules also showed that the number of lung nodules in the EV@IFNγ+PD-L1 group was the least ( Figure 13 b, c).

[0088] Subsequently, the PDX model was used to verify the tumor metastasis treatment model. First, the evolution screening module in the 3E platform was used to screen out more invasive cells from the tumor tissue, and then a tumor xenograft model was constructed using these cells to mimic the tumor metastasis lesions. Subsequently, the mice were randomly divided into 5 groups and treated with the same reagents as in the CDX model. As the treatment process of the mice in each group progressed, compared with the tumor volumes of the mice in the other four groups, the inhibitory effect of the EV@IFNγ+PD-L1 group on the tumor volume of the mice was the most obvious ( Figure 14 ).

[0089] It should be noted that in this article, the terms "including", "comprising" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements not only includes those elements, but also includes other elements not expressly listed, or further includes elements inherent to such process, method, article or device. Without further limitations, an element defined by the statement "including one..." does not exclude the existence of additional identical elements in the process, method, article or device including that element.

[0090] The embodiments of the present invention have been described above in conjunction with the accompanying drawings, but the present invention is not limited to the above specific embodiments. The above specific embodiments are merely illustrative and not restrictive. Under the inspiration of the present invention, those of ordinary skill in the art can also make many forms without departing from the purpose of the present invention and the scope protected by the claims. These all belong to the protection scope of the present invention.

Claims

1. An integrated chip device for screening and functional modification of highly invasive cells, characterized in that, The integrated chip device is provided with two modules of cell screening and functional transformation, including multiple functional layers; Among them, multiple micropores are provided on the first functional layer 100 for sample loading or sample output, including the first micropore 110, the second micropore 120, the third micropore 130 and the fourth micropore 140; A microchannel 210 is provided on the second functional layer 200 for liquid circulation; A cell electro-remodeling region 310 and a cell evolutionary screening region 320 with a hollow circle structure are provided on the third functional layer 300; wherein, the edge of the hollow circle of the cell evolutionary screening region 320 has a slope to form two inner and outer circular structures, and the slope range is set to 20°-80°; the inner circular diameter in the hollow circle structure of the cell evolutionary screening region 320 is smaller than the outer circular diameter; a nano-film 330 is also provided on the third functional layer 300 and attached to the bottom of the cell electro-remodeling region 310; A reagent storage region 410 and a cell culture region 420 with a hollow circle structure are provided on the fourth functional layer 400; wherein the reagent storage region 410 is arranged below the cell electro-remodeling region 310; the cell culture region 420 is arranged below the cell evolutionary screening region 320; and the diameter of the cell culture region 420 is equal to the inner circular diameter of the cell evolutionary screening region 320; The fifth functional layer 500 is set as an electrode; at least one pair of micropores are also provided on the fifth functional layer 500 for sample loading or sample output.

2. The integrated chip device according to claim 1, characterized in that The slope of the cell evolutionary screening region 320 is set to 30°-50°.

3. The integrated chip device according to claim 1, characterized in that The cell electro-remodeling region 310 is set to deliver target substances to target cells by using the nano-pore focused electric field technology; the target substances include immunotherapy molecules and / or drugs.

4. The integrated chip device according to claim 3, characterized in that, The target cells include tumor cells, immune cells and / or stem cells.

5. The integrated chip device according to claim 1, characterized in that, The material of the integrated chip device is a transparent material; the transparent material includes polydimethylsiloxane and / or polymethyl methacrylate.

6. The integrated chip device according to claim 1, wherein, The first functional layer 100 can seal the microchannel 210 provided on the second functional layer 200; or, multiple micropores are provided on the second functional layer 200 and the third functional layer 300 and are connected and penetrated with the micropores on the first functional layer 100.

7. The integrated chip device according to claim 1, characterized in that, The fifth functional layer 400 is also provided with a fifth micropore 510 and a sixth micropore 520 for sample loading or sample output; or, multiple drainage holes are also provided on the third functional layer 300 and the fourth functional layer 400 for draining liquid into the microchannel 210.

8. A method for preparing extracellular vesicles with enhanced tumor targeting and therapeutic functions, characterized in that, The method is implemented based on the integrated chip device according to any one of claims 1-7, and the specific steps are as follows: S01: Prepare a tumor cell suspension, inject the tumor cell suspension through the micropores on the first functional layer 100, and the tumor cells sequentially enter the cell culture region 420 through the through micropores provided on the second functional layer 200, the third functional layer 300 and the fourth functional layer 400; when it is observed that the tumor cells complete adherent growth, supplement the cell culture medium to the cell screening region 320; S02: After observing that the tumor cells adhere and grow on the slope of the cell screening region 320, aspirate the non-adherent tumor cells and the excess cell culture medium through the micropores on the first functional layer 100; S03: Flip the integrated chip device, inject a small amount of trypsin from the sixth micropore 520 so that the trypsin liquid level is only in the region of the cell screening region 320, and briefly incubate with the tumor cells at room temperature to digest the tumor cells, and continue to inject fresh cell culture medium from the sixth micropore 520 to terminate the digestion; S04: The tumor cells in the cell screening region 320 enter the microchannel 210 through the drainage holes on the fourth functional layer 400 and the third functional layer 300, and flow into the cell electro-remodeling region 310 through the microchannel 210; S05: Flip the integrated chip device so that the tumor cells entering the cell electro-remodeling region 310 adhere to the nanofilm 330; S06: Inject the reagent containing the target substance to be delivered by electroporation through the micropores on the first functional layer 100 into the reagent storage region 410, connect the electrodes of the electroporator on the first functional layer 100 and the fifth functional layer 500 respectively, and apply a pulsed electric field to the tumor cells in the cell electro-remodeling region 310 for electroporation; S07: After applying electroporation, continue to culture the tumor cells in situ, and aspirate the supernatant of the tumor cell culture from the fifth micropore 510 for extracting functionalized extracellular vesicles.

9. The method according to claim 8, characterized in that, The target substance includes immunotherapeutic molecules and / or drugs.

10. The method according to claim 9, characterized in that, The immunotherapeutic molecules include IFNγ and / or PD-L1 siRNA.

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